AR080229A1

Fibronectin based scaffold domain proteins that bind il-23

Abstract

A fibronectin-based framework domain protein that binds interleukin 23 (IL-23). specifically to the p19 subunit of IL-23. Also to the use of innovative proteins in therapeutic applications to treat autoimmune diseases. In addition to cells comprising such proteins. polynucleotides encoding such proteins or fragments thereof and to vectors comprising the polynucleotides encoding the innovative proteins. Claim 1: A polypeptide characterized in that it comprises a tenth fibronectin type III (10Fn3) domain in which 10Fn3 has at least one loop selected from the BC, DE and FG loop with an altered amino acid sequence in relation to the corresponding sequence loop of the human 10Fn3 domain and binding the polypeptide to the p19 subunit of IL-23 with a KD of less than 500 nM.

AR080229A1, drawing sheet 1 — Sheet 1 of 145

Term

No projected expiry on record.

16 claims: 10 independent, 6 dependent

1
A polypeptide characterized in that it comprises a tenth fibronectin type III domain (10Fn3) in which the 10Fn3 has at least one loop selected from the BC, DE and FG loop with an amino acid sequence altered in relation to the sequence of the corresponding domain loop 10Human Fn3 and binding the polypeptide to the p19 subunit of IL-23 with a KDless than 500 nM. 1. Un polipéptido caracterizado porque comprende un dominio dècimo de fibronectina de tipo III (10Fn3) en el que el 10Fn3 tiene al menos un bucle seleccionado del bucle BC, DE y FG con una secuencia de aminoàcidos alterada en relación a la secuencia del correspondiente bucle del dominio 10Fn3 humano y uniéndose el polipéptido a la subunidad p19 de IL-23 con una KDde menos de 500 nM.
2
Un polipéptido que comprende un dominio dècimo de fibronectina de tipo III (10Fn3) caracterizado porque el 10Fn3 tiene al menos un bucle seleccionado del bucle BC, DE y FG con una secuencia de aminoàcidos alterada en relación a la secuencia del correspondiente bucle del dominio 10Fn3 humano y uniéndose el polipéptido al epitopo estructural de la subunidad p19 de IL-23. two. A polypeptide comprising a tenth fibronectin type III domain (10Fn3) characterized in that the 10Fn3 has at least one loop selected from the BC, DE and FG loop with an amino acid sequence altered in relation to the sequence of the corresponding domain loop 10Human Fn3 and binding the polypeptide to the structural epitope of the p19 subunit of IL-23.
3
The polypeptide of claims 1 or 2 characterized in that the BC loop is selected from SEQ ID NO:2-6. 3. El polipéptido de las reivindicaciones 1 ό 2 caracterizado porque el bucle BC esta seleccionado de las SEC ID N°: 2-6.
4
El polipéptido de las reivindicaciones 1 ό 2 caracterizado porque el bucle DE esta seleccionado de las SEC ID N°:7-48. Four. The polypeptide of claims 1 or 2 characterized in that the DE loop is selected from SEQ ID NO: 7-48.
5
The polypeptide of claims 1 or 2 characterized in that the FG loop is selected from SEQ ID NO:49-59. 5. El polipéptido de las reivindicaciones 1 ό 2 caracterizado porque el bucle FG esta seleccionado de las SEC ID N°: 49-59.
9
The polypeptide of claims 1 or 2 characterized in that the remainder of the BC loop sequence is GHYPXil · ^ shown in SEQ ID NO:257, wherein Xi is either methionine or leucine and X2 It is either isoleucine or valine. 9. El polipéptido de las reivindicaciones 1 ό 2 caracterizado porque el resto de secuencia del bucle BC es GHYPXil·^ mostrado en la SEC ID N°: 257, en el que Xi es bien metionina o bien leucina y X2 es bien isoleucina o bien valina.
10
The polypeptide of claims 1 or 2 characterized in that the rest of the sequence of the FG loop is YYX3X3X3X3YX3X3I shown in SEQ ID NO:258, in which X3 It can be any amino acid. 10. El polipéptido de las reivindicaciones 1 ό 2 caracterizado porque en el que el resto de secuencia del bucle FG es YYX3X3X3X3YX3X3I mostrado en la SEC ID N°: 258, en el que X3 puede ser cualquier aminoâcido.
11
El polipéptido de las reivindicaciones 1 ό 2 caracterizado porque comprende adicionalmente uno o mâs restos farmacocinéticos (PK) seleccionados del grupo constituido por polietilenglicol, àcido siâlico, Fc, fragmento de Fc, transferrina, seroalbùmina, una proteina de unión a seroalbùmina y una proteina de unión a inmunoglobulina. eleven. The polypeptide of claims 1 or 2 characterized in that it additionally comprises one or more pharmacokinetic (PK) moieties selected from the group consisting of polyethylene glycol, silica, Fc, Fc fragment, transferrin, seroalbumin, a serum albumin binding protein and a protein of immunoglobulin binding.
13
The polypeptide of claims 1 or 2 characterized in that it further comprises a cistern crimp. 13. El polipéptido de Ias reivindicaciones 1 ó 2 caracterizado porque comprende adicionalmente un engarce de cisterna.
15
Una composición farmacèuticamente aceptable caracterizado porque comprende el polipéptido de una cualquiera de Ias reivindicaciones 1-14, en la que la composición està esencialmente libre de endotoxinas. fifteen. A pharmaceutically acceptable composition characterized in that it comprises the polypeptide of any one of claims 1-14, wherein the composition is essentially free of endotoxins.

Independent claims10

2,666 paragraphs in 205 sections, as filed

FIBRONECTINE HAMMER DOMAIN PROTEINS THAT JOIN IL-23

FIELD OF THE INVENTION

The present invention relates to a fibronectin-based framework domain protein that binds interleukin 23 (IL-23), specifically to the p19 subunit of IL-23. The invention also relates to the use of innovative proteins in therapeutic applications to treat autoimmune diseases. The invention further relates to cells that comprise such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising polynucleotides encoding the innovative proteins.

INTRODUCTION

IL-23 is a member of the heterodimeric cytokine family of IL-12. It contains the p40 subunit, which is common to IL-12 and a unique p19 subunit. The IL-23 sends signals through a heterodimeric receptor complex consisting of IL-12R1 and IL-23R (Aggarwal S et al. Interleukin-23 promotes a distinct CD4 T celi activation state characterized by the production of interleukin-17. J Biol Chem. 278: 1910-4, 2003). IL-23 is a potential target for the treatment of chronic inflammatory disorders such as multiple sclerosis, rheumatoid arthritis, psoriasis and Crohn's disease.

Fibronectin-based frameworks are a family of proteins capable of developing to bind to any compound of interest. These proteins, which generally make use of a framework derived from a type III fibronectin (Fn3) or domain similar to Fn3, function in a characteristic manner of natural or modified antibodies (i.e., polyclonal, monoclonal, or single chain antibodies) and, in addition, they have structural advantages. Specifically, the structure of these antibody mimics has been designed for optimal folding, stability and solubility, even under conditions that normally lead to loss of structure and function in antibodies. An example of fibronectin-based framework proteins are Adnectins ™ (Adnexns, a Bristol-Myers Squibb R&D Company).

Type III fibronectin (Fn3) domains comprise, in order from the N-terminal to the C-terminal, a beta or beta-like chain, A; A loop,

AB; a beta chain or similar to beta, B; a loop, BC; a beta chain or similar to

ί beta C; a CD loop; a beta chain or similar to beta D; a DE loop; a beta or beta-like chain, E; a loop, EF; a beta chain or similar to beta F; an FG loop; and a beta or similar chain to beta G. Any of all loops AB, BC, CD, DE, EF and FG can participate in the target union. The BC, DE and FG loops are both functionally structurally analogous to the complementary complementarity determining regions (CDRs) of immunoglobulins. US Patent No. 7,115,396 describes the Fn3 domain proteins in which alterations in the BC, DE and FG loops result in high affinity TNFa binders. US Publication No. 2007/0148126 describes Fn3 domain proteins in which alterations in the BC, DE and FG loops result in high affinity VEGFR2 binders.

It would be advantageous to obtain improved fibronectin domain framework proteins for the therapeutic treatment of autoimmune disorders. A subset of effector T lymphocytes that produce interleukin 17 (IL-17; 'Th17 cells') are highly proinflammatory and induce severe autoimmunity. Th 17 lymphocytes express a distinct subgroup of cytokines and chemokines compared to Th1 and Th2 lymphocytes, including IL-6, tumor necrosis factor (TNF), IL-22, IL-17A and IL-17F as well as the CCR6 chemokine receptor . IL-23 promotes the production of IL-17 by activated T lymphocytes (Aggarwal S et al. Lnterleukin-23 promotes a distinct CD4 T celi activation state characterized by the production of interleukin-17. J Biol Chem. 278: 1910-4, 2003) and is a key cytokine to induce expansion of CD4 + T lymphocytes that produce IL17. Exposure to IL-23 seems to be the key characteristic that determines the pathogenicity of Th17 lymphocytes.

SUMMARY OF THE INVENTION

The application provides Adnectin against the specific p19 subunit of IL23. One aspect of the invention provides polypeptides comprising Fn3 domain in which one or more of the solvent-accessible loops has been randomly altered or mutated. In some embodiments, the Fn3 domain is an Fn3 domain derived from the tenth nature module of the human type III fibronectin domain (1° Fn3). In some embodiments, the polypeptide10Fn3 of the invention is at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the domain 10Human Fn3

In some embodiments, one or more selected loops of BC, DE and FG may be extended or shortened in length relative to the corresponding human fibronectin loop.

In some embodiments, the polypeptides of the invention comprise a tenth fibronectin domain of the Ili type (10Fn3), in which the domain 10Fn3 comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; an EF loop; and an FG loop; and have at least one loop selected from the BC, DE and FG loops with an amino acid sequence altered in relation to the corresponding loop sequence of the domain10Human Fn3

In some embodiments, the polypeptide of the invention comprises an Fn3 domain comprising an amino acid sequence at least in 80, 85, 90, 95, 98, 99 or 100% identical to regions that are not loops.

In some embodiments, the BC loop of the protein of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO.s: 2-6.

In some embodiments, the DE loop of the protein of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO.s: 7-48.

In some embodiments, the FG loop of the protein of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO.s: 49-59.

In some embodiments, the domain 10Fn3 can start and / or end with amino acid substitutions, insertions or deletions.

In some embodiments, the protein of the invention comprises a loop sequence of the BC loop sequences shown in SEQ ID NO.s: 2-6, a DE loop sequence shown in SEQ ID NO.s: 7-48 and an FG loop sequence shown in SEQ ID NO.s: 49-59.

In some embodiments, the protein of the invention comprises a sequence of loop amino acids BC, DE and FG in at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% identical to any one of the SEC ID No.s: 2-59.

In some embodiments, the anti-IL-23 Adnectin comprises the amino acid sequence of any one of SEQ ID NO.s: 60-100.

In some embodiments, the anti-IL-23 Adnectin comprises the amino acid sequence from position 3-96 of any one of SEQ ID Nyou: 60-100.

In some embodiments, the anti-IL-23 Adnectin comprises the identical amino acid sequence at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% to any one of SEQ ID NO.s: 60-100.

In one aspect, the anti-IL-23 Adnectin additionally comprises a pharmacokinetic moiety (PK). In some embodiments, the rest of PK comprises

polyethylene glycol (PEG).

In one aspect, the application provides an anti-IL-23 Ad Adnectin useful in the treatment of autoimmune diseases.

In one aspect, the present invention provides a fusion polypeptide comprising a tenth fibronectin type III domain (10Fn3) and Adnectin anti-lL-23, in which the domain 10Fn3 binds to HSA with a Kd of 1 μΜ or less. In certain embodiments, the domain10Fn3 comprises a sequence of amino acids at least 70% identical to SEQ ID NO: 103. In one embodiment, the domain 10Fn3 comprises a BC loop having the amino acid sequence set forth in SEQ ID NO: 104, a DE loop having the amino acid sequence set forth in SEQ ID NO: 105, and an FG loop having the amino acid sequence set forth in SEQ ID NO: 106. In another embodiment, the domain 10Fn3 comprises one or more of a BC loop having the amino acid sequence set forth in SEQ ID NO: 104, a DE loop having the amino acid sequence set forth in SEQ ID NO: 105, and a DE loop having the amino acid sequence set forth in SEQ ID NO: 106.

In one embodiment, the domain 10Fn3 of the fusion polypeptide also binds to one or more of the Indian macaque serum albumin (RhSA), Java macaque serum albumin (CySA), or murine serum albumin (MuSA). In other embodiments, the domain10Fn3 does not cross-react with one or more of RhSA, CySA or MuSA.

In certain embodiments, the domain 10Fn3 of the fusion polypeptide binds to HSA with a Kd of 1 μΜ or less. In certain embodiments, the domain10Fn3 binds to HSA with a Kd of 500 nM or less. In other embodiments, the domain10Fn3 binds to HSA with a Kd of at least 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 5 nM.

In other embodiments, the domain 10Fn3 of the fusion polypeptide binds to the I ο II domain of HSA. In one embodiment, the domain10Fn3 binds to both domains I and II of HSA. In some embodiments, the domain10Fn3 binds to HSA in a pH range of 5.5 to 7.4. In certain embodiments, the domain10Fn3 binds to HSA with a Kd of 200 nM or less at pH 5.5. In another embodiment, the domain10Fn3 binds to HSA with a Kd of at least 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 5 nM in a pH range of 5.5 to 7.4. In one embodiment, the domain10Fn3 binds to HSA with a Kd of at least 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, or 5 nM at pH 5.5.

In some embodiments, the serum half-life of the fusion polypeptide is at least 5 times greater than the serum half-life of the polypeptide in the absence of serum albumin. In certain embodiments, the serum half-life of the fusion polypeptide is at least 2 times, 5 times, 7 times, 10 times, 12 times, 15 times, 20 times, 22 times, 25 times, 27 times, or 30 times greater than the serum half-life of the polypeptide in the absence of serum albumin. In some embodiments, bovine serum albumin is any one of HSA, RhSA, CySA, or MuSA.

In certain embodiments, the serum half-life of the fusion polypeptide in the presence of serum albumin is at least 20 hours. In certain embodiments, the serum half-life of the fusion polypeptide in the presence of seroalbumin is at least 10 hours, 12 hours, 15 hours, 20 hours, 25 hours, 30 hours, 40 hours, 50 hours, 75 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 150 hours, 170 hours, or 200 hours. In some embodiments, the half-life of the fusion polypeptide is observed in a primate (eg, human or monkey) or a mouse.

In any of the preceding aspects and embodiments, the domain 1° Fn3 comprises a sequence of SEQ ID NO: 8, 12, 16, 20 and 24-44.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the full length DNA sequence alignment of the anti-IL-23 Adnectin of the invention.

Figure 2: pBMS2008 / ATI001044 protein expression vector as described in Example 2.

Figure 3 shows the full length amino acid sequence alignment of the anti-IL-23 Adnectin of the invention.

Figure 4 shows Cl curves5or representative of inhibition of PBMC pSTAT3 by adnectin anti-lL-23 as described in Example 4.

Figure 5 shows IC curves50 Representative for IL-23-dependent inhibition of IL-23 by anti-IL-23 adnectins and anti-p40 monoclonal antibody (MAB1510) as described in Example 4.

Figure 6 shows inhibition of IL-17 production of IL-17 induced by IL-23 by donor PBMC 228 (one of 4 donors tested as described in Example 4).

Figure 7 shows representative selectivity data for antiIL-23 adnectins. The sensograms to which the buffer has been removed illustrating the association and dissociation phases of 10 nM IL-23 and 1 μΜ IL-12 to ATI001016 as described in Example 4.

Figure 8 shows that anti-L-23 adnectins do not inhibit the production of IL-12-induced IFN-γ in NK-92 lymphocytes as described in Example 4.

Figure 9a shows that ATI001045 inhibits serum IL-17 levels in mouse pharmacodynamic models of IL-23 as described in Example 4.

Figure 9b shows a comparison of anti-IL-23 adnectin inhibitory activities in a mouse pharmacodynamic model as described in Example 4.

Figure 10a shows dose response of ATI000934 in acanthosis induced by human IL-23 as described in Example 4.

Figure 10b shows dose response of ATI001045 in acanthosis induced by human IL-23 as described in Example 4.

Figure 11 shows half-life of HSA in vivo in mice. HSA was injected into mice at 20 mg / kg (Figure 11A) or at 50 mg / kg (Figure 11B).

Figures 12a-b show half-life determination of SABA1-SABA4 in mice.

Figure 13a shows a graphical summary of half-life potentiation in SABA1-4 mice when injected with HSA. Figure 13b compares data from Java macaque and mice.

Figures 14A-B show the half-life determination for SABA1.1 and SABA5.1 in Java macaque.

Figure 15 shows the binding of SABA1.2 to human, mouse and rat albumin by direct binding ELISA assay.

Figure 16 shows the stoichiometry determination of SABA1.1 and HSA. Figure 17 shows the Biacore analysis of binding SABA1.2 to recombinant domain fragments of HSA.

Figure 18 shows the pharmacokinetic profile for SABA1.2 in monkeys administered at 1 mpk and 10 mpk.

Figure 19 shows the pharmacokinetic profile for SABA1.2 in monkeys administered intravenously or subcutaneously at 1 mpk.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

By a polypeptide is meant any sequence of two or more amino acids, regardless of length, post-translational modification, or function. Polypeptide, peptide and protein are used interchangeably herein. The polypeptides may include natural amino acids and non-natural amino acids such as those described in US Patent No. 6,559,126, incorporated herein by reference. The polypeptides can be modified in any of a variety of standard chemical routes (for example, an amino acid can be modified by a protective group; the carboxy terminal amino acid can be made within a terminal amide group; the amino acid residue)

terminal can be modified with groups to, for example, enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or half-life in vivo). Polypeptide modifications may include the binding of another structure such as a cyclic compound or another molecule to the polypeptide and may also include polypeptides containing one or more amino acids in an altered configuration (ie, R or S; or, L or D). The peptides of the invention are proteins derived from the tenth type III fibronectin domain that have been modified to specifically bind to the p19 subunit of IL-23 and are referred to herein as "Adnectin or" Adnectin anti-IL-23.

The term PK is an acronym for pharmaceutics (a) and encompasses properties of a compound that include, by way of example, absorption, distribution, metabolism and elimination by a subject. A PK modulation protein or PK moiety refers to any protein, peptide or moiety that affects the famakinetic properties of a biologically active molecule when condensed to or when co-administered with the biologically active molecule. Examples of a PK modulation protein include PEG, bovine serum albumin binders (HSA) (as described in U.S. Publications No.s 20050287153 and 20070003549), bovine serum albumin, Fc or Fc fragments and sugars (for example, sialic acid).

Amino acid sequence identity in percent (%) herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a given sequence, after aligning the sequences and entering the gaps, if necessary, to achieve the maximum percentage of sequence identity, and not to consider any conservative substitutions as part of the sequence identity. The alignment in order to determine the percentage of amino acid sequence identity can be carried out in several ways that are within the skill in the art using, for example, publicly available computer software such as the BLAST, BLAST-2, ALIGN software, ALIGN-2 or Megalign (ADNSTAR). Those skilled in the art can determine the appropriate parameters to measure the alignment, including the algorithms necessary to achieve maximum alignment along the entire length of the sequences being compared.

An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Pollutant components of the natural environment are materials that could interfere with the uses

diagnostic or therapeutic for the polypeptide and may include enzymes, hormones and other protein or non-protein solutes. In preferred embodiments, the polypeptide will be purified (1) to more than 95% by weight of polypeptide as determined by the Lowry method and most preferably to more than 99% by weight, (2) to a degree sufficient to obtain the less residues of N-terminal sequence or internal amino acid sequence through the use of a rotating cup sequencer, or (3) to homogenize by SDS-PAGE in reducing or non-reducing condition using Coomassie blue or, preferably, staining with piata. The isolated polypeptide includes the polypeptide in situ within the recombinant cells since at least one component of the natural environment of the polypeptide will not be present. Ordinarily, however, usually the isolated antibody will be prepared in at least one purification step.

The "half-life of an amino acid sequence or a compound can generally be defined as the time taken by the serum concentration of the polypeptide to be reduced to 50%, in vivo, for example due to the degradation of the sequence or compound and / or the evacuation or capture of the sequence or composed of natural mechanisms. The half-life can be determined in any way known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art and may, for example, generally involve the steps of properly administering to the primate an adequate dose of the amino acid sequence or compound of the invention; collect blood samples or other samples of said primate at regular intervals; determining the concentration level of the amino acid sequence or the compound of the invention in said blood sample; and calculate from (a batch of) the data thus obtained, the time until the concentration level of the amino acid sequence or of the compounds of the invention has been reduced by 50% compared to the initial level after administering doses. Reference is made, for example, to standard manuals, such as Kenneth, A et al .: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinete analysis: A Practical Approach (1996). Reference is also made to Pharmacokinetics, M Gibaldi & D Perron, published by Marcel Dekker, 2to rev edition (1982).

The half-life can be expressed using parameters such as t1 / 2-alpha, t1 / 2-beta and the area under the curve (AUC). In the present specification, an increase in half-life refers to an increase in any one of these parameters, any two of these parameters, or all these three parameters. An increase in half-life refers in particular to an increase in t1 / 2-beta, either with or without an increase in t1 / 2-alpha and / or in the AUC or both.

General Vision

The application provides Adnectin against the specific p19 subunit of IL23. In order to identify the specific antagonist of IL-23, IL-23 was presented to large synthetic Adnectin libraries using anti-p40 mAb. Adnectins that bound the p19 subunit of IL-23 were screened for binding to human IL-23, competition for IL-23 / IL-23R interaction and inhibition of IL-23 induced signaling in a line of T lymphocytes. Anti-IL-23 Adnectins were subjected to additional selective pressure by lowering the target concentration and selecting for Anti-IL-23 Adnectins with slow dissociation rates. From this optimization process, a family of adnectins were identified as specific inhibitors of IL-23 with favorable biochemical and biophysical properties.

Fibronectin-based frameworks

One aspect of the application provides polypeptides comprising Fn3 domain in which one or more of the solvent-accessible loops has been randomly altered or mutated. In some embodiments, the Fn3 domain is an Fn3 domain derived from the tenth nature module of the human fibronectin domain of the Ili type (10Fn3). VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATI SGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1). In the sequence of10Fn3 above, the BC, DE and FG loops are underlined.

A variety of frameworks of 10Fn3 mutants. In one aspect, one or more of Asp 7, Glu 9 and Asp 23 is replaced by another amino acid, such as, for example, a non-negatively charged amino acid residue (eg, Ash, Lys, etc.). It has been reported that these mutations have the effect of promoting greater stability of the10Fn3 mutant at neutral pH as compared to the natural form (see, PCT Publication No. W002 / 04523). A variety of additional alterations have been revealed in the framework of10Fn3 that are either beneficial or neutral. See, for example, Batori et al., Protein Eng. December 2002; 15 (12): 1015-20; Koide et al., Biochemistry August 28, 2001; 40 (34): 10326-33.

Both protein variants of 10Fn3 as natural proteins from 10Fn3 are characterized by the same structure, namely seven sequences of beta chain domains designated from A to G and six loop regions (AB loop, BC loop, CD loop, DE loop, EF loop and FG loop) that connect the seven sequences of beta chain domains. The beta chains positioned closer to the ends

N- and C-terminals can adopt a conformation similar to beta in solution. In the

SEQ ID NO: 1, the AB loop corresponds to residues 15-16, the BC loop corresponds to residues 21-30, the CD loop corresponds to residues 39-45, the DE loop corresponds to residues 51-56, the EF loop corresponds to residues 60-66 and the FG loop corresponds to residues 76-87 (Xu et al., Chemistry & Biology 2002 9: 933-942).

In some embodiments, the polypeptide 10Fn3 can be at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the domain 10Human Fn3, shown in SEQ ID NO: 1. Much of the variability generally occurs in one or two of the loops. Each of the beta sheets or each of the beta-like sheets of a polypeptide10Fn3 may consist essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the sequence of a corresponding beta or beta-like chain of SEQ ID NO: 1, given that such variation does not alter the stability of the polypeptide under physiological conditions.

In some embodiments, the disclosure polypeptides comprise a tenth fibronectin type III domain (10Fn3), in which the domain 10Fn3 comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; an EF loop; and an FG loop; and has at least one loop selected from the BC, DE and FG loops with an amino acid sequence altered in relation to the corresponding loop sequence of the domain10Human Fn3 In some embodiments, the BC and FG loops are altered, in some embodiments, the BC, DE and FG loops are altered, that is, the Fn3 domains comprise domains that do not occur in nature. By altered it is meant one or more amino acid sequence alterations relative to the piantine sequence (corresponding human fibronectin domain) and includes additions, deletions and amino acid substitutions. Altering an amino acid sequence can be achieved through intentional, blind or spontaneous sequence variation, generally of a nucleic acid coding sequence, and can occur by any technique, for example, PCR, error-prone PCR, or DNA synthesis chemistry.

In some embodiments, one or more selected loops of BC, DE and FG may be extended or shortened in length relative to the corresponding human fibronectin loop. In some embodiments, the length of the loop can be extended from 2 to 25 amino acids. In some embodiments, the length of the loop can be decreased by 1-11 amino acids. To optimize antigen binding, therefore, the length of a loop of10Fn3 can be altered as well in the sequence

to obtain the greatest possible flexibility and affinity in antigen binding.

In some embodiments, the polypeptide comprises an Fn3 domain comprising an amino acid sequence at least in 80, 85, 90, 95, 98, 99 or 100% identical to the non-loop regions of SEQ ID NO: 1 , in which at least one loop selected from BC, DE and FG is altered. In some embodiments, the altered BC loop has up to 10 amino acid substitutions, up to 4 amino acid deletions, up to 10 amino acid insertions, or a combination thereof. In some embodiments, the altered DE loop has up to 6 amino acid substitutions, up to 4 amino acid deletions, up to 13 amino acid insertions or a combination thereof. In some embodiments, the altered FG loop has up to 12 amino acid substitutions, up to 11 amino acid deletions, up to 25 amino acid insertions or a combination thereof.

In some embodiments, the BC loop of the protein of the invention comprises an amino acid sequence selected from the group consisting of GHYPMHV (SEQ ID NO: 2), GHYPLHV (SEQ ID NO: 3), GHYPMHI (SEQ ID NO: 4), GHYPLHI (SEQ ID NO; 5) and GHYPLHL (SEQ ID NO; 6).

In some embodiments, the DE loop of the protein of the invention comprises an amino acid sequence selected from the group consisting of HRTH (SEQ ID NO: 7), YYHY (SEQ ID NO: 8), SKQH (SEQ ID NO: 9), SNVH (SEQ ID NO: 10), NRAH (SEQ ID NO: 11), RKTY (SEQ ID NO: 12), RSRY (SEQ ID NO: 13), SRYY (SEQ ID NO; 14), PHRY (SEQ ID NO: 15), RSTH (SEQ ID NO: 16), SRIY (SEQ ID NO: 17), HQRY (SEQ ID NO 18), KQVY (SEQ ID NO: 19 ), AHRY (SEQ ID NO: 20), RSRH (SEQ ID NO: 21), ARQY (SEQ ID NO: 22), RTQY (SEQ ID NO: 23), PRYH (SEQ ID NO: 24 ), MRQH (SEQ ID NO; 25), SRKY (SEQ ID NO; 26), RQKY (SEQ ID NO: 27), HAKY (SEQ ID NO: 28), SNRY (SEQ ID NO: 29), NTSH (SEQ ID NO: 30), SQVY (SEQ ID NO: 31), NRVY (SEQ ID NO: 32), PRSH (SEQ ID NO: 33), RTKY (SEQ ID NO: 34), SRYH (SEQ ID NO: 35), PRRY (SEQ ID NO: 36), RQKY (SEQ ID NO; 37), RYKY (SEQ ID NO: 38), VPRH (SEQ ID NO: 39), TPKH (SEQ ID NO: 40), RSKY (SEQ ID NO: 41), SRKY (SEQ ID NO; 42), VPRY (SEQ ID NO: 43), PRRY (SEQ ID NO: 44), RMRH (SEQ ID NO: 45), PPRH (SEQ ID NO: 46), RQIY (SEQ ID NO: 47) and MRQH (SEQ ID NO; 48).

In some embodiments, the FG loop of the protein of the invention comprises an amino acid sequence selected from the group consisting of YYNEADYSQI (SEQ ID NO; 49), YYQEYEYRYI (SEQ ID NO: 50), YYMEEKYAVI (SEQ ID NO: 51), YYAQENYKEI (SE ID NO: 52), YYKEANYREI (SEQ ID NO: 53), YYAQEEYHII (SEQ ID NO: 54), YYKEADYSQI (SEQ ID NO; 55), YYEQVEYREI (SEQ ID

N °: 56), YYEQPIYATI (SEQ ID N °: 57), YYEQVEYREI (SEQ ID N °: 58) and YYSEELYKYI (SEQ ID N °: 59).

The Dominion 10Fn3 can start with amino acid alterations. For example, an additional MG sequence may be located at the N-terminal end of an Fn3 domain. The M will usually be cleaved, leaving the G at the terminal end. In some embodiments, the sequences may be located at the Cterminal end of the domain.10Fn3. For example, site directed PEGylation where a cysteine-containing linker such as GSGC horn (SEQ ID NO: 101) is added to the C-terminal end. Alternatively, the PEGylation of the tail of the C-terminal end that occurs in nature that has been mutated by changing the Ser to a Cys for a crimp containing EIDKPÇK cysteine (SEQ ID NO: 102). Examples of the anti-IL-23 adnectin of the invention comprising the GSGC linker include ATI001014, ATI001015, ATI001016, ATI001044, ATI001045 and ATI001047. ATI000934 is an example of the anti-IL-23 adnectin of the invention comprising the EIDKPCQ linker.

In some embodiments, the protein of the invention comprises a loop sequence of the BC loop sequences shown in SEQ ID N,you: 2-6, a DE loop sequence shown in SEQ ID NO.s: 7-48, an FG loop sequence shown in SEQ ID NO.s: 49-59. In some embodiments, the protein of the invention comprises a sequence of BC, DE and FG loop amino acids of at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% identical to any one of the SEC ID No.s: 2-59.

Additionally, a person skilled in the art will recognize that the BC loop sequences shown in SEQ ID NO.s: 2-6 share a remainder of the common GHYPXiHXa sequence (SEQ ID N °: 257) where ΧΊ is either Μ or L and X2 is either I or V and the FG loop sequences shown in SEQ ID NO.s: 49-59 share a common sequence remainder YYX3X3X3X3Yx3X3l (SEQ ID NO: 258) where X3 It can be any amino acid. It would therefore be possible to generate additional Adnectins that bind to IL-23 with BC loops that conform to the GHYPX consensus sequence.1Hx2 and / or with other FG loops, outside those explicitly listed in SEQ ID NO.s: 49-59, which fit the YYX pattern3X3X3X3Yx3X3I.

In some embodiments, the anti-IL-23 Adnectin comprises the amino acid sequence of any one of SEQ ID NO.s: 60-100. In some embodiments, the anti-IL-23 Adnectin comprises the amino acid sequence of the Fn3 domain of position 3-96 of any one of SEQ ID NO.s: 60-100. In some embodiments, the anti-1 L-23 Adnectin comprises the identical amino acid sequence in at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% at any one of the StC; ffit No.s: 60100. In some embodiments, the anti-IL-23 Adnectin comprises the amino acid sequence identical in at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% to the amino acid sequence of position 3-96 of any one of SEQ ID NO.s:

60-100.

In some embodiments, the anti-IL-23 Adnectin may be pegylated and / or contain a His tag. As used herein, ATI000934 refers to a protein in which the loop sequences are identical to those of construction 1571G06 (Sec ID 87) and the protein contains EIDKPÇQ residues at the C-terminal end where the protein It is pegylated and contains a His mark. ATI001014 refers to a protein in which the loop sequences are identical to those of construction 1571G04 (Sec ID 86) and the protein contains a GSGC linker at the C-terminal end where the protein is pegylated and contains a His mark. . ATI001015 refers to a protein in which the loop sequences are identical to those of construction 1572G06 (Sec ID 91) and the protein contains a GSGC linker at the C-terminal end where the protein is pegylated and contains a His mark. . ATI001016 refers to a protein in which the loop sequences are identical to those of construction 1490B03 (Sec ID 79) and the protein contains a GSGC linker at the C-terminal end where the protein is pegylated and contains a His mark. . ATI001044 refers to a protein in which the loop sequences are identical to those of construction 1490B03 (Sec ID 79) and the protein contains a GSGC linker at the C-terminal end, but the protein is not pegylated and there is no label of His. ATI001045 refers to a protein in which the loop sequences are identical to those of construction 1490B03 (Sec ID 79) and the protein contains a GSGC linker at the C-terminal end where the protein is pegylated; and there is no mark of His. ATI001047 refers to a protein in which the loop sequences are identical to those of construction 1571G04 (Sec ID 86) and the protein contains a GSGC linker at the Cterminal end where the protein is pegylated; and there is no mark of His.

Fibronectin naturally binds to certain types of integrins because of its binding to integrins, arginine-glycine-aspartic acid (RGD). In some embodiments, the polypeptide comprises a domain of10Fn3 lacking the integrin binding motif (RGD).

Pharmacokinetic Remains

In one aspect, the application provides anti-IL-23 Adnectin comprising

additionally a pharmacokinetic moiety (PK). Enhanced pharmacokinetics can be evaluated according to the perceived therapeutic need. It is often desirable to increase bioavailability and / or increase the time between doses, possibly increasing the time that a protein remains available in the serum after dosing. In some examples, it is desirable to improve the continuity of serum protein concentration over time (for example, decrease the difference in serum protein concentration just after administration and just before the next administration). Anti-23-Adnectin can bind to a moiety that reduces the clearance rate of the polypeptide in a mammal (eg, mouse, rat, or human) more than three times in relation to unmodified Adnectin. Other measures of increased pharmacokinetic values include serum half-life, which is often divided between an alpha phase and a beta phase. Either one of them or both phases can be significantly improved by adding an appropriate residue.

Residues that tend to slow the clearance of a blood protein, referred to herein as "PK residues", include polyoxyalkylene residues, for example, polyethylene glycol, sugars (eg, silica acid) and well tolerated protein residues (for example, Fc, Fc fragments, transferrin, or serum albumin). Adnectin can be fused with albumin or a fragment (portion) or variant of albumin as described in United States Publication No. 20070048282.

In some embodiments, the PK moiety is a serum albumin binding protein such as those described in United States Publication N.you 2007/0178082 and 2007/0269422.

In some embodiments, the PK moiety is a serum immunoglobulin binding protein such as those described in United States Publication No. 2007/0178082.

In some embodiments, the Adnectin moiety comprises polyethylene glycol (PEG). One or more PEG molecules can bind at different positions in the protein and such binding can be achieved by reaction with amines, thiols or other suitable reactive groups. The amine moiety can be, for example, a primary amine that is at the N-terminal end of a polypeptide or an amine group present in an amino acid, such as lysine or arginine. In some embodiments, the rest of PEG is attached at a position in the polypeptide selected from the group consisting of: a) the N-terminal end; b) between the N-terminal end and the beta chain or the beta-like sheet plus N-terminal; c) a loop located on one side of the polypeptide opposite the target binding site; d) between the C-terminal end and the beta chain or the beta-like chain plus C-terminal; and e) at the C-terminal end.

Pegylation can be carried out by site directed pegylation, in which a suitable reactive group is introduced into the protein to create a site where pegylation takes place preferentially. In some embodiments, the protein is modified to introduce a cistern residue in the desired position, allowing site directed pegylation in the cysteine. PEG can vary widely in molecular weight and can be branched or linear.

In some embodiments, the Adnectin comprises an Fn3 domain and a PK moiety. In some embodiments, the Fn3 domain is a domain.10Fn3. In some embodiments, the PK moiety increases the serum half-life of the polypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800 , 1000% or more in relation to the Fn3 domain alone.

In some embodiments, the rest PK is a polymeric sugar. In some embodiments, the rest PK is a polymeric sugar. In some embodiments, the PK moiety is a serum albumin binding protein. In some embodiments, the PK moiety is a human serum albumin. In some embodiments, the PK moiety is an immunoglobulin binding protein. In some embodiments, the PK moiety is transferrin. In some embodiments, the PK moiety is another specific Adnectin for a whey protein.

Biophysical and Biochemical Characterization

The application provides Adnectin comprising an Fn3 domain that binds to the p19 subunit of IL-23. As shown in Table 1 and in Example 4, polypeptide binding to a target molecule can be evaluated in terms of equilibrium constants (eg, dissociation, KD) and in terms of kinetic constants (for example, association rate constant, Kon and dissociation rate constant, koff). An adnectin will usually bind to a molecule with a KD less than 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, although K values can be toleratedD higher when the Koff is low enough or when the Kon It is tall enough.

The BC, DE and FG loop sequences of the anti-IL-23 Adnectin family of the invention are present in Table 1 below, as well as SEQ ID No. of corresponding total length.

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CN ¢ 0 ω- O CD

Ν 'CO CN CO <QO OO Tr-'0 C0 C

TO TO C c

N- TO TO co ”CC

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c '2

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LJLULUUJUJLUUJLUra.h = UJUJLUra. ± ra.bra.i = UJlJJUJLUUJLJJ 'OOCN' ^ N-CNCOOC-aiOCMCNp'DCXJP'DCON-OOtOT-CO N · oo τ- ιη ÿ ra cq cq raq qq oo cq o> cq cq rJ n F τ »· ' co ro n F τι F n F lo m · 'co in oj _, CN C0 b iq cq N cq vco' co 'co' co 'co co' co c

'2 υ

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ra>

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Table 1: Anti-IL-23 Adnectin Family

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Association Speed Dissociation Speed Affinity pSTAT3 Don ID Loop BC Loop Loop FG (ka, (kd, s) (KD, M) of kit 225 SEQ ID N?

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m σ> ο co r- oo 03 03 Ο m ττ ο oo m ο οο c ο 'θ θ ο OJ <0 ο

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CM TJ EDE TJ E CM Ό E

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The characterization of additional anti-IL-23 Adnectin is described in Table 2.

Table 2: Adnectin Anti-IL-23 IC50 / EC50

IC50 of pSTAT3 of

<td>Gift ID</td><td>BC</td><td>FROM</td><td>FG</td><td>PBMNC (nM)</td><td>IL50 EC50 (nM)</td><td>IL50 EC50 (nM)</td>

<td>1571G04</td><td>GHYPLHV</td><td>PRSH</td><td>YYAQENYKEI</td><td> 0,23±,05</td><td> 1,4 ±0,3</td><td> 1,3 ±0,7</td>

<td>1490B03</td><td>GHYPLHV</td><td>SRKY</td><td>YYKEANYREI</td><td> 0,09±0,01</td><td> 1,4 ±0,1</td><td> 1,7 ±0,8</td>

<td>1572G06</td><td>GHYPLHV</td><td>RYKY</td><td>YYAQENYKEI</td><td> 0,21±0,03</td><td> 1,6 ±0,3</td><td> 1,9 ±0,3</td>

<td>1550E06</td><td>GHYPMHV</td><td>PPRH</td><td>YYAQENYKEI</td><td> 1,15±0,5</td><td> 1,5 ±0,6</td><td> 2,1 ±0,8</td>

<td>1571H03</td><td>GHYPLHV</td><td>NRVY</td><td>YYAQEEYHII</td><td>nd</td><td> 2,9 ±0,8</td><td> 2,3 ± 0,4</td>

<td>1490H05</td><td>GHYPLHV</td><td>MRQH</td><td>YYAQENYKEI</td><td>nd</td><td> 1,8 ± 2,1</td><td> 2,9 ±1,0</td>

<td>1571G06</td><td>GHYPLHL</td><td>RTKY</td><td>YYKEADYSQI</td><td> 0,93±0,5</td><td> 3,5 ± 1,4</td><td> 5,1 ±4,3</td>

<td>1572C09</td><td>GHYPMHI</td><td>TPKH</td><td>YYNEADYSQI</td><td>nd</td><td> 7,9 ±6,1</td><td> 5,3 ±4,5</td>

(nd: not determined) (Detailed procedures described in Example 4).

Technology of fusion of nucleic acids-proteins

In one aspect, the application provides Adnectin comprising fibronectin domains of type III that bind to the p19 subunit of IL-23. One way to quickly manufacture and test Fn3 domains with specific binding properties is Adnexus nucleic acid-protein fusion technology, from Bristol-Myers Squibb R&D Company. This disclosure uses in vitro expression and labeling technology, called PROfusion ™, that exploits fusions of nucleic acid proteins (RNA-protein and DNA-protein fusions) to identify novel polypeptides and amino acid residues that are important to bind proteins. . The nucleic acid-protein fusion technology is a technology that covalently couples a protein with its encoded genetic information. For a detailed description of RNA-protein fusion technology and fibronectin-based scaffold protein screening procedures see Szostak et al., U.S. Pat.s: 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; 6,518,018; 6,818,418; and Roberts and Szostak, Proc. Nati Acad. Sci. 94: 12297-12302, 1997, incorporated herein by reference.

Vector and Polynucleotide Embodiments

Nucleic acids encoding any of the various proteins or polypeptides disclosed herein can be chemically synthesized. The use of codon can be selected to improve expression in a cell. Such use of the codon will depend on the selected cell type. Codon usage patterns

specialized have been developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See for example: Mayfield et al., Proc. Nati Acad Sci USA January 21, 2003; 100 (2): 43842; Sinclair et al. Protein Expr Purif. October 2002; 26 (l): 96-105; Connell ND. Curr Opin Biotechnol. October 2001; 12 (5): 446-9; Makrides et al. Microbiol Rev. September 1996; 60 (3): 512-38; and Sharp et al. Yeast October 1991 Oct; 7 (7): 657-78.

General techniques for handling nucleic acids are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, volumes 1-3, Cold Spring Harbor Laboratory Press, 2to ed., 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-lnterscience: New York, 1987) and periodic updates, incorporated herein by reference. Generally, the DNA encoding the polypeptide is operably linked to suitable transcriptional or translational elements derived from mammalian, viral, or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding sites and sequences that control the termination of transcription and translation. The ability to replicate in a host, usually contained by an origin of replication and a selection gene to facilitate recognition of transformants is further incorporated.

The proteins described herein can be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptides having a specific cleavage site at the N-terminal end of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (ie, cleaved by a peptidase signal) by the host cell.

For prokaryotic host cells that do not recognize or process a native signal sequence, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, Ipp, or stable enterotoxin II leader sequences.

For yeast secretion the native signal sequence can be substituted by, for example, the yeast invertase leader sequence, a leading factor (including alpha Saccharomyces and Kluyveromyces leading factors), or acidic phosphatase leader sequence, the glucoamylase leader sequence of C. albicans, or the signal described

in US Pat. 5,631,144. In mammalian cell expression, the mammalian ace signal sequences! as the sequences of viral secretory leaders, for example, the gD signal of the herpes simplex gD virus, are available. The DNA for such precursor regions may be linked in reading phase to the DNA encoding the protein.

Both the cloning and expression vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Generally, in the cloning vectors this sequence is one that allows the vector to replicate independently of the host's chromosomal DNA, and includes replication origins of sequences that replicate autonomously. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication of plasmid pBR322 is suitable for gram-negative bacteria, the origin of the 2 micrometer plasmid is suitable for yeast origin and for various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors into mammalian cells Generally, the replication origin component is not necessary for mammalian expression vectors (the SV40 origin can typically be used only because it contains the early promoter).

Expression and cloning vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate, or tracycline, (b) autotrophic complement deficiencies, or (c) critical supply nutrients not available from complex means, for example, the racemase gene encoding D-alanine for Bacilli.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the protein of the invention, for example, a fibronectin-based framework protein. Promoters suitable for use with prokaryotic hosts include the phoA promoter, beta-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system and hybrid promoters such as the Tan promoter. However, others are suitable known bacterial promoters. Promoters for use in bacterial systems will contain a Shine-Dalgarno (SD) sequence operatively linked to the DNA encoding the protein for the invention. Promoter sequences are also known for eukaryotes. Virtually all eukaryotic genes have a region rich in

AT approximately 25 to 30 bases above in the chain of the site where the transcription begins. Another sequence found 70 to 80 bases before the transcription origin of many genes is a CNCAAT region where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence that can be a signal for adding poly-A tail to the 3' end of the coding sequence. All these sequences are properly inserted into eukaryotic expression vectors.

Examples of promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, taies corno enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, priruvate kinase, triosaphosphate isomerase, phosphoglucose isomerase and glucokinase.

Transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of taies corno virus, polyoma virus, avian smallpox virus, adenovirus (taies corno Adenovirus 2), bovine papillomavirus , avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Apes Virus 40 (SV40), from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, from thermal shock promoters, since such promoters are compatible with host cell systems.

The transcription of DNA coding proteins of the invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetal protein and insulin). Typically, however, someone will use an eukaryotic cell virus enhancer. Examples include the SV40 enhancer on the forward side of the origin of replication (base pairs 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the forward side of the origin of replication and the enhancers of replication. adenovirus See also Yaniv, Nature 297: 17-18 (1982) in enhancer elements for activation of eukaryotic promoters. The enhancer can be adjusted in the vector in a 5 'or 3' position to the sequence encoding the peptide, but is preferably located at a 5 'site from the promoter.

Expression vectors used in eukaryotic host cells (for example, yeast cells, fungi, insects, plants, animals, humans, or cells

nucleated from other multicellular organisms) will also contain sequences necessary for termination of transcription and to stabilize mRNA. Such sequences are commonly available from untranslated regions of DNA or eukaryotic cDNA in 5 ', and, occasionally, in 3', or from untranslated regions of viral DNA or cDNA in 5 ', and, occasionally , in 3'. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the untranslated part of mRNA encoding the protein of the invention. A transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector described in the above.

Recombinant DNA can also include any type of protein brand sequence that may be useful for purifying the protein. Examples of protein brands include but are not limited to a histidine brand, a FLAG brand, a myc brand, an HA brand, or a GST brand. Cloning and expression vectors can be found for use with bacterial, fungal, yeast and mammalian cell hosts in Cloning Vectors: A Laboratory Manual, (Elsevier, New York, 1985), the revelation that makes the case of which it is incorporated in this document by reference.

The expression construct is introduced into the host cell using an appropriate procedure for the host cell, as will be apparent to someone skilled in the art. A variety of methods for introducing nucleic acids into host cells, including, but not limited to, electroporation, are known in the art; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; bombardment with microprojectiles; lipofection; and infection (where the vector is an infectious agent).

Suitable host cells include prokaryotes, yeasts, mammalian cells or bacterial cells. Suitable bacteria include gram negative or gram positive organisms, for example, E. coli or Bacillus spp. It can also be used for the production of yeast polypeptides, preferably from the Saccharomyces genus, taies corno S. cerevisiae. Various insect or mammalian cell culture systems can also be used to express recombinant proteins. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, (Bin / Technology, 6: 47, 1988). Examples of host mammalian cell lines include cell lines of endothelial cells, monkey kidney cells CO8-7, CV-1, L cells,

C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T and BHK. Purified polypeptides are prepared by culturing host / vector systems suitable for expressing recombinant proteins. For many applications, the small size of many of the polypeptides disclosed herein would make the expression in E. coli the preferred method for expression. The protein is then purified from culture media or cell extracts.

Protein Production

Host cells are transformed with the expression described herein in cloning vectors for protein production and are grown in conventional nutrient media modified for protein production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences. In the examples shown here, the host cells used for high-performance protein production (HTPP) and medium scale production were the bacterial strain BL21 DE3 plysS. The host cells used to produce the proteins of this invention can be cultured in a variety of media, such as those described in Ham et al., Meth. Enz 58; 44 (1979), Barites et al., Anal. Biochem 102: 255 (1980), U.S. Pat.you 4,767,704; 4,657,866; 4,927,762; 4,560,655; 5,122,469; 6,048,728; 5,672,502; o United States Patent No. Re. 30,985. Any other necessary supplements may be included in appropriate concentrations that would be known to those skilled in the art. The culture conditions such as temperature, pH and the like are those previously used with the host cell selected for expression, and will be apparent to the skilled worker.

The proteins described herein can also be produced using cellular translation systems. For such purposes the nucleic acids encoding the polypeptide should be modified to allow in vitro transcription to produce mRNA and to allow free translation of mRNA cells in the particular cell-free system being used (eukaryotic such as cell-free translation system of mammals or yeast or prokaryotic such as a bacterial cell free translation system).

The proteins of the invention can also be produced by chemical synthesis (for example, by the procedures described in Solid Phase Peptide Synthesis, 2to ed., 1984, The Pierce Chemical Co., Rockford, IL). Modifications to the protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified by isolation / purification procedures for proteins generally known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, precipitation of protein by salts (for example with ammonium sulfate or with sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, single exchange chromatography, hydrophobic chromatography, normal phase chromatography , reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations thereof. After purification, the polypeptides can be exchanged in different buffers and / or can be concentrated by any of a variety of procedures known in the art, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, or is preferably at least 95% pure and most preferably at least 98% pure. Regardless of the exact numerical value of purity, the polypeptide is pure enough to use as a pharmaceutical product.

A platform development process was used to prepare Adnectin antiIL-23. Example 1 describes an example of the manufacturing process. Adnectin is produced in Escherichia celi (E. coli). E. coli MG1655 cells were transformed with expression vector (pBMS2008 / ATI001044) that produces the protein in an insoluble form as inclusion bodies. The recombinant strain is grown in agitated tank fermenters. At the end of the fermentation the inclusion bodies are collected, solubilized and returned to paste in the preparation for purification. The purified Adnectin is conjugated with a 40 kDa branched methoxy-PEG using a maleimide linker. The conjugated material is subsequently repurified to remove free PEG, free Adnectin and product related impurities. Quality control tests are carried out on most drug substances.

Therapeutic uses in vivo

In one aspect, the application provides Adnectin anti-l L-23 useful in the treatment of autoimmune diseases taies corno lupus (e.g. lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, gastritis autoimmune atrophy, Addison's disease, diabetes (for example insulin-dependent diabetes mellitus, diabetes mellitus of

type I), Goodpasture syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic-acting hepatitis, ulcerative colitis, Sjog's disease rheumatic (eg rheumatoid arthritis), polymyositis, scleroderma and mixed connective tissue disease.

The application also provides procedures for administering anti-IL-23 adnectins to a subject. In some embodiments, the subject is a human being. In some embodiments, anti-IL-23 Adnectins are pharmaceutically acceptable to a mammal, in particular to a human being. A pharmaceutically acceptable polypeptide refers to a polypeptide that is administered to an animal without significant adverse medical consequences, such as essentially free of endotoxins or having very low levels of endotoxins.

Formulation and Administration

The application further provides pharmaceutically acceptable compositions comprising the anti-IL-23 Adnectin described herein, in which the composition is essentially free of endotoxin. Therapeutic formulations comprising Adnectin anti-IL-23 are prepared for storage by mixing the described Adnectin having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16to edition, Osol, A. Ed. (1980)), in the form of aqueous, lyophilized solutions or other dried formulations. Acceptable vehicles, excipients or stabilizers are non-toxic for the recipients at the dosages and concentrations employed, and include tampons such as phosphate, citrate and other organic acids; antioxidants that include ascorbic acid and methionine; preservatives (taies such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabenos taies as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, taies as seroalbumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents taies corno EDTA, taies corno sucrose sugars, mannitol, trehalose or sorbitol; salt forming counterparts taies as sodium; metal complexes (for example, Zn-protein complexes); and / or non-isonic surfactants taies as TWEEN ™ 'PLURONICS ™ or polyethylene glycol (PEG).

The formulations herein may also contain more than one active compound as necessary for the particular condition being treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the desired purposes.

The formulations to be used for in vivo administration must be sterile. This can easily be achieved by filtration through sterile filtration membranes.

The skilled technician will understand that the dosage of each therapeutic agent will be dependent on the identity of the agent.

For therapeutic applications, Adnectin anti-IL-23 is administered to a subject, in a pharmaceutically acceptable dosage form. They can be administered intravenously in the form of a rapid intravenous injection or by continuous infusion over a period of time, or by subcutaneous routes. Suitable pharmaceutically acceptable carriers, diluents and / or excipients are well known and can be determined by those skilled in the art according to the guarantees of the clinical situation. Examples of suitable vehicles, diluents and / or excipients include: (1) Dulbecco phosphate buffered saline solution, (2) 0.9% saline solution (0.9% NaCl w / v) and (3) dextrose at 5% (p / v).

The process of the present invention can be practiced in vitro, in vivo, or ex vivo.

Administration of Adnectin anti-IL-23 and one or more additional therapeutic agents, if co-administered or if administered sequentially, may take place as described above for therapeutic applications. Vehicles, diluents and excipients for co-administration will be understood by the skilled technician who depend on the identity of the particular therapeutic agent being administered.

When present in an aqueous dosage form, rather than lyophilized, the protein will typically be formulated at a concentration of approximately 0.1 mg / ml to 100 mg / ml, although wide variation is allowed outside these ranges. For the treatment of the disease, the appropriate dosage of Adnectin anti-IL-23 will depend on the type of disease to be treated, the severity and the course of the disease, if the Adnectin is administered for preventive or therapeutic purposes, on the course of the previous therapy, the patient's medical history and the patient's response to Adnectin and the doctor's discretion. The protein is properly administered to the patient at one time or during a series of treatments.

Adnectin ™ fusions from Union to Seroalbùmina (SABA)

In certain aspects, the application provides fusion proteins comprising domain-fused Adnectin anti-IL23 10Fn3 that bind to human serum albumin (an Adnectin ™ of Seroalbumin binding (domibnium 10Fn3) or SABA). Such fusion proteins have prolonged serum half-lives in the presence of albumin in relation to the anti-IL23 Adnectin alone.

In certain aspects, the application provides fusion proteins comprising domains 10Fn3 that specifically bind to serum albumin, for example, human serum albumin (HSA) to prolong t1/2 of the fusion protein.

In certain embodiments, the serum half-life of the anti-IL-23 adnectin condensed to the SABA is increased in relation to the half-life of the antiIL-23 Adnectin when it is not conjugated to the SABA. In certain embodiments, the serum half-life of the SABA merger is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800, 1900, 2000, 2500, or 3000% longer in relation to the serum half-life of the anti-IL-23 Adnectin when it is not condensed to the SABA. In other embodiments, the serum half-life of the SABA fusion is at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 10 times, 12 times, 13 times, 15 times, 17 times, 20 times, 22 times, 25 times, 27 times, 30 times, 35 times, 40 times, or 50 times larger than the half-life serum of the Adnectin anti-IL-23 when it is not condensed to the SABA. In some embodiments, the serum half-life of the SABA fusion is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

According to elio, the SABA fusion molecules described herein are useful for increasing the half-life of Adnectin anti-IL23 creating a fusion between Adnectin anti-IL23 and SABA. Such fusion molecules can be used to treat conditions that respond to the biological activity of IL23. The present invention contemplates the use of SABA fusion molecules in diseases caused by the deregulation of IL-23.

Fusion can be formed by attaching Adnectin anti-IL23 to each end of the SABA molecule, that is, provisions SABA-Adnectin anti-IL23 or Adnectin antiIL23-SABA.

In one aspect, the disclosure provides fusion proteins comprising

Anti-IL23 adnectin comprising a domain 10Fn3 binding to serum albumin. In exemplary embodiments, the proteins of10Seroalbumin binding Fn3 described herein bind to HSA with a KD of less than 3 μΜ, 2.5 μΜ, 2 μΜ, 1.5 μΜ, 1 μΜ, 500 ηΜ, 100 ηΜ, 50 ηΜ, 10 ηΜ , 1 ηΜ, 500 ρΜ, 100 ρΜ, 100 ρΜ, 50 ρΜ ο 10 ρΜ. In certain exemplary embodiments, the proteins of10Seroalbumin binding Fn3 described herein bind to HSA with a KD of less than 3 μΜ, 2.5 μΜ, 2 μΜ, 1.5 μΜ, 1 μΜ, 500 ηΜ, 100 ηΜ, 50 ηΜ, 10 ηΜ , 1 ηΜ, 500 ρΜ, 100 ρΜ. 100 ρΜ, 50 ρΜ ο 10 ρΜ at a pH range of 5.5 to 7.4 at 25 ° C or 37 ° C. In some embodiments, the proteins of10Seroalbumin binding Fn3 described herein binds very closely to HSA at a pH of less than 7.4 according to the binding affinity for HSA at a pH of 7.4 or higher.

In some embodiments, fusion proteins that comprise domains 1° Fn3 binding to HSA described herein may also bind seroalbumin from one or more monkey, rat, or mouse. In certain embodiments, the proteins of10Seroalbumin-binding Fn3 described herein bind to Indian macaque seroalbumin (RhSA) or Java macaque seroalbumin (CySA) with a KD of less than 3 μΜ, 2.5 μΜ, 2 μΜ, 1.5 μΜ, 1 μΜ, 500 ηΜ, 100 ηΜ, 50 ηΜ, 10 ηΜ, 1 ηΜ, 500 ρΜ ο 100 ρΜ.

In certain embodiments, fusion proteins that comprise domains 10Seroalbumin binding Fn3 described herein bind to domain I and / or domain II of the HSA. In one embodiment, fusion proteins comprising domains10Seroalbumin binding Fn3 described herein do not bind to domain III of the HSA.

In certain embodiments, the part 10Seroalbumin-binding (SABA) Fn3 of the fusion proteins comprises a sequence that has at least 40%, 50%, 60%, 70%, 75%, 80% or 85% identity with the domain 10Fn3 naturai (SEQ ID NO: 1). In one embodiment, at least one of the BC, DE, or FG loops is modified in relation to the domain of10Fn3 nature In another embodiment, at least two of the BC, DE, or FG loops are modified in relation to the domain of10Fn3 nature In another embodiment, all three of the BC, DE, or FG loops are modified in relation to the domain of10Fn3 nature In other embodiments, a SABA comprises a sequence that has at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% identity to any one of the 26 sequences Core SABAs shown in Table 3 (ie, SEQ ID NO: 103, 107, 111, 115, 119 and 123-143) or any of the SABA sequences

prolonged shown in Table 3 (ie, SEQ ID NO: 188-215, minus the 6 x HIS mark).

In certain embodiments, the amino acid residues of the nucleus are fixed and any substitutions, conservative substitutions, deletions or additions take place in residues other than the amino acid residues of the nucleus. In exemplary embodiments, the BC, DE and FG loops are replaced with polypeptides comprising the BC, DE and FG loop sequences of any of the HSA-binding substances shown in Table 3 below (ie, SEQ ID N . °s: 103, 107, 111, 115, 119 and 123-143 in Table 3).

In certain embodiments, an SABA (for example, a SABA core sequence or a sequence based on it as described above) can be modified to comprise an N-terminal extension sequence and / or a C-terminal extension sequence. Exemplary extension sequences are shown in Table 3. For example, SEQ ID NO: 188 designated as SABA1.1 comprises the core SABA 1 sequence (SEQ ID NO: 103) with a terminal sequence MGVSDVPRDLE (SEQ ID NO: 144, designated as AdNT1) and a sequence C-terminal EIDKPSQ (SEQ ID NO: 153). SABA1.1 additionally comprises a His6 mark at the C-terminal end, however, it would be understood that the His6 mark is completely optional and can be located anywhere within the Ν- or C-terminal extension sequences. Additionally, any of the exemplary Ν- or C-terminal extension sequences provided in Table 3 (SEQ ID NO: 144-163), and any variants, can be used to modify any given SABA core sequence provided in Table 3 .

In other embodiments, the tail sequences can be combined with other known linker sequences (eg, SEQ ID NO.s: 164-187 in Table 3) as necessary when designing a SABA fusion molecule.

Conjugation / Crimping

SABA mergers can be covalently or non-covalently linked. In some embodiments, a10Seroalbumin binding Fn3 may be directly or indirectly bound to a heterologous molecule by means of a polypeptide linker. Suitable linkers for joining Fn3 are those that allow separate domains to independently stroll from each other forming a three-dimensional structure that allows high affinity binding to a target molecule.

The disclosure provides a series of suitable linkers that meet

these requirements, including glycine-serine-based linkers, glycine-proline-based linkers, as well as the linker having the PSTSTST amino acid sequence (SEQ ID NO: 184). The Examples described herein demonstrate that the Fn3 domains linked by means of polypeptide linkers maintain their function of binding to the target. In some embodiments, the linker is a glycine-serine-based linker. These ligands comprise glycine and serine residues and can be between 8 and 50, 10 and 30 and 10 and 20 amino acids in length. Examples include linkers having an amino acid sequence (GS)7 (SEQ ID NO: 171), G (GS)6 (SEQ ID NO: 166), and G (GS)6 (SEQ ID NO: 168). Other linkers contain glutamic acid, and include, for example, (GSE)5 (SEQ ID NO: 173) and GGSE GGSE (SEQ ID NO: 177). Other exemplary glycine-serine linkers include (GS)4 (SEQ ID NO: 170), (GGGGS)7(SEQ ID NO: 179), (GGGGS)5(SEQ ID NO: 180), (GGGGS)3G (SEQ ID NO: 181). In some embodiments, the linker is a glycine-proline-based linker. These ligands comprise glycine and proline residues and can be between 3 and 50, 10 and 30 and 3 and 20 amino acids in length. Examples include linkers having an amino acid sequence (GS)3G (SEQ ID NO: 182), (GP)5G (SEQ ID NO: 183). In other embodiments, the linker may be a proline-alanine-based linker that is between 3 and 30, 10 and 30, and 3 and 20 amino acids in length. Examples of proline alanine-based linkers include, for example, (PA)3 (SEQ ID NO: 185), (PA)6 (SEQ ID NO: 186), and (PA)9 (SEQ ID NO: 187). It is contemplated that the optimum crimp length and the optimal amino acid composition can be determined by routine experimentation by procedures well known in the art.

In some embodiments, the fusions described herein are linked by means of a polypeptide linker having a protease site that is cleavable by a protease in the blood or in the target tissue. Such embodiments can be used to release a therapeutic protein for better supply or for better therapeutic properties or for more efficient production.

Additional linkers or spacers can be introduced at the Cterminal end of an Fn3 domain between the Fn3 domain and the polypeptide linker. Additional linkers or spacers can be introduced at the N-terminal end of an Fn3 domain between the Fn3 domain and the polypeptide linker.

In some embodiments, a therapeutic moiety may be directly or indirectly linked to a SABA by means of a polymeric linker. Polymeric linkers can be used to optimally vary the distance between each

Fusion component to create a fusion protein with one or more of the following characteristics: 1) reduced or increased steric hindrance of one or more protein domains when bound to a protein of interest, 2) increased protein stability or solubility, 3) decreased protein aggregation and 4) increased avidity or increased protein affinity.

In some embodiments, a therapeutic moiety is bound to SABA by means of a biocompatible polymer such as a polymer sugar. Polymeric sugar may include an enzyme cleavage site that is cleavable by an enzyme in the blood or in the target tissue. Such embodiments can be used to release therapeutic proteins for better delivery or for better therapeutic properties or for more efficient production.

Summary of Sequences of Adnectins ™ from Seroalbumin Bonding (SABA)

Many of the sequences referenced in this application are summarized in Table 3 below. Unless otherwise specified, all N-terminal extensions are indicated with a simple underline, all C-terminal queues / extensions are indicated with a double underline, and the crimp sequences are within a rectangle. The BC, DE and FG loop regions are shaded for each SABA core sequence.

Table 3. Summary of exemplary sequences of SABA

N °:

<td></td><td>SPVQE TO SGLKPGVDYTITVYAVYG ISINYR</td>

<td></td><td>SPVQE LKPGVDYTITVYAVSK ITEM</td>

<td></td><td>SPVQ VDYTITVYAVSY ISI T</td>

<td>it contains a mutation (highlighted in negrità); the frame version SABA5</td><td>SPVQEFTVPSDLYT SGLKPGVDYTITVYAVTY DVTDLIMHEPISINYRT</td>

<td>see description for SAVA4 'the residue</td><td>SPVQE S VDYTITVYAVTY I INYRT</td>

Sequence

<td>SEC</td><td>Name of</td><td>Description</td>

<td>ID N °:</td><td>Sequence</td><td></td>

<td></td><td></td><td>corrected is highlighted in</td>

<td> 123</td><td>SABA6</td><td>Adnectin ”from Nucleus 6</td>

<td> 124</td><td>SABA7</td><td>Adnectin '1 of Core 7</td>

<td> 125</td><td>SABA8</td><td>Adnectin ”from Core 8</td>

<td> 126</td><td>SABA9</td><td>Adnectin ”from Core 9</td>

<td> 127</td><td>SABA10</td><td>Core 10 adnectin</td>

<td> 128</td><td>SABA11</td><td>Core 11 adnectin</td>

<td> 129</td><td>SABA12</td><td>Core 12 adnectin</td>

<td> 130</td><td>SABA13</td><td>Adnectin ”from Core 13</td>

<td> 131</td><td>SABA14</td><td>Adnectin '™ of Core 14</td>

<td> 132</td><td>SABA15</td><td>Adnectin '™ Nucleus 15</td>

<td> 133</td><td>SABA16</td><td>Adnectin ”from Core 16</td>

<td> 134</td><td>SABA17</td><td>Core 17 'adnectin</td>

<td> 135</td><td>SABA18</td><td>Adnectin ”from Core 18</td>

<td> 136</td><td>SABA19</td><td>Adnectin '™ of Core 19</td>

<td> 137</td><td>SABA20</td><td>Core 20 adnectin</td>

<td> 138</td><td>SABA21</td><td>Core 21 Adnectin</td>

<td> 139</td><td>SABA22</td><td>Adnectin ”from Core 22</td>

<td> 140</td><td>SABA23</td><td>Core 23 adnectin</td>

<td> 141</td><td>SABA24</td><td>Core 24 adnectin</td>

<td> 142</td><td>SABA25</td><td>Adnectin ”from Core 25</td>

<td> 143</td><td>SABA26</td><td>Adnectin1™ of Core 26</td>

EDDSYYSR

SDLY

YDVTDLIMHE

SPVQEFTVPNYYNTATISGLKPGVDYTITVYAVTR

IKANNYMYGPISINYRT

EVVÄÄTpTS'LLISWNHLEHVARYYRITYÖETGGN

SPVQEFTVPEYPTTATISGLKPGVDYTITVYAVTiT

MLKYPTQSPISINYRT

EWAATPTSLLISWGHYRRSGHYYRITYGETGGN

SPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSK

DDYYPHEHRPISINYRT

ËVVÂATP'TSLLISWDASHYERRYYRITYGETÔÔN

SPVQEFTVPRYHHTATISGLKPGVDYTITVYAVTQ

AQEHYQPPISINYRT

EWAATPTSLLIS YYRITYGETGGN

SPVQEFTVPYPPTTATISGLKPGVDYTITVYAVYS AKSYYPISINYRT

EW PT LLI W RITY ET N

SPVQEFTVPSGNATATISGLKPGVDYTITVYAVED

TNDYPHTHRPISINYRT ewaatpts'Lliswhgepdqtryyritygetggn

SPVQEFTVPPYRRTATISGLKPGVDYTITVYAVTS

GYTGHYQPISINYRT ewaatptslliswskysKHgHYyritygetggn spvqeftvdpssytatisglkpgvdytitvyavsk

DDYYPHEHRPISINYRT

EWAATPTèLLISWYEPYÎPIHYYRITYGET'GGNS

PVQEFTVPGYYGTATISGLKPGVDYTITVYAVYGY

YQYTPISINYRT

SPVQEFTVPSGNATATISGLKPGVDYTITVYAVSD

DNKYYHQHRPISINYRT

EWAATPTSLLISW YYRITYGETGGN

SPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSK DDYYPHEHRPISINYRT

EWAATPTSLLISWSKYSKHGHYYRITYGETGGN

SPVQEFTVPSGNATATISGLKPGVDYTITVYAVED

TNDYPHTHRPISINYRT

EVVÄÄTPTSLLISWYEPGASVYYYRITYGETGGN

SPVQEFTVPSYYHTATISGLKPGVDYTITVYAVYG

YYEYEPISINYRT

EVVAATPTSLLISWQSYYAHSDYYRITYGETGGN

SPVQEFTVPYPPQTATISGLKPGVDYTITVYAVYA

GSSYYPISINYRT

E AAT T LLI YYRITY ET N

SPVQEFTVDPSSYTATISGLKPGVDYTITVYAVSK DDYYPHEHRPISINYRT

EVVÄÄTPTSLLISWPEPGTPVYYYRITYGETGGN

SPVQEFTVPAYYGTATISGLKPGVDYTITVYAVYG

YYDYSPISINYRT

SPVQEFTVPPESGTATISGLKPGVDYTITVYAVYA

GYEYPHTHRPISINYRT

EWAATPT LL I ET N

SPVQEFTVPYYVHTATISGLKPGVDYTITVYAVTE

YYYAGAWSVPISINYRT

EWAATPTSLLISWYDPYTYGSYYRITYGETGGN

SPVQEFTVGPYTTTATISGLKPGVDYTITVYAVSY

YYSTQPISINYRT

SPVQEFTVPSSQTTATISGLKPGVDYTITVYAVSY YTKKAYSAGPISINYRT ewaätptSlliswPqpyykpdYyritygetggn spvqeftvprdyttatisglkpgvdytitvyavys, 1. - _ YYGYYPISINYRT

Sequences of N-Terminal Extensions of Adnectlnä '^ Tjempla

144 | ÄdNT 1 | Exemplary leader sequence I MGVSDVPRDI ilar

ID N °:

GGGGS

<td>Adnectin</td><td>what's wrong with it</td>

<td>sequences</td><td>terminals</td>

<td colspan="2">His6 brand</td>

<td>Adnectin</td><td>what's wrong with it</td>

<td>sequences</td><td>terminals</td>

<td>Adnectin</td><td>what's wrong with it</td>

<td>sequences</td><td>terminals</td>

<td colspan="2">His6 brand</td>

<td>Adnectin</td><td>what's wrong with it</td>

<td>sequences</td><td>terminals</td>

<td colspan="2">His6 brand</td>

<td>Adnectin</td><td>what's wrong with it</td>

<td>sequences</td><td>terminals</td>

EFTVPYSQTTATISGLKPGV

DYTITVYAVYGSKYYYPISINYRTEIDKPSQHHHH

H H

PVQEFTVPYSQTTATISGLKPGV

DYTITVYAVYGSKYYYPISINYRTEIEDEDEDEDED

EFTVPYSQTTATISGLKPGV

DYTITVYAVYGSKYYYPISINYRTEIEDEDEDEDED

HHHHHH

PVQEFTVPTRQTTATISGLKPGV

DYTITVYAVSKDDYYPHEHRPISINYRTEIDKPSQ

HHHHHH

PVQEFTVPSSQTTATISGLKPGV

DYTITVYAVSYYTKKAYSAGPISINYRTEIDKPSQH

HHHHH

Sequence

Sequence Name

SABA4.1

SABA5.1

SABA6.1

SABA7.1

SABA8.1

SABA9.1

SABA10.1

SABA11.1

SABA12.1

SABA13.1

SABA14.1

SABA15.1

SABA16.1

SABA17.1

His6 brand description Adnectin ™ nucleus 4 sequence having AdNT1 and AdCT1 terminal sequences with His6 label Adnectin ™ nucleus 5 sequence having AdNT1 and AdCT1 terminal sequences with His6 label Adnectin ™ nucleus sequence 6 having sequences AdNT 1 and AdCT 1 terminals with His6 mark Adnectin ™ core sequence 7 that has AdNT1 and AdCT1 terminal sequences with His6 label Adnectin ™ core sequence 8 that has terminal sequences AdNT1 and AdCT1 with His6 Sequence mark. of nucleus 9 of Adnectin which has AdNT1 and AdCT1 terminal sequences with His6 brand Adnectin nucleus sequence 10 that has AdNT1 and AdCT1 terminal sequences with His6 label Adnectin ™ nucleus 11 sequence that has AdNT1 and AdCT1 terminal sequences with His6 Adnectin ™ nucleus 12 sequence having AdNT1 and AdCT1 terminal sequences with His6 label Adnectin ™ nucleus sequence 13 having AdNT1 and AdCT1 terminal sequences with His6 label Sequence of nucleus 14 of Adnectin ™ having terminal sequences AdNT1 and AdCT1 with His6 mark Sequence of nucleus Adnectin 15 having terminal sequences AdNT1 and AdCT1 with mark of His6 Sequence of nucleus Adnectin 16 having terminal sequences AdNT1 and AdCT1 with His6 tag Sequence of nucleus 17 of Adnectin having terminal sequences AdNT1 and AdCT1 with His6 tag

MGVSDVPRDLEMVAAIHI sLLISWEDDSYYSRY

YRITYGETGGNSPVQEFTVPSDLYTATISGLKPGV

DYTITVYAVTYDVTDLIMHEPISINYRTEIDKPSQH

HHHHH

MGVSDVPRDLEWAATPTSLLISWEDDSYYSRYY

RITYGETGGNSPVQEFTVPSDLYTATISGLKPGV

DYTITVYAVTYDVTDLIMHEPISINYRTEIDKPSQH

HHHHH

MGVSDVPRDLEWAATPTSLLISWYMDEYDVRY

YRITYGETGGNSPVQEFTVPNYYNTATISGLKPG

VDYTITVYAVTRIKANNYMYGPISINYRTEIDKPSQ

HHHHHH

MGVSDVPRDLEWAATPTbLLISWNHLEHVARYY

RITYGETGGNSPVQEFTVPEYPTTATISGLKPGV

DYTITVYAVTITMLKYPTQSPISINYRTEIDKPSQH

HHHHH

MGVSDVPRDLEWAATPTSLLISWGHYRRSGHY

YRITYGETGGNSPVQEFTVDPSSYTATISGLKPG

VDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPS

QHHHHHH

MGVSDVPRDLEWAATPTSLLISWDASHYERRYY

RITYGETGGNSPVQEFTVPRYHHTATISGLKPGV

HHHH

MGVSDVPRDLEWAATPTSLLISWNSYYHSADYY

RITYGETGGNSPVQEFTVPYPPTTATISGLKPGV

DYTITVYAVYSAKSYYPISINYRTEIDKPSQHHHH

H H

MGVSDVPRDLEWAATPTSLLISWSKYSKHGHYY

RITYGETGGNSPVQEFTVPSGNATATISGLKPGV

DYTITVYAVEDTNDYPHTHRPISINYRTEIDKPSQ

HHHHHH

MGVSDVPRDLEWAATPTSLLISWHGEPDQTRY

YRITYGETGGNSPVQEFTVPPYRRTATISGLKPG

VDYTITVYAVTSGYTGHYQPISINYRTEIDKPSQH

HHHHH

MGVSDVPRDLEWAATPTSLLISWSKYSKHGHYY

RITYGETGGNSPVQEFTVDPSSYTATISGLKPGV

DYTITVYAVSKDDYYPHEHRPISINYRTEIDKPSQ

HHHHHH

MGVSDVPRDLEWAATPTSLLISWYEPYTPIHYY

RITYGETGGNSPVQEFTVPGYYGTATISGLKPGV

DYTITVYAVYGYYQYTPISINYRTEIDKPSQHHHH

H H

MGVSDVPRDLEWAATPTSLLISWSKYSKHGHYY

RITYGETGGNSPVQEFTVPSGNATATISGLKPGV

DYTITVYAVSDDNKYYHQHRPISINYRTEIDKPSQ

HHHHHH

MGVSDVPRDLEWAATPTSLLISWGHYRRSGHY

YRITYGETGGNSPVQEFTVDPSSYTATISGLKPG

VDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPS

QHHHHHH

MGVSDVPRDLEWAATPTSLLISWSKYSKHGHYY

RITYGETGGNSPVQEFTVPSGNATATISGLKPGV

DYTITVYAVEDTNDYPHTHRPISINYRTEIDKPSQ

HHHHHH

<td>SEC ID N °:</td><td>Sequence Name</td><td>Description</td><td>Sequence</td>

<td> 207</td><td>SABA18.1</td><td>Sequence of nucleus 18 of Adnectin that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWYEPGASVWY RITYGETGGNSPVQEFTVPSYYHTATISGLKPGV DYTITVYAVYGYYEYEPISINYRTEIDKPSQHHHH H H</td>

<td> 208</td><td>SABA19.1</td><td>Sequence of nucleus 19 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWQSYYAHSDYY RITYGETGGNSPVQEFTVPYPPQTATISGLKPGV DYTITVYAVYAGSSYYPISINYRTEIDKPSQHHHH H H</td>

<td> 209</td><td>SABA20.1</td><td>Sequence of nucleus 20 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWGHYRRSGHY YRITYGETGGNSPVQEFTVDPSSYTATISGLKPG VDYTITVYAVSKDDYYPHEHRPISINYRTEIDKPS QHHHHHH</td>

<td> 210</td><td>SABA21.1</td><td>SequencigLde nucleus 21 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWPEPGTPVYYY RITYGETGGNSPVQEFTVPAYYGTATISGLKPGV DYTITVYAVYGYYDYSPISINYRTEIDKPSQHHHH H H</td>

<td> 211</td><td>SABA22.1</td><td>SequenceJe nucleus 22 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWYRYEKTQHYY RITYGETGGNSPVQEFTVPPESGTATISGLKPGV DYTITVYAVYAGYEYPHTHRPISINYRTEIDKPSQ HHHHHH</td>

<td> 212</td><td>SABA23.1</td><td>Sequence of nucleus 23 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWVKSEEYYRYY RITYGETGGNSPVQEFTVPYYVHTATISGLKPGV DYTITVYAVTEYYYAGAWSVPISINYRTEIDKPSQ HHHHHH</td>

<td> 213</td><td>SABA24.1</td><td>Sequence of nucleus 24 of Adnectin that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWYDPYTYGSYY RITYGETGGNSPVQEFTVGPYTTTATISGLKPGV DYTITVYAVSYYYSTQPISINYRTEIDKPSQHHHH H H</td>

<td> 214</td><td>SABA25.1</td><td>Sequence of nucleus 25 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWSNDGPGLSYY RITYGETGGNSPVQEFTVPSSQTTATISGLKPGV DYTITVYAVSYYTKKAYSAGPISINYRTEIDKPSOH HHHHH</td>

<td> 215</td><td>SABA26.1</td><td>Sequence of nucleus 26 of Adnectin ™ that has terminal sequences AdNT1 and AdCT1 with His6 brand</td><td>MGVSDVPRDLEWAATPTSLLISWPDPYYKPDYY RITYGETGGNSPVQEFTVPRDYTTATISGLKPGV DYTITVYAVYSYYGYYPISINYRTEIDKPSOHHHH H H</td>

EXAMPLES

Example 1: Fermentation and Collection Preparation Process

A production fermentation is prepared with sterile base medium. Thaw and use a vial to inoculate a transfer vessel containing growth medium. The inoculum is transferred immediately to the production fermentation. The culture is maintained at a temperature of 34 ° C with stirring and allowed to grow until an OD is reached.600 5-10 (one unit of DO. is approximately 1x109 cells / ml). The addition of feeding medium begins to this DO Fermentation takes place to DOeoo= The point at which the culture is induced by producing Adnectin by the addition of isopropyl β-Dl-thiogalactopyranoside (IPTG). The temperature of the container increases from 34 ° C to

39 ° C at the time of induction. Samples are taken aseptically every hour and tested for cell density.

After 9-12 hours of induced fermentation the vessel is prepared for collection by reducing the temperature to 25 ° C, by adding ethylenediaminetetraacetic acid (EDTA) to a final concentration of sodium hydroxide and by reduction of agitation. After a retention period of one hour, the fermenter content is emptied into a collection vessel.

Preparation of Inclusion Bodies

The cell disruption of the collection accumulation is done by passing the material through a microfluidifier that disrupts the cells and releases their contents. After cell disruption the inclusion bodies are collected using a disk centrifuge separating solid and liquid phases in a continuous process by extremely high centrifugal forces. The inclusion bodies were then washed twice with buffer (20-25 ° C) and twice with water (20-25 ° C). Each time the inclusion bodies washed are collected by centrifugation. The inclusion bodies washed were recovered as a suspension.

Solubilization of Protein Inclusion and Replenishment Bodies

The solubilization buffer is added to the inclusion body suspension followed by stirring at room temperature for 1 hour. A D.0 is marked as objective during this process.28o = 20.

Protein refolding is carried out using a two stage dilution procedure. Dilution buffer is added to solubilized inclusion bodies at a proportion of one part of solubilized inclusion bodies versus half a part of dilution buffer (v / v). A second dilution is carried out by adding solubilized inclusion bodies for refolding buffer marking as target a D.0.28o = 0.7 (total protein). Dilutions are carried out while stirring while at room temperature. After careful mixing for one hour, the stirring stops and the protein solution is kept at room temperature overnight. The solubilized and refolded Adnectin is passed through a 0.8 μηι-0.22 pm filter and tested for protein content by A28o and HPLC in reverse phase.

Purification and Conjugation to PEG

The refolded and filtered Adnectin is loaded directly onto a cation exchange column (CEX1) for initial capture. The bound material is washed with wash buffer and eluted with 50 mM sodium acetate, 500 mM sodium chloride,

1.5% propylene glycol, pH 5.5. The eluate accumulation is subjected to tests of purity, identity, concentration and endotoxin.

The eluate from the capture chromatography is further purified using hydrophobic interaction chromatography (HIC). The eluate of CEX1 is loaded directly into the HIC column, washed and subsequently eluted with 50 mM sodium acetate, 30% propylene glycol, pH 5.5. The eluted accumulation is subjected to tests of purity, identity and concentration.

The purified Adnectin is then directly formatted with a maleimide derivative of a 40 kDa branched PEG (mPEG2-MAL). The HIC eluate is stirred at room temperature and mPEG2-MAL is added. After 1 hour of mixing at room temperature, the reaction mixture is allowed to incubate overnight at the same temperature. The PEGylation solution was then processed in the final CEX column (CEX2). Samples were taken for protein content, purity and endotoxin.

The pH and conductivity of the PEGylation solution are adjusted to 4.0 and 1.0 ms / cm respectively, with 75 mM acetic acid before loading into the cation exchange column (CEX2) for repurification. Once loaded, the bound material was washed with buffer and subsequently eluted with 50 mM sodium acetate, 25 mM sodium chloride, pH 5.0. Samples were taken for protein content, purity and endotoxin.

The eluate of CEX2 is concentrated at 15 mg / ml in a tangential flow filtration unit equipped with a 30 kDa nominal molecular weight limit membrane with a V sieve. The raw drug substance in 50 mM sodium acetate, 25 mM sodium chloride, pH 5.0, is passed through a 0.22 pm filter and frozen at -80 ° C.

Example 2: Gene, Vector and Host Cell

A plasmid encoding the protein under the control of the T7 promoter was generated for use in strain construction. This plasmid DNA was used to transform competent E. coli K-12 MG 1655 cells (F-lambda-, ilvG-rfb-50 rph-1). The host strain was designed to allow induction of expression from genes after addition of IPTG. The transformed MG1655 strain is resistant to kanamycin. The protein expression vector is shown in Figure 2. A simple selection of plaque colonies is used by inoculating a fermentation culture from which aliquots are taken and frozen using a bank of research cells.

Example 3: Biophysical and Biochemical Characterization

The structure and quality of the protein of the invention was examined by several comprehensive analytical procedures.

EM-MALDI

Mass spectral profiles were analyzed by MALDI. To assess the accuracy of the MALDI analyzes in the samples, 20 individual points were placed on the steel plate for each sample and analyzed sequentially. A total of 20 spectra were generated.

Peptide Mapping

Peptide mapping was confirmed by confirming the correct expression of the amino acid sequence (primary structure) predicted from the cDNA sequence for the protein of the invention as well as for the corresponding dePEGylated protein. In order to obtain full sequence coverage, trypsin (cleavage at the C-terminal side of the Lys and Arg residues) and Glu-C endoprotease (cleavage at the C-terminal side of the Glu residues) were used to provide two overlapping groups of peptide fragments. Peptide mapping was used determining covalent post-translational modifications that include residual N-terminal methionine, disulfide bridge formation, asparagine deamidation, methionine oxidation (etc.). The peptides were identified and characterized by liquid chromatography-mass spectrometry (LC-MS) by means of molecular weight and tandem mass spectrometry (EMEM) that provides partial sequence information by means of collision-induced dissociation (CID).

SDS-PAGE

Polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) was used visualizing patterns of formation of stripped and PEGylated anti-IL-23 adnectin bands. Samples were prepared in a sample buffer with or without a reducing agent. After heating in SDS, samples and molecular weight markers were analyzed electrophoretically in gradient polyacrylamide gels (4-20%), pre-molded. After electrophoresis, the gels were fixed and tineron using Coomassie Blue. The equivalence of sample band formation patterns was assessed visually.

Exclusion Chromatography by Size / Light Dispersion of Multi-Angle (SECMALS)

Exclusion chromatography per size (SEC) was used for quantitative analysis of monomeric, High Molecular Weight (HMW) and Low Weight species

Molecular (LMW). After separation of SEC, the molecular mass of species separated by multi-angle light scattering in tandem with a differential refractomer was determined.

Example 4: In Vitro Non-Clinical Pharmacology

KD by SPR

Binding characteristics were characterized by Surface Plasmon Resonance (SPR). Human IL-23 was immobilized at two different levels in one dimension of Proteon XPR (Bio-Rad) chip surfaces and exposed to 6 different concentrations of anti-IL-23 adnectins in the other dimension of the same surface of splinter of SPR. This allowed kinetic determination in the absence of regeneration. Duplicate chips were used for kinetic determinations at 25 ° C and 37 ° C. Kinetic parameter evaluation was carried out using the Langmuir interaction model and the constant parameter setting with ProteOn Manager software.

As shown in Table 4 below, the dissociation rates for these anti-IL-23 adnectins are slow (of the order of 10 '5 s'1) at 25 ° C. Even at 37 ° C the dissociation rates are close to the detection limit for SPR technologies such that it is possible that the dissociation constants measurements are underestimated.

Table 4: Kinetic parameters of anti-IL-23 adnectin versus directly immobilized human IL-23

IL-23 (° C) ±

± ±

Solution Phase Affinity

The solution affinity of ATI001045 for human IL-23 was measured using a Kinetic Exclusion Assay (KinExA). In one format the duplicate assessments of hlL-23 were carried out for each of three concentrations. The relative unbound ATI001045 concentration was measured by capture on a solid human IL-23 matrix followed by detection with a fluorescently labeled antibody that recognizes the Adnectin framework. Due to technical limitations, the lower concentration

that can be tested was 0.75 nM. So while the analysis of KD Overall shown in Table 5, gives an estimate of 51 pM for the KD, the affinity could be as low as a single digit of pM or such a high horn as 150 pM in a confidence interval of

95%.

Table 5: Phase affinity measurements in solution for ATI001045

The solution affinity of ATI001045 and ATI001047 for human IL-23 was also measured using an alternate format in KinExA. Duplicate titrations of adnectins were performed for each of three concentrations (ATI001045) or for individual concentrations (ATI001047) of human IL-23 (quadrupled for the lowest concentration). The concentration of relative unbound human IL-23 was measured by capture on a solid matrix of non-pegylated ATI001045 followed by detection with a fluorescently labeled antibody that recognizes the p40 subunit of IL-23. K analysisD overall shown in Table 6 gives a KD 9.4 pM with a 95% confidence interval of 22-2.4 pM for ATI001045 and a KD 36.3 pM with a 95% confidence interval of 60.1 to 19.4 pM.

Table 6: Phase affinity measurements in solution

Phosphorylation of STAT3 in Kit225 Cells

Parham et al. (A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rbeta1 and a novel cytokine receptor subunit, IL-23R. J Immunol. June 1, 2002; 168 (11): 5699-708) cloned the IL-23R from the T-lymphocyte line dependent on human IL-2, Kit225. These cells have been characterized for expression of both IL-12RB1 and IL-23R by FACS analysis and responded to IL-23 by stimulation of pSTAT3 and IL-12 by stimulation of pSTAT4. Kit225 cells were seeded in 96-well plates and quiescent in the absence of FBS and IL-2 for 3 hours at 37 ° C. After this incubation, the recombinant IL-23 (or IL-23 preincubated with antagonist for 1 hour) was applied and the cells were returned to the incubator for 15 minutes at 37 ° C to stimulate phosphorylation of

STAT3 (abbreviated as p-STAT3). Each condition was tested in duplicate in 96-well plates. Stimulation was stopped by placing the cells on ice and by adding ice-cold PBS. Finally, the cells were sedimented and lysed following standard protocols and the production of pSTAT3 was detected by ELISA.

The optimal concentration of IL-23 for stimulation was 35 pM. Inhibition of pSTAT3 induced by 11-23 was demonstrated by an anti-p40 monoclonal antibody titre (mAb1510) as well as an anti-p19 polyclonal antibody (AF1716). ATI001045, ATI001047, ATI001014 and ATI001016 have equivalent activity with an IC50 of ~ 300 pM, approximately 150 times more potent than the polyclonal anti-p19 antibody while ATI001015 has an IC50 of ~ 1.2 nM, about 40 times more potent anti-p19 monoclonal antibody. The Adnectin ATI00093 4 is 1/3 of the power of ATI001045, with an IC50 of 1 nM (Table 7).

Table 7: Inhibition of IL-23 induced phosphorylation of STAT3 by antiIL-23 ± antagonists. .

± ±

+ ±

Phosphorylation of STAT3 in Human PBMC

A secondary cell-based confirmatory assay was developed with the goal of evaluating STAT3 phosphorylation as a mechanism of action in primary human cells. Peripheral human mononuclear cells (PBMC) of healthy humans consist mainly of T lymphocytes that have not been previously treated and quiescent that nominally express low levels of IL-23R and do not respond appreciably when stimulated with exogenous IL-23. However, polyclonal activation of PBMC that have not been previously treated with IL-2 results in activation and differentiation of T lymphocytes that have not been previously treated with subsequent increased IL-23R expression of IL-23R. These activated lymphocytes are then susceptible to stimulation with exogenous IL-23 that activates the STAT pathway, resulting in STAT3 phosphorylation.

Commercially available antibodies (AF1716, an anti-p19 pAb and

Mab1510, an anti-p40 mAb, both from R&D Systems), was used as positive controls for inhibition of IL-23-induced STAT3 phosphorylation. Activity

Six adnectin inhibitor was compared in ten separate experiments using multiple donor blood (summarized in Table 8). Exemplary data for a subgroup are shown in Figure 4. Anti-IL-23 adnectins were significantly (> 150 times) more potent than anti-p19 in inhibition of STAT3 phosphorylation but similar to 5 times less potent than antibody. monoclonal anti-p40.

Table 8: Inhibition of PBMC pSTAT3 by anti-IL-23 ± antagonists

± ±

Production of Cytokines Induced by IL-23 by Mouse Splenocytes

Initial cell assays were designed with primary cells to assess the ability of anti-IL-23 adnectins to inhibit IL-23-dependent cytokine secretion from murine Th17 lymphocytes. To differentiate murine Th17 lymphocytes by analysis, CD4 + T lymphocytes were enriched with magnetic beads, co-cultured with irradiated splenocytes and activated with anti-CD3 in the presence of TGF-β and IL-6 and neutralizing antibodies by IL-4 and IFN -γ. After 6 days in culture, polarized Th17 lymphocytes were collected, re-seeded in a 96-well plate and stimulated with human IL-23 at 100 ng / ml and with murine IL-2 at 5 ng / ml. The addition of IL-2 was required to maintain cell viability and to allow production of solid cytokines in response to IL-23 but did not strongly induce production of IL17A or IL-22 alone. Because IL-2 induces a low level of cytokine secretion, each group of samples included cells stimulated with IL-2 alone controlling basal levels of cytokine produced in the absence of IL-23. The IL-23 dependent response was evaluated by calcining the difference between the level of cytokine induced by the combination of IL-2 and IL-23 and the baseline level induced by IL-2 alone. A dose interval of adnectins was added during the re-stimulation of Th17 lymphocytes with IL-2 and IL-23 to test their potential inhibitor. A dosage range of human anti-p40 antibody (MAB1510 from R&D Systems) was run in parallel in the form of positive controls evaluating inhibition of!

I

IL-23 Each condition was tested in triplicate wells of a 96) well plate. After 4 days, the conditioned media from the triplicates will be j!

i ί

I

stored, emptied of cell debris and tested for both concentrations of IL-17A as for concentrations of IL-22 by ELISA.

Stimulation of Th17 lymphocytes with IL-2 and IL-23 induced a 2 to 3-fold increase in IL-17A and at least a 5-fold potentiation of IL-22 compared to levels induced by IL-2 alone. ATI000934, ATI001014, ATI001015, ATI001016, ATI001045 and the positive control anti-p40 monoclonal antibody mediated a dose-dependent decrease in secretion of IL-17A and IL-22-dependent IL23. CI values50 for inhibition of secretion of both IL-17A and IL-22 were calculated for each adnectin as well as the anti-p40 control and these data were summarized in Table 9. All tested adnectins were twice as potent as the control. anti-p40 for inhibition of IL-17A-dependent IL-17A secretion and were within 2 to 3 times as potent for inhibition of IL-23-dependent IL-22 production.

Table 9: Inhibition of IL-23-dependent cytokines by anti-IL-23 adnectins.

± . . 50 ± ·

Adnectin / Ac IL-17 IL-22 ± ± ±. .

± ± n = ± ± ± ± ± ±

Production of Cytokines Induced by IL-23 by Human T Lymphocytes

PBMC were obtained by separating density gradient from whole blood treated with EDTA from normal healthy donors. T lymphocytes were prepared from E + fractions of PBMC surrounded with sheep red blood cells (SRBC). T lymphocytes were plated at 100,000 cells per well in 96-well flat bottom plates that were coated with anti-CD3 (OKT at 10 pg / ml) for 1 hour at 37 ° C and washed with PBS. Mixtures of RPMI-FCS containing anti-CD28 (9.3 to 1 pg / ml) and IL-1β (10 ng / ml) or IL-1ß + IL-23 (1 ng / ml) were prepared. This combination of cytokines has been shown to promote differentiation of human T lymphocytes into IL-17 secretory T lymphocytes. Starting concentration of ATI001045 of 1 pg / ml was added to the mixture containing IL-1β + IL-23. IL-17 was detected in supernatants using Duoset ELISA development kits (R&D Systems). ATI001045 inhibited production of IL-17 with an EC50 2.0 ± 1.6 nM (n = 4 different donors), using the IL-1 ß alone as antecedent. Commercial anti-p40 antibody (MAB1510) was used as an internal control and inhibited the production of IL-17

with an EC50 2.2 ± 1.4 nM (n = 3). Exemplary donor data 228 is shown in Figure 6.

Selectivity of Adnectin Anti-IL-23 for IL-23 over IL-12

ATI001016, the version marked His6 of ATI001045, was used by examining biochemical selectivity for 1L-23 / IL-12. Binding analysis involved the capture of anti-IL-23 Adnectins in immobilized anti-His antibody followed by flow of IL-23 or IL-12 onto the Adnectin. Adnectin selectivity for IL-23 was evaluated by comparing the binding signal for a 100 times higher concentration of IL-12 over IL-23. As shown in Figure 7, ATI001016 showed solid binding (~ 40 RU) to 10 nM human IL-23 while no detectable binding was observed for 1 μΜ human IL-12.

NK-92 lymphocytes are a cell line of human natural cytolytic lymphocytes that respond to IL-12 in an IL-2 dependent manner by secreting IFNγ. The lymphocytes are typically washed by removing IL-2, then seeded in 96-well plates, then treated with 25 pM recombinant human IL-12 (or with IL-12 pre-incubated with antagonists) and incubated for an additional 20 hours. The clarified supernatants were tested for IFN-γ by ELISA.

A 5-fold dilution series was prepared starting at 5 μΜ, 4 points, of each of the adnectin clones listed in Table 2 and incubated with 25 pM IL-12 for 30 minutes at 37 ° C before adding to NK-92 cells. Serial 5-fold dilutions were incubated starting at 5 μΜ, 12-point, ATI001045 and ATI001016 with 25 pM IL-12 for 30 minutes at 37 ° C before addition to NK-92 cells. None of the clones listed in Table 2 or ATI001045 or ATI001016 detectably inhibited IFN-γ secretion at any of the concentrations tested by demonstrating that these anti-IL-23 adnectins do not inhibit the interaction of IL-12 with receptors on the surface of NK92 lymphocytes. They seem equivalent to a negative control and a 100 nM antip19 polyclonal antibody. As a positive control, the anti-p40 monoclonal antibody (mAb1510) inhibited IL-2-induced IFN-γ secretion with an IC50 0.07 nM (Figure 8). Adnectin Anti-IL-23 Blocks IL-17 induced by IL-23 in a Pharmacodynamic Model

Female C57BI / 6 mice were injected intraperitoneally (IP) with recombinant murine IL-2 and with human IL-23 according to the following program.

μα

Table 8: Dosing and Injection Program

Time = -24 h Time = 0 h Time = 7 h

IL-2murina 5pg_ 5 pg 10 pg

Human IL-23 0 10 pg 10 pg

All mice were euthanized 7-8 hours after the final dose of IL-2 and IL-23 at Time = 30 h. Serum was collected and tested for IL-17 and IL-23 by ELISA.

Human IL-23 binds to the mouse receptor and induces the production of cytokines taies as IL-17 and IL-22 Splenocytes from animals to which intraperitoneally (IP) doses of human IL-2 and IL-23 are secreted IL-17 when stimulated in ex vivo culture with mouse anti-CD3e. Significant levels of IL-17 can be detected in the serum of animals that underwent the treatment regimen described in Table 14 in which C57BI / 6 mice are primed with IL-2 24 hours before 3 dual injections of IL- 2 + IL-23 for an additional 24 hours. Presumably, IL-2 activates and polyclonally expands Th populations in situ and regulates upward expression of the IL-23 receptor. This provides a procedure where the mechanisms of drug action and the relationship between drug concentration and the effect in an in vivo framework can be investigated. The model was validated with an anti-p40 monoclonal antibody, mAb1510 (data not shown). In eight separate experiments, five anti-IL-23 adnectins were tested for their ability to inhibit murine IL-17 production when administered subcutaneously at 0.5, 0.15, 0.05 and 0.015 mg / kg 2 hours before the initial dose of IL-2 + IL-23 in two separate experiments. Exemplary dose response data for ATI001045 is shown in Figure 9a (DE50 calculated average of 0.03 mg / kg). All tested anti-IL-23 adnectins showed two-dependent inhibition of human serum IL-17 murine IL-17 production although the extent of inhibition was variable by adnectins.

Activity of ATI001045 in the Cutanea Acanthosis Model Induced by IL-23

Intradermal injection of IL-23 into the skin of the back or into the ear pavilion of the outer ear of mice induced dermal inflammation and hyperplasia of the epidermis (acanthosis) (Yang Zheng lnterlueukin-22, at TH17 cytoquine, media IL-23 -induced fermai inflammation and acanthosis, Nature Vol. 445/8 February 2007). In these studies, recombinant human IL-23 (rHulL-23) was injected into the ears of mice exploring the subsequent consequences of aberrant skin exposure to II-23.

C57BL / 6 female mice were injected six to eight weeks of age with 5 45

pg of recombinant, double-stranded human IL-23 into the right ear every two days until Day 12. PBS was injected into the contra-lateral ear as a control. In one study, treatment with ATI001045 began approximately 2 hours before the first injection of IL-23 and was continued 3 times per week until Day 12. ATI001045 CS was administered at doses of 0.1, 0.3, 1, 3 mg / kg In a second study, IP vehicle ο ATI000934-123 (1753E02) was administered at 1, 3, or 10 mg / kg approximately 1 hour before administration of IL-23 and three times per week thereafter until Day 10. gave IP Anti-HUIL-12 / IL-23 p40 antibody (mAb1510 of R&D) at 10 mg / kg on Day 0 and Day 4 as a positive control. Ear thickness (in thousandths of an inch) was measured every two days, before the first injection in the next ear, using a Mitutoyo dial gauge (No. 2412F). Ear thickness was calculated by subtracting the value of the ear control from the measure for the ear injected by IL-23 for each animal. At the end of the study (Day 14 for ATI01045 and Day 12 for ATI000934), after euthanasia with CO gas2, the ears were split at the birth of the hair and the formalin-fixed / paraffin-embedded tissues were examined histologically on slides held with H&E.

Overall, doses of 1, 3 and 10 mg / kg of ATI000934 provided a similar level of inhibition of ear thickening induced by IL-23 in this study (Figure 10). Ear thickness in all treatment groups was significantly (p <0.01 ANOVA / Dunnette) less than with Vehicle, including the anti-p40 group, from Day 5 until the end of the study on Day 12 . On Day 12, terminal plasma samples were obtained 48 hours after the last dose and analyzed for circulating levels of ATI000934 which were determined to be 11, 18, 36 pg / ml respectively.

After the last measurement on Day 12, the ears were collected at necropsy for routine histological examination from 10 animals per group. Most of the animals administered with ATI000934 had acanthosis and dermal infiltrates, but the histological severity assessment was reduced from that observed in animals treated with vehicles. There was no response to patent dose. All animals administered with anti-p40 also had acanthosis and dermal infiltrates, but the histological severity assessment was also reduced from that observed in animals treated with vehicle.

ATI001045 (at 1 mg / kg and at 3 mg / kg) reduced the thickness of the ear in a two-dependent manner compared to the animals treated with Vehicle (PBS) from Day 5 to Day 14 (p <0.01 versus ANOVA / Dunnett's vehicle, Figure 10). In

In contrast, the dose level of 0.1 mg / kg was not statistically different (p> 0.05) from vehicle treatment on a study day. Serum samples collected 48 hours after the last dose were evaluated for circulating levels of ATI01045 which were determined to be 0.698, 2.72, 8, 22.5 pg / ml for doses of 0.1, 0.3, 1, 3 mg / kg respectively. Histological analyzes revealed that administration of ATI001045 resulted in a dose-dependent reduction of cell infiltrates and acanthosis induced by! L-23 with ear thickness assessment.

Example 5: Material and Procedures Used In This Document Production of High Performance Proteins (HTPP)

The selected binder was cloned into the PET9d vector and transformed into E. coli BL21 DE3 pLysS cells that were inoculated into 5 ml of LB medium containing 50 pg / ml of kanamycin in a 24-well format and grown at 37 ° C for a whole night. Cultures of 5 ml of fresh LB medium (kanamycin at 50 pg / ml) were prepared for inducible expression by aspiration of 200 pl from the culture overnight and were dispensed into the appropriate well. The cultures were grown at 37 ° C to Αβοο 0.6-0.9. After induction with isopropyl β-thiogalactoside (IPTG) with 1 mM the culture was expressed for 6 hours at 30 ° C and collected by centrifugation for 10 minutes at 2750 g at 4 ° C.

Cell pellets were lysed (in 24-well format) by resuspension in 450 pl of Lisis buffer (NaH2PO4 50 mM, 0.5 M NaCl, Complete Protease Inhibitor Cocktail ™ -free 1x EDTA (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1 mg / ml lysozyme, 30 pg / ml DNase, Aprotonin at 2 pg / ml, pH 8.0) and stirred at room temperature for 1-3 hours. The lysates were clarified and re-shaken in a 96-well format by transfer in a 97-well Whatman GF / D Unifilter fitted with a 12-well, 96-well collection plate and filtered by positive pressure. The cleared lysates were transferred to a 96-well Ni Chelating Plate that had been equilibrated with equilibration buffer (NaH2PO4 50 mM, 0.5 M NaCl, 40 mM Imidazole, pH 8.0) and incubated for 5 min. Unbound material was removed by positive pressure. The resin was washed 2 x 0.3 ml / well with Wash Buffer N °: 1 (NaH2PO4 50 mM, 0.5 M NaCl, 5 mM CHAPS, 40 mM Imidazole, pH 8.0) with each wash removed by positive pressure. Before elution, each well was washed with 50 pi Elution Buffer (20 mM PBS + EDTA), incubated for 5 minutes and this wash was removed by positive pressure. The protein was eluted by applying an additional 100 pi of Elution Buffer to each well. After a 30 minute elution at room temperature the plate (s) were centrifuged / centrifuged for 5 minutes at

200 g and eluted protein is collected in 96-well collection plates containing 5 μΙ of MgCI2 0.5 M added to the bottom of the collection plate before elution. The eluted protein was quantified using a BCA assay with SGE as the protein standard.

Medium-scale expression and purification of insoluble fibronectin-based framework protein binders

For expression, the selected clone (s), followed by the HIS mark6, was cloned / cloned into a pET9d vector and expressed / expressed in E.coli BL21 DE3 pLysS cells. Twenty ml of an inoculum culture (generated from an individual plated colony) was inoculated by inoculating 1 liter of LB medium or Expression Media All Night of TB (auntoinduction) containing carbenicillin at 50 pg / ml and chloramphenicol at 34 pg.ml. Cultures in LB medium were incubated at 37 ° C to A6or 0.6-1.0 time at which they were induced with isopropyl-β-thiogalactoside (IPTG) and cultured for 4 hours at 30 ° C. Cultures grown in Expression Media All Night of TB were incubated at 37 ° C for 5 hours at which time the temperature was lowered to 18 ° C for 19 hours. Cultures were collected by centrifugation for 30 minutes at 10,000g at 4 ° C. The cell pellets were frozen at -80 ° C. The cell pellet was resuspended in 25 ml of lysis buffer (NaH2PO4 20 mM, 0.5 M NaCl, Complete Protease Inhibitor Cocktail ™ -free EDTA 1x (Roche), pH 7.4) using an Ultraturrax homogenizer (IKA works) on ice. Cell lysis was achieved by high pressure homogenization (> 124,110 103 pascals (18,000 psi)) using a Model M11 OS Microfluidifier (Microfluidics). The insoluble fraction was separated by centrifugation for 30 minutes at> 23,300 g at 4 ° C. The insoluble sediment recovered by centrifugation of the lysate was washed with 20 mM sodium phosphate / 500 mM NaCl, pH 7.4. The sediment was resolubilized in 6.0 M guanidine hydrochloride in 20 mM sodium phosphate / 500 M NaCl pH 7.4 with sonication followed by incubation at 37 degrees for 1-2 hours. The resolubilized sediment was filtered at 0.45 pm and loaded onto a Histrap column equilibrated with pH 7.4 buffer of 20 mM sodium phosphate / 500 M NaCl / guanidine. After loading, the column was washed for an additional 25 hp with the same buffer. The bound protein was eluted with 50 mM Imidazole in 20 mM sodium phosphate / 500 mM NaCl / 6.0 M guan-HCI pH 7.4. The purified protein was reattached by dialysis against 50 mM sodium acetate / 150 mM NaCl pH 4.5 or PBS pH 7.2.

Medium-scale expression and purification of framework protein binders

based on soluble fibronectin

As an alternative to the purification of insoluble binders, the purification of soluble binders can also be used. For expression, the selected clone (s), followed by the HIS mark6, were cloned / cloned into a pET9d vector and expressed / expressed in E.coli BL21 DE3 pLysS cells. Twenty ml of an inoculum culture (generated from an individual plated colony) was inoculated by inoculating 1 liter of LB medium or Expression Media During A Night of TB (self-induction) containing carbenicillin at 50 pg / ml and chloramphenicol at 34 pg.ml. Cultures in LB medium were incubated at 37 ° C to A6or 0.6-1.0 time at which they were induced with isopropyl-β-thiogalactoside (IPTG) and cultured for 4 hours at 30 ° C. Cultures grown in Expression Media All Night of TB were incubated at 37 ° C for 5 hours at which time the temperature was lowered to 18 ° C for 19 hours. Cultures were collected by centrifugation for 30 minutes at £ 10,000 at 4 ° C. The cell pellets were frozen at -80 ° C. The cell pellet was resuspended in 25 ml of lysis buffer (NaH2PO4twenty mM, 0.5 M NaCl, Complete Protease Inhibitor Cocktail ™ -free EDTA 1x (Roche), pH 7.4) using an Ultra-turrax homogenizer (IKA works) on ice. Cell lysis is achieved by high pressure homogenization (> 124,110103 pascals (18,000 psi)) using a Model M-110S Microfluidifier (Microfluidics). The soluble fraction is separated by centrifugation for 30 minutes at> 23,300 g at 4 ° C. The supernatant is clarified by means of a 0.45 pm filter. The cleared lysate is loaded onto a Histrap (GE) column pre-equilibrated with 20 mM sodium phosphate / 500 M BaCI pH 7.4. The column is then washed with 25 column volumes of the same buffer, followed by 20 column volumes of 20 mM sodium phosphate / 500 M NaCI / 25 mM lmidazole, pH 7.4 and then 35 column volumes of sodium phosphate 20 mM / 500 M NaCI / 40 mM lmidazole, pH 7.4. The protein is eluted with 15 column volumes of 20 mM sodium phosphate / 500 M NaCI / 500 mM lmidazole, pH 7.4, the fractions were stored based on absorbance at A280 and dialyzed against 1 x PBS, 50 mM Tris, 150 mM NaCl, pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH 4.5. Any precipitate was removed by filtering at 0.22 pm.

Fibronectin-based framework proteins (Adnectins) can be pegylated with various sizes and types of PEG. To allow pegylation, the EIDKPSQ residues that occur in nature, found at the C-terminal end of 10FN3 proteins can be modified by an individual point mutation of an amino acid, typically a serine, to a cysteine. PEGylation of the protein in the individual tank residue is carried out by conjugating various forms of PEG derivatized from maleimide, combining the PEG reagent with the protein solution and incubating. An alternative procedure is to replace the EIDKPSQ tail with a GSGC crimp and similarly use the cistern residue for PEGylation. Adnectins containing a cistern residue were conjugated with PEG by means of Michael's addition chemistry between the thiol group in the cistern and the PEG reagent maleimide functional group. Briefly, 40 kDa of PEG are added in a molar excess for protein solution under slightly acidic to neutral conditions. The reaction is then applied to a single exchange column to separate the PEGylated Adnectin from the unreacted PEG-maleimide and the non-PEGylated Adnectin. SE / HPLC procedures can also be used. Purified PEGylated Adnectin is typically analyzed by SDS-PAGE and size exclusion chromatography.

Example 6

Tracking and selection of candidate seroalbumin binding (SABA) Adnectin ™

A known selection technique as PROfusión (see for example, Roberts and Szostak, Proc Nati Acad Sci USA 94 (23): 12297-302, 1997 and WO 2008/066752) were applied to a DNA library with variable regions designed within of the loops of BC, DE and FG of 10Fn3. A random library of more than 1013 Molecules were created from this design, and the selection pressure was applied against a biotinylated form of HSA isolating candidate serum albumin-binding Adnectin (SABA) with desirable binding properties.

Production process of high performance proteins (HTTP)

The various HSA binding Adnectins ™ were purified using a high yield protein production (HTPP) process. The selected binders were cloned into pET9d vector containing an HIS6 label and transformed into E. coli BL21 (DE3) pLysS cells. Transformed lymphocytes were inoculated into 5 ml of LB medium containing 50 pg / ml Kanamycin in a 24-well format and cultured at 37 ° C overnight. Cultures of 5 ml of freshly prepared LB medium (50 pg / ml Kanamycin) were prepared for inducible expression by aspirating 200 μl of the culture overnight and dispensing it into the appropriate well. The cultures were grown at 37 ° C to A600 0.6-0.9. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), the culture was grown for another 4 hours at 30 ° C and collected by centrifugation for 10 minutes at 3220 xga at 4 ° C. Cellular Sediments were frozen at -80 ° C.

The cell sediments (in 24-well format) were lysed by resuspension in 450 μΙ of Lysis Buffer (NaH2PO4 50 mM, 0.5 M NaCl, Cocktail Complete Protease Inhibitor ™ -free 1x EDTA (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1 mg / ml lysozyme, 30 pg / ml DNase, 2 pg / ml aprotonin, pH 8.0) and stirred at room temperature for 1 hour. The lysates were clarified and resuscitated in a 96-well format by transfer in a 96-well Whatman GF / D Unifilter fitted with a 650-well 96-well pickup plate and centrifuged for 5 minutes at 200 x g. The cleared lysates were transferred to a 96-well Ni Chelating Plate that has been equilibrated with equilibration buffer (NaH2PO4fifty mM, 0.5 M NaCl, 10 mM CHAPS, 40 mM Imidazole, pH 8.0) and incubated for 5 minutes. Unbound material was removed. The resin was washed 2 x 0.3 ml / well with Wash Buffer N °: 1 (NaH2PO4 50 mM, 0.5 M NaCl, 5 mM CHAPS, 40 mM Imidazole, pH 8.0). The resin was then washed with 3 x 0.3 ml / well with PBS. Before elution, each well was washed with 50 pl Elution Buffer (20 mM PBS + EDTA), incubated for 5 minutes and this wash was removed by vacuum. The protein was eluted by applying 100 pl of additional elution buffer to each well. After incubation for 30 minutes at room temperature the plate (s) were centrifuged for 5 minutes at 200 xg and the eluted protein was collected in 96-well plates containing 5 pl of MgCI2 0.5 M fixed to the bottom of the Ni. The eluted protein was quantified using a BCA Protein Assay with SGE (Adnectin Control) as the protein standard. SGE Adenectin is a domain of10Natural Fn3 (SEQ ID NO: 1) in which the integrin binding domain (RGD amino acids at positions 78-80) has been replaced with SGE.

Direct union ELISA to HSA, RhSA and MuSA

To submit direct binding tests to HSA, MaxiSorp ™ plates (Nunc International, Rochester, NY) were coated with 10 pg / ml of HSA (Sigma, St. Louis, MO) in PBS at 4 ° C overnight followed by their blockade in casein blocking buffer (Thermo Scientific, Rockford, IL) for 1-3 hours at room temperature. For single point screening assays, Adnectins ™ HTPP 1:20 were diluted in casein blocking buffer and allowed to bind HSA in each well for 1 hour at room temperature. For dose response assays, concentrations ranging from 0.1 nM to 1 pM were used. After washing in PBST by removing unbound Adnectins ™, anti-His mAb-HRP conjugate (R&D Systems, MN) diluted 1: 2500 in casein blocking buffer labeled with His labeled Adnectin ™ was added for 1 hour at room temperature. Excess conjugate was removed by washing with PBST and bound Adnectins ™ were detected using TMB detection reagents (BD Biosciences) according to the manufacturer's instructions.

Identification of bovine serum albumin binding (SABA)

As a result of screening for HSA / RhSA / MuSA binding and biophysical criteria, four unique seroalbumin binding Adnectins (SABA) were identified and chosen to have their half-lives evaluated in mice. In order to carry out the characterization in vitro and in vivo, intermediate scales were assumed for the four SABAs. Table 3 provided the sequences of twenty-six unique SABA core sequences identified from PROfusión, designated as SABA 1-26. SABA4 has a framework mutation that was fixed before performing the intermediate scale. The perfect version in the SABA4 framework is SABA5. SABA4 and SABA5 have identical sequences in the loops of BC, DE and FG.

Example 7

Production and formulation of SAAS candidates

SABA medium-scale protein production

Selected SABAs followed by the His brand6, were cloned into a pET 9d t vector and expressed in E. coli BL21 (DE3) pLysS cells (see Table 3 for each SABA sequence labeled with His designated SABA1.1, SABA2.1, SABA3.1 and SABA5.1 ). 20 ml of an inoculum culture (generated from an individual plate colony) was used by inoculating 1 liter of LB medium containing 50 pg / ml Kanamycin. The culture is grown at 37 ° C to A600 0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG) the culture was grown for another 4 hours at 30 ° C and collected by centrifugation for 30 minutes at ä 10,000 xga 4 ° C. Cellular Sediments were frozen at -80 ° C. The cell pellet was resuspended in 25 ml of lysis buffer (NaH2PO4 20 mM, 0.5 M NaCl, Complete Protease Inhibitor ™ -free EDTA 1x (Roche), pH 7.4) using an Ultraturrax homogenizer (IKA works) on ice. Cell lysis was achieved by high pressure homogenization (à 124,110 103 pascals (18,000 psi)) using a Model M110S Microfluidifier (Microfluidics). The soluble fraction was separated by centrifugation for 30 minutes at 23,300 xga at 4 ° C. The supernatant was clarified by means of a 0.45 pm filter. The cleared lysate was cleared on a HisTrap (GE) column pre-equilibrated with NaH2PO4twenty mM, 0.5 M NaCl, pH 7.4. The column was then washed with 25 column volumes of NaH2PO4 20 mM, 0.5 M NaCl, pH 7.4, followed by 20 column volumes of NaH2PO4 20 mM, 0.5 M NaCl, 25 mM imidazole pH 7.4 and then 35 column volumes of NaH2PO4 20 mM, 0.5 M NaCl, 40 mM imidazole pH 7.4. The protein was eluted with 15 column volumes of NaH2PO4 20 mM, 0.5 M NaCl, 500 mM imidazole pH 7.4, stored fractions based on absorbance at A280 and dialyzed against 1x PBS, 50 mM Tris, 150 mM NaCl pH 8.5 or 50 mM NaOAc; NaCl

150 mM; pH 4.5. Any precipitate was removed by filtering at 0.22 pm.

Medium-scale expression and purification produced highly pure and active Adnectins ™ that were expressed in a soluble form and purified from the soluble fraction of the bacterial cytosol. SEC analysis in a Superdex 200 or Superdex 75 10 / 30GL in a mobile phase of NaPO4 100 mM, NaSO4 100 mM, 150 mM NaCl, pH 6.8 (GE Healthcare) demonstrated predominantly monomeric Adnectins ™.

SABA1.2 formulation

A specific SABA, SABA1.2 (SEQ ID NO: 80), was chosen for a preliminary formulation scan. SABA1.2 comprises an extension (DE)5 of the sequence “core 1” of 10Fn3. For SABA1.2, a stable formulation of 10 mM succinic acid, 8% sorbitol, 5% glycine at pH 6.0 and a product concentration of 5 mg / ml was identified. In this formulation the protein fusion temperature was 75 ° C as determined by Differential Scanning Calorimetry (DSC) using a protein concentration of 1.25 mg / ml. The formulation provided satisfactory physical and chemical stability at 4 ° C and 25 ° C, with an initial aggregate level of 1.2%. After a month of stability, the level of aggregation was very low (1.6% at 4 ° C and 3.8% at 25 ° C). The protein was also stable in this formulation after five freezing-melting eidos as it transitioned from -80 ° C and -20 ° C to room temperature. In addition, in this formulation SABA1.2 was soluble at least 20 mg / ml of protein concentration at 4 ° C and room temperature with no precipitation or with no increase in aggregation.

Example 8

Biophysical characterization of SABA candidates

Exclusion chromatography by size

Standard size exclusion chromatography (SEC) was carried out in the SABA candidates that result from the medium-scale manufacturing process. The SEC of medium-sized material prepared was carried out using a Superdex 200 10/30 column or a Superdex 75 10/30 column (GE Healthcare) in an Agilent 1100 or 1200 HPLC system with UV detection at A214 nm already A280 nm and with fluorescence detection (excitation = 280 nm, emission = 350 nm). I start a

100 mM sodium sulfate buffer, 100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8 at the appropriate flow rate of the SEC column used. Gel filtration standards (Bio-Rad Laboratories, Hercules, CA) were used for molecular weight calibration.

The results of the SEC in the purified medium-scale SABAs showed predominantly monomeric Adnectin ™ and elution in the approximate 10 kDa range between standards of Globular Gel Filtration (BioRad) as shown.

Thermostability

Differential Exploration Calorimetry (DSC) analyzes of medium-scale SABAs were carried out by determining their Tm respective. A solution of 1 mg / ml was explored in an N-DSC II calorimeter (Calorimetry Sciences Corp) by raising the temperature from 5 ° C to 95 ° C at a rate of 1 degree per minute at a pressure of 303975 pascals (3 atmospheres). The data was analyzed for a control operation in the appropriate buffer using a better fit using Orgin Software (OrginLab Corp). The results of SEC and DSC analysis are summarized in Table 1.

Table 1. Summary of analysis of SEC and DSC in the SABA candidates.

mere

Example 9

Characterization of union of SABA1 candidate for serum albumin

The kinetic properties of selected SABA clones purified from HTPP and / or medium-sized material were determined by capturing the respective serum albumin (HSA / RhSA / MuSA) on the surface of a CM5 Biasensor chip and flowing a series of concentrations of SABAs both on the reference cell flow as on the captured albumin. In addition, albumin binding was carried out under various pH conditions ranging from pH 5.5 to pH 7.4. Adnectins ™ that bind to HSA SABA2.1, SABA3.1, SABA4.1 (SABA5.1) and SABA1.1 were cross-reacted with RhSA but do not cross-react with MuSA. Binding to SABA2 and SABA4 is pH sensitive while clone SABA3 demonstrated resistance to binding HSA below pH 6.0. SABA1.1 adjusts biochemical criteria for pH resistance and

affinity / kinetic properties below pH 5.5.

Domain mapping was determined by Biacore. Selected SABA clones purified from HTPP and / or medium scale material were determined by capturing HSA or a construct consisting of only HSA domain I and II or HSA domain III on the surface of a CM5 Biasensor chip and making A series of concentrations of the SABAs flow over both the reference cell flow and the captured albumin. The SABA2 and SABA1 clones join HSA and the construction of domains l-ll of HSA but not the construction of domain III of HSA. The SABA3 and SABA4 clones bind to HSA but not to any of the constructions of the l-ll domains of HSA or the ili domain of HSA. The results are summarized in table 12.

Table 12. Affinity and Union Kinetics of SABA Candidates (SABA1.1, 2.1, 3.1 and 4.1).

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Example 10

T test1/2 SABA candidates live

The half-life of HSA in mice was determined allowing evaluation of

HSA-binding adnectins ™ in mice, taking into account that HSA-binding Adnectins ™ do not cross-react with MuSA. HSA was injected into the tail vein of atimic female mice approximately 6 weeks of age at a dose of 20 mg / kg (Figure 10A) and at a dose of 50 mg / kg (Figure 10B) and the concentration of HSA in samples of Blood taken at intervals after injection was determined by ELISA. The t1/2 of HSA injected into mice at 20 mg / kg and at 50 mg / kg was determined to be ~ 24 hours and ~ 20 hours, respectively.

Half-life determination of SABA1-4 in mice

One liter of E. coli culture of SABA1.1, SABA2.1, SABA3.1 and SABA4.1 of HSA binding clones was prepared, purified and endotoxins removed. Each variant of SABA was injected into the mouse tail vein, and the concentration

in blood samples taken at intervals after injection, it was determined by ELISA.

The pharmacokinetic profiles of each SABA were compared in the presence or absence of HSA in Ncr athletic female mice approximately 6 weeks of age. The mice that were injected with HSA had the HSA premixed with each of the SABAs (HSA in a molar excess of 3-4) because the binding gift was selective for HSA and RhSA and did not bind bovine serum albumin. The half-life of SABA1.1 in mice plasma was 0.56 hours while the half-life of SABA1.1 co-injected with HSA was 5.6 hours, an increase of ~ 10 times in half-life (Figure 12A). The SABA2.1 half-life in mice plasma was 0.24 hours while the SABA2.1 half-life co-injected with HSA was 2.8 hours, an increase of ~ 12 times in half-life (Figure 12B). The SABA3.1 half-life in mice plasma was 0.28 hours while the SABA3.1 half-life co-injected with HSA was 0.53 hours, an increase of ~ 2 times in half-life (Figure 12C). The SABA4.1 half-life in mice plasma was 0.66 hours while the half-life of SABA4 co-injected with HSA was 4.6 hours, an increase of ~ 7 times in half-life (Figure 12D). A summary of the present example is shown in Figure 13A.

Half-life determination of SABA1.1 and SABA5.1 in Java macaques

A single dose test of three weeks of concept study of SABA1.1 and SABA5.1 was conducted in Java macaques evaluating pharmacokinetics at an intravenous dose of 1 mg per kg (mpk) in two Java macaques. Pharmacokinetics were evaluated using a quantitative ELISA based assay that was developed to detect Adnectin ™ in plasma samples. SABA1.1 has a half-life in the 96-137 hour interval. SABA5.1 has a half-life of approximately 12 hours and was only measurable in the ELISA for up to 120 hours. Figures 14 A and B summarize data for these clones and compare Java macaque data.

Example 11

Characterization of binding of SABA1 to serum albumin

SABA1.1 and 1.2 join HSA and RhSA

SABA1.2, a 10"Core 1" Fn3 comprising an extension of (DE) 5 (SEQ ID NO: 190) was bound to human serum albumin (HSA) at neutral pH and at 25 ° C with an average association speed constant (ka) of 8.21 E + 03 M '1s'1 and an average dissociation rate constant (kd) of 4.43E-04 s'1, for a calculated average Kd of 55.3 nM (Table 13). For rhesus serum albumin (RhSA), the measured association rate constant was 6.6E + 03 M '1s'1, and the average dissociation rate constant was 3.78E-03 s1, giving a Kd calculated average of 580 nM. No measurable interaction between SABA1.2 and mouse or rat serum albumin was observed up to 1 μΜ (Table 13 and Figure 15). At 37 ° C, ka and kd were increased between 2 to 5 times, leading to a ~ 2-fold increase in affinity for HSA and a 1/2 increase in affinity for RhSA (Table 13).

Table 13. Kinetic parameters for binding to SABA1.2 for albumins, in HBS-P buffer.

<td>Albumin Human</td><td>Temp ('C)</td><td>ka (1 / Ms) 8.21 ± 1.19 E + 03</td><td>kd (1 / s) 4.43 ± 0.65 E-04</td><td>KD (nM) 55.3 ± 13.7</td>

<td>Macaque from India</td><td> 25</td><td>6.60 ± 1.18 E + 03</td><td>3.78 ± 0.45 E-03</td><td> 580 ± 62,6</td>

<td>Mouse</td><td></td><td colspan="2">no observable union</td><td></td>

<td>Human</td><td></td><td>3.38E + 04</td><td>8.15E-04</td><td> 24,1</td>

<td>Macaque from India</td><td> 37</td><td>1.89E + 04</td><td>1.85E-02</td><td> 977,4</td>

<td>Mouse</td><td></td><td colspan="2">no observable union</td><td></td>

Additionally, a calorimetric titration was carried out determining the stoichiometry between SABA1 and HSA. For this study, SABA1.1, a10Fn3 "of core 1 comprising an extension of His6 (SEQ ID NO: 189). HSA (10 μΙ was injected by 115 μΜ protein solution injection) into the calorimetric cells containing SABA1.1 at a concentration of 8.1 μΜ. The experiment was carried out at 37 ° C in pH 7.4 PBS buffer. Figure 16 shows that SABA1.1 binds to HSA with 1: 1 stoichiometry.

SABA1.2 binds potently to HSA at low pH

The long half-life of albumins (for example, t1/2 of HSA is 19 days) is due in large part to the fact that they are recycled from an endocytic pathway by binding to the neonatal Fc receptor, FcRn, according to the low pH conditions that exist within the endosome. As shown in Table 4, SABA1.2 potently binds HSA to the endosomal pH of 5.5 pH, suggesting that t1/2 SABA1, once joined to HSA, would benefit from the FcRn recycling mechanism.

Table 14. Comparison of albumin binding kinetics at pH 7.4 and 5.5, in MES buffer.

<td>albumin</td><td>PH</td><td>ka (1 / Ms)</td><td>kd (1 / s)</td><td>KD (nM)</td>

<td rowspan="2">Human</td><td> 7,4</td><td>9.26E + 03</td><td>3.88E-04</td><td> 41,9</td>

<td>Macaque</td><td> 7,4</td><td>6.16E + 03</td><td>2.95E-03</td><td> 479</td>

<td>india</td><td> 5,5</td><td>7.57E + 03</td><td>2.72E-03</td><td> 359</td>

SABA1.2 joins domains I and II of HSA, but not Ili domain

The SABA1.2 binding site in albumin was mapped into the N-terminal domains I or II using recombinant HSA fragments and there is no binding detectable to domain III (Figure 17). Because domain III is the HSA domain that interacts primarily with FcRn, SABA1.2 is less likely to compete by joining HSA with FcRn, again increasing the possibility of boosting the recycling mechanism for enhanced half-life.

Example 12

In vivo pharmacology of SABA1.2

A four-week single dose pre-toxicological study of SABA1.2 was carried out in Java macaques evaluating pharmacokinetic characteristics and toxicity at two different dose levels. Pharmacokinetic characteristics and immunogenicity were also evaluated in a single dose, three-week pretoxicological study that included both intravenous administration and cutaneous administration. Additionally, pharmacokinetics of SABA1.2 were evaluated in two separate pretoxicological studies in Java macaques using a quantitative ELISA based assay that was administered to detect SABA1.2 in plasma samples.

SABA1.2 was administered to monkeys at 1 mpk and 10 mpk intravenously. As shown in Figure 18 and in the parameters described below, the Cmâx and the AUC were increased approximately in line with doses. Non-compartmental analyzes using WinNonlin software were carried out evaluating pharmacokinetic parameters. The clearance (CL) for SABA1.2 at 10 mpk was 0.15 ml / hr / kg, the half-life (t1/2) of the beta phase was 143 hours, the volume of distribution (Vz) was 30 ml / kg and the total drug exposure (AUCall) was 5,609,457 hr x nmol / l (Table 15). The clearance (CL) for SABA1.2 at 1 mpk was 0.4 ml / hr / kg, the half-life (t1/2) was 124 hours, the volume of distribution (Vz) was 72 ml / kg and the total drug exposure (AUCall) was 214,636 hr x nmol / l (Table 15).

After subcutaneous or intravenous administration of SABA1.2, the beta phase pharmacokinetic profiles were similar (Figure 19). Non-compartmental analyzes using WinNonlin software were carried out evaluating pharmacokinetic parameters. The clearance (CL) for SABA1.2 at 1 mpk intravenously was 0.22 ml / hr / kg, the half-life (tV2) of the beta phase was 125 hours, the volume of distribution (Vz) was 40 ml / kg and the total drug exposure (AUCall) was 357,993 hr x nmol / l (Table 15). The clearance (CL) for SABA1.2 at 1 mpk subcutaneously was 0.32 ml / hr / kg, the half-life (t1/2) of the beta phase was 134 hours,

the volume of distribution (Vz) was 62 ml / kg and the total drug exposure (AUCall) was 251,339 hr x nmol / l (Table 15). The relative subcutaneous bioavailability (F) compared with intravenous was 0.7.

Table 15. Pharmacokinetic Parameters for SABA1.2 in Mice.

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LIST OF SEQUENCES <110> Bristol-Myers Squibb Company Schneeweis, Lumelle Bush, Alex

Dasgupta, Ruchira Engle, Linda <120> Fibronectin framework domain proteins that bind to IL-23 <130> 11563 PCT <150> 61/305566 <151> 2010-02-18 <150> 61/330706 <151 > 2010-05-03 <160> 258 <170> Patentln version 3.5 <210> 1 <211> 94 <212> PRT <213> Homo sapiens <400> 1

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Tyr Lys Glu

<td> <221></td><td>MISC_FEATURE</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<td> <221></td><td>MISC_FEATURE</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<td> <221></td><td>MISC_FEATURE</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

Asn

Glu

Tyr Arg Glu

Tyr His Ile

Ile

Ile ί

<220>

<221> MISC_FEATURE <223> FG loop <400> 58

Tyr Tyr Glu Gin Val Glu Tyr Arg Glu Ile 15 10 <210> 59 <211> 10 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> FG loop <400> 59

Tyr Tyr Ser Glu Glu Leu Tyr Lys Tyr Ile 15 10 <210> 60 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1434A08 <400> 60

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

I î

I

Item

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Met His Val Arg 20 25 30 i

I »

I

Tyr Tyr Arg 35

Gly Glu 40

Thr gly

Gly asn

Pro Val Gin

Ile

Thr Tyr r

f / f / Ì / · ' ; ·-, J; I 1

His His His

<td> <213></td><td colspan="2">artificial</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="2">Polypeptide</td><td></td><td></td><td></td><td></td><td></td>

<td> <221></td><td colspan="2">MISC FEATURE</td><td></td><td></td><td></td><td></td><td></td>

Glu Val Val Wing Wing Thr 15

Tyr Pro Met His Val Arg 30

Gly Asn Ser Pro Val Gin 45

Wing Thr Ile Ser Gly Leu 60

Tyr Ala Val Thr Tyr Tyr 75 80

Ser Ile Asn Tyr Arg Thr 95

His His His t!

<210> 63 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin

40 45

Glu Phe Thr Val Pro Asn Arg Ala His Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80

Asn Glu Wing Asp Tyr Ser Gin Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His His 100 105 <210> 65 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1438B02 <400> 65

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Thr Wing

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Met His Val Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Arg Lys Thr Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80 <220>

<td> <223></td><td>Polypeptide</td>

<td> <221></td><td>MISC_FEATURE</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<td> <221></td><td>MISC_FEATURE</td>

His His His

Glu Val Val Wing Wing Thr I

I fc

Tyr Pro Met His Val Arg 3 0

Gly Asn Ser Pro Val Gin 45

Wing Thr Ile Ser Gly Leu 60

Tyr Ala Val Thr Tyr Tyr 75 80

Ser Ile Asn Tyr Arg Thr 95

Glu Val Val Wing Ala Thr | 15 î

K

I

I ί

Lys

Pro Gly Val Asp Tyr Thr 70

Ile Thr Val

Tyr Ala Val 75

Thr Tyr Tyr 80

Asn Glu Wing Asp Tyr Ser Gin Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro 100

Be Gin His

His

105

His His His His <210> 70 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1486B05 <400> 70

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr

Be Leu Leu

Ile

Be Trp Gly His Tyr 25

Pro Met

His Val Arg 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Ser Arg Ile Tyr Thr Ala Thr Ile Ser Gly Leu f

55 60 ί

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80

Asn Glu Wing Asp Tyr Ser Gin Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys 100

Pro

Be Gin His

His His His His 105 <210> 71

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Leu His Ile Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Lys Gin Val Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80

Asn Glu Wing Asp Tyr Ser Gin Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His His 100 105 <210> 73 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1487G03 <400> 73

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Leu His Val Arg 20 25 30

Tyr Tyr Arg 35

Ile Thr Tyr Gly Glu 40

Thr gly

Gly Asn Ser 45

Pro Val

Gin

100

105 <210> 75 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1487H04 <400> 75

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Met His Val Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Ala Arg Gin Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80

Gin Glu Tyr Glu Tyr Arg Tyr Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His 105

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<220>

Thr Gly Gly Asn Ser Pro Val Gin

and

<td> <210> <211> <212> <213></td><td colspan="3">78 109 PRT artificial</td><td></td><td></td><td></td>

<td> <220> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td>

<td> <220> <221> <223></td><td colspan="3">MISC_FEATURE 1490H05</td><td></td><td></td><td></td>

<td>Pro Thr</td><td>Be</td><td>Leu twenty</td><td>Leu</td><td>Ile</td><td>Be</td><td>Trp</td>

<td>Glu Phe fifty</td><td>Thr</td><td>Val</td><td>Pro</td><td>Met</td><td>Arg 55</td><td>Gin</td>

His Thr Ala Thr Ile Ser Gly Leu 60

Thr Val Tyr Ala Val Thr Tyr Tyr 75 80

Pro Ile Ser Ile Asn Tyr Arg Thr 90 95

His His His His 105

Asp Leu Glu Val Val Wing Wing Thr 10 15

Gly His Tyr Pro Leu His Val Arg 25 30

Thr Gly Gly Asn Ser Pro Val Gin

His Thr Ala Thr Ile Ser Gly Leu 60

Thr Val Tyr Ala Val Thr Tyr Tyr ji

80 I%

I

Pro Ile Ser Ile Asn Tyr Arg Thr

I

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1490H06 <400> 80

<td>Pro</td><td>Thr</td><td>Be</td><td>Leu twenty</td><td>Leu</td><td>Ile</td><td>Be</td><td>Trp</td><td>Gly 25</td><td>His</td>

<td>Giu</td><td>Phe fifty</td><td>Thr</td><td>Val</td><td>Pro</td><td>Arg</td><td>Gin 55</td><td>Lys</td><td>Tyr</td><td>Thr</td>

I ί

ff:

Val Val Ala Ala Thr { 15 I f

I

Pro Leu His Ile Arg 5

0 Î

F fc t

Asn Ser Pro Val Gin 1

Thr Ile Ser Gly Leu

Wing Val Thr Tyr Tyr 80

Ile Asn Tyr Arg Thr 95

His His î · f

I <210> 81 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1490A07 <400> 81

Met Gly Val Ser Asp Val Pro Arg Asp Leu 15 10

Val Val Wing Wing Thr 15

I ί

• t

I

I ί

70 75 80

Asn Glu Wing Asp Tyr Ser Gin Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His His 100 105 <210> 83 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<td colspan="16"><221> MISC FEATURE</td>

Asn Glu Ala Asp

Tyr

Be gin

Ile

Pro

Ile

Ile Asn Tyr Arg Thr 95

Glu Ile Asp

Lys Pro Ser Gin His 100

His His 105

His His His jί <210> 84 <211> 109 î t

Item

<td>Met one</td><td>Gly</td><td>Val</td><td>Be</td><td>Asp 5</td><td>Val</td><td>Pro</td><td colspan="5">Arg Asp Leu Glu Val 10</td><td>Val</td><td>To</td><td>To fifteen</td><td>Thr</td>

<td> <213</td><td> ></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Yeah

<220>

<td> <223></td><td>Polypeptide</td>

<td> <221></td><td>MISC_FEATURE</td>

I ι

lì ί

I ι

ι ί

<210> 88 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1571F10 <400> 88

Met Gly Val Ser Asp Val Pro Arg 1 5

Pro Thr Ser Leu Leu Ile Ser Trp

Tyr Tyr Arg Ile Thr Tyr Gly Glu 35 40

Glu Phe Thr Val Pro Ser Arg Tyr 50 55

Lys Pro Gly Val Asp Tyr Thr Ile 65 70

Glu Gin Val Glu Tyr Arg Glu Ile

Glu Ile Asp Lys Pro Ser Gin His 100 <210> 89 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide

Asp Leu 10

Gly His 25

Thr gly

His Thr

Thr val

Pro Ile 90

His His 105

<220>

<221> MISC FEATURE

<td> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <221></td><td>MISC</td><td colspan="2">_FEATURE</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<220>

<221> MISC_FEATURE <223> 1572C09 <400> 93

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Met His Ile Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Thr Pro Lys His Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80

Asn Glu Wing Asp Tyr Ser Gin Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His His 100 105 <210> 94 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1572H05 <400> 94

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Leu His Ile Arg

<td> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <221></td><td>MISC</td><td colspan="2">FEATURE</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

f ί

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Leu His Ile Arg

25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Arg Met Arg His Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80

Ser Glu Glu Leu Tyr Lys Tyr Ile Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His His 100 105 <210> 99 <211> 109 <212> PRT <213> artificial <220>

<223> Polypeptide <220>

<221> MISC_FEATURE <223> 1550E06 <400> 99

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Gly His Tyr Pro Met His Val Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Pro Pro Arg His Thr Ala Thr Ile Ser Gly Leu 50 55 60

<td> <213></td><td>artificial</td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td></td>

<td> <221></td><td>MISC_FEATURE</td><td></td><td></td>

<td> <223></td><td>Crimp GSGC</td><td></td><td></td>

<td> <213></td><td>artificial</td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td></td>

<td> <221></td><td>MISC_FEATURE</td><td></td><td>Ì t</td>

<td> <223></td><td>Crimp EIDKPÇQ</td><td></td><td>i</td>

<td> <213></td><td>artificial</td><td></td><td>ί j. one</td>

<td> <223></td><td>Polypeptide</td><td></td><td>i ί F</td>

10 15

Tyr Tyr Glu Gin Asn Ser Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30

<210> 107 <211> 86 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 107

Glu Val Val Wing Wing Thr Pro Thr Ser Leu Leu Ile Ser Trp Pro Lys 15 10 15

Tyr Asp Lys Thr Gly His Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30

Gly Asn Ser Pro Val Gin Glu Phe Thr Val Pro Thr Arg Gin Thr Thr 35 40 45

Wing Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60

Tyr Ala Val Ser Lys Asp Asp Tyr Tyr Pro His Glu His Arg Pro Ile 65 70 75 80

Be Ile Asn Tyr Arg Thr J

J (

<td> <210> <211> <212> <213></td><td>108 8 PRT artificial</td><td>t r F one I ·</td>

<td> <220> <223></td><td>Polypeptide</td><td>£ F</td>

hee

<td> <213></td><td>artificial</td><td>î</td>

<td> <213></td><td>artificial</td><td></td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td></td><td></td>

<td> <213></td><td>artificial</td><td></td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td></td><td></td>

<td> <213></td><td>artificial</td><td></td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td></td><td></td>

<td> <213></td><td>artificial</td><td></td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td></td><td></td>

Be Wing Gly 10

Be Leu Leu Ile Be Trp Glu Asp 10 15

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

Asp Ser Tyr Tyr Ser Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30

Gly Asn Ser Pro Val Gin Glu Phe Thr Val Pro Ser Asp Leu Tyr Thr 35 40 45

Wing Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60

Tyr Ala Val Thr Tyr Asp Val Thr Asp Leu Ile Met His Glu Pro Ile 65 70 75 80

Be Ile Asn Tyr Arg Thr 85 <210> 120 <211> 8 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 120

Glu Asp Asp Ser Tyr Tyr Ser Arg 1 5 <210> 121

<td> <213></td><td colspan="3">artificial</td>

<td> <223></td><td colspan="3">Polypeptide</td>

<td> <213></td><td colspan="3">artificial</td>

<td> <223></td><td colspan="3">Polypeptide</td>

.Χ · 'ί rX / <220>

<223> Polypeptide <400> 127

<td> <213></td><td colspan="3">artificial</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> <223></td><td colspan="3">Polypeptide</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>

Gly Asn Ser Pro Val Gin Glu Phe Thr Val Asp Pro Ser Ser Tyr Thr

40 45

Wing Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60

Tyr Ala Val Ser Lys Asp Asp Tyr Tyr Pro His Glu His Arg Pro Ile 65 70 75 80

Ser Ile Asn Tyr Arg Thr 85

<td> <213></td><td>artificial</td><td></td><td></td>

<td> <223></td><td>Polypeptide</td><td></td><td> !</td>

Pro Tyr Thr Pro Ile His Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30

Gly Asn Ser Pro Val Gin Glu Phe Thr Val Pro Gly Tyr Tyr Gly Thr f

40 45 you

tea

Wing Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val j

55 60 î

Tyr Ala Val Tyr Gly Tyr Tyr Gin Tyr Thr Pro Ile Ser Ile Asn Tyr f

70 75 80 {r

I

Arg Thr {

Be Ile Asn Tyr Arg Thr 85 <210> 134 <211> 86 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 134

Glu Val Val Wing Wing Thr Pro Thr Ser Leu Leu Ile Ser Trp Ser Lys 15 10 15

Tyr Ser Lys His Gly His Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30

Gly Asn Ser Pro Val Gin Glu Phe Thr Val Pro Ser Gly Asn Ala Thr 35 40 45

Wing Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55 60

Tyr Ala Val Glu Asp Thr Asn Asp Tyr Pro His Thr His Arg Pro Ile 65 70 75 80

Be Ile Asn Tyr Arg Thr 85 <210> 135 <211> 82 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 135

Glu Val Val Wing Wing Thr Pro Thr Ser Leu Leu Ile Ser Trp Tyr Glu 15 10 15 <211> 86 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 137

<td> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

Arg Thr hee

Wing Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val

55 60

Tyr Ala Val Tyr Ser Tyr Tyr Gly Tyr Tyr Pro Ile Ser Ile Asn Tyr 65 70 75 80

Arg Thr <210> 144 <211> 10 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 144

Met Gly Val Ser Asp Val Pro Arg Asp Leu 15 10 <210> 145 <211> 9 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 145

Gly Val Ser Asp Val Pro Arg Asp Leu <210> 146 <211> 8 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 146

Val Ser Asp Val Pro Arg Asp Leu 1 5 <210> 147

<td> <213></td><td>artificial</td>

<td> <220> <223></td><td>Polypeptide</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Polypeptide</td>

<211> Ί <212> PRT <213> artificial <220>

<223> Polypeptide <400> 159

Glu Ile Glu Lys Pro Ser Gin <210> 160 <211> 9 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 160

Glu Ile Asp Lys Pro Ser Gin Leu Glu <210> 161 <211> 12 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 161

Glu Ile Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp 15 10 <210> 162 <211> 17 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 162

Glu Ile Glu Lys Pro Ser Gin Glu Asp Glu Asp Glu Asp Glu Asp Glu 15 10 15

Asp

10 <210> 167 <211> 15 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 167

Gly Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 15 10 15 <210> 168 <211> 16 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 168

Gly Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly 15 10 15 <210> 169 <211> 4 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 169 i

Gly Ser Gly Ser Ì f

t ï

<210> 170 j <211> 8 {<212> PRT ί f

<213> artificial jt

I <220> f <223> Polypeptide i <400> 170 ί

Gly Ser Gly Ser Gly Ser Gly Ser f

10 15 <210> 175 <211> 15 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 175

Gly Ser Pro Gly Ser Pro Gly Ser Pro Gly Ser Pro Gly Ser Pro 15 10 15 <210> 176 <211> 14 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 176

Gly Ser Thr Val Wing Pro Wing Thr Val Wing Pro Wing Ser 15 10 <210> 177 <211> 8 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 177

Gly Gly Ser Glu Gly Gly Ser Glu 1 5 <210> 178 <211> 7 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 178

Ser Thr Ser Thr Ser Thr Gly

<210> 182 <211> 7 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 182

Gly Pro Gly Pro Gly Pro Gly 1 5 <210> 183 <211> 11 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 183

Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 15 10 <210> 184 <211> 7 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 184

Pro Ser Thr Ser Thr Ser Thr 1 5 <210> 185 <211> 6 <212> PRT <213> artificial <220>

<223> Polypeptide <400> 185

Pro Wing Pro Wing Pro Wing 1 5

<td colspan="2"> <213></td><td colspan="3">artificial</td>

<td colspan="2"> <223></td><td colspan="3">Polypeptide</td>

<212> PRT

105

<td>Glu</td><td>Phe fifty</td><td>Thr</td><td>Val</td><td>Pro</td><td>Thr</td><td>Arg 55</td><td>Gin</td><td>Thr</td><td>Thr</td><td>To</td><td>Thr 60</td><td>Ile</td><td>Be</td><td>Gly</td><td>Leu</td><td>i one</td>

<td colspan="2"> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>t- one</td>

<td colspan="2"> <223></td><td colspan="3">Polypeptide</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</td>

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Ser Tyr Tyr 65 70 75 80

Thr Lys Lys Ala Tyr Ser Ala Gly Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His 100 105 <210> 193

<td> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

100 105

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin

40 45

Glu Phe Thr Val Asp Pro Ser Ser Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Ser Lys Asp 65 70 75 80

Asp Tyr Tyr Pro His Glu His Arg Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His 100 105

<td> <213></td><td>artificial</td><td></td><td></td><td>F; L</td>

<td> <223></td><td>Polypeptide</td><td></td><td></td><td>i</td>

Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Ser His Tyr Glu Arg Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Arg Tyr His His Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gin Ala 65 70 75 80

Gin Glu His Tyr Gin Pro Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95

Asp Lys Pro Ser Gin His His His His His His

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Ser Gly Asn Ala Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Glu Asp Thr 65 70 75 80

Asn Asp Tyr Pro His Thr His Arg Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95

Glu Ile Asp Lys Pro Ser Gin His His His His His 100 105

<td>one j l</td><td> <210> <211> <212> <213></td><td colspan="3">201 107 PRT artificial</td>

<td>i one</td><td> <220> <223></td><td>Polypeptide</td><td></td><td></td>

Pro Thr Ser Leu Leu Ile Ser Trp His Gly Glu Pro Asp Gin Thr Arg 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Pro Tyr Arg Arg Thr Wing Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Gly 65 70 75 80

Tyr Thr Gly His Tyr Gin Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95

<td> <213> ,</td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>j</td><td>Pro</td><td>Thr</td><td>Be</td><td>Leu twenty</td><td>Leu</td><td>Ile</td><td>Be</td><td>Trp</td><td>Be 25</td><td>Lys</td><td>Tyr</td><td>Be</td><td>Lys</td><td>His 30</td><td>Gly</td><td>His</td>

<td>î ί</td><td colspan="2"> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>one j</td><td colspan="2"> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <213> ,</td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

ι s

Item

ί l

I

To go

I i

<td> <213></td><td colspan="3">artificial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="3">Polypeptide</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

101 * I

<220>

<223> Polypeptide <400> 215

Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Wing Wing Thr 15 10 15

Pro Thr Ser Leu Leu Ile Ser Trp Pro Asp Pro Tyr Tyr Lys Pro Asp 20 25 30

Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gin 35 40 45

Glu Phe Thr Val Pro Arg Asp Tyr Thr Thr Ala Thr Ile Ser Gly Leu 50 55 60

Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Tyr Ser Tyr 65 70 75 80

Tyr Gly Tyr Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys 85 90 95

Pro Ser Gin His His His His His 100 105 <210> 216 <211> 330 <212> DNA <213> Artificial <220>

<223> Oligonucleotide

<td colspan="2"><220> <221> MISC_FEATURE <223> 1434A08 <400> 216 atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>atcgtactca</td><td>tacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

103 ccatcccagc accatcacca ccaccactga <210> 219 <211> 330 <212> DNA <213> artificial <220>

<223> Oligonucleotide

<td colspan="2"><220> <221> tnisc_f eature <223> 1438E05 <400> 219 atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctt</td><td>ctaacgttca</td><td>tacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 220 <211> 330 <212> DNA <213> artificial <220>

<223> Oligonucleotide

<td colspan="2"><220> <221> misc_feature <223> 1438D01 <400> 220 atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgccta</td><td>accgtgctca</td><td>tacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

105

<211> 330 <212> Artificial <213> DNA <2 2 0> J <223> Oligonucleotide st

ί {

<2 2 0> I <221> misc_feature f <223> 1486G03 î <400> 223 '

<td>atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctt</td><td>ctcgttacta</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 224 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide <220> j <221> misc_feature (<223> 1486C04 |

<td><400> 224 atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td><td>j i L</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td><td> ?</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>cgcatcgtta</td><td>cacagctacc</td><td> 180</td><td>ί one</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td><td>i ï ï.</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td><td>î k î; F</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td><td>i</td>

f <210> 225 <211> 330 <212> DNA

107 <220>

<223> Oligonucleotide

<td colspan="2"><221> misc_feature <223> 1486D05</td><td></td><td></td><td></td><td></td><td></td>

<td><400> 227 atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgctgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>atcagcgtta</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 228 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide

<td><221> misc_feature</td>

<td>atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60</td>

<td>ctgatcagct ggggtcatta cccgctgcat atccgatatt accgcatcac ttacggcgaa 120</td>

<td>acaggaggca atagccctgt ccaggagttc actgtgccta aacaggttta cacagctacc 180</td>

<td>atcagcggcc ttaaacctgg cgttgattat accatcactg tgtatgctgt cacttactac 240</td>

<td>aacgaagctg actactctca gatcccaatt tccattaatt accgcacaga aattgacaaa 300</td>

<td>ccatcccagc accatcacca ccaccactga 330</td>

<210> 229 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide

109

<td colspan="2"><221> misc_feature <223> 1487H04</td><td></td><td></td><td></td><td></td><td></td>

<td><400> 231 atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctg</td><td>ctcgtcagta</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>caggaatacg</td><td>aataccgtta</td><td>cataccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 232 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide

<td colspan="2"><221> mise feature <223> 1490E02</td><td></td><td></td><td></td><td></td><td></td>

<td><400> 232 atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>gtactcagta</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<td> <213></td><td>artificial</td>

<td> <223></td><td>Oligonucleot</td>

<td> <221></td><td>misc_feature</td>

111

<400> 235

<td>atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgctgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctt</td><td>ctcgtaaata</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aaagaagcta</td><td>actatcgtga</td><td>aatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 236 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide <220>

<221> misc_feature <223> 1490H06 <400> 236

<td>atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgctgcat</td><td>atccgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>gtcagaaata</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 237 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide <220>

<221> misc_feature <223> 1490A07 <400> 237 atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60

113

<td colspan="2">acaggaggca atagccctgt</td><td>ccaggagttc</td><td>actgtgccta</td><td>acacttctca</td><td>tacagctacc</td><td> 180</td>

<td colspan="2">atcagcggcc ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td colspan="2">aacgaagctg actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacgga</td><td>aattgacaaa</td><td> 300</td>

<td colspan="2">ccatcccagc accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<td> <213></td><td>artificial</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="2">Oligonucleotide</td><td></td><td></td><td></td><td></td>

<td> <221></td><td>misc_feature</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td colspan="2">ctgatcagct ggggtcatta</td><td>cccgctgcat</td><td>gttegatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td colspan="2">acaggaggca atagccctgt</td><td>ccaggagttc</td><td>actgtgcctt</td><td>ctcaggttta</td><td>cacagctacc</td><td> 180</td>

<td colspan="2">atcagcggcc ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td colspan="2">gctcaggaaa actacaaaga</td><td>aatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td colspan="2">ccatcccagc accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<td> <213></td><td>artificial</td><td></td><td></td><td></td><td></td><td></td>

<td> <223></td><td colspan="2">Oligonucleotide</td><td></td><td></td><td></td><td></td>

<td> <221></td><td>mise feature</td><td></td><td></td><td></td><td></td><td></td>

<td colspan="2">atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td colspan="2">ctgatcagct ggggtcatta</td><td>cccgctgcat</td><td>gttegatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td colspan="2">acaggaggca atagccctgt</td><td>ccaggagttc</td><td>actgtgccta</td><td>accgtgttta</td><td>cacagctacc</td><td> 180</td>

115

aaagaagctg actactctca gatcccaatt tccattaatt accgcacaga aattgacaaa 300 ccatcccagc accatcacca ccaccactga 330 <210> 244 <211> 330 <212> DNA <213> artificial <220>

<223> Oligonucleotide

<td colspan="2"><220> <221> mise feature <223> 1571F10 <400> 244 atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgctgcat</td><td>atccgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctt</td><td>ctcgttacca</td><td>tacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>gaacaggttg</td><td>aataccgtga</td><td>aatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 245 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide

<td colspan="2"><220> <221> mise feature <223> 1572D04 <400> 245 atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>cgcgtcgtta</td><td>cacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>gaacagccga</td><td>tctacgccac</td><td>tatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

117

<210> 248 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide

<td colspan="2"><220> <221> misc_feature <223> 1572B10 <400> 248 atgggagttt ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctg</td><td>ttccgcgtca</td><td>tacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

<210> 249 <211> 330 <212> Artificial <213> DNA <220>

<223> Oligonucleotide

<td colspan="2"><221> mise feature <223> 1572C09</td><td></td><td></td><td></td><td></td><td></td>

<td><400> 249 atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>ateegatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgccta</td><td>ctccgaaaca</td><td>tacagctacc</td><td> 180</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td>

119

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<td>atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td><td>i £</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td><td>ί F F</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctg</td><td>ttccgcgtta</td><td>cacagctacc</td><td> 180</td><td>i. one one</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td><td>i r 'i</td>

<td>gctcaggaaa</td><td>actacaaaga</td><td>aatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td><td> 1 1 «</td>

<td>ccatcccagc</td><td>accatcacca</td><td>ccaccactga</td><td></td><td></td><td></td><td> 330</td><td> 1</td>

t <210> 253 I <211> 330 f <212> DNA f <213> artificial ï <220>

<223>

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<td>atgggagttt</td><td>ctgatgtgcc</td><td>gcgcgacctg</td><td>gaagtggttg</td><td>ctgccacccc</td><td>caccagcctg</td><td> 60</td><td>» i t</td>

<td>ctgatcagct</td><td>ggggtcatta</td><td>cccgatgcat</td><td>gttcgatatt</td><td>accgcatcac</td><td>ttacggcgaa</td><td> 120</td><td>one F t | -</td>

<td>acaggaggca</td><td>atagccctgt</td><td>ccaggagttc</td><td>actgtgcctc</td><td>cgcgtcgtta</td><td>cacagctacc</td><td> 180</td><td> 1</td>

<td>atcagcggcc</td><td>ttaaacctgg</td><td>cgttgattat</td><td>accatcactg</td><td>tgtatgctgt</td><td>cacttactac</td><td> 240</td><td> [</td>

<td>aacgaagctg</td><td>actactctca</td><td>gatcccaatt</td><td>tccattaatt</td><td>accgcacaga</td><td>aattgacaaa</td><td> 300</td><td></td>

<td colspan="2">ccatcccagc accatcacca ccaccactga</td><td colspan="2">330 f i one</td>

<td> <213></td><td>artificial</td><td></td><td> 1</td>

121

<223> Oligonucleotide <220>

<221> misc_feature <223> 1550H05 <400> 256 atgggagttt ctgatgtgcc gcgcgacctg gaagtggttg ctgccacccc caccagcctg 60 ctgatcagct ggggtcatta cccgctgcat gttcgatatt accgcatcac ttacggcgaa 120 acaggaggca atagccctgt ccaggagttc actgtgcctc gtcagatcta cacagctacc 180 atcagcggcc ttaaacctgg cgttgattat accatcactg tgtatgctgt cacttactac 240 aacgaagctg actactctca tccattaatt gatcccaatt accgcacaga aattgacaaa 300

<td colspan="2">ccatcccagc accatcacca ccaccactga</td><td> 330</td>

<td> <213></td><td>artificial</td><td></td>

<td> <223></td><td>Polypeptide</td><td></td>

<td> <221></td><td>misc_feature</td>

<td> <223></td><td>Reason for the common sequence of the BC loop</td>

<td> <220> <221></td><td>mise feature</td>

<td> <221></td><td>mise feature</td>

<td> <213></td><td>artificial</td>

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Priority claims
Document	Office	Kind	Date
30556610	United States of America	P	2010-02-18
33070610	United States of America	P	2010-05-03
61305566	–	–	–
61330706	–	–	–
US20100305566P	–	–	–
US20100330706P	–	–	–