Human cgrp receptor binding proteins

HUMAN CGRP RECEPTOR BIND PROTEINS

CONTEXT

This application contains a sequence listing that was submitted by EFS-Web and is incorporated herein by reference in its entirety. Said ASCII copy, created on December 14, 2009, is called A1472PCT.txt, and its size is 312,447 bytes.

The calcitonin peptide superfamily includes at least five known members: calcitonin, amylin, adrenomedullin, and two calcitonin gene-related peptides (CGRP ”, calcitonin gene-related peptides), CGRP1 (also called ctCGRP, or CGRP) and CGRP2 (also known as GAAP). CGRP is a vasoactive 37 amino acid neuropeptide expressed in the central and peripheral nervous systems, which has been shown to be a potent vasodilator at the periphery, with neurons containing CGRP being closely associated with blood vessels. CGRP-mediated vasodilation is also associated with neurogenic inflammation, as part of a cascade of events that results in extravasation of plasma and vasodilation of the microvascular system and is present in migraine. Amylin also has specific binding sites in the CNS and is thought to regulate gastric emptying and play a role in carbohydrate metabolism,! - Adrenomedullin is a potent vasodilator. Adrenomedullin has specific receptors on astrocytes and its messenger RNA is upregulated in CNS tissues affected by ischemia (Zimmermann, et al .. Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastoma glioma hybrid cells (NG1O8- 15), Brain Res., 724: 238-245 (1996); Wang et al., Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage, Proc. Natl. Acad. Sci. USA, 92: 11480-11484 (1995)).

Calcitonin is involved in the regulation of bone metabolism and is also active in the central nervous system (CNS). The biological activities of CGRP include the regulation of neuromuscular junctions, antigen presentation in the immune system, vascular tone and sensory neurotransmission (Poyner, DR, Calcitonin gene-related peptide: multiple actions, multiple receptors, Pharmacol . Shoot., 56: 23-51 (1992); Muff et al., Calcitonin, calcitonin gene related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions, Eur. j. Endocrinol., 133: 17-20 (1995)). Three calcitonin receptor stimulating peptides (CRSP) have also been identified in several species of mammals: CRSPs could form a new subfamily in the CGRP family (Katafuchi, T and Minamino, N, Structure and biological properties ofthree calcitonin receptorΜΑ 32982Β1

A-1472-WO-PCT stimulating peptides, novel members of the calcitonin gene-related peptide family, Peptides, 25 (11): 2039-2045 (2004)).

Peptides of the calcitonin superfamily act through paired G-protein receptors (GPCRs) at seven transmembrane domains. Calcitonin receptors ("CT", "CTR" or "CT receptor") and CGRP receptors are type II GPCRs (family B), this family comprising other GPCRs which recognize regulatory peptides such as secretin , glucagon, and vasoactive intestinal polypeptide (VIP). The best characterized human calcitonin receptor splice variants differ depending on whether the 16 amino acids in the first intracellular loop are present (formerly CTR „٠ or CTR1, now referred to as CT) or absent (the splice variant major, previously called CTRu- or CTR2, and now CT (a)) (Gom et al .. Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell ttimor of bone: the first intracellular domain modulates ligand binding and signal ttansduction, J. Clin. Invest., 95: 2680-2691 (1995); Hay et al., Amylin receptors: molecular composition and pharmacology, Biochem. Soc. Trans., 32: 865-867 (2004); Poyner et al., 2002). The existence of at least two subtypes of CGRP receptors has been proposed based on the affinities of differential antagonists and the efficacies of agonists in various in vivo and in vitro biological tests (Dennis et al., CGRP8- 37, A calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery, I Pharmacol. Exp. Ther., 254: 123-128 (1990); Dennis et al., Sttucttre-activity profile of calcitonin generelated peptide in peripheral and brain tissues. Evidence for multiplicity, J. Pharmacol. Exp. Ther., 251: 718-725 (1989); Dumont et al., Apotent and selective CGRP2 agonist, [Cys (Et) 2,7] hCGRE: comparison in prototypical CGRP, and CGRP2 in vitro assays, Can. J. Physiol. Pharmacol., 75: 671-676 (1997)).

The CGRPi receptor subtype has been shown to be sensitive to the CGRP antagonist fragment (8-37). (Chiba et al., Calcitonin gene-related peptide receptor antagonist human CGRP (8-37), Am. J. Physiol., 256: Ε331-Ε335 (1989); Dennis et al. (1990); Mimeault et al .. Comparative affinities and antagonistic potencies of various human calcitonin gene-related peptide fragments on calcitonin gene-related peptide receptors in brain and periphery, I. Pharmacol. Exp. Ther., 258: 1084-1090 (1991)). In contrast, the CGRP2 receptor was sensitive to linear human CGRP analogs (hCGRP), in which the cysteine ​​residues at positions 2 and 7 were derived (eg, with the acetoaminomethyl group [Cys (ACM)²'?] or the ethylamide group [Cys (Et)²’⁷]), but the CGRP2 receptor was /

ΜΑ 32982Β1

A-1472-WO-PCT insensitive to the CGRP (8-37) fragment (Dennis et al. (1989); Dennis et al. (1990); Dumont et al. (1997)).

The ligand specificity of a calcitonin receptor and a calcitonin-like receptor ("CL", "CLR" or "CRLR") depends on the co-expression of members of a family of accessory proteins called proteins modifying. receptor activity rdifying proteins (RAMP). The RAMP family comprises three polypeptides (RAMP1, RAMP2 and RAMP3) which act as receptor modulators which determine receptor specificity for members of the calcitonin family. RAMPs are type I transmembrane proteins that share an amino acid sequence identity of about 30٠ / ο and a common predicted topology, with short cytoplasmic terminal ends, a ttansmembrane domain, and large extracellular terminal ends that are responsible. of the specificity (McLatchie et al., (1998) RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature, 393: 333-339; Fraser et al., (1999) The amino terminus of receptor activity modifying proteins is a critical determinant of glycosylation State and ligand binding of calcitonin receptor-like receptor, Molecular Pharmacology, 55: 1054-1059).

In 1998, the CGRPi receptor was identified as a heterodimer composed of a novel accessory protein to a single transmembrane domain, the protein modifying the activity of receptors 1 (RAMP1) and CRLR. (McLatchie et al., Supra). Cross-linking experiments suggest that the CGRP receptor consists of a one-to-one stoichiometric arrangement of CRLR and RAMP1 (Hilair et al. JBC 276, 42182-42190 (2001)), more recent studies using several methodologies such as BRET and BiFC have revealed that the functional CGRP receptor complex may be composed of an asymmetric homooligomer of CRLR and a RAMP1 monomer (Heroux et al. JBC 282, 31610-31620 (2007)).

A purified CRLR N-terminal domain has been shown to specifically bind ICGRP (Chauhan et al. Biochemistry 44, 782 (2005)), confirming the strong and direct interaction between CRLR and the CGRP ligand. In particular, Leu 24 and Leu 34 of CRLR are believed to constitute the docking site of the Phe37 c-terminus of CGRP (Banerjee et al. BMC Phannacol. 6, 9 (2006)). In addition, Koller et al. (FEBS Lett. 531, 464-468 ​​(2002)) obtained evidence that the 18 N-terminal amino acid residues of CRLR contribute to the selective interaction, with CGRP or adrenomedullin, and Ittner et al (Biochemistry 44, 57495754 ( 2005)) suggested that N-terminal amino acid residues 23-60 of CRLR mediate association with RAMP1.

ΜΑ 32982Β1

A-1472-WO-PCT

Analysis of the structure and function of RAMP1a identifies residues 91-103, which correspond to "helix 3" (Simms et al. Biophys. J. 91, 662 - 669 (2006)), as potentially significant in interaction with CRLR, and residues Trp74 and Phe92 as potentially interacting with the CGRP ligand in connection with its binding to the CGRP receptor complex. Ligand binding studies using a human / rat RAMP1 chimera suggest that the binding site for some CGRP R inhibitory small molecules (eg, BIBN4096BS), is located in a region that includes amino acids 66- 102 of RAMP1 (Mallee et al. JBC 277, 14294-14298 (2002)).

CRLR shows an overall sequence identity of 55٠ / with CTR, but the transmembrane domains are nearly 80٠ / ο identical (McLatchie et al. (1998); Poyner et al., International union of pharmacology. XXXII. The mammalian calcitonin. gene-related peptides, adrenomedullin, amylin and calcitonin receptors, Pharmacol. Rev., 54: 233-246 (2002)).

CRLR has been shown to form a high affinity receptor for CGRP, when associated with RAMP1, or preferentially bind adrenomedullin when associated with RAMP2 or RAMP3 (McLatchie et al. (1998); Sexton et al., Receptor activity modifying proteins. Cellular Signaling, 13: 73-83 (2001); Conner et al .. Interaction of calcitonin-generelated peptide with its receptors, Biochemical Society Transactions 30 (Part 4): 451 -454 ( 2002)). The glycosylation state of CRLR is associated with its pharmacology. RAMP 1,2 and 3 transport CRLR to the plasma membrane with similar efficiencies, but RAMP1 presents CRLR as a mature glycosyl-terminated glycoprotein, and a CGRP receptor, while RAMP 2 and 3 presents CRLR as a centrally glycosylated, immature adrenomedullin receptor ("AM" or "AMR" or "AM receptor" (Fraser et al. (1999)). Characterization of CRLR / RAMP2 and CRLR / RAMP3 receptors in ΗΕΚ293Τ cells by radioactive ligand binding ('I-adrenomedullin as radioactive ligand), functional test (measurement of cAMP), or biochemical analysis (SDS-polyacrylamide gel electrophoresis ) revealed that they could not be distinguished, even though RAMP 2 and 3 share an amino acid sequence identity which is only 30% (Fraser et al. 1999)). However, differences were observed in the pharmacology for CRLR expressed with RAMP 2 versus RAMP 3. CGRP and CGRP8-37, as well as adrenomedullin and the peptide derived from adrenomedullin AM 22-52, are active at level 1. 'RAMP 3 heterodimer, indicating that this complex could act as both CGRP receptor and AM receptor (Howitt et al., British Journal of Pharmacology, 140: 477-486 (2003); Muffet al., Hypertens, Res., 26: S3-S8 (2003)). Co-expression of human CRLR with RAMP1 of

ΜΑ 32982Β1

A-1472-WO-PCT rat, and vice-versa, suggests that the RAMP1 species determines the pharmacological characteristics of the CRTHAMP1 complex against several small molecule CGRE receptor antagonists tested. (Mallee et al., Receptor ActivityModifying Protein 1 determines the species selectivity of ηοη-peptide CGRP receptor antagonists, j. Biol. Chem., 277 (16): 14294-14298 (2002)). Unless associated with RAMP, no binding of CRLR to any endogenous ligand is known; it is currently the only GPCR to behave in this way (Conner et al., A key role for transmembrane prolines in calcitonin receptor-like agonist binding and signaling: hnplications for family B G-proteincoupled receptors, Molec. Pharmacol., 67 (1): 20-31 (2005)).

A calcitonin receptor (CT) has also been shown to form heterodimeric complexes with RAMPs, which are known as amylin receptors ("AMY", "AMY R" or "AMY receptor"). Generally, the CTRAMP1 receptors (designated by “AMY 1” or “AMY 1”) exhibit a high affinity for salmon calcitonin, amylin and CGRP and a lower affinity for mammalian calcitonins. For CT / RAMP2 receptors ("ΑΜΥ2" or "ΑΜΥ2") and CT / RAMP3 receptors ("ΑΜΥ3" or "ΑΜΥ3"), a similar pattern is mainly observed, although the affinity for CGRP is lower, and may not be significant at physiologically relevant ligand concentrations. The precise phenotype of the receptor depends on the type of cells and the CTR splice variant (CT (a) or CT (b)) ١ in particular for the amylin receptors generated by RAMP2. For example, a pure population of osteoclast-like cells has been reported to express RAMP2, CTR, and CRLR, but not RAMP1 or RAMP3 (Hay et al.

(2004) ; Christopoulos et al .. Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product, Molecular Pharmacology, 56: 235-242 (1999); Muff et al .. An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-l or -3, Endocrinology, 140: 29242927 (1999); Sexton et al. (2001); Leuthauser et al., Receptor-activity-modifying protein 1 forms heterodimers with two G-protein-coupled receptors to define ligand recognition, Biochem. j., 351: 347-351 (2000); Tilakaratne et al., Amylin receptor phenotypes derived from human calcitonin receptor / RAMP co-expression exhibit pharmacological differences depend on receptor isofonn and host cell environment, j. Pharmacol. Exp. Ther., 294: 61-72 (2000); Nakamura et al., Osteoclastlke cells express receptor activity modifying protein 2: application of laser capttrre microdissection, j. Molec. Endocrinol., 34: 257-261 (2005)).

Table 1 below summarizes the relationships of receptor components discussed above.

ΜΑ 32982Β1

A-1472-WO-PCT

Table 1

<td>Component of receiver</td><td>CRLR (CL)</td><td>CT (receptor of calcitonin)</td>

<td>RAMPl</td><td>CGRP Receiver</td><td>AMY1 receiver</td>

<td>RAMP2</td><td>AMI Receiver</td><td>Receiver ΑΜΥ2</td>

<td>RAMP3</td><td>Receiver ΑΜ2</td><td>Receiver ΑΜΥ3</td>

Therapeutic uses of CGRP antagonists have been proposed. Noda et al. describe the use of CGRP or CGRP derivatives to inhibit platelet aggregation and for the treatment or prevention of arteriosclerosis or thrombosis (EP 0385712 B1). Liu et al. describe therapeutic agents which modulate the activity of CTR, in particular peptides conjugated to a vehicle such as calcitonin and human aCGRP (WO 01/83526 Α2; US 2002/0090646 A1). Vasoactive CGRP peptide antagonists and their use in a method of inhibiting the binding of CGRP to CGRP receptors has been described by Smith et al. ; These CGRP peptide antagonists have been shown to inhibit binding to coronary artery membranes and relax capsaicin-treated porcine coronary arteries (US Patent No. 6,268,474 B1; and US Patent No. 6,756,205 Β2). Rist et al. describe peptide analogs with CGRP receptor antagonist activity and their use in a medicament for the treatment and prophylaxis of various rubles (DE 19732944 A1).

CGRP is a potent vasodilator which has been implicated in the pathology of several vasomotor symptoms, such as all forms of vascular headache, including migraines (with or without aura) and cluster headache. Durham, N. Engl. j. Med. 350: 1073-1075, 2004. The pathophysiology of migraine involves activation of the trigeminal ganglia, in which CGRP has been localized, and CGRP levels increase significantly during a migraine attack. This action promotes the dilation of cranial blood vessels and neurogenic inflammation and sensitization (Doods, H., Curr. Opin. Investig. Drugs, 2: 1261-1268 (2001)). In addition, serum CGRP levels in the external jugular vein are elevated in patients with migraine headache. Goadsby et al., Ann. Neurol. 28: 183-7, 1990. Intaveinous administration of human ci-CGRP has induced headache and migraine in migraine-free patients without aura, confirming the hypothesis that CGRP has a causal role in migraine (Lassen et al, Cephalalgia 22:54 -61, 2002).

Migraine is a common complex neurological condition characterized by severe and episodic headache attacks and associated features, which may

<img file="MA32982B1_D0001.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT understands nausea, vomiting, sensitivity to light, noise or movement. In some patients, the headache is preceded or accompanied by an aura. Headache pain can be severe and can also be one-sided in some patients. Migraine attacks disrupt everyday life. In the United States of America and Western Europe, the overall prevalence of migraine sufferers is 11% in the general population (6% of men; 15-18% of women). In addition, the median frequency of seizures in an individual is 1.5 / month. While there are several treatments available to relieve or reduce symptoms, preventative therapy is recommended for patients with more than 3-4 migraine attacks per month. Goadsby, et al. New Engl. J. Med. 346 (4): 257-275,2002.

Some migraine patients have been treated with topiramate, an anticonvulsant that blocks voltage-dependent calcium channels, and certain glutamate receptors (AMPAkainate), potentiates the activity of the GABA-A receptor, and blocks carbonic anhydrase. The relatively recent success of 5ΗΤ-Ι Β / ID and / or 5ΗΤ-1a serotonin receptor agonists, such as sumariptan, in some patients has led researchers to propose a seratonergic etiology for the disorder. Unfortunately, although some patients respond well to this treatment, aures are relatively resistant to its effects.

The possible involvement of CGRP in migraine has served as the basis for the development and testing of many compounds which inhibit the release of CGRP (eg, sumatriptan), exert an antagonistic effect on the CGRP receptor (eg, dipeptide derivative BIBN4096BS (Boehringer Ingelheim); CGRP (8-37)), or interact with one or more receptor-associated proteins, such as RAMP1. Brain, s. et al., Trends in Pharmacological Sciences 23: 51-53, 2002. Alpha-2 adrenoeceptor subtypes and adenosine A1 receptors also control (inhibit) CGRP release and trigeminal activation (Goadsby et al., Brain 125: 1392-401, 2002). On the other hand, treatment with compounds which exclusively inhibit neurogenic activation (eg, tachykinin ΝΚ1 receptor antagonists) or trigeminal activation (eg, 5ΗΤ10 receptor agonists) appears to be relatively ineffective as an acute treatment for migraine. which raises the question of whether the inhibition of the release of CGRP is indeed the basis of effective anti-migraine treatments. Aralmani et al., Eur. I. Pharmacol. 500: 315-330, 2004.

Although the precise pathophysiology of migraine is not yet well understood, the therapeutic use of CGRP antagonists and aptamers targeting CGRP has been proposed for the treatment of migraine and other disorders (e.g., Olesen et al. ., Calcitonin gene-related peptide receptor antagonist BIBN 4096 BS for the acute treatment ofmigraine.

/

ΜΑ 32982Β1

A-1472-WO-PCT

New Engl. j. Med.) 350: 1104-1110 (2004); Perspective: CGRP-receptor antagonists - a fresh appreach to migraine, New Engl. j. Med., 350: 1075 (2004); Vater et al .. Short bioactive Spiegelmers to migraine-associated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-SELEX, Nue. Acids Res., 31 (21-130): 1-7 (2003); wo 96/03993).

Additionally, a potent, small molecule CGRP antagonist has been shown to relieve moderate to severe migraine attacks, including migraine pain and migraine-associated symptoms, in a recent Phase III clinical trial (Connor, et al. Efficacy and Safety of telcagep nt (ΜΚ-0974), a Novel Oral CGRP Receptor Antagonist, for Acute Migraine Attacks. Poster, European Headache and Migraine Trust International Congress,

London, England, September 2008).

CGRP can also be involved in chronic pain syndromes other than migraine. In rodents, CGRP administered intrathecally induces severe pain, and CGRP levels increase in several pain models. In addition, CGRP antagonists partially block nociception in acute pancreatitis in rodents (Wick, et al., (2006) Surgery, Volume 139, Issue 2, Pages 197-201). Taken together, these observations imply that a potent and selective CGRP receptor antagonist could be an effective therapeutic substance for the treatment of chronic pain, especially migraine.

ABSTRACT

Isolated antibodies, their antigen-binding fragments, and other isolated antigen-binding proteins that bind CGRP R, particularly CGRP R from primates, eg, human CGRP R, are described herein. These isolated antigen-binding proteins can selectively inhibit primate CGRP R (compared to AMI, ΑΜ2, CT, or primate amylin receptors) and can bind both the CRLR and RAMP1 components of CGRP R. CGRP R binding proteins have been shown to inhibit, interfere with, or modulate at least one of the biological responses related to CGRP R, and as such, are therefore useful for ameliorating the effects of diseases or disorders associated with CGRP R. The binding of certain antigen-binding proteins to CGRP R may therefore have one or more of the following activities: inhibit, interfere with or modulate CGRP R, inhibit vasodilation, reduce neurogenic inflammation, and alleviate, enhance, treat, prevent or reduce symptoms of chronic pain or migraine.

In an illustrative aspect, the isolated antigen-binding proteins selectively inhibit the human CGRP receptor (relative to AMI, ΑΜ2 or human amylin receptors). In some embodiments, the isolated antigen-binding protein selectively inhibits the

<img file="MA32982B1_D0002.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT human CGRP receptor with a selectivity ratio of 50 or more, 75 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 400 or more, 500 or more, 750 or more or 1000 or more. The degree of selective inhibition can be determined using any suitable method, for example using a cAMP assay as described in the examples given herein. In some embodiments, the isolated antigen-binding protein binds specifically to both human CRLR and human RA MPI, and does not specifically bind human AMI, human ΑΜ2, or a human amylin receptor (e.g. , AMY1 or ΑΜΥ2). For example, the isolated antigen-binding protein can specifically bind human CGRP R with a K il μΜ, 00 nM, iio nM or 5 ؛ nM. In some embodiments, the isolated antigen-binding protein specifically binds to human CGRP R with a Kd 00 nM, nM, or 5 ؛ nM, as determined by a FACS binding assay and analysis, for example, using the methods described in Rathanaswami, et al., Biochemical and BiophysicalResearch Communications 334 (2005) 10041013. In some embodiments, the binding protein an isolated antigen has a Ki 00 nM, 10 ؛ nM, 1 ؛ nM, 0.5 ؛ nM or

0.1 ؛ nM in a competition assay for binding to CGRP. In some embodiments, the antigen-binding protein isolated aunKi 100 ؛ nM, 50 ؛ nM, 20 ؛ nM, 10 ؛ nM, 1 ؛ nM, 0.5 ؛ nM or 0.1 ؛ nM in a competitive assay for the binding of radiolabeled) Ι-CGRP to membranes of cells expressing CGRP R, e.g., e.g., the assay described in Example 5 presented herein.

In another illustrative aspect, isolated antigen-binding proteins compete for binding to human CGRP R, e.g., the extracellular portion of CGRP R, with an antibody of reference comprising a heavy chain variable region comprising a sequence selected from. the set consisting of SEQ ID ΝΟ: 158-170 and a light chain variable region comprising a sequence selected from the set consisting of SEQ ID ΝΟ: 137-153. In some embodiments, competition for binding is assessed using binding assays, e.g., using Biacore assay, e.g., as described in Example 7 presented herein. In some embodiments, the isolated antigen-binding protein competes for binding to human CGRP R with a reference antibody, the reference antibody comprising (i) a heavy chain variable region comprising a sequence selected from the reference antibody. set consisting of SEQ ID NOs: 161, 163, 164, 166 and 168; and (ii) a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NOs: 140, 143, 146, 148 and 150. In certain embodiments, the reference antibody comprises (i) a heavy chain defined by a sequence selected from the group consisting of SEQ ID NOs: 32, 34, 35, 37 and 39; and (ii) a string /

ΜΑ 32982Β1

Light A-1472-WO-PCT defined by a sequence selected from the group consisting of SEQ ID NOs: 15, 18, 21, 23 and 25. In more specific embodiments, the antibody of referenced comprises a heavy chain and a light chain defined by one of the following sequence pairs: (i) SEQ ID NO: 32 and SEQ ID NO: 15; (ii) SEQ ID NO: 34 and SEQ ID NO: 18; (iii) SEQ ID NO: 35 and SEQ ID NO: 21; (iv) SEQ ID NO: 37 and SEQ ID NO: 23; and (v) SEQ ID NO: 39 and SEQ ID NO: 25. In such an embodiment, the antibody of reference comprises a heavy chain comprising SEQ ID NO: 32 and a light chain comprising SEQ ID NO: 15. In another of these embodiments, the anticoips of reference comprises a chain heavy chain comprising SEQ ID NO: 34 and a light chain comprising SEQ ID NO: 18. In another of these embodiments, the reference antibody comprises a heavy chain comprising SEQ ID NO: 35 and a light chain comprising SEQ ID NO: : 21. In another of these embodiments, the reference antibody comprises a heavy chain comprising SEQ ID NO: 37 and a light chain comprising SEQ ID NO: 23. In another of these embodiments, the reference antibody comprises a heavy chain comprising SEQ ID NO: 39 and a light chain comprising SEQ ID NO: 25.

In some embodiments, isolated antigen-binding proteins that compete for binding to human CGRP R also selectively inhibit the human CGRP receptor, for example with a selectivity ratio of 100 or more, 250 or more, 500 or more. more, 750 or more, 1,000 or more, 2,500 or more, 5,000 or more or 10,000 or more, and this selectivity can be determined, for example, by means of a cAMP test as described in the examples given here. In related embodiments, isolated antigen-binding proteins that compete for binding to human CGRP R specifically bind to human CGRP R with a Kd1 ؛ μΜ, 00 nM, &lt;10 nM, or &lt;5 nM, for example, as determined by a FACS binding assay and analysis, for example, using the methods described in Rathanaswami, et al., Biochemical سع Biophysical Research Communications 334 (2005) 10041013. In related embodiments, the isolated antigen-binding proteins that compete for binding to human CGRP R have a Ki of 100 ؛ nM, 10 ؛ nM, 1 ؛ nM, 0.5 ؛ nM or 0.1 ؛ nM in a competition assay for binding to CGRP, e.g., in a competition assay for the binding of radiolabeled 'Ι-CGRP to membranes of cells expressing CGRP R, e.g., e.g., the assay described in Example 5 present here.

In any of the above embodiments, the isolated antigen-binding proteins which compete for binding to human CGRP R may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antibody. human

ΜΑ 32982Β1

A-1472-WO-PCT (eg, fully human), a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof. In addition, the antibody fragment of the isolated antigen-binding protein which competes for binding with human CGRP R may be an Fab fragment, and a Fab 'fragment, an F (ab') 2 fragment, an Fv fragment. , a diabody, or a single chain antibody molecule, and may be, for example, a human monoclonal antibody, for example, an antibody of the IgG1, IgG2, IgG3 or IgG4 type. In some embodiments, the isolated antigen-binding proteins that compete for binding with human CGRP R can be neutralizing antigen-binding proteins.

In certain illustrative aspects, the isolated antigen-binding proteins described, for example, isolated antibodies or fragments thereof comprise (A) one or more heavy chain complementarity determining regions (CDRH) selected from the group consisting of: (i) a CDRH1 having SEQ ID NO: 134; (ii) a CDRH2 having SEQ ID NO: 135; (iii) a CDRH3 having SEQ ID NO: 136; and optionally (iv) a CDRH of (i), (ii) and (iii) which contains one or more substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids which collectively do not add up to more than four amino acids; (B) one or more light chain complementarity determining regions (CDRL) selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NOs: 107, 111 and 118; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 108, 112 and 119; (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 109,113 and 120; and optionally (iv) a CDRL of (i), (ii) and (iii) which contains one or more, eg, one, two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids or more which collectively do not total more than four amino acids; or (C) one or more heavy chain CDRHs from (A) and one or more light chain CDRLs from (B).

In some embodiments, the CDRHs are among others selected from the group consisting of: (i) a CDRH1 having SEQ ID ΝΟ.-131; (ii) a CDRH2 having SEQ ID NO: 132; (iii) a CDRH3 having SEQ ID NO: 133; and optionally (iv) a CDRH of (i), (ii) and (iii) which contains one or more, for example one, two, three, four substitutions (for example, conservative amino acid substitutions), deletions or insertions of amino acids or more which collectively do not add up to more than three amino acids. In related embodiments, the CDRHs are further selected from the group consisting of: (i) a CDRH1 selected from the group consisting of SEQ ID NO: 76, 88, 100, 121, 125 and 128; (ii) a CDRH2 selected from the group consisting of SEQ ID NO: 89, 101, 122, 124,

126 and 129; (iii) a CDRH3 selected from the group consisting of SEQ ID NO: 78, 90, 102,

ΜΑ 32982Β1

A-1472.WO-PCT

123, 127 and 130; and optionally (iv) a CDRH of (i), (ii) and (iii) which contains one or more, eg one, two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids or more that collectively total no more than two amino acids. In other related embodiments, the CDRHs are further selected from the group consisting of: (i) a CDRH1 selected from the group consisting of SEQ ID NO: 73, 76, 79, 82, 85, 88, 92, 97 and 100; (ii) CDRH2 selected from the group consisting of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101 and 129; (iii) a CDRH3 selected from the group consisting of SEQ ID NO: 75,

78, 81, 84, 87, 90, 96, 99, 102 and 123; and optionally (iv) a CDRH of (i), (ii) and (iii) which contains one or more, eg one, two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids or more that collectively total no more than two amino acids.

In some embodiments, the CDRLs are further selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NOs: 107, 111 and 115; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 108, 112 and 116;

(iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 109, 113 and 117; and optionally (iv) a CDRL of (i), (ii) and (iii) which contains one or more, for example a , two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids or more that collectively do not add up to more than three amino acids per CDRL. In some embodiments, the amino acid substitutions, deletions or insertions collectively do not add up to more than two amino acids per CDRL. In some embodiments, amino acid substitutions, deletions, or insertions collectively total no more than two amino acids per CDRL. In linked embodiments, the CDRLs are further selected from the set consisting of: (i ) a CDRL1 selected from the group consisting of SEQ ID NOs: 42, 45, 51, 57, 62, 69, 103 and 110; (ii) a CDRL2 chosen from the set consisting of SEQ ID NOs:

43, 52, 55, 58, 63, 70, 104, 108 and 114; (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 44.47, 53, 56, 59, 64, 105 and 106; and optionally (iv) a CDRL of (i), (ii) and (iii) which contains one or more, eg one, two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids or more that collectively total no more than two amino acids. In other related embodiments, the CDRLs are further selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NOs: 42, 45, 48, 51, 54, 57, 62, 65, 66, and 69; (ii) a CDRL2 selected from the group consisting of SEQ ID NOs: 43,46,

ΜΑ 32982Β1

A-1472-WO-PCT

49, 52, 55, 58, 61, 63, 67, and 70; (iii) a CDRL3 selected from the group consisting of SEQ ID NOs: 44, 47, 50, 53, 56, 59, 64, 68, 71, and 72; and optionally (iv) a CDRL of (i), (ii) and (iii) which contains one or more, eg one, two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids or more. In one embodiment, the total number of amino acid substitutions, deletions or insertions is not more than two amino acids per CDR. In another embodiment, the amino acid substitutions are conservative substitutions.

In another embodiment, the isolated antigen binding protein comprises at least one or two CDRHs of any of (A) mentioned above and at least one or two CDRLs of any of (B) mentioned. above. In yet another embodiment, the isolated antigen-binding protein comprises (i) at least three CDRHs which are any of those shown in (A), the three CDRHs including CDRH1, CDRH2 and CDRH3, and ( ii) at least three CDRLs which are any of those shown in (B), the three CDRLs including CDRL1, one CDRL2 and one CDRL3. In other embodiments, the isolated antigen-binding proteins described above comprise a first amino acid sequence comprising at least one CDRH and a second amino acid sequence comprising at least one CDRL. In one embodiment, the first and second amino acid sequences are covalently linked to each other.

In another aspect, the isolated antigen binding protein comprises CDRH1, CDRH2 and CDRH3. In one embodiment, CDRH1 comprises SEQ ID NO: 73, CDRH2 comprises SEQ ID NO: 74 and CDRH3 comprises SEQ ID NO: 75. In another embodiment, CDRH1 comprises SEQ ID ΝΟ.76, CDRH2 comprises SEQ ID NO: 77 and CDRH3 comprises SEQ ID NO: 78. In another embodiment, CDRH1 comprises SEQ ID 079, CDRH2 comprises SEQ ID NO: 80 and CDRH3 comprises SEQ ID NO: 81.

In another embodiment, CDRH1 comprises SEQ ID NO: 82, CDRH2 comprises SEQ ID NO: 83 and CDRH3 comprises SEQ ID NO: 84. In another embodiment, CDRH1 comprises SEQ ID NO: 85, CDRH2 comprises SEQ ID NO: 86 and CDRH3 comprises SEQ ID NO: 87. In another embodiment, CDRH1 comprises SEQ ID NO: 88, CDRH2 comprises SEQ ID NO: 89 and CDRH3 comprises SEQ ID NO: 90. In another embodiment, CDRH1 comprises SEQ ID NO: 76, CDRH2 comprises SEQ ID NO: 91 and CDRH3 comprises SEQ ID NO: 78. In another embodiment, CDRH1 comprises SEQ ID NO: 92, CDRH2 comprises SEQ ID NO: 93 and CDRH3 comprises SEQ ID NO: 94.

In another embodiment, CDRH1 comprises SEQ ID NO: 76, CDRH2 comprises

SEQ ID NO: 95 and CDRH3 comprises SEQ ID NO: 78. In another embodiment.

ΜΑ 32982Β1

A-1472-WO-PCT

CDRH1 includes SEQ ID NO: 73, CDRH2 includes SEQ ID 0:74 and CDRH3 includes

SEQ ID NO: 96. In another embodiment, CDRH1 comprises SEQ ID NO: 97,

CDRH2 comprises SEQ ID NO: 98 and CDRH3 comprises SEQ ID NO: 99. In another embodiment, CDRH1 comprises SEQ ID NO: 100, CDRH2 comprises SEQ ID NO: 101 and

CDRH3 comprises SEQ ID NO: 102.

In another aspect, the isolated antigen binding protein comprises a CDRL1 sequence, a CDRL2 sequence and a CDRL3 sequence. In one embodiment, CDRL1 comprises SEQ ID NO: 42, CDRL2 comprises SEQ ID NO: 43 and CDRL3 comprises SEQ ID NO: 44. In another embodiment, CDRL1 comprises SEQ ID NO: 45, CDRL2 comprises SEQ ID NO: 46 and CDRL3 comprises SEQ ID NO: 47. In another embodiment, CDRL1 comprises SEQ ID NO: 48, CDRL2 comprises SEQ ID NO: 49 and CDRL3 comprises SEQ ID NO: 50. In another embodiment, CDRL1 comprises SEQ ID NO: 51, CDRL2 comprises SEQ ID NO: 52 and CDRL3 comprises SEQ ID NO: 53. In another embodiment, CDRL1 comprises SEQ ID NO: 54, CDRL2 comprises SEQ ID NO: 55 and CDRL3 comprises SEQ ID NO: 56. In another embodiment, CDRL1 comprises SEQ ID NO: 57, CDRL2 comprises SEQ ID NO: 58 and CDRL3 comprises SEQ ID NO: 59. In another embodiment, CDRL1 comprises SEQ ID NO: 60, CDRL2 comprises SEQ ID NO: 55 and CDRL3 comprises SEQ ID NO: 56. In another embodiment, CDRL1 comprises SEQ ID NO: 45, CDRL2 comprises SEQ ID NO: 61 and CDRL3 comprises SEQ ID NO: 47. In another embodiment, CDRL1 comprises SEQ ID NO: 62, CDRL2 comprises SEQ ID NO: 63 and CDRL3 comprises SEQ ID NO: 64. In another embodiment, CDRL1 comprises SEQ ID NO: 65, CDRL2 comprises SEQ ID NO: 55 and CDRL3 comprises SEQ ID NO: 56. In another embodiment, CDRL1 comprises SEQ ID NO: 66, CDRL2 comprises SEQ ID NO: 67 and CDRL3 comprises SEQ ID NO: 68. In another embodiment, CDRL1 comprises SEQ ID NO: 69, CDRL2 comprises SEQ ID NO: 70 and CDRL3 comprises SEQ ID NO: 71. In another embodiment, CDRL1 comprises SEQ ID NO: 69, CDRL2 comprises SEQ ID NO: 70 and CDRL3 comprises SEQ ID NO: 72.

In another aspect, the isolated antigen binding protein comprises a CDRL1 sequence, a CDRL2 sequence, a CDRL3 sequence, a CDRH1 sequence, a CDRH2 sequence and a CDRH3 sequence. In one embodiment, CDRL1 includes SEQ ID ΝΟ-.42, CDRL2 includes SEQ ID NO: 43, CDRL3 includes SEQ ID NO: 44, CDRH1 includes SEQ ID NO: 73, CDRH2 includes SEQ ID NO: 74 and CDRH3 includes SEQ ID NO: 75. In another embodiment, CDRL1 comprises SEQ ID ΝΟ.-45, CDRL2 comprises SEQ ID NO: 46, CDRL3 comprises SEQ ID NO: 47, CDRH1 comprises SEQ ID

ΜΑ 32982Β1

A-1472-WO-PCT

ΝΟ: 76, CDRH2 includes SEQ ID NO: 77 and CDRH3 includes SEQ ID NO: 78. In another embodiment, CDRL1 includes SEQ ID NO: 48, CDRL2 includes SEQ ID NO: 49, CDRL3 includes SEQ ID NO: 50, CDRH1 includes SEQ ID NO: 79, CDRH2 includes SEQ ID NO: 80 and CDRH3 includes SEQ ID NO: 81. In another embodiment, CDRL1 includes SEQ ID ΝΟ: 51, CDRL2 includes SEQ ID NO: 52, CDRL3 includes SEQ ID NO: 53, CDRH1 includes SEQ ID NO: 82, CDRH2 includes SEQ ID NO: 83 and CDRH3 includes SEQ ID NO: 84. In another embodiment, CDRL1 includes SEQ ID NO: 54, CDRL2 includes SEQ ID NO: 55, CDRL3 includes SEQ ID NO: 56, CDRH1 includes SEQ ID NO: 85, CDRH2 includes SEQ ID 0:86 and CDRH3 includes SEQ ID NO: 87. In another embodiment, CDRL1 includes SEQ ID NO: 57, CDRL2 includes SEQ ID NO: 58, CDRL3 includes SEQ ID NO: 59, CDRH1 includes SEQ ID NO: 88, CDRH2 includes SEQ ID NO: 89 and CDRH3 includes SEQ ID NO: 90. In another embodiment, CDRL1 comprises SEQ ID NO: 60, CDRL2 comprises SEQ ID NO: 55, CDRL3 comprises SEQ ID NO: 56, CDRH1 comprises SEQ ID

NO: 85, CDRH2 comprises SEQ ID NO: 86 and CDRH3 comprises SEQ ID NO: 87. In another embodiment, CDRL1 includes SEQ ID NO: 45, CDRL2 includes SEQ ID NO: 61, CDRL3 includes SEQ ID NO: 47, CDRH1 includes SEQ ID NO: 76, CDRH2 includes SEQ ID NO: 91 and CDRH3 includes SEQ ID NO: 78. In another embodiment, CDRL1 includes SEQ ID NO: 62, CDRL2 includes SEQ ID NO: 63, CDRL3 includes SEQ ID 0:64, CDRH1 includes SEQ ID NO: 92, CDRH2 includes SEQ ID NO: 93 and CDRH3 includes SEQ ID NO: 94. In another embodiment, CDRL1 includes SEQ ID NO: 45, CDRL2 includes SEQ ID NO: 61, CDRL3 includes SEQ ID NO: 47, CDRH1 includes SEQ ID NO: 76, CDRH2 includes SEQ ID NO: 95 and CDRH3 includes SEQ ID NO: 78. In another embodiment, CDRL1 comprises SEQ ID

065, CDRL2 includes SEQ ID NO: 55, CDRL3 includes SEQ ID NO: 56, CDRH1 includes SEQ ID NO: 85, CDRH2 includes SEQ ID NO: 86 and CDRH3 includes SEQ ID NO: 87. In another embodiment, CDRL1 includes SEQ ID NO: 42, CDRL2 includes SEQ ID NO: 43, CDRL3 includes SEQ ID NO: 44, CDRH1 includes SEQ ID NO: 73, CDRH2 includes SEQ ID NO: 74 and CDRH3 includes SEQ ID NO: 96. In another embodiment, CDRL1 includes SEQ ID 0:66, CDRL2 includes SEQ ID NO: 67, CDRL3 includes SEQ ID NO: 68, CDRH1 includes SEQ ID NO: 97, CDRH2 includes SEQ ID NO: 98 and CDRH3 includes SEQ ID NO: 99. In another embodiment, CDRL1 includes SEQ ID NO: 69, CDRL2 includes SEQ ID NO: 70, CDRL3 includes SEQ ID NO: 71, CDRH1 includes SEQ ID 0: 100, CDRH2 includes SEQ ID

ΜΑ 32982Β1

A-1472-WO-PCT

NO: 101 and CDRH3 includes SEQ ID NO: 102. In another embodiment, CDRL1 includes SEQ ID NO: 69, CDRL2 includes SEQ ID NO: 70, CDRE3 includes SEQ ID NO: 72, CDRH1 includes SEQ ID NO: 100 , CDRH2 comprises SEQ ID NO: 101 and CDRH3 comprises SEQ ID NO: 102.

In any of the sequence-defined embodiments mentioned above, the isolated antigen-binding protein may be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody (e.g., fully human), a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof. Further, the antibody fragment of the isolated antigen-binding proteins may be an Fab fragment, and an Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, a diabody, or an antibody molecule to an antibody. single string. For example, the isolated antigen binding protein can be a human monoclonal antibody and can be, for example, an antibody of the IgG1, IgG2, IgG3, or IgG4 type. In addition, isolated antigen-binding proteins can neutralize antigen-binding proteins.

In any of the above-mentioned sequenced embodiments, the isolated antigen-binding protein can specifically bind to both human CRLR and human RAMP1, and does not specifically bind AMI, ΑΜ2, or a. human amylin receptor (eg, ΑΜΥ1). For example, the isolated antigen-binding protein can specifically bind to human CGRP R with a Kd1 ؛ μΜ, 100 ؛ nM, 10 ؛ nM, or 5 ؛ nM, for example, as determined by a FACS binding assay and analysis, for example, using the methods described in Rathanaswami, et al., Biochemical andBiophysical Research Communications 334 (2005) 10041013. In any of the sequence-defined embodiments mentioned above, the isolated antigen-binding protein can selectively inhibit human CGRP R, relative to human AMI, ΑΜ2 or ΑΜΥ1 receptors, for example, with a ratio of selectivities of 100 or more, 250 or more, 500 or more, 750 or more, 1,000 or more, 2,500 or more, 5,000 or more or 10,000 or more, the degree of selective inhibition can be determined by means of a any suitable method, for example, by means of a cAMP test as described in the examples given here. In any of the sequence-defined embodiments mentioned above, the isolated antigen-binding protein may have in Ki 100 ؛ nM, 10 ؛ nM, 1 ؛ nM, 0.5 ؛ nM or 0.1 ؛ nM in a CGRP binding competition assay, e.g., in a competition assay for binding of radiolabeled CGRP to membranes of cells expressing CGRP R, e.g., e.g., the assay described in Example 5 presented herein .

ΜΑ 32982Β1

A-1472-WO-PCT

Another set of embodiments include isolated antigen-binding proteins which comprise one or more CDRs having the consensus sequences described below, and optionally, bind human CGRP R. Consensus sequences are derived from phylogenetically related CDR sequences. In one aspect, the CDRs from the various sets can be mixed and matched into any particular isolated antigen-binding protein that binds human CGRP R. In another aspect, the antigen binding protein comprises heavy and light chain CDRs which are derived from the same group of phylogenetically related antibody clones. Examples of CDR consensus sequences are as follows:

Kl Consensus

CDR1 RASQGIRXiDLG (SEQ ID NO: 103), Xi being selected from the set consisting of N and K.

CDR2 XiASSLQS (SEQ ID NO: 104), Xi being chosen from the set consisting of A and G.

CDR3 LQYNXXPWT (SEQ ID ΝΟ: 105), X, being selected from the set consisting of I and s, and Χ2 being selected from the set consisting of Y and F.

Κ4 Consensus

CDR3 QQYGNSLXjR (SEQ ID NO: 106), Xi being selected from the group consisting of S and c.

Kl, 4 Consensus

CDR1 RASQX1X2X3X4GX5LX6 (SEQ ID NO: 107), Xi being selected from the set consisting of s and G, Χ2 being selected from the set consisting of V and I, Χ3 being selected from the set consisting of s and R, Χ4 being selected from the set consisting of s, N and K, Χ5 being selected from the set consisting of Y and D, and Χό being selected from the set consisting of T and G.

CDR2 X1ASSX2X3X4 (SEQ ID NO: 108), X, being selected from the set consisting of G and A, Χ2 being selected from the set consisting of R and L, Χ3 being selected from the set consisting of A and Q, and Χ4 being selected from the set consisting of T and s.

CDR3 X1QYX2X3X4X5X6X7 (SEQ ID NO: 109), Xi being selected from the set consisting of Q and L, Χ2 being selected from the set consisting of G and N, Χ3 being selected from the set consisting of N and τ, Χ4 being selected from the set consisting of s, Y and F, Χ5 being selected from the set consisting of L and p, x₆ being selected from the set consisting of C, W and S, and Χ7 being chosen from the set consisting of R and T.

ΜΑ 32982Β1

A-1472-WO-PCT

Κ3 Consensus

CDR1 KSSQSLLHSX1GX2X3YLY (SEQ ID NO: 110), Xi being selected from the set consisting of D and A, Χ2 being selected from the set consisting of R and K, and Χ3 being selected from the set consisting of N and T.

Κ2.3 Consensus

CDR1 X1SSQSLLHSX2GX3X4YLX5 (SEQ ID NO: 111), Xi being selected from the set consisting of R and K, Χ2 being selected from the set consisting of F, D and A, Χ3 being selected from the set consisting of Y, R and K, Χ4 being selected from the set consisting of N and T, and X ؛ being selected from the set consisting of D and Y.

CDR2 X1X2SNRX3S (SEQ ID NO: 112), Xi being selected from the set consisting of L and E, Χ2 being selected from the set consisting of G and V, and Χ3 being selected from the set consisting of A and F.

CDR3 MQX1X2X3XX5T (SEQ ID 0: 113), Xi being selected from the set consisting of A and s, Χ2 being selected from the set consisting of L and F, Χ3 being selected from the set consisting of Q and p, Χ4 being selected from the set consisting of T and L, and Χ5 being selected from the set consisting of F and L.

Lm3 Consensus

CDR2 RXiNQRPS (SEQ ID NO: 114), Xi being selected from the group consisting of N and S.

Lml. 2 3 Consensus

CDR1 SGSSSNIGX1NX2VX3 (SEQ ID NO: 115), Xj being selected from the set consisting of N and s, Χ2 being selected from the set consisting of Y and T, and Χ3 being selected from the set consisting of s, N and Y.

CDR2 X1X2NX3RPS (SEQ ID NO: 116), Xi being selected from the set consisting of D, T and R, Χ2 being selected from the set consisting of N and S, and Χ3 being selected from the set consisting of K and Q.

CDR3 X1X2X3DX4X5LXXW (SEQ ID NO: 117), Xi being selected from the set consisting of G and A, Χ2 being selected from the set consisting of T and A, Χ3 being selected from the set consisting of w and R, Χ4 being selected from the set consisting of S and D, Χ5 being selected from the set consisting of R and s, X6 being selected from the set consisting of s and N, and Χ7 being selected from the set consisting of A and G.

LmAll Consensus

CDRl X1GX2X3SX4X5X6X7X8X9X10X11 (SEQ ID NO: 118), Xi being chosen from the set consisting of s and Q, Χ2 being present or absent, and if present, representing s, Χ3

ΜΑ 32982Β1

A-1472-WO-PCT being selected from the set consisting of s and D, Χ4 being present or absent, and if present, representing N, Χ5 being selected from the set consisting of I and L, Xe being selected from the set consisting of G and R, Χ7 being selected from the set consisting of N and s, Xs being selected from the set consisting of N and F, Χ9 being selected from the set consisting of Y and T, Χ10 being chosen from the set consisting of V and A, and Χ11 being selected from the set consisting of s, N and Y.

CDR2 X1X2NX3RPS (SEQ ID NO: 119), Xi being selected from the set consisting of D, G, T, and R, Χ2 being selected from the set consisting of N, K and s, and Χ3 being selected from set consisting of K, N and Q.

CDR3 X1X2X3DX4X5X6X7X8X9V (SEQ ID NO: 120), Xi being selected from the set consisting of G, N and A, Χ2 being selected from the set consisting of T, s and A, Χ3 being selected from the set consisting of w and R, Χ4 being selected from the set consisting of s and D, X ؛ being selected from the set consisting of R and s, Xfi being selected from the set consisting of L and V, Χ7 being selected from the set consisting of s, Y and N, Xs being selected from the set consisting of A , H and G, and Xg being selected from the set consisting of V and L.

HCl Consensus

CDRl Χ1ΥΥΜΧ2 (SEQ ID ΝΟ: 121), X, being selected from the set consisting of G and D, Χ2 being selected from the set consisting of H and Y.

CDR2 WIXiPNSGGTNYAQKFQG (SEQ ID ΝΟ: 122), X | being selected from the set consisting of N and s.

CDR3 X1X2X3SX XXXXioXuXYYXirGMDV (SEQ IDNO: 123), X | being selected from the set consisting of D and G, Χ2 being selected from the set consisting of Q and G, Χ3 being selected from the set consisting of M and Y, Χ4 being selected from the set consisting of I and G , Χ5 being selected from the group consisting of I and Y, x٥ being selected from the group consisting of M and A, Χ7 being present or absent, and if present, representing L, Xg being present or absent, and if he is present, representing R, Xg being chosen from the set consisting of V and L, Χ10 being chosen from the set consisting of F and Y, Χ11 being chosen from the set consisting of p and s, Χ12 being chosen from the set consisting of in p and H, and Χ13 being present or absent, and if present, representing Y.

HC2 Consensus

CDR2 RIKSX1TDGGTTDYX2APVKG (SEQ ID ΝΟ: 124), X) being chosen from the set consisting of K and T, and Χ2 being chosen from the set consisting of T and A.

ΜΑ 32982Β1

A-1472-WO-PCT

HC3 Consensus

CDR1 Χ1ΥΧ2ΜΧ3 (SEQ ID 0: 125), Xi being selected from the set consisting of T and S, Χ2 being selected from the set consisting of s and A, and Χ3 being selected from the set consisting of N and s.

CDR2 X1ISX2SX3X4XsXeYYADSVKG (SEQ ID ΝΟ126), Xi being selected from the set consisting of s and A, Χ2 being selected from the set consisting of s and G, Χ3 being selected from the set consisting of s and G, Χ4 being chosen in the set consisting of s and G, Χ5 being chosen from the set consisting of Y and R, and Χδ being chosen from the set consisting of R and T.

CDRS X1X2X3X4X5X6X7PYSX8X9WYDYYYGMDV (SEQ ID ΝΟ127), Xi being chosen from the set consisting of E and D, Χ2 being chosen from the set consisting of G and Q, Χ3 being chosen from the set consisting of V and R, Χ4 being chosen in the set consisting of s and E, Χ5 being selected from the set consisting of G and V, Xf) being selected from the set consisting of s and G, Χ7 being present or absent, and if present, representing s, Xs being chosen from the set consisting of I and s, and Χ9 being chosen from the set consisting of S and G.

HC4 Consensus

CDRl SXiGMH (SEQ ID ΝΟ: 128), X, being chosen from the set consisting of F and

CDR2 VISX1DGSX2KYX3X4DSVKG (SEQ ID NO: 129), Xi being selected from the set consisting of F and Y, Χ2 being selected from the set consisting of I and H, Χ3 being selected from the set consisting of s and Y, and Χ4 being chosen from the set consisting of V

CDR3 X1RX2X3X4X5X6SX7X8YYX9X10X11YYGX12X13V (SEQ ID NO: 130), X] being selected from the set consisting of D and E, Χ2 being selected from the set consisting of L and K, Χ3 being selected from the set consisting of N and R, Χ4 being selected from the set consisting of Y and V, Χ5 being selected from the set consisting of Y and T, x٥ being selected from the set consisting of D and Μ, Χ7 being selected from the set consisting of s and T, Xs being chosen from the set consisting of G and L, Χ9 being selected from the set consisting of H and Y, Χ10 being present or absent, and if present, representing Y, Χ11 being selected from the set consisting of K. and F, Χ12 being selected from the set consisting of M and L, and Χ13 being chosen from the set consisting of A and D.

ΜΑ 32982Β1

A-1472-WO-PCT

HCA Consensus

CDRl Χ1Χ2Χ3ΜΧ4 (SEQ ID NO: 131), Xi being selected from the set consisting of N and S, Χ2 being selected from the set consisting of A, Y and F, Χ3 being selected from the set consisting of w, A and G, and Χ4 being selected from the set consisting of s and HCDR2 X1IX2X3X4X5X6GX7X8X9X10X11X12X13X14VKG (SEQ ID ΝΟ: 132), X] being selected from the set consisting of R, A and V, Χ2 being selected from the set consisting of K, S and W, Χ3 being chosen from the set consisting of s. G, F and Y, Χ4 being present or absent, and if present, being selected from the set consisting of K and T, Χ5 being present or absent, and if present, representing T, x &lt;5 being selected from the set consisting of D and s, Χ7 being selected from the set consisting of G and s, Xs being selected from the set consisting of T, R, I, N and H, Χ9 being selected from set consisting of T and K, Χ10 being chosen from the set consisting of D and Y, Χ11 being selected from the set consisting of Y and s, Χ12 being selected from the set consisting of T, A and V, Χ13 being selected from the set consisting of A and D, and Χ14 being selected from the set consisting in P and s.

CDR3 XjXXXiXXeXjXioXiiXXX ^ XisXKiXGXigXV (SEQ ID NO: 133), Xi being chosen from the set consisting of D, A and E, Χ2 being chosen from the set consisting of R, Q and G, Χ3 being chosen from the set consisting in T, R, L, G and K, Χ4 being selected from the set consisting of G, E, N, I and R, Χ5 being selected from the set consisting of Y, V and A, x٥ being chosen from l 'set consisting of s. G, Y, A and T, Χ7 being selected from the set consisting of I, P, D, A and M, Xg being present or absent, and if present, being selected from the set consisting of s and Y, Χ9 being present or absent, and if present, being selected from the set consisting of w, s and T, Χ10 being selected from the set consisting of s, G and L, Χ11 being selected from set consisting of s. G, L and Y, X ، 2 being present or absent, and if present, being selected from the set consisting of w and Y, Χ13 being selected from the set consisting of Y and H, X | 4 being present or absent, and if present, being selected from the group consisting of Y and D, Χ15 being selected in the group consisting of Y, K and F, X) 6 being present or absent, and s' it is present, representing Y, Χ17 being present or absent, and if it is present, representing Y, x, 8 being selected from the set consisting of M and L, and x, 9 being selected from the set consisting of D and A.

HCB Consensus

CDRl Χ1Χ2Χ3Χ4Χ5 (SEQ ID ΝΟ: 134), X, being chosen from the set consisting of N, G, D, S and A, Χ2 being selected from the set consisting of A, F and Y, Χ3 being chosen from l 'set consisting of w. Y, A and G, Χ4 being chosen from the set consisting of M and L, and X ؛ being chosen from the set consisting of s and H.

ΜΑ 32982Β1

A-1472-WO-PCT

CDR2 XiIX2X3X4X5X6X7X8X9XioXiiXi2Xi3Xi4Xi5XiôXi7G (SEQIDNO: 135), Xi being chosen from the set consisting of R, w, A, V, s and F, Χ2 being chosen from the set consisting of K, N, s, w and R, Χ3 being chosen from the set consisting of s, p. G, F and Y, Χ4 being present or absent, and if present, being selected from the group consisting of K, T and R, Χ5 being present or absent, and if present, being selected from l 'set consisting of T and A, x٥ being selected from the set consisting of D, N, H, s and Y, Χ7 being selected from the set consisting of G and s, Xg being selected from the set consisting of G and s, X? being selected from the set consisting of T, G, R, I, N, H and Y, Χ10 being selected from the set consisting of T, K, R and P, Χ11 being selected from the set consisting of D, N, Y and E, Χ12 being selected from the set consisting of Y and s, Χ13 being chosen in the set consisting of T, A and V, Χ14 being selected from the set consisting of A, Q and D, Χ15 being selected from the set consisting of p, K and s, Χ16 being selected from the set consisting in V and F, and Χ17 being chosen from the set consisting of K and Q.

CDR3XiX2X3X4X5SX6X7X8X9Xl0XllXl2Xl3Xl4Xl5Xl6GXi7Xl8V (SEQIDNO: 136), Xi being chosen from the set consisting of D, G, A and E, Χ2 being chosen from the set consisting of R, G and Q, the set Χ3 being chosen from the set M, Y, R, L, G and K, Χ4 being selected from the set consisting of G, s, E, N, I and R, Χ5 being selected from the set consisting of Y, I, G, V and A, Χό being chosen from the set consisting of s, I, Y, G, A and T,

Χ7 being selected from the set consisting of I, M, A, p and D, Xg being present or absent, and if present, being selected from the set consisting of s, L and Y, Χ9 being present or absent, and if present, being selected from the set consisting of w, R, s and T, Χ10 being selected from the set consisting of s, G and L, Χ11 being selected from the set consisting of s , V, L, G and Y, x, 2 being present or absent, and if present, being selected from the set consisting of F, Y and W, x! 3 being selected from the set consisting of Y, p, s and H, Χ14 being present or absent, and if present, being selected from the set consisting of Y, p, D and H, Χ15 being chosen from the set consisting of Y, K and F, Χ16 being present or absent, and if present, representing Y, Χ17 being present or absent, and if present, representing Y and X | g being chosen in the set consisting of M and L.

In any of the embodiments defined by consensus sequences mentioned above, the isolated antigen-binding protein can be, for example, an AVIMER polypeptide, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody ( e.g., fully human), a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof. Additionally, the antibody fragment of the isolated antigen-binding proteins may be an Fab fragment, and a

ΜΑ 32982Β1

A-1472-WO-PCT Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, a diacoips, or a single chain antibody molecule. For example, the isolated antigen-binding protein may be a human monoclonal antibody, and may be, for example, an antibody of the IgG1, IgG2, IgG3, or IgG4 type.

In addition, the isolated antigen-binding proteins may neutralize antigen-binding proteins.

In any of the embodiments defined by consensus sequences mentioned above, the isolated antigen-binding proteins can specifically bind to both human CRLR and human RAMP1, and not specifically bind to AMI, ΑΜ2 or to a human amylin receptor (eg, ΑΜΥ1), eg, isolated antigen binding protein can specifically bind human CGRF R with a Kd &lt;1 μΜ, &lt;100 nM, &lt;10 nM, 0U &lt;5 nM, for example as determined by a FACS binding assay and analysis, for example using the methods described in Rathanaswami, et al., Biochemical and BiophysicalResearch Communications 334 (2005) 10041013. In any of the embodiments defined by consensus sequences mentioned above, the isolated antigen-binding protein can selectively inhibit human CGRF R, relative to human AMI, ΑΜ2 or AMY1 receptors, for example, with a ratio of selectivities of 100 or more, 250 or more, 500 or more, 750 or more, 1,000 or more, 2,500 or more, 5,000 or more or 10,000 or more, the degree of selective inhibition can be determined by means of any suitable process, for example, using a cAMP test as described in the examples given here. In any of the embodiments defined by consensus sequences mentioned above, the isolated antigen binding protein may have a Ki 00 nM, 10 ك nM, &lt;1 nM, 0 5 nM or 0.1 nM in an assay. competition for binding to CGRF, for example, in a competition assay for the binding of radiolabeled ا I CGRP to membranes of cells expressing CGRF R, for example, for example the test described in Example 5 presented here.

Some of the isolated antigen-binding proteins described include a heavy chain variable region (٧H) sequence that has sequence identity of at least 80%, 85 ٠/٥,

٥/٥ or 95 ٥/٥ with an amino acid sequence selected from the group consisting of SEQ ID NOs: 158-170. Some of the isolated antigen-binding proteins described comprise a light chain variable region (Vl) sequence which has sequence identity of at least 80 ٠/٥, 85 ٥/٥, 90 ٠/٥ or 95 ٥/٥ with an amino acid sequence selected from the group consisting of SEQ ID NOs: 137-153. Some of the isolated antigen-binding proteins described comprise a Vh sequence which has sequence identity of at least 80%, 85 ٠/٥, 90 ٠/٥ or 95 ٥/٥ with an amino acid sequence selected from the following group: set consisting of SEQ ID Nos: 158-170, and a VI which has a sequence identity of at least 80 ٥/٥, 85 ٥/٥, 90 ٠/٥ or 95 ٠/٥

ΜΑ 32982Β1

A-1472-WO-PCT with an amino acid sequence selected from the group consisting of SEQ ID NOs: 137153. In some embodiments, the isolated antigen-binding proteins comprise (A) a heavy chain variable region (Vh) comprising a sequence (i) selected from the group consisting of SEQ ID Nos: 158-170, or (ii) as defined by (i) and containing one or more (eg, five, ten, fifteen or twenty) substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; (B) a VI comprising a sequence (iii) selected from the group consisting of SEQ ID NOs: 137-153, or (iv) as defined by (iii) containing one or more (eg, five, ten, fifteen or twenty) substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; or (C) a Vh of (A) and a Vl of (B). In some embodiments, the isolated antigen-binding proteins comprise a heavy chain variable region (Vh) comprising a sequence selected from the group consisting of SEQ ID NOs: 158170 and a Vl comprising a sequence selected from the set consisting of SEQ ID Nos: 137-153.

In one embodiment, the isolated antigen-binding protein comprises a heavy chain variable region (Vh) comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 158, (ii) a sequence which is at least 90 ٠/٥ or 95 ٠/٥ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., substitutions of conservative amino acids), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vh comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 159, (ii) a sequence which is identical to at least 90% or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vh comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 160, (ii) a sequence which is identical to at least 90 ٠/٠ or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vh comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 161, (ii) a sequence which is identical to at least 90 ٠/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions).

<img file="MA32982B1_D0003.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises an ٧H comprising an amino acid sequence selected from the group consisting of (i) SEQ ID ΝΟ162, (ii) a sequence which is at least 90 ٠ identical / ٥ or 95 ٠/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the isolated antigen binding protein comprises a ٧H comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 163, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٠/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a ٧H comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 164, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a ٧H comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 165, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٠/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vh comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 166, (ii) a sequence which is identical to at least 90 ٠/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vh comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 167, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vh comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 168, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i)

ΜΑ 32982Β1

A-1472-WO-PCT containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vfj comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 169, (ii) a sequence which is identical to at least 90 ٠/٠ or 95 ٠/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vu comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 170, (ii) a sequence which is identical to at least 90 ٥/٠ or 95 ٥/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids.

In one embodiment, the isolated antigen-binding protein comprises a light chain variable region (Vl) comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 137, (ii) a sequence which is the same as arr minus 90 ٠/٠ or 95 ٥/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., substitutions of amino acids conservattices), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 138, (ii) a sequence which is identical to at least 90 ٥/٠ or 95 ٥/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 139, (ii) a sequence which is identical to at least 90 ٥/٠ or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 140, (ii) a sequence which is identical to at least 90 ٥/٠ or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the isolated antigen-binding protein comprises a V1 comprising an amino acid sequence selected from /

ΜΑ 32982 Β1

A-1472-WO-PCT the set consisting of (i) SEQ ID NO: 141, (ii) a sequence which is at least 90 ٠/٠ or 95 ٠/٠ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 142, (ii) a sequence which is identical to at least 90 ٥/٠ or 95 ٠/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a Vl comprising an amino acid sequence selected from the group consisting of (i) SEQ ID ΝΟ143, (ii) a sequence which is at least 90 ٥ identical / ٠ or 95 ٥/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the isolated antigen binding protein comprises a V1 comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 144, (ii) a sequence which is identical to at least 90 ٠/٠ or 95 ٥/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 145, (ii) a sequence which is identical to at least 90% or 95 ٠/٠ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 146, (ii) a sequence which is identical to at least 90 ٠/٠ or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 147, (ii) a sequence which is identical to at least 90 ٠/٠ or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the protein binding a

ΜΑ 32982Β1

A-1472-WO-PCT isolated antigen includes a V_L comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 148, (ii) a sequence which is at least 90 ٥/٥ or 95 ٥/٥ identical to the sequence defined by (i )) and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 149, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٠/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 150, (ii) a sequence which is identical to at least 90 ٥/٥ or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 151, (ii) a sequence which is identical to at least 90 ٠/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID ΝΟ.-152, (ii) a sequence which is identical to minus 90 ٥/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, conservative amino acid substitutions), deletions or insertions of amino acids. In another embodiment, the isolated antigen-binding protein comprises a VI comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 153, (ii) a sequence which is identical to at least 90 ٥/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids.

In any of the embodiments defined by the V1 and Vh sequences mentioned above, the isolated antigen-binding protein can be, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody (e.g. example, fully human), a humanized anticoips, a chimeric antibody, an anticoips

ΜΑ 32982Β1

Multispecific A-1472-WO-PCT, or an antigenic fragment thereof. Further, the antibody fragment of the isolated antigen-binding proteins may be an Fab fragment, and an Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, a diabody, or an antibody molecule to an antibody. single string. For example, the isolated antigen-binding protein can be a human monoclonal antibody, and can be, for example, an antibody of the IgG1, IgG2١ IgG3, or IgG4 type. In addition, the isolated antigen-binding proteins can be neutralizing antigen-binding proteins.

In any of the V1 and Vh sequence-defined embodiments mentioned above, the isolated antigen-binding protein can specifically bind to both human CRFR and human RAMP1, and not specifically bind to AMI. , ΑΜ2 or to a human amylin receptor (eg, AMY1), eg, isolated antigen-binding protein can specifically bind to human CGRP R with a Kd1 ؛ μΜ, 100 nM, &lt;10 nM, or &lt;5 nM, for example according to determination by a FACS binding assay and analysis, for example using the methods described in Rathanaswami, et al., Biochemical and BiophysicalResearch Communications 334 (2005) 1004-1013. In any one of the embodiments defined by the sequences defined by sequences ٧L and Vf! mentioned above, the isolated antigen-binding protein can selectively inhibit human CGRP R, relative to human AMI, ΑΜ2 or AMY 1 receptors, for example, with a selectivity ratio of 100 or more, 250 or more, 500 or more , 750 or more, 1,000 or more, 2,500 or more,

000 or more or 10,000 or more, the degree of selective inhibition being determinable by any suitable method, for example, by means of a cAMP assay as described in the examples given herein. In any of the embodiments defined by Vl and Vh sequences mentioned above, the isolated antigen binding protein may have a Ki &lt;100 nM, &lt;10 nM, اة nM, 0.5 ة nM or 0.1 ة nM in a competitive assay by binding to CGRP, e.g., in a competition assay for the binding of radiolabeled 'Ι-CGRP to membranes of cells expressing CGRP R, e.g., e.g., the assay described in Example 5 present here.

In one aspect, the isolated antigen-binding proteins comprise a heavy chain sequence that has sequence identity of at least 80%, 85 ٠/٠, 90%, or 95% with an amino acid sequence selected from the following. set consisting of SEQ ID NOs: 29-41. Some of the isolated antigen-binding proteins described comprise a light chain sequence which has sequence identity of at least 80%, 85%, 90% or 95% with an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-28. Some of the isolated antigen-binding proteins comprise a heavy chain sequence which has a sequence identity of at least 80 ٠/٥, 85 نره 90, نره, or 95 نره with an amino acid sequence selected from

ΜΑ 32982Β1

A-1472-WO-PCT the set consisting of SEQ ID NOs: 29-41, and a light chain sequence which has a sequence identity of at least 80 ٥/٥, 85%, 90 ٠/٥ or 95 ٠/٥ with an amino acid sequence chosen from the group consisting of SEQ ID NOs: 12-28. In certain embodiments, the isolated antigen-binding proteins comprise (A) a heavy chain comprising a sequence (i) selected from the group consisting of SEQ ID NOs: 29-41, or (ii) as defined by (i ) and containing one or more (eg, five, ten, fifteen or twenty) substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; (B) a light chain comprising a sequence (iii) selected from the group consisting of SEQ ID NOs: 12-28, or (iv) as defined by (iii) containing one or more (eg, five, ten, fifteen or twenty) substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; or (C) a heavy chain of (A) and a light chain of (B). In some embodiments, the isolated antigen-binding proteins comprise a heavy chain comprising a sequence selected from the group consisting of SEQ ID NOs: 2941 and a light chain comprising a sequence selected from the group consisting of SEQ ID NOs: 12 -28.

In one embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 29, (ii) a sequence which is identical at least 90 ٠/٥ or 95 ٥/٥ to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, conservative amino acid substitutions ), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 12, (ii) a sequence which is at least 90 ٠/٠ or 95 ٥/٥ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID ΝΟ-.30, (ii) a sequence which is at least 90 ٠/٠ or 95 ٠/٠ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, acid substitutions conservative amines), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 13, (ii) a sequence which is at least 90 ٠/٥ or 95 ٠/٥ identical to the sequence defined by (i), and (iii) to a sequence as defined by

ΜΑ 32982Β1

A-1472-WO-PCT (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 31, (ii) a sequence which is at least 90 ٠ / o or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, conservative amino acid substitutions) , deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 14, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 32, (ii) a sequence which is at least 90% or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 15, (ii) a sequence which is at least 90% or 95 ٠/٥ identical to sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33, (ii) a sequence which is at least 90% or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 16, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of:

ΜΑ 32982Β1

A-1472-WO-PCT in (i) SEQ ID NO: 29, (ii) a sequence which is at least 90 ٥/٠ or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 17, (ii) a sequence which is at least 90% or 95 ٥/٠ identical to sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 34, (ii) a sequence which is at least 90 ٠/٠ or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, conservative amino acid substitutions) , deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 18, (ii) a sequence which is at least 90 ٠/٠ or 95 ٠/٠ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33, (ii) a sequence which is at least 90% or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 19, (ii) a sequence which is at least 90 ٥/٠ or 95 ٠/٠ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 29, (ii) a sequence which is at least 90 ٠/٠ or 95 ٠/٠ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, amino acid substitutions conservative), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence

ΜΑ 32982Β1

A-1472-WO-PCT selected from the group consisting of (i) SEQ ID NO: 20, (ii) a sequence which is at least 90% or 95 ٥/٥ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 35, (ii) a sequence which is at least 90 ٥ / o or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, conservative amino acid substitutions) , deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 21, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 36, (ii) a sequence which is at least 90 ٠/٠ or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions) , deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 22, (ii) a sequence which is at least 90 ٥ / o or 95 ٥/٥ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 37, (ii) a sequence which is at least 90 ٥/٠ or 95 ٥ / o identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, amino acid substitutions conservative), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 23, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

ΜΑ 32982Β1

A-1472-WO-PCT

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 38, (ii) a sequence which is at least 90% or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 23, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 33, (ii) a sequence which is identical at least 90 ٠ / o or 95% to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 24, (ii) a sequence which is at least 90 ٠ / o or 95% identical to sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 39, (ii) a sequence which is at least 90 ٥/٠ or 95 ٠/٠ identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (for example, substitutions of amino acids conservative), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 25, (ii) a sequence which is at least 90% or 95 ٥/٠ identical to sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 40, (ii) a sequence which is at least 90% or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten

ΜΑ 32982Β1

A-1472-WO-PCT substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 26, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 41, (ii) a sequence which is at least 90 ٠/٠ or 95% identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions) , deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID 0:27, (ii) a sequence which is at least 90 ٥/٠ or 95% identical to sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In another embodiment, the isolated antigen-binding protein comprises (A) a heavy chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 41, (ii) a sequence which is at least 90% or 95 ٠ / û identical to the sequence defined by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (e.g., conservative amino acid substitutions) , deletions or insertions of amino acids; and (B) a light chain comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 28, (ii) a sequence which is at least 90% or 95% identical to the defined sequence by (i), and (iii) a sequence as defined by (i) containing up to ten substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids.

In any of the embodiments defined by heavy and light chain sequences mentioned above, the isolated antigen-binding protein may comprise the specified heavy and / or light chain sequence, but with a different signal peptide or without. signal peptide. In any of the embodiments defined by heavy and light chain sequences mentioned above, the isolated antigen-binding protein can be, for example, a monoclonal antibody, a polyclonal anticoips, a recombinant anticoips, a human antibody. (eg, fully human), a humanized anti-tip, a chimeric anti-tip, a multi-specific anti-tip, or an antigen-binding moiety thereof. In addition, the /

ΜΑ 32982Β1

A-1472-WO-PCT antibody fragment of the isolated antigen-binding proteins may be an Fab fragment, and an Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, a diabody, or a d molecule. single chain antibody. For example, the isolated antigen-binding protein may be a human monoclonal antibody, and may be, for example, an antibody of the IgG1, IgG2, IgG3١ or IgG4 type.

In addition, the isolated antigen-binding proteins can be neutralizing antigen-binding proteins.

In any of the heavy and light chain sequence-defined embodiments mentioned above, the isolated antigen-binding protein can specifically bind to both human CRLR and human RAMP1, and not not specifically bind to AMI, ΑΜ2 or a human amylin receptor (e.g., AMY1), for example, isolated antigen-binding protein can specifically bind human CGRF R with a κ1 ؛ ٥ μΜ, 100 ؛ nM, 10 ؛ nM, or 5 ؛ nM, for example, as determined by a FACS binding assay and analysis, for example, using the methods described in ymmi, et al., Biochemical andBiophysical Research Communications (15-10 1004-1013. In any of the embodiments defined by heavy and light chain sequences mentioned above, the isolated antigen-binding protein can selectively inhibit human CGRF R, relative to human AMI, ΑΜ2 or ΑΜΥ1 receptors, for example, with a selectivity ratio of 100 or more, 250 or more, 500 or more, 750 or more, 1,000 or more, 2,500 or more, 5,000 or more or 10,000 or more, the degree of selective inhibition can be determined by any suitable method, for example, by means of a cAMP test as described in the examples given herein. In any of the embodiments defined by heavy and light chain sequences mentioned above, the isolated antigen-binding protein may have a Ki 100 ؛ nM, 10 ؛ nM, 1 ؛ nM, 0.5 ؛ nM or 0.1 ؛ nM in a competition assay for binding to CGRF, e.g., in a competition assay for the binding of radiolabeled ICGRP to membranes of cells expressing CGRF R, e.g., e.g., the assay described in present Example 5 here.

In one aspect, the invention also relates to isolated nucleic acid polynucleotides which encode any of the CGRF R antigen-binding proteins summarized above. In one embodiment, the isolated polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 175, 176, 178, 179, 180, 181, 182, 183, 186, 187, 188, 189, 191, 192 , 193,194,195,196, 197, 200, 201, 202, 203, 204, 205, 206, 207,208,209 and 210. In another embodiment, the isolated polynucleotide comprises a sequence selected from the group consisting of SEQ ID 0: 224-258. In another embodiment, the isolated polynucleotide comprises a sequence capable of hybridizing in

ΜΑ 32982Β1

A-1472-WO-PCT sttingent hybridization conditions with a sequence selected from the group consisting of SEQ ID 10: 224-258. In another embodiment, the isolated polynucleotide comprises a sequence which is about 80 ٥ / o, 85 ٥ / o, 90 ٠/٠ or 95 ٠/٠ or more identical to a sequence selected from the set consisting of SEQ ID Nos: 224-258 In some instances, the isolated nucleic acid molecules are operably linked to a control sequence. In related embodiments, the isolated polynucleotides are incorporated into an expression vector.

The invention also includes cell lines transformed with expression vectors comprising isolated polynucleotides described above. In a related aspect, the invention also relates to expression vectors and host cells transformed or transfected with the expression vectors which comprise the above-mentioned nucleic acid molecules which encode the CGRP R antigen binding proteins. described above.

In another aspect, the invention also relates to a method of preparing the antigen-binding protein, which comprises the step of preparing the antigen-binding protein from a host cell which secretes the antigen-binding protein. In some embodiments, the antigen binding protein is produced using an immunogen comprising the soluble CGRP receptor. In certain embodiments, this soluble CGRP receptor is obtained by co-expression and purification of the N-terminal extracellular domain (DEC) of human CRLR and a DEC of human RAMP1, for example a DEC of human CRLR comprising SEQ ID NO: 6 and a DEC of RAMP1 comprising SEQ ID NO: 8, for example, as described in Examples 1 and 2 herein.

In yet another aspect, the invention relates to a pharmaceutical composition comprising at least one of the antigen-binding proteins summarized above and a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition may comprise an active agent blade which is selected from the group consisting of a radioactive isotope, a radionuclide, a toxin or a therapeutic or chemotherapeutic group.

In one aspect, the isolated antigen-binding protein is effective in inhibiting vasodilation and / or reducing neurogenic inflammation when administered to a patient. In one embodiment, the isolated antigen-binding protein is effective in reducing the frequency and / or severity of headaches, eg, migraine headaches. For example, the antigen binding protein can be used as an acute treatment for migraine and / or prophylactic treatment to prevent or reduce the frequency and / or severity of symptoms, particularly painful symptoms, associated with a migraine attack.

ΜΑ 32982Β1

A-1472.WO-PCT

Further aspects further relate to methods of treating or preventing a condition associated with CGRP R in a patient, comprising administering to a patient an effective amount of at least one of the isolated antigen-binding proteins summarized herein. -above. In one embodiment, the condition is headache or cluster headache or some other type of pain, eg, chronic pain; in another embodiment, it is diabetes sucks (type II); in another embodiment, it is inflammation, in particular neurogenic inflammation; in another embodiment, it is a cardiovascular disorder; in another embodiment, it is a hemodynamic disturbance with endotoxemia and sepsis; in another embodiment, it is vasodilation.

In another aspect, the invention also relates to a method of inhibiting the binding of CGRR to human CGRP R, for example, to the extracellular portion of CGRP R, in a patient, comprising administering an effective amount of at least one of the antigen-binding proteins provided herein and / or summarized above.

These and other aspects will be described in more detail here. Each aspect can encompass various embodiments provided herein. It is therefore envisioned that each embodiment involving an element or combinations of elements may be included in each aspect described, and all such combinations of the above aspects and embodiments are expressly considered. Other characteristics, objects and advantages of the invention appear in the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows an alignment of human, cynomolgus monkey and rat RAMP-1 sequences.

Fig. 2 shows an alignment of human, cynomolgus monkey and rat CRLR sequences.

Figs 3Α and 3Β show sequence alignments based on CDR light chain phylogenesis of the indicated anti-CGRP receptor antibody clones having kappa light chains, and some corresponding consensus sequences.

Fig. 4 shows sequence alignments based on the light chain CDR phylogenesis of the indicated anti-CGRP receptor antibody clones having lambda light chains, and some corresponding consensus sequences.

Figs. 5A, 5Β, 5C, 5D, and 5Ε show sequence alignments based on the heavy chain CDR phylogenesis of the indicated anti-CGRP receptor antibody clones, and some corresponding consensus sequences.

ΜΑ 32982Β1

A-1472-WO-PCT

Fig. 5F shows consensus sequences of exemplary heavy chain CDRs of anti-CGRP receptor antibodies described herein.

Fig. 6 presents a curve of the data obtained from two experiments representing the percentage of inhibition of the binding of labeled ligand to CGRP R by 1092 supernatants of anti-CGRP R hybridomas (diamonds) and 68 supernatants of negative controls (squares).

Figs. 7A-D show examples of IC50 data of cAMP assay on cells expressing the hCGRP receptor (Fig. 7Α), hAM1 (Fig. 7Β), hAM2 (Fig. 7C) and human amylin receptors ( Fig. 7D) for the three indicated anti-CGRP R mAbs.

Fig. 8 shows an example of ICGRP binding data that can be used to determine the Ki of mAbs against the human CGRP receptor.

Figs. 9A-D present Biacore competition data for selected antibodies described herein.

Fig. 10 shows a FACS determination of Kd of mAb 12G8.

Fig. 11 shows an alignment of cynomolgus, human, human, rat, and rhesus RAMP1 sequences.

Figs. 12Α-Β show an alignment of CRLR sequences from cynomolgus, human, human chimeras, rat and rhesus.

Figs. 13A-13C present representative FACS data of binding of different chimeric CGRP receptors binding to anti-CGRP R antibodies.

Fig. 14 shows peptide maps derived from digestion by AspN of CGRP R alone (chromatogram A) and from digestion of a control sample of the anti-CGRP R 12G8 monoclonal anticoqrs (chromatogram B).

Fig. 15 shows AspN digestions of CGRP R in the presence of different concentrations of CGRP R neutralizing antibody.

Fig. 16 shows AspN digestions of CGRP R in the presence of different concentrations of the neutralizing antibody CGRP R, 4Ε4.

Fig. 17 shows the immunohistochemical staining intensity of cells expressing various receptor components with the 32Η7 antibody.

DETAILED DESCRIPTION

The chapter titles used here are for organizational purposes only and should not be construed as limiting the subject matter described here.

Unless defined otherwise, the technical and scientific terms used in relation to the present application have the meanings which are commonly understood by men of the

<img file="MA32982B1_D0004.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT trade of ordinary skill. In addition, unless the context requires otherwise, singular terms must include pluralities and plural terms must include singular.

In general, the nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and chemistry of proteins and nucleic acids and hybridization, and related techniques, described here are well known to those skilled in the art and commonly used in the field. The methods and techniques of the present application are generally carried out in accordance with conventional methods well known to those skilled in the art and described in various general and more specific references which are cited and discussed throughout the present description, unless otherwise indicated. See, e.g., Sambrook al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν.Υ. (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν.Υ. (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as is commonly practiced in the art or as described herein. The technology used in combination and the laboratory protocols and techniques of analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyzes, pharmaceutical preparation, formulation and administration, and treatment of patients.

It should be understood that this invention is not limited to methodology, protocols and reagents etc. particulars described here, which may therefore vary. The terminology used here has simple purposes of describing particular embodiments, and is not intended to limit the scope of the present invention, which is exclusively defined by the claims.

Except in the working examples, or unless otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions are to be understood as being modified in all cases by the term about. The term about, when used in conjunction with percentages means ± 1 ٥/٥.

Definitions

The term "polynucleotide" or "nucleic acid" includes single-stranded and double-stranded nucleotide polymers. The nucleotides constituting the polynucleotide

ΜΑ 32982Β1

A-1472-WO-PCT can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include basic modifications, such as bromo-uridine and inosine derivatives, modifications of riboses, such as 2 ', 3'didesoxyribose, and modifications of intemucleotide bonds such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate and phosphoroamidate.

The term "oligonucleotide" refers to a polynucleotide comprising 200 nucleotides or less. In some embodiments, the oligonucleotides are 10 to 60 bases in length. In other embodiments, the oligonucleotides are 12,13,14, 15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. The oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing a mutant gene. The oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, especially a radioactive label, a fluorescent label, a hapten or an antigen label, for detection tests. The oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.

An "isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or any combination thereof, which is not associated with all or part of it. a polynucleotide in which the polynucleotide isolates is found in nature, or which is linked to a polynucleotide to which it is not linked in the nahire. For purposes of this description, it is to be understood that a "nucleic acid molecule comprising" a particular nucleotide sequence does not include intact chromosomes. Isolated nucleic acid molecules "comprising" specified nucleic acid sequences may comprise, in addition to the specified sequences, coding sequences of up to ten and even up to twenty other proteins or parts thereof, or may include operably linked regulatory sequences which control expression of the coding region of the cited nucleic acid sequences, and / or may include vector sequences.

Unless otherwise specified, the left end of any single stranded polynucleotide sequence is referred to as a 5 'end; the left direction of the polynucleotide sequences is referred to as the 5 'direction. The direction of 5 'to 3' addition of nascent RNA transcripts is referred to as the direction of transcription; regions of sequence on the DNA strand having the same sequence as the RNA transcript which are 5 'to the 5' end of the RNA transcript are referred to as "upstream sequences"; the regions of /

ΜΑ 32982Β1

A-1472-WO-PCT sequenced on the DNA strand having the same sequence as the RNA transcript which is 3 'to the 3' end are referred to as "downstream sequences".

The term "control sequence" refers to a polynucleotide sequence which can affect the expression and processing of the coding sequences to which it is ligated. The number of these control sequences may depend on the host organism. In particular embodiments, the control sequences for prokaryotes can include a promoter, a ribosome binding site, and a transcription termination sequence. For example, control sequences for eukaryotes may include promoters comprising one or more recognition sites for tanscription factors, transcription enhancer sequences and a transcription termination sequence. "Control sequences" can include leader sequences and / or fusion partner sequences.

The term "vector" refers to any molecule or entity (eg, nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.

The term "expression vector" or "expression construct" refers to a vector which is suitable for transformation of a host cell and contains nucleic acid sequences which direct and / or control (in combination with the host cell) expression of one or more heterologous coding regions which are operably linked to it. An expression construct can include, but are not limited to, sequences which affect or control transcription, translation and, if introns are present, which affect RNA splicing of a coding region linked to it. operationally.

Used herein, "operably linked" means that the components to which the term applies are in a relationship that enables them to perform their inherent functions under appropriate conditions. For example, a control sequence in a vector which is "operably linked" to a protein encoding sequence is ligated thereto such that expression of the protein encoding sequence takes place under conditions compatible with it. the transcription activity of the control sequences.

The term "host cell" refers to a cell which has been transformed, or which can be transformed, with a nucleic acid sequence and thus expresses a gene of interest. The term includes the progeny of the parent cell, whether or not that progeny is morphologically or genetically identical to the original parent cell, as long as the gene of interest is present.

ΜΑ 32982Β1

A-1472-WO-PCT

The term "transduction" refers to the transfer of genes from one bacterium to another, usually by a bacteriophage. Transduction ”also refers to the acquisition and transfer of eukaryotic cell sequences by replication of defective etroviruses.

The term "transfection" refers to the integration of foreign or exogenous DNA by a cell, and a cell has been transfected when the exogenous DNA has been introduced inside the cell membrane. Numerous transfection techniques are well known to those skilled in the art and are described here. See, e.g., Graham et al., 1973, Virology 456: جج; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, 3: 197 ح „ح ٠. These techniques can be used to introduce one or more fragments of exogenous DNA into appropriate host cells.

The term "transformation" refers to a change in the genetic characteristics of a cell, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed when it has been genetically modified from its native state by the introduction of new genetic material, through ttansfection, transduction, or other techniques. After transfection or transduction, the transforming DNA may recombine with that of the cell by physical integration into a chromosome of the cell, or it may transiently maintain itself as an episomal element without being replicated, or it may become. replicate independently as a plasmid. A cell is considered to have been "stably formed" when the transforming DNA replicates upon division of the cell.

The terms "polypeptide" or "protein" are used interchangeably herein to denote a polymer of amino acid residues. The terms also apply to polymers of amino acids in which one or more amino acid residues are analogs or mimetics of a corresponding natural amino acid, as well as to polymers of natural amino acids. The terms can also encompass polymers of amino acids which have been modified, for example by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. The polypeptides or proteins can be produced by a nahirelle cell and not recombinant; or they are produced by a genetically modified or recombinant cell, and comprise molecules having the amino acid sequence of the native protein, or molecules comprising deletions, additions and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" or "protein" specifically encompass antigen-binding proteins, for example CGRR R antigen-binding proteins, CGRP R-binding proteins, or antibodies.

ΜΑ 32982Β1

A-1472-WO-PCT sequences which include deletions, additions and / or substitutions of one or more amino acids of an antigen-binding protein. The term "polypeptide moiety" refers to a polypeptide which has an amino-terminal deletion, a carboxy-terminal deletion and / or an internal deletion relative to the full-length protein. In some embodiments, the fragments are about 5 to 500 amino acids in length. For example, the fragments can be at least 5, 6, 8, 10, 14,20, 50, 70,100,

110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Useful polypeptide fragments include immunologically functional antibody fragments, including binding domains. In the case of a CGRP R binding antibody, useful fragments include, but are not limited to, a CDR region, a heavy or light chain variable domain, part of an antibody chain or just its variable domain including two CDRs, and similar items. The "CGRP receptor", or "CGRPR" is understood to include RAMP1 and CRLR.

The term "isolated protein" (eg, an isolated antigen-binding protein), "isolated polypeptide" or "isolated antibody" means that a subject protein, polypeptide or anticoipsis (1) is free from at least some other proteins. with which it is normally found, (2) is essentially free from other proteins of the same source, for example of the same species, (3) is expressed by a cell of a different species, (4) has been separated from at least about 50 percent of the polynucleotides, lipids, carbohydrates or other materials with which it associates in nafirre, (5) is operably associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nafirre, or (6) is not found in nafirre. Typically, an "isolated protein", "isolated polypeptide" or "isolated antibody" constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a sample. given. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof may encode such isolated protein. Preferably, the isolated protein, polypeptide or antibody is substantially free from other proteins or other polypeptides or other contaminants which are found in its nafiral environment and which could interfere with its therapeutic, diagnostic, prophylactic, or therapeutic use. research or whatever.

A "variant" of a polypeptide (eg, an antigen-binding protein, or an antibody) comprises an amino acid sequence, one or more amino acids being inserted, deleted and / or replaced in the amino acid sequence. relative to another polypeptide sequence. Variants include fiision proteins.

ΜΑ 32982Β1

A-1472-WO-PCT

A "derivative" of a polypeptide is a polypeptide (eg, an antigen or antibody binding protein) that has been chemically modified in some way distinct from that used for insertion, deletion, or substitution variants, eg. by conjugation to another chemical residue.

The term "natural" as used throughout this specification in connection with biological materials such as polypeptides, nucleic acids, host cells and the like, refers to materials which are found in the nose.

An "antigen-binding protein" as the term is used herein refers to a protein that specifically binds a specified target antigen, such as CGRP R, particularly from primate, eg, human CGRR R. A CGRR R antigen-binding protein specifically binds the human CGRP R receptor.

An antigen-binding protein is said to “specifically bind” its target when the dissociation constant (Ko) is &lt;10⁶ M. The antibody specifically binds the target antigen with "high affinity" when the Ko is &lt;1 x 10 M. In one embodiment, the antibodies will bind to CGRP R, or human CGRP R with a K٥ &lt;5x 10 '? ; in another embodiment, the antibodies will bind with a Ko &lt;1χ 10? ; in another embodiment, the antibodies will bind with a Kd ix 10'8; in another embodiment, the antibodies will bind with a Ko six 108; in another embodiment, the antibodies will bind with a Ko S5x 10٠; in another embodiment, the antibodies will bind with a Ko six 10'5; in another embodiment, the antibodies will bind with a Ko S5x 10! ٥; in another embodiment, the antibodies will bind with a Kd six 10''٠.

An antibody, one of its antigen-binding fragments, or an antigen-binding protein "selectively inhibits" a specific receptor over other receptors when the IC50 of the antigen, its antibody-binding moiety or the antigen-binding protein in a specific receptor inhibition assay is at least 50 times lower than the IC50 in an inhibition assay of another "benchmark" receptor. The "selectivity ratio" is the IC50 of the reference receptor divided by the IC50 of the specific receptor. An antibody, one of its antigen-binding fragments or an antigen-binding protein selectively inhibits the human CGRP receptor if the IC50 of the antibody, its antigen-binding fragment or the antigen-binding protein in a cAMP test, for example the cAMP inhibition test described in example 4 given here, is at least 50 times lower than the IC50 of the same antibody, antigen-binding fragment thereof or protein binding the antigen in an AM1 inhibition test, AM2 or a human amylin receptor (eg, AMY1). By way of non-limiting example, if the IC50 of a specific anti-CGRP R antibody in a hCGRP R cAMP assay is, for example

ΜΑ 32982Β1

A-1472-WO-PCT example, between 0.1 nM and 20 nM, and that the IC50 of the same antibody in a cAMP test of hAM1, hAM2 or of the human AMY1 receptor is equal to 1000 nM or more, this antibody selectively inhibits the hCGRP receptor. An antigen-binding protein which selectively inhibits a specific receptor is also considered to be a neutralizing antigen-binding protein to that receptor.

An "antigen-binding region" refers to a protein or part of a protein, which specifically binds a specified antigen. For example, that part of an antigen-binding protein which contains the amino acid residues which interact with an antigen and which confer on the antigen-binding protein its specificity and affinity for the antigen is referred to as "binding region". 'antigen'. An antigen binding region generally comprises one or more "complementarity determining regions" ("CDR"). Some antigen binding regions also include one or more "framework" regions. A "CDR" is an amino acid sequence that contributes to the specificity and affinity of binding to the antigen. "Framework" regions can help maintain the correct conformation of CDRs to promote binding between the antigen binding region and an antigen.

In certain aspects, the invention relates to recombinant antigen-binding proteins that bind the human CGRP R protein, or CGRP R. In this context, a "recombinant protein" is a protein produced using recombinant techniques, i.e. by the expression of recombinant nucleic acid as described herein. Methods and techniques for producing recombinant proteins are well known to those skilled in the art.

The term "antibody" refers to intact immunoglobulin of any isotype, or an antigen-binding moiety thereof, and includes, for example, chimeric, humanized, fully human, and bispecific antibodies. An "antibody" as such is an antigen binding protein. An intact antibody usually consists of two full length heavy chains and two full length light chains, but in some cases it may have fewer chains, like natural antibodies in camels which only have heavy chains. The antibodies may be derived only from a single source, or they may be "chimeric", i.e. different parts of the antibody may be derived from two different antibodies, as described in more detail herein. below. Antigen-binding proteins, antibodies or binding fragments can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two heavy chains of full length and two light chains

ΜΑ 32982Β1

Full length A-1472-WO-PCT, derivatives, variants, fragments, and mutations thereof, examples of which are described below.

The term "light chain" includes a full length light chain and its fragments having sufficient variable region sequence to confer binding specificity. A full length light chain includes a variable region domain, V1, and a constant region domain. Cl. The variable region domain of the light chain binds to the amino terminus of the polypeptide. The light chains include kappa and lambda chains.

The term "heavy chain" includes a full length heavy chain and its fragments having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes one variable region domain, Vh, and three constant region domains, c, c, and c. The Vh domain is located at the amino terminus of the polypeptide, and the Ch domains are located at the carboxy terminus, C3 being closest to the carboxy terminus of the polypeptide. The heavy chains can have any isotype, including IgG (in particular, IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (in particular IgAl and IgA2 subtypes), IgM and IgE.

The terms "signal sequence", "leader sequence" or "signal peptide" refer to a short peptide chain (3-60 amino acids in length) which directs the transport of a protein.

Signal peptides can also be referred to as targeting signals, signal sequences, transit peptides, or localization signals. Some signal peptides are cleaved from the protein by a signal peptidase after protein transport, and the biologically active form of the protein (eg, an antigen-binding protein described herein) is therefore the shorter cleaved form. Therefore terms like "antibody comprising a heavy chain ... "," Antibody comprising a light chain ... , Etc., wherein the antibody is characterized as having a heavy chain and / or a light chain with a particular identified sequence, are understood to include antibodies having the specific identified sequences, antibodies having the specific identified sequences, but the signal sequences of which are replaced by different signal sequences, as well as antibodies having the identified sequences without any signal sequence.

The term "antigen-binding fragment" (or simply "fragment") of an antibody or immunoglobulin chain (heavy or light chain), as used herein, includes a part (however which part of which is obtained or synthesized) of an antibody which lacks at least some of the amino acids present in a full length chain, but which is capable of binding specifically to an antigen. These fragments i

ΜΑ 32982Β1

A-1472-WO-PCT are biologically active in the sense that they bind specifically to the target antigen and can compete with other antigen-binding proteins, especially intact antibodies, for specific binding to a given epitope. . In one aspect, such a component retains at least one of the CDRs present in the full length light or heavy chain, and in some embodiments, it comprises a single heavy chain and / or light chain or part thereof. These biologically active fragments can be produced by recombinant DNA techniques, or they can be produced by enzymatic or chemical cleavage of antigen-binding proteins, especially intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab,

Fab ', F (ab') 2, Fv, domain antibodies and single chain antibodies, and they may be derived from any mammalian source, including, without limitation, human, murine, rat, camelid or rabbit. It is further contemplated that a functional portion of the antigen-binding proteins described herein, for example, one or more CDRs, may be covalently linked to a second protein or to a small molecule to create a therapeutic agent directed to a particular target. in the body, which has bifunctional therapeutic properties, or which has an extended half-life in serum.

An "Fab fragment" consists of a light chain and C1 and variable regions of a heavy chain. The heavy chain of an Fab molecule cannot form a disulfide bond with another heavy chain molecule.

An "Fc" region contains two heavy chain fragments comprising the ChI and Ch2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interactions between the c domains.

An "Fab 'fragment" contains a light chain and part of a heavy chain which contains the Vfi domain and the c domain and also the region between the C1 and C2 domains, so that a disulfur bond can form between the two heavy chains of the two Fab 'fragments to form an F (ab') 2 molecule.

An "F (ab ') 2 fragment" contains two light chains and two heavy chains containing part of the constant region between the C1 and c domains, such that an interchain disulfide bond forms between the two heavy chains. An F (ab ') 2 fragment is therefore composed of two Fab' fragments which are held together by a disulfur bond between the two heavy chains.

The "Fv Tegion" comprises the variable regions of the heavy and light chains, nrais does not contain the constant regions.

ΜΑ 32982Β1

A-1472-WO-PCT

"Single chain anticotps" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in International Patent Application Publication No. WO 88/01649 and US Patent Nos. 4,946,778 and No. 5,260,203, the disclosures of which are incorporated herein by reference.

A "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some cases, two or more Vh regions are covalently joined with a linker peptide to create a bivalent domain antibody. The two Vh regions of bivalent domain antibodies can target the same antigen or different antigens.

A "bivalent antigen-binding protein" or a "bivalent antibody" includes two antigen binding sites. In some cases, the two binding sites have the same antigen specificities. Bivalent antigen-binding proteins and bivalent antibodies can be bispecific, see below.

A "multispecific antigen binding protein" or a "multispecific antibody" is a protein or antibody that targets more than one antigen or epitope.

A "bispecific," "dual-specific" or "bifunctional" antigen or anticoip-binding protein is an antigen-binding protein or a hybrid antibody, respectively, having two different antigen-binding sites. Antigen-binding proteins and bispecific antibodies constitute a species of multispecific antigen-binding protein or antibody and can be produced by a variety of methods, including, but not limited to, hybridoma binding or binding of Fab 'fragments. . See, for example,

Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 22: 315-321; Kostelnye1992,7, / ٥ ؛. Immunol. 1553-1547: قئ. The two binding sites of an antigen-binding protein or a bispecific anticoips bind to two different epitopes, which may be located on the same protein target or on different targets.

The term "neutralizing antigen-binding protein" or "neutralizing antibody" refers to an antigen-binding protein or an antibody, respectively, which binds to a ligand, prevents binding of the ligand to its binding partner, and interrupts the biological response that would have. was otherwise produced by binding of the ligand to its binding partner. When evaluating the binding and specificity of an antigen-binding protein, for example, an antibody or one of its immunologically functional antigen-binding fragments, an antibody or its fragment substantially inhibits the binding of a ligand to its binding partner when an excess

ΜΑ 32982Β1

Antibody A-1472-WO-PCT reduces the amount of binding partner bound to ligand by at least about 20%,

٥/٠, 40 ٥/٠, 50 ٥/٠, 60 ٠/٠, 70 ٠/٠, 80 ٥/٠, 85%, 90 ٥/٥, 95 ٠/٠, 97 ٥/٠, 99 ٠ / ٠ or more (measured in competitive in vitro binding assay). In the case of a CGRP R binding protein, this neutralizing molecule will decrease the ability of CGRP R to bind CGRP.

The term "competition," used in the context of antigen-binding proteins which can bind the same region on a target antigen, refers to competition between antigen-binding proteins which is determined by an assay in which the tested antigen-binding protein ( e.g. antibody or one of its immunologically functional antigen-binding fragments) prevents or inhibits the specific binding of a protein binding a reference antigen (e.g., ligand or reference antibody) to a common antigen (eg, CGRP R or an antigen-binding fragment thereof). Any number of competitive binding assays can be used, for example: direct or indirect solid phase radioimmunoassay (RIA), direct or indirect solid phase enzyme immunoassay (EIA), competition sandwich assay (see , for example, Stahli et al., 1983, Methods in Enzymology 9: 242253); Solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986,7. Immunol. 132: 3614-3619), direct label solid phase assay, direct label solid phase sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); Direct RIA with solid phase label using label 1-125 (see, eg, Morel et al., 1988, Molec. Immunol. 22: 7-15); Solid phase dhect biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology No .: 546-552); RIA marked direct (Moldenhauer et al., 1990, Scand. j. Immunol. 22: 77-82). Such an assay may involve the use of a purified antigen bound to a solid surface or cells bearing any of them, an unlabeled test antigen binding protein and a labeled reference antigen binding protein. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or to cells in the presence of the antigen binding protein being tested. Antigen-binding proteins identified by competition assays (competitor antigen-binding proteins) include antigen-binding proteins binding to the same epitope as the proteins binding a reference antigen and proteins binding an antigen binding to an adjacent epitope sufficiently close to the epitope to which the protein binding a reference antigen binds so that there is steric hindrance. Usually when a competitor antigen-binding protein is present in excess, it inhibits the specific binding of a reference antigen-binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60% , 65%, 70% or 75%. In some cases the binding is inhibited at least 80 ٠/٠, 85%, 90 ٥/٠ 95, نره, or 97% or more. Competitive inhibition can also

ΜΑ 32982Β1

A-1472-WO-PCT can be measured by immobilizing a protein binding a reference antigen on a substrate, for example, a detector chip, which captures the antigen on the substrate by binding to the reference antibody, and assessing whether a different antigen-binding protein (a competitor antigen-binding protein) may additionally bind to the antigen. An example of the latter competitive binding assay employs Biacore assay, and is described in Example 7 given here.

The term "antigen" or "immunogen" refers to a molecule or part of a molecule capable of being bound by a selective binding agent, such as an antigen-binding protein (including, for example, an antibody or one of its fragments binding the immunologically functional antigen), and moreover capable of being used in an animal to produce antibodies capable of binding to the antigen. An antigen may have one or more epitopes which are capable of interacting with different antigen-binding proteins, for example, antibodies.

The term "epitope" refers to the part of a molecule which is bound by an antigen-binding protein (eg, an antibody). The term includes any determinant capable of binding specifically to an antigen-binding protein, such as an antibody or a T cell receptor. An epitope can be contiguous or non-contiguous (e.g., (i) in a single chain polypeptide, amino acid residues which are not contiguous to each other in the polypeptide sequence, but which, in the context of molecule, are linked by the antigen-binding protein, or (ii) in a multimeric receptor, e.g., CGRP R, comprising two or more individual components, e.g., RAMP1 and CRLR, amino acid residues present on two or more individual components, but which, in the context of a multimeric receptor, are linked by the antigen-binding protein). In some embodiments, the epitopes may be mimetic in that they comprise a three-dimensional structure which is similar to that of an epitope used to produce the antigen-binding protein, yet do not include any or only some of the residues of amino acids that are found in the epitope used to produce the antigen-binding protein. Most often, epitopes are located on proteins, but in some cases, they can split onto other types of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groups such as amino acids, carbohydrate side chains, and phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics, and / or specific charge characteristics. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

ΜΑ 32982Β1

A-1472-WO-PCT

The term "identity" refers to a relationship between the sequences of two or more polypeptide molecules, or between two or more nucleic acid molecules, as determined by alignment and comparison of the sequences. "Percent identity" refers to the percentage of identical residues between amino acids or nucleotides in the molecules being compared and is calculated based on the size of the smallest of the molecules being compared. For these calculations, the gaps in the alignments (if any) must be addressed by a mathematical model or a particular computer program (ie an "algorithm"). Methods which can be used to calculate the identity of nucleic acids or aligned polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. w., ed.), 1993, New York: Academie Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, H.

G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academie Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M, Stockton Press; and Carillo et al., 1988, SIAMJ. Applied Math. tAI.

When calculating the percentage identity, the sequences to be compared are aligned so as to obtain the greatest match between the sequences. The computer program used to determine percent identity is the GCG software package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res.12: 387; Genetics Computer Group, University of Wisconsin, Madison, WI). The GAP computer algorithm is used to align the two polypeptides or polynucleotides whose percent sequence identity is to be determined. The sequences are aligned for optimal pairing of their respective amino acids or nucleotides ("paired extent" determined by the algorithm). A breach introduction penalty (which is calculated to be equal to 3x the mean diagonal, the “mean diagonal” being the mean of the diagonal of the comparison matrix used; the diagonal is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is typically 1 / 10th of the gap introduction penalty), as well that a comparison matrix like PAM 250 or BLOSUM 62 are used in combination with the algorithm. In some embodiments, a standard comparison matrix (see, Daylroff et al., 1978, Atlas of Protein Sequence and Structure §: 345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992, Proc. Natl Acad Sci USA §2: 10915-10919 for the BLOSUM comparison matrix 62 is also used by the algorithm.

ΜΑ 32982Β1

A-1472-WO-PCT

The recommended parameters for determining percent identity for polypeptide or nucleotide sequences using the GAP program are as follows:

Algorithm: Needlemaneia /., 1970.7 Mol. Biol. 48: 443-453;

Comparison matrix: BLOSUM 62 of Henikoff et al., 1992, supra;

Breach penalty: 12 (but without penalty for end breaches)

Breach length penalty: 4

Similarity threshold: 0

Some alignment schemes for aligning two amino acid sequences may allow pairing of only a short region of two sequences, and this small aligned region may have very strong sequence identity even though there is no significant relationship between the two full length sequences. Therefore, the chosen alignment method (GAP program) can be adjusted if desired to achieve alignment that spans at least 50 contiguous amino acids of the target polypeptide.

As used herein, the term "substantially pure" means that the species of molecule described is the predominant species present, i.e., on a molar basis, it is the most abundant of all. species in the same mixture. In some embodiments, a substantially pure molecule is a composition in which the subject species constitutes at least 50% (on a molar basis) of all macromolecular species present. In other embodiments, in a substantially pure composition, it will constitute at least 80%, 85%, 90%, 95%, or 99% of all the macromolecular species present in the composition. In other embodiments, the subject species is purified essentially to homogeneity, the contaminating species cannot be detected in the composition by conventional detection methods, and the composition therefore consists of a single detectable macromolecular species. .

The term "taiter" refers to any indication of success in the treatment, or improvement of a lesion, pathology or condition, in particular any objective or subjective parameter such as lowering, remission. , decreasing symptoms, or making the injury, pathology or condition more tolerable to the patient; slowing down degeneration or decline; obtaining a less debilitating final point of degeneration; improving the patient's physical or mental well-being. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examinations, and / or psychiatric evaluation. For example, some methods presented here successfully treat migraine headaches as a prophylaxis or as an acute treatment, reducing the frequency

ΜΑ 32982Β1

A-1472-WO-PCT migraine headache, reducing the severity of migraine headache, and / or improving a symptom associated with migraine headache.

An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying cause, prevent the onset of symptoms, and / or their underlying cause, and / or ameliorate or repair damage due or associated with migraine headache. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to cure a disease condition (eg, migraine headache) or symptoms, particularly a condition or symptoms associated with the disease condition, or to prevent, hinder, delay or reverse it. another way the course of the medical condition or any other unwanted symptom relates to the disease in any way.

A "prophylactically effective amount" is an amount of the pharmaceutical composition which, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of. migraine headache, or reduced likelihood of onset (or recurrence) of migraine headache or migraine headache symptoms. The full therapeutic or prophylactic effect does not necessarily follow administration of a single dose, and may be exerted only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

"Amine acid" includes its normal meaning in the field. The twenty naturally occurring amino acids and their abbreviations conform to conventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. s. Golub and DR Green, eds.), Sinauer Associates: Sunderland, Mass. (1991), incorporated here by reference for convenience. Stereoisomers (e.g. D-amino acids) of the twenty conventional amino acids, non-conventional amino acids such as α-, α-disubstituted amino acids, Ν-alkylated amino acids, and other unconventional amino acids can also constitute suitable components for polypeptides and are included within the term "amino acid". Examples of unconventional amino acids include the following: 4-hydroxyproline, y-carboxyglutamate, ε-Ν, Ν, Ν-trimethyllysine, ε-Ν-acetylysine, O-phosphoserine, N-acetylserine, Nformylmethionine, 3-methylhistidine, 5-hydroxylysine, oN-methylarginine, and other similar amino acids and imino acids (eg, 4-hydroxyproline). In the polypeptide notation used here, the direction to the left is the amino-terminal direction and the direction

ΜΑ 32982Β1

A-1472-WO-PCT direction to the right is the carboxy-terminal direction, in accordance with standard usage and conventions.

General summary

The present invention provides antigen-binding proteins that bind the CGRP R protein, including human CGRP R (hCGRP R). It relates to antigen-binding proteins which are polypeptides in which one or more complementarity determining regions (CDRs) described herein are included and / or joined. In some antigen-binding proteins, the CDRs are included in a "framework" region, which orients the CDR (s), so as to obtain the correct antigen-binding properties of the CDRs. In general, the antigen-binding proteins of the invention can interfere with, block, reduce or modulate the interaction between CGRP and CGRP R.

Certain antigen-binding proteins described herein are antibodies or are derived from antibodies. In some embodiments, the polypeptide structure of the antigen-binding proteins is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to as here by "antibody mimetics"), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as "antibody conjugates"), and their fragments. The various structures are described in more detail below.

The antigen-binding proteins of the invention have been shown to bind to CGRR R, in particular to human CGRP R. As described in more detail below, certain antigen-binding proteins have been tested and shown to bind to epitopes different from those linked by a number of other antibodies directed against one or the other. 'other component of CGRP R. The proteins binding an antigen of the invention compete with CGRP and thus prevent CGRP from binding to its receptor. Therefore, the antigen binding proteins of the invention are able to inhibit the activity of CGRP R. In particular, the proteins binding an antigen binding to these epitopes can have one or more of the following activities: inhibition, inter alia of the induction of the CGRP R signal transduction pathways, inhibition of vasodilation, initiation of vasoconstriction, reduction of inflammation, e.g. neurogenic inflammation, and other physiological effects induced by CGRP R after CGRP binding.

The antigen-binding proteins described herein have a variety of uses.

Some of the antigen-binding proteins, for example, are useful in specific binding assays, affinity purification of CGRP R, especially hCGRP R or its

ΜΑ 32982Β1

A-1472-WO-PCT ligands and in screening assays to identify other antagonists of the activity of

CGRP R. Some of the antigen-binding proteins are useful in inhibiting the binding of CGRP to CGRP R.

Antigen binding proteins can be used in a variety of processing applications, as explained herein. For example certain CGRP R antigen binding proteins are useful for treating conditions associated with signaling mediated by CGRP R, such as reducing, alleviating or curing the frequency and / or severity of migraine headaches, reduction, relief or treatment of cluster headache, reduction, relief or treatment of chronic pain, relief or treatment of diabetes sucks (type II), reduction, the relief or treatment of hemodynamic disorders associated with endotoxemia and sepsis in a patient. Other uses of the antigen-binding proteins include, for example, the diagnosis of diseases or conditions associated with CGRP R and screening assays for the presence or absence of CGRP R. Some of the antigen binding proteins described herein are useful in the treatment of consequences, symptoms and / or pathology associated with CGRP R activity. They include, but are not limited to, various types of migraine headache.

CGRP Receiver

The antigen-binding proteins described herein bind to CGRP R, in particular to human CGRP R. CGRP R is a multimere which includes CRLR and RAMP1. The nucleotide sequence of human CRLR is given here by SEQ ID ΝΟ: 1. The amino acid sequence of human CRLR is given herein by SEQ ID NO: 2. The nucleotide sequence of human RAMP1 is given herein by SEQ ID NO: 3. The amino acid sequence of human RAMP1 is given herein by SEQ ID NO: 4. The antigen-binding proteins described herein bind the extracellular part of CGRP R, which includes the extracellular parts of CRLR and RAMP1. An exemplary extracellular domain ("DEC") of human CRLR is encoded by the nucleotide sequence shown by SEQ ID NO: 5, and has the amino acid sequence shown by SEQ ID NO: 6. This sequence comprises a signal peptide; an example of a DEC of the material CRLR (minus the signal peptide) has the amino acid sequence shown by SEQ ID NO: 10. An example of a DEC of human RAMP1 is encoded by the nucleotide sequence shown by SEQ ID NO: 7, and has the amino acid sequence shown by SEQ ID NO: 8. This sequence comprises a peptide, signal; an example of a mature RAMP1 DEC (minus the signal peptide) has the amino acid sequence shown by SEQ ID NO: 11. As described below, CGRP R proteins can also include fragments. Used here, the terms are

ΜΑ 32982Β1

A-1472-WO-PCT are used interchangeably to designate a receptor, in particular, unless otherwise indicated, a human receptor which binds specifically to CGRP.

The term CGRP R also includes post-translational modifications of the amino acid sequence of CGRP R, eg, possible N-linked glycosylation sites. Thus, antigen-binding proteins can bind to or be produced from proteins glycosylated at one or more of the positions.

CGRP receptor binding proteins

Various selective binding agents useful for regulating CGRP R activity are provided. These agents include, for example, antigen-binding proteins that contain an antigen-binding domain (eg, single chain antibody, antibody domains, immunoadhesions and polypeptides with an antigen binding region) and which specifically bind to CGRP R, in particular to human CGRP R. Certain agents, for example, are useful for inhibiting the binding of CGRP to CGRP R, and therefore may be used to inhibit, interfere with or modulate one or more of the activities associated with CGRP R signaling.

In general, the antigen-binding proteins of the invention usually comprise one or more CDRs described herein (eg, 1,2,3,4,5 or 6). In some cases, the antigen-binding protein comprises (a) a polypeptide structure and (b) one or more CDRs which are inserted into and / or joined to the polypeptide structure. The polypeptide structure can take a variety of different forms. For example, it can be, or include, the char-slope of a nahrrel antibody or a fiagment or variant thereof, or it can be entirely synthetic in nature. Examples of various polypeptide structures are described in detail below.

In some embodiments, the polypeptide structure of the antigen-binding proteins is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, antibodies. synthetic antibodies (sometimes referred to herein as antibody mimetics), chimeric antibodies, humanized antibodies, antibody firsions (sometimes referred to herein as antibody conjugates), and parts and fiagments of each of them, respectively. In some cases, the antigen-binding protein is an immunologic fragment of an antibody (eg, Fab, Fab ', F (ab') 2, or scFv). The various structures are described in detail and defined here.

Some of the antigen-binding proteins of the invention bind specifically to CGRE

Human R. In a specific embodiment, the antigen-binding protein binds

ΜΑ 32982Β1

A-1472-WO-PCT specifically to the human CGRP R protein comprising human CRLR having the amino acid sequence of SEQ ID NO: 2 and human RAMP1 having the amino acid sequence of SEQ ID NO: 4.

In embodiments in which the antigen-binding protein is used in therapeutic applications, an antigen-binding protein can inhibit, interfere with, or modulate one or more biological activities of CGRP R. In this case, an antigen-binding protein specifically binds and substantially inhibits the binding of human CGRP R to CGRP when an excess of antibody reduces the amount of human CGRP R bound to CGRP, or vice versa, by at least about 20 ٥/٠, 30%, 40 ٠/٥, 50 ٠/٥, 60 ٠/٥, 70 ٠/٠, 80 ٠/٠, 85 ٥/٠, 90 ٥/٠, 95 ٠/٠,

٠/٠, 99 ٠/٥ or more (eg, based on measurement of binding in a competitive in vitro binding assay).

Natural antibody strucfare

Some of the antigen-binding proteins of the invention have the structure generally associated with nataral antibodies. The structural units of these antibodies generally include one or more tetramers, each consisting of two identical pairs of polypeptide chains, although some mammalian species also produce antibodies having only one heavy chain. In a typical antibody, each pair or pair comprises a full length "light" chain (in some embodiments, about 25 kDa) and a full length "heavy" chain (in some embodiments, about 50-70 kDa). ). Each individual immunoglobulin chain is composed of several "immunoglobulin domains", each constituting approximately 90 to 110 amino acids, and expressing a characteristic folding pattern. These domains are the basic units of which the anticoips polypeptides are composed. The amino-terminal part of each chain generally comprises a variable domain which is responsible for the recognition of the antigen. The carboxy-terminus is more evolutionarily conserved than the other end of the chain and is referred to as a "constant region" or "c-region". Human light chains are generally classified into kappa and lambda light chains, and each contains a variable domain and a constant domain. Heavy chains are generally classified as mu, delta, gamma, alpha, or epsilon chains, which define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively. IgGs have several subtypes including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM, and IgM2. The IgA subtypes include IgAl and IgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; IgG and IgE isotypes contain two heavy chains and

ΜΑ 32982Β1

A-1472-WO-PCT two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain c region generally includes one or more domains which may be responsible for effector function. The number of heavy chain constant region domains depends on the isotype. IgG heavy chains, for example, contain three c-region domains called ChI, Ch2 and Ch. The antibodies of the invention can have any of these isotypes and subtypes. In some embodiments, the CGRP R antibody is of the IgG1, IgG2, or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regions are joined by an "ل" region of about twelve or more amino acids, the heavy chain comprising a "D" region of about ten or more amino acids. See, eg, Fundamental Immunology, 2nd ed. Ch. 7 (Paul, w.) Ed.) 1989, New York: Raven Press (incorporated herein by reference in its entirety for convenience). The variable region of each heavy / light chain pair generally forms the antigen binding site.

An example of an IgG2 heavy chain constant domain of an example anti-CGRP R monoclonal antibody has the amino acid sequence:

<img file="MA32982B1_D0005.tif" />

YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (the last 326 residues of the sequence shown by SEQ. ID NO: 29).

An example of a kappa light chain constant domain of an example anti-CGRP R monoclonal antibody has the amino acid sequence:

<img file="MA32982B1_D0006.tif" />

last 107 residues of the sequence presented by SEQ ID NO: 14).

Variable regions of immunoglobulin chains generally exhibit the same overall strachira, comprising relatively conserved framework regions (FRs) joined by three hypervariable regions, most often referred to as "complementarity determining regions" or CDRs. The CDRs of the two chains of each heavy chain / light chain pair mentioned above are generally aligned by the framework regions to form a strachira which specifically binds with a specific epitope on the target protein (eg, CGRP R). From the C-terminus to the N-terminus,

ΜΑ 32982Β1

A-1472-WO-PCT natural light and heavy chain variable regions generally conform to the following order of elements: FRI, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised to assign numbers to amino acids which occupy positions in each of these domains. This numbering system is defined in Kabat

Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia &amp; Lesk, 1987,2. Rio /. ٥: 901-917; Chothia e ؛ ai., 1989, Atowre 342.:878-883.

The various heavy and light chain variable regions given herein are shown in Table 3. Each of these variable regions can be attached to the heavy and light chain constant regions above to form an antibody heavy and light chain. complete, respectively. Additionally, each heavy and light chain sequence so produced can be combined to form a complete antibody structure. It should be understood that the heavy chain and light chain variable regions, shown herein, may also be attached to other constant domains having different sequences from the example sequences shown above.

Specific examples of some of the full length heavy and light chains of the antibodies of the invention and their corresponding amino acid sequences are summarized in Tables 2Α and 2B. Table 2Α shows examples of light chain sequences and Table 2Β shows examples of heavy chain sequences.

Table 2Α - Example of antibody light chain amino acid sequences

<td>SEQ ID NQ:</td><td>Called ion</td><td>Content in the clone</td><td>Sequence</td>

<td> 12</td><td>١ ا</td><td>OIEIIIC</td><td>MDMRVPAQLIGLLLLWLRGARCQSVITQPPSVSEAPGQKVTISC SGSSSNIGNNYVSWYQQIPGTAPKUIYDNNKRPSGIPDRFSGS KSGTSATLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL GQPKANPTVTLFPPSSEEIQANKATVCLISDFYPGAVTVAWKAD GSPVKAGVETTKPSKQSNNKYAASSYISLTPEQWKSHRSYSCQ VT HEGSTVEKTVAPTECS</td>

<td> 13</td><td> 12</td><td>01Η7 LC</td><td>MDMRVPAQUGLLLLWIRGARCQSVLTQPPSASGTPGQRVTISC GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKAD GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VT HEGSTVEKTVAPTECS</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td> 14</td><td> 13</td><td>02Ε7 LC</td><td>MDMR٧PAQLLGLLLLWLRGARCDIQMlQSPSSLSAS٧GDRVH1 GSGTEFll-riSSLQPEDLAlYYCIQYNIYPWlFGQGLKVEIKRTVA APSVFIFPPSDEQLKSGIASVVCLLNNFYPREAKVQWKVDAQ SGNSQESVIEQDSKDSTYSISSTLTLSKADYEKHKVYACÉVIHQ GLSSPVLKSFNRGEC</td>

<td> 15</td><td>L4</td><td>03B6LC</td><td>MDMRVPAQLLGLLLLWLRGARCSSELTQDPTVSVALGQLVKIC SGNIASLTGAQAEDEADYYCNSRDSSVYHLVLGGGIKLTVLG QPKANPTTLFPPSSEELQANKATIVCLISDFYPGAVTVAWKADG SPVKAGVEUKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPFECS</td>

<td> 16</td><td>L5</td><td>O3C8 IC</td><td>MDMRYPAQLLGLLLLWLRGARCDIIIAQTPISLSTPGQPASISC RIVAAPSVFIFPPSDEQLKSGASWCI.LNNFYPREAKVQWKVD VTHQGLSSPVTKSFNRGEC</td>

<td> 17</td><td>IR</td><td>04Ε4 LC</td><td>MDMRVPAQllGlLllWLRGARCQSVLLQPPSVSAAPGQKVTISC SGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGS KSGLSTLGITGLQTGDEADYYCGTWDSRLSAWFGGGTKLLVL GQPKANPTVTLFPPSSEELQANKALIVCLISDFYPGAWVAWKAD GSPVKAGVETKPSKQSNNKYAASSYISLFPEQWKSHRSYSCQ VTHEGSFVEKTVAPTECS</td>

<td> 18</td><td> 17</td><td>04Η6 LC</td><td>MDMRVPAQLIGLILLWLRGARCDIVMTQSPISLPVLPGEPASISC RSSQSLLHSFGYNYLDWYLQKPGQSPQLYLGSNRASGVPDRF SGSGSGDFTLKISRVEAEDVGVYYCMQALQIPF LFGPGLKVDI KRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLLSKADYEKHKVYAC ÉWHQGLSSPVTKSFNRGEC</td>

<td> 19</td><td> 18</td><td>O5F5 LC</td><td>MDMRVPAQLLGLLLIWIRGARCDIILTQTPLSLSVTPGQPASISC KSSQSLLHSDGKIYLYWYLQKPGQPPQLUYEVSNRFSGEPDRF SGSGSGÏDFILKISRVEAEDVGTYYCMQSFPLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGLASWCILNNFYPREAKVQWKVD</td>

ا.

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td></td><td>NALQSGNSQESVLEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VT HQGLSSPVTKSFNRGEC</td>

<td> 20</td><td> 19</td><td>09D41C</td><td>MDMRVPAQUGLLILWLRGARCQSVLTQPPSVSAAPGQKVTISC KSGTSATLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL GQPKANPJVLLFPPSSEELQANKATLVCLISDFYPGAVTAWKAD VLHEGSLVEKAPLECS</td>

<td> 21</td><td>IOL</td><td>O9F5 LC</td><td>MDMRVPAQLLGLLLWLRGARCQSVLTQSPSASGTPGQRVSC KSGTSASLTISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL GQPKANPIVTLFPPSSEEIQANKATLVCLISDFYPGAVLVAWKAD VTHEGSTVEKTVAPTECS</td>

<td> 22</td><td>READ</td><td>10Ε4 LC</td><td>MDMRVPAQLLGLLLLWLRGARCQSVLrQPPSASGGQRVÏlSC SGSSSNIGSNLVNWYQQLPGTAPKLLIYÏNNQRPSGVPDRFSGS GQPKANPTVTIFPPSSEELQANKATLVCLSDFYPGAVTVAWKAD VTHEGSTEKLVAPTECS</td>

<td> 23</td><td> 112</td><td>11D11HL 11Η9 LC</td><td>MDMRVPAQLLGlllLWLRGARCQSVITQPPSASGIPGQRVLISC GQPKANPTVTLFPPSSEELQANKATLVCUSDFYPGAVTVAWKAD GSPVKAGVETTKPSKQSNNKYAASSYLSITPEQWKSHRSYSCQ VTHEGSTVEKVAPTECS</td>

<td> 24</td><td> 113</td><td>12Ε8 LC</td><td>MDMRVPAQLLGLLLLWLRGARCDITTQTPLSLSVSPGQPASISC KSSQSILHSDGRNYLYWYLQKPGQPPQLLIYEVSNRFSGLPDRF SGSGSGTDFTLKISRVEAEDVGIYYCMQSFPLPLTFGGGTVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSIYSLSSTITSKADYEKHKVYACEA VT HQGLSSPVTKSFNRGEC</td>

<td> 25</td><td> 114</td><td>12G8 HL</td><td>MDMRVPAQU-GLLLLWLRGARCQSVLTQPPSVSAAPGQKVTSC SGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGS</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td></td><td>KSGTSATLGITGLQTGDEADYYCGTWDSRLSAWFGGGKVL GQPKANPTVTLFPPSSEEIQANKATLVCLISDFYPGAVTVAWKAD GSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VHEGSTEKTVAPÏECS</td>

<td> 26</td><td> 115</td><td>13Η2 LC</td><td>MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTIT GSGTEFILPSSLQPEDFAIYYCLQYNSFPWTFGQGTKVELKRTV AAPSVFIFPPSDEQLKSGIASVVCINNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACÉVTH QGLSSPVIKSFNRGEC</td>

<td> 27</td><td> 116</td><td>32Η7 LC</td><td>METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLSPGERATLSCRA SQSVSSGYLTWYQQKPGQAPRLLIYGASSRAGIPDRFSGSGSG TDFTLTISRLEPEDFAVYYCQQYGNSLCRFGQGTKLEIKRTVAAP NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACÉVTHQGL SSPVTKSFNRGEC</td>

<td> 28</td><td>ί١٦</td><td>32Η7 CS LC</td><td>METPAQUFLLLLWIPDTTGEIVTQSPGTLSISPGERATLSCRA TDFTLTISRLEPEDFAVYYCQQYGNSISRFGQGTKLEIKRTVAAP SVFIFPPSDEQIKSGTASWCLLNNFYPREAKVQWKVALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACÉVTHQGL SSPVTKSFNRGEC</td>

Table 2Β - Examples of amino acid sequence of heavy chains of antibodies

<td>SEQ ID NO:</td><td>Called tion</td><td>Content in the clone</td><td>Sequence</td>

<td> 29</td><td>Η1</td><td>01E11HC 04E4HC 09D4HC</td><td>MDMRVPAQILGLLILWLRGARCQVQLVESGGGWQPGRSLRLS GRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDRLNYYDSSGY YHYKYYGMAVWGQGTTVTVSSASTKGPSVFPIAPCSRSTSEST AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVIQSSGIYSLS SWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP APPVAGPSVFLFPPKPKDTIMISRTPEVTCVWDVSHEDPEVQFN</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td></td><td>WYVDGVEVHNAKTKPREEQFNSTFRSLTVVHQDWLNGKEY KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVS LCVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFIYS KITVDKSRWQQGNVFSCSVMHEAIHNHYTQKSLSISPGK</td>

<td> 30</td><td>Η2</td><td>01Η7 HC</td><td>MDMRYPAQLIGLLILWIRGARCEVQLVESGGGLVKPGGSLRLS CAASGFTFSNAWMSWOAPGKGLEWYGRIKSrtDGGrtDYAA PVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTDRTGYSISW SSYYYYYGMDVWGQGTl / SSASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSl SSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVFLFPPKPKDTIMISRTPEVTCWVDVSHEDPEVQF SLCLVKGFYPSDIAVEWESNGQPENNYKDPPMIDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEAIHNHYTQKSLSLSPGK</td>

<td> 31</td><td>Η3</td><td>02Ε7 HC</td><td>MDMRVPAQILGLLLLWLRGARCEVQLLESGGGLVQPGESIRLS GWYDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSES TAAIGCIVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGIYSL SSVVTVPSSNFGGIYTCNVDHKPSNKVDKTVERKCCVECPPC PAPPVAGPSVFIFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQF YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV SLTCIGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFIY SKITDKSRWQQGNVFSCSVMHEALHNHYTQKSLSISPGK</td>

<td> 32</td><td>Η4</td><td>03Β6 HC</td><td>MDMRVPAQLLGLHLWLRGARCQVQIVQSGAEVKKPGASVKVS CKASGY7FTGYYMHWVRQAPGQGLEWMGWINPNSGGNYAQK FQGR ^ MTRDTSISTAYMELSRLRSDDTAVYFCARDQMSIIMLRG VFPPYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSEST AALGCLVKDYFPEPVrt / SWNSGALISGVHTFPAVIQSSGLYSLS SWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP APPVAGPSVFIFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTFRVVSVITWHQDWLNGKEY</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td></td><td>KCKVSNKGIPAPIEKTISK٢KGQPREPQVY٦-LPPSREEMTKNQVS ITCIVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYS KTDKSRWQQGNVFSCSVMHEALHNHYTQKSLSISPGK</td>

<td> 33</td><td>Η5</td><td>O3C8 HC O5F5 HC 12E8HC</td><td>MDMRVPAQUGLILLWIRGARCQVQIVESGGGWQPGRSLRLS CAASGFJFSSYGMHWVRQAPGKGLEWAVISYDGSHESYADSV KGRFTISRDISKNT LYLQMNSLRAEDTAVYFCARERKRYTMSTLY YYFYYGMDVWGQGUVTSSASTKGPSVFPIAPCSRSTSESJAA LGCIVKDYFPEPVTSWNSGALSGVHTFPAVLQSSGLYSISSV VTVPSSNFGTQTYTCNVDHKPSKVDKERKCCVECPPCPAP PVAGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTFRWSVLTVVHQDWLNGKEYK CKVSNKGIPAPIEKBSK GQPREPQVYTLPPSREEMFKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFIYSK LVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</td>

<td> 34</td><td>Η6</td><td>04Η6 HC</td><td>MDMRVPAQLGULIWLRGARCEVQIVESGGGIVKPGRSLRLS CTASGFTFGDYAMSWFRQAPGKGLEWIGFIRSRAYGGÏPEYAAS VKGRFPISRDDSKTIAYLQMNSIKTEDFAVYFCARGRGIAARWDY WGQGTTVSSASTKGPSVFPIAPCSRSFSESTAAIGCLVKDYF PEPVTSWNSGALTSGVHJFPAVLQSSGLYSISSWTVPSSNFG TQIYICNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFL AKTKPREEQFNSTFRWSVtTVVHQDWLNGKEYKCKVSNKGLPA PIEKTISKKGQPREPQVYTIPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPMIDSDGSFFLYSKLTVDKSRWQ QGNVFSCSYMHEALHNHYTQKSISLSPGK</td>

<td> 35</td><td>Η7</td><td>O9F5 HC</td><td>MDMRVPAQLIGILWLRGARCEVQIVESGGGI-VKPGGSLRLS CAASGFTFSNAWMSIQAPGKGLEWVGRIKSKTDGGnDYTA PVKGRFflSRDDSKNT LYLQMNSLKAEDIAVYYC DRTGYSISW SSYYYYYGMDVWGQG٢T٧7VSSASKGPSVFPIAPCSRSTSES TAALGCLVKDYFPEP ^ VSWNSGALTSGVHTFPAVLQSSGLYSl SSVVTVPSSNFG7QTYICNVDHKPSNTKVDKTVERKCCVECPPC PAPPVAGPSVFIFPPKPKLMISRTPEVTCWVDVSHEDPEVQF YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQV</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td></td><td>SLTCLYKGFYPSDIAVEWESNGQPENNYKnPPMIDSDGSFFLY SKLFVDKSRWQQGNVFSCSVMHEALHNHYIQKSLSISPGK</td>

<td> 36</td><td>Η8</td><td>10Ε4 HC</td><td>MDMRVPAQLLGLLLIWLRGARCQVQIYQSGAEVKKPGASVKVS CKASGYlFTDYYMY١WVRQAPGQGLEWMGWlSPNSGGTNYAQK FQGRVTITRDTSISTAYMELSRLRSDDTAVYYCVRGGYSGYAGL YSHYYGMDVWGQGTTVFVSSASTKGPSVFPLAPCSRSTSESTA ALGCKYFPEPVTVSWNSGAIISGVHFFPAVLQSSGI-YSISS WTVPSSNFGFQTYCNVDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFIFPPKPKDTLMISRIPEVTCVWDVSHEDPEYQFN WYVDGVEVHNAKTKPREEQFNSFFRVVSVTVHQDWLNGKEY KCKVSNKGLPAPIEKTISKKGQPREPQVYTIPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTPPMLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYLQKSLSISPGK</td>

<td> 37</td><td>Η9</td><td>11D11HC</td><td>CAASGFIFGNAWMSWRQAPGKGLEWVGRIKSKTDGGHDYA APVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYFDRTGYSIS SIAALGCIVKDYFPEPVTVSWNSGALTSGVHTFPAVIQSSGLYS ISSWTVPSSNFGFQTYTCNVDHKPSNTKVDKTVERKCCVECPP CPAPPYAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQ FNWYVDGVEVHNAKFKPREEQFNSTFRWSVITVHQDWINGK EYKCKVSNKGLPAPIEKSKTKGQPREPQVYTPPSREEMIKNQ VSITCIVKGFYPSDIAVEWESNGQPENNYKTPPMIDSDGSFFL YSKTDKSRWQQGNVFSCSVMHEALHNHYFQKSLSLSPGK</td>

<td> 38</td><td>Η10</td><td>11Η9 HC</td><td>MDMRVPAQLIGLLLLWIRGARCEVQLVESGGGLVKPGGSLRLS APVKGRFTISRDDSKNTLYLQMNSLKTEDTAWYCTTDRTGYSIS WSSYYYYYGMDVWGQGTTVTSSASTGPSVFPLAPCSRSTSE SLAALGCLVKDYFPEPVTSWNSGALTSGVHTFPAVIQSSGLYS LSSWTVPSSNFGIQTYTCNVDHKPSNTKVDKTVERKCCVECPP CPAPPVAGPSVFIFPPKPKDTLMISRFPEVCWVSHEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGK EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFL</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td></td><td>YSKLTVDKSRWQQGNVFSCSVMHEAIHNHYIQKSISLSPGK</td>

<td> 39</td><td>Η11</td><td>12G8 HC</td><td>MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGWQPGRSIRIS GRFTISRDNSKNTFIQMNSIRAEDIAVYYCARDRINYYDSSGY YHYKYYGIAVWGQGTTVTVSSASIKGPSVFPLAPCSRSTSESIA AIGCLVKDYFPEPVTSWNSGALTSGVHTFPAVIQSSGLYSISS VVTVPSSNFGTQTY'TCtDHKPSNTKVDKTVERKCCVECPPCPA PPVAGPSVFIFPPKPKDFIMISRTPEVTCVWDVSHEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTFRVVSVITWHQDWLNGKEY KCKVSNKGIPAPIEKTISKTKGQPREPQVYTLPPSREEMIKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTPPMIDSDGSFFIYS KLVDKSRWQQGNVFSCSVMHEALHNHYTQKSISISPGK</td>

<td> 40</td><td>Η12</td><td>13Η2 HC</td><td>MDMRVPAQILGLUIWLRGARCEVQLVESGGGLVKPGGSLRIS AALGCIVKDYFPEPVTVSWNSGALTSGVHTFPAVIQSSGIYSL SSVV7VPSSNFGTQTYCNVDHKPSNTVDKTVERKCCVECPPC PAPPVAGPSVFIFPPKPKDILMISRTPEVTCWVDVSHEDPEVQF YKCKVSNKGLPAPIEKTISKTKGQPREPQVYTPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSISPGK</td>

<td> 41</td><td>Η13</td><td>32Η7 HC</td><td>MDMRVPAQLIGILILWIRGARCQVQIVESGGGWQPGRSLRIS YYYGMDVWGQGTTVTSSASKGPSVFPLAPCSRSTSESAAl GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW IVPSSNFGIQTYTCNVDHKPSNIKVDKTVERKCCVECPPCPAPP VAGPSVFIFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNW VDGVEVHNAKTKPREEQFNSIFRWSVLTWHQDWINGKEYKC KVSNKGLPAPIEKTISKTKGQPREPQVYTIPPSREEMTKNQVSIT CLVKGFYPSDIAVEWESNGQPENNYKTIPPMLDSDGSFFIYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSISPGK</td>

ΜΑ 32982Β1

A-1472-WO-PCT

The first 22 amino acids of each light chain sequence in Table 2Α, except 32Η7 and 32Η7 CS, constitute a signal sequence. In the case of 32Η7 and 32Η7 CS, the signal sequence comprises 20 amino acids. Likewise, the first 22 amino acids of each heavy chain sequence in Table 2Β constitute a signal sequence. Signal peptides can be replaced by signal peptides having different sequences, for example, for more optimal expression in certain host cells. It should therefore be understood that the invention also includes antibodies having light and / or heavy chain sequences specified in Tables 2Α and 2Β, but with different signal sequences.

Again, each heavy chain example (H1, Η2, Η3 etc.) shown in Table 2Β can be combined with any one of the light chain examples shown in Table 2Α to form an antibody. Examples of such combinations include H1 combined with any of L1 through L17; Η2 combines with any one from L1 to L17; Η3 combines with any one from L1 to L17, etc. In some cases, the antibody comprises at least one heavy chain and one light chain from those shown in Tables 2Α and 2Β. In some cases the antibodies comprise two different heavy chains and two different light chains shown in Tables 2Α and 2Β. In other cases, the antibodies contain two identical light chains and two identical heavy chains. For example, an antibody or one of its immunologically functional fragments can comprise two H1 heavy chains and two L1 light chains, or two Η2 heavy chains and two L2 light chains, or two Η3 heavy chains and two L3 light chains and other similar combinations of light chain pairs and heavy chain pairs shown in Tables 2Α and 2Β.

Other antigen-binding proteins of the invention are antibody variants formed by combining the heavy and light chains presented in Tables 2Α and 2Β and comprise light and / or heavy chains which each have an identity of at least 70 ٠/٠, 75 ٠/٥, 80 ٠/٠, 85 ٠/٠, 90%, 95%, 97% or 99% with the amino acid sequence of these chains. In some cases, these antibodies include at least one heavy chain and one light chain, while in other cases, the variant forms contain two identical light chains and two identical heavy chains.

Variable areas of anticoms

Also disclosed are antigen-binding proteins which contain an antibody heavy chain variable region selected from the group consisting of Vfjl, Vh2, ٧η3, Vh4, Vh5, Vh6, ​​Vh7, Vh8, Vh9, Vh10, VhI 1 , Vh12, and Vh13, and / or a variable region

ΜΑ 32982Β1

A-1472.WOPCT of antibody light chain selected from the group consisting of Vl, Vl2, Vl3, Vl4, Vl5,

Vl6, Vl7, Vl8, Vl9, Vl10, VlI 1, Vl12, Vl13, Vl14, Vl15, Vl16, and Vl17, as presented in Table 3 below and the immunologically functional fragments, derivatives, muteins and variants of these light chain and heavy chain variable regions.

The sequence alignments of the various heavy chain and light chain variable regions, respectively, are shown in Figs. IA and IB.

Proteins binding an antigen of this type may be generally designated by the formula "Vhx / V_Ly ”, where“ X ”corresponds to the number of heavy chain variable regions and“ y ”corresponds to the number of light chain variable regions.

Table 3: Examples of amino acid sequences of Vh and Vl chains

<td>Content in the clone</td><td>Naming</td><td>SEQ ID NO.</td><td>Amino acid sequence</td>

<td>1Ε11</td><td>١ الآ</td><td> 137</td><td>QSVLIQPPSVSEAPGQKVTISCSGSSSNIGNNYVSWYQQLP GTAPKLLIYDNNKRPSGIPDRFSGSKSGISATIGITGLQTGDE ADYYCGIWDSRISAWFGGGKVL</td>

<td>1Η7</td><td>ها \</td><td> 138</td><td>QSVLGPPSASGTPGQRVHSCSGSSSNIGSNYVYWYQQLP EADYYCAAWDDSLSGWVFGGGTTVL</td>

<td>2Ε7</td><td>٦ الآ</td><td> 139</td><td>DIQMTQSPSSLSASVGDRVTCRASQGIRNDLGWFQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDLA TYYCLQYNIYPWTFGQGTKVEIK</td>

<td>3Β6</td><td>4 أ ٧</td><td> 140</td><td>SSELTQDPTVSVALGQTVKITCQGDSLRSFYASWYQQKPGQ APVIVFYGKNNRPSGIPDRFSGSSSGNÏASLIGAQAEDEA DYYCNSRDSSVYHLVIGGGTKLTVL</td>

<td>3C8</td><td>5 ط ٧</td><td> 141</td><td>DIIIAQTPLSLSVGQPASISCKSSQSLIHSAGKTYIYIA / YIQ KPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEA EDVGIYYCMQSFPLPIGGGKVEIK</td>

<td>4Ε4</td><td>Vl6</td><td> 142</td><td>QSVLIQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQIP GTAPKUIYDNNKRPSGIPDRFSGSKSGTSTTGITGIQTGDE ADYYCGTWDSRLSAWFGGGfKLTVL</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Content in the cione</td><td>Naming</td><td>SEQ ID NO.</td><td>Amino acid sequence</td>

<td>4Η6</td><td>٦ اا \</td><td> 143</td><td>DIVMTQSPISLPVTPGEPASISCRSSQSLLHSFGYNYLDWYI QKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCMQAIQTPFTFGPGTVDIK</td>

<td>5F5</td><td>Vl8</td><td> 144</td><td>DIILTQTPLSLSVTPGQPASISCKSSQSILHSDGKTYWYIQ KPGQPPQLLIYEVSNRFSGEPDRFSGSGSGTDFTLKISRVEA EDVGTYYCMQSFPIPLTFGGGKVEIK</td>

<td>9D4</td><td>Vi9</td><td> 145</td><td>QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQFP GTAPKLIYDNNKRPSGIPDRFSGSKSGTSATIGGIQTGDE ADYYCGTWDSRLSAWFGGGTKITVI.</td>

<td>9F5</td><td>Viio</td><td> 146</td><td>QSVLTQSPSASGTPGQR١ / TISCSGSSSNIGSNY١ / YWYQQLP GAAPKLLILRNNQRPSGVPDRFSGSKSGTSASITISGLRSED EADYYCAAWDDSLSGWVFGGGTTVL</td>

<td>10Ε4</td><td>١١ الآ</td><td> 147</td><td>QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLP GTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASIAISGLQSED EADFYCAARDESLNGWFGGGTKITI</td>

<td>IIIDII 11Η9</td><td>Vl12</td><td> 148</td><td>QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLP EADYYCAAWDDSISGWVFGGGTTVL</td>

<td>12Ε8</td><td>Vl13</td><td> 149</td><td>SAYS QTPLSLSVSPGQPASISCKSSQSLLHSDGRNYLYWYL QKPGQPPQlllYEVSNRFSGLPDRFSGSGSGTDFTIKISRVE AEDVGIYYCMQSFPLPITFGGGTKVEIK</td>

<td>12G8</td><td>Vi14</td><td> 150</td><td>QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLP GTAPKIIYDNNKRPSGIPDRFSGSKSGTSATLGITGOGDE ADYYCGTWDSRLSAWFGGGTKLTVL</td>

<td>13Η2</td><td>Vt! 5</td><td> 151</td><td>DIQMTQSPSSLSASVGDRVTITCRASQGIRKDLGWYQQKPG KAPKRLIYGASSLQSGVPSRFSGSGSGTEFTITISSLQPEDFA TYYCIQYNSFPWTFGQGTKVEIK</td>

ΜΑ 32982Β1

A.-1472-WO-PCT

<td>Content in the clone</td><td>Naming</td><td>SEQ ID NO.</td><td>Amino acid sequence</td>

<td>32Η7</td><td>Vl16</td><td> 152</td><td>ETQSPGTLSISPGERAFLSCRASQSVSSGYITWYQQKP GQAPRUIYGASSRATGIPDRFSGSGSGTDFTIISRIEPEDF AVYYCQQYGNSLCRFGQGTKLEIK</td>

<td>32Η7 CS</td><td></td><td> 153</td><td>EIVLQSPGTLSLSPGERATLSCRASQSVSSGYLTWYQQKP GQAPRLIIYGASSRATGIPDRFSGSGSGTDFTITISRIEPEDF AVYYCQQYGNSLSRFGQGIKLEIK</td>

<td>32Η8</td><td>Vl * 18</td><td> 154</td><td>QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTUISSl QAEDYAVYYCQQYYNTPFTFGPGTKVDIK</td>

<td>33Β5</td><td> 7,19</td><td> 155</td><td>KAPKRLIYVASSLQSGVPSRFSGSGSGTEFTLTISSIQPEDFA TYYCLQYNTYPllFGGGTKVEIK</td>

<td>33Ε4</td><td>٧l2O</td><td> 156</td><td>EIVMGSPATISVSPGERATSCRASQSVRSNLAWYQQKPG QAPRILIHDASPRTAGIPARFSGSGSGTEFTLTINSLQSEDFA VYYCQQYNYWTPITFGQGTRLEIK</td>

<td>34Ε3</td><td>ة ة ١ لا</td><td> 157</td><td>GTAPKUIYDNNKRPSGIPDRFSGSKSGTSATIGITGLQTGDE ANYCCGTWDIGISVW / VFGGGTKLTVL</td>

<td>4Ε4 9D4 1Ε11</td><td>VhI</td><td> 158</td><td>PGKGIEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQ MNSLRAEDTAWYCARDRLNYYDSSGYYHYKYYGMAVWGQ GTTVTVSS</td>

<td>1Η7</td><td>٧h2</td><td> 159</td><td>EVQLVESGGGLVKPGGSLRLSCAASGFIFSNAWMSIWRQA PGKGLEWVGRIKSTTDGGTTDYAAPVKGRFTISRDDSKNTLY LQMNSIKTEDTAVYYCTTDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Content in the clone</td><td>Naming</td><td>SEQ ID NO.</td><td>Amino acid sequence</td>

<td>2Ε7</td><td>٧h3</td><td> 160</td><td>EVQtlESGGGLVQPGESLRLSCAASGFTFSSYAMSWRQA PGKGIEWVSAISGSGGRTYYADSVKGRFTISRDNSKNTLYI GQGITVTVSS</td>

<td>3Β6</td><td>Vh4</td><td> 161</td><td>QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQA PGQGLEWMGWINPNSGGTNYAQKFQGRVTMFRDSSTAY QGTVIVSS</td>

<td>3C8 12Ε8 5F5</td><td>Vh5</td><td> 162</td><td>PGKGIEWVAVISYDGSHESYADSVKGRFTISRDISKNTYLQ MNSLRAEDTAVYFCARERKRVTMSTIYYYFYYGMDVWGQG TTVSS</td>

<td>4Η6</td><td>Vh6</td><td> 163</td><td>EVQLVESGGGLVKPGRSLRLSCASGFTFGDYAMSWFRQA PGKGLEWIGFIRSRAYGGTPEYAASVKGRFTISRDDSKTIAYI QMNSLKIEDTAVYFCARGRGIAARWDYWGQGTI-VTVSS</td>

<td>9F5</td><td></td><td> 164</td><td>PGKGLEWVGRIKSKTDGGTTDYTAPVKGRFTISRDDSKNTLY QGTTVTVSS</td>

<td>10Ε4</td><td>٧h8</td><td> 165</td><td>QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMYWVRQA PGQGIEWMGWISPNSGGTNYAQKFQGRVTMTRDTSISTAY MELSRLRSDDTAVYYCVRGGYSGYAGLYSHYYGMDVWGQ GTTVTVSS</td>

<td> 11011</td><td>٧h9</td><td> 166</td><td>EVQIVESGGGLVKPGGSLRLSCAASGFTFGNAWMSWVRQA PGKGLEWRKSKTDGGTDYAAPVKGRFTSRDDSKNTIY IQMNSLKTEDTAVYFCT TDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS</td>

٦2! Β١

A-1472-WO-PCT

<td>Content in the clone</td><td>Naming</td><td>SEQ ID NO.</td><td>Amino acid sequence</td>

<td>11Η9</td><td>M</td><td> 167</td><td>EVQIVESGGGKPGGSLRLSCAASGFIFGNAWMSWVRQA PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLY LQMNSLKTEDTAVYYCTTDRTGYSISWSSYYYYYGMDVWG QGTTVTVSS</td>

<td>12G8</td><td>VhII</td><td> 168</td><td>QVQLVESGGGWQPGRSLRLSCAASGFTFSSFGMHWVRQA PGKGIEWVAVISFDGSIKYSVDSVKGRFIISRDNSKNFIFIQ GHVTVSS</td>

<td>13Η2</td><td>Vh12</td><td> 169</td><td>EVQLVESGGGLVKPGGSLRLSCAASGYTFSTYSMIfRQA QGTTVTVSS</td>

<td>32Η7</td><td>Vh13</td><td> 170</td><td>PGKGIEWVAVIWYDGSNKYYADSVKGRFIISRDKSKNTLYIQ TVSS</td>

<td>32Η8</td><td>٧η14</td><td> 171</td><td>QVQLVQSGAEVKKPGASVKVSCKASGYTFTAYYIHIWVRQA PGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAY MEISRIRSDDTAVFYCARGRQWIGFDYWGQGTLVTVSS</td>

<td>33Ε4</td><td>Vh15</td><td> 172</td><td>PGKGIEWIGEINHSGGTKYNPSLKSRVTISVDTSKNQFSLKL SSVTAADTAVYFCARGDVVGFFDYWGQGTLVTVSS</td>

<td>33Β5</td><td>٧η16</td><td> 173</td><td>QVQLVQSGAEVKKSGASVKVSCKASGYTFTGYYMHWVRQA PGQGLEWMGWINPNSGGTNYVQKFQGRVTMTRDTSISTAY MELSRIRSDDTAVYYCARNEYSSAWPLGYWGQGTLVTVSS</td>

<td>34Ε3</td><td>٧η17</td><td> 174</td><td>QITLKESGPTLVKPTQTITITCTFSGFSISTSGVGVAWIRQPP GKALEWLAIIYWTDDKRYSPSIKSRLTITKDTSKNQVVLRMT NMDPLDTATYFCAHRPGGWFDPWGQGTLVTVSS</td>

ا

ΜΑ 32982Β1

A-1472-WO-PCT

Each heavy chain variable region shown in Table 3 can be combined with any of the light chain variable regions shown in Table 3 to form an antigen binding protein. Examples of such combinations include Vff1 combined with any one of Vl, Vl2, Vl, Vl4, Vl5, Vl6, Vl7, Vl8, Vl9, Vl10, VlI 1, ν٤12, Vl13, Vl14, Vl15, Vl16, or Vl17; Vh2 combined with any one of VlI, Vl2, Vl3, Vl4, Vl5, Vl6, Vl7, Vl8, v٤9, VlIO, VlII, Vl12, Vl13, Vl14, Vl15٠ Vl16, or Vl17; V3 combined with any one of VlI, Vl2, Vl3, Vl4, Vl5, Vl6, Vl7, Vl8, Vl9, Vl10, Vlll, Vl12, Vl13, Vl14, Vl15, Vl16, or Vl17; etc.

In some cases, the antigen-binding protein comprises at least one heavy chain variable region and / or a light chain variable region from those shown in Table 3. In some cases, the antigen-binding protein comprises at least two variable regions. heavy chain and / or light chain variable regions different from those shown in Table 3. An example of such an antigen binding protein comprises (a) a Vfil, and (b) one of Vh2, Vh3, Vh4, Vh5, Vh6, ​​Vh7, Vh8, Vh9, vio, VhII, V_h12, or V3. Another example includes (a) a Vfj2, and (b) one of VhIj Vff3, Vh4, Vjj5, Vh6, ​​Vh7, Vh8, ٧h9, VhIO, VhI 1, Vh12, or Vh13. Still another example includes (a) a V 3, and (b) one of Vfl, Vh2, Vh4, Vh5, Vh6, ​​Vh7, Vh8, Vh9, VhIO, VhI 1, Vh12, or Vh13, etc. Yet another example of such an antigen binding protein comprises (a) a VII, and (b) one of V12, Vt.3. V, 4. Vl5, Vl6, Vl7, Vl8, Vl9, VlIO, VlII, V_l12, V13, V) 14. ν, 15- ν, 16. or V, 17. ٧l18, Vl19, Vl20, or Vl21. Yet another example of such an antigen binding protein comprises (a) a Vl2, and (b) one of VlI, Vl3, Vl4, Vl5, Vl6, Vl7, Vl8, Vl9, Vl10, VlII, Vl12, Vl13, Vl14 , Vl15, Vl16, Vl17, Vl18, Vl19, Vl20, or Vl21. Yet another example of such an antigen-binding protein comprises (a) a Vl3, and (b) one of VlI, Vl2, Vl4,

Vl5, Vl6, Vl7, Vl8, Vl9, VlIO, VlII, Vl12, V13٠ Vl14, Vl15, Vl16, Vl17, Vl18, ν, 19. Vl20, 0uVL21, etc.

The various combinations of heavy chain variable regions can be combined with any of the various combinations of light chain variable regions as will be apparent to those skilled in the art.

In other cases, the antigen-binding protein contains two identical light chain variable regions and / or two identical heavy chain variable regions. For example, the antigen-binding protein can be an antibody or an immunologically functional fragment that comprises two light chain variable regions and two heavy chain variable regions in combinations of light chain variable region pairs and variable chain region pairs. heavy values ​​shown in Table 3.

ΜΑ 32982Β1

A-1472-WO-PCT

Certain antigen-binding proteins provided herein comprise a heavy chain variable domain comprising an amino acid sequence which differs from the sequence of a heavy chain variable domain selected from Vl, Vh2, Vh3, Vh4, Vh5, Vh6, ​​v ”7 , VS, Vh9, VhIO, VhII, Vh12, and Vh13 by only 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues, this difference in sequence being independently a deletion, an insertion or a substitution of an amino acid, deletions, insertions and / or substitutions resulting in no more than 15 amino acid changes from the preceding variable domain sequences. The heavy chain variable region in some antigen-binding proteins comprises an amino acid sequence that has sequence identity of at least 70%, 75 ٠/٥, 80%, 85%, 90 ٥/٥, 95% , 97% or 99% with the amino acid sequences of the heavy chain variable region of VhI, Vh2, Vh3, Vh4, Vh5, Vh6, ​​Vh7, Vh8, Vh9, Vh10, Vh11, Vh12, andVH13.

Some antigen-binding proteins include a light chain variable domain comprising an amino acid sequence that differs from the sequence of a light chain variable domain selected from Vl, Vl2, V3, Vl4, Vl5, Vl6, Vl7, Vl8, Vl9, VlIO, VlI 1, V12, Vl13, Vl14, Vl15, Vl16, or Vl17 by only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14 or 15 amino acid residues, this difference in sequence being independently a deletion, an insertion or a substitution of an amino acid, the deletions, insertions and / or substitutions not causing more than 15 changes of amino acids relative to the preceding variable domain sequences. The heavy chain variable region in some antigen-binding proteins comprises an amino acid sequence that has sequence identity of at least 70%, 75 ٠/٥, 80 ٠/٥, 85%, 90%, 95% , 97 ٠/٥ or 99 ٥/٥ with the amino acid sequences of the light chain variable region of VlI, Vl2, Vl3, Vl4, Vl5, Vl6, Vl7, Vl8, Vl9, VlIO, VlII, Vl12, Vl13 , Vl14, Vl15, Vl16, or Vl17.

In other cases, the antigen-binding proteins include the following pairings of light chain and heavy chain variable domains: VLI with VHI, VL2 with VH2, VL3 with VH3, VL4 with VH4, VL5 with VH5, VL6 with VHI, VL7 with VH6, VL8 with VH5, VL9 with VHI, VLIO with VH7, VLll with, Η8, VL12 with VH9, VL12 with VHIO, VL13 with VH5, VL14 with VHI 1, VL15 with VH12, VL16 withVH13, and VL17 with VH13. In some cases, the antigen-binding proteins in the above pairings may include amino acid sequences that have sequence identity of 70 ٠/٥, 75%, 80%, 85%, 90%, 95%, 97 ٠ / ٥ or 99% with the variable domains specified.

ΜΑ 32982Β1

A-1472-WO-PCT

Still other antigen binding proteins, for example, antibodies or immunologically functional fragments, include variant forms of a variant heavy chain and a variant light chain described above.

CDR

The antigen-binding proteins described herein are polypeptides into which one or more CDRs are grafted, inserted and / or joined. An antigen binding protein can include 1, 2, 3, 4, 5 or 6 CDRs. An antigen-binding protein can therefore comprise, for example, a CDR1 heavy chain (CDRH1 ”), and / or a CDR2 heavy chain (“ CDRH2 ”), and / or a CDR3 heavy chain (“ CDRH3 ”), and / or a CDR1 light chain (CDRL1 ”), and / or a CDR2 light chain (“ CDRL2 ”), and / or a CDR3 light chain (CDRL3”). Some antigen binding proteins include both CDRH3 and CDRL3. Specific heavy and light chain CDRs are identified in Tables 4Α and 4Β, respectively.

Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody can be identified using the system described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., us Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242,1991. Certain antibodies which are described herein comprise one or more amino acid sequences which are identical to or have substantial sequence identity with the amino acid sequence of one or more of the CDRs shown in Table 4Α (CDRH) and the table 4Β (CDRL).

Table 4Α: Examples of heavy chain CDR amino acid sequence

<td>Num ay</td><td>SEQ ID NO:</td><td>Content in referenced</td><td>Appellation</td><td>Sequence</td>

<td> 42</td><td> 73</td><td>1E11HCDR1 4E4HCDR1 9D4HCDR1 12G8HCDR1</td><td>CDRH 1-1</td><td>SFGMH</td>

<td> 43</td><td> 76</td><td>1H7HCDR1 9F5HCDR1 11D11HCDR1 11H9HCDR1</td><td>CDRH 1-2</td><td>NAWMS</td>

<td> 44</td><td> 70</td><td>2E7HCDR1</td><td>CDRH 1-3</td><td>SYAMS</td>

/

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Num ay</td><td>SEQ ID NO:</td><td>Content in referenced</td><td>Appellation</td><td>Sequence</td>

<td> 45</td><td> 82</td><td>3B6HCDR1</td><td>CDRH 1-4</td><td>GYYMH</td>

<td> 46</td><td> 85</td><td>3C8HCDR1 5F5HCDR1 12E8HCDR1</td><td>CDRH 1-5</td><td>SYGMH</td>

<td> 47</td><td> 88</td><td>4H6HCDR1</td><td>CDRH 1-6</td><td>DYAMS</td>

<td> 48</td><td> 92</td><td>10E4HCDR1</td><td>CDRH 1-7</td><td>DYYMY</td>

<td> 49</td><td> 97</td><td>13H2HCDR1</td><td>CDRH 1-8</td><td>TYSMN</td>

<td> 50</td><td> 100</td><td>32H7HCDR1</td><td>CDRH 1-9</td><td>SYGMH</td>

<td> 51</td><td> 74</td><td>1E11HCDR2 4E4HCDR2 9D4HCDR2 12G8HCDR2</td><td>CDRH 2-1</td><td>VISFDGSIKYSVDSVKG</td>

<td> 52</td><td> ٦٦</td><td>1H7HCDR2</td><td>CDRH 2-2</td><td>RIKSTTDGGT٦DYAAPVKG</td>

<td> 53</td><td> 80</td><td>2E7HCDR2</td><td>CDRH 2-3</td><td>AISGSGGRTYYADSVKG</td>

<td> 54</td><td> 83</td><td>3B6HCDR2</td><td>CDRH 2-4</td><td>WINPNSGGTNYAQKFQG</td>

<td> 55</td><td> 86</td><td>3C8HCDR2 5F5HCDR2 12E8HCDR2</td><td>CDRH 2-5</td><td>VISYDGSHESYADSVKG</td>

<td> 56</td><td> 89</td><td>4H6HCDR2</td><td>CDRH 2-6</td><td>FIRSRAYGGTPEYAASVKG</td>

<td> 57</td><td> 91</td><td>9F5HCDR2</td><td>CDRH 2-7</td><td>RIKSKTDGGTTDYTAPVKG</td>

<td> 58</td><td> 93</td><td>10E4HCDR2</td><td>CDRH 2-8</td><td>WISPNSGGTNYAQKFQG</td>

<td> 59</td><td> 95</td><td>11D11HCDR2 11H9HCDR2</td><td>CDRH 2-9</td><td>RIKSKDGGTTDYAAPVKG</td>

<td> 60</td><td> 98</td><td>13H2HCDR2</td><td>CDRH 2-10</td><td>SISSSSSYRYYADSVKG</td>

<td> 61</td><td> 101</td><td>32H7HCDR2</td><td>CDRH 2-11</td><td>VIWDGSNKYYADSVKG</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Num ay</td><td>SEQ ID NO:</td><td>Contained in reference</td><td>Appellation</td><td>Sequence</td>

<td> 62</td><td> 75</td><td>1E11HCDR3 4E4HCDR3 9D4HCDR3</td><td>CDRH 3-1</td><td>DRLNYYDSSGYYHYKYYGMAV</td>

<td> 63</td><td> 78</td><td>1H7HCDR3 9F5HCDR3 11D11HCDR3 11H9HCDR3</td><td>CDRH 3-2</td><td>DRTGYSISWSSYYYYYGMDV</td>

<td> 64</td><td> 81</td><td>2E7HCDR3</td><td>CDRH 3-3</td><td>DQREVGPYSSGWYDYYYGMDV</td>

<td> 65</td><td> 84</td><td>3B6HCDR3</td><td>CDRH 3-4</td><td>DQMSIIMIRGVFPPYYYGMDV</td>

<td> 66</td><td> 87</td><td>3C8HCDR3 5F5HCDR3 12E8HCDR3</td><td>CDRH 3-5</td><td>ERKRVIMSHYYYFYYGMDV</td>

<td> 67</td><td> 90</td><td>4H6HCDR3</td><td>CDRH 3-6</td><td>GRGIAARWDY</td>

<td> 68</td><td> 94</td><td>10E4HCDR3</td><td>CDRH 3-7</td><td>GGYSGYAGLYSHYYGMDV</td>

<td> 69</td><td> 96</td><td>12G8HCDR3</td><td>CDRH 3-8</td><td>DRLNYYDSSGYYHYKYYGIAV</td>

<td> 70</td><td> 99</td><td>13H2HCDR3</td><td>CDRH 3-9</td><td>EGVSGSSPYSISWYDYYYGMDV</td>

<td> ٦١</td><td> 102</td><td>32H7HCDR3</td><td>CDRH 3-10</td><td>AGGIAAAGIYYYYGMDV</td>

Table 4Β: Examples of CDR amino acid sequence of light chains

<td>Num ay</td><td>SEQ ID NO:</td><td>Contained in reference</td><td>Appellation</td><td>Sequence</td>

<td> 72</td><td> 42</td><td>1E11LCD1 4E4LCD1 9D4LCD1 12G81CD1</td><td>CDRL 1-1</td><td>SGSSSNIGNNYVS</td>

<img file="MA32982B1_D0007.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Num ay</td><td>SEQ ID NQ:</td><td>Contained in reference</td><td>Appellation</td><td>Sequence</td>

<td> 73</td><td> 45</td><td>1H7LCD1 9F5LCD1 11D11LC1 11H9LCD1</td><td>CDRl 1-2</td><td>SGSSSNIGSNYVY</td>

<td> 74</td><td> 48</td><td>2E7LCD1</td><td>CDRL 1-3</td><td>RASQGIRNDLG</td>

<td> 75</td><td> 51</td><td>3B6LCD1</td><td>CDRl 1-4</td><td>QGDSLRSFYAS</td>

<td> 76</td><td> 54</td><td>3C8LCD1</td><td>CDRL 1-5</td><td>KSSQSLLHSAGKTYLY</td>

<td>Π</td><td> 57</td><td>4H6LCD1</td><td>CDRL 1-6</td><td>RSSQSLLHSFGYNYLD</td>

<td> 78</td><td> 60</td><td>5F5LCD1</td><td>CDRl 1-7</td><td>KSSQSLLHSDGKTYLY</td>

<td> 79</td><td> 62</td><td>10E4LCD1</td><td>CDRL 1-8</td><td>SGSSSNIGSNTVN</td>

<td> 80</td><td> 65</td><td>12E8LCD1</td><td>CDRl 1-9</td><td>KSSQSLLHSDGRNYLY</td>

<td> 81</td><td> 66</td><td>13H21CD1</td><td>CDRl 1-10</td><td>RASQGIRKDLG</td>

<td> 82</td><td> 69</td><td>32Η7 ICDI 32H7m LCDI</td><td>CDRL 1-11</td><td>RASQSVSSGYLI</td>

<td> 83</td><td> 43</td><td>1E11LCD2 4E4LCD2 9D41CD2 12G8LCD2</td><td>CDRl 2٠1</td><td>DNNKRPS</td>

<td> 84</td><td> 46</td><td>1H7LCD2</td><td>CDRL 2-2</td><td>RSNQRPS</td>

<td> 85</td><td> 49</td><td>2E71CD2</td><td>CDRL 2-3</td><td>AASSLQS</td>

<td> 86</td><td> 52</td><td>3B6LCD2</td><td>CDRL 2-4</td><td>GKNNRPS</td>

<td> 87</td><td> 55</td><td>3C81CD2 5F51CD2 12E8LCD2</td><td>CDRl 2-5</td><td>EVSNRFS</td>

<td> 88</td><td> 58</td><td>4H6LCD2</td><td>CDRL 2-6</td><td>LGSNRAS</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Num ay</td><td>SEQ ID NO:</td><td>Content in referenced</td><td>Appellation</td><td>Sequence</td>

<td> 89</td><td> 61</td><td>9F5LCD2 11D11LC2 11H9LCD2</td><td>CDRL2-7</td><td>RNNQRPS</td>

<td> 90</td><td> 63</td><td>10E4LCD2</td><td>CDRl 2-8</td><td>fNNQRPS</td>

<td> 91</td><td> 67</td><td>13H2LCD2</td><td>CDRl 2-9</td><td>GASSLQS</td>

<td> 92</td><td> 70</td><td>32Η7LCD2 32H7m 1CD2</td><td>CDRL 2-10</td><td>GASSRAL</td>

<td> 93</td><td> 44</td><td>1E11LCD3 4E41CD3 9D41CD3 12G8LCD3</td><td>CDRL 3-1</td><td>GTWDSRLSAW</td>

<td> 94</td><td> 47</td><td>1H71CD3 9F5LCD3 11D1HC3 11H91CD3</td><td>CDRl 3-2</td><td>AAWDDSLSGWV</td>

<td> 95</td><td> 50</td><td>2E71CD3</td><td>CDRL 3-3</td><td>LQYNIYPWT</td>

<td> 95</td><td> 53</td><td>3B61CD3</td><td>CDRL 3-4</td><td>NSRDSSVYHLV</td>

<td> 97</td><td> 56</td><td>3C8LCD3 5F51CD3 12E8LCD3</td><td>CDRL 3-5</td><td>MQSFPLPLT</td>

<td> 98</td><td> 59</td><td>4H6LCD3</td><td>CDRl 3-6</td><td>MQALQTPFT</td>

<td> 99</td><td> 64</td><td>10E41CD3</td><td>CDRL 3-7</td><td>AARDESLNGW</td>

<td> 100</td><td> 68</td><td>13H2LCD3</td><td>CDRL 3-8</td><td>LQYNSFPWT</td>

<td> 101</td><td> ٦٩</td><td>32Η71CD3</td><td>CDRl 3-9</td><td>QQYGNSLCR</td>

<td> 102</td><td> 72</td><td>32H7m LCD3</td><td>CDRl 3-10</td><td>QQYGNSISR</td>

The structure and properties of CDRs in a natural antibody have been described above. Briefly, in a classicjue antibody, CDRs are embedded within a framework

ΜΑ 32982Β1

A-1472-WO-PCT in the heavy and light chain variable region where they constitute the regions responsible for antigen binding and recognition. A variable region comprises at least three heavy or light chain CDRs, see, above (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service Ν.Ι.Η., Bethesda, MD; see also Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342 .: 877-883), in a framework region (called framework regions 14, FRI, FR2, FR3, and FR4, by Kabat et al., 1991, supra; see also Chothia and Lesk , 1987, supra). However, the CDRs of the invention can not only be used to define the antigen binding domain of a typical antibody structure, but can also be included in various other polypeptide templates, as described herein.

In one aspect, the CDRs of the invention are (a) a CDRH selected from the group consisting of (i) a CDRH1 selected from the group consisting of SEQ ID NO: 73, 76, 79, 82, 85, 88 , 92, 97 and 100; (ii) CDRH2 selected from the group consisting of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101 and 129; (iii) CDRH3 selected from the group consisting of SEQ ID NO: 75, 78, 81, 84, 87, 90, 96, 99, 102 and 123; and (iv) a CDRH of (i), (ii) and (iii) which contains one or more, eg, one, two, three, four substitutions (eg, conservative amino acid substitutions), deletions or insertions of amino acids of not more than five, four, three, two or one amino acids; (B) a CDRL selected from the set consisting of (i) a CDRL1 selected from the set consisting of SEQ ID

NO: 42.45, 48, 51, 54, 57, 62, 65, 66 and 69; (ii) a CDRL2 selected from the group consisting of SEQ ID NO: 43, 46, 49, 52, 55, 58, 61, 63, 67 and 70; (iii) CDRL3 selected from the group consisting of SEQ ID NO: 44.47, 50, 53, 56, 59, 64, 68, 71, and 72; and (iv) a CDRL of (i), (ii) and (iii) which contains one or more, for example ine, two three, four substitutions (for example, conservative amino acid substitutions), deletions or insertions of Amino acids of no more than five, four, three, two or one amino acids.

In another aspect, an antigen binding protein comprises 1, 2, 3, 4, 5, or 6 variant forms of the CDRs shown in Tables 4Α and 4Β, each having a sequence identity of 80 ٥/٠, 85 ٠/. , 90 ٥ / o or 95 ٠/٥ with a CDR sequence shown in Tables 4Α and 4Β. Some antigen-binding proteins include 1, 2, 3,4, 5, or 6 of the CDRs shown in Tables 4Α and 4Β, each differing by no more than 1, 2, 3, 4, or 5 amino acids from the CDRs shown in these tables.

In yet another aspect, the CDRs disclosed herein comprise consensus sequences derived from groups of related monoclonal antibodies. As described herein, a "consensus sequence" refers to amino acid sequences having amino acids

ΜΑ 32982Β1

A-1472-WO-PCT conserves many sequences and variable amino acids that vary in given amino acid sequences. Consensus sequences of the invention include CDRs corresponding to each of CDRH1, CDRH2, CDRH, CDRL1, CDRL2 and CDRL3.

In yet another aspect, an antigen binding protein comprises the following combinations of CDRL1, CDRL2 and CDRL3: SEQ ID NOs: 42,43, and 44; SEQ ID NOs: 45.46, and 47; SEQ ID NOs: 48.49, and 50; SEQ ID NOs: 51.52, and 53; SEQ ID NOs: 54, 55, and 56; SEQ ID NOs: 57, 58, and 59; SEQ ID NOs: 60, 55, and 56; SEQ ID NOs: 45.61, and 47; SEQ ID NOs: 62, 63, and 64; SEQ ID NOs: 65, 55, and 56; SEQ ID NOs: 66, 67, and 68; SEQ ID NOs:

69, 70, and 71; and SEQ ID NOs: 69, 70, and 72.

In another aspect, an antigen binding protein comprises the following combinations of CDRH1, CDRH2 and CDRH3: SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 76.77, and 78; SEQ ID NOs: 79, 80, and 81; SEQ ID NOs: 82, 83, and 84; SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 76.91, and 78; SEQ ID NOs: 92, 93, and 94; SEQ ID NOs: 76, 95, and 78; SEQ ID NOs: 73, 74, and 96; SEQ ID NOs: 97, 98, and 99; and SEQ ID NOs: 100, 101, and 102.

In another aspect, an antigen binding protein comprises the following associations of CDRL1, CDRL2, and CDRL3 with CDRH1, CDRH2 and CDRH3: SEQ ID NOs: 42, 43, and 44 with SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 45, 46, and 47 with SEQ ID NOs: 76, 77, and 78; SEQ ID NOs: 48, 49, and 50 with SEQ ID NOs: 79, 80, and 81; SEQ ID NOs: 51, 52, and 53 with SEQ ID NOs: 82, 83, and 84; SEQ ID NOs: 54, 55, and 56 with SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 57, 58, and 59 with SEQ ID NOs: 88, 89, and 90; SEQ ID NOs: 60, 55, and 56 with SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 45, 61, and 47 with SEQ ID NOs: 76.91, and 78; SEQ ID NOs: 62, 63, and 64 with SEQ ID NOs: 92, 93, and 94; SEQ ID NOs: 45, 61, and 47 with SEQ ID NOs: 76, 95, and 78; SEQ ID NOs: 65.55, and 56 with SEQ ID NOs: 85, 86, and 87; SEQ ID NOs: 42.43, and 44 with SEQ ID NOs: 73, 74, and 96; SEQ ID NOs: 66, 67, and 68 with SEQ ID NOs: 97, 98, and 99; SEQ ID NOs: 69, 70, and 71 with SEQ ID NOs: 100, 101, and 102; and SEQ ID NOs: 69, 70, and 72 with SEQ ID NOs: 100, 101, and 102.

The consensus sequences were determined using standard phylogenic analyzes of the CDRs corresponding to the Vh and to the Vl of the anti-CGRP R antibodies. The consensus sequences were determined by maintaining the contiguous CDRs in the same sequence corresponding to a Vh or a Vl.

As illustrated in Figs. 3A, 3Β, 4, 5A, 5Β, 5C, 5D and 5Ε, line analysis of various antigen-binding proteins of the invention yielded groups of sequences

ΜΑ 32982Β1

Related A-1472-WO-PCT referred to as K1, Κ2, Κ3 and Κ4 light chain CDR groups (Figs. 3Α and 3Β), L1, L2, L3, and L4 light chain CDR groups (Fig. 4) , and heavy chain CDR groups HCl (Fig. 5Α), HC2 (Fig. 5Β), HC3 (Fig. 5C), HC4 (Fig. 5C), HC5 (Fig. 5D) and HC6 (Fig. 5Ε). Some of the above groups were used to generate additional consensus sequences, as shown in Figs. 3Α, 3Β, 4 and 5F, to give the light chain CDR groups K1, 4 (Fig. 3Α), Κ2.3 (Fig. 3Β), L1, 2.3 (Fig, 4), and LA11 (Fig. . 4), and the heavy chain CDR groups HCA and HCB (Fig. 5F).

The consensus sequences of the various CDR region groups are given below: Kl Consensus

CDRl RASQGIRXiDLG (SEQ ID ΝΟ: 103), X) being selected from the set consisting of N and K.

CDR2 XiASSLQS (SEQ ID NO: 104), Xi being chosen from the set consisting of A and G.

CDR3 LQYNXXPWT (SEQ ID NO: 105), Xi being selected from the set consisting of I and S, and Χ2 being selected from the set consisting of Y and F.

Κ4 Consensus

CDR3 QQYGNSLXiR (SEQ ID NO: 106), Xi being selected from the group consisting of S and c.

Κ1.4 Consensus

CDR1 RASQX1X2X3X4GX5LX6 (SEQ IDNO: 107), Xj being chosen from the set consisting of s and G, Χ2 being chosen from the set consisting of V and I, Χ3 being chosen from the set consisting of s and R, Χ4 being selected from the set consisting of s, N and K, Χ5 being selected from the set consisting of Y and D, and Χή being selected from the set consisting of T and G.

CDR2 Xi ASSX2X3X4 (SEQ ID NO: 108), Xi being chosen from the set consisting of

G and A, Χ2 being selected from the set consisting of R and F, Χ3 being selected from the set consisting of A and Q, and Χ4 being selected from the set consisting of T and s.

CDR3 X1QYX2X3X4X5X6X7 (SEQ ID NO: 109), Xi being selected from the set consisting of Q and L, Χ2 being selected from the set consisting of G and N, Χ3 being selected from the set consisting of N and T, Χ4 being selected from the set consisting of s, Y and F, Χ5 being selected from the set consisting of L and p, Xs being selected from the set consisting of C, W and S, and Χ7 being selected from the set consisting of R and T.

/

ΜΑ 32982Β1

A-1472-WO-PCT

Κ3 Consensus

CDR1 KSSQSLLHSX1GX2X3YLY (SEQ ID NO: 110), Xi being selected from the set consisting of D and A, Χ2 being selected from the set consisting of R and K, and Χ3 being selected from the set consisting of N and T.

Κ2.3 Consensus

CDR1 X1SSQSLLHSX2GX3X4YLX5 (SEQ IDNO: 111), X, being selected from the set consisting of R and K, Χ2 being selected from the set consisting of F, D and A, Χ3 being selected from the set consisting of Y, R and K, Χ4 being selected from the group consisting of N and T, and Χ5 being selected from the group consisting of D and Y.

CDR2 X1X2SNRX3S (SEQ ID NO: 112), Xi being selected from the set consisting of L and E, Χ2 being selected from the set consisting of G and V, and Χ3 being selected from the set consisting of A and F.

CDR3 MQXXXXXT (SEQ ID ΝΟ: 113), Χ٠ being selected from the set consisting of A and s, Χ2 being selected from the set consisting of L and F, Χ3 being selected from the set consisting of Q and p, Χ4 being selected from the set consisting of T and L, and Χ5 being selected from the set consisting of F and L.

Lm3 Consensus

CDR2 RXiNQRPS (SEQ ID NO: 114), Xi being selected from the group consisting of N and S.

Lml.2.3 Consensus

CDR1 SGSSSNIGX1 NX2VX3 (SEQ ID NO: 115), Xi being selected from the set consisting of N and s, Χ2 being selected from the set consisting of Y and T, and Χ3 being selected from the set consisting of s, N and Y.

CDR2 X1X2NX3RPS (SEQ ID NO: 116), Xi being selected from the set consisting of D, T and R, Χ2 being selected from the set consisting of N and s, and Χ3 being selected from the set consisting of K and Q.

CDR3 X1X2X3DX4X5IXX7VV (SEQ ID NO: 117), Xi being selected from the set consisting of G and A, Χ2 being selected from the set consisting of T and A, Χ3 being selected from the set consisting of w and R, Χ4 being selected from the set consisting of s and D, Χ5 being selected from the set consisting of R and s, Χδ being selected from the set consisting of s and N, and Χ7 being selected from the set consisting of A and G.

LAll Consensus

CDRl X1GX2X3SX4X5X6X7X8X9X10X11 (SEQ ID NO: 118), Xi being chosen from the set consisting of s and Q, Χ2 being present or absent, and if present, representing s, Χ3

ΜΑ 32982Β1

A-1472-WO-PCT being selected from the set consisting of s and D, Χ4 being present or absent, and if present, representing N, Χ5 being selected from the set consisting of I and L, x &lt; 5 being selected from the set consisting of G and R, Χ7 being selected from the set consisting of N and s, Xs being selected from the set consisting of N and F, Χ9 being selected from the set consisting of Y and T, Χ10 being chosen from the set consisting of V and A, and Χ11 being selected from the set consisting of s, N and Y.

CDR2 X1X2NX3RPS (SEQ ID NO: 119), Xi being selected from the set consisting of D, G, T, and R, Χ2 being selected from the set consisting of N, K and s, and Χ3 being selected from set consisting of K, N and Q.

CDR3 X1X2X3DXX X6X7X8X9V (SEQ ID NO: 120), Xi being selected from the set consisting of G, N and A, Χ2 being selected from the set consisting of T, s and A, Χ3 being selected from the set consisting of w and R, Χ4 being chosen from the set consisting of s and D, X ؛ being selected from the set consisting of R and s, Χό being selected from the set consisting of L and V, Χ7 being selected from the set consisting of s, Y and N, Xg being selected from the set consisting of A , H and G, and Χ9 being chosen from the set consisting of V and L.

HCl Consensus

CDRl Χ1ΥΥΜΧ2 (SEQ ID NO: 121), Xi being selected from the set consisting of G and D, Χ2 being selected from the set consisting of H and Y.

CDR2 WlXiPNSGGTNYAQKFQG (SEQ ID ΝΟ: 122), X] being chosen from ؛ 'the set consisting of N and s.

CDR3 X1XX3SX4X5X6X7X8GX9X10X11X12YYXGMDV (SEQ IDNO: 123), Xi being chosen from the set consisting of D and G, Χ2 being chosen from the set consisting of Q and G, Χ3 being chosen from the set consisting of M and Y, Χ4 being selected from the set consisting of I and G, Χ5 being selected from the set consisting of I and Y, x٥ being selected in the set consisting of M and A, Χ7 being present or absent, and if present , representing L, X؟ being present or absent, and if present, representing R, Χ9 being chosen from ؛ 'the set consisting of V and L, Χ10 being chosen from the set consisting of F and Y, X, I being chosen from the set consisting of p and s, Χ12 being chosen from the set consisting of p and H, and Χ13 being present or absent, and if present, representing Y.

HC2 Consensus

CDR2 RIKSX1TDGGTTDYX2APVKG (SEQ ID NO: 124), Xi being selected from the set consisting of K and T, and Χ2 being selected from the set consisting of T and A.

HC3 Consensus

ΜΑ 32982Β1

A-1472-WO-PCT

CDRl Χ1ΥΧ2ΜΧ3 (SEQ ID NO: 125), Xi being selected from the set consisting of T and S, Χ2 being selected from the set consisting of s and A, and Χ3 being selected from the set consisting of N and S.

CDR2 X1ISX2SX3X4X5XsYYADSVKG (SEQ ID ΝΟ.-126), Xi being selected from the set consisting of S and A, Χ2 being selected from the set consisting of Set G, Χ3 being selected from the set consisting of s and G, Χ4 being selected from the set consisting of s and G, Χ5 being selected from the set consisting of Y and R, and x٥ being selected from the set consisting of R and T.

CDR3X1X2X3X4X5X6X7PYSX8X9WYDYYYGMDV (SEQ IDNO: 127), Xi being selected from the set consisting of E and D, Χ2 being selected from the set consisting of G and

Q, Χ3 being selected from the set consisting of ٧ and R, Χ4 being selected from the set consisting of s and E, Χ5 being selected from the set consisting of G and V, Χό being selected from the set consisting of s and G, Χ7 being present or absent, and if present, representing s, Xs being chosen from the set consisting of I and s, and Χ9 being chosen from the set consisting of S and G.

HC4 Consensus

CDRl SXiGMH (SEQ ID NO: 128), X, being selected from the group consisting of F and Y.

CDR2 VISX1DGSX2KYX3X4DSVKG (SEQ ID NO: 129), Xj being selected from the set consisting of F and Y, Χ2 being selected from the set consisting of I and H, Χ3 being selected from the set consisting of s and Y, and Χ4 being chosen from the set consisting of V

CDR3 X1RX2X3X4X5X6SX7X8YYX9X10X11YYGX12X13V (SEQ ID NO: 130), Xi being selected from the set consisting of D and E, Χ2 being selected from the set consisting of L and

K, Χ3 being selected from the set consisting of Net R, Χ4 being selected from the set consisting of Y and V, Χ5 being selected from the set consisting of Y and T, Χό being selected from the set consisting of D and Μ, Χ7 being selected from the group consisting of s and T, Xs being selected from the group consisting of G and L, Χ9 being selected from the group consisting of H and Y, Χ10 being present or absent, and s 'it is present, representing Y, Χ11 being chosen from the set consisting of K and F, Χ12 being chosen from the set consisting of M and L, and Χ13 being chosen from the set consisting of A and D.

<img file="MA32982B1_D0008.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

HCA Consensus

CDRl ΧΧΧ3ΜΧ4 (SEQ ID ΝΟ: 131), X] being selected from the set consisting of N and S, Χ2 being selected from the set consisting of A, Y and F, Χ3 being selected from the set consisting of w, A and G, and Χ4 being chosen from the set consisting of s and H.

CDR2 X1IX2X3X4X5X6GX7X X Xi0XuXi2Xi3Xi4١ / KG (SEQ ID NO: 132), where Xl is selected from the set consisting of R, A and V, Χ2 being selected from the set consisting of K, S and W, Χ3 being selected from set consisting of s. G, F and Y, Χ4 being present or absent, and if present, being selected from the set consisting of K and T, Χ5 being present or absent, and if present, representing T, X6 being chosen in the set consisting of D and s, Χ7 being selected from the set consisting of G and s, Xg being selected from the set consisting of T, R, I, N and H, Χ9 being selected from the set consisting of in T and K, Χ10 being chosen from the set consisting of D and Y, Χ11 being selected from the set consisting of Y and s, Χ12 being selected from the set consisting of T, A and V, Χ13 being selected from the set consisting of A and D, and Χ14 being selected from the set consisting in P and s.

CDR3 X, X2X3XXX6X7X8XXoXl | X, 2Xl3Xl4Xl5Xl6Xl7GX, 8Xl9V (SEQ ID NO: 133), Xl being selected from the set consisting of D, A and E, Χ2 being selected from the set consisting of R, Q and G, Χ3 being selected from the set consisting of T, R, L, G and K, Χ4 being selected from the set consisting of G, E, N, I and R, Χ5 being selected from the set consisting of Y, V and A , X6 being chosen from the set consisting of s. G, Y, A and T, Χ7 being selected from the set consisting of I, P, D, A and M, Xg being present or absent, and if present, being selected from the set consisting of s and Y, Χ9 being present or absent, and if present, being selected from the set consisting of w, s and T, Χ10 being selected from the set consisting of s, G and L, Xu being selected from the group consisting of set consisting of s. G, L and Y, Χ12 being present or absent, and if present, being selected from the set consisting of w and Y, Χ13 being selected from the set consisting of Y and H, Χ14 being present or absent, and if present, being selected from the set consisting of Y and D, Χ15 being selected from the set consisting of Y, K and F, Χ16 being present or absent, and if present, representing Y, Χ17 being present or absent, and if present, representing Y, Χ18 being selected from the set consisting of M and L, and Χ19 being selected from the set consisting of D and A.

HCB Consensus

CDRl Χ1Χ2Χ3Χ4Χ5 (SEQ ID NO: 134), Xl being selected from the group consisting of N, G, D, S and A, Χ2 being selected from the group consisting of A, F and Y, Χ3 being selected from the set consisting of w. Y, A and G, Χ4 being selected from the set consisting of M and L, and Χ5 being selected from the set consisting of s and H.

ΜΑ 32982Β1

A-1472-WO-PCT

CDR2XiIX2X3X &lt;X5X6X7X8XXioXiXi2Xj3X٠4Xi5X٠6Xi7٠ (SEQIDNO: 135), Xi being chosen from the set consisting of R, w. A, V, s and F, Χ2 being selected from the set consisting of K, N, s, w and R, Χ3 being selected from the set consisting of s, P, G, F and Y, Χ4 being present or absent, and if present, being selected from the set consisting of K, T and R, Χ5 being present or absent, and if present, being selected from the set consisting of T and A, x٥ being chosen from the set consisting of D, N, H, s and Y, Χ7 being chosen from the set consisting of G and s, Xs being selected from the set consisting of G and s, Χ9 being selected from the set consisting of T, G, R, I, N, H and Y, Χ10 being selected from the set consisting of T, K, R and P, Χ11 being selected from the set consisting of D, N, Y and E, Χ12 being selected from the set consisting of Y and s, Χ13 being selected from the set consisting of T, A and V, Χ14 being selected from the set consisting of A, Q and D, Χ15 being selected from the set consisting of P, K and s, Χ16 being chosen from the set consisting of V and F, and Χ17 being chosen from the set consisting of K and Q.

CDR3 X1X2X3X4X5SX6X7X8X9X10X11X12X13X14X15X16GX17X18V (SEQ ID NO: 136), Xj being chosen from the set consisting of D, G, A and E, Χ2 being chosen from the set consisting of R, G and Q, Χ3 being chosen from the set consisting in T, M, Y, R, L, G and K, Χ4 being selected from the set consisting of G, s, E, N, I and R, Χ5 being selected from the set consisting of Y, I, G , V and A, Xê being chosen from the set consisting of s, I, Y, G, A and T, Χ7 being chosen from the set consisting of I, M, A, P and D, Xg being present or absent, and if present, being selected from the group consisting of s, L and Y, Xg being present or absent, and if present, being selected in the set consisting of w, R, s and τ, Χ10 being selected from the set consisting of s, G and L, Χ11 being selected from the set consisting of s, V, L, G and Y, Χ12 being present or absent, and if present, being selected from the set consisting of F, Y and w, Χ13 being selected from the set consisting of Y, P, s and H, Χ14 being present or absent, and if present, being selected from the set consisting of Y, P, D and H, Χ15 being selected from the set consisting of Y, K and F, Xi6 being present or absent, and if present, representing Y, Χ17 being present or absent, and if present, representing Y, and Χ18 being chosen from the set consisting of M and L.

In some instances, the antigen binding protein comprises at least one heavy chain CDR1, CDR2, or CDR3 having one of the above consensus sequences. In some cases, the antigen binding protein comprises at least one light chain CDR1, CDR2, or CDR3 having one of the above consensus sequences. In other cases, the antigen binding protein comprises at least two heavy chain CDRs conforming to the above consensus sequences and / or at least two light chain CDRs conforming to the above consensus sequences.

ΜΑ 32982Β1

A-1472-WO-PCT

In yet other cases, the antigen-binding protein comprises at least three heavy chain CDRs conforming to the above consensus sequences, and / or at least three light chain CDRs conforming to the above consensus sequences.

Examples of monks binding an antigen

In one aspect, the invention relates to an isolated antigen binding protein which binds CGRP R, comprising (A) one or more heavy chain complementarity determining regions (CDRH) selected from the group consisting of: (i) a selected CDRH1 in the set consisting of SEQ ID NO: 73, 76, 79, 82, 85, 88, 92, 97 and 100; (ii) CDRH2 selected from the group consisting of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101 and 129; (iii) CDRH3 selected from the group consisting of SEQ ID NO: 75, 78, 81, 84, 87, 90, 96, 99, 102 and 123; and (iv) a CDRH of (i), (ii) and (iii) which contains one or more, for example, one, two, three, four or more amino acid substitutions, deletions or insertions of not more than five , four, three, two or one amino acids; (B) one or more light chain complementarity determining regions (CDRL) selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NO: 42,45,48, 51, 54, 57, 62, 65, 66 and 69; (ii) a CDRL2 selected from the group consisting of SEQ ID ΝΟ-.43, 46, 49, 52, 55, 58, 61,63, 67 and 70; (iii) a CDRL3 selected from the group consisting of SEQ ID NO: 44,47,

50, 53, 56, 59, 64, 68, 71 and 72; and (iv) a CDRL of (i), (ii) and (iii) which contains one or more, for example, one, two, three, four or more amino acid substitutions, deletions or insertions of not more than five , four, three, two or one amino acids; or (C) one or more heavy chain CDRH (A) and one or more light chain CDRL (B).

In yet another embodiment, the isolated antigen binding protein may comprise (A) a CDRH selected from the group consisting of (i) a CDRH1 selected from the group consisting of SEQ ID NO: 73, 76, 79, 82, 85, 88, 92, 97 and 100; (ii) CDRH2 selected from the group consisting of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101 and 129; and (iii) CDRH3 selected from the group consisting of SEQ ID NO: 75, 78, 81, 84, 87, 90, 96, 99, 102 and 123; (B) a CDRL selected from the group consisting of (i) a CDRL1 selected from the group consisting of SEQ ID NO: 42, 45,48,51, 54, 57, 62, 65, 66 and 69; (ii) a CDRL2 selected from the group consisting of SEQ ID NO: 43,46,49, 52, 55, 58, 61, 63, and 70; and (iii) a CDRL3 selected from the group consisting of SEQ ID NO: 44, 47, 50, 53, 56, 59, 64, 68, 71, and 72; or (C) one or more heavy chain CDRH (A) and one or more light chain CDRL (B). In one embodiment, the isolated antigen-binding protein may comprise (A) a CDRH1 of SEQ ID NO: 73, 76, 79, 82, 85, 88, 92, 97 and 100, ine CDRH2 of SEQ IDNO: 74, 77, 80, 83, 86, 89, 91,93, 95, 98,101, and 129, and one

ΜΑ 32982Β1

A-1472-WO-PCT

CDRH3 of SEQ ID NO: 75) 78, 81, 84, 87, 90,96,99, 102 and 123, and (B) a CDRL1 of SEQ IDNO: 42, 45, 48, 51, 54, 57, 62, 65, 66 and 69, a CDRL2 of SEQ ID NO: 43, 46, 49, 52, 55, 58, 61, 63, 67 and 70, and a CDRL3 of SEQ IDNO: 44,47, 50, 53, 56, 59, 64, 68, 71 and 72.

In another embodiment, the heavy chain variable region (Vh) has a sequence identity of at least 70 ٠ / o, 75 ٠/٠, 80 ٠ / o, 85 ٠/٠, 90%, 95% , 97% or 99% with an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 158-170, and / or V1 has a sequence identity of at least 70 ٥/٠, 75 ٥ / ٠, 80 ٥/٠, 85 ٠/٥, 90%, 95 ٥ / o, 97 ٥/٠ or 99 ٥/٥ with an amino acid sequence selected from the group consisting of SEQ ID ΝΟ: 137-153 . In another embodiment, Vh is selected from the set consisting of SEQ ID NO: 158-170, and or V1 is selected from the set consisting of SEQ ID NO: 137-153.

In another aspect, the invention also relates to an isolated antigen-binding protein that specifically binds to an epitope forming amino acid residues of the CRLR and RAMP1 components of CGRP R.

In yet another embodiment, the isolated antigen-binding protein described above comprises a first amino acid sequence comprising at least one of the consensus CDRH sequences described herein, and a second amino acid sequence comprising at least one of the consensus sequences. CDRL described here. In one aspect, the first amino acid sequence comprises at least two of the CDRH consensus sequences, and / or the second amino acid sequence comprises at least two of the CDRL consensus sequences.

In some embodiments, the first and second amino acid sequences are covalently linked to each other.

In another embodiment, the first amino acid sequence of the isolated antigen-binding protein comprises CDRH3s of SEQ ID NOs: 75, 78, 81, 84, 87, 90, 96, 99,

102 and 123, CDRH2 of SEQ ID NO: 74, 77, 80, 83, 86, 89, 91, 93, 95, 98, 101 and 129, and CDRH1 of SEQ ID NO: 73, 76, 79, 82, 85, 88, 92, 97 and 100, and / or the second amino acid sequence of the isolated antigen-binding protein comprises the CDRL3s of SEQ ID NO: 44, 47, 50, 53, 56, 59, 64, 68,71 and 72, CDRL2 of SEQ ID NO: 43, 46, 49, 52, 55, 58,61, 63, 67 and 70, and CDRL1 of SEQ IDNO: 42,45,48, 51, 54, 57, 62, 65 , 66 and 69.

In another embodiment, the antigen-binding protein comprises at least two CDRH sequences of heavy chain sequence Hl, Η2, Η3, Η4, Η5, Η6, Η7, Η8, Η9, Η10, Hl 1, Η12 or Η13, presented in Table 5A. In yet another embodiment, the antigen-binding protein comprises at least two CDRL sequences of light chain sequence L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, Lll, L12, L13, L14, L15, /

ΜΑ 32982Β1

A-1472-WO-PCT

L16 or L17, shown in Table 5Β, In yet another embodiment, the antigen-binding protein comprises at least two CDRH sequences of heavy chain sequence H1, Η2, Η3, Η4, Η5, Η6, Η7, Η8 , Η9, Η10, Hll, Η12 or Η13, shown in Table 5A, and at least two CDRLs of light chain sequence L1, L2, L3, L4, L5, L6, L7,

L8, L9, LIO, Lll, L12, L13, L14, L15, L16 or L17, presented in Table 5Β.

In yet another embodiment, the antigen-binding protein comprises the CDRH1, CDRH2 and CDRH3 heavy chain sequence sequences H1, Η2, Η3, Η4, Η5, Η6, Η7, Η8, Η9, Η10, Hl 1, Η12 or Η13, shown in Table 5A. In yet another embodiment, the antigen-binding protein comprises the CDRL1, CDRL2, and CDRL3 light chain sequence sequences L1, L2, L3, L4, L5, L6, L7, L8, L9, L10, L11, L12, L13, L14, L15, L16 or L17, presented in table 5Β.

In yet another embodiment, the antigen-binding protein comprises the six CDRs of L1 and Hl, or L2 and Η2, or L3 and Η3, or L4 and Η4, or L5 and Η5, or L6 and Hl, or L7 and Η6, or L8 and Η5, or L9 and Hl, or LIO and Η7, or Lll and Η8, or L12 and Η9, or L12 and Η10, or

L13 and Η5, or L14 and Hll, or L15 and Η12, or L16 and Η13, or L17 and Η13, presented in Tables 5Α and 5Β.

Table 5Α - Examples of regions of amino acid sequences

<td>Φ o vs 2 S ٠ كاً</td><td>Group of channels heavy whole</td><td>Full heavy chain SEQID NO</td><td>magnifier ae regions string variables heavy</td><td>Kegion variaoie ae heavy chain SEQ ID NO</td><td>٠ Z o q READ لى Ξ Οί ه ü</td><td>CDRH2 SEQ ID NO</td><td>CDRH3SEQID NO</td>

<td>1Ε11</td><td>Hl</td><td> 29</td><td>VhI</td><td> 158</td><td> 73</td><td> 74</td><td> 75</td>

<td>1Η7</td><td>Η2</td><td> 30</td><td>Vh2</td><td> 159</td><td> 76</td><td>IT</td><td> 78</td>

<td>2Ε7</td><td>Η3</td><td> 31</td><td>Vh3</td><td> 160</td><td> 79</td><td> 80</td><td> 81</td>

<td>3Β6</td><td>Η4</td><td> 32</td><td>Vh4</td><td> 161</td><td> 82</td><td> 83</td><td> 84</td>

<td>3C8</td><td>Η5</td><td> 33</td><td>Vh5</td><td> 162</td><td> 85</td><td> 86</td><td> 87</td>

<td>4Ε4</td><td>Hl</td><td> 29</td><td>VhI</td><td> 158</td><td> 73</td><td> 74</td><td> 75</td>

<td>4Η6</td><td>Η6</td><td> 34</td><td>Vh6</td><td> 163</td><td> 88</td><td> 89</td><td> 90</td>

<td>5F5</td><td>Η5</td><td> 33</td><td>٧h5</td><td> 162</td><td> 85</td><td> 86</td><td> 87</td>

<td>9D4</td><td>Hl</td><td> 29</td><td>VhI</td><td> 158</td><td> 73</td><td> 74</td><td> 75</td>

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>Group of channels heavy whole</td><td>Full heavy chain SEQ ID NO</td><td>Group oe regions string variables heavy</td><td>Q ٠ ٠ SS "V) ο β" ta · Β ؤة ة 2 Ο ء «SZ ن ة</td><td>٠ Ζ ع ٠ ... m Ξ كه ه دلم</td><td>CDRH2 SEQ ID NO</td><td>٠ Z ع σ read لى لم I كه ه ى</td>

<td>9F5</td><td>Η7</td><td> 35</td><td>١١٦ لا</td><td> 164</td><td> 76</td><td> 91</td><td> 78</td>

<td>10Ε4</td><td>Η8</td><td> 36</td><td>٧η8</td><td> 165</td><td> 92</td><td> 93</td><td> 94</td>

<td>IIIDII</td><td>Η9</td><td> 37</td><td>Vh9</td><td> 166</td><td> 76</td><td> 95</td><td> 78</td>

<td>11Η9</td><td>Η10</td><td> 38</td><td>VhIO</td><td> 167</td><td> 76</td><td> 95</td><td> 78</td>

<td>12Ε8</td><td>Η5</td><td> 33</td><td>٧η5</td><td> 162</td><td> 85</td><td> 86</td><td> 87</td>

<td>12G8</td><td>Η11</td><td> 39</td><td>VhII</td><td> 168</td><td> 73</td><td> 74</td><td> 96</td>

<td>13Η2</td><td>Η12</td><td> 40</td><td>Vh12</td><td> 169</td><td> 97</td><td> 98</td><td> 99</td>

<td>32Η7</td><td>Η13</td><td> 41</td><td>Vh13</td><td> 170</td><td> 100</td><td> 101</td><td> 102</td>

<td>32Η7 CS</td><td>Η13</td><td> 41</td><td>٧η13</td><td> 170</td><td> 100</td><td> 101</td><td> 102</td>

<td>32Η8</td><td></td><td></td><td>٧η14</td><td> 171</td><td></td><td></td><td></td>

<td>33Β5</td><td></td><td></td><td>٧η15</td><td> 172</td><td></td><td></td><td></td>

<td>33Ε4</td><td></td><td></td><td>٧η16</td><td> 173</td><td></td><td></td><td></td>

<td>34Ε3</td><td></td><td></td><td>٧η17</td><td> 174</td><td></td><td></td><td></td>

Table 5 - Examples of Light Chain Amino Acid Sequence Regions

<td>Reference</td><td>Group of channels light whole</td><td>SEQ light chain Integer ID NO</td><td>Group of regions variables of light chains</td><td>Kegion variant ae light chain SEQ ID NO</td><td>O Z Q ٠ CO ت ٥ دلم</td><td>٠ Z at at ... co د ٥ ٥ ى</td><td>CDRL3SEQ ID NO ١</td>

<td>1Ε11</td><td>Ll</td><td> 12</td><td>VlI</td><td> 137</td><td> 42</td><td> 43</td><td> 44</td>

<td>1Η7</td><td>L2</td><td> 13</td><td>Vl2</td><td> 138</td><td> 45</td><td> 46</td><td> 47</td>

<td>2Ε7</td><td>L3</td><td> 14</td><td> 3,٧</td><td> 139</td><td> 48</td><td> 49</td><td> 50</td>

<img file="MA32982B1_D0009.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>Group of channels light whole</td><td>SEQ light chain Integer ID NO</td><td>troop ae regions variables of light chains</td><td>Kegion variaoie ae light chain SEQ ID NO</td><td>O z ح σ UJ لى ة ه ى</td><td>٠ أ ح ٠ UJ لى ت Οί ه at</td><td>٠ ة ح σ UJ لن ثآ عاً ه O</td>

<td>3Β6</td><td> 14</td><td> 15</td><td> ٧_L4</td><td> 140</td><td> 51</td><td> 52</td><td> 53</td>

<td>3C8</td><td> 15</td><td> 16</td><td>٧l5</td><td> 141</td><td> 54</td><td> 55</td><td> 56</td>

<td>4Ε4</td><td> 16</td><td> 17</td><td>Vi 6</td><td> 142</td><td> 42</td><td> 43</td><td> 44</td>

<td>4Η6</td><td>not</td><td> 18</td><td>١٦ لا</td><td> 143</td><td> 57</td><td> 58</td><td> 59</td>

<td>5F5</td><td> 18</td><td> 19</td><td>Vi8</td><td> 144</td><td> 60</td><td> 55</td><td> 56</td>

<td>9D4</td><td>IQ</td><td> 20</td><td>لآ_الا</td><td> 145</td><td> 42</td><td> 43</td><td> 44</td>

<td>9F5</td><td> 110</td><td> 21</td><td>VlIO</td><td> 146</td><td> 45</td><td> 61</td><td> 47</td>

<td>10Ε4</td><td> 111</td><td> 22</td><td>VII</td><td> 147</td><td> 62</td><td> 63</td><td> 64</td>

<td>IIIDII</td><td>L12</td><td> 23</td><td>12 أ ٧</td><td> 148</td><td> 45</td><td> 61</td><td> 47</td>

<td>11Η9</td><td> 112</td><td> 23</td><td>٧ι12</td><td> 148</td><td> 45</td><td> 61</td><td> 47</td>

<td>12Ε8</td><td> 113</td><td> 24</td><td>V, 13</td><td> 149</td><td> 65</td><td> 55</td><td> 56</td>

<td>12G8</td><td>L14</td><td> 25</td><td>Vi 14</td><td> 150</td><td> 42</td><td> 43</td><td> 44</td>

<td>13Η2</td><td> 115</td><td> 26</td><td>Vi 15</td><td> 151</td><td> 66</td><td> 67</td><td> 68</td>

<td>32Η7</td><td> 116</td><td> 27</td><td>V. 16</td><td> 152</td><td> 69</td><td> 70</td><td> ٦١</td>

<td>32Η7 CS</td><td> 117</td><td> 28</td><td>Vi 17</td><td> 153</td><td> 69</td><td> 70</td><td> 72</td>

<td>32Η8</td><td></td><td></td><td>VilB</td><td> 154</td><td></td><td></td><td></td>

<td>33Β5</td><td></td><td></td><td>Vt19</td><td> 155</td><td></td><td></td><td></td>

<td>33Ε4</td><td></td><td></td><td>Vl20</td><td> 156</td><td></td><td></td><td></td>

<td>34Ε3</td><td></td><td></td><td>Vi 21</td><td> 157</td><td></td><td></td><td></td>

In one aspect, the isolated antigen-binding proteins provided herein may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or a fragment thereof. antigen binding antibody.

ΜΑ 32982Β1

A-1472-WO-PCT

In another embodiment, the antibody fragment of the isolated antigen-binding proteins provided herein may be an Fab fragment, an Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, a diabody, or a molecule. of single chain antibodies.

In another embodiment, the isolated antigen binding protein provided herein is a human antibody and may be of the IgG1, IgG2, IgG3 or IgG4 type.

In another embodiment, the antigen-binding protein consists only of a light or heavy chain polypeptide shown in Tables 5Α-5Β. In some embodiments, the antigen-binding protein is only comprised of a light or heavy chain variable domain shown in Tables 5Α-5Β. These antigen-binding proteins can be pegylated with one or more PEG molecules.

In yet another aspect, the isolated antigen-binding protein can be coupled to a tag group and can compete for binding to the extracellular portion of human CGRP R with an antigen-binding protein of one of the antigen-binding proteins. isolated from the invention. In one embodiment, the isolated antigen-binding protein provided herein can reduce monocyte chemotaxis, inhibit monocyte migration into tameurs, or inhibit the accumulation and function of tumor-associated macrophages in a tumor, when is administered to the patient.

As will be appreciated by those skilled in the art, for any antigen binding protein with more than one CDR of the sequences described, any combination of CDRs independently selected from the sequences described is useful. Thus, proteins binding an antigen with one, two, three, four, five or six independently selected CDRs can be produced. However, as will be appreciated by those skilled in the art, specific embodiments will employ combinations of CDRs which are non-repeating, eg, antigen binding proteins generally do not consist of two CDRH2 regions, etc.

Certain antigen-binding proteins provided herein are discussed in more detail below.

Antigen-binding proteins and binding epitopes and binding domains

When we say that an antigen-binding protein binds to an epitope, such as one or both components of CGRP R, or the extracellular domain of CGRP R, for example, it means that the protein binds an antigen specifically binds to a specified part of CGRP R, which can be on CRLR, RAMP1, or spans parts of CRLR and RAMP1. In cases where the antigen-binding protein only binds CRLR (and not RAMP1), the antigen-binding protein is not expected to selectively bind CGRP R because CRLR is shared between

ΜΑ 32982Β1

A-1472.WO-PCT others with AMI and AM1 receptors. Likewise, in cases where the antigen-binding protein only binds RAMP1 (and not CRLR), the antigen-binding protein is not expected to selectively bind CGRP R because RAMP1 is shared with an AMY1 receptor among others. . In instances where the antigen-binding protein interacts with CRLR and RAMP1, the antigen-binding protein is expected to bind residues or residue sequences, or regions of CRLR and RAMP1. In none of the embodiments, is it intended that an antigen binding protein will contact all of the residues in CRLR or RAMP1. Likewise, not all amino acid substitutions or deletions in CRLR, RAMP1 or their extracellular domains are expected to significantly affect binding affinity.

The methods detailed, for example, in Example 10, can be used to determine which regions of multimeric receptors, such as CGRP R, may be involved in binding to selected antigen-binding proteins.

Comnétitatrice antigen-binding proteins

In another aspect, the invention relates to antigen-binding proteins that compete with one of the example, or "referenced" antibodies or functional epitope-binding fragments described above for specific binding to CGRP R. These antigen-binding proteins can also bind to the same epitope as the example or reference antigen-binding proteins, or an overlapping epitope. Antigen-binding proteins and the moieties which compete or bind to the same epitope as the example or reference antigen-binding proteins are expected to exhibit similar functions. Examples of antigen-binding proteins and fiagments include those with heavy and light chains, Vl- V17 and VhI-V3 variable region domains, and CDRs included in Tables 2A, 2Β, 3,4A, 4Β, 5Α. and 5Β. Thus, as a specific example, the antigen-binding proteins which are provided include those which compete with an antibody having: (a) the 6 CDRs indicated for an antibody indicated in Tables 5Α and 5Β; (b) a Vh and a Vl selected from VlI-Vl17 and VhI-Vh13 and indicates for an antibody indicated in Tables 5Α and 5Β; or (c) two light chains and two heavy chains specified for an antibody indicated in Tables 5Α and 5Β. Other examples of suitable reference antibodies include those which have a heavy chain variable region having a sequence corresponding to any of the sequences identified as SEQ ID ΝΟ: 158-170 and a light chain variable region having a sequence corresponding to any one of the sequences identified as SEQ ID 0: 137-153.

Competition for binding can be assessed, for example, using binding assays, such as the Biacore assay described in Example 7, below. In this example, 19 antibodies

ΜΑ 32982Β1

A-1472-WO-PCT described herein were tested against each of six "reference" antibodies - five neutralizing antibodies (11D11, 3Β6,4Η6, 12G8, and 9F5) and one non-neutralizing antibody (34Ε3). The results of the test, shown in Table 13, indicate that all neutralizing antibodies tested (1Ε11,1Η7,2Ε7, 3Β6, 3C8,4E4,4H6, 5F5, 9D4, 9F5, 10Ε4, HDll, 11Η9, 12Ε8,12G8, 13Η2 and 32Η7) bind essentially to the same region of CGRP R, which is distinct from the region of CGRP R which is bound by the neutralizing antibodies tested (32Η8.33Β5, 33Ε4 and 34Ε3). From these data, any of the non-neutralizing antibodies could constitute an antigen-binding protein of example referenced in a competition assay, in particular any of the neutralizing antibodies which were immobilized in the assay described in example 7 - llDll, 3Β6, 4Η6, 12G8, and 9F5.

Monoclonal antibody

The antigen-binding proteins provided include monoclonal anticoips which bind to CGRP R. The monoclonal antibody can be produced using any of the techniques known to those skilled in the art, for example, by immortalizing spleen cells harvested from the spleen. transgenic animal after completion of the immunization program. Spleen cells can be immortalized using any technique known to those skilled in the art, for example, by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion protocols are preferably non-antibody-producing, have high filtration efficiency, and enzyme deficiencies which render them unable to grow in certain selective media which only allow the growth of the desired fired cells (hybridomas). Examples of cell lines suitable for use in mouse fijsions include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NSl / l.Ag4 1, Sp210-Agl4, FO, NSO / υ, MPC-11, MPCU-X45-GTG 1.7 and S194 / 5XXO Bul; examples of cell lines used in rat fiisions include R210.RCY3, Y3-Ag 1.2.3, IR983F and 4Β210. Other useful cell lines for cell division are υ-266, GM 1500-GRG2, LICR-LON-HMy2 and UC729-6. An exemplary process for preparing monoclonal antibodies is described in Example 2, below.

In some cases, a hybridoma cell line is produced by immunizing an animal (eg, a transgenic animal having human immunoglobulin sequences) with a CGRP R immunogen; harvesting the spleen cells from the animal immunizes; splitting the harvested spleen cells to a myeloma cell line, thereby producing hybridoma cells; establishing hybridoma cell lines from the hybridoma cells and identifying a hybridoma cell line which

م

ΜΑ 32982Β1

A-1472-WO-PCT produces an antibody which binds CGRP R (eg, as described in Examples 1-3, below). These hybridoma cell lines, and the anti-CGRP R monoclonal antibodies they produce, are aspects of the present application.

Monoclonal antibodies secreted by a hybridoma cell line can be purified using any technique known to those skilled in the art. Hybridomas or mAbs can then be screened to identify mAbs with particular properties, such as the ability to bind CGRP-expressing cells, the ability to block or interfere with, the binding of CGRP ligand or CGRPs 37 peptide, or ability to functionally block the receptor, for example by means of a cAMP test, for example, as described herein.

Chimeric and humanized antibodies

The invention also relates to chimeric and humanized antibodies based on the above sequences. Monoclonal antibodies for use as therapeutic agents can be modified in various ways before use. An example is a chimeric antibody, which is an anticoips composed of protein segments from different antibodies which are covalently joined to produce light or heavy chains of immunoglobulin or immunologically functional parts thereof. Usually, part of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (s) is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another class or subclass of anticoips. For methods relating to chimeric antibodies, see, eg, US Patent No. 4,816,567; and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA £ 1: 6851-6855, which are colorless here by reference. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370, No. 5,693,762, No. 5,693,761, No. 5,585,089 and No. 5,530,101.

Generally, the objective of preparing an antibody is to create a chimera in which the number of amino acids of the species of the patient for which it is intended is maximum. An example is the "CDR-grafted" antibody, wherein the antibody comprises one or more complementarity determining regions (CDRs) of a particular species or belonging to a particular class or subclass of antibody, while that the remainder of the chain (s) of anticoips is identical or homologous to a corresponding sequence in antibodies derived from another species or belonging to another class or subclass of antibodies. For use in humans, variable region or selected CDRs

ΜΑ 32982Β1

A-1472-WO.PCT of a rodent antibody is often grafted into a human antibody, replacing the natural variable regions or CDRs of the human antibody.

Another useful type of chimeric antibody is a "humanized" antibody.

Generally, a humanized antibody is produced from a monoclonal antibody initially produced in a non-human animal. Certain amino acid residues in this monoclonal antibody, usually parts of the antibody not recognizing the antigen, are modified to be homologous to corresponding residues in a human antibody of the corresponding isotype. Humanization can be carried out, for example, by means of various methods by replacing at least part of a rodent variable region with the corresponding regions of a human antibody (see, for example, US Patent No. 5,585. 089 and No. 5,693,762; Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988,

Nature 332: 323-27; Verhoeyen and “/., 1988, Science 239: 1534-1536),

In one aspect, the CDRs of the light and heavy chain variable regions of the antibody of the invention (see Table 4) are grafted onto the framework regions (FR) of antibodies of the same phylogenic species or of a different species. . For example, CDRs of heavy and light chain variable regions VhI, Vh2, Vh3, ٧h4, Vh5, Vh6, ​​Vh7, Vh8, Vh9, VhIO, VhII, Vh12 and Vh13, and / or Vl٠ Vl2, Vl3, Vl4, Vl5 , Vl6, Vl7, Vl8, Vl9, vio. Vil, V_l12, V13, V_l14, Vl15, Vl16 and V_l17 can be grafted onto consensus human FRs. To create consensus human FRs, the FRs of multiple heavy chain or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence. In other embodiments, FRs of a heavy chain or light chain described herein are replaced by FRs of a different heavy chain or light chain. In one aspect, rare amino acids in the heavy and light chain RFs of an anti-CGRP R antibody are not replaced while the other amino acids of the RF are replaced. A "rare amino acid" is a specific amino acid that is in a position where that particular amino acid is generally not found in an RF. Variable regions grafted to a heavy or light chain can also be used with a constant region that is different from the constant region of that particular heavy or light chain described herein. In other embodiments, the grafted variable regions are part of a single chain Fv antibody.

In some embodiments, the constant regions of species other than human can be used with the human variable region (s) to produce hybrid antibodies.

ΜΑ 32982Β1

A-1472-WO-PCT

Fully human antibodies

The invention also relates to fully human antibodies. There are methods of preparing fully human antibodies specific for a given antigen without exposing humans to the antigen ("fully human antibody"). A specific means of enabling the production of fully human antibodies is "humanization" of the mouse humoral immune system. Introducing human immunoglobulin (Ig) loci into mice in which endogenous Ig genes have been inactive is a means of producing fully human monoclonal anticoips (mAbs) in mice, an animal that can be immunized. with any desired antigen. The use of fully human antibodies can reduce the immunogenic and allergic responses which are sometimes elicited by administration of mouse mAb or mouse derivatives to humans as therapeutic agents.

Fully Hunan antibodies can be produced by immunizing transgenic animals (usually mice) which are capable of producing a human antibody repertoire in the absence of endogenous immunoglobulin production. The antigens used for this purpose generally consist of six or more contiguous amino acids, and are obviously conjugated to a carrier, such as a hapten. See, e.g., Jakobovits and ٥ /., 1993, Proc. Natl. Acad. Sci. USA 0: 2551-2555; Jakobovits et al., 1993, Nature 3: 255258; and Bruggermann et al., 1993, Year in Immunol. 7:33. In one example of such a method, transgenic animals are produced by disabling endogenous mouse imminoglobulin loci encoding mouse immunoglobulin heavy and light chains, and inserting large fragments of DNA into the mouse genome. genome containing loci that encode human heavy and light chain proteins. Partially modified animals, which contain less than the full complement of human immunoglobulin loci, are then crossed to obtain an animal containing all of the desired changes in the immune system. When administered with an immunogen, these fransgenic animals produce antibodies which are immunospecific for the immunogen but have human rather than murine amino acid sequences, including variable regions. For further details on these methods, see, for example, WO96 / 33735 and WO94 / 02602. Other methods related to transgenic mice for the preparation of human antibodies are described in US Patent Nos. 5,545,807; No. 6713,610; No. 6,673,986; No. 6,162,963; No. 5,545,807; No. 6,300,129; No. 6,255,458; No. 5,877,397; No. 5,874,299 and No. 5,545,806; PCT publications WO91 / 10741, WO90 / 04036, and in EP 546073Β1 and ΕΡ 546073Α1.

ΜΑ 32982Β1

A-1472-WO-PCT

The transgenic mice described herein, referred to as "HuMab" mice, contain a human immunoglobulin gene minilocus which encodes unrearranged heavy chain ([mu] and [gamma]) and light chain [kappa] immunoglobulin sequences, associated with targeted mutations which inactivate the endogenous [mu] and [kappa] chain loci (Lonberg et al.,

1994, Nature 3: 856-859). Therefore, mice exhibit reduced expression of IgM or mouse [kappa] in response to immunization, and introduced human heavy and light chain transgenes undergo class change and somatic mutation to produce monoclonal antibodies. High affinity human IgG [kappa] (Lonberg et al., Supra .; Lonberg and Huszar, 1995, Intern. Rev. lmmunol.93-65: ثل; Harding and Lonberg,

1995, Ann. NY Acad. Sci. 764: 536-546). The preparation of HuMab mice is described in detail in Taylor et al., 1992, Nucleic Acids Research 0: 6287-6295; Chen et al., 1993, InternationalImmunology 5: 647-656; Tuaillonera /., 1994, j. lmmunol. 152: 2912-2920; Lonberg et al., 1994, Nature s 856-859; Lonberg, 1994, Handbook ofExp. Pharmacology

3: 49-101; Taylor and "7, 1994, International Immunology 6: 579-591; Lonberg and Huszar, 1995, Intern. Rev. lmmunol. 3: 65-93; Harding and Lonberg, 1995, Ann. N. Y Acad. Sci. 764: 536-546; Fishwildera /., 1996, Nature Biotechnology 851-4: 845 ل; the foregoing references are incorporated herein by reference in their entirety for convenience. See, other US Patents Nos. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425; No.

789 650; No. 5,877,397; No. 5,661,016; No. 5,814318; No. 5,874,299; and No. 5,770,429; as well as U.S. Patent No. 5,545,807; International publications Nos. wo 93/1227; wo 92/22646; and wo 92/03918, the descriptions of which are incorporated herein by reference in their entirety for convenience. The technologies used to generate the human antibodies in these transgenic mice are also described in wo 98/24893, and Mendez et /., 1997, Nature Genetics 156-146: ئ, which are incorporated herein by reference. For example, the HCo7 and HCol2 transgenic mice can be used to produce an antiCGRP R antibody. Further details regarding the production of human antibodies using transgenic mice are given in the examples below.

Using hybridoma technology, antigen-specific human mAbs having the desired specificity can be produced and selected from transgenic mice such as those described above. These anticoips can be cloned and expressed using an appropriate vector and host cell, or the antibodies can be harvested from the hybridoma cells in culture.

Fully human antibodies can also be derived from phage display libraries (as described in Hoogenboom et al., 1991, U. Mol. Biol.

100

ΜΑ 32982Β1

A-1472-WO-PCT

227 : 381; and Marks et al., 1991, 7. Mol. Biol. 222: 581). Phage display techniques mimic immune selection by display of antibody repertoires on the surface of filamentous bacteriophages, and subsequent selection of phage by binding to an antigen of interest. One of these techniques is described in PCT Publication No. wo 99/10494 (incorporated herein by reference) which describes the isolation of functional and high affinity agonist antibodies for MPL and msk receptors using this approach.

Bispecific or bifunctional antigen-binding proteins

The antigen-binding proteins of the invention also include bispecific and bifunctional antibodies which comprise one or more CDRs or one or more variable regions described above. A bispecific or bifunctional antibody is in some cases an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or binding of Fab 'fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 22: 315-321; Kostelny et al., 1992,7. Immunol. 143: 1547-1553.

Various other shapes

Some of the antigen-binding proteins of the invention are variant forms of the antigen-binding proteins described above (eg, those which have the sequences shown in Tables 2-5). For example, some of the antigen-binding proteins have one or more conservative amino acid substitutions in one or more of the heavy or light chains, variable regions or CDRs noted above.

Natural amino acids can be divided into classes based on common side chain properties:

1) hydrophobic: norleucine. Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acid: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues which influence the orientation of the chains: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions can involve the exchange of a member of one of these classes for another member of the same class. Conservative amino acid substitutions can include non-nataral amino acid residues, which are generally incorporated by chemical synthesis of peptides rather than by synthesis ./

101

ΜΑ 32982Β1

A-1472-WO-PCT in biological systems. They include peptidomimetics and other inverted forms of amino acid fragments.

Non-conservative substitotions can involve swapping one of the members of one class above with a member of another class. These substituted residues can be introduced into regions of the antibody which are homologous to human antibodies, or into non-homologous regions of the molecule.

To effect these changes, in some embodiments, the hydropathy index of amino acids can be considered. The hydropathic profile of a protein is calculated by assigning each amino acid a numerical value ("hydropathy index") and then repeatedly averaging these values ​​along the peptide chain. A hydropathy index can be assigned to each amino acid depending on its hydrophobicity and charging characteristics. These are: isoleucine (t4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​/ cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic profile in assigning an interactive biological function to a protein is understood by those skilled in the art (see, e.g., Kyte et al., 1982, j. Mol. Biol. 157: 105-131). . It is known that certain amino acids can replace other amino acids having a similar hydropathy index or score and yet retain similar biological activity. When changes are made based on the hydropathy index, in some embodiments, substitution of amino acids with hydropathy index other than ± 2 is included. In some aspects those different from ± 1 are included and in other aspects those different from ± 0.5 are included.

It is also accepted in the art that the substitution of similar amino acids can be effected efficiently on the basis of hydrophilicity, particularly when the biologically functional protein or peptide thus created is intended for use in immunological embodiments. , as in this case. In some embodiments, the higher local hydrophilicity of a protein, determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and binding to antigen or immunogenicity, i.e. that is, to a biological property of the protein.

The following hydrophilicity values ​​have been assigned to the indicated amino acid residues: arginine (+ -3.0); lysine (+3.0); aspartate (+ 3, o ± 1); glutamate (+301); serine

102

ΜΑ 32982Β1

A-1472-WO-PCT (0.3 ؛); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). When changes are made based on similar hydrophilicity values, in some embodiments the substitution of amino acids with hydrophilicity values ​​different from ± 2 is included, in other embodiments those which differ by ± 1 are included and in other aspects those which differ by ± 0.5 are included. In some cases, epitopes of primary amino acid sequences can also be identified from hydrophilicity. These regions are also referred to as the epitope core regions.

Examples of conservative amino acid substitutions are shown in Table 6.

Table 6: Conservative amino acid substitutions

<td>Original residue</td><td>Substitution example</td>

<td>To the</td><td>Ser</td>

<td>Arg</td><td>Lilies</td>

<td>Asn</td><td>Gin, His</td>

<td>Asp</td><td>Glue</td>

<td>Cys</td><td>Ser</td>

<td>Gin</td><td>Asn</td>

<td>Glue</td><td>Asp</td>

<td>Gly</td><td>Pro</td>

<td>His</td><td>Asn, Gin</td>

<td>Isle</td><td>Leu, Val</td>

<td>Leu</td><td>Island, Val</td>

<td>Lilies</td><td>Arg, Gin, Glu</td>

<td>Met</td><td>Leu, Island</td>

<td>Phe</td><td>Met, Leu, Tyr</td>

<td>Ser</td><td>Thr</td>

<td>Thr</td><td>Ser</td>

103

<img file="MA32982B1_D0010.tif" />

ΜΑ 32982Β1

A-1472-WO-FCT

<td>Original residue</td><td>Substitution example</td>

<td>Trp</td><td>Tyr</td>

<td>Tyr</td><td>Trp, Ehe</td>

<td>Val</td><td>Island, Leu</td>

Those skilled in the art will be able to determine suitable variants of the polypeptides described herein using techniques well known in the art. Those skilled in the art can identify suitable areas of the molecule which can be modified without destroying activity by targeting regions believed to be not important for activity. Those skilled in the art will also be able to identify residues and parts of molecules which are conserved between similar polypeptides. In other embodiments, even areas which may be important for biological activity or structure may be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting the structure of the polypeptide.

Additionally, one skilled in the art can rely on structure-function studies identifying residues in similar polypeptides which are important for activity or structure. Using this comparison, one can predict the importance of amino acid residues in a protein, since they correspond to amino acid residues important for activity or structure in a similar protein. Those skilled in the art can opt for chemically similar amino acid substitutions for these presumably important amino acid residues.

Those skilled in the art can also analyze the three-dimensional structure and the amino acid sequence in relation to this structure in similar polypeptides. Based on this information, one skilled in the art can predict the alignment of the amino acid residues of an antibody relative to its three-dimensional structure. Those skilled in the art may choose not to make radical changes in amino acids expected to be on the surface of the protein, as these residues may be involved in important interactions with other molecules. Additionally, one skilled in the art can produce test variants containing a single amino acid substitution in each desired amino acid residue. These variants can be screened using CGRE R neutralizing activity assays (see examples below), which provides information about which amino acids can be changed and which should not be changed. In other words, from the information gathered by these routine experiments, a person skilled in the art can

104

ΜΑ 32982Β1

A-1472-WO-PCT readily determine the amino acid positions in which substitutions should be avoided alone or in combination with other mutations.

Several specific publications have been devoted to the prediction of secondary structure. See, Moult, 1996, Curr. Op. In Biotech. 2: 422427; Cabbage e1974, ٥٤ ؛,

Biochem. 1: 222-245; Cabbage e1974, ٥٤ ؛, Biochemistry 113: 211-222; Chou et٥٤, 1978, AA ,. Enzymol. Relat. Areas Mol. Biol. 42: 45-148; Chou and ٥٤, 1979, Ann. Rev. Biochem. 42: 251276; and Chou et ٥٤, 1979, Biophys. J. 2: 367-384. In addition, computer programs are currently available to aid in the prediction of secondary structure. A method of predicting secondary structure relies on homology modeling. For example, two polypeptides or proteins which have sequence identity greater than 30 ٠/٠ or similarity greater than 40 1½ may have similar structural topologies. Recent development of the Protein Structure Database (PDB) has made it possible to better predict secondary structure, including the potential number of folds in a polypeptide or protein structure. See, Holm et ٥٤, 1999, Nucl. Acid. Res. 22: 244-247. It has been suggested (Brenner et ٥٤, 1997, Curr. Op. Struct. Biol. 2: 369-376) that there is a limited number of folds in a given polypeptide or protein, and that once a critical number of structures have been resolved, structural prediction becomes much more accurate.

Other methods of predicting secondary structure include "threading" (Jones, 1997, Curr. Opin. Struct. Biol. 2: 377-387; Sippl et ٥٤, 1996, Structure 4: 15- 19), “profile analysis” (Bowie and ٥٤, 1991, Science 23: 164-170; ÜQ \ and αΙ., ΙΕ Meth. Enzym. L١AG- \ ٦ 9 &lt;؟ Gririshi et al., N, p١î . Nat. Acad. Sci. §4: 4355-4358), and "evolutionary linking" (see, Holm, 1999, supra; and Brenner, 1997, supra).

In some embodiments, amino acid substitutions are made in order to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity to form protein complexes, (4) modify binding affinities for the ligand or antigen, and / or (5) confer or modify other physicochemical or functional properties on these polypeptides. For example, single or multiple amino acid substitutions (in some embodiments, conservative amino acid substitutions) can be made in the natural sequence. Substitutions can be made in the part of the antibody that binds outside of the domain (s) forming intermolecular contacts. In these embodiments, one can use conservative amino acid substitutions which do not substantially alter the structural features of the parent sequence (e.g., one or more

105

ΜΑ 32982Β1

A-1472-WO-PCT amino acid replacements which do not disrupt the secondary structure which characterizes the protein binding a parent or native antigen). Examples of secondary and tertiary polypeptide structures recognized in the art are described in Proteins, Stnrctures and

Molecular Principles (Creighton, Ed.), 1984, w. H. New York: Freeman and Company;

Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland

Publishing; and Thomton e ؛ al., 1991, Nature 354: 105. all of which are incorporated herein by reference.

Other preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues in the parent or native amino acid sequence are deleted or replaced with another amino acid (eg, serine). Cysteine ​​variants are useful, inter alia, when antibodies are to be refolded into a biologically active conformation. Cysteine ​​variants may have fewer cysteine ​​residues than the native antibody, and they usually have an even number to reduce interactions due to unpaired cysteine ​​residues.

The heavy and light chains, variable region domains and CDRs that are described can be used to prepare polypeptides which contain an antigen-binding region which can specifically bind to CGRP R. For example, one or more CDRs indicated in Tables 4 and 5 can be incorporated into a molecule (eg, a polypeptide) by covalent or non-covalent interaction to achieve immunoadhesion. An immunoadhesion can incorporate the CDR (s) as part of a larger polypeptide chain, can covalently link the CDR (s) to another polypeptide chain, or can incorporate the CDR (s) by non-covalent interaction. The CDR (s) allow the immunoadhesion molecule to specifically bind to an antigen of particular interest (eg, CGP R or one of its epitopes).

The invention also relates to mimetics (eg, "peptide mimics" or "peptidomimetics") based on the variable region domains and CDRs which are described herein. These analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchere, 1986, Adv. DrugRes. 29: ئ; Veber and Freidinger, 1985, TINS ​​p. 392; and Evans et al., 1987, J. Med. Chem. 0: 1229, which are incorporated herein by reference for convenience. Peptide mimetics which are structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. These compounds are often developed using computerized molecular modeling. Generally, peptidomimetics are proteins which are structurally similar to an antibody exhibiting a desired biological activity, such as here the ability to specifically bind CGRP.

106

ΜΑ 32982Β1

A-1472-WO-PCT

R, but which comprise one or more peptide bonds optionally replaced by a bond chosen from: -CHNH-, -CHS-, -CH2-CH2-, -CHCHfcis and trans), -COCH-, CH (OH) CH2-, and -CHSO-, by methods well known to those skilled in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (eg, D-lysine in place of L-lysine) can be used in some embodiments to produce proteins. more stable. Further, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be produced by methods known to those skilled in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61: 387). incorporated herein by reference), for example, by addition of internal cysteine ​​residues capable of forming intramolecular disulfide bridges which cycle the peptide.

The invention also relates to derivatives of the antigen-binding proteins described herein. The derived antigen-binding proteins can comprise any molecule or substance which confers a desired property on an antibody or fragment, so as to increase the half-life in a particular use. The antigen-binding protein may include, for example, a detectable (or labeling) residue (eg, a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (such as a magnetic or electron-dense (eg gold) bead) , or a molecule that binds to another molecule (eg, biotin or streptavidin), a therapeutic or diagnostic residue (eg, a radioactive, cytotoxic or pharmaceutically active residue), or a molecule which enhances the adaptation of the antigen binding protein for a particular use (eg, administration to a subject, such as a human subject, or other uses in vivo or in vitro). Examples of molecules which can be used to derive a protein in an antigen include albumin (eg, human serum albumin) and polyethylene glycol (PEG). Albumin and PEGyl related derivatives of antigen binding proteins can be prepared using techniques well known to those skilled in the art. Certain antigen-binding proteins include a pegylated single chain polypeptide described herein. In one embodiment, the antigen binding protein is conjugated or otherwise linked to transthyetine (TTR) or a variant of TTR. The TTR or the TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly (n-vinylpyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide ethylene oxide copolymers, polyoxyethylated polyols and polyvinyl alcohols.

107 /

ΜΑ 32982Β1

A-1472-WO-PCT

Other derivatives include covalent or aggregation conjugates of CGRP R-binding proteins with other proteins or polypeptides, for example by expression of recombinant fusion proteins comprising heterologous polypeptides fused at the N-terminus or c-terminus of a protein binding CGRP R. For example, the conjugate peptide can be a heterologous signal (leader) polypeptide, eg, the yeast alpha factor leader, or a peptide such as an epitope tag. Fusion proteins containing the antigen-binding protein include peptides added to facilitate the purification or identification of the CGRP R-binding protein (eg, poly-His). A CGRP R binding protein can also be linked to the FLAG peptide described in Hopp et al., 1988, Bio / Technology £: 1204; and US Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides a reversibly linked epitope by monoclonal antibody (mAb) which allows rapid testing and easy purification of the expressed recombinant protein. Reagents useful for preparing binding proteins in which the FLAG peptide is bound to a given polypeptide are commercially available (Sigma, St. Louis, MO).

Oligomers which contain one or more CGRR R binding proteins can be employed as CGRP R antagonists. Oligomers can be in the form of dimers, trimers or higher oligomers covalently linked or linked by a non-covalent linkage. Oligomers comprising two or more CGRP R binding proteins are contemplated for this use, and one example is a homodimer. Other oligomers include heterodimers, homotrimeres, heterotrimers, homotetramers, heterotetramers, etc.

One embodiment relates to oligomers comprising multiple CGRP R binding polypeptides joined by covalent or non-covalent interactions between peptide residues fused to CGRP R binding proteins. These peptides may be peptide linkers (spacers), or peptides which have the same property to promote oligomerization. Leucine zippers and certain polypeptides derived from anticoips are among the peptides capable of promoting the oligomerization of the CGRP R binding proteins attached thereto, as is described in more detail below.

In particular embodiments, the oligomers comprise from two to four CGRP R binding proteins. The CGRP R binding protein portions of the oligomer may be in any of the forms described above, e.g., variants or fragments. . Preferably, the oligomers comprise CGRP R binding proteins which have CGRP R binding activity.

108

ΜΑ 32982Β1

A-1472.WO-PCT

In one embodiment, an oligomer is prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various parts of antibody-derived polypeptides (especially the Fc domain) has been described, for example, by Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 3: 10535; Bym et al., 1990, Nature 344: 677; and Hollenbaugh et al., 1992 "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.

One embodiment relates to a dimer comprising two fusion proteins created by fusion of a CGRP R binding protein to the Fc region of an antibody. The dimer can be prepared, for example, by inserting a fusion of genes encoding a fusion protein into an appropriate expression vector, by expressing the fusion of genes in host cells transformed with the recombinant expression vector, and by allowing the expressed fusion protein to assemble much like antibody molecules, allowing disulfide bonding between the Fc fragments to give the dimer.

The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are forms of such polypeptides containing the hinge region which promote dimerization. Fusion proteins comprising Fc portions (and oligomers formed therefrom) have the advantage of easy purification by affinity chromatography on Protein A and Protein G columns.

A suitable Fc polypeptide, described in PCT application wo 93/10151 and US Pat. Nos. 5,426,048 and 5,262,522, is a single chain polypeptide extending from the N-terminal hinge region to 1 '. native c-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein disclosed in the US patent.

No. 5,457,035, and in Baum et al., 1994, EMBO J. 3: 39924001. The amino acid sequence of this mutein is identical to that of the native Fc sequence shown in wo 93/10151, except amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 was changed from Gly to Ala. Mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable part of the heavy and / or light chains of a CGRP R binding protein described herein can replace the variable part of a heavy and / or light chain of an antibody.

The oligomer can also be a binding protein comprising several CGRP R binding proteins, with or without peptide linkers (spacer peptides). Among theirs

109

ΜΑ 32982Β1

Suitable peptide A-1472.WO-PCT, there are those described in US Pat. Nos. 4,751,180 and

No. 4,935,233.

Another method of preparing oligomeric CGRP R binding protein derivatives involves the use of a leucine zipper. Leucine zipper domains are peptides that promote the oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA binding proteins (Landschulz et al., 1988, Science 240: 1759). and have since been discovered in various different proteins. Among the leucine zippers, there are natural peptides and their derivatives which dimerize or trimerize. Examples of leucine zipper domains suitable for the production of soluble oligomeric proteins are described in PCT application wo 94/10308, and the pulmonary surfactant D protein (SPD) leucine zipper described in Hoppe et al., 1994, FEBSLetters 344: 191, incorporated here by reference. The use of a modified leucine zipper which allows the stable trimerization of a heterologous protein matched to it is described in Fanslow et al., 1994, Semin. Immunol. £: 267-278. In one approach, the recombinant fusion proteins comprising a fragment or derivative of a CGRP R binding protein fitted to a leucine zipper peptide are expressed in appropriate host cells, and the fragments or derivatives of CGRP R binding protein which form are recovered in the cell. the culhire supernatant.

In some embodiments, the antigen-binding protein has a Ko (equilibrium binding affinity) of less than 1 pM, 10 pM, 100 pM, 1 nM, 2 nM, 5 nM, 10 nM, 25 nM, or 50 nM.

Another aspect relates to an antigen-binding protein having a half-life of at least one day in vitro or in vivo (eg, when administered to a human subject). In one embodiment, the antigen binding protein has a half-life of at least three days. In another embodiment, the antibody or part of it has a half-life of four days or more. In another embodiment, the antibody or part of it has a half-life of eight days or more. In another embodiment, the antibody or an antigen-binding moiety thereof is derived or altered so as to increase its half-life relative to that of an un-derived or unmodified antibody. In another embodiment, the antigen-binding protein contains point mutations in order to increase its half-life in serum, as described in wo 00/09560, published February 24, 2000, incorporated herein by reference.

Glycosylation

The antigen-binding protein may have a glycosylation profile that is different or altered from that seen in native species. As the men of the

110

ΜΑ 32982Β1

A-1472-WO-PCT art, glycosylation profiles may depend on the protein sequence (eg, the presence or absence of glycosylated amino acid residues, discussed below), or on the cell or of the host organism in which the protein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is usually N-linked or O-linked. N-linked refers to the attachment of the carbohydrate residue to the side chain of an asparagine residue. The asparagine-X-serine and asparagine-X-thronine tripeptide sequences, where X represents any amino acid except pro line, are the recognition sequences for the enzymatic attachment of the carbohydrate residue to the side chain of asparagine. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars Nacetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most often serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition of glycosylation sites to the antigen-binding protein is easily accomplished by altering the amino acid sequence so that it contains one or more of the tripeptide sequences described above (for the linked glycosylation sites ). An alteration can also be effected by the addition of, or substitution with, one or more serine or threonine residues on the starting sequence (for O-linked glycosylation sites). For convenience, the amino acid sequence of the antigen-binding protein can be altered by changes in the DNA, in particular by mutating the DNA encoding the target polypeptide at preselected bases so as to produce codons which will be translated into the desired amino acids.

Another way to increase the number of carbohydrate residues on the antigen-binding protein is by chemical or enzymatic coupling of glycosides to the protein. These methods are advantageous in that they do not require the production of the protein in a host cell which has N- and ο-linked glycosylation capabilities. Depending on the coupling mode used, the sugar (s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups like those of serine, threonine, or hydroxyproline, (e) aromatic residues like those of aphenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in wo 87/05330 published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev, Biochem., PP. 259-306.

], the removal of carbohydrate residues present on the starting antigen-binding protein can be accomplished chemically or enzymatically. Chemical deglycosylation

111

<img file="MA32982B1_D0011.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all of the sugars except the binding sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described in Hakimuddin et al., 1987,

Arch. Biochem. Biophys. 222: 52 and parEdge et al., 1981, Anal. Biochem. 118: 131. Enzymatic cleavage of carbohydrate residues on polypeptides can be accomplished using various endo- and exo-glycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138: 350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound hmicamycin as described by Duskin et al., 1982, j. Biol.

Chem. 257: 3105. Hmicamycin blocks the formation of protein-N-glycoside bonds.

Thus, certain aspects include glycosylation variants of antigen-binding proteins in which the number and / or type of the glycosylation site (s) has been altered from the amino acid sequence of the parent polypeptide. In some embodiments, the antibody protein variants have a higher or lower number of N-linked glycosylation sites than that of the native antibody. An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, in which the amino acid residue denoted by X can be any amino acid except proline. Substituting amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Substitutions which remove or modify this sequence will also prevent the addition of an N-linked carbohydrate chain present in the native polypeptide For example, glycosylation can be reduced by deleting an Asn or replacing Asn with an Asn. different amino acid. In other embodiments, one or more new N-linked sites are created. Antibodies generally have an N-linked glycosylation site in the Fc region.

Markers and effector groups

In some embodiments, binding to the antigen includes one or more markers. The term "label group" or "label" refers to any detectable label. Examples of suitable marker groups include, but are not limited to, the following: radioactive isotopes or radionuclides (e.g., لاة, 'c'⁵NOT, ³⁵s, ⁹⁰Y, ⁹⁹Te, ؛ In, ؛ 2 ؛ I, ؛³؛ I), fluorescent groups (eg, FITC, rhodamine, lanthanide phosphors), enzymatic groups (eg, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predetermined polypeptide epitopes recognized by a secondary reporter (for example, leucine zipper pair sequences, secondary antibody binding sites,

112

ΜΑ 32982Β1

A-1472-WO-PCT metal, epitope labels). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known to those skilled in the art and can be used when they appear to be suitable.

The term "effector group" refers to any group coupled to an antigen-binding protein which acts as a cytotoxic agent. Examples of suitable effector groups are radioactive isotopes or radionuclides (eg, لاد, 'c¹⁵N, s,

Y, ⁹⁹Te, “) In, '²⁵I, ا I). Other suitable groups include toxins, therapeutic groups or chemotherapeutic groups. Examples of suitable groups include calicheamicin, auristatins, geldanamycin and maytansin. In some embodiments, the effector group is coupled to the antigen-binding protein via spacer arms to reduce potential steric hindrance.

In general, markers are listed in various classes, depending on the assay in which they are to be detected: a) isotopic markers, which can be radioactive or heavy isotopes; b) magnetic markers (eg magnetic particles); active redox residues; d) optical dyes; enzymatic groups (eg, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinyl groups; and predetermined polypeptide epitopes recognized by a secondary reporter (eg, leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known to those skilled in the art.

Specific markers include optical dyes including, but not limited to, chromophores, phosphors, and fluorophores, the latter being specific in many instances. Fluorophores can be small molecule fluorophores or protein fluorophores.

A "fluorescent label" refers to any molecule which can be detected by its inherent fluorescence properties. Suitable fluorescent markers include, but are not limited to, fluorescein, rhodamine, tettamethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarins, pyrene, malachite green, stilbene, yellow. Lucifer, Cascade Blue I, Texas Red, IAEDANS, ED ANS, BODIPY FL, LC 640 Red, Cy 5, Cy 5.5, LC 705 Red, Oregon Green, Alexa-Fluor Dyes (Alexa Fluor

113

ΜΑ 32982Β1

A-1472-WO-PCT

350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), waterfall blue, waterfall yellow, and R- phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, rhodamine, and Texas red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, especially fluorophores, are described in the Molecular Probes Handbook by Richard p. Haugland, expressly incorporated here by reference.

Protein fluorescent markers also include, but are not limited to, the Renilla, Ptilosarcus, or Aequorea species of GFP (Chalfie et al., 1994, Science 263: 802-805), EGFP (Clontech Labs., Inc., number d. Genbank access U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec, Canada; Stauber, 1998, Biotechniques 24: 462471; Heim et al., 1996, Curr. Biol. 6: 178-182), enhanced yellow fluorescent protein (EYFP, Clontech Labs., Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 0: 5408-5417), β galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA £ 6: 2603-2607) and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / ​​49019, US Patents No. 5292658 , No. 5418155, No. 5683888, No. 5741668, No. 5777079, No. 5804387, No. 5874304, No. 5876995, No. 5925558).

Nucleic Acid Sequences Encoding CGRP Antigen Binding Proteins

The invention also relates to nucleic acids which encode the antigen-binding proteins described herein, or parts thereof, in particular nucleic acids encoding one or more chains of an antibody, or of one of their fragments, derivatives, muteins or variants, polynucleotides encoding heavy chain variable regions or only CDRs, polynucleotides sufficient to be used as hybridization probes, PCR primers or sequencing primers to identify, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide, antisense nucleic acids to inhibit the expression of a polynucleotide, and sequences complementary to the above. Nucleic acids can be of any length. Their length can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450 , 500, 750, 1000, 1500 nucleotides or more and / or they can comprise one or more additional sequences, for example regulatory sequences, and / or be part of a larger nucleic acid, for example, a vector. Nucleic acids can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides and their artificial variants (eg, peptide nucleic acids).

114

ΜΑ 32982Β1

A-1472-WO-PCT

Table 7 shows examples of the nucleic acid sequences encoding an IgG2 heavy chain constant region, a kappa light chain constant region, and an hCL-1 light chain constant region. Any variable region provided herein can be attached to these constant regions to form complete heavy and light chain sequences.

However, it should be understood that these constant region sequences are offered by way of specific examples only, and those skilled in the art may employ other constant regions, in particular an IgG1 heavy chain constant region, constant regions of IgG1. IgG3 or IgG4, any one of the seven lambda light chain constant regions including hCL-1, hCL-2, hCL-3 and hCL-7; constant regions which have been modified to achieve improved stability, expression, manufacturability or other characteristics, and similar effects. In some embodiments, the variable region sequences are appended to other constant region sequences which are known to those skilled in the art. Examples of nucleic acid sequences encoding variable regions of light chains and heavy chains are shown in Table 8.

Table 7: Examples of nucleic acid sequences of heavy and light chain constant regions

<td>Type</td><td>.EO ID NO. ؛ Number of cid. IW٠I٠٠U ؛ .٩ ، s</td>

<td>heavy chain from lgG2</td><td>بل'كك'.ا .٢ ؛ ' ٠ ·: '/</td>

115

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Type</td><td>Nucleic acid sequence G ID NO.</td>

<td>light chain kappa from lgG2</td><td>cacaaagagcttcaacaggggagagtgttag (SEQ ID NOll</td>

<td>light chain lambda hCL-1 from lgG2</td><td>ggaga ^ gacagg ECDNO: 261]</td>

Table 8 shows examples of nucleic acid sequences encoding heavy and light chain variable regions, in which the various sequences CDRL1, CDRL2 and CDRL3, or CDRH1, CDRH2 and CDRH3, are included.

Table 8: Examples of nucleic acid sequences of heavy and light chain variable regions

<td>Reference</td><td>SEQ ID NO.</td><td>Nucleic acid sequence</td>

<td>2Ε7٧ι</td><td> 175</td><td>cgggcaagtcagggcattagaaatgatttaggctggtttcagcagaaaccagggaaagcccctaa gcgcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctg ggacagaattcactctcacaatcagcagcctgcagcctgaagatttagcaacttattactgtctacagt ataatatttacccgtggacgttcggccaagggaccaaggtggaaatcaaa</td>

<td>13Η2 Vl</td><td> 176</td><td>gggacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtctacag tataatagtttcccgtggacgttcggccaagggaccaaggtggaaatcaaa</td>

116

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>SEQ ID NO.</td><td>Nucleic acid sequence</td>

<td>33B5Vt</td><td> ١11</td><td>agggaaccctggtcaccgtctctagt</td>

<td>4H6Vt</td><td> 178</td><td>ggtctagtcagagcctcctgcatagttttgggtacaactatttggattggtacctgcagaagccagggc catgcaagctctacaaactccattcactttcggccctgggaccaaagtggatatcaaa</td>

<td>3C8Vi</td><td> 179</td><td>gatattatactggcccagactccactttctctgtccgtcacccctggacagccggcctccatctcctgca actgcatgcaaagttttccgcttccgctcactttcggcggagggaccaaggtggagatcaaa</td>

<td>5F5Vl</td><td> 180</td><td>gatattattctgacccagactccactttctctgtccgtcacccctggacagccggcctccatctcctgcaa gtctagtcagagcctcctgcacagtgatggaaagacctatttgtattggtacctgcagaagcccggcc ttgcatgcaaagttttccgcttccgctcactttcggcggagggaccaaggtggagatcaaa</td>

<td>12E8Vl</td><td> 181</td><td>gatattacactgacccagactecactttctctgtccgtctcccctggacagccggcctccatctcctgca tactgcatgcaaagttttccgcttccgctcactttcggcggagggaccaaggtggagatcaaa</td>

117

ΜΑ 32982Β1

A-1472-WO-PCT

<img file="MA32982B1_D0012.tif" />

118

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>SEQ ID NQ.</td><td>Nucleic acid sequence</td>

<td>9D4 Vl</td><td> 188</td><td>aaactcctcatttatgacaataataagcgaccclcagggattcctgaccgattctctggctccaagtctg acatgggatagccgcctgagtgctgtggttttcggcggagggaccaagctgaccgtccta</td>

<td>12G8Vl</td><td> 189</td><td>acatgggatagccgcctgagtgctgtggttttcggcggagggaccaagctgaccgtccta</td>

<td>34Ε3 Vt</td><td> 190</td><td>acatgggatatcggcctgagtgtttgggtgttcggcggagggaccaaactgaccgtccta</td>

<td>10Ε4 ٧1</td><td> 191</td><td>ggcacctcagcctccctggccatcagtggactccagtctgaggatgaggctgatttttactgtgcagcg</td>

<td>IIDIIVt 11H9٧L</td><td> 192</td><td>aaacCcatctttaggaateatcagcggccctcgggtcccgaccgcttccggeccaagtct</td>

<td>1Η7 Vl</td><td> 193</td><td>aaactcctcatctttaggagtaatcagcggccctcaggggtccctgaccgattctctggctccaagtct</td>

119

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>SEQ ID NO.</td><td>Nucleic acid sequence</td>

<td>9F5Vl</td><td> 194</td><td> =</td>

<td>3B6٧l</td><td> 195</td><td>ggagacagcctcagaagtttttatgcaagclggtaccagcagaagccaggacaggcccctgtactt gacagcagtgtttaccatctggtactcggcggagggaccaaggccgtccta</td>

<td>3Β6 Vh</td><td> 196</td><td>acgacacggccgtgatttCgtgcgagagatcaaatgagtaltatatgcttoggggagtttttccccctt</td>

<td>10E4٧h</td><td> 197</td><td>acggtaiggacgtctggggccaagggaccacggtcaccgtcictagt</td>

<td>32Η8 Vh</td><td> 198</td><td>aaggcttctggatacaccttcaccgcctactatttacactgggtgcgacaggcccctggacaagggct tgagtggatgggatggatcaaccctcacagtggtggcacaaactatgcacagaagtttcagggcag gaaccctggteaccgtctctagt</td>

120

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>SEQID NO.</td><td>Nucleic acid sequence</td>

<td>33Β5 Vh</td><td> 199</td><td>gacagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtctacagtat aacacttacccgctcactttcggcggagggaccaaggtggagatcaag</td>

<td>IIDIIVh</td><td> 200</td><td>¹::: ح :::</td>

<td>9F5 Vh</td><td> 201</td><td>ΞΞΞΞ ==</td>

<td>11Η9 Vh</td><td> 202</td><td>تتتتة</td>

<td>1Η7 Vh</td><td> 203</td><td></td>

121

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>SEQ ID NO.</td><td>Nucleic acid sequence</td>

<td>13H2Vh</td><td> 204</td><td>acacggggtattacgtgcgagagaaggggtgtctggcagttogccgtatagcatcagctggtac</td>

<td>2Ε7 ٧ H</td><td> 205</td><td>agocc gttcacCttagcagcatgocatgagctgggtccgccaggctccagggaaggggctg</td>

<td>3C8Vh 12Ε8 Vh 5F5٧h</td><td> 206</td><td>caKB ^ ag-a.gàa ^ l ؛ Hlo ^ mt٠a.H ^^^ agga</td>

<td>4E4Vh 9D4٧h IEIIVh</td><td> 207</td><td>taoggtatggccgtctggggccaagggacacggtcaccgtcagt</td>

<td>12G8 Vh</td><td> 208</td><td>cagcctctggattcaccttcagtagctttggcatgcattgggtccgccaggctccaggcaaggggctg tacggtctggccgtctggggccaagggaccacggtcagctatagt</td>

122

ΜΑ 32982Β1

A-1472-WO-PCT

<td>Reference</td><td>SEQID NO.</td><td>Nucleic acid sequence</td>

<td>4H6Vh</td><td> 209</td><td>gggaacccggtcaccgtctctagt</td>

<td>32Η7 Vh</td><td> 210</td><td>gtatggacgtctggggccaagggaccacggtcaccgtctctagt</td>

<td>33Ε4 Vh</td><td> 211</td><td>tcaccgcagt</td>

Table 9 shows SEQ ID NOs of examples of nucleic acid sequences encoding full heavy and light chains, as well as heavy and light chain variable regions, of examples of isolated antigen-binding proteins, specifically the binding proteins. CGRP R, described here.

Table 9 - Example of nucleic acid sequence of HC, LC, Vh and Vl SEQ ID NOs

<img file="MA32982B1_D0013.tif" />

123

ΜΑ 32982Β1

A-1472-WO-PCT

<td>2Ε7</td><td> 175</td><td> 205</td><td> 226</td><td> 244</td>

<td>13Η2</td><td> 176</td><td> 204</td><td> 239</td><td> 257</td>

<td>4Η6</td><td> 178</td><td> 209</td><td> 230</td><td> 248</td>

<td>3C8</td><td> 179</td><td> 206</td><td> 228</td><td> 246</td>

<td>5F5</td><td> 180</td><td> 206</td><td> 231</td><td> 249</td>

<td>12Ε8</td><td> 181</td><td> 206</td><td> 237</td><td> 255</td>

<td>1Ε11</td><td> 186</td><td> 207</td><td> 224</td><td> 242</td>

<td>4Ε4</td><td> 187</td><td> 207</td><td> 229</td><td> 247</td>

<td>9D4</td><td> 188</td><td> 207</td><td> 232</td><td> 250</td>

<td>12G8</td><td> 189</td><td> 208</td><td> 238</td><td> 256</td>

<td>10Ε4</td><td> 191</td><td> 197</td><td> 234</td><td> 252</td>

<td>IIIDII</td><td> 192</td><td> 200</td><td> 235</td><td> 253</td>

<td>11Η9</td><td> 192</td><td> 202</td><td> 236</td><td> 254</td>

<td>1Η7</td><td> 193</td><td> 203</td><td> 225</td><td> 243</td>

<td>9F5</td><td> 194</td><td> 201</td><td> 233</td><td> 251</td>

<td>3Β6</td><td> 195</td><td> 196</td><td> 227</td><td> 245</td>

<td>32Η7</td><td> 182</td><td> 210</td><td> 240</td><td> 258</td>

<td>32Η7 CS</td><td> 183</td><td> 210</td><td> 241</td><td> 258</td>

<td>32Η8</td><td> 185</td><td> 198</td><td></td><td></td>

<td>33Β5</td><td> 177</td><td> 199</td><td></td><td></td>

<td>33Ε4</td><td> 184</td><td> 211</td><td></td><td></td>

<td>34Ε3</td><td> 190</td><td> 212</td><td></td><td></td>

Nucleic acids encoding certain antigen-binding proteins, or parts thereof (eg, full length antibody, heavy or light chain, variable domain, or CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3) can be isolated from B cells of mice that have been immunized with CGRP R or immunogenic components of

124

ΜΑ 32982Β1

A-1472-WO-PCT this, for example, by immunization with full-length CGRP R (including both CRLR and RAMP1), with the extracellular domain of CGRP R (including the extracellular domains of CRLR and RAMP1), with whole cells expressing CGRP R, with membranes prepared from cells expressing CGRP R, with fusion proteins, for example, fusions of Fc, including CRLR, RAMP1 (or their exttacellular domains) fused to Fc, and other methods known to those skilled in the art, for example, as described in Examples 1-3 given herein. Nucleic acid can be isolated by conventional methods such as polymerase chain reaction (PCR). Phage display is another example of a known technique by which derivatives of antibodies and other antigen-binding proteins can be prepared. In one approach, polypeptides which are components of an antigen-binding protein of interest are expressed in any suitable recombinant expression system, and the expressed polypeptides may assemble to form antigen-binding protein molecules.

The nucleic acids shown in Tables 7-9 are only examples. Due to the degeneracy of the genetic code, each polypeptide sequence shown in Tables 2-5 or described elsewhere herein is also encoded by a large number of other nucleic acid sequences in addition to those provided. Those of ordinary skill in the art will appreciate that the present application therefore gives an adequate written description and gives all the possibilities of degenerate nucleotide sequences encoding each protein binding an antigen.

The invention further relates in one aspect to nucleic acids which hybridize to other nucleic acids (e.g. nucleic acids comprising a nucleotide sequence shown in Table 7, Table 8, Table 9 and / or SEQs. ID NOs: 224258) under special hybridization conditions. Nucleic acid hybridization methods are well known to those skilled in the art; see, e.g., Current Protocols in Molecular Biology, John Wiley &amp; Sons, Ν.Υ. (1989), 6.3.1-6.3.6. As defined here, moderately stringent hybridization conditions correspond to a prewash solution containing sodium chloride / sodium citrate (SSC) 5x, SDS 0.5%, EDTA 1.0 mM (pH8.0), hybridization buffer at about 50 ٠/٠ formamide, SSC 6x, and a hybridization temperature of 55 ٠c (or other similar hybridization solutions, such as a solution containing about 50 ٠/٥ formamide, with a hybridization temperature of 42 ٠C), and washing conditions of 60٥c, in 0.5x SSC, 0.1% SDS. Stringent hybridization conditions hybridize in 6x SSC at 45 ٠c, followed by one or more washes in Ojlx SSC, 0.2 ٥/٥ SDS at 68 ٥c. In addition, those skilled in the art can manipulate the conditions

125

ΜΑ 32982Β1

A-1472-WO-PCT hybridization and / or washing to increase or decrease the stingency of hybridization such that nucleic acids comprising sequences which are at least 65 ٥/٠, 70 ٥/٥ identical , 75%, 80 ٠ / ο, 85٥ / ο, 90%, 95٠ / ο, 98% or 99% to each other remain hybridized to each other.

The basic parameters affecting the choice of hybridization conditions and guidelines for developing suitable conditions are present, for example, by Sambrook, Fritsch, and Maniatis (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν.Υ., supra; and Curent Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley &amp; Sons, Inc., Chapters 2.10 and 6.3-6.4), and can be readily determined by one of ordinary skill in the art from , for example, the length and / or the base composition of the nucleic acid.

Changes can be introduced by mutation in a nucleic acid, thereby making changes in the amino acid sequence of a polypeptide (eg, an antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known to those skilled in the art. In one embodiment, one or more residues are modified using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, random mutagenesis. Regardless of how it is prepared, a mutant polypeptide can be expressed and screened for a desired property.

Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide it encodes. For example, substitutions resulting in amino acid substitutions can be made on non-essential amino acid residues. One or more mutations can also be introduced into a nucleic acid which selectively modifies the biological activity of a polypeptide which it encodes. For example, the mutation can quantitatively or qualitatively alter biological activity. Examples of quantitative changes include increasing, reducing, or eliminating activity. Examples of qualitative changes include changing the specificity for the antigen of an antibody. In one embodiment, nucleic acid encoding any antigen-binding protein described herein can be mutated to alter the amino acid sequence using molecular biology techniques which are well established in the art.

Another aspect relates to nucleic acid molecules which are suitable for use as primers or hybridization probes for the detection of acid sequences.

126

ΜΑ 32982Β1

Nucleic A-1472-WO-PCT. A nucleic acid molecule may comprise only a part of a nucleic acid sequence encoding a full length polypeptide, for example, a fragment which can be used as a probe or primer or a fragment encoding an active part (eg, a CGRP R binding portion of a polypeptide.

Probes based on the sequence of a nucleic acid can be used to detect nucleic acid or similar nucleic acids, for example, transcripts encoding a polypeptide. The probe can include a marker group, eg, a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme co-factor. These probes can be used to identify a cell which expresses the polypeptide.

Another aspect relates to vectors comprising a nucleic acid encoding a polypeptide or a part thereof (eg, a fragment containing one or more CDRs, or one or more variable region domains). Exemplary vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors, eg, recombinant expression vectors. Recombinant expression vectors comprise nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Recombinant expression vectors comprise one or more regulatory sequences, selected depending on the host cells to be used for expression, which are operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct the constitutive expression of a nucleotide sequence in many types of host cells (eg, SV40 early gene activator, Rous sarcoma virus promoter, and cytomegalovirus promoter), those which direct expression of the nucleotide sequence only in certain host cells (eg, tissue specific regulatory sequences, see, Voss et al., 1986, Trends Biochem. Sci. 1 : 287, Maniatis et al., 1987, Science 236: 1237, incorporated herein by reference in their entirety), and those which direct the inducible expression of a nucleotide sequence in response to a particular treatment or condition (e.g. , the metallothionin promoter in mammalian cells and the promoter that responds to tet and / or that responds to streptomycin in prokaryotic and eukaryotic systems (see, id.). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of protein expression desired, etc. Expression vectors can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides encoded by nucleic acids described herein.

127 /

ΜΑ 32982Β1

A-1472-WO-PCT

Another aspect relates to host cells into which the recombinant expression vector has been introduced. A host cell can be any prokaryotic cell (eg, E. coli) or a eukaryotic cell (eg, yeast, insect, or mammalian (eg, CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. For the stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of the cells can integrate the foreign DNA into their genome. In order to identify and select those cells which have effected the integration, a gene which encodes a selectable marker (for example, for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to substances, such as G418, hygromycin, and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by selection for the substance (eg, cells which have incorporated the selectable marker survive, while the rest die), among other methods.

Preparation of antigen-binding proteins

The non-human antibodies which are provided herein may be, for example, derived from any animal producing antibodies, such as a mouse, rat, rabbit, goat, donkey, or non-human primate (such as a monkey ( for example, cynomolgus or rhesus monkey) or a great ape (for example, chimpanzee)). Non-human antibodies can be used, for example, in in vitro cell culture and in cell culture applications, or any other application in which an immune response to the antibody does not occur or is insignificant. , can be prevented, is not a problem, or is desired. In some embodiments, the antibody can be produced by immunizing animals using methods known to those skilled in the art, as described above and / or in Examples 1-3 below. The examples describe the production of anti-CGRP Ren antibodies using three different immunogen preparations - (i) whole cells expressing full length versions of two major components of CGRP R-RAMP1 and CRLR; (ii) membrane extracts from these cells; and (iii) soluble CGRP R obtained by co-expression and purification of the N-terminal extracellular domains of CRLR and RAMPL. Anticoips can be polyclonal, monoclonal, or they can be synthesized in host cells by expression of recombinant DNA. Fully human antibodies can be prepared as described above by immunizing transgenic animals containing loci.

128 /

ΜΑ 32982Β1

A-1472-WO-PCT human immunoglobulin or by selecting a phage display library which expresses a repertoire of human antibodies.

Monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, eg, the standard somatic cell hybridization technique of Kohler and Milstein, 1975, Nature 256: 495. Other techniques for producing monoclonal antibodies can also be employed, for example viral or oncogenic transformation of B lymphocytes. Another suitable animal system for preparing hybridomas is the murine system which is a very well established process. Immunization protocols and techniques for isolating splenocytes immunized for fusion are known to those skilled in the art and exemplary approaches are described in the Examples below. For these protocols, B cells from immunized mice are typically fused with an appropriate immortalized fusion partner, such as a murine myeloma cell line. If desired, rats or other mammals can be immunized instead of mice and the B cells from these animals can be fused with the murine myeloma cell line to form hybridomas. A myeloma cell line from a source other than a mouse can also be used. Fusion protocols for preparing hybridomas are also well known.

The single chain antibodies which are provided can be formed by linking variable domain fragments of heavy and light chains (Fv region) through an amino acid bridge (short peptide linker) resulting in a single peptide chain. These single chain Fvs can be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (Vl and Vh). The resulting polypeptides can fold on themselves to form antigen-binding monomers, or they can form multimeres (eg, dimers, trimers or tetramers) depending on the length of the flexible linker between the two variable domains (Kortt et al. , 1997, Prot. Eng. 10: 423; Kortt et al., 2001, Biomol. Eng. 108-95: ؤإ). By combining different peptides including V1 and Vh, one can form multimeric scFvs which bind to different epitopes (Kriangkum et al., 2001, Biomol. Eng. S: 31-40). Techniques developed for the production of single chain antibodies include those described in US Patent No. 4,946,778; Bird, 1988,

Science 242.:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 3: 5879; Ward et al., 1989, Nature 334: 544, de Graaf et al., 2002, Methods Mol Biol. 3: 379-387. The antibody-derived single chain antibodies provided include, but are not limited to, scFvs comprising the variable domain combinations of heavy and light chain variable regions

129

<img file="MA32982B1_D0014.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT shown in Table 3, or heavy and light chain variable domain combinations which include the CDRs shown in Tables 4Α and 4Β.

Antibodies provided herein which belong to one subclass can be replaced with antibodies from another subclass using subclass exchange methods. Thus IgG antibodies can be derived from an IgM antibody, for example, and vice versa. These techniques allow the preparation of new antibodies which possess the antigen binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with a different isotype or subclass of antibody. than that of the parent antibody. Recombinant DNA techniques can be used. Cloned DNA encoding particular antibody polypeptides can be employed in these methods, for example, DNA encoding the constant domain of an antibody of the desired isotype. See, for example, Lantto et al., 2002, Methods Mol. Biol. 178: 303-316

Therefore, the antibodies which are provided include those comprising, for example, the combinations of variable domains described above, having the desired isotype (eg, IgA, IgG1, IgG2, IgG3, IgG4, IgE, and IgD) as well. as Fab or F (ab ') 2 fragments thereof. Further, if IgG4 is desired, it may be desirable to introduce a point mutation (CPSCPCPPCP) into the hinge region, as described in Bloom et al., 1997, Protein Science 6: 407, incorporated herein as reference), to attenuate the tendency for the formation of intra-heavy chain disulfide bonds which can lead to heterogeneity in IgG4 antibodies.

In addition, techniques for deriving antibodies having different properties (ie varying affinities for the antigen to which they bind) are also known. One such technique, referred to as chain rearrangement, involves the presentation of gene repertoires of variable domain immunoglobulins on the surface of filamentous bacteriophages, often referred to as phage display. The chain rearrangement has been used to prepare high affinity antibodies against the 2phenyloxazol-5-οηε hapten, as described by Marks et al., 1992, BioTechnology 0: 779.

Conservative modifications can be performed on the heavy and light chain variable regions described in Table 3, or the CDRs described in Tables 4Α and 4Β (and corresponding to modifications on the coding nucleic acids) to produce a CGRP R binding protein. having certain desirable functional and biochemical characteristics. Methods of obtaining these modifications are described above.

The CGRF antigen-binding proteins can then be modified in various ways. For example, if they are to be used for therapeutic purposes, they may

130

ΜΑ 32982Β1

A-1472-WO-PCT be conjugated with polyethylene glycol (pegylees) in order to prolong the half-life in serum or to improve the delivery of proteins. The V region of antibodies or fragments thereof can also be fused with the Fc region of a different anticoips molecule. The Fc region used for this purpose can be modified so that it does not bind to complement, thereby reducing the likelihood of inducing cell lysis in the patient when the binding protein is used as a therapeutic agent. In addition, the subject anticoips or functional fragments thereof may be conjugated to human serum albumin in order to increase the serum half-life of the antibody or its antigen binding moiety. Another useful binding partner for the binding of antigen-binding proteins or fragments thereof is transthyretin (TTR). TTR has the ability to form a tetramer, and an antibody-TTR binding protein can therefore form a multivalent antibody which can increase its binding avidity.

Significant changes in the functional and / or biochemical characteristics of the antigen-binding proteins described herein can also be achieved by creating substitutions in the amino acid sequence of heavy and light chains whose effects differ significantly on the maintenance of (a) protein. stnictorium of the molecular skeleton in the region of the substitution, for example a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. A "conservative amino acid substitution" can involve a substitution of a native amino acid residue for a non-native residue which has little or no effect on the polarity or charge of the amino acid residue in this. position. See Table 4, supra. In addition, any native residue in the polypeptide can also be replaced by an alanine, as previously described for the systematic mutagenesis of each residue to alanine (alanine scanning mutagenesis).

Amino acid substitutions (whether conservative or non-conservative) in subject antibodies can be made by those skilled in the art by application of routine techniques. The amino acid substitutions can be used to identify important residues of the antibodies provided herein, or to increase or decrease the affinity of these antibodies for human CGRP R or to modify the binding affinity of other antigen-binding proteins described. here.

Methods of Expression of Antigen Binding Proteins

Expression systems and constructs in the form of plasmids, expression vectors, registration or expression cassettes which comprise at least one

131

<img file="MA32982B1_D0015.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT polynucleotide described above are also provided herein, as well as host cells comprising such expression systems or constructs.

The antigen-binding proteins provided herein can be prepared by any of several conventional techniques. For example, the CGRP R antigen binding proteins can be produced by recombinant expression systems, using any technique known to those skilled in the art. See, for example. Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Kennet al. (eds.) Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν.Υ. (1988).

Antigen-binding proteins can be expressed in hybridoma cell lines (e.g., particular antibodies can be expressed in hybridomas) or in cell lines other than hybridomas. Expression constructs encoding the antibodies can be used to transform a microbial, insect or mammalian host cell. Transformation can be carried out using any known method of introducing polynucleotides into a host cell, including, for example, packaging the polynucleotide into a virus or bacteriophage and transducing a host cell with the construct by protocols. transfection known to those skilled in the art, as illustrated by US Pat. Nos. 4,399,216;

No. 4,912,040; No. 4,740,461; No. 4,959,455. The optimal transformation protocol used will depend on the type of host cell to be transformed. Methods of introducing heterologous polynucleotides into mammalian cells are well known to those skilled in the art and include, but are not limited to, dextran mediated transfection, calcium phosphate precipitation, polybene mediated transfection, fosion of protoplasts, electroporation, encapsulation of the polynucleotide (s) in liposomes, mixing nucleic acid with positively charged lipids and direct microinjection of DNA into nuclei.

Recombinant expression constructs generally comprise a nucleic acid molecule encoding a polypeptide comprising one or more of the following: one or more CDRs provided herein; a light chain constant region; a light chain variable region; a heavy chain constant region (eg. C11, c and or C); and / or another structural part of a protein binding the CGRE R antigen. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In one embodiment, the heavy or light chain constant region is added to the C-terminus of the specific heavy or light chain variable region.

132

ΜΑ 32982Β1

A-1472.WO-PCT of Tanti-CGRP R and is ligated into an expression vector. The vector is generally chosen to be functional in the particular host cell employed (i.e., the vector is compatible with the machinery of the host cell, allowing amplification and / or expression of the gene. to take place). In some embodiments, vectors are used which employ protein-fragment complementation assays using protein reporters, such as dihydrofolate eductase (see, e.g., US Patent No. 6,270,964, which is incorporated herein by reference. ). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies or BD Biosciences (formerly "Clontech"). Other vectors useful for the cloning and expression of antibodies and fragments include those described in Bianchi and McGrew, 2003, Biotech. Biotechnol. Bioeng. £ 4: 43944, which is incorporated herein by reference. Other suitable expression vectors are discussed, for example, in Methods Enzymol., Vol. 185 (DV Goeddel, ed.), 1990, New York: Academie Press.

Generally, expression vectors used in any of the host cells contain sequences for maintenance of plasmids and for cloning and expression of exogenous nucleotide sequences. These sequences, collectively referred to as "flanking sequences" in certain embodiments, generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing an acceptor and donor splice site, a sequence encoding a leader sequence for the secretion of polypeptide, a ribosome binding site, a polyadenylation sequence, a polylinker region for the insertion of a nucleic acid encoding the polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

Optionally, the vector may contain a sequence encoding a "tag", ie an oligonucleotide molecule located at the 5 'or 3' end of the sequence encoding the CGRP R binding protein; the oligonucleotide sequence encodes a polyHis (for example a hexaHis), or another "tag", such as FLAG®, HA (influenza virus hemaglutinin), or myc, for which there are commercially available antibodies. This tag is generally fused to the polypeptide after expression of the polypeptide, and can serve as a means of affinity purification or detection of the CGRP R binding protein from the host cell. Affinity purification can be carried out, for example, by column chromatography using anti-clips against the label as a template.

133

ΜΑ 32982Β1

Affinity A-1472-WO-PCT. Optionally, the tag can then be removed from the CGRP R binding protein by various means such as using certain peptidases for cleavage.

The flanking sequences can be homologous (that is to say come from the same species and / or strain as that of the host cell), heterologous (that is to say come from a species different from that of the host cell). host cell or strain), hybrid (that is, a combination of flanking sequences from several sources), synthetic or native. Thus, the source of a flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by the machinery of, the host cell.

Flanking sequences useful in vectors can be obtained by any of several methods well known to those skilled in the art. Generally, flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion and therefore can be isolated from the appropriate source tissue using the appropriate restriction endonucleases. In some cases, the entire nucleotide sequence of a flanking sequence can be known. The flanking sequence can then be synthesized using the methods described herein for the synthesis or cloning of nucleic acids.

Whether all or only part of the flanking sequence is known, it can be obtained by means of a polymerase chain reaction (PCR) and / or by screening a genomic library with a suitable probe such as an oligonucleotide and / or or a fragment of a flanking sequence of the same species or another. When the flanking sequence is not known, a DNA fragment containing a flanking sequence can be isolated from a larger piece of DNA which may contain, for example, one coding sequence or even another or other genes. Isolation can be done by restriction endonuclease digestion to produce the appropriate DNA fragment, followed by isolation by agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods. known to those skilled in the art. The choice of suitable enzymes to achieve this objective will be obvious to those of ordinary skill in the art.

An origin of replication is generally one of the expression vectors purchased commercially, and the origin contributes to the amplification of the vector in a host cell. If the vector of choice does not contain an origin site of replication, it can be chemically synthesized from a known sequence, and ligated into the vector. For example, the origin of replication of plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for

لم

134

ΜΑ 32982Β1

A-1472.WO-PCT most Gram-negative bacteria, and various viral origins (eg, SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus such as HPV or BPV) are useful for cloning vectors in mammalian cells. Usually, the replication origin component is not required for mammalian expression vectors (eg, the SV40 origin is often used only because it contains the early promoter of the virus).

A transcription termination sequence is generally located 3 'to the end of a region encoding a polypeptide and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a GC-rich fragment followed by a poly-Τ sequence. Although it is easy to clone the sequence from a library or to purchase it commercially as part of a vector, it can also be easily synthesized using nucleic acid synthesis methods such as those described here.

A selectable marker gene encodes a protein necessary for the survival and growth of a host cell cultured in a selective growth medium. Typical selection marker genes encode proteins which (a) confer resistance to antibiotics or other toxins, eg, ampicillin, tetracycline or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) provide essential nutrients not available in a complex or defined medium. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. A neomycin resistance gene can also be advantageously used for selection in prokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene to be expressed. Amplification is the process by which genes which are necessary for the production of a protein essential for cell growth or survival are reiterated in tandem in the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. The transformed mammalian cells are placed under selection pressure, only the transformants being specially adapted to survive thanks to the selectable gene present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is gradually increased, which allows amplification of the selectable gene.

135

ΜΑ 32982Β1

A-1472-WO-PCT and DNA which encodes another gene, such as an antigen-binding protein that binds CGRP R. Thus, increased amounts of a polypeptide such as an antigen-binding protein are synthesized from of DNA amplifies.

A ribosome binding site is generally required for the initiation of mRNA translation and is characterized by a Shine-Dalgamo sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is generally located 3 'from the promoter and 5' from the coding sequence of the polypeptide to be expressed.

In some cases, for example when glycosylation is desired in the eukaryotic host cell expression system, the various pre- or ρο-sequences can be manipulated in order to improve glycosylation or yield. For example, one can modify the peptidase cleavage site of a particular signal peptide, or add prosequences, which can also affect glycosylation. The final protein product may include, in position -1 (relative to the first amino acid of the mafirre protein), one or more additional expression-linked amino acids, which may not have been completely removed. For example, the final protein product may contain one or more amino acid residues which are found in the peptidase cleavage site, attached at the amino terminus. The use of certain enzymatic cleavage sites can also provide a truncated form of the desired polypeptide if the enzyme cuts into this region of the mature polypeptide.

Expression and cloning will generally involve a promoter which is recognized by the host organism and operably linked to the molecule encoding a CGRP R binding protein. Promoters are untranscribed upstream sequences (i.e. say in 5 ') of the start codon of a structtrral gene (generally about 100 to 1000 bp) which control transcription of the structural gene. Promoters are usually grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of DNA transcription under their control in response to certain changes in cultivation conditions, such as the presence or absence of a nutrient or a change in temperature. The constitutive promoters, for their part, uniformly transcribe a gene to which they are operably linked, that is to say with little or no control over the expression of the gene. A large number of promoters, recognized by various potential host cells, are well known. A suitable promoter is operably linked to DNA encoding a heavy chain or a light chain comprising a CGRP R binding protein by removing the promoter from the source DNA by restriction enzyme digestion and insertion of the desired promoter sequence into it. the vector.

136

ΜΑ 32982Β1

A-1472-WO-PCT

Promoters suitable for use in yeast containers are well known to those skilled in the art. Yeast activators are advantageously used with yeast promoters. Promoters suitable for use with mammalian host cells are well known and include, but are not limited to, those obtained from genomes of viruses such as polyomaviras, fowl pox virus, adenovirus (such as adenovirus 2 ), bovine papillomaviras, avian sarcoma virus, cytomegalovirus, retro viras, hepatitis B virus, and simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example heat shock protein promoters and the actin promoter.

Other promoters which may be of interest include, but are not limited to: the SV40 early promoter (Benoist and Chambon, 1981, Nature 0: 304-310); the CMV promoter (Thomsen et al., 1984, Proc. Natl. Acad. USA s: 659-663); the promoter contained in the long terminal repeat of the Rous sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 26: 1444-1445); the promoter and regulatory sequences of the metallothionin gene (Prinster et al., 1982, Nature 226: 3942); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 26: 3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA O: 21-25). The following animal transcription control regions, which also show tissue specificity and have been used in transgenic animals, are also of interest: the elastase I gene control region which is active in cells pancreatic acinar cells (Swift et al., 1984, Cel 66: 639-646; Omitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 0: 399-409; MacDonald, 1987, Hepatology 2: 425-515); the insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 3: 115-122); the control region of the immunoglobulin gene which is active in lymphoid cells (Grosschedl et al., 1984, Cell 1.641-6¾% 'ةعاتلخ ١ et al., \ 1, Nature Ί6 ١٦-Ί -, Mexsàî and αΐ., π, Mol. Cell. Biol. 2: 1436-1444); the control region of the mouse mammary tamer virus gene which is active in testis, breast, lymphoid and mast cells (Leder et al., 1986, Cell 46: 485-495); the control region of the albumin gene which is active in the liver (Pinkert et al., 1987, Genes andDevel. 1: 268-276); the control region of the alphafeto-protein gene which is active in the liver (Kramlauf et al., 1985, Mol. Cell. Biol. 6 : 1639-1648; Hammer et al., 1987, Science 266: 53-58); the control region of the antitrypsin gene

137

ΜΑ 32982Β1

A-1472-WO-PCT alpha-1 which is active in the liver (Kelsey et al., 1987, Genes andDevel. 1: 161-171); the control region of the beta-globin gene which is active in myeloid cells (Mogram et al., 1985, Nature 3: 338-340; Kollias et al., 1986, Cell 42: 89-94); the control region of the myelin basic protein gene, which is active in oligodendrocytic cells in the brain (Readhead et al., 1987, Ce 43: 703-712); the control region of the myosin light chain 2 gene which is active in skeletal muscle (Sani, 1985, Nature 314: 283286); and the control region of the gonadorophin releasing hormone gene which is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

An activator sequence can be inserted into the vector to increase transcription of DNA encoding a light or heavy chain comprising a human CGRP R binding protein by higher eukaryotes. Activators are cis-acting pieces of DNA, usually about 10-300 bp in length, which act on the promoter to increase transcription. The activators are relatively independent of orientation and position, having been observed at the 5 'and 3' positions of the transcription unit. Several activator sequences available on mammalian genes are known (eg, globin, elastase, albumin, alpha-fetoprotein and insulin). Usually, however, a virus activator is used. SV40 activator, cytomegalovirus early promoter activator, polyoma activator and adenovirus activators known to those skilled in the art are examples of activation of eukaryotic promoters. Although an activator can be placed in the vector 5 'or 3' of a coding sequence, it is generally located at a site 5 'of the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector, in order to promote the extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides which are functional in mammalian host cells include the following: the signal sequence for the interleukin 7 (IL-7) receptor described in US Patent No. 4,965,195; the signal sequence for the interleukin 2 receptor described in Cosman et ٥ /., 1984, Nature 312: 768; the signal sequence for the interleukin 4 receptor described in EP Patent No. 0367 566; the signal sequence for the interleukin-1 type I receptor disclosed in US Patent No. 4,968,607; the signal sequence for the interleukin 1 type II receptor described in EP Patent No. 0 460 846.

The expression vectors which are provided are constructed from a starting vector such as a commercially available vector. These vectors may or may not contain all

138

ΜΑ 32982Β1

A-1472-WO-PCT the desired flanking sequences. When one or more of the flanking sequences described herein are not already present in the vector, they can be individually obtained and ligated into the vector. Methods used to obtain each flanking sequence are well known to those skilled in the art.

After constructing the vector and inserting a nucleic acid molecule encoding a light chain and a heavy chain, or a light chain and a heavy chain comprising a sequence binding the CGRP R antigen into the appropriate site of the vector, the vector can terminate. be inserted into a suitable host cell for amplification and / or expression of the polypeptide. Transformation of a selected host cell by an expression vector for an antigen-binding protein can be carried out by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, etc. microinjection, lipofection, transfection mediated by DEAEdextran or other known techniques. The method chosen will depend in part on the type of host cell to be used. These and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al, 2001, supra.

A host cell, when cultured under suitable conditions, synthesizes an antigen-binding protein which can then be collected from the culture medium (if the host cell secretes it into the culture medium) or directly from the culture medium. producer host cell (if not secreted). Selection of an appropriate host cell depends on a variety of factors, such as desired expression levels, modifications of the polypeptide that are desirable or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a molecule biologically. active.

Mammalian cell lines available as hosts for expression are well known to those skilled in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including without limitation. Limit it, Chinese hamster ovary cells (CHO), HeLa cells, newborn hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and many other cell lines. In some embodiments, cell lines can be selected by determining which cell lines have high expression levels and constitutively produce antigen-binding proteins with CGRP R binding properties. In another embodiment, a cell line of the B cell line which does not make its own antibody but has the ability to produce and secrete a heterologous antibody can be chosen.

139

<img file="MA32982B1_D0016.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

Use of CGRP antigen-binding proteins for diagnostic and therapeutic uses

The antigen-binding proteins are useful for detecting CGRP R in biological samples and the identification of cells or tissues that produce CGRP R. For example, CGRP antigen-binding proteins can be used in diagnostic tests, for example, diagnostic tests. binding assays to detect and / or quantify CGRP R expresses in a tissue or cell. Antigen-binding proteins which specifically bind CGRP R can also be used in the treatment of CGRP R related diseases in a patient in need thereof. Additionally, CGRP R antigen binding proteins can be used to inhibit CGRP R formation of a complex with its CGRP ligand, thereby modulating the biological activity of CGRP R in a cell or tissue. Examples of activities which can be modulated include, but are not limited to, inhibiting vasodilation and / or reducing neurogenic inflammation. Antigen-binding proteins that bind to CGRP R may therefore modulate and / or block the interaction with other binding compounds, and thus may have therapeutic use in ameliorating CGRP R-related diseases.

Idications

A disease or condition associated with human CGRP R includes any disease or condition the onset of which in a patient is caused, at least in part, by the interaction of CGRP R with its ligand, CGRP. The severity of the disease or condition may also be increased or decreased by Interaction of CGRP R with CGRP. Examples of diseases or conditions that can be treated with the antigen-binding proteins described herein include headache, such as cluster headache, migraine, including migraine headache, chronic pain, type II sugar diabetes, severe pain. inflammation, e.g. neurogenic inflammation, cardiovascular disorders, and hemodynamic disturbance associated with endotoxemia and sepsis.

In particular, the antigen-binding proteins described herein can be used to treat migraine, as an acute treatment initiated after the onset of the migraine attack and / or as a prophylactic treatment administered, for example, once a day, once a week. , twice a week, once a month, twice a month, twice a year, etc., to prevent or reduce the frequency and / or severity of symptoms, for example pain symptoms, associated with migraine attacks.

Diagnostic procedures

The antigen-binding proteins described herein can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or conditions.

140

<img file="MA32982B1_D0017.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT associated with CGRP. The invention also relates to methods of detecting the presence of CGRP R in a sample using immunohistochemical methods known to those skilled in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds RH Burdon et al. Ρ.Η. van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et a /., 1985,2. Ce / (Α ; 'ΰ /. 101: 976-985; Alkanen and ٥/٠١ 1987.2. Cell Biol. 105: 30873096). Detection of CGRP R can be performed in vivo or in vitro.

Diagnostic applications provided herein include the use of antigen binding proteins to detect expression of CGRP R and binding of ligands to CGRP R. Examples of methods useful in detecting the presence of CGRP R include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein is generally labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioactive isotopes or radionuclides (eg, 5H, 'c⁵ ل N, s, 9٥Y, Te, “'in,' 1, '5 (1), fluorescent groups (eg, FITC, rhodamine, lanthanide-type phosphors), enzymatic groups (eg, horseradish peroxidase, p-galactosidase , luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope labels). In some embodiments, the labeling group is coupled to the antigen-binding protein via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known to those skilled in the art and can be used.

In another aspect, an antigen-binding protein can be used to identify a cell or cells which express CGRP R. In a specific embodiment, the antigen-binding protein is labeled with a tag group and the binding of the protein. binding of a labeled antigen is detected. In another specific embodiment, the binding of the antigen-binding protein to CGRP R is detected in vivo. In another specific embodiment, the CGRP antigen binding protein is isolated and measured using techniques known to those skilled in the art. See, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual٠ New York: Cold Spring Harbor (ed. 1991 and supplements

141

ΜΑ 32982Β1

A-1472-WO-PCT periodicals); John E. Coligan, eri., 1993, Current Protocols In Immunology New York: John Wiley &amp; Sons.

Another aspect allows the detection of the presence of a test molecule which competes for binding to CGRP R with the antigen binding proteins of the invention. An example of such a test involves the detection of the amount of protein binding a free antigen in a solution containing an amount of CGRP R in the presence or absence of the test molecule. An increase in the amount of free antigen binding protein (i.e., antigen binding protein not bound to CGRP R) indicates that the tested molecule is able to compete for binding to CGRP R with the binding protein. an antigen.

In one embodiment, the antigen binding protein is labeled with a labeling group. The tested molecule can also be labeled and the amount of free test molecule is monitored in the presence and absence of an antigen binding protein.

Treatment methods: pharmaceutical formulations and routes of administration

Methods of using the antigen binding proteins are also provided. In some methods, an antigen binding protein is administered to a patient. The antigen-binding protein inhibits the binding of CGRP to human CGRP R.

Pharmaceutical compositions which comprise a therapeutically effective amount of one or a plurality of antigen binding proteins and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant are also provided. Additionally, methods of treating a patient, eg, migraine, by administering such a pharmaceutical composition are included. The term "patient" includes human patients.

Acceptable formulation materials are non-toxic to recipients at the doses and concentrations employed. In specific embodiments, concentrations comprising a therapeutically effective amount of human CGRP R antigen binding proteins are provided.

In some embodiments, acceptable formulation materials are preferably non-toxic to recipients at the doses and concentrations employed. In some embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, color. sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In these embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); of

142

<img file="MA32982B1_D0018.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogensulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); diluting agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or thydroxypropyl-beta-cyclodextrin); charges ; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); agents which improve stability (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol or sorbitol); administration vehicles; diluents; pharmaceutical excipients and / or adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

In some embodiments, the optimum pharmaceutical composition will be determined by those skilled in the art, depending, for example, on the intended route of administration, the format of administration and the desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In some embodiments, these compositions can influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antigen binding proteins described. In some embodiments, the vehicle or primary carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline

143

<img file="MA32982B1_D0019.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT or saline mixed with serum albumin are other examples of vehicles. In specific embodiments, the pharmaceutical compositions comprise Tris buffer at pH about 7.0-8.5, or acetate buffer at pH about 4.0-5.5, and may further comprise sorbitol or a substitute. appropriate. In certain embodiments, human CGRP R antigen-binding protein compositions can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form a lyophilized cake or an aqueous solution. Further, in certain embodiments, the human CGRP R antigen binding protein can be formulated as a lyophilizate using suitable excipients such as sucrose.

Pharmaceutical compositions can be selected for parenteral administration. The compositions can also be selected for inhalation or administration via the digestive tract, for example, orally. The preparation of these pharmaceutically acceptable compositions is within the skill of those skilled in the art.

The compounds of the formulation are preferably present in concentrations which are acceptable at the site of administration. In some embodiments, buffers are used to maintain the composition at physiological pH or slightly lower, generally within a pH range of about 5 to about 8.

When parenteral administration is contemplated, the therapeutic compositions can be provided in the form of a parenterally acceptable pyrogen-free aqueous solution of the desired human CGRP R binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the human CGRP R antigen-binding protein is formulated as a suitably stored sterile isotonic solution. In some embodiments, the preparation may involve formulating the desired molecule with an agent, such as injectable microspheres, bioerodable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes. , which can provide controlled or prolonged release of the product which can be administered by depot injection. In some embodiments, hyaluronic acid can also be used, and it has the effect of promoting a prolonged duration in the circulation. In some embodiments, implantable drug delivery devices can be used to introduce the desired antigen binding protein.

144

ΜΑ 32982Β1

A-1472-WO-PCT

Some pharmaceutical compositions are formulated for inhalation. In some embodiments, human CGRP R antigen-binding proteins are formulated as a dry powder for inhalation. In specific embodiments, solutions for inhalation of human CGRP R antigen-binding protein can also be formulated with a propellant for aerosol delivery. In some embodiments, solutions can be nebulized. Pulmonary administration and formulation methods therefor are described in detail in Patent Application No. PCT / US94 / 001875, which is incorporated herein by reference and describes pulmonary administration of chemically modified proteins. Some formulations can be administered orally. The human CGRP R antigen-binding proteins which are administered in this manner can be formulated with or without the carriers commonly used in the composition of solid dosage forms such as tablets and capsules. In some embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract where bioavailability is maximum and presystemic degradation is minimal. Other agents may be included to facilitate absorption of the human CGRP R antigen binding protein. Diluents, flavoring agents, low-melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and binders can also be employed.

Certain pharmaceutical compositions comprise an effective amount of one or more human CGRP R antigen binding proteins in admixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other suitable vehicle, solutions can be prepared in unit dosage form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents such as starch, gelatin or gum arabic; or lubricating agents such as magnesium stearate, stearic acid or talc.

Other pharmaceutical compositions will be obvious to those skilled in the art, including formulations of human CGRP R antigen binding proteins in sustained or controlled delivery formulations. Techniques for formulating various other means of sustained or controlled delivery, such as liposomal carriers, bioerodible particles or porous beads and delayed acting injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which is incorporated by reference and describes the release

145

ΜΑ 32982Β1

A-1472-WO-PCT controlled porous polymeric microparticles for the administration of pharmaceutical compositions. Sustained release preparations can comprise semi-permeable polymeric matrices in the form of shaped articles, for example, films or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (as described in US Patent No. 3,773,919 and European Patent Application Publication No. EP 058481, which are each incorporated by reference), copolymers of! -Glutamic acid and gamma-ethyl L-glutamate (Sidman et al.,

1983, Biopolymers 2547-556), poly (2-hydroxyethyl methacrylate) (Langer et al., 1981, j. Biomed. Mater. Res. 277-167: فإ and Langer, 1982, Chem. Tech.105- 98: ظ), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D (-) - 3-hydroxybutyric acid (European patent application publication No. EP 133,988). Sustained release compositions can also include liposomes which can be prepared by any of the methods known to those skilled in the art. See, for example, Eppstein e ؛ al., 1985, Proc. Natl. Acad. Sci. USA 3: 3688-3692; European patent application publications Nos.

EP 036,676; EP 088 046 and EP 143 949, incorporated referenced.

Pharmaceutical compositions used for administration in Vi'o are generally supplied as sterile preparations. Sterilization can be carried out on sterile filtration membranes. When the composition is lyophilized, sterilization by this method may be carried out before or after lyophilization and reconstitution.

Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are generally placed in a container provided with a sterile access port, for example, an intravenous solution bag or a vial provided with a stopper to be pierced by a hypodermic injection syringe.

In certain embodiments, cells expressing an antigen binding protein described herein are encapsulated for administration (see, Invest. Ophthalmol Vis Sci 43: 3292-3298, 2002 and Proc. Natl. Acad. Sciences 103: 3896-3901. , 2006).

In some formulations, an antigen-binding protein has a concentration of at least 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml , 80 mg / ml, 90 mg / ml, 100 mg / ml or 150 mg / ml. Some formulations contain buffer, sucrose, and polysorbate. An example of a formulation is a formulation containing 50,100 mg / ml of the antigen-binding protein, 5-20 mM sodium acetate acetate, 5-10 ٥/٥ w / v of sucrose, and 0.002 - 0.008 ٠ / ٥ w / v of polysorbate. Some formulations, for example, contain 65-75 mg / ml of an antigen binding protein in 911 mM sodium acetate buffer, 8-10% w / v sucrose, and 0.005-0.006% w / v polysorbate. The pH of some of these

146

<img file="MA32982B1_D0020.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT formulations is in the range of 4.5-6. Other formulations have a pH of 5.0-5.5 (eg, pH 5.0, 5.2, or 5.4).

Once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal or liquid. lyophilized or dehydrated powder. These formulations can be stored in a ready-to-use form or in a form which is reconstituted before use (eg, lyophilized). Single dose administration unit production kits are also provided. Some kits contain a first container containing a dried protein and a second container containing an aqueous formulation. In some embodiments, kits containing single and multiple compartment pe-filled syringes (eg, liquid syringes and lyosyringes) are provided. The therapeutically effective amount of a pharmaceutical composition containing a human CGRR R antigen binding protein to be employed will depend on the context and the therapeutic objectives. Those skilled in the art will recognize that dose levels for treatment will vary depending, in part, on the molecule delivered, the indication for which the human CGRP R antigen-binding protein is used, the route of administration. , and the dhnensions (body weight, body surface area or organ size) and / or condition (age and general health) of the patient. In some embodiments, the clinician can titrate the dose and modify the route of administration in order to obtain the optimal therapeutic effect.

A typical dose can be in the range of about 1 μg / kg up to about mg / kg or more, depending on the factors mentioned above. In specific embodiments, the dose can range from about 10 μg / kg up to about 30 mg / kg, optionally from 0.1 mg / kg up to about 30 mg / kg, or still about 0.3 mg / kg up to about 20 mg / kg. In some applications the dose is between

0.5 mg / kg and 20 mg / kg. In some cases, an antigen-binding protein is assayed at 0.3 mg / kg,

0.5mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, or 20 mg / kg. The dosage regimen in some treatment regimens is a dose of 0.3 mg / kg per week, 0.5 mg / kg per week, 1 mg / kg per week, 3 mg / kg per week, 10 mg / kg per week , or 20 mg / kg per week.

The frequency of dosing depends on the pharmacokinetics of the particular human CGRP R antigen binding protein in the formulation used.

Typically, a clinician will administer the composition until a dose is reached which exerts the desired effect. The composition may therefore be administered in a single dose, or in two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion, via the medium. a device

147

ΜΑ 32982Β1

A-1472-WO-PCT implantation or catheter. Appropriate doses can be determined by the use of appropriate dose response data. In some embodiments, the antigen binding proteins can be administered over an extended period of time. Chronic administration of an antigen-binding protein reduces the immune or allergic response commonly associated with antigen-binding proteins that are not completely human, for example an antibody raised against a human antigen in a non-human animal, for example an antibody entirely human or a non-human antibody produced in a non-human species.

The route of administration of the pharmaceutical composition is according to known methods, for example, oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, Intraportal, or intralesion injection; by sustained release systems or by implantation devices. In some embodiments, the compositions may be administered by bolus injection or continuously by infosion or through an implantation device.

The composition can also be administered topically, by implanting a membrane, sponge or other suitable material onto which the desired molecule has been absorbed or encapsulated. In some embodiments, when an implantation device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffosion, timed-release bolus, or administration. keep on going.

It may also be desirable to use pharmaceutical compositions of protein binding the human CGRF R antigen ex vivo. In these cases, cells, tissues or organs which have been taken from the patient are exposed to the pharmaceutical compositions of protein binding the CGIF R antigen, and then the cells, tissues and / or organs are then reimplanted into the patient.

In particular, human CGRF R antigen-binding proteins can be administered by implanting certain cells which have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. In some embodiments, these cells can be minimal or human cells, and can be autologous, heterologous, or xenogenic. In some embodiments, cells can be immortalized. In other embodiments, in order to reduce the risk of an immunological response, the cells can be encapsulated to prevent infiltration into the surrounding stem cells. In other embodiments, the encapsulation materials are

148

<img file="MA32982B1_D0021.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT usually contain biocompatible, semi-permeable polymeric enclosures or membranes which allow the release of the protein product (s) but prevent the destruction of cells by the patient's immune system or other harmful factors from surrounding tissues .

The following examples, in particular the experiments carried out and the results obtained, are offered by way of illustration only and should not be considered as limiting the scope of the appended claims.

EXAMPLE 1

PRODUCTION OF CGRP RECEIVER AS AN ANTIGEN

A. Molecular cloning of human CRLR and RAMP1

Human CRLR cDNA (GenBank Accession No. U17473; SEQ ID NO: 1) and RAMP1 cDNA (GenBank Accession No. AJOO1014; SEQ ID NO: 3) were cloned into the vectors. mammalian expression pcDNA3.1-Zeo and pcDNA3.1-Hyg (Invitrogen, Carlsbad, CA), respectively for HEK 293ΕΒΝΑ cell transfections (Invitrogen) as described below. The hCRLR and hRAMP1 cDNAs were also cloned into the vector pDSRa24 (Kim, HY et al. J. Inv. Derm. Symp. Proc. (2007) 49-48: ئ) for ΑΜ-1 CHO cell transfections (US Patent No. 6,210,924).

B. Stably transfected cell lines

Stable expression of human CGRP R in 293ΕΒΝΑ cells

HEK 293ΕΒΝΑ cells (available from ATCC or Invitrogen) were seeded at a density of 1.5χ10٥ cells per 100 mm dish. After 24 h, cells were co-transfected with 6 µg of linearized DNA from huRAMP1 / pcDNA3.LHyg and huCRLR / pcDNA31Zeo with FuGene6 (Invitrogen, Carlsbad, CA) following the instructions provided by Invitrogen. After two days the cells were trypsinized and subcultured in growth medium containing 400 µg / ml hygromycin + 250 µg / ml zeocin. After two weeks, the colonies resistant to the substances obtained were trypsinized and combined into pools. The pools were subjected to four rounds of FACS sorting an Alexa 647-labeled CGRP8-37 peptide analog (described above). The 5 ٠/٠ of cells with the highest expression were harvested at each cycle.

Stable expression of human CGRP R in ΑΜ-1 CHO cells

ΑΜ-1 CHO cells (a variant adapted to serum-free growth medium derived from the DHFR-deficient CHO cell line described in Urlaub and Chasin, Proc. Natl. Acad. Sci. 77, 4216 (1980)), were seeded. to 1.5xio٥ cells per 100 mm dish. After 24 h, cells were co-transfected with 4 µg of linearized DNA from

149

ΜΑ 32982Β1

A-1472-WO-PCT pDSRa24 / huRAMP1 and pDSRa24 / huCRLR with FuGeneé (Invitrogen, Carlsbad, CA) following the instructions provided by Invitrogen. The transfected cells were trypsinized 2 days after transfection and seeded on selective CHO DHFR growth medium containing 10 dial/y of dialyzed FBS, without hypoxanthine / thymidine supplementation. After two weeks, the resulting transfected colonies were trypsinized and pooled. The pools were subjected to FACS sorting analysis.

3. Stable expression of human adrenomedullin (ΆΜ11 in HEK 293ΕΒΝΑ cells

293ΕΒΝΑ cells were seeded in 100 mm dishes at 1.5 × 0 cells / dish in DMEM (high glucose content) -t- FBS 5 ؛ ٠/٠ non-essential amino acids MEM 1 ب ٥/٠ sodium pyruvate 1 ٥/٠. The next day, cells were COtransfected using the FuGENE 6 transfection reagent (Roche) with pcDNA3.1 / zeocin / huCRLRplus pcDNA3.1 / hygromycin / huRAMP2. Both cDNA constructs were linearized with Fsp1. After 48 h, the cells were subcultured in 100 mm dishes at 3 cell densities (8x103.2, ؛ x10 ؛, and 8x0 cells / dish) in growth medium containing 200 gg / ml of zeocin . The medium was changed twice a week. After one week, the dishes were fed with medium containing 200 μg / ml of hygromycin + 200 gg / ml of zeocin. After two weeks, 96 colonies were isolated with cloning rings. The rest of the colonies were put together into a single pool culhire. The response of clones and pools to stimulation by an agonist receptor or forskolin was tested. Several clones showed a good response and one of them was selected for use in the following experiments.

4. Stable expression of cvnoCGRP R in HEK 293ΕΒΝΑ cells

293ΕΒΝΑ cells were seeded in 100 mm dishes at 1.5χ10٥ cells / dish in DMEM (high glucose content) + FBS 5 ٥z -t- non-essential amino acids MEM 1% + sodium pyruvate 1%. The next day, cells were COtransfected using FuGENE 6 with pcDNA3.1 / zeocin / cynoCRLR plus pcDNA3.1 / hygromycin / cynoRAMP1. Both cDNA constructs were linearized with Fsp1. After 48 h, cells were subcultured in growth medium containing 200 gg / ml zeocin 1400 pg / ml hygromycin at dilutions of 1:20, 1:40, 1: 100 and 1: 200 . The medium was changed twice a week. After two weeks, 96 transfected colonies were isolated with cloning rings. The response of the clones to stimulation by the CGRP ligand was tested. Several clones showed high levels

150

ΜΑ 32982Β1

Similar response A-1472-WO-PCT and one of them was selected for use in the following experiments.

C. Isolation of cells expressing the high level CGRP receptor

A CGRP 8-37 peptide analog was synthesized (Midwest Bio-Tech Inc. Fisher, IN) with the following sequence:

The peptide was labeled with Alexa 647-NHS following the manufacturer's instructions (Molecular Probes, Inc. Cat A 2006). Alexa 647 labeled CGRPs-37 showed specific staining of cells transfected with the CGRP receptor and not untransfected cells and was used as a FACS reagent.

The pools of 293ΕΒΝΑ and ΑΜ-1 CHO cells transfected with the huCGRP receptor (produced as above) were repeatedly screened up to four times using the CGRPs-37 peptide labeled with Alexa 647. Cells at high expression were collected at each sorting, multiplied and after final sorting, frozen in vials. ΑΜ-1 CHO / huCGRP R cells were used for immunization as described below, and 293EBNA / huCGRP R cells were used to titrate mouse sera after imminization and in supernatant binding screens. of hybridomas.

D. Production of soluble CGRP receptor

Soluble CGRP receptor polypeptides containing the N-terminal extracellular (DEC) domains of human CRLR (SEQ ID NO: 6) and human RAMP1 (SEQ ID NO: 8) were produced by transient co-transfection of 293 6Ε cells ( Durocher, et al., Nucleic Acids Res. 30: E9 (2002)) with vectors comprising the corresponding cDNAs (SEQ ID NO: 5 or SEQ ID NO: 7) as described below. Commonly used tags (polyHis, Flag, HA, and or Fc) have been employed to facilitate subsequent secretion and / or purification.

A soluble heterodimeric CGRP R DEC fused to Fc was prepared by PCR cloning with the appropriate primers in the transient vector ρΤΤ5 (Durocher, et al., Supra).

The DEC of the N-terminus of CRLR-Fc consists of the Nterminal extracellular domain of CRLR (SEQ ID NO: 6) fused to the Fc of human IgG1. The DEC of RAMP1-Fc contains the extracellular domain of RAMP1 (SEQ ID NO: 8) fused to the Fc of human IgG1. In both cases, there is a linker made up of five consecutive glycine residues between the DEC domain and Fc.

The heterodimeric CGRP receptor was expressed by co-transfection of the two constructs as follows. 293-6Ε cells at Ix10 cells / ml in flasks

151

ΜΑ 32982Β1

Shaking A-1472.WO.PCT were transfected with 0.5 mg / l DNA (hCRLRN-ter ECD-F٠ / pTT5 and huRAMP1 ECD-Fc / pTT5) with 3 ml ΡΕΙ / mg DNA in FreeStyle 293 medium (Invitrogen). Cells were grown in suspension in FreeStyle 293 expression medium supplemented with 0.1% Pluronic F68 and 50 µg / ml geneticin for 7 days and harvested for purification.

Purifications from conditioned medium ("MC") were performed by buffering MC with the addition of 50 mM Tris, 400 mM sodium citrate, and adjusting the pH to 8.5. The buffered MC was then passed through a Protein A affinity column equilibrated with 50 mM Tris dns and 400 mM sodium citrate, the pH having been adjusted to pH 8.5.

The Protein A affinity column was washed with PBS and the Fc binding protein was eluted with 0.1N HOAc. The peak eluted contained the CRLR and RAMP1 components when tested by blot. Western using individual antibodies specific for CRLR or RAMP1. Then, CLMS and N-terminal sequencing confirmed the presence of the CRLR: RAMP1 heterodimer and the CRLR.CRLR homodimer at about a (2/3) ratio.

This soluble CGRP receptor was shown to compete for the binding of Alexa647-labeled CGRPs-37 to recombinant cells expressing the CGRP receptor in the FMAT assay, although it could not bind the CGRP ligand according to. a determination by means of the Biacore test. Fe material was used as an immunogen as described in the example, despite, among other things, its heterogeneity and the absence of binding to the CGRP ligand.

E. Production of membrane extracts from cells expressing the recombinant CGRP receptor

The membrane extracts were prepared from cells expressing the CGRP receptor by means of a method described by Bossé, R. and ٥/٠٠ (Journal of Biomolecular Screening, 3 (4); 285-292 (1998)). Briefly, about 5 grams of cell paste was pelletized in 50 ml of PBS at 3000 rpm for 10 min at 4 Ce resuspended in 30 ml of cold lysis buffer (25 mM HEPES, pH 7.4, MgCl 3 mM plus one Roche protease inhibitor cocktail tablet / 50 ml). The lysate was homogenized with Glas-Col (Teflon-glass homogenizer) with ~ 20 strokes at 5,000 rpm and centrifuged in a JA21 rotor at 20,000 rpm for 15 min at 4 ٥c. This process was repeated once more, and the final pellet was resuspended in -1-5 ml of "final pellet" buffer (25mM HEPES, pH 7.4, 3mM MgCl, 10 ٠/٠ ( w / v) sucrose plus one Roche protease inhibitor cocktail tablet / 50 ml). The membrane extracts were sheared by passage through 6 G and 25 G needles 2-3 times. The total concentration of membrane protein was determined with a Microplate BCA protein assay (Pierce).

152

<img file="MA32982B1_D0022.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

EXAMPLE 2

ANTIBODY PRODUCTION AGAINST CGRP RECEIVER

A. Immunization

Immunizations were performed using the following forms of CGRP receptor antigens, prepared as described in Example 1:

(i) CHO ΑΜ-1 transfectants expressing full length human CRLR and RAMP1 at the cell surface, obtained by cotransfection of CHO cells with full length human CRLR cDNA (SEQ ID NO: 1) encoding a polypeptide having the sequence SEQ ID NO: 2, and the cDNA of RAMP1 (SEQ ID NO: 3) encoding a polypeptide having the sequence SEQ ID ΝΟ.-4 (ii) membrane extract from the cells described in (i) above; and (iii) soluble CGRP receptor obtained by co-expression and purification of the DEC Nterminal of CRLR (SEQ ID NO: 6) and the extraccllular domain (DEC) of RAMP1 (SEQ ID NO: 8) described in Example 1.

XENOMOUSE animals were immunized with soluble CGRP receptor protein and purified CGRP R bearing membranes prepared from ΑΜ-1 CHO cells stably expressing CGRP R in the same manner using doses of 10 gg / mouse and of 150 gg / mouse, respectively. CGRP membranes were prepared using the methods described above.

Consecutive boosters were administered at doses of 10 gg / mouse of soluble CGRP R or 75 gg of membranes carrying purified CGPR R. The XENOMOUSE animals were also immunized with cells expressing the CGRP receptor using doses of 3.4 X 10٥ cells transfected with CGRP / mouse. The injection sites used were basic combinations of the tail subcutaneously and intraperitoneally. Immunizations were performed according to the methods described in US Patent No. 7,064,244, filed February 19, 2002, the disclosure of which is incorporated herein by reference. The adjuvants TiterMax Gold (Sigma ؛ cat. # Τ2684), alum (S.Μ. Sergent Pulp and Chemical Co., Clifiton, NJ, cat. # 1452-250) were prepared according to the manufacturer's instructions and mixed accordingly. 1/1 of adjuvant emulsion to antigen solution.

The sera were collected 4-6 weeks after the first injection and the specific titers were determined by FACS staining of 293ΕΒΝΑ cells expressing the recombinant CGRP receptor.

Mice were immunized with cells / membranes expressing full length CGPR R or the soluble CGRP R extracellular domain, with a series of 11-17

153

ΜΑ 32982Β1

A-1472-WO-PCT immunizations over a period of about one to three and a half months. The mice with the highest serum titer were identified and prepared for hybridoma production. Immunizations were performed in groups of several mice, usually ten. Popliteal and inguinal lymph nodes and splenic tissues were usually gathered from each group to produce firsions.

B. Preparation of monoclonal antibodies

Animals with appropriate titers were identified, and lymphocytes were obtained from draining lymph nodes, and, if necessary, pooled for each cohort. Lymphocytes were dissociated from lymphoid tissue in an appropriate medium (e.g., Dulbecco's Modified Eagle's Medium; DMEM; available from

Invitrogen, Carlsbad, CA) to liberate cells from tissue, and suspended in DMEM. B cells were selected and / or grown using an appropriate method, and fused with an appropriate fusion partner, for example, non-secreting P3X63Ag8.653 myeloma cells (American Type Culture Collection CRL 1580;

Keamey et al, J. Immunol. 123, 1979, 1548-1550).

Lymphocytes were mixed with firsion partner cells in a ratio of 1/4. The cell mixture was gently pelletized by centrifugation at 400 X g for 4 minutes, the supernatant was decanted and the cell mixture gently mixed using a 1 ml pipette. Fusion was induced with PEG / DMSO (polyethylene glycol / dimethyl sulfoxide; obtained from Sigma-Aldrich, St. Louis MO; 1 ml per million lymphocytes). PEG / DMSO was added slowly with gentle stirring over one minute, followed by one minute of mixing. IDMEM (DMEM without glutamine; 2 ml per million B cells), was then added slowly over 2 minutes, followed by additional IDMEM (8 ml per million B cells), which was added for 3 minutes.

The fused cells were gently pelletized (400χg 6 minutes) and resuspended in 20 mL of selection medium (e.g., DMEM containing azaserine and hypoxanthine [HA) and other additional materials if required) per million B cells. The cells were incubated for 20-30 minutes at 37 c, then resuspended in 200 ml of selection medium and cultured for three to four days in T175 flasks before plating on 96-well plates.

The cells were distributed in 96-well plates using standard techniques in order to maximize the cloning capacity of the colonies obtained. After several days of culture, the hybridoma supernatants were harvested and screened as described in detail in the examples below, including confirmation of

154

<img file="MA32982B1_D0023.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT binding to the human CGRP receptor, identification of blocking antibodies by competitive assay for ligand binding, and evaluation of cross-reactivity with other receptors bound to the CGRP receptor ( for example, human adrenomedullin receptor). Positive cells were re-selected and subjected to standard cloning and subcloning techniques. The clonal lines were multiplied in vitro and secreted human antibodies were obtained for analysis.

C. Sequence Analysis of Selected Monoclonal Antibodies

Selected subcloned monoclonal antibodies were sequenced using standard RT-PCR methods. Table 2Α shows the amino acid sequence of the illustrative antibody light chains described herein. Table 2Β shows the amino acid sequence of the illustrative anticoips heavy chains described herein.

The amino acid sequences corresponding to the CDR regions of the sequenced antibodies were aligned and the alignments were used to group the clones by similarity.

The CDR sequence alignments of light chains of clones having kappa light chains, and some corresponding consensus sequences are shown in Figs 3Α and 3Β.

The CDR sequence alignments of light chains of clones having lambda light chains, and some corresponding consensus sequences are shown in FIG. 4.

The heavy chain CDR sequence alignments of exemplary anticoips described herein, and some corresponding consensus sequences are shown in Figs. 5A, 5Β, 5C, 5D and 5Ε.

Some illustrative heavy chain CDR consensus sequences described herein are shown in FIG. 5F.

EXAMPLE 3

IDENTIFICATION OF SPECIFIC RECEIVER ANTIBODIES

CGRP

A. Selection of Antibodies Specifically Binding CGRP by FMAT

After 14 days in culture, the hybridoma supernatants were screened for CGRP R specific monoclonal antibodies by microvolume fluorimetric assay (FMAT) technology (Applied Biosystems, Foster City, CA). The supernatants were screened against ΑΜ-1 CHO huCGRP R cells or recombinant HEK 293 cells which had been transfected with human CGRP R and screened against parental ΗΕΚ293 cells (prepared as described in Example 1).

155

ΜΑ 32982Β1

A-1472-WO-PCT

Briefly, cells in Freestyle medium (Invitrogen, Carlsbad, CA) were seeded in 384-well FMAT plates in a volume of 50 μΐ / well at a density of about 4000 cells / well for stable transfectants and at a density of about 16,000 cells / well for the parental cells, and the cells were incubated overnight at 37 c. Then, 10 μΐ / well of supernatant was added and the plates were incubated for about an hour at 4 ٥c, then 10 μΐ / well of anti-human IgG secondary antibody٠Cy5 (Jackson Immunoresearch, West Grove, PA) were added. at a concentration of 2.8 μg / ml (400 ng / ml final concentration). The plates were then incubated for one hour at 4 ٥c, and the fluorescence was read on an FMAT macroconfoncal analyzer (Applied Biosystems, Foster City, CA).

For the count-screens, ΑΜ-1 CHO cells or HEK 293 cells were seeded in a similar manner and the supernatants screened by FMAT on these cells in parallel in order to differentiate and eliminate hybridomas binding to cellular proteins, but no to the CGRP receptor.

B. Identification of blocking antibodies by competitive binding assay by FMAT

A competitive method for ligand binding has been developed to identify antibodies (in hybridoma supernatants) that bind to the CGRP receptor and block the binding of the CGRP ligand. 384-well plates (Corning Costar, Cat: # 3712) were prepared with 5,000 cells from the 2ΑΜ-1 huCGRP R pool and 20,000 untransfected CHO-S cells in each well, and the plates were incubated for 1 hour. ambient temperature. Ten microliters of Alexa647-CGRPs.37 peptide at 2.8 µg / ml was then added to each well and the plates were then incubated for a further 3 hours at room temperature. The amount of Alexa647-CGRPs.37 bound to cells was tested on an FMAT 8200 cell detection system (Applied Biosystems). The data obtained was a digital signal strength FL1 value (higher FL1 values ​​indicate higher signal strength) and also an image of the cells.

The experiments included supernatants from negative control hybridomas. The mean FL1 value observed in these negative control experiments was adopted as the maximum possible signal for the test. The experimental supernatants were compared to this maximum signal and a percentage inhibition was calculated for each well (% Inhibition = (1- (FL1 of the anti-CGRF R hybridoma supernatant / maximum FL1 signal)).

A summary of the data is presented in fig. 6. In this experiment, 1092 anti-CGPRR supernatant was tested using the receptor binding assay. The data

156

ΜΑ 32982Β1

A-1472-WO.PCT were ordered using percent inhibition. Ninety supernatants showed a mean inhibition> 25 ٥ / o, the mean inhibition of 31 of these was> 50% and for 7 it was> 70 ٠ / o.

A summary data set is presented in Table 10, below. The 5 samples ID Nos. 1-5 illustrate examples of anti-CGRP R hybridoma supernatants which inhibited the binding of A) exa647-CGRPs peptide. 37 to the CGRP receptor and samples ID Nos 536 - 540 illustrate examples of anti-hybridoma supernatants. -CGRP R which did not inhibit the binding of Alexa647-peptide CGRPs-37 to the CGRP receptor.

157

ΜΑ 32982Β1

A-1472-WO-PCT

Table 10 - Illustrative Competition Assay Data for Ligand Binding by FMAT

<td>Sample ID</td><td> ٢١٠٩</td><td colspan="3">Expt. # 1 Picture</td><td>% Inhibition</td><td>FLI</td><td>Expt. # 2 Picture</td><td>٥/٠ Inhibition</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> . .</td><td></td>

<td></td><td></td><td colspan="3">. ٠ μ ...</td><td></td><td></td><td>- ٠ ' .... ، 'د μ</td><td></td>

<td></td><td></td><td colspan="3"></td><td></td><td></td><td>. ٠ 'ج -</td><td></td>

<td></td><td></td><td colspan="3"></td><td></td><td></td><td>د '' .- 'ع' ;؛ ورك</td><td></td>

<td></td><td></td><td colspan="3"></td><td></td><td></td><td>ك ': حدذ' '·</td><td></td>

<td></td><td></td><td colspan="3"> ٠</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>K ١،٠ ا :.) ؛</td><td colspan="2"> -</td><td></td><td></td><td> ٠</td><td></td>

<td></td><td></td><td> .</td><td colspan="2"> !</td><td></td><td></td><td>٠ ة</td><td></td>

<td></td><td></td><td colspan="2">not</td><td>لأ</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td colspan="3">'.كئي'١ي -٠ ة</td><td></td><td></td><td></td><td></td>

Based on competition assays for binding, approximately 30 supernatants were selected for further characterization.

158

ΜΑ 32982Β1

A-1472-WO-PCT

EXAMPLE 4

ACTIVITY OF MONCLONAL BLOCKING ANTIBODIES SPECIFIC

CGRP RECEPTOR IN A FUNCTIONAL cAMP TEST A. Activity of Antibodies Against the CGRP Receptor

Selected anti-CGRP receptor antibodies were screened in an in vitro CGRP receptor-mediated cAMP assay to determine intrinsic efficacy. The in vitro cAMP assay employed a human neuroblastoma-derived cell line (SK-N-MC; Spengler, et al., (1973) In Vitro 8: 410) obtained from ATCC (A TCC number ΗΤΒ-10 ; cells ΗΤΒ-10 ”). ΗΤΒ-10 cells expressed CRLR and RAMP1, which form the CGRP receptor (LM McLatchie et al, 1998). A 293ΕΒΝΑ cell line expressing CGRP R from recombinant cynomolgus was produced as described in Example 1, and a rat L6 cell line expressing the rat CGRP receptor was obtained from ATCC (CRL-1458) .

The LANCE cAMP assay kit (PerkinElmer, Boston, MA) was used in the screening. The tests were performed in white 96-well plates in a total volume of 60 μΐ. Briefly, on the day of the test, frozen ΗΤΒ-10 cells were thawed at 37 ٠c, the cells were washed once with test buffer and 12 μΐ of cell suspension containing 10,000 cells mixed of the anti- antibody! A2MPC branded with Alexa were added in 96 half-zone white plates. After addition of 12 μl of anti-CGRP receptor antibody, the mixture was incubated for 30 minutes at room temperature. Then, 12 μΐ of human α-CGRP CGRP receptor agonist (final concentration 1 nM) were added and then incubated for 15 min at room temperature. After stimulation with human α-CGRP, 24 μΐ of detection mixture were added and the whole was incubated for 60 minutes at room temperature and the dishes were read on an instrument.

EnVision (PerkinElmer, Boston, MA) at Em665 nm. Data was processed and analyzed by Prizm (GraphPad Inc. software) or ActivityBase (IDBS).

Fig. 7Α shows illustrative data obtained as described above using the cell line expressing the ΗΤΒ-10 hCGRP receptor for three antibodies 3C8, 13Η2 etlEll. The data are plotted on a curve in percentage relative to the control (“POC”) as a function of the concentration of antibody (3C8, 13Η2 or 1Ε11) and are fitted with standard nonlinear regression curves to obtain the IC50 values. shown at the bottom of the figure.

159

<img file="MA32982B1_D0024.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

B. Lack of activity of the antibody in related receptors.

Cells expressing related AMI receptors (HEK 293 cells expressing hCRLRhRAMP2; DR Poyner, et al, Pharmacological review, 54: 233-246, 2002), ΑΜ2 (CHO cells expressing hCRLR٠hRAMP3; DR Poyner, et al, Pharmacological review,

54: 233-246, 2002) or the human amylin receptor AMY1 (MCF-7 hCTRthRAMP1 cells; Wen-Ji Chen, et al, Molecular pharmacology, 52: 1164-1175, 1997) were used to determine the selectivity antibodies tested. The HEK 293 cell line expressing AMI was produced as described in Example 1, above. The CHO cell line expressing ΑΜ2 was purchased from EuroScreen (now PerkinElmer,

Inc.); and the MCF-7 cell line expressing the human amylin receptor AMY 1 (Zimmermann, et al, Journal of Endocrinology, 423-431, 1997), was obtained from ATCC (ΗΤΒ-22). Illustrative results, plotted on curves as described above, are present in figs. 7Β (hAM1-ΗΕΚ cells), 7C (hAM2-CH0 cells) and 7D (hAMY-MCF-7 cells). It should be noted that none of the antibodies tested had inhibitory activity against the hAM1, hAM2 or hAMY1 receptors over the range tested.

Similar experiments were carried out using recombinant HEK cells expressing cynomolgus CGRP receptors and rat L6 cells expressing CGRP receptor (ATCC). The data from these studies, as well as additional IC50 data obtained as described in part A of this example, are presented in the "cAMP" columns in Table 11, below. It should be noted that the IC50 values ​​against human CGRP and cynomolgus receptors are in the nanomolar range, while activities against the rat CGRP receptor and human AMI, I2 and AMY 1 receptors, as well as in MCF7 cells expressing calcitonin (data not shown) are all greater than 1 micromolar. The difference in IC50 between the human CGRP receptor and human AMI, ΑΜ2, amylin, and calcitonin receptors illustrates the high selectivity of these antibodies for the CGRP receptor over related receptors which in part form the same receptor components. . The IC50s obtained using human CGRP and cynomolgus CGRP receptors were similar, while the antibodies tested did not appear to cross-react with the rat receptor.

160

ΜΑ 32982Β1

A-1472-WO-PCT

Table 11

<td rowspan="2">Clone</td><td colspan="6">CAMP test</td><td>Test of</td>

<td>hCGRP R CI50 (nM)</td><td>cynoCGR PR CI50 (nM)</td><td>CGRP R rat IC50 (nM)</td><td>hAmylin e 1 CI50 (nM)</td><td>hAMl CI50 (nM)</td><td>hAM2 CI50 (nM)</td><td>CGRP human Ki (nM)</td>

<td>01Ε11.2</td><td> ١١٦٦</td><td> 2,79</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,030</td>

<td>01Η7.2</td><td> ٦١٦٦</td><td> 4,74</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,079</td>

<td>02Α10.1</td><td> 11,81</td><td> 17,6</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,291</td>

<td>02Ε7.2</td><td> 6,30</td><td> 5,51</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,117</td>

<td>03Α5.1</td><td> 9,89</td><td> 28,9</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,093</td>

<td>03Β6.2</td><td> 2,74</td><td> 2,22</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,033</td>

<td>O3C8.2</td><td> 6,66</td><td> 5,32</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,044</td>

<td>03Η8.2</td><td> 10,84</td><td> 10,6</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,111</td>

<td>04Ε4.2</td><td> 2,38</td><td> 3,52</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,015</td>

<td>04Η6.1</td><td> 3,78</td><td> 5,59</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,052</td>

<td>O5F5.1</td><td> 4,79</td><td> 4,78</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,147</td>

<td>07Β2.1</td><td> 8,96</td><td> ٦٦١٦</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,116</td>

<td>07Β3.1</td><td> 10,2</td><td> 14,1</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,127</td>

<td>O7F1.1</td><td> 8,92</td><td> 10,5</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,140</td>

<td>08Β11.2</td><td> 10,7</td><td> 17,0</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,118</td>

<td>O9D4.2</td><td> 1,40</td><td> 2,46</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,023</td>

<td>O9F5.2</td><td> 3,06</td><td> 4,44</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,043</td>

<td>10Ε4.2</td><td> 3,08</td><td> 3,23</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,100</td>

<td> 11491</td><td> 16,1</td><td> 47,8</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,157</td>

<td>llDll.l</td><td> 4,93</td><td> 3,85</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,044</td>

<td>11Η9.1</td><td> 4,56</td><td> 5,07</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,057</td>

<td>12Ε8.2</td><td> 2,93</td><td> 4,13</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,097</td>

<td>12G8.2</td><td> 2,14</td><td> 2,74</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,017</td>

<td>13D6.2</td><td> 8,23</td><td> 11,8</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,055</td>

<td>13Ε2.2</td><td> 18,3</td><td> 49,2</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,128</td>

<td>13Η2.2</td><td> 1,95</td><td> 8,41</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td> 0,033</td>

<td>32H7.1G</td><td></td><td> 1,93</td><td></td><td> &gt;1000</td><td> &gt;1000</td><td> &gt;1000</td><td></td>

161

ΜΑ 32982Β1

A-1472-WO-PCT

EXAMPLE 5

RADIOLIGAND CGRP BIND TEST FOR YOUR DETERMINATION

Ki OF ANTIBODIES BLOCKING THE RECEIVER

CGRP branded by ،²⁵I (Amersham Biosciences, Piscataway, NJ) and cell membranes from ΗΤΒ-10 cells (PerkinElmer Inc., Waltham, Mass.) Were used for a radioligand binding experiment in the presence of various concentrations of the antibodies tested to determine the corresponding Ki values. The CGRP binding assay was performed at room temperature in 96-well plates containing:

110 μΐ of binding buffer (20 mM Tris-HCl, pH 7.5, 5.0 mM MgSO4, 0.2% BSA (Sigma),

1 tablet of CompleteTMO ml of buffer (a protease inhibitor)); 20 μΐ of test compound (10Χ); 20 μΐ of ؛²؛ I-haCGRP (Amersham Biosciences; 10Χ); and 50 μl of Hunan neuroblastoma cell membrane suspension (ΗΤΒ-10) (10 μg per well, PerkinElmer). The plates were incubated at room temperature for 2 hours with shaking at 60 rpm, then the contents of each well were filtered on a 96-well GF / C filtration plate treated with 0.5 ٠/٠ polyethyleneimine (PEI) ( for at least one hour) on filter plates

96-well GF / C. The GF / C filter plates were washed six times with ice-cold 50 mM Tris, pH 7.5 and dried in an oven at 55 ° C for 1 hour. The bottoms of the GF / C plates were then sealed. Forty microliters of Microscint ™ 20 were added to each well, the tops of the GF / C plates were sealed with TopSeal ™ -A (a pressure-sensitive adhesive sealant film), and GF / C plates were counted with TopCount. NXT (Packard). Data were analyzed with the Prizm (GraphPad Inc. software)

Illustrative data and Ki values ​​obtained using the 3C8, 12Η2 and 1Ε11 antibodies are shown in fig. 8.

The rightmost column of Table 11, above, gives the Ki values ​​of the mAbs indicated in the competition binding assay of CGRP radiolabeled with ،²⁵I to ΗΒΤ-10 cell membranes. The data demonstrate that the anti-CGRP receptor antibodies were very competitive (all within the sub-nanomole range) against CGRP binding.

EXAMPLE 6

FACS BIND TEST FOR KD DETERMINATION

ANTIBODIES BLOCKING THE CGRP RECEIVER

Anti-CGRP R mAb affinities for CGRP receptors expressed on cells were determined using a FACS method. Briefly, ΑΜ-1 CHO cells expressing huCGRP R, prepared as described above, were plated on plates.

162

ΜΑ 32982Β1

A-1472-WO-PCT wells at densities of 16,000 or 160,000 cells per well in DMEM medium containing 10% FBS, NEAA, PS, L Glut, NaPyr and 0.05% azide sodium. CGRP receptor antibodies were titrated in the same medium from 50 nM to 1 µM and incubated with cells. After incubation overnight at 4 4c in a total volume of 120 μΐ, on a plate shaker, the cells were washed 2Χ with 2% PBStFBS, by centrifuging and discarding the supernatant each time. One hundred microliters per well of G anti-Hu Fc Cy5 (5 gg / ml; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) containing 7AAD (5 μΐ / well) were then added and incubated at 4 ٥c for 40 min . The cells were washed 2Χ with 2٠ / PBSEBS, centrifuging and discarding the supernatant each time. One hundred microliters of 2% PBStFBS buffer were then added and analyzed by FACS in order to determine the geometric mean of binding. Kd was calculated using KinExA software by taking the negative geometric mean at each concentration of anticoips as the amount of free Ac present Rathanaswami, et al., Biochemical and Biophysical Research Communications 334 (2005) 1004-1013. The data obtained at the two different cell concentrations were analyzed by analysis of n curves in order to determine the KD and the 95 ٠/٠ confidence interval as described in Rathanaswami, ef al ,, Biochemical and Biophysical Research Communications ل ٦ لااج 1 ١ لآ 1 ع

Illustrative data with corresponding curve fits are shown in fig. 10 for the 12G8.2 antibody. Data from eight anticoip blockers produced are shown in Table 12. One of the antibodies (3Β6) was analyzed on two different days.

The ratio of 0.9 obtained for the experiment with 16Κ cells indicates that the concentration of the antigen is predicted to be 0.9X the Kd, and the curve obtained by this experiment is therefore a curve controlled by Kd. It can be appreciated that the Kd values ​​obtained in this way were located in the low single digit nanomolar range for all anticoips tested.

Table 12

<td></td><td colspan="5">١nal.١١ ، of n curves</td>

<td></td><td>Kd (nM)</td><td>Low Kd (nM)</td><td>High Kd (nM)</td><td>error &lt;؛ '٠</td><td>Report 16Κ</td>

<td>1Η7</td><td> 1,9</td><td> 1,5</td><td> 3</td><td> 3,8</td><td> 0,001</td>

<td>2Ε7</td><td> 1,5</td><td> 0,7</td><td> 3,4</td><td> 6,3</td><td> 0,19</td>

<td>3Β6 (a)</td><td> 1,7</td><td> 1,1</td><td> 2,7</td><td> 5,3</td><td> 0,060</td>

<td>3Β6 (b)</td><td> 2,0</td><td> 1,6</td><td> 2,6</td><td> 3,2</td><td> 0,21</td>

163

ΜΑ 32982Β1

A-1472-WO-PCT

<td>4Ε4</td><td> 1,3</td><td> 0,9</td><td> 2,05</td><td> 3,9</td><td> 0,16</td>

<td>4Η6</td><td> 2,4</td><td> 1,78</td><td> 4,35</td><td> 3,8</td><td> 0,070</td>

<td>9D4</td><td> 2,5</td><td> 1.8</td><td> 4,39</td><td> 4,3</td><td> 0,060</td>

<td>12Ε8</td><td> 2,3</td><td> 1,58</td><td> 3,36</td><td> 3,7</td><td> 0,55</td>

<td>12G8</td><td> 1,4</td><td> 0,92</td><td> 2,21</td><td> 3,6</td><td> 0,94</td>

EXAMPLE 7

BINDING ANTIBODIES BLOCKING THE CGRP RECEIVER BY

COMPETITION FOR THE BIACORE LIAISON

Biacore analyzes (Karlsson, R. et al., Methods; A Companion to Methods in Enzymology, 6: 99-110 (1994) were carried out as follows. The immobilization of anti-CGRP receptor antibodies to Surface of CM5 sensor chips was performed according to the manufacturer's instructions, using a continuous flow of 10 mM HEPES, 0.15 M NaCl, 3.4 mM EDTA, 0.005% Ρ-20, pH 7.4 (HBS buffer -ΕΡ). The carboxyl groups on the sensor chip surfaces were activated by injecting 60 μΐ of a mixture containing 0.2 N of N-ethyl-N '- (dimethylaminopropyl) carbodiimide (EDC) and 0.05 M of Nhydroxysuccinimide (NHS ). Specific surfaces were obtained by injection of 180 μΐ of anti-CGRP receptor antibody diluted in 10 mM acetate, pH 4.0 at a concentration of 30 μg / ml. Excess reactive groups on the surfaces were deactivated by injection of 60 μΐ of 1 M ethanolamine. The final immobilized levels for the individual antibodies were as follows:

Antibody Resonance units (UR) llDll -5 900

3Β6 -7 700

4Η6 -8,000

12G8 -7,800

9F5 6,600

34Ε3 -3 700

A dummy-coupled reference surface serving as a blank was also prepared on the sensor chip. The soluble huCGRP receptor at a concentration of 100 nM was captured on sensor chips having one of the six immobilized antibodies referenced above (1 IDl 1, 3Β6, 4Η6, 12G8, 9F5 or 34Ε3). Each of the twenty anti-CGRP R antibodies tested was then injected onto the capture huCGRP receptor. If the injected antibody recognizes a

164

<img file="MA32982B1_D0025.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT epitope distinct from that recognized by the immobilized antibody, a second binding event should be observed. If the antibody recognizes the same epitope or very similar epitopes, only the binding of the huCGRP receptor will be observed.

Illustrative data obtained using a sensor chip coated with the immobilized 3Β6 antibody is shown in Fig. 9A. The four plots are data obtained using the 1Ε11,4Ε4,2Ε7 and 12G8 antibodies in the injected solution. The events during the experiment are represented by letters, "A" corresponding to the injection of huCGRP R-Fc, "B" corresponding to the end of the injection of huCGRP R-Fc injection, "c" corresponding to the secondary mAb injection, and "D" corresponding to the end of the secondary mAb injection and to the start of the buffer wash. Note that there is no indication of any binding signal from any of the injected anticoips on the immobilized antibody surface, indicating that all four injected antibodies recognized the same epitope or one or more epitopes similar to those recognized by the immobilized antibody. Almost the same results were observed with all of the blocking antibodies tested applied to each of the five immobilized neutralizing antibody surfaces, indicating that the five anti-huCGRP blocking antibodies all recognized the same epitope or very similar and strongly overlapping antibody (s). .

On the other hand, as shown in part in Figs. 9Β, 9C and 9D, the four nonblocking CGRP receptor specific antibodies tested 32Η8, 33Β5, 33Ε4 and 34Ε3 failed in competition with 11D11 (data not shown), 3Β6 (Fig. 9Β), 12G8 (Fig. 9C) and 9F5 (data not shown), although 34Ε3 may have competed with 4Η6 (Fig. 9D) and weakly with 32Η7 (data not shown). 32Η8 failed to compete with 3Β6,4Η6, 12G8, 9F5 or nonblocking antibody 34Ε3, but 33Β5 and 33Ε4 were able to compete with nonblocking antibody 34Ε3. Data for all blocking and non-blocking antibodies are summarized in Table 13, below. “NL” indicates no link; “؛” indicates a significant binding; and weak ”indicates weak binding.

Table 13

<td></td><td colspan="6">Immobilized antibodies</td>

<td>Ac in solution</td><td>llDll</td><td>3Β6</td><td>4Η6</td><td>12G8</td><td>9F5</td><td>34Ε3</td>

<td>1Ε11</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td> +</td>

<td>1Η7</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>2Ε7</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>3Β6</td><td>NL</td><td>NL</td><td>BORN</td><td>NL</td><td>NL</td><td> +</td>

<td>3C8</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

165

ΜΑ 32982Β1

A-1472-WO-PCT

<td>4Ε4</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>4Η6</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td>

<td>5F5</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>9D4</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>9F5</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>10Ε4</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>UDU</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>11Η9</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>12Ε8</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td> *</td>

<td>12G8</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td></td>

<td>13Η2</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>ب</td>

<td>32Η7</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>NL</td><td>Low</td>

<td>32Η8</td><td>ب</td><td> +</td><td>ب</td><td>ب</td><td>ب</td><td> +</td>

<td>33Β5</td><td></td><td>ب</td><td>ب</td><td>ب</td><td>ب</td><td>NL</td>

<td>33Ε4</td><td> +</td><td>ب</td><td></td><td>ب</td><td>ب</td><td>NL</td>

As will be appreciated from reading the data, all the blocking or neutralizing antibodies tested bind to the same region as the five immobilized blocking antibodies, i.e. all the neutralizing anticoips tested bind the same region of the molecule.

CGRP R. On the other hand, non-blocking antibodies generally do not compete with immobilized blocking antibodies, indicating that non-blocking antibodies primarily bind a region other than CGRP R.

EXAMPLE 8

BINDING ANTIBODY PE CGRP ANTI-RECEIVER TO RECEIVER

FROM SOLUBUE CGRP TO WESTERN TRANSFER

Three representative CGRP receptor blocking antibodies were tested using Western blots for binding of a solublemuFc CGRP receptor firsion protein.

One hundred nanograms of purified R-muFc CGRP (produced and purified as described above for CGRP R-huFc, except that mouse Fc was used and the linker between DEC of RAMP1 or CRLR and muFc was replaced. by “GGGGGVDGGGGGV” (SEQ ID „were diluted in PBS with PAGE sample buffer with (reduced) or without (unreduced) beta-mercaptoethanol (βΜΕ) at the concentration of 13.3%. The sample containing βΜΕ was then boiled for 4 min. Reduced and unreduced samples were loaded

166

<img file="MA32982B1_D0026.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT on 4-20% Tris -glycine gels (Invitrogen) with alternating tracks of CGRP R-Fc protein and molecular weight markers (Invitrogen). Gels were electroblotted through 0.2 µm nitrocellulose filters (Invitrogen). The prints were washed with saline solution buffered with 1 ٠/٠ Tris ب Tween 20 (TBST) and then blocked with TBST + 5 ٠/٠ dried milk powder for 30 min. The prints were cut into strips along the molecular weight marker tracks. The bands, each with reduced and unreduced R-muFc CGRP, were incubated with the purified anti-huCGRP R antibodies 4Ε4, 9F5 or 3Β6 (dilution 1/500 dilution in TBST + 5 ٥/٠ milk), goat antihuRAMP1 Ν-20 (1/500; Santa Cruz Biotechnology, Inc), anti-rabbit mouse IgG-Fc-HRP (1 / 10,000) (Pierce), or anti-human goat IgG-Fc-HRP (1/10 000) (Pierce). The prints were incubated with the antibodies for one hour, then washed 3x10min with TBST f 1% milk. The fingerprints treated with the anti-huCGRP R antibody were then incubated with the goat mouse antiIgG-Fc-HRP (1: 10,000 in TBST t milk 1 ٠/٠) and the fingerprints shaved with anti-huRAMP1 (goat anti-RAMP1 polyclonal antibody Ν-20, Santa Cruz Biotech, CA) were incubated with anti-rabbit goat IgG-Fc-HRP (1: 10,000) for 20 min. The impressions were washed 3x15 min with TBST. The anti-huCGRP R and anti-huRAMP1 antibody fingerprints were treated with the Pierce Supersignal West Pico Detection detection reagent, and the anti-mouse IgG and anti-human IgGFc-HRP fingerprints were treated with the reagent. standard Pierce detection (1 min.). The impressions were then exposed on Kodak Biomax MS X-ray film.

The three anti-CGRP receptor antibodies, 4Ε4, 9F5 and 3Β6 were all able to detect soluble CGRP R-muFc (containing the DECs of RAMP1-ECD and CRLR) under non-reducing conditions, but n under reducing conditions. indicates that the binding epitopes of these anti-CGRP R antibodies were conformational and sensitive to disulfide bonds (3 pairs in the DEC of RAMP1 and 3 pairs in the DEC N-terde CRLR). In contrast, the commercial anti-RAMP1 Ν-20 antibody (Santa Cruz Biotech) bound RAMP1 a las under reduced and unreduced conditions, indicating that the binding site for the Ν-20 antibody was predominantly linear and insensitive to disulfide bonds.

EXAMPLE 9

BINDING ANTIBODIES BLOCKING THE CGRP RECEIVER TO

CHEMICAL RECEIVERS

CGRP receptors forms of native RAMP1 with chimeric CRLR, or native CRLR with chimeric RAMP1, were used to identify CGRP receptor sequences involved in antibody binding. All antibodies blocking the

167

ΜΑ 32982Β1

A-1472-WO-PCT

As human CGRF tested failed to exhibit functional activity on the rat CGRF receptor, the chimeric components contained regions of rat sequence in human sequence context. The following chimeras were prepared for FACS binding analysis:

Chimera # 1 of RAMP1 (028 to Α341: SEQ ID 0: 217

The amino acid residues Q28 to Α34 in human RAMP1 were replaced with the corresponding sequences from rat RAMP1. This sequence included five amino acid residues which are different between human and rat RAMP1.

Chimera # 2 of RAMP1 (043 to Ε531: SEQ ID 0: 218

The amino acid residues Q43 to Ε53 in human RAMP1 were replaced by the corresponding sequences from rat RAMP1. This sequence included six amino acid residues which are different between human and rat RAMP1.

Chimera # 3 of RAMP1 R7 to Ε78); SEQ ID NO: 219

The amino acid residues R67 to Ε78 in human RAMP1 were replaced by the corresponding sequences from rat RAMP1. This sequence included seven amino acid residues which are different between human and rat RAMP1.

Chimera#! from ÇRLRfL24 to 033): SEQ ΙΡ1223

The amino acid residues L24 to Q33 in human CRLR were replaced with the corresponding sequences from rat CRLR. This sequence included eight amino acid residues that are different between human and rat CRLRs.

Fig. 11 shows an alignment of the amino acid sequence of RAMP1 from cynomolgus monkey (SEQ ID NO: 215), human (SEQ ID NO: 4), rat (SEQ ID NO: 214) and rhesus monkey (SEQ ID ΝΟ -.216), with the sequences of RAMP1 chimeric # 1 (SEQ ID NO: 217), chimera # 2 (SEQ ID NO: 218) and chimera # 3 (SEQ ID NO: 219). The frgs. 12Α and 12Β show an alignment of the amino acid sequence of human CRLR (SEQ IDN0: 2), cynomolgus monkey (SEQ ID NO: 221), rhesus monkey (SEQ ID NO: 222), rat ( SEQ ID NO: 220), as well as the amino acid sequence of chimeric CRLR # 1 (SEQ ID NO: 223).

293-6Ε cells were transiently transfected with CGRP R chimera DNA constructs (CRLRwt - RAMP1 Q28-A34; CRLR wt t RAMP1 Q4330 Ε53; CRLR wt ؛ RAMP1 R67-E78; CRLR L24-Q33 t RAMP wt; CRLR wt t RAMPl wt;

control vector ρΊΤ5). The cells were harvested after 72 h, washed with PBS ؛ BSA 0.5 ٠/٥, and counted. Each transfected cell line was resuspended at a dilution of 5x10 ؛ cells per 100 μΐ of PBS / BSA. A 100 μl aliquot of cell suspension was taken per well of a 96-well round-bottom plate (Falcon). The cells were

168

ΜΑ 32982Β1

A-1472.WO.PCT pelleted at 1200 rpm for 5 min. The supernatant was removed and replaced with 100 μΐ containing 0.5 μg of anti-huCGRP R antibody purified 1Η7, 2Ε7, 3Β6, 9F5, 4Η6, 12G8, 3C8,10Ε4, 1 lDll, 32H8, OR 33Β5. Control wells were treated with anti-DNP huIgG2 (0.5 µg), Alexa647-pepüde CGRP (0.5 µg), or PBS / BSA alone. The cells were incubated on ice for 1 h, then washed twice with PBS / BSA. Cells were resuspended in 100 µl / well of PBS / BSA containing Tanti-hug-Fc-FITC (0.5 µg) (except for cells treated with Aexa647-CGRP). Cells were incubated on ice in the dark for 1 hr, then washed twice with PBS / BSA. Cells were resuspended in 200 µl PBS / BSA and analyzed using FACS Calibur.

Ten representative blocking antibodies (3Β6, 9F5, 4Η6, 12G8, 3C8, 10Ε4, 32Η7, 4Ε4, llDllet 1Η7) and two non-blocking antibodies (32Η8 and 33Β5) were tested. Representative data (9F5 antibody) are shown in Figs. 13Α, 13Β and 13C. Fig. 13Α exhibits binding to wild type CGRP receptors; fig. 13Β shows the binding to CGRP receptors containing the CRLR L24-Q33 chimera, and FIG. 13C shows binding to CGRP receptors containing the RAMP1 Q28-A34 chimera. FACS analysis showed that all 12 antibodies bound the control wild type CGRP receptor as expected. All 12 antibodies show significantly reduced binding to any of the three RAMP1 chimeras (Q28A34), (Q43-E53) and (R67 - Ε78). This could be due to (1) the much lower chimeric receptor expression level, (2) the alteration of CRLR folding by the RAMP1 chimera and the change in the conformation of the receptor complex, and / or ( 3) the direct involvement of these three selected regions on RAMP1 on the binding of these antibodies to the CGRP receptor.

When FCS follow-ups were adjusted to include only very small "expressing" populations, non-blocking antibodies 33Β5 and 32Η8 appeared to bind consistently less well (weaker geometric averages) to the RAMP1 Q43-E53 chimera than blocking antibodies, suggesting that binding to the Q43-E53 region of RAMP1 might be more important for non-blocking antibodies. On the other hand, 33Β5 and 32Η8 consistently bound better to the RAMP1 chimera R67-E78 than the blocking antibodies, suggesting that the R67-E78 sequence of RAMP1 might be more important for non-blocking antibodies.

All of the CGRP receptor antibodies tested bound reasonably well to the CRLR chimera (L24-Q33), suggesting that this site is not essential for binding of the blocking antibodies.

169

<img file="MA32982B1_D0027.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT

In summary, the data show that three discontinuous regions on RAMP1 - (Q28Α34), (Q43 - Ε53) and (R67-E78) - could be involved in the binding of the antibody to the CGRP receptor, (R67-E78 ) was more important for blocking antibodies. The involvement of the N-terminal sequences (L24-Q33) of CRLR does not appear to be critical in the binding of anti-CGRP receptor antibodies according to the analysis by this method. This approach does not exclude the existence of other binding sites with identical or similar sequences shared between human and rat CGRP receptors which are not targeted in this assay.

EXAMPLE 10

IDENTIFICATION OF HUMAN CGRP R EPITOPES FOR UES

ΑΝΤΙ-CGRP R NEUTRAUIZING ANTIBODIES BY A PROTECTION TEST

AGAINST PROTEASES

The CRLR portion in the mature form of the CRLR-Fc fusion molecule (with the signal peptide removed as described herein by SEQ ID NO: 10) contains 116 amino acids (preceding the glycine linker) and has three large loop structures created by the formation of three disulfide bonds. The three disulfide bonds in CRLR are Cys1 at sequence position 26 (all CRLR sequence positions indicated in this paragraph refer to the sequence shown by SEQ ID NO: 10) linked to Cys3 at sequence position 52 (designated by CRLR C1-C3), Cys2 at sequence position 43 linked to Cys5 at sequence position 83 (designated as CRLR C2-C5), Cys4 at sequence position 66 linked to Cys6 at sequence position 105 (designated as CRLR C4-C6 ). The RAMP1 part in the master form of the RAMP1-Fc fusion molecule contains 91 amino acids (SEQ ID ΝΟ: 11) preceding the glycine linker, which also form three intramolecular disulfide bonds. The three disulfide bonds in RAMP1 are Cys1 at sequence position 1 (all RAMP1 sequence positions shown in this paragraph refer to the sequence material shown by SEQ ID NO: 11) linked to Cys5 at sequence position 56 (designated by RAMP1 ClC5), Cys2 in sequence position 14 linked to Cys4 in sequence position 46 (designated by RAMP1 C2-C4), Cys3 in sequence position 31 linked to Cys6 in sequence position 78 (designated by RAMP1 C3-C6) .

Regions of human CGRP receptor protein bound by neutralizing anti-CGRP monoclonal antibodies were identified by fragmenting h CGRP R into peptides with specific proteases and determining the sequence of the resulting h CGRP R peptides (i.e. that is, the fragments containing or not containing disulfore bonds for the CRLR and RAMP1 parts). A protease protection test was then carried out to determine the proteolytic digestion of hCGRP R in the presence of the monoclonal antibodies.

170

<img file="MA32982B1_D0028.tif" />

ΜΑ 32982Β1

A-1472-WO-PCT binding. The general principle! of this test is that binding of an mAb to CGRP R may allow protection of certain specific protease cleavage sites and this information can be used to determine the region or part of CGRP R to which the mAb binds. .

Briefly, the peptide digests were subjected to peptide mapping by HPLC; individual peaks were collected, and peptides identified and mapped by LC-MS analyzes, in-line electrospray ionization (ESICL-MS) and / or N-terminal sequencing analyzes. All HPLC analyzes for these studies were performed using a reverse phase C18 column (di 2.1mm X 15cm long; Zorbax 300SB, 5 µm, Agilent Technologies) for off-line analysis and using a capillary reversed phase C18 column (0.5 mm id X 25 cm Vydac C18 MS, 5 µm; The Separation Group) for LC-MS. The mapping of peptides by HPLC was carried out with a linear gradient of 0.05% trifluoroacetic acid (mobile phase A) to 90 ٠/٠ acetonitrile in 0.05% trifluoroacetic acid. Columns were grown for 90 minutes at a flow rate of 0.25 ml / min for small bore HPLC for on-line and off-line LC-MS analyzes, and 0.018 ml / min for capillary HPLC for analyzes. of CL-SM online.

The mahire form of human CGRP R was digested with AspN (which cleaves after aspartic acid and some glutamic acid residues at the amino terminus) by incubating approximately 100 µg of 1.0 mg / ml CGRP R in sodium phosphate. 0.1 M sodium (pH 6.5) for 20 h at 37 ° C with 2 µg AspN.

HPLC chromatography of the AspN digests produced a peptide profile shown in FIG. 14 (injection of 30 µg of each sample), the chromatogram mark A for CGRP R alone (concentration 1 mg / ml), while a control digestion with a similar amount of CGRP R neutralizing antibody, clone 12G8, shows that the The antibody is essentially resistant to the endoproteinase AspN (chromatogram mark B; CGRP R / antibody ratio, 100/2; 100/7; 100/20, w / w, respectively). Sequence analyzes were performed by on-line LC-MS and by Edman sequencing on peptide peaks recovered by HPLC. ESI CL-SM online analyzes of the digests were performed to determine the precise mass and sequence of the peptides which were separated by HPLC. The identities of several peptides present in the peaks of peptides obtained from digestion with AspN were therefore determined (indicated by the peaks numbered in FIG. 14). Table 14 below shows the locations of these peptide sequences in the corresponding component (CRLR or RAMP1) of hCGRP R. A capital letter c followed by a number or by X represents a peptide identified as a CRLR peptide; a capital letter R followed by a number or X represents a peptide identified as a RAMP1 peptide and

171

ΜΑ 32982Β1

A-1472-WO-PCT "Fc" represents the large undigested Fc fragment released by the CRLR-Fc and RAMP1-Fc fusion molecules.

Table 14

CRLR and RAMP1 Peptides Identified by CGRP R Digest Peptide Mapping by AspN

<td>Peptide</td><td colspan="2">Disulfide location (#) of the sequence</td><td>Intact mass</td><td>Origin</td>

<td>Cl</td><td>E1U-V122</td><td> 0</td><td> 1059</td><td>CRLR</td>

<td>C2</td><td>D33-A38</td><td> 0</td><td> 619</td><td>CRLR</td>

<td>C3</td><td>D55M63</td><td> 0</td><td> 880</td><td>CRLR</td>

<td>C4</td><td>D68-Q71</td><td> 0</td><td> 571</td><td>CRLR</td>

<td>C5</td><td>D55-P67 / D86-HU0</td><td></td><td>nd</td><td>CRLR</td>

<td>C6</td><td>D8-Y24</td><td> 0</td><td> 1938</td><td>CRLR</td>

<td>CT</td><td>E25-Q32 / D48-N54</td><td> 1</td><td> 1933</td><td>CRLR</td>

<td>CX</td><td></td><td> 3</td><td>nd</td><td>CRLR</td>

<td>RI</td><td>D32-A44</td><td> 0</td><td> 1622</td><td>RAMPl</td>

<td>R2</td><td>E12-V2O / D45-A51</td><td> 1</td><td> 1939</td><td>RAMPl</td>

<td>RX</td><td>C1-R86</td><td> 3</td><td> 10049</td><td>RAMPl</td>

<td>Fc</td><td></td><td></td><td> 20500</td><td>RAMPl / CRLR</td>

Fig. 15 presents a comparison between an experiment of AspN digestion (injection of 30 µg of each sample) of CGRP alone (chromatogram marked A) and an experiment carried out in the presence of the neutralizing antibody 12G8 (chromatogram marked B). The CGRP R / antibody weight ratio was 1/1. Several peaks (C5, C6, and C7) show a decrease in peak height in chromatogram B compared to chromatogram A, while two other peaks (CX and RX) show an increase in peak height in chromatogram B compared to chromatogram A. A similar peptide map pattern was also observed when a different neutralizing anti-CGRP R antibody (10Ε4, 3Β6, 3C8 or 4Ε4) in a similar amount was present in

172

Γ

ΜΑ 32982Β1

A-1472-WO-PCT the digestion sample as shown in the chromatogram mark c. C6 and C7 are both disulfide-bonded peptides that span a major part of the CRLR molecule, while C5 is a CRLR peptide that does not contain a disulfore bond located at the N-terminus of the molecule and is positron penultimate of the C7 disulfore peptide. The cx peak contains three CRLR disulfor bonds with multiple sequences, indicating that at least two of the three peptides are linked together by disulfor bonds. The fact that the CX peak shows higher peaks in a CGPR R digest in the presence of CGPR neutralizing antibody indicates that the antibody protected CGPR R from AspN digestion at several cleavage sites located in Glu25 and Asp55. The antibody does not appear to have a significant protective effect on Asp33 and Asp72, as the intensity of the peaks for the C2 and C4 peptides does not decrease at all. Therefore, the antibody appears to bind to a region of CRLR that includes the CRLR C1-C3 and CRLR C4-C6 disulforces region with the loop region between Cys53 and Cys66.

AspN mapping of hCGRP R a also identified a RAMP1 disulfore peptide (R2) and a RAMP1 non-disulfore peptide (RI) (see Table 14 and Fig. 14). In the presence of any of the neutralizing antibodies mentioned above (12G8, 10Ε4, 4Ε4, 3Β6 or 3C8), the RX peptide was recovered at a peak intensity significantly higher than that obtained by digestion without anticoips in the 'sample. Mass and sequence analyzes showed that RX contains a single polypeptide chain corresponding to the RAMP1 sequence between CysI and Arg86. These experiments indicate that the CGPR R neutralizing antibody can protect a significant region of RAMP1 from proteolytic digestion with AspN.

In order to verify whether the protective effect against proteolysis by AspN of CGPR R was specific for CGPR R neutralizing (blocking) anticoips (compared to non-neutralizing antiCGPR R anticoips), an AspN digestion of CGRP R was carried out in the presence of of a non-apparent control monoclonal antibody which does not neutralize the activity of CGPR R. The results are presented in fig. 15 in chromatogram D. The non-neutralizing antibody shows no significant blocking effect on the proteolysis of CGPR R by AspN; indeed, the peptide map profile (chromatogram D) is virtually indistinguishable in its relevant aspects from the profile derived from the digestion of CGPR R alone (chromatogram A).

The protective effect against proteolysis was dependent on the concentration added to the digest sample. As seen in fig. 16, an amount of CGRP R fixed in the sample (100 pg) with varying amounts of neutralizing anti-CGPR R 4Ε4 antibody

173

ΜΑ 32982Β1

A-1472-WO-PCT (CGRP R / antibody ratio in micrograms, 100/2; 100/7; 100/20, w / w respectively) was used for the proteolysis by AspN. The protection profile can be observed and the protection is dependent on the antibody concentration.

Combined, these data demonstrate that the described anti5 CGRP R blocking or neutralizing antibodies can block CGRP R (on the CRLR and RAMP1 components) from proteolysis by AspN, suggesting that the blocking antibodies bind to both CRLR and RAMP1 when these antibodies bind to the CGRP receptor. Further, the protective effect is dependent on the antibody concentration. These results also indicate that CGPR R neutralizing antibodies bind to common regions on human CGRP R which are close to AspN cleavage sites.

EXAMPLE 11

ANTIBODIES ΑΝΤΙ-RAMPl AND ANTICRLR AVAILABLE IN THE

TRADE IN A WORKING AMP TEST

Commercially available antibodies directed against either of the components (RAMP1 or CRLR) of the human CGRP receptor were screened in the CGRP receptor-mediated cAMP assay using ΗΤΒ-10 cells. as described in Example 4, above, to determine if the antibodies had biological activity. The data are presented in Table 15, below. The antibodies exhibited undetectable ("ND"), very low "TF"), or low ("F") biological activity over a range of concentrations in which the illustrative antibodies described herein have high biological activity.

Table 15 Activity of commercially available antibodies

<td>Last name</td><td>Source</td><td>Antigen or epitope</td><td>Seller</td><td>Activity ΗΤΒ-10</td>

<td>Anticoips CRLR (abl3164)</td><td>Polyclonal Ac rabbit</td><td>DEC N-terminal dehCRLR</td><td>Abcamlnc., Cambridge, MA</td><td>ND</td>

<td>Anticoips CALCRL</td><td>Polyclonal Ac rabbit</td><td>DEC N-terminal by hCRLR</td><td>GenWay Biotech, Inc., San Diego, CA</td><td>ND</td>

<td>CRLR (N-18)</td><td>Polyclonal Ac Goat</td><td>Mapping epitope near The N- end terminal of</td><td>Santa Cruz Biotech</td><td>TF</td>

174

ΜΑ 32982Β1

A-1472-WO-PCT

<td></td><td></td><td>hCRLR</td><td></td><td></td>

<td>CRLR (H-42)</td><td>Polyclonal Ac rabbit</td><td>aa23-64 from hCRLR</td><td>Santa Cruz Biotech</td><td>ND</td>

<td>Antibody CALCRL (Α01)</td><td>Polyclonal Ac mouse</td><td>aa23-133 from hCRLR</td><td>Novus Biologicals, Inc.</td><td>TF</td>

<td>RAMPl (N- 20)</td><td>Polyclonal Ac Goat</td><td>Mapping epitope at The N- end terminal of hCRLR</td><td>Santa Cruz Biotech</td><td>ND</td>

<td>Antibody RAMPl (ME)</td><td>Polyclonal Ac mouse</td><td>aa27-U8 from hRAMPl</td><td>Novus Biologicals, Inc., Littleton, CO</td><td>F</td>

<td>Antibody RAMPl (IFI)</td><td>Monoclonal Ac mouse</td><td>aa27-U8 from hRAMPl</td><td>Novus Biologicals, Inc.</td><td>ND</td>

<td>Antibody RAMPl (ab67151)</td><td>Polyclonal Ac mouse</td><td>hRAMPl from total length</td><td>Abcam, Inc.</td><td>F</td>

<td>RAMPl (FL- 148)</td><td>Polyclonal Ac rabbit</td><td>hRAMPl from total length</td><td>Santa Cruz Biotech, Santa Cruz, CA</td><td>ND</td>

EXAMPLE 12

CoaionniuhhBifldeuleg £ XQi-imantdifferent receptor components

2-4x10٥ cells were injected by collagen buffer (Integra LifeSciences Co.,

Plainsboro, NJ), Collagen buffers were embedded in OCT medium (Sakura Finetek Inc., Torrance, CA), frozen at -20 ٥c and cut into 20 µm sections using a cryostat. Sections were fixed with paraformaldehyde 4 نره for 1 hour at room temperature (RT) and then washed in phosphate buffered saline (PBS).

Endogenous peroxidase was blocked with 3% Η2Ο2 / PBS for 15 min and sections were incubated in blocking solution (PBS with 3% normal goat serum

175

ΜΑ 32982Β1

A-1472-WO-PCT (Vector Labs, Burlingame, CA) and triton Χ-100 0.3 ٥/٥) for 1 hour. Then the sections were incubated in a primary anti-human CGPR receptor antibody (32Η7, 0.03 0.1 µg / ml) at 4 ° C overnight, washed in PBS and incubated in a secondary antibody (fragment Biotinylated goat anti-human IgG fc, 1/800, Jackson Immunoresearch, West

Grove, PA) in 1% normal goat serum / PBS for 1 h at RT. Immunoreactivity was amplified using the Vector Elite kit according to the manufacturer's instructions (Vector Labs, Burlingame, CA) and the stain was developed using 3,3'diaminobenzidine-nickel as a chromogen (Sigma-Aldrich, St. Louis, MO). Sections were cleaned with xylene and covered with a coverslip with Permount (Fisher Chemicals,

Fair Lawn, NJ). Immunoreactivity was analyzed with a Nikon Ε-800 microscope and associated software (Nikon, Melville, NY).

Data from cells expressing different receptor components (identified below) using the 32Η7 anticoips described above revealed pronounced staining of CHO cells expressing recombinant human CGPR receptor (CRLRiRAMP1; "CHO ​​/ CGRP R cells ») And lower staining of SK-NMC cells which endogenously express CGRP receptors (due to a much lower receptor density). No coloration was observed in the parent CHO cell line, the CHO cells expressing an unrelated recombinant protein (TRPM8), the CHO / CGRP R cells after pre-absorption with the corresponding 32Η7 antigen, the cells

CHO expressing the recombinant human adrenomedullin receptor 2 (CRLR - + - RAMP3), the MCF-7 cells expressing amylin receptors in the endogenous medium, the HEK cells expressing the recombinant human adrenomedullin 1 receptor (CRLRAMP2), or the parent HEK cells. The data from these experiments are summarized in Table 16, below.

Table 16 Immunohistochemical staining intensity of indicated cells

<td>الفئغثعفا</td><td>، Intensity of coloring (visual score) ،</td>

<td></td><td></td>

<td>CGRP / CHO</td><td> 4+</td>

<td>SK-N-MC</td><td> 1+</td>

<td>CHO</td><td> 0</td>

<td>TRPM8 / CH0</td><td> 0</td>

<td>CGRP / CHO pre-adsorbed</td><td> 0</td>

<td>AM2 / CH0</td><td> 0</td>

<td>MCF-7</td><td> 0</td>

<td>ΑΜ1 / ΗΕΚ</td><td> 0</td>

٩6\

ΜΑ 32982Β1

A-1472-WO-PCT

<td>HEK</td><td> 0</td>

All identified patents and other publications are expressly incorporated herein by reference for the purposes of description and presentation, for example, the methodologies described in such publications which may be used in connection with the subject described herein. These publications are offered only for their description before the filing date of the present application. Nothing in this regard can be considered as amounting to admitting that the inventors are not entitled to backdate this description by reason of an earlier invention or for another reason. All statements concerning the date or representation of the contents of these documents are based on the information available to the applicants and in no way constitute an admission of the accuracy of the dates or contents of these documents.

١٦٦

ΜΑ 32982Β1

A-1472-PCT Sequence_French LIST OF SEQUENCES <110>

<120>

<130>

<140>

&lt;141

AMGEN INC.

IIANF IE PROTEINS

A-1472-WO-PCT

HUMAN CGRP RECEIVER <150>

<151>

<150>

<151>

<160>

<170>

<210>

<211>

<212>

<213>

<400>

61/264,622

25-11-2009

61/203,569

23-12-2008

261

Patentin version 3.5

419؛

DNA

Homo sapiens 1

<td>atgttataca</td><td>gcatatttca</td><td>ttttggctta</td><td>atgatggaga</td><td>aaaagtgtac</td><td>cctgtatttt</td><td> 60</td>

<td>ctggttctct</td><td>tgcctttttt</td><td>tatgattctt</td><td>gttacagcag</td><td>aattagaaga</td><td>gagtcctgag</td><td> 120</td>

<td>gactcaattc</td><td>agttgggagt</td><td>tactagaaat</td><td>aaaatcatga</td><td>cagctcaata</td><td>tgaatgttac</td><td> 180</td>

<td>caaaagatta</td><td>tgcaagaccc</td><td>cattcaacaa</td><td>gcagaaggcg</td><td>tttactgcaa</td><td>cagaacctgg</td><td> 240</td>

<td>gatggatggc</td><td>tctgctggaa</td><td>cgatgttgca</td><td>gcaggaactg</td><td>aatcaatgca</td><td>gctctgccct</td><td> 300</td>

<td>gattactttc</td><td>aggactttga</td><td>tccatcagaa</td><td>aaagttacaa</td><td>agatctgtga</td><td>ccaagatgga</td><td> 360</td>

<td>aactggttta</td><td>gacatccagc</td><td>aagcaacaga</td><td>acatggacaa</td><td>attataccca</td><td>gtgtaatgtt</td><td> 420</td>

<td>aacacccacg</td><td>agaaagtgaa</td><td>gactgcacta</td><td>aatttgtttt</td><td>acctgaccat</td><td>aattggacac</td><td> 480</td>

<td>ggattgtcta</td><td>ttgcatcact</td><td>gcttatctcg</td><td>cttggcatat</td><td>tcttttattt</td><td>caagagccta</td><td> 540</td>

<td>agttgccaaa</td><td>ggattacctt</td><td>acacaaaaat</td><td>ctgttcttct</td><td>catttgtttg</td><td>taactctgtt</td><td> 600</td>

<td>gtaacaatca</td><td>ttcacctcac</td><td>tgcagtggcc</td><td>aacaaccagg</td><td>ccttagtagc</td><td>cacaaatcct</td><td> 660</td>

<td>gttagttgca</td><td>aagtgtccca</td><td>gttcattcat</td><td>ctttacctga</td><td>tgggctgtaa</td><td>ttacttttgg</td><td> 720</td>

<td>atgctctgtg</td><td>aaggcaitta</td><td>cctacacaca</td><td>ctcattgtgg</td><td>tggccgtgtt</td><td>tgcagagaag</td><td> 780</td>

<td>caacatttaa</td><td>tgtggtatta</td><td>ttttcttggc</td><td>tggggatttc</td><td>cactgattcc</td><td>tgcttgtata</td><td> 840</td>

<td>catgccattg</td><td>ctagaagctt</td><td>atattacaat</td><td>gacaattgct</td><td>ggatcagttc</td><td>tgatacccat</td><td> 900</td>

<td>ctcctctaca</td><td>ttatccatgg</td><td>cccaatttgt</td><td>gctgctttac</td><td>tggtgaatct</td><td>ttttttcttg</td><td> 960</td>

<td>ttaaatattg</td><td>tacgcgttct</td><td>catcaccaag</td><td>ttaaaagtta</td><td>cacaccaagc</td><td>ggaatccaat</td><td> 1020</td>

<td>ctgtacatga</td><td>aagctgtgag</td><td>agctactctt</td><td>atcttggtgc</td><td>cattgcttgg</td><td>cattgaattt</td><td> 1080</td>

<td>gtgctgattc</td><td>catggcgacc</td><td>tgaaggaaag</td><td>attgcagagg</td><td>aggtatatga</td><td>ctacatcatg</td><td> 1140</td>

<td>cacatcctta</td><td>tgcacttcca</td><td>gggtcttttg</td><td>gtctctacca</td><td>ttttctgctt</td><td>ctttaatgga</td><td> 1200</td>

<td>gaggttcaag</td><td>caatictgag</td><td>aagaaactgg</td><td>aatcaataca</td><td>aaatccaatt</td><td>tggaaacagc</td><td> 1260</td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French ttttccaact cagaagctct tcgtagtgcg tcttacacag tgtcaacaat cagtgatggt ccaggttata gtcatgactg tcctagtgaa cacttaaatg gaaaaagcat ccatgatatt gaataaa2ta210 ttc tattaaaaaaaaaaaaaagcat ccatgatatt gaataaa2ta210> tatta <2 <211

1320

1380

1419

<td>&lt;213&gt; Homo sapiens &lt;400&gt; 2 Met leu Tyr Ser Ile 1 5</td><td>Phe</td><td>His</td><td>Phe</td><td>Gly</td><td>leu 10</td><td>Met</td><td>Met</td><td>Glue</td><td>lily</td><td>lily 15</td><td>s لاح</td>

<td>Thr Leu Tyr Phe Leu 20</td><td>val</td><td>leu</td><td>Leu</td><td>Pro 25</td><td>Phe</td><td>Phe</td><td>Met</td><td>Isle</td><td>leu 30</td><td>٦ ةلآ</td><td>Thr</td>

<td>Ala Glu leu Glu Glu</td><td>Ser</td><td>pro</td><td>Glue 40</td><td>Asp</td><td>Ser</td><td>Isle</td><td>Gin</td><td>leu 45</td><td>Gly</td><td>Val</td><td>Thr</td>

<td>Arg Asn Lys Ile Met 50</td><td>Thr</td><td>To the 55</td><td>Gin</td><td>Tyr</td><td>Glue</td><td>Cys</td><td>Tyr 60</td><td>Gin</td><td>lily</td><td>Isle</td><td>Met</td>

<td>Gin Asp Pro Ile Gin 65</td><td>Gin 70</td><td>To the</td><td>Glue</td><td>Gly</td><td>val</td><td>Tyr 75</td><td>Cys</td><td>Asn</td><td>Arg</td><td>Thr</td><td>Trp 80</td>

<td>Asp Gly Trp leu Cys 85</td><td>Trp</td><td>Asn</td><td>Asp</td><td>val</td><td>To the 90</td><td>To the</td><td>Gly</td><td>Thr</td><td>Glue</td><td>Ser 95</td><td>Met</td>

<td>Gin Leu cys Pro Asp 100</td><td>Tyr</td><td>Phe</td><td>Gin</td><td>Asp 105</td><td>Phe</td><td>Asp</td><td>Pro</td><td>ser</td><td>Glue 110</td><td>Lilies</td><td>Val</td>

<td>Thr Lys Ile Cys Asp 115</td><td>Gin</td><td>Asp</td><td>Gly 120</td><td>Asn</td><td>Trp</td><td>Phe</td><td>Arg</td><td>His 125</td><td>Pro</td><td>To the</td><td>Ser</td>

<td>Asn Arg Thr Trp Thr 30 ؛ h</td><td>Asn</td><td>Tyr 135</td><td>Thr</td><td>Gin</td><td>Cys</td><td>Asn</td><td>Val 140</td><td>Asn</td><td>Thr</td><td>Hi s</td><td>Glue</td>

<td>Lily Val Lily Thr Ala 145</td><td>leu 150</td><td>Asn</td><td>leu</td><td>Phe</td><td>Tyr</td><td>Leu 155</td><td>Thr</td><td>Isle</td><td>Isle</td><td>Gly</td><td>His 160</td>

<td>Gly leu Ser Ile Ala 165</td><td>Ser</td><td>Leu</td><td>leu</td><td>Isle</td><td>Ser 170</td><td>Leu</td><td>Gly</td><td>Isle</td><td>Phe</td><td>Phe 175</td><td>Tyr</td>

<td>Phe Lys ser Leu Ser 180</td><td>(؟ لاح</td><td>Gin</td><td>Arg</td><td>Isle 185</td><td>Thr</td><td>Leu</td><td>His</td><td>lily</td><td>Asn 190</td><td>lou</td><td>Phe</td>

<td>Phe Ser Phe Val Cys ؛ 19</td><td>Asn</td><td>Ser</td><td>Val 200</td><td>val</td><td>Thr</td><td>Isle</td><td>Isle</td><td>His 205</td><td>leu</td><td>Thr</td><td>To the</td>

<td>Val Ala Asn Asn Gin 210</td><td>To the</td><td>Leu 215</td><td>val</td><td>To the</td><td>Thr</td><td>Asn</td><td>Pro 220</td><td>Val</td><td>ser</td><td>Cys</td><td>lily</td>

I /

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>val</td><td>ser</td><td>Gin</td><td>Phe</td><td>Isle</td><td>His</td><td>leu</td><td>Tyr</td><td>Leu</td><td>Met</td><td>Gly</td><td>Cys</td><td>Asn</td><td>Tyr</td><td>Phe</td><td>Trp</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Met</td><td>leu</td><td>s لاح</td><td>Glue</td><td>Gly</td><td>Isle</td><td>Tyr</td><td>leu</td><td>His</td><td>Thr</td><td>leu</td><td>Isle</td><td>Val</td><td>Val</td><td>To the</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Phe</td><td>To the</td><td>Glue</td><td>lily</td><td>Gin</td><td>His</td><td>leu</td><td>Met</td><td>Trp</td><td>Tyr</td><td>Tyr</td><td>Phe</td><td>Leu</td><td>Gly</td><td>Trp</td><td>Gly</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>leu</td><td>Isle</td><td>Pro</td><td>To the</td><td>Cys</td><td>Isle</td><td>His</td><td>To the</td><td>Isle</td><td>To the</td><td>Arg</td><td>Ser</td><td>leu</td><td>Tyr</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Tyr</td><td>Asn</td><td>Asp</td><td>Asn</td><td>لآلاح</td><td>Trp</td><td>Isle</td><td>ser</td><td>Ser</td><td>Asp</td><td>Thr</td><td>His</td><td>Leu</td><td>Leu</td><td>Tyr</td><td>Isle</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Isle</td><td>His</td><td>Gly</td><td>Pro</td><td>Isle</td><td>Cys</td><td>To the</td><td>To the</td><td>Leu</td><td>leu</td><td>Val</td><td>Asn</td><td>Leu</td><td>Phe</td><td>Phe</td><td>Leu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>Asn</td><td>Isle</td><td>Val</td><td>Arg</td><td>val</td><td>Leu</td><td>Isle</td><td>Thr</td><td>Lilies</td><td>Leu</td><td>lily</td><td>Val</td><td>Thr</td><td>His</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>To the</td><td>Glue</td><td>Ser</td><td>Asn</td><td>Leu</td><td>Tyr</td><td>Met</td><td>Lilies</td><td>To the</td><td>val</td><td>Arg</td><td>To the</td><td>hr</td><td>leu</td><td>Isle</td><td>leu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>val</td><td>Pro</td><td>Leu</td><td>leu</td><td>Gly</td><td>Isle</td><td>Glue</td><td>Phe</td><td>Val</td><td>leu</td><td>Isle</td><td>Pro</td><td>Trp</td><td>Arg</td><td>Pro</td><td>Glue</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Gly</td><td>Lilies</td><td>Isle</td><td>To the</td><td>Glue</td><td>Glue</td><td>val</td><td>Tyr</td><td>Asp</td><td>Tyr</td><td>Isle</td><td>١٨and</td><td>His</td><td>Isle</td><td>leu</td><td>Met</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>His</td><td>Phe</td><td>Gin</td><td>Gly</td><td>leu</td><td>leu</td><td>٦ ةتي ١</td><td>Ser</td><td>Thr</td><td>Isle</td><td>Phe</td><td>Cys</td><td>Phe</td><td>Phe</td><td>Asn</td><td>Gly</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Glue</td><td>val</td><td>Gin</td><td>To the</td><td>Isle</td><td>leu</td><td>Arg</td><td>Arg</td><td>Asn</td><td>Trp</td><td>Asn</td><td>Gin</td><td>٢yr</td><td>Lilies</td><td>Isle</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Phe</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Phe</td><td>Ser</td><td>Asn</td><td>Ser</td><td>Glue</td><td>To the</td><td>Leu</td><td>Arg</td><td>Ser</td><td>To the</td><td>Ser</td><td>Tyr</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Thr</td><td>٦ ةلآ</td><td>Ser</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Asp</td><td>Gly</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>Ser</td><td>His</td><td>Asp</td><td>Cys</td><td>Pro</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Ser</td><td>Glue</td><td>His</td><td>Leu</td><td>Asn</td><td>Gly</td><td>ةلا 1</td><td>Ser</td><td>Isle</td><td>His</td><td>Asp</td><td>Isle</td><td>Glue</td><td>Asn</td><td>val</td><td>leu</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Lilies</td><td>Pro</td><td>Glue</td><td>Asn</td><td>leu</td><td>Tyr</td><td>Asn</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 3 <211> 447

٩ ر

ΜΑ 32982Β1

A-1472-PCT Sequence_French <21 DNA <213> Homo sapiens <400> 3 atggcccggg ccctgtgccg cctcccgcgg cgcggcctct ggctgctcct ggcccatcac ctcttcatga ccactgcctg ccaggaggct aactacggtg ccctcctccg ggagctctgc ctcacccagt tccaggtaga catggaggcc gtcggggaga cgctgiggtg tgactggggc aggaccatca ggagctacag ggagctggcc gactgcacct ggcacatggc ggagaagctg ggctgcttct ggcccaatgc agaggtggac aggttcttcc tggcagtgca tggccgctac ttcaggagct gccccatctc aggcagggcc gtgcgggacc cgcccggcag catcctctac cccttcatcg tggtccccat cacggtgacc ctgctggtga cggcactggt ggtctggcag agcaagcgca ctgagggcat tgtgtag

120

180

240

300

360

420

447 <211> 148 <212> PRT <213> Homo sapiens <400> 4

<td>Met</td><td>To the</td><td>Arg</td><td>To the</td><td>Leu 5</td><td>Cys</td><td>Arg</td><td>leu</td>

<td>leu</td><td>To the</td><td>His</td><td>His 20</td><td>Leu</td><td>Phe</td><td>Met</td><td>Thr</td>

<td>Gly</td><td>To the</td><td>Leu 35</td><td>U</td><td>Aru</td><td>Glue</td><td>Leu</td><td>Cys 40</td>

<td>Glue</td><td>To the 50</td><td>١ ةلا</td><td>Gly</td><td>Glue</td><td>Thr</td><td>Leu 55</td><td>Trp</td>

<td>Ser 65</td><td>Tyr</td><td>Arg</td><td>Glue</td><td>Leu</td><td>To the 70</td><td>Asp</td><td>Cys</td>

<td>Gly</td><td>Cys</td><td>Phe</td><td>Trp</td><td> ٢٥؟ 85</td><td>Asn</td><td>To the</td><td>Glue</td>

<td>His</td><td>Gly</td><td>Arq</td><td>Tyr 100</td><td>Phe</td><td>Arg</td><td>Ser</td><td>Cys</td>

<td>Asp</td><td>Pro</td><td>pro 115</td><td>Gly</td><td>Ser</td><td>Isle</td><td>Leu</td><td>Tyr 120</td>

<td>val</td><td>Thr 130</td><td>Leu</td><td>leu</td><td>Val</td><td>Thr</td><td>To the 135</td><td>leu</td>

<td>Glue 145</td><td>Gly</td><td>Isle</td><td>val</td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;210&gt; 5</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Arg</td><td>Arg</td><td>Gly</td><td>Leu</td><td>Trp</td><td>leu</td><td>leu</td>

<td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Thr</td><td>To the</td><td>Cys</td><td>Gin</td><td>Glue</td><td>To the</td><td>Asn</td><td rowspan="2">Tyr</td>

<td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td>

<td>leu</td><td>Thr</td><td>G٦n</td><td>Phe</td><td>Gin</td><td>val</td><td rowspan="2">Asp</td><td>Met</td>

<td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>Cys</td><td>Asp</td><td>Trp</td><td>Gly 60</td><td>Arg</td><td>Thr</td><td>Isle</td><td>Arg</td>

<td>Thr</td><td>Trp</td><td>His</td><td>Met</td><td>To the</td><td>Glue</td><td rowspan="2">lily</td><td>leu</td>

<td></td><td></td><td> 75</td><td></td><td></td><td></td><td> 80</td>

<td>val</td><td>Asp</td><td rowspan="2">A٣q</td><td>Phe</td><td>Phe</td><td>Leu</td><td>To the</td><td>val</td>

<td></td><td> 90</td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Pro</td><td>Isle</td><td>Ser</td><td>Gly</td><td rowspan="2">Arg</td><td>To the</td><td>val</td><td rowspan="2">Arg</td>

<td> 105</td><td></td><td></td><td></td><td> 110</td><td></td>

<td>Pro</td><td>Phe</td><td>Isle</td><td>val</td><td>val</td><td>Pro</td><td>Isle</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>val</td><td>val</td><td>Trp</td><td>Gin</td><td>Ser</td><td rowspan="2">Lilies</td><td rowspan="2">Arg</td><td>Thr</td>

<td></td><td></td><td></td><td> 140</td><td></td><td></td>

ΛΖΛ

ΜΑ 32982Β1

A-1472-PCT Sequence_French <211> 414 <212> DNA <213> Homo <400> 5 atggagaaaa acagcagaat atcatgacag gaaggcgttt ggaactgaat gttacaaaga tggacaaatt sapiens agtgtaccct tagaagagag ctcaatatga actgcaacag caatgcagct tctgtgacca atacccagtg gtattttctg tcctgaggac atgttaccaa aacctgggat ctgccctgat agatggaaac taatgttaac gttctcttgc tcaattcagt aagattatgc ggatggctct tactttcagg tggtttagac acccacgaga ctttttttat tgggagttac aagaccccat gctggaacga actttgatcc atccagcaag aagtgaagac gattcttgtt tagaaataaa tcaacaagca tgttgcagca atcagaaaaa caacagaaca tgca

120

180

240

300

360

414 <20 6 <211 138 <212> PRT <213> Homo sapiens <400> 6

<td>Met</td><td>Glue</td><td>Lilies</td><td>Lilies</td><td>Cys</td><td>Ihr</td><td>leu</td><td>Tyr</td><td>Phe</td><td>Leu</td><td>٦ ةلا</td><td>leu</td><td>leu</td><td>Pro</td><td>Phe</td><td>Phe</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Met</td><td>Isle</td><td>leu</td><td>١ ةلا</td><td>Ihr</td><td>To the</td><td>Glue</td><td>leu</td><td>Glue</td><td>Glue</td><td>Ser</td><td>Pro</td><td>Glue</td><td>Asp</td><td>Ser</td><td>Isle</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30^P</td><td></td><td></td>

<td>Gin</td><td>leu</td><td>Gly</td><td>val</td><td>Thr</td><td>Arg</td><td>Asn</td><td>Lilies</td><td>Isle</td><td>Met</td><td>Thr</td><td>To the</td><td>Gin</td><td>Tyr</td><td>Glue</td><td>s لاح</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>Gin</td><td>Lilies</td><td>Isle</td><td>Met</td><td>Gin</td><td>Asp</td><td>Pro</td><td>Isle</td><td>Gin</td><td>Gin</td><td>To the</td><td>Glue</td><td>Gly</td><td>val</td><td>lyr</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Cys</td><td>Asn</td><td>Arg</td><td>Thr</td><td>Trp</td><td>Asp</td><td>Gly</td><td>Trp</td><td>Leu</td><td>Cys</td><td>Irp</td><td>Asn</td><td>Asp</td><td>val</td><td>To the</td><td>To the</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td>75Ρ</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Gly</td><td>Thr</td><td>Glue</td><td>Ser</td><td>Met</td><td>Gin</td><td>Leu</td><td>Cys</td><td>Pr٥</td><td>Asp</td><td>Tyr</td><td>Phe</td><td>Gin</td><td>Asp</td><td>Phe</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Pro</td><td>Ser</td><td>Glue</td><td>Lilies</td><td>Val</td><td>Thr</td><td>ةلاأ</td><td>Isle</td><td>s لاح</td><td>Asp</td><td>Gin</td><td>Asp</td><td>Gly</td><td>Asn</td><td>Trp</td><td>Phe</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Arg</td><td>His</td><td>Pro</td><td>To the</td><td>Ser</td><td>Asn</td><td>Arg</td><td>Thr</td><td>Trp</td><td>Thr</td><td>Asn</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Cys</td><td>Asn</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>val</td><td>Asn</td><td>Thr</td><td>His</td><td>Glue</td><td>Lilies</td><td>Val</td><td>lily</td><td>Thr</td><td>To the</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

أا ١ ر <210> 7 <211 351 <212> DNA <213> Homo sapiens <400> 7

ΜΑ 32982Β1

A-1472-PCT Sequence_French atggcccggg ccctgtgccg cctcccgcgg cgcggcctct ggctgctcct ggcccatcac ctcttcatga ccacigcctg ccaggaggct aactacggtg ccctcctccg ggagctctgc ctcacccagt tccaggtaga catggaggcc gtcggggaga cgctgtggtg tgactggggc aggaccatca ggagctacag ggagctggcc gactgcacct ggcacatggc ggagaagctg ggctgcttct ggcccaatgc agaggtggac aggttcttcc tggcagtgca tggccgctac ttcaggagct gccccatctc aggcagggcc gtgcgggacc cgcccggcag c

120

180

240

300

351 <211> 117 <212> PRT <21 Homo sapiens

<td colspan="2">&lt;400&gt; 8 Met Ala</td><td>Arg</td><td>To the</td><td>Leu 5</td><td>Cys</td><td>Macaw</td><td>Leu</td><td>Pro</td><td>Arg 10</td><td>Arg</td><td>Gly</td><td>wish</td><td>Trp</td><td>leu 15</td><td>leu</td>

<td>leu</td><td>To the</td><td>His</td><td>His 20</td><td>Leu</td><td>Phe</td><td>Met</td><td>Thr</td><td>Thr 25</td><td>To the</td><td>Cys</td><td>Gin</td><td>Glue</td><td>To the 30</td><td>Asn</td><td>Tyr</td>

<td>Gly</td><td>To the</td><td>Leu 35</td><td>Leu</td><td>Arq</td><td>Glue</td><td>leu</td><td>s لاح 40</td><td>leu</td><td>Thr</td><td>Gin</td><td>Phe</td><td>Gin 45</td><td>Val</td><td>Asp</td><td>Met</td>

<td>Glue</td><td>To the 50</td><td>Val</td><td>Gly</td><td>Glue</td><td>Thr</td><td>leu 55</td><td>Trp</td><td>Cys</td><td>Asp</td><td>Trp</td><td>Gly 60</td><td>Arg</td><td>Thr</td><td>Isle</td><td>Arg</td>

<td>Ser 65</td><td>Tyr</td><td>Arg</td><td>Glue</td><td>Leu</td><td>To the 70</td><td>Asp</td><td>Cys</td><td>Thr</td><td>Trp</td><td>His 75</td><td>Met</td><td>To the</td><td>Glue</td><td>lily</td><td>leu 80</td>

<td>Gly</td><td>Cys</td><td>Phe</td><td>Trp</td><td>Pro 85</td><td>Asn</td><td>To the</td><td>Glue</td><td>val</td><td>Asp 90</td><td>Arg</td><td>Phe</td><td>Phe</td><td>Leu</td><td>To the 95</td><td>val</td>

<td>His</td><td>Gly</td><td>Arg</td><td>lyr 100</td><td>Phe</td><td>Arg</td><td>Ser</td><td>Cys</td><td>Pro 105</td><td>Isle</td><td>Ser</td><td>Gly</td><td>Arg</td><td>To the 110</td><td>Val</td><td>Arg</td>

<td>Asp</td><td>Pro</td><td>Pro 115</td><td>Gly</td><td>Ser</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 9 <211 31 <212> PRT <213> Homo sapiens <400> 9

<td>Trp</td><td>٦ ةلا</td><td>Thr</td><td>His</td><td>Arg</td><td>Leu</td><td>To the</td><td>Gly</td><td>leu</td><td>Leu</td><td>Ser</td><td rowspan="2">Arg</td><td>Ser</td><td rowspan="2">Gly</td><td>Gly</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td>15Υ</td>

<td>Val</td><td>Arg</td><td>Cys</td><td>Asn</td><td>Phe</td><td>Val</td><td>Pro</td><td>Thr</td><td>Asp</td><td>Val</td><td rowspan="2">Gly</td><td>Pro</td><td>Phe</td><td>To the</td><td>Phe</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td>25Ρ</td><td></td><td></td><td></td><td> 30</td><td></td>

<210> 10 <211> 116 <212> PRT aV>

ΜΑ 32982Β1

A-1472-PCT Sequence_French <213> Homo sapiens <400> 10

<td>Glue</td><td>leu</td><td>Glue</td><td>Glue</td><td>Ser</td><td>Pro</td><td>Glue</td><td>Asp</td><td>Ser</td><td>Isle</td><td>Gin</td><td>leu</td><td>Gly</td><td>val</td><td>Thr</td><td>Arg</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Asn</td><td>Lilies</td><td>Isle</td><td>Met</td><td>Thr</td><td>To the</td><td>Gin</td><td>Tyr</td><td>Glue</td><td>cys</td><td>Tyr</td><td>Gin</td><td>Lilies</td><td>Isle</td><td>Met</td><td>Gin</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Asp</td><td>Pro</td><td>Isle</td><td>Gin</td><td>Gin</td><td>To the</td><td>Glue</td><td>Gly</td><td>Val</td><td>Tyr</td><td>Cys</td><td>Asn</td><td>Arg</td><td>Thr</td><td>Trp</td><td>Asp</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45⁹</td><td></td><td></td><td></td>

<td>Gly</td><td>Trp</td><td>Leu</td><td>Cys</td><td>Trp</td><td>Asn</td><td>Asp</td><td>Val</td><td>To the</td><td>To the</td><td>Gly</td><td>Thr</td><td>Glue</td><td>Ser</td><td>Met</td><td>Gin</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td>55Ρ</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Leu</td><td>Cys</td><td>Pro</td><td>Asp</td><td>Tyr</td><td>Phe</td><td>Gin</td><td>Asp</td><td>Phe</td><td>Asp</td><td>Pro</td><td>ser</td><td>Glue</td><td>Lilies</td><td>val</td><td>Thr</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Lilies</td><td>Isle</td><td>Cys</td><td>Asp</td><td>Gin</td><td>Asp</td><td>Gly</td><td>Asn</td><td>Trp</td><td>Phe</td><td>Arg</td><td>His</td><td>Pro</td><td>To the</td><td>Ser</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Arg</td><td>Thr</td><td>Trp</td><td>Thr</td><td>Asn</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Cys</td><td>Asn</td><td>Val</td><td>Asn</td><td>Thr</td><td>His</td><td>Glue</td><td>Lilies</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

Val Lys Thr Ala <210> 11 <211> 91 <212> PRT <213> Homo sapiens

<td>&lt;400&gt; 11 Cys Gin Glu</td><td>To the</td><td>Asn 5</td><td>Tyr</td><td>Gly</td><td>To the</td><td>Leu</td><td>Leu 10</td><td>Arg</td><td>Glue</td><td>Leu</td><td>cys</td><td>leu 15</td><td>Thr</td>

<td>ل Gin Phe Gin</td><td>val</td><td>Asp</td><td>Met</td><td>Glue</td><td>To the</td><td>١ ةلا</td><td>Gly</td><td>Glue</td><td>Thr</td><td>Leu</td><td>Trp</td><td>Cys</td><td>Asp</td>

<td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td>30Ρ</td><td></td><td></td>

<td>Trp Gly Arg</td><td>Thr</td><td>Isle</td><td></td><td>Ser</td><td>Tyr</td><td>Arg</td><td>Glue</td><td>Leu</td><td>To the</td><td>Asp</td><td>Cys</td><td>Thr</td><td>Trp</td>

<td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td>45Ρ</td><td></td><td></td><td></td>

<td>His Met Ala</td><td>Glue</td><td>Lilies</td><td>Leu</td><td>Gly</td><td>Cys</td><td>Phe</td><td>Trp</td><td>P٣o</td><td>Asn</td><td>To the</td><td>Glue</td><td>٦ ةلآ</td><td>Asp</td>

<td> 50</td><td></td><td></td><td></td><td>55Υ</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Arg Phe Phe</td><td>Leu</td><td>To the</td><td>val</td><td>His</td><td>Gly</td><td>Arg</td><td>Tyr</td><td>Phe</td><td>Arg</td><td>Ser</td><td>Cys</td><td>Pro</td><td>Isle</td>

<td> 65</td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Ser Gly Arg</td><td>To the</td><td>val</td><td>Macaw</td><td>Asp</td><td>Pro</td><td>Pro</td><td>Gly</td><td>Ser</td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;20 12</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;211&gt; 238</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>&lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

/1$¼

ΜΑ 32982Β1

A-1472-PCT Sequence_French

Artifi cial SZquence source / note = Description of artificial SZquence: synthetic polypeptide 12 <21 <220>

<221>

<223>

<400>

<td>١٨el</td><td>Asp</td><td>Met</td><td>Arg</td><td>Val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Gly</td><td>leu</td><td>leu</td><td>leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>R</td><td>Cys</td><td>Gin</td><td>Ser</td><td>Val</td><td>leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>ser</td><td>val</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>Glue</td><td>To the</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Lilies</td><td>val</td><td>Ihr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>val</td><td>Ser</td><td>Trp</td><td>٣ لآ ٦</td><td>Gin</td><td>Gin</td><td>Leu</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> ؛5</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>thr</td><td>To the</td><td>Pro</td><td>lily</td><td>leu</td><td>leu</td><td>Isle</td><td>Tyr</td><td>Asp</td><td>Asn</td><td>Asn</td><td>lily</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Isle</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>ser</td><td>ةلا 1</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Ser</td><td>To the</td><td>Thr</td><td>leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Isle</td><td>Thr</td><td>Gly</td><td>leu</td><td>Gin</td><td>Thr</td><td>Gly</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>thr</td><td>Trp</td><td>Asp</td><td>Ser</td><td>Arg</td><td>leu</td><td>Ser</td><td>To the</td><td>Val</td><td>val</td><td>Phe</td><td>Gly</td><td colspan="2">Gly Gly</td><td>Thr</td><td>lily</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>lily</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>Val</td><td>Thr</td><td>leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>Leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>s لاط</td><td>To the</td><td>Thr</td><td>Leu</td><td>val</td><td>Cys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Isle</td><td>Ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>Val</td><td>Thr</td><td>Val</td><td>To the</td><td>Trp</td><td>Lilies</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp</td><td>Gly</td><td>Ser</td><td>Pro</td><td>Val</td><td>lily</td><td>To the</td><td>Gly</td><td>Val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>lily</td><td>Pro</td><td>Ser</td><td>Lilies</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin</td><td>Ser</td><td>Asn</td><td>Asn</td><td>lily</td><td>lyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>leu</td><td>Ser</td><td>Leu</td><td>thr</td><td>Pro</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>Trp</td><td>lily</td><td>Ser</td><td>His</td><td>Arg</td><td>Ser</td><td>Tyr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>val</td><td>Thr</td><td>His</td><td>Glue</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ser</td><td>Thr</td><td>Val</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>val</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<210> 13

5 ىر

ΜΑ 32982Β1

A-1472-PCT sequence_French <211 <21 <213>

238

PRT Artificial Sequence Synthetic Polypeptide Artificial Sequence:

<220>

<221> source <223> / note = Description of <400> 13

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Leu</td><td>leu</td><td>leu</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>1 PII</td><td>Arg</td><td>Gly</td><td>To the</td><td>Macaw</td><td>Cys</td><td>Gin</td><td>Ser</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ser</td><td>To the</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>Gly</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Arg</td><td>Val</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Ser</td><td>Asn</td><td>lyr</td><td>Val</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>leu</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>To the</td><td>To the</td><td>Pro</td><td>lily</td><td>leu</td><td>Leu</td><td>Isle</td><td>Phe</td><td>Arg</td><td>Ser</td><td>Asn</td><td>Gin</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>lily</td><td>Ser</td><td>Gly</td><td>Thr</td><td>ser</td><td>To the</td><td>Ser</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>To the</td><td>Isle</td><td>Ser</td><td>Gly</td><td>leu</td><td>Arg</td><td>Ser</td><td>Glue</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>To the</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>To the</td><td>Trp</td><td>Asp</td><td>Asp</td><td>Ser</td><td>leu</td><td>Ser</td><td>Gly</td><td>Irp</td><td>val</td><td>Phe</td><td colspan="3">Gly Gly Gly</td><td>Thr</td><td>lily</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>leu</td><td>Thr</td><td>val</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>lily</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Thr</td><td>leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Pro</td><td>ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>Leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>lily</td><td>To the</td><td>Thr</td><td>leu</td><td>val</td><td>Cys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>leu</td><td>Isle</td><td>ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>٦ ةلآ</td><td>Thr</td><td>val</td><td>To the</td><td>Trp</td><td>lily</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Asp</td><td>Gly</td><td>Ser</td><td>Pro</td><td>Val</td><td>lily</td><td>To the</td><td>Gly</td><td>Val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>Ser</td><td>Lilies</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin</td><td>ser</td><td>Asn</td><td>Asn</td><td>lily</td><td>Tyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>leu</td><td>Ser</td><td>leu</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>Trp</td><td>lily</td><td>Ser</td><td>His</td><td>Arg</td><td>Ser</td><td>Tyr</td><td>ser</td><td>Cys</td><td>Gin</td><td>val</td><td>Thr</td><td>His</td><td>Glue</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ser</td><td>Thr</td><td>val</td><td>Glue</td><td>lily</td><td>Thr</td><td>val</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

Λ

ΜΑ 32982Β1

A-1472-PCT Sequence-French Synthetic Polypeptide <210> 14 <211> 236 <212> PRT <213 ح Sequence Artificial Sequence Artificial:

<220>

<221 source <223> / note = Description of <400> 14

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>leu</td><td>Gly</td><td>leu</td><td>leu</td><td>leu</td><td>Leu</td><td>trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Asp</td><td>Isle</td><td>Gin</td><td>Met</td><td>Thr</td><td>Gin</td><td>ser</td><td>pro</td><td>Ser</td><td>ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>leu</td><td>Ser</td><td>To the</td><td>Ser</td><td>val</td><td>Gly</td><td>Asp</td><td>K٢a</td><td>val</td><td>Thr</td><td>Isle</td><td>Thr</td><td>Cys</td><td>Arg</td><td>To the</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> ؛4</td><td></td><td></td><td></td>

<td>Gin</td><td>Gly</td><td>Isle</td><td>Arg</td><td>Asn</td><td>Asp</td><td>Leu</td><td>Gly</td><td>trp</td><td>Phe</td><td>Gin</td><td>Gin</td><td>Lilies</td><td>Pro</td><td>Gly</td><td>lily</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>To the</td><td>P٣o</td><td>lily</td><td>Arg</td><td>1 PII</td><td>Isle</td><td>tyr</td><td>To the</td><td>To the</td><td>ser</td><td>ser</td><td>leu</td><td>Gin</td><td>ser</td><td>Gly</td><td>Val</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Pro</td><td>ser</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Glue</td><td>Phe</td><td>Thr</td><td>leu</td><td>thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Isle</td><td>ser</td><td>Ser</td><td>Leu</td><td>Gin</td><td>Pro</td><td>Glue</td><td>Asp</td><td>leu</td><td>To the</td><td>Thr</td><td>tyr</td><td>Tyr</td><td>لآلاح</td><td>Leu</td><td>Gin</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>tyr</td><td>Asn</td><td>Isle</td><td>Tyr</td><td>Pro</td><td>Trp</td><td>Thr</td><td>phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>s لاط</td><td>val</td><td>Glue</td><td>Isle</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>lily</td><td>Arg</td><td>Thr</td><td>val</td><td>To the</td><td>To the</td><td>Pro</td><td>ser</td><td>Val</td><td>Phe</td><td>Isle</td><td>Phe</td><td>Pro</td><td>Pro</td><td>ser</td><td>Asp</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>leu</td><td>Lilies</td><td>ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>٦ ةلآ</td><td>val</td><td>Cys</td><td>Leu</td><td>leu</td><td>Asn</td><td>Asn</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>Tyr</td><td>Pro</td><td>Arg</td><td>Glue</td><td>To the</td><td>Lilies</td><td>val</td><td>Gin</td><td>Trp</td><td>lily</td><td>٦ جلآ</td><td>Asp</td><td>Asn</td><td>To the</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Gin</td><td>Ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>Ser</td><td>val</td><td>Thr</td><td>Glue</td><td>Gin</td><td>Asp</td><td>Ser</td><td>lily</td><td>Asp</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ser</td><td>Thr</td><td>Tyr</td><td>Ser</td><td>leu</td><td>Ser</td><td>ser</td><td>Thr</td><td>leu</td><td>Thr</td><td>Leu</td><td>Ser</td><td>lily</td><td>To the</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glue</td><td>s لالم</td><td>His</td><td>Lilies</td><td>Val</td><td>Tyr</td><td>To the</td><td>cys</td><td>Glue</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Pro</td><td>Val</td><td>Thr</td><td>lily</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glue</td><td>Cys</td><td></td><td></td><td></td><td></td>

ج $ ١ ر

ΜΑ 32982Β1

225

230

A-1472-PCT sequence_French

235 <21 15 <211 236 <212> PRT <213> Artificial Sequence Synthetic Polypeptide Artificial Sequence:

<220>

<221 source <223> / note = Description of <400> 15

<td>Met</td><td>Asp</td><td>Met</td><td>Arq</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>A٢q</td><td>Cys</td><td>Ser</td><td>Ser</td><td>Glue</td><td>leu</td><td>Thr</td><td>Gin</td><td>Asp</td><td>Pro</td><td>Thr</td><td>٦ ةلآ</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>val</td><td>To the</td><td>1 PII</td><td>Gly</td><td>Gin</td><td>Thr</td><td>Val</td><td>Lilies</td><td>Isle</td><td>Thr</td><td>Cys</td><td>Gin</td><td>Gly</td><td>Asp</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Leu</td><td>Arg</td><td>Ser</td><td>Phe</td><td>Tyr</td><td>To the</td><td>Ser</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>lily</td><td>Pro</td><td>Gly</td><td>Gin</td><td>To the</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>val</td><td>Leu</td><td>Val</td><td>Phe</td><td>tyr</td><td>Gly</td><td>lily</td><td>Asn</td><td>Asn</td><td>Arg</td><td>Pro</td><td>ser</td><td>Gly</td><td>Isle</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td>و 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td><td>Ser</td><td>Gly</td><td>Asn</td><td>Thr</td><td>To the</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Isle</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Gly</td><td>To the</td><td>Gin</td><td>To the</td><td>Glue</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Asn</td><td>Ser</td><td>Arg</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Asp</td><td>Ser</td><td>Ser</td><td>val</td><td>Tyr</td><td>His</td><td>Leu</td><td>val</td><td>Leu</td><td colspan="3">Gly Gly Gly</td><td>Thr</td><td>lily</td><td>Leu</td><td>Thr</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Val</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>lily</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Thr</td><td>Leu</td><td>Phe</td><td>Pro</td><td>Pro</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>Lilies</td><td>To the</td><td>Thr</td><td>Leu</td><td>val</td><td>Cys</td><td>leu</td><td>Isle</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>val</td><td>Thr</td><td>val</td><td>To the</td><td>Trp</td><td>Lilies</td><td>To the</td><td>Asp</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Ser</td><td>Pro</td><td>val</td><td>Lilies</td><td>To the</td><td>Gly</td><td>val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>Ser</td><td>lily</td><td>Gin</td><td>Ser</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Asn</td><td>Asn</td><td>lily</td><td>Tyr</td><td>To the</td><td>To the</td><td>ser</td><td>Ser</td><td>Tyr</td><td>leu</td><td>ser</td><td>Leu</td><td>Thr</td><td>Pro</td><td>Glue</td><td>Gin</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Irp</td><td>lily</td><td>Ser</td><td>His</td><td>Arg</td><td>Ser</td><td>Tyr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>Val</td><td>Thr</td><td>His</td><td>Glue</td><td>Gly</td><td>ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<img file="MA32982B1_D0029.tif" />

»ألر

ΜΑ 32982Β1

A-1472-PCT Sequence_French Thr Val Glu lys Thr val Ala ΡΓΟ lhr Glu cys Ser 225 y 230 235 <20 16 <211 241 <21 PRT <213> Sequence artificial synthetic polypeptide '' artificial sequence:

<220>

<221 source <223> / note = Descri pti on of <400> 16

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>pro</td><td>To the</td><td>Gin</td><td>leu</td><td>Leu</td><td>Gly</td><td>leu</td><td>leu</td><td>leu</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Asp</td><td>Isle</td><td>Isle</td><td>1 PII</td><td>To the</td><td>Gin</td><td>Thr</td><td>Pro</td><td>leu</td><td>Ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>leu</td><td>ser</td><td>val</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td><td>To the</td><td>Ser</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Lilies</td><td>ser</td><td>ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td>؟ ة</td><td></td><td></td><td></td>

<td>Gin</td><td>Ser</td><td>Leu</td><td>leu</td><td>His</td><td>ser</td><td>To the</td><td>Gly</td><td>Lilies</td><td>lhr</td><td>Tyr</td><td>leu</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Lilies</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gin</td><td>leu</td><td>leu</td><td>Isle</td><td>Tyr</td><td>Glue</td><td>val</td><td>Ser</td><td>Asn</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>val</td><td>Pro</td><td>Asp</td><td>لآينإ</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Asp</td><td>Phe</td><td>lhr</td><td>leu</td><td>lily</td><td>Isle</td><td>Ser</td><td>Arg</td><td>٦ ةل ١</td><td>Glue</td><td>To the</td><td>Glue</td><td>Asp</td><td>val</td><td>Gly</td><td>Isle</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Met</td><td>Gin</td><td>ser</td><td>Phe</td><td>Pro</td><td>leu</td><td>Pro</td><td>leu</td><td>Thr</td><td>Phe</td><td>Gly</td><td colspan="2">Gly Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>lily</td><td>val</td><td>Glue</td><td>Isle</td><td>lily</td><td>Arg</td><td>Thr</td><td>٦ ةلآ</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>val</td><td>Phe</td><td>Isle</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 13?</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Glue</td><td>Gin</td><td>leu</td><td>Lilies</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>Val</td><td>val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> ؛15</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>cys</td><td>leu</td><td>leu</td><td>Asn</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Arq</td><td>Glue</td><td>To the</td><td>lily</td><td>٦ ةلا</td><td>Gin</td><td>Trp</td><td>Lilies</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td>Asp</td><td>Asn</td><td>To the</td><td>leu</td><td>Gin</td><td>ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>ser</td><td>val</td><td>Thr</td><td>Glue</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>GTn</td><td>Asp</td><td>Ser</td><td>ys</td><td>Asp</td><td>ser</td><td>Thr</td><td>Tyr</td><td>Ser</td><td>leu</td><td>Ser</td><td>Ser</td><td>Thr</td><td>leu</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>ser</td><td>lily</td><td>AT has</td><td>Asp</td><td>lyr</td><td>Glue</td><td>Lilies</td><td>His</td><td>lily</td><td>val</td><td>Tyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>val</td><td>Thr</td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French

210

215

220

His

225

Gin Gly Leu ser

Ser

230

Pro Val Thr Lys

Ser

235

Phe Asn Arg Gly

Glue

240

Cys <20 17 <211 238 <212> PRT <213> Sequence artificial synthetic polypeptide artificial sequence:

<220>

<221> source <223> / note = Description of <400> 17

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>leu</td><td>leu</td><td>Gly</td><td>leu</td><td>leu</td><td>Leu</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Gin</td><td>Ser</td><td>Val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ser</td><td>val</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>To the</td><td>To the</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Lilies</td><td>Val</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>val</td><td>Ser</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Leu</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> ؛5</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>To the</td><td>Pro</td><td>Lilies</td><td>leu</td><td>leu</td><td>Isle</td><td>Tyr</td><td>Asp</td><td>Asn</td><td>Asn</td><td>ةلاأ</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Isle</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>ةلاأ</td><td>ser</td><td>Gly</td><td>Thr</td><td>ser</td><td>Thr</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Isle</td><td>Thr</td><td>Gly</td><td>leu</td><td>Gin</td><td>Thr</td><td>Gly</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>s لاح</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Thr</td><td>Trp</td><td>Asp</td><td>Ser</td><td>Arg</td><td>Leu</td><td>Ser</td><td>To the</td><td>val</td><td>val</td><td>Phe</td><td colspan="3">Gly Gly Gly</td><td>Thr</td><td>Lilies</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>leu</td><td>Thr</td><td>Val</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Lilies</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Thr</td><td>leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>Leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>ةلاأ</td><td>To the</td><td>Thr</td><td>Leu</td><td>val</td><td>Cys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Isle</td><td>Ser</td><td>Asp</td><td>Phe</td><td>lyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>val</td><td>Thr</td><td>١ ةلآ</td><td>To the</td><td>Trp</td><td>lily</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp</td><td>Gly</td><td>Ser</td><td>Pro</td><td>Val</td><td>Lilies</td><td>To the</td><td>Gly</td><td>Val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>lily</td><td>Pro</td><td>Ser</td><td>Lilies</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

/

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Gin</td><td>Ser</td><td>Asn</td><td>Asn</td><td>lily</td><td>Tyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>Leu</td><td>Ser</td><td>leu Thr</td><td>Pro</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>Trp</td><td>lily</td><td>Ser</td><td>His</td><td>Arg</td><td>Ser</td><td>lyr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>٦ لآل ١</td><td>Thr His</td><td>Glue</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td>

<td>Gly</td><td>ser</td><td>Thr</td><td>val</td><td>Glue</td><td>lily</td><td>Thr</td><td>١ ةلا</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td>

225 230 235 &lt;21 18 &lt;211 241 &lt;21 PRT &lt;213 ^ artificial sequence Synthetic polypeptide '' artificial sequence (^:

<220>

<221 ح source <223> / note = Description of <400> 18

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>you</td><td>you</td><td>Gly</td><td>you</td><td>you</td><td>you</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Leu</td><td>لأمبع</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Asp</td><td>Isle</td><td>٦ ةلا</td><td>Met</td><td>Thr</td><td>Gin</td><td>Ser</td><td>Pro</td><td>you</td><td>Ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>leu</td><td>Pro</td><td>val</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Glue</td><td>Pro</td><td>To the</td><td>Ser</td><td>Isle</td><td>Ser</td><td></td><td>لأمبع</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> ؛4</td><td></td><td></td><td></td>

<td>Gin</td><td>Ser</td><td>leu</td><td>leu</td><td>His</td><td>Ser</td><td>Phe</td><td>Gly</td><td>Tyr</td><td>Asn</td><td>Tyr</td><td>leu</td><td>Asp</td><td>Trp</td><td>Tyr</td><td>Leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>lily</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Ser</td><td>Pro</td><td>Gin</td><td>you</td><td>you</td><td>Isle</td><td>٢yr</td><td>leu</td><td>Gly</td><td>ser</td><td>Asn</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg</td><td>To the</td><td>Ser</td><td>Gly</td><td>Val</td><td>Pi-fl</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>ser</td><td>Gly</td><td>ser</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95٧</td><td></td>

<td>Asp</td><td>Phe</td><td>Thr</td><td>you</td><td>lily</td><td>Isle</td><td>Ser</td><td>لأمبع</td><td>Val</td><td>Glue</td><td>To the</td><td>Glue</td><td>Asp</td><td>٦ ةا \</td><td>Gly</td><td>Val</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Met</td><td>Gin</td><td>To the</td><td>leu</td><td>Gin</td><td>Thr</td><td>Pro</td><td>Phe</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Pro</td><td>Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>lily</td><td>Val</td><td>Asp</td><td>Isle</td><td>Lilies</td><td>Arg</td><td>Thr</td><td>Val</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>٦ ةلا</td><td>Phe</td><td>Isle</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Ksp</td><td>Glue</td><td>Gin</td><td>you</td><td>Lilies</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>ser</td><td>val</td><td>٦ ةى ١</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Cys</td><td>leu</td><td>leu</td><td>Asn</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Arg</td><td>Glue</td><td>To the</td><td>Lilies</td><td>val</td><td>Gin</td><td>Trp</td><td>Lilies</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Val</td><td>Asp</td><td>Asn</td><td>To the</td><td>leu</td><td>Gin</td><td>Ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>Ser</td><td>val</td><td>Thr</td><td>Glue</td>

Λ٩٨

ΜΑ 32982Β1

A-1472-PCT Sequence_French

180

185 190

<td>Gin</td><td rowspan="2">Asp</td><td>Ser</td><td rowspan="2">lily</td><td rowspan="2">Asp</td><td>Ser</td><td>Thr</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser</td><td>Ihr</td><td>Leu</td><td>Thr</td><td>Leu</td>

<td></td><td> 195</td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Ser</td><td>Lilies</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Glue</td><td>Lilies</td><td>His</td><td>Lilies</td><td>val</td><td>Tyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>val</td><td>Thr</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>His</td><td>Gin</td><td rowspan="2">Gly</td><td>Leu</td><td>Ser</td><td>Ser</td><td>Pro</td><td>Val</td><td>Thr</td><td rowspan="2">Lilies</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glue</td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

Cys <20 19 <211> 241 <21 PRT <213> Artificial Sequence Synthetic Polypeptide Artificial Sequence:

<220>

<221> source <223> / note = Descri pti on of <400> 19

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Gly</td><td>leu</td><td>leu</td><td>Leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Asp</td><td>Isle</td><td>Isle</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Thr</td><td>Pro</td><td>1 PII</td><td>ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>Ser</td><td>val</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td><td>To the</td><td>Ser</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Lilies</td><td>Ser</td><td>ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Gin</td><td>Ser</td><td>leu</td><td>Leu</td><td>His</td><td>Ser</td><td>Asp</td><td>Gly</td><td>Lilies</td><td>Thr</td><td>lyr</td><td>Leu</td><td>lyr</td><td>Trp</td><td>Tyr</td><td>leu</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>ةلا 1</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Isle</td><td>lyr</td><td>Glue</td><td>Val</td><td>Ser</td><td>Asn</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Glue</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>ser</td><td>Gly</td><td>ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95٧</td><td></td>

<td>Asp</td><td>Phe</td><td>Thr</td><td>leu</td><td>Lilies</td><td>Isle</td><td>Ser</td><td>Arg</td><td>Val</td><td>Glue</td><td>To the</td><td>Glue</td><td>Asp</td><td>val</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Tyr</td><td>٢ لاآ</td><td>Cys</td><td>Met</td><td>Gin</td><td>Ser</td><td>Phe</td><td>Pro</td><td>Leu</td><td>Pro</td><td>Leu</td><td>Thr</td><td>Phe</td><td colspan="3">Gly Gly Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>Lilies</td><td>Val</td><td>Glue</td><td>Isle</td><td>lily</td><td>Arg</td><td>Thr</td><td>Val</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>val</td><td>Phe</td><td>Isle</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Glue</td><td>Gin</td><td>leu</td><td>lily</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>val</td><td>val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

كأود

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Cys</td><td>leu</td><td>leu</td><td>Asn</td><td>Asn 165</td><td>Phe</td><td>Tyr</td><td>Pro</td>

<td>Val</td><td>Asp</td><td>Asn</td><td>To the</td><td>leu</td><td>Gin</td><td>Ser</td><td>Gly</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Asp</td><td>Ser</td><td>Lilies</td><td>Asp</td><td>Ser</td><td>Thr</td><td>Tyr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td>

<td>Ser</td><td>Lilies</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Glue</td><td>Lilies</td><td>His</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td>

<td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>ser</td><td>Ser</td><td>Pro</td><td>val</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td>

<td>Arg</td><td>Glue</td><td>To the</td><td>lily</td><td>val</td><td>Gin</td><td>Trp</td><td>Lilies</td>

<td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asn</td><td>Ser</td><td>Gin</td><td>G٦u</td><td>Ser</td><td>Val</td><td>Thr</td><td>Glue</td>

<td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ser</td><td>Leu</td><td>ser</td><td>Ser</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Lilies</td><td>val</td><td>Tyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>val</td><td>Thr</td>

<td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Lilies</td><td>ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glue</td>

<td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

Cys <20 20 <21 238 <212> PRT <213> Artificial Sequence Synthetic Polypeptide Artificial Sequence:

<220>

<221 source <223> / note = Description of <400> 20

<td>Met</td><td>Asp</td><td>v \ el</td><td>Macaw</td><td>Val</td><td>Pro</td><td>To the</td><td>Gin</td><td>leu</td><td>Leu</td><td>Gly</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Gin</td><td>Ser</td><td>val</td><td>leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ser</td><td>٦ ةلآ</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>To the</td><td>To the</td><td>pro</td><td>Gly</td><td>Gin</td><td>lily</td><td>val</td><td>Thr</td><td>Isle</td><td>ser</td><td>IMS نمل</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>٦ ةلآ</td><td>Ser</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Phe</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>To the</td><td>Pro</td><td>lily</td><td>Leu</td><td>Leu</td><td>Isle</td><td>Tyr</td><td>Asp</td><td>Asn</td><td>Asn</td><td>Lilies</td><td>ا ٢0 ح</td><td>Pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Isle</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Lilies</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Ser</td><td>To the</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Isle</td><td>Thr</td><td>Gly</td><td>1 PII</td><td>Gin</td><td>Thr</td><td>Gly</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>lily</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Thr</td><td>Trp</td><td>Asp</td><td>ser</td><td>ا ٢0 ح</td><td>leu</td><td>Ser</td><td>To the</td><td>val</td><td>val</td><td>Phe</td><td colspan="2">Gly Gly</td><td>Gly</td><td>Thr</td><td>Lilies</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Leu</td><td>Thr</td><td>Val</td><td>Leu</td><td>Gly</td><td>Gin</td><td>Pre</td><td>Lilies</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Thr</td><td>Leu</td><td>Phe</td>

ة ١٠ ر

ΜΑ 32982Β1

A-1472-PCT Sequence_French

140

135

130

<td>Pro</td><td>Pro</td><td>Ser</td><td>ser</td><td>Glue</td><td>Glue</td><td>leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>lily</td><td>To the</td><td>Thr</td><td>leu</td><td>val</td><td>Cys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>leu</td><td>Tl</td><td>Ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>Val</td><td>Thr</td><td>val</td><td>To the</td><td>Trp</td><td>lily</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp</td><td>Gly</td><td>Ser</td><td>Pro</td><td>Val</td><td>Lilies</td><td>To the</td><td>Gly</td><td>Val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>Ser</td><td>lily</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin</td><td>Ser</td><td>Asn</td><td>Asn</td><td>lily</td><td>Tyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>Leu</td><td>Ser</td><td>Leu</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>Trp</td><td>Lilies</td><td>Ser</td><td>His</td><td>Arg</td><td>Ser</td><td>Tyr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>Val</td><td>Thr</td><td>His</td><td>Glue</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Ser</td><td>Thr</td><td>val</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>Val</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>ser</td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<20 21 <211 238 <212> PRT <213> artificial sequence Synthetic polypeptide artificial sequence:

<220>

<221 source <223> / note = Descri pti on of <400> 21

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>Val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>leu</td><td>Gly</td><td>leu</td><td>leu</td><td>leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Gin</td><td>Ser</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Ser</td><td>pro</td><td>Ser</td><td>To the</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>Gly</td><td>Thr</td><td>pro</td><td>Gly</td><td>Gin</td><td>Arq</td><td>val</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Ser</td><td>Asn</td><td>Tyr</td><td>val</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>leu</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>To the</td><td>To the</td><td>Pro</td><td>lily</td><td>leu</td><td>Leu</td><td>Isle</td><td>leu</td><td>Arg</td><td>Asn</td><td>Asn</td><td>Gin</td><td>Aro ها</td><td>pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Lilies</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Ser</td><td>To the</td><td>Ser</td><td>leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>Isle</td><td>Ser</td><td>Gly</td><td>leu</td><td>Arg</td><td>ser</td><td>Glue</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>To the</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>To the</td><td>Trp</td><td>Asp</td><td>Asp</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Gly</td><td>Trp</td><td>val</td><td>Phe</td><td colspan="2">Gly Gly</td><td>Gly</td><td>Thr</td><td>lily</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Leu</td><td>Ihr</td><td>val</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>lily</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>Val</td><td>Ihr</td><td>leu</td><td>Phe</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>pro</td><td>Ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>Lilies</td><td>To the</td><td>Thr</td><td>leu</td><td>val</td><td>Cys</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu</td><td>Isle</td><td>Ser</td><td>Asp</td><td>Phe</td><td>lyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>Val</td><td>Thr</td><td>val</td><td>To the</td><td>Trp</td><td>Lilies</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp</td><td>Gly</td><td>Ser</td><td>Pro</td><td>Val</td><td>lily</td><td>To the</td><td>Gly</td><td>val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>ser</td><td>Lilies</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin</td><td>ser</td><td>Asn</td><td>Asn</td><td>lily</td><td>Tyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>Leu</td><td>Ser</td><td>leu</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>Irp</td><td>Lilies</td><td>Ser</td><td>His</td><td>Arg</td><td>Ser</td><td>٢yr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>val</td><td>Thr</td><td>His</td><td>Glue</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>ser</td><td>Thr</td><td>val</td><td>Glue</td><td>lily</td><td>Thr</td><td>val</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<20 22 <211 238 <212> PRT <213> Artificial Sequence Synthetic Polypeptide Artificial Sequence:

<220>

<221 source <223> / note = Description of <400> 22

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>leu</td><td>Gly</td><td>leu</td><td>Leu</td><td>leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>cys</td><td>Gin</td><td>ser</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>ser</td><td>To the</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>Gly</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Arg</td><td>val</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Ser</td><td>Asn</td><td>Thr</td><td>Val</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Leu</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>To the</td><td>Pro</td><td>lily</td><td>Leu</td><td>Leu</td><td>Isle</td><td>Tyr</td><td>Thr</td><td>Asn</td><td>Asn</td><td>Gin</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>val</td><td>Pro</td><td>Asp</td><td>Macaw</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>lily</td><td>Ser</td><td>Gly</td><td>Thr</td><td>ser</td><td>To the</td><td>ser</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>To the</td><td>Isle</td><td>Ser</td><td>Gly</td><td>leu</td><td>Gin</td><td>Ser</td><td>Glue</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Cys</td><td>To the</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>To the</td><td>Arg</td><td>Asp</td><td>Glue</td><td>Ser</td><td>Leu</td><td>Asn</td><td>Gly</td><td>val</td><td>val</td><td>Phe</td><td colspan="2">Gly Gly</td><td>Gly</td><td>Thr</td><td>lily</td>

هالآد

ΜΑ 32982Β1

Α-1472-ΡΤ sequence_French 120 125

115

<td>Leu Thr</td><td>val</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Lilies</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Thr</td><td>Leu</td><td>Phe</td>

<td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro Pro</td><td>Ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>Leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>Lilies</td><td>To the</td><td>Thr</td><td>Leu</td><td>val</td><td>cys</td>

<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu Island</td><td>Ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>val</td><td>Thr</td><td>Val</td><td>To the</td><td>Trp</td><td>Lilies</td><td>To the</td>

<td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp Gly</td><td>Ser</td><td>Pro</td><td>val</td><td>Lilies</td><td>To the</td><td>Gly</td><td>val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>Ser</td><td>Lilies</td>

<td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin Ser</td><td>Asn</td><td>Asn</td><td>Lilies</td><td>Tyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>Leu</td><td>Ser</td><td> 1*11</td><td>Thr</td><td>Pl *</td>

<td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glu Gin</td><td>Trp</td><td>Lilies</td><td>ser</td><td>His</td><td>Arg</td><td>ser</td><td>Tyr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>val</td><td>Thr</td><td>His</td><td>Glue</td>

<td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly Ser</td><td>Thr</td><td>val</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>val</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;210&gt; 23</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;21 238</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="6">&lt;213&gt; Artificial SZquence</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3">&lt;221&gt; source</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;223&gt; / note =</td><td colspan="4">:Description of</td><td colspan="6">Artificial SZquence:</td><td colspan="3">polypeptide</td>

<td colspan="2"> &lt;400&gt; 23</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Met Asp</td><td>Met</td><td>Krq</td><td>Val</td><td>Pro</td><td>To the</td><td>Gin</td><td>leu</td><td>leu</td><td>Gly</td><td>Leu</td><td>had</td><td>leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu A٣q</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Gin</td><td>ser</td><td>Val</td><td>leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ser</td><td>To the</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser Gly</td><td>Thr</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Arg</td><td>val</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>ser</td>

<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser Asn</td><td>Isle</td><td>Gly</td><td>Ser</td><td>Asn</td><td>Tyr</td><td>val</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Leu</td><td>Pl *</td><td>Gly</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Ala Ala</td><td>Pro</td><td>Lilies</td><td>leu</td><td>Leu</td><td>Isle</td><td>Phe</td><td>Arg</td><td>Asn</td><td>Asn</td><td>Gin</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Gly</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Val Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>ser</td><td>Lilies</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Ser</td><td>To the</td><td>Ser</td><td>Leu</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ala Island</td><td>Ser</td><td>Gly</td><td>leu</td><td>Arg</td><td>Ser</td><td>Glue</td><td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>To the</td>

<td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

.الآد

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Ala Trp</td><td>Asp</td><td>Asp</td><td>ser</td><td>leu</td><td>Ser</td><td>Gly</td><td>Trp</td><td>val</td><td>Phe</td><td>Gly</td><td>Gly</td><td>Gly</td><td>Thr</td><td>ةلا 1</td>

<td></td><td>ll</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Leu Thr</td><td>val</td><td>leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>lily</td><td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Ttir</td><td>leu</td><td>Phe</td>

<td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>pro pro</td><td>ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>leu</td><td>Gin</td><td>To the</td><td>Asn</td><td>ةلاأ</td><td>To the</td><td>Thr</td><td>leu</td><td>go to</td><td>cys</td>

<td> 145</td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Leu Island</td><td>Ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td><td>To the</td><td>Val</td><td>Thr</td><td>val</td><td>To the</td><td>Trp</td><td>Lilies</td><td>To the</td>

<td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Asp Gly</td><td>Ser</td><td>Pro</td><td>Val</td><td>Lilies</td><td>To the</td><td>Gly</td><td>Val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>Ser</td><td>Lilies</td>

<td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin Ser</td><td>Asn</td><td>Asn</td><td></td><td>Tyr</td><td>To the</td><td>To the</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>leu</td><td>Ser</td><td>leu</td><td>Thr</td><td>Pro</td>

<td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glu Gin</td><td>Trp</td><td>lily</td><td>Ser</td><td>His</td><td>Macaw</td><td>ser</td><td>Tyr</td><td>Ser</td><td>cys</td><td>Gin</td><td>val</td><td>Thr</td><td>His</td><td>Glue</td>

<td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Gly Ser</td><td>Thr</td><td>Val</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>val</td><td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td><td></td>

<td> ؛22</td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;20 24</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;211 241</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="6">&lt;213&gt; artificial sequence</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3">&lt;221 source</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;223&gt; / note =</td><td colspan="4">:"Description of</td><td colspan="6">artificial sequence:</td><td colspan="3">synthetic polypeptide</td>

<td colspan="2"> &lt;400&gt; 24</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Met Asp</td><td>TAel</td><td>لأيع</td><td>Val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>leu</td><td>Gly</td><td>leu</td><td>had</td><td>had</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu Arg</td><td>Gly</td><td>To the</td><td></td><td>Cys</td><td>Asp</td><td>Isle</td><td>Thr</td><td>leu</td><td>Thr</td><td>Gin</td><td>Thr</td><td>Pro</td><td>leu</td><td>ser</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu Ser</td><td>Val</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td><td>To the</td><td>Ser</td><td>Isle</td><td>Ser</td><td>ةلاأ</td><td>lily</td><td>Ser</td><td>Ser</td>

<td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> ؛4</td><td></td><td></td><td></td>

<td>Gin Ser</td><td>leu</td><td>leu</td><td>His</td><td>Ser</td><td>Asp</td><td>Gly</td><td>Arg</td><td>Asn</td><td>Tyr</td><td>leu</td><td>Tyr</td><td>Trp</td><td>Tyr</td><td>leu</td>

<td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gin Lily</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Gin</td><td>leu</td><td>leu</td><td>Isle</td><td>Tyr</td><td>Glue</td><td>val</td><td>Ser</td><td>Asn</td>

<td> 65</td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Arg Phe</td><td>ser</td><td>Gly</td><td>Leu</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td>

<td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95٧</td><td></td>

<td>Asp Phe</td><td>Thr</td><td>leu</td><td>lily</td><td>Isle</td><td>Ser</td><td>Aro</td><td>Val</td><td>Glue</td><td>To the</td><td>Glue</td><td>Asp</td><td>val</td><td>Gly</td><td>Isle</td>

جكحدد

ΜΑ 32982Β1

A-1472-PCT Sequence_French 105 110

100

<td>Tyr</td><td>Tyr</td><td>Cys</td><td>Met</td><td>Gin</td><td>Ser</td><td>Phe</td><td>Pro</td><td>Leu</td><td>Pro</td><td>leu</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gly</td><td>Gly</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Thr</td><td>Lilies</td><td>Val</td><td>Glue</td><td>Isle</td><td>Lilies</td><td>Arg</td><td>Thr</td><td>val</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>val</td><td>Phe</td><td>Isle</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Glue</td><td>Gin</td><td>Leu</td><td>ةلاأ</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>val</td><td>val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Cys</td><td>Leu</td><td>leu</td><td>Asn</td><td>Asn</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Arg</td><td>Glue</td><td>To the</td><td>Lilies</td><td>Val</td><td>Gin</td><td>Trp</td><td>ةلا 1</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Val</td><td>Asp</td><td>Asn</td><td>To the</td><td>Leu</td><td>Gin</td><td>Ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>Ser</td><td>val</td><td>Thr</td><td>Glue</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Gin</td><td>Asp</td><td>Ser</td><td>Lilies</td><td>Asp</td><td>Ser</td><td>Thr</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Ser</td><td>Lilies</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Glue</td><td>lily</td><td>His</td><td>lily</td><td>Val</td><td>lyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>val</td><td>Thr</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>ser</td><td>Ser</td><td>Pro</td><td>val</td><td>Thr</td><td>Lilies</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glue</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

Cys <210> 25 <211 238 <212> PRT <213> Artificial SZquence <220>

<221> source <223> / note = Description of artificial sequence: synthetic polypeptide <400> 25

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Leu</td><td>leu</td><td>leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Gin</td><td>Ser</td><td>val</td><td>Leu</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Pro</td><td>Ser</td><td>val</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Ser</td><td>To the</td><td>To the</td><td>Pro</td><td>Gly</td><td>Gin</td><td>Lilies</td><td>val</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Gly</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Ser</td><td>Asn</td><td>Isle</td><td>Gly</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>٦ ةلآ</td><td>Ser</td><td>Trp</td><td>Fyr</td><td>Gin</td><td>Gin</td><td>Leu</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>To the</td><td>Pro</td><td>Lilies</td><td>leu</td><td>Leu</td><td>Isle</td><td>Tyr</td><td>Asp</td><td>Asn</td><td>Asn</td><td>Lilies</td><td>Arg</td><td>Pro</td><td>Ser</td><td>Gly</td>

أؤم ./

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Isle</td><td>Pro</td><td>Asp</td><td>Arg</td><td>Phe 85</td><td>ser</td><td>Gly</td><td>Ser</td>

<td>Gly</td><td>Isle</td><td>Thr</td><td>Gly</td><td>Leu</td><td>Gin</td><td>Thr</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td>

<td>Thr</td><td>Trp</td><td>Asp</td><td>Ser</td><td>Arg</td><td>Leu</td><td>ser</td><td>To the</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td>

<td>Leu</td><td>Thr</td><td>val</td><td>Leu</td><td>Gly</td><td>Gin</td><td>Pro</td><td>ةلا 1</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td>

<td>Pro</td><td>Pro</td><td>Ser</td><td>Ser</td><td>Glue</td><td>Glue</td><td>leu</td><td>Gin</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td>

<td>leu</td><td>Isle</td><td>Ser</td><td>Asp</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td>

<td>Asp</td><td>Gly</td><td>Ser</td><td>Pro</td><td>val</td><td>Lilies</td><td>To the</td><td>Gly</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Ser</td><td>Asn</td><td>Asn</td><td>Lilies</td><td>Tyr</td><td>To the</td><td>To the</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td>

<td>Glue</td><td>Gin</td><td>Trp</td><td>لا</td><td>ser</td><td>His</td><td>Arg</td><td>Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td>

<td>Gly</td><td>Ser</td><td>Thr</td><td>val</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>val</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td>

<td>ةلا_ \</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Ser</td><td>To the</td><td>Thr</td><td>Leu</td>

<td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Asp</td><td>Glue</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gly</td>

<td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Val</td><td>val</td><td>Phe</td><td colspan="3">Gly Gly Gly</td><td>Thr</td><td>Lilies</td>

<td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>To the</td><td>Asn</td><td>Pro</td><td>Thr</td><td>val</td><td>Thr</td><td>leu</td><td>Phe</td>

<td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>To the</td><td>Asn</td><td>Lilies</td><td>To the</td><td>Thr</td><td>leu</td><td>١ ة ؛ \</td><td>Cys</td>

<td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>To the</td><td>Val</td><td>Thr</td><td>val</td><td>To the</td><td>Trp</td><td>Lilies</td><td>To the</td>

<td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td>Glue</td><td>Thr</td><td>Thr</td><td>Lilies</td><td>Pro</td><td>Ser</td><td>Lilies</td>

<td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ser</td><td>Ser</td><td>Tyr</td><td>leu</td><td>Ser</td><td>leu</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Tyr</td><td>Ser</td><td>Cys</td><td>Gin</td><td>val</td><td>Thr</td><td>His</td><td>Glue</td>

<td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>To the</td><td>Pro</td><td>Thr</td><td>Glue</td><td>Cys</td><td>Ser</td><td></td><td></td>

<td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<210> 26 <211> 236 <212> PRT <213> artificial sequence <220>

<221> source

<td colspan="3">&lt;223&gt; / note:</td><td colspan="4">^ 'Description of</td><td colspan="5">artificial sequence</td><td>theÊ:</td><td colspan="3">synthetic polypeptide</td>

<td colspan="3"> &lt;400&gt; 26</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>leu</td><td>Leu</td><td rowspan="2">Gly</td><td>Leu</td><td>leu</td><td>leu</td><td>Leu</td><td rowspan="2">Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td>

<td>Leu</td><td>A٣٩</td><td>Gly</td><td>To the</td><td></td><td rowspan="2">Cys</td><td>Asp</td><td>Isle</td><td>Gin</td><td>Met</td><td>Thr</td><td>Gin</td><td>Ser</td><td>Pro</td><td>Ser</td><td>Ser</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>leu</td><td>Ser</td><td>To the</td><td>ser</td><td>val</td><td>Gly</td><td>Asp</td><td>Arg</td><td>Val</td><td>Thr</td><td>Isle</td><td>Thr</td><td>لا</td><td rowspan="2">Arg</td><td>To the</td><td>ser</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td>

<td>Gin</td><td>Gly 50</td><td>Isle</td><td>Arg</td><td>Lilies</td><td>Asp</td><td>leu 55</td><td>Gly</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin 60</td><td>lily</td><td>Pro</td><td>Gly</td><td>Lilies</td>

<td>To the</td><td>Pro</td><td>ةلاأ</td><td></td><td>leu</td><td>Isle</td><td>Tyr</td><td>Gly</td><td>To the</td><td>Ser</td><td>Ser</td><td>Leu</td><td>Gin</td><td>Ser</td><td>Gly</td><td>Val</td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Pro</td><td>Ser</td><td>Arg</td><td>Phe</td><td>ser</td><td>Gly</td><td>ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Glue</td><td>Phe</td><td>Thr</td><td>leu</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Isle</td><td>Ser</td><td>Ser</td><td>Leu</td><td>Gin</td><td>Pro</td><td>Glue</td><td>Asp</td><td>Phe</td><td>To the</td><td>Thr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Leu</td><td>Gin</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Tyr</td><td>Asn</td><td>Ser</td><td>Phe</td><td>pro</td><td>Trp</td><td>Thr</td><td>Phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lily</td><td>val</td><td>Glue</td><td>Isle</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Lilies</td><td>Arg</td><td>Thr</td><td>val</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>val</td><td>Phe</td><td>Isle</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Asp</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Glue</td><td>Gin</td><td>leu</td><td>Lilies</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>val</td><td>val</td><td>Cys</td><td>leu</td><td>leu</td><td>Asn</td><td>Asn</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>Tyr</td><td>Pro</td><td>Arg</td><td>Glue</td><td>To the</td><td>Lilies</td><td>val</td><td>Gin</td><td>Trp</td><td>Lilies</td><td>val</td><td>Asp</td><td>Asn</td><td>To the</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Gin</td><td>Ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>Ser</td><td>Val</td><td>Thr</td><td>Glue</td><td>Gin</td><td>Asp</td><td>Ser</td><td>Lilies</td><td>Asp</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ser</td><td>Thr</td><td>Tyr</td><td>Ser</td><td>leu</td><td>Ser</td><td>ser</td><td>Thr</td><td>leu</td><td>Thr</td><td>leu</td><td>Ser</td><td>lily</td><td>To the</td><td>Asp</td><td>Tyr</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Glue</td><td>lily</td><td>His</td><td>Lilies</td><td>val</td><td>Tyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Pro</td><td>٦ ةي ١</td><td>Thr</td><td>lily</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>GTu</td><td>Cys</td><td></td><td></td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<2 0 27 <211 235 <212> PRT <213> artificial sequence <220>

<221 source <223 = ► / note = Descri pti on of artificial sequences: synthetic polypeptide <400> 27

Met Glu Thr Pro Ala Gin Leu leu Phe Leu leu Leu Leu Trp Leu Pro

10 15

Asp Thr Thr Gly Glu Ile Val leu Thr Gin Ser Pro Gly Thr Leu Ser 20 25 30 leu Ser Pro Gly Glu Arg Ala Thr leu Ser Cys Arg Ala Ser Gin Ser

5 45 5 ٧ 40 5 ٧ ؛3

Val Ser Ser Gly Tyr leu Thr Trp Tyr Gin Gin lys Pro Gly Gin Ala 50 yy 55 py 60 y ٦

00

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Pro</td><td>Macaw</td><td>Leu</td><td>Leu</td><td>Isle</td><td>ryr</td><td>Gly</td><td>To the</td><td>Ser</td><td>Ser</td><td>Macaw</td><td>To the</td><td>Thr</td><td>Gly</td><td>Isle</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td>لا 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Phe</td><td>Thr</td><td>leu</td><td>Thr</td><td>Isle</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ser</td><td>MO</td><td>leu</td><td>Glue</td><td>Pro</td><td>Glue</td><td>Asp</td><td>Phe</td><td>To the</td><td>Val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Asn</td><td>Ser</td><td>Leu</td><td>Cys</td><td>Arg</td><td>Phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lily</td><td>leu</td><td>Glue</td><td>Isle</td><td>Lilies</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Arg</td><td>hr</td><td>val</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>Val</td><td>Phe</td><td>Isle</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Glue</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>G٦n</td><td>Leu</td><td>lily</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>val</td><td>val</td><td>Cys</td><td>leu</td><td>leu</td><td>Asn</td><td>Asn</td><td>Phe</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Tyr</td><td>Pro</td><td>Arg</td><td>Glue</td><td>To the</td><td>Lilies</td><td>val</td><td>Gin</td><td>Trp</td><td>Lilies</td><td>val</td><td>Asp</td><td>Asn</td><td>To the</td><td>leu</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>Ser</td><td>val</td><td>Thr</td><td>Glue</td><td>Gin</td><td>Asp</td><td>Ser</td><td>Lilies</td><td>Asp</td><td>ser</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Thr</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser</td><td>Thr</td><td>Leu</td><td>Thr</td><td>leu</td><td>ser</td><td>Lilies</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Glue</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Lilies</td><td>His</td><td>Lilies</td><td>val</td><td>Tyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>Val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Ser</td><td>ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Val</td><td>hr</td><td>lily</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glue</td><td>Cys</td><td></td><td></td><td></td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<210> 28 <211 235 <212> PRT <213> Artificial SZquence <220>

<221 source <223> / note = Description of artificial sequence: synthetic polypeptide

<td>&lt;400&gt; 28 Met Glu Thr</td><td>Pro</td><td>To the</td><td>Gin</td><td>leu</td><td>leu</td><td>Phe</td><td>Leu 10</td><td>leu</td><td>Leu</td><td>Leu</td><td>Irp</td><td>leu 15</td><td>Pro</td>

<td>Asp Thr Thr</td><td>Gly</td><td>Glue</td><td>Isle</td><td>٦ ةي ١</td><td>Leu</td><td>Thr</td><td>Gin</td><td>ser</td><td>Pro</td><td>Gly</td><td>Thr</td><td>leu</td><td>ser</td>

<td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>leu Ser Pro</td><td>Gly</td><td>Glue</td><td>fvrq</td><td>To the</td><td>Thr</td><td>leu</td><td>Ser</td><td>cys</td><td>Arg</td><td>To the</td><td>Ser</td><td>Gin</td><td>Ser</td>

<td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>val Ser Ser</td><td>Gly</td><td>Tyr</td><td>leu</td><td>Thr</td><td>Trp</td><td>Tyr</td><td>Gin</td><td>Gin</td><td>Lilies</td><td>Pro</td><td>Gly</td><td>Gin</td><td>To the</td>

/ م 0 و

ΜΑ 32982Β1

A-1472-PC Sequence_French

<td>Pro</td><td>Arg</td><td>leu</td><td>Leu</td><td>Isle</td><td>Tyr</td><td>Gly</td><td>To the</td><td>Ser</td><td>Ser</td><td>Arg</td><td>To the</td><td>Thr</td><td>Gly</td><td>Isle</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Asp</td><td>Arg</td><td>Phe</td><td>Ser</td><td>Gly</td><td>ser</td><td>Gly</td><td>Ser</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Isle</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Ser</td><td>Arg</td><td>leu</td><td>Glue</td><td>Pro</td><td>Glue</td><td>Asp</td><td>Phe</td><td>To the</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>lyr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>Asn</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Arg</td><td>Phe</td><td>Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>lily</td><td>leu</td><td>Glue</td><td>Isle</td><td>lily</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Arg</td><td>Thr</td><td>val</td><td>To the</td><td>To the</td><td>Pro</td><td>Ser</td><td>val</td><td>Phe</td><td>Isle</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Glue</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Leu</td><td>lily</td><td>Ser</td><td>Gly</td><td>Thr</td><td>To the</td><td>Ser</td><td>٦ ةلآ</td><td>Val</td><td>cys</td><td>Leu</td><td>leu</td><td>Asn</td><td>Asn</td><td>Phe</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Tyr</td><td>Pro</td><td>Arg</td><td>Glue</td><td>To the</td><td>lily</td><td>val</td><td>Gin</td><td>Trp</td><td>Lilies</td><td>val</td><td>Asp</td><td>Asn</td><td>To the</td><td>leu</td><td>Gin</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>ser</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Gin</td><td>Glue</td><td>Ser</td><td>Val</td><td>Thr</td><td>Glue</td><td>Gin</td><td>Asp</td><td>Ser</td><td>Lilies</td><td>Asp</td><td>Ser</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Thr</td><td>Tyr</td><td>Ser</td><td>leu</td><td>Ser</td><td>Ser</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Ser</td><td>Lilies</td><td>To the</td><td>Asp</td><td>Tyr</td><td>Glue</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>lily</td><td>His</td><td>Lilies</td><td>val</td><td>Tyr</td><td>To the</td><td>Cys</td><td>Glue</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>leu</td><td>Ser</td><td>Ser</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>val</td><td>Thr</td><td>lily</td><td>Ser</td><td>Phe</td><td>Asn</td><td>Arg</td><td>Gly</td><td>Glue</td><td>Cys</td><td></td><td></td><td></td><td></td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td></td>

<210> 29 <211 478 <212> PR٢ <213> SZquence artificial <220>

<221 source <223> / note = Description of artificial sequences: synthetic <400> 29 polypeptide

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>al</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu 10</td><td>Gly</td><td>Leu</td><td>Leu</td><td>leu</td><td>Leu 15</td><td>Trp</td>

<td>Leu</td><td>Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Gin</td><td>Val</td><td>Gin</td><td>Leu</td><td>Val</td><td>Glue</td><td>Ser</td><td>Gly</td><td>Gly</td><td>Gly</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30٧</td><td></td><td></td>

<td>val</td><td>٦ ةلآ</td><td>Gin</td><td>Pro</td><td>Gly</td><td>Arg</td><td>Ser</td><td>Leu</td><td>Arg</td><td>Leu</td><td>Ser</td><td>Cys</td><td>To the</td><td>To the</td><td>Ser</td><td>Gly</td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Phe</td><td>Thr</td><td>Phe</td><td>Ser</td><td>ser</td><td>Phe</td><td>Gly</td><td>Met</td><td>His</td><td>Trp</td><td>val</td><td>Arg</td><td>Gin</td><td>To the</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55٧</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>lily</td><td>Gly</td><td>Leu</td><td>Glue</td><td>Trp</td><td>val</td><td>To the</td><td>val</td><td>Isle</td><td>Ser</td><td>Phe</td><td>Asp</td><td>Gly</td><td>Ser</td><td>Isle</td><td>lily</td>

<td></td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Tyr</td><td>Ser</td><td>٦ ةلآ</td><td>Asp</td><td>ser</td><td>val</td><td>lily</td><td>Gly</td><td>A٢٩</td><td>Phe</td><td>Thr</td><td>Isle</td><td>ser</td><td>Arg</td><td>Asp</td><td>Asn</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>ser</td><td>lily</td><td>Asn</td><td>Thr</td><td>leu</td><td>Phe</td><td>Leu</td><td>Gin</td><td>Met</td><td>Asn</td><td>Ser</td><td>Leu</td><td>AT</td><td>To the</td><td>Glue</td><td>Asp</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Thr</td><td>To the</td><td>val</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>To the</td><td>A٣Q</td><td>Asp</td><td>Arg</td><td>leu</td><td>Asn</td><td>Tyr</td><td>Tyr</td><td>Asp</td><td>Ser</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>ser</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>His</td><td>Tyr</td><td>lily</td><td>Tyr</td><td>Tyr</td><td>Gly</td><td>Met</td><td>To the</td><td>val</td><td>Trp</td><td>Gly</td><td>Gin</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Thr</td><td>val</td><td>Thr</td><td>val</td><td>ser</td><td>Ser</td><td>To the</td><td>Ser</td><td>Thr</td><td>lily</td><td>Gly</td><td>Pro</td><td>ser</td><td>val</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Phe</td><td>Pro</td><td>leu</td><td>To the</td><td>Pro</td><td>Cys</td><td>Ser</td><td>A٢٩ 9</td><td>ser</td><td>Thr</td><td>Ser</td><td>Glue</td><td>ser</td><td>Thr</td><td>To the</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Leu</td><td>Gly</td><td>Cys</td><td>leu</td><td>val</td><td>lily</td><td>Asp</td><td>Tyr</td><td>Phe</td><td>Pro</td><td>Glue</td><td>Pro</td><td>١ ةلا</td><td>Thr</td><td>val</td><td>Ser</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Trp</td><td>Asn</td><td>Ser</td><td>Gly</td><td>To the</td><td>leu</td><td>Thr</td><td>Ser</td><td>Gly</td><td>Val</td><td>His</td><td>Thr</td><td>Phe</td><td>Pro</td><td>To the</td><td>val</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>leu</td><td>Gin</td><td>Ser</td><td>ser</td><td>Gly</td><td>Leu</td><td>Tyr</td><td>Ser</td><td>Leu</td><td>Ser</td><td>Ser</td><td>val</td><td>٦ ةلآ</td><td>Thr</td><td>val</td><td>Pro</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>ser</td><td>Ser</td><td>Asn</td><td>Phe</td><td>Gly</td><td>Thr</td><td>Gin</td><td>Thr</td><td>Tyr</td><td>Thr</td><td>Cys</td><td>Asn</td><td>val</td><td>Asp</td><td>His</td><td>Lilies</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Pro</td><td>Ser</td><td>Asn</td><td>Thr</td><td>Lilies</td><td>val</td><td>Asp</td><td>Lilies</td><td>Thr</td><td>val</td><td>Glue</td><td>Arg</td><td>lily</td><td>Cys</td><td>Cys</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Glue</td><td>Cys</td><td>Pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>To the</td><td>Pro</td><td>Pro</td><td>val</td><td>To the</td><td>Gly</td><td>Pro</td><td>Ser</td><td>val</td><td>Phe</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Leu</td><td>Phe</td><td>Pro</td><td>Pro</td><td>lily</td><td>Pro</td><td>lily</td><td>Asp</td><td>Thr</td><td>leu</td><td>Met</td><td>Isle</td><td>ser</td><td>Arg</td><td>Thr</td><td>Pro</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Glue</td><td>Val</td><td>Thr</td><td>Cys</td><td>val</td><td>val</td><td>val</td><td>Asp</td><td>val</td><td>Ser</td><td>His</td><td>Glue</td><td>Asp</td><td>Pro</td><td>Glue</td><td>val</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>val</td><td>Asp</td><td>Gly</td><td>Val</td><td>Glue</td><td>Val</td><td>His</td><td>Asn</td><td>To the</td><td>Lilies</td><td>Thr</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>IMS نمد</td><td>Pro</td><td>Ntu ف؟</td><td>Glue</td><td>Glue</td><td>Gin</td><td>Phe</td><td>Asn</td><td>Ser</td><td>thr</td><td>Phe</td><td>Arg</td><td>val</td><td>val</td><td>ser</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>leu</td><td>Thr</td><td>val</td><td>٦ ةلآ</td><td>His</td><td>Gin</td><td>Asp</td><td>Trp</td><td>Leu</td><td>Asn</td><td>Gly</td><td>ةلا 1</td><td>Glue</td><td>Tyr</td><td>lily</td><td>cys</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Lilies</td><td>val</td><td>Ser</td><td>Asn</td><td>Lilies</td><td>Gl y</td><td>leu</td><td>Pro</td><td>To the</td><td>Pro</td><td>Isle</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>Isle</td><td>ser</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Lilies</td><td>Thr</td><td>Lilies</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Ntq</td><td>Glue</td><td>Pro</td><td>Gin</td><td>val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Ser</td><td>Arg</td><td>Glue</td><td>Glue</td><td>Met</td><td>thr</td><td>Lilies</td><td>Asn</td><td>Gin</td><td>val</td><td>ser</td><td>Leu</td><td>Thr</td><td>لآلاء</td><td>Leu</td><td>val</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Lilies</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>Ser</td><td>Asp</td><td>Isle</td><td>To the</td><td>val</td><td>Glue</td><td>Trp</td><td>Glue</td><td>Ser</td><td>Asn</td><td>Gly</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Gin</td><td>Pro</td><td>Glue</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>لا</td><td>thr</td><td>thr</td><td>Pto</td><td>pro</td><td>Met</td><td>leu</td><td>Asp</td><td>ser</td><td>Asp</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Gly</td><td>Ser</td><td>Phe</td><td>Phe</td><td>Leu</td><td>fyr</td><td>Ser</td><td>Lilies</td><td>leu</td><td>Thr</td><td>val</td><td>Asp</td><td>ةلا 1</td><td>Ser</td><td>Arg</td><td>Trp</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Gin</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser</td><td>Cys</td><td>Ser</td><td>Val</td><td>Met</td><td>His</td><td>Glue</td><td>To the</td><td>Leu</td><td>His</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lilies</td><td>Ser</td><td>Leu</td><td>Ser</td><td>leu</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Lilies</td><td></td><td></td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td></td>

<210> 30 <211 479 <212> PRT <213> Artificial SZquence <220>

<221> source <223> / note = Descri pti on of artificial sequences: synthetic polypeptide ”

<td>Met</td><td>Asp</td><td>Met</td><td>Arg</td><td>Val</td><td>Pro</td><td>To the</td><td>Gin</td><td>Leu</td><td>Leu 10</td><td>Gly</td><td>Leu</td><td>leu</td><td>Leu</td><td>Leu 15</td><td>Trp</td>

<td>Leu</td><td>Ntq</td><td>Gly</td><td>To the</td><td colspan="2">Arg Cys</td><td>Glue</td><td>val</td><td>Gin</td><td>leu</td><td>٦ ةل ١</td><td>Glue</td><td>Ser</td><td>Gly</td><td colspan="2">Gly Gly</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 3(3</td><td></td><td></td>

<td>leu</td><td>Val</td><td>ةلاأ</td><td>Pro</td><td colspan="2">Gly Gly</td><td>Ser</td><td>leu</td><td>Arg</td><td>leu</td><td>Ser</td><td>Cys</td><td>To the</td><td>To the</td><td>ser</td><td>Gly</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Phe</td><td>Thr</td><td>Phe</td><td>Ser</td><td>Asn</td><td>To the</td><td>Trp</td><td>Met</td><td>Ser</td><td>Trp</td><td>val</td><td>Arg</td><td>Gin</td><td>To the</td><td>Pro</td><td>Gly</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>ةلاأ</td><td>Gly</td><td>Leu</td><td>Glue</td><td>Trp</td><td>val</td><td>Gly</td><td>Ntq</td><td>Isle</td><td>Lilies</td><td>Ser</td><td>Thr</td><td>Thr</td><td>Asp</td><td>Gly</td><td>Gly</td>

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Thr</td><td>Thr</td><td>Asp</td><td>Tyr</td><td>To the</td><td>To the</td><td>Pro</td><td>Val</td><td>lily</td><td>Gly</td><td>Arg</td><td>Phe</td><td>Thr</td><td>Isle</td><td>Ser</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Asp</td><td>Asp</td><td>Ser</td><td>lily</td><td>Asn</td><td>Thr</td><td>leu</td><td>Tyr</td><td>Leu</td><td>Gin</td><td>Met</td><td>Asn</td><td>Ser</td><td>leu</td><td>lily</td><td>Thr</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>G٦u</td><td>Asp</td><td>Thr</td><td>To the</td><td>Val</td><td>Tyr</td><td>٢ لا ٦</td><td>Cys</td><td>Thr</td><td>Thr</td><td>Asp</td><td>Arg</td><td>Thr</td><td>Gly</td><td>Tyr</td><td>ser</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Isle</td><td>Ser</td><td>٢rp</td><td>Ser</td><td>Ser</td><td>Tyr</td><td>Tyr</td><td>Tyr</td><td>Tyr</td><td>Tyr</td><td>Gly</td><td>Met</td><td>Asp</td><td>val</td><td>Trp</td><td>Gly</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>Gly</td><td>Thr</td><td>Thr</td><td>val</td><td>Thr</td><td>val</td><td>Ser</td><td>Ser</td><td>To the</td><td>Ser</td><td>Thr</td><td>Lilies</td><td>Gly</td><td>Pro</td><td>ser</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>٦ ةلآ</td><td>Phe</td><td>Pro</td><td>leu</td><td>To the</td><td>Pro</td><td>Cys</td><td>Ser</td><td>A٣Q</td><td>Ser</td><td>Thr</td><td>Ser</td><td>Glue</td><td>Ser</td><td>Thr</td><td>To the</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>To the</td><td>leu</td><td>Gly</td><td>Cys</td><td>leu</td><td>٦ ةلآ</td><td>lily</td><td>Asp</td><td>tyr</td><td>Phe</td><td>Pro</td><td>Glue</td><td>Pro</td><td>١ ةلآ</td><td>Thr</td><td>Val</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Ser</td><td>Trp</td><td>Asn</td><td>Ser</td><td>Gly</td><td>To the</td><td>leu</td><td>Thr</td><td>Ser</td><td>Gly</td><td>val</td><td>His</td><td>Thr</td><td>Phe</td><td>Pro</td><td>To the</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>val</td><td>leu</td><td>Gin</td><td>Ser</td><td>Ser</td><td>Gly</td><td>leu</td><td>Tyr</td><td>Ser</td><td>leu</td><td>Ser</td><td>Ser</td><td>val</td><td>val</td><td>Thr</td><td>val</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Ser</td><td>Ser</td><td>Asn</td><td>Phe</td><td>Gly</td><td>Thr</td><td>Gin</td><td>Thr</td><td>Tyr</td><td>Thr</td><td>Cys</td><td>Asn</td><td>val</td><td>Asp</td><td>His</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Lilies</td><td>Pro</td><td>Ser</td><td>Asn</td><td>Thr</td><td>لا</td><td>val</td><td>Asp</td><td>lily</td><td>Thr</td><td>val</td><td>Glue</td><td>Arg</td><td>lily</td><td>Cys</td><td>cys</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Val</td><td>Glue</td><td>Cys</td><td>Pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>To the</td><td>Pro</td><td>Pro</td><td>Val</td><td>To the</td><td>Gly</td><td>Pro</td><td>ser</td><td>val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>Phe</td><td>leu</td><td>Phe</td><td>Pro</td><td>Pro</td><td>Lilies</td><td>Pro</td><td>Lilies</td><td>Asp</td><td>Thr</td><td>leu</td><td>Met</td><td>Isle</td><td>Ser</td><td>Arg</td><td>Thr</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Pro</td><td>Glue</td><td>val</td><td>Thr</td><td>Cys</td><td>٦ ةلآ</td><td>١ جلآ</td><td>val</td><td>Asp</td><td>go٦</td><td>Ser</td><td>His</td><td>Glue</td><td>Asp</td><td>Pro</td><td>Glue</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Val</td><td>Gin</td><td>Phe</td><td>Asn</td><td>Trp</td><td>Tyr</td><td>Val</td><td>Asp</td><td>Gly</td><td>val</td><td>Glue</td><td>Val</td><td>His</td><td>Asn</td><td>To the</td><td>لا</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Thr</td><td>lily</td><td>Pro</td><td>Arg</td><td>Glue</td><td>Glue</td><td>Gin</td><td>Phe</td><td>Asn</td><td>ser</td><td>Thr</td><td>Phe</td><td>Arg</td><td>val</td><td>val</td><td>ser</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>Val</td><td>leu</td><td>Thr</td><td>Val</td><td>val</td><td>His</td><td>Gin</td><td>Asp</td><td>Trp</td><td>e</td><td>Asn</td><td>Gly</td><td>lily</td><td>Glue</td><td>Tyr</td><td>ةلاأ</td>

<img file="MA32982B1_D0030.tif" />

ة

ΜΑ 32982Β1

A-1472-PCT Sequence_French

<td>Cys lily</td><td>Val</td><td>Ser</td><td>Asn</td><td>Lilies</td><td>Gly</td><td>leu</td><td>Pro</td><td>To the</td><td>Pro</td><td>Isle</td><td>Glue</td><td>Lilies</td><td>Thr</td><td>Isle</td>

<td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>Ser Lys</td><td>Thr</td><td>Lilies</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glue</td><td>Pro</td><td>Gin</td><td>Val</td><td>Tyr</td><td>Thr</td><td>Leu</td><td>Pro</td>

<td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Pro Ser</td><td>Arg</td><td>Glue</td><td>Glue</td><td>Met</td><td>Thr</td><td>ةلاأ</td><td>Asn</td><td>Gin</td><td>val</td><td>Ser</td><td>leu</td><td>Thr</td><td>s لاح</td><td>leu</td>

<td> 385</td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Val Lys</td><td>Gly</td><td>Phe</td><td>Tyr</td><td>Pro</td><td>ser</td><td>Asp</td><td>Isle</td><td>To the</td><td>val</td><td>Glue</td><td>Trp</td><td>Glue</td><td>Ser</td><td>Asn</td>

<td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Gly Gin</td><td>Pro</td><td>Glue</td><td>Asn</td><td>Asn</td><td>Tyr</td><td>Lilies</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>Met</td><td>Leu</td><td>Asp</td><td>Ser</td>

<td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>Asp Gly</td><td>Ser</td><td>Phe</td><td>Phe</td><td>leu</td><td>Tyr</td><td>Ser</td><td>ةلا 1</td><td>leu</td><td>Thr</td><td>val</td><td>Asp</td><td>lily</td><td>Ser</td><td>Arg</td>

<td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>Trp Gin</td><td>Gin</td><td>Gly</td><td>Asn</td><td>Val</td><td>Phe</td><td>Ser</td><td>cys</td><td>Ser</td><td>١ ةي ١</td><td>Met</td><td>His</td><td>Glue</td><td>To the</td><td>leu</td>

<td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>His Asn</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>Lilies</td><td>Ser</td><td>leu</td><td>Ser</td><td>leu</td><td>Ser</td><td>Pro</td><td>Gly</td><td>Lilies</td><td></td>

<td> 455</td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;20 31</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2"> &lt;211&gt; 478</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;212&gt; PRT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="6">&lt;213&gt; artificial sequence</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="3">&lt;221&gt; source</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">&lt;223&gt; / note =</td><td colspan="4">/'Description of</td><td colspan="6">artificial sequence:</td><td colspan="3">synthetic polypeptide</td>

<td colspan="2"> &lt;400&gt; 31</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Met Asp</td><td>Met</td><td>Arg</td><td>val</td><td>Pro</td><td>To the</td><td>Gin</td><td>leu</td><td>Leu</td><td>Gly</td><td>leu</td><td>Leu</td><td>Leu</td><td>leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>leu Arg</td><td>Gly</td><td>To the</td><td>Arg</td><td>Cys</td><td>Glue</td><td>val</td><td>Gin</td><td>leu</td><td>leu</td><td>Glue</td><td>Ser</td><td colspan="3">Gly Gly Gly</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>