Covalent diabodies and uses thereof.

The reference patent is granted based on articles 1 * 2 “fraction V, 6th fraction Hf, and 59 date of the Industrial Property Law.

Pursuant to article 23 of Industrial Property, the heavy patent is valid for twenty years, which cannot be extended, counted from the date of filing of the document and will be subject to the payment of the fee to keep the rights in force.

Whoever signs this title does so based on the provisions of articles 6®fraccionestll γ, ΛΗβΛ of the Industrial Property Law (Official Gazette of the Federation (DOF) 08/27/1991. Amended on 08/02/1994 , 10/25/1998, 12/26/1997, 05/17/1999, 01/26/2004, 06/16/2005, 01/25/2006, 05/06/2009, 06/01/2010,18 / 06/2010, 28/06 / 2010,27 / 01/2012 and 09/04/2012); Articles i °, 3 ° fraction v subsection a), 4^or and 12^or Sections I and III of the Regulations of the Mexican Institute of Industrial Property (D, OF 12/14/1999, -reformed etO1 / 07/2002, 07/15/2004, 07/28/2004 and 09/07/2007); items 1^or, 3°, 4^or, 5^or fraction V part a), 16 fractions! and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007); 1 *; 4? and 5^or ineteO'á) of the Agreement that delegates powers to the Deputy General Directors, Coordinator, Divisional Directors, Heads of Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Industrial Property. (DOF 12/15/1999, amended on 02/04/2000, 07/29/2004, 08/04/2004 and 09/13/2007).

This document is signed with an advanced electronic signature (FIEL), based on articles 7 BIS 2 of the Industrial Property Law; 3 of its Regulations, and 1 section III, 2 section V, 26 BIS and 26 TER of the Agreement establishing the guidelines for the use of the Payment and Electronic Services Portal (PASE) of the Mexican Institute of Industrial Property, in the procedures indicated.

THE DIVISIONAL DIRECTOR OF PATENTS

NAHANNY CANAL REYES

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Tax | 1695 || MX / 2017/43588 | MX / a / 2013/010955 | Normal patent title with divisional PCT | 1488 | IAR | Page (s) 2 | 7eKJna7DRd3bhcvCZ + 5KWHCTrMA =

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UvYZGcynEzwkzESndRH // HQXI7sP6oYA + GNC2eryqyTygRRaYfwmf6NwphVrmtwZIRBrdR6büxuo0ERaUtG7Fd / y1X / REeGMtZu1VD2tM2eDt1K + cDv7crGbdwBArrdvanOYbB2NeNuLSTjDYTyn4IOobXwCojOJ3i / UnDJX9LkBÍI1 + pqqX 6Oq30gEeFF2VBf5OznlR7NNdgyjXCPo1f9DSziudcgpN4Cb7pULI8Y9km01qBkNrqjKTcrVRh747mnyYUwjrRu6E / 4 fMmT91wLADgxsu8MVfEsZGIIheYVvGZ84SfbWvGnXCMwy / + / njtgE02UbC17zVdKDnNrUE / A == * Additional information on the back

Arenal No 550. Floor 1 I can Santa Mana Tepepan. Xochimilco, 16020,

Mexico City (55) 53340700 www.gob mx / iinpi

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COVALENT DIACBODIES AND THEIR USES -------------

Field of Invention

The present invention is directed to diabody molecules, instead referred to as dual affinity redirection reagents (DARTS), and their uses in treating a variety of diseases and disorders including immune disorders and cancers. The diabody molecules of the invention comprise at least two polypeptide chains that associate to form at least two epitope binding sites, which can recognize the same or different epitopes. Additionally, the epitopes can be of the same or different molecules or localized in the same or different cells. The individual polypeptide chains of the diabody molecule can be covalently linked through non-peptide binding covalent bonds, such as, but not limited to, disulfide attachment to cysteine ​​residues located within each polypeptide chain. In particular embodiments, the diabody molecules of the present invention further comprise an Fe region, which allows an antibody-like functionality to be modified in the molecule.

Background of the Invention

The design of covalent diabodies is based on a single chain Fv (scFv) construct (Holliger et al.

<img file="MX348166B_D0004.tif" />

IMPIí wsrmrro müícaj® DE LA PROBIDAD (1993) 'Diabodies'

Fragments, Proc.

INDUSTRIAL

Small Bivalent And Bispecific Antibody

Nati. Acad. Sci. USA 90: 6444-6448;

incorporated herein by reference in its entirety). In intact unmodified IgG, the VL and VH domains are located on separate polypeptide chains, ie, the light chain and the heavy chain, respectively. The interaction of an antibody light chain and an antibody heavy chain and, in particular, the interaction of the VL and VH domains that form one of the epitope binding sites of the antibody. In contrast, the scFv construct comprises a VL and VH domain of an antibody contained in a single polypeptide chain wherein the domains are separated through a flexible linker of sufficient length to allow the self-assembly of two domains into one binding site. functional epitope binding. When self-assembly is impossible due to a linker such as insufficient length (less than about 12 amino acid residues), two of the scFv constructs interact with each other to form a divalent molecule, the VL of a chain that associates with the VH of the other (reviewed in Marvin et al. (2005) Recombinant

Approaches To IgG-Like Bispecific Antibodies, Acta Pharmacol. Without. 26: 649-658). Furthermore, the addition of a cysteine ​​residue to the C-terminus of the construct has been shown to allow disulfide bonding of polypeptide chains, stabilizing the resulting dimer without interfering with the

<img file="MX348166B_D0005.tif" />

IMPI

1NSTHVTO MBXICAMO ΠΪ LA HtOHEDA * binding characteristics of the molecule d ± val ^ xü ^ e — 4u ££ ^ -— PAK For example, Olafsen et al. (2004) Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, Prot. Engr. Des. Sel. 17: 21-27). Furthermore, when VL and VH domains of different specificity are selected, not only a divalent but also a bispecific molecule can be constructed.

The . Bivalent diabodies have a wide range of applications including therapy and immunodiagnosis. Bivalence allows great flexibility in the design and modification of diabodies in various applications, providing improved avidity to multimeric antigens, crosslinking of different antigens, and targeting specific cell types that are based on the presence of both. target antigens. Due to their increased valence, low degrees of dissociation, and rapid clearance from the circulation (for small-sized diabodies, at or below ~ 50 kDa), diabody molecules that are known in the art have also shown particular use in the field of tumor imaging (Fitzgerald et al. (1997) Improved Tumor Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris ”, Protein Eng. 10: 1221). Of particular importance is the intertwining of the different cells,

<img file="MX348166B_D0006.tif" />

. LMPI iMsTrnnuMiBGANO nt LA MOnFDAD 'NDnsTWIAL for example the entanglement of cytotoxic T cells and tumor cells (Staerz et al. (1985) Hybrid Antibodies Can Target Sites For Attack By T Cells, Nature 314: 628-631, and Holliger et al. (1996) Specific Killing Of Lymphoma Cells

By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9: 299-305). The epitope binding domains of the diabody can also target a determinant on the surface of any immune-effect cell such as CD3, CD16, CD32, or CD64, which are expressed on lymphocytes.

T, natural killer (NK) cells, or other mononuclear cells. In many studies, the binding of diabody to effector cell determinants (e.g., Fcy receptors (FcyR), is also found to activate the effector cell (Holliger et al. (1996) Specific

Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, Protein Eng. 9: 299-305; Holliger et al.

(1999) Carcinoembryonic Antigen (CEA) -Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3 x Anti-CEA Bispecific Diabodies And B7 x Anti-CEA Bispecific Fusion

Proteins, Cancer Res. 59: 2909-2916). Normally, effector cell activation is directed through the binding of an antigen-binding antibody to an effector cell through the Fc-Fc and R interaction; thus, in this regard, the diabody molecules of the invention can exhibit Ig-type functionality independent of⁵ ΙΜΡΙ ^ 5 f ^ MFIICAN SUlUTE

DF LA PHOFIFDAD &lt;V «m3UÍ iwnnrrwiAt j * do comprise an Fe domain (eg, as assayed in any effector function assay known in the art or exemplified herein (eg, the ADCC assay)). Through the entanglement of the effector tumor cells, the diabody not only brings the effector cell into proximity with the tumor cells but leads to effective tumor killing (see, for example, Cao et al. (2003) Bispecific Antibody Conjugates In Therapeutics ”, Adv. Drug. Deliv. Rev. 55: 171-197, incorporated herein by reference in its entirety).

EFFECT CELL RECEPTORS AND THEIR ROLE IN THE IMMUNE SYSTEM

In traditional immune function the interaction of antibody-antigen complexes with cells of the immune system results in a wide array of responses, in the range of effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to immunomodulatory signals such as regulation of lymphocyte proliferation and antibody secretion. All of these interactions are initiated through the binding of the Fe domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses activated by antibodies and immune complexes⁶IKTTTUTOMKlCANr χΤλ ot LA MtOVIKDAO 'νπκ; τ »ιαι results from the structural heterogeneity of Fe receptors. Fe receptors split structurally related to antigen-binding domains that presumably mediate intracellular signaling.

Fcy receptors, members of the immunoglobulin gene superfamily of proteins, are surface glycoproteins that bind to the Fcy portion of immunoglobulin molecules. Each member of the family recognizes immunoglobulins of one or more isotypes through a recognition domain on the alpha chain of the Fcy receptor. Fcy receptors are defined by their specificity for immunoglobulin subtypes. The Fcy receptors for IgG are referred to as FcyR, for IgE as FceR, and for IgA as FcaR. Different accessory cells carry Fcy receptors for antibodies of different isotype, and the isotype of the antibody determines which accessory cells will connect in a given response (reviewed by Ravetch JV et al. (1991) Fe Receptors, Annu. Rev. Immunol. 9: 457-92; Gerber JS et al. (2001) Stimulatory And Inhibitory Signáis Originating From

The Macrophage Fcgamma Receptors, Microbes and Infection, 3: 131-139; Billadeau DD et al. (2002), ITAMs Versus ITIMs: Striking A Balance During Cell Regulation, The Journal of Clinical Investigation, 2 (109): 161-1681; Ravetch JV et al. (2000) Immune Inhibitory Receptors, Science, 290: 84-89;

Ravetch JV et al., (2 001) IgG Fe Receptors, Annu. Rev.

Mexican iNsrmrro r * IA PltORBDAD iK'n «'&lt;T» UL

Immunol. 19: 275-90; Ravetch JV (1994) Fe Receptors: Rubor Redux, Cell, 78 (4): 553-60). The different Fcy receptors, the cells that express them, and their isotype specificity are summarized in Table 1 (adapted from Immunobiology: The Immune System in Health and Disease, 4^to ed. 1999, Elsevier Science Ltd / Garland Publishing, New York).

Fcy receptors

Each member of this family is an integral membrane glycoprotein, processing extracellular domains related to a C2 group of immunoglobulin-related domains, a single membrane extension domain, and an intracytoplasmic domain of variable length. There are three known FcyRs, designated FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16). All three receptors are encoded by different genes; however, the extensive homology between the three family members suggests that they arise from a common parent perhaps through gene duplication.

FcyRII (CD32)

FcyRII proteins are 40 kDa integral membrane glycoproteins that bind only complex IgG due to low affinity for Ig (10⁶ M '¹). This receptor is the most widely expressed FcyR, present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets.

<img file="MX348166B_D0007.tif" />

IMPI nmrn ^ o MEXICAN INCHIMAL PROPERTY

FcyRII has only two regions of ti ^ e-immunoglobulrrta · in its immunoglobulin binding chain and therefore a much lower affinity for IgG than FcyRI. There are three human FcyRII genes (FcyRII-A, FcyRII-B, FcyRII-C), all of which bind IgG in aggregates and immune complexes.

The various differences between the cytoplasmic domains of FcyRII-A and FcyRII-B create two functionally heterogeneous responses to receptor ligation. The fundamental difference is that isoform A initiates intracellular signaling that leads to cell activation such as phagocytosis and respiratory burst, while isoform B initiates inhibitory signals, for example, inhibition of B cell activation.

FcyRIII (CDU)

Due to heterogeneity within this class, the size of FcyRIII is in the range of 40 to 80 kDa in mouse and man. The two human genes encode two transcripts, FcyRIIIA, an integral membrane glycoprotein, and FcyRIIIB, a version that binds to glycosylphosphatidyl-inositol (GPI). A murine gene encodes an FcyRIII homolog to human membrane-spanning FcyRIIIA. FcyRIII shares structural characteristics with each of the other two FcyRs. Like FcyRII, FcyRIII binds IgG with low affinity and contains the two domains of

<img file="MX348166B_D0008.tif" />

corresponding extracellular Ig types. FcyRIIIA is in macrophages, mast cells and FcyR only in NK cells. GPI-linked FcyRIIIB is currently known to be expressed only in human neutrophils.

Signaling through FcyRs

Both activation and inhibition signals are transduced through FcyRs after ligation. These diametrically opposite functions result in structural differences between the different isoforms of the receptor. Two different domains within the receptor's cytoplasmic signaling domains called immuno-tyrosine receptor-based activation motifs (ITAM) or immuno-tyrosine receptor-based inhibitory motifs (ITIMS) represent the different responses. The recruitment of different cytoplasmic enzymes to these structures dictates the outcome of FcyR-mediated cellular responses. ITAM complexes containing FcyR include FcyRI, FcyRIIA, FcyRIIIA, while complexes containing ITIM only include FcyRIIB.

Human neutrophils express the FcyRIIA gene. Clustering of FcyRIIA through immune complexes or specific antibody crosslinking serves to aggregate ITAM along with receptor-associated kinases that facilitate phosphorylation of ITAM. ITAM phosphorylation serves as a docking site for Syk kinase, the₁₀ IMPI ^

OF INDUSTRIAL REOFMDAD activation of which results in activation ·, of — the — amstiaLUg¹downstream (for example, PI₃K). Cell activation leads to the release of pro-inflammatory mediators.

The FcyRIIB gene is expressed in B lymphocytes; its extracellular domain is 96% identical to FcyRIIA and it binds to IgG complexes in an indistinguishable way. The presence of an ITIM in the cytoplasmic domain of FcyRIIB defines its inhibitory subclass of FcyR. Recently, the molecular basis for this inhibition was established. When co-ligated together with an FcyR, the ITIM in FcyRIIB becomes phosphorylated and attracts the SH2 domain of inositol polyphosphate 5'-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers released as a consequence of tyrosine activation. FcyR-mediated kinase containing ITAM, consequently preventing Ca influx⁺⁺ intracellular. In this way, the crosslinking of FcyRIIB hinders the activation of the response for FcyR ligation and inhibits the degree of cellular reaction. B cell activation, B cell proliferation, and antibody secretion are thereby aborted.

TABLE 1. Receptors for the Fe Regions of Immunoglobulin Isotypes

<td>Receiver</td><td>Union</td><td>Cell type</td><td>Ligation effect</td>

<td>FcyRI (CD64)</td><td>IgGl 10⁸ M '¹</td><td>Macrophages Neutrophils Eosinophils Dendritic cells</td><td>Absorption stimulation Activation of respiratory burst Induction of</td>

<img file="MX348166B_D0009.tif" />

<td>Receiver</td><td>Union</td><td>Cell type</td><td>Ligation effect</td>

<td></td><td></td><td></td><td>annihilation. - --------</td>

<td>FcyRII-A (CD32)</td><td>IgGl 2 x 10⁶ M '¹</td><td>Macrophages Neutrophils Eosinophils Dendritic cells Platelets Langerhan cells</td><td>Absorption of Granule Release</td>

<td>FcyRII-B2 (CD32)</td><td>IgGl 2 x 10⁶ M '¹</td><td>Macrophages Neutrophils Eosinophils</td><td>Inhibition of Absorption of Stimulation</td>

<td>FcyRII-Bl (CD32)</td><td>IgGl 2 x 10⁶ M '¹</td><td>B cells Mast cells</td><td>No absorption Stimulation inhibition</td>

<td>FcyRIII (CD16)</td><td>IgGl 5 X 10⁵ M '¹</td><td>NK cells Eosinophils Macrophages Neutrophils Mast cells</td><td>Induction of annihilation</td>

<td>FcDRI</td><td>IgE 10¹⁰ M '¹</td><td>Mast cells Eosinophils Basophils</td><td>Granule secretion</td>

<td>FcORI (CD89)</td><td>IgGAl, IgA2 10⁷ M ¹</td><td>Macrophages Neutrophils Eosinophils</td><td>Annihilation Absorption Induction</td>

Brief Description of the Invention

The present invention relates to covalent diabodies and / or covalent diabody molecules and their use in the treatment of a variety of diseases and disorders including cancer, autoimmune disorders, allergy disorders, and infectious diseases caused by bacteria, fungi, or viruses. Preferably, the diabody of the present

<img file="MX348166B_D0010.tif" />

IMPI nwrmnc mmjcano DE ΙΑ PM9IÍDAD • HousntiAi invention can unite two different ^ '^ pf LOpus a। lili ιηί different cells where the first first epitope is expressed in a different cell type than the second epitope, in such a way that the diabody can put two cells in contact.

In one embodiment, the present invention is directed to a covalent bispecific diabody, the antibody of which comprises a first and a second polypeptide chain, wherein the first polypeptide chain comprises (i) a first domain comprising a domain binding region light chain variable of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope, and, optionally, (iii) a third domain comprising at least one residue of cysteine, the first and second domains of which are covalently linked such that the first and second domains do not associate to form an epitope binding site; whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a variable domain of heavy chain of the first immunoglobulin (VH1), and optionally (iii)

<img file="MX348166B_D0011.tif" />

IMPI rwmiiro mMjcamo nElAPROntOAD INPITJTIAI a sixth domain comprising at least one cysteine ​​residue, the fourth and fifth domains of which are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked, wherein the first domain and the fifth domain associate to form a first binding site (VL1) (VH1) that binds the first epitope;

wherein the second domain and the fourth domain associate to form a second binding site (VL2) (VH2) that binds the second epitope.

In another embodiment, the present invention is directed to a covalent bispecific diabody, which diabody comprises a first and a second polypeptide chain, the first polypeptide chain comprising (i) a first domain comprising a binding region of a variable domain of light chain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fe domain or a portion thereof, which first and second domains are covalently linked such that the first and second domains do not associate to form a binding site

<img file="MX348166B_D0012.tif" />

epitope; whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a variable domain of heavy chain of the first immunoglobulin (VH1), the fourth and fifth domains of which are covalently linked such that the third and fourth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked, provided that the covalent linkage is not a peptide linkage; wherein the first domain and the fifth domain associate to form a first binding site (VL1) (VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2) (VH2) that binds the second epitope.

In certain aspects, the present invention is directed to the diabody molecule, the molecule of which comprises a first and a second polypeptide chain, the first polypeptide chain of which comprises (i) a first domain comprising a binding region of a variable domain of light chain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a

ΙΝΪΤΠΤΠΌ MBtICAMü ηε laíhomídad t2 * ~ »¿3njo! Nr&gt; 'STstAi second epitope and (iii) a third domain comprising ^ a, Fe domain or one of its portions, the first and second domains of which are covalently linked in such a way that the first and second domains do not associate to form an epitope binding site; wherein the second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a domain heavy chain variable of the first immunoglobulin (VH1), and (iii) a sixth domain comprising at least one cistern residue, which fourth and fifth domains are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked, provided that the covalent linkage is not a peptide linkage; wherein the first domain and the fifth domain associate to form a first binding site (VL1) (VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2) (VH2) that binds the second epitope.

In certain embodiments, the present invention is directed to a covalent bispecific diabody, which diabody is a dimer of diabody molecules, each

<img file="MX348166B_D0013.tif" />

IMPI nrjfHyro muicaho OiUMDPinAD INDUJTWIAf diabody molecule comprises a first and a second polypeptide chain, the first polypeptide chain of which comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fe domain or a portion thereof, which first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site; and whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii ) a fifth domain comprising a binding region of a variable domain heavy chain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising at least one cysteine ​​residue, which fourth and fifth domains are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain of each diabody molecule are covalently linked, provided that the covalent linkage is not a peptide linkage;

¹⁷ innnvTOMtxiCANo

Dt THE mOPltDAÍ INDtTHtU!

wherein the first domain and the fifth domain of each diabody molecule associate to form a first binding site (VL1) (VH1) that binds the first epitope; wherein the second domain and the fourth domain of each diabody molecule associate to form a second binding site (VL2) (VH2) that binds the second epitope.

In still other embodiments, the present invention is directed to a covalent tetra-specific diabody, whose diabody is a dimer of diabody molecules, the first diabody molecule comprising a first and a second polypeptide chain, the first polypeptide chain comprising (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fe domain or a portion thereof, which first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site; and whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a second immunoglobulin light chain variable domain (VL2), (ii) a fifth domain comprising ¹⁸IWSTTTOTO MEXICANO Pf. LA ΧΒΟΜΕΟΑΟ INm'ÜTXIAL a binding region of a heavy chain variable domain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising at least one cysteine ​​residue, the fourth and fifth domains of which are covalently linked from such that the fourth and fifth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked, provided that the covalent bond is not a peptide bond; wherein the first domain and the fifth domain associate to form a first binding site (VL1) (VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2) (VH2) that binds the second epitope; and the second diabody molecule comprises a first and a second polypeptide chain, the first polypeptide chain of which comprises (i) a first domain comprising a binding region of a light chain variable domain of a third immunoglobulin (VL3) specific for a third epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a fourth immunoglobulin (VH4) specific for a fourth epitope and (iii) a third domain comprising an Fe domain or a portion thereof, which first and second domains are covalently linked such that the first and second domains do not associate to form a binding site ¹⁹ IMPI ι Nsrmrro mixica no

OE LA PHOWIDAO IN »PST» IAL of epitope; and whose second polypeptide chain comprises

<img file="MX348166B_D0014.tif" />

(i) a fourth domain comprising a binding region of a fourth immunoglobulin light chain variable domain (VL4), (ii) a fifth domain comprising a binding region of a third immunoglobulin heavy chain variable domain (VH3), and (iii) a sixth domain comprising at least one cysteine ​​residue, which fourth and fifth domains are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked, provided that the covalent linkage is not a peptide linkage; wherein the first domain and the fifth domain associate to form a first binding site (VL3) (VH3) that binds the third epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL4) (VH4) that binds the fourth epitope.

In certain aspects of the invention the first epitope, the second epitope, and where applicable, the third epitope and the fourth epitope may be the same. In other aspects, the first epitope, the second epitope, and where applicable, the third epitope and the fourth epitope may each be different from each other. In certain aspects of the invention comprising a third binding domain of₂o rNffmVTOMlXlGANi DÍ LA PtOHRDAD vV ^ »i £ L £ 2f rNpiimiAL epitope, the first epitope and the third epitope may be the same. In certain aspects of the invention that comprise a fourth epitope binding domain, the first epitope and the fourth epitope may be the same. In certain aspects of the invention that comprise a third epitope binding domain, the second epitope and the third epitope may be the same. In certain aspects of the invention that comprise a fourth epitope binding domain, the second epitope and the fourth epitope may be the same. In preferred aspects of the invention, the first epitope and the second epitope are different. In still other aspects of the invention comprising a third epitope-binding domain and a fourth epitope-binding domain, the third epitope and the fourth epitope may be different. Any combination of the foregoing is understood to be encompassed by the present invention.

In particular aspects of the invention, the first domain and the fifth domain of the diabody or the diabody molecule can be derived from the same immunoglobulin. In another aspect, the second domain and the fourth domain of the diabody or the diabody molecule can be derived from the same immunoglobulin. In still another aspect, the first domain and the fifth domain of the diabody or the diabody molecule can be derived from a different immunoglobulin. In still another aspect, the second domain and the fourth domain of the

<img file="MX348166B_D0015.tif" />

IMPI nWllWI »MUICAXO · € MOISTURE • iwstwiai diabody or the diabody molecule may be derived from a different immunoglobulin. Any combination of the foregoing is understood to be encompassed by the present invention.

In certain aspects of the invention, the covalent bond between the first polypeptide chain and the second polypeptide chain of the diabody or the diabody molecule can be through a disulfide bond between at least one cysteine ​​residue in the first chain. polypeptide and at least one cysteine ​​residue in the second polypeptide chain. Cysteine ​​residues in the first or second polypeptide chain that are responsible for disulfide bonding can be found anywhere in the polypeptide chain including within the first, second, third, fourth, fifth, and sixth domains. In a specific embodiment the cysteine ​​residue in the first polypeptide chain is in the first domain and the cysteine ​​residue in the second polypeptide chain is in the fifth domain. The first, second, fourth, and fifth domains correspond to variable regions responsible for binding. In preferred embodiments, the cysteine ​​residues responsible for the disulfide bond between the first and second polypeptide chains are located within the third and sixth domains, respectively. In a particular aspect of this

ΙΝΠΤΤυΤΟ MEJUCAMoij

MLAMORtBAO »

In embodiment, the third domain of the first polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23), which can be encoded through the amino acid sequence (SEQ ID NO: 17 ). In another aspect of this embodiment, the sixth domain of the second polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23), which can be encoded through the amino acid sequence (SEQ ID NO: 17). In yet another aspect of this embodiment, the third domain of the first polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77), derived from the hinge domain of a human IgG, and which can be encoded through the sequence nucleotide (SEQ ID NO: 78). In another aspect of this embodiment, the sixth domain of the second polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77), derived from the hinge domain of a human IgG, and which can be encoded through the sequence of nucleotide (SEQ ID NO: 78). In certain aspects of this embodiment, the third domain of the first polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain of the second polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77). In other aspects of this modality, the sixth domain of the second

<img file="MX348166B_D0016.tif" />

IMPI

ΙΚίΠΤυΤΌ ΜβΚΛΗΟ

DK LA PfcOFftDAl '1NPUJTWIAI polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO: 77). In still other aspects of this embodiment, the third domain of the first polypeptide chain comprises 6 amino acids C terminal of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain of the second polypeptide chain comprises a hinge domain. In other aspects of this embodiment, the sixth domain of the second polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptide chain comprises the hinge domain. In still other aspects of this embodiment, the third domain of the first polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the sixth domain of the first polypeptide chain comprises an Fe domain, or a portion thereof. In still other aspects of this embodiment, the sixth domain of the second polypeptide chain comprises 6 C-terminal amino acids of the human kappa light chain, FNRGEC (SEQ ID NO: 23); and the third domain of the first polypeptide chain comprises an Fe domain, or a portion thereof.

In other embodiments, cysteine ​​residues in

INSTITUTO MEXICANO DI LA PROPIEDAD INDCSTRIAl * »the first or second polypeptides that are responsible for the disulfide bond can be located outside the first, second or third domains in the first polypeptide chain and outside the fourth, fifth and sixth domains in the second chain of polypeptide. In particular, the cysteine ​​residue in the first polypeptide chain can be N-terminal to the first domain or they can be C-terminal to the first domain. The cysteine ​​residue in the first polypeptide chain can be N-terminal to the second domain or they can be C-terminal to the second domain. The cysteine ​​residue in the first polypeptide chain can be N-terminal to the third domain or they can be C-terminal to the third domain. Furthermore, the cysteine ​​residue in the second polypeptide chain can be N-terminal to the fourth domain or can be C-terminal to the fourth domain. The cysteine ​​residue in the second polypeptide chain can be N-terminal to the fifth domain or it can be C-terminal to the fifth domain. Accordingly, the cysteine ​​residue in the second polypeptide chain can be C-terminal to the sixth domain or it can be N-terminal to the sixth domain. In a particular aspect, the disulfide bond can be between at least two cysteine ​​residues in the first polypeptide chain and at least two cysteine ​​residues in the second polypeptide chain. In a particular aspect, where the third domain and the sixth domain do not comprise an Fe domain, or one of its portions,

<img file="MX348166B_D0017.tif" />

IMPI twsTrtvro MeocAHo Di LA MOPIBDAD INMBT1IAL The cistern residue may be at the C-terminal of -la. first polypeptide chain and at the C-terminus of the second polypeptide chain. Any combination of the foregoing is understood to be encompassed by the present invention.

In specific embodiments of the invention described supra, the covalent diabody of the invention encompasses dimers of diabody molecules, wherein each diabody molecule comprises a first and a second polypeptide chain. In certain aspects of this embodiment the diabody molecules can be covalently linked to form the dimer, provided that the covalent bond is not a peptide bond. In preferred aspects of this embodiment, the covalent bond is a disulfide bond between at least one cysteine ​​residue in the first polypeptide chain of each diabody molecule of the dimer. In still other preferred aspects of this invention, the covalent bond is a disulfide bond between at least one cysteine ​​residue in the first polypeptide chain of each diabody molecule that forms the dimer, wherein at least one cysteine ​​residue it is located in the third domain of each first polypeptide chain.

In certain aspects of the invention, the first domain in the first polypeptide chain can be N26

<img file="MX348166B_D0018.tif" />

IMPI

ΙΝ5ΠΊ LITO MEXICAN

OF LA F1OP1SBAD ΓΝΤ&gt; 'STWIAJI terminal to the second domain or may be C-terminal to the second domain. The first domain in the first polypeptide chain can be N-terminal to the third domain or it can be C-terminal to the third domain. The second domain in the first polypeptide chain can be N-terminal to the first domain or they can be C-terminal to the first domain. Furthermore, the second domain in the first polypeptide chain can be N-terminal to the third domain or they can be C-terminal to the third domain. Accordingly, the third domain in the first polypeptide chain can be N-terminal to the first domain or can be C-terminal to the first domain. The third domain in the first polypeptide chain can be N-terminal to the second domain or they can be C-terminal to the second domain. With respect to the second polypeptide chain, the fourth domain can be N-terminal to the fifth domain or they can be C-terminal to the fifth domain. The fourth domain can be N-terminal to the sixth domain or they can be C-terminal to the sixth domain. The fifth domain in the second polypeptide chain can be N-terminal to the fourth domain or they can be C-terminal to the fourth domain. The fifth domain in the second polypeptide chain can be N-terminal to the sixth domain or they can be C-terminal to the sixth domain. Therefore the sixth domain in the second polypeptide chain may be N-terminal to the fourth domain or it may be C-terminal to the fourth domain. The sixth domain in the iWTnyroMgtjcANo or noniDAD 'mdustwial second polypeptide chain may be N-terminal to the fifth domain or may be C-terminal to the fifth domain. Any combination of the foregoing is understood to be encompassed by the present invention.

In certain embodiments, the first domain and the next domain can be located C-terminal to the third domain in the first polypeptide chain; or the first domain and the second domain can be located N-terminal to the third domain in the first polypeptide chain. With respect to the second polypeptide chain, the fourth domain and the fifth domain can be located C-terminal to the sixth domain, or the fourth domain and the fifth domain can be located N-terminal to the sixth domain. In certain aspects of this embodiment, the present invention is directed to a covalent bispecific diabody, whose diabody is a dimer of diabody molecules, each diabody molecule comprising a first and a second polypeptide chain, the first polypeptide chain of which comprises (i ) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fe domain or a portion thereof,

IMPI ^. πβτπυτο M «) UCAN &lt;;

Μ LA ΜΟηίΟΛΠ ϊν »! rtourruiAt Xj ?.

wherein the first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site and wherein the third domain is located N-terminal to both the first domain and the second domain; and whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a variable domain heavy chain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising at least one cysteine ​​residue, which fourth and fifth domains are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; and wherein the first polypeptide chain and the second polypeptide chain of each diabody molecule are covalently linked, provided that the covalent linkage is not a peptide linkage; wherein the first domain and the fifth domain of each diabody molecule associate to form a first binding site (VL1) (VH1) that binds the first epitope; wherein the second domain and the fourth domain of each diabody molecule associate to form a second binding site (VL2) (VH2) that binds the second epitope.

In yet another embodiment, the present invention is directed to a covalent tetra-specific diabody, whose

IMPI

ΓΗΓΠΤΪΠΓΟ MK1CANE OF ΙΛ INDUSTRIAL MOCEDAD diabody is a dimer of diabody molecules, the first diabody molecule comprises a first and a second polypeptide chain, the first polypeptide chain of which comprises (i) a first domain comprising a binding region of a light chain variable domain of a first immunoglobulin (VL1) specific for a first epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for a second epitope and (iii) a third domain comprising an Fe domain or a portion thereof, wherein the first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site and wherein the third domain is located N-terminal to both the first domain and the second domain; and whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a light chain variable domain of the second immunoglobulin (VL2), (ii) a fifth domain comprising a binding region of a variable domain heavy chain of the first immunoglobulin (VH1), and (iii) a sixth domain comprising at least one cysteine ​​residue, which fourth and fifth domains are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site; and where the first string of³⁰

IWSTrnrTOMBUCANO ~ Ξ ^ ΒΪμΓΪ ^ _Γ1 Ο (U PROREDITY - .. Hee?

INDUSTRIAL polypeptide and the second polypeptide chain are · covalently linked, wherein the first domain and the fifth domain associate to form a first binding site (VL1) (VH1) that binds the first epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL2) (VH2) that binds the second epitope; and the second diabody molecule comprises a first and a second polypeptide chain, the first polypeptide chain of which comprises (i) a first domain comprising a binding region of a light chain variable domain of a third immunoglobulin (VL3) specific for a third epitope, (ii) a second domain comprising a binding region of a heavy chain variable domain of a fourth immunoglobulin (VH4) specific for a fourth epitope and (iii) a third domain comprising an Fe domain or a portion thereof, wherein the first and second domains are covalently linked such that the first and second domains do not associate to form an epitope binding site and wherein the third domain is located N-terminal to both the first domain and the second domain; and whose second polypeptide chain comprises (i) a fourth domain comprising a binding region of a fourth immunoglobulin light chain variable domain (VL4), (ii) a fifth domain comprising a binding region of a variable domain heavy chain third ³¹

Π'.πιινίυ M1X1CA Ν · DF THE PUOMÍDAD rwsruiiu.

immunoglobulin (VH3), and (iii) a sixth domain comprising at least one cistern residue, the fourth and fifth domains of which are covalently linked such that the fourth and fifth domains do not associate to form an epitope binding site ; and wherein the first polypeptide chain and the second polypeptide chain are covalently linked, provided that the covalent bond is not a peptide bond; wherein the first domain and the fifth domain associate to form a first binding site (VL3) (VH3) that binds the third epitope; wherein the second domain and the fourth domain associate to form a second binding site (VL4) (VH4) that binds the fourth epitope.

As explained above, the domains in the individual polypeptide chains are covalently linked. In specific aspects, the covalent bond between the first and second domain, the first and third domain, second and third domain, the fourth and fifth domain, the fourth and sixth domain, and / or the fifth and sixth domain they can be a peptide bond. In particular, the first and second domains, and the fourth and fifth domains can be separated by the third domain and the sixth domain, respectively, or through additional amino acid residues, while the first and the second, and the fourth and the fifth domains do not associate to form a binding site. The number of amino acid residues can be "IMF! ®" Mexican twsTnvro

OI LA MOM EDA D INntfSTRIAL

0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues. In a preferred aspect, the number of amino acid residues between the domains is 8.

In certain aspects of the invention, the first and second polypeptide chain domains comprise an Fe domain, that is, optionally, the third and sixth domains, respectively, may further comprise a hinge domain such that the domain comprises a Fe region of hinge. In alternative embodiments, the first polypeptide chain or the second polypeptide chain can comprise a hinge domain without also comprising an Fe domain. The heavy chains, light chains, hinge regions, Fe domains, and / or hinge-Fc domains for use in the invention can be derived from any type of immunoglobulin including IgA, IgD, IgE, IgG or IgM. In a preferred aspect, the immunoglobulin type is IgG, or any of its subtypes, i.e., IgGi, IgG₂, IgG₃ or IgG₄. In other aspects, the immunoglobulin from which the heavy and light chains are derived is humanized or chimerized.

Furthermore, the first epitope and the second epitope, and, where applicable, the third epitope and the fourth epitope, to which the diabody or diabody molecule binds may be different epitopes of the same antigen or they may be different epitopes of different antigens. . Antigens³³

Ιίβτπνπ? MEXICAN OF THE MIDDLE cy-ni tNDWKIAL can be any molecule from which an antibody can be generated. For example, proteins, nucleic acids, bacterial toxins, cell surface markers, autoimmune markers, viral proteins, drugs, etc. In particular aspects, at least one diabody epitope binding site is specific for an antigen on a particular cell, such as a B cell, a T cell, a phagocytic cell, a natural killer (NK) cell, or a dendritic cell. .

In certain aspects of the present embodiment, at least one epitope binding site of the diabody or the diabody molecule is specific for an Fe receptor, which Fe receptor can be an activating Fe receptor or an inhibitory Fe receptor. In particular aspects, the Fe receptor is an Fcy receptor, and the Fcy receptor is a receptor.

FcyRI, FcyRII or FcyRIII. In more preferred aspects, the FcyRIII receptor is a FcyRIIIA receptor (CD16A) or the FcyRIIIB receptor (CD16B), and more preferably the FcyRIII receptor is the FcyRIIIA receptor (CD16A). In another preferred aspect, the FcyRII receptor is the FcyRIIA receptor (CD32A) or the FcyRIIB receptor (CD32B), and more preferably the FcyRIIB receptor (CD32B). In a more particularly preferred aspect, one binding site of the antibody is specific for CD32B and the other binding site is specific for CD16A. In a specific modality of the

V V- Ή v 1 1 \ Á PI INSTITUTO M4XICA NO DE LA MIOME Da Ο. . „_. According to the invention, at least one epitope binding site on the diabody or the diabody molecule is specific for an activating Fe receptor and at least one other site specific for the inhibitory Fe receptor. In certain aspects of this embodiment the activating Fe receptor is CD32B. In other aspects of this embodiment the activating Fe receptor is BCR and the inhibitory Fe receptor is CD32B. In still other aspects of this embodiment, the activating Fe receptor is IgERI and the inhibitory Fe receptor is CD32B.

In cases where an epitope binding site is specific for CD16A, the VL and VH domains may be the same as or similar to the VL and VH domains of the 3G8 mouse antibody, the sequence of which has been cloned and is established in the present. In other cases where an epitope binding site is specific for CD32A, the VL and VH domains may be the same as or similar to the VL and VH domains of the mouse IV antibody. 3. In still other cases where the epitope binding site is specific for CD32B, the VL and VH domains may be the same as or similar to the VL and VH domains of the mouse antibody 2B6, the sequence of which has been cloned and established in the present. It is understood that any of the VL or VH domains of antibodies 3G8, 2B6 and IV. 3 can be used in any combination. The present invention is also directed to a diabody

<img file="MX348166B_D0019.tif" />

rmmuTc mexicai *? DE LA MOREDAI IND '.' MtAt

<img file="MX348166B_D0020.tif" />

specific as that of the diabody where the first epitope is specific for CD32B, and the second epitope is specific for CD16A.

In other aspects, an epitope binding site can be specific for a pathogenic antigen. As used herein, a pathogenic antigen is an antigen involved in specific pathogenic diseases, including cancer, infection, and autoimmune disease. Thus, the pathogenic antigen can be a tumor antigen, a bacterial antigen, a viral antigen, or an autoimmune antigen. Illustrative pathogenic antigens include, but are not limited to, lipopolysaccharides, viral antigens selected from the group of viral antigens for human immunodeficiency virus, adenovirus, respiratory syncytial virus, West Nile virus (eg, the E16 and / or E53 antigens) of the hepatitis virus, nucleic acids (DNA and RNA) and collagen. Preferably, the pathogenic antigen is a neutralizing antigen. In a preferred aspect, where one epitope binding site is specific for CD16A or CD32A, the other epitope binding site is specific for a pathogenic antigen that excludes autoimmune antigens. In yet another preferred aspect, wherein one epitope binding site is specific for CD32B, the other epitope binding site

<img file="MX348166B_D0021.tif" />

IMPI mrmrru Mexican □ · INDUSTRIAL PROPERTY · pathogenic antigen. In is specific for any specific modalities, the diabody molecule of the invention binds to two different antigens on the same cell, for example, one antigen-binding site is specific for an activating Fe receptor while the other is receptor-specific. Inhibitory faith. In other embodiments, the diabody molecule binds to two different viral neutralizing epitopes, for example, but not limited to, West Nile Virus E16 and E53.

In yet another embodiment of the present invention, the diabodies of the invention can be used to treat a variety of diseases and disorders. Accordingly, the present invention is directed to a method of treating a disease or disorder which comprises administering to a patient in need thereof an effective amount of a diabody or covalent diabody molecule of the invention wherein at least one site of binding is specific for a pathogenic antigen, such as an antigen expressed on the surface of a cancer cell or on the surface of a bacterium or virion and at least one other binding site that is specific for the Fe receptor, eg, CD16A.

In yet another embodiment, the invention is directed to a method of treating a disease or disorder comprising administering to a patient in need of the

IMPIOS MEXICAN FNSTTTUTO

OF THE PROPERTY itself an effective amount of a diabody or^r'Ta^AI mocen it from the diabody of the invention, in which at least one binding site is specific for CD32B and at least one other binding site is specific for CD16A.

In still another embodiment, the invention is directed to a method for inducing immune tolerance to a pathogenic antigen comprising administering to a patient in need thereof an effective amount of a covalent diabody or covalent diabody molecule of the invention, wherein at At least one binding site is specific for CD32B and at least one other binding site is specific for the pathogenic antigen. In aspects of this embodiment, the pathogenic antigen can be an allergen or another molecule for which immune tolerance is desired, such as a protein expressed in transplanted tissue.

In yet another embodiment, the present invention is directed to a method for detoxification comprising administering to a patient in need thereof an effective amount of a covalent diabody or the diabody molecule of the invention, wherein at least one site The binding site is specific for a cell surface marker such as Fe and the other binding site is specific for a bacterial toxin or drug. In one aspect, the surface marker

<img file="MX348166B_D0022.tif" />

cell is not found in red blood cells.

DEFINITIONS

Unless otherwise defined, all art terms, annotations, and other scientific terms or terminology used herein are intended to have the meanings commonly obtained by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference from what is generally understood in the present. technique. The practice of the present invention will utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are within the skill of the art. The technique. Such techniques are fully explained in the literature, such as Current Protocols in Immunology (JE Coligan et al., Eds., 1999, including 2001 supplements); Current Protocols in Molecular Biology (FM Ausubel et al., Eds., 1987, including 2001 supplements); Molecular Cloning: A Laboratory Manual, Third Edition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction, (Mullís et³⁹ LM P i INCTIFVTO MEXICANO rr LA PROPfEDAC ΙΝΓΗ · ΓΠΗΑΙ 3 ^^ al., Eds., 1994); The Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996); Methods of Immunological Analysis (R. Masseyeff, WH Albert, and NA Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow and Lañe Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999; and Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley &amp; Sons, Inc., New York, 2000).

As used herein, the terms antibody and antibodies refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single chain Fvs (scFv), Fab fragments, F (ab ') fragments, bispecific disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (antiId) antibodies (including for example, anti-Id and antianti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (ie, molecules that contain an antigen-binding site). Immunoglobulin molecules can be of any type (for example,

<img file="MX348166B_D0023.tif" />

(for example, IgG_lz

IgG, IgE, IgM, IgD, IgA and IgY), class

IgG₂, IgG₃, IgG₄, IgAi and IgA₂) or subclass.

As used herein, the terms binds immunospecifically, recognizes immunospecifically, binds specifically, recognizes specifically, and like terms refer to molecules that specifically bind to an antigen (eg, an epitope or immune complex) and do not bind. specifically to another molecule. A molecule that specifically binds to an antigen can bind to other peptides or polypeptides with lower affinity as determined through, for example, immunoassays, BIAcore, or other assays known in the art. Preferably, molecules that specifically bind to an antigen do not cross-react with other proteins. Molecules that specifically bind to an antigen can be identified, for example, through immunoassays, BIAcore, or other techniques known to those of skill in the art.

As used herein, "immune complex" refers to a structure that forms when at least one other target molecule and at least one polypeptide containing the heterologous Fcy region bind to another to form a larger molecular weight complex. Examples of immune complexes are antigen-antibody complexes that can be either soluble in particles (e.g.

<img file="MX348166B_D0024.tif" />

IMPI

IWSrrtVTC 'MEXICAN OI LA ItOHEDAD INnt'STRUl example, an antigen / ant.íniifirpo complex. on the cell surface).

As used herein, the terms heavy chain, light chain, variable region, main chain region, constant domain, and the like, have their ordinary meaning in the immunological art and refer to domains in naturally occurring immunoglobulin, and corresponding domains. synthetic (eg recombinant) binding proteins (eg humanized antibodies, single chain antibodies, chimeric antibodies, etc.). The basic structural unit of naturally occurring immunoglobulins (eg, IgG) is a tetramer that has two light chains and two heavy chains, usually expressed as a glycoprotein of approximately 150,000 Da. The amino-terminal (N) portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal (C) portion of each chain defines a constant region, with light chains having a single constant domain and heavy chains usually having three constant domains and a hinge region. In this way, the structure of the light chains of an IgG molecule is V_l-Cl-c and IgG heavy chain structure is nV_HiHC_H2-CH3-c (where H is the hinge region). The variable regions of a molecule

<img file="MX348166B_D0025.tif" />

IgG consists of the complementarity determining regions (CDR), which contain the residues in contact with the antigen and the non-CDR segments, referred to as main chain segments, where in general the structure is maintained and is determined in the placement of the CDR loops (although certain backbone residues may also be in contact with the antigen). In this way, the domains of V_L and V_H they have the structure n-FRl, CDR1, FR2, CDR2, FR3, CDR3, FR4-C.

When referring to binding proteins or antibodies (as broadly defined herein), the assignment of amino acids to each domain is according to the definitions of Kabat, Protein Sequences of Immune Interest (National Institutes of Health, Bethesda , Md., 1987 and 1991). The amino acids in the variable regions of the mature immunoglobulin heavy and light chains are designated by the position of an amino acid in the chain. The numerous amino acid sequences described by Kabat for antibodies, identify a consensus amino acid sequence for each subgroup, and are assigned a residue number for each amino acid. The Kabat numbering scheme is extended to unaffected antibodies in its digest through alignment of the antibody in question with one of the Kabat consensus sequences through reference to conserved amino acids.

<img file="MX348166B_D0026.tif" />

This method of assigning residue numbers has become standard in the field and easily identifies amino acids at equivalent positions in different antibodies, including humanized chimeric variants. For example, an amino acid at position 50 of a human antibody light chain occupies the equivalent position for an amino acid at position 50 of the mouse antibody light chain.

As used herein, the term heavy chain is used to define the heavy chain of an IgG antibody. In a native, intact IgG, the heavy chain comprises the immunoglobulin domains VH, CH1, hinge, CH2 and CH3. Throughout this specification, the numbering of residues in an IgG heavy chain is that of the EU index as in Kabat et al, Sequences of Proteins of Immunological Interest, 5^to Ed. Public Health Service, NH1, MD (1991), expressly incorporated herein by reference. The EU index as in Kabat refers to the numbering of the human IgGl antibody EU. Examples of amino acid sequences containing the IgGl, CH2 and CH3 hinge domains are shown in Figures 1A and IB as described,

Infra. Figures 1A and IB also establish amino acid sequences of the hinge, CH2 and CH3 domains of the heavy chains of IgG2, IgG3 and IgG4. The amino acid sequences of the IgG2, IgG3 and IgG4 isotypes align with

ΙΝτητυτο MEXICAN DE LA PROnEKAI? IND'.lSTIjlAL. _ _,, IND'JSTIJIAL the IgGl sequence by placing the first and last and last cysteine ​​residue in the respective hinge regions, which form the SS inter-heavy chain linkages, in the same positions. For the IgG2 and IgG3 hinge region, not all residues are listed by the EU index.

The hinge region or the hinge domain is generally defined as the Glu216 to Pro230 elongation of human IgGl. An example of the amino acid sequence of the human IgGl hinge region is shown in Figure 1A (amino acid residues in Figure 1A are listed according to the Kabat system). The hinge regions of other IgG isotypes can be aligned to the IgGl sequence by placing the first and last cysteine ​​residues that form the SS inter-heavy chain linkages in the same positions as shown in Figure 1A.

As used herein, the term region

Fe, Fe domain or analogous terms are used to define the C-terminal region of the IgG heavy chain. An example of the amino acid sequence that human IgGl contains is shown in Figure IB. Although the limits may vary slightly, a number according to the Kabat system, the Fe domain extends from amino acid 231 to amino acid 246 (amino acid residues in Figure IB are listed according to the Kabat system). Figure IB

<img file="MX348166B_D0027.tif" />

it also provides examples of the amino acid sequences of the Fe regions of the IgGl, IgG2, IgG3, and IgG4 isotypes.

The Fe region of an IgG comprises two constant domains, CH2 and CH3. The CH2 domain of a human Fe IgG region usually extends from amino acids 231 to amino acid 341 according to the Kabat numbering system (Figure IB). The CH3 domain of a human Fe IgG region usually extends from amino acids 342 to 447 according to the Kabat numbering system. The CH2 domain of the human Fe IgG region (also referred to as the Cy2 domain) is unique in that it does not pair closely with another domain. Rather, two N-linked branched carbohydrate chains are interposed between two CH2 domains of an intact native IgG.

As used herein the terms FcyR binding protein, FcyR antibody, and anti-FcyR antibody are used interchangeably and refer to a variety of immunoglobulin-like or immunoglobulin-derived proteins. FcyR binding proteins bind FcyR through an interaction with the V domains_L and / or V_H (as different from Fcy-mediated binding). Examples of FcyR binding proteins include fully human, polyclonal, chimeric, and humanized antibodies (eg, they comprise two heavy and two light chains). Its fragments (for example, Fab, Fab ', F (ab')₂, and fragments

<img file="MX348166B_D0028.tif" />

IMPI INSTITUTE ΜβΠΟΑΛΚ. ΓΛ LA NtOHEDAO INOUSTFIAl

Fv), bifunctional or multifunctional antibodies (see, for example, Lanzavecchia et al. (1987) The Use Of Hybrid

Hybridomas To Target Human Cytotoxic T Lymphocytes, Eur. J. Immunol. 17: 105-111), single chain antibodies (see, eg, Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242: 423-26), fusion proteins (eg, fusion proteins phage display), minibody (see, for example, US Patent No. 5,837,821), and other antigen-binding proteins that comprise a V domain_L and / or V_H or one of its fragments. In one aspect, the FcyRIIIA binding protein is a tetrameric antibody that is, it generally has the structure of a naturally occurring IgG and comprises the variable and constant domains, that is, two light chains, that comprise the V domain._L and a constant domain of light chain and two heavy chains comprising a V domain_H and a heavy chain constant and hinge domain.

As used herein the term FcyR antagonists and like terms refer to protein and non-protein substances, including small molecules that antagonize at least one biological activity of an FcyR, eg, blocking signaling. For example, the molecule of the invention blocks signaling by blocking the binding of IgGs to an FcyR.

As used herein, the term "derivative" in the context refers to a polypeptide or amino acid sequence that has

IMPIAS (NHTTUTO MtílCANO

OF THE PROPERTY. AC-

INfimiAt polypeptides or proteins are proteins that comprise a been altered through the introduction of substitutions, deletions or additions of amino acid residues. The term "derivative" as used herein also refers to a polypeptide or protein that has been modified, that is, through the covalent attachment of any type of molecule to the polypeptide or protein. For example, but not by way of limitation, an antibody can be modified, for example, through glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization through known protecting / blocking groups, proteolytic cleavage, cell binding to an antigen or other protein, etc. A derived polypeptide or protein can be produced through chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Furthermore, a derived polypeptide or protein possesses a function similar or identical to that of the polypeptide or protein from which it was derived.

As used herein, the term "derivative" in the context of a non-protein derivative refers to a second organic or inorganic molecule that is formed based on the structure of a first molecule.

IMPI

MLAPWKDAD ΙΝηΐ ^ ΤΚΙ »!

organic or inorganic. A derivative of an organic molecule includes, but is not limited to, a molecule modified, for example, through the addition or removal of a hydroxyl, methyl, ethyl, carboxyl, or amine group. An organic molecule can also be esterified, alkylated, and / or phosphorylated.

As used herein the term "diabody molecule" refers to a complex of two or more polypeptide chains or proteins, each comprising at least one VL domain and one VH domain or one of its fragments, where both domains are comprised within a single polypeptide chain. In certain embodiments "diabody molecule" includes molecules that comprise an Fe domain or a hinge-Fc domain. Such polypeptide chains in the complex can be the same or different, that is, the diabody molecule can be a homo-multimer or a heteromultimer. In specific aspects, "diabody molecule" includes dimers or tetramers or polypeptide chains that contain either the VL or VH domain. Individual polypeptide chains comprise multimeric proteins that can be covalently linked to at least one other peptide of the multimer through interchain disulfide bonding.

As used herein, the terms disorder and disease are used interchangeably.

<img file="MX348166B_D0029.tif" />

IMPI

MUICAN IHSTnVTO

M THE «CRITY

INDtrSTRIAl interchangeable to refer to an afArr-ínn ^ n _1in subject. In particular, the term autoimmune disease is used interchangeably with the term autoimmune disorder to refer to a condition in a subject characterized by brain, tissue and / or organ damage caused by an immune reaction of the subject to his own cells, cultures and / or organs. The term inflammatory disease is used interchangeably with the term inflammatory disorder to refer to a condition in a subject characterized by inflammation, preferably chronic inflammation. An immune disorder may or may not be associated with inflammation. Also, the inflammation may or may not be caused by an autoimmune disorder. In this way, certain disorders can be characterized as both autoimmune and inflammatory disorders.

Identical polypeptide chains as used herein also refers to polypeptide chains that have nearly identical amino acid sequences, for example, including chains that have one or more amino acid differences, preferably conservative amino acid substitutions, such as activity. of two polypeptide chains is not significantly different.

As used herein, the term cancer refers to a neoplasm or tumor that results from the growth «rrrrvro μΛγλμο Ο | Μ «C * I! DAO uncontrolled abnormal cells. As is 'WTTlza' in ^ the present, cancer explicitly inulLiyt !, lehóemias y lyfornas. In some modalities, cancer refers to a malignant tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed adjoining body structures, and has hidden at a distant site. In some embodiments, the cancer is associated with a specific cancer antigen.

As used herein, the term "immunoregulatory agent" and its variations can be an agent that modulates a host immune system. In certain embodiments, an immunomodulatory agent is an immunosuppressive agent. In certain other embodiments, an immunoregulatory agent is an immunostimulatory agent. Immunomodulatory agents include, but are not limited to, small molecules, peptides, polypeptides, fusion proteins, antibodies, inorganic molecules, mimetic agents, and organic molecules.

As used herein, the term "epitope" refers to a fragment of a polypeptide or protein or a non-protein molecule that has antigenic or immunogenic activity in an animal, preferably a mammal, and more preferably a human. An epitope having immunogenic activity is a polypeptide fragment or

IMPI

ΙΝΠΊΐυΤΟ Μ RUCANO MUPBOtEDA · fNIX * T »l * L protein that is an antibody response in an animal. An epitope having antigenic activity within a polypeptide or protein to which an antibody immunospecifically binds as determined by any method well known to one of skill in the art, for example, through immunoassay. Antigenic epitopes do not necessarily have to be immunogenic.

As used herein, the term "fragment" refers to a peptide or polypeptide that comprises an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, as least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous 15 amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues 20, at least 125 contiguous amino acid residues, at least least 150 contiguous contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues from the amino acid sequence of another polypeptide. In a specific modality, a fragment of a

<img file="MX348166B_D0030.tif" />

ffHWTO MEXICANO DI LA HOnMDAR “HJU

INDUSTRIAL polypeptide retains at least one function of the polypeptide.

As used herein, the term nucleic acids and nucleotide sequences include DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), combinations of DNA and RNA molecules, or DNA molecules. / RNA hybrids, and analogs of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, including, but not limited to, inosine or tritylated bases. Such analogs may also comprise ADR or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cell membranes. The nucleic acid or nucleotide sequences may be single-stranded, double-stranded, and may contain both single-stranded and double-stranded portions, and may contain triple-stranded portions, but is preferably double-stranded DNA.

As used herein, a therapeutically effective amount refers to the amount of a therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount can refer to the amount of the therapeutic agent sufficient to delay or minimize the onset of the disease, for example, delay or minimize the extent of the disease.

IMPI

DVHfVTO MEXICAN ocla non wao • «• umiAL

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also cancer.

A therapeutically effective amount can refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Furthermore, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of the therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in treating or managing a disease.

As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to any agent (s) that can be used in the prevention and disorder, or the prevention of the recurrence or spread of a disorder. A prophylactically effective amount can refer to the amount of prophylactic agent sufficient to prevent the recurrence or spread of hyperproliferative disease, particularly cancer, or the appearance of such cancer in a patient, including but not limited to those predisposed to hyperproliferative disease, for example, those genetically predisposed to cancer or previously exposed to the carcinogen. A "prophylactically effective amount" can also refer to the amount of the prophylactic agent that provides a prophylactic benefit in preventing disease. Also, a prophylactically effective amount

<img file="MX348166B_D0032.tif" />

with respect to a prophylactic agent of the invention means the amount of prophylactic agent alone, or in combination with other agents, that provides a prophylactic benefit in preventing disease.

As used herein, the terms "prevent, anticipate and prevent" refer to the prevention of the recurrence or initiation of one or more of the symptoms of a disorder in a subject as a result of the administration of a prophylactic or therapeutic agent.

As used herein, the term in combination refers to the use of more than one prophylactic and / or therapeutic agent. The use of the term in combination does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequently (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes , 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later) to the administration of a

IMPI INSTTTUT · MSXICANC DE LA FROriEDAU second prophylactic or therapeutic agent to a subject disorder.

Effector function as used herein means a biochemical event that results from the interaction of an Fe region of the antibody with an Fe receptor or an antigen. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and dependent cytotoxicity. Supplement (CDC). Effector functions include both those that operate after antigen binding and those that operate independently of antigen binding.

Effector cell as used herein means a cell of the immune system that expresses one or more Fe receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and can be from any organism including but not limited to not limited to humans, mice, rats, rabbits, and monkeys.

As used herein, the term joins

<img file="MX348166B_D0033.tif" />

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INSTITUTO MEXICANO □ t la moniioAc c * · y-'wv. And specifically to an immune complex and analogous terms refer to molecules that specifically bind to an immune complex and do not specifically bind to another molecule. A molecule that specifically binds to an immune complex can bind to other peptides or polypeptides with lower affinity as determined through, for example, immunoassays, BIAcore, or other assays known in the art. Preferably, molecules that specifically bind to an immune complex do not react reciprocally on other proteins. Molecules that specifically bind to an immune complex can be identified, for example, through immunoassays, BIAcore, or other techniques known to those of skill in the art.

A stable fusion protein as used herein refers to a fusion protein that undergoes a minimal undetectable level of degradation during production and / or storage as evaluated using common biochemical and functional assays known to those of skill in the art. and can be stored for an extended period of time without loss of biological activity, eg, binding to FcyR.

Brief Description of Figures

Figures 1A-1B. Amino acid sequence of the human Fe and hinge IgG CH1 regions.

<img file="MX348166B_D0034.tif" />

The amino acid sequences of the IgGl, IgG2, IgG3 and hinge domains IgG4 (Fig. 1A) and Fe (Fig. IB) are provided. (IgGl hinge domain (SEQ ID NO: 1); IgG2 hinge domain (SEQ ID NO: 2); IgG3 hinge domain (SEQ ID NO: 3); IgG4 hinge domain (SEQ ID NO: 4); domain Fe IgGl (SEQ ID NO: 5); Fe IgG2 domain (SEQ ID NO: 6); Fe IgG3 domain (SEQ ID NO: 7); Fe IgGl domain (SEQ ID NO: 8)). The amino acid residues shown in Figures 1A and IB are listed according to the Kabat EU numbering system. The isotype sequences are aligned with the IgGl sequence by placing the first and last cysteine ​​residues of the respective hinge regions, which form the heavy interchain SS bonds, in the same positions. For Figure IB, residues in the CH2 domain are indicated by +, while residues in the CH3 domain are indicated by ~.

Figure 2. Schematic representation of polypeptide chains of covalent bifunctional diabodies.

Polypeptides of a covalent, bifunctional diabody consist of an antibody VL domain and an antibody VH domain separated through a short peptide linker. The 8 amino acid residue linker prevents the self-assembly of a single polypeptide chain in the scFv constructs, and rather the integration between the VL and VH domains of different chains predominates.

<img file="MX348166B_D0035.tif" />

DE LA PROPfEDA I INDUSTRIAI polypeptide. 4 constructs were created (each construct is described from the amino-terminal (n), left side of the construct, to the carboxy-terminal (c), right side of the figure): construct (1) (SEQ ID NO: 9) comprising, n-the Hu2B6 VL-linker domain (GGGSGGGG (SEQ ID NO: 10)) - the {VH domain Hu3G8 - and a C-terminal sequence (LGGC) -c; construct (2) (SEQ ID NO: 11) comprising n-the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - and a C-terminal sequence (LGGC) -c; construct (3) (SEQ ID NO: 12) comprising n-the VL · Hu3G8 linker domain (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu3G8 - and a C-terminal sequence (LGGC) -c; construct (4) (SEQ ID NO: 13) comprising n-the VL domain of Hu2B6 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - and a C-terminal sequence (LGGC) -c.

Figure 3. SDS-PAGE analysis of affinity purified diabodies.

Affinity purified diabodies were subjected to SDS-PAGE analysis under reducing (lanes 1-3) or non-reducing (lanes 4-6) conditions. Approximate molecular weights of the standard (between lanes 3 and 4) are indicated. Lanes 1 and 4, h3G8 CMD; Lanes 2 and 5, h2B6 CMD; and Lanes 3 and 6, h2B6-h3G8 CBD.

Figures 4A-4B. affinity purified.

Analysis

SEC de diacuerpos tfwrrRno mexican &lt;Ot LA KORitlMI: IMniJSTtIAl

Affinity purified diabodies were subjected to SEC analysis. (Figure 4A) Known standard elution profile: full length IgG (-150 kDa), IgG Fab fragment (~ 50 kDa), and scFv (-30 kDa); (Figure 4B) Elution profile of h2b6 CMD, h3G8 CMD, and h2B6-h3G8 CBD.

Figure 5. Binding of h2B6-h3G8 CBD to SCD32B and SCD16A.

The binding of h2B6-h3G8 CBD to SCD32B and sCD16A was assayed in an ELISA sandwich. SCD32B was used as the target protein. The secondary probe was sCD16A conjugated to HRP. h3G8 CMD, which binds CD16A, was used as a control.

Figures 6A-6C. BIACORE analysis of diabody binding to sCD15A, SCD32B and SCD32B.

The binding of h2B6-h3G8 CBD, h2B6 CMD, and H3G8 CMD to SCD16A, sCD32B, and sCD32A (negative control) was tested through SPRs analysis. h3G8 scFv was also tested as a control. (Figure 6A) binding to sCD16; (Figure 6B) binding to SCD32B and (Figure 6C) binding to sCD32A. Diabodies were injected at a concentration of 100 NM, and scFv at a concentration of 200 nM, onto receptor surfaces at a flow rate of 50 ml / min for 60 seconds.

Figures 7A-7E. BIACORE analysis of diabody binding to sCDISA and SCD32B.

The binding of h2B6-h3G8 CBD, h2B6 CMD and h3G8 CMD to

SCD16A, and sCD32B was tested via SPR analysis. h3G8 scFv was also tested as a control.

IMPI • NSTmiTO MEXICANc 'nEUmerfFDAI;

(Figure 7A) Union of

<img file="MX348166B_D0036.tif" />

h3G8 CMD SCD16A; (Figure 7B) Binding of h2B6-h3G8 CBD to SCD16A7 (Figure 7C) Binding of h3G8 scFv to SCD16A; (Figure 7D) Binding of h2B6 CMD to sCD32B; and (Figure 7E) Binding of h2B6-h3G8 CBD to SCD32B. The diabodies were injected at concentrations of 6.25-200 nM onto receptor surfaces at a flow rate of 70 ml / min for 180 seconds.

Figure 8. Schematic description of the interaction of polypeptide chains comprising the VL and VH domains to form a covalent bispecific diabody molecule.

NH₂ and COOH represent the amino-terminal and carboxy-terminal, respectively, of each polypeptide chain. S represents the C-terminal cysteine ​​residue in each polypeptide chain. VL and VH indicate the variable light domain and the variable heavy domain, respectively. The dotted and broken lines are to distinguish between two polypeptide chains and, in particular, represent the linker portions of such chains. h2B6 Fv and h3G8 Fv indicate a specific epitope binding site for CD32B and CD16, respectively.

Figure 9. Schematic representation of polypeptide chains containing Fe domains of covalent bispecific diabodies.

Representation of polypeptide constructs of the ⁶¹ .IMPI iNSTrnrro μϊχιοανο Dt LA MOREDA D (ΝΟ'ΤΠ, ΙΑΙ diabody molecules of the invention (each construct is described from the amino-terminal (n), left side of the construct, to the carboxy-terminal (c), right side of the figure). Construct (5) (SEQ ID NO: 14) comprising, n VL domain Hu2B6 - a first linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu3G8 - a second linker (LGGC) and a C-terminal Fe domain human IgGl-c; construct (6) (SEQ ID NO: 15) comprising n-the VL domain Hu3G8 linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - and the second linker (LGGC) - and an Fe C- domain terminal of human IgGl-c; construct (7) (SEQ ID NO: 16) comprising n-the VL domain Hu2B6 - a first linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu3G8 - and a C-terminal sequence (LGGCFNRGEC) (SEQ ID NO: 17) -c; construct (8) (SEQ ID NO: 18) comprising n-the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - and the second linker (LGGC) - and a hinge domain / C-terminal human IgGl Fc (with A215V amino acid substitution) -c.

Figure 10. Binding of diabody molecules comprising Fe domains to SCD32B and SCD16A.

The binding of the diabody molecules comprising Fe domains to sCD32B and sCDISA was assayed in an ELISA sandwich. The tested diabodies were produced through 3 recombinant expression systems: co-

<img file="MX348166B_D0037.tif" />

<img file="MX348166B_D0038.tif" />

transfection of pMGX669 and pMGX674, expressing loe 1 and 6, respectively; co-transfection of pMGX667 and pMGX676, expression of constructs 2 and 5, respectively; and cotransfection of pMGX674 and pMGX676, expressing constructs 5 and 6, respectively. SCD32B was used as the target protein. The secondary probe was SCD16A conjugated to HRP.

Figure 11. Schematic description of the interaction of two polypeptide chains each comprising an Fe domain to form a covalent-bivalent diabody.

NH₂ and COOH represent the amino-terminal and carboxy-terminal respectively for each polypeptide chain. S represents at least one disulfide bond between a cysteine ​​residue in the second linker sequence of each polypeptide chain. VL and VH indicate the variable light domain and the variable heavy domain, respectively. The dotted and broken lines are to distinguish between two polypeptide chains and, in particular, represent the first linker portions of such chains. CH2 and CH3 represent the constant CH2 and CH3 domains of an Fe domain. H2B6 Fv and h3G8 Fv indicate a specific binding site for CD32B and CD16, respectively.

Figure 12. Binding of diabody molecules comprising hinge / Fc domains sCD32B and sCD16A.

The binding of the diabody molecules that

<img file="MX348166B_D0039.tif" />

comprising Fe domains for SCD32B and sCDIGA were tested in a sandwich ELISA. The tested diabodies were produced through 4 recombinant expression systems: cotransfection of pMGX669 + pMGX674, expression of constructs 1 and 6, respectively; co-transfection of pMGX669 + pMGX678, expression of constructs 2 and 8, respectively; co-transfection of pMGX677 + pMGX674, expression of constructs 7 and 6, respectively; and cotransfection of pMGX677 + pMGX678, expression of constructs 7 and 8, respectively. SCD32B was used as the target protein. The secondary probe was SCD16A conjugated to HRP.

Figure 13. Schematic description of the interaction of polypeptide chains to form a tetrameric diabody molecule.

NH₂ and COOH represent the amino-terminal and carboxy-terminal, respectively, of each polypeptide chain. S represents at least one disulfide bond between a cysteine ​​residue in the second Fe linker sequence that carries a heavier polypeptide chain and a cysteine ​​residue in the C-terminal sequence of the lighter polypeptide chain that does not carry Fe VL and VH indicate the variable light domain and the variable heavy domain, respectively. Dotted and broken lines are to distinguish between polypeptide chains and, in ⁶⁴ IMPI iNSTmrro Mexican of the particular promida or INDUSTRIAL, they represent the first linker portions of the heavier chains or the linker of the lighter chains. CH2 and CH3 represent the constant CH2 and CH3 domains of an Fe domain. H2B6 Fv and h3G8 Fv indicate a specific epitope binding site for CD32B and CD16, respectively.

Figure 14. Schematic representation of polypeptide chains containing Fe domains that form covalent bispecific diabodies.

The representation of the polypeptide constructs that form the diabody molecules of the invention (each construct is described from the amino-terminal (n), left side of the construct, to the carboxy-terminal (c), right side of the figure). Construct (9) (SEQ ID NO: 19) comprising n-a hinge / Fc domain of human IgGl - the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - linker (GGGSGGGG (SEQ ID NO: 10)) and a C-terminal LGGC c-sequence; construct (10) (SEQ ID NO: 20) comprising n-a Fe domain of human IgGl - the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 - linker (GGGSGGGG (SEQ ID NO: 10)) and a C-terminal LGGC c-sequence; construct (11) (SEQ ID NO: 21) comprising n-the VL domain Hu2B6 (G105C) linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu3G8 - and a C-terminal hinge / Fc domain of the Human IgGl with

<img file="MX348166B_D0040.tif" />

<img file="MX348166B_D0041.tif" />

the A215V-C amino acid substitution; construct (12) (SEQ ID NO: 22) comprising n-the VL domain Hu3G8 - linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6 (G44C) and a C-terminal FNRGEC c-sequence (SEQ ID NO: 23).

Figures 15A-15B. SDS-PAGE and Western Blot affinity analysis of tetrameric diabodies.

Diabodies produced through the recombinant expression system were co-transfected with vectors expressing constructs 10 and 1, constructs 9 and 1, and constructs 11 and 12 that were subjected to SDS-PAGE analysis under non-reducing conditions ( Figure 15A) and Western Blot analysis using H + L goat anti-human IgGl as the probe (Figure 15B). Proteins on SDS-PAGE gel visualized with Simple Blue Sapstain (Invitrogen). For both panels of Figure 15A and Figure 15B, the diabody molecules comprised constructs 10 and 1, constructs 9 and 11, and constructs 11 and 12A which are in lanes 1, 2, and 3, respectively.

Figure 16. Binding of diabody molecules comprising Fe domains and modified inter-chain disulfide bonds for SCD32B and sCD16A.

The binding of diabody molecules comprising Fe domains and modified disulfide bonds between the lighter and heavier polypeptide chains for sCD32B and SCD16A were tested in an ELISA sandwich. The

- IMPIOS nWTffWTO MKXICANO A • fu «omedad iNDí'rnifAi tested diabodies were produced through three recombinant expression systems: expression of constructs 1 and 10, expression of constructs 1 and 9, and expression of constructs 11 and 12 , respectively. sCD32B was used as the target protein. The secondary probe was sCD16A conjugated to HRP. The h3G8 binding was used as a control.

Figure 17. Schematic representation of the diabody molecule polyprotein precursor and schematic representation of the polypeptide chains containing the Lambda light chain and / or hinge domains.

The representation of the polypeptide constructs that comprise the diabody molecules of the invention (each construct is described from the amino-terminal (n), left side of the construct, to the carboxy-terminal (c), right side of the figure). Construct (13) (SEQ ID NO: 95) comprising, n-domain VL 3G8 - a first linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of 2.4G2VH - a second linker (LGGC) - recognition site of furin (RAKR (SEQ ID NO: 93)) - VL domain of 2.4G2 - a third linker (GGGSGGG (SEQ ID NO: 10) - VH domain of 3G8 - and a C-terminal LGGC domain; (nucleotide sequence that encodes SEQ ID NO: 95 is provided in SEQ ID NO: 96). Construct (14) (SEQ ID NO: 97) comprising n-domain VL 3G8 - a first linker (GGGSGGGG (SEQ ID NO: 10)) - the

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2.4G2VH VH domain a second linker (LGGC) furin recognition site (RAKR (SEQ ID NO: 93)) - FMD (Protease

Foot and Mouth Disease Virus C3) site-VL domain of 2.4G2- a third linker (GGGSGGG (SEQ ID NO: 10) VH domain of 3G8- and a C-terminal LGGC domain; (nucleotide sequence encoding SEQ ID NO: 97 is provided in SEQ ID NO: 98). Construct (15) (SEQ ID NO: 99) comprising a VL domain Hu2B6 - a linker (GGGSGGGG (SEQ ID NO: 10)) the VH domain of Hu3G8- and a C-terminal FNRGEC domain (SEQ ID NO: 23); (nucleotide sequence encoding SEQ ID NO: 99 is provided in SEQ ID NO: 100). Construct (16) (SEQ ID NO: 101) comprising n-VL domain Hu3G8 - a linker (GGGSGGGG (SEQ ID NO: 10)) - the VH domain of Hu2B6- and a C-terminal VEPKSC domain (SEQ ID NO: 77 ); (nucleotide sequence encoding SEQ ID NO: 101 is provided in SEQ ID NO: 102).

Figure 18. Binding of diabody molecules derived from a polyprotein precursor molecule to mCD32B and SCD16A.

The binding of diabody molecules derived from polyprotein precursor molecule construct 13 (SEQ ID NO: 95) to murine CD32B (mCD32B) and soluble CD16A (sCD16A) were tested in a sandwich ELISA. mCD32B was used as the target protein. The secondary probe was SCD16A conjugated to biotin.

IMPI ΠΤΠΙΙΙΓΓΟ MEXICAN _ 5Ε U RKOPltTY INDUSTRIAL ___ ____

Figure 19. Union of the diabody molecules comprising the Lambda chain and / or hinge domains for sCD32B and SCD16A.

The binding of diabody molecules comprising the C-terminal derived domains of the IgG hinge and / or human lambda light chain domain to sCD32B and sCD16A were tested and compared to the diabody comprising constructs 1 and 2 ( Figure 5) in an ELISA sandwich. Test diabodies were produced through recombinant expression systems expressing constructs 15 and 16 (SEQ ID NO: 99 and SEQ ID NO: 101, respectively). sCD32B was used as the target protein. The secondary probe was sCD16A conjugated to HRP. The bars with small squares represent the combination of construct 15/16 while the bars with larger squares represent the combination of construct 1/2.

Figure 20. Schematic representation of the DART binding of 2B6 / 4420 to CD32B located on the surface of a cell of a molecule and a molecule conjugated to fluorescein.

The diagram shows the flexibility of the DART fluorescein arm universal adapter as well as the ability to substitute for other 2B6 arm specificities. The V regions are shown as linker boxes GGGSGGGG (SEQ ID NO: 10) are shown as lines, and the link

<img file="MX348166B_D0043.tif" />

IMPI ^

INSTITUTO MEXICANf of'ιλ ncpifon '»ΙΝΝιςτηι, of disulfide is shown connecting the two chains. The constituents of one chain are shown in blue while the other is in pink. N, amino-terminal; C, carboxy terminal, FL, fluorescein, VL, light chain variable region; and VH, heavy chain variable region.

Figures 21A and 2IB. DART 2B6 / 4420 specifically binds fluorescein-conjugated molecules and can simultaneously bind CD32B.

(Figure 21A) 2B6 / 4420 or 2B6 / 3G8 were bound to ELISA plates coated with the FITC-S protein. Binding and function of the 2B6 arm was detected through soluble CD32B binding, followed by a CD32B-specific antibody and a HRP-conjugated secondary detection antibody. (Figure 21B) 2B6 / 4420 or 2B6 / 3G8 were bound to ELISA plates coated with HuIgG or FITC-HuIgG (fluorescein conjugated). Binding was detected through connection with a 2B6 Fv-specific polyclonal serum followed by a HRP-conjugated secondary antibody.

Figures 22A and 22B. Activation of purified B cells using anti-human CD79B antibodies.

Purified B cells were activated using increasing concentrations of CB3.1-FITC (Figure 22A) or CB3.2-FITC (Figure 22B) conjugated with FITC anti-human CD79b antibodies and 50 pg / ml of the F (ab ') fragment 2 Fe IgG GAM specific (x-axis). B cells were activated in the⁷⁰fNSTTTVTÜ MlXlCAMC. PF LA mOPlSDAI · IHDtimtAL presence of PBS (white bars) or 5 pg / ml of either aFITCaCD32BDART (black bars) or aCD16aCD32BDART (gray bars). Reactions were carried out in triplicate and standard deviations were calculated.

Figures 23A and 23B Activation of purified B cells.

Purified B cells from a healthy second donor were activated as described in Figure 22B. The proliferation index was measured in activated cells in the presence of CB3.2-FITC conjugated to the anti-CD79b antibody FITC (Figure 23A) and compared with the proliferation index of activated cells in the presence of CB3.2 antibody without mark (Figure 23B).

Figures 24A-24B. In vivo mouse B cell depletion in HCD16A / B transgenic mouse using

MGD261.

The mCD32 - / - hCD16A + C57B1 / 6, mCD32- / hCD32B + C57B1 / 6 and mCD32 - / - hCD16A + hCD32B + C57B1 / 6 mice were injected IV on days 0, 3, 7, 10, 14 and 17 with MGD261 (10, 3, 1 or 0.3 mg / kg), or an irrelevant antibody (hE16 10 mg / kg). Blood was collected on days -19 (pre-bleed), 4, 11, 18, 25, and 32 for FACS analysis. The health and activity of the animals were recorded three times a week. Fig. 24A: h2B6-3G8 and WNV mAb; Fig. 24B: h2B6-3G8 -hCD16A or -hCD32B mice and ⁷¹ IMPIAS

INSTITUTO MOITANt, I heard about WSTFfAL mice WNV mAb -hCD16A or -hCD32B.

Figure 25. In vivo mouse B cell depletion in HCD16A / B transgenic mice using 2.4G2-3G8 DB.

MCD16 - / -, mCD16 - / - hCD16A + C57B1 / 6, mCD16 - / - hCD16B + and mCD16 - / - hCD16A + hCD16B + mice from the MacroGenics breeding colony were injected IP on days 0, 2, 4, 7, 9 , 11, 14, 16 and 18 with 2.4G2-3G8 DB (75 ug / mice), or PBS. Blood was collected on days -10 (prebleed), 4, 11 and 18 for FACS analysis. The health and activity of the animals were recorded 3 times a week.

Figure 26. Demonstration of the antitumor activity of MGD261 using an intravenous (IV) model of the human tumor cell line RAJI.

Twelve 20 week old mCD16 - / -, hCD16A +, RAG1- / C57B1 / 6 mice from the breeding colony MacroGenics IV were injected on day 0 with 5x10⁶ Raji cells. On days 6, 9, 13, 16, 20, 23, 27 and 30 the mice were also treated intraperitoneally (IP) with 250, 25 or 2.5ug MGD261 or with PBS (negative control). The mice were then observed daily and body weight was recorded twice a week. Mice that developed palsy were euthanized.

Figure 27. DART expression in a non-mammalian host.

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BL21DE3 cells (Novagen) were transformed with plasmid pET25b (+) T7-lac + 3G8 / 3G8 and an amp resistant colony was used to seed the broth culture. When the culture reached 0.5 OD600 units, 0.5 mM IPTG was added to induce expression. The culture was grown at 30 ° C for 2 hours and harvested in cell-free medium.

Figure 28. DART ELISA.

The h3G8-h3G8 DART binding ELISA was conducted using Maxisorp 96 well plates. After the reaction the plate was washed with PBS-T three times and developed with 8 0 µΐ / well of TMB substrate. After 5 min incubation, the reaction was stopped through 40 μΐ / well of 1% H₂SW₄. OD450nm was read using a 96 well plate reader and SOFTmax software. The reading was plotted using GraphPadPrism 3.03 software.

Figure 29. DART-induced human B cell death.

Human PBMC was incubated overnight with indicated molecules. Apoptosis was assayed through FACS analysis as the percentage of PI⁺Annexin-V⁺ population of B cells (CD20 + cells) in the total non-barrier FSC / SSC population.

Figure 30. DART 8B5-CB3.1 constructs.

Multiple 8B5-CB3.1 DART constructs were produced ⁷³ IMPI institoto m »xican (, Γί LA FROHEDAi.

INDI imiAl to illustrate the present invention. Construct 5 and 6, or 6 and 7, Ó8y9, or 9 and 10, encoding expression plasmids were co-transfected into HEK-293 cells to express 8B5-CB3.1 with or without anti-flag tag using Lipofectamine 2000 ( Invitrogen). The conditioned medium was harvested for every three days for three times. The conditioned medium was then purified using a CD32B affinity column.

Figure 31. DART 8B5-CB3.1 ELISA.

The competition ELISA 8B5-CB3.1 DART / ch8B5 was conducted using Maxisorp 96 well plates. After reaction, the plate was washed with PBS-T three times and developed with 80 µΐ / well TMB substrate. After 5 min incubation, the reaction was stopped through 40 μΐ / well of 1% H₂SW₄. OD450nm was eluted using a 96 well plate reader and SOFTmax software. The reading was plotted using GraphPadPrism 3.03 software.

Figure 32. Schematic illustration of the tetravalent DART structure.

The general structure of a DART species produced through the assembly of four polypeptide chains is illustrated. The four Ig-type DART antigen-binding domains are shown as striped ellipses and dark gray.

Figure 33. Ig-type tetravalent DART.

A schematic of the sites of

<img file="MX348166B_D0044.tif" />

<img file="MX348166B_D0045.tif" />

<td>epitope binding</td><td>of a tetravalent DART of t ipn Tg .______</td>

<td>(A) +</td><td>(E) = Epitope 1 binding site</td>

<td>(D) +</td><td>(B) = Epitope 2 binding site</td>

<td>(C) similar to an Ig</td><td>+ (F) = C-kappa / lambda + CHi (association raditional)</td>

hinge - CH2 - CH3 - will associate to form an Fe Figure 34. mCD32-hCD16A binding ELISA.

The ELISA result is provided demonstrating that the Ig-type tetravalent DART species of Examples 6-10 bind antigen with higher affinity than control antibody (ch-mCD32 mAb) or other DART species.

Figures 35A-35K. Schematic illustration of Ig DART molecules.

A schematic of Ig DART molecules is provided. Specificity is indicated by reaction shaded, patterned, or colored in white, constant regions are shown in black, and disulfide bonds are indicated by black dotted lines. The N-terminus of all protein chains is oriented towards the top of the figure, while the C-terminus of all protein chains is oriented towards the bottom of the figure. Figures 35A-35E are bispecific and Figures 35F-35J are trispecific. Figures 35A and 35E are⁷⁵ IMPI iwsniirro mcucanc PELA PROPERTY INDD ^ RiAL tetravalent. Figures 35B, 35C, 35F, 351, and 35J are hexavalent. Figures 35D, 35G, and 35H are octavalent. Refer to Figures 1A, IB, 2, 9, 14 and 17 and Section 3.1 for detailed descriptions of the individual domains.

Figure 36. Binding ability of the biospecific diabody HU2B6 4.5-HU3G8 5.1.

The ability of the Hu2B6 4.5-Hu3G8 5.1 (squares) biospecific diabody to bind CD32b and CD16a relative to the Hu2B6 4.5 or Hu3G8 5.1 diabodies (triangles) is shown.

Figure 37. Schematic of the E-coil and K-coil DART derivatives.

The general conformation of the DART E-coil and K-coil derivatives is illustrated.

Figure 38. Helix configuration of preferred E-coil and K-coil spacers.

Figure 38 shows the helix configuration of the preferred E-spiral sequence (EVAALEK)₄ (SEQ ID NO: 299) and the preferred K-coil sequence (KVAALKE)₄ (SEQ ID NO: 300).

Figure 39. DART derivatives containing Spiral and K-spiral Fe.

Different species of derivatives are illustrated

<img file="MX348166B_D0046.tif" />

DART containing E-spiral and K-spiral Fe that can

<img file="MX348166B_D0047.tif" />

be formed through chain exchange.

IMPI *

INSTTTUTO MSXICANO PE! .A PROHEDAI 'irwsTRiAt

Figure 40. Exclusion chromatography of f'állldTlJ in E-coil and / or K-coil derivatives and derivatives containing Fe E-coil and / or K-coil of DARTS h2B6YAhCB3.

Size exclusion chromatography on E-coil and / or K-coil derivatives and derivatives containing Fe E-coil and / or K-coil of DARTS h2B6YAhCB3 is shown. Four species of each molecule were analyzed; all had an E-coil and a K-coil: EK (no Fe region), 2.1 mg; EFc / K (E-spiral bound Fe), 2.7 mgs; E / KFc (K-spiral bound Fe), 1.8 mgs; EFc / KFc (K-spiral bound Fe and Spiral of the same DART), 2.0 mg.

Figure 41. Structure of produced dimer molecules.

The possible structure of the produced dimer molecule is shown and explained in size exclusion chromatography of Figure 40.

Figure 42. Electropractic polyacrylamide SDS gel analysis of E-coil and / or K-coil derivatives and derivatives containing Fe E-coil and / or K-coil of DARTS h2B6YAhCB3.

The results of an SDS-polyacrylamide gel electrophoretic analysis of the fractions obtained from the size exclusion chromatography (Figure 40) of derivatives of E-spiral and / or K-spiral and of derivatives containing

Rails

Faith

E-spiral and / or K-spiral

IMPI

Mexican wllHyto La moreda i

<img file="MX348166B_D0048.tif" />

by DART h2B6YAhCB3.

flanking: molecular marker controls;

Lane 1: EK (no Fe region); Lane 2: EFc / K, aggregate fraction; Lane 3: EFc / K, monomer fraction; Lane4:

E / KFc, aggregate fraction; Lane 5: E / KFc, monomer fraction; Lane 6: EFc / KFc, aggregate fraction; Lane7:

EFc / KFc, dimer fraction; Lane 8: EFc / KFc, monomer fraction.

Figure 43. Specific binding ELISA analysis.

The result of a specific binding ELISA comparing h2B6YAhCB3 DART derivatives containing Fe E-coil / K-coil (EFc / K or E / KFc), DART h2B6YAhCB3, control and a DART derivative EFc / KFc h2B6YAhCB3 is shown.

Figure 44. Ability of DART h2B6YAhCB3 Spiral and / or K-coil derivatives and Fe-containing derivatives E-coil and / or K-coil to inhibit T cell proliferation

The ability of E-coil and / or K-coil derivatives and Fe containing E-coil and / or K-coil derivatives of DART h2B6YAhCB3 to inhibit T cell proliferation is shown.

Figure 45. ABD-DART hCD16-hCD32B.

Shown is a schematic of a recombinant antibody molecule, hCD16- hCD32B ABD-DART composed of the ABD3 domain of fused streptococcal G protein vs IMPIAS

MEJtlCANC INSTmjTO

DI LA MOHEDAL J #

INniVntAt. WgE with a recombinant bispecific DART that immunoreactive fcs with hCD16 and HCD32B antigens.

Figures 46A-46B. HCD16hCD32B ABD-DART binding affinity using dual specific ELISA.

ELISA plates were coated with either CD16 antigen (Figure 46A) or human serum albumin (Figure 46B) at a concentration of 2 pg / ml. Varying concentrations of hCD16-h.CD32B ABD-DART () and control hCD16hCD32B DART (o) starting at 2 pg / ml were pooled. Biotinylated SCD32B antigen was added to the plate followed by HRP-conjugated Streptavidin for detection.

Figure 47. PBMC-mediated cytotoxicity of DART proteins.

The PBMC-mediated cytotoxicity of DART proteins. ADCC assays were carried out using human B cell lines, Daudi as target cells incubated with PBMC as effector cells. The individual assays were done in triplicate in a 20: 1 effector-to-target ratio and a tritation of antibodies: hCD16A-hCD32B DART (·) and hCD16A -hCD32B ABD DART (). Cell-mediated cytotoxicity was measured through the LDH release assay. The curve below 10 ° is hCD16A-hCD32B ABD DART ().

Figure 48. Enhanced pharmacokinetic properties of hCD 16-hCD32B ABD-DART in C57B1 / 6 mice.

The mice (n = 3) were injected with a single injection ”IMPI ^ s instituto mexjcano k

OF PROPERTY Z

Intravenous INrumiAl of (A) hCD16- hCD32B ABD-DART (·) and (B) hCD16hCD32B DART (A) at 5 mg / kg. Mouse serum was collected at various points in time and serum protein concentrations were quantified by ELISA. Pharmacokinetic calculations were carried out using WinNonlin Professional 5.1.

Figures 4 9A-4 9E. HER2 x TCRb DART activity in panel of low HER2 expressing cell lines.

DART molecules that have Her2 and T cell receptor (TCR) binding domains were tested for their ability to mediate cytotoxicity in multiple breast cancer, colon cancer, and bladder cancer cell lines that had previously been characterized as exhibiting low levels of HER2 expression (and thus being refractory to treatments with the antiHer2 / neu antibody, Herceptin®. The breast cancer cell lines tested are ZR75-1 (HER2 2+) (Figure 49A), MCF-7 (HER2 1+) (Figure 49B) and MDA-MB468 (HER2-ve) (Figure 49C). The non-breast cancer cell lines tested are HT-29 (colon cancer cell line) (Figure 49D) and SW780 (bladder cancer cell line) (Figure 49E).

Detailed description of the invention

Each polypeptide chain of the diabody molecule comprises a VL domain and a VH domain, which are covalently linked such that the domains are ⁸⁰ IMPI n * STI! A&gt; «ΜΚΑΝΟ rFUPSOMtoAD INDIÍSTRIAL Restricted Self-Assembly. The interaction of two of the polypeptide chains will produce two VLVH pairings, forming two epitope binding sites, ie, one divalent molecule. Neither of the VL or VL domains is limited to any position within the polypeptide chain, that is, restricted to the amino-terminal (N) or carboxy (C), and to the restricted domains in their positions relative to each other, that is, the domain VL can be Nterminal to domain VH and vice versa. The only restriction is that a complementary polypeptide chain is available in order to form a functional diabody. When the VL and VH domains are derived from the same antibody, the two complementary polypeptide chains can be identical. For example, when the binding domains are derived from an antibody specific for the A epitope (i.e., the binding domain is formed from a VL interaction_TO-VH_TO), each polypeptide will result in the formation of two VL binding sites_TO-VH_TO, resulting in a bivalent monospecific antibody. When the VL and VH domains are derived from antibodies specific for different antigens, the formation of a functional bispecific diabody requires the interaction of two different polypeptide chains, that is, the formation of a heterodimer. For example, for a bispecific diabody, a polypeptide chain will comprise

<img file="MX348166B_D0049.tif" />

VL_to and a VL_b; homodimerization of such a chain will give as

<img file="MX348166B_D0050.tif" />

resulted in the formation of two sites of ηηίδη ~ Λ7Έ_Α^ ΗΒΤΎ * ~ ^ β ^ - non-binding or unpredictable binding. In contrast, when two different polypeptide chains are free to interact, for example in a recombinant expression system, one comprises VL_TO and a VH_B and the other comprises VL_b and VH_to, two different binding sites will form: VL_TO-VH_TO and VIj_B-VHb. For all pairs of diabody polypeptide chains the possibility of a misalignment or a bad union of the two chains is a possibility, that is, the interaction of the domain VL-VL or VH-VH; however, purification of functional diabodies is easily handled based on the immunospecificity of the appropriately dimerized binding site using any affinity-based method known in the art or exemplified herein, eg, affinity chromatography.

In other embodiments, one or more of the diabody polypeptide chains comprise an Fe domain. Fe domains on the polypeptide chains of diabody molecules preferably dimerize, resulting in the formation of a diabody molecule that exhibits type properties. immunoglobulin, eg, Fc-Fc and R interactions. Fe comprising a diabodies can be dimers, for example, comprised of two polypeptide chains, each comprising a VH domain, a VL domain, and an Fe domain. Dimerization of such polypeptide chains gives as

<img file="MX348166B_D0051.tif" />

IMPI i wrmTv mlxican · of u non e mu indi'STuiai resulted in a divalent diabody comprising an Fe domain, despite having a different structure from that of an unmodified divalent antibody (Figure 11). Such diabody molecules will exhibit altered phenotypes relative to wild-type immunoglobulin, eg, altered serum half-life, binding properties, etc. In other embodiments, the diabody molecules comprising Fe domains can be tetramers. Such tetramers comprise two heavier polypeptide chains, i.e., a polypeptide chain, comprising a VL, a VH, and a Fe domain, and two lighter polypeptide chains, i.e., a polypeptide chain comprising a VL and VH. . Such lighter and heavier chains interact to form a monomer, and such monomers interact through their unpaired Fe domains to form an Ig-like molecule. Such an Ig-type diabody is tetravalent and can be monospecific, bispecific, or tetra-specific.

At least two binding sites on the diabody molecule can recognize the same or different epitopes. The different epitopes can be from different antigens or epitopes from different antigens. In one embodiment, the epitopes are from different cells. In another embodiment, the epitopes are cell surface antigens on the same cell or virus. Epitope binding sites can

<img file="MX348166B_D0052.tif" />

recognize any antigen from which Ufi 'antibody can be generated. For example, proteins, nucleic acids, bacterial toxins, cell surface markers, autoimmune markers, viral proteins, drugs, etc. In particular aspects, at least one diabody epitope binding site is specific for an antigen on a particular cell, such as a B cell or T cell, a phagocytic cell, a natural killer (NK) cell, or a dendritic cell.

Each domain of the diabody polypeptide chain, ie, the VL, VH, and FC domain can be separated through a peptide linker. The peptide linker can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. In certain embodiments, the amino acid linker sequence is GGGSGGGG (SEQ ID NO: 10) encoded by the nucleic acid sequence (SEQ ID NO: 74).

In certain embodiments, each polypeptide chain of the diabody molecule is modified to comprise at least one cysteine ​​residue that will interact with a counterpart on at least one cysteine ​​residue in a second polypeptide chain of the invention to form linkage of interchain disulfide. Such inter-chain disulfide bonds serve to stabilize the diabody molecule, improving expression and recovery in recombinant systems, resulting in a formulation

<img file="MX348166B_D0053.tif" />

Consistent stable as well as improving -stability of the isolated and / or purified product in vivo. Such at least one cysteine ​​residue can be introduced as a single amino acid or as part of a larger amino acid sequence, eg, the hinge domain, in any portion of the polypeptide chain. In a specific embodiment, the at least one cysteine ​​residue is modified to occur at the C-terminus of the polypeptide chain. In some embodiments, the at least one cysteine ​​residue is introduced into the polypeptide chain within the LGGC amino acid sequence. In a specific embodiment, the C-terminus of the polypeptide chain comprising the diabody molecule of the invention comprises the amino acid sequence LGGC. In another embodiment, the at least one cysteine ​​residue is introduced into the polypeptide within an amino acid sequence comprising a hinge domain, for example SEQ ID NO: 1 or SEQ ID NO: 4. In a specific embodiment, C -terminal of a polypeptide chain of the diabody molecule of the invention comprises the amino acid sequence of an IgG hinge domain, for example SEQ ID NO: 1. In another embodiment, the C-terminus of a polypeptide chain of a diabody molecule of the invention comprises the amino acid sequences VEPKSC (SEQ ID NO: 77), which can be encoded through the nucleotide sequence (SEQ ID NO: 78). In other modalities, the at least⁸⁵

INSTITUTO MEXICANi '

OF THE PROPERTY

INDUSTRIAL 'a cysteine ​​residue is introduced into the polypeptide chain of the amino acid sequence LGGCFNRGEC (SEQ ID NO: 17), which can be encoded through the nucleotide sequence (SEQ ID NO: 76). In a specific embodiment, the C-terminus of a polypeptide chain comprising the diabody of the invention comprises the amino acid sequence LGGCFNRGEC (SEQ ID NO: 17), which can be encoded through the nucleotide sequence (SEQ ID NO: 76). In still other embodiments, the at least one cysteine ​​residue is introduced into the polypeptide chain within the amino acid sequence FNRGEC (SEQ ID NO: 23), which can be encoded through the nucleotide sequence (SEQ ID NO: 75 ). In a specific embodiment, the C-terminus of the polypeptide chain comprising the diabody of the invention comprises the amino acid sequence FNRGEC (SEQ ID NO: 23), which can be encoded through the nucleotide sequence (SEQ ID NO: 75).

In certain embodiments, the diabody molecule comprises at least two polypeptide chains, each of which comprises the LGGC amino acid sequence and are covalently linked via a disulfide bond between cysteine ​​residues in LGGC sequences. In another specific embodiment, the diabody molecule comprises at least two polypeptide chains, one of which comprises the sequence FNRGEC (SEQ ID NO: 23) while the other comprises a hinge domain (containing at least one cysteine ​​residue) , wherein at least two polypeptide chains are covalently linked through a disulfide bond between the cysteine ​​residue in FNRGEC (SEQ ID NO: 23) and the cysteine ​​residue in the hinge domain. In particular aspects, the cysteine ​​residue responsible for the disulfide bond located in the hinge domain is Cys-128 (as listed according to Kabat EU; located in the hinge domain of an intact, unmodified IgG heavy chain) and the Cysteine ​​residue in the counterpart in SEQ ID NO: 23 is Cys-214 (as listed with Kabat EU; located at the C-terminus of an intact, unmodified IgG light chain) (Elkabetz et al. (2005) Cysteines In CH1 Underlie Retention Of Unassembled Ig Heavy Chains, J. Biol. Chem. 280: 14402-14412; incorporated herein by reference in its entirety). In still other embodiments, the at least one cysteine ​​residue is modified to occur at the N-terminus of the amino acid chain. In still another embodiment, the at least one cysteine ​​residue is modified to occur in the linker portion of the polypeptide chain of the diabody molecule. In additional embodiments, the VH or VL domain is modified to comprise at least one amino acid modification relative to the parent VL or VL domain such that the amino acid modification comprises a substitution of a parent amino acid with cysteine.

AF * 2 *

<img file="MX348166B_D0054.tif" />

The invention encompasses molecule ^ "¹ de-antibody comprising an Fe domain or a portion thereof (eg a CH2 domain, or a CH3 domain). The Fe domain or a portion thereof can be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE, and IgM. In preferred embodiments, the Fe domain (or a portion thereof) is derived from IgG. In specific embodiments, the IgG isotype is IgGll, IgG2, IgG3, or IgG4 or an allotype thereof. In one embodiment, the diabody molecule comprises an Fe domain, which domain comprises a CH2 domain and a CH3 domain independently selected from any immunoglobulin isotype (i.e., an Fe domain comprising the IgG-derived CH2 domain and a CH3 domain derived from IgE, or the CH2 domain derived from IgGl and a CH3 domain derived from IgG2, etc.). Such Fe domain can be modified into a polypeptide chain comprising the diabody molecule of the invention at any position relative to other domains or portions of the polypeptide chain (for example, the Fe domain, or one of its portions, may be c-terminal for both VL and VH domains of the chain polypeptide, can be N-terminal for both VL and VH domains; or it can be N-terminal for one domain and C-terminal for another (ie, between two domains of the polypeptide chain)).

<img file="MX348166B_D0055.tif" />

The present invention also encompasses molecules that comprise a hinge domain. The hinge domain is derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE, and IgM. In preferred embodiments, the hinge domain is derived from IgG, where the IgG isotype is IgGl, IgG2, IgG3, or IgG4, or one of their allotypes. The hinge domain can be modified into a polypeptide chain comprising the diabody molecule together with an Fe domain such that the diabody molecule comprises a hinge-Fc domain. In certain embodiments, the hinge domain and Fe are independently selected from any immunoglobulin isotype known in the art or exemplified herein. In other embodiments the hinge domain and Fe are separated into at least one other domain of the polypeptide chain, eg, the VL domain. The hinge domain, or optionally the hinge-Fc domain can be modified in a polypeptide of the invention at any position relative to other domains or portions of the polypeptide. In certain embodiments, a polypeptide chain of the invention comprises a hinge domain, which hinge domain is at the C-terminus of the polypeptide chain, wherein the polypeptide chain does not comprise an Fe domain. In still other embodiments, A polypeptide chain of the invention comprises a hinge-Fc domain, which domain of

IMPI

INSTITUTO MEXICANO r&gt; t LA PÍOP1TDAD ΙΝΓΊΚΤΕΙΑΙ.

<img file="MX348166B_D0056.tif" />

hinge-Fc is at the C-terminus of the polypeptide chain.

In further embodiments a polypeptide chain of the invention comprises a hinge-Fe domain, which hinge-Fc domain is at the N-terminus of the polypeptide chain.

As explained above, the invention encompasses polypeptide chain multimers, each of the polypeptide chains comprising a VH and VL domain. In certain aspects, the polypeptide chains in such multimers further comprise an Fe domain. The dimerization of the Fe domains leads to the formation of a diabody molecule that exhibits immunoglobulin-like functionality, i.e., Fe-mediated function (e.g. , Fc-Fc and R interaction, whole binding, etc.). In certain embodiments, the VL and VH domains comprise each polypeptide chain that has the same specificity, and such a diabody molecule is bivalent and monospecific. In other embodiments, the VL and VH domains comprise each polypeptide chain that has a different specificity and the diabody is bivalent and bispecific.

In still other embodiments, the diabody molecules of the invention encompass polypeptide chain tetramers, each polypeptide chain comprising a VH and VL domain. In certain embodiments, two tetramer polypeptide chains further comprise an Fe domain. The tetramer⁹⁰ IMPIAS

MEXICAN INSTITUTE

OF THE PROPERTY 7&gt; ι ^ 33ύ £ INrRISTRIAI V. »ΓΤ therefore is comprised of two heavier polypeptide chains each comprising a VL domain, VH and Fe, and two lighter polypeptide chains, comprising a VL and VH domain . The interaction of a heavier and lighter chain on a coupled divalent monomer with dimerization of the monomers through the Fe domains of the heavier chains will lead to the formation of a tetravalent immunoglobulin-like molecule (exemplified in Example 6.2 and in Example 6.3). In certain respects the monomers are the same, and the tetravalent diabody molecule is monospecific or bispecific. In other respects the monomers are different, and the tetravalent molecule is bispecific or tetra-specific.

The formation of a tetra-specific diabody molecule as described supra requires the interaction of four different polypeptide chains. Such interactions are difficult to achieve efficiently within a single cell recombinant production system, due to the many potential chain mismatch variants. One solution to increase the probability of mismatches is to modify button-like mutations into holes in the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization. For example, with respect to Fc-Fc interactions, a substitution-

<img file="MX348166B_D0057.tif" />

interactions, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group that forms a 'button', for example tryptophan) can be introduced into the CH2 or CH3 domain in such a way that spherical interference will prevent interaction with a similarly mutated domain and will force the mutated domain to pair with a domain whose complementary, or adaptive mutation has been modified, i.e. 'the hole' (for example, a glycine substitution). Such groups of mutations can be modified in any pair of polypeptide chains in the pair. Methods for modifying proteins to promote heterodimerization over homodimerization are well known in the art, particularly with respect to modifying immunoglobulin-like molecules, and are encompassed herein (see, for example, Ridgway et al. (1996) 'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, Protein Engr. 9: 617-621, Atwell et al. (1997) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. MoL. Biol. 270: 26-35, and Xie et al. (2005) A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis, J. Immunol. Methods 296 ^ ΒΙΟΙ; each of which is incorporated herein by reference in its entirety).

₉two EMPI

INSTITUTO MÍtlCAMO • E LA FROPIFDaD industrial

The invention also encompasses diabody molecules comprising the variant Fe domain or the variant hinge-Fc domain (or a portion thereof), the variant Fe domain of which comprises at least one amino acid modification (eg substitution, insertion, deletion) relative to a comparable wild-type Fe domain or hinge Fe domain (or a portion thereof). Molecules comprising variant Fe domains or hinge-Fc domains (or a portion thereof) (eg, antibodies) typically have altered phenotypes relative to molecules comprising the wild-type Fe domain or hinge domains. -Fc or its portions. The variant phenotype can be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function as assayed in an NK-dependent or macrophage-dependent assay. The Fe domain variants identified as altered effector function are described in International Application WO04 / 063351, US Patent Application 2005/0037000 and 2005/0064514, US Provisional Application 60 / 626,510, filed November 10, 2004, 60 / 636,663, filed December 15, 2004, and 60 / 781,564, filed March 10, 2006, and US Patent Application 11/271, 140, filed on November 10, 2005, and

<img file="MX348166B_D0058.tif" />

11 / 305,787, filed on December 15, 2005, ₃3 MEXICAN INSTTTUTq · 'LA «OPfiüA Γ, fnn stp। _{At t} Inventors' concurrent applications -, - each of which is incorporated herein by reference in its entirety.

The bispecific diabodies of the invention can simultaneously bind two separate and different epitopes. In certain embodiments the epitopes are from the same antigen. In other embodiments, the epitopes are from different antigens. In preferred embodiments, at least one epitope binding site is specific for a determinant expressed on an immune effector cell (e.g., CD3, CD16, CD32, CD64, etc.) that are expressed on T lymphocytes, natural killer cells ( NK) or other mononuclear cells. In one embodiment, the diabody molecule binds to the effector cell determinant and also activates the effector cell. In this regard, the diabody molecules of the invention may exhibit self-independent Ig-like functionality further comprising an Fe domain (e.g., as assayed in any effector function assay known in the art or exemplified herein (e.g. example, ADCC assay). In certain embodiments, the bispecific diabody of the invention binds to both a cancer antigen on a tumor cell and a determinant on the effector cell while activating the cell. In alternative embodiments, the bispecific diabody or the diabody molecule of the invention can inhibit the activation of a target, for example,_S4 íMSTrTTrTQ MlUlCANG -. 2

RE THE MYOFIETY 'W7 -,' st_R|_{TO THE} effector cell via q-ί binding, v of this binding an activating and inhibitory receptor in the same cell (e.g., binding of both CD32A and CD32B, BCR and CD32B, or IgER1 and CD32B) as described supra (see, Background of the Invention Section). In a further aspect of this embodiment, the bispecific diabody can exhibit anti-viral properties through the simultaneous binding of two neutralizing epitopes on a virus (eg, RSV epitopes; WNV epitopes such as E16 and E53).

In certain embodiments, the bispecific diabody molecules of the invention offer unique opportunities to activate specific cell types. For example, the bispecific diabody or the diabody molecule can be modified to comprise a combination of epitope binding sites that recognize a group of antigens unique to a target cell or tissue type. Additionally, when either or both of the individual antigens are widely separated in common with other tissues and / or cell types, the low-skill binding domains can be used to build the diabody or the diabody molecule. Such low affinity binding domains will be unable to bind to the individual epitope or antigen with sufficient avidity for therapeutic purposes. However, when both epitopes or antigens are present on a single target cell or tissue, the avidity of the diabody or the

ΙΜΡΙ «^

INHTHrrOMMtCANC

OF THE mOFIEDAI

INr&gt;! &lt;STTUA | The diabody for the target cell, relative to a cell or tissue expressing only one of the 'antigens, will increase such that the cell or tissue can be effectively activated through the invention. Such a bispecific molecule may exhibit improved binding to one or both of its target antigens in cells expressing either antigens relative to the monospecific diabody or an antibody with a specificity to only one of the antigens.

Preferably, the binding properties of the diabodies of the invention are characterized by in vitro functional assays to determine the binding activity and / or one or more functions of the effector cell of the mediator.

FcyR (mediated through Fc-FcyR interactions or through immunospecific binding of a diabody molecule to a

FcyR) (See Section 5.4.2 and 5.4.3). The affinities and binding properties of the molecule, eg diabodies, of the invention for an FcyR can be determined using in vitro assays (biochemical or immunological based assays) known in the art to determine domain-antigen binding or interactions. Fc-FcyR, that is, the specific binding of an antigen to a binding domain or the specific binding of an Fe region to an FcyR, respectively, including but not limited to the ELISA assay, to the surface plasmon resonance test,

ΙΜΡΪ ^ MEXICAN INSTITUTE

OF ΙΛ OWNER INDST »Ia | immunoprecipitation (See Section 5.4.2). In more preferred embodiments, the molecules of the invention have similar binding properties in in vivo models (such as those described and explained herein) to in vitro-based assays. However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro-based assays but exhibit the desired phenotype in vivo.

In some embodiments, the molecules of the invention are modified to comprise an altered glycosylation pattern or an altered glycoform relative to the comparable portion of the template molecule. Modified glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing effector function. The modified glycoforms can be generated by any method known to those skilled in the art, for example by using modified or variant expression strains, through co-expression with one or more enzymes, for example DI Nacetylglucosaminyltransferase III (GnTIll ), through the expression of a diabody of the invention in various organisms or cell lines of various organisms, or through a modifying carbohydrate (s) after the diabody has been expressed and purified. Methods for generating modified glycoforms are known in the art and

IMPI

TNSTTPTTO MUiCANO

OF THE IBUSTRIAN MORITY

<img file="MX348166B_D0059.tif" />

include but are not limited to those described in Umana et al.

(1999) Engineered Glycoforms Of An Antineuroblastoma IgGl

With Optimized Antibody-Dependent Cellular Cytotoxic

Activity, Nat. Biotechnol 17: 176-180; Davies et al. (2001) Expression Of GnTIII In A Recombinant Anti ~ CD20 CHO Production Cell Line: Expression Of Antibodies With Alterad

Glycoforms Leads To An lacrease In Adcc ​​Through Higher

Affinity For Fe Gamma RUI, Biotechnol Bioeng 74: 288-294;

Shields et al. (2002) Lack Of Fucose On Human IgGl N-Linked

Oligosaccharide Improves Binding To Human Fcgamma RUI And Antibody-Dependent Cellular Toxicity, J Biol Chem 277: 2673326740; Shinkawa et al. (2003) The Absence Of Fucose But Not The Presence Of Galactose Or Bisecting N-Acetylglucosamine Of Human IgGl Complex-Type Oligosaccharides Shows The Critical Role Of Enhancing Antibody-Dependent Cellular Cytotoxicity, J Biol Chem 278: 3466-3473) US 6,602,684; USSN 10 / 277,370; USSN 10 / 113,929; PCT WO 00 / 61739A1; PCT WO 01 / 292246A1; PCT WO 02 / 311140A1; PCT WO 02 / 30954A1; Potillegent ™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb ™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, for example, WO 00061739; EA01229125; US 20030115614; Okazaki et al. (2004) Fucose Depletion From Human IgGl Oligosaccharide Enhances Binding Enthalpy And Association Rate Entre IgGl And FcGammaRIIIA,

ΙΜΤΙΠΠΌ MEXICAN ΟΕ LA MOREDA D

JMB, 336: 1239-49 each of which is incorporated herein by reference in its entirety.

The invention further encompasses the incorporation of non-natural amino acids to generate the diabodies of the invention. Such methods are known to those skilled in the art such as those that use natural biosynthetic machinery to allow the incorporation of unnatural amino acids into proteins, see, for example, Wang et al. (2002) Expanding The Genetic Cede, Chem. Comm. 1: 111; Wang et al. (2001) Expanding The Genetic Code Of Escherichia coli, Science, 292: 498-500; van Hest et al. (2001) Protein-Based Materials, Toward A New Level Of

Structural Control, Chem. Comm. 19: 1897-1904, each of which is incorporated herein by reference in its entirety. Alternative strategies focus on the enzymes responsible for amino acyl-tRNA biosynthesis, see, for example, Tang et al. (2001) Biosynthesis Of A Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine In An Engineered Bacterial Host, J. Am. Chem. Soc. 123 (44): 1108911090; Kiick et al. (2001) Identification Of An Expanded Set Of Translationally Active Methionine Analogues In Escherichia coli, FEBS Lett. 502 (1-2): 25-30, - each of which is incorporated herein by reference in its entirety.

In some embodiments, the invention encompasses methods for modifying a VL, VH, or Fe domain of a molecule of the ⁹⁹ IMPI ^ í

MU'CANO INSTITUTE OF INDITTRIAL PROPERTY invention by adding or removing a glycosylation site. Methods for modifying the carbohydrate of proteins are well known in the art and are encompassed within the invention, see, for example, US Pat.

No. 6,218,149; EP 0 359 096 Bl; US Publication No. US 2002/0028486; WO 03/035835; US Publication No. 2003/0115614; US Patent No. 6,218,149; US Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety.

The diabody molecules of the present invention can be constructed to comprise a domain that is a binding ligand for the natural killer group 2D receptor (NKG2D). Such binding ligands, and particularly those not expressed in normal cells, include the histocompatibility molecule 60 (H60), the retinoic acid early inducible gene-1 product (RAE-I), and the type 1 transcript. murine UL16 binding protein (MULT1) (Raulet DH (2 003) Roles Of The NKG2D Imunoreceptor And Its Ligands, Nature Rev. Immunol. 3: 781-790; Coudert, JD et al. (2005) Altered

NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells, Blood 106: 17111717). Additional human NKG2D reactive ligands include the MHC class I chain-related polymorphic MICA molecules and MICB (Diefenbach, A. et al. (1999)

100

ZMPI

MSTIIIITU ΜβΟΟΛΝΟ DELA PROPERTY

IND'JSTWIAJ.

<img file="MX348166B_D0060.tif" />

Natural Killer Cells: Stress Out, Turn On, Tune In, Curr.

Biol. 9 (22): R851-R8533; Bauer, S. et al. (1999) Activation

Of NK Cells And T Cells By NKG2D, A Receptor For StressInducible MICA, Science 285 (5428): 727-729; Stephens, HA (2001) MICA and MICB genes: can the enigma de lair polymorphism be resolved ?, Trends Immunol. 22: 378-385.

The MICA sequence is SEQ ID NO: 311:

MGLGPVFLLL AGIFPFAPPG AAAEPHSLRY NLTVLSWDGS VQSGFLTEVH LDGQPFLRCD RQKCRAKPQG QWAEDVLGNK TWDRETRDLT GNGKDLRMTL AHIKDQKEGL HSLQEIRVCE IHEDNSTRSS QHFYYDGELF LSQNLETKEW TMPQSSRAQT LAMNVRNFLK EDAMKTKTHY HAMHADCLQE LRRYLKSGW LRRTVPPMVN VTRSEASEGN ITVTCRASGF YPWNITLSWR QDGVSLSHDT QQWGDVLPDG NGTYQTWVAT RICQGEEQRF TCYMEHSGNH STHPVPSGKV LVLQSHWQTF HVSAVAAAAI FVIIIFYVRC CKKKTSAAEG PELVSLQVLD QHPVGTSDHR DATQLGFQPL EGA MSDLGSTGST

The sequence of MICB is SEQ ID NO: 312:

PHSLRYNLMV LSQDGSVQSG FLAEGHLDGQ PFLRYDRQKR RAKPQGQWAE DVLGAKTWDT ETEDLTENGQ DLRRTLTHIK DQKGGLHSLQ EIRVCEIHED SSTRGSRHFY YDGELFLSQN LETQESTVPQ SSRAQTLAMN VTNFWKEDAM KTKTHYRAMQ ADCLQKLQLP PMVNVICSEV SEGNITVTCR ASSFYPRNIT LTWRQDGVSL SHNTQQWGDV LPDGNGTYQT WVATRIRQGE EQRFTCYMEH SGNHGTHPVP SGKALVLQSQ RTDFPYVSAA MPCFVIIII LCVPCCKKKTS AAEGP

Antibodies that specifically bind to the T cell receptor include the anti-TCR BMA antibody. ₁₀₁ IMPI ^ mkican institute · DE LA ntOVIEDA »INDUSTRIAL

031 (Kurrle, R. et al. (1989) BMA 031 -A TCR-Sp ^ cific Monoclonal Antibody For Clinical Application, Transplant Proc. 21 (1 Pt 1): 1017-1019; Nashan, B. et al. (1987) Fine Specificity Of A Panel Of Antibodies Against The TCR / CD3 Complex, Transplant Proc. 19 (5): 4270-4272, - Shearman, CW. Et al. (1991) Construction, Expression, And Biologic Activity Of Murine / Human Chimeric Antibodies With Specificity For The Human α / β T Cell, J. Immunol. 146 (3): 928-935; Shearman, CW. Et al. (1991) Construction, Expression And Characterization of Humanized Antibodies Directed Against The Human α / β T Cell Receptor, J. Immunol. 147 (12): 4366-4373). Antibodies that specifically bind to the NKG2D receptor include KYK2.0 (Kwong, KY et al. (2008) Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic

Activity, J. Mol. Biol. 384: 1143-1156; and PCT / US09 / 54911).

Through the use of such an antibody, the target cells are now redirected to be a cell that can bind through cells that make up the receptor (NKG2D). The NKG2D receptor is expressed in all natural human (and other mammalian) killer cells (Bauer,

S. et al. (1999) Activation Of NK Cells And T Cells By

NKG2D, A Receptor For Stress-Inducible MICA Science

285 (5428): 727-729, - Jamieson, AM et al. (2002) The Role Of

The NKG2D Immunoreceptor In Immune Cell Activation And

IMPI rwsTrnrro mbucano Dt INDUSTRIAL PROPERTY

19-29) as well as cells

<img file="MX348166B_D0061.tif" />

102

Natural Killing, Immunity 17 (1): T CD8⁺ (Groh, V. et al. (2001) Cells By NKG2D Via Engagement

Costimulation Of CD8aβ T

By MIC Induced On VirusInfected Cells, Nat. Immunol. 2 (3): 255-260, - Jamieson, AM

et al. (2002) The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing Immunity 17 (1): 19-29).

Alternatively, the diabody molecules of the present invention can be constructed to comprise a domain that is a binding ligand for the T cell receptor (TCR). TCR is natively expressed through CD4 + or CD8 + T cells, and enables such cells to recognize antigenic peptides that are bound and presented by MHC class I or class II proteins of antigen-presenting cells. Recognition of a pMHC (peptide-MHC) complex through TCR initiates the propagation of a cellular immune response leading to cytokine production and lysis of the antigen-presenting cell (see, for example, Armstrong, KM et al. . (2008) Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide-MHC Complexes, Biochem. J. 415 (Pt 2): 183-196, - Willemsen, R. (2008) Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy, Cytometry A. 73 (11): 1093-1099, - Beier, KC et al. (2007) Master Switches Of T-Cell Activation And Differentiation, Eur. Respir. J. 29: 804-812; Mallone, R. et

103

ΙΝΓΤΤΠΓΓΟΜβΚΛΝΟ 'C ^ Sz ^' Xj DE LA FROFIEDAO al. (2005) Targeting T Lymphocytes For Immune iVSn ^^ kring ^ And Intervention In Autoimmune Diabetes, Am. TT. '¹ΤΪΤ5I ·. T2 (b): 5 3 4 550).

By constructing such diabody molecules to further comprise at least one epitope binding domain capable of binding to, for example, a receptor present on the surface of a target cell, such diabody molecules will be DART molecules and thus are capable of binding to target cells and thereby causing the target cells to describe the binding ligand for the natural killer group receptor 2D (NKG2D) or the TCR (whichever is present in the target cell-binding diabody) (see, for example, Germain, C. et al. (2008) Redirecting NK Cells Mediated Tumor Cell Lysis By A New Recombinant Bifunctional Protein, Prot. Engineer. Design Selection 21 (11): 665-672).

Such diabodies can be used to redirect any desired target cell into a cell that is either NK cell mediated cell lysis or T cell mediated cytotoxicity. In one embodiment, the epitope-binding domain of the diabody capable of binding to a receptor present on the surface of the target cell is an epitope that binds to a tumor-associated antigen to thereby redirect cancer cells into substrates for cell lysis. NK cell mediated or cytotoxicity mediated ¹⁰⁴ ΣΜΡΙ

ΙΝΠΤΤυΤΟ MEXICANO DE LA EKOriEDAP IND''STX1AI by the T cell. Of particular interest are the tumor associated antigens which is a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen, a colon cancer antigen, a testicular cancer antigen, a glioblastoma antigen, an antigen associated with a B-cell malignancy, an antigen associated with multiple myeloma, an antigen, associated with non-Hodgkins lymphoma, or an antigen associated with chronic lymphocytic leukemia.

<img file="MX348166B_D0062.tif" />

Suitable tumor associated antigens for which use include A33 (a colorectal carcinoma antigen; Almqvist, Y. 2006, Nucí Med Biol. Nov; 33 (8): 991998); B1 (Egloff, AM et al. 2006, Cancer Res. 66 (1): 6-9), BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2 (6): 577-84), beta-catenin (Frange W. et al. 2003 J Pathol. 201 (2): 250-9), CA125 (Bast, RC Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3: 274-81); CD5 (Calin, GA et al. 2006 Semin Oncol. 33 (2): 167-73; CD19 (Troussard, X. et al. 1998 Hematol Cell Ther. 40 (4): 139-48); CD20 (Thomas, DA et al. 2006 Hematol Oncol Clin North Am. 20 (5): 1125-36); CD22 (Kreitman, RJ 2006 AAPS J. 18; 8 (3): E532-51); CD23 (Rosati, S. et al. 2005 Curr Top Microbiol Immunol. 5, -294: 91-107); CD25 (Troussard, X. et al. 1998 Hematol Cell Ther. 40 (4): 139-48), - CD27

IMPI

Mexican WSIIIVtO

OF THE PRWlF.DAn INDUSTRIAL

91 (9): 1234-40); CD28 (Bataille, R. 2006 Haematologica (Bataille, R. 2006 Haematologica 91 (9): 1234-40); CD36 (Ge, Y. 2005 Lab Hematol. 1 1 (1): 31-7), - CD40 / CD154 (Messmer, D. et al. 2005 Ann NY Acad Sci. 1062: 51-60); CD45 (Jurcic, JG 2005 Curr Oncol Rep. 7 (5): 339-46); CD56 (Bataille, R. 2006 Haematologica 91 (9): 1234-40); CD79a / CD79b (Troussard, X. et al. 1998 Hematol Cell Ther. 40 (4): 139-48; Chu, PG et al. 2001 Appl Immunohistochem Mol Morphol. 9 (2) : 97-106); CD103 (Troussard, X. et al. 1998 Hematol Cell Ther. 40 (4): 139-48); CDK4 (Lee, YM et al. 2006 Cell Cycle 5 (18): 2110-4); CEA (carcinoembryonic antigen; Mathelin, C. 2006 Gynecol Obstet Fertil. 34 (7-8): 638-46; Tellez-Avila, FI et al. 2005 Rev Invest Clin. 57 (6): 814-9), - CTLA4 (Peggs, KS et al. 2006 Curr Opin Immunol. 18 (2): 206-13), - EGF-R (epidermal growth factor receptor; Adenis, A. et al. 2003 Bull Cancer. 90 Spec No: S228 -32), · Erb (ErbBl; ErbB3; ErbB4; Zhou, H. et al. 2002 Oncogene 21 (57): 8732-40, - Rimon, E. et al. 2004 Int J Oncol. 24 (5): 1325-38); GAGE (GAGE-I; GAGE-2;

Akcakanat, A. et al. 2006 Int J Cancer. 118 (1): 123-8);

GD2 / GD3 / GM2 (Livingston, PO et al. 2005 Cancer Immunol Immunother. 54 (10): 1018-25), - gplOO (Lotem, M. et al. 2006 J Immunother. 29 (6): 616-27) , - HER-2 / neu (Kumar, Pal S et al. 2006 Semin Oncol. 33 (4): 386-91), - human papillomavirus E6 / human papillomavirus E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66: 125-59; KSA (17-1A) (Ragupathi, G. 2005 Cancer

Treat Res. 123: 157-80); MAGE (MAGE-I; MAGE-3; (Bodey, B. 2002

Expert Opin Biol Ther. 2 (6): 577-84); MART (Kounalakis, N. et al. 2005 Curr Oncol Rep. 7 (5): 377-82, - MUC-I (Mathelin, C. 2006 Gynecol Obstet Fertile. 34 (7-8): 638-46), - MUM-I (Castelli, C. et al. 2000 J Cell Physiol. 182 (3): 323-31), - Nacetylglucosaminyltransferase (Dennis, JW 1999 Biochim Biophys Acta. 6, -1473 (1): 21-34), - pl5 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol. 7 (9): 667-77), - PSA (prostate specific antigen; Cracco, CM. et al. 2005 Minerva Urol Nefrol. 57 (4): 301-1 1); PSMA (Ragupathi, G. 2005 Cancer Treat Res. 123: 157-80); sTn (Holmberg, LA 2001 Expert Opin Biol Ther. 1 (5): 881-91), - TNF receptor (TNF-α receptor, TNF-β receptor; or TNF-γ receptor; van Horssen, R. et al. 2006 Oncologist. 11 (4): 397-408, - Gardnerova, M. et al. 2000 Curr Drug Targets. 1 (4): 327-64); or VEGF receptor (O'Dwyer. PJ. 2006 Oncologist. 11 (9): 992-8).

Additional tumor associated antigens for such use (and publications describing antibodies specifically reactive for such antigens) include ADAM-9 (US Patent Publication No. 2006/0172350; PCT Publication No. WO 06/084075); ALCAM (PCT Publication No. WO 03/093443); Carboxypeptidase M (US Patent Publication No. 2006/0166291); CD46 (US Patent No. 7,148,038;

PCT Publication No.

WO 03/032814); Cytokeratin 8

<img file="MX348166B_D0063.tif" />

107 IMPI trtSTTRrro m * ican DE LA MOREDA D iwdvstuiai (PCT Publication No. WO 03/024191); Ephrin receptors (and in particular EphA2 (US Patent No. 7,569,672; PCT Publication No. WO 06/084226); Integrin Alpha-V-Beta-6 (PCT Publication No. WO 03/087340); JAM-3 (Publication PCT No. WO 06/084078); KID3 (PCT Publication No. WO 05/028498); KID31 (PCT Publication No. WO 06/076584); LUCA-2 (United States Patent Publication No. 2006/0172349; PCT Publication No. WO 06/083852); Oncostatin M (Oncostatin Beta Receptor) (US Patent No.

7,572,896; PCT Publication No. WO 06/084092); PIPA (US Patent No. 7,405,061; PCT Publication No. WO 04/043239); RAAG10 (US Patent No. 7,527,969; PCT Publication No. WO 04/001381); ROR1 (US Patent No. 5,843,749); TES7 (PCT Publication No. WO 08/066691); and the Transferrin Receptor (US Patent No. 7,572,895; PCT Publication No. WO 05/121179).

Also of interest are specific antigens for particular infectious agents, for example viral agents including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza, human papillomavirus (HPV). ), feet and mouth (coxsackie virus), rabies virus, herpes simplex virus (HSV), and causative agents of gastroenteritis, including rotavirus, adenovirus, calici virus, astrovirus, and Norwalk virus; Bacterial agents including, but not limited to,

INCITOJTD MflbCAMO OS FEOMITY limited to, E. coli, Salmonella thyphimurium, ^^ 'Ps'é'udoTtron ^ s aeruginosa, Vibrio cholerae, Neisseria goiióírVKJtíae, Helicobacter pylori, Hemophilus influenzae, Shigella aureusobac eptococcus tuberculosis, and Staphylococcus tuberculosis pneumoniae, fungal agents, and parasites such as Giardi.

Alternatively, such an epitope may bind to an Fe receptor (eg, FcyRI or FcyRII), such as for example to redirect acute monocyclic leukemic cells into substrates for NK cell-mediated cell lysis.

UNION DOMAINS OF THE DIACBODY

The diabodies of the present invention comprise antigen-binding domains generally derived from immunoglobulins or antibodies. The antibodies from which the binding domains used in the methods of the invention are derived can be of any animal origin including birds and mammals (for example humans, non-human primates, murine, donkey, sheep, rabbit, from goat, guinea pig, camel, horse, or chicken). Preferably, the antibodies are human or humanized monoclonal antibodies. As used herein, human antibodies include antibodies that have the amino acid sequence of human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or libraries of human immunoglobulin sequences.

109

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1Ν5ΤΓΠ.ΓΓΟ MMUCAHC Γ · Ε INDUSTRIAL FROHITY

<img file="MX348166B_D0065.tif" />

libraries of synthetic human or mouse immunoglobulin ii * m - i - »B · * --- ^ 1 coding sequences expressing antibodies to human genes.

The invention contemplates the use of any antibody known in the art for the treatment and / or prevention of cancer, autoimmune disease, inflammatory disease, or infectious disease as the source of the binding domains for the diabodies of the invention. Non-limiting examples of known carcinogenic antibodies are provided in section 5.7.1, as well as other specific antibodies for the listed target antigens and antibodies against the carcinogenic antigens listed in section 5.6.1; non-limiting examples of known antibodies for the treatment and / or prevention of autoimmune diseases and inflammatory disease are provided in section 5.7.2, as well as antibodies against the target antigens listed in the antibodies against the antigens listed in section 5.6.2; in other embodiments antibodies to epitopes associated with infectious diseases as listed in section 5.6.3 may be used. In certain embodiments, the antibodies comprise a variant Fe region comprising one or more amino acid modifications, which have been identified by the methods of the invention to have a conferred effector function and / or enhanced affinity for FcyRIIB, and an affinity ¹¹⁰ institute mlx'can '&gt; DE LA FRÍinEOAl' lUPU.craiAl decreased for FcyRIIIA relative to a compared molecule comprising a wild-type Fe region. A non-limiting example of antibodies that are used for the treatment or prevention of inflammatory disorders that can be modified in accordance with the invention are presented in Table 9, and a non-limiting example of antibodies that are used for the treatment or prevention of Autoimmune disorder is presented in Table 10.

For some uses, including in vivo use of human antibodies and in vitro detection assays, it may be preferable to use diabodies with variable domains derived from human, chimeric, or humanized antibodies. Variable domains for fully human antibodies are particularly desirable for the therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, US Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

A humanized antibody is an antibody, a

111

IίΤΤΓΠ.ΤΤ 'Μ SXJCA Ni i ΠΕ LA PROÍAFRAI iíwstrial variant of one of its fragments that is capable of binding to a predetermined antigen and that comprises a backbone region having substantially the amino acid sequence of a human immunoglobulin and a CDR that it has substantially the amino acid sequence of a non-human immunoglobulin. A humanized antibody may comprise substantially all of at least, and typically two, variable domains wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the chain regions. The main ones are that of a human immunoglobulin consensus sequence.

The main chain and regions

CDRs of a humanized antibody need not precisely match the parent sequences, for example, the donor CDR or consensus backbone can be mutagenized through infusion, insertion or deletion of at least one residue such that the CDR or the Backbone residue at the site corresponds to neither consensus nor donor antibody. Such mutations, however, preferably do not spread. Usually, at least 75% of the residues of the humanized antibody will correspond to those in the parent backbone region (FR) and the CDR sequence, generally 90%, and more preferably greater than 95%. Humanized antibodies can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP 239,400; International Publication No. WO 91/09967; and US Patent Nos. 5,225,539, 5,530,101, and 5,585,089), plating or coating (European Patent Nos. EP 592,106 and EP 519,596; Padlan (1991) A Possible Procedure By Reducing The Immunogenicity Of Antibody Variable Domains While Preserving Their Ligand-Binding Properties, Molecular Immunology 28 (4 / 5): 489-498, - Studnicka et al. (1994) HumanEngineered Monoclonal Antibodies Retain Full Specific Binding Activity By Preserving Non-CDR Complementarity-Modulating Residues ”, Protein Engineering 7 (6): 805-814, - and Roguska et al. (1994) Humanization Of Murine Monoclonal Antibodies Through Variable Domain Resurfacing ", Proc Nati Acad Sci USA 91: 969973), chain rearrangement (US Patent No. 5,565,332), and the techniques described in, for example. US Patent Nos. 6,407,213, 5,766,886, 5,585,089, International Publication No. WO 9317105, Tan et al. (2002) 'Super humanized' Antibodies: Reduction Of Immunogenic Potential By Complementarity-Determining Region Grafting With Human Germline Sequences: Application To An Anti-CD28, J. Immunol. 169: 1119-25, Caldas et al. (2000) Design And Synthesis Of Germline-Based Hemi-Humanized Single-Chain Fv Against The CD 18 Surface Antigen '', Protein Eng. 13: 353-60,

INSTnVT * MUUCAN ^ £ '

FROM THE MKW »TO Π,. , INDUSTRY!

Morea et al. (2000) Antibody Modelmg: Implications For Engineering And Design, Methods 20: 267-79, Baca et al. (1997) Antibody Humanization Using Monovalent Phage Display, J. Biol. Chem. 272: 10678-84, Roguska et al. (1996) A Compari son Of Two Murine Monoclonal Antibodies Humanized By CDR-Grafting And Variable Domain Resurfacing, Protein Eng. 9: 895-904, Couto et al. (1995) Designing Human Consensúa Antibodiea With Minimal Positional Templates, Cancer Res. 55 (23 Sup): 5973s-5977s, Couto et al. (nineteen ninety five)

Anti-BA46 Monoclonal Antibody Mc3: Humanization Uaing A Novel Poaitional Consensus And In Vivo And In vitro Characterization, Cancer Res. 55: 1717-22, Sandhu (1994) A Rapid Procedure For The Humanization Of Monoclonal Antibodiea ”, Gene 150: 409- 10, Pedersen et al. (1994) Comparison Of Surface Accessible Residues In Human And Murine Immunoglobulin Fv Domains. Implication For Humanization Of Murine Antibodies, J. Mol. Biol. 235: 959973, Jones et al. (1986) Replacing The Complementarity Determining Regions In A Human Antibody With Those From A Mouse, Nature 321: 522-525, Riechmann et al. (1988) Reshaping Human Antibodies For Therapy, Nature 332: 323-327, and Presta (1992) Antibody Engineering, Curr. Op. Biotech. 3 (4): 394-398. Typically, the backbone residues in the backbone regions will be replaced with the corresponding residue from the antibody

<img file="MX348166B_D0066.tif" />

<img file="MX348166B_D0067.tif" />

114 donor CDR to alter, preferably enhance, antigen binding. These backbone substitutions are identified through methods well known in the art, for example, through modeling the interactions of the CDR and backbone residues to identify backbone residues important for antigen binding and sequence comparison to identify unusual backbone residues at particular positions. (see, for example, Queen et al, US Patent No. 5,585,089; US Publication Nos. 2004/0049014 and 2003/0229208; US Patent Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761; 5,585,089; and 5,530,101 and Riechmann et al. al. (1988) Reshaping Human Antibodies For Therapy, Nature 332: 323-327, all of which are incorporated herein by reference in their entirety).

In a more preferred embodiment, the humanized binding domain specifically binds to the same epitope as the donor murine antibody. It will be appreciated by those skilled in the art that the invention encompasses CDR grafting of antibodies in general. In this way, the donor and acceptor antibodies can be derived from animals of the same species and still the same class or subclass of the antibody. More usually, however, the donor and acceptor antibodies are derived from animals of different species. Typically the donor antibody is a non-human antibody, such as a

115

IMPI ίΚΠΤΤνΤη MEXICAN PE LA PROníUAl. INRUSTRIAL

<img file="MX348166B_D0068.tif" />

rodent mAb, and an acceptor antibody is ^ a, human antibody.

In some embodiments, at least one CDR from the donor antibody is grafted onto the human antibody. In other embodiments, at least two and preferably all three CDRs from each of the heavy and / or light chain variable regions are grafted onto the human antibody. The

CDRs can comprise Kabat CDRs, structural loop CDRs, or a combination thereof. In some embodiments, the invention encompasses a humanized FcyRIIB antibody comprising at least one CDR-grafted heavy chain and at least one CDR-grafted light chain.

The diabodies used in the methods of the invention include derivatives that are modified, that is, through the covalent attachment of any type of molecule to the diabody. For example, but not by way of limitation, diabody derivatives include diabodies that can be modified, for example, through glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization through known protecting / blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of the numerous chemical modifications can be carried out through known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally ^ the derivative may contain one or more non-classical amino acids.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies that have a variable region derived from a non-human antibody and a constant region from human immunoglobulin. Known methods for producing antibodies are known in the art. See, for example, Morrison (1985) Transfectomas Provide Novel Chimeric Antibodies, Science 229: 1202-1207; Oi et al. (1986) Chimeric Antibodies, BioTechniques 4: 214-221; Gillies et al. (1989) High-Level Expression Of Chimeric Antibodies Using Adapted cDNA Variable Region Cassettes, J. Immunol. Methods 125: 191-202; and US Patent Nos. 6,311,415, 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

Generally, the backbone residues in the backbone regions will be replaced with the corresponding residue from the CDR donor antibody to alter, preferably enhance, antigen binding. These backbone substitutions are identified through methods well known in the art, for example, through the modeling of the interactions of the CDR and

IMPI ^ tNSTTRrre Mexican 'Kc ^ qsYes¿' r&gt; E THE PROPERTY

INDUSTRIAL &gt;&gt; r ^ backbone residues to identify backbone residues important for antigen binding and sequence comparison to identify unusual backbone residue at particular positions. (See, for example, US Patent No. 5,585,089; and Riechmann et al. (1988) Reshaping Human Antibodies For Therapy, Nature 332: 323-327, which is incorporated herein by reference in its entirety).

Monoclonal antibodies from which the binding domains of the diabodies of the invention can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination of these. For example, monoclonal antibodies can be produced using hybridoma techniques including those known and taught in the art, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Coid Spring Harbor Laboratory Press, 2^to ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, NY, 1981) (both of which are incorporated by reference in their entirety). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single

<img file="MX348166B_D0069.tif" />

prokaryotic or 3e

118 clone, which includes any eukaryotic clone, phage, and not the method through which it is produced.

Methods for producing and classifying specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell that expresses such antigen.

Once the immune response is detected, for example, for example, antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and the splenocytes are inflated. The splenocytes are then fused through well known techniques to any suitable myeloma cell. Hybridomas are selected and cloned through limiting dilution. The hybridoma clones are then tested by methods known in the art for cells that secrete antibodies capable of binding antigen. Ascites fluid, generally containing high levels of antibodies, can be generated through inoculation of mice intraperitoneally with positive hybridoma clones. Antigens of interest include, but are not limited to, cancer-associated antigens provided in section 5.8.1, autoimmune disease-associated antigens, and inflammatory diseases provided in section 5.8.2, disease-associated antigens

INSTITUTO MEXICANO ΙΈ LA INDI 'STUIAL PROPERTY infectious diseases provided in section 5.8.3, and toxins provided in section 5.8.4.

Antibodies can also be generated using phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequence that encodes them. In a more particular embodiment, such a phage can be used to display antigen-binding domains, such as Fab and Fe, or disulfide-stabilized Fv, expressed from a repertoire or collection of combination antibody (eg, human or murine). Phage expression in the antigen-binding domain that binds to the antigen of interest can be selected or identified with antigen, for example, using a labeled antigen or an antigen bound or captured on a solid surface or granule. The phage used in these methods is a typically filamentous phage, which includes fd and M13. The antigen-binding domains are expressed as a recombinantly fused protein to either a phage III gene or a gene VIII gene. Examples of the phage display method that can be used to make the immunoglobulins, or their fragments, of the present invention include those described in Brinkmann et al. (1995) Phage Display Of Disulfide-Stabilized Fv Fragmenta, J. Immunol. Methods,

<img file="MX348166B_D0070.tif" />

¹²⁰ LMPI .'NflWrOMÜUCANo de LA moheda d INDUSTRIAL _____

182: 41-50; Ames et al. (1995) Conversion Of Murine Fabs

Isolated From A Combinatorial! Phage Display Library To Fu 11

Length Immunoglobulins, J. Immunol. Methods, 184: 177-186; Kettleborough et al. (1994) Isolation Of Tumor CellSpecific Single-Chain Fv From Immunized Mice Using PhageAntibody Librarles And The Re-Construction Of Whole Antibodies From These Antibody Fragments, Eur. J. Immunol., 24: 952-958; Persic et al. (1997) An Integrated Vector System For The Eukaryotic Expression Of Antibodies Or Their Fragments After Selection From Phage Display Libraries ”, Gene, 187: 9-18; Burton et al. (1994) Human Antibodies From Combinatorics! Libraries ", Advances in Immunology, 57: 191-280; PCT Application No. PCT / GB91 / 01134; PCT applications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and US Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

Phage display technology can be used to increase the affinity of an antibody for its antigen. This technique would be useful in obtaining high affinity antibodies. The technology, referred to as affinity maturation, uses mutagenesis or CDR displacement and re-selection using the cognate antigen to

<img file="MX348166B_D0071.tif" />

identify antibodies that bind with a higher affinity to the antigen when compared to the initial or parent antibody (see, for example, Glaser et al. (1992) Dissection Of The Combining Site In A Humanized Anti-Tac Antibody, J. Immunology 149: 2607-2614). Mutagenizing entire codons instead of individual codons results in a semi-random repertoire of amino acid mutations. Libraries can be constructed consisting of a group of variant clones each differing by a single amino acid alteration in a single CDR and containing variants representing every possible amino acid substitution for each CDR residue. Mutants with increased binding affinity for the antigen can be classified through contact of the immobilized mutants with the labeled antigen. Any classification method known in the art can be used to identify mutant antibodies with increased avidity for the antigen (eg ELISA) (see, Wu et al. (1998) Stepwise In vitro Affinity Maturation Of Vitaxin, An Alphav Beta3-Specific Humanized mAb, Proc Nati. Acad Sci. USA 95: 6037-6042; Yelton et al. (1995) Affinity Maturation Of The Br96 Anti-Carcinoma Antibody By Codon-Based Mutagenesis ", J. Immunology 155: 1994-2004). CDR displacement that randomly distributes the light chain is also possible (See Schier et al. (1996) Isolation Of Picomolar Affinity

122

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Anti-C-ErbB-2 Single-Chain Fv By Molecular. Evolution Of The

Complementarity Determining Regions In The Center Of The

Antibody Binding Site, J. Mol. Bio. 263: 551-567).

The present invention also encompasses the use of binding domains comprising amino acid sequences of any of the binding domains described herein known in the art with mutations (eg, one or more amino acid substitutions) in the backbone or the CDR region. Preferably, mutations in these binding domains maintain or enhance the avidity and / or affinity of the binding domains for FcyRIIB to which they immunospecifically bind. Standard techniques known to those of skill in the art (eg, immunoassay) can be used to test the affinity of an antibody for a particular antigen.

. Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody, or one of its fragments, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, resulting in amino acid substitutions. Preferably, the derivatives include less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less.

123 of 2 amino acid substitutions relative to

IMPI iNfiuuít) mMicani

M THE MOHITY

INDUSTRIAL

<img file="MX348166B_D0073.tif" />

original antibody or one of its fragments. In a preferred embodiment, the derivatives have conservative amino acid substitutions made at one or more predicted nonessential amino acid residues.

Diabodies Comprising Epitope Binding Sites that Bind Immunospecifically to FcyRIIB

In a particular embodiment, at least one of the binding domains of the diabodies of the invention agonizes at least one FcyRIIB activity. In one embodiment of the invention, such a B cell receptor-mediated signaling inhibition activity. In another embodiment, the binding domain inhibits B cell activation, B cell proliferation, antibody production, intracellular calcium influx from B cells, cell site advancement, or the activity of one or more downstream signaling molecules in the FcyRIIB signal transduction pathway. In yet another embodiment, the binding domain enhances FcyRIIB phosphorylation or SHIP recruitment. In a further embodiment of the invention the binding domain inhibits MAP kinase activity or Akt recruitment into the B cell receptor-mediated signaling pathway. In another embodiment, the binding domain agonizes mediated inhibition by FcyRIIB is FceRI signaling. In a modality

<img file="MX348166B_D0074.tif" />

In particular, such a binding domain inhibits FceRI-induced mast cell activation, calcium mobilization, dysregulation, cytokine production, or serotonin release. In another embodiment, the binding domain of the invention stimulates FcyRIIB phosphorylation, stimulates SHIP recruitment, stimulates SHIP phosphorylation and its association with Shc, or inhibits activation of MAP kinase family media (e.g. example, Erkl, Erk2, JNK, p38, etc.). In yet another embodiment, the binding domains of the invention enhance tyrosine phosphorylation of p62dok and its association with SHIP and rasGAP. In another embodiment, the binding domains of the invention inhibit FcyR-mediated phagocytosis in monocytes or macrophages.

In another embodiment, the binding domains antagonize at least one FcyRIIB activity. In one embodiment, such activity is the activation of B cell receptor-mediated signaling. In a particular embodiment, the binding domains enhance B cell activity, B cell proliferation, antibody production, intracellular calcium influx, or the activity of one or more downstream signaling molecules in the cell. trajectory of its FcyRIIB signal transduction. In yet another particular embodiment, the binding domains decrease FcyRIIB phosphorylation or SHIP recruitment. In a further embodiment of the invention, the binding domains

125

<img file="MX348166B_D0075.tif" />

they enhance MAP kinase activity or Akt recruitment into the B cell receptor-mediated signaling pathway. In another embodiment, the binding domains agonize FcyRIIB-mediated inhibition of FcsRI signaling. In a particular embodiment, the binding domains enhance FctRI-induced mast cell activation, calcium mobilization, dysregulation, cytokine production, or serotonin release. In another embodiment, the binding domains inhibit FcyRIIB phosphorylation, inhibit SHIP recruitment, inhibit SHIP phosphorylation and its association with Shc, enhance activation of MAP kinase family media (e.g., Erkl, Erk2, JNK, p38, etc.). In yet another embodiment, the binding domains inhibit tyrosine phosphorylation of p62dok and its association with SHIP and rasGAP. In another embodiment, the binding domains enhance FcyR-mediated phagocytosis in monocytes or macrophages. In another embodiment, the binding domains prevent phagocytosis, the clearance of opsonized particles via splenic macrophages.

In other embodiments, at least one of the binding domains can be used to activate the diabodies of the invention to cells expressing FcyRIIB.

In a particular embodiment, one of the binding domains is derived from a mouse monoclonal antibody produced by clone 2B6 or 3H7, which has the numbers of

126

IMPI wrrnvto mBucanc DE M INDUSTRIAL CURRENCY

<img file="MX348166B_D0076.tif" />

access ATCC PTA-4591 and PTA-4592, respectively. Hybridomas producing antibodies 2B6 and 3H7 have been deposited with the American Type Culture Collection (10801 University Blvd., Manassas, VA. 20110-2209) on August 13, 2002 under the provisions of the Budapest Treaty on Recognition International Deposit

Microorganisms for Patent Procedure Purposes, and assigned accession numbers PTA-4591 (for the hybridoma producing 2B6) and PTA-4592 (for the hybridoma producing 3H7), respectively, and are incorporated herein by reference. In a preferred embodiment, the binding domains are human or have been humanized, preferably derived from a humanized version of the antibody produced by clone 3H7 or 2B6.

The invention also encompasses diabodies with binding domains of other antibodies, which specifically bind to FcyRIIB, preferably human FcyRIIB, more preferably native human FcyRIIB, which are derived from clones including but not limited to 1D5, 2E1, 2H9, 2D11, and 1F2 which have accession numbers ATCC, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. The hybridomas producing the clones identified above were deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (10801 University Blvd., Manassas, VA. 20110-2209) on May 7, 2004, and are incorporated herein ™ Mexican rrrrvro ni la rsopieoad

INDirsTRIAL by reference. In preferred embodiments, the binding domains of the antibodies described above are humanized.

In a specific embodiment, the binding domains used in the diabodies of the present invention are from an antibody or one of its antigen-binding fragments (e.g., comprising one or more complementarity determining regions (CDRs), preferably the 6 CDR) of the antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9,

2D11, or 1F2. In another embodiment, the binding domain binds the same epitope as the mouse monoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, respectively, and / or competes with the mouse monoclonal antibody produced from clone clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 as determined, for example, in an ELISA or other appropriate competitive immunoassay, and also binds to FcyRIIB with a higher affinity than the binding domain binds to FcyRIIA .

The present invention also encompasses diabodies with binding domains that comprise an amino acid sequence of a variable heavy chain and / or variable light chain that is at least 45%, at least 50%, at least 55%, at least 60%, at least. less 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the variable heavy chain amino acid sequence me

<img file="MX348166B_D0077.tif" />

^1st IMPI rWÍTHVTO MEXICANO DE LA mÓWF.DAD IWDU'THIAI light chain of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. The present invention further encompasses diabodies with binding domains that specifically bind to FcyRIIB with higher affinity than the antibody or one of its fragments binds to FcyRIIA, and which comprise an amino acid sequence of one or more CDRs that is at least 45 %, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, or at least 99% identical to the amino acid sequence of one or more CDRs of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. Determining the percent identity of two amino acid sequences can be determined by any method known to those skilled in the art, including BLAST protein searches.

The present invention also encompasses the use of diabodies that contain binding domains that specifically bind to FcyRIIB with higher affinity than the binding domains that bind to FcyRIIA, which are encoded by a nucleotide sequence that hybridizes the nucleotide sequence of the antibody. mouse monoclonal produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 under severe conditions. In a preferred embodiment, the binding domain specifically binds to FcyRIIB with a higher affinity than FcyRIIA, and comprises a variable light chain and / or chain

WICKED

INSTTTVTO MMCANC Jb ot u noRE * Ao ΙΜΙΗΈΤΧΙΑΙ and variable weight encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of the variable light chain and / or variable heavy chain of the mouse monoclonal antibody produced by clone 2B6 , 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2 under severe conditions. In another preferred embodiment, the binding domains specifically bind to FcyRIIB with higher affinity than FcyRIIA, and comprise one or more CDRs encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of one or more CDRs of the monoclonal antibody. mouse produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. Stringent hybridization conditions include, but are not limited to, hybridization for a filter-bound DNA in 6X sodium chloride / sodium citrate (SSC) at approximately 45 ° C followed by one or more washes in 0.2X SSC / 0.1 % SDS at about 50-65 ° C, highly stringent conditions such as 6X filter-bound DNA hybridization

SSC at approximately 45 ° C followed by one or more washes at 0. IX SSC / 0.2% SDS at approximately 60 ° C, or any other severe hybridization condition known to one of skill in the art (see, for example, Ausubel, FM et al, eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green

Publishing Associates, Inc. and John Wiley and Sons, Inc., NY, pages 6.3.1 to 6.3.6 and 2.10.3, incorporated herein by reference).

130

IMPI iwrm / το MEXiCANt ns LA PíOPIEDAl 'INDUSTXIAl

<img file="MX348166B_D0078.tif" />

The present invention also encompasses the use of binding domains comprising an amino acid sequence of any of the binding domains described above with mutations (eg, one or more amino acid substitutions) in the backbone or CDR regions. Preferably, mutations in these binding domains maintain or enhance the avidity and / or affinity of the binding domains for FcyRIIB to which it binds immunospecifically. Standard techniques known to those of skill in the art (eg, immunoassays) can be used to test the affinity of an antibody for a particular antigen.

Standard techniques known to those skilled in the art can be used to introduce mutations in the nucleotide sequence encoding an antibody, or one of its fragments, including, site-directed and mutagenesis resulting in the substitutions of, for example, PCR-mediated mutagenesis. , which gives as amino acid. Preferably, the derivatives include less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the antibody. original or one of its fragments. In a preferred embodiment, the derivatives have amino acid substitutions

131

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Say LA WOHSOAI iNnuSTWiAi

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conservatives made at one or more predicted nonessential amino acid residues.

In preferred embodiments, the binding domains are derived from humanized antibodies. A humanized FcyRIB specific antibody may comprise substantially all, and at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of the non-human immunoglobulin (i.e., the donor antibody) and all or substantially all the backbone regions are those of the human immunoglobulin consensus sequence.

The diabodies of the present invention comprise humanized variable domains specific for FcyRIIB wherein one or more regions of one or more CDRs of the heavy and / or light chain regions of a human antibody (the receptor antibody) have been replaced by analogous parts of one or more CDRs of a donor monoclonal antibody that specifically binds to FcyRIIB, with a higher affinity than FcyRIIA, for example, a monoclonal antibody produced clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In other embodiments, humanized antibodies bind to the same epitope as 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, respectively.

In a preferred embodiment, the CDR regions of the humanized FcyRIIB binding domain are derived from a

132

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WTTTWO MUBCANO DI LA FROREDAD INDUSTRIA!

Murine antibody specific for some embodiments, the humanized antibodies described herein comprise alterations, including but not limited to deletions, insertions, amino acid modifications, the acceptor antibody, i.e., the variable domain backbone regions of human, heavy and / or light chain that are necessary to retain the binding specificity of the donor monoclonal antibody. In some embodiments, the backbone regions of the humanized antibodies described herein do not necessarily consist of the precise amino acid sequence of the backbone regions of a naturally occurring human antibody variable region, but contain various alterations, including but are not limited to amino acid deletions, insertions, modifications that alter the property of the humanized antibody, for example, they enhance the binding properties of a region of the humanized antibody that is specific for the same target as the murine FcyRIIB specific antibody. In more preferred embodiments, a minimal number of backbone region alterations are made in order to avoid large-scale introductions of non-human backbone residues and to ensure minimal immunogenicity of the humanized antibody in human. The donor monoclonal antibody is preferably an antibody

133

IMPIí

IRSWTUTO MEXICANO 0 * INDUSTRIAL PROPERTY

<img file="MX348166B_D0081.tif" />

monoclonal produced by clones 2B6, 3H7, 1D5, 2E1, 2H9,

2D11, or 1F2.

In a specific embodiment, the binding domain encompasses variable domains of a CDR-grafted antibody that specifically binds to FcyRIIB with a higher affinity than the antibody that binds to FcyRIIA, wherein the CDR-grafted antibody comprises a variable region domain of heavy chain comprising receptor antibody main chain residues and donor monoclonal antibody residues, that specifically binds to FcyRIIB with a higher affinity than the antibody binds to FcyRIIA, eg, the monoclonal antibody produced in clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In another specific embodiment, the diabodies of the invention comprise variable domains of a CDR grafted antibody that specifically binds to FcyRIIB with a higher affinity of the antibody that binds FcyRIIA, when the CDR grafted antibody comprises a light chain variable region domain comprising receptor antibody backbone residues and donor monoclonal antibody residues, that specifically binds FcyRIIB with a higher affinity than the antibody binds FcyRIIA, for example, the monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2.

The humanized anti-FcyRIIB variable domains used in the invention may have a variable region

<img file="MX348166B_D0082.tif" />

<img file="MX348166B_D0083.tif" />

chain comprising the amino acid sequence ^^ g, CDR1 (SEQ ID NO: 24 or SEQ ID NO: 25) and / or CDR2 (SEQ ID NO: 26 or SEQ ID NO: 27) and / or CDR3 (SEQ ID NO: 27). ID NO: 28 or SEQ ID NO: 29) and / or the light chain variable region comprising the amino acid sequence of CDR1 (SEQ ID NO: 30 or SEQ ID NO: 31) and / or a CDR2 (SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35) and / or CDR3 (SEQ ID NO: 3-6 or SEQ ID NO: 37).

In a specific embodiment, the diabody comprises variable domains of a humanized 2B6 antibody, wherein the VH region of the FR segments of the human germline VH segment VH1-18 (Matsuda et al. (1998) The Complete Nucleotide Sequence Of The Human Immunoglobulin Heavy Chain Variable Region Locus, J. Exp. Med. 188: 2151-2162) and JH6 (Ravetch et al. (1981) Structure Of The Human Immunoglobulin Mu Locus: Characterization Of Embryonic And Rearranged J And D Genes, Cell 27 (3 Pt. 2): 583-91), and one or more VH 2B6 CDR regions, having the amino acid sequences of SED ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. In one embodiment, the VH 2B6 has the amino acid sequence of SEQ ID NO: 38. In another embodiment the VH domain 2B6 has the amino acid sequence of Hu2B6VH, SEQ ID NO: 85, and can be encoded through the nucleotide sequence of SEQ ID NO: 86 . In another specific embodiment, the diabody further comprises a VL region, consisting of the FR segments of the VK- segment.

<img file="MX348166B_D0084.tif" />

Α26 VL of the human germ line (Lautner-Ri ^ ske. Et al. (1992) The Human Immunoglobulin Kappa Locus. Characterization Of The Duplicated A Regions ”, Eur. J. Immunol. 22: 1023-1029) and JK4 (Hieter et al. (1982) Evolution Of Human Immunoglobulin Kappa J Region Genes ", J. Biol. Chem. 257: 1516-22), and one or more CDR regions of 2B6VL, having the amino acid sequence of SEQ ID NO: 30 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 36. In one embodiment, VL 2B6 VL has the amino acid sequence of SEQ ID NO: 39; SEQ ID NO: 40, or SEQ ID NO: 41. In a specific embodiment, VL 2B6 VL has the amino acid sequence of Hu2B6VL, SEQ ID NO: 87, and can be encoded through the nucleotide sequence provided in SEQ ID NO: NO: 88.

In another specific embodiment, the diabody has variable domains from a humanized 3H7 antibody, wherein the VH region consists of the FR segments of a human germline VH segment and the 3H7 VH CDR regions, which has the amino acid sequence of SEQ ID NO. 35. In another specific embodiment, the humanized 3H7 antibody further comprises a VL region, consisting of the FR segments of a human germline VL segment and the CDR regions of 3H7VL, having the amino acid sequence of SEQ ID NO: 42.

In particular, the binding domains

<img file="MX348166B_D0085.tif" />

immunospecifically they bind to dominions. jjYt-ranel ulgxes of native human FcyRIIB, and comprises (or alternatively consists of) CDR sequences from 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, in any of the following combinations: a CDR1 VH and a CDR1 VL; a CDR1 VH and a CDR2 VL; a CDR1 VH and a CDR3 VL; a CDR2 VH and a CDR1 VL; CDR2 VH and CDR2 VL; a CDR2 VH and a CDR3 VL; a CDR3 VH and a CDR1 VH; a CDR3 VH and a CDR2 VL; a CDR3 VH and a CDR3 VL; a CDR1 VH1, a CDR2 VH and a CDR1 VL; a CDR1 VH, a CDR2 VH and a CDR2 VL; a CDR1 VH, a CDR2 VH and a CDR3 VL; a VH CDR2, a VH CDR3 and a VL CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; a CDR2 VH, a CDR2 VH and a CDR3 VL; a CDR1 VH, a CDR1 VL and a CDR2 VL; a CDR1 VH, a CDR1 VL and a CDR3 VL; a CDR2 VH, a CDR1 VL and a CDR2 VL; a CDR2 VH, a CDR1 VL and a CDR3 VL; a CDR3 VH, a CDR1 VL and a CDR2 VL; a CDR3 VH, a CDR1 VL and a CDR3 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a CDR1 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a CDR2 VL; a CDR1 VH, a CDR2 VH, a CDR3 VH and a CDR3 VL; a CDR1 VH, a CDR2 VH, a CDR1 VL and a CDR2 VL; a CDR1 VH, a CDR2 VH, a CDR1 VL and a CDR3 VL; a CDR1 VH, a CDR3 VH, a CDR1 VL and a CDR2 VL; a CDR1 VH, a CDR3 VH, a CDR1 VL and a CDR3 VL; a CDR2 VH, a CDR3 VH, a CDR1 VL and a CDR2 VL; a CDR2 VH, a CDR3 VH, a CDR1 VL and a CDR3 VL; a CDR2 VH, a CDR3 VH, a CDR2 VL and a CDR3 VL; a CDR1 VH, a CDR2 VH, a CDR3

137

<img file="MX348166B_D0086.tif" />

VH, a CDR1 VL and a CDR2 VL; a CDR1 VH, -unn a CDR3 VH, a CDR1 VL and a CDR3 VL; a CDR1 VH, a CDR2 VH, a CDR1 VL, a CDR2 VL, and a CDR3 VL; a CDR1 VH, a CDR3 VH, a CDR1 VL, a CDR2 VL, and a CDR3 VL; a CDR2 VH, a CDR3 VH, a CDR1 VL, a CDR2 VL, and a CDR3 VL; or any combination of these of the VH CDRs and the VL CDRs described herein.

Antibodies to derive binding domains to be included in the diabodies of the invention can further be characterized by epitope mapping, such that the antibodies can be selected to have the highest specificity for FcyRIIB compared to FcyRIIA. Methods of epitope mapping of antibodies are well known in the art and are encompassed within the methods of the invention. In certain embodiments fusion proteins comprising one or more regions of FcyRIIB can be used for epitope mapping of an antibody of the invention. In a specific embodiment, the fusion protein contains the amino acid sequence of a region of an FcyRIIB fused to the Fe portion of human IgG2. Each fusion protein may further comprise amino acid substitutions and / or replacements of certain regions of the receptor with the corresponding region of a homologous receptor, for example, FcyRIIA, as shown in Table 2 below, pMGX125 and pMGX132 contain the binding site FcyRIIB receptor IgG,

<img file="MX348166B_D0087.tif" />

¹³⁸ IMPL · INSTTFrTOMUiCANC 'f DE LA PROPIEDAI INDUSTRIAL the former with the C-terminal of FcyRIIB and the latter with the

C-terminus of FcyRIIA and can be used to differentiate the binding of the C-terminus. The others have FcyRIIA substitutions at the IgG binding site and either at the N-terminus FcylIA or FcylIB. These molecules can help determine the part of the receptor molecule where antibodies bind.

Table 2. List of fusion proteins that can be used to investigate the epitope of monoclonal anti-FcyRIIB antibodies. Residues 172 to 180 belonging to the IgG binding site of FcyRIIA and B. Specific amino acids of the sequence FcyRIIA are in bold.

<td>Plasmid</td><td>Receiver</td><td>Nterminal</td><td> 172-180</td><td>SEQ ID NO.</td><td>C-terminal</td>

<td>pMGX125</td><td>RHb</td><td>Ilb</td><td>KKFSRSDPN</td><td> 43</td><td>APS ---- SS (IIb)</td>

<td>PMGX126</td><td>Rlla / b</td><td>Ha</td><td>QKFSRLDPN</td><td> 44</td><td>APS —- SS (Hb)</td>

<td>PMGX127</td><td></td><td>Ha</td><td>QKFSRLDPT</td><td> 45</td><td>APS ---- SS (Ilb)</td>

<td>PMGX128</td><td></td><td>Ilb</td><td>KKFSRLDPT</td><td> 46</td><td>APS ----- SS (Ilb)</td>

<td>PMGX129</td><td></td><td>lia</td><td>QKFSHLDPT</td><td> 47</td><td>APS — SS (Hb)</td>

<td>pMGXBO</td><td></td><td>Ilb</td><td>KKFSHLDPT</td><td> 48</td><td>APS ---- SS (IIb)</td>

<td>pMGX131</td><td></td><td>Ha</td><td>QKFSRLDPN</td><td> 49</td><td>VPSMGSSS (lIa)</td>

<td>pMGX132</td><td></td><td>Ilb</td><td>KKFSRSDPN</td><td> 50</td><td>VPSMGSSS (Ha)</td>

<td>pMGX133</td><td>RIIa-131R</td><td>Ha</td><td>QKFSRLDPT</td><td> 51</td><td>VPSMGSSS (IIa)</td>

<td>pMGX134</td><td>Rlla-ISIH</td><td>Ha</td><td>QKFSHLDPT</td><td> 52</td><td>VPSMGSSS (Ha)</td>

<td>pMGX135</td><td></td><td>Ilb</td><td>KKFSRLDPT</td><td> 53</td><td>VPSMGSSS (IIa)</td>

<td>PMGX136</td><td></td><td>Ilb</td><td>KKFSHLDPT</td><td> 54</td><td>VPSMGSSS (lIa)</td>

<td colspan="5">Note: APSSS is SEQ ID NO: 309, VPSMGSSS is SEQ ID NO: </td><td> 110</td>

The fusion proteins can be used in any biochemical assay for the determination of the binding of an anti-FcyRIIB antibody of the invention, for example an ELISA. In other embodiments, further confirmation by the use of epitope specificity can be made, peptides with specific residues replaced with those of the FcyRIIA sequence. .

Antibodies can be characterized using assays to identify the function of the antibodies of the invention, particularly the activity to modulate FcyRIIB signaling. For example, the characterization assays of the invention can measure the phosphorylation of tyrosine residues in the ITIM motif of FcyRIIB, or measure the inhibition of calcium mobilization generated by the B cell receptor. The characterization assays of the invention can be cell-based assays or cell-free assays.

It has been well established in the art that coaggregation of FcyRIIB mast cells with the high affinity IgE receptor, FceRI, leads to inhibition of antigen-induced degranulation, calcium mobilization, and cytokine production (Metcalfe DD et al. (1997) Mast Cells, Physiol. Rev. 77: 1033-1079; Long EO (1999) Receptor Regulation Of Immune Responses Through Inhibitory, Annu. Rev. Immunol. 17: 875-904). The molecular details of this signaling pathway have recently been interpreted (Ott VL (2002) Downstream Of Kinase, p62 (dok), Is A Mediator Of FcgammalIB Inhibition Of Fe Epsilon RI Signaling, J. Immunol. 162 (9): 4430- 4439). A

140

<img file="MX348166B_D0088.tif" />

Once aggregated with FcsRI, FcyRIIB are rapidly tyrosine phosphorylated in their ITIM motif, and then recruit Src with

Homology-2 containing inositol-5-phosphatase (SHIP), an SH2 domain containing inositol polyphosphate 5-phosphatase, which in turn is phosphorylated and associates with She and p62^dok (p62^dok is the prototype of a family of adapter molecules, including termination domains such as the amino-terminal plecstrin homology domain (pH domain), a PTB domain, a carboxy-terminal region containing motifs

PXXP and numerous phosphorylation sites (Carpino et al.

(1997) p62 (dok): A Constitutively Tyrosine-Phosphorylated, GAP-Associated Protein In Chronic Myelogenous Leukemia Progenitor Celia, Cell, 88: 197-204; Yamanshi et al. (1997) Identification Of The Abl-And rasGAP-Aasociated 62 kDa

Protein As A Docking Protein, Dok, Cell, 88: 205-211).

Anti-FcvRIIB antibodies for use in the invention can also be characterized by the ability to modulate one or more IgE-mediated responses. Preferably, cell lines that co-express the high affinity receptor for IgE and the low affinity receptor for FcyRIIB will be used in the characterization of anti-FcyRIIB antibodies in modulating IgE-mediated responses. In a specific embodiment, cells from a rat basophilic leukemia cell line (RBL-H23; Barsumian EX. Et al. (1981) IgE-Induced Histamine

<img file="MX348166B_D0089.tif" />

Release From Rat Basophilic Leukemia Cell Lines: Isolation Of Releasing And Nonreleasing Clones, Eur. J. Immunol. 11: 317323, incorporated herein by reference in its entirety) transfected with full-length human FcyRIIB will be used. RBL-2H3 is a well characterized rat cell line that has been used extensively to study signaling mechanisms following IgE-mediated cell activation. When expressed in RBL-2H3 cells and co-aggregated with FcsRI, FcyRIIB inhibits FceRI-induced calcium mobilization, degranulation, and cytokine production (Malbec et al. (1998) Fe Epsilon Receptor I-Associated Lyn-Dependent Phosphorylation Of Fe Gamma Receptor HB During Negative Regulation Of Mast Cell Activation, J. Immunol. 160: 16471658; Daeron et al. (1995) Regulation Of High-Affinity IgE Receptor-Mediated Mast Cell Activation By Murine LowAffinity IgG Receptors, J. Clin . Invest. 95: 577; Ott VL (2002) Downstream Of Kinase, p62 (dok), Is A Mediator Of FcgammalIB Inhibition Of Fe Epsilon RI Signaling, J. Immunol. 162 (9): 4430-4439).

Antibodies for use in the invention can also be characterized by inhibition of FceRI-induced mast cell activation, For example, cells from a rat basophilic leukemia cell line (RBL-H23; Barsumian EX. Et al. (1981 ) IgE-Induced

142

<img file="MX348166B_D0090.tif" />

Histamine Release From Rat Basophilic Leukemia · £ β · ί! —Lin &amp; e i Isolation Of Releasing And Ndnreleasing Clones, Eur. J. Immunol. 11: 317-323) that have been transfected with FcyRIIB are primed with IgE and stimulated with either the F (ab ') fragments₂ of rabbit anti-mouse IgG, to add FcsRI alone, or with whole rabbit anti-mouse IgG to co-aggregate FcyRIIB and FcsRI. In this system, indirect modulation of downstream signaling molecules can be assayed after addition of the antibodies of the invention to sensitize and stimulate cells. For example, the tyrosine phosphorylation of FcyRIIB and the recruitment and phosphorylation of SHIP, the activation of members of the MAP kinase family, including but not limited to Erkl, Erk2, JNK, or p38; and tyrosine phosphorylation of p62^dok and its association with SHIP and RasGAP can be tested.

An illustrative assay to determine the inhibition of FceRI-induced mast cell activation through the antibodies of the invention may comprise the following: transfecting RBL-H23 cells with human FcyRIIB; priming RBL-H23 cells with IgE; stimulate RBL-H23 cells with either F (ab ')₂ of rabbit anti-mouse IgG (to add FcsRI alone and elicit FceRI-mediated signaling, as a control), or stimulate RBL-H23 cells with whole rabbit anti-mouse IgG to (to co-add FcyRIIB and

143

<img file="MX348166B_D0091.tif" />

IMPI IWSTm / TO MWUCANO Γϊ LA PHOWLDA ».

FceRI, resulting in inhibition of the one mediated by FceRI). Cells that have been 'tested' ^ crrrr whole rabbit anti-mouse IgG antibodies can furthermore be pre-incubated with the antibodies of the invention. Measurement of the FceRI-dependent activity of cells that have been pre-incubated with the antibodies of the invention and cells that have not been pre-incubated with the antibodies of the invention, and compare the levels of FceRI-dependent activity in these cells, would indicate a modulation of FceRI-dependent activity through the antibodies of the invention.

The illustrative assay described above can, for example, be used to identify antibodies that block ligand (IgG) binding to the FcyRIIB receptor and antagonize FcyRIIB-mediated inhibition of FceRI signaling by preventing co-aggregation of FcyRIIB and FceRI. This assay also identifies antibodies that enhance FcyRIIB and FceRI co-aggregation and agonize FcyRIIB-mediated inhibition of FceRI signaling by promoting FcyRIIB and FceRI co-aggregation.

In some embodiments, the, diacuer.pos-antiFcyRIIB, comprising the epitope binding domains of the identified anti-FcyRIIB antibodies described herein or known in the art, of the invention are

<img file="MX348166B_D0092.tif" />

characterized by their ability to modulate an IgE-mediated response through monitoring and / or measuring degranulation of mast cells or basophils, preferably in a cell-based assay. Preferably, the mast cells or basophils for use in such assays have been modified to contain human FcyRIIB using standard recombinant methods known to those of skill in the art. In a specific embodiment the antiFcyRIIB antibodies of the invention are characterized by their ability to modulate an IgE-mediated response in a cell-based β-hexosaminidase release assay (enzyme contained in the granules). The release of β-hexosaminidase from basophilic mast cells is a primary event in an acute and inflammatory allergic condition (Aketani et al. (2001) Correlation Between Cytosolic Calcium Concentration And Degranulation In RBL-2H3 Cells In The Presence Of Various Concentrations Of Antigen-Specific IgEs, Immunol. Lett. 75: 185-189; Aketani et al. (2000) A Screening Method For Antigen-Specific IgE Using Mast Cells Based On Intracellular Calcium Signaling, Anal. Chem. 72: 2653-2658). The release of other inflammatory mediators including but not limited to serotonin and histamine can be assayed to measure an IgE-mediated response according to the methods of the invention. Although it is not intended to be linked to any particular mechanism of action,

145

<img file="MX348166B_D0093.tif" />

The release of granules such as those containing β-hexosaminidase from mast cells and basophils is a process dependent on intracellular calcium concentration that is initiated through crosslinking of FcyRIs with the multivalent antigen.

The ability to study human mast cells has been limited by the absence of adequate long-term human mast cell cultures. Recently two stem cell factor dependent human mast cell lines , designated LAD1 and LAD2, were established from spinal cord aspirates from a patient with mast cell sarcoma / leukemia (Kirshenbaum et al. (2003) Characterization Of Novel Stem Cell Factor Responsive Human Mast Cell Lines LAD 1 And 2 Established From A Patient With Mast Cell Sarcoma / Leukemia; Activation Following Aggregation Of FcRI Or FcyRI, Leukemia research, 27: 677-82, which is incorporated herein by reference in its entirety). Both cell lines have been described to express FcsRI and human mast cell marker barios. LAD cells 1 and 2 can be used to assess the effect of the antibodies of the invention on IgE-mediated responses. In a specific embodiment, cell-based β-hexosaminidase release assays such as those described supra can be used in LAD cells to determine any modulation of the IgE-mediated response through

INSTO FTO MIXICANC) M LA PROFIWAr INIMISTIIAl of the anti-FcYRIIB antibodies of the invention. In an illustrative assay, human mast cells, eg, LAD 1, were primed with chimeric human anti-nitrophenol IgE (NP) and boosted with BSA-NP, the polyvalent antigen, and cell degranulation was monitored by measuring the β-hexosaminidase released in the supernatant (Kirshenbaum et al. (2003) Characterization Of Novel Stem Cell Factor Responsive Human Mast Cell Lines LAD 1 And 2 Established From A Patient With Mast Cell Sarcoma / Leukemia; Activation Following Aggregation Of FcRI Or FcyRI, Leukemia research, 27: 677-82, which is incorporated herein by reference in its entirety).

In some embodiments if human mast cells have low expression of endogenous FcyRIIB, as determined using standard methods known in the art, for example, FACS staining, it may be difficult to monitor and / or detect differences in activation of the mediated inhibitory pathway. by the antiFcyRIIB diabodies of the invention. The invention thus encompasses alternative methods, whereby the expression of FcyRIIB can be upregulated using particular cytokines and culture conditions. FcyRIIB has been described as being highly upregulated in human monocyte cell lines, e.g. THP1 U937, (Tridandapani et al. (2002) Regulated

147

<img file="MX348166B_D0094.tif" />

IMPI irwrnyTo mijucano

FROM THE «ONSDAC INNUST_RIAl

Expression

And Inhibitory Function Of Fcgamma RIIB In Human

Monocytic Cells, J. Biol. Chem., 277 (7): 5082-5089) and primary human monocytes (Pricop et al. (2001)

Differential Modulation Of Stimulatory And Inhibitory Fe

Gamma Receptors On Human Monocytes By Thl And Th2 Cytokines,

J. of Immunol, 166: 531-537) by IL4. Differentiation of U937 cells with cyclic dibutyryl AMP has been described to increase the expression of FcyRII (Cameron et al. (2002) Differentiation Of The Human Monocyte Cell Line, U937, With Dibutyryl Cy die AMP Induces The Expression Of The Inhibitory Fe Receptor , FcgammaRIIB, Immunology Letters 83, 171-179). In this way the expression of endogenous FcyRIIB in human mast cells for use in the methods of the invention can be upregulated using cytokines, eg IL-4, IL-13, in order to improve the sensitivity of detection.

Anti-FcyRIIB diabodies can also be tested for inhibition of B cell receptor (BCR) mediated signaling. BCR-mediated signaling can include at least one or more downstream biological responses, such as B-cell activation and proliferation, antibody production, etc. Co-aggregation of FcyRIIB and BCR leads to inhibition of cell site advancement and cell survival. Furthermore, the co-aggregation of FcyRIIB and BCR leads to

<img file="MX348166B_D0095.tif" />

148 inhibition of BCR-mediated signaling.

IMPI '' ^^ MíXIGANt

THE PKCPIMJAI

Specifically, mediated signaling '^ pcrr · —BCR comprises at least one or more of the following: modulation of downstream signaling molecules (eg, FcyRIIB phosphorylation listing, SHIP recruitment, and Btk localization and / or PLCy, MAP kinase activity, Akt recruitment (anti-apoptotic signal), calcium mobilization, cell site advancement, and cell proliferation.

Although numerous effector functions of FcyRIIB- mediated inhibition of BCR signaling are mediated through SHIP, it has recently been shown that B cells, lipopolysaccharide (LPS) activities, from SHOP-deficient mice exhibit significant FcyRIIB-mediated inhibition of calcium mobilization, Ins (1,4,5) P production₃, and Erk and Akt phosphorylation (Brauweiler et al. (2001) Partially Distinct Molecular Mechanisms Mediate Inhibitory FcgammaRIIB Signaling In

Resting And Activated B Cells, Journal of Immunology, 167 (1): 204-211). Therefore, ex vivo B cells from SHIP deficient mice can be used to characterize the antibodies of the invention. An illustrative assay to determine FcyRIIB-mediated inhibition of BCR signaling through the antibodies of the invention may comprise the following: isolation of spleen B cells from SHIP deficient mice, activation of the¹⁴⁹ IMPI ΙΑΜΤΓΠ ΓΤΟ MEXICANO 'DE IA INDUSTRIAL PROPERTY cells with lipopolysaccharides and stimulation of cells with either F (ab')₂ anti-IgM to add BCR or with anti-IgM to co-add BCR with FcyRIIB. Cells that have been stimulated with intact anti-IgM to co-aggregate BCR with FcyRIIB can furthermore be pre-incubated with the antibodies of the invention. FcyRIIB-dependent cell activity can be measured by standard techniques known in the art. Comparison of the level of FcyRIIB-dependent activity in cells that have been pre-incubated with the antibodies and cells that have not been pre-incubated and comparing the levels could indicate a modulation of FcyRIIB-dependent activity through the antibodies.

Measurement of FcyRIIB-dependent activity can include, for example, measuring intracellular calcium mobilization via flow cytometry, measuring Akt and / or Erk phosphorylation, measuring PI (3,4,5) P accumulation₃ BCR-mediated, or measure FcyRIIB-mediated B cell proliferation.

The assays can be used, for example, to identify anti-FcyRIIB diabodies or antibodies for use in the invention that modulate the FcyRIIB-mediated inhibition of BCR signaling by blocking the ligand (IgG) binding site for the FcyRIIB receptor and antagonize FcyRIIB-mediated inhibition of

<img file="MX348166B_D0096.tif" />

<img file="MX348166B_D0097.tif" />

ΐ5ο IMPI

MEXICAN ΙΜΓΓΠΠ rro DE LA PROHEDAD IWimiAt BCR signaling preventing the co-aggregation of FcyRIIB and BCR. The assays can also be used to identify antibodies that enhance the co-aggregation of FcyRIIB and BCR and agonize the FcyRIIB-mediated inhibition of BCR signaling.

Anti-FcyRIIB antibodies can also be tested for FcyRII-mediated signaling in human monocytes / macrophages. Co-aggregation of FcyRIIB with a receptor carrying the immuno-receptor tyrosine-based activation motif (ITAM) acts to down-regulate FcyR-mediated phagocytosis using SHIP as its effector (Tridandapani et al. (2002) Regulated Expression And Inhibitory Function Of Fcgamma RIIB In Human Monocytic Cells ”, J. Biol. Chem., 277 (7): 5082-5089). Coaggregation of FcyRIIA with FcyRIIB results in the rapid phosphorylation of tyrosine residue in the ITIM motif of FcyRIIB, leading to improved phosphorylation of SHIP, association of SHIP with Shc, and phosphorylation of proteins having a molecular weight 120 and 60-65 kDa. Furthermore, the co-aggregation of FcyRIIA with FcyRIIB results in the down-regulation of phosphorylation of Akt, which is a serine-threonine kinase that is involved in cell regulation and that serves to suppress apoptosis.

Anti-FcyRIIB diabodies can also be tested for inhibition of FcyR-mediated phagoGITR in human mocytes / macrophages. For example, cells from a human monocyte cell line, THP-1 can be stimulated with either Fab fragments of antibody IV. 3 mouse monoclonal against FcyRII and goat anti-mouse antibody (to add FcyRIIA alone), or with complete mouse monoclonal antibody IV.3 and goat anti-mouse antibody (to co-add FcyRIIA and FcyRIIB). In this system, modulation of downstream signaling molecules, such as tyrosine phosphorylation of FcyRIIB, SHIP phosphorylation, association of SHIP with Shc, Akt phosphorylation, and phosphorylation of proteins having a molecular weight of 120 and 60-65 kDa can be tested after addition of the molecules of the invention to the stimulated cells. Furthermore, the FcyRIIB-dependent phagocytic efficiency of the monocytic cell line can be directly measured in the presence and absence of antibodies of the invention.

Another illustrative assay to determine inhibition of FcyR-mediated phagocytosis in human monocytes / macrophages through the antibodies of the invention may comprise the following: stimulating THP-1 cells with either Fab from the mouse anti-FcyRII antibody IV.3 and the goat anti-mouse antibody (to add FcyRIIA alone and elicit FcyRIIA-mediated signaling); or with

152 mouse anti-FcvRII antibody and goat ° a® £ i - ^^ 5 antibody (to co-add FcyRIIA and FcyRIIB and inhibit FcyRIIA-mediated signaling. Cells that have been stimulated with anti-FcyRII antibody Mouse and goat anti-mouse antibodies can also be pre-incubated with the molecules of the invention. Measurement of FcyRIIA-dependent activity of stimulated cells that have been pre-incubated with molecules of the invention and cells that have not been pre-incubated with antibodies of the invention and comparing the levels of FcyRIIA-dependent activity in these cells indicate a modulation of FcyRIIA-dependent activity through the antibodies of the invention.

The illustrative assay described can be used, for example, to identify binding domains that block FcyRIIB receptor ligand binding and antagonize FcyRIIB-mediated inhibition of FcyRIIA signaling by preventing co-aggregation of FcyRIIB and FcyRIIA. This assay also identifies the binding domains that enhance the co-aggregation of FcyRIIB and FcyRIIA and agonize the FcyRIIB-mediated inhibition of FcyRIIA signaling.

The FcyRIIB binding domains of interest can be assayed while comprising antibodies I by measuring the ability of THP-1 cells to IgG with sheep red blood cell phagocytosis fluorescein.

153 ητπτυτο opsonized mbucanu (SRBC) through the prév ^ men ^^^^ methods described (Tridandapani et al. (2000) The Adapt-e ^ ~ --Brobein- ~~_n-. ~ 9 · ^ LAT Enhances Fcgamma Receptor-Mediated Signal Transduction In Myeloid Cells ”, J. Biol. Chem. 275: 20480-7). For example, an illustrative assay to measure phagocytosis comprises: treating THP-1 cells with the antibodies of the invention or with a control antibody that binds to FcyRII, comprising levels of activities of such cells, wherein a difference in the activities of the cells (eg, rosette activity (the number of THP-1 cells that bind to IgG, coated SRBC), adhesion activity (the total number of SRBC bound to THP-1 cells), and the phagocytic degree) would indicate a modulation of FcyRIIA-dependent activity through the antibodies of the invention. This assay can be used to identify, for example, antibodies that block FcyRIIB receptor ligand binding and antagonize FcyRIIB-mediated inhibition of phagocytosis. This assay can also identify antibodies that enhance FcyRIIB mediated inhibition of FcyRIIA signaling.

In a preferred embodiment, the binding domains modulate FcyRIIB-dependent activity in human monocytes / macrophages in at least one or more of the following ways: modulation of downstream signaling molecules (modulation of state of

<img file="MX348166B_D0098.tif" />

154 IA4PJ

ΙΗΠιιιΓΠΙ MIXJCAHO r »LA noHEDAD INDÍimiAi phosphorylation of FcyRIIB, modulation of phosphorylation, ________________________________

SHIP, modulation of SHIP and Shc association, modulation of Akt phosphorylation, modulation of phosphorylation of additional proteins around 120 and 60-65 kDa) and modulation of phagocytosis.

UNION CD16A DOMAINS

The next section explains the CD16A binding proteins that can be used as sources for heavy and light chain variable regions for covalent diabody production. In the present invention, CD16A binding proteins include molecules that comprise the VL and VH domains of CD16A antibodies, the VH and VL domains of which are used in the production of the diabodies of the present invention.

A variety of CD16A binding proteins can be used in connection with the present invention. Suitable CD16A binding proteins include human or humanized monoclonal antibodies as well as CD16A binding antibody fragments (eg, scFv, or single chain antibodies, Fab fragments, minibodies) and other antibody-like proteins that bind CD16A through of an interaction with the light chain variable region domain, the heavy chain variable region domain, or both.

In some embodiments, the CD16A binding protein

<img file="MX348166B_D0099.tif" />

155

IMPI iNSTrn το mbhcanc r&gt; £ THE INDUSTRIAL PROPERTY to be used according to the invention comprises a VL and / or VH domain having one or more CDRs with sequences derived from a non-human anti-CD16A antibody, such as an anti-CD16A antibody mouse, and one or more backbone regions that are derived from the backbone sequences of one or more human immunoglobulins. A number of non-human anti-CD16A monoclonal antibodies, from which CDRs and other sequences can be obtained, are known (see, for example, Tamm et al. (1996) The Binding Epitopes Of Human CD16 (Fe gamma RUI) Monoclonal Antibodies .

Implications For Ligand Binding, J. Imm. 157: 1576-81; Fleit et al. (1989) p.159; LEUKOCYTE TYPING II: HUMAN MYELOID AND HEMATOPOIETIC CELLS, Reinherz et al, eds. New York: SpringerVerlag; (1986); LEUCOCYTE TYPING III: WHITE CELL DIFFERENTIATION ANTIGENS McMichael AJ, ed., Oxford: Oxford University Press, 1986); LEUKOCYTE TYPING IV: WHITE CELL DIFFERENTIATION ANTIGENS, Kapp et al., Eds. Oxford Univ. Press, Oxford; LEUKOCYTE TYPING V: WHITE CELL DIFFERENTIATION ANTIGENS, Schlossman et al., Eds. Oxford Univ. Press, Oxford; LEUKOCYTE TYPING VI: WHITE CELL DIFFERENTIATION ANTIGENS, Kishimoto, ed. Taylor &amp; Francis. Furthermore, as shown in the examples, the new CD16A binding proteins that recognize human CD16A are expressed in cells that can be obtained using well known methods for the production and selection of monoclonal antibodies or binding proteins.

<img file="MX348166B_D0100.tif" />

related (eg, hybridoma technology, phage display, and the like). See, for example O'Connell et al. (2002) Phage Versus Phagemid Librarles For Generation Of Human Monoclonal Antibodies, J. Mol. Biol. 321: 49-56; Hoogenboom et al. (2,000) Natural And Designer Binding Sites Made By Phage Display Technology, Imm. Today 21: 371078; Rrebs et al. (2001) High-Throughput Generation And Engineering Of Recombinant Human Antibodies, J. Imm. Methods 254: 67-84; and other references cited herein. Monoclonal antibodies from the non-human species can be chimerized or humanized using antibody humanization techniques known in the art.

Alternatively, fully human antibodies to CD16A can be produced using transgenic animals bearing elements of a human immune system (see, for example, US Patent Nos. 5,569,825 and 5,545,806), using human peripheral blood cells (Casali et al. (1986) Human Monoclonals From AntigenSpecific Selection Of B Lymphocytes And Transformation By EBV, Science 234: 476-479), through the classification of a DNA collection of human B cells according to the general protocol obtained through Huse et al. to the. (1989) Generation Of A Large Combinatorics! Library Of The

Immunoglobulin Repertoire In Phage Lambda, Science 246: 12751281, and by other methods.

157

IMPI

INSTITUTO MEXICANO DE ΙΑ ηΟΗΜΑΟ ΙΝΓΗ IST »I ​​Al

<img file="MX348166B_D0101.tif" />

In a preferred embodiment, the binding donor is the 3G8 antibody or one of its humanized versions, for example, such as those described in US Patent Application Publication 2004/0010124 which is incorporated herein by reference in its entirety. It is contemplated that, for some purposes, it may be advantageous to use CD16A binding proteins that bind to the CD16A receptor on the same epitope linked through 3G8, or at least close enough to this epitope to block binding through 3G8. . Methods for epitope mapping and competitive binding experiments to identify binding proteins with the desired binding properties are well known to those skilled in the art of experimental immunology. See, for example, Harlow and Lane, cited above; Stáhli et al. (1983) Distinction Of Epitopes By Monoclonal Antibodies, Methods in Enzymology 92: 242-253; Kirkland et al. (1986) Analysis Of The Fine Specificity And Cross-Reactivity Of Monoclonal AntiLipid A Antibodies, J. Immunol. 137: 3614-3619; Morel et al. (1988) Monoclonal Antibodies To Bovine Serum Albumin:

Affinity And Specificity Determinations, Molec. Immunol. 25: 7-15; Cheung et al. (1990) Epitope-Specific Antibody Response To The Surface Antigen Of Duck Hepatitis B Virus In Infected Ducks, Virology 176: 546-552; and Moldenhauer et al. (1990) Identity Of HML-I Antigen On Intestinal Intraepithelial T Cells And Of B-ly7 Antigen On Hairy Cell

158

<img file="MX348166B_D0102.tif" />

IMPI ^

W LA nopno_AD

Leukemia, Scand. J.

possible to determine if

Immunol. 32: 77-82. For example, two antibodies are bound at the same site by using one of the antibodies to capture the antigen on an ELISA plate and then measuring the ability of the second antibody to bind to the captured antigen. Comparison of epitopes can also be achieved through labeling of a first antibody, directly or indirectly, with a radionuclide or fluorophore enzyme, inhibiting the ability of an unlabeled second antibody to inhibit the binding of the first antibody to antigen in cells. , in solution, or in solid phase.

It is also possible to measure the ability of antibodies to block the binding of the CD16A receptor to immune complexes formed on ELISA plates. Such immune complexes are formed by first coating the plate with an antigen such as fluorescein, then applying a specific anti-fluorescein antibody to the plate. The immune complex then serves as the ligand for soluble Fe receptors such as sFcRIIIa. Alternatively, a soluble immune complex can be formed and labeled, directly or indirectly, with an enzyme, radionuclides, or fluorophore. The ability of antibodies to inhibit the binding of these Fe receptor-labeled immune complexes in cells, in solution, in solid phase can then be measured.

OF THE níüPIRDAC

The CD16A binding proteins of the invention may or may not comprise a human immunoglobulm Fe region. Fe regions are not present, for example, in scFv binding proteins. Fe regions are present, for example, in human or humanized tetrameric monoclonal IgG antibodies. As described supra, in some embodiments of the present invention, CD16A binding proteins include an Fe region that has altered effector function, eg, reduced affinity for an effect ligand such as the Fe receptor or the C1 complement component compared. with the Fe region unaltered (eg, Fe from naturally occurring IgGl proteins). In one embodiment, the Fe region is not glycosylated at the amino acid of the Fe region corresponding to position 297. Such antibodies lack the Fe effector function.

Thus, the CD16A binding protein may not exhibit Fe-mediated binding to an effector ligand such as an Fe receptor or the C1 component of complement due to the absence of the Fe domain in the binding protein while, in other cases, the lack of binding or effector function is due to an alteration in the constant region of the antibody.

CD16A Binding Proteins Comprising CDR Sequences Similar to a 3G8 mAb CDR Sequence

CD16A binding proteins can also ¹⁶⁰ IMPI 'STiTirro msxjcano OF INDIVIDUAL PROPERTY used in the practice of the invention including proteins comprising CDRs derived from (ie, having a sequence that is the same as or similar to) the CDRs of the mouse monoclonal antibody 3G8. The complementary cDNAs encode the heavy chain and light chain variable regions of the mouse monoclonal antibody 3G8, including the CDR coding sequences, where they are cloned and sequenced as described. The nucleic acid and protein sequences of 3G8 are provided below. Using the mouse variable region and CDR sequences, a large number of chimeric and humanized monoclonal antibodies, comprising complementarity determining regions derived from CDR 3G8 are produced and their properties analyzed. To identify humanized antibodies that bind to CD16A with high affinity and have other desirable properties, heavy chains and antibodies comprising the VH region with 3G8-derived CDRs were produced and combined (via co-expression) with light chains. of the antibody comprising a VL region with 3G8-derived CDRs to produce a tetrameric antibody for analysis. The properties of the resulting tetrameric antibodies were determined as described below. As described below, CD16A binding proteins comprising 3G8 CDRs, such as the humanized antibody proteins described herein,

<img file="MX348166B_D0103.tif" />

can be used according ¹⁶¹ IMPI

ΙΝΓΠΤΌΤΟ MEXICAN FROM IA ROM AGE

IND! rSTPIAl with the invention.

<img file="MX348166B_D0104.tif" />

REGION VH

In one aspect, the CD16A binding protein of the invention may comprise a heavy chain variable domain wherein at least one CDR (and usually three CDRs) have the sequence of one CDR (and more typically all three

CDR) of the 3G8 heavy chain of the mouse monoclonal antibody and for which the remaining portions of the binding protein are substantially human (derived from and substantially similar to the heavy chain variable region of the human antibody (s)).

In one aspect, the invention provides a humanized 3G8 antibody or CDR-containing antibody fragment derived from 3G8 antibody on a substantially human backbone, but in which at least one of the heavy chain variable domain CDRs differs in sequence. of the corresponding mouse 3G8 antibody heavy chain CDR. For example, in one embodiment, the CDR (S) differs from the CDR 3G8 sequence by at least one that has one or more substitutions shown known in the art that affect the binding of 3G8 to CD16A, as known in the art or as it is described in Tables 3 and 4A-4H. Suitable CD16 binding proteins can comprise 0, 1, 2, 3, or 4, or more of these functions (they generally have 1 to 4 of these substitutions), and

162

<img file="MX348166B_D0105.tif" />

optionally have additional substitutions also Table 3. Substitution of Domino V_H

<td>NOT.</td><td>Kabat position</td><td>Region</td><td>Substitutions</td>

<td> 1</td><td> 2</td><td>FR1</td><td>lie</td>

<td> 2</td><td> 5</td><td>FR1</td><td>Lys</td>

<td> 3</td><td> 10</td><td>FR1</td><td>Thr</td>

<td> 4</td><td> 30</td><td>FR1</td><td>Arg</td>

<td> 5</td><td> 34</td><td>CDR1</td><td>Val</td>

<td> 6</td><td> 50</td><td>CDR2</td><td>Leu</td>

<td> 7</td><td> 52</td><td>CDR2</td><td>Phe or Tyr or Asp</td>

<td>Not.</td><td>Kabat position</td><td>Region</td><td>Substitutions</td>

<td> 8</td><td> 54</td><td>CDR2</td><td>Asn</td>

<td> 9</td><td> 60</td><td>CDR2</td><td>To be</td>

<td> 10</td><td> 62</td><td>CDR2</td><td>To be</td>

<td> 11</td><td> 70</td><td>FR3</td><td>Thr</td>

<td> 12</td><td> 94</td><td>FR3</td><td>Gln or Lys or Wing o His</td>

<td> 13</td><td> 99</td><td>CDR3</td><td>Tyr</td>

<td> 14</td><td> 101</td><td>CDR3</td><td>Asp</td>

163

ΙΝΤΤΤΠΓΓΟ MEXICAN

PROPRIETARY

Table 4A _ ........._ _n.tniSTRiAi

Sequence V_H derivative of V_H 3G8 *

<td></td><td>FR1</td><td>CDR1</td><td>FR2</td><td>CDR2</td><td>FR3</td><td>CDR3</td><td>FR4</td>

<td>3G8VH</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td>

<td>Ch3G8VH</td><td>TO___</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>B</td>

<td>HxC</td><td>B</td><td>B</td><td>B</td><td>TO</td><td>TO</td><td>TO</td><td>B</td>

<td>CxH</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-1</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-2</td><td>C</td><td>TO</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-3</td><td>D</td><td>TO</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-4</td><td>B</td><td>TO</td><td>B</td><td>TO___</td><td>C</td><td>TO</td><td>B</td>

<td>HU358VH-5</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>C</td><td>B</td><td>B</td>

<td>HU358VH-6</td><td>B</td><td>B</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu358VH-7</td><td>B</td><td>B</td><td>B</td><td>TO</td><td>B</td><td>C</td><td>B</td>

<td>Hu358VH-8</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td><td>B</td><td>B</td>

<td>HU358VH-9</td><td>B</td><td>TO</td><td>B</td><td>B</td><td>B</td><td>B</td><td>B</td>

<td>HU358VH-10</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu358VH-ll</td><td>B</td><td>TO</td><td>B</td><td>B</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-12</td><td>B</td><td>TO</td><td>B</td><td>C</td><td>B</td><td>TO</td><td>B</td>

<td>Hu358VH-13</td><td>B</td><td>TO</td><td>B</td><td>D</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-14</td><td>B</td><td>TO</td><td>B</td><td>AND</td><td>B</td><td>TO</td><td>B</td>

<td>HU358VH-15</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>p</td><td>TO</td><td>B</td>

<td>HU358VH-16</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>AND</td><td>TO</td><td>B</td>

<td>HU358VH-17</td><td>B</td><td>TO</td><td>B</td><td>TO</td><td>F</td><td>TO</td><td>B</td>

<td></td><td>FR1</td><td>CDR1</td><td>FR2</td><td>CDR2</td><td>FR3</td><td>nr The pNpiíl, INIHNTRI CDR3</td><td>^rFR4</td><td></td>

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

<td>HU358VH-18</td><td>Β</td><td>Α</td><td>Β</td><td>Α</td><td>G</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-19</td><td>Β</td><td>Α</td><td>Β</td><td>Α</td><td>C</td><td>C</td><td>B</td><td></td>

<td>HU358VH-20</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>Β</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-21</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>ρ</td><td>B</td><td>B</td><td></td>

<td>HU.358VH-22</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>Β</td><td>C</td><td>B</td><td></td>

<td>HU358VH-23</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>Ε</td><td>c</td><td>B</td><td></td>

<td>HU358VH-24</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>F</td><td>c</td><td>B</td><td></td>

<td>HU358VH-25</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>G</td><td>c</td><td>B</td><td></td>

<td>HU358VH-26</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>C</td><td>c</td><td>B</td><td></td>

<td>HU358VH-27</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>Ε</td><td>p</td><td>B</td><td></td>

<td>HU358VH-28</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>F</td><td>p</td><td>B</td><td></td>

<td>HU358VH-29</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>G</td><td>p</td><td>B</td><td></td>

<td>H11358VH-30</td><td>Β</td><td>Β</td><td>Β</td><td>C</td><td>C</td><td>p</td><td>B</td><td></td>

<td>HU358VH-31</td><td>Ε</td><td>Β</td><td>Β</td><td>C</td><td>Β</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-32</td><td>Ε</td><td>Β</td><td>Β</td><td>Η</td><td>Β</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-33</td><td>Ε</td><td>Β</td><td>Β</td><td>Η</td><td>Β</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-34</td><td>Ε</td><td>Β</td><td>Β</td><td>C</td><td>Β</td><td>C</td><td>B</td><td></td>

<td>HU358VH-35</td><td>Ε</td><td>Β</td><td>Β</td><td>C</td><td>C</td><td>C</td><td>B</td><td></td>

<td>HU358VH-36</td><td>Ε</td><td>Β</td><td>Β</td><td>Η</td><td>C</td><td>p</td><td>B</td><td></td>

<td>HU358VH-37</td><td>Ε</td><td>Β</td><td>Β</td><td>Η</td><td>Ε</td><td>c</td><td>B</td><td></td>

<td>HU358VH-38</td><td>Ε</td><td>Β</td><td>Β</td><td>F</td><td>Β</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-39</td><td>Ε</td><td>Β</td><td>Β</td><td>I</td><td>Β</td><td>TO</td><td>B</td><td></td>

<td>HU358VH-40</td><td>Ε</td><td>Β</td><td>Β</td><td>G</td><td>Β</td><td>TO</td><td>B</td><td></td>

<img file="MX348166B_D0106.tif" />

165

INSTITUTO MEXICANO CE LA ΡΚΟΠΗίΑΠ

<td></td><td>FR1</td><td>CDR1</td><td>FR2</td><td>CDR2</td><td>FR3</td><td>—- CDR3</td><td>FR4</td>

<td>Hu358VH-41</td><td>AND</td><td>B</td><td>B</td><td>J</td><td>B</td><td>TO</td><td>B</td>

<td></td><td></td><td></td><td> —' —</td><td></td><td> —</td><td></td><td></td>

<td>Hu358VH-42</td><td>AND</td><td>B</td><td>B</td><td>c</td><td>H</td><td>TO</td><td>B</td>

<td></td><td> —-</td><td></td><td> —</td><td> ·—·</td><td> —</td><td> — </td><td></td>

<td>Hu358VH-43</td><td>AND</td><td>B</td><td>B</td><td>c</td><td>H</td><td>c</td><td>B</td>

<td></td><td> ——</td><td> 1</td><td></td><td>M · '</td><td></td><td> —</td><td> —</td>

<td>Hu358VH-44</td><td>AND</td><td>B</td><td>B</td><td>c</td><td>I</td><td>D</td><td>B</td>

<td></td><td> —</td><td> —</td><td> ·&gt;*·</td><td></td><td> ——</td><td> —</td><td> —</td>

<td>Hu358VH-45</td><td>AND</td><td>B</td><td>B</td><td>c</td><td>J</td><td>D</td><td>B</td>

<td></td><td> —</td><td></td><td></td><td> —</td><td></td><td></td><td> —</td>

* The letters in Table 4A refer to sequences in the

Tables 4B-4H.

Table 4B: FR1

<td>TO</td><td>B</td><td>c</td><td>D</td><td>AND</td><td></td><td>RESIDUE</td>

<td>Q</td><td>Q</td><td>Q</td><td>Q</td><td> 0</td><td></td><td> 1</td>

<td>V</td><td>V</td><td>V</td><td>V</td><td>I</td><td></td><td> 2</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td></td><td> 3</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td></td><td> 4</td>

<td>K</td><td>R</td><td>K</td><td>R</td><td>K</td><td></td><td> 5</td>

<td>AND</td><td>AND</td><td>AND</td><td>AND</td><td>AND</td><td></td><td> 6</td>

<td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td></td><td> 7</td>

<td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td></td><td> 8</td>

<td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td></td><td> 9</td>

<td>G</td><td>TO</td><td>TO</td><td>TO</td><td>T</td><td></td><td> 10</td>

<td>I</td><td>L</td><td>L</td><td>L</td><td>L</td><td></td><td> 11</td>

<td>L</td><td>V</td><td>V</td><td>V</td><td>V</td><td></td><td> 12</td>

<img file="MX348166B_D0107.tif" />

166

INSTtn / ΤΌ MEXJGAbKj “. . .--: iAi

<td>Q</td><td>K</td><td>K</td><td>K</td><td>K</td><td></td><td>INONTl 13</td>

<td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td></td><td> 14</td>

<td>s</td><td>T</td><td>T</td><td>T</td><td>T</td><td></td><td> 15</td>

<td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td></td><td> 16</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td></td><td> 17</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td> 11</td><td></td><td> 18</td>

<td>S</td><td>T</td><td>T</td><td>T</td><td>T</td><td></td><td> 19</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td></td><td> 20</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td></td><td> 21</td>

<td>C</td><td>C</td><td>C</td><td>c</td><td>c</td><td></td><td> 22</td>

<td>S</td><td>T</td><td>T</td><td>T</td><td>T</td><td></td><td> 23</td>

<td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td></td><td> 24</td>

<td>S</td><td>S</td><td>Ξ</td><td>s</td><td>S</td><td></td><td> 25</td>

<td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td></td><td> 26</td>

<td>F</td><td>F</td><td>F</td><td>F</td><td>F</td><td></td><td> 27</td>

<td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td></td><td> 28</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td></td><td> 29</td>

<td>R</td><td>S</td><td>S</td><td>R</td><td>S</td><td></td><td> 30</td>

<td> 103</td><td> 104</td><td> 105</td><td> 106</td><td> 107</td><td></td><td>SEQ ID NOT.</td>

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 103</td><td>QVTLKESGPGILQPSQTLSLTCSFSGFSLR</td>

<td> 104</td><td>QVTLRESGPALVKPTQTLTLTCTFSGFSLS</td>

<td> 105</td><td>QVTLKESGPALVKPTQTLTLTCTFSGFSLS</td>

<td> 106</td><td>0νΤΙΚΕ56ΡΑΐνΚΡΤ0Τ1ΤΣΤ0ΤΡ3ΘΓ31Κ</td>

<td> 107</td><td>QITLKESGPTLVKPTQTLTLTCTFSGFSLS</td>

<img file="MX348166B_D0108.tif" />

167

Table 4C: CDR1

IMPI ⁿ* 'fnjnVMtUCAM_OR

MU ntOMIDAi ·&gt; INM'STKfAL

<td>TO</td><td>B</td><td></td><td>RESIDUE</td>

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

<td>T</td><td>T</td><td></td><td> 31</td>

<td>s</td><td>S</td><td></td><td> 32</td>

<td>G</td><td>G</td><td></td><td> 33</td>

<td>M</td><td>V</td><td></td><td> 34</td>

<td>G</td><td>G</td><td></td><td> 35</td>

<td>V</td><td>V</td><td></td><td>35A</td>

<td>G</td><td>G</td><td></td><td>35B</td>

<td> 108</td><td> 109</td><td></td><td>SEQ ID NO.</td>

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 108</td><td>TSGMGVG</td>

<td> 109</td><td>TSGVGVG</td>

Table 4D: FR2

<td>TO</td><td>B</td><td></td><td>RESIDUE</td>

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

<td>W</td><td>W</td><td></td><td> 36</td>

<td>I</td><td>I</td><td></td><td> 37</td>

<td>R</td><td>R</td><td></td><td> 38</td>

<td>Q</td><td>Q</td><td></td><td> 39</td>

<td>P</td><td>P</td><td></td><td> 40</td>

<td>s</td><td>P</td><td></td><td> 41</td>

<td>G</td><td>G</td><td></td><td> 42</td>

<td>K</td><td>K</td><td></td><td> 43</td>

<td>G</td><td>TO</td><td></td><td> 44</td>

<td>L</td><td>L</td><td></td><td> 45</td>

<td>AND</td><td>AND</td><td></td><td> 46</td>

<td>W</td><td>W</td><td></td><td> 47</td>

<img file="MX348166B_D0109.tif" />

168

IMPIOUS*

INSTITUTO MEXICANO ΠΕ THE PROPERTY

<td>L</td><td>L</td><td></td><td> 48</td>

<td>TO</td><td>TO</td><td></td><td> 49</td>

<td> 108</td><td> 109</td><td></td><td>SEQ ID NO.</td>

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 110</td><td>WIRQPSGKGLEWLA</td>

<td> 111</td><td>WIRQPPGKALEWLA</td>

Table 4E. CDR2

<td>TO</td><td>B</td><td>c</td><td>D</td><td>AND</td><td>F</td><td>G</td><td>H</td><td>I</td><td>J</td><td>RESIDUE</td>

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

<td>H</td><td>H</td><td>H</td><td>H</td><td>H</td><td>L</td><td>H</td><td>L</td><td>H</td><td>L</td><td> 50</td>

<td>I</td><td> 1</td><td> 1</td><td> 1</td><td>I</td><td>I</td><td>I</td><td> 1</td><td>I</td><td>I</td><td> 51</td>

<td>w</td><td>Y</td><td>W</td><td>Y</td><td>w</td><td>D</td><td>F</td><td>w</td><td>D</td><td>w</td><td> 52</td>

<td>w</td><td>W</td><td>w</td><td>w</td><td>w</td><td>W</td><td>W</td><td>w</td><td>W</td><td>w</td><td> 53</td>

<td>D</td><td>N</td><td>D</td><td>D</td><td>N</td><td>D</td><td>D</td><td>D</td><td>D</td><td>N</td><td> 54</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 55</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 56</td>

<td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td> 57</td>

<td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td> 58</td>

<td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td> 59</td>

<td>N</td><td>N</td><td>S</td><td>N</td><td>N</td><td>s</td><td>S</td><td>S</td><td>s</td><td>S</td><td> 60</td>

<td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td> 61</td>

<td>TO</td><td>TO</td><td>s</td><td>TO</td><td>TO</td><td>s</td><td>S</td><td>S</td><td>s</td><td>s</td><td> 62</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td> 63</td>

<td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td> 64</td>

<td>S</td><td>S</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>S</td><td>s</td><td>s</td><td> 65</td>

<td> 112</td><td> 113</td><td> 114</td><td> 115</td><td> 116</td><td> 117</td><td> 118</td><td> 119</td><td> 120</td><td> 121</td><td>SEQ ID NO</td>

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 112</td><td>HIWDDDKRYNPALKS</td>

<td> 113</td><td>ΗIXWNDDKRYNPALKS</td>

<td> 114</td><td>HIWWDDDKRYSPSLKS</td>

<td>US</td><td>HIYWDDDKRYNPALKS</td>

<td> 116</td><td>HIWWNDDKRYHPALKS</td>

<td> 117</td><td>LIDWDDDKRYSPSLKS</td>

<td> 118</td><td>HIFWDDDKRYSPSLKS</td>

<td> 119</td><td>LIWWDDDKRYSPSLKS</td>

<td> 120</td><td>HIDWDDDKRYSPSLKS</td>

<td> 121</td><td>LIWWNDDKRYSPSLKS</td>

169

Table 4F: FR3

IMPL

MEXICAN INSTITUTE 1

DE LA FROREDAO 'INDUSTRIAL

<td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>G</td><td>H</td><td>I</td><td>J</td><td>RESIDUE</td>

<td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td>R</td><td> 66</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td> 67</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 68</td>

<td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td> 69</td>

<td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td> 70</td>

<td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td> 71</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 72</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 73</td>

<td>S</td><td>s</td><td>S</td><td>S</td><td>S</td><td>S</td><td>s</td><td>S</td><td>s</td><td>S</td><td> 74</td>

<td>S</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td> 75</td>

<td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td> 76</td>

<td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td> 77</td>

<td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td> 78</td>

<td>F</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td> 79</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td> 80</td>

<td>K</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 81</td>

<td>I</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td> 82</td>

<td>TO</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>82A</td>

<td>S</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>82B</td>

<td>V</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>M</td><td>82C</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 83</td>

<img file="MX348166B_D0110.tif" />

170

IMPI nwnvro MEXICAN

DS LA rSOPIIDAO

<td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>G</td><td>H</td><td>I _</td><td>J</td><td>RESIDUE</td>

<td>T</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td> 84</td>

<td>TO</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td> 85</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 86</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 87</td>

<td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td> 88</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 89</td>

<td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td> 90</td>

<td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td> 91</td>

<td>C</td><td>c</td><td>C</td><td>c</td><td>c</td><td>c</td><td>C</td><td>C</td><td>c</td><td>c</td><td> 92</td>

<td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td> 93</td>

<td>Q</td><td>R</td><td>Q</td><td>T</td><td>K</td><td>TO</td><td>H</td><td>R</td><td>H</td><td>Q</td><td> 94</td>

<td> 122</td><td> 123</td><td> 124</td><td> 125</td><td> 12 6</td><td> 127</td><td> 128</td><td> 129</td><td> 130</td><td> 131</td><td>82 SEC ID NO.</td>

<td> 132</td><td> 133</td><td> 134</td><td> 135</td><td> 13 6</td><td> 137</td><td> 138</td><td> 139</td><td> 140</td><td> 141</td><td>82A SEC ID NO.</td>

<td> 142</td><td> 143</td><td> 144</td><td> 145</td><td> 146</td><td> 147</td><td> 148</td><td> 149</td><td> 150</td><td> 151</td><td>82B SEC ID NO.</td>

<td> 152</td><td> 153</td><td> 154</td><td> 155</td><td> 156</td><td> 157</td><td> 158</td><td> 159</td><td> 160</td><td> 161</td><td>82C SEC ID NO.</td>

171

<img file="MX348166B_D0111.tif" />

<td>SEQ ID NO.</td><td>Sequence ---—</td>

<td> 122</td><td>RLTISKDTSSNQVFLKIDTADTATYYCAQ</td>

<td> 123</td><td>RLTISKDTSKNQVVLTMDPVDTATYYCAR</td>

<td> 124</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAQ</td>

<td> 125</td><td>RLTISKDTSKNQWLTMDPVDTATY YCAT</td>

<td> 126</td><td>RLTISKDTSKNQVVLTMDPVDTATYYCAK</td>

<td> 127</td><td>RLTI SKDTSKNQWLTMDPVDTAT YYCAA</td>

<td> 128</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAH</td>

<td> 129</td><td>RLTITKDTSKNQWLTMDPVDTATYYCAR</td>

<td> 130</td><td>RLTITKDTSKNQWLTMDPVDTATYYCAH</td>

<td> 131</td><td>RLTITKDTSKNQWLTMDPVDTAT AND YCAQ</td>

<td> 132</td><td>RLTISKDTSSNQVFLKADTADTATYYCAQ</td>

<td> 133</td><td>RLTISKDTSKNQWLTTDPVDTATYYCAR</td>

<td> 134</td><td>RLTISKDTSKNQWLTTDPVDTATYYCAQ</td>

<td> 135</td><td>RLTISKDTSKNQVVLTTDPVDTATYYCAT</td>

<td> 136</td><td>RLTISKDTSKNQWLTTDPVDTATYYCAR</td>

<td> 137</td><td>RLTISKDTSKNQWLTTDPVDTATYYCAA</td>

<td> 138</td><td>RLTISKDTSKNQWLTTDPVDTATYYCAH</td>

<td> 139</td><td>RLTITKDTSKNQWLTTDPVDTATYYCAR</td>

<td> 140</td><td>RLTITKDTSKNQWLTTDPVDTATYYCAH</td>

<td> 141</td><td>RLTITKDTSKNQWLTTDPVDTATYYCAQ</td>

<td> 142</td><td>RLTISKDTSSNQVFLKSDTADTATYYCAQ</td>

<td> 143</td><td>RLTISKDTSKNQVVLTMDPVDTATYYCAR</td>

<td> 144</td><td>RLTISKDTSKNQWLTMDPVDTAT AND YCAQ</td>

<td> 145</td><td>RLTISKDTSKNQWLTNDPVDTATY YCAT</td>

<td> 146</td><td>RLTISKDTSKNQVVLTNDPVDTATYYCAK</td>

<td> 147</td><td>RLTI SKDTSKNQWLTNDPVDTATY YCAA</td>

<td> 148</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAH</td>

<td> 149</td><td>RLTITKDTSKNQWLTNDPVDTATYYCAR</td>

<td> 150</td><td>RLTITKDTSKNQWLTNDPVDTATYYCAH</td>

<td> 151</td><td>RLTITKDTSKNQWLTNDPVDTATYYCAQ</td>

<td> 152</td><td>RLTISKDTSSNQVFLKVDTADTATYYCAQ</td>

<td> 153</td><td>RLTISKDTSKNQVVLTMDPVDTATYYCAR</td>

<td> 154</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAQ</td>

<td> 155</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAT</td>

<td> 156</td><td>RLTISKDTSKNQVVLTMDPVDTATYYCAR</td>

<td> 157</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAA</td>

<td> 158</td><td>RLTISKDTSKNQWLTMDPVDTATYYCAH</td>

<td> 159</td><td>RLTITKDTSKNQWLTMDPVDTATYYCAR</td>

<td> 160</td><td>RLTITKDTSKNQWLTMDPVDTATYYCAH</td>

<td> 161</td><td>RLTITKDTSKNQWLTMDPVDTATYYCAQ</td>

172

<img file="MX348166B_D0112.tif" />

IMPI ίΝΤΓΤηΝΌ MUlCANí

GIVE OWNERSHIP

ΤΝπντηίΑΐ

Table 4G. CDR3

<td>TO</td><td>B</td><td>C</td><td>D</td><td>RESIDUE</td>

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

<td> 1</td><td>I</td><td>I</td><td>I</td><td> 95</td>

<td>N</td><td>N</td><td>N</td><td>N</td><td> 96</td>

<td>P</td><td>P</td><td>P</td><td>P</td><td> 97</td>

<td>TO</td><td>TO</td><td>TO</td><td>TO</td><td> 98</td>

<td>w</td><td>W</td><td>Y</td><td>Y</td><td> 99</td>

<td>F</td><td>F</td><td>F</td><td>F</td><td> 100</td>

<td>TO</td><td>D</td><td>TO</td><td>D</td><td> 101</td>

<td>Y</td><td>Y</td><td>Y</td><td>Y</td><td> 102</td>

<td> 162</td><td> 163</td><td> 164</td><td> 165</td><td>SEQ ID NO</td>

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 162</td><td>INPAWFAY</td>

<td> 163</td><td>INPAWFDY</td>

<td> 164</td><td>INPAYFAY</td>

<td> 165</td><td>INPAYFDY</td>

Table 4H. FRA

<td>TO</td><td>B</td><td></td><td>RESIDUE</td>

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

<td>W</td><td>W</td><td></td><td> 103</td>

<td>G</td><td>G</td><td></td><td> 104</td>

<td>Q</td><td>Q</td><td></td><td> 105</td>

<td>G</td><td>G</td><td></td><td> 106</td>

<td>T</td><td>T</td><td></td><td> 107</td>

<td>L</td><td>L</td><td></td><td> 108</td>

<td>V</td><td>V</td><td></td><td> 109</td>

<td>T</td><td>T</td><td></td><td> 110</td>

<td>V</td><td>V</td><td></td><td> 111</td>

<td>s</td><td>s</td><td></td><td> 112</td>

<td>TO</td><td>s</td><td></td><td> 113</td>

<td> 166</td><td> 167</td><td></td><td>SEQ ID NO</td>

<td>SEC NO.</td><td>Sequence</td>

<td> 166</td><td>WGQGTLVTVSA</td>

<td> 167</td><td>WGQGTLVTVSS</td>

rr ιΓ ¿ZJÜSAno ¹¹ THE HtOPfEDAp

In one embodiment, a CDT®A binding protein can comprise a sequence of the variable domain of ^ heavy-chain .., which is equal to or similar to, the VH domain of the Hu3G8VH-1 construct, the sequence of which is provided in SEQ ID NO: 68. For example, the invention provides a CD16A binding protein comprising a VH domain with the sequence that (1) differs from the VH domain of Hu3G8VH-1 (SEQ ID NO: 68) by zero, one, or more than one of the CDR substitutions. established in Table 1; (2) differs from the VH domain of Hu3G8VH-1 by zero, one or more than one of the backbone substitutions set forth in Table 1; and (3) is at least about 80% identical, generally at least about 90% and sometimes at least about 95% identical, or even at least 98% identical to the Hu3G8VH-1 sequence in the remaining positions.

Illustrative VH domains of the CD16 binding proteins of the invention have the sequence of 3G8VH, HU3G8VH-5 and Hu3G8VH-22 (SEQ ID NO: 79, SEQ ID NO: 69 and SEQ ID NO: 70, respectively). Illustrative nucleotide sequences encoding the 3G8VH and Hu3G8VH5 sequences (SEQ ID NO: 79 and SEQ ID NO: 69, respectively) are provided with SEQ ID NO: 80 and SEQ ID NO: 81, respectively.

The VH domain may have a sequence that differs from Hu3G8VH-1 (SEQ ID NO: 68) by at least one, at least two, at least three, at least 4, at least 5, or at least 6 of

<img file="MX348166B_D0113.tif" />

IMPI INSTITUTO MEXICANO DE LA MOntOAD The substitutions shown in the Table Substitutions are believed to result in increased activity for CD16A and / or reduce the immunogenicity of the CD16A binding protein when administered to humans. In certain embodiments, the degree of sequence identity to the VH domain Hu3G8VH-1 at the remaining positions is at least about 80%, at least about 90%, at least about 95%, or at least about 98%.

For illustration and not limitation, the sequences of a number of VH domains of the CD16A binding protein are shown in Table 4. Heavy chains comprise these sequences fused to a human Cyl constant region that were co-expressed with the hu3G8VL light chain -l (described below) to form tetrameric antibodies, and the binding of the antibodies to CD16A was measured to evaluate the effect of amino acid substitutions compared to the VH domain hu3G8VH-1. Constructs where the VH domain has the sequence of hu3G8VH-1, 2, 3, 4, 5, 8, 12, 14, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 42, 43, 44 and 45 showed high affinity binding, with the VH domains hu3G8VH-6 and -40 showing intermediate binding. CD16A binding proteins comprising the VH domains of hu3G8VH-5 and hu3G8VH-22 (SEQ ID NO: 69 and SEQ ID NO: 70, respectively) are considered to have particularly favorable binding properties.

<img file="MX348166B_D0114.tif" />

Similar studies were conducted to identify^- light chain variable domain sequences with favorable binding properties. In one aspect, the invention provides a CD16A binding protein containing a light chain variable domain wherein at least one CDR (and usually 3 CDRs) have the sequence of one light chain CDR (more typically all three CDRs). of the mouse monoclonal 3G8 antibody and for which the remaining portions of the binding protein are substantially human (derived from and substantially similar to, the heavy chain variable region of a human antibody or antibodies).

In one aspect, the invention provides a fragment of a CDR containing the humanized 3G8 antibody derived from the 3G8 antibody on a substantially human backbone, but in which at least one of the light chain variable domain CDRs differs in sequence from the Mouse monoclonal antibody 3G8 light chain CDR. In one embodiment, the CDR (s) differ from the 3G8 sequence by at least having one or more amino acid substitutions in a CDR, such as, one or more substitutions shown in Table 12 (e.g., arginine at position 24 and in CDR1; serine at position 25 in

CDR1; tyrosine at position 32 on CDR1; leucine in

176

<img file="MX348166B_D0115.tif" />

position 33 in CDR1; Aspartic acid

MEXICAN INSTITUTE ÜE LA PXOREDAJ3. _ industrial, tryptophan or serine at position 50 on CDR2; serine at position 53 in CUR2;¹ alanine or glutamine at position 55 on CDR2; threonine at position 56 on CDR2; serine at position 93 in CDR3; and / or threonine at position 94 in CDR3). In various embodiments, the variable domain may have 0, 1, 2, 3, 4, 5, or more of these substitutions (and generally has 1 to 4 of these substitutions) and optionally, it may have additional substitutions as well.

In one embodiment, a suitable CD16A binding protein may comprise a light chain variable domain sequence that is the same or similar to the VL domain of the Hu3G8VL-1 construct (SEQ ID NO: 71), the sequence of which is provided. in Table 6. For example, the invention provides a CD16A binding protein comprising a VL domain with a sequence that (1) differs from the VJ domain of Hu3G8VL-1 (SEQ ID NO: 71) by zero, one, or more of the established CDR substitutions. in Table 5; (2) differs from the VL domain of Hu3G8VL-2 by zero, one or more of the backbone substitutions set forth in Table 5; and (3) is at least about 80% identical, usually at least about 90%, and sometimes at least 95% identical, or even at least 98% identical to the Hu3G8VL-I VL sequence (SEQ ID NO: 71 ) in the remaining positions.

<img file="MX348166B_D0116.tif" />

Table 5.

177

Domain V Substitutions_L

<td>Not.</td><td>Kabat position</td><td>Region</td><td>Substitutions</td>

<td> 1</td><td> 24</td><td>CDR1</td><td>Arg</td>

<td> 2</td><td> 25</td><td>CDR1</td><td>To be</td>

<td> 3</td><td> 32</td><td>CDR1</td><td>Tyr</td>

<td> 4</td><td> 33</td><td>CDR1</td><td>Leu</td>

<td> 5</td><td> 50</td><td>CDR2</td><td>Aspo Trpo Ser</td>

<td> 6</td><td> 51</td><td>CDR2</td><td>To</td>

<td> 7</td><td> 53</td><td>CDR2</td><td>To be</td>

<td> 8</td><td> 55</td><td>CDR2</td><td>Wing or Gln</td>

<td> 9</td><td> 56</td><td>CDR2</td><td>Thr</td>

<td> 10</td><td> 93</td><td>CDR3</td><td>To be</td>

<td> 11</td><td> 94</td><td>CDR3</td><td>Thr</td>

Table 6. Sequences derived from V_L 3G8 *

<td></td><td>FR1</td><td>CDR1</td><td>FR2</td><td>CDR2</td><td>FR3</td><td>CDR3</td><td>FR4</td>

<td>3G8VL</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td>

<td>Ch3G8VL</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td>

<td>Hu3G8VL-l</td><td>B</td><td>TO</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-2</td><td>B</td><td>B</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-3</td><td>B</td><td>C</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-4</td><td>B</td><td>D</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-5</td><td>B</td><td>AND</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-6</td><td>B</td><td>F</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-7</td><td>B</td><td>G</td><td>TO</td><td>TO</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-8</td><td>B</td><td>TO</td><td>TO</td><td>B</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-9</td><td>B</td><td>TO</td><td>TO</td><td>C</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-10</td><td>B</td><td>TO</td><td>TO</td><td>D</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-ll</td><td>B</td><td>TO</td><td>TO</td><td>AND</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-12</td><td>B</td><td>TO</td><td>TO</td><td>F</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-13</td><td>B</td><td>TO</td><td>TO</td><td>G</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-14</td><td>B</td><td>TO</td><td>TO</td><td>TO</td><td>B</td><td>B</td><td>B</td>

<td>Hu3G8VL-15</td><td>B</td><td>TO</td><td>TO</td><td>TO</td><td>B</td><td>C</td><td>B</td>

<td>Hu3G8VL-16</td><td>B</td><td>TO</td><td>TO</td><td>TO</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-17</td><td>B</td><td>TO</td><td>TO</td><td>TO</td><td>B</td><td>AND</td><td>B</td>

<td>Hu3G8VL-18</td><td>B</td><td>B</td><td>TO</td><td>D</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-19</td><td>B</td><td>B</td><td>TO</td><td>D</td><td>B</td><td>D</td><td>B</td>

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<td></td><td>FRI</td><td>CDR1</td><td>FR2</td><td>CDR2</td><td>FR3</td><td>cW</td><td> -</td>

<td>Hu3G8VL-20</td><td>B</td><td>B</td><td>.......TO........</td><td>D</td><td></td><td>AND</td><td>B</td>

<td>Hu3G8VL-21</td><td>B</td><td>C</td><td>TO</td><td>D</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-22</td><td>B</td><td>C</td><td>TO</td><td>D</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-23</td><td>B</td><td>C</td><td>TO</td><td>D</td><td>B</td><td>AND</td><td>B</td>

<td>Hu3G8VL-24</td><td>B</td><td>D</td><td>TO</td><td>D</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-25</td><td>B</td><td>D</td><td>TO</td><td>D</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-26</td><td>B</td><td>D</td><td>TO</td><td>D</td><td>B</td><td>AND</td><td>B</td>

<td>Hu3G8VL-27</td><td>B</td><td>AND</td><td>TO</td><td>D</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-28</td><td>B</td><td>AND</td><td>TO</td><td>D</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-29</td><td>B</td><td>AND</td><td>TO</td><td>D</td><td>B</td><td>AND</td><td>B</td>

<td>Hu3G8VL-30</td><td>B</td><td>TO</td><td>TO</td><td>D</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-31</td><td>B</td><td>TO</td><td>TO</td><td>D</td><td>B</td><td>AND</td><td>B</td>

<td>Hu3G8VL-32</td><td>B</td><td>TO</td><td>TO</td><td>H</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-33</td><td>B</td><td>TO</td><td>TO</td><td>I</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-34</td><td>B</td><td>TO</td><td>TO</td><td>J</td><td>B</td><td>TO</td><td>B</td>

<td>Hu3G8VL-35</td><td>B</td><td>B</td><td>TO</td><td>H</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-36</td><td>B</td><td>C</td><td>TO</td><td>H</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-37</td><td>B</td><td>AND</td><td>TO</td><td>H</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-38</td><td>B</td><td>B</td><td>TO</td><td>I</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-39</td><td>B</td><td>C</td><td>TO</td><td>I</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-40</td><td>B</td><td>AND</td><td>TO</td><td>I</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-41</td><td>B</td><td>B</td><td>TO</td><td>J</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-42</td><td>B</td><td>C</td><td>TO '</td><td>J</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-43</td><td>B</td><td>AND</td><td>TO</td><td>J</td><td>B</td><td>D</td><td>B</td>

<td>Hu3G8VL-44</td><td>B</td><td>TO</td><td>TO</td><td>K</td><td>B</td><td>TO</td><td>B</td>

* The letters in Table 6A refer to sequences in Tables 6B-H.

Table 6B. FRI

<td>TO</td><td>B</td><td>RESIDUE</td>

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

<td>D</td><td>D</td><td> 1</td>

<td>T</td><td>I</td><td> 2</td>

<td>V</td><td>V</td><td> 3</td>

<td>L</td><td>M</td><td> 4</td>

<td>T</td><td>T</td><td> 5</td>

<td>Q</td><td>Q</td><td> 6</td>

<td>s</td><td>s</td><td> 7</td>

<td>P</td><td>P</td><td> 8</td>

<td>TO</td><td>D</td><td> 9</td>

<td>S</td><td>S</td><td> 10</td>

<td>L</td><td>L</td><td> 11</td>

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<td>TO</td><td>TO</td><td> 12</td>

<td>V</td><td>V</td><td> 13</td>

<td>S</td><td>S</td><td> 14</td>

<td>L</td><td>L</td><td> 15</td>

<td>G</td><td>G</td><td> 16</td>

<td>Q</td><td>AND</td><td> 17</td>

<td>R</td><td>R</td><td> 18</td>

<td>TO</td><td>TO</td><td> 19</td>

<td>T</td><td>T</td><td> 20</td>

<td>I</td><td>I</td><td> 21</td>

<td>S</td><td>N</td><td> 22</td>

<td>c</td><td>C</td><td> 23</td>

<td> 168</td><td> 169</td><td>SEQ ID NO</td>

<td>SEQ ID NO.</td><td>Sequence</td>

<td> 168</td><td>DTVLTQSPASLAVSL</td>

<td> 169</td><td>DIVMTQSPDSLAVSL</td>

Table 6C. CDR1

<td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>G</td><td>RESIDUE</td>

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

<td>K</td><td>R</td><td>K</td><td>K</td><td>K</td><td>K</td><td>K</td><td> 24</td>

<td>TO</td><td>TO</td><td> 8</td><td>TO</td><td>TO</td><td>TO</td><td>TO</td><td> 25</td>

<td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td> 26</td>

<td>Q</td><td>Q</td><td> 9</td><td>Q</td><td>Q</td><td> 9</td><td> 9</td><td> 27</td>

<td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>s</td><td>27A</td>

<td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>27B</td>

<td>D</td><td>D</td><td> 0</td><td>D</td><td>D</td><td>D</td><td>D</td><td>27C</td>

<td>F</td><td>F</td><td></td><td>F '</td><td>F</td><td>F</td><td>F</td><td>': 27i&gt;,</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 28</td>

<td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td>G</td><td> 29</td>

<td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td>D</td><td> 30</td>

<td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td> 31</td>

<td>F</td><td>F</td><td>F</td><td>Y</td><td>F</td><td>p</td><td>Y</td><td> 32</td>

<td>M</td><td>M</td><td>M</td><td>M</td><td>L</td><td>M</td><td>L</td><td> 33</td>

<td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>TO</td><td>TO</td><td> 34</td>

<td> 170</td><td> 171</td><td> 172</td><td> 173</td><td> 174</td><td> 175</td><td> 176</td><td>27 SEQ ID NO</td>

<td> 177</td><td> 178</td><td> 179</td><td> 180</td><td> 181</td><td> 182</td><td> 183</td><td>27ASEC ID NO</td>

<td> 184</td><td> 185</td><td> 186</td><td> 187</td><td> 188</td><td> 189</td><td> 190</td><td>27BSEC ID NO</td>

<td> 191</td><td> 192</td><td> 193</td><td> 194</td><td> 195</td><td> 196</td><td> 197</td><td>27CSEC ID NO</td>

<td> 198</td><td> 199</td><td> 200</td><td> 201</td><td> 202</td><td> 203</td><td> 204</td><td>27DSEC ID NO</td>

<img file="MX348166B_D0118.tif" />

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<td>SEQ ID NO.</td><td></td>

<td> 170</td><td>KASQDGDSFMN</td>

<td> 171</td><td>RASQDGDSFMN</td>

<td> 172</td><td>KSSQDGDSFMN</td>

<td> 173</td><td>KASQDGDSYMN</td>

<td> 174</td><td>KASQDGDSFLN</td>

<td> 175</td><td>KASQDGDSFMA</td>

<td> 176</td><td>KASQDGDSYLA</td>

<td> 177</td><td>KASSDGDSFMN</td>

<td> 178</td><td>RASSDGDSFMN</td>

<td> 179</td><td>KSSSDGDSFMN</td>

<td> 180</td><td>KASSDGDSYMN</td>

<td> 181</td><td>KASSDGDSFLN</td>

<td> 182</td><td>KASSDGDSFMA</td>

<td> 183</td><td>KASSDGDSYLA</td>

<td> 184</td><td>KASVDGDSFMN</td>

<td> 185</td><td>RASVDGDSFMN</td>

<td> 186</td><td>KSSVDGDSFMN</td>

<td> 187</td><td>KASVDGDSYMN</td>

<td> 188</td><td>KASVDGDSFLN</td>

<td> 189</td><td>KASVDGDSFMA</td>

<td> 190</td><td>KASVDGDSYLA</td>

<td> 191</td><td>KASDDGDSFMN</td>

<td> 192</td><td>RASDDGDSFMN</td>

<td> 193</td><td>KSSDDGDSFMN</td>

<td> 194</td><td>KASDDGDSYMN</td>

<td> 195</td><td>KASDDGDSFLN</td>

<td> 196</td><td>KASDDGDSFMA</td>

<td> 197</td><td>KASDDGDSYLA</td>

<td> 198</td><td>KASFDGDSFMN</td>

<td> 199</td><td>RASFDGDSFMN</td>

<td> 200</td><td>KSSFDGDSFMN</td>

<td> 201</td><td>KASFDGDSYMN</td>

<td> 202</td><td>KASFDGDSFLN</td>

<td> 203</td><td>KASFDGDSFMA</td>

<td> 204</td><td>KASFDGDSYLA</td>

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Table 6D. FR2

<td>TO</td><td>RESIDUE</td>

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

<td>W</td><td> 35</td>

<td>Y</td><td> 36</td>

<td>Q</td><td> 37</td>

<td>Q</td><td> 38</td>

<td>K</td><td> 39</td>

<td>P</td><td> 40</td>

<td>G</td><td> 41</td>

<td>Q</td><td> 42</td>

<td>P</td><td> 43</td>

<td>P</td><td> 44</td>

<td>K</td><td> 45</td>

<td>L</td><td> 46</td>

<td>L</td><td> 47</td>

<td>I</td><td> 48</td>

<td>Y</td><td> 49</td>

<td> 205</td><td>ID NO</td>

<td>SECID NO.</td><td>Sequence</td>

<td> 205</td><td>WYQQKAPGQPPKLLIY</td>

Table 6E. CDR2

<td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>G</td><td>H</td><td>I</td><td>J</td><td>K</td><td>RESIDUE</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>T</td><td>D</td><td>W</td><td>T</td><td>D</td><td>D</td><td>S</td><td>S</td><td>s</td><td>T</td><td>T</td><td> 50</td>

<td>T</td><td>TO</td><td>TO</td><td>T</td><td>TO</td><td>TO</td><td>TO</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 51</td>

<td>S</td><td>S</td><td>S</td><td>s</td><td>S</td><td>S</td><td>S</td><td>S</td><td>s</td><td>S</td><td>S</td><td> 52</td>

<td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td>N</td><td> 53</td>

<td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td>L</td><td> 54</td>

<td>AND</td><td>AND</td><td>AND</td><td>Ξ</td><td>AND</td><td>TO</td><td>Q</td><td>AND</td><td>Q</td><td>Q</td><td>Q</td><td> 55</td>

<td>S</td><td>S</td><td>S</td><td>T</td><td>T</td><td>T</td><td>S</td><td>S</td><td>S</td><td>S</td><td>S</td><td> 54</td>

<td> 206</td><td> 207</td><td> 208</td><td> 209</td><td> 210</td><td> 211</td><td> 212</td><td> 213</td><td> 214</td><td> 215</td><td> 216</td><td>SEQ ID NOT</td>

<img file="MX348166B_D0120.tif" />

<td>SEQ ID NOT.</td><td>Sequence</td>

<td> 206</td><td>TTSNLES</td>

<td> 207</td><td>DASNLES</td>

<td> 208</td><td>WASNLES</td>

<td> 209</td><td>TTSNLET</td>

<td> 210</td><td>DASNliET</td>

<td> 211</td><td>DASNLAT</td>

<td> 212</td><td>SASNLQS</td>

<td> 213</td><td>STSNLES</td>

<td> 214</td><td>STSNLQS</td>

<td> 215</td><td>TTSNLQS</td>

<td> 16</td><td>TTSSLQS</td>

Table 6F: FR3

<td>TO</td><td>B</td><td>RESIDUE</td>

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

<td>G</td><td>G</td><td> 57</td>

<td>I</td><td>V</td><td> 58</td>

<td>P</td><td>P</td><td> 59</td>

<td>TO</td><td>D</td><td> 60</td>

<td>R</td><td>R</td><td> 61</td>

<img file="MX348166B_D0121.tif" />

<td>TO</td><td>B</td><td>RESIDUE___</td>

<td>F</td><td>F</td><td> 62</td>

<td>S</td><td>S</td><td> 63</td>

<td>TO</td><td>G</td><td> 64</td>

<td>S</td><td>S</td><td> 65</td>

<td>G</td><td>G</td><td> 66</td>

<td>S</td><td>S</td><td> 67</td>

<td>G</td><td>G</td><td> 68</td>

<td>T</td><td>T</td><td> 69</td>

<td>D</td><td>D</td><td> 70</td>

<td>F</td><td>F</td><td> 71</td>

<td>T</td><td>T</td><td> 72</td>

<td>L</td><td>L</td><td> 73</td>

<td>N</td><td>T</td><td> 74</td>

<td>I</td><td>I</td><td> 75</td>

<td>H</td><td>s</td><td> 76</td>

<td>TO</td><td>B</td><td>RESIDUE</td>

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

<td>P</td><td>S</td><td> 77</td>

<td>V</td><td>L</td><td> 78</td>

<td>AND</td><td>Q</td><td> 79</td>

<td>AND</td><td>TO</td><td> 80</td>

<td>AND</td><td>AND</td><td> 81</td>

<td>D</td><td>D</td><td> 82</td>

<td>T</td><td>V</td><td> 83</td>

<td>TO</td><td>TO</td><td> 84</td>

<td>T</td><td>V</td><td> 85</td>

<td>Y</td><td>Y</td><td> 86</td>

<td>Y</td><td>Y</td><td> 87</td>

<td>c</td><td>c</td><td> 88</td>

<td> 217</td><td> 218</td><td>SEQ ID NO</td>

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<td>SEQ ID NO.</td><td>SEQUENCE ---------</td>

<td> 217</td><td>GIPARFSASGSGTDFTLNIHFVEEEDTATYYC</td>

<td> 218</td><td>GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC</td>

Table 6G: CDR3

<td>TO.</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>RESIDUE</td>

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

<td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td> 89</td>

<td>Q</td><td>Q</td><td>Q</td><td>Q</td><td>Q</td><td> 90</td>

<td>s</td><td>S</td><td>s</td><td>s</td><td>s</td><td> 91</td>

<td>N</td><td>Y</td><td>Y</td><td>N</td><td>N</td><td> 92</td>

<td>AND</td><td>s</td><td>AND</td><td>S</td><td>AND</td><td> 93</td>

<td>D</td><td>T</td><td>D</td><td>D</td><td>T</td><td> 94</td>

<td>P</td><td>P</td><td>P</td><td>P</td><td>P</td><td> 95</td>

<td>Y</td><td>Y</td><td>Y</td><td>Y</td><td>Y</td><td> 96</td>

<td>T</td><td>T</td><td>T</td><td>T</td><td>T</td><td> 97</td>

<td> 219</td><td> 220</td><td> 221</td><td> 222</td><td> 223</td><td>SEQ ID NOT</td>

<td>SEQ ID NO.</td><td>SEQUENCE</td>

<td> 219</td><td>QQSNEDPYT</td>

<td> 220</td><td>QQSYSTPYT</td>

<td> 221</td><td>QQSYEDPYT</td>

<td> 222</td><td>QQSNSDPYT</td>

<td> 223</td><td>QQSNETPYT</td>

<td>TO</td><td>B</td><td>RESIDUE</td>

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

<td>F</td><td>P</td><td> 98</td>

<td>G</td><td>G</td><td> 99</td>

<td>G</td><td>Q</td><td> 100</td>

<td>Q</td><td>G</td><td> 101</td>

<td>T</td><td>T</td><td> 102</td>

<td>K</td><td>K</td><td> 103</td>

<td>L</td><td>L</td><td> 104</td>

<td>AND</td><td>AND</td><td> 105</td>

<td>I</td><td>I</td><td> 106</td>

<td>K</td><td>K</td><td> 107</td>

<td> '224</td><td> 225</td><td>SÉQ ID NO</td>

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<td>SEQID NO,</td><td>ΙΝΓΛ SEQUENCE</td><td>rrn iai</td>

<td> 224</td><td>FGGGTKLEIK</td><td rowspan="2"></td>

<td> 225</td><td>FGQGTKLEIK</td>

Illustrative VL domains of the CD16 binding proteins of the invention have the sequence of 3G8VL, HU3G8VL-1 or Hu3G8VL-43, (SEQ ID NO: 82, SEQ ID NO: 71 and SEQ ID NO: 72, respectively) as shown shown in Tables 5 and 6. Illustrative nucleotide sequences encoding 3G8VL (SEQ ID NO: 82) and Hu3G8VL-1 (SEQ ID NO: 71) are provided in SEQ ID NO: 83 and SEQ ID NO: 84, respectively. .

The VL domain can have a sequence that differs from that of Hu3G8VL-1 (SEQ ID NO: 71) by zero, one, or at least two, at least 3, at least 4, at least 5, at least 6, at least least 7, at least 8, or at least 9 of the substitutions shown in Table 2. These substitutions are believed to result in increased affinity for CD16A and / or reduce the immunogenicity of the CD16A binding protein when administered to human. In certain embodiments, the degree of sequence identity at the remaining positions is at least about 80%, at least about 90%, at least about 95%, or at least about 98%.

For illustration and not limitation, the sequences of a number of VL domains of CD16A binding proteins are shown in Table 6. Light chains comprise these sequences fused to a human Ck constant domain, which

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INSTITUTE MlüCANC Jk '

OF THE NO. 7TH SO

INDUSTRIAL ¹⁸¹ are co-expressed with a Hu3G8VH heavy chain (described above) to form tetrameric antibodies, and the binding of the antibodies to CD16A was measured to evaluate the effect of amino acid substitutions compared to the VL domain Hu3G8VL-1 (SEQ ID NO: 71). Constructs where the VL domain has the sequence of hu3G8VL-1, 2, 3, 4, 5, 10, 16, 18, 19, 21, 22, 24, 27, 28, 32, 33, 34, 35, 36 , 37, and 42 showed high affinity binding and hu3G8VL-15, 17, 20, 23, 25, 26, 29, 30, 31, 38, 39, 40, and 41 showed intermediate binding CD16A-binding proteins comprising the VL domains of hu3G8VL-1, hu3G8VL-22, and hu3G8VL-43 which are considered to have particularly favorable binding properties (SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 72, respectively).

COMBINATION OF THE VL AND / 0 VH DOMAINS

As is known in the art and described elsewhere herein, immunoglobulin heavy and light chains can be recombinantly expressed under conditions where they associate to produce a diabody, which can be combined in vitro. Thus it will be appreciated that a 3G8-derived VL domain described herein may be a combined 3G8-derived VL domain described herein to produce a CD16A binding diabody and all such combinations are contemplated.

For illustration and not limitation, the examples

187 IMPI ^

MEXICAN INSTITUTE

OF THE PHOMBDAD CnWÍ

INr * »miAl ^^” &lt;12 of useful CD16A diabodies are those comprising at least one VH domain and at least one VL domain, where the VH domain is from hu3G8VH-l, hu3G8VH-22, or hu3G8VH-5 (SEQ ID NO: 68, SEQ ID NO: 70 and SEQ ID NO: 69, respectively) and the VL domain is from hu3G8VL-1, hu3G8VL-22 or hu3G8VL-43 (SEQ ID NO: 71, SEQ ID NO: 73 and SEQ ID NO: 41, respectively). In particular, humanized antibodies comprising hu3G8VH-22 (SEQ ID NO: 22) and any, hu3G8VL-1, hu3G8VL-22 OR hu3G8VL-43 (SEQ ID NO: 71, SEQ ID NO: 70 and SEQ ID NO: 72 , respectively), or hu3G8VH-5 (SEQ ID NO: 69) and hu3G8VL-1 (SEQ ID NO: 71) have favorable properties.

It will be appreciated by one of skill in the art that the sequences of the VL and VH domains described herein can further be modified through known methods such as affinity maturation (see, Schier et al. (1996) Isolation Of Picomolar Affinity Anti- C-ErbB-2 Single-Chain Fv By Molecular Evolution Of The Complementarity Determining Regions In The Center Of The Antibody Binding Site, J. Mol. Biol. 263: 551-567; Daugherty et al. (1998) Antibody Affinity Maturation Using Bacterial Surface Display, Protein Eng. 11: 825-832; Boder et al. (1997) Yeast Surface Display For Screening Combinatorial! Polypeptide Librarles, Nat. Biotechnol. 15: 553-557; Boder et al. (2000) Directed

Evolution Of Antibody Fragmente With Monovalent Femtomolar Antigen-Binding Affinity, Proc. Nati. Acad. Sci. USA¹⁸⁸ IMPIOUS"

MEXICAN INSTITUTE »E LA TtOPIEDA L

INnUSTUIAI

97: 10701-10705; Hudson et al. (2003) Engineered Antibodies, Nature Medicine 9: 129-39). For example, CD16A binding proteins can be modified using affinity maturation techniques to identify proteins with increased affinity for CD16A and / or decreased affinity for CD16B.

An exemplary CD16 binding protein is the mouse 3G8 antibody. The amino acid sequence comprising the VH and CL domains of humanized 3G8 are described in Figures 2, 9, 14 and are set out in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72.

Diabodies Comprising Faith or Its Portions

The invention encompasses molecules and diabody comprising Fe domains or portions thereof (eg, a CH2 or CH3 domain). In certain embodiments, the Fe domain, or portions thereof, comprise one or more constant domains (s) from the Fe region of IgG2, IgG3, or IgG4 (eg, CH2 or CH3). In other embodiments, the invention encompasses molecules that comprise an Fe domain or one of its portions, wherein the Fe domain or one of its portions comprises at least one amino acid modification (eg, substitution) relative to the Fe domain of the type comparable wild type or one of

<img file="MX348166B_D0124.tif" />

your portions. Variable Fe domains are well known in the art, and are used primarily to alter the phenotype of the antibody comprising the variant Fe domain as tested in any binding activity or effector function assays known in the art, eg ELISA, SPR analysis, or ADCC. Such variant Fe domains, or portions thereof, have use in the present invention by conferring or modifying the effector function exhibited by a diabody molecule of the invention comprising an Fe domain (or a portion thereof) as functionally assayed, for example, in an NK or macrophage dependent assay. Fe domain variants identified as altering effector function are described in international application WO04 / 063351, US Patent Application Publications 2005/0037000 and 2005/0064514, US Provisional Applications 60 / 626,510, filed November 10. 2004, 60 / 636,663, filed December 15, 2004, and 60 / 781,564, filed March 10, 2006, and U.S. Patent Applications 11/271, 140, filed November 10, 2005, and 11 / 305,787, filed December 15, 2005, concurrent applications from the Inventors, each of which is incorporated by reference in its entirety.

In other embodiments, the invention encompasses the use of any Fe variant known in the art, such as the ¹⁹⁰INSTITUTO MIXlCANO DE LA rtOKEDAO C ^ MgEjLlK ^ INDUSTRIA!

described in Duncan et al. (1988) Localization Of The Binding Site For The Human High-Affinity Fe Receptor On IgG, Nature 332: 563-564; Lund et al. (1991) Human Fe Gamma RI And Fe Gamma RII Interact With Distinct But Overlapping Sites On Human IgG, J. Immunol. 147: 2657-2662; Lund et al. (1992) Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fe Gamma RII, Mol. Immunol. 29: 53-59; Alegre et al. (1994) A Non-Activating Humanized Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo, Transplantation 57: 1537-1543; Hutchins et al. (1995) Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-1H, Proc. Nati. Acad. Sci. USA 92: 11980-11984; Jefferis et al. (1995) Recognition Sites On Human IgG For Fe Gamma Receptors: The Role Of Glycosylation, Immunol. Lett. 44: 111-117; Lund et al. (1995) Oligosaccharide-Protein Interactions In IgG Can Modulate Recognition By Fe Gamma Receptors, FASEB J. 9: 115-119; Jefferis et al. (1996) Modulation Of Fe (Gamma) R And Human Complement Activation By IgGS-Core Oligosaccharide Interactions, Immunol. Lett. 54: 101-104; Lund et al. (1996) Multiple Interactions Of Igg With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fe Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains, J. Immunol. 157: 4963-4969; Armor et al. (1999) Recombinant Human IgG

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IMPI ΙΜΤΠντοΜβΚΑΝΟ

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I Binding And Mon

Immunol

The C1Q Binding Site On

A Human IgGl Fe, J.

191

Molecules Lacking Fcgamma Receptor Triggering Activities, Eur. J. Idusogie et al. (2000) Mapping Of Rituxan, A Chimeric Antibody With

Immunol. 164: 4178-4184; Reddy et al. (2000) Elimination Of Fe Receptor-Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4, J. Immunol. 164: 1925-1933; Xu et al. (2000) In vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol. 200: 16-26; Idusogie et al. (2001) Engineered Antibodies With Increased Activity To Recruit Complement, J. Immunol. 166: 2571-2575, - Shields et al. (2001) High Resolution Mapping Of The Binding Site On Human IgGl For Fe gamma RI, Fe gamma RII, Fe gamma RUI, And FcRn And Design Of IgGl Variants With Improved Binding To The Fe gamma R, J. Biol. Chem. 276: 65916604; Jefferis et al. (2002) Interaction Sites On Human IgGFe For FcgammaR: Current Models, Immunol. Lett. 82: 57-65; Presta et al. (2002) Engineering Therapeutic Antibodies For Improved Function, Biochem. Soc. Trans. 30: 487-490); US

5,624,821; US 5,885,573; US 6,194,551; PCT WO 00/42072; PCT WO 99/58572; each of which is incorporated herein by reference in its entirety.

In certain embodiments, the one or more modifications to the amino acids of the Fe region reduce the affinity and avidity of the Fe region and thus the Fe region molecule.

<img file="MX348166B_D0126.tif" />

192

IMPÍ IIwrnvro mocano INDUSTRIAL OtUmOHIDAD diabody of the invention, a specific modality, for one or more Fc and R receptors. The invention encompasses diabodies comprising a variant Fe region, or a portion thereof, wherein the variant Fe region comprises at least one amino acid modification relative to the wild-type Fe region, the variant Fe region of which only binds to a FcyR, where the FcyR is FcyRIIIA. In another specific embodiment, the invention encompasses diabodies comprising a variant Fe region, or a portion thereof, wherein the variant Fe region comprises at least one amino acid modification relative to the wild-type Fe region, the variant Fe region of which only binds to a FcyR, where the FcyR is FcyRIIA. In another specific embodiment, the invention encompasses diabodies comprising a variant Fe region, or a portion thereof, wherein the variant Fe region comprises at least one amino acid modification relative to the wild-type Fe region, the variant Fe region of which only binds to an FcyR, where the FcyR is FcyRIIB. In certain embodiments, the invention encompasses molecules that comprise a variant Fe domain wherein the variant confers or mediates increased ADCC activity and / or increased binding to FcyRIIA (CD32A), relative to a molecule that does not comprise an Fe domain or that comprises a wild-type Fe domain, as measured using methods known to those skilled in the art and described in

<img file="MX348166B_D0127.tif" />

Present. In alternative embodiments, the invention encompasses --------—— i * j - - x_-i— --j — m — lj— molecules comprising a variant Fe domain wherein the variant confers or mediates activity Decreased ATCC (or other effector function) and / or increased binding to FcyRIIB (CD32B), relative to a molecule that does not comprise a CF domain or that comprises a wild-type domain, as measured using methods known to those skilled in the art. technique and described herein.

The invention also encompasses the use of an Fe domain that comprises domains or regions of two or more IgG isotypes. As is known in the art, amino acid modifications of the Fe region can profoundly affect Fe-mediated effector function and / or binding activity. However, these alterations in functional characteristics can be further refined and / or manipulated when implemented in the context of selected IgG isotypes. Similarly, the native characteristics of Fe isotype can be manipulated through one or more amino acid modifications. Multiple IgG isotypes (i.e., IgGl, IgG2, IgG3, and IgG4) exhibit different physical and functional properties including serum half-life, complement fixation, FcyR binding affinity, and effector function affinities (e.g., ADCC, CDC ) due to differences in the amino acid sequences of their hinge and / or Fe domains. In certain embodiments, the amino acid modification and the Fe region independently select on the basis of their respective activities, separate binding, and / or effector function in order to modify a diabody with the desired characteristics. In most embodiments, such amino acid modifications and those of the IgG / Fc hinge regions have been separately tested for binding activity and / or effector function as described herein or known herein. technique in the context of an IgGl. In certain embodiments, the amino acid modification and the hinge / Fc IgG region display similar functionality, eg, increased affinity for FcyRIIA, when separately tested for FcyR binding by effector function in the context of the diabody molecule. or another molecule that contains Fe (for example, and immunoglobulin). The combination of the amino acid modification and the selected Fe IgG region then act additively or, more preferably, synergistically to modify functionality in the diabody molecule of the invention, relative to the diabody molecule of the invention comprising a region Wild-type faith. In other embodiments, amino acid modification and Fe IgG regions display opposite functionalities, eg, affinity for FcyRIIA, increased and decreased, respectively, when separately tested for

195

<img file="MX348166B_D0128.tif" />

IMPI

MEXICAN INSTIIUTE

DB THE PROPERTY

IND »» STRIAL FcyR binding and / or effector function in the context of the diabody molecule or other Fe-containing molecule (eg, an immunoglobulin) comprising a wild-type Fe region as described herein or as I know in the technique; the combination of the opposite amino acid modification and the selected IgG region then act to selectively anneal or reduce a specific functionality in the diabody of the invention relative to a diabody of the invention that contains an Fe region or that comprises an Fe region of the wild of the same isotype. Alternatively, the invention encompasses variant Fe regions comprising combinations of art-known amino acid modifications and selected IgG regions exhibiting new properties, the properties of which were not detectable when the modifications and / or regions were independently tested as described in Present.

The functional characteristics of the multiple IgG isotypes, and their domains, are well known in the art. The amino acid sequences of IgGl, IgG2, IgG3 and IgG4 are presented in Figures 1A-1B. Selection and / or combination of two or more specific IgG isotype domains for use in the methods of the invention can be based on any known parameter of the parent isotypes including affinity to FcyR (Table 7; Flesch et al. (2000)

1 «IMPI ^

WSTTWTO M »1CANC

Di LA paopif.DAr tVZSHSl industrial

Functions Of The Fe Receptors For Immunoglobulin G, J.

Clin. Lab. Anal. 14: 141-156; Chappel et al. (1993) Identification Of A Secondary Fe Gamma RI Binding Site Within A Genetically Engineered Human IgG Antibody, J. Biol.

Chem. 33: 25124-25131; Chappel et al. (1991) Identification Of The Fe Gamma Receptor Class I Binding Site In Human IgG Through The Use Of Recombinant IgGl / IgG2 Hybrid And PointMutated Antibodies, Proc. Nati. Acad. Sci. USA 88: 9036-9040, each of which is incorporated herein by reference in its entirety). For example, the use of IgG regions or domains and isotypes that exhibit limited or no binding to FcyRIIB, eg, IgG2 or IgG4, may find particular use when it is desired to modify a diabody to maximize binding to an activating receptor and minimize binding to an inhibitory receptor. Similarly, the use of Fe regions or domains of IgG isotypes are known to preferentially bind Clq or FcyRIIIA, eg, IgG3 (Bruggemann et al. (1987) Comparison Of The Effector Functions Of Human Immunoglobulins Using A Matched Set Of Chimeric Antibodies, J. Exp. Med. 166: 1351-1361), can be combined with art-known Fe amino acid modifications to enhance ADCC, to modify a diabody molecule such as the activity of the effector function, eg, complement activation or ADCC, is maximized.

197

<img file="MX348166B_D0129.tif" />

Table 7. General characteristics of IgG binding to FcyR, adapted from Flesch and Neppert, 1999, J. Clin. Lab.

Anal. 14: 141-156

<td>Receiver</td><td>Affinity for IgG estimated (M¹)</td><td>Relative Affinity</td>

<td>FcyRI</td><td> 10⁸ - 10⁹</td><td>IgG3&gt; IgGl &gt;&gt; IgG4 non-binding: IgG2</td>

<td>FcyRIIA R^{131 TO}</td><td> &lt;10⁷</td><td>IgG3&gt; IgGl non-binding: IgG2, IgG4</td>

<td>FcyRIIA H^{131 TO}</td><td> &lt;10⁷</td><td>IgG3&gt; IgGl&gt; IgG2 non-binding: IgG4</td>

<td>FcyRIIB ^TO</td><td> &lt;10⁷</td><td>IgG3&gt; IgGl&gt; IgG4 non-binding: IgG2</td>

<td>FcyRI II</td><td> &lt;10⁷</td><td>IgG3 = IgGl non-binding: IgG2, IgG4</td>

binds only to IgG in complex

MOLECULAR CONJUGATES

The diabody molecules of the invention can be recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to heterologous polypeptides (i.e., an unrelated polypeptide; or a portion thereof, preferably at least 10, at least 20, to minus 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least

198

<img file="MX348166B_D0130.tif" />

DELA MOFIEDAP 1NDVST1IAI

100 amino acids of the polypeptide to generate the fusion proteins. Fusion does not necessarily need to be direct, but can occur through linker sequences.

In addition, the diabody molecules of the invention (ie, polypeptide) can be conjugated to a therapeutic agent or a drug moiety that modifies a biological response. As an alternative to direct conjugation, due to the multiple epitope binding sites on the multivalent, e.g., tetravalent, diabody molecules of the invention, at least one diabody binding region can be designed to bind the therapeutic agent or the desired drug fraction without affecting diabody binding.

The therapeutics of the drug moieties are not construed as limiting the classical chemical therapeutics. For example, the drug moiety can be a protein or polypeptide that processes a desired biological activity. Such proteins may include, for example, a toxin such as abrin, resin A, pseudomonas exotoxin (i.e. PE-40) or diphtheria toxin, resin, gelonin, and the carmine grass antiviral protein, a protein such as tumor necrosis factor, interferons including, but not limited to, interferon (IFN-a), β-interferon (IFN-β), nerve growth factor (NGF), growth factor derived from

199

<img file="MX348166B_D0131.tif" />

IMPI

INTUI UTO ΐβΜΚΝ i nt THE PROPERTY IHDUSTffFA platelet (PDGF), tissue plasminogen activator (TPA), an apoptotic agent (e.g. TNF-a, TNF-β, AIM I as described in PCT Publication No. WO 97 / 33899), AIM II (see, PCT Publication No. WO 97/34911), the Fas ligand, and VEGI (PCT Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent (for example angiostatin or endostatin), or a biological response modifier such as, for example, a lymphokine (for example interleukin-1 (IL-1 ), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte ghoul colony stimulating factor (GM-CSF), and granulocyte colony stimulating factors (G-CSF), macrophage colony, (MCSF), or growth factor (eg, growth hormone (GH); proteases or ribonucleases.

The diabody molecules of the invention (ie, polypeptide) can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequences is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available. As described in Gentz ​​et al. (1989) Bioassay For Trans Activation Using Purified Human Immunodeficiency Virus TATEncoded Protein: Trans-Activation Requires mRNA Synthesis,

200

<img file="MX348166B_D0132.tif" />

IMPI institutomexicUno ne industrial Property

Proc. Nati. Acad. Sci. USA, 86: 821-824, _ For example, hexa-histidine provides for convenient purification of the fusion protein. Other useful peptide tags for purification include, but are not limited to, the hemagglutinin tag, HA, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al. (1984) The Structure of An Antigenic Determinant In A Protein, Cell, 37: 767-778) and the Bandera label (Knappik et al. (1994) An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments ”, Biotechniques, 17 (4): 754761).

Additional fusion proteins can be generated through gene swapping, motif swapping, exon swapping, and / or codon swapping techniques (collectively referred to as DNA swapping). DNA swapping can be used to alter the activities of the molecules of the invention (eg, epitope binding sites with higher affinities and lower degrees of dissociation). See generally US Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al. (1997) Applications Of DNA Shuffling To Pharmaceuticals And Vaccines, Curr. Biotechnol Opinion. 8: 724-733; Harayama (1998) Artificial Evolution By DNA Shuffling, Trends

<img file="MX348166B_D0133.tif" />

201

IMPI

IMWWUTO MIXICANO

MU INDUSTRIAL PROPERTY

Biotechnol. 16: 76-82; Hansson et al. (1999) Evolution Of

Differential Substrate Specificities In Mu Class Glutathione

Transferases Probed By DNA Shuffling, J. Mol. Biol. 287: 265

276, - and Lorenzo et al. (1998) PCR-Based Method For The Introduction Of Mutations In Genes Cloned And Expressed In Vaccinia Virus, BioTechniqu.es 24: 308-313 (each of these patents and publications are incorporated herein by reference in their entirety). The diabody molecules of the invention, or the nucleic acids encoding the molecules of the invention, can further be altered by being subjected to random mutagenesis via error prone PCR, random nucleotide insertion or other methods prior to recombination. One or more portions of the polynucleotide that encodes a molecule of the invention can be recombined with one or more components, motifs, sections, parts, domains, fragments, etc., of one or more heterologous molecules.

The present invention also encompasses diabody molecules of the invention conjugated to or that immunospecifically recognize a diagnostic or therapeutic agent or any other molecule for which it is desired that the serum half-life is increased / decreased and / or activated towards a particular subgroup. cell. The molecules of the invention can be used diagnostically to, for example, monitor the development or progress of a

<img file="MX348166B_D0134.tif" />

202

IMPI Mexican Institute

DE u PROP1HMO INDUSTRIAD disease, disorder or infection as -paT-tQ of a clinical test procedure, for example, to determine the efficiency of a given treatment regimen. Detection can be facilitated through coupling of the molecules of the invention with a detectable substance or through immunospecific recognition of the molecules for detectable substances. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated either directly to the molecules of the invention or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art, or the molecule can recognize Immospecifically the detectable substance: immunospecific binding of the substance. See, for example, US Patent No. 4,741,900, for metal ions that can be conjugated to antibodies to be used as diagnostics in accordance with the present invention. Such diagnosis and detection can be accomplished by designating the molecules to immunospecifically recognize the detectable substance or through coupling the molecules of the invention to detectable substances that include, but are not limited to,

IMPÍ • NrmUTOMEXtCANO limit to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholine esterase; prosthetic group complexes such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to luminol, - bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to bismuth (²¹³Bi), coal (¹⁴C), chrome (⁵¹Cr), cobalt (⁵⁷Co), fluorine (^1SF), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), Indian (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹³Ιη), iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetio (¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorus (³²P), praseodynium (¹⁴²Pr), promised (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (^10SRh), ruthenium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron-using metals using various positron emission tomography and non-radioactive paramagnetic metal ions.

204

MEXICAN INSTITUTE

OF THE PRÜPIEOAD VTSo ^ íiía. /

INDUSTRIAL SaMrb

The diabody molecules of the invention can be immunospecifically recognized or they can be conjugated to the therapeutic moiety such as a cytotoxin (eg, a cytostatic or cytocidal agent), a therapeutic agent or a radioactive element (eg, alpha emitters, gamma emitters, etc. ). Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthrazine dione, mitoxandehydrochlorine, actin-glutamicide, mitomycin, actin-glutamicide tetracaine, lidocaine, propanolol, and puromycin and their analogs or homologues. Therapeutic agents include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, mechlortamine, chlorambucil thioepa, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and platinum (II) cisdichlorodiamine (DDP) cisplatin), anthracyclines (for example, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (for example, dactinomycin (formerly actinomycin), bleomycin,

205 WICKED

IΝΓΓΤΠΛ · MEXICANO M LA MIOPIUttD industrial mithramycin, and anthramycin (AMC), and anti-mitotic agents (eg, vincristine and vinblastine).

Furthermore, a diabody molecule of the invention can be conjugated or designed to immunospecifically recognize therapeutic moieties such as radioactive materials or monocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials). In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecan-Ν, Ν ', N, N'-tetraacetic acid (DOTA) which can bind to the polypeptide through a linker molecule. Such linker molecules are commonly known in the art and are described in Denardo et al. (1998) Comparison Of 1,4,7,10-TetraazacyclododecaneN, N ', N, N'-Tetraacetic ñcid (DOTA) -Peptide-ChL6, A Novel Immunoconjugate With Catabolizable Linker, To 2Iminothiolane-2- [p- (bromoacetamido ) benzyl] -DOTA-ChL6 In

Breast Cancer Xenografts, Clin. Cancer Res. 4: 2483-2490; Peterson et al. (1999) Enzymatic Cleavage Of Peptide Linked Radiolabels From Immunoconjugates, Bioconjug. Chem. 10: 553-; and Zimmerman et al, (1999) A Triglycine Linker Improves Tumor Uptake And Biodistributions Of 67-Cu-Labeled Anti-Neuroblastoma mAb chCE7 F (ab) '2 Fragments ”, Nucí. Med. Biol. 26: 943-950 each of which is incorporated herein by reference in its entirety.

206

<img file="MX348166B_D0135.tif" />

Techniques for conjugating therapeutic moieties to polypeptides include, for example, Fe domains, which are well known, see, for example, Arnon et al., Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), 1985, pp. 243-56, Alan R. Liss, Inc.); Hellstrom et al, Antibodies For Drug Delivery, in Controlled Drug Delivery (2^to Ed.), Robinson et al. (eds.),

1987, pp. 623-53, Marcel Dekker, Inc.); Thorpe, Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies'84: Biological And Clinical Applications, Finchera et al. (eds.), 1985, pp. 475-506); Analysis, Resulte, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), 1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, Immunol. Rev., 62: 119-158.

The diabody molecule of the invention can be administered with or without a therapeutic moiety conjugated to it, administered alone, or in combination with a cytotoxic factor (s), and / or cytokine (s) to be used as a treatment. When administered alone, at least one epitope of a multivalent diabody molecule, for

207 iwtttut · Mexican DE LA PROHIBA · INDUSTRY * • tetravalent example can be designed to immunospecifically recognize a therapeutic agent, for example, a cytotoxic factor (s) and / or cytokine (s) that can be administered concurrently or subsequently to the molecule of the invention. In this form, the diabody molecule can specifically activate the therapeutic agent in a manner similar to direct conjugation. Alternatively, a molecule of the invention can be conjugated to an antibody to form a heteroconjugate antibody as described by Segal in US Patent No. 4,676,980, which is incorporated herein by reference in its entirety. The diabody molecules of the invention can also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Characterization of the Union of Diabody Molecules

The diabody molecules of the present invention can be characterized in a variety of ways. In particular, the molecules of the invention can be tested for the ability to immunospecifically bind to an antigen, for example, FcRIIIA or FcRIIB, or, when the molecule comprises an Fe domain (or a portion thereof)

208

ΓΜΡΙ ^^.

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for the ability to exhibit Fc-FcvR interactions, that is, the specific binding of an Fe domain (or one of its portions) to FcyR. Such an assay can be carried out in solution (for example, Houghten (1992) The Use Of Synthetic Peptide Combinatoria! Librarles For The Identification Of Bioactive Peptides, BioTechniques, 13: 412-421), in beads (Lam (1991) A New Type Of Synthetic Peptide Library For Identifying Ligand-Binding Activity, Nature, 354: 82-84, in chips (Fodor (1993) Multiplexed Biochemical Assays With Biologica! Chips, Nature, 364: 555-556), in bacteria (US Patent No. 5,223,409), in spores (US Patents Nos. 5,571,698; 5,403,484; and 5,223,409), in plasmids (Culi et al. (1992) Screening For Receptor Ligands Using Large Librarles Of Peptides Linked To The C Terminus Of The Lac Repressor, Proc. Nati. Acad. Sci. USA, 89: 1865-1869) or on phage (Scott et al. (1990) Searching For Peptide Ligands With An Epitope Library, Science, 249: 386-390; Devlin (1990) Random Peptide Librarles: A Source Of Specific Protein Binding Molecules, Science, 249: 404-406 ; Cwirla et al. (1990) Peptides On Phage: A Vast Library Of Peptides For Identifying Ligands, Proc. Nati. Acad. Sci. USA, 87: 63786382; and Felici (1991) Selection Of Antibody Ligands From A Large Library Of Oligopeptides Expressed On A Multivalent

Exposition Vector, J. Mol. Biol., 222: 301-310) (each of these references is incorporated by reference herein

<img file="MX348166B_D0136.tif" />

₂₀₉ ΪΜΡΪ

INSTITUTO ΜIXICAN Ο Of INDUSTRIAL PROPERTY in its entirety). Molecules that have -elent-iiii ^ ade — qjAe. immunospecifically bind to an antigen, eg, Fc and RIIIA, can be tested for their specificity and affinity for the antigen.

Molecules of the invention that have been modified to comprise multiple epitope-binding domains can be tested to immunospecifically bind to one or more antigens (eg, cancer antigen and reciprocal activity with other antigens (eg, FcyR)) or , when the molecule comprises an Fe domain (or one of its portions, for Fc-Fc and R interactions by any method known in the art. Immunoassays that can be used to test for immunospecific binding, reciprocal reactivity, and Fc-FcyR interactions include, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (linked immunosorbent assay). to enzyme), sandwich immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name a few. Such tests are routine and well known in the art (see,

<img file="MX348166B_D0137.tif" />

210

ΪΜΡΙ

MEíICANC INSTITUTE

DI LA HúüHEOAD INDUSTRIAL for example, Ausubel et al., Eds, 1994, Ciarrent ^ ProEo'colé ITT Molecular Biology, Vol. 1, John Wiley &amp; Sons, Inc., New York, which is incorporated herein by reference in its entirety) .

The binding affinity and the degree of deactivation of the antigen-binding domain interaction or the Fc-FcyR interaction can be determined through competitive binding assays. An example of a competitive binding assay is a radioimmunoassay comprising incubation of the labeled antigen, such as tetrameric FcyR (e.g.,³H o ¹²⁵I, see Section 5.4.1) with a molecule of interest (for example, the molecules of the present invention comprise multiple epitope-binding domains in the presence of increasing amounts of the unlabeled epitope, such as tetrameric FcyR (see Section 5.4 .1), and the detection of the molecule bound to the labeled antigen. The affinity of the molecule of the present invention for an antigen and the degrees of non-binding can be determined from the saturation data through Scatchard analysis.

The affinities and binding properties of the molecules of the invention for an antigen or FcyR can initially be determined using in vitro assays (biochemical or immunological assays) known in the art for antigen-binding domain or Fc-FcyR, interactions, which include but are not limited to ELISA assay, surface plasmon resonance assay,

211

INíTITUTO MEJtCAHC wjjSS'Γ&gt; Ε LA FROHEDAr

IWUSTItlAt.

immunoprecipitation. Preferably, the binding properties of the molecules of the invention are also characterized by in vitro functional assays to determine one or more functions of the mediator effector cell Fc and R, as described in Section 5.4.2. In more preferred embodiments, the molecules of the invention have similar binding properties in in vivo models (such as described and described herein) as in vitro based assays). However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro based assays but exhibit the desired phenotype in vivo.

In some embodiments, the classification and identification of molecules comprise multiple epitope binding domains, and optionally Fe domains (or portions thereof) that are done through functional assays, preferably in a high throughput manner. Functional assays can be any assay known in the art for the characterization of one or more of the FcR-mediated effector cell functions such as those described herein in Sections 5.4.2 and 5.4.3. Non-limiting examples of effector cell functions that can be used in accordance with the methods of the invention include but are not limited to, antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis₂₁₂ ΪΜΡΙ ^ ικπτυτη mbucant i sla PRomt.AL ΐΝηικτ »|_ΑιAntibody-dependent, phagocytosis ·, —opsonization, opsonophagocytosis, cell binding, rosettes, Clq binding, and complement-dependent cell-mediated cytotoxicity.

In a preferred embodiment, BIAcore kinetic analysis is used to determine the degrees of activation and deactivation of the binding of the molecules of the present invention to an antigen or FcyR. BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen or FcyR from the chips with immobilized molecules (eg, molecules comprising epitope-binding domains or Fe domains (or portions thereof) respectively) on their surface. The BIAcore analysis is described in Section 5.4.3.

Preferably, fluorescence activity cell sorting (FACS), using any of the techniques known to those skilled in the art, is used for an immunological or functional-based assay to characterize molecules of the invention. Flow sorters are capable of rapidly examining large numbers of individual cells that have been attached, for example, opsonized, through the molecules of the invention (for example, 10-100 million cells per hour) (Shapiro et al. . (1995) Practical Flow Cytometry).

Additionally, the specific parameters used for the ²¹³ IMPI iwrrrurc méxicanc DI LA FROPIf DAO INDUSTFIAI optimization of diabody behavior, including but not limited to, antigen concentration (i.e., FcyR tetrameric complex, see Section 5.4.1), kinetic competition time, or FACS severity, for a from which it can vary in order to select diabody molecules comprising molecules of the invention that exhibit specific binding properties, eg, concurrent binding to multiple epitopes. Flow cytometers for classifying and examining biological cells are well known in the art. Known flow cytometers are written, for example, in US Patent Nos. 4,347,935;

5,464,581; 5,483,469; 5,602,039; 5,643,796; and 6,211,477; the full content of which is incorporated herein by reference. Other known flow cytometers are the FACS Vantage ™ system manufactured by Becton Dickinson and Company, and the COPAS ™ system manufactured by Union Biometrica.

Characterization of target antigen binding affinity or Fc-FcyR binding affinity, and evaluation of target antigen or FcyR density at the cell surface can be done through methods well known in the art such as assay. Scatchard through the use of kits through the manufacturer's instructions, such as Quantum ™ Simply Cellular ® (Bangs Laboratories, Inc., Fishers, IN). The one or more functional assays can be any assay known in the art.

<img file="MX348166B_D0138.tif" />

to characterize one or more FcyR-mediated — La— functions as known to one of ordinary skill in the art or described herein. In specific embodiments, the molecules of the invention comprise multiple epitope binding domains and, optionally, an Fe domain (or a portion thereof) is assayed in an ELISA assay for binding to one or more target antigens or one or more Fc and Rs. , for example, FcyRIIIA, FcyRIIA, FcyRIIA; followed by one or more ADCC trials. In some embodiments, the molecules of the invention are further tested using a surface plasmon resonance based assay, eg, BIAcore. Surface plasmon resonance-based assays are well known in the art, and are further explained in Section 5.4.3, and exemplified herein, for example, in Example 6.1.

In more preferred embodiments, molecules of the invention comprising multiple epitope binding domains and optionally an Fe domain (or a portion thereof) are further characterized in an animal model for interaction with a target antigen (eg, an FcyR) or the Fc-FcyR interaction. When Fc-FcyR interactions are to be evaluated, preferred animal models for use in the methods of the invention are, for example, transgenic mice expressing FcyRs, for example, any mouse model described in US Patent Nos.

215 INSTITUTO MEXICANO DE LA mor · DA P C &lt;w2iLA »INDUSTWIAl

5,877,397, and 6,676,927 which is incorporated herein by reference in its entirety. Additional transgenic mice for use in such methods include, but are not limited to, nude transgenic FcyRIIIA mice, carrying human FcyRIIIA; nude transgenic FcyRIIIA mice bearing FcyRIIA; nude transgenic FcyRIIIA mice bearing human FcyRIIB and human FcyRIIIA; nude transgenic FcyRIIIA mice bearing human FcyRIIB and human FcyRIIA; Naked transgenic FcyRIIIA and FcyRIIA mice bearing human FcyRIIIA and FcyRIIA and nude transgenic FcyRIIIA, FcyRIIA and FcyRIIB mice bearing human FcyRIIIA, FcyRIIA and FcyRIIB.

JOINT TESTS INCLUDING FCyR

The characterization of binding for FcyR through molecules that comprise an Fe domain (or one of its portions) and / or that comprise specific epitope binding domains for an FcyR can be done using any FcyR, including but not limited to variants polymorphic of FcyR. In some embodiments, a polymorphic variant of FcyRIIIA is used, containing alanine at 158. In other embodiments, characterization is done using a polymorphic variant of FcyRIIIA containing a valine at position 158. FcyRIIIA 158V displays higher affinity for 158F IgGl and increased ADCC activity (see, for example, Koene et al. ( 1997) Fe gammaRIIIa-158V / F ₂₁₆ MPI!

i! * rrmrrc mxjcano ¿ESCL-jí re la «ολεολο tVZsSr industrial

Polymorphism Influences The Binding Of IgG By ^ NaturaT ~~ ífi-ldre ^ · -Cell Fe gammaRIIIa, Independently Of The Fe gammaRIIIa48L / R / H Phenotype, Blood, 90: 1109-14; Wu et al. (1997) A Novel Polymorphism Of FcgammaRIIIa (CDI 6) Altere Receptor Function And Predisposes To Autoimmune Disease, J. Clin. Invest. 100: 1059-70, both of which are incorporated herein by reference in their entirety); this residue in fact acts directly with the lower hinge region of IgGl as recently shown by co-crystallization studies of IgGl-FcyRIIIA, see, for example, Sondermann et al. (2000) The 3.2 -A Crystal Structure Of The Human IgGl Fe Fragment-Fc gammaRIII complex, Nature, 406 (6793): 267-273, which is incorporated herein by reference in its entirety. Studies have shown that in some cases, therapeutic antibodies have improved efficacy, in homozygous FcyRIIIA-158V patients. For example, the humanized anti-CD20 monoclonal antibody Rituximab was more therapeutically effective in homozygous FcyRIIIA158V patients compared to homozygous FcyRIIIA 158F patients (see, for example, Cartron et al. (2002) Therapeutic Activity Of Humanized Anti-CD20 Monoclonal Antibody And Polymorphism In IgG Fe Receptor FcgammaRIIIA Gene, Blood, 99 (3): 754-758). In other embodiments, therapeutic molecules that comprise this region will also be more effective in patients heterozygous for FcyRIIIA-158V and

<img file="MX348166B_D0139.tif" />

217

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nr i * mcmDAD INWSTWIAl

FcyRIIIA-158F, and in patients with FcyRIIA-13 lH7 ~ Although it is not intended to be bound through any particular mechanism of action, the selection of the molecules of the invention with alternative allotypes can provide variants that once modified into therapeutic diabodies will be clinically more effective for patients homozygous for such an allotype.

Any FcyR binding assay was developed to determine the binding of the molecules of the invention to FcyR, and, in particular, to determine the binding of Fe domains to FcyR. The assay allowed the detection and quantification of Fc-FcyR interactions, despite the inherent weak receptor affinity for this ligand, eg, in the micromolar range for FcyRIIB and FcyRIIIA. The method is described in detail in International Application WO04 / 063351 and US Patent Application Publications 2005/0037000 and 2005/0064514, each of which is incorporated herein by reference in its entirety. In summary, the method involves the formation of a FcyR complex that can be used in any standard immunoassay known in the art, eg, FACS, ELISA, surface plasmon resonance, etc. Additionally, the FcyR complex has improved avidity for an Fe region relative to an FcyR not a complex. According to the invention, the preferred molecular complex is a tetrameric immune complex comprising: (a) the soluble region of FcyR (for example, the

218

IMPI

MEXICAN INSTITUTE OF THE MOR AGE INDI 'STPIAI

<img file="MX348166B_D0140.tif" />

soluble region of FcyRIIIA, FcyRIIA or FcyRIIB); (b) a biotinylated 15 amino acid sequence AVITAG (AVITAG) operably linked to the C-terminus of the soluble region of FcyR (eg, the soluble region of FcyRIIIA, FcyRIIA or FcyRIIB); and (c) streptavidin-phycoerythrin (SA-PE); in a molar ratio to form a tetrameric FcyR complex (preferably in a 5: 1 molar ratio). The fusion protein is biotinylated enzymatically, using, for example, the Bir A enzyme from E. coli, a biotin ligase that specifically biotinylates a Usine residue in the AVITAG sequence at amino acid 15. Biotinylated soluble FcyR proteins are then mixed with SA-PE in a ratio of SA-PE IX biotinylated soluble FcyR: 5X to form a tetrameric FcyR complex.

Polypeptides comprising FC regions have been shown to bind to tetrameric FcyR complexes with at least an 8-fold greater affinity than non-monomeric complexed FcyR. The binding of polypeptides comprising Fe regions to tetrameric FcyR complexes can be determined using standard techniques known to those skilled in the art, such as, for example, fluorescence activated cell sorting (FACS), radioimmunoassays, ELISA assays, etc.

The invention encompasses the use of immune complexes comprising molecules of the invention, and is

219 INSTITUTE MMtlCANC DE LA HMMIDAD Ο · βί3ίΐΛ. ' INDf "T" IALs formed according to the methods described above, to determine the functionality of molecules comprising an Fe region in a cell-based or cell-free assay.

As a matter of convenience, reagents can be provided in an assay, ie, a combination of packaged reagents to test the ability of molecules comprising Fe regions to bind Fc and R tetrameric complexes. Other forms of molecular complexes for use in determining Fc-Fc and R interactions are also contemplated for use in the methods of the invention, for example fusion proteins formed as described in US Provisional Application 60 / 439,709, filed 13 January 2003, which is incorporated herein by reference in its entirety.

FUNCTIONAL TESTS OF MOLECULES WITH HEAVY CHAINS VARIANTS

The invention encompasses the characterization of the molecules of the invention comprising multiple epitope binding domains and, optionally, Fe domains (or portions thereof) using assays known to those skilled in the art to identify the effector cellular function of the molecules. In particular, the invention encompasses the characterization of the molecules of the invention for FcyR-mediated effector cell function.

<img file="MX348166B_D0141.tif" />

Additionally, when at least one of the target antigens of the diabody molecule of the invention is an FcyR, the binding of FcyR through the diabody molecule can serve to activate FcyR-mediated pathways similar to those activated by the binding of FcyR-Fc. Thus, when at least one epitope binding domain of the diabody molecule recognizes an FcyR, the diabody molecule can elicit FcyR-mediated effector cell function without containing an Fe domain (or a portion thereof), or without concomitant Fc-FcyR binding. Examples of effector cell functions that can be assayed in accordance with the invention include, but are not limited to, antibody-dependent cell-mediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis, Clq binding, and complement-dependent cell-mediated cytotoxicity. Any cell-free cell-based assay known to the person skilled in the art to determine the activity of effector cell function can be used (for effector cell assays, see Perussia et al. (2000) Assays For Antibody-Dependent Cell -Mediated Cytotoxicity (ADCC) And Reverse ADCC (Redirected Cytotoxicity) In Human Natural Killer Cells, Methods Mol. Biol. 121: 179-92; Baggiolini et al. (1988) Cellular Models For The Detection And Evaluation Of Drugs That Modulate Human Phagocyte Activity, Experientia, 44 (10): 841-848, - Lehmann et al. (2000)

221

<img file="MX348166B_D0142.tif" />

IMPI

MEXICAN INSTITUTE

Dt THE PBXJMSDAC INDUSTRY!

Phagocytosis: Measurement By Flow Cytometry, J. Immunol.

Methods, 243 (1-2): 229-42; Brown (1994) In vitro Assays Of

Phagocytic Function Of Human Peripheral Blood Leukocytes: Receptor Modulation And Signal Transduction, Methods Cell Biol, 45: 147-64; Munn et al. (1990) Phagocytosis Of Tumor Cells By Human Monocytes Cultured In Recombinant Macrophage Colony-Stimulating Factor, J. Exp. Med., 172: 231-237, AbdulMajid et al. (2002) Fe Receptors Are Critical For Autoimmune Inflammatory Damage To The Central Nervous System In Experimental Autoimmune Encephalomyelitis, Scand. J. Immunol. 55: 70-81; Ding et al. (1998) Two Human T Cell Receptors Bind In A Similar Diagonal Mode To The HLA-A2 / Tax Peptide Complex Using Different TCR Amino Acids, Immunity 8: 403-411, each of which is incorporated herein by reference in its entirety) .

In one embodiment, the molecules of the invention can be tested for FcyR-mediated phagocytosis in human mocytes. Alternatively, FcyR-mediated phagocytosis of the molecules of the invention can be assayed in other phagocytes, for example neutrophils (polymorphonuclear leukocytes; PMN); human peripheral blood monocytes, monocyte derived macrophages, obtainable using standard procedures known to those skilled in the art (for example see Brown (1994) In vitro

Assays Of Phagocytic Function Of Human Peripheral Blood

222

<img file="MX348166B_D0143.tif" />

IMPI ^

MEXICAN INSTITUTE OF INDUSTRIAL NOPIEOAD

Leukocytes: Receptor Modulation And Signal Transduction

Methods Cell Biol., 45: 147-164). In one embodiment, the function of the molecules of the invention is characterized by measuring the ability of THP-1 cells to carry out phagocytosis of fluorescein IgG opsonized sheep red blood cells (SRBC) through methods previously described (Tridandapani et al. (2000) The Adapter Protein LAT Enhances Fcgamma Receptor-Mediated Signal Transduction In Myeloid Cells, J. Biol. Chem. 275: 2048020487).

Another illustrative assay to determine phagocytosis of the molecules of the invention is an antibody-dependent opsonophagocytosis (ADCP) assay that may comprise the following: coating a target bioparticle such as FITC labeled with Escherichia coli (Molecular Probes) or Staphylococcus aureus-FTTC with (i) the wild-type 4-4-20 antibody, an antibody to fluorescein (See Bedzyk et al. (1989) Comparison Of Variable Region Primary Structures Within An Anti-Fluorescein Idiotype Family, J. Biol. Chem, 264 (3): 1565-1569, incorporated herein by reference in its entirety), as the control antibody for FcyR-dependent ADCP; or (ii) the 4-4-20 antibody harboring the D265A mutation that kills FcyRIII binding, as a background control for FcyR-dependent ADCP (iii) a diabody comprising the epitope binding domain of 4- 4-20 and a Fe domain and / or an epitope binding domain specific for FcyRIII; and form the opsonized particle; add any of the described opsonized particles (i-iii) for THP-1 effector cells (a monocytic cell line available from ATCC) at a ratio of 1: 1, 10: 1, 30: 1, 60: 1, 75 : 1 or 100: 1 to allow FcyR-mediated phagocytosis to occur, preferably a clone of the cell and E. coli-FITC / antibody at 37 ° C for 1.5 hour; add trypan blue after incubation (preferably at room temperature for 2-3 minutes) to the cells to quench the fluorescence of the bacteria that adhered to the outside of the cell surface without internalizing; transfer the cell into a FACS pH buffer (eg 0.1%, BSA in PBS, 0.1%, sodium azide), analyze THP1 cells for fluorescence using FACS (eg BD FACS Calibur). Preferably the THP-1 cells used in the assay are analyzed via FACS for expression of FcyR on the cell surface. THP-1 cells express both CD32A and CD64. CD64 is high affinity FcyR that is blocked in conducting the ADCP assay according to the methods of the invention. THP-1 cells are preferably blocked with 100 ug / ml soluble IgGl or 10% human serum. To analyze the ADCP extract, the gate is preferably established in THP-1 cells and the intensity is measured.

224 medium individual fluorescent. Activity

<img file="MX348166B_D0144.tif" />

is calculated and reported as a normalized value for wild-type chMab 4-4-20 obtained. The opsonized particles are added to the THP-1 cells in such a way that the ratio of the opsonized particles to the THP-1 cells is 30: 1 or 60: 1. In more preferred embodiments, the ADCP assay is conducted with controls, such as E. coli-FITC in medium, E. coli-FITC and THP-1 cells (to serve as ADCP activity independently Fc and R), E. coli-FITC, THP-1 cells and the wild-type 4-4-20 antibody (to serve as the ADCP FcyR activity), E coliFITC, THP-1 cells, 4-4-20 D265A (to serve as the control of background for FcyR-dependent ADCP activity).

In another embodiment, the molecules of the invention can be assayed for FcyR-mediated ADCC activity in effector cells, eg, natural killer cells, using any of the standard methods known to those skilled in the art (see, for example, Perussia et al. al. (2000) Assays For Antibody-Dependent CellMediated Cytotoxicity (ADCC) And Reverse ADCC (Redirected Cytotoxicity) In Human Natural Killer Cells, Methods Mol. Biol. 121: 179-92, - Weng et al. (2003) Two Immunoglobulin G Fragment C Receptor Polymorphisms Independently Predict Response To Rituximab In Patients With Follicular Lymphoma, J. Clin. Oncol. 21: 3940-3947; Ding et al. (1998) Two Human T²²⁵ nti-M-WR ^ Ap^: ν r&gt; ι -m ι λ। QgRfg

Cell Receptor Bind In A Similar Diagonal Mode To The HLAA2 / Tax Peptide Complex Using Different TCR Amino Acid, Immunity 8: 403-411). An illustrative assay to determine the ADCC activity of the molecules of the invention is based on a release assay of⁵¹Cr comprising: labeling of target cells with [⁵¹Cr] Na₂CrO₄ (This permeable cell membrane molecule is commonly used for labeling as it binds cytoplasmic proteins and although it is spontaneously released from cells with slow kinetics, it massively releases the next target cell necrosis); opsonizing target cells for molecules of the invention comprising variable heavy chains; combining opsonized radiolabeled target cells with effector cells in a microtiter plate at an appropriate ratio of target cells to effector cells; incubate the cell mixture for 16-18 hours at 37 ° C; collect the supernatants; and analyze radioactivity. The cytotoxicity of the molecules of the invention can then be determined, for example using the following formula:% lysis = (experimental cpm - target leak cpm) / (detergent lysis cpm - target leak cpm) x 100%. Alternatively,% lysis = (ADCC-AICC) / (maximum release - spontaneous release). Specific lysis can be calculated using the formula: specific lysis =% lysis with the molecules of the invention -% lysis in the

<img file="MX348166B_D0145.tif" />

226

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ΙΝΠ! ΚΤ »|_Α| absence of the molecules of the invention ^ A graph can be generated through the ratio of any variable target: effector cell or antibody concentration.

Preferably, the effector cells used in the ADCC assays of the invention are peripheral blood mononuclear cells (PBMC) which are preferably purified from normal human blood, using standard methods known to those skilled in the art, for example, using gradient centrifugation. FicollPaque density. Preferred effector cells for use in the methods of the invention express different FcyR activation receptors. The invention encompasses effector cells, THP1, which express FcyRI, FcyRIIA and FcyRIIB, and macrophages and various monocyte derivatives derived from human whole blood expressing both FcyRIIIA and FcyRIIB, to determine whether the heavy chain antibody mutants showed ADCC activity increased and a phagocytosis related to wild-type IgGl antibodies.

The human monocyte cell line, THP-1, activates phagocytosis through the expression of the high affinity receptor FcyRI and the low affinity receptor FcyRIIA (Fleit et al. (1991) The Human Monocyte-Like Cell Line THP-1 Expresses Fe Gamma RI And Fe Gamma RII, J. Leuk. Biol. 49: 556-565). THP-1 cells do not constitutively express FcyRIIA or FcyRIIB. Stimulation of these cells with

227

IMPI

IFBTnVTC MIXJCAMC t * INDUSTRIAL MOLD cytokines affect the FcR expression pattern (Pricop et al. (2001) Differential Modulation Of Stimulatory And Inhibitory Fe Gamma Receptors On Human Monocytes By Thl And Th2 Cytokines, J. of Immunol., 166: 531- 537). The growth of THP-1 cells in the presence of IL4 cytokine induces the expression of FcyRIIB and causes the reduction in the expression of FcyRIIA and FcyRI. The expression of FcyRIIB can also be enhanced by increasing cell density (Tridandapani et al. (2002) Regulated Expression And Inhibitory Function Of Fcgamma RIIB In Human Monocytic Cells, J. Biol. Chem., 277 (7): 5082-5089). In contrast, IFNy has been reported to lead to the expression of FcyRIIIA (Pearse et al. (1993) Interferon Gamma-Induced Transcription Of The High Affinity Fe Receptor For IgG Requires Assembly Of A Complex That Ineludes The 91-kDa Subunit Of Transcription Factor ISGF 3, Proc. Nat. Acad. Sci. USA 90: 4314-4318). The presence or absence of cell surface receptors can be determined via FACS using common methods known to those of skill in the art. Cytokine-induced expression of FcyR on the cell surface provides a system for testing both activation and inhibition in the presence of FcyRIIB. If THP-1 cells are unable to express FcyRIIB the invention also encompasses another human monocyte cell line, U937. These cells have been shown to terminally differentiate macrophages in the

228

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M LA MOR ID_Arindustrial presence of IFNy and TNF (Koren et al. (1979) In vitro

Activation Of A Human Macrophage-Like Cell Line, Nature 279:

328-331).

FcyR-dependent tumor cell killing is mediated by macrophage and NK cells in mouse tumor models (Clynes et al. (1998) Fe Receptor Are Required In Passive And Active Immunity To Melanoma ”, Proc. Nat. Acad. Sci USA 95: 652-656). The invention encompasses the use of washed monocytes from donors as effector cells to test the efficiency of Fe mulants to activate cellular cytotoxicity of target cells in both phagocytosis and ADCC assays. The expression patterns of FcyRI, FcyRIIIA, and FcyRIIB are affected by different culture conditions. The expression of FcyR from frozen washed monocytes, fresh washed monocytes, monocytes maintained in 10% FBS, and monocytes cultured in FBS + GMCSF and / or in human serum can be determined using common methods known to those of skill in the art. For example, cells can be stained with FcR specific antibodies and analyzed via FACS to determine FcR profiles. Conditions that best mimic macrophage FcyR expression in vivo are then used for the methods of the invention.

In some embodiments, the invention encompasses the use of mouse cells especially when the cells of ²²⁹ IMPI ητ la Ρ »ορΐίο_ΑΓ;INDUSTRIAL humans with adequate FcyR profiles are unable to be obtained. In some embodiments, the invention encompasses the mouse macrophage cell line RAW264.7 (ATCC) which can be transfected with human FcyRIIIA and isolated stable transfectants using methods known in the art, see, for

<img file="MX348166B_D0147.tif" />

example, Ralph et al. (1977) Antibody-Dependent Killing Of

Erythrocyte And Tumor Targets By Macrophage-Related Cell

Lines: Enhancement By PPD And LPS, J. Immunol. 119: 950-4).

Transfectants can be quantified for FcyRIIIA expression via FACS analysis using routine experimentation and high expressions can be used in ADCC assays of the invention. In other embodiments, the invention encompasses the isolation of spleen peritoneal macrophage expressing human FcyR from genetically engineered transgenic mice such as those described herein.

Lymphocytes can be harvested from donor peripheral blood (PBM) using a FicollPaque gradient (Pharmacia). Within the isolated mononuclear population of cells most of the ADCC activity occurs through natural killer (NK) cells that contain FcyRIIIA but not FcyRIIB on their surface. The results with these cells indicate the efficacy of the mutants in activating the ADCC of the NK cell and establish the reagents to test with washed monocytes.

Target cells used in ADCC assays

IMPI

MKICANO of the invention include, but are not limited to, breast cancer cell lines, eg, accession number SK-BR-3 with accession number HTB-30 (see, eg, Tremp et al. (1976) Human Breast Cancer In Culture, Recent Results Cancer Res. 33-41); B-lymphocytes; cells derived from Burkitts lymphoma, eg, Raji cells with ATCC accession number CCL-86 (see, eg, Epstein et al. (1965) Characteristics And Mode Of Growth Of Tissue Culture Strain (EB1) Of Human Lymphoblasts From Burkitt's Lymphoma, J. Nati. Cancer Inst. 34: 231-240), and Daudi cells with accession number ATCC CCL-213 (see, for example, Klein et al. (1968) Surface IgM-Kappa Specificity On A Burkitt Lymphoma Cell In Vivo And In Derived Culture Lines ”, Cancer Res. 28: 1300-1310). Target cells must be recognized through the antigen-binding site of the diabody molecule to be tested.

The ADCC assay is based on the ability of NK cells to mediate cell death through an apoptotic pathway. NK cells mediate cell death in part through FcyRIIIA recognition of an Fe IgG domain bound to an antigen on the cell surface. The ADCC assays used in accordance with the methods of the invention can be radioactive-based assays or fluorescence-based assays. The essays

Using ADCC to characterize the molecules of the

231 ΙΜΡΙΟ ^

MIJCCAHG INSTITUTE Yg- &lt;í- ^ csí &lt;j Dt u noniDAD Cta- ^ feiLiíS ΙΝΓ '· ΤΤ »ΙΛΙ invention include various Fe-¿¡utL ^ mpr regions in the marking of target cells, for example, SK-BR-3 , MCF7, OVC AR3, Raji, Daudi cells, opsonization of target cells with an antibody that recognizes a receptor on the target cell's cell surface through its antigen-binding site; combining the labeled opsonized target cells and the effector cells with an appropriate ratio, which can be determined through your routine experimentation; harvest the cells; detect the tag in the lysed target cell supernatant, using an appropriate detection scheme based on the tag used. Target cells can be labeled with either a radioactive tag or a fluorescent tag, using standard methods known in the art. For example, tags include, but are not limited to, [⁵¹Cr] Na₂CrO₄; and acetoxymethyl ester of the ligand that improves fluorescence, 2,2¹: 6 ', 2-terpyridine-6-6-dicarboxylate (TDA).

In a specific preferred embodiment, a time-resolved fluorometric assay is used to measure ADCC activity against target cells that have been labeled with acetoxymethyl ester of the fluorescence enhancing ligand, 2,2 ': 6', 2-terpyridin-6- 6-dicarboxylate (TDA). Such fluorometric assays are known in the art, for example, see Blomberg et al. (1996) Time²³² IMPI ^

INSTITUTO MEXICANO DB tu * frohedai, INDUSTRIA!

Resolved Fluorometric Assay For Natural Killer Activity Using Target Cells Labeled With A Fluorescence Enhancing Ligand, Journal of Immunological Methods, 193: 199-206; which is incorporated herein by reference in its entirety. In summary, the target cells are labeled with the permeable acetoxymethyl diether membrane of TDA (bis (acetoxymethyl) 2,2 ': 6', 2terpiridine-6-6-dicarboxylate, (BATDA), which rapidly diffuses through the cell membrane of the viable ones. Cluster intracellular esterase cleavage and the regenerated membrane impermeable TDA molecule are entrapped within the cell. After incubation of target and effector cells, for example, for at least 2 hours, up to 3.5 hours, at 37 ° C, under 5% CO₂, the TDA released from the lysed target cells is chelated with Eu3 + and the fluorescence of the Europium TDA chelates formed is quantified on a time-resolved fluorometer (eg, Victor 1420, Perkin Elmer / Wallace).

In another specific embodiment, the ADCC assay used to characterize the molecules of the invention comprise multiple epitope binding sites and, optionally, an Fe domain (or a portion thereof) comprising the following steps: preferably 4-5x10⁶target cells (e.g. SK-BR-3, MCF-7, OVCAR3, Raji cells) that are labeled with bis (acetoxymethyl) 2,2 ': 6', 2terpiridin-t-6-dicarboxylate (DELFIA BATDA Reagent, Perkin

233

<img file="MX348166B_D0148.tif" />

Elmer / Wallac). For efficient optimal labeling, the number of target cells used in the ADCC assay should preferably not exceed 5x10.⁶. The BATDA reagent is added to the cells and the mixture is incubated at 37 ° C preferably under 5% C0₂, for at least 30 minutes. The cells are then washed with a physiological pH buffer, for example PBS with 0.125 mM sulf inpyrazole, and a medium containing 0.125 mM sulfinpyrazole. The labeled target cells are then opsonized (coated) with a molecule of the invention comprising an epitope binding domain specific for FcyRIIA and, optionally, an Fe domain (or a portion thereof). In preferred embodiments, the molecule used in the ADCC assay is also specific for the cell surface receptor, a tumor antigen, or a cancer antigen. The diabody molecule of the invention can specifically bind to any cancer or tumor antigen, such as those listed in section 5.6.1. Target cells in the ADCC assay are selected according to modified epitope binding sites on the diabody of the invention, such that the diabody specifically binds to the cell surface receptor of the target cell.

Target cells are added to effector cells, e.g. PBMC, to produce effector: target ratios of approximately 1: 1, 10: 1, 30: 1, 50: 1,

<img file="MX348166B_D0149.tif" />

234

IMPI Mexican mrrmrro et LA INDUSTRIAL CURRENCY

75: 1, or 100: 1. Effector cells and obj6 ± jjíq__sa-juiouhaii ----—. for at least 2 hours, up to 3.5 hours, at 37 ° C, under 5% C0₂. Cell supernatant is harvested and added to acidic europium solution (eg, DELFIA Europium Solution, Perkin Elmer / Wallac). The fluorescence of the formed carated EuropioTDA is quantified on a time-resolved fluorometer (eg, Victor 1420, Perkin Elmer / Wallac). Maximum release (MR) and spontaneous release (SR) are determined through incubation of target cells with 1% TX-100 and medium alone, respectively. Antibody independent cellular cytotoxicity (AICC) is measured through incubation of target and effector cells in the absence of a test molecule, eg, the diabody of an invention. Each test is preferably carried out in triplicate. The mean percentage specific lysis is calculated as: Experimental release (ADCC) - AICC) / (MR-SR) x 100.

The invention encompasses assays known in the art and exemplified herein, to characterize Clq binding and mediation of complement dependent cytotoxicity (CDC) through molecules of the invention comprising Fe domains (or portions thereof). To determine Clq binding, a Clq binding ELISA is performed. An illustrative assay may comprise the following: assay plates that can be covered overnight

235

<img file="MX348166B_D0150.tif" />

at 4 ° C with a polypeptide comprising a molecule of the invention or a starting (control) polypeptide in a control pH buffer. The plates can then be washed and blocked. After washing, an aliquot of human Clq can be added to each well and incubated for 2 hours at room temperature. After one more wash, 100 ul of sheep anti-complement Clq peroxidase conjugated antibody can be added to each well and incubated for 1 hour at room temperature. The plate can again be washed with a wash buffer and 100 ul OPD (0-phenylenediamine dihydrochloride (SIGMA) containing the substrate pH buffer can be added to each well. The oxidation reaction, observed by the appearance of the yellow color, can be allowed to proceed for 30 minutes and stopped by adding 100 ul of 4.5 NH2SO4. The absorbance can then be read at (492-405) nm.

To assess complement activation, a complement-dependent cytotoxicity (CDC) assay can be carried out, for example, as described in GazzanoSantoro et al. (1997) A Non-Radioactive Complement-Dependent Cytotoxicity Assay For Anti-CD20 Monoclonal Antibody, J. Immunol. Methods 202: 163-171, which is incorporated herein by reference in its entirety. In summary, various concentrations of the molecule comprising an Fe domain (variant) (or a portion thereof) and a complement can ²³⁶ ΙΜΡΙ®&gt;

INSTITUTO MEXICANt

DE LA MOHEDA O CtaoíSEi-® INDUSTRIAL&gt; *.

be diluted with pH regulator. Cells expressing the antigen to which the diabody molecule binds can be diluted to a density of approximately Ix10.⁶ cells / ml. Mixtures of the diabody molecule comprising an Fe (variant) domain (or a portion thereof), diluted human complement, and antigen-expressing cells can be added to a flat 96-well tissue culture plate and allowed to incubate for 2 hours. at 37 ° C and 5% CO2 to facilitate complement-mediated cell lysis. Then 50 ul Alamar Blue (Accumed International) can be added to each well and incubated overnight at 37 ° C. Absorbance is measured using a 96 well fluorometer with excitation at 530 nm and emission at 590 nm. Results can be expressed in relative fluorescence units (RFU). The sample concentrations can be calculated from a standard curve and the percentage activity as compared to the non-variant molecule, that is, the molecule that does not comprise an Fe domain or that comprises a non-variant Fe domain, are reported for the variant of interest.

OTHER TESTS

Molecules of the invention comprising multiple epitope-binding domains and optionally an Fe domain can be assayed using any known surface plasmon resonance-based assays in the art.

<img file="MX348166B_D0151.tif" />

237

ΓΜΡΙ iwnvro m * icano DI LA mOHEBAD rNDUmtlAL technique for the characterization of the kinetic parameters of an antigen-binding domain to Fc-FcyR binding. Any commercially available SPR instruments including, but not limited to, BIAcore Instruments, available from Biacore AB (Uppsala, Sweden); lAsys instruments available from Affinity Sensors (Franklin, MA.); The IBIS system available from Windsor Scientific Limited (Berks, UK), the SPR-CELLIA systems available from Nippon Laser and Electronics Lab (Hokkaido, Japan), and the SPR Detector Spreeta available from Texas Instruments (Dallas, TX) can be used herein. invention. For a review of SPR-based technology see Mullet et al. (2000) Surface Plasmon Resonance-Based Immunoassays, Methods 22: 77-91; Dong et al. (2002) Some new aspects in biosensors, Reviews in Mol. Biotech. 82: 303-23; Fivash et al. (1998) BIAcore For Macromolecular Interaction, Current Opinion in Biotechnology 9: 97-101; Rich et al. (2000) Advances In Surface Plasmon Resonance Biosensor Analysis, Current Opinion in Biotechnology 11: 54-61; all of which are incorporated herein by reference in their entirety. Additionally any SPR instrument and SPR-based method for measuring protein-protein interactions described in US Patent Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215; 6,268,125, all of which are incorporated herein by reference in their entirety, are contemplated in the methods of the

238 invention.

IMPI ^

ΙΝΤΤΤΤΤΟ MEXICAN

MU FtOriíDAO ®¡Kü • musruiAi VánEn

In summary, the áPR 'ThvolTrCLdli-based assays immobilize one member of the binding pair on a surface, and monitor its interaction with another member of the binding pair in real-time solutions. SPR is based on the measurement of the change in the refractive index of the solvent near the surface that occurs after complex formation or dissociation. The surface on which immobilization occurs in the sensor chip, which is at the heart of SPR technology, consists of a glass surface coated with a thin layer of gold and forms the basis for a range of specialized surfaces designed to optimize the binding of a surface molecule. A variety of sensor chips are commercially available especially from companies listed above, all of which can be used in the methods of the invention. Examples of sensor chips include those available from BIAcore AB, Inc., eg, Sensor Chip CM5, SA, NTA, and HPA. A molecule of the invention can be immobilized on the surface of a sensor chip using any of the chemical and immobilization methods known in the art, including but not limited to, direct covalent coupling through amine groups, direct covalent coupling through the group. sulfhydryl, surface coated biotin binding to avidin, aldehyde coupling ²³⁹ IMPI INSTITUTO MEXICANO PE LA PROHSbAD tNr&gt; H5TKiAi to carbohydrate groups, and binding through a histidine tag with NTA chips.

In some embodiments, the kinetic parameters of the binding of molecules of the invention comprising multiple epitope binding sites, and optionally, an Fe domain, to an antigen, or an FcyR can be determined using the BIAcore instrument (e.g., BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ). As explained supra, see Section 5.4.1, any FcyR can be used to assess the binding of the molecules of the invention either where at least one epitope binding site of the diabody molecule immunospecifically recognizes an FcyR, and / or wherein the diabody molecule comprises an Fe domain (or a portion thereof). In a specific embodiment, FcyR is FcyRIIIA, preferably a soluble monomeric FcyRIIIA. For example, in one embodiment, the soluble monomeric FcyRIIIA from the extracellular region of FcyRIIIA linked to the linked AVITAG sequence (see, US Provisional Application No. 60 / 439,498, filed January 9, 2003, and US Provisional Application No. 60 / 456,041, filed March 19, 2003, which are incorporated herein by reference in their entirety). In another specific embodiment, FcyR is FcyRIIB, preferably a soluble chimeric FcyRIIB. For example, in one mode, the

<img file="MX348166B_D0152.tif" />

Soluble chimeric FcyRIIB protein is prepared according to ²⁴⁰ IMPIf ^

INSTITUTO MEXICANO Dt LA PROPERTY CV¿2 “! 5 INDomiAl technology described in US Provisional Application No. 60 / 439,709, filed on January 13, 2003, which is incorporated herein by reference in its entirety.

For all immunogenic assays, the recognition / binding of FcyR through a molecule of the invention can be affected by multiple domains: in certain embodiments, the molecules of the invention immunospecifically recognize FcyR through one of the multiple binding domains of the invention. epitope; in other embodiments, where the molecule of the invention comprises an Fe domain (or a portion thereof) the diabody molecule can immunospecifically recognize FcyR through FcFcyR interactions; In still other embodiments where the molecule of the invention comprises both an Fe domain (or a portion thereof) and an epitope binding site that immunospecifically recognizes the FcyR, the diabody molecule can recognize FcyR through one or both the epitope binding domain and the Fe domain (or a portion thereof). An illustrative assay for determining the kinetic parameters of a molecule comprising multiple epitope-binding domains and optionally a domain of Fe (or a portion thereof) to an antigen and / or an FcyR using the BIAcore instrument comprises the following: a first antigen is immobilized on one of the four flow cells of the sensor chip surface, preferably at

241 ΙΜΡΙ63 ^ iKnTUTüMUiCANc OF THE industrial r &amp; OmPAD through amine -Hq coupling chemistry. This way, 5000 response units (RU) of the first antigen are immobilized on a surface. Once the appropriate surface is removed, the molecules of the invention that immunospecifically recognize the first antigen are passed onto the surface, preferably through one minute injections of a 20 pg / ml solution at 5 μΐ / ml range. flow. Levels of the molecules of the invention bound to the surface of this step typically range between 400 and 700 RU. Next, the inventive series of a second antigen (for example, FcyR) or the FcyR receptor in pH buffer HBS-P (20 mM HEPES, 150 mM NaCl, 3mM EDTA, pH 7.5) are injected into the surface at 100 μΐ / min. The regeneration of the molecules between the different dilutions of the second antigen or receptor is preferably carried out through individual 5 second injections of 100 mM NaHC0.₃ pH

9.4; 3M NaCl. Any regeneration technique known in the art is contemplated in the method of the invention.

Once the complete data set is collected, the resulting junction curves are globally tailored using computer algorithms supplied by the manufacturer of the SPR instrument, eg, BIAcore, Inc. (Piscataway, NJ). These algorithms calculate both K_acti_hed_OR and deactivated / of which the constant Mexican institute.

FROM THE MtOPlEOAD ¿industrial junction in apparent equilibrium, K¿ follows to a protection of two speed constants T say 'K off / Kactivated) · More detailed treatments of how individual speed constants are derived can be found in the Book of Text from BIAevaluaion Software (BIAcore, Inc., Piscataway, NJ). Analysis of the data generated can be done using the method known in the art. For a review of the various methods of interpretation of the generated kinetic data see Myszka (1997) Kinetic Analysis Of Macromolecular Interactions Using Surface Plasmon Resonance Biosensors, Current Opinion in Biotechnology 8: 50-7; Fisher et al. (1994) Surface Plasmon Resonance Based Methods For Measuring The Kinetics And Binding Affinities Of Biomolecular Interactions, Current Opinion in Biotechnology 5: 389-95; O'Shannessy (1994) Determination Of Kinetic Rate And Equilibrium Binding Constants For Macromolecular Interactions: A Critique Of The Surface Plasmon Resonance Literature, Current Opinion in Biotechnology, 5: 65-71; Ch &amp; iken et al. (1992) "Analysis Of Macromolecular Interactions Using Immobilized Ligands", Analytical Biochemistry, 201: 197-210; Norton et al. (1995) Interpreting Complex Binding Kinetics From Optical Biosensors: A Comparison Of Analysis By Linearization, The Integrated Rate Eguation, And Numerical Integration, Analytical Biochemistry 227: 176-85; O'Shannessy et al.,

<img file="MX348166B_D0153.tif" />

1996, Analytical Biochemistry 236: 275-83; all of which are incorporated herein by reference in their entirety.

In preferred embodiments, kinetic parameters determined using SPR analysis, eg, BIAcore, can be used as a measurement to predict how a molecule of the invention will perform in a functional assay, eg, ADCC. The illustrative method for predicting the efficacy of a molecule of the invention based on kinetic parameters obtained from an SPR analysis may comprise the following: determining the K-inactivated values ​​for binding of a molecule of the invention to FcyRIIIA and FcyRIIB (via a epitope binding domain and / or an Fe domain (or a portion thereof); plot (1) Koff (ps) / Kd_{that C}activated (mut) for FcyRIIIA; (2) K off (mut) / K off (ps) for FcyRIIB against ADCC data. Numbers higher than one show a decreased degree of dissociation for FcyRIIIA and an increased degree of dissociation for FcyRIIB relative to wild type; and they possess an enhanced ADCC function.

METHODS TO PRODUCE DIACBODY MOLECULES OF THE

INVENTION

The diabody molecules of the present invention can be produced using a variety of methods well known in the art, including synthesis of the novel protein and recombinant expression of nucleic acids that ²⁴⁴ ΪΜΡΙ ^^

ΙΝϊΤΠυΤΟ MEDICAN 'DE LA PMHEDAD ΙΝΠΙΤΓΚΙΑΙ encode binding proteins. The desired nucleic acid sequences can be produced through recombinant methods (eg, PCR mutagenesis of a previously prepared variant of the desired polynucleotide) or through solid phase DNA synthesis. Recombinant expression methods are usually used. In one aspect the invention provides a polynucleotide comprising a VH and / or VL CD16A coding sequence; In another aspect, the invention provides a polynucleotide comprising a sequence encoding VH and / or VL CD32B. Due to the degeneracy of the genetic code, a variety of nucleic acid sequences encode each immunoglobulin amino acid sequence, and the present invention includes all nucleic acids encoding the binding proteins described herein.

POLYNUCLEOTIDES THAT CODE MOLECULES OF THE INVENTION

The present invention also includes polynucleotides encoding the molecules of the invention, including antibody polypeptides. The polynucleotides encoding the molecules of the invention can be obtained and the nucleotide sequence of the polynucleotides determined, by any method known in the art.

Once the nucleotide sequence is determined

IMPI INíTmWO MIXICANC DE LA MOHEDA! * · 7? ^ · »ΒΓ'Α * δ ^ of the molecules that are identified through the methods of the invention, the * nucleotide sequence can be monitored using methods well known in the art, for example, in recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, 3^to Ed., Coid Spring Harbor Laboratory, Coid Spring Harbor, NY; and Ausubel et al, eds. , 1998,

Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY, both incorporated herein by reference in their entirety). To generate, for example, antibodies having different amino acid sequences, for example, by generating amino acid substitutions or deletions and / or insertions.

In one embodiment, human libraries, or any other library available in the art, can be classified by standard techniques known in the art, to clone the nucleic acids encoding the molecules of the invention.

RECOMBINANT EXPRESSION OF MOLECULES OF THE INVENTION

Once the nucleic acid sequence encoding the molecules of the invention (i.e., antibody) has been obtained, the vector for the production of the molecules can be produced through recombinant DNA technology using techniques well known in the art.

<img file="MX348166B_D0154.tif" />

technique. Methods that are well known to those of skill in the art can be used to construct expression vectors containing the coding sequences for the molecules of the invention and appropriate transcriptional and translational control signals. These methods include, for example, in vivo recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, for example, the techniques described in Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2^to Ed., Coid Spring Harbor Laboratory, Coid Spring Harbor, NY and Ausubel et al. eds., 1998, Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY).

An expression vector comprising the nucleotide sequence of the molecule identified by the methods of the invention can be transferred into a host cell through conventional techniques (eg, electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells. they were then cultured through conventional techniques to produce the molecules of the invention. In specific embodiments, the expression of the molecules of the invention is regulated through a specific, constitutive, or inducible or tissue promoter.

The host cells used to express the molecules identified by the methods of the invention

IMPI can be either bacterial cells such as Escherichia coli, or, preferably eukaryotic cells, especially for the expression of the entire recombinant immunoglobulin molecule. In particular, mammalian cells, such as Chinese hamster ovary (CHO) cells, together with a vector such as the promoter element of the human cytomegalovirus major early intermediate gene is an effective expression system for immunoglobulins (Foecking et al. . (1986) Powerful And Versatile Enhancer Promoter Unit For Mammalian Expression Vector, Gene 45: 101106; Cockett et al. (1990) High Level Expreasion Of Tiaaue Inhibitor Of Metalloproteinase In Chineae Hamater Ovary Celia Uaing Glutamine Synthetase Gene Amplification ”, Biotechnology 8: 662-667).

A variety of host expression vector systems can be used to express the molecules identified by the methods of the invention. Such host expression systems represent vehicles through which the coding sequences of the molecules of the invention can be produced and subsequently purified, but also represent cells that can, when transformed or transfected with the appropriate nucleotide coding sequences, expressing the molecules of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and

248

B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmic DNA expression vectors, which contain coding sequences for the molecules identified by the methods of the invention; yeast (eg, Saccharomyces pichia) transformed with recombinant yeast expression vectors containing sequences encoding the molecules identified by the method of the invention; insect cell systems infected with recombinant virus (eg, baculovirus) expression vectors containing the coding sequences for the molecules identified by the methods of the invention; Plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., of plasmid Ti) that contains coding sequences of the molecules identified by the methods of the invention; mammalian cell systems (e.g. COS, CHO, BHK, 293, 293T, 3T3 cells, lymphocytic cells) (see US 5,807,715), Per C.6 cells (human retinal cells developed by Crucell) harboring constructs of recombinant expression containing promoters derived from the genome of mammalian cells (for example, the metallothionein promoter) or mammalian virus (for example, the late promoter of ²⁴⁹ IMPI iNsTrnrro Mexican property INDUSTRY!

adenovirus; the 7.5K promoter of the vaccinia virus).

In bacterial systems, a number of expression vectors can advantageously be selected depending on the intended use for the molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody, vectors that induce the expression at high levels of the easily purified fusion protein products may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Rüther et al. (1983) Easy Identification Of cDNA Clones, EMBO J. 2: 1791-1794), in which the coding sequence of the antibody can be individually ligated into the vector in structure with the lac Z coding region such that the fusion protein is produced; pIN vectors (Inouye et al. (1985) Up-Promoter Mutations In The Ipp Gene Of Escherichia Coli, Nucleic Acids Res. 13: 3101-3110; Van Heeke et al. (1989) Expression Of Human Asparagine Synthetase In Escherichia Coli, J. Biol. Chem. 24: 55035509); and the like. The pGEX vectors can also be used to express foreign polypeptides as glutathione S-transferase (GST) fusion proteins. In general, such fusion proteins are soluble and can easily be purified from lysed cells through adsorption and binding to a glutathione-agarose bead matrix followed by

<img file="MX348166B_D0155.tif" />

250

INSTITUTO MEXICANO DE LA PHOHEDAI INDUSTRIAL avoidance in the presence of free glutathione. The pGEX vectors are designed to include thrombin or protease factor Xa cleavage sites such that the target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is grown in Spodoptera frugiperda cells. The antibody coding sequence can be individually cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter).

In mammalian host cells, a number of virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the coding sequence of the antibody of interest can be ligated into an adenovirus transcription / translation control complex, eg, the tripartite leader and late promoter sequence. This chimeric gene can then be inserted into the adenovirus genome through in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (for example, the El or E3 region) will result in the recombinant virus that is viable and capable of expressing the immunoglobulin molecule in infected hosts (see, for example,

25ΐ

MEXICAN INSTITOR OF RAOHEDAD CV.USKJÁW INDUSTRIAL

Logan et al., (1984) Adenovirus Tripartite Leader Sequence Enhances Translation Of mRNAs Late After Infection, Proc.

Nati. Acad. Sci. USA 81: 3655-3659). Specific initiation signals may also require efficient translation of the inserted antibody coding sequences. These signals include the ATG start codon and adjacent sequences. In addition, the start codon may be in frame with the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be improved through the inclusion of appropriate transcription enhancing elements, transcription terminators, etc. (see Bitter et al. (1987) Expression And Secretion Vectors For Yeast, Methods in Enzymol. 153: 516-544).

In addition, a host cell strain can be selected that modulates the expression of the inserted sequences, modifies and processes the gene product in a specific manner desired. Such modifications (eg, glycosylation) and processing (eg, separation) of protein products to be important for protein function. For example, in certain embodiments, polypeptides comprise a diabody molecule of the invention that can be expressed as a single gene product (eg, as a single polypeptide chain, i.e., as a polyprotein precursor), requiring separation proteolytic through native or recombinant cellular mechanisms to form the separate polypeptides of the diabody molecules of the invention. The invention thus encompasses modifying the nucleic acid sequence to encode a polyprotein precursor molecule comprising the polypeptides of the invention, including coding sequences capable of directing post-translational separation of the polyprotein precursor. Post-translational separation of the polyprotein precursor results in polypeptides of the invention. Post-translational separation of the precursor molecule comprises the polypeptides of the invention that can occur in vivo (i.e., within the host cell through recombinant native cell systems / mechanisms, e.g., furin cleavage at the appropriate site ), which can occur in vitro (for example, incubation of the polypeptide chain in a composition comprising proteases or peptidases of known activity and / or in a composition comprising known conditions or reagents to promote the desired proteolytic action. Purification and modification of recombinant protein are well known in the art such that precursor design of

253

IMPI

MBílCAHC INSTITUTE

Say THE INGUSTRIAL PROPERTY

<img file="MX348166B_D0156.tif" />

protein could include the number of modalities readily appreciated by the skilled worker. Any protease or peptidase known in the art can be used for the desired modification of the precursor molecule, for example, thrombin (which recognizes the amino acid sequence LVPR'gS (SEQ ID NO: 89)), or factor Xa (which recognizes the amino acid sequence I (E / D) GR^TO (SEQ ID NO: 90) (Nagai et al. (1985) Oxygen Binding Properties Of Human Mutant Hemoglobin Synthesized In Escherichia Coli, Proc. Nat. Acad. Sci. USA

82: 7252-7255, and reviewed by Jenny et al. (2003) A Critical Review Of The Methods For Cleavage Of Fusion Proteins With Thrombin And Factor Xa, Protein Expr. Purif. 31: 1-11, each of which is incorporated herein by reference in its entirety)), enterokinase (which recognizes the amino acid sequence DDDDK (SEQ ID NO: 91) (Collins-Racie et al. (1995) Production Of Recombinant Bovine Enterokinase Catalytic Subunit In Escherichia Coli Using The Novel Secretory Fusion Partner DsbA, Biotechnology 13: 982-987 incorporated herein by reference in its entirety)), furin (which recognizes the amino acid sequence ΡΧΧΡ^Λ, with a preference for ΡΧ (Κ / Ρ) Ε^λ (SEQ ID NO: 92 and SEQ ID NO: 93, respectively) (additional R at position P6 appears to improve separation)), and AcTEV (which recognizes the amino acid sequence ENLYFQ^TOG (SEQ ID NO: 94) (Parks et al. (1994) Release Of Proteins And Peptides From Fusion Proteins Using

A Recombinant Plant

<img file="MX348166B_D0157.tif" />

216: 413-417 incorporated herein by reference in its entirety)) and Foot and Mouth Disease Virus Protease C3. See, for example, Section 6.4, supra.

Different host cells have specific characteristic mechanisms for post translation processing and modification of proteins and gene products. Appropriate cell lines from the host systems can be selected to ensure correct modification and processing of the expressed foreign protein. At this point, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are limited to

CHO, VERY, BHK, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483,

HS578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For high-throughput, long-term production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express an antibody of the invention can be modified. Instead of using expression vectors containing viral origins of replication, host cells are transformed with DNA controlled through appropriate expression control elements (e.g., promoter,²⁵⁵ ”S Ά INDUSTRIAL HROPFÍTY (enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. After introduction of the foreign DNA, the modified cells can be grown for 1-2 days in an enriched medium, and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form the site which in turn can be cloned and expanded into cell lines. This method can advantageously be used to modify cell lines expressing the antibodies of the invention. Such modified cell lines can be particularly useful in the classification and evaluation of compounds that interact directly or indirectly with the molecules of the invention.

A number of selection systems can be used, including but not limited to herpes simplex virus thymidine kinase genes from (Wigler et al. (1977) Transfer Of Purified Herpes Virus Thymidine Kinase Gene To Cultured Mouse Cells, Cell 11: 223-232), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al. (1992) Use Of The HPRT Gene And The HAT Selection Technigue In DNA-Mediated Transformation Of Mammalian Cells: First Steps Toward Developing Hybridoma Techniques And Gene

Therapy, Bioessays 14: 495-500), and phosphoribosyltransferase ²⁵⁶MIXICAN X

OF THE PROPERTY indintriai adenine (Lowy et al. (1980) Isolation Of Transforming DNA:

Cloning The Hamster aprt Gene, Cell 22: 817-823) that can be used in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used according to the selection bases of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. (1980) Transformation Of Mammalian Cells With An Amplifiable Dominant-Acting Gene, Proc. Nati. Acad Sci. USA 77: 35673570, O'Hare et al. (1981) Transformation Of Mouse Fibroblasts To Methotrexate Resistance By A Recombinant Plasmid Expressing A Prokaryotic Dihydrofolate Redactase, Proc. Nati. Acad. Sci. USA 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan et al. (1981) Selection For Animal Cells That Express The Escherichia coli Gene Coding For Xanthine-Guanine Phosphoribosyltransferase, Proc. Nati. Acad. Sci. USA 78: 2072-2076); neo, which confers resistance to aminoglycoside G-418 (Tolstoshev (1993) Gene Therapy, Concepts, Current Triais And Future Directions, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan (1993) The Basic Science Of Gene Therapy , Science 260: 926-932; and Morgan et al. (1993) Human Gene Therapy, Ann. Rev. Biochem. 62: 191-217) and hygro, which confers resistance to hygromycin (Santerre et al. (1984) Expression Of Prokaryotic Genes For Hygromycin B And G418 Resistance As Dominant-Selection Markers In Mouse L Cells, Gene 30: 147-156). Methods

<img file="MX348166B_D0158.tif" />

commonly known in the art for recombinant DNA technology that can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley &amp; Sons, NY .; Colberre-Garapin et al. (1981) A New Dominant Hybrid Selective Marker For Higher Eukaryotic Cells, J. Mol. Biol. 150: 1-14.

The expression levels of a molecule of the invention can be increased through vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning. Vol. 3 (Academic Press, New York, 1987). When a marker in the vector system that expresses an antibody is amplifiable, increasing the level of the inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the nucleotide sequence of a polypeptide of the diabody molecule, the production of the polypeptide will also increase (Crouse et al. (1983) Expression And Amplification Of Engineered Mouse Dihydrofolate Redactase Minigenes, Mol. Cell. Biol. 3: 257-266).

LMPI ^

INSTITUTE MtXICANt * Λλ

DE ΙΛ PROPISdai ^ raSr- ^ U _T_&gt; ____ 1 _ i INOL-miA ·

The host cell can be co-transfected with the expression vectors of the invention, the first vector encodes the first polypeptide of the diabody molecule and the second vector encodes the second polypeptide of the diabody molecule. The two vectors can contain identical selectable markers that allow equal expression of both polypeptides. Alternatively, a single vector can be used that encodes both polypeptides. The coding sequences for the polypeptides of the molecules of the invention may comprise genomic cDNA or cDNA.

Once the molecule of the invention (i.e., diabodies) has been recombinantly expressed, it can be purified by any method known in the art for the purification of polypeptides, polyproteins, or diabodies (for example, analogous to purification schemes of the antibody based on antigen selectivity, e.g., via chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen (optionally after selection of protein A where the diabody molecule comprises an Fe domain (or one of its portions)), and sizing column chromatography), centrifugation, differential solubility or through from any other standard technique for the purification of polypeptides, polyproteins

259 or diabodies.

INSTITUTO MEXICANO OE LA PRGHEOAD PiLlUSTSIAl

<img file="MX348166B_D0159.tif" />

PROPHYLACTIC AND THERAPEUTIC METHOD

The molecules of the invention are particularly useful for the treatment and / or prevention of a disease, disorder or infection where an effector cell function (eg ADCC) mediated by FcyR is desired (eg cancer, infectious disease) . As explained supra, the diabodies of the invention may exhibit antibody-like functionality in eliciting effector function even though the diabody molecule does not comprise an Fe domain. By comprising at least one epitope binding domain that immunospecifically recognizes an FcyR, the diabody molecule can exhibit FcyR binding and activity analogous to Fc-FcyR interactions. For example, the molecules of the invention can bind a cell surface antigen and an FcyR (eg,

FcyRIIIA) into an immune effector cell (eg, NK cell), stimulating effector function (eg,

ADCC, CDC, phagocytosis, opsonization, etc.) against such a cell.

In other embodiments, the diabody molecule of the invention comprises an Fe domain (or a portion thereof). In such embodiments, the Fe domain may further comprise at least one amino acid modification relative to the wild-type Fe domain (or one of its

IMPI portions) and / or may comprise domains from one or more IgG isotypes (eg, IgGl, IgG2, IgG3 or IgG4). Molecules of the invention comprising variant Fe domains may exhibit conferred or altered phenotypes relative to molecules comprising a wild-type Fe domain such as altered or transferred effector function activity (eg, as assayed in an assay). NK-dependent or macrophage-dependent). In such embodiments, molecules of the invention with conferred or altered effector function activity are useful for the treatment and / or prevention of a disease, disorder, or infection where improved efficacy of the activity between effector function is desired. In certain embodiments, the diabody molecules of the invention comprise an Fe domain (or a portion thereof) that mediates the complement-dependent cascade. Variants of the Fe domain identified as altering effector function are described in International Application WO 04/063351, US Patent Application Publications 2005/0037000 and 2005/0064514, US Provisional Applications 60 / 626,510, filed 10/10 November 2004, 60 / 636,663, filed December 15, 2004, and 60 / 781,564, filed March 10, 2006, and US Patent Applications 11 / 271,140, ​​filed November 10, 2005

11 / 305,787, filed on December 15, 2005, concurrent applications^{To the} of '^^ inventors, each of which is incorporated by reference in its entirety.

The invention encompasses methods and compositions for the treatment, prevention, or administration of a cancer in a subject, comprising administering to the subject a therapeutically effective amount of one or more molecules comprising one or more epitope binding sites, and optionally, a domain. Fe (or one of its portions) modified according to the invention, the molecule of which also binds to the cancer antigen. The molecules of the invention are particularly useful for the prevention, inhibition, reduction of growth or advancement of primary tumors, cancer cell metastases, and infectious diseases. Although not intended to be bound through a particular mechanism of action, the molecules of the invention mediate effector function that results in tumor clearance, tumor shrinkage, or a combination of these. In alternative embodiments, the diabodies of the invention mediate therapeutic activity through crosslinking of cell surface antigens and / or receptors and enhanced apoptosis or negative growth regulatory signaling.

Although not intended to be bound through a particular mechanism of action, the diabody molecules of ²⁶²INSTI-ΠΛΟ M SICA) *&gt; DJ LA M ^ mClAD INDUSTRIAL the invention exhibits improved therapeutic efficacy relative to therapeutic antibodies known in the art, in part, due to the ability of the diabody to immunospecifically bind to the target cell that expresses a particular antigen (e.g. Fc and R) at reduced levels, e.g. by virtue of the ability of the diabody to stay in the target cell longer due to an improved avidity of the diabodyepitope interaction.

Diabodies of the invention with improved affinity and avidity for antigens (eg, FcyRs) are particularly useful for the treatment, prevention, or management of a cancer, or other disease or disorder, in a subject, where FcyRs are expressed at low levels. in the target cell populations. As used in the present invention, FcyR expression in cells is defined in terms of the density of such molecules per cell as measured using common methods known to those of skill in the art. Molecules of the invention comprising multiple epitope binding sites and optionally and FcyR (or a portion thereof) preferably also have conferred or enhanced avidity and affinity and / or effector function in cells expressing effective function. , for example, a cancer antigen, at a density of 30,000 to 20,000 molecules / cell, at a density of 20,000 to

10,000 molecules / cell, at a density of 10,000 molecules / cell or less, at a density of 5,000 molecules / cell or less, or a density of 1,000 molecules / cell or less. The molecules of the invention have particular utility in the treatment, prevention, or management of a disease or disorder, such as cancer, in a sub-population, where the target antigen is expressed at low levels in the target cell population.

The molecules of the invention can also advantageously be used in combination with other therapeutic agents known in the art for the treatment and prevention of diseases, such as cancer, autoimmune disease, inflammatory disorders, and infectious diseases. In a specific embodiment, the molecules of the invention can be used in combination with monoclonal or chimeric antibodies, lymphokines, or hematopoietic growth factors (such as, for example, IL-2, IL-3 and IL-7), which, for example, they serve to increase the number of effector cell activity that interact with the molecules and increase the immune response. The molecules of the invention can also advantageously be used in combination with one or more drugs used to treat a disease, disorder or infection such as, for example, anti-cancer agents, anti²⁶⁴ IMPI® ft * sTFTvro muucanc

DELA MOHEDA D

WDVSTRlAl ^ sZV ^ LJÍ inflammatory, or anti-viral agents, for example, as detailed in Section 5.7.

CANCERES

The invention encompasses methods and compositions for treatment and prevention in a subject comprising administering to the subject a therapeutically effective amount of one of more molecules comprising multiple epitope-binding domains. In some embodiments, the invention encompasses methods and compositions for the treatment or prevention of cancer in a subject with an FcyR polymorphism such as those homozygous for FyRIIIA-158V or FcyRllIA-158F alleles. In some embodiments, the invention encompasses modifying at least one epitope binding domain of the diabody molecule to immospecifically bind FcyRIIIA (158F). In other embodiments, the invention encompasses modifying at least one epitope binding domain of the diabody molecule to immunospecifically bind FcyRIIIA (158V).

The efficacy of standard monoclonal antibody therapy depends on the subject's FcyR polymorphism (Cartron et al. (2002) Therapeutic Activity Of Humanized Anti-CD20 Monoclonal Antibody And Polymorphism In IgG Fe Receptor FcRIIIa Gene, Blood 99: 754-758; Weng et al. (2003) Two Immunoglobulin G Fragment C Receptor Polymorphisms Independently Predict Response To Rituximab In Patients With Follicular Lymphoma ”, J Clin Oncol. 21 (21): 3940-3947, both

<img file="MX348166B_D0160.tif" />

of which are incorporated herein by reference in their entirety). These receptors are expressed on the surface of effector cells and mediate ADCC. High affinity alleles of low affinity activating receptors enhance the ability of effector cells to mediate ADCC. In contrast to relying on Fc-Fc and R interactions to effect effector function, the methods of the invention encompass modified molecules to immunospecifically recognize low affinity activation receptors, allowing the molecules to be engineered for a specific polymorphism. Alternatively or additionally, the molecule of the invention can be modified to comprise a variant Fe domain that exhibits enhanced affinity to FcyR (relative to a wild-type Fe domain) in effector cells. The modified molecules of the invention provide better immunotherapy reagents for patients regardless of their FcyR polymorphism.

Modified diabody molecules according to the invention are assayed via ADCC using either a cell culture line or a patient derived PMBC cell to determine the ability of Fe mutations to enhance ADCC. Standard ADCC is carried out using the methods described herein. Lymphocytes are harvested from peripheral blood using a

266

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ΟΓ THE INDUSTRIAL ROHEDAD

<img file="MX348166B_D0161.tif" />

Ficoll-Paque gradient (Pharmacia). Target cells, i.e., cultured lines or patient derived cells, are loaded with Europium (PerkinElmer) and incubated with effectors for 4 hours at 37 ° C. Released Europium is detected using the fluorescent plate reader (Wallac). The resulting ADCC data indicates the efficacy of the molecules of the invention in activating NK cell mediated cytotoxicity and establishes which molecules can be tested with both patient samples and washed monocytes. Diabody molecules that show a greater potential to elicit ADCC activity, these are tested in an ADCC assay using PBMCs from patients. PBMC from healthy donors are used as effector cells.

Accordingly, the invention provides methods for preventing or treating cancer characterized by a carcinogenic antigen by modifying the diabody molecule to immunospecifically recognize the cancer antigen such that the diabody molecule itself is cytotoxic (e.g., through crosslinking of surface receptors that drive increased apoptosis or down-regulation of proliferative signals) and / or comprises an Fe domain, according to the invention, and / or mediates one or more of the effector functions (for eg, ADCC, phagocytosis). Diabodies that have been modified in accordance with the

ΙΝΙΤΠΤΓΤΟ MEXICANO M LA «OflEDAU industrial YrerVy invention are useful for the prevention or treatment of cancer, as they have a cytotoxic activity (eg enhanced and / or enhanced tumor cell killing eg ADCC activity or CDC activity).

Cancers associated with a carcinogenic antigen can be treated or prevented through the administration of a diabody that binds a carcinogenic antigen and the cytotoxic, and / or has been modified according to the methods of the invention to exhibit an effector function. For example, and not by way of limitation, cancers associated with the following carcinogenic antigens can be treated or prevented through the methods and compositions of the invention: KS 1/4 pan-carcinoma antigen (Perez et al., (1989) Isolation And Characterization Of A Cdna Encoding The Ksl / 4 Epithelial Carcinoma Marker, J. Immunol. 142: 36623667; Móller et al. (1991) Bispecific-Monoclonal-Antibody-Directed Lysis Of Ovarian Carcinoma Cells By Activated Human T Lymphocytes, Cancer Immunol. Immunother. 33 (4): 210-216), Ovarian Carcinoma Antigen (CA125) (Yu et al. (1991) Coexpression Of Different Antigenic Markers On Moieties That Bear CA 125 Determinant, Cancer Res. 51 (2): 468-475) , Prosthetic acid phosphate (Tailor et al. (1990) Nucleotide Sequence Of Human Prostatic Acid Phosphatase Determined From A Full-Length cDNA Clone, Nucí. Acids Res. 18 (16): 4928), Prostate Specific Antigen (Henttu et al. (1989) cDNA²⁶⁸ WICKED

INSTrtUTO MEXICANO de ia moriWAi ¿aura

INDUSTRY! Coding For The Entire Human Prostate Specific Antigen Shows High Homologies To The Human Tissue Kallikrein Genes, Biochem. Biophys. Res. Ate. 10 (2): 903-910, - Israeli et al. (1993) Molecular Cloning Of A Complementary DNA Encoding A Prostate-Specific Membrane Antigen, Cancer Res. 53: 227-230), p97 antigen associated with melanoma p97 (Estin et al. (1989) Tr ansfected Mouse Melanoma Lines That Express Various Levels Of Human Melanoma-Associated Antigen p97, J. Nati Cancer Instit. 81 (6): 445-454), melanoma antigen gp75 (Vijayasardahl et al. (1990) The Melanoma Antigen Gp75 Is The Human Homologue Of The Mouse B (Brown) Locus Gene

Product, J. Exp. Med. 171 (4): 1375-1380), High Molecular Weight Melanoma Antigen (HMW-MAA) (Natali et al. (1987) Immunohistochemical Detection Of Antigen In Human Primary And Metastatic Melanomas By The Monoclonal Antibody 140,240 And Its Possible Prognostic Significance, Cancer 59: 55-63; Mittelman et al. (1990) Active Specific Immunotherapy In Patients With Melanoma. A Clinical Trial With Mouse Antiidiotypic Monoclonal Antibodies Elicited With Syngeneic Anti-High-Molecular-Weight-Melanoma-Associated Antigen Monoclonal Antibodies, J. Clin. Invest. 86: 21362144)), prostate specific membrane antigen, carcinoembryonic antigen (CEA) (Foon et al. (1995) Immune Response To The Carcinoembryonic Antigen In Patients Treated With An Anti-Idiotype Antibody Vaccine, J. Clin. Invest. 96 (1): 334269 IMPI ^ Mexican institute

OF THE INDUSTRIAL IROnSDAI

42), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor associated antigens such as: CEA, TAG-72 (Yokota et al. (1992) Rapid Tumor Penetration Of A Single-Chain Fv And Comparison With Other Immunoglobulin Forms, Cancer Res. 52: 3402-3408), CO17-1A (Ragnhammar et al. (1993) Effect Of Monoclonal Antibody 17- IA And GM-CSF In Patients With Advanced Colorectal Carcinoma - Long-Lasting, Complete Remissions Can Be Induced, Int. J. Cancer 53: 751-758); GICA 19-9 (Herlyn et al. (1982) Monoclonal Antibody Detection Of A Circulating Tumor-Associated Antigen. I. Presence Of Antigen In Sera Of Patients With Colorectal, Gastric, And Pancreatic Carcinoma, J. Clin. Immunol. 2: 135 -140), CTA-1 and LEA, Burkitt's lymphoma CD19 antigen-38.13 (Ghetie et al. (1994) Anti-CD 19 Inhibits The Growth Of Human B-Cell Tumor Lines In Vitro And Of Daudi Cells In SCID Mice By Inducing Cell Cycle Arrest, Blood 83: 1329-1336), human B lymphoma CD20 antigen (Reff et al. (1994) Depletion Of B Cells In Vivo By A Chimeric Mouse Human Monoclonal Antibody To CD20, Blood 83: 435-445), CD33 (Sgouros et al. (1993) Modeling And Dosimetry Of Monoclonal Antibody M195 (Anti-CD33) In Acute Myelogenous Leukemia, J. Nucí. Med. 34: 422-430), melanoma specific antigens such as ganglioside GD2 (Saleh et al. (1993) Generation Of A Human Anti-Idiotypic Antibody

That Mimics The GD2 Antigen, J. Immunol., 151, 3390-3398),

270 ΙΜΡΙ ^^_; ΐΝϊπτντο mbucano Df LA PRQWiDA Ο IWWSTWIAL gangliosida GD3 (Shitara et al. (1993) A Mouse / Human Chimeric Anti- (Ganglioside GD3) Antibody With Enhanced Antitumor Activities, Cancer Immunol. Immunother. 36: 373380), ganglioside et al GM2 (Livingston et al. (1994) Improved Survival In Stage III Melanoma Patients With GM2 Antibodies: A Randomized Trial Of Adjuvant Vaccination With GM2 Ganglioside, J. Clin. Oncol. 12: 1036-1044), GM3 ganglioside (Hoon et al. (1993) Molecular Cloning Of A Human Monoclonal Antibody Reactive To Ganglioside GM3 Antigen On Human Cancers, Cancer Res. 53: 5244-5250), type of cell surface antigen (TSTA) tumor-specific transplantation such as virally induced tumor antigens including T antigen DNA tumor virus and RNA tumor virus envelope antigens, oncofetal antigen-alpha-fetoprotein such as colon CEA, bladder tumor oncofetal antigen (Hellstróm et al. (1985) Monoclonal Antibodies To Cell Surface Antigens Shared By Chemically Induced Mouse Bladder Carcinomas, Cancer. Res. 45: 2210-2188), differentiation antigen such as human lung carcinoma antigen L6, L2 0 (Hellstróm et al. (1986) Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma, Cancer Res. 46: 3917-3923 ), fibrosarcoma antigen, human leukemia T-cell antigen-Gp37 (Bhattacharya-Chatterjee et al. (1988) Idiotype Vaccines Against Human T Cell Leukemia. II. Generation And²⁷¹ 'ΙΜΡΙ ^ wsrninu mujcanc ^ γ ** ®® -? **

I heard LA PROSUDA ​​D fNn ”miAi

Characterization Of A Monoclonal Idiotype Cascade (Abl, Ab2, and Ab3), J. Immunol. 141: 1398-1403), neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR (epidermal growth factor receptor), HER2 antigen (pl8^HER2), polymorphic epithelial mucin (PEM) (Hilkens et al. (1992) Cell Membrane-Associated Mucins And Their Adhesion-Modulating Property, Trends in Biochem. Sci. 17: 359-363), APO-1 malignant human lymphocyte antigen ( Trauth et al. (1989) Monoclonal Antibody-Mediated Tumor Regression By Induction Of Apoptosis, Science 245: 301-304), differentiation antigen (Feizi (1985) Demonstrate ion By Monoclonal Antibodies That Carbohydrate Structures Of Glycoproteins And Glycolipids Are Onco-Developmental Antigens, Nature 314 : 53-57) such as antigen I found in fetal erythrocytes and primary endoderma, I (Ma) found in gastric adenocarcinomas, M18 and M39 found in chest epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl, VIM-D5, and 0 ^ 6-22 found in colorectal cancer, TRA-1-85 (blood group H), C14 found in colonic adenocarcinoma, F3 found in adenocarcinoma lung, AH6 found in gastric cancer, AND hapten, Le^Y found in embryonic carcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells, Ei series (blood group B) found in pancreatic cancer, FC 10.2 found in

INSTITUTE MLXJCANCi

OF THE ΛΟΜΪΟΑΠ

272 embryonal carcinoma cells, gastric adenocarcinoma, CO-514 (blood group Le^to) found in adenocarcinoma, NS-10 found in adenocarcinomas, CO-43 (blood group Le^b), G49, EGF receptor, (blood group ALe^b/You^Y) found in colonic adenocarcinoma, 19.9 found in colon cancer, gastric cancer mucins, T₅TO₇ found in myeloid cells, R₂₄ found in melanoma, 4.2, G_D3, Dl.l, OFA-1, Gm₂, OFA-2, G_D2, MI: 22: 25: 8 found in embryonic carcinoma cells, and SSEA-3, SSEA-4 found in 4-8 cell stage embryos. In another embodiment, the antigen is a peptide-derived T-cell receptor from a cutaneous T-cell lymphoma (see, Edelson (1998) Cutaneous T-Cell Lymphoma: A Model For Selective Immunotherapy, Cancer J Sci Am. 4: 62- 71).

Cancers and related disorders that can be treated or prevented through the methods and compositions of the present invention include, but are not limited to, the following: leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic granulocytic leukemia ), chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not²⁷³ IMPIAS

ΤΝΠΠΙΓΓΟ IND1-STR1AL ROUNDED MMUCAN limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to incandescent multiple myeloma, non-secretory myeloma, osteoclerotic myeloma, plasma cell leukemia, solitary plasmacytoma, and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammoplasty of undetermined significance; benign monoclonal gamopathy; heavy chain disease; sarcomas of bone and connective tissue such as but not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemoma, rhabdomyosarcoma, synovial sarcoma, brain tumors including but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neuroma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma, breast cancer including, but not limited to, adenocarcinoma, lobular carcinoma (small cell), intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, including but not limited to

274

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OF THE PROMISE INDUSTRY *

<img file="MX348166B_D0162.tif" />

a, pheochromocytoma and adenocortical carcinoma; thyroid cancer such as, but not limited to, papillary or follicular thyroid cancer, medullary thyroid cancer, and anaplastic thyroid cancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers, including but not limited to Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; cancers of the eye including but not limited to ocular melanoma, such as iris melanoma, choroidal melanoma and ciliary body melanoma, and retinoblastoma; vaginal cancers, including but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma, vulvar cancer, including but not limited to squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and disease of Paget; cervical cancer including, but not limited to, squamous cell carcinoma and adenocarcinoma; uterine cancers including, but not limited to, carcinoma of the en dometrial and uterine sarcoma; ovarian cancers including but not limited to ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including but not limited to squamous cancer, adenocarcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma,

275 mexican institute

Dt THE MHOPIEOAL · ^ 1 j TÍSC

INDUSTRIAL&gt; ¿^^ 35 adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, wart carcinoma, oat cell carcinoma (small cell), stomach cancers including but not limited to, adenocarcinoma, fungus (polypoid), ulceration, de superficial extension, diffuse extension, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers, rectal cancers, liver cancers including but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including but not limited to adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including but not limited to non-small cell lung cancer, squamous cell carcinoma (squamous cell carcinoma), adenocarcinoma, large cell carcinoma, and small cell lung cancer; Testicular cancers including but not limited to, germ cell tumor, seminoma, anaplastic, classic (typical), spermatocytic, non-seminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk sac tumor), prostate cancers including but They are not limited to adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; oral cancers, penile cancers including but not limited to squamous cell carcinoma; basal cancers, salivary gland cancers including but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; cancers of₂₇₆ IMPIAS

INSITOMfeUCAHC;

OF THE INDUSTRIAL ΡΚΟΠΒΟΛΠ OfttoPtW Pharynx including but not limited to, heal ^ e — cell ™, »squamous, and warts; skin cancer including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, malignant lentigo melanoma, acral lentiginous melanoma; Kidney cancers including but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, temporal cell cancer (renal pelvis and / or uterus), Wilms tumor; bladder cancers including but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, carcinoma of the papal gland, and carcinoma of the pacinoma se disorders, see Fishman et al. (1985) Medicine, 2^to Ed., JB Lippincott Co., Philadelphia; and Murphy et al. (1997) Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America).

Accordingly, the methods and compositions are also useful in the treatment or prevention of a variety of cancers or other proliferative diseases.

<img file="MX348166B_D0163.tif" />

abnormal including (but not limited to) the following: carcinoma, including bladder, chest, colon, kidney, liver, lung, ovary, pancreas, stomach, prostate, cervix, thyroid, and skin; including squamous cell carcinoma, hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, and glioma; central and peripheral nervous system tumors, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosa, keratoactanthoma, seminoma, follicular thyroid cancer, and teratocarcinoma. Cancers caused by aberrations in apoptosis which can also be treated by the methods and compositions of the Invention are also contemplated. Such cancers may include but are not limited to follicular lymphomas, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.

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<img file="MX348166B_D0164.tif" />

In specific embodiments, malignancy or disproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, are treated or avoided through the methods and compositions of the invention in the ovary, bladder, chest, colon, lung, skin, pancreas, and uterus. In other specific embodiments, sarcoma, melanoma, or leukemia are treated or prevented by methods and compositions of the invention.

In a specific embodiment, a molecule of the invention (eg, a diabody comprising multiple epitope-binding domains and optionally an Fe domain (or a portion thereof)) inhibits or reduces cancer cell growth by at least 99 %, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40 %, at least 45%, at least 35%, at least 30%, at least 25%, at least

20%, or at least 10% relative to the growth of cancer cells in the absence of the molecule of the invention.

In a specific embodiment, a molecule of the invention (eg, a diabody comprising multiple epitope-binding domains and optionally an Fe domain (or a portion thereof)) kills cells or inhibits or reduces the growth of cancer cells. by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least

279

<img file="MX348166B_D0165.tif" />

35%, at least 40%, at least at least 7 0%, at least 75%

45%, at least 50%, at least 60%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% better than the parent molecule.

AUTOIMMUNE DISEASE AND INFLAMMATORY DISEASES

In some diseases, the molecules of the invention comprise a specific epitope binding domain for FcyRIIB and / or a variant Fe domain (or one of its portions), modified according to the methods of the invention, the Fe domain of which exhibits a greater affinity for

FcyRIIB and a decreased affinity for FcyRIIIA and / or FcyRIIA relative to a wild-type Fe domain. Molecules of the invention with such binding characteristics are useful in the regulation of the immune response, for example, in the inhibition of the immune response in connection with autoimmune diseases or inflammatory diseases.

Although not intended to be linked to any particular mechanism of action, molecules of the invention with an affinity for FcyRIIB and / or comprising an Fe domain with an increased affinity for FcyRIIB and a decreased affinity for FcyRIIIA and / or FcyRIIA can drive to the de-stimulation of the activation of the response to FcyR and the inhibition of cellular sensitivity, and thus has a therapeutic efficacy to treat and / or prevent a

280 autoimmune disorder.

IMPI

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OF THE INDUSTRIAL MOHEDAL

<img file="MX348166B_D0166.tif" />

The invention also provides methods for preventing, treating or managing one or more symptoms associated with an inflammatory disorder in a subject which further comprises administering to the subject a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents. The invention also provides methods for preventing, treating or managing one or more symptoms associated with an autoimmune disease which further comprises administering to the subject a therapeutically or prophylactically effective amount of one or more immunomodulatory agents. See section 5.7 which provides non-limiting examples of anti-inflammatory agents and immunomodulatory agents.

Examples of autoimmune disorders that can be treated during administration of the molecules of the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia. , autoimmune hepatitis, inflammation of the ovaries and autoimmune orchitis, autoimmune thrombocytopenia, Behcet's disease, pemphigus bullous, cardiomyopathy, celiac dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS),

281

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Chronic inflammatory demyelizing polyneuropathy, Churg-Strauss syndrome, scar pemphigus, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgiafibromiositis, glomerulonephritis, GravesBasedow's disease, Guillainitis-Barré syndrome, Hashimoto's, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agamaglobulinemia , psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjógren's syndrome, Stiff man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu arteritis, temporal arteritis, giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vasculitis herpetiformis, dermatitis, vitiligo, and Wegener's granulomatosis. Examples of inflammatory disorders include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD),

<img file="MX348166B_D0168.tif" />

allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic bacterial or viral infections. As described herein in Section 2.2.2, some autoimmune disorders are associated with an inflammatory condition. Thus, there is an overlap between what is considered an autoimmune disorder and an inflammatory disorder. Consequently, some autoimmune disorders can also be characterized as inflammatory disorders. Examples of inflammatory disorders that can be prevented, treated, or managed according to the methods of the invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock. , pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation resulting from chronic viral or bacterial infections.

Molecules of the invention comprising at least one epitope binding domain specific for FcyRIIB and / or a variant FC domain with improved affinity for FcyRIIB and decreased affinity for FcyRIIIA can also be used to reduce the inflammation experienced.

283

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by animals, particularly mammals, inflammatory corr - ^ - tTrfte-fzemas. In a specific embodiment, a molecule of the invention reduces inflammation in an animal by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least minus 10% relative to inflammation with an animal, which does not administer the molecule.

The molecules of the invention comprise at least one epitope binding domain specific for FcyRIIB and / or a variant Fe domain with improved affinity for FcyRIIB and decreased affinity for FcyRIIIA can also bind to prevent transplant rejection.

INFECTIOUS DISEASES

The invention also encompasses methods for treating or preventing an infectious disease in a subject comprising administering a therapeutically or prophylactically effective amount of one or more molecules of the invention comprising at least one epitope binding domain specific for an infectious agent associated with infectious disease. In certain embodiments, the molecules of the invention are toxic to the infectious agent, enhance the immune response against the agent, or enhance the effector function against the agent, relative to the immune response in

284

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absence of the molecule. Diseases Tnfgocee-ao · quo. can be treated or prevented through the molecules of the invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, and viruses.

Viral diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention include, but are not limited to, those caused by type A hepatitis, type B hepatitis, type C hepatitis, influenza, chickenpox, adenovirus, herpes simplex type I (HSV-I), herpes simplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papota virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, smallpox virus, Epstein Barr virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-2), and agents of viral diseases such as viral meningitis, encephalitis, dengue or smallpox.

Bacterial diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, which are caused by bacteria include, but are not limited to, myobiacterial richesia, mycoplasma, neisseria, S. pneumonia, Borrelia

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INSTITUID MÍXICANO DE LA nCPIEDAt: · industrial

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burgdorferi (Lyme disease), Bacillus anthracis (anthrax), tetanus, streptococcus, staphylococcus, mycobacterium, tetanus, typhoid, cholera, plague, diphtheria, chlamydia, S. aureus, and legionella.

Protozoal diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, that are caused by protozoa include, but are not limited to, leishmania, coczidia, tripnosome, or malaria.

Parasitic diseases that can be treated or prevented using the molecules of the invention in conjunction with the methods of the present invention, which are caused by parasites including, but not limited to, Chlamydia and Richetsia.

According to one aspect of the invention, molecules of the invention that comprise at least one epitope-binding domain specific for an infectious agent exhibit an effector function of the antibody towards the agent, eg, the pathogenic protein. Examples of infectious agents include but are not limited to (eg, Escherichia coli, Klebsiella pneumonia, Staphylococcus aureus, Enterococcus faecials, Candida albicans, Proteus vulgaris, Staphylococcus viridans, and Pseudotomonas aeruginosa), a pathogen (eg lymphoid Bone virus). (LPV), Bordetella pertussis; virus

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236 IMPI

INSTITUTE M * ¡CAN &lt;. FROM THE INDUSTRIAL PROPERTY of Boma's disease (BDV), bovine coronavirus, choriomeningitis virus; dengue virus; a virus, E. coli, Ebola, Ecovirus 1; Ecovirus-11 (EV); endotoxin (LPS), enteric bacteria, enteric orphan virus, enterovirus; feline leukemia virus; foot and mouth disease virus; Gibbon monkey leukemia virus (GALV); Gram-negative bacteria; Helicobacter pylori, hepatitis B virus (HBV), herpes simplex virus, HIV-1; Human cytomegalovirus; human coronaviruses; influenza A, B and C; Legionella; Leishmania mexicana, Listeria monocytogenes, measles virus, meningococcus; Morbillivirus, mouse hepatitis virus; murine leukemia virus; murine gamma herpes virus; murine retrovirus; mouse hepatitis virus murine coronavirus; Avian Mycobacterium, Neisseria gonorrhea; Newcastle disease virus; parvovirus B19; Plasmodium falciparum; Smallpox virus; Pseudomonas; rotavirus; Salmonella typhiurium; Shigella, Streptococcus; T-cell lymphotropic virus 1; vaccine virus).

Detoxification

The invention also encompasses methods for detoxifying a subject exposed to a toxin (eg, a toxic drug molecule) comprising administering a therapeutically or prophylactically effective amount of one or more or more molecules of the invention that

IMPI

INSTITUTO MEXICANO DE LA PROPIEDAD INIMISTRIAL comprises at least one epitope-binding domain specific for a toxic drug molecule. In certain embodiments, binding of the molecule of the invention to the toxin reduces or eliminates the adverse physiological effect of such toxin. In still other embodiments, the binding of the diabody of the invention to the toxin enhances or enhances the elimination, degradation or neutralization of the toxin relative to elimination to elimination, degradation or neutralization of the absence of the diabody. Immunotoxicotherapy according to the methods of the invention can be used to treat overdose or exposure to drugs including, but not limited to, digixin, PCP, cocaine, conchycin, and tricyclic antidepressants.

COMBINATION THERAPY

The invention further encompasses the administration of the molecules of the invention in combination with therapies known to those skilled in the art, for the treatment or prevention of cancer, autoimmune disease, infectious disease or intoxication, including but not limited to standard chemotherapies. current experimental, hormonal therapies, biological therapies, immunotherapies, radiation therapies or surgery. In some embodiments, the molecules of the invention can be administered in combination with a therapeutically or prophylactically effective amount of one or more

288 ! Ν5ΤΓΓυΤ · MEXICAN plus agents, therapeutic antibodies or ofFós¹''¹ Agents known in the art for the treatment and / or prevention of cancer, autoimmune disease, infectious disease or intoxication.

In certain embodiments, one or more of the molecules of the invention are administered to a mammal, preferably a human, concurrently with one or more other therapeutic agents useful for the treatment of cancer. The term concurrently is not limited to the administration of prophylactic or therapeutic agents at exactly the same time, but rather means that a molecule of the invention and the other agent are administered to a mammal in a sequence and within a time interval of such that the molecule of the invention can act in conjunction with the other agent to provide an increased benefit than if administered otherwise. For example, each prophylactic or therapeutic agent (eg, chemotherapy, radiation therapy, hormonal therapy, or biological therapy) can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, it should be administered close enough in time to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered separately, in any appropriate form and through ²⁸⁹ fNSTm nX 'MEXICAN Dt LA PROBIDAD FND »» 5TRLAl any suitable route. In various embodiments, the prophylactic or therapeutic agents are administered less than 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, from about 5 hours to about 6 hours apart, from about 6 hours to about 7 hours apart, from about 7 hours to about 8 hours apart, from about 8 hours to about 9 hours apart, from about 9 hours to about 10 hours apart, from about 10 hours to about 11 hours apart, from about 11 hours to about 12 hours apart, no more than 24 hours 24 hours apart or no more than 48 hours apart. In preferred embodiments, two or more components are administered within the same patient visit.

In other embodiments, the prophylactic or therapeutic agents are administered about 2 to 4 days apart, about 4 to 6 days apart, about 1 week apart, about 1 to 2 weeks apart, or more than 2 weeks apart. In preferred embodiments, the prophylactic or therapeutic variants are administered in a time frame where

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ÍMPI iwhwtd mbxicamt, DE LA MOHEDAL industrial

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both agents are still active. An expert will be able to determine such a time frame by determining the half-life of the administered agents.

In certain embodiments, the prophylactic or therapeutic agents of the invention are administered cyclically to a subject. Cyclic therapy involves the administration of the first agent over a period of time, followed by the administration of a second agent and / or a third agent over a period of time and repeating this sequential administration. Cyclic therapy can reduce the development of resistance to one or more of the therapies, avoid or reduce the side effects of one of the therapies, and / or improve the efficacy of the treatment.

In certain embodiments, the prophylactic or therapeutic agents are administered on a cycle of less than about 3 weeks, about once or twice every two weeks, about once every 10 days, or about once every week. A cycle may comprise the administration of a therapeutic or prophylactic agent via infusion for approximately 90 minutes each cycle, approximately 1 hour each cycle, approximately 45 minutes each cycle. Each cycle can comprise at least 1 week off, at least 2 weeks off, at least 3 weeks off. The number of cycles administered is 1 to approximately 12 cycles, plus

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typically from about 2 to aprnxi madgpnA-nt-g io ninlna, and more typically from about 2 to about 8 cycles.

In still other embodiments, the therapeutic and prophylactic agents of the invention are administered in metronomic dosage regimens, either through continuous infusion or frequent administration without extended rest periods. Such metronomic administrations may involve dosing at constant intervals without rest periods. Typically therapeutic agents, in particular cytotoxic agents, are used at lower doses. Typically therapeutic agents, in particular cytotoxic agents, are used at lower doses. Such dosage regimens encompass the chronic viable administration of relatively low doses for extended periods of time. In preferred embodiments, the use of lower doses can minimize toxic side effects and eliminate rest periods. In certain embodiments, the therapeutic and prophylactic agents are delivered via chronic or continuous low-dose infusion in the range of from about 24 hours to about 2 days, from about 1 week, from about 2 weeks, from about 3 weeks to about 1 month to about 2 months, from about 3 months, from about 4 months, from

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KTTIWO MKlCANL OF INDUSTRIAL HtShRDAD

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about 5 months, about 6 months. Scheduling of dosage regimens can be optimized by the skilled oncologist.

In other embodiments, courses of treatment are administered concurrently to a mammal, that is, individual doses of the therapeutics that are administered separately yet within a time interval such that the molecules of the invention can work together with the another agent or agents. For example, a component can be administered once a week in combination with other components that can be administered once every two weeks or once every 3 weeks. In other words, the dosing regimens for the therapeutics are carried out concurrently even though the therapeutics are either not administered simultaneously or within the same patient visit.

When used in combination with other prophylactic and / or therapeutic agents, the molecules of the invention and the prophylactic and / or therapeutic agent can act additively or, more preferably, synergistically. In one embodiment, a molecule of the invention is administered concurrently with one or more other therapeutic agents in the same pharmaceutical composition. In another embodiment, a molecule of the invention is administered concurrently with one or more therapeutic agents in pharmaceutical compositions.

293

MCaCANO INSTITUTE

DE ΙΑ ΡΛΟΡΙΕΟΑΠ &lt;^ - 2S5fJ * U IND '' STWIAI.

separated. In yet another embodiment, the mnl ^ nnla Ha of the invention is administered prior to or subsequent to the administration of another prophylactic agent or therapeutic agent. The invention contemplates the administration of a molecule of the invention in combination with other prophylactic or therapeutic agents via the same or different routes of administration, for example oral and parenteral. In certain embodiments, when a molecule of the invention is administered concurrently with another prophylactic or therapeutic agent that potentially produces adverse side effects including, but not limited to, toxicity, the prophylactic or therapeutic agent can advantageously be administered at a dose that falls by below the threshold at which the adverse side effect occurs.

Dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dose and frequency will also typically vary according to factors specific to each patient depending on the specific therapeutic or prophylactic agents administered, the severity and type of cancer, the route of administration as well as age, body weight, response and past medical history. of the patient's past. Suitable regimens can be selected by one of ordinary skill in the art considering such

INSTITUTO MEXICANO DE LA FROPIEDAL · factors and through the following, for example, the doses reported in the literature and recommended by the Pliysician's Desk Reference (56^to ed., 2002).

ANTI-CANCER AGENTS

In a specific embodiment, the methods of the invention encompass the administration of one or more molecules of the invention with one or more therapeutic agents used for the treatment and / or prevention of cancer. In one embodiment, the angiogenesis inhibitors can be administered in combination with the molecules of the invention. Angiogenesis inhibitors that can be used in the methods and compositions of the invention include but are not limited to: Angiostatin (plasminogen fragment); antiangiogenic antithrombin III; Angioenzyme; ABT-627; Bay 12-9566; Benefina; Bevacizumab; BMS-275291; cartilage derived inhibitor (CDI); CAI; complement fragment CD59; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagen fragment XVIII); Fibronectin fragment; Gro-beta; Halofuginone; Heparinases; Heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); IM-862; Alpha / beta / gamma interferon; Interferon-inducible protein (IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat; metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS

27023 TO); MoAb IMC-IC11; Neovastat; NM-3; Panzem; PI-88;

295

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MIÜCANO INSTHVTO

OF THE MOHEDAL »INDUSTRIAL

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Placenta ribonuclease inhibitor; Plasminogen activator inhibitor; platelet factor-4 (PF4); Prinomastat;

16 kDa fragment of prolactin; proliferin-related protein (PRP); PTK 787 / ZK 222594; Retinoids; Solimastat; squalamine; SS 3304; SU5416; SU6668; SU11248; Tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; Thrombospondin-1 (TSP-1); TNP-470; transforming growth factor-beta (TGF-b); Vasculostatin; Vasostatin (Calreticulin fragment); ZD6126; ZD 6474; farnesyl transfersa inhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with the molecules of the invention and the various embodiments of the invention include pharmaceutical compositions and dosage forms and kits of the invention, including, but not limited to: acivicin; aclarubicin; Acodazole Hydrochloride; acronin; adozelesin; aldesleucine; altretamine; ambomycin; methantrone acetate; aminoglutethimide; amsacrine; anastrozole; Anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; Bisanthrene Hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropyrimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; Carubicin Hydrochloride;

carzelesin;

cedefingol;

chlorambucil;

Cyrolemycin;

296

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INSTITUTO MLJUCANO

Pt LA MOHEDAL INDUSTRIAL

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cisplatin; cladribine; chrysnatol mesylate; cyclophosphamide;

cytarabine; dacarbazine; dactinomycin; Daunorubicin Hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; Doxorubicin Hydrochloride; droloxifene; Droloxifene Citrate; Dromostanolone Propionate; duazomycin; edatrexate; Eflornithine Hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; Epirubicin Hydrochloride; erbulozole; Esorubicin Hydrochloride; estramustine; estramustine sodium phosphate; ethanidazole; etoposide; Etoposide Phosphate; ethoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; Fludarabine Phosphate; fluorouracil; flurocitabine; phoschidone; sodium phostriecin; gemcitabine; Gemcitabine Hydrochloride; hydroxyurea; Idarubicin Hydrochloride; ifosfamide; ilmophosine; interleukin II (including interleukin II, or recombinant rIL2), interferon alpha-2a; interferon alfa-2b; interferon alpha-nl; interferon alpha-n3; interferon beta-la; interferon gamma-Ib; iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; lomustine; Losoxantrone Hydrochloride; masoprocol; maytansine; Mechlorethamine Hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; sodium methotrexate; metoprine; meturedepa; mitindomide;

- IMPI fwrrrrvro Mexican DEMKOKEDAO INDfSTRIAI mitocarcina; mitochromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; Mitoxantrone Hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxysuran; paclitaxel; pegaspargasa; peliomycin; pentamustine; peplomycin sulfate; perfosphamide; pipobroman; piposulfan; Pyroxantrone Hydrochloride; plicamycin; plomestane; porfimer sodium; porphyromycin; prednimustine; Procarbazine Hydrochloride; puromycin; Puromycin Hydrochloride; pyrazofurin; riboprine; rogletimide; safmgol; Safmgol Hydrochloride; semustine; sytrazene; sodium sparphosphate; sparsomycin; Spirogermanium Hydrochloride; spiromustine; spiroplatin; streptonigrin;

streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; Teloxantrone Hydrochloride; temoporphan; teniposide; teroxyrone; testolactone; thiamiprine; thioguanine; thiotepa; thiazofurine; tirapazamine; toremifen citrate; trestolon acetate; triciribine phosphate; trimetrexate; glucuronate trimetrexate; triptorelin; Tubulozole Hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatine; zinostatin; zorubicin hydrochloride. Other anticancer drugs include, but are not limited to: 20-

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IMPI ^ ΐΗππυτυ Mexican

DE LA KOm »At&gt; INDUSTRIAL epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abirateroña; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleucine; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;

angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azathyrosine; Baccatin III derivatives; balanol; batimastat; BCR / ABL antagonists; benzochlorines; benzoylstaurosporine; derivatives of beta lactam; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantreno; bisaziridinylpermine; bisnafide; bistratene A; bizelesin; breflate; bropyrimine; budotitan; butionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; Capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorines;

chloroquinoxaline sulfonamide; cicaprost; cis-porfriña;

IMPI

INSTITUTO MÍX1CAW DE LAttOREDAD INDUSTRIAL cladribine; Clomiphene Analogs; clotrimazole; colismycin A; colismycin B; combretastatin A4; combretastatin analog; conagenina; crambescidin 816; crisnatol; cryptophycin 8; Cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocphosphate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexyphosphamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnospermine; dihydro-5azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxyfluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelphosine; edrecolomab; eflornithine; elemene; emitter; epirubicin; epriesteride; estramustine analog; estrogen agonists; estrogen antagonists; ethanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastima; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; Fluorodaunorunicin Hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocytabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; Hexamethylene Bisacetamide; hypericin; Ibandronic acid; idarubicin; idoxifen; idramantone; ilmophosine; ilomastat; imidazoacridones; imiquimod; peptides

3οο ΙΜΡΙ ^ 5 | Mexican institute of rsom »A d C'enrÑF-W? / INDUSTRIAL immunostimulants; insulin-like growth factor 1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguan; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazol;

isohomohalicondrin B; itasetron; jasplaquinolide; cahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstima; lentinan sulfate; leptolestatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen-progesterone; leuprorelin; levamisole; liarozole; linear polyamine analog; lipophilic disaccharide peptide; lipophilic platinum compounds; lysoclinamide 7; lobaplatina; earthworm; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetio texaphyrin; lysophylline; lytic peptides; maytansine; mannostatin A; marimastat; masoprocol; maspina; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarona; metherelin; methioninase;

metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; Mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogs; mitonafida; mitotoxin-saporin fibroblast growth factor; mitoxantrone; Mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotropin; monophosphoryl-bacterium lipid A cell wall sk; mopidamole; multiple drug resistant gene inhibitor; therapy to³⁰¹ IMPI ^

ΙΝΓΓΠνΤΌ MEXICAN ΟΙ Μ ttOfllDAD ί ^ - ™, industrial * * Multiple tumor suppressor 1 base; mustard anticancer agent; mycaperoxide B; Mycobacterian cell wall extract; myriaporone; N-acetyldinaline;

N-substituted benzamides; nafarelin; nagrestip; naloxone + pentazocine; napavine; nafterpine; nartograstima; nedaplatin; nemorubicin; Neridronic Acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrulline; 06benzylguanine; octreotide; oquicenone; oligonucleotides;

onapristone; ondansetron; ondansetron; orazine; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogs; aclitaxel derivatives; palauamina; palmitoylrhizoxin; Pamidronic acid; panaxytriol; panomiphene; parabactin; pazeliptin; pegaspargasa; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosphamide; perilic acid; phenazinomycin; Phenylacetate; phosphatase inhibitors; picibanil; Pilocarpine Hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; plathion complex, platinum-triamine complex, porfimer sodium; porphyromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; Protein A-based immune modulator; protein kinase inhibitor C; protein kinase inhibitors C, microalgal; protein tyrosine inhibitors

302

IMPI

IWHTUTO LUOUCAHC OF WtlSTRIAL rtOPIETY phosphatase; purine nucleoside phosphorylase inhibitors; glitter; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; farnseyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; demethylated reteliptin; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitucin; romurtida; roquinimex; rubiginone Bl; ruboxyl; safmgol; saintopine; SarCNU; sarcophytol A; sargramostima; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; individual chain antigen-binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermina; sparphosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitors; stipiamide; stromelysin inhibitors; sulfmosine; superactive vasoactive intestinal peptide antagonist; suradista; suramina; swainsonin; synthetic glycosaminoglycans; talimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrillium; telomerase inhibitors; temoporfm; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; taliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;

303

IMPI ^ iwrrruTOMDucANo

Of LA MtOfVDAD thymalfasin; 'T'fffltt ^ oySfrnTa receptor agonist; thymothrinan; thyroid stimulating hormone, tin — etM * etiopurpurine; tirapazamine; titanocene bichloro; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; thyphostines; UBC inhibitors; ubenimex; growth inhibitory factor derived from the urogenital sinus; urokinase receptor antagonists; vapreotide; variolin B; erythrocyte gene therapy, vector system; velaresol; veramin; verdinas; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatine; zilascorb; and zinostatin stimalamer. Preferred additional anti-cancer drugs are 5-fluorouracil and leucovorin. Additional preferred anticancer drugs are 5-fluorouracil and leucovorin.

Examples of therapeutic antibodies that can be used in the methods of the invention include but are not limited to ZENAP AX® (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressant, humanized anti-CD25 monoclonal antibody for the prevention of rejection of the acute renal allograft; PANOREX ™ which is a murine anti-17-ΙΑ cell surface antigen IgG2a antibody (Glaxo Wellcome / Centocor); BEC2 which is an anti-idiotype IgG antibody (GD3 epitope) (ImClone System); What is IMC-C225

304 Ι_ΜΡΙ

IWHTVTO MEXICANO DE LA HWlUMr INDUSTRIAL a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN ™ which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution / Medlmmune); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab / Kanebo); LYMPHOCIDE ™ which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDEC Pharm / Mitsubishi); IDEC-131 is a humanized anti-CD40L antibody (IDEC / Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC / Seikagaku); SMART is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 antibody (C5) (Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT / BASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC151 is a primatized anti-CD4 IgGl antibody (IDEC Pharm / SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex / Eisai / Genmab); CDP571 is a humanized anti-TNF-a IgG4 antibody (Celltech); LDP-02 is a humanized anti-o¡437 antibody (LeukoSite / Genentech); OrthoClone 0KT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA ™ is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN ™ is an anti-VLA-4 IgG antibody (Elan); and CAT-152 is an anti-TGF-3 antibody₂ human (Cambridge Ab Tech). Other examples of therapeutic antibodies that can be used in accordance with the invention are presented in Table 8.

<img file="MX348166B_D0179.tif" />

305

<img file="MX348166B_D0180.tif" />

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Table 8: Therapeutic antibodies AnHnan&gt; '»rigid» nng.

<td></td><td>Company</td><td>Product</td><td>Disease</td><td>objective</td>

<td></td><td>Abgenix</td><td>ABX-EGF</td><td>Cancer</td><td>EGF receptor</td>

<td> 5</td><td>AltaRex</td><td>OvaRex</td><td>Ovarian cancer</td><td>antigen of</td>

<td></td><td></td><td></td><td></td><td>CA125 tumor</td>

<td></td><td></td><td>BravaRex</td><td>Metastatic cancers</td><td>antigen of</td>

<td></td><td></td><td></td><td></td><td>tumor</td>

<td></td><td></td><td></td><td></td><td>MUC1</td>

<td> 10</td><td></td><td></td><td></td><td></td>

<td></td><td>Antisorna</td><td>Teragin</td><td>Ovarian cancer</td><td>PEM antigen</td>

<td></td><td></td><td>(pemtumomabitrio-</td><td></td><td></td>

<td></td><td></td><td> 90)</td><td></td><td></td>

<td></td><td></td><td>Therex</td><td>Breast cancer</td><td>PEM antigen</td>

<td> 15</td><td>Boehringer</td><td>Blvatuzumab</td><td>Head cancer and</td><td>CD44</td>

<td></td><td>Ingelheim</td><td></td><td>neck</td><td></td>

<td></td><td>Centocor / J &amp; J</td><td>Panorex</td><td>Colorectal cancer</td><td>17-1A</td>

<td></td><td></td><td>ReoPro</td><td>PTCA</td><td>gp Illb / IIIa</td>

<td></td><td></td><td>ReoPro</td><td>MI sharp</td><td>gp Illb / IIIa</td>

<td rowspan="2"> 20</td><td></td><td>ReoPro</td><td>Ischemic shock</td><td>gp Illb / IIIa</td>

<td>Corixa</td><td>Bexocar</td><td>NHL</td><td>CD20</td>

<td></td><td>CRC</td><td>MAb, idiotypic</td><td>Cancer vaccine</td><td>gp72</td>

<td></td><td>Technology</td><td>105AD7</td><td>colorectal</td><td></td>

<td></td><td>Crucell</td><td>Anti-EpCAM</td><td>Cancer</td><td>Ep-CAM</td>

<td> 25</td><td>Cytoclonal</td><td>Lung cancer</td><td>Lung cancer</td><td>NA</td>

306

<td colspan="4">_______ IMPIf ^</td>

<td></td><td></td><td colspan="2">INSTmn®MSXICANO</td>

<td></td><td></td><td colspan="2">BE LA ΡΒΟΠΕΒΑΡ</td>

<td>Company</td><td>Product</td><td>Disease</td><td>^INr06Wivo^Sa-^--'</td>

<td></td><td>MAb</td><td></td><td></td>

<td>Genentech</td><td>Herceptin</td><td>Breast cancer</td><td>HER-2</td>

<td></td><td></td><td>metastatic</td><td></td>

<td></td><td>Herceptin</td><td>Breast cancer</td><td>HER-2</td>

<td></td><td></td><td>early stage</td><td></td>

<td></td><td>Rituxan</td><td>NHL's</td><td>CD20</td>

<td></td><td></td><td>recidivism / refractory</td><td></td>

<td></td><td></td><td>or low grade or</td><td></td>

<td></td><td></td><td>follicular</td><td></td>

<td></td><td>Rituxan</td><td>NHL intermediate and</td><td>CD20</td>

<td></td><td></td><td>high grade</td><td></td>

<td></td><td>MAb-VEGF</td><td>NSCLC, metastatic</td><td>VEGF</td>

<td></td><td>MAb-VEGF</td><td>Colorectal cancer,</td><td>VEGF</td>

<td></td><td></td><td>metastatic</td><td></td>

<td></td><td>AMD Fab</td><td>Macular degeneration</td><td>CD18</td>

<td></td><td></td><td>age related</td><td></td>

<td></td><td>E-26 (2^to gen. IgE)</td><td>Allergic asthma and rhinitis</td><td>IgE</td>

IDEC

Zevalin (Rituxan + low CD20 ytio-90 B-cell NHL) follicular grade, relapse or refractory CD20-positive, and

Ri tuximab-NHL

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KU nOHIDAP ------- INBUmiAl

<td>Company</td><td>Product</td><td>Disease</td><td>objective</td>

<td></td><td></td><td>refractory</td><td></td>

<td>ImClone</td><td>Cetuximab +</td><td>Colorectal carcinoma</td><td>EGF receptor</td>

<td></td><td>inotecan</td><td>refractory</td><td></td>

<td></td><td>Cetuximab +</td><td>Head cancer and</td><td></td>

<td></td><td>cisplatin and</td><td>neck recently</td><td>EGF receptor</td>

<td></td><td>radiation</td><td>diagnosed or</td><td></td>

<td></td><td></td><td>recurrent</td><td></td>

<td></td><td>Cetuximab +</td><td>Pancreatic carcinoma</td><td>EGF receptor</td>

<td></td><td>Gemcitabine</td><td>metastatic</td><td></td>

<td></td><td></td><td>Recently</td><td></td>

<td></td><td></td><td>diagnosed</td><td></td>

<td></td><td>Cetuximab +</td><td>Head cancer and</td><td>EGF receptor</td>

<td></td><td>cisplatin +</td><td>recurrent neck or</td><td></td>

<td></td><td>5FU or Taxol</td><td>metastatic</td><td></td>

<td></td><td>Cetuximab +</td><td>Lung carcinoma</td><td>EGF receptor</td>

<td></td><td>carboplatin +</td><td>not small cell</td><td></td>

<td></td><td>paclitaxel</td><td>newly diagnosed</td><td></td>

<td></td><td>Cetuximab +</td><td>Head cancer and</td><td>EGF receptor</td>

<td></td><td>Cisplatin</td><td>neck (disease</td><td></td>

<td></td><td></td><td>local / regional</td><td></td>

<td></td><td></td><td>incurable extensive and</td><td></td>

<td></td><td></td><td>distant metastasis)</td><td></td>

<td></td><td>Cetuximab +</td><td>Head carcinoma and</td><td>EGF receptor</td>

<td></td><td>radiation</td><td>neck locally</td><td></td>

ΙΜΡΙ ^^ mxxjcano institute

FROM ΙΑ ΕΕΟΤΙΕΟΛΙ - ^ ÍJWa

<td>Company</td><td>Product</td><td>Disease _____</td><td>objective</td>

<td></td><td></td><td>advanced</td><td></td>

<td></td><td>BEC2 + Bacillus</td><td>Lung carcinoma</td><td>Mimics CD3's</td>

<td></td><td>Calmette guerin</td><td>small cell</td><td>ganglioside</td>

<td></td><td>BEC2 + Bacillus</td><td>Melanoma</td><td>Mita GD3 of ganglioside</td>

<td></td><td>Calmette guerin IMC-1C11</td><td>Colorectal cancer with liver metastasis</td><td>VEGF receptor</td>

<td>ImmunoGen</td><td>nuC242-DMl</td><td>Colorectal cancer gastric and pancreatic</td><td>nuC242</td>

<td>ImmunoMedics</td><td>LymphoCide</td><td>Non-Hodgkins lymphoma</td><td>CD22</td>

<td></td><td>LymphoCide Y-90</td><td>Non-Hodgkins lymphoma</td><td>CD22</td>

<td></td><td>CEA-Cide</td><td>Solid tumors metastatic</td><td>CEA</td>

<td></td><td>Cide Y-90</td><td>Solid tumors</td><td>CEA</td>

metastatic

<td>CEA-Scan (Tc-99m</td><td>Colorectal cancer</td><td>CEA</td>

<td>Marked as</td><td>(radio image)</td><td></td>

<td>arcitumomab)</td><td></td><td></td>

<td>CEA-Scan (Tc-99m</td><td>Breast cancer</td><td>CEA</td>

<td>Marked as</td><td>(radio image)</td><td></td>

<td>arcitumomab)</td><td></td><td></td>

<td>CEA-Scan (Tc-99m</td><td>Lung cancer</td><td>CEA</td>

<td>Marked as</td><td>(radio image)</td><td></td>

arcitumomab)

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'.ΝΤΤΓΤυΤΟ MSX1CANC. 1 ^ * 63 ^

OS LA ΡβΟΗΕΟΑΙ) industrial -

<td>Company</td><td>Product</td><td>Disease</td><td></td>

<td></td><td>CEA-Sean (Tc-99m Marked as arcitumomab)</td><td>Intraoperative tumors (radio image)</td><td>CEA</td>

<td></td><td>LeukoScan (Tc-99m Marked as sulesomab)</td><td>Mild tissue infection (radio image)</td><td>CEA</td>

<td></td><td>LymphoScan (marked Tc-99m)</td><td>Lymphomas (radioimaging)</td><td>CD22</td>

<td></td><td>AFP-Scan (marked Tc-99m</td><td>7 cell cancers liver gene</td><td>AFP</td>

<td>Intracel</td><td>HumaRAD-HN (+ itio-90)</td><td>Head and neck cancer</td><td>NA</td>

<td></td><td>HumaSPECT</td><td>Imaging</td><td>NA</td>

colorectals

<td>Medarex</td><td>MDX-101 (CTLA-4)</td><td>Prostate cancer and</td><td>CTLA-4</td>

<td></td><td>MDX-210</td><td>others</td><td></td>

<td></td><td>(overexpression</td><td>Prostate cancer</td><td>HER-2</td>

<td></td><td>her-2)</td><td></td><td></td>

<td>Medlmmune</td><td>MDX-210 / MAK</td><td>Cancer</td><td>HER-2</td>

<td>Merck KGaA</td><td>Vitaxin</td><td>Cancer</td><td>Ανβ</td>

<td></td><td>MAb 425</td><td>Various cancers</td><td>EGF receptor</td>

<td></td><td>IS-IL-2</td><td>Various cancers</td><td>Ep-CAM</td>

<td>Millennium</td><td>Campath</td><td>Lymphocytic leukemia</td><td>CD52</td>

<td></td><td>(alemtuzumab)</td><td>chronicle</td><td></td>

310

IMPI

<td></td><td>Imil TUTO MEX1CAN * DE U PROREDAI</td>

<td>Company Product</td><td>Disease ^IHb¿ ^ É¿Ívo</td>

<td>NeoRx CD20-streptavidin</td><td>Non-HodLfltlliy LU2U Lymphoma '' </td>

(+ biotin-yttrium

90)

<td>Avidicin</td><td>Metastatic cancer NA</td>

(albumin +

NANRLU13)

<td>Peregrine</td><td>Oncolima (+ iodine- 131) Cotara (+ iodine- 131)</td><td>Non-Hodgkins lymphoma Malignant glioma no amputable</td><td>HLA-DR 10 beta Proteins associated with DNA</td>

<td>Pharmacia</td><td>C215 (+</td><td>Pancreatic cancer</td><td>NA</td>

<td></td><td>enterotoxin</td><td></td><td></td>

<td></td><td>staphylococcus)</td><td></td><td></td>

<td>Corporaton</td><td>MAb, cancer of</td><td></td><td></td>

<td></td><td>lung / kidney</td><td>Lung cancer and</td><td>NA</td>

<td></td><td>nacolomab</td><td>kidney</td><td></td>

<td></td><td>tafenatox</td><td></td><td>NA</td>

<td></td><td>(C242 +</td><td>Cancer and pancreatic</td><td></td>

<td></td><td>enterotoxin</td><td></td><td></td>

<td></td><td>staphylococcus)</td><td></td><td></td>

<td>Protein Design</td><td>Nuvion</td><td>Malignancies of the</td><td>CD3</td>

<td>Labs</td><td></td><td>T cell</td><td></td>

<td></td><td>SMART MI95</td><td>AML</td><td>CD33</td>

311

<img file="MX348166B_D0181.tif" />

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<td>Company</td><td>Product</td><td>Disease _____</td><td>objective</td>

<td>Titan</td><td>SMART ID10</td><td>NHL</td><td>HLA-DR antigen</td>

<td></td><td>CEA Vac</td><td>Colorectal cancer,</td><td>CEA</td>

<td></td><td></td><td>advanced</td><td></td>

<td></td><td>TriGem</td><td>Metastatic melanoma and</td><td>GD2-ganglioside</td>

<td></td><td></td><td>lung cancer</td><td></td>

<td></td><td></td><td>small cell</td><td></td>

<td></td><td>TriAb</td><td>Breast cancer</td><td>MUC-1</td>

metastatic

<td>Trilex</td><td>CEAVac</td><td colspan="2">Colorectal cancer,</td><td>CEA</td>

<td></td><td></td><td>advanced</td><td></td><td></td>

<td></td><td>TriGem TriAb</td><td>Metastatic melanoma Small cell lung cancer Metastatic breast cancer</td><td>Y</td><td>GD2-ganglioside MUC-1</td>

<td>Viventia Biotech</td><td>NovoMAbradimarked G2</td><td colspan="2">Non-Hodgkins lymphoma</td><td>NA</td>

<td></td><td>Monopharm C GlioMab-H (+ gelonin toxin)</td><td>Colorectal carcinoma pancreatic Glioma, melanoma neuroblastoma</td><td>Y</td><td>SK-1 antigen NA</td>

<td>Xoma</td><td>Rituxan</td><td>NHL repeat offender</td><td></td><td>CD2 0</td>

low-grade refractory or follicular

<img file="MX348166B_D0182.tif" />

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CtUnOPIEDAt: wtxirntiAi

<td>Company Product Disease</td><td>,, nhjnt., .. », --------</td>

<td>Rituxan NHL intermediate</td><td>and CD20</td>

high grade

<td>ING-1 Adenocarcinoma</td><td>Ep-CAM</td>

IMMUNOMODULATING AGENTS AND ANTI-INFLAMMATORY AGENTS

The present invention provides methods of treatment for autoimmune diseases and inflammatory diseases that comprise the administration of the molecules of the invention in conjunction with other treatment agents. Examples of immunomodulatory agents include, but are not limited to, methotrexate, ENBREL, REMICADE ™, leflunomide, cyclophosphamide, cyclosporine A, and macrolide antibiotics (eg, FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, spheroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindas (eg, leflunamide), T cell receptor modulators, and cytokine receptor modulators.

Anti-inflammatory agents have shown success in treating inflammatory and autoimmune disorders and are now common and standard treatment for such disorders. Any anti-inflammatory agent well known to one of ordinary skill in the art can

<img file="MX348166B_D0183.tif" />

³¹³ IMPI

INSTmlTO ΜΒΛΛΛ1 * 7 Ρϊ tA INDUSTRIAL PROPERTY used in the methods of the invention. Non-limiting examples and anti-inflammatory agents include non-spheroid anti-inflammatory drugs (NSAIDs), spheroidal anti-inflammatory drugs, beta-agonists, anticholinergic agents, and methyl xanthine. Examples of NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREX ™), diclofenac (VOLTAREN ™), etodolac (LODINE ™), fenoprofen (NALFON ™), indomethacin (INDOCIN ™), ketoralac (TORADOL ™ ), oxaprozin (DAYPRO ™), nabumentone (RELAFEN ™), sulindac (CLINORIL ™), tolmentine (TOLECTIN ™), rofecoxib (VIOXX ™), naproxen (ALEVE ™, NAPROSYN ™), ketoprofen (ACTRON ™) and nabumetone (RELAFEN ™) ™). Such NSAIDs work through the inhibition of an enzyme cyclooxygenase (eg, COX-1 and / or COX-2). Examples of spheroid anti-inflammatory drugs include, but are not limited to, glucocorticosteroids, dexamethasone (DECADRON ™), cortisone, hydrocortisone, prednisone (DELTASONE ™), prednisolone, triamcinolone, azulfidine, and eicosanoids such as prostaglandins, thromboxanes leukotrienes.

A non-limiting example of antibodies that can be used for the treatment or prevention of inflammatory disorders in conjunction with the molecules of the invention are presented in Table 9, and a non-limiting example of antibodies that can be used for the treatment or prevention of the immune disorder are presented in the Table.

314

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<img file="MX348166B_D0184.tif" />

Table 9: Therapeutic antibodies for the treatment of inflammatory diseases

<td>Name of Antibody</td><td>Target Antigen</td><td>Kind of product</td><td>Isotype</td><td>Sponsors</td><td>Indication</td>

<td>5G1.1</td><td>Complement (C5)</td><td>Humanized</td><td>IgG</td><td>Alexion pharm Inc.</td><td>Arthritis Rheumatoid</td>

<td>5G1.1</td><td>Complement (C5)</td><td>Humanized</td><td>IgG</td><td>Alexion pharm Inc.</td><td>SLE</td>

<td>5G1.1</td><td>Complement (C5)</td><td>Humanized</td><td>IgG</td><td>Alexion pharm Inc.</td><td>Nephritis</td>

<td>5GL1-SC</td><td>Complement (C5)</td><td>Humanized</td><td>ScFc</td><td>Alexion pharm Inc.</td><td>Bypass Cardiopulmonary</td>

<td>5GL1-SC</td><td>Complement (C5)</td><td>Humanized</td><td>ScFc</td><td>Alexion Pharm inc.</td><td>Heart attack Myocardium</td>

<td>5GL1-SC</td><td>Complement (C5)</td><td>Humanized</td><td>ScFc</td><td>Alexion pharm Inc.</td><td>Angioplasty</td>

<td>ABX-CBL</td><td>CBL</td><td>Human</td><td></td><td>Abgenix Inc</td><td>GvHD</td>

<td>ABX-CBL</td><td>CD147</td><td>Murine</td><td>IgG</td><td>Abgenix Inc</td><td>Rejection of Allograft</td>

<td>ABX-IL8</td><td>IL-8</td><td>Human</td><td>IgG2</td><td>Abgenix Inc</td><td>Psoriasis</td>

<td>Antegren</td><td>VLA-4</td><td>Humanized</td><td>IgG</td><td>Athena / Elan</td><td>Multiple sclerosis</td>

<td>Anti-CDlla</td><td>CDlla</td><td>Humanized</td><td>IgGl</td><td>Genentech Inc / Xoma</td><td>Psoriasis</td>

<td>Anti-CD18</td><td>CD18</td><td>Humanized</td><td>Fac '2</td><td>Genentech Inc</td><td>Heart attack Myocardium</td>

<td>Anti-LFAl</td><td>CD18</td><td>Murine</td><td>Fab'2</td><td>Pasteur Merieux / Immunotech</td><td>Rejection of Aloinj erto</td>

<td>Antova</td><td>CD40L</td><td>Humanized</td><td>IgG</td><td>Biogen</td><td>Allograft rejection</td>

<td>Antova</td><td>CD4 0L</td><td>Humanized</td><td>igG</td><td>Biogen</td><td>SLE</td>

<td>BTI-322</td><td>CD2</td><td>Rat</td><td>IgG</td><td>Medimmune Inc</td><td>GvHD, Psoriasis</td>

<td>CDP571</td><td>TNF-Alpha</td><td>Humanized</td><td>IgG4</td><td>Celltech</td><td>Crohn</td>

<td>CDP571</td><td>TNF-Alpha</td><td>Humanized</td><td>IgG4</td><td>Celltech</td><td>Arthritis</td>

315 IMPI ΐκπτππ · MEXICAN Ct LA HK) F! »AD

<td>Antibody Name</td><td>Antigen objective</td><td>Kind of product</td><td>Isotype</td><td>Sponsors</td><td>Indication</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>Rheumatoid</td><td rowspan="21"></td>

<td>CDP850</td><td>Selectin E</td><td>Humanized</td><td></td><td>Celltech</td><td>Psoriasis</td>

<td>Corsevin M</td><td>Fact VII</td><td>Chimerical</td><td></td><td>Centocor</td><td>Anticoagulant</td>

<td>D2E7</td><td>TNF-Alpha</td><td>Human</td><td></td><td>CAT / BASF</td><td>Arthritis Rheumatoid</td>

<td>Hu23F2G</td><td>CD11 / 18</td><td>Humanized</td><td></td><td>ICOS Pharm Inc</td><td>Multiple sclerosis</td>

<td>Hu23F2G</td><td>CD11 / 18</td><td>Humanized</td><td>IgG</td><td>ICOS Pharm Inc</td><td>Heart attack</td>

<td>IC14</td><td>CD14</td><td></td><td></td><td>ICOS Pharm Inc</td><td>Toxic shock</td>

<td>ICM3</td><td>ICAM-3</td><td>Humanized</td><td></td><td>ICOS Pharm Inc</td><td>Psoriasis</td>

<td>IDEC-114</td><td>CD80</td><td>Primatized</td><td></td><td>IDEC Pharm / Mitsubishi</td><td>Psoriasis</td>

<td>IDEC-131</td><td>CD40L</td><td>Humanized</td><td></td><td>IDEC Pharm / eisai</td><td>SLE</td>

<td>IDEC-131</td><td>CD40L</td><td>Humanized</td><td></td><td>IDEC Pharm / eisai</td><td>Multiple sclerosis</td>

<td>IDEC-151</td><td>CD4</td><td>Primatized</td><td>IgGl</td><td>IDEC Pharm / Glaxo SmithKline</td><td>Arthritis Rheumatoid</td>

<td>IDEC-152</td><td>CD23</td><td>Primatized</td><td></td><td>IDEC Pharm</td><td>Asthma / Allergy</td>

<td>Infliximab</td><td>TNF-Alga</td><td>Chimerical</td><td>IgGl</td><td>Centocor</td><td>Arthritis Rheumatoid</td>

<td>Infliximab</td><td>TNF-Alga</td><td>Chimerical</td><td>IgGl</td><td>Centocor</td><td>Crohn</td>

<td>LDP-01</td><td>Beta 2integrin</td><td>humanized</td><td>IgG</td><td>Millennium Inc (LeukoSite Inc.)</td><td>Heart attack</td>

<td>LDP-01</td><td>Beta 2integrin</td><td>Humanized</td><td>IgG</td><td>Millennium Inc (LeukoSite Inc.)</td><td>Rejection of Allograft</td>

<td>LDP-02</td><td>Alpha4beta7</td><td>Humanized</td><td></td><td>Millennium Inc (LeukoSite Inc.)</td><td>Colitis Ulcerative</td>

<td>MAK-195F</td><td>TNF-Alpha</td><td>Murine</td><td>Fab '2</td><td>Knoll Pharm, BASF</td><td>Toxic Shock</td>

<td>MDX-33</td><td>CD64 (FcR)</td><td>Human</td><td></td><td>Medarex / Hundred</td><td>Hematologic Autoimmune Disorders</td>

<td>MDX-CD4</td><td>CD4</td><td>Human</td><td>IgG</td><td>Medarex / Eisai /</td><td>Arthritis</td>

<img file="MX348166B_D0185.tif" />

IMPI

316 Dtsri ferro mhjcano of LA MOHEDAP INDUSTRIAL

<td>Name of Antibody</td><td>Antigen objective</td><td>Kind of product</td><td>Isotype</td><td>Sponsors</td><td>Indication</td>

<td></td><td></td><td></td><td></td><td>Genmab</td><td>Rheumatoid</td>

<td>MEDI-507</td><td>CD2</td><td>Humanized</td><td></td><td>Medimmune Inc</td><td>Psoriasis</td>

<td>MEDI-507</td><td>CD2</td><td>Humanized</td><td></td><td>Medimmune Inc</td><td>GvHD</td>

<td>OKT4A</td><td>CD4</td><td>Humanized</td><td>IgG</td><td>Ortho Biotech</td><td>Rejection of Allograft</td>

<td>Ortho Clone OKT4A</td><td>CD4</td><td>Humanized</td><td>IgG</td><td>Ortho Biotech</td><td>Autoimmune disease</td>

<td>Orthoclone / anti-CD3 OKT3</td><td>CD3</td><td>Murine</td><td>mIG2a</td><td>Ortho Biother</td><td>Rejection of Allograft</td>

<td>Repair/ Abciximab</td><td>gplibiia</td><td>Chimerical</td><td>Fab</td><td>Centocor / Lilly</td><td>Complications of coronary angioplasty</td>

<td>rhuMab-E25</td><td>igE</td><td>Humanized</td><td>IgGl</td><td>Genentech / Novart is / Tanox Biosystems</td><td>Asthma / Allergy</td>

<td>SB-240563</td><td>IL5</td><td>Humanized</td><td></td><td>GlaxoSmithKline</td><td>Asthma / Allergy</td>

<td>SB-240683</td><td>IL-4</td><td>Humanized</td><td></td><td>GlaxoSmithKline</td><td>Asthma / Allergy</td>

<td>SCH55700</td><td>IL-5</td><td>Humanized</td><td></td><td>Celltech / Schlering</td><td>Asthma / Allergy</td>

<td>Simulect</td><td>CD25</td><td>Chimerical</td><td>IgGl</td><td>Norvartis Pharm</td><td>Rejection of Allograft</td>

<td>SMART a- CD3</td><td>CD3</td><td>Humanized</td><td></td><td>Protein Design Lab</td><td>Autoimmune disease</td>

<td>SMART a- CD3</td><td>CD3</td><td>Humanized</td><td></td><td>Protein Design Lab</td><td>Allograft rejection</td>

<td>SMART a- CD3</td><td>CD3</td><td>Humanized</td><td>IgG</td><td>Protein Design Lab</td><td>Psoriasis</td>

<td>Zenapax</td><td>CD25</td><td>Humanized</td><td>IgGl</td><td>Protein Design Lab / Hoffman-La Roche</td><td>Rejection of Allograft</td>

Table 10:

317

Antibodies

IMPI

INSTITUTO MEXICANO ΙΛ LA PEOMEDAI INDUSTRIAL

<img file="MX348166B_D0186.tif" />

therapeutics for the treatment of autoimmune disorders

<td>Antibody</td><td>Indication</td><td>Target Antigen</td>

<td>ABX-RB2</td><td></td><td>Antibody to the CDL antigen on T cells, B cells and NK cells of the Xeno-Mouse fully human antibody</td>

<td>5c8 (an anti-CD-40 antigen antibody)</td><td>Phase II trial stopped in October 1999 that examined adverse events</td><td>CD-40</td>

<td>IDEC 131</td><td>Systemic lupus erythematosus (SLE)</td><td>Anti-CD40 humanized</td>

<td>IDEC 151</td><td>Rheumatoid arthritis</td><td>Privatized; anti-CD40</td>

<td>IDEC 152</td><td>Asthma</td><td>Privatized; anti-CD23</td>

<td>IDEC-507</td><td>Psoriasis</td><td>Privatized anti-CD80</td>

<td>MEDI-507</td><td>Rheumatoid arthritis, multiple sclerosis Crohn's disease Psoriasis</td><td>Anti-CD20</td>

<td>LDP-02 (anti-b7 znAb)</td><td>Inflammatory bowel disease Crohn's disease Ulcerative colitis</td><td>Interim a4b7 receptor on white blood cells (leukocytes)</td>

<td>Anti-Gamut Interferon SMART</td><td>Autoimmune disorders</td><td>Anti-Gamma Interferon</td>

<td>Verteportina</td><td>Rheumatoid arthritis</td><td></td>

<td>MDX-33</td><td>Blood disorders caused by autoimmune reactions Idiopathic thrombocytopenia purpura (ITP) Autoimmune hemolytic anemia</td><td>Monoclonal antibody against FcRI receptors</td>

<td>MDX-CD4</td><td>Treat rheumatoid arthritis and other autoimmunities</td><td>Monoclonal antibody against the CD4 receptor molecule</td>

IMPI ^ nwffwro mixicanc

<td>Antibody</td><td>Indication</td><td>___________ DtUFBQHiPAP— Obj etf! W&gt; S ™ Antigen^{AL ντχ}·—-—</td>

<td>VX-497</td><td>Autoimmune disorders Multiple Sclerosis Rheumatoid Arthritis Inflammatory bowel disease Lupus Psoriasis</td><td>Inosine monophosphate dehydrogenase inhibitor (enzyme necessary to make new RNA and DNA used in the production of the nucleotides necessary for the proliferation of lymphocytes</td>

<td>VX-740</td><td>Rheumatoid arthritis</td><td>ICE inhibitor Interleukin-1 beta (pathways that control enzyme conversion leading to an aggressive immune response)</td>

<td>VX-745</td><td>Specific for inflammation involved in chemical signaling of the initiation of the immune response and the progression of inflammation</td><td>Mitogen-activated protein kinase P38MAP kinase inhibitor</td>

<td>Enbrel (etanercept)</td><td></td><td>Target TNF (tumor necrosis factor)</td>

<td>IL-8</td><td></td><td>Fully human monoclonal antibody to IL-8 (interleukin 8)</td>

<td>Apogen MP4</td><td></td><td>Recombinant antigen that selectively kills C-cell-associated disease induces apoptosis of T-cells killed by programmed cell death that already attacks the body's own cells of target-specific T-cells of apogens</td>

AGENTS TO BE USED IN THE TREATMENT OF

INFECTIOUS DISEASE

In some embodiments, the molecules of the

3ΐ9 IMPIAS Mexican institute

Df LA VA0B2DAD VVwZML, ® INDUSTRIAL invention can be administered in combination with a therapeutically or prophylactically effective amount of one or more additional therapeutic agents known to those skilled in the art for the treatment and / or prevention of an infectious disease. The invention contemplates the use of the molecules of the invention in combination with antibiotics known to those skilled in the art for the treatment and / or prevention of infectious disease. Antibiotics that can be used in combination with the molecules of the invention include, but are not limited to, a macrolide (eg, tobramycin (Tobi®)), a cephalosporin (eg, cephalexin (Keflex®), cephradine (Velosef®) , cefuroxime (Ceftin®), cefprozil (Cefzil®), cefaclor (Ceclor®), cefixime (Suprax®), or cefadroxil (Duricef®)), a clarithromycin (for example, clarithromycin (Biaxin®)), an erythromycin (for example , erythromycin (EMycin®)), a penicillin (for example, penicillin V (V-Cillin K® or Pen Vee K®)) or a quinolone (for example, ofloxacin (Floxin®), ciprofloxacin (Cipro®), or norfloxacin (Noroxin®)), aminoglycoside antibiotics (for example, apramycin, arbecacin, Bambermycin, butyrosine, dibecacin, neomycin, neomycin, undecycinate, netilmicin, paromomycin, ribostamycin, sisomycin, and spectinomycin), amphenicol antibiotics (for example, azidanphenicol, chloramphenicol, floranphenicol, and ansanphenicol antibiotics),

320 IMPI ^

INSTITUTO MEXICANO DI LA MOMIDAt) IND / 'STUIAL (for example, rifamide and rifampicin), carbacephems (for example, loracarbef), carbapenems (for example, biapenem and imipenem), cephalosporins (for example, cefachlor, cefadroxil, cefamandol, cefamandol cefazedone, cefozopran, cefpimizole, cefpyramide, and cefpirome), cefamycins (for example, cefbuperazone, cefmetazole, and cefminox), monobactams (for example, aztreonam, carumonam, and tigemonam), oxyacephems (for example, flomoctam), and moxaphems (for example, flomoctam), and moxa penicillins (for example, amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, epicillin, phenbenicillin, floxacillin, penamcillin, penetamate iodidehydrate, penicillin o-Villin, penicillin 0-benzyl penicillin, penicillin, Vamin, penicillin, penicillin, Vamine, penicillin, Vamyl penicillin, penicillin hydride , penimepicycline and phencyhicillin potassium), lincosamides (for example, clindamycin and lincomycin), ampomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, tetracyclines (eg, apycycline, chlortetracycline, clomocycline, and demeclocycline), 2,4-diaminopyrimidines (eg, brodimoprim), nitrofurans (eg, furaltadone, and furazolium chloride), quinolones and their analogs (eg, cinoxacin, clinafloxacin , flumequine, and grepagloxacin), sulfonamides (for example, acetyl sulfamethoxypyrazine, benzylsulfamide, noprylsulfamide, phthalylsulfacetamide, sulfacrisoidine, and sulfacytin), sulfones

<img file="MX348166B_D0187.tif" />

321

IMPI iwhtuto Mexicano ML * HlOn * AD industry * (for example, diathimosulfone, glucosulfOlid budica and ...... solasulfone), cycloserine, mupirocin and tuberin.

In certain embodiments, the molecules of the invention can be administered in combination with a therapeutically or prophylactically effective amount of one or more antifungal agents. Antifungal agents can be used in combination with the molecules of the invention including but not limited to amphotericin B, itraconazole, ketoconazole, fluconazole, intrathecal, flucytosine, miconazole, butoconazole, clotrimazole, nystatin, terconazole, tioconazole, cyclopyrox, econazole, haloprogrin , naphthyphin, terbinafa, undecylenate, and griseofuldin.

In some embodiments, the molecules of the invention can be administered in combination with a therapeutically or prophylactically effective amount of one or more anti-viral agents. Useful anti-viral agents that can be used in combination with the molecules of the invention include, but are not limited to, protease inhibitors, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and nucleoside analogs. Examples of antiviral agents include, but are not limited to, zidovudine, acyclovir, gangciclovir, vidarabine, idoxuridine, trifluridine, and ribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir, the

322 IMPI

INjTfr &lt;WMW «* MO ÍÍIML · DÉ LA ΠΚΜίβΛΟ

IÑtXJStRtAl - alpha-interferons; adefovir, clevadine ,. __ _-gntecaviy ,, pleconaril.

VACCINE THERAPY

The invention further encompasses the use of compositions of the invention to induce an immune response against an antigenic or immunogenic agent, including but not limited to cancer antigens and infectious disease antigens (examples of which are described Infra). Vaccine compositions of the invention comprise one or more antigenic or immunogenic agents for which an immune response is desired, wherein the one or more antigenic or immunogenic agents is coated with variant antibody of the invention having improved affinity for FcyRIIIA. The vaccine compositions of the invention are particularly effective in eliciting immune responses, preferably a protective immune response against the antigenic or immunogenic agent.

In some embodiments, the antigenic or immunogenic agent in the vaccine compositions of the invention comprise a virus against which an immune response is desired. Viruses can be recombinant or chimeric, preferably they are attenuated. The production of attenuated, chimeric, recombinant virus can be carried out using standard methods known to those of skill in the art. The invention encompasses a recombinant viral vaccine

323

<img file="MX348166B_D0188.tif" />

live or an inactivated recombinant viral vaccine to be formulated according to the invention. A vaccine may be preferred because of the multiplication in the host that leads to prolonged challenge of a kind and magnitude similar to that which occurs in natural infections, and therefore confers substantial, long-term immunity. Production of live recombinant virus vaccine formulations can be accomplished using conventional methods involving coagulation of the virus in cell culture or in chicken embryo allantois followed by purification.

In a specific embodiment, the recombinant virus is non-pathogenic to the subject to which it is administered. In this regard, the use of genetically modified virus for vaccine purposes may require the presence of attenuation characteristics in this strain. The introduction of appropriate mutations (eg deletions) into the templates used for transfection can provide new viruses with attenuation characteristics. For example, specific nonsense mutations that are associated with temperature sensitivity or cold adaptation can be made in mutation clearance. These mutations should be more stable than the point mutations associated with cold or temperature sensitive mutants and the frequencies of inversion should be

324

IMPI

<img file="MX348166B_D0189.tif" />

extremely low.

Recombinant DNA technologies for the modification of recombinant viruses are known in the art and are encompassed by the invention. For example, techniques for modifying negative strand structure RNA viruses are known in the art, see, for example, US Patent No. 5,166,057, which is incorporated herein by reference in its entirety.

Alternatively, chimeric viruses with suicidal characteristics can be constructed for use in the intradermal vaccine formulations of the invention.

Such viruses will only pass through a few rounds of replication within the host. When used as a vaccine, the recombinant virus will go through a limited replication cycle (s) and induce a sufficient level of immune response but will not be able to go further in the human host and cause disease. Alternatively, the inactivated (killed) virus can be formulated in accordance with the invention. Inactivated vaccine formulations can be prepared using conventional techniques to kill the chimeric virus. Inactivated vaccines are dead in the sense that their ineffectiveness has been destroyed. Ideally, the ineffectiveness of the virus is destroyed without affecting its immunogenicity. In order to prepare inactivated vaccines, the chimeric virus can be cultured in cell culture or in allantois from

<img file="MX348166B_D0190.tif" />

chick embryos, purified by zone ultracentrifugation, inactivated with formaldehyde or β-propriolactone, and pooled.

In certain embodiments, completely foreign epitopes, including antigens derived from other viral or non-viral pathogens, can be modified in the virus for use in the intradermal vaccine formulations of the invention. For example, unrelated virus antigens such as HIV parasite antigens (gpl60, gpl20, gp41) (eg, malaria), bacterial or fungal antigens, or tumor antigens can be modified into an attenuated strain.

Virtually any heterologous gene sequence can be constructed in the chimeric viruses of the invention for use in intradermal vaccine formulations. Preferably, the heterologous gene sequences are fractions and peptides that act as modifiers of the biological response. Preferably, epitopes that induce a protective immune response to any of a variety of pathogens, or antigens that bind to neutralizing antibodies can be expressed through or as part of the chimeric virus. For example, heterologous gene sequences that can be constructed in chimeric viruses of the invention include, but are not limited to, influenza and parainfluenza hemagglutinin neuraminidase,₃₂₆

ΙΝΪΤΓΓυΤΟ Mexican μ la puonsDAi: Λϋη »2Ηυ

INDUÍTRlAl · χ .2? and fusion glycoproteins such as the PIV3 HN and F genes. In yet another embodiment, heterologous gene sequences can be modified in the chimeric virus to include those encoding proteins with immunomodulatory activities. Examples of immunomodulatory proteins include, but are not limited to, cytokines, type 1 interferon, gamma-interferon, colony stimulating factors, interleukin -1, -2, -4, -5, -6, -12, and antagonists of these agents.

In still other embodiments, the invention encompasses pathogenic cells or viruses, preferably attenuated viruses, that express the variant antibody on their surface.

In alternative embodiments, the vaccine compositions of the invention comprise a fusion polypeptide wherein an antigenic or immunogenic agent is operably linked to a variant antibody of the invention that has improved affinity for FcyRIIIA. Modification of fusion polypeptides for use in the vaccine compositions of the invention is carried out using methods of recombinant DNA technology that are routine and within the level of technical skill.

The invention further encompasses methods of inducing tolerance in a subject through administration of a composition of the invention. Preferably a composition suitable for inducing tolerance in a subject comprises a₃₂₇ ΙΜΡ1

INyTHVTOM £ JUCAN &lt;

de la mold or kYrt.uüWtijar INDUSTRIAL · * 3 antigenic or immunogenic agent coated with — axit.iGuewn, a variant of the invention, where the variant antibody has a higher affinity to FcyRIIB. Although intended to be bound through no particular mechanism of action, such compositions are effective in inducing tolerance through activation of the inhibitory pathway mediated by FcyRIIB.

COMPOSITIONS AND METHODS OF ADMINISTRATION

The invention provides methods and pharmaceutical compositions comprising molecules of the invention (ie, dibodies) comprising multiple epitope-binding domains and, optionally, an Fe domain (or a portion thereof). The invention also provides methods of treating, prophylaxis, and ameliorating one or more of the symptoms associated with a disease, disorder, or infection through the administration to a subject of an effective amount of a fusion protein or a conjugated molecule of the invention. , or a pharmaceutical composition comprising a fusion protein or a conjugated molecule of the invention. In a preferred aspect, an antibody, a fusion protein, or a conjugated molecule is substantially purified (ie, substantially free of substances that limit its effect or produce unwanted side effects). In a specific embodiment, the subject is an animal, preferably a mammal such as a non-primate (e.g., cows,

328 IMPI ^

MÜbCANO INSTITUTE

OE Ι.Λ FtUFIEDAL '* AíZZza INDUSTRIAL pigs, horses, cats, dogs, rats, etc.) and a primate (for example, a monkey, such as for example, a cynomolgous monkey and a human). In a preferred embodiment, the subject is a human. In yet another preferred embodiment, the antibody of the invention is of the same species as the subject.

Various delivery systems are known and can be used to deliver a composition comprising molecules of the invention, for example encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or fusion protein, receptor-mediated endocytosis (see , for example, Wu et al. (1987) Receptor-Mediated In Vitro Gene Transition By A Soluble DNA Carrier System, J. Biol.

Chem. 262: 4429-4432), nucleic acid construct as part of the retroviral vector or other vector, etc. Methods for administering a molecule of the invention include, but are not limited to, parenteral (eg, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, mucosal (eg, intranasal, and oral routes) administration. In a specific embodiment, the molecules of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, through instituto mejucamo DE LA FtOnfiMÍ ^ e ^ SUiF fNDtmrRíAL absorption through epithelium or mucocutaneous linings (by * · M 11 « ·! * »*»? * ««. —Γ1Μ ['- &lt;| W1MW example, oral mucosa, rectal and intestinal mucosa, etc.), and can be administered in conjunction with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be used, for example, through the use of an inhaler or nebulizer, and formulation with an aerosol agent. See, for example, US Patent Nos. 6,019,968; 5,985,320; 5,985,309;

5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, each of which is incorporated herein by reference in its entirety.

The invention also provides that the molecules of the invention are packaged in a hermetically sealed container such as an ampoule or a sachet indicating the amount of the antibody. In one embodiment, the molecules of the invention are supplied as a dry-sterilized or concentrated lyophilized powder without water in a hermetically sealed container and can be reconstituted, for example, with water or saline at the appropriate concentration for administration to a subject. Preferably, the molecules of the invention are supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dose of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, al

330

<img file="MX348166B_D0191.tif" />

minus 45 mg, at least 5 0 mg, or at least 75. jng · The lyophilized mulémldü of the invention should be stored at between 2 and 8 ° C in their original container and the molecules should be administered within 12 hours, preferably within 5 within 6 hours, within 5 hours, within 3 hours, or within 1 hour after reconstitution. In an alternative embodiment, the molecules of the invention are supplied in liquid form in a hermetically sealed container that indicates the amount and concentration of the molecule, fusion protein, or conjugated molecule. Preferably, the liquid form of the molecules of the invention are supplied in a hermetically sealed container at at least 1 mg / ml, more preferably at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 100 mg / ml, at least 150 mg / ml, at least 200 mg / ml of the molecules.

The amount of the composition of the invention that will be effective in treating, preventing or ameliorating one or more of the symptoms associated with a disorder can be determined through standard clinical techniques. The precise dose to be used in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be said with the judgment of the practitioner and each patient's circumstance. Effective doses can be extrapolated from the derived dose and response curves.

331

IMPI ¡wrnruTOMBDCAf * 'ΠΈ THE INDUSTRIAL FUCWEDAD

<img file="MX348166B_D0192.tif" />

of in vitro or animal model test systems.

For the diabodies encompassed by the invention, the dose administered to a patient is typically 0.0001 mg / kg to 100 mg / kg of the patient's body weight. Preferably, the dose administered to a patient is between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to 0.25 mg / kg, or 0.01 to 0.10 mg / kg of the patient's body weight. The dose and frequency of administration of the diabodies of the invention can be reduced or altered by improving absorption and tissue penetration of the diabodies through modifications such, for example, lipidation.

In one embodiment, the dose of the molecules of the invention administered to a patient can be from 0.01 mg to 1000 mg / day when used as a single agent therapy. In another embodiment the molecules of the invention are used in combination with other therapeutic compositions and the dose administered to a patient is less than when such molecules are used with single agent therapy.

In a specific embodiment, it may be desired to administer the pharmaceutical compositions of the invention

332

<img file="MX348166B_D0193.tif" />

FROM RlKMEDAD locally to the area in need of treatment; p be achieved through, for example, and ττσ — a — nnaiiera de · limitation, local infusion, through injection or by means of an implant, the implant being of porous, non-porous or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a molecule of the invention, care must be taken to use materials that the molecule does not absorb.

In another embodiment, the compositions can be delivered in a vesicle, in particular a liposome (see, Langer (1990) New Methods Of Drug Delivery, Science 249: 1527-1533); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, López -Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); López Berestein, ibid. , pp. 3 17-327, - see generally ibid.).

In yet another embodiment, the compositions may be delivered in a sustained release or controlled release system. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more molecules of the invention. See, for example, US Patent No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 96/20698; Ning et al. (1996) Intratumoral

Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A

Sustained-Release Gel, Radiotherapy &amp; Oncology 39: 179-189,

Song et al. (1995) Antibody Mediated Lung Targeting Of Long Circulating Emulsifier, PDA Journal of Pharmaceutical Science &amp; Technology 50: 372-397; Cleek et al. (1997) Biodegradable Polymeric Carriers For A bF GF Antibody For Cardiovascular Application, Pro. Int'l. Symp. Control. I laughed. Bioact. Mater. 24: 853-854; and Lam et. to the. (1997) Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int'l. Symp. Control I laughed. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, (1987) Implantable Pumps, CRC Crit. Rev. Biomed. Eng. 14: 201-240; Buchwald et al. (1980 ) Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis, Surgery 88: 507-516; and Saudek et al. (1989) A Preliminary Trial of The Programmable Implantable Medication System For Insulin Delivery, N. Engl. J. Med. 321: 574-579). In another embodiment, polymeric materials can be used to achieve controlled release of the antibodies (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Levy et al. (1985) Inhibition Of ³³⁴ IMPIS

INSTITUTO MEXICANO ΠΕ LA FWHEDAD INDUSTRIAL

Calcification Of Bioprosthetic Heart Valves By Local Controlled-Release Diphosphonate, Science 228: 190-192;

During et al. (1989) Controlled Release Of Dopamine From A Polymeric Brain Implant: In Vivo Characterization, Ann.

Neurol. 25: 351-356; Howard et al. (1989) Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits, J.

Neurosurg. 7 (1): 105-112); US Patent No. 5,679,377;

US Patent No. 5,916,597; US Patent No.

5,912,015; US Patent No. 5,989,463; US patent

No. 5,128,326; PCT Publication No. WO 99/15154; PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-vinyl acetate), poly ( methacrylic acid), polyglycolides (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactides (PLA), poly (lactide-co-glycolides) (PLGA), polyorthoesters . In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic agent (eg, the lungs), thus requiring only a fraction of the systemic dose (see, eg, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). In another embodiment, polymer compositions useful as controlled release implants are

335 used according to Dunn et al.

(See UST ^ s;

IMPI

INSTITUTO MEJKAHO DF LA FBOPleDAD

<img file="MX348166B_D0194.tif" />

This particular method is based on the specific effect of the controlled in situ release of the bioactive material from the polymeric system. The implant can generally occur anywhere within the body of the patient in need of therapeutic treatment. In another embodiment, a non-polymeric sustained delivery system is used, through which a non-polymeric implement in the subject's body is used as the drug delivery system. After implantation in the body, the organic solvent from the implant will dissipate, disperse, or leach from the composition into the surrounding tissue fluid, and the non-polymeric material will gradually coagulate or precipitate to form a solid, microporous matrix (see, US 5,888,533). .

Controlled release systems are explained in the review by Langer (1990, New Methods Of Drug Delivery, Science 249: 1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more therapeutic agents of the invention. See, for example, US Patent No. 4,526,938; International Publication Nos. WO 91/05548 and WO 96/20698; Ning et al. (1996) Intratumoral Radioimmunotheraphy Of A Human Colon Cancer

Xenograft Using A Sustained-Release Gel, Radiotherapy &amp;

Oncology 39: 179-189, Song et al. (1995) Antibody Mediated

336

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INSTITUTO MUBCAN · OEIAMKNIWAD IISOUSTIUAl

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Lung Targeting Of Long-Circulating Emulsions, PDA Journal of

Pharmaceutical Science &amp; Technology 50: 372-397; Cleek et al.

(1997) Biodegradable Polymeric Carriers For A bF GF Antibody For Cardiovascular Application, Pro. Int'1. Symp. Control. I laughed. Bioact. Mater. 24: 853-854; and Lam et al. (1997) Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, Proc. Int¹1. Symp. Control I laughed. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in its entirety.

In a specific embodiment where the composition of the invention is a nucleic acid encoding a diabody of the invention, the nucleic acid can be administered in vivo to promote the expression of its encoded diabody, through construction as part of a vector of expression of appropriate nucleic acid and administering it in such a way that it becomes intracellular, for example through the use of a retroviral vector (see, US Patent No. 4,980,286), or through direct injection or through the use of a microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or by coating with lipids or cell surface receptors or transfection agents, or through delivery in the bond to a homeobox-like peptide that is known to enter the nucleus (See, for example, Joliot et al. (1991) Antennapedia Homeobox Peptide Regulates Neural i ΝΓΓΠυτΟ MEXICAN »Ε LA MONEDAD

Morphogenesis, Proc. Nati. Acad. Sci. USA 88: 18ET &lt;L868T7 etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated into the DNA of the host cell for expression through homologous recombination.

Treatment of a subject with a therapeutically or prophylactically effective amount of molecules of the invention may include a single treatment or, preferably, may include a series of treatments. In a preferred example, a subject is treated with molecules of the invention in the range of about 0.1 to 30 mg / kg of body weight, once a week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5 or 6 weeks. In other embodiments, the pharmaceutical compositions of the invention are administered once a day, twice a day, or three times a day. In other modalities, the pharmaceutical compositions are administered once a week, twice a week, one week yes and another week no, once a month, once every six months, once every two months, twice a year or once a year. It will also be appreciated that the effective dose of the molecules used for treatment can be increased or decreased depending on the course of a particular treatment.

³³⁸ IMPI INSTITUTE MtXlCANí; OF THE ΝΟΕΙ AGE INDUSTRY!

PHARMACEUTICAL COMPOSITIONS

Compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (eg, impure or non-sterile compositions) and pharmaceutical compositions (ie compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactic or therapeutically effective amount of a prophylactic and / or therapeutic agent described herein or a combination of the agents and a pharmaceutically acceptable carrier. Preferably, the compositions of the invention comprise a prophylactically or therapeutically effective amount of one or more of the molecules of the invention and a pharmaceutically acceptable carrier.

The invention also encompasses pharmaceutical compositions comprising a diabody molecule of the invention and a therapeutic antibody (eg, a tumor-specific monoclonal antibody) that is specific for a particular cancer antigen, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or State Government or listed.

<img file="MX348166B_D0196.tif" />

<img file="MX348166B_D0197.tif" />

³³⁹ IMPI “« ibs in the US Pharmacopoeia or other generally recognized Pharmacopoeia for use in animals, and more particularly in humans. The term "carrier" refers to an adjuvant diluent (eg, Freund's adjuvant (complete or incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry fat-free milk, glycerol, propylene, glycol , water, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH regulating agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, and the like.

³⁴⁰iNmFT »* UKiCANc Γί PROPERTY Vy-nr ^ LJK * INDUSTRY! X Generally, the ingredients of the compositions of the invention are dispensed either separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or as a waterless concentrate in a hermetically sealed container such as a ampoule or sachet indicating the amount of the active agent. When the compositions are to be administered by infusion, they can be dispensed from an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided such that the ingredients can be mixed prior to administration.

The compositions of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, etc. calcium, ferric hydroxides, isopropylamine, trethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

GEN THERAPY

In a specific embodiment, nucleic acids comprising sequences encoding the molecules of the invention are administered to treat, prevent, or ameliorate a

341

<img file="MX348166B_D0198.tif" />

or more of the symptoms associated with the disease, disorder or infection, through gene therapy 'Gene therapies refer to therapy carried out through the administration to a subject of an expressed nucleic acid or is to be can express. In this embodiment of the invention, the nucleic acids produce their encoded antibody or fusion protein that mediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be used in accordance with the present invention. Illustrative methods are described below.

For a general review of gene therapy methods, see Goldspiel et al. (1993) Human Gene Therapy, Clinical Pharmacy 12: 488-505; Wu et al. (1991) Delivery Systems For Gene Therapy, Biotherapy 3: 87-95, Tolstoshev (1993,) Gene Therapy, Concepts, Current Triais And Future Directions, Ann. Rev. Pharmacol. Toxicol. 32: 573596; Mulligan (1993) The Basic Science Of Gene Therapy, Science 260: 926-932; and Morgan et al. (1993) Human Gene Therapy, Ann. Rev. Biochem. 62: 191-217. Methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &amp; Sons, NY (1993); and Kriegler, Gene Transfer and

IMPIAS *

INSTITUTO MlfclCANo ns u ntoviEDAn, INDUSTRY! , -Expression, A Laboratory Manual. Stockton Press, NY (1990) T

In a preferred aspect, a composition of the invention comprises nucleic acids that encode a diabody of the invention, the nucleic acids are part of an expression vector that express the antibody in a suitable host. In particular, nucleic acids have promoters, preferably heterologous promoters, operably linked to an antibody coding region, the promoter is inducible or constitutive, and optionally tissue specific. In another particular embodiment, nucleic acid molecules are used where the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thereby providing intrachromosomal expression of the nucleic acid encoding the antibody (Koller et al. (1989) Inactivating The Beta 2Microglobulin Locus In Mouse Embryonic Stem Celia By Homologoua Recombination, Proc. Nati. Acad. Sci. USA 86: 8932-8935; and Zijlstra et al. (1989) Germ-Line Tranamisaion Of A Diarupted Beta 2-Microglobulin Gene Produced By Homologoua Recombination In Embryonic Stem Celia, Nature 342: 435-438).

In another preferred aspect, a composition of the invention comprises nucleic acids encoding a ³⁴³

MBtlCANc INSTITUTE

DE * V - ZSa 2 * INDUSTStAI fusion protein, nucleic acids that are part of an expression vector that express the fusion protein in a suitable host. In particular, such nucleic acids have promoters, preferably heterologous promoters, operably linked to the coding region of the fusion protein, the promoter being induced constitutively, and optionally tissue specific. In another particular embodiment, nucleic acid molecules are used where the coding sequence of a fusion protein and any other desired sequence are flanked by regions that promote homologous recombination at the desired site in the genome, in this way providing intrachromosomal expression of the fusion protein.

The distribution of nucleic acids in a subject can be either direct, in the case where the subject is exposed directly to the nucleic acid or vectors that carry the nucleic acid, or indirect, in which case, the cells are first transformed with acids. nucleic acids in vitro, then transplanted into the subject. These two methods are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, nucleic acid sequences are administered directly in vivo where they are expressed to produce the encoded product. This can be accomplished through any number of known methods in³⁴⁴ IMPI ¡ίττττυτο mkilano DB THE INDUSTRIAL PROPERTY ^ &lt;r7 — the technique, for example, through the construction of these as part of an appropriate nucleic acid expression vector and administering them in such a way that they become intracellular, for example, to via infection using defective or attenuated retroviral vectors or other viral (see, for example, US Patent No. 4,980,286), or through direct injection of naked DNA, or through the use of microparticle bombardment (eg, gene gun; Biolistic, DuPont), or coating with lipids or cell surface receptors or transient agents, encapsulation in liposomes, microparticles, or microcapsules, or through the administration thereof in coupling with a peptide that is known to enter the nucleus, to via coupling administration to a subject of the antigen for receptor-mediated endocytosis (see, for example, Wu et al. (1987) Receptor-Mediated In Vitro Gene

Transformation By A Soluble DNA Carrier System, J. Biol. Chem. 262: 4429-4432) (which can be used for target cell types that specifically express the receptors), etc. In another embodiment, nucleic acid-antigen complexes can be formed where the antigen comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to prevent lysosomal degradation. In yet another embodiment, the nucleic acid can be activated in vivo for specific absorption.

<img file="MX348166B_D0199.tif" />

WHTHrro mkxicani i DE LA PROMEDAT tNniiSTWIAL of the cell and expression, through specific receptor activation (see, for example, PCT Publications WO 92/06180; WO 92/22635; WO 92/20316; WO93 / 14188; WO 93 / 20221).

Alternatively, nucleic acid can be introduced intracellularly and incorporated into host cell DNA for expression, through homologous recombination (Koller et al. (1989) Inactivating The Beta 2 -Microglobulin Locus In Mouse Embryonic Stem Cells By Homologous Recombination, Proc Nati Acad Sci USA 86: 8932-8935, and Zijlstra et al. (1989) Germ-Line Transmission Of A Disrupted Beta 2-Microglobulin Gene Produced By Homologous Recombination In Embryonic Stem Cells, Nature 342: 435-438).

In a specific embodiment, viral vectors containing nucleic acid sequences encoding a molecule of the invention (eg, a diabody or a fusion protein) are used. For example, a retroviral vector can be used (see Miller et al. (1993) Use Of Retroviral Vectors For Gene Transfer And Expression, Meth. Enzymol. 217: 581-599). These retroviral vectors contain the necessary components for the correct packaging of the viral genome and integration into the DNA of the host cell. The nucleic acid sequences encoding the antibody or a fusion protein to be used in gene therapy are cloned into one or more vectors, which facilitate

346 Mexican institute DE LA ΛΟΡΙΕΟΑΙ nucleotide sequence distribution — aa to -cujotor More details about retroviral vectors can be found in Boesen et al. (1993) Circumvention Of Chemotherapy-Induced Myelosuppression By Transfer Of The Mdrl Gene, Biotherapy 6: 291-302), describing the use of a retroviral vector to deliver the mdrl gene for hematopoietic stem cells in order to make stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al. (1994) Long-Term Biological Response Of Injured Rat Carotid Artery Seeded With Smooth Muscle Cells Expressing Retrovirally Introduced Human Genes, J. Clin. Invest. 93: 644-651; Keim et al. (1994) Retrovirus-Mediated Gene Transduction Into Canine Peripheral Blood Repopulating Cells, Blood 83: 1467-1473; Salmons et al. (1993) Targeting Of Retroviral Vectors For Gene Therapy, Human Gene Therapy 4: 129-141; and Grossman et al. (1993) Retroviruses: Delivery Vehicle To The Liver, Curr. Opin. Genetics and Devel. 3: 110114.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses can naturally infect the respiratory epithelium where they cause mild illness. Other objectives of the

<img file="MX348166B_D0200.tif" />

Adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscles. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky et al. (1993, Gene Therapy: Adenovirus Vectors, Current Opinion in Genetics and Development 3: 499-503) presents a review of adenovirus-based gene therapy. Bout et al. (1994, Lung Gene Therapy: In Vivo Adenovirus-Mediated Gene Transfer To Rhesus Monkey Airway Epithelium, Human Gene Therapy, 5: 3-10) demonstrates the use of adenovirus vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other instances of the use of adenovirus in gene therapy can be found in Rosenfeld et al. (1991) Adenovirus Mediated Transfer Of A Recombinant Alpha 1 -Antitrypsin Gene To The Lung Epithelium In Vivo, Science 252: 431-434; Rosenfeld et al. (1992) In Vivo Transfer Of The Human Cystic Fibrosis Transmembrane Conductance Regulator Gene To The Airway Epithelium, Cell 68: 143-155; Mastrangeli et al. (1993) Diversity Of Airway Epithelial Cell Targets For In Vivo Recombinant Adenovirus-Mediated Gene Transfer, J. Clin. Invest. 91: 225-234; PCT Publication WO94 / 12649; and Wang et al. (1995) A Packaging Cell Line For Propagation Of Recombinant Adenovirus Vectors Containing Two Lethal Gene-Region Deletions, Gene Therapy 2: 775-783. In a preferred embodiment, adenovirus vectors are used.

<img file="MX348166B_D0201.tif" />

Adeno-associated virus (AAV) has been proposed for use in gene therapy (see, for example, Walsh et al. (1993) Gene Therapy For Human Hemoglobinopathies, Proc. Soc. Exp. Biol. Med. 204: 289- 300 and US Patent No. 5,436,146).

Another method for gene therapy involves the transfer of a gene into cells in tissue culture through methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Usually, the method of transfer includes transferring a selectable marker to the cells. The cells are then placed under selection to isolate cells that have been taken up and are expressing the transferred gene. These cells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be carried out via any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a vector or bacteriophage, containing nucleic acid sequences, cell fusion, transfer chromosome-mediated gene transfer, microcell-mediated gene transfer, steroplast fusion, etc. Numerous techniques are known in the art for the introduction

<img file="MX348166B_D0202.tif" />

of foreign genes in cells (see, for example, Loeffler et al. (1993) Gene Transfer Into Primary And Established Mammalian Cell Lines With Lipopolyamine-Coated DNA, Meth. Enzymol. 217: 599-618, Cotten et al. (1993) ) Receptor-Mediated Transport Of DNA Into Eukaryotic Cells, Meth. Enzymol. 217: 618-644) and can be used in accordance with the present invention, provided that the necessary development and physiological functions of the recipient cells are not altered. The technique should provide for a stable transfer of the nucleic acid into the cell, such that the nucleic acid can be expressed throughout the cell and preferably inherited and expressed through its cell line.

The resulting recombinant cells can be delivered to a subject by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells intended for use depends on the desired effect, the condition of the patient, etc., and can be determined by the person skilled in the art.

Cells into which nucleic acid can be introduced for gene therapy purposes encompass any desired cell type, available including but not limited to epithelial cells, endothelial cells,

350

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keratinocytes, fibroblasts, cells. ^ hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic progenitor stem cells, for example, as obtained from the spinal cord, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the subject.

In an embodiment where recombinant cells are used in gene therapy, the acid sequences encoding an antibody or a fusion protein are introduced into the cells in such a way that they can be expressed by the cells or their progeny, and the recombinant cell are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and / or progenitor cell that can be isolated and maintained in vitro can potentially be used in accordance with another embodiment of the present invention (see, for example, PCT Publication WO 94/08598; Stemple et al. (1992) Isolation Of Ά Stem Cell For Neurons And GHa From The Mammalian Neural Crest, Cell 71: 973-985, Rheinwald (1980) Serial Cultivation Of Normal Human Epidermal Keratinocytes, Meth Cell Bio 21A: 229-254;

³⁵¹

INSTITUTO MEXICANO ΙΈ LA MlOWEOAO iwsnm and Pittelkow et al. (1986) New Techniques For The In Vitro Culture Of Human Skin Keratinocytes And Perspectives On Their Use For Grafting Of Patients With Extensive Burns, Mayo Clinic Proc. 61: 771-777).

In a specific embodiment, the nucleic acid to be introduced for gene therapy purposes comprises an inducible promoter operably linked to the coding region, such that the expression of the nucleic acid is controllable by controlling the presence or absence of the inducer. appropriate transcription.

KITS

The invention provides a pharmaceutical pack or kit comprising one or more containers filled with the molecules of the invention. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease are also included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container (s) may be a notice in the prescribed form through a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, the notice of which reflects approval through the agency of the manufacture, use or ³⁵² WICKED

NSTtTUTC M EXJC AN &lt;

'nr ΙΛ mohedal · sJSBSttí ινγ &gt;&gt; «τ» ιαι --— sale for human administration.

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises one or more molecules of the invention. In another embodiment, a kit further comprises one or more other prophylactic or therapeutic agents useful for treating cancer, in one or more containers. In another embodiment, a kit further comprises one or more cytotoxic antibodies that bind to one or more cancer-associated cancer antigens. In certain embodiments, the other prophylactic or therapeutic agents are a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

CHARACTERIZATION AND DEMONSTRATION OF THERAPEUTIC UTILITY

In various aspects of the pharmaceutical compositions, prophylactic or therapeutic agents of the invention are preferably tested in vitro, in a cell culture system, and in an animal model organism, such as a rodent animal model system, for therapeutic activity. desired before use in humans. For example, assays that can be used to determine whether administration of a specific pharmaceutical composition is desired include cell culture assays in which the patient's tissue sample is grown in culture, and exposed to

353 or on the contrary it is brought into contact with a pharmaceutical composition of the invention, and the effect of such composition on the tissue sample is observed. The tissue sample can be obtained through a patient biopsy. This test allows the identification of the most therapeutically effective prophylactic or therapeutic molecule (s) for each individual patient. In various embodiments, in vitro assays can be carried out with cells representative of the cell types involved in inflammatory autoimmune disorder (eg, T cells), to determine whether a pharmaceutical composition of the invention has the desired effect on such cell types. .

Combinations of prophylactic and / or therapeutic agents can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well known in the art can be used. In a specific embodiment of the invention, combinations of prophylactic and / or therapeutic agents are tested in a mouse system model. Such model systems are widely used and well known to those of skill in the art. Prophylactic and / or therapeutic agents can be administered repeatedly. Various aspects of the procedure may vary. Such aspects

354

<img file="MX348166B_D0204.tif" />

they include the temporal regimen of administration of the prophylactic and / or therapeutic agents, and whether such agents are administered separately or as a mixture.

Preferred animal models for use in the methods of the invention are, for example, human FcyRs expressing transgenic mice in mouse effector cells, for example, any mouse model described in US 5,877,396 (incorporated herein by reference in its whole) can be used in the present invention. Transgenic mice for use in the methods of the invention include, but are not limited to, mice bearing human FcyRIIIA; mice bearing human FcyRIIA; mice bearing human FcyRIIB and human FcyRIIIA; mice bearing human FcyRIIB and FcyRIIA. Human. Preferably, the mutations showing the highest levels of activity in the functional assays described above will be tested for use in studies in animal models for use in humans. Sufficient amounts of antibodies for use in animal models can be prepared using the methods described supra, for example using mammalian expression systems and purification methods described and exemplified herein.

Mouse xenograft models can be used to examine the efficacy of antibodies to ³⁵⁵iMfflTUIC MEXICA * t'f ia puctiedac INDUSTRIAL - mouse for generated against a specific target of _____ tumor based on the affinity and specificity of the epitope binding domains of the diabody molecule of the invention and the ability of diabody to provoke an immune response (Wu et al. (2001) Mouse Model For Multistep Tumorigenesis, Trends Cell Biol. 11: 82-9). Human FcyRs expressing transgenic mice in single mouse effector cells and adapted to animal models to test the efficacy of human FcFcyR interactions. The pairs of transgenic mouse lines FcyRIIIA, FcyRIIIB and FcyRIIA generated in the laboratory of Dr. Jeffrey Ravetch (through a license agreement with Rockefeller U. and Sloan Kettering Cancer center) can be used as listed in Table 11 below.

Table 11: Mouse Strains

<td>Antecedent strain</td><td>Human FcR</td>

<td>Naked / CD16A KO</td><td>None</td>

<td>Naked / CD16A KO</td><td>FcyRIIIA</td>

<td>Naked / CD16A KO</td><td>FcyR IIA</td>

<td>Naked / CD16A KO</td><td>FcyR IIA and IIIA</td>

<td>Nude / CD32B KO</td><td>None</td>

<td>Nude / CD32B KO</td><td>FcyR IIB</td>

356

The anti-inflammatory activity of

<img file="MX348166B_D0205.tif" />

The combination therapies of the invention can be determined through the use of various experimental animal models of inflammatory arthritis known in the art and described in Crofford LJ and Wilder RL, Arthritis and Autoimmunity in Anime, in Arthritis and Allied Conditions: A Textbook of

Rheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993). Spontaneous and experimental animal models of inflammatory arthritis and autoimmune rheumatic diseases can also be used to evaluate the anti-inflammatory activity of the combination therapies of the invention. The following are some tests intended as examples and not by limitation.

Animal models in principle for arthritis or inflammatory disease known in the art and widely used include: adjuvant-induced arthritis rat models, collagen-induced arthritis rat and mouse models, and arthritis-induced rat, rabbit and hamster models. by antigen, all described in Crofford LJ and Wilder RL, Arthritis and Autoimmunity in Animáis, in Arthritis and Allied Conditions: A Textbook of Rheumatology, McCarty et al. (eds.), Chapter 30 (Lee and Febiger, 1993), incorporated herein by reference in its entirety.

The anti-inflammatory activity of the therapy

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The combination of the invention can be evaluated using a rat model with carrageenan-induced arthritis. Carrageenan-induced arthritis also used in rabbits, dogs and pigs in studies of chronic arthritis or inflammation. Quantitative histomorphometric evaluation is used to determine therapeutic efficacy. Methods for using such a carrageenan-induced arthritis model are described in Hansra P. et al. (2000) Carrageenan-induced Arthritis In The Rat, Inflammation, 24 (2): 141-155. Animal models with zymosan-induced inflammation are also commonly used as known and described in the art.

The anti-inflammatory activity of the combination therapies of the invention can also be evaluated by measuring the inhibition of carrageenan-induced foot edema in rats, using a modification of the method described in Winter CA et al. (1962) Carrageenan-induced Edema In Hind Paw Of The Rat As An Assay For Anti-Inflammatory Drugs Proc. Soc. Exp. Biol Med. 111, 544-547. This assay has been used as a primary in vivo classification for the anti-inflammatory activity of most NSAIDs, and is considered to be predictive of human efficacy. The anti-inflammatory activity of the prophylactic or therapeutic agent test is expressed as the percentage of inhibition of the³⁵⁸ IΜ ΡI iwsttti γγο muucan * INI'IIETRIAl PROPERTY _ Increase in the weight of the hind leg of the test group in relation to the control group dosed with vehicle.

Additionally, animal models for inflammatory bowel disease as well. can be used to evaluate the efficacy of the combination therapies of the invention (Kim et al. (1992) Experimental Colitis In Animal Models, Scand. J. Gastroentrol. 27: 52953 7; Strober (1985) Animal Models Of Inflammatory Bowel Dis ease —An OverView, Dig. Dis. Sci. 30 (12 Suppl): 3S-10S). Ulcerative colitis and Crohn's disease are human inflammatory bowel diseases that can be induced in animals. Sulfated polysaccharides include, but are not limited to, amylopectin, carrageenan, amylopectin sulfate, and dextrin sulfate or chemical irritants including but not limited to trinitrobenzenesulfonic acid (TNBS) and acetic acid can be administered to animals orally to induce inflammatory diseases of the intestine.

Animal models for autoimmune disorders can also be used to evaluate the efficacy of the combination therapies of the invention. Animal models for autoimmune disorders such as type 1 diabetes, thyroid autoimmunity, systemic lupus erythematosus, and glomerulonephritis have been developed (Flanders et al. (1999) Prevention Of Type 1 Diabetes From Laboratory To Public Health '', Autoimmunity

359

29: 235-246; Rasmussen et al. (1999)

Pathogenesis Of Thyroid Autoimmunity

<img file="MX348166B_D0207.tif" />

Models To Study The

B i ochimie 81: 511-515;

Foster (1999) Relevance Of Systemic Lupus Erythematosus Nephritis Animal Models To Human Disease, Semin. Nephrol. 19: 12-24).

Furthermore, any assay known to those of skill in the art can be used to evaluate the prophylactic and / or therapeutic utility of the combination therapies described herein for autoimmune and / or inflammatory diseases.

The toxicity and efficacy of the present prophylactic and / or therapeutic protocols can be determined through standard pharmaceutical procedures in cell cultures or experimental animals, for example to determine LD.₅₀ (the lethal dose to 50% of the population) and ED₅₀ (the therapeutically effective dose in 50% of the population). The dose relationship between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD ratio.₅o / ED₅or. Prophylactic and / or therapeutic agents that exhibit high therapeutic indices are preferred. Although prophylactic and / or therapeutic agents that exhibit toxic side effects can be used, care must be taken to design a delivery system that activates such agents at the affected tissue site in order to minimize potential damage.

360 r «Trrvro mbuca h &lt;OI LA FROM SDA Γ of uninfected cells, and therefore side effects.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage of prophylactic and / or therapeutic agents for use in humans. The dose of such agents is based primarily within a range of circulating concentrations including ED.₅₀ with little or no toxicity. The dose can vary within this range depending on the dosage form used and the route of administration used. For any agent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a concentration range in circulating plasma that includes IC₅or (ie, the concentration of the test compound that achieves mean maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more precisely determine useful doses in humans. Levels in plasma can be measured, for example, through high performance liquid chromatography.

The anti-cancer activity of the therapies used in accordance with the present invention can also be determined through the use of various animal models.

361

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<img file="MX348166B_D0208.tif" />

experimental tests for the study of cancer such as the model «* t 1 ···· ~ .ι ··· ι .ι II« Jua of SCID mice or transgenic mice or nude mice with human xenografts, animal models, such as hamsters , rabbits, etc., known in the art and described in Relevance of Tumor Model for Anticancer Drug Development (1999, eds. Fiebig and Burger); Contribution to Oncology (1999, Karger); The Nude Mouse in Oncology Research (1991, eds. Boven and Winograd); and Anticancer Drug Development Guide (1997 ed. Teicher), incorporated herein by reference in its entirety.

The preferred animal models for determining the therapeutic efficacy of the molecules of the invention are mouse xenograft models. Tumor cell lines that can be used as a source for xenograft tumors include but are not limited to SKBR3 and MCF7 cells, which can be derived from patients with adenocarcinoma of the breast. These cells have both erbB2 and prolactin receptors. SKBR3 cells have been routinely used in the art as ADCC and xenograft tumor models. Alternatively, 0VCAR3 cells derived from human ovarian adenocarcinoma can be used as a source of the xenograft tumors.

The protocols and compositions of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity, prior to

362

<img file="MX348166B_D0209.tif" />

use in humans. Therapeutic agents and methods can be classified using cells from a tumor or malignant cell line. Many standard tests can be used in the art to assess such survival and / or growth; For example, cell proliferation can be assayed to measure the incorporation of³H-thymidine, through direct cell counting, by detecting changes in the transcriptional activity of known genes such as proto-oncogenes (eg, fos, myc) or cell cycle markers; Cell viability can be assessed through trypan blue staining, differentiation can be visually assessed based on changes in morphology, decreased growth and / or colony formation on soft agar or tubular network formation in three-dimensional basement membrane or the preparation of the extracellular matrix, etc.

Compounds for use in therapy can be tested in suitable animal model systems prior to human testing, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, hamsters, etc., for example , the animal models described above. The compounds can then be used in appropriate clinical trials.

In addition, any test known to the person skilled in the art can be used to assess the utility.

363

INSTITUTO MEXICANt. ,. of THE PROPERTY, prophylactic and / or therapeutic of the therapies of^NH5Wftbin? Pe ¥ en described herein for the treatment --- o — pgavaweién of · cancer, inflammatory disorder, or autoimmune disease.

DESIGN AND CHARACTERIZATION EXAMPLES OF COVALENT BISPECIFIC DIACBODIES

A monospecific covalent diabody and a bispecific covalent diabody were constructed to assess the recombinant production, purification, and binding characteristics of each. The affinity purified diabody molecules were then produced through the recombinant expression systems described herein found through SDSPAGE and SEC analysis to consist of a single dimeric species. ELISA and SPR analysis further revealed that the covalent bispecific diabody exhibited affinity for both target antigens and can bind to both antigens simultaneously.

Materials and methods:

Construction and Design of Polypeptide Molecules:

Nucleic acid expression vectors were designed to produce four polypeptide constructs, schematically depicted in FIG. 2. Construct 1 (SEQ ID NO: 9) comprises the VL domain of the humanized 2B6 antibody, which recognizes FcyRIIB, and the VH domain of the³⁶⁴ IΜ ΡI

INSTITUTO MEXICANI DE LA PROPIEDAD INnUSTWIAI humanized 3G8 antibody, which recognizes FcyRIIIA. Construct 2 (SEQ ID NO: 11) comprising the VL domain of Hu3G8 and the VH domain of Hu2B6. Construct 3 (SEQ ID NO: 12) comprising the VL domain of Hu3G8 and the VH domain of Hu3G8. Construct 4 (SEQ ID NO: 13) comprising the VL domain of Hu2B6 and the VH domain of Hu2B6.

PCR and Expression Vector Construction: The coding sequences for the VL or VH domains were amplified from the template DNA using the forward and reverse primers designed in such a way that the initial PCR products would contain overlapping sequences, which allow PCR overlapping generates the coding sequences for the desired polypeptide constructs.

Initial PCR amplification of template DNA ·.

About 35 ng of template DNA, eg light chain and heavy chain of the antibody of interest; 1 ul of 10 uM of contiguous and reverse primers; 2.5 ul 10x pH buffer pfuUltra (Stratagene, Inc.); 1 ul of 10 mM dNTP; 1 ul of 2.5 units / ul DNA polymerase pfuUltra (Stratagene, Inc.); and distilled water for a total volume of 25 ul was mixed lightly in a microfuge tube and briefly centrifuged in a microfuge to collect the reaction mixture at the bottom of the tube. PCR reactions were carried out using the 9700 System

<img file="MX348166B_D0210.tif" />

PCR GeneAmp (PE Applied Biosystem) and the following

<img file="MX348166B_D0211.tif" />

setting: 94 ° C, 2 minutes; 25 cycles of 94 C, every 15 seconds; 58 ° C, 30 seconds; and 72 ° C, 1 minute.

The Hu2B6 VL was amplified from the Hu2B6 light chain using the forward and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 56, respectively. The Hu2B6 VH was amplified from the Hu2B6 heavy chain using the forward and reverse primers of SEQ ID NO: 57 and SEQ ID NO: 58, respectively. The Hu3G8 VL was amplified from the Hu3G8 light chain using the forward and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 59, respectively. The Hu3G8 VH was amplified from the Hu3G8 heavy chain using the forward and reverse primers of SEQ ID NO: 60 and SEQ ID NO: 61, respectively.

The PCR products were electrophored on 1% agarose gel for 30 minutes at 120 volts. The PCR products were cut from the gel and purified using the MinElute GEL Extraction Kit (Qiagen, Inc.).

Overlap PCR: The initial PCR products were combined as described below and amplified using the same PCR conditions described for the initial template DNA amplification. The overlap PCR products were also purified as described supra.

The nucleic acid sequence encoding Construct 1, SEQ ID NO: 9 (shown schematically in

366

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<img file="MX348166B_D0212.tif" />

FIG. 2), was amplified by combining the products pcp * ** * amplifications of VL Hu2B6 and VH Hu3G8, and the contiguous and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 61, respectively. The nucleic acid sequence encoding construct 2, SEQ ID NO: 11 (shown schematically in FIG. 2), was amplified by combining the PCR products of VL Hu3G8 and VH Hu2B6 amplifications, and contiguous and reverse primers of SEQ ID. NO: 55 and SEQ ID NO: 58, respectively. The nucleic acid sequence encoding construct 3, SEQ ID NO: 12 (shown schematically in FIG. 2), was amplified by combining the PCR products of the VL Hu3G8 and VH Hu3G8 amplifications, and the contiguous and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 61, respectively. The nucleic acid sequence encoding construct 4, SEQ ID NO: 13 (shown schematically in FIG. 2), was amplified by combining the PCR products of the VL Hu2B6 and VH Hu2B6 amplifications, and the contiguous and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 58, respectively.

The forward primers of the VL domains (i.e. SEQ ID NO: 55) and reverse primers of the VH domains (i.e. SEQ ID NO: 58 and SEQ ID NO: 61) contained unique restriction sites to allow cloning of the final product in an expression vector. The purifier overlapped PCR products were digested with endonucleases of

367

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INWSTH.IAI

<img file="MX348166B_D0213.tif" />

Nhe I and EcoR I restriction, and cloned into pCIneo mammalian expression (Promega, Inc.). The plasmids encoding the constructs were designed as identified in Table 12:

Table 12. PLASMID CONSTRUCTS

<td>Coding of construct</td><td>Designation of plasmid</td><td>Insert</td>

<td> 1</td><td>pMGX0669</td><td>hu2B6VL-hu3G8VH</td>

<td> 2</td><td>pMGX0667</td><td>hu3G8VL-hu2B6VH</td>

<td> 3</td><td>pMGX0666</td><td>hu3G8VL-hu3G8VH</td>

<td> 4</td><td>pMGX0668</td><td>hu2 B 6VL-hu2 B 6VH</td>

Polypeptide / diabody expression: pMGX0669, encoding construct 1, was co-transfected with pMGX0667, encoding construct 2, in HEk-293 cells using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). Co-transfection of these two plasmids was designed to drive the expression of a covalent bispecific diabody (CBD) immunospecific for both FcyRIIB and FcyRIIIA (the h2B6-h3G8 diabody). pMGX0666 and pMGX0668, encoding constructs 3 and 4, respectively, were transfected separately into HEK-293 cells for the expression of a covalent monospecific diabody (CMD), immunospecific for

FcyRIIIA (h3G8 diabody) and FcyRIIB (h2B6 diabody), respectively. After 3 days in culture, the secreted products were purified from the conditioned medium.

Purification: Diabodies were captured from conditioned medium using relevant antigens coupled to CNBr activated Sepharose 4B. The affinity of the Sepharose resin was equilibrated in 20 mM Tris / HCl, pH 8.0 before loading. After loading, the resin was washed with an equilibrium buffer before elution. Diabodies were eluted from the washed resin using 50 mM Glycine pH 3.0. Eluted diabodies were immediately neutralized with 1M Tris / HCl pH 8.0 and concentrated using a spin-type concentrator. The concentrated diabodies were further purified by size exclusion chromatography using a Superdex 200 column equilibrated in PBS.

SEC:

Size exclusion chromatography used to analyze the approximate size and heterogeneity of the diabodies eluted from the column. SEC analysis was carried out on a GE healthcare Superdex 200HR 10/30 column equilibrated with PBS. Comparison with the elution profiles of a full length IgG (-150 kDa), a Fab fragment (-50 kDa) and a single chain Fv (-30 kDa) were used as controls).

ELISA: The binding of the eluted diabodies and

369

Purified IMPI was characterized by the ELISA assay, as described in 5.4.2. 50 ul / well of a 2 ug / ml solution of sCD32B-Ig was coated on a 96 well Maxisorp plate in Carbonate pH buffer at 4 ° C overnight. The plate was washed three times with PBS-T (PBS, 0.1% Tween 20) and blocked by 0.5% BSA in PBS-T for 30 minutes at room temperature. Subsequently, h2B6-h3G8 CBD, h2B6 CMD, or h3G8 CMD were diluted in blocking buffer in a series of two-fold dilutions to generate a range of diabody concentrations, from 0.5 pg / ml to 0.001 pg / ml. The plate was then incubated at room temperature for 1 hour. After washing with PBS-T three times, 50 ul / well of 0.2 ug / ml sCD16A-Biotin was added to each well. The plate was re-incubated at room temperature for 1 hour. After washing with PBS-T three times, · 50 ul / well of a 1: 5000 dilution of HRP-conjugated streptavidin (Amersham Pharmacia Biotech) was used for detection. HRP-streptavidin was allowed to incubate for 45 minutes at room temperature. The plate was washed with PBS-T three times and developed using 80 ul / well TMB substrate. After 10 minutes of incubation, the HRP-TMB reaction was stopped by the addition of 40 ul / well of 1% H₂SW₄. OD450 nm was read using a 96 well plate reader and SOFTmax software, and results were plotted using GraphPadPrism software.

370

3.03.

IMPI

INSTITUTO MEXICANA · PE LA FROMEOAU INDUSTRIAI

<img file="MX348166B_D0214.tif" />

BIAcore Assay: The -Trretricuy '' · Ϊ3 · binding parameters of the eluted and purified diabodies were analyzed using a BIAcore assay (BIAcore instrument 1000, BIAcore Inc., Piscataway, NJ) and associated software as described in section 5.4 .3.

SCD16A, sCD32B, or sCD32A (negative control) were immobilized on one of the four flow cells (flow cell 2) on the surface of the sensor chip via amine coupling chemistry (by modifying carboxymethyl groups with the mixture of NHS / EDC) such that approximately 1000 response units (RU) from any receptor were immobilized on the surface. After this, the unreacted active esters were capped with an injection of 1M Et-NH2. Once a suitable surface is prepared, specific covalent diabodies (h2B6-h3G8CBD) or monospecific covalent diabodies (h2B6 CMD or h3G8 CMB) are passed over the surface by 180 second injections of a 6.25-200 nM solution at a flow rate of 70 ml / min. h3G8 scFV was also tested for comparison.

Once the complete data set was collected, the resulting binding curves were globally fitted using computer algorithms supplied by the manufacturer, BIAcore, Inc. (Piscataway,

371

NJ). These algorithms calculated both

IMPI

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INDUSTRIAL

<img file="MX348166B_D0215.tif" />

^ on Y ^ off / &amp;

from which the apparent equilibrium junction constant, K_D is derived as a ratio of the two rate constants (i.e., K_acti_Vado / Koff) · More detailed treatments of how individual rate constants are derived can be found in the BIAevaluation Software Textbook (BIAcore, Inc., Piscataway, NJ).

The association and dissociation phases were adapted separately. The dissociation degree constants were obtained for 32-34 second intervals of the 180 second dissociation phase; the adaptation of the association phase was obtained through the Langmuir 1: 1 model and the adaptation of the phase was selected on the basis of the R criterion_max and chi² for specific diabodies and scFv; adaptation of the bivalent analyte for CMD binding was used.

Results

SDS-PAGE analysis under non-reducing conditions revealed that the purified product of the h3G8 CMD, h2B6 CMD and h2B6-h3G8 CBD expression systems were each a single species with an estimated molecular weight of approximately 50 kDa (FIG. 3, lanes 4, 5 and 6, respectively). Under reducing conditions, the purified product of any of the CMD expression systems

372

<img file="MX348166B_D0216.tif" />

IMPI __ · '____ __ ___Ί_ Τ ___ ·. / __¹ ι „ίΝΤΠΤυΤΟ. MEXICAN as a single band (lanes 1 and 2), onanenraoira purified product of the h2B6-h3G8 CBD system was revealed as being 2 separate proteins. (FIG. 3, lane 3). All polypeptides purified from the expression system and visualized by SDS-PAGE under reducing conditions migrated at approximately 28 kDa.

SEC analysis of each of the expression system products also revealed a single molecular species (FIG. 4B), each of which eluted at approximately the same time as an IgG Fab fragment (-50 kDa) (FIG. 4A). The results indicate that the affinity of the purified product was a homogeneous covalent homodimer for the case of the CMD expression system and a homogeneous covalent heterodimer for the case of h2B6-h3G8 CBD.

An ELISA sandwich assay was used to test the binding of h2B6-h3G8 CBD for the specificity of either or both CD32B and / or CD16A (FIG. 5). CD32B served as the target antigen and CD16A was used as the secondary probe. The positive signal in ELIZA revealed that heterodimeric h2B6-h3G8 CBD had specificity for both antigens. A similar h3G8 CMD test (which should not bind CD32B) showed no signal.

SPR analysis indicated that h3G8 CMD immunospecifically recognized sCD16 but not SCD32B, that h2B6 CMD immunospecifically recognized sCD32B but not ³⁷³

MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY

SCD16, and that h2B6-h3G8 CBD immunospecifically recognized both sCD16 and SCD32B (FIGS. 6A-6B). None of the diabodies tested bound to the control receptor, sCD32A (FIG. 6C).

SPR analysis was also used to estimate the kinetic and equilibrium constants of CMDs and h2B6-h3G8 CBD at sCD16 and / or sCD32B. The results were compared with the same constants calculated for h3G8 scFV. FIGS. 7A-7E show the graphical results of the SPR analysis. The degrees of kinetic on and off, as well as the equilibrium constant, were calculated from the results described in FIGS. 7A to 7E and are provided in Table 13.

Table 13. Kinetic and Equilibrium Constants Calculated from BIAcore Data.

<td>Receptor / Analyte</td><td>k-activated</td><td>k-off</td><td>Kd</td>

<td>SCD16 / h3G8 diabody</td><td>2.3 X 10⁵</td><td> 0.004</td><td> 18.0</td>

<td>CBD SCD16 / h2B6-h3G8</td><td>4.6 X 10⁵</td><td> 0.010</td><td> 22.7</td>

<td>scFv SCD16 / h3B6-h3G8</td><td>3.2 x 10⁵</td><td> 0.013</td><td> 38.7</td>

<td>CBD SCD32B / h2B6-h3G8</td><td>3.6 X 10⁵</td><td> 0.005</td><td> 15.0</td>

<td>sCD32B / h2B6 diabody</td><td>6.2 x 10⁵</td><td> 0.013</td><td> 21.0</td>

Coupled with the results of the ELISA analysis, the studies confirm that the covalent heterodimer h2B625 h3G8 retained specificity for both CD32B and CD16, and was ³⁷⁴ IMPI® fWmTtfw »mexkxnc l tA MU.W.DAL Ow» - M-aLJ iMDUSTRIAt Z?

capable of binding both antigens simultaneously. The molecule is schematically represented in FIG. 8.

DESIGN AND CHARACTERIZATION OF COVALENT BI-SPECIFIC DIACBODIES THAT INCLUDE Domains Fe

In an effort to create an IgG-like molecule, that is, comprising an Fe domain, one of the polypeptides comprising the heterodimeric CBD molecule presented in Example 6.1 was modified to further comprise an Fe domain (creating a 'plus chain heavy ', and' lighter ', analogs of an antibody heavy and light chain). The heterodimeric bispecific molecule would then contain an Fe domain that will dimerize with a homologous molecule forming a tetrameric IgG-like molecule with tetravalence (ie, formed by dimerization through the Fe domains of heterodimeric bispecific molecules). Interestingly, such tetrameric molecules were not detected in the conditioned medium of recombinant expression systems using functional assays, eg, testing the conditioned medium for immunospecific binding to target antigens. Rather, only one dimeric molecule, comprising monomers consisting of a VL, VH, and Fe domain, was detected in such functional assays. To test whether the stability of the theoretical tetrameric structure was a problem, the polypeptides comprising the Fe domain were modified to

375

IMPIAS

INSTITUTO MEXICANO also comprises a hinge region of polypeptides comprising the 'lighter' chain modified to further comprise the 6 amino acids Cterminal of the constant domain of the human kappa light chain. When such modified 'heavier' and 'lighter' chains were co-expressed in recombinant expression systems, functional assays detected diabody molecules that were able to immunospecifically bind both target antigens and anti-Fc antibodies.

Materials and methods

Construction and Design of Polypeptide Molecules:

The nucleic acid expression vectors were designed to produce modified versions of constructs 1 and 2 presented in Example 6.1. Construct 5 (SEQ ID NO: 14) and 6 (SEQ ID NO: 15), were created by modifying construct 1 and 2, respectively to further comprise an Fe domain. Construct 7 (SEQ ID NO: 16) was created by modifying the Construct 1 to further comprise the sequence FNRGEC (SEQ ID NO: 23) at its C-terminus. Construct 8 (SEQ ID NO: 18) was created by modifying construct 2 to further comprise a hinge region and the Fe domain (comprising the V215A mutation). The schematic representation of constructs 5-8 is shown in FIG. 9.

PCR and Expression Vector Construction: All

ΙΝΤΓΓΤυτΟ Μ EX ΙΟ. Ν '' from μ ηοτι εολ ι C&gt; «5l INDUSTRIAL _ the PCR purification protocols and the PCR product were as described in Example 6. í^- Plasmids pMGX0669 and pMGX0667 served as templates for the coding sequences of constructs 1 and 2, respectively. The coding sequences for the Fe HuIgG domain and / or hinge domain were

SEQ ID NO: 5 or SEQ ID NO: 1 and SEQ ID NO: 5, respectively. The coding sequences of the template DNAs were amplified using the forward and reverse primers such that the PCR products would contain overlapping sequences, allowing the overlapping PCR to generate the coding sequences for the desired products.

The coding sequence of construct 1 was amplified from pMGX0669 using the forward and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 62, respectively. The coding sequence for construct 2 was amplified from pMGX0667 using the forward and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 63, respectively. HuIgG hinge-Fc was amplified using the forward and reverse primers of SEQ ID NO: 65 and SEQ ID NO: 66, respectively. Construct 7 (SEQ ID NO: 16) was amplified from pMGX0669 using the forward and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 67.

377

<img file="MX348166B_D0217.tif" />

PCR overlap. The initial PCR products were combined as described below, amplified and purified, and as described in Example 6.1.

The nucleic acid sequence encoding construct 5, SEQ ID NO: 14 (shown schematically in FIG. 9), was amplified by combining the PCR products of construct 1 and HuIgG Fe amplifications, and contiguous and reverse primers of SEQ ID. NO: 55 and SEQ ID NO: 64, respectively. The nucleic acid sequence encoding construct 6, SEQ ID NO: 15 (shown schematically in FIG. 9), was amplified by combining the PCR products of construct 2 and HuIgG Fe amplifications, and contiguous and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 66, respectively. The nucleic acid sequence encoding construct 8, SEQ ID NO: 18 (shown schematically in FIG. 9), was amplified by combining the PCR products of construct 2 and HuIgG hinge-Fc amplifications, and contiguous and reverse primers of SEQ ID NO: 55 and SEQ ID NO: 66, respectively.

The final products were cloned into the mammalian expression vector pCIneo (Promega, Inc.) as previously described. The plasmid encoding the constructs was designed as identified in Table 14:

378

Table 14. PLAMID CONSTRUCTS

<img file="MX348166B_D0218.tif" />

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<td>Construct coding</td><td>Designation of plasmid</td><td>Insert</td>

<td> 5</td><td>pMGX0676</td><td>hu2B6VL-hu3G8VH-huFc</td>

<td> 6</td><td>pMGX0674</td><td>hu3G8VL-hu2B6VH-huFc</td>

<td> 7</td><td>pMGX0677</td><td>hu3G8VL-hu3G8VH-FNRGEC</td>

<td> 8</td><td>PMGX0678</td><td>hu2B6VL-hu2B6VH-hu- hinge-Fc (A215V)</td>

Polypeptide / diabody expression: Four separate co-transfections were performed in HEK-293 cells using Lipofectamine 2000, as described in section 6.1: pMGX0669 and pMGX0674, encoding constructs 1 and 6, respectively; pMGX0667 and pMGX0676, encoding constructs 2 and 5, respectively; and pMGX0677 and pMGX0678, which encode constructs 7 and 8, respectively.

Co-transfection of these plasmids was designed to drive the expression of a tetravalent bispecific diabody (CBD) with IgG-like structure, immunospecific for both FcyRIIB and FcyRIIIA. An additional cotransfection was also performed: pMGX0674 and pMGX0676, encoding constructs 6 and 5, respectively. After three days in culture, the conditioned medium was harvested. The amount of product secreted in the conditioned medium was quantified at³⁷⁹ iwrrruro »eiiCAMv DCLAnOMTOM) tNDUmUl - through ELISA Fe anti IgG using purified Fe as a standard. Product concentrations in the samples were then normalized based on quantitation, and the normalized samples were used in the remaining assays.

ELISA: The binding of secreted diabody molecules in the medium was assayed via an ELISA sandwich as described, supra. Unless noted, CD32B was used to cover the plate, ie, as the target protein, and HRP-conjugated CD16 was used as the probe.

Results

An ELISA assay was used to test standard samples from recombinant expression systems comprising constructs 1 and 6 (pMGX669, pMGX674), constructs 2 and 5 (pMGX667-pNGX676), and constructs 5 and 6 (pMGX674-pMGX676) for the expression of diabody molecules capable of binding CD32B and CD16A simultaneously (FIG. 10). ELISA data indicated that co-transfection with constructs 1 and 6 or cotransfection with constructs 2 and 5 failed to produce a product that could bind to either or both antigens (FIG. 10, D, and A, respectively) . However, cotransf ection of constructs 5 and 6 led to the secretion of a product capable of binding to both CD32B and CD16 antigens. The above product was a dimer of constructs 5 and 6, containing a binding site for each antigen with a ³⁸⁰ IMPI iNsmuTo mkucanc ns THE DEPTH 'Nrwrrwur structure schematically described in FIG. eleven.

In order to drive the formation of an IgG-like heterotetrameric structure, the coding sequence for six additional amino acids was coupled to the C-terminal end of construct 1, generating construct 7 (SEQ ID NO: 16 and shown schematically in FIG . 9). The additional six amino acids, FNRGEC (SEQ ID NO: 23), were derived from the C-terminus of the Kappa light chain and normally interacted with the upper hinge domain of the heavy chain on an IgG molecule. A hinge domain was then modified in construct 6, generating construct 8 (SEQ ID NO: 18 and FIG. 9). Construct 8 additionally comprises an amino acid mutation in the upper hinge region, A215V. Expression plasmids encoding construct 7 and construct 8, pMGX677 and pMGX678, respectively, were then co-transfected into HEK-293 cells and expressed as described.

Diabody molecules produced from recombinant expression systems comprising constructs 7 and 8 (pMGX0677 + pMGX0678), were compared in an ELISA for binding of CD32B and CD16A to diabody molecules produced from expression systems comprising the constructs 1 and 6 (pMGX669 + pMGX674), constructs 2 and 8 (pMGX669 + pMGX678), and constructs 6 and 7 (pMGX677 + pMGX674) (FIG. 12).

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OF THE MOHEDAL 'CjMdgWSy

As before, the molecule produced TclS¹*¹ by ~ the expression system comprising uunsLrLKJLUÜ '± and 6 ~ (pMGX669 + pMGX674) proved unable to bind both CD32A and CD16A (FIG. 10 and FIG. 12). In contrast, the product of the co-expression of any of constructs 7 and 6 (pMGX0677 + pMGX0674) or of the co-expression of constructs 7 and 8 (pMGX0677-pMGX0678) was able to bind both CD32B and CD16 (FIG 12). Construct 7 is seen to be analogous to construct 1, with the exception that construct 7 comprises the C-terminal sequence FNRGEC (SEQ ID NO: 23); and that construct 8 is analogous to construct 6, except that construct 8 comprises a hinge domain and the A215V mutation. The data indicate that the addition of 6 extra amino acids from the C-terminus of the C-kappa light chain (FNRGEC; SEQ ID NO: 23) to the 'lighter' chain that does not carry Fe, helped to stabilize the formation of the molecules. diabody, tetrameric IgG type, regardless of whether the corresponding heavier chain comprises a hinge domain (ie, pMGX0677 + pMGX0674 and pMGX0677-pMGX0678, FIG. 12). The addition of the hinge domain to the Fe-bearing 'heavier' polypeptide, without the addition of FNRGEC (SEQ ID NO: 23) C-terminal sequence for the corresponding 'lighter' chain, was apparently unable to effect similar stabilization ( that is, lack of binding by the co-transfection product of constructs 2 and 8 ³⁸² ΙΜΡΙ intotuto mjjucanc

D »u nonWAD -C&gt;

INDUSnUAL (pMGX669 + pMGX678)). The structure of the molecule^{1 1} M · * »tetrameric diabody is represented schematically in the

FIG. 13.

EFFECT OF DOMAIN ORDER AND ADDITIONAL DISULFIDE LINKS ON THE FORMATION OF THE TETRAMERIC IgG TYPE DIACBODY

The effect of additional stabilization between the 'lighter' and 'heavier' polypeptide chains of the tetrameric IgG-like diabody molecule was investigated by substituting the selected residues in the polypeptide chains with cysteines. The additional cistern residues provide additional disulfide bonds between the 'heavier' and 'lighter' chains. Additionally, the domain order in binding activity was investigated by moving the Fe domain or the hinge-Fc domain from the C-terminal end of the N-terminal polypeptide chain. Although the binding activity of the molecule comprises the additional disulfide bonds was not altered relative to diabody molecules previously constructed with such bonds, the transfer of the Fe or hinge-Fe domain to the N-terminus of the heavier 'polypeptide chain 'comprises the surprisingly improved binding affinity and / or avidity of the diabodies of the bispecific molecule to both of their target antigens.

383

Materials and methods

IMPI tirsTmjTo Mexican

OF INDUSTRIAL PROPERTY

<img file="MX348166B_D0219.tif" />

Construction and Design of Molecules of * PoTipepEídcT ·

The nucleic acid expression vectors were designed to produce modified versions of constructs 5, 6 and 8 presented in Example 6.2. Construct 9 (SEQ ID NO: 19) and construct 10 (SEQ ID NO: 20) (both shown schematically in FIG. 13) were analogous to constructs 8 and 6, with the exception that the Fe or hinge domain -Fc, respectively, alternated from the C terminal of the polypeptide to the N-terminus. Additionally, all the Fe domains used were wild-type IgGl domain Fe. Construct 11, SEQ ID NO: 21, (shown schematically in FIG. 14) was analogous to construct 2 of Example 6.1 except that the C-terminal was designed to further comprise the sequence FNRGEC (SEQ ID NO: 23). Construct 12, SEQ ID NO: 22 (shown schematically in FIG. 14) was analogous to construct 5 of Example 6.2 except that the Fe domain further comprised a hinge region. Also, for constructs 11 and 12, the VL 2B6 domain and VH 2B6 domain comprised a single amino acid modification (G105C and G44C, respectively) such that one glycine in each domain was replaced by cysteine.

PCR and Expression Vector Construction: All PCR product and PCR purification protocols were as described in Example 6.1 and 6.2

IRJTTTUTO MEXICANO r »LA MOHEDAL

INtV'STRIAl

PCR overlap: The final products were constructed, amplified and purified using the methods described in Example 6.1 and Example 6.2.

The final products were cloned into the mammalian expression vector pCIneo (Promega, Inc.) as previously described. The plasmid encoding the constructs was designed as identified in Table 15:

Table 15. PLASMID CONSTRUCTS

<td>Coding of construct</td><td>Designation of plasmid</td><td>Insert</td>

<td> 9</td><td>pMGX0719</td><td>Hubisagra / Fc-hu3G8VL- hu2B6VH</td>

<td> 10</td><td>pMGX0718</td><td>huFc-hu2B6VL-hu3G8VH</td>

<td> 11</td><td>pMGX0716</td><td>hu2B6VL (G / C) -hu3G8VH- hubisagraFC</td>