WO2011140151A1

WO2011140151A1 - Antibodies against epidermal growth factor receptor (egfr)

Info

Publication number: WO2011140151A1
Application number: PCT/US2011/035073
Authority: WO
Inventors: David Buckler; Arumugam Muruganandam; Ulrik Nielsen
Original assignee: Dyax Corp.; Merrimack Pharmaceuticals, Inc.
Priority date: 2010-05-04
Filing date: 2011-05-03
Publication date: 2011-11-10

Abstract

Anti-EGFR antibodies are disclosed.

Description

ANTIBODIES AGAINST EPIDERMAL GROWTH FACTOR

RECEPTOR (EGFR)

This application claims priority to U.S. Serial Number 61/331,333, filed May 4, 2010. The disclosures of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

Background of the Invention

The Epidermal Growth Factor Receptor (EGFR) is the cell-surface receptor for members of the epidermal growth factor family (EGF-family) of extracellular protein ligands. EGFR is believed to be an oncogene and as such, anticancer therapeutics which bind to and inhibit EGFR function are of interest.

Summary of the Invention

This invention provides novel antibodies which bind to EGFR and inhibit various EGFR functions, which may provide the basis for novel cancer therapeutics and diagnostics.

Accordingly, in one embodiment, the present disclosure includes an antibody comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% identical thereto, and/or a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12,16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, or an amino acid sequence at least 80%, 85%, 90%, 95%, 98%, 99% identical thereto. Particular antibodies of the present disclosure also include those which comprise a heavy and light chain variable region having the following amino acid sequences:

(a) SEQ ID NOs: 2 and 4, respectively;

(b) SEQ ID NOs: 6 and 8, respectively, or SEQ ID NOs: 6 and 181, respectively;

(c) SEQ ID NOs: 10 and 12, respectively;

(d) SEQ ID NOs: 14 and 16, respectively, or SEQ ID NOs: 14 and 182, respectively; (e) SEQ ID NOs: 18 and 20, respectively;

(f) SEQ ID NOs: 22 and 24, respectively;

(g) SEQ ID NOs: 26 and 28, respectively;

(h) SEQ ID NOs: 30 and 32, respectively;

(i) SEQ ID NOs: 34 and 36, respectively;

G) SEQ ID NOs: 38 and 40, respectively;

(k) SEQ ID NOs: 42 and 44, respectively;

(1) SEQ ID NOs: 46 and 48, respectively;

(m) SEQ ID NOs: 50 and 52, respectively;

(n) SEQ ID NOs: 54 and 56, respectively;

(o) SEQ ID NOs: 58 and 60, respectively;

(p) SEQ ID NOs: 62 and 64, respectively;

(q) SEQ ID NOs: 66 and 68, respectively; or

(r) SEQ ID NOs: 70 and 72, respectively.

Other particular antibodies of the present invention include those which comprise the following heavy and light chain CDR3, CDR2, and CDRl, sequences:

(a) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 73, 74, and 75, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 76, 77, and 78, respectively;

(b) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 79, 80, and 81 , respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 82, 83, and 84, respectively, or SEQ ID NOs: 82, 183, and 84, respectively;

(c) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 85, 86, and 87, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 88, 89, and 90, respectively;

(d) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 91, 92, and 93, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 94, 95, and 96, respectively, or SEQ ID NOs: 94, 184, and 96, respectively; (e) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 97, 98, and 99, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 100, 101 , and 102, respectively;

(f) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 103, 104, and 105, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 106, 107, and 108, respectively;

(g) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 109, 110, and 1 11, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 1 12, 1 13, and 114, respectively;

(h) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 115, 116, and 1 17, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 1 18, 1 19, 120, respectively;

(i) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 121 , 122, and 123, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 124, 125, and 126, respectively;

(j) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 127, 128, and 129, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 130, 131 , and 132, respectively;

(k) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 133, 134, and 135, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 136, 137, and 138, respectively;

(1) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 139, 140, and 141, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 142, 143, and 144, respectively;

(m) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 145, 146, and 147, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 48, 149, and 150, respectively;

(n) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 151 , 152, and 153, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 154, 155, and 156, respectively; (o) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 157, 158, and 159, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 160, 161 , and 162, respectively;

(p) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 163, 164, and 165, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 166, 167, and 168, respectively;

(q) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 169, 170, and 171 , respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 172, 173, and 174, respectively; and

(r) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 175, 176, and 177, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 178, 179, and 180, respectively.

Also encompassed by the present invention are monoclonal antibodies that bind to the same or overlapping epitopes bound by any of the particular antibodies described herein (e.g., as determined by standard competition assays).

Antibodies of the present invention include all known forms of antibodies and other protein scaffolds with antibody-like properties. For example, the antibody can be a human antibody, a humanized antibody, a bispecific antibody, an immunoconjugate, a chimeric antibody or a protein scaffold with antibody-like properties, such as fibronectin or Ankyrin repeats. The antibody also can be a Fab, Fab'2, ScFv, affibody, nanobody, or a domain antibody. The antibody also can have any of the following isotypes: IgGl , IgG2, IgG3, IgG4, IgM, IgAl , IgA2, IgAsec, IgD, and IgE.

The antibodies of the present disclosure may further be administered with an additional therapeutic agent. Such compositions can be administered sequentially or together with other therapeutic agents, such as anti-cancer agents, e.g., other antibodies, chemo therapeutic agents and/or radiation.

Kits comprising one or more antibody of the present disclosure are also provided, optionally, contained within a container and/or with instructions for use in treating or diagnosing a disease associated with EGFR, such as cancers.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Detailed Description

In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

I. Definitions

The terms "EGFR," "ERB1," "EGF receptor," and "HER1 receptor," as used interchangeably herein, refer to human EGFR protein, as described in (1984) Nature 309:418-425(1984); see, also UniProtKB/Swiss-Prot entry P00533 (SEQ ID NO: 181).

The term "inhibition" as used herein, refers to any statistically significant decrease in biological activity, including full blocking of the activity. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.

The term "antibody" refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')₂, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39.)) as well as complete antibodies. Examples of binding fragments encompassed within the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains;

(iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,

(v) a dAb including VH and VL domains; (vi) a dAb fragment (Ward et al. (1989) Nature 341 , 544-546), which consists of a VH domain; (vii) a dAb which consists of a VH or a VL domain; and (viii) an isolated complementarity determining region (CDR) or (ix) a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242, 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 58795883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen- binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be from any source, but primate (human and non- human primate) and primatized are preferred.

The VH and VL regions can be further subdivided into regions of

hypervariability, termed "complementarity determining regions" ("CDR"), interspersed with regions that are more conserved, termed "framework regions" ("FR"). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, see also www.hgmp.mrc.ac.uk). Kabat definitions are used herein. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino -terminus to carboxy-terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4.

The VH or VL chain of the antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively. In one embodiment, the antibody is a tetramer of two heavy

immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds. In IgGs, the heavy chain constant region includes three immunoglobulin domains, CHI , CH2 and CH3. The light chain constant region includes a CL domain. The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The light chains of the immunoglobulin may be of types, kappa or lambda. In one embodiment, the antibody is glycosylated. An antibody can be functional for antibody-dependent cytotoxicity and/or complement-mediated cytotoxicity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional

(polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies can be prepared using any art recognized technique and those described herein such as, for example, a hybridoma method, as described by Kohler et al. (1975) Nature, 256:495, a transgenic animal, as described by, for example, (see e.g., Lonberg, et al (1994) Nature 368(6474): 856-859), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or using phage antibody libraries using the techniques described in, for example, Clackson et ah, Nature, 352:624- 628 (1991) and Marks et al, J. Mol. Biol, 222:581-597 (1991). Monoclonal antibodies include chimeric antibodies, human antibodies and humanized antibodies and may occur naturally or be recombinantly produced.

The term "recombinant antibody," refers to antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal {e.g., a mouse) that is transgenic or transchromosomal for immunoglobulin genes (e.g., human immunoglobulin genes) or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library (e.g., containing human antibody sequences) using phage display, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences (e.g., human immunoglobulin genes) to other DNA sequences. Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The term "chimeric immunoglobulin" or antibody refers to an immunoglobulin or antibody whose variable regions derive from a first species and whose constant regions derive from a second species. Chimeric immunoglobulins or antibodies can be constructed, for example by genetic engineering, from immunoglobulin gene segments belonging to different species.

The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences as described, for example, by Kabat et al. (See Kabat, et al. (1991) Sequences of proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The human antibody can have at least one or more amino acids replaced with an amino acid residue, e.g., an activity enhancing amino acid residue which is not encoded by the human germline immunoglobulin sequence. Typically, the human antibody can have up to twenty positions replaced with amino acid residues which are not part of the human germline immunoglobulin sequence. In a particular embodiment, these replacements are within the CDR regions as described in detail below.

The term "humanized immunoglobulin" or "humanized antibody" refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term "humanized immunoglobulin chain" or "humanized antibody chain" (i.e., a "humanized

immunoglobulin light chain" or "humanized immunoglobulin heavy chain") refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term "humanized variable region" (e.g. , "humanized light chain variable region" or "humanized heavy chain variable region") refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody.

A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy /light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol. 79, 315- 321 ; Kostelny et al. (1992) J. Immunol. 148, 1547-1553.

As used herein, a "heterologous antibody" is defined in relation to the transgenic nonhuman organism or plant producing such an antibody.

An "isolated antibody," as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to EGFR is substantially free of antibodies that specifically bind antigens other than EGFR). In addition, an isolated antibody is typically substantially free of other cellular material and/or chemicals. As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes. In one embodiment, an antibody or antigen binding portion thereof is of an isotype selected from an IgGl , an IgG2, an IgG3, an IgG4, an IgM, an IgAl , an IgA2, an IgAsec, an IgD, or an IgE antibody isotype. In some embodiments, a monoclonal antibody of the invention is of the IgGl isotype. In other embodiments, a monoclonal antibody of the invention is of the IgG2 isotype. As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes. As used herein, "nonswitched isotype" refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence regions in a gene encoding an antibody. Non-classical isotype switching may occur by, for example, homologous recombination between human αμ and human∑μ (ς-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.

As used herein, the term "switch sequence" refers to those DNA sequences responsible for switch recombination. A "switch donor" sequence, typically a μ switch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch recombination. The "switch acceptor" region will be between the construct region to be deleted and the replacement constant region (e.g., γ, ε, etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct.

An "antigen" is an entity (e.g. , a proteinaceous entity or peptide) to which an antibody or antigen-binding portion thereof binds. In various embodiments of the present invention, an antigen is EGF. In a particular embodiment according to the invention, an antigen is human EGFR.

The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents.

An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from EGFR are tested for reactivity with the given anti-EGFR antibody. Methods of determining spatial conformation of epitopes are also well known in the art and include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

Accordingly, also encompassed by the present invention are antibodies that bind to an epitope on EGFR which comprise all or a portion of an epitope recognized by the particular antibodies described herein (e.g. , the same or an overlapping region or a region between or spanning the region).

In another embodiment, the invention provides antibodies that compete for binding to EGFR with the antibodies described herein. Competing antibodies and antibodies that recognize the same or an overlapping epitope can be identified using routine techniques such as an immunoassay, for example, by showing the ability of one antibody to block the binding of another antibody to a target antigen, i.e., a competitive binding assay. Competitive binding is determined in an assay in which the

immunoglobulin under test inhibits specific binding of a reference antibody to an antigen, such as EGFR. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et ah, (1983) Methods in Enzymology 9:242); solid phase direct biotin-avidin EIA (see Kirkland et al., (1986) J. Immunol. 137:3614); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see Morel et al., (1988) Mol. Immunol. 25(1):7); solid phase direct biotin-avidin EIA (Cheung et al., (1990) Virology 176:546); and direct labeled RIA. (Moldenhauer et al, (1990) Scand. J. Immunol. 32:77). Typically, such an assay involves the use of purified antigen (e.g., EGFR) bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60- 65%, 65-70% 70-75% or more.

As used herein, the terms "specific binding," "specifically binds," "selective binding," and "selectively binds," mean that an antibody or antigen-binding portion thereof, exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant crossreactivity with other antigens and epitopes.

"Appreciable" or preferred binding includes binding with an affinity of at least 10⁶, 10⁷; 10⁸, 10⁹ M^"1, or 10¹⁰ M^"1. Affinities greater than 10⁷ M^"1, preferably greater than 10⁸ M¹ are more preferred. Values intermediate of those set forth herein are also intended to be within the scope of the present invention and a preferred binding affinity can be indicated as a range of affinities, for example, 10⁶ to 10¹⁰ M^"1, preferably 10⁷ to 10¹⁰ M^"1, more preferably 10⁸ to 10¹⁰ M ¹. An antibody that "does not exhibit significant cross-reactivity" is one that will not appreciably bind to an undesirable entity (e.g., an undesirable proteinaceous entity). For example, in one embodiment, an antibody or antigen-binding portion thereof that specifically binds to EGFR will appreciably bind that EGFR molecule but will not significantly react with other ErbB molecules and non-ErbB proteins or peptides. Specific or selective binding can be determined according to any art- recognized means for determining such binding, including, for example, according to Scatchard analysis and/or competitive binding assays.

The term "K_D," as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction or the affinity of an antibody for an antigen. In one embodiment, the antibody or antigen binding portion thereof according to the present invention binds an antigen (e.g., EEGFR) with an affinity (KD) of 100 nM or better (i.e., or less) (e.g., 90 nM, 80 nM, 70 nM, 60nM, 50nM, 40nM, 30nM, 20nM, or 10 nM or less), as measured using a surface plasmon resonance assay or a cell binding assay. In a particular embodiment, an antibody or antigen binding portion thereof according to the present invention binds EGFR with an affinity (KD) of 8 nM or better (e.g., 7 nM, 6 nM, 5 nM, 4 nM, 2 nM, 1.5 nM, 1.4 nM, 1.3 nM, InM or less), as measured by a surface plasmon resonance assay or a cell binding assay. In other embodiments, an antibody or antigen binding portion thereof binds an antigen (e.g., EGFR) with an affinity (KD) of approximately less than 10^"7M, such as approximately less than 10^"8 M, 10^"9 M or 10^"10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE 3000 instrument using recombinant EGFR as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a nonspecific antigen (e.g. , SA, casein) other than the predetermined antigen or a closely-related antigen. Other methods for determining KD include equilibrium binding to live cells expressing EGFR via flow cytometry (FACS) or in solution using KinExA® technology.

The term "K_<,_ff ," as used herein, is intended to refer to the off rate constant for the dissociation of an antibody from the antibody/antigen complex.

The terms "IC50" and "IC90," as used herein, refer to the measure of the effectiveness of a compound (e.g., an anti-EGFR antibody) in inhibiting a biological or biochemical function (e.g., the function or activity of EGFR) by 50% and 90%, respectively. For example, IC50 indicates how much of an anti-EGFR antibody is needed to inhibit the activity of EGFR (e.g., the growth of a cell expressing EGFR) by half. That is, it is the half maximal (50%) inhibitory concentration (IC) of an anti-EGFR antibody (50% IC, or IC50). According to the FDA, IC50 represents the concentration of a drug that is required for 50% inhibition in vitro. The IC50 and IC90 can be determined by techniques known in the art, for example, by constructing a dose-response curve and examining the effect of different concentrations of the antagonist (i.e., the anti-EGFR antibody) on reversing EGFR activity. As used herein, "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein.

The term "naturally-occurring" as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

The term "rearranged" as used herein refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element.

The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

The term "modifying," or "modification," as used herein, is intended to refer to changing one or more amino acids in the antibodies or antigen-binding portions thereof. The change can be produced by adding, substituting or deleting an amino acid at one or more positions. The change can be produced using known techniques, such as PCR mutagenesis. For example, in some embodiments, an antibody or an antigen-binding portion thereof identified using the methods of the invention can be modified, to thereby modify the binding affinity of the antibody or antigen-binding portion thereof to EGFR. The present invention also encompasses "conservative amino acid substitutions" in the sequences of the antibodies of the invention, i.e., nucleotide and amino acid sequence modifications which do not abrogate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence, to the antigen, i.e., EGFR. Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains have been categorized and include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gin, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in an anti-EGFR antibody is preferably replaced with another amino acid residue from the same class. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

The term "non-conservative amino acid substitution" refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.

Alternatively, in another embodiment, mutations (conservative or non- conservative) can be introduced randomly along all or part of an anti-EGFR antibody coding sequence, such as by saturation mutagenesis, and the resulting modified anti- EGFR antibodies can be screened for binding activity.

A "consensus sequence" is a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a family of proteins, each position in the consensus sequence is occupied by the amino acid occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence. A "consensus framework" of an antibody refers to a framework region in the consensus antibody sequence.

Similarly, the consensus sequence for the CDRs of can be derived by optimal alignment of the CDR amino acid sequences of EGFR antibodies of the present invention.

For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

As used herein, the term "therapeutic agent" is intended to encompass any and all compounds that have an ability to decrease or inhibit the severity of the symptoms of a disease or disorder, or increase the frequency and/or duration of symptom- free or symptom-reduced periods in a disease or disorder, or inhibit or prevent impairment or disability due to a disease or disorder affliction, or inhibit or delay progression of a disease or disorder, or inhibit or delay onset of a disease or disorder, or inhibit or prevent infection in an infectious disease or disorder. Non-limiting examples of therapeutic agents include small organic molecules, monoclonal antibodies, bispecific antibodies, recombinantly engineered biologies, RNAi compounds, tyrosine kinase inhibitors, and commercial antibodies.

Various aspects of the invention are described in further detail in the following subsections.

II. Methods for Producing Antibodies of the Invention

(i) Monoclonal Antibodies

Monoclonal antibodies of the invention can be produced using a variety of known techniques, such as the standard somatic cell hybridization technique described by Kohler and Milstein (1975) Nature 256: 495, viral or oncogenic transformation of B lymphocytes or phage display technique using libraries of human antibody genes. In particular embodiments, the antibodies are fully human monoclonal antibodies. Accordingly, in one embodiment, a hybridoma method is used for producing an antibody that binds EGFR. In this method, a mouse or other appropriate host animal can be immunized with a suitable antigen in order to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes can then be fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103 (Academic Press, 1986)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

In another embodiment, antibodies (and antibody portions) that bind EGFR can be isolated from antibody phage libraries generated using the techniques described in, for example, McCafferty et al, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991), Marks et al, J. Mol. Biol, 222:581-597 (1991) and Hoet et al (2005) Nature Biotechnology 23, 344-348 ; U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al; and U.S. Patent Nos. 5,885,793; 6,521 ,404; 6,544,731 ; 6,555,313; 6,582,915 and 6,593,081 to Griffiths et al. Additionally, production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21 :2265-2266 (1993)) may also be used. In a particular embodiment, the monoclonal antibody or antigen binding portion thereof that binds EGFR is produced using the phage display technique described by Hoet et al., supra. This technique involves the generation of a human Fab library having a unique combination of immunoglobulin sequences isolated from human donors and having synthetic diversity in the heavy-chain CDRs is generated. The library is then screened for Fabs that bind to EGFR.

In yet another embodiment, human monoclonal antibodies directed against EGFR can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859; Lonberg,N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 1 13:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci. 764:536-546. See further, U.S. Patent os. 5,545,806; 5,569,825;

5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay; U.S. Patent No. 5,545,807 to Surani et al; PCT Publication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT Publication No. WO 01/14424 to Korman et al).

In another embodiment, human antibodies of the invention can be raised using a mouse that carries human immunoglobulin sequences on transgenes and

transchromosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome (see e.g., PCT Publication WO 02/43478 to Ishida et al.). Still further, alternative transgenic animal systems expressing human

immunoglobulin genes are available in the art and can be used to raise anti-EGFR antibodies of the invention. For example, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Patent Nos. 5,939,598; 6,075,181 ; 6,114,598; 6, 150,584 and 6,162,963 to

Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-EGFR antibodies of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome can be used; as described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727 ^'. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used to raise anti- EGFR antibodies of the invention.

In yet another embodiment, antibodies of the present invention can be prepared using a transgenic plant and/or cultured plant cells (such as, for example, tobacco, maize and duckweed) that produce such antibodies. For example, transgenic tobacco leaves expressing antibodies or antigen binding portions thereof can be used to produce such antibodies by, for example, using an inducible promoter (see, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95 118 (1999)). Also, transgenic maize can be used to express such antibodies and antigen binding portions thereof (see, e.g., Hood et al., Adv.Exp. Med. Biol. 464: 127 147 (1999)). Antibodies can also be produced in large amounts from transgenic plant seeds including antibody portions, such as single chain antibodies (scFv's), for example, using tobacco seeds and potato tubers (see, e.g., Conrad et ah, Plant Mol. Biol. 38: 101 109 (1998)). Methods of producing antibodies or antigen binding portions in plants can also be found in, e.g., Fischer et ah, Biotechnol. Appl. Biochem. 30:99 108 (1999), Ma et ah, Trends Biotechnol. 13:522 7 (1995); Ma et ah, Plant Physiol. 109:341 6 (1995); Whitelam et ah, Biochem. Soc. Trans. 22:940 944 (1994) and U.S. Patent Nos. 6,040,498 and 6,815,184.

The binding specificity of monoclonal antibodies (or portions thereof) that bind EGFR prepared using any technique including those disclosed here, can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzymelinked immunoabsorbent assay (ELISA). The binding affinity of a monoclonal antibody or portion thereof also can be determined by the Scatchard analysis of Munson et ah, Anal. Biochem., 107:220 (1980).

In certain embodiments, an EGFR antibody produced using any of the methods discussed above may be further altered or optimized to achieve a desired binding specificity and/or affinity using art recognized techniques, such as those described herein.

In one embodiment, partial antibody sequences derived from an EGFR antibody may be used to produce structurally and functionally related antibodies. For example, antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et ah, 1998, Nature

332:323327; Jones, P. et al, 1986, Nature 321 :522-525; and Queen, C. et al, 1989, Proc. Natl. Acad. See. U.S.A. 86: 10029-10033). Such framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. Thus, one or more structural features of an anti-EGFR antibody of the invention, such as the CDRs, can be used to create structurally related anti-EGFR antibodies that retain at least one functional property of the antibodies of the invention, e.g., inhibiting growth of cells expressing EGFR.

In a particular embodiment, one or more CDR regions selected from SEQ ID NOs: 73-180 is combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, anti-EGFR antibodies of the invention. The heavy and light chain variable framework regions can be derived from the same or different antibody sequences.

It is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity/affinity of an antibody for an antigen (see, Hall et al., J. Imunol., 149: 1605-1612 (1992); Polymenis et ah, J. Immunol., 152:5318-5329 (1994); Jahn et ah, Immunobioh, 193:400-419 (1995); Klimka et ah, Brit. J. Cancer, 83:252-260 (2000); Beiboer et ah, J. Mol. Biol, 296:833-849 (2000); Rader et ah, Proc. Natl. Acad. Sci. USA, 95:8910-8915 (1998); Barbas et ah, J. Am. Chem. Soc, 116:2161-2162 (1994); Ditzel et al., J. Immunol., 157:739-749 (1996)). Accordingly, in certain embodiments, antibodies are generated that include the heavy and/or light chain CDR3s of the particular antibodies described herein. The antibodies can further include the heavy and/or light chain CDR1 and/or CDR2s of the antibodies of the present invention.

The CDR1 , 2, and/or 3 regions of the engineered antibodies described above can comprise the exact amino acid sequence(s) as those disclosed herein. However, the ordinarily skilled artisan will appreciate that some deviation from the exact CDR sequences may be possible while still retaining the ability of the antibody to bind EGFR effectively (e.g., conservative amino acid substitutions). Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98%, 99% or 99.5% identical to one or more CDRs of an antibody described herein.

In another embodiment, one or more residues of a CDR may be altered to modify binding to achieve a more favored on- rate of binding. Using this strategy, an antibody having ultra high binding affinity of, for example, 10¹⁰M^_1or more, can be achieved. Affinity maturation techniques, well known in the art and those described herein, can be used to alter the CDR region(s) followed by screening of the resultant binding molecules for the desired change in binding. Accordingly, as CDR(s) are altered, changes in binding affinity as well as immunogenicity can be monitored and scored such that an antibody optimized for the best combined binding and low immunogenicity are achieved. In addition to, or instead of, modifications within the CDRs, modifications can also be made within one or more of the framework regions, FR1, FR2, FR3 and FR4, of the heavy and/or the light chain variable regions of an antibody, so long as these modifications do not eliminate the binding affinity of the antibody.

In another embodiment, the antibody is further modified with respect to effector function, so as to enhance the effectiveness of the antibody in treating cancer, for example. For example cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, J. Exp Med. 176: 1 191-1195 (1992) and Shopes, B. J. Immunol.

148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

Also encompassed by the present invention are bispecific antibodies and

immunoconjugates, as discussed below.

(ii) Bispecific Antibodies

Bispecific antibodies of the present invention include binding specificities for EGFR and additional binding specificities, e.g., binding specificity for another ErbB receptor {e.g., ErbB3) or another antigen, such as the product of an oncogene. Bispecific antibodies can be prepared as full length antibodies or antibody fragments {e.g. F(ab')2 bispecific antibodies).

Methods for making bispecific antibodies are well known in the art (see, e.g., WO 051 17973 and WO 06091209). For example, production of full length bispecific antibodies can be based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (see, e.g., Millstein et ah, Nature, 305:537-539 (1983)). Further details of generating bispecific antibodies can be found, for example, in Suresh et al., Methods in Enzymology, 121 :210 (1986) and in Brennan et al., Science, 229: 81 (1985), which describes a chemical linkage process for making bispecific antibodies. Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (see, e.g., Kostelny et ah, J. Immunol., 148(5): 1547-1553 (1992)). Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported (see, e.g., Gruber et ah, J. Immunol., 152:5368 (1994)).

In a particular embodiment, the bispecific antibody comprises a first antibody (or binding portion thereof) which binds to EGFR derivatized or linked to another functional molecule, e.g., another peptide or protein {e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody of the invention may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific molecules comprising at least one first binding specificity for EGFR and a second binding specificity for a second target epitope. In a particular embodiment of the invention, the second target epitope is an Fc receptor, e.g., human Fc_RI (CD64) or a human Fc_ receptor (CD89). Therefore, the invention includes bispecific molecules capable of binding both to Fc_R, Fc_R or Fc_R expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing EGFR. These bispecific molecules target EGFR expressing cells to effector cell and trigger Fc receptor-mediated effector cell activities, such as phagocytosis of an EGFR expressing cells, antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release, or generation of superoxide anion.

In one embodiment, the bispecific molecules of the invention comprise a binding specificity at least one antibody, or an antibody fragment thereof, including, e.g., an Fab, Fab', F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as described in Ladner et al. U.S. Patent No. 4,946,778, the contents of which is expressly incorporated by reference.

The bispecific molecules of the present invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-EGFR binding specificities, using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross- linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA),

5,5'dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM),

Nsuccinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4- (Nmaleimidomethyl) cyclohaxane- 1 -carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan ei a/. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificities are antibodies, they can be conjugated via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific molecule of the invention can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described for example in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091 ,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.

Binding of the bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the FcR-antibody complexes can be detected using, e.g., an enzyme-linked antibody or antibody fragment which recognizes and specifically binds to the antibody- FcR complexes. Alternatively, the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of

Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ-counter or a scintillation counter or by autoradiography.

(iii) Immunoconjugates

Immunoconjugates of the present invention can be formed by conjugating the antibodies or antigen binding portions thereof described herein to another therapeutic agent. Suitable agents include, for example, a cytotoxic agent (e.g., a chemotherapeutic agent), a toxin (e.g. an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), and/or a radioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of such immunoconjugates have been described above. Enzymatically active toxins and fragments thereof which can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. A variety of radionuclides are available for the

212 131 131 production of radioconjugated anti-EGFR antibodies. Examples include Bi, I, In, ⁹⁰Y and ¹⁸⁶ Re.

Immunoconjugates of the invention can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as

glutareldehyde), bis-azido compounds (such as bis (pazidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),

diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et ah, Science 238: 1098 (1987). Carbon- 14-labeled 1- isothiocyanatobenzy 1-3 -methyldi ethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see, e.g., WO94/11026). III. Methods for Screening Antibodies of the Invention

Subsequent to producing antibodies (or antigen binding portions) that bind EGFR, such antibodies (or portions thereof) can be screened for various properties using a variety of assays that are well known in the art.

In one embodiment, the antibodies (or antigen binding portions) are screened (e.g., by flow cytometry) for binding to EGFR using, for example, purified EGFR and/or EGFR-expressing cells, such as A431 cells.

An antibody that competes for binding with an anti-EGFR antibody, e.g., an anti- EGFR antibody described herein, can be determined using routine techniques. Such techniques include, for example, an immunoassay, which shows the ability of one antibody to block (or not block) the binding of another antibody to a target antigen, i.e., a competitive binding assay.

Competitive binding is determined in an assay in which the antibody under test inhibits specific binding of a reference antibody to a common antigen, such as EGFR. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli et ah, Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et ah, J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1): 7 (1988)); solid phase direct biotin-avidin EIA (Cheung et ah, Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et ah, Scand. J. Immunol. 32:77 (1990)).

Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 6065%, 65-70% 70-75% or more. Other screening techniques for determining the epitope bound by antibodies of the present invention include, for example, x-ray analyses of crystals of antigen: antibody complexes which provides atomic resolution of the epitope. Other methods monitor the binding of the antibody to antigen fragments or mutated variations of the antigen where loss of binding due to a modification of an amino acid residue within the antigen sequence is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping can also be used.

These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed which have been shown to map conformational discontinuous epitopes.

Antibodies also can be screened (tested) for their binding affinity. This can be done, for example, using known methods in the art (and described herein), such as Biacore Plasmon resonance assay (Biacore, Upsula, Sweden).

Antibodies also can be screened for their ability to inhibit signaling through EGFR using routine assays, such as, those described in Horst et al. supra. For example, the ability to inhibit EGFR- ligand mediated phosphorylation of EGFRs can be assessed by treating cells expressing EGFR with EGFR ligand (e.g., EGF) in the presence and absence of the antibody. The cells can then be lysed, crude lysates centrifuged to remove insoluble material, and EGFR phosphorylation measured, for example, by Western blotting followed by probing with an anti-phosphotyrosine antibody as described in Kim et al., supra.

Alternatively, the ability of an antibody to inhibit downstream signaling through EGFR can be measured by kinase assays for known substrates of EGFR such as, for example, AKT and/or ERK, as described in, for example, by Horst et al. supra, Sudo et al, (2000) Methods Enzymol, 322:388-92; and Morgan et al. (1990) Eur. J. Biochem., 191 :761-767, following EGFR stimulation by EGF ligand. For example, cells expressing EGFR can be stimulated with EGF ligand and incubated with a candidate antibody (or antigen-binding portion thereof). Cell lysates subsequently prepared from such cells can be immunoprecipitated with an antibody for a substrate of EGFR (or a protein in a cellular pathway involving EGFR) such as, an anti-AKT antibody, and assayed for kinase activity (e.g., AKT kinase activity) using art recognized techniques. A decrease in or complete disappearance in level or activity (e.g., kinase activity) of a EGFR substrate or protein in a pathway involving EGFR in the presence of the antibody, relative to the level or activity in the absence of the antibody is indicative of an antibody which inhibits EGFR signaling.

Antibodies that decrease levels of EGFR on cell surfaces can be identified by their ability to downregulate or inhibit EGFR expression on tumor cells. In certain embodiments, the antibodies decrease EGFR on cell surfaces by inducing internalization (or increasing endocytosis) of EGFR (e.g., by internalization and recycling of the receptor and/or internalization and degradation of the receptor). To test this, EGFR can be biotinylated and the number of EGFR molecules on the cell surface can be readily determined, for example, by measuring the amount of biotin on a monolayer of cells in culture in the presence or absence of an antibody or antigen binding portion thereof, for example, as described in, e.g., Waterman et ah, J. Biol. Chem. (1998), 273: 13819-27, followed by immunoprecipitation of EGFR and probing with streptavidin. A decrease in detection of biotinylated EGFR over time in the presence of an antibody is indicative of an antibody which decreases EGFR levels on cell surfaces.

Antibodies of the present invention can also be tested for their ability to inhibit growth of cells expressing EGFR (either in vivo or in vitro), such as tumor cells, using art recognized techniques, including a Cell Titer Glow Assay and Tritiumlabeled thymidine incorporation assay (also see, e.g., Macallan et al., Proc. Natl. Acad. Sci. (1998)

20;95(2):708-13; Perez et al. (1995) Cancer Research 55, 392-398. Antibodies also can be screened for the ability to inhibit spheroid growth of cells expressing EGFR. This can be done by using an assay which approximates conditions of a developing tumor growth (see, e.g., Herman et al. (2007) Journal of Biomolecular Screening Electronic

publication) as described herein.

Pharmaceutical Compositions In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, containing an antibody (or antigen-binding portion), of the present invention, formulated together with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19).

Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenylsubstituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, Nmethylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

Compositions of the invention can be administered alone or in combination therapy, i.e., combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one or more additional therapeutic agents, such as the anti-cancer agents described herein. The compositions of the invention can also be administered in conjunction with radiation therapy and/or surgery.

Compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The antibodies can be prepared with carriers that will protect the antibodies against rapid release, such as a controlled release formulation, including implants, transdermal patches, and

microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York,1978.

To administer compositions of the invention by certain routes of administration, it may be necessary to coat the constituents, e.g., antibodies, with, or co-administer the compositions with, a material to prevent its inactivation. For example, the compositions may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the antibodies, use thereof in the compositions of the invention is contemplated.

Supplementary active constituents can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the monoclonal antibodies in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the antibodies into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, the human antibodies of the invention may be administered once or twice weekly by subcutaneous injection or once or twice monthly by subcutaneous injection. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of antibodies calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The

specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the antibodies and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such antibodies for the treatment of sensitivity in individuals. Examples of pharmaceutically - acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of antibodies which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of antibodies which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.001 percent to about ninety percent of active ingredient, preferably from about 0.005 percent to about 70 percent, most preferably from about 0.01 percent to about 30 percent.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal,

intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Particular examples of adjuvants which are well-known in the art include, for example, inorganic adjuvants (such as aluminum salts, e.g., aluminum phosphate and aluminumhydr oxide), organic adjuvants (e.g., squalene), oil-based adjuvants, virosomes (e.g., virosomes which contain a membrane- bound heagglutinin and neuraminidase derived from the influenza virus).

Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

When the antibodies of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Regardless of the route of administration selected, the antibodies of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the antibodies in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required. For example, the physician or veterinarian could start doses of the antibodies of the invention employed in the compositions at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of compositions of the invention will be that amount of the antibodies which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851 , 5,312,335, 5,064,413, 4,941 ,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4. ,486, 194, which discloses a therapeutic device for administering medications through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic antibodies of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery {see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et ah); mannosides (Umezawa et ah, (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et ah (1995) FEBS Lett. 357: 140; M. Owais et ah (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et ah (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; pl20 (Schreier et ah (1994) J. Biol. Chem.

269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; J.J.

Killion; I.j. Fidler (1994) Immunomethods 4:273.

Other embodiments of the present invention are described in the following Examples.

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Examples

Materials and Methods

Throughout the examples, the following materials and methods were used unless otherwise stated.

In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in polypeptide preparation. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology) , 510, Paul, S., Humana Pr (1996); Antibody

Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et ah, C.S.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et ah, John Wiley & Sons (1992). In vitro and in vivo model systems for assaying HCV biology are described, for example, in Cell culture models and animal models of viral hepatitis. Part II:

hepatitis C, Lab. Anim. ( Y);34(2):39-47 (2005) and in The chimpanzee model of hepatitis C virus infections, ILAR J.;42(2): l 17-26 (2001).

Example 1: Production of Antibodies Using Phage Display

In order to obtain human anti-EGFR antibodies, a human Fab-phage library including a unique combination of immunoglobulin sequences obtained from human donors (Hoet et al. supra, incorporated by reference in its entirety herein) was initially screened for EGFR binders.

Briefly, the immobilized target protein (described further below) was exposed to the phage library. Each phage expresses a unique Fab and carries its DNA sequence. Because of diversity, some phage bind to the target stronger than others. The

immobilized target was then washed to remove unbound or loosely bound phage. Phage that bind are then further purified by binding to A431 cells and then protein again. The remaining bound Fabs were then isolated, amplified and sequenced.

This selection process included the use of immobilized rEGFR alone or combined with competitor antibody (or competitor molecule) which elutes phage that recognize the same epitope. Accordingly, Fabs identified by this selection process have non- overlapping epitopes.

The selection process also included A431 cells, biotinylated EGFR, or both. Another selection process included biotinylated EGFR in the presence of EGF ligand to identify Fabs which bind only the activated form of EGFR.

Example 2: Binding Affinity / Epitope Binding

Surface Plasmon Resonance (SPR) was used to analyze binding affinity and epitope binding. Specifically, one of the proteins (antibody or target) is immobilized on the surface of the chip (as described herein) and the other protein is added. The association /dissociation interaction of the two proteins is measured. As the protein in solution associates with the immobilized protein, an increase in refractive index results which is captured by the resonance signal. As the protein dissociates, a decrease in signal results. Epitope binding (EGF blocking) was analyzed using the Flexchip system.

Briefly, one of the Fabs is immobilized on the surface of the chip. As EGFR associates with the Fab, the resonance signal increases. The chip is then regenerated and a mixture of EGFR and another antibody (e.g., Erbitux) is injected. If the Fab binds overlapping epitopes with the injected antibody, then the signal will be less compared to EGFR injected alone. The chip is then regenerated again and a mixture of EGF ligand and EGFR is injected. Resonance signal is measured. A decrease in signal indicates overlapping epitopes with EGF ligand. The chip is regenerated for a final time and injected with EGFR to confirm the activity of the Fab.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims. Any combination of the embodiments disclosed in the dependent claims are contemplated to be within the scope of the invention.

Incorporation by Reference

All publications, patents, and pending patent applications referred to herein are hereby incorporated by reference in their entirety.

SUMMARY OF SEQUENCE LISTING

DNA sequences = lower case

Amino acid sequences = upper case

Leader sequences = broken underline

CDRs = underlines

DX 1-4 IgGl

Heavy chain SEP ID NO: l :

atgggcttcggcctgtcctggctgtttctggtcgccatcctgacgcgtgtccagtgcgaggtgcaactgctggaaagcggagg aggcctggtgcaacccggtggatctctgagactgagttgtgctgcatcagggttcacattttccacatatgaaatggattgggtcc ggcaggcccctggcaaggggctggaatgggtgagcgtcatttctccaagtggcgggtggaccggttatgctgattcagtgaa aggccgcttcacaatctcccgagacaacagcaagaatactctgtatctgcagatgaactctctgcgtgcagaggataccgccgt ctattactgcgctaaaacacccggatatgacagttcagggtactatggttcctggtttgattactggggccagggaactctggtga ccgtgagctctgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctc tgggctgcctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacac cttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacct acatctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccaca cctgccctccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgat ctcccggacccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacg gcgtggaggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccg tgctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaaga ccatctccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaacc aggtgtccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaac aactacaagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtgg cagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccc tggcaag

SEP ID NO: 2

MGFGLSWLFLVAILTRVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSTYEMD

WVROAPGKGLEWVSVISPSGGWTGYADSVKGRFTISRDNSKNTLYLQMNSLRA

EDTAVYYCAKTPGYDSSGYYGSWFDYWGOGTLVTVSSASTKGPSVFPLAPSSK

STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWT

VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTC A^VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light Chain

SEP ID NO:3

atggccctgcagacccaagtgttcatctccctgctgctgtggattagcggcgcctacggagacatcgtcatgacacaatcaccc gatagtctggcagtgtcactgggtgagagggcaactattaactgcaagtccagccagtctgtcctgtatagttcaaataacaaga attacctggcctggtatcaacagaagcctggtcaaccacccaaactgctgatctactgggcttccaccagagaaagcggcgtg cctgaccggttttctggaagtgggagtgggactgatttcactctgactatctcttcactgcaagcagaggacgtggccgtctatta ctgccaacaatatggctctagtccaatcacatttggacaggggactcgtctggaaattaagcgtacggtggccgctccctccgtc tttatctttcctccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaag ccaaagtgcaatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatag tacttattcactgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcagg ggctgagttcacctgtgaccaaatcctttaacagaggtgagtgc

SEP ID NO: 4

MALPTPVFISLLLWISGAYGDIVMTPSPDSLAVSLGERATINCKSSPSVLYSSNN

KNYLAWYPPKPGPPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLPAEDV

A YCPPYGSSPITFGPGTRLEIKRTVAAPSVFIFPPSDEPLKSGTASWCLLNNF

YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

DX 1-6 IgGl

Heavy chain

SEP ID NP:5

atgggcttcggcctgtcctggctgtttctggtcgccatcctgacgcgtgtccagtgcgaggtgcaactgctggaaagcggaggt ggactggtccaacccggtggatctctgagactgagttgtgctgcatcagggttcacattttccgagtacgccatgatgtgggtgc ggcaggctcctggcaaggggctggaatgggtgagctccatctggccaagtggcgggtatacttcatacgctgattccgtcaaa ggtcgcttcaccatcagccgagataactctaagaatacactgtatctgcagatgaattcactgcgggcagaggacactgctgtgt actattgcgcaaaagactactatggttcaggaagttattacggggtcgactactggggtcagggcacactggtgaccgtgagta gcgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgc ctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgc cgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgc aacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccct ccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccgg acccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtgg aggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgc accaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatct ccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgt ccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactac aagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagca gggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggca ag

SEP ID N0:6

MGFGLSWLFLVAILTRVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSEYAM

MWVROAPGKGLEWVSSIWPSGGYTSYADSVKGRFTISRDNSK TLYLQMNSLR

AEDTA YCAKDYYGSGSYYGVDYWGOGTLVTVSSASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTVP

SSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:7

atggccatgagggtgcccgcccagctgctggggctgctgctgctgtggctgcctggagctagatgcgcaatccaaatgaccc aatccccaagctctctgagtgcttcagtcggtgacagagtgacaattacttgtcgggccaaccacgggatccgcaatgatctgg gctggtaccagcagaagcccggaaaagcacctaaactgctgatctatggggcctccagcctgcagtctggtgtcccaagtcg attctcaggctccggaagcgggaccgactttacactgactatctctagtctgcagcccgaggatttcgctacctactattgcctgc aagaatactcatttcctctgacattcggtggtggaactaaagtggagattaagcgtacggtggccgctccctccgtctttatctttc ctccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtg caatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattca ctgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagt tcacctgtgaccaaatcctttaacagaggtgagtgc

SEP ID NO: 8

MAMRVPAOLLGLLLLWLPGARCAIOMTOSPSSLSASVGDRVTITCRANHGIRND

LGWYOOKPGKAPKLLIYGASSLOSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY

CLOEYSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEV

THQGLSSPVTKSFNRGEC

SEQ ID NO: 181

MAMRVPAOLLGLLLLWLPGARCAIOMTOSPSSLSASVGDRVTITCRANHGIRND

LGWYOOKPGKAPKLLIYGASSLXSGVPSRFSGSGSGTDFTLTISSLOPEDFATYY

CLOEYSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEV

THQGLSSPVTKSFNRGEC

wherein X is Q or E

DX 1-10 IgGl

Heavy chain

SEP ID NO:9

atgggcttcggcctgtcctggctgtttctggtcgccatcctgacgcgtgtccagtgcgaggtgcaactgctggaaagcggaggt ggactggtccaacccggtggatctctgcgactgtcttgcgctgcatcagggttcacattttcctggtacactatgttctgggtgcg gcaggcccctggcaaggggctggagtgggtgagctctatctatcccagtgggggcatcaccaaatacgctgactctgtgaaa ggtcgctttacaatttcccgagataacagcaagaatactctgtatctgcagatgaactctctgcgtgcagaagataccgccgtgta ctattgcgctacaatctactgttccgggggatcatgctatggggaggatgcattcgacatttggggtcagggcactatggtcacc gtgtccagcgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctg ggctgcctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacacctt ccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctac atctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacct gccctccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctc ccggacccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggc gtggaggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtg ctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagacc atctccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccag gtgtccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaa ctacaagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggca gcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctg gcaag

SEP ID NO: 10

MGFGLS WLFLV AILTRVQCE VQLLE S GGGL VQPGG SLRLS CAAS GFTF S WYTMF

WVROAPGKGLEWVSSIYPSGGITKYADSVKGRFTISRDNSKNTLYLQMNSLRAE

DTAVYYCATIYCSGGSCYGEDAFDIWGOGTMVTVSSASTKGPSVFPLAPSSKST

SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP

SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP

PKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO: 11

atggccatgagggtgcccgcccagctgctggggctgctgctgctgtggctgagaggagctagatgcgacatccaaatgaccc aatcccctagctctctgagtgcatcagtcggggatcgggtgacaattacttgtcgcgcctcccagagcatctctagttacctgaa ctggtaccagcaaaagcctgggaaggctcccaaactgctgatctacgcagcctcaaccctgcagtccggcgtccctagccga ttctctggaagtgggagtgggactgactttactctgaccatctccagcctgcaaccagaggatttcgctacatattactgccagca gtcttatagtactccccctacctttggcggagggacaaaagtggaaattaagcgtacggtggccgctccctccgtctttatctttcc tccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgc aatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcac tgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagtt cacctgtgaccaaatcctttaacagaggtgagtgc

SEP ID NO: 12

MAMRVPAOLLGLLLLWLRGARCDIOMTOSPSSLSASVGDRVTITCRASOSISSY

LNWYOOKPGKAPKLLIYAASTLOSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY

COOSYSTPPTFGGGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASWCLLNNFYPRE

AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

DX 1-17 IgGl

Heavy chain

SEP ID NO: 13

atgggcttcggcctgtcctggctgtttctggtcgccatcctgacgcgtgtccagtgcgaggtgcaactgctggaaagcggaggt ggactggtccaacccggtggatctctgagactgagttgtgctgcatccggttttacattttcctactatcacatgctgtgggtgcgg caggcccctggcaaggggctggagtgggtgagctggatttctagttcaggcgggtacactgaatatgctgactccgtcaaggg gcgcttcaccatcagccgagataactctaagaatacactgtatctgcagatgaacagtctgcgtgcagaggacaccgctgtgta ttactgcgctagggataatgactgggcatcagattattggggccagggtaccctggtcacagtcagtagcgctagcactaagg gcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaaggactactt ccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgcagtcctccg gcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaaccacaagc cttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctgcccctga gctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgaggtgacctg cgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaacgctaag accaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaac ggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccag cctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgtctggtgaa gggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccacccctcctgtg ctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgc tccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag SEQ ID NO: 14

MGFGLSWLFLVAILTRVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSYYHML

WVROAPGKGLEWVSWISSSGGYTEYADSVKGRFTISRDNSKNTLYLOMNSLRA

EDTAVYYCARDNDWASDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ

TYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQYNSTYRWS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO: 15

Atggccctgcccgcccaactgctgggtctgctgatgctgtgggtgtccggaagctctggtgacatcgtcatgacccaaagtcc tctgagcctgccagtcacccccggagagccagcttctatttcctgtcgttcaagtcaatctctgctgcactccagcgggtacactt atctggattggtatctgcagaagcccggccaatctccacagctgctgatctatctgagaagtaaccgggcatcaggggtccctg accgcttctccggtagcggctctggaaccgactttacactgaaaattagtcgagtggaagccgaggacgtgggggtctactatt gtatgcagggtactcaaaccccatacacattcggccagggaactaagctggaaatcaaacgtacggtggccgctccctccgtc tttatctttcctccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaag ccaaagtgcaatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatag tacttattcactgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcagg ggctgagttcacctgtgaccaaatcctttaacagaggtgagtgc

SEQ ID NO: 16

MALPAOLLGLLMLWVSGSSGDIVMTOSPLSLPVTPGEPASISCRSSOSLLHSSGY

TYLDWYLOKPGOSPOLLIYLRSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVG YCMOGTOTPYTFGOGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNF

YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 182 MALPAOLLGLLMLWVSGSSGDIVMTOSPLSLPVTPGEPASISCRSSOSLLHSSGY

TYLXWYLQKPGOSPOLLIYLRSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVG YCMOGTOTPYTFGOGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNF

YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

wherein X is D or E.

DX 1-18 IgGl

Heavy chain

SEOID NO: 17

atgggcttcggcctgtcctggctgtttctggtcgccatcctgacgcgtgtccaatgcgaggtgcaactgctggaaagcggaggt ggactggtccaacccggtggatctctgagactgagttgtgctgcatcagggttcacattttcctggtacgtgatgctgtgggtgc ggcaggcccctggcaaggggctggaatgggtgtcatctattagtccatcaggcgggtatactaactacgctgactccgtgaaa ggtcgcttcaccatcagccgagataattctaagaacacactgtatctgcagatgaatagtctgcgtgcagaggacactgccgtct actattgcgctaggcacggcgagtactcatccagctggtttgattattggggacatggcacactggtgacagtgtctagtgctag cactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaa ggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgc agtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaa ccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccct gcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgag gtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcaca acgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggact ggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggcca agggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacct gtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccac ccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgt gttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO: 18

MGFGLSWLFLVAILTRVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSWYVM LWVROAPGKGLEWVSSISPSGGYTNYADSVKGRFTISRDNSK TLYLQMNSLR

AEDTA YCARHGEYSSSWFDYWGHGTLVTVSSASTKGPSVFPLAPSSKSTSG

GT AALGCLVKD YFPEP VT VS W S G ALTS G VHTFP AVLQ S S GLYSLS S WT VP S S

SLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTC A^VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ST

YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPP

SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO: 19

atggcctgggccctgctgctgctgactctgctgactcaagatacaggcagctgggcacagtccgtgctgactcagcccgccag cgtctctggctcccctgggcaatctatcaccatcagctgcaccggcacatcctctaacgtgggtaattacaacctggtgtcctggt atcagcagcaccccggcaaggctccaaagctgattatctatgaagacaataagaggcctagcggggtgtccaacaggttctct gtgtctaaaagcggttccactgcaagcctgaccattagcggcctgcagaccaaggacgaggctcagtactactgttgctcttat gccggaggtggaacatgggtgtttgggggaggaacaaaagtcaccgtcctaggacagcctaaggccgctccttccgtgacc ctgttccctccttcctccgaggagctgcaggccaacaaggctaccctggtgtgcctggtgtccgacttctaccctggcgccgtg accgtggcttggaaggccgacggctcccctgtgaaggtgggcgtggagaccaccaagccttccaagcagtccaacaacaag tacgccgcctcctcctacctgtccctgacccctgagcagtggaagtcccaccggtcctacagctgccgggtgacccacgagg gctccaccgtggaaaagaccgtggcccctgccgagtgctcc

SEP ID NO:20

MAWALLLLTLLTPDTGSWAPSVLTPPASVSGSPGPSITISCTGTSSNVGNYNLV

SWYPPHPGKAPKLIIYEDNKRPSGVSNRFSVSKSGSTASLTISGLPTKDEAPYYC

CSYAGGGTWVFGGGTKVTVLGPPKAAPSVTLFPPSSEELPANKATLVCLVSDF

YPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYS

CRVTHEGSTVEKTVAPAECS

DX 10-4 IgGl

Heavy chain SEP ID NO:21

atgggctttggcttgtcctggttgtttctcgtggctatcctcaaaggtgttcagtgcgaagttcagctcctcgaaagcggtggcgg actggtgcagccagggggtagcctgaggctgagttgtgccgcttcaggcttcaccttcagtcggtactccatgatctgggtgag acaagcccctggaaagggcctggagtgggtatcatacataagctcatccggcggctggaccttttacgctgactccgtgaagg ggaggttcacaatttctcgcgacaactctaaaaacactctgtatctgcagatgaattctcttcgagccgaggacacagcagtcta ctattgcgcacgtgattgggggggacccagcgattattgggggcagggaacccttgtcactgtgagttctgctagcactaagg gcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaaggactactt ccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgcagtcctccg gcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaaccacaagc cttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctgcccctga gctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgaggtgacctg cgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaacgctaag accaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaac ggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccag cctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgtctggtgaa gggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccacccctcctgtg ctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgc tccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO: 22

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVOPGGSLRLSCAASGFTFSRYSMI

WVROAPGKGLEWVSYISSSGGWTFYADSVKGRFTISRDNSKNTLYLQMNSLRA

EDTA YCARDWGGPSDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ

TYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQYNSTYRWS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain SEP ID NO:23

atgggcactcccgcacaacttctgttcctcctcctcttgtggttgccagacacaactggggatatccaaatgactcagtctccttcc tccctctccgcttccgtgggcgaccgggttacaattacctgcagggccagtcaatctatcagcagttaccttaactggtaccagc agaagccaggtaaggctcccaagctgctgatatatgcagccagcagcctgcagagtggggtcccttcaagattttctggctctg gctctggtaccgactttaccttgaccatcagctcactgcagcctgaggatttcgccacctactattgtcagcagagctattcaactc ccgatacattcggacagggaacaaaactggaaattaaacgtacggtggccgctccctccgtctttatctttcctccaagcgacga gcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgcaatggaaagtgg ataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcactgtccagcaccc tgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagttcacctgtgacca aatcctttaacagaggtgagtgc

SEP ID NO: 24

MGTPAOLLFLLLLWLPDTTGDIOMTOSPSSLSASVGDRVTITCRASOSISSYLNW

YOOKPGKAPKLLIYAASSLOSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCOOS

YSTPDTFGOGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV

QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEVTHQ

GLSSPVTKSFNRGEC

DX 10-6 IgGl

Heavy chain

SEP ID NO:25

atgggcttcggactgagctggctcttcctcgtggcaattctgaagggtgttcaatgtgaggtgcagcttctggaatccggcggcg gtcttgtgcagcccggcggcagtctgcggctgagctgcgccgcctccggttttaccttcagccactatactatgctgtgggtgag gcaggctccagggaagggcctcgaatgggtgagttccatctccccctcagggggtgagacacaatatgccgattctgttaaag gaagatttaccatctctcgagacaactctaaaaacaccctctacttgcagatgaattcattgcgcgcagaggacactgcagtctat tactgcgctacctggcctggagggtactacgatagctcaggatactacgcttttgactattggggccaggggacattggtaaca gtcagttctgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgg gctgcctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttc cctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctaca tctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacct gccctccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctc ccggacccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggc gtggaggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtg ctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagacc atctccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccag gtgtccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaa ctacaagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggca gcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctg gcaag

SEQ ID NO:26

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVOPGGSLRLSCAASGFTFSHYTML

WVRQAPGKGLEWVSSISPSGGETQYADSVKGRFTISRDNSK TLYLQMNSLRA

EDTA YCATWPGGYYDSSGYYAFDYWGOGTLVTVSSASTKGPSVFPLAPSSK

STSGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWT

VPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL

FPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQ

Y STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV

YTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:27

atgggctgggcactgcttctcctgaccctccttacacagggcactgggagttgggctcagagcgccttgacccaacctgcttct gtatctggaagccctggacagtccattacaatcagctgtaccggtactagcagtgatgtgggtgcatataattacgtttcttggtat cagcaacaccccggcaaggtccccaaattgatgatatacgaggtgtcaaacaggccatctggggtctccaatagattctcagg atccaaatcagggaacacagctagcctgacaatcagtggcctgcaggccgaagacgaggccgactactactgcaactcctat actacctctgccacactggtgtttggcggcggaaccaagctcactgtcctaggacagcctaaggccgctccttccgtgaccctg ttccctccttcctccgaggagctgcaggccaacaaggctaccctggtgtgcctggtgtccgacttctaccctggcgccgtgacc gtggcttggaaggccgacggctcccctgtgaaggtgggcgtggagaccaccaagccttccaagcagtccaacaacaagtac gccgcctcctcctacctgtccctgacccctgagcagtggaagtcccaccggtcctacagctgccgggtgacccacgagggct ccaccgtggaaaagaccgtggcccctgccgagtgctcc SEP ID NO:28

MGWALLLLTLLTOGTGSWAOSALTOPASVSGSPGOSITISCTGTSSDVGAY YV

SWYOOHPGKVPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLOAEDEADYY

CNSYTTSATLVFGGGTKLTVLGOPKAAPSVTLFPPSSEELQANKATLVCLVSDF

YPGAVTVAWKADGSPVKVGVETTKPSKQS KYAASSYLSLTPEQWKSHRSYS

CRVTHEGSTVEKTVAPAECS

DX 10-9 IgGl

Heavy chain SEP ID NO:29

atgggcttcgggttgtcatggctctttctcgtagcaatcctcaaaggcgtgcagtgcgaggtgcagctgctggaatccggcggc gggctggtgcagcccggcgggtcccttcggcttagttgcgctgcttcaggatttacattctccccctacggtatggtctgggtca ggcaagccccagggaaaggattggagtgggttagctacatatctcctagcggcggatatactgactatgccgacagcgtcaa gggccgtttcaccatctctagagacaatagtaagaacaccctgtacctgcagatgaacagcctgcgcgcagaagatactgccg tttattactgtgctcgagatttgggagacattcacgattattggggtcagggtacactggtgaccgtgtcttctgctagcactaagg gcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaaggactactt ccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgcagtcctccg gcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaaccacaagc cttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctgcccctga gctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgaggtgacctg cgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaacgctaag accaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaac ggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccag cctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgtctggtgaa gggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccacccctcctgtg ctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgc tccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:30

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSPYGM VWVROAPGKGLEWVSYISPSGGYTDYADSVKGRFTISRDNSK TLYLQMNSLR AEDTA YCARDLGDIHDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA

LGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ

TYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL

MISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQYNSTYRWS

VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL

TK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDGSFFLYSKLTV

DKSRWQQG VFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:31

atgggcacaccagctcagttgttgtttctcctcctcctttggcttccagacaccaccggcgatatccagatgacccagtctcccgg caccctgagcctgagccctggtgagagggccacactctcttgtcgagccagtcagtccgtttctcgctcctcactggcatggta ccaacaaaagcctggacaggcaccccggctgctgatttacggggcttctagtcgtgccacaggcattcccgacagattcagcg gatccggaagtgggacagatttcactttgactatcagcaggctggaacctgaggactttgctgtgtactattgccagcagtatgg caactccccaggtggcactttcggacaggggaccaaagtcgaaatcaagcgtacggtggccgctccctccgtctttatctttcct ccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgc aatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcac tgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagtt cacctgtgaccaaatcctttaacagaggtgagtgc

SEP ID NO:32

MGTPAOLLFLLLLWLPDTTGDIOMTOSPGTLSLSPGERATLSCRASOSVSRSSLA WYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAWYCQ OYGNSPGGTFGOGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASWCLL FYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLS SP VTKSFNRGEC

DX 10-11 IgGl

Heavy chain SEP ID NO:33

atgggctttggtctctcttggttgtttttggtcgccatcctcaagggggtccagtgcgaggtacagcttctggaaagcggaggag gtctggtgcaacctggggggagcctccggctgtcctgcgctgcatccggcttcaccttcagcaagtacccaatgcagtgggtc cgccaggcacccggaaaaggcctggaatgggtgtcttctatctattcttccggcgattggaccgcttacgcagatagtgttaaag gcaggttcacaatatccagagacaattcaaagaacaccctttatctgcagatgaactcactgcgagccgaggacacagccacc tactactgtgctaaagctgccgtgtattattgcacaggcggtatttgtagttatagttactacatggacgtgtggggcaagggaact actgttactgtgagctcagctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccg ccgctctgggctgcctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtg cacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcaccc agacctacatctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaaga cccacacctgccctccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccc tgatgatctcccggacccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacg tggacggcgtggaggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgc tgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcg aaaagaccatctccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgacca agaaccaggtgtccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcct gagaacaactacaagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcc cggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccct gagccctggcaag

SEP ID NO:34

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSKYPM

OWVROAPGKGLEWVSSIYSSGDWTAYADSVKGRFTISRDNSK TLYLQMNSLR

AEDT ATYYC AKAAVYYCTGGIC S Y S YYMD V WGKGTT VT VS S AS TKGP S VFPL A

PSSKSTSGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSS

WTVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPR

EEQY STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QWTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain

SEP ID NO:35

atgggcactcctgctcagttgctgttcctgctcctcttgtggcttcccgacactacaggcgacattcaaatgacccaaagtccatct tctgtcagtgcttctgttggagaccgggtaactattaactgcagagcttcacgcgatgtgagcagctggttggcatggtaccagc agaagccaggtaaagcccctaagcttctcatctactctgcaagtggcctgcagagcggcgtgccctccaggttctccggatca gggtccgggaccgattttacacttaccatctccagcctgcagcccgaggatttcgccacctatttctgtcagcaggccaagacct ttcctctgacatttggaggtggcacaaaggtggaaatcaaacgtacggtggccgctccctccgtctttatctttcctccaagcgac gagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgcaatggaaagt ggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcactgtccagcac cctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagttcacctgtgac caaatcctttaacagaggtgagtgc

SEP ID NO:36

MGTP AOLLFLLLLWLPDTTGDIOMTQ SPS S VS AS VGDRVTINCRASRD VS S WLA

WYOOKPGKAPKLLIYSASGLOSGVPSRFSGSGSGTDFTLTISSLOPEDFATYFCO

OAKTFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL FYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

DX 10-12 IgGl

Heavy chain

SEP ID NO:37

atgggcttcggactttcttggctgtttctggtggctatcttgaaaggtgttcagtgtgaagttcagcttctggagtctggcggcggct tggtccagccaggtggtagcctgagactctcttgcgcagcaagtggattcaccttttctcactataatatgtcctgggtccgacag gccccaggcaagggcctggaatgggtgtcctccatctggcccagtgggggacatacatggtatgctgactccgtgaagggga ggttcacaattagccgggacaacagtaaaaacaccctgtacctgcagatgaatagcctccgcgccgaggataccgctgtctac tactgcgccagccccgaccctcaatttccttattactactatggcatggatgtgtgggggcaaggaacaactgtaactgtgtcatc agctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcct ggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccg tgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaa cgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctcc ctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggac ccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggag gtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcacc aggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctcca aggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccc tgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaag accacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcaggg caacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:38

MGFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSHY M

SWVROAPGKGLEWVSSIWPSGGHTWYADSVKGRFTISRDNSK TLYLOMNSLR

AEDTA YCASPDPOFPYYYYGMDVWGOGTTVTVSSASTKGPSVFPLAPSSKS

TSGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTV

PSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQY

NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:39

atgggcaccccggcgcagctgctgtttctgctgctgctgtggctgccggataccaccggcgatattcagatgacccagagccc ggcgaccctgagcctgagcccgggcgaaaccgcgaccctgagctgccgcgcgagccagagcgtgagctattatctggcgt ggtatcagcagaaaccgggccaggcgccgcgcctgctgatttatgatacctttaaccgcgcgaccggcattccggcgcgcttt agcggcagcggcagcggcaccgattttaccctgaccattagcagcctggaagcggaagattttgcggtgtattattgccagca gtttaacagctatccgcgcacctttggccagggcaccaaactggaaattaaacgtacggtggccgctccctccgtctttatctttc ctccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtg caatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattca ctgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagt tcacctgtgaccaaatcctttaacagaggtgagtgc SEP ID NO:40

MGTPAOLLFLLLLWLPDTTGDIOMTOSPATLSLSPGETATLSCRASOSVSYYLA

WYOOKPGOAPRLLIYDTFNRATGIPARFSGSGSGTDFTLTISSLEAEDFA YCO

OFNSYPRTFGOGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

DX 10-13 IgGl

Heavy chain

SEP ID NO:41

atgggctttggtctgagttggttgttcctggtcgctattctcaaaggtgtccagtgcgaagtacagttgttggagtcaggcggcgg gctcgtgcagcccggaggcagcctccggctgtcctgcgcagcttccgggttcacatttagtgactacaccatgaactgggtgc gacaagcaccaggcaagggacttgaatgggtttctagtatcgttcctagcggtggctggaccacctatgccgactccgccaag ggaaggttcactatctctagagacaattctaagaacacactgtaccttcagatgaatagcctgcgcgctgaggatacagccgtgt actattgtgcacgtgattggaaatactcttcatcctggtattggtgggattattgggggcagggaactctggtgactgtgtcaagc gctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctg gtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgt gctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaac gtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccct gccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggaccc ctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggt gcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcacca ggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaa ggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccct gacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaag accacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcaggg caacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO: 42

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSDYTM N WVRO APGKGLE WVS SI VP S GG WTTY AD S AKGRFTI SRDNS K TLYLQMNSLR

AEDTAVYYCARDWKYSSSWYWWDYWGOGTLVTVSSASTKGPSVFPLAPSSKS

TSGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTV

PSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTC A^VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY

TLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDGSF

FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:43

atgggcactccagcacagctgctgtttttgctcctcctctggctgcccgatacaacaggcgatatccagatgacacagagtcca agtaccttgtccgcctccgtgggggaccgcgtcacaatcacctgccgggcaagtcaaaggatcgggagctggctggcctggt accaacagcgacctggtaaagctcctaagcttctgatatatggagccagcaccctggcttcaggcgtgcccagcagattcagc ggatctggctctgggaccgaattcactttgacaatttcttccctgcagcccgacgactttgccacttattactgtcagcaggctaac tcattcccccttacctttggtggcggaactaaagttgagattaagcgtacggtggccgctccctccgtctttatctttcctccaagc gacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgcaatggaa agtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcactgtccag caccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagttcacctgt gaccaaatcctttaacagaggtgagtgc

SEP ID NO: 44

MGTPAOLLFLLLLWLPDTTGDIOMTOSPSTLSASVGDRVTITCRASORIGSWLA

WYOORPGKAPKLLIYGASTLASGVPSRFSGSGSGTEFTLTISSLOPDDFATYYCO

OANSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT

HQGLSSPVTKSFNRGEC

DX 10-15 IgGl

Heavy chain SEP ID NO:45

atgggctttggcctctcttggctgtttctggtagcaattcttaagggagttcagtgtgaagtacaacttctggaaagtggcggcggt ttggtccagcctggaggctctttgcggctgagctgcgctgcatccgggtttacattctctatgtatgacatgctctgggtcaggca ggccccaggtaagggcctggagtgggttagccgaattagcccctccggtgggcacacacattacgcagattcagtgaaagga cgcttcaccatctctcgtgataacagcaaaaacaccctgtatttgcagatgaatagtctcagagccgaggatacagctgtgtact actgcgctactgacatcgtggtggtggtcgccgccacctatctggacgccttcgacatctgggggcagggaactatggtgacc gtttcatccgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgg gctgcctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttc cctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctaca tctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacct gccctccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctc ccggacccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggc gtggaggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtg ctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagacc atctccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccag gtgtccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaa ctacaagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggca gcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctg gcaag

SEP ID NO:46

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSMYDM

LWVROAPGKGLEWVSRISPSGGHTHYADSVKGRFTISRDNSK TLYLQMNSLR

AEDTAVYYCATDIVVVVAATYLDAFDIWGQGTMVTVSSASTKGPSVFPLAPSS

KSTSGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSW

TVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF

LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREE

QY STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

WTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSD

GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Light chain

SEP ID NO:47

atgggctgggccctgcttctgcttacattgttgactcagggaacaggatcttgggcccagtatgagctgacccagcctccatccg ttagtgtcagccccggtcaaaccgcaacaatcacctgctttggctacgatctgtgggataaagacattagctggtatcagagaaa gcccggccagtctcctctgctcataatctaccaggataccaaaaggcccagcggcatccctgagcggttctccgggtcaaacc caggaaataccgccacactcaccattaatggcatccaaactatggacgaagctgattactattgtctcgtgtgggacaacgaca aggctgtgttcgggactggtactaaggtaacagtcctaggacagcctaaggccgctccttccgtgaccctgttccctccttcctc cgaggagctgcaggccaacaaggctaccctggtgtgcctggtgtccgacttctaccctggcgccgtgaccgtggcttggaag gccgacggctcccctgtgaaggtgggcgtggagaccaccaagccttccaagcagtccaacaacaagtacgccgcctcctcct acctgtccctgacccctgagcagtggaagtcccaccggtcctacagctgccgggtgacccacgagggctccaccgtggaaa agaccgtggcccctgccgagtgctcc

SEP ID NO:48

MGWALLLLTLLTOGTGSWAOYELTOPPSVSVSPGOTATITCFGYDLWDKDISW

YORKPGOSPLLIIYODTKRPSGIPERFSGSNPGNTATLTINGIOTMDEADYYCLV

WDNDKAVFGTGTKVTVLGOPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPG

AVTVAWKADGSPVKVGVETTKPSKQS KYAASSYLSLTPEQWKSHRSYSCR

VTHEGSTVEKTVAPAECS

DX 10-19 IgGl

Heavy chain SEQ ID NO:49

atgggcttcggcctgagttggttgtttctggtggccattctcaaaggggttcaatgtgaggtgcaactccttgagagcggtggcg gcttggtgcagcctggcggctcacttcggctgtcctgcgcagcatctggcttcacctttagtcagtacagcatgggatgggttag gcaggctcccggaaaagggctcgaatgggtatcttggatcaggtccagcggaggtgccacattttacgctgactctgtcaagg gacgattcaccatctctcgcgacaactcaaagaatactctgtatcttcagatgaacagcttgcgtgctgaagataccgccgtgtat tattgcgccagagtgggggcctattacggggactacgtcgattactggggtcagggtacactggtcactgtgtcctccgctagc actaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaag gactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgca gtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaac cacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctg cccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgagg tgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaa cgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactg gctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaa gggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgt ctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccaccc ctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgt tctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:50

MGFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSQYSM

GWVROAPGKGLEWVSWIRSSGGATFYADSVKGRFTISRDNSK TLYLOMNSLR

AEDTA YCARVGAYYGDYVDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSG

GT AALGCLVKD YFPEP VT VS W S G ALTS G VHTFP AVLQ S S GLYSLS S WT VP S S

SLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK

PKDTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQYNST

YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPP

SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:51

atgggcactccagctcagttgctgttcctccttcttctttggctcccagacactaccggtgacatccagatgacacagagtccttcc tctctctctgccagtatcggcgactctgttgctatcacatgcagggccagtcagtccatctccatacacttgaattggtaccaacag aaacccggcaaggcccccaagttcctgatttatgcagcaagcaagctgcagagcggggtcccctcacggttttctgggtcagt gtccggaactgatttcaccctgaccattagctcattgcagcctgaagatttcgctacatacttttgtcaacagacctatagcaaccc cagcacctttggcctgggaacaagactggagattaaacgtacggtggccgctccctccgtctttatctttcctccaagcgacgag cagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgcaatggaaagtgga taacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcactgtccagcaccct gacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagttcacctgtgaccaa atcctttaacagaggtgagtgc SEQ ID NO:52

MGTPAOLLFLLLLWLPDTTGDIOMTOSPSSLSASIGDSVAITCRASOSISIHLNWY

OOKPGKAPKFLIYAASKLOSGVPSRFSGSVSGTDFTLTISSLOPEDFATYFCOOTY

SNPSTFGLGTRLEIKRTVAAPSVFIFPPSDEOLKSGTASWCLL FYPREAKVO

WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEVTHQG

LSSPVTKSFNRGEC

DX 10-22 IgGl

Heavy chain

SEP ID NO:53

atgggctttggcctgagctggctgtttctggtggcgattctgaaaggcgtgcagtgcgaagtgcagctgctggaaagcggcgg cggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttacctttagcgattatccgatgcagtgg gtgcgccaggcgccgggcaaaggcctggaatgggtgagcagcatttatccgagcggcggccgcaccctgtatgcggatag cgcgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagat accgcggtgtattattgcgcgcgcgatgatgaagtgcgctattttgattggatttattattattatggcatggatgtgtggggccag ggcaccaccgtgaccgtgagcagcgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccgg cggcaccgccgctctgggctgcctggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgacc agcggcgtgcacaccttccctgccgtgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccc tgggcacccagacctacatctgcaacgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctg cgacaagacccacacctgccctccctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaa ggacaccctgatgatctcccggacccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttca attggtacgtggacggcgtggaggtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtgg tgtccgtgctgaccgtgctgcaccaggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgc ccccatcgaaaagaccatctccaaggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacga gctgaccaagaaccaggtgtccctgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacg gccagcctgagaacaactacaagaccacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtgg acaagtcccggtggcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtc cctgtccctgagccctggcaag

SEP ID NO:54 MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSDYPM

OWVROAPGKGLEWVSSIYPSGGRTLYADSAKGRFTISRDNSK TLYLQMNSLR

AEDTAVYYCARDDEVRYFDWIYYYYGMDVWGOGTTVTVS SASTKGP SVFPLA

PSSKSTSGGTAALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSS

WTVPSSSLGTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS

VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPR

EEQY STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QWTLPPSRDELTK QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:55

atgggcacacctgcccagttgcttttccttttgttgctctggttgcctgacaccactggcgacattcagatgacacagtcaccacttt ctctgcccgtgactcccggtgaaccagccagcatctcctgcagatctagccaaagtctgctccacaccaacggctacaactatg ttgattggtacctgcagaaacccggacagtcccctcagcttctcatttatctggggtctactcgggcatccggggtaccagatcg cttttcaggcagcggaagtgacaccgacttcaccctgaagatcaatagggtcgaggctgaggatgtgggactgtattactgtat gcaagctctccagacacccctgacatttgggggcggtaccaaagtggaaatcaagcgtacggtggccgctccctccgtctttat ctttcctccaagcgacgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaa agtgcaatggaaagtggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtactt attcactgtccagcaccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggct gagttcacctgtgaccaaatcctttaacagaggtgagtgc

SEQ ID NO:56

MGTPAOLLFLLLLWLPDTTGDIOMTOSPLSLPVTPGEPASISCRSSOSLLHTNGY

NYVDWYLQKPGOSPOLLIYLGSTRASGVPDRFSGSGSDTDFTLKINRVEAEDVG

LYYCMOALOTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL F

YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY

ACEVTHQGLSSPVTKSFNRGEC

DX 10-23 IgGl

Heavy chain SEP ID NO:57

atgggctttgggctgtcttggctcttcctcgtcgccatccttaagggtgttcagtgcgaagtgcagcttctcgagagcggcgggg gcctggtgcaacccggtggaagcctgagacttagttgcgccgcttccggcttcaccttctcttactatatgatgatgtgggtacga caggcacctggaaaagggttggagtgggtttcaagcattggaccatccggcggatttaccccctacgctgatagcgtcaaggg ccgcttcactatctctagggacaatagtaaaaacacactgtatctgcagatgaactctctgcgggcagaagacactgccgtgtac tattgtgctcgtgggcacttttggtcaggtttggattactggggtcagggcaccttggtgacagtgagttccgctagcactaaggg cccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaaggactacttc cctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgcagtcctccgg cctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaaccacaagcct tccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctgcccctgag ctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgaggtgacctgc gtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaacgctaaga ccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctgaacg gcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggccagc ctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgtctggtgaa gggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccacccctcctgtg ctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcctgc tccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:58

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSYYMM

MWVROAPGKGLEWVSSIGPSGGFTPYADSVKGRFTISRDNSK TLYLQMNSLR

AEDTA YCARGHFWSGLDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL

GTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTC\^/YVDVSHEDPEVKFNWYVDGVEVFINAKTKPREEQY STYR

WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain SEP ID NO:59

atgggcactccagctcaactgctctttctgctgcttctctggctgcccgataccaccggggatatccagatgactcagtccccttc tactttgtccgcctcagttggtgacagagtcaccatgacatgccgcgcaagccaaaatatctcccggtggttggcctggtatcaa cagaagcccggtaacgcacccaaacttctcatctacaaggcttctaccctgcagagcggcgtgccaactaggttcagtggcag cgggagtggcacagaattcaccttgaccattagttctcttcagcctgacgatttcgccacatattactgtcagcagtacgactcag acagcacatttggacagggaaccaaactggagattaagcgtacggtggccgctccctccgtctttatctttcctccaagcgacg agcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgcaatggaaagtg gataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcactgtccagcacc ctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagttcacctgtgacc aaatcctttaacagaggtgagtgc

SEP ID NO:60

MGTPAOLLFLLLLWLPDTTGDIOMTOSPSTLSASVGDRVTMTCRASONISRWLA

WYOOKPGNAPKLLIYKASTLOSGVPTRFSGSGSGTEFTLTISSLOPDDFATYYCO

OYDSDSTFGOGTKLEIKRTVAAPSVFIFPPSDEOLKSGTASWCLLNNFYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKWACEVTH

QGLSSPVTKSFNRGEC

DX 10-27 IgGl

Heavy chain SEP ID NO:61

atgggctttggcctctcctggctgtttcttgttgctattcttaagggcgtgcaatgtgaagtccagctccttgagtctggtggcggg ctggtgcagcccggcggaagcctgcgcctgagttgcgccgcttcaggtttcaccttttcattgtacccaatgcagtgggtgcga caagcacccggcaagggtttggaatgggtcagctcaatatactcctctggagggatgacaccttatgccgatagcgtgaaagg ccggttcactatcagtagggacaacagtaaaaataccttgtatctccagatgaactccctgagagcagaggacacagctgtcta ctattgcgtacgtgagggagtgtccagcgatgccttcgacatctggggacaggggactatggttaccgtgtcttctgctagcact aagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaagga ctacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgcagtc ctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaaccac aagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctgccc ctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgaggtga cctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaacgc taagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggctg aacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagggc cagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgtctggt gaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccacccctcct gtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttctcc tgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:62

MGFGLSWLFLVAILKGVQCEVQLLESGGGLVQPGGSLRLSCAASGFTFSLYPMQ

WVROAPGKGLEWVSSIYSSGGMTPYADSVKGRFTISRDNSK TLYLOMNSLRA

EDTA YCVREGVSSDAFDIWGOGTMVTVSSASTKGPSVFPLAPSSKSTSGGTA

ALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT

QTYICNWHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT

LMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQYNSTYRV

VSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQWTLPPSRD

ELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDGSFFLYSKL

TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:63

atgggcacacccgctcaactcctgttcctccttttgttgtggcttccagacacaaccggggatatccagatgactcagagtccctc ctctctcagcgcttcagtcggagatcgggttacaattacatgtagagcatcccaaggcatccgcaacgacctgggctggtttca gcagaagccaggaaaggcccctaaaaggctgatctacgcagcctcttccctgcagtctggtgtgcccagccgattcagtggat ctgggtccggtaccgagtttactcttaccattagctcactgcagcctgaagacttcgccagttactattgcttgcagcacataagct acccttatacctttggcgggggcactaaagtggagatcaagcgtacggtggccgctccctccgtctttatctttcctccaagcga cgagcagctgaagtctggcaccgcaagtgtggtgtgtctgctgaacaatttctaccccagggaagccaaagtgcaatggaaag tggataacgctctgcagtcaggaaattcccaggagagcgtcacagaacaagactctaaagatagtacttattcactgtccagca ccctgacactgtctaaggccgattatgagaaacacaaggtgtatgcctgtgaagtcactcatcaggggctgagttcacctgtga ccaaatcctttaacagaggtgagtgc SEQ ID NO:64

MGTPAOLLFLLLLWLPDTTGDIOMTOSPSSLSASVGDRVTITCRASOGIRNDLG

WFOOKPGKAPKRLIYAASSLOSGVPSRFSGSGSGTEFTLTISSLOPEDFASYYCLO

HISYPYTFGGGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASWCLL FYPREAK

VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH

QGLSSPVTKSFNRGEC

DX 10-28 IgGl

Heavy chain SEP ID NO:65

atgggcttcggtctctcttggctgtttctggttgctattcttaaaggcgtgcaatgcgaagtgcagttgctggagtcaggcggcgg actcgtccagcccggcggaagcctgcgtctgtcctgtgctgctagtgggttcacctttagcggttatgtaatgcagtgggtgcgc caagcacccggaaaggggctggaatgggtcagctggatcgggtcatctgggggttatacaagctacgcagacagtgtgaag ggcagattcacaatctcccgggataactccaaaaacaccctctacttgcagatgaattccttgcgagccgaggacactgccgttt actattgcgccaggactaggggcaattggaacccacctaataactggggacagggtacccttgtgacagtctcttctgctagca ctaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcctggtgaagg actacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccgtgctgcagt cctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaacgtgaacca caagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctccctgccctgcc cctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggacccctgaggtg acctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggaggtgcacaacg ctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcaccaggactggct gaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaaggg ccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccctgacctgtctg gtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaagaccacccctc ctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcagggcaacgtgttct cctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:66

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSGYVM OWVROAPGKGLEWVSWIGSSGGYTSYADSVKGRFTISRDNSK TLYLOMNSLR AEDTA YCARTRGNWNPPNNWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGG

TAALGCLVKDYFPEP VTVS WNSGALTS GVHTFP AVLQS SGLYSLS S WTVPS S SL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:67

atgggctgggccttgcttctgttgacattgcttacacaaggtactggtagttgggcccagtacgaactcacccagcctccctccg cctccggtactcccggacaacgcgtgactatcagctgttccggatcaaattccgacatagggagcaactacgtttattggtacca gcagctgcctggcaccgcacccaagctgctcatttatcgaaacaatcagcggccatcaggcgtccccgatagattcagcggct ctaagagcggcacaagtgcctctcttgcaatcagtgggctgaggtcagaggatgaggctgactatcactgcgctgcttgggac gattctctgtctggaccagtgtttggcggagggaccaaactcaccgtcctaggacagcctaaggccgctccttccgtgaccctg ttccctccttcctccgaggagctgcaggccaacaaggctaccctggtgtgcctggtgtccgacttctaccctggcgccgtgacc gtggcttggaaggccgacggctcccctgtgaaggtgggcgtggagaccaccaagccttccaagcagtccaacaacaagtac gccgcctcctcctacctgtccctgacccctgagcagtggaagtcccaccggtcctacagctgccgggtgacccacgagggct ccaccgtggaaaagaccgtggcccctgccgagtgctcc

SEP ID NO:68

MGWALLLLTLLTPGTGSWAPYELTPPPSASGTPGPRVTISCSGSNSDIGSNYVY

WYPPLPGTAPKLLIYRNNPRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYHC

AAWDDSLSGPVFGGGTKLTVLGPPKAAPSVTLFPPSSEELPANKATLVCLVSDF

YPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYS

CRVTHEGSTVEKTVAPAECS

DX 10-30 IgGl

Heavy chain SEP ID NP:69

atgggctttggcctgagctggctgtttctggtggcgattctgaaaggcgtgcagtgcgaagtgcagctgctggaaagcggcgg cggcctggtgcagccgggcggcagcctgcgcctgagctgcgcggcgagcggctttacctttagctattatatgatggcgtgg gtgcgccaggcgccgggcaaaggcctggaatgggtgagcggcattggcccgagcggcggcagcaccatttatgcggatag cgtgaaaggccgctttaccattagccgcgataacagcaaaaacaccctgtatctgcagatgaacagcctgcgcgcggaagat accgcggtgtattattgcgcgcgcgaacagctgtggagccattttgattattggggccagggcaccctggtgaccgtgagcag cgctagcactaagggcccttccgtgttccctctggccccttcctccaagtccacctccggcggcaccgccgctctgggctgcct ggtgaaggactacttccctgagcctgtgaccgtgagctggaactccggcgctctgaccagcggcgtgcacaccttccctgccg tgctgcagtcctccggcctgtactccctgtcctccgtggtgacagtgccttcctcctccctgggcacccagacctacatctgcaa cgtgaaccacaagccttccaacaccaaggtggacaagaaggtggagcctaagtcctgcgacaagacccacacctgccctcc ctgccctgcccctgagctgctgggcggaccctccgtgttcctgttccctcctaagcctaaggacaccctgatgatctcccggac ccctgaggtgacctgcgtggtggtggacgtgtcccacgaggatcctgaggtgaagttcaattggtacgtggacggcgtggag gtgcacaacgctaagaccaagcctcgggaggagcagtacaactccacctaccgggtggtgtccgtgctgaccgtgctgcacc aggactggctgaacggcaaggaatacaagtgcaaggtctccaacaaggccctgcctgcccccatcgaaaagaccatctcca aggccaagggccagcctcgcgagcctcaggtgtacaccctgcctccctcccgggacgagctgaccaagaaccaggtgtccc tgacctgtctggtgaagggcttctacccttccgacatcgccgtggagtgggagtccaacggccagcctgagaacaactacaag accacccctcctgtgctggactccgacggctccttcttcctgtactccaagctgaccgtggacaagtcccggtggcagcaggg caacgtgttctcctgctccgtgatgcacgaggccctgcacaaccactacacccagaagtccctgtccctgagccctggcaag

SEP ID NO:70

MGFGLSWLFLVAILKGVOCEVOLLESGGGLVQPGGSLRLSCAASGFTFSYYMM

AWVROAPGKGLEWVSGIGPSGGSTIYADSVKGRFTISRDNSK TLYLQMNSLR

AEDTA YCAREQLWSHFDYWGOGTLVTVSSASTKGPSVFPLAPSSKSTSGGT

AALGCLVKDYFPEPVTVSW SGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL

GTQTYICNV HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

DTLMISRTPEVTCVWDVSHEDPEVKFN VDGVEVHNAKTKPREEQYNSTYR

WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

DELTK QVSLTCLVKGFYPSDIAVEWESNGQPE YKTTPPVLDSDGSFFLYSK

LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Light chain

SEP ID NO:71

atgggctgggcactgcttctcctgaccctccttacacagggcactgggagttgggctcagagcgccttgacccaacctgcttct gtatctggaagccctggacagtccattacaatcagctgtaccggtactagcagtgatgtgggtgcatataattacgtttcttggtat cagcaacaccccggcaaggtccccaaattgatgatatacgaggtgtcaaacaggccatctggggtctccaatagattctcagg atccaaatcagggaacacagctagcctgacaatcagtggcctgcaggccgaagacgaggccgactactactgcaactcctat actacctctgccacactggtgtttggcggcggaaccaagctcactgtcctaggacagcctaaggccgctccttccgtgaccctg ttccctccttcctccgaggagctgcaggccaacaaggctaccctggtgtgcctggtgtccgacttctaccctggcgccgtgacc gtggcttggaaggccgacggctcccctgtgaaggtgggcgtggagaccaccaagccttccaagcagtccaacaacaagtac gccgcctcctcctacctgtccctgacccctgagcagtggaagtcccaccggtcctacagctgccgggtgacccacgagggct ccaccgtggaaaagaccgtggcccctgccgagtgctcc

SEP ID NO:72

MGWALLLLTLLTOGTGSWAOSALTQPASVSGSPGOSITISCTGTSSDVGAY YV

SWYOOHPGKVPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLOAEDEADYY

CNSYTTSATLVFGGGTKLTVLGOPKAAPSVTLFPPSSEELOANKATLVCLVSDF

YPGAVTVAWKADGSPVKVGVETTKPSKQS KYAASSYLSLTPEQWKSHRSYS

CRVTHEGSTVEKTVAPAECS

CDR sequences

DX 1-4 IgGl

Heavy chain

SEP ID NO:73

TYEMD (CDR1)

SEP ID NO:74

VISPSGGWTGYADSVKG (CDR2) SEP ID NO:75

TPGYDSSGYYGSWFDY (CDR3) Light chain SEP ID NO:76

KSSQSVLYSSNNKNYLA (CDRl)

SEP ID NO:77

WASTRES (CDR2)

SEP ID NP:78

QQYGSSPIT (CDR3)

DX 1-6 IgGl

Heavy chain

SEP ID NP:79

EYAMM (CDRl)

SEP ID NP:80

SIWPSGGYTSYADSVKG (CDR2) SEP ID NP:81

DYYGSGSYYGVDY (CDR3)

Light chain

SEP ID NP:82

RANHGIRNDLG (CDRl)

SEP ID NP:83

GASSLQS (CDR2)

SEP ID NP: 183 GASSLXS (CDR2), wherein X is Q or E

SEP ID NO: 84

LQEYSFPLT (CDR3)

DX 1-10 IgGl

Heavy chain SEP ID NO: 85

WYTMFWVRQAPGKGLEWVS (CDRl) SEQ SEP ID NP:86

SIYPSGGITKYADSVKG (CDR2) SEQ ID NP:87

IYC S GGS CYGED AFDI (CDR3)

Light chain

SEP ID NP:88

RASQSISSYLN (CDRl)

SEQ ID NP:89

AASTLQS (CDR2)

SEP ID NP:90

QQSYSTPPT (CDR3)

DX 1-17 IgGl

Heavy chain SEP ID NO:91

YYHML (CDRl)

SEP ID NO:92

WISSSGGYTEYADSVKG (CDR2)

SEP ID NP:93

DNDWASDY (CDR3)

Light chain

SEP ID NP:94

RSSQSLLHSSGYTYLD (CDRl) SEP ID NP: 184

RSSQSLLHSSGYTYLX (CDRl), wherein X

SEP ID NP:95

LRSNRAS (CDR2)

SEP ID NP:96

MQGTQTPYT (CDR3)

DX 1-18 IgGl

Heavy chain

SEP ID NP:97

WYVML (CDRl)

SEP ID NP:98 SISPSGGYTNYADSVKG (CDR2)

SEP ID NO:99

HGEYSSSWFDY (CDR3)

Light chain

SEP ID NO: 100

TGTSSNVGNYNLVS (CDRl)

SEP ID NO: 101

EDNKRPS (CDR2)

SEP ID NO: 102

CSYAGGGTWV (CDR3)

DX 10-4 IgGl

Heavy chain

SEP ID NP: 103

RYSMI (CDRl)

SEP ID NP: 104

YISSSGGWTFYADSVKG (CDR2)

SEP ID NP: 105

DWGGPSDY (CDR3)

Light chain SEP ID NO: 106

RASQSISSYLN (CDRl)

SEP ID NO: 107

AASSLQS (CDR2)

SEP ID NP: 108

QQSYSTPDT (CDR3)

DX 10-6 IgGl

Heavy chain

SEP ID NP: 109

HYTML (CDRl)

SEP ID NO: 110

SISPSGGETQYADSVKG (CDR2) SEP ID NP: 11 1

WPGGYYDSSGYYAFDY (CDR3)

Light chain

SEP ID NO: 112

TGTSSDVGAYNYVS (CDRl)

SEP ID NO: 113

EVSNRPS (CDR2) SEP ID NO: 114

NSYTTSATLV (CDR3)

DX 10-9 IgGl

Heavy chain

SEP ID NO: 115

PYGMV (CDRl)

SEP ID NO: 116

YISPSGGYTDYADSVKG (CDR2)

SEP ID NO: 117

DLGDIHDY (CDR3)

Light chain

SEP ID NO: 118

RASQSVSRSSLA (CDRl)

SEP ID NO: 119

GASSRAT (CDR2)

SEP ID NP: 120

QQYGNSPGGT (CDR3)

DX 10-11 IgGl

Heavy chain SEP ID NO: 121

KYPMQ (CDR1)

SEP ID NO: 122

SIYSSGDWTAYADSVKG (CDR2) SEP ID NP: 123

AAWYCTGGICSYSYYMDV (CDR3)

Light chain

SEP ID NP: 124

RASRDVSSWLA (CDR1)

SEP ID NP: 125

SASGLQS (CDR2)

SEP ID NP: 126

QQAKTFPLT (CDR3)

DX 10-12 IgGl

Heavy chain

SEP ID NP: 127

HYNMS (CDR1)

SEP ID NP: 128

SIWPSGGHTWYADSVKG (CDR2)

SEP ID NP: 129

PDPQFPYYYYGMDV (CDR3) Light chain

SEP ID NO: 130

RASQSVSYYLA (CDRl)

SEP ID NO: 131

DTFNRAT (CDR2)

SEP ID NP: 132

QQFNSYPRT (CDR3)

DX 10-13 IgGl

Heavy chain

SEP ID NP: 133

DYTMN (CDRl)

SEP ID NP: 134

SIVPSGGWTTYADSAKG (CDR2) SEP ID NP: 135

DWKYSSSWYWWDY (CDR3)

Light chain

SEP ID NP: 136

RASQRIGSWLA (CDRl)

SEP ID NP: 137

GASTLAS (CDR2) SEP ID NO: 138

QQANSFPLT (CDR3)

DX 10-15 IgGl

Heavy chain

SEP ID NO: 139

MYDML (CDRl)

SEP ID NP: 140

RISPSGGHTHYADSVKG (CDR2) SEP ID NO: 141

DIVVWAATYLDAFDI (CDR3)

Light chain

SEP ID NP: 142

FGYDLWDKDIS (CDRl)

SEP ID NP: 143

QDTKRPS (CDR2)

SEP ID NP: 144

LVWDNDKAV (CDR3)

DX 10-19 IgGl

Heavy chain

SEP ID NP: 145

QYSMG (CDRl) SEP ID NO: 146

WIRSSGGATFYADSVKG (CDR2)

SEP ID NO: 147

VGAYYGDYVDY (CDR3)

Light chain

SEP ID NP: 148

RASQSISIHLN (CDRl)

SEP ID NP: 149

AASKLQS (CDR2)

SEP ID NP: 150

QQTYSNPST (CDR3)

DX 10-22 IgGl

Heavy chain

SEP ID NO: 151

DYPMQ (CDR1)

SEP ID NP: 152

SIYPSGGRTLYADSAKG (CDR2) SEP ID NP: 153

DDEVRYFDWIYYYYGMDV (CDR3)

Light chain

SEP ID NP: 154 RSSQSLLHTNGYNYVD (CDRl)

SEP ID NO: 155

LGSTRAS (CDR2)

SEP ID NO: 156

MQALQTPLT (CDR3)

DX 10-23 IgGl

Heavy chain

SEP ID NP: 157

YYMMM (CDRl)

SEP ID NP: 158

SIGPSGGFTPYADSVKG (CDR2) SEP ID NP: 159

GHFWSGLDY (CDR3)

Light chain

SEQ ID NC: 160

RASQNISRWLA (CDRl)

SEQ ID NO: 161

KASTLQS (CDR2)

SEQ ID NC: 162

QQYDSDST (CDR3)

DX 10-27 IgGl Heavy chain

SEP ID NO: 163

LYPMQ (CDRl)

SEP ID NP: 164

SIYSSGGMTPYADSVKG (CDR2)

SEP ID NP: 165

EGVSSDAFDI (CDR3)

Light chain SEP ID NP: 166

RASQGIRNDLG (CDRl)

SEP ID NP: 167

AASSLQS (CDR2)

SEP ID NP: 168

LQHISYPYT (CDR3)

DX 10-28 IgGl

Heavy chain

SEP ID NP: 169

GYVMQ (CDRl)

SEP ID NP: 170

WIGSSGGYTSYADSVKG (CDR2)

SEP ID NO: 171

TRGNWNPPNN (CDR3) Light chain

SEP ID NO: 172

SGSNSDIGSNYW (CDRl)

SEP ID NO: 173

RNNQRPS (CDR2)

SEP ID NP: 174

AAWDDSLSGPV (CDR3)

DX 10-30 IgGl

Heavy chain

SEP ID NP: 175

YYMMA (CDRl)

SEP ID NP: 176

GIGPSGGSTIYADSVKG (CDR2)

SEP ID NP: 177

EQLWSHFDY (CDR3)

Light chain

SEP ID NP: 178

TGTSSDVGAYNYVS (CDRl)

SEP ID NP: 179

5 EVSNRPS (CDR2) SEP ID NO: 180

NSYTTSATLV (CDR3)

SEP ID NP: 185 (EGFR)

10 20 30 40 50 60

MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCEV

70 80 90 100 110 120

VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQI IRGN MYYENSYALA

130 140 150 160 170 180

VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF

190 200 210 220 230 240

QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC

250 260 270 280 290 300

TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV

310 320 330 340 350 360

VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK

370 380 390 400 410 420

NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF

430 440 450 460 470 480

ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL

490 500 510 520 530 540

FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDKCN

550 560 570 580 590 600

LLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM

610 620 630 640 650 660

GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLW

670 680 690 700 710 720

ALGIGLFMRR RHIVRKRTLR RLLQERELVE PLTPSGEAPN QALLRILKET EFKKIKVLGS

730 740 750 760 770 780

GAFGTVYKGL WIPEGEKVKI PVAIKELREA TSPKANKEIL DEAYVMASVD NPHVCRLLGI

790 800 810 820 830 840

CLTSTVQLIT QLMPFGCLLD YVREHKDNIG SQYLLNWCVQ IAKGMNYLED RRLVHRDLAA

850 860 870 880 890 900

RNVLVKTPQH VKITDFGLAK LLFAEEKEYH AEFFKCPIKW MALESILHRI YTHQSDVWSY

910 920 930 940 950 960

GVTVWELMTF GSKPYDGIPA SEISSILEKG ERLPQPPICT IDVYMIMVKC WMIDADSRPK

970 980 990 1000 1010 1020 FRELI IEFSK MARDPQRYLV IQGDERMHLP SPTDSNFYRA LMDEEDMDDV VDADEYLIPQ

1030 1040 1050 1060 1070 1080

QGFFSSPSTS RTPLLS SLSA TSNNSTVACI DRNGLQSCPI KEDSFLQRYS SDPTGALTED

1090 1000 1110 1120 1130 1140

SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPELYN

1150 1160 1170 1180 1190 1200

TVQPTCVNST FDSPAHWAQK GSHQI SLDNP DYQQDFFPKE AKPNGIFKGS TAENAEYLRV

1210

APQSSEFIGA

Claims

What is claimed is:

1. An isolated antibody that binds to EGFR, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, and 70.

2. An isolated antibody that binds to EGFR, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, and 72.

3. An isolated antibody that binds to EGFR, comprising a heavy and light chain variable region having an amino acid sequence selected from the group consisting of:

(a) SEQ ID NOs: 2 and 4, respectively;

(b) SEQ ID NOs: 6 and 8, respectively, or SEQ ID NOs:6 and 181, respectively;

(c) SEQ ID NOs: 10 and 12, respectively;

(d) SEQ ID NOs: 14 and 16, respectively, or SEQ ID NOs: 14 and 182, respectively;

(e) SEQ ID NOs: 18 and 20, respectively;

(f) SEQ ID NOs: 22 and 24, respectively;

(g) SEQ ID NOs: 26 and 28, respectively;

(h) SEQ ID NOs: 30 and 32, respectively;

(i) SEQ ID NOs: 34 and 36, respectively;

G) SEQ ID NOs: 38 and 40, respectively;

(k) SEQ ID NOs: 42 and 44, respectively;

(1) SEQ ID NOs: 46 and 48, respectively;

(m) SEQ ID NOs: 50 and 52, respectively;

(n) SEQ ID NOs: 54 and 56, respectively;

(o) SEQ ID NOs: 58 and 60, respectively;

(p) SEQ ID NOs: 62 and 64, respectively;

(q) SEQ ID NOs: 66 and 68, respectively; and

(r) SEQ ID NOs: 70 and 72, respectively.

4. An isolated antibody which binds EGFR, wherein the heavy and light chain CDR3, CDR2, and CDRl, sequences are selected from the group consisting of:

(b) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 79, 80, and 81 , respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 82, 83, and 84, respectively, or a light chain CDR3, CDR2, and CDRl sequence as shown in SEQ ID NOs.: 82, 183, and 84, respectively;

(d) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 91 , 92, and 93, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 94, 95, and 96, respectively, or a light chain CDR3, CDR2, and CDRl sequence as shown in SEQ ID NOs.: 184, 95, and 96, respectively;;

(e) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 97, 98, and 99, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 100, 101, and 102, respectively;

(h) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 115, 116, and 1 17, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 1 18, 1 19, 120, respectively; (i) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 121 , 122, and 123, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 124, 125, and 126, respectively;

(n) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 151 , 152, and 153, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 154, 155, and 156, respectively;

(o) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 157, 158, and 159, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 160, 161 , and 162, respectively;

(q) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 169, 170, and 171, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 172, 173, and 174, respectively; and

(r) heavy chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 175, 176, and 177, respectively, and light chain CDR3, CDR2, and CDRl sequences as shown in SEQ ID NOs: 178,179, and 180, respectively.

5. An isolated antibody that competes for binding to EGFR with the antibody of any one of the preceding claims.

6. An isolated antibody of any one of the preceding claims, wherein the antibody is human, chimeric, or humanized.

7. An isolated antibody of any one of the preceding claims, wherein the antibody is selected from the group consisting of a bispecific, immunoconjugate, Fab, Fab'2, ScFv, affibody, avimer, nanobody, and a domain antibody.

8. The composition of any of the preceeding claims, wherein the antibodies bind to EGFR with a K_D of at least 1 OOnm.