Dual-epitope tetravalent antibody targeting EGFR

Abstract

A dual-epitope tetravalent antibody having binding affinity for at least two epitopes of EGFR. The antibody comprises an antibody scaffold and an scFv domain linked to the antibody scaffold. The two epitopes of EGFR include an EGFR wild-type (EGFRwt) epitope and an EGFRvIII epitope. The antibody has stronger binding affinity for the EGFRvIII epitope than for the EGFRwt epitope.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. 119(e) of the filing date of U.S. Provisional Application No. 63 / 402,945, filed August 31, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Technical Field FIELD OF THE DISCLOSURE The present disclosure relates generally to the field of antibody cancer therapeutics, and more particularly to dual epitope tetravalent antibodies. [Background technology]

[0003] Glioblastoma multiforme is an aggressive cancer that accounts for the majority of malignant tumors that occur in the brain, and has a poor prognosis with an average survival time of 14 to 15 months after diagnosis. 1 The majority of glioblastoma cases (34-63%) involve amplification of the EGFR gene. 2 In addition to overexpression of wild-type EGFR protein, mutant EGFR forms occur in 63–75% of cases, the most common of which is called EGFRvIII, occurring in 25–64% of cases. 2 Structurally, the EGFRvIII mutation results in a deletion of 267 residues in the extracellular domain of the protein, and functionally, it may result in inefficient receptor endocytosis, thereby inducing constitutive signaling. 2 These changes lead to increased proliferation, increased angiogenesis, and decreased apoptosis in EGFRvIII-expressing tumors. 2 In addition to their association with glioblastoma multiforme, wild-type EGFR and mutant EGFRvIII may also be overexpressed in other solid tumors, including ovarian, breast, and lung cancers. 2 .

[0004] Several antibodies targeting EGFR, including cetuximab, panitumumab, and necitumumab, have been approved by the FDA for the treatment of epithelial tumors. Nimotuzumab is also approved in some countries for the treatment of solid tumors. The lower affinity of nimotuzumab for EGFR compared with cetuximab or panitumumab may explain its reduced skin toxicity. 3 Meanwhile, antibody-based therapies targeting specifically mutant EGFRvIII are being evaluated in clinical trials, including the monoclonal antibody depatuxizumab (Phase I) and the antibody-drug conjugate depatuxizumab-mafodotin (Phase III). Notably, a Phase III trial of an ADC targeting EGFRvIII was terminated after failing to meet its primary endpoint of overall survival in newly diagnosed glioblastoma patients. 4 T cell engagers targeting EGFRvIII and CD3 are also being evaluated in preclinical studies, further strengthening the promise of EGFRvIII as a therapeutic target. 5,6 Although EGFRvIII-targeted therapies will not be available anytime soon, no single therapeutic agent simultaneously targets tumor cells expressing oncogenic EGFR, including both overexpressed (wt) and mutant EGFR (vIII).

[0005] A potential drawback of cetuximab and ABT806-derived antibodies is that the variable regions of these antibodies originate from mice and maintain non-human sequences. It has been shown that chimeric antibodies may have increased immunogenic potential when compared to humanized or human antibodies. 7 On the other hand, humanization not only reduces immunogenicity but also increases antibody stability by better matching the variable and constant regions. 8 .

[0006] Cetuximab's high affinity for its antigen, EGFR, means that it may have on-target and off-tumor binding. As a result, toxicities such as severe skin irritation are often observed. On the other hand, nimotuzumab, which binds to EGFR with lower affinity, is thought to have fewer of these unwanted side effects. 9Therefore, it may be beneficial to reduce the affinity of an antibody such as cetuximab to reduce binding to low-expressing healthy tissues and thereby increase the therapeutic index while maintaining its antigen specificity. For bispecific antibodies, weakening the affinity may be useful to balance the relative strengths of the two binding domains and effectively optimize the overall specificity of the antibody. Summary of the Invention

[0007] The following summary is for illustrative purposes only and is not intended to be in any way limiting. In addition to the exemplary aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

[0008] This disclosure provides dual epitope tetravalent antibodies that target at least two epitopes of EGFR, as well as methods for making and using the antibodies.

[0009] In one aspect, the present application provides a dual epitope tetravalent antibody having binding affinity for at least two epitopes of EGFR. The antibody may comprise an antibody scaffold comprising an antibody light chain having an antibody light chain variable (VL) domain and an antibody heavy chain having an antibody heavy chain variable (VH) domain, wherein the antibody VL domain and the antibody VH domain form a Fab region. The antibody may further comprise an scFv domain having an scFv light chain variable (VL) domain and an scFv heavy chain variable (VH) domain, wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain via an interdomain linker.

[0010] In one embodiment, each scFv domain has the structural order N-terminus-VH-linker-VL-C-terminus, or N-terminus-VL-linker-VH-C-terminus. In one embodiment, the linker comprises a flexible GS linker having about 20 amino acids. In one embodiment, the linker comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, where m is an integer of at least 3. In one embodiment, m is 4.

[0011] In one embodiment, the interdomain linker comprises from about 10 to about 30 amino acids. In one embodiment, the interdomain linker comprises the amino acid sequence (Gly-Gly-Gly-Gly-Ser)m, where m is an integer of at least 2. In one embodiment, m is an integer from 2 to 6.

[0012] In one embodiment, the two epitopes of EGFR include an EGFR wild-type (EGFRwt) epitope and an EGFRvIII epitope, and the dual epitope tetravalent antibody has stronger binding affinity for the EGFRvIII epitope than for the EGFRwt epitope. In one embodiment, the antibody has binding affinity for the EGFRwt epitope at a first KD and has binding affinity for the EGFRvIII epitope at a second KD, the first KD being higher than the second KD.

[0013] In one embodiment, the first KD is not less than 1E-11 M and the second KD is not more than 1E-06 M. In one embodiment, the first KD is between 1E-10 M and 1E-06 M and the second KD is between 1E-11 M and 1E-07 M. In one embodiment, the first KD is 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, or 70-fold stronger than the second KD. In one embodiment, the first KD is 1.1-fold to 1000-fold stronger than the second KD.

[0014] In one embodiment, the antibody has a first ADCC EC50 against cells expressing EGFRwt only and a second ADCC EC50 against cells expressing EGFRwt and EGFRvIII, wherein the first ADCC EC50 is greater than the second ADCC EC50. In one embodiment, the first ADCC EC50 is 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or greater than the second ADCC EC50.

[0015] In one embodiment, the EGFRwt epitope comprises an epitope with binding affinity for cetuximab. In one embodiment, the EGFRvIII epitope comprises an epitope with binding affinity for ABT-806.

[0016] In one embodiment, the scFv domain of the dual epitope tetravalent antibody has binding affinity for the EGFRwt epitope and the Fab domain has binding affinity for the EGFRvIII epitope, hi one embodiment, the scFv domain has binding affinity for the EGFRvIII epitope and the Fab domain has binding affinity for the EGFRwt epitope.

[0017] In one embodiment, the scFv domain is linked to a dual epitope tetravalent antibody light chain at its C-terminus. In one embodiment, the scFv domain is linked to an antibody light chain at its N-terminus. In one embodiment, the scFv domain is linked to an antibody heavy chain at its C-terminus. In one embodiment, the scFv domain is linked to an antibody heavy chain at its N-terminus.

[0018] In one embodiment, the antibody scaffold may be derived from cetuximab, humanized cetuximab, desaturated cetuximab, or humanized desaturated cetuximab (i.e., a scaffold derived from cetuximab), or nimotuzumab, and the scFv domain may comprise a binding domain derived from ABT-806 or humanized ABT-806.

[0019] Alternatively, the antibody scaffold may be derived from ABT-806 or humanized ABT-806, and the scFv domain may comprise the binding domain from cetuximab, humanized cetuximab, desaturated cetuximab, or humanized desaturated cetuximab, or nimotuzumab.

[0020] In one embodiment, the humanized ABT-806 is ABT-806 V1, V2, V3, V4, V5, V6, V7, V8, or V9.

[0021] Cetuximab-based dual epitope tetravalent antibody In one embodiment, the present application provides a dual epitope tetravalent antibody in which the antibody scaffold is derived from cetuximab, humanized cetuximab, de-matured cetuximab, or humanized de-matured cetuximab (i.e., a scaffold derived from cetuximab). In one embodiment, the scFv domain comprises a binding domain derived from the variable region of ABT-806 or humanized ABT-806.

[0022] In one embodiment, the antibody backbone comprises cetuximab dematuration mutations (sequential numbering) selected from VH-Y101, VH-Y102, VH-D103, VH-Y104, VL-N92, or a combination thereof. In one embodiment, the cetuximab dematuration mutations (sequential numbering) comprise VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL-N92F, VL-N92K, or a combination thereof.

[0023] In one embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 1, 13, 79, 97, 101, 103, 105, 107, 123, 127, 129, 131, or 133. In one embodiment, the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 3, 81, 83, 99, 109, 125, or 135.

[0024] In one embodiment, the antibody scaffold comprises humanized cetuximab. In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 237. In one embodiment, the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 239.

[0025] In one embodiment, the antibody scaffold comprises de-matured cetuximab. In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 247, 251, 253, 259, 261, 263, 265, or 267. In one embodiment, the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 243 or 269.

[0026] In one embodiment, the antibody scaffold comprises humanized de-matured cetuximab. In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 245, 249, 255, or 257. In one embodiment, the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 241.

[0027] In one embodiment, the humanized ABT-806 is ABT-806 V1, V2, V3, V4, V5, V6, V7, V8, or V9.

[0028] In one embodiment, the scFv domain comprises the variable regions of ABT-806 or humanized ABT-806.

[0029] In one embodiment, the scFv VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 209, 225, 229, or 233. In one embodiment, the scFv VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 211, 227, 231, or 235.

[0030] In one embodiment, an antibody VH domain comprises three complementarity determining regions (CDRs) of SEQ ID NO: 201, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267. In one embodiment, an antibody VL domain comprises three CDRs of SEQ ID NO: 203, 239, 241, 243, or 269. In one embodiment, an scFv VH domain comprises three CDRs of SEQ ID NO: 209. In one embodiment, an scFv VL domain comprises three CDRs of SEQ ID NO: 211.

[0031] In one embodiment, the dual epitope tetravalent antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 1, 3, 13, 79, 81, 83, 97, 99, 101, 103, 105, 107, 109, 123, 125, 127, 129, 131, 133, or 135.

[0032] ABT-806-based dual epitope tetravalent antibody In one embodiment, the present application provides a dual epitope tetravalent antibody, wherein the antibody scaffold is derived from ABT-806 or humanized ABT-806, and the scFv domain comprises binding domains derived from the variable regions of cetuximab, humanized cetuximab, desaturated cetuximab, or humanized desaturated cetuximab. In one embodiment, the scFv domain comprises a binding domain derived from nimotuzumab.

[0033] In one embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 9, 17, 19, 21, 23, 25, 27, 29, 31, 75, 93, 113, 115, 117, 119, 121, 137, 139, 141, 143, 145, 147, or 179.

[0034] In one embodiment, the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 11, 33, 35, 37, 39, 41, 43, 77, 91, 95, or 111.

[0035] In one embodiment, the antibody scaffold comprises ABT-806, and the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 209. In one embodiment, the VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 211.

[0036] In one embodiment, the antibody scaffold comprises humanized ABT-806. In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 225, 229, or 233. In one embodiment, the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 227, 231, or 235.

[0037] In one embodiment, the scFv VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 201, 205, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267.

[0038] In one embodiment, the scFv VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 203, 207, 239, 241, 243, or 269.

[0039] In one embodiment, the scFv domain comprises a binding domain derived from the variable region of de-matured cetuximab or humanized de-matured cetuximab. In one embodiment, the de-matured cetuximab or humanized de-matured cetuximab comprises cetuximab de-maturation mutations (sequential numbering) selected from Y101, Y102, D103, Y104, N92, and combinations thereof. In one embodiment, the cetuximab de-maturation mutations (sequential numbering) comprise VH-Y101, VH-Y102, VH-D103, VH-Y104, VL-N92, or combinations thereof. In one embodiment, the cetuximab dematuration mutations (sequential numbering) include VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL-N92F, VL-N92K, or a combination thereof.

[0040] In one embodiment, a dual epitope tetravalent antibody VH domain comprises the three complementarity determining regions (CDRs) of SEQ ID NO: 209. In one embodiment, a dual epitope tetravalent antibody VL domain comprises the three CDRs of SEQ ID NO: 211. In one embodiment, an scFv VH domain comprises the three CDRs of SEQ ID NO: 201, 205, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267. In one embodiment, an scFv VL domain comprises the three CDRs of SEQ ID NO: 203, 207, 239, 241, 243, or 269.

[0041] In one embodiment, the dual epitope tetravalent antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 37, 41, 43, 75, 77, 91, 93, 95, 111, 113, 115, 117, 119, 121, 137, 139, 141, 143, 145, 147, or 179.

[0042] Nimotuzumab backbone antibody In one embodiment, the present application provides a dual epitope tetravalent antibody, wherein the antibody scaffold comprises nimotuzumab and the scFv domain comprises binding domains derived from the variable regions of ABT-806 or humanized ABT-806. In one embodiment, the scFv domain comprises the variable regions of ABT-806.

[0043] In one embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 5, 15, 45, 47, 49, 51, 53, 55, or 85.

[0044] In one embodiment, the antibody light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 7, 57, 59, 61, 63, 65, 67, 69, 71, or 73.

[0045] In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO:205.

[0046] In one embodiment, the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO:207.

[0047] In one embodiment, the scFv VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 209. In one embodiment, the scFv VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 211.

[0048] In one embodiment, an antibody VH domain comprises the three complementarity determining regions (CDRs) of SEQ ID NO: 205. In one embodiment, an antibody VL domain comprises the three CDRs of SEQ ID NO: 207. In one embodiment, an scFv VH domain comprises the three CDRs of SEQ ID NO: 209. In one embodiment, an scFv VL domain comprises the three CDRs of SEQ ID NO: 211.

[0049] In one embodiment, the dual epitope tetravalent antibody comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 5, 7, 15, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 85.

[0050] Dematured cetuximab monoclonal antibody In one aspect, the application provides a desaturated cetuximab monoclonal antibody, comprising a light chain having a light chain variable (VL) domain, and a heavy chain having a heavy chain variable (VH) domain, wherein the VL domain and the VH domain form a Fab region, and wherein the monoclonal antibody comprises a cetuximab desaturation mutation (sequential numbering) selected from VH-Y101, VH-Y102, VH-D103, VH-Y104, VL-N92, or a combination thereof.

[0051] In one embodiment, the cetuximab dematuration mutations (sequential numbering) include VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL-N92F, VL-N92K, or a combination thereof.

[0052] In one embodiment, the desaturated cetuximab monoclonal antibody is not humanized.

[0053] In one embodiment, the antibody VH domain comprises three complementarity determining regions (CDRs) having amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 247, 251, 253, 259, 261, 263, 265, or 267. In one embodiment, the antibody VL domain comprises three CDRs having amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 243 or 269.

[0054] In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 1, 155, 157, 159, 161, 163, 165, 167, or 169. In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 247, 251, 253, 259, 261, 263, 265, or 267.

[0055] In one embodiment, the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 3, 151, or 153. In one embodiment, the VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 243 or 269.

[0056] In one embodiment, the desaturated cetuximab monoclonal antibody is humanized.

[0057] In one embodiment, the antibody VH domain comprises three complementarity determining regions (CDRs) having amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 245, 249, 255, or 257. In one embodiment, the antibody VL domain comprises three complementarity determining regions (CDRs) having amino acid sequences with at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 241.

[0058] In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 123, 127, 129, 131, or 133. In one embodiment, the antibody VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 245, 249, 255, or 257.

[0059] In one embodiment, the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 99 or 109. In one embodiment, the antibody VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 241.

[0060] In one embodiment, the present application provides a multispecific antibody comprising an antibody scaffold and at least one scFv domain linked to the antibody scaffold, wherein the antibody scaffold comprises a desaturated cetuximab monoclonal antibody disclosed herein.

[0061] In one embodiment, the present application provides a multispecific antibody comprising an antibody scaffold and at least one scFv domain linked to the antibody scaffold, wherein the scFv comprises the variable region of a desmatured cetuximab monoclonal antibody disclosed herein.

[0062] Humanized anti-EGFRvIII monoclonal antibody In one aspect, the present application provides a humanized anti-EGFRvIII monoclonal antibody, the humanized anti-EGFRvIII monoclonal antibody comprising a light chain having a light chain variable (VL) domain and a heavy chain having a heavy chain variable (VH) domain, wherein the VL domain and the VH domain form a Fab region. In one embodiment, the monoclonal antibody is ABT0806 or humanized ABT-806.

[0063] In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 171, 149, or 177. In one embodiment, the light chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 111, 173, or 175. In one embodiment, the VH domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 225, 229, or 233. In one embodiment, the VL domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 227, 231, or 235.

[0064] In one embodiment, the present application provides a multispecific antibody comprising an antibody scaffold and at least one scFv domain linked to the antibody scaffold, wherein the antibody scaffold comprises a humanized anti-EGFRvIII monoclonal antibody disclosed herein.

[0065] In one embodiment, the present application provides a multispecific antibody comprising an antibody scaffold and at least one scFv domain linked to the antibody scaffold, wherein the scFv comprises the variable region of a humanized anti-EGFRvIII disclosed herein.

[0066] tetravalent antibody In one aspect, the present application provides a tetravalent antibody with binding affinity to EGFR, comprising a light chain having an antibody light chain variable (VL) domain, and a heavy chain having an antibody heavy chain variable (VH) domain, and an scFv domain having an scFv light chain variable (VL) domain and an scFv heavy chain variable (VH) domain, wherein the antibody VL domain and the antibody VH domain form a Fab region, and wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain.

[0067] In one embodiment, the antibody VH domain comprises the amino acid sequence having SEQ ID NO: 201. In one embodiment, the antibody VL domain comprises the amino acid sequence having SEQ ID NO: 203. In one embodiment, the antibody heavy chain comprises the amino acid sequence having SEQ ID NO: 89. In one embodiment, the antibody light chain comprises the amino acid sequence having SEQ ID NO: 3. In one embodiment, the scFv domain comprises a light chain variable (VL) domain having the amino acid sequence having SEQ ID NO: 203. In one embodiment, the scFv domain comprises a heavy chain variable (VH) domain having the amino acid sequence having SEQ ID NO: 201.

[0068] In one embodiment, the antibody heavy chain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% sequence identity to SEQ ID NO: 89. In one embodiment, the antibody light chain comprises SEQ ID NO: 3. In one embodiment, the antibody VH comprises SEQ ID NO: 201. In one embodiment, the antibody VL comprises SEQ ID NO: 203. In one embodiment, the antibody scFv VH comprises SEQ ID NO: 201. In one embodiment, the antibody scFv VL comprises SEQ ID NO: 203.

[0069] In one embodiment, the antibody VH comprises the three complementarity determining regions (CDRs) of SEQ ID NO: 201. In one embodiment, the antibody VL comprises the three CDRs of SEQ ID NO: 203. In one embodiment, the antibody scFv VH comprises the three CDRs of SEQ ID NO: 201. In one embodiment, the three CDRs of the antibody scFv VL comprise SEQ ID NO: 203.

[0070] In one embodiment, the tetravalent antibody is represented by SI-95X42, as shown in the Examples below.

[0071] In another embodiment, the present application provides a tetravalent antibody having binding affinity for EGFRvIII, comprising a light chain having an antibody light chain variable (VL) domain, and a heavy chain having an antibody heavy chain variable (VH) domain, and an scFv domain having an scFv light chain variable (VL) domain and an scFv heavy chain variable (VH) domain, wherein the antibody VL domain and the antibody VH domain form a Fab region, and wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain.

[0072] In one embodiment, the antibody VH domain comprises the amino acid sequence having SEQ ID NO: 209. In one embodiment, the antibody VL domain comprises the amino acid sequence having SEQ ID NO: 211.

[0073] In one embodiment, the antibody heavy chain comprises an amino acid sequence having SEQ ID NO: 87. In one embodiment, the antibody light chain comprises an amino acid sequence having SEQ ID NO: 11. In one embodiment, the scFv domain comprises a light chain variable (VL) domain having an amino acid sequence having SEQ ID NO: 211. In one embodiment, the scFv domain comprises a heavy chain variable (VH) domain having an amino acid sequence having SEQ ID NO: 209.

[0074] In one embodiment, the antibody heavy chain comprises SEQ ID NO: 87. In one embodiment, the antibody light chain comprises SEQ ID NO: 11. In one embodiment, the antibody VH domain comprises SEQ ID NO: 209. In one embodiment, the antibody VL domain comprises SEQ ID NO: 211. In one embodiment, the antibody scFv VH domain comprises SEQ ID NO: 209. In one embodiment, the antibody scFv VL domain comprises SEQ ID NO: 211.

[0075] In one embodiment, an antibody VH domain comprises the three complementarity determining regions (CDRs) of SEQ ID NO: 209. In one embodiment, an antibody VL domain comprises the three CDRs of SEQ ID NO: 211. In one embodiment, an antibody scFv VH domain comprises the three CDRs of SEQ ID NO: 209. In one embodiment, an antibody scFv VL domain comprises the three CDRs of SEQ ID NO: 211.

[0076] In one embodiment, the intraspecific antibody is represented by SI-95X41, as shown in the Examples below.

[0077] In another aspect, the present application provides an isolated nucleic acid sequence encoding a dual epitope tetravalent antibody, monoclonal antibody, tetrabody, or multispecific antibody disclosed herein.

[0078] In a further aspect, the present application provides an expression vector expressible in a cell, comprising a nucleic acid sequence disclosed herein.

[0079] In a further aspect, the application provides a host cell comprising a nucleic acid disclosed herein. In one embodiment, the host cell comprises an expression vector disclosed herein. In one embodiment, the host cell is a prokaryotic or eukaryotic cell.

[0080] In a further aspect, the application provides a method of producing the antibodies disclosed herein. In one embodiment, the method comprises culturing a host cell disclosed herein to produce the disclosed antibodies. In one embodiment, the antibody is a dual epitope tetravalent antibody. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a tetraspecific antibody. In one embodiment, the antibody is a multispecific antibody.

[0081] In a further aspect, the present application provides an immunoconjugate comprising the dual epitope tetravalent antibody disclosed herein and a cytotoxic agent. In one embodiment, the cytotoxic agent comprises a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioisotope.

[0082] In a further aspect, the present application provides a pharmaceutical composition comprising an antibody disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further comprises a radioisotope, radionuclide, toxin, therapeutic agent, chemotherapeutic agent, or a combination thereof. In one embodiment, the antibody is a dual epitope tetravalent antibody. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody is a tetraspecific antibody. In one embodiment, the antibody is a multispecific antibody.

[0083] In a further aspect, the present application provides a pharmaceutical composition comprising an immunoconjugate disclosed herein and a pharmaceutically acceptable carrier.

[0084] In a further aspect, the present application provides a method of treating a subject suffering from cancer, comprising administering to the subject an effective amount of a dual epitope tetravalent antibody disclosed herein. In one embodiment, the cancer comprises cells expressing EGFR, EGFRvIII, or both. In one embodiment, the cancer comprises breast cancer, colon cancer, pancreatic cancer, head and neck cancer, melanoma, ovarian cancer, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioma, esophageal cancer, nasopharyngeal cancer, kidney cancer, gastric cancer, liver cancer, bladder cancer, cervical cancer, brain tumor, lymphoma, leukemia, and myeloma.

[0085] In one embodiment, the method of treatment further comprises co-administering an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof. In one embodiment, the therapeutic agent comprises capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, vorozole, formestane, fadrozole, letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, nab-paclitaxel, derivatives, or combinations thereof.

[0086] In one embodiment, the subject is a human.

[0087] In a further aspect, the present application provides a solution comprising an effective concentration of the dual epitope tetravalent antibody disclosed herein, wherein the solution is plasma in a subject. [Brief explanation of the drawings]

[0088] The foregoing and other features of the present disclosure may become more apparent from the following description and appended claims, taken in conjunction with the accompanying drawings, in which the present disclosure may be described with additional specificity and detail through the use of the accompanying drawings, with the understanding that these drawings represent only some embodiments constructed in accordance with the present disclosure and therefore should not be considered limiting of its scope.

[0089] [Figure 1]FIG. 1 shows the configuration of a dual-epitope tetravalent antibody capable of targeting two epitopes of EGFR, comprising two anti-EGFR Fab domains and two anti-EGFRvIII scFv domains (A) or two anti-EGFRvIII Fab domains and two anti-EGFR scFv domains (B), where the scFv domains may be linked to the N- or C-terminus of the antibody heavy or light chain, and the binding domains may be derived from cetuximab (which may be humanized and / or desmatured) or nimotuzumab (which may be humanized) conjugated to ABT-806.

[0090] [Figure 2] Figure 2 shows the structural arrangement of cetuximab (PDB 1YY9), panitumumab (PDB 5SX4), and necitumumab (PDB 6B3S) complexed with EGFR or domain III of EGFR, demonstrating that all three antibodies bind to overlapping epitopes within domain III of EGFR (EGFRwt).

[0091] [Figure 3] Figure 3 shows the mass spectrometry results for wild-type and humanized forms of ABT806, with the wild-type containing two purified fractions corresponding to the intact mAb and a heavy chain dimer impurity (3A), and the two humanized forms showing a single pure fraction containing the intact mAb of interest but without the heavy chain dimer impurity (3B).

[0092] [Figure 4] Figure 4 shows the results of an ADCC assay measuring the efficacy of SI-95m4 (4A) and SI-95m5 (4B) against EGFRwt-expressing U87MG-EGFRwt(GFP) cells and EGFRwt / EGFRvIII-expressing U87MG-EGFRvIII(NR) cells, as well as separate or mixed target cells (T), in the presence of NK effector cells (E:T = 5:1).

[0093] [Figure 5]Figure 5 shows the results of an ADCC assay measuring the potency of a panel of anti-EGFRwt monoclonal antibodies (mAbs), including dematuration mutants SI-95m6, SI-95m7, SI-95m8, SI-95m9, and SI-95m10, as well as anti-EGFRvIII and anti-EGFRwt antibody controls, against U87MG-EGFRwt(GFP) cells expressing EGFRwt (5A) or U87MG-EGFRvIII(NR) cells expressing EGFRwt / EGFRvIII (5B) in the presence of NK effector cells (E:T = 5:1). The EC50 values ​​of the antibodies are also shown.

[0094] [Figure 6] Figure 6 shows the results of an ADCC assay measuring the potency of a panel of dual epitope tetravalent antibodies (i.e., bispecific antibodies) targeting EGFRwt and EGFRvIII, including SI-95X44 and dematured variants of SI-95X44, including, but not limited to, SI-95X52, SI-95X53, SI-95X54, SI-95X55, and SI-95X56, against U87MG-EGFRwt(GFP) cells expressing EGFRwt (6A) and U87MG-EGFRvIII(NR) cells expressing EGFRwt / EGFRvIII (6B) in the presence of NK effector cells (E:T = 5:1). The EC50 values ​​of the antibodies are also shown.

[0095] [Figure 7]Figure 7 shows the effect of de-matured cetuximab variants VH-Y101A (7A) or VH-Y102A (7B) on SI-95X44, a dual epitope tetravalent antibody (also known as a bispecific antibody) targeting EGFRwt and EGFRvIII, and the effect of having the humanized anti-EGFRvIII antibody ABT-806 backbone in SI-95X53 and SI-95X54 compared to SI-95X65 and SI-95X66, respectively, as measured by potency against EGFRwt-expressing U87MG-EGFRwt(GFP) cells in the presence of NK effector cells (E, ET = 5:1), demonstrating that both SI-95X53 and SI-95X54 have more complete killing effects and superior EC50 values ​​against EGFRwt-expressing cells, as shown; and

[0096] [Figure 8] Figure 8 shows the potential increase in therapeutic index obtained by introducing the dematuration mutation Y102A into the mature cetuximab-binding domain of a dual-epitope tetravalent antibody (or simply bispecific antibody) targeting both EGFRwt and EGFRvIII, as demonstrated by measuring their ADCC activity against U87MG-EGFRwt- and U87MG-EGFRwt / EGFRvIII-expressing cells as either separate or mixed populations. SI-95X44, with its mature cetuximab binding, yields overlapping killing curves with similar ED values, while SI-95X54, with the Y102A mutation, differentiates killing of EGFRwt-expressing cells in the mixed versus separated populations, mimicking glioblastoma versus normal tissue in patients. DETAILED DESCRIPTION OF THE INVENTION

[0097] Because wild-type and mutant EGFR (i.e., overexpressed EGFRwt protein and EGFRvIII mutant protein) are frequently expressed in several types of solid tumors, developing effective therapeutic strategies that simultaneously target both EGFR types remains an unmet need. This strategy would involve a primary specificity for EGFRvIII, which may allow preferential targeting of the most aggressive cells, and a secondary specificity for the wild-type receptor, which may allow targeting of a broad range of tumors. Such bispecific antibodies would represent a new class of dual-epitope tetravalent antibodies, characterized by dual binding specificity for two epitopes of the same EGFR target molecule.

[0098] To construct a bispecific tetravalent antibody capable of binding to both wild-type and mutant EGFR proteins and preferentially binding to EGFRvIII, scFv domains were attached to various ends of the antibody's heavy and light chains. As shown in Figure 1, an EGFRvIII-targeting scFv was attached to the N- or C-terminus (via the heavy or light chain) of an EGFR-targeting antibody (Figures 1A, 1B, 1C, and 1D). Alternatively, an EGFRvIII-targeting scFv was attached to the N- or C-terminus (via the heavy or light chain) of the antibody. Several parameters, including the length of the interdomain linker and the orientation of the scFv VH / VL domains, were optimized for stability and binding affinity (Table 1).

[0099] This application relates to the production and use of bispecific tetravalent antibodies, particularly dual epitope tetravalent antibodies.

[0100] The term "epitope" defines the region of an antigen that binds to an antibody. The term "antibody" is used in the broadest sense and specifically encompasses single monoclonal antibodies (including agonist and antagonist antibodies), antibodies with polyepitopic specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), so long as they exhibit the desired biological activity. In some embodiments, antibodies may be monoclonal, polyclonal, chimeric, single-chain, bispecific or bi-effective, human, and humanized antibodies, as well as active fragments thereof. Examples of active fragments of known antigen-binding molecules include Fab, F(ab')2, scFv, and the products of Fab immunoglobulin expression libraries, and Fv fragments containing epitope-binding fragments of any of the foregoing antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and molecules that contain immunologically active sites of immunoglobulin molecules, i.e., the binding site that immunospecifically binds an antigen. Immunoglobulins may be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. In one embodiment, antibodies may be whole antibodies and any antigen-binding fragment derived from whole antibodies. A typical antibody generally refers to a heterotetrameric protein comprising two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain comprises a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions are further subdivided into hypervariable complementarity-determining regions (CDR) domains and more conserved regions called framework regions (FR). Each variable domain (either VH or VL) typically consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Within the variable regions of both the light and heavy chains are binding regions that interact with an antigen.

[0101] As used herein, the term "monoclonal antibody" 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 minor naturally occurring mutations. Monoclonal antibodies are highly specific because they are directed against a single common 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. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins.

[0102] The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. In the context of antibodies that bind to multiple common antigenic sites, the term "monoclonal antibody" refers to a group of monoclonal, monospecific antibodies in early stages of development. For example, monoclonal antibodies used in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or may be produced by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In the latter case, the modifier "monoclonal" from the term "monoclonal polyspecific antibody" is often, of course, omitted.

[0103] Monoclonal antibodies may also include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0104] Monoclonal antibodies can be produced by a variety of methods, including mouse hybridoma and phage display (see Siegel. Transfus. Clin. Biol. 9:15-22 (2002)), or by molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28:453-7 (2011)). Antibody variable genes are isolated using recombinant DNA technology, and the resulting antibodies are recombinantly expressed and screened for desired properties, such as the ability to inhibit binding of wild-type or mutant EGFR. This typical method of antibody discovery is similar to that described in Seeber et al. PLOS One. 9:e86184 (2014). Once monoclonal antibody genes are discovered, they can be humanized, engineered, recombined, and otherwise modified to generate a wide variety of multispecific antibodies. Cetuximab (anti-EGFR) and ABT-806 (anti-EGFRvIII) were both discovered using mouse hybridomas. 9、10 .

[0105] The term "antigen- or epitope-binding portion or fragment" refers to a fragment of an antibody capable of binding to an antigen, such as EGFR, in this application. These fragments may be capable of antigen-binding function as well as additional functions of an intact antibody. Examples of binding fragments include, but are not limited to, a single-chain Fv fragment (scFv), consisting of the VL and VH domains of a single antibody arm linked into a single polypeptide chain by a synthetic linker, or a Fab fragment, which is a monovalent fragment consisting of the VL, light chain constant (CL), VH, and heavy chain constant 1 (CH1) domains. Antibody fragments may also be smaller subfragments, consisting of a single CDR domain, particularly a small domain such as the CDR3 region of the VL and / or VH (see, e.g., Beiboer et al., J. Mol. Biol. 296:833-49 (2000)). Antibody fragments are produced using conventional methods well known to those skilled in the art. Antibody fragments can be screened for utility using the same techniques as used with intact antibodies.

[0106] "Antigen- or epitope-binding fragments" can also be obtained from the antibodies of the present disclosure by several techniques well known to those skilled in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Appropriate fractions containing Fab fragments can then be collected and concentrated by membrane filtration or the like. For further description of typical techniques for isolating active fragments of antibodies, see, for example, Khaw, BA et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0107] Digestion of antibodies with papain produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects the ability to crystallize readily. Treatment with pepsin produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0108] Fab fragments may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH, as used herein, refers to Fab' in which the cysteine ​​residues in the constant domains bear free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0109] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs from each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs determine the antigen-binding specificity of the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site.

[0110] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0111] Immunoglobulins are classified into different classes depending on the amino acid sequence of the constant domain of the heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4, IgA-1, and IgA-2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, delta, epsilon, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0112] A "humanized antibody" refers to a type of genetically engineered antibody that has CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule being derived from one or more human immunoglobulins. Furthermore, framework support residues may be modified to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (See, for example, Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989); Hodgson et al., Bio / Technology, 9:421 (1991)).

[0113] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and are defined to mean a biomolecule composed of amino acids joined by peptide bonds.

[0114] As used herein, the terms "a," "an," and "the" are defined to mean "one or more," and also include the plural forms unless the context is inappropriate.

[0115] "Isolated" refers to a biological molecule that is free from at least some of its naturally occurring components. When used to describe various polypeptides of the present disclosure, the term "isolated" refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen-binding specificities.

[0116] By "recombinant" it is meant that the antibody is made using recombinant nucleic acid techniques in a foreign host cell.

[0117] The term "antigen" refers to an entity or fragment thereof that is capable of inducing an immune response in an organism, particularly an animal, more particularly a mammal, including a human. The term includes immunogens and antigenic regions or determinants.

[0118] Also, as used herein, the term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells against the immunogenic substance and contributes to an immune response in humans or animals. An immune response occurs when an individual produces enough antibodies, T cells, and other reactive immune cells against the administered immunogenic composition of the present disclosure to alleviate or ameliorate the disease being treated. When producing humanized antibodies, the term "immunogenicity" refers to the ability of a therapeutic agent, such as a non-humanized antibody, to provoke an undesired immune response against the therapeutic agent.

[0119] "Specific binding" or "binds specifically" or "specific" for a particular antigen or epitope means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule, which is generally a molecule of similar structure that has no binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0120] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 This can be demonstrated by an antibody having a KD for an antigen or epitope of M or greater, where KD refers to the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a KD for a control molecule that is 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10000-fold, or greater than that of the antigen or epitope.

[0121] Specific binding to a particular antigen or epitope can also be demonstrated by an antibody having a K A or K A for the antigen or epitope that is at least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5000-fold, 10000-fold, or greater than that for the epitope compared to a control, where K A or K A refers to the on-rate of a particular antibody-antigen interaction.

[0122] The "homology" between two sequences is determined by sequence identity. When the two sequences to be compared are of different lengths, sequence identity is preferably expressed as the percentage of nucleotide residues in the shorter sequence that match the nucleotide residues in the longer sequence. Sequence identity can usually be determined using a computer program. Differences that occur in the comparison between any sequence and the above sequences of the present disclosure may be caused by, for example, addition, deletion, substitution, insertion, or recombination.

[0123] The term "dematuration" refers to a process in which a mature antibody (such as cetuximab in this application) is engineered to have mutations (e.g., point mutations) in its binding domain. The resulting antibody is called a "dematured" antibody and exhibits altered, yet desirable, binding activity that is characteristically different from that of a "mature" antibody.

[0124] The term "ADCC" refers to antibody-dependent cellular cytotoxicity, an adaptive immune response in which Fc receptor-bearing effector cells recognize and kill target cells expressing antibody-coated tumor-associated or pathogen-derived antigens on their surface. ADCC is widely used to characterize and compare monoclonal antibody therapeutic candidates. Classical ADCC is mediated by natural killer (NK) cells.

[0125] The present disclosure may be more readily understood by reference to the detailed descriptions of certain embodiments and examples contained herein. Although the present disclosure has been described with reference to specific details of certain embodiments, such details should not be construed as limiting the scope of the disclosure. [Example]

[0126] Example 1. Design of a dual epitope tetravalent antibody targeting EGFR Genes encoding the antibody heavy and light chains (preceded by Kozak and secretory signal peptides) were cloned into the pTT5 vector using standard molecular biology techniques. Antibodies and bispecific antibodies were expressed by transiently transfecting the heavy and light chain expression plasmids in the ExpiCHO system (Thermo Fisher). Briefly, 10 μg of each expression plasmid was diluted in 1 ml of OptiPRO SFM medium. 1 ml of OptiPRO SFM medium containing 80 μl of Expifectamine CHO reagent was added to the DNA and incubated at room temperature for 2.5 minutes. The resulting mixture was then transfected into 25 ml of ExpiCHO cells (6x10 6Cells were added to a 125 ml Erlenmeyer flask (1000 cells / ml) and cultured at 37°C, 5% CO2, and 150 rpm. At 24 hours post-transfection, 8.75 ml of ExpiCHO feed and 150 μl of CHO enhancer were added, and the temperature was shifted to 32°C, 5% CO2, and 150 rpm. At 48 hours post-transfection, the cells were again fed with 8.75 ml of ExpiCHO feed. Culture supernatants were harvested 9 days post-transfection and centrifuged at 4500 rpm for 1 hour to pellet the cells, then passed through a 0.2 mm filter. Expression titers were quantified using biolayer interferometry on the Octet384 system using a Protein A sensor and a calibration curve prepared with purified bispecific antibody proteins.

[0127] Proteins were purified from the collected supernatant using a 5 ml MabSelect PrismA Protein A column (GE Healthcare) equilibrated with phosphate-buffered saline on an AKTA Avant system. The supernatant was then passed through the column at a flow rate of 5 ml / min and washed with 25 ml of PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8). The protein of interest was then eluted by passing 15 ml of 50 mM sodium acetate (pH 3.5) through the column. The eluted protein was immediately neutralized by adding 0.5 ml of 1 M Tris-Cl (pH 8.0).

[0128] Immediately following the first step of Protein A purification, the protein was analyzed by analytical SEC using an ACQUITY UPLC® Protein BEH SEC 200Å, 4.6 mm x 150 mm, 1.7 μm column controlled by a Waters Acquity UPLC H-Class. 10 μg of protein was injected in a PBS mobile phase and run at 0.3 ml / min for 10 minutes. The protein was further purified by preparative SEC using a Superdex Increase 10 / 300 GL column on an AKTA Pure system with a mobile phase of 25 mM sodium acetate, 125 mM NaCl, pH 5.5, and finally buffer-exchanged to 25 mM sodium acetate, 125 mM NaCl, 10% sucrose, pH 5.5. The final sample contained >95% of the target protein as assessed by analytical SEC and was used for subsequent assays.

[0129] To quantify the binding kinetics of bispecific antibodies against EGFR and EGFRvIII, BioLayer Interferometry (Octet) binding assays (affinity) were performed on an Octet384 instrument. Antibodies were captured onto an anti-human Fc (AHC) sensor chip by loading at 5 μg / ml for 180 seconds. After a 60-second baseline step, a 180-second binding step was performed using serial dilutions (0-100 nM; 1:2 dilution factor) of His-tagged EGFR or EGFRvIII (expressed and purified in-house) diluted in assay buffer (PBS containing 0.1% BSA and 0.05% Tween 20), followed by a 300-second dissociation step in assay buffer. Reconstitution was performed using 10 mM glycine, pH 1.5. Binding curves were globally fitted to a 1:1 model to determine the dissociation constant, K D , and the association and dissociation rates were calculated.

[0130] To quantify the bivalent binding kinetics of bispecific antibodies to immobilized EGFR and EGFRvIII, BioLayer Interferometry (Octet) binding assays (avidity) were performed on an Octet384 instrument. Biotinylated EGFR or EGFRvIII was captured onto a streptavidin (SA) sensor chip by loading at 1 μg / ml for 120 seconds. After a 60-second baseline step, a 180-second binding step was performed using serial dilutions of antibody (0-25 nM; 1:2 dilution factor) diluted in assay buffer, followed by a 300-second dissociation step in assay buffer. Reconstitution was performed using 10 mM glycine, pH 1.5. Binding curves were globally fit to a 1:1 model to determine the dissociation constant, K D , and the association and dissociation rates were calculated.

[0131] Epitope binning studies were performed using Biolayer Interferometry (Octet). Biotinylated EGFR-WT (Acro EGR-H82E3) or EGFRvIII (Acro EGR-H82E0) was immobilized on a streptavidin sensor at 1 μg / ml for 180 seconds in assay buffer (PBS containing 0.1% BSA and 0.05% Tween 20). After a 30-second baseline, binding with 100 nM primary antibody was performed for 300 seconds. Finally, binding with 100 nM secondary antibody was performed for 300 seconds. Epitope binning matrices were generated using Octet Analysis 12.0 software.

[0132] Example 2: Expression titers Protein stability is an important parameter defined by the difference in free energy between the folded and unfolded states. For protein therapeutics, stability can affect immunogenicity, pharmacokinetics, and even efficacy. Reducing aggregation can help develop therapeutics that are easier to manufacture and safer for patients. Furthermore, expression efficiency and protein yield directly determine the cost of protein therapeutics. If proteins can be efficiently expressed and achieve higher titers and increased yields of purified protein, production costs can be significantly reduced.

[0133] After transient expression in ExpiCHO cells, titers were quantified using biolayer interferometry (Table 2). Monoclonal antibodies containing simple structures (EGFR or EGFRvIII) were produced with the highest efficiency, while bispecific antibodies (EGFR x EGFRvIII, SI-95X1-31, SI-95X35-39) were produced with variable efficiency, primarily depending on the composition of the binding domain. Bispecific antibodies using nimotuzumab scFv (SI-95X4-16) were produced with significantly lower titers, indicating the instability of the nimotuzumab VH and VL in this scFv format. On the other hand, constructs incorporating nimotuzumab into the Fab region of the antibody and ABT-806 scFv (SI-95X2, SI-95X17-31) tended to exhibit higher titers on average.

[0134] Example 3: Stability by analytical size exclusion chromatography Immediately after the first step of Protein A purification, the stability and aggregation of the bispecific EGFR x EGFRvIII antibodies were evaluated by analytical size-exclusion chromatography on a Waters UPLC system (Table 2). Antibodies containing nimotuzumab scFv (SI-95X4-16) typically exhibited low signal due to low expression levels, and in addition to the main peak corresponding to the protein of interest (POI), they tended to exhibit high abundances of high-molecular-weight (HMW) or low-molecular-weight (LMW) contaminants. This suggests that nimotuzumab scFv is not stable when incorporated into bispecific antibodies. Other bispecific antibodies exhibited, on average, more protein of interest and fewer HMW and LMW species. The protein in which the C-terminal scFv was fused to the heavy chain (SI-95X1-3) exhibited the highest % POI, indicating good stability. This also suggests that the orientation of the scFv affects the SEC profile. The ABT-806 scFv-containing antibodies showed complex split profiles in the HL orientation (SI-95X17-19, SI-95X23-25, SI-95X29-31), but no corresponding structure was observed when the ABT-806 scFv was in the LH orientation (SI-95X20-22, SI-95X26-28). This result suggests that the LH orientation of this scFv may be more stable and / or structurally uniform. The length of the interdomain linker had little effect on aggregation.

[0135] Example 4: Binding of Octet to EGFR WT (EGFRwt) Biolayer interferometry was used to evaluate the affinity of the bispecific antibodies for wild-type EGFR (Table 3). Cetuximab and nimotuzumab antibodies showed strong binding to wild-type EGFR, with reactivity values ​​of 0.75 and 0.32 nm, respectively. In contrast, ABT-806 (an EGFRvIII-specific antibody) showed a much lower reactivity value of 0.07 nm. Most of the EGFR x EGFRvIII bispecific antibodies, except those containing nimotuzumab scFv and ABT-806 Fab (SI-95X4-16), bound strongly to wild-type EGFR. This result indicates that nimotuzumab exhibits reduced function when present in the scFv format. On the other hand, when nimotuzumab was present in the Fab region (SI-95X2, SI-95X17-31) or when cetuximab was present in the FAb (SI-95X1, SI-95X37-39) or scFv (SI-95X3, SI-95X35-36) regions, good binding to wild-type EGFR was observed. Affinity data showed that antibodies with cetuximab domains showed stronger affinity (lower KD) than antibodies with nimotuzumab domains.

[0136] Example 5: Binding of Octet to EGFRvIII Biolayer interferometry was used to evaluate the affinity of the bispecific antibodies for wild-type EGFRvIII (Table 4). As expected, all monoclonal and bispecific antibodies targeting wild-type EGFR and / or EGFRvIII showed detectable binding to EGFRvIII, consistent with the presence of both binding epitopes on the EGFRvIII protein. Cetuximab showed stronger binding activity and affinity than nimotuzumab. Meanwhile, ABT-806 binding was intermediate. For the bispecific antibody containing nimotuzumab scFv and ABT-806 Fab (SI-95X4-16), the observed binding was similar to that of the ABT-806 monoclonal antibody, indicating that the nimotuzumab scFv does not confer additional functionality. On the other hand, bispecific antibodies containing nimotuzumab Fab and ABT-806 scFv (SI-95X2, SI-95X17-31) showed lower KD values, slower dissociation rates, and in some cases, improved affinity compared to the component mAbs. Similar affinity improvements were also observed with bispecific antibodies containing cetuximab and the ABT-806 domain (SI-95X1, SI-95X3, SI-95X35-39). These results suggest that nimotuzumab / cetuximab and the ABT-806 domain may simultaneously bind to two epitopes on the same EGFRvIII molecule, thereby generating a multivalent avidity effect that increases apparent affinity.

[0137] Example 6: Epitope binning (EGFRwt epitopes) Biolayer interferometry was used to perform epitope binning of a panel of monoclonal antibodies against wild-type EGFR (Table 5). The antibodies used were EGFR-specific cetuximab (Cetu), panitumumab (Pani), nimotuzumab (Nimo), and necitumumab (Neci), as well as EGFRvIII-specific ABT-806. A large reactivity value indicates that the secondary antibody can bind to EGFR even after saturation with the primary antibody has already occurred (suggesting a different epitope). Conversely, a small value indicates that the secondary antibody is blocked by the primary antibody through competitive inhibition (suggesting the same epitope).

[0138] As expected, ABT-806 consistently showed low reactivity as a secondary antibody because it barely binds to wild-type EGFR. After ABT-806 binding, all EGFR-targeting antibodies showed high reactivity as secondary antibodies because ABT-806 was unable to bind and block the secondary antibodies.

[0139] On the other hand, the four EGFR-targeting antibodies each blocked each other to some extent, suggesting identical or overlapping epitopes. Of note, the four EGFR-targeting antibodies showed detectable but reduced binding when nimotuzumab was used as the primary antibody. This is likely due to the slow binding kinetics of nimotuzumab, which was unable to fully saturate the EGFR in the first 300-second step. In conclusion, this data confirms that ABT-806 does not significantly bind to wild-type EGFR and indicates that all four EGFR-targeting antibodies (cetuximab, panitumumab, nimotuzumab, and necitumumab) share a conserved epitope. This latter conclusion is reinforced by other studies showing that panitumumab, cetuximab, and nimotuzumab have overlapping epitopes within EGFR domain III. The crystal structures of EGFR complexed with the Fabs of cetuximab (1YY9), panitumumab (5SX4), and necitumumab (6B3S) were structurally aligned using the MatchMaker function in Chimera 1.13 to compare the epitopes of these EGFR-targeting antibodies (Figure 2). This alignment showed that all three antibodies bind to overlapping epitopes within domain III of EGFR, consistent with the epitope binning data from Octet. 11-12 .

[0140] Example 7: Epitope binning (EGFRvIII epitope) Biolayer interferometry was used for epitope binning of the same panel of monoclonal antibodies against EGFRvIII (Table 6). A large reactivity value indicates that the secondary antibody is able to bind to EGFR even after saturation with the primary antibody has already occurred (suggesting a different epitope). Conversely, a small value indicates that the secondary antibody is blocked by the primary antibody through competitive inhibition (suggesting the same epitope).

[0141] As observed in wild-type EGFR binning experiments, all four EGFR-targeting antibodies (cetuximab, panitumumab, nimotuzumab, and necitumumab) exhibited competitive binding with each other, as indicated by decreased binding of the secondary antibody after saturation of the primary antibody. This indicates that these four antibodies share overlapping epitopes on EGFRvIII that may not be specific for EGFRvIII, i.e., may be EGFRwt epitopes. A slightly higher secondary antibody response was observed when nimotuzumab was used as the first (blocking) antibody, but this can be explained by the slower binding kinetics of nimotuzumab, which did not fully saturate the EGFRvIII protein during the first 300-second blocking step.

[0142] On the other hand, ABT-806 clearly classified itself as an epitope distinct from the four EGFR-targeting antibodies, i.e., an EGFRvIII-specific epitope. When ABT-806 was used as the primary antibody, each of the other antibodies showed significant binding activity in the second step. Similarly, when ABT-806 was used as the secondary antibody, a significant additional activity was observed. The lack of competitive binding between ABT-806 and the other antibodies suggests that ABT-806 targets a unique epitope on EGFRvIII and that ABT-806 and the other EGFR-binding antibodies may simultaneously interact with different epitopes on the EGFRvIII protein. The fact that ABT-806 binds to an epitope on EGFRvIII distinct from the EGFR-targeting antibodies is consistent with ABT-806 binding to a conformation of the EGFR protein that is restricted to mutant EGFRvIII and not present in wild-type EGFR.

[0143] Example 8: Generation and characterization of humanized ABT-806 To enhance the degree of humanization of the ABT-806 variable regions and reduce potential immunogenicity, the murine VH and VK domains were engineered into more human-like frameworks. All versions used scFv models generated from the antibody modeling feature in Discovery Studio based on the ABT-806 variable domain sequences. All humanized versions were designed using the Discovery Studio 2022 suite. VH1, VH2, VK1, and VK2 were designed using the "Humanization Mutation Prediction Protocol," which set an identity threshold of 50, a tolerance for frequent residue substitutions of 20, a tolerance for germline substitutions of 0, and exclusion of substitutions of Kabat CDR residues, IMGT CDR residues, Vernier zone residues, and human germline residues. This protocol was based solely on the ABT-806 sequence as the query sequence. VH1 and VK1 were constructed based on germline substitutions, while VH2 and VK2 versions used frequent residue substitutions. For VH3 and VK3, the Fv model of ABT806 was used as input, and "calculate mutation energy" was set to true to generate the "best single mutation" sequence (CHARMm force field). The query structure was a model of ABT-806 generated by the antibody modeling cascade in Discovery Studio. The input sequences were ABT-806 VH and VL. The CDR loop definition was set to Honegger, and the number of templates for each loop was set to a maximum of 3, with the optimization level set to high.

[0144] Each humanized chain was combined to generate nine versions: V1 (VH1, VK1), V2 (VH1, VK2), V3 (VH1, VK3), V4 (VH2, VK1), V5 (VH2, VK2), V6 (VH2, VK3), V7 (VH3, VK1), V8 (VH3, VK2), and V9 (VH3, VK3) (Table 7). Heavy and light chain genes were cloned and expressed as in Example 1, and the antibody stability and functional properties were then evaluated.

[0145] After transient expression in ExpiCHO cells, titers were quantified using biolayer interferometry (Table 7). The optimal humanized version was selected (in part) based on overall expression.

[0146] Antibodies were purified by Protein A and SEC chromatography as described in Example 1. After purification with Protein A, many antibodies tended to have high abundances of high molecular weight (HMW) or low molecular weight (LMW) contaminants in addition to the main peak corresponding to the protein of interest (POI). This suggests that these antibodies are less stable, more prone to forming other species (HC dimers), and / or more prone to aggregation. V3 and V9 appear to be the most stable humanized antibodies based on the above indicators (Table 7). Samples used for subsequent testing were purified to 95% or greater purity by preparative SEC.

[0147] Biolayer Interferometry (Octet) binding assays were performed on an Octet384 instrument to quantify the binding kinetics of the humanized antibodies to EGFR and EGFRvIII, as described in Example 1. Table 8 shows that all monoclonal antibodies targeting EGFRvIII exhibited detectable binding to EGFRvIII. All antibodies exhibited similar binding kinetics, but the humanized V3 and V9 antibodies exhibited high reactivity comparable to that of ABT-806 and superior to that of competitor depatuxizumab, AbbVie's humanized ABT-806 (in-house purified).

[0148] To confirm antibody binding on cells, purified primary antibodies were added to EGFR-, EGFRvIII-, or uPAR-transfected CHO cells. A fluorescent secondary antibody against the primary antibody was used to measure antigen binding on the cell surface. All tested antibodies maintained their binding properties to cells mimicking cancer cell surfaces (Table 9). Furthermore, all antibodies bound significantly more strongly to EGFRvIII-expressing CHO cells than to EGFR-expressing CHO cells. This indicates that these antibodies bind to the ABT-806 epitope, not the cetuximab epitope.

[0149] Preparative SEC (pSEC) was used to partially separate full mAb molecules (pSEC fraction 1) from heavy chain dimer molecules (pSEC fraction 2) prior to intact mass analysis. For mass analysis and identification of the pSEC fractions, samples were diluted to 0.5 mg / mL in 50 mM Tris pH 7.5. 1.0 μg of the prepared sample was injected onto an online analytical SEC column for LC-MS intact mass analysis under denaturing conditions. Data were processed using the MaxEnt 1 algorithm in Unifi (Waters Corp). As shown in Figure 3A, pSEC fraction 1 of depatuzumab contains almost exclusively intact full mAb molecules, while the later-eluting pSEC fraction 2 contains a mixture of full mAb and heavy chain dimers. Figure 3B shows that both purified hABT-806 V3 and V9 antibodies contain primarily intact full mAb. Thus, humanization of ABT806 produced an antibody that is more stable and expressed with fewer problematic contaminants.

[0150] The variable domains of the lead heavy and light chains were compared to the LENS database for sequence similarity, which showed that the variable region sequences of VH3, VK3, and VH1 were unique (Table 10).

[0151] Example 9. Production and characterization of desmatured cetuximab The cetuximab mutations that dematurate EGFR binding were identified using an in silico approach. Based on the high-resolution crystal structure of cetuximab-bound EGFR (1YY9), the contact area between cetuximab and EGFR was calculated using the protein interface analysis method in Discovery Studio (Biovia). The contact surface area was 20 Å. 2Nine cetuximab residues were identified as being strongly involved in binding to EGFR. The residues of interest were as follows (consecutive numbering): W94, N91, and N92 from the light chain; W52, N56, Y101, Y102, D103, and Y104 from the heavy chain. Each residue of interest was individually mutated to one of 20 naturally occurring amino acids. A total of 180 (9 residues x 20 amino acids) independent mutations were generated in silico. The effect of mutations on cetuximab stability was calculated using the Calculate Mutation Energy (Stability) function in Discovery Studio, while the effect of mutations on EGFR binding was calculated using the Calculate Mutation Energy (Binding) function, with EGFR as the ligand molecule. Briefly, the stability function calculates the energy difference between WT cetuximab and mutant cetuximab (ΔΔG Stability), while the binding function calculates the binding energy between cetuximab and EGFR (ΔΔG WT), then calculates the binding energy between mutant cetuximab and EGFR (ΔΔG Mutant), and reports the energy difference as the binding energy change due to the mutation (ΔΔG Binding = ΔΔG Mutant - ΔΔG WT). Energies were calculated using CHARMm in pH-independent mode. For all mutations, ΔΔG Stability was plotted against the corresponding ΔΔG Binding to identify mutations in the range that may dematurate the bond but do not strongly destabilize cetuximab. Ten mutations were selected for further analysis: N92K, N92F from the light chain; Y101A, Y101W, Y102V, Y102A, D103W, D103Y, D103F, and Y104L from the heavy chain (Table 11).

[0152] Dematured cetuximab variants were prepared as follows: The codon-optimized coding region preceding the Kozak and secretory signal peptide sequences was cloned into the pTT5 vector using standard molecular biology techniques. The antibody was expressed by transiently transfecting the light and heavy chains in the ExpiCHO system (ThermoFischer Scientific) as described in Example 1, but the culture volume was reduced to 2 ml in a 6-well plate. Protein was purified from the harvested supernatant using a Captureem Protein A 24-Well Plate (Takara). The supernatant was loaded onto a plate equilibrated with phosphate-buffered saline (PBS, 125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8). After washing the plate with 10 ml of PBS, bound proteins were eluted with 0.5 ml of 50 mM sodium acetate (pH 3.5). The eluted protein was immediately neutralized by adding 0.1 ml of 1 M Tris-Cl (pH 8.0). Immediately after purification with Protein A, the proteins were analyzed by analytical SEC as described in Example 1. Biolayer Interferometry (Octet) binding assays were performed as described in Example 1.

[0153] Biolayer interferometry and analytical size-exclusion chromatography were used to evaluate the stability of de-matured cetuximab variants. After transient expression in ExpiCHO cells, titers were quantified using biolayer interferometry (Table 12). Selection of the optimal de-matured variant was determined in part based on overall expression levels. Immediately following the first step of Protein A purification, antibodies were evaluated for stability and aggregation by analytical size-exclusion chromatography on a Waters UPLC system. Many antibodies tended to exhibit high molecular weight (HMW) species in addition to the main peak corresponding to the protein of interest (POI) (Table 12). The high abundance of HMW species suggests that these antibodies may be less stable and more prone to aggregation.

[0154] Biolayer interferometry was used to evaluate the affinity and binding activity of de-matured cetuximab to EGFR and EGFRvIII. Table 13 shows that most de-matured cetuximab variants have reduced binding affinity and / or binding response to EGFR compared to wild-type cetuximab, indicating that selected mutations alter the binding ability of cetuximab to EGFR. However, these antibodies maintain good binding activity to immobilized EGFR.

[0155] Table 14 shows that most dematured cetuximab variants also exhibited reduced binding affinity and / or binding response to EGFRvIII. Despite this reduction, all selected dematured cetuximab variants maintained the ability to bind to EGFRvIII with similar binding kinetics. This data reaffirms that cetuximab binds to the same epitope on EGFRvIII and EGFR. Although the variants had weaker affinity for EGFR and EGFRvIII, they maintained good binding activity to EGFR and EGFRvIII through bivalent binding. Because binding activity depends on the level of antigen on the surface, this result suggests that dematured cetuximab may be more selective for tumors that express high amounts of EGFR and / or EGFRvIII.

[0156] To confirm that the de-matured cetuximab variants maintained cell binding activity, purified primary antibodies were added to CHO cells transfected with EGFR, EGFRvIII, or uPAR. A fluorescent secondary antibody against the primary antibody was used to measure antigen binding on the cell surface. Table 15 shows that all tested antibodies (except HC:D103W) maintained their binding properties to cells mimicking the cancer cell surface. This result indicates that these antibodies maintain the binding properties desired for antibody drug manufacturing.

[0157] Example 10. Production and characterization of a dual epitope tetravalent antibody targeting EGFR Nimotuzumab was approved in several countries for the treatment of solid tumors after the approval of cetuximab. At least one of its improvements is reduced skin toxicity, which appears to correlate with its lower affinity for EGFR, i.e., KD value of 9.1 nM compared to 3.77 nM (Table 3). 9 In this context, the most kinetically similar cetuximab mutant is Y104L (KD 9.2 nM). Meanwhile, all other mutants, including Y101A (KD 67 nM), Y102A (97 nM), and N92F (34 nM), exhibited even weaker EGFR affinity than nimotuzumab. These dematuration mutants were incorporated into a dual-epitope tetravalent antibody to screen for potential therapeutic candidates that effectively target tumors expressing EGFRvIII in the presence of EGFRwt.

[0158] Dual-epitope tetravalent antibodies targeting EGFR and EGFRvIII (also known as anti-EGFR x EGFRvIII) were constructed by fusing scFvs to the N- or C-termini of antibody heavy or light chains. Figure 1 shows four exemplary structures, where the EGFR-binding domains may be derived from humanized and / or de-matured cetuximab variants (hDC-EGFR) and may be formed as Fabs or scFvs. Meanwhile, ABT-806 or humanized ABT-806 may be reciprocally formed as scFvs or Fabs.

[0159] Table 16 lists four configuration formats of anti-hDC-EGFR x hABT-806 antibodies: SI-95X45-49, SI-95X52-56, SI-95X58-62, and SI-95X64-68. Their EGFR-binding domains are derived from a humanized, de-matured cetuximab variant (hDC-EGFR), i.e., SI-95m4-10. SI-95X45-49 contains a C-terminal fusion of anti-ABT-806 scFv in the antibody heavy chain and anti-hDC-EGFR in the Fab region. SI-95X52-56 contains an N-terminal fusion of anti-hDC-EGFR scFv in the antibody heavy chain and ABT-806 V9 in the Fab region. SI-95X58-62 contains a C-terminal fusion of ABT-806 V9 scFv in the antibody light chain and anti-hDC-EGFR in the Fab region. SI-95X64-68 contains a C-terminal fusion of anti-hDC-EGFR scFv in the antibody heavy chain and ABT-806 in the Fab region. The materials and methods used to generate the anti-EGFR x EGFRvIII antibodies were the same as or similar to those described in Example 1.

[0160] After transient expression in ExpiCHO cells, titers were quantified using biolayer interferometry (Table 17). As expected, these bispecific antibodies (SI-95X40-68) were produced with variable efficiency, depending primarily on the composition and configuration of each binding domain. For example, molecules in which the anti-hDC-EGFR scFv was fused to the C-terminus of the antibody heavy chain (SI-95X63-68) appeared to be expressed in lower amounts than molecules in which the anti-hDC-EGFR scFv was fused to the N-terminus of the antibody heavy chain (SI-95X51-56). Furthermore, molecules containing the anti-hDC-EGFR Y101A domain (SI-95X47, 53, 58, 65) tended to have high titers, whereas molecules containing the anti-hDC-EGFR Y104L domain (SI-95X46, 56, 61, 68) tended to have low titers.

[0161] Immediately after first-step purification with Protein A, the anti-EGFR x EGFRvIII bispecific antibody was evaluated for stability and aggregation by analytical size-exclusion chromatography on a Waters UPLC system (Table 17). The molecular configuration appears to play the largest role in the percentage of target protein after first-step purification. The abundance of high-molecular-weight aggregates indicates instability of the SI-95X57-62 molecule. This trend indicates that fusion of ABT-806 V9 scFv to the antibody light chain typically produces an unstable molecule. Conversely, SI-95X51-56 showed very high protein yields after prepurification, indicating that N-terminal fusion of anti-hDC-EGFR scFv to the antibody heavy chain typically produces a stable molecule.

[0162] Biolayer interferometry was used to evaluate the affinity of the bispecific antibodies for EGFR (Table 18). Molecules containing the anti-hDC-EGFR D103Y (SI-95X45, 55, 60, 67) domain showed no or very little binding to EGFR, indicating that the affinity of this domain for EGFR is very low or absent. All other molecules containing the anti-hDC-EGFR domain exhibited reduced affinity for EGFR compared with the control molecule in the "mature" configuration. The reduced affinity varied depending on both the molecular configuration and the type of anti-hDC-EGFR domain.

[0163] Biolayer interferometry was used to evaluate the affinity of the bispecific antibodies for EGFRvIII (Table 19). Molecules containing the anti-hDC-EGFR D103Y (SI-95X45, 55, 60, and 67) domains were shown to bind to EGFRvIII but not to EGFR (Tables 18 and 19). Taken together, these results suggest that the ABT-806 domain (or domain derivatives) is functional, whereas the anti-hDC-EGFR D103Y domain is either not functional or only marginally functional depending on the molecular configuration. All molecules were able to bind to EGFRvIII (albeit with slightly lower affinity) compared to control molecules in the mature configuration. All bispecific antibodies showed significantly improved binding compared to the monospecific mAbs in Table 14. Therefore, these molecules, despite possessing the anti-hDC-EGFR domain, are able to tightly bind to EGFR and EGFRvIII. Indeed, the affinity of several bispecific antibodies for EGFRvIII was improved compared to wild-type cetuximab and ABT806 mAb, recapitulating the multivalent avidity effect observed in Example 5 and suggesting a mechanism by which the mature cetuximab and ABT806 domains simultaneously bind to the same EGFRvIII molecule. Furthermore, binding of several antibodies was stronger to EGFRvIII (Table 19) than to EGFRwt (Table 18), indicating that they preferentially bind to EGFRvIII while maintaining good binding to EGFRwt.

[0164] Example 11: Dual epitope tetravalent antibodies exhibit ADCC activity against cells expressing EGFRvIII. To analyze the varying potency of a dual-epitope tetravalent antibody targeting tumor cells expressing EGFRwt and EGFRvIII, ADCC assays were performed using NK cells (one donor) cocultured with U87-MG-GFP cells expressing EGFRwt, U87-MG-EGFRvIII-NR cells expressing both EGFRvIII and EGFRwt (i.e., mKATE2+), or a mixed culture of these two. The mixed cell cultures were seeded and initiated with a 50:50 green / red signal. The green signal from U87-MG-GFP cells correlated with live cells expressing EGFRwt, while the red signal from U87-MG-EGFRvIII-NR cells correlated with cells expressing EGFRwt and EGFRvIII. After spheroid formation, NK cells were seeded at a 5:1 effector-to-target cell ratio, and antibodies were added according to an 8-point log10 dose-response curve. Cell fluorescence was measured after 60 hours to quantify viable cells and normalized to the value at time 0. This ADCC assay was used to screen monoepitope bivalent antibodies (i.e., mAbs) and dual epitope tetravalent antibodies for therapeutic candidates (Table 16).

[0165] Both the SI-95m4 and SI-95m5 antibodies are monoepitope bivalent antibodies targeting EGFRvIII and EGFRwt, respectively. As shown in Figure 4A, SI-95m4 failed to kill U87-MG-EGFRwt-GFP cells in ADCC assays. This confirms that this humanized anti-EGFRvIII antibody maintains the exclusive binding specificity of the ABT-806 antibody. However, when mixed with U87-MG-EGFRvIII-NR cells, SI-95m4 exerted ADCC activity against U87-MG-EGFRwt-GFP cells (i.e., EGFRwt target cells, Figure 4A). This surprising result may be interpreted as a bystander effect of activated NK cells against EGFRwt-expressing cells. In contrast, SI-95m5 was able to kill both U87-MG-EGFRwt and U87-MG-EGFRvIII cells, either in separate or mixed states, consistent with the ability of this antibody to bind to both EGFRwt and EGFRvIII (Fig. 4B).

[0166] To analyze the effects of dematuration, a panel of monoepitope bivalent antibodies bearing either mature EGFRwt (cetuximab) or dematured EGFRwt binding domains was subjected to ADCC assays using U87-MG-GFP cells expressing EGFRwt and U87-MG-NR cells expressing EGFRwt / EGFRvIII, respectively. As shown in Figure 5, the panel of monoepitope bivalent antibodies (Table 16), consisting of SI-95m6, SI-95m7, SI-95m8, SI-95m9, and SI-95m10, exhibited a range of reduced ADCC activity against cells expressing EGFRwt when compared with the parent anti-hDC-EGFR antibody, SI-95m5 (Figure 5A). The EC50 values ​​of the variants were increased by up to 12-fold (SI-95m6) compared with that of the parent mAb, SI-95m5, indicating reduced potency. As expected, the test anti-EGFRvIII control antibody, SI-95m4, showed no activity. When cells expressing EGFRwt / EGFRvIII were used, SI-95m6, SI-95m9, and SI-95m10 exerted ADCC activity at levels similar to that of the positive control antibody, SI-95m4 (Figure 5). Of this panel of antibodies, SI-95m7 exhibited a right-shifted curve with a 3-fold increased EC50, indicating a characteristic decrease in ADCC activity. On the other hand, SI-95m8 no longer exhibited any activity against EGFR (Figures 5A and 5B), likely due to the specific D103Y mutation in the EGFR-binding domain. Although limited data exist, there does not appear to be a correlated decrease in ADCC between EGFRwt- and EGFRvIII-expressing cells when compared with a panel of mAbs with different dematuration mutations. Although limited data exist, there does not appear to be a correlated decrease in ADCC between EGFRwt- and EGFRvIII-expressing cells when compared with a panel of mAbs with different dematuration mutations. This result may be explained by differences in the density or geometric arrangement of EGFRwt / EGFRvIII in the two cell populations, resulting in differences in avidity and initiation of ADCC.

[0167] To screen for bispecific candidates, a panel of dual epitope tetravalent antibodies was constructed based on the panel of monoepitope bivalent antibodies (Table 16, Figures 4 and 5) and subjected to the same ADCC assay using cells expressing EGFRwt and cells expressing EGFRwt / EGFRvIII, respectively.

[0168] The panel of dual-epitope tetravalent antibodies included SI-95X52, SI-95X53, SI-95X54, SI-95X55, and SI-95X56, each containing an anti-EGFRvIII Fab binding domain, i.e., the hABT-806(V9) antibody backbone, and an anti-EGFRwt scFv binding domain, sharing a common antibody backbone from its parent mAb, SI-94m4. These EGFRwt-binding domains were derived from anti-hDC-EGFR scFv variants and corresponded to a panel of de-matured anti-EGFRwt mAbs, namely, SI-95m10 (corresponding to SI-95X52), SI-95m6 (SI-95X53), SI-95m7 (SI-95X54), SI-95m8 (SI-95X55), and SI-95m9 (SI-95X56), with SI-95m5 as one of the parent antibodies (Table 16, Figure 5B). In this regard, SI-95X44 was used as a matured dual-epitope tetravalent antibody control (Table 16). These dual-epitope tetravalent antibodies exhibited a range of ADCC activity against EGFRwt-expressing cells (Figure 6A) and apparently overlapping curves against EGFRvIII-expressing tumor cells (Figure 6B). Notably, SI-95X55 showed significantly reduced activity against EGFRwt-expressing cells. The EC50 values ​​for these overlapping curves were calculated and summarized in Figure 6B. The EC50 value was lower than that of the parent antibody, SI-95m4, at 19.3 nM. A lower EC50 value indicates improved ADCC activity. This observation may suggest a synergistic effect of simultaneous binding to two EGFR epitopes on EGFRwt / EGFRvIII-expressing cells to enhance ADCC activity. It is noteworthy that different configurations of dual-epitope antibodies, such as the geometric arrangement and distance of the anti-EGFR and anti-EGFRvIII binding domains, may alter the efficacy of this simultaneous binding mechanism in cells, which may affect the degree of ADCC not only against EGFRwt-expressing tumor cells but also against EGFRwt / EGFRvIII-expressing tumor cells.In conjunction with the findings from the mAb format, the synergistic effect of the dual-epitope tetravalent antibody is likely influenced by structural changes resulting from humanization, dematuration, and / or configuration. Furthermore, SI-95X55 exhibited improved potency against cells expressing EGFRwt / EGFRvIII, as shown by its EC50 (12.37 nM) compared to either SI-95m4 (19.39 nM) or SI-95X44 (14.72 nM) in Figure 6B. This observation indicates that even residual affinity / avidity for EGFRwt (Table 15) contributed to improved ADCC efficacy. This also indicates an improved therapeutic index due to increased potency against cells expressing EGFRwt / EGFRvIII while decreasing potency against cells expressing EGFRwt.

[0169] Example 12: Dual epitope tetravalent antibody targeting both EGFRwt and EGFRvIII expressing tumors. The results of Example 11 suggest that the ADCC activity of dematurated monoepitope antibodies may not be the same even for dual epitope antibodies with the same dematuration mutation. For example, SI-95m8, SI-95X55, and SI-95X67 (the opposite configuration to SI-95X55, see Table 16) have the same D103Y mutation. The loss of ADCC activity against EGFRwt-expressing cells makes SI-95X55 a candidate for targeting only EGFRvIII tumors. In this context, screening for dual epitope tetrameric antibodies that can efficiently target both EGFRwt- and EGFRvIII-expressing tumors focused on antibodies with the dematuration mutations Y101A and Y102A.

[0170] The Y101A mutation is a common feature of the dematured antibodies SI-95m6 and SI-95X53 / SI-95X65, of which the antibody backbone is not humanized (Table 16). As shown in Figure 7, SI-95X65 contains an anti-EGFR scFv at the opposite heavy chain end to SI-95X53, and exhibited overlapping but incomplete killing curves compared to SI-95X53 (Figure 7A). Meanwhile, SI-95m6 had an approximately 10-fold higher EC50 (199.9 nM) against EGFRwt cells than the parent antibody (i.e., mature binding to EGFRwt) SI-95m5 (EC50 = 16.06 nM) (see table in Figure 7B). This means that when the dematuration mutation Y101A was introduced into the dual epitope tetravalent antibody, the EC50 (7.659 nM) was similar to that of the parent SI-95m5 antibody and a bispecific antibody (SI-95X44) containing the parent mAb backbone structure (EC50 = 14.38 nM). In this regard, SI-95X52, which carries the N92F mutation (Table 16), showed similar potency and complete killing capacity (Figure 6B).

[0171] The Y102A mutation is a common feature of the dematured antibodies SI-95m7, SI-95X54, and SI-95X66, of which SI-95X66 has a non-humanized antibody backbone and contains an anti-EGFR scFv domain at the opposite end of the heavy chain compared to SI-95X54 (Table 16). Compared to the overlapping curves of SI-95m5 and SI-95X44, both SI-95m7 and SI-95X66 exhibited right-shifted curves indicating incomplete killing, whereas SI-95X54 exhibited a curve that completely killed EGFRwt-expressing cells in the 10 nM to 100 nM range (see table in Figure 7B). The right-shift by SI-95X54 was measured to be approximately 5-fold higher than the EC50 values ​​of either SI-95m5 or SI-95X44. SI-95X54 is a dual-epitope tetravalent antibody with an scFv domain derived from the anti-hDC-EFGR mutant with Y102A. SI-95X54 therefore killed EGFRwt tumors with at least a 5-fold higher EC50 value, while demonstrating EGFRvIII-biased cell killing.

[0172] To further compare changes in ADCC activity, we used a panel of isolated and mixed EGFRwt and EGFRvIII cells. As a control, SI-95X44 showed overlapping curves and similar ED50 values, as expected (Figure 8A). Bispecific candidates such as SI-95X54 showed a characteristic right-shifted curve with complete killing of mixed cells (Figure 8B). This result indicates that SI-95X54 is an excellent example of a dual-epitope tetravalent antibody that can efficiently kill mixed EGFRwt and EGFRvIII cell populations, potentially mimicking the heterogeneity of glioblastoma in human patients. Furthermore, the reduced sensitivity of killing ability to EGFRwt cells may mean reduced toxicity to healthy tissues in patients.

[0173] EGFRwt and EGFRvIII play complex and interactive roles in brain tumor formation. While EGFR overexpression is widespread in tumor cells in most cases of glioblastoma, EGFRvIII typically shows patchy tumor positivity in only a minority of tumor cells when stained with a pan-EGFR antibody. This tumor heterogeneity remains an unresolved challenge in the development of effective and meaningful treatments for brain tumors. Therefore, therapeutic antibodies that simultaneously target both EGFRwt and EGFRvIII cells and have EGFRvIII-biased binding may be a desirable solution. In this context, this application presents proof-of-concept for a therapeutic strategy that creates a dual-epitope tetrameric antibody that effectively eradicates mixed tumor cell populations expressing EGFRwt and / or EGFRvIII, while reducing toxicity to normal tissues.

[0174] table

[0175] Table 1. Configurations of bispecific tetravalent antibodies [Table 1]

[0176] Table 2. Stability assessment of bispecific tetravalent antibodies targeting EGFR-WT x EGFRvIII by expression titer and analytical size exclusion chromatography after first-step Protein A purification. [Table 2]

[0177] Table 3. Binding kinetics of bispecific tetravalent antibodies against EGFRwt [Table 3]

[0178] Table 4. Binding kinetics of bispecific tetravalent antibodies against EGFRvIII [Table 4]

[0179] Table 5. EGFRwt epitope binning of EGFRwt- and EGFRvIII-targeting antibodies. [Table 5]

[0180] Table 6. EGFRvIII epitope binning of EGFRwt- and EGFRvIII-targeting antibodies [Table 6]

[0181] Table 7. Characterization of humanized ABT-806 variants, including expression titer and purity assessed using analytical size exclusion chromatography after protein A purification. [Table 7]

[0182] Table 8. Binding affinity of humanized ABT-806 antibody to EGFRvIII. [Table 8]

[0183] Table 9. On-cell binding assay verifying that antibodies maintained binding to antigen on cells. All values ​​reported are MFI at 1.1 μg / mL for each antibody. [Table 9]

[0184] Table 10. Similarity of the lead sequence of humanized ABT-806 to all sequences in the LENS database. [Table 10]

[0185] Table 11. Energy calculations of dematuration cetuximab mutations [Table 11]

[0186] Table 12. Characterization of desaturated cetuximab variants, including expression titer and purity assessed using analytical size exclusion chromatography after Protein A purification. [Table 12]

[0187] Table 13. Binding affinity and avidity of de-matured cetuximab to EGFR [Table 13]

[0188] Table 14. Binding affinity and avidity of de-matured cetuximab to EGFRvIII [Table 14]

[0189] Table 15. On-cell binding assay verifying that antibodies maintained binding to antigen on cells. All values ​​reported are MFI at 10 μg / mL for each antibody. [Table 15]

[0190] Table 16. Configurations of anti-EGFR x EGFRvIII bispecific antibodies and component mAbs containing humanized desmature EGFR binding domain and humanized EGFRvIII binding domain. [Table 16] JPEG2025532482000018.jpg153163

[0191] Table 17. Characterization of anti-EGFR x EGFRvIII bispecific antibodies comprising a humanized desmature EGFR binding domain and a humanized EGFRvIII binding domain, including expression titer and purity assessed using analytical size exclusion chromatography after Protein A purification. [Table 17]

[0192] Table 18. Binding kinetics of anti-EGFR x EGFRvIII dual epitope tetravalent antibodies containing humanized de-matured EGFR binding domain and humanized EGFRvIII binding domain to EGFR-WT [Table 18]

[0193] Table 19. Binding kinetics of anti-EGFR x EGFRvIII bispecific antibodies containing a humanized de-matured EGFR binding domain and a humanized EGFRvIII binding domain to EGFRvIII. [Table 19]

[0194] References 1. Hanif F, Muzaffar K, Perveen K, Malhi SM, Simjee SU. Glioblastoma multiforme: A review of its epidemiology and pathogenesis through clinical presentation and treatment. Asian Pacific J Cancer Prev 2017; 18:3 - 9. 2. Gan HK, Cvrljevic AN, Johns TG. The epidermal growth factor receptor variant III (EGFRvIII): Where wild things are altered. FEBS J 2013; 280:5350 - 70. 3. Ramakrishnan MS, Eswaraiah A, Crombet T, Piedra P, Saurez G, Iyer H, Arvind AS. Nimotuzumab, a promising therapeutic monoclonal for treatment of tumors of epithelial origin. MAbs [Internet] 2009; 1:41 - 8. Available from: www.landesbioscience.com 4.AbbVie Provides Update on Depatuxizumab Mafodotin (Depatux-M), an Investigational Medicine for Newly Diagnosed Glioblastoma, an Aggressive Form of Brain Cancer [Internet]. AbbVie Press Release2019 [cited 2022 Mar 31]; Available from:https: / / news.abbvie.com / news / press-releases / abbvie-provides-update-on-depatuxizumab-mafodotin-depatux-m-an-investigational-medicine-for-newly-diagnosed-glioblastoma-an-aggressive-form-brain-cancer.htm 5.Ellwanger K, Reusch U, Fucek I, Knackmuss S, Weichel M, Gantke T, Molkenthin V, Zhukovsky EA, Tesar M, Treder M. Highly specific and effective targeting of EGFRvIII-positive tumors with TandAb antibodies. Front Oncol 2017; 7:1-17. 6.Gedeon PC, Schaller TH, Chitneni SK, Choi BD, Kuan CT, Suryadevara CM, Snyder DJ, Schmittling RJ, Szafranski SE, Cui X, et al. A rationally designed fully human EGFRvIII:CD3-targeted bispecific antibody redirects human T cells to treat patient-derived intracerebral malignant glioma. Clin Cancer Res 2018; 24:3611-31. 7.Li, S.; Schmitz, K.R.; Jeffrey, P.D.; Wiltzius, J.J.W.; Kussie, P.; Ferguson, K.M. Structural basis for inhibition of the epidermal growth factor receptor by Cetuximab. Cancer Cell 2005, 7, 301-311, doi:10.1016 / j.ccr.2005.03.003. 11.Sickmier, E.A.; Kurzeja, R.J.M.; Michelsen, K.; Vazir, M.; Yang, E.; Tasker, A.S. The panitumumab EGFR complex reveals a binding mechanism that overcomes Cetuximab induced resistance. PLoS One 2016, 11, 1-11, doi:10.1371 / journal.pone.0163366. 12. Bagchi, A.; Haidar, J.N.; Eastman, S.W.; Vieth, M.; Topper, M.; Iacolina, M.D.; Walker, J.M.; Forest, A.; Shen, Y.; Novosiadly, R.D.; et al. Molecular basis for necitumumab inhibition of EGFR variants associated with acquired Cetuximab resistance. Mol. Cancer Ther. 2018, 17, 521 - 531, doi:10.1158 / 1535 - 7163.MCT - 17 - 0575.

[0195] Sequence Listing JPEG2025532482000022.jpg229158JPEG2025532482000023.jpg225158JPEG2025532482000024.jpg72158JPEG2025532482000025.jpg192118 >Seq ID 1: Cetuximab heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 2: Cetuximab heavy chain nucleotide sequence >Seq ID 3: Cetuximab light chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 4: Cetuximab light chain nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Seq ID 5: Nimotuzumab heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 6: Nimotuzumab heavy chain nucleotide sequence >Seq ID 7: Nimotuzumab light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 8: Nimotuzumab light chain nucleotide sequence GATATTCAAATGACTCAATCTCCTTCTTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTACTTGTCGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGATTGGTATCAACAAACTCCTGGTAAAGCTCCTAAACTTCTTATTTATAAAGTTTCTAATCGTTTTTCTGGTGTTCCTTCTCGTTTTTCTGGTTCTGGTTCTGGTACTGATTTTACTTTTACTATTTCTTCTCTTCAACCTGAAGATATTGCTACTTATTATTGTTTTCAATATTCTCATGTTCCTTGGACTTTTGGTCAAGGTACTAAACTTCAAATTACTCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Seq ID 9: ABT-806 heavy chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Seq ID 10: ABT-806 heavy chain nucleotide sequence >Seq ID 11: ABT-806 light chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 12: ABT-806 light chain nucleotide sequence GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATTAACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGACGATGAAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Seq ID 13: SI-95X1 heavy chain amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK >Seq ID 14: SI-95X1 heavy chain nucleotide sequence >Seq ID 15: SI-95X2 heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK >Seq ID 16: SI-95X2 heavy chain nucleotide sequence >Seq ID 17: SI-95X3 heavy chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK >Seq ID 18: SI-95X3 heavy chain nucleotide sequence >Seq ID 19: SI-95X4 heavy chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQIT >Seq ID 20: SI-95X4 heavy chain nucleotide sequence >Seq ID 21: SI-95X5 heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 22: SI-95X5 heavy chain nucleotide sequence >Seq ID 23: SI-95X6 heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 24: SI-95X6 heavy chain nucleotide sequence >Seq ID 25: SI-95X7 heavy chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 26: SI-95X7 heavy chain nucleotide sequence >Seq ID 27: SI-95X8 heavy chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 28: SI-95X8 heavy chain nucleotide sequence >Seq ID 29: SI-95X9 heavy chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 30: SI-95X9 heavy chain nucleotide sequence >Seq ID 31: SI-95X10 heavy chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 32: SI-95X10 heavy chain nucleotide sequence >Seq ID 33: SI-95X11 light chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 34: SI-95X11 light chain nucleotide sequence >Seq ID 35: SI-95X12 light chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 36: SI-95X12 light chain nucleotide sequence >Seq ID 37: SI-95X13 light chain amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 38: SI-95X13 light chain nucleotide sequence >Seq ID 39: SI-95X14 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 40: SI-95X14 light chain nucleotide sequence >Seq ID 41: SI-95X15 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 42: SI-95X15 light chain nucleotide sequence >Seq ID 43: SI-95X16 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 44: SI-95X16 light chain nucleotide sequence >Seq ID 45: SI-95X17 heavy chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKSGSGSGGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 46: SI-95X17 heavy chain nucleotide sequence >Seq ID 47: SI-95X18 heavy chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGSGSGGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 48: SI-95X18 heavy chain nucleotide sequence >Seq ID 49: SI-95X19 heavy chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 50: SI-95X19 heavy chain nucleotide sequence >Seq ID 51: SI-95X20 heavy chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSASGSGSGGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 52: SI-95X20 heavy chain nucleotide sequence >Seq ID 53: SI-95X21 heavy chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGSGSGGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 54: SI-95X21 heavy chain nucleotide sequence >Seq ID 55: SI-95X22 heavy chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIYWVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITADESSTTAYMELSSLRSEDTAFYFCTRQGLWFDSDGRGFDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 56: SI-95X22 heavy chain nucleotide sequence >Seq ID 57: SI-95X23 light chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKSGSGSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 58: SI-95X23 light chain nucleotide sequence >Seq ID 59: SI-95X24 light chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGSGSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 60: SI-95X24 light chain nucleotide sequence >Seq ID 61: SI-95X25 light chain amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 62: SI-95X25 light chain nucleotide sequence >Seq ID 63: SI-95X26 light chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSASGSGSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 64: SI-95X26 light chain nucleotide sequence >Seq ID 65: SI-95X27 light chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGSGSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 66: SI-95X27 light chain nucleotide sequence >Seq ID 67: SI-95X28 light chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 68: SI-95X28 light chain nucleotide sequence >Seq ID 69: SI-95X29 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK >Seq ID 70: SI-95X29 light chain nucleotide sequence >Seq ID 71: SI-95X30 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK >Seq ID 72: SI-95X30 light chain nucleotide sequence >Seq ID 73: SI-95X31 light chain amino acid sequence DIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGNTYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK >Seq ID 74: SI-95X31 light chain nucleotide sequence >Seq ID 75: SI-95X35 heavy chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 76: SI-95X35 heavy chain nucleotide sequence >Seq ID 77: SI-95X36 light chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSSGSGSGGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 78: SI-95X36 light chain nucleotide sequence >Seq ID 79: SI-95X37 heavy chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSASGSGSGGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 80: SI-95X37 heavy chain nucleotide sequence >Seq ID 81: SI-95X38 light chain amino acid sequence DILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIKGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSASGSGSGGGGGSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 82: SI-95X38 light chain nucleotide sequence >Seq ID 83: SI-95X39 light chain amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSGSGSGGGGGSGGGGSGGGGSDVQLQESGPSLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYISYSGNTRYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCVTAGRGFPYWGQGTLVTVSAGGGGSGGGGSGGGGSDILMTQSPSSMSVSLGDTVSITCHSSQDINSNIGWLQQRPGKSFKGLIYHGTNLDDEVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPWTFGGGTKLEIK >Seq ID 84: SI-95X39 light chain nucleotide sequence >Seq ID 85: SI-95X40 heavy chain amino acid sequence >Seq ID 86: SI-95X40 heavy chain nucleotide sequence >Seq ID 87: SI-95X41 heavy chain amino acid sequence >Seq ID 88: SI-95X41 heavy chain nucleotide sequence >Seq ID 89: SI-95X42 heavy chain amino acid sequence >Seq ID 90: SI-95X42 heavy chain nucleotide sequence >Seq ID 91: SI-95X43 light chain amino acid sequence >Seq ID 92: SI-95X43 light chain nucleotide sequence >Seq ID 93: SI-95X44 heavy chain amino acid sequence >Seq ID 94: SI-95X44 heavy chain nucleotide sequence >Seq ID 95: SI-95X44 light chain amino acid sequence >Seq ID 96: SI-95X44 light chain nucleotide sequence >Seq ID 97: SI-95X45 heavy chain amino acid sequence >Seq ID 98: SI-95X45 heavy chain nucleotide sequence >Seq ID 99: SI-95m5 light chain amino acid sequence >Seq ID 100: SI-95m5 light chain nucleotide sequence >Seq ID 101: SI-95X46 heavy chain amino acid sequence >Seq ID 102: SI-95X46 heavy chain nucleotide sequence >Seq ID 103: SI-95X47 heavy chain amino acid sequence >Seq ID 104: SI-95X47 heavy chain nucleotide sequence >Seq ID 105: SI-95X48 heavy chain amino acid sequence >Seq ID 106: SI-95X48 heavy chain nucleotide sequence >Seq ID 107: SI-95X49 heavy chain amino acid sequence >Seq ID 108: SI-95X49 heavy chain nucleotide sequence >Seq ID 109: SI-95X49 light chain amino acid sequence >Seq ID 110: SI-95X49 light chain nucleotide sequence >Seq ID 111: SI-95m4 light chain amino acid sequence >Seq ID 112: SI-95m4 light chain nucleotide sequence >Seq ID 113: SI-95X52 heavy chain amino acid sequence >Seq ID 114: SI-95X52 heavy chain nucleotide sequence >Seq ID 115: SI-95X53 heavy chain amino acid sequence >Seq ID 116: SI-95X53 heavy chain nucleotide sequence >Seq ID 117: SI-95X54 heavy chain amino acid sequence >Seq ID 118: SI-95X54 heavy chain nucleotide sequence >Seq ID 119: SI-95X55 heavy chain amino acid sequence >Seq ID 120: SI-95X55 heavy chain nucleotide sequence >Seq ID 121: SI-95X56 heavy chain amino acid sequence >Seq ID 122: SI-95X56 heavy chain nucleotide sequence >Seq ID 123: SI-95m5 heavy chain amino acid sequence >Seq ID 124: SI-95m5 heavy chain nucleotide sequence >Seq ID 125: SI-95X57 light chain amino acid sequence >Seq ID 126: SI-95X50 light chain nucleotide sequence >Seq ID 127: SI-95X58 heavy chain amino acid sequence >Seq ID 128: SI-95X58 heavy chain nucleotide sequence >Seq ID 129: SI-95X59 heavy chain amino acid sequence >Seq ID 130: SI-95X59 heavy chain nucleotide sequence >Seq ID 131: SI-95X60 heavy chain amino acid sequence >Seq ID 132: SI-95X60 heavy chain nucleotide sequence >Seq ID 133: SI-95X61 heavy chain amino acid sequence >Seq ID 134: SI-95X61 heavy chain nucleotide sequence >Seq ID 135: SI-95X62 light chain amino acid sequence >Seq ID 136: SI-95X62 light chain nucleotide sequence >Seq ID 137: SI-95X63 heavy chain amino acid sequence >Seq ID 138: SI-95X63 heavy chain nucleotide sequence >Seq ID 139: SI-95X64 heavy chain amino acid sequence >Seq ID 140: SI-95X64 heavy chain nucleotide sequence >Seq ID 141: SI-95X65 heavy chain amino acid sequence >Seq ID 142: SI-95X65 heavy chain nucleotide sequence >Seq ID 143: SI-95X66 heavy chain amino acid sequence >Seq ID 144: SI-95X66 heavy chain nucleotide sequence >Seq ID 145: SI-95X67 heavy chain amino acid sequence >Seq ID 146: SI-95X67 heavy chain nucleotide sequence >Seq ID 147: SI-95X68 heavy chain amino acid sequence >Seq ID 148: SI-95X68 heavy chain nucleotide sequence >Seq ID 149: SI-95m4 heavy chain amino acid sequence >Seq ID 150: SI-95m4 heavy chain nucleotide sequence >Seq ID 151: SI-95m11 light chain amino acid sequence >Seq ID 152: SI-95m11 light chain nucleotide sequence >Seq ID 153: SI-95m12 light chain amino acid sequence >Seq ID 154: SI-95m12 light chain nucleotide sequence >Seq ID 155: SI-95m13 heavy chain amino acid sequence >Seq ID 156: SI-95m13 heavy chain nucleotide sequence >Seq ID 157: SI-95m14 heavy chain amino acid sequence >Seq ID 158: SI-95m14 heavy chain nucleotide sequence >Seq ID 159: SI-95m15 heavy chain amino acid sequence >Seq ID 160: SI-95m15 heavy chain nucleotide sequence >Seq ID 161: SI-95m16 heavy chain amino acid sequence >Seq ID 162: SI-95m16 heavy chain nucleotide sequence >Seq ID 163: SI-95m17 heavy chain amino acid sequence >Seq ID 164: SI-95m17 heavy chain nucleotide sequence >Seq ID 165: SI-95m18 heavy chain amino acid sequence >Seq ID 166: SI-95m18 heavy chain nucleotide sequence >Seq ID 167: SI-95m19 heavy chain amino acid sequence >Seq ID 168: SI-95m19 heavy chain nucleotide sequence >Seq ID 169: SI-95m20 heavy chain amino acid sequence >Seq ID 170: SI-95m20 heavy chain nucleotide sequence >Seq ID 171: ABT-806 V1 heavy chain amino acid sequence >Seq ID 172: ABT-806 V1 heavy chain nucleotide sequence >Seq ID 173: ABT-806 V1 light chain amino acid sequence >Seq ID 174: ABT-806 V1 light chain nucleotide sequence >Seq ID 175: ABT-806 V2 light chain amino acid sequence >Seq ID 176: ABT-806 V2 light chain nucleotide sequence >Seq ID 177: ABT-806 V4 heavy chain amino acid sequence >Seq ID 178: ABT-806 V4 heavy chain nucleotide sequence >Seq ID 179: SI-95X51 heavy chain amino acid sequence >Seq ID 180: SI-95X51 heavy chain nucleotide sequence >Seq ID 201: Cetuximab VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDYEFAY WGQGTLVTVSS >Seq ID 202: Cetuximab VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 203: Cetuximab VL amino acid sequence DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNNNWPTT FGAGTKLELK >Seq ID 204: Cetuximab VL nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA >Seq ID 205: Nimotuzumab VH amino acid sequence QVQLQQSGAEVKKPGSSVKVSCKASGYTFT NYYIY WVRQAPGQGLEWIG GINPTSGGSNFNEKFKT RVTITADESSTTAYMELSSLRSEDTAFYFCTR QGLWFDSDGRGFDF WGQGTTVTVSS >Seq ID 206: Nimotuzumab VH nucleotide sequence CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACTACATCTACTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATCGGCGGCATCAACCCCACCAGCGGCGGCAGCAACTTCAACGAGAAGTTCAAGACCCGGGTGACCATCACCGCCGACGAGAGCAGCACCACCGCCTACATGGAGCTGAGCAGCCTGCGGAGCGAGGACACCGCCTTCTACTTCTGCACCCGGCAGGGCCTGTGGTTCGACAGCGACGGCCGGGGCTTCGACTTCTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC >Seq ID 207: Nimotuzumab VL amino acid sequence DIQMTQSPSSLSASVGDRVTITC RSSQNIVHSNGNTYLD WYQQTPGKAPKLLIY KVSNRFS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC FQYSHVPWT FGQGTKLQIT >Seq ID 208: Nimotuzumab VL nucleotide sequence GATATTCAAATGACTCAATCTCCTTCTTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTACTTGTCGTTCTTCTCAAAATATTGTTCATTCTAATGGTAATACTTATCTTGATTGGTATCAACAAACTCCTGGTAAAGCTCCTAAACTTCTTATTTATAAAGTTTCTAATCGTTTTTCTGGTGTTCCTTCTCGTTTTTCTGGTTCTGGTTCTGGTACTGATTTTACTTTTACTATTTCTTCTCTTCAACCTGAAGATATTGCTACTTATTATTGTTTTCAATATTCTCATGTTCCTTGGACTTTTGGTCAAGGTACTAAACTTCAAATTACT >Seq ID 209: ABT-806 VH amino acid sequence DVQLQESGPSLVKPSQSLSLTCTVTGYSIT SDFAWN WIRQFPGNKLEWMG YISYSGNTRYNPSLKS RISITRDTSKNQFFLQLNSVTIEDTATYYCVT AGRGFPY WGQGTLVTVSA >Seq ID 210: ABT-806 VH nucleotide sequence GATGTGCAGCTTCAGGAGTCGGGACCTAGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTTTGCCTGGAACTGGATTCGGCAGTTTCCAGGAAACAAGCTGGAGTGGATGGGCTACATAAGTTATAGTGGTAACACTAGGTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGCGACACATCCAAGAACCAATTCTTCCTGCAGTTGAACTCTGTGACTATTGAGGACACAGCCACATATTACTGTGTAACGGCGGGACGCGGGTTTCCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA >Seq ID 211: ABT-806 VL amino acid sequence DILMTQSPSSMSVSLGDTVSITC HSSQDINSNIG WLQQRPGKSFKGLIY HGTNLDD EVPSRFSGSGSGADYSLTISSLESEDFADYYC VQYAQFPWT FGGGTKLEIK >Seq ID 212: ABT-806 VL nucleotide sequence GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCATTCAAGTCAGGACATTAACAGTAATATAGGGTGGTTGCAGCAGAGACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAACTTGGACGATGAAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCCGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGCTCAGTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA >Seq ID 213: (G4S)2 linker amino acid sequence GGGGSGGGGS >Seq ID 214: (G4S)2 linker nucleotide sequence GGCGGTGGAGGGTCCGGCGGTGGTGGATCC >Seq ID 215: (G4S)4 linker amino acid sequence GGGGSGGGGSGGGGS >Seq ID 216: (G4S)4 linker nucleotide sequence GGTGGAGGAGGCAGTGGTGGTGGAGGAAGCGGAGGTGGTGGCAGC >Seq ID 217: (G4S)5 linker amino acid sequence GGGGSGSGSGGGGGSGGGGSGGGGS >Seq ID 218: (G4S)5 linker nucleotide sequence GGTGGCGGTGGCTCCGGATCCGGTTCTGGAGGAGGCGGTGGAAGCGGAGGCGGTGGCTCTGGAGGAGGCGGTTCG >Seq ID 219: (G4S)6 linker amino acid sequence GGGGSGGGGSGSGSGGGGGSGGGGSGGGGS >Seq ID 220: (G4S)6 linker nucleotide sequence GGAGGCGGTGGTAGCGGTGGCGGTGGCTCCGGATCCGGTTCTGGAGGAGGCGGTGGAAGCGGAGGCGGTGGCTCTGGAGGAGGCGGTTCG >Seq ID 221: Human IgG1 amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Seq ID 222: Human IgG1 nucleotide sequence GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT >Seq ID 223: Human CK amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Seq ID 224: Human CK nucleotide sequence CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT >Seq ID 225: ABT-806 V1 VH amino acid sequence QVQLQESGPGLVKPSQSLSLTCTVSGYSITSDFAWNWIRQPPGKGLEWIGYISYSGNTRYNPSLKSRVTISRDTSKNQFSLQLNSVTAEDTAVYYCVTAGRGFPYWGQGTLVTVSS >Seq ID 226: ABT-806 V1 VH nucleotide sequence CAAGTGCAGTTGCAGGAGAGTGGTCCGGGCCTGGTGAAGCCTTCTCAGTCACTGAGCCTCACGTGCACCGTAAGCGGTTATAGTATCACCAGTGATTTTGCCTGGAACTGGATTCGGCAGCCGCCCGGGAAGGGACTGGAGTGGATCGGATACATTTCCTACAGTGGAAACACTCGATATAATCCTAGTCTTAAAAGTCGGGTAACCATCTCTAGGGATACCAGCAAGAATCAGTTCTCATTGCAACTCAATTCAGTGACCGCAGAAGACACCGCAGTCTACTATTGCGTCACAGCCGGACGCGGTTTCCCGTACTGGGGCCAAGGAACACTAGTCACCGTGTCTTCC >Seq ID 227: ABT-806 V1 VL amino acid sequence DIQMTQSPSSVSASVGDRVTITCHSSQDINSNIGWLQQKPGKAPKGLIYHGTNLDDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPWTFGQGTKVEIK >Seq ID 228: ABT-806 V1 VL nucleotide sequence GACATTCAGATGACACAGAGCCCAAGTAGCGTTTCTGCCAGCGTGGGGGACCGAGTCACCATTACTTGTCACAGTTCACAAGATATTAACTCTAACATCGGCTGGCTGCAGCAAAAACCAGGTAAAGCACCAAAAGGGCTCATTTATCATGGGACCAACCTGGATGACGGAGTCCCGAGTCGATTCAGCGGGAGTGGAAGCGGAGCTGATTATACCCTGACGATCTCAAGCTTGCAGCCAGAGGATTTCGCCACATACTATTGTGTCCAGTATGCCCAGTTCCCCTGGACATTTGGCCAGGGTACGAAGGTGGAGATTAAG >Seq ID 229: ABT-806 V2 VH amino acid sequence QVQLQESGPGLVKPSQSLSLTCTVSGYSITSDFAWNWIRQAPGKGLEWVGYISYSGNTRYNPSLKSRFTISRDTSKNQFYLQLNSVTAEDTAVYYCVTAGRGFPYWGQGTLVTVSS >Seq ID 230: ABT-806 V2 VH nucleotide sequence CAAGTCCAGCTGCAGGAAAGTGGCCCTGGACTCGTTAAGCCTAGTCAGAGCTTGTCATTGACTTGCACAGTCAGTGGTTATTCCATCACAAGTGACTTTGCCTGGAATTGGATTCGGCAGGCTCCCGGGAAGGGGCTGGAATGGGTCGGGTATATTTCCTACAGCGGAAATACTCGATATAACCCATCCCTTAAAAGTCGCTTCACCATATCTCGTGACACCAGCAAAAATCAATTTTATCTTCAACTCAATTCAGTGACAGCCGAGGATACAGCTGTTTACTATTGTGTGACAGCTGGCAGAGGATTTCCCTATTGGGGACAGGGAACCCTTGTAACGGTCTCCAGC >Seq ID 231: ABT-806 V2 VL amino acid sequence DIVMTQSPSSLSVSPGDRVTITCHSSQDINSNIGWLQQKPGKAPKGLIYHGTNLDDGVPSRFSGSGSGADYTLTISSLEPEDFAVYYCVQYAQFPWTFGQGTKVEIK >Seq ID 232: ABT-806 V2 VL nucleotide sequence GACATAGTGATGACACAGTCTCCAAGTTCTTTGAGCGTATCCCCAGGCGACCGCGTAACAATTACCTGTCACAGCTCCCAGGATATTAATAGTAATATTGGCTGGCTTCAACAGAAGCCAGGGAAGGCACCAAAAGGACTGATCTACCACGGAACTAATCTGGACGATGGGGTTCCTTCACGGTTCTCCGGGTCCGGCTCTGGTGCCGACTACACTCTGACAATTAGCTCCCTGGAGCCCGAGGACTTCGCAGTGTACTATTGCGTGCAGTACGCACAATTTCCCTGGACATTTGGTCAAGGCACCAAAGTCGAAATTAAA >Seq ID 233: ABT-806 V3 VH amino acid sequence QVQLQESGPSLVKPSQTLSLTCTVSGYSITSDFAWNWIRQFPGRKLEWMGYISYSGNTRYNPSLKSRFTISRDTSKNQFYLKLRSVTIADTAVYYCVTAGRGFPYWGQGTLVTVSS >Seq ID 234: ABT-806 V3 VH nucleotide sequence CAAGTGCAGCTCCAGGAGTCAGGCCCTAGCCTCGTGAAGCCCTCCCAGACCCTGAGCCTGACCTGCACCGTATCTGGTTATAGTATTACAAGTGACTTCGCCTGGAACTGGATTAGGCAGTTTCCAGGTCGCAAGCTGGAGTGGATGGGGTATATCTCCTACTCCGGTAACACTCGATACAACCCTTCCCTCAAAAGCAGATTTACCATTTCCAGAGACACATCTAAGAACCAATTTTATCTGAAGTTGAGATCTGTTACCATCGCTGATACTGCTGTGTACTATTGCGTTACCGCCGGACGCGGCTTCCCCTATTGGGGGCAGGGAACCCTGGTGACTGTATCATCC >Seq ID 235: ABT-806 V3 VL amino acid sequence DIVMTQSPSSVSLSLGDRVTITCHSSQDINSNIGWLQQKPGKAFKGLIYHGTNLDDGVPSRFSGSGSGADYTLTISSLQAEDFATYYCVQYAQFPWTFGPGTKLEIK >Seq ID 236: ABT-806 V3 VL nucleotide sequence GACATCGTGATGACCCAATCTCCAAGCTCAGTGAGTCTGTCACTAGGCGACCGTGTCACCATTACATGTCATAGTTCCCAGGATATCAACAGTAATATTGGGTGGCTTCAGCAAAAGCCTGGTAAAGCCTTTAAAGGACTGATTTATCACGGGACCAACTTGGACGATGGAGTTCCCTCCCGGTTTTCTGGCTCCGGGTCCGGTGCAGACTATACTTTGACAATTAGCTCTCTCCAGGCAGAGGACTTCGCTACATATTACTGCGTCCAGTATGCCCAGTTCCCTTGGACCTTTGGACCCGGCACTAAACTGGAAATTAAA >Seq ID 237: SI-95m5 (humanized Cetuximab) VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Seq ID 238: SI-95m5 (humanized Cetuximab) VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Seq ID 239: SI-95m5 (humanized Cetuximab) VL amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVL >Seq ID 240: SI-95m5 (humanized Cetuximab) VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACCGTGCTG >Seq ID 241: Cetuximab N92F VL amino acid sequence EIVLTQSPSTLSVSPGERATFSC RASQSIGTNIH WYQQKPGKPPRLLIK YASESIS GIPDRFSGSGSGTEFTLTISSVQSEDFAVYYC QQNFNWPTT FGPGTKLTVL >Seq ID 242: Cetuximab N92F VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACTTCAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACCGTGCTG >Seq ID 243: Cetuximab N92K VL amino acid sequence DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNKNWPTT FGAGTKLELK >Seq ID 244: Cetuximab N92K VL nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAAGAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA >Seq ID 245: Humanized Cetuximab Y101A VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLT NYGVH WIRQAPGKGLEWLG VIWSGGNTDYNTPFTS RFTITKDNSKNQVYFKLRSVRADDTAIYYCAR ALTAYDYEFAY WGQGTLVTVSS >Seq ID 246: Humanized Cetuximab Y101A VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCGCCTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Seq ID 247: Cetuximab Y101W VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTWYDYEFAY WGQGTLVTVSS >Seq ID 248: Cetuximab Y101W VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTGGTATGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 249: Humanized Cetuximab Y102A VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLT NYGVH WIRQAPGKGLEWLG VIWSGGNTDYNTPFTS RFTITKDNSKNQVYFKLRSVRADDTAIYYCAR ALTYADYEFAY WGQGTLVTVSS >Seq ID 250: Humanized Cetuximab Y102A VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACGCCGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Seq ID 251: Cetuximab D103F VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYFYEFAY WGQGTLVTVSS >Seq ID 252: Cetuximab D103F VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATTTCTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 253: Cetuximab D103W VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYWYEFAY WGQGTLVTVSS >Seq ID 254: Cetuximab D103W VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATTGGTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 255: Humanized Cetuximab D103Y VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLT NYGVH WIRQAPGKGLEWLG VIWSGGNTDYNTPFTS RFTITKDNSKNQVYFKLRSVRADDTAIYYCAR ALTYYYYEFAY WGQGTLVTVSS >Seq ID 256: Humanized Cetuximab D103Y VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACTACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Seq ID 257: Humanized Cetuximab Y104L VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLT NYGVH WIRQAPGKGLEWLG VIWSGGNTDYNTPFTS RFTITKDNSKNQVYFKLRSVRADDTAIYYCAR ALTYYDLEFAY WGQGTLVTVSS >Seq ID 258: Humanized Cetuximab Y104L VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACCTCGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Seq ID 259: Cetuximab Y101A VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTAYDYEFAY WGQGTLVTVSS >Seq ID 260: Cetuximab Y101A VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCGCCTATGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 261: Cetuximab Y102A VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYADYEFAY WGQGTLVTVSS >Seq ID 262: Cetuximab Y102A VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACGCCGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 263: Cetuximab Y102V VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYVDYEFAY WGQGTLVTVSS >Seq ID 264: Cetuximab Y102V VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACGTGGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 265: Cetuximab D103Y VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYYYEFAY WGQGTLVTVSS >Seq ID 266: Cetuximab D103Y VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATTACTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 267: Cetuximab Y104L VH amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGNTDYNTPFTS RLSINKDNSKSQVFFKMNSLQSNDTAIYYCAR ALTYYDLEFAY WGQGTLVTVSS >Seq ID 268: Cetuximab Y104L VH nucleotide sequence CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATGATCTCGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTAGC >Seq ID 269: Cetuximab N92F VL amino acid sequence DILLTQSPVILSVSPGERVSFSC RASQSIGTNIH WYQQRTNGSPRLLIK YASESIS GIPSRFSGSGSGTDFTLSINSVESEDIADYYC QQNFNWPTT FGAGTKLELK >Seq ID 270: Cetuximab N92F VL nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATTTCAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

Claims

1. A biepitope tetravalent antibody having binding affinity to at least two epitopes of EGFR, Antibody light chain having a variable (VL) domain, The antibody backbone comprises an antibody heavy chain having a variable (VH) domain, Here, the antibody VL domain and the antibody VH domain form a Fab region, and The scFv domain comprises an scFv light chain variable (VL) domain and an scFv heavy chain variable (VH) domain, wherein the scFv domain is linked to at least one end of the antibody light chain or the antibody heavy chain via an interdomain linker. Bivalent epitope tetravalent antibody.

2. The bi-epitope tetravalent antibody according to claim 1, wherein the two EGFR epitopes include an EGFR wild-type (EGFRwt) epitope and an EGFRvIII epitope, and the antibody has a stronger binding affinity to the EGFRvIII epitope than to the EGFRwt epitope.

3. The biepitope tetravalent antibody according to claim 2, wherein the antibody has binding affinity to the EGFRwt epitope at the first KD and binding affinity to the EGFRvIII epitope at the second KD, where the first KD is higher than the second KD, the first KD is not less than 1E-11 M, and the second KD is not greater than 1E-06 M.

4. The bi-epitope tetravalent antibody according to claim 2, wherein the EGFRwt epitope comprises an epitope having binding affinity to cetuximab, or the EGFRvIII epitope comprises an epitope having binding affinity to ABT-806.

5. The scFv domain has binding affinity to the EGFRwt epitope, and the Fab domain has binding affinity to the EGFRvIII epitope, or The scFv domain has binding affinity to the EGFRvIII epitope, and the Fab domain has binding affinity to the EGFRwt epitope. The biepitopic tetravalent antibody according to claim 2.

6. The biepitope tetravalent antibody according to claim 1, wherein the scFv domain is linked to the antibody light chain at its C-terminus, to the antibody light chain at its N-terminus, to the antibody heavy chain at its C-terminus, or to the antibody heavy chain at its N-terminus.

7. The biepitope tetravalent antibody according to claim 1, wherein the antibody skeleton comprises cetuximab, humanized cetuximab, dematured cetuximab, or humanized dematured cetuximab, and the scFv domain comprises a binding domain derived from the variable region of ABT-806 or humanized ABT-806.

8. The biepitopic tetravalent antibody according to claim 7, wherein the antibody backbone comprises cetuximab dematuration mutations (sequentially numbered) including VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL-N92F, VL-N92K, or combinations thereof.

9. The biepitopic tetravalent antibody according to claim 7, wherein the antibody heavy chain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs: 1, 13, 79, 97, 101, 103, 105, 107, 123, 127, 129, 131, or 133, or the antibody light chain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs: 3, 81, 83, 99, 109, 125, or 135.

10. The biepitope tetravalent antibody according to claim 7, wherein the antibody skeleton comprises humanized cetuximab, the antibody VH domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 237, or the antibody VL domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

239.

11. The biepitope tetravalent antibody according to claim 7, wherein the antibody skeleton comprises dematured cetuximab, and the antibody VH domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NOs: 247, 251, 253, 259, 261, 263, 265, or 267, or the antibody VL domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NOs: 243 or 269.

12. The biepitope tetravalent antibody according to claim 7, wherein the antibody skeleton comprises the humanized demature cetuximab, and the antibody VH domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 245, 249, 255, or 257, or the antibody VL domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

241.

13. The biepitope tetravalent antibody according to claim 7, wherein the scFv domain comprises the variable region of ABT-806 or humanized ABT-806.

14. The biepitope tetravalent antibody according to claim 7, wherein the scFv VH domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs. 209, 225, 229, or 233, or the scFv VL domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs. 211, 227, 231, or 235.

15. The antibody VH domain includes three complementarity-determining regions (CDRs) of sequence numbers 201, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267. The antibody VL domain comprises three CDRs, SEQ ID NOs. 203, 239, 241, 243, or 269. The scFv VH domain includes three CDRs of sequence number 209, and The scFv VL domain contains three CDRs of sequence number 211, The biepitopic tetravalent antibody according to claim 7.

16. The biepitope tetravalent antibody according to claim 1, wherein the antibody skeleton comprises ABT0806 or humanized ABT-806, and the scFv domain comprises a binding domain derived from the variable region of cetuximab, humanized cetuximab, demature cetuximab, humanized demature cetuximab, or nimotuzumab.

17. The biepitope tetravalent antibody according to claim 16, wherein the antibody heavy chain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs: 9, 17, 19, 21, 23, 25, 27, 29, 31, 75, 93, 113, 115, 117, 119, 121, 137, 139, 141, 143, 145, 147, or 179, or the antibody light chain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs: 11, 33, 35, 37, 39, 41, 43, 77, 91, 95, or 111.

18. The biepitope tetravalent antibody according to claim 16, wherein the antibody skeleton comprises ABT-806, and the antibody VH domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 209, or the antibody VL domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

211.

19. The biepitope tetravalent antibody according to claim 16, wherein the antibody skeleton comprises humanized ABT-806, and the antibody VH domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 225, 229, or 233, or the antibody skeleton comprises humanized ABT-806, and the antibody VL domain comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 227, 231, or 235.

20. The biepitope tetravalent antibody according to claim 16, wherein the scFv VH domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs. 201, 205, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267, or the scFv VL domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs. 203, 207, 239, 241, 243, or 269.

21. The dual epitope tetravalent antibody according to claim 16, wherein the scFv domain comprises a binding domain derived from the variable region of the demature cetuximab or the humanized demature cetuximab, and the demature cetuximab or the humanized demature cetuximab comprises cetuximab demature mutations (sequentially numbered) including VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL-N92F, VL-N92K, or combinations thereof.

22. The antibody VH domain includes three complementarity-determining regions (CDRs) of SEQ ID NO: 209, The antibody VL domain contains three CDRs of Sequence ID No. 211, The scFv VH domain includes three CDRs of sequence numbers 201, 205, 237, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, or 267, and The scFv VL domain includes three CDRs, number 203, 207, 239, 241, 243, or 269. The biepitopic tetravalent antibody according to claim 16.

23. The biepitope tetravalent antibody according to claim 1, wherein the antibody skeleton comprises nimotuzumab, and the scFv domain comprises a binding domain derived from the variable region of ABT-806 or humanized ABT-806.

24. The biepitopic tetravalent antibody according to claim 23, wherein the antibody heavy chain contains an amino acid sequence having 99% sequence identity with SEQ ID NOs. 5, 15, 45, 47, 49, 51, 53, 55, or 85, or the antibody light chain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NOs. 7, 57, 59, 61, 63, 65, 67, 69, 71, or 73.

25. The biepitopic tetravalent antibody according to claim 23, wherein the antibody VH domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 205, or the antibody VL domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

207.

26. The biepitope tetravalent antibody according to claim 23, wherein the scFv VH domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 209, or the scFv VL domain contains an amino acid sequence having at least 99% sequence identity with SEQ ID NO:

211.

27. A demature cetuximab monoclonal antibody comprising a light chain having a variable light chain (VL) domain and a heavy chain having a variable heavy chain (VH) domain, wherein the VL domain and the VH domain form a Fab region, and the monoclonal antibody comprises a cetuximab demature mutation (sequential number) selected from VH-Y101A, VH-Y101W, VH-Y102A, VH-D103F, VH-D103W, VH-D103Y, VH-Y104L, VL-N92F, VL-N92K, or a combination thereof.

28. A humanized anti-EGFRvIII monoclonal antibody comprising a light chain having a variable light chain (VL) domain and a heavy chain having a variable heavy chain (VH) domain, wherein the VL domain and the VH domain form a Fab region, and the heavy chain contains an amino acid sequence having at least 99% sequence identity to SEQ ID NOs. 171, 149, or 177, or the light chain contains an amino acid sequence having at least 99% sequence identity to SEQ ID NOs. 111, 173, or 175.

29. An isolated nucleic acid encoding a biepitopic tetravalent antibody as described in claim 1.

30. A method for producing a biepitope tetravalent antibody, comprising culturing host cells such that the biepitope antibody is produced, wherein the host cells comprise the nucleic acid described in claim 29.

31. An immune complex comprising a double-epitope tetravalent antibody and a cytotoxic agent as described in claim 1, wherein the cytotoxic agent comprises a chemotherapeutic agent, a growth inhibitor, a toxin, or a radioisotope.

32. A pharmaceutical composition comprising a biepitopic tetravalent antibody as described in claim 1, and a pharmaceutically acceptable carrier.

33. The pharmaceutical composition according to claim 32, further comprising a radioisotope, a radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent, or a combination thereof.

34. A pharmaceutical composition comprising the immune complex described in claim 31 and a pharmaceutically acceptable carrier.

35. A pharmaceutical composition for treating cancer, comprising an effective amount of the bivalent epitope tetravalent antibody described in Claim 1, wherein the cancer comprises cells expressing EGFR, EGFRvIII, or both.

36. The pharmaceutical composition according to claim 35, wherein the treatment comprises the combined administration of a bivalent epitope tetravalent antibody and a therapeutic agent according to claim 1, and the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

37. A pharmaceutical composition for treating cancer comprising an effective amount of a therapeutic agent, wherein the treatment comprises the combined administration of a bivalent epitope tetravalent antibody and the therapeutic agent as described in Claim 1, the cancer comprises cells expressing EGFR, EGFRvIII, or both, and the therapeutic agent comprises an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

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