Anti-glyco-CMET antibodies and uses thereof

Abstract

The present disclosure relates to anti-glyco-cMET antibodies and antigen-binding fragments thereof that specifically bind to cancer-specific glycosylation variants of cMET, as well as related fusion proteins and antibody-drug conjugates, as well as nucleic acids encoding such biomolecules. The present disclosure further relates to the use of the antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates, and nucleic acids for cancer therapy.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 63 / 240,761, filed September 3, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] 2.Background Therapies using chimeric antigen receptors (CARs) to redirect T cell responses have emerged as a powerful tool in cancer immunotherapy and have proven highly effective in hematological cancers, targeting nonessential tissue-shared antigens such as CD19 in B cell malignancies (Brentjens et al., 2013, Sci Transl Med. 5(177):177ra38-177ra38; Grupp et al., 2013, N Engl J Med. 368(16):1509-1518; Kalos et al., 2011, Sci Transl Med. 3(95):95ra73-95ra73; Kochenderfer et al., 2010, Blood. 116(20):4099-4102; Porter et al., 2011, N Engl J Med. 365(8):725-733). However, the adoption of CAR therapy in solid tumors has been difficult because the majority of CAR targets are normal self-antigens that are overexpressed in solid tumors. Therefore, studies targeting solid tumors with CAR T cells often report adverse effects due to cross-reactivity with essential healthy tissues (Bin Hou et al., 2019, Dis Markers, Article ID 3425291). To overcome the challenges of adopting CAR therapy in solid tumors, new cancer-specific antigens that enable selective targeting are needed.

[0003] Many cancers express aberrantly glycosylated proteins that differ from those in healthy tissues. These aberrantly glycosylated proteins contain glycopeptide epitopes that may be suitable for immunotherapy of solid tumors, but only a few such glycopeptide epitopes have been identified.

[0004] The MET proto-oncogene encodes a receptor tyrosine kinase that is widely expressed by cells of epithelial-endothelial origin (Brand-Saberi et al., 1996, Dev Biol. 179(1):303-308; Heymann et al., 1996, Devel Biol. 180(2):566-578; Bladt et al., 1995, Nature. 376(6543):768-771). Under normal conditions, c-MET signaling elicits a wide variety of biological effects, including increased cell proliferation, scattering and motility, invasiveness, protection from apoptosis, and angiogenesis (Sierra et al., 2008, J Exp Biol. 205(7):1673-1655; Conrotto et al., 2005, Blood. 105(11):4321-4329; Yi and Tsao, 2000, Neoplasia. 2(3):226-236; Silvagno et al., 1995, Arterioscler Thromb Vasc Biol. 15(11):1857-1865). However, in transformed epithelia, inappropriate activation of c-MET supports the proliferation and invasive potential of cancer cells (Benvenuti and Comoglio, 2007, J Cell Physiol. 213(2):316-325; Danilkovitch-Miagkova and Zbar, 2002, J Clin Invest. 109(7):863-867). Many studies have reported that c-MET is overexpressed in various types of cancer.This includes carcinomas of the lung, breast, ovary, kidney, colon, thyroid, liver, and stomach (Knowles et al., 2009, Clin Cancer Res. 15(11):3740-3750; Lengyel et al., 2005, Int J Cancer. 113(4):678-682; Tokunou et al., 2001, Am J Pathol. 158(4):1451-1463; Ramirez et al., 2000, Clin Endocrinol (Oxf). 53(5):635-644; Tsao et al., 1998, Lung Cancer. 20(1):1-16; Koochekpour et al., 1997, Cancer Res. 57(23):5391-5398; Olivero et al., 1996, Br J Cancer. 74(12):18621868;Tuck et al., 1996, Am J Pathol. 148(1):225-232;Di Renzo et al., 1995, Cancer Res. 55(5):1129-1138;Furukawa et al., 1995, Am J Pathol. 147(4):889-895;Liu et al., 1992, Oncogene. 7(1):181-185;Soman et al., 1991, Proc Natl Acad Sci USA. 88(11):4892-4896;Houldsworth et al., 1990, Cancer Res. 50(19):6417-6422). Due to the importance of c-MET in carcinogenesis and cancer progression, c-MET is considered an important target for anticancer therapy (Trusolino et al., 2010, Nat Rev Mol Cell Bio. 11(12):834-848; Migliore and Giordano, 2008, Eur J Cancer. 44(5):641-651; Peschard and Park, 2007, Oncogene. 26(9):1276-1285; Corso et al., 2005, Trends Mol Med. 11(6):284-292).Although several monoclonal antibodies have shown promising results in tumors with high HGF / c-MET levels, most of these are primarily hindered by HGF-mediated c-MET activation and are not suitable for immunotherapy targeted by cytotoxic strategies due to the prominent expression of c-MET in healthy tissues. Therefore, the identification of glyco-cMET epitopes overexpressed in cancer cells and new therapeutic modalities, such as antibodies and CARs, that target such glyco-cMET epitopes are needed. Summary of the Invention

[0005] 3. Summary The present disclosure captures the tumor specificity of glycopeptide variants by providing therapeutic and diagnostic agents based on antibodies and antigen-binding fragments selective for cancer-specific epitopes of glyco-cMET, which advantageously bind to both the cMET backbone and its cancer-specific O-linked glycans, but do not bind to cMET in healthy tissues.

[0006] Thus, the present disclosure provides anti-glyco-cMET antibodies and antigen-binding fragments thereof that bind to cancer-specific glycosylation variants of cMET. The present disclosure further provides fusion proteins and antibody-drug conjugates comprising the anti-glyco-cMET antibodies and antigen-binding fragments, as well as nucleic acids encoding the anti-glyco-cMET antibodies, antigen-binding fragments, and fusion proteins.

[0007] The present disclosure further provides methods of using anti-glyco-cMET antibodies, antigen-binding fragments, fusion proteins, antibody-drug conjugates and nucleic acids for cancer therapy.

[0008] In certain embodiments, the present disclosure provides bispecific and other multispecific anti-glyco-cMET antibodies and antigen-binding fragments that bind to a cancer-specific glycosylation variant of cMET and a second epitope. The second epitope may be on cMET itself, on another protein co-expressed with cMET on cancer cells, or on another protein presented on a different cell, such as activated T cells. Additionally, nucleic acids encoding such antibodies are also disclosed, including nucleic acids containing codon-optimized coding regions and nucleic acids containing coding regions that are not codon-optimized for expression in a particular host cell.

[0009] Anti-glyco-cMET antibodies and binding fragments can be in the form of fusion proteins containing fusion partners. Fusion partners can be useful for providing a second function, such as the signaling function of a signaling domain of a T cell signaling protein, a peptide modulator of T cell activation, or an enzymatic component of a labeling system. Exemplary T cell signaling proteins include 4-1BB, CD28, CD2, and fusion peptides such as CD28-CD3-zeta, 4-1BB-CD3-zeta, CD2-CD3-zeta, CD28-CD2-CD3-zeta, and 4-1BB-CD2-CD3-zeta. 4-1BB, also known as CD137, is a costimulatory receptor for T cells; CD2 is a costimulatory receptor for T and NK cells; and CD3-zeta is a signaling component of the T cell antigen receptor. The moiety providing the second function may be a modulator of T cell activation, e.g., IL-15, IL-15Rα, or an IL-15 / IL-15Rα fusion; it may be an MHC class I chain-related (MIC) protein domain useful for generating MicAbodies; or it may encode a label or enzymatic element of a labeling system useful for monitoring the degree and / or location of binding in vivo or in vitro. Constructs encoding these prophylactic and therapeutic biomolecules in the context of T cells, e.g., autologous T cells, provide, in some embodiments of the present disclosure, a powerful platform for replenishing adoptively transferred T cells to prevent or treat various cancers.

[0010] In certain embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy and / or light chain CDR sequences (defined by Kabat, Chothia, IMGT, or combined regions of overlap thereof) of anti-glyco-cMET antibody 15C4.1D8.1G2 (sometimes referred to herein as "15C4"), 8H3.2B9.2C7 (sometimes referred to herein as "8H3"), 16E12.1D9.1B11 (sometimes referred to herein as "16E12"), 14E9, 19H2, or 39A3, or a humanized counterpart of any one thereof. In some embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises (or is encoded by) the heavy and / or light chain variable sequences of anti-glycoed cMET antibodies 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3, or their humanized counterparts. The CDRs and variable sequences (and their coding sequences) of anti-glycoed cMET antibodies 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3, respectively, are set forth in Tables 1A-1F. In certain aspects, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises (or is encoded by) the heavy and / or light chain variable sequences set forth in Tables 1A-1F. For clarity, when the term "anti-glyco-cMET antibody" is used in this document, it is intended to include, unless the context dictates otherwise, monospecific and multispecific (including bispecific) anti-glyco-cMET antibodies, antigen-binding fragments of monospecific and multispecific antibodies, and fusion proteins and conjugates containing the antibodies and antigen-binding fragments. Similarly, when the term "anti-glyco-cMET antibody or antigen-binding fragment" is used, it is also intended to include, unless the context dictates otherwise, monospecific and multispecific (including bispecific) anti-glyco-cMET antibodies and antigen-binding fragments thereof, as well as fusion proteins and conjugates containing such antibodies and antigen-binding fragments.

[0011] In other embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure comprises (or is encoded by) the heavy and / or light chain CDR sequences set forth in Tables 1A-3H. The CDR sequences set forth in Tables 1A-1F include CDR sequences defined according to the IMGT (Lefranc et al., 2003, Dev Comparat Immunol 27:55-77), Kabat (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and Chothia (Al-Lazikani et al., 1997, J. Mol. Biol 273:927-948) schemes for defining CDR boundaries. The CDR sequences listed in Tables 1G-1I are consensus sequences obtained from the CDR sequences listed in Tables 1A-1C ("Group 1" antibodies: 15C4, 8H3, 16E12) according to the definitions of IMGT, Kabat, and Chothia, respectively. The CDR sequences listed in Tables 1J-1L are consensus sequences obtained from the CDR sequences listed in Tables 1D-1F ("Group 2" antibodies: 14E9, 19H2, and 39A3) according to the definitions of IMGT, Kabat, and Chothia, respectively. The CDR sequences listed in Tables 2A-2F are combined regions of overlap of the CDR sequences listed in Tables 1A-1F, respectively, with the IMGT, Kabat, and Chothia sequences shown in bold and underlined. The CDR sequences listed in Table 2G are combined regions of overlap of the consensus CDR sequences listed in Tables 2A-2C ("Group 1" antibodies: 15C4, 8H3, 16E12). The CDR sequences listed in Table 2H are the combined regions of overlap of the consensus CDR sequences listed in Tables 2D-2F ("Group 2" antibodies: 14E9, 19H2, and 39A3). The CDR sequences listed in Tables 3A-3F are the common regions of overlap of the CDR sequences shown in Tables 1A-1F, respectively.The CDR sequences listed in Table 3G are the common regions of overlap of the CDR sequences listed in Tables 3A-3D ("Group 1" antibodies: 15C4, 8H3, 16E12). The CDR sequences listed in Table 3H are the common regions of overlap of the CDR sequences listed in Tables 3D-3F ("Group 2" antibodies: 14E9, 19H2, and 39A3). The framework sequences of such anti-glyco-cMET antibodies and antigen-binding fragments may be the native murine framework sequences of the VH and VL sequences listed in Tables 1A-1F, or may be non-native (e.g., humanized or human) framework sequences.

[0012] In other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the disclosure comprises the heavy and / or light chain variable sequence of humanized anti-glyco-cMET antibody 8H3, as set forth in Tables 4A-4G.

[0013] [Table 1-1]

[0014] [Table 1-2]

[0015] [Table 2-1]

[0016] [Table 2-2]

[0017] [Table 3-1]

[0018] [Table 3-2]

[0019]

Table 4-1

[0020]

Table 4-2

[0021]

Table 5-1

[0022]

Table 5-2

[0023]

Table 6-1

[0024]

Table 6-2

[0025]

Table 7

[0026]

Table 8

[0027]

Table 9

[0028]

Table 10

[0029]

Table 11

[0030]

Table 12

[0031]

Table 13

[0032]

Table 14

[0033]

Table 15

[0034] Table 16

[0035] Table 17

[0036] Table 18

[0037] Table 19

[0038] Table 20

[0039] Table 21

[0040] Table 22

[0041] Table 23

[0042] Table 24

[0043] Table 25

[0044] Table 26

[0045] Table 27

[0046] Table 28

[0047] Table 29

[0048] Table 30

[0049] Table 31

[0050] [Table 32]

[0051] [Table 33]

[0052] [Table 34]

[0053] [Table 35]

[0054] In certain aspects, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a CDR comprising the amino acid sequence of any of the CDR combinations set forth in Tables 1A-3H. In certain embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 253, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 254, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 255, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 256, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 257, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 258. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 253. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 254. In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 255. In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 256. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 257. In some embodiments, the CDR-L3 comprises the amino acid sequence of SEQ ID NO:258.

[0055] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 259, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 260, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 261, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 262, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 263, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 342. In some embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 259. In some embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 260. In some embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 262. In some embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 263. In some embodiments, CDR-L3 comprises the amino acid sequence of SEQ ID NO: 342.

[0056] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 3-5 and a light chain CDR of SEQ ID NO: 6-8. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 9-11 and a light chain CDR of SEQ ID NO: 12-14. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 15-17 and a light chain CDR of SEQ ID NO: 18-20. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 169-171 and a light chain CDR of SEQ ID NO: 172-174.

[0057] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 25-27 and a light chain CDR of SEQ ID NO: 28-30. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 31-33 and a light chain CDR of SEQ ID NO: 32-34. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 35-37 and a light chain CDR of SEQ ID NO: 38-40. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 175-177 and a light chain CDR of SEQ ID NO: 178-180.

[0058] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 47-49 and the light chain CDRs of SEQ ID NOs: 50-52. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 53-55 and the light chain CDRs of SEQ ID NOs: 56-58. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 59-61 and the light chain CDRs of SEQ ID NOs: 62-64. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 181-183 and the light chain CDRs of SEQ ID NOs: 184-186.

[0059] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 69-71 and the light chain CDRs of SEQ ID NOs: 72-74. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 75-77 and the light chain CDRs of SEQ ID NOs: 78-80. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 81-83 and the light chain CDRs of SEQ ID NOs: 84-86. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 187-189 and the light chain CDRs of SEQ ID NOs: 190-192.

[0060] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 91-93 and a light chain CDR of SEQ ID NO: 94-96. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 97-99 and a light chain CDR of SEQ ID NO: 100-102. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 103-105 and a light chain CDR of SEQ ID NO: 106-108. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises a heavy chain CDR of SEQ ID NO: 193-195 and a light chain CDR of SEQ ID NO: 196-198.

[0061] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 113-115 and the light chain CDRs of SEQ ID NOs: 116-118. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 119-121 and the light chain CDRs of SEQ ID NOs: 122-124. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 125-127 and the light chain CDRs of SEQ ID NOs: 128-130. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 199-201 and the light chain CDRs of SEQ ID NOs: 202-204.

[0062] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 133-135 and the light chain CDRs of SEQ ID NOs: 136-138. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 139-141 and the light chain CDRs of SEQ ID NOs: 142-144. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 145-147 and the light chain CDRs of SEQ ID NOs: 148-150.

[0063] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 151-153 and the light chain CDRs of SEQ ID NOs: 154-156. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 157-159 and the light chain CDRs of SEQ ID NOs: 160-162. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the present disclosure comprises the heavy chain CDRs of SEQ ID NOs: 163-165 and the light chain CDRs of SEQ ID NOs: 166-168.

[0064] In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the disclosure comprises the heavy chain CDRs of SEQ ID NOs: 205-207 and the light chain CDRs of SEQ ID NOs: 208-210. In other embodiments, an anti-glycoed cMET antibody or antigen-binding fragment of the disclosure comprises the heavy chain CDRs of SEQ ID NOs: 211-213 and the light chain CDRs of SEQ ID NOs: 214-216.

[0065] In certain embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the disclosure comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 133, 139, 145, 169, 175, 181, 205, 217, 223, 229, or 253; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 134, 140, 146, 170, 176, 182, 206, 218, 224, 230, or 254; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 135, 141, 147, 171, 177, 183, 207, 219, 225, 231, or 25 CDR-H3 comprising the amino acid sequence of SEQ ID NO: 136, 142, 148, 172, 178, 184, 208, 220, 226, 232, or 256; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 137, 143, 149, 173, 179, 185, 209, 221, 227, 233, or 257; and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 138, 144, 150, 174, 180, 186, 210, 222, 228, 234, or 258.

[0066] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the disclosure comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 151, 157, 163, 187, 193, 199, 211, 235, 241, 247, or 259; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 152, 158, 164, 188, 194, 200, 212, 236, 242, 248, or 260; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 153, 159, 165, 189, 195, 201, 213, 237, 243, 249, or 260; CDR-H3 comprising the amino acid sequence of SEQ ID NO:1; CDR-L1 comprising the amino acid sequence of SEQ ID NO:154, 160, 166, 190, 196, 202, 214, 238, 244, 250, or 262; CDR-L2 comprising the amino acid sequence of SEQ ID NO:155, 161, 167, 191, 197, 203, 215, 239, 245, 251, or 263; and CDR-L3 comprising the amino acid sequence of SEQ ID NO:156, 162, 168, 192, 198, 204, 216, 240, 246, 252, or 342.

[0067] The antibodies and antigen-binding fragments of the present disclosure may be murine, chimeric, humanized, or human.

[0068] In further embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NOs: 1 and 2, respectively. In still other embodiments, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 1 and 2, respectively.

[0069] In yet other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NOs: 23 and 24, respectively. In still other embodiments, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 23 and 24, respectively.

[0070] In yet other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NOs: 45 and 46, respectively. In still other embodiments, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 45 and 46, respectively.

[0071] In yet other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NOs: 67 and 68, respectively. In still other embodiments, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 67 and 68, respectively.

[0072] In yet other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NOs: 89 and 90, respectively. In still other embodiments, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 89 and 90, respectively.

[0073] In yet other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of SEQ ID NOs: 111 and 112, respectively. In still other embodiments, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 111 and 112, respectively.

[0074] In yet another embodiment, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising a heavy chain variable region of any one of SEQ ID NOs: 133-144 and a light chain variable region of any one of SEQ ID NOs: 145-153. In yet another embodiment, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having a heavy chain variable region having at least 95%, 98%, 99%, or 99.5% sequence identity to any one of SEQ ID NOs: 133-134 and a light chain variable region having at least 95%, 98%, 99%, or 99.5% sequence identity to any one of SEQ ID NOs: 145-153.

[0075] In yet another embodiment, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with an antibody or antigen-binding fragment comprising a heavy chain variable region of any one of SEQ ID NOs: 264-275 and a light chain variable region of any one of SEQ ID NOs: 276-284. In yet another embodiment, the present disclosure provides an anti-cMET antibody or antigen-binding fragment having a heavy chain variable region having at least 95%, 98%, 99%, or 99.5% sequence identity to any one of SEQ ID NOs: 264-275 and a light chain variable region having at least 95%, 98%, 99%, or 99.5% sequence identity to any one of SEQ ID NOs: 276-284.

[0076] In yet another aspect, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure is a single-chain variable fragment (scFv). Exemplary scFvs include a heavy chain variable fragment N-terminal to a light chain variable fragment. In some embodiments, the scFv heavy chain variable fragment and light chain variable fragment are covalently linked by a linker sequence of 4 to 15 amino acids. The scFv may be in the form of a bispecific T cell engager or within a chimeric antigen receptor (CAR).

[0077] The anti-glyco-cMET antibodies and antigen-binding fragments may be in the form of single-chain variable fragment multimers, bispecific single-chain variable fragments, and bispecific single-chain variable fragment multimers. In some embodiments, the single-chain variable fragment multimers are selected from bivalent single-chain variable fragments, tribodies, or tetrabodies. In some of these embodiments, the bispecific single-chain variable fragment multimers are bispecific T cell engagers.

[0078] Other aspects of the present disclosure extend to nucleic acids encoding the anti-glycoed cMET antibodies and antigen-binding fragments of the present disclosure. In some embodiments, the portion of the nucleic acid encoding the anti-glycoed cMET antibody or antigen-binding fragment is codon-optimized for expression in human cells. In certain aspects, the present disclosure provides an anti-glycoed cMET antibody or antigen-binding fragment having heavy and light chain variable regions encoded by a heavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 21, 43, or 65, and a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 22, 44, or 66. In other aspects, the present disclosure provides an anti-glyco-cMET antibody or antigen-binding fragment having heavy and light chain variable regions encoded by a heavy chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 87, 109, or 131, and a light chain nucleotide sequence having at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NO: 88, 110, or 132. Vectors (e.g., viral vectors, such as lentiviral vectors) and host cells containing nucleic acids are also within the scope of the present disclosure. The sequences encoding the heavy and light chains may be present on a single vector or on separate vectors.

[0079] In yet another aspect of the present disclosure is a pharmaceutical composition comprising an anti-glyco-cMET antibody, antigen-binding fragment, nucleic acid (or pair of nucleic acids), vector (or pair of vectors) or host cell according to the present disclosure, and a physiologically suitable buffer, adjuvant, or diluent.

[0080] Yet another aspect of the present disclosure is a method of making a chimeric antigen receptor, the method comprising incubating a cell containing a nucleic acid or vector according to the present disclosure under conditions suitable for expression of the coding region, and harvesting the chimeric antigen receptor.

[0081] Another aspect of the present disclosure is a method for detecting cancer, the method comprising contacting a biological sample (e.g., a cell, a tissue sample, or an extracellular vesicle) with an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure and detecting whether the antibody binds to the biological sample (e.g., a cell, a tissue sample, or an extracellular vesicle).

[0082] Yet another aspect of the present disclosure is an anti-glyco-cMET antibody or antigen-binding fragment according to the present disclosure for use in detecting cancer.

[0083] Yet another aspect of the present disclosure is a method for treating cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of an anti-glyco-cMET antibody, antigen-binding fragment, nucleic acid, vector, host cell, or pharmaceutical composition according to the present disclosure.

[0084] Yet another aspect of the present disclosure is an anti-glyco-cMET antibody, antigen-binding fragment, nucleic acid, vector, host cell or pharmaceutical composition according to the present disclosure for use in the treatment of cancer.

[0085] Yet another aspect of the present disclosure is the use of an anti-glyco-cMET antibody, antigen-binding fragment, nucleic acid, vector, host cell or pharmaceutical composition according to the present disclosure for the manufacture of a medicament for the treatment of cancer.

[0086] Glyco-cMET peptides are also provided herein. The peptides may be 13-30 amino acids in length and may be selected from the group consisting of amino acids 1-11, 1-12, 1-13, 1-14, 1-15, 1-16, 1-17, 1-18, 1-19, 1-20, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 3-11, 3-12, 3-13, 3-14, 3-15, 3-16, 3-17, 3-18, 3-19, 3-20, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16 of SEQ ID NO: 285 (PTKSFISGGSTITGVGKNLN, glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined). , 4-17, 4-18, 4-19, 4-20, 5-11, 5-12, 5-13, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-11, 6-12, 6-13, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-11, 7-12, 7-13, 7-14, The glyco-cMET peptide may comprise 7-15, 7-16, 7-17, 7-18, 7-19, 7-20, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, or 9-20. The glyco-cMET peptide is described in Section 5.10 and numbered embodiments 894-920. The peptide may be included in a composition as described in Section 5.10.1 and numbered embodiments 921 and 922. The glyco-cMET peptide can be used in methods for producing antibodies in an animal and / or eliciting an immune response in an animal. Methods for using glyco-cMET peptides are described in Section 5.10.2 and numbered embodiments 923-926. [Brief explanation of the drawings]

[0087] [Figure 1] 1 is a graph showing ELISA of 14E9, 19H2, and 39A3 rabbit antibodies against 50 ng of Tn-glycosylated cMET and Syndecan2 peptides. [Figure 2-1] Figure 2 shows flow cytometry analysis of cMET mouse antibodies in A549 COSMC-KO and A549 cells. Figure 2A: Representative histograms of 15C4.1D8.1G2, 8H3.2B9.2C7, and 16E12.1D9.1B11, anti-Golgi, mouse IgG isotype control, and anti-cMET antibodies in A549 COSMC-KO and A549 cells. Figures 2B1-2B-4: Titration of 15C4.1D8.1G2, 8H3.2B9.2C7, and 16E12.1D9.1B11 against cell surface antigens found in A549 COSMC-KO and A549 cells. Figure 2B-5: Legend for Figures 2B-1-2B-4. [Figure 2-2] (As mentioned above.) [Figure 2-3] (As mentioned above.) [Figure 3-1] Flow cytometry analysis of cMET rabbit antibody in A549 COSMC-KO and A549 cells. Figure 3A: Representative histograms of staining of 14E9, 19H2, and 39A3, anti-Golgi, mouse IgG isotype control, and anti-cMET antibody in A549 COSMC-KO and A549 cells. Figures 3B-1 to 3B-4: Titration of 14E9, 19H2, and 39A3 against cell surface antigens found in A549 COSMC-KO and A549 cells. Figure 3B-5: Legend for Figures 3B-1 to 3B-4. [Figure 3-2] (As mentioned above.) [Figure 3-3] (As mentioned above.) [Figure 4-1] Immunofluorescence staining of cMET mouse and rabbit antibodies. Figures 4A-4B: Immunofluorescence staining of 15C4.1D8.1G2, 8H3.2B9.2C7, 16E12.1D9.1B11, anti-cMET, and anti-Tn antibodies in A549 and A549 COSMC-KO cells (Figure 4A) and / or T47D and T47D COSMC-KO cells (Figure 4B). Figure 4C: Immunofluorescence staining of 14E9, 19H2, 39A3, anti-cMET, and anti-Tn antibodies in A549 COSMC-KO and A549 cells. [Figure 4-2] (As mentioned above.) [Figure 4-3] (As mentioned above.) [Figure 5-1] Immunohistochemistry of cMET mouse and rabbit antibodies. Figure 5A: Staining of 15C4.1D8.1G2, 8H3.2B9.2C7, 16E12.1D9.1B11, 14E9, 19H2, 39A3, and IgG control antibodies in colon cancer and normal tissues (TMA-T051b Biomax). Figure 5B: Statistics of positive and negative staining of tissues. [Figure 5-2] (As mentioned above.) [Figure 6-1] Immunohistochemistry of the cMET mouse antibody. Figure 6A-1: Staining of the 8H3.2B9.2C7 ("GO-8H3") antibody in ovarian cancer (TMA-OV1502), pancreatic cancer (TMA-PA2082), lung cancer (TMA-LC121b), and cholangiocarcinoma (TMA-GA802a). Statistics are shown in Figure 6A-2. Positive samples had approximately 70% of cancer cells with strong cell surface staining. Approximately 10-20% of the analyzed cancer tissues had specific cell surface staining in approximately 70% of cancer cells. Figure 6B-1: Staining of the 8H3.2B9.2C7 ("GO-8H3") in normal tissue (TMA-FDA999x). Statistics are shown in Figure 6B-2. No specific cell surface staining was observed in normal tissue. [Figure 6-2] (As mentioned above.) [Figure 7-1]Figure 7 shows cell killing assay of cMET CART. Figure 7A: Killing of cMET CART (8H3.2B9.2C7) against A673 COSMC-KO and A673 target cells by titration of T cell to target cell ratios (1, 5, and 10). Figure 7B: Summary of the time for cMET-CART to kill 50% of A673 COSMC-KO (KT50) target cells. N / A = not killed 50% of the cells. Figure 7C: Killing of cMET CART (8H3.2B9.2C7) in various cell lines with low cMET-Tn expression (<500 receptors per cell) and high cMET-Tn expression (2 × 10 receptors per cell). cMET-CART demonstrated cytotoxicity in cells with low levels of cMET-Tn expression (A549-wt cells). Experiments were performed by titrating the T cell to target cell ratio (1, 5, and 10). [Figure 7-2] (As mentioned above.) [Figure 8] In vivo activity of cMET-CART (8H3) in solid tumor mouse models. Figure 8A: A549 solid tumor model (lung cancer cell line) established by flank injection (CDx). Tumor volume at the time of CART injection was 88 mm3. Mice were treated with second-generation 8H3-CAR-T by IT injection (2 doses of 1 x 107 cells). Tumor volume was measured by calipers. Figure 8B: Lung cancer solid tumor model (Champions model CTG-2823) established by flank injection (PDx). Tumor volume at the time of CART injection was 200 mm3, and CART was delivered by IV injection (4 doses of 1 x 107 cells). [Figure 9-1] 9A-9C show exemplary cMET-CART constructs 15C4-CART (FIG. 9A), 16E12-CART (FIG. 9B), and 8H3-CART (FIG. 9C). Testing of the constructs is described in Example 5. [Figure 9-2] (As mentioned above.) [Figure 10] Figure 1 shows the cytotoxicity of hu8H3-CART in A673 (Tn+) and (Tn-) cells at an E:T ratio of 2:1. DETAILED DESCRIPTION OF THE INVENTION

[0088] 5. Detailed Description 5.1 Antibodies The present disclosure provides novel antibodies directed against glycoforms of cMET displayed on tumor cells. These are exemplified by antibodies 15C4.1D8.1G2 (hereinafter "15C4"), 8H3.2B9.2C7 (hereinafter "8H3"), 16E12.1D9.1B11 (hereinafter "16E12"), 14E9, 19H2, and 39A3. 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 were identified in a screen for antibodies that bind to a glycosylated peptide present in cMET, PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), which is glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined to mimic the glycosylation pattern of cMET present on tumor cells.

[0089] The anti-glyco-cMET antibodies of the present disclosure, exemplified by antibodies 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3, are useful as tools in cancer diagnosis and therapy.

[0090] Thus, in certain aspects, the present disclosure provides antibodies and antigen-binding fragments that bind to the glycoform of cMET present in tumor cells (referred to herein as "glyco-cMET"), preferably the peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285) glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined.

[0091] Anti-glyco-cMET antibodies of the present disclosure may be polyclonal, monoclonal, genetically engineered, and / or otherwise modified in nature, including, but not limited to, chimeric antibodies, humanized antibodies, human antibodies, primatized antibodies, single-chain antibodies, bispecific antibodies, dual variable domain antibodies, etc. In various embodiments, the antibody comprises all or a portion of an antibody constant region. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM. In a specific embodiment, an anti-glyco-cMET antibody of the present disclosure comprises a constant region isotype of IgG1.

[0092] The term "monoclonal antibody," as used herein, is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies are derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For many uses of the present disclosure, including in vivo use of anti-glyco-cMET antibodies in humans, chimeric, primatized, humanized, or human antibodies may be suitably used.

[0093] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties.

[0094] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions (FR) are those of a human immunoglobulin sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; Queen et al., U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; EP 239400; PCT Publication WO 91 / 09967; U.S. Patent No. 5,225,539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332.

[0095] Exemplary humanized sequences are set forth in numbered embodiments 8 through 115. Variable region sequences of exemplary humanized antibodies and antigen-binding fragments thereof of the present disclosure are set forth in Tables 4A through 4G.

[0096] "Human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin, and further include antibodies isolated from a human immunoglobulin library or from an animal transgenic for one or more human immunoglobulins and that does not express endogenous immunoglobulins. Human antibodies can be produced by various methods known in the art, such as phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO 96 / 33735; and WO 91 / 10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be produced by using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., PCT Publication Nos. WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference in their entireties. Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection." In this approach, a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (see Jespers et al., 1988, Biotechnology 12:899-903).

[0097] A "primatized antibody" comprises a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570; 5,681,722; and 5,693,780, which are incorporated by reference in their entireties.

[0098] Anti-glyco-cMET antibodies of the present disclosure include both full-length (intact) antibody molecules and antigen-binding fragments capable of binding to glyco-cMET. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, single-chain Fv fragments, and single-domain fragments.

[0099] Fab fragments contain the constant domain of the light chain (CL) and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')1 fragments lack the Fc fragment of intact antibodies, are rapidly cleared from the animal's circulation, and may have less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0100] An "Fv" fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (V H -V L In this configuration, the three CDRs of each variable domain interact to form a V H -VL A target binding site is defined on the surface of the dimer. Often, the six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may have the ability to recognize and bind to the target, albeit with lower affinity than the entire binding site.

[0101] "Single-chain Fv" or "scFv" antigen-binding fragments are fragments of the V H and V L Fv polypeptides generally comprise V domains, where these domains are present in a single polypeptide chain. H Domains and V L It further comprises a polypeptide linker between the domains which enables the scFv to form the desired structure for target binding.

[0102] "Single domain antibodies" are single V domain antibodies that exhibit sufficient affinity for glycosylated cMET. H or V L In a specific embodiment, a single domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0103] The anti-glyco-cMET antibodies of the present disclosure may also be bispecific and other multispecific antibodies. Bispecific antibodies are monoclonal antibodies, often human or humanized, that have binding specificities for two different epitopes on the same or different antigens. In the present disclosure, one binding specificity may be directed to glyco-cMET, and the other may be directed to any other antigen, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterial protein, or a bacterial surface protein. In certain embodiments, bispecific and other multispecific anti-glyco-cMET antibodies and antigen-binding fragments specifically bind to a second cMET epitope, an epitope on another protein co-expressed with cMET on cancer cells, or an epitope on another protein present on a different cell, such as an activated T cell. Bispecific antibodies of the present disclosure include IgG-format bispecific antibodies and single-chain-based bispecific antibodies.

[0104] The IgG-format bispecific antibodies of the present disclosure may be any of the various types of IgG-format bispecific antibodies known in the art, such as quadroma bispecific antibodies, "knobs-in-holes" bispecific antibodies, CrossMab bispecific antibodies (i.e., bispecific domain-swapped antibodies), charge-paired bispecific antibodies, general light chain bispecific antibodies, one-arm single chain Fab-immunoglobulin gamma bispecific antibodies, disulfide-stabilized Fv bispecific antibodies, DuetMab, controlled Fab arm-swapped bispecific antibodies, chain-swapped engineered domain body bispecific antibodies, two-arm leucine zipper heterodimeric monoclonal bispecific antibodies, kappa lambda body bispecific antibodies, dual variable domain bispecific antibodies, and crossed dual variable domain bispecific antibodies.See, e.g., Kohler and Milstein, 1975, Nature 256:495-497; Milstein and Cuello, 1983, Nature 305:537-40; Ridgway et al., 1996, Protein Eng. 9:617-621; Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92; Gunasekaran et al., 2010, J Biol Chem 285:19637-46; Fischer et al., 2015 Nature Commun 6:6113; Schanzer et al., 2014, J Biol Chem 289:18693-706; Metz et al., 2012 Protein Eng Des Sel 25:571-80;Mazor et al., 2015 MAbs 7:377-89;Labrijn et al., 2013 Proc Natl Acad Sci USA 110:5145-50;Davis et al., 2010 Protein Eng Des Sel 23:195-202;Wranik et al., 2012, J Biol Chem 287:43331-9;Gu et al., 2015, PLoS One 10(5):e0124135;Steinmetz et al., 2016, MAbs 8(5):867-78;Klein et al., 2016, mAbs, 8(6):1010-1020;Liu et al., 2017, Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48.

[0105] In some embodiments, the bispecific antibody of the present disclosure is a domain-exchanged antibody, which is referred to as CrossMab in scientific and patent literature.See, for example, Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92.CrossMab technology is described in detail in WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253, WO 2009 / 080254, WO 2013 / 026833, WO 2016 / 020309, and Schaefer et al., 2011, Proc Natl Acad Sci USA 108:11187-92, which are incorporated herein by reference in their entirety. Briefly, CrossMab technology is based on domain crossover between the heavy and light chains within one Fab arm of a bispecific IgG, which promotes correct chain association. The CrossMab bispecific antibody of the present disclosure is a "CrossMab" antibody in which the heavy and light chains of the Fab portion of one arm of a bispecific IgG antibody are swapped. FAB In other embodiments, the CrossMab bispecific antibody of the present disclosure may be a "CrossMab" antibody in which only the heavy and light chain variable domains of the Fab portion of one arm of a bispecific IgG antibody are exchanged. VH-VL In yet another embodiment, the CrossMab bispecific antibody of the present disclosure may be a "CrossMab" antibody in which only the heavy and light chain constant domains of the Fab portion of one arm of the bispecific IgG antibody are exchanged. CH1-CL " antibody. CrossMab CH1-CL The antibody is CrossMab FAB and CrossMab VH-VL In contrast to CH1-CL Bispecific antibodies are preferred, see Klein et al., 2016, mAbs, 8(6):1010-1020.

[0106] In some embodiments, the bispecific antibody of the present disclosure is a controlled Fab arm exchange bispecific antibody. Methods for producing Fab arm exchange bispecific antibodies are described in PCT Publication No. WO 2011 / 131746 and Labrijn et al., 2014 Nat Protoc. 9(10):2450-63, which are incorporated herein by reference in their entireties. Briefly, a controlled Fab arm exchange bispecific antibody can be produced by separately expressing two parent IgG1s containing single matching point mutations in the CH3 domain, mixing the parent IgG1s in vitro under redox conditions to allow recombination of half molecules, and removing the reductant to reoxidize the interchain disulfide bonds, thereby forming the bispecific antibody.

[0107] In some embodiments, the bispecific antibodies of the present disclosure are bispecific antibodies of the "bottle opener," "mAb-Fv," "mAb-scFv," "core-scFv," "core-Fv," "one-arm core-scFv," or "dual-scFv" format. These formats of bispecific antibodies are described in PCT Publication WO 2016 / 182751, the contents of which are incorporated herein by reference in their entirety. Each of these formats relies on the self-assembling nature of the Fc domain of the antibody heavy chain, whereby two Fc subunit containing "monomers" assemble into an Fc domain containing "dimer."

[0108] In the bottle opener format, the first monomer comprises an scFv covalently linked to the N-terminus of the Fc subunit, optionally via a linker, and the second monomer comprises a heavy chain (comprising a VH, a CH1, and a second Fc subunit). The bottle opener format bispecific antibody further comprises a light chain capable of pairing with the second monomer to form a Fab.

[0109] The mAb-Fv bispecific antibody format relies on an "extra" VH domain attached to the C-terminus of one heavy chain monomer and an "extra" VL domain attached to the other heavy chain monomer, forming a third antigen-binding domain. In some embodiments, the mAb-Fv bispecific antibody comprises a first monomer comprising a first VH domain, a CH1 domain, and a first Fc subunit, with a VL domain covalently attached to the C-terminus. The second monomer comprises a VH domain, a CH1 domain, a second Fc subunit, and a VH covalently attached to the C-terminus of the second monomer. The two variable domains attached to the C-terminus constitute an Fv. The mAb-Fv further comprises two light chains, which, when associated with the first and second monomers, form Fab.

[0110] The mAb-scFv bispecific format relies on the C-terminal attachment of an scFv to one of the mAb monomers to form a third antigen-binding domain. Thus, the first monomer comprises a first heavy chain (comprising a VH, CH1, and the first Fc subunit) with an scFv covalently attached to its C-terminus. The mAb-scFv bispecific antibody further comprises a second monomer (comprising a VH, CH1, and the first Fc subunit) and two light chains, which, when associated with the first and second monomers, form Fab.

[0111] The core-scFv bispecific format relies on the use of an inserted scFv domain within a mAb to form a third antigen-binding domain. The scFv domain is inserted between the Fc subunit and the CH1 domain of one of the monomers to provide the third antigen-binding domain. Thus, the first monomer may comprise a VH domain, a CH1 domain (and optional hinge), and the first Fc subunit, and the scFv may be covalently linked between the C-terminus of the CH1 domain and the N-terminus of the first Fc subunit using an optional domain linker. The other monomer may be a standard Fab-side monomer. The core-scFv bispecific antibody further comprises two light chains, which, when associated with the first and second monomers, form an Fab.

[0112] The core-Fv bispecific format relies on the use of an inserted Fv domain to form a third antigen-binding domain. Each monomer can contain the components of an Fv (e.g., one monomer contains a variable heavy chain domain and the other contains a variable light chain domain). Thus, one monomer can contain a VH domain, a CH1 domain, a first Fc subunit, and a VL domain covalently linked between the C-terminus of the CH1 domain and the N-terminus of the first Fc subunit, optionally using a domain linker. The other monomer can contain a VH domain, a CH1 domain, a second Fc subunit, and an additional VH domain covalently linked between the C-terminus of the CH1 domain and the N-terminus of the second Fc domain, optionally using a domain linker. Core-Fv bispecific antibodies further contain two light chains, which, when associated with the first and second monomers, form Fab.

[0113] The one-arm core-scFv bispecific format comprises one monomer containing only an Fc subunit, and the other monomer contains an inserted scFv domain, thus forming the second antigen-binding domain. Thus, one monomer may contain a VH domain, a CH1 domain, and a first Fc subunit, optionally using a domain linker, with the scFv covalently linked between the C-terminus of the CH1 domain and the N-terminus of the first Fc subunit. The second monomer may contain an Fc domain. This embodiment further utilizes a light chain comprising a variable light domain and a constant light domain, which associates with the first monomer to form an Fab.

[0114] The dual-scFv bispecific format comprises a first monomer comprising an scFv covalently linked, optionally via a linker, to the N-terminus of a first Fc subunit, and a second monomer comprising an scFv covalently linked, optionally via a linker, to the N-terminus of a second Fc subunit.

[0115] The bispecific antibody of the present disclosure may comprise an Fc domain composed of a first and a second subunit. In one embodiment, the Fc domain is an IgG Fc domain. In a specific embodiment, the Fc domain is an IgG1 Fc domain. In another embodiment, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228, particularly the amino acid substitution S228P (Kabat EU index numbering). Unless otherwise specified herein, the numbering of amino acid residues in the Fc domain or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., 2010, Drug Metabolism and Disposition 38:84-91). In a further specific embodiment, the Fc domain is a human Fc domain. In an even more specific embodiment, the Fc domain is a human IgG1 Fc domain. An exemplary sequence of a human IgG1 Fc region is:

[0116] [ka] is.

[0117] In certain embodiments, the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0118] In a specific embodiment, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which involves a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain. Knob-into-hole technology is described, for example, in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., 1996, Prot Eng 9:617-621; and Carter, J, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protrusion ("knob") into the boundary of a first polypeptide and a corresponding cavity ("hole") into the boundary of a second polypeptide, so that the protrusion can enter the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing small amino acid side chains from the boundary of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a corresponding cavity of the same or similar size as the protrusion is created in the boundary of the second polypeptide.

[0119] Thus, in some embodiments, replacing an amino acid residue in the CH3 domain of a first subunit of an Fc domain with an amino acid residue having a larger side chain volume creates a protrusion in the CH3 domain of the first subunit that can fit into a cavity in the CH3 domain of a second subunit, and replacing an amino acid residue in the CH3 domain of a second subunit of an Fc domain with an amino acid residue having a smaller side chain volume creates a cavity in the CH3 domain of the second subunit that can fit the protrusion in the CH3 domain of the first subunit. Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis, or by peptide synthesis.

[0120] In certain such embodiments, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the second subunit of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to the Kabat EU index). In a further embodiment, in the first subunit of the Fc domain, in addition, the serine residue at position 354 is replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced with a cysteine ​​residue), and in the second subunit of the Fc domain, in addition, the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbering according to the Kabat EU index). In a specific embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the Kabat EU index).

[0121] In some embodiments, electrostatic steering (e.g., as described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to promote association of the first and second subunits of the Fc domain.

[0122] In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and / or effector function.

[0123] In a specific embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, and most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and cytokine secretion. In a specific embodiment, the effector function is ADCC.

[0124] Typically, the same amino acid substitution(s) are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid substitution(s) reduce the binding affinity of the Fc domain to an Fc receptor. In one embodiment, the amino acid substitution(s) reduce the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold.

[0125] In one embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of E233, L234, L235, N297, P331 and P329 (numbering according to Kabat EU index). In a more specific embodiment, the Fc domain comprises an amino acid substitution at a position selected from the group of L234, L235 and P329 (numbering according to Kabat EU index). In some embodiments, the Fc domain comprises amino acid substitutions L234A and L235A (numbering according to Kabat EU index). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbering according to Kabat EU index). In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and an additional amino acid substitution at a position selected from E233, L234, L235, N297, and P331 (numbering according to the Kabat EU index). In a more specific embodiment, the additional amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc domain comprises amino acid substitutions at positions P329, L234, and L235 (numbering according to the Kabat EU index). In a more particular embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G (which can be referred to using the shorthand terminology "P329G LALA," "PGLALA," or "LALAPG").Specifically, in certain embodiments, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), i.e., in each of the first and second subunits of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 is replaced with an alanine residue (L235A), and the proline residue at position 329 is replaced with a glycine residue (P329G) (Kabat EU index numbering). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain.

[0126] The single-chain-based bispecific antibodies of the present disclosure can be any of the various types of single-chain-based bispecific antibodies known in the art, such as bispecific T cell engagers (BiTEs), diabodies, tandem diabodies (tandabs), dual affinity retargeting molecules (DARTs), and bispecific killer cell engagers. See, e.g., Loffler et al., 2000, Blood 95:2098-103; Holliger et al., 1993, Proc Natl Acad Sci USA, 90:6444-8; Kipriyanov et al., 1999, Mol Biol 293:41-56; Johnson et al., 2010, Mol Biol 399:436-49; Wiernik et al., 2013, Clin Cancer Res 19:3844-55; Liu et al., 2017, Front. Immunol. 8:38; and Yang et al., 2017, Int. J. Mol. Sci. 18:48, which are incorporated by reference in their entireties.

[0127] In some embodiments, the bispecific antibody of the present disclosure is a bispecific T cell engager (BiTE). BiTEs are single polypeptide chain molecules with two antigen-binding domains, one of which binds to a T cell antigen and the second of which binds to an antigen displayed on the surface of a target (see PCT Publication WO 05 / 061547; Baeuerle et al., 2008, Drugs of the Future 33: 137-147; Bargou, et al., 2008, Science 321:974-977, which are incorporated by reference in their entireties). Thus, BiTEs of the present disclosure have an antigen-binding domain that binds to a T cell antigen and a second antigen-binding domain directed against glyco-cMET.

[0128] In some embodiments, the bispecific antibody of the present disclosure is a dual affinity retargeting molecule (DART). A DART comprises at least two polypeptide chains that associate (particularly through covalent interactions) to form at least two epitope-binding sites, which may recognize the same epitope or different epitopes. Each of the DART polypeptide chains comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope-binding site. Instead, the immunoglobulin heavy chain variable region of one (e.g., a first) DART polypeptide chain interacts with the immunoglobulin light chain variable region of a different (e.g., a second) DART™ polypeptide chain to form an epitope-binding site. Similarly, the immunoglobulin light chain variable region of one (e.g., a first) DART polypeptide chain interacts with the immunoglobulin heavy chain variable region of a different (e.g., a second) DART polypeptide chain to form an epitope-binding site. DARTs may be monospecific, bispecific, trispecific, etc., and thus can simultaneously bind to one, two, three, or more different epitopes (which may be of the same or different antigens). DARTs may also be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., and thus can simultaneously bind to one, two, three, four, five, six, or more molecules. These two properties of DARTs (i.e., degree of specificity and valency) can be combined to produce, for example, tetravalent (i.e., capable of binding to four sets of epitopes) bispecific antibodies (i.e., capable of binding to two epitopes). DART molecules are disclosed in PCT Publication Nos. WO 2006 / 113665, WO 2008 / 157379, and WO 2010 / 080538, the entire contents of which are incorporated herein by reference.

[0129] In some embodiments of the bispecific antibodies of the present disclosure, one binding specificity is directed against glyco-cMET, and the other is directed against an antigen expressed on an immune effector cell. The term "immune effector cell" or "effector cell," as used herein, refers to a cell within the natural repertoire of cells in the mammalian immune system that can be activated to affect the viability of a target cell. Immune effector cells include lymphoid cells, such as natural killer (NK) cells, T cells, including cytotoxic T cells, or B cells. In addition, myeloid cells, such as monocytes or macrophages, dendritic cells, and neutrophilic granulocytes, can also be considered immune effector cells. Thus, the effector cells are preferably NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophilic granulocytes. The recruitment of effector cells to abnormal cells means that the immune effector cells are brought into close proximity to the abnormal target cells, so that the effector cells can directly kill or indirectly initiate the death of the recruited abnormal cells. To avoid non-specific interactions, the bispecific antibodies of the present disclosure preferably specifically recognize antigens on immune effector cells that are at least overexpressed by these immune effector cells compared to other cells in the body. Target antigens presented on immune effector cells can include CD3, CD8, CD16, CD25, CD28, CD64, CD89, NKG2D, and NKp46. Preferably, the antigen on immune effector cells is CD3, which is expressed on T cells.

[0130] As used herein, unless otherwise specified, "CD3" refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term encompasses all forms of CD3 resulting from intracellular processing, in addition to "full-length" unprocessed CD3. The term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants. The most preferred antigen on immune effector cells is the CD3 epsilon chain. This antigen has been shown to be highly effective in recruiting T cells to abnormal cells. Therefore, the bispecific antibodies of the present disclosure preferably specifically recognize CD3 epsilon. The amino acid sequence of human CD3 epsilon is available at UniProt (www.uniprot.org) accession number P07766 (version 144) or at NCBI (ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey (Macaca fascicularis) CD3 epsilon is available at NCBI GenBank number BAB71849.1. For human therapeutic use, bispecific antibodies whose CD3-binding domain specifically binds to human CD3 (e.g., human CD3 epsilon chain) can be used. For preclinical studies in non-human animals or cell lines, bispecific antibodies whose CD3-binding domain specifically binds to CD3 of the species being used in the preclinical study (e.g., cynomolgus monkey CD3 for primate studies) can be used.

[0131] As used herein, a binding domain that "specifically binds to" or "specifically recognizes" a target antigen from a particular species does not exclude binding to or recognition of antigens from other species, and thus encompasses antibodies in which one or more of the binding domains have species cross-reactivity. For example, a CD3 binding domain that "specifically binds to" or "specifically recognizes" human CD3 can also bind to or recognize cynomolgus monkey CD3, and vice versa.

[0132] In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody H2C (described in PCT Publication WO 2008 / 119567) for binding to an epitope on CD3. In other embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody V9 (described in Rodrigues et al., 1992, Int J Cancer Suppl 7:45-50 and U.S. Pat. No. 6,054,297) for binding to an epitope on CD3. In yet other embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody FN18 (described in Nooij et al., 1986, Eur J Immunol 19:981-984) for binding to an epitope on CD3. In yet other embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody SP34 (described in Pessano et al., 1985, EMBO J 4:337-340) for binding to an epitope on CD3.

[0133] In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody mAb1 (described in U.S. Pat. No. 10,730,944) for binding to an epitope on CD8. In other embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody YTS169 (described in U.S. Pat. No. 2015 / 0191543) for binding to an epitope on CD8. In other embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody 4C9 5F4 (described in WO 1987 / 005912) for binding to an epitope on CD8.

[0134] In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody 3G8 (described in WO 2006 / 064136) for binding to an epitope of CD16. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody VEP13 (described in Ziegler-Heitbrock et al., 1984, Clin. Exp. Immunol. 58:470-477) for binding to an epitope of CD16. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody B73.1 (described in Perussia et al., 1983, J. Immunol. 130(5):2142-2148) for binding to an epitope of CD16.

[0135] In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody daclizumab and variants thereof (described in WO 2014 / 145000) for binding to an epitope on CD25. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibodies AB1, AB7, AB11, or AB12 (described in WO 2004 / 045512) for binding to an epitope on CD25. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibodies ALD25H1, ALD25H2, or ALD25H4 (described in WO 2020 / 234399) for binding to an epitope on CD25.

[0136] In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody FR104 (described in WO 2017 / 103003) for binding to an epitope on CD28. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody hCD28.3 (described in WO 2011 / 101791) for binding to an epitope on CD28.

[0137] In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibodies MS or 21 F2 (described in WO 2009 / 077483) for binding to an epitope of NKG2D. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibodies 5C5, 320, 230, 013, 296, or 395 (described in WO 2021 / 009146) for binding to an epitope of NKG2D. In some embodiments, bispecific antibodies of the present disclosure can compete with monoclonal antibody KYK-2.0 (described in WO 2010 / 017103) for binding to an epitope of NKG2D.

[0138] The anti-glyco-cMET antibodies of the present disclosure include derivatized antibodies. For example, but not limited to, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or linkage to a cellular ligand or other protein. Any of a variety of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, or metabolic synthesis of tunicamycin. In addition, derivatives may contain one or more unnatural amino acids, for example, using ambrx technology (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0139] An anti-glyco-cMET antibody or binding fragment may be an antibody or fragment whose sequence has been modified to alter at least one constant region-mediated biological effector function. For example, in some embodiments, an anti-glyco-cMET antibody may be modified to reduce at least one constant region-mediated biological effector function, e.g., to reduce Fc receptor (FcγR) binding, compared to an unmodified antibody. FcγR binding can be reduced by mutating specific regions of the antibody's immunoglobulin constant region segment that are required for FcγR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662). Reducing the FcγR binding ability of an antibody can also reduce other effector functions that rely on FcγR interactions, such as opsonization, phagocytosis, and antigen-dependent cellular cytotoxicity ("ADCC").

[0140] The anti-glyco-cMET antibodies or binding fragments described herein include antibodies and / or binding fragments that have been modified to acquire or improve at least one constant region-mediated biological effector function compared to the unmodified antibody, e.g., to enhance FcγR interactions (see, e.g., U.S. Patent Application Publication No. 2006 / 0134709). For example, an anti-glyco-cMET antibody of the present disclosure may have a constant region that binds FcγRIIA, FcγRIIB, and / or FcγRIIIA with greater affinity than the corresponding wild-type constant region.

[0141] Thus, the antibodies of the present disclosure may have alterations in biological activity that result in increased or decreased opsonization, phagocytosis, or ADCC. Such alterations are known in the art. For example, modifications in antibodies that reduce ADCC activity are described in U.S. Pat. No. 5,834,597. An exemplary variant that reduces ADCC corresponds to "mutant 3," in which residue 236 is deleted and residues 234, 235, and 237 (using EU numbering) are substituted with alanine (shown in Figure 4 of U.S. Pat. No. 5,834,597). Another exemplary variant that reduces ADCC contains the amino acid mutations L234A, L235A, and P329G (which can be referred to as "P329G LALA" using the shorthand term). The "P329G LALA" combination of amino acid substitutions almost completely abolishes Fcγ receptor (and complement) binding of human IgG1 Fc domains, as described in PCT Publication WO 2012 / 130831, which is incorporated herein by reference in its entirety. WO 2012 / 130831 also describes methods for preparing such mutant Fc domains and determining their properties, such as Fc receptor binding or effector function.

[0142] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have low levels of fucose or lack fucose. Antibodies lacking fucose have been shown to correlate with enhanced ADCC activity, particularly at low antibody doses. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740; Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods for preparing antibodies with reduced fucose include growth in rat myeloma YB2 / 0 cells (ATCC CRL1662). YB2 / 0 cells express low levels of FUT8 mRNA, which encodes α-1,6-fucosyltransferase, an enzyme required for polypeptide fucosylation.

[0143] In some embodiments, the anti-glyco-cMET antibody or binding fragment comprises a bisected oligosaccharide, e.g., a biantennary oligosaccharide attached to the Fc domain is bisected by GlcNAc. Such variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., 1999, Nat Biotechnol 17:176-180; Ferrara et al., 2006, Biotechn Bioeng 93: 851-861; WO 99 / 54342; WO 2004 / 065540; and WO 2003 / 011878.

[0144] In yet another aspect, the anti-glyco-cMET antibody or binding fragment comprises a modification that increases or decreases its binding affinity to the fetal Fc receptor, FcRn, e.g., by mutating specific regions of the immunoglobulin constant region segment involved in FcRn interaction (see, e.g., WO 2005 / 123780). In a specific embodiment, the anti-glyco-cMET antibody of the IgG class is mutated so that at least one of amino acid residues 250, 314, and 428 in the heavy chain constant region is substituted, e.g., at positions 250 and 428, or 250 and 314, or 314 and 428, or 250, 314, and 428, alone or in any combination thereof, with a particular combination being positions 250 and 428. In the case of position 250, the substituted amino acid residue may be any amino acid residue other than threonine, such as, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. In the case of position 314, the substituted amino acid residue may be any amino acid residue other than leucine, such as, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. In the case of position 428, the substituted amino acid residue may be any amino acid residue other than methionine, such as, but not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. Specific combinations of suitable amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797, which is incorporated herein by reference.Such mutations increase binding to FcRn, thereby protecting the antibody from degradation and increasing its half-life.

[0145] In yet other embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure has one or more amino acids inserted into one or more of its hypervariable regions, e.g., as described in Jung and Pluckthun, 1997, Protein Engineering 10:9, 959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9. Epub 2004 Aug. 17; and U.S. Patent Application No. 2007 / 0280931.

[0146] In yet other embodiments particularly useful for diagnostic applications, the anti-glyco-cMET antibodies or antigen-binding fragments of the present disclosure are attached to a detectable moiety, including a radioactive moiety, a colorimetric molecule, a fluorescent moiety, a chemiluminescent moiety, an antigen, an enzyme, a detectable bead (e.g., a magnetic or electron-dense (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin).

[0147] Radioisotopes or radionuclides are 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I can be mentioned.

[0148] Fluorescent labels can include rhodamine, lanthanide fluorescein and its derivatives, fluorescent dyes, GFP (GFP stands for "Green Fluorescent Protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

[0149] Enzyme labels can include horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase, and peroxidase.

[0150] Chemiluminescent labels or chemiluminescers, such as isoluminol, luminol, and dioxetanes.

[0151] Other detectable moieties include molecules such as biotin, digoxigenin, or 5-bromodeoxyuridine.

[0152] In yet other embodiments, the anti-glyco-cMET antibodies or antigen-binding fragments of the present disclosure can be used in detection systems to detect biomarkers in a sample, such as a biological sample from a patient. The biomarker can be a protein biomarker (e.g., a tumor-associated glycoform of cMET, e.g., a glycoform of cMET containing the amino acid sequence PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined, present on or within cancer cells (e.g., from tissue biopsies or circulating tumor cells) or cancer-derived extracellular vesicles).

[0153] Extracellular vesicles (EVs) are lipid membrane vesicles released from almost all cell types. EVs carry complex molecular cargoes such as proteins, RNA (e.g., mRNA and non-coding RNAs (microRNA, transfer RNA, circular RNA, and long non-coding RNA)), and DNA fragments. The molecular content of EVs primarily reflects the cell of origin and therefore exhibits cell-type specificity. In particular, cancer-derived EVs contain cancer-specific molecules expressed by the parent cancer cells and display them on their surface (see, for example, Yanez-Mo et al., 2015, J Extracell Vesicles. 4:27066; and Li et al., 2015, Cell Res. 25:981-984).

[0154] In one embodiment, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure is used in a method for detecting a biomarker in a sample containing EVs (e.g., liquid cytology). In such an embodiment, the biomarker is recognized by the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure. The biomarker may be displayed on the surface of EVs. Exemplary methods for detecting the biomarker include, but are not limited to, immunoassays such as immunoprecipitation; Western blot; ELISA; immunohistochemistry; immunocytochemistry; flow cytometry; and immuno-PCR. In some embodiments, the immunoassay may be a chemiluminescent immunoassay. In some embodiments, the immunoassay may be a high-throughput and / or automated immunoassay platform.

[0155] In some embodiments, methods for detecting a biomarker in a sample include contacting the sample with an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure. In some embodiments, such methods further include contacting the sample with one or more detection labels. In some embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure is labeled with one or more detection labels.

[0156] In some embodiments, a capture assay is performed to selectively capture EVs from a sample, such as a liquid cytology sample. Illustrative examples of capture assays for EVs are described in U.S. Patent Application Publication No. 2021 / 0214806, which is incorporated herein by reference in its entirety. In some embodiments, a capture assay is performed to selectively capture EVs of a certain size range and / or certain characteristics, e.g., EVs associated with cancer (e.g., tumor-associated glycoforms of cMET, e.g., glycoforms of cMET containing the amino acid sequence PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285) glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined). In some such embodiments, prior to performing the capture assay, the sample may be pretreated to remove non-EVs, including, but not limited to, soluble proteins and interfering substances such as, for example, cellular debris. In some embodiments, EVs are purified from the sample using size-exclusion chromatography.

[0157] In some embodiments, the method for detecting biomarkers involves analyzing individual EVs (e.g., single EV assays). For example, such an assay may include (i) a capture assay, such as an antibody capture assay, and (ii) one or more detection assays for at least one or more additional biomarkers, where the capture assay is performed before the detection assay. See, e.g., U.S. Patent Application Publication No. 2021 / 0214806.

[0158] In some embodiments, the capture assay involves contacting the sample with at least one capture agent comprising an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure. The capture agent may be immobilized on a solid substrate. The solid substrate may be provided in a form suitable for capturing EVs and that does not interfere with downstream manipulation, processing, and / or detection. For example, in some embodiments, the solid substrate may be or comprise beads (e.g., magnetic beads). In some embodiments, the solid substrate may be or comprise a surface. For example, in some embodiments, such a surface may be the capture surface of an assay chamber (e.g., a tube, well, microwell, plate, filter, membrane, matrix, etc.). In some embodiments, the capture agent is or comprises a magnetic bead comprising a capture moiety (e.g., an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure) conjugated to the magnetic bead. See, e.g., U.S. Patent Application Publication No. 2021 / 0214806.

[0159] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with 15C4, or an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 15C4 (SEQ ID NOs: 1 and 2, respectively).

[0160] In certain embodiments, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with 8H3, or an antibody or antigen-binding fragment comprising the heavy chain variable region of murine or humanized 8H3 (e.g., SEQ ID NO: 23 (murine) and SEQ ID NOs: 264-275 (exemplary humanized sequences)), and the light chain variable region of murine or humanized 8H3 (e.g., SEQ ID NO: 24 (murine) and SEQ ID NOs: 276-284 (exemplary humanized sequences)).

[0161] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with 16E12, or an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 16E12 (SEQ ID NOs: 45 and 46, respectively).

[0162] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with 14E9, or an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 14E9 (SEQ ID NOs: 67 and 68, respectively).

[0163] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with 19H2, or an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 19H2 (SEQ ID NOs: 89 and 90, respectively).

[0164] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure competes with 39A3, or an antibody or antigen-binding fragment comprising the heavy and light chain variable regions of 39A3 (SEQ ID NOs: 111 and 112, respectively).

[0165] Competition can be assayed on cells expressing the glycosylated cMET epitope bound by 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3, or on a glycosylated cMET peptide containing the epitope bound by 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3, such as the peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined. Cells not expressing the epitope or an unglycosylated peptide can be used as a control.

[0166] Cells that can be used for the competition assay include, but are not limited to, lung, breast, kidney, and liver cell lines (e.g., breast cancer cell line T47D; adenocarcinoma human alveolar basal epithelial cell line A549), and recombinant cells engineered to express the glycosylated cMET epitope. In one non-limiting example, T47D cells, which are inherently Tn-negative but express cMET, are engineered to express the cMET Tn antigen by knockout of the COSMC chaperone. Wild-type T47D cells expressing the unglycosylated form of cMET can be used as a negative control. In another non-limiting example, A549 cells, which are inherently Tn-negative but express cMET, are engineered to express the cMET Tn antigen by knockout of the COSMC chaperone. Wild-type A549 cells expressing the unglycosylated form of cMET can be used as a negative control.

[0167] Assays for competition include, but are not limited to, radioactive immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), sandwich ELISA, fluorescence-activated cell sorting (FACS) assays, surface plasmon resonance (e.g., Biacore) assays, and biolayer interferometry (BLI) assays. In some embodiments, antibody competition assays can be performed using BLI (e.g., using the Octet-HTX system (Molecular Devices)). Antibody competition or epitope binning of monoclonal antibodies can be evaluated in tandem against their specific antigens using BLI. In BLI assays, antigens can be immobilized on a biosensor and presented to two competing antibodies in successive steps. Binding to non-overlapping epitopes occurs when saturation by the first antibody does not prevent binding of the second antibody. In some embodiments, antibody competition assays can be performed using surface plasmon resonance (e.g., using the Biacore system (Cytiva)). In a surface plasmon resonance assay, one or more antibodies may be immobilized on a biosensor and presented with an analyte (e.g., a glycosylated cMET peptide of SEQ ID NO: 285 or a negative control analyte such as the unglycosylated cMET peptide of SEQ ID NO: 286). In some embodiments, the antibody is contacted with the analyte at a saturating concentration, e.g., a concentration of at least about 0.5 μM. In some embodiments, the saturating concentration is about 1 μM, about 1.5 μM, or about 2 μM. When comparing the binding affinities of two antibodies, the affinities of both antibodies are preferably measured using the same concentration of both antibodies, e.g., using a concentration of each antibody of 1 μM.

[0168] In performing an antibody competition assay between a reference antibody and a test antibody (regardless of species or isotype), the reference may first be labeled with a detectable label, such as a fluorophore, biotin, or enzyme label (or even a radioactive label), to allow for subsequent identification. In this case, cells expressing glyco-cMET are incubated with unlabeled test antibody, labeled reference antibody is added, and the intensity of bound label is measured. If the test antibody competes with the labeled reference antibody by binding to an overlapping epitope, the intensity is expected to be reduced compared to a control reaction performed without the test antibody.

[0169] In certain embodiments of this assay, the concentration of labeled reference antibody that results in 80% of maximal binding under the assay conditions (e.g., at a particular density of cells) ("concentration"). 80% ") was first determined and compared to the unlabeled test antibody by 10-fold conc. 80% , and the conc of the labeled reference antibody 80% A competition assay is performed.

[0170] Inhibition is expressed as the inhibition constant, or K i which can be expressed as: K i =IC 50 / (1+[reference Ab concentration] / K d ) where IC 50 is the concentration of test antibody that results in a 50% reduction in binding of the reference antibody, and K d is the dissociation constant of the reference antibody, which is a measure of its affinity for glycated cMET. Antibodies that compete with the anti-glycated cMET antibodies disclosed herein have a K of 10 pM to 10 nM under the assay conditions described herein. i may have:

[0171] In various embodiments, a test antibody is considered to compete with a reference antibody if, at a reference antibody concentration that is 80% of maximal binding under the particular assay conditions used, and a test antibody concentration that is 10-fold greater than the reference antibody concentration, the test antibody reduces binding of the reference antibody by at least about 20% or more, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even more, or a percentage range between any of the aforementioned values.

[0172] In one example of a competitive assay, glycosylated cMET peptide of SEQ ID NO: 285 is attached to a solid surface, e.g., a microwell plate, by contacting the plate with a solution of the peptide (e.g., at a concentration of 1 μg / mL in PBS, overnight at 4° C.). The plate is washed (e.g., with 0.1% Tween 20 in PBS) and blocked (e.g., with Superblock, Thermo Scientific, Rockford, IL). A mixture of subsaturating amounts of biotinylated 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 (e.g., at a concentration of 80 ng / mL) and serially diluted (e.g., at concentrations of 2.8 μg / mL, 8.3 μg / mL, or 25 μg / mL) unlabeled 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 ("reference" antibody) or competing anti-glyco-cMET antibody ("test" antibody) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20 in PBS) is added to the wells, and the plate is incubated for 1 hour with gentle shaking. The plate is washed, and 1 μg / mL of HRP-conjugated streptavidin diluted in ELISA buffer is added to each well, and the plate is incubated for 1 hour. The plate is washed, and bound antibody is detected by adding a substrate (e.g., TMB, Biofx Laboratories Inc., Owings Mills, MD). The reaction is stopped by adding a stop buffer (e.g., Bio FX Stop Reagent, Biofx Laboratories Inc., Owings Mills, MD), and the absorbance is measured at 650 nm using a microplate reader (e.g., VERSAmax, Molecular Devices, Sunnyvale, CA).

[0173] Variations on this competition assay can also be used to test the competition of 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 with another anti-glyco-cMET antibody. For example, in certain embodiments, an anti-glyco-cMET antibody is used as the reference antibody, and 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 is used as the test antibody. Additionally, membrane-bound glyco-cMET expressed on the cell surface (e.g., on the surface of one of the cell types mentioned above) during culture can be used in place of the glycosylated cMET peptide of SEQ ID NO: 285. Generally, approximately 10 4 From 10 6 transformants, for example, about 10 5 Other formats of competitive assays are known in the art and can be employed.

[0174] In various embodiments, when the anti-glyco-cMET antibodies of the present disclosure are used at a concentration of 0.08 μg / mL, 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, or at a concentration range between any of the aforementioned values ​​(e.g., at a concentration ranging from 2 μg / mL to 10 μg / mL), the anti-glyco-cMET antibodies of the present disclosure exhibit a similar activity to labeled 15C4, 8H3, 16E12, 14E9, or 16E12 antibodies. , 19H2, or 39A3 by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or a range of percentages between any of the aforementioned values ​​(e.g., anti-glyco-cMET antibodies of the present disclosure reduce binding of labeled 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 by 50% to 70%).

[0175] In other embodiments, when 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 is used at a concentration of 0.4 μg / mL, 2 μg / mL, 10 μg / mL, 50 μg / mL, 250 μg / mL, or at a concentration range between any of the aforementioned values ​​(e.g., at a concentration ranging from 2 μg / mL to 10 μg / mL), ... reduces binding of a labeled anti-glyco-cMET antibody of the present disclosure by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or a percentage range between any of the aforementioned values ​​(e.g., 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 reduces binding of a labeled anti-glyco-cMET antibody of the present disclosure by 50% to 70%).

[0176] In the foregoing assays, the 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 antibody can be replaced with any antibody or antigen-binding fragment containing the CDRs or heavy and light chain variable regions of 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3, such as the humanized or chimeric counterparts of 3C7, 13C3, or 13G2. Exemplary humanized heavy and light chain variable regions of 8H3 are provided in Tables 4A-4G.

[0177] In certain embodiments, an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure has an epitope that is the same as or similar to that of 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3. The epitope of an anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure can be characterized, for example, by performing alanine scanning. A library of glycopeptides is created, each of which differs from the cMET glycopeptide (SEQ ID NO: 285) by an alanine point mutation at one amino acid position of SEQ ID NO: 285 (or by a glycine point mutation, if the cMET peptide has an alanine). The epitope of the antibody or antigen-binding fragment can be mapped by measuring the binding of the antibody or antigen-binding fragment to each of the peptides by ELISA.

[0178] In certain embodiments, anti-glyco-cMET antibodies or antigen-binding fragments of the present disclosure comprise (or are encoded by) the heavy and / or light chain variable sequences set forth in Tables 1A-1F (murine) and 4A-4G (humanized). In other embodiments, anti-glyco-cMET antibodies or antigen-binding fragments of the present disclosure comprise (or are encoded by) the heavy and / or light chain CDR sequences set forth in Tables 1A-3H. The framework sequences of such anti-glyco-cMET antibodies and antigen-binding fragments may be the native murine framework sequences of the VH and VL sequences set forth in Tables 1A-1F or non-native (e.g., humanized or human) framework sequences. The humanized framework sequences of the VH and VL sequences of 8H3 are set forth in Tables 4A-4G.

[0179] In yet other aspects, the disclosure provides anti-cMET antibodies or antigen-binding fragments having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 1 and 2, respectively.

[0180] In yet other aspects, the disclosure provides anti-cMET antibodies or antigen-binding fragments having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 23 and 24, respectively.

[0181] In yet other aspects, the disclosure provides anti-cMET antibodies or antigen-binding fragments having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 45 and 46, respectively.

[0182] In yet other aspects, the disclosure provides anti-cMET antibodies or antigen-binding fragments having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 67 and 68, respectively.

[0183] In yet other aspects, the disclosure provides anti-cMET antibodies or antigen-binding fragments having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 89 and 90, respectively.

[0184] In yet other aspects, the disclosure provides anti-cMET antibodies or antigen-binding fragments having heavy and light chain variable regions with at least 95%, 98%, 99%, or 99.5% sequence identity to SEQ ID NOs: 111 and 112, respectively.

[0185] In yet another aspect, the disclosure provides an anti-cMET antibody or antigen-binding fragment having a heavy chain variable region having at least 95%, 98%, 99%, or 99.5% sequence identity to one of SEQ ID NOs: 264-275, and a light chain variable region having at least 95%, 98%, 99%, or 99.5% sequence identity to one of SEQ ID NOs: 276-284.

[0186] In yet another aspect, the anti-glyco-cMET antibody or antigen-binding fragment of the present disclosure is a single-chain variable fragment (scFv). An exemplary scFv comprises a heavy chain variable fragment N-terminal to a light chain variable fragment. Another exemplary scFv comprises a light chain variable fragment N-terminal to a heavy chain variable fragment. In some embodiments, the heavy and light chain variable fragments of the scFv are covalently linked by a linker sequence of 4 to 15 amino acids. The scFv may be in the form of a bispecific T cell engager or within a chimeric antigen receptor (CAR).

[0187] 5.1.1. Antibody specificity In some embodiments, anti-glyco-cMET antibodies of the disclosure specifically bind to the cMET glycoprotein PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), glycosylated with GalNAc at the serine and threonine residues shown in bold and underlined. In certain embodiments, anti-glyco-cMET antibodies of the present disclosure specifically bind to the cMET glycoprotein and are capable of isolating any of the following peptides: the unglycosylated cMET peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 286) ("unglycosylated cMET peptide"); the MUC1 tandem repeat (VTSAPDTRPAPGSTAPPAHG)3 (SEQ ID NO: 288) glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4 ("first MUC1 glycopeptide"); the MUC1 peptide TAPPAHGVTSAPDTRPAPGSTAPPAHGVT (SEQ ID NO: 289) glycosylated in vitro with GalNAc at the serine and threonine residues shown in bold and underlined ("second MUC1 glycopeptide"); and the MUC1 peptide TAPPAHGVTSAPDTRPAPGSTAPPAHGVT (SEQ ID NO: 289) glycosylated in vitro with GalNAc at the threonine residue shown in bold and underlined ("second MUC1 glycopeptide"). It does not specifically bind to one or more of the following: the in vitro glycosylated podoplanin peptide ERGTKPPLEELSGK (SEQ ID NO: 290) ("PDPN glycopeptide"); the CD44v6 peptide GYRQTPKEDSHSTTGTAAA (SEQ ID NO: 345) ("CD44v6 glycopeptide"), which was glycosylated in vitro with GalNAc at the threonine and serine residues shown in bold and underlined ("CD44v6 glycopeptide"); the MUC4 peptide CTIPSTAMHTRSTAAPIPILP (SEQ ID NO: 291) ("MUC4 glycopeptide"), which was glycosylated in vitro with GalNAc at the serine and threonine residues shown in bold and underlined ("MUC4 glycopeptide"); and the LAMP1 peptide CEQDRPSPTTAPPAPPSPSP (SEQ ID NO: 292) ("LAMP1 glycopeptide"), which was glycosylated in vitro with GalNAc at the serine and threonine residues shown in bold and underlined ("LAMP1 glycopeptide").

[0188] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have a binding affinity to a cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody to an unglycosylated cMET peptide.

[0189] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have a binding affinity to a cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody to a first MUC1 glycopeptide.

[0190] In some embodiments, an anti-glyco-cMET antibody of the present disclosure has a binding affinity to a cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody to a second MUC1 glycopeptide.

[0191] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have a binding affinity for cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody for PDPN glycopeptide.

[0192] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have a binding affinity to a cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody to a CD44v6 glycopeptide.

[0193] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have a binding affinity for cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody for MUC4 glycopeptide.

[0194] In some embodiments, the anti-glyco-cMET antibodies of the present disclosure have a binding affinity to a cMET glycopeptide that is at least 3 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, or at least 1000 times greater than the binding affinity of the anti-glyco-cMET antibody to a LAMP1 glycopeptide.

[0195] Assays for determining affinity, including relative affinity, include, but are not limited to, radioactive immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), sandwich ELISA, fluorescence-activated cell sorting (FACS) assays, surface plasmon resonance (e.g., Biacore) assays, and biolayer interferometry (BLI) assays. In some embodiments, affinity is measured by surface plasmon resonance (e.g., Biacore). In other embodiments, affinity is measured by surface plasmon resonance (e.g., Biacore).

[0196] Exemplary anti-glyco-cMET antibodies and fragments thereof are described in numbered embodiments 1-657.

[0197] 5.2 Antibody-drug conjugates Another aspect of the present disclosure relates to antibody-drug conjugates (ADCs) comprising the anti-glyco-cMET antibodies and antigen-binding fragments of the present disclosure. The ADCs generally comprise the anti-glyco-cMET antibodies and / or binding fragments described herein linked by one or more linkers to one or more cytotoxic and / or cytostatic agents. In specific embodiments, the ADCs have structural formula (I): [DL-XY] n-Ab or a salt thereof, wherein each "D" independently represents a cytotoxic and / or cytostatic agent ("drug"); each "L" independently represents a linker; "Ab" represents an anti-glycoed cMET antigen binding domain, e.g., an anti-glycoed cMET antibody or binding fragment described herein; and each "XY" represents a functional group R on the linker. x and a "complementary" functional group R on the antibody y where n represents the number of drugs linked to the ADC, or the drug-to-antibody ratio (DAR) of the ADC.

[0198] Specific embodiments of the various antibodies (Abs) that can comprise the ADC include the various embodiments of the anti-glyco-cMET antibodies and / or binding fragments described above.

[0199] In some specific embodiments of the ADCs and / or salts of structural formula (I), each D is the same and / or each L is the same.

[0200] Specific embodiments of the cytotoxic and / or cytostatic agents (D) and linkers (L) that may comprise the anti-glyco-cMET ADCs of the disclosure, as well as the number of cytotoxic and / or cytostatic agents linked to the ADC, are described in more detail below.

[0201] 5.2.1. Cytotoxic and / or cytostatic substances Cytotoxic and / or cytostatic substances can be any substance known to inhibit the growth and / or replication of cells, particularly cancer and / or tumor cells, and / or kill such cells. Numerous substances with cytotoxic and / or cytostatic properties are known in the literature. Non-limiting examples of classes of cytotoxic and / or cytostatic substances include, by way of example only, but not limited to, radionuclides, alkylating agents, topoisomerase I inhibitors, topoisomerase II inhibitors, DNA intercalators (e.g., minor groove binders), RNA / DNA antimetabolites, cell cycle modulators, kinase inhibitors, protein synthesis inhibitors, histone deacetylase inhibitors, mitochondrial inhibitors, and antimitotic agents.

[0202] Specific non-limiting examples of substances within certain of these various classes are provided below.

[0203] Alkylating agents: Asare ((L-leucine, N-[N-acetyl-4-[bis-(2-chloroethyl)amino]-DL-phenylalanyl]-, ethyl ester; NSC 167780; CAS Registry Number 3577897)); AZQ ((1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-, diethyl ester; NSC 182986; CAS Registry Number 57998682)); BCNU ((N,N'-bis(2-chloroethyl)-N-nitrosourea; NSC 409962; CAS Registry Number 15 4938); Busulfan (1,4-butanediol dimethanesulfonate; NSC 750; CAS Registry Number 55981); (Carboxyphthalato)platinum (NSC 27164; CAS Registry Number 65296813); CBDCA ((cis-(1,1-cyclobutanedicarboxylato)diammineplatinum(II)); NSC 241240; CAS Registry Number 41575944); CCNU ((N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea; NSC 79037; CAS Registry Number 13010474)); CHIP (Iproplatin) ;NSC256927); chlorambucil (NSC3088; CAS Registry Number 305033); chlorozotocin ((2-[[[(2-chloroethyl)nitrosamino]carbonyl]amino]-2-deoxy-D-glucopyranose; NSC178248; CAS Registry Number 54749905)); cisplatin (cisplatin; NSC119875; CAS Registry Number 15663271); clomesone (NSC338947; CAS Registry Number 88343720); cyanomorpholinodoxorubicin (NSC357704; CAS Registry Number 88254073) ; cyclodisone (NSC348948; CAS Registry Number 99591738); dianhydrogalactitol (5,6-diepoxydulcitol; NSC132313; CAS Registry Number 23261203); fluorodopan ((5-[(2-chloroethyl)-(2-fluoroethyl)amino]-6-methyl-uracil; NSC73754; CAS Registry Number 834913); hepsulfame (NSC329680; CAS Registry Number 96892578); hycanthone (NSC142982; CAS Registry Number 23255938); melphalan (NSC8806;CAS Registry Number 3223072; Methyl CCNU ((1-(2-chloroethyl)-3-(trans-4-methylcyclohexane)-1-nitrosourea; NSC 95441; 13909096); Mitomycin C (NSC 26980; CAS Registry Number 50077); Mitozolomide (mitozolamide) (NSC 353451; CAS Registry Number 85622953); Nitrogen Mustard ((Bis(2-chloroethyl)methylamine hydrochloride; N SC762; CAS Registry Number 55867; PCNU ((1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea; NSC95466; CAS Registry Number 13909029)); piperazine alkylating agent ((1-(2-chloroethyl)-4-(3-chloropropyl)-piperazine dihydrochloride; NSC344007)); piperazinedione (NSC135758; CAS Registry Number 41109802); pipobroman ((N,N-bis( (3-bromopropionyl)piperazine; NSC 25154; CAS Registry Number 54911); porfiromycin (N-methylmitomycin C; NSC 56410; CAS Registry Number 801525); spirohydantoin mustard (NSC 172112; CAS Registry Number 56605164); teroxylon (triglycidyl isocyanurate; NSC 296934; CAS Registry Number 2451629); tetraplatin (NSC 363812; CAS Registry Number 6281 6982); Thiotepa (N,N',N''-tri-1,2-ethanediylthiophosphoramide; NSC 6396; CAS Registry Number 52244); Triethylenemelamine (NSC 9706; CAS Registry Number 51183); Uracil Nitrogen Mustard (Desmethyldopane; NSC 34462; CAS Registry Number 66751); Yoshi-864 ((Bis(3-mesyloxypropyl)amine hydrochloride; NSC 102627; CAS Registry Number 3458228);

[0204] Topoisomerase I inhibitors: camptothecin (NSC94600; CAS Registry Number 7689-03-4); various camptothecin derivatives and analogues (e.g., NSC100880, NSC603071, NSC107124, NSC643833, NSC629971, NSC295500, NSC249910, NSC606985, NSC74028, NSC176323, NSC295501 , NSC606172, NSC606173, NSC610458, NSC618939, NSC610457, NSC610459, NSC606499, NSC610456, NSC364830, and NSC606497); morpholine isoxorubicin (NSC354646; CAS Registry Number 89196043); SN-38 (NSC673596; CAS Registry Number 86639-52-3).

[0205] Topoisomerase II inhibitors: doxorubicin (NSC 123127; CAS Registry Number 25316409); amonafide (benzisoquinolinedione; NSC 308847; CAS Registry Number 69408817); m-AMSA ((4'-(9-acridinylamino)-3'-methoxymethanesulfonanilide; NSC 249992; CAS Registry Number 51264143)); anthrapyrazole derivatives (NSC 355644); etoposide (VP-16; NSC 141540; CAS Registry Number 33419420); pyrazoloacridine (pyrazolo[3,4,5-kl]acridine-2(6H)-propanamine, 9-methoxy-N,N-dimethyl-5-nitro-, monomethanesulfonate; NSC 366140; CAS Registry Number 99009219) ; Bisantrene hydrochloride (NSC337766; CAS Registry Number 71439684); Daunorubicin (NSC821151; CAS Registry Number 23541506); Deoxydoxorubicin (NSC267469; CAS Registry Number 63950061); Mitoxantrone (NSC301739; CAS Registry Number 70476823); Menogaril (NSC269148; C AS Registry No. 71628961); N,N-dibenzyldaunomycin (NSC268242; CAS Registry No. 70878512); oxantrazole (NSC349174; CAS Registry No. 105118125); rubidazone (NSC164011; CAS Registry No. 36508711); teniposide (VM-26; NSC122819; CAS Registry No. 29767202).

[0206] DNA intercalators: anthramycin (CAS Registry Number 4803274); ticamycin A (CAS Registry Number 89675376); tomaymycin (CAS Registry Number 35050556); DC-81 (CAS Registry Number 81307246); sibiromycin (CAS Registry Number 12684332); pyrrolobenzodiazepine derivatives (CAS Registry Number 945490095); SGD-1882 ((S)-2-(4-aminophenyl)-7-methoxy-8-(3-(4(S)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-5,11a-diamino ... Hydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)propoxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one; SG2000 (SJG-136; (11aS,11a'S)-8,8'-(propane-1,3-diylbis(oxy))bis(7-methoxy-2-methylene-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-5(11aH)-one); NSC694501; CAS Registry Number 232931576).

[0207] RNA / DNA antimetabolites: L-alanosine (NSC153353; CAS Registry Number 59163416); 5-azacytidine (NSC102816; CAS Registry Number 320672); 5-fluorouracil (NSC19893; CAS Registry Number 51218); acivicin (NSC163501; CAS Registry Number 42228922); aminopterin derivative N-[2-chloro-5-[[(2,4-diamino-5-methyl-6-quinazolinyl)methyl]amino] ]benzoyl-]L-aspartic acid (NSC132483); aminopterin derivative N-[4-[[(2,4-diamino-5-ethyl-6-quinazolinyl)methyl]amino]benzoyl]L-aspartic acid (NSC184692); aminopterin derivative N-[2-chloro-4-[[(2,4-diamino-6-pteridinyl)methyl]amino]benzoyl]L-aspartic acid monohydrate (NSC134033); antifolate (antifolate) ((N α- (4-amino-4-deoxypteroyl)-N 7-hemiphthaloyl-L-ornithine; NSC 623017); Baker's soluble antifolate (NSC 139105; CAS Registry Number 41191042); dichlorallyl lawsone (2-(3,3-dichloroallyl)-3-hydroxy-1,4-naphthoquinone; NSC 126771; CAS Registry Number 36417160); brequinar (NSC 368390; CAS Registry Number 96201886); ftorafur (prodrug; 5-fluoro-1-(tetrahydro-2-furyl)-uracil; NSC 148958; CAS Registry Number 37076689); 5,6- Dihydro-5-azacytidine (NSC 264880; CAS Registry Number 62402317); methotrexate (NSC 740; CAS Registry Number 59052); methotrexate derivative (N-[[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]-1-naphthalenyl]carbonyl]L-glutamic acid; NSC 174121); PALA ((N-(phosphonoacetyl)-L-aspartate; NSC 224131; CAS Registry Number 603425565); pyrazofurin (NSC 143095; CAS Registry Number 30868305); trimetrexate (NSC 352122; CAS Registry Number 82952645).

[0208] DNA antimetabolites: 3-HP (NSC95678; CAS Registry Number 3814797); 2'-deoxy-5-fluorouridine (NSC27640; CAS Registry Number 50919); 5-HP (NSC107392; CAS Registry Number 19494894); α-TGDR (α-2'-deoxy-6-thioguanosine; NSC71851; CAS Registry Number 2133815); aphidicolin glycinate (NSC303812; CAS Registry Number 92802822); ara C (cytosine arabinoside; NSC63878; CAS Registry Number 69749); 5-aza-2'-deoxycytidine (NSC127716; CAS Registry Number 2353335); β-TGDR (β-2'-deoxy-6-thioguanosine; NSC71261; CAS Registry Number 789617); cyclocytidine (NSC145668; CAS Registry Number 10212256); guanazole (NSC1895; CAS Registry Number 1455772); hydrazine roxyurea (NSC 32065; CAS Registry Number 127071); inosine glycodialdehyde (NSC 118994; CAS Registry Number 23590990); macbecin II (NSC 330500; CAS Registry Number 73341738); pyrazoloimidazole (NSC 51143; CAS Registry Number 6714290); thioguanine (NSC 752; CAS Registry Number 154427); thiopurine (NSC 755; CAS Registry Number 50442).

[0209] Cell cycle modulators: silibinin (CAS Registry Number 22888-70-6); epigallocatechin gallate (EGCG; CAS Registry Number 989515); procyanidin derivatives (e.g., procyanidin A1 [CAS Registry Number 103883030], procyanidin B1 [CAS Registry Number 20315257], procyanidin B4 [CAS Registry Number 29106512], arekatanin B1 [CAS Registry Number 79763283]); isoflavones (e.g., genistein [4% 5,7-trihydroxyisoflavone; CAS Registry Number 446720], daidzein) Indolone [4',7-dihydroxyisoflavone, CAS Registry Number 486668]; indole-3-carbinol (CAS Registry Number 700061); quercetin (NSC 9219; CAS Registry Number 117395); estramustine (NSC 89201; CAS Registry Number 2998574); nocodazole (CAS Registry Number 31430189); podophyllotoxin (CAS Registry Number 518285); vinorelbine tartrate (NSC 608210; CAS Registry Number 125317397); cryptophycin (NSC 667642; CAS Registry Number 124689652).

[0210] Kinase inhibitors: afatinib (CAS Registry Number 850140726); axitinib (CAS Registry Number 319460850); ARRY-438162 (binimetinib) (CAS Registry Number 606143899); bosutinib (CAS Registry Number 380843754); cabozantinib (CAS Registry Number 1140909483); ceritinib (CAS Registry Number 1032900256); crizotinib (CAS Registry Number 877399525); dabrafenib (CAS Registry Number 1195765457); dasatinib (NSC732517; CAS Registry Number 302 962498); erlotinib (NSC718781; CAS Registry Number 183319699); everolimus (NSC733504; CAS Registry Number 159351696); fostamatinib (NSC745942; CAS Registry Number 901119355); gefitinib (NSC715055; CAS Registry Number 184475352); ibrutinib (CAS Registry Number 936563961); imatinib (NSC716051; CAS Registry Number 220127571); lapatinib (CAS Registry Number 388082788); lenvatinib (CAS Registry Number 857 890392); mubritinib (CAS 366017096); nilotinib (CAS 923288953); nintedanib (CAS 656247175); palbociclib (CAS 571190302); pazopanib (NSC 737754; CAS 635702646); pegaptanib (CAS 222716861); ponatinib (CAS 1114544318); rapamycin (NSC 226080; CAS 53123889); regorafenib (CAS 755037037); AP 23573 (ridaforolimus) (CAS Registry Number 572924540); INCB018424 (ruxolitinib) (CAS Registry Number 1092939177); ARRY-142886 (selumetinib) (NSC741078; CAS Registry Number 606143-52-6); sirolimus (NSC226080; CAS Registry Number 53123889); sorafenib (NSC724772; CAS Registry Number 475207591); sunitinib (NSC736511; CAS Registry Number 341031547); tofacitinib (CAS Registry Number 477600752);Temsirolimus (NSC683864; CAS Registry Number 163635043); trametinib (CAS Registry Number 871700173); vandetanib (CAS Registry Number 443913733); vemurafenib (CAS Registry Number 918504651); SU6656 (CAS Registry Number 330161870); CEP-701 (lesaurtinib) (CAS Registry Number 11135888 4); XL019 (CAS Registry Number 945755566); PD-325901 (CAS Registry Number 391210109); PD-98059 (CAS Registry Number 167869218); ATP-competitive TORC1 / TORC2 inhibitors, such as PI-103 (CAS Registry Number 371935749), PP242 (CAS Registry Number 1092351671), PP30 (CAS Registry Number 1092788094), Torin 1 (CAS Registry Number 1222998368), LY294002 (CAS Registry Number 154447366), XL-147 (CAS Registry Number 934526893), CAL-120 (CAS Registry Number 870281348), ETP-45658 (CAS Registry Number 1198357797), PX866 (CAS Registry Number 502632668), GDC-0941 (CAS Registry Number 957054307), BGT226 (CAS Registry Number 1245537681), BEZ235 (CAS Registry Number 915019657), XL-765 (CAS Registry Number 934493762), etc.;

[0211] Protein synthesis inhibitors: acriflavine (CAS Registry Number 65589700); amikacin (NSC177001; CAS Registry Number 39831555); arbekacin (CAS Registry Number 51025855); astromycin (CAS Registry Number 55779061); azithromycin (NSC643732; CAS Registry Number 83905015); bekanamycin (CAS Registry Number 4696768); chlortetracycline (NSC13252; CAS Registry Number 64722); clarithromycin (NSC643733; CAS Registry Number 81103) 119); Clindamycin (CAS Registry Number 18323449); Chromocycline (CAS Registry Number 1181540); Cycloheximide (CAS Registry Number 66819); Dactinomycin (NSC3053; CAS Registry Number 50760); Dalfopristin (CAS Registry Number 112362502); Demeclocycline (CAS Registry Number 127333); Dibekacin (CAS Registry Number 34493986); Dihydrostreptomycin (CAS Registry Number 128461); Dirithromycin (CAS Registry Number 62013041); Doxycycline phospholipid (CAS Registry Number 17086281); emetine (NSC 33669; CAS Registry Number 483181); erythromycin (NSC 55929; CAS Registry Number 114078); flurithromycin (CAS Registry Number 83664208); framycetin (neomycin B; CAS Registry Number 119040); gentamicin (NSC 82261; CAS Registry Number 1403663); glycylcyclines, such as tigecycline (CAS Registry Number 220620097); hygromycin B (CAS Registry Number 31282049); isepamicin (C AS Registry No. 67814760; josamycin (NSC 122223; CAS Registry No. 16846245); kanamycin (CAS Registry No. 8063078); ketolides, such as telithromycin (CAS Registry No. 191114484), cethromycin (CAS Registry No. 205110481), and solithromycin (CAS Registry No. 760981837); lincomycin (CAS Registry No. 154212); lymecycline (CAS Registry No. 992212); meclocycline (NSC 78502; CAS Registry No. 2013583);methacycline (londomycin; NSC 356463; CAS Registry Number 914001); midecamycin (CAS Registry Number 35457808); minocycline (NSC 141993; CAS Registry Number 10118908); miokamycin (CAS Registry Number 55881077); neomycin (CAS Registry Number 119040); netilmicin (CAS Registry Number 56391561); oleandomycin (CAS Registry Number 3922905); oxazolidinones, such as eperezolid (CAS Registry Number 165800044) and linezolid (CAS Registry Number 165800033), pozizolid (CAS Registry Number 252260029), radezolid (CAS Registry Number 869884786), lambezolid (CAS Registry Number 392659380), stezolid (CAS Registry Number 168828588), tedizolid (CAS Registry Number 856867555); oxytetracycline (NSC9169; CAS Registry Number 2058460); paromomycin (CAS Registry Number 7542372); penimepicycline (CAS Registry Number 4599604); peptidyl transferase inhibitors anti-inflammatory drugs, such as chloramphenicol (NSC3069; CAS Registry Number 56757) and derivatives of azidamfenicol (CAS Registry Number 13838089), florfenicol (CAS Registry Number 73231342), and thiamphenicol (CAS Registry Number 15318453), and pleuromutilins, such as retapamulin (CAS Registry Number 224452668), tiamulin (CAS Registry Number 55297955), valnemulin (CAS Registry Number 101312929); pirlimycin (CAS Registry Number 7954 8735); puromycin (NSC3055; CAS Registry Number 53792); quinupristin (CAS Registry Number 120138503); ribostamycin (CAS Registry Number 53797356); rokitamycin (CAS Registry Number 74014510); rolitetracycline (CAS Registry Number 751973); roxithromycin (CAS Registry Number 80214831); sisomicin (CAS Registry Number 32385118); spectinomycin (CAS Registry Number 1695778); spiramycin (CAS Registry Number 8025818);Streptogramins, such as pristinamycin (CAS Registry Number 270076603), quinupristin / dalfopristin (CAS Registry Number 126602899), and virginiamycin (CAS Registry Number 11006761); streptomycin (CAS Registry Number 57921); tetracycline (NSC108579; CAS Registry Number 60548); tobramycin (CAS Registry Number 32986564); troleandomycin (CAS Registry Number 2751099); tylosin (CAS Registry Number 1401690); verdamycin (CAS Registry Number 49863481);

[0212] Histone deacetylase inhibitors: abexinostat (CAS Registry Number 783355602); belinostat (NSC 726630; CAS Registry Number 414864009); chidamide (CAS Registry Number 743420022); entinostat (CAS Registry Number 209783802); gibinostat (CAS Registry Number 732302997); mocetinostat (CAS Registry Number 726169739); panobinostat (CAS Registry Number 404950807); xinostat (CAS Registry Number 875320299); resminostat (CAS Registry Number 864814880); romidepsin (CAS Registry Number 128517077); sulforaphane (CAS Registry Number 4478937); thioureide Butyronitrile (Kevetrin™; CAS Registry Number 6659890); valproic acid (NSC 93819; CAS Registry Number 99661); vorinostat (NSC 701852; CAS Registry Number 149647789); ACY-1215 (rosilinostat; CAS Registry Number 1316214524); CUDC-101 (CAS Registry Number 1012054599); CHR-2845 (tefinostat; CAS Registry Number 914382608); CHR-3996 (CAS Registry Number 1235859138); 4SC-202 (CAS Registry Number 910462430); CG200745 (CAS Registry Number 936221339); SB939 (prasinostat; CAS Registry Number 929016966).

[0213] Mitochondrial inhibitors: Pancratistatin (NSC349156; CAS Registry Number 96281311); Rhodamine-123 (CAS Registry Number 63669709); Edelfosine (NSC324368; CAS Registry Number 70641519); d-alpha-tocopherol succinate (NSC173849; CAS Registry Number 4345033); Compound 11β (CAS Registry Number 865070377); Aspirin (NSC406186; CAS Registry Number 50782); Ellipticine (CAS Registry Number 519233); Berberine (CAS Registry Number 633658); Cerulenin (C AS Registry No. 17397896; GX015-070 (Obatoclax®; 1H-indole, 2-(2-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-3-methoxy-2H-pyrrol-5-yl)-; NSC 729280; CAS Registry No. 803712676); celastrol (tripterine; CAS Registry No. 34157830); metformin (NSC 91485; CAS Registry No. 1115704); Brilliant Green (NSC 5011; CAS Registry No. 633034); ME-344 (CAS Registry No. 1374524556).

[0214] Antimitotic agents: allocolchicine (NSC 406042); auristatins, such as MMAE (monomethylauristatin E; CAS Registry Number 474645-27-7) and MMAF (monomethylauristatin F; CAS Registry Number 745017-94-1); halichondrin B (NSC 609395); colchicine (NSC 757; CAS Registry Number 64868); colchicine derivative (N-benzoyl-deacetylbenzamide; NSC 33410; CAS Registry Number 63989753); dolastatin 10 (NSC 376128; CAS Registry Number 110417-88-4); maytansine (NSC 153858; CAS Registry Number No. 35846-53-8); rhozoxin (NSC 332598; CAS Registry Number 90996546); taxol (NSC 125973; CAS Registry Number 33069624); taxol derivative ((2'-N-[3-(dimethylamino)propyl]glutaramate)taxol; NSC 608832); thiocolchicine (3-demethylthiocolchicine; NSC 361792); trityl cysteine ​​(NSC 49842; CAS Registry Number 2799077); vinblastine sulfate (NSC 49842; CAS Registry Number 143679); vincristine sulfate (NSC 67574; CAS Registry Number 2068782).

[0215] Any of these substances that contain a site for attachment to an antibody, or that can be modified to contain a site for attachment to an antibody, can be included in the ADCs disclosed herein.

[0216] In a specific embodiment, the cytotoxic and / or cytostatic agent is an antimitotic agent.

[0217] In another specific embodiment, the cytotoxic and / or cytostatic agent is an auristatin, eg, monomethylauristatin E ("MMAE") or monomethylauristatin F ("MMAF").

[0218] Linker In the anti-glyco-cMET ADCs of the present disclosure, the cytotoxic and / or cytostatic agent is linked to the antibody by a linker. The linker linking the cytotoxic and / or cytostatic agent to the antibody of the ADC may be short, long, hydrophobic, hydrophilic, flexible, or rigid, or may be composed of segments each independently having one or more of the above-mentioned properties, such that the linker may contain segments with different properties. Linkers may be multivalent, such that they covalently link more than one agent to a single site on the antibody, or monovalent, such that they covalently link a single agent to a single site on the antibody.

[0219] As will be understood by those skilled in the art, a linker links a cytotoxic and / or cytostatic agent to an antibody by forming a covalent linkage to the cytotoxic and / or cytostatic agent at one position and a covalent linkage to the antibody at the other position. The covalent linkage is formed by reaction of functional groups on the linker with functional groups on the drug and antibody. As used herein, the term "linker" is intended to include (i) an unconjugated form of the linker, which includes a functional group capable of covalently linking the linker to a cytotoxic and / or cytostatic agent and a functional group capable of covalently linking the linker to an antibody; (ii) a partially conjugated form of the linker, which includes a functional group capable of covalently linking the linker to an antibody and covalently linking the cytotoxic and / or cytostatic agent, or vice versa; and (iii) a fully conjugated form of the linker, which is covalently linked to both the cytotoxic and / or cytostatic agent and the antibody. In certain specific embodiments of the linkers and anti-glyco-cMET ADCs of the present disclosure, as well as the synthons used to conjugate the linker-agent to the antibody, the moieties comprising the functional group on the linker and the covalent linkage formed between the linker and the antibody are each R xand XY.

[0220] The linker is preferably, but not necessarily, chemically stable to conditions outside the cell and may be designed to be cleaved, destroyed, and / or otherwise specifically degraded inside the cell. Alternatively, a linker not designed to be specifically cleaved or degraded inside the cell may be used. The choice between a stable and an unstable linker may depend on the toxicity of the cytotoxic and / or cytostatic agent. For drugs that are toxic to normal cells, a stable linker is preferred. Drugs that are selective for or target normal cells, reducing toxicity, may be utilized, and the chemical stability of the linker to the extracellular environment is less important. A variety of linkers useful for linking drugs to antibodies in ADCs are known in the art. Any of these linkers, as well as others, can be used to link cytotoxic and / or cytostatic agents to the antibodies of the anti-glyco-cMET ADCs of the present disclosure.

[0221] Exemplary multivalent linkers that can be used to link multiple cytotoxic and / or cytostatic agents to a single antibody molecule are described, for example, in WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; and WO 2014 / 093640, the contents of which are incorporated by reference in their entireties. For example, the Fleximer linker technology developed by Mersana et al. has the potential to enable high-DAR ADCs with excellent physicochemical properties. As shown below, the Mersana technology is based on incorporating drug molecules into a soluble polyacetal backbone via an array of ester bonds. This approach results in highly loaded ADCs (DAR up to 20) while maintaining excellent physicochemical properties.

[0222] Further examples of dendritic linkers are described in U.S. Patent Application Publication No. 2006 / 116422; U.S. Patent Application Publication No. 2005 / 271615; de Groot et al. (2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al. (2003) Angew. Chem. Int. Ed. 42:4494-4499; Shamis et al. (2004) J. Am. Chem. Soc. 126:1726-1731; Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768; King ... al. (2002) Tetrahedron Letters 43:1987-1990.

[0223] Exemplary monovalent linkers that can be used are described, for example, in Nolting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013, CROs / CMOs--Chemica Oggi--Chemistry Today 31(4):30-38; Ducrry et al., 2010, Bioconjugate Chem. 21:5-13; Zhao et al., 2011, J. Med. Chem. 54:3606-3623; U.S. Pat. No. 7,223,837; U.S. Pat. No. 8,568,728; U.S. Pat. No. 8,535,678; and WO 2004010957, each of which is incorporated herein by reference.

[0224] By way of example, and without limitation, some cleavable and non-cleavable linkers that can be included in the anti-glyco-cMET ADCs of the disclosure are described below.

[0225] 5.2.3. Cleavable Linkers In certain embodiments, the selected linker is cleavable in vivo. Cleavable linkers may contain chemically or enzymatically unstable or degradable linkages. Cleavable linkers generally rely on internal cellular processes to release the drug, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers generally incorporate one or more chemical bonds that are cleavable either chemically or enzymatically, while the remainder of the linker is non-cleavable. In certain embodiments, the linker contains a chemically labile group, such as a hydrazone and / or disulfide group. Linkers containing chemically labile groups take advantage of the different properties between plasma and some cytoplasmic compartments. The intracellular conditions that facilitate drug release for hydrazone-containing linkers are the acidic environment of endosomes and lysosomes, while disulfide-containing linkers are reduced in the cytosol, which contains high thiol concentrations, such as glutathione. In certain embodiments, the plasma stability of linkers containing chemically labile groups can be increased by introducing steric hindrance using substituents near the chemically labile group.

[0226] Acid-labile groups, such as hydrazones, remain intact during systemic circulation in the neutral pH environment of blood (pH 7.3–7.5) but undergo hydrolysis to release the drug upon internalization of the ADC into the weakly acidic endosomal (pH 5.0–6.5) and lysosomal (pH 4.5–5.0) compartments of cells. This pH-dependent release mechanism is associated with nonspecific drug release. To increase the stability of the hydrazone group in the linker, the linker may be modified by chemical modifications, such as substitutions, that can be tailored to achieve more efficient release in lysosomes while minimizing loss in the circulation.

[0227] Hydrazone-containing linkers may contain additional cleavage sites, such as additional acid-labile and / or enzyme-labile cleavage sites. Exemplary ADCs containing hydrazone-containing linkers include the following structures:

[0228] [ka] In the formula, D and Ab represent a cytotoxic and / or cytostatic substance (drug) and Ab, respectively, and n represents the number of drug-linkers linked to the antibody. In certain linkers, such as linker (Ig), the linker contains two cleavable groups: a disulfide moiety and a hydrazone moiety. For such linkers, effective release of the unmodified free drug requires acidic pH or disulfide reduction and acidic pH. Linkers such as (Ih) and (Ii) have been shown to be effective with a single hydrazone cleavage site.

[0229] Additional linkers that remain intact during systemic circulation but undergo hydrolysis to release the drug upon internalization of the ADC within acidic cellular compartments include carbonates, which may be useful when cytotoxic and / or cytostatic agents can be covalently attached via oxygen.

[0230] Other acid-labile groups that can be included in the linker include linkers containing cis-aconityl. Cis-aconityl chemistry uses a carboxylic acid juxtaposed to the amide bond to promote amide hydrolysis under acidic conditions.

[0231] The cleavable linker may also contain a disulfide group. Disulfides are designed to be thermodynamically stable at physiological pH and release the drug upon internalization into cells, where the cytosol provides a significantly more reducing environment than the extracellular environment. Dissolution of the disulfide bond generally requires the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), so that disulfide-containing linkers are reasonably stable in circulation and selectively release the drug in the cytosol. In addition, intracellular enzyme proteins such as disulfide isomerase or similar enzymes capable of cleaving disulfide bonds may also contribute to preferential cleavage of disulfide bonds inside cells. In approximately five tumor cells, GSH has been reported to be present in cells at concentrations ranging from 0.5 to 10 mM, compared with significantly lower concentrations of GSH or cysteine ​​(the most abundant low-molecular-weight thiol) in the circulation, where irregular blood flow causes hypoxia, enhancing the activity of reductive enzymes and therefore leading to higher glutathione concentrations. In certain embodiments, the in vivo stability of disulfide-containing linkers can be enhanced by chemical modification of the linker, for example, by the use of steric hindrance adjacent to the disulfide bond.

[0232] Exemplary ADCs with disulfide-containing linkers include the following structures:

[0233] [ka] where D and Ab represent a drug and an antibody, respectively, n represents the number of drug-linkers linked to the antibody, and R is independently selected for each occurrence from, for example, hydrogen or alkyl. In certain embodiments, increasing the steric hindrance adjacent to the disulfide bond increases the stability of the linker. Structures such as (Ij) and (Il) exhibit increased in vivo stability when one or more R groups are selected from lower alkyl, for example, methyl.

[0234] Another type of cleavable linker that can be used is a linker that is specifically cleaved by enzymes. Such linkers are typically peptide-based or contain peptide regions that act as enzyme substrates. Peptide-based linkers tend to be more stable in plasma and extracellular environments than chemically unstable linkers. Lysosomal proteolytic enzymes have very low activity in blood due to endogenous inhibitors and the unfavorably high pH value of blood compared to lysosomes, so peptide bonds generally have excellent stability in serum. Drug release from antibodies is specifically due to the action of lysosomal proteases, such as cathepsin and plasmin. These proteases may be present at high levels in certain tumor cells.

[0235] In exemplary embodiments, the cleavable peptide is a tetrapeptide such as Gly-Phe-Leu-Gly (SEQ ID NO: 343), Ala-Leu-Ala-Leu (SEQ ID NO: 344), or a peptide such as Val-Cit, Val-Ala, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, Phe-Lys, Ile-Val, Asp-Val, His-Val, NorVal-(D)Asp, Ala-(D)Asp, Met-Lys, Asn-Lys, Ile-Pro, Me3Lys-Pro, phenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, Met-(D)Lys, Asn-(D)Lys, AM Met-(D)Lys, Asn-(D)Lys, AW It is selected from dipeptides such as Met-(D)Lys, and Asn-(D)Lys. In certain embodiments, dipeptides are preferred over longer polypeptides because longer peptides are more hydrophobic.

[0236] A variety of dipeptide-based cleavable linkers useful for linking drugs such as doxorubicin, mitomycin, camptothecin, pyrrolobenzodiazepines, tallysomycin, and auristatin / auristatin family members to antibodies have been described (Dubowchik et al., 1998, J. Org. Chem. 67:1866-1872; Dubowchik et al., 1998, Bioorg. Med. Chem. Lett. 8(21):3341-3346; Walker et al., 2002, Bioorg. Med. Chem. Lett. 12:217-219; Walker et al., 2004, Bioorg. Med. Chem. Lett. 14:4323-4327; Sutherland et al., 2013, Blood 122: 1455-1463; and Francisco et al., 2003, Blood 102:1458-1465). All of these dipeptide linkers, or modified versions of these dipeptide linkers, can be used in the anti-glyco-cMET ADCs of the disclosure. Other dipeptide linkers that can be used include those found in ADCs such as Seattle Genetics' brentuximab vedotin SGN-35 (Adcetris™), Seattle Genetics' SGN-75 (anti-CD-70, Val-Cit-monomethylauristatin F (MMAF), Seattle Genetics' SGN-CD33A (anti-CD-33, Val-Ala-(SGD-1882)), Celldex Therapeutics' glenbatumumab (CDX-011) (anti-NMB, Val-Cit-monomethylauristatin E (MMAE), and Cytogen PSMA-ADC (PSMA-ADC-1301) (anti-PSMA, Val-Cit-MMAE).

[0237] The enzymatically cleavable linker can include a self-immolative spacer that separates the drug from the enzymatic cleavage site.Direct attachment of the drug to the peptide linker can cause the release of amino acid adducts of the drug through proteolysis, thereby impairing its activity.The use of a self-immolative spacer allows the release of a fully active, chemically unmodified drug after the hydrolysis of the amide bond.

[0238] One self-immolative spacer is the bifunctional para-aminobenzyl alcohol group, which is linked to the peptide via the amino group to form an amide bond, while amine-containing drugs may be attached to the benzyl hydroxyl group of the linker (PABC) via a carbamate functionality. The resulting prodrug is activated by protease-mediated cleavage, resulting in a 1,6-elimination reaction that releases the unmodified drug, carbon dioxide, and the remainder of the linker group. The following scheme depicts the fragmentation of the p-amidobenzyl ether and release of the drug.

[0239] [ka] where XD represents the unmodified drug.

[0240] Heterocyclic variants of this self-immolative group have also been described, see, for example, U.S. Patent No. 7,989,434, incorporated herein by reference.

[0241] In some embodiments, the enzymatically cleavable linker is a β-glucuronic acid-based linker. The easy release of the drug can be achieved through the cleavage of the β-glucuronide glycosidic bond by the lysosomal enzyme β-glucuronidase. This enzyme is abundant in lysosomes and is overexpressed in some tumor types, but its enzymatic activity outside the cell is low. β-glucuronic acid-based linkers can be used to avoid the tendency of ADCs to undergo aggregation due to the hydrophilic nature of β-glucuronides. In some embodiments, β-glucuronic acid-based linkers are preferred as linkers for ADCs linked to hydrophobic drugs. The following scheme depicts the release of a drug from an ADC containing a β-glucuronic acid-based linker.

[0242] [ka]

[0243] A variety of cleavable β-glucuronic acid-based linkers useful for linking drugs, such as auristatins, camptothecin and doxorubicin analogs, CBI minor groove binders, and psymberin, to antibodies have been described (Nolting, Chapter 5 “Linker Technology in Antibody-Drug Conjugates,” In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2013; Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; each of which is incorporated herein by reference). 2005, J. Am. Chem. Soc. 127:11254-11255). All of these β-glucuronic acid-based linkers can be used in the anti-glyco-cMET ADCs of the present disclosure.

[0244] Additionally, cytotoxic and / or cytostatic agents containing a phenolic group can be covalently attached to the linker via the phenolic oxygen. One such linker is described in WO 2007 / 089149 and relies on the use of a diamino-ethane "SpaceLink" with a conventional "PABO"-based self-immolative group to deliver the phenol. Linker cleavage is depicted schematically below, where D represents the cytotoxic and / or cytostatic agent bearing the phenolic hydroxyl group.

[0245] [ka]

[0246] A cleavable linker may contain a non-cleavable moiety or segment, and / or a cleavable segment or moiety may be included in an otherwise non-cleavable linker to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone. For example, polyethylene glycol or a polymer linker may contain one or more cleavable groups, such as a disulfide, hydrazone, or dipeptide.

[0247] Other degradable linkages that can be included in the linker include ester linkages formed by the reaction of a carboxylic acid of PEG or an activated PEG with an alcohol group on a bioactive agent, in which case the ester group typically hydrolyzes under physiological conditions to release the bioactive agent. Hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine linkages resulting from the reaction of an amine with an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; acetal linkages, which are the reaction product of an aldehyde with an alcohol; orthoester linkages, which are the reaction product of a formate with an alcohol; and oligonucleotide linkages, which are formed by a phosphoramidite group at the end of a polymer and the 5' hydroxyl group of an oligonucleotide.

[0248] In certain embodiments, the linker is an enzymatically cleavable peptide moiety, such as that represented by structural formula (IVa) or (IVb):

[0249] [ka] or a salt thereof, wherein peptide represents a peptide (exemplified C→N, carboxy and amino "termini" not shown) cleavable by a lysosomal enzyme; T represents a polymer comprising one or more ethylene glycol units or alkylene chains, or a combination thereof; ais selected from hydrogen, alkyl, sulfonate, and methylsulfonate; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

[0250] [ka] represents the point of attachment of the linker to the cytotoxic and / or cytostatic agent; * represents the point of attachment to the rest of the linker.

[0251] In certain embodiments, the peptide is selected from a tripeptide or a dipeptide. In certain embodiments, the dipeptide is selected from Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Val-Lys; Ala-Lys; Phe-Cit; Leu-Cit; Ile-Cit; Phe-Arg; and Trp-Cit. In certain embodiments, the dipeptide is selected from Cit-Val and Ala-Val.

[0252] Specific exemplary embodiments of linkers according to structural formula (IVa) that may be included in the anti-glyco-cMET ADCs of the disclosure include the linkers illustrated below (as illustrated, the linker comprises a group suitable for covalently linking the linker to an antibody):

[0253] [ka]

[0254] [ka]

[0255] Specific exemplary embodiments of linkers according to structural formula (IVb) that may be included in the anti-glyco-cMET ADCs of the disclosure include the linkers illustrated below (as illustrated, the linker comprises a group suitable for covalently linking the linker to an antibody):

[0256] [ka]

[0257] [ka]

[0258] [ka]

[0259] [ka]

[0260] [ka]

[0261] In certain embodiments, the linker is an enzymatically cleavable peptide moiety, such as a peptide represented by structural formula (IVc) or (IVd):

[0262] [ka] or a salt thereof, wherein peptide represents a peptide (exemplified C→N, carboxy and amino "termini" not shown) cleavable by a lysosomal enzyme; T represents a polymer comprising one or more ethylene glycol units or alkylene chains, or a combination thereof; ais selected from hydrogen, alkyl, sulfonate, and methylsulfonate; p is an integer ranging from 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

[0263] [ka] represents the point of attachment of the linker to the cytotoxic and / or cytostatic agent; * represents the point of attachment to the rest of the linker.

[0264] Specific exemplary embodiments of linkers according to structural formula (IVc) that can be included in the anti-glyco-cMET ADCs of the disclosure include the linkers illustrated below (as illustrated, the linker comprises a group suitable for covalently linking the linker to an antibody):

[0265] [ka]

[0266] Specific exemplary embodiments of linkers according to structural formula (IVd) that may be included in the anti-glyco-cMET ADCs of the disclosure include the linkers illustrated below (as illustrated, the linker comprises a group suitable for covalently linking the linker to an antibody):

[0267] [ka]

[0268] [ka]

[0269] [ka]

[0270] In certain embodiments, the linker comprising structural formula (IVa), (IVb), (IVc), or (IVd) further comprises a carbonate moiety cleavable by exposure to acidic media. In certain embodiments, the linker is attached to the cytotoxic and / or cytostatic agent via oxygen.

[0271] 5.2.4. Non-cleavable linkers Although cleavable linkers may offer certain advantages, the linkers used in the anti-glyco-cMET ADCs of the present disclosure do not necessarily have to be cleavable. With non-cleavable linkers, drug release does not depend on differential properties between plasma and certain cytoplasmic compartments. Drug release occurs after internalization of the ADC via antigen-mediated endocytosis and delivery to the lysosomal compartment, where the antibody is hypothesized to be degraded to the amino acid level via intracellular proteolysis. This process releases the drug, the linker, and a drug derivative formed by the amino acid residue to which the linker is covalently attached. Amino acid drug metabolites from conjugates with non-cleavable linkers are more hydrophilic and generally have lower membrane permeability than conjugates with cleavable linkers, resulting in fewer bystander effects and less nonspecific toxicity. Generally, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleavable linkers. The non-cleavable linker may be an alkylene chain or may be polymeric in nature, e.g., based on a polyalkylene glycol polymer, an amide polymer, or may contain segments of alkylene chains, polyalkylene glycols, and / or amide polymers.

[0272] A variety of non-cleavable linkers have been described for use in linking drugs to antibodies. See Jeffrey et al., 2006, Bioconjug. Chem. 17; 831-840; Jeffrey et al., 2007, Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al., 2005, J. Am. Chem. Soc. 127:11254-11255, each of which is incorporated herein by reference. Any of these linkers may be included in the anti-glyco-cMET ADCs of the present disclosure.

[0273] In certain embodiments, the linker is not cleavable in vivo, e.g., a linker according to structural formula (VIa), (VIb), (VIc), or (VId) (as exemplified), and the linker comprises a group suitable for covalently linking the linker to an antibody:

[0274] [ka] or a salt thereof, wherein R a is selected from hydrogen, alkyl, sulfonate, and methylsulfonate; R x is a moiety containing a functional group capable of covalently linking the linker to the antibody;

[0275] [ka] represents the point of attachment of the linker to the cytotoxic and / or cytostatic agent.

[0276] Specific exemplary embodiments of linkers according to structural formulas (VIa)-(VId) that may be included in the anti-glyco-cMET ADCs of the present disclosure include the linkers exemplified below (as exemplified, the linker comprises a group suitable for covalently linking the linker to an antibody:

[0277] [ka] represents the point of attachment to a cytotoxic and / or cytostatic agent).

[0278] [ka]

[0279] 5.2.5. Groups Used to Attach the Linker to the Antibody A variety of groups can be used to attach linker-drug synthons to antibodies to yield ADCs. The attachment group can be electrophilic in nature, including maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, formic acid halides, acid halides, alkyl and benzyl halides, such as haloacetamides. As discussed below, there are also emerging technologies for "self-stabilizing" maleimides and "cross-linking disulfides" that can be used in accordance with the present disclosure. The specific group used will depend, in part, on the site of attachment to the antibody.

[0280] An example of a "self-stabilizing" maleimide group that spontaneously hydrolyzes under antibody conjugation conditions to yield an ADC species with improved stability is depicted in the following schematic: U.S. Patent Application Publication No. 20130309256; see also Lyon et al., Nature Biotech published online, doi:10.1038 / nbt.2968.

[0281] Normal system

[0282] [ka]

[0283] Over time, this leads to "loss of DAR."

[0284] SGN MalDPR (maleimidodipropylamino)

[0285] [ka]

[0286] Polytherics discloses a method for cross-linking pairs of sulfhydryl groups derived from the reduction of native hinge disulfide bonds. See Badescu et al., 2014, Bioconjugate Chem. 25:1124-1136. This reaction is depicted in the following schematic diagram. The advantage of this approach is the ability to synthesize enriched DAR4 ADCs by complete reduction of IgG (yielding four pairs of sulfhydryls) followed by reaction with four equivalents of an alkylating agent. ADCs containing "bridged disulfides" are also said to have increased stability.

[0287] [ka]

[0288] Similarly, maleimide derivatives (1, below) have been developed that are capable of cross-linking pairs of sulfhydryl groups, as depicted below. See WO 2013 / 085925.

[0289] [ka]

[0290] 5.2.6. Linker Selection Considerations As known to those skilled in the art, the selected linker for a particular ADC can be influenced by various factors, including, but not limited to, the site of attachment to the antibody (e.g., lys, cys, or other amino acid residue), the structural constraints of the drug's pharmacophore, and the lipophilicity of the drug. The selected linker for a particular ADC will require balancing these various factors for the particular antibody / drug conjugate. For a review of factors influenced by linker selection in ADCs, see Nolting, Chapter 5 "Linker Technology in Antibody-Drug Conjugates," In: Antibody-Drug Conjugates: Methods in Molecular Biology, vol. 1045, pp. 71-100, Laurent Ducry (Ed.), Springer Science & Business Medica, LLC, 2013.

[0291] For example, ADCs have been observed to kill bystander antigen-negative cells present near antigen-positive tumor cells. The mechanism of bystander cell killing by ADCs has shown that metabolic products formed during the intracellular processing of ADCs may play a role. Neutral cytotoxic metabolic products generated by the metabolism of ADCs in antigen-positive cells appear to play a role in bystander cell killing, while charged metabolic products may be prevented from diffusing into the medium through membranes and therefore cannot affect bystander killing. In certain embodiments, linkers are selected to weaken the bystander killing effect caused by cellular metabolic products of ADCs. In certain embodiments, linkers are selected to increase bystander killing effect.

[0292] Linker characteristics may also affect ADC aggregation under use and / or storage conditions. Typically, ADCs reported in the literature contain no more than 3-4 drug molecules per antibody molecule (see, e.g., Chari, 2008, Acc Chem Res 41:98-107). Attempts to achieve higher drug-to-antibody ratios ("DAR") often fail due to ADC aggregation, especially when both the drug and linker are hydrophobic (King et al., 2002, J Med Chem 45:4336-4343; Hollander et al., 2008, Bioconjugate Chem 19:358-361; Burke et al., 2009 Bioconjugate Chem 20:1242-1250). In many instances, a DAR higher than 3-4 may be beneficial as a means of increasing efficacy. In instances where the cytotoxic and / or cytostatic agent is hydrophobic in nature, particularly in instances where a DAR greater than 3-4 is desired, it may be desirable to select a relatively hydrophilic linker as a means of reducing ADC aggregation. Thus, in certain embodiments, the linker incorporates a chemical moiety that reduces ADC aggregation during storage and / or use. To reduce ADC aggregation, the linker may incorporate polar or hydrophilic groups, e.g., charged groups or groups that become charged at physiological pH. For example, the linker may incorporate charged groups, e.g., salts or groups that deprotonate, e.g., carboxylic acids, or protonate, e.g., amines, at physiological pH.

[0293] Exemplary multivalent linkers that can be used to link numerous cytotoxic and / or cytostatic agents to antibodies, reportedly resulting in DARs as high as 20, are described in WO 2009 / 073445; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; WO 2014 / 093640, the contents of which are incorporated by reference in their entireties.

[0294] In certain embodiments, aggregation of the ADC during storage or use is less than about 10% as determined by size exclusion chromatography (SEC). In certain embodiments, aggregation of the ADC during storage or use is less than 10%, e.g., less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.1%, or even lower, as determined by size exclusion chromatography (SEC).

[0295] 5.2.7. Methods for Making Anti-Glyco-cMET ADCs The anti-glyco-cMET ADCs of the present disclosure can be synthesized using well-known chemical methods. The chemical method selected will depend, among other things, on the nature of the cytotoxic and / or cytostatic agent, the linker, and the group used to attach the linker to the antibody. Generally, ADCs according to Formula (I) can be prepared according to the following scheme: DLR x +Ab-R y →[DL-XY] n -Ab (I)

[0296] where D, L, Ab, XY and n are as previously defined, and R xand R y represents complementary groups capable of forming a covalent linkage with one another, as discussed above.

[0297] R x and R y The nature of the group is determined by the synthon DLR xThe binding characteristics of the conjugated antibody will depend on the chemical method used to link the antibody to the antibody. Generally, the chemical method used should not alter the integrity of the antibody, e.g., its ability to bind to its target. Preferably, the binding characteristics of the conjugated antibody will be very similar to those of the unconjugated antibody. A variety of chemical methods and techniques for conjugating molecules to biomolecules such as antibodies are known in the art, particularly for conjugation to antibodies.For example, Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in: Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. Eds., Alan R. Liss, Inc., 1985;Hellstrom et al., “Antibodies For Drug Delivery,” in: Controlled Drug Delivery, Robinson et al. Eds., Marcel Dekker, Inc., 2nd Ed. 1987;Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in: Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., Eds., 1985;“Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy,” in: Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. al., Eds., Academic Press, 1985; Thorpe et al., 1982, Immunol. Rev. 62:119-58; PCT Publication WO 89 / 12624. Any of these chemical methods can be used to link the synthon to the antibody.

[0298] Some functional groups R useful for linking synthons to accessible lysine residues x Chemical methods are known and include, but are not limited to, NHS-esters and isothiocyanates.

[0299] Some functional groups R useful for linking synthons to accessible free sulfhydryl groups of cysteine ​​residues. x and chemical methods are known, examples include, but are not limited to, haloacetyls and maleimides.

[0300] However, the conjugation chemistry is not limited to the available side chain groups. Side chains such as amines can be converted to other useful groups, such as hydroxyls, by linking an appropriate small molecule to the amine. This strategy can be used to increase the number of available linkage sites on an antibody by conjugating multifunctional small molecules to the side chains of accessible amino acid residues of the antibody. A suitable functional group R is then added to covalently link a synthon to these "converted" functional groups. x is included in the synthon.

[0301] Antibodies can also be engineered to contain amino acid residues for conjugation. Approaches for engineering antibodies to contain non-genetically encoded amino acid residues useful for conjugating drugs in the form of ADCs, along with chemical methods and functional groups useful for linking synthons to non-encoded amino acids, are described by Axup et al., 2012, Proc Natl Acad Sci USA. 109(40):16101-16106.

[0302] Typically, synthons are linked to the side chains of amino acid residues of antibodies, such as the primary amino group of an accessible lysine residue or the sulfhydryl group of an accessible cysteine ​​residue. Free sulfhydryl groups can be obtained by reducing interchain disulfide bonds.

[0303] R y is a sulfhydryl group (e.g., R x For linkages using carboxyl groups (where is a maleimide), the antibody is generally first fully or partially reduced to disrupt interchain disulfide bridges between cysteine ​​residues.

[0304] Cysteine ​​residues that do not participate in disulfide bridges can be engineered into the antibody by mutation of one or more codons. Reduction of these unmatched cysteines generates sulfhydryl groups suitable for conjugation. Preferred positions for incorporating an engineered cysteine ​​include, by way of example and not limitation, positions S112C, S113C, A114C, S115C, A176C, 5180C, S252C, V286C, V292C, S357C, A359C, S398C, S428C (Kabat numbering) on ​​the human IgG1 heavy chain, and positions V110C, S114C, S121C, S127C, S168C, V205C (Kabat numbering) on ​​the human Ig kappa light chain (see, e.g., U.S. Pat. Nos. 7,521,541, 7,855,275, and 8,455,622).

[0305] As will be understood by those skilled in the art, the number of cytotoxic and / or cytostatic agents linked to antibody molecules can vary, resulting in an inherently heterogeneous population of ADCs, with some antibodies containing one linked agent, some containing two linked agents, some containing three linked agents, and so on (some antibodies containing no linked agents). The degree of heterogeneity is expected to depend, among other things, on the chemical method used to link the cytotoxic and / or cytostatic agents. For example, when antibodies are reduced to create sulfhydryl groups for attachment, heterogeneous mixtures of antibodies with 0, 2, 4, 6, or 8 linked agents per molecule are often produced. Furthermore, limiting the molar ratio of the attachment compounds often produces antibodies with 0, 1, 2, 3, 4, 5, 6, 7, or 8 linked agents per molecule. Therefore, it will be understood that, depending on the context, the DAR stated may be an average for a population of antibodies. For example, "DAR4" can refer to an ADC preparation that has not been subjected to purification to isolate a specific DAR peak and that may contain a heterogeneous mixture of ADC molecules with different numbers of cytostatic and / or cytotoxic agents attached per antibody (e.g., 0, 2, 4, 6, 8 agents per antibody), but with an average drug-to-antibody ratio of 4. Similarly, in some embodiments, "DAR2" refers to a heterogeneous ADC preparation with an average drug-to-antibody ratio of 2.

[0306] If an enriched preparation is desired, antibodies having a predetermined number of linked cytotoxic and / or cytostatic substances can be obtained through purification of a heterogeneous mixture, e.g., via column chromatography, e.g., hydrophobic interaction chromatography.

[0307] Purity can be assessed by various methods, as known in the art. As a specific example, an ADC preparation may be analyzed via HPLC or other chromatography, and purity may be assessed by analyzing the area under the curve of the resulting peak.

[0308] 5.3 Chimeric Antigen Receptors The present disclosure provides chimeric antigen receptors (CARs) comprising the anti-glyco-cMET antibodies or antigen-binding fragments described herein. In some embodiments, the CARs comprise one or more scFvs (e.g., one or two) described herein. For example, the CARs may comprise two scFvs covalently connected by a linker sequence (e.g., of 4 to 15 amino acids). Exemplary linkers include GGGGS (SEQ ID NO: 293) and (GGGGS)3 (SEQ ID NO: 346).

[0309] The CAR of the present disclosure typically comprises an extracellular domain operably linked to a transmembrane domain, which in turn is operably linked to an intracellular domain for signal transduction. The CAR may further comprise a signal peptide at the N-terminus of the extracellular domain (e.g., human CD8 signal peptide). In some embodiments, the CAR of the present disclosure comprises a human CD8 signal peptide comprising the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 294).

[0310] The extracellular domain of a CAR of the present disclosure comprises the sequence of an anti-glyco-cMET antibody or antigen-binding fragment (e.g., as described in Section 5.1 or numbered embodiments 689-724).

[0311] Exemplary transmembrane and intracellular domain sequences are described in Sections 5.3.1 and 5.3.2, respectively.

[0312] Numerous fusion proteins described herein (e.g., numbered embodiments 664-688) are CARs, and the disclosure related to CARs (e.g., numbered embodiments 689-724) applies to such fusion proteins. Other fusion proteins described herein (e.g., numbered embodiments 735-834) are chimeric T cell receptors, and the disclosure related to chimeric TCRs applies to such fusion proteins.

[0313] 5.3.1. Transmembrane Domain With respect to the transmembrane domain, the CAR can be designed to include a transmembrane domain operably linked (e.g., fused) to the extracellular domain of the CAR.

[0314] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane region of particular use in this disclosure may be derived from (i.e., may include at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some cases, various human hinges, such as human Ig (immunoglobulin) hinges, may also be employed.

[0315] In one embodiment, the transmembrane domain is synthetic (i.e., not naturally occurring). An example of a synthetic transmembrane domain is a peptide containing primarily hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet is expected to be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2 to 10 amino acids in length, can form a link between the transmembrane domain and the CAR cytoplasmic signaling domain. A glycine-serine duo provides a particularly suitable linker.

[0316] In one embodiment, the transmembrane domain in a CAR of the present disclosure is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence YLHLGALGRDLWGPSPVTGYHPLL (SEQ ID NO: 295).

[0317] In one embodiment, the transmembrane domain in a CAR of the present disclosure is a CD28 transmembrane domain. In one embodiment, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 296).

[0318] In some cases, the transmembrane domain of a CAR of the present disclosure is linked to the extracellular domain by a CD8a hinge domain. In one embodiment, the CD8a hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC (SEQ ID NO: 297). In another embodiment, the CD8a hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 298). In another embodiment, the CD8a hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 349).

[0319] In some cases, the transmembrane domain of a CAR of the present disclosure is linked to the extracellular domain by a human IgG4-short hinge. In one embodiment, the human IgG4-short hinge comprises the amino acid sequence ESKYGPPCPSCP (SEQ ID NO: 299).

[0320] In some cases, the transmembrane domain of a CAR of the present disclosure is linked to the extracellular domain by a human IgG4-long hinge. In one embodiment, the human IgG4-long hinge has the amino acid sequence

[0321] [ka] Includes:

[0322] 5.3.2. Intracellular Domain The intracellular signaling domain of the CAR of the present disclosure is involved in activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain can be employed, but in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transduces the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain that is sufficient to transduce the effector function signal.

[0323] Preferred examples of intracellular signaling domains for use in the CARs of the present disclosure include the cytoplasmic sequences of T cell receptors (TCRs) and coreceptors that act cooperatively to initiate signal transduction after binding to an antigen receptor, as well as any derivatives or variants of these sequences, and any synthetic sequences that have the same functional capability.

[0324] Signals generated by the TCR alone may be insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0325] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex either stimulatory or inhibitory. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0326] Examples of primary cytoplasmic signaling sequences containing ITAMs that have particular utility in the CARs of the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CARs of the present disclosure comprises a cytoplasmic signaling sequence derived from CD3-zeta.

[0327] In a preferred embodiment, the cytoplasmic domain of the CAR is designed to include an ITAM-containing primary cytoplasmic signaling sequence domain (e.g., that of CD3-zeta) by itself, or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present disclosure. For example, the cytoplasmic domain of the CAR may include a CD3 zeta chain portion and a costimulatory signaling region.

[0328] The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, DAP10, and GITR.

[0329] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present disclosure can be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably 2 to 10 amino acids in length, can form the linkage. Glycine-serine duplexes provide particularly suitable linkers.

[0330] In one embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta and the signaling domain of CD28. In some embodiments, the signaling domain of CD3-zeta has the amino acid sequence

[0331] [ka] In some embodiments, the signaling domain of CD28 comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 302).

[0332] In another embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta and the signaling domain of 4-1BB.

[0333] In another embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta and the signaling domain of CD2. In some embodiments, the signaling domain of CD2 has the amino acid sequence

[0334] [ka] Includes:

[0335] In another embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta, the signaling domain of CD28, and the signaling domain of CD2.

[0336] In another embodiment, the cytoplasmic domain comprises the signaling domain of CD3-zeta, the signaling domain of 4-1BB, and the signaling domain of CD2.

[0337] Inclusion of a CD2 signaling domain in the cytoplasmic domain allows for the modulation of cytokine production of CAR T cells (see U.S. Pat. No. 9,783,591, the contents of which are incorporated herein by reference in their entirety). As disclosed in U.S. Pat. No. 9,783,591, inclusion of a CD2 signaling domain in the CAR cytoplasmic domain significantly alters cytokine production of CAR T cells, both positively and negatively, and its effect depends on the presence and identity of other costimulatory molecules in the costimulatory signaling region of the cytoplasmic domain. For example, in some embodiments, inclusion of a CD2 signaling domain and a CD28 signaling domain in the costimulatory signaling region of the cytoplasmic domain results in significantly less IL2 release than T cells expressing a CAR that has CD28 but not CD2. CAR T cells that release less IL2 can result in reduced proliferation of immunosuppressive Treg cells. In some embodiments, inclusion of a CD2 signaling domain in the costimulatory signaling region of the cytoplasmic domain significantly reduces calcium influx in CAR T cells. This was shown to reduce activation-induced CAR T cell death.

[0338] 5.4 Chimeric T-cell receptors The present disclosure provides chimeric T cell receptors (TCRs) comprising the anti-glycoed cMET antibodies or antigen-binding fragments described herein. The chimeric TCRs provide antibodies specific for anti-glycoed cMET and TCR chimeras that specifically bind to anti-glycoed cMET and are capable of recruiting at least one TCR-associated signaling molecule (e.g., CD3γε, CD3δε, and ζζ). In some embodiments, the chimeric TCRs comprise one or more antigen-binding fragments capable of binding to glycoed cMET. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments (scFV), and single-domain fragments. In some embodiments, the antigen-binding fragment of the chimeric T cell receptor comprises at least one anti-glycoed cMET variable heavy chain and at least one anti-glycoed cMET variable light chain described herein.

[0339] TCRs occur as either αβ or γδ heterodimers, and T cells express either the αβ or γδ forms of TCRs on the cell surface. Each of the four chains (α, β, γ, and δ) has a characteristic extracellular structure consisting of a highly polymorphic "immunoglobulin variable region"-like N-terminal domain and a second "immunoglobulin constant region"-like domain. Each of these domains has characteristic intradomain disulfide bridges. The constant region is located proximal to the cell membrane, connecting the peptide, the transmembrane region, and the short intracytoplasmic tail. The covalent link between the two chains of a heterodimeric TCR is formed by cysteine ​​residues located within a short connecting peptide sequence bridging the extracellular constant domain and the transmembrane region, which then form disulfide bonds with corresponding cysteine ​​residues in the corresponding TCR chain (Lefranc and Lefranc, "The T Cell Receptor Facts Book," Academic Press, 2001).

[0340] Numerous examples of chimeric TCRs are known in the art (see, e.g., Kuwana et al., Biochem Biophys Res Commun. 149(3):960-968; Gross et al., 1989, Proc Natl Acad Sci USA. 86:10024-10028; Gross & Eshhar, 1992, FASEB J. 6(15):3370-3378; Liu et al., 2021, Sci Transl Med, 13:eabb5191, WO 2016 / 187349, WO 2017 / 070608, WO 2020 / 029774, and U.S. Patent No. 7,741,465, the contents of each of which are incorporated herein by reference in their entirety.

[0341] A chimeric TCR generally comprises a first polypeptide chain comprising a first TCR domain, a second polypeptide chain comprising a second TCR domain, and an anti-glyco-cMET antigen-binding fragment, as described herein. In some embodiments, a chimeric TCR comprises a single anti-glyco-cMET antigen-binding fragment. In other embodiments, a chimeric TCR comprises two or more anti-glyco-cMET antigen-binding fragments. In a specific embodiment, a chimeric TCR comprises two anti-glyco-cMET antigen-binding fragments.

[0342] In some embodiments, the anti-glyco-cMET antigen-binding fragment is an scFv described herein. In embodiments in which the chimeric TCR comprises a single anti-glyco-cMET antigen-binding fragment, the single anti-glyco-cMET scFv may be comprised in either the first or second polypeptide chain of the chimeric TCR. In embodiments in which the chimeric TCR comprises, for example, two anti-glyco-cMET antigen-binding fragments, the two anti-glyco-cMET scFvs may be comprised in either the first or second polypeptide chain of the chimeric TCR, or the first scFv may be comprised in the first polypeptide chain and the second scFv may be comprised in the second polypeptide chain. In embodiments in which two scFvs are comprised in either the first or second polypeptide chain of the chimeric TCR, the two scFvs may be linked via a peptide linker. In some embodiments, the chimeric TCR comprises two or more anti-glyco-cMET scFvs having the same amino acid sequence. In other embodiments, the chimeric TCR comprises two or more anti-glyco-cMET scFvs with different amino acid sequences.

[0343] In other embodiments, the anti-glyco-cMET antigen-binding fragment is an Fv fragment. In some embodiments, the anti-glyco-cMET variable heavy chain (VH) described herein is included in one of the two polypeptide chains that associate to form the chimeric TCR. The anti-glyco-cMET variable light chain (VL) described herein may be included in the polypeptide chain that does not include the anti-glyco-cMET VH. When the first and second polypeptide chains dimerize, the anti-glyco-cMET VH and VL combine to form the anti-glyco-cMET Fv fragment. In some embodiments, the VH is included in the first polypeptide chain and the VL is included in the second polypeptide chain. In other embodiments, the VH is included in the second polypeptide chain and the VL is included in the first polypeptide chain.

[0344] In other embodiments, the anti-glycoed cMET antigen fragment is a Fab domain comprising VH, VL, CH1, and CL domains. In some embodiments, the anti-glycoed cMET variable heavy chain (VH) and CH1 domain described herein are comprised in a first or second polypeptide chain. In some embodiments, the anti-glycoed cMET variable light chain (VL) and CL domain described herein are comprised in a first or second polypeptide chain that does not comprise anti-glycoed cMET VH and CH1. In other embodiments, the anti-glycoed cMET variable heavy chain (VH) and CL domain are comprised in a first or second polypeptide chain. In some embodiments, the anti-glycoed cMET variable light chain (VL) and CH1 domain are comprised in a polypeptide chain that does not comprise anti-glycoed cMET VH and CL. When the first and second polypeptide chains dimerize, the anti-glycoed cMET VH and VL, and CH1 and CL, combine to form the anti-glycoed cMET Fab domain. In some embodiments, the VH and CH1 or CL are comprised in a first polypeptide chain and the VL and CL or CH1 are comprised in a second polypeptide chain, hi other embodiments, the VH and CH1 or CL are comprised in a second polypeptide chain and the VL and CH1 or CL are comprised in the first polypeptide chain.

[0345] In other embodiments, the anti-glyco-cMET VH and CH1 or CL are contained in a first or second polypeptide chain, and the chimeric TCR further comprises a third polypeptide comprising a VL and either a CL domain or a CH1 domain. The third polypeptide is capable of associating with the VH and CH1 or CL of the first or second polypeptide chain, thereby forming a Fab domain. In some embodiments, both the first and second polypeptide chains comprise a VH and a CH1 domain or a CL domain. When both the first and second polypeptide chains comprise a VH and a CH1 or CL, the third polypeptide comprising the VL and CL or CH1 associates with the first polypeptide chain to form a first Fab domain, and the fourth polypeptide comprising the VL and CL or CH1 associates with the second polypeptide chain to form a second Fab domain.

[0346] The first and second TCR domains are comprised in first and second polypeptide chains, respectively, and the first TCR domain comprises a first TCR transmembrane domain from a first TCR subunit, and the second TCR domain comprises a second TCR transmembrane domain from a second TCR subunit. In some embodiments, the first TCR subunit is a TCR α chain and the second TCR subunit is a TCR β chain. In other embodiments, the first TCR subunit is a TCR β chain and the second TCR subunit is a TCR α chain. In some embodiments, the first TCR subunit is a TCR γ chain and the second TCR subunit is a TCR δ chain. In other embodiments, the first TCR subunit is a TCR δ chain and the second TCR subunit is a TCR γ chain. The TCR transmembrane domains from the TCR subunits may be native TCR transmembrane domains, natural or engineered variants thereof, or fragments of native or variant TCR transmembrane domains. In some embodiments, the first and / or second TCR transmembrane domains individually comprise the amino acid sequence of a TCR transmembrane domain contained in one of SEQ ID NOS: 77-80 of WO 2017 / 070608, which is incorporated by reference in its entirety. In other embodiments, the first and / or second TCR transmembrane domains individually comprise the amino acid sequence of SEQ ID NOS: 1-4 of WO 2017 / 070608.

[0347] In some embodiments, in addition to the first and second TCR transmembrane domains, the first and second TCR domains also comprise first and second connecting peptides, respectively. The first and second connecting peptides are located at the N-termini of the first and second TCR transmembrane domains, respectively. In some embodiments, the first connecting peptide comprises all or a portion of the connecting peptide of a first TCR subunit, and / or the second connecting peptide comprises all or a portion of the connecting peptide of a second TCR subunit. In some embodiments, the first transmembrane domain and the first connecting peptide are derived from different TCR subunits, and / or the second transmembrane domain and the second connecting peptide are derived from different TCR subunits. The connecting peptides from the TCR subunits may be native TCR connecting peptides, natural or engineered variants thereof, or fragments of native or variant TCR connecting peptides. In some embodiments, the first and / or second connecting peptides individually comprise the amino acid sequence of a connecting peptide contained in one of SEQ ID NOS: 77-80 of WO 2017 / 070608. In other embodiments, the first and / or second connecting peptides individually comprise the amino acid sequence of SEQ ID NOS: 5-12 of WO 2017 / 070608.

[0348] In some embodiments, the first and second TCR domains comprise first and second TCR constant domains, respectively. The first and second TCR constant domains are located C-terminal to the first and second TCR transmembrane domains, respectively. When the first and / or second TCR domains comprise a TCR-connecting peptide, the TCR constant domain may be located C-terminal to the TCR-connecting peptide. In some embodiments, the first TCR constant domain comprises all or a portion of the constant domain of a first TCR subunit, and / or the second TCR constant domain comprises all or a portion of the constant domain of a second TCR subunit. For example, in some embodiments, the first and / or second TCR constant domains are derived from TCR α and β subunit constant domains, or TCR γ and δ subunit constant domains. The TCR constant domains from the TCR subunits may be native TCR internal constant cell domains, natural or engineered variants thereof, or fragments of native or variant TCR constant domains. In some embodiments, the first and / or second TCR constant domains individually comprise the amino acid sequence of SEQ ID NO: 172, 174, 176, 178, 180, or 182, or their wild-type equivalents.

[0349] In some embodiments, the first and second TCR domains comprise first and second TCR intracellular domains, respectively. The first and second TCR intracellular domains are located C-terminal to the first and second TCR transmembrane domains, respectively. In some embodiments, the first TCR intracellular domain comprises all or a portion of the intracellular domain of a first TCR subunit, and / or the second TCR intracellular domain comprises all or a portion of the intracellular domain of a second TCR subunit. The TCR intracellular domain from a TCR subunit may be a native TCR intracellular domain, a natural or engineered variant thereof, or a fragment of a native or variant TCR intracellular domain. In some embodiments, the first and / or second TCR intracellular domains individually comprise the amino acid sequence of a TCR intracellular domain contained in one of SEQ ID NOs: 77-80 of WO 2017 / 070608. In other embodiments, the first and / or second TCR intracellular domains individually comprise the amino acid sequences of SEQ ID NOs: 13-14 of WO 2017 / 070608.

[0350] In some embodiments, the first polypeptide chain of the chimeric TCR further comprises a first accessory intracellular domain C-terminal to the first TCR transmembrane domain, and / or the second polypeptide chain of the chimeric TCR further comprises a second accessory intracellular domain C-terminal to the second transmembrane domain. In some embodiments, the first and / or second accessory intracellular domain comprises a TCR costimulatory domain. In some embodiments, the TCR costimulatory domain comprises all or part of the amino acid sequence of SEQ ID NO: 70 or 71 of WO 2017 / 070608.

[0351] In some embodiments, the first TCR domain is a fragment of a first TCR subunit and / or the second TCR subunit is a fragment of a second TCR subunit.

[0352] The first and second polypeptide chains forming the chimeric TCR are linked. In some embodiments, the first and second polypeptide chains forming the chimeric TCR are linked by a disulfide bond. In some embodiments, the first and second polypeptide chains forming the chimeric TCR are linked by a disulfide bond between a residue in the first connecting peptide and a residue in the second connecting peptide.

[0353] In some embodiments, the first and second polypeptide chains are linked or otherwise associated. In some embodiments, the associated first and second polypeptide chains are capable of recruiting at least one TCR-associated signaling module, such as CD3δε, CD3γε, and ζζ. In certain embodiments, the associated first and second polypeptide chains are capable of recruiting each of CD3δε, CD3γε, and ζζ to form a TCR-CD3 complex.

[0354] In some embodiments, the first polypeptide chain comprises a first linker between the first TCR domain and the anti-glyco-cMET VH or VL of the scFv, Fv, or Fab fragment comprised in the first polypeptide chain. In some embodiments, the second polypeptide chain comprises a second linker between the second TCR domain and the anti-glyco-cMET VH or VL of the scFv, Fv, or Fab fragment comprised in the second polypeptide chain. In some embodiments, the first peptide linker and / or the second peptide linker comprise about 5 to about 70 amino acids. In some embodiments, the first and / or second linker comprises a constant domain or fragment thereof from an immunoglobulin or T cell receptor subunit. In some embodiments, the first and / or second linker comprises an immunoglobulin constant domain or fragment thereof. For example, in embodiments comprising a CH1 or CL domain described above, the CH1 or CL domain functions as a linker between the TCR domain and the anti-glyco-cMET binding fragment, or a subpart thereof (e.g., VH or VL). The immunoglobulin constant domain may also be a CH2, CH3, or CH4 domain or fragment thereof in addition to CH1 or CL. The immunoglobulin constant domain may be derived from an IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain. In some embodiments, the constant domain may be derived from a human (e.g., IgG1, IgG2, IgG3, or IgG4), IgA (e.g., IgA1 or IgA2), IgD, IgM, or IgE heavy chain. In other embodiments, the above-described TCR constant domain or fragment thereof functions as a linker between the TCR domain and the anti-glyco-cMET-binding fragment, or a subpart thereof (e.g., VH or VL). In some embodiments, the first and second linkers are capable of binding to each other.

[0355] In some embodiments, the first and second polypeptide chains are at least temporarily connected by a cleavable peptide linker. In some embodiments, the cleavable peptide linker is a furin-p2A cleavable peptide. The cleavable peptide linker can facilitate expression of the two polypeptide chains. The cleavable peptide linker may be designed to temporarily associate the first polypeptide chain with the second polypeptide chain during and / or immediately after protein translation.

[0356] In some embodiments, the chimeric TCR is a synthetic T cell receptor and antigen receptor (STAR), as described in Liu et al., 2021, Sci Transl Med, and WO 2020 / 029774, the contents of each of which are incorporated by reference in their entirety.

[0357] In some embodiments, a STAR comprises, from N-terminus to C-terminus, a first polypeptide chain comprising an anti-glycoed cMET variable heavy chain and a TCR alpha chain constant region domain; a cleavable peptide linker; and a second polypeptide chain comprising an anti-glycoed cMET variable light chain and a TCR beta constant region domain (configuration STAR1).

[0358] In other embodiments, a STAR comprises, from N- to C-terminus, a first polypeptide chain comprising an anti-glycoed cMET variable heavy chain and a TCR β chain constant region domain; a cleavable peptide linker; and a second polypeptide chain comprising an anti-glycoed cMET variable light chain and a TCR α constant region domain (configuration STAR2).

[0359] In other embodiments, a STAR comprises, from N-terminus to C-terminus, a first polypeptide chain comprising an anti-glycoed cMET variable light chain and a TCR alpha chain constant region domain; a cleavable peptide linker; and a second polypeptide chain comprising an anti-glycoed cMET variable heavy chain and a TCR beta constant region domain (configuration STAR3).

[0360] In other embodiments, a STAR comprises, from N-terminus to C-terminus, a first polypeptide chain comprising an anti-glycoed cMET variable light chain and a TCR β chain constant region domain; a cleavable peptide linker; and a second polypeptide chain comprising an anti-glycoed cMET variable heavy chain and a TCR α constant region domain (configuration STAR4).

[0361] In certain embodiments, the TCR alpha and beta chain constant region domains of any one of the configurations STAR1 to STAR4 may be replaced with TCR gamma and TCR delta constant region domains, respectively.

[0362] The chimeric TCRs of the present disclosure can form complexes with TCR-associated signaling molecules (e.g., CD3γε, CD3δε, and ζζ) that are endogenously expressed in T cells. These complexes result in TCR signaling that is regulated by binding of the anti-glyco-cMET variable heavy and light chains by their targets.

[0363] The chimeric TCRs of the present disclosure are further described in numbered embodiments 735 to 834.

[0364] TCR Constant Domains With respect to the TCR constant domains, chimeric TCRs can be designed to include constant regions derived from, for example, human peripheral blood T cells. The nucleotide and corresponding amino acid sequences of TCR constant regions for use in chimeric TCRs according to the present disclosure are provided in Table 5.

[0365] [Table 36-1]

[0366] [Table 36-2]

[0367] In certain embodiments, the TCR constant domains of the chimeric TCR may be modified to provide additional bonds between the two TCR constant domains of the chimeric TCR. In some embodiments, as shown in Table 5, the residue corresponding to position 48 of the wild-type human TCR α constant domain is mutated to cysteine, and the residue corresponding to position 57 of the wild-type human TCR β constant domain is mutated to cysteine. This results in the formation of a disulfide linkage between the TCR α and TCR β constant domains, resulting in a disulfide bond between the first and second polypeptide chains of the chimeric TCR. In some embodiments, as shown in Table 5, the residue corresponding to position 85 of the wild-type human TCR α constant domain is mutated to alanine, and the residue corresponding to position 88 of the wild-type human TCR β constant domain is mutated to glycine. Again, this results in the formation of a disulfide linkage between the TCR α and TCR β constant regions.

[0368] 5.4.2. Cleavable Linkers In some embodiments, the two polypeptide chains of the chimeric TCR of the present disclosure are linked via a cleavable peptide linker. In some embodiments, the two polypeptide chains of the chimeric TCR are linked via a furin-P2A peptide linker, thereby providing a protease cleavage site between the two polypeptide chains. Thus, the two polypeptide chains can be transcribed and translated into a fusion protein, which is then cleaved into separate protein subunits by a protease. In some embodiments, the resulting two protein subunits are covalently linked via a disulfide bond and then form a complex with the T cell's endogenous CD3 subunit.

[0369] In some embodiments, the furin-P2A peptide linker comprises the sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 316).

[0370] In some embodiments, the furin-P2A peptide linker comprises the sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 317).

[0371] 5.5 Neuraminidase Sialic acid, the terminal sugar of glycans on either glycoproteins or glycolipids on the cell surface, has been shown to be aberrantly expressed during tumor transformation and malignant progression. Excessive sialylation frequently occurs in tumor tissues due to the aberrant expression of sialytransferases / sialidases, which may lead to accelerated cancer progression. Sialylation facilitates immune evasion, enhances tumor growth and metastasis, aids tumor angiogenesis, and promotes apoptosis and resistance to cancer therapy.

[0372] Host cells (e.g., T cells, NK cells) expressing the CAR of the present disclosure can be engineered to co-express cell surface or secreted neuraminidase (sialidase) along with the CAR. The cell surface neuraminidase is anchored to the cell surface via a heterologous transmembrane and confers glycoediting activity to the host cell. This enhances the cytotoxicity and antitumor efficacy of CAR-T cells and immune cells, such as innate NK cells and monocytes. Host cells co-expressing a CAR and engineered neuraminidase are described in PCT Publication No. WO 2020 / 236964, the entire contents of which are incorporated herein by reference.

[0373] Neuraminidase can be co-expressed with a CAR described herein in a host cell. Exemplary host cells co-expressing neuraminidase and a CAR are described in specific embodiments.

[0374] Neuraminidase may be included as a domain in the fusion proteins described herein.

[0375] In certain embodiments, the neuraminidase is EC 3.2.1.18 or EC 3.2.1.129.

[0376] In some embodiments, the neuraminidase is derived from Micromonospora viridifaciens.

[0377] In some embodiments, the neuraminidase is

[0378] [ka] and amino acid sequences having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of the present invention.

[0379] Neuraminidase may be carried on the surface of a host cell engineered to express neuraminidase or may be secreted by a host cell engineered to express neuraminidase. Hose cells engineered to express neuraminidase may, for example, contain a vector encoding neuraminidase.

[0380] 5.6 MicA Body The present disclosure provides MicAbodies comprising the anti-glyco-cMET antibodies and antigen-binding fragments of the present disclosure. MicAbodies are fusion proteins comprising an antibody or antigen-binding fragment and an engineered MHC class I chain-related (MIC) protein domain. The MIC protein is a natural ligand for the human NKG2D receptor, which is expressed on the surface of NK cells, and the α1-α2 domain of the MIC protein provides the binding site for the NKG2D receptor. By fusing the engineered MIC protein domain (e.g., the engineered α1-α2 domain) to a cancer-targeting antibody or antigen-binding fragment, T cells expressing an engineered NKG2D receptor capable of binding to the engineered MIC protein domain can be targeted to cancer cells. Engineered MIC protein domains that may be included in the MicA bodies of the present disclosure, as well as NKG2D receptors capable of binding to engineered MIC protein domains, CARs and CAR T cells comprising the NKG2D receptor, are described in U.S. Patent Application Publication Nos. 2011 / 0183893, 2011 / 0311561, 2015 / 0165065, and 2016 / 0304578, and PCT Publication Nos. WO 2016 / 090278, WO 2017 / 024131, WO 2017 / 222556, and WO 2019 / 191243, the contents of which are incorporated herein by reference in their entireties.

[0381] In some embodiments, a MicAbody of the present disclosure comprises an α1-α2 domain that is at least 80% identical or homologous to the α1-α2 domain of an NKG2D ligand (e.g., MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or OMCP). Exemplary amino acid sequences of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and OMCP are set forth as SEQ ID NOs: 1-9, respectively, in WO 2019 / 191243, and these sequences are incorporated herein by reference. In other embodiments, the α1-α2 domain is 85% identical to the native or naturally occurring α1-α2 domain of an NKG2D ligand. In yet other embodiments, the α1-α2 domain is 90% identical to the native or naturally occurring α1-α2 domain of a natural NKG2D ligand protein and binds to a non-native NKG2D.

[0382] In some embodiments, the MicAbodies of the present disclosure comprise an α1-α2 domain at least 80% identical or homologous to the native or naturally occurring α1-α2 domain of a human MICA or MICB protein and bind to NKG2D. In some embodiments, the α1-α2 domain is 85% identical to the native or naturally occurring α1-α2 domain of a human MICA or MICB protein and binds to NKG2D. In other embodiments, the α1-α2 domain is 90%, 95%, 96%, 97%, 98%, or 99% identical to the native or naturally occurring α1-α2 platform domain of a human MICA or MICB protein and binds to NKG2D.

[0383] In some embodiments, specific mutations in the α1-α2 domain of an NKG2D ligand can be engineered to create a non-native α1-α2 domain that binds to a non-native NKG2D receptor that has itself been engineered to have reduced affinity for the native NKG2D ligand. This can be achieved, for example, through genetic engineering. Such modified non-native NKG2D receptors can be used on the surface of NK or T cells of the immune system to create NKG2D-based CARs that preferentially bind to and are activated by molecules composed of the non-native α1-α2 domain. These pairs of non-native NKG2D receptors and their cognate non-native NKG2D ligands can offer important safety, efficacy, and manufacturing advantages for treating cancer and viral infections compared to conventional CAR-T cells and CAR-NK cells. The activation of CAR-T cells and CAR-NK cells bearing NKG2D-based CARs can be controlled by the administration of MicAbodies. In the event of an adverse event, the dosage regimen of MicAbodies can be modified, rather than requiring the activation of an induced suicide mechanism to destroy the infused CAR cells.

[0384] MicA bodies can be generated by attaching an antibody or antigen-binding fragment to an engineered α1-α2 domain via a linker, such as APTSSSGGGGS (SEQ ID NO: 319), GGGS (SEQ ID NO: 320), or GGGGS (SEQ ID NO: 293). For example, the α1-α2 domain may be fused to the C-terminus of an IgG heavy or light chain, as described, for example, in WO 2019 / 191243.

[0385] In some embodiments, the MicA body of the present disclosure comprises the amino acid sequence

[0386] [ka] It contains an engineered α1-α2 domain containing

[0387] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0388] [ka] It contains an engineered α1-α2 domain containing

[0389] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0390] [ka] It contains an engineered α1-α2 domain containing

[0391] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0392] [ka] It contains an engineered α1-α2 domain containing

[0393] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0394] [ka] It contains an engineered α1-α2 domain containing

[0395] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0396] [ka] It contains an engineered α1-α2 domain containing

[0397] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0398] [ka] It contains an engineered α1-α2 domain containing

[0399] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0400] [ka] It contains an engineered α1-α2 domain containing

[0401] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0402] [ka] It contains an engineered α1-α2 domain containing

[0403] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0404] [ka] It contains an engineered α1-α2 domain containing

[0405] In other embodiments, the MicA body of the present disclosure has the amino acid sequence

[0406] [ka] It contains an engineered α1-α2 domain containing

[0407] An exemplary engineered NKG2D receptor has the amino acid sequence

[0408] [ka] wherein the tyrosine at position 73 is replaced with another amino acid, for example, alanine.

[0409] Another exemplary engineered NKG2D receptor has the amino acid sequence

[0410] [ka] wherein the tyrosines at positions 75 and 122 are replaced by another amino acid, e.g., alanine at position 75 and phenylalanine at position 122.

[0411] 5.7 Nucleic Acids, Recombinant Vectors and Host Cells The present disclosure encompasses nucleic acid molecules encoding immunoglobulin light and heavy chain genes of anti-glycated cMET antibodies, vectors containing such nucleic acids, and host cells capable of producing the anti-glycated cMET antibodies of the present disclosure. In certain aspects, the nucleic acid molecules encode, and host cells are capable of expressing, the anti-glycated cMET antibodies and antigen-binding fragments of the present disclosure (e.g., those described in Section 5.1 and numbered embodiments 1-657), as well as fusion proteins (e.g., those described in numbered embodiments 664-668), chimeric antigen receptors (e.g., those described in Section 5.3 and numbered embodiments 689-724), and chimeric TCRs containing them (e.g., those described in Section 5.4 and numbered embodiments 735-834). Exemplary vectors of the present disclosure are described in numbered embodiments 837-839, and exemplary host cells are described in numbered embodiments 840-846.

[0412] The anti-glyco-cMET antibodies of the present disclosure can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in host cells. To recombinantly express an antibody, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, allowing the light and heavy chains to be expressed in the host cells and, optionally, secreted into the medium in which the host cells are cultured, from which the antibody can be recovered. Standard recombinant DNA techniques are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce vectors into host cells, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch, and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989), and U.S. Pat. No. 4,816,397.

[0413] To generate a nucleic acid encoding such an anti-glyco-cMET antibody, first obtain DNA fragments encoding the light and heavy chain variable regions. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding the light and heavy chain variable sequences, for example, using the polymerase chain reaction (PCR). Germline DNA sequences of human heavy and light chain variable region genes are known in the art (see, for example, the "VBASE" human germline sequence database; see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J. Immunol. 24:827-836; the contents of each of which are incorporated herein by reference).

[0414] Anti-glyco-cMET antibody-related V H and V L Once the DNA fragments encoding the segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. H or V L The DNA fragment encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that the two DNA fragments are joined together so that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0415] V HThe isolated DNA encoding the region is V H The DNA encoding the heavy chain constant region (CH1, CH2, CH3, and, optionally, CH4) can be operably linked to another DNA molecule encoding the heavy chain constant region (CH1, CH2, CH3, and, optionally, CH4) to convert it into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but in specific embodiments, is an IgG1 or IgG4 constant region. In the case of a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain CH1 constant region may be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0416] V L The isolated DNA encoding the region is V L The DNA encoding the light chain constant region (CL) can be operably linked to another DNA molecule encoding the light chain constant region, CL, to form a full-length light chain gene (as well as a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but in certain embodiments is a kappa constant region.

[0417] To generate the scFv gene, H and V L The DNA fragment encoding V H and V L The sequence is V H and V L The regions may be operably linked to another fragment encoding a flexible linker, e.g., another fragment encoding the amino acid sequence (Gly4 to Ser)3, so that they can be expressed as a contiguous single-chain protein joined by the flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0418] To express the anti-glyco-cMET antibody of the present disclosure, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0419] The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Prior to insertion of the anti-glyco-cMET antibody-related light or heavy chain sequences, the expression vector may already contain antibody constant region sequences. For example, the V H and V L One approach to converting sequences into full-length antibody genes is to H segment operably linked to a CH segment in a vector, V L The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0420] In addition to the antibody chain genes, the recombinant expression vectors of the disclosure carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. One of ordinary skill in the art will understand that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of host cell to be transformed, the level of expression of protein desired, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high-level protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), promoters and / or enhancers derived from simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), promoters and / or enhancers derived from adenovirus (e.g., adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyoma. For further description of viral regulatory elements and their sequences, see, for example, U.S. Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 by Bell et al., and U.S. Patent No. 4,968,615 by Schaffner et al.

[0421] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (DHFR - The gene encoding the light and heavy chains may include a ribozyme (for use in host cells with methotrexate selection / amplification) and a neo gene (for G418 selection). For expression of the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass various techniques commonly used for introducing exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0422] It is contemplated that the antibodies of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of antibodies, or optimal secretion of properly folded and immunologically active antibodies, is carried out in eukaryotic cells, e.g., mammalian host cells. Exemplary mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells (e.g., DHFR-CHO cells, as described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220). -Examples of suitable host cells include CHO cells (used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells grow. The antibody can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations of the above procedures are within the scope of the present disclosure. For example, it may be desirable to transfect host cells with DNA encoding either the light chain or the heavy chain (but not both) of the anti-glyco-cMET antibody of the present disclosure.

[0423] For expression of a CAR of the present disclosure, the host cell is preferably a T cell, preferably a human T cell, e.g., as described in Section 5.3 and numbered embodiments 689-724. In some embodiments, the host cell exhibits anti-tumor immunity when the cell is crosslinked with cMET on tumor cells. Detailed methods for producing the T cells of the present disclosure are described in Section 5.7.1.

[0424] For expression of the chimeric TCRs of the present disclosure, e.g., as described in Section 5.4 and numbered embodiments 735-834, the host cells are T cells, preferably human T cells. In some embodiments, the host cells exhibit anti-tumor immunity when the cells are crosslinked with glyco-cMET on tumor cells. Detailed methods for producing the T cells of the present disclosure are described in Section 5.7.1.

[0425] Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to glycosylated cMET. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present disclosure.

[0426] For recombinant expression of the anti-glyco-cMET antibodies of the present disclosure, host cells may be co-transfected with two expression vectors of the present disclosure: a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable marker, or they may each contain a different selectable marker. Alternatively, a single vector may be used that encodes both heavy and light chain polypeptides.

[0427] Once a nucleic acid encodes one or more portions of an anti-glyco-cMET antibody, further modifications or mutations can be introduced into the coding sequence to generate, for example, nucleic acids encoding antibodies with different CDR sequences, antibodies with reduced affinity for Fc receptors, or antibodies of a different subclass.

[0428] The anti-glyco-cMET antibodies of the present disclosure can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be generated using cell-free platforms (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17:420-426).

[0429] Once produced by recombinant expression, the anti-glycoed cMET antibodies of the present disclosure can be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential lysis, or any other standard protein purification technique. Additionally, the anti-glycoed cMET antibodies and / or binding fragments of the present disclosure can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0430] Once isolated, the anti-glyco-cMET antibody may be further purified, if desired, by, for example, high performance liquid chromatography (see Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980) or gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

[0431] 5.7.1. Recombinant Production of CARs and Chimeric TCRs in T Cells In some embodiments, the nucleic acid encoding the anti-glyco-cMET CAR or chimeric TCR of the present disclosure is delivered to cells using a retroviral or lentiviral vector. Retroviral and lentiviral vectors expressing CAR or chimeric TCR can be delivered to different types of eukaryotic cells, and even to tissues and whole organisms, using transduced cells as carriers, or using cell-free local or systemic delivery of encapsulated, bound, or naked vectors. The method used can be for any purpose where stable expression is necessary or sufficient.

[0432] In other embodiments, CAR or chimeric TCR sequences are delivered to cells using in vitro transcribed mRNA. In vitro transcribed mRNA CAR or chimeric TCR can be delivered to different types of eukaryotic cells, and even to tissues and whole organisms, using transfected cells as carriers, or using cell-free local or systemic delivery of encapsulated, bound, or naked mRNA. The method used can be for any purpose where transient expression is necessary or sufficient.

[0433] In another embodiment, the desired CAR, or chimeric TCR, can be expressed in the cell by a transposon.

[0434] One advantage of the disclosed RNA transfection method is that RNA transfection is essentially transient and vector-free; that is, the RNA transgene is delivered to lymphocytes and, after a short period of in vitro cell activation, is expressed there as a minimal expression cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cell cloning does not necessarily depend on the transfection efficiency of the RNA or its ability to uniformly modify an entire lymphocyte population.

[0435] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which are currently being tested in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. Preferably, IVT-RNA is stabilized using various modifications to achieve sustained expression of the transfected IVT-RNA.

[0436] It is well known in the literature that some IVT vectors are used in a standardized manner as templates for in vitro transcription and have been genetically modified to produce stabilized RNA transcripts. Currently, protocols used in the industry are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest flanked by untranslated regions (UTRs) at either the 3' and / or 5' ends, and a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette with a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). The polyadenylation cassette therefore corresponds to the late poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is unclear whether this unphysiological overhang affects the amount of protein produced in cells from such constructs.

[0437] RNA has several advantages over more traditional plasmid or viral approaches. Gene expression from RNA sources does not require transcription, and protein products are produced immediately after transfection. Furthermore, because RNA only needs to enter the cytoplasm, not the nucleus, typical transfection methods result in extremely high transfection rates. In addition, plasmid-based approaches require an active promoter to drive expression of the gene of interest in the cells under study.

[0438] In another embodiment, RNA constructs can be delivered to cells by electroporation.See, for example, the formulations and methods of electroporating nucleic acid constructs into mammalian cells as taught in US Patent Application Publication No. 2004 / 0014645, US Patent Application Publication No. 2005 / 0052630, US Patent Application Publication No. 2005 / 0070841, US Patent Application Publication No. 2004 / 0059285, and US Patent Application Publication No. 2004 / 0092907.Various parameters, such as the electric field strength required for electroporation of any known cell type, are generally known in many patents and applications, as well as in the relevant research literature in this field.See, for example, US Patent No. 6,678,556, US Patent No. 7,171,264, and US Patent No. 7,173,116. Devices for therapeutic applications of electroporation, such as the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), are commercially available and are described in patents such as U.S. Pat. Nos. 6,567,694; 6,516,223; 5,993,434; 6,181,964; 6,241,701; and 6,233,482. Electroporation can also be used to transfect cells in vitro, as described, for example, in U.S. Patent Application Publication No. 20070128708. Electroporation can also be used to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, to cells using any of the many available devices and electroporation systems known to those skilled in the art provides an exciting new means for delivering RNA of interest to target cells.

[0439] 5.7.1.1 T cell sources The source of T cells is obtained from a subject before being expanded and genetically modified. The term "subject" is intended to include organisms (e.g., mammals) that can elicit an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human.

[0440] T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, various T cell lines available in the art can be used. In certain embodiments of the present disclosure, T cells can be obtained from a blood unit collected from a subject using various techniques known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place them in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the present disclosure, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, may lack magnesium, or may lack, partially or substantially, divalent cations. Again, surprisingly, an initial activation step in the absence of calcium results in further activation. As would be readily understood by one of ordinary skill in the art, the wash step may be accomplished by methods known to those skilled in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or other salt solutions with or without buffers. Alternatively, unwanted components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0441] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation. Specific subpopulations of T cells, such as CD3 + , CD28', CD4 + , CD8 + , CD45RA + and CD45RO +T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubating with anti-CD3 / anti-CD28 (i.e., 3 x 28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28T, for a period sufficient to positively select the desired T cells. In one embodiment, the period is about 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. In the case of isolating T cells from patients with leukemia, the use of a longer incubation time, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process simply by shortening or lengthening the time T cells are bound to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein). Additionally, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces. Those skilled in the art will recognize that multiple rounds of selection can also be used in the context of the present disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion process.The "unselected" cells can also be subjected to additional rounds of selection.

[0442] Enrichment of T cell populations by negative selection can be achieved by combining antibodies against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers displayed on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, typically CD4 + , CD25 + , CD62L hi , G.I.T.R. + , and FoxP3 + It may be desirable to enrich for or positively select for regulatory T cells that express T-cell receptor 2 (TCR2). Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0443] For isolation of a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) may be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of 2 billion cells per ml is used. In one embodiment, a concentration of 1 billion cells per ml is used. In a further embodiment, greater than 100 million cells per ml are used. In a further embodiment, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells per ml are used. In yet another embodiment, cell concentrations from 75, 80, 85, 90, 95, or 100 million cells per ml are used. In further embodiments, a concentration of 125 million or 150 million cells per ml may be used. The use of a high concentration can result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of a high cell concentration allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or capture from samples containing many tumor cells (i.e., leukemia blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are expected to be desirable to obtain. For example, the use of a high concentration of cells can be advantageous for the capture of CD8 T cells, which typically have relatively weak CD28 expression. + Allows for more efficient selection of T cells.

[0444] In related embodiments, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surfaces (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express large amounts of the desired antigen to be bound to the particles. For example, CD4 + T cells express higher levels of CD28 and, at dilute concentrations, CD8 +In one embodiment, the cell concentration used is 5×10 6 In another embodiment, the concentration used is about 1 x 10 5 / ml to 1 × 10 6 / ml and any integer range therebetween.

[0445] In other embodiments, the cells may be incubated on a rotator for various lengths of time at various speeds at either 2° C.-10° C. or room temperature.

[0446] T cells for stimulation may also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step provides a more homogenous product by removing granulocytes and, to some extent, monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and are expected to be useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1° / min and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can also be used, as well as immediate freezing at -20°C or uncontrolled freezing in liquid nitrogen.

[0447] In certain embodiments, cryopreserved cells are thawed, washed as described herein, and allowed to sit at room temperature for 1 hour prior to activation using the methods of the present disclosure.

[0448] It is also contemplated within the context of the present disclosure that a blood sample or apheresis product may be collected from a subject at a time prior to the time when the expanded cells described herein are expected to be needed. Thus, the source of cells to be expanded can be collected at any time point necessary, and the desired cells, e.g., T cells, can be isolated and frozen for later use in T cell therapy for various diseases or conditions expected to benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the desired cells are isolated and frozen for later use. In certain embodiments, the T cells can be expanded, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient immediately after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, cells are isolated from a blood sample or apheresis from a subject prior to treatment with various relevant treatments, including, but not limited to, natalizumab, efalizumab, antiviral agents, chemotherapeutic agents, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signal transduction (rapamycin). (Liu et al., Cell 66:807-815, 1991;Henderson et al., Immun. 73:316-321, 1991;Bierer et al., Curr. Opin. Immun. 5:763-773, 1993).In a further embodiment, cells are isolated and frozen for individual patients for use after combined (e.g., prior to, concurrent with, or following) bone marrow or stem cell transplantation or T-cell depletion therapy using any of the chemotherapeutic agents, such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide.

[0449] In a further embodiment of the present disclosure, T cells are obtained from a patient immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly those with drugs that damage the immune system, the quality of the obtained T cells may be optimal or improved in terms of their ex vivo expansion capacity immediately after treatment, during the period during which patients are typically expected to recover from treatment. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Thus, within the context of the present disclosure, it is anticipated that blood cells, including T cells, dendritic cells, or other hematopoietic lineage cells, may be collected during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that favor the repopulation, recirculation, regeneration, and / or expansion of specific cell types, particularly during a defined time frame following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other immune system cells.

[0450] 5.7.1.2 T cell activation and proliferation T cells are generally activated and expanded using methods such as those described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Publication No. 20060121005.

[0451] Generally, the T cells of the present disclosure are expanded by contacting them with a surface to which an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the T cell surface are attached. In particular, the T cell population can be stimulated as described herein, for example, by contacting them with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD2 antibody immobilized on the surface, or by contacting them with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. To costimulate accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, the T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. CD4 + T cells or CD8 +To stimulate the proliferation of either T cells, anti-CD3 antibodies and anti-CD28 antibodies, such as 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), can be used, as can other methods generally known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

[0452] In certain embodiments, the primary stimulatory signal and the costimulatory signal for T cells can be provided by various protocols. For example, the agents providing each signal can be in solution or coupled to a surface. If coupled to a surface, the agents can be coupled to the same surface (i.e., in a "cis" configuration) or to separate surfaces (i.e., in a "trans" configuration). Alternatively, one agent can be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then crosslinked to a surface, such as a cell expressing an Fc receptor, or to an antibody or other binding agent that will bind to the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810, regarding artificial antigen-presenting cells (APCs) contemplated for use in activating and expanding T cells in the present disclosure.

[0453] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, and both agents are co-immobilized on the same bead in equivalent molecular amounts. In one embodiment, CD4 + For T cell expansion and T cell proliferation, a 1:1 ratio of each antibody bound to beads is used. In certain embodiments of the present disclosure, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell expansion is observed compared to the increase observed using a 1:1 ratio. In one particular embodiment, an increase of about 1 to about 3-fold is observed compared to the increase observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one embodiment of the present disclosure, more anti-CD28 antibodies than anti-CD3 antibodies are bound to the particles, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the present disclosure, the ratio of anti-CD28 antibodies bound to beads is greater than 2:1. In one particular embodiment, a CD3:CD28 ratio of 1:100 is used. In another embodiment, antibody bound to beads at a CD3:CD28 ratio of 1:75 is used. In a further embodiment, antibody bound to beads at a CD3:CD28 ratio of 1:50 is used. In another embodiment, antibody bound to beads at a CD3:CD28 ratio of 1:30 is used. In a preferred embodiment, antibody bound to beads at a CD3:CD28 ratio of 1:10 is used. In another embodiment, antibody bound to beads at a CD3:CD28 ratio of 1:3 is used. In yet another embodiment, antibody bound to beads at a CD3:CD28 ratio of 3:1 is used.

[0454] Particle to cell ratios of 1:500 to 500:1, and all integer values ​​therebetween, can be used to stimulate T cells or other target cells. As one of ordinary skill in the art will readily appreciate, the particle to cell ratio can depend on the particle size relative to the target cells. For example, small beads can only bind a few cells, while larger beads can bind many cells. In certain embodiments, the cell to particle ratio ranges from 1:100 to 100:1, and all integer values ​​therebetween, and in further embodiments, the ratio includes 1:9 to 9:1, and all integer values ​​therebetween, which can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 coupled particles to T cells at which T cell stimulation occurs can vary as described above, but certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least a 1:1 particle to T cell ratio. In one embodiment, a particle to cell ratio of 1:1 or lower is used. In one particular embodiment, a preferred particle:cell ratio is 1:5. In a further embodiment, the particle to cell ratio can be varied depending on the day of stimulation. For example, in one embodiment, the particle to cell ratio is 1:1 to 10:1 on day 1, and then, up to day 10, additional particles are added to the cells daily or every other day for a final ratio of 1:1 to 1:10 (based on the cell number on the day of addition). In one particular embodiment, the particle to cell ratio is 1:1 on day 1 of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day for a final ratio of 1:1 on day 1 and 1:5 on days 3 and 5 of stimulation. In another embodiment, the particle to cell ratio is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation.In another embodiment, particles are added daily or every other day to a final ratio of 1:1 on day 1 and 1:10 on days 3 and 5 of stimulation. Those skilled in the art will appreciate that various other ratios may be suitable for use in the present disclosure. In particular, the ratio will be expected to vary depending on the particle size and cell size and type.

[0455] In another embodiment of the present disclosure, cells (for example, T cells) are combined with drug-coated beads, then the beads and cells are separated, and then the cells are cultured.In an alternative embodiment, drug-coated beads and cells are not separated before culture, but are cultured together.In another embodiment, the beads and cells are first concentrated by applying force such as magnetic force, which results in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0456] As an example, cell surface proteins can be ligated by contacting T cells with paramagnetic beads (3 x 28 beads) to which anti-CD3 and anti-CD28 are attached. In one embodiment, cells (e.g., 10 4 From 10 9T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a 1:1 ratio) are combined in a buffer, preferably PBS (free of divalent cations such as calcium and magnesium). Again, one of skill in the art will readily appreciate that any cell concentration can be used. For example, the target cells may be very rare in the sample, constituting only 0.01% of the sample, or the entire sample (i.e., 100%) may be comprised of the desired target cells. Thus, any cell number is within the scope of the present disclosure. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the concentration of cells) to ensure maximum contact between the cells and the particles. For example, in one embodiment, a concentration of approximately 2 billion cells per ml is used. In another embodiment, greater than 100 million cells per ml is used. In further embodiments, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per ml are used. In yet another embodiment, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per ml are used. In further embodiments, concentrations of 125 million or 150 million cells per ml may be used. The use of higher concentrations can result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of higher cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and are expected to be desirable in certain embodiments. For example, the use of high cell concentrations may result in CD8 cells that typically have relatively weak CD28 expression. + Allows for more efficient selection of T cells.

[0457] In one embodiment of the present disclosure, the mixture can be cultured for a few hours (about 3 hours) to about 14 days, or any integer value therebetween. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the present disclosure, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may be desirable, allowing the T cells to be cultured for 60 days or longer. Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo15 (Lonza)), which may contain factors necessary for growth and survival, such as serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, X-Vivo 20, and Optimizer, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines sufficient for T cell proliferation and expansion, supplemented with amino acids, sodium pyruvate, and vitamins. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and are not included in the culture of cells to be infused into subjects. Target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0458] T cells exposed to various stimulation times can exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product may exhibit either cytotoxic or suppressor T cell populations (TC , CD8 + ) a larger helper T cell population (T H , CD4 + Ex vivo stimulation of CD3 and CD28 receptors leads to an expansion of T cells, primarily T H After about 8-9 days, the T cell population becomes larger and larger. C Therefore, depending on the purpose of the treatment, it is possible to use a population of cells, primarily T H It may be advantageous to infuse a population of T cells containing T C Once an antigen-specific subset of cells has been isolated, it may be beneficial to further enrich for this subset.

[0459] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers change significantly, but largely reproducibly, during the course of the cell expansion process. Such reproducibility therefore allows for the ability to tailor activated T cell products to specific purposes.

[0460] 5.8 Composition The anti-glyco-cMET antibodies, fusion proteins, and / or anti-glyco-cMET ADCs of the present disclosure may be in the form of a composition comprising the anti-glyco-cMET antibody, fusion protein, and / or ADC and one or more carriers, excipients, and / or diluents. The composition may be formulated for a particular use, e.g., for veterinary use or human pharmaceutical use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and excipients, diluents, and / or carriers used will depend on the intended use of the antibody, fusion protein, and / or ADC, as well as the therapeutic use and mode of administration.

[0461] For therapeutic use, the composition may be provided as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any suitable form (depending on the desired method of administering it to a patient). The pharmaceutical composition can be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical, or local. The most suitable administration route in any given case will depend on the particular antibody and / or ADC, the subject, and the nature and severity of the subject's disease and physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0462] Pharmaceutical compositions can be conveniently provided in unit dosage forms containing a predetermined amount of the anti-glyco-cMET antibody and / or anti-glyco-cMET ADC of the present disclosure per dose. The amount of antibody and / or ADC included in a unit dose will depend on the disease being treated, as well as other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing an amount of antibody and / or ADC suitable for a single administration, or in liquid form. Dry powder unit dosage forms can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. Liquid unit dosages can conveniently be supplied in the form of a syringe pre-filled with an amount of antibody and / or ADC suitable for a single administration.

[0463] Pharmaceutical compositions may also be supplied in bulk form containing an amount of the ADC suitable for multiple administration.

[0464] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing antibodies, fusion proteins, and / or ADCs having the desired purity with any pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as "carriers") typically employed in the art, i.e., buffers, stabilizers, preservatives, tonicity agents, non-ionic detergents, antioxidants, and other compounded additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0465] Buffers help maintain pH in a range close to physiological conditions. Buffers may be present in a variety of concentrations, but will typically be present at a concentration ranging from about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include, for example, citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-disodium fumarate mixtures, fumaric acid-monosodium fumarate mixtures, fumaric acid-monosodium fumarate mixtures, fumaric acid-monosodium citrate mixtures, etc.), and the like. Examples of suitable buffers include organic and inorganic acids and their salts, such as monobasic sodium fumarate-disodium fumarate mixtures, gluconic acid buffers (e.g., gluconic acid-sodium gluconate mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium gluconate mixtures, etc.), oxalic acid buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). In addition, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0466] Preservatives may be added to prevent microbial growth, and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity agents, sometimes known as "stabilizers," can be added to ensure the isotonicity of the liquid compositions of the present disclosure, including polyhydric sugar alcohols, such as sugar alcohols with three or more hydroxyl groups, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, which can vary in function from bulking agents to additives that solubilize the therapeutic agent or help prevent denaturation or adhesion to the container wall.Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol, etc., cyclitols, e.g., inositol; polyethylene glycol; amino acid polymers; e.g., urea, glutathione, thioctic acid, sodium thioglycolate, ... The stabilizer may be a sulfur-containing reducing agent such as monothioglycerol, α-monothioglycerol, and sodium thiosulfate; a low molecular weight polypeptide (e.g., a peptide of 10 or fewer residues); a protein such as human serum albumin, bovine serum albumin, gelatin, or an immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; a monosaccharide such as xylose, mannose, fructose, glucose, or the like; a disaccharide such as lactose, maltose, sucrose, and trehalose; and a trisaccharide such as raffinose; and a polysaccharide such as dextran. The stabilizer may be present in an amount ranging from 0.5 to 10 wt % based on the weight of the ADC.

[0467] In addition to aiding in solubilization of glycoproteins, non-ionic surfactants or detergents (also known as "wetting agents") may be added to protect the glycoproteins from agitation-induced aggregation, thereby allowing the formulation to be subjected to stressful shear surfaces without causing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), and pluronic polyols. The non-ionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, e.g., about 0.07 mg / mL to about 0.2 mg / mL.

[0468] Additional composite excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0469] 5.9 How to use The anti-glyco-cMET antibodies or binding fragments described herein can be used in a variety of diagnostic assays and therapeutic methods. In some embodiments, a patient can be diagnosed with cancer using any of the methods described herein (e.g., as described in Section 5.9.1) and then treated using any of the methods described herein (e.g., as described in Section 5.9.2). The diagnostic methods described herein (e.g., as described in Section 5.9.1) can be used to monitor a patient's cancer status during or after cancer therapy (such as, but not limited to, the cancer therapies described in Section 5.9.2).

[0470] 5.9.1. Diagnostic Methods Anti-glyco-cMET antibodies or binding fragments (including immunoconjugates and labeled antibodies and binding fragments) can be used in diagnostic assays. For example, the antibodies and binding fragments can be employed in immunoassays, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, such as immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), and Western blot.

[0471] The anti-glyco-cMET antibodies or antigen-binding fragments of the present disclosure can be used in methods for detecting biomarkers in samples (e.g., liquid biopsies) containing one or more EVs. In such embodiments, EV surface biomarkers are recognized by the anti-glyco-cMET antibodies or antigen-binding fragments of the present disclosure. Exemplary methods for detecting biomarkers include, but are not limited to, capture assays, immunoassays, such as immunoprecipitation; Western blots; ELISAs; immunohistochemistry; immunocytochemistry; flow cytometry; and immuno-PCR. In some embodiments, the immunoassay may be a chemiluminescent immunoassay. In some embodiments, the immunoassay may be a high-throughput and / or automated immunoassay platform.

[0472] The anti-glyco-cMET antibodies or binding fragments described herein are also useful for in vivo imaging by radiography, in which an antibody labeled with a detectable moiety, such as a radiopaque substance or a radioisotope, is administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed. This imaging technique is useful in the staging and treatment of malignant tumors.

[0473] 5.9.2. Treatment method The anti-glyco-cMET antibodies or binding fragments, fusion proteins, ADCs and CARs, and chimeric TCRs described herein are useful for treating cancers that express glyco-cMET, such as lung cancer, breast cancer, pancreatic cancer, ovarian cancer, bile duct cancer, colon cancer, thyroid cancer, liver cancer, or gastric cancer.

[0474] Thus, the present disclosure provides anti-glyco-cMET antibodies, binding fragments, fusion proteins, ADCs, CARs, and chimeric TCRs described herein for use as medicaments, e.g., for use in the treatment of cancer, e.g., any of the cancers identified in the previous paragraph, for use in diagnostic assays, and for use in in vivo imaging by radiography. The present disclosure further provides the use of anti-glyco-cMET antibodies, binding fragments, fusion proteins, ADCs, CARs, and chimeric TCRs described herein in the manufacture of a medicament, e.g., for the treatment of cancer, e.g., any of the cancers identified in the previous paragraph.

[0475] When a CAR or chimeric TCR of the present disclosure is used in therapy, the treatment methods of the present disclosure include administering to a subject having a tumor that expresses glyco-cMET an effective amount of genetically modified cells engineered to express a CAR or chimeric TCR of the present disclosure, e.g., a CAR described in Section 5.3 or numbered embodiments 689-724, a chimeric TCR described in Section 5.4 or numbered embodiments 735-834, or a MicA body described in Section 5.6. Methods of modifying cells, particularly T cells, to express a CAR or chimeric TCR are described in Section 5.7.1.

[0476] When the MicAbodies of the present disclosure are used in therapy, the treatment methods of the present disclosure include administering to a subject having a tumor that expresses glycosylated cMET a therapeutically effective amount of a MicAbody of the present disclosure, e.g., a MicAbody described in Section 5.6, and genetically modified T cells engineered to express a CAR comprising an NKG2D receptor capable of specifically binding to the MicAbody.

[0477] 5.10 cMET peptide Also provided is an isolated cMET glycopeptide or glyco-cMET peptide, or a fragment thereof, comprising the amino acids PTKSFISGGSTITGVGKNLN (SEQ ID NO: 286). In some embodiments, the cMET glycopeptide is glycosylated with O-linked GalNAc at the serine residue at amino acid position 10 and the threonine residue at amino acid position 11 of PTKSFISGGSTITGVGKNLN (SEQ ID NO: 286). In some embodiments, the cMET glycopeptide comprises the amino acids PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285) or a fragment thereof, having O-linked GalNAc at the serine and threonine residues shown in bold and underlined. Exemplary isolated cMET glycopeptides are set forth in numbered embodiments 894-920.

[0478] The present disclosure encompasses the synthesis of the isolated cMET glycoprotein and recombinant methods for producing the isolated cMET glycoprotein.

[0479] In certain embodiments, the isolated cMET peptide is synthesized using a solid phase peptide synthesis (SPPS) strategy. SPPS methods are known in the art. SPPS provides for the rapid assembly of polypeptides through sequential reactions of amino acid derivatives on a solid support. Successive amino acid derivatives are added to the polypeptide through repeated cycles of alternating N-terminal deprotection and coupling reactions. In other embodiments, the isolated cMET peptide is synthesized using a solution phase peptide synthesis strategy. Solution phase peptide synthesis methods are known in the art.

[0480] To ensure proper O-linked glycosylation with GaINAc at the serine at amino acid position 10 of SEQ ID NO:285 and the threonine at amino acid position 11 of SEQ ID NO:285, presynthesized glycosylated amino acids may be used in the extension reaction.

[0481] Nucleic acid molecules encoding the isolated cMET glycopeptide, vectors containing such nucleic acids, and host cells capable of producing the isolated cMET glycopeptide of the present disclosure are provided. In certain embodiments, the nucleic acid molecule encodes, and the host cell is capable of expressing, a fusion protein comprising the cMET glycopeptide as well as the cMET glycoprotein.

[0482] The isolated cMET glycopeptide of the present disclosure can be prepared by recombinant expression in a host cell. To recombinantly express a cMET glycopeptide, a host cell is transfected with a recombinant expression vector carrying DNA encoding the glycopeptide, such that the glycopeptide is expressed in the host cell and, optionally, secreted into the medium in which the host cell is cultured, from which the glycoprotein can be recovered (i.e., isolated). Standard recombinant DNA techniques are used to obtain the cMET glycoprotein gene, incorporate the gene into a recombinant expression vector, and introduce the vector into a host cell, such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch, and Maniatis (eds), Cold Spring Harbor, NY, 1989), 122 Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989), and U.S. Pat. No. 4,816,397.

[0483] The cMET glycoprotein of the present disclosure can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of the cMET glycoprotein is carried out in eukaryotic cells, such as mammalian host cells. To produce the isolated cMET glycoprotein of the present disclosure, host cells are selected based on their ability to glycosylate the serine at amino acid position 10 of SEQ ID NO: 285 and the threonines at amino acids 10 and 11 of SEQ ID NO: 285. An exemplary host cell is a COSMC HEK293 cell.

[0484] 5.10.1. cMET Peptide Compositions The cMET glycopeptides of the present disclosure may be in the form of a composition comprising a cMET glycopeptide and one or more carriers, excipients, diluents, and / or adjuvants. The composition can be formulated for a specific use, for example, for veterinary use or for human pharmaceutical use. The form of the composition (e.g., dry powder, liquid formulation, etc.) and excipients, diluents, and / or carriers used will depend on the intended use of the cMET glycopeptide, and in the case of therapeutic use, the mode of administration.

[0485] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition that includes a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant. This composition may take any suitable form (depending on the desired method of administering it to a patient). Pharmaceutical compositions can be administered to a patient by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intratumoral, intrathecal, topical, or local. The most suitable route of administration in any given case will depend on the particular cMET glycopeptide to be administered, the subject, and the nature and severity of the disease, as well as the physical condition of the subject. Typically, pharmaceutical compositions will be administered intravenously or subcutaneously.

[0486] The pharmaceutical composition can be conveniently provided in a unit dosage form containing a predetermined amount of the cMET glycopeptide of the present disclosure per dose. The amount of cMET glycopeptide contained in the unit dose will depend on the disease being treated as well as other factors well known in the art. Such unit dosages can be in the form of a lyophilized dry powder containing an amount of cMET glycopeptide suitable for a single administration, or in liquid form. The dry powder unit dosage form can be packaged in a kit with a syringe, a suitable amount of diluent, and / or other components useful for administration. The liquid unit dosage can be conveniently provided in the form of a syringe pre-filled with an amount of cMET glycopeptide suitable for a single administration.

[0487] The pharmaceutical composition may also be supplied in bulk form containing an amount of cMET glycopeptide suitable for multiple administration.

[0488] Pharmaceutical compositions may be prepared for storage as lyophilized formulations or aqueous solutions by mixing cMET glycopeptide having the desired purity with any pharmaceutically acceptable carrier, excipient, adjuvant, or stabilizer (all of which are referred to herein as "carriers") typically employed in the art, i.e., buffers, stabilizers, preservatives, isotonicity agents, non-ionic detergents, antioxidants, and other compounded additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosages and concentrations employed.

[0489] In some embodiments, the composition contains one or more pharmaceutically acceptable adjuvants. Examples of adjuvants include aluminum salts (e.g., amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum)), dsRNA analogs, lipid A analogs, flagellin, imidazoquinoline, CpG ODN, saponins (e.g., QS21), C-type lectin ligands (e.g., TDB), CD1d ligands (α-galactosylceramide), MF59, AS01, AS02, AS03, AS04, AS15, AF03, GLA-SE, IC31, CAF01, and virosomes. Other adjuvants known in the art, including chemical adjuvants, genetic adjuvants, protein adjuvants, and lipid adjuvants, may also be included in the composition.

[0490] Buffering agents help maintain pH in a range close to physiological conditions and may be present in various concentrations, but will typically be present in a concentration ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include, for example, citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.), gluconate buffers (e.g., gluconate-sodium gluconate mixtures, Examples of suitable buffers include organic and inorganic acids and their salts, such as a mixture of oxalic acid and sodium oxalate, a mixture of oxalic acid and sodium hydroxide, a mixture of gluconic acid and potassium gluconate, an oxalic acid buffer (e.g., a mixture of oxalic acid and sodium oxalate, a mixture of oxalic acid and sodium hydroxide, a mixture of oxalic acid and potassium oxalate, an oxalic acid buffer, a lactic acid buffer (e.g., a mixture of lactic acid and sodium lactate, a mixture of lactic acid and sodium hydroxide, a mixture of lactic acid and potassium lactate, an acetic acid buffer, an acetic acid buffer and an acetic acid buffer (e.g., a mixture of acetic acid and sodium acetate, an acetic acid buffer and sodium hydroxide), ...

[0491] Preservatives may be added to prevent microbial growth, and can be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Tonicity agents, sometimes known as "stabilizers," can be added to ensure the isotonicity of the liquid compositions of the present disclosure, including polyhydric sugar alcohols, such as sugar alcohols with three or more hydroxyl groups, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, which can vary in function from bulking agents to additives that solubilize the therapeutic agent or help prevent denaturation or adhesion to the container wall.Typical stabilizers include polyhydric sugar alcohols (as listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inositol, galactitol, glycerol, etc., cyclitols, e.g., inositol; polyethylene glycol; amino acid polymers; e.g., urea, glutathione, thioctic acid, thioglycol; The stabilizer may be a sulfur-containing reducing agent such as sodium phosphate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; a low molecular weight polypeptide (e.g., a peptide of 10 or fewer residues); a protein such as human serum albumin, bovine serum albumin, gelatin, or an immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; a monosaccharide such as xylose, mannose, fructose, or glucose; a disaccharide such as lactose, maltose, sucrose, and trehalose; and a trisaccharide such as raffinose; and a polysaccharide such as dextran. The stabilizer may be present in an amount ranging from 0.5 to 10 wt % based on the weight of the cMET peptide.

[0492] In addition to aiding in solubilization of glycoproteins, non-ionic surfactants or detergents (also known as "wetting agents") may be added to protect the glycoproteins from agitation-induced aggregation, thereby allowing the formulation to be subjected to stressful shear surfaces without causing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), and pluronic polyols. The non-ionic surfactant may be present in a range of about 0.05 mg / mL to about 1.0 mg / mL, e.g., about 0.07 mg / mL to about 0.2 mg / mL.

[0493] Additional composite excipients include bulking agents (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and cosolvents.

[0494] Exemplary cMET peptide compositions of the present disclosure are set forth in numbered embodiments 921 and 922.

[0495] 5.10.2. Methods Using cMET Peptide The cMET peptide described herein can be used in the production of antibodies against tumor-associated forms of cMET. The cMET peptide can be administered to an animal. The amount of peptide administered can be an amount effective to cause the animal to produce antibodies against the peptide. As used herein, "animal" refers to a multicellular eukaryotic organism from the biological kingdom Animalia. In some embodiments, the animal is a mammal. In some embodiments, the animal is a mouse or rabbit. The resulting antibodies can then be collected from the animal. The cMET peptide can be administered as a purified peptide or as part of a composition provided herein.

[0496] The cMET peptides described herein can be used to elicit an immune response against tumor-associated forms of cMET. The cMET peptides can be administered to an animal in an amount effective to cause the animal to mount an immune response (e.g., produce antibodies) against the peptide.

[0497] Exemplary methods for using the cMET peptides of the present disclosure are described in numbered embodiments 923-926.

[0498] 6. Working Example 6.1 Example 1: Identification and Characterization of Anti-Glyco-cMET Antibodies Overview Glycans are essential membrane components, and neoplastic transformation of human cells is virtually always associated with aberrant glycosylation of proteins and lipids. There are numerous types of protein glycosylation, including N-glycosylation and many types of O-glycosylation. One of the most diverse types is mucin-type GalNAc-type O-glycosylation (hereafter referred to as O-glycosylation). Cancer-associated alterations in O-glycans are particularly interesting, and the most frequently observed aberrant glycophenotypes are the expression of the most immature truncated O-glycan structures, designated Tn (GalNAcα1-O-Ser / Thr), STn (NeuAcα2-6GalNAcα1-O-Ser / Thr), and T (Galβ1-3GalNAcα1-O-Ser / Thr) antigens. Truncated O-glycans are observed in almost all epithelial cancer cells and are strongly correlated with poor prognosis. In addition, it is becoming increasingly clear that glycans also play an important role in cancer development, with truncated O-glycans affecting differentiation, cell-cell and cell-matrix interactions, and directly inducing tumorigenic characteristics in susceptible cells.

[0499] The inventors identified cMET glycopeptide epitopes in human cancer cells and used the defined glycopeptides to develop cancer-specific anti-glyco-cMET monoclonal antibodies.

[0500] 6.1.2. Materials and Methods 6.1.2.1 Synthesis of Tn cMET glycopeptide The cMET glycopeptide, PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), with O-linked GalNAc on the serine and threonine residues shown in bold underlined, was synthesized using a standard FMOC peptide synthesis strategy. Presynthesized glycosylated amino acids were coupled to the growing peptide at specific positions using stepwise solid- or solution-phase peptide chemistry. After full sequencing was completed and all protecting groups were removed, the resulting glycopeptide was purified by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry (electrospray ionization in positive mode).

[0501] 6.1.2.2 Synthesis of Recombinant Tn-Glycosylated cMET 1 x 10 in 30 mL of Opti-MEM 6 COSMC KO HEK293 cells were transfected with 30 μg of a plasmid encoding his-tagged human cMET and 60 μL of 293fectin™ transfection reagent (Gibco). After 48 hours of culture, the cells were spun down, and the his-tagged recombinant cMET protein was purified from the supernatant using a 50% Ni-NTA agarose slurry column (Invitrogen) and eluted with 250 mM imidazole. This purification step was repeated to increase purity. The recombinant SC-cMET protein was concentrated in PBS using an Amicon Ultra centrifugal filter.

[0502] 6.1.2.3 Mouse Immunization Protocol Female Balb / c mice were subcutaneously immunized with Tn-glycosylated cMET glycopeptide conjugated to KLH (keyhole limpet hemocyanin) via a maleimide linker. Mice were immunized with 50 μg, 45 μg, and 45 μg of KLH-glycopeptide on days 0, 14, and 35, respectively. The first immunization was in Freund's complete adjuvant. All subsequent immunizations were in Freund's incomplete adjuvant. On day 45, tail bleeds were evaluated for polyclonal responses. On day 56 or later, mice to be fused were boosted with 15 μg of KLH-glycopeptide in Freund's incomplete adjuvant 3–5 days prior to hybridoma fusion. Splenocytes from the mice were fused with SP2 / 0-Ag14 (ATCC, catalog number CRL-1581) myeloma cells using an Electro Cell Manipulator (ECM2001) from BTX Harvard Apparatus. Hybridomas were seeded into 96-well plates, cultured, weighed, evaluated, and selected for specificity to cMET-Tn using ELISA, flow cytometry, and immunofluorescence to obtain monoclonal antibodies with specificity for cMET-Tn.

[0503] 6.1.2.4 Rabbit Immunization Protocol New Zealand White rabbits were immunized with Tn-glycosylated cMET glycopeptide conjugated to KLH via a maleimide linker. Rabbits were immunized with 50–200 μg of KLH-glycopeptide on days 0, 28, and 47. On day 58, blood samples were collected to assess polyclonal responses. On or after day 66, rabbits were boosted with 50–200 μg of KLH-glycopeptide 12 days before peripheral blood collection for B cell collection. B cells were enriched, seeded in 96-well plates, cultured, and assessed for specificity against cMET-Tn using ELISA and flow cytometry. The B cells were cloned, expressed, and screened by ELISA, flow cytometry, and immunofluorescence to obtain monoclonal antibodies specific for cMET-Tn.

[0504] 6.1.2.5 ELISA 96-well Corning high-bind microplates (Fisher) were coated with various concentrations of proteins, peptides, or glycopeptides in 0.2 M bicarbonate-carbonate buffer (pH 9.4) overnight at 4°C. Plates were then blocked with phosphate-buffered saline (PBS) (pH 7.4) containing 2.5% BSA for 1 hour at room temperature. The plate contents were discarded, and purified antibodies, hybridoma supernatants, or serum for polyclonal responses were added at various concentrations and incubated for 2 hours at room temperature. The plates were washed with Tris-buffered saline containing 0.05% Tween-20 and then incubated with a 1:3000 dilution of HRP-conjugated goat anti-mouse IgG Fcγ (Sigma) for 1 hour at room temperature. The plates were washed again and developed with TMB chromogenic substrate. After appropriate color development (approximately 2–3 minutes), the reaction was stopped with 0.2 N H2SO4, and the absorbance was read at 450 nm. Data were analyzed with GraphPad Prism software.

[0505] 6.1.2.6 Flow cytometry Adherent cells were dissociated with TrypLE select (Gibco) and washed off the flask surface with cell culture medium (RPMI w / L-glutamine, 1% PenStrep, 1x Glutamax, and 10% FBS). Cells were washed several times by centrifugation at 300 x g for 5 min at 4°C, and then resuspended in PBS containing 1% BSA (PBS / 1% BSA). Cells were cultured at a concentration of 5 x 10 cells. 5 cells / ml ~ cells 2×10 6The cells were resuspended at 0.25-2 μg / ml and then distributed into 96-well U-bottom plates. For polyclonal responses, diluted commercial antibodies (0.25-2 μg / ml), hybridoma supernatants, or serum were added to the cells and incubated on ice for 1 hour. After several washes with PBS / 1% BSA, the cells were incubated with a 1:1600 dilution of AlexaFluor 647-conjugated F(ab)2 goat anti-mouse IgG Fcγ (Jackson ImmunoResearch) on ice for 30 minutes. The cells were washed again with PBS / 1% BSA and then fixed in 1% formaldehyde in PBS / 1% BSA. The cells were analyzed using either a 2- or 4-laser Attune NXT flow cytometer. Data were processed using FlowJo software.

[0506] 6.1.2.7 Immunofluorescence Cells were seeded to 50% confluence in glass-bottom 96-well plates (Greiner Bio) and incubated for 12–18 hours at 37°C and 5% CO2. After overnight growth, the medium was removed from the slides, and the cells were fixed with 4% formaldehyde in PBS (pH 7.4) for 10 minutes at room temperature. The slides were washed in PBS. For polyclonal responses, diluted commercial antibodies (1–4 μg / ml), hybridoma supernatants, or serum were added to the slides, and the slides were incubated overnight at 4°C. The slides were washed in PBS and stained with a 1:800 dilution of AlexaFluor 488-conjugated F(ab)2 rabbit anti-mouse IgG (H+L) (Invitrogen) for 45 minutes at room temperature. The slides were washed in PBS and incubated with 4 μg / ml DAPI. After removing DAPI and adding PBS, the slides were imaged on a Nikon Ti LTTL microscope.

[0507] 6.1.3.Results 6.1.3.1 Glycopeptide-specific antibodies against Tn-cMET Antibodies reactive with glycopeptides were generated using Tn-glycosylated cMET glycopeptides. Mouse antibodies 15C4, 8H3, and 16E12 and rabbit antibodies 14E9, 19H2, and 39A3 showed excellent selectivity. These six antibodies were carried forward for further characterization.

[0508] 5.1.3.2 Characterization of mAb 14E9, 19H2, and 39A3 Binding Specificity To characterize the binding specificity of 14E9, 19H2, and 39A3, we performed ELISAs against Tn-glycosylated cMET and Tn-glycosylated Syndecan2 peptides. In the context of the ELISA, we found that all three rabbit cMET mAbs reacted exclusively with the Tn-glycosylated cMET peptide (Figure 1).

[0509] 6.2 Example 2: Functional Characterization of 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 Antibodies by Octet and Biacore Overview 15C4, 8H3, and 16E12 were characterized by Biacore to test the reactivity of the anti-CMET mAbs to titrated CMET peptides. 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 were also characterized by Octet to test the reactivity of the anti-CMET mAbs to peptides with different glycosylated residues (including non-glycosylated peptides), as shown in Table 6.

[0510] [Table 37]

[0511] 6.2.2 Materials and Methods 6.2.2.1 Surface plasmon resonance Antibody affinity assays can be performed using surface plasmon resonance (e.g., using a Biacore system (Cytiva)). In a surface plasmon resonance assay, one or more antibodies can be immobilized on a biosensor and presented with an analyte (e.g., cMET-Tn peptide biotin-PTKSFISGGSTITGVGKNLN (the amino acid portion of which is SEQ ID NO: 285; the underlined, bolded residues indicate the GalNAc glycosylation site) or a negative control analyte such as unglycosylated cMET peptide biotin-PTKSFISGGSTITGVGKNLN (the amino acid portion of which is SEQ ID NO: 286)). The antibodies are contacted with various concentrations of analyte, e.g., 2.5 nM, 7.4 nM, 22 nM, 66 nM, and 200 nM. The affinity is measured using a 100-μL NMR spectroscopy (MRI) assay. Multicycle kinetics of 1 minute association and 5 minutes dissociation are measured in triplicate per concentration. When comparing the binding affinity of two antibodies, the same concentration of both antibodies was used (e.g., both antibodies were measured at 1 μM concentration). Affinity is determined by fitting the binding curve to a specific model: kinetic fitting (1:1 model) or, if applicable, a heterogeneous ligand binding model. Errors (>95% confidence) were calculated based on how closely the generated curve matched the model.

[0512] 6.2.2.2 Biolayer Interferometry (Octet) The antibody affinity and epitope binning of monoclonal antibodies can be assessed for specific antigens using biolayer interferometry (BLI). In a BLI assay, the antigen (e.g., cMET-Tn peptide biotin-PTKSFISGGSTITGVGKNLN (amino acid portion of which is SEQ ID NO: 285) or a negative control analyte, such as the nonglycosylated cMET peptide biotin-PTKSFISGGSTITGVGKNLN (amino acid portion of which is SEQ ID NO: 286)) is immobilized on a biosensor and presented to a single antibody for affinity measurements, or to two competing antibodies in tandem (or sequential steps) for epitope binning. Binding to non-overlapping epitopes occurs when saturation with the first antibody does not block binding of the second antibody. Affinity is determined by fitting the binding curve to a specific model: a 1:1 monovalent model or a 2:1 bivalent model. The error (>95% confidence) is calculated based on how closely the generated curve matches the model.

[0513] 6.2.2.1 Flow cytometry Adherent cells were dissociated with TrypLE Select (Gibco) and washed off the flask surface with cell culture medium (RPMI w / L-glutamine, 1% PenStrep, and 10% FBS). Cells were washed several times by centrifugation at 300 × g for 5 min at 4 °C, followed by resuspension in PBS containing 1% BSA (PBS / 1% BSA). Cells were collected at a concentration of 5 × 10 cells. 5 cells / ml ~ cells 2×10 6The cells were resuspended at 0.25-2 μg / ml and then distributed into 96-well U-bottom plates. For polyclonal responses, diluted commercial antibodies (0.25-2 μg / ml), hybridoma supernatants, or serum were added to the cells and incubated on ice for 1 hour. After several washes with PBS / 1% BSA, the cells were incubated with a 1:1600 dilution of AlexaFluor 647-conjugated F(ab)2 goat anti-mouse IgG Fcγ (Jackson ImmunoResearch) on ice for 30 minutes. The cells were washed again with PBS / 1% BSA and then fixed in 1% formaldehyde in PBS / 1% BSA. The cells were analyzed using either a 2- or 4-laser Attune NXT flow cytometer. Data were processed using FlowJo software.

[0514] 6.2.2.2 Immunofluorescence Cells were seeded in glass chamber slides (nunc) to 50% confluence and incubated for 12–18 hours at 37°C and 5% CO2. After overnight growth, the medium was removed from the slides, and the cells were fixed with 4% formaldehyde in PBS (pH 7.4) for 10 minutes at room temperature. The slides were washed in PBS and blocked with PBS / 2% BSA for 1 hour. For polyclonal responses, diluted commercial antibodies (1–4 μg / ml), hybridoma supernatants, or serum were added to the slides, and the slides were incubated overnight at 4°C. The slides were washed in PBS and stained with a 1:800 dilution of AlexaFluor 488-conjugated F(ab)2 rabbit anti-mouse IgG (H+L) (Invitrogen) for 45 minutes at room temperature. The slides were washed in PBS, mounted using Prolong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific), and examined using an Olympus FV3000 confocal microscope.

[0515] 6.2.3.Results 6.2.3.1 Glycopeptide-specific antibodies against Tn-cMET Antibodies reactive with glycopeptides were generated using Tn-glycosylated cMET glycopeptides, including 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3. Antibodies generated using cMET glycopeptides demonstrated excellent selectivity.

[0516] 6.2.3.2 Binding Specificity of mAbs 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 The affinity of 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 for various cMET glycopeptides was determined by Biacore and Octet. Table 7 shows the dissociation constants (K) of 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 for various glycoforms of the cMET peptide, as well as for unglycosylated cMET and MUC1-Tn. d ) is summarized below.

[0517] [Table 38]

[0518] To further evaluate the specificity of 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 in a more native conformation, we used 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 to stain A549 cells for flow cytometry and A549 and T47D cells for immunofluorescence. The T47D and A549 cell lines are inherently Tn-negative but can be induced to express Tn antigen by knockout of the COSMC chaperone. When using 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3 to stain for flow cytometry, we found that each antibody selectively stained COSMC KO A549 cells but not their wild-type counterparts, even though both cells stained positive for CMET (Figures 2A-3B-5). Consistent with these results, immunofluorescence demonstrated that CMET + Tn +T47D COSMC KO and CMET + Tn + Only T47D COSMC KO A549 cells stained with 15C4, 8H3, 16E12, 14E9, 19H2, or 39A3, whereas CMET + Tn - T47D WT cells showed no staining (Figures 4A to 4C).

[0519] 6.3 Example 3: Tissue Expression of Tn Glycosylated CMET Epitopes Recognized by 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 Overview 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 were characterized by immunohistochemistry in a variety of normal and cancer tissues.

[0520] 6.3.2 Materials and Methods Paraffin-embedded tissue microarrays (TMAs) or tissue sections were deparaffinized with xylene and ethanol, followed by antigen retrieval with citrate buffer (pH 6.0) and microwave heating for 18 minutes. TMAs were obtained from USBIOMAX and stained with the UltraVison Quanto Detection System HRP DAB. The TMAs were briefly washed in TBS and incubated with mAb supernatant for 2 hours. After two TBS washes, the TMAs were incubated with Primary Antibody Amplifier Quanto for 10 minutes. After washing in TBS, the TMAs were incubated with HRP polymer quanto (10 minutes), followed by DAB chromogen. Slides were counterstained with hematoxylin, dehydrated, and mounted.

[0521] 6.3.3.Results Immunohistochemistry of formalin-fixed, paraffin-embedded tissue sections revealed positive staining for 15C4, 8H3, 16E12, 4E9, 19H2, and 39A3 in 8 / 8 colon cancers (Figures 5A-5B). 8H3 showed positive cell surface staining in ovarian cancer (17%), pancreatic cancer (13%), lung cancer (14%), and cholangiocarcinoma (11%; Figures 6A-1-6A-2). This staining pattern correlated with normal CMET expression, indicating that CMET expression in these carcinomas predicts reactivity to 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3. Importantly, when 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 were used to stain healthy adjacent tissue, no reactivity was observed on the surface of the cells (Figures 5A-5B and 6B-1-6B-2). 15C4, 8H3, 16E12, 14E9, 19H2, and 39A3 were found to react positively with numerous cancer tissue sections but not with their healthy counterparts.

[0522] The identification of each tissue in the TMA is shown in Tables 8 to 14.

[0523] [Table 39]

[0524] [Table 40]

[0525] [Table 41-1]

[0526] [Table 41-2]

[0527] [Table 41-3]

[0528]

Table 42-1

[0529]

Table 42-2

[0530]

Table 42-3

[0531]

Table 42-4

[0532]

Table 42-5

[0533]

Table 43-1

[0534]

Table 43-2

[0535]

Table 43-3

[0536]

Table 44-1

[0537]

Table 44-2

[0538] [Table 44-3]

[0539] [Table 45-1]

[0540] [Table 45-2]

[0541] [Table 45-3]

[0542] 6.4 Example 4: Tn-CMET-based CAR Overview Chimeric antigen receptors (CARs) containing the VH and VL domains of 15C4, 8H3, and 16E12 were designed and evaluated in target-specific cytotoxicity assays.

[0543] 6.4.2 Materials and Methods 6.4.2.1 Vector design Various CAR constructs were designed with scFvs containing the VH and VL domains of 15C4, 8H3, and 16E12 (Figures 9A-9C). In these constructs, the VH and VL domains are linked to a CD8a hinge, followed by a second-generation CAR-T (CD28 intracellular signaling domain and CD3-zeta intracellular chain) with a single long linker (GGGGS)3 (SEQ ID NO: 346). The N-terminus of the scFv was l...

Claims

1. An anti-glyco-cMET antibody or antigen-binding fragment, VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 3 to 5, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 6 to 8, respectively; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 9 to 11, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 12 to 14; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 15 to 17, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 18 to 20, respectively; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 25-27, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 28-30, respectively; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 31 to 33, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 34 to 36; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 37 to 39, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 40 to 42, respectively; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 47-49, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 50-52, respectively; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 53 to 55, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 56 to 58; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 59 to 61, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 62 to 64; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 69 to 71, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 72 to 74; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 75-77, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 78-80; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 81 to 83, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 84 to 86; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 91 to 93, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 94 to 96; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 97 to 99, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 100 to 102, respectively; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 103 to 105, respectively, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 106 to 108; VH includes CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 113 to 115, and VL includes CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 116 to 118; VH containing CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 119 to 121, respectively, and VL containing CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs. 122 to 124, respectively; or VH contains CDR-H1, CDR-H2, and CDR-H3, each having the amino acid sequences of SEQ ID NOs. 125-127, and VL contains CDR-L1, CDR-L2, and CDR-L3, each having the amino acid sequences of SEQ ID NOs. 128-130. Antiglyco-cMET antibodies or antigen-binding fragments containing these.

2. The anti-glyco-cMET antibody or antigen-binding fragment according to Claim 1, comprising VH, which includes CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 25 to 27, respectively, and VL, which includes CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs. 28 to 30, respectively.

3. The anti-glyco-cMET antibody or antigen-binding fragment according to Claim 1, comprising VH, which includes CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 31 to 33, respectively, and VL, which includes CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs. 34 to 36, respectively.

4. The anti-glyco-cMET antibody or antigen-binding fragment according to Claim 1, comprising VH, which includes CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences of SEQ ID NOs. 37 to 39, respectively, and VL, which includes CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences of SEQ ID NOs. 40 to 42, respectively.

5. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 2, comprising one heavy chain variable (VH) sequence from sequence numbers 264 to 275 and one light chain variable (VL) sequence from sequence numbers 276 to 284.

6. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 2, wherein VH comprises the sequence of SEQ ID NO: 264 and VL comprises the sequence of SEQ ID NO:

276.

7. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 2, wherein VH comprises the sequence of SEQ ID NO: 265 and VL comprises the sequence of SEQ ID NO:

276.

8. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which is a chimeric or humanized antibody, or an antigen-binding fragment of a chimeric or humanized antibody.

9. VH comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and VL comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 2; VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 23, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 24; VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 45, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 46; VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 67, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 68; VH containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 89, and VL containing an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 90; or VH contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 111, and VL contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

112. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, comprising:

10. A heavy chain variable (VH) sequence of Sequence ID No. 1 and a light chain variable (VL) sequence of Sequence ID No. 2; The heavy chain variable (VH) sequence of SEQ ID NO: 23 and the light chain variable (VL) sequence of SEQ ID NO: 24; The heavy chain variable (VH) sequence of sequence number 45 and the light chain variable (VL) sequence of sequence number 46; The heavy chain variable (VH) sequence of SEQ ID NO: 67 and the light chain variable (VL) sequence of SEQ ID NO: 68; The heavy chain variable (VH) sequence of SEQ ID NO: 89 and the light chain variable (VL) sequence of SEQ ID NO: 90; or The heavy chain variable (VH) sequence of SEQ ID NO: 111 and the light chain variable (VL) sequence of SEQ ID NO: 112 The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, comprising:

11. The antiglyco-cMET antibody or antigen-binding fragment according to claim 2, comprising VH having an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 23, and VL having an amino acid sequence having at least 95% sequence identity with SEQ ID NO:

24.

12. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 2, comprising the VH sequence of SEQ ID NO: 23 and the VL sequence of SEQ ID NO:

24.

13. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, which preferentially binds to glyco-cMET epitopes that are overexpressed in cancer cells compared to normal cells.

14. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, which specifically binds to the cMET peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), which is glycosylated with STn at serine and threonine residues indicated in underlined bold.

15. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind to the cMET peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285), which is glycosylated with STn at the serine and threonine residues indicated in underlined bold.

16. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, which binds to the cMET glycopeptide with a binding affinity (KD) of 1 nM to 200 nM as measured by surface plasmon resonance or biolayer interferometry.

17. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind to the non-glycosylated cMET peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 286) ("non-glycosylated cMET peptide").

18. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind to the MUC1 tandem repeat (VTSAPDTRPAPGSTAAPPAHG) 3 (SEQ ID NO: 288) ("first MUC1 glycopeptide") glycosylated in vitro using purified recombinant human glycosyltransferases GalNAc-T1, GalNAc-T2, and GalNAc-T4.

19. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind to the MUC1 peptide TAPPAHGVTSAPDTTRPAPGSTAAPPAHGVT (SEQ ID NO: 289) ("second MUC1 glycopeptide"), which is glycosylated with GalNAc at serine and threonine residues indicated in underlined bold in vitro.

20. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind in vitro to the CD44v6 peptide GYRQTPKEDSHSTTGTAAAA (SEQ ID NO: 345) ("CD44v6 glycopeptide") glycosylated with GalNAc at the threonine and serine residues indicated in underlined bold.

21. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind in vitro to the MUC4 peptide CTIPSTAMHTRSTAAPIPILP (SEQ ID NO: 291) ("MUC4 glycopeptide") glycosylated with GalNAc at serine and threonine residues indicated in underlined bold.

22. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which does not specifically bind to the LAMP1 peptide CEQDRPSPTTAPPAPPPSPSP (SEQ ID NO: 292) ("LAMP1 glycopeptide") glycosylated with GalNAc at serine and threonine residues indicated in underlined bold in vitro.

23. A polyvalent anti-glyco-cMET antibody or antigen-binding fragment according to claim 1.

24. The anti-glyco cMET antibody or antigen-binding fragment according to claim 1, which is an antigen-binding fragment.

25. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 24, wherein the antigen-binding fragment is in the form of a single-chain variable fragment (scFv).

26. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, which is in the form of a multispecific antibody.

27. ​​The anti-glyco-cMET antibody or antigen-binding fragment according to claim 26, wherein the multispecific antibody is a bispecific antibody that binds to a second epitope different from the first epitope.

28. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 27, wherein the bispecific antibody is a bottle opener, mAb-Fv, mAb-scFv, center-scFv, one-arm center-scFv, or bi-scFv bispecific antibody.

29. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 27, wherein the bispecific antibody is a bispecific domain exchange antibody (e.g., CrossMab), a Fab-arm exchange antibody, a bispecific T cell engager (BiTE), or a biaffinity retargeting molecule (DART).

30. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 27, wherein the second epitope is a cMET epitope.

31. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 27, wherein the second epitope is a cMET epitope that is overexpressed in cancer cells compared to normal cells.

32. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 27, wherein the second epitope is a T cell epitope.

33. The anti-glyco-cMET antibody or antigen-binding fragment according to claim 32, wherein the T cell epitope comprises a CD3 epitope, a CD8 epitope, a CD16 epitope, a CD25 epitope, a CD28 epitope, or an NKG2D epitope.

34. A fusion protein comprising the amino acid sequence of the anti-glyco cMET antibody or antigen-binding fragment according to claim 1, operably linked to at least a second amino acid sequence.

35. A chimeric antigen receptor (CAR) comprising one or more antigen-binding fragments as described in Claim 24.

36. A chimeric antigen receptor (CAR) wherein its amino acid sequence comprises the amino acid sequence of SEQ ID NO: 348, SEQ ID NO: 339, SEQ ID NO: 340, or SEQ ID NO:

341.

37. An antibody-drug conjugate comprising the anti-glyco-cMET antibody or antigen-binding fragment according to claim 1, conjugated to a cytotoxic substance.

38. The antigen-binding fragment according to claim 24; A first polypeptide chain comprising a first TCR domain comprising a first TCR transmembrane domain from a first TCR subunit; and A second polypeptide chain containing a second TCR domain, which includes a second TCR transmembrane domain derived from a second TCR subunit. Chimeric T cell receptors (TCRs), including...

39. A nucleic acid comprising an antiglyco-cMET antibody or antigen-binding fragment according to any one of claims 1 to 33, a fusion protein according to claim 34, a CAR according to claim 35 or 36, or a coding region for a chimeric TCR according to claim 38.

40. A vector comprising the nucleic acid described in claim 39.

41. A host cell manipulated to express the nucleic acid described in claim 39.

42. (a) an antiglyco-cMET antibody or antigen-binding fragment according to any one of claims 1 to 33, a fusion protein according to claim 34, a CAR according to claim 35 or 36, an antibody-drug conjugate according to claim 37, or a chimeric TCR according to claim 38, and (b) a physiologically suitable buffer, adjuvant, diluent, or combination thereof.

43. The pharmaceutical composition according to claim 42, wherein the composition is used to treat cancer.

44. The pharmaceutical composition according to claim 43, wherein the cancer is lung cancer, breast cancer, pancreatic cancer, ovarian cancer, bile duct cancer, colon cancer, thyroid cancer, liver cancer, or stomach cancer.

45. A method for detecting cancer in a biological sample, comprising contacting the sample with an anti-glyco-cMET antibody or antigen-binding fragment according to any one of claims 1 to 33, and detecting the binding of the anti-glyco-cMET antibody or antigen-binding fragment.

46. The method according to claim 45, wherein the cancer is lung cancer, breast cancer, pancreatic cancer, ovarian cancer, bile duct cancer, colon cancer, thyroid cancer, liver cancer, or stomach cancer.

47. A cMET peptide comprising PTKSFISGGSTITGVGKNLN (SEQ ID NO: 286) or a peptide of 13 to 30 amino acids in length comprising a fragment thereof comprising amino acids corresponding to amino acids 9 and 10 of PTKSFISGGSTITGVGKNLN (SEQ ID NO: 286).

48. A peptide of 13 to 30 amino acids in length, comprising a fragment of the cMET peptide PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285) or PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285) that is O-glycosylated at serine and threonine residues indicated in underlined bold, and containing amino acids corresponding to amino acids 9 and 10 of PTKSFISGGSTITGVGKNLN (SEQ ID NO: 285).

49. A composition comprising the peptide and adjuvant according to claim 47 or 48.

50. A method for producing antibodies against tumor-associated forms of cMET, comprising administering the composition according to claim 49 to an animal.

51. (a) the nucleic acid according to claim 39, and (b) a pharmaceutical composition comprising a physiologically suitable buffer, adjuvant, diluent, or combination thereof.

52. (a) the vector according to claim 40, and (b) a pharmaceutical composition comprising a physiologically suitable buffer, adjuvant, diluent, or combination thereof.

53. (a) the host cells described in Claim 41, and (b) a pharmaceutical composition comprising a physiologically suitable buffer, adjuvant, diluent, or combination thereof.