Maytansine antibody conjugates and methods of using same - Patents.com

Abstract

An antibody drug conjugate (ADC) is described, which comprises an anti-TM4SF1 antibody, an antigen-binding fragment thereof, a linker, and a drug. The drug is an inhibitor of tubulin polymerization. Methods of using the ADC are also described.

Description

[Technical field]

[0001] 1. Cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 166,793, filed March 26, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] 2.Background technology

[0002] There remains a need for cancer therapeutics, particularly therapeutics with improved therapeutic potential that can cause regression of primary tumors as well as invasive tumor cells and metastases.

[0003]

[0003] Cancer treatments designed to destroy tumor vasculature have failed in clinical trials in the past due to toxicity. Examples include vascular disrupting agents such as combretastatin (CA4P). See, e.g., Grisham et al. Clinical trial experience with CA4P anticancer therapy: focus on efficacy, cardiovascular adverse events, and hypertension management. Gynecol Oncol Res Pract. 2018;5:1. In the Phase II FALCON trial, CA4P reduced overall survival by 16.2 to 13.6 months, and seven patients experienced a heart attack while being treated with CA4P. Id. Coronary heart disease and stroke are the leading causes of death, and any vascular-targeting toxic treatment may pose a risk of fatal toxicity.

[0004]

[0004] TM4SF1 is an endothelial cell marker that has a functional role in angiogenesis. See, for example, Shih et al. The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis. Cancer Res. 2009;69(8):3272-7. Antibody drug conjugates targeting TM4SF1 have been previously explored (see, for example, Visintin et al. Novel Anti-TM4SF1 Antibody-Drug Conjugates with Activity against Tumor Cells and Tumor Vasculature, Mol Cancer Ther 2015(14)(8)1868-1876) so that anti-TM4SF1 antibody drug conjugates fulfill their promise as treatments for solid tumors, but there is a need for TM4SF1-targeting ADCs with reduced toxicity to normal blood vessels, especially arteries. Summary of the Invention [Means for solving the problem]

[0005] 3. Overview of the Invention

[0005] Maytansine, a benzanthamocalolide, is a highly potent microtubule-targeting compound that induces mitotic arrest and kills tumor cells at nanomolar or subnanomolar concentrations. However, maytansine does not exhibit tumor specificity in the clinical setting and exhibits unacceptable systemic toxicity. The potent cell killing potential of maytansine may be used in the treatment of multiple myeloma. Targeted ADC therapy using maytansine with minimal vascular toxicity is needed.

[0006] One aspect of the present disclosure relates to an antibody drug conjugate (ADC) comprising: i) an anti-TM4SF1 antibody or an antigen-binding fragment thereof; and ii) a tubulin polymerization inhibitor. In some embodiments, the inhibitor of tubulin polymerization is maytansine or a derivative thereof. In some embodiments, the inhibitor of tubulin polymerization is maytansine.

[0007] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof and the inhibitor of tubulin polymerization are conjugated by a linker in a one-step or multi-step protocol. In some embodiments, the linker comprises a cleavable linker, a non-cleavable linker, a hydrophilic linker, a pro-charged linker, or a dicarboxylic acid-based linker. In some embodiments, the cleavable linker comprises a cleavable covalent or non-covalent linker. In other embodiments, the linker comprises a non-cleavable covalent or non-covalent linker. In some embodiments, the cleavable linker comprises an acid-labile linker, a protease-sensitive linker, a photolabile linker, or a disulfide-containing linker. In some embodiments, the linker comprises a cysteine ​​linker or a non-cysteine ​​linker. In certain embodiments, the non-cysteine ​​linker comprises a lysine linker.In some embodiments, the linker is selected from the group consisting of MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 5-ethyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclobutyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopentyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclohexyl-4-(pyridin-2-ylthio)butanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), or SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate).In some embodiments, the linker is derived from a cross-linking reagent, which may be N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), 2,5-dioxopyrrolidin-1-yl 3-cyclopropyl-3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 3-cyclobutyl-3-(pyridin-2-yldisulfanyl)propanoate, N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), 2,5-dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-yldisulfanyl)butanoate, 2,5-dioxopyrrolidin-1-yl 4-Cyclobutyl-4-(pyridin-2-yldisulfanyl)butanoate, N-Succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), 2,5-Dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-yldisulfanyl)butanoate, 2,5-Dioxopyrrolidin-1-yl 4-cyclobutyl-4-(pyridin-2-yldisulfanyl)butanoate, N-Succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (Sulfo-SPDB), N-Succinimidyl Iodoacetate (SIA), N-Succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), Maleimide PEG NHS, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC), or 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate (CX1-1).

[0008]

[0008] In some embodiments, the linker comprises a non-cleavable covalent linker. In some embodiments, the non-cleavable covalent linker is attached to the N-terminus, C-terminus or internal amino acid position of the anti-TM4SF1 antibody or antigen-binding fragment thereof. In some embodiments, the non-cleavable covalent linker is covalently attached to an inhibitor of tubulin polymerization. In some embodiments, the non-cleavable covalent linker is covalently attached to a heteroatom, including N, O, or S, or a carbon atom of the inhibitor of tubulin polymerization.

[0009] In some embodiments, the non-cleavable covalent linker is a C1-C6 alkylene, alkenylene, cycloalkylene having 3-7 ring members, alkynylene, arylene, heteroarylene, heterocyclene having 5-12 ring members containing 1-3 atoms of N, O or S, -O-, -NH-, -S-, -N(C 1~6 In some embodiments, the non-cleavable linker is selected from the group consisting of C1-C6 alkylene, alkenylene, cycloalkylene having 3-7 ring members, arylene, heteroarylene, and heterocyclene having 5-12 ring members containing 1-3 atoms of N, O, or S, which are unsubstituted or substituted with halide, amino, -CF3, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkoxy, C1-C3 alkoxy, or C1-C3 alkylthio. In some embodiments, the non-cleavable linker is selected from the group consisting of C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkoxy, C1-C3 alkoxy, and C1-C3 alkylthio.

[0010] [ka]

[0011] (In the formula, the integer m is 0 to 3, the integer q is 0 to 12, and the integer r is 1 to 3.) Includes.

[0012] In some embodiments, the non-cleavable linker is

[0013] [ka]

[0014] wherein each Y and Y is independently a bond, O, S, or NR 100 ;R 100 are independently H, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkyl; 12 Aryl, 5-12 membered heteroaryl, C3-C 12 Cycloalkyl, or a 3- to 12-membered heteroalicyclic group, or R 100 R together with the nitrogen to which it is attached 100 and another atom of the non-cleavable linker, the anti-TM4SF1 antibody or antigen-binding fragment thereof, or the inhibitor of tubulin polymerization combine to form a 3-12 membered heteroalicyclic or 5-12 membered heteroaryl group, which may contain 1-3 additional heteroatoms selected from the group consisting of N, O, and S; the integer m is 0-3, the integer q is 0-12, and the integer r is 1-3. Includes.

[0015] In some embodiments, the antibody drug conjugate has formula (I), a stereoisomer or a pharma- ceutically acceptable salt thereof:

[0016] [ka]

[0017] (In the formula: R 1 is -LL-AA; LL is a linker between the anti-TM4SF1 antibody or antigen-binding fragment thereof and the inhibitor of tubulin polymerization; AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof. has.

[0018] In some embodiments, the antibody drug conjugate has formula (II) or a pharma- ceutically acceptable salt thereof:

[0019] [ka]

[0020] (In the formula: R 1 is -LL-AA; LL is a linker between the anti-TM4SF1 antibody or antigen-binding fragment thereof and the inhibitor of tubulin polymerization; AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof. has.

[0021] In some embodiments, the antibody drug conjugate has formula (III) or a pharma- ceutically acceptable salt thereof:

[0022] [ka]

[0023] (In the formula: The integer n is 1 to 12; Y is MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 5-ethyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclobutyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopentyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclohexyl-4-(pyridin-2-ylthio)butanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), or SIAB (N-succinimidyl(4-iodoacetyl)aminobenzoate); AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof. has.

[0024] In some embodiments, the antibody drug conjugate has formula (IV) or a pharma- ceutically acceptable salt thereof:

[0025] [ka]

[0026] (In the formula: The integer n is 1 to 7; AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof. has.

[0027] In some embodiments, the antibody drug conjugate has formula (V) or a pharma- ceutically acceptable salt thereof:

[0028] [ka]

[0029] (In the formula: AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof. has.

[0030]

[0016] In some embodiments, the antibody drug conjugate comprises a covalent bond between an engineered amino acid residue of the anti-TM4SF1 antibody or antigen-binding fragment thereof and a linker attached to the inhibitor of tubulin polymerization.

[0031]

[0017] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a modified IgG Fc region, the modified IgG Fc region comprises one or more substitutions compared to the wild-type IgG Fc region. In some embodiments, the wild-type IgG Fc region is a wild-type IgG1, IgG2, IgG3, or IgG4 Fc region. In some embodiments, the wild-type Fc region is an IgG1 Fc region, the modified IgG1 Fc region comprises an IgG1 Fc region comprising mutations at one or more positions selected from the group consisting of E233, L234, L235, G237, M252, S254, T250, T256, D265, N297, K322, P331, M428, N434 of the wild-type IgG1 Fc region, as numbered by the EU index as described in Kabat. In some embodiments, the modified IgG1 Fc region further comprises a mutation at position N297. In some embodiments, the mutation at position N297 is a cysteine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position E233. In some embodiments, the mutation at position E233 is a proline. In some embodiments, the modified IgG1 Fc region comprises a mutation at position L234. In some embodiments, the mutation at position L234 is an alanine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position L235. In some embodiments, the mutation at position L235 is an alanine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position G237. In some embodiments, the mutation at position G237 is an alanine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position M252. In some embodiments, the mutation at position M252 is a tyrosine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position S254. In some embodiments, the mutation at position S254 is a threonine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position T256. In some embodiments, the mutation at position T256 is glutamic acid.In some embodiments, the modified IgG1 Fc region comprises a mutation at position M428.In some embodiments, the mutation at position M428 is a leucine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position N434. In some embodiments, the mutation at position N434 is a serine or an alanine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position T250. In some embodiments, the mutation at position T250 is a glutamine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position D265. In some embodiments, the mutation at position D265 is an alanine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position K322. In some embodiments, the mutation at position K322 is an alanine. In some embodiments, the modified IgG1 Fc region comprises a mutation at position P331. In some embodiments, the mutation at position P331 is a glycine.

[0032] In some embodiments, the wild-type Fc region is an IgG4 Fc region and the modified IgG4 Fc region comprises an IgG4 Fc region comprising mutations at one or more positions selected from the group consisting of S228, F234, L235, G237, P238, F243, T250, M252, S254, T256, E258, D259, V264, D265, K288, T299, T307, V308, Q311, K322, L328, P329, A330, P331, T356, K370, A378, R409, V427, M428, H433, N434, H435 of the wild-type IgG4 Fc region, when numbered by the EU index as described in Kabat.

[0033]

[0019] In some embodiments, one or more amino acid residues after position K447 are independently selected from the group consisting of lysine, proline, arginine, or any combination thereof.

[0034]

[0020] In some embodiments, one or more amino acid residues after position K447 are independently selected from the group consisting of lysine and proline.

[0035] In some embodiments, the modified IgG Fc region comprises a mutation at position S228. In some embodiments, the mutation at position S228 comprises S228P.

[0036]

[0022] In some embodiments, the modified IgG4 Fc region comprises a mutation at position F234.

[0037]

[0023] In some embodiments, the mutation at position F234 includes F234A.

[0038]

[0024] In some embodiments, the modified IgG4 Fc region comprises a mutation at position L235.

[0039]

[0025] In some embodiments, the mutation at position L235 includes L235E.

[0040]

[0026] In some embodiments, the mutation at position L235 includes L235E.

[0041]

[0027] In some embodiments, the modified IgG4 Fc region comprises S228P, L235E, and N297C.

[0042]

[0028] In some embodiments, the modified IgG4 Fc region comprises S228P, F234A, L235E, and N297C.

[0043]

[0029] In some embodiments, the modified IgG4 Fc region comprises M428L and N434S.

[0044]

[0030] In some embodiments, the modified IgG4 Fc region comprises mutations at L235 and F234.

[0045]

[0031] In some embodiments, the modified IgG4 Fc region comprises mutations at positions L328, A330, and T299.

[0046]

[0032] In some embodiments, the modified IgG4 Fc region comprises S228P, F234A, L235A, G237A, and P238S.

[0047] In some embodiments, the modified IgG4 Fc region comprises F243A and V264A. In some embodiments, the modified IgG4 Fc region comprises S228P and L235A.

[0048]

[0034] In some embodiments, the modified IgG4 Fc region comprises M252Y and M428L; D259I and V308F; or N434S.

[0049]

[0035] In some embodiments, the modified IgG4 Fc region comprises T307Q and N434S; M428L and V308F; Q311V and N434S; H433K and N434F; E258F and V427T; or T256D, Q311V, and A378V.

[0050] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises: (i) a CDR3 domain comprising an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 96, 118, 119, 120, or 121; a CDR2 domain comprising an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NO: 7, 19, 31, 43, 55, 67, 79, 95, 116, or 117; and a CDR2 domain comprising an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 94, or 115. and (ii) a CDR3 domain comprising an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 14, 26, 38, 50, 62, 74, 86, 110, or 129; a CDR2 domain comprising an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 13, 25, 37, 49, 61, 73, 85, 109, or 128; and a light chain comprising a CDR1 comprising an amino acid sequence having at least 75% identity to a sequence selected from the group consisting of SEQ ID NOs: 12, 24, 36, 48, 60, 72, or 84, 107, 108, 124, 125, 126, or 127. Includes.

[0051]

[0037] In some embodiments, the heavy chain comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 3, 15, 27, 39, 51, 63, 75, 90, 92, 112, 114, 130, or 132, and the light chain comprises an amino acid sequence having at least 75% identity to SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 97, 99, 101, 122, 131, or 133.

[0052]

[0038] In some embodiments, the heavy chain comprises a sequence set forth in SEQ ID NO: 3, 15, 27, 39, 51, 63, 75, 90, 92, 112, 114, 130, or 132, and the light chain variable domain comprises a sequence set forth in SEQ ID NO: 9, 21, 33, 45, 57, 69, 81, 97, 99, 101, 122, 131, or 133.

[0053]

[0039] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:8, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:7, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:6; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:14, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:13, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:12.

[0054]

[0040] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 20, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 19, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 18; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 26, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 25, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 24.

[0055]

[0041] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 32, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 31, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 30; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 38, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 37, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 36.

[0056]

[0042] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 44, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 43, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 42; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 50, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 49, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 48.

[0057]

[0043] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:56, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:55, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:54; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:62, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:61, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:60.

[0058]

[0044] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 68, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 67, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 66; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 74, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 73, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 72.

[0059]

[0045] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 80, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 79, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 78; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 86, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 85, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 84.

[0060]

[0046] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 96, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 95, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 94; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 111 or SEQ ID NO: 110, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 109, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 108.

[0061]

[0047] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 96, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 95, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 94; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 110, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 109, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 107.

[0062]

[0048] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 96, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 95, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 94; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 110, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 109, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 108.

[0063]

[0049] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 96, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 95, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 94; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 111, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 109, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 107.

[0064]

[0050] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 96, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 95, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 94; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 111, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 109, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 108.

[0065]

[0051] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 118, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 116, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 115; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO: 129, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO: 128, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO: 124.

[0066]

[0052] In some embodiments, the heavy chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, or SEQ ID NO:121, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:116 or SEQ ID NO:117, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:115; the light chain comprises a CDR3 domain having the amino acid sequence set forth in SEQ ID NO:129, a CDR2 domain having the amino acid sequence set forth in SEQ ID NO:128, and a CDR1 domain having the amino acid sequence set forth in SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, or SEQ ID NO:127.

[0067]

[0053] In some embodiments, the antigen-binding fragment includes a Fab, Fab', F(ab')2, Fv, VHH, or scFv.

[0068]

[0054] In some embodiments, the inhibitor of tubulin polymerization or a derivative thereof is configured to be rendered ineffective by lysosomal internalization.

[0069]

[0055] In some embodiments, the inhibitor of tubulin polymerization or a derivative thereof is configured to be effective following nuclear translocation.

[0070]

[0056] In some embodiments, the inhibitor of tubulin polymerization or a derivative thereof is configured to be degraded in the lysosome.

[0071]

[0057] In some embodiments, the inhibitor of tubulin polymerization or a derivative thereof is configured to be trapped within a lysosome.

[0072]

[0058] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate of the present disclosure and a pharma ceutically acceptable excipient.

[0073]

[0059] Another aspect of the present disclosure relates to a method for treating or preventing a disease or disorder in a subject, wherein the disease is characterized by abnormal endothelial cell (EC) interactions, the method comprising the step of administering a pharmaceutical composition of the present disclosure to the subject.

[0074]

[0060] In some embodiments, the EC cell interactions include one or more of EC-mesenchymal stem cell, EC-fibroblast, EC-smooth muscle, EC-tumor cell, EC-leukocyte, EC-adipocyte, and EC-neuronal cell interactions.

[0075] In some embodiments, the disease or disorder comprises an inflammatory disease or cancer.

[0076]

[0062] Another aspect of the present disclosure relates to a method of treating or preventing inflammation in a subject, the method comprising the step of administering to the subject a pharmaceutical composition of the present disclosure.

[0077]

[0063] Another aspect of the present disclosure relates to a method of treating or preventing metastasis in a subject, comprising administering to the subject a pharmaceutical composition of the present disclosure, in some embodiments, the subject is in partial or complete remission from cancer.

[0078]

[0064] Another aspect of the present disclosure relates to a method of treating a subject having cancer with a high risk of metastasis, comprising administering a pharmaceutical composition of the present disclosure, hi some embodiments, the subject having cancer with a high risk of metastasis.

[0079]

[0065] Another aspect of the present disclosure relates to a method of treating or preventing metastasis in a subject having cancer, the method comprising the step of administering to the subject having cancer a pharmaceutical composition of the present disclosure.

[0080]

[0066] Yet another aspect of the present disclosure relates to a method of treating or preventing metastasis in a subject, the method being for treating a subject with cancer with high risk of metastasis and for treating or preventing metastasis in a subject with cancer. In some embodiments, the subject is undergoing a treatment that may induce metastasis. In some embodiments, the treatment includes surgery, radiation treatment, or chemotherapy.

[0081]

[0067] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the cancer is a carcinoma or sarcoma. In some embodiments, the carcinoma is breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, liver cancer, gastric cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, or ovarian cancer. In some embodiments, the sarcoma comprises angiosarcoma, osteosarcoma, or soft tissue sarcoma. In some embodiments, the cancer is glioblastoma.

[0082]

[0068] Another aspect of the present disclosure relates to a method of treating or preventing lymphatic or hematogenous metastasis in a human subject, comprising administering to the human subject a pharmaceutical composition according to the present disclosure.

[0083] 4. Incorporation by Reference

[0069] All publications, patents, and patent applications mentioned in this specification are incorporated by reference into this specification to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0084] [Figure 1]

[0070] Figure 1 shows an illustrative example of forming an anti-TM4SF1 antibody-drug conjugate (ADC) by attaching a payload (maytansine) to an anti-TM4SF1 antibody (A07-YTEC) via a linker, resulting in an ADC (A07-YTEC-M conjugate). [Figure 2A] FIG. 2A shows the results of a capillary electrophoresis SDS (CE-SDS) stability test of the A07 antibody with or without mutations. [Figure 2B] FIG. 2B shows the results of a stability test of the A07-YTEC antibody by size exclusion chromatography-HPLC (SEC-HPLC) analysis. [Diagram 3] FIG. 3 shows the SEC-HPLC chromatograms of A07-YTEC and A07-YTEC-M conjugates. [Figure 4] FIG. 4 shows hydrophobic interaction chromatography-HPLC (HIC-HPLC) chromatograms of the A07-YTEC antibody and the A07-YTEC-M conjugate. [Diagram 5]

[0074] FIG. 5 shows the results of reverse-phase HPLC analysis of the non-reduced A07-YTEC antibody and the non-reduced A07-YTEC-M conjugate. [Figure 6] FIG. 6 shows the results of reverse-phase HPLC analysis of the reduced A07-YTEC antibody and the reduced A07-YTEC-M conjugate. [Figure 7]

[0076] Figure 7 shows the deconvoluted LC-MS spectra of the A07-YTEC antibody and the A07-YTEC-M conjugate. Full mass range deconvolution was performed. [Figure 8A] FIG. 8A shows the deconvoluted LC-MS spectrum of the 70 kDa A07-YTEC antibody. [Figure 8B] FIG. 8B shows the deconvoluted LC-MS spectrum of the 150 kDa A07-YTEC antibody. [Figure 8C] FIG. 8C shows the deconvoluted LC-MS spectrum of the A07-YTEC-M conjugate at 70 kDa. [Figure 8D] FIG. 8D shows the deconvoluted LC-MS spectrum of the A07-YTEC-M conjugate at 150 kDa. [Figure 9] FIG. 9 provides deconvoluted LC-MS spectra for estimating the DAR value of the A07-YTEC-M conjugate. [Figure 10] FIG. 10 shows experimental tumor regression analysis of A07 YTEC-M conjugates (6 or 12 mg / kg in one injection) targeting pancreatic (MiaPaca2) and ovarian cancer (SKOV3). [Figure 11A]

[0080] Figures 11A and 11B provide in vivo pharmacokinetic profiles of the ADC in monkeys. Figure 11A shows the plasma concentration-time profiles of the total antibody and the ADC. [Figure 11B] FIG. 11B shows the % ADC across all antibodies in the plasma-time profile. [Figure 12] FIG. 12 provides a schematic of how the payload can be separated from the linker-payload complex in vivo. [Figure 13A]FIG. 13A shows the deconvoluted LC-MS spectrum of the A07-YTEC-M conjugate in plasma on day 4 after injection into cynomolgus monkeys. [Figure 13B] FIG. 13B shows the deconvoluted LC-MS spectrum of the A07-YTEC-M conjugate in plasma 8 days after injection into cynomolgus monkeys. [Figure 13C] Figure 13C shows the deconvoluted LC-MS spectrum of the A07-YTEC-M conjugate in plasma 12 days after injection into cynomolgus monkeys. Peak percentages were calculated based on total peak height. [Figure 14A] FIG. 14A shows electrocardiogram (ECG) diagrams of individual heart rates and individual QRS durations of test subjects. [Figure 14B] FIG. 14B shows electrocardiogram (ECG) diagrams of individual QT intervals and individual corrected QT intervals of test subjects. [Figure 14C] FIG. 14C shows electrocardiogram (ECG) diagrams of individual RR and PR intervals of a test subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] 6. MODE FOR CARRYING OUT THEINVENTION

[0084] Transmembrane-4L6 family member-1 (TM4SF1) is a small membrane glycoprotein with the form of a tetraspanin that is highly expressed on many human epithelial tumor cells and in endothelial cells, particularly in endothelial cells of neovascular or hyperplastic new blood vessels.

[0086]

[0085] In one embodiment, provided herein is an antibody drug conjugate (ADC) for vascular targeting therapy, which can cause primary tumor regression, for example by killing endothelial cells of tumor blood vessels. This therapy can include various attractive features. In particular, (1) angiogenesis is a hallmark of cancer, and therapy that destroys angiogenic blood vessels can be a common treatment for solid tumors; (2) vascular endothelium is an unmutated host system and would not be able to evolve into therapy resistance. Thus, vascular targeting therapy can overcome the common problem associated with tumor cell targeting therapy, where the target tissue evolves and becomes therapy resistant; (3) the tumor's vascular endothelium is directly exposed to intravenously injected drugs, and can therefore be made accessible to drugs that cannot reach tumor cells. Inaccessibility to tumor cells can be a significant problem in cancer, for example pancreatic cancer, which has a dense fibrous stroma that limits the access of drugs to tumor cells. Vascular targeting therapy using ADCs that include anti-TM4SF1 antibodies can advantageously reach the tumor's vascular endothelium.

[0087] In one embodiment, the disclosure provides antibody drug conjugates (ADCs) comprising TM4SF1 binding proteins, such as anti-TM4SF1 antibodies and antigen-binding fragments thereof. The disclosure includes, in some examples, methods of using the ADCs to treat or prevent cancer. The disclosure includes, in some embodiments, an ADC in which the drug payload conjugated to the antibody comprises maytansine.

[0088] 6.1 Definition

[0087] Unless otherwise defined herein, scientific and technical terms used in connection with the disclosure of the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in case of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or non-essential definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is non-limiting.

[0089]

[0088] In general, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art, unless otherwise specified, and as described in the various general and more detailed references cited and discussed herein. Enzymatic reactions and purification techniques are carried out according to detailed manufacturer's instructions, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with, and experimental procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0090]

[0089] In order to make the disclosure of the present invention easier to understand, selected terms are defined below. The term "transmembrane-4L6 family member-1" or "TM4SF1" as used herein refers to a polypeptide of the transmembrane 4 superfamily / tetraspanin family that is highly expressed on tumor vasculature endothelial cells (EC), tumor cells (TC), retinal vasculature and ECs developing angiogenic blood vessels. TM4SF1 has two extracellular loops (ECL1 and ECL2), N- and C-termini, and an intracellular loop (ICL) separated by four transmembrane domains (M1, M2, M3, and M4). ECL2 contains two N-glycosylation sites. The amino acid sequence of human TM4SF1 (hTM4SF1) is set forth in SEQ ID NO: 90 (see also NCBI reference SEQ ID NO: NP_055035.1).

[0091]

[0090] The term "antibody" as used herein means any antigen-binding molecule that contains at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., TM4SF1). The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further divided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more highly conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present disclosure, the FRs of the anti-TMS4F1 antibody (or its antigen-binding site) can be identical to human germline sequences or can be naturally or artificially modified. An amino acid consensus sequence can be defined based on the parallel analysis of two or more CDRs.

[0092]

[0091] The term "YTEC," as used herein, generally refers to an anti-TM4SF1 antibody having modifications in the Fc region (eg, the A07-YTEC antibody).

[0093]

[0092] The term "intact antibody" refers to an antibody that comprises four polypeptide chains, i.e., two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. In one embodiment, the anti-TM4SF1 antibody is an intact antibody. In one embodiment, the intact antibody is an intact human IgG1, IgG2 or IgG4 isotype. In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is a human IgG1, IgG2, or IgG4 isotype.

[0094]

[0093] Terms such as "antigen-binding site" of an antibody, "antigen-binding fragment" of an antibody, or "antibody fragment", as used herein, include any naturally occurring, enzymatically accessible, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding the variable and optionally constant domains of an antibody. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (e.g., phage-antibody libraries, etc.), or can be synthesized. The DNA can be sequenced, chemically, or manipulated by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains in a suitable configuration, or to introduce codons, to create cysteine ​​residues, to modify, add or delete amino acids, etc.

[0095]

[0094] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) a minimal recognition unit consisting of amino acid residues that mimic a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR), such as a CDR3 peptide), or a restrictive FR3-CDR3-FR4 peptide).

[0096]

[0095] The term "variable region" or "variable domain" of an antibody or fragment thereof, as used herein, refers to the portion of the light and heavy chains of an antibody molecule, including the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR-1, CDR-2, and CDR-3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. According to the method used in this disclosure, the amino acid positions assigned to the CDRs and FRs can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). The numbering of the amino acids of an antibody or antigen-binding fragment also follows the Kabat numbering.

[0097]

[0096] The term "complementarity determining region" or "CDR" as used herein refers to the complementarity determining region in an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, which are designated CDR1, CDR2 and CDR3 for each of the variable regions. The term "CDR set" as used herein refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991))) provides an unambiguous residue numbering system that is applicable to any variable region of an antibody, as well as providing the exact residue boundaries that define the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Chothia and coworkers (Chothia et al., J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite great diversity at the level of amino acid sequence. These subportions were designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions, sometimes referred to as Chothia CDRs, have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)).Still other CDR boundary definitions may not strictly follow either of the above systems, but are still expected to overlap with the Kabat CDRs, and they may nevertheless be shortened or lengthened in view of predictions or experimental findings that particular residues or groups of residues, or even the CDR as a whole, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use the CDRs defined by Kabat or Chothia.

[0098]

[0097] The term "framework region" (hereinafter FR), as used herein, refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. Common structural features among the variable regions of antibodies or functional fragments thereof are well known in the art. DNA sequences encoding specific antibodies can generally be found according to well-known methods, for example, those described in Kabat et al., 1987 Sequence of Proteins of Immunological Interest, USDepartment of Health and Human Services, Bethesda MD, which is incorporated herein by reference. In addition, general methods for cloning functional variable regions from antibodies can be found in Chaudhary, VK et al., 1990 Proc. Natl. Acad. Sci. USA 87:1066, which is incorporated herein by reference.

[0099]

[0098] The term "Fc region" as used herein is used to define the C-terminal region of an antibody heavy chain, and includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of an antibody heavy chain Fc region may vary, the Fc region of a human IgG heavy chain is often defined as an extension from the amino acid residue at Cys226, or from Pro230, to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system described in Kabat et al.) may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, a composition of intact antibodies may include an antibody population with all K447 residues removed, an antibody population with the K447 residue not removed, and an antibody population with a mixture of antibodies with and without the K447 residue. Additionally, a composition of intact antibodies of the present disclosure may include an antibody population with an extension of residues after the C-terminal lysine, K447.

[0100]

[0099] The term "humanized antibody", as used herein, refers to an antibody or variant, derivative, analog or fragment thereof that immunospecifically binds to an antigen of interest (e.g., human TM4SF1) and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will contain 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 FR regions are those of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, for example, Riechmann et al., 1988, Nature 1999, ... 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. Patent No. 5,565,332.

[0101]

[0100] The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible mutations, e.g., naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be the selection of a unique clone from a plurality of clones, e.g., from a pool of hybridoma clones, phage clones, or recombinant DNA clones. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.

[0102]

[0101] The term "chimeric antibody," as used herein, refers to antibodies (immunoglobulins) which have portions of the heavy and / or light chains which are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as the desired biological activity is exhibited (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

[0103]

[0102] The term "epitope" as used herein refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on the antigen and may have different biological actions. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational epitopes, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments may have specific three-dimensional structural features, and / or specific charge characteristics.

[0104]

[0103] The term "effective amount" as used herein refers to an effective amount at the dosage and time required to achieve the desired result in terms of treating a disease. For example, in treating dry eye disease, an agent (i.e., a compound, pharmaceutical composition, antibody drug conjugate) that reduces, prevents, delays, or inhibits or stops any symptoms of cancer is effective. An effective amount of an agent is not necessary for the treatment of a disease or condition, but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the symptoms of the disease or condition are alleviated. An effective amount may be divided into one, two, or more doses in a suitable form and administered once, twice, or more times over a specified time period.

[0105]

[0104] The terms "payload," "drug payload," "therapeutic molecule," "therapeutic payload," "therapeutic agent," and "therapeutic moiety" are used interchangeably herein and refer to chemical or biological moieties that are conjugated to an anti-TMSF1 antibody or antigen-binding fragment (e.g., an anti-TM4SF1 antibody or antigen-binding fragment disclosed herein), and may include maytansine and its derivatives.

[0106]

[0105] The term "polypeptide" or "peptide" may refer to two or more naturally or non-naturally occurring amino acids joined by a covalent bond (e.g., an amide bond). Polypeptide or peptide, as described herein, includes full-length proteins (e.g., fully processed proteins) as well as short amino acid sequences (e.g., fragments of naturally occurring proteins or synthetic polypeptide fragments). A tetrapeptide has four amino acids covalently joined by a covalent bond.

[0107] The term "substituent" may refer to a group that replaces a second atom or group, such as a hydrogen atom, on any molecule, compound, or moiety. Suitable substituents may include, but are not limited to, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, and cyano groups.

[0108] The term "drug-to-antibody ratio" or "DAR" may refer to the number of drugs (also referred to herein as therapeutic molecules, therapeutic agents, or therapeutic moieties) attached to an anti-TM4SF1 antibody or antigen-binding fragment thereof of an ADC disclosed herein. The DAR of an ADC typically ranges from 1 to 12, although higher loadings, e.g., 16, are possible depending on the number of binding sites on the antibody or the use of multivalent linkages in which multiple drug payloads are attached to one binding site. The term DAR may be used in reference to the number of drug molecules loaded onto an individual antibody, or in addition, may be used in reference to the average or median DAR of a group of ADCs, which reflects the average drug loading. A composition, batch, and / or formulation of multiple ADCs may be characterized by an average DAR. DAR and average DAR may be determined by a variety of conventional means, such as UV spectroscopy, mass spectrometry, ELISA assay, radiometric methods, hydrophobic interaction chromatography (HIC), electrophoresis, and HPLC.

[0109] The term "binding affinity" generally refers to the strength of the total non-covalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). The affinity of a binding molecule X (e.g., an anti-TM4SF1 antibody) to its binding partner Y (e.g., human TM4SF1) can generally be represented by a dissociation constant (KD). Affinity can be measured by common methods known in the art, such as those described herein. Low affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present disclosure. Certain exemplary embodiments include the following. In one embodiment, the "KD" or "KD value" can be measured by an assay known in the art, such as a binding assay. KD can be measured by RIA, for example, using the Fab version of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). KD can also be measured by using surface plasmon resonance assays by FACS, or BIACORE, for example, using BIACORE2000 or BIACORE3000, or by biolayer interferometry, for example, using OCTET QK384 system. In certain embodiments, the KD of anti-TM4SF1 antibodies is determined using standard flow cytometry assays with HUVEC cells. Also, "on rate" or "rate of association" or "association rate" or "kon" and "off rate" or "rate of dissociation" or "dissociation rate" or "koff" can be determined with the same surface plasmon resonance or biolayer interferometry techniques described above, for example, using BIACORE2000 or BIACORE3000, or OCTET QK384 system.

[0110]

[0109] The term "k on " as used herein is intended to refer to the on rate constant for the association of an antibody to an antigen to form an antibody / antigen complex, as known in the art.

[0111]

[0110] The term "koff," as used herein, is intended to refer to the off rate constant for dissociation of an antibody from the antibody / antigen complex, as known in the art.

[0112]

[0111] The terms "inhibition" or "inhibit" as used herein refer to partial (e.g., 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (i.e., 100%) inhibition.

[0113]

[0112] The term "cancer," as used herein, refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0114]

[0113] The term "cancer with high risk of metastasis," as used herein, refers to a cancer associated with at least one factor known to increase the risk that a subject with cancer will develop metastatic cancer. Examples of factors associated with increased risk of metastasis include, but are not limited to, the number of cancerous lymph nodes a subject has at the time of initial cancer diagnosis, tumor size, histological grading, and stage of the cancer at the time of initial diagnosis.

[0115]

[0114] The term "hematogenous metastasis," as used herein, refers to the ability of cancer cells to invade the blood vessel wall and then circulate via the bloodstream (circulating tumor cells) to other sites and tissues in the body.

[0116]

[0115] The term "lymphatic metastasis," as used herein, refers to the ability of cancer cells to enter lymphatic vessels and drain into the bloodstream.

[0117]

[0116] In the context of this disclosure, the term "treat" or "treatment" as used herein means ameliorating, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition. The term "treat cancer" as used herein means inhibiting the proliferation and / or growth of cancer cells. In one embodiment, the compositions and methods described herein are used to treat metastasis in a subject with metastatic cancer.

[0118] The term "preventing cancer" or "cancer prevention" refers to delaying, inhibiting, or preventing the onset of cancer in a mammal in which the occurrence of carcinogenesis or tumorigenesis has not been proven, but in which a predisposition to cancer has been identified, whether determined by, for example, genetic screening or otherwise. The term also encompasses treating a mammal having a pre-malignant condition to halt or reverse the progression of a pre-malignant condition to a malignant condition. Examples of pre-malignant conditions include hyperplasia, dysplasia, and dysplasia. In some embodiments, cancer prevention is used in reference to a subject in remission from cancer.

[0119]

[0118] A variety of cancers, such as malignant or benign and / or primary or secondary, can be treated or prevented by the methods disclosed herein. Examples of such cancers are known to those skilled in the art and are listed in standard textbooks such as the Merck Manual of Diagnosis and Therapy (published by Merck).

[0120]

[0119] The term "subject," as used herein, refers to a mammal (eg, a human).

[0121]

[0120] The term "administering" as used herein refers to a method of giving a dosage of an antibody or fragment thereof, or a composition (e.g., a pharmaceutical composition) to a subject. The method of administration can vary depending on various factors (e.g., the binding protein or pharmaceutical composition being administered and the severity of the condition, disease, or disorder being treated).

[0122]

[0121] The term "effective amount," as used herein, refers to an amount of an antibody or pharmaceutical composition provided herein sufficient to effect a desired outcome.

[0123]

[0122] The terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0124]

[0123] The terms "identity" or "homology" are used interchangeably herein and may be a calculation of "identity", "homology" or "percent homology" between two or more nucleotide or amino acid sequences that can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps may be introduced into the sequence of the first sequence). The nucleotides at corresponding positions can then be compared, and the percent identity between the two sequences can be a function of the number of identical positions that the sequences share (i.e., % homology = number of identical positions / total number of positions x 100). For example, a position in a first sequence may be occupied by the same nucleotide as the corresponding position in a second sequence, in which case the molecules are identical at that position. The percent homology between two sequences can be a function of the number of identical positions that the sequences share, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. In some embodiments, the length of the aligned sequences for comparison purposes can be at least about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 95% of the length of the reference sequence. A BLAST® search can determine the homology between two sequences. The two sequences can be genes, nucleotide sequences, protein sequences, peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. Non-limiting examples of such mathematical algorithms can be described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such an algorithm may be incorporated into the NBLAST and XBLAST programs (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997).When utilizing BLAST and Gapped BLAST programs, any relevant parameters of the respective programs (e.g., NBLAST) can be used. For example, parameters for sequence comparison can be set at score=100, word length=12, or can be altered (e.g., W=5 or W=20). Other examples include the algorithm of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In another embodiment, the percent identity between two amino acid sequences can be achieved, for example, using the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0125]

[0124] The term "manufacturability" as used herein refers to the stability of a particular protein during recombinant expression and purification of that protein. Manufacturability is believed to be due to the inherent properties of the molecule under the conditions of expression and purification. Examples of improved manufacturability characteristics include uniform glycosylation of the protein, increased cell titer, growth and protein expression during recombinant production of the protein, improved purification characteristics, low or no tendency to aggregate, and improved stability, including but not limited to, thermostability and stability at low pH. In some embodiments, TM4SF1 binding proteins are provided that exhibit manufacturability with retention of activity in vitro and in vivo compared to other TM4SF1 antibodies. In some embodiments, humanization of the parent TM4SF1 binding protein by making amino acid substitutions in the CDR or framework regions can confer additional manufacturability benefits.

[0126] In some embodiments, TM4SF1 binding proteins are provided that exhibit improved developability characteristics, such as, but not limited to, improved purification yields after Protein A purification or size exclusion chromatography, improved homogeneity after purification, improved thermal stability, etc. In some cases, the improvement is with respect to the anti-TM4SF1 antibody produced by the hybridoma mouse cell line 8G4-5-13-13F (PTA-120523) as determined by HLA molecule binding.

[0127]

[0126] In some examples, binding affinity is determined by Scatchard analysis, which involves generating a Scatchard plot, which is a plot of the ratio of the concentration of bound ligand to the concentration of bound ligand.

[0128] The term "vascular toxicity" refers to any effect of an anti-TM4SF1 antibody-therapeutic molecule conjugate (also referred to herein as an anti-TM4SF1 ADC or a TM4SF1-targeted ADC) that results in vascular injury, either directly by the antibody or therapeutic molecule acting on antigen-bearing cells, or indirectly by activation of the immune system or resulting inflammation. Such vascular injury may include, but is not limited to, damage or inflammation affecting vascular endothelial cells or underlying smooth muscle cells or pericytes, or the basement membrane of any blood vessel, including the endocardium (inner lining of the heart). Such vascular injury may affect arteries, including major arteries, such as the aorta, elastic arteries (such as the aorta), muscular arteries of various sizes, such as coronary arteries, pulmonary arteries, carotid arteries, arterioles, capillaries, cerebral or renal arteries; venules, veins; or angiogenic blood vessels, including those supplying hair follicles, gastrointestinal tract, and bone marrow. Such vascular injuries may include microvascular dysfunction or damage in the heart, lungs, kidneys, retina, brain, skin, liver, gastrointestinal tract, bone marrow, endocrine glands, testes or ovaries, endometrium, and other target organs, as well as dysfunction of the renal, retinal, or cerebrovascular circulation.

[0129] The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" as used herein refers to the killing of antibody-coated target cells by cytotoxic effector cells through a non-phagocytic process characterized by the release of the contents of cytotoxic granules or by the expression of cell death-inducing molecules. ADCC is initiated through the interaction of target-bound antibodies (belonging to the IgG or IgA or IgE class) with specific Fc receptors (FcR), glycoproteins present on the effector cell surface that bind to the Fc region of immunoglobulins (Ig). Effector cells that mediate ADCC include natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils and dendritic cells. ADCC is a rapid effector mechanism whose efficacy depends on a number of parameters (density and stability of antigens on the surface of target cells; antibody affinity and FcR binding affinity). PBMC-based ADCC assays and natural killer cell-based ADCC assays can be used to detect ADCC. The readout in these assays is endpoint driven (target cell lysis).

[0130] The term "complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (belonging to the appropriate subclass) that binds to its cognate antigen. To assess complement activation, a CDC assay (see, e.g., Gazzano-Santoro et al., 1996, J. Immunol. Methods 202:163) may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with variant Fc regions) and increased or decreased C1q binding capacity have been described (see, e.g., U.S. Pat. No. 6,194,551; WO 1999 / 51642; Idusogie et al., 2000, J. Immunol. 164:4178-84). Antibodies (or fragments) with little to no CDC activity may also be selected for use.

[0131]

[0130] The term "effector function", as used herein, refers to a function contributed by the Fc effector domain of IgG (e.g., the Fc region of an immunoglobulin). Such a function can be achieved, for example, by the Fc effector domain binding to an Fc receptor on an immune cell with phagocyte or lytic activity, or by the Fc effector domain binding to an element of the complement system. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); down-regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0132] The terms "reduce" or "eliminate" as used herein refer to the ability to cause a total decrease, preferably of 20% or more, more preferably of 50% or more, and most preferably of 75%, 85%, 90%, 95% or more. Reduction or elimination may refer to the binding affinity of two molecules, e.g., the binding of an immunoglobulin to a C1q or Fc receptor; or may refer to a symptom of the disorder (e.g., cancer) being treated, e.g., the presence or size of metastases or the size of a primary tumor.

[0133]

[0132] The term "reduced ADCC / CDC function" as used herein refers to a reduction in a specific effector function, e.g., ADCC and / or CDC, of ​​at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to a control (e.g., an antibody having an Fc region that does not contain a mutation).

[0134]

[0133] The term "cytotoxin," as used herein, refers to a molecule that, when released within a cancer cell, is toxic to that cell. Cytotoxins of particular interest in the present invention are tubulysins (e.g., natural tubulysins or their derivatives), auristatins (e.g., monomethylauristatin E and monomethylauristatin F), maytansinoids (e.g., mertansine), and calicheamicins (e.g., calicheamicin gamma).

[0135] For all amino acid positions discussed in this disclosure, in the context of an antibody or antigen-binding fragment thereof, numbering is according to the EU index. "EU index" or "EU index as described in Kabat et al." or "EU numbering scheme" refers to the numbering of the EU antibody (see Edelman et al., 1969; Kabat et al., 1991).

[0136] 6.2 Antibody Drug Conjugates Containing Anti-TM4SF1 Antibodies or Antigen-Binding Fragments With Altered Fc and / or CDR Regions

[0135] One embodiment of the present disclosure provides an antibody drug conjugate (ADC) comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof linked to a therapeutic molecule, where the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a modified Fc region, e.g., a modified IgG region (e.g., IgG1, IgG2, IgG3, IgG4) comprising one or more mutations. In some cases, one or more mutations in the Fc region result in an improvement of the drug comprising such a modified Fc region in areas such as 1) reduced effector function, 2) half-life modulation, 3) stability, and 4) downstream process improvements. In some cases, the modified Fc region may comprise one or more mutations that reduce or eliminate interactions between the antibody and the immune system. Important interactions may include the interaction of the antibody Fc with Fcγ receptors on white blood cells and platelets, and C1q of the complement system, which results in complement-dependent cytotoxicity.

[0137] The present disclosure provides, in some cases, ADCs comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof, e.g., comprising an immunoabolishing mutation in the Fc region, in such cases a modified Fc region, e.g., a modified IgG Fc region. In some embodiments, the modified Fc region comprises a modification at position N297. In some embodiments, the modified Fc region comprises a modified IgG Fc region (e.g., a modified IgG1, IgG2, IgG3, or IgG4 Fc region) comprising one or more mutations at positions E233, L234 or F234, L235, G237, P238, F243, T250, M252, S254, T256, E258, D259, V264, D265, K288, N297, T299, T307, V308, Q311, K322, L328, P329, A330, P331, T356, K370, A378, R409, V427, M428, H433, N434, and H435, or any combination thereof. In some embodiments, the Fc region comprises an extension of residues at its C-terminus such that a positive charge is maintained at the C-terminus (e.g., in some cases, when an anti-TM4SF1 antibody or antigen-binding fragment comprises two heavy chains, at least one heavy chain comprises an extension of residues at the C-terminus). Such an extension of residues may include the addition of one or more amino acids, such as arginine, lysine, proline, or any combination thereof. In some cases, the extended C-terminus of the Fc region results in a reduction in the CDC function of the anti-TM4SF1 antibody or antigen-binding fragment thereof, and ADCs comprising the anti-TM4SF1 antibody or antigen-binding fragment thereof. Such an effect is seen in some cases by the addition of KP residues after K447 of the Fc in IgG1 or IgG4, alone or in combination with other mutations (e.g., K322A, P331G-IgG1).

[0138] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may include an antibody with reduced effector function, comprising one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (see, e.g., U.S. Pat. No. 6,737,056). In some cases, such mutations in the Fc region may include substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, for example, substitutions of residues 265 and 297 to alanine (DANA mutations, i.e., D265A and N297A) (see, e.g., U.S. Pat. No. 7,332,581). In some cases, mutations in the Fc region may include substitutions at one or more of amino acid positions E233, L234, L235, G237, D265, N297, K322, and P331. In some cases, the mutations in the Fc region may include at least one of E233P, L234A, L235A, G237A, D265A, N297A, K322A, and P331G, or any combination thereof. For example, the mutations in the Fc region may include L234A / L235A / G237A (IgG1), or F234A / L235E (IgG4), and an anti-TM4SF1 antibody or antigen-binding fragment containing such mutations may exhibit altered FcgRI interactions.

[0139] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an Fc variant comprising the following mutations: amino acid substitutions at positions M428 and N434 (M428L, N434S) (see, e.g., US9803023). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an Fc variant comprising the following mutations: amino acid substitutions at positions T250 and M428 (T250Q, M428L) (see, e.g., US9803023).

[0140] In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof may contain the mutations D265A and N297A. In some cases, the proline at position 329 (P329) of the wild-type human Fc region may be replaced with glycine or arginine, or an amino acid residue large enough to disrupt the proline sandwich in the Fc / Fcy receptor interface formed between P329 of Fc and tryptophan residues W87 and WHO of FcgRIII (see, e.g., Sondermann et al., Nature 406, 267-273 (20 July 2000)). In further embodiments, the mutations in the Fc region may include one or more amino acid substitutions such as S228P (IgG4), E233P, L234A, L235A, L235E, N297A, N297D, or P331S, and in yet other embodiments, L234A and L235A in a human IgG1 Fc region or S228P and F234A, L235A, or L235E in a human IgG4 Fc region.

[0141] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an Fc variant of a wild-type human IgG Fc region in which P329 of the human IgG Fc region is replaced by glycine, and the Fc variant may comprise a modified Fc region comprising at least two additional amino acid substitutions at L234A and L235A of the human IgG1 Fc region or S228P and L235E of the human IgG4 Fc region, with the residues numbered according to EU numbering (see, e.g., US8969526). Polypeptides comprising the P329G, L234A and L235A substitutions may exhibit reduced affinity to human FcyRIIIA and FcyRIIA for downregulation of ADCC and / or downregulation of ADCP to at least 20% of that induced by a polypeptide comprising a wild-type human IgG Fc region (see, e.g., US8969526).

[0142]

[0141] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an Fc variant comprising a triple mutation: an amino acid substitution at position P329, L234A and L235A mutations (P329 / LALA) (see, for example, US8969526).

[0143]

[0142] Certain anti-TM4SF1 antibodies or antigen-binding fragments of the present disclosure may, in some embodiments, contain mutations that exhibit improved or diminished binding to FcR (see, e.g., US6737056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0144] In some examples, an anti-TM4SF1 antibody or antigen-binding fragment may include an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334. For example, modifications may be made to the Fc region that result in altered (i.e., improved or diminished) C1q binding and / or complement dependent cytotoxicity (CDC), as described in US6194551, WO99 / 51642, and Idusogie et al. (2000) J. Immunol. 164:4178-4184.

[0145] Antibodies with increased half-life and improved binding to neonatal Fc receptor (FcRn). Named for its function for the transfer of maternal IgG to the fetus, FcRn also contributes to protecting antibodies from degradation in lysosomes by trapping them in endosomes and returning them to circulation. (See, for example, Guyer et al., J.Immunol.117:587 (1976) and Kim et al., J.Immunol.24:249 (1994)), as described in US2005 / 0014934. Without being bound to any particular theory, it is believed that antibodies with improved binding to FcRn will detach from TM4SF1, bind to FcRn, and then recycle the ADC back to circulation, thus reducing vascular toxicity. In some embodiments, provided herein are anti-TM4SF1 antibodies or antigen-binding fragments comprising an Fc region with one or more substitutions that enhance FcRn recycling. In some embodiments, provided herein is an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising an Fc region having one or more substitutions that improve binding of the Fc region to FcRn, e.g., a substitution at one or more of positions: 238, 250, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 428, 424, 434, and 435, e.g., a substitution at Fc region residue 434 according to EU numbering (US7371826). For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); US5648260; US5624821; US2005 / 0014934 and WO94 / 29351, which are incorporated by reference in their entireties.

[0146] In some embodiments, the present specification provides an anti-TM4SF1 antibody or antigen-binding fragment thereof with pH-dependent FcRn binding affinity. Without being bound to any particular theory, it is believed that an ADC antibody or antigen-binding fragment thereof with pH-dependent FcRn binding affinity detaches from FcRn at pH>7 and binds to FcRn at pH6. Thus, FcRn in intracellular organelles at acidic pH, such as endosomes, binds such antibodies, transports them back to the cell membrane, and releases them into plasma at pH>7, recycling the antibodies and avoiding lysosomal release of the ADC payload.

[0147] In certain embodiments, provided herein is an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising an Fc region with one or more substitutions that modulate FcRn recycling. In some embodiments, provided herein is an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising one or more substitutions that enhance FcRn binding at acidic pH, e.g., pH 6, and do not affect FcRn binding at neutral or basic pH, e.g., pH 7. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise a substitution at one or more of positions 250, 252, 254, 256, 428, and 434 according to EU numbering. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an Fc variant comprising one or more of the substitutions T250Q, M252Y, S254T, T256E, M428L, and N434S. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an IgG1 Fc variant comprising substitutions T250Q and M428L ("QL mutation"). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an IgG4 Fc variant comprising substitutions T250Q and M428L ("QL mutation"). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an IgG1 Fc variant comprising substitutions M252Y, S254T, and T256E ("YTE mutation"). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an IgG1 Fc variant comprising substitutions M428L and N434S ("LS mutation"). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof may comprise an IgG4 Fc variant comprising substitutions M428L and N434S ("LS mutation").The effect of amino acid substitutions in the Fc region to modulate FcRn recycling is described, for example, in Hamblett et al., Mol. Pharm. 13(7):2387-96 (2016); Dall'Acqua et al., J. Biol. Chem. 281(33):23514-24 (2006), Hinton et al., J. Biol. Chem. 279(8):6213-6 (2003), Hinton et al., J. Immunol., 176(1):346-56 (2006), US20080181887, US7361740, and EP2235059, which are incorporated by reference in their entireties.

[0148] In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, M428L, and N434S. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region comprising one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, M428L, and N434S. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising substitutions T250Q and M428L. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc variant comprising substitutions M252Y, S254T, and T256E. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc variant comprising substitutions M252Y, S254T, and T256E. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc variant comprising substitutions M428L and N434S. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc variant comprising substitutions M428L and N434S.

[0149]

[0148] In certain embodiments, the ADCs disclosed herein exhibit reduced vascular toxicity, reduced lysosomal toxicity, improved efficacy, and / or improved therapeutic margin. In some embodiments, the ADCs disclosed herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a mutated Fc region having increased FcRn binding affinity and increased serum half-life. In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a mutated Fc region has a serum half-life of at least 10 days, at least 15 days, at least 20 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days or more. In some embodiments, In certain embodiments, the ADCs of the present disclosure exhibit reduced vascular toxicity, improved therapeutic margins, or both. In certain embodiments, the ADCs of the present disclosure comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that comprises a mutated Fc region that has reduced or eliminated affinity for Fc ligands involved in promoting effector function, compared to an antibody having the same amino acid sequence as an antibody of the present disclosure but that does not include an addition, substitution, or deletion of at least one amino acid residue in the Fc region (also referred to herein as an "unmodified antibody").

[0150] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises an Fc region comprising at least two mutations that reduce or eliminate ADCC and / or CDC effector function of the antibody or antigen-binding fragment thereof. In a further embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises an Fc region comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more mutations that reduce or eliminate ADCC and / or CDC effector function of the antibody or antigen-binding fragment thereof.

[0151]

[0151] In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region containing one or more mutations selected from the group consisting of E233P, L234V, L234A, L235A, G236delta (deletion), G237A, V263L, N297A, N297D, N297G, N297Q, K322A, A327G, P329A, P329G, P329R, A330S, P331A, P331G, and P331S.

[0152] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region that includes L234A / L235A mutations, with or without a G237A mutation. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region that includes L234A, L235A, and G237A mutations.

[0153]

[0153] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising A327G / A330S / P331S mutations.

[0154]

[0154] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising E233P / L234V / L235A / delta G236 (deletion) mutations that provide reduced binding to FcγRI (also referred to herein as FcgRI), FcγRIIA (also referred to herein as FcgRIIA), FcγRIIIA (also referred to herein as FcgRIIIAI), and reduced ADCC and CDC effector function, e.g., as described in An Z et al., Mabs 2009 Nov-Ec;1(6):572-9, which is incorporated herein by reference in its entirety.

[0155]

[0155] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is of the IgG1 isotype and comprises an Fc region that includes an N297x mutation (wherein x = A, D, G, Q).

[0156]

[0156] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising A327G / A330S / P331S mutations.

[0157]

[0157] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region containing one or more of the following mutations that provide for reduced binding to C1q: K322A, P329A, and P331A, e.g., as described in Canfield & Morrison. J Exp Med (1991) 173(6):1483-91.10.1084, the entire contents of which are incorporated herein by reference.

[0158]

[0158] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region that includes a V263L mutation that provides enhanced binding to FcγRIIB (also referred to herein as FcgRIIB) and enhanced ADCC, e.g., as described in Hezareh et al., J Virol. 2001 Dec;75(24):12161-8, which is incorporated herein by reference in its entirety.

[0159]

[0159] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising an L234A / L235A, G237A or L235E mutation.

[0160]

[0160] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG1 isotype and comprises an Fc region comprising an L234F, an L235E or a P331S mutation.

[0161]

[0161] In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG2 isotype and comprises an Fc region containing one or more mutations selected from the group consisting of V234A, G237A, P238S, H268A or H268Q, V309L, A330S and P331S.

[0162]

[0162] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG2 isotype and comprises an Fc region comprising A330S / P331S mutations.

[0163]

[0163] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG2 isotype and comprises an Fc region comprising A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S or H268Q / V309L / A330S / P331S mutations.

[0164]

[0164] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region containing one or more mutations selected from the group consisting of S228P, E233P, F234A, F234V, L235E, L235A, G236delta (deletion), N297A, N297D, N297G, N297Q, P329G, P329R.

[0165]

[0165] In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region that contains an S228P mutation that provides for reduced Fab-arm exchange and reduced aggregation, e.g., as described in Chappel et al., Proc Natl Acad Sci USA (1991) 88(20):9036-40, which is incorporated by reference in its entirety.

[0166]

[0166] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is of the IgG4 isotype and comprises an Fc region comprising S228P / L235E mutations.

[0167]

[0167] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is of IgG4 isotype and comprises an Fc region comprising S228P / E233P / F234V / L235A / delta G236 (deletion) mutations.

[0168]

[0168] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is of the IgG4 isotype and comprises an Fc region that includes an N297x mutation (wherein x = A, D, G, Q).

[0169]

[0169] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region comprising S228P / F234A / L235A mutations.

[0170]

[0170] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region comprising an L235E mutation that provides reduced binding to FcγRI, FcγRIIA, FcγRIIIA and reduced ADCC and CDC effector activity, e.g., as described in Saxena et al., Front Immunol. 2016 Dec 12;7:580.

[0171]

[0171] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region comprising S228P / F234A / L235A or E233P / L235A / G236 delta mutations.

[0172] In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is of IgG4 isotype and comprises an Fc region containing at least an S228P mutation. Angal et al. (Mol Immunol. 1993 Jan;30(1):105-8) describe the analysis of the hinge sequence of human IgG4 heavy chain to determine that the presence of a serine at residue 241 (according to the EU numbering system, here corresponding to residue 228 in Kabat numbering) is responsible for heterogeneity of inter-heavy chain disulfide bridges in the hinge region in a given percentage of secreted human IgG4. Silva et al. (J Biol Chem. 2015 Feb 27;290(9):5462-9) describe the S228P mutation in human IgG4 that prevents IgG4 Fab-arm exchange in vivo and in vitro.

[0173]

[0173] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 isotype and comprises an Fc region comprising an L235E or S228P mutation.

[0174]

[0174] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 or IgG1 isotype and comprises an Fc region that includes an N297A, N297D or N297G mutation.

[0175]

[0175] In other embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is an IgG4 or IgG1 isotype and comprises an Fc region comprising a P329G, P329R mutation.

[0176] In one exemplary embodiment, the mutated Fc region of any IgG isotype contains one or more mutations at positions 234, 235, 236, 237, 297, 318, 320, 322 (as described in WO1988007089, which is incorporated herein by reference in its entirety). Other possible mutations in the Fc region, such as substitutions, deletions and additions, are also described, for example, in US20140170140, WO2009100309, US20090136494 and US8969526, which are incorporated herein by reference in their entireties.

[0177] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction or abolishment of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody has lost FcγR binding (and thus potentially lost ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, RII and RIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82 (1985) 1499-1502; U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assay methods may be employed (see, e.g., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be measured in vivo, e.g., as described in, for example, Clynes et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656. A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and thus loses CDC activity. See, for example, the C1q and C3c binding ELISAs described in WO2006 / 029879 and WO2005 / 100402.To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202 (1996) 163; Cragg, MS et al., Blood 101 (2003) 1045-1052; and Cragg, MS, and Glennie, MJ, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12) (2006) 1759-1769).

[0178]

[0178] In some embodiments, the mutated Fc region of any IgG isotype includes a mutation at position L328, e.g., L328M, L328D, L328E, L328N, L328Q, L328F, L328I, L328V, L328T, L328H, L328A (see, e.g., US20050054832).

[0179] In one embodiment, the antibodies or antigen-binding fragments thereof of the disclosure exhibit reduced or abolished ADCC effector function compared to the unmodified antibody. In another embodiment, the antibodies or antigen-binding fragments thereof of the disclosure exhibit at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold reduced ADCC effector function compared to the unmodified antibody. In yet another embodiment, the antibodies of the disclosure exhibit at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100% reduced ADCC effector function compared to the unmodified antibody. In further aspects of the disclosure, the reduction or downregulation of ADCC effector function induced by an antibody or antigen-binding fragment thereof of the present disclosure is a 0, 2.5, 5, 10, 20, 50, or 75% reduction of that observed for induction of ADCC by the unmodified antibody. In certain embodiments, the reduction and / or loss of ADCC activity may be due to reduced affinity of the antibody or antigen-binding fragment thereof of the present disclosure for Fc ligands and / or receptors.

[0180] CDR substitutions that modulate pH-dependent TM4SF1 binding of anti-TM4SF1 antibodies or antigen-binding fragments thereof

[0181] One embodiment of the present disclosure provides an ADC comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof linked to a therapeutic molecule or payload, wherein the anti-TM4SF1 antibody or antigen-binding fragment thereof exhibits pH-dependent binding affinity to TM4SF1. In some examples, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to TM4SF1 with higher affinity in a particular pH range compared to other pH ranges. For example, the anti-TM4SF1 antibody or antigen-binding fragment thereof may bind to TM4SF1 with a different affinity at acidic pH than at neutral or basic pH. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to TM4SF1 with higher affinity at acidic pH than at neutral or basic pH. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to TM4SF1 with lower affinity at acidic pH than at neutral or basic pH. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to TM4SF1 at acidic pH and dissociates from TM4SF1 at neutral or basic pH. In some embodiments, the anti-TM4SF1 antibody or its antigen-binding fragment binds to TM4SF1 at pH 7 or higher and releases from TM4SF1 at pH 6 or lower. In intracellular compartments such as plasma, cytosol, and nucleus, pH is neutral or basic. In lysosomes or endosomes, pH is acidic. Without being bound to any particular theory, the anti-TM4SF1 antibody or its antigen-binding fragment binds to antigen and then internalizes in the membrane of endosome. The pH-dependent anti-TM4SF1 antibody or its antigen-binding fragment can release from TM4SF1 in endosome, bind to FcRn receptor in endosome, and can be recycled by FcRn receptor and returned to circulation rather than being degraded in lysosomes where endosomes proceed. Thus, the pH-dependent anti-TM4SF1 antibody or its antigen-binding fragment can bind to TM4SF1 antigen multiple times.Thus, the pH-dependent anti-TM4SF1 antibody and the therapeutic molecule or payload associated therewith can be recycled by the FcRn receptor without releasing the payload in the lysosome.

[0181]

[0182] Target-mediated drug disruption, or TMDD, occurs when an antigen carries bound antibody and / or any associated ADC payload to the lysosome, where the ADC is degraded and the payload is released. Lysosomal toxicity associated with TMDD is described in Grimm et al., J. Pharmacokinet. Pharmacodyn. 36(5):407-20 (2009), which is incorporated herein by reference in its entirety. In some embodiments, provided herein is an ADC comprising an anti-TM4SF1 antibody, or antigen-binding fragment thereof, linked to a therapeutic molecule that exhibits reduced vascular toxicity, increased serum half-life, and / or improved therapeutic margin. In some embodiments, the anti-TM4SF1 antibody, or antigen-binding fragment thereof, comprises one or more histidine amino acid residue substitutions in the CDR residues. Without being bound to any particular theory, the introduction of histidine residues at suitable positions of the anti-TM4SF1 antibody may enable pH-modulated binding affinity to TM4SF1. For example, ADCs with pH-dependent anti-TM4SF1 antibodies can dissociate from TM4SF1 in acidic lysosomal or endosomal environments, and then recycle to circulation via FcRn binding.Compared with other comparable wild-type anti-TM4SF1 antibodies or their antigen-binding fragments, pH-dependent anti-TM4SF1 antibodies can show increased serum half-life and reduced degradation rate or payload release rate in lysosomes.In some cases, ADCs with pH-dependent anti-TM4SF1 antibodies or their antigen-binding fragments can show increased serum half-life, reduced vascular toxicity, improved therapeutic limit, and / or improved or at least approximately equivalent in vivo efficacy.

[0182]

[0183] Disclosed herein are methods of making ADCs comprising anti-TM4SF1 antibodies or antigen-binding fragments thereof with increased half-life and / or pharmacodynamic effects by modulating antibody-TM4SF1 binding affinity in a pH-dependent manner, comprising selecting antibody CDR histidine or other residues that optimize the microenvironment that affects the antibody's pKa, such that the antibody-TM4SF1 binding has a Kd ratio and / or Koff ratio at pH 6.0 / pH 7.4 that is at least 2, 3, 4, 8, 10, 16 or more, or in a range between 2, 3, 4, 8, 10, 16 or more. In some embodiments, the method comprises introducing amino acid substitutions into the anti-TM4SF1 antibody or antigen-binding fragment thereof to achieve TM4SF1 affinity with a KD at pH 7.4 of at least 100 nM as measured at 25° C. In certain embodiments, the method comprises generating an antibody library enriched for histidines in CDR or other residues that optimize the microenvironment that affects the pKa. In some embodiments, the antibody library comprises anti-TM4SF1 antibodies or antigen-binding fragments thereof with histidine residues introduced at CDR positions. In some embodiments, the antibody library comprises a series of anti-TM4SF1 antibodies or antigen-binding fragments thereof, where each anti-TM4SF1 antibody of the antibody library comprises a single histidine substitution at a different CDR position. In some embodiments, the antibody library comprises a series of anti-TM4SF1 antibodies or antigen-binding fragments thereof, each of which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutations at histidine residues. In some embodiments, in at least one of the TM4SF1 antibodies or antigen fragments of the antibody library, all CDR positions are mutated to histidine.

[0183]

[0184] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one, two, three, four, five or more histidine substitutions in the CDR region. Histidine residues can be engineered into different positions of the anti-TM4SF1 antibody light chain (LC) or heavy chain (HC) for pH-dependent binding affinity. Thus, in some embodiments, ADCs having histidine engineered anti-TM4SF1 antibodies or antigen-binding fragments thereof are provided herein. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR1, CDR2, and / or CDR3 of the light chain variable region (VL). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR1 of the light chain variable region (VL). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR2 of the light chain variable region (VL). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR3 of the light chain variable region (VL). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR1, CDR2, and / or CDR3 of the heavy chain variable region (VH). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR1 of the heavy chain variable region (VH). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR2 of the heavy chain variable region (VH). In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR3 of the heavy chain variable region (VH). Thus, in some embodiments, the ADC of the present disclosure comprises a histidine engineered anti-TM4SF1 antibody or antigen-binding fragment thereof.

[0184]

[0185] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR1, CDR2, and / or CDR3 of the light chain, e.g., at one or more of positions 30 (S30H), 92 (S92H), and 93 (N93H) of SEQ ID NO:101 or SEQ ID NO:131. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises one or more histidine residues in CDR1, CDR2, and / or CDR3 of the heavy chain, e.g., at one or more of positions 28 (T28H), 31 (N31H), 32 (Y32H), 52 (N52H), 54 (Y54H), 57 (N57H), 100 (Q100H), and 101 (Y101H) of SEQ ID NO:92 or SEQ ID NO:130.

[0185]

[0186] Substitution at Position N297 (Asn297) and Conjugation of One or More Therapeutic Molecules to the Anti-TM4SF1 Antibody or Antigen-Binding Fragment thereof

[0187] Human IgG molecules have a conserved glycosylation site at each N297 residue of the CH2 domain, making these pendant N-glycans convenient targets for site-specific conjugation. This glycosylation site is far enough away from the variable region that conjugation of a drug moiety to the attached glycan does not affect antigen binding. In some embodiments of the present disclosure, therapeutic molecules are linked to the glycans using an exemplary method involving oxidative cleavage of vicinal diol moieties contained in these glycans with periodate to generate aldehydes that can be reductively aminated and conjugated to hydrazides and aminooxy compounds. (See, e.g., O'Shannessy et al. (1984) Immunol. Lett. 8:273-77).

[0186]

[0188] Another method may involve increasing the fucosylation of N-acetylglucosamine residues in these glycans. Oxidation of these fucose residues may produce carboxylic acid and aldehyde moieties that can be used to link drugs and fluorescers to these specific sites on the antibody (see, e.g., Zuberbuhler et al. (2012) Chem. Commun. 48:7100-02). Another method may involve modifying the sialic acid of these glycans (as well as increasing the sialic acid content in these glycans), followed by oxidation of the sialic acid and conjugation with an aminooxy drug to form an oxime-linked conjugate (see, e.g., Zhou et al. (2014) Bioconjugate Chem. 25:510-20).

[0187]

[0189] Alternatively, sialyltransferases can be used to incorporate engineered sialic acid residues containing orthogonal functional groups into these glycans, which can then be engineered to attach therapeutic molecules to sites on the glycans (see, e.g., Li et al. (2014) Angew. Chem. Int. 53:7179-82). Another approach to modifying these glycan sites is the use of glycosyltransferases to link galactose, or ketone- or azide-containing galactose analogs, to the N-acetylglucosamine of these glycans and link drugs or radionucleotides to the galactose molecules (see, e.g., Khidekel et al. (2003) J. Am. Chem. Soc. 125:16162-63; Clark et al., (2008) J. Am. Chem. Soc. 130:11576-77; Boeggeman et al. (2007) Bioconjugate Chem. 18:806-14). Another approach relies on the introduction of modified sugars onto these glycans during expression of the antibody by metabolic oligosaccharide engineering (see, e.g., Campbell et al. (2007) Mol. BioSyst. 3:187-94; Agard et al., (2009) Acc. Chem. Res. 42:788-97).

[0188]

[0190] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is conjugated to a therapeutic molecule by site-specific conjugation. Several natural or engineered amino acids, including cysteine ​​and glutamine, may be selected as sites of conjugation.

[0189]

[0191] In some instances, a cysteine ​​residue may be engineered into different positions in the antibody heavy chain (HC) or light chain (LC) for linkage, such as, for example, at position N297, i.e., N297C. Thus, in some embodiments, the ADCs of the disclosure comprise a cysteine ​​engineered anti-TM4SF1 antibody or antigen-binding fragment thereof.

[0190]

[0192] Introduction of cysteine ​​residues at suitable positions of anti-TM4SF1 antibodies can allow control of the site of conjugation, and the resulting site-specific conjugates can be more uniform than those obtained via wild-type conjugation, i.e., conjugation via reduced interchain cysteines. In some cases, ADCs comprising at least one conjugation via cysteine ​​can exhibit at least equivalent in vivo potency, improved pharmacokinetics (PK), and expanded therapeutic window compared to wild-type conjugates. In some embodiments, the ADCs comprise a cleavable dipeptide linker (i.e., valine-alanine) and a DNA-bridged pyrrolobenzodiazepine (PBD) dimer as a drug linked to a cysteine ​​at the heavy chain N297C position of the Fc portion of the anti-TM4SF1 antibody or antigen-binding fragment thereof. In some cases, the ADCs have an average drug-to-antibody ratio (DAR) of 1 or more, for example, a DAR of about 2, 6, 10, etc.

[0191]

[0193] Without being bound to any particular theory, it is believed that site-specific conjugation via unpaired cysteines can be relatively simple and scalable. For example, the attachment of therapeutic molecules can be performed without the need for special reagents. In some cases, ADCs prepared via site-specific cysteines can exhibit strong in vivo antitumor activity and be more tolerable than conventional conjugates. In some embodiments, the N297 position of an anti-TM4SF1 antibody or antigen-binding fragment thereof can be mutated to cysteine, i.e., N297C, and the cysteine ​​residue can be conjugated to a therapeutic molecule. In some cases, the N297C mutation is combined with further mutations of nearby residues to add stabilizing residues (e.g., arginine, lysine) and / or remove glutamic acid. In some cases, in addition to N297C, one or more positions from residues 292-303 are modified. The sequence at positions 292 to 303 can be REEQYCSTYRVV (IgG1), and REEQFCSTYRVV (IgG4).

[0192]

[0194] In some embodiments, the anti-TM4SF1 antibody or its antigen-binding fragment is conjugated to a therapeutic molecule by site-specific conjugation via glutamine residue. In some cases, microbial transglutaminase (mTG) can be used to transfer amine containing drug-linker or reactive spacer to the Q295 residue of the heavy chain of TM4SF1 antibody or its antigen-binding fragment, for example, deglycosylated anti-TM4SF1 antibody or its antigen-binding fragment. The conjugation was optimized using a two-step chemoenzymatic approach, whereby a reactive spacer containing a synthetic orthogonal azide or thiol functional linker was attached to the antibody by mTG, followed by reaction with maleimide containing dibenzocyclooctyne (DBCO) or MMAE. By using strain-promoted azide-alkyne cycloaddition (SPAAC) or thiol-maleimide chemistry, ADCs can be produced, for example, with a DAR of about 2.

[0193]

[0195] In some examples, the anti-TM4SF1 antibody or its antigen-binding fragment is conjugated to a therapeutic molecule by site-specific conjugation via glutamine residue (e.g., Q295) and cysteine ​​at position 297, N297C. This combination of mutations opens up two conjugation handles in the anti-TM4SF1 antibody or its antigen-binding fragment, and can obtain ADCs with higher DAR. Thus, in some embodiments of the present disclosure, an ADC is provided in which more than one therapeutic molecule (e.g., two therapeutic molecules) is conjugated to the anti-TM4SF1 antibody or its antigen-binding fragment via site-specific conjugation at N297C and Q295. For example, the cysteine ​​conjugation can be maleimide, haloacetamide, or another partner.

[0194]

[0196] Increased DAR may result in efficient ADC construction, minimal destabilization of antibody structure, and enhanced ADC efficacy. Cysteine ​​conjugation-based dual-charged linkers have been developed recently that allow modular payload introduction (Levengood et al., 2017). Thus, there remains a need for ADCs that can deliver multiple payloads.

[0195]

[0197] In addition, ADC linker structure and antibody-payload conjugation mode affect ADC homogeneity, cytotoxicity, tolerance, and pharmacokinetics (PK). These important parameters can contribute greatly to overall in vivo therapeutic efficacy (see, for example, Lu et al., 2016; Hamblett et al., 2004; Junutula et al., 2008; and Behrens et al., 2015). Thus, refining linker and conjugation chemistry is crucial to maximizing the therapeutic potential and safety profile of ADCs.

[0196]

[0198] Bioconjugation modes and methods can be optimized for improved ADC stability and efficacy. In some embodiments, one or more therapeutic and / or diagnostic agents are conjugated to anti-TM4SF1 antibodies or antigen-binding fragments thereof via maleimide, such as cysteine-maleimide conjugation. Other functional groups other than maleimide are reactive in some cases with thiol groups of anti-TM4SF1 antibodies, such as cysteine-engineered anti-TM4SF1 antibodies, and include iodoacetamide, bromoacetamide, vinylpyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. In some embodiments, therapeutic and / or diagnostic agents are conjugated to anti-TM4SF1 antibodies or antigen-binding fragments thereof via acetamide. For example, therapeutic agents can be conjugated to anti-TM4SF1 antibodies or antigen-binding fragments thereof via bromoacetamide conjugation. In some cases, ADCs comprising bromoacetamide-conjugated anti-TM4SF1 antibodies or antigen-binding fragments thereof exhibit increased stability, increased half-life, reduced toxicity, and / or improved therapeutic margins. Exemplary ADC structures are provided in Figures 1-6.

[0197] 6.3 Anti-TM4SF1 Antibodies or Antigen-Binding Fragments Thereof

[0199] TM4SF1 is a small plasma membrane glycoprotein with tetraspanin morphology but no homology (NCBI reference sequence NP_055035.1) (Wright et al., Protein Sci. 9:1594-1600, 2000). It forms a TM4SF1 enriched domain (TMED) on the plasma membrane where, like true tetraspanins, it serves as a molecular facilitator that recruits functionally related membrane and cytosolic molecules (Shih et al., Cancer Res. 69:3272-3277, 2009; Zukauskas et al., Angiogenesis. 14:345-354, 2011), playing an important role in cancer cell proliferation (Hellstrom et al., Cancer Res. 46:3917-3923, 1986), motility (Chang et al., Int J Cancer. 116:243-252, 2005), and metastasis (Richman et al., Cancer Res. 5916s-5920s, 1995). The amino acid sequence of the human TM4SF1 protein (NCBI reference sequence NP_055035.1) is shown below as SEQ ID NO: 134.

[0198]

[0200] MCYGKCARCI GHSLVGLALL CIAANILLYF PNGETKYASE NHLSRFVWFF SGIVGGGLLM LLPAFVFIGL EQDDCCGCCG HENCGKRCAM LSSVLAALIG IAGSGYCVIV AALGLAEGPLCLDSLGQWNYTFASTEGQYLLDTSTWSECTEPKHIVEWNVSLFSILLALG GIEFILCLIQVINGVLGGIC GFCCSHQQQY DC (SEQ ID NO: 91)

[0201] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure is specific to the ECL2 domain of TM4SF1. The amino acid sequence of the ECL2 domain of human TM4SF1 is EGPLCLDSLGQWNYTFASTEGQYLLDTSTWSECTEPKHIVEWNVSLFS (SEQ ID NO: 92).

[0199]

[0202] Included in the present disclosure are novel antibodies specific for TM4SF1, as described in Table 5 below. The antibodies described in Table 5 are monoclonal mouse antibodies AGX-A03, AGX-A04, AGX-A05, AGX-A07, AGX-A08, AGX-A09, and AGX-A11, each of which was identified in the screening described in the Examples and binds to the ECL2 region of TM4SF1. Further provided in Table 5 below are humanized antibodies hAGX-A07 and hAGX-A01.

[0200]

[0203] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises an IgG heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 87 or 88, or a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 73 or 74.

[0201]

[0204] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO:89 or a light chain constant region comprising a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO:89.

[0202]

[0205] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises an amino acid sequence set forth in SEQ ID NO: 3, 15, 27, 39, 51, 63, or 75, or a heavy chain variable domain comprising a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO: 3, 15, 27, 39, 51, 63, or 75.

[0203]

[0206] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 90 or 92, or a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO: 90 or 92.

[0204]

[0207] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:112 or 114, or a sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO:112 or 114.

[0205]

[0208] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises an amino acid sequence set forth in SEQ ID NO: 9, 21, 33, 45, 57, 69, or 81, or a light chain variable domain comprising a sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO: 9, 21, 33, 45, 57, 69, or 81.

[0206]

[0209] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 97, 99, 101, 103, or 105, or a sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO: 97, 99, 101, 103 or 105. In another embodiment, the antibody or antigen-binding fragment thereof is humanized and comprises a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 97, 99, or 101, or a sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO: 97, 99, or 101.

[0207]

[0210] In another embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:122 or a sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical, or 100% identical to SEQ ID NO:122.

[0208]

[0211] In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 comprising an amino acid sequence that is at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO: 6, 18, 30, 42, 54, 66, or 78. In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a heavy chain CDR2 that comprises an amino acid sequence that is at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO: 7, 19, 31, 43, 55, 67, or 79.In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a heavy chain CDR3 comprising an amino acid sequence that is at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:8, 20, 32, 44, 56, 68, or 80.

[0209]

[0212] In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a light chain CDR1 comprising an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO: 12, 24, 36, 48, 60, 72, or 84. In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a light chain CDR2 comprising an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO: 13, 25, 37, 49, 61, 73, or 85.In some embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a light chain CDR3 comprising an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO: 14, 26, 38, 50, 62, 74, or 86.

[0210]

[0213] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a heavy chain CDR1 comprising an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:94 or SEQ ID NO:115. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a heavy chain CDR2 that comprises an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:95, SEQ ID NO:116, or SEQ ID NO:117.In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a heavy chain CDR3 that comprises an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:96, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, or SEQ ID NO:121.

[0211]

[0214] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a light chain CDR1 comprising an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, or SEQ ID NO:127. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a light chain CDR2 that comprises an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:109, or SEQ ID NO:128.In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a light chain CDR3 that comprises an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:110, SEQ ID NO:111, or SEQ ID NO:129. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized and comprises a light chain CDR3 that comprises an amino acid sequence at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 91%, at least about 91% to at least about 92%, at least about 92% to at least about 93%, at least about 93% to at least about 94%, at least about 94% to at least about 95%, at least about 95% to at least about 96%, at least about 96% to at least about 97%, at least about 97% to at least about 98%, at least about 98% to at least about 99%, or at least about 99% to 100% identical to SEQ ID NO:110 or SEQ ID NO:129.

[0212]

[0215] The amino acid sequence of mouse monoclonal antibody AGX-A03 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 6, 7, and 8 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 12, 13, and 14 (CDR1, CDR2, and CDR3). Anti-TM4SF1 antibodies or antigen-binding fragments comprising a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 6, 7, and 8, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 12, 13, and 14, are included in the present disclosure. Humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A03 are included in the present disclosure. Additionally, the heavy chain variable amino acid sequences and the light chain variable amino acid sequences of AGX-A03 are set forth in SEQ ID NOs: 3 and 9, respectively.

[0213]

[0216] The amino acid sequence of mouse monoclonal antibody AGX-A04 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 18, 19, and 20 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 24, 25, and 26 (CDR1, CDR2, and CDR3). Anti-TM4SF1 antibodies or antigen-binding fragments comprising a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 18, 19, and 20, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 24, 25, and 26 are included in the present disclosure. Humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A04 are included in the present disclosure. Additionally, the heavy chain variable amino acid sequences and the light chain variable amino acid sequences of AGX-A04 are set forth in SEQ ID NOs: 15 and 21, respectively.

[0214]

[0217] The amino acid sequence of the murine monoclonal antibody AGX-A05 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 30, 31, and 32 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 36, 37, and 38 (CDR1, CDR2, and CDR3). Anti-TM4SF1 antibodies or antigen-binding fragments comprising a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 30, 31, and 32, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 36, 37, and 38 are included in the present disclosure. Humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A05 are included in the present disclosure. Additionally, the heavy chain variable amino acid sequence and the light chain variable amino acid sequence of AGX-A05 are set forth in SEQ ID NOs: 27 and 33, respectively. The amino acid sequence of the murine monoclonal antibody AGX-A07 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 42, 43, and 44 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 48, 49, and 50 (CDR1, CDR2, and CDR3). Included in the present disclosure are anti-TM4SF1 antibodies or antigen-binding fragments comprising a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 42, 43, and 44, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 48, 49, and 50. Included in the present disclosure are humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A07. Additionally, the heavy and light chain variable amino acid sequences of AGX-A07 are set forth in SEQ ID NOs: 39 and 45, respectively.

[0215]

[0218] In one embodiment, a humanized AGX-A07 (hAGX-A07) antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain sequence set forth in the amino acid sequence of SEQ ID NO: 90. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 (hmAGX-A07) antibody or antigen-binding fragment thereof, which comprises a heavy chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 90. As shown in Table 5, the heavy chain sequence set forth in SEQ ID NO: 90 is also referred to herein as AGX-A07H2. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof, which comprises a heavy chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 90, wherein the one or more substitutions are at amino acid positions 1, 44, and 80 of SEQ ID NO: 90. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises E1Q (substitution of glutamic acid to glutamine at position 1 of SEQ ID NO:90, heavy chain). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises D44G (substitution of aspartic acid to glycine at position 44 of SEQ ID NO:90, heavy chain). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises F80Y (substitution of phenylalanine to tyrosine at position 80 of SEQ ID NO:90, heavy chain). In some embodiments, a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof is provided that comprises a heavy chain sequence set forth in the amino acid sequence of SEQ ID NO:92. As shown in Table 5, the heavy chain sequence set forth in SEQ ID NO:92 is also referred to herein as AGX-A07H2v1. In some embodiments, a humanized AGX-A07 antibody or antigen-binding fragment thereof is provided that comprises a light chain sequence set forth in the amino acid sequence of SEQ ID NO:97. As shown in Table 5, the light chain sequence set forth in SEQ ID NO:97 is also referred to herein as AGX-A07L5.In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 97. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 97, wherein the one or more substitutions are at amino acid positions 3, 26, 62, and 90 of SEQ ID NO: 97. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises I3V (light chain, isoleucine to valine substitution at position 3 of SEQ ID NO: 97). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises N26Q (light chain, asparagine to glutamine substitution at position 26 of SEQ ID NO: 97). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises N26S (light chain, asparagine to serine substitution at position 26 of SEQ ID NO: 97). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises G62S (light chain, glycine to serine substitution at position 62 of SEQ ID NO: 97). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises W90Y (light chain, tryptophan to tyrosine substitution at position 90 of SEQ ID NO: 97). In some embodiments, a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof is provided that comprises a light chain sequence set forth in an amino acid sequence selected from the group consisting of SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, and SEQ ID NO: 105. As shown in Table 5, the light chain sequence set forth in SEQ ID NO: 99 is also referred to herein as AGX-A07L5v1, the light chain sequence set forth in SEQ ID NO: 101 is also referred to herein as AGX-A07L5v2, the light chain sequence set forth in SEQ ID NO: 103 is also referred to herein as AGX-A07L5v3, and the light chain sequence set forth in SEQ ID NO: 105 is also referred to herein as AGX-A07L5v4.An exemplary coding sequence for the heavy chain of a humanized AGX-A07 antibody or antigen-binding fragment thereof is provided in SEQ ID NO: 91. An exemplary coding sequence for the heavy chain of a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof is provided in SEQ ID NO: 93. An exemplary coding sequence for the light chain of a humanized AGX-A07 antibody or antigen-binding fragment thereof is provided in SEQ ID NO: 98 (AGX-A07L5). An exemplary coding sequence for the light chain of a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof is provided in SEQ ID NO: 100 (AGX-A07L5v1), SEQ ID NO: 102 (AGX-A07L5v2), SEQ ID NO: 104 (AGX-A07L5v3), and SEQ ID NO: 106 (AGX-A07L5v4).

[0216]

[0219] In one embodiment, a humanized AGX-A07 antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable domain sequence set forth in the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 132. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 130 or SEQ ID NO: 132. In one embodiment, a humanized AGX-A07 antibody or antigen-binding fragment thereof is provided, comprising a light chain variable domain sequence set forth in the amino acid sequence of SEQ ID NO: 131 or SEQ ID NO: 133. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain variable domain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 131 or SEQ ID NO: 133.

[0217]

[0220] In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain variable domain sequence comprising the sequence set forth in the amino acid sequence of SEQ ID NO: 131, and a heavy chain variable domain sequence comprising the sequence set forth in the amino acid sequence of SEQ ID NO: 130. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain variable domain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 131, and a heavy chain variable domain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 130. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain variable domain sequence comprising the sequence set forth in the amino acid sequence of SEQ ID NO: 133, and a heavy chain variable domain sequence comprising the sequence set forth in the amino acid sequence of SEQ ID NO: 132. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a light chain variable domain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 133, and a heavy chain variable domain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 132. In some embodiments, the humanized AGX-A07 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprising a heavy chain sequence comprising the sequence set forth in the amino acid sequence of SEQ ID NO: 156, or a sequence comprising one or more substitutions in the amino acid sequence of SEQ ID NO: 156.

[0218]

[0221] In some cases, the humanized AGX-A07 antibody or antigen-binding fragment thereof comprises a heavy chain CDR sequence set forth in SEQ ID NOs: 94, 95, and 96 (CDR1, CDR2, and CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NOs: 94, 95, and 96 (CDR1, CDR2, and CDR3). In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a heavy chain CDR sequence set forth in SEQ ID NOs: 94, 95, and 96 (CDR1, CDR2, and CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NOs: 94, 95, and 96 (CDR1, CDR2, and CDR3).

[0219]

[0222] In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 sequence set forth in SEQ ID NO: 94, or a heavy chain CDR1 sequence containing one or more substitutions in the sequence set forth in SEQ ID NO: 94. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a heavy chain CDR2 sequence set forth in SEQ ID NO: 95, or a heavy chain CDR2 sequence containing one or more substitutions in the sequence set forth in SEQ ID NO: 95. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a heavy chain CDR3 sequence set forth in SEQ ID NO: 96, or a heavy chain CDR3 sequence containing one or more substitutions in the sequence set forth in SEQ ID NO: 96.

[0220]

[0223] In some cases, the humanized AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR sequence set forth in SEQ ID NOs: 107, 109, and 110 (CDR1, CDR2, and CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NOs: 107, 109, and 110 (CDR1, CDR2, and CDR3). In some cases, the humanized AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR sequence set forth in SEQ ID NOs: 107, 109, and 111 (CDR1, CDR2, and CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NOs: 107, 109, and 111 (CDR1, CDR2, and CDR3). In some cases, the humanized AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR sequence set forth in SEQ ID NOs: 108, 109, and 110 (CDR1, CDR2, and CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NOs: 108, 109, and 110 (CDR1, CDR2, and CDR3). In some cases, the humanized AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR sequence set forth in SEQ ID NOs: 108, 109, and 111 (CDR1, CDR2, and CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NOs: 108, 109, and 111 (CDR1, CDR2, and CDR3).

[0221]

[0224] In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR1 sequence set forth in SEQ ID NO: 107 or 108, or a light chain CDR1 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 107 or 108. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR2 sequence set forth in SEQ ID NO: 109, or a light chain CDR2 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 109. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR3 sequence set forth in SEQ ID NO: 110 or 111, or a light chain CDR1 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 110 or 111. In some cases, the humanized mutant AGX-A07 antibody or antigen-binding fragment thereof comprises a light chain CDR3 sequence set forth in SEQ ID NO: 110, or a light chain CDR1 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 110.

[0222]

[0225] In some embodiments, the humanized mutant AGX-A07 comprises a heavy chain variable region comprising the following amino acid substitutions in SEQ ID NO: 132: Q1E, D44G, F80Y (also referred to herein as AGX-A07H2), and a light chain variable region comprising the following amino acid substitutions in SEQ ID NO: 133: I3V, N26Q, G62S (also referred to herein as AGX-A07L5). In some embodiments, the humanized mutant AGX-A07 comprises a heavy chain variable region comprising the following amino acid substitutions in SEQ ID NO: 132: Q1E, D44G, F80Y, and a light chain variable region comprising the following amino acid substitutions in SEQ ID NO: 133: I3V, N26Q, G62S, wherein the heavy chain comprises CDR1 (SEQ ID NO: 94), CDR2 (SEQ ID NO: 95), and CDR3 (SEQ ID NO: 96), and wherein the light chain comprises CDR1 (SEQ ID NO: 108), CDR2 (SEQ ID NO: 109), and CDR3 (SEQ ID NO: 110). In some embodiments, the humanized mutant AGX-A07 is AGX-A07H2v1L5v2 and comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 130 (also referred to herein as AGX-A07H2v1), and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 131 (also referred to herein as AGX-A07L5v2). In some embodiments, the humanized mutant AGX-A07 comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 92, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 101.

[0223]

[0226] The amino acid sequence of mouse monoclonal antibody AGX-A08 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 54, 55, and 56 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 60, 61, and 62 (CDR1, CDR2, and CDR3). Anti-TM4SF1 antibodies or antigen-binding fragments comprising a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 54, 55, and 56, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 60, 61, and 62, are included in the present disclosure. Humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A08 are included in the present disclosure. Additionally, the heavy chain variable amino acid sequences and the light chain variable amino acid sequences of AGX-A08 are set forth in SEQ ID NOs: 51 and 57, respectively.

[0224]

[0227] The amino acid sequence of the murine monoclonal antibody AGX-A09 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 66, 67, and 68 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 72, 73, and 74 (CDR1, CDR2, and CDR3). Included in the present disclosure are anti-TM4SF1 antibodies or antigen-binding fragments that include a heavy chain variable region that includes the CDRs set forth in the amino acid sequences of SEQ ID NOs: 66, 67, and 68, and / or a light chain variable region that includes the CDRs set forth in the amino acid sequences of SEQ ID NOs: 72, 73, and 74. Included in the present disclosure are humanized antibodies or antigen-binding fragments that include the CDRs of AGX-A09. Additionally, the heavy chain variable amino acid sequence and the light chain variable amino acid sequence of AGX-A09 are set forth in SEQ ID NOs: 63 and 69, respectively.

[0225]

[0228] The amino acid sequence of mouse monoclonal antibody AGX-A11 is set forth in Table 5. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 78, 79, and 80 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 84, 85, and 86 (CDR1, CDR2, and CDR3). Anti-TM4SF1 antibodies or antigen-binding fragments comprising a heavy chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 78, 79, and 80, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 84, 85, and 862 are included in the present disclosure. Humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A11 are included in the present disclosure. Additionally, the heavy chain variable amino acid sequence and the light chain variable amino acid sequence of AGX-A11 are set forth in SEQ ID NOs: 75 and 81, respectively.

[0226]

[0229] The amino acid sequence of humanized antibody AGX-A01 (hAGX-A01) is set forth in Table 5. As shown in Table 5, the heavy chain sequence set forth in SEQ ID NO: 112 is also referred to herein as AGX-A01H1. Specifically, the heavy chain CDR sequences are set forth in SEQ ID NOs: 115, 116, and 118 (CDR1, CDR2, and CDR3), and the light chain CDR amino acid sequences are set forth in SEQ ID NOs: 124, 128, and 129 (CDR1, CDR2, and CDR3). Additionally, exemplary humanized AGX-A01 heavy and light chain amino acid sequences are set forth in SEQ ID NOs: 112 and 122, respectively. Exemplary coding sequences for the humanized AGX-A01 heavy and light chains are set forth in SEQ ID NOs: 113 and 123, respectively.

[0227]

[0230] In some embodiments, the humanized AGX-A01 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A01 (hmAGX-A01) antibody or antigen-binding fragment thereof comprising a heavy chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 112. In some embodiments, the humanized AGX-A01 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprising a heavy chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 112, wherein the one or more substitutions are at amino acid positions 63 and 106 of SEQ ID NO: 112. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises G63S (heavy chain, glycine to serine substitution at position 63 of SEQ ID NO: 112). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises D106E (heavy chain, aspartic acid to glutamic acid substitution at position 106 of SEQ ID NO: 112). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises D106S (aspartic acid to serine substitution at position 106 of heavy chain, SEQ ID NO: 112). In some embodiments, a humanized mutant AGX-A01 antibody or antigen-binding fragment is provided that comprises a heavy chain sequence set forth in the amino acid sequence of SEQ ID NO: 114. As shown in Table 5, the heavy chain sequence set forth in SEQ ID NO: 114 is also referred to herein as AGX-A01H1v1.

[0228]

[0231] In some embodiments, a humanized AGX-A01 antibody or antigen-binding fragment is provided that comprises a light chain sequence set forth in the amino acid sequence of SEQ ID NO: 122. As shown in Table 5, the light chain sequence set forth in SEQ ID NO: 122 is also referred to herein as AGX-A01L10. In some embodiments, the humanized AGX-A01 antibody or antigen-binding fragment is a humanized mutant AGX-A01 antibody or antigen-binding fragment thereof that comprises a light chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 122. In some embodiments, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A01 antibody or antigen-binding fragment thereof that comprises a light chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 122, wherein the one or more substitutions are at one or more amino acid positions selected from amino acid positions 1, 33, 42, 51, 86, and 90 of SEQ ID NO: 122. In some embodiments, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof is a humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprising a light chain sequence comprising one or more substitutions in the sequence set forth in the amino acid sequence of SEQ ID NO: 122, wherein the one or more substitutions are at one or more amino acid positions selected from amino acid positions 1, 33, 42, 51, and 86 of SEQ ID NO: 122. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises A1E (light chain, alanine to glutamic acid substitution at position 1 of SEQ ID NO: 122). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises N33S (light chain, asparagine to serine substitution at position 33 of SEQ ID NO: 122). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises M42Q (light chain, methionine to glutamine substitution at position 42 of SEQ ID NO: 122). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises V51L (light chain, substitution of valine to leucine at position 51 of SEQ ID NO: 122).In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises D86E (light chain, aspartic acid to glutamic acid substitution at position 86 of SEQ ID NO: 122). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises I90V (light chain, isoleucine to valine substitution at position 90 of SEQ ID NO: 122).

[0229]

[0232] In some cases, the humanized AGX-A01 antibody or antigen-binding fragment thereof comprises a heavy chain CDR sequence set forth in SEQ ID NO: 115 (CDR1); 116 (CDR2); and 118 (CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 115 (CDR1); 116 (CDR2); and 118 (CDR3). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a heavy chain CDR sequence set forth in SEQ ID NO: 115 (CDR1); 116 or 117 (CDR2); and 118, 119, 120, or 121 (CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 115 (CDR1); 116 or 117 (CDR2); and 118, 119, 120, or 121 (CDR3).

[0230]

[0233] In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 sequence set forth in SEQ ID NO: 115, or a heavy chain CDR1 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 115. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a heavy chain CDR2 sequence set forth in SEQ ID NO: 116, or a heavy chain CDR2 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 116. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a heavy chain CDR2 sequence set forth in SEQ ID NO: 117, or a heavy chain CDR2 sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 117. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a heavy chain CDR3 sequence set forth in a sequence selected from SEQ ID NOs: 118, 119, 120, and 121, or a heavy chain CDR3 sequence comprising one or more substitutions in the sequence selected from SEQ ID NOs: 118, 119, 120, and 121.

[0231]

[0234] In some cases, the humanized AGX-A01 antibody or antigen-binding fragment thereof comprises a light chain CDR sequence set forth in SEQ ID NO: 124 (CDR1); 128 (CDR2); and 129 (CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 124 (CDR1); 128 (CDR2); and 129 (CDR3). In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a light chain CDR sequence set forth in SEQ ID NO: 124, 125, 126, or 127 (CDR1); 128 (CDR2); and 129 (CDR3), or a CDR sequence comprising one or more substitutions in the sequence set forth in SEQ ID NO: 124, 125, 126, or 127 (CDR1); 128 (CDR2); and 129 (CDR3).

[0232]

[0235] In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a light chain CDR1 sequence set forth in SEQ ID NO: 125, 126, 127, or 128, or a light chain CDR1 sequence containing one or more substitutions in the sequence set forth in SEQ ID NO: 125, 126, 127, or 128. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a light chain CDR2 sequence set forth in SEQ ID NO: 129, or a light chain CDR2 sequence containing one or more substitutions in the sequence set forth in SEQ ID NO: 129. In some cases, the humanized mutant AGX-A01 antibody or antigen-binding fragment thereof comprises a light chain CDR3 sequence set forth in SEQ ID NO: 130, or a light chain CDR1 sequence containing one or more substitutions in the sequence set forth in SEQ ID NO: 130.

[0233]

[0236] In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:3, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:9. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:15, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:21. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:27, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:33. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:39, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:45. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:51, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:57. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:63, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:69. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:75, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:81. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:90, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:97.In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:90, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:99. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:90, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:101. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:90, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:103. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:90, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:105. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:92, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:97. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:92, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:99. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:92, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:101. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:92, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:103.In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:92, and a light chain variable domain encoded by the nucleic acid sequence set forth in SEQ ID NO:105.

[0234]

[0237] In one embodiment, the present disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof having a heavy chain variable domain sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:112, or SEQ ID NO:114; and a light chain variable domain sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:122. In one embodiment, the present disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof having a heavy chain variable domain sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:112, or SEQ ID NO:114; and a light chain variable domain sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, or SEQ ID NO:122.In one embodiment, the present disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof having a heavy chain variable domain sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:63, SEQ ID NO:75, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:112, or SEQ ID NO:114; and a light chain variable domain sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, or SEQ ID NO:122.

[0235]

[0238] In one embodiment, the disclosure includes an anti-TM4SF1 antibody that is an IgG and comprises four polypeptide chains including two heavy chains each comprising a heavy chain variable domain and a heavy chain constant region CH1, CH2 and CH3, and two light chains each comprising a light chain variable domain and a light chain constant region (CL). In a specific embodiment, the antibody is human IgG1, IgG2, or IgG4. In a specific embodiment, the antibody is human IgG1. In other embodiments, the antibody is IgG2. The heavy and light chain variable domain sequences may contain the CDRs set forth in Table 5.

[0236]

[0239] Complementarity determining regions (CDRs) are known as hypervariable regions in both light and heavy chain variable domains. The more highly conserved parts of the variable domain are called frameworks (FRs). The CDRs and framework regions (FRs) of a given antibody can be identified using the systems described by Kabat et al., supra; Lefranc et al., supra, and / or Honegger and Pluckthun, supra. The numbering system described in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.) is also familiar to those skilled in the art. In this regard, Kabat et al. have defined a numbering system for variable domain sequences that includes the identification of CDRs that is applicable to any antibody.

[0237]

[0240] One or more CDRs can also be incorporated, either covalently or non-covalently, into a molecule, making it an antigen binding protein.

[0238]

[0241] An antigen-binding protein may incorporate CDRs as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may incorporate CDRs non-covalently. CDRs allow antigen-binding proteins to specifically bind to a particular antigen of interest. In particular, CDR3 has been found to play an important role in antigen-binding of antibodies or antibody fragments.

[0239]

[0242] In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain comprising a CDR3 domain set forth in any one of SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:68, or SEQ ID NO:80, and comprising a variable domain comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity to a sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:63, or SEQ ID NO:75. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a light chain comprising a CDR3 domain set forth in any one of SEQ ID NO:14, SEQ ID NO:26, SEQ ID NO:38, SEQ ID NO:50, SEQ ID NO:62, SEQ ID NO:74, or SEQ ID NO:86, and having a light chain variable domain comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to a sequence set forth in any one of SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, or SEQ ID NO:81. Thus, in certain embodiments, variability may be introduced into the remaining CDR and / or framework regions of the heavy and / or light chain while the CDR3 domain is kept constant, while the antibody or antigen-binding fragment thereof retains the ability to bind TM4SF1 and retains a functional characteristic, e.g., binding affinity, of the parent or has an improved functional characteristic, e.g., binding affinity, compared to the parent.

[0240]

[0243] In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain comprising a CDR2 domain set forth in any one of SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:67, or SEQ ID NO:79, and comprising a variable domain comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to a sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:51, SEQ ID NO:63, or SEQ ID NO:75. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a light chain comprising a CDR2 domain set forth in any one of SEQ ID NO:13, SEQ ID NO:25, SEQ ID NO:37, SEQ ID NO:49, SEQ ID NO:61, SEQ ID NO:73, or SEQ ID NO:85, and having a light chain variable domain comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to a sequence set forth in any one of SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, or SEQ ID NO:81. Thus, in certain embodiments, variability may be introduced into the remaining CDR and / or framework regions of the heavy and / or light chain while the CDR2 domain is kept constant, while the antibody or antigen-binding fragment thereof retains the ability to bind TM4SF1 and retains a functional characteristic, e.g., binding affinity, of the parent or has an improved functional characteristic, e.g., binding affinity, compared to the parent.

[0241]

[0244] In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a heavy chain comprising a CDR1 domain set forth in any one of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:54, SEQ ID NO:66, or SEQ ID NO:78, and comprising a variable domain comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to a sequence set forth in any one of SEQ ID NO:3, SEQ ID NO:15, SEQ ID NO:27, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:69, or SEQ ID NO:81. In one embodiment, the disclosure provides an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a light chain comprising a CDR1 domain set forth in any one of SEQ ID NO:12, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:72, or SEQ ID NO:84, and having a light chain variable domain comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% identity to a sequence set forth in any one of SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, or SEQ ID NO:81. Thus, in certain embodiments, the CDR1 domain may be kept constant and variability introduced into the remaining CDR and / or framework regions of the heavy and / or light chain, while the antibody or antigen-binding fragment thereof retains the ability to bind TM4SF1 and retains the functional characteristics, e.g., binding affinity, of the parent.

[0242]

[0245] In some embodiments, an anti-TM4SF1 antibody of the disclosure comprises a heavy chain comprising an Fc region, wherein the Fc region comprises a sequence selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:151, SEQ ID NO:152, and SEQ ID NO:153, or the Fc region comprises a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:151, SEQ ID NO:152, and SEQ ID NO:153. For example, in some embodiments, an anti-TM4SF1 antibody of the disclosure comprises an Fc region, wherein the Fc region is substituted with at least about 70% to about 100%, such as at least about 70%, at least about 75%, or at least a small proportion of a sequence selected from the group consisting of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:151, SEQ ID NO:152, and SEQ ID NO:153. The sequence may be at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical.

[0243]

[0246] In some embodiments, an anti-TM4SF1 antibody of the disclosure comprises a heavy chain comprising a sequence selected from the group consisting of SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156, wherein the heavy chain comprises a sequence containing one or more substitutions in a sequence selected from the group consisting of SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156. For example, in some embodiments, an anti-TM4SF1 antibody of the disclosure comprises a heavy chain comprising a sequence that is at least about 70% to about 100%, e.g., at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156.

[0244]

[0247] The anti-TM4SF1 antibodies and fragments described in Table 5 may also be humanized. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically obtained from an "import" variable domain. Humanization can be performed, for example, by replacing hypervariable region sequences with the corresponding sequences of a human antibody according to the methods of Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-27; and Verhoeyen et al., 1988, Science 239:1534-36.

[0245]

[0248] In some cases, humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six CDRs of a parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. determined that only about one-third of the residues in CDRs actually contact antigen, and named them "specificity determining residues," or SDRs (Padlan et al., 1995, FASEB J. 9:133-39). In the technique of SDR grafting, only SDR residues are grafted onto a human antibody framework (see, for example, Kashmiri et al., 2005, Methods 36:25-34).

[0246]

[0249] The selection of human variable domains, both light and heavy chains, used in the production of humanized antibodies can be important to reduce antigenicity. For example, according to the so-called "best-fit" method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence can be selected as the human framework of the humanized antibody (Sims et al., 1993, J.Immunol. 151:2296-308; and Chothia et al., 1987, J.Mol.Biol. 196:901-17). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; and Presta et al., 1993, J. Immunol. 151:2623-32). In some cases, the framework is derived from the consensus sequence of the most abundant human subclasses, VL6 subgroup I (VL6I) ​​and VH subgroup III (VHIII). In other methods, human germline genes are used as the source of the framework regions.

[0247]

[0250] Moreover, it is generally desirable for an antibody to be humanized while retaining its affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, 2000, Protein Eng. 13:819-24), Modeller (Sali and Blundell, 1993, J. Mol. Biol. 234:779-815), and Swiss PDB Viewer (Guex and Peitsch, 1997, Electrophoresis 18:2714-23). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, for example, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen, is achieved. In general, the hypervariable region residues are directly, and most substantially, involved in influencing antigen binding.

[0248]

[0251] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151 (1993) 2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 4285; and Presta et al., J. Immunol., 151 (1993) 2623). reference); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro, and Fransson, Front. Biosci. 13 (2008) 1619-1633); and framework regions obtained by screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272 ​​(1997) 10678-10684 and Rosok et al., J. Biol. Chem. 271 (1996) 22611-22618).

[0249]

[0252] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13 (2008) 1619-1633, and further described, e.g., in Riechmann et al., Nature 332 (1988) 323-329; Queen et al., Proc. Nat'l Acad. Sci. USA 86 (1989) 10029-10033; U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36 (2005) 25-34 (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28 (1991) 489-498 (describing "resurfacing"); Dall'Acqua et al., Methods 36 (2005) 43-60 (describing "FR shuffling"); and Osbourn et al., Methods 36 (2005) 61-68 and Klimka et al., Br. J. Cancer, 83 (2000) 252-260 (describing a "guided selection" approach to FR shuffling).

[0250]

[0253] In one embodiment, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure binds to cynomolgus TM4SF1 with a KD of about 1×10 −6 M or less.

[0251]

[0254] In certain embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure binds to an epitope on the ECL2 loop of human TM4SF1 with a KD of about 5×10−8 M or less, as determined by a standard flow cytometry assay using HUVEC cells.

[0252]

[0255] In certain embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure binds to human TM4SF1 with a KD of about 1×10 −8 M or less in a standard flow cytometry assay using HUVEC cells.

[0253]

[0256] In certain embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments thereof of the present disclosure are from about 1x10-3M to about 1x10-4M, from about 1x10-4M to about 1x10-5M, from about 1x10-5M to about 1x10-6M, from about 1x10-6 to about 1x10-7M, from about 1x10-7 to about 1x10-8M, from about 1x10-8M to about 1x10-9M, from about 1x10-9M to about 1x10-10M, from about 1x10-10M to about 1x10-11M, from about 1x10-11M to about 1x10-12M, from about 2x10-3M to about 2x10-4M, from about 2x10-4M to about 2x1 0-5M, about 2x10-5M to about 2x10-6M, about 2x10-6 to about 2x10-7M, about 2x10-7 to about 2x10-8M, about 2x10-8M to about 2x10-9M, about 2x10-9M to about 2x10-10M, about 2x10-10M to about 2x10-11M, about 2x10-11M to about 2x10-12M, about 3x10-3M to about 3x10-4M, about 3x10-4M to about 3x10-5M, about 3x10-5M to about 3x10-6M, about 3x10-6 to about 3x10-7M, about 3x10-7 to about 3x10-8M, about 3x10-8M to about 3x10 -9M, about 3x10-9M to about 3x10-10M, about 3x10-10M to about 3x10-11M, about 3x10-11M to about 3x10-12M, about 4x10-3M to about 4x10-4M, about 4x10-4M to about 4x10-5M, about 4x10-5M to about 4x10-6M, about 4x10-6 to about 4x10-7M, about 4x10-7 to about 4x10-8M, about 4x10-8M to about 4x10-9M, about 4x10-9M to about 4x10-10M, about 4x10-10M to about 4x10-11M, about 4x10-11M to about 4x10-12M, about 5x10-3M or from about 5x10-4M, from about 5x10-4M to about 5x10-5M, from about 5x10-5M to about 5x10-6M, from about 5x10-6 to about 5x10-7M, from about 5x10-7 to about 5x10-8M, from about 5x10-8M to about 5x10-9M, from about 5x10-9M to about 5x10-10M, from about 5x10-10M to about 5x10-11M, from about 5x10-11M to about 5x10-12M, from about 5x10-7M to about 5x10-11M, about 5x10-7M, about 1x10-7M, about 5x10-8M, about 1x10-8M, about 5x10-9M, about 1x10-9M, about 5x10-10M,It binds to human TM4SF1 with a KD of about 1×10 M, about 5×10 M, or about 1×10 M. In some embodiments, the KD is determined by a standard flow cytometry assay using HUVEC cells.

[0254]

[0257] In certain embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure binds to human TM4SF1 with a KD of about 5×10 −10 M or less in a standard flow cytometry assay using HUVEC cells.

[0255]

[0258] In certain embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments of the present disclosure bind to cynomolgus TM4SF1 with a KD of about 1×10-6M or less in a standard flow cytometry assay using HEK293 overexpressing cells. In one embodiment, the HEK293 cells are transfected to express cynomolgus TM4SF1. In a further embodiment, the HEK293 cells express cynomolgus TM4SF1 at about 600 mRNA copies per 106 copies of 18S rRNA.

[0256]

[0259] Methods for determining the KD of an antibody or antibody fragment are known in the art. For example, surface plasmon resonance can be used to determine the KD of an antibody for an antigen (e.g., using a BIACORE2000 or BIACORE3000 (BIAcore, Inc., Piscataway, NJ) with immobilized antigen or Fc receptor CM5 chip at about 10 response units (RU) at 25°C). In certain embodiments, FACS or flow cytometry is used to determine the KD, and cells expressing TM4SF1, such as HEK293 cells or HUVEC cells, are used to bind the antibody or fragment and measure the KD according to standard methods. Antibody affinity determination using flow cytometry is described, for example, in Geuijen et al. (2005) J Immunol Methods. 302(1-2):68-77. In certain embodiments, FACS is used to determine the affinity of an antibody.

[0257]

[0260] In one embodiment, the disclosure features an anti-TM4SF1 antibody or antigen-binding fragment thereof having a CDR amino acid sequence that includes a conservative amino acid substitution as described herein, such that the anti-TM4SF1 antibody or antigen-binding fragment thereof includes an amino acid sequence of a CDR that is at least 95% identical (or at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical) to the CDR amino acid sequence set forth in Table 5. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, a conservative amino acid substitution is one that does not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine.

[0258]

[0261] The disclosure further features, in one aspect, an anti-TM4SF1 antibody or antigen-binding fragment thereof that binds to an epitope on the ECL2 loop of human TM4SF1 with a KD of about 5×10-8 M or less as determined by a standard flow cytometry assay using HUVEC cells, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a human IgG framework region and a heavy chain variable region comprising a human IgG framework region. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is humanized. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof cross-reacts with cynomolgus monkey TM4SF1.

[0259]

[0262] In another aspect of the disclosure, the anti-TM4SF1 antibody or antigen-binding fragment thereof is a humanized anti-TM4SF1 antibody or antigen-binding fragment thereof that binds to an epitope on the ECL2 loop of human TM4SF1 with a KD of about 5×10 M or less as determined in a standard flow cytometry assay using HUVEC cells. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to cynomolgus monkey TM4SF1 with a KD of about 1×10 M or less in a standard flow cytometry assay using HEK293 overexpressing cells. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to human TM4SF1 with a KD of about 1×10 M or less in a standard flow cytometry assay using HUVEC cells. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is from 1x10-3M to about 1x10-4M, from about 1x10-4M to about 1x10-5M, from about 1x10-5M to about 1x10-6M, from about 1x10-6 to about 1x10-7M, from about 1x10-7 to about 1x10-8M, from about 1x10-8M to about 1x10-9M, from about 1x10-9M to about 1x10-10M, about 1x10-10M to about 1x10-11M, about 1x10-11M to about 1x10-12M, about 2x10-3M to about 2x10-4M, about 2x10-4M to about 2x10-5M, about 2x10-5M to about 2x10-6M, about 2x10-6 to about 2x10-7M, about 2x10-7 to about 2x10-8M, about 2x10-8M to about 2x1 0-9M, about 2x10-9M to about 2x10-10M, about 2x10-10M to about 2x10-11M, about 2x10-11M to about 2x10-12M, about 3x10-3M to about 3x10-4M, about 3x10-4M to about 3x10-5M, about 3x10-5M to about 3x10-6M, about 3x10-6 to about 3x10-7M, about 3x10-7 to about 3x10-8 M, about 3x10-8M to about 3x10-9M, about 3x10-9M to about 3x10-10M, about 3x10-10M to about 3x10-11M, about 3x10-11M to about 3x10-12M, about 4x10-3M to about 4x10-4M, about 4x10-4M to about 4x10-5M, about 4x10-5M to about 4x10-6M, about 4x10-6 to about 4x10-7M,about 4x10-7 to about 4x10-8M, about 4x10-8M to about 4x10-9M, about 4x10-9M to about 4x10-10M, about 4x10-10M to about 4x10-11M, about 4x10-11M to about 4x10-12M, about 5x10-3M to about 5x10-4M, about 5x10-4M to about 5x10-5M, about 5x10-5M to about 5x10-6M, about 5x10-6 to about 5x10-7M, about 5x10-7 to about 5x10-8M, about 5x10-8M to about It binds to human TM4SF1 with a KD of 5x10-9 M, about 5x10-9 M to about 5x10-10 M, about 5x10-10 M to about 5x10-11 M, about 5x10-11 M to about 5x10-12 M, about 5x10-7 M to about 5x10-11 M, about 5x10-7 M, about 1x10-7 M, about 5x10-8 M, about 1x10-8 M, about 5x10-9 M, about 1x10-9 M, about 5x10-10 M, about 1x10-10 M, about 5x10-11 M or about 1x10-11 M. In some embodiments, the KD is determined by a standard flow cytometry assay using HUVEC cells. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to human TM4SF1 with a KD of about 5×10 M or less in a standard flow cytometry assay using HUVEC cells expressing TM4SF1.

[0260]

[0263] In one embodiment, binding of an anti-TM4SF1 antibody or antigen-binding fragment of the present disclosure to human TM4SF1 is not dependent on glycosylation of the ECL2 loop of human TM4SF1, i.e., antibody binding is independent of glycosylation of TM4SF1 within the ECL2 loop (sequence number 77).

[0261]

[0264] The anti-TM4SF1 antibodies or antigen-binding fragments thereof of the present disclosure can be of any isotype (such as, but not limited to, IgG, IgM, and IgE). In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are of the IgG isotype. In specific embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure are from the IgG1, IgG2, or IgG4 isotype. In certain embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments thereof are of the human IgG1, human IgG2, or human IgG4 isotype.

[0262]

[0265] IgG2 naturally has the lowest ADCC and / or CDC activity (An et al., MAbs. 2009 Nov-Dec;1(6):572-579). Thus, in certain embodiments, IgG2 is advantageously used. However, IgG2 has two extra cysteines (resulting in four inter-hinge disulfide bonds), which makes it more susceptible to aggregation via the formation of inter-antibody disulfide bonds. In related embodiments, mutations to the IgG2 cysteines are made to reduce aggregation.

[0263]

[0266] The present disclosure provides antibody fragments that bind to TM4SF1. In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies. The small size of the fragments allows for rapid clearance, which can provide improved access to cells, tissues, or organs. For a review of specific antibody fragments, see Hudson et al., 2003, Nature Med. 9:129-34.

[0264]

[0267] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, for example, Morimoto et al., 1992, J. Biochem. Biophys. Methods 24:107-17; and Brennan et al., 1985, Science 229:81-83). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed and secreted in E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., 1992, Bio / Technology 10:163-67). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described, for example, in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to the skilled artisan. In certain embodiments, the antibody is a single chain Fv fragment (scFv) (see, for example, WO 93 / 16185; U.S. Pat. Nos. 5,571,894 and 5,587,458). Fv and scFv have intact binding sites that lack constant regions. Thus, they may be suitable for reducing non-specific binding during in vivo use. scFv fusion proteins can be constructed to provide fusion of effector proteins at either the amino or carboxy terminus of the scFv (see, for example, Borrebaeck, ed., supra). Antibody fragments may also be "linear antibodies", such as those described in the references cited above. Such linear antibodies may be monospecific or multispecific, e.g., bispecific.

[0265]

[0268] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, and scFv.

[0266]

[0269] For example, anti-TM4SF1 antibodies (and fragments) having high affinity for human TM4SF1 can be identified using screening techniques known in the art. For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., 1975, Nature 256:495-97, or can be made by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).

[0267]

[0270] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized, for example, by using the ECL2 loop of human TM4SF1 or a cell expressing TM4SF1 (whereby the ECL2 loop is expressed on the cell surface) to elicit lymphocytes that produce or are capable of producing antibodies that are expected to specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (1986)).

[0268]

[0271] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which, in certain embodiments, contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells have lost the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then a culture medium selective for hybridomas would typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which would prevent the growth of HGPRT-deficient cells.

[0269]

[0272] Exemplary fusion partner myeloma cells are those that fuse efficiently, maintain stable high levels of antibody production by the selected antibody-producing cells, and are sensitive to selective media that select for unfused parent cells. Exemplary myeloma cell lines are mouse myeloma lines, such as SP-2 and derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection (Manassas, Va.), and MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center (San Diego, Calif.). Human myeloma and mouse-human heteromyeloma cell lines have also been described for human monoclonal antibody production (Kozbor, 1984, Immunol. 133:3001-05; and Brodeur et al., Monoclonal Antibody Production Techniques and Applications 51-63 (1987)).

[0270]

[0273] The culture medium in which hybridoma cells grow is assayed for the production of monoclonal antibodies directed against the antigen. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as RIA or ELISA. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson et al., 1980, Anal.Biochem.107:220-39.

[0271]

[0274] Once hybridoma cells that produce the antibody with desired specificity, affinity, and / or activity are identified, the clones are subcloned by limiting dilution and grown by standard methods (Goding, supra).Suitable culture media for this purpose include, for example, DMEM or RPMI-1640 medium.In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by ip injection of cells into mice.

[0272]

[0275] The monoclonal antibodies secreted by the subclones are preferably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures, such as, for example, affinity chromatography (e.g., with Protein A or Protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or the like.

[0273]

[0276] DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells may serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that do not otherwise produce antibody protein, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to achieve synthesis of monoclonal antibodies in recombinant host cells. Reviews on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., 1993, Curr. Opinion in Immunol. 5:256-62 and Pluckthun, 1992, Immunol. Revs. 130:151-88.

[0274]

[0277] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using techniques described, for example, in Antibody Phage Display: Methods and Protocols (O'Brien and Aitken, eds., 2002). In principle, synthetic antibody clones are selected by screening phage libraries containing phages displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are screened against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional cycles of antigen adsorption / elution.

[0275]

[0278] The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which VH and VL are covalently linked via a short flexible peptide, or as Fab fragments in which they are fused to constant domains and interact non-covalently, e.g., as described in Winter et al., 1994, Ann. Rev. Immunol. 12:433-55.

[0276]

[0279] Repertoires of VH and VL genes can be cloned separately by PCR and randomly recombined in phage libraries, which can then be searched for antigen-binding clones, as described in Winter et al., supra. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies to diverse non-self and even self antigens without any immunization, as described in Griffiths et al., 1993, EMBO J 12:725-34. Finally, naive libraries can also be synthetically generated by cloning unrearranged V-gene segments from stem cells and using PCR primers containing random sequences to code for highly variable CDR3 regions and achieve in vitro rearrangement, as described, for example, by Hoogenboom and Winter, 1992, J. Mol. Biol. 227:381-88.

[0277]

[0280] Screening of the library can be accomplished by various techniques known in the art. For example, TM4SF1 (e.g., soluble form of the ECL2 loop or cells expressing the loop) can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate, or used in cell sorting, conjugated to biotin for capture on streptavidin-coated beads, or any other method for panning the display library. Selection of antibodies with slow dissociation kinetics (e.g., good binding affinity) can be improved by using long washes and monovalent phage display as described in Bass et al., 1990, Proteins 8:309-14 and WO92 / 09690, and further by using low coating density of antigen as described in Marks et al., 1992, Biotechnol.10:779-83.

[0278]

[0281] Anti-TM4SF1 antibodies can be obtained by designing a suitable antigen screening procedure to select a phage clone of interest, followed by constructing a full-length anti-TM4SF1 antibody clone using VH and / or VL sequences (e.g., Fv sequences), or various CDR sequences from the VH and VL sequences, from the phage clone of interest, and suitable constant region (e.g., Fc) sequences as described in Kabat et al., supra.

[0279]

[0282] Screening of anti-TM4SF1 antibodies can be carried out using binding assays known in the art and described herein to determine whether the antibody has therapeutic affinity for the ECL2 loop of TM4SF1. The ability of the antibody to inhibit or reduce metastatic cell activity can be measured using standard assays in the art and as described herein. Preclinical assays generally require the use of animal models of metastasis that are one of three types: (i) the injection of metastatic mouse tumor cells, e.g., B16F10 melanoma TCs, into mice, typically via tail vein injection to generate lung metastases, via portal vein or intrasplenic injection to generate liver metastases, or via injection into the left ventricle of the heart to generate bone and other metastases; (ii) the orthotopically implantation of metastatic tumor cells or intact tumor fragments into mice, a method that often requires subsequent surgical resection of the primary tumor to limit morbidity associated with primary tumor growth; and (iii) genetically engineered mouse models of spontaneous metastasis, the most common of which is the MMTV-Pyt (Mouse Mammary Tumor Virus-Polyomavirus Middle T Antigen) mouse breast cancer model, which provides an extremely realistic mouse model of human cancer metastasis, with more than 85% of hemizygous MMTV-PyMT females spontaneously developing palpable breast cancer that metastasizes to the lungs by 8–16 weeks of age. It is expected that the metastatic burden in the lungs will be quantified, either by imaging live animals or by directly counting metastatic nodules in the lungs of sacrificed animals as a function of the degree of TM4SF1 immunoblockade, and that a therapeutic level, e.g., at least a 50% reduction in lung metastases, will be achieved, indicating that the therapeutic antibody can be used, e.g., in the methods of the present disclosure. Additionally, cross-species reactivity assays are known in the art.Examples of assays that can be used are described, for example, in Khanna and Hunter (Carcinogenesis. 2005 March;26(3):513-23) and Saxena and Christofori (Mol Oncol. 2013 April;7(2):283-96), which are incorporated by reference in their entireties.

[0280]

[0283] In some embodiments, the anti-TM4SF1 antibodies and antigen-binding fragments thereof can be used, for example, to treat or prevent cancer. In certain embodiments, the anti-TM4SF1 antibodies and antigen-binding fragments of the present disclosure can be used to prevent metastasis of tumor cells. The anti-TM4SF1 antibodies and antigen-binding fragments of the present disclosure, in some instances, prevent metastasis of tumor cells by disrupting the interaction between tumor cells and vascular endothelial cells.

[0281] 6.4 Maytansine

[0284] Maytansinoids are derivatives of maytansine, which are cell division inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (see, e.g., U.S. Pat. No. 3,896,111). It has also been discovered that certain microorganisms can produce maytansinoids, such as maytansinol and C-3 maytansinol esters (see, e.g., U.S. Pat. No. 4,151,042). Synthetic maytansinoids are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,320,111; 4,330,112; 4,340,111; 4,350,111; 4,360,112; 4,370,113; 4,380,114; 4,390,122; 4,392,123; 4,393,124; 4,394,137; 4,395,141; 4,396,142; 4,397,151; 4,398,161; 4,399,170; 4,400,182; 4,401,197; 4,402,197; 4,403,198; Nos. 315929; 4317821; 4322348; 4331598; 4361650; 4364866; 4424219; 4450254; 4362663; and 4371533, each of which is incorporated by reference herein in its entirety.

[0282]

[0285] Maytansinoids are attractive agents for antibody-drug conjugates because they are: (i) relatively accessible for preparation by fermentation or chemical modification or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation to antibodies with non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0283]

[0286] Certain maytansinoids can be isolated from natural sources by known methods or can be produced using genetic engineering techniques (see, e.g., Yu et al. (2002) PNAS 99:7968-7973, which is incorporated herein by reference in its entirety). Maytansinoids can also be prepared synthetically by known methods.

[0284]

[0287] Examples of maytansinoids include, but are not limited to, those with modified aromatic rings, such as: C-19 dechloro (U.S. Pat. No. 4,256,746) (prepared, e.g., by lithium aluminum hydride reduction of ansamytocin P2); C-20-hydroxy (or C-20-demethyl) + / - C-19 dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared, e.g., by demethylation using Streptomyces or Actinomyces, or by dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / - dechloro (U.S. Pat. No. 4,294,757) (prepared, e.g., by acylation utilizing acyl chlorides), as well as those with modifications at other positions on the aromatic ring. Each of the patents cited in this paragraph is incorporated herein by reference in its entirety.

[0285]

[0288] Examples of maytansinoids include modified maytansinoids such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared, for example, by reaction of maytansinol with H2S or P2S5); C-14-alkoxymethyl (demethoxy / CH2OR) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH2OH or CHOAc) (U.S. Pat. No. 4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared, for example, by conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared, for example, from Trewia nudiflora nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared, for example, by demethylation of maytansinol by Streptomyces sp.); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by titanium trichloride / LAH reduction of maytansinol). Each of the patents cited in this paragraph is incorporated herein by reference in its entirety.

[0286]

[0289] As described in this invention, chemical conjugation of maytansine to antibodies is expected to selectively deliver the cytotoxic agent to tumor vasculature and surrounding cells.

[0287] 6.5 Maytansine in ADCs

[0290] In some embodiments, the ADCs of the present disclosure comprise one or more therapeutics (also referred to herein as therapeutic molecules or therapeutic agents) conjugated to an anti-TM4SF1 antibody or antigen-binding fragment thereof. In some embodiments, the ADCs are directed to the nucleus and are known as ND-ADCs. In some embodiments, the agent is maytansine or a derivative thereof.

[0288]

[0291] Without being bound by theory, maytansine-conjugated anti-TM4SF1 antibodies and derivatives thereof are expected to reduce drug-induced toxicity.

[0289] 6.6 Linker

[0292] One or more agents (e.g., therapeutic and / or diagnostic agents) may be indirectly conjugated to the anti-TM4SF1 antibody or antigen-binding fragment (e.g., by using a linker in a direct covalent or non-covalent interaction). The linker may be a chemical linking agent, such as homobifunctional and heterobifunctional cross-linking agents, which are available from many commercial sources. Regions available for cross-linking can be found on the binding proteins (e.g., anti-TM4SF1 antibodies) of the present disclosure. The linker may include flexible arms, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Exemplary linkers include BS3 ([bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS / EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodiimide; NHS / EDC allows for conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([Ne-maleimidocaproic acid]hydrazide; sulfo-EMCS is a heterobifunctional reactive group (maleimide and NHS-ester) that is reactive to sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates and hydrazides are useful reagents for linking carboxyl groups to primary amines), and SATA (N-succinimidyl-S-acetylthioacetate; SATA is reactive to amines and adds protected sulfhydryl groups). For forming covalent bonds, the chemically reactive groups are various activated carboxyl groups (eg, esters) where the hydroxyl moieties are physiologically acceptable at the level required to modify the peptide.Specific agents include N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimido-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxysuccinimide ester (GMBS), maleimidopropionic acid (MPA), maleimidohexanoic acid (MHA), and maleimidoundecanoic acid (MUA). Primary amines are the primary targets of NHS esters. Accessible a-amino groups present at the N-terminus of proteins and ε-amines of lysines react with NHS esters. Amide bonds are formed when the conjugation reactant of the NHS ester reacts with a primary amine to release N-hydroxysuccinimide. These succinimide-containing reactive groups are referred to herein as succinimidyl groups. In certain embodiments of the present disclosure, the functional group on the protein is expected to be a thiol group and the chemically reactive group is expected to be a maleimide-containing group, such as gamma-maleimide-butyrylamide (GMBA or MPA). Such maleimide-containing groups are referred to herein as maleimide groups. Maleimide groups are most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is between 6.5 and 7.4. At pH 7.0, the reaction rate of maleimide groups with sulfhydryls (e.g., thiol groups on proteins such as serum albumin or IgG) is 1000 times faster than amines. Thus, stable thioether bonds between maleimide groups and sulfhydryls can be formed.

[0290]

[0293] In other embodiments, the linker comprises at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino acid, e.g., Cys). In other embodiments, a glycine-rich peptide, e.g., a peptide, can be used. In some cases, the linker may be a single amino acid (e.g., any amino acid, e.g., Gly or Cys). Examples of suitable linkers are succinic acid, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys. When the linker is succinic acid, one of its carboxyl groups can form an amide bond with the amino group of an amino acid residue, and the other carboxyl group can form an amide bond with, for example, the amino group of a peptide or a substituent. When the linker is Lys, Glu or Asp, their carboxyl group can form an amide bond with the amino group of the amino acid residue, and their amino group can form an amide bond with, for example, the carboxyl group of the substituent. When Lys is used as a linker, a further linker may be inserted between the ε-amino group of Lys and the substituent. In one particular embodiment, the further linker is succinic acid, which forms an amide bond with, for example, the ε-amino group of Lys and the amino group present in the substituent. In one embodiment, the further linker is Glu or Asp (e.g., which forms an amide bond with the ε-amino group of Lys and forms another amide bond with the carboxyl group present in the substituent), i.e., the substituent is a NE-acylated lysine residue.

[0291]

[0294] In some embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments thereof described herein and oligonucleotides (e.g., nucleic acid molecules such as RNA or DNA molecules) may be conjugated using a variety of approaches, e.g., genetic conjugation, enzymatic conjugation, chemical conjugation, or any combination thereof.

[0292]

[0295] In some embodiments, the RNA molecules in the ADC can be conjugated to the anti-TM4SF1 antibody or its antigen-binding fragment using an enzymatic site-specific conjugation method, including the use of mammalian or bacterial transglutaminase enzymes. Microbial transglutaminase (mTG) is a versatile tool in modern research and biotechnology. The availability of large amounts of relatively pure enzyme, ease of use, and lack of regulation by calcium and guanosine-5' triphosphate (GTP) have propelled mTG to become the main cross-linking enzyme used in both the food industry and biotechnology. Currently, mTG is used in many applications to attach proteins and peptides to small molecules, polymers, surfaces, DNA, and other proteins. See, for example, Pavel Strp, Veracity of microbial transglutaminase, Bioconjugate Chem. 25, 5, 855-862).

[0293]

[0296] In some embodiments, the RNA molecule in the conjugate may be conjugated to the anti-TM4SF1 antibody or antigen-binding fragment thereof by using a linker in a direct covalent or non-covalent interaction. The linker may be an amino acid or peptide-based linker, or a chemical linking agent, such as homobifunctional and heterobifunctional cross-linking agents, which are available from many commercial sources. The region available for cross-linking may be found on the anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure. The linker may include flexible arms, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Exemplary linkers include cleavable, non-cleavable, covalent, or non-covalent linkers, or any combination thereof. In some embodiments, the cleavable linker includes an acid-labile linker, a protease-sensitive linker, a photolabile linker, or a disulfide-containing linker. In some embodiments, the linker comprises a non-cysteine ​​linker, such as a cysteine ​​linker or a lysine linker. In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a non-natural amino acid, and the antibody or antibody fragment and the oligonucleotide are linked / conjugated via the non-natural amino acid.

[0294]

[0297] In some embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a natural amino acid, and the antibody or antibody fragment and the oligonucleotide are linked / conjugated via the natural amino acid. The non-natural amino acid may be inserted between two naturally occurring amino acids in the antibody or antibody fragment. One or more non-natural amino acids may replace one or more naturally occurring amino acids in the antibody or antibody fragment. One or more non-natural amino acids may be incorporated at the N-terminus of the antibody or antibody fragment. One or more non-natural amino acids may be incorporated at the C-terminus of the antibody or antibody fragment. The non-natural amino acid may be incorporated away from the binding region of the antibody or antibody fragment. The non-natural amino acid may be incorporated near the binding region of the antibody or antibody fragment. The non-natural amino acid may be incorporated at the binding region of the antibody or antibody fragment.

[0295]

[0298] The one or more unnatural amino acids may be encoded by a codon that does not encode one of the 20 amino acids. The one or more unnatural amino acids may be encoded by a nonsense codon (stop codon). The stop codon may be an amber codon. The amber codon may comprise a UAG sequence. The stop codon may be an ochre codon. The ochre codon may comprise a UAA sequence. The stop codon may be an opal or amber codon. The opal or amber codon may comprise a UGA sequence. The one or more unnatural amino acids may be encoded by a four-base codon.

[0296]

[0299] The one or more unnatural amino acids can be p-acetylphenylalanine (pAcF or pAcPhe). The one or more unnatural amino acids can be selenocysteine. The one or more unnatural amino acids can be p-fluorophenylalanine (pFPhe). The one or more unnatural amino acids may be selected from the group including p-azidophenylalanine (pAzF), p-azidomethylphenylalanine (pAzCH2F), p-benzoylphenylalanine (pBpF), p-propargyloxyphenylalanine (pPrF), p-iodophenylalanine (pIF), p-cyanophenylalanine (pCNF), p-carboxymethylphenylalanine (pCmF), 3-(2-naphthyl)alanine (NapA), p-boronophenylalanine (pBoF), o-nitrophenylalanine (oNiF), (8-hydroxyquinolin-3-yl)alanine (HQA), selenocysteine, and (2,2'-bipyridin-5-yl)alanine (BipyA). The one or more unnatural amino acids may be 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine.

[0297]

[0300] The one or more unnatural amino acids can be β-amino acids (β3 and β2), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, N-methyl amino acids, or combinations thereof.

[0298]

[0301] Additional examples of unnatural amino acids include, but are not limited to: 1) various substituted tyrosine and phenylalanine analogs, such as O-methyl-L-tyrosine, p-amino-L-phenylalanine, 3-nitro-L-tyrosine, p-nitro-L-phenylalanine, m-methoxy-L-phenylalanine, and p-isopropyl-L-phenylalanine; 2) amino acids with aryl azide and benzophenone groups that can be photocrosslinked; 3) amino acids with unique chemical reactivity, including acetyl-L-phenylalanine and m-acetyl-L-phenylalanine, O-aryl-L-tyrosine, O-(2-propynyl)-L-tyrosine, p-ethylthiocarbonyl-L-phenylalanine, and p-(3-oxobutanoyl)-L-phenylalanine. amino acids; 4) heavy atom containing amino acids for phasing in X-ray crystallography including p-iodo- and p-bromo-L-phenylalanine; 5) redox-active amino acid dihydroxy-L-phenylalanine; 6) glycosylated amino acids including bN-acetylglucosamine-O-serine and N-acetylgalactosamine-O-threonine; 7) fluorescent amino acids with naphthyl, dansyl, and 7-aminocoumarin side chains; 8) photocleavable and photoisomerizable amino acids with azobenzene and nitrobenzyl Cys, Ser, and Tyr side chains; 9) phosphotyrosine mimic p-carboxymethyl-L-phenylalanine; 10) glutamine homolog homoglutamine; and 11) 2-aminooctanoic acid. Unnatural amino acids can be modified to incorporate chemical groups. Unnatural amino acids can be modified to incorporate ketone groups.

[0299]

[0302] The one or more unnatural amino acids may comprise at least one oxime, carbonyl, dicarbonyl, hydroxylamine group, or a combination thereof. The one or more unnatural amino acids may comprise at least one carbonyl, dicarbonyl, alkoxy-amine, hydrazine, acyclic alkene, acyclic alkyne, cyclooctyne, aryl / alkyl azide, norbornene, cyclopropene, trans-cyclooctene, or tetrazine functional group, or a combination thereof.

[0300]

[0303] One or more unnatural amino acids can be incorporated into an antibody or antibody fragment by methods known in the art. Cell-based or cell-free systems can be used to modify the genetic sequence of an antibody or antibody fragment, thereby producing an antibody or antibody fragment with one or more unnatural amino acids. Auxotrophic strains can be used in place of engineered tRNAs and synthetic enzymes. One or more unnatural amino acids can be produced through a selection reaction of one or more natural amino acids. The selection reaction can be mediated by one or more enzymes. In one non-limiting example, a selection reaction of one or more cysteines with formylglycine generating enzyme (FGE) can produce one or more formylglycines as described in Rabuka et al., Nature Protocols 7:1052-1067 (2012).

[0301]

[0304] One or more unnatural amino acids can participate in a chemical reaction to form a linker. The chemical reaction to form the linker can be a bioorthogonal reaction. The chemical reaction to form the linker can be click chemistry.

[0302]

[0305] Additional unnatural amino acids are disclosed in Liu et al. (Annu Rev Biochem, 79:413-44, 2010), Wang et al. (Angew Chem Int Ed, 44:34-66, 2005) and PCT Application Nos. PCT / US2012 / 039472, PCT / US2012 / 039468, PCT / US2007 / 088009, PCT / US2009 / 058668, PCT / US2007 / 089142, PCT / US2007 / 088011, PCT / US2007 / 001485, PCT / US2006 / 049397, PCT / US2006 / 047822, and PCT / US2006 / 044682, all of which are incorporated by reference in their entireties.

[0303]

[0306] The one or more non-natural amino acids can replace one or more amino acids in the antibody or antibody fragment. The one or more non-natural amino acids can replace any natural amino acid in the antibody or antibody fragment.

[0304]

[0307] One or more unnatural amino acids may be incorporated into the light chain of the antibody or antibody fragment. One or more unnatural amino acids may be incorporated into the heavy chain of the antibody or antibody fragment. One or more unnatural amino acids may be incorporated into the heavy and light chains of the antibody or antibody fragment. One or more unnatural amino acids may replace amino acids in the light chain of the antibody or antibody fragment. One or more unnatural amino acids may replace amino acids in the heavy chain of the antibody or antibody fragment. One or more unnatural amino acids may replace amino acids in the heavy chain of the antibody or antibody fragment. One or more unnatural amino acids may replace amino acids in the heavy and light chains of the antibody or antibody fragment.

[0305]

[0308] Anti-TM4SF1 antibody or antigen-binding fragment thereof. In some embodiments, the linker comprises a small molecule fragment, a spacer, a non-covalent linker, or a combination thereof. In some embodiments, the linker comprises one or more small molecule fragments. In some embodiments, the linker comprises a spacer.

[0306]

[0309] In some embodiments, the linker comprises one or more reactive moieties, hi some embodiments, the linker comprises a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with the antibody fragment and / or therapeutic agent.

[0307]

[0310] In some embodiments, the small anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a reactive moiety, hi some embodiments, the small molecule fragment comprises a reactive moiety selected from a Michael acceptor moiety, a leaving group moiety, or a moiety capable of forming a covalent bond with a thiol group of a cysteine ​​residue.

[0308]

[0311] In some embodiments, the Michael acceptor moiety comprises an alkene or alkyne moiety. In some embodiments, the small molecule fragment is obtained from a compound library. In some embodiments, the compound library comprises ChemBridge fragment library, Pyramid Platform fragment-based drug discovery, Maybridge fragment library, FRGx from AnalytiCon, TCI-Frag from AnCoreX, Bio Building Blocks from ASINEX, BioFocus 3D from Charles River, Fragments of Life (FOL) from Emerald Bio, Enamine fragment library, IOTA Diverse 1500, BIONET fragment library, Life Chemicals Fragments Collection, OTAVA fragment library, Prestwick fragment library, Selcia fragment library, TimTec fragment-based library, Allium from Vitas-M Laboratory, or Zenobia fragment library.

[0309]

[0312] In some embodiments, the small molecule fragment comprises a carbodiimide, N-hydroxysuccinimide (NHS) ester, imidoester, pentafluorophenyl ester, hydroxymethylphosphine, maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, vinyl sulfone, hydrazide, alkoxyamine, alkyne, azide, or isocyanate group. In some embodiments, the small molecule fragment comprises an alkyne or azide group. In some embodiments, the small molecule fragment comprises an alkyne group. In some embodiments, the small molecule fragment comprises an azide group.

[0310]

[0313] In some embodiments, the small molecule fragment interacts with the spacer through a covalent bond. In some embodiments, the spacer comprises an amide moiety, an ester moiety, an ether moiety, a substituted or unsubstituted C1-C6 alkylene moiety, a substituted or unsubstituted C1-C6 haloalkylene moiety, a substituted or unsubstituted C1-C6 heteroalkylene moiety, a substituted or unsubstituted C3-C8 cycloalkylene moiety, a substituted or unsubstituted C2-C7 heterocycloalkylene moiety, a substituted or unsubstituted arylene moiety, a substituted or unsubstituted heteroarylene moiety, or any combination thereof.

[0311]

[0314] In some embodiments, the linker is MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), or aryloxycarbonyl (aryloxycarbonyl). Further examples of linkers include: BS3 ([bis(sulfosuccinimidyl)suberate]; BS3 is a homobifunctional N-hydroxysuccinimide ester that targets accessible primary amines), NHS / EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodiimide; NHS / EDC allows for conjugation of primary amine groups with carboxyl groups), sulfo-EMCS ([Ne-maleimide]), [N-docaproic acid]hydrazide; sulfo-EMCS is a heterobifunctional reactive group (maleimide and NHS-ester) that is reactive to sulfhydryl and amino groups), hydrazide (most proteins contain exposed carbohydrates, and hydrazides are useful reagents for linking carboxyl groups to primary amines), and SATA (N-succinimidyl-S-acetylthioacetate; SATA is reactive to amines and adds protected sulfhydryl groups). To form covalent bonds, the chemically reactive groups are A variety of active carboxyl groups (e.g., esters) where the hydroxyl moiety is physiologically acceptable at the level required to modify the peptide. Specific agents include N-hydroxysuccinimide (NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimido-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxysuccinimide ester (GMBS), maleimidopropionic acid (MPA), maleimidohexanoic acid (MHA), and maleimidoundecanoic acid (MUA).Primary amines are the primary targets for NHS esters. Accessible a-amino groups present at the N-terminus of proteins and the ε-amines of lysines react with NHS esters. Amide bonds are formed when the NHS ester conjugation reactant reacts with a primary amine to release N-hydroxysuccinimide. These succinimide-containing reactive groups are referred to herein as succinimidyl groups. In certain embodiments of the present disclosure, the functional groups on the protein are expected to be thiol groups and the chemically reactive groups are expected to be maleimide-containing groups, e.g., gamma-maleimide-butyrylamide (GMBA or MPA). Such maleimide-containing groups are referred to herein as maleimide groups. Maleimide groups are most selective for sulfhydryl groups on peptides when the pH of the reaction mixture is between 6.5 and 7.4. At pH 7.0, the rate of reaction of maleimide groups with sulfhydryls (e.g., thiol groups on proteins such as serum albumin or IgG) is 1000 times faster than amines. Thus, a stable thioether bond between the maleimide group and the sulfhydryl can be formed.

[0312]

[0315] In other embodiments, the linker comprises at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino acid, such as Cys or Lys). In other embodiments, a glycine-rich peptide, such as a peptide, can be used. In some cases, the linker may be a single amino acid (e.g., any amino acid, such as Gly or Cys or Lys). Examples of suitable linkers are succinic acid, Lys, Glu, and Asp, or a dipeptide such as Gly-Lys. When the linker is succinic acid, one of its carboxyl groups can form an amide bond with the amino group of the amino acid residue, and the other carboxyl group can form an amide bond with, for example, the amino group of the peptide or substituent. When the linker is Lys, Glu, or Asp, their carboxyl group can form an amide bond with the amino group of the amino acid residue, and their amino group can form an amide bond with, for example, the carboxyl group of the substituent. When Lys is used as a linker, a further linker may be inserted between the ε-amino group of Lys and the substituent. In one particular embodiment, the further linker is succinic acid, which forms an amide bond with, for example, the ε-amino group of Lys and the amino group present in the substituent. In one embodiment, the further linker is Glu or Asp (e.g., which forms an amide bond with the ε-amino group of Lys and another amide bond with the carboxyl group present in the substituent), i.e., the substituent is a NE-acylated lysine residue. In some embodiments, the linker comprises a single amino acid peptide consisting of lysine. In some embodiments, the linker comprises a LysLys dipeptide. In some embodiments, the linker is * Lys and / or Lys * In some embodiments, the linker comprises a dipeptide. * and / or * LysLys, Lys *Lys tripeptide In some embodiments, the linker comprises a LysLysLys tripeptide.

[0313]

[0316] In some embodiments, the conjugation of the anti-TM4SF1 antibody or antigen-binding fragment thereof to the RNA molecule is performed in a manner that produces a ring-tethered molecule. In some embodiments, the spacer further comprises a macrocycle. In some embodiments, the macrocycle comprises a non-covalent macrocycle. In some embodiments, the macrocycle comprises a covalent macrocycle.

[0314]

[0317] In some embodiments, the macrocycle comprises a cucurbit[X]uril, where X is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the macrocycle comprises a cucurbit[X]uril, where X is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the macrocycle comprises a cucurbit[X]uril, where X is 5, 6, 7, or 8. In some embodiments, the cucurbit[X]uril is

[0315] [ka]

[0316] wherein x is 5, 6, 7, or 8. The compound has a structure represented by:

[0317]

[0318] In some embodiments, x is 5. In some embodiments, x is 6. In some embodiments, x is 7. In some embodiments, x is 8.

[0318]

[0319] In some embodiments, the macrocycle comprises a cucurbit[6]uril (CB6). In some embodiments, the macrocycle comprises a cucurbit[7]uril (CB7). In some embodiments, the cucurbit[7]uril is

[0319] [ka]

[0320] The compound has a structure represented by:

[0321] In some embodiments, the macrocycle comprises a cyclodextrin (CD). In some embodiments, the cyclodextrin is

[0322] [ka]

[0323] (wherein n is 5, 6, 7, or 8). The compound has a structure represented by:

[0324] In some embodiments, the macrocycle comprises a beta-cyclodextrin (n=7). In some embodiments, the macrocycle comprises a gamma-cyclodextrin (n=8). In some embodiments, the beta-cyclodextrin comprises

[0325] [ka]

[0326] The compound has a structure represented by:

[0327] In some embodiments, the macrocycle comprises a polypeptide. In some embodiments, the polypeptide comprises:

[0328] [ka]

[0329] (In the formula, R 1is H, D, F, -CN, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 fluoroalkyl, substituted or unsubstituted C1-C6 heteroalkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; m is 5, 6, 7, or 8). The compound has a structure represented by:

[0330] In some embodiments, the macrocycle comprises cycloglycine. In some embodiments, the macrocycle comprises cyclo(glycylglycylglycylglycylglycylglycyl). In some embodiments, the macrocycle comprises cyclo(glycylglycylglycylglycylglycylglycylglycyl). In some embodiments, the macrocycle comprises cyclo(glycylglycylglycylglycylglycylglycylglycyl). In some embodiments, cyclo(glycylglycylglycylglycylglycylglycylglycylglycyl) is

[0331] [ka]

[0332] The compound has a structure represented by:

[0333]

[0324] In some embodiments, the macrocycle comprises a crown ether. In some embodiments, the crown ether is 15-crown-5, 18-crown-6, dibenzo-18-crown-6, or diaza-18-crown-6.

[0334]

[0325] In some embodiments, the macrocycle comprises a cycloalkane, hi some embodiments, the cycloalkane is cyclopentadecane, cyclohexadecane, cycloheptadecane, or cyclooctadecane.

[0335] In some embodiments, the macrocycle comprises cyclobis(paraquat-p-phenylene) (CBPQT4+). In some embodiments, cyclobis(paraquat-p-phenylene) (CBPQT4+) is

[0336] [ka]

[0337] The compound has a structure represented by:

[0338] In some embodiments, the linker comprises a quaternary nitrogen.

[0339] [ka]

[0340] (wherein each R is independently H or C1-C6 alkyl). In some embodiments, the linker is

[0341] [ka]

[0342] (wherein each R is independently H or C1-C6 alkyl). In some embodiments, the linker is

[0343] [ka]

[0344] (wherein each R is independently H or C1-C6 alkyl). It is.

[0345]

[0328] In some embodiments, the linker is

[0346] [ka]

[0347] It is.

[0348] In some embodiments, the conjugate is produced by linking a first portion of the linker to the anti-TM4SF1 antibody or antigen-binding fragment thereof and a second portion of the linker to the oligonucleotide. The step of conjugating the linker to the anti-TM4SF1 antibody or antigen-binding fragment thereof or therapeutic molecule may include the production of an ionic bond, a covalent bond, a non-covalent bond, or a combination thereof, between the linker and the antibody, the antigen-binding fragment thereof, or the therapeutic agent. The step of conjugating the linker to the anti-TM4SF1 antibody or antigen-binding fragment thereof or oligonucleotide may, in some cases, be performed as described in Roberts et al., Advanced Drug Delivery Reviews 54:459-476 (2002). The linker may be selected from a bifunctional linker, a cleavable linker, a non-cleavable linker, an ethylene glycol linker, a bifunctional ethylene glycol linker, a flexible linker, or a non-flexible linker. The linker may comprise a chemical group selected from cyclooctyne, cyclopropene, aryl / alkyl azide, transcyclooctene, norborene, and tetrazine. In some embodiments, the terminus of the linker comprises an alkoxyamine. In some embodiments, the terminus of the linker comprises an azide or cyclooctyne group. In some embodiments, the antibody or antibody fragment or therapeutic agent may be attached to the linker by a chemical group selected from cyclooctyne, cyclopropene, aryl / alkyl azide, transcyclooctene, norborene, and tetrazine. Linking the anti-TM4SF1 antibody or antigen-binding fragment thereof or oligonucleotide to the linker may comprise carrying out one or more copper-free reactions. Linking the antibody or antibody fragment or oligonucleotide to the linker may comprise carrying out one or more copper-containing reactions. Linking the anti-TM4SF1 antibody or antigen-binding fragment thereof or oligonucleotide to the linker may comprise one or more cycloadditions.The step of linking the anti-TM4SF1 antibody or antigen-binding fragment thereof or oligonucleotide to the linker may comprise one or more Huisgen cycloadditions. The step of linking the anti-TM4SF1 antibody or antigen-binding fragment thereof or oligonucleotide to the linker may comprise one or more Diels-Alder reactions. The step of linking the anti-TM4SF1 antibody or antigen-binding fragment thereof or oligonucleotide to the linker may comprise one or more hetero Diels-Alder reactions. In some embodiments, the terminus of the linker comprises a leaving group.

[0349] In some embodiments, the first portion of the linker covalently interacts with a cysteine-containing anti-TM4SF1 antibody or antigen-binding fragment thereof as described herein. In some embodiments, the first portion of the linker covalently interacts with a cysteine-containing anti-TM4SF1 antibody or antigen-binding fragment thereof as described herein. In some embodiments, the oligonucleotide described herein covalently interacts with a second portion of the linker. In some embodiments, the oligonucleotide described herein non-covalently interacts with a second portion of the linker.

[0350] In some embodiments, a viral protein p19-based siRNA carrier is contemplated, which has been shown to have high affinity for siRNA. See, for example, Yang et al. Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins, Nucleic Acids Res. 2017 Jul 27; 45(13): 7602-7614. In some examples, a p19-siRNA complex is generated and fused to an anti-TM4SF1 antibody or antigen-binding fragment thereof. In further embodiments, the statistical or random conjugation method is via Cys, Lys, or arginine residues in the antibody or antigen-binding fragment thereof.

[0351] Synthesis of ADCs Comprising Anti-TM4SF1 Antibody or Antigen-Binding Fragment thereof and siRNA In one embodiment, a conjugate comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof and an oligonucleotide is developed by covalent conjugation of the antibody or antigen-binding fragment and an RNA molecule (e.g., siRNA). As a first step in such an exemplary process, an engineered anti-TM4SF1 antibody is generated in which a cysteine ​​residue is introduced into the heavy chain (thereby producing anti-TM4SF1 HC THIOMAB). In some examples, the anti-TM4SF1 thiomab provides at least two separate positions for binding to an RNA molecule, such as to an siRNA. For example, one siRNA molecule can be attached to each heavy chain of the anti-TM4SF1 thiomab. In a separate or subsequent step of the conjugation process, a chemically stable siRNA (e.g., synthesized using siSTABLE chemistry) is generated that is modified with a 3'-amine for binding to a passenger strand having a sequence targeting peptidylpropyl isomerase B (PPIB, cyclophilin B). In some embodiments, the conjugation further comprises a reducible N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) or non-reducible succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) (SMCC) NHS (N-hydroxysuccinimide) linker. In some embodiments, using anti-TM4SF1 thiomab, the exemplary conjugate molecules described in the present disclosure are produced in a multi-step process that includes at least two main steps: (i) reaction of amine-tagged siRNA with the NHS linker to form a thiol-reactive siRNA linker adduct, and (ii) reacting the adduct with the thiol group of THIOMAB, which covalently links the siRNA via a thiol-ester bond. The exemplary ADC is subsequently purified using anion exchange chromatography to remove free siRNA, followed by size exclusion chromatography to remove unbound antibody.Additional techniques, such as gel electrophoresis and electrospray TOF mass spectrometry, can then be used to evaluate the yield of exemplary ADCs, as well as characteristics such as monomeric conjugates with one or two linked siRNAs per antibody.Additional methods that can be used for conjugation include using chemical or peptide-based linkers, chemical or enzymatic conjugation methods (e.g., using mammalian or bacterial transglutaminase), or any combination thereof.Any of the above linkers and / or methods can be used to link the anti-TM4SF1 antibody or its antigen-binding fragment and oligonucleotide of the conjugate.

[0352] Using appropriate conjugation methods, it is possible to generate ADCs of the present disclosure that include, for example, about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 or more ratios of anti-TM4SF1 antibodies or antigen-binding fragments thereof to oligonucleotides. In some embodiments, the ADCs include anti-TM4SF1 antibodies or antigen-binding fragments thereof to oligonucleotides at a 1:1 ratio. This can be accomplished, for example, by using antigen-binding fragments or portions of antibodies, such as half antibodies, Fabs, or other fragments, including cysteine-engineered THIOMAB. In some examples, ADCs can be designed to include a 1:1 ratio of anti-TM4SF1 antibodies or antigen-binding fragments thereof to oligonucleotides using whole antibodies that are conjugated to oligonucleotides by conjugation methods that utilize multi-metalloproteins (e.g., hexarhodium metallopeptides) to allow modification of the protein based on molecular recognition. For example, anti-TM4SF1 antibody or its antigen-binding fragment and oligonucleotide can be conjugated using site-specific antibody functionalization based on molecular recognition of antibody Fc domain constant region by multimetalloprotein. In some embodiments, multimetalloprotein comprises three rhodium complexes that attach to specific sites of protein that bind to antibody Fc domain. Upon binding, multimetalloprotein can catalyze site-specific conjugation of oligonucleotide to antibody. An advantage of using multimetalloprotein can be minimal disruption of antibody, such as by avoiding manipulation of residues in antibody during conjugation.

[0353] Non-cleavable linker In some embodiments, the non-cleavable linker is between the anti-TM4SF1 antibody or antigen-binding fragment thereof and maytansine or a derivative thereof. In some embodiments, the non-cleavable linker is a covalent linker. In some embodiments, the non-cleavable linker is attached to the N-terminus, C-terminus or an internal amino acid position of the anti-TM4SF1 antibody or antigen-binding fragment thereof. In some embodiments, the non-cleavable linker is covalently attached to maytansine or a derivative thereof. In some embodiments, the non-cleavable linker is attached to the N, O, or S of maytansine or a derivative thereof, or to a C1-C6 alkyl, alkenyl, alkynyl, C3 ... 12 Cycloalkyl, C5-C 12 Cycloalkenyl, C8-C 16 Cycloalkynyl, C6-C 12 It is covalently attached to a heteroatom that includes a carbon atom of an aryl, 5- to 12-membered heteroaryl, or 3- to 12-membered heteroalicyclic group.

[0354]

[0337] In some embodiments, the non-cleavable linker is a C1-C6 alkylene, alkenylene, a cycloalkylene having 3-7 ring members, an alkynylene, an arylene, a heteroarylene, a heterocyclene having 5-12 ring members containing 1-3 atoms of N, O, or S, -O-, -NH-, -S-, -N(C 1~6 alkyl)-, -C(=O)-, -C(=O)NH-, or a combination thereof, and C1-C6 alkylene, alkenylene, cycloalkylene having 3-7 membered ring, arylene, heteroarylene, and heterocyclene having 5-12 membered ring containing 1-3 atoms of N, O or S are unsubstituted or substituted by halide, amino, -CF3, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkoxy, C1-C3 alkoxy, or C1-C3 alkylthio.

[0355] In some embodiments, the non-cleavable linker is

[0356] [ka]

[0357] (In the formula, the integer m is 0 to 3, the integer q is 0 to 12, and the integer r is 1 to 3.) Includes.

[0358]

[0339] In some embodiments, the non-cleavable linker is

[0359] [ka]

[0360] wherein each Y and Y is independently a bond, O, S, or NR 100 and; R 100 are independently H, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkyl; 12 Aryl, 5-12 membered heteroaryl, C3-C 12 cycloalkyl, or a 3- to 12-membered heteroalicyclic group, or R1 and another atom of the non-cleavable linker, together with the nitrogen to which R1 is attached, the anti-TM4SF1 antibody or antigen-binding fragment thereof, or maytansine combine to form a 3- to 12-membered heteroalicyclic or 5- to 12-membered heteroaryl group which may contain 1-3 additional heteroatoms selected from the group consisting of N, O, and S; integer m is 0-3, integer q is 0-12, and integer r is 1-3). Includes.

[0361] Synthesis of Antibody Drug Conjugates Containing Non-Cleavable Linkers

[0341] The attachment of the antibody to the drug via a non-cleavable linker can be carried out in various ways. For example, one method is to prepare the antibody and the drug separately. Then, a linker reagent is prepared with two different activated / activatable groups at its termini. The two different activated / activatable groups can selectively react with the antibody and the drug, respectively, thereby completing the synthesis of the antibody drug conjugate. For example, one activated / activatable group can be BrCH2C(=O)-, which can react with the thiol groups of the antibody, especially the surface thiol groups, and attach the linker to the antibody. Another activated / activatable group can be an activated carbonyl, such as an acyl chloride or a mixed anhydride or simply a carboxylic acid, which can react with the amino group of the drug. A covalent bond forming reaction can be used to attach the linker to the antibody or the drug.

[0362] Another method incorporates a linker group or a fragment thereof during the synthesis of the drug. After the synthesis of the drug is completed, modification of the incorporated linker group or a fragment thereof can result in the addition / unmasking of an activated / activatable group at the free end of the linker. The newly revealed activated / activatable group of the linker can react with a surface reactive group (e.g., a thiol group) of the antibody.

[0363]

[0343] Figure 1 shows examples of maytansine-associated ADCs and exemplary pathways leading to their synthesis.

[0364] 6.7 Polynucleotides Also provided in some embodiments are polynucleotides encoding anti-TM4SF1 antibodies or antigen-binding fragments thereof. In some embodiments, the polynucleotide molecules are provided as DNA constructs. In other embodiments, the polynucleotide molecules are provided as messenger RNA transcripts.

[0365] In some examples, an anti-TM4SF1 antibody of the present disclosure comprises a heavy chain variable domain encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs: 4, 16, 28, 40, 52, 64, or 76. In some examples, an anti-TM4SF1 antibody of the present disclosure comprises a light chain variable domain encoded by a nucleic acid sequence set forth in any one of SEQ ID NOs: 10, 22, 34, 46, 58, 70, or 82.

[0366] In some embodiments, a nucleic acid sequence is provided that is codon-optimized for expression in a host cell, e.g., a bacterium such as E. coli, or a eukaryotic cell such as a CHO cell. In some examples, the nucleic acid sequence is codon-optimized for expression in a CHO cell. In some examples, the anti-TM4SF1 antibody of the present disclosure comprises a heavy chain variable domain encoded by a codon-optimized nucleic acid sequence set forth in any one of SEQ ID NOs: 5, 17, 29, 41, 53, 65, or 77. In some examples, the anti-TM4SF1 antibody of the present disclosure comprises a light chain variable domain encoded by a codon-optimized nucleic acid sequence set forth in any one of SEQ ID NOs: 11, 23, 35, 47, 59, 71, or 83. In certain cases, the nucleic acid sequence of any one of SEQ ID NOs: 5, 17, 29, 41, 53, 65, or 77 is a nucleic acid sequence that is codon-optimized for expression in a CHO cell. In certain cases, the nucleic acid sequence of any one of SEQ ID NOs: 11, 23, 35, 47, 59, 71, or 83 is a nucleic acid sequence that is codon-optimized for expression in a CHO cell.

[0367]

[0347] The polynucleotide molecule is constructed by known methods, for example by incorporating the gene encoding the binding protein into a genetic construct linked to a suitable promoter and, optionally, a suitable transcription terminator, and expressing it in bacteria or other suitable expression systems, such as CHO cells. Depending on the vector system and host utilized, a variety of suitable transcription and translation elements can be used, including constitutive and inducible promoters. The promoter is selected such that it drives expression of the polynucleotide in the respective host cell.

[0368] In some embodiments, the polynucleotides described herein are inserted into a vector, preferably an expression vector, which represents a further embodiment. The recombinant vector can be constructed according to known methods. Particularly interesting vectors include plasmids, phagemids, phage derivatives, virii (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0369]

[0349] A variety of expression vector / host systems can be utilized to contain and express the polynucleotides encoding the polypeptides of the described TM4SF1 binding proteins. An example of an expression vector for expression in E. coli is pSKK (Le Gall et al., J Immunol Methods. (2004) 285(1):111-27), or for expression in mammalian cells, pcDNA5 (Invitrogen).

[0370]

[0350] Thus, in some embodiments, the TM4SF1 binding proteins described herein are produced by introducing a vector encoding the above-mentioned protein into a host cell and culturing the host cell under conditions in which the protein domain is expressed, which may be isolated and optionally further purified.

[0371] 6.8 Treatment Methods The present disclosure further provides a method of inhibiting cell-cell interactions that are endothelial cell (EC) specific, but not limited to, for example, EC-EC, EC-mesenchymal stem cell, EC-fibroblast, EC-smooth muscle cell, EC-tumor cell, EC-leukocyte, EC-adipocyte, and EC-neuronal cell interactions. In certain embodiments, ADCs containing the anti-TM4SF1 antibodies and fragments of the present disclosure can be used to treat any human disease or disorder having a pathology characterized by abnormal EC-cell interactions. In certain embodiments, the EC-cell interaction is an EC-leukocyte interaction, and inhibition of EC-leukocyte interaction is used to prevent inflammation.

[0372] In other embodiments, the disclosure focuses on a method of treating or preventing a disease or disorder in a subject, the disease or disorder being characterized by abnormal endothelial cell (EC)-cell interactions, the method comprising administering an antibody or antigen-binding fragment thereof described herein. In certain embodiments, the EC-cell interactions include one or more of EC-mesenchymal stem cells, EC-fibroblasts, EC-smooth muscle cells, EC-tumor cells, EC-leukocytes, EC-adipocytes, and EC-neuronal cell interactions. In an exemplary embodiment, the disease is an inflammatory disease or disorder, and the antibodies and fragments of the disclosure are used to inhibit EC-leukocyte interactions. In another exemplary embodiment, the disease or disorder is selected from inflammatory diseases or cancer. Adhesion of leukocytes to the vascular endothelium is a hallmark of the inflammatory process. Thus, in one embodiment, ADCs containing the disclosed anti-TM4SF1 antibodies or antigen-binding fragments thereof are used to treat inflammatory diseases, where inhibition of leukocyte adhesion to endothelial cells or leukocyte transmigration through the endothelium is useful for treatment (see, e.g., Rychly et al., Curr Pharm Des. 2006;12(29):3799-806, which is incorporated herein by reference in its entirety). Examples include, but are not limited to, sepsis, inflammatory bowel disease, psoriasis, or multiple sclerosis.

[0373] In the United States alone, approximately 500,000 patients die from cancer each year. Tumor metastasis is responsible for approximately 90% of these deaths. There are no known therapies that block metastasis. The present disclosure provides a novel target, an antibody and its antigen-binding fragment that can treat cancer and inhibit metastatic cells based on immune blockade of tumor cell (TC)-endothelial cell (EC) interactions mediated by TM4SF1.

[0374] As noted above, TM4SF1 is a small, tetraspanin-like, cell surface glycoprotein originally discovered as a TC antigen with a role in TC invasion and metastasis. TM4SF1 is selectively expressed by TCs and ECs. TM4SF1 is expressed at low levels in vascular ECs that supply normal tissues in both mice and humans. TM4SF1 has been shown to be expressed at approximately 10-20 fold higher levels in vascular ECs lining the blood vessels that supply many human cancers, and at comparable high levels in cultured ECs. TM4SF1-enriched microdomains (TMEDs) recruit cell surface proteins like integrin and aid in the formation of nanopodia, thin-film channels extending from the cell surface, mediating cell-cell interactions. Thus, in certain instances, ADCs containing the anti-TM4SF1 antibodies and fragments described herein interfere with nanopodia-mediated interactions and inhibit TCs' interactions with ECs, which are necessary for TC extravasation.

[0375] The ADCs of the disclosure may be formulated to treat a subject (e.g., a human) having a disorder associated with pathological angiogenesis (e.g., cancer, such as breast cancer, ovarian cancer, renal cancer, colorectal cancer, liver cancer, gastric cancer, and lung cancer; obesity; macular degeneration; diabetic retinopathy; psoriasis; rheumatoid arthritis; cell-mediated immunity; and rosacea.

[0376] TM4SF1 is highly expressed on the surface of most epithelial TCs, and also on ECs lining tumor blood vessels and in cultured ECs. It is expressed at approximately 10-20 fold lower levels on the surface of normal vascular ECs. In mouse models, tumor metastasis to the lung is associated with TM4SF1 expression on both ECs and TCs. Metastasis requires initial adhesion of TCs to vascular ECs and their subsequent migration through ECs to invade the lung or other metastatic sites. The following examples show that in some instances, the anti-TM4SF1 antibodies of the present disclosure can interfere with TC-EC interactions in culture and also inhibit tumor metastasis in vivo.

[0377]

[0357] Thus, the ADCs of the present disclosure can be used to block one or both of the early steps of metastasis, i.e., TC adhesion to vascular ECs and / or TC transmigration through ECs, thereby preventing metastasis or substantially reducing the number of metastases in at-risk cancer patients.

[0378]

[0358] The present disclosure further provides a method for preventing metastasis. Human tumors typically shed TCs into the blood and lymph at the beginning of growth; therefore, early treatment of primary tumors does not provide a guarantee that metastasis has not yet occurred. Therefore, immune blockade of TM4SF1 can be used to treat or prevent hematogenous metastasis or to treat or prevent lymphatic metastasis.

[0379]

[0359] The methods of the present disclosure, in some embodiments, are directed to inhibiting metastatic cells in a subject. In one embodiment, the subject has cancer, such as a cancer associated with metastasis or a cancer that has already metastasized. In other embodiments, the subject has already been treated for cancer and is in remission or partial remission, and the advantage of administering ADCs containing anti-TM4SF1 antibodies or fragments described herein is that they act to prevent metastasis and maintain remission or partial remission.

[0380]

[0360] In certain embodiments, the present disclosure provides methods of treating humans at high risk of developing metastases, where administration of ADCs containing the anti-TM4SF1 antibodies and fragments described herein can be used to inhibit or delay the onset of metastases.

[0381]

[0361] The present disclosure includes a method of blocking tumor metastasis, particularly metastasis to the lung, by administering an anti-TM4SF1 antibody to a subject in need thereof. In some examples, the anti-TM4SF1 antibody is a human anti-TM4SF1 antibody, also referred to herein as anti-hTM4SF1. In certain embodiments, the method includes administering to a subject in need thereof an ADC containing an effective amount of an anti-hTM4SF1 antibody, wherein the effective amount of the antibody prevents adhesion of tumor cells (TC) to and migration through vascular endothelial cells (EC).

[0382] In certain embodiments, an ADC containing an anti-TM4SF1 antibody is administered to a subject with cancer or at risk of having metastasis at a dosage and frequency that maintains long-term TM4SF1 immune blockade. The dosing regimen maximally inhibits TM4SF1-mediated metastasis by administering an ADC containing an anti-TM4SF1 antibody to a subject in an amount sufficient to saturate TM4SF1 expressed on normal vascular ECs in the subject.

[0383]

[0363] In certain embodiments, the effective amount of the ADC containing an anti-TM4SF1 antibody, or antigen-binding fragment thereof, administered is an amount sufficient to achieve a circulating antibody concentration of >1 μg / ml for one week.

[0384]

[0364] In certain embodiments, the effective amount of the ADC containing an anti-TM4SF1 antibody, or antigen-binding fragment thereof, administered is an amount sufficient to maintain a serum concentration of the antibody at 1 μg / ml or greater for about one continuous month.

[0385]

[0365] In one embodiment, the disclosure provides a method of treating or preventing metastasis in a human subject comprising administering to the subject an ADC containing an effective amount of an anti-TM4SF1 antibody, or antigen-binding fragment thereof, wherein the effective amount of the antibody, or antigen-binding fragment thereof, comprises an amount of the antibody, or antigen-binding fragment thereof, between 1 and 80 mg / kg.

[0386]

[0366] The mode of administration for therapeutic uses of the ADCs of this disclosure can be any suitable route that delivers the antibody to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using tablets, capsules, solutions, powders, gels, particles, and formulations contained in syringes, implantable devices, osmotic pumps, cartridges, micropumps, or other means recognized by those of skill in the art and known in the art. Site-specific administration can be achieved, for example, by intraarticular, intrabronchial, intraperitoneal, intracapsular, intrachondral, intracoelastic, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intraperitoneal, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

[0387] In some embodiments, the ADCs of this disclosure may be administered to a subject by any suitable route, for example, parenterally, intramuscularly or subcutaneously or intraperitoneally by intravenous (iv) infusion or bolus injection. The iv infusion may be given, for example, over 15, 30, 60, 90, 120, 180, or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In some embodiments, the dose given to the subject is about 0.005 mg to about 100 mg / kg, such as about 0.05 mg to about 30 mg / kg or about 5 mg to about 25 mg / kg, or about 4 mg / kg, about 8 mg / kg, about 16 mg / kg or about 24 mg / kg, or for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. In certain embodiments, the dose given to the subject is, for example, about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / kg. In some examples, the dose of the antibody of the present disclosure given to the subject may be about 0.1 mg / kg to 10 mg / kg via intravenous administration. In some examples, the dose of the antibody of the present disclosure given to the subject is about 0.1 mg / kg to 10 mg / kg via subcutaneous administration. In some examples, the dose of the antibody of the present disclosure given to the subject is about 0.1 mg / kg via intravenous administration. In some examples, the dose of the antibody of the present disclosure given to the subject is about 0.1 mg / kg via subcutaneous administration. In some embodiments, the dose of the antibody of the present disclosure given to the subject is about 0.3 mg / kg via intravenous administration. In some examples, the dose of the antibody of the present disclosure given to the subject is about 0.3 mg / kg via subcutaneous administration. In some examples, the dose of the antibody of the present disclosure given to the subject is about 1.0 mg / kg via intravenous administration. In some examples, the dose of the antibody of the present disclosure given to the subject is about 1.0 mg / kg via subcutaneous administration. In some examples, the dose of an antibody of the disclosure given to a subject is about 3.0 mg / kg via intravenous administration.In some examples, the dose of the antibody of the present disclosure given to a subject is about 3.0 mg / kg via subcutaneous administration.In some examples, the dose of the antibody of the present disclosure given to a subject can be about 10.0 mg / kg via intravenous administration.In some examples, the dose of the antibody of the present disclosure given to a subject is about 10.0 mg / kg via subcutaneous administration.

[0388] In certain embodiments, a fixed unit dose of an antibody of the disclosure is given, e.g., 50, 100, 200, 500, or 1000 mg, or the dose can be based on the surface area of ​​the patient, e.g., 500, 400, 300, 250, 200, or 100 mg / m2. In some instances, between 1 and 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) are administered to treat a patient, although 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses are given.

[0389]

[0369] Administration of the ADCs of the disclosure described herein is repeated in some embodiments after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or more. Repeated courses of treatment are also possible for chronic administration. Repeated administrations are at the same dose or different doses. In some examples, the ADCs of the disclosure described herein are administered at 8 mg / kg or 16 mg / kg at weekly intervals for 8 weeks, followed by 8 mg / kg or 16 mg / kg every 2 weeks for an additional 16 weeks, followed by 8 mg / kg or 16 mg / kg every 4 weeks by intravenous infusion. Alternatively, in some embodiments, the ADCs of the disclosure described herein are administered at between 0.1 mg / kg and about 10 mg / kg at weekly intervals for 17 weeks. For example, in some cases, the antibodies of the disclosure may be administered as a daily dose using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, for at least one of the following days: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initiation of treatment, or alternatively, for at least one of the following days: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initiation of treatment. For at least one week of week 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, the dose is provided in an amount of about 0.1 to 100 mg / kg, e.g., 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg per day. In some embodiments, the ADCs of the disclosure described herein are administered prophylactically to reduce the risk of developing an inflammatory disease, e.g., RA, psoriatic arthritis or psoriasis, or to delay the onset of the occurrence of events in the progression of an inflammatory disease, e.g., RA, psoriatic arthritis or psoriasis.In some cases, the ADC of the present disclosure is lyophilized for storage and reconstituted with a suitable carrier before use. In some cases, the antibody of the present disclosure is supplied as a sterile frozen liquid in a glass vial with a flip-off cap stopper and an aluminum seal. In some cases, each vial may contain an ADC containing 3.3 mL of a 50 mg / mL solution of antibody (including 10% excess) in a formulation of 10 mM histidine, 8.5% (w / v) sucrose, and 0.04% (w / v) polysorbate 80 at pH 5.8. In some cases, the vial does not contain a preservative and is for single use. The vial is stored frozen and protected from light. To prepare for IV administration, the ADC formulation is in some cases filtered through a 0.22 micron filter before being diluted into a sterile diluent. In some cases, the diluted ADC, volumetrically adjusted to approximately 100 mL, is administered by IV infusion over a period of at least 30 minutes using an in-line 0.22 micron filter. Alternatively, in some embodiments, the ADC is administered as one or two subcutaneous injections containing about 50 mg / mL antibody in about 3.3 mL. The subcutaneous injection site may be, for example, in the abdominal region.

[0390]

[0370] Patient Treatment

[0371] The compositions or unit dosage forms of the present disclosure may be administered simultaneously to patients with a form of cancer. In some embodiments, the compositions are administered to inhibit tumor growth. In some embodiments, the compositions are administered for their protective effects, even in the absence of clear evidence or diagnosis of a tumor. Because elderly people may develop some types of tumors as they age, preventative treatment using the ADCs of the present disclosure may be beneficial in this patient population for their anti-tumor effects.

[0391] In some embodiments, the present disclosure is directed to the treatment of patients with cancer. In some embodiments, the cancer is hemangiosarcoma. As used herein, the term "hemangiosarcoma" (HSA) may refer to a cancer that arises in the vascular endothelium. HSA may arise inside any of the heart, blood vessels, lymphatics, and spleen. However, HSA prefers the heart, pericardium, and spleen. In addition to humans, the methods of the present disclosure may treat hemangiosarcoma in any species using the ADCs described in this disclosure.

[0392] In some embodiments, other types of cancer occurring in humans may be targeted for treatment as well. Examples of types of human cancer that may be treated by the ADCs of the present disclosure may include, but are not limited to, sarcomas, carcinomas, and adenocarcinomas. Cancers or malignant tumors may be classified by the type of tissue in which they develop. A broad division of cancer separates carcinomas, tumors that arise from epithelial tissue, and sarcomas that arise from all other tissues. Epithelium is tissue that covers the internal or external surfaces of the body. Thus, for example, the skin, the lining of the mouth, the stomach, the intestines, and the bladder are all epithelial tissues.

[0393]

[0374] Within the category of carcinomas, there can be many subdivisions corresponding to the different types of epithelium from which they may originate. Thus, skin, which is made up of a type of epithelium called squamous epithelium, can give rise to squamous cell carcinoma. There are also other epithelial cells present in the skin, such as basal cells, which can give rise to basal cell carcinoma, and melanocytes, which can give rise to melanoma.

[0394] Adenocarcinoma is a cancer that arises in glandular cells. Adenocarcinoma can arise in the lungs, from small glands in the bronchi; in the stomach, from one of several types of glands inside it; in the colon, breast, ovaries, testes, prostate, lungs, and other locations. Adenocarcinomas arising from different organs are often identified under a microscope by a pathologist, even when taken from different locations they have metastasized to, such as the liver. Thus, it can refer to an adenocarcinoma of the stomach that has metastasized to the liver, or one from the colon that has metastasized to the lungs.

[0395]

[0376] The compounds, pharmaceutical compositions, or unit dosage forms of the present invention may be administered alone at appropriate dosages as determined by routine testing to obtain optimal activity while minimizing any potential toxicity.

[0396] Treatment Dosage The daily dosage of the ADC of the present disclosure may vary depending on various factors, such as the underlying disease state, the individual type, species, and health condition, body weight, sex, age, the severity of the condition being treated; the route of administration; and the specific compound used. A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the effective amount of drug required to prevent, counter, or stop the progression of the condition. The optimal formulation to achieve a concentration of drug within the range that produces efficacy without causing toxicity may require a regimen based on the kinetics of the drug's availability to the target site. This may include consideration of the drug's absorption, distribution, metabolism, and excretion, which are easily quantified.

[0397]

[0379] The pharmaceutical compositions or unit dosage forms may be administered in a single daily dose, or the total daily dosage may be administered in distributed doses two, three, or four times daily. In some embodiments, administration of the dosing regimen may be daily, every other day, every third day, weekly, biweekly, monthly, bimonthly, semi-annually, or yearly.

[0398]

[0380] In addition, co-administration or sequential administration of other active agents may be desirable. For combination treatment with more than one active agent, the active agents are in separate dosage formulations and the active agents may be administered simultaneously or separately at different times. Dosage amounts may be adjusted to achieve the desired effect when combined with other active agents as described above. Alternatively, the unit dosage forms of these various active agents may be optimized independently and combined to achieve synergistic results in which lesions are reduced more than when each active agent is used alone.

[0399]

[0381] According to the present disclosure, administration should be continuous and regular so as to sustain the anti-metastatic effect of the formulation for at least the given treatment window. In some embodiments, administration is extended indefinitely.

[0400] The formulations of the present disclosure are administered at any time prior to diagnosis as a prophylactic for tumor development, and preferably after splenectomy to inhibit metastasis. In the present disclosure, the lifespan of patients is extended with a median survival time of more than 80 days, preferably more than 400 days, and most preferably more than 500 days.

[0401]

[0383] As used herein, "extended life span" may refer to the prognosis of cancer only.

[0402] Clinical Benefits and Uses

[0385] The present disclosure provides a first-line treatment regimen for cancer, particularly hemangiosarcoma. The regimen not only extends the lifespan of animals by inhibiting metastasis, but can also be used as a treatment for tumor recurrence associated with cancer. Furthermore, the present invention promotes tumor regression and normal hematological profile. Upon administration of a composition comprising the ADS disclosed herein, animals may not experience weight loss and may have improved coat texture. Additionally, administration of the ADC disclosed herein is not associated with neurological symptoms.

[0403] 6.9 Pharmaceutical Compositions

[0386] In some embodiments, the ADCs of this disclosure may be included in a composition (e.g., a pharmaceutical composition). The pharmaceutical composition of this disclosure may further include a pharma- ceutical acceptable carrier, excipient, or diluent.

[0404]

[0387] As used herein, the term "pharmaceutical composition" refers to a composition containing a TM4SF1 binding protein described herein formulated with a pharma- ceutically acceptable carrier and manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. The pharmaceutical composition may be formulated, for example, for oral administration in a unit dosage form (e.g., tablet, capsule, caplet, gel cap, or syrup), for topical administration (e.g., as a cream, gel, lotion, or ointment), for intravenous administration (e.g., as a sterile solution free of particulate plugs and in a solvent system suitable for intravenous use), or in any other formulation described herein.

[0405]

[0388] As used herein, the term "pharmaceutical acceptable carrier" refers to a carrier that is physiologically acceptable to the mammal (e.g., human) being treated while retaining the therapeutic properties of the protein administered therewith. One exemplary pharmaceutical acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to those of skill in the art and are described, for example, in Remington's Pharmaceutical Sciences (18th edition, A. Gennaro, 1990, Mack Publishing Company, Easton, PA), which is incorporated herein by reference.

[0406]

[0389] In some embodiments, a pharmaceutical composition containing an ADC containing an anti-TM4SF1 antibody or antigen-binding fragment thereof is prepared as an inhalable dosage form, as an intranasal dosage form, as a liposomal formulation, a dosage form comprising nanoparticles, a dosage form comprising microparticles, a polymeric dosage form, or any combination thereof, as a solution, a dispersion in glycerol, liquid polyethylene glycol, and in oil, or any combination thereof in a solid dosage form.

[0407]

[0390] Pharmaceutically acceptable excipients, in some instances, are those excipients described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 1986. Non-limiting examples of suitable excipients include buffers, preservatives, stabilizers, binders, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweeteners, and coloring agents.

[0408]

[0391] In some embodiments, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As buffering agents, in some embodiments, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts or combinations thereof are used in the pharmaceutical compositions of the present disclosure.

[0409]

[0392] In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha tocopherol and ascorbate, and antimicrobial agents, such as parabens, chlorobutanol, and phenol. In some examples, the antioxidant further includes, but is not limited to, EDTA, citric acid, ascorbic acid, butyl hydroxytoluene (BHT), butyl hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some examples, preservatives include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethylketone, Na-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitors, phosphatase inhibitors, caspase inhibitors, granzyme inhibitors, cell adhesion inhibitors, cell division inhibitors, cell cycle inhibitors, lipid signaling inhibitors, protease inhibitors, reducing agents, alkylating agents, antibacterial agents, oxidase inhibitors, or other inhibitors.

[0410]

[0393] In some embodiments, the pharmaceutical compositions described herein include a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohols, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof. Binders used in pharmaceutical formulations are in some instances selected from starches, such as potato starch, corn starch, wheat starch; sugars, such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives, such as crystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols, such as sorbitol, xylitol, mannitol, and water, or any combination thereof.

[0411]

[0394] In some embodiments, the pharmaceutical compositions described herein include a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. In some embodiments, the lubricant used in the pharmaceutical formulation is selected from metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metallic lauryl sulphates (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0412]

[0395] In some embodiments, the pharmaceutical formulation comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersion agents include, in some cases, starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as a high HLB emulsifier surfactant.

[0413]

[0396] In some embodiments, the pharmaceutical compositions described herein include a disintegrant as an excipient. In some embodiments, the disintegrant is a non-foaming disintegrant. Non-limiting examples of suitable non-foaming disintegrants include starches, such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, gums, such as agar, gaur, carob, karaya, pecitin, and tragacanth. In some embodiments, the disintegrant is a foaming disintegrant. Non-limiting examples of suitable foaming disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0414]

[0397] In some embodiments, the excipient comprises a flavoring agent. The flavoring agent incorporated in the outer layer is in some cases selected from synthetic flavor oils and flavoring fragrances; natural oils; extracts from plants, leaves, flowers and fruits, and combinations thereof. In some embodiments, the flavoring agent can be selected from the group consisting of cinnamon oil; oil of wintergreen; peppermint oil; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oils, such as lemon oil, orange oil, grape and grapefruit oil; and fruit extracts, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot.

[0415]

[0398] In some embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as the sodium salt; dipeptide sweeteners, such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (stevioside); chloro derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, sylitol, and the like.

[0416]

[0399] In some examples, the pharmaceutical compositions described herein include colorants. Non-limiting examples of suitable colorants include Food, Drug and Cosmetic Colors (FD&C), Drug and Cosmetic Colors (D&C), and External Drug and Cosmetic Colors (Ext. D&C). Colorants can be used as dyes or their corresponding lakes.

[0417]

[0400] In some instances, the pharmaceutical compositions described herein include a chelating agent. In some instances, the chelating agent is a fungicidal chelating agent. Examples include, but are not limited to, ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N',N'-tetraacetic acid; 1,3-diaminopropane-N,N,N',N'-tetraacetic acid; ethylenediamine-N,N'-diacetic acid; ethyl ethylenediamine-N,N'-dipropionic acid dihydrochloride;ethylenediamine-N,N'-bis(methylenephosphonic acid) hemihydrate;N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid;ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic acid);O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid;N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid;1,6-hexamethylenediamine-N,N,N',N'-tetraacetic acid;N-(2-hydroxyethyl)iminodiacetic acid;Iminodiacetic acid;1,2-diaminopropane-N,N,N',N'-tetraacetic acid;Nitrilotriacetic acid;Nitrilotripropionic acid;Nitrilotris(methylenephosphonic acid(methylenephosphoric trisodium salt of 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide; or triethylenetetramine-N,N,N',N'',N''',N'''-hexaacetic acid.

[0418]

[0401] Also contemplated are combination products comprising an anti-TM4SF1 antibody disclosed herein and one or more other antibacterial or antifungal agents, such as polyenes, e.g., amphotericin B, amphotericin B lipid complex (ABCD), liposomal amphotericin B (L-AMB) and liposomal nystatin, azoles and triazoles, such as voriconazole, fluconazole, ketoconazole, itraconazole, pozaconazole, and the like; glucan synthase inhibitors, e.g., caspofungin, micafungin (FK463), and V-echinocandin (LY303366); griseofulvin; allylamines, e.g., terbinafine; flucytosine or other antifungal agents, including those described herein. In addition, it is contemplated that the peptides may be combined with topical antifungal agents, such as ciclopirox olamine, haloprogin, tolnaftate, undecylenate, topical nysatin, amorolfine, butenafine, naftifine, terbinafine and other topical agents. In some instances, the pharmaceutical composition includes an additional agent. In some instances, the additional agent is present in the pharmaceutical composition in a therapeutically effective amount.

[0419]

[0402] Under normal conditions of storage and use, the pharmaceutical compositions described herein contain a preservative to prevent the growth of microorganisms. In certain examples, the pharmaceutical compositions described herein do not contain a preservative. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Pharmaceutical compositions include a carrier which is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and / or vegetable oils, or any combination thereof. Good fluidity is maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms is brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, an isotonic agent, for example, sugar or sodium chloride, is included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0420] For parenteral administration in aqueous solution, for example, the liquid dosage form is suitably buffered if necessary and the liquid diluent is made isotonic with sufficient saline or glucose. The liquid dosage form is particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, a single dose may be dissolved in 1 mL to 20 mL of isotonic NaCl solution in certain cases and added to 100 mL to 1000 mL of liquid, for example sodium bicarbonate buffered saline, or injected at the proposed site of injection.

[0421]

[0404] In certain embodiments, sterile injectable solutions are prepared by incorporating the immunotherapeutic agent in the required amount in an appropriate solvent with various other ingredients as enumerated above, if necessary, and sterile filtering. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. The compositions disclosed herein are in some instances formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups are in some cases derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, and the like, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, the pharmaceutical compositions are, in some embodiments, administered in a manner and in an amount that is effective for the treatment for which the dosage formulation is administered.

[0422] In certain embodiments, the pharmaceutical composition of the present disclosure comprises an effective amount of an anti-TM4SF1 antibody disclosed herein in combination with a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable" includes any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and / or is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, e.g., oil / water emulsions, various types of wetting agents, and sterile solutions. Further non-limiting examples of pharma- ceutically compatible carriers may include gels, bioabsorbable matrix materials, implantable elements, delivery or dispensing means or materials containing immunotherapeutic agents or any other suitable vehicle. Such carriers are formulated, for example, by conventional methods, and administered to the subject in an effective amount.

[0423] 6.10 Combination Therapy In certain embodiments, the methods of the disclosure include administering an ADC disclosed herein following, preceding, or in combination with one or more additional therapies. Examples of additional therapies may include, but are not limited to, chemotherapy, radiation, anti-cancer agents, or any combination thereof. The additional therapies may be administered simultaneously or sequentially with the administration of the immunotherapy. In certain embodiments, the methods of the disclosure include administering an immunotherapy disclosed herein following, preceding, or in combination with one or more anti-cancer agents or cancer therapies. Anti-cancer agents include, but are not limited to, chemotherapeutic agents, radiotherapeutic agents, cytokines, immune checkpoint inhibitors, anti-angiogenic agents, apoptosis inducers, anti-cancer antibodies, and / or anti-cyclin-dependent kinase agents. In certain embodiments, the cancer therapies include chemotherapy, biological therapy, radiation therapy, immunotherapy, hormonal therapy, anti-vascular therapy, cryotherapy, toxic therapy, and / or surgery, or a combination thereof. In certain embodiments, the method of the present disclosure comprises administering the immunotherapy disclosed herein that is followed by, preceded by, or combined with one or more additional immunomodulators.Immunomodulators include any compound, molecule, or substance that can suppress antiviral immunity associated with tumor or cancer in some cases.Non-limiting examples of additional immunomodulators include anti-CD33 antibody or their variable regions, anti-CD11b antibody or their variable regions, COX2 inhibitors such as celecoxib, cytokines such as IL-12, GM-CSF, IL-2, IFN3 and IFNγ, and chemokines such as MIP-1, MCP-1, and IL-8.

[0424] In certain instances, the additional treatment may have an exemplary radiation dose of 5,000 Rads (50 Gy) to 100,000 Rads (1000 Gy), or 50,000 Rads (500 Gy), or other suitable dose within the ranges described. Alternatively, the radiation dose may be about 30 to 60 Gy, about 40 to about 50 Gy, about 40 to 48 Gy, or about 44 Gy, or other suitable dose within the ranges described, for example, depending on the dose determined by the dosimetry studies described above. As used herein, "Gy" may refer to a specific absorbed dose unit of radiation equal to 100 Rads. Gy is an abbreviation for "Gray."

[0425]

[0408] In certain instances, the additional treatment is chemotherapy, and exemplary chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., nitrogen mustard derivatives, ethylenimines, alkyl sulfonates, hydrazines and triazines, nitrosurea, and metal salts), plant alkaloids (e.g., benca alkaloids, taxanes, podophyllotoxins, and camptothecan analogs), antitumor antibiotics (e.g., anthracyclines, chromomycins, etc.), metabolic antagonists (e.g., folate antagonists, pyrimidine antagonists, purine antagonists, and adenosine deaminase inhibitors), topoisomerase I inhibitors, topoisomerase II inhibitors, and various antineoplastic agents (e.g., ribonucleotide reductase inhibitors, corticosteroid inhibitors, enzymes, microtubule inhibitors, and retinoids). Exemplary chemotherapeutic agents include, but are not limited to, anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Le ustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®,Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea®), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), These include cyclosporine (registered trademark), mitoxantrone (Novantrone®), Mylotarg, paclitaxel (Taxol®), Phoenix (Yttrium 90 / MX-DTPA), pentostatin, carmustine implant with polipheprosan 20 (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®), ibrutinib, idelalisib, and brentuximab vedotin.

[0426] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, and triazenes: uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard (Aminouracil Mustard®), Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), chlorambucil (Leukeran®), pipobacter These include romane (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®).Further exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as actinomycin D, Cosmegen®); melphala...

Claims

1. Formula (III) or (IV) or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 (In the formula: m is an integer from 1 to 12; n is an integer from 1 to 7; Y is MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), ala-phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)hexanoate, 2,5-dioxopyrrolidin-1-yl 5-methyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 5-ethyl-4-(pyridin-2-ylthio)heptanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopropyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclobutyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclopentyl-4-(pyridin-2-ylthio)butanoate, 2,5-dioxopyrrolidin-1-yl 4-cyclohexyl-4-(pyridin-2-ylthio)butanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), or SIAB (N-succinimidyl (4-iodo-acetyl)aminobenzoate); AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof.

2. An antibody-drug conjugate comprising a payload having the formula:

2. Formula (IV) or a pharma- ceutically acceptable salt thereof: 【Chemistry 2】 (In the formula: The integer n is 1 to 7; AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof.

2. The antibody drug conjugate of claim 1, having the formula:

3. Formula (V) or a pharma- ceutically acceptable salt thereof: 【Chemistry 3】 (In the formula: AA is an anti-TM4SF1 antibody or an antigen-binding fragment thereof.

2. The antibody drug conjugate of claim 1, having the formula:

4. 2. The antibody drug conjugate of claim 1, wherein the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a modified IgG1 Fc region, the modified IgG1 Fc region comprises one or more substitutions compared to a wild-type IgG1 Fc region, the modified IgG1 Fc region further comprises a mutation at position N297, the mutation at position N297 is a cysteine, and the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain.

5. The modified IgG1 Fc region comprises: (a) T250Q and M428L; or (b) L234A, L235A, and G237A; or (c) L234A, L235A, G237A, and P331G; or (d) L234A, L235A, G237A, N297C, and P331G; or (e) E233P, L234A, L235A, G237A, and P331G; or (f) E233P, L234A, L235A, G237A, and N297C; or (g) L234A, L235A, G237A, N297C, K322A, and P331G; or (h) E233P, L234A, L235A, G237A, D265A, N297C, K322A, and P331G; or (i) E233P and D265A; or (j) M252Y, S254T, and T256E; or (k) M252Y, S254T, T256E, and N297C; or (l) Combinations thereof The antibody drug conjugate of claim 4, comprising:

6. The antibody drug conjugate of claim 5, wherein the modified IgG1 Fc region comprises the mutations M252Y, S254T, T256E, and N297C.

7. The anti-TM4SF1 antibody or antigen-binding fragment thereof (a) a heavy chain comprising: a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 96, 118, 119, 120, or 121; a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, 19, 31, 43, 55, 67, 79, 95, 116, or 117; and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 94, or 115; and (b) SEQ ID NO: 14, 26, 38, 50, 62, 74, 86, 110, or 129 a light chain comprising: a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 13, 25, 37, 49, 61, 73, 85, 109, or 128; and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 12, 24, 36, 48, 60, 72, 84, 107, 108, 124, 125, 126, or 127. The antibody drug conjugate of claim 1 , comprising:

8. 8. The antibody drug conjugate of claim 7, wherein the heavy chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 3, 15, 27, 39, 51, 63, 75, 90, 92, 112, 114, 130, or 132, and the light chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9, 21, 33, 45, 57, 69, 81, 97, 99, 101, 122, 131, or 133. (i) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO:8, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:7, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:6, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO:14, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:13, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:12; or (ii) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 20, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 19, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 18, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 25, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 24; or (iii) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 32, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 31, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 30, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 38, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 37, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 36; or (iv) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 44, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 43, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 42, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 50, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 49, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 48; or (v) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO:56, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:55, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:54, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO:62, a CDR2 domain comprising the amino acid sequence of SEQ ID NO:61, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO:60; or (vi) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 68, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 67, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 66, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 74, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 73, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 72; or (vii) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 80, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 79, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 78, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 86, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 85, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 84; or (viii) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 96, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 95, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 94, and the light chain comprises the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO:

110. a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 109, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108; or (ix) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 96, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 95, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 94, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 110, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 109, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 107; or (x) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 96, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 95, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 94, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 110, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 109, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 108; or (xi) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 96, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 95, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 94, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 111, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 109, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 107; or (xii) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 96, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 95, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 94, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 111, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 109, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 108; or (xiii) the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 118, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 116, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 115, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 129, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 128, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 124; or (xiv) The antibody drug conjugate of claim 8, wherein the heavy chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, or SEQ ID NO: 121, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 116 or SEQ ID NO: 117, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 115, and the light chain comprises a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 129, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 128, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, or SEQ ID NO:

127.

10. The antibody-drug conjugate described in claim 9, comprising an inhibitor of tubulin polymerization or a derivative thereof, wherein the inhibitor of tubulin polymerization or a derivative thereof is configured to be rendered ineffective by intralysosomal translocation.

11. (1) An inhibitor of tubulin polymerization or a derivative thereof is configured to be effective after nuclear translocation; or (2) the inhibitor of tubulin polymerization or a derivative thereof is configured to be degraded in the lysosome; or (3) The antibody-drug conjugate of claim 10, wherein the inhibitor of tubulin polymerization or a derivative thereof is configured to be trapped in a lysosome.

12. A pharmaceutical composition comprising the antibody-drug conjugate of claim 1 and a pharma- ceutically acceptable excipient.

13. 13. The pharmaceutical composition of claim 12 for use in the treatment or prevention of a disease or disorder in a subject, wherein the disease is characterized by abnormal endothelial cell (EC) interactions, and the EC interactions include one or more of EC-mesenchymal stem cell, EC-fibroblast, EC-smooth muscle cell, EC-tumor cell, EC-leukocyte, EC-adipocyte, and EC-neuronal cell interactions.

14. (1) The disease or disorder comprises an inflammatory disease or cancer, the cancer being a carcinoma or sarcoma, the carcinoma comprising breast cancer, lung cancer, colon cancer, prostate cancer, pancreatic cancer, liver cancer, gastric cancer, kidney cancer, bladder cancer, uterine cancer, cervical cancer, or ovarian cancer, and the sarcoma comprising angiosarcoma, osteosarcoma, or soft tissue sarcoma; or (2) The pharmaceutical composition according to claim 13, wherein the disease or disorder includes lymphatic or hematogenous metastasis.

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