Treatment and prevention of cancer using VISTA antigen binding molecules

Abstract

Disclosed are VISTA antigen binding molecules. Also disclosed are nucleic acids and expression vectors encoding the VISTA antigen binding molecules, compositions including the VISTA antigen binding molecules, and methods of using the VISTA antigen binding molecules.

Description

[0001] This application claims the priority of U.S. Application No. 63 / 409,003, filed on September 22, 2022, the content and elements of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to the field of molecular biology, and more particularly to antibody technology and medical and prophylactic methods.

[0003] Background

[0004] Myeloid-derived suppressor cell (MDSC)-mediated suppression of immune responses has been found in a variety of solid tumors and lymphomas. MDSCs are elevated in advanced colorectal cancer (Toor et al., Front Immunol. 2016; 7:560). MDSCs can also be observed in breast cancer, and the proportion of MDSCs in the peripheral blood of patients with advanced breast cancer increases (Markowitz et al., Breast Cancer Res Treat. July 2013; 140(1):13 - 21). The abundance of MDSCs is also associated with poor prognosis of solid tumors (Charoentong et al., Cell Rep. January 3, 2017; 18(1):248 - 262).

[0005] MDSCs inhibit T cells through multiple mechanisms, including the production of reactive oxygen species, nitric oxide, and arginase. These mechanisms ultimately inhibit the activities of DCs, NK cells, and T cells, increasing tumor burden (Umansky et al., Vaccines (Basel) (2016) 4(4):36). MDSCs also promote angiogenesis, invasion, proliferation, and metastasis by producing soluble factors such as matrix metalloproteinases, VEGF, bFGF, TGF-β, and S100A8 / A9, thereby leading to tumor development and metastasis.

[0006] V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is an immune checkpoint molecule mainly expressed on MDSCs, and targeting VISTA is an attractive therapeutic strategy for eliminating MDSC-mediated suppression of effector immune cell function.

[0007] WO 2017 / 137830 A1 discloses the anti-VISTA antibody VSTB174, for example, it is disclosed in paragraph

[00221] that this antibody comprises the variable regions of the anti-VISTA antibody VSTB112. Paragraph

[00362] discloses that VSTB123 comprises the variable regions of VSTB174. Paragraph

[0417] of Example 25 of WO 2017 / 137830 A1 and Figure 42A disclose that in the MB49 tumor model, the mIgG2a antibody VSTB123 can inhibit tumor growth. Paragraph

[0418] and Figure 42A indicate that, in contrast, VSTB124 (the same antibody provided in the IgG2a LALA form) cannot inhibit tumor growth (see paragraph

[0408] ). Based on these results, the conclusion drawn in paragraph

[0419] of Example 25 is that the efficacy of anti-VISTA antibody therapy may require active Fc. Therefore, the proposed mechanism of action of the anti-VISTA antibody is schematically represented in Figure 47 (see the legend of Figure 47 in paragraph

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

[0008] Le Mercier et al., "Cancer Res." (2014) 74(7):1933 - 44 disclose the hamster monoclonal anti-VISTA antibody mAb13F3, which inhibits tumor growth in the B16OVA and B16 - BL6 melanoma models. The paragraph spanning the left and right columns on page 1942 states that the immunogenicity and FcR binding activity of VISTA mAbs may be key limiting factors for achieving optimal target neutralization and efficacy. VISTA-binding antibodies are also disclosed in, for example, WO 2019 / 185879 A1.

[0009] Overview

[0010] In a first aspect, the present disclosure provides an antigen-binding molecule that binds to VISTA for use in a method of treating or preventing cancer in a subject, wherein the treatment or prevention comprises:

[0011] (i) increasing the number and / or proportion of antigen-specific CD8+ T cells;

[0012] (ii) increasing CD8+ T cell activity;

[0013] (iii) reducing the level of T cell exhaustion;

[0014] (iv) reducing the number and / or proportion of tumour-associated macrophages (TAM);

[0015] (v) increasing the number and / or proportion of M1 macrophages; and / or

[0016] (vi) Increase the activity of M1 macrophages.

[0017] The present disclosure also provides the use of an antigen-binding molecule that binds to VISTA in the preparation of a medicament for treating or preventing cancer in a subject, wherein the treatment or prevention includes:

[0018] (i) Increase the number and / or proportion of antigen-specific CD8+ T cells;

[0019] (ii) Increase the activity of CD8+ T cells;

[0020] (iii) Reduce the level of T cell exhaustion;

[0021] (iv) Reduce the number and / or proportion of tumor-associated macrophages (TAMs);

[0022] (v) Increase the number and / or proportion of M1 macrophages; and / or

[0023] (vi) Increase the activity of M1 macrophages.

[0024] The present disclosure also provides a method for treating or preventing cancer in a subject, wherein the method includes administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule that binds to VISTA, wherein the treatment or prevention includes:

[0025] (i) Increase the number and / or proportion of antigen-specific CD8+ T cells;

[0026] (ii) Increase the activity of CD8+ T cells;

[0027] (iii) Reduce the level of T cell exhaustion;

[0028] (iv) Reduce the number and / or proportion of tumor-associated macrophages (TAMs);

[0029] (v) Increase the number and / or proportion of M1 macrophages; and / or

[0030] (vi) Increase the activity of M1 macrophages.

[0031] In some embodiments, the cancer includes a tumor containing cells that express VISTA.

[0032] The present disclosure also provides a method for selecting a subject to be treated with an antigen-binding molecule that binds to VISTA, including:

[0033] (a) Analyze the cancer of the subject to determine whether the cancer has the following characteristics:

[0034] (i) a low number and / or proportion of antigen-specific CD8+ T cells;

[0035] (ii) low CD8+ T cell activity;

[0036] (iii) a high presence and / or level of exhausted T cells;

[0037] (iv) a high presence and / or number and / or proportion of TAMs;

[0038] (v) a low number and / or proportion of M1 macrophages; and / or

[0039] (vi) low M1 macrophage activity; and

[0040] (b) When it is determined in step (a) that the cancer of the subject has one or more of (i) to (vi), select the subject for treatment with an antigen-binding molecule that binds to VISTA.

[0041] The present disclosure also provides a method for determining a patient's response to treatment with an antigen-binding molecule that binds to VISTA, comprising:

[0042] (a) Analyze the cancer of the subject at a first time point to determine:

[0043] (i) the number and / or proportion of antigen-specific CD8+ T cells;

[0044] (ii) the activity of CD8+ T cells;

[0045] (iii) the level of exhausted T cells;

[0046] (iv) the number and / or proportion of tumor-associated macrophages (TAMs);

[0047] (v) the number and / or proportion of M1 macrophages; and / or

[0048] (vi) the activity of M1 macrophages;

[0049] (b) Analyze the cancer of the subject at a subsequent time point to determine one or more of (i) to (vi); and

[0050] (c) Determine the difference between (a) and (b), wherein:

[0051] (i) the number and / or proportion of antigen-specific CD8+ T cells is increased;

[0052] (ii) the activity of CD8+ T cells is increased;

[0053] (iii) the level of exhausted T cells is decreased;

[0054] (iv) a decrease in the number and / or proportion of tumor-associated macrophages (TAMs);

[0055] (v) an increase in the number and / or proportion of M1 macrophages; and / or

[0056] (vi) an increase in the activity of M1 macrophages,

[0057] The values in (b) represent a positive response to treatment with an antigen-binding molecule that binds to VISTA, compared to (a).

[0058] In some embodiments, the antigen-binding molecule comprises:

[0059] (i) a heavy-chain variable (VH) region comprising the following CDRs:

[0060] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:305

[0061] HC-CDR2 having the amino acid sequence shown in SEQ ID NO:306

[0062] HC-CDR3 having the amino acid sequence shown in SEQ ID NO:307; and

[0063] (ii) a light-chain variable (VL) region comprising the following CDRs:

[0064] LC-CDR1 having the amino acid sequence shown in SEQ ID NO:41

[0065] LC-CDR2 having the amino acid sequence shown in SEQ ID NO:308

[0066] LC-CDR3 having the amino acid sequence shown in SEQ ID NO:43.

[0067] In some embodiments, the antigen-binding molecule comprises:

[0068] (i) a heavy-chain variable (VH) region comprising the following CDRs:

[0069] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:290

[0070] HC-CDR2 having the amino acid sequence shown in SEQ ID NO:291

[0071] HC-CDR3 having the amino acid sequence shown in SEQ ID NO:278; and

[0072] (ii) a light-chain variable (VL) region comprising the following CDRs:

[0073] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0074] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 295

[0075] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43.

[0076] In some embodiments, the antigen-binding molecule comprises:

[0077] a VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289; and

[0078] a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 297.

[0079] In some embodiments, the antigen-binding molecule comprises:

[0080] a VH region comprising the following framework regions (FR):

[0081] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 63

[0082] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 292

[0083] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 293

[0084] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 281.

[0085] In some embodiments, the antigen-binding molecule comprises:

[0086] a VL region comprising the following framework regions (FR):

[0087] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 288

[0088] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 298

[0089] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 284

[0090] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47.

[0091] In some embodiments, the antigen-binding molecule comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 331.

[0092] In some embodiments, the antigen-binding molecule comprises a light chain having the amino acid sequence shown in SEQ ID NO: 317.

[0093] In some embodiments, the cancer is selected from: hematological malignancies, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, multiple myeloma, mesothelioma, epithelioid mesothelioma, solid tumors, lung cancer, non-small cell lung cancer, gastric cancer, gastric malignancy, colorectal cancer, colorectal tumor, colorectal adenocarcinoma, uterine cancer, endometrial cancer, breast cancer, triple-negative breast cancer, triple-negative invasive breast cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, thyroid cancer, thymoma, skin cancer, melanoma, cutaneous melanoma, kidney cancer, renal cell carcinoma, renal papillary cell carcinoma, head and neck cancer, head and neck squamous cell carcinoma (SCCHN), ovarian cancer, ovarian tumor, ovarian serous cystadenocarcinoma, prostate cancer, and / or prostatic adenocarcinoma.

[0094] In some embodiments, the cancer is selected from: colorectal cancer, pancreatic cancer, breast cancer, triple-negative breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, leukemia, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC), and solid tumors.

[0095] In some embodiments, the cancer is epithelioid mesothelioma.

[0096] Description

[0097] The present disclosure relates to VISTA-binding molecules that can alter the tumor microenvironment.

[0098] Aspects and embodiments of the present disclosure particularly relate to antigen-binding molecules that bind to VISTA and affect tumor microenvironment remodeling. Such antigen-binding molecules can be used for the treatment / prevention of cancer

[0099] VISTA, Interaction Partners, and VISTA-Mediated Signaling

[0100] T cell activation inhibitory factor containing V-type immunoglobulin domain (VISTA; also known as B7-H5, SISP1, PD-1H) is a protein identified by UniProt Q9H7M9 and has the amino acid sequence shown in SEQ ID NO:1 (Q9H7M9-1, v3). The structure and function of VISTA are as described by Lines et al. in "Cancer Res." (2014) 74(7):1924-1932, the entire content of which is incorporated herein by reference. VISTA is a single-pass type I transmembrane protein of approximately 50 kDa with immune checkpoint function and is encoded by the C10orf54 gene. The extracellular domain of VISTA is homologous to PD-L1.

[0101] The N-terminal 32 amino acids of SEQ ID NO:1 constitute a signal peptide. 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. The 33rd to 194th positions of SEQ ID NO:1 form the extracellular domain (SEQ ID NO:3), the 195th to 215th positions form the transmembrane domain (SEQ ID NO:4), and the 216th to 311th positions form the cytoplasmic domain (SEQ ID NO:5). The extracellular domain includes an Ig-like V-type domain (the 33rd to 168th positions of SEQ ID NO:1, shown as SEQ ID NO:6).

[0102] In this specification, "VISTA" refers to VISTA from any species, including VISTA isotypes, fragments, variants (including mutants), or homologs from any species.

[0103] As used herein, "fragment", "variant", "homolog" 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 (such as a reference isotype). In some embodiments, fragments, variants, isotypes, and homologs of a reference protein may be characterized by the ability to perform the functions performed by the reference protein.

[0104] "Fragment" generally refers to a part of a reference protein. "Variant" generally refers to a protein whose amino acid sequence has one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of the reference protein, but retains a substantial degree of sequence identity (such as at least 60%) with the amino acid sequence of the reference protein. "Isotype" generally refers to a variant of the reference protein expressed by the same species as the reference protein. "Homolog" generally refers to a variant of the reference protein produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0105] A "fragment" can be of any length (counted by the 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 any of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length of the reference protein. A VISTA fragment can have a minimum length of any of 10, 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids, and can have a maximum length of any of 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids.

[0106] In some embodiments, the VISTA is a VISTA from a mammal (such as a VISTA from a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) and / or a rodent (such as a rat or mouse)). An isotype, fragment, variant or homolog of VISTA can optionally be characterized as having at least 70%, preferably any of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence of an immature or mature VISTA isotype of a particular species (such as human).

[0107] An isotype, fragment, variant or homolog can optionally be a functional isotype, fragment, variant or homolog, such as, determined by appropriate assay analysis of functional properties / activities, having the functional properties / activities of the reference VISTA. For example, an isotype, fragment, variant or homolog of VISTA can show an association with LRIG1, VSIG3, PSGL-1 and / or VSIG8.

[0108] In some embodiments, the 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, the VISTA fragment 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 any one of SEQ ID NO:2, 3 or 6.

[0109] VISTA is a member of the B7 protein family and is mainly expressed by leukocytes, especially CD14+ monocytes (including monocyte-derived suppressor cells (MDSC)) and CD33+ myeloid cells. CD56+ NK cells, dendritic cells, and a small number of CD4+ and CD8+ T cells also express VISTA. VISTA is highly expressed on MDSC (especially tumor-infiltrating MDSC), tumor-infiltrating myeloid DC (LeMercier et al., Cancer Res. (2014) 74(7):1933-44), and tumor-associated macrophages (TAM) and neutrophils.

[0110] Evidence has shown that VISTA on T cells can act as both a ligand and a receptor, thereby inhibiting T cell effector functions and maintaining peripheral tolerance; edited tumors overexpressing VISTA can evade immune control and grow faster than tumors not overexpressing VISTA (Wang et al., J. Exp. Med. (2011) 208(3):577–92; Lines et al., Cancer Res. (2014) 74(7):1924-1932). Studies have shown that VISTA is a co-inhibitory receptor for CD4+ T cells or a co-inhibitory ligand for T cells. It has been reported that compared with wild-type CD4+ T cells, VISTA - / - CD4+ T cells showed stronger antigen-specific proliferation and cytokine production capabilities, indicating that VISTA functions as an inhibitory receptor on CD4+ T cells. Blocking VISTA function using monoclonal anti-VISTA antibodies has been shown to enhance the infiltration, proliferation, and effector functions of tumor-reactive T cells in the tumor microenvironment (Le Mercier et al., Cancer Res. (2014) 74(7):1933-4).

[0111] VISTA has been proposed to interact with VSIG3 (IGSF11), see, e.g., as described in Wang et al., J Immunol (2017), 198 (Suppl 1) 154.1, the entire content of which is incorporated herein by reference. VSIG3 binds to activated T cells via VISTA and can inhibit T cell proliferation and reduce the production of cytokines and chemokines such as IFN-γ, IL-2, IL-17, CCL5 / RANTES, CCL3 / MIP-1a, and CXCL11 / I-TAC.

[0112] VSIG3 is a protein identified by UniProt Q5DX21. Alternative splicing of the mRNA encoded by the human IGSF11 gene gives rise to 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 different from SEQ ID NO:7 at positions 1 to 17; and isoform 3 (UniProt: Q5DX21-3; SEQ ID NO:9), which contains a sequence different from SEQ ID NO:7 at positions 1 to 17 and also contains a sequence different from SEQ ID NO:7 at positions 211 to 235.

[0113] The N-terminal 22 amino acids of SEQ ID NOs: 7, 8, and 9 constitute a signal peptide. Thus, the mature forms of VSIG3 isoforms 1, 2, and 3 (i.e., after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively. The amino acids at positions 23 to 241 of SEQ ID NOs: 7 and 8 constitute the extracellular domain of VSIG3 isoforms 1 and 2 (SEQ ID NO:13), and the amino acids at positions 23 to 216 of SEQ ID NO:9 constitute the extracellular domain of VSIG3 isoform 3 (SEQ ID NO:14). The transmembrane domain of VSIG3 is shown in SEQ ID NO:15, and the cytoplasmic domain is shown in SEQ ID NO:16. The extracellular domain includes an Ig-like V-type domain (shown in SEQ ID NO:17), and the extracellular domain of VSIG3 isoforms 1 and 2 also includes an Ig-like C2-type domain (shown in SEQ ID NO:18).

[0114] In this specification, "VSIG3" refers to VSIG3 from any species, including VSIG3 isoforms, fragments, variants (including mutants), or homologs from any species.

[0115] The VSIG3 fragment can have a minimum length of any one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids and can have a maximum length of any one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0116] In some embodiments, the VSIG3 is a VSIG3 from a mammal (such as a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or a rodent (such as a rat or mouse)). The isotype, fragment, variant, or homolog of VSIG3 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 VSIG3 isotype of a particular species (such as a human).

[0117] The isotype, fragment, variant, or homolog can optionally be a functional isotype, fragment, variant, or homolog, such as having the functional properties / activities of a reference VSIG3 as determined by appropriate assay analysis of the functional properties / activities. For example, the isotype, fragment, variant, or homolog of VSIG3 can show an association with VISTA.

[0118] In some embodiments, the VSIG3 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 any one of SEQ ID NOs: 7 to 12. In some embodiments, the VSIG3 fragment 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 any one of SEQ ID NOs: 10 to 14, 17, or 18.

[0119] VISTA has been proposed to interact with VSIG8, see, e.g., WO / 2016 / 090347A1. The VSIG8 protein is the protein identified by UniProt P0DPA2 (SEQ ID NO:19). The N-terminal 21 amino acids of SEQ ID NO:19 constitute a signal peptide. Thus, the mature form of VSIG8 (i.e., after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO:20. Amino acids 22 to 263 of SEQ ID NO:19 constitute the extracellular domain of VSIG8 (SEQ ID NO:21). The transmembrane domain of VSIG8 is shown in SEQ ID NO:22, and the cytoplasmic domain is shown in SEQ ID NO:23. The extracellular domain includes Ig-like V-type domain 1 (shown in SEQ ID NO:24) and Ig-like V-type domain 2 (shown in SEQ ID NO:25).

[0120] In this specification, "VSIG8" refers to VSIG8 from any species, including VSIG8 isotypes, fragments, variants (including mutants) or homologs from any species.

[0121] A VSIG8 fragment can have a minimum length of any one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids, and can have a maximum length of any one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids.

[0122] In some embodiments, the VSIG8 is VSIG8 from a mammal (such as a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) and / or a rodent (such as a rat or mouse)). An isotype, fragment, variant or homolog of VSIG8 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 VSIG8 isotype of a particular species (such as human).

[0123] An isotype, fragment, variant or homolog can optionally be a functional isotype, fragment, variant or homolog, e.g., as determined by appropriate assay analysis of functional properties / activities, having the functional properties / activities of a reference VSIG8. For example, an isotype, fragment, variant or homolog of VSIG8 can show an association with VISTA.

[0124] In some embodiments, the VSIG8 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, the VSIG8 fragment 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 any one of SEQ ID NO:20, 21, 24 or 25.

[0125] VISTA has been proposed to interact with PSGL-1, see, e.g., WO 2018 / 132476 A1. Johnston et al. in Nature (2019) 574:565-570 disclose that PSGL-1 binds to VISTA through interactions involving positions Y46, Y48, Y51, E56 and T57 of PSGL-1 and positions H98, H100, H153, H154 and H155 of VISTA.

[0126] PSGL-1 isoform 1 is the protein identified by UniProt Q14242-1 (SEQ ID NO:323). PSGL-1 isoform 2 is the protein identified by UniProt Q14242-2 (SEQ ID NO:324), which differs from PSGL-1 isoform 1 in that it contains an additional 16 amino acids after position 1 of SEQ ID NO:323.

[0127] The N-terminal 17 amino acids of SEQ ID NO:323 constitute a signal peptide. Thus, the mature form of PSGL-1 (i.e., after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO:325. Positions 18 to 320 of SEQ ID NO:323 constitute the extracellular domain of PSGL-1 (SEQ ID NO:326). The transmembrane domain of PSGL-1 is shown in SEQ ID NO:327 and the cytoplasmic domain is shown in SEQ ID NO:328. The extracellular domain includes tandem repeats of 12, 10 amino acids; the repeat sequence region is shown in SEQ ID NO:329.

[0128] In this specification, "PSGL-1" refers to PSGL-1 from any species, including PSGL-1 isoforms, fragments, variants (including mutants) or homologs from any species.

[0129] The PSGL-1 fragment can have a minimum length of any one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids, and can have a maximum length of any one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids.

[0130] In some embodiments, the PSGL-1 is PSGL-1 from a mammal (such as a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) and / or a rodent (such as a rat or mouse)). The isotype, fragment, variant or homolog of PSGL-1 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 with the amino acid sequence of an immature or mature PSGL-1 isotype of a particular species (such as a human).

[0131] The isotype, fragment, variant or homolog can optionally be a functional isotype, fragment, variant or homolog, e.g., as determined by appropriate assay analysis of functional properties / activities, having the functional properties / activities of a reference PSGL-1. For example, an isotype, fragment, variant or homolog of PSGL-1 may display an association with VISTA.

[0132] In some embodiments, the 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 with SEQ ID NO: 323 or 324. In some embodiments, the PSGL-1 fragment 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 with any one of SEQ ID NO: 325, 326 or 329.

[0133] VISTA has been proposed to interact with LRIG1, see, e.g., WO / 2019 / 165233A1. WO / 2019 / 165233A1 discloses that LRIG1 binds to VISTA through an interaction involving amino acids 245 to 260 of LRIG1 and amino acids 68 to 92 of VISTA.

[0134] LRIG1 isoform 1 is the protein identified by UniProt Q96JA1-1 (SEQ ID NO: 332). LRIG1 isoform 2 is the protein identified by UniProt Q96JA1-2 (SEQ ID NO: 334), which differs from LRIG1 isoform 1 in that it contains an additional 14 amino acids after position 387 of SEQ ID NO: 332 and is substituted with Q at positions 644 to 691 of SEQ ID NO: 332.

[0135] The N-terminal 34 amino acids of SEQ ID NO: 332 constitute the signal peptide. Thus, the mature forms of LRIG1 isoform 1 and 2 (i.e., after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NO: 333 and 335, respectively. The extracellular domain of LRIG1 isoform 1 is shown in SEQ ID NO: 336, and the extracellular domain of LRIG1 isoform 2 is shown in SEQ ID NO: 337. The transmembrane domain of LRIG1 is shown in SEQ ID NO: 338, and the cytoplasmic domain is shown in SEQ ID NO: 339. The extracellular domain includes 15 leucine-rich repeats, followed by three Ig-like domains proximal to the transmembrane domain (see, e.g., Xu et al., J Mol Biol. (2015) 427(10):1934-1948).

[0136] In this specification, "LRIG1" refers to LRIG1 from any species, including LRIG1 isoforms, fragments, variants (including mutants), or homologs from any species.

[0137] An LRIG1 fragment can have a minimum length of any of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, or 700 amino acids and can have a maximum length of any of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, or 700 amino acids.

[0138] In some embodiments, the LRIG1 is LRIG1 from a mammal (such as LRIG1 from a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) and / or a rodent (such as a rat or mouse)). Isoforms, fragments, variants or homologs of LRIG1 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 LRIG1 isoform of a particular species (such as human).

[0139] The isoform, fragment, variant or homolog may optionally be a functional isoform, fragment, variant or homolog, such as, determined by appropriate assay analysis of functional properties / activities, having the functional properties / activities of a reference LRIG1. For example, an isoform, fragment, variant or homolog of LRIG1 may show an association with VISTA.

[0140] In some embodiments, the LRIG1 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: 332, 333, 334 or 335. In some embodiments, the LRIG1 fragment 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 any one of SEQ ID NO: 336 or 337.

[0141] As described in the experimental examples of the present disclosure, both VSIG3 and LRIG1 are considered to bind to VISTA by interacting with the C-C’ loop region of VISTA (the amino acid sequence of which is shown in SEQ ID NO: 344).

[0142] Regions of Particular Interest on Target Molecules

[0143] The antigen-binding molecules of the present disclosure are specifically designed to target regions of VISTA of particular interest. Using a two-step method, the VISTA regions to be targeted are selected after analysis of predicted antigenicity, function and safety. Then, a peptide corresponding to the target region is used as an immunogen to prepare specific antibodies against the VISTA target region to cultivate specific monoclonal antibodies, which are subsequently screened to identify antibodies capable of binding to VISTA in its native state. This method allows precise control of the antibody epitope.

[0144] The antigen-binding molecules of the present disclosure can be defined with reference to the VISTA region to which they bind. The antigen-binding molecules of the present disclosure can bind to a particular region of interest of VISTA. In some embodiments, the antigen-binding molecule can bind to a linear epitope of VISTA, which is composed of a continuous amino acid sequence (i.e., the amino acid primary sequence). In some embodiments, the antigen-binding molecule can bind to a conformational epitope of VISTA, which is composed of discontinuous amino acid sequences of the amino acid sequence.

[0145] In some embodiments, the antigen-binding molecules of the present disclosure bind to VISTA. In some embodiments, the antigen-binding molecule binds to the extracellular region of VISTA (such as the region shown in SEQ ID NO: 3). In some embodiments, the antigen-binding molecule binds to the Ig-like V domain of VISTA (such as the region shown in SEQ ID NO: 6). In some embodiments, the antigen-binding molecule binds to VISTA in a region corresponding to positions 61 to 162 of SEQ ID NO: 1 (such as SEQ ID NO: 31).

[0146] In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 322. In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 26. In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 27. In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 28. In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 29. In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 30.

[0147] In some embodiments, the antigen-binding molecule does not bind to the VISTA region shown in SEQ ID NO: 271. In some embodiments, the antigen-binding molecule does not bind to the VISTA region shown in SEQ ID NO: 272. In some embodiments, the antigen-binding molecule does not bind to the VISTA region shown in SEQ ID NO: 273. In some embodiments, the antigen-binding molecule does not bind to the VISTA region shown in SEQ ID NO: 274. In some embodiments, the antigen-binding molecule does not bind to the VISTA region shown in SEQ ID NO: 275.

[0148] The region where the antibody binds to the peptide / polypeptide can be determined by those skilled in the art using various methods known in the art, including X-ray co-crystallography analysis of the antibody-antigen complex, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange mass spectrometry, phage display, competitive ELISA, and proteolysis-based "protection" methods. These methods are described in Gershoni et al., BioDrugs 2007, 21(3):145-156, the entire content of which is incorporated herein by reference.

[0149] In a preferred embodiment, the region where the antigen-binding molecule according to the present disclosure binds to the peptide / polypeptide is evaluated by hydrogen-deuterium exchange mass spectrometry (HDXMS), for example, as described in the experimental examples of the present disclosure.

[0150] In some embodiments, the antigen-binding molecule binds to the C-C’ region of VISTA. In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO:344. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:344. In some embodiments, the antigen-binding molecule contacts the VISTA region shown in SEQ ID NO:344. In some embodiments, the antigen-binding molecule binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO:344. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:344.

[0151] In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO:340. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:340. In some embodiments, the antigen-binding molecule contacts the VISTA region shown in SEQ ID NO:340. In some embodiments, the antigen-binding molecule binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO:340. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:340.

[0152] In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 341. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 341. In some embodiments, the antigen-binding molecule contacts the VISTA region shown in SEQ ID NO: 341. In some embodiments, the antigen-binding molecule binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO: 341. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 341.

[0153] In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 342. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 342. In some embodiments, the antigen-binding molecule contacts the VISTA region shown in SEQ ID NO: 342. In some embodiments, the antigen-binding molecule binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO: 342. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 342.

[0154] In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO: 341, and / or binds to the VISTA region shown in SEQ ID NO: 342. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 341, and / or binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 342. In some embodiments, the antigen-binding molecule contacts the VISTA region shown in SEQ ID NO: 341, and / or contacts the VISTA region shown in SEQ ID NO: 342. In some embodiments, the antigen-binding molecule binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO: 341, and / or binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO: 342. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO: 341, and / or the amino acid sequence shown in SEQ ID NO: 342.

[0155] In some embodiments, the antigen-binding molecule binds to the VISTA-binding region through a VISTA interaction partner, and the interaction partner binds to the C-C’ region of VISTA (such as LRIG1 or VSIG3). In some embodiments, the antigen-binding molecule binds to the VISTA-binding region through LRIG1. In some embodiments, the antigen-binding molecule binds to the VISTA-binding region through VSIG3.

[0156] In some embodiments, the antigen-binding molecule binds to the VISTA region shown in SEQ ID NO:343. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:343. In some embodiments, the antigen-binding molecule contacts the VISTA region shown in SEQ ID NO:343. In some embodiments, the antigen-binding molecule binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO:343. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:343.

[0157] In some embodiments, the antigen-binding molecule is capable of binding the same or overlapping region of VISTA to the VISTA region, where the VISTA region binds to an antibody comprising the VH and VL sequences of any one of the 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 described herein. In some embodiments, the antigen-binding molecule is capable of binding the same or overlapping region of VISTA to the VISTA region, where the VISTA region binds to an antibody comprising the VH and VL sequences of any one of the antibody clones 4M2-C12, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31. In some embodiments, the antigen-binding molecule is capable of binding the same or overlapping region of VISTA to the VISTA region that binds to an antibody comprising the VH and VL sequences of V4-C26.

[0158] As used herein, "peptide" refers to a chain of two or more amino acid monomers linked by peptide bonds. The length of a peptide is typically in the range of about 2 to 50 amino acids. "Polypeptide" is a polymeric chain of two or more peptides. The length of a polypeptide typically exceeds about 50 amino acids.

[0159] In some embodiments, the antigen-binding molecules of the present disclosure are capable of binding to a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NO: 1, 2, 3, 6, or 31.

[0160] In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence shown in 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 shown in 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 shown in 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 shown in 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 shown in 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 shown in SEQ ID NO: 30.

[0161] In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence shown in 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 shown in 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 shown in 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 shown in 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 shown in SEQ ID NO: 275.

[0162] The ability of an antigen-binding molecule to bind to a specific peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including by ELISA, immunoblotting (such as Western blotting), 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) and other methods.

[0163] In embodiments where 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 ends of the reference amino acid sequence. In some embodiments, the peptide / polypeptide comprises, at one or both ends of the reference amino acid sequence, such as 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids.

[0164] In some embodiments, the additional amino acids provided at one or both ends (i.e., the N-terminus and C-terminus) of the reference sequence correspond to the positions at both ends of the reference sequence in the VISTA amino acid sequence. For example, if the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising the sequence shown in SEQ ID NO:26, and there are two additional amino acids at the C-terminus of SEQ ID NO:26, then these two additional amino acids may be arginine and asparagine, corresponding to positions 90 and 91 of SEQ ID NO:1.

[0165] In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide bound by an antibody comprising the VH and VL sequences of any one of the 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 described herein.

[0166] Myeloid-Derived Suppressor Cells (MDSC)

[0167] Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune cells within the myeloid lineage that have an immunosuppressive phenotype. The physiological properties of MDSC are reviewed in Kumar et al., Trends Immunol. (2016); 37(3):208–220, the entire contents of which are incorporated herein by reference.

[0168] MDSC have a number of biochemical and genomic features that distinguish these cells from mature myeloid cells (i.e., macrophages, dendritic cells, and neutrophils), such as increased expression of NADPH oxidase (Nox2) and increased production of reactive oxygen species (ROS) (such as superoxide anion (O 2-)), increased production of hydrogen peroxide (H2O2) and peroxynitrite (PNT; ONOO - )); increased expression of arginase 1 and nitric oxide synthase 2 (nos2), increased production of nitric oxide (NO); increased expression of c / EBPβ and STAT3; decreased expression of IRF8; increased production of S100A8 / 9 protein.

[0169] 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 in, for example, Marvel and Gabrilovich, J Clin Invest. September 1, 2015; 125(9):3356–3364, the entire content of which is incorporated herein by reference. In mice, MDSCs are generally considered to be CD11b + Gr1 + cells. Gr-1 hi cells are mostly PMN-MDSCs, and Gr-1 lo cells are mostly M-MDSCs. These subsets can be more accurately identified based on the Ly6C and Ly6G markers; M-MDSCs are CD11b + Ly6C hi Ly6G – , and PMN-MDSCs are CD11b + Ly6C lo Ly6G + . In humans, MDSCs are found within monocytes. PMN-MDSCs are CD14 – CD11b + CD33 + CD15 + or CD66b + cells, and M-MDSCs are CD14 + HLA-DR – / lo cells. Lin – HLA-DR – CD33 + The MDSC population represents a heterogeneous cell population enriched in myeloid progenitors.

[0170] Factors related to MDSC-mediated immunosuppression include the expression of arginase (ARG1), inducible NOS (iNOS), TGF-β, IL-10, and COX2, chelation of cysteine, reduction of T cell type I selectin expression, and induction of Tregs. M-MDSC and PMN-MDSC employ different immunosuppressive mechanisms. M-MDSC inhibits antigen-specific and non-specific T cell responses by producing NO and cytokines, and its immunosuppressive effect is stronger than that of PMN-MDSC. PMN-MDSC inhibits the immune response in an antigen-specific manner by producing ROS. From a pathological perspective, MDSC is associated with the development and progression of cancer and infectious diseases. The role of MDSC in human diseases is reviewed in, for example, 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, the entire contents of which are incorporated herein by reference.

[0171] MDSC is abundant in tumor tissues and promotes the development and deterioration of cancer through multiple mechanisms, as reviewed in Umansky et al., Vaccines (Basel) (2016) 4(4):36. MDSC is recruited to the tumor site through the expression of chemokines, and pro-inflammatory factors in the tumor microenvironment lead to a significant upregulation of the immunosuppressive function of MDSC. MDSC causes tumor development, angiogenesis, and metastasis by inhibiting the functions of effector immune cells (such as effector T cell and NK cell functions), promoting the production / activity of regulatory T cells, the production of growth factors such as VEGF and bFGF, and the production of ECM modifying factors such as matrix metalloproteinases.

[0172] The characteristics of MDSC can be referenced by the expression of VISTA. In embodiments of various aspects of the present disclosure, MDSC can be "VISTA-expressing MDSC" or "VISTA+MDSC". MDSC can express VISTA on the cell surface (i.e., VISTA can be expressed within or on the cell membrane).

[0173] Antigen-Binding Molecules

[0174] The present disclosure relates to the therapeutic and prophylactic uses of antigen-binding molecules that bind to VISTA.

[0175] "Antigen-binding molecule" refers to a molecule capable of binding to a target antigen. Antigen-binding molecules include, for example, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (such as bispecific antibodies), and antibody fragments (such as Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single-domain antibodies (such as VhH), etc.), provided that they can bind to the relevant target molecule.

[0176] Antigen-binding molecules according to the present disclosure also include antibody-derived molecules, for example, molecules comprising an antigen-binding region / domain derived from an antibody. Antibody-derived antigen-binding molecules can include an antigen-binding region / domain that comprises or consists of the antigen-binding region of an antibody (such as an antigen-binding fragment of an antibody). In some embodiments, the antigen-binding region / domain of the antibody-derived antigen-binding molecule can be or include the Fv (such as provided in the form of scFv) or Fab region of an antibody, or the whole antibody. For example, antigen-binding molecules according to the present disclosure include antibody-drug conjugates (ADCs) containing a (cytotoxic) drug moiety (as described below). Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules, such as immune cell attracting molecules comprising a domain for recruiting (effector) immune cells (as reviewed in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17:418–434 and Ellerman, Methods (2019) 154:102-117, the entire contents of both are incorporated herein by reference), including BiTEs, BiKEs and TriKEs. Antigen-binding molecules according to the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors having both antigen-binding and T cell activation functions (the structure, function and processing of CARs are reviewed in, for example, Dotti et al., Immunol Rev (2014) 257(1), the entire contents of which are incorporated herein by reference).

[0177] Antigen-binding molecules according to the present disclosure include a portion capable of binding to a target antigen. In some embodiments, the portion capable of binding to a target antigen includes the heavy-chain variable region (VH) and the light-chain variable region (VL) of an antibody that can specifically bind to the target antibody. In some embodiments, the portion capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, such as a nucleic acid aptamer (reviewed in Zhou and Rossi, Nat Rev Drug Discov. 2017;16(3):181-202). In some embodiments, the portion 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, as reviewed in Reverdatto et al., Curr Top Med Chem. 2015;15(12):1082–1101, the entire contents of which are incorporated herein by reference (see also, e.g., Boersma et al., J Biol Chem (2011) 286:41273-85 and Emanuel et al., Mabs (2011) 3:38-48).

[0178] Antigen-binding molecules of the present disclosure generally include an antigen-binding domain containing the VH and VL of an antibody that can specifically bind to a target antigen. The antigen-binding domain formed by VH and VL may also be referred to herein as the Fv region.

[0179] The antigen-binding molecule may be or may include an antigen-binding polypeptide or an antigen-binding polypeptide complex. The antigen-binding molecule may include more than one polypeptide that together form an antigen-binding domain. The polypeptides may be bound covalently or non-covalently. In some embodiments, the polypeptides form part of a larger polypeptide that includes the polypeptides (such as in the case of an scFv that includes VH and VL, or an scFab that includes VH-CH1 and VL-CL).

[0180] The antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (such as 2, 3, 4, 6, or 8 polypeptides), such as an IgG-like antigen-binding molecule that includes two heavy-chain polypeptides and two light-chain polypeptides.

[0181] The antigen-binding molecules of the present disclosure can be designed and prepared using monoclonal antibody (mAb) sequences 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 / provided. An “antigen-binding region” is any fragment of an antibody that is capable of binding to a target specific to the given antibody.

[0182] Antibodies generally include six complementarity-determining regions CDR; 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. The six CDRs together determine the paratope of the antibody, which is the part of the antibody that binds to the target antigen.

[0183] The VH and VL regions include framework regions (FR) flanking each CDR, providing a scaffold for the CDRs. From the N-terminus to the C-terminus, the VH region has the following structure: N-terminus - [HC-FR1] - [HC-CDR1] - [HC-FR2] - [HC-CDR2] - [HC-FR3] - [HC-CDR3] - [HC-FR4] - C-terminus; the VL region has the following structure: N-terminus - [LC-FR1] - [LC-CDR1] - [LC-FR2] - [LC-CDR2] - [LC-FR3] - [LC-CDR3] - [LC-FR4] - C-terminus.

[0184] There are several different conventions for defining antibody CDRs and FRs, such as those described by 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, as described by 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 are defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described by Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0185] 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 and VL regions of an antigen-binding molecule capable of binding to VISTA.

[0186] 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 the VISTA-binding antibody clones described herein (i.e., 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).

[0187] In some embodiments, the antigen-binding molecule comprises any one of the following VH regions (1) to (18):

[0188] (1) (4M2-C12-derived consensus sequence) A VH region comprising the following CDRs:

[0189] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 305

[0190] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 306

[0191] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 307,

[0192] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0193] (2) (V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31) A VH region comprising the following CDRs:

[0194] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 290

[0195] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 291

[0196] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 278,

[0197] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0198] (3) (V4-C1) The VH region comprising the following CDRs:

[0199] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33

[0200] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 277

[0201] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 278,

[0202] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0203] (4) (V4-C9) The VH region comprising the following CDRs:

[0204] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33

[0205] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 286

[0206] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 278,

[0207] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0208] (5) (4M2-C12 / V4H1 / V4H2 consensus sequence) The VH region comprising the following CDRs:

[0209] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 244

[0210] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 34

[0211] An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 35,

[0212] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0213] (6) (4M2-C12, 4M2-B4, V4H2) comprises a VH region having the following CDRs:

[0214] An HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33

[0215] An HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 34

[0216] An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 35,

[0217] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0218] (7) (V4H1) comprises a VH region having the following CDRs:

[0219] An HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 53

[0220] An HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 34

[0221] An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 35,

[0222] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0223] (8) (2M1-B12, 2M1-D2) comprises a VH region having the following CDRs:

[0224] An HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 72

[0225] An HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 73

[0226] An HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 74,

[0227] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0228] (9) (4M2-C9, 5M1-A11) comprises a VH region having the following CDRs:

[0229] HC-CDR1 having the amino acid sequence as shown in SEQ ID NO:88

[0230] HC-CDR2 having the amino acid sequence as shown in SEQ ID NO:89

[0231] HC-CDR3 having the amino acid sequence as shown in SEQ ID NO:90,

[0232] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0233] (10) (4M2-D9) comprises a VH region having the following CDRs:

[0234] HC-CDR1 having the amino acid sequence as shown in SEQ ID NO:33

[0235] HC-CDR2 having the amino acid sequence as shown in SEQ ID NO:107

[0236] HC-CDR3 having the amino acid sequence as shown in SEQ ID NO:108,

[0237] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0238] (11) (1M2-D2) comprises a VH region having the following CDRs:

[0239] HC-CDR1 having the amino acid sequence as shown in SEQ ID NO:120

[0240] HC-CDR2 having the amino acid sequence as shown in SEQ ID NO:121

[0241] HC-CDR3 having the amino acid sequence as shown in SEQ ID NO:122,

[0242] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0243] (12) (4M2-D5) comprises the VH region of the following CDRs:

[0244] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 144

[0245] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 145

[0246] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 146,

[0247] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0248] (13) (4M2-A8) comprises the VH region of the following CDRs:

[0249] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 158

[0250] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 159

[0251] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 160,

[0252] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0253] (14) (9M2-C12) comprises the VH region of the following CDRs:

[0254] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 169

[0255] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 170

[0256] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 171,

[0257] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0258] (15) (derived from 13D5) comprises the VH region of the following CDRs:

[0259] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:72

[0260] HC-CDR2 having the amino acid sequence shown in SEQ ID NO:184

[0261] HC-CDR3 having the amino acid sequence shown in SEQ ID NO:246,

[0262] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0263] (16)(13D5p) comprises a VH region having the following CDRs:

[0264] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:72

[0265] HC-CDR2 having the amino acid sequence shown in SEQ ID NO:184

[0266] HC-CDR3 having the amino acid sequence shown in SEQ ID NO:185,

[0267] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0268] (17)(13D5-1) comprises a VH region having the following CDRs:

[0269] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:72

[0270] HC-CDR2 having the amino acid sequence shown in SEQ ID NO:184

[0271] HC-CDR3 having the amino acid sequence shown in SEQ ID NO:195,

[0272] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0273] (18)(13D5-13) comprises a VH region having the following CDRs:

[0274] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:72

[0275] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 184

[0276] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 200,

[0277] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid.

[0278] In some embodiments, the antigen-binding molecule comprises any of the VH regions in (19) to (35) below:

[0279] (19) (V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31) VH regions comprising the following FRs:

[0280] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 63

[0281] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 292

[0282] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 293

[0283] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 281,

[0284] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0285] (20) (V4-C1, V4-C9) VH regions comprising the following FRs:

[0286] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 63

[0287] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 279

[0288] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 280

[0289] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 281,

[0290] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0291] (21) (4M2-C12) comprises the VH region of the following FR:

[0292] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 36

[0293] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 37

[0294] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 38

[0295] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 39,

[0296] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0297] (22) (4M2-B4) comprises the VH region of the following FR:

[0298] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 49

[0299] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 37

[0300] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 38

[0301] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 39,

[0302] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0303] (23) (V4H1) comprises the VH region of the following FR:

[0304] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 54

[0305] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 55

[0306] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 56

[0307] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 39

[0308] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0309] (24) (V4H2) comprises the VH region of the following FRs:

[0310] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 63

[0311] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 64

[0312] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 65

[0313] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 39

[0314] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0315] (25) (2M1-B12) comprises the VH region of the following FRs:

[0316] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 75

[0317] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 76

[0318] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 77

[0319] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 78

[0320] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0321] (26) (4M2-C9) comprises the VH region of the following FRs:

[0322] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 91

[0323] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 92

[0324] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 93

[0325] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 94,

[0326] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0327] (27) (2M1-D2) comprises the VH region of the following FRs:

[0328] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 103

[0329] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 76

[0330] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 77

[0331] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 78,

[0332] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0333] (28) (4M2-D9) comprises the VH region of the following FRs:

[0334] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 109

[0335] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 110

[0336] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 111

[0337] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 112,

[0338] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0339] (29) (1M2-D2) includes the VH regions of the following FRs:

[0340] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 123

[0341] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 124

[0342] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 125

[0343] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 78,

[0344] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0345] (30) (5M1-A11) includes the VH regions of the following FRs:

[0346] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 134

[0347] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 92

[0348] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 93

[0349] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 135,

[0350] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0351] (31) (4M2-D5) includes the VH regions of the following FRs:

[0352] HC-FR1 having the amino acid sequence shown in SEQ ID NO: 147

[0353] HC-FR2 having the amino acid sequence shown in SEQ ID NO: 148

[0354] HC-FR3 having the amino acid sequence shown in SEQ ID NO: 149

[0355] HC-FR4 having the amino acid sequence shown in SEQ ID NO: 135,

[0356] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0357] (32)(4M2-A8) comprises the VH region of the following FR:

[0358] HC-FR1 having the amino acid sequence shown in SEQ ID NO:161

[0359] HC-FR2 having the amino acid sequence shown in SEQ ID NO:162

[0360] HC-FR3 having the amino acid sequence shown in SEQ ID NO:163

[0361] HC-FR4 having the amino acid sequence shown in SEQ ID NO:135,

[0362] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0363] (33)(9M2-C12) comprises the VH region of the following FR:

[0364] HC-FR1 having the amino acid sequence shown in SEQ ID NO:172

[0365] HC-FR2 having the amino acid sequence shown in SEQ ID NO:173

[0366] HC-FR3 having the amino acid sequence shown in SEQ ID NO:174

[0367] HC-FR4 having the amino acid sequence shown in SEQ ID NO:175,

[0368] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0369] (34)(13D5p, 13D5-1) comprises the VH region of the following FR:

[0370] HC-FR1 having the amino acid sequence shown in SEQ ID NO:103

[0371] HC-FR2 having the amino acid sequence shown in SEQ ID NO:186

[0372] HC-FR3 having the amino acid sequence shown in SEQ ID NO:187

[0373] HC-FR4 having the amino acid sequence shown in SEQ ID NO:86,

[0374] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0375] (35)(13D5-13) comprises a VH region of the following FRs:

[0376] HC-FR1 having the amino acid sequence shown in SEQ ID NO:103

[0377] HC-FR2 having the amino acid sequence shown in SEQ ID NO:186

[0378] HC-FR3 having the amino acid sequence shown in SEQ ID NO:201

[0379] HC-FR4 having the amino acid sequence shown in SEQ ID NO:86,

[0380] or a variant thereof, wherein one or two or three amino acids in one or more of HC-FR1, HC-FR2, HC-FR3 or HC-FR4 are replaced by another amino acid.

[0381] In some embodiments, the antigen-binding molecule comprises a VH region containing any one of the CDRs as described above in (1) to (18), and any one of the FRs as described above in (19) to (35).

[0382] In some embodiments, the antigen-binding molecule comprises any one of the following VH regions in (36) to (57):

[0383] (36) A VH region comprising the CDR as shown in (1) and the FR as shown in (19), (20), (21), (22), (23) or (24).

[0384] (37) A VH region comprising the CDR as shown in (2) and the FR as shown in (19).

[0385] (38) A VH region comprising the CDR as shown in (3) and the FR as shown in (20).

[0386] (39) A VH region comprising the CDR as shown in (4) and the FR as shown in (20).

[0387] (40) The VH region includes the CDR as shown in (5) and the FR as shown in (21), (22), (23), or (24).

[0388] (41) The VH region includes the CDR as shown in (6) and the FR as shown in (21).

[0389] (42) The VH region includes the CDR as shown in (6) and the FR as shown in (22).

[0390] (43) The VH region includes the CDR as shown in (6) and the FR as shown in (24).

[0391] (44) The VH region includes the CDR as shown in (7) and the FR as shown in (23).

[0392] (45) The VH region includes the CDR as shown in (8) and the FR as shown in (25).

[0393] (46) The VH region includes the CDR as shown in (8) and the FR as shown in (27).

[0394] (47) The VH region includes the CDR as shown in (9) and the FR as shown in (26).

[0395] (48) The VH region includes the CDR as shown in (9) and the FR as shown in (30).

[0396] (49) The VH region includes the CDR as shown in (10) and the FR as shown in (28).

[0397] (50) The VH region includes the CDR as shown in (11) and the FR as shown in (29).

[0398] (51) The VH region includes the CDR as shown in (12) and the FR as shown in (31).

[0399] (52) The VH region includes the CDR as shown in (13) and the FR as shown in (32).

[0400] (53) The VH region includes the CDR as shown in (14) and the FR as shown in (33).

[0401] (54) The VH region includes the CDR as shown in (15) and the FR as shown in (34) or (35).

[0402] (55) The VH region includes the CDR as shown in (16) and the FR as shown in (34).

[0403] (56) The VH region includes the CDR as shown in (17) and the FR as shown in (34).

[0404] The VH region includes a CDR as shown in (18) and an FR as shown in (35).

[0405] In some embodiments, the antigen-binding molecule includes any one of the following VH regions (58) to (76):

[0406] (58) A VH region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 276, 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 with any of the sequences.

[0407] (59) A VH region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 285, 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 with any of the sequences.

[0408] (60) A VH region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289, 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 with any of the sequences.

[0409] (61) A VH region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, 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 with any of the sequences.

[0410] (62) A VH region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 48, 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 with any of the sequences.

[0411] (63)A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:52, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0412] (64)A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:62, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0413] (65)A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:71, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0414] (66)A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:87, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0415] (67)A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:102, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0416] (68) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 106, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0417] (69) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 119, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0418] (70) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 133, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0419] (71) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 143, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0420] (72) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 157, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0421] (73) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 168, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0422] (74) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 183, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0423] (75) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 194, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0424] (76) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 199, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0425] In some embodiments, the antigen-binding molecule comprises any one of the following VL regions (77) to (96):

[0426] (77) (4M2-C12-derived consensus sequence) A VL region comprising the following CDRs:

[0427] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0428] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 308

[0429] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0430] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0431] (78) (C24 / C26 / C27 consensus sequence) The VL region comprising the following CDRs:

[0432] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO: 41

[0433] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO: 309

[0434] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO: 43;

[0435] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0436] (79) (V4-C24, V4-C26) The VL region comprising the following CDRs:

[0437] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO: 41

[0438] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO: 295

[0439] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO: 43;

[0440] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0441] (80) (V4-C27, V4-C30, V4-C31) The VL region comprising the following CDRs:

[0442] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO: 41

[0443] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO: 300

[0444] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO: 43;

[0445] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0446] (81) (4M2-C12 / V4H1 / V4H2 consensus sequence) The VL region comprising the following CDRs:

[0447] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0448] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 245

[0449] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0450] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0451] (82) (4M2-C12, 4M2-B4, V4-C1, V4-C9, V4-C28) The VL region comprising the following CDRs:

[0452] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0453] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 42

[0454] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0455] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0456] (83) (V4H1) The VL region comprising the following CDRs:

[0457] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0458] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 58

[0459] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0460] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0461] (84)(V4H2) The VL region comprising the following CDRs:

[0462] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO:41

[0463] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO:67

[0464] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO:43;

[0465] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0466] (85)(2M1-B12, 2M1-D2) The VL region comprising the following CDRs:

[0467] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO:80

[0468] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO:81

[0469] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO:82;

[0470] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0471] (86)(4M2-C9) The VL region comprising the following CDRs:

[0472] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO:96

[0473] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO:97

[0474] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO:98;

[0475] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0476] (87)(4M2-D9) includes the VH region of the following CDRs:

[0477] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 114

[0478] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 67

[0479] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 115;

[0480] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced by another amino acid.

[0481] (88)(1M2-D2) includes the VL region of the following CDRs:

[0482] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 127

[0483] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 128

[0484] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 129;

[0485] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced by another amino acid.

[0486] (89)(5M1-A11) includes the VL region of the following CDRs:

[0487] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 137

[0488] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 138

[0489] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 139;

[0490] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2, or LC-CDR3 are replaced by another amino acid.

[0491] (90)(4M2-D5) includes the VL region of the following CDRs:

[0492] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 151

[0493] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 152

[0494] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 153;

[0495] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0496] (91) (4M2-A8) comprises the VL region of the following CDRs:

[0497] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 165

[0498] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 152

[0499] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 153;

[0500] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0501] (92) (9M2-C12) comprises the VL region of the following CDRs:

[0502] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 177

[0503] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 178

[0504] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 179;

[0505] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0506] (93) (derived from 13D5p) comprises the VL region of the following CDRs:

[0507] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 247

[0508] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 178

[0509] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 190;

[0510] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0511] (94) (13D5p) comprises the VL region of the following CDRs:

[0512] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 189

[0513] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 178

[0514] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 190;

[0515] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0516] (95) (13D5-1) comprises the VL region of the following CDRs:

[0517] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 197

[0518] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 178

[0519] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 190;

[0520] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0521] (96) (13D5-13) comprises the VL region of the following CDRs:

[0522] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 203

[0523] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 178

[0524] An LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 190;

[0525] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0526] In some embodiments, the antigen-binding molecule comprises any of the following VL regions (97) to (120):

[0527] (97)(V4-C1) The VL region comprising the following FRs:

[0528] An LC-FR1 having the amino acid sequence shown in SEQ ID NO: 59

[0529] An LC-FR2 having the amino acid sequence shown in SEQ ID NO: 283

[0530] An LC-FR3 having the amino acid sequence shown in SEQ ID NO: 284

[0531] An LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0532] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0533] (98)(V4-C9) The VL region comprising the following FRs:

[0534] An LC-FR1 having the amino acid sequence shown in SEQ ID NO: 288

[0535] An LC-FR2 having the amino acid sequence shown in SEQ ID NO: 283

[0536] An LC-FR3 having the amino acid sequence shown in SEQ ID NO: 284

[0537] An LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0538] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0539] (99)(V4-C24) The VL region comprising the following FRs:

[0540] LC-FR1 having the amino acid sequence shown in SEQ ID NO:288

[0541] LC-FR2 having the amino acid sequence shown in SEQ ID NO:283

[0542] LC-FR3 having the amino acid sequence shown in SEQ ID NO:296

[0543] LC-FR4 having the amino acid sequence shown in SEQ ID NO:47,

[0544] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0545] (100)(V4-C26) comprises the VL region of the following FRs:

[0546] LC-FR1 having the amino acid sequence shown in SEQ ID NO:288

[0547] LC-FR2 having the amino acid sequence shown in SEQ ID NO:298

[0548] LC-FR3 having the amino acid sequence shown in SEQ ID NO:284

[0549] LC-FR4 having the amino acid sequence shown in SEQ ID NO:47,

[0550] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0551] (101)(V4-C27) comprises the VL region of the following FRs:

[0552] LC-FR1 having the amino acid sequence shown in SEQ ID NO:288

[0553] LC-FR2 having the amino acid sequence shown in SEQ ID NO:283

[0554] LC-FR3 having the amino acid sequence shown in SEQ ID NO:284

[0555] LC-FR4 having the amino acid sequence shown in SEQ ID NO:47,

[0556] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0557] (102) (V4-C28) comprises the VL region of the following FR:

[0558] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 288

[0559] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 283

[0560] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 296

[0561] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0562] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0563] (103) (V4-C30) comprises the VL region of the following FR:

[0564] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 288

[0565] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 283

[0566] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 296

[0567] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0568] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0569] (104) (V4-C31) comprises the VL region of the following FR:

[0570] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 288

[0571] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 283

[0572] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 304

[0573] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0574] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0575] (105)(4M2-C12) comprises the VL region of the following FR:

[0576] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 44

[0577] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 45

[0578] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 46

[0579] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0580] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0581] (106)(4M2-B4) comprises the VL region of the following FR:

[0582] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 51

[0583] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 45

[0584] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 46

[0585] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0586] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0587] (107)(V4H1) comprises the VL region of the following FR:

[0588] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 59

[0589] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 60

[0590] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 61

[0591] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0592] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0593] (108)(V4H2) comprises the VL region of the following FR:

[0594] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 68

[0595] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 69

[0596] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 70

[0597] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 47,

[0598] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0599] (109)(2M1-B12) comprises the VL region of the following FR:

[0600] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 83

[0601] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 84

[0602] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 85

[0603] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0604] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0605] (110)(4M2-C9) comprises the VL region of the following FR:

[0606] LC-FR1 having the amino acid sequence shown in SEQ ID NO:99

[0607] LC-FR2 having the amino acid sequence shown in SEQ ID NO:100

[0608] LC-FR3 having the amino acid sequence shown in SEQ ID NO:101

[0609] LC-FR4 having the amino acid sequence shown in SEQ ID NO:86,

[0610] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0611] (111)(2M1-D2) comprises the VL region of the following FR:

[0612] LC-FR1 having the amino acid sequence shown in SEQ ID NO:105

[0613] LC-FR2 having the amino acid sequence shown in SEQ ID NO:84

[0614] LC-FR3 having the amino acid sequence shown in SEQ ID NO:85

[0615] LC-FR4 having the amino acid sequence shown in SEQ ID NO:86,

[0616] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0617] (112)(4M2-D9) comprises the VL region of the following FR:

[0618] LC-FR1 having the amino acid sequence shown in SEQ ID NO:116

[0619] LC-FR2 having the amino acid sequence shown in SEQ ID NO:117

[0620] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 118

[0621] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0622] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0623] (113)(1M2-D2) comprises the VL region of the following FR:

[0624] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 130

[0625] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 131

[0626] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 132

[0627] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0628] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0629] (114)(5M1-A11) comprises the VL region of the following FR:

[0630] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 140

[0631] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 141

[0632] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 142

[0633] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0634] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0635] (115)(4M2-D5) comprises the VL region of the following FR:

[0636] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 154

[0637] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 155

[0638] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 156

[0639] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0640] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0641] (116)(4M2-A8) comprises the VL region of the following FR:

[0642] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 166

[0643] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 155

[0644] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 167

[0645] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0646] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0647] (117)(9M2-C12) comprises the VL region of the following FR:

[0648] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 180

[0649] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 181

[0650] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 182

[0651] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0652] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0653] (118)(13D5p) comprises the VL region of the following FR:

[0654] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 191

[0655] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 192

[0656] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 193

[0657] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0658] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0659] (119)(13D5-1) comprises the VL region of the following FR:

[0660] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 191

[0661] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 198

[0662] LC-FR3 having the amino acid sequence shown in SEQ ID NO: 193

[0663] LC-FR4 having the amino acid sequence shown in SEQ ID NO: 86,

[0664] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0665] (120)(13D5-13) comprises the VL region of the following FR:

[0666] LC-FR1 having the amino acid sequence shown in SEQ ID NO: 191

[0667] LC-FR2 having the amino acid sequence shown in SEQ ID NO: 192

[0668] LC-FR3 having the amino acid sequence as set forth in SEQ ID NO: 204

[0669] LC-FR4 having the amino acid sequence as set forth in SEQ ID NO: 86,

[0670] or a variant thereof, wherein one or two or three amino acids in one or more of LC-FR1, LC-FR2, LC-FR3 or LC-FR4 are replaced by another amino acid.

[0671] In some embodiments, the antigen-binding molecule comprises a VL region comprising any one of the CDRs as above (77) to (96) and any one of the FRs as above (97) to (120).

[0672] In some embodiments, the antigen-binding molecule comprises any one of the following VL regions (121) to (148):

[0673] (121) A VL region comprising the CDR as shown in (77) and the FRs as shown in (97), (98), (99), (100), (101), (102), (103), (104), (105), (106), (107) or (108).

[0674] (122) A VL region comprising the CDR as shown in (78) and the FRs as shown in (99), (100) or (101).

[0675] (123) A VL region comprising the CDR as shown in (79) and the FR as shown in (99).

[0676] (124) A VL region comprising the CDR as shown in (79) and the FR as shown in (100).

[0677] (125) A VL region comprising the CDR as shown in (80) and the FR as shown in (101).

[0678] (126) A VL region comprising the CDR as shown in (82) and the FR as shown in (97).

[0679] (127) A VL region comprising the CDR as shown in (82) and the FR as shown in (98).

[0680] (128) A VL region comprising the CDR as shown in (82) and the FR as shown in (102).

[0681] (129) A VL region comprising the CDR as shown in (80) and the FR as shown in (103).

[0682] (130) The VL region includes the CDR shown in (80) and the FR shown in (104).

[0683] (131) The VL region includes the CDR shown in (81) and the FR shown in (105), (106), (107), or (108).

[0684] (132) The VL region includes the CDR shown in (82) and the FR shown in (105).

[0685] (133) The VL region includes the CDR shown in (82) and the FR shown in (106).

[0686] (134) The VL region includes the CDR shown in (83) and the FR shown in (107).

[0687] (135) The VL region includes the CDR shown in (84) and the FR shown in (108).

[0688] (136) The VL region includes the CDR shown in (85) and the FR shown in (109).

[0689] (137) The VL region includes the CDR shown in (85) and the FR shown in (111).

[0690] (138) The VL region includes the CDR shown in (86) and the FR shown in (110).

[0691] (139) The VL region includes the CDR shown in (87) and the FR shown in (112).

[0692] (140) The VL region includes the CDR shown in (88) and the FR shown in (113).

[0693] (141) The VL region includes the CDR shown in (89) and the FR shown in (114).

[0694] (142) The VL region includes the CDR shown in (90) and the FR shown in (115).

[0695] (143) The VL region includes the CDR shown in (91) and the FR shown in (116).

[0696] (144) The VL region includes the CDR shown in (92) and the FR shown in (117).

[0697] (145) The VL region includes the CDR shown in (93) and the FR shown in (118), (119), or (120).

[0698] (146) The VL region includes a CDR as shown in (94) and an FR as shown in (118).

[0699] (147) The VL region includes a CDR as shown in (95) and an FR as shown in (119).

[0700] (148) The VL region includes a CDR as shown in (96) and an FR as shown in (120).

[0701] In some embodiments, the antigen-binding molecule includes any one of the following VL regions (149) to (173):

[0702] (149) A VL region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 310, 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.

[0703] (150) A VL region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 282, 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.

[0704] (151) A VL region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 287, 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.

[0705] (152) A VL region that includes an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 294, 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.

[0706] (153) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 297, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0707] (154) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 299, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0708] (155) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 301, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0709] (156) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 302, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0710] (157) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 303, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0711] (158) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0712] (159) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 50, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0713] (160) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 57, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0714] (161) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 66, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0715] (162) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 79, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0716] (163) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 95, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0717] (164) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 104, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0718] (165) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 113, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0719] (166) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 126, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0720] (167) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 136, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0721] (168) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 150, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0722] (169) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 164, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0723] (170) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 176, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0724] (171) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 188, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0725] (172) A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 196, more preferably having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0726] (173) A VL region, which comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 202, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0727] In some embodiments, the antigen-binding molecule comprises any one of the VH regions as described above in (1) to (76), and any one of the VL regions as described above in (77) to (173).

[0728] In some embodiments, the antigen-binding molecule comprises the CDRs of a VISTA-binding antibody clone or comprises the VH and VL of a VISTA-binding antibody clone, and the VISTA-binding antibody clone is selected from: 4M2-C12, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30 or V4-C31. In some embodiments, the antigen-binding molecule comprises the CDRs of V4-C26, or the VH and VL of V4-C26.

[0729] In some embodiments, the antigen-binding molecule comprises:

[0730] (A) A VH region comprising the following CDRs:

[0731] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 305

[0732] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 306

[0733] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 307,

[0734] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0735] (B) A VL region comprising the following CDRs:

[0736] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0737] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 308

[0738] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0739] or a variant thereof, wherein one, two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0740] (B) a VH region comprising the following CDRs:

[0741] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 290

[0742] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 291

[0743] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 278,

[0744] or a variant thereof, wherein one, two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0745] a VL region comprising the following CDRs:

[0746] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0747] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 295

[0748] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0749] or a variant thereof, wherein one, two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0750] (C) a VH region comprising the following CDRs:

[0751] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 53

[0752] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 34

[0753] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 35,

[0754] or a variant thereof, wherein one, two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0755] The VL region including the following CDRs:

[0756] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0757] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 58

[0758] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0759] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0760] (D) The VH region including the following CDRs:

[0761] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33

[0762] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 34

[0763] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 35,

[0764] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0765] The VL region including the following CDRs:

[0766] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0767] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 67

[0768] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0769] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0770] (E) The VH region including the following CDRs:

[0771] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33

[0772] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 277

[0773] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 278,

[0774] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0775] The VL region comprising the following CDRs:

[0776] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0777] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 42

[0778] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0779] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0780] (F) The VH region comprising the following CDRs:

[0781] HC-CDR1 having the amino acid sequence shown in SEQ ID NO: 33

[0782] HC-CDR2 having the amino acid sequence shown in SEQ ID NO: 286

[0783] HC-CDR3 having the amino acid sequence shown in SEQ ID NO: 278,

[0784] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0785] The VL region comprising the following CDRs:

[0786] LC-CDR1 having the amino acid sequence shown in SEQ ID NO: 41

[0787] LC-CDR2 having the amino acid sequence shown in SEQ ID NO: 42

[0788] LC-CDR3 having the amino acid sequence shown in SEQ ID NO: 43;

[0789] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0790] (G) The VH region comprising the following CDRs:

[0791] HC-CDR1 having the amino acid sequence as shown in SEQ ID NO: 290

[0792] HC-CDR2 having the amino acid sequence as shown in SEQ ID NO: 291

[0793] HC-CDR3 having the amino acid sequence as shown in SEQ ID NO: 278,

[0794] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0795] The VL region comprising the following CDRs:

[0796] LC-CDR1 having the amino acid sequence as shown in SEQ ID NO: 41

[0797] LC-CDR2 having the amino acid sequence as shown in SEQ ID NO: 300

[0798] LC-CDR3 having the amino acid sequence as shown in SEQ ID NO: 43;

[0799] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0800] (H) The VH region comprising the following CDRs:

[0801] HC-CDR1 having the amino acid sequence as shown in SEQ ID NO: 290

[0802] HC-CDR2 having the amino acid sequence as shown in SEQ ID NO: 291

[0803] HC-CDR3 having the amino acid sequence as shown in SEQ ID NO: 278,

[0804] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0805] The VL region comprising the following CDRs:

[0806] LC-CDR1 having the amino acid sequence shown in SEQ ID NO:41

[0807] LC-CDR2 having the amino acid sequence shown in SEQ ID NO:42

[0808] LC-CDR3 having the amino acid sequence shown in SEQ ID NO:43;

[0809] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0810] (I) A VH region comprising the following CDRs:

[0811] HC-CDR1 having the amino acid sequence shown in SEQ ID NO:33

[0812] HC-CDR2 having the amino acid sequence shown in SEQ ID NO:34

[0813] HC-CDR3 having the amino acid sequence shown in SEQ ID NO:35,

[0814] or a variant thereof, wherein one or two or three amino acids in one or more of HC-CDR1, HC-CDR2 or HC-CDR3 are replaced by another amino acid; and

[0815] A VL region comprising the following CDRs:

[0816] LC-CDR1 having the amino acid sequence shown in SEQ ID NO:41

[0817] LC-CDR2 having the amino acid sequence shown in SEQ ID NO:42

[0818] LC-CDR3 having the amino acid sequence shown in SEQ ID NO:43;

[0819] or a variant thereof, wherein one or two or three amino acids in one or more of LC-CDR1, LC-CDR2 or LC-CDR3 are replaced by another amino acid.

[0820] In some embodiments, the antigen-binding molecule comprises:

[0821] (J) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 297, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0822] (K) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 52, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 57, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0823] (L) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 62, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 66, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0824] (M) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 276, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 282, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0825] (N) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 285, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 287, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0826] (O) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 294, more preferably an amino acid sequence having any one of the sequence identities of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0827] (P) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 299, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0828] (Q) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 301, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0829] (R) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 289, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 302, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0830] (S) A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 32, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; and a VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 40, more preferably an amino acid sequence having any one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0831] In some embodiments, the antigen-binding molecule comprises, or consists of:

[0832] (i) one or more (such as two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 331; and

[0833] (ii) one or more (such as two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 317.

[0834] In embodiments according to the present disclosure, one or more amino acids are replaced by another amino acid, and these replacements can be conservative replacements, for example according to the following table. In some embodiments, amino acids in the same block in the middle column are replaced. In some embodiments, amino acids in the same column in the rightmost column are replaced:

[0835]

[0836] In some embodiments, the replacement can be functionally conservative. That is, in some embodiments, compared with the equivalent un-replaced molecule, the replacement may not affect (or may not substantially affect) one or more functional properties (such as target binding) of the antigen-binding molecule comprising the replacement.

[0837] The VH and VL regions of the antibody antigen-binding domain together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present disclosure comprises or consists of an Fv region that binds to VISTA. In some embodiments, the VH and VL regions of the Fv are a single polypeptide linked by a linker region, i.e., a single-chain Fv (scFv).

[0838] In some embodiments, the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE or IgM. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from the heavy chain constant sequence of IgG4.

[0839] In some embodiments, the immunoglobulin heavy chain constant sequence is human immunoglobulin G1 constant (IGHG1; UniProt: P01857-1, v1; SEQ ID NO:205). The 1st to 98th positions of SEQ ID NO:205 form the CH1 region (SEQ ID NO:206). The 99th to 110th positions of SEQ ID NO:205 form the hinge region between the CH1 and CH2 regions (SEQ ID NO:207). The 111th to 223rd positions of SEQ ID NO:205 form the CH2 region (SEQ ID NO:208). The 224th to 330th positions of SEQ ID NO:205 form the CH3 region (SEQ ID NO:209).

[0840] Exemplary antigen-binding molecules can be prepared using pFUSE-CHIg-hG1, the CH3 region of which comprises substitutions of D356E, L358M (numbered according to EU numbering). The amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO:210. It is understood that the CH3 region can be further substituted according to the modifications of the Fc region of the antigen-binding molecule described herein.

[0841] In some embodiments, the CH1 region comprises or consists of the sequence shown in 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 shown in SEQ ID NO: 206. In some embodiments, the CH1-CH2 hinge region comprises or consists of the sequence shown in 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 shown in SEQ ID NO: 207. In some embodiments, the CH2 region comprises or consists of the sequence shown in 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 shown in SEQ ID NO: 208. In some embodiments, the CH3 region comprises or consists of the sequence shown in 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 shown in SEQ ID NO: 209 or 210.

[0842] In some embodiments, the antigen-binding molecule of the present disclosure comprises the sequence shown in SEQ ID NO: 345, 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 shown in SEQ ID NO: 345. In some embodiments, the antigen-binding molecule of the present disclosure comprises the sequence shown in SEQ ID NO: 346, 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 shown in SEQ ID NO: 346.

[0843] In some embodiments, the antigen-binding molecules of the present disclosure include 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 (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 (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the CL region comprises or consists of the sequence shown in 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 shown in SEQ ID NO: 211.

[0844] The VL and light chain constant (CL) regions of the antibody antigen-binding region, and the VH region and the heavy chain constant 1 (CH1) region together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region containing VH, CH1, VL, and CL (such as Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide containing VH and CH1 (such as a VH-CH1 fusion polypeptide) and a polypeptide containing VL and CL (such as a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide containing VH and CL (such as a VH-CL fusion polypeptide) and a polypeptide containing VL and CH (such as a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of the Fab or CrossFab are single polypeptides linked by a linker region, i.e., a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0845] In some embodiments, the antigen-binding molecules of the present disclosure comprise or consist of a Fab region that binds to VISTA.

[0846] In some embodiments, the antigen-binding minutes described herein comprise or consist of a full antibody that binds to VISTA. As used herein, "full antibody" refers to an antibody having a structure substantially similar to an immunoglobulin (Ig) structure. Different types of immunoglobulins and their structures are described in Schroeder and Cavacini, J Allergy Clin Immunol. (2010) 125(2 Suppl 2):S41-S52, the entire contents of which are incorporated herein by reference.

[0847] Immunoglobulin G (i.e., IgG) is a ~150 kDa glycoprotein that includes two heavy chains and two light chains. From the N- to the C-terminus, the heavy chain includes a VH, followed by a heavy chain constant region that includes three constant domains (CH1, CH2, and CH3). Similarly, the light chain includes a VL, followed by a CL. Immunoglobulins can be classified as IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE, or IgM based on the heavy chain. The light chain can be kappa (κ) or lambda (λ).

[0848] In some embodiments, the antigen-binding molecules described herein comprise or consist of IgG (such as IgG1, IgG2, IgG3, IgG4), IgA (such as IgA1, IgA2), IgD, IgE, or IgM that binds to VISTA. In a preferred embodiment, the antigen-binding molecule is IgG4.

[0849] In some embodiments, the antigen-binding molecules of the present disclosure have at least monovalent binding affinity for VISTA. Binding valence refers to the number of binding sites for a particular antigenic determinant in an antigen-binding molecule. Thus, in some embodiments, the antigen-binding molecule comprises at least one VISTA-binding site.

[0850] In some embodiments, the antigen-binding molecule comprises more than one VISTA-binding site, e.g., 2, 3, or 4 binding sites. The binding sites can be the same or different. In some embodiments, the antigen-binding molecule is, e.g., bivalent, trivalent, or tetravalent for VISTA.

[0851] Aspects of the present disclosure relate to multispecific antigen-binding molecules. The term "multispecific" refers to an antigen-binding molecule having specific binding affinity for 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, e.g., comprising non-identical VH and VL).

[0852] In some embodiments, the antigen-binding molecule binds to VISTA and another target (such as an antigen other than VISTA), and thus is at least bispecific. The term "bispecificity" refers to an antigen-binding molecule being able to specifically bind to at least two different antigenic determinants.

[0853] It will be understood that antigen-binding molecules according to the present disclosure (such as multispecific antigen-binding molecules) can include antigen-binding molecules capable of binding to targets specific to the antigen-binding molecule. For example, antigen-binding molecules capable of binding to antigens other than VISTA and VISTA can include: (i) antigen-binding molecules capable of binding to VISTA, and (ii) antigen-binding molecules capable of binding to antigens other than VISTA.

[0854] It will also be understood that antigen-binding molecules according to the present disclosure (such as multispecific antigen-binding molecules) can include antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to targets specific to the antigen-binding molecule. For example, antigen-binding molecules according to the present disclosure can include, for example, (i) an antigen-binding polypeptide complex capable of binding to VISTA, including a light chain polypeptide (including a VL-CL structure) and a heavy chain polypeptide (including a VH-CH1-CH2-CH3 structure); and (ii) an antigen-binding polypeptide complex capable of binding to antigens other than VISTA, including a light chain polypeptide (including a VL-CL structure) and a heavy chain polypeptide (including a VH-CH1-CH2-CH3 structure).

[0855] In some embodiments, the antigen-binding molecule component in a larger antigen-binding molecule (such as a multispecific antigen-binding molecule) can refer to, for example, the "antigen-binding domain" or "antigen-binding region" of the larger antigen-binding molecule.

[0856] In some embodiments, the antigen-binding molecule includes 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 an immune cell surface molecule. In some embodiments, the antigen other than VISTA is a tumor cell antigen. In some embodiments, the antigen other than VISTA is a receptor molecule, such as a cell surface receptor. In some embodiments, the antigen other than VISTA is a cell signaling molecule, such as a cytokine, chemokine, interferon, interleukin, or lymphokine. In some embodiments, the antigen other than VISTA is a growth factor or hormone.

[0857] A cancer cell antigen is an antigen expressed or overexpressed by a cancer cell. A cancer cell antigen can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or a fragment thereof. The expression of a cancer cell antigen may be associated with cancer. A cancer cell antigen may be abnormally expressed by a cancer cell (e.g., a cancer cell antigen may be expressed in an abnormally localized manner), or expressed by a cancer cell with an abnormal structure. A cancer cell antigen can elicit an immune response. In some embodiments, the antigen is expressed on the cell surface of a cancer cell (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, the portion of the antigen bound by an antigen-binding molecule described herein is located on the outer surface (i.e., extracellular) of a cancer cell. A cancer cell antigen can be a cancer-associated antigen. In some embodiments, the cancer cell antigen is an antigen whose expression is associated with the development, progression, or severity of symptoms of cancer. The cancer-associated antigen may be related to the etiology or pathology of cancer or may be abnormally expressed due to cancer. In some embodiments, the cancer cell antigen is an antigen that is upregulated (e.g., at the RNA and / or protein level) in cancer cells, such as compared to the expression level in comparable non-cancer cells (e.g., non-cancer cells from the same tissue / cell type). In some embodiments, the cancer-associated antigen may be preferentially expressed by cancer cells while comparable non-cancer cells (e.g., non-cancer cells from the same tissue / cell type) do not express it. In some embodiments, the cancer-associated antigen may be the product of a mutated oncogene or a mutated tumor suppressor gene. In some embodiments, the cancer-associated antigen may be an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, a carcinoembryonic antigen, or a product of a cell surface glycolipid or glycoprotein.

[0858] An immune cell surface molecule can be any peptide / polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or a fragment thereof expressed in or on the cell surface of an immune cell. In some embodiments, the portion of the immune cell surface molecule bound by an antigen-binding molecule of the present disclosure is located on the outer surface (i.e., extracellular) of the immune cell. 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 hematopoietic cell, such as a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. The lymphocyte can be, for example, a T cell, B cell, natural killer (NK) cell, NKT cell, or innate lymphoid cell (ILC), or a precursor thereof (e.g., a thymocyte or pre-B cell). In some embodiments, the immune cell surface molecule can be a co-stimulatory molecule (e.g., CD28, OX40, 4-1BB, ICOS, or CD27) or its ligand. 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 its ligand.

[0859] Multispecific antigen-binding molecules according to the present disclosure can be provided in any suitable form, such as those described in Brinkmann and Kontermann MAbs (2017) 9(2):182-212, the entire content of which is incorporated herein by reference. Suitable forms include antibody conjugates shown in Figure 2 of Brinkmann and Kontermann MAbs (2017) 9(2):182-212, such as IgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, such as IgG, chimeric IgG, κλ co-body HC; CH1 / CL fusion proteins, such as scFv2-CH1 / CL, VHH2-CH1 / CL; “variable domain only” bispecific antigen-binding molecules, such as tandem scFv (taFV), triabody, diabody (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, trisome, tandem dAb / VHH, tetravalent dAb.VHH; non-Ig fusion proteins, such as scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, minibody, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, such as 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), TriFabs; Fc fusions, such as Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, such as Dia-diabody, scDb-CH3; IgE / IgM CH2 fusions, such as scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, such as Fab-scFv (diabody), Fab-scFv2 (triabody), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, such as DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine2;Asymmetric IgG or IgG-like molecules, such as 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-YscFab-IgG, FcFc*; additional and Fc-modified IgGs, 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 and CH3 fusion proteins, such as Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv-Fc-SEEDbody, TriFab; additional 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(CαCβFab), Fab-IgG(CR3), Fab-hinge-IgG(CR3); additional IgG-LC fusions, such as IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG; additional IgG-HC and LC fusions, such as DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, Zybody; Fc fusions, such as Fab-scFv-Fc, scFv4-Ig; F(ab’)2 fusions, such as F(ab’)2-scFv2; CH1 / CL fusion proteins, such as scFv2-CH1-hinge / CL; modified IgGs, such as DAF (two-in-one IgG), DutaMab, Mab; 2 ; and non-Ig fusions, such as DNL-Fab4-IgG.

[0860] A person skilled in the art can design and prepare bispecific antigen-binding molecules. Methods for preparing bispecific antigen-binding molecules include chemical cross-linking of antigen-binding molecules or antibody fragments, such as using reducible disulfide bonds or non-reducible thioether bonds, as reviewed 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, the entire content of which is incorporated herein by reference. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically cross-link Fab fragments through the hinge region SH-groups to generate disulfide-linked bispecific F(ab)2 heterodimers.

[0861] Other methods for generating bispecific antigen-binding molecules include fusing antibody-producing hybridomas with, for example, polyethylene glycol to generate cell hybridomas that are capable of secreting bispecific antibodies, as described 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.

[0862] The bispecific antigen-binding molecules according to the present disclosure can also be produced recombinantly, for example, by expressing a nucleic acid construct encoding an antigen-binding molecule polypeptide, as in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), Chapter 40: Production of Bispecific Antigen-Binding Molecules: Bispecific Antibodies and Tandem scFv (Hornig and )), or French, How to Prepare Bispecific Antigen-Binding Molecules, Methods Mol. Med. 2000; 40:333-339, the entire content of both of which is incorporated herein by reference. For example, a DNA construct encoding the light and heavy chain variable domains of two antigen-binding fragments (i.e., the light and heavy chain variable domains of the antigen-binding fragment that can bind to VISTA, and the light and heavy chain variable domains of the antigen-binding fragment that can bind to another target protein) can be prepared by molecular cloning techniques and include sequences encoding appropriate linkers or dimerization domains between the antigen-binding fragments. Thereafter, a recombinant bispecific antibody can be produced by expressing (such as in vitro expression) the construct in a suitable host cell (such as a mammalian host cell), and then optionally purifying the expressed recombinant bispecific antibody.

[0863] Fc Region

[0864] In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region.

[0865] In the IgG, IgA, and IgD isotypes, the Fc region consists of the CH2 and CH3 regions of one polypeptide and the CH2 and CH3 regions of another polypeptide. The CH2 and CH3 regions from the two polypeptides together constitute the Fc region. In the IgM and IgE isotypes, the Fc region contains three constant domains (CH2, CH3, and CH4), and the CH2 to CH4 from two polypeptides together constitute the Fc region.

[0866] The Fc region can interact with Fc receptors and other molecules of the immune system, thereby generating functional effects. IgG Fc-mediated effector functions are reviewed in, e.g., Jefferis et al., Immunol Rev 1998, 163:59-76 (the entire content of which is incorporated herein by reference), and are achieved by the recruitment and activation of Fc-mediated immune cells (such as macrophages, dendritic cells, NK cells, and T cells) through the interaction between the Fc region and Fc receptors expressed on immune cells, the recruitment of complement pathway components by the binding of the Fc region to the complement protein C1q, and the subsequent activation of the complement cascade.

[0867] Fc-mediated functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, production of cytokines and / or chemokines, and antigen processing and presentation.

[0868] Modifications of the antibody Fc region that affect Fc-mediated functions are known in the art, as described in, e.g., Wang et al., Protein & Cell (2018) 9(1):63-73, the entire content of which is incorporated herein by reference. In particular, Table 1 of Wang et al., Protein & Cell (2018) 9(1):63-73 summarizes examples of known Fc region modifications that affect antibody effector functions. Modifications of the Fc region that affect antibody effector activity are described below.

[0869] When the Fc region / CH2 / CH3 is described as containing a modification “corresponding to” a reference substitution, it can be considered an isosubstitution in the homologous Fc / CH2 / CH3. For example, the L234A / L235A substitution in human IgG1 (as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991, numbered according to EU numbering) corresponds to the L to A substitution at positions 117 and 118 of the C region A allele of the murine Ig gamma-2A chain, numbered according to SEQ ID NO:256.

[0870] When the Fc region is described as containing a modification, the modification may be present in one or both of the polypeptide chains that together constitute the Fc region.

[0871] In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region containing a modification. In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region containing a modification in one or more CH2 and / or CH3 regions.

[0872] In some embodiments, the Fc region includes a modification that enhances Fc-mediated function. In some embodiments, the Fc region includes a modification that enhances ADCC. In some embodiments, the Fc region includes a modification that enhances ADCP. In some embodiments, the Fc region includes a modification that enhances CDC. Compared to an antigen-binding molecule that includes a corresponding unmodified Fc region, an antigen-binding molecule that includes an Fc region that includes a modification that enhances Fc-mediated function (such as ADCC, ADCP, CDC) induces an increase in the level of the associated effector function.

[0873] In some embodiments, the Fc region includes a modification that increases binding to an Fc receptor. In some embodiments, the Fc region includes a modification that increases binding to an Fcγ receptor. In some embodiments, the Fc region includes a modification that increases binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIIa. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIa. In some embodiments, the Fc region includes a modification that increases binding to FcγRIIb. In some embodiments, the Fc region includes a modification that increases binding to FcRn. In some embodiments, the Fc region includes a modification that increases binding to a complement protein. In some embodiments, the Fc region includes a modification that increases binding to C1q. In some embodiments, the Fc region includes a modification that enhances hexamerization of the antigen-binding molecule. In some embodiments, the Fc region includes a modification that enhances the half-life of the antigen-binding molecule. In some embodiments, the Fc region includes a modification that enhances co-binding.

[0874] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination F243L / R292P / Y300L / V305I / P396L 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 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 described in Shields et al., J Biol Chem. (2001) 276:6591–6604. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A described in Mimot et al., MAbs. (2013):5:229–236, and modifications of other heavy chain polypeptides corresponding to the substitution combination D270E / K326D / A330M / K334E. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination G236A / S239D / I332E described in Richards et al., Mol Cancer Ther. (2008) 7:2517–2527.

[0875] 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 described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. 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.

[0876] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M252Y / S254T / T256E as 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 as described in Zalevsky et al., Nat Biotechnol. (2010) 28:157–159.

[0877] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S267E / L328F as described in Chu et al., Mol Immunol. (2008) 45:3926–3933. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination N325S / L328F as described in Shang et al., Biol Chem. (2014) 289:15309–15318.

[0878] In some embodiments, the Fc region comprises modifications that reduce / prevent Fc-mediated functions. In some embodiments, the Fc region comprises modifications that reduce / prevent ADCC. In some embodiments, the Fc region comprises modifications that reduce / prevent ADCP. In some embodiments, the Fc region comprises modifications that reduce / prevent CDC. Compared to an antigen-binding molecule comprising the corresponding unmodified Fc region, an antigen-binding molecule comprising a modified Fc region can reduce / prevent Fc-mediated functions (such as ADCC, ADCP, CDC), thereby reducing the level of associated effector functions.

[0879] In some embodiments, the Fc region comprises modifications that reduce / prevent binding to Fc receptors. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to Fcγ receptors. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to FcγRIIIa. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to FcγRIIa. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to FcγRIIb. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to complement proteins. In some embodiments, the Fc region comprises modifications that reduce / prevent binding to C1q. In some embodiments, the Fc region comprises modifications that reduce / prevent glycosylation of the amino acid residue corresponding to N297.

[0880] In some embodiments, the Fc region is unable to reduce one or more Fc-mediated functions (i.e., lacks the ability to elicit the relevant Fc-mediated functions). Thus, an antigen-binding molecule comprising such an Fc region also lacks the ability to induce the relevant functions. Such an antigen-binding molecule can be described as not having the relevant functions.

[0881] In some embodiments, the Fc region is unable to induce ADCC. In some embodiments, the Fc region is unable to induce ADCP. In some embodiments, the Fc region is unable to induce CDC. In some embodiments, the Fc region is unable to induce ADCC and / or is unable to induce ADCP and / or is unable to induce CDC.

[0882] In some embodiments, the Fc region is unable to bind to an Fc receptor. In some embodiments, the Fc region is unable to bind to an Fcγ receptor. In some embodiments, the Fc region is unable to bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region is unable to bind to FcγRIIIa. In some embodiments, the Fc region is unable to bind to FcγRIIa. In some embodiments, the Fc region is unable to bind to FcγRIIb. In some embodiments, the Fc region is unable to bind to FcRn. In some embodiments, the Fc region is unable to bind to a complement protein. In some embodiments, the Fc region is unable to bind to C1q. In some embodiments, the amino acid residue corresponding to N297 of the Fc region is not glycosylated.

[0883] In some embodiments, the Fc region comprises a modification corresponding to N297A or 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 combination 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 a modification corresponding to the substitution combination L234A / L235A / P329G as described in Lo et al., J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination as described in Rother et al., Nat Biotechnol. (2007) 25:1256–1264. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination S228P / L235E as described in Newman et al., Clin Immunol. (2001) 98:164–174. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination H268Q / V309L / A330S / P331S as described in An et al., MAbs. (2009) 1:572–579. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination V234A / G237A / P238S / H268A / V309L / A330S / P331S as described in Vafa et al., Methods (2014) 65:114–126. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S as described in US2015 / 0044231 A1.

[0884] The known substitution combination "L234A / L235A" and corresponding substitutions (such as F234A / L235A in human IgG4) disrupt the binding of Fc to Fcγ receptors, inhibit ADCC, ADCP, and reduce C1q binding to thereby inhibit CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457–466, the entire content of which is incorporated herein by reference). Substitutions "P329G" and "P329A" reduce C1q binding (thereby inhibiting CDC). Substitution of "N297" with "A", "G", or "Q" is known to abolish glycosylation, reduce Fc binding to C1q and Fcγ receptors, thereby inhibiting CDC and ADCC. Lo et al., J. Biol. Chem. (2017) 292(9):3900-3908 (the entire content of which is incorporated herein by reference) reported that the substitution combination L234A / L235A / P329G abolishes complement binding and fixation and Fcγ receptor-dependent, antibody-dependent, and cell-mediated cytotoxicity of murine IgG2a and human IgG1.

[0885] US2015 / 0044231 A1 discloses a substitution combination of L234A / L235E / G237A / A330S / P331S in the IgG1 Fc to abolish the induction of phagocytosis, ADCC, and CDC.

[0886] In some embodiments, the Fc region comprises a modification corresponding to the S228P substitution described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The S229P substitution in IgG4 Fc reduces Fab arm exchange (Fab arm exchange may be undesirable).

[0887] 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 P329G substitution. In some embodiments, the Fc region comprises a modification corresponding to the N297Q substitution.

[0888] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G.

[0889] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235A / P329G / N297Q.

[0890] In some embodiments, the Fc region comprises a modification corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S.

[0891] In some embodiments, the Fc region comprises a modification corresponding to the S228P substitution as in IgG4.

[0892] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region containing one or more modifications in the CH2 and CH3 regions to enhance binding of the Fc region. Recombinant co-expression of the component polypeptides of the antigen-binding molecule and subsequent binding can result in several possible combinations. To increase the yield of the desired polypeptide combination of the antigen-binding molecule in the recombinant product, it is advantageous to introduce modifications in the Fc region that promote binding of the desired heavy chain polypeptide combination. For example, the modification can promote hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. Suitable modifications are described in Ha et al., Front. Immunol. (2016) 7:394, the entire content of which is incorporated herein by reference.

[0893] In some embodiments, the antigen-binding molecules of the present disclosure comprise an Fc region containing paired substitutions in the CH3 region of the Fc region, the substitutions being in one of the following forms as shown in Table 1 of Ha et al., Front. Immunol. (2016) 7:394: KiH, KiH s-s , HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT s-s , SEED or A107.

[0894] In some embodiments, the Fc region comprises a "knob-into-hole" or "KiH" modification as described in US 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001). In these 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 each located within the CH3 region such that the "knob" can fit into the "hole" to promote heterodimerization of the polypeptides (and inhibit homodimerization) and / or stabilize the heterodimer. The knob can be formed by substituting an amino acid with a smaller side chain (such as alanine or threonine) with an amino acid having a larger side chain (such as tyrosine or tryptophan). The hole can be formed by substituting an amino acid with a larger side chain with an amino acid having a smaller side chain.

[0895] In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule of the present disclosure comprises a T366W substitution (the numbering of positions / substitutions in the Fc, CH2, and CH3 regions herein is according to the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991), and the other CH3 regions of the Fc region comprise a Y407V substitution. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises a T366W substitution, and the other CH3 regions of the Fc region comprise T366S and L368A substitutions. In some embodiments, one of the CH3 regions of the Fc region of the antigen-binding molecule comprises a T366W substitution, and the other CH3 regions of the Fc region comprise Y407V, T366S, and L368A.

[0896] In some embodiments, the Fc region comprises the "DD-KK" modification as described in WO 2014 / 131694 A1. In some embodiments, one of the CH3 regions comprises K392D and K409D substitutions, and the other CH3 regions of the Fc region comprise E356K and D399K substitutions. The modification enhances the electrostatic interaction between the CH3 regions.

[0897] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region modified in the form of a "Duobody" as described in Labrijn et al., Proc Natl Acad Sci U S A. (2013) 110(13):5145-50. In some embodiments, one of the CH3 regions comprises a K409R substitution, and the other CH3 regions of the Fc region comprise a K405L substitution.

[0898] In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region containing the "EEE-RRR" modification as described in Strop et al., J Mol Biol. (2012) 420(3):204-19. In some embodiments, one of the CH3 regions comprises D221E, P228E, and L368E substitutions, and the other CH3 regions of the Fc region comprise D221R, P228R, and K409R substitutions.

[0899] In some embodiments, the antigen-binding molecule comprises an Fc region containing the "EW-RVT" modification as described in Choi et al., Mol Cancer Ther (2013) 12(12):2748–59. In some embodiments, one of the CH3 regions comprises K360E and K409W substitutions, and the other CH3 regions of the Fc region comprise Q347R, D399V, and F405T substitutions.

[0900] In some embodiments, one of the CH3 regions includes an S354C substitution, and the other CH3 region of the Fc region includes a Y349C substitution. Introduction of these cysteine residues can form a disulfide bond between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001), Journal of Immunological Methods 248, 7-15).

[0901] In some embodiments, the Fc region includes "KiH" S-S " substitutions. In some embodiments, one of the CH3 regions includes T366W and S354C substitutions, and the other CH3 region of the Fc region includes T366S, L368A, Y407V, and Y349C substitutions.

[0902] In some embodiments, the antigen-binding molecule of the present disclosure includes an Fc region containing a "SEED" modification as described in Davis et al., Protein Eng Des Sel (2010) 23(4):195–202, wherein the β-sheet fragments of human IgG1 CH3 and IgA CH3 are exchanged.

[0903] In some embodiments, one of the CH3 regions includes S364H and F405A substitutions, and the other CH3 region of the Fc region includes Y349T and T394F substitutions (see, e.g., Moore et al., MAbs (2011) 3(6):546–57).

[0904] In some embodiments, one of the CH3 regions includes T350V, L351Y, F405A, and Y407V substitutions, and the other CH3 region of the Fc region includes T350V, T366L, K392L, and T394W substitutions (see, e.g., Von Kreudenstein et al., MAbs (2013) 5(5):646–54).

[0905] In some embodiments, one of the CH3 regions includes K360D, D399M, and Y407A substitutions, and the other CH3 region of the Fc region includes E345R, Q347R, T366V, and K409V substitutions (see, e.g., Leaver-Fay et al., Structure (2016) 24(4):641–51).

[0906] In some embodiments, one of the CH3 regions includes K370E and K409W, and the other CH3 region of the Fc region includes E357N, D399V, and F405T (see, e.g., Choi et al., PLoS One (2015) 10(12):e0145349).

[0907] In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region that does not bind to Fcγ receptors. In some embodiments, the antigen-binding molecule includes 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 molecule includes an Fc region that does not bind to one or more of FcγRIIa, FcγRIIb, and FcγRIIIa. In some embodiments, the antigen-binding molecule includes an Fc region that does not bind to one or both of FcγRIIa and FcγRIIb.

[0908] The ability of an Fc region or an antigen-binding molecule comprising an Fc region to bind to a reference protein 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).

[0909] As used herein, an Fc region that "does not bind" to a reference protein can be shown to bind substantially not to the reference protein, as determined by, for example, ELISA, immunoblotting (such as Western blotting), immunoprecipitation, SPR, or BLI. "Substantially not bind" can mean that the level of interaction is not significantly higher than the level of interaction determined for proteins that do not bind to each other in a particular assay. "Substantially not bind" can mean that in a particular assay, the level of interaction is ≤ 5 - fold, such as ≤ 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.

[0910] In some embodiments, the antigen-binding molecule includes an Fc region that binds to FcRn.

[0911] In some embodiments, the antigen-binding molecule includes 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 includes an Fc region that binds to FcRn but does not bind to one or both of FcγRIIa and FcγRIIb.

[0912] In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region that does not induce ADCC. In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region that does not induce ADCP. In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region that does not induce CDC. In some embodiments, the antigen-binding molecules of the present disclosure include an Fc region that does not induce ADCC, ADCP, or CDC.

[0913] As used herein, an Fc region / antigen-binding molecule that does not induce (i.e., is unable to induce) ADCC / ADCP / CDC substantially does not cause ADCC / ADCP / CDC activity, as determined by an appropriate assay for the relevant activity. "Substantially no ADCC / ADCP / CDC activity" means that in a particular assay, the ADCC / ADCP / CDC level is not significantly higher than the ADCC / ADCP / CDC level measured for an appropriate negative control molecule (such as an antigen-binding molecule lacking an Fc region, or an antigen-binding molecule containing a "silent" Fc region (as described in Schlothauer et al., Protein Engineering, Design & Selection (2016), 29(10):457–466, the entire content of which is incorporated herein by reference)). "Substantially inactive" can be a relevant activity level ≤5-fold, such as ≤4-fold, ≤3-fold, ≤2.5-fold, ≤2-fold, or ≤1.5-fold the activity level measured for an appropriate negative control molecule in a particular assay.

[0914] The ability of an Fc region or an antigen-binding molecule comprising an Fc region to induce ADCC can be determined according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (the entire content of which is incorporated herein by reference), or by the 51 Cr release assay described in Jedema et al., Blood (2004) 103:2677–82 (the entire content of which is incorporated herein by reference). The ability of an Fc region or an antigen-binding molecule comprising an Fc region to induce ADCP can be analyzed according to the method described in Kamen et al., J Immunol (2017) 198 (Suppl 1) 157.17 (the entire content of which is incorporated herein by reference). The ability of an Fc region or an antigen-binding molecule comprising an Fc region to induce CDC can be analyzed using the C1q binding assay described in Schlothauer et al., Protein Engineering, Design & Selection (2016), 29(10):457–466 (the entire content of which is incorporated herein by reference).

[0915] In some embodiments, the Fc region contained in the antigen-binding molecule comprises an amino acid sequence having at least 70%, preferably any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 254. In some embodiments, the Fc region contained in the antigen-binding molecule comprises an amino acid sequence having at least 70%, preferably any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 257. In some embodiments, the Fc region contained in the antigen-binding molecule comprises an amino acid sequence having at least 70%, preferably any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 259. In some embodiments, the Fc region contained in the antigen-binding molecule comprises an amino acid sequence having at least 70%, preferably any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 260.

[0916] In some embodiments, the Fc region contained in the antigen-binding molecule comprises an amino acid sequence having at least 70%, preferably any one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 347.

[0917] In some embodiments, the antigen-binding molecule of the present disclosure lacks an Fc region.

[0918] Fc Receptors

[0919] Fc receptors are polypeptides that bind to the Fc region of immunoglobulins. Fc receptor structures and functions are reviewed in Masuda et al., Inflammatory & Allergy Drug Targets (2009) 8(1):80–86 and Bruhns, Blood (2012) 119:5640-5649, the entire contents of which are incorporated herein by reference.

[0920] Fc receptors are expressed on the surface of hematopoietic cells, including macrophages, neutrophils, dendritic cells, eosinophils, basophils, mast cells, and NK cells. They include Fcγ receptors that bind IgG, the high-affinity receptor for IgE (FcεRI), IgA receptors, and the polymeric Ig receptor for IgA and IgM. The neonatal Fc receptor (FcRn) is another Fc receptor for IgG that is involved in the transport of IgG across epithelial barriers (transcytosis), prevention of IgG degradation, and antigen presentation.

[0921] There are six different types of Fcγ receptors in humans (mouse homologs in parentheses): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV), and FcγRIIIb

[0922] The intracellular domains of FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa contain immunoreceptor tyrosine-based activation motifs (ITAMs) that, upon

[0923] Fc binding, activate the cells expressing the receptor. The intracellular domain of FcγRIIb contains an immunoreceptor tyrosine-based activation motif (ITAM) that, upon

[0924] Fc binding, negatively regulates cell activation and degranulation, cell proliferation, endocytosis, and phagocytosis.

[0925] In this specification, "Fcγ receptor" can be from any component, including any isotype, fragment, variant (including mutants) or homolog of any component. Similarly, "FcγRI", "FcγRIIa", "FcγRIIb", "FcγRIIc", "FcγRIIIa", and "FcγRIIIb" respectively refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb from any component, and include any isotype, fragment, variant (including mutants) or homolog of any component.

[0926] In some embodiments, the Fcγ receptors (such as FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb) are from mammals (such as primates (rhesus monkey, cynomolgus monkey, non-human primates or humans) and / or rodents (such as rats or mice)). The isotypes, 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 with the amino acid sequence of the immature or mature isotype of the Fcγ receptor from a specific species (such as human) (such as FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb).

[0927] The isotypes, fragments, variants or homologs can optionally be functional isotypes, fragments, variants or homologs, such as having the functional properties / activities of the reference Fcγ receptor, which can be determined by appropriate functional property / activity assays. For example, the isotypes, fragments, variants or homologs of FcγRI can show binding to human IgG1 Fc.

[0928] In this specification, the "FcRn receptor" can be from any component, including isotypes, fragments, variants (including mutants) or homologs from any component.

[0929] In some embodiments, the FcRn receptor is from mammals (such as primates (rhesus monkey, cynomolgus monkey, non-human primates or humans) and / or rodents (such as rats or mice)). The isotypes, 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 with the amino acid sequence of the immature or mature isotype of the FcRn receptor from a specific species (such as human).

[0930] The isotypes, fragments, variants or homologs can optionally be functional isotypes, fragments, variants or homologs, such as having the functional properties / activities of the reference FcRn receptor, which can be determined by appropriate functional property / activity assays. For example, the isotypes, fragments, variants or homologs of FcRn can show binding to human IgG1 Fc.

[0931] Polypeptides

[0932] The present disclosure also provides a polypeptide component of an antigen-binding molecule. The polypeptide can be provided in isolated or substantially purified form.

[0933] The antigen-binding molecules of the present disclosure can be or can include polypeptide complexes.

[0934] In this specification, when a polypeptide includes more than one domain or region, it is understood that multiple domains / regions are preferably present in the same polypeptide chain. That is, a polypeptide including more than one domain or region is a fusion polypeptide including multiple domains / regions.

[0935] In some embodiments, the polypeptide according to the present disclosure comprises or consists of the VH described herein. In some embodiments, the polypeptide according to the present disclosure comprises or consists of the VL described herein.

[0936] In some embodiments, the polypeptide further comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide further comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises the CH1, CH2 region and / or CH3 region of an immunoglobulin (Ig).

[0937] In some embodiments, the polypeptide comprises one or more immunoglobulin heavy chain constant sequence regions. In some embodiments, the polypeptide comprises the CH1 region described herein. In certain embodiments, the polypeptide comprises the CH1-CH2 hinge region described herein. In some embodiments, the polypeptide comprises the CH2 region described herein. In certain embodiments, the polypeptide comprises the CH3 region described herein.

[0938] In some embodiments, the polypeptide comprising the CH2 and / or CH3 region comprises any one of the following amino acid substitutions / amino acid substitution combinations: 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 / E430G / 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.

[0939] In some embodiments, the polypeptide comprising the CH3 region comprises any one of the following amino acid substitutions / amino acid substitution combinations (as shown in Table 1 of Front. Immunol (2016) 7:394, the entire content of which is 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.

[0940] In some embodiments, the CH2 and / or CH3 region of the polypeptide comprises one or more amino acid substitutions to promote the binding of the polypeptide to another polypeptide comprising a CH2 and / or CH3 region.

[0941] In some embodiments, the polypeptide comprises one or more regions of the immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises the CL region described herein.

[0942] 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.

[0943] In some embodiments, the polypeptide according to the present disclosure comprises one of the following structures from the N-terminus to the C-terminus:

[0944] (i) VH

[0945] (ii) VL

[0946] (iii) VH-CH1

[0947] (iv) VL-CL

[0948] (v) VL-CH1

[0949] (vi) VH-CL

[0950] (vii) VH-CH1-CH2-CH3

[0951] (viii) VL-CL-CH2-CH3

[0952] (ix) VL-CH1-CH2-CH3

[0953] (x) VH-CL-CH2-CH3

[0954] The present disclosure also provides an antigen-binding molecule composed of the polypeptides of the present disclosure. In some embodiments, the antigen-binding molecule of the present disclosure comprises one of the following polypeptide combinations:

[0955] (A) VH + VL

[0956] (B) VH-CH1 + VL-CL

[0957] (C) VL-CH1 + VH-CL

[0958] (D) VH-CH1-CH2-CH3 + VL-CL

[0959] (E) VH-CL-CH2-CH3 + VL-CH1

[0960] (F) VL-CH1-CH2-CH3 + VH-CL

[0961] (G) VL-CL-CH2-CH3 + VH-CH1

[0962] (H) VH-CH1-CH2-CH3 + VL-CL-CH2-CH3

[0963] (I) VH-CL-CH2-CH3 + VL-CH1-CH2-CH3

[0964] In some embodiments, the antigen-binding molecule comprises a combination of more than one polypeptide as shown in (A) to (I) above. For example, referring to (D) above, in some embodiments, the antigen-binding molecule comprises two polypeptides consisting of the VH-CH1-CH2-CH3 structure and two polypeptides consisting of the VL-CL structure.

[0965] In some embodiments, the antigen-binding molecule of the present disclosure comprises one of the following polypeptide combinations:

[0966] (J) VH(anti-VISTA) + VL(anti-VISTA)

[0967] (K) VH(anti-VISTA)-CH1 + VL(anti-VISTA)-CL

[0968] (L) VL (anti-VISTA)-CH1 + VH (anti-VISTA)-CL

[0969] (M) VH (anti-VISTA)-CH1-CH2-CH3 + VL (anti-VISTA)-CL

[0970] (N) VH (anti-VISTA)-CL-CH2-CH3 + VL (anti-VISTA)-CH1

[0971] (O) VL (anti-VISTA)-CH1-CH2-CH3 + VH (anti-VISTA)-CL

[0972] (P) VL (anti-VISTA)-CL-CH2-CH3 + VH (anti-VISTA)-CH1

[0973] (Q) VH (anti-VISTA)-CH1-CH2-CH3 + VL (anti-VISTA)-CL-CH2-CH3

[0974] (R) VH (anti-VISTA)-CL-CH2-CH3 + VL (anti-VISTA)-CH1-CH2-CH3

[0975] Wherein: "VH (anti-VISTA)" means that the VH of the antigen-binding molecule can bind to VISTA as described herein, as defined in any one of (1) to (76); "VL (anti-VISTA)" means that the VL of the antigen-binding molecule can bind to VISTA as described herein, as defined in any one of (77) to (173).

[0976] 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 any one of the amino acid sequences of SEQ ID NO: 212 to 243, 248 to 250, 258, 266 or 311 to 321.

[0977] Linkers and Spacer Sequences

[0978] In some embodiments, the antigen-binding molecules and polypeptides of the present disclosure include a hinge region. In some embodiments, there is a hinge region between the CH1 region and the CH2 region. In some embodiments, there is a hinge region 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 shown in SEQ ID NO: 207.

[0979] In some embodiments, the antigen-binding molecules and polypeptides of the present disclosure include one or more linker sequences between amino acid sequences. Linker sequences can be provided at one or both ends of one or more VH, VL, CH1-CH2 hinge regions, CH2 regions and CH3 regions of the antigen-binding molecule / polypeptide.

[0980] Linker sequences are known to those skilled in the art, as described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker sequence can be a flexible linker sequence. A flexible linker sequence allows relative movement of the amino acid sequences joined by the linker sequence. Flexible linkers are known to those skilled in the art, and several have been identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10):1357-1369. Flexible linker sequences generally include a high proportion of glycine and / or serine residues.

[0981] In some embodiments, the linker sequence includes at least one glycine and / or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the length of the linker sequence is 1-2, 1-3, 1-4, 1-5 or 1-10 amino acids.

[0982] The antigen-binding molecules and polypeptides of the present disclosure can also include additional amino acids or amino acid sequences. For example, the antigen-binding molecules and polypeptides can include amino acid sequences to facilitate the expression, folding, transport, processing, purification or detection of the antigen-binding molecule / polypeptide. For example, the antigen-binding molecule / polypeptide can include sequences encoding His (such as 6XHis), Myc, GST, MBP, FLAG, HA, E or biotin tags, optionally located at the N-terminus or C-terminus of the antigen-binding molecule / polypeptide. In some embodiments, the antigen-binding molecule / polypeptide includes a detectable moiety, such as a fluorescent, luminescent, immunodetection, radioactive, chemical, nucleic acid or enzyme label.

[0983] The antigen-binding molecules and polypeptides of the present disclosure may also include a signal peptide (also referred to as a leader sequence or signal sequence). Signal peptides generally consist of 5-30 hydrophobic amino acid sequences that form a single α helix. Secreted proteins and proteins expressed on the cell surface typically consist of signal peptides.

[0984] The signal peptide may be located at the N-terminus of the antigen-binding molecule / polypeptide and may be present in the newly synthesized antigen-binding molecule / polypeptide. The signal peptide facilitates the efficient transport and secretion of the antigen-binding molecule / polypeptide. The signal peptide is typically removed by cleavage and thus is not included in the mature antigen-binding molecule / polypeptide secreted by the cell expressing the antigen-binding molecule / polypeptide.

[0985] Signal peptides of many proteins are known 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 by amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0986] Labels and Conjugates

[0987] In some embodiments, the antigen-binding molecules of the present disclosure further include a detectable moiety.

[0988] In some embodiments, the antigen-binding molecule includes a detectable moiety such as a fluorescent label, a phosphorescent label, a luminescent label, an immunoassay label (e.g., an epitope tag), a radioactive label, a chemical label, a nucleic acid label, or an enzyme label. The antigen-binding molecule may be labeled covalently or non-covalently with a detectable molecule.

[0989] Fluorescent labels include, for example, fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP) chelates of rare earths (such as europium (Eu), terbium (Tb), and samarium (Sm)), tetramethylrhodamine, Texas red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5. Radioactive labels include radioactive isotopes such as iodine 123 、iodine 125 、iodine 131 、iodine 133 、bromine 77 、technetium 99m 、indium 111 、indium 113m 、gallium67 , gallium 68 , ruthenium 95 , ruthenium 97 , 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 , antimony 211 . Luminescent labels include radioluminescence, chemiluminescence (such as acridinium esters, luminol, isoluminol) and bioluminescent tags. Detectable immunological tags include haptens, peptides / polypeptides, antibodies, receptors and ligands, such as biotin, avidin, streptavidin or digoxin. Nucleic acid tags include aptamers. Enzyme tags include peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase and luciferase, etc.

[0990] In some embodiments, the antigen-binding molecules of the present disclosure are conjugated to a chemical moiety. The chemical moiety can be a moiety that provides therapeutic efficacy. Antibody-drug conjugates are reviewed in Parslow et al., Biopharmaceutics 2016 Sep;4(3):14. In some embodiments, the chemical moiety can be a drug moiety (such as a cytotoxic agent). In some embodiments, the drug moiety can be a chemotherapeutic agent. In some embodiments, the drug moiety can be selected from calicheamicin, DM1, DM4, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, azithromycin, duocarmycin, D6.5 and PBD.

[0991] Specific Exemplary Embodiments of Antigen-Binding Molecules

[0992] In some embodiments, the antigen-binding molecule comprises or consists of:

[0993] (i) two polypeptides, which comprise or consist of amino acid sequences 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 shown in SEQ ID NO:212; and

[0994] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 213.

[0995] In some embodiments, the antigen-binding molecule comprises or consists of:

[0996] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 214; and

[0997] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 215.

[0998] In some embodiments, the antigen-binding molecule comprises or consists of:

[0999] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 216; and

[1000] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 217.

[1001] In some embodiments, the antigen-binding molecule comprises or consists of:

[1002] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 218; and

[1003] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 219.

[1004] In some embodiments, the antigen-binding molecule comprises or consists of:

[1005] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 220; and

[1006] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 221.

[1007] In some embodiments, the antigen-binding molecule comprises or consists of:

[1008] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 222; and

[1009] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 223.

[1010] In some embodiments, the antigen-binding molecule comprises or consists of:

[1011] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 224; and

[1012] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 225.

[1013] In some embodiments, the antigen-binding molecule comprises or consists of:

[1014] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 226; and

[1015] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 227.

[1016] In some embodiments, the antigen-binding molecule comprises or consists of:

[1017] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 228; and

[1018] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one amino acid sequence identity of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence shown in SEQ ID NO:229.

[1019] In some embodiments, the antigen-binding molecule comprises or consists of:

[1020] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one amino acid sequence identity of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence shown in SEQ ID NO:230; and

[1021] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one amino acid sequence identity of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence shown in SEQ ID NO:231.

[1022] In some embodiments, the antigen-binding molecule comprises or consists of:

[1023] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one amino acid sequence identity of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence shown in SEQ ID NO:232; and

[1024] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one amino acid sequence identity of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the amino acid sequence shown in SEQ ID NO:233.

[1025] In some embodiments, the antigen-binding molecule comprises or consists of:

[1026] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 234; and

[1027] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 235.

[1028] In some embodiments, the antigen-binding molecule comprises or consists of:

[1029] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 236; and

[1030] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 237.

[1031] In some embodiments, the antigen-binding molecule comprises or consists of:

[1032] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 238; and

[1033] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 239.

[1034] In some embodiments, the antigen-binding molecule comprises or consists of:

[1035] (i) two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:240; and

[1036] (ii) two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:241.

[1037] In some embodiments, the antigen-binding molecule comprises or consists of:

[1038] (i) two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:242; and

[1039] (ii) two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:243.

[1040] In some embodiments, the antigen-binding molecule comprises or consists of:

[1041] (i) two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:248; and

[1042] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:250.

[1043] In some embodiments, the antigen-binding molecule comprises or consists of:

[1044] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:249; and

[1045] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:250.

[1046] In some embodiments, the antigen-binding molecule comprises or consists of:

[1047] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:258; and

[1048] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:250.

[1049] In some embodiments, the antigen-binding molecule comprises or consists of:

[1050] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 266; and

[1051] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 250.

[1052] In some embodiments, the antigen-binding molecule comprises or consists of:

[1053] (i) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 330; and

[1054] (ii) Two polypeptides, which comprise or consist of an amino acid sequence having at least 70%, preferably any one of 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with the amino acid sequence shown in SEQ ID NO: 213.

[1055] Functional Properties of Antigen-Binding Molecules

[1056] The antigen-binding molecules described herein can be characterized by certain functional properties. In some embodiments, the antigen-binding molecules described herein can have one or more of the following properties:

[1057] Bind to VISTA (such as human, murine and / or cynomolgus VISTA);

[1058] Do not bind to PD-L1 and / or HER3;

[1059] Do not bind to Fcγ receptors;

[1060] Do not bind to C1q;

[1061] Do not induce ADCC;

[1062] Do not induce ADCP;

[1063] Does not induce CDC;

[1064] Binds to the FcRn receptor;

[1065] Binds to VISTA with similar affinity at pH 5.5 to pH 7.5;

[1066] Binds to cells expressing VISTA;

[1067] Inhibits the interaction between VISTA and its interaction partners (such as LRIG1, PSGL-1, VSIG3 or VSIG8);

[1068] Inhibits VISTA-mediated signaling;

[1069] Inhibits VISTA-mediated signaling independently of Fc-mediated function;

[1070] Enhances the killing of cells expressing VISTA;

[1071] Does not induce / enhance the killing of cells expressing VISTA;

[1072] Reduces the number / proportion of cells expressing VISTA;

[1073] Does not reduce the number / proportion of cells expressing VISTA;

[1074] Increases the number / activity of effector immune cells;

[1075] Reduces the number / activity of inhibitory immune cells;

[1076] Reduces the proliferation of inhibitory immune cells;

[1077] Reduces immune suppression mediated by cells expressing VISTA;

[1078] Enhances antigen presentation by antigen-presenting cells;

[1079] Increases the production of IL-6 by immune cells;

[1080] Increases the production of IFN-γ, IL-2 and / or IL-17 in a mixed lymphocyte reaction (MLR) assay;

[1081] Enhances T cell proliferation, IFN-γ production, TNFα production and / or T cell-mediated tumor cell lysis;

[1082] Inhibits cancer development and / or progression in vivo;

[1083] Does not induce cytokine release syndrome in vivo;

[1084] Increase the number and / or proportion of antigen-specific CD8+ T cells;

[1085] Increase the activity of CD8+ T cells;

[1086] Upregulate one or more cytotoxicity-related markers (such as granzyme B, CX3CR1, ICOS, CD27);

[1087] Upregulate one or more genes related to pro-inflammatory macrophage activation;

[1088] Upregulate one or more genes related to T cell cytotoxic activity;

[1089] Increase the production of granzyme B;

[1090] Reduce the level of T cell exhaustion;

[1091] Reduce the number and / or proportion of tumor-associated macrophages (TAM);

[1092] Reduce the activity of tumor-associated macrophages;

[1093] Increase the number and / or proportion of M1 macrophages;

[1094] Increase the activity of M1 macrophages.

[1095] As used herein, the "proportion" of a cell type / subtype can be the proportion of that cell type / subtype in a cell population, such as CD45+ cells, such as CD45+ cells obtained from a tumor.

[1096] It is understood that a particular antigen-binding molecule may exhibit more than one of the aforementioned properties. Suitable detection methods can be used to evaluate the aforementioned properties of a particular antigen-binding molecule. These detection methods can be in vitro detections, which can be cell-free or cell-based detections. Alternatively, they can also be in vivo detections, i.e., tests conducted in non-human animals.

[1097] When the detection is a cell-based detection, it can include contacting the cells with a particular antigen-binding molecule to determine whether the antigen-binding molecule exhibits one or more of the listed properties. The detection can employ components labeled with detectable entities to facilitate detection. The detection can include evaluating the listed properties after treating the cells with a certain number / concentration of antigen-binding molecules (such as a dilution series). It should be understood that the cells are preferably cells expressing VISTA, such as MDSCs.

[1098] Analysis of such assay results can include determining the concentration at which 50% of the maximum relevant activity is achieved. The concentration at which an antigen-binding molecule reaches 50% of the maximum relevant activity can be referred to as the "half-maximal effect concentration" of the antigen-binding molecule with respect to the relevant activity, or it can also be referred to as "EC 50 ". For example, the EC 50 of a particular antigen-binding molecule binding to VISTA can be the concentration at which 50% of the maximum level of binding to the relevant species is achieved.

[1099] Depending on the property, EC 50 can also be referred to as the "half-maximal inhibitory concentration" or "IC 50 ", which is the concentration of the antigen-binding molecule at which 50% of the maximum inhibitory level of a particular property can be observed. For example, the IC 50 of a particular antigen-binding molecule inhibiting the interaction between VISTA and its interaction partners (such as LRIG1, PSGL-1, VSIG3 or VSIG8) can be the concentration at which 50% of the maximum inhibitory level is achieved.

[1100] The antigen-binding molecules described herein bind to VISTA. In a preferred embodiment, the antigen-binding molecule exhibits specific binding to VISTA. As used herein, "specific binding" refers to the selective binding of an antigen, which can be distinguished from non-specific binding to non-target antigens. An antigen-binding molecule that specifically binds VISTA preferably has a stronger and / or longer-lasting affinity for binding to VISTA than other non-target molecules.

[1101] The ability of a specific polypeptide to specifically bind to a specific molecule can be determined 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) or biolayer interferometry (BLI; see, e.g., Lad et al. (2015) J Biomol Screen 20(4):498-507), flow cytometry, or by radiolabeled antigen-binding assays (RIA), enzyme-linked immunosorbent assays. Through such assays, the binding to a specific molecule can be measured and quantified. In some embodiments, the binding can be a reaction detected in a specific assay.

[1102] In some embodiments, the degree of binding of the antigen-binding molecule to non-target molecules is less than about 10% of the degree of binding of an antibody to the target molecule, as determined by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, binding specificity can be reflected by the binding affinity, i.e., the binding dissociation constant (K D ) of the antigen-binding molecule is at least lower than the K D of the antigen-binding molecule for non-target moleculesGreater than 0.1 order of magnitude (i.e., 0.1x10 n , where n is an integer representing the order of magnitude). One of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 can be selected.

[1103] In some embodiments, the antigen-binding molecule according to the present disclosure binds to VISTA with an affinity in the micromolar range, i.e., K D = 9.9x10 -4 to 1x10 -6 M. In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the submicromolar range, i.e., KD < 1x10 -6 M. In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the nanomolar range, i.e., K D = 9.9x10 -7 to 1x10 -9 M. In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the subnanomolar range, i.e., K D < 1x10 -9 M. In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the picomolar range, i.e., K D = 9.9x10 -10 to 1x10 -12 M. In some embodiments, the antigen-binding molecule binds to VISTA with an affinity in the subpicomolar range, i.e., K D < 1x10 -12 M.

[1104] In some embodiments, the K D of the antigen-binding molecule according to the present disclosure binding to VISTA is 10 μM or less, preferably any 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, ≤500 pM, ≤400 pM, ≤300 pM, ≤200 pM, ≤100 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤10 pM, or ≤1 pM. In some embodiments, the K D(As measured by SPR (Biocore), such as the SPR analysis in the examples described in the present disclosure) ≤ 1 nM (such as any one of ≤ 900 pM, ≤ 800 pM, ≤ 700 pM, ≤ 600 pM, ≤ 500 pM, ≤ 400 pM, ≤ 300 pM).

[1105] In some embodiments, the antigen-binding molecule according to the present disclosure binds to VISTA with a K D (As measured by SPR (Biocore), such as the SPR analysis in the examples described in the present disclosure) ≤ 1 nM (such as any one of ≤ 900 pM, ≤ 800 pM, ≤ 700 pM, ≤ 600 pM, ≤ 500 pM). In some embodiments, the antigen-binding molecule according to the present disclosure binds to cynomolgus VISTA with a K D (As measured by SPR (Biocore), such as the SPR analysis in the examples described in the present disclosure) ≤ 1 nM (such as any one of ≤ 900 pM, ≤ 800 pM, ≤ 700 pM, ≤ 600 pM, ≤ 500 pM, ≤ 400 pM). In some embodiments, the antigen-binding molecule according to the present disclosure binds to rat VISTA with a K D (As measured by SPR (Biocore), such as the SPR analysis in the examples described in the present disclosure) ≤ 1 nM (such as any one of ≤ 900 pM, ≤ 800 pM, ≤ 700 pM, ≤ 600 pM, ≤ 500 pM, ≤ 400 pM). In some embodiments, the antigen-binding molecule according to the present disclosure binds to mouse VISTA with a K D (As measured by SPR (Biocore), such as the SPR analysis in the examples described in the present disclosure) ≤ 1 nM (such as any one of ≤ 900 pM, ≤ 800 pM, ≤ 700 pM, ≤ 600 pM).

[1106] In some embodiments, the antigen-binding molecule according to the present disclosure binds to VISTA with an EC 50 (As measured by ELISA, such as the ELISA in the examples described in the present disclosure) is 1 μM or less, such as any 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.

[1107] In some embodiments, the antigen-binding molecule exhibits binding to human VISTA, murine (e.g., mouse) VISTA, rat VISTA, and / or cynomolgus macaque (Macaca fascicularis) VISTA. In some embodiments, the antigen-binding molecule binds human VISTA and mouse VISTA and rat VISTA and cynomolgus macaque VISTA. In some embodiments, the antigen-binding molecule is cross-reactive with human VISTA, mouse VISTA, rat VISTA, and cynomolgus macaque VISTA. In some embodiments, the antigen-binding molecules of the present disclosure are cross-reactive with VISTA of non-human primates. Cross-reactivity with VISTA in model species allows for the in vivo exploration of the efficacy of synthetic models without relying on surrogate molecules.

[1108] In some embodiments, the EC of the antigen-binding molecule according to the present disclosure binding to human VISTA 50 (as determined by ELISA, such as the ELISA in the examples described in the present disclosure) ≤ 20 pM (any one of ≤ 15 pM, ≤ 12.5 pM, ≤ 10 pM, ≤ 7.5 pM). In some embodiments, the EC of the antigen-binding molecule according to the present disclosure binding to rat VISTA 50 (as determined by ELISA, such as the ELISA in the examples described in the present disclosure) ≤ 20 pM (any one of ≤ 15 pM, ≤ 12.5 pM, ≤ 10 pM, ≤ 7.5 pM). In some embodiments, the EC of the antigen-binding molecule according to the present disclosure binding to mouse VISTA 50 (as determined by ELISA, such as the ELISA in the examples described in the present disclosure) ≤ 20 pM (any one of ≤ 15 pM, ≤ 12.5 pM, ≤ 10 pM, ≤ 7.5 pM, ≤ 5 pM).

[1109] In some embodiments, the antigen-binding molecule according to the present disclosure binds to VISTA (such as human VISTA) with similar affinities at pH 5.5 to pH 7.5. For example, in some embodiments, the VISTA affinity of the antigen-binding molecule at pH 5.5 is similar to the VISTA affinity at pH 7.5.

[1110] Herein, a binding affinity "similar" to a reference binding affinity means that the binding affinity measured under comparable conditions is within 50% of the reference binding affinity, such as 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%.

[1111] The K of binding to VISTA (such as human VISTA)D May be similar at pH 5.5 to pH 7.5. The EC that binds to VISTA (such as human VISTA) 50 May be similar at pH 5.5 to pH 7.5.

[1112] Here, the K that is "similar" to the reference value D or EC 50 Can be ≥0.5-fold and ≤2-fold, such as ≥0.7-fold and ≤1.5-fold, ≥0.75-fold and ≤1.25-fold, ≥0.8-fold and ≤1.2-fold, ≥0.85-fold and ≤1.15-fold, ≥0.9-fold and ≤1.1-fold, ≥0.91-fold and ≤1.09-fold, ≥0.92-fold and ≤1.08-fold, ≥0.93-fold and ≤1.07-fold, ≥0.94-fold and ≤1.06-fold, ≥0.95-fold and ≤1.05-fold, ≥0.96-fold and ≤1.04-fold, ≥0.97-fold and ≤1.03-fold, ≥0.98-fold and ≤1.02-fold, or ≥0.99-fold and ≤1.01-fold, any one of which is relative to the reference value.

[1113] In some embodiments, the antigen-binding molecule does not exhibit specific binding to PD-L1 (such as human PD-L1). In some embodiments, the antigen-binding molecule does not exhibit specific binding to HER3 (such as human HER3). In some embodiments, the antigen-binding molecule does not exhibit specific binding to another member of the B7 protein family (such as human HER3) (i.e., does not cross-react with it). 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.

[1114] 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.

[1115] In some embodiments, the antigen-binding molecule is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to elicit related Fc-mediated functions). Such an antigen-binding molecule can be described as not having the related functions.

[1116] As described above, an Fc region / antigen-binding molecule that cannot induce (i.e., is unable to induce) ADCC / ADCP / CDC basically does not cause ADCC / ADCP / CDC activity, for example, as determined by an appropriate assay for the related activity. Similarly, an antigen-binding molecule that "does not bind" to a reference protein (such as a specific Fc receptor or complement protein) may also exhibit basically no binding to the reference protein in an appropriate assay.

[1117] In some embodiments, the antigen-binding molecule does not induce ADCC. In some embodiments, the antigen-binding molecule does not induce ADCP. In some embodiments, the antigen-binding molecule does not induce CDC. In some embodiments, the antigen-binding molecule does not induce ADCC and / or does not induce ADCP and / or does not induce CDC.

[1118] In some embodiments, the antigen-binding molecule does not bind to Fc receptors. In some embodiments, the antigen-binding molecule does not bind to Fcγ receptors. In some embodiments, the antigen-binding molecule does not bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antigen-binding molecule does not bind to FcγRIII (such as FcγRIIIa and / or FcγRIIIb). In some embodiments, the antigen-binding molecule does not bind to FcγRIIIa. In some embodiments, the antigen-binding molecule does not bind to FcγRIIa. In some embodiments, the antigen-binding molecule does not bind to FcγRIIb. In some embodiments, the antigen-binding molecule binds to FcRn. In some embodiments, the antigen-binding molecule does not bind to complement proteins. In some embodiments, the antigen-binding molecule does not bind to C1q. In some embodiments, the amino acid residue corresponding to N297 of the antigen-binding molecule is not glycosylated.

[1119] In some embodiments, the antigen-binding molecule binds to human VISTA, murine VISTA, and / or cynomolgus monkey VISTA; and does not bind to PD-L1, PD-1, B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7), and / or CTLA4 (such as human PD-L1 / PD-1 / B7H3 / VTCN1 / NCR3LG1 / HHLA2 / CTLA4).

[1120] In some embodiments, the K of the antigen-binding molecule described herein binding to VISTA (such as human VISTA, murine VISTA) D is 10 μM or less, preferably ≤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. In some embodiments, the K of the antigen-binding molecule described herein binding to VISTA (such as human VISTA, murine VISTA)D = ≤ 10 nM, ≤ 9 nM, ≤ 8 nM, ≤ 7 nM or ≤ 6 nM, ≤ 5 nM, ≤ 4 nM, ≤ 3 nM, ≤ 2 nM or ≤ 1 nM. In some embodiments, the antigen-binding molecule binds to VISTA (such as human VISTA, mouse VISTA) with a K D = ≤ 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.

[1121] The antigen-binding molecules of the present disclosure can bind to specific target regions of VISTA. According to the art, the antigen-binding regions of antigen-binding molecules can bind to linear epitopes of VISTA, which are composed of contiguous amino acid sequences (i.e., amino acid primary sequences). In some embodiments, the antigen-binding region molecule can bind to a conformational epitope of VISTA, which is composed of discontinuous amino acid sequences of an amino acid sequence.

[1122] In some embodiments, the antigen-binding molecules of the present disclosure are capable of binding to VISTA. In some embodiments, the antigen-binding molecule is capable of binding to VISTA in the extracellular region of VISTA. In some embodiments, the antigen-binding molecule is capable of binding to the Ig-like V-type domain of VISTA (such as the region shown in SEQ ID NO: 6). In some embodiments, the antigen-binding molecule is capable of binding to the region shown in SEQ ID NO: 31 in VISTA.

[1123] In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown 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 shown 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 shown 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 shown 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 shown 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 shown 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 shown in SEQ ID NO:30.

[1124] In some embodiments, the antigen-binding molecule does not bind to the VISTA region that does not bind to IGN175A (as described in WO 2014 / 197849A2). In some embodiments, the antigen-binding molecule does not bind to the VISTA region that binds to the antigen-binding molecule composed of the polypeptide consisting of SEQ ID NO:267 and the polypeptide consisting of SEQ ID NO:268.

[1125] In some embodiments, the antigen-binding molecule does not compete with IGN175A (as described in WO 2014 / 197849A2) for binding to VISTA. In some embodiments, the antigen-binding molecule does not compete with the antigen-binding molecule composed of the polypeptide consisting of SEQ ID NO:267 and the polypeptide consisting of SEQ ID NO:268 for binding to VISTA.

[1126] The ability of a specific antigen-binding molecule to compete with IGN175A or the antigen-binding molecule composed of the polypeptide consisting of SEQ ID NO:267 and the polypeptide consisting of SEQ ID NO:268 for binding to VISTA can be analyzed by, for example, competitive ELISA or epitope sorting (as described in Abdiche et al., J Immunol Methods (2012) 382(1-2):101-116, the entire content of which is incorporated herein by reference). Epitope sorting can be performed by methods such as BLI analysis, as described in Example 8 herein.

[1127] In some embodiments, the antigen-binding molecule is unable to bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO:275.

[1128] As used herein, "peptide" refers to a chain formed by two or more amino acid monomers linked by peptide bonds. The length of a peptide is typically between 2 and 50 amino acids. "Polypeptide" is a polymeric chain composed of two or more peptides. The length of a polypeptide is typically greater than about 50 amino acids.

[1129] The ability of an antigen-binding molecule to bind to a specific peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (such as Western blotting), immunoprecipitation, surface plasmon resonance, and biolayer interferometry.

[1130] In some embodiments, the antigen-binding molecule is capable of binding the same region of VISTA or an overlapping region of VISTA to the region of VISTA that binds to an antibody comprising the VH and VL sequences of any one of the 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.

[1131] In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that is different from the region of VISTA that binds IGN175A (as described in WO 2014 / 197849 A2). In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA that is different from the region of VISTA that binds an antigen-binding molecule comprising a polypeptide consisting of the sequence shown in SEQ ID NO:267 and a polypeptide consisting of the sequence shown in SEQ ID NO:268.

[1132] 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 that binds IGN175A (as described in WO 2014 / 197849 A2). 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 that binds an antigen-binding molecule comprising a polypeptide consisting of the sequence shown in SEQ ID NO:267 and a polypeptide consisting of the sequence shown in SEQ ID NO:268.

[1133] In some embodiments, the antigen-binding molecule binds to VISTA by contacting a residue of VISTA that is different from the VISTA residue contacted by VSTB112 (as described in WO 2015 / 097536 A2). In some embodiments, the antigen-binding molecule binds to VISTA by contacting a residue of VISTA that is different from the VISTA residue contacted by an antigen-binding molecule comprising a polypeptide consisting of the sequence shown in SEQ ID NO: 269 and a polypeptide consisting of the sequence shown in SEQ ID NO: 270).

[1134] In some embodiments, the epitope of the antigen-binding molecule is different from the epitope of VSTB112. In some embodiments, the epitope of the antigen-binding molecule is different from the epitope of an antigen-binding molecule comprising a polypeptide consisting of the sequence shown in SEQ ID NO: 269 and a polypeptide consisting of the sequence shown in SEQ ID NO: 270).

[1135] The region of the antibody that binds to the peptide / polypeptide can be determined by those skilled in the art using various methods known in the art, including X-ray co-crystallography analysis of the antibody-antigen complex, peptide scanning, mutagenesis mapping, mass spectrometry hydrogen-deuterium exchange analysis, phage display, competitive ELISA, and proteolysis-based "protection" methods. These methods are described in, for example, Gershoni et al., BioDrugs 2007, 21(3): 145-156, the entire contents of which are incorporated herein by reference.

[1136] In some embodiments, when VISTA is expressed on the cell surface (i.e., within or on the cell membrane), the antigen-binding molecule of the present disclosure binds to VISTA in a region accessible to the antigen-binding molecule (i.e., the extracellular antigen-binding molecule). In some embodiments, the antigen-binding molecule is capable of binding to VISTA expressed on the cell surface of a cell that expresses VISTA. In some embodiments, the antigen-binding molecule is capable of binding to cells that express VISTA (such as CD14+ monocytes (such as monocyte-derived suppressor cells (MDSC)) and / or CD33+ myeloid cells, tumor-associated macrophages (TAM), and neutrophils).

[1137] When analyzing the ability of an antigen-binding molecule to bind to a specific cell type, the antigen-binding molecule can be contacted with the cells, and then the antigen-binding molecule bound to the cells can be detected, for example, after a washing step to remove unbound antigen-binding molecules. The ability of the antigen-binding molecule to bind to cells expressing immune cell surface molecules and / or cancer cell antigen-expressing cells can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[1138] The antigen-binding molecules of the present disclosure can be antagonists of VISTA. In some embodiments, the antigen-binding molecules are capable of inhibiting a function or process (such as interaction, signal transduction, or other functions) mediated by VISTA and / or a VISTA interaction partner (such as LRIG1, VSIG3, PSGL-1, VSIG8). Herein, "inhibit" means a reduction, decrease, or attenuation relative to a control condition. An antigen-binding molecule that inhibits a particular interaction / activity / process can be referred to as an inhibitor or antagonist of that interaction / activity / process, and can also be said to "block" or "neutralize" that interaction / activity / process.

[1139] The antigen-binding molecules that bind to VISTA described herein are capable of inhibiting VISTA-mediated functions / processes by a mechanism that does not require Fc-mediated functions (such as ADCC, ADCP, and CDC). This means that the antigen-binding molecules that bind to VISTA described herein are capable of inhibiting the immunosuppressive activity of VISTA-expressing cells without inducing ADCC, ADCP, and / or CDC.

[1140] In particular, the antigen-binding molecules that bind to VISTA described herein are capable of inhibiting VISTA by a mechanism that does not require binding to Fcγ receptors and / or binding to C1q.

[1141] In some embodiments, the antigen-binding molecules of the present disclosure are capable of inhibiting the interaction between VISTA and a VISTA interaction partner (such as LRIG1, VSIG3, PSGL-1, VSIG8).

[1142] In some embodiments, the antigen-binding molecules are capable of inhibiting the interaction between VISTA and a VISTA interaction partner that binds to the C-C' region of VISTA.

[1143] In some embodiments, a VISTA interaction partner that binds to the C-C' region of VISTA binds to the VISTA region shown in SEQ ID NO: 344. In some embodiments, a VISTA interaction partner that binds to the C-C' region of VISTA binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 344. In some embodiments, a VISTA interaction partner that binds to the C-C' region of VISTA contacts the VISTA region shown in SEQ ID NO: 344. In some embodiments, a VISTA interaction partner that binds to the C-C' region of VISTA binds to VISTA by contacting one or more amino acids in the region shown in SEQ ID NO: 344.

[1144] In some embodiments, the VISTA interaction partner that binds to the C-C’ region of VISTA is selected from: LRIG1 and VSIG3. In some embodiments, the VISTA interaction partner is LRIG1. In some embodiments, the VISTA interaction partner is VSIG3.

[1145] In some embodiments, the antigen-binding molecules of the present disclosure are capable of inhibiting the interaction between VISTA and LRIG1. In some embodiments, the antigen-binding molecules of the present disclosure are capable of inhibiting the interaction between VISTA and PSGL-1. In some embodiments, the antigen-binding molecules of the present disclosure are capable of inhibiting the interaction between VISTA and VSIG3.

[1146] The ability of an antigen-binding molecule to inhibit the interaction between two factors can be determined by analyzing the interaction in the presence of the antibody / fragment, or the interaction after one or both of the interaction partners are incubated with the antibody / fragment. Assays for determining whether a particular antigen-binding molecule can inhibit the interaction between two interaction partners include competitive ELISA and SPR assays.

[1147] The ability of an antigen-binding molecule to inhibit a particular interaction (e.g., the interaction between VISTA and a VISTA interaction partner) can be determined by observing a decrease in the level of interaction of the interaction partners in the presence of the antigen-binding molecule, or after one or both of the interaction partners are incubated 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). Suitable assays can be performed in vitro, for example, using recombinant interaction partners or cells expressing the interaction partners. The cells expressing the interaction partners can be endogenously expressed or expressed by nucleic acids introduced into the cells. For such assays, one or both of the interaction partners and / or the antigen-binding molecule can be labeled or used in conjunction with a detectable entity to detect and / or measure the level of interaction.

[1148] 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 an interaction. For example, downstream functional consequences of the interaction between VISTA and a VISTA interaction partner may include VISTA-mediated signal transduction. For example, the ability of an antigen-binding molecule to inhibit the interaction between VISTA and a VISTA interaction partner can be determined by analyzing the production of IL-2, IFN-γ, and / or IL-17 in an MLR assay.

[1149] In some embodiments, the antigen-binding molecules of the present disclosure are capable of inhibiting the interaction between VISTA and its interaction partners to less than 1-fold, such as ≤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 level of interaction between VISTA and its binding partner in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1150] In some embodiments, the antigen-binding molecule inhibits VISTA-mediated signal transduction. In some embodiments, VISTA-mediated signal transduction may be signal transduction mediated by a polypeptide complex comprising VISTA. In some embodiments, VISTA-mediated signal transduction may be signal transduction mediated by a polypeptide complex comprising VISTA and its interaction partners (such as LRIG1, VSIG3, PSGL-1, VSIG8). In some embodiments, VISTA-mediated signal transduction may be signal transduction mediated by a polypeptide complex comprising VISTA and an interaction partner of VISTA that binds to the C-C’ region of VISTA (such as LRIG1 or VSIG3). In some embodiments, VISTA-mediated signal transduction may be signal transduction mediated by a polypeptide complex comprising VISTA and LRIG1. In some embodiments, VISTA-mediated signal transduction may be signal transduction mediated by a polypeptide complex comprising VISTA and VSIG3.

[1151] VISTA-mediated signal transduction can be analyzed by methods such as the detection of the number / activity of effector immune cells, such as the MLR assay described in the exemplary embodiments herein. Inhibition of VISTA-mediated signal transduction can be determined by detecting an increase in the number and / or activity of effector immune cells, for example, by an increase in the production of IL-2, IFN-γ, and / or IL-17.

[1152] In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signal transduction by a mechanism that does not require or involve Fc-mediated functions. In some embodiments, the antigen-binding molecule inhibits VISTA-mediated signal transduction independent of Fc-mediated functions. This means that, in some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signal transduction in a manner independent of the Fc region.

[1153] The ability of an antigen-binding molecule to inhibit VISTA-mediated signaling through a mechanism that does not require / does not involve Fc-mediated functions can be evaluated, for example, by analyzing the ability of an antigen-binding molecule provided in a form lacking a functional Fc region to inhibit VISTA-mediated signaling. For example, an antigen-binding molecule containing a "silent" Fc region (such as containing the LALAPG substitution) or an antigen-binding molecule provided in a form lacking an Fc region (such as scFv, Fab, etc.) can be used to study the effect on VISTA-mediated signaling.

[1154] In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not involve ADCC. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not involve ADCP. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not involve CDC.

[1155] In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through 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 through 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 through 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 through a mechanism that does not require binding of the antigen-binding molecule to FcγRIIIa. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not require binding of the antigen-binding molecule to FcγRIIa. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not require binding of the antigen-binding molecule to FcγRIIb. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not require binding of the antigen-binding molecule to a complement protein. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not require binding of the antigen-binding molecule to C1q. In some embodiments, the antigen-binding molecule is capable of inhibiting VISTA-mediated signaling through a mechanism that does not require N297 glycosylation.

[1156] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the killing of VISTA-expressing cells. The killing of VISTA-expressing cells can be increased through the effector functions of the antigen-binding molecules. In embodiments of antigen-binding molecules that include an Fc region, the antigen-binding molecules can increase 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).

[1157] An antigen-binding molecule capable of increasing the killing of VISTA-expressing cells can be determined by observing an increase in the killing of VISTA-expressing cells in the presence of the antigen-binding molecule, or after incubation of VISTA-expressing cells with the antigen-binding molecule, compared to the cell killing detected in the absence of the antigen-binding molecule (or with a suitable control antigen-binding molecule) in an appropriate assay. Methods for detecting 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 VISTA-expressing cells that are alive and / or not alive after exposure to different treatment conditions.

[1158] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of killing of VISTA-expressing cells (such as VISTA-expressing MDSC) to more than 1-fold, such as ≥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 with a suitable control antigen-binding molecule).

[1159] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the number of VISTA-expressing cells (such as VISTA-expressing MDSC) to less than 1-fold, such as ≤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 (such as VISTA-expressing MDSC, TAM, neutrophils) detected after incubation in the absence of the antigen-binding molecule (or after incubation with a suitable control antigen-binding molecule) in a comparable experiment.

[1160] In some embodiments, the antigen-binding molecule is a non-depleting antigen-binding molecule. This means that, 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 / increase ADCC, ADCP, and / or CDC against VISTA-expressing cells.

[1161] In some embodiments, the antigen-binding molecules of the present disclosure do not induce / increase the killing of VISTA-expressing cells, such as in embodiments where the antigen-binding molecule lacks an Fc region, or in embodiments where the antigen-binding molecule comprises an Fc region that cannot induce Fc-mediated antibody effector functions. In some embodiments, the antigen-binding molecules of the present disclosure cannot reduce the number / proportion of VISTA-expressing cells.

[1162] In some embodiments, the antigen-binding molecules of the present disclosure (i) inhibit VISTA-mediated signal transduction and (ii) do not induce / increase the killing of VISTA-expressing cells. In some embodiments, the antigen-binding molecules of the present disclosure (i) inhibit VISTA-mediated signal transduction and (ii) do not reduce the number / proportion of VISTA-expressing cells.

[1163] This can be particularly advantageous because VISTA is expressed by cells that should not be depleted. For example, VISTA is expressed at low levels in immune cells such as certain types of T cells and dendritic cells, and thus it is not advisable to kill or reduce their number / proportion.

[1164] In some embodiments, relative to negative control conditions, the antigen-binding molecules of the present disclosure are capable of increasing the number and / or activity of effector immune cells, such as in appropriate in vitro or in vivo experiments. By way of explanation, the antigen-binding molecules of the present disclosure may be capable of rendering effector immune cells resistant to MDSC-mediated inhibition 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+ CTL), CD4+ T cells, CD4+ T helper cells, NK cells, IFNγ-producing cells, memory T cells, central memory T cells, antigen-experienced T cells, or CD45RO+ T cells.

[1165] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the number of effector immune cells to greater than 1-fold the number observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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. In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of a factor associated with effector immune cell activity to greater than 1-fold the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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.

[1166] Cell numbers and proportions can be determined by methods such as flow cytometry, using antibodies that detect cell types. Cell proliferation can be analyzed by in vitro assays 3 such as H-thymidine incorporation or CFSE dilution assays, as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, the entire content of which is incorporated herein by reference. Effector immune cell activity can be analyzed by measuring factors associated with such activity. In some embodiments, effector immune cell activity can be determined by analyzing the production of IL-2, IFN-γ, and / or IL-17.

[1167] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of immunosuppression mediated by VISTA-expressing cells. Changes in the level of immunosuppression can be determined by measuring the expression of arginase 1 and / or the production of reactive oxygen species (ROS) by VISTA-expressing cells, as described in Ochoa et al., Ann Surg. 2001, March; 233(3):393–399 and Dikalov and Harrison, Antioxid Redox Signal. 2014, January 10; 20(2):372–382.

[1168] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing antigen presentation by antigen-presenting cells, as determined by appropriate assays for antigen presentation. In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing phagocytosis by phagocytic cells (such as neutrophils, monocytes, macrophages, mast cells, and / or dendritic cells), as determined using appropriate assays for phagocytosis levels.

[1169] In some embodiments, relative to a negative control condition, the antigen-binding molecules of the present disclosure are capable of increasing the number and / or activity of antigen-presenting cells (such as CD11b+ MHCII+ cells), such as in appropriate in vitro or in vivo (such as in a tumor) experiments. In some embodiments, relative to a negative control condition, the antigen-binding molecules of the present disclosure are capable of increasing the number and / or activity of macrophages (such as CD11b+ F4 / 80+ cells), such as in appropriate in vitro or in vivo (such as in a tumor) experiments. In some embodiments, relative to a negative control condition, the antigen-binding molecules are capable of increasing the number and / or activity of dendritic cells (such as CD11c+ cells), such as in appropriate in vitro or in vivo (such as in a tumor) experiments.

[1170] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the number of the cell types described above to greater than 1-fold the number observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), such as ≥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. In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of a cell type activity-related substance to greater than 1-fold the level observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), such as ≥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.

[1171] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the production of IL-6 by immune cells. The immune cells can be, for example, PBMC, 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 the production of IL-6 by immune cells after stimulation with, for example, LPS. The ability of the antigen-binding molecules to increase the production of IL-6 by immune cells can be analyzed in in vitro assays, as described in Example 10 herein. Such an assay can include stimulating monocytes (such as THP1 cells) with LPS and incubating the stimulated cells with the antigen-binding molecules.

[1172] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the production of IL-6 by immune cells to greater than 1-fold, such as ≥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, of the level observed in the absence of the antigen-binding molecules (or in the presence of a suitable control antigen-binding molecule).

[1173] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the number and / or activity of Th1 / Th17 cells. In some embodiments, the antigen-binding molecules are capable of upregulating the Th1 / Th17 response. In some embodiments, the antigen-binding molecules favor the Th1 / Th17 response over the Th2 response. In some embodiments, in a mixed lymphocyte reaction (MLR) assay, the antigen-binding molecules of the present disclosure are capable of increasing the proliferation of T cells, the production of IL-2, the production of IFN-γ, the production of TNFα, and / or the production of IL-17A. The MLR assay can be performed as described by Bromelow et al., J. Immunol. Methods, 2001, Jan. 1; 247(1-2):1-8 or as described in the exemplary embodiments herein. The production of IL-2, IFNγ, and / or IL-17 can be analyzed by antibody-based methods well known to those skilled in the art, such as immunoblotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter factor-based methods.

[1174] In some embodiments, in an MLR experiment, the antigen-binding molecules of the present disclosure are capable of increasing the proliferation of T cells (such as Th1 / Th17 cells), IL-2 production, IFN-γ production, and / or IL-17A production by more than 1-fold, such as ≥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 over the levels observed in the absence of the antigen-binding molecules (or in the presence of a suitable control antigen-binding molecule).

[1175] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the proliferation of T cells (such as Th1 / Th17 cells), the production of IFN-γ, and / or the production of TNFα, such as in the presence of VISTA / VISTA-expressing cells. These properties of the antigen-binding molecules can be evaluated in in vitro experiments as described in the exemplary embodiments herein.

[1176] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing (such as in the presence of VISTA / VISTA-expressing cells) the proliferation of T cells (such as Th1 / Th17 cells), IFN-γ production, and / or TNFα production by more than 1-fold over the levels observed in the absence of the antigen-binding molecules (or in the presence of a suitable control antigen-binding molecule), such as ≥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.

[1177] In some embodiments, compared to VISTA-binding antibodies disclosed in the prior art (such as VSTB112, as described in WO 2015 / 097536A2), the antigen-binding molecules of the present disclosure are capable of enhancing T cell (such as CD4+ T cells and / or CD8+ T cells, such as Th1 / Th17 cells) proliferation to a greater extent. T cell proliferation can be evaluated in in vitro experiments, as described in Example 9 herein, by culturing to stimulate T cell proliferation in the presence of an agonist anti-CD3 antibody. In some embodiments, the antigen-binding molecules of the present disclosure are capable of enhancing T cell proliferation in such experiments to greater than 1-fold the level induced by VISTA-binding antibodies (such as VSTB112) in the prior art, such as ≥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.

[1178] In some embodiments, the antigen-binding molecules of the present disclosure are capable of enhancing T cell-mediated cancer cell lysis to greater than 1-fold the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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.

[1179] In some embodiments, the antigen-binding molecules of the present disclosure are capable of enhancing T cell-mediated cancer cell lysis, such as in the presence of VISTA / VISTA-expressing cells. These properties of the antigen-binding molecule can be evaluated in in vitro experiments, as described in the exemplary embodiments herein.

[1180] In some embodiments, the antigen-binding molecules of the present disclosure are capable of enhancing T cell-mediated lysis (such as in the presence of VISTA / VISTA-expressing cells) to greater than 1-fold the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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.

[1181] In some embodiments, compared to VISTA-binding antibodies (such as VSTB112 as described in WO 2015 / 097536A2) disclosed in the prior art, the antigen-binding molecules of the present disclosure are capable of increasing IL-6 production by THP1 cells to a greater extent. The production of IL-6 by THP1 cells can be evaluated in in vitro experiments, as described in Example 10 herein, and may involve stimulating THP1 cells with LPS. In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing IL-6 production in such experiments to greater than 1-fold the level induced by VISTA-binding antibodies (such as VSTB112) in the prior art, such as ≥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.

[1182] In some embodiments, the antigen-binding molecules of the present disclosure are capable of: reducing the number and / or activity of inhibitory immune cells, inhibiting the proliferation of inhibitory immune cells, and / or reducing the proportion of inhibitory immune cells in a cell population (such as CD45+ cells, such as CD45+ cells obtained from a tumor) relative to a control condition, as determined in a suitable in vitro or in vivo experiment.

[1183] The inhibitory immune cells can be, for example, VISTA-expressing cells, Arg-1-expressing cells, MDSCs, granulocytic MDSCs (g-MDSCs) or monocytic MDSCs (m-MDSCs). In some embodiments, the inhibitory immune cells are CD11b+GR1+MHCII− cells.

[1184] In some embodiments, reducing the number / activity / proliferation / proportion to less than 1-fold the number / activity / proliferation / proportion observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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.

[1185] In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / percentage of inhibitory immune cells by a mechanism that does not involve Fc-mediated functions. In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / percentage of inhibitory immune cells in a manner independent of Fc-mediated functions (e.g., in a manner unrelated to the Fc region). In some embodiments, the antigen-binding molecule is capable of reducing the number / activity / proliferation / percentage of inhibitory immune cells by 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 / percentage of inhibitory immune cells by a mechanism that does not involve the depletion of VISTA-expressing cells.

[1186] In some embodiments, the antigen-binding molecules of the present disclosure inhibit the development and / or progression of cancer in vivo.

[1187] In some embodiments, the antigen-binding molecule can increase the killing of cancer cells by effector immune cells and the like. In some embodiments, the antigen-binding molecule can reduce the number of cancer cells in vivo, e.g., as compared to appropriate control conditions. In some embodiments, the antigen-binding molecule can inhibit tumor growth, e.g., as determined by measuring tumor size / volume over time.

[1188] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the levels of IFN-γ and / or IL-23 in the sera of mice treated with the antigen-binding molecule. The levels of IFN-γ and / or IL-23 in the sera can be analyzed by methods such as ELISA on sera from blood samples obtained from the mice. In some embodiments, administration of the antigen-binding molecules of the present disclosure increases the levels of IFN-γ and / or IL-23 in the sera to greater than 1-fold the levels observed in the absence of administration of the antigen-binding molecule (or the levels observed in the presence of administration of an appropriate control antigen-binding molecule), such as ≥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.

[1189] The ability of the disclosed antigen-binding molecules to inhibit cancer development and / or progression could be analyzed in a suitable in vivo model, such as a cell line-derived xenograft model, e.g., a CT26 cell-derived model, a 4T-1 cell-derived model, an LL2 cell-derived model, a B16 cell-derived model, or an EL4 cell-derived model. The cancer can be a cancer that expresses VISTA and / or is pathologically associated with MDSCs (such as VISTA-expressing MDSCs, TAMs, neutrophils). Cancers that are "pathologically associated" with MDSCs include cancers in which an increase in the number / proportion of MDSCs is positively correlated with the occurrence, development, or progression of cancer and / or the severity of one or more symptoms of cancer, or cancers in which an increase in the number / proportion of MDSCs is a risk factor for the occurrence, development, or progression of cancer. The cancer can include MDSCs in the affected organ / tissue (such as the organ / tissue showing the disease / symptoms) or tumor.

[1190] In some embodiments, administration of the disclosed antigen-binding molecules can result in one or more of the following: inhibition of cancer development / progression, delay / prevention of cancer occurrence, reduction / delay / prevention of tumor growth, reduction / delay / prevention of metastasis, alleviation of the severity of cancer symptoms, reduction of the number of cancer cells, reduction of tumor size / volume, and / or improvement of survival rate (such as progression-free survival), as determined in a CT26 cell, 4T-1 cell, LL2 cell, B16 cell, or EL4 cell-derived xenograft model.

[1191] In some embodiments, administration of the disclosed antigen-binding molecules is capable of inhibiting tumor growth to greater than 5% of that observed in the absence of administration of the antigen-binding molecule (or after administration of a suitable control antigen-binding molecule), such as ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90%, or ≥95%.

[1192] In some embodiments, in the CT26 cell-derived model of the exemplary embodiments of the present disclosure, the antigen-binding molecule is administered at the doses and cycles described in the examples, inhibiting tumor growth to greater than 5% of that observed in the absence of administration of the antigen-binding molecule (or after administration of an appropriate control antigen-binding molecule), such as ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75% or ≥80%. In some embodiments, in the 4T-1 cell-derived model of the exemplary embodiments of the present disclosure, the antigen-binding molecule is administered at the doses and cycles described in the examples, inhibiting tumor growth to greater than 5% of that observed in the absence of administration of the antigen-binding molecule (or after administration of an appropriate control antigen-binding molecule), such as ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45% or ≥50%.

[1193] In some embodiments, administration of the antigen-binding molecule according to the present disclosure is not associated with cytokine release syndrome. In some embodiments, administration of the antigen-binding molecule according to the present disclosure is not associated with systemic activation of leukocytes such as B cells, T cells, NK cells, macrophages, dendritic cells, and / or monocytes. In some embodiments, administration of the antigen-binding molecule is not associated with systemic upregulation of expression of inflammatory cytokines and / or chemokines such as IL-6, IFN-γ, IL-8, IL-10, GM-CSF, MIP-1α / β, MCP-1, CXCL9, and / or CXCL10.

[1194] Aspects and embodiments of the present disclosure particularly relate to antigen-binding molecules capable of inhibiting the interaction between VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C' region of VISTA.

[1195] In some embodiments, an antigen-binding molecule according to the present disclosure binds to VISTA in a region that is bound by a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA. In some embodiments, the antigen-binding molecule is a VISTA competitive inhibitor that binds to a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA. In some embodiments, the antigen-binding molecule is a VISTA allosteric inhibitor that binds to a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA. In some embodiments, the antigen-binding molecule displaces a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA. In some embodiments, the antigen-binding molecule does not bind to a complex containing VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA.

[1196] The ability of an antigen-binding molecule to inhibit the interaction between VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA can be determined, for example, by analyzing the interaction in the presence of the antigen-binding molecule or after incubating one or both of the interaction partners with the antigen-binding molecule. Assays for determining whether a particular antigen-binding molecule is capable of inhibiting the interaction between VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA include competitive ELISA assays and SPR analyses.

[1197] An antigen-binding molecule capable of inhibiting the interaction between VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA can be determined by observing a decrease in the level of interaction between the interaction partners in the presence of the antigen-binding molecule or after incubating 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 analyses can be performed in vitro, for example, using recombinant interaction partners or cells expressing the interaction partners. The cells expressing the interaction partners can be endogenous or can be obtained by introducing nucleic acid into the cells. For performing such assays, one or both of the interaction partners and / or the antigen-binding molecule can be labeled or conjugated to a detectable entity to detect and / or measure the level of interaction.

[1198] For example, Example 2 of the present disclosure describes an ELISA experiment of VISTA in combination with an antigen-binding molecule, and analyzes its ability to inhibit the interaction between VISTA and LRIG1. Briefly, the wells of a plate are coated with Fc-labeled human VISTA protein. After blocking and incubation with the VISTA antigen-binding molecule, HIS-labeled LRIG1 is added to the wells. The captured HIS-labeled LRIG1 is detected using an HRP-conjugated anti-HIS antibody, and then developed with 3,3’,5,5’-tetramethylbenzidine to detect the VISTA-LRIG1 complex. In this detection method, based on the detected HRP activity level being lower than the HRP activity level observed for an isotype-matched antigen-binding molecule that does not bind to VISTA under control conditions, it can be inferred that a specific VISTA-binding antigen-binding molecule inhibits the interaction between VISTA and LRIG1.

[1199] In some embodiments, in a specific assay, an antigen-binding molecule according to the present disclosure inhibits the interaction between VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA to less than 1-fold, such as ≤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 interaction level observed in the absence of the antigen-binding molecule or in the presence of an equivalent amount of a suitable control antigen-binding molecule known not to inhibit the interaction between VISTA and the VISTA interaction partner.

[1200] In some embodiments, an antigen-binding molecule according to the present disclosure inhibits the interaction between VISTA and a VISTA interaction partner (such as LRIG1 or VSIG3) that binds to the C-C’ region of VISTA, and the IC 50 (as determined by ELISA, such as the ELISA described in the examples of the present disclosure) is 1 μM or less, such as any one of ≤500 nM, ≤100 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤10 nM, ≤5 nM, ≤4 nM.

[1201] Aspects and embodiments of the present disclosure particularly relate to antigen-binding molecules capable of inhibiting the interaction between VISTA and LRIG1.

[1202] In some embodiments, an antigen-binding molecule according to the present disclosure binds to VISTA in the region bound by LRIG1. In some embodiments, the antigen-binding molecule is a competitive inhibitor of the binding of LRIG1 to VISTA. In some embodiments, the antigen-binding molecule is an allosteric inhibitor of the binding of LRIG1 to VISTA. In some embodiments, the antigen-binding molecule replaces LRIG1 in a complex containing VISTA and LRIG1. In some embodiments, the antigen-binding molecule does not bind to a complex containing VISTA and LRIG1.

[1203] In some embodiments, in a specific assay, an antigen-binding molecule according to the present disclosure inhibits the interaction between VISTA and LRIG1 to less than 1-fold, such as ≤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 interaction level observed in the absence of the antigen-binding molecule or in the presence of an equivalent amount of a suitable control antigen-binding molecule known not to inhibit the interaction between VISTA and LRIG1.

[1204] In some embodiments, an antigen-binding molecule according to the present disclosure inhibits the interaction between VISTA and LRIG1 with an IC 50 (as determined by ELISA, such as the ELISA described in the examples of the present disclosure) of 1 μM or less, such as any one of ≤500 nM, ≤100 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, ≤10 nM, ≤5 nM, ≤4 nM.

[1205] Aspects and embodiments of the present disclosure particularly relate to antigen-binding molecules capable of inhibiting the interaction between VISTA and VSIG3.

[1206] In some embodiments, an antigen-binding molecule according to the present disclosure binds to VISTA in the region bound by VSIG3. In some embodiments, the antigen-binding molecule is a competitive inhibitor of the binding of VSIG3 to VISTA. In some embodiments, the antigen-binding molecule is an allosteric inhibitor of the binding of VSIG3 to VISTA. In some embodiments, the antigen-binding molecule replaces VSIG3 in a complex containing VISTA and VSIG3. In some embodiments, the antigen-binding molecule does not bind to a complex containing VISTA and VSIG3.

[1207] In some embodiments, in a particular assay, an antigen-binding molecule according to the present disclosure inhibits the interaction between VISTA and VSIG3 to less than 1-fold, such as ≤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 interaction level observed in the absence of the antigen-binding molecule or in the presence of an equivalent amount of a suitable control antigen-binding molecule known not to inhibit the interaction between VISTA and VSIG3.

[1208] In some embodiments, the antigen-binding molecule according to the present disclosure inhibits the interaction between VISTA and VSIG3 with an IC 50 (as determined by ELISA, such as the ELISA described in the examples of the present disclosure) of 1 μM or less, such as any 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, ≤900 pM, ≤800 pM, ≤700 pM.

[1209] In some embodiments, relative to a negative control condition, the antigen-binding molecule of the present disclosure is capable of increasing the number and / or proportion of antigen-specific CD8+ T cells (such as gp70+ CD8+ T cells), such as in a suitable in vitro or in vivo experiment. The ability of the antigen-binding molecule to increase the number and / or proportion of antigen-specific CD8+ T cells can be analyzed by an assay method, such as that described in Example 8 herein. Such a method can include performing an immune profiling of a tumor in a non-human animal cancer model (such as a cell line-derived mouse model) after administration of the antigen-binding molecule.

[1210] In some embodiments, the antigen-binding molecule of the present disclosure is capable of increasing the number of antigen-specific CD8+ T cells to exceed the number observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1211] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the number or proportion of antigen-specific CD8+ T cells to greater than 1-fold, such as ≥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, ≥10-fold, ≥20-fold, ≥30-fold, ≥40-fold, or ≥50-fold of the number / proportion of antigen-specific CD8+ T cells observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule). In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the number or proportion of antigen-specific CD8+ T cells to greater than 1%, such as at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the number / proportion of antigen-specific CD8+ T cells observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1212] The cell number and proportion can be determined by methods such as flow cytometry, which uses antibodies that can detect cell types. Antigen-specific CD8+ T cells can be determined by the presence and / or absence of one or more cell markers (such as gp70+, CD8+). For example, Example 8 illustrates determining the proportion of tumor antigen-specific CD8+ T cells by flow cytometry. Antigen-specific CD8+ T cells can be identified by using peptide-MHC multimers (pMHC multimers) that contain the antigen of interest, such as an antigen expected to be found in cancer / tumor.

[1213] In some embodiments, relative to a negative control condition, the antigen-binding molecules of the present disclosure are capable of increasing the activity of antigen-specific CD8+ T cells (such as cytotoxic CD8+ T cells), such as in a suitable in vitro or in vivo experiment. The ability of the antigen-binding molecule to increase CD8+ T cell activity can be analyzed by detection methods, as described in Example 8 herein. Such methods can include performing an immunoprofile analysis of tumors in a non-human animal cancer model (such as a cell line-derived mouse model) after administration of the antigen-binding molecule.

[1214] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing CD8+ T cell activity to greater than 1-fold the CD8+ T cell activity observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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, ≥10-fold. In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of factors related to CD8+ T cell activity to greater than 1-fold the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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, ≥10-fold.

[1215] In some embodiments, CD8+ T cell activity can be analyzed by measuring factors related to such activity. In some embodiments, CD8+ T cell activity can be determined by analyzing the production of granzyme B, CX3CR1, ICOS, CD27. For example, Example 8 illustrates determining CD8+ T cell activity by flow cytometry to measure the proportion of cells expressing granzyme B, CX3CR1, ICOS, and / or CD27.

[1216] In some embodiments, relative to negative control conditions, the antigen-binding molecules of the present disclosure are capable of upregulating one or more cytotoxicity-related markers, such as in a suitable in vitro or in vivo experiment. The ability of the antigen-binding molecule to upregulate one or more cytotoxicity-related markers can be analyzed by a detection method, such as that described in Example 8 herein. Such a method can include performing an immunoprofile analysis of tumors in a non-human animal cancer model (such as a cell line-derived mouse model) after administration of the antigen-binding molecule or a suitable control antigen-binding molecule.

[1217] In some embodiments, the antigen-binding molecules of the present disclosure are capable of upregulating one or more cytotoxicity-related markers to a level greater than 1-fold, such as ≥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, ≥10-fold of the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule). In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of one or more cytotoxicity-related markers to a level greater than 1-fold, such as ≥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, ≥10-fold of the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule).

[1218] Cytotoxicity-related markers are well known to those skilled in the art and include granzyme B (i.e., GZMB), CX3CR1, ICOS, CD27, TNFa, INFγ, IL-2, CXCR3, TBX21, IL-4, CCR4, GATA3, IL-9, IL-10, IRF4, CCR6, KLRB1, IL-17, IRF4, RORc.

[1219] In some embodiments, the antigen-binding molecules of the present disclosure are capable of upregulating one or more genes related to pro-inflammatory macrophage activation, such as in a suitable in vitro or in vivo experiment. The ability of the antigen-binding molecule to upregulate one or more genes related to pro-inflammatory macrophage activation can be analyzed by detection methods, such as those described in Example 9 herein. Such methods can include analyzing the transcriptome of a tumor cell line or tumor after administration of the antigen-binding molecule or a suitable control antigen-binding molecule.

[1220] In some embodiments, the antigen-binding molecules of the present disclosure are capable of upregulating one or more pro-inflammatory macrophage activation-related genes to a level greater than 1-fold of the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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, ≥10-fold.

[1221] In some embodiments, the one or more pro-inflammatory macrophage activation-related genes may be selected from those Figure 18 as shown. Gene expression can be determined by methods well known to those skilled in the art. The RNA levels encoding one or more pro-inflammatory macrophage activation-related genes can be determined by techniques such as RT-qPCR, northern blot hybridization, etc.

[1222] In some embodiments, the antigen-binding molecules of the present disclosure are capable of upregulating one or more cytotoxicity-related genes of T cells, such as in suitable in vitro or in vivo experiments. The ability of the antigen-binding molecule to upregulate one or more cytotoxicity-related genes of T cells can be analyzed by detection methods, such as those described in Example 9 herein. Such methods may include analyzing the transcriptome of a tumor cell line or tumor after administration of the antigen-binding molecule or a suitable control antigen-binding molecule.

[1223] In some embodiments, the antigen-binding molecules of the present disclosure are capable of upregulating one or more cytotoxicity-related genes of T cells to a level greater than 1-fold of the level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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, ≥10-fold.

[1224] In some embodiments, the one or more cytotoxicity-related genes of the T cells may be selected from those Figure 18 as shown. Gene expression can be determined by methods well known to those skilled in the art. The RNA levels encoding one or more cytotoxicity-related genes of T cells can be determined by techniques such as RT-qPCR, northern blot hybridization, etc.

[1225] In some embodiments, relative to a negative control condition, the antigen-binding molecules of the present disclosure are capable of increasing granzyme B produced by immune cells, such as in a suitable in vitro or in vivo experiment. The ability of the antigen-binding molecule to increase granzyme B production by immune cells can be analyzed by a detection method, such as that described in Example 8 herein. Such a method can include performing an immune profiling of a tumor in a non-human animal cancer model (such as a cell line-derived mouse model) after administration of the antigen-binding molecule or a suitable control antigen-binding molecule.

[1226] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing granzyme B produced by immune cells to greater than 1-fold of the granzyme B produced by immune cells observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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, ≥10-fold. In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the level of granzyme B produced by immune cells to greater than 1-fold of the granzyme B level observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≥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, ≥10-fold.

[1227] In some embodiments, the antigen-binding molecules of the present disclosure are capable of increasing the production of granzyme B in a mixed lymphocyte reaction (MLR) assay. The MLR assay can be performed according to the method described in Bromelow et al., J. Immunol Methods, 2001, Jan. 1; 247(1-2):1-8 (the entire content of which is incorporated herein by reference). The production of Granzyme B can be analyzed by antibody-based methods well known to those skilled in the art, such as immunoblotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter factor-based methods.

[1228] In some embodiments, relative to negative control conditions, the antigen-binding molecules of the present disclosure are capable of reducing the level of T cell exhaustion, such as in suitable in vitro or in vivo experiments. The ability of the antigen-binding molecules to reduce the level of T cell exhaustion can be analyzed by detection methods, as described in Example 10 herein. Such methods can include performing an immune profiling of tumors in a non-human animal cancer model (such as a cell line-derived mouse model) after administration of the antigen-binding molecule or a suitable control antigen-binding molecule.

[1229] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the level of T cell exhaustion to less than 1-fold of the level of T cell exhaustion observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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. In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the level of a T cell exhaustion-related factor to less than 1-fold of the level of the T cell exhaustion-related factor observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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.

[1230] In some embodiments, T cell exhaustion can be analyzed by determining the number and / or proportion of exhausted T cells. The cell number and proportion can be determined by methods such as flow cytometry analysis, and the antibodies used in flow cytometry can detect cell types. Exhausted T cells can be identified by the presence and / or absence of one or more cell markers, such as gp70+, CD8+, PD-1+, IL-7Ra-. For example, Example 8 illustrates the use of flow cytometry analysis to detect gp70+, CD8+, PD-1+, IL-7Ra- cells to determine the proportion of exhausted T cells.

[1231] In some embodiments, T cell exhaustion can be analyzed by measuring T cell exhaustion-related factors. In some embodiments, T cell exhaustion can be determined by the presence and / or absence of T cell exhaustion cell markers, such as PD-1+, LAG-3+, TIM-3+, IL-7Ra-.

[1232] In some embodiments, relative to negative control conditions, the antigen-binding molecules of the present disclosure are capable of reducing the number and / or proportion of tumor-associated macrophages (TAMs), such as in a suitable in vitro or in vivo assay. The ability of the antigen-binding molecules to reduce the number and / or proportion of tumor-associated macrophages can be analyzed by detection methods, such as those described in Example 8 herein. Such methods can include performing an immune profiling of tumors in a non-human animal cancer model (such as a cell line-derived mouse model) after administration of the antigen-binding molecule or a suitable control antigen-binding molecule.

[1233] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the number of tumor-associated macrophages to less than 1-fold the number of tumor-associated macrophages observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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.

[1234] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the proportion of tumor-associated macrophages to less than 1-fold the proportion of tumor-associated macrophages observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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.

[1235] The cell number and proportion can be determined by methods such as flow cytometry analysis. The antibodies used can detect cell types, and tumor-associated macrophages can be identified by the presence and / or absence of one or more cell markers (such as CD45+, F4 / 80+, MHCII-). TAM can present as M2-phenotype acquired macrophages, such as showing the characteristics of M2 macrophages (also known as alternatively activated macrophages). TAM can secrete anti-inflammatory cytokines (such as IL-10, IL-13, and IL-4), express arginase-1, express mannose receptor (MR, CD206), and / or express scavenger receptors such as MARCO. Tumor-associated macrophages are as described in Lin et al., Journal of Hematology & Oncology (2019) 12:76 (the entire content of which is incorporated herein by reference). For example, Example 8 illustrates the use of flow cytometry analysis to detect CD45+F4 / 80+MHCII- cells to determine the proportion of tumor-associated macrophages.

[1236] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the activity of tumor-associated macrophages. In some embodiments, relative to a negative control condition, the antigen-binding molecules of the present disclosure are capable of reducing the activity of tumor-associated macrophages, such as in a suitable in vitro or in vivo experiment.

[1237] In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the activity of tumor-associated macrophages to less than 1-fold of the activity of tumor-associated macrophages observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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. In some embodiments, the antigen-binding molecules of the present disclosure are capable of reducing the level of a relevant factor of tumor-associated macrophage activity to less than 1-fold of the level of the relevant factor of tumor-associated macrophage activity observed in the absence of the antigen-binding molecule (or in the presence of a suitable control antigen-binding molecule), such as ≤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.

[1238] In some embodiments, tumor-associated macrophage activity can be analyzed by measuring factors associated with tumor-associated macrophage activity. In some embodiments, the activity of tumor-associated macrophages can be determined by analyzing the production of, e.g., IL-10, IL-13, IL-4.

[1239] In some embodiments, relative to a negative control condition, the antigen-binding molecules of the present disclosure are capable of increasing the number and / or proportion of M1 macrophages (i.e., M1 macrophages), e.g., in a suitable in vitro or in vivo assay. The ability of the antigen-binding molecules to increase the number and / or proportion of M1 macrophages can be analyzed by detection methods, such as those described in Example 8 herein. Such methods can includ...

Claims

1. An antigen-binding molecule that binds to VISTA in a method for treating or preventing cancer in a subject, characterized in that, The treatment or prevention includes: (i) increasing the number and / or proportion of antigen-specific CD8+ T cells; (ii) increasing the activity of CD8+ T cells; (iii) reducing the level of T cell exhaustion; (iv) reducing the number and / or proportion of tumor-associated macrophages (TAMs); (v) increasing the number and / or proportion of M1 macrophages; and / or (vi) increasing the activity of M1 macrophages.

2. Use of an antigen-binding molecule conjugated to VISTA in the preparation of a medicament for treating or preventing cancer in a subject, characterized in that, The treatment or prevention includes: (i) increasing the number and / or proportion of antigen-specific CD8+ T cells; (ii) increasing the activity of CD8+ T cells; (iii) reducing the level of T cell exhaustion; (iv) reducing the number and / or proportion of tumor-associated macrophages (TAMs); (v) increasing the number and / or proportion of M1 macrophages; and / or (vi) increasing the activity of M1 macrophages.

3. A method of treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of an antigen-binding molecule that binds to VISTA, wherein, The treatment or prevention includes: (i) increasing the number and / or proportion of antigen-specific CD8+ T cells; (ii) increasing the activity of CD8+ T cells; (iii) reducing the level of T cell exhaustion; (iv) reducing the number and / or proportion of tumor-associated macrophages (TAMs); (v) increasing the number and / or proportion of M1 macrophages; and / or (vi) increasing the activity of M1 macrophages.

4. The antigen-binding molecule according to claim 1, the use according to claim 2, or the method according to claim 3, wherein The cancer includes tumors containing VISTA-expressing cells.

5. A method of selecting a subject for treatment with an antigen-binding molecule that binds to VISTA, comprising: (a) analyzing a subject's cancer to determine whether the cancer has the following characteristics: (i) a low number and / or proportion of antigen-specific CD8+ T cells; (ii) low CD8+ T cell activity; (iii) a high presence and / or level of exhausted T cells; (iv) a high presence and / or number and / or proportion of TAMs; (v) a low number and / or proportion of M1 macrophages; and / or (vi) low M1 macrophage activity; and (b) selecting the subject for treatment with an antigen-binding molecule that binds to VISTA when it is determined in step (a) that the subject's cancer has one or more of (i) to (vi).

6. A method of determining a patient's response to treatment with an antigen-binding molecule that binds to VISTA, comprising: (a) analyzing a subject's cancer at a first time point to determine: (i) the number and / or proportion of antigen-specific CD8+ T cells; (ii) the activity of CD8+ T cells; (iii) the level of exhausted T cells; (iv) the number and / or proportion of tumor-associated macrophages (TAMs); (v) the number and / or proportion of M1 macrophages; and / or (vi) the activity of M1 macrophages; (b) analyzing the subject's cancer at a subsequent time point to determine one or more of (i) to (vi); and (c) determining the differences between (a) and (b), wherein: (i) the number and / or proportion of antigen-specific CD8+ T cells increases; (ii) the activity of CD8+ T cells increases; (iii) the level of exhausted T cells decreases; (iv) the number and / or proportion of tumor-associated macrophages (TAMs) decreases; (v) An increase in the number and / or proportion of M1 macrophages; and / or (vi) An increase in the activity of M1 macrophages, Compared with (a), the values in (b) represent a positive response to treatment with an antigen-binding molecule that binds to VISTA.

7. The antigen-binding molecule, use or method according to any one of claims 1 to 6, characterized in that The antigen-binding molecule comprises: (i) A heavy-chain variable (VH) region comprising the following CDRs: HC-CDR1 having the amino acid sequence as shown in SEQ ID NO:305 HC-CDR2 having the amino acid sequence as shown in SEQ ID NO:306 HC-CDR3 having the amino acid sequence as shown in SEQ ID NO:307; and (ii) A light-chain variable (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence as shown in SEQ ID NO:41 LC-CDR2 having the amino acid sequence as shown in SEQ ID NO:308 LC-CDR3 having the amino acid sequence as shown in SEQ ID NO:

43.

8. The antigen-binding molecule, its use or method according to any one of claims 1 to 7, characterized in that, The antigen-binding molecule comprises: (i) A heavy-chain variable (VH) region comprising the following CDRs: HC-CDR1 having the amino acid sequence as shown in SEQ ID NO:290 HC-CDR2 having the amino acid sequence as shown in SEQ ID NO:291 HC-CDR3 having the amino acid sequence as shown in SEQ ID NO:278; and (ii) A light-chain variable (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence as shown in SEQ ID NO:41 LC-CDR2 having the amino acid sequence as shown in SEQ ID NO:295 LC-CDR3 having the amino acid sequence as shown in SEQ ID NO:

43.

9. The antigen-binding molecule, its use or method according to any one of claims 1 to 8, characterized in that, The antigen-binding molecule comprises: A VH region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:289; and A VL region comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:

297.

10. The antigen-binding molecule, its use or method according to any one of claims 1 to 9, characterized in that The antigen-binding molecule comprises: A VH region comprising the following framework regions (FRs): HC-FR1 having the amino acid sequence as shown in SEQ ID NO:63 HC-FR2 having the amino acid sequence as shown in SEQ ID NO:292 HC-FR3 having the amino acid sequence as shown in SEQ ID NO:293 HC-FR4 having the amino acid sequence as shown in SEQ ID NO:

281.

11. The antigen-binding molecule, its use or method according to any one of claims 1 to 10, characterized in that, The antigen-binding molecule comprises: A VL region comprising the following framework regions (FRs): LC-FR1 having the amino acid sequence as shown in SEQ ID NO:288 LC-FR2 having the amino acid sequence as shown in SEQ ID NO:283 LC-FR3 having the amino acid sequence as shown in SEQ ID NO:284 LC-FR4 having the amino acid sequence as shown in SEQ ID NO:

47.

12. The antigen-binding molecule, its use or method according to any one of claims 1 to 11, characterized in that, The antigen-binding molecule comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO:

331.

13. The antigen-binding molecule, its use or method according to any one of claims 1 to 12, characterized in that, The antigen-binding molecule comprises a light chain containing the amino acid sequence shown in SEQ ID NO:

317.

14. The antigen-binding molecule, use or method according to any one of claims 1 to 13, wherein The cancer is selected from: hematological malignancies, leukemia, acute myeloid leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, multiple myeloma, mesothelioma, epithelioid mesothelioma, solid tumors, lung cancer, non-small cell lung cancer, gastric cancer, gastric malignancy, colorectal cancer, colorectal tumor, colorectal adenocarcinoma, uterine cancer, endometrial cancer, breast cancer, triple-negative breast cancer, triple-negative invasive breast cancer, liver cancer, hepatocellular carcinoma, pancreatic cancer, pancreatic ductal adenocarcinoma, thyroid cancer, thymoma, skin cancer, melanoma, cutaneous melanoma, renal cancer, renal cell carcinoma, renal papillary cell carcinoma, head and neck cancer, head and neck squamous cell carcinoma (SCCHN), ovarian cancer, ovarian tumor, ovarian serous cystadenocarcinoma, prostate cancer, and / or prostatic adenocarcinoma.

15. The antigen-binding molecule, its use or method according to claim 14, characterized in that, The cancer is selected from: colorectal cancer, pancreatic cancer, breast cancer, triple-negative breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, leukemia, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC), and solid tumors.

16. The antigen-binding molecule, its use or method according to claim 14, characterized in that, The cancer is epithelioid mesothelioma.

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