Treatment and prevention of cancer using VISTA antigen binding molecules

Abstract

The present disclosure provides antigen binding molecules that bind to VISTA, compositions comprising said molecules, and therapeutic and prophylactic methods using said molecules for the treatment or prevention of cancer.

Description

[Technical Field]

[0001] This application claims priority from US 63 / 244,986, filed September 16, 2021, the contents and elements of which are incorporated herein by reference in their entirety for all purposes. FIELD OF THE INVENTION The present invention relates to the field of molecular biology, more particularly to the field of antibody technology. The present invention also relates to methods of medical treatment and prophylaxis. [Background technology]

[0002] Myeloid-derived suppressor cell (MDSC)-mediated suppression of immune responses has been identified in multiple solid tumors and lymphomas. MDSCs are elevated in advanced colorectal cancer (Toor et al., Front Immunol., 2016, 7:560). MDSCs are also observed in breast cancer, and the percentage of MDSCs in peripheral blood increases in patients with late-stage breast cancer (Markowitz et al., Breast Cancer Res Treat., July 2013, 140(1):13-21). MDSC abundance also correlates with poor prognosis in solid tumors (Charoentong et al., Cell Rep., January 3, 2017, 18(1):248-262).

[0003] MDSCs exert suppression on T cells through multiple mechanisms, including the production of reactive oxygen species, nitric oxide, and arginase, which ultimately result in the suppression of DC, NK, and T cell activity and an increase in tumor burden (Umansky et al., Vaccines (Basel) (2016), 4(4):36). MDSCs also contribute to tumor initiation and metastasis through the production of soluble factors such as matrix metalloproteinases, VEGF, bFGF, TGF-β, and S100A8 / A9, which promote angiogenesis, invasion, proliferation, and metastasis.

[0004] Targeting V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), an immune checkpoint molecule primarily expressed on MDSCs, is an attractive therapeutic strategy to remove MDSC-mediated suppression of effector immune cell function.

[0005] For example, WO2017 / 137830A1 discloses, in paragraph

[0221] , an anti-VISTA antibody, VSTB174, which is disclosed to contain the variable region of the anti-VISTA antibody, VSTB112. Paragraph

[0362] discloses that VSTB123 contains the variable region of VSTB174. In paragraph

[0417] and Figure 42A, Example 25 of WO2017 / 137830A1 discloses that the mIgG2a antibody, VSTB123, was able to inhibit tumor growth in an MB49 tumor model. Paragraph

[0418] and Figure 42A, in contrast, disclose that VSTB124 (the same antibody provided in an IgG2a LALA format; see paragraph

[0408] ) did not inhibit tumor growth. Based on these results, Example 25 in paragraph

[0419] concludes that efficacy of treatment with anti-VISTA antibodies may require active Fc. Thus, the proposed mechanistic action of anti-VISTA antibodies, depicted schematically in Figure 47 (see the caption to Figure 47 in paragraph

[0053] ), involves Fc-mediated engagement of FcγRIII expressed by NK cells.

[0006] The hamster monoclonal anti-VISTA antibody, mAb13F3, is disclosed in Le Mercier et al., Cancer Res. (2014), 74(7):1933-44, to inhibit tumor growth in B16OVA and B16-BL6 melanoma models. The paragraph spanning page 1942 teaches that the immunogenicity and FcR binding activity of VISTA mAbs may be critical limiting factors for achieving optimal target neutralization and therapeutic efficacy. Summary of the Invention [Means for solving the problem]

[0007] In a first aspect, the present invention provides antigen binding molecules, optionally isolated antigen binding molecules, that are capable of binding to VISTA and inhibiting VISTA-mediated signaling independent of Fc-mediated function.

[0008] In some embodiments, the antigen-binding molecule is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 290 HC-CDR2 having the amino acid sequence of SEQ ID NO: 291 HC-CDR3 having the amino acid sequence of SEQ ID NO: 278 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 309 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43 The light chain variable (VL) region comprises:

[0009] In some embodiments, the antigen-binding molecule is (i) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 290 HC-CDR2 having the amino acid sequence of SEQ ID NO: 291 HC-CDR3 having the amino acid sequence of SEQ ID NO: 278 a heavy chain variable (VH) region incorporating (ii) the following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 295 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43 The light chain variable (VL) region comprises:

[0010] In some embodiments, the antigen-binding molecule is a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 289; and It comprises a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 310.

[0011] In some embodiments, the antigen-binding molecule is a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 289; and It comprises a VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 297.

[0012] In some embodiments, the antigen-binding molecule is The following framework regions (FR): HC-FR1 having the amino acid sequence of SEQ ID NO: 63 HC-FR2 having the amino acid sequence of SEQ ID NO: 292 HC-FR3 having the amino acid sequence of SEQ ID NO: 293 HC-FR4 having the amino acid sequence of SEQ ID NO: 281 It contains a VH region incorporating

[0013] In some embodiments, the antigen-binding molecule is The following framework regions (FR): LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 298 LC-FR3 having the amino acid sequence of SEQ ID NO: 284 LC-FR4 having the amino acid sequence of SEQ ID NO: 47 It contains a VL region incorporating

[0014] In some embodiments, the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 331. In some embodiments, the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 317.

[0015] In another aspect, the present invention provides a composition comprising an antigen-binding molecule according to the present disclosure.

[0016] In some embodiments, the composition comprises: (i) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate 80, and a pH of 4.0 to 7.0; or (ii) containing 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate 20, and having a pH of 4.0 to 7.0; or (iii) 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, a pH of 4.0 to 7.0, and optionally, 0.001% to 0.1% (w / v) polysorbate 20 or polysorbate 80; or (iv) containing 2 mM to 200 mM histidine, 0.001% to 0.1% (w / v) polysorbate 20 or polysorbate 80, and having a pH of 4.0 to 7.0; or (v) 2 mM to 200 mM acetate, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate 80, with a pH of 4.0 to 7.0; or (vi) 2 mM to 200 mM acetate, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate 20, with a pH of 4.0 to 7.0; or (vii) 2 mM to 200 mM succinate, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate 80, with a pH of 4.0 to 7.0; or (viii) 2 mM to 200 mM succinate, 2% to 20% (w / v) sucrose, and 0.001% to 0.1% (w / v) polysorbate 20, with a pH of 4.0 to 7.0.

[0017] In some embodiments, the composition comprises: (i) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.5; or (ii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.8; or (iii) 20 mM histidine, 4% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.8; or (iv) 20 mM histidine, 2% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.8; or (v) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 6.3; or (vi) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 20, pH 5.8; or (vii) 20 mM acetate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.5; or (viii) 20 mM succinate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.5; or (ix) 20 mM histidine, 0.02% (w / v) polysorbate 80, and a pH of 5.8; or (x) 20 mM histidine, 150 mM sodium chloride, pH 5.8.

[0018] In some embodiments, the composition comprises 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, and has a pH of 5.5.

[0019] In some embodiments, the composition comprises about 50 mg / mL (e.g., 50 mg / mL) of the antigen-binding molecule.

[0020] In some aspects, there is provided an antigen-binding molecule or composition according to the present disclosure for use as a medicament.

[0021] In some aspects, there is provided an antigen-binding molecule or composition according to the present disclosure for use in a method of treating or preventing cancer in a subject.

[0022] In some aspects, there is provided the use of an antigen-binding molecule or composition according to the present disclosure in the manufacture of a medicament for treating or preventing cancer in a subject.

[0023] In some aspects, methods of treating or preventing cancer in a subject are provided, comprising administering a therapeutically or prophylactically effective amount of an antigen-binding molecule or composition according to the present disclosure.

[0024] In some embodiments, the cancer is characterized by the presence of cells that express VISTA and / or signaling mediated by complexes that include VISTA.

[0025] In some embodiments, the cancer is selected from blood cancer, leukemia (e.g., T-cell leukemia), acute myeloid leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, multiple myeloma, mesothelioma, solid tumor, lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, gastric cancer, colorectal cancer, colorectal adenocarcinoma, uterine cancer, endometrial cancer, breast cancer, triple-negative breast cancer (TBNC), triple-negative invasive breast cancer, invasive ductal carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, thyroid cancer, thymoma, skin cancer, melanoma, cutaneous melanoma, renal cancer, renal cell carcinoma, papillary renal cell carcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian cancer, ovarian serous cystadenocarcinoma, bladder cancer, prostate cancer, and / or prostate cancer. In some embodiments, the cancer is triple-negative breast cancer (TBNC), non-small cell lung carcinoma (NSCLC), and / or a solid tumor.

[0026] In some embodiments, treating or preventing cancer additionally comprises administering an agent capable of inhibiting signaling mediated by an immune checkpoint molecule other than VISTA, for example, the immune checkpoint molecule other than VISTA is PD-1 and / or PD-L1. The agent may be an anti-PD-1 or anti-PD-L1 antibody.

[0027] In some embodiments, the treatment or prevention, or methods thereof, comprises detecting the presence of cells expressing VISTA and / or signaling mediated by a complex comprising VISTA, hi some embodiments, a subject is selected for treatment with the antigen-binding molecule or composition if the presence of cells expressing VISTA and / or signaling mediated by a complex comprising VISTA is detected.

[0028] In some embodiments, the antigen-binding molecule is administered weekly, for example, with a composition according to the present disclosure. In some embodiments, the antigen-binding molecule is administered 1, 2, or 3 times within a 21-day administration cycle, optionally, the treatment includes up to 35 administration cycles. In some embodiments, the antigen-binding molecule is administered on days 1, 8, and / or 15 within a 21-day administration cycle, optionally, the treatment includes up to 35 administration cycles. In some embodiments, the antigen-binding molecule is administered on days 1, 8, 15, and / or 22 within a 28-day administration cycle, optionally, the treatment includes up to 35 administration cycles.

[0029] In some embodiments, the treatment or prevention or method comprises administering between 3.5 mg and 2200 mg of the antigen-binding molecule per administration.

[0030] In some embodiments, the treatment or prevention, or methods thereof, comprises administering (at most or at least) 3.5 mg, 7 mg, 10.5 mg, 17.5 mg, 20 mg, 21 mg, 40 mg, 60 mg, 72 mg, 120 mg, 180 mg, 240 mg, 360 mg, 400 mg, 800 mg, 1200 mg, 1600 mg, 1900 mg, or 2200 mg of antigen-binding molecule (e.g., in a composition in accordance with the present disclosure), per administration, e.g., according to a dosing schedule of the present disclosure.

[0031] In some embodiments, the treatment or prevention, or methods thereof, comprises administering up to 10.5 mg, up to 21 mg, up to 31.5 mg, up to 52.5 mg, up to 60 mg, up to 63 mg, up to 120 mg, up to 180 mg, up to 216 mg, up to 360 mg, up to 540 mg, up to 720 mg, up to 1080 mg, up to 1200 mg, up to 2400 mg, up to 3600 mg, up to 4800 mg, up to 5700 mg, or up to 6600 mg of antigen-binding molecule (e.g., in a composition according to the present disclosure) per 21-day administration cycle. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to novel VISTA binding molecules that have new and / or improved properties compared to known anti-VISTA antibodies.

[0033] The present inventors have generated antigen-binding molecules that bind to regions of particular interest within the extracellular domain of VISTA, and the VISTA-binding molecules of the present invention possess a combination of desirable biophysical and functional properties compared to VISTA-binding antigen-binding molecules disclosed in the prior art.

[0034] In particular, it is demonstrated that the VISTA-binding molecules described herein are capable of antagonizing VISTA-mediated signaling through a mechanism that does not require Fc-mediated function. The present inventors demonstrate that the VISTA-binding molecules described herein, which comprise an Fc that lacks the ability to bind to Fcγ receptors and / or C1q, are capable of providing anti-cancer therapeutic effects in vivo.

[0035] The inventors establish for the first time that it is possible to directly antagonize VISTA-mediated signaling through a mechanism that does not require Fc-mediated effector functions (e.g., ADCC / ADCP / CDC against VISTA-expressing cells).

[0036] The VISTA-binding molecules of the present disclosure target regions of VISTA that are distinct from those targeted by known anti-VISTA antibodies. Antigen-binding molecules that target specific regions of VISTA are capable of antagonizing VISTA-mediated signaling without requiring Fc-mediated effector functions.

[0037] Therefore, the VISTA-binding molecules disclosed herein are useful for inhibiting VISTA-mediated signaling without depleting VISTA-expressing cells. This is important because VISTA is expressed on cells that are not desired to be depleted. Thus, the VISTA-binding molecules disclosed herein are capable of inhibiting VISTA-mediated signaling while minimizing undesired side effects.

[0038] Advantageously, the VISTA binding molecules disclosed herein have also been shown to be capable of relieving T cells from VISTA-mediated suppression. Specifically, the VISTA binding molecules disclosed herein have been shown to be capable of increasing T cell proliferation and the production of IFNγ and TNFα, for example, from T cells cultured in the presence of VISTA or VISTA-expressing cells. VISTA, binding partners, and VISTA-mediated signaling V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA; also known as B7-H5, SISP1, and PD-1H) is a protein identified by UniProt:Q9H7M9, having the amino acid sequence set forth in SEQ ID NO:1 (Q9H7M9-1, v3). The structure and function of VISTA are described, for example, in Lines et al., Cancer Res. (2014), 74(7):1924-1932, incorporated herein by reference in its entirety. VISTA functions as an immune checkpoint and is a type I single-pass transmembrane protein of approximately 50 kDa encoded by the C10orf54 gene. The extracellular domain of VISTA is homologous to PD-L1.

[0039] The N-terminal 32 amino acids of SEQ ID NO: 1 constitute a signal peptide, and therefore the mature form of VISTA (i.e., after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO: 2. Positions 33 to 194 of SEQ ID NO: 1 form the extracellular domain (SEQ ID NO: 3), positions 195 to 215 form the transmembrane domain (SEQ ID NO: 4), and positions 216 to 311 form the cytoplasmic domain (SEQ ID NO: 5). The extracellular domain comprises an Ig-like V-type domain (positions 33 to 168 of SEQ ID NO: 1, shown in SEQ ID NO: 6).

[0040] As used herein, "VISTA" refers to VISTA from any species, and includes isoforms, fragments, variants (including mutants), or homologs of VISTA from any species.

[0041] As used herein, a "fragment," "variant," or "homologue" of a protein may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of a reference protein (e.g., a reference isoform). In some embodiments, fragments, variants, isoforms, and homologues of a reference protein may be characterized by their ability to perform a function performed by the reference protein.

[0042] A "fragment" generally refers to a portion of a reference protein. A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but retains a significant degree of sequence identity (e.g., at least 60%) to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. A "homologue" generally refers to a variant of a reference protein that is produced by a different species compared to the species of the reference protein. Homologues include orthologs.

[0043] A "fragment" can be any length (by number of amino acids), but can optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment is derived), and can have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. A fragment of VISTA can have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, or 300 amino acids, and can have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, or 300 amino acids.

[0044] In some embodiments, VISTA is mammalian-derived VISTA (e.g., primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or rodent (e.g., rat or mouse) VISTA). Isoforms, fragments, variants, or homologs of VISTA may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VISTA isoform from a given species, e.g., human.

[0045] The isoform, fragment, variant, or homologue may optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of the reference VISTA, e.g., as determined by analysis via an appropriate assay for that functional property / activity. For example, an isoform, fragment, variant, or homologue of VISTA may exhibit association with, e.g., VSIG-3, LRIG1, VSIG8, and / or PSGL-1.

[0046] In some embodiments, VISTA comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1 or 2. In some embodiments, a fragment of VISTA comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 2, 3, or 6.

[0047] VISTA is a member of the B7 family of proteins and is primarily expressed by leukocytes, particularly CD14+ monocytes (including monocyte-derived suppressor cells (MDSCs)) and CD33+ myeloid cells. VISTA is also expressed by CD56+ NK cells, dendritic cells, and, to a lesser extent, CD4+ and CD8+ T cells. VISTA is highly expressed on MDSCs, particularly tumor-infiltrating MDSCs, and also on tumor-infiltrating myeloid DCs (Le Mercier et al., Cancer Res. (2014), 74(7):1933-44), as well as on tumor-associated macrophages (TAMs) and neutrophils.

[0048] VISTA acts on T cells as both a ligand and receptor, inhibiting T cell effector functions and maintaining peripheral tolerance, and there is evidence that tumors engineered to overexpress VISTA evade immune control and grow more rapidly than tumors that do not overexpress VISTA (Wang et al., Journal of Experimental Medicine. (2011), 208(3):577-92; Lines et al., Cancer Res. (2014), 74(7):1924-1932). VISTA has been shown to be a co-inhibitory receptor on CD4+ T cells or a co-inhibitory ligand for T cells. VISTA - / -VISTA has been reported to exhibit stronger antigen-specific proliferation and cytokine production than wild-type CD4+ T cells, suggesting that VISTA functions as an inhibitory receptor on CD4+ T cells. Blocking VISTA function using a monoclonal anti-VISTA antibody has been shown to enhance the infiltration, proliferation, and effector function of tumor-reactive T cells in the tumor microenvironment (Le Mercier et al., Cancer Res. (2014), 74(7):1933-44).

[0049] VISTA has been shown to interact with VSIG-3 (IGSF11) (see, e.g., Wang et al., J Immunol (2017), 198(Suppl. 1), 154.1, incorporated herein by reference in its entirety). Engagement of VSIG-3 through VISTA on activated T cells inhibits T cell proliferation and reduces production of cytokines and chemokines, such as IFN-γ, IL-2, IL-17, CCL5 / RANTES, CCL3 / MIP-1a, and CXCL11 / I-TAC.

[0050] VSIG-3 is a protein identified by UniProt:Q5DX21. Alternative splicing of the mRNA encoded by the human IGSF11 gene results in three different isoforms: isoform 1 (UniProt:Q5DX21-1, v3; SEQ ID NO:7); isoform 2 (UniProt:Q5DX21-2; SEQ ID NO:8), which contains a sequence that differs from SEQ ID NO:7 at positions 1-17; and isoform 3 (UniProt:Q5DX21-3; SEQ ID NO:9), which contains a sequence that differs from SEQ ID NO:7 at positions 1-17 and also at positions 211-235.

[0051] The N-terminal 22 amino acids of SEQ ID NOs: 7, 8, and 9 constitute a signal peptide, and thus mature forms of VSIG-3, isoforms 1, 2, and 3 (i.e., after processing to remove the signal peptide), have the amino acids set forth in SEQ ID NOs: 10, 11, and 12, respectively. Positions 23-241 of SEQ ID NOs: 7 and 8 form VSIG-3 extracellular domain isoforms 1 and 2 (SEQ ID NO: 13), and positions 23-216 of SEQ ID NO: 9 form VSIG-3 extracellular domain isoform 3 (SEQ ID NO: 14). The transmembrane domain of VSIG-3 is set forth in SEQ ID NO: 15, and the cytoplasmic domain is set forth in SEQ ID NO: 16. The extracellular domain contains an Ig-like V-type domain (set forth in SEQ ID NO: 17), and VSIG-3 extracellular domain isoforms 1 and 2 additionally contain an Ig-like C2-type domain (set forth in SEQ ID NO: 18).

[0052] As used herein, "VSIG-3" refers to VSIG-3 from any species, and includes isoforms, fragments, variants (including mutants), or homologs of VSIG-3 from any species.

[0053] A fragment of VSIG-3 can have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids, and can have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0054] In some embodiments, the VSIG-3 is mammalian-derived VSIG-3 (e.g., primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or rodent (e.g., rat or mouse) VSIG-3). VSIG-3 isoforms, fragments, variants, or homologs may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VSIG-3 isoform from a given species, e.g., human.

[0055] The isoform, fragment, variant, or homologue may optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of the reference VSIG-3, e.g., as determined by analysis via an appropriate assay for that functional property / activity. For example, an isoform, fragment, variant, or homologue of VSIG-3 may exhibit, e.g., association with VISTA.

[0056] In some embodiments, VSIG-3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 7-12. In some embodiments, a fragment of VSIG-3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 10-14, 17, or 18.

[0057] VISTA has also been proposed to interact with VSIG-8 (see, e.g., WO2016 / 090347A1). VSIG-8 is a protein identified by UniProt:P0DPA2 (SEQ ID NO: 19). The N-terminal 21 amino acids of SEQ ID NO: 19 constitute a signal peptide, and thus the mature form of VSIG-8 (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO: 20. Positions 22 to 263 of SEQ ID NO: 19 form the extracellular domain of VSIG-8 (SEQ ID NO: 21). The transmembrane domain of VSIG-8 is set forth in SEQ ID NO: 22, and the cytoplasmic domain is set forth in SEQ ID NO: 23. The extracellular domain includes Ig-like V-domain 1 (set forth in SEQ ID NO: 24) and Ig-like V-domain 2 (set forth in SEQ ID NO: 25).

[0058] As used herein, "VSIG-8" refers to VSIG-8 from any species, and includes isoforms, fragments, variants (including mutants), or homologs of VSIG-8 from any species.

[0059] A fragment of VSIG-8 can have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids, and can have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0060] In some embodiments, the VSIG-8 is mammalian-derived VSIG-8 (e.g., primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or rodent (e.g., rat or mouse) VSIG-8). VSIG-8 isoforms, fragments, variants, or homologs may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VSIG-8 isoform from a given species, e.g., human.

[0061] The isoform, fragment, variant, or homologue may optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of the reference VSIG-8, e.g., as determined by analysis via an appropriate assay for that functional property / activity. For example, an isoform, fragment, variant, or homologue of VSIG-8 may exhibit, e.g., association with VISTA.

[0062] In some embodiments, VSIG-8 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 19 or 20. In some embodiments, a fragment of VSIG-8 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 20, 21, 24, or 25.

[0063] VISTA has also been proposed to interact with PSGL-1 (see, e.g., WO2018 / 132476A1). PSGL-1 isoform 1 is a protein identified by UniProt: Q14242-1 (SEQ ID NO: 323). PSGL-1 isoform 2 is a protein identified by UniProt: Q14242-2 (SEQ ID NO: 324) and differs from PSGL-1 isoform 1 in that it contains an additional 16 amino acids after position 1 of SEQ ID NO: 323.

[0064] The N-terminal 17 amino acids of SEQ ID NO:323 constitute a signal peptide, and thus the mature form of PSGL-1 (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO:325. Positions 18-320 of SEQ ID NO:323 form the extracellular domain of PSGL-1 (SEQ ID NO:326). The transmembrane domain of PSGL-1 is set forth in SEQ ID NO:327, and the cytoplasmic domain is set forth in SEQ ID NO:328. The extracellular domain contains tandem repeats of 12, 10 amino acids, and the repeat region is set forth in SEQ ID NO:329.

[0065] As used herein, "PSGL-1" refers to PSGL-1 from any species, and includes isoforms, fragments, variants (including mutants), or homologs of PSGL-1 from any species.

[0066] A fragment of PSGL-1 can have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids and can have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0067] In some embodiments, the PSGL-1 is mammalian-derived PSGL-1 (e.g., primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or rodent (e.g., rat or mouse) PSGL-1). PSGL-1 isoforms, fragments, variants, or homologs can optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature PSGL-1 isoform from a given species, e.g., human.

[0068] The isoform, fragment, variant, or homologue can optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of the reference PSGL-1, e.g., as determined by analysis via an assay appropriate for that functional property / activity. For example, an isoform, fragment, variant, or homologue of PSGL-1 can exhibit, e.g., association with VISTA.

[0069] In some embodiments, PSGL-1 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 323 or 324. In some embodiments, a fragment of PSGL-1 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 325, 326, or 329. A region of particular interest on the target molecule The antigen-binding molecules of the present invention were specifically designed to target specific regions of VISTA. In a two-step approach, the VISTA region to be targeted was selected based on predicted antigenicity, function, and safety analyses. Then, peptides corresponding to the target region were used as immunogens to elicit specific monoclonal antibodies, thereby preparing antibodies specific to the target region of VISTA. Subsequent screening identified antibodies capable of binding to VISTA in the naive state. This approach allows for precise control over the antibody epitope.

[0070] Antigen-binding molecules of the present invention can be defined by reference to the region of VISTA to which they bind. Antigen-binding molecules of the present invention may bind to a region of VISTA of particular interest. In some embodiments, antigen-binding molecules may bind to a linear epitope of VISTA consisting of a consecutive sequence of amino acids (i.e., a primary sequence of amino acids). In some embodiments, antigen-binding molecules may bind to a conformational epitope of VISTA consisting of a discontinuous sequence of amino acids within the amino acid sequence.

[0071] In some embodiments, the antigen-binding molecule of the present invention binds to VISTA. In some embodiments, the antigen-binding molecule binds to the extracellular region of VISTA (e.g., the region shown in SEQ ID NO: 3). In some embodiments, the antigen-binding molecule binds to the Ig-like V-type domain of VISTA (e.g., the region shown in SEQ ID NO: 6). In some embodiments, the antigen-binding molecule binds to VISTA within a region corresponding to positions 61 to 162 of SEQ ID NO: 1 (shown in SEQ ID NO: 31).

[0072] In some embodiments, the antigen-binding molecule binds to the region of VISTA set forth in SEQ ID NO: 322. In some embodiments, the antigen-binding molecule binds to the region of VISTA set forth in SEQ ID NO: 26. In some embodiments, the antigen-binding molecule binds to the region of VISTA set forth in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule binds to the region of VISTA set forth in SEQ ID NO: 28. In some embodiments, the antigen-binding molecule binds to the region of VISTA set forth in SEQ ID NO: 29. In some embodiments, the antigen-binding molecule binds to the region of VISTA set forth in SEQ ID NO: 30.

[0073] In some embodiments, the antigen-binding molecule does not bind to the region of VISTA set forth in SEQ ID NO: 271. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA set forth in SEQ ID NO: 272. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA set forth in SEQ ID NO: 273. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA set forth in SEQ ID NO: 274. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA set forth in SEQ ID NO: 275.

[0074] The region of a peptide / polypeptide to which an antibody binds can be determined by those skilled in the art using a variety of methods known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometric hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is incorporated herein by reference in its entirety.

[0075] In some embodiments, the antigen-binding molecule is capable of binding to the same region of VISTA as, or an overlapping region of VISTA as, an antibody comprising the VH and VL sequences of one of the antibody clones described herein, 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31, 2M1-B12, 2M1-D2, 1M2-D2, 13D5p, 13D5-1, 13D5-13, 5M1-A11, or 9M2-C12.

[0076] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides are typically about 2 to 50 amino acids in length. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides are typically greater than about 50 amino acids in length.

[0077] In some embodiments, an antigen-binding molecule of the invention is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of one of SEQ ID NOs: 1, 2, 3, 6, or 31.

[0078] In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 322. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 30.

[0079] In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO: 271. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO: 272. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO: 273. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO: 274. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO: 275.

[0080] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblot (e.g., Western blot), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012), 907:411-442), or biolayer interferometry (see, e.g., Lad et al. (2015), J Biomol Screen, 20(4):498-507).

[0081] In embodiments in which the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising a reference amino acid sequence, the peptide / polypeptide may comprise one or more additional amino acids at one or both termini of the reference amino acid sequence. In some embodiments, the peptide / polypeptide comprises, for example, 1 to 5, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 1 to 50, 5 to 10, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 20 to 30, 20 to 40, or 20 to 50 additional amino acids at one or both termini of the reference amino acid sequence.

[0082] In some embodiments, in the context of the amino acid sequence of VISTA, the additional amino acid(s) provided at one or both ends (i.e., the N-terminus and C-terminus) of the reference sequence correspond to positions at the ends of the reference sequence. By way of example, if an antigen-binding molecule is capable of binding to a peptide comprising the sequence of SEQ ID NO:26 and two additional amino acids at the C-terminus of SEQ ID NO:26, the two additional amino acids may be arginine and asparagine, which correspond to positions 90 and 91 of SEQ ID NO:1.

[0083] In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide to which an antibody comprising the VH and VL sequences of one of the antibody clones described herein: 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31, 2M1-B12, 2M1-D2, 1M2-D2, 13D5p, 13D5-1, 13D5-13, 5M1-A11, or 9M2-C12. Myeloid-derived suppressor cells (MDSCs) Myeloid-derived suppressor cells (MDSCs) are a non-allogeneic immune cell population of myeloid lineage cells characterized by an immunosuppressive phenotype. The biology of MDSCs is reviewed in Kumar et al., Trends Immunol. (2016), 37(3):208-220, which is incorporated herein by reference in its entirety.

[0084] MDSCs exhibit increased expression of NADPH oxidase (Nox2), reactive oxygen species (ROS) and superoxide anion (O 2- ), hydrogen peroxide (H2O2), and peroxynitrite (PNT; ONOO - These cells are characterized by numerous biochemical and genomic features that distinguish them from mature myeloid cells (i.e., macrophages, dendritic cells, and neutrophils), including increased production of cytokines such as erythrocyte myeloid leukemia (ERL), inflammatory bowel disease (IGL), and inflammatory bowel disease (IGS)-associated cytokines (e.g., inflammatory bowel disease, inflammatory bowel disease, and inflammatory bowel disease); increased expression of arginase 1 and nitric oxide synthase 2 (nos2), and increased production of nitric oxide (NO); increased expression of c / EBPβ and STAT3; decreased expression of IRF8; and increased production of S100A8 / 9 proteins.

[0085] There are two distinct types of MDSCs: polymorphonuclear MDSCs (PMN-MDSCs), which are morphologically and phenotypically similar to neutrophils, and monocytic MDSCs (M-MDSCs), which are more similar to monocytes. The morphological and phenotypic characteristics of MDSCs are described, for example, in Marvel and Gabrilovich, J Clin Invest., September 1, 2015, 125(9):3356-3364, which is incorporated herein by reference in its entirety. In mice, MDSCs are widely expressed as CD11b + Gr1 + Identified as Gr-1 cells hi The majority of cells were PMN-MDSCs and Gr-1 lo These subsets may be more accurately identified based on the Ly6C and Ly6G markers, and M-MDSCs express CD11b + Ly6C hi Ly6G - and PMN-MDSCs express CD11b + Ly6C lo Ly6G + In humans, MDSCs are identified within the mononuclear fraction. PMN-MDSCs express CD14 - CD11b + CD33 + CD15 + or CD66b + M-MDSCs are CD14 + HLA-DR - / lo It is a cell. - HLA-DR - CD33 + The MDSC population represents a mixed cell population enriched for myeloid progenitor cells.

[0086] Factors involved in MDSC-mediated immunosuppression include expression of arginase (ARG1), inducible NOS (iNOS), TGF-β, IL-10, and COX2, cysteine ​​blockade, reduced expression of l-selectin by T cells, and induction of Tregs. M-MDSCs and PMN-MDSCs utilize different mechanisms of immunosuppression. M-MDSCs suppress both antigen-specific and non-specific T cell responses through the production of NO and cytokines and are more immunosuppressive than PMN-MDSCs. PMN-MDSCs suppress immune responses in an antigen-specific manner through the production of ROS.

[0087] MDSCs are pathologically involved in the development and progression of cancer and infectious diseases. The role of MDSCs in human diseases is reviewed, for example, in Kumar et al., Trends Immunol. (2016), 37(3):208-220 (incorporated herein by reference); and Greten et al., Int Immunopharmacol. (2011), 11(7):802-807, which are incorporated herein by reference in their entirety.

[0088] MDSCs are abundant in tumor tissue and contribute to cancer development and progression through multiple mechanisms, as reviewed, for example, in Umansky et al., Vaccines (Basel) (2016), 4(4):36. MDSCs are recruited to tumor sites via expression of chemokines, and proinflammatory factors within the tumor microenvironment result in a significant upregulation of immunosuppressive functions by MDSCs. MDSCs contribute to tumor development, angiogenesis, and metastasis through suppression of effector immune cell function (e.g., effector T cell and NK cell function), promotion of regulatory T cell production / activity, production of growth factors such as VEGF and bFGF, and production of ECM modifiers such as matrix metalloproteinases.

[0089] MDSCs may be characterized for expression of VISTA. In embodiments of various aspects of the invention, MDSCs may be "VISTA-expressing MDSCs" or "VISTA+ MDSCs." MDSCs may express VISTA on the cell surface (i.e., VISTA may be expressed within or at the cell membrane). antigen binding molecule The present invention provides antigen-binding molecules capable of binding to VISTA.

[0090] The term "antigen-binding molecule" refers to a molecule capable of binding to a target antigen, and includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g., VhH), etc.), so long as they exhibit binding to the intended target molecule(s).

[0091] The antigen-binding molecules of the present invention comprise a moiety capable of binding to a target antigen(s). In some embodiments, the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specifically binding to the target antigen. In some embodiments, the moiety capable of binding to the target antigen comprises or consists of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (reviewed, e.g., in Zhou and Rossi, Nat Rev Drug Discov., 2017, 16(3):181-202). In some embodiments, the moiety capable of binding to a target antigen comprises or consists of an antigen-binding peptide / polypeptide, such as a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (i.e., single domain antibody (sdAb)), affilin, armadillo repeat protein (ArmRP), OBody, or fibronectin (e.g., reviewed in Reverdatto et al., Curr Top Med Chem., 2015, 15(12):1082-1101, which are incorporated herein by reference in their entireties (see also, e.g., Boersma et al., J Biol Chem (2011), 286:41273-85; and Emanuel et al., Mab (2011), 3:38-48)).

[0092] The antigen-binding molecules of the present invention generally comprise an antigen-binding domain, comprising an antibody VH and VL, capable of specifically binding to a target antigen. Herein, the antigen-binding domain formed by the VH and VL is also referred to as an Fv region.

[0093] An antigen-binding molecule may be or comprise an antigen-binding polypeptide or antigen-binding polypeptide complex. An antigen-binding molecule may comprise more than one polypeptide that together form an antigen-binding domain. The polypeptides may be covalently or non-covalently associated. In some embodiments, the polypeptide forms part of a larger polypeptide that comprises the polypeptide (e.g., in the case of an scFv comprising a VH and a VL, or in the case of an scFab comprising a VH-CH1 and a VL-CL).

[0094] An antigen-binding molecule may refer to a non-covalent or covalent complex of an IgG-like antigen-binding molecule that includes more than one polypeptide (e.g., two, three, four, six, or eight polypeptides), e.g., two heavy chain polypeptides and two light chain polypeptides.

[0095] Antigen-binding molecules of the present invention can be designed and prepared using the sequence of a monoclonal antibody (mAb) capable of binding to VISTA. Antigen-binding regions of antibodies, such as single-chain variable fragments (scFv), Fab, and F(ab')2 fragments, can also be used / derived. An "antigen-binding region" is any fragment of an antibody capable of binding to a target for which a given antibody is specific.

[0096] Antibodies generally contain six complementarity-determining regions, or CDRs: three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, the six CDRs define the paratope of the antibody, the portion of the antibody that binds to the target antigen.

[0097] The VH and VL regions comprise, on either side of each CDR, framework regions (FRs) that provide a scaffold for the CDRs. From N- to C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0098] There are several different conventions for defining the CDRs and FRs of antibodies, such as the convention described in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991); Chothia et al., J. Mol. Biol., 196:901-917 (1987), and VBASE2, described in Retter et al., Nucl. Acids Res., (2005), 33(Suppl. 1):D671-D674. The CDRs and FRs of the VH and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015), 43(Database Issue):D413-22), using the IMGT V-domain numbering convention described in Lefranc et al., Dev. Comp. Immunol. (2003), 27:55-77.

[0099] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule capable of binding to VISTA. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule capable of binding to VISTA. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule capable of binding to VISTA. That is, in some embodiments, the antigen-binding molecule comprises the VH region and VL region of an antigen-binding molecule capable of binding to VISTA.

[0100] In some embodiments, the antigen-binding molecule comprises a VH region and a VL region that are or are derived from the VH / VL regions of a VISTA-binding antibody clone described herein (i.e., the anti-VISTA antibody clones 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31, 2M1-B12, 2M1-D2, 1M2-D2, 13D5p, 13D5-1, 13D5-13, 5M1-A11, or 9M2-C12).

[0101] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (1) to (18) below.

[0102] (1) (Consensus derived from 4M2-C12) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 305 HC-CDR2 having the amino acid sequence of SEQ ID NO: 306 HC-CDR3 having the amino acid sequence of SEQ ID NO: 307; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0103] (2) (V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31) The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 290 HC-CDR2 having the amino acid sequence of SEQ ID NO: 291 HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0104] (3)(V4-C1) CDR below: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33 HC-CDR2 having the amino acid sequence of SEQ ID NO: 277 HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0105] (4)(V4-C9) CDR below: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33 HC-CDR2 having the amino acid sequence of SEQ ID NO: 286 HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0106] (5) (4M2-C12 / V4H1 / V4H2 consensus) CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 244 HC-CDR2 having the amino acid sequence of SEQ ID NO: 34 HC-CDR3 having the amino acid sequence of SEQ ID NO: 35; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0107] (6) (4M2-C12, 4M2-B4, V4H2) CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33 HC-CDR2 having the amino acid sequence of SEQ ID NO: 34 HC-CDR3 having the amino acid sequence of SEQ ID NO: 35; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0108] (7)(V4H1) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 53 HC-CDR2 having the amino acid sequence of SEQ ID NO: 34 HC-CDR3 having the amino acid sequence of SEQ ID NO: 35; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0109] (8) (2M1-B12, 2M1-D2) CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 72 HC-CDR2 having the amino acid sequence of SEQ ID NO: 73 HC-CDR3 having the amino acid sequence of SEQ ID NO: 74; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0110] (9) (4M2-C9, 5M1-A11) CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 88 HC-CDR2 having the amino acid sequence of SEQ ID NO: 89 HC-CDR3 having the amino acid sequence of SEQ ID NO: 90; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0111] (10)(4M2-D9) CDR below: HC-CDR1 having the amino acid sequence of SEQ ID NO: 33 HC-CDR2 having the amino acid sequence of SEQ ID NO: 107 HC-CDR3 having the amino acid sequence of SEQ ID NO: 108; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0112] (11)(1M2-D2) CDR below: HC-CDR1 having the amino acid sequence of SEQ ID NO: 120 HC-CDR2 having the amino acid sequence of SEQ ID NO: 121 HC-CDR3 having the amino acid sequence of SEQ ID NO: 122; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0113] (12)(4M2-D5) CDR below: HC-CDR1 having the amino acid sequence of SEQ ID NO: 144 HC-CDR2 having the amino acid sequence of SEQ ID NO: 145 HC-CDR3 having the amino acid sequence of SEQ ID NO: 146; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0114] (13)(4M2-A8) CDR below: HC-CDR1 having the amino acid sequence of SEQ ID NO: 158 HC-CDR2 having the amino acid sequence of SEQ ID NO: 159 HC-CDR3 having the amino acid sequence of SEQ ID NO: 160; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 have been replaced with another amino acid.

[0115] (14)(9M2-C12) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 169 HC-CDR2 having the amino acid sequence of SEQ ID NO: 170 HC-CDR3 having the amino acid sequence of SEQ ID NO: 171; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0116] (15) (derived from 13D5) the following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 72 HC-CDR2 having the amino acid sequence of SEQ ID NO: 184 HC-CDR3 having the amino acid sequence of SEQ ID NO: 246; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0117] (16)(13D5p) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 72 HC-CDR2 having the amino acid sequence of SEQ ID NO: 184 HC-CDR3 having the amino acid sequence of SEQ ID NO: 185; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0118] (17)(13D5-1) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 72 HC-CDR2 having the amino acid sequence of SEQ ID NO: 184 HC-CDR3 having the amino acid sequence of SEQ ID NO: 195; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0119] (18)(13D5-13) The following CDR: HC-CDR1 having the amino acid sequence of SEQ ID NO: 72 HC-CDR2 having the amino acid sequence of SEQ ID NO: 184 HC-CDR3 having the amino acid sequence of SEQ ID NO: 200; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-CDR1, HC-CDR2, or HC-CDR3 are replaced with different amino acids.

[0120] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (19) to (35) below.

[0121] (19)(V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31) Below FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 63 HC-FR2 having the amino acid sequence of SEQ ID NO: 292 HC-FR3 having the amino acid sequence of SEQ ID NO: 293 HC-FR4 having the amino acid sequence of SEQ ID NO: 281; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0122] (20)(V4-C1, V4-C9) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 63 HC-FR2 having the amino acid sequence of SEQ ID NO: 279 HC-FR3 having the amino acid sequence of SEQ ID NO: 280 HC-FR4 having the amino acid sequence of SEQ ID NO: 281; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with another amino acid.

[0123] (21)(4M2-C12) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 36 HC-FR2 having the amino acid sequence of SEQ ID NO: 37 HC-FR3 having the amino acid sequence of SEQ ID NO: 38 HC-FR4 having the amino acid sequence of SEQ ID NO: 39; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with another amino acid.

[0124] (22)(4M2-B4) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 49 HC-FR2 having the amino acid sequence of SEQ ID NO: 37 HC-FR3 having the amino acid sequence of SEQ ID NO: 38 HC-FR4 having the amino acid sequence of SEQ ID NO: 39; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with another amino acid.

[0125] (23)(V4H1) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 54 HC-FR2 having the amino acid sequence of SEQ ID NO: 55 HC-FR3 having the amino acid sequence of SEQ ID NO: 56 HC-FR4 having the amino acid sequence of SEQ ID NO: 39; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0126] (24)(V4H2) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 63 HC-FR2 having the amino acid sequence of SEQ ID NO: 64 HC-FR3 having the amino acid sequence of SEQ ID NO: 65 HC-FR4 having the amino acid sequence of SEQ ID NO: 39; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0127] (25)(2M1-B12) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 75 HC-FR2 having the amino acid sequence of SEQ ID NO: 76 HC-FR3 having the amino acid sequence of SEQ ID NO: 77 HC-FR4 having the amino acid sequence of SEQ ID NO: 78; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0128] (26)(4M2-C9) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 91 HC-FR2 having the amino acid sequence of SEQ ID NO: 92 HC-FR3 having the amino acid sequence of SEQ ID NO: 93 HC-FR4 having the amino acid sequence of SEQ ID NO: 94; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0129] (27)(2M1-D2) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 103 HC-FR2 having the amino acid sequence of SEQ ID NO: 76 HC-FR3 having the amino acid sequence of SEQ ID NO: 77 HC-FR4 having the amino acid sequence of SEQ ID NO: 78; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0130] (28)(4M2-D9) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 109 HC-FR2 having the amino acid sequence of SEQ ID NO: 110 HC-FR3 having the amino acid sequence of SEQ ID NO: 111 HC-FR4 having the amino acid sequence of SEQ ID NO: 112; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0131] (29)(1M2-D2) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 123 HC-FR2 having the amino acid sequence of SEQ ID NO: 124 HC-FR3 having the amino acid sequence of SEQ ID NO: 125 HC-FR4 having the amino acid sequence of SEQ ID NO: 78; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0132] (30)(5M1-A11) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 134 HC-FR2 having the amino acid sequence of SEQ ID NO: 92 HC-FR3 having the amino acid sequence of SEQ ID NO: 93 HC-FR4 having the amino acid sequence of SEQ ID NO: 135; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0133] (31)(4M2-D5) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 147 HC-FR2 having the amino acid sequence of SEQ ID NO: 148 HC-FR3 having the amino acid sequence of SEQ ID NO: 149 HC-FR4 having the amino acid sequence of SEQ ID NO: 135; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0134] (32)(4M2-A8) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 161 HC-FR2 having the amino acid sequence of SEQ ID NO: 162 HC-FR3 having the amino acid sequence of SEQ ID NO: 163 HC-FR4 having the amino acid sequence of SEQ ID NO: 135; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0135] (33)(9M2-C12) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 172 HC-FR2 having the amino acid sequence of SEQ ID NO: 173 HC-FR3 having the amino acid sequence of SEQ ID NO: 174 HC-FR4 having the amino acid sequence of SEQ ID NO: 175; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0136] (34) (13D5p, 13D5-1) FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 103 HC-FR2 having the amino acid sequence of SEQ ID NO: 186 HC-FR3 having the amino acid sequence of SEQ ID NO: 187 HC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0137] (35)(13D5-13) The following FR: HC-FR1 having the amino acid sequence of SEQ ID NO: 103 HC-FR2 having the amino acid sequence of SEQ ID NO: 186 HC-FR3 having the amino acid sequence of SEQ ID NO: 201 HC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 are replaced with different amino acids.

[0138] In some embodiments, the antigen-binding molecule comprises a VH region comprising a CDR according to one of (1) to (18) above and a FR according to one of (19) to (35) above.

[0139] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (36) to (57) below.

[0140] (36) A VH region comprising a CDR according to (1) and a FR according to (19), (20), (21), (22), (23), or (24).

[0141] (37) A VH region comprising a CDR according to (2) and a FR according to (19).

[0142] (38) A VH region comprising a CDR according to (3) and a FR according to (20).

[0143] (39) A VH region comprising a CDR according to (4) and a FR according to (20).

[0144] (40) A VH region comprising a CDR according to (5) and a FR according to (21), (22), (23), or (24).

[0145] (41) A VH region comprising a CDR according to (6) and a FR according to (21).

[0146] (42) A VH region comprising a CDR according to (6) and a FR according to (22).

[0147] (43) A VH region comprising a CDR according to (6) and a FR according to (24).

[0148] (44) A VH region comprising a CDR according to (7) and a FR according to (23).

[0149] (45) A VH region comprising a CDR according to (8) and a FR according to (25).

[0150] (46) A VH region comprising a CDR according to (8) and a FR according to (27).

[0151] (47) A VH region comprising a CDR according to (9) and a FR according to (26).

[0152] (48) A VH region comprising a CDR according to (9) and a FR according to (30).

[0153] (49) A VH region comprising CDRs according to (10) and FRs according to (28).

[0154] (50) A VH region comprising a CDR according to (11) and a FR according to (29).

[0155] (51) A VH region comprising a CDR according to (12) and a FR according to (31).

[0156] (52) A VH region comprising a CDR according to (13) and a FR according to (32).

[0157] (53) A VH region comprising a CDR according to (14) and a FR according to (33).

[0158] (54) A VH region comprising a CDR according to (15) and a FR according to (34) or (35).

[0159] (55) A VH region comprising a CDR according to (16) and a FR according to (34).

[0160] (56) A VH region comprising a CDR according to (17) and a FR according to (34).

[0161] (57) A VH region comprising a CDR according to (18) and a FR according to (35).

[0162] In some embodiments, the antigen-binding molecule comprises a VH region according to one of (58) to (76) below.

[0163] (58) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 276.

[0164] (59) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 285.

[0165] (60) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 289.

[0166] (61) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32.

[0167] (62) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48.

[0168] (63) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 52.

[0169] (64) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62.

[0170] (65) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0171] (66) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87.

[0172] (67) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 102.

[0173] (68) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106.

[0174] (69) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119.

[0175] (70) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 133.

[0176] (71) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 143.

[0177] (72) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 157.

[0178] (73) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 168.

[0179] (74) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 183.

[0180] (75) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 194.

[0181] (76) A VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 199.

[0182] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (77) to (96) below.

[0183] (77) (consensus derived from 4M2-C12) CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 308 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0184] (78) (C24 / C26 / C27 consensus) CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 309 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0185] (79) (V4-C24, V4-C26) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 295 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0186] (80) (V4-C27, V4-C30, V4-C31) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 300 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0187] (81) (4M2-C12 / V4H1 / V4H2 consensus) CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 245 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0188] (82) (4M2-C12, 4M2-B4, V4-C1, V4-C9, V4-C28) CDRs below: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 42 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0189] (83)(V4H1) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 58 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0190] (84)(V4H2) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 43; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0191] (85) (2M1-B12, 2M1-D2) CDRs below: LC-CDR1 having the amino acid sequence of SEQ ID NO: 80 LC-CDR2 having the amino acid sequence of SEQ ID NO: 81 LC-CDR3 having the amino acid sequence of SEQ ID NO: 82; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0192] (86)(4M2-C9) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 96 LC-CDR2 having the amino acid sequence of SEQ ID NO: 97 LC-CDR3 having the amino acid sequence of SEQ ID NO: 98; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0193] (87)(4M2-D9) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 114 LC-CDR2 having the amino acid sequence of SEQ ID NO: 67 LC-CDR3 having the amino acid sequence of SEQ ID NO: 115; or a VH region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0194] (88)(1M2-D2) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 127 LC-CDR2 having the amino acid sequence of SEQ ID NO: 128 LC-CDR3 having the amino acid sequence of SEQ ID NO: 129; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0195] (89)(5M1-A11) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 137 LC-CDR2 having the amino acid sequence of SEQ ID NO: 138 LC-CDR3 having the amino acid sequence of SEQ ID NO: 139; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0196] (90)(4M2-D5) CDR below: LC-CDR1 having the amino acid sequence of SEQ ID NO: 151 LC-CDR2 having the amino acid sequence of SEQ ID NO: 152 LC-CDR3 having the amino acid sequence of SEQ ID NO: 153; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0197] (91)(4M2-A8) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 165 LC-CDR2 having the amino acid sequence of SEQ ID NO: 152 LC-CDR3 having the amino acid sequence of SEQ ID NO: 153; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0198] (92)(9M2-C12) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 177 LC-CDR2 having the amino acid sequence of SEQ ID NO: 178 LC-CDR3 having the amino acid sequence of SEQ ID NO: 179; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0199] (93) (derived from 13D5p) CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 247 LC-CDR2 having the amino acid sequence of SEQ ID NO: 178 LC-CDR3 having the amino acid sequence of SEQ ID NO: 190; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0200] (94)(13D5p) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 189 LC-CDR2 having the amino acid sequence of SEQ ID NO: 178 LC-CDR3 having the amino acid sequence of SEQ ID NO: 190; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with different amino acids.

[0201] (95)(13D5-1) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 197 LC-CDR2 having the amino acid sequence of SEQ ID NO: 178 LC-CDR3 having the amino acid sequence of SEQ ID NO: 190; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0202] (96)(13D5-13) The following CDR: LC-CDR1 having the amino acid sequence of SEQ ID NO: 203 LC-CDR2 having the amino acid sequence of SEQ ID NO: 178 LC-CDR3 having the amino acid sequence of SEQ ID NO: 190; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced with another amino acid.

[0203] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (97) to (120) below.

[0204] (97)(V4-C1) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 59 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 284 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0205] (98)(V4-C9) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 284 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0206] (99)(V4-C24) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 296 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0207] (100)(V4-C26) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 298 LC-FR3 having the amino acid sequence of SEQ ID NO: 284 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0208] (101)(V4-C27) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 284 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0209] (102)(V4-C28) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 296 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0210] (103)(V4-C30) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 296 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0211] (104)(V4-C31) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 283 LC-FR3 having the amino acid sequence of SEQ ID NO: 304 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0212] (105)(4M2-C12) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 44 LC-FR2 having the amino acid sequence of SEQ ID NO: 45 LC-FR3 having the amino acid sequence of SEQ ID NO: 46 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0213] (106)(4M2-B4) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 51 LC-FR2 having the amino acid sequence of SEQ ID NO: 45 LC-FR3 having the amino acid sequence of SEQ ID NO: 46 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0214] (107)(V4H1) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 59 LC-FR2 having the amino acid sequence of SEQ ID NO: 60 LC-FR3 having the amino acid sequence of SEQ ID NO: 61 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0215] (108)(V4H2) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 68 LC-FR2 having the amino acid sequence of SEQ ID NO: 69 LC-FR3 having the amino acid sequence of SEQ ID NO: 70 LC-FR4 having the amino acid sequence of SEQ ID NO: 47; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0216] (109)(2M1-B12) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 83 LC-FR2 having the amino acid sequence of SEQ ID NO: 84 LC-FR3 having the amino acid sequence of SEQ ID NO: 85 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0217] (110)(4M2-C9) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 99 LC-FR2 having the amino acid sequence of SEQ ID NO: 100 LC-FR3 having the amino acid sequence of SEQ ID NO: 101 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0218] (111)(2M1-D2) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 105 LC-FR2 having the amino acid sequence of SEQ ID NO: 84 LC-FR3 having the amino acid sequence of SEQ ID NO: 85 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0219] (112)(4M2-D9) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 116 LC-FR2 having the amino acid sequence of SEQ ID NO: 117 LC-FR3 having the amino acid sequence of SEQ ID NO: 118 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0220] (113)(1M2-D2) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 130 LC-FR2 having the amino acid sequence of SEQ ID NO: 131 LC-FR3 having the amino acid sequence of SEQ ID NO: 132 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0221] (114)(5M1-A11) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 140 LC-FR2 having the amino acid sequence of SEQ ID NO: 141 LC-FR3 having the amino acid sequence of SEQ ID NO: 142 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0222] (115)(4M2-D5) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 154 LC-FR2 having the amino acid sequence of SEQ ID NO: 155 LC-FR3 having the amino acid sequence of SEQ ID NO: 156 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0223] (116)(4M2-A8) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 166 LC-FR2 having the amino acid sequence of SEQ ID NO: 155 LC-FR3 having the amino acid sequence of SEQ ID NO: 167 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0224] (117)(9M2-C12) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 180 LC-FR2 having the amino acid sequence of SEQ ID NO: 181 LC-FR3 having the amino acid sequence of SEQ ID NO: 182 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0225] (118)(13D5p) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 191 LC-FR2 having the amino acid sequence of SEQ ID NO: 192 LC-FR3 having the amino acid sequence of SEQ ID NO: 193 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0226] (119)(13D5-1) FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 191 LC-FR2 having the amino acid sequence of SEQ ID NO: 198 LC-FR3 having the amino acid sequence of SEQ ID NO: 193 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0227] (120)(13D5-13) The following FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 191 LC-FR2 having the amino acid sequence of SEQ ID NO: 192 LC-FR3 having the amino acid sequence of SEQ ID NO: 204 LC-FR4 having the amino acid sequence of SEQ ID NO: 86; or a VL region incorporating these variants in which one, two, or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 are replaced with another amino acid.

[0228] In some embodiments, the antigen-binding molecule comprises a VL region comprising a CDR according to one of (77) to (96) above and a FR according to one of (97) to (120) above.

[0229] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (121) to (148) below.

[0230] (121) A VL region comprising CDRs according to (77) and FRs according to (97), (98), (99), (100), (101), (102), (103), (104), (105), (106), (107) or (108).

[0231] (122) A VL region comprising a CDR according to (78) and a FR according to (99), (100), or (101).

[0232] (123) A VL region comprising CDRs according to (79) and FRs according to (99).

[0233] (124) A VL region comprising CDRs according to (79) and FRs according to (100).

[0234] (125) A VL region comprising CDRs according to (80) and FRs according to (101).

[0235] (126) A VL region comprising CDRs according to (82) and FRs according to (97).

[0236] (127) A VL region comprising CDRs according to (82) and FRs according to (98).

[0237] (128) A VL region comprising CDRs according to (82) and FRs according to (102).

[0238] (129) A VL region comprising CDRs according to (80) and FRs according to (103).

[0239] (130) A VL region comprising CDRs according to (80) and FRs according to (104).

[0240] A VL region comprising a CDR according to (131)(81) and a FR according to (105), (106), (107), or (108).

[0241] (132) A VL region comprising CDRs according to (82) and FRs according to (105).

[0242] (133) A VL region comprising CDRs according to (82) and FRs according to (106).

[0243] (134) A VL region comprising CDRs according to (83) and FRs according to (107).

[0244] (135) A VL region comprising CDRs according to (84) and FRs according to (108).

[0245] (136) A VL region comprising CDRs according to (85) and FRs according to (109).

[0246] (137) A VL region comprising CDRs according to (85) and FRs according to (111).

[0247] (138) A VL region comprising CDRs according to (86) and FRs according to (110).

[0248] (139) A VL region comprising CDRs according to (87) and FRs according to (112).

[0249] (140) A VL region comprising CDRs according to (88) and FRs according to (113).

[0250] (141) A VL region comprising CDRs according to (89) and FRs according to (114).

[0251] (142) A VL region comprising CDRs according to (90) and FRs according to (115).

[0252] (143) A VL region comprising CDRs according to (91) and FRs according to (116).

[0253] (144) A VL region comprising CDRs according to (92) and FRs according to (117).

[0254] (145) A VL region comprising CDRs according to (93) and FRs according to (118), (119), or (120).

[0255] (146) A VL region comprising CDRs according to (94) and FRs according to (118).

[0256] (147) A VL region comprising CDRs according to (95) and FRs according to (119).

[0257] (148) A VL region comprising CDRs according to (96) and FRs according to (120).

[0258] In some embodiments, the antigen-binding molecule comprises a VL region according to one of (149) to (173) below.

[0259] (149) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 310.

[0260] (150) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 282.

[0261] (151) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 287.

[0262] (152) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 294.

[0263] (153) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 297.

[0264] (154) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 299.

[0265] (155) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 301.

[0266] (156) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 302.

[0267] (157) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 303.

[0268] (158) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0269] (159) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50.

[0270] (160) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 57.

[0271] (161) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66.

[0272] (162) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79.

[0273] (163) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0274] (164) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 104.

[0275] (165) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113.

[0276] (166) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126.

[0277] (167) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 136.

[0278] (168) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 150.

[0279] (169) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 164.

[0280] (170) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176.

[0281] (171) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 188.

[0282] (172) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 196.

[0283] (173) A VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 202.

[0284] In some embodiments, the antigen-binding molecule comprises a VH region according to any one of (1) to (76) above and a VL region according to any one of (77) to (173) above.

[0285] In some embodiments, the antigen-binding molecule is a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 289; and a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 297. Includes.

[0286] In some embodiments, the antigen-binding molecule is (i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 315; and (ii) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 317. It comprises or consists of:

[0287] In some embodiments, the antigen-binding molecule is (i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 331; and (ii) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 317. It comprises or consists of:

[0288] In embodiments according to the invention in which one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example, according to the following table: In some embodiments, amino acids in the same block in the middle column are substituted; In some embodiments, amino acids in the same stretch in the rightmost column are substituted.

[0289] [Table 1] In some embodiments, the substitution(s) may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule comprising the substitution compared to a comparable unsubstituted molecule.

[0290] The VH and VL regions of the antigen-binding region of an antibody together constitute an Fv region. In some embodiments, an antigen-binding molecule according to the present invention comprises or consists of an Fv region that binds to VISTA. In some embodiments, the VH and VL regions of the Fv are provided as a single polypeptide, i.e., a single-chain Fv (scFv), joined by a linker region.

[0291] In some embodiments, the antigen-binding molecules of the present invention comprise one or more regions of an immunoglobulin heavy chain constant sequence, which is or is derived from an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM heavy chain constant sequence.

[0292] In some embodiments, the immunoglobulin heavy chain constant sequence is the human immunoglobulin G1 constant sequence (IGHG1; UniProt: P01857-1, v1; SEQ ID NO: 205). Positions 1 to 98 of SEQ ID NO: 205 form the CH1 region (SEQ ID NO: 206). Positions 99 to 110 of SEQ ID NO: 205 form the hinge region between the CH1 and CH2 regions (SEQ ID NO: 207). Positions 111 to 223 of SEQ ID NO: 205 form the CH2 region (SEQ ID NO: 208). Positions 224 to 330 of SEQ ID NO: 205 form the CH3 region (SEQ ID NO: 209).

[0293] An exemplary antigen-binding molecule can be prepared using pFUSE-CHIg-hG1, which contains the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region. The amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO: 210. It will be understood that the CH3 region can be further substituted in accordance with the modifications to the Fc region of the antigen-binding molecule described herein.

[0294] In some embodiments, the CH1 region comprises or consists of the sequence of SEQ ID NO: 206, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 206. In some embodiments, the hinge region between CH1 and CH2 comprises or consists of the sequence of SEQ ID NO: 207, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207. In some embodiments, the CH2 region comprises or consists of the sequence of SEQ ID NO: 208, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 208. In some embodiments, the CH3 region comprises or consists of the sequence of SEQ ID NO: 209 or 210, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 209 or 210.

[0295] In some embodiments, the antigen-binding molecules of the present invention comprise one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin kappa constant sequence (IGKC; Cκ; UniProt: P01834-1, v2; SEQ ID NO: 211). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant sequence (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the CL region comprises or consists of the sequence of SEQ ID NO: 211 or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 211.

[0296] The VL and light chain constant (CL) regions, and the VH region and heavy chain constant 1 (CH1) region of the antigen-binding region of an antibody, together constitute a Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising a VH, CH1, VL, and CL (e.g., CK or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CH1 (e.g., a VH-CH1 fusion polypeptide) and a polypeptide comprising a VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising a VH and CL (e.g., a VH-CL fusion polypeptide) and a polypeptide comprising a VL and CH (e.g., a VL-CH1 fusion polypeptide); i.e., in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of a Fab or CrossFab are provided as a single polypeptide joined by a linker region, i.e., a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0297] In some embodiments, the antigen binding molecules of the invention comprise or consist of a Fab region that binds to VISTA.

[0298] In some embodiments, the antigen-binding molecules described herein comprise or consist of a whole antibody that binds to VISTA. As used herein, "whole antibody" refers to an antibody that has a structure substantially similar to that of an immunoglobulin (Ig). Different types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini, J Allergy Clin Immunol. (2010), 125(202):S41-S52, which is incorporated herein by reference in its entirety.

[0299] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa that contain two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a VH followed by a heavy chain constant region containing three constant domains (CH1, CH2, and CH3); similarly, the light chain contains a VL followed by a CL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be either a kappa (κ) chain or a lambda (λ) chain.

[0300] In some embodiments, the antigen-binding molecules described herein comprise or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to VISTA.

[0301] In some embodiments, the antigen-binding molecules of the present invention are at least monovalent binding molecules for VISTA. Valency refers to the number of binding sites within an antigen-binding molecule for a given antigenic determinant. Thus, in some embodiments, the antigen-binding molecule comprises at least one binding site for VISTA.

[0302] In some embodiments, the antigen-binding molecule comprises more than one binding site for VISTA, e.g., two, three, or four. The binding sites may be the same or different. In some embodiments, the antigen-binding molecule is, for example, bivalent, trivalent, or tetravalent for VISTA.

[0303] Aspects of the present invention relate to multispecific antigen-binding molecules. "Multispecific" means that an antigen-binding molecule specifically binds to more than one target. In some embodiments, the antigen-binding molecule is a bispecific antigen-binding molecule. In some embodiments, the antigen-binding molecule comprises at least two different antigen-binding domains (i.e., at least two antigen-binding domains comprising, for example, non-identical VH and VL).

[0304] In some embodiments, the antigen-binding molecule is at least bispecific, as it binds to VISTA and another target (e.g., an antigen other than VISTA). The term "bispecific" means that the antigen-binding molecule is capable of specifically binding to at least two distinct antigenic determinants.

[0305] It will be understood that antigen-binding molecules (e.g., multispecific antigen-binding molecules) according to the present invention may include antigen-binding molecules capable of binding to a target for which the antigen-binding molecule is specific. For example, antigen-binding molecules capable of binding to VISTA and antigens other than VISTA may include (i) antigen-binding molecules capable of binding to VISTA, and (ii) antigen-binding molecules capable of binding to antigens other than VISTA.

[0306] It will also be understood that antigen-binding molecules (e.g., multispecific antigen-binding molecules) according to the present invention may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to a target for which the antigen-binding molecule is specific. For example, antigen-binding molecules according to the present invention may include, for example, (i) antigen-binding polypeptide complexes capable of binding to VISTA, comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3), and (ii) antigen-binding polypeptide complexes capable of binding to antigens other than VISTA, comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3).

[0307] In some embodiments, an antigen-binding molecule that is a component of a larger antigen-binding molecule (e.g., a multispecific antigen-binding molecule) may be referred to as, for example, an "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0308] In some embodiments, the antigen-binding molecule comprises an antigen-binding molecule capable of binding to VISTA and an antigen-binding molecule capable of binding to an antigen other than VISTA. In some embodiments, the antigen other than VISTA is a surface molecule of an immune cell. In some embodiments, the antigen other than VISTA is a cancer cell antigen. In some embodiments, the antigen other than VISTA is a receptor molecule, e.g., a cell surface receptor. In some embodiments, the antigen other than VISTA is a cell signaling molecule, e.g., a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen other than VISTA is a growth factor or hormone.

[0309] A cancer cell antigen is an antigen that is expressed or overexpressed by cancer cells. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof. Expression of a cancer cell antigen can be associated with cancer. A cancer cell antigen can be aberrantly expressed by cancer cells (e.g., the cancer cell antigen can be expressed with abnormal localization) or with abnormal structure by cancer cells. A cancer cell antigen can be capable of eliciting an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen that is bound by the antigen-binding molecule of the present invention is displayed on the external surface of the cancer cell (i.e., is extracellular). A cancer cell antigen can be a cancer-associated antigen. In some embodiments, a cancer cell antigen is an antigen whose expression is associated with the onset, progression, or severity of symptoms of cancer. Cancer-associated antigens may be associated with the cause or pathology of cancer, or may be aberrantly expressed as a consequence of cancer. In some embodiments, a cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA level and / or protein level) by cancer cells, e.g., compared to the expression level by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be preferentially expressed by cancerous cells and not expressed by comparable non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, a cancer-associated antigen may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, a cancer-associated antigen may be the product of an overexpressed intracellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.

[0310] The immune cell surface molecule can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed on the cell surface of the immune cell or on the cell surface. In some embodiments, the portion of the immune cell surface molecule bound by the antigen-binding molecule of the present invention is on the external surface of the immune cell (i.e., extracellular). The immune cell surface molecule can be expressed on the cell surface of any immune cell. In some embodiments, the immune cell can be a cell of hematopoietic origin, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, a B cell, a natural killer (NK) cell, a NKT cell, or an innate lymphoid cell (ILC), or a precursor cell thereof (e.g., a thymocyte or a pre-B cell). In some embodiments, the immune cell surface molecule can be a costimulatory molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or a ligand thereof. In some embodiments, the immune cell surface molecule can be a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, or BTLA) or a ligand thereof.

[0311] Multispecific antigen-binding molecules according to the present invention may be provided in any suitable format, such as those described in Brinkmann and Kontermann, MAbs (2017), 9(2):182-212, which is incorporated herein by reference in its entirety. Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann, MAbs (2017), 9(2):182-212: antibody conjugates, e.g., IgG2, F(ab')2, or CovX-Body; IgG or IgG-like molecules, e.g., common HC of IgG, chimeric IgG, κλ-body; CH1 / CL fusion proteins, e.g., scFv2-CH1 / CL, VHH2-CH1 / CL; "variable domain-only" bispecific antigen-binding molecules, e.g., tandem scFv (taFv), triple body, diabody (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAb, triple head, tandem dAb / VHH, tetravalent dAb.VHH; non-Ig fusion proteins, e.g., scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibodies, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2, ImmTAC (TCR-scFv); modified Fc / CH3 fusion proteins, e.g., scFv-Fc(kih), scFv-Fc(CH3 charge pair), scFv-Fc(EW-RVT), scFv-fc(HA-TF), scFv-Fc(SEEDbody), taFv-Fc(kih), scFv -Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc(SEEDbody), DART-Fc, scFv-CH3(kih), TriFab; Fc fusions, e.g., di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, e.g., dia-diabody, scDb-CH3; IgE / IgM CH2 fusions, e.g., scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, e.g., Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, e.g., DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g., IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab, CrossMab(kih), scFab-IgG(kih), Fab-scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pair + CH1 / CL charge pair, hinge / CH3 charge pair, SEED-body, duobody, four-in-one-CrossMab(kih), LUZ-Y common LC; LUZ-Y scFab-IgG, FcFc. *appended / Fc-modified IgG, such as IgG(kih)-Fv, IgG HA-TF-Fv, IgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-Ig, DVI-Ig(four-in-one), CrossMab-Fab; modified Fc / CH3 fusion proteins, such as Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; appended IgG-HC fusions, such as IgG-HC, scFv, IgG-dAb, IgG-taFV, IgG-CrossFab, IgG-orthogonal Fab, IgG-(CαCβ)Fab, scFv-HC-IgG, tandem Fab-IgG (orthogonal Fab) Fab-IgG(CαCβ) Fab), Fab-IgG(CR3), Fab-hinge-IgG(CR3); appended IgG-LC fusions, e.g., IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; appended IgG-HC / LC fusions, e.g., DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, e.g., Fab-scFv-Fc, scFv4-Ig; F(ab')2 fusions, e.g., F(ab')2-scFv2; CH1 / CL fusion proteins, e.g., scFv2-CH1-hinge / CL; modified IgGs, e.g., DAF (two-in-one-IgG), DutaMab, Mab 2 and non-Ig fusions, such as DNL-Fab4-IgG.

[0312] Those skilled in the art can design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include chemically crosslinking antigen-binding molecules or antibody fragments, for example, via a reducible disulfide bond or a non-reducible thioether bond, as described, for example, in Segal and Bast, 2001, "Production of Bispecific Antigen-binding Molecules," Current Protocols in Immunology, 14:IV:2.13:2.13.1-2.13.16, which is incorporated herein by reference in its entirety. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically crosslink Fab fragments, for example, via SH groups in the hinge region, to create disulfide-linked, bispecific F(ab)2 heterodimers.

[0313] Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas, for example with polyethylene glycol, to generate quadroma cells capable of secreting bispecific antibodies, as described, for example, in D. M. and Bast, B. J., 2001, "Production of Bispecific Antigen-binding Molecules," Current Protocols in Immunology, 14:IV:2.13:2.13.1-2.13.16.

[0314] Bispecific antigen-binding molecules according to the present invention may also be produced by recombinant expression from nucleic acid constructs encoding polypeptides for the antigen-binding molecules, e.g., as described in "Antibody Engineering: Methods and Protocols", 2nd Edition (Humana Press, 2012), Chapter 40: "Production of Bispecific Antigen-binding Molecules: Diabodies and Tandem scFv" (Hornig and Faerber-Schwarz); or French, "How to make bispecific antigen-binding molecules", Methods Mol. Med., 2000, 40:333-339, the entire contents of both of which are incorporated herein by reference. For example, a DNA construct encoding the light and heavy chain variable domains for two antigen-binding fragments (i.e., the light and heavy chain variable domains for the antigen-binding fragment capable of binding to VISTA, and the light and heavy chain variable domains for the antigen-binding fragment capable of binding to another target protein), and containing sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments, can be prepared by molecular cloning methods. Recombinant bispecific antibodies can then be produced by expression (e.g., in vitro) of the constructs in suitable host cells (e.g., mammalian host cells), and the expressed recombinant bispecific antibodies can then, optionally, be purified. Fc area In some embodiments, the antigen-binding molecules of the invention comprise an Fc region.

[0315] In IgG, IgA, and IgD isotypes, the Fc region is composed of a CH2 and CH3 region derived from one polypeptide and a CH2 and CH3 region derived from another polypeptide. The CH2 and CH3 regions derived from the two polypeptides together form the Fc region. In IgM and IgE isotypes, the Fc region contains three constant domains (CH2, CH3, and CH4), and the CH2 to CH4 regions derived from the two polypeptides together form the Fc region.

[0316] The Fc region mediates interactions with Fc receptors and other molecules of the immune system to produce functional effects. IgG Fc-mediated effector functions are reviewed, for example, in Jefferis et al., Immunol Rev, 1998, 163:59-76 (incorporated herein by reference in its entirety), and include Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, NK cells, and T cells) through the interaction of the Fc region with Fc receptors expressed by immune cells, recruitment of complement pathway components through binding of the Fc region to the complement protein C1q, and consequent activation of the complement cascade.

[0317] Fc-mediated functions include binding to Fc receptors, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cellular degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0318] Modifications to the Fc region of antibodies that affect Fc-mediated function are known in the art, such as those described in Wang et al., Protein Cell (2018), 9(1):63-73, which is incorporated herein by reference in its entirety. In particular, exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018), 9(1):63-73. Hereinafter, modifications to the Fc region that affect antibody effector activity are described.

[0319] When an Fc region / CH2 / CH3 is described as containing modification(s) "corresponding to" a reference substitution(s), the equivalent substitution(s) in the homologous Fc / CH2 / CH3 are assumed. By way of example, the L234A / L235A substitution in human IgG1 (numbered according to the EU numbering system as described in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991) corresponds to the L to A substitution at positions 117 and 118 of the mouse Ig gamma 2 A chain C region, with the A allele numbered according to SEQ ID NO: 256.

[0320] When an Fc region is described as including a modification, the modification may be present in one or both of the polypeptide chains that together form the Fc region.

[0321] In some embodiments, the antigen-binding molecules of the invention comprise an Fc region comprising a modification, hi some embodiments, the antigen-binding molecules of the invention comprise an Fc region comprising a modification in one or more of the CH2 and / or CH3 regions.

[0322] In some embodiments, the Fc region comprises a modification that increases an Fc-mediated function. In some embodiments, the Fc region comprises a modification that increases ADCC. In some embodiments, the Fc region comprises a modification that increases ADCP. In some embodiments, the Fc region comprises a modification that increases CDC. Antigen-binding molecules comprising an Fc region comprising a modification that increases an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce increased levels of the associated effector function compared to a corresponding antigen-binding molecule comprising an unmodified Fc region.

[0323] In some embodiments, the Fc region comprises a modification that increases binding to an Fc receptor. In some embodiments, the Fc region comprises a modification that increases binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification that increases binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification that increases binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification that increases binding to FcγRIIa. In some embodiments, the Fc region comprises a modification that increases binding to FcγRIIb. In some embodiments, the Fc region comprises a modification that increases binding to FcRn. In some embodiments, the Fc region comprises a modification that increases binding to a complement protein. In some embodiments, the Fc region comprises a modification that increases binding to C1q. In some embodiments, the Fc region comprises a modification that promotes hexamerization of the antigen-binding molecule. In some embodiments, the Fc region comprises a modification that increases the half-life of the antigen-binding molecule, hi some embodiments, the Fc region comprises a modification that increases co-engagement.

[0324] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination F243L / R292P / Y300L / V305I / P396L, as described in Stavenhagen et al., Cancer Res. (2007), 67:8882-8890. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S239D / I332E or S239D / I332E / A330L, as described in Lazar et al., Proc Natl Acad Sci USA. (2006), 103:4005-4010. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S298A / E333A / K334A, as described in Shields et al., J Biol Chem. (2001), 276:6591-6604. In some embodiments, the Fc region comprises a modification to one of the heavy chain polypeptides corresponding to the substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A and a modification to the other heavy chain polypeptide corresponding to the substitution combination D270E / K326D / A330M / K334E, as described in Mimoto et al., MAbs. (2013):5:229-236. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination G236A / S239D / I332E, as described in Richards et al., Mol Cancer Ther. (2008)7:2517-2527.

[0325] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination K326W / E333S described in Idusogie et al., J Immunol. (2001), 166(4):2571-5. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S267E / H268F / S324T described in Moore et al., MAbs. (2010), 2(2):181-9. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination E345R / E430G / S440Y described in Diebolder et al., Science (2014), 343(6176):1260-3.

[0326] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M252Y / S254T / T256E described in Dall'Acqua et al., J Immunol. (2002), 169:5171-5180. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M428L / N434S described in Zalevsky et al., Nat Biotechnol. (2010), 28:157-159.

[0327] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination S267E / L328F described in Chu et al., Mol Immunol. (2008), 45:3926-3933. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination N325S / L328F described in Shang et al., Biol Chem. (2014), 289:15309-15318.

[0328] In some embodiments, the Fc region comprises a modification that reduces / prevents an Fc-mediated function. In some embodiments, the Fc region comprises a modification that reduces / prevents ADCC. In some embodiments, the Fc region comprises a modification that reduces / prevents ADCP. In some embodiments, the Fc region comprises a modification that reduces / prevents CDC. Antigen-binding molecules comprising an Fc region comprising a modification that reduces / prevents an Fc-mediated function (e.g., ADCC, ADCP, CDC) induce a reduced level of the associated effector function compared to an antigen-binding molecule comprising a corresponding unmodified Fc region.

[0329] In some embodiments, the Fc region comprises a modification that reduces / prevents binding to an Fc receptor. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIa. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIb. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to a complement protein. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to C1q. In some embodiments, the Fc region comprises a modification that reduces / prevents glycosylation of the amino acid residue corresponding to N297.

[0330] In some embodiments, the Fc region is not capable of inducing one or more Fc-mediated functions (i.e., lacks the ability to induce the relevant Fc-mediated function(s)). Accordingly, antigen-binding molecules comprising such Fc regions also lack the ability to induce the relevant function(s). Such antigen-binding molecules may be described as lacking the relevant function(s).

[0331] In some embodiments, the Fc region is not capable of inducing ADCC. In some embodiments, the Fc region is not capable of inducing ADCP. In some embodiments, the Fc region is not capable of inducing CDC. In some embodiments, the Fc region is not capable of inducing ADCC and / or is not capable of inducing ADCP and / or is not capable of inducing CDC.

[0332] In some embodiments, the Fc region is not capable of binding to an Fc receptor. In some embodiments, the Fc region is not capable of binding to an Fcγ receptor. In some embodiments, the Fc region is not capable of binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region is not capable of binding to FcγRIIIa. In some embodiments, the Fc region is not capable of binding to FcγRIIa. In some embodiments, the Fc region is not capable of binding to FcγRIIb. In some embodiments, the Fc region is not capable of binding to FcRn. In some embodiments, the Fc region is not capable of binding to complement proteins. In some embodiments, the Fc region is not capable of binding to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0333] In some embodiments, the Fc region comprises a modification corresponding to N297A, N297Q, or N297G, as described in Leabman et al., MAbs. (2013), 5:896-903. In some embodiments, the Fc region comprises a modification corresponding to L235E, as described in Alegre et al., J Immunol. (1992), 148:3461-3468. In some embodiments, the Fc region comprises a modification corresponding to the substitution combinations L234A / L235A or F234A / L235A, as described in Xu et al., Cell Immunol. (2000), 200:16-26. In some embodiments, the Fc region comprises a modification corresponding to P329A or P329G, as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235A / P329G described in Lo et al., J. Biol. Chem (2017), 292(9):3900-3908. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination Rother et al., Nat. Biotechnol. (2007), 25:1256-1264. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S228P / L235E described in Newman et al., Clin. Immunol. (2001), 98:164-174. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination H268Q / V309L / A330S / P331S described in An et al., MAbs. (2009), 1:572-579. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination V234A / G237A / P238S / H268A / V309L / A330S / P331S described in Vafa et al., Methods. (2014), 65:114-126. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S described in US2015 / 0044231A1.

[0334] The substitution combination "L234A / L235A" and corresponding substitutions (e.g., F234A / L235A in human IgG4) are known to disrupt Fc binding to Fcγ receptors, inhibit ADCC, ADCP, and also reduce binding to C1q, thereby reducing CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, incorporated herein by reference in its entirety). The substitutions "P329G" and "P329A" reduce binding to C1q (and thereby reduce CDC). Substitution of "N297" with "A," "G," or "Q" is known to eliminate glycosylation, thereby reducing Fc binding to C1q and Fcγ receptors, thereby reducing CDC and ADCC. Lo et al., J. Biol. Chem (2017), 292(9):3900-3908 (incorporated herein by reference in its entirety) reported that the substitution combination L234A / L235A / P329G abolished complement binding and fixation, and Fcγ receptor-dependent, antibody-dependent, cell-mediated cytotoxicity in both mouse IgG2a and human IgG1.

[0335] The combination of substitutions in IgG1 Fc, L234A / L235E / G237A / A330S / P331S, has been disclosed in US2015 / 0044231A1 to abolish the induction of phagocytosis, ADCC, and CDC.

[0336] In some embodiments, the Fc region comprises a modification corresponding to the substitution S228P described in Silva et al., J Biol Chem. (2015), 290(9):5462-5469. The substitution S228P in IgG4 Fc reduces Fab arm exchange, which may be undesirable.

[0337] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A. In some embodiments, the Fc region comprises a modification corresponding to the substitution P329G. In some embodiments, the Fc region comprises a modification corresponding to the substitution N297Q.

[0338] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235A / P329G.

[0339] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235A / P329G / N297Q.

[0340] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S.

[0341] In some embodiments, the Fc region includes a modification corresponding to, for example, S228P, a substitution in IgG4.

[0342] In some embodiments, the antigen-binding molecules of the present invention comprise an Fc region containing modifications in one or more of the CH2 and CH3 regions that promote Fc region assembly. Recombinant coexpression of constituent polypeptides of an antigen-binding molecule and subsequent assembly results in several possible combinations. To improve the yield of a desired combination of polypeptides within an antigen-binding molecule during recombinant production, it is advantageous to introduce a modification(s) in the Fc region that promotes the assembly of a desired combination of heavy chain polypeptides. Modifications may, for example, promote hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described, for example, in Ha et al., Front. Immunol (2016), 7:394, incorporated herein by reference in its entirety.

[0343] In some embodiments, the antigen-binding molecules of the invention are in the following formats: KiH, KiH, as shown in Table 1 of Ha et al., Front. Immunol (2016), 7:394. S-S , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT S-S , SEED, or A107.

[0344] In some embodiments, the Fc region comprises a "knob-into-hole (KIH)" or "KiH" modification, e.g., as described in U.S. Pat. No. 7,695,936; and Carter, J. Immunol. Meth., 248, 7-15 (2001). In such embodiments, one CH3 region of the Fc region comprises a "knob" modification, and the other CH3 region comprises a "hole" modification. The "knob" and "hole" modifications are positioned within the respective CH3 regions such that the "knob" is positioned within the "hole" to promote heterodimerization (and inhibit homodimerization) of the polypeptides and / or stabilize heterodimers. The knob is constructed by substituting an amino acid with a larger side chain (e.g., tyrosine or tryptophan) for an amino acid with a larger side chain (e.g., alanine or threonine). The hole is created by substituting an amino acid with a larger side chain (e.g., alanine or threonine).

[0345] In some embodiments, one CH3 region of the Fc region of an antigen-binding molecule of the invention contains the substitution T366W (position / substitution numbering of the Fc, CH2, and CH3 regions herein is according to the EU numbering system as set forth in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991), and the other CH3 region of the Fc region contains the substitution Y407V. In some embodiments, one CH3 region of the Fc region of an antigen-binding molecule contains the substitution T366W, and the other CH3 region of the Fc region contains the substitutions T366S and L368A. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S, and L368A.

[0346] In some embodiments, the Fc region comprises a "DD-KK" modification, e.g., as described in WO2014 / 131694A1. In some embodiments, one of the CH3 regions comprises substitutions K392D and K409D, and the other CH3 region of the Fc region comprises substitutions E356K and D399K. The modifications promote electrostatic interactions between the CH3 regions.

[0347] In some embodiments, the antigen-binding molecules of the present invention comprise Fc regions modified as described in Labrijn et al., Proc Natl Acad Sci USA. (2013), 110(13):5145-50, referred to as a "duobody" format. In some embodiments, one of the CH3 regions comprises the substitution K409R, and the other CH3 region of the Fc region comprises the substitution K405L.

[0348] In some embodiments, antigen-binding molecules of the invention comprise an Fc region containing the "EEE-RRR" modification described in Strop et al., J Mol Biol. (2012), 420(3):204-19. In some embodiments, one of the CH3 regions comprises the substitutions D221E, P228E, and L368E, and the other CH3 region of the Fc region comprises the substitutions D221R, P228R, and K409R.

[0349] In some embodiments, the antigen-binding molecule comprises an Fc region comprising the "EW-RVT" modification described in Choi et al., Mol Cancer Ther (2013), 12(12):2748-59. In some embodiments, one of the CH3 regions comprises the substitutions K360E and K409W, and the other CH3 region of the Fc region comprises the substitutions Q347R, D399V, and F405T.

[0350] In some embodiments, one of the CH3 regions contains the substitution S354C and the other CH3 region of the Fc region contains the substitution Y349C. The introduction of these cysteine ​​residues results in the formation of disulfide bridges between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001) J Immunol Methods 248:7-15).

[0351] In some embodiments, the Fc region is S-S In some embodiments, one of the CH3 regions includes the substitutions T366W and S354C, and the other CH3 region of the Fc region includes the substitutions T366S, L368A, Y407V, and Y349C.

[0352] In some embodiments, the antigen-binding molecules of the invention comprise an Fc region comprising the "SEED" modification, in which the β-strand segment of human IgG1 CH3 is swapped for the β-strand segment of human IgA CH3, as described by Davis et al., Protein Eng Des Sel (2010), 23(4):195-202.

[0353] In some embodiments, one of the CH3 regions contains the substitutions S364H and F405A, and the other CH3 region of the Fc region contains the substitutions Y349T and T394F (see, e.g., Moore et al., MAbs (2011), 3(6):546-57).

[0354] In some embodiments, one of the CH3 regions contains the substitutions T350V, L351Y, F405A, and Y407V, and the other CH3 region of the Fc region contains the substitutions T350V, T366L, K392L, and T394W (see, e.g., Von Kreudenstein et al., MAbs (2013), 5(5):646-54).

[0355] In some embodiments, one of the CH3 regions includes the substitutions K360D, D399M, and Y407A, and the other CH3 region of the Fc region includes the substitutions E345R, Q347R, T366V, and K409V (see, e.g., Leaver-Fay et al., Structure (2016), 24(4):641-51).

[0356] In some embodiments, one of the CH3 regions contains the substitutions K370E and K409W, and the other CH3 region of the Fc region contains the substitutions E357N, D399V, and F405T (see, e.g., Choi et al., PLoS One (2015), 10(12):e0145349).

[0357] In some embodiments, the antigen-binding molecules of the present invention comprise an Fc region that does not bind to an Fcγ receptor. In some embodiments, the antigen-binding molecules comprise an Fc region that does not bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antigen-binding molecules comprise an Fc region that does not bind to one or more of FcγRIIa, FcγRIIb, and FcγRIIIa. In some embodiments, the antigen-binding molecules comprise an Fc region that does not bind to one or both of FcγRIIa and FcγRIIb.

[0358] The ability of an Fc region, or an antigen-binding molecule comprising an Fc region, to bind to a reference protein (e.g., an Fc receptor) can be analyzed according to methods known in the art, such as ELISA, immunoblotting, immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012), 907:411-442), or biolayer interferometry (BLI; see, e.g., Lad et al. (2015), J Biomol Screen, 20(4):498-507).

[0359] As used herein, an Fc region that "does not bind" to a reference protein may exhibit substantially no binding to the reference protein, as determined, for example, by ELISA, immunoblot (e.g., Western blot), immunoprecipitation, SPR, or BLI. "Does not substantially bind to" can be a level of interaction that does not significantly exceed the level of interaction determined for proteins that do not bind to each other in a given assay. "Does not substantially bind to" can be a level of interaction that is ≦5-fold, e.g., ≦4-fold, ≦3-fold, ≦2.5-fold, ≦2-fold, or ≦1.5-fold the level of interaction determined for proteins that do not bind to each other in a given assay.

[0360] In some embodiments, the antigen-binding molecule comprises an Fc region that binds to FcRn.

[0361] In some embodiments, the antigen-binding molecule comprises an Fc region that binds to FcRn but does not bind to one or more of FcγRIIa, FcγRIIb, and FcγRIIIa. In some embodiments, the antigen-binding molecule comprises an Fc region that binds to FcRn but does not bind to one or both of FcγRIIa and FcγRIIb.

[0362] In some embodiments, antigen-binding molecules of the present invention comprise an Fc region that does not induce ADCC. In some embodiments, antigen-binding molecules of the present invention comprise an Fc region that does not induce ADCP. In some embodiments, antigen-binding molecules of the present invention comprise an Fc region that does not induce CDC. In some embodiments, antigen-binding molecules of the present invention comprise an Fc region that does not induce ADCC, ADCP, or CDC.

[0363] As used herein, an Fc region / antigen-binding molecule that does not induce (i.e., is not capable of inducing) ADCC / ADCP / CDC does not substantially induce ADCC / ADCP / CDC activity, for example, as determined by analysis in an assay appropriate for the activity of interest. "Substantially free of ADCC / ADCP / CDC activity" refers to a level of ADCC / ADCP / CDC in a given assay that does not significantly exceed the level of ADCC / ADCP / CDC determined for an appropriate negative control molecule (e.g., an antigen-binding molecule lacking an Fc region or an antigen-binding molecule comprising a "silent" Fc region (e.g., as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, incorporated herein by reference)). "Substantially no activity" can be a level of activity of interest that is ≦5-fold, e.g., ≦4-fold, ≦3-fold, ≦2.5-fold, ≦2-fold, or ≦1.5-fold the level of activity determined for an appropriate negative control molecule in a given assay.

[0364] The ability of an Fc region, or an antigen-binding molecule comprising an Fc region, to induce ADCC may be analyzed, for example, according to the method described in Yamashita et al., Scientific Reports (2016), 6:19772 (incorporated herein by reference in its entirety), or by the method described, for example, in Jedema et al., Blood (2004), 103:2677-82 (incorporated herein by reference in its entirety). 51 The ability of an Fc region or an antigen-binding molecule comprising an Fc region to induce ADCP can be analyzed, for example, according to the method described in Kamen et al., J Immunol (2017), 198 (Suppl. 1) 157.17 (incorporated herein by reference in its entirety). The ability of an Fc region or an antigen-binding molecule comprising an Fc region to induce CDC can be analyzed, for example, using a C1q binding assay, for example, as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (incorporated herein by reference above).

[0365] In some embodiments, the antigen-binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence with at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 254. In some embodiments, the antigen-binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence with at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 257. In some embodiments, the antigen-binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence with at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 259. In some embodiments, the antigen-binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence with at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 260.

[0366] In some embodiments, the antigen-binding molecules of the invention lack an Fc region. Fc receptors Fc receptors are polypeptides that bind to the Fc region of immunoglobulins. The structure and function of Fc receptors are reviewed, for example, in Masuda et al., Inflamm Allergy Drug Targets (2009), 8(1):80-86 and Bruhns, Blood (2012), 119:5640-5649, both of which are incorporated herein by reference in their entirety.

[0367] Fc receptors are expressed on the surface of hematopoietic cells, including macrophages, neutrophils, dendritic cells, eosinophils, basophils, mast cells, and NK cells. Fc receptors include IgG-binding Fcγ receptors, high-affinity receptors for IgE (FcεRI), IgA receptors, and polymeric Ig receptors for IgA and IgM. The neonatal Fc receptor (FcRn) is an additional Fc receptor for IgG that is involved in IgG transport across epithelial barriers (transcytosis), protection of IgG from degradation, and antigen presentation.

[0368] Humans have six different classes of Fcγ receptors (murine orthologues are shown in parentheses): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV), and FcγRIIIb.

[0369] FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa contain immunoreceptor tyrosine-based activation motifs (ITAMs) within their intracellular domains, and ligation by Fc leads to activation of cells expressing the receptor. FcγRIIb contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) within its intracellular domain, which upon ligation by Fc negatively regulate cell activation and degranulation, cell proliferation, endocytosis, and phagocytosis.

[0370] As used herein, "Fcγ receptor" can be derived from any species and can include isoforms, fragments, variants (including mutants), or homologs derived from any species. Similarly, "FcγRI," "FcγRIIa," "FcγRIIb," "FcγRIIc," "FcγRIIIa," and "FcγRIIIb" refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb, respectively, derived from any species, and include isoforms, fragments, variants (including mutants), or homologs derived from any species.

[0371] In some embodiments, the Fcγ receptor (e.g., FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb) is derived from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or a rodent (e.g., rat or mouse). Isoforms, fragments, variants, or homologs are optionally derived from a given species, e.g., human. They may be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature isoform of a cγ receptor (e.g., FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb).

[0372] The isoform, fragment, variant, or homologue may optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of the reference Fcγ receptor, e.g., as determined by analysis via an appropriate assay for that functional property / activity. For example, an isoform, fragment, variant, or homologue of FcγRI may exhibit, e.g., association with human IgG1 Fc.

[0373] As used herein, an "FcRn receptor" can be derived from any species and can include isoforms, fragments, variants (including mutants), or homologs derived from any species.

[0374] In some embodiments, the FcRn receptor is derived from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or a rodent (e.g., a rat or mouse). Isoforms, fragments, variants, or homologs may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature Fcγ receptor isoform from a given species, e.g., human.

[0375] The isoform, fragment, variant, or homologue can optionally be a functional isoform, fragment, variant, or homologue that has a functional property / activity of the reference FcRn, e.g., as determined by analysis via an appropriate assay for that functional property / activity. For example, an isoform, fragment, variant, or homologue of FcRn can exhibit, e.g., association with human IgG1 Fc. Polypeptides The present invention also provides polypeptide components of antigen-binding molecules, which may be provided in isolated or substantially purified form.

[0376] The antigen-binding molecules of the present invention may be or may comprise a complex of polypeptides.

[0377] It will be understood that where a polypeptide comprises more than one domain or region, it is preferred herein that multiple domains / regions are present in the same polypeptide chain, i.e., a polypeptide comprising more than one domain or region is a fusion polypeptide comprising the domains / regions.

[0378] In some embodiments, a polypeptide in accordance with the invention comprises or consists of a VH described herein. In some embodiments, a polypeptide in accordance with the invention comprises or consists of a VL described herein.

[0379] In some embodiments, the polypeptide additionally comprises one or more heavy chain constant regions (CH) of an antibody. In some embodiments, the polypeptide additionally comprises one or more light chain constant regions (CL) of an antibody. In some embodiments, the polypeptide comprises a CH1, CH2, and / or CH3 region of an immunoglobulin (Ig).

[0380] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a hinge region between CH1 and CH2 as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein.

[0381] In some embodiments, the polypeptide has the following amino acid substitutions / combinations of amino acid substitutions: F243L / R292P / Y300L / V305I / P396L; S239D / I332E; S239D / I332E / A330L; S298A / E333A / K334A; L234Y / L235Q / G236W / S239M / H268D / D270E / S298A; D270E / K326D / A330M / K334E; G236A / S239D / I332E; K326W / E333S; S267E / H268F / S324T; E345R / E430 G / S440Y; M252Y / S254T / T256E; M428L / N434S; S267E / L328F; N325S / L328F; N297A; N297Q; N297G; L235E; L234A / L235A; F234A / L235A; P329A; P329G; L234A / L235A / P329G; H268Q / V309L / A330S / P331S; and V234A / G237A / P238S / H268A / V309L / A330S / P331S, comprising a CH2 and / or CH3 region.

[0382] In some embodiments, the polypeptides have the following amino acid substitutions / combinations of amino acid substitutions (e.g., as shown in Table 1 of Ha et al., Front. Immunol (2016), 7:394, incorporated herein by reference): T366W; T366S, L368A, and Y407V; T366W and S354C; T366S, L368A, Y407V, and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A, and Y407V; T350V, T366L , K392L, and T394W; K360D, D399M, and Y407A; E345R, Q347R, T366V, and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V, and F405T; K360E, K409W, and Y349C; Q347R, D399V, F405T, and S354C; K370E and K409W; and E357N, D399V, and F405T.

[0383] In some embodiments, the CH2 and / or CH3 region of the polypeptide comprises one or more amino acid substitutions to promote association of the polypeptide with another polypeptide that comprises a CH2 and / or CH3 region.

[0384] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence, hi some embodiments, the polypeptide comprises a CL region described herein.

[0385] In some embodiments, the polypeptide lacks one or more regions of the immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide lacks the CH2 region. In some embodiments, the polypeptide lacks the CH3 region. In some embodiments, the polypeptide lacks the CH2 region and also lacks the CH3 region.

[0386] In some embodiments, a polypeptide in accordance with the invention comprises, from N-terminus to C-terminus, the following: (i) VH (ii) VL (iii) VH-CH1 (iv) VL-CL (v) VL-CH1 (vi) VH-CL (vii) VH-CH1-CH2-CH3 (viii) VL-CL-CH2-CH3 (ix) VL-CH1-CH2-CH3 (x)VH-CL-CH2-CH3 It contains a structure that conforms to one of the following:

[0387] The present invention also provides antigen-binding molecules composed of the polypeptides of the present invention. In some embodiments, the antigen-binding molecules of the present invention comprise a combination of the following polypeptides: (A) VH+VL (B) VH-CH1+VL-CL (C) VL-CH1+VH-CL (D) VH-CH1-CH2-CH3+VL-CL (E) VH-CL-CH2-CH3+VL-CH1 (F) VL-CH1-CH2-CH3+VH-CL (G)VL-CL-CH2-CH3+VH-CH1 (H)VH-CH1-CH2-CH3+VL-CL-CH2-CH3 (I)VH-CL-CH2-CH3+VL-CH1-CH2-CH3 Contains one of the following:

[0388] In some embodiments, an antigen-binding molecule comprises more than one of the polypeptide combinations set forth in (A) through (I) above. By way of example, and referring to (D) above, in some embodiments, an antigen-binding molecule comprises two polypeptides comprising the structure VH-CH1-CH2-CH3 and two polypeptides comprising the structure VL-CL.

[0389] In some embodiments, the antigen-binding molecules of the invention comprise a combination of the following polypeptides: (J)VH (anti-VISTA) + VL (anti-VISTA) (K)VH(Anti-VISTA)-CH1+VL(Anti-VISTA)-CL (L)VL(Anti-VISTA)-CH1+VH(Anti-VISTA)-CL (M)VH(Anti-VISTA)-CH1-CH2-CH3+VL(Anti-VISTA)-CL (N)VH(Anti-VISTA)-CL-CH2-CH3+VL(Anti-VISTA)-CH1 (O)VL(Anti-VISTA)-CH1-CH2-CH3+VH(Anti-VISTA)-CL (P)VL(Anti-VISTA)-CL-CH2-CH3+VH(Anti-VISTA)-CH1 (Q)VH(Anti-VISTA)-CH1-CH2-CH3+VL(Anti-VISTA)-CL-CH2-CH3 (R)VH(Anti-VISTA)-CL-CH2-CH3+VL(Anti-VISTA)-CH1-CH2-CH3 In this case, "VH (anti-VISTA)" refers to the VH of an antigen-binding molecule capable of binding to VISTA as described herein, for example, as defined in one of (1) to (76); and "VL (anti-VISTA)" refers to the VL of an antigen-binding molecule capable of binding to VISTA as described herein, for example, as defined in one of (77) to (173).

[0390] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 212-243, 248-250, 258, 266, or 311-321. Linkers and further sequences In some embodiments, the antigen-binding molecules and polypeptides of the present invention comprise a hinge region. In some embodiments, the hinge region is provided between the CH1 region and the CH2 region. In some embodiments, the hinge region is provided between the CL region and the CH2 region. In some embodiments, the hinge region comprises or consists of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207.

[0391] In some embodiments, the antigen-binding molecules and polypeptides of the present invention comprise one or more linker sequences between amino acid sequences, which may be provided at one or both ends of one or more of the VH, VL, hinge region between CH1 and CH2, CH2 region, and CH3 region of the antigen-binding molecule / polypeptide.

[0392] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013), 65(10):1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence may be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences connected by the linker sequence. Linker sequences are known to those skilled in the art, and several linker sequences are identified in Chen et al., Adv Drug Deliv Rev (2013), 65(10):1357-1369. Flexible linker sequences often contain a high proportion of glycine and / or serine residues.

[0393] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 10 amino acids.

[0394] The antigen-binding molecules and polypeptides of the present invention may additionally comprise additional amino acids or amino acid sequences. For example, the antigen-binding molecules and polypeptides may comprise an amino acid sequence(s) that facilitates expression, folding, trafficking, processing, purification, or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecule / polypeptide may optionally comprise a sequence encoding His (e.g., 6xHis), Myc, GST, MBP, FLAG, HA, E, or biotin tag at the N- or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide comprises a detection moiety, such as a fluorescent, luminescent, immunodetection, radioactive, chemical, nucleic acid, or enzyme label.

[0395] The antigen-binding molecules and polypeptides of the present invention may additionally contain a signal peptide (also known as a leader sequence or signal sequence). A signal peptide usually consists of a sequence of 5 to 30 hydrophobic amino acids that forms a single alpha helix. Proteins that are secreted and expressed on the cell surface often contain a signal peptide.

[0396] A signal peptide can be present at the N-terminus of an antigen-binding molecule / polypeptide and can be present in a newly synthesized antigen-binding molecule / polypeptide. The signal peptide ensures efficient trafficking and secretion of the antigen-binding molecule / polypeptide. The signal peptide is often removed by cleavage and is therefore not included in the mature antigen-binding molecule / polypeptide secreted from cells expressing the antigen-binding molecule / polypeptide.

[0397] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as, for example, SignalP (Petersen et al., 2011, Nature Methods, 8:785-786) or Signal-BLAST (Frank and Sippl, 2008, Bioinformatics, 24:2172-2176). Labels and conjugates In some embodiments, the antigen-binding molecules of the invention additionally comprise a detectable moiety.

[0398] In some embodiments, the antigen-binding molecule comprises a detectable moiety, such as a fluorescent label, a phosphorescent label, a luminescent label, an immunodetectable label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzymatic label. The antigen-binding molecule may be covalently or non-covalently labeled with the detectable moiety.

[0399] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), rare earth chelators such as europium (Eu), terbium (Tb), and samarium (Sm), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include iodine 123 , iodine 125 , iodine 126 , iodine 131 , iodine 133 ,bromine 77 , technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium97 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 , tellurium 121m , tellurium 122m , tellurium 125m ,thulium 165 ,thulium 167 ,thulium 168 ,copper 67 , fluorine 18 ,yttrium 90 ,palladium 100 , bismuth 217 , and antimony 211 Examples of suitable labels include radioisotopes such as fluorophore, ...

[0400] In some embodiments, the antigen-binding molecules of the present invention are conjugated to a chemical moiety. The chemical moiety can be a moiety that provides a therapeutic effect. Antibody-drug conjugates are reviewed, for example, in Parslow et al., Biomedicines, September 2016, 4(3):14. In some embodiments, the chemical moiety can be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety can be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5, and PBD. Certain exemplary embodiments of antigen-binding molecules In some embodiments, the antigen-binding molecule is (i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 331; and (ii) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 317. It comprises or consists of:

[0401] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 212; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 213. It comprises or consists of:

[0402] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 214; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 215. It comprises or consists of:

[0403] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 216; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 217. It comprises or consists of:

[0404] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 218; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 219. It comprises or consists of:

[0405] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 220; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 221. It comprises or consists of:

[0406] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 222; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 223. It comprises or consists of:

[0407] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 224; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 225. It comprises or consists of:

[0408] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 226; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 227. It comprises or consists of:

[0409] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 228; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 229. It comprises or consists of:

[0410] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 230; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 231. It comprises or consists of:

[0411] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 232; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 233. It comprises or consists of:

[0412] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 234; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 235. It comprises or consists of:

[0413] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 236; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 237. It comprises or consists of:

[0414] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 238; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 239. It comprises or consists of:

[0415] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 240; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241. It comprises or consists of:

[0416] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 242; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 243. It comprises or consists of:

[0417] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 248; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 250. It comprises or consists of:

[0418] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 249; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 250. It comprises or consists of:

[0419] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 258; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 250. It comprises or consists of:

[0420] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 266; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 250. It comprises or consists of:

[0421] In some embodiments, the antigen-binding molecule is (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 330; and (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 213. It comprises or consists of:

[0422] In some embodiments, the antigen-binding molecule is produced by cells of the cell line deposited on May 7, 2021, as ATCC Patent Accession No. PTA-127063, e.g., as described in GB2108446.2, which is incorporated herein by reference in its entirety. Functional properties of antigen-binding molecules The antigen-binding molecules described herein may be characterized by reference to certain functional properties. In some embodiments, the antigen-binding molecules described herein have the following properties: binding to VISTA (e.g., human, mouse, and / or cynomolgus VISTA); lack of binding to PD-L1 and / or HER3; no binding to Fcγ receptors; no binding to C1q; failure to induce ADCC; Do not induce ADCP; Not leading the CDC; binding to the FcRn receptor; binding to VISTA-expressing cells; inhibiting the interaction of VISTA with a binding partner of VISTA (e.g., PSGL-1, VSIG-3, VSIG-8, or LRIG1); inhibiting VISTA-mediated signaling; inhibiting VISTA-mediated signaling independently of Fc-mediated functions; increasing killing of VISTA-expressing cells; does not induce / enhance killing of VISTA-expressing cells; Reducing the number / proportion of VISTA-expressing cells; Does not reduce the number / proportion of VISTA-expressing cells; Increasing the number / activity of effector immune cells; reducing the number / activity of immunosuppressive cells; reducing the proliferation of immunosuppressive cells; reducing immunosuppression mediated by VISTA-expressing cells; increasing antigen presentation by antigen-presenting cells; increasing the production of IL-6 by immune cells; increasing the production of IFN-γ, IL-2, and / or IL-17 in a mixed lymphocyte reaction (MLR) assay; Increasing T cell proliferation, IFN-γ production, and / or TNFα production; and inhibiting the onset and / or progression of cancer in vivo; and Does not induce cytokine release syndrome in vivo The ion channel may have one or more of:

[0423] It will be understood that a given antigen-binding molecule may exhibit more than one of the properties listed in the preceding paragraph. A given antigen-binding molecule can be assessed for the properties listed in the preceding paragraph using an appropriate assay. The assay can be, for example, an in vitro assay, which can be a cell-free assay or a cell-based assay. Alternatively, the assay can be, for example, an in vivo assay performed in a non-human animal.

[0424] If the assay is cell-based, the assay may include contacting cells with a given antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the listed properties. The assay may utilize molecular species labeled with a detection entity to facilitate their detection. The assay may include individually assessing the listed properties after treatment of the cells with a range of numbers / concentrations of the antigen-binding molecule (e.g., a dilution series). It will be understood that the cells are preferably cells that express VISTA, e.g., MDSC.

[0425] Analysis of the results of such assays may include determining the concentration at which 50% of the maximal level of the desired activity is achieved. The concentration of the antigen-binding molecule at which 50% of the maximal level of the desired activity is achieved may be referred to as the "50% effective concentration" of the antigen-binding molecule in relation to the desired activity, which may also be referred to as the "EC 50 For illustrative purposes, the EC of a given antigen-binding molecule for binding to VISTA may be 50 may be the concentration at which 50% of the maximal level of binding to the relevant molecular species is achieved.

[0426] Depending on the characteristics, EC 50 is also referred to as the "50% inhibitory concentration" or "IC", which is the concentration of an antigen-binding molecule at which 50% of the maximal level of inhibition of a given property is observed. 50For illustrative purposes, the IC of a given antigen-binding molecule for inhibiting the interaction of VISTA with an interaction partner of VISTA (e.g., LRIG1, PSGL-1, VSIG3, or VSIG8) may be expressed as: 50 may be the concentration at which 50% of the maximal level of inhibition is achieved.

[0427] The antigen-binding molecules described herein preferably exhibit specific binding to VISTA. As used herein, "specific binding" refers to binding that is selective for the antigen and can be distinguished from non-specific binding to non-target antigens. An antigen-binding molecule that specifically binds to a target molecule preferably binds to the target with greater affinity and / or longer duration than it binds to other non-target molecules.

[0428] The ability of a given polypeptide to specifically bind to a given molecule can be determined by analysis according to methods known in the art, such as ELISA, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012), 907:411-442), biolayer interferometry (see, e.g., Lad et al. (2015), J Biomol Screen, 20(4):498-507), flow cytometry, or radiolabeled antigen binding assay (RIA), enzyme-linked immunosorbent assay. By such analysis, binding to a given molecule can be measured and quantified. In some embodiments, binding can be a response detected in a given assay.

[0429] In some embodiments, the extent of binding of the antigen-binding molecule to the non-target molecule is less than about 10% of the binding of the antibody to the target molecule, as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, binding specificity can be measured by the K D by at least 0.1 orders of magnitude (i.e., 0.1 × 10 n where n is an integer representing the number of digits]) D), which may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

[0430] In some embodiments, the antigen-binding molecules exhibit binding to human VISTA, murine (e.g., mouse) VISTA, and / or cynomolgus monkey (Macaca fascicularis) VISTA. That is, in some embodiments, the antigen-binding molecules are cross-reactive with human VISTA, murine VISTA, and / or cynomolgus monkey VISTA. In some embodiments, the antigen-binding molecules of the present invention are cross-reactive with VISTA of non-human primates. Cross-reactivity to VISTA in model species allows for testing of efficacy in in vivo syngeneic models without relying on surrogate molecules.

[0431] In some embodiments, the antigen-binding molecule does not provide specific binding to PD-L1 (e.g., human PD-L1). In some embodiments, the antigen-binding molecule does not provide specific binding to HER3 (e.g., human HER3). In some embodiments, the antigen-binding molecule does not provide specific binding to (i.e., does not cross-react with) other members of the B7 family of proteins. In some embodiments, the antigen-binding molecule does not provide specific binding to PD-1, PD-L2, CD80, CD86, ICOSLG, CD276, VTCN1, NCR3LG1, HHLA2, and / or CTLA4.

[0432] In some embodiments, the antigen-binding molecule does not exhibit specific binding to PD-1, PD-L1, B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7), and / or CTLA4.

[0433] In some embodiments, the antigen-binding molecule is not capable of inducing one or more Fc-mediated functions (i.e., lacks the ability to trigger the relevant Fc-mediated function(s). Such antigen-binding molecules may be described as lacking the relevant function(s).

[0434] As explained hereinabove, an Fc region / antigen-binding molecule that does not induce (i.e., is not capable of inducing) ADCC / ADCP / CDC does not substantially induce ADCC / ADCP / CDC activity, as determined, for example, by analysis in an assay appropriate for the activity of interest. Similarly, an antigen-binding molecule that does not "bind" to a reference protein (e.g., a given Fc receptor or complement protein) may exhibit substantially no binding to the reference protein in an appropriate assay.

[0435] In some embodiments, the antigen-binding molecule is not capable of inducing ADCC. In some embodiments, the antigen-binding molecule is not capable of inducing ADCP. In some embodiments, the antigen-binding molecule is not capable of inducing CDC. In some embodiments, the antigen-binding molecule is not capable of inducing ADCC and / or is not capable of inducing ADCP and / or is not capable of inducing CDC.

[0436] In some embodiments, the antigen-binding molecule is not capable of binding to an Fc receptor. In some embodiments, the antigen-binding molecule is not capable of binding to an Fcγ receptor. In some embodiments, the antigen-binding molecule is not capable of binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antigen-binding molecule is not capable of binding to FcγRIIIa. In some embodiments, the antigen-binding molecule is not capable of binding to FcγRIIa. In some embodiments, the antigen-binding molecule is not capable of binding to FcγRIIb. In some embodiments, the antigen-binding molecule binds to FcRn. In some embodiments, the antigen-binding molecule is not capable of binding to complement proteins. In some embodiments, the antigen-binding molecule is not capable of binding to C1q. In some embodiments, the antigen-binding molecule is not glycosylated at the amino acid residue corresponding to N297.

[0437] In some embodiments, the antigen-binding molecule binds to human VISTA, mouse VISTA, and / or cynomolgus VISTA, and does not bind to PD-L1, PD-1, B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7), and / or CTLA4 (e.g., human PD-L1 / PD-1 / B7H3 / VTCN1 / NCR3LG1 / HHLA2 / CTLA4).

[0438] In some embodiments, the antigen-binding molecule does not exhibit specific binding to PD-L1 (e.g., human PD-L1). In some embodiments, the antigen-binding molecule does not exhibit specific binding to HER3 (e.g., human HER3). In some embodiments, the antigen-binding molecule does not exhibit specific binding to (i.e., does not cross-react with) other members of the B7 family of proteins. In some embodiments, the antigen-binding molecule does not exhibit specific binding to PD-L1, PD-L2, CD80, CD86, ICOSLG, CD276, VTCN1, NCR3LG1, HHLA2, and / or CTLA4.

[0439] In some embodiments, the antigen-binding molecule does not exhibit specific binding to (PD-1, PD-L1, B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7), and / or CTLA4.

[0440] In some embodiments, antigen-binding molecules according to the present disclosure bind to VISTA with an affinity in the micromolar range, i.e., K D =9.9×10 -4 ~1×10 -6 In some embodiments, the antigen-binding molecule binds to VISTA with submicromolar affinity, i.e., K D <1×10 -6 In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the nanomolar range, i.e., K D =9.9×10 -7 ~1×10 -9 In some embodiments, the antigen-binding molecule binds to VISTA with subnanomolar affinity, i.e., K D <1×10 -9 In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the picomolar range, i.e., K D =9.9×10 -10 ~1×10 -12 In some embodiments, the antigen-binding molecule binds to VISTA with sub-picomolar affinity, i.e., K D <1×10 -12 Join with M.

[0441] In some embodiments, the antigen-binding molecules described herein have a K of 10 μM or less, preferably one of: ≦5 μM, ≦2 μM, ≦1 μM, ≦500 nM, ≦100 nM, ≦75 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦15 nM, ≦12.5 nM, ≦10 nM, ≦9 nM, ≦8 nM, ≦7 nM, ≦6 nM, ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, or ≦500 pM. DIn some embodiments, the antigen-binding molecule binds to VISTA (e.g., human VISTA, mouse VISTA) at K D In some embodiments, the antigen-binding molecule binds to VISTA (e.g., human VISTA, mouse VISTA) with an affinity of ≦10 nM, ≦9 nM, ≦8 nM, ≦7 nM, or ≦6 nM, ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, or ≦1 nM. D In some embodiments, an antigen-binding molecule according to the present disclosure binds to VISTA (e.g., human VISTA, mouse VISTA) with an affinity of ≦500 pM, ≦100 pM, ≦90 pM, ≦80 pM, ≦70 pM, or ≦60 pM, ≦50 pM, ≦40 pM, ≦30 pM, ≦20 pM, ≦10 pM, ≦9 pM, ≦8 pM, ≦7 pM, or ≦6 pM, ≦5 pM, ≦4 pM, ≦3 pM, ≦2 pM, or ≦1 pM. In some embodiments, an antigen-binding molecule according to the present disclosure binds to VISTA with a K of ≦1 nM (e.g., one of ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM, ≦400 pM, ≦300 pM). D (e.g., as determined by SPR (Biocore) analysis, such as the SPR analysis described in the Examples of the present disclosure).

[0442] In some embodiments, an antigen-binding molecule according to the present disclosure has a K for human VISTA of ≦1 nM (e.g., one of ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM). D (e.g., as determined by SPR (Biocore) analysis, such as the SPR analysis described in the Examples of the present disclosure). In some embodiments, an antigen-binding molecule in accordance with the present disclosure binds to cynomolgus monkey VISTA with a K of ≦1 nM (e.g., one of ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM, ≦400 pM). D (e.g., as determined by SPR (Biocore) analysis, such as the SPR analysis described in the Examples of the present disclosure). In some embodiments, the antigen-binding molecule according to the present disclosure binds to rat VISTA with a K of ≦1 nM (e.g., one of ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM, ≦500 pM, ≦400 pM). D(e.g., as determined by SPR (Biocore) analysis, such as the SPR analysis described in the Examples of the present disclosure). In some embodiments, the antigen-binding molecule according to the present disclosure binds to mouse VISTA with a K of ≦1 nM (e.g., one of ≦900 pM, ≦800 pM, ≦700 pM, ≦600 pM). D (e.g., as determined by SPR (Biocore) analysis, such as the SPR analysis described in the Examples of the present disclosure).

[0443] In some embodiments, the antigen-binding molecules according to the present disclosure have an EC 50 (e.g., as determined by ELISA, such as the ELISAs described in the Examples of the present disclosure), for example, at one of ≦500 nM, ≦100 nM, ≦50 nM, ≦40 nM, ≦30 nM, ≦20 nM, ≦10 nM, ≦5 nM, ≦4 nM, ≦3 nM, ≦2 nM, ≦1 nM, ≦500 pM, ≦400 pM, ≦300 pM, ≦200 pM, ≦100 pM, ≦50 pM, ≦40 pM, ≦30 pM, ≦20 pM, ≦15 pM, ≦10 pM, ≦5 pM, or ≦1 pM.

[0444] In some embodiments, the antigen-binding molecule exhibits binding to human VISTA, murine (e.g., mouse) VISTA, rat VISTA, and / or cynomolgus monkey (Macaca fascicularis) VISTA. In some embodiments, the antigen-binding molecule binds to human VISTA, mouse VISTA, rat VISTA, and cynomolgus monkey VISTA. In some embodiments, the antigen-binding molecule is cross-reactive with human VISTA, mouse VISTA, rat VISTA, and cynomolgus monkey VISTA. In some embodiments, the antigen-binding molecule of the present disclosure provides cross-reactivity with VISTA of non-human primates. Cross-reactivity to VISTA in model species allows for testing of efficacy in in vivo syngeneic models without relying on surrogate molecules.

[0445] In some embodiments, an antigen-binding molecule according to the present disclosure has an EC of ≦20 pM (e.g., one of ≦15 pM, ≦12.5 pM, ≦10 pM, ≦7.5 pM) for human VISTA. 50 In some embodiments, an antigen-binding molecule in accordance with the present disclosure binds to cynomolgus monkey VISTA with an EC of ≦50 pM (e.g., one of ≦25 pM, ≦20 pM, ≦15 pM) (e.g., as determined by ELISA, such as the ELISA described in the Examples of the present disclosure). 50 In some embodiments, an antigen-binding molecule in accordance with the present disclosure binds to rat VISTA with an EC50 of ≦20 pM (e.g., one of ≦15 pM, ≦12.5 pM, ≦10 pM, ≦7.5 pM). 50 In some embodiments, an antigen-binding molecule in accordance with the present disclosure binds to mouse VISTA with an EC of ≦20 pM (e.g., one of ≦15 pM, ≦12.5 pM, ≦10 pM, ≦7.5 pM, ≦5 pM). 50 (eg, as determined by ELISA, such as the ELISAs described in the Examples of this disclosure).

[0446] In some embodiments, an antigen-binding molecule according to the present disclosure binds to VISTA (e.g., human VISTA) with similar affinity at pH 5.5 to pH 7.5. For example, in some embodiments, an antigen-binding molecule exhibits similar affinity for VISTA at pH 5.5 as it does for VISTA at pH 7.5.

[0447] As used herein, a binding affinity that is "similar" to a reference binding affinity means a binding affinity that is within 50%, e.g., within one of 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, of the reference binding affinity, determined under equivalent conditions.

[0448] K for binding to VISTA (e.g., human VISTA) D The EC for binding to VISTA (e.g., human VISTA) can be similar at pHs between 5.5 and 7.5. 50 can be similar at pH values ​​between 5.5 and 7.5.

[0449] As used herein, a K that is "similar" to a reference value D or EC 50 The value can be ≧0.5 times and ≦2 times, for example, ≧0.7 times and ≦1.5 times, ≧0.75 times and ≦1.25 times, ≧0.8 times and ≦1.2 times, ≧0.85 times and ≦1.15 times, ≧0.9 times and ≦1.1 times, ≧0.91 times and ≦1.09 times, or ≧0.92 times the reference value. It may be one of ≦1.08 times, ≧0.93 times and ≦1.07 times, ≧0.94 times and ≦1.06 times, ≧0.95 times and ≦1.05 times, ≧0.96 times and ≦1.04 times, ≧0.97 times and ≦1.03 times, ≧0.98 times and ≦1.02 times, or ≧0.99 times and ≦1.01 times.

[0450] The antigen-binding molecules of the present invention may bind to a region of particular interest in VISTA. The antigen-binding region of an antigen-binding molecule of the present invention may bind to a linear epitope of VISTA consisting of a consecutive sequence of amino acids (i.e., a primary sequence of amino acids). In some embodiments, the antigen-binding region may bind to a conformational epitope of VISTA consisting of a discontinuous sequence of amino acids within the amino acid sequence.

[0451] In some embodiments, the antigen-binding molecules of the present invention are capable of binding to VISTA. In some embodiments, the antigen-binding molecules are capable of binding to VISTA within the extracellular region of VISTA. In some embodiments, the antigen-binding molecules are capable of binding to VISTA within the Ig-like V-type domain (e.g., the region set forth in SEQ ID NO: 6). In some embodiments, the antigen-binding molecules are capable of binding to VISTA within the region set forth in SEQ ID NO: 31.

[0452] In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:322. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:30.

[0453] In some embodiments, the antigen-binding molecule does not bind to the region of VISTA to which IGN175A binds (e.g., as described in WO2014 / 197849A2). In some embodiments, the antigen-binding molecule does not bind to the region of VISTA to which an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268 binds.

[0454] In some embodiments, the antigen-binding molecule does not compete for binding to VISTA with IGN175A (e.g., as described in WO2014 / 197849A2). In some embodiments, the antigen-binding molecule does not compete for binding to VISTA with an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268.

[0455] The ability of a given antigen-binding molecule to compete for binding to VISTA with IGN175A or an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268 can be analyzed by competitive ELISA, or epitope binning, for example, as described in Abdiche et al., J Immunol Methods (2012), 382(2):101-116 (incorporated herein by reference in its entirety). Epitope binning can be performed, for example, by BLI analysis, for example, as described in Example 8 of the present application.

[0456] In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence set forth in SEQ ID NO:275.

[0457] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. Peptides are typically about 2 to 50 amino acids in length. A "polypeptide" is a polymeric chain of two or more peptides. Polypeptides are typically greater than about 50 amino acids in length.

[0458] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including ELISA, immunoblot (e.g., Western blot), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[0459] In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that is the same as or overlaps with a region of VISTA bound by an antibody comprising the VH and VL sequences of one of the following clones: 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31, 2M1-B12, 2M1-D2, 1M2-D2, 13D5p, 13D5-1, 13D5-13, 5M1-A11, or 9M2-C12.

[0460] In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that is different from the region of VISTA to which IGN175A binds (e.g., as described in WO2014 / 197849A2). In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that is different from the region of VISTA to which an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268 binds.

[0461] In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that does not overlap with the region of VISTA to which IGN175A binds (e.g., as described in WO2014 / 197849A2). In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that does not overlap with the region of VISTA to which an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268 binds.

[0462] In some embodiments, the antigen-binding molecule binds to VISTA through contacts with residues of VISTA that are not identical to the residues of VISTA contacted by VSTB112 (e.g., as described in WO2015 / 097536A2). In some embodiments, the antigen-binding molecule binds to VISTA through contacts with residues of VISTA that are not identical to the residues of VISTA contacted by an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 269 and a polypeptide consisting of the sequence of SEQ ID NO: 270.

[0463] In some embodiments, the epitope of the antigen-binding molecule is not identical to the epitope of VSTB112. In some embodiments, the epitope of the antigen-binding molecule is not identical to the epitope of an antigen-binding molecule consisting of a polypeptide consisting of the sequence of SEQ ID NO: 269 and a polypeptide consisting of the sequence of SEQ ID NO: 270.

[0464] The region of a peptide / polypeptide to which an antibody binds can be determined by those skilled in the art using a variety of methods known in the art, including X-ray cocrystallography of antibody-antigen complexes, peptide scanning, mutagenesis mapping, mass spectrometric hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is incorporated herein by reference in its entirety.

[0465] In some embodiments, the antigen-binding molecules of the present invention bind to VISTA within a region accessible to antigen-binding molecules (i.e., extracellular antigen-binding molecules) when VISTA is expressed on the cell surface (i.e., within or on the cell membrane). In some embodiments, the antigen-binding molecules are capable of binding to VISTA expressed on the cell surface of VISTA-expressing cells. In some embodiments, the antigen-binding molecules are capable of binding to VISTA-expressing cells (e.g., CD14+ monocytes (such as monocyte-derived suppressor cells (MDSCs)) and / or CD33+ myeloid cells, tumor-associated macrophages (TAMs), and neutrophils).

[0466] The ability of an antigen-binding molecule to bind to a given cell type can be analyzed, for example, by contacting the cells with the antigen-binding molecule and detecting the antigen-binding molecule bound to the cells, after a washing step to remove unbound antigen-binding molecules. The ability of an antigen-binding molecule to bind to immune cell surface molecule-expressing cells and / or cancer cell antigen-expressing cells can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0467] Antigen-binding molecules of the present invention may be antagonists of VISTA. In some embodiments, antigen-binding molecules are capable of inhibiting a function or process (e.g., an interaction, signal transduction, or other activity) mediated by VISTA and / or a VISTA binding partner (e.g., PSGL-1, VSIG-3, VSIG-8, LRIG1). As used herein, "inhibition" refers to a decrease, reduction, or decrease compared to a control condition. An antigen-binding molecule that inhibits a given interaction / activity / process may be referred to as an inhibitor or antagonist of the interaction / activity / process, and may be said to "block" or "neutralize" the interaction / activity / process.

[0468] The VISTA-binding antigen binding molecules described herein are capable of inhibiting VISTA-mediated functions / processes by mechanisms that do not require Fc-mediated functions such as ADCC, ADCP, and CDC, i.e., the VISTA-binding antigen binding molecules described herein are capable of inhibiting the immunosuppressive activity of VISTA-expressing cells without the need to induce ADCC, ADCP, and / or CDC.

[0469] In particular, the VISTA-binding antigen binding molecules described herein are capable of inhibiting VISTA through a mechanism that does not require binding to Fcγ receptors and / or binding to C1q.

[0470] In some embodiments, antigen-binding molecules of the present invention are capable of inhibiting the interaction of VISTA with its binding / interaction partners (e.g., PSGL-1, VSIG-3, VSIG-8, LRIG1). In some embodiments, antigen-binding molecules of the present invention are capable of inhibiting the interaction of VISTA with PSGL-1. In some embodiments, antigen-binding molecules of the present invention are capable of inhibiting the interaction of VISTA with VSIG-3. In some embodiments, antigen-binding molecules of the present invention are capable of inhibiting the interaction of VISTA with LRIG1.

[0471] The ability of an antigen-binding molecule to inhibit the interaction between two factors can be determined, for example, by analyzing the interaction in the presence of an antibody / fragment, or after incubation with an antibody / fragment of one or both of the interaction partners. Assays for determining whether a given antigen-binding molecule is capable of inhibiting the interaction between two interaction partners include competitive ELISA assays and SPR analysis.

[0472] Antigen-binding molecules capable of inhibiting a given interaction (e.g., the interaction between VISTA and a binding partner of VISTA) can be identified by observing a reduced / diminished level of interaction between the interaction partners in the presence of the antigen-binding molecule (or after incubation of one or both of the interaction partners with the antigen-binding molecule) compared to the level of interaction in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule). Suitable analyses can be performed in vitro, for example, using recombinant interaction partners or using cells expressing the interaction partners. Cells expressing the interaction partners may express them endogenously or from nucleic acids introduced into the cells. For purposes of such assays, one or both of the interaction partners and / or the antigen-binding molecule may be labeled with or used in conjunction with a detectable entity for the purpose of detecting and / or measuring the level of interaction.

[0473] The ability of an antigen-binding molecule to inhibit the interaction between two binding partners can also be determined by analyzing the downstream functional consequences of such interaction. For example, a downstream functional consequence of the interaction of VISTA with its binding partner can include VISTA-mediated signal transduction. For example, the ability of an antigen-binding molecule to inhibit the interaction of VISTA with its binding partner can be determined by analyzing the production of IL-2, IFN-γ, and / or IL-17 in an MLR assay.

[0474] In some embodiments, the antigen-binding molecules of the present invention are capable of inhibiting the interaction of VISTA with a binding partner of VISTA (e.g., PSGL-1, VSIG-3, VSIG-8, LRIG1) to less than 1-fold, for example, <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the level of interaction of VISTA with its binding partner in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0475] In some embodiments, the antigen-binding molecule inhibits VISTA-mediated signaling. In some embodiments, VISTA-mediated signaling may be signaling mediated by a polypeptide complex comprising VISTA. In some embodiments, VISTA-mediated signaling may be signaling mediated by a polypeptide complex comprising VISTA and a VISTA interaction partner (e.g., LRIG1, VSIG3, PSGL-1, VSIG8). VISTA-mediated signaling may be analyzed using an assay for the number / activity of effector immune cells, such as the MLR assay described in the Examples herein. Inhibition of VISTA-mediated signaling may be identified by detecting an increase in the number and / or activity of effector immune cells, as determined, for example, by increased production of IL-2, IFN-γ, and / or IL-17.

[0476] The ability of an antigen-binding molecule to inhibit the interaction of VISTA with its interaction partner can be determined, for example, by analyzing the interaction in the presence of the interaction partner, or after incubation of one or both of the interaction partners with the antigen-binding molecule. Assays for determining whether a given antigen-binding molecule is capable of inhibiting the interaction of VISTA with its interaction partner include competitive ELISA assays and analysis by SPR.

[0477] Antigen-binding molecules capable of inhibiting the interaction of VISTA with its interaction partners can be identified by observing a reduction / diminished level of interaction between the interaction partners in the presence of the interaction partner (or after incubation of one or both of the interaction partners with the antigen-binding molecule) compared to the level of interaction in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule known not to inhibit such interaction). Suitable assays can be performed in vitro, for example, using recombinant interaction partners or using cells expressing the interaction partner. Cells expressing the interaction partner may express it endogenously or from a nucleic acid introduced into the cell. For purposes of such assays, one or both of the interaction partners and / or the antigen binding may be labeled with or used in conjunction with a detection entity for the purpose of detecting and / or measuring the level of interaction.

[0478] In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require or involve Fc-mediated function. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling without relying on Fc-mediated function. That is, in some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling without relying on the Fc region.

[0479] The ability of an antigen-binding molecule to inhibit VISTA-mediated signaling by a mechanism that does not require / involve Fc-mediated function can be assessed, for example, by analyzing the ability of an antigen-binding molecule prepared in a format lacking a functional Fc region to inhibit VISTA-mediated signaling. For example, the effect on VISTA-mediated signaling may be examined using an antigen-binding molecule that includes a "silent" Fc region (e.g., containing a LALA PG substitution), or may be examined using an antigen-binding molecule prepared in a format lacking an Fc region (e.g., scFv, Fab, etc.).

[0480] In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not involve ADCC. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not involve ADCP. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not involve CDC.

[0481] In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding of the antigen-binding molecule to an Fc receptor. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding of the antigen-binding molecule to an Fcγ receptor. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding of the antigen-binding molecule to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding to FcγRIIIa. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding to FcγRIIa. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding to FcγRIIb. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding to complement proteins. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding to C1q. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling by a mechanism that does not require glycosylation of N297.

[0482] In some embodiments, the antigen-binding molecules of the present invention are capable of enhancing the killing of VISTA-expressing cells. The enhancing killing of VISTA-expressing cells may be achieved through the effector functions of the antigen-binding molecules. In embodiments in which the antigen-binding molecule comprises an Fc region, the antigen-binding molecule may enhance the killing of VISTA-expressing cells through one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0483] Antigen-binding molecules capable of increasing the killing of VISTA-expressing cells can be identified by observing an increased level of killing of VISTA-expressing cells in the presence of the antigen-binding molecule (or following incubation of VISTA-expressing cells with the antigen-binding molecule) compared to the level of cell killing detected in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule) in an appropriate assay. Assays for CDC, ADCC, and ADCP are well known to those skilled in the art. The level of killing of VISTA-expressing cells can also be determined by measuring the number / proportion of surviving and / or non-viable VISTA-expressing cells after exposure to different treatment conditions.

[0484] In some embodiments, the antigen binding molecules of the invention are capable of increasing killing of VISTA-expressing cells (e.g., VISTA-expressing MDSCs) by more than 1-fold, for example, ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold the level of killing observed in the absence of the antigen binding molecule (or in the presence of a suitable control antigen binding molecule).

[0485] In some embodiments, the antigen-binding molecules of the present invention are capable of reducing the number of VISTA-expressing cells (e.g., VISTA-expressing MDSCs) in an equivalent assay to less than 1-fold, for example, <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, the number of VISTA-expressing cells (e.g., MDSCs, TAMs, neutrophils expressing VISTA) detected after incubation in the absence of the antigen-binding molecule (or after incubation in the presence of an appropriate control antigen-binding molecule).

[0486] In some embodiments, the antigen-binding molecule is a non-depleting antigen-binding molecule. That is, in some embodiments, the antigen-binding molecule does not cause substantial depletion of VISTA-expressing cells. In some embodiments, the antigen-binding molecule does not induce / enhance ADCC, ADCP, and / or CDC against VISTA-expressing cells.

[0487] In some embodiments, the antigen-binding molecules of the invention do not induce / increase killing of VISTA-expressing cells, e.g., in embodiments in which the antigen-binding molecule lacks an Fc region or in embodiments in which the antigen-binding molecule comprises an Fc region that is not capable of inducing Fc-mediated antibody effector functions. In some embodiments, the antigen-binding molecules of the invention do not reduce the number / proportion of VISTA-expressing cells.

[0488] In some embodiments, antigen-binding molecules of the invention (i) inhibit VISTA-mediated signaling and (ii) do not induce / increase killing of VISTA-expressing cells. In some embodiments, antigen-binding molecules of the invention (i) inhibit VISTA-mediated signaling and (ii) do not reduce the number / proportion of VISTA-expressing cells.

[0489] This can be particularly advantageous because VISTA is expressed by cells that are not desired to be depleted, for example, VISTA is expressed at low levels by immune cells (e.g., certain types of T cells and dendritic cells) that are not desired to be killed or reduced in number / proportion.

[0490] In some embodiments, the antigen-binding molecules of the present invention can increase the number and / or activity of effector immune cells, for example, in a suitable in vitro assay or in vivo, compared to negative control conditions. To illustrate, the antigen-binding molecules of the present invention can relieve effector immune cells from MDSC-mediated suppression of effector immune cell proliferation and function. In some embodiments, the effector immune cells can be, for example, CD8+ T cells, CD8+ cytotoxic T lymphocytes (CD8+ CTLs), CD4+ T cells, CD4+ helper T cells, NK cells, IFNγ-producing cells, memory T cells, central memory T cells, antigen-experienced T cells, or CD45RO+ T cells.

[0491] The number and proportion of cells can be determined, for example, by flow cytometry analysis using antibodies that allow for the detection of cell types. 3 The activity of effector immune cells may be analyzed by in vitro analysis of H-thymidine incorporation, for example, by the CFSE dilution assay described in Fulcher and Wong, Immunol Cell Biol (1999), 77(6):559-564, which is incorporated herein by reference in its entirety. The activity of effector immune cells may be analyzed by measuring correlates of such activity. In some embodiments, the activity of effector immune cells may be determined, for example, by analysis of the production of IL-2, IFN-γ, and / or IL-17.

[0492] In some embodiments, antigen-binding molecules of the invention are capable of increasing the number of effector immune cell types by more than 1-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, above the number observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule). In some embodiments, antigen-binding molecules of the invention are capable of increasing the level of a correlate of effector immune cell activity by more than 1-fold, e.g., ≥ 1.01-fold, ≥ 1.02-fold, ≥ 1.03-fold, ≥ 1.04-fold, ≥ 1.05-fold, ≥ 1.1-fold, ≥ 1.2-fold, ≥ 1.3-fold, ≥ 1.4-fold, ≥ 1.5-fold, ≥ 1.6-fold, ≥ 1.7-fold, ≥ 1.8-fold, ≥ 1.9-fold, ≥ 2-fold, ≥ 3-fold, ≥ 4-fold, ≥ 5-fold, ≥ 6-fold, ≥ 7-fold, ≥ 8-fold, ≥ 9-fold, or ≥ 10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0493] In some embodiments, the antigen-binding molecules of the present invention are capable of reducing the level of immunosuppression mediated by VISTA-expressing cells. Changes in the level of immunosuppression can be determined using methods measuring the expression of arginase 1 and / or the production of reactive oxygen species (ROS) by VISTA-expressing cells, as described, for example, in Ochoa et al., Ann Surg., March 2001, 233(3):393-399; and Dikalov and Harrison, Antioxid Redox Signal., January 10, 2014, 20(2):372-382.

[0494] In some embodiments, antigen-binding molecules of the invention are capable of increasing antigen presentation by antigen-presenting cells, e.g., as determined using a suitable assay for antigen presentation. In some embodiments, antigen-binding molecules of the invention are capable of increasing phagocytosis by phagocytes (e.g., neutrophils, monocytes, macrophages, mast cells, and / or dendritic cells), e.g., as determined using a suitable assay for the level of phagocytosis.

[0495] In some embodiments, an antigen-binding molecule of the present disclosure is capable of increasing, for example, the number and / or activity of antigen-presenting cells (e.g., CD11b+ MHCII+ cells) compared to negative control conditions, e.g., in a suitable in vitro assay or in vivo (e.g., in a tumor). In some embodiments, an antigen-binding molecule is capable of increasing, for example, the number and / or activity of macrophages (e.g., CD11b+ F4 / 80+ cells) compared to negative control conditions, e.g., in a suitable in vitro assay or in vivo (e.g., in a tumor). In some embodiments, an antigen-binding molecule is capable of increasing, for example, the number and / or activity of dendritic cells (e.g., CD11c+ cells) compared to negative control conditions, e.g., in a suitable in vitro assay or in vivo (e.g., in a tumor).

[0496] In some embodiments, an antigen-binding molecule of the disclosure is capable of increasing the number of cell types listed in the preceding paragraph by more than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, the number observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule). In some embodiments, an antigen-binding molecule of the disclosure is capable of increasing the level of a correlate of activity of a cell type listed in the preceding paragraph by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0497] In some embodiments, the antigen-binding molecules of the present invention are capable of increasing IL-6 production by immune cells. The immune cells can be, for example, PBMCs, lymphocytes, T cells, B cells, NK cells, or monocytes. In some embodiments, the immune cells are monocytes. In some embodiments, the antigen-binding molecules are capable of increasing IL-6 production by immune cells after stimulation with, for example, LPS. The ability of an antigen-binding molecule to increase IL-6 production by immune cells can be analyzed, for example, in the in vitro assay described in Example 10 herein. Such a method can include stimulating monocytes (e.g., THP1 cells) with LPS and incubating the stimulated cells with the antigen-binding molecule.

[0498] In some embodiments, antigen-binding molecules of the invention are capable of increasing IL-6 production by immune cells (e.g., THP1 cells stimulated with LPS) by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0499] In some embodiments, antigen-binding molecules of the present disclosure are capable of increasing the number and / or activity of Th1 / Th17 cells. In some embodiments, antigen-binding molecules are capable of upregulating Th1 / Th17 responses. In some embodiments, antigen-binding molecules prioritize Th1 / Th17 responses over Th2 responses. In some embodiments, antigen-binding molecules of the present disclosure are capable of increasing T cell proliferation, IL-2 production, IFN-γ production, TNFα production, and / or IL-17A production in a mixed lymphocyte reaction (MLR) assay. MLR assays can be performed as described in Bromelow et al., J. Immunol Methods, 2001 Jan. 1;247(1-2):1-8 (incorporated herein by reference in its entirety), or as described in the Examples herein. The production of IL-2, IFNγ, and / or IL-17 may be analyzed by antibody-based methods well known to those skilled in the art, such as, for example, Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or by reporter-based methods.

[0500] In some embodiments, the antigen-binding molecules of the present invention are capable of increasing T cell (e.g., Th1 / Th17 cell) proliferation, IL-2 production, IFN-γ production, and / or IL-17 production in a mixed lymphocyte reaction (MLR) assay. The MLR assay can be performed as described in Bromelow et al., J. Immunol Methods, 2001 Jan. 1;247(1-2):1-8 (incorporated herein by reference in its entirety), or as described in the Examples herein. IL-2, IFN-γ, and / or IL-17 production can be analyzed by antibody-based methods well known to those skilled in the art, such as, for example, Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0501] In some embodiments, antigen-binding molecules of the invention are capable of increasing T cell proliferation, IL-2 production, IFN-γ production, and / or IL-17 production in an MLR assay by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0502] In some embodiments, antigen-binding molecules of the present invention are capable of increasing T cell proliferation, IFN-γ production, and / or TNFα production, for example, in the presence of VISTA / VISTA-expressing cells. Antigen-binding molecules can be evaluated for such properties, for example, in in vitro assays described in the Examples herein.

[0503] In some embodiments, antigen-binding molecules of the invention are capable of increasing T cell (e.g., Th1 / Th17 cell) proliferation, IFN-γ production, and / or TNFα production (e.g., in the presence of cells expressing VISTA / VISTA) by more than 1-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0504] In some embodiments, the antigen-binding molecules of the present invention are capable of increasing the proliferation of T cells (e.g., CD4+ T cells and / or CD8+ T cells, e.g., Th1 / Th17 cells) to a greater extent than VISTA-binding antibodies disclosed in the prior art (e.g., VSTB112, described in WO2015 / 097536A2). T cell proliferation may be assessed in an in vitro assay, for example, as described in Example 9 herein, which may involve stimulating T cell proliferation by culture in the presence of an agonistic anti-CD3 antibody. In some embodiments, the antigen binding molecules of the invention are capable of increasing T cell proliferation in such assays by more than 1-fold, for example, >1.01-fold, >1.02-fold, >1.03-fold, >1.04-fold, >1.05-fold, >1.1-fold, >1.2-fold, >1.3-fold, >1.4-fold, >1.5-fold, >1.6-fold, >1.7-fold, >1.8-fold, >1.9-fold, >2-fold, >3-fold, >4-fold, >5-fold, >6-fold, >7-fold, >8-fold, >9-fold, or >10-fold the proliferation level induced by a prior art VISTA binding antibody (e.g., VSTB112).

[0505] In some embodiments, an antigen-binding molecule of the disclosure is capable of increasing T-cell-mediated lysis of cancer cells to a level of more than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0506] In some embodiments, antigen-binding molecules of the present disclosure are capable of increasing T cell-mediated lysis of cancer cells, for example, in the presence of VISTA / VISTA-expressing cells. Antigen-binding molecules can be assessed for such properties, for example, in the in vitro assays described in the Examples herein.

[0507] In some embodiments, antigen-binding molecules of the disclosure are capable of increasing T cell-mediated lysis (e.g., in the presence of VISTA / VISTA-expressing cells) to a level of more than 1-fold, e.g., ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, above the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0508] In some embodiments, the antigen-binding molecules of the invention are capable of increasing IL-6 production by THP1 cells to a greater extent than VISTA-binding antibodies disclosed in the prior art (e.g., VSTB112 described in WO2015 / 097536A2). IL-6 production by THP1 cells may be assessed in an in vitro assay, for example, as described in Example 10 herein, which may involve stimulating THP1 cells with LPS. In some embodiments, the antigen-binding molecules of the invention are capable of increasing IL-6 production in such assays by more than 1-fold, for example, ≧1.01-fold, ≧1.02-fold, ≧1.03-fold, ≧1.04-fold, ≧1.05-fold, ≧1.1-fold, ≧1.2-fold, ≧1.3-fold, ≧1.4-fold, ≧1.5-fold, ≧1.6-fold, ≧1.7-fold, ≧1.8-fold, ≧1.9-fold, ≧2-fold, ≧3-fold, ≧4-fold, ≧5-fold, ≧6-fold, ≧7-fold, ≧8-fold, ≧9-fold, or ≧10-fold, above the level induced by a prior art VISTA-binding antibody (e.g., VSTB112).

[0509] In some embodiments, the antigen-binding molecules of the invention are capable of reducing the number and / or activity of immunosuppressive cells, inhibiting the proliferation of immunosuppressive cells, and / or reducing the proportion of immunosuppressive cells within a population of cells (e.g., CD45+ cells, e.g., CD45+ cells obtained from a tumor), e.g., in a suitable in vitro assay or in vivo, relative to control conditions.

[0510] The immunosuppressive cells can be, for example, VISTA-expressing cells, Arg1-expressing cells, MDSCs, granulocytic MDSCs (g-MDSCs), or monocytic MDSCs (m-MDSCs). In some embodiments, the immunosuppressive cells are CD11b+GR1+MHCII- cells.

[0511] In some embodiments, the reduction in number / activity / proliferation / ratio is less than 1-fold, e.g., <0.99-fold, <0.95-fold, <0.9-fold, <0.85-fold, <0.8-fold, <0.75-fold, <0.7-fold, <0.65-fold, <0.6-fold, <0.55-fold, <0.5-fold, <0.45-fold, <0.4-fold, <0.35-fold, <0.3-fold, <0.25-fold, <0.2-fold, <0.15-fold, <0.1-fold, <0.05-fold, or <0.01-fold, of the number / activity / proliferation / ratio observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[0512] In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / ratio of immunosuppressive cells through a mechanism that does not involve an Fc-mediated function. In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / ratio of immunosuppressive cells independently of an Fc-mediated function (i.e., independent of the Fc region). In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / ratio of immunosuppressive cells through a mechanism that does not involve ADCC, ADCP, and / or CDC. In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / ratio of immunosuppressive cells through a mechanism that does not involve depletion of VISTA-expressing cells.

[0513] In some embodiments, the antigen-binding molecules of the invention inhibit the onset and / or progression of cancer in vivo.

[0514] In some embodiments, the antigen-binding molecule causes increased killing of cancer cells, e.g., by effector immune cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g., compared to appropriate control conditions. In some embodiments, the antigen-binding molecule inhibits tumor growth, e.g., as determined by measuring tumor size / volume over time.

[0515] In some embodiments, the antigen-binding molecules of the present invention are capable of increasing serum levels of IFN-γ and / or IL-23 in mice treated with the antigen-binding molecules. Serum levels of IFN-γ and / or IL-23 can be analyzed, for example, by ELISA on serum derived from blood samples obtained from the mice. In some embodiments, administration of an antigen-binding molecule of the invention increases serum levels of IFN-γ and / or IL-23 by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, above the level observed in the absence of the antigen-binding molecule (or the level observed after administration of a suitable control antigen-binding molecule).

[0516] The antigen-binding molecules of the present invention can be analyzed for their ability to inhibit cancer development and / or progression in appropriate in vivo models, such as cell line-derived xenograft models, such as CT26 cell-derived models, 4T-1 cell-derived models, LL2 cell-derived models, B16 cell-derived models, or EL4 cell-derived models. The cancer can be one in which VISTA-expressing cells and / or MDSCs (e.g., VISTA-expressing MDSCs, TAMs, and neutrophils) are pathologically involved. Cancers in which MDSCs are "pathologically involved" include cancers in which an increase in MDSCs or the number / proportion of MDSCs is positively associated with the onset, development, or progression of cancer and / or the severity of one or more symptoms of cancer, or cancers in which an increase in MDSCs or the number / proportion of MDSCs is a risk factor for the onset, development, or progression of cancer. Cancers can contain MDSCs in organs / tissues affected by disease (e.g., organs / tissues where symptoms of a disease / condition are manifested) or within tumors.

[0517] In some embodiments, administration of an antigen-binding molecule according to the invention may result in one or more of: inhibiting cancer onset / progression, delaying cancer onset / preventing cancer onset, reducing tumor growth / delaying tumor growth / preventing tumor growth, reducing metastasis / delaying metastasis / preventing metastasis, reducing the severity of cancer symptoms, reducing cancer cell number, reducing tumor size / volume, and / or increasing survival (e.g., progression-free survival), for example, as determined in xenograft models derived from CT26 cells, 4T-1 cells, LL2 cells, B16 cells, or EL4 cells.

[0518] In some embodiments, administration of an antigen-binding molecule of the invention is capable of inhibiting more than 5%, e.g., ≥ 10%, ≥ 15%, ≥ 20%, ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, ≥ 50%, ≥ 55%, ≥ 60%, ≥ 65%, ≥ 70%, ≥ 75%, ≥ 80%, ≥ 85%, ≥ 90%, or ≥ 95%, of tumor growth observed in the absence of administration of the antigen-binding molecule (or after administration of a suitable control antigen-binding molecule).

[0519] In some embodiments, administration of an antigen-binding molecule at the doses and with the regularity described in the experiments using a CT26 cell-derived model in the Examples of the present disclosure inhibits more than 5% of tumor growth observed in the absence of administration of the antigen-binding molecule (or after administration of an appropriate control antigen-binding molecule), e.g., ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, or ≥80% of tumor growth observed in the absence of administration of the antigen-binding molecule (or after administration of an appropriate control antigen-binding molecule). In some embodiments, administration of an antigen-binding molecule at the doses and with the regularity described in the experiments using a 4T-1 cell-derived model in the Examples of the present disclosure inhibits more than 5% of tumor growth observed in the absence of administration of the antigen-binding molecule (or after administration of an appropriate control antigen-binding molecule), e.g., ≥ 10%, ≥ 15%, ≥ 20%, ≥ 25%, ≥ 30%, ≥ 35%, ≥ 40%, ≥ 45%, ≥ 50%.

[0520] In some embodiments, administration of an antigen-binding molecule according to the present disclosure is not associated with cytokine release syndrome. In some embodiments, administration of an antigen-binding molecule is not associated with systemic activation of leukocytes (e.g., B cells, T cells, NK cells, macrophages, dendritic cells, and / or monocytes). In some embodiments, administration of an antigen-binding molecule is not associated with systemic upregulation of inflammatory cytokine and / or chemokine expression by leukocytes (e.g., IL-6, IFN-γ, IL-8, IL-10, GM-CSF, MIP-1α / β, MCP-1, CXCL9, and / or CXCL10).

[0521] In some embodiments, treatment of a subject with an antigen-binding molecule or other article (e.g., composition, nucleic acid, etc.) disclosed herein, e.g., wherein the antigen-binding molecule / article is administered to the subject at a dosage and / or according to a dosing schedule described herein, results in the following outcomes: · Absence of adverse events (AEs); · Absence of serious adverse events (SAEs); ·Minimized or reduced frequency of adverse events (AEs), e.g., compared with other therapeutic anti-VISTA antibodies; · Minimal or reduced frequency of serious adverse events (SAEs), e.g., compared with other therapeutic anti-VISTA antibodies; Absence of dose-limiting toxicities (DLTs); ·Minimized or reduced frequency of dose-limiting toxicities (DLTs) compared to other therapeutic anti-VISTA antibodies, for example, W0180 and CA-170; A reduction in circulating tumor markers (e.g., cell-free (cf) DNA mutant allele fraction / tumor fraction, ctDNA) after treatment, e.g., compared to the same subject before treatment; Absence, minimal or reduced frequency of infusion-related reactions (IRRs), e.g., cytokine release syndrome and associated cytokine increases, compared to other therapeutic anti-VISTA antibodies, e.g., W0180 and CA-170; - absence, minimal or reduced incidence of gastrointestinal toxicities compared to other therapeutic anti-VISTA antibodies, e.g., W0180 and CA-170; - absence, minimal or reduced frequency of redness or dermatitis compared to other therapeutic anti-VISTA antibodies, e.g., W0180 and CA-170; - absence or minimal or reduced frequency of hematological changes compared to other therapeutic anti-VISTA antibodies, e.g., W0180 and CA-170; the absence, minimal incidence, or reduced incidence of cardiac toxicity compared to other therapeutic anti-VISTA antibodies, such as W0180 and CA-170; and / or Absence, minimal frequency, or reduced frequency of albumin elevation compared to other therapeutic anti-VISTA antibodies, e.g., W0180 and CA-170 may be associated with one or more of:

[0522] An adverse event (AE) may be defined as any undesired, unwanted, or unplanned medical occurrence in a patient administered an investigational medicinal product (IMP), comparator, or approved drug. An AE may be a sign, symptom, disease, and / or laboratory or physiological observation that may or may not be related to the IMP or comparator. AEs include, but are not limited to, the AEs in the following list:

[0523] A clinically significant worsening of a pre-existing condition. This includes conditions that may resolve completely and then become abnormal again.

[0524] -AEs resulting from accidental or intentional overdose of IMP.

[0525] AEs resulting from lack of efficacy of an IMP, for example, when the investigator suspects that a drug batch is not effective, or when the investigator suspects that an IMP contributed to disease progression.

[0526] A serious adverse event (SAE) is any of the following AEs, regardless of dose, causality, or foreseeability: AEs resulting in death; - AEs that are fatal, i.e., events for which the patient is at substantial risk of death at the time of the adverse event or with continued use of the device or other medicinal product that could have resulted in the patient's death; AEs requiring inpatient hospitalization or prolonging an existing inpatient hospitalization (some hospitalizations, e.g., hospital admissions planned before the patient entered the study; overnight stays for planned procedures, such as blood transfusions, are exempt from SAE reporting); AEs that result in incapacity or disability, whether prolonged or significant; ·AEs that are congenital anomalies or birth defects; Any other medically significant event, i.e., any event that may be patient-threatening and may require intervention to prevent one of the outcomes listed above.

[0527] In some embodiments, response to treatment according to the present disclosure may be characterized by reference to tumor / lesion response, which may be assessed according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria, e.g., RECIST 1.1 criteria, as described in Eisenhauer et al., Eur J Cancer. 2009 Jan;45(2):228-47, which is incorporated herein by reference in its entirety.

[0528] In some embodiments, treatment of a subject with an antigen-binding molecule or article described herein, e.g., where the antigen-binding molecule / article is administered to the subject at a dosage described herein and / or according to a dosing schedule described herein, may be associated with one or more of the following outcomes (assessed according to RECIST 1.1 criteria, as appropriate; see Example 19.9 for details and assessment methods), e.g., at 12 and / or 24 months from the start of treatment: Antitumor response; Complete response (CR): CR refers to the complete disappearance of all target and / or non-target tumors at the visual level. CR may be associated with normalization of tumor marker levels; an increase in the likelihood of a complete response (CR), e.g., compared to the likelihood of a CR in the same subject without treatment with the antigen binding molecule, in a subject who has not received the antigen binding molecule, or in a subject who has been treated with a different anti-VISTA antigen binding molecule; an increase in the proportion of subjects experiencing a complete response (CR), e.g., compared to the proportion of subjects experiencing a CR who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; · Overall survival (OS): OS is defined as the time from the start of treatment to death due to any cause; an increase in the likelihood of overall survival (OS), e.g., compared to the likelihood of OS in the same subject without treatment with the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject treated with a different anti-VISTA antigen binding molecule; an increase in the proportion of subjects exhibiting overall survival (OS), e.g., compared to the proportion of subjects exhibiting OS who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; · Progression-free survival (PFS): PFS refers to the time from the start of treatment to disease progression or death; an increase in the likelihood of progression-free survival (PFS), e.g., compared to the likelihood of PFS in the same subject without treatment with the antigen binding molecule, a subject who does not receive the antigen binding molecule, or a subject who is treated with a different anti-VISTA antigen binding molecule; an increase in the proportion of subjects experiencing progression-free survival (PFS), e.g., compared to the proportion of subjects experiencing PFS who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; Progression-free survival at 6 months (PFS6): PFS6 refers to the percentage of patients who are alive and progression-free 6 months (26 weeks) after the start of treatment; an increase in the likelihood of progression-free survival at 6 months (PFS6), e.g., compared to the likelihood of PFS in the same subject without treatment with the antigen binding molecule, a subject who did not receive the antigen binding molecule, or a subject who was treated with a different anti-VISTA antigen binding molecule; an increase in the percentage of subjects exhibiting progression-free survival at 6 months (PFS6) compared to, for example, the percentage of subjects exhibiting PFS6 who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; Partial response (PR): PR refers to a reduction of at least 30% in the sum of all target tumor diameters compared to the baseline sum of diameters calculated before treatment; an increase in the likelihood of a partial response (PR), e.g., compared to the likelihood of a PR in the same subject without treatment with the antigen binding molecule, in a subject who has not received the antigen binding molecule, or in a subject who has been treated with a different anti-VISTA antigen binding molecule; an increase in the proportion of subjects experiencing a partial response (PR), e.g., compared to the proportion of subjects experiencing a PR who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; MR (mixed response): MR refers to one or more tumor lesions meeting the criteria for PR and other tumor lesion(s) meeting the criteria for progression (at least a 20% increase in the sum of all tumor diameters from the smallest tumor size and / or the appearance of new tumor lesions); the likelihood of a mixed response (MR), e.g., an increased likelihood compared to MR in the same subject without treatment with the antigen binding molecule, in a subject not receiving the antigen binding molecule, or in a subject treated with a different anti-VISTA antigen binding molecule; an increase in the proportion of subjects exhibiting mixed response (MR), e.g., compared to the proportion of subjects exhibiting MR who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; Stable disease (SD): SD is neither a partial response nor progression compared to the tumor burden at the start of treatment.

[0529] an increase in the likelihood of stable disease (SD), e.g., compared to the likelihood of SD in the same subject without treatment with the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject treated with a different anti-VISTA antigen binding molecule; an increase in the proportion of subjects exhibiting stable disease (SD), e.g., compared to the proportion of subjects exhibiting SD who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; - Prolongation of duration of response (DoR) or CR duration, e.g., compared to the duration in the same subject without treatment with the antigen binding molecule, in subjects who did not receive the antigen binding molecule, or in subjects treated with a different anti-VISTA antigen binding molecule: Duration of response (DoR) is defined as the time from the date when metrics for PR or CR were first met to the date when metrics for progressive disease (PD) were first met. Duration of CR (DoCR) is defined as the time from the date when metrics for CR were met to the date when metrics for PD were first met; an increase in the proportion of subjects exhibiting an extended duration of response (DoR) or an extended duration of CR, e.g., compared to the proportion of subjects exhibiting an extended DoR or DoCR without treatment with the antigen binding molecule or treated with a different anti-VISTA antigen binding molecule; · Overall response (OR): OR is defined as the achievement of a complete response (CR) or partial response (PR); · Overall response rate (ORR): ORR is defined as the proportion of patients who achieve a complete response (CR) or partial response (PR); an increase in ORR, e.g., compared to the proportion of subjects exhibiting OR who are not treated with the antigen binding molecule or who are treated with a different anti-VISTA antigen binding molecule; Tumor response can be assessed using appropriate imaging methods, such as CT scans, MRI scans, and FDG-PET, depending on the tumor and its location. Those skilled in the art are familiar with appropriate techniques, which are described in Eisenhauer et al., supra; and / or in Example 19.9 herein.

[0530] The subject can be a subject as defined herein, eg, a subject having or determined to have a cancer or solid tumor according to the present disclosure. Chimeric antigen receptor (CAR) The present invention also provides a chimeric antigen receptor (CAR) comprising an antigen-binding molecule or polypeptide of the present invention.

[0531] CAR is a recombinant receptor that provides both antigen binding and T cell activation function.The structure and operation of CAR are reviewed in, for example, Dotti et al., Immunol Rev (2014), 257(1), which is incorporated herein by reference in its entirety.CAR comprises an antigen binding region that is connected to a cell membrane anchor region and a signal transduction region.Optionally, hinge region can separate the antigen binding region from the cell membrane anchor region, and can act as a flexible linker.

[0532] The CAR of the present invention comprises an antigen-binding region that comprises or consists of an antigen-binding molecule of the present invention or that comprises or consists of a polypeptide according to the present invention.

[0533] The cell membrane anchor region is provided between the antigen-binding region and the signaling region of the CAR, and allows for anchoring of the CAR, with the antigen-binding region in the extracellular space and the signaling region inside the cell, to the cell membrane of the cell expressing the CAR. In some embodiments, the CAR comprises a cell membrane anchor region that comprises, consists of, or is derived from the amino acid sequence of the transmembrane region of one of CD3-zeta, CD4, CD8, or CD28. As used herein, a region "derived from" a reference amino acid sequence includes an amino acid sequence that has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference sequence.

[0534] The signaling region of a CAR enables T cell activation. The signaling region of a CAR can include the amino acid sequence of the intracellular domain of CD3-zeta, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells. CARs have also utilized signaling regions containing sequences from other ITAM-containing proteins, such as FcγRI (Haynes et al., 2001, J Immunol, 166(1):182-187). The signaling region of a CAR can also include a costimulatory sequence derived from the signaling region of a costimulatory molecule to facilitate activation of CAR-expressing T cells upon binding to a target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, CARs are engineered to provide costimulation of different intracellular signaling pathways. For example, signal transduction associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas 4-1BB-mediated signal transduction is mediated through the TNF receptor-associated factor (TRAF) adaptor protein. Thus, the signal transduction region of the CAR may contain costimulatory sequences derived from the signal transduction region of more than one costimulatory molecule. In some embodiments, the CAR of the present invention comprises one or more costimulatory sequences that comprise, consist of, or are derived from the amino acid sequence of one or more intracellular domains of CD28, OX40, 4-1BB, ICOS, and CD27.

[0535] The optional hinge region can separate the antigen-binding domain from the transmembrane domain and can act as a flexible linker. The hinge region can be derived from IgG1. In some embodiments, the CAR of the present invention comprises a hinge region that comprises, consists of, or is derived from the amino acid sequence of the hinge region of this IgG1.

[0536] Also provided are cells comprising a CAR according to the present invention. A CAR according to the present invention can be used to generate CAR-expressing immune cells, such as CAR-T cells or CAR-NK cells. Engineering a CAR into immune cells can be performed during in vitro culture.

[0537] The antigen-binding region of the CAR of the present invention can be provided in any suitable format, e.g., scFv, scFab, etc. Nucleic acids and vectors The invention provides a nucleic acid or nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR in accordance with the invention.

[0538] In some embodiments, the nucleic acid may be purified or isolated, for example, from other nucleic acids, or from naturally occurring biological material, hi some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0539] The present invention also provides a vector or vectors comprising a nucleic acid or nucleic acids in accordance with the invention.

[0540] The nucleotide sequence may be contained within a vector, for example, an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle to introduce exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a cell. Such a vector may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from a vector according to the present invention.

[0541] The term "operably linked" can include the situation where a selected nucleic acid sequence is covalently linked to a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) such that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) can then be translated into the desired peptide(s) / polypeptide(s).

[0542] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[0543] In some embodiments, the vector can be a eukaryotic vector, e.g., a vector containing elements necessary for expression of a protein from a vector in a eukaryotic cell, hi some embodiments, the vector can be a mammalian vector, e.g., containing a cytomegalovirus (CMV) or SV40 promoter driving expression of a protein.

[0544] The component polypeptides of an antigen-binding molecule according to the present invention can be encoded by different nucleic acids from among a plurality of nucleic acids or by different vectors from among a plurality of vectors. Antigen-binding molecules and cells containing / expressing polypeptides The present invention also provides cells comprising or expressing an antigen-binding molecule, polypeptide, or CAR according to the invention. Also provided are cells comprising or expressing a nucleic acid, multiple nucleic acids, single vector, or multiple vectors according to the invention.

[0545] The cell can be a eukaryotic cell, e.g., a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human), or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Murine), cat, dog, pig, sheep, goat, cattle (including bovine, e.g., dairy cow, or any animal in the order Bovidae), horse (including any animal in the order Equine), donkey, and non-human primate).

[0546] The present invention also provides methods for producing a cell comprising a nucleic acid(s) or vector(s) in accordance with the invention, the method comprising introducing into a cell a nucleic acid, multiple nucleic acids, a vector, or multiple vectors in accordance with the invention. In some embodiments, introducing into a cell the isolated nucleic acid(s) or isolated vector(s) in accordance with the invention comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0547] The present invention also provides methods for producing cells that express / comprise an antigen-binding molecule, polypeptide, or CAR according to the present invention, comprising introducing into the cell a nucleic acid, multiple nucleic acids, single vector, or multiple vectors according to the present invention. In some embodiments, the method further comprises culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell. In some embodiments, the method is performed in vitro.

[0548] The present invention also provides a cell obtained or obtainable by a method according to the present invention. Production of antigen-binding molecules and polypeptides The antigen-binding molecules and polypeptides according to the present invention can be prepared according to methods for producing polypeptides known to those skilled in the art.

[0549] Polypeptides can be prepared by chemical synthesis, e.g., liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using the methods described in, e.g., Chandrudu et al., Molecules (2013), 18:4373-4388, which is incorporated herein by reference in its entirety.

[0550] Alternatively, antigen-binding molecules and polypeptides can be produced by recombinant expression. Molecular biology methods suitable for the recombinant production of polypeptides are well known in the art, such as those disclosed in Green and Sambrook, "Molecular Cloning: A Laboratory Manual" (4th ed.), Cold Spring Harbor Press, 2012; and Nat Methods. (2008), 5(2):135-146, all of which are incorporated herein by reference in their entirety. Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013), 4:217; and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016), 100:3451-3461, all of which are incorporated herein by reference in their entirety.

[0551] In some cases, an antigen-binding molecule of the present invention is composed of more than one polypeptide chain. In such cases, production of the antigen-binding molecule may involve transcription and translation of more than one polypeptide and subsequent assembly of the polypeptide chains to form the antigen-binding molecule.

[0552] For recombinant production according to the present invention, any cell suitable for expressing a polypeptide can be used. The cell can be a prokaryotic or eukaryotic cell. In some embodiments, the cell is a prokaryotic cell, such as an archaeal or bacterial cell. In some embodiments, the bacterium can be a gram-negative bacterium, such as a bacterium of the Enterobacteraceae family, e.g., Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell, or a mammalian cell, e.g., a CHO, HEK (e.g., HEK293), HeLa, or COS cell. In some embodiments, the cell is a CHO cell that transiently or stably expresses a polypeptide.

[0553] In some cases, the cells are not prokaryotic because some prokaryotic cells do not allow the same folding or post-translational modifications as eukaryotic cells. In addition, extremely high expression levels are possible in eukaryotic cells, and proteins are easy to purify from eukaryotic cells using appropriate tags. Specific plasmids that enhance protein secretion into the medium can also be used.

[0554] In some embodiments, polypeptides may be prepared by cell-free protein synthesis (CFPS), for example, using the system described in Zemella et al., Chembiochem (2015), 16(17):2420-2431, which is incorporated herein by reference in its entirety.

[0555] Production may involve the culture or fermentation of eukaryotic cells that have been modified to express the polypeptide(s) of interest. Cultivation or fermentation may be carried out in a bioreactor with an appropriate supply of nutrients, air / oxygen, and / or growth factors. Secreted proteins may be recovered by fractionating the culture medium / fermentation broth from the cells, extracting the protein content, separating the individual proteins, and isolating the secreted polypeptide(s). Cultivation, fermentation, and isolation methods are well known to those of skill in the art and are described, for example, in Green and Sambrook, "Molecular Cloning: A Laboratory Manual" (4th ed.; incorporated herein by reference).

[0556] A bioreactor contains one or more reaction vessels in which cells can be cultured. Cultivation in a bioreactor can occur continuously, with a continuous inflow of reactants into the reactor and a continuous outflow of cultured cells from the reactor. Alternatively, cultivation can occur in batches. Bioreactors monitor and control environmental conditions, such as pH, oxygen, flow rates into and out of the reactor, and agitation within the reactor, to provide optimal conditions for the cultured cells.

[0557] After culturing cells expressing the antigen-binding molecule / polypeptide(s), the polypeptide(s) of interest can be isolated. Any suitable method known in the art for separating proteins from cells can be used. To isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide(s) of interest are secreted from the cells, the cells can be separated from the culture medium containing the secreted polypeptide(s) of interest by centrifugation. If the polypeptide(s) of interest are collected intracellularly, protein isolation can include centrifugation to separate the cells from the cell culture medium, treatment of the cell pellet with a lysis buffer, and disruption of the cells by sonication, rapid freeze-thawing, or osmotic lysis.

[0558] It may then be desirable to isolate the polypeptide(s) of interest from the supernatant or culture medium, which may contain other proteins and non-protein components. A common technique for separating protein components from the supernatant or culture medium is precipitation. Proteins with different solubility are precipitated with different concentrations of precipitants, such as ammonium sulfate. For example, low concentrations of precipitant extract water-soluble proteins. Therefore, by adding increasing concentrations of precipitant, proteins with different solubility can be distinguished. Dialysis can then be used to remove ammonium sulfate from the separated proteins.

[0559] Other methods for distinguishing between different proteins are known in the art, such as ion exchange chromatography and size chromatography, which may be used as alternatives to precipitation or may be performed subsequent to precipitation.

[0560] Once the polypeptide(s) of interest have been isolated from the culture, it may be desirable or necessary to concentrate the polypeptide(s). Numerous methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization. composition The present invention also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[0561] The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may include pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The compositions may be formulated for local, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral, or transdermal administration routes, which may include injection or infusion.

[0562] Suitable formulations may include antigen-binding molecules in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in a fluid, including in gel form. Fluid formulations may be formulated for administration by injection or infusion (e.g., via a catheter) to a selected area within the human or animal body.

[0563] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel or tumor.

[0564] Also provided in accordance with the present invention are methods for making the pharmaceutically useful compositions described herein, which may include one or more steps selected from: making an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein; and / or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0565] For example, a further aspect of the invention described herein relates to a method of formulating or making a medicament or pharmaceutical composition for use in treating a disease / condition (e.g., cancer), comprising the step of formulating the pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), or cell(s) described herein with a pharmaceutically acceptable carrier, adjuvant, excipient, or diluent.

[0566] In aspects and embodiments of the present disclosure, the antigen-binding molecule may be provided in a composition containing specific chemical moieties at specified concentrations / ratios.

[0567] In some embodiments, the antigen-binding molecule is provided in a buffer. As used herein, "buffer" refers to a buffer that resists pH changes due to the action of its acid-base conjugate component. Buffers of the present disclosure preferably have a pH in the range of about 4.5 to about 7.0, preferably about 5.0 to about 6.5. Examples of buffers that control the pH within this range include acetate, succinate, histidine, histidine-arginine, histidine-methionine, and other organic acid buffers.

[0568] In some embodiments, the composition comprising the antigen-binding molecule has a pH of 4.0 to 7.0, e.g., one of pH 4.5 to 6.8, pH 4.6 to 6.5, pH 4.8 to 6.3, or pH 5.0 to 6.3. In some embodiments, the composition has a pH of about 5.5. In some embodiments, the composition has a pH of about 5.8. In some embodiments, the composition has a pH of about 6.3.

[0569] In some embodiments, the antigen-binding molecule is provided in an acetate buffer, i.e., a buffer containing acetate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising acetate at a final concentration of 2 mM to 200 mM acetate, for example, one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM acetate. The buffer may contain sodium acetate.

[0570] In some embodiments, the antigen-binding molecule is provided in a histidine buffer, i.e., a buffer containing histidine ions. In some embodiments, the antigen-binding molecule is provided in a composition containing histidine at a final concentration of 2 mM to 200 mM histidine, for example, one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM histidine.

[0571] In some embodiments, the antigen-binding molecule is provided in a succinate buffer, i.e., a buffer containing succinate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising succinate at a final concentration of 2 mM to 200 mM succinate, for example, one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM succinate. The buffer may contain sodium succinate.

[0572] In some embodiments, the antigen-binding molecule is provided in a sodium phosphate buffer, i.e., a buffer containing sodium ions and phosphate ions. In some embodiments, the antigen-binding molecule is provided in a composition containing sodium phosphate at a final concentration of 2 mM to 200 mM sodium phosphate, for example, one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM sodium phosphate.

[0573] In some embodiments, the antigen-binding molecule is provided in a sodium acetate buffer, i.e., a buffer containing sodium and acetate ions. In some embodiments, the antigen-binding molecule is provided in a composition comprising sodium acetate at a final concentration of 2 mM to 200 mM sodium acetate, for example, one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM sodium acetate.

[0574] In some embodiments, the antigen-binding molecule is provided in an arginine buffer, i.e., a buffer containing arginine ions. In some embodiments, the antigen-binding molecule is provided in a composition containing arginine at a final concentration of 1 mM to 250 mM arginine, for example, one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM. In some embodiments, the composition may contain about 20 mM arginine.

[0575] In some embodiments, the antigen-binding molecule is provided in a histidine-arginine buffer, i.e., a buffer containing histidine ions and arginine ions. In some embodiments, the antigen-binding molecule is provided in a composition containing histidine at a final concentration of 2 mM to 200 mM histidine, e.g., 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 mM to 25 mM, or 18 mM to 22 mM, and arginine at a final concentration of 1 mM to 300 mM arginine, e.g., 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 mM to 175 mM. In some embodiments, the composition may contain about 20 mM histidine and about 150 mM arginine.

[0576] In some embodiments, a composition comprising an antigen-binding molecule comprises an isotonic agent. The isotonic agent can be used to create an isotonic formulation. Examples of isotonic agents include salts (e.g., sodium chloride, potassium chloride) and sugars (e.g., sucrose, glucose, trehalose).

[0577] In some embodiments, the antigen-binding molecule is provided in a composition comprising sodium chloride, i.e., sodium ions and chloride ions. The sodium chloride component of the composition is provided at a final concentration of 1 mM to 250 mM sodium chloride, e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 mM to 175 mM. In some embodiments, the composition may contain about 150 mM sodium chloride.

[0578] In some embodiments, the antigen-binding molecule is provided in a composition comprising methionine. The methionine component of the composition is provided at a final concentration of 1 mM to 250 mM methionine, e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 mM to 175 mM. In some embodiments, the composition may contain about 150 mM methionine.

[0579] In some embodiments, the antigen-binding molecule is provided in a composition comprising sucrose. The sucrose component of the composition is provided at a final concentration (mass per volume) of 2% to 20%, e.g., 2% to 15%, 3% to 12%, or 4% to 10%. In some embodiments, the composition may contain about 2, about 4, about 6, or about 8% (w / v) sucrose. The sucrose component of the composition may be provided at a final concentration of 200 to 300 nM, e.g., about 240 mM.

[0580] In some embodiments, a composition comprising an antigen-binding molecule comprises a surfactant. As used herein, "surfactant" refers to an agent that reduces interfacial tension. The surfactant is preferably a nonionic surfactant. Examples of surfactants include polysorbates (polysorbate 20, polysorbate 80), poloxamers (poloxamer 188), and Triton X-100. The surfactant is preferably present in the composition in a range of about 0.001% (w / v) to about 0.5% (w / v).

[0581] In some embodiments, the antigen-binding molecule is provided in a composition comprising polysorbate 20. The polysorbate 20 component of the composition is provided at a final concentration (by mass per volume) of 0.001% to 0.1%, e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise about 0.02% (w / v) polysorbate 20. In some embodiments, the composition may comprise about 0.05% (w / v) polysorbate 20.

[0582] In some embodiments, the antigen-binding molecule is provided in a composition comprising polysorbate 80. The polysorbate 80 component of the composition is provided at a final concentration (by mass per volume) of 0.001% to 0.1%, e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%. In some embodiments, the composition may comprise about 0.01% (w / v) polysorbate 80. In some embodiments, the composition may comprise about 0.02% (w / v) polysorbate 80.

[0583] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) (w / v) sucrose, more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.5 The composition is provided by the method of claim 1.

[0584] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) (w / v) sucrose, more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.8 The composition is provided by the method of claim 1.

[0585] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 1% to 15%, 2% to 10%, or 3% to 8%) (w / v) sucrose, more preferably about 4% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.8 The composition is provided by the method of claim 1.

[0586] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 0.2% to 20% (e.g., one of 0.5% to 15%, 0.75% to 10%, or 1% to 5%) (w / v) sucrose, more preferably about 2% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.8 The composition is provided by the method of claim 1.

[0587] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) (w / v) sucrose, more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 6.3 The composition is provided by the method of claim 1.

[0588] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) (w / v) sucrose, more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 20, more preferably about 0.02% (w / v) polysorbate 20; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.8 The composition is provided by the method of claim 1.

[0589] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) acetate, e.g., sodium acetate, more preferably about 20 mM acetate; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) (w / v) sucrose, more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.5 The composition is provided by the method of claim 1.

[0590] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) succinate, e.g., sodium succinate, more preferably about 20 mM succinate; 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) (w / v) sucrose, more preferably about 8% (w / v) sucrose; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.5 The composition is provided by the method of claim 1.

[0591] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) (w / v) polysorbate 80, more preferably about 0.02% (w / v) polysorbate 80; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.8 The composition is provided by the method of claim 1.

[0592] In some embodiments, the antigen-binding molecule is 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) histidine, more preferably about 20 mM histidine; 1 mM to 250 mM (e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM) sodium chloride, more preferably about 150 mM sodium chloride; and pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably, pH about 5.8 The composition is provided by the method of claim 1.

[0593] The composition may contain about 0.025 mg / mL to about 100 mg / mL of the antigen-binding molecule. The composition may contain about 0.05 mg / mL to about 80 mg / mL of the antigen-binding molecule. The composition may contain about 0.06 mg / mL to about 70 mg / mL of the antigen-binding molecule. The composition may contain about 0.07 mg / mL to about 60 mg / mL of the antigen-binding molecule. The composition may contain about 0.07 mg / mL to about 50 mg / mL of the antigen-binding molecule.

[0594] The antigen-binding molecule may be formulated in a composition according to the present disclosure at a concentration of, for example, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, or about 70 mg / mL, or any range therein. The antigen-binding molecule may be formulated in a composition according to the present disclosure at a concentration of, for example, about 50 mg / mL.

[0595] As used herein, "about" refers to the specified concentration and concentrations within ±10% of that concentration. For example, a concentration of "about 50 mg / mL" refers to a concentration in the range of 45 mg / mL to 55 mg / mL, inclusive of 50 mg / mL.

[0596] The antigen-binding molecule may, for example, be formulated in a composition according to the present disclosure at a concentration of at least 0.05 mg / mL, at least 0.1 mg / mL, at least 0.15 mg / mL, at least 0.2 mg / mL, at least 0.25 mg / mL, at least 0.3 mg / mL, at least 0.35 mg / mL, at least 0.4 mg / mL, at least 0.5 mg / mL, at least 0.8 mg / mL, at least 1 mg / mL, at least 1.4 mg / mL, at least 2 mg / mL, at least 2.4 mg / mL, at least 4 mg / mL, at least 6 mg / mL, at least 8 mg / mL, at least 10 mg / mL, at least 12 mg / mL, at least 14 mg / mL, or at least 16 mg / mL.

[0597] A 50 mg / mL antigen-binding molecule solution can be diluted using any suitable excipient before administration. In some embodiments, a 50 mg / mL antigen-binding molecule solution is diluted in 5% dextrose for administration. In some embodiments, a 50 mg / mL antigen-binding molecule solution is diluted to a concentration of at least 0.07 mg / mL, at least 0.14 mg / mL, at least 0.21 mg / mL, at least 0.35 mg / mL, at least 0.42 mg / mL, at least 0.8 mg / mL, at least 1.4 mg / mL, or at least 2.4 mg / mL in a volume of, for example, 50 mL for administration. In some embodiments, a 50 mg / mL antigen-binding molecule solution is diluted to a concentration of at least 2.4 mg / mL, at least 4 mg / mL, at least 8 mg / mL, at least 12 mg / mL, or at least 16 mg / mL in a volume of, for example, 100 mL for administration. In some embodiments, the diluted antigen-binding molecule solution is then administered to a subject, e.g., via intravenous administration, e.g., using a dose / dosing regimen according to the present disclosure, e.g., to treat a disease / condition according to the present disclosure. In some embodiments, the diluted antigen-binding molecule solution is administered to a subject within 48 hours of the initial dilution step.

[0598] For example, using the dose / dosage regimens described herein, an antigen-binding molecule according to the present disclosure may be administered in combination with an agent capable of inhibiting PD-1-mediated signaling. The agent capable of inhibiting PD-1-mediated signaling may be a PD-1 or PD-L1 targeting agent. The agent capable of inhibiting PD-1-mediated signaling may be, for example, an antibody capable of binding to PD-1 or PD-L1 and inhibiting PD-1-mediated signaling. In some embodiments, the agent is an antagonistic anti-PD-1 antibody. In some embodiments, the agent is an antagonistic anti-PD-L1 antibody. In some embodiments, the agent is pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). Such medications may be prepared and administered according to the medication preparation instructions provided with the medication(s). Therapeutic and prophylactic applications The antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells, and compositions described herein are used in therapeutic and prophylactic methods.

[0599] The present invention provides an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein for use in a method of medical treatment or prevention. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition. Also provided is a method of treating or preventing a disease or condition, comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid(s), expression vector(s), cell, or composition described herein. All of the therapeutic and prophylactic methods / uses described herein can be performed on a subject.

[0600] The method may be effective in reducing the onset or progression of the disease / condition, alleviating the symptoms of the disease / condition, or reducing the pathology of the disease / condition. The method may be effective in preventing the progression of the disease / condition, e.g., preventing the worsening of the disease / condition or slowing the rate of onset of the disease / condition. In some embodiments, the method may result in an improvement of the disease / condition, e.g., a reduction in the symptoms of the disease / condition, or a reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the method may prevent the onset of a later stage of the disease / condition (e.g., chronic or metastatic).

[0601] It will be appreciated that the articles of the present invention can be used to treat / prevent any disease / condition that derives therapeutic or prophylactic benefit from a reduction in the number and / or activity of VISTA-expressing cells (e.g., MDSCs). It will also be apparent that the therapeutic and prophylactic utility of the present invention extends to essentially any disease / condition that would benefit from a reduction in the number or activity of MDSCs and / or other VISTA-expressing cells, such as tumor-associated macrophages (TAMs) and neutrophils. Antagonism to VISTA effectively relieves effector immune cells from suppression by MDSCs and / or other VISTA-expressing cells.

[0602] For example, the disease / condition may be one in which cells expressing VISTA (e.g., MDSC) are pathologically involved, for example, one in which an increase in the number / proportion of cells expressing VISTA (e.g., MDSC) is positively associated with the occurrence, development, or progression of the disease / condition, and / or the severity of one or more symptoms of the disease / condition, or one in which an increase in the number / proportion of cells expressing VISTA (e.g., MDSC) is a risk factor for the occurrence, development, or progression of the disease / condition.

[0603] In some embodiments, the disease / condition treated / prevented in accordance with the present invention is a disease / condition characterized by an increase in the number / proportion / activity of cells expressing VISTA (e.g., MDSC), e.g., compared to the number / proportion / activity of cells expressing VISTA (e.g., MDSC) in the absence of the disease / condition.

[0604] In some embodiments, a subject may be selected for a treatment described herein based on, for example, detection of an increase in the number / proportion / activity of VISTA-expressing cells (e.g., MDSCs) in the periphery or in an organ / tissue affected by the disease / condition (e.g., an organ / tissue where symptoms of the disease / condition are manifested), or may be selected for this by the presence of VISTA-expressing cells (e.g., MDSCs or tumor-associated macrophages) within a tumor. The disease / condition may affect any tissue or organ or organ system. In some embodiments, the disease / condition may affect several tissues / organs / organ systems.

[0605] In some embodiments, a subject may be selected for treatment / prevention in accordance with the present invention based on a determination that the subject has an increased number / proportion / activity of VISTA-expressing cells (e.g., MDSCs) in the periphery or organ / tissue compared to the number / proportion / activity of such cells in a healthy subject, or based on a determination that the subject has a tumor containing VISTA-expressing cells (e.g., MDSCs).

[0606] In some embodiments, the disease / condition to be treated / prevented is cancer.

[0607] It will be understood that the antigen-binding molecules of the present invention are useful for relieving effector immune cells from MDSC-mediated suppression or suppression by cells expressing VISTA, thereby enhancing anti-cancer immune responses, and therefore are useful in the treatment of cancer in general.

[0608] Cancer can be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm, or tumor. Cancer can be benign or malignant, primary, or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. The cancer can be, for example, a cancer of tissue / cells derived from the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gallbladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblast, jaw, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissue, spleen, stomach, testis, thymus, thyroid, tongue, tonsils, trachea, uterus, vulva, and / or leukocytes.

[0609] The tumors to be treated may be nervous system tumors or non-nervous system tumors. Nervous system tumors, such as glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, neurofibrosarcoma, astrocytoma, and oligodendroglioma, may originate from the central nervous system or the peripheral nervous system. Non-nervous system cancers / tumors may originate from any other non-nervous tissue, including melanoma, mesothelioma, lymphoma, myeloma, leukemia, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), liver cancer, epidermoid carcinoma, prostate cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, pancreatic cancer, thymic cancer, NSCLC, blood cancer, and sarcoma.

[0610] MDSCs are elevated in advanced colorectal cancer (Toor et al., Front Immunol., 2016, 7:560). MDSCs are also observed in breast cancer, and the percentage of MDSCs in peripheral blood is increased in patients with late-stage breast cancer (Markowitz et al., Breast Cancer Res Treat., July 2013, 140(1):13-21). MDSC abundance also correlates with poor prognosis in solid tumors (Charoentong et al., Cell Rep., January 3, 2017, 18(1):248-262), and MDSCs are enriched in liver cancer models (Connolly et al., J Leukoc Biol. (2010), 87(4):713-25). Prostate and breast cancer, melanoma, colorectal cancer, and Lewis lung cancer have been reported to produce chemokines that attract MDSCs and contribute to immunosuppression (Umansky et al., Vaccines (Basel) (2016), 4(4):36), and MDSCs in pancreatic cancer patients are positively correlated with tumor burden (Xu et al., Hepatobiliary Pancreat Dis Int. (2016), 15(1):99-105). VISTA has also been reported to be a target for the treatment of ovarian cancer (see, e.g., US9,631,018B2) and lymphoma (see, e.g., WO2017 / 023749A1).

[0611] Blando et al., Proc Natl Acad Sci USA. (2019), 116(5):1692-1697, recently reported significant infiltration of VISTA-expressing myeloid cells into pancreatic cancer, and in prostate cancer, expansion of VISTA-expressing myeloid cells was observed after treatment with a CTLA4 antagonist, and in melanoma, expansion of VISTA-expressing myeloid cells was observed both before and after treatment with a PD-L1 antagonist.

[0612] In some embodiments, the cancer is a cancer comprising cells expressing VISTA, a cancer comprising infiltration of cells expressing VISTA, a cancer comprising cancer cells expressing VISTA, a ...

Claims

1. A composition comprising an antigen-binding molecule capable of binding to VISTA and inhibiting VISTA-mediated signaling, wherein the antigen-binding molecule is one of the following: (a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 331; and (b) a light chain comprising the amino acid sequence of SEQ ID NO:

317. Including, The composition further comprises: (i) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.5; or (ii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.8; or (iii) 20 mM histidine, 4% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.8; or (iv) 20 mM histidine, 2% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.8; or (v) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 6.3; or (vi) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 20, pH 5.8; or (vii) 20 mM acetate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.5; or (viii) 20 mM succinate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, pH 5.5; or (ix) 20 mM histidine, 0.02% (w / v) polysorbate 80, pH 5.8; or (x) The composition comprising 20 mM histidine, 150 mM sodium chloride, and having a pH of 5.

8.

2. 2. The composition of claim 1, comprising 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate 80, and having a pH of 5.

5.

3. 10. The composition of claim 1, comprising about 50 mg / mL of the antigen-binding molecule.

4. A pharmaceutical for treating or preventing cancer in a subject, comprising the composition of any one of claims 1 to 3 as an active ingredient.

5. The pharmaceutical composition of claim 4, wherein the cancer is characterized by the presence of cells expressing VISTA and / or signal transduction mediated by a complex containing VISTA.

6. 5. The medicament of claim 4, wherein the cancer is selected from blood cancer, leukemia (e.g., T-cell leukemia), acute myeloid leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, multiple myeloma, mesothelioma, solid tumor, lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, gastric cancer, colorectal cancer, colorectal adenocarcinoma, uterine cancer, endometrial cancer, breast cancer, triple-negative breast cancer (TBNC), triple-negative invasive breast cancer, invasive ductal carcinoma, liver cancer, hepatocellular carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, thyroid cancer, thymoma, skin cancer, melanoma, cutaneous melanoma, renal cancer, renal cell carcinoma, papillary renal cell carcinoma, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian serous cystadenocarcinoma, bladder cancer, prostate cancer, and / or prostate cancer.

7. The pharmaceutical composition of claim 4, wherein the cancer is triple-negative breast cancer (TBNC), non-small cell lung carcinoma (NSCLC), and / or a solid tumor.

8. The pharmaceutical described in claim 4, wherein treating or preventing cancer in a subject includes detecting the presence of cells expressing VISTA and / or signal transduction mediated by a complex containing VISTA.

9. The pharmaceutical agent of claim 8, wherein the subject is selected for treatment with the antigen-binding molecule or composition when the presence of cells expressing VISTA and / or signaling mediated by a complex comprising VISTA is detected.

Images