Combinations Comprising Anti-TM4SF1 Antibodies and Immunotherapeutic Agents and Methods of Using Same

Abstract

Anti-TM4SF1 antibodies, and antigen-binding fragments thereof, antibody drug conjugates containing same, and combinations of such antibodies and ADCs with immunotherapies, such as immunomodulatory agents, are described.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 166,805, filed March 26, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002]

[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, e.g., Shih et al. The L6 protein TM4SF1 is critical for endothelial cell function and tumor angiogenesis. Cancer Res. 2009;69(8):3272-7. Summary of the Invention [Means for solving the problem]

[0005]

[0005] One embodiment provides a combination comprising: a. an anti-TM4SF1 binding protein; and b. an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is a cytokine, an adjuvant, or an immune checkpoint inhibitor. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against a target present on a cell of myeloid lineage, a tumor cell, a cell of lymphoid lineage, or a protein present in the tumor microenvironment. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of PD-1, CTLA-4, CD40, CSF1 / CSF1R, SIRPα, CLEC-1. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of CCR4, CTLA-4, A1R, A2AR, A3R, TIM-3, BTLA, VISTA, TIGIT, LAG-3, ILRa / CD25, ITGB1 / CD29, Ly 24 / CD44, CD48, CEACAM1 / CD66a, Nt5e / CD73, CD94 / NKG2A, FAS / CD95, SLAF1 / CD150, NRP1 / CD304, GITR / CD357, ICOS, Tnfrs4 / OX40, Folr4 / JUNO, P2X7, ANXA2, IDO, B7-H6, KIR, GARP (LRRC32), TNFR2. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of PD-L1, PD-L2, B7-H3, B7-H4, CD47, TDO, DcR3. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of exosomes, cytokines, interleukins, or chemokines. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of PS, PD-L1, STING.

[0006]

[0006] In one embodiment, the present invention relates to: a. an anti-TM4SF1 binding protein; and b. ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, spartalizumab, avelumab, sintilimab, toripalimab, MGA012, MGD013, MGD019, enoblitzumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatlimab. ab) (BMS986016), LAG525, IMP321, REGN2810 (cemiplimab), REGN3767, pexidartinib, LY3022855, FPA008, BLZ945, GDC0919, epacadostat, indoximide, BMS986205, CPI-444, MEDI9447, PBF509, lirilumab, IMC-001, monalizumab, and combinations thereof. In some embodiments, an anti-TM4SF1 binding protein comprises a CDR3 domain comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 96, 118, 119, 120, 121, or 162; a CDR2 domain comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 7, 19, 31, 43, 55, 67, 79, 95, 116, 117, or 161; and a CDR1 domain comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 94, 115, or 160. a light chain variable domain; and a CDR3 domain comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 14, 26, 38, 50, 62, 74, 86, 110, 129, or 159; a CDR2 domain comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 13, 25, 37, 49, 61, 73, 85, or 109, 128, or 158; and a CDR1 comprising an amino acid sequence having at least 75% identity to SEQ ID NO: 12, 24, 36, 48, 60, 72, 84, 107, 108, 124, 125, 126, 127, or 157.

[0007] In some embodiments, the anti-TM4SF1 binding protein comprises an IgG Fc region, the IgG Fc region comprising mutations at at least one of the following positions, as numbered by the EU index as described in Kabat: E233, L234, L235, G237, M252, S254, T250Q, T256E, D265, N297, K322, P331, M428, and N434. In some embodiments, the IgG Fc region comprises at least one of the following mutations, as numbered by the EU index as described in Kabat: E233P, L234A, L235A, G237A, M252Y, S254T, T250Q, T256E, D265A, N297C, K322A, P331G, M428L, N434A, and N434S.

[0008] In some embodiments, the anti-TM4SF1 binding protein is conjugated to a therapeutic molecule to form an antibody-drug conjugate, in some embodiments, the therapeutic molecule is selected from the group consisting of a proteasome inhibitor, a calicheamicin, a pyrrolobenzodiazepine, an auristatin, a duocarmycin, a maytansinoid, and any combination thereof. In some embodiments, the therapeutic molecule comprises a proteasome inhibitor, which is selected from the group consisting of: bortezomib (Velcade, PS-341), PR-171 (carfilzomib), ixazomib (Ninlaro®), delanzomib, marizomib, oprozomib, VR23, PI-1840, (benzyloxycarbonyl)-Leu-Leu-phenylalanine, 2,3,5a,6-tetrahydro-6-hydroxy-3-(hydroxymethyl)-2-methyl-10H-3a,10a-epidithio-pyrazinol[1,2α]indole-1,4-Dione, 4-hydroxy-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone, sapojargon, Ac-hFLFL-epoxide, aclacinomycin A, aclarubicin, ACM, AdaK(Bio)Ahx3L3VS, AdaLys(Bio)Ahx3L3VS, adamantane-acetyl-(6-aminohexanoyl)-3-(leucunyl)-3-vinyl-(methyl)-sulfone, ALLM , ALLN, calpain inhibitor I, calpain inhibitor II, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal, gliotoxin, isovaleryl-L-tyrosyl-L-valyl-DL-tyrosinal, clasto-lactacystin-β-lactone, Z-LL-Nva-CHO, ubiquitin aldehyde, YU101, MP-LLL-VS, LDN-5 7444, Z-GPFL-CHO, Z-LLL-CHO, lovastatin, α-methyl-clasto-lactacystin-β-lactone, mevinolin, MK-803, NIP-L3VS, NP-LLL-VS, NPI-0052 (salinosporamide A), MLN519 (PS-519), NLVS (trileucine vinyl sulfone), ritonavir, Ro106-9920, Z-LLF-CHO, Z-LL-B(OH)2, RRRPRPPYLP R, tyropeptin A, ZL3VS, PR-11, PR-39, 0106-9920, proteasome inhibitor I, proteasome inhibitor II, proteasome inhibitor III, proteasome inhibitor IV, AdaAhx3L3VS, efrapeptin, MG-132, MG-262, MG-115, alpha-methyl omuralide, MG-101, epoxomicin, omuralide, lactacystin, and NEOSH101.

[0009]

[0009] One embodiment provides a kit comprising any of the combinations of the above embodiments and instructions for administering an anti-TM4SF1 binding protein and an immunotherapeutic agent to a subject. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are in a single composition. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are in separate compositions.

[0010]

[0010] One embodiment provides a kit comprising a combination of any one of the above embodiments and an anti-TM4SF1 binding protein and an immunotherapeutic agent, and instructions for administering to a subject. In some embodiments, the binding protein and the immunotherapeutic agent are in a single composition. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are in separate compositions. One embodiment provides a kit comprising any one of a. a composition comprising a therapeutically effective amount of an anti-TM4SF1 binding protein; and b. a composition comprising a therapeutically effective amount of an immunotherapeutic agent, and instructions for administering a. and b., either sequentially or simultaneously, to a subject.

[0011]

[0011] In one embodiment, a composition comprising: a. a therapeutically effective amount of an anti-TM4SF1 binding protein; and b. an anti-TM4SF1 antibody, ... and combinations thereof, and instructions for administering a. and b., either sequentially or simultaneously, to a subject.

[0012]

[0012] An embodiment provides a pharmaceutical composition comprising the combination of any one of the above embodiments or the kit of any one of the above embodiments and at least one of a pharma- ceutically acceptable carrier, excipient, diluent, or any combination thereof. An embodiment provides a method of treating a subject, comprising administering a combination comprising: a. an anti-TM4SF1 binding protein; and b. an immune checkpoint inhibitor. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are administered to the subject simultaneously. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are administered to the subject sequentially. In some embodiments, the anti-TM4SF1 binding protein is administered first and the immunotherapeutic agent is administered second. In some embodiments, the immunotherapeutic agent is administered first and the anti-TM4SF1 binding protein is administered second. In some embodiments, the first and second administrations are 1, 2, 3, 6, 12, 24, 48, or more hours apart. In some embodiments, the administration results in an improvement of T cell function. In some embodiments, the improved T cell function comprises increased T cell infiltration in the tumor microenvironment (TME). In some embodiments, the improved T cell function comprises increased expression of ICAM-1 and VCAM-1 on tumor vasculature.

[0013]

[0013] One embodiment is a method of treating a subject comprising: a. an anti-TM4SF1 binding protein; and b. ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, spartalizumab, avelumab, sintilimab, toripalimab, MGA012, MGD013, MGD019, enoblitzumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatlimab. ab) (BMS986016), LAG525, IMP321, REGN2810 (cemiplimab), REGN3767, pexidartinib, LY3022855, FPA008, BLZ945, GDC0919, epacadostat, indoximide, BMS986205, CPI-444, MEDI9447, PBF509, lirilumab, IMC-001, monalizumab, and combinations thereof. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are administered to the subject simultaneously. In some embodiments, the anti-TM4SF1 binding protein and the immunotherapeutic agent are administered to the subject sequentially. In some embodiments, the anti-TM4SF1 binding protein is administered first and the immunotherapeutic agent is administered second. In some embodiments, the immunotherapeutic agent is administered first and the anti-TM4SF binding protein is administered second. In some embodiments, the first and second administrations are 1, 2, 3, 6, 12, 24, 48, or more hours apart. In some embodiments, the administration results in an improvement in T cell function. In some embodiments, the improvement in T cell function comprises increased T cell infiltration in the tumor microenvironment (TME). In some embodiments, the improvement in T cell function comprises increased expression of ICAM-1 and VCAM-1 on tumor vasculature.

[0014]

[0014] One embodiment provides a method of improving a T cell response in a subject, comprising administering a combination according to any one of the above embodiments, wherein the improved response is associated with improved T cell function. In some embodiments, the improved T cell function comprises increased T cell infiltration in the tumor microenvironment (TME). In some embodiments, the improved T cell function comprises increased expression of ICAM-1 and VCAM-1 in tumor vasculature. In some embodiments, the step of administering the combination therapeutic comprises a first treatment and a second treatment. In some embodiments, the first treatment comprises an anti-TM4SF1 binding protein and the second treatment comprises an immune checkpoint inhibitor. In some embodiments, the first treatment comprises an anti-TM4SF1 binding protein and the second treatment comprises a therapeutic agent. In some embodiments, the first treatment and the second treatment are performed within 1, 2, 3, 4, 5, or 7 days. In some embodiments, the combination is administered weekly, biweekly, monthly, or semi-annually.

[0015]

[0015] In some embodiments, the subject is undergoing a treatment that may induce metastasis. In some embodiments, the treatment includes surgery, radiation treatment, and chemotherapy. In some embodiments, the subject has cancer. In some embodiments, the cancer is prostate cancer, liver cancer, colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, kidney cancer, lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS). In some embodiments, the method of any one of claims 26 to 55, wherein the subject is a human.

[0016]

[0016] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0017] [Figure 1]

[0017] Figure 1 shows the binding affinity of capped anti-TM4SF1 antibodies of exemplary antibody group 1 in mouse tumor and endothelial cells. Typically, the binding affinity of antibodies of exemplary antibody group 1 is 3-7 nM EC50 in cell-based assays. [Figure 2-1]

[0018] 2A-2B show the results of in vivo B16F10 (syngeneic to C57Bl / 6 mice, FIG. 2A) and CT26 (syngeneic to BALB / c mice, FIG. 2B) tumor lytic activity using exemplary antibody 1-L1, an anti-TM4SF1 antibody drug conjugate, alone or in combination with an immune checkpoint inhibitor, anti-PD-1 antibody (Clone RMP1-14; BioXCell). In both syngeneic tumor models, the anti-PD-1 antibody alone showed no tumor lytic activity, but when the antibody was co-injected, it enhanced the tumor lytic activity of exemplary antibody 1-L1. [Figure 2-2]

[0018] Figures 2A-2B show the results of in vivo tumor regression activity of B16F10 (syngeneic with C57Bl / 6 mice, Figure 2A) and CT26 (syngeneic with BALB / c mice; Figure 2B) using an exemplary antibody 1-L1, an anti-TM4SF1 antibody-drug conjugate, alone or in combination with an immune checkpoint inhibitor, an anti-PD-1 antibody (Clone RMP1-14; BioXCell). [Diagram 3]

[0019] FIG. 3 shows binding affinity by an exemplary group of capped anti-TM4SF1 antibodies in mouse tumor and endothelial cells. [Figure 4-1]

[0020] Figures 4A-4H show the results of in vivo CT26 (syngeneic to BALB / c mice) tumor growth activity with a single injection of vehicle or isotype-matched control (IS-Ctl), or using an exemplary anti-TM4SF1 antibody drug conjugate, antibody 1-L1, alone or in combination with immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). Figure 4A represents tumor growth with vehicle control and isotype-matched control antibody drug conjugate (IS-Ctl), while Figure 4E shows tumor growth in the presence of an exemplary anti-TM4SF1 antibody drug conjugate, antibody 1-L1. Figures 4B-4D show results from three different doses (2.5, 5, or 10 mpk) of anti-mouse CTLA-4 antibody alone, while Figures 4F-4H show results using three different doses of anti-mouse CTLA-4 antibody in combination with the exemplary anti-TM4SF1 antibody 1-L1. [Figure 4-2]

[0020] Figures 4A-4H show the results of in vivo CT26 (syngeneic to BALB / c mice) tumor growth activity with a single injection of vehicle or isotype-matched control (IS-Ctl), or using an exemplary anti-TM4SF1 antibody drug conjugate, antibody 1-L1, alone or in combination with an immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). [Figure 4-3]

[0020] Figures 4A-4H show the results of in vivo CT26 (syngeneic to BALB / c mice) tumor growth activity with a single injection of vehicle or isotype-matched control (IS-Ctl), or using an exemplary anti-TM4SF1 antibody drug conjugate, antibody 1-L1, alone or in combination with an immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). [Figure 4-4]

[0020] Figures 4A-4H show the results of in vivo CT26 (syngeneic to BALB / c mice) tumor growth activity with a single injection of vehicle or isotype-matched control (IS-Ctl), or using an exemplary anti-TM4SF1 antibody drug conjugate, antibody 1-L1, alone or in combination with an immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). [Figure 5-1]

[0021] Figures 5A-5D show the results of rechallenge of BALB / c mice showing tumor-free (TF) 60-70 days after the first challenge with freshly prepared CT26 tumor cells (as shown in Figures 4A-4H). Eight-week-old naive BALB / c mice and 6- to 8-month-old retired BALB / c breeders were both used as tumor growth controls and were inoculated with the same number of CT26 tumor cells as the rechallenged mice. [Figure 5-2]

[0021] Figures 5A-5D show the results of rechallenge of BALB / c mice with freshly prepared CT26 tumor cells (as shown in Figures 4A-4H), demonstrating tumor-free (TF) 60-70 days after initial administration. [Figure 6-1]

[0022] Figures 6A-6F show the results of in vivo Renca (syngeneic to BALB / c mice) tumor regression activity using the exemplary antibody 1-L1 anti-TM4SF1 antibody drug conjugate alone or in combination with an immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). Figure 6A represents tumor growth with a vehicle control and an isotype-matched control antibody drug conjugate (IS-Ctl), while Figure 6D shows tumor growth in the presence of the exemplary antibody 1-L1 anti-TM4SF1 antibody drug conjugate. Figures 6B-6C show results from two different doses (5, or 10 mpk) of anti-mouse CTLA-4 antibody alone, while Figures 6E-6F show results using the exemplary anti-TM4SF1 antibody 1-L1 combination with two different doses of anti-mouse CTLA-4 antibody. [Figure 6-2]

[0022] Figures 6A-6F show the results of in vivo Renca (syngeneic to BALB / c mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). [Figure 6-3]

[0022] Figures 6A-6F show the results of in vivo Renca (syngeneic to BALB / c mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint anti-mouse CTLA-4 antibody (Clone9H10). [Figure 7-1]

[0023] 7A-7D show BALB / c mice showing no tumors (TF) (as shown in FIGS. 6A-6H) and the results of rechallenge with Renca, a retired BALB / c breeder. [Figure 7-2] 7A-7D show BALB / c mice showing no tumors (TF) (as shown in FIGS. 6A-6H) and the results of rechallenge with retired BALB / c breeder Renca. [Figure 8-1]

[0024] Figures 8A-8H show in vivo tumor regression activity results of B16F10 (syngeneic to C57Bl / 6 mice) using the exemplary antibody 1-L1 anti-TM4SF1 antibody drug conjugate alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). Figure 8A represents tumor growth with a vehicle control and an isotype-matched control antibody drug conjugate (IS-Ctl), while Figure 8E shows tumor growth in the presence of the exemplary antibody 1-L1 anti-TM4SF1 antibody drug conjugate. Figures 8B-8D show results from three different doses (2.5, 5, or 10 mpk) of anti-mouse CTLA-4 antibody alone, while Figures 8F-8H show results using the exemplary anti-TM4SF1 antibody 1-L1 combination with three different doses of anti-mouse CTLA-4 antibody. [Figure 8-2]

[0024] Figures 8A-8H show the results of in vivo B16F10 (syngeneic to C57Bl / 6 mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). [Figure 8-3]

[0024] Figures 8A-8H show the results of in vivo B16F10 (syngeneic to C57Bl / 6 mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). [Figure 8-4]

[0024] Figures 8A-8H show the results of in vivo B16F10 (syngeneic to C57Bl / 6 mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). [Figure 9-1]

[0025] Figures 9A-9H show in vivo LLC (syngeneic to C57Bl / 6 mice) tumor regression activity results using the exemplary antibody 1-L1 anti-TM4SF1 antibody drug conjugate alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). Figure 9A represents tumor growth with vehicle control and isotype-matched control antibody drug conjugate (IS-Ctl), while Figure 9E shows tumor growth in the presence of the exemplary antibody 1-L1 anti-TM4SF1 antibody drug conjugate. Figures 9B-9D show results from three different doses (2.5, 5, or 10 mpk) of anti-mouse CTLA-4 antibody alone, while Figures 9F-9H show results using the exemplary anti-TM4SF1 antibody 1-L1 combination with three different doses of anti-mouse CTLA-4 antibody. [Figure 9-2]

[0025] Figures 9A-9H show the results of in vivo LLC (syngeneic to C57Bl / 6 mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). [Figure 9-3]

[0025] Figures 9A-9H show the results of in vivo LLC (syngeneic to C57Bl / 6 mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). [Figure 9-4]

[0025] Figures 9A-9H show the results of in vivo LLC (syngeneic to C57Bl / 6 mice) tumor regression activity using an anti-TM4SF1 antibody drug conjugate, exemplary antibody 1-L1, alone or in combination with an immune checkpoint CTLA-4 antibody (Clone9H10). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018]

[0026] Transmembrane-4L6 family member-1 (TM4SF1) is a small membrane glycoprotein with the form of a tetraspanin that is highly expressed on most human tumors of solid origin and in endothelial cells, particularly in activated endothelial cells of angiogenic blood vessels.

[0019]

[0027] In one embodiment, provided herein is a combination that can improve tumor regression through the use of vascular targeting therapy (e.g., an antibody that interacts with an endothelial cell biomarker, e.g., an anti-angiogenic antibody, e.g., an anti-TM4SF1 antibody or an antigen-binding fragment thereof, and a payload, e.g., an antibody conjugated to a cytotoxic payload) and an immunotherapeutic agent (e.g., an immune checkpoint inhibitor, cytokine, adjuvant). This combination can include a variety of attractive features. For example, anti-angiogenic antibodies may provide some benefit because at least (1) angiogenesis is a hallmark of cancer, and therapies that destroy angiogenic blood vessels may be a common treatment for solid tumors; (2) vascular endothelium is an unmutated host system and cannot evolve into therapy resistance; thus, vascular targeted therapy may overcome the common problem associated with tumor cell targeted therapy, where the target tissue evolves and becomes therapy resistant; (3) tumor vascular endothelium is directly exposed to drugs injected intravenously (IV), and thus may be accessible to drugs that cannot reach tumor cells; and (4) tumor vascular destruction may provide some advantage, since it promotes immune cell infiltration for wound healing. Immune checkpoint receptors act as T cell receptor (TCR) co-signaling partners that deliver negative signals to T lymphocytes. For example, programmed death protein-1 (PD-1) is a negative co-stimulatory receptor that delivers negative signals to cytotoxic T lymphocytes. PD-1 is important for self-immune tolerance under normal conditions, but when it interacts with its ligands PD-L1 and PD-L2 expressed on tumor cells, it can allow tumors to escape anti-tumor immune responses, leading to increased T cell exhaustion and reduced anti-tumor responses. Blockade of PD-1 with anti-PD-1 antibodies or blockade of PD-L1 / L2 with anti-PD-L1 / PD-L2 antibodies can block the negative signal and help restore immune function of cytotoxic T cells. Inaccessibility of blocking antibodies, such as PD-L1, to reach tumor cells can be a significant challenge in targeting solid tumors, especially tumors that resemble the phenotype of pancreatic cancer, which has a dense fibrous stroma that limits drug access to tumor cells.Therefore, combination therapy using anti-angiogenic antibodies that cause tumor vascular damage and deliver immune blocking antibodies that enhance leukocyte infiltration or help T cells to target tumor cells is an advantageous approach to make cold tumors hot and improve immunotherapy. In another embodiment, the disclosure provides a combination comprising an antibody drug conjugate (ADC) that includes an anti-angiogenic ADC (e.g., an anti-TM4SF1 antibody or antigen-binding fragment thereof conjugated with a payload, e.g., a cytotoxic payload; and an immunotherapeutic agent (e.g., an immune checkpoint inhibitor). The disclosure includes, in some examples, methods of using the above combinations to treat or prevent cancer.

[0020] I. Definition

[0028] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure 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.

[0021]

[0029] 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.

[0022]

[0030] The term "transmembrane-4L6 family member-1" or "TM4SF1" as used herein refers to a polypeptide of the transmembrane 4 superfamily / tetraspanin family expressed on tumor vasculature endothelial cells (EC), EC of the developing retinal vasculature, and more highly expressed in tumor EC, tumor cells (TC), and 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. An exemplary amino acid sequence of human TM4SF1 (hTM4SF1) is set forth in SEQ ID NO: 166 (see also NCBI reference SEQ ID NO: NP_055035.1).

[0023]

[0031] 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.

[0024]

[0032] 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.

[0025]

[0033] The terms "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 an antibody can be obtained from an intact antibody molecule 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 the 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.

[0026]

[0034] 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).

[0027]

[0035] The term "variable region" or "variable domain" of an antibody or its fragment, as used herein, refers to the light and heavy chain portions 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.

[0028]

[0036] 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 as 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.

[0029]

[0037] 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. The common structural features of the variable regions of antibodies or functional fragments thereof are well known in the art. The DNA sequence encoding a particular antibody can generally be found according to well-known methods, for example, the methods 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, a general method for cloning functional variable regions from an antibody can be found in Chaudhary, VK et al., 1990 Proc.Natl.Acad.Sci.USA 87:1066, which is incorporated herein by reference.

[0030]

[0038] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments and a residual "Fc" fragment, a designation reflecting their ability to crystallize readily. The Fab fragment consists of the entire L chain along with the variable region domain (VH) of the H chain and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and are capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having an additional few residues at the carboxy terminus of the CH1 domain that includes one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment contains the carboxy-terminal portions of both H chains linked by disulfide bonds. The effector functions of an antibody are determined by the sequence of the Fc region, which is also the portion recognized by Fc receptors (FcRs) found on certain types of cells. The term "Fc region" is used herein 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 the Fc region of an antibody heavy chain may vary, the Fc region of a human IgG heavy chain is often defined as the extension from the amino acid residue at position 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 antibody populations having all K447 residues removed, antibody populations in which the K447 residues have not been removed, and antibody populations having 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 having an extension of residues after the C-terminal lysine, K447.

[0031]

[0039] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one "amino acid modification" as defined herein. A variant Fc region has at least one amino acid substitution, e.g., about 1 to about 10 amino acid substitutions, about 1 to about 5 amino acid substitutions, compared to a native sequence Fc region or the Fc region of a parent polypeptide. In one embodiment, a variant Fc region herein can have a sequence that has at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, or at least about 99% homology to a native sequence Fc region. According to another embodiment, a variant Fc region herein can have a sequence that has at least about 80% homology, at least about 85% homology, at least about 90% homology, at least about 95% homology, or at least about 99% homology to a parent polypeptide Fc region.

[0032]

[0040] 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, 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.

[0033]

[0041] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up 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 characteristic of the antibody that it is not a mixture of isolated antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence was 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.

[0034]

[0042] The term "chimeric antibody," as used herein, refers to antibodies (immunoglobulins) which have portions of the heavy and / or light chains that 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)).

[0035]

[0043] 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 effects. 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 (non-contiguous) 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. Epitopes can be formed both from contiguous amino acids (usually linear epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of a protein (usually structural epitopes). Epitopes formed from contiguous amino acids are typically, but not always, maintained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or contiguous peptides (e.g., TREM-1) are tested for reactivity with a given antibody (e.g., anti-TREM-1 antibody). Methods for determining the spatial structure of epitopes include those known in the art and described herein, e.g., X-ray crystallography, antigenic variation analysis, two-dimensional nuclear magnetic resonance and HDX-MS (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0036]

[0044] The terms "payload," "drug payload," "therapeutic molecule," "therapeutic payload," "therapeutic agent," and "therapeutic moiety" are used interchangeably herein and refer to a chemical or biological moiety that is conjugated to an anti-TMSF1 antibody or antigen-binding fragment (e.g., an anti-TM4SF1 antibody or antigen-binding fragment disclosed herein) and include any therapeutic or diagnostic agent, including, but not limited to, small molecules for cancer and non-cancer angiogenesis indications; V-ATPase inhibitors; proapoptotic agents; Bcl2 inhibitors; MCL1 inhibitors; HSP90 inhibitors; IAP inhibitors; mTor inhibitors; microtubule stabilizers; microtubule destabilizers; auristatins; dolastatins; maytansinoids; MetAP (methionine aminopeptidase); inhibitors of nuclear export of protein CRM1; DPPIV inhibitors; proteasome inhibitors; inhibitors of phosphoryl transfer reactions in mitochondria; tacrolimus inhibitors; Protein synthesis inhibitors; kinase inhibitors (e.g., CDK2 inhibitors, CDK9 inhibitors); kinesin inhibitors; HDAC inhibitors; DNA damaging agents; DNA alkylating agents; DNA intercalators; DNA minor groove binders; DHFR inhibitors; nucleic acids; CRISPR enzymes; degraders (e.g., agents that induce protein degradation, (e.g., HSP90 inhibitors, selective estrogen receptor degraders (SERDs), selective androgen receptor degraders (SARDs); hydrophobic tags that can be used to recruit chaperones to a protein of interest, e.g., adamantane, Arg-Boc3; ligands that recruit E3 ligases, e.g., Nutlin-3a (MDM2 ligand), bestatin (cIAP ligand), VHL ligand, pomalidomide (CRBN ligand); proteolysis-inducing chimeras (PROTACs) that can utilize different D3 ligases to target a protein of interest for degradation) (e.g., Lai AC,Crews CM.Induced protein degradation: an emerging drug discovery paradigm.Nat Rev Drug Discov.2016;16(2):101-114); antisense oligonucleotides; RNAi agents (e.g., siRNA); CRISPR-Cas9 gene editing systems; RNA molecules; DNA, e.g., plasmids; anti-cancer agents, anti-inflammatory agents, anti-infective agents (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral), anesthetic agents; RNA polymerase II inhibitors; DNA intercalating agents, DNA crosslinking agents; anti-tubulin agents; cytotoxic agents, tumor vaccines, antibodies, peptides, peptibodies, chemotherapeutic agents, cytotoxic agents; cytostatic agents; immunomodulatory agents, interferons, interleukins, immunostimulatory growth hormones, cytokines, vitamins, minerals, aromatase inhibitors, histone deacetylases (HDACs), HDAC inhibitors, lipid nanoparticles encapsulating one or more therapeutic molecules.

[0037]

[0045] 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 where 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.

[0038]

[0046] 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, "KD" or "KD value" can be measured by assays known in the art, such as by binding assays. 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.

[0039]

[0047] The term "k" 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.

[0040]

[0048] 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.

[0041]

[0049] 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.

[0042]

[0050] The term "cancer," as used herein, refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0043]

[0051] The term "cancer with high metastatic risk" as used herein refers to cancer associated with at least one factor that is known to increase the risk that a subject with cancer will develop metastatic cancer. Examples of factors associated with increased metastatic risk include, but are not limited to, the number of cancerous lymph nodes that a subject has at the time of first cancer diagnosis, tumor size, histological grade, and stage of cancer at the time of first diagnosis.

[0044]

[0052] 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.

[0045]

[0053] The term "lymphatic metastasis," as used herein, refers to the ability of cancer cells to enter lymphatic vessels and drain into the bloodstream.

[0046]

[0054] In the context of this disclosure, the term "treat" or "treatment" as used herein means to ameliorate, alleviate, inhibit the progression of, or prevent 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 to inhibit 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.

[0047]

[0055] 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.

[0048]

[0056] 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).

[0049]

[0057] The term "subject," as used herein, refers to a mammal (e.g., a human).

[0050]

[0058] 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).

[0051]

[0059] 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.

[0052]

[0060] 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.

[0053]

[0061] The terms "identity" or "homology" are used interchangeably herein and may be the 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 in 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).

[0054]

[0062] 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 features include uniform glycosylation of the protein, increased cell titer, growth and protein expression during recombinant production of the protein, improved purification properties, 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.

[0055]

[0063] 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 relative to the anti-TM4SF1 antibody produced by the hybridoma mouse cell line 8G4-5-13-13F (PTA-120523) as determined by HLA molecule binding.

[0056]

[0064] In some instances, 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.

[0057]

[0065] 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 activating 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.

[0058]

[0066] 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 the target cells; antibody affinity as well as FcR binding affinity). PBMC-based and natural killer cell-based ADCC assays can be used to detect ADCC. The readout in these assays is end-point driven (target cell lysis).

[0059]

[0067] 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.

[0060]

[0068] The term "effector function" as used herein refers to the function contributed by the Fc effector domain of IgG (e.g., the Fc region of an immunoglobulin). Such function can be influenced, for example, by the binding of the Fc effector domain to an Fc receptor on an immune cell with phagocytic or lytic activity, or by the binding of the Fc effector domain 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.

[0061]

[0069] The term "reduce" or "eliminate" as used herein refers to the ability to cause a total decrease, preferably 20% or more, more preferably 50% or more, and most preferably 75%, 85%, 90%, 95% or more. Reduction or elimination may refer to the binding affinity of two molecules, for example, the binding of immunoglobulin to C1q or Fc receptors; or may refer to the symptoms of the disorder (e.g., cancer) being treated, such as the presence or size of metastases or the size of the primary tumor.

[0062]

[0070] 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).

[0063]

[0071] For all amino acid positions discussed in the present 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).

[0064] II. Anti-TM4SF1 Antibodies or Antigen-Binding Fragments Thereof

[0072] In some embodiments, provided herein is a combination comprising an anti-angiogenic antibody or antigen-binding fragment thereof, wherein the anti-angiogenic antibody or antigen-binding fragment thereof comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof described herein.

[0065]

[0073] 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: 165. MCYGKCARCI GHSLVGLALL CIAANILLYF PNGETKYASE NHLSRFVWFF SGIVGGGLLM LLPAFVFIGL EQDDCCGCCG HENCGKRCAM LSSVLAALIG IAGSGYCVIV AALGLAEGPLCLDSLGQWNYTFASTEGQYLLDTSTWSECTEPKHIVEWNVSLFSILLALG GIEFILCLIQVINGVLGGIC GFCCSHQQQY DC (Sequence number 165)

[0074] 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: 166).

[0066]

[0075] Examples of antibodies that are part of the combinations provided herein include the TM4SF1-specific antibodies provided in Table 2. Examples of anti-TM4SF1 antibodies listed in Table 2 are monoclonal mouse antibodies AGX-A03, AGX-A04, AGX-A05, AGX-A07, AGX-A08, AGX-A09, and AGX-A11, which can bind to the ECL2 region of TM4SF1. Further provided in Table 2 below are humanized versions of some of these antibodies, hAGX-A07 and hAGX-A01.

[0067]

[0076] 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: 87 or 88.

[0068]

[0077] 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.

[0069]

[0078] 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.

[0070]

[0079] 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.

[0071]

[0080] 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.

[0072]

[0081] 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.

[0073]

[0082] 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.

[0074]

[0083] 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.

[0075]

[0084] 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, 78, 94, 115, or 160. 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, 79, 95, 116, 117, or 161.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, 80, 96, 118, 119, 120, 121, or 162.

[0076]

[0085] 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, 84, 107, 108, 124, 125, 126, 127, or 157. 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, 85, 109, 128, or 158.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, 86, 110, 129, or 159.

[0077]

[0086] 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.

[0078]

[0087] 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.

[0079]

[0088] 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:157. 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:158. 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 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:159.

[0080]

[0089] 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:160. 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 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:161. 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 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:162.

[0081]

[0090] The amino acid sequence of mouse monoclonal antibody AGX-A03 is set forth in Table 2. 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.

[0082]

[0091] The amino acid sequence of mouse monoclonal antibody AGX-A04 is set forth in Table 1. 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.

[0083]

[0092] The amino acid sequence of the mouse monoclonal antibody AGX-A05 is set forth in Table 1. 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 mouse monoclonal antibody AGX-A07 is set forth in Table 1. 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.

[0084]

[0093] 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 1, 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 1, 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 1, 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 1, 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).

[0085]

[0094] 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.

[0086]

[0095] 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.

[0087]

[0096] 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).

[0088]

[0097] 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.

[0089]

[0098] 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).

[0090]

[0099] 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.

[0091]

[0100] 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.

[0092]

[0101] The amino acid sequence of mouse monoclonal antibody AGX-A08 is set forth in Table 1. 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.

[0093]

[0102] The amino acid sequence of the murine monoclonal antibody AGX-A09 is set forth in Table 1. 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). 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: 66, 67, and 68, and / or a light chain variable region comprising the CDRs set forth in the amino acid sequences of SEQ ID NOs: 72, 73, and 74, are included in the present disclosure. Humanized antibodies or antigen-binding fragments comprising the CDRs of AGX-A09 are included in the present disclosure. Additionally, the heavy chain variable amino acid sequences and the light chain variable amino acid sequences of AGX-A09 are set forth in SEQ ID NOs: 63 and 69, respectively.

[0094]

[0103] The amino acid sequence of mouse monoclonal antibody AGX-A11 is set forth in Table 1. 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.

[0095]

[0104] The amino acid sequence of humanized antibody AGX-A01 (hAGX-A01) is set forth in Table 1. As shown in Table 1, 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.

[0096]

[0105] 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 the 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 1, the heavy chain sequence set forth in SEQ ID NO: 114 is also referred to herein as AGX-A01H1v1.

[0097]

[0106] 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 1, 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 instances, 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).

[0098]

[0107] 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).

[0099]

[0108] 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.

[0100]

[0109] 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).

[0101]

[0110] 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.

[0102]

[0111] 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.

[0103]

[0112] 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.

[0104]

[0113] 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 1.

[0105]

[0114] 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.

[0106]

[0115] One or more CDRs can also be incorporated, either covalently or non-covalently, into a molecule, making it an antigen binding protein.

[0107]

[0116] 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.

[0108]

[0117] 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.

[0109]

[0118] 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.

[0110]

[0119] 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.

[0111]

[0120] 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 wherein the Fc region comprises a sequence comprising 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%, to 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 sequences include sequences that are 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.

[0112]

[0121] 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 comprising 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.

[0113]

[0122] The anti-TM4SF1 antibodies and fragments described in Table 1 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.

[0114]

[0123] 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).

[0115]

[0124] 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.

[0116]

[0125] 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.

[0117]

[0126] 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).

[0118]

[0127] 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).

[0119]

[0128] 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.

[0120]

[0129] 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.

[0121]

[0130] In certain embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure exhibits a cell proliferation rate of about 1×10 in a standard flow cytometry assay using HUVEC cells. -8 K below M D It binds to human TM4SF1 at

[0122]

[0131] In certain embodiments, the anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure is administered in an amount of about 1×10 -3 M to approx. 1×10 -4 M, about 1 x 10 -4 M to approx. 1×10 -5 M, about 1 x 10 -5 M to approx. 1×10 -6 M, about 1 x 10 -6 From about 1×10 -7 M, about 1 x 10 -7 From about 1×10 -8 M, about 1 x 10 -8 M to approx. 1×10 -9 M, about 1 x 10 -9 M to approx. 1×10 -10 M, about 1 x 10 -10 M to approx. 1×10 -11 M, about 1 x 10 -11 M to approx. 1×10 -12 M, approx. 2 x 10 -3 M to approx. 2 x 10 -4 M, approx. 2 x 10 -4 M to approx. 2 x 10 -5 M, approx. 2 x 10 -5 M to approx. 2 x 10 -6 M, approx. 2 x 10 -6 From about 2 × 10 -7 M, approx. 2 x 10 -7 From about 2 × 10 -8 M, approx. 2 x 10 -8 M to approx. 2 x 10 -9 M, approx. 2 x 10 -9 M to approx. 2 x 10 -10 M, approx. 2 x 10 -10 M to approx. 2 x 10 -11 M, approx. 2 x 10 -11 M to approx. 2 x 10 -12 M, about 3 x 10 -3 M to approx. 3 x 10 -4 M, about 3 x 10 -4 M to approx. 3 x 10 -5 M, about 3 x 10 -5 M to approx. 3 x 10 -6 M, about 3 x 10 -6 From about 3 × 10 -7 M, about 3 x 10 -7 From about 3 × 10 -8 M, about 3 x 10-8 M to approx. 3 x 10 -9 M, about 3 x 10 -9 M to approx. 3 x 10 -10 M, about 3 x 10 -10 M to approx. 3 x 10 -11 M, about 3 x 10 -11 M to approx. 3 x 10 -12 M, approx. 4 x 10 -3 M to approx. 4 x 10 -4 M, approx. 4 x 10 -4 M to approx. 4 x 10 -5 M, approx. 4 x 10 -5 M to approx. 4 x 10 -6 M, approx. 4 x 10 -6 From about 4 × 10 -7 M, approx. 4 x 10 -7 From about 4 × 10 -8 M, approx. 4 x 10 -8 M to approx. 4 x 10 -9 M, approx. 4 x 10 -9 M to approx. 4 x 10 -10 M, approx. 4 x 10 -10 M to approx. 4 x 10 -11 M, approx. 4 x 10 -11 M to approx. 4 x 10 -12 M, about 5 x 10 -3 M to approx. 5 x 10 -4 M, about 5 x 10 -4 M to approx. 5 x 10 -5 M, about 5 x 10 -5 M to approx. 5 x 10 -6 M, about 5 x 10 -6 From about 5 × 10 -7 M, about 5 x 10 -7 From about 5 × 10 -8 M, about 5 x 10 -8 M to approx. 5 x 10 -9 M, about 5 x 10 -9 M to approx. 5 x 10 -10 M, about 5 x 10 -10 M to approx. 5 x 10 -11 M, about 5 x 10 -11 M to approx. 5 x 10 -12 M, about 5 x 10 -7 M to approx. 5 x 10 -11 M, about 5 x 10 -7 M, about 1 x 10 -7 M, about 5 x 10 -8 M, about 1 x 10-8 M, about 5 x 10 -9 M, about 1 x 10 -9 M, about 5 x 10 -10 M, about 1 x 10 -10 M, about 5 x 10 -11 M or approx. 1×10 -11 K of M D In some embodiments, the K D is determined in a standard flow cytometry assay using HUVEC cells.

[0123]

[0132] In certain embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure exhibits a cell proliferation rate of about 5×10 in a standard flow cytometry assay using HUVEC cells. -10 K below M D It binds to human TM4SF1 at

[0124]

[0133] In certain embodiments, an anti-TM4SF1 antibody or antigen-binding fragment thereof of the present disclosure exhibits a cytotoxicity of about 1×10 in a standard flow cytometry assay using HEK293 overexpressing cells. -6 K below M D In one embodiment, HEK293 cells are transfected to express cynomolgus TM4SF1. In a further embodiment, HEK293 cells are transfected at 10 6 Cynomolgus TM4SF1 is expressed at approximately 600 mRNA copies per 18S rRNA copy.

[0125]

[0134] K of an antibody or antibody fragment D Methods for determining the K of an antibody to an antigen are known in the art. For example, surface plasmon resonance can be used to determine the K of an antibody to an antigen. D(e.g., using a BIACORE2000 or BIACORE3000 (BIAcore, Inc., Piscataway, NJ) at 25° C. with immobilized antigen or Fc receptor CM5 chips at about 10 response units (RU). In certain embodiments, FACS or flow cytometry can be used to determine the K D To determine the K, cells expressing TM4SF1, such as HEK293 cells or HUVEC cells, are bound to the antibody or fragment and assayed for K according to standard methods. D 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 antibody affinity.

[0126]

[0135] 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 a CDR amino acid sequence 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 1. 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.

[0127]

[0136] The disclosure further provides, in one embodiment, a method for producing a medicament for the treatment of a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of at least about 5×10 mAb to a patient in need thereof, as determined by a standard flow cytometry assay using HUVEC cells. -8 K below M Dand wherein the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises 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 TM4SF1.

[0128]

[0137] In another embodiment of the disclosure, the anti-TM4SF1 antibody or antigen-binding fragment thereof has a titer of about 5×10 as determined by a standard flow cytometry assay using HUVEC cells. -8 K below M D and a humanized anti-TM4SF1 antibody or antigen-binding fragment thereof that binds to an epitope on the ECL2 loop of human TM4SF1. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to an epitope on the ECL2 loop of human TM4SF1 at about 1×10 -6 K below M D In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to cynomolgus monkey TM4SF1 at about 1×10 -8 K below M D In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof binds to human TM4SF1 at a concentration of 1×10 -3 M to approx. 1×10 -4 M, about 1 x 10 -4 M to approx. 1×10 -5 M, about 1 x 10 -5 M to approx. 1×10 -6 M, about 1 x 10 -6 From about 1×10 -7 M, about 1 x 10 -7 From about 1×10 -8 M, about 1 x 10 -8 M to approx. 1×10 -9 M, about 1 x 10-9 M to approx. 1×10 -10 M, about 1 x 10 -10 M to approx. 1×10 -11 M, about 1 x 10 -11 M to approx. 1×10 -12 M, approx. 2 x 10 -3 M to approx. 2 x 10 -4 M, approx. 2 x 10 -4 M to approx. 2 x 10 -5 M, approx. 2 x 10 -5 M to approx. 2 x 10 -6 M, approx. 2 x 10 -6 From about 2 × 10 -7 M, approx. 2 x 10 -7 From about 2 × 10 -8 M, approx. 2 x 10 -8 M to approx. 2 x 10 -9 M, approx. 2 x 10 -9 M to approx. 2 x 10 -10 M, approx. 2 x 10 -10 M to approx. 2 x 10 -11 M, approx. 2 x 10 -11 M to approx. 2 x 10 -12 M, about 3 x 10 -3 M to approx. 3 x 10 -4 M, about 3 x 10 -4 M to approx. 3 x 10 -5 M, about 3 x 10 -5 M to approx. 3 x 10 -6 M, about 3 x 10 -6 From about 3 × 10 -7 M, about 3 x 10 -7 From about 3 × 10 -8 M, about 3 x 10 -8 M to approx. 3 x 10 -9 M, about 3 x 10 -9 M to approx. 3 x 10 -10 M, about 3 x 10 -10 M to approx. 3 x 10 -11 M, about 3 x 10 -11 M to approx. 3 x 10 -12 M, approx. 4 x 10 -3 M to approx. 4 x 10 -4 M, approx. 4 x 10 -4 M to approx. 4 x 10 -5 M, approx. 4 x 10 -5 M to approx. 4 x 10 -6 M, approx. 4 x 10 -6 From about 4 × 10-7 M, approx. 4 x 10 -7 From about 4 × 10 -8 M, approx. 4 x 10 -8 M to approx. 4 x 10 -9 M, approx. 4 x 10 -9 M to approx. 4 x 10 -10 M, approx. 4 x 10 -10 M to approx. 4 x 10 -11 M, approx. 4 x 10 -11 M to approx. 4 x 10 -12 M, about 5 x 10 -3 M to approx. 5 x 10 -4 M, about 5 x 10 -4 M to approx. 5 x 10 -5 M, about 5 x 10 -5 M to approx. 5 x 10 -6 M, about 5 x 10 -6 From about 5 × 10 -7 M, about 5 x 10 -7 From about 5 × 10 -8 M, about 5 x 10 -8 M to approx. 5 x 10 -9 M, about 5 x 10 -9 M to approx. 5 x 10 -10 M, about 5 x 10 -10 M to approx. 5 x 10 -11 M, about 5 x 10 -11 M to approx. 5 x 10 -12 M, about 5 x 10 -7 M to approx. 5 x 10 -11 M, about 5 x 10 -7 M, about 1 x 10 -7 M, about 5 x 10 -8 M, about 1 x 10 -8 M, about 5 x 10 -9 M, about 1 x 10 -9 M, about 5 x 10 -10 M, about 1 x 10 -10 M, about 5 x 10 -11 M or approx. 1×10 -11 K of M D In some embodiments, the K Dis determined in a standard flow cytometry assay using HUVEC cells. In one embodiment, the anti-TM4SF1 antibody or antigen-binding fragment thereof is at least about 5×10 in a standard flow cytometry assay using HUVEC cells expressing TM4SF1. -10 K below M D It binds to human TM4SF1 at

[0129]

[0138] 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).

[0130]

[0139] 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.

[0131]

[0140] 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.

[0132]

[0141] 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.

[0133]

[0142] 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.

[0134]

[0143] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, and scFv.

[0135]

[0144] 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).

[0136]

[0145] 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)).

[0137]

[0146] 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.

[0138]

[0147] 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)).

[0139]

[0148] 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.

[0140]

[0149] 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.

[0141]

[0150] 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.

[0142]

[0151] 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.

[0143]

[0152] 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.

[0144]

[0153] 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.

[0145]

[0154] 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.

[0146]

[0155] Screening of the library can be accomplished by various techniques known in the art. For example, TM4SF1 (e.g., the soluble form of the ECL2 loop or cells expressing said 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 a 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 by using low coating density of antigen as described in Marks et al., 1992, Biotechnol.10:779-83.

[0147]

[0156] 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.

[0148]

[0157] 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.

[0149]

[0158] 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.

[0150]

[0159] One embodiment of the present disclosure provides a combination comprising an anti-angiogenic antibody, such as an anti-TM4SF1 antibody or antigen-binding fragment thereof, and an immunotherapeutic agent, wherein the anti-TM4SF1 antibody or antigen-binding fragment thereof comprises a modified Fc region, such as a modified IgG region (e.g., IgG1, IgG2, IgG3, IgG4) comprising one or more mutations. In some cases, the 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 regulation, 3) stability, and 4) downstream process improvement. In some cases, the modified Fc region may comprise one or more mutations that reduce or eliminate the interaction between the antibody and the immune system. Important interactions may include the interaction of antibody Fc with Fcγ receptors on white blood cells and platelets, and C1q of the complement system, which results in complement-dependent cytotoxicity.

[0151]

[0160] The present disclosure provides, in some cases, for example, a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof, which comprises an immunoabolishing mutation in the Fc region, in such case 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 examples, 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, including 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).

[0152]

[0161] In some embodiments, a combination comprising an anti-TM4SF1 antibody or an antigen-binding fragment thereof is provided, which 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.

[0153]

[0162] In some embodiments, a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof is provided and 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, an 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).

[0154]

[0163] In some embodiments, a combination is provided that includes an anti-TM4SF1 antibody or antigen-binding fragment thereof, comprising the mutations D265A and N297A. In some cases, the proline at position 329 (P329) of the wild-type human Fc region can 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 W110 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.

[0155]

[0164] In some embodiments, a combination is provided comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof, which comprises 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 comprises 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 show 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).

[0156]

[0165] In some embodiments, a combination is provided comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising an Fc variant comprising a triple mutation: an amino acid substitution at position P329, L234A and L235A mutations (P329 / LALA) (see, e.g., US8969526).

[0157]

[0166] Certain anti-TM4SF1 antibodies or antigen-binding fragments of the present disclosure may contain mutations that exhibit improved or diminished binding to FcR in some embodiments of the combinations provided herein (see, e.g., US6737056, WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0158]

[0167] In some examples, combinations are provided that include anti-TM4SF1 antibodies or antigen-binding fragments that may include an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334. For example, modifications can 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.

[0159]

[0168] Antibodies with increased half-life and improved binding to neonatal Fc receptor (FcRn) are also provided herein in some combination embodiments. Named for its function for the transfer of maternal IgG to 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 are able to detach from TM4SF1, bind to FcRn, and then recycle the antibody back to circulation, thus reducing vascular toxicity. In some embodiments, combinations are provided herein that include anti-TM4SF1 antibodies or antigen-binding fragments that include an Fc region with one or more substitutions that enhance FcRn recycling. In some embodiments, provided herein is a combination comprising 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.

[0160]

[0169] In some embodiments, the present disclosure provides a combination comprising an anti-TM4SF1 antibody or its antigen-binding fragment, which has pH-dependent FcRn binding affinity. Without being bound to any particular theory, it is believed that an antibody or its antigen-binding fragment, which has pH-dependent FcRn binding affinity, will leave FcRn at pH>7 and bind to FcRn at pH6. Thus, FcRn in intracellular organelles with acidic pH, such as endosomes, will bind such antibody, transport the antibody back to the cell membrane, and release the antibody into plasma at pH>7, recycling the antibody.

[0161]

[0170] In certain embodiments, the present disclosure provides a combination comprising 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, the present disclosure provides a combination comprising 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 present disclosure provides a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a substitution at one or more of positions 250, 252, 254, 256, 428, and 434 according to EU numbering. In some embodiments, the present disclosure provides a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising an Fc variant comprising one or more of the following substitutions: T250Q, M252Y, S254T, T256E, M428L, and N434S. In some embodiments, the combinations provided herein include an anti-TM4SF1 antibody or antigen-binding fragment thereof, which includes an IgG1 Fc variant including substitutions T250Q and M428L ("QL mutation"). In some embodiments, the combinations provided herein include an anti-TM4SF1 antibody or antigen-binding fragment thereof, which may include an IgG4 Fc variant including substitutions T250Q and M428L ("QL mutation"). In some embodiments, the combinations provided herein include an anti-TM4SF1 antibody or antigen-binding fragment thereof, which includes an IgG1 Fc variant including substitutions M252Y, S254T, and T256E ("YTE mutation"). In some embodiments, the combinations provided herein include an anti-TM4SF1 antibody or antigen-binding fragment thereof, which includes an IgG1 Fc variant including substitutions M428L and N434S ("LS mutation"). In some embodiments, the combination provided herein comprises an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that comprises an IgG4 Fc variant comprising the substitutions M428L and N434S (the "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.

[0162]

[0171] In certain embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG1 isotype and comprises an Fc region that comprises one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, M428L, and N434S. In some embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG4 isotype and comprises an Fc region that comprises one or more substitutions selected from the group consisting of T250Q, M252Y, S254T, T256E, M428L, and N434S. In some embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG1 isotype and comprises an Fc region that comprises substitutions T250Q and M428L. In some embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG1 isotype and comprises an Fc variant that includes substitutions M252Y, S254T, and T256E. In some embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG4 isotype and comprises an Fc variant that includes substitutions M252Y, S254T, and T256E. In some embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG1 isotype and comprises an Fc variant that includes substitutions M428L and N434S. In some embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG4 isotype and comprises an Fc variant that includes substitutions M428L and N434S.

[0163]

[0172] In some embodiments, the combinations disclosed herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a mutated Fc region with increased FcRn binding affinity and increased serum half-life. In certain embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising a mutated Fc region with 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.

[0164]

[0173] In certain embodiments, the combination comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that exhibits reduced vascular toxicity, improved therapeutic margins, or both. In certain embodiments, the combination of the present disclosure comprises 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").

[0165]

[0174] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising 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 combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof comprising 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.

[0166]

[0175] In certain embodiments, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of IgG1 isotype and comprises an Fc region that includes 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.

[0167]

[0176] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG1 isotype and comprises an Fc region that includes L234A / L235A mutations, with or without the 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.

[0168]

[0177] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is of the IgG1 isotype and comprises an Fc region that includes the A327G / A330S / P331S mutations.

[0169]

[0178] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG1 isotype and comprises an Fc region that comprises 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.

[0170]

[0179] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of IgG1 isotype and comprises an Fc region that includes an N297x mutation (wherein x=A, D, G, Q).

[0171]

[0180] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is of the IgG1 isotype and comprises an Fc region that includes the A327G / A330S / P331S mutations.

[0172]

[0181] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of the IgG1 isotype and comprises an Fc region that includes 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, which is incorporated herein by reference in its entirety.

[0173]

[0182] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that 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.

[0174]

[0183] In other embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is of the IgG1 isotype and comprises an Fc region that includes an L234A / L235A, G237A, or L235E mutation.

[0175]

[0184] In other embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is of the IgG1 isotype and comprises an Fc region that includes an L234F, an L235E, or a P331S mutation.

[0176]

[0185] In certain embodiments, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof, which is an IgG2 isotype and comprises an Fc region comprising one or more mutations selected from the group consisting of V234A, G237A, P238S, H268A or H268Q, V309L, A330S and P331S.

[0177]

[0186] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody, or an antigen-binding fragment thereof, that is of the IgG2 isotype and comprises an Fc region that includes the A330S / P331S mutations.

[0178]

[0187] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG2 isotype and comprises an Fc region that includes A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S or H268Q / V309L / A330S / P331S mutations.

[0179]

[0188] In other embodiments, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of IgG4 isotype and comprises an Fc region that contains one or more mutations selected from the group consisting of S228P, E233P, F234A, F234V, L235E, L235A, G236delta (deletion), N297A, N297D, N297G, N297Q, P329G, P329R.

[0180]

[0189] In certain embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of an IgG4 isotype and comprises an Fc region that includes 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 herein by reference in its entirety.

[0181]

[0190] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody, or an antigen-binding fragment thereof, that is of the IgG4 isotype and comprises an Fc region that includes S228P / L235E mutations.

[0182]

[0191] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of IgG4 isotype and comprises an Fc region that includes S228P / E233P / F234V / L235A / delta G236 (deletion) mutations.

[0183]

[0192] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is of IgG4 isotype and comprises an Fc region that includes an N297x mutation (wherein x=A, D, G, Q).

[0184]

[0193] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody, or an antigen-binding fragment thereof, that is of the IgG4 isotype and comprises an Fc region that includes the S228P / F234A / L235A mutations.

[0185]

[0194] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG4 isotype and comprises an Fc region that includes a 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.

[0186]

[0195] In other embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody or antigen-binding fragment thereof that is an IgG4 isotype and comprises an Fc region that includes S228P / F234A / L235A or E233P / L235A / G236D mutations.

[0187]

[0196] In one embodiment, the combination provided herein comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof, which is of IgG4 isotype and comprises an Fc region containing at least an S228P mutation.See, for example, Angal et al. (Mol Immunol. 1993 Jan;30(1):105-8) who describe the analysis of the hinge sequence of human IgG4 heavy chain to determine that the presence of serine at residue 241 (according to the EU numbering system, here corresponding to residue 228 in Kabat numbering) is responsible for the heterogeneity of inter-heavy chain disulfide bridges in the hinge region in a given percentage of secreted human IgG4.See, Silva et al. (J Biol Chem. 2015 Feb 27;290(9):5462-9) who describe the S228P mutation in human IgG4 that prevents IgG4 Fab-arm exchange in vivo and in vitro.

[0188]

[0197] In other embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is of the IgG4 isotype and comprises an Fc region that includes an L235E or S228P mutation.

[0189]

[0198] In other embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is an IgG4 or IgG1 isotype and comprises an Fc region that includes an N297A, N297D, or N297G mutation.

[0190]

[0199] In other embodiments, the combinations provided herein comprise an anti-TM4SF1 antibody, or antigen-binding fragment thereof, that is an IgG4 or IgG1 isotype and comprises an Fc region that includes a P329G, P329R mutation.

[0191]

[0200] In an exemplary embodiment, the mutated Fc region of any IgG isotype comprises one or more mutations at positions 234, 235, 236, 237, 297, 318, 320, 322 (as described in WO1988007089, which is incorporated by reference in its entirety). Other possible mutations in the Fc region, such as substitutions, deletions and additions, are also described in, for example, US20140170140, WO2009100309, US20090136494 and US8969526, which are incorporated by reference in their entireties.

[0192]

[0201] 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).

[0193]

[0202] In some embodiments, the mutated Fc region of any IgG isotype comprises a mutation at position L328, e.g., L328M, L328D, L328E, L328N, L328Q, L328F, L328I, L328V, L328T, L328H, L328A (see, e.g., US20050054832).

[0194]

[0203] In one embodiment, the antibodies or antigen-binding fragments thereof of the combinations provided herein exhibit reduced or abolished ADCC effector function compared to the unmodified antibody. In another embodiment, the antibodies or antigen-binding fragments thereof of the combinations provided herein 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 or antigen-binding fragments thereof of the combinations provided herein 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 combinations provided herein, the reduction or downregulation of ADCC effector function induced by the antibodies or antigen-binding fragments 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 antibodies or antigen-binding fragments thereof of the present disclosure for Fc ligands and / or receptors.

[0195]

[0204] One embodiment of the present disclosure provides a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof and an immunotherapeutic agent, 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 by any 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.

[0196]

[0205] Also disclosed herein is a method of making an anti-TM4SF1 antibody or antigen-binding fragment thereof for the combinations provided herein with increased half-life and / or pharmacodynamic effect 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 an 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.

[0197]

[0206] In some embodiments, the combinations provided herein include anti-TM4SF1 antibodies or antigen-binding fragments thereof that contain one, two, three, four, five or more histidine substitutions in the CDR regions. 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, combinations with histidine-engineered anti-TM4SF1 antibodies or antigen-binding fragments thereof are provided herein. In some embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments thereof contain one or more histidine residues in CDR1, CDR2, and / or CDR3 of the light chain variable region (VL). In some embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments thereof contain one or more histidine residues in CDR1 of the light chain variable region (VL). In some embodiments, the anti-TM4SF1 antibodies or antigen-binding fragments thereof contain 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 combination of the present disclosure comprises a histidine engineered anti-TM4SF1 antibody or antigen-binding fragment thereof.

[0198]

[0207] 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.

[0199] III. Antibody Drug Conjugates

[0208] In one embodiment, provided herein is a combination comprising an immunotherapeutic agent and an anti-angiogenic antibody or antigen-binding fragment thereof linked to a payload. In some embodiments, the antibody is an anti-TM4SF1 antibody or antigen-binding fragment thereof. In some embodiments, the payload is a small molecule, a protein, a peptide, an antibody, or any combination thereof. In some embodiments, provided is a combination comprising an ADC comprising an anti-angiogenic antibody (e.g., an anti-TM4SF1 antibody or antigen-binding fragment thereof) conjugated to a therapeutic or diagnostic agent.

[0200]

[0209] In some embodiments, the therapeutic agent is a biologically active moiety. In some embodiments, biologically active moieties include radioisotopes, cytotoxic agents, chemotherapeutic agents, proteins, peptides, antibodies, growth inhibitors, prodrug activating enzymes, and antihormonal agents. In some embodiments, the therapeutic molecule is a small molecule (e.g., for both cancer and non-cancer angiogenesis indications); V-ATPase inhibitors; proapoptotic agents; Bcl2 inhibitors; MCL1 inhibitors; HSP90 inhibitors; IAP inhibitors; mTor inhibitors; microtubule stabilizing agents; microtubule destabilizing agents; auristatins; dolastatins; maytansinoids; MetAP (methionine aminopeptidase); inhibitors of nuclear export of protein CRM1; DPPIV inhibitors; proteasome inhibitors; inhibitors of phosphoryl transfer reactions in mitochondria; protein synthesis inhibitors; kinase inhibitors; (e.g., CDK2 inhibitors, CDK9 inhibitors); kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA interfering agents, and the like. degraders (e.g., drugs that induce protein degradation, (e.g., HSP90 inhibitors, selective estrogen receptor degraders (SERDs), selective androgen receptor degraders (SARDs); hydrophobic tags that can be used to recruit chaperones to a protein of interest, e.g., adamantane, Arg-Boc3; ligands that recruit E3 ligases, e.g., Nutlin-3a (MDM2 ligand), bestatin (cIAP ligand), VHL ligand, pomalidomide (CRBN ligand); proteolysis-inducing chimeras (PROTACs) that can utilize different D3 ligases to target a protein of interest for degradation) (e.g., Lai AC, Crews CM. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov.2016;16(2):101-114); antisense oligonucleotides; RNAi agents (e.g., siRNA); CRISPR-Cas9 gene editing systems; RNA molecules; DNA, e.g., plasmids; anti-cancer agents, anti-inflammatory agents, anti-infective agents (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral), anesthetic agents; RNA polymerase II inhibitors; DNA intercalating agents, DNA crosslinking agents; anti-tubulin agents; cytotoxic agents, tumor vaccines, antibodies, peptides, peptibodies, chemotherapeutic agents, cytotoxic agents; cytostatic agents; immunomodulatory agents, interferons, interleukins, immunostimulatory growth hormones, cytokines, vitamins, minerals, aromatase inhibitors, histone deacetylases (HDACs), HDAC inhibitors, lipid nanoparticles encapsulating one or more therapeutic molecules.

[0201]

[0210] In some embodiments, the radioisotope is 211 At, 131 I, 125 I, 90 Y, 186 Re, 153 Sm, 212 Bi, 32 The prodrug activating enzyme may be, but is not limited to, one or more species selected from the group consisting of: P, and radioactive isotopes of Lu. In some embodiments, the prodrug activating enzyme is, but is not limited to, one or more species selected from the group consisting of: alkaline phosphatase, arylsulfatase, cytosine deaminase, protease, D-alanyl carboxypeptidase, carbohydrate cleaving enzyme, P-lactamase, and penicillin amidase.

[0202]

[0211] In some embodiments, the cytotoxic agent includes, but is not limited to, one or more selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, Pseudomonas aeruginosa toxin, and the like. Cytotoxic agents, in some instances, include cisplatin, carboplatin, oxaliplatin, paclitaxel, melphalan, doxorubicin, methotrexate, 5-fluorouracil, etoposide, mechlorethamine, cyclophosphamide, bleomycin, calicheamicin, maytansine, trichothene, CC1065, diphtheria A chain, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii protein, dianthin protein, Phytolaca americana protein, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogenin, restrictocin, phenomycin, enomycin, trichothecene, ribonuclease and deoxyribonuclease. In some embodiments, the cytotoxic agent is one or more species selected from the group consisting of, but not limited to, duocarmycin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1), PBD (pyrrolobenzodiazepine) dimer, duocarmycin, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF). In some embodiments, cytotoxic agents include ribosome-inactivating proteins, histone deacetylase (HDAC) inhibitors, tubulin inhibitors, alkylating agents, antibiotics, anti-neoplastic agents, anti-proliferative agents, antimetabolites, topoisomerase I or II inhibitors, hormonal agonists or antagonists, immunomodulators, DNA minor groove binders, and radioactive agents.In certain embodiments, the ribosome-inactivating protein is saporin. In some embodiments, the diagnostic agent is a label. In some embodiments, the label is a fluorescent label, a chromogenic label, or a radiolabel. In some embodiments, the agent is directly conjugated to the anti-TM4SF1 antibody or antigen-binding fragment thereof. In other embodiments, the agent is indirectly conjugated to the anti-TM4SF1 antibody or antigen-binding fragment thereof, optionally via a linker.

[0203]

[0212] In some embodiments, an ADC of the disclosure comprises an anti-TM4SF1 antibody or antigen-binding fragment thereof and one or more agents (e.g., one, two, three, or four or more agents), e.g., a therapeutic agent that acts additively or synergistically with the anti-TM4SF1 antibody or antigen-binding fragment thereof to kill or inhibit tumor cells (TCs) and / or tumor vasculature endothelial cells (ECs), e.g., in the treatment of a disorder associated with pathological angiogenesis, e.g., cancer. For example, the therapeutic agent can be a biologically active moiety, e.g., a cytotoxic agent, a chemotherapeutic agent, a protein, a peptide, an antibody, a growth inhibitory agent, and / or an anti-hormonal agent.

[0204]

[0213] Examples of tubulin inhibitors that can be conjugated either directly or indirectly to an anti-TM4SF1 antibody or antigen-binding fragment thereof can include, but are not limited to, polymerization inhibitors (e.g., vinblastine, vincristine, vinorelbine, vinflunine, cryptophycin 52, halichondrin, dolastatin, hemiasterlin, which can bind to the vinca domain of tubulin; colchine, combretastatin, 2-methoxy-estradiol, E7010, which can bind to the cholchicine domain of tubulin; depolymerization inhibitors, e.g., paclitaxel, docetaxel, epothilone, discodermolide, which can bind to the taxane site).

[0205]

[0214] Exemplary chemotherapeutic agents include, but are not limited to, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents; enzymes and fragments thereof, such as nucleases, antibiotics, and toxins, e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, and fragments and / or variants thereof. Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0206]

[0215] In addition, various radionuclides can be used for conjugation of anti-TM4SF1 antibodies or antigen-binding fragments thereof to therapeutic agents to produce the ADCs of the present disclosure. Examples include At211, I131, I125, Y90, Re186, Sm153, Bi212, P32, and Lu radioisotopes. Alternatively, anti-TM4SF1 antibodies or antigen-binding fragments can be conjugated to one or more smaller molecule toxins, such as calicheamicin, maytansinoids, dolastatins, aurostatins, trichothecenes, and CC1065, and derivatives of these toxins that have toxin activity are also contemplated herein. Other therapeutic agents that can be conjugated to the TM4SF1 binding proteins of the present disclosure include, in various examples, BCNU, streptozocin, vincristine, and 5-fluorouracil.

[0207]

[0216] In some embodiments, the diagnostic agent for conjugation is a label, such as a fluorescent label, a chromogenic label, or a radiolabel. Thus, the label can be used for detection purposes and can be a fluorescent compound, an enzyme, a prosthetic group, a luminescent material, a bioluminescent material, or a radioactive material. The radiolabel can include, for example, a radioactive atom for scintigraphy studies, such as Tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0208]

[0217] One or more agents (e.g., therapeutic and / or diagnostic agents) may be directly conjugated to the anti-TM4SF1 antibody or antigen-binding fragment (e.g., by direct covalent or non-covalent interaction methods) such that the agent is conjugated adjacent to the protein. An agent may be directly conjugated to a binding protein of the disclosure, for example, by a direct peptide bond. In other examples, direct conjugation is by direct non-covalent interaction methods, for example, interaction between an anti-TM4SF1 antibody or antigen-binding fragment and an agent that specifically binds to the anti-TM4SF1 antibody or antigen-binding fragment.

[0209]

[0218] In some embodiments, the therapeutic agent is: bortezomib (Velcade, PS-341), PR-171 (carfilzomib), ixazomib (Ninlaro®), delanzomib, marizomib, oprozomib, VR23, PI-1840, (benzyloxycarbonyl)-Leu-Leu-phenylalanine, 2,3,5a,6-tetrahydro-6-hydroxy-3-(hydroxymethyl)-2-methyl-10H-3a,10a-epidithio-pyrazinol[1,2α]indole-1,4-Dione, 4-hydroxy-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone, sapojargon, Ac-hFLFL-epoxide, aclacinomycin A, aclarubicin, ACM, AdaK(Bio)Ahx3L3VS, AdaLys(Bio)Ahx3L3VS, adamantane-acetyl-(6-aminohexanoyl)-3-(leucunyl)-3-vinyl-(methyl)-sulfone, ALLM, ALL N, calpain inhibitor I, calpain inhibitor II, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal, gliotoxin, isovaleryl-L-tyrosyl-L-valyl-DL-tyrosinal, clasto-lactacystin-β-lactone, Z-LL-Nva-CHO, ubiquitin aldehyde, YU101, MP-LLL-VS, LDN-57444, Z-GP FL-CHO, Z-LLL-CHO, lovastatin, α-methyl-clasto-lactacystin-β-lactone, mevinolin, MK-803, NIP-L3VS, NP-LLL-VS, NPI-0052 (salinosporamide A), MLN519 (PS-519), NLVS (trileucine vinyl sulfone), ritonavir, Ro106-9920, Z-LLF-CHO, Z-LL-B(OH)2, RRRPRPPYLPR, tyropeptin A, ZL3V S, PR-11, PR-39, 0106-9920, proteasome inhibitor I, proteasome inhibitor II, proteasome inhibitor III, proteasome inhibitor IV, AdaAhx3L3VS, efrapeptin, MG-132, MG-262, MG-115, alpha-methyl omuralide, MG-101, epoxomicin, omuralide, lactacystin, NEOSH101, or an analog thereof. In certain embodiments, the proteasome inhibitor is carfilzomib.

[0210]

[0219] In some embodiments, the therapeutic agent is an AKT kinase inhibitor, including but not limited to, ATP-competitive AKT kinase inhibitors, isoquinoline-5-sulfonamides (H-8, H-89, and NL-71-101), azepane derivatives (derived from (-)-balanol), aminofurazans (GSK690693), heterocycles (7-azaindole derivatives, 6-phenylpurine derivatives, pyrrolo[2,3-d]pyrimidinamides, derivatives, CCT128930, 3-aminopyrrolidine, aminotriazole derivatives, spiroindoline derivatives, AZD5363, ipatasertib (GDC-0068, RG7440), A-674563, and A443654), phenylpyrazole derivatives (AT7867, AT13148), thiophenecarboxamide derivatives (afuresertib (GSK2110183), uprosertib (GSK2141795), 2 -pyrimidyl-5-amidothiophene derivatives (DC120)), allosteric AKT kinase inhibitors, 2,3-diphenylquinoxaline analogues (2,3-diphenylquinoxaline derivatives, triazolo[3,4-f][1,6]naphthyridin-3(2H)-one derivatives (MK-2206)), alkylphospholipids (edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18- OCH3), irmofosine (BM41.440), miltefosine (hexadecylphosphocholine, HePC), erucylphosphocholine (ErPC), elfosine (ErPC3), erucylphosphohomocholine), sulfonamide derivatives (PH-315, PHT-427), indole-3-carbinol analogues (indole-3-carbinol, 3-chloroacetylindole, diindolylmethane, OSU-A9, diethyl 6-Methoxy-5,7-dihydroindolo[2,3-b]carbazole-2,10-dicarboxylate (SR13668)), thiourea derivatives (N-[(1-methyl-1H-pyrazol-4-yl)carbonyl]-N'-(3-bromophenyl)-thiourea, PIT-1, PIT-2, DM-PIT-1), purine derivatives (triciribine (TCN, NSC154020), triciribine monophosphate active analogue (TCN-P), 4-amino-pyrido[2,3-d]pyrimidine derivatives API-1, ARQ 092, 3-phenyl-3H-imidazo[4,5-b]pyridine derivatives), BAY1125976, 3-methyl-xanthine, quinoline-4-carboxamide, 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenyl, 3-oxo-tirucaric acid, 3α- and 3β-acetoxy-tirucaric acid, acetoxy-tirucaric acid, irreversible inhibitors (lactoquinomycin, frenolicin B, calafungin, medermicin, Boc-Phe-vinyl ketone, 4-hydroxynonenal (4-HNE), 1,6-naphthyridinone derivatives, imidazo-1,2-pyridine derivatives), and more. Some examples of small molecule AKT inhibitors are GSK690693, Capiversertib, Ipatasertib, Afuresertib, Uprosertib, Milansertib, Miltefosine, Triciribine, Perifosine, Tehranolide, (E)-1-(2,4-dihydroxyphenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (Isoliquiritigenin), 7-(β-D-glucopyranuronosyloxy)-5,6-dihydropyran-2-one (D-glucuronidase), 5-(β- ... These are hydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (scutellarin), 7a,20-epoxy-1a,6b,7,14-tetrahydroxy-kaur-16-en-15-one (oridonin), 3,5,7,8-tetrahydroxy-2-(4-hydroxyphenyl)chromen-4-one (herbacetin), and (E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one ([6]-shogaol).

[0211]

[0220] In some embodiments, the therapeutic agent is an antimitotic tetrapeptide. In some embodiments, the antimitotic tetrapeptide may include, but is not limited to, tubulysin AI, U, V, Y, z, and pretubulysin, and derivatives thereof. As used herein, the term "derivative of tubulin" generally refers to an analog of tubulysin, including, but not limited to, an oxazole analog of tubulysin, in which an oxazole ring replaces a thiazole ring. In some embodiments, the antimitotic tetrapeptide is tubulysin A or D, or a derivative thereof. Without being bound by theory, it is believed that anti-TM4SF1 antibodies and derivatives thereof conjugated to tubulysin reduce drug-related toxicity.

[0212] IV. Polynucleotides

[0221] 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.

[0213]

[0222] 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.

[0214]

[0223] 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.

[0215]

[0224] 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, various suitable transcription and translation elements, including constitutive and inducible promoters, can be used. The promoter is selected so that it drives the expression of the polynucleotide in the respective host cell.

[0216]

[0225] In some embodiments, the polynucleotide described herein is inserted into a vector, preferably an expression vector, which corresponds to one of further embodiments.The recombinant vector can be constructed according to known methods.Particularly interesting vectors include plasmids, phagemids, phage derivatives, viruses (e.g., retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, lentiviruses, etc.), and cosmids.

[0217]

[0226] 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 is pcDNA5 (Invitrogen).

[0218]

[0227] Thus, in some embodiments, the TM4SF1 binding proteins described herein are produced by introducing vectors encoding the above-mentioned proteins into host cells and culturing the host cells under conditions in which the protein domains are expressed, which may be isolated and optionally further purified.

[0219] V. Immunotherapeutic Agents

[0228] In some embodiments, the combination of the present disclosure comprises i) an anti-TM4SF1 antibody conjugated with a cytotoxic payload and ii) an immunotherapeutic agent. The combination of an anti-angiogenic ADC and an immunotherapy may improve the therapeutic outcome of the immunotherapeutic agent. The anti-angiogenic ADC may promote immune cell infiltration into the tumor microenvironment, leading to improved therapeutic outcome of the immunotherapeutic agent by turning a cold tumor into a hot one. Such therapeutic outcome may include, but is not limited to, increased immune cell infiltration or decreased T-reg cell population. The ADC may also be used in some instances in the context of vascular injury in wound healing.

[0220]

[0229] In some embodiments, the immunotherapeutic agent is an antibody, or an antigen-binding fragment thereof, directed against targets present in cells of the myeloid lineage, tumor cells, cells of the lymphoid system, or molecules present in the tumor microenvironment, hi some embodiments, the immunotherapeutic agent is an antibody, or an antigen-binding fragment thereof, directed against at least one of: PD-1, CTLA-4, CD40, CSF1 / CSF1R, SIRPα, CLEC-1. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of: CCR4, CTLA-4, A1R, A2AR, A3R, TIM-3, BTLA, VISTA, TIGIT, LAG-3, ILRa / CD25, ITGB1 / CD29, Ly 24 / CD44, CD48, CEACAM1 / CD66a, Nt5e / CD73, CD94 / NKG2A, FAS / CD95, SLAF1 / CD150, NRP1 / CD304, GITR / CD357, ICOS, Tnfrs4 / OX40, Folr4 / JUNO, P2X7, ANXA2, IDO, B7-H6, KIR, GARP (LRRC32), TNFR2. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of: PD-L1, PD-L2, B7-H3, B7-H4, CD47, TDO, DcR3. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of: exosomes, cytokines, interleukins, or chemokines. In some embodiments, the immunotherapeutic agent is an antibody or antigen-binding fragment thereof against at least one of: PS, STING.

[0221]

[0230] Immunotherapeutic agents, in some embodiments, are cytokines, such as IL-2 and IL-15, which may function in supporting the proliferation and differentiation of B cells, T cells, and NK cells. In some embodiments, immunotherapeutic agents include cell therapy, cancer vaccines, hormones, epitopes, cytokines, tumor antigens, CD4 cell stimulators, NKT cell agonists, or adjuvants. For example, immunotherapeutic agents include interferons, interleukins, tumor necrosis factors, ovalbumin, Neuvenge, Oncophage, CimaVax-EGF, Mobilan, a-Gal glycolipid, a-galactosylceramide (a-GalCer), β-mannosylceramide (β-ManCer), adenovirus-derived vaccines, Celldex's CDX1307 and CDX1401; GRNVAC1, viral based vaccines, MVA-BN, PROSTVAC®, Advaxis'; ADXS11-001, ADXS31-001, ADXS31-164, BiovaxID, folate binding protein (E39), granulocyte macrophage colony stimulating factor (GM-CSF) with and without E75 (NeuVax) or OncoVEX, trastuzumab, Ae-37, IMA901, SC1B 1, Stimuvax, peptides capable of eliciting a cytotoxic lymphocyte response, peptide vaccines including telomerase peptide vaccines (GV1001), survivin peptides, MUC1 peptides, ras peptides, TARP29-37-9V peptide epitope enhanced peptides, DNA Vector pPRA-PSM with synthetic peptides E-PRA and E-PSM.

[0222]

[0231] In some embodiments, the immunotherapeutic agent comprises adoptive immunotherapy, which involves the transfer of autoantigen-specific T cells generated ex vivo. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or by redirection of T cells by genetic engineering. Isolation and transfer of tumor-specific T cells have been shown to be effective in the treatment of melanoma. Novel specificities of T cells have been effectively generated by gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs). CARs are synthetic receptors consisting of a targeting moiety that combines with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of the antigen-binding domain of a single chain antibody (scFv), which comprises the light chain and variable fragment of a monoclonal antibody connected by a flexible linker. Binding moieties based on receptor or ligand domains have also been effectively used. The signaling domain of first generation CARs is derived from the cytoplasmic region of CD3 zeta or Fc receptor gamma chain. CARs have effectively redirected T cells against antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors.

[0223]

[0232] In some embodiments, the immunotherapeutic agent is an inhibitor of an inhibitory molecule of an immune checkpoint molecule. The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4 and CD8 T cells. These molecules can effectively serve as "brakes" that downregulate or inhibit antitumor immune responses. Inhibition of inhibitory molecules can be performed by inhibition at the DNA, RNA, or protein level. In embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit the expression of inhibitory molecules. In other embodiments, the inhibitor of inhibitory signals is a polypeptide, such as a soluble ligand, or an antibody or antigen-binding fragment thereof that binds to the inhibitory molecule. Immune checkpoint inhibitors can suppress antitumor immunity and / or enhance the visibility of tumor neo-antigens.

[0224]

[0233] In some embodiments, the immunotherapeutic agent may be an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment against a target expressed on myeloid cells, lymphocytes, tumor cells, or other small particles of the tumor microenvironment. In some embodiments, the small particles of the tumor microenvironment include exosomes, cytokines, interleukins, or chemokines. Exemplary targets expressed on myeloid cells may be, but are not limited to, PD-1, CD40, CSF1, CSF1-R, SIRPα, and / or CLEC1. Exemplary targets expressed on lymphoid cells can be, but are not limited to, CCR4, CTLA-4, A1R, A2AR, A3R, TIM-3, BTLA, VISTA, TIGIT, LAG-3, ILRa / CD25, ITGB1 / CD29, Ly 24 / CD44, CD48, CEACAM1 / CD66a, Nt5e / CD73, CD94 / NKG2A, FAS / CD95, SLAF1 / CD150, NRP1 / CD304, GITR / CD357, ICOS, Tnfrs4 / OX40, Folr4 / JUNO, P2X7, ANXA2, IDO, B7-H6, KIR, GARP (LRRC32), or TNFR2. Exemplary targets expressed on tumor cells may be, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD47, TDO, or DcR3. Exemplary small particles in tumor microenvironment may be, but are not limited to, PS, or STING. In some embodiments, the immune checkpoint inhibitor is a protein that inhibits T cell activation, such as CTLA-4, PD-1, PD-L1, PD-L2, GITR, and LAG-3, galectin-9, CEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, TIM-3, and TIM-4. In some embodiments, the anti-LAG-3 antibody is BMS-986016. BMS-986016 (also called BMS986016; Bristol-Myers Squibb) is a monoclonal antibody that binds to LAG-3.BMS-986016 and other humanized anti-LAG-3 antibodies are disclosed in U.S. Patent Application Publication No. 2011 / 0150892, WO 2010 / 019570, and WO 2014 / 008218.

[0225]

[0234] Other exemplary immunotherapeutic agents include, but are not limited to, alemtuzumab, rituximab, tosituimomab, obinutuzumab, ofatumumab, ibritumomab tiuxetan, dinutuximab, blinatumomab, daratumumab, isatuximab, ipilimumab, cetuximab-irfc, elotuzumab, cetuximab, panitumumab, necitumumab, catumaxomab, trastuzumab, pertuzumab, olaratumab, bevacizumab, ramucinumab, imiquimod, tocilzumab, nivolumab, pembrolizumab, ipilimumab, tremelimumab, nivolumab, cemiplimab, pembrolizumab ... Lizumab, avelumab, atezolizumab, durvalumab, tremelimumab, spartalizumab, avelumab, sintilimab, toripalimab, NKTR-214, tisagenlecluecel, axicabtadine, ciloreucel, aldesleukin, interferon alpha-3a / 2b, peridaratinib, sipuleucel-T, HIOI, talimogene Laherparepvec, MGA012, MGD013, MGD019, enoblituzumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatlinab (BMS986016), LAG525, IMP321, REGN2810 (cemiplimab), REGN3767, pexidartic The antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. The immunotherapeutic agent may be an antibody-drug conjugate.Exemplary antibody drug conjugates can include, but are not limited to, moxetumomab, bretuximab, trastuzumab, inotuzumab, gemtuzumab, tagraxofusp-erza, polatuzumab, enfortumab vedotin-ejfv, trastuzumab deruxtecan, sacituzumab govitecan-hziy.

[0226]

[0235] Exemplary immunotherapeutic agents include, but are not limited to, YERVOY® (ipilimumab) or tremelimumab (against CTLA-4), galiximab (against B7.1), BMS-936558 (against PD-1), MK-3475 (against PD-1), atezolizumab (TECENTRIQ®), AMP224 (against B7DC), BMS-936559 (against B7-H1 (against B7-H1), MPDL3280A (against B7-H1), MEDI-570 (against ICOS), AMG557 (against B7H2), MGA271 (against B7H3), IMP321 (against LAG-3), BMS-663513 (against CD137), PF-05082566 (against CD137), CDX-1127 (against CD27), anti-OX40 (Providence Health Services), huMAbOX40L (against OX40L), atacicept (against TACT), CP-870893 (against CD40), lucatumumab (against CD40), dacetuzumab (against CD40), muromonab-CD3 (against CD3); anti-GITR antibodies MK4166, TRX518, Medi1873, INBRX-110, LK2-145, GWN-323, GITRL-Fc, or any combination thereof.

[0227]

[0236] Exemplary immunotherapeutic agents include, but are not limited to, ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, spartalizumab, avelumab, sintilimab, toripalimab, MGA012, MGD013, MGD019, enoblitzumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatlinab ) (BMS986016), LAG525, IMP321, REGN2810 (cemiplimab), REGN3767, pexidartinib, LY3022855, FPA008, BLZ945, GDC0919, epacadostat, indoximid, BMS986205, CPI-444, MEDI9447, PBF509, lirilumab, IMC-001, monalizumab, and combinations thereof.

[0228] VI. Treatment Method

[0237] Provided herein are methods of treatment that include administering a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment, or an ADC containing such an antibody and an immunotherapeutic agent.

[0229]

[0238] In some embodiments, the present disclosure provides methods for inhibiting cell-cell interactions that are endothelial cell (EC) specific, for example, but not limited to, EC-EC, EC-mesenchymal stem cell, EC-fibroblast, EC-smooth muscle cell, EC-tumor cell, EC-leukocyte, EC-adipocyte, and EC-neuronal cell interactions.

[0230]

[0239] By "combination" or "in combination with" it is not intended to mean that the treatments or therapeutic agents must be administered at the same time and / or formulated for delivery together (e.g., in the same composition), but these methods and compositions are within the scope described herein. The immunomodulatory agent and the second therapeutic agent may be administered simultaneously with, prior to, or subsequent to one or more other additional treatments or therapeutic agents. The agents in the combination may be administered in any order. Generally, each agent is administered at a dose and / or time schedule determined for that agent. It is further contemplated that the additional therapeutic agents utilized in the combination may be administered together in a single composition or administered separately in different compositions. In general, it is contemplated that the additional therapeutic agents utilized in combination may be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than those utilized individually.

[0231]

[0240] In some embodiments, the methods disclosed herein include administering an anti-TM4SF1 antibody or antigen-binding fragment or ADC described herein in combination with one or more immunotherapeutic agents (e.g., an anti-PD-1 or PD-L1 antibody molecule, an anti-LAG-3, or an anti-TIM-3 antibody molecule).

[0232]

[0241] The components of the combination, anti-TM4SF1 antibodies or antigen-binding fragments thereof, or ADCs containing such antibodies, in combination with immunotherapeutic agents, are administered simultaneously in some cases. Simultaneous administration means that each component is administered at the same time or within 8-12 hours of each other. Administration of the second component 12 hours or more after the first component may be considered sequential administration. The components of the combination, anti-TM4SF1 antibodies or antigen-binding fragments thereof, or ADCs containing such antibodies, in combination with immunotherapeutic agents, are administered sequentially in any order and in any combination in some cases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 days later; 1, 2, 3, or 4 weeks later; 1, 2, 3, 4, 5, 6, 7, 8, or 9 weeks later; or 1, 2, 3, 4, 5, 6 years or more later; etc.). In some embodiments, when administered in sequence, the anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, is administered first and the immunotherapeutic agent is administered second. In other embodiments, the immunotherapeutic agent is administered first and the anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, is administered second.

[0233]

[0242] Further drugs

[0243] In some embodiments, the combination may further comprise additional agents, such as tyrosine kinase inhibitors.Exemplary tyrosine kinase inhibitors include, but are not limited to, epidermal growth factor (EGF) pathway inhibitors (e.g., epidermal growth factor receptor (EGFR) inhibitors), vascular endothelial growth factor (VEGF) pathway inhibitors (e.g., vascular endothelial growth factor receptor (VEGFR) inhibitors (e.g., VEGFR-1 inhibitors, VEGFR-2 inhibitors, VEGFR-3 inhibitors)), platelet-derived growth factor (PDGF) pathway inhibitors (e.g., platelet-derived growth factor receptor (PDGFR) inhibitors (e.g., PDGFR-β inhibitors)), RAF-1 inhibitors, KIT inhibitors and RET inhibitors. In some embodiments, the anti-cancer agents used in combination with the hedgehog inhibitor are: axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®, ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), alemtuzumab (CAMPATH®), gemtuzumabOzogamicin (MYLOTARG®), ENMD-2076, PCI-32765, AC220, Dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (V ARGATEF (registered trademark)), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11 981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, XL228, AEE788, Ag-490, AST- 6, BMS-599626, CUDC-101, PD153035, pelitinib (EKB-569), vandetanib (Zactima), WZ3146, WZ4002, WZ8040, ABT-869 (linifanib), AEE788, AP24534 (ponatinib), AV-951 (tivozanib), axitinib, BAY 73-4506 (regorafenib), brevanib alanine (BMS-582664), brivanib (BMS-540215), cediranib (AZD2171), CHIR-258 (dovitinib), CP 673451, CYC116, E7080, ΚI8751, masitinib (ABIOIO), MGCD-265, motesanib diphosphate (AMG-706), MP-470, OSI-930, pazopanib hydrochloride, PD173074, sorafenib tosylate (Bay 43-9006), SU 5402, TSU-68 (SU6668), vatalanib, XL880 (GSK1363089, EXEL-2880). Further examples of hedgehog inhibitors include, but are not limited to, vismodegib (2-chloro-N-[4-chloro-3-(2-pyridinyl)phenyl]-4-(methylsulfonyl)-benzamide, GDC-0449, described in PCT Publication WO06 / 028958); l-(4-chloro-3-(trifluoromethyl)phenyl)-3-((3-(4-fluorophenyl)-3,4-dihydro-4-oxo-2-quinazolinyl)methyl)-urea (CAS330796-24-2;N-[(2S,3R,3'R,3aS,4'aR,6S,6'aR,6'bS,7aR,12'aS,12'bS)-2',3',3a,4,4',4'a,5,5',6,6',6'a,6'b,7,7',7a,8',10',12',12'a,12'b-eicosahydro-3,6,l l',12'b-tetramethylspiro[furo[3,2-b]pyridine-2(3H),9'(l'H)-naphtha[2,la]azulen]-3'-yl]-methanesulfonamide (IPI926, CAS 1037210-93-7); and 4-fluoro-N-methyl-N-[1-[4-(1-methyl-1H-pyrazol-5-yl)-1-phthalazinyl]-4-piperidinyl]-2-(trifluoromethyl)-benzamide (LY2940680, CAS 1258861-20-9); and erismodegib (LDE225). Selected tyrosine kinase inhibitors include gefitinib; erlotinib hydrochloride (Tarceva®); linifanib (available from Genentech, ABT 869; sunitinib malate (Sutent®); bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-l-yl)propoxy]quinoline-3-carbonitrile, also known as SKI-606, described in U.S. Pat. No. 6,780,996; dasatinib (Sprycel®); pazopanib (Votrient®); sorafenib (Nexavar®); Zactima (ZD6474); and imatinib or imatinib mesylate (Gilvec® and Gleevec®).

[0234]

[0244] In some embodiments, the combination may further comprise an additional agent, such as a vascular endothelial growth factor (VEGF) receptor inhibitor. Examples of VEGF inhibitors include, but are not limited to, bevacizumab (Avastin®), axitinib (Inlyta®); brivanib alaninate (BMS-582664, (5)-((R)-l-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-5-methylpyrrolo[2,l- / ][l,2,4]triazin-6-yloxy)propan-2-yl) 2-aminopropanoate); sorafenib (Nexavar®); pazopanib (Votrient®); sunitinib malate (Sutent®); Registered trademark); Cediranib (AZD2171, CAS288383-20-1); Balagatev (BIBF1120, CAS928326-83-4); Foretinib (GSK1363089); Telatinib (BAY57-9352, CAS332012-40-5); Apatinib (YN968D1, CAS811803-05-1); Imatinib (Gleevec®); Ponatinib (AP24534, CAS943319-70-8); Tivozanib (AV951, CAS475108-18-0); Regorafenib (BAY73-4506, CAS 755037-03-7), vatalanib dihydrochloride (PTK787, CAS 212141-51-0); brivanib (BMS-540215, CAS 649735-46-6); vandetanib (Caprelsa® or AZD6474); motesanib diphosphate (described in PCT Publication WO 02 / 066470, AMG 706, CAS 857876-30-3, N-(2,3-dihydro-3,3-diphenyl ether) Methyl-lH-indol-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide; Dovitinib dilactate (TKI258, CAS852433-84-2); Linfanib (ABT869, CAS796967-16-3); Cabozantinib (XL184, CAS849217-68-1); Lestaurtinib (CAS111358-88-4);N-[5-[[[5-(l,l-dimethylethyl)-2-oxazolyl]methyl]thio]-2-thiazolyl]-4-piperidinecarboxamide (BMS38703, CAS345627-80-7);(3R,4R)-4-amino-l-((4-((3-methoxyphenyl)amino)pyrrolo[2,lf][l,2,4]triazin-5-yl)methyl)piperidin-3-ol (BMS690514);N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3 aa,5p,6aa)-Octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-aminoquinazoline (XL647, CAS 781613-23-8); 4-Methyl-3-[[1-methyl-6-(3-pyridinyl)-1H-pyrazolo[3,4-J]pyrimidin-4-yl]amino]-N-[3 trifluoromethyl)phenyl]-benzamide (BHG712, CAS 940310-85-0); and aflibercept (Eylea®). Exemplary anti-VEGF antibodies may include, but are not limited to, a monoclonal antibody that binds to the same epitope as monoclonal anti-VEGF antibody A4.6.1, produced by hybridoma ATCC HB 10709;The present invention includes recombinant humanized anti-VEGF monoclonal antibodies produced according to Presta et al. (1997) Cancer Res. 57:4593-4599. In one embodiment, the anti-VEGF antibody is bevacizumab (BV), also known as rhuMAb VEGF or AVASTIN®. It contains mutated human IgG1 framework regions and antigen-binding complementarity determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1, which blocks binding of human VEGF to its receptor. Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Patent No. 6,884,879, filed February 26, 2005. Further antibodies include the G6 or B20 series antibodies (eg, G6-31, B20-4.1) described in PCT Publication No. WO2005 / 012359, PCT Publication No. WO2005 / 044853, the contents of which are expressly incorporated herein by reference. For additional antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6,703,020, 6,054,297, WO 98 / 45332, WO 96 / 30046, WO 94 / 10202, EP 0666868B1, U.S. Patent Application Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and 20050112126; and Popkov et al., Journal of Immunological Methods 288:149-164 (2004). Other antibodies include those that bind to a functional epitope of human VEGF that includes residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104, or, alternatively, that includes residues F17, Y21, Q22, Y25, D63, 183, and Q89;

[0235]

[0245] In some embodiments, the combination may further comprise an additional agent, such as a PI3K inhibitor. Examples of PI3K inhibitors are inhibitors of the delta and gamma isoforms of PI3K. Further examples of PI3K inhibitors include, but are not limited to, 4-[2-(lH-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-l-yl]methyl]thieno[3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941, described in PCT Publications WO09 / 036082 and WO09 / 055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-l-yl]phenyl]propionitrile (BEZ235 or NVP-BEZ, described in PCT Publication WO06 / 122806). 235); 4-(triiluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine (described in PCT Publication WO 2007 / 084786, also known as BKM120 or NVP-BKM120); tozasertib (VX680 or MK-0457, CAS 639089-54- 6);(5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS958852-01-2);(lE,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-l-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro-l l-Hydroxy-4-(methoxymethyl)-4a,6a-dimethyl-cyclopenta[5,6]naphtho[l,2-c]pyran-2,7,10(lH)-trione (PX866, CAS 502632-66-8); 8-Phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6); 2-Amino-8-ethyl-4-methyl-6-(lH-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-7(8H)-one (SAR 245409 or XL 765);l,3-Dihydro-8-(6-methoxy-3-pyridinyl)-3-methyl-1-[4-(1-piperazinyl)-3-(trifluoromethyl)phenyl]-2H-imidazo[4,5-c]quinolin-2-one, (2Z)-2-butenedioic acid (1:1) (BGT 226); 5-Fluoro-3-phenyl-2-[(lS)-l-(9H-purin-6-ylamino)ethyl]-4(3H)-quinazolinone (CAL101); 2-Amino-N-[3-[N-[3-[(2-chloro-5-methoxyphenyl)amino]quinoxalin-2-yl]sulfamoyl]phenyl]-2-methylpropanamide (SAR 245408 or XL 147); and (S)-pyrrolidine-l,2-dicarboxylic acid 2-amide l-({4-methyl-5-[2-(2,2,2-trifluoro-1,1-dimethyl-ethyl)-pyridin-4-yl]-thiazol-2-yl}-amide) (B YL719).

[0236]

[0246] In some embodiments, the combination includes an additional agent, such as an mTOR inhibitor, such as rapamycin, temsirolimus (TORISEL®), AZD8055, BEZ235, BGT226, XL765, PF-4691502, GDC0980, SF1126, OSI-027, GSK1059615, KU-0063794, WYE-354, Palomid 529 (P529), PF-04691502, or PKI-587, ridaforolimus (formally known as deferolimus, PCT Publication WO 2014 / 023366), or combination therapy with rituximab. (lR,2R,4S)-4-[(2R)-2[(1R,9S,12S,15R,16E,1SR,19R,21R,23S,24E,26E,2SZ,30S,32S,35R)-1,18-dihydroxy-l 9,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-ll,36-dioxa-4-azatricyclo[30.3.1.04'9]Hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate; everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS164301-51-3); emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-JJ pyrimidin-7-yl]-2-methoxyphenyl}methanol (AZD8055); 2-amino-8-[iraw5,-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2 ,3-JJ pyrimidin-7(8H)-one (PF04691502, CAS1013101-36-4); and N2-[l,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-l-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-La-aspartylL-serine-, inner salt (SF1126, CAS936487-67-1), (lr,4r)-4-(4-amino-5-(7-methoxy-lH-indol-2-yl)imidazo[l,5-f][l,2,4]triazin-7-yl)cyclohexanecarboxylic acid (OSI-027); and XL765.

[0237]

[0247] In some embodiments, the combination may further comprise an additional agent, such as a BRAF inhibitor, such as GSK2118436, RG7204, PLX4032, GDC-0879, PLX4720, and sorafanib tosylate (Bay 43-9006). In further embodiments, the BRAF inhibitor may be, but is not limited to, regorafenib (BAY73-4506, CAS755037-03-7); tuvizanib (AV951, CAS475108-18-0); vemurafenib (Zelboraf®, PLX-4032, CAS918504-65-1); encorafenib (also known as LGX818); 1-methyl-5-[[2-[5-(trifluoromethyl)-1 H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl-lH-benzimidazol-2-amine (RAF265, CAS 927880-90-8); 5-[l-(2-hydroxyethyl)-3-(pyridin-4-yl)-lH-pyrazol-4-yl]-2,3-dihydroinden-l-one oxime (GDC-0879, CAS 905281-76-7); 5-[2-[4-[2-(dimethylamino)ethoxy]phenyl]-5-(4-pyridinyl)-lH-imidazol-4-yl]-2,3-dihydro-lH-inden-l-one Oxime (GSK2118436 or SB590885); (+ / -)-methyl(5-(2-(5-chloro-2-methylphenyl)-l-hydroxy-3-oxo-2,3-dihydro-lH-isoindol-l-yl)-lH-benzimidazol-2-yl)carbamate (also known as XL-281 and BMS908662) and N-(3-(5-chloro-lH-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl)propane-l-sulfonamide (also known as PLX4720).

[0238]

[0248] In some embodiments, the combination may further comprise an additional agent, such as a MEK inhibitor. Examples include, but are not limited to, selumetinib (5-[(4-bromo-2-chlorophenyl)amino]-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzimidazole-6-carboxamide, described in PCT Publication WO2003077914 and also known as AZD6244 or ARRY142886); trametinib dimethylsulfoxide (GSK-1120212, CA S1204531-25-80;RDEA436;N-[3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3-dihydroxypropyl]-cyclopropanesulfonamide (described in PCT Publication WO2007014011, also known as RDEA119 or BAY869766);AS703026;BIX 02188; BIX 02189; 2-[(2-chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluoro-benzamide (described in PCT Publication WO2000035436, also known as CI-1040 or PD184352); N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (described in PCT Publication WO2002006213, also known as PD0325901); 2'-amino-3'-methoxyflavone (also known as PD98059, Biaffin GmbH&co.,kg, Germany); 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also known as U0126 and described in U.S. Pat. No. 2,779,780); XL-518 (also known as GDC-0973, Cas No. 1029872-29-4, available from ACC Corp.); G-38963; and G02443714 (also known as AS703206), or a pharma- ceutically acceptable salt or solvent thereof, or any MEK inhibitor may be used in combination.Further examples of MEK inhibitors are disclosed in WO 2013 / 019906, WO 03 / 077914, WO 2005 / 121142, WO 2007 / 04415, WO 2008 / 024725, and WO 2009 / 085983, the contents of which are incorporated herein by reference. Further examples of MEK inhibitors include, but are not limited to, benimetinib (6-(4-bromo-2-fluorophenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxyethyoxy)-amide, also known as MEK162, CAS1073666-70-2, described in PCT Publication WO2003077914; 2,3-bis[amino[(2-aminophenyl)thio]methylene]-butanedinitrile (also known as U0126, described in U.S. Patent No. 2,779,780); (3S,4R,5Z,8S,9S,10S,11S,12S,13S,14S,15S,16S,17S,18S,19S,20S,21S,22S,23S,24S,25S,26S,27S,28S,29S,30S,31S,32S,33S,34S,35S,36S,37S,38S,39S,40S,41S,42S,43S,44S,45S,50S,51S,52S,53S,54S,55S,66S,77S,88S,99S,100S,1111, and described in U.S. Patent No. 2,779,780); (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-((R)-1,4-dimethyl-1H-2-benzoxacyclotetradecyne-1,7(8H)-dione] (also known as E6201 and described in PCT Publication WO2003076424); vemurafenib (PLX-4032, CAS 918504-65-1); (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-((R)-1,4-dimethyl-1H-2-benzoxacyclotetradecyne-1,7(8H)-dione) (also known as E6201 and described in PCT Publication WO2003076424); 2-Fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (TAK-733, CAS 1035555-63-5); pimasertib (AS-703026, CAS 1204531-26-9); 2-(2-Fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-l,5-dimethyl-6-oxo-l,6-dihydropyridine-3-carboxamide (AZD 8330); and 3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-5-[(3-oxo-[l,2]oxazinan-2-yl)methyl]benzamide (CH 4987655 or Ro 4987655).

[0239]

[0249] In some embodiments, the combination further comprises an additional agent, e.g., a JAK inhibitor, e.g., examples of JAK inhibitors include, but are not limited to, rCEP-701, INCB18424, CP-690550 (tasocitinib), uxolitinib (Jakafi®); tofacitinib (CP690550); axitinib (AG013736, CAS319460-85-0); 5-chloro-N2-[(lS)-l-(5-fluoro-2-pyrimidinyl)ethoxy]ethyl]ethyl] yl]-N4-(5-methyl-lH-pyrazole-3-y)-12,4-pyrimidinediamine (AZD1480, CAS 935666-88-9);(9E)-15-[2-(l-pyrrolidinyl)ethoxy]-7,12,26-trioxa-19,21,24-triazatetracyclo[18.3.1.12,5.114,18]-hexacosa-l(24),2,4,9,14,16,18(25),20,22-nonane (SB-1578, CAS 937273-04-6);momelotinib (CYT 387); baricitinib (INCB-028050 or LY-3009104); pacritinib (SB1518); (16E)-14-methyl-20-oxa-5,7,14,27-tetraazatetracyclo[19.3.1.12,6.18,12]heptacosa-l(25),2,4,6(27),8,10,12(26),16,21,23-decane (SB 1317); gandotinib (LY 2784544); and N,N-cyclopropyl-4-[(l,5-dimethyl-lH-pyrazol-3-yl)amino]-6-ethyl-l,6-dihydro-l-methyl-imidazo[4,5-d]pyrrolo[2,3-b]pyridine-7-carboxamide (BMS 911543).

[0240]

[0250] In some embodiments, the combination may further include an additional agent, such as an antibody against a killer cell immunoglobulin-like receptor (also referred to herein as an "anti-KIR antibody"). In some embodiments, the combination may further include administering a cellular immunotherapy (e.g., Provenge (e.g., Sipuleucel)), optionally in combination with cyclophosphamide. In some embodiments, the combination may further include an additional agent, such as a vaccine, such as a dendritic cell renal cancer (DC-RCC) vaccine. In some embodiments, the combination may further include an additional agent, such as paclitaxel or a paclitaxel agent, such as TAXOL®, protein-bound paclitaxel (e.g., ABRAXANE®). Examples of paclitaxel agents include, but are not limited to, nanoparticle albumin-bound paclitaxel (ABRAXANE, marketed by Abraxis Bioscience), docosahexaenoic acid-bound paclitaxel (DHA-paclitaxel, Taxoplexin, marketed by Protarga), polyglutamic acid-bound paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX, Cell Signaling, Inc., Sanofi, Calif. These include, for example, -paclitaxel methyl 2-glucopyranosyl succinate (e.g. ...

[0241]

[0251] In some embodiments, the combination may further comprise an additional agent, e.g., chemotherapy, and / or immunotherapy. For example, a combination comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof or an ADC comprising the same in combination with an immunotherapy agent can be used to treat myeloma, alone or in combination with one or more of chemotherapy or other anti-cancer agents (e.g., thalidomide analogs, e.g., lenalidomide), tumor antigen-pulsed dendritic cells, fusion of tumor cells with dendritic cells (e.g., electrofusion), or vaccination with immunoglobulin idiotypes produced by malignant plasma cells.

[0242]

[0252] cancer

[0253] In some embodiments of the method, the cancer treated by the combination is selected from the group consisting of prostate cancer, liver cancer, colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, stomach (gastric) cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), sarcoma (soft tissue sarcoma and osteosarcoma), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cancer, lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS). In some embodiments of the method, the cancer is selected from the group consisting of triple-negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumor, adult T-cell leukemia / lymphoma, and myelodysplastic syndrome (MDS). In some embodiments of the method, the cancer is selected from the group consisting of triple-negative breast cancer, stomach (gastric) cancer, lung cancer (small cell lung, non-small cell lung), Merkel cell carcinoma, and MSI-high cancer. In certain embodiments, the cancer comprises a BRAF mutation (e.g., a BRAF V600E mutation), a BRAF wild type, a KRAS wild type, or an activating KRAS mutation. The cancer can be early stage, intermediate stage, or late stage.

[0243]

[0254] In one embodiment, the combination provided herein is further combined with chemotherapy to treat lung cancer, for example, non-small cell lung cancer. In one embodiment, the combination is used in platinum doublet therapy to treat lung cancer. In yet another embodiment, the combination is used to treat kidney cancer, for example, renal cell carcinoma (RCC) (for example, renal clear cell carcinoma (CCRCC) or metastatic RCC). The combination is administered in combination with additional agents, including one or more of immune-based strategies (for example, interleukin-2 or interferon-a), targeting agents (for example, VEGF inhibitors, for example, monoclonal antibodies against VEGF); VEGF tyrosine kinase inhibitors, for example, sunitinib, sorafenib, axitinib and pazopanib; RNAi inhibitors), or inhibitors of downstream mediators of VEGF signaling, for example, inhibitors of mammalian target of rapamycin (mTOR), for example, everolimus and temsirolimus.Examples of suitable therapies for use in combination for the treatment of small cell lung cancer include, but are not limited to, chemotherapeutic agents (e.g., etoposide, carboplatin, cisplatin, irinotecan, topotecan, gemcitabine, liposomal SN-38, bendamustine, temozolomide, belotecan, NK012, FR901228, flavopiridol); tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib, gefitinib, cetuximab, panitumumab); multikinase inhibitors (e.g., sorafenib, sunitinib); VEGF inhibitors (e.g., bevacizumab, vandetanib); cancer vaccines (e.g., GVAX); Bcl-2 inhibitors (e.g., oblimersen sodium, ABT-263); proteasome inhibitors (e.g., bortezomib (Velcade), NPI-0052), panitumumab, serovar IgE, and serovar IgE. These include clitaxel or paclitaxel agents, docetaxel, IGF-1 receptor inhibitors (e.g., AMG479); HGF / SF inhibitors (e.g., AMG102, MK0646); chloroquine; Aurora kinase inhibitors (e.g., MLN8237); radioimmunotherapy (e.g., TF2); HSP90 inhibitors (e.g., tenespimycin, STA-9090); mTOR inhibitors (e.g., everolimus); Ep-CAM- / CD3-bispecific antibodies (e.g., MT110); CK-2 inhibitors (e.g., CX-4945); HDAC inhibitors (e.g., belinostat); SMO antagonists (e.g., BMS833923); peptide cancer vaccines, and radiation therapy (e.g., intensity-modulated radiation therapy (IMRT), hypofractionation, hypoxia-guided radiation therapy), surgery, and combinations thereof.

[0244]

[0255] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of non-small cell lung cancer include, but are not limited to, chemotherapeutic agents, such as vinorelbine, cisplatin, docetaxel, pemetrexed disodium, etoposide, gemcitabine, carboplatin, liposomal SN-38, TLK286, temozolomide, topotecan, pemetrexed disodium, azacitibine, irinotecan, tegafur-gimeracil-oteracil potassium, sapacita, vin; tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib, gefitinib, cetuximab, panitumumab, necitumumab, PF-00299804, nimotuzumab, RO5083945), MET inhibitors (e.g., PF-02341066, ARQ197), PDK kinase inhibitors (e.g., XL147, GDC-0941), Raf / MEK dual kinase inhibitors (e.g., R05126766), PI3K / mTOR dual kinase inhibitors (e.g., XL765), SRC inhibitors (e.g., dasatinib), dual inhibitors (e.g., BIBW2992, GSKI363089, ZD6474, AZD0530, AG-013736, lapatinib, MEHD7945A, linifanib), multikinase inhibitors (e.g., sorafanib, sunitinib, pazopanib, AMG706, XL184, MGCD265, BMS-690514, R935788), VEGF inhibitors (e.g., endostar, endostatin, bevacizumab, cediranib, BIBF1120, axitinib, tivozanib, AZD2171), cancer vaccines (e.g., BLP25 liposomal vaccine, GV AX, recombinant DNA and adenovirus expressed L523S protein), Bcl-2 inhibitors (e.g., oblimersen sodium), proteasome inhibitors (e.g., bortezomib, carfilzomib, NPI-0052, MLN9708), paclitaxel or paclitaxel agents, docetaxel, IGF-1 receptor inhibitors (e.g., cixutumumab, MK-0646, OSI906, CP-751871, BIIB022), hydroxychloroquinone, HSP90 inhibitors (e.g., tanespimycin, STA-9090, AUY922, XL888),mTOR inhibitors (e.g., everolimus, temsirolimus, ridaforolimus), Ep-CAM- / CD3-bispecific antibodies (e.g., MT110), CK-2 inhibitors (e.g., CX-4945), HDAC inhibitors (e.g., MS275, LBH589, vorinostat, valproic acid, FR901228), DHFR inhibitors (e.g., pralatrexate), retinoids (e.g., bexarotene, tretinoin), antibody drug conjugates (e.g., SGN-15), bisphosphonates (e.g., zoledronic acid), cancer vaccines (e.g., beragen pumascel-L), low molecular weight heparins (LMWHs) (e.g., tinzaparin, enoxapari ), GSK1572932A, melatonin, talactoferrin, dimesna, topoisomerase inhibitors (e.g., amrubicin, etoposide, karenitecin), nelfinavir, cilengitide, ErbB3 inhibitors (e.g., MM-121, U3-1287), survivin inhibitors (e.g., YM155, LY2181308), eribulin mesylate, COX-2 inhibitors (e.g., celecoxib), pegfilgrastim, Polo-like kinase 1 inhibitors (e.g., BI6727), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), CNGRC peptide-TNF alpha conjugate, dichloroacetate (DC A), HGF inhibitors (e.g., SCH900105), SAR240550, PPAR-gamma agonists (e.g., CS-7017), gamma-secretase inhibitors (e.g., RO4929097), epigenetic therapy (e.g., 5-azacytidine), nitroglycerin, MEK inhibitors (e.g., AZD6244), cyclin-dependent kinase inhibitors (e.g., UCN-01), cholesterol Fusl, anti-Tul rabulin agents (e.g., E7389), farnesyl-OH-transferase inhibitors (e.g., lonafarnib), immunotoxins (e.g., BB-10901, SSI(dsFv)PE38), fondaparinux, vascular disrupters (e.g., AVE8062), PD-L1 inhibitors (e.g., MDX-1105, MDX-1106), beta-glucans, NGR-hTNF, EMD521873, MEK inhibitors (e.g., GSK1 120212), epothilone analogs (e.g., ixabepilone),These include kinesin-spindle inhibitors (e.g., 4SC-2005), telomere targeting agents (e.g., KML-001), P70 pathway inhibitors (e.g., LY2584702), AKT inhibitors (e.g., MK-2206), angiogenesis inhibitors (e.g., lenalidomide), Notch signaling inhibitors (e.g., OMP-21M18), radiation therapy, surgery, and combinations thereof. Examples of suitable therapies for use in combination for the treatment of ovarian cancer include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents; docetaxel; carboplatin; gemcitabine; doxorubicin; topotecan; cisplatin; irinotecan, TLK286, ifosfamide, olaparib, oxaliplatin, melphalan, pemetrexed disodium, SJG-136, cyclophosphamide, etoposide, decitabine); Ghrelin antagonists (e.g., AEZS-130), immunotherapy (e.g., APC8024, oregovomab, OPT-821), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), dual inhibitors (e.g., E7080), multikinase inhibitors (e.g., AZD0530, JI-101, sorafenib, sunitinib, pazopanib), ON01910.Na), VEGF inhibitors (e.g., bevacizumab, BIBF 1120, cediranib, AZD2171), PDGFR inhibitors (e.g., IMC-3G3), paclitaxel, topoisomerase inhibitors (e.g., karenitecin, irinotecan), HDAC inhibitors (e.g., valproic acid, vorinostat), folate receptor inhibitors (e.g., farletuzumab), angiopoietin inhibitors (e.g., AMG386), epothilone analogues (e.g., ixabepilone), proteasome inhibitors (e.g., carfilzomib), IGF-1 receptor inhibitors ( For example, OSI906, AMG479), PARP inhibitors (for example, veliparib, AG014699, iniparib, MK-4827), Aurora kinase inhibitors (for example, MLN8237, ENMD-2076), angiogenesis inhibitors (for example, lenalidomide), DHFR inhibitors (for example, pralatrexate), radioimmunotherapeutic agents (for example, Hu3S193), statins (for example, lovastatin), topoisomerase 1 inhibitors (for example, NKTR-102), cancer vaccines (for example,p53 synthetic long peptide vaccine, autologous OC-DC vaccine), mTOR inhibitors (e.g., temsirolimus, everolimus), BCR / ABL inhibitors (e.g., imatinib), ET-A receptor antagonists (e.g., ZD4054), TRAIL receptor 2 (TR-2) agonists (e.g., CS-1008), HGF / SF inhibitors (e.g., AMG102), EGEN-001, Polo-like kinase 1 inhibitors (e.g., BI6727), gamma-secretase inhibitors (e.g., RO4929097), Wee-1 inhibitors (e.g., MK-1775), antitubulin agents (e.g., vinorelbine, E7389), immunotoxins (e.g., denileukin diftitox), SB-485232, vascular disrupters (e.g., AVE8062), integrin inhibitors (e.g., EMD525797), kinesin-spindle inhibitors (e.g., 4SC-205), Revlimid, HER2 inhibitors (e.g., MGAH22), ErrB3 inhibitors (e.g., MM-121), radiation therapy, and combinations thereof.

[0245]

[0256] Examples of additional agents for use with anti-TM4SF1 antibodies or antigen-binding fragments thereof, or ADCs containing such antibodies, in combination with immunotherapeutic agents for the treatment of myeloma include, but are not limited to, chemotherapeutic agents, including but not limited to: chemotherapeutic agents or other anti-cancer agents (e.g., thalidomide analogs, e.g., lenalidomide), HSCT (Cook, R. (2008) J Manag Care Pharm. 14(7 Suppl):19-25), anti-TIM3 antibodies (Hallett, WHD et al. (2011) / of American Society for Blood and Marrow Transplantation 17(8):1133-145), tumor antigen-pulsed dendritic cells, fusion (e.g., electrofusion) of tumor cells with dendritic cells, or vaccination with immunoglobulin idiotypes produced by malignant plasma cells (reviewed in Yi, Q. (2009) Cancer J. 15(6):502-10).

[0246]

[0257] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of renal cell carcinoma or metastatic renal cell carcinoma include, but are not limited to, chemotherapeutic agents, examples of which are provided above.

[0247]

[0258] Anti-PD-1 antibody molecules have been used in combination with immune-based strategies (e.g., interleukin-2 or interferon a), targeted agents (e.g., VEGF inhibitors, such as monoclonal antibodies against VEGF, e.g., bevacizumab (Rini, BI et al. (2010) J. Clin. Oncol. 28(13):2137-2143)); VEGF tyrosine kinase inhibitors, such as sunitinib, sorafenib, axitinib and pazopanib (Pal. SK et al. (2014) Clin. Advances in Hematology & Oncology 12(2):90-99); RNAi inhibitors), or inhibitors of downstream mediators of VEGF signaling, such as inhibitors of the mammalian target of rapamycin (mTOR), such as everolimus and temsirolimus (Hudes, G. et al. (2007) N. Engl. J. Med. 356(22):2271-2281; Motzer, RJ et al. (2008) Lancet 372:449-456).

[0248]

[0259] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of chronic myeloid leukemia (AML) according to the invention include, but are not limited to, chemotherapy (e.g., cytarabine, hydroxyurea, clofarabine, melphalan, thiotepa, fludarabine, busulfan, etoposide, cordycepin, pentostatin, capecitabine, azacitidine, ribavirin ... tyrosine kinase inhibitors (e.g., BCR / ABL inhibitors (e.g., imatinib, nilotinib), ON01910.Na, dual inhibitors (e.g., dasatinib, bosutinib), multikinase inhibitors (e.g., DCC-2036, ponatinib, sorafenib, sunitinib, RGB-286638)), interferon alpha, steroids, apoptotic agents (e.g., omacetaxine mepesuccinate), immunotherapy (e.g., allogeneic CD4+ memory Th1-like T cells / microparticle-bound anti-CD3 / anti-CD28, autologous cytokine-induced killer cells (CIK), AHN-12), CD52 targeting agents (e.g., alemtuzumab), HSP90 inhibitors (e.g., tenespimycin, STA-9090, AUY922, XL888), mTOR inhibitors (e.g., everolimus), SMO antagonists (e.g., BMS833923), ribonucleotide reductase inhibitors (e.g., 3-AP), JAK-2 inhibitors (e.g., INCB018424), hydroxychloroquine, retinoids (e.g., fenretinide), cyclin-dependent kinase inhibitors (e.g., UCN-01), HDAC inhibitors (e.g., belinostat, vorinostat, JNJ-26481585), PARP inhibitors (e.g., veliparib), MDM2 antagonists (e.g., RO5045337), Aurora B kinase inhibitors (e.g., TAK-901), radioimmunotherapy (e.g., actinium-225-labeled anti-CD33 antibody HuM195), hedgehog inhibitors (e.g., PF-04449913), STAT3 inhibitors (e.g., OPB-31121), KB004, cancer vaccines (e.g., AG858), bone marrow transplantation, stem cell transplantation, radiation therapy, and combinations thereof.

[0249]

[0260] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of chronic lymphocytic leukemia (CLL) include, but are not limited to, chemotherapeutic agents (e.g., fludarabine, cyclophosphamide, doxorubicin, vincristine, chlorambucil, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pentostatin, mitoxantrone, 5-azacytidine, pemetrexed disodium), tyrosine kinase inhibitors (e.g., , EGFR inhibitors (e.g., erlotinib), BTK inhibitors (e.g., PCI-32765), multikinase inhibitors (e.g., MGCD265, RGB-286638), CD20 targeting agents (e.g., rituximab, ofatumumab, RO5072759, LFB-R603), CD52 targeting agents (e.g., alemtuzumab), prednisolone, darbepoetin alfa, lenalidomide, Bcl-2 inhibitors (e.g., ABT-263), immunotherapy (e.g., allogeneic CD4+ memory Th1-like T cells / microparticle-bound anti-CD3 / anti-CD28, autologous Cytokine-induced killer cells (CIK)), HDAC inhibitors (e.g., vorinostat, valproic acid, LBH589, JNJ-26481585, AR-42), XIAP inhibitors (e.g., AEG35156), CD-74 targeting agents (e.g., milatuzumab), mTOR inhibitors (e.g., everolimus), AT-101, immunotoxins (e.g., CAT-8015, anti-Tac(Fv)-PE38(LMB-2)), CD37 targeting agents (e.g., TRU-016), radioimmunotherapy (e.g., 131-tositumomab), hydroxychloroquine, perifosine, SR C inhibitors (e.g., dasatinib), thalidomide, PI3K delta inhibitors (e.g., CAL-101), retinoids (e.g., fenretinide), MDM2 antagonists (e.g., RO5045337), plerixafor, Aurora kinase inhibitors (e.g., MLN8237, TAK-901), proteasome inhibitors (e.g., bortezomib), CD19 targeting agents (e.g., MEDI-551, MOR208), MEK inhibitors (e.g., ABT-348), JAK-2 inhibitors (e.g., INCB018424), hypoxia-activated prodrugs (e.g.,TH-302), paclitaxel or paclitaxel agents, HSP90 inhibitors, AKT inhibitors (e.g., MK2206), HMG-CoA inhibitors (e.g., simvastatin), GNKG186, radiation therapy, bone marrow transplantation, stem cell transplantation, and combinations thereof.

[0250]

[0261] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of acute lymphoblastic leukemia (ALL) include, but are not limited to, chemotherapeutic agents (e.g., prednisolone, dexamethasone, vincristine, asparaginase, daunorubicin, cyclophosphamide, cytarabine, etoposide, thioguanine, mercaptopurine, clofarabine, liposomal annamin, sirolimus, busulfan, etoposide, capecitabine, decitabine, azacitidine, topotecan, temozolomide), tyrosine kinase inhibitors (e.g., BCR / ABL inhibitors (e.g., imatinib, nilotinib), ON01910.Na, multikinase inhibitors (e.g., sorafenib), CD20 targeting agents (e.g., rituximab), CD52 targeting agents (e.g., alemtuzumab), HSP90 inhibitors (e.g., STA-9090), mTOR inhibitors (e.g., everoli mus, rapamycin), JAK-2 inhibitors (e.g., INCB018424), HER2 / neu receptor inhibitors (e.g., trastuzumab), proteasome inhibitors (e.g., bortezomib), methotrexate, asparaginase, CD22 targeting agents (e.g., epratuzumab, inotuzumab), immunotherapy (e.g., autologous cytokine-induced killer cells (CIK), AHN-12), blinatumomab, cyclin-dependent kinase inhibitors (e.g., UCN-01), CD4 5 Targeting agents (e.g., BC8) MDM2 antagonists (e.g., RO5045337), immunotoxins (e.g., CAT-8015, DT2219ARL), HDAC inhibitors (e.g., JNJ-26481585), JVRS-100, paclitaxel or paclitaxel agents, STAT3 inhibitors (e.g., OPB-31121), PARP inhibitors (e.g., veliparib), EZN-2285, radiation therapy, steroids, bone marrow transplant, stem cell transplant, or combinations thereof.

[0251]

[0262] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of acute myeloid leukemia (AML) include, but are not limited to, chemotherapeutic agents (e.g., cytarabine, daunorubicin, idarubicin, clofarabine, decitabine, vosaroxin, azacitidine, clofarabine, ribavirin, CPX-351, treosulfan, elacitarabine, azacitidine), tyrosine kinase inhibitors (e.g., BCR / ABL inhibitors (e.g., imatinib, nilotinib), ON01910.Na, multikinase inhibitors (e.g., midostaurin, SU11248, quizartinib, sorafinib)), immunotoxins (e.g., gemtuzumab, ozogamicin), DT388IL3 fusion protein, HDAC inhibitors (e.g., vorinostat, LBH589), plerixafor, mTOR inhibitors (e.g., everolimus), SRC inhibitors (e.g., dasatinib), HSP90 inhibitors (e.g., STA-9090), retinoids (e.g., bexarotene, Aurora kinase inhibitors (e.g., BI811283), JAK-2 inhibitors (e.g., INCB018424), Polo-like kinase inhibitors (e.g., For example, BI6727), cenersen, CD45 targeting agents (e.g., BC8), cyclin dependent kinase inhibitors (e.g., UCN-01), MDM2 antagonists (e.g., RO5045337), mTOR inhibitors (e.g., everolimus), LY573636-sodium, ZRx-101, MLN4924, lenalidomide, immunotherapy (e.g., AHN-12), histamine dihydrochloride, radiation therapy, bone marrow transplant, stem cell transplant, and combinations thereof.

[0252]

[0263] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of multiple myeloma (MM) include, but are not limited to, chemotherapeutic agents (e.g., melphalan, amifostine, cyclophosphamide, doxorubicin, clofarabine, bendamustine, fludarabine, adriamycin, SyB L-0501), thalidomide, lenalidomide, dexamethasone, prednisone, pomalidomide, proteasome inhibitors (e.g., bortezomib, carfilzomib, MLN9708), cancer vaccines (e.g., GVAX), CD-40 targeting agents (e.g., SGN-40, CHIR-12.12), perifosine, zoledronic acid, immunotherapy (e.g., MAGE-A3, NY-ESO-1, HuMAX-CD38), HDAC inhibitors (e.g., vorinostat, LBH589, AR-42), aplidine, cyclin-dependent quinine inhibitors (e.g., PD-0332991, dinaciclib), arsenic trioxide, CB3304, HSP90 inhibitors (e.g., KW-2478), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., cetuximab), multikinase inhibitors (e.g., AT9283)), VEGF inhibitors (e.g., bevacizumab), plerixafor, MEK inhibitors (e.g., AZD6244), IPH2101, atorvastatin, immunotoxins (e.g., BB-10901), NPI-0052, radioimmunotherapy (e.g., yttrium Y90 ibritumomab tiuxetan), STAT3 inhibitors (e.g., OPB-31121), MLN4924, Aurora kinase inhibitors (e.g., ENMD-2076), IMGN901, ACE-041, CK-2 inhibitors (e.g., CX-4945), radiation therapy, bone marrow transplant, stem cell transplant, and combinations thereof.

[0253]

[0264] Examples of additional agents for use with an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, in combination with an immunotherapeutic agent for the treatment of prostate cancer include, but are not limited to, chemotherapeutic agents (e.g., docetaxel, carboplatin, fludarabine), abiraterone, hormone therapy (e.g., flutamide, bicalutamide, nilutamide, cyproterone acetate, ketoconazole, aminoglutimide, abarelix, degarelix, leuprolide, goserelin, triptorelin, buserelin), tyrosine kinase inhibitors (e.g., dual kinase inhibitors (e.g., lapatanib), multi-kinase inhibitors (e.g., sorafenib, sunitinib)), VEGF inhibitors (e.g., bevacizumab, ), TAK-700, cancer vaccines (e.g., BPX-101, PEP223), lenalidomide, TOK-001, IGF-1 receptor inhibitors (e.g., cixutumumab), TRC105, Aurora kinase inhibitors (e.g., MLN8237), proteasome inhibitors (e.g., bortezomib), OGX-011, radioimmunotherapy (e.g., HuJ591-GS), HDAC inhibitors (e.g., valproic acid, SB939, LBH589), hydroxychloroquine, mTOR inhibitors (e.g., everolimus), dovitinib lactate, diindolylmethane, efavirenz, OGX-427, genistein, IMC-3G3, bafetinib, CP-675,206, radiation therapy, surgery, or a combination thereof.

[0254] VII. Pharmaceutical Compositions

[0265] In some embodiments, the anti-TM4SF1 antibodies, ADCs, and immunotherapeutic agents of the present disclosure may be included in a single or separate compositions (e.g., pharmaceutical compositions). The pharmaceutical compositions of the present disclosure may further include a pharma- ceutical acceptable carrier, excipient, or diluent.

[0255]

[0266] The term "pharmaceutical composition" as used herein refers to a composition containing a TM4SF1 binding protein described herein, formulated with a pharma- ceutical acceptable carrier, and manufactured or sold by approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in 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 embolism and in a solvent system suitable for intravenous use), or in any other formulation described herein.

[0256]

[0267] The term "pharmaceutical acceptable carrier" as used herein 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 skilled 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.

[0257]

[0268] The pharmaceutical compositions, in some embodiments, are prepared as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, as nanoparticle-containing dosage forms, as microparticle-containing dosage forms, as polymeric dosage forms, or any combination thereof, as solutions, dispersions in glycerol, liquid polyethylene glycols, and in oils, as solid dosage forms, or any combination thereof.

[0258]

[0269] 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, flavors, sweeteners, and colorants.

[0259]

[0270] 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.

[0260]

[0271] In some embodiments, excipient comprises preservative.Non-limiting examples of suitable preservative include antioxidant, such as alpha tocopherol and ascorbate, and antibacterial agent, such as paraben, chlorobutanol, and phenol.In some examples, 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.

[0261]

[0272] 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.

[0262]

[0273] 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.

[0263]

[0274] In some embodiments, pharmaceutical formulations include dispersion enhancers as excipients.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 crystalline cellulose as high HLB emulsifier surfactant.

[0264]

[0275] In some embodiments, the pharmaceutical composition described herein comprises 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, pectin, 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.

[0265]

[0276] In some embodiments, the excipient comprises flavoring agent.The flavoring agent incorporated in the outer layer is in some cases selected from synthetic flavor oils and flavor 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; wintergreen oil; 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.

[0266]

[0277] In some embodiments, excipient comprises sweetener.Non-limiting examples of suitable sweetener include glucose (corn syrup), dextrose, invert sugar, fructose and their mixtures (when not used as carrier); saccharin and its various salts, such as sodium salt; dipeptide sweetener, such as aspartame; dihydrochalcone compound, glycyrrhizin; Stevia rebaudiana (stevioside); chloro derivative of sucrose, such as sucralose; and sugar alcohol, such as sorbitol, mannitol, sylitol, etc.

[0267]

[0278] In some examples, the pharmaceutical compositions described herein comprise colorants.Non-limiting examples of suitable colorants include Food, Drug and Cosmetic Colorants (FD&C), Drug and Cosmetic Colorants (D&C), and External Drug and Cosmetic Colorants (Ext.D&C).Colorants can be used as dyes or their corresponding lakes.

[0268]

[0279] In some cases, the pharmaceutical compositions described herein include a chelating agent. In some cases, 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'-di ... 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.

[0269]

[0280] Compositions that include one or more other antibacterial or antifungal agents are also contemplated, such as polyenes, such as 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, such as caspofungin, micafungin (FK463), and V-echinocandin (LY303366); griseofulvin; allylamines, such as 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.

[0270]

[0281] Under normal conditions of storage and use, the pharmaceutical compositions described herein contain a preservative to prevent the growth of microorganisms. In certain instances, 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 carriers that are solvents or dispersion media containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and / or vegetable oils, or any combination thereof. Good fluidity is maintained, for example, by the use of coatings, 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, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, isotonic agents, such as sugars or sodium chloride, are included. Prolonged absorption of the injectable compositions can be brought about by the use of agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0271]

[0282] 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, the sterile aqueous medium 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, added to 100 mL to 1000 mL of liquid, for example sodium bicarbonate buffered saline, or injected at the proposed site of injection.

[0272]

[0283] 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 listed above, if necessary, and sterile filtering. Usually, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from those listed 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 free amino groups of proteins), 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. When formulated, the pharmaceutical compositions are administered in some embodiments in a manner compatible with the dosage formulation and in an amount that is effective for treatment.

[0273]

[0284] In certain embodiments, the pharmaceutical composition of the present disclosure includes a pharma- ceutically acceptable carrier. As used herein, "pharma-ceutically 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 which it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solution, water, emulsions, such as 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, implant elements containing immunotherapeutic agents or any other suitable vehicle, delivery or distribution means or materials. Such carriers are, for example, formulated by conventional methods and administered to subjects in effective amounts.

[0274] VIII. Kit

[0285] In some embodiments, the disclosure provides a kit comprising a composition (e.g., a pharmaceutical composition) of the disclosure (e.g., a composition comprising an anti-TM4SF1 antibody or antigen-binding fragment thereof, or an ADC containing such an antibody, and an immunotherapeutic agent). The kit includes instructions that enable a clinician (e.g., a doctor or nurse) to administer the composition contained therein to a subject to treat a disorder associated with pathological angiogenesis (e.g., cancer).

[0275]

[0286] In certain embodiments, the kit comprises a package of a single-dose pharmaceutical composition containing an effective amount of the antibody of the present disclosure.Optionally, the kit may comprise the instrument or device required for administering the pharmaceutical composition.For example, the kit of the present disclosure may provide one or more pre-filled syringes containing an effective amount of the vaccine, vector, stabilized trimer, or optimized viral polypeptide of the present disclosure.In addition, the kit may also comprise additional components, such as instructions on the administration schedule for subjects with disorders associated with pathological angiogenesis (e.g., cancer), and may use the pharmaceutical composition containing the TM4SF1 binding protein or polynucleotide of the present disclosure. EXAMPLES

[0276] Example 1: Characterization of anti-TM4SF1 antibodies In vitro binding to cell lines

[0287] Antigen binding of the exemplary anti-TM4SF1 antibody 1, which contains a human IgG1 constant region containing a YTEC mutation, was assessed using a cell-based flow cytometry assay in different mouse cell lines: MS1 (a mouse islet endothelial-derived cell line), CT26 (a mouse colon carcinoma-derived cell line), and B16F10 (a mouse melanoma-derived cell line).

[0277]

[0288] Binding EC 50 Values ​​are shown in the legend to Figure 1. Maximal binding of exemplary Antibody 1 indicated that expression of TM4SF1 was highest in MS1, followed by CT26, followed by B16F10.

[0278] In vitro cell proliferation inhibition assay

[0289] The effect of antibody drug conjugates containing a drug (maytansine) and either Exemplary Antibody 1 or Exemplary Antibody 2 (drug to antibody ratio (DAR) of about 2) was evaluated in cultured mouse cells (MS1, CT26, B16F10) and human cells (MiaPaCa2, A549, SKOV3, and HUVEC), respectively. Both Exemplary Antibody 1 and Exemplary Antibody 2 contain a human IgG1 constant region with a YTEC mutation. Cells were incubated and treated with the antibody drug conjugates for 5 days before assessing cell survival on day 5.

[0279]

[0290] Table 1 shows representative ECs from the cell proliferation inhibitory activity of exemplary antibody 1 conjugated to a maytansine payload via linker 1 (L1) (exemplary antibody 1-L1) and exemplary antibody 2 conjugated to a maytansine payload via linker 1 (L1) (exemplary antibody 2-L1), or antibodies via other linkers, in different cell lines. 50 Indicates the value. Summary: Exemplary Antibody 1-L1 killed MS1 (0.05 nM), B16F10 (0.12 nM) and CT26 (no killing), and Exemplary Antibody 2-L1 killed MiaPaCa2 (0.04 nM), A549 (0.06 nM), SKOV3 (0.81 nM), and HUVEC (0.10 nM). TM4SF1 is highly expressed in CT26 cells, but the cells were resistant to Exemplary Antibody 1-L1 antibody-mediated killing.

[0280]

[0291] Table 1: Exemplary antibody 1 targets only cells of mouse origin. Conversely, exemplary antibody 2 targets only cells of human origin. Variants of the exemplary antibodies were generated, producing different antibodies that were capped and naked or conjugated with a maytansine payload via seven different linkers. The exemplary antibody 1-L1 ADC was subsequently used for all immuno-oncology (IO) related studies in mice.

[0281] [Table 1]

[0282] The data presented above shows the EC50 from seven different linkers conjugated to a maytansine payload.

[0283] Linker 1: PEG4-maytansine; BA-PEG4-N-methyl-alanine-maytansine Linker 2: PEG4Ahx-Maytansine; BA-PEG4-Ahx-N-methyl-alanine-Maytansine Linker 3: Glc-maytansine; BA-glucuronide-N-methyl-alanine-maytansine Linker 4: Glu(tb)PEG4-Glc-Maytansine; BA-Glu-(Ot-Butyl)-Glucoronide-N-Methyl-Glycine-N-Methyl-Alanine-Maytansine Linker 5: Glu-tb-Maytansine; BA-PEG4-D-Glu-Ot-Butyl-Ahx-N-Methyl-Alanine-Maytansine Linker 6: GluAhx-Maytansine; BA-PEG4-D-Glu-Ahx-N-methyl-alanine-Maytansine Linker 7: PEG4Glu-Maytansine; BA-PEG4-D-Glu-N-methyl-alanine-Maytansine Example 2: Tumor regression using anti-TM4SF1 ADC and anti-mouse PD-1 antibody

[0292] For this study, cell-derived syngeneic mouse models were used to evaluate the efficacy of treatments involving a combination of an anti-TM4SF1 ADC (exemplary antibody 1-L1) and an anti-PD-1 antibody. The syngeneic models evaluated were B16F10-derived tumors in C57BL / 6 mice (FIG. 2A) and a CT26-derived tumor model in BALB / c mice (FIG. 2B).

[0284]

[0293] In Figure 2A, the B16F10 C57BL / 6 syngeneic model was divided into multiple treatment groups and tumor volume (mm 3) were evaluated: 1) vehicle (control), 2) anti-mouse PD-1 antibody (BioXcell, clone RMP1-14, 10 mg / kg), 3) exemplary antibody 1-L1 (20 mg / kg), and 4) exemplary antibody 1-L1 (20 mg / kg) + anti-mouse PD1 (10 mg / kg). Exemplary antibody 1-L1 was administered once at the beginning of the study. Anti-mouse PD1 antibody was administered three times on days 0, 5, and 10 after treatment. During treatment, tumor volumes were measured on days 4, 6, 9, 13, and 15. Within 10 days, treatment using anti-mouse PD-1 antibody alone (treatment 2) did not inhibit tumor growth compared to the vehicle control (treatment 1). Treatment containing 20 mg / kg exemplary antibody 1-L1 delayed tumor growth (treatment 3) and showed synergistic tumor regression activity in the presence of anti-mouse PD-1 antibody (treatment 4).

[0285]

[0294] In Figure 2B, the CT26 BALB / c syngeneic model was divided into multiple treatment groups and tumor volume (mm ) was measured in the presence of different treatments. 3 ) were evaluated: 1) vehicle (control), 2) anti-mouse PD-1 antibody (BioXcell, clone RMP1-14, 10 mg / kg), 3) exemplary antibody 1-L1 (20 mg / kg), and 4) exemplary antibody 1-L1 (20 mg / kg) + anti-mouse PD1 antibody (10 mg / kg). As in FIG. 2A, anti-TM4SF1 antibody treatment was administered once at the start of treatment, while anti-mouse PD1 antibody injections were administered three times on days 0, 7, and 14. Treatment with anti-mouse PD-1 antibody alone resulted in tumors that progressed similarly to the vehicle control, and animals were sacrificed on day 14. Treatment of mice with 20 mg / kg exemplary antibody 1-L1 improved tumor regression over vehicle control, but the addition of administration of anti-mouse PD-1 antibody further increased inhibition of tumor growth by 28 days after the start of treatment.

[0286]

[0295] Figures 2A and 2B show that the tested ADC (exemplary antibody 1-L1) has synergistic tumor regression effects with anti-mouse PD-1 antibody in both B16F10 (C57BL / 6 mice) and CT26 (Balb / c mice) syngeneic tumor models, respectively. This synergistic tumor growth delay result indicates that tumor vascular targeting by exemplary antibody 1-L1 appears to activate immune responses in tumors by promoting leukocyte infiltration into tumors through tumor vascular damage. The combo approach of immune checkpoint blockade and tumor vascular targeting is expected to provide a great opportunity to convert many different types of solid tumors from immunologically inactive to active states.

[0287]

[0296] As well as due to spindle checkpoint mutations, some tumor cell lines, such as CT26, are resistant to maytansine payloads. Thus, the results of tumor regression by the exemplary antibody 1-L1 antibody in CT26 syngeneic tumors (Figure 2B) are due solely to targeting tumor vasculature. Such tumor regression was further improved in the presence of anti-mouse PD-1 immune blocking antibodies.

[0288] Example 4: Characterization of anti-TM4SF1 antibodies In vitro binding to cell lines

[0297] Antigen binding of an exemplary anti-TM4SF1 antibody-drug conjugate (maytansine) containing a human IgG1 constant region containing the YTEC mutation was assessed using a cell-based flow cytometry assay in different mouse cell lines: MS1 (mouse islet endothelial cell line), CT26 (mouse colon carcinoma cell line), B16F10 (mouse melanoma cell line), and LLC (mouse lung carcinoma). The RENCA cell line was used as a negative control due to the absence of TM4SF1 expression.

[0289]

[0298] Binding EC 50Values ​​are shown in the legend of Figure 3. Maximum binding of Exemplary Antibody 1 showed that expression of TM4SF1 was highest in MS1, followed by CT26, then B16F10, followed by LLC. Renca does not express TM4SF1. Typically, the binding affinity of antibodies in the Exemplary Antibody 1 group is an EC50 of 3-7 nM in cell-based assays.

[0290] In vitro cell proliferation inhibition assay

[0299] The effect of antibody drug conjugates containing a drug (maytansine) and either Exemplary Antibody 1 or Exemplary Antibody 2 (drug to antibody ratio (DAR) of about 2) was evaluated in cultured mouse cells (MS1, CT26, B16F10) and human cells (MiaPaCa2, A549, SKOV3, and HUVEC), respectively. Both Exemplary Antibody 1 and Exemplary Antibody 2 contain a human IgG1 constant region with a YTEC mutation. Cells were incubated and treated with the antibody drug conjugates for 4 days before assessing cell survival on day 5.

[0291] Table 3 shows representative EC values ​​from the cell proliferation inhibitory activity of exemplary antibody 1 conjugated to a maytansine payload via linker 1 (L1) (exemplary antibody 1-L1), exemplary antibody 2 conjugated to a maytansine payload via linker 1 (L1) (exemplary antibody 2-L1), or antibodies via other linkers, in different cell lines. 50 Indicates the value. Summary: Exemplary antibody 1-L1 killed MS1 (0.05 nM), B16F10 (0.12 nM), RENCA (no killing) and CT26 (no killing), and exemplary antibody 2-L1 killed MiaPaCa2 (0.04 nM), A549 (0.06 nM), SKOV3 (0.81 nM), and HUVEC (0.10 nM). TM4SF1 is highly expressed in CT26 cells, but the cells were resistant to exemplary antibody 1-L1 antibody-mediated killing.

[0292] [Table 2]

[0293]

[0300] The data presented above demonstrate the EC 50 Shows.

[0294]

[0301] Linker 1: PEG4-maytansine; BA-PEG4-N-methyl-alanine-maytansine

[0302] Linker 2: PEG4Ahx-Maytansine; BA-PEG4-Ahx-N-methyl-alanine-Maytansine

[0303] Linker 3: Glc-maytansine; BA-glucuronide-N-methyl-alanine-maytansine

[0304] Linker 4: Glu(tb)PEG4-Glc-Maytansine; BA-Glu-(Ot-Butyl)-Glucoronide-N-Methyl-Glycine-N-Methyl-Alanine-Maytansine

[0305] Linker 5: Glu-tb-Maytansine; BA-PEG4-D-Glu-Ot-Butyl-Ahx-N-Methyl-Alanine-Maytansine

[0306] Linker 6: GluAhx-Maytansine; BA-PEG4-D-Glu-Ahx-N-methyl-alanine-Maytansine

[0307] Linker 7: PEG4Glu-Maytansine; BA-PEG4-D-Glu-N-methyl-alanine-Maytansine Example 5: Tumor regression using anti-TM4SF1 ADC and anti-mouse CTLA-4 antibody in the CT26 syngeneic model

[0308] For this study, a cell-derived syngeneic CT26 mouse model was used to evaluate the effects of treatments including a combination of an anti-TM4SF1 ADC (exemplary antibody 1-L1) and / or an anti-mouse CTLA-4 antibody (clone 9H10; BioXCell).

[0295]

[0309] CT26 murine colon carcinoma derived tumor model in BALB / c mice (FIGS. 4A-4H). In FIGS. 4A-4D, 0 mpk of anti-TM4SF1 ADC (exemplary antibody 1-L1) was administered, and anti-mouse CTLA-4 antibody was administered at 0, 2.5, 5, and 10 mpk, respectively. FIGS. 4E-4H show a syngeneic model in which 20 mpk of anti-TM4SF1 ADC (exemplary antibody 1-L1) was administered, and anti-mouse CTLA-4 antibody was administered at 0, 2.5, 5, and 10 mpk, respectively. All doses were administered once on day 0 of the study.

[0296]

[0310] In Figure 4A, CT26 BALB / c syngeneic models (n=40) were divided into two treatment groups and tumor volume (mm ) was plotted in the presence of different treatments. 3) were evaluated: 1) vehicle (control) and 2) isotype-matched control (IS-Ctl), administered once on day 0 of the study. During treatment, tumor volumes were measured 2-3 times per week as indicated by dots in Figure 4A. None of the treatments resulted in tumor regression. In Figure 4B, a third CT26 BALB / c treatment group (n=5) received anti-mouse CTLA-4 antibody at 2.5 mpk once on day 0 of the study. Treatment with anti-mouse CTLA-4 antibody alone resulted in some level of tumor growth delay, with one animal being tumor-free (TF) by day 14 (20% TF) and persisting until at least day 50 ...

Claims

(a) an anti-TM4SF1 binding protein; and (b) immunotherapeutic agent A combination comprising Immunotherapeutic agents, (i) an antibody or antigen-binding fragment thereof against at least one of PD-1, CD40, CTLA-4, CSF1 / CSF1R, SIRPα, CLEC-1; or (ii) an antibody or antigen-binding fragment thereof against at least one of CCR4, CTLA-4, A1R, A2AR, A3R, TIM-3, BTLA, VISTA, TIGIT, LAG-3, ILRa / CD25, ITGB1 / CD29, Ly 24 / CD44, CD48, CEACAM1 / CD66a, Nt5e / CD73, CD94 / NKG2A, FAS / CD95, SLAF1 / CD150, NRP1 / CD304, GITR / CD357, ICOS, Tnfrs4 / OX40, Folr4 / JUNO, P2X7, ANXA2, IDO, B7-H6, KIR, GARP (LRRC32), TNFR2; or (iii) an antibody or antigen-binding fragment thereof against at least one of PD-L1, PD-L2, B7-H3, B7-H4, CD47, TDO, and DcR3; or (iv) ipilimumab, nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, spartalizumab, avelumab, sintilimab, toripalimab, MGA012, MGD013, MGD019, enoblitzumab, MGD009, MGC018, MEDI0680, PDR001, FAZ053, TSR022, MBG453, relatlinab (BMS986 016), LAG525, IMP321, REGN2810 (cemiplimab), REGN3767, pexidartinib, LY3022855, FPA008, BLZ945, GDC0919, epacadostat, indoximide, BMS986205, CPI-444, MEDI9447, PBF509, lirilumab, IMC-001, monalizumab, and combinations thereof. The combination agent.

2. The anti-TM4SF1 binding protein is: a heavy chain variable domain comprising: a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 8, 20, 32, 44, 56, 68, 80, 96, 118, 119, 120, 121, or 162; a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 7, 19, 31, 43, 55, 67, 79, 95, 116, 117, or 161; and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 6, 18, 30, 42, 54, 66, 78, 94, 115, or 160; and a light chain variable domain comprising: a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 14, 26, 38, 50, 62, 74, 86, 110, 129, or 159; a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 13, 25, 37, 49, 61, 73, 85, or 109, 128, or 158; and a CDR1 comprising the amino acid sequence of SEQ ID NO: 12, 24, 36, 48, 60, 72, 84, 107, 108, 124, 125, 126, 127, or 157.

2. The combination of claim 1 , comprising:

3. An anti-TM4SF1 binding protein comprising: (i) a heavy chain variable domain comprising: 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 (ii) a light chain variable domain comprising: a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 110 or 111; a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 109; and a CDR1 comprising the amino acid sequence of SEQ ID NO: 107 or 108.

2. The combination of claim 1 , comprising:

4. An anti-TM4SF1 binding protein comprising: (i) a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 92, 130 and 132; and (ii) a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 99, 101, 103, 105, 131, and 133.

2. The combination of claim 1 , comprising:

5. The anti-TM4SF1 binding protein, comprising an IgG1 Fc region, the IgG1 Fc region comprising the following mutations: (i) N297C, L234A, L235A, and G237A; or (ii) N297C, L234A, L235A, G237A, and P331G; or (iii) N297C, L234A, L235A, G237A, and E233P. The combination according to any one of claims 2 to 4, comprising:

6. The anti-TM4SF1 binding protein, comprising an IgG1 Fc region, the IgG1 Fc region comprising the following mutations: N297C, M252Y, S254T, and T256E The combination according to any one of claims 2 to 4, comprising:

7. 7. The combination of claim 6, wherein the anti-TM4SF1 binding protein is conjugated to a therapeutic molecule to form an antibody drug conjugate, and the therapeutic molecule is selected from the group consisting of a proteasome inhibitor, a calicheamicin, a pyrrolobenzodiazepine, an auristatin, a duocarmycin, a maytansinoid, and any combination thereof.

8. The therapeutic molecule comprises a proteasome inhibitor, the proteasome inhibitor being: bortezomib (Velcade, PS-341), PR-171 (carfilzomib), ixazomib ( Ninlaro®), delanzomib, marizomib, oprozomib, VR23, PI-1840, (benzyloxycarbonyl)-Leu-Leu-phenylalanine, 2,3,5a,6-tetrahydro-6-hydroxy-3-(hydroxymethyl)-2-methyl-10H-3a,10a-epidithio-pyrazinol[1,2α]indole-1,4-dione, 4-hydroxy-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone, sapojargon, Ac-hFLFL -epoxide, aclacinomycin A, aclarubicin, ACM, AdaK(Bio)Ahx3L3VS, AdaLys(Bio)Ahx3L3VS, adamantane-acetyl-(6-aminohexanoyl)-3-(leucunyl)-3-vinyl-(methyl)-sulfone, ALLM, ALLN, calpain inhibitor I, calpain inhibitor II, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal, gliotoxin, isovalery-L-tyrosyl- L-valyl-DL-tyrosinal, clasto-lactacystin-β-lactone, Z-LL-Nva-CHO, ubiquitin aldehyde, YU101, MP-LLL-VS, LDN-57444, Z-GPFL-CHO, Z-LLL-CHO, lovastatin, α-methyl-clasto-lactacystin-β-lactone, mevinolin, MK-803, NIP-L3VS, NP-LLL-VS, NPI-0052 (salinosporamide A), MLN519 (PS-519), NLVS (trileucine vinyl sulfone), ritonavir, Ro106-992 8. The combination of claim 7, wherein the compound is selected from the group consisting of 0, Z-LLF-CHO, Z-LL-B(OH)2, RRRPRPPYLPR, tyropeptin A, ZL3VS, PR-11, PR-39, 0106-9920, proteasome inhibitor I, proteasome inhibitor II, proteasome inhibitor III, proteasome inhibitor IV, AdaAhx3L3VS, efrapeptin, MG-132, MG-262, MG-115, alpha-methyl omuralide, MG-101, epoxomicin, omuralide, lactacystin, and NEOSH101.

9. 9. A pharmaceutical composition comprising the combination of any one of claims 1 to 8 and at least one of a pharma- ceutically acceptable carrier, excipient, diluent, or any combination thereof.

10. 9. A combination according to any one of claims 1 to 8 for use in the treatment of cancer in a subject, wherein administration of the combination to a subject improves T cell function.

11. 11. The combination of claim 10, wherein the improved T cell function comprises increased T cell infiltration in the tumor microenvironment (TME) or wherein the improved T cell function comprises increased expression of ICAM-1 and VCAM-1 in tumor vasculature.

12. 11. The combination of claim 10, wherein the cancer is selected from the group consisting of prostate cancer, liver cancer, colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple negative breast cancer, pancreatic cancer, gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial cancer, lung cancer (small cell lung, non-small cell lung), melanoma, non-melanoma skin cancer (squamous cell carcinoma and basal cell carcinoma), glioma, renal cancer, lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumor, mesothelioma, esophageal cancer, Merkel cell carcinoma, MSI-high cancer, KRAS mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS).

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