EGFR-binding complex, its manufacturing method and use

Abstract

To provide a protein-based therapeutic agent that reduces the risk of immunogenicity caused by cetuximab in humans. [Solution] The binding domain having binding specificity to human EGFR (epidermal growth factor receptor) has a VH domain and a VL domain, and the VH and VL domains each independently contain a sequence having at least 90% sequence identity with a specific amino acid sequence. The present invention further provides an antibody containing the said binding domain.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims the priority of U.S. Provisional Application No. 63 / 081,315, filed on September 21, 2020, and U.S. Provisional Application No. 63 / 109,877, filed on November 5, 2020, under 35 U.S.C. 119(e), and the entire disclosures of these are incorporated herein by reference.

[0002] This disclosure relates to the technical field of cancer treatment using antibodies, and more specifically, to the production and use of multispecific antibodies.

Background Art

[0003] Cetuximab is a chimeric (mouse / human) monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). It was approved as a therapeutic agent for colorectal cancer in the United States and the EU in 2004 and is also used in the treatment of head and neck cancers 1 - 3. In addition to mAb, cetuximab has also been used in T - cell redirecting bispecific antibodies 4, antibody - peptide fusions, and antibody - drug conjugates 5. Cetuximab can bind to domain III of the extracellular domain of EGFR, which is normally overexpressed in tumor cells. Binding of cetuximab to tumor cells competitively inhibits the binding of EGF and other ligands, preventing EGFR dimerization and suppressing receptor tyrosine autophosphorylation. As a result of inhibition and reduced EGFR-mediated signaling, cetuximab binding effectively downregulates tumor cell proliferation, angiogenesis, and metastasis while inducing apoptosis. In addition to targeting EGFR, the Fc domain of cetuximab can bind to CD16a and other Fc receptors, mobilizing and activating immune mechanisms such as antibody-dependent cell-mediated cytotoxicity.4 These antitumor properties make cetuximab highly desirable for development as a monotherapy or as part of a combination regimen. However, there is concern that the variable region (VH / Vk) of cetuximab remains in the mouse framework after isolation from mouse hybridomas. Studies have shown that using mouse sequences such as mouse VH / Vk increases the incidence of immunogenicity when the protein is administered to human patients. Therefore, protein-based therapies using humanized VH / Vk regions may reduce the risk of cetuximab-induced immunogenicity in humans. [Overview of the project]

[0004] The following summary is illustrative and not intended to be limiting. Further embodiments, features, and characteristics beyond those described above will become apparent by reference to the drawings and the detailed description below.

[0005] The present invention provides a binding domain and peptide having binding specificity to the human epidermal growth factor receptor (EGFR), an antibody-like protein incorporating the anti-EGFR binding domain and peptide disclosed herein, an immune complex and pharmaceutical composition incorporating the anti-EGFR binding domain and peptide disclosed herein, a method for producing and using such an anti-EGFR binding domain, a peptide and an antibody-like protein. In one embodiment, the anti-EGFR antibody-like protein includes an antibody, a monoclonal antibody, a humanized antibody, or a chimeric antibody. In one embodiment, the anti-EGFR antibody may be monospecific or multispecific. In one embodiment, the multispecific anti-EGFR antibody may be bispecific, triplicate, quadruple, quintuple, or hexaspecific. In one embodiment, the anti-EGFR antibody may be symmetric or asymmetric.

[0006] In one embodiment, the present invention provides a human EGFR-binding peptide having binding specificity to human EGFR. The peptide may include an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 57, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or a combination thereof.

[0007] In one embodiment, the EGFR-binding peptide comprises a variable heavy (VH) chain and a variable light (VL) chain. In one embodiment, the VH chain comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, and 41. In one embodiment, the VL chain comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, and 43.

[0008] In one embodiment, the EGFR-binding peptide comprises an scFv domain, the scFv domain comprising the VH and VL chains disclosed herein.

[0009] In one embodiment, the present invention provides an anti-EGFR scFv domain or a peptide forming such scFv domain. In one embodiment, the scFv domain comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 57, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, and 83. In one embodiment, the scFv domain comprises a VH chain and a VL chain as disclosed herein.

[0010] In one embodiment, the EGFR-binding peptide may include a histidine residue bound to at least one terminus of the scFv domain (e.g., ScFV-HIS). In one embodiment, the EGFR-binding peptide may include an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 57.

[0011] In one embodiment, the EGFR-binding peptide may include a Fab domain, the Fab domain comprising the VH and VL chains disclosed herein. In one embodiment, to provide a Fab-monoFc fusion protein, the EGFR-binding peptide may further include an Fc domain bound to the Fab domain. In one embodiment, the Fc domain includes a sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from SEQ ID NOs. 45 and 47.

[0012] In another embodiment, the present invention provides an antibody-like protein having binding specificity to human EGFR. The antibody-like protein may include an EGFR-binding domain having a variable heavy (VH) chain and a variable light (VL) chain. In one embodiment, the VH chain includes an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, or 41. In one embodiment, the VL chain may include an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, or 43.

[0013] In one embodiment, the antibody-like protein may include an scFv domain having an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, or 83.

[0014] In one embodiment, the antibody-like protein may be a monospecific antibody. In one embodiment, the antibody may contain an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 137, 139, 141, 143; 141, 149, 151, 139, 145, 147 or a combination thereof. In one embodiment, the monospecific antibody may contain a pair of light chains and heavy chains or fragments thereof selected from the sequence combinations of SEQ ID NOs: 137 and 139, 141 and 143, 141 and 149, 151 and 139, and 145 and 147.

[0015] In one embodiment, the antibody-like protein may have binding specificity to at least two different antigens selected from tumor antigens, immune signaling antigens, or combinations thereof.

[0016] In one embodiment, the antibody-like protein may be a bispecific antibody. In one embodiment, the bispecific antibody may contain an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 137, 145, 139, 147, 141, 145, 149, 147, 151, 145, 139, 147 or a combination thereof. In one embodiment, the bispecific antibody may contain a combination of light chains and heavy chains (or fragments thereof) selected from the sequence combinations of SEQ ID NOs: 137, 145, 139, 147, 141, 145, 143, 147, 141, 145, 149, 147, and 151, 145, 139, 147.

[0017] In one embodiment, the antibody-like protein may have binding specificity to at least three different antigens selected from tumor antigens, immune signaling antigens, or combinations thereof.

[0018] In one embodiment, the antibody-like protein may have binding specificity to at least four different antigens selected from tumor antigens, immune signaling antigens, or combinations thereof.

[0019] In one embodiment, the antibody-like protein may have binding specificity to at least five different antigens selected from tumor antigens, immune signaling antigens, or combinations thereof. In one embodiment, the antibody-like protein may be a quintuple-specific antibody.

[0020] In one embodiment, the quintuple-specific antibody may include amino acid sequences having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 85 and 87, 89 and 91, 93 and 95, 97 and 99, 101 and 103, and 105 and 107. In one embodiment, the quintuple-specific antibody may include pairs of light and heavy chains or fragments thereof selected from sequence combinations of SEQ ID NOs. 85 and 87, 89 and 91, 93 and 95, 97 and 99, 101 and 103, and 105 and 107.

[0021] In one embodiment, the antibody-like protein may have binding specificity to at least six different antigens selected from tumor antigens, immune signaling antigens, or combinations thereof. In one embodiment, the antibody-like protein may be a hexaspecific antibody.

[0022] In one embodiment, the hexaspecific antibody may include an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 109, 111, 113, 115, 117, 119, or a combination thereof. In one embodiment, the hexaspecific antibody may include a combination of light and heavy chains or fragments thereof selected from the combinations of SEQ ID NOs: 109 and 111, 113 and 115, and 117 and 119.

[0023] In one embodiment, the antibody-like protein may include a heavy chain (HC) and a light chain (LC). In one embodiment, the HC includes an amino acid sequence having at least 98%, 95%, or 92% sequence identity with SEQ ID NOs. 85, 89, 93, 97, 101, 105, 109, 113, 117, 137, 141, 145, and 151. The LC includes an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs. 87, 91, 95, 99, 103, 107, 111, 115, 119, 139, 143, 147, and 149.

[0024] In one embodiment, the antibody-like protein may comprise a heavy chain monomer and a light chain monomer. The heavy chain monomer has an N-terminus and a C-terminus and comprises, in tandem from the N-terminus to the C-terminus, an optional first binding domain (D1) at the N-terminus, a Fab region as a second binding domain (D2) containing a light chain, an Fc domain, an optional third binding domain (D3), and an optional fourth binding domain (D4) at the C-terminus. The light chain may comprise an optional fifth binding domain (D5) covalently bonded to the C-terminus, an optional sixth binding domain (D6) covalently bonded to the N-terminus, or a combination thereof. At least one of D1, D2, D3, D4, D5, and D6 comprises an EGFR binding domain as disclosed herein.

[0025] In one embodiment, D1 contains an EGFR-binding domain. In one embodiment, D2 contains an EGFR-binding domain. In one embodiment, D3, D4, D5, and D6 each contain an EGFR-binding domain. In one embodiment, D1, D2, D3, D4, D5, and D6 each have binding specificity to different antigens. The antigens are tumor antigens, immune signaling antigens, or combinations thereof.

[0026] In one embodiment, the antibody-like protein may be a bispecific antibody. In one embodiment, the bispecific antibody is asymmetric with respect to D2, which contains an EGFR-binding domain, and D3 has binding specificity to CD3.

[0027] In one embodiment, the bispecific antibody may contain amino acid sequences having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 137, 145, 139, 147, 141, 145, 149, 147, 151, 145, 139, 147, or combinations thereof. In one embodiment, the bispecific antibody may contain light and heavy chains or fragments thereof selected from sequence combinations of 147, 141, 145, 149, and 147, and 151, 145, 139, and 147.

[0028] In one embodiment, the antibody-like protein may comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 137, 139, 141, 143, 141, 149, 151, 139, 145, 147 or a combination thereof. In one embodiment, the bispecific antibody may comprise a combination of a light chain and a heavy chain or fragments thereof selected from the sequence combinations of SEQ ID NO: 137 and 139, 141 and 143, 141 and 149, 151 and 139, and 145 and 147.

[0029] In one aspect, the present invention provides an antibody-like protein having a Fab-Fc structure comprising one or more binding domains bound to the Fab-Fc structure. In one embodiment, the antibody-like protein has an N-terminus and a C-terminus and comprises a first monomer and a second monomer. The first monomer comprises, from the N-terminus to the C-terminus, a first binding domain (mD1), a variable heavy (VH) chain, a CH1 domain, a first hinge, a first CH2 domain, a first CH3 domain, and a fourth binding domain (mD4). The second monomer comprises, from the N-terminus to the C-terminus, a second binding domain (mD2), a variable light (VL) chain, a CL domain, a second hinge, a second CH2 domain, a second CH3 domain, and a fifth binding domain (mD5). The CH chain and the CL chain constitute a third binding domain (mD3). The first monomer and the second monomer can be paired by covalent bonds via at least one disulfide bond between the CH1 domain and the CL domain and at least one disulfide bond between the first hinge and the second hinge, and the antibody-like protein is at least bispecific.

[0030] In one embodiment, at least one of mD1, mD2, mD3, mD5, and mD5 in the antibody-like protein may include an EGFR-binding domain as disclosed herein. In one embodiment, the mD3 domain includes an EGFR-binding domain. In one embodiment, the mD2 domain includes an EGFR-binding domain. In one embodiment, mD2, mD4, and mD5 each include an EGFR-binding domain.

[0031] In one embodiment, the antibody-like protein may contain an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 121, 123, 125, 127, 129, 131, 133, 135 or a combination thereof. In one embodiment, the antibody-like protein may contain a combination of peptides or fragments thereof selected from the sequence combinations of 121 and 123, 125 and 127, 129 and 131, and 133 and 135.

[0032] In one embodiment, the present invention provides a heavy chain. In one embodiment, the heavy chain may include an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 121, 125, 129, and 133.

[0033] In one embodiment, the present invention provides a light chain. In one embodiment, the light chain may include an amino acid sequence having at least 70%, 80%, 85%, 90%, 92%, 95%, 98%, 99%, or 100% sequence identity with SEQ ID NOs: 123, 127, 131, and 135.

[0034] In one embodiment, the present invention provides isolated nucleic acid sequences encoding antibody-like proteins, light chains, heavy chains, and peptide sequences disclosed herein.

[0035] In one embodiment, the present invention provides an expression vector comprising an isolated nucleic acid sequence disclosed herein.

[0036] In one embodiment, the present invention provides host cells for producing antibody-like proteins, light chains, heavy chains, or combinations thereof. In one embodiment, the host cells comprise isolated nucleic acid sequences disclosed herein. In one embodiment, the host cells may be prokaryotic or eukaryotic cells.

[0037] In one embodiment, the present invention may include an immune complex. In one embodiment, the immune complex may include an antibody-like protein, an antibody, an anti-EGFR binding domain or peptide as disclosed herein, and a cytotoxic agent. In one embodiment, the cytotoxic agent may include a chemotherapeutic agent, a growth inhibitor, a toxin, or a radioisotope.

[0038] In one embodiment, the present invention provides a pharmaceutical composition for treating a disease or health condition. In one embodiment, the pharmaceutical composition may comprise an antibody-like protein, antibody, immune complex, anti-EGFR binding domain or peptide disclosed herein and a pharmaceutically acceptable carrier.

[0039] In one embodiment, the pharmaceutical composition may further comprise a therapeutic agent. In one embodiment, the therapeutic agent may be a chemotherapeutic agent, a growth inhibitor, a toxin, a radioisotope, or a combination thereof. In one embodiment, the therapeutic agent may be, for example, capecitabine, cisplatin, trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole, aminoglutethimide, testolactone, borozole, formestan, fadrozole, letrozole, erlotinib, afatinib, dasatinib, gefitinib, imatinib, pazopanib, lapatinib, sunitinib, nilotinib, sorafenib, nabuparitaxel, derivatives thereof, or combinations thereof.

[0040] In one embodiment, the present invention provides a method for treating or preventing a target cancer, autoimmune disease, or infection. In one embodiment, the method may include the step of administering to the target a pharmaceutical composition comprising a purified antibody-like protein, antibody, anti-EGFR domain, or peptide disclosed herein. In one embodiment, the target is a mammal. In one embodiment, the target is a human.

[0041] In one embodiment, the method may further include a step of co-administering an effective amount of the therapeutic agent. In one embodiment, the therapeutic agent may be an antibody, a chemotherapeutic agent, an enzyme, or a combination thereof.

[0042] In one embodiment, the cancer may include cells expressing HER3 or EGFR. In one embodiment, the cancer may be, for example, breast cancer, colorectal 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, or myeloma. In one embodiment, the present invention provides a method for preparing an antibody-like protein, antibody, anti-EGFR domain, or peptide disclosed herein. In one embodiment, the method may include the steps of culturing host cells to express a DNA sequence encoding an antibody-like protein, anti-EGFR domain, or peptide disclosed herein, and purifying the multispecific antibody-like protein, anti-EGFR domain, or peptide disclosed herein.

[0043] In one embodiment, the present invention provides a solution comprising an effective concentration of an antibody-like protein, anti-EGFR domain, or peptide disclosed herein. In one embodiment, the solution is the plasma of a subject. In one embodiment, the subject is a human. [Brief explanation of the drawing]

[0044] The aforementioned and other features of the disclosure herein will become more fully apparent from the following description and the appended claims, together with the accompanying drawings. The drawings show only a few embodiments prepared in accordance with the disclosure herein and should therefore not be considered limiting, and further specificities and details of the disclosure herein may be illustrated through the use of the accompanying drawings.

[0045] [Figure 1] The alignment (Kabat numbering) of the VH(A) and VL(B) sequences derived from cetuximab is shown. In Figure 1, N85E is aglycosylated cetuximab, and H1 to H11 are humanized variants.

[0046] [Figure 2] (C) The predicted number of MHCII-binding peptides in the variable region of cetuximab is shown, along with the T20 humanness score of the VH(A) and Vk(B) domains based on the MixMHC2pred algorithm.

[0047] [Figure 3] The SEC profile of the His-tagged anti-EGFR scFv domain is shown, indicating that aggregation was reduced by humanizing SI-79R1 (cetuximab) to SI-79R2 (H1).

[0048] [Figure 4] We have shown Octet binding analysis of the His-tagged anti-EGFR scFv domain, demonstrating that humanized cetuximab H1 binds to human EGFR in the same way as mouse scFv.

[0049] [Figure 5] The results of thermal stability analysis of the His-tagged anti-EGFR scFv domain are shown, demonstrating that humanized cetuximab SI-79R2 is significantly more stable (unfolds at higher temperatures) than SI-79R1, as measured by DLS.

[0050] [Figure 6] The results of chemical denaturation stability analysis of the His-tagged anti-EGFR scFv domain are shown, indicating that humanized cetuximab SI-79R2 is significantly more stable than SI-79R1 (requiring higher concentrations of guanidine / urea for unfolding), as measured by denaturation of guanidine and urea.

[0051] [Figure 7] Analytical size exclusion chromatography of scFv-monoFc (A) and mAb proteins (B) immediately after the first step of protein A purification is shown, as well as unreduced SDS-PAGE (C) of the purified scFv-monoFc protein. The data are representative of two independent expression and purifications, where N85E is aglycosylated cetuximab and H1 to H11 are humanized variants.

[0052] [Figure 8] The binding dynamics of cetuximab-derived scFv-monoFc protein (A) and mAb (B), determined by biolayer interferometry using an anti-human Fc (AHC) sensor and a soluble recombinant extracellular domain of human EGFR, are shown. The data are representative of two independent experiments, and the KD values ​​are shown in Table 6-8, where N85E is aglycosylated cetuximab and H1 to H11 are humanized variants.

[0053] [Figure 9] The thermal stability of cetuximab-derived scFv-monoFc protein (A) and mAb (B) as determined by dynamic light scattering is shown. The data are representative of two independent experiments, and the unfolding temperature (point where the radius exceeds 10 nm) is shown in Table 7-8, where N85E is aglycosylated cetuximab and H1 to H11 are humanized variants.

[0054] [Figure 10]This shows the cation exchange chromatography of αCD3 × αEGFR bispecific antibodies and their parental αCD3 and αEGFR antibodies, along with their characteristic retention times.

[0055] [Figure 11] This study demonstrates T cell-dependent cell-mediated cytotoxicity (TDCC) of αCD3 × αEGFR bispecific antibodies. Here, luciferase-treated EGFR-supported BxPC-3 cell lines were incubated with activated T cells and αEGFR arms as target cells (including wild-type cetuximab, aglycosylated cetuximab (N85E), and humanized version H7), and luminescence signals were used as the endpoint for live BxPC-3 cells after 72 hours (EC50 values ​​are shown in Table 8).

[0056] [Figure 12] A schematic diagram of a GNC antibody is shown, illustrating: 1) the variable region (D2) of Fab shown in black, and both the constant region and Fc region of Fab shown in white; 2) additional antigen-binding domains shown in shaded boxes (each replaceable by receptor-ligand binding); 3) a heavy chain monomer to which D1 is attached to the N-terminus and / or D3 and / or D4 are tandemly attached to the C-terminus via D4; and 4) a light chain monomer to which D5 and / or D6 are attached to its N-terminus and C-terminus to obtain hexa-GNC antibody and penta-GNC antibody, respectively.

[0057] [Figure 13] The analytical SEC profile of anti-huEGFR pentaGNC antibodies containing humanized anti-EGFR scFv (SI-55P3, H1 scFv; SI-55P4, H1 scFv; SI-79P2, H4 scFv; SI-79P3, H4 scFv; and SI-55P9, H7 scFv) or Fab regions (SI-77P1, H7 Fab) is shown, indicating minimal aggregation after protein A purification.

[0058] [Figure 14]Octet binding analysis of anti-huEGFR pentaGNC antibodies shows that pentaGNC antibodies containing humanized anti-EGFR scFv (SI-55P3, H1 scFv; SI-79P2, H4 scFv; and SI-79P3, H4 scFv) or Fab regions (SI-77P1, H7 Fab) maintain tight binding.

[0059] [Figure 15] We have demonstrated that penta-GNC antibodies containing humanized anti-EGFR scFv (SI-55P9, H7 scFv) or Fab (SI-77P1, H7 Fab) induce potent TDCC against EGFR-expressing tumor cells.

[0060] [Figure 16] The analytical SEC profiles of anti-huEGFR hexa-GNC antibodies are shown, indicating that hexa-GNC antibodies with humanized anti-EGFR scFv (SI-55H11, H7 scFv) or Fab regions (SI-77H4, H7 Fab) exhibit less aggregation than hexa-GNC antibodies with anti-EGFR domains derived from cetuximab.

[0061] [Figure 17] Octet binding analysis of anti-huEGFR hexa-GNC antibodies shows that hexa-GNC antibodies possessing humanized anti-EGFR scFv (SI-55H11, H7 scFv) or Fab regions (SI-77H4, H7 Fab) maintain similar binding to EGFR as the cetuximab-derived hexa-GNC antibody SI-77H4.

[0062] [Figure 18] This study demonstrates that hexa-GNC antibodies containing humanized anti-EGFR scFv (SI-55H11, H7 scFv) induce potent TDCC against EGFR-expressing tumor cells.

[0063] [Figure 19]Figure 19A shows a schematic diagram of an asymmetric bispecific antibody, where the EGFR Fab is derived from one of three cetuximab Fabs (wild-type with or without N85E, or wild-type with humanized VH / VL), the second Fab is the CD3 Fab, and the CH3 domain contains the K409R mutation. Figure 19B shows a schematic diagram of a miniGNC antibody-like protein, whose heterodimer composition includes: 1) a variable region of a single Fab (mD3) shown in black, and both the constant region and Fc region of the Fab shown in white; 2) an additional antigen-binding domain shown in a shaded box (each replaceable by receptor-ligand binding); 3) a chain A monomer with mD1 attached to its N-terminus and mD4 attached to its C-terminus; and 4) a chain B monomer with mD2 attached to its N-terminus and mD5 attached to its C-terminus.

[0064] [Figure 20] The analytical SEC profiles of anti-huEGFR pentaminiGNC antibodies are shown, indicating that pentaminiGNC antibodies containing humanized anti-EGFR scFv (SI-68P7, H1 scFv; SI-79P1, H4 scFv; and SI-68P13, H7 scFv) or Fab domains (SI-68P17, H7 Fab) exhibit less aggregation.

[0065] [Figure 21] Octet binding analysis of anti-huEGFR pentaminiGNC antibodies shows that pentaminiGNC antibodies possessing humanized anti-EGFR scFv (SI-709P1, H4 scFv; SI-68P13, H7 scFv) or Fab regions (SI-68P17, H7 Fab) maintain binding to EGFR.

[0066] [Figure 22] We have shown that pentaminiGNC antibodies containing humanized anti-EGFR scFv (SI-68P13, H7 scFv) or Fab region (SI-68P17, H7 Fab) induce potent TDCC in EGFR-expressing tumor cells. [Modes for carrying out the invention]

[0067] The following detailed description refers to the accompanying drawings, which form part of this specification. In the drawings, similar symbols generally identify similar components unless otherwise indicated in the context. The exemplary embodiments described in the detailed description, drawings, and claims are not intended to limit. Other embodiments may be utilized and other modifications made without departing from the spirit or scope of the subject matter presented herein. It will be readily apparent that the aspects of this disclosure generally described herein and shown in the drawings can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations. All of these are explicitly discussed herein.

[0068] This disclosure provides isolated antibodies, methods for producing the antibodies, monoclonal and / or recombinant monospecific antibodies, multispecific antibodies, antibody-drug conjugates, and / or immune complexes composed of the antibodies or antigen-binding fragments; pharmaceutical compositions comprising the antibodies, monoclonal and / or recombinant monospecific antibodies, multispecific antibodies, antibody-drug conjugates, and / or immune complexes; methods for producing the antibodies and compositions; and methods for treating cancer using the antibodies and compositions. Specifically, this disclosure provides isolated monoclonal antibodies (mAbs) or antigen-binding fragments thereof that have binding specificity to human EGFR (Table 1, Figure 1). The isolated mAb or antigen-binding fragments mentioned above are SEQ ID NOs: 1 and 3; 5 and 7; 9 and 11; 13 and 15; 17 and 19; 21 and 23; 25 and 27; 29 and 31; 33 and 35; 37 and 39; 41 and 43; 55; 57; 59; 61; 63; 65; 67; 69; 71; 73; 75; 77; 79; 81; 83; 85 and 87 ;Includes amino acid sequences identical to sequences selected from 89 and 91; 93 and 95; 97 and 99; 101 and 103; 105 and 107; 109 and 111; 113 and 115; 117 and 119; 121 and 123; 125 and 127; 129 and 131; 133 and 135; 137 and 139; 141 and 143; 141 and 149; 151 and 139; 145 and 147; 137, 145, 139 and 147; 141, 145, 143 and 147; 141, 145, 149 and 147; 151, 145, 139 and 147.

[0069] As used herein, the terms “a,” “an,” and “the” are defined as meaning “one or more,” and include the plural form unless the context is appropriate.

[0070] As used herein, the terms “polypeptide,” “peptide,” and “protein” are interchangeable and are defined to mean biomolecules composed of amino acids linked by peptide bonds.

[0071] The term "antigen" refers to an entity or fragment thereof that can elicit an immune response in living organisms, particularly animals, and more specifically mammals, including humans. This term includes immunogens and their regions involved in antigenicity or antigenic determinants.

[0072] The terms “antigen or epitope binding portion or fragment,” “variable region,” “variable region sequence,” or “binding domain” refer to fragments of an antibody that can bind to an antigen (in this invention, for example, EGFR). An antigen-binding fragment (Fab) is a region on an antibody (Fab region) that binds to an antigen. These fragments may have the ability to perform antigen-binding function and additional functions of an intact antibody. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) consisting of variable light chain (VL) and variable heavy chain (VH) domains of a single arm of an antibody linked to a single polypeptide chain by a synthetic linker, or Fab fragments that are monovalent fragments consisting of VL, constant light chain (CL), VH, and constant heavy chain 1 (CH1) domains.

[0073] Antibody fragments are even smaller subfragments and can consist of domains as small as a single CDR domain, particularly the CDR3 region from either the VL and / or VH domains (see, e.g., Beiboer et al., J.Mol.Biol.296:833-49(2000)). Antibody fragments are produced using conventional methods known to those skilled in the art. Antibody fragments can be screened for utility using the same techniques used for intact antibodies.

[0074] The “antigen, epitope-binding region or fragment,” “variable region,” “variable region sequence,” or “binding domain” can be derived from the antibodies of this disclosure by many techniques known in the art. For example, a purified monoclonal antibody can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Papain digestion of the antibody produces two identical antigen-binding fragments (called “Fab” fragments), each having a single antigen-binding site, and the remaining “Fc” fragment, whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still capable of crosslinking antigens. A suitable fraction containing the Fab fragment can then be collected and concentrated by membrane filtration or the like. For further explanation of general techniques for isolating antibody active fragments, see, for example, Khaw, BA et al. J. Nucl. Med. 23:1011-1019 (1982) and Rousseaux et al. Methods Enzymology, 121:663-69, Academic Press, 1986.

[0075] The term “antibody” is used in its broadest sense and specifically covers single monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions having polyepitope specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv) as long as they exhibit desirable biological activity. In some embodiments, the antibody may be monoclonal, polyclonal, chimeric, single-chain, multispecific or multi-effective, human and humanized antibodies, and their active fragments. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, as well as products of Fab immunoglobulin expression libraries, and epitope-binding fragments of any of the antibodies and fragments described above.

[0076] The term "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of one heavy-chain variable domain and one light-chain variable domain, tightly bound together by non-covalent bonds. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only three antigen-specific CDRs) may also recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0077] In some embodiments, antibodies may comprise an immunoglobulin molecule and a molecule containing the immunologically active portion of the immunoglobulin molecule, i.e., a binding site that immunely binds to an antigen. A typical antibody refers to a heterotetramer protein typically having two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The light chains of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on the amino acid sequence of their constant domains. The VH and VL regions may be further subdivided into domains of hypervariable complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs arranged in the following order: The regions from the amino terminus to the carboxyl terminus are designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Within the variable regions of the light and heavy chains are binding regions that interact with antigens.

[0078] Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, and some of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, delta, epsilon, γ, and μ, respectively. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known.

[0079] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies. That is, individual antibodies within the population are identical except for any spontaneous mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage of being synthesized by hybridoma culture and not contaminated with other immunoglobulins. The modifier “monoclonal” indicates the characteristic of the antibody that it is obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring antibody production by a specific method. For example, monoclonal antibodies used in accordance with the disclosure herein may be produced by the hybridoma method first described by Kohler & Milstein, Nature, 256:495 (1975), or by the recombinant DNA method (see, e.g., USPat. No. 4, 816, 567). "Recombinant" means the production of antibodies by recombinant nucleic acid technology in exogenous host cells.

[0080] Monoclonal antibodies can be prepared by various methods. These methods include molecular cloning of antibodies directly from mouse hybridomas, phage displays, recombinant DNA, and primary B cells, as well as antibody discovery methods (Siegel.Transfus.Clin.Biol.2002; Tiller.New Biotechnol.2011; Seeber et al.PLOS One.2014). Monoclonal antibodies may contain “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to the corresponding sequence of an antibody derived from a different species or belonging to a different antibody class or subclass, and of a fragment of such an antibody, insofar as it exhibits the desired biological activity (USPat. No. 4, 816, 567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0081] As used herein, the term “multispecific” antibody refers to an antibody having at least two binding sites, each of which has a binding affinity to an antigen epitope. As used herein, the terms “bispecific, triplicate, tetraspecific, quinticate, or hexaticate” antibody refer to an antibody having two, three, four, five, or six antigen-binding sites. For example, an antibody described herein is quinticate if it has five binding sites, and hexaticate if it has six binding sites.

[0082] The term “Guidance and Navigation Control (GNC)” refers to a multispecific protein capable of binding to at least one effector cell (e.g., immune cell) antigen and at least one target cell (e.g., tumor cell, immune cell, or microbial cell) antigen (WO2019191120A1; the entire term is incorporated herein by reference). The GNC protein may employ an antibody core structure comprising a Fab region and an Fc region having various binding domains bound to the antibody core. In this case, the GNC protein is also called a GNC antibody. The GNC protein may employ an antibody-like structure. In this case, the Fv fragment may be replaced with a non-antibody-based binding domain (e.g., NKG2D, 4-1BBL (4-1BB receptor ligand), 4-1BBL trimer for 4-1BB) or receptor.

[0083] The term "GNC antibody" refers to a GNC protein having an antibody structure capable of simultaneously binding to at least one effector cell (e.g., an immune cell) and at least one target cell (e.g., a tumor cell, immune cell, or microbial cell). As used herein, "bi-GNC, tri-GNC, tetra-GNC, penta-GNC, or hexa-GNC" antibodies refer to GNC antibodies having two, three, four, five, or six antigen-binding sites, where at least one antigen-binding site has binding affinity to immune cells and at least one antigen-binding site has binding affinity to tumor cells. In one embodiment, the GNC antibodies described herein each have four to six binding sites (or binding domains) and are tetra-GNC, penta-GNC, and hexa-GNC antibodies. In some embodiments, the GNC antibody includes an antibody-binding domain (e.g., Fab and scFv) and does not require separate protein engineering in the Fc region. In one embodiment, the GNC antibody has the further advantage of retaining the bivalent nature of each target antigen. In a further embodiment, the GNC antibody has the advantage of an avidity effect, resulting in high affinity and low dissociation rate for the antigen. This bivalent nature for each antigen differs from many multispecific platforms, which are monovalent for each target antigen and often lose the beneficial avidity effect that would otherwise lead to very strong antibody binding.

[0084] A “humanized antibody” refers to a type of modified antibody that has a CDR derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portion of the molecule 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)).

[0085] "Isolated" or "purified" means a biomolecule that does not contain at least some of its naturally occurring components. When used to describe the various polypeptides disclosed herein, "isolated" or "purified" means a polypeptide identified, isolated, and / or recovered from the cells or cell culture from which it was expressed. Typically, purified polypeptides are prepared by at least one purification step. "Isolated or purified antibody" means an antibody that is substantially free of other antibodies having different antigen-binding specificities.

[0086] The term "immunogenic" refers to a substance that induces or enhances the production of antibodies, T cells, or other reactive immune cells to an immunogenic substance, thereby contributing to the immune response in a human or animal. An immune response occurs when an individual produces sufficient antibodies, T cells, and other reactive immune cells to the immunogenic composition of the present invention administered to alleviate or reduce the disorder being treated. Immunogenic responses generally include both the cellular (T cell) and humoral (antibody) arms of the immune response, and antibodies against therapeutic proteins (anti-drug antibodies, ADAs) consist of IgM, IgG, IgE, and / or IgA isotypes.

[0087] "Specific binding" or "specific" to a particular antigen or epitope means a binding that is clearly different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule by comparing it to the binding of a control molecule, which is a similarly structured molecule that generally does not exhibit binding activity. For example, specific binding can be determined by competition with a regulatory molecule similar to the target.

[0088] The term "affinity" refers to a measure of the attractiveness between two polypeptides, such as antibody / antigen or receptor / ligand. The inherent attractiveness between two polypeptides can be expressed as the binding affinity equilibrium dissociation constant (KD) for a particular interaction. The KD binding affinity constant can be measured, for example, by biolayer interferometry. Here, KD is the ratio of kdis (dissociation rate constant) to kon (binding rate constant), i.e., KD = kdis / kon.

[0089] Specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody having a KD of 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, at least about 10⁻⁹ M, at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, at least about 10⁻¹² M, or greater, for the antigen or epitope. Here, KD refers to the equilibrium dissociation constant of the particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen may have a KD of 20, 50, 100, 500, 1000, 5000, 10000, or greater than that of the control molecule for the antigen or epitope.

[0090] Furthermore, specific binding to a particular antigen or epitope can be demonstrated, for example, by antibodies having a KA or Ka ratio of at least 20, 50, 100, 500, 1000, 5000, or greater than 20-, 50-, 10000-, or greater than the epitope relative to the control. Here, KA or Ka refers to the association rate of a particular antibody-antigen interaction.

[0091] A potential drawback of cetuximab is that its variable regions were found in mice, and these regions retain non-human sequences. Chimeric antibodies have been shown to potentially have higher immunogenicity compared to humanized or human antibodies.6 Humanization, on the other hand, can increase antibody stability by improving the fit of framework regions.7 Another concern is the glycan-occupied site of VH N85 (Kabat), where Fab glycosylation may affect the biological properties of the antibody and could introduce glycan heterogeneity that needs to be carefully controlled during manufacturing.8,9 The immunogenicity of cetuximab is low, with a low incidence of anti-cetuximab IgG response (5%), although hypersensitivity is common and is mainly due to pre-existing IgE antibodies against galactose-α-1,3-galactose oligosaccharides that modify VH expressed in SP2 / 0 cells.10-12

[0092] To overcome these drawbacks, cetuximab was humanized with the aim of removing post-translational modification sites, stabilizing the antibody, and reducing the potential for immunogenicity while maintaining high affinity for EGFR. Humanization strategies included direct CDR transplantation into a stable human framework, sequence-inducible transplantation into the most similar germline or consensus framework, and a structure-inducible approach based on the predicted stability effect of humanized mutations. As a result, a panel of humanized cetuximab sequences with excellent biophysical properties was obtained. The structure modeling approach was most successful in generating a stable binder without compromising EGFR affinity.

[0093] Humanization of antibodies discovered in non-human species is a common method not only to reduce immunogenicity but also to increase stability and eliminate sequence defects. In this study, cetuximab scFv was humanized using three different strategies, including unbiased CDR grafting, sequence-guided humanization, and model-guided humanization (Table 2). Each approach successfully produced an EGFR binder with improved humanity, but there was a clear trend in stability and affinity retention across all humanization strategies. Simple CDR grafts resulted in destabilization and the largest (4-fold) loss of antigen affinity, while the sequence homology approach resulted in stabilization and a more gradual (2-fold) decrease in affinity, and the model-guided approach was the most successful, resulting in significant stabilization and no change in antigen affinity.

[0094] When expressed as an mAb, the humanized version H7, when converted to the bispecific αEGFRxαCD3 form, showed increased potency and thermal stability compared to cetuximab, with no change in binding dynamics and similar TDCC efficacy (Table 8).

[0095] In addition to superior biophysical properties, the humanized version eliminated sequence defects associated with the mouse variable region of cetuximab. VH and VL humanization was significantly increased in all humanizations, and the presence of immunogenic peptides appeared to decrease based on the predicted affinity for the MHCII allele (Table 1). Predicting the immunogenicity of therapeutic antibodies is difficult, but increased humanization and decreased T cell epitope numbers may significantly reduce the incidence of immunogenicity.17,18,20 Furthermore, the removal of glycosylation and deamidation sites reduces the complexity of lot-to-lot characterization and eliminates the possibility of immunogenic Fab saccharides, even when expressed in non-human cells.

[0096] After humanization, additional modifications to the three C-terminal residues of VL were attempted as a means of reducing scFv aggregation.13,15,16 Because the CH1 / CL domain is absent, the surface of scFv is unnaturally exposed to the solvent. Human Vλ has fewer charged residues and a more hydrophobic C-terminus than Vκ (LTVL vs. LEIK), which may result in better packing of the last β-sheet. Indeed, this adoption reduced aggregation in all three cases (an average of 8%) and improved titer and thermal stability in two of the three cases (Table 6).

[0097] Furthermore, IgG antibodies possess a conserved N-glycosylation site located at N297 in the CH2 domain within the upper Fc domain. Additionally, a small number of antibodies are glycosylated in the Fab region due to N-glycosylation motifs that may be present within the variable region (8). The glycan profile of antibody drugs needs to be regularly characterized batch by batch to ensure consistent and homogeneous proteins during large-scale expression and purification. When glycan sites are present in both the Fab and Fc domains of an antibody, challenges arise because the molecule needs to be digested or deconvoluted to clearly distinguish the glycan profile of each site. However, removing glycans within the Fab domain often impairs affinity to the target antigen because the glycans can stabilize the active antigen-binding conformation. In the case of cetuximab, the antibody is glycosylated at N99 (AHo) in the VH domain (9). Glycosylation increases the cost of the product because it requires an additional quality control step to characterize that glycan site. Removing Fab glycans while retaining their original affinity for EGFR represents a significant leap forward in the production of antibodies that are easy to characterize while maintaining complete efficacy.

[0098] Cetuximab has been previously humanized using other strategies. In one CDR transplantation study of cetuximab, an antibody capable of binding to EGFR-overexpressing cells was produced, but with a 9-fold decrease in affinity.21,22 This increase in KD reflects the affinity changes observed for CDR transplantation in this study. Cetuximab has also been glycosylated to remove the α-1,3-galactose epitope, representing another approach to reduce the immunogenicity of this antibody.23 In summary, the data presented here demonstrate that protein engineering of cetuximab can improve its stability and immunogenic properties, and more generally, suggest that sequence, particularly structural guiding methods, can produce humanized antibodies with superior stability and binding properties.

[0099] This disclosure may be more readily understood by referring to the following detailed descriptions of the specific embodiments and examples contained herein. Although this disclosure has been described with reference to certain details of its specific embodiments, such details are not intended to be considered limitations on the scope of this disclosure.

[0100] Examples Example 1: Humanized EGFR binding sequence The humanizability of the sequences was calculated using the Lake Pharma Antibody Analyzer (https: / / dm.lakepharma.com / bioinformatics / ), which provides a T20 score (ranging from 0 to 100, with 100 indicating optimal humanizability) (Table 1, Figure 2C). In particular, wild-type mouse sequences had low scores of 66.44 (VH) and 70.38 (VK) when only the framework region was scored. In contrast, the humanized variant sequences (H1-H11) had significantly higher T20 humanizability scores (76.95 to 88.10 (VH) and 81.44 to 91.04 (VK)). Therefore, it is predicted that the humanized variant sequences will be less immunogenic than the mouse sequences due to their lower MHCII binding and higher humanizability.

[0101] To enhance the humanization of the cetuximab variable region and reduce its immunogenic potential, the mouse VH and Vk domains were converted to a more humanized framework (Figure 1). Version H1 was based on a simple transplantation of Kabat CDR residues into a stable human framework.13 Versions H8, H9, H10, and H11 were designed based on sequence identity with human germline sequences. In particular, in versions H10 and H11, framework residues were mutated to match the most similar human germline sequences. In versions H8 and H9, framework residues were mutated to consensus residues of the human antibody. The remaining humanized versions (H2, H3, H4, H5, H6, H7) were designed based on the structural analysis of cetuximab by mutating framework residues to residues occurring at a frequency of at least 5% in the human germline that resulted in the most stable structure in silico. Since the energy analysis of this type of humanization is dependent on the input model, several input structures were investigated. Version H2 used the cetuximab crystal structure 1YY914. Versions H3, H4, H5, H6, and H7 used scFv models generated from the antibody modeling function of Discovery Studio based on the sequence of the cetuximab variable domain. In versions H4, H5, H6, and H7, modifications were incorporated into the input sequence to increase the similarity between the VH C-terminus and the human consensus sequence, or to make the Vκ C-terminus more Vλ-like. After humanization in Discovery Studio, H7, H9, and H11 were further modified by converting the last three residues of the Vκ domain to the corresponding residues of the λJ gene. The above modifications were evaluated due to the known importance of the final VLβ chain in determining the stability and aggregation tendency of scFv, and the higher hydrophobic nature of the Vλ terminus, which can provide packing energy to stabilize the interaction13,15,16. All humanization strategies are summarized in Table 2.

[0102] In humanized version H1, cetuximab Kabat CDR was transplanted into the stable framework described above.13 All other humanized versions were designed using the Discovery Studio 2020 suite. Versions H8–H11 were designed using a Predict Humanizing Mutations protocol based solely on the amino acid sequence of cetuximab as the query sequence. The Identity Threshold was set to 50, Frequent Residue Substitution Tolerance to 20, and Germline Substitution Tolerance to 0, excluding substitutions of Kabat CDR residues, IMGT CDR residues, Vernier Zone residues, and human germline residues. Versions H10 and H11 were generated based on germline substitutions, while versions H8 and H9 used frequent residue substitutions. To generate the best single-mutation sequences, versions H2–H7 were designed using various input models of cetuximab, with Calculate Mutation Energy set to True (CHARMm force field). The query structures were various models of cetuximab, as shown in Table 2. In version H2, the cetuximab component of PDB 1YY9 (cetuximab complexed with EGFR) was used to obtain the pose of the bound state CDR. Versions H3–H7 used cetuximab models generated by Discovery Studio's Antibody Modeling Cascade. For input sequences, H3 used cetuximab VH and VL; H4 and H7 used cetuximab VH (ending in TVSS instead of TVSA) and VL; H5 used cetuximab VH and VL (ending in LTVL instead of LELK); and H6 used cetuximab VH (ending in TVSS instead of TVSA) and VL (ending in LTVL instead of LELK). The top five framework templates were used with 10 sequence similarity cutoffs.The CDR loop definition was set to Honegger, the maximum number of templates per loop was set to 3, and the optimization level was set to high. After producing the humanized sequence, versions H10, H8, and H4 were changed to H11, H9, and H7, respectively, by substituting the last four residues of VL with LTVL to mimic the stable FR4 of the lambda antibody.

[0103] Figure 1 shows the sequences of the variable domain of cetuximab and its humanized version. Figures 1A and 1B show the alignment of the VH and VL sequences, respectively. Vernier zone residues adjacent to the CDR region and vernier zone residues in structurally important framework regions were conserved and maintained antigen binding. Amino acid identity testing between sequences (Table 3) revealed that the humanized VH sequence had 84–87% identity with cetuximab and 79–100% identity with each other. The maximum identity between humanized VH sequences was 95%, except for a comparison with a version having a modified λJ region that, by definition, had 100% VH identity with the corresponding unmodified humanized version. The humanized VL sequence had 79–86% identity with cetuximab and 76–98% identity with each other. Furthermore, when comparing only the framework region, sequence identity decreased (identity was 70-82% between cetuximab VH and humanized VH, and 60-82% between cetuximab VL and humanized VL).

[0104] In addition to the Fc glycan, wild-type cetuximab has two potential glycosylation sites each in the VH domain (Kabat N85; known to be glycosylated) and the VK domain (Kabat N41; part of the NGS glycosylation motif). Furthermore, this same VK N49 may be deamidated as it forms an NG deamidation motif. To eliminate the disadvantages associated with the stability and manufacturing evaluation of these post-translational modification sites, three disadvantages (two glycosylations and one deamidation) were removed in all humanized sequences H1-H11 (Figure 1). In particular, the Kabat residue VH N85, which contains the occupied NDT glycosylation motif in cetuximab, was modified to an A, D, or E amino acid in humanization to remove this known glycan site. Similarly, VL N41, which contains the NGS glycosylation motif in cetuximab, was changed to a more typical G residue in all humanizations. The humanizability of wild-type cetuximab and its humanized variable region was calculated using a T20 humanizability score based on the framework region sequence.17 Cetuximab is a chimeric antibody with a mouse variable region, and its T20 scores were low, at 66.44 (VH) and 70.38 (Vκ). The T20 score of the humanized VH domain increased from 66.44 to a range of 76.95-88.10 (Table 1, Figure 2A), and the score of the humanized Vκ domain increased from 70.38 to a range of 81.44-91.04 (Table 1, Figure 2B). Therefore, humanization of the cetuximab variable region significantly improves the humanizability of these sequences, and the increased sequence identity with human germline cells can reduce immunogenicity.

[0105] The presence of non-human sequences in biologics can trigger immunogenicity in the form of anti-drug antibodies (ADAs), but potent, high-affinity ADAs only occur when the B cells in question are activated and undergo class-switch recombination to the IgG subtype. This B-cell activation requires the presented MHCII-peptide to bind to a suitable T cell receptor on CD4+ T cells. Therefore, if a therapeutic antibody contains a peptide that stably binds to MHC class II, the likelihood of an undesirable ADA response increases.

[0106] The MixMHC2pred algorithm (https: / / github.com / GfellerLab / MixMHC2pred) was used to predict MHCII-binding ligands in antibody sequences.18 This algorithm can detect the number of "core" peptides in specific amino acid sequences that bind to MHCII with sufficient affinity to form stable T cell epitopes. The more MHCII-binding peptides identified in a sequence, the more potential T cell epitopes it contains. Note that the algorithm cannot distinguish between immunogenic and tolerogenic peptides. However, a higher number of core peptides increases the likelihood of the presence of pro-immunogenic peptides. The MixMHC2pred algorithm was purchased and downloaded from the GitHub repository. After running the algorithm on VL / VH scFv sequences containing the (G4S)4 linker, the number of core peptides for different sequences was calculated and compiled into a table. Scoring was performed across multiple alleles to allow sequences to be evaluated for the presence of potent ligands for any given MHCII allele. The number of core peptides was calculated based on the number of peptides in the sequence that could bind to any MHCII allele with the top 0.2% interaction scores.

[0107] To assess the presence of MHCII epitopes within the cetuximab variable region, VH and VL sequences were subjected to a computer that predicts MHCII binding affinity. The algorithm MixMHC2pred is based on the binding of approximately 100,000 peptides to different HLA-II alleles.18 Based on the input sequence, MixMHC2pred assesses the binding of each peptide in the sequence to each HLA-II allele and returns a ranked score for each residue based on the strongest interaction with any given allele. The number of core peptides binding to MHC was calculated based on the number of unique peptides ranked within the top 0.2% of all interactions. To simplify the scoring system to one value per VH-VL pair, sequences were run as scFv[VL-(G4S)3-VH], consisting of peptides in both VH and VL. Using this system, the number of MHCII core peptides in cetuximab and the humanized sequence was calculated (Table 1, Figure 2C). Although MHCII binding was not used as a criterion for humanization, the number of peptides scored within 0.2% of interactions across all humanized sequences decreased. Cetuximab has 12 core peptides, while humanized sequences had 7–11 core peptides. This reduction in MHCII binding, when combined with more human sequences, may reduce the potential immunogenicity of the humanized variable region. Furthermore, in mouse sequences, 12 residues within the CDR predicted to be part of the MHCII-binding peptide were identified. In many humanized variants (H2, H3, H4, H5, H6, H7, H10, and H11), the number of CDR residues in the MHCII-binding peptide decreased.

[0108] Example 2: Method for producing and characterizing humanized EGFR-binding peptides, domains, antibodies, and antibody-like proteins To characterize the humanized anti-EGFR variable region in the form of various single therapeutic agents, the following were produced and characterized using the methods described below: EGFR binding complex, huEGFR variant (huEGFR), His-tagged scFv protein (scFv-6His), recombinant scFv-monoFc monomer (scFv-monoFc), monoclonal antibody (mAb), bispecific antibody (bispecific), pentaGNC antibody (pentaGNC), hexaGNC antibody (HexaGNC), and pentaminiGNC antibody (miniGNC) (Table 4).

[0109] 2a. Expression and purification of the EGFR-binding complex Protein stability is a crucial parameter defined by the difference in free energy between the folded and unfolded states. In the case of protein-based therapeutics, stability can affect immunogenicity, pharmacokinetics, and even efficacy (7), and reduced aggregation helps in the development of therapeutics that are easier to manufacture and safer for patients. Furthermore, expression efficiency and protein yield directly determine the cost of protein-based therapeutics. If proteins can be expressed more efficiently to achieve higher titers and increase the yield of purified protein, manufacturing costs can be significantly reduced.

[0110] The protein is expressed by transfection with a His-tagged scFv or scFv-monoFc (single plasmid) expression plasmid, or by co-transfection of the heavy and light chains (in other forms) using the ExpiCHO system (Thermo Fisher), and is collectively referred to as the EGFR-binding complex. Briefly, 10 μg of each expression plasmid (or 20 μg of unpaired plasmid) was mixed with 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 added to 25 ml of ExpiCHO cells in a 125 ml Erlenmeyer flask at a rate of 6 × 10⁶ cells / ml and incubated at 37°C, 5% CO₂, and 150 rpm. 24 hours after transfection, the cells were supplied with 8.75 ml of ExpiCHO feed and 150 μl of CHO enhancer, and the temperature was changed to 32°C, 5% CO2, and 150 rpm. 48 hours after transfection, the cells were supplied with 8.75 ml of ExpiCHO feed. Eight days after transfection, the culture supernatant was collected, the cells were pelleted by centrifugation at 4500 rpm for 1 hour, and the supernatant was passed through a 0.2 mm filter.

[0111] Fc-containing proteins were purified from the recovered supernatant using a 1 ml MAbSelect PrismA Protein A column (GE Healthcare). The column was equilibrated with phosphate-buffered saline. The supernatant was then passed through the column at a flow rate of 2 ml / min. The column was washed with 10 ml of PBS + 0.1% Triton X-100, followed by 10 ml of PBS + 300 mM NaCl, and finally 10 ml of PBS. Next, the protein was eluted by passing 5 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).

[0112] His-tagged scFv proteins were purified from the recovered supernatant using a 1 ml HisTrap HP column or a 1 ml Protein L (CaptoL) column (GE). The column was equilibrated with phosphate-buffered saline (pH 7.4) (HisTrap) or PBS (Protein L) containing 0.5 M NaCl and 20 mM imidazole. The supernatant was spiked with 10x binding buffer to reach 0.5 M NaCl and 20 mM imidazole (His Trap only) and passed through the column at a flow rate of 2 ml / min. The column was washed with PBS or PBS (Protein L) containing 10 column volumes of 0.5 M NaCl and 20 mM imidazole (His Trap). Next, the protein was eluted with PBS (pH 7.4) (HisTrap) containing 0.5 M NaCl and 500 mM imidazole, or with 50 mM sodium acetate (pH 3.5), and then neutralized with 0.5 ml of 1 M Tris (pH 8.0) (Protein L).

[0113] Immediately after the first-stage protein A or His-tagged purification, scFv-monoFc proteins were analyzed by Analytical SEC using Waters Acquity UPLC H-Class and ACQUITY UPLC® Protein BEH SEC 200A, 4.6 mm x 150 mm, 1.7 μm column. PBS (125 mM sodium phosphate, 137 mM sodium chloride, pH 6.8) was used as the mobile phase, running at 0.3 ml / min for 10 minutes and injecting 10 μg of protein. For higher resolution, mAbs were analyzed by Analytical SEC instead using Acquity Arc Waters HPLC with XBridge BEH SEC 300A, 7.8 x 300 mm, 3.5 μm column. PBS (150 mM sodium phosphate, 100 mM sodium chloride, pH 6.8) was used as the mobile phase, running at 0.714 ml / min for 20 minutes and injecting 50 μg of protein. Two separate purification methods were evaluated for each protein, and the percentage values ​​of the target peaks were reported as mean ± standard deviation.

[0114] 2b. Assay for characterizing the binding specificity and affinity of EGFR-binding complexes Biolayer interferometry (Octet) binding assays were performed using Octet96 or Octet384 instruments to confirm that proteins containing the humanized cetuximab binding domain retained binding to their congener antigens. Fc-containing proteins were loaded at 10 μg / ml for 180 seconds and captured on anti-human Fc (AHC) sensor chips. Alternatively, His-tagged proteins were covalently bound to AR2G chips at 10 ug / ml using the manufacturer's protocol. After a 60-second baseline step, a 180–300-second association phase was performed using serial dilutions (0–200 nM; 1:2.5 dilution factor) or a single 100 nM concentration of purified human EGFR in assay buffer, followed by a 300–600-second association phase in assay buffer. Regeneration was achieved using 10 mM glycine (pH 1.5). Binding curves were globally fitted to a 1:1 model to extract the dissociation constant KD. The binding dynamics of each protein were evaluated twice, and the values ​​summarized in the table were reported as mean ± standard deviation.

[0115] 2c. Assay for characterizing TDCC of EGFR-binding complexes The tumor targeting properties of humanized anti-EGFR domains in multispecific antibodies (collectively known as GNC antibodies) were evaluated by testing their ability to induce tumor-specific cytotoxicity while involving T cell activation, redirecting T cell-mediated cytolysis, and ultimately killing target cells. The extent of antibody-induced cytotoxicity was measured by quantifying cell viability through constitutive expression of luciferase using a luminescence-based T cell-dependent cell-mediated cytotoxicity (TDCC) assay.

[0116] Luciferase-treated BXPC3 tumor cells (ATCC) were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cell viability was monitored using a Vi-CELL automated cell counter (Beckman Coulter). 500 tumor cells (20 μL) per well were seeded into a 384-well white flat-bottom polystyrene TC-treated microplate (Corning) and incubated at 37°C and 5% CO2. After 24 hours, human pan-T cells were added to achieve an effector-to-target (ET) ratio of 5:1, and antibodies were added in a 5-fold dilution series (0-30 nM). Cells were distributed using a Multidrop bulk liquid dispenser (BIOTEK). Antibody dilution (10 μL / well) was added, and the plates were further incubated at 37°C and 5% CO2 for 72 hours, after which luminescence-based cell viability was quantified.

[0117] To quantify the luminescence induced by constitutively expressed firefly luciferase, the Bright-Glo Luciferase Assay System (Promega) was used. BrightGlo reagent was added at room temperature (20 μL per well), and luminescence was quantified using a luminescence detection plate reader (BMG Labtech). The EC50 of the antibody was determined by converting the data to Microsoft Excel and analyzing it using GraphPad Prism 6 software, "log(agonist) vs. response-variable gradient (4 parameters)". The obtained EC50 values ​​are reported. The TDCC assay was performed in a quadruplicate configuration, which provided good inter-plate reproducibility, and no significant variation was observed from different positions on the plate.

[0118] Example 3: His-tagged EGFR-binding scFv protein

[0119] The sequence encoding the humanized (H1) anti-EGFR binding domain was cloned into a His-tagged scFv expression format containing the residue GSHHHHHH at the C-terminus of scFv. The expression vector was transfected into 25 mL of ExpiCHO and expressed for 8 days, after which it was recovered and purified by protein L affinity chromatography. The H1 variant showed significantly higher titers than the mouse version (Table 5).

[0120] Analysis of SEC data after protein L purification showed that humanized scFv exhibited significantly less aggregation than scFv encoded by the wild-type mouse sequence (Figure 3, Table 5). Furthermore, the main peak shifted to the right, consistent with modification of the glycosylation site and aglycosylation of the resulting VH.

[0121] Octet confirmed that humanized scFv proteins can bind to human EGFR (Figure 4). His-tagged scFv proteins were covalently loaded onto an AR2G sensor at 10 ug / ml and bound to His-tagged human EGFR at serial dilutions (maximum 200 nM, 1:2.5 dilution). Global fitting to the resulting 1:1 binding model showed that both wild-type mouse scFv and humanized scFv proteins bind to EGFR with low nanomolar affinity (Table 5).

[0122] The thermal stability of scFv proteins was compared using dynamic light scattering (Figure 5). The temperature was increased from 25°C to 75°C at a rate of 0.5°C / min, and the radius of the scFv protein (1 mg / ml) was monitored using a Wyatt DynaPro Plate Reader III. The data showed a significantly higher Tm value for the H1 version (Table 5), consistent with improved stability.

[0123] Stability against chemical denaturation was also investigated by guanidine and urea unfolding assays (Figure 6). Proteins at a concentration of 0.1 mg / ml were incubated overnight at 24-guanidine HCl concentrations of 0 to 5.4 M or urea concentrations of 0 to 7.2 M. Fluorescence intensity (excitation 295 nm, emission 360 nm) was measured using a CLARIOstar plate reader, and the fluorescence intensity was normalized to represent the percentage of unfolded protein. EC50 values ​​were extracted by sigmoid fitting for stability comparison. According to the obtained EC50 values, the humanized variant H1 was more resistant to unfolding due to guanidine denaturation than the wild type (Table 5).

[0124] Example 4: Humanized EGFR-binding scFv-monoFc fusion protein To evaluate the biophysical properties of the humanized cetuximab VH and VL domains, sequences were cloned into scFv formatted cells and fused to monoFc cells to facilitate purification and Octet analysis.19 The scFv domain was oriented in the VL-VH direction and contained a (G4S)4 linker between the VH and VL domains. Notably, generating the scFv panel was more efficient than generating the corresponding mAb panel, which requires separate cloning of the heavy and light chains. As controls, wild-type cetuximab scFv and an aglycosylated version (VH N85E) generated by mutations in modified asparagine residues were also produced.

[0125] Plasmids encoding wild-type, aglycosylated (N85E), and humanized scFv-monoFc proteins were transiently transfected into ExpiCHO cells, and the proteins were purified from the cell supernatant using protein A affinity chromatography. As shown in Table 6, the majority of the humanized proteins exhibited superior expression titers compared to the wild-type and simple aglycosylated versions of cetuximab, despite containing the same monoFc domain and using the same algorithm for codon optimization. The mean titers of wild-type and aglycosylated cetuximab were 163 and 116 μg / ml, respectively, while the mean titers of the humanized versions ranged from 220 to 506 μg / ml at H8 and H7, respectively.

[0126] Following the initial step of protein A purification, the aggregation of scFv-monoFc proteins was evaluated using analytical size exclusion chromatography (SEC) (Figure 7A, Table 6). Wild-type and aglycosylated cetuximab had 93.6% and 94.9% of the target protein, respectively, with low levels of aggregation, while humanized versions had similar aggregation levels on average. The least aggregated version (H9) contained 97.4% of the target protein, while the most aggregated version (H4) contained 82.6% of the target protein. Modification of the VκC terminus to include a sequence from Vλ appeared to reduce aggregation. Humanized versions containing these Vλ residues (H11, H9, and H7) aggregated less than their corresponding versions using the original Vκ residues (H10, H8, and H4, respectively). Aggregates were removed, replaced with storage buffer, and preparative SEC was performed on all proteins to ensure accuracy for subsequent biophysical assays. SDS-PAGE of purified scFv-monoFc protein showed increased mobility of N85E and all humanized versions compared to wild-type cetuximab, confirming the lack of glycosylation in these variants (Figure 7C).

[0127] scFv-monoFc proteins were analyzed by SDS-PAGE using NuPAGE 4-12% Bis-Tris gel (Thermo Fisher, NP0323BOX) and MES running buffer (Thermo Fisher, NP0002). 3 μg of each protein was prepared in LDS sample buffer (Thermo Fisher, NP0007) with or without 10 mM DTT and heated at 70°C for 10 minutes. The gels were run at 150 V for 50 minutes, stained with Simply Blue (Thermo Fisher, LC6065), and destained with water before imaging.

[0128] The binding of the scFv-monoFc protein to human EGFR was evaluated using a biolayer interferometer to determine whether the humanization process altered the binding dynamics (Table 6, Figure 8A). After loading the protein into an anti-human Fc (AHC) sensor using the monoFc domain, scFv was conjugated to serial dilutions of the extracellular domain of human EGFR. Wild-type cetuximab scFv had an affinity of 3.18 nM, consistent with previous reports. The aglycosylated variant (N85E) showed very similar binding dynamics with a KD of 3.16 nM, indicating that glycosylation is not essential for antigen binding.

[0129] The KD values ​​of the humanized versions were categorized into three main groups. In humanized versions using direct CDR transplantation into a stable human framework (H1), binding affinity decreased fourfold due to increased dissociation rate. In humanization based on sequence identity with a single human germline (H10, H11) or a global dataset of human germlines (H8, H9), binding affinity consistently decreased twofold, with faster dissociation being the dynamic determinant. Finally, in humanization based on structural homology (H2 to H7), there was no significant decrease in binding affinity.

[0130] The thermal stability of the scFv-monoFc protein was assessed by dynamic light scattering (Table 6, Figure 9A) by observing the increase in hydrodynamic radius as the temperature increased from 25°C to 85°C.

[0131] Because the shape of the unfolding curve is complex and not uniform from sample to sample, protein stability was objectively compared using temperatures with a radius exceeding 10 nm. Using this metric, wild-type cetuximab protein unfolded at 47.2°C, while the aglycosylated N85E variant was slightly less stable, unfolding at 44.5°C. Therefore, the occupied glycosylation site may help stabilize the folded conformation of wild-type cetuximab scFv.

[0132] Similar to the binding results, stability was observed across three categories. Humanization based on CDR transplantation into an unrelated human framework showed slightly reduced stability compared to wild-type cetuximab. Five humanized versions showed similar or slightly higher stability compared to cetuximab. Two of these were based on sequence identity to a global dataset of human germline cells (H8, H9), and three (H4, H5, H7) were based on structural modeling. Finally, the five humanized versions appeared significantly more stable than the other proteins. H10 and H11 were generated by CDR transplantation into the most sequencely homologous human framework, while H2, H3, and H6 were based on homology models. In contrast to SEC data showing a systematic reduction in aggregation by using the C-terminal residue of the λJ gene, DLS data did not show a consistent effect of these residues on stability. Versions H11 and H7 showed slightly improved stability compared to H10 and H4, respectively, while H9 was actually less stable than its associated H8.

[0133] Example 5: Humanized anti-EGFR monoclonal antibody To understand whether the properties of the scFv-monoFc protein can be converted to IgG format, we generated mAbs of wild-type cetuximab, the aglycosylated variant N85E, and a humanized version of cetuximab. Based on the best protein expression, low aggregation, improved thermal stability, and invariant binding affinity, the humanized version H7 was selected and converted to mAb format. These three mAb proteins were produced by transient transfection in ExpiCHO cells and recovered after 9 days of expression.

[0134] As a result of the scFv-monoFc formulation, the difference in titer was not as pronounced as with the scFv-mFc format, but the expression titer of humanized H7 was increased compared to that of wild-type or non-glycosylated cetuximab (Table 8). After purification of protein A, all proteins were evaluated by SEC analysis, and the purity was over 99% (Table 8, Figure 7B). Notably, mAb aggregation was significantly less than with the corresponding scFv-mFc protein, which may be due to the inherent stability of the IgG backbone to the scFv and monoFc domains. SEC data also showed that the retention time of wild-type cetuximab was significantly shorter than that of either the aglycosylated N85E or humanized H7 version. This apparent difference in molecular size may be due to the glycosylation of cetuximab that is not present in the N85E and humanized versions.

[0135] Biolayer interferometry (BIO) analysis of the binding kinetics of the mAb to human EGFR (Table 8, Figure 8B) showed no differences in binding affinity or kinetics between versions. These results support the results for the scFv-monoFc protein and indicate that the humanized aglycosylation mutations N85E and H7 do not interfere with the interaction between cetuximab CDR and its antigen. The binding affinity of the mAb was similar to that of the corresponding scFv-monoFc protein.

[0136] Finally, DLS experiments were repeated to evaluate the stability of the mAb (Table 8, Figure 9B). The stability of wild-type and aglycosylated cetuximab was very similar (unfolding temperatures of 68.3°C and 68.1°C, respectively), with the H7 version showing a higher unfolding temperature of 72.5°C. Therefore, the humanized H7 version was more stable than wild-type cetuximab in both scFv and mAb formats.

[0137] Example 6: Bispecific antibody with T cell engagement and humanized EGFR binding Bispecific versions of cetuximab were generated and their functional activity was finalized by a T cell-dependent cell-mediated cytotoxicity (TDCC) assay. Three versions of cetuximab mAbs (wild-type, N85E, and H7) contained the K409R mutation in the CH3 domain, allowing for controlled Fab arm exchange when incubated with an anti-CD3 antibody containing the complementary F405L mutation. The formation of αEGFR × αCD3 bispecific antibodies from complementary anti-EGFR and anti-CD3 mAbs was confirmed by cation exchange chromatography (Figure 10). Antibodies were analyzed by cation exchange chromatography using an Agilent 1260 Infinity Quaternary HPLC and a Thermo Scientific ProPac™ SCX-10 HPLC column (4x250 mm, 10 μm, 35°C). Thermo Scientific CX-1 pH gradient buffer was used as the mobile phase (the gradient step is shown in Table 7). A 50 μg protein sample was loaded, separated at a flow rate of 0.5 ml / min, and eluted for 35 minutes according to the gradient shown in the table below.

[0138] To evaluate TDCC activity, serial dilutions of bispecific antibodies and control mAbs were incubated with activated T cells and lucifectorized EGFR-supported BxPC3 target cells in 384-well plates at a 5:1 effector:target ratio (Figure 11, Table 8). After incubation at 37°C for 3 days, BrightGlo reagent was added and luminescence was read in proportion to the number of remaining target cells. The bispecific cetuximab xαCD3 antibody showed potent tumor cell killing with an EC50 value of 24.6 nM. Aglycosylated N85E and humanized H7 showed similar EC50 values ​​(30.7 and 20.3 pM, respectively) with overlapping 95% confidence intervals. In contrast, none of the control mAbs (αCD3, cetuximab, or cetuximab H7) showed BxPC3 killing up to 30 nM. This indicates that cytotoxicity requires simultaneous targeting of both tumor cells and T cells. Therefore, as tested using the TDCC assay, the humanized version of cetuximab retained the biological function of cetuximab.

[0139] Example 7: Humanized anti-EGFR scFv or pentaGNC antibody having a Fab domain Humanized EGFR-binding variants H1, H4, and H7 were constructed and cloned into PentaGNC format at one of four scFv or Fab positions (Figure 12, D1 or D2 position). The proteins were transfected into 25 mL of ExpiCHO and expressed for 8 days, after which they were recovered and purified by protein A affinity chromatography. The proteins were expressed with good titers (Table 9).

[0140] Analysis of SEC data after purification of protein A indicates that pentaGNC antibodies containing humanized anti-EGFR domains can be expressed with low aggregation as scFv or Fab (Figure 13, Table 9).

[0141] Octe confirmed that pentaGNC antibodies possessing humanized anti-EGFR domains (e.g., H1, H4, H7) can bind to human EGFR (Figure 14). PentaGNC antibodies were loaded at 10 ug / ml via an AHC sensor and conjugated to His-tagged human EGFR at serial dilutions (up to 200 nM, 1:2.5 dilution) or a single 100 nM concentration. Global fit to the resulting 1:1 binding model indicates that pentaGNC antibodies bind to EGFR with affinity in the low nanomolar range (Table 10).

[0142] TDCC activity for two pentaGNC antibodies was tested using luciferase-treated (luciferized) BXPC3 cells as target cells (Figure 15). Five-fold serial dilutions (0–30 nM) of pentaGNC antibodies were administered to a mixture of 500 BxPC3 cells and 2500 activated T cells (5:1 effector:target), incubated for 72 hours, and luminescence readings corresponding to target cell viability were measured. Fitting to the resulting sigmoid function revealed that the EGFR-binding domain (H7) of the pentaGNC antibody efficiently targeted BxPC3 tumor cells for killing by co-cultured T cells, as demonstrated by sub-picomolecular EC50 values ​​(Table 9).

[0143] Example 8: Humanized anti-EGFR scFv or hexa-GNC antibody having a Fab domain Humanized anti-EGFR binding variant H7 was constructed and cloned into hexa-GNC format at either one of the five scFv positions or the Fab position (Figure 12, D1 or D2 position). The protein was transfected into 25 mL of ExpiCHO and expressed for 8 days, after which it was recovered and purified by protein A affinity chromatography. The protein was expressed with good titer (Table 11).

[0144] Analysis of SEC data after purification of protein A indicates that hexa-GNC molecules containing either a humanized anti-EGFR domain, scFv, or Fab can be expressed with low aggregation (Figure 16, Table 11). Furthermore, proteins containing version H7 showed significantly reduced aggregation compared to proteins containing cetuximab.

[0145] Octet confirmed that hexa-GNC antibodies containing a humanized anti-EGFR domain can bind to human EGFR (Figure 17). Hexa-GNC proteins were loaded at 10 ug / ml via an AHC sensor and conjugated to His-tagged human EGFR at serial dilutions (maximum 200 nM, 1:2.5 dilution) or a single 100 nM concentration. Global fit to the resulting 1:1 binding model indicates that hexa-GNC antibodies bind to EGFR with affinity in the low nanomolar range (Table 11).

[0146] Activity of a single HexaGNC was tested using a TDCC bioassay with luciferase-treated BXPC3 cells as target cells (Figure 18). Five-fold serial dilutions (0-30 nM) of the hexaGNC antibody were administered to a mixture of 500 BxPC3 cells and 2500 activated T cells. After incubation for 72 hours, luminescence readings corresponding to target cell viability were measured. Fitting to the resulting sigmoid function revealed that the EGFR-binding domain (H7) of the hexaGNC antibody efficiently targeted BxPC3 tumor cells for killing by co-cultured T cells, as demonstrated by EC50 values ​​within the sub-picomolecal range (Table 11).

[0147] Example 9: Humanized anti-EGFR scFv or pentaminiGNC antibody having a Fab domain Humanized EGFR-binding variants H1, H4, and H7 were constructed and cloned into pentaminiGNC format at either one of four scFv positions (mD1, mD2, mD4, mD5) or the Fab (mD3) position (PCT / US2021 / 022847; the entire file is incorporated herein by reference) (Figure 19). The proteins were transfected into 25 mL of ExpiCHO and expressed for 8 days, after which they were recovered and purified by protein A affinity chromatography. The proteins were expressed with good titers (Table 12).

[0148] Analysis of SEC data after purification of protein A indicates that pentaminiGNC molecules containing humanized anti-EGFR domains can be expressed with low aggregation (Figure 20, Table 12).

[0149] Octet confirmed that pentaminiGNC antibodies containing humanized anti-EGFR domains (H4, H7) can bind to human EGFR (Figure 21). PentaminiGNC antibodies were loaded at 10 ug / ml via an AHC sensor and bound to His-tagged human EGFR at serial dilutions (maximum 200 nM, 1:2.5 dilution) or a single 100 nM concentration. Global fit to the resulting 1:1 binding model indicates that pentaminiGNC antibodies bind to EGFR with affinity in the low nanomolar range (Table 12).

[0150] The TDCC activity of two pentaminiGNC antibodies was tested using luciferase-treated BXPC3 cells as target cells (Figure 22). Five-fold serial dilutions (0–30 nM) of the pentaminiGNC antibody were administered to a mixture of 500 BxPC3 cells and 2500 activated T cells (5:1 effector:target), incubated for 72 hours, and luminescence readings corresponding to target cell viability were measured. Fitting to the resulting sigmoid function revealed that the EGFR-binding variant H7 of the pentaminiGNC antibody efficiently targeted BxPC3 tumor cells for killing by co-cultured T cells, as demonstrated by its sub-picomolar EC50 value (Table 12).

[0151] table Table 1: Humanization of the VH / VK region leads to decreased immunogenicity and improved humanity score (framework region only). [Table 1]

[0152] Table 2: Methods for producing humanized cetuximab variants [Table 2]

[0153] Table 3: Sequence identity matrices of the entire VH domain (A), VH framework region (entire VH domain excluding Kabat CDR residues) (B), entire VL domain (C), and VL framework region (entire VL domain excluding Kabat CDR residues) (D) for wild-type cetuximab, aglycosylated cetuximab (N85E), and humanized cetuximab versions (H1-H11). [Table 3] Table 4: EGFR-binding complexes in the form of humanized EGFR-binding sequence variants (variable regions H1-H11), His-tagged scFv protein (scFv-6His), recombinant scFv-monoFc monomer (scFv-monoFc), monoclonal antibody (mAb), bispecific antibody (bispecific), pentaGNC antibody (pentaGNC), hexaGNC antibody (hexaGNC), and pentaminiGNC antibody (miniGNC). [Table 4]

[0154] Table 5. Characterization of His-tagged humanized anti-EGFR scFv [Table 5]

[0155] Table 6: Biophysical properties of cetuximab-derived scFv-monoFc protein The values ​​are the mean and standard deviation of two independent experiments. [Table 6]

[0156] Table 7: Gradient method for cation exchange separation of αEGFR and αCD3 antibodies [Table 7]

[0157] Table 8: Biophysical properties of cetuximab-derived monoclonal antibodies The values ​​are the mean and standard deviation of two independent experiments. [Table 8] *EC50 values ​​when EGFR+BxPC3 cells were depleted using an αCD3×αEGFR bispecific antibody. The 95% confidence interval is shown in parentheses.

[0158] Table 9: Characterization of pentaGNC antibodies containing humanized anti-EGFR scFv domain or humanized anti-EGFR Fab region [Table 9]

[0159] Table 10: Octet binding analysis of EGFR-binding complexes [Table 10]

[0160] Table 11: Characterization of hexa-GNC antibodies containing humanized anti-EGFR scFv domain or humanized anti-EGFR Fab region [Table 11]

[0161] Table 12: Characterization of pentaminiGNC antibodies containing humanized anti-EGFR scFv domain or humanized anti-EGFR Fab domain [Table 12]

[0162] Sequence List

[0163] Sequences of humanized EGFR binding sequence variants (H1-H11) [Table 13]

[0164] Sequence of antibody constant region, linker motif, and tag [Table 14]

[0165] Sequence of the αEGFR scFv-His protein [Table 15]

[0166] Sequence of αEGFR scFv-monoFc protein [Table 16]

[0167] Sequence of pentaGNC protein containing humanized EGFR-binding domain [Table 17]

[0168] Sequence of hexaGNC protein containing a humanized EGFR-binding domain [Table 18]

[0169] Sequence of the pentaminiGNC protein containing the humanized EGFR-binding domain [Table 19]

[0170] Sequences of αEGFR mAb and αCD3 mAb [Table 20]

[0171] Sequence of the αEGFRxαCD3 bispecific antibody

Table 21

[0172] >Sequence ID 1: Cetuximab H1 VH amino acid sequence EVQLVESGGGLVQPGGSLRLSCKVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 2: Cetuximab H1 VH nucleotide sequence GAAGTTCAGCTGGTGGAATCCGGCGGAGGATTGGTTCAACCTGGCGGCTCTCTGAGACTGTCCTGTAAGGTGTCTGGCTTCTCCCTGACCAACTACGGCGTGCACTGGGTCCGACAGGCACCTGGAAAAGGACTGGAATGGGTCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCTCTCGGGACACCTCCAAGAACACCGTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTAGAGCCCTGACCTACTATGACTACGAGTTCGCCTATTGGGGCCAGGGAACCCTGGTCACAGTCTCCTCT >Sequence ID 3: Cetuximab H1 VL amino acid sequence EIVMTQSPSTLSASVGDRVIITCRASQSIGTNIHWYQQKPGKAPKLLIYYASESISGIPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQNNNWPTTFGQGTKLTVL >Sequence ID 4: Cetuximab H1 VL nucleotide sequence GAGATCGTGATGACCCAGTCTCCTTCCACACTGTCCGCCTCTGTGGGCGACAGAGTGATCATCACCTGTAGAGCCAGCCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGATCTGGCGCTGAGTTTACCCTGACAATCTCCAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAACTGACAGTTCTT >Sequence ID 5: Cetuximab H2 VH amino acid sequence QVQLQQSGPGLVKPSQTLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYFKLRSLRAEDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 6: Cetuximab H2 VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTCGTGAAGCCTTCTCAGACCCTGTCTATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCTCCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGTCCCTGAGAGCCGAGGACACCGCCATCTACTACTGTGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTCTCTTCT >Sequence ID 7: Cetuximab H2 VL amino acid sequence EIVLTQSPSILSVSPGERATFSCRASQSIGTNIHWYQQRPGKPPRLLIKYASESISGIPSRFSGSGSGTEFTLTITSVQSEDIAVYYCQQNNNWPTTFGPGTKLELK >Sequence ID 8: Cetuximab H2 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCATCCTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGCGGCCTGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGATCTGGCACCGAGTTCACCCTGACCATCACCTCCGTGCAGTCCGAGGATATCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGGAATTGAAA >Sequence ID 9: Cetuximab H3 VH amino acid sequence QVQLVQSGPGLVKPSQTLSLTCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 10: Cetuximab H3 VH nucleotide sequence CAAGTTCAGCTGGTTCAGTCTGGCCCTGGCCTCGTGAAGCCTTCTCAGACCCTGTCTCTGACCTGCACCGTGTCTGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGAGATCCGTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTCTCTTCT >Sequence ID 11: Cetuximab H3 VL amino acid sequence EIVLTQSPSILSVSPGERVTFSCRASQSIGTNIHWYQQRPGKPPRLLIKYASESISGIPARFSGSGSGTEFTLTISSVQSEDFATYYCQQNNNWPTTFGPGTKLELK >Sequence ID 12: Cetuximab H3 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCATCCTGTCTGTGTCTCCCGGCGAGAGAGTGACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGCGGCCTGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGCCAGATTTTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCACCTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGGAATTGAAA >Sequence ID 13: Cetuximab H4 VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 14: Cetuximab H4 VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Sequence ID 15: Cetuximab H4 VL amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLELK >Sequence ID 16: Cetuximab H4 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGGAATTGAAA >Sequence ID 17: Cetuximab H5 VH amino acid sequence QVQLLQSGPGLVKPSETLSLTCTVSGFSLTNYGVHWIRQAPGKGLEWIGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYFKLRSLTSEDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 18: Cetuximab H5 VH nucleotide sequence CAAGTTCAGCTGTTGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCCGAAACACTGTCTCTGACCTGCACCGTGTCCGGCTTCTCCCTGACCAATTATGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGATCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCTCCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGTCCCTGACCTCTGAGGACACCGCCATCTACTACTGCGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGCACACTGGTCACAGTCTCTTCT >Sequence ID 19: Cetuximab H5 VL amino acid sequence EIQLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKTGQPPRLLIKYASESISGIPDRFSGSGSGTEFTLSITSVQSEDFAVYYCQQNNNWPTTFGPGTKLEIL >Sequence ID 20: Cetuximab H5 VL nucleotide sequence GAGATCCAGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAAACCGGCCAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTCACCCTGTCTATCACCTCCGTGCAGTCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGGAAATTCTT >Sequence ID 21: Cetuximab H6 VH amino acid sequence QVQLLQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWIGVIWSGGNTDYNTPFTSRFTITKDNSKNQVFFKLRSVRAEDTALYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 22: Cetuximab H6 VH nucleotide sequence CAAGTTCAGCTGTTGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCCGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAATGGATCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTTCTTCAAGCTGCGGAGCGTGCGCGCTGAGGATACCGCTCTGTACTATTGCGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT >Sequence ID 23: Cetuximab H6 VL amino acid sequence EIVLTQSPSTLSVSPGERVSFSCRASQSIGTNIHWYQQRTGQPPRLLIKYASESISGIPARFSGSGSGTEFTLTITSVQSEDFAVYYCQQNNNWPTTFGQGTKLDIK >Sequence ID 24: Cetuximab H6 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGTGTCCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAGAACCGGCCAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGCCAGATTTTCCGGCTCTGGCTCTGGCACCGAGTTCACCCTGACCATCACCTCCGTGCAGTCTGAGGACTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAGCTGGATATCAAA >Sequence ID 25: Cetuximab H7 VH amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 26: Cetuximab H7 VH nucleotide sequence CAAGTTCAGTTGCAGCAGTCTGGCCCTGGCCTGGTCAAGCCTTCTGAGACACTGTCCATCACCTGTACCGTGTCCGGCTTCTCCCTGACCAATTACGGCGTGCACTGGATCAGACAGGCCCCTGGCAAAGGACTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCACCAAGGACAACTCCAAGAACCAGGTGTACTTCAAGCTGCGGAGCGTGCGGGCTGATGACACCGCCATCTACTACTGTGCTCGGGCCCTGACCTACTACGACTACGAGTTTGCTTACTGGGGCCAGGGCACCCTGGTCACAGTTTCTTCT >Sequence ID 27: Cetuximab H7 VL amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVL >Sequence ID 28: Cetuximab H7 VL nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACAGTTCTT >Sequence ID 29: Cetuximab H8 VH amino acid sequence QVQLVESGPGLVQPSGSLSLTCTVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYLKMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 30: Cetuximab H8 VH nucleotide sequence CAAGTTCAGCTGGTGGAATCTGGCCCTGGCCTGGTTCAGCCTTCTGGCTCTCTGTCTCTGACCTGCACCGTGTCTGGCTTCTCCCTGACCAATTACGGCGTGCACTGGGTTCGACAGGCTCCAGGCAAAGGACTGGAATGGGTCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCTCCAAGGACAACTCCAAGAACCAGGTGTACCTGAAGATGAACTCCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGAACCCTGGTCACAGTCTCCTCT >Sequence ID 31: Cetuximab H8 VL amino acid sequence DIVLTQSPSSLSVSPGERVTISCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVESEDFAVYYCQQNNNWPTTFGQGTKLEIK >Sequence ID 32: Cetuximab H8 VL nucleotide sequence GACATCGTGCTGACCCAGTCTCCATCCAGCCTGTCTGTGTCTCCTGGCGAGAGAGTGACCATCTCTTGCCGGGCCTCTCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACAGACTTTACCCTGACCATCAGCTCCGTGGAATCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAGCTGGAAATCAAA >Sequence ID 33: Cetuximab H9 VH amino acid sequence QVQLVESGPGLVQPSGSLSLTCTVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYLKMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 34: Cetuximab H9 VH nucleotide sequence CAAGTTCAGCTGGTGGAATCTGGCCCTGGCCTGGTTCAGCCTTCTGGCTCTCTGTCTCTGACCTGCACCGTGTCTGGCTTCTCCCTGACCAATTACGGCGTGCACTGGGTTCGACAGGCTCCAGGCAAAGGACTGGAATGGGTCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCAGCCGGTTCACCATCTCCAAGGACAACTCCAAGAACCAGGTGTACCTGAAGATGAACTCCCTGAGAGCCGAGGACACCGCTGTGTATTACTGTGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGAACCCTGGTCACAGTCTCCTCT >Sequence ID 35: Cetuximab H9 VL amino acid sequence DIVLTQSPSSLSVSPGERVTISCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVESEDFAVYYCQQNNNWPTTFGQGTKLTVL >Sequence ID 36: Cetuximab H9 VL nucleotide sequence GACATCGTGCTGACCCAGTCTCCATCCAGCCTGTCTGTGTCTCCTGGCGAGAGAGTGACCATCTCTTGCCGGGCCTCTCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACAGACTTTACCCTGACCATCAGCTCCGTGGAATCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAGCTGACAGTTCTT >Sequence ID 37: Cetuximab H10 VH amino acid sequence QVQLQESGPGLVKPSESLSLTCTVSGFSLTNYGVHWVRQPPGKGLEWIGVIWSGGNTDYNTPFTSRVTISKDNSKNQVSLKMNSLTAADTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 38: Cetuximab H10 VH nucleotide sequence CAAGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCTTCCGAATCTCTGTCTCTGACCTGCACCGTGTCCGGCTTCTCCCTGACCAATTATGGCGTGCACTGGGTTCGACAGCCTCCAGGCAAAGGCCTGGAATGGATCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCTCTAGAGTGACCATCAGCAAGGACAACTCCAAGAACCAGGTGTCCCTGAAGATGAACAGCCTGACCGCTGCCGACACCGCTGTGTACTATTGTGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGAACCCTGGTCACAGTCTCCTCT >Sequence ID 39: Cetuximab H10 VL amino acid sequence DIVLTQSPATLSVSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVQSEDFAVYYCQQNNNWPTTFGQGTKLEIK >Sequence ID 40: Cetuximab H10 VL nucleotide sequence GACATCGTGCTGACCCAGTCTCCAGCCACACTGAGTGTGTCTCCAGGCGAGAGAGCTACCCTGTCCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACAGACTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAGCTGGAAATCAAA >Sequence ID 41: Cetuximab H11 VH amino acid sequence QVQLQESGPGLVKPSESLSLTCTVSGFSLTNYGVHWVRQPPGKGLEWIGVIWSGGNTDYNTPFTSRVTISKDNSKNQVSLKMNSLTAADTAVYYCARALTYYDYEFAYWGQGTLVTVSS >Sequence ID 42: Cetuximab H11 VH nucleotide sequence CAAGTTCAGCTGCAAGAGTCTGGCCCTGGCCTGGTCAAGCCTTCCGAATCTCTGTCTCTGACCTGCACCGTGTCCGGCTTCTCCCTGACCAATTATGGCGTGCACTGGGTTCGACAGCCTCCAGGCAAAGGCCTGGAATGGATCGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCTTTCACCTCTAGAGTGACCATCAGCAAGGACAACTCCAAGAACCAGGTGTCCCTGAAGATGAACAGCCTGACCGCTGCCGACACCGCTGTGTACTATTGTGCTAGAGCCCTGACCTACTACGACTACGAGTTCGCCTATTGGGGCCAGGGAACCCTGGTCACAGTCTCCTCT >Sequence ID 43: Cetuximab H11 VL amino acid sequence DIVLTQSPATLSVSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVQSEDFAVYYCQQNNNWPTTFGQGTKLTVL >Sequence ID 44: Cetuximab H11 VL nucleotide sequence GACATCGTGCTGACCCAGTCTCCAGCCACACTGAGTGTGTCTCCAGGCGAGAGAGCTACCCTGTCCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCTGGACAGGCCCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACAGACTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCCAGGGCACCAAACTGACAGTTCTT >Sequence ID 45: MonoFc amino acid sequence GSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 46: MonoFc nucleotide sequence GGATCCGGCGGCTCTCCCGTCGCTGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCTAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAGATGCCACGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGAAGCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCACCCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGCTCCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGCAAATGA >Sequence ID 47: Human IgG1 amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Sequence ID 48: Human IgG1 nucleotide sequence GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTAA >Sequence ID 49: Human C kappa amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 50: Human C kappa amino acid sequence CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Sequence ID 51: (G4S)4 linker amino acid sequence GGGGSGGGGSGGGGSGGGGS >Sequence ID 52: (G4S)4 linker nucleotide sequence GGCGGCGGAGGATCTGGCGGAGGTGGAAGCGGAGGCGGAGGAAGCGGTGGCGGCGGATCT >Sequence ID 53: His tag amino acid sequence GSHHHHHH >Sequence ID 54: His tag nucleotide sequence GGATCCCATCATCACCATCACCATTGA >Sequence ID 55: SI-79R1 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGSHHHHHH >Sequence ID 56: SI-79R1 nucleotide sequence GACATCTTGCTGACTCAGTCTCCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGTATTGGCACAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGTCTATCTCTGGGATTCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAATAATAACTGGCCAACCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCCGGCGGTGGAGGATCACAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAACACAGACTATAATACACCTTTCACATCCAGACTGAGCATCAACAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAATCTAATGACACAGCCATATATTACTGTGCCAGAGCCCTCACCTACTATGATTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCGAGTGGATCCCATCATCACCATCACCATTGA >Sequence ID 57: SI-79R2 amino acid sequence EIVMTQSPSTLSASVGDRVIITCRASQSIGTNIHWYQQKPGKAPKLLIYYASESISGIPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQNNNWPTTFGQGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSHHHHHH >Sequence ID 58: SI-79R2 nucleotide sequence GAAATCGTTATGACACAGTCCCCATCCACTCTTAGCGCTTCTGTAGGGGATCGAGTGATTATCACATGCCGGGCCTCCCAATCCATAGGAACCAACATACACTGGTATCAACAAAAACCAGGCAAAGCGCCAAAACTGCTTATCTACTACGCCTCCGAGAGTATTTCTGGAATCCCGAGTCGCTTCTCAGGTTCTGGAAGCGGCGCTGAGTTTACCCTCACAATTTCTTCACTCCAACCGGATGACTTCGCTACATATTACTGCCAACAAAACAATAATTGGCCGACGACCTTTGGCCAGGGCACGAAACTTACGGTACTTGGCGGTGGCGGTAGTGGGGGAGGCGGTTCTGGCGGCGGAGGGTCCGGCGGTGGAGGATCAGAAGTACAGCTTGTCGAGTCCGGTGGGGGGCTTGTTCAGCCAGGGGGTTCCTTGAGGCTTTCCTGCACCGTCTCTGGGTTTAGCTTGACGAATTACGGCGTTCACTGGGTTAGACAAGCACCGGGGAAGGGGCTGGAATGGGTCGGTGTGATATGGTCCGGGGGTAATACGGATTACAATACACCTTTCACGTCACGCTTTACGATTAGCAGGGACACGTCAAAAAATACAGTCTACTTGCAGATGAACTCTCTTAGGGCGGAAGATACTGCAGTTTATTACTGCGCAAGGGCTCTGACATACTACGATTATGAATTTGCATATTGGGGCCAGGGGACTTTGGTCACGGTCTCGAGTGGATCCCATCATCACCATCACCATTGA >Sequence ID 59: SI-79SF1 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 60: SI-79SF1 nucleotide sequence >Sequence ID 61: SI-79SF2 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSEDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 62: SI-79SF2 nucleotide sequence >Sequence ID 63: SI-79SF3 amino acid sequence EIVMTQSPSTLSASVGDRVIITCRASQSIGTNIHWYQQKPGKAPKLLIYYASESISGIPSRFSGSGSGAEFTLTISSLQPDDFATYYCQQNNNWPTTFGQGTKLTVLGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 64: SI-79SF3 nucleotide sequence >Sequence ID 65: SI-79SF4 amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 66: SI-79SF4 nucleotide sequence >Sequence ID 67: SI-79SF5 amino acid sequence DIVLTQSPSSLSVSPGERVTISCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVESEDFAVYYCQQNNNWPTTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLVESGPGLVQPSGSLSLTCTVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYLKMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 68: SI-79SF5 nucleotide sequence >Sequence ID 69: SI-79SF6 amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 70: SI-79SF6 nucleotide sequence >Sequence ID 71: SI-79SF7 amino acid sequence DIVLTQSPSSLSVSPGERVTISCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVESEDFAVYYCQQNNNWPTTFGQGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLVESGPGLVQPSGSLSLTCTVSGFSLTNYGVHWVRQAPGKGLEWVGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYLKMNSLRAEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 72: SI-79SF7 nucleotide sequence >Sequence ID 73: SI-79SF8 amino acid sequence DIVLTQSPATLSVSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVQSEDFAVYYCQQNNNWPTTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSESLSLTCTVSGFSLTNYGVHWVRQPPGKGLEWIGVIWSGGNTDYNTPFTSRVTISKDNSKNQVSLKMNSLTAADTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 74: SI-79SF8 nucleotide sequence >Sequence ID 75: SI-79SF9 amino acid sequence EIVLTQSPSTLSVSPGERVSFSCRASQSIGTNIHWYQQRTGQPPRLLIKYASESISGIPARFSGSGSGTEFTLTITSVQSEDFAVYYCQQNNNWPTTFGQGTKLDIKGGGGSGGGGSGGGGSGGGGSQVQLLQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWIGVIWSGGNTDYNTPFTSRFTITKDNSKNQVFFKLRSVRAEDTALYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 76: SI-79SF9 nucleotide sequence >Sequence ID 77: SI-79SF10 amino acid sequence EIVLTQSPSILSVSPGERVTFSCRASQSIGTNIHWYQQRPGKPPRLLIKYASESISGIPARFSGSGSGTEFTLTISSVQSEDFATYYCQQNNNWPTTFGPGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLVQSGPGLVKPSQTLSLTCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRSEDTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 78: SI-79SF10 nucleotide sequence >Sequence ID 79: SI-79SF11 amino acid sequence EIQLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKTGQPPRLLIKYASESISGIPDRFSGSGSGTEFTLSITSVQSEDFAVYYCQQNNNWPTTFGPGTKLEILGGGGSGGGGSGGGGSGGGGSQVQLLQSGPGLVKPSETLSLTCTVSGFSLTNYGVHWIRQAPGKGLEWIGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYFKLRSLTSEDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 80: SI-79SF11 nucleotide sequence >Sequence ID 81: SI-79SF12 amino acid sequence EIVLTQSPSILSVSPGERATFSCRASQSIGTNIHWYQQRPGKPPRLLIKYASESISGIPSRFSGSGSGTEFTLTITSVQSEDIAVYYCQQNNNWPTTFGPGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSQTLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTISKDNSKNQVYFKLRSLRAEDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 82: SI-79SF12 nucleotide sequence >Sequence ID 83: SI-79SF13 amino acid sequence DIVLTQSPATLSVSPGERATLSCRASQSIGTNIHWYQQKPGQAPRLLIKYASESISGIPSRFSGSGSGTDFTLTISSVQSEDFAVYYCQQNNNWPTTFGQGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSESLSLTCTVSGFSLTNYGVHWVRQPPGKGLEWIGVIWSGGNTDYNTPFTSRVTISKDNSKNQVSLKMNSLTAADTAVYYCARALTYYDYEFAYWGQGTLVTVSSGSGGSPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSTLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK >Sequence ID 84: SI-79SF13 nucleotide sequence >Sequence ID 85: SI-55P3 heavy chain amino acid sequence >Sequence ID 86: SI-55P3 heavy chain nucleotide sequence >Sequence ID 87: SI-55P3 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 88: SI-55P3 light chain nucleotide sequence >Sequence ID 89: SI-55P4 heavy chain amino acid sequence >Sequence ID 90: SI-55P4 heavy chain nucleotide sequence >Sequence ID 91: SI-55P4 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 92: SI-55P4 light chain nucleotide sequence >Sequence ID 93: SI-79P2 heavy chain amino acid sequence >Sequence ID 94: SI-79P2 heavy chain nucleotide sequence >Sequence ID 95: SI-79P2 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 96: SI-79P2 light chain nucleotide sequence >Sequence ID 97: SI-79P3 heavy chain amino acid sequence >Sequence ID 98: SI-79P3 heavy chain nucleotide sequence >Sequence ID 99: SI-79P3 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDPVLTQSPSSLSASVGDRVTISCQSSQSVAKNNNLAWFQQKPGQAPKLLIYSASTLAAGVPSRFSGSGSGTDFTLTISSVQPEDFATYYCSARDSGNIQSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 100: SI-79P3 light chain nucleotide sequence >Sequence ID 101: SI-55P9 heavy chain amino acid sequence >Sequence ID 102: SI-55P9 heavy chain nucleotide sequence >Sequence ID 103: SI-55P9 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLRLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 104: SI-55P9 light chain nucleotide sequence >Sequence ID 105: SI-77P1 heavy chain amino acid sequence >Sequence ID 106: SI-77P1 heavy chain nucleotide sequence >Sequence ID 107: SI-77P1 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSEIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 108: SI-77P1 light chain nucleotide sequence >Sequence ID 109: SI-77H5 heavy chain amino acid sequence >Sequence ID 110: SI-77H5 heavy chain nucleotide sequence >Sequence ID 111: SI-77H5 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGCGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 112: SI-77H5 light chain nucleotide sequence >Sequence ID 113: SI-55H11 heavy chain amino acid sequence >Sequence ID 114: SI-55H11 heavy chain nucleotide sequence >Sequence ID 115: SI-55H11 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGCGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 116: SI-55H11 light chain nucleotide sequence >Sequence ID 117: SI-77H4 heavy chain amino acid sequence >Sequence ID 118: SI-77H4 heavy chain nucleotide sequence >Sequence ID 119: SI-77H4 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTVLGGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLSTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYYCARMELWSYYFDYWGQGTLVTVSSGGGGSGGGGSEIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGCGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNFVSWYQQHPGKAPKLMIYDVSDRPSGVSDRFSGSKSGNTASLIISGLQADDEADYYCSSYGSSSTHVIFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSQVQLQESGGGLVKPGGSLSLSCAASGFTFSSYWMSWVRQAPGKGLEWVANINRDGSASYYVDSVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARDRGVGYFDLWGRGTLVTVSS >Sequence ID 120: SI-77H4 light chain nucleotide sequence >Sequence ID 121: SI-68P7 heavy chain amino acid sequence >Sequence ID 122: SI-68P7 heavy chain nucleotide sequence >Sequence ID 123: SI-68P7 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTLGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYCARMELWSYYFDYWGQGT LVTVSSGGGGSGGGGSDPQLTQSPSSLSATVGQRVTINCQSSQSVAKNNNLAWFQQKPGKPKLLIYSASTLAAGVPSRFSGSGSGTQFTLTITRVQSEDFATYYCSARDSGNIQSFGGGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECGGDKTHTC PPCPAPEAAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLWCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGGGGGSGGGGSDIQMTQSPSTL SASVGDRVTITCQASQSISSHLNWYQQKPGKAPCLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYSWGNVDNVFGGGTKVTVLGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLSLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCARSAFSFDYAMDLWGQGTLVTVSS >Sequence ID 124: SI-68P7 light chain nucleotide sequence >Sequence ID 125: SI-79P1 heavy chain amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNWPTTFGPGTKLELKGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDDTAIYYCARALTYYDYEFAYWGQ GTLVTVSSGGGGSGGGGSQVQLQESGGRLVQPGEPLSLTCKTSGIDLSSNAIGWVRQAPGKGLEWIGVIFGSGNTYYASWAKGRFTISRSTSTVYLKMNSLRSEDTAIYYCARGGYSSDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPSCDKTHT CPPCPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGGTDFTLTISDLEPGDAATYYCQSTYLGTDYVGGAFGGGTKLTVLGGGGSGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPGKGLEYIGTISSGGVNVYYASSARGRFTISRPSSKNTVDLQMNSLRAEDTAVYYCARDSGYSDPMWGQGTLVTVSS >Sequence ID 126: SI-79P1 heavy chain nucleotide sequence >Sequence ID 127: SI-79P1 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTLGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYCARMELWSYYFDYWGQGT LVTVSSGGGGSGGGGSDPQLTQSPSSLSATVGQRVTINCQSSQSVAKNNNLAWFQQKPGKPKLLIYSASTLAAGVPSRFSGSGSGTQFTLTITRVQSEDFATYYCSARDSGNIQSFGGGTRVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECGGDKTHTC PPCPAPEAAGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLWCLVKGPYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGGGGGSGGGGSDIQMTQSPSTL SASVGDRVTITCQASQSISSHLNWYQQKPGKAPCLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYSWGNVDNVFGGGTKVTVLGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLSLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCARSAFSFDYAMDLWGQGTLVTVSS >Sequence ID 128: SI-79P1 light chain nucleotide sequence >Sequence ID 129: SI-68P13 heavy chain amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGGSGTEFTLISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVLGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFISTNAMSWVRQAPGKGLEWIGVITGRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGGTDFTLTISDLEPGDAATYYCQSTYLGTDYVGGAFGGGTKLTVLGGGGSGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPGKGLEYIGTISSGGVNVYYASSARGRFTISRPSSKNTVDLQMNSLRAEDTAVYYCARDSGYSDPMWGQGTLVTVSS >Sequence ID 130: SI-68P13 heavy chain nucleotide sequence >Sequence ID 131: SI-68P13 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTLGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAADTAVYCARMELWSYYFDYWGQGTL VTVSSGGGGSGGGGSDVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECGGDKTTH CPPCPAEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGGGGGSGGGGSDIQMTQSPST LSASVGDRVTITCQASQSISSHLNWYQQKPGKAPCLLIYKASTLASGVPSRFSGSGSGTETLTISSLQPDDFATYYCQQGYSWGNVDNVFGGGTKVTVLGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLSLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCARSASFDYAMDLWGQGTLVTVSS >Sequence ID 132: SI-68P13 light chain nucleotide sequence >Sequence ID 133: SI-68P17 heavy chain amino acid sequence DVVMTQSPSTLSASVGDRVTINCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGGGTKVEIKGGGGSGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFISTNAMSWVRQAPGKGLEWIGVITGRDITYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSGGGSGGGSGGGSQVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS CDKTHTCPPCPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGGSGGGSDVVMTQSPSSVSASVGDRVTITCQASQNIRTYLSWYQQKPGKAPKLLIYAAANLASGVPSRFSGSGGTDFTLTISDLEPGDAATYYCQSTYLGTDYVGGAFGGGTKLTVLGGGGSGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCTASGFTISSYHMQWVRQAPGKGLEYIGTISSGGVNVYYASSARGRFTISRPSSKNTVDLQMNSLRAEDTAVYYCARDSGYSDPMWGQGTLVTVSS >Sequence ID 134: SI-68P17 heavy chain nucleotide sequence >Sequence ID 135: SI-68P17 light chain amino acid sequence ENVLTQSPASLSASPGERVTITCSASSSVSYMHWYQQKPGQAPKLWIYDTSKLASGVPSRFSGSGSGNDHTLTISSMEPEDFATYYCFQGSVYPFTFGQGTKVTLGGGSGGGGSGGGGSGGGGSQVTLKESGPGLVQPGQTLSLTCAFSGFSLTSGMGVGWIRQPPGKGLEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVYLQMNSLDAEDTAVYCARMELWSYYFDYWGQGT LVTVSSGGGGSGGGGSEIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNWPTTFGPGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECGGDKTHTCPP CAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLPGGGGGSGGGGSDIQMTQSPSTLS ASVGDRVTITCQASQSISSHLNWYQQKPGKAPCLLIYKASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQGYSWGNVDNVFGGGTKVTVLGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLSLSCAASGFSFSSGYDMCWVRQAPGKGLEWIACIAAGSAGITYDANWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYCARSAFSFDYAMDLWGQGTLVTVSS >Sequence ID 136: SI-68P17 light chain nucleotide sequence >Sequence ID 137: SI-79C1 HC and SI-79X1 HC1 amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Sequence ID 138: SI-79C1 HC and SI-79X1 HC1 nucleotide sequence >Sequence ID 139: SI-79C1 LC, SI-79C5 LC, SI-79X1 LC1, and SI-79X5 LC1 amino acid sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 140: SI-79C1 LC, SI-79C5 LC, SI-79X1 LC1, and SI-79X5 LC1 nucleotide sequence GACATCCTGCTGACCCAGTCTCCAGTGATCCTGTCCGTGTCTCCTGGCGAGAGAGTGTCCTTCAGCTGCAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACGGCTCCCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCTATCAGCGGCATCCCCTCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGTCCATCAACTCCGTGGAATCCGAGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGCGCTGGCACCAAGCTGGAATTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Sequence ID 141: SI-79C2 HC, SI-79C3 HC, SI-79X2 HC1, SI-79X3 HC1 amino acid sequence QVQLQQSGPGLVKPSETLSITCTVSGFSLTNYGVHWIRQAPGKGLEWLGVIWSGGNTDYNTPFTSRFTITKDNSKNQVYFKLRSVRADDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Sequence ID 142: SI-79C2 HC, SI-79C3 HC, SI-79X2 HC1, SI-79X3 HC1 nucleotide sequence >Sequence ID 143: SI-79C2 LC and SI-79X2 LC1 amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 144: SI-79C2 LC and SI-79X2 LC1 nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGGAATTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Sequence ID 145: SI-9C21 HC, SI-79X1 HC2, SI-79X2 HC2, SI-79X3 HC2, and SI-79X5 HC2 amino acid sequence EVQLVESGGGLVQPGGSLRLSCTASGFTISTNAMSWVRQAPGKGLEWVGVITGRDITYYASWAKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARDGGSSAITSNNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Sequence ID 146: SI-9C21 HC, SI-79X1 HC2, SI-79X2 HC2, SI-79X3 HC2, and SI-79X5 HC2 nucleotide sequence >Sequence ID 147: SI-9C21 LC, SI-79X1 LC2, SI-79X2 LC2, SI-79X3 LC2, and SI-79X5 LC2 amino acid sequence EIVMTQSPSTLSSASVGDRVIITCQASESISSWLAWYQQKPGKAPKLLIYEASKLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQGYFYFISRTYVNSFGQGTKL TVLRTVAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 148: SI-9C21 LC, SI-79X1 LC2, SI-79X2 LC2, SI-79X3 LC2, and SI-79X5 LC2 nucleotide sequence GAAATCGTTATGACGCAGAGTCCCTCCACGCTCTCCGCTAGTGTCGGGGATCGCGTCATTATCACATGCCAGGCCTCCGAGTCAATCAGCAGCTGGCTTGCATGGTATCAACAGAAGCCGGGAAAAGCTCCTAAATTGCTGATCTATGAAGCGTCAAAATTGGCGTCTGGTGTCCCATCTAGGTTCTCCGGCTCTGGGTCTGGTGCGGAATTTACTTTGACAATCTCCAGTCTTCAACCAGACGATTTCGCTACCTACTACTGCCAAGGGTATTTCTATTTTATAAGCCGGACATATGTAAACTCCTTCGGCCAAGGAACAAAGTTGACTGTTCTTCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Sequence ID 149: SI-79C3 LC and SI-79X3 LC1 amino acid sequence EIVLTQSPSTLSVSPGERATFSCRASQSIGTNIHWYQQKPGKPPRLLIKYASESISGIPDRFSGSGSGTEFTLTISSVQSEDFAVYYCQQNNNWPTTFGPGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >Sequence ID 150: SI-79C3 LC and SI-79X3 LC1 nucleotide sequence GAGATCGTGCTGACCCAGTCTCCTTCCACACTGTCTGTGTCTCCCGGCGAGAGAGCCACCTTCAGCTGTAGAGCCTCTCAGTCCATCGGCACCAACATCCACTGGTATCAGCAGAAGCCCGGCAAGCCTCCTCGGCTGCTGATTAAGTACGCCTCCGAGTCCATCAGCGGCATCCCTGACAGATTCTCCGGCTCTGGCTCTGGCACCGAGTTTACCCTGACCATCTCCTCCGTGCAGTCCGAGGATTTCGCCGTGTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTTGGACCCGGCACCAAGCTGACCGTGCTGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG >Sequence ID 151: SI-79C5 HC and SI-79X5 HC1 amino acid sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSEDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG >Sequence ID 152: SI-79C5 HC and SI-79X5 HC1 nucleotide sequence

[0173] References 1. Graham J, Muhsin M, Kirkpatrick P. Cetuximab. Nat Rev Drug Discov 2004; 3:549-50. 2. Bou-Assaly W, Mukherji S. Cetuximab (Erbitux). Am J Neuroradiol 2010; 31:626-7. 3. Hanck-Silva G, Fatori Trevizan LN, Petrilli R, de Lima FT, Eloy JO, Chorilli M. A Critical Review of Properties and Analytical / Bioanalytical Methods for Characterization of Cetuximab. Crit Rev Anal Chem [Internet] 2020; 50:125-35. Available from: https: / / doi.org / 10.1080 / 10408347.2019.1581984 4. Garcia-Foncillas J, Sunakawa Y, Aderka D, Wainberg Z, Ronga P, Witzler P, Stintzing S. Distinguishing Features of Cetuximab and Panitumumab in Colorectal Cancer and Other Solid Tumors. Front Oncol 2019; 9:1-16. 5. Kaplon H, Reichert JM. Antibodies to watch in 2021. MAbs [Internet] 2021; 13:1-34. Available from: https: / / doi.org / 10.1080 / 19420862.2020.1860476 6. Hwang WYK, Foote J. Immunogenicity of engineered antibodies. Methods 2005; 36:3-10. 7. Park SS, Kim J, Brandts JF, Hong HJ. Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus. Biologicals 2003; 31:295-302. 8. Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, Baessmann C, Wagner-Rousset E, Suckau D, Beck A. Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques. MAbs 2013; 5:699-710. 9. van de Bovenkamp FS, Hafkenscheid L, Rispens T, Rombouts Y. The Emerging Importance of IgG Fab Glycosylation in Immunity. J Immunol 2016; 196:1435-41. 10. Chung CH, Mirakhur B, Chan E, Le Q-T, Berlin J, Morse M, Murphy BA, Satinover SM, Hosen J, Mauro D, et al. Cetuximab-Induced Anaphylaxis and IgE Specific for Galactose-α-1,3-Galactose. N Engl J Med 2008; 358:1109-17. 11. Kuriakose A, Chirmule N, Nair P. Immunogenicity of Biotherapeutics: Causes and Association with Posttranslational Modifications. J Immunol Res 2016; 2016. 12. Zhang P, Woen S, Wang T, Liau B, Zhao S, Chen C, Yang Y, Song Z, Wormald MR, Yu C, et al. Challenges of glycosylation analysis and control: An integrated approach to producing optimal and consistent therapeutic drugs. Drug Discov Today [Internet] 2016; 21:740-65. Available from: http: / / dx.doi.org / 10.1016 / j.drudis.2016.01.006 13. Borras L, Gunde T, Tietz J, Bauer U, Hulmann-Cottier V, Grimshaw JPA, Urech DM. Generic approach for the generation of stable humanized single-chain Fv fragments from rabbit monoclonal antibodies. J Biol Chem 2010; 285:9054-66. 14. Li S, Schmitz KR, Jeffrey PD, Wiltzius JJW, Kussie P, Ferguson KM. Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell 2005; 7:301-11. 15. Ewert S, Huber T, Honegger A, Pluckthun A. Biophysical properties of human antibody variable domains. J Mol Biol 2003; 325:531-53. 16. Nieba L, Honegger A, Krebber C, Pluckthun A. Disrupting the hydrophobic patches at the antibody variable / constant domain interface: Improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng 1997; 10:435-44. 17. Gao SH, Huang K, Tu H, Adler AS. Monoclonal antibody humanness score and its applications. BMC Biotechnol 2013; 13. 18. Racle J, Michaux J, Rockinger GA, Arnaud M, Bobisse S, Chong C, Guillaume P, Coukos G, Harari A, Jandus C, et al. Robust prediction of HLA class II epitopes by deep motif deconvolution of immunopeptidomes. Nat Biotechnol [Internet] 2019; 37:1283-6. Available from: http: / / dx.doi.org / 10.1038 / s41587-019-0289-6 19. Wang C, Wu Y, Wang L, Hong B, Jin Y, Hu D, Chen G, Kong Y, Huang A, Hua G, et al. Engineered soluble monomeric IgG1 Fc with significantly decreased non-specific binding. Front Immunol 2017; 8:1-8. 20. Ulitzka M, Carrara S, Grzeschik J, Kornmann H, Hock B, Kolmar H. Engineering therapeutic antibodies for patient safety: Tackling the immunogenicity problem. Protein Eng Des Sel 2020; 33:1-12. 21. Banisadr A, Safdari Y, Kianmehr A, Pourafshar M. Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells. Hum Vaccines Immunother [Internet] 2018; 14:856-63. Available from: https: / / doi.org / 10.1080 / 21645515.2017.1407482 22. Choi Y, Ndong C, Griswold KE, Bailey-Kellogg C. Computationally driven antibody engineering enables simultaneous humanization and thermostabilization. Protein Eng Des Sel 2016; 29:419-26. 23. Giddens JP, Lomino J V., DiLillo DJ, Ravetch J V., Wang LX. Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody. Proc Natl Acad Sci U S A 2018; 115:12023-7.

Claims

1. A human epidermal growth factor receptor (EGFR) binding peptide having binding specificity to human EGFR, EGFR-conjugated peptides containing amino acid sequences having at least 98%, 95%, or 92% sequence identity with SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 57, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83.

2. It includes variable heavy (VH) chains and variable light (VL) chains, The VH chain comprises an amino acid sequence having at least 98%, 95%, or 92% sequence identity with SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, or 41. The EGFR-conjugated peptide according to claim 1, wherein the VL chain comprises an amino acid sequence having at least 98%, 95%, or 92% sequence identity with SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, or 43.

3. Includes the scFv domain, The scFv domain includes the VH chain and the VL chain, The EGFR-binding peptide according to claim 2, wherein the scFv domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 57, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, or 83.

4. The scFv domain comprises a histidine residue bound to at least one terminal, The EGFR-binding peptide according to claim 3, wherein the EGFR-binding peptide comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO:

57.

5. Including the Fab domain, The EGFR-binding peptide according to claim 2, wherein the Fab domain comprises the VH chain and the VL chain.

6. The Fab-monoFc fusion protein further comprises an Fc domain bound to the Fab domain, The EGFR-binding peptide according to claim 5, wherein the Fc domain comprises a sequence having at least 98% sequence identity with an amino acid sequence selected from SEQ ID NOs. 45 and 47.

7. An antibody-like protein having binding specificity to human EGFR, It contains an EGFR-binding domain having a variable heavy (VH) chain and a variable light (VL) chain, The VH chain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, or 41. The VL chain is an antibody-like protein comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, or 43.

8. Includes the scFv domain, The antibody-like protein according to claim 7, wherein the scFv domain comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 or any combination thereof.

9. The antibody-like protein according to claim 7, wherein the antibody-like protein is a monospecific antibody and comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 137, 139; 141, 143; 141, 149, 151, 139; 145, 147 or a combination thereof.

10. The antibody-like protein according to claim 7, wherein the antibody-like protein is a bispecific antibody and comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 137, 145, 139, 147, 141, 145, 149, 147, 151, 145, 139, 147 or a combination thereof.

11. The antibody-like protein according to claim 7, wherein the antibody-like protein is a quintuplicate antibody and comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 85, 87; 89, 91; 93, 95, 97, 99, 101, 103, 105, 107 or a combination thereof.

12. The antibody-like protein according to claim 7, wherein the antibody-like protein is a hexaspecific antibody and comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 109, 111, 113, 115, 117, 119 or a combination thereof.

13. It contains a heavy chain (HC) and a light chain (LC), The HC comprises an amino acid sequence having at least 98%, 95%, or 92% sequence identity with SEQ ID NOs: 85, 89, 93, 97, 101, 105, 109, 113, 117, 137, 141, 145, or 151. The antibody-like protein according to claim 7, wherein the LC comprises an amino acid sequence having at least 98%, 95%, or 92% sequence identity with SEQ ID NOs: 87, 91, 95, 99, 103, 107, 111, 115, 119, 139, 143, 147, or 149.

14. It contains heavy chain monomers and light chain monomers. The heavy chain monomer has an N-terminus and a C-terminus, and from the N-terminus to the C-terminus An arbitrary first binding domain (D1) located at the N-terminus, The Fab domain containing the light chain serves as the second binding domain (D2), FC domain and, Any third binding domain (D3) and An arbitrary fourth binding domain (D4) located at the C-terminus, This includes tandem, The light chain includes an arbitrary fifth binding domain (D5) covalently bonded to the C-terminus, an arbitrary sixth binding domain (D6) covalently bonded to the N-terminus, or a combination thereof. The antibody-like protein according to claim 7, wherein at least one of D1, D2, D3, D4, D5, and D6 includes the EGFR-binding domain.

15. The antibody-like protein according to claim 14, wherein at least one of D1 or D2 includes the EGFR-binding domain.

16. The antibody-like protein according to claim 14, wherein D3, D4, D5, and D6 each contain the EGFR-binding domain.

17. The antibody-like protein according to claim 14, wherein the antibody-like protein is a bispecific antibody and comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 137, 145, 139, 147; 141, 145, 143, 147; 141, 145, 149, 147; 151, 145, 139, or 147.

18. The antibody-like protein according to claim 17, wherein the bispecific antibody is asymmetric with respect to D2, which includes the EGFR binding domain, and D3 has binding specificity to CD3.

19. The N-terminus and the C-terminus are A first monomer comprising a first binding domain (mD1), a variable weight (VH) chain, a CH1 domain, a first hinge, a first CH2 domain, a first CH3 domain, and a fourth binding domain (mD4) from the N-terminus to the C-terminus, The second monomer comprises a second binding domain (mD2), a variable light (VL) chain, a CL domain, a second hinge, a second CH2 domain, a second CH3 domain, and a fifth binding domain (mD5) from the N-terminus to the C-terminus, Includes, The CH chain and CL chain form a third binding domain (mD3), The first monomer and the second monomer are paired by covalent bonds via at least one disulfide bond between the CH1 domain and the CL domain, and at least one disulfide bond between the first hinge and the second hinge. The antibody-like protein according to claim 7, wherein the multispecific antibody-like protein is at least bispecific.

20. The antibody-like protein according to claim 19, wherein at least one of mD1, mD2, mD3, mD5, and mD5 includes the EGFR-binding domain.

21. The antibody-like protein according to claim 19, wherein at least one of the mD3 or mD2 domains includes the EGFR-binding domain.

22. The antibody-like protein according to claim 19, wherein mD2, mD4, and mD5 each contain the EGFR-binding domain.

23. The antibody-like protein according to claim 19, comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 121, 123, 125, 127, 129, 131, 133, 135 or any combination thereof.

24. A heavy chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 121, 125, 129, or 133.

25. A light chain comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NOs: 123, 127, 131, or 135.

26. An isolated nucleic acid sequence encoding the antibody-like protein described in claim 7.

27. An expression vector comprising the isolated nucleic acid sequence described in claim 26.

28. A host cell comprising the isolated nucleic acid sequence described in claim 26.

29. A pharmaceutical composition comprising the antibody-like protein described in claim 7 and a pharmaceutically acceptable carrier.

30. An immune complex comprising the antibody-like protein described in claim 7 and a cytotoxic agent.

31. A pharmaceutical composition comprising the immune complex described in claim 30 and a pharmaceutically acceptable carrier.

32. A method for treating or preventing a target cancer, autoimmune disease, or infectious disease, A method comprising the step of administering a pharmaceutical composition comprising a purified antibody-like protein according to claim 7 to the subject.

33. A method for producing an antibody-like protein according to claim 7, The steps of culturing host cells so that a DNA sequence encoding the antibody-like protein described in claim 9 is expressed, The step of purifying the multispecific antibody-like protein, Methods that include...

34. A solution comprising the antibody-like protein according to claim 7, with an effective concentration, The aforementioned solution is a solution containing the target plasma.

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