Treatment and prevention of cancer using VISTA antigen binding molecules

Abstract

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

Description

[0001] This application claims priority to US63 / 244986, filed September 16, 2021, the contents and elements of which are incorporated herein by reference for all purposes. Technical Field

[0002] The present invention relates to the field of molecular biology, in particular to antibody technology. The present invention also relates to medical drug treatment and prevention methods. Background Art

[0003] Myeloid Derived Suppressor Cell (MDSC)-mediated suppression of immune responses has been demonstrated in a variety of solid tumors and lymphomas. MDSCs are elevated in advanced colorectal cancer (Toor et al., Front Immunol. 2016; 7: 560). MDSCs have also been observed in breast cancer, and the percentage of MDSCs in the peripheral blood of patients with advanced breast cancer is elevated (Markowitz et al., Breast Cancer Res Treat. 2013 July; 140(1): 13-21). MDSC abundance is also associated with poor prognosis in solid tumors (Charoentong et al., Cell Rep. 2017 January 3; 18(1): 248-262).

[0004] MDSCs suppress T cells through multiple mechanisms, including the production of reactive oxygen species, nitric oxide, and arginase. These ultimately lead to the suppression of DC, NK, and T cell activity and increased tumor burden (Umansky et al., Vaccines (Basel) (2016) 4(4):36). MDSCs also promote tumor development and metastasis by producing soluble factors such as matrix metalloproteinases, VEGF, bFGF, TGF-β, and S100A8 / A9, which promote angiogenesis, invasion, proliferation, and metastasis.

[0005] VISTA, targeting V-type immunoglobulin domain-containing inhibitor of T-cell activation, is an immune checkpoint molecule primarily expressed on MDSCs and deserves attention as a therapeutic strategy for eliminating MDSC-mediated suppression of effector immune cell function.

[0006] WO 2017 / 137830A1 discloses an anti-VISTA antibody VSTB174, for example, in paragraph

[00212] , which discloses that it comprises the variable region of the anti-VISTA antibody VSTB112. Paragraph

[00362] discloses that VSTB123 comprises the variable region of VSTB174. Example 25 of WO 2017 / 137830A1 discloses in paragraphs

[0417] and

[0418] Figure 42A discloses that the mIgG2a antibody VSTB123 can inhibit tumor growth in the MB49 tumor model. Figure 42 A publicly showed that, in contrast, the same antibody VSTB124 provided in the form of IgG2a-LALA could not inhibit tumor growth; see paragraph

[0408] . Based on these results, Example 25 concluded in paragraph

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

[0053] ), involving the participation of FcγRIII mediated by Fc expressed by NK cells.

[0007] Le Mercier et al., Cancer Res. (2014) 74(7): 1933-44 discloses that hamster monoclonal anti-VISTA antibody mAb13F3 inhibits tumor growth in B16OVA and B16-BL6 melanoma models. The left and right paragraphs on page 1942 respectively illustrate that the immunogenicity and FcR binding activity of VISTA mAb may be key limiting factors in achieving optimal target neutralization and therapeutic efficacy. Summary of the Invention

[0008] In a first aspect, the present invention provides an optionally isolated antigen binding molecule that is capable of binding to VISTA and inhibiting VISTA-mediated signal transduction independent of Fc-mediated function.

[0009] In some embodiments, the antigen binding molecule comprises:

[0010] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0011] HC-CDR1 having the amino acid sequence of SEQ ID NO: 290

[0012] HC-CDR2 having the amino acid sequence of SEQ ID NO: 291

[0013] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; and

[0014] (ii) a light chain variable (VL) region comprising the following CDRs:

[0015] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0016] LC-CDR2 having the amino acid sequence of SEQ ID NO: 309

[0017] LC-CDR3 having the amino acid sequence of SEQ ID NO:43.

[0018] In some embodiments, the antigen binding molecule comprises:

[0019] (i) a heavy chain variable (VH) region comprising the following CDRs:

[0020] HC-CDR1 having the amino acid sequence of SEQ ID NO: 290

[0021] HC-CDR2 having the amino acid sequence of SEQ ID NO: 291

[0022] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; and

[0023] (ii) a light chain variable (VL) region comprising the following CDRs:

[0024] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0025] LC-CDR2 having the amino acid sequence of SEQ ID NO: 295

[0026] LC-CDR3 having the amino acid sequence of SEQ ID NO:43.

[0027] In some embodiments, the antigen binding molecule comprises:

[0028] a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 289; and

[0029] A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 310.

[0030] In some embodiments, the antigen binding molecule comprises:

[0031] a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 289; and

[0032] A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 297.

[0033] In some embodiments, the antigen binding molecule comprises:

[0034] A VH region comprising the following framework regions (FRs):

[0035] HC-FR1 having the amino acid sequence of SEQ ID NO: 63

[0036] HC-FR2 having the amino acid sequence of SEQ ID NO: 292

[0037] HC-FR3 having the amino acid sequence of SEQ ID NO: 293

[0038] HC-FR4 having the amino acid sequence of SEQ ID NO: 281.

[0039] In some embodiments, the antigen binding molecule comprises:

[0040] A VL region comprising the following framework regions (FRs):

[0041] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0042] LC-FR2 having the amino acid sequence of SEQ ID NO: 298

[0043] LC-FR3 having the amino acid sequence of SEQ ID NO: 284

[0044] LC-FR4 having the amino acid sequence of SEQ ID NO:47.

[0045] In some embodiments, the antigen binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 331. In some embodiments, the antigen binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO: 317.

[0046] In another aspect, the present invention provides a composition comprising an antigen binding molecule of the present disclosure.

[0047] In some embodiments, the composition comprises:

[0048] (i) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 80, pH 4.0 to 7.0; or

[0049] (ii) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 20, pH 4.0 to 7.0; or

[0050] (iii) 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, pH 4.0 to 7.0, optionally containing 0.001% to 0.1% (w / v) polysorbate 20 or polysorbate 80; or

[0051] (iv) 2 mM to 200 mM histidine, 0.001% to 0.1% (w / v) polysorbate 20 or polysorbate 80, pH 4.0 to 7.0; or

[0052] (v) 2 mM to 200 mM acetate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 80, pH 4.0 to 7.0; or

[0053] (vi) 2 mM to 200 mM acetate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 20, pH 4.0 to 7.0; or

[0054] (vii) 2 mM to 200 mM succinate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 80, pH 4.0 to 7.0; or

[0055] (viii) 2 mM to 200 mM succinate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 20, pH 4.0 to 7.0.

[0056] In some embodiments, the composition comprises:

[0057] (i) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and having a pH of 5.5; or

[0058] (ii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or

[0059] (iii) 20 mM histidine, 4% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or

[0060] (iv) 20 mM histidine, 2% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or

[0061] (v) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 6.3; or

[0062] (vi) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-20, and having a pH of 5.8; or

[0063] (vii) 20 mM acetate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and having a pH of 5.5; or

[0064] (viii) 20 mM succinate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and having a pH of 5.5; or

[0065] (ix) 20 mM histidine, 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or

[0066] (x) 20 mM histidine, 150 mM sodium chloride, and having a pH of 5.8.

[0067] In some embodiments, the composition comprises 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and has a pH of 5.5.

[0068] In some embodiments, the composition comprises about 50 mg / mL (eg, 50 mg / mL) of the antigen binding molecule.

[0069] In some aspects, the antigen binding molecules or compositions according to the present disclosure are provided for use as a medicament.

[0070] In some aspects, the antigen binding molecules or compositions according to the present disclosure are provided for use in a method of treating or preventing cancer in a subject.

[0071] In some aspects, there is also provided use of an antigen binding molecule or composition according to the present disclosure for the preparation of a medicament for treating or preventing cancer in a subject.

[0072] In some aspects, a method of treating or preventing cancer in a subject is also provided, the method comprising administering a therapeutically or prophylactically effective amount of an antigen binding molecule or composition according to the present disclosure.

[0073] In some embodiments, the cancer is characterized by the presence of cells expressing VISTA and / or signaling mediated by a complex comprising VISTA.

[0074] In some embodiments, the cancer is selected from the group consisting of hematological cancers, leukemias (e.g., T-cell leukemias), acute myeloid leukemias, lymphomas, B-cell lymphomas, T-cell lymphomas, multiple myeloma, mesothelioma, solid tumors, lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, gastric malignancies, colorectal cancer, colorectal tumors, colorectal adenocarcinoma, uterine cancer, endometrial cancer, breast cancer, triple-negative breast cancer (TBNC), triple-negative invasive breast cancer, infiltrating ductal carcinoma, 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, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian cancer, ovarian serous cystadenocarcinoma, bladder cancer, prostate cancer, and / or prostate adenocarcinoma. In some embodiments, the cancer is triple-negative cancer (TBNC), non-small cell lung cancer (NSCLC), and / or a solid tumor.

[0075] In some embodiments, the treatment or prevention of the cancer further comprises administering an agent capable of inhibiting signal transduction mediated by immune checkpoint molecules other than VISTA, for example, an immune checkpoint inhibitor other than VISTA is PD-1 and / or PD-L1, and the agent may be an anti-PD-1 or anti-PD-L1 antibody.

[0076] In some embodiments, the treatment or prevention, or a method thereof, includes detecting the presence of cells expressing VISTA and / or signal transduction mediated by a complex comprising VISTA. In some embodiments, when the presence of cells expressing VISTA and / or signal transduction mediated by a complex comprising VISTA is detected, the subject is selected to be treated with the antigen binding molecule or composition.

[0077] In some embodiments, the antigen binding molecules are administered weekly, for example, with compositions according to the present disclosure. In some embodiments, the antigen binding molecules are administered once, twice, or three times within a 21-day dosing cycle, and optionally the treatment includes up to 35 dosing cycles. In some embodiments, the antigen binding molecules are administered on the 1st, 8th, and / or 15th day within a 21-day dosing cycle, and optionally the treatment includes up to 35 dosing cycles. In some embodiments, the antigen binding molecules are administered on the 1st, 8th, 15th, and / or 21st day within a 28-day dosing cycle, and optionally the treatment includes up to 35 dosing cycles.

[0078] In some embodiments, the treatment or prevention, or method thereof, comprises administering 3.5 mg to 2200 mg of the antigen binding molecule per administration.

[0079] In some embodiments, the treatment or prevention, or method thereof, comprises administering (at most or at least) 3.5 mg, 7 mg, 10.5 mg, 17.5 mg, 20 mg, 21 mg, 40 mg, 60 mg, 72 mg, 120 mg, 180 mg, 240 mg, 360 mg, 400 mg, 800 mg, 1200 mg, 1600 mg, 1900 mg or 2200 mg of an antigen binding molecule (e.g., in a composition according to the disclosure), e.g., according to a dosing schedule according to the disclosure.

[0080] In some embodiments, the treatment or prevention, or method thereof, comprises administering up to 10.5 mg, up to 21 mg, up to 31.5 mg, up to 52.5 mg, up to 60 mg, up to 63 mg, up to 120 mg, up to 180 mg, up to 216 mg, up to 360 mg, up to 540 mg, up to 720 mg, up to 1080 mg, up to 1200 mg, up to 2400 mg, up to 3600 mg, up to 4800 mg, up to 5700 mg, or up to 6600 mg of an antigen binding molecule (e.g., in a composition according to the disclosure) per 21 day dosing cycle.

[0081] illustrate

[0082] The present invention relates to novel VISTA-binding molecules with novel and / or improved properties compared to known anti-VISTA antibodies.

[0083] The present inventors have prepared antigen binding molecules that bind to specific target regions in the extracellular region of VISTA. Compared with the VISTA binding antigen binding molecules disclosed in the prior art, the VISTA binding molecules of the present invention have a combination of desired biophysical and functional properties.

[0084] In particular, the VISTA-binding molecules described herein have been shown to antagonize VISTA-mediated signal transduction by a mechanism that does not require Fc-mediated function. The inventors have demonstrated that the Fc-containing VISTA-binding molecules described herein lack the ability to bind to Fcγ receptors and / or C1q and can provide therapeutic anti-cancer effects in vivo.

[0085] The inventors have established for the first time that VISTA-mediated signaling can be directly antagonized by a mechanism that does not require Fc-mediated effector functions (e.g., ADCC / ADCP / CDC against VISTA-expressing cells).

[0086] The VISTA-binding molecules disclosed herein target a region of VISTA that is different from the region targeted by known anti-VISTA antibodies. Antigen-binding molecules targeting a specific region of VISTA are able to antagonize VISTA-mediated signaling without the need for Fc-mediated effector functions.

[0087] Therefore, the VISTA binding molecules disclosed herein can be used to inhibit VISTA-mediated signal transduction without consuming cells expressing VISTA. This is important because VISTA is expressed on cells that do not wish to be consumed. Therefore, the VISTA binding molecules disclosed herein can inhibit VISTA-mediated signal transduction while minimizing adverse side effects.

[0088] It has also been advantageously shown that the VISTA-binding molecules disclosed herein can release T cells from VISTA-mediated inhibition. Specifically, the VISTA-binding molecules disclosed herein have been shown to increase T cell proliferation and the production of, for example, IFNγ and TNFα, by T cells cultured in the presence of cells expressing VISTA or VISTA.

[0089] VISTA, binding partners, and VISTA-mediated signaling

[0090] V-type immunoglobulin domain-containing inhibitor of T cell activation (VISTA; also known as, for example, B7-H5, SISP1, PD-1H) is a protein identified by UniProt Q9H7M9, which has an amino acid sequence as shown in SEQ ID NO: 1 (Q9H7M9-1, v3). The structure and function of VISTA are described in Lines et al., Cancer Res. (2014) 74 (7): 1924-1932, which is incorporated herein by reference in its entirety. VISTA is a type I single-pass transmembrane molecule of approximately 50 kDa that acts as an immune checkpoint and is encoded by the C10orf54 gene. The extracellular domain of VISTA is homologous to PD-L1.

[0091] The N-terminal 32 amino acids of SEQ ID NO: 1 constitute a signal peptide, so the mature form of VISTA (i.e., after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO: 2. Positions 33 to 194 of SEQ ID NO: 1 form the extracellular domain (SEQ ID NO: 3), positions 195 to 215 form the transmembrane domain (SEQ ID NO: 4), and positions 216 to 311 form the cytoplasmic domain (SEQ ID NO: 5). The extracellular domain contains an Ig-like V-type domain (positions 33 to 168 of SEQ ID NO: 1, as shown in SEQ ID NO: 6).

[0092] In this specification, "VISTA" refers to VISTA from any species, and includes VISTA isoforms, fragments, variants (including mutants) or homologs from any species.

[0093] As used herein, a "fragment," "variant," or "homolog" of a protein can optionally be characterized as having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of a reference protein (e.g., a reference isoform). In some embodiments, fragments, variants, isoforms, and homologs of a reference protein can be characterized by their ability to perform a function performed by the reference protein.

[0094] "Fragment" generally refers to a portion of a reference protein. "Variant" generally refers to a protein having an amino acid sequence comprising one or more amino acid substitutions, insertions, deletions or other modifications relative to the amino acid sequence of a reference protein, but retaining a considerable degree of sequence identity (e.g., at least 60%) with the amino acid sequence of the reference protein. "Isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein. "Homolog" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologs include orthologs.

[0095] A "fragment" can be of any length (in terms of the number of amino acids), although it can optionally be at least 20% of the length of a reference protein (i.e., the protein from which the fragment is derived) and can have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. A fragment of VISTA can have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, or 300 amino acids and a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, or 300 amino acids.

[0096] In some embodiments, VISTA is VISTA from a mammal, e.g., a primate (rhesus macaque, cynomolgus macaque, non-human primate, or human), and / or VISTA from a rodent, e.g., rat or murine. An isoform, fragment, variant, or homolog of VISTA can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VISTA isoform from a given species, e.g., human.

[0097] The isoform, fragment, variant or homologue can optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity with reference to VISTA, as determined by a suitable assay. For example, an isoform, fragment, variant or homologue of VISTA can, for example, be shown to be associated with VSIG-3, LRIG1, VSIG8 and / or PSGL-1.

[0098] In some embodiments, VISTA comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1 or 2. In some embodiments, a fragment of VISTA comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NO: 2, 3 or 6.

[0099] VISTA is a member of the B7 protein family and is primarily expressed by leukocytes, particularly CD14+ monocytes (including monocyte-derived suppressor cells (MDSCs)) and CD33+ myeloid cells. VISTA is also expressed by CD56+ NK cells and dendritic cells, and is expressed to a lesser extent on CD4+ and CD8+ T cells. VISTA is highly expressed on MDSCs, particularly tumor-infiltrating MDSCs and tumor-infiltrating myeloid DCs (Le Mercier et al., Cancer Res. (2014) 74(7): 1933-44), as well as tumor-associated macrophages (TAMs) and neutrophils.

[0100] There is evidence that VISTA can act as both a ligand and a receptor for T cells, thereby inhibiting T cell effector function and maintaining peripheral tolerance; tumors that overexpress VISTA escape immune control and grow faster than tumors that do not overexpress VISTA (Wang et al., Journal of Experimental Medicine. (2011) 208 (3): 577-92; Lines et al., Cancer Res. (2014) 74 (7): 1924-1932). VISTA has been shown to be a co-inhibitory receptor on CD4+ T cells or a co-inhibitory ligand for T cells. It is reported that VISTA- / -CD4+ T cells show stronger antigen-specific proliferation and cytokine production than wild-type CD4+ T cells, suggesting that VISTA acts as an inhibitory receptor on CD4+ T cells. Blockade of VISTA function using monoclonal anti-VISTA antibodies has been shown to enhance the infiltration, proliferation, and effector function of tumor-reactive T cells in the tumor microenvironment (Le Mercier et al., Cancer Res. (2014) 74(7): 1933-4).

[0101] VISTA has been proposed to interact with VSIG-3 (IGSF11) - see, for example, Wang et al., J Immunol (2017), 198 (1 Supplement) 154.1, which is incorporated herein by reference in its entirety. Binding of VSIG-3 to activated T cells via VISTA can inhibit T cell proliferation and reduce the production of cytokines and chemokines (e.g., IFN-γ, IL-2, IL-17, CCL5 / RANTES, CCL3 / MIP-1a, and CXCL11 / I-TAC).

[0102] VSIG-3 is a protein identified by UniProt Q5DX21. The mRNA encoded by the human IGSF11 gene is alternatively spliced ​​to produce three different isoforms: isoform 1 (UniProt: Q5DX21-1, v3; SEQ ID NO: 7); isoform 2 (UniProt: Q5DX21-2; SEQ ID NO: 8), which contains a sequence that differs from SEQ ID NO: 7 at positions 1 to 17; and isoform 3 (UniProt: Q5DX21-3; SEQ ID NO: 9), which contains a sequence that differs from SEQ ID NO: 7 at positions 1 to 17 and further contains a sequence that differs from SEQ ID NO: 7 at positions 211 to 235.

[0103] The N-terminal 22 amino acids of SEQ ID NOs: 7, 8, and 9 constitute the signal peptide, so that the mature forms of VSIG-3 isoforms 1, 2, and 3 (i.e., after processing to remove the signal peptide) have the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. Positions 23 to 241 of SEQ ID NOs: 7 and 8 form the extracellular domain of VSIG-3 isoforms 1 and 2 (SEQ ID NO: 13), while positions 23 to 216 of SEQ ID NO: 9 form the extracellular domain of VSIG-3 isoform 3 (SEQ ID NO: 14). The transmembrane domain of VSIG-3 is set forth in SEQ ID NO: 15, and the cytoplasmic domain is set forth in SEQ ID NO: 16. The extracellular domain contains an Ig-like V-type domain (set forth in SEQ ID NO: 17), and the extracellular domains of VSIG-3 isoforms 1 and 2 additionally contain an Ig-like C2-type domain (set forth in SEQ ID NO: 18).

[0104] In the present specification, “VSIG-3” refers to VSIG-3 from any species, and includes VSIG-3 isoforms, fragments, variants (including mutants), or homologs from any species.

[0105] The minimum length of a VSIG-3 fragment can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids, and the maximum length can be 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0106] In some embodiments, the VSIG-3 is a VSIG-3 from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or a VSIG-3 from a rodent (e.g., rat or murine). The isoform, fragment, variant, or homolog of VSIG-3 can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VSIG-3 isoform from a given species (e.g., human).

[0107] The isoform, fragment, variant or homologue can optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of a reference VSIG-3, as determined by a suitable assay. For example, an isoform, fragment, variant or homologue of VSIG-3 can, for example, be shown to be associated with VISTA.

[0108] In some embodiments, VSIG-3 comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs: 7 to 12. In some embodiments, a fragment of VSIG-3 comprises or consists of an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs: 10 to 14, 17 or 18.

[0109] VISTA has been proposed to interact with VSIG-8 - see, for example, WO / 2016 / 090347A1. VSIG-8 is a protein identified by UniProt P0DPA2 (SEQ ID NO: 19). The N-terminal 21 amino acids of SEQ ID NO: 19 constitute the signal peptide, so the mature form of VSIG-8 (i.e., after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO: 20. Positions 22 to 263 of SEQ ID NO: 19 form the extracellular domain of VSIG-8 (SEQ ID NO: 21). The transmembrane domain of VSIG-8 is shown in SEQ ID NO: 22, while the cytoplasmic domain is shown in SEQ ID NO: 23. The extracellular domain contains Ig-like V-type domain 1 (as shown in SEQ ID NO: 24) and Ig-like V-type domain 2 (as shown in SEQ ID NO: 25).

[0110] In the present specification, "VSIG-8" refers to VSIG-8 of any species, including VSIG-8 isoforms, fragments, variants (including mutants) or homologs from any species.

[0111] The minimum length of a VSIG-8 fragment can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0112] In some embodiments, VSIG-8 is VSIG-8 from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or VSIG-8 from a rodent (e.g., rat or murine). An isoform, fragment, variant, or homolog of VSIG-8 can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature VSIG-8 isoform from a given species (e.g., human).

[0113] The isoform, fragment, variant or homologue can optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of a reference VSIG-8, as determined by a suitable assay. For example, an isoform, fragment, variant or homologue of VSIG-8 can, for example, be shown to be associated with VISTA.

[0114] In some embodiments, VSIG-8 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 19 or 20. In some embodiments, a fragment of VSIG-8 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs: 20, 21, 24 or 25.

[0115] VISTA has been proposed to interact with PSGL-1 - see, for example, WO 2018 / 132476 A1. PSGL-1 isoform 1 is a protein identified by UniProt Q14242-1 (SEQ ID NO: 323). PSGL-1 isoform 2 is a protein identified by UniProt Q14242-2 (SEQ ID NO: 324) and differs from PSGL-1 isoform 1 in that it contains 16 additional amino acids after position 1 of SEQ ID NO: 323.

[0116] The N-terminal 17 amino acids of SEQ ID NO:323 constitute a signal peptide, so the mature form of PSGL-1 (i.e., after processing to remove the signal peptide) has the amino acid sequence set forth in SEQ ID NO:325. Positions 18 to 320 of SEQ ID NO:323 form the extracellular domain of PSGL-1 (SEQ ID NO:326). The transmembrane domain of PSGL-1 is set forth in SEQ ID NO:327, and the cytoplasmic domain is set forth in SEQ ID NO:328. The extracellular domain contains 12 and 10 tandem repeats of amino acid sequences; the repeat region is set forth in SEQ ID NO:329.

[0117] In the present specification, "PSGL-1" refers to PSGL-1 from any species, and includes PSGL-1 isoforms, fragments, variants (including mutants), or homologs from any species.

[0118] The minimum length of a PSGL-1 fragment can be one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids, and the maximum length can be one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, or 400 amino acids.

[0119] In some embodiments, PSGL-1 is PSGL-1 from a mammal, e.g., a primate (rhesus monkey, cynomolgus monkey, non-human primate, or human) and / or PSGL-1 from a rodent, e.g., rat or murine. An isoform, fragment, variant, or homolog of PSGL-1 can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature PSGL-1 isoform from a given species, e.g., human.

[0120] The isoform, fragment, variant or homologue can optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of a reference PSGL-1, as determined by a suitable assay. For example, an isoform, fragment, variant or homologue of PSGL-1 can, for example, be shown to be associated with VISTA.

[0121] In some embodiments, PSGL-1 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 323 or 324. In some embodiments, a fragment of PSGL-1 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NO: 325, 326 or 329.

[0122] Regions of particular interest on the target molecule

[0123] The antigen binding molecules of the present invention are specially designed to target the VISTA region of particular interest. Using a two-step method, the VISTA region to be targeted is selected by analyzing the predicted antigenicity, function and safety. The peptide corresponding to the target region is then used as an immunogen to produce specific monoclonal antibodies to prepare antibodies specific to the VISTA target region, which are then screened to identify antibodies capable of binding to the VISTA of the native state. The method provides precise control of antibody epitopes.

[0124] The antigen binding molecules of the present invention can be defined with reference to the VISTA region they are combined.The antigen binding molecules of the present invention can be combined with the specific region of VISTA of interest.In some embodiments, antigen binding molecules can be combined with the linear epitope of VISTA, which is composed of a continuous sequence of amino acids (i.e., amino acid primary sequence).In some embodiments, antigen binding molecules can be combined with the conformational epitope of VISTA, which is composed of a discontinuous sequence of amino acids of amino acid sequence.

[0125] In some embodiments, the antigen binding molecules of the present invention bind to VISTA. In some embodiments, the antigen binding molecules bind to the extracellular region of VISTA (e.g., the region shown in SEQ ID NO: 3). In some embodiments, the antigen binding molecules bind to the Ig-like V-type domain of VISTA (e.g., the region shown in SEQ ID NO: 6). In some embodiments, the antigen binding molecules bind to VISTA in the region corresponding to SEQ ID NO: 1, 61 to 162 (as shown in SEQ ID NO: 31).

[0126] In some embodiments, the antigen binding molecule binds to the VISTA region set forth in SEQ ID NO: 322. In some embodiments, the antigen binding molecule binds to the VISTA region set forth in SEQ ID NO: 26. In some embodiments, the antigen binding molecule binds to the VISTA region set forth in SEQ ID NO: 27. In some embodiments, the antigen binding molecule binds to the VISTA region set forth in SEQ ID NO: 28. In some embodiments, the antigen binding molecule binds to the VISTA region set forth in SEQ ID NO: 29. In some embodiments, the antigen binding molecule binds to the VISTA region set forth in SEQ ID NO: 30.

[0127] In some embodiments, the antigen binding molecule does not bind to the VISTA region set forth in SEQ ID NO: 271. In some embodiments, the antigen binding molecule does not bind to the VISTA region set forth in SEQ ID NO: 272. In some embodiments, the antigen binding molecule does not bind to the VISTA region set forth in SEQ ID NO: 273. In some embodiments, the antigen binding molecule does not bind to the VISTA region set forth in SEQ ID NO: 274. In some embodiments, the antigen binding molecule does not bind to the VISTA region set forth in SEQ ID NO: 275.

[0128] The region of the peptide / polypeptide to which the antibody binds can be determined by a skilled artisan using various methods well known in the art, including X-ray co-crystal analysis of the antibody-antigen complex, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange mass spectrometry, phage display, competitive ELISA, and proteolytic "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3): 145-156, which is incorporated herein by reference in its entirety.

[0129] In some embodiments, the antigen binding molecule can bind to the same or overlapping VISTA region as the VISTA region bound by an antibody comprising the VH and VL sequences of 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 as described herein.

[0130] As used herein, "peptide" refers to a chain of two or more amino acid monomers connected by peptide bonds. Peptides generally range in length from about 2 to 50 amino acids. A "polypeptide" is a polymer chain of two or more peptides. Polypeptides generally have a length greater than about 50 amino acids.

[0131] In some embodiments, the antigen binding molecules of the present invention are capable of binding to a polypeptide comprising or consisting of an amino acid sequence of one of SEQ ID NOs: 1, 2, 3, 6, or 31.

[0132] In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 322. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 27. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 28. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 29. In some embodiments, the antigen-binding molecule is capable of binding to a peptide / polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 30.

[0133] In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 271. In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 272. In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 273. In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 274. In some embodiments, the antigen-binding molecule cannot bind to a peptide consisting of the amino acid sequence of SEQ ID NO: 275.

[0134] The ability of an antigen-binding molecule to bind a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blot), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol Biol (2012) 907: 411-442), or biofilm interferometry (see, e.g., Lad et al., (2015) J Biomol Screen 20(4): 498-507).

[0135] 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, for example, 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 amino acids additional at one or both ends of the reference amino acid sequence.

[0136] In some embodiments, the additional amino acids provided at one or both ends of the reference sequence (i.e., the N-terminus and the C-terminus) correspond to the positions of the ends of the reference sequence in the context of the amino acid sequence of VISTA. For example, if the antigen binding molecule is capable of binding to a peptide / polypeptide comprising the sequence of SEQ ID NO: 26 and two additional amino acids at the C-terminus of SEQ ID NO: 26, the two additional amino acids can be arginine and asparagine, corresponding to positions 90 and 91 of SEQ ID NO: 1.

[0137] 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 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 as described herein.

[0138] Myeloid-derived suppressor cells (MDSCs)

[0139] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of immune cells of the myeloid lineage characterized by an immunosuppressive phenotype. MDSC biology is reviewed in Kumar et al., Trends Immunol. (2016); 37(3): 208-220, which is incorporated herein by reference in its entirety.

[0140] MDSCs are characterized by 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 production of reactive oxygen species (ROS) such as superoxide anion (O2-), hydrogen peroxide (H2O2), and peroxynitrite (PNT; ONOO-); increased expression of arginase 1 and nitric oxide synthase 2 (NOS2) and production of nitric oxide (NO); increased expression of c / EBPβ and STAT3; decreased expression of IRF8; and increased production of S100A8 / 9 proteins.

[0141] There are two different types of MDSCs: polymorphonuclear MDSCs (PMN-MDSCs), which are morphologically and phenotypically similar to neutrophils, and mononuclear MDSCs (M-MDSCs), which are more similar to monocytes. The morphological and phenotypic characteristics of MDSCs are described in Marvel and Gabrilovich, J Clin Invest. 2015 Sep 1; 125(9): 3356-3364, which is incorporated herein by reference in its entirety. In mice, MDSCs are broadly identified as CD11b+Gr1+ cells. Gr-1hi cells are primarily PMN-MDSCs, while Gr-1lo cells are primarily M-MDSCs. These subsets can be more accurately identified based on Ly6C and Ly6G markers; M-MDSCs are CD11b+Ly6ChiLy6G–, while PMN-MDSCs are CD11b+Ly6CloLy6G+. In humans, MDSCs are identified in the mononuclear fraction. PMN-MDSCs are CD14–CD11b+CD33+CD15+ or CD66b+ cells, while M-MDSCs are CD14+HLA-DR– / lo cells. The Lin–HLA-DR–CD33+MDSC population represents a mixed cell population enriched for myeloid progenitor cells.

[0142] Factors associated with MDSC-mediated immunosuppression include expression of arginase (ARG1), inducible NOS (iNOS), TGF-β, IL-10, and COX2, cysteine ​​sequestration, reduced expression of l-selectin on T cells, and induction of Tregs. MMDSC and PMN-MDSC employ different immunosuppressive mechanisms. M-MDSC suppress antigen-specific and nonspecific T cell responses by producing NO and cytokines and have a stronger immunosuppressive effect than PMN-MDSC. PMN-MDSC suppresses immune responses in an antigen-specific manner by producing ROS. MDSC is pathologically implicated in the occurrence and development of cancer and infectious diseases. The role of MDSC in human diseases is reviewed in Kumar et al., Trends Immunol. (2016); 37(3): 208-220 (incorporated herein by reference) and Greten et al., Int Immunopharmacol. (2011) 11(7): 802-807, which are incorporated herein by reference in their entirety.

[0143] MDSCs are abundant in tumor tissues and promote the development and progression of cancer through various mechanisms, as reviewed, for example, in Umansky et al., Vaccines (Basel) (2016) 4(4): 36. MDSCs are recruited to tumor sites through chemokine expression, and proinflammatory factors in the tumor microenvironment lead to a significant upregulation of the immunosuppressive function of MDSCs. MDSCs promote tumor development, neovascularization, and metastasis by inhibiting the function of effector immune cells (e.g., effector T cells and NK cells), promoting the generation / activation of regulatory T cells, the production of growth factors (e.g., VEGF and bFGF), and the production of ECM-modifying factors (e.g., matrix metalloproteinases).

[0144] MDSCs can be characterized by reference to the expression of VISTA. In embodiments of various aspects of the present invention, MDSCs can be "MDSCs expressing VISTA" or "VISTA+MDSCs." MDSCs can express VISTA on the cell surface (i.e., VISTA can be expressed in or on the cell membrane).

[0145] Antigen-binding molecules

[0146] The present invention provides antigen-binding molecules capable of binding to VISTA.

[0147] "Antigen-binding molecules" refer to molecules that can bind to target antigens, including monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies) and antibody fragments (e.g., Fv, scFv, Fab, scFab, F(ab')2, Fab2, diabodies, triabodies, scFv-Fc, miniantibodies, single domain antibodies (e.g., VhH), etc.), as long as they show binding to the relevant target molecule.

[0148] The antigen binding molecules of the present invention include a portion capable of binding to a target antigen. In some embodiments, the portion capable of binding to a target antigen includes an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) that can specifically bind to an antibody of the target antigen. In some embodiments, the portion capable of binding to a target antigen includes an aptamer that can bind to the target antigen or is composed of an aptamer, such as a nucleic acid aptamer that can bind to the target antigen (as reviewed in Zhou and Rossi, Nat Rev Drug Discov 2017 16(3):181-202). In some embodiments, the portion capable of binding to the target antigen comprises or consists of an antigen-binding peptide / polypeptide, such as a peptide aptamer, a thioredoxin, a monomer, anticalin, a Kunitz domain, an avimer, a knottin, a fynomer, an atrimer, a DARPin, an affibody, a nanobody (i.e., a single domain antibody (sdAb)), an avidin, an armadillo repeat protein (ArmRP), an OBody, or a fibronectin—as reviewed in Reverdatto et al., Curr Top Med Chem. 2015; 15(12): 1082-1101, which is incorporated herein by reference in its entirety (see also, e.g., Boersma et al., J Biol Chem (2011) 286: 41273-85 and Emanuel et al., Mabs (2011) 3: 38-48).

[0149] The antigen binding molecules of the present invention generally include an antigen binding domain comprising the VH and VL of an antibody capable of specifically binding to a target antigen. The antigen binding domain formed by VH and VL may also be referred to herein as an Fv region. The antigen binding molecules may be or may include an antigen binding polypeptide or an antigen binding polypeptide complex.

[0150] Antigen binding molecules may comprise more than one polypeptide, which together form an antigen binding domain. The polypeptides may be covalently or non-covalently bound. In some embodiments, the polypeptides form part of a larger polypeptide comprising the polypeptide (e.g., in the case of an scFv comprising VH and VL, or in the case of an scFab comprising VH-CH1 and VL-CL).

[0151] An antigen binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (eg, 2, 3, 4, 6, or 8 polypeptides), such as an IgG-like antigen binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.

[0152] The antigen binding molecules of the present invention can be designed and prepared using sequences of monoclonal antibodies (mAbs) that can bind 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 for a given antibody.

[0153] Antibodies typically contain six complementarity-determining regions (CDRs); three in the heavy chain variable (VH) region: HC-CDR1, HC-CDR2, and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1, LC-CDR2, and LC-CDR3. Together, the six CDRs define the antibody's paratope, which is the part of the antibody that binds to its target antigen.

[0154] The VH and VL regions contain framework regions (FRs) on both sides of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, the VH region comprises the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HCFR3]-[HC-CDR3]-[HC-FR4]-C-terminus; and the VL region comprises the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0155] There are several different conventions for defining antibody CDRs and FRs, such as those described in Kabat et al., Protein Sequences of Immunological Significance, 5th ed., National Institutes of Health, Public Health Service, Bethesda, Maryland (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and in VBASE2, Retter et al., Nucl. Natl. Acad. Sci. (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 convention as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27: 55-77.

[0156] In some embodiments, the antigen binding molecules include CDRs of antigen binding molecules capable of binding to VISTA. In some embodiments, the antigen binding molecules include FRs of antigen binding molecules capable of binding to VISTA. In some embodiments, the antigen binding molecules include CDRs and FRs of antigen binding molecules capable of binding to VISTA. That is, in certain embodiments, the antigen binding molecules include VH and VL regions of antigen binding molecules capable of binding to VISTA.

[0157] In some embodiments, the antigen binding molecule comprises a VH region and a VL region that are or are derived from the VH / VL regions of a VISTA-binding antibody clone described herein (i.e., 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).

[0158] In some embodiments, the antigen binding molecule comprises a VH region according to one of the following (1) to (18):

[0159] (1) (4M2-C12 derived consensus sequence) VH region containing the following CDRs:

[0160] HC-CDR1 having the amino acid sequence of SEQ ID NO: 305

[0161] HC-CDR2 having the amino acid sequence of SEQ ID NO: 306

[0162] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 307,

[0163] 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 substituted with another amino acid.

[0164] (2) (V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31) a VH region comprising the following CDRs:

[0165] HC-CDR1 having the amino acid sequence of SEQ ID NO: 290

[0166] HC-CDR2 having the amino acid sequence of SEQ ID NO: 291

[0167] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278,

[0168] 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 substituted with another amino acid.

[0169] (3) (V4-C1) a VH region comprising the following CDRs:

[0170] HC-CDR1 having the amino acid sequence of SEQ ID NO: 33

[0171] HC-CDR2 having the amino acid sequence of SEQ ID NO: 277

[0172] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278,

[0173] 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 substituted with another amino acid.

[0174] (4) (V4-C9) VH region comprising the following CDRs:

[0175] HC-CDR1 having the amino acid sequence of SEQ ID NO: 33

[0176] HC-CDR2 having the amino acid sequence of SEQ ID NO: 286

[0177] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278,

[0178] 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 substituted with another amino acid.

[0179] (5) (4M2-C12 / V4H1 / V4H2 consensus sequence) VH region comprising the following CDRs:

[0180] HC-CDR1 having the amino acid sequence of SEQ ID NO: 244

[0181] HC-CDR2 having the amino acid sequence of SEQ ID NO: 34

[0182] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 35,

[0183] 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 substituted with another amino acid.

[0184] (6) (4M2-C12, 4M2-B4, V4H2) VH region containing the following CDRs:

[0185] HC-CDR1 having the amino acid sequence of SEQ ID NO: 33

[0186] HC-CDR2 having the amino acid sequence of SEQ ID NO: 34

[0187] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 35,

[0188] 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 substituted with another amino acid.

[0189] (7) (V4H1) VH region comprising the following CDRs:

[0190] HC-CDR1 having the amino acid sequence of SEQ ID NO: 53

[0191] HC-CDR2 having the amino acid sequence of SEQ ID NO: 34

[0192] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 35,

[0193] 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 substituted with another amino acid.

[0194] (8) (2M1-B12, 2M1-D2) VH region containing the following CDRs:

[0195] HC-CDR1 having the amino acid sequence of SEQ ID NO: 72

[0196] HC-CDR2 having the amino acid sequence of SEQ ID NO: 73

[0197] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 74,

[0198] 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 substituted with another amino acid.

[0199] (9) (4M2-C9, 5M1-A11) VH region containing the following CDRs:

[0200] HC-CDR1 having the amino acid sequence of SEQ ID NO: 88

[0201] HC-CDR2 having the amino acid sequence of SEQ ID NO: 89

[0202] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 90,

[0203] 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 substituted with another amino acid.

[0204] (10) (4M2-D9) VH region comprising the following CDRs:

[0205] HC-CDR1 having the amino acid sequence of SEQ ID NO: 33

[0206] HC-CDR2 having the amino acid sequence of SEQ ID NO: 107

[0207] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 108,

[0208] 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 substituted with another amino acid.

[0209] (11)(1M2-D2) VH region comprising the following CDRs:

[0210] HC-CDR1 having the amino acid sequence of SEQ ID NO: 120

[0211] HC-CDR2 having the amino acid sequence of SEQ ID NO: 121

[0212] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 122,

[0213] 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 substituted with another amino acid.

[0214] (12)(4M2-D5) VH region comprising the following CDRs:

[0215] HC-CDR1 having the amino acid sequence of SEQ ID NO: 144

[0216] HC-CDR2 having the amino acid sequence of SEQ ID NO: 145

[0217] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 146,

[0218] 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 substituted with another amino acid.

[0219] (13) (4M2-A8) VH region comprising the following CDRs:

[0220] HC-CDR1 having the amino acid sequence of SEQ ID NO: 158

[0221] HC-CDR2 having the amino acid sequence of SEQ ID NO: 159

[0222] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 160,

[0223] 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 substituted with another amino acid.

[0224] (14) (9M2-C12) VH region comprising the following CDRs:

[0225] HC-CDR1 having the amino acid sequence of SEQ ID NO: 169

[0226] HC-CDR2 having the amino acid sequence of SEQ ID NO: 170

[0227] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 171,

[0228] 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 substituted with another amino acid.

[0229] (15) (13D5 derived) VH region comprising the following CDRs:

[0230] HC-CDR1 having the amino acid sequence of SEQ ID NO: 72

[0231] HC-CDR2 having the amino acid sequence of SEQ ID NO: 184

[0232] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 246,

[0233] 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 substituted with another amino acid.

[0234] (16)(13D5p) VH region comprising the following CDRs:

[0235] HC-CDR1 having the amino acid sequence of SEQ ID NO: 72

[0236] HC-CDR2 having the amino acid sequence of SEQ ID NO: 184

[0237] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 185,

[0238] 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 substituted with another amino acid.

[0239] (17)(13D5-1) VH region comprising the following CDRs:

[0240] HC-CDR1 having the amino acid sequence of SEQ ID NO: 72

[0241] HC-CDR2 having the amino acid sequence of SEQ ID NO: 184

[0242] an HC-CDR3 having the amino acid sequence of SEQ ID NO: 195,

[0243] 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 substituted with another amino acid.

[0244] (18)(13D5-13) VH region comprising the following CDRs:

[0245] HC-CDR1 having the amino acid sequence of SEQ ID NO: 72

[0246] HC-CDR2 having the amino acid sequence of SEQ ID NO: 184

[0247] HC-CDR3 having the amino acid sequence of SEQ ID NO: 200,

[0248] 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 substituted with another amino acid.

[0249] In some embodiments, the antigen binding molecule comprises a VH region according to one of the following (19) to (35):

[0250] (19) (V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31) contains the following VH region:

[0251] HC-FR1 having the amino acid sequence of SEQ ID NO: 63

[0252] HC-FR2 having the amino acid sequence of SEQ ID NO: 292

[0253] HC-FR3 having the amino acid sequence of SEQ ID NO: 293

[0254] HC-FR4 having the amino acid sequence of SEQ ID NO: 281,

[0255] 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 substituted with another amino acid.

[0256] (20) (V4-C1, V4-C9) contains the following VH region:

[0257] HC-FR1 having the amino acid sequence of SEQ ID NO: 63

[0258] HC-FR2 having the amino acid sequence of SEQ ID NO: 279

[0259] HC-FR3 having the amino acid sequence of SEQ ID NO: 280

[0260] HC-FR4 having the amino acid sequence of SEQ ID NO: 281,

[0261] 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 substituted with another amino acid.

[0262] (21)(4M2-C12) has a VH region comprising the following FRs:

[0263] HC-FR1 having the amino acid sequence of SEQ ID NO: 36

[0264] HC-FR2 having the amino acid sequence of SEQ ID NO: 37

[0265] HC-FR3 having the amino acid sequence of SEQ ID NO: 38

[0266] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0267] 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 substituted with another amino acid.

[0268] (22)(4M2-B4) contains the following VH region:

[0269] HC-FR1 having the amino acid sequence of SEQ ID NO: 49

[0270] HC-FR2 having the amino acid sequence of SEQ ID NO: 37

[0271] HC-FR3 having the amino acid sequence of SEQ ID NO: 38

[0272] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0273] 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 substituted with another amino acid.

[0274] (23)(V4H1) contains the following VH region:

[0275] HC-FR1 having the amino acid sequence of SEQ ID NO: 54

[0276] HC-FR2 having the amino acid sequence of SEQ ID NO: 55

[0277] HC-FR3 having the amino acid sequence of SEQ ID NO: 56

[0278] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0279] 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 substituted with another amino acid.

[0280] (24)(V4H2) has a VH region comprising the following FRs:

[0281] HC-FR1 having the amino acid sequence of SEQ ID NO: 63

[0282] HC-FR2 having the amino acid sequence of SEQ ID NO: 64

[0283] HC-FR3 having the amino acid sequence of SEQ ID NO: 65

[0284] HC-FR4 having the amino acid sequence of SEQ ID NO: 39,

[0285] 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 substituted with another amino acid.

[0286] (25)(2M1-B12) contains the following FR VH region:

[0287] HC-FR1 having the amino acid sequence of SEQ ID NO: 75

[0288] HC-FR2 having the amino acid sequence of SEQ ID NO: 76

[0289] HC-FR3 having the amino acid sequence of SEQ ID NO: 77

[0290] HC-FR4 having the amino acid sequence of SEQ ID NO: 78,

[0291] 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 substituted with another amino acid.

[0292] (26)(4M2-C9) contains the following VH region:

[0293] HC-FR1 having the amino acid sequence of SEQ ID NO: 91

[0294] HC-FR2 having the amino acid sequence of SEQ ID NO: 92

[0295] HC-FR3 having the amino acid sequence of SEQ ID NO: 93

[0296] HC-FR4 having the amino acid sequence of SEQ ID NO: 94,

[0297] 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 substituted with another amino acid.

[0298] (27)(2M1-D2) contains the following VH region:

[0299] HC-FR1 having the amino acid sequence of SEQ ID NO: 103

[0300] HC-FR2 having the amino acid sequence of SEQ ID NO: 76

[0301] HC-FR3 having the amino acid sequence of SEQ ID NO: 77

[0302] HC-FR4 having the amino acid sequence of SEQ ID NO: 78,

[0303] 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 substituted with another amino acid.

[0304] (28)(4M2-D9) contains the following FR VH region:

[0305] HC-FR1 having the amino acid sequence of SEQ ID NO: 109

[0306] HC-FR2 having the amino acid sequence of SEQ ID NO: 110

[0307] HC-FR3 having the amino acid sequence of SEQ ID NO: 111

[0308] HC-FR4 having the amino acid sequence of SEQ ID NO: 112,

[0309] 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 substituted with another amino acid.

[0310] (29)(1M2-D2) contains the following VH region:

[0311] HC-FR1 having the amino acid sequence of SEQ ID NO: 123

[0312] HC-FR2 having the amino acid sequence of SEQ ID NO: 124

[0313] HC-FR3 having the amino acid sequence of SEQ ID NO: 125

[0314] HC-FR4 having the amino acid sequence of SEQ ID NO: 78,

[0315] 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 substituted with another amino acid.

[0316] (30)(5M1-A11) has a VH region comprising the following FRs:

[0317] HC-FR1 having the amino acid sequence of SEQ ID NO: 134

[0318] HC-FR2 having the amino acid sequence of SEQ ID NO: 92

[0319] HC-FR3 having the amino acid sequence of SEQ ID NO: 93

[0320] HC-FR4 having the amino acid sequence of SEQ ID NO: 135,

[0321] 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 substituted with another amino acid.

[0322] (31)(4M2-D5) contains the following VH region:

[0323] HC-FR1 having the amino acid sequence of SEQ ID NO: 147

[0324] HC-FR2 having the amino acid sequence of SEQ ID NO: 148

[0325] HC-FR3 having the amino acid sequence of SEQ ID NO: 149

[0326] HC-FR4 having the amino acid sequence of SEQ ID NO: 135,

[0327] 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 substituted with another amino acid.

[0328] (32)(4M2-A8) contains the following VH region:

[0329] HC-FR1 having the amino acid sequence of SEQ ID NO: 161

[0330] HC-FR2 having the amino acid sequence of SEQ ID NO: 162

[0331] HC-FR3 having the amino acid sequence of SEQ ID NO: 163

[0332] HC-FR4 having the amino acid sequence of SEQ ID NO: 135,

[0333] 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 substituted with another amino acid.

[0334] (33)(9M2-C12) has a VH region containing the following FRs:

[0335] HC-FR1 having the amino acid sequence of SEQ ID NO: 172

[0336] HC-FR2 having the amino acid sequence of SEQ ID NO: 173

[0337] HC-FR3 having the amino acid sequence of SEQ ID NO: 174

[0338] HC-FR4 having the amino acid sequence of SEQ ID NO: 175,

[0339] 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 substituted with another amino acid.

[0340] (34) (13D5p, 13D5-1) contains the following FR VH region:

[0341] HC-FR1 having the amino acid sequence of SEQ ID NO: 103

[0342] HC-FR2 having the amino acid sequence of SEQ ID NO: 186

[0343] HC-FR3 having the amino acid sequence of SEQ ID NO: 187

[0344] HC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0345] 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 substituted with another amino acid.

[0346] (35)(13D5-13) has a VH region comprising the following FRs:

[0347] HC-FR1 having the amino acid sequence of SEQ ID NO: 103

[0348] HC-FR2 having the amino acid sequence of SEQ ID NO: 186

[0349] HC-FR3 having the amino acid sequence of SEQ ID NO: 201

[0350] HC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0351] 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 substituted with another amino acid.

[0352] In some embodiments, the antigen-binding molecule comprises a VH region comprising a CDR according to one of (1) to (18) above and a FR according to one of (19) to (35) above.

[0353] In some embodiments, the antigen binding molecule comprises a VH region according to one of the following (36) to (57):

[0354] (36) A VH region comprising CDRs according to (1) and FRs according to (19), (20), (21), (22), (23) or (24).

[0355] (37) A VH region comprising CDRs according to (2) and FRs according to (19).

[0356] (38) A VH region comprising CDRs according to (3) and FRs according to (20).

[0357] (39) A VH region comprising CDRs according to (4) and FRs according to (20).

[0358] (40) A VH region comprising CDRs according to (5) and FRs according to (21), (22), (23) or (24).

[0359] (41) A VH region comprising CDRs according to (6) and FRs according to (21).

[0360] (42) A VH region comprising CDRs according to (6) and FRs according to (22).

[0361] (43) A VH region comprising CDRs according to (6) and FRs according to (24).

[0362] (44) A VH region comprising CDRs according to (7) and FRs according to (23).

[0363] (45) A VH region comprising CDRs according to (8) and FRs according to (25).

[0364] (46) A VH region comprising CDRs according to (8) and FRs according to (27).

[0365] (47) A VH region comprising CDRs according to (9) and FRs according to (26).

[0366] (48) A VH region comprising CDRs according to (9) and FRs according to (30).

[0367] (49) A VH region comprising CDRs according to (10) and FRs according to (28).

[0368] (50) A VH region comprising CDRs according to (11) and FRs according to (29).

[0369] (51) A VH region comprising CDRs according to (12) and FRs according to (31).

[0370] (52) A VH region comprising CDRs according to (13) and FRs according to (32).

[0371] (53) A VH region comprising CDRs according to (14) and FRs according to (33).

[0372] (54) A VH region comprising CDRs according to (15) and FRs according to (34) or (35).

[0373] (55) A VH region comprising CDRs according to (16) and FRs according to (34).

[0374] (56) A VH region comprising CDRs according to (17) and FRs according to (34).

[0375] (57) A VH region comprising CDRs according to (18) and FRs according to (35).

[0376] In some embodiments, the antigen binding molecule comprises a VH region according to one of the following (58) to (76):

[0377] (58) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 276.

[0378] (59) A VH region comprising an amino acid sequence that has at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 285.

[0379] (60) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 289.

[0380] (61) A VH region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32.

[0381] (62) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 48.

[0382] (63) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 52.

[0383] (64) A VH region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 62.

[0384] (65) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71.

[0385] (66) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87.

[0386] (67) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 102.

[0387] (68) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 106.

[0388] (69) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119.

[0389] (70) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 133.

[0390] (71) A VH region comprising an amino acid sequence having at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 143.

[0391] (72) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 157.

[0392] (73) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 168.

[0393] (74) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 183.

[0394] (75) A VH region comprising an amino acid sequence that has at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 194.

[0395] (76) A VH region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 199.

[0396] In some embodiments, the antigen binding molecule comprises a VL region according to one of the following (77) to (96):

[0397] (77) (4M2-C12 derived consensus sequence) contains the following CDRs in the VL region:

[0398] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0399] LC-CDR2 having the amino acid sequence of SEQ ID NO: 308

[0400] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0401] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0402] (78) (C24 / C26 / C27 consensus sequence) contains the following CDRs in the VL region:

[0403] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0404] LC-CDR2 having the amino acid sequence of SEQ ID NO: 309

[0405] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0406] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0407] (79) (V4-C 24, V4-C26) contains the following CDRs in the VL region:

[0408] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0409] LC-CDR2 having the amino acid sequence of SEQ ID NO: 295

[0410] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0411] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0412] (80) (V4-C27, V4-C30, V4-C31) contains the following CDRs in the VL region:

[0413] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0414] LC-CDR2 having the amino acid sequence of SEQ ID NO: 300

[0415] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0416] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0417] (81) (shared by 4M2-C12 / V4H1 / V4H2) VL region containing the following CDRs:

[0418] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0419] LC-CDR2 having the amino acid sequence of SEQ ID NO: 245

[0420] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0421] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0422] (82) (4M2-C12, 4M2-B4, V4-C1, V4-C9, V4-C28) contains the following CDRs in the VL region:

[0423] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0424] LC-CDR2 having the amino acid sequence of SEQ ID NO: 42

[0425] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0426] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0427] (83)(V4H1) has a VL region comprising the following CDRs:

[0428] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0429] LC-CDR2 having the amino acid sequence of SEQ ID NO: 58

[0430] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0431] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0432] (84)(V4H2) has a VL region comprising the following CDRs:

[0433] LC-CDR1 having the amino acid sequence of SEQ ID NO: 41

[0434] LC-CDR2 having the amino acid sequence of SEQ ID NO: 67

[0435] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 43;

[0436] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0437] (85) (2M1-B12, 2M1-D2) contains the following CDRs in the VL region:

[0438] LC-CDR1 having the amino acid sequence of SEQ ID NO: 80

[0439] LC-CDR2 having the amino acid sequence of SEQ ID NO: 81

[0440] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 82;

[0441] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0442] (86)(4M2-C9) contains the following CDRs in the VL region:

[0443] LC-CDR1 having the amino acid sequence of SEQ ID NO: 96

[0444] LC-CDR2 having the amino acid sequence of SEQ ID NO: 97

[0445] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 98;

[0446] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0447] (87)(4M2-D9) contains the following CDRs in the VH region:

[0448] LC-CDR1 having the amino acid sequence of SEQ ID NO: 114

[0449] LC-CDR2 having the amino acid sequence of SEQ ID NO: 67

[0450] LC-CDR3 having the amino acid sequence of SEQ ID NO: 115,

[0451] 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 substituted with another amino acid.

[0452] (88)(1M2-D2) contains the following CDRs in the VL region:

[0453] LC-CDR1 having the amino acid sequence of SEQ ID NO: 127

[0454] LC-CDR2 having the amino acid sequence of SEQ ID NO: 128

[0455] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 129;

[0456] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0457] (89)(5M1-A11) contains the following CDRs in the VL region:

[0458] LC-CDR1 having the amino acid sequence of SEQ ID NO: 137

[0459] LC-CDR2 having the amino acid sequence of SEQ ID NO: 138

[0460] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 139;

[0461] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0462] (90)(4M2-D5) contains the following CDRs in the VL region:

[0463] LC-CDR1 having the amino acid sequence of SEQ ID NO: 151

[0464] LC-CDR2 having the amino acid sequence of SEQ ID NO: 152

[0465] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 153;

[0466] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0467] (91)(4M2-A8) contains the following CDRs in the VL region:

[0468] LC-CDR1 having the amino acid sequence of SEQ ID NO: 165

[0469] LC-CDR2 having the amino acid sequence of SEQ ID NO: 152

[0470] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 153;

[0471] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0472] (92)(9M2-C12) has a VL region comprising the following CDRs:

[0473] LC-CDR1 having the amino acid sequence of SEQ ID NO: 177

[0474] LC-CDR2 having the amino acid sequence of SEQ ID NO: 178

[0475] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 179;

[0476] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0477] (93) (13D5p derived) VL region containing the following CDRs:

[0478] LC-CDR1 having the amino acid sequence of SEQ ID NO: 247

[0479] LC-CDR2 having the amino acid sequence of SEQ ID NO: 178

[0480] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;

[0481] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0482] (94)(13D5p) contains the following CDRs in the VL region:

[0483] LC-CDR1 having the amino acid sequence of SEQ ID NO: 189

[0484] LC-CDR2 having the amino acid sequence of SEQ ID NO: 178

[0485] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;

[0486] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0487] (95)(13D5-1) has a VL region comprising the following CDRs:

[0488] LC-CDR1 having the amino acid sequence of SEQ ID NO: 197

[0489] LC-CDR2 having the amino acid sequence of SEQ ID NO: 178

[0490] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;

[0491] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0492] (96)(13D5-13) has a VL region comprising the following CDRs:

[0493] LC-CDR1 having the amino acid sequence of SEQ ID NO: 203

[0494] LC-CDR2 having the amino acid sequence of SEQ ID NO: 178

[0495] an LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;

[0496] or a variant thereof, wherein one or two or three amino acids in one or more LC-CDR1, LC-CDR2 or LC-CDR3 are substituted with another amino acid.

[0497] In some embodiments, the antigen binding molecule comprises a VL region according to one of the following (97) to (120):

[0498] (97)(V4-C1) contains the following FR VL region:

[0499] LC-FR1 having the amino acid sequence of SEQ ID NO: 59

[0500] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0501] LC-FR3 having the amino acid sequence of SEQ ID NO: 284

[0502] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0503] 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 substituted with another amino acid.

[0504] (98)(V4-C9) contains the following FR VL region:

[0505] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0506] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0507] LC-FR3 having the amino acid sequence of SEQ ID NO: 284

[0508] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0509] 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 substituted with another amino acid.

[0510] (99)(V4-C24) contains the following FR VL region:

[0511] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0512] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0513] LC-FR3 having the amino acid sequence of SEQ ID NO: 296

[0514] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0515] 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 substituted with another amino acid.

[0516] (100)(V4-C26) contains the following FR VL region:

[0517] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0518] LC-FR2 having the amino acid sequence of SEQ ID NO: 298

[0519] LC-FR3 having the amino acid sequence of SEQ ID NO: 284

[0520] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0521] 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 substituted with another amino acid.

[0522] (101)(V4-C27) contains the following FR VL region:

[0523] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0524] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0525] LC-FR3 having the amino acid sequence of SEQ ID NO: 284

[0526] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0527] 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 substituted with another amino acid.

[0528] (102)(V4-C28) contains the following FR VL region:

[0529] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0530] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0531] LC-FR3 having the amino acid sequence of SEQ ID NO: 296

[0532] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0533] 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 substituted with another amino acid.

[0534] (103)(V4-C30) contains the following FR VL region:

[0535] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0536] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0537] LC-FR3 having the amino acid sequence of SEQ ID NO: 296

[0538] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0539] 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 substituted with another amino acid.

[0540] (104)(V4-C31) contains the following FR VL region:

[0541] LC-FR1 having the amino acid sequence of SEQ ID NO: 288

[0542] LC-FR2 having the amino acid sequence of SEQ ID NO: 283

[0543] LC-FR3 having the amino acid sequence of SEQ ID NO: 304

[0544] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0545] 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 substituted with another amino acid.

[0546] (105)(4M2-C12) has a VL region comprising the following FRs:

[0547] LC-FR1 having the amino acid sequence of SEQ ID NO: 44

[0548] LC-FR2 having the amino acid sequence of SEQ ID NO: 45

[0549] LC-FR3 having the amino acid sequence of SEQ ID NO: 46

[0550] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0551] 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 substituted with another amino acid.

[0552] (106)(4M2-B4) has a VL region comprising the following FRs:

[0553] LC-FR1 having the amino acid sequence of SEQ ID NO: 51

[0554] LC-FR2 having the amino acid sequence of SEQ ID NO: 45

[0555] LC-FR3 having the amino acid sequence of SEQ ID NO: 46

[0556] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0557] 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 substituted with another amino acid.

[0558] (107)(V4H1) has a VL region comprising the following FRs:

[0559] LC-FR1 having the amino acid sequence of SEQ ID NO: 59

[0560] LC-FR2 having the amino acid sequence of SEQ ID NO: 60

[0561] LC-FR3 having the amino acid sequence of SEQ ID NO: 61

[0562] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0563] 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 substituted with another amino acid.

[0564] (108)(V4H2) has a VL region comprising the following FRs:

[0565] LC-FR1 having the amino acid sequence of SEQ ID NO: 68

[0566] LC-FR2 having the amino acid sequence of SEQ ID NO: 69

[0567] LC-FR3 having the amino acid sequence of SEQ ID NO: 70

[0568] LC-FR4 having the amino acid sequence of SEQ ID NO: 47,

[0569] 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 substituted with another amino acid.

[0570] (109)(2M1-B12) A VL region comprising the following FRs:

[0571] LC-FR1 having the amino acid sequence of SEQ ID NO: 83

[0572] LC-FR2 having the amino acid sequence of SEQ ID NO: 84

[0573] LC-FR3 having the amino acid sequence of SEQ ID NO: 85

[0574] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0575] 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 substituted with another amino acid.

[0576] (110)(4M2-C9) contains the following FR VL region:

[0577] LC-FR1 having the amino acid sequence of SEQ ID NO: 99

[0578] LC-FR2 having the amino acid sequence of SEQ ID NO: 100

[0579] LC-FR3 having the amino acid sequence of SEQ ID NO: 101

[0580] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0581] 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 substituted with another amino acid.

[0582] (111)(2M1-D2) contains the following FR VL region:

[0583] LC-FR1 having the amino acid sequence of SEQ ID NO: 105

[0584] LC-FR2 having the amino acid sequence of SEQ ID NO: 84

[0585] LC-FR3 having the amino acid sequence of SEQ ID NO: 85

[0586] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0587] 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 substituted with another amino acid.

[0588] (112)(4M2-D9) contains the following FR VL region:

[0589] LC-FR1 having the amino acid sequence of SEQ ID NO: 116

[0590] LC-FR2 having the amino acid sequence of SEQ ID NO: 117

[0591] LC-FR3 having the amino acid sequence of SEQ ID NO: 118

[0592] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0593] 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 substituted with another amino acid.

[0594] (113)(1M2-D2) contains the following FR VL region:

[0595] LC-FR1 having the amino acid sequence of SEQ ID NO: 130

[0596] LC-FR2 having the amino acid sequence of SEQ ID NO: 131

[0597] LC-FR3 having the amino acid sequence of SEQ ID NO: 132

[0598] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0599] 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 substituted with another amino acid.

[0600] (114)(5M1-A11) contains the following FR VL region:

[0601] LC-FR1 having the amino acid sequence of SEQ ID NO: 140

[0602] LC-FR2 having the amino acid sequence of SEQ ID NO: 141

[0603] LC-FR3 having the amino acid sequence of SEQ ID NO: 142

[0604] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0605] 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 substituted with another amino acid.

[0606] (115)(4M2-D5) contains the following FR VL region:

[0607] LC-FR1 having the amino acid sequence of SEQ ID NO: 154

[0608] LC-FR2 having the amino acid sequence of SEQ ID NO: 155

[0609] LC-FR3 having the amino acid sequence of SEQ ID NO: 156

[0610] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0611] 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 substituted with another amino acid.

[0612] (116)(4M2-A8) contains the following FR VL region:

[0613] LC-FR1 having the amino acid sequence of SEQ ID NO: 166

[0614] LC-FR2 having the amino acid sequence of SEQ ID NO: 155

[0615] LC-FR3 having the amino acid sequence of SEQ ID NO: 167

[0616] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0617] 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 substituted with another amino acid.

[0618] (117)(9M2-C12) contains the following FR VL region:

[0619] LC-FR1 having the amino acid sequence of SEQ ID NO: 180

[0620] LC-FR2 having the amino acid sequence of SEQ ID NO: 181

[0621] LC-FR3 having the amino acid sequence of SEQ ID NO: 182

[0622] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0623] 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 substituted with another amino acid.

[0624] (118)(13D5p) contains the following FR VL region:

[0625] LC-FR1 having the amino acid sequence of SEQ ID NO: 191

[0626] LC-FR2 having the amino acid sequence of SEQ ID NO: 192

[0627] LC-FR3 having the amino acid sequence of SEQ ID NO: 193

[0628] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0629] 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 substituted with another amino acid.

[0630] (119)(13D5-1) has a VL region comprising the following FRs:

[0631] LC-FR1 having the amino acid sequence of SEQ ID NO: 191

[0632] LC-FR2 having the amino acid sequence of SEQ ID NO: 198

[0633] LC-FR3 having the amino acid sequence of SEQ ID NO: 193

[0634] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0635] 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 substituted with another amino acid.

[0636] (120)(13D5-13) has a VL region comprising the following FRs:

[0637] LC-FR1 having the amino acid sequence of SEQ ID NO: 191

[0638] LC-FR2 having the amino acid sequence of SEQ ID NO: 192

[0639] LC-FR3 having the amino acid sequence of SEQ ID NO: 204

[0640] LC-FR4 having the amino acid sequence of SEQ ID NO: 86,

[0641] 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 substituted with another amino acid.

[0642] In some embodiments, the antigen binding molecule comprises a VL region comprising a CDR according to one of (77) to (96) above and a FR according to one of (97) to (120) above.

[0643] In some embodiments, the antigen binding molecule comprises a VL region according to one of the following (121) to (148):

[0644] (121) A VL region comprising CDRs according to (77) and FRs according to (97), (98), (99), (100), (101), (102), (103), (104), (105), (106), (107) or (108).

[0645] (122) A VL region comprising CDRs according to (78) and FRs according to (99), (100) or (101).

[0646] (123) A VL region comprising CDRs according to (79) and FRs according to (99).

[0647] (124) A VL region comprising CDRs according to (79) and FRs according to (100).

[0648] (125) A VL region comprising CDRs according to (80) and FRs according to (101).

[0649] (126) A VL region comprising CDRs according to (82) and FRs according to (97).

[0650] (127) A VL region comprising CDRs according to (82) and FRs according to (98).

[0651] (128) A VL region comprising CDRs according to (82) and FRs according to (102).

[0652] (129) A VL region comprising CDRs according to (80) and FRs according to (103).

[0653] (130) A VL region comprising CDRs according to (80) and FRs according to (104).

[0654] (131) A VL region comprising CDRs according to (81) and FRs according to (105), (106), (107) or (108).

[0655] (132) A VL region comprising CDRs according to (82) and FRs according to (105).

[0656] (133) A VL region comprising CDRs according to (82) and FRs according to (106).

[0657] (134) A VL region comprising CDRs according to (83) and FRs according to (107).

[0658] (135) A VL region comprising CDRs according to (84) and FRs according to (108).

[0659] (136) A VL region comprising CDRs according to (85) and FRs according to (109).

[0660] (137) A VL region comprising CDRs according to (85) and FRs according to (111).

[0661] (138) A VL region comprising CDRs according to (86) and FRs according to (110).

[0662] (139) A VL region comprising CDRs according to (87) and FRs according to (112).

[0663] (140) A VL region comprising CDRs according to (88) and FRs according to (113).

[0664] (141) A VL region comprising CDRs according to (89) and FRs according to (114).

[0665] (142) A VL region comprising CDRs according to (90) and FRs according to (115).

[0666] (143) A VL region comprising CDRs according to (91) and FRs according to (116).

[0667] (144) A VL region comprising CDRs according to (92) and FRs according to (117).

[0668] (145) A VL region comprising CDRs according to (93) and FRs according to (118), (119) or (120).

[0669] (146) A VL region comprising CDRs according to (94) and FRs according to (118).

[0670] (147) A VL region comprising CDRs according to (95) and FRs according to (119).

[0671] (148) A VL region comprising CDRs according to (96) and FRs according to (120).

[0672] In some embodiments, the antigen binding molecule comprises a VL region according to one of the following (149) to (173):

[0673] (149) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 310.

[0674] (150) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 282.

[0675] (151) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 287.

[0676] (152) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 294.

[0677] (153) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 297.

[0678] (154) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 299.

[0679] (155) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 301.

[0680] (156) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 302.

[0681] (157) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 303.

[0682] (158) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0683] (159) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 50.

[0684] (160) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 57.

[0685] (161) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 66.

[0686] (162) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79.

[0687] (163) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0688] (164) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 104.

[0689] (165) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113.

[0690] (166) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 126.

[0691] (167) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 136.

[0692] (168) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 150.

[0693] (169) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 164.

[0694] (170) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 176.

[0695] (171) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 188.

[0696] (172) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 196.

[0697] (173) A VL region comprising an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 202.

[0698] In some embodiments, the antigen binding molecule comprises the VH region according to any one of (1) to (76) above and the VL region according to any one of (77) to (173) above.

[0699] In some embodiments, the antigen binding molecule comprises:

[0700] and a VL region comprising an amino acid sequence that is at least 70%, more preferably at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 297.

[0701] In some embodiments, the antigen binding molecule comprises or consists of:

[0702] (i) one or more (e.g., two) polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 315; and

[0703] (ii) comprising one or more (e.g., two) polypeptides or a composition thereof having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 317.

[0704] In some embodiments, the antigen binding molecule comprises or consists of:

[0705] (i) comprising one or more (e.g., two) polypeptides or a composition thereof having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 331; and

[0706] (ii) comprising one or more (e.g., two) polypeptides or a composition thereof having at least 70%, more preferably at least one of 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 317.

[0707] In embodiments according to the present invention where one or more amino acids are substituted with another amino acid, the substitution may be a conservative substitution, such as a substitution according to the table below. In some embodiments, amino acids in the same block in the middle column are substituted. In some embodiments, amino acids in the same row in the rightmost column are substituted:

[0708]

[0709] In some embodiments, the substitutions may be functionally conservative. That is, in some embodiments, the substitutions may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of the antigen binding molecule comprising the substitutions, compared to an equivalent unsubstituted molecule.

[0710] The VH and VL regions of the antigen-binding region of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecules according to the present invention comprise or consist of an Fv region that is bound to VISTA. In some embodiments, the VH and VL regions of Fv are provided as a single polypeptide connected by a connecting region, i.e., a single-chain Fv (scFv).

[0711] In some embodiments, the antigen binding molecules of the present invention comprise 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 (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM.

[0712] In some embodiments, the immunoglobulin heavy chain constant sequence is the human immunoglobulin G1 constant sequence (IGHG1; UniProt: P01857-1, v1; SEQ ID NO: 205). Positions 1 to 98 of SEQ ID NO: 205 form the CH1 region (SEQ ID NO: 206). Positions 99 to 110 of SEQ ID NO: 205 form the hinge region between the CH1 and CH2 regions (SEQ ID NO: 207). Positions 111 to 223 of SEQ ID NO: 205 form the CH2 region (SEQ ID NO: 208). Positions 224 to 330 of SEQ ID NO: 205 form the CH3 region (SEQ ID NO: 209).

[0713] Exemplary antigen-binding molecules can be prepared using pFUSE-CHIg-hG1, which contains D356E, L358M substitution mutations in the CH3 region (positions encoded 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 will be appreciated that further substitutions can be provided for the CH3 region, depending on the modification of the Fc region of the antigen-binding molecules described herein.

[0714] In some embodiments, the CH1 region comprises or consists of the sequence of SEQ ID NO: 206, or comprises or consists of an amino acid sequence that has at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 206. In some embodiments, the CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID NO: 207, or comprises or consists of an amino acid sequence that has at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207. In some embodiments, the CH2 region comprises or consists of the sequence of SEQ ID NO: 208, or comprises or consists of an amino acid sequence that has at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 208. In some embodiments, the CH3 region comprises or consists of the sequence of SEQ ID NO: 209 or 210, or comprises or consists of an amino acid sequence that has at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 209 or 210.

[0715] In some embodiments, the antigen-binding molecules of the present invention comprise one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin kappa constant sequence (IGKC; Cκ; UniProt: P01834-1, v2; SEQ ID NO: 211). In some embodiments, the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant sequence (IGLC; Cλ), such as IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the CL region comprises or consists of the sequence of SEQ ID NO: 211, or comprises or consists of an amino acid sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 211.

[0716] The VL and light chain constant (CL) regions of the antibody's antigen-binding region, as well as the VH region and heavy chain constant 1 (CH1) region, together constitute the Fab region. In some embodiments, the antigen-binding molecule comprises a Fab region comprising VH, CH1, VL, and CL (e.g., Cκ or Cλ). In some embodiments, the Fab region comprises a polypeptide comprising VH and CH1 (e.g., a VH-CH1 fusion polypeptide), and a polypeptide comprising VL and CL (e.g., a VL-CL fusion polypeptide). In some embodiments, the Fab region comprises a polypeptide comprising VH and CL (e.g., a VH-CL fusion polypeptide), and a polypeptide comprising VL and CH (e.g., 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 a Fab or CrossFab are provided as a single polypeptide connected by a linker region, i.e., a single-chain Fab (scFab) or a single-chain CrossFab (scCrossFab).

[0717] In some embodiments, the antigen-binding molecules of the present invention comprise or consist of a Fab region that binds to VISTA.

[0718] In some embodiments, the antigen binding molecules described herein comprise or consist of a complete antibody that binds to VISTA. As used herein, a "complete antibody" refers to an antibody having a structure substantially similar to that of an immunoglobulin (Ig). For example, different types of immunoglobulins and their structures are described in Schroeder and Cavacini, J Allergy Clin Immunol. (2010) 125 (202): S41-S52, which is incorporated herein by reference in its entirety.

[0719] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa, consisting of two heavy chains and two light chains. From N-terminus to C-terminus, the heavy chain comprises VH, followed by a heavy chain constant region comprising three constant domains (CH1, CH2, and CH3). Similarly, the light chain comprises VL, followed by CL. Based on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be either kappa (κ) or lambda (λ).

[0720] In some embodiments, the antigen binding molecules described herein comprise or consist of IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that binds to VISTA.

[0721] In some embodiments, the antigen binding molecules of the present invention are at least monovalently bound to VISTA. Binding valence refers to the number of binding sites in the antigen binding molecules of a given antigenic determinant. Therefore, in some embodiments, the antigen binding molecules include at least one VISTA binding site.

[0722] In some embodiments, the antigen binding molecule comprises multiple VISTA binding sites, such as 2, 3 or 4 binding sites. The binding sites can be the same or different. In some embodiments, the antigen binding molecule is, for example, divalent, trivalent or tetravalent for VISTA.

[0723] The present invention relates to aspects of multispecific antigen binding molecules. "Multispecific" refers to an antigen binding molecule that exhibits specific binding to more than one target. In some embodiments, the antigen binding molecule is a bispecific antigen binding molecule. In some embodiments, the antigen binding molecule comprises at least two different antigen binding domains (i.e., at least two antigen binding domains, e.g., comprising different VH and VL).

[0724] In some embodiments, the antigen binding molecule binds to VISTA and another target (e.g., an antigen other than VISTA) and is therefore at least bispecific. The term "bispecific" refers to an antigen binding molecule that is able to specifically bind to at least two different antigenic determinants.

[0725] It should be understood that the antigen binding molecules according to the present invention (e.g., multispecific antigen binding molecules) can include antigen binding molecules that can be bound to the specific target of the antigen binding molecules. For example, antigen binding molecules that can be combined with antigens other than VISTA and VISTA can include: (i) antigen binding molecules that can be combined with VISTA, and (ii) antigen binding molecules that can be combined with antigens other than VISTA.

[0726] It should also be understood that the antigen binding molecules according to the present invention (e.g., multispecific antigen binding molecules) may include antigen binding polypeptides or antigen binding polypeptide complexes that can be bound to the specific target of the antigen binding molecule. For example, the antigen binding molecules according to the present invention may include, for example, (i) antigen binding polypeptide complexes capable of binding to VISTA, comprising light chain polypeptides (comprising VL-CL structure) and heavy chain polypeptides (comprising VH-CH1-CH2-CH3 structure), and ii) antigen binding polypeptide complexes capable of binding to antigens other than VISTA, comprising light chain polypeptides (comprising VL-CL structure) and heavy chain polypeptides (comprising VH-CH1-CH2-CH3 structure).

[0727] In some embodiments, a component antigen binding molecule of a larger antigen binding molecule (e.g., a multispecific antigen binding molecule) can be referred to as, for example, an "antigen binding domain" or "antigen binding region" of the larger antigen binding molecule.

[0728] In some embodiments, the antigen binding molecules include antigen binding molecules capable of binding to VISTA and antigen binding molecules capable of binding to antigens other than VISTA. In some embodiments, the antigens other than VISTA are immune cell surface molecules. In some embodiments, the antigens other than VISTA are cancer cell antigens. In some embodiments, the antigens other than VISTA are receptor molecules, such as cell surface receptors. In some embodiments, the antigens other than VISTA are cell signaling molecules, such as cytokines, chemokines, interferons, interleukins, or lymphokines. In some embodiments, the antigens other than VISTA are growth factors or hormones.

[0729] Cancer cell antigens are antigens expressed or overexpressed by cancer cells. Cancer cell antigens can be any peptide / polypeptide, glycoprotein, lipoprotein, polysaccharide, glycolipid, lipid or its fragment. The expression of cancer cell antigens may be related to cancer. Cancer cell antigens can be abnormally expressed by cancer cells (e.g., cancer cell antigens can be abnormally positioned and expressed), or can be expressed by cancer cells with abnormal structures. Cancer cell antigens may be able to trigger an immune response. In some embodiments, the antigen is expressed on the cell surface of cancer cells (i.e., the cancer cell antigen is a cancer cell surface antigen). In some embodiments, a portion of the antigen bound to the antigen binding molecules described herein is displayed on the outer surface of the cancer cell (i.e., extracellular). Cancer cell antigens may be cancer-associated antigens. In some embodiments, the cancer cell antigen is an antigen whose expression is related to the development, progression or severity of cancer symptoms. Antigens associated with cancer may be related to the cause or pathology of cancer, or may be abnormally expressed due to cancer. In some embodiments, the cancer cell antigen is an antigen whose expression is upregulated (e.g., at the RNA and / or protein level) by cancer cells compared to the expression level of corresponding non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). In some embodiments, the cancer associated antigen may be preferentially expressed by cancer cells, but not by corresponding non-cancerous cells (e.g., non-cancerous cells derived from the same tissue / cell type). 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 oncovirus, a carcinoembryonic antigen, or a product of a cell surface glycolipid or glycoprotein.

[0730] Immune cell surface molecules can be any peptide / polypeptide, glycoprotein, lipoprotein, polysaccharide, glycolipid, lipid or its fragment on the cell surface or surface expression of immune cell.In some embodiments, a part for the immune cell surface molecules combined by the antigen binding molecules of the present invention is on the outer surface of immune cell (i.e., extracellular). Immune cell surface molecules can be expressed on the cell surface of any immune cell.In some embodiments, immune cell can be a cell of hematopoietic origin, such as neutrophil, eosinophil, basophil, dendritic cell, lymphocyte or monocyte. Lymphocyte can be, such as T cell, B cell, natural killer (NK) cell, NKT cell or innate lymphoid cell (ILC) or its precursor (such as thymocyte or B precursor cell).In some embodiments, immune cell surface molecules can be costimulatory molecules (such as CD28, OX40, 4-1BB, ICOS or CD27) or its part. In some embodiments, the immune cell surface molecule can be a checkpoint molecule (e.g., PD-1, CTLA-4, LAG-3, TIM-3, TIGIT, or BTLA) or a ligand thereof.

[0731] The multispecific antigen-binding molecules according to the present invention may be provided in any suitable format, such as those described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, which is incorporated herein by reference in its entirety. Suitable formats include those described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212. Figure 2Formats shown in: antibody conjugates, such as IgG2, F(ab')2 or CovX-antibodies; IgG or IgG-like molecules, such as IgG, chimeric IgG, κλ-antibody common 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), triabodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAb, three-head antibodies, tandem dAb / VHH, trivalent dAb.VHH; non-Ig fusion proteins, such as scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibodies, DNL-Fab2, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine 2, ImmTAC (TCR-scFv); modified F c and CH3 fusion proteins, such as scFv-Fc (kih), scFv-Fc (CH3 charge pair), scFv-Fc (EW-RVT), scFv-fc (HA-TF), scFv-Fc (SEED antibody), taFv-Fc (kih), scFv-Fc (kih) -Fv, Fab-Fc (kih) -scFv, Fab-scFv-Fc (kih), Fab-scFv-Fc (BE AT), Fab-scFv-Fc (SEED antibody), DART-Fc, scFv-CH3 (kih), TriFabs; Fc fusions, such as diabodies, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab-scFv-Fc, scFv4-Ig, scFv2-Fcab; CH3 fusions, such as diabodies, scDb-CH3; IgE / IgM CH2 fusions, such as scFv-EHD2-scFv, scFvMHD2-scFv; Fab fusion proteins, such as Fab-scFv (diabodies), Fab-scFv2 (tribodies), Fab-Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-Ig fusion proteins, such as DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG-cytokine 2;Asymmetric IgG or IgG-like molecules, such as IgG(kih), IgG(kih) universal LC, ZW1 IgG universal LC, Biclonics universal 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-antibody, bispecific antibody, four-in-one CrossMab(kih), LUZ-Y universal LC; LUZ-Y scFab-IgG, FcFc*; appended and Fc-modified IgG, such as IgG(kih)-Fv, IgG HA-TF-Fv, IgG (kih) scFab, scFab-Fc (kih) -scFv2, scFab-Fc (kih) -scFv, half DVD-Ig, DVI-Ig (four in one), CrossMab-Fab; modified Fc and CH3 fusion proteins, such as Fab-Fc (kih) -scFv, Fab-scFv-Fc (kih), Fab-scFv-Fc (BEAT), Fab-scFv-Fc-SEED antibody, TriFab; additional IgG-HC fusion proteins, 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, Zy antibodies; Fc fusions, such as Fab-scFv-Fc, scFv4-Ig; F(ab')2 fusions, such as F(ab')2-scFv2; CH1 / CL fusion protein scFv2-CH1-hinge / CL; modified IgG, such as DAF (two-in-one IgG), DutaMab, Mab2; and non-Ig fusions, such as DNL-Fab4-IgG.;

[0732] Technicians are able to design and prepare bispecific antigen-binding molecules. Methods for producing bispecific antigen-binding molecules include chemical cross-linking of antigen-binding molecules or antibody fragments, for example, by having reducible disulfide bonds or non-reducible thioether bonds, as described in Segal and Bast in 2001 in "Generation of Bispecific Antigen-Binding Molecules", Current Protocols in Immunology 14: IV: 2.13: 2.13.1–2.13.16, which is incorporated herein by reference in its entirety. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used for chemical cross-linking, such as Fab fragments via hinge region SH-groups to produce disulfide-linked bispecific F(ab)2 heterodimers.

[0733] Other methods for producing bispecific antigen-binding molecules include fusing antibody-producing hybridomas, for example, with polyethylene glycol to produce a quadrivalent cell capable of secreting the bispecific antibody, as described in DM and Bast, BJ, 2001, "Generation of Bispecific Antigen-Binding Molecules," Current Protocols in Immunology, 14: IV: 2.13: 2.13.1–2.13.16.

[0734] The bispecific antigen-binding molecules according to the present invention can also be produced recombinantly, for example by expression from a nucleic acid construct encoding the polypeptide of the antigen-binding molecule, for example in Antibody Engineering: Methods and Protocols, 2nd Edition (Humana Press, 2012), Chapter 40: Generation of Bispecific Antibodies: Diabodies and Tandem scFv (Hornig -Schwarz), or in French, How to make bispecific antigen binding molecules, Methods in Molecular Medicine 2000, 40: 333-339, which are incorporated herein by reference in their entirety. For example, a DNA construct encoding the light chain and heavy chain variable domains of two antigen binding fragments (i.e., a light chain and heavy chain variable domain of an antigen binding fragment capable of binding to VISTA, and a light chain and heavy chain variable domain of an antigen binding fragment capable of binding to another target protein), and a sequence encoding a suitable connector or dimerization domain between the antigen binding fragments can be prepared by molecular cloning techniques. Recombinant bispecific antibodies can be produced by expression (e.g., in vitro) in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibodies can then be selectively purified.

[0735] Fc region

[0736] In some embodiments, the antigen binding molecules of the present invention comprise an Fc region.

[0737] In IgG, IgA, and IgD isotypes, the Fc region is composed of the CH2 and CH3 regions of one polypeptide and the CH2 and CH3 regions of another polypeptide. The CH2 and CH3 regions from both polypeptides together form the Fc region. In IgM and IgE isotypes, the Fc region contains three constant domains (CH2, CH3, and CH4), and the CH2 to CH4 regions from both polypeptides together constitute the Fc region.

[0738] The Fc region provides for interactions with Fc receptors and other molecules of the immune system to produce functional effects. For example, IgG Fc-mediated effector functions are reviewed in Jefferis et al., Immunol Rev 1998 163:59-76 (incorporated herein in its entirety by reference), and lead to Fc-mediated recruitment and activation of immune cells (e.g., macrophages, dendritic cells, NK cells, and T cells), through the interaction between the Fc region and Fc receptors expressed by immune cells, through binding of the Fc region to the complement protein C1q, recruiting complement pathway components, and thereby activating the complement cascade.

[0739] Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), membrane attack complex (MAC) formation, cell degranulation, cytokine and / or chemokine production, and antigen processing and presentation.

[0740] Modifications of antibody Fc regions that affect Fc-mediated functions are known in the art, such as those described in Wang et al., Protein Cell (2018) 9(1): 63-73, which is incorporated herein by reference in its entirety. In particular, exemplary Fc region modifications known to affect antibody effector function are summarized in Table 1 of Wang et al., Protein Cell (2018) 9(1): 63-73. Modifications of the Fc region that affect antibody effector activity are described below.

[0741] When an Fc region / CH2 / CH3 is described as comprising a modification "corresponding to" a reference substitution, equivalent substitutions in the homologous Fc / CH2 / CH3 are contemplated. For example, the L234A / L235A substitution in human IgG1 (numbered according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Public Health Service, Bethesda, MD, 1991) corresponds to an L to A substitution at positions 117 and 118 of the A allele of the C region of mouse Ig gamma-2 A chain, which is numbered as SEQ ID NO: 256.

[0742] When an Fc region is described as comprising a modification, the modification may be present in one or both polypeptide chains that together form the Fc region.

[0743] In some embodiments, the antigen-binding molecules of the present invention comprise an Fc region comprising a modification. In some embodiments, the antigen-binding molecules of the present invention comprise an Fc region comprising a modification in one or more CH2 and / or CH3 regions.

[0744] In some embodiments, the Fc region contains modifications to increase Fc-mediated functions. In some embodiments, the Fc region contains modifications to increase ADCC. In some embodiments, the Fc region contains modifications to increase ADCP. In some embodiments, the Fc region contains modifications to increase CDC. Antigen binding molecules comprising an Fc region that contains modifications to increase Fc-mediated functions (e.g., ADCC, ADCP, CDC) induce increased levels of related effector functions compared to antigen binding molecules comprising a corresponding unmodified Fc region.

[0745] In some embodiments, the Fc region contains modifications to increase binding to Fc receptors. In some embodiments, the Fc region contains modifications to increase binding to Fcγ receptors. In some embodiments, the Fc region contains modifications to increase binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region contains modifications to increase binding to FcγRIIIa. In some embodiments, the Fc region contains modifications to increase binding to FcγRIIa. In some embodiments, the Fc region contains modifications to increase binding to FcγRIIb. In some embodiments, the Fc region contains modifications to increase binding to FcRn. In some embodiments, the Fc region contains modifications to increase binding to complement proteins. In some embodiments, the Fc region contains modifications to increase binding to C1q. In some embodiments, the Fc region contains modifications to promote hexamerization of the antigen-binding molecule. In some embodiments, the Fc region contains modifications to increase the half-life of the antigen binding molecule. In some embodiments, the Fc region contains modifications to increase co-binding.

[0746] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination F243L / R292P / Y300L / V305I / P396L, as described in Stavenhagen et al., Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S239D / I332E or S239D / I332E / A330L, as described in Lazar et al., Proc Natl Acad Sci USA. (2006) 103:4005-4010. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination S298A / E333A / K334A, as described in Shields et al., J Biol Chem. (2001) 276:6591-6604. In some embodiments, the Fc region comprises a modification of one of the heavy chain polypeptides corresponding to the substitution combination L234Y / L235Q / G236W / S239M / H268D / D270E / S298A and a modification of the other heavy chain polypeptide corresponding to the substitution combination D270E / K326D / A330M / K334E, as described in Mimoto et al., MAbs. (2013): 5: 229–236. In some embodiments, the Fc region comprises a modification corresponding to the substitution combination G236A / S239D / I332E, as described in Richards et al., Mol Cancer Ther. (2008) 7: 2517-2527.

[0747] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination K326W / E333S, as 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, as described in Moore et al., MAbs. (2010) 2(2):181-9. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination 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, as described in Diebolder et al., Science (2014) 343(6176):1260-3.

[0748] In some embodiments, the Fc region comprises modifications corresponding to the substitution combination M252Y / S254T / T256E, as described in DallAcqua 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.

[0749] In some embodiments, the Fc region comprises a modification 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 a modification corresponding to the substitution combination N325S / L328F, as described in Shang et al., Biol Chem. (2014) 289: 15309-15318.

[0750] In some embodiments, the Fc region comprises a modification that reduces / prevents Fc-mediated functions. In some embodiments, the Fc region comprises a modification that reduces / prevents ADCC. In some embodiments, the Fc region comprises a modification that reduces / prevents ADCP. In some embodiments, the Fc region comprises a modification that reduces / prevents CDC. Compared to an antigen-binding molecule comprising a corresponding unmodified Fc, an antigen-binding molecule comprising an Fc region that contains a modification that reduces / prevents Fc-mediated functions (e.g., ADCC, ADCP, CDC) induces a reduction in the level of the relevant effector function.

[0751] In some embodiments, the Fc region comprises a modification that reduces / prevents binding to an Fc receptor. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to an Fcγ receptor. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIIa. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIa. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to FcγRIIb. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to a complement protein. In some embodiments, the Fc region comprises a modification that reduces / prevents binding to C1q. In some embodiments, the Fc region comprises a modification that reduces / prevents glycosylation of the amino acid residue corresponding to N297.

[0752] In some embodiments, the Fc region is unable to induce one or more Fc-mediated functions (i.e., lacks the ability to induce the relevant Fc-mediated function). Therefore, the antigen binding molecules comprising such Fc regions also lack the ability to induce the relevant function. Such antigen binding molecules can be described as lacking the relevant function.

[0753] In some embodiments, the Fc region is incapable of inducing ADCC. In some embodiments, the Fc region is incapable of inducing ADCP. In some embodiments, the Fc region is incapable of inducing CDC. In some embodiments, the Fc region is incapable of inducing ADCC and / or incapable of inducing ADCP and / or incapable of inducing CDC.

[0754] 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 Fc region is not glycosylated at the amino acid residue corresponding to N297.

[0755] 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 modifications 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 modifications corresponding to the substitution combination described in Rother et al., Nat Biotechnol. (2007) 25:1256-1264. In some embodiments, the Fc region comprises modifications 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 modifications 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 modifications corresponding to the substitution combination V234A / G237A / P238S / H268A / V309L / A330S / P331S as described in Vafa et al., (2014) 65: 114-126. In some embodiments, the Fc region comprises modifications corresponding to the substitution combination L234A / L235E / G237A / A330S / P331S as described in US 2015 / 0044231A1.

[0756] It is known that the substitution mutations L234A / L235A and combinations of corresponding substitution mutations (e.g., F234A / L235A in human IgG4) disrupt Fc binding to Fcγ receptors and inhibit ADCC, ADCP, and also reduce C1q binding, thereby reducing CDC (Schlothauer et al., Protein Engineering, Design & Selection (2016), 29(10): 457–466, incorporated herein by reference in its entirety). The P329G and P329A substitution mutations reduce C1q binding (thereby reducing CDC). It is known that substitution of "N297" with A, G, or Q eliminates glycosylation, thereby reducing Fc binding to C1q and Fcγ receptors, and thereby reducing CDC and ADCC binding. Lo et al., J. Biol. Chem. Chem (2017) 292(9): 3900-3908 (incorporated herein by reference in its entirety) reported that the substitution combination L234A / L235A / P329G abolished complement binding and fixation as well as Fcγ receptor-dependent, antibody-dependent cell-mediated cytotoxicity of murine IgG2a and human IgG1.

[0757] In some embodiments, the Fc region comprises a modification of the S228P substitution as described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469. The S228P substitution in IgG4 Fc reduces Fab arm exchange, which may be undesirable.

[0758] In some embodiments, the Fc region comprises a modification corresponding to a combination of L234A / L235A substitutions. In some embodiments, the Fc region comprises a modification corresponding to a P329G substitution. In some embodiments, the Fc region comprises a modification corresponding to a N297Q substitution.

[0759] In some embodiments, the Fc region comprises modifications corresponding to a combination of L234A / L235A / P329G substitutions.

[0760] In some embodiments, the Fc region comprises modifications corresponding to a combination of L234A / L235A / P329G / N297Q substitutions.

[0761] In some embodiments, the Fc region comprises modifications corresponding to a combination of L234A / L235E / G237A / A330S / P331S substitutions.

[0762] In some embodiments, the Fc region comprises a modification corresponding to an S228P substitution, eg, in IgG4.

[0763] In some embodiments, the antigen binding molecules of the present invention are included in one or more CH2 and CH3 regions containing modified Fc regions that promote the association of the Fc region. The recombinant co-expression and subsequent association of the constituent polypeptides of the antigen binding molecules result in several possible combinations. In order to increase the yield of the desired polypeptide combination in the antigen binding molecules in recombinant production, it is advantageous to introduce modifications in the Fc region that promote the association of the desired combination of heavy chain polypeptides. Modifications can promote, for example, hydrophobic and / or electrostatic interactions between the CH2 and / or CH3 regions of different polypeptide chains. For example, suitable modifications are described in Ha et al., Front.Immnol (2016) 7: 394, which is incorporated herein by reference in its entirety.

[0764] In some embodiments, the antigen-binding molecules of the invention comprise an Fc region comprising paired substitutions in the CH3 region of the Fc region according to one of the following formats, as shown in Ha et al., Immnol (2016) 7:394: KiH, KiHs-s, HA-TF, ZW1, 7.8.60, DD-KK, EW-RVT, EW-RVT ss, SEED or A107 in Table 1.

[0765] In some embodiments, the Fc region comprises a "knob-into-hole" or "KiH" modification, for example, as described in U.S. Patent No. 7,695,936 and Carter, J. Immunol. Meth. 248, 7-15 (2001). In such embodiments, one CH3 region of the Fc region comprises a "knob" modification, while the other CH3 region comprises a "hole" modification. The "knob" and "hole" modifications are located within the corresponding CH3 regions, so that the "knob" can be positioned within the "hole" to promote heterodimerization (and inhibit homodimerization) and / or stabilize heterodimerization of the polypeptide. The knob is constructed by replacing amino acids with small side chains with amino acids with larger side chains (e.g., tyrosine or tryptophan). The hole is created by replacing amino acids with large side chains with amino acids with smaller side chains (e.g., alanine or threonine).

[0766] In some embodiments, a CH3 region in the Fc region of the antigen binding molecules of the present invention comprises a T366W substitution (the numbering of the positions / substituents in the Fc, CH2, and CH3 regions herein is according to the EU numbering system in Kabat et al., "Protein Sequences of Interest in Immunology," 5th edition, National Institutes of Health, Public Health, Bethesda, Maryland, 1991 publication), and another CH3 region in the Fc region comprises a Y407V substitution. In some embodiments, a CH3 region in the Fc region of the antigen binding molecules comprises a T366W substitution, and another CH3 region in the Fc region comprises a T366S and L368A substitution. In some embodiments, a CH3 region in the Fc region of the antigen binding molecules comprises a T366W substitution, and another CH3 region in the Fc region comprises a Y407V, T366S, and L368A substitution.

[0767] In some embodiments, the Fc region comprises a "DD-KK" modification as described in WO 2014 / 131694 A1. In some embodiments, one CH3 region comprises K392D and K409D substitutions, and the other CH3 region of the Fc region comprises E356K and D399K substitutions. These modifications promote electrostatic interactions between the CH3 regions.

[0768] In some embodiments, the antigen binding molecules of the present invention comprise an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci USA. (2013) 110(13): 5145-50, referred to as a "diabody" format. In some embodiments, one CH3 region comprises a K409R substitution and the other CH3 region in the Fc region comprises a K405L substitution.

[0769] In some embodiments, the antigen binding molecules of the present invention comprise an Fc region comprising an "EEE-RRR" modification as described in Strop et al., J Mol Biol. J. Biol. (2012) 420(3): 204-19. In some embodiments, one CH3 region comprises D221E, P228E, and L368E substitutions, and the other CH3 region of the Fc region comprises D221R, P228R, and K409R substitutions.

[0770] In some embodiments, the antigen binding molecule comprises an Fc region comprising the "EW-RVT" modification described in Choi et al., Mol Cancer Ther (2013) 12(12): 2748-59. In some embodiments, one CH3 region comprises K360E and K409W substitutions, and the other CH3 region of the Fc region comprises Q347R, D399V, and F405T substitutions.

[0771] In some embodiments, one CH3 region comprises an S354C substitution and the other CH3 region of the Fc region comprises a Y349C substitution. The introduction of these cysteine ​​residues results in the formation of a disulfide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001), J Immunol Methods 248, 7-15).

[0772] In some embodiments, the Fc region comprises a "KiH SS" modification. In some embodiments, one CH3 region comprises T366W and S354C substitutions, and the other CH3 region of the Fc region comprises T366S, L368A, Y407V, and Y349C substitutions.

[0773] In some embodiments, the antigen binding molecules of the invention comprise an Fc region comprising a "SEED" modification as described in Davis et al., Protein Eng Des Sel (2010) 23(4): 195-202, wherein the β-segments of human IgG1 CH3 and IgA CH3 are exchanged.

[0774] In some embodiments, one CH3 region comprises S364H and F405A substitutions, and the other CH3 region of the Fc region comprises Y349T and T394F substitutions (see, e.g., Moore et al., MAbs (2011) 3(6):546-57).

[0775] In some embodiments, one CH3 region comprises T350V, L351Y, F405A, and Y407V substitutions, and the other CH3 region of the Fc region comprises T350V, T366L, K392L, and T394W substitutions (e.g., see Von Kreudenstein et al., MAbs (2013) 5(5):646-54).

[0776] In some embodiments, one CH3 region comprises K360D, D399M, and Y407A substitutions, and the other CH3 region of the Fc region comprises E345R, Q347R, T366V, and K409V substitutions (see, e.g., Leaver-Fay et al., Structure (2016) 24(4): 641-51).

[0777] In certain embodiments, one CH3 region comprises K370E and K409W substitutions, and the other CH3 region of the Fc region comprises E357N, D399V, and F405T substitutions (see, e.g., Choi et al., PLoS One (2015) 10(12): e0145349).

[0778] In some embodiments, the antigen binding molecules of the present invention comprise an Fc region that does not bind to Fcγ receptors. In some embodiments, the antigen binding molecules comprise an Fc region that does not bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb. In some embodiments, the antigen binding molecules comprise an Fc region that does not bind to one or more of FcγRIIa, FcγRIIb, and FcγRIIIa. In some embodiments, the antigen binding molecules comprise an Fc region that does not bind to one or more of FcγRIIa and FcγRIIb.

[0779] The ability of an Fc region or an antigen-binding molecule comprising an Fc region to bind to a reference protein (e.g., an Fc receptor) can be analyzed according to methods well 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).

[0780] As used herein, an Fc region that "does not bind" a reference protein can be shown to be substantially free of binding to the reference protein as determined by, for example, ELISA, immunoblotting (e.g., Western blot), immunoprecipitation, SPR, or BLI. "Substantially free of binding" can mean that the level of interaction is not significantly greater than the level of interaction determined for proteins that do not bind to each other in a given assay. "Substantially free of binding" can mean that the level of interaction is ≤5-fold, e.g., ≤4-fold, ≤3-fold, ≤2.5-fold, ≤2-fold, or ≤1.5-fold greater than the level of interaction determined for proteins that do not bind to each other in a given assay.

[0781] In some embodiments, the antigen binding molecule comprises an Fc region that binds to FcRn.

[0782] In some embodiments, the antigen binding molecule comprises an Fc region that binds to FcRn and does not bind to one or more of FcγRIIa, FcγRIIb, and FcγRIIIa. In some embodiments, the antigen binding molecule comprises an Fc region that binds to FcRn and does not bind to one or both of FcγRIIa and FcγRIIb.

[0783] In some embodiments, the antigen binding molecules of the present invention comprise an Fc region that does not induce ADCC. In some embodiments, the antigen binding molecules of the present invention comprise an Fc region that does not induce ADCP. In some embodiments, the antigen binding molecules of the present invention comprise an Fc region that does not induce CDC. In some embodiments, the antigen binding molecules of the present invention comprise an Fc region that does not induce ADCC, ADCP or CDC.

[0784] As used herein, an Fc region / antigen binding molecule that does not induce (i.e., is unable to induce) ADCC / ADCP / CDC does not substantially cause ADCC / ADCP / CDC activity, such as the relevant activity determined by an assay in an appropriate assay. "Substantially no ADCC / ADCP / CDC activity" refers to an ADCC / ADCP / CDC level that is not significantly greater than the level of ADCC / ADCP / CDC determined by an appropriate negative control molecule in a given assay (e.g., an antigen binding molecule lacking an antigen binding molecule Fc region or comprising a "silent" Fc region (e.g., as described in Schlothauer et al., Protein Engineering, Design & Selection (2016), 29(10): 457-466, incorporated herein by reference). "Substantially no activity" can be a level of relevant activity in a given assay that is ≤5 times, e.g., ≤4 times, ≤3 times, ≤2.5 times, ≤2 times, or ≤1.5 times, the level of activity determined by an appropriate negative control molecule.

[0785] The ability of an Fc region or an antigen binding molecule comprising an Fc region to induce ADCC can be analyzed, for example, according to the method described in Yamashita et al., Scientific Reports (2016) 6: 19772 (incorporated herein by reference in its entirety), or by the 51Cr release method described, for example, in Jedema et al., Blood (2004) 103: 2677–82 (incorporated herein by reference in its entirety). The ability of an Fc region or an antigen binding molecule comprising an Fc region to induce ADCP can be analyzed, for example, according to the method described in Kamen et al., J Immunol (2017) 198 (1 Suppl.) 157.17 (incorporated herein by reference in its entirety). The ability of an Fc region or an antigen binding molecule comprising an Fc region to induce CDC can be analyzed, for example, using a C1q binding assay, such as described in Schlothauer et al., Protein Engineering, Design & Selection (2016), 29 (10): 457-466 (incorporated herein by reference).

[0786] In some embodiments, the antigen binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence that is at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 254. In some embodiments, the antigen binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence that is at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 257. In some embodiments, the antigen binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence that is at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 259. In some embodiments, the antigen binding molecule comprises an Fc region comprising a polypeptide having an amino acid sequence that is at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 260.

[0787] In some embodiments, the antigen binding molecules of the invention lack an Fc region.

[0788] Fc receptors

[0789] Fc receptors are polypeptides that bind to the Fc region of immunoglobulins. The structure and function of Fc receptors are reviewed in, for example, Masuda et al., Inflamm Allergy Drug Targets (2009) 8(1):80-86 and Bruhns, Blood (2012) 119:5640-5649, both of which are incorporated herein by reference in their entirety.

[0790] Fc receptors are expressed on the surface of hematopoietic cells, including macrophages, neutrophils, dendritic cells, eosinophils, basophils, mast cells, and NK cells. They include the Fcγ receptor, which binds to IgG, the high-affinity receptor for IgE (FcεRI), the IgA receptor, and the polymeric Ig receptor for IgA and IgM. The neonatal Fc receptor (FcRn) is another Fc receptor for IgG and is involved in the transport of IgG across epithelial barriers (endocytosis), protecting IgG from degradation and antigen presentation. Humans have six different classes of Fcγ receptors (mouse orthologs are shown in parentheses): FcγRI (mFcγRI), FcγRIIa (mFcγRIII), FcγRIIb (mFcγRIIb), FcγRIIc, FcγRIIIa (mFcγRIV), and FcγRIIIb.

[0791] FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa contain an immunoreceptor tyrosine-based activation motif (ITAM) in their intracellular domains and lead to activation of cells expressing the receptors through Fc ligation. FcγRIIb contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its intracellular domain and, upon Fc ligation, negatively regulates cellular activation and degranulation, cell proliferation, endocytosis, and phagocytosis.

[0792] In this specification, "Fcγ receptor" can be from any species and includes isoforms, fragments, variants (including mutants) or homologs from any species. Similarly, "FcγRI", "FcγRIIa", "FcγRIIb", "FcγRIIc", "FcγRIIIa" and "FcγRIIIb" refer to FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb of any species, respectively, and include isoforms, fragments, variants (including mutants) or homologs from any species.

[0793] In some embodiments, the Fcγ receptor (e.g., FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb) is from a mammal (e.g., a primate (rhesus monkey, macaque, non-human primate, or human) and / or a rodent (e.g., rat and mouse). The isoform, fragment, variant, or homolog can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature isoform of an Fcγ receptor (e.g., FcγRI / FcγRIIa / FcγRIIb / FcγRIIc / FcγRIIIa / FcγRIIIb) of a given species (e.g., human).

[0794] The isoform, fragment, variant or homolog can optionally be a functional isoform, fragment, variant or homolog, e.g., having a functional property / activity of a reference Fcγ receptor as determined by an appropriate functional property / activity assay. For example, an isoform, fragment, variant or homolog of FcγRI can, e.g., be shown to be related to human IgG1 Fc.

[0795] In the present specification, the “FcRn receptor” may be derived from any species, and includes isoforms, fragments, variants (including mutants), or homologs derived from any species.

[0796] In some embodiments, the FcRn receptor is from a mammal, e.g., a primate (rhesus monkey, macaque, non-human primate, or human) and / or a rodent (e.g., rat or mouse). The isoform, fragment, variant, or homolog can optionally be characterized as having at least 70%, preferably one of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of an immature or mature isoform of the FcRn receptor from a given species (e.g., human).

[0797] The isoform, fragment, variant or homologue may optionally be a functional isoform, fragment, variant or homologue, e.g., having a functional property / activity of a reference FcRn receptor as determined by an appropriate functional property / activity assay. For example, an isoform, fragment, variant or homologue of FcRn may, for example, be shown to be related to human IgG1 Fc.

[0798] peptides

[0799] The present invention also provides polypeptide components of antigen binding molecules. The polypeptides can be provided in isolated or substantially purified form.

[0800] The antigen-binding molecule of the present invention may be or may comprise a complex of polypeptides.

[0801] In this specification, when a polypeptide comprises more than one domain or region, it should be understood that the multiple domains / regions are preferably present in the same polypeptide chain. That is, a polypeptide comprising more than one domain or region is a fusion polypeptide comprising these domains / regions.

[0802] In some embodiments, the polypeptide according to the present invention comprises or consists of a VH as described herein. In some embodiments, the polypeptide according to the present invention comprises or consists of a VL as described herein.

[0803] 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, and / or CH3 regions of an immunoglobulin (Ig).

[0804] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide comprises a CH1 region as described herein. In some embodiments, the polypeptide comprises a CH1-CH2 hinge region as described herein. In some embodiments, the polypeptide comprises a CH2 region as described herein. In some embodiments, the polypeptide comprises a CH3 region as described herein.

[0805] In some embodiments, the polypeptide comprises a CH2 and / or CH3 region comprising any of the following amino acid substitutions / combinations of amino acid substitutions: F243L / R292P / Y300L / V305I / P396L; S239D / I332E; S239D / I332E / A330L; S298A / E333A / K334A; L234Y / L235Q / G236W / S239M / H268D / D270E / S298A; D270E / K326D / A330M / K334E; G236A / S239D / I332E; K326W / E333S; S267E / H 268F / 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.

[0806] In some embodiments, the polypeptide comprises a CH3 region comprising any of the following amino acid substitutions / combinations of amino acid substitutions (e.g., as shown in Table 1 of Ha et al., Front. Immunol (2016) 7:394, incorporated herein by reference): T366W; T366S, L368A, and Y407V; T366W and S354C; T366S, L368A, Y407V, and Y349C; S364H and F405A; Y349T and T394F; T350V, L351Y, F405A, and Y407V; T350V, T366L, K392L and T394W; K360D, D399M and Y407A; E345R, Q347R, T366V and K409V; K409D and K392D; D399K and E356K; K360E and K409W; Q347R, D399V and F405T; K360E, K409W and Y349C; Q347R, D399V, F405T and S354C; K370E and K409W; and E357N, D399V and F405T.

[0807] In some embodiments, the CH2 and / or CH3 region of a polypeptide comprises one or more amino acid substitutions to promote association of the polypeptide with another polypeptide comprising a CH2 and / or CH3 region.

[0808] In some embodiments, the polypeptide comprises one or more regions of an immunoglobulin light chain constant sequence. In some embodiments, the polypeptide comprises a CL region as described herein.

[0809] In some embodiments, the polypeptide lacks one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the polypeptide lacks a CH2 region. In some embodiments, the polypeptide lacks a CH3 region. In some embodiments, the polypeptide lacks a CH2 region and also lacks a CH3 region.

[0810] In some embodiments, the polypeptide according to the present invention comprises, from N-terminus to C-terminus, a structure according to one of the following:

[0811] (i)VH

[0812] (ii) VL

[0813] (iii) VH-CH1

[0814] (iv)VL-CL

[0815] (v)VL-CH1

[0816] (vi) VH-CL

[0817] (vii) VH-CH1-CH2-CH3

[0818] (viii)VL-CL-CH2-CH3

[0819] (ix)VL-CH1-CH2-CH3

[0820] (x)VH-CL-CH2-CH3

[0821] The present invention also provides antigen-binding molecules composed of the polypeptides of the present invention. In some embodiments, the antigen-binding molecules of the present invention comprise a polypeptide combination of one of the following:

[0822] (A)VH+VL

[0823] (B)VH-CH1+VL-CL

[0824] (C)VL-CH1+VH-CL

[0825] (D)VH-CH1-CH2-CH3+VL-CL

[0826] (E)VH-CL-CH2-CH3+VL-CH1

[0827] (F)VL-CH1-CH2-CH3+VH-CL

[0828] (G)VL-CL-CH2-CH3+VH-CH1

[0829] (H)VH-CH1-CH2-CH3+VL-CL-CH2-CH3

[0830] (I)VH-CL-CH2-CH3+VL-CH1-CH2-CH3

[0831] In some embodiments, the antigen-binding molecule comprises a plurality of polypeptides in the combination shown in (A) to (I) above. For example, referring to (D) above, in some embodiments, the antigen-binding molecule comprises two polypeptides comprising a VH-CH1-CH2-CH3 structure, and two polypeptides comprising a VL-CL structure.

[0832] In some embodiments, the antigen binding molecules of the present invention comprise one of the following polypeptide combinations:

[0833] (J) VH (anti-VISTA) + VL (anti-VISTA)

[0834] (K)VH(anti-VISTA)-CH1+VL(anti-VISTA)-CL

[0835] (L)VL(anti-VISTA)-CH1+VH(anti-VISTA)-CL

[0836] (M) VH(anti-VISTA)-CH1-CH2-CH3+VL(anti-VISTA)-CL

[0837] (N)VH(anti-VISTA)-CL-CH2-CH3+VL(anti-VISTA)-CH1

[0838] (O)VL(anti-VISTA)-CH1-CH2-CH3+VH(anti-VISTA)-CL

[0839] (P)VL(anti-VISTA)-CL-CH2-CH3+VH(anti-VISTA)-CH1

[0840] (Q) VH (anti-VISTA)-CH1-CH2-CH3 + VL (anti-VISTA)-CL-CH2-CH3

[0841] (R)VH(anti-VISTA)-CL-CH2-CH3+VL(anti-VISTA)-CH1-CH2-CH3

[0842] Wherein: "VH(anti-VISTA)" refers to a VH of an antigen-binding molecule capable of binding to VISTA as described herein, for example, as defined in any one of (1) to (76); "VL(anti-VISTA)" refers to a VL of an antigen-binding molecule capable of binding to VISTA as described herein, for example, as defined in any one of (77) to (173).

[0843] In some embodiments, the polypeptide comprises or consists of an amino acid sequence having at least 70%, preferably 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 of one of SEQ ID NOs: 212 to 243, 248 to 250, 258, 266 or 311 to 321.

[0844] Adapters and other sequences

[0845] In some embodiments, the antigen-binding molecules and polypeptides of the present invention comprise a hinge region. In some embodiments, the hinge region is provided between the CH1 region and the CH2 region. In some embodiments, the hinge region is provided between the CL region and the CH2 region. In some embodiments, the hinge region comprises or consists of an amino acid sequence having at least 70%, preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 207.

[0846] In some embodiments, the antigen binding molecules and polypeptides of the present invention comprise one or more linker sequences between the amino acid sequences. A linker sequence can be provided at one or both ends of one or more of the VH, VL, CH1-CH2 hinge region, CH2 region, and CH3 region of the antigen binding molecules / polypeptide.

[0847] Linker sequences are known to those skilled in the art and are described, for example, in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the linker sequence can be a flexible linker sequence. Flexible linker sequences allow relative movement of the amino acid sequences connected 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 typically contain a high proportion of glycine and / or serine residues.

[0848] In some embodiments, the linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence is composed of glycine and serine residues. In some embodiments, the linker sequence has 1-2, 1-3, 1-4, 1-5 or 1-10 amino acid length.

[0849] The antigen binding molecules and polypeptide of the present invention can additionally include other amino acids or amino acid sequences.For example, the antigen binding molecules and polypeptide can include amino acid sequences to promote expression, folding, transport, processing, purification or detection of antigen binding molecules / polypeptides.For example, the antigen binding molecules / polypeptides can include sequences encoding His (such as 6XHis), Myc, GST, MBP, FLAG, HA, E or biotin tags optionally at the N- or C-terminal ends of antigen binding molecules / polypeptides.In some embodiments, the antigen binding molecules / polypeptides include detectable moieties, such as fluorescence, luminescence, immunoassay, radiation, chemistry, nucleic acid or enzyme labeling.

[0850] The antigen binding molecules and polypeptides of the present invention may further comprise a signal peptide (also referred to as a leader sequence or signal sequence). A signal peptide is typically composed of a sequence of 5-30 hydrophobic amino acids that form a single α helix. Secreted proteins and proteins expressed on the cell surface typically comprise a signal peptide.

[0851] The signal peptide may be present at the N-terminus of the antigen-binding molecule / polypeptide and may be present in newly synthesized antigen-binding molecules / polypeptides. The signal peptide provides for efficient transport and secretion of the antigen-binding molecule / polypeptide. The signal peptide is typically removed by cleavage and is therefore not included in the mature antigen-binding molecule / polypeptide secreted by the cell expressing the antigen-binding molecule / polypeptide.

[0852] Signal peptides are known for many proteins and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8:785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24:2172-2176).

[0853] Tags and conjugates

[0854] In some embodiments, the antigen binding molecules of the invention further comprise a detectable moiety.

[0855] In some embodiments, antigen binding molecules include detectable moieties, such as fluorescent markers, phosphorescent markers, luminescent markers, immune detectable markers (such as epitope markers), radioactive markers, chemicals, nucleic acids, or enzyme markers. Described antigen binding molecules can be covalently or non-covalently labeled with detectable moieties.

[0856] 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 radioisotopes such as iodine-123, iodine-125, iodine-126, iodine-131, iodine-133, bromine-77, technetium-99m, indium-111, indium-113m, gallium-67, 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, and antimony-211. Luminescent labels include radioluminescent, chemiluminescent (e.g., apidinium esters, luminol, isoluminol), and bioluminescent labels. Immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands, such as biotin, avidin, streptavidin, or digoxigenin. Nucleic acid labels include aptamers. Enzyme labels include, for example, peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, and luciferase.

[0857] In some embodiments, the antigen binding molecules of the present invention are conjugated to a chemical moiety. The chemical moiety can be a moiety for providing a therapeutic effect. Antibody-drug conjugates are reviewed in Parslow et al., Biomedicines. 2016 Sep; 4(3): 14. In some embodiments, the chemical moiety can be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety can be a chemotherapeutic agent. In some embodiments, the drug moiety is selected from calicheamicin, DM1, DM4, monomethyl autamicin E (MMAE), monomethyl autamicin F (MMAF), SN-38, doxorubicin, ducathiamycin, D6.5, and PBD.

[0858] Specific Exemplary Embodiments of Antigen-Binding Molecules

[0859] In some embodiments, the antigen binding molecule comprises or consists of:

[0860] (i) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 331; and

[0861] (ii) one or more (e.g., two) polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 317.

[0862] In some embodiments, the antigen binding molecule comprises or consists of:

[0863] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 212; and

[0864] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 213.

[0865] In some embodiments, the antigen binding molecule comprises or consists of:

[0866] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 214; and

[0867] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 215.

[0868] In some embodiments, the antigen binding molecule comprises or consists of:

[0869] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 216; and

[0870] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 217.

[0871] In some embodiments, the antigen binding molecule comprises or consists of:

[0872] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 218; and

[0873] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 219.

[0874] In some embodiments, the antigen binding molecule comprises or consists of:

[0875] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 220; and

[0876] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 221.

[0877] In some embodiments, the antigen binding molecule comprises or consists of:

[0878] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 222; and

[0879] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 223.

[0880] In some embodiments, the antigen binding molecule comprises or consists of:

[0881] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 224; and

[0882] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 225.

[0883] In some embodiments, the antigen binding molecule comprises or consists of:

[0884] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 226; and

[0885] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 227.

[0886] In some embodiments, the antigen binding molecule comprises or consists of:

[0887] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 228; and

[0888] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 229.

[0889] In some embodiments, the antigen binding molecule comprises or consists of:

[0890] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 230; and

[0891] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 231.

[0892] In some embodiments, the antigen binding molecule comprises or consists of:

[0893] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 232; and

[0894] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 233.

[0895] In some embodiments, the antigen binding molecule comprises or consists of:

[0896] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 234; and

[0897] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 235.

[0898] In some embodiments, the antigen binding molecule comprises or consists of:

[0899] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 236; and

[0900] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 237.

[0901] In some embodiments, the antigen binding molecule comprises or consists of:

[0902] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 238; and

[0903] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 239.

[0904] In some embodiments, the antigen binding molecule comprises or consists of:

[0905] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 240; and

[0906] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 241.

[0907] In some embodiments, the antigen binding molecule comprises or consists of:

[0908] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 242; and

[0909] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 243.

[0910] In some embodiments, the antigen binding molecule comprises or consists of:

[0911] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 248; and

[0912] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 250.

[0913] In some embodiments, the antigen binding molecule comprises or consists of:

[0914] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 249; and

[0915] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 250. In some embodiments, the antigen binding molecule comprises or consists of:

[0916] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 258; and

[0917] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 250.

[0918] In some embodiments, the antigen binding molecule comprises or consists of:

[0919] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 266; and

[0920] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 250. In some embodiments, the antigen binding molecule comprises or consists of:

[0921] (i) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 330; and

[0922] (ii) two polypeptides comprising or consisting of an amino acid sequence having at least 70%, preferably 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 of SEQ ID NO: 213.

[0923] In some embodiments, the antigen binding molecule is produced by cells of the cell line deposited on May 7, 2021, with ATCC patent deposit number PTA-127063, which is described in, for example, GB 2108446.2, which is incorporated herein by reference in its entirety.

[0924] Functional properties of antigen-binding molecules

[0925] The antigen binding molecules described herein can be characterized by reference to certain functional properties. In some embodiments, the antigen binding molecules described herein can have one or more of the following properties:

[0926] In combination with VISTA (e.g., human, murine and / or cynomolgus monkey VISTA);

[0927] Does not bind to PD-L1 and / or HER3;

[0928] Does not bind to Fcγ receptors;

[0929] does not bind C1q;

[0930] Does not induce ADCC;

[0931] Does not induce ADCP;

[0932] Does not induce CDC;

[0933] Binds to FcRn receptor;

[0934] Binds to cells expressing VISTA;

[0935] Inhibiting the interaction between VISTA and VISTA's binding partners (e.g., PSGL-1, VSIG-3, VSIG-8, or LRIG1);

[0936] Inhibit VISTA-mediated signaling;

[0937] Inhibits VISTA-mediated signaling independent of Fc-mediated function;

[0938] Increased killing of VISTA-expressing cells;

[0939] does not induce / increase killing of VISTA-expressing cells;

[0940] Reducing the number / proportion of cells expressing VISTA;

[0941] Do not reduce the number / proportion of cells expressing VISTA;

[0942] Increase the number / activity of effector immune cells;

[0943] reducing the number / activity of suppressive immune cells;

[0944] Reduce the proliferation of suppressive immune cells;

[0945] Reduce immune suppression mediated by VISTA-expressing cells;

[0946] Increase antigen presentation by antigen-presenting cells;

[0947] Increase the ability of immune cells to produce IL-6;

[0948] Increased production of IFN-γ, IL-2, and / or IL-17 in mixed lymphocyte reaction (MLR) assays;

[0949] Increase T cell proliferation, IFN-γ production and / or TNFa production; and

[0950] Inhibiting the development and / or progression of cancer in the body; and

[0951] Does not induce cytokine release syndrome in vivo.

[0952] It will be appreciated that a given antigen binding molecule may exhibit more than one of the properties described in the preceding paragraph. A given antigen binding molecule may be evaluated using a suitable test to assess the properties described in the preceding paragraph. The test may be an in vitro test, which may be a cell-free test or a cell-based test. Alternatively, the test may be, for example, an in vivo test, i.e., performed in a non-human animal.

[0953] When the test is based on the test of cell, they can include contacting cells with given antigen binding molecules to determine whether antigen binding molecules show one or more described characteristics. The test can be performed using species labeled with detectable entities for ease of detection. The test can include assessing the characteristic after treating cells with a certain amount / concentration of antigen binding molecules (such as dilution series). It should be understood that the cell is preferably a cell expressing VISTA, such as MDSCs.

[0954] Analysis of the results of such assays may include determining the concentration at which 50% of the maximal level of the relevant activity is achieved. The concentration of the antigen-binding molecule that achieves 50% of the maximal level of the relevant activity may be referred to as the "half-maximal effective concentration" of the antigen-binding molecule for the relevant activity, also referred to as the "EC 50 For example, the EC of a given antigen-binding molecule binding to VISTA 50 This may be the concentration at which 50% of the maximum binding level to the species of interest is achieved.

[0955] According to the different nature, EC 50 It may also be called the "half-maximal inhibitory concentration" or "IC 50This is the concentration of the antigen-binding molecule at which 50% of the maximal level of inhibition of a given property is observed. For example, the IC of a given antigen-binding molecule that inhibits the interaction between VISTA and an interacting partner of VISTA, such as LRIG1, PSGL-1, VSIG3, or VSIG8, is 50 This may be the concentration that achieves 50% of the maximum inhibition level.

[0956] The antigen binding molecules described herein preferably show specific binding to VISTA. As used herein, "specific binding" refers to selective binding to an antigen and can be distinguished from non-specific binding to a non-target antigen. Compared with binding to other, non-target molecules, antigen binding molecules that specifically bind to a target molecule preferably have a greater affinity for the target molecule and / or a longer duration.

[0957] The ability of a given polypeptide to specifically bind to a given molecule can be determined by analysis according to methods known in the art, such as by ELISA, surface plasmon resonance (SPR; see, for example, Hearty et al., Methods Mol Biol (2012) 907: 411-442), biolayer interferometry (see, for example, Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by radiolabeled antigen binding assay (RIA) enzyme-linked immunosorbent assay. By such analysis, binding to a given molecule can be measured and quantified. In some embodiments, the binding can be a response detected in a given assay.

[0958] In some embodiments, the extent of binding of the antigen binding molecule to the non-target molecule is less than about 10% of the extent of binding of the antibody to the target molecule as measured, for example, by ELISA, SPR, biolayer interferometry, or RIA. Alternatively, binding specificity can be reflected as binding affinity, where the dissociation constant (K) for binding of the antigen binding molecule is D ) is greater than the K of the antigen-binding molecule to the non-target molecule D At least 0.1 orders of magnitude (i.e. 0.1×10 n , where n is an integer representing an order of magnitude). This may optionally be at least one of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 or 2.0.

[0959] In some embodiments, antigen binding molecules show a combination with people VISTA, murine (such as mouse) VISTA and / or cynomolgus macaque (cynomolgus macaque) VISTA. That is, in some embodiments, antigen binding molecules have cross reactivity to people VISTA and murine VISTA and / or cynomolgus macaque VISTA. In some embodiments, the antigen binding molecules of the present invention show cross reactivity with the VISTA of non-human primates. In model species, the cross reactivity with VISTA can be explored in vivo in a homologous model for efficacy, without relying on alternative molecules.

[0960] In some embodiments, the antigen binding molecules do not show specific binding to PD-L1 (e.g., human PD-L1). In some embodiments, the antigen binding molecules do not show specific binding to HER3 (e.g., human HER3). In some embodiments, the antigen binding molecules do not show specific binding to another member of the B7 protein family (i.e., do not cross-react with it). In some embodiments, the antigen binding molecules do not show specific binding to PD-L1, PD-L2, CD80, CD86, ICOLSG, CD276, VTCN1, NCR3LG1, HHLA2, and / or CTLA4.

[0961] In certain 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.

[0962] In some embodiments, the antigen binding molecule is unable to induce one or more Fc-mediated functions (ie, lacks the ability to elicit the relevant Fc-mediated function). Such antigen binding molecules can be described as lacking the relevant function.

[0963] As explained above, an Fc region / antigen binding molecule that does not induce (i.e., is unable to induce) ADCC / ADCP / CDC does not substantially elicit ADCC / ADCP / CDC activity, such as the relevant activity determined by an assay in an appropriate assay. Similarly, an antigen binding molecule that "does not bind" a reference protein (e.g., a given Fc receptor or complement protein) may not substantially bind to the reference protein in an appropriate assay.

[0964] In some embodiments, the antigen binding molecules are unable to induce ADCC. In some embodiments, the antigen binding molecules are unable to induce ADCP. In some embodiments, the antigen binding molecules are unable to induce CDC. In some embodiments, the antigen binding molecules are unable to induce ADCC and / or unable to induce ADCP and / or unable to induce CDC.

[0965] In some embodiments, the antigen binding molecule does not bind to an Fc receptor. In some embodiments, the antigen binding molecule does not bind to an Fcγ receptor. 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 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 antigen binding molecule is not glycosylated at the amino acid residue corresponding to N297.

[0966] In some embodiments, the antigen binding molecule binds to human VISTA, murine VISTA and / or cynomolgus macaque VISTA; and does not bind to PD-L1, PD-1, B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7) and / or CTLA4 (e.g., human PD-L1 / PD-1 / B7H3 / VTCN1 / NCR3LG1 / HHLA2 / CTLA4).

[0967] In some embodiments, the antigen binding molecules do not show specific binding to PD-L1 (e.g., human PD-L1). In some embodiments, the antigen binding molecules do not show specific binding to HER3 (e.g., human HER3). In some embodiments, the antigen binding molecules do not show specific binding to another member of the B7 protein family (i.e., do not cross-react with it). In some embodiments, the antigen binding molecules do not show specific binding to PD-L1, PD-L2 CD80, CD86, ICOSLG, CD276, VTCN1, NCR3LG1, HHLA2, and / or CTLA4.

[0968] In some embodiments, the antigen binding molecule does not show specific binding to PD-1, PD-L1, B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7) and / or CTLA4.

[0969] In some embodiments, the antigen binding molecules according to the present invention bind to VISTA with an affinity in the micromolar range, e.g., K D =9.9×10 -4 -1×10 -6In some embodiments, the antigen binding molecule binds to VISTA with submicromolar affinity, e.g., K D <1×10 -6 In some embodiments, the antigen binding molecules bind to VISTA with an affinity in the nanomolar range, e.g., K D =9.9×10 -7 -1×10 -9 In some embodiments, the antigen binding molecule binds to VISTA with a subnanomolar affinity, e.g., K D <1×10 -9 In some embodiments, the antigen binding molecule binds to VISTA with an affinity in the picomolar range, e.g., K D =9.9×10 -10 -1×10 -12 In some embodiments, the antigen binding molecule binds to VISTA with a sub-picomolar affinity, e.g., K D <1×10 -12 M.

[0970] In some embodiments, the antigen binding molecules described herein bind to the K of VISTA (e.g., human VISTA, mouse VISTA). D 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 affinity K of the antigen binding molecule for binding to VISTA (e.g., human VISTA, mouse VISTA) is D =≤10nM, ≤9nM, ≤8nM, ≤7nM or ≤6nM, ≤5nM, ≤4nM, ≤3nM, ≤2nM or ≤1nM. In some embodiments, the affinity K of the antigen binding molecule binding to VISTA (e.g., human VISTA, mouse VISTA) is D =≤500pM, ≤100pM, ≤90pM, ≤80pM, ≤70pM or ≤60pM, ≤50pM, ≤40pM, ≤30pM, ≤20pM, ≤10pM, ≤9pM, ≤8pM, ≤7pM or ≤6pM, ≤5pM, ≤4pM, ≤3pM, ≤2pM or ≤1pM. In some embodiments, the antigen binding molecules according to the present disclosure have a K of ≤1 nM (e.g., one of ≤900pM, ≤800pM, ≤700pM, ≤600pM, ≤500pM, ≤400pM, ≤300pM). DBinding to VISTA (e.g., determined by SPR (Biocore) analysis, e.g., the SPR analysis described in the Examples of the present disclosure).

[0971] In some embodiments, antigen binding molecules according to the present disclosure have a K of ≤1 nM (e.g., one of ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM). D Binding to human VISTA (e.g., determined by SPR (Biocore) analysis, e.g., the SPR analysis described in the Examples of the present disclosure). In some embodiments, the antigen binding molecules according to the present disclosure have a K of ≤1 nM (e.g., ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM). D Binding to cynomolgus monkey VISTA (e.g., determined by SPR (Biocore) analysis, e.g., the SPR analysis described in the Examples of the present disclosure). In some embodiments, the antigen binding molecules according to the present disclosure have a K of ≤1 nM (e.g., ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM, ≤500 pM, ≤400 pM). D Binding to rat VISTA (e.g., determined by SPR (Biocore) analysis, e.g., the SPR analysis described in the Examples of the present disclosure). In some embodiments, the antigen binding molecules according to the present disclosure have a K of ≤1 nM (e.g., ≤900 pM, ≤800 pM, ≤700 pM, ≤600 pM). D Binding to mouse VISTA (e.g., determined by SPR (Biocore) analysis, e.g., the SPR analysis described in the Examples of the present disclosure).

[0972] In some embodiments, the antigen binding molecules according to the present disclosure have an EC of 1 μM or less, for example, 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. 50 Binding to VISTA (e.g., determined by ELISA, e.g., the ELISA described in the Examples of the present disclosure).

[0973] In certain embodiments, antigen binding molecules are shown to be combined with human VISTA, murine (e.g., mouse) VISTA, rat VISTA, and / or cynomolgus monkey (long-tailed macaque) VISTA. In certain embodiments, antigen binding molecules are combined with human VISTA and mouse VISTA and rat VISTA and cynomolgus monkey VISTA. In certain embodiments, antigen binding molecules have cross-reactivity to human VISTA and mouse VISTA and rat VISTA and cynomolgus monkey VISTA. In certain embodiments, antigen binding molecules of the present disclosure show cross-reactivity with the VISTA of non-human primates. In model species, the cross-reactivity with VISTA can be explored in vivo in a homologous model for efficacy, without relying on alternative molecules.

[0974] In some embodiments, antigen binding molecules according to the present disclosure have an EC of ≤ 20 pM (e.g., one of ≤ 15 pM, ≤ 12.5 pM, ≤ 10 pM, ≤ 7.5 pM). 50 Binds to human VISTA (e.g., as determined by ELISA, e.g., the ELISA described in the Examples of the present disclosure). In some embodiments, the antigen binding molecules according to the present disclosure bind to human VISTA with an EC of ≤50 pM (e.g., one of ≤25 pM, ≤20 pM, ≤15 pM). 50 Binding to cynomolgus monkey VISTA (e.g., determined by ELISA, e.g., the ELISA described in the Examples of the present disclosure). In some embodiments, antigen binding molecules according to the present disclosure bind to cynomolgus monkey VISTA with an EC of ≤ 20 pM (e.g., ≤ 15 pM, ≤ 12.5 pM, ≤ 10 pM, ≤ 7.5 pM). 50 Binding to rat VISTA (e.g., determined by ELISA, e.g., the ELISA described in the Examples of the present disclosure). In some embodiments, antigen binding molecules according to the present disclosure bind to rat VISTA with an EC of ≤20 pM (e.g., ≤15 pM, ≤12.5 pM, ≤10 pM, ≤7.5 pM, ≤5 pM). 50 Binding to mouse VISTA (e.g., determined by ELISA, e.g., the ELISA described in the Examples of the present disclosure).

[0975] In some embodiments, the antigen binding molecules according to the present disclosure bind to VISTA (e.g., human VISTA) with similar affinity at pH from 5.5 to pH 7.5. For example, in some embodiments, the affinity of the antigen binding molecules for VISTA at pH 5.5 is similar to the affinity for VISTA at pH 7.5.

[0976] As used herein, a binding affinity that is "similar" to a reference binding affinity refers to a binding affinity that is within 50%, for example, 40%, 45%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the reference binding affinity determined under comparable conditions.

[0977] K binding to VISTA (such as human VISTA) D The EC binding to VISTA (e.g. human VISTA) is likely to be similar between pH 5.5 and 7.5. 50 It may be similar between pH 5.5 and 7.5.

[0978] In this article, reference values ​​of "similar" K D or EC 50 The value may be ≥0.5 times and ≤2 times, such as between ≥0.7 times and ≤1.5 times, between ≥0.75 times and ≤1.25 times, between ≥0.8 times and ≤1.2 times, between ≥0.85 times and ≤1.15 times, between ≥0.9 times and ≤1.1 times, between ≥0.91 times and ≤1.09 times, between ≥0.92 times and ≤1.08 times, between ≥0.93 times and ≤1.07 times, between ≥0.94 times and ≤1.06 times, between ≥0.95 times and ≤1.05 times, between ≥0.96 times and ≤1.04 times, between ≥0.97 times and ≤1.03 times, between ≥0.98 times and ≤1.02 times, or between ≥0.99 times and ≤1.01 times the reference value.

[0979] The antigen binding molecules of the present invention can be combined with the specific region of interest of VISTA. According to the antigen binding region of the antigen binding molecules of this domain, the linear epitope of VISTA consisting of a continuous sequence of amino acids (i.e., amino acid primary sequence) can be combined. In some embodiments, the antigen binding region molecule can be combined with the conformational epitope of VISTA, which is composed of a discontinuous sequence of discontinuous amino acids in the amino acid sequence. In some embodiments, the antigen binding molecules of the present invention can be combined with VISTA. In some embodiments, the antigen binding molecules can be combined with VISTA in the extracellular domain of VISTA. In some embodiments, the antigen binding molecules can be combined with VISTA in the Ig-like V-type domain (e.g., the region shown in SEQ ID NO: 6). In some embodiments, the antigen binding molecules can be combined with VISTA in the region shown in SEQ ID NO: 31.

[0980] In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of 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 of 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 of 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 of 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 of 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 of 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 of SEQ ID NO: 30.

[0981] In some embodiments, the antigen binding molecule does not bind to the VISTA region bound by IGN175A (e.g., as described in WO 2014 / 197849 A2). In some embodiments, the antigen binding molecule does not bind to the VISTA region bound by the following antigen binding molecules, the antigen binding molecule comprising a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268.

[0982] In some embodiments, the antigen binding molecule does not compete with IGN175A (e.g., as described in WO 2014 / 197849A2) for binding to VISTA. In some embodiments, the antigen binding molecule does not compete with the following antigen binding molecules for binding to VISTA: the antigen binding molecule comprising a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268.

[0983] The ability of a given antigen binding molecule to compete with IGN175A or the following antigen binding molecules for binding to VISTA can be analyzed by competition ELISA or by epitope binding, such as Abdiche et al., J Immunol Methods (2012) 382 (-2): 101-116 (incorporated herein by reference in its entirety), wherein the antigen binding molecule includes a polypeptide consisting of SEQ ID NO: 267 sequence and a polypeptide consisting of SEQ ID NO: 268 sequence. Antigenic determinant merging can be performed, for example, by BLI analysis, for example, as described in Example 8 of the present application.

[0984] In some embodiments, the antigen binding molecule cannot bind to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 275.

[0985] As used herein, "peptide" refers to a chain of two or more amino acid monomers connected by peptide bonds. Peptides generally range in length from about 2 to 50 amino acids. A "polypeptide" is a polymer chain of two or more peptides. Polypeptides generally have a length greater than about 50 amino acids.

[0986] The ability of an antigen binding molecule to bind a given peptide / polypeptide can be assayed by methods well known to those skilled in the art, including ELISA, immunoblotting (e.g., Western blot), immunoprecipitation, surface plasmon resonance, and bio-layer interferometry.

[0987] In some embodiments, the antigen binding molecules are capable of binding to the same or overlapping VISTA regions as those bound by the following antibodies comprising the VH and VL sequences of one of the following clones: 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1, V4H2, V4-C1, V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31, 2M1-B12, 2M1-D2, 1M2-D2, 13D5p, 13D5-1, 13D5-13, 5M1-A11, or 9M2-C12.

[0988] In some embodiments, the antigen binding molecule is capable of binding to a VISTA region different from the VISTA region bound by IGN175A (e.g., as described in WO 2014 / 197849 A2). In some embodiments, the antigen binding molecule is capable of binding to a VISTA region different from the VISTA region bound by the following antigen binding molecule, the antigen binding molecule comprising a polypeptide consisting of the sequence of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268.

[0989] In some embodiments, the antigen binding molecule is capable of binding to a VISTA region that does not overlap with the VISTA region bound by IGN175A (e.g., as described in WO 2014 / 197849 A2). In some embodiments, the antigen binding molecule is capable of binding to a VISTA region that does not overlap with the VISTA region bound by the following antigen binding molecule, the antigen binding molecule comprising a polypeptide consisting of SEQ ID NO: 267 and a polypeptide consisting of the sequence of SEQ ID NO: 268.

[0990] In some embodiments, the antigen binding molecule binds to VISTA by contacting a residue of VISTA that is different from the residue of VISTA contacted by VSTB112 (e.g., 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 residue of VISTA contacted by an antigen binding molecule comprising a polypeptide consisting of the sequence of SEQ ID NO: 269 and a polypeptide consisting of the sequence of SEQ ID NO: 270.

[0991] 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 of SEQ ID NO: 269 and a polypeptide consisting of the sequence of SEQ ID NO: 270.

[0992] The region of the peptide / polypeptide to which the antibody binds can be determined by a skilled artisan using various methods well known in the art, including X-ray co-crystal analysis of the antibody-antigen complex, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange mass spectrometry, phage display, competitive ELISA, and proteolytic "protection" methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21(3): 145-156, which is incorporated herein by reference in its entirety.

[0993] In some embodiments, when VISTA is expressed on the cell surface (i.e., in a cell membrane or on a cell membrane), the antigen binding molecules of the present invention bind to VISTA in an accessible region of an antigen binding molecule (i.e., an extracellular antigen binding molecule). In some embodiments, the antigen binding molecules can be combined with the cell surface of cells expressing VISTA. In some embodiments, the antigen binding molecules can be combined with cells expressing VISTA (e.g., CD14+ monocytes (e.g., monocyte-derived suppressor cells (MDSC)) and / or CD33+ myeloid cells, tumor-associated macrophages (TAMs) and neutrophils).

[0994] The ability of antigen binding molecules to bind to a given cell type can be analyzed by contacting the cells with antigen binding molecules and detecting the antigen binding molecules bound to the cells (e.g., after a washing step, removing unbound antigen binding molecules). The ability of antigen binding molecules to bind to cells expressing immune cell surface molecules and / or cells expressing cancer cell antigens can be analyzed by methods such as flow cytometry and immunofluorescence microscopy.

[0995] The antigen binding molecules of the present invention can be antagonists of VISTA. In some embodiments, the antigen binding molecules can inhibit the function or process (such as interaction, signal transduction or other activity) mediated by VISTA and / or VISTA's binding partner (such as PSGL-1, VSIG-3, VSIG-8, LRIG1). Here, "inhibition" refers to a reduction, decrease or relief relative to a control condition.

[0996] Antigen binding molecules that inhibit a given interaction / activity / process may be referred to as inhibitors or antagonists of the interaction / activity / process, and may be said to "block" or "neutralize" the interaction / activity / process.

[0997] The antigen binding molecules described herein in conjunction with VISTA can suppress the function / process of VISTA mediation by mechanisms such as ADCC, ADCP and CDC that do not require Fc-mediated functions. That is, the VISTA-binding antigen binding molecules described herein can suppress the immunosuppressive activity of cells expressing VISTA without causing ADCC, ADCP and / or CDC.

[0998] 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.

[0999] In some embodiments, the antigen binding molecules of the present invention can suppress the interaction between VISTA and the binding partner of VISTA (such as PSGL-1, VSIG-3, VSIG-8, LRIG1). In some embodiments, the antigen binding molecules of the present invention can suppress the interaction between VISTA and PSGL-1. In some embodiments, the antigen binding molecules of the present invention can suppress the interaction between VISTA and VSIG-3. The antigen binding molecules of the present invention can suppress the interaction between VISTA and LRIG1.

[1000] The ability of an antigen binding molecule to inhibit the interaction between two factors can be determined, for example, by analyzing the interaction in the presence or absence of one or both interaction partners after incubation with the antibody / fragment. Assays for determining whether a given antigen binding molecule is able to inhibit the interaction between two interaction partners include competitive ELISA assays and SPR analysis.

[1001] By observing the interaction level compared with the absence of antigen binding molecules (or the presence of appropriate control antigen binding molecules), the reduction / reduction of the interaction level in the presence of antigen binding molecules or between the interaction partners after incubation of one or two interaction partners and antigen binding molecules, it is possible to identify the antigen binding molecules (such as between the binding partners of VISTA and VISTA) that can suppress a given interaction. Suitable analysis can be carried out in vitro, for example, using a recombinant interaction partner or using cells expressing an interaction partner. The cells expressing the interaction partner may be endogenous or may be expressed by introducing a nucleic acid of a cell. For the purpose of this assay, one or two interaction partners and / or antigen binding molecules can be marked or used in combination with a detectable entity to detect and / or measure the level of the interaction.

[1002] 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 this interaction. For example, the downstream functional consequences of the interaction between VISTA and a binding partner of VISTA may include VISTA-mediated signal transduction. For example, the ability of an antigen binding molecule to inhibit the interaction between VISTA and a binding partner of VISTA can be determined by analyzing the production of IL-2, IFN-γ, and / or IL-17 in an MLR assay.

[1003] In some embodiments, the antigen binding molecules of the present invention are capable of inhibiting the interaction between VISTA and a binding partner of VISTA (e.g., PSGL-1, VSIG-3, VSIG-8, LRIG1) to less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, 3-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, compared to the level of interaction between VISTA and a binding partner in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1004] In certain embodiments, antigen binding molecules suppress the signal transduction mediated by VISTA.In certain embodiments, the signal transduction mediated by VISTA can be the signal transduction mediated by the polypeptide complex comprising VISTA.In certain embodiments, the signal transduction mediated by VISTA can be the signal transduction mediated by the polypeptide complex comprising VISTA and an interaction partner (such as LRIG1, VSIG3, PSGL-1, VSIG8) of VISTA. The signal transduction mediated by VISTA can be analyzed using, for example, the MLR assay method described in the experimental examples herein for the determination of effector immune cell number / activity. The inhibition of the signal transduction mediated by VISTA can be identified by detecting the increase in the number and / or activity of effector immune cells, for example, by determining the increase in the production of IL-2, IFN-γ and / or IL-17.

[1005] The ability of an antigen binding molecule to inhibit the interaction between VISTA and an interacting partner of VISTA can be determined, for example, by analyzing the interaction in the case of or after incubation of one or both interacting partners with the antigen binding molecule. Tests for determining whether a given antigen binding molecule is capable of inhibiting the interaction between VISTA and a VISTA interacting partner include competitive ELISA assays and SPR analysis.

[1006] The ability of the antigen-binding molecule to inhibit the interaction between VISTA and a mutual partner of VISTA, as compared to the level of interaction in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule known not to inhibit such interaction), can be determined by observing a decrease / reduction in the level of interaction between the interacting partners in the presence of the antigen-binding molecule or after incubation of one or both of the interacting partners with the antigen-binding molecule.

[1007] Suitable assays can be performed in vitro, for example, using recombinant interaction partners or using cells expressing the interaction partner. The cells expressing the interaction partner may be endogenous or may be expressed by introducing nucleic acid into the cells. For the purposes of these assays, one or both of the interaction partner and / or antigen-binding molecule may be labeled or used with a detectable entity to detect and / or measure the level of interaction.

[1008] In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require or involve Fc-mediated function. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction independently of Fc-mediated function. That is, in some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction in a non-Fc region-dependent manner.

[1009] The ability of an antigen binding molecule to inhibit VISTA-mediated signaling by a mechanism that does not require / involve Fc-mediated function can be assessed, for example, by analyzing the ability of an antigen binding molecule provided in the absence of a functional Fc region to inhibit VISTA-mediated signaling. For example, the effect on VISTA-mediated signaling can be studied using an antigen binding molecule comprising a "silent" Fc region (e.g., comprising a LALA PG substitution) or using an antigen binding molecule provided in the absence of an Fc region (e.g., scFv, Fab, etc.).

[1010] In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not involve ADCC. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not involve ADCP. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not involve CDC.

[1011] In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require the antigen binding molecules to bind to Fc receptors. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require the antigen binding molecules to bind to Fcγ receptors. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require the antigen binding molecules to bind to one or more of FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require binding to FcγRIIIa. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require binding to FcγRIIa. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require binding to FcγRIIb. In some embodiments, the antigen binding molecules can inhibit VISTA-mediated signal transduction by a mechanism that does not require binding to complement proteins. In some embodiments, the antigen binding molecules are capable of inhibiting VISTA-mediated signaling by a mechanism that does not require binding to C1q. In some embodiments, the antigen binding molecules are capable of inhibiting VISTA-mediated signaling by a mechanism that does not require glycosylation of N297.

[1012] In some embodiments, the antigen binding molecules of the present invention can increase the killing of cells expressing VISTA.The killing of cells expressing VISTA can be increased by the effector function of antigen binding molecules.In the embodiment in which antigen binding molecules include Fc district, the antigen binding molecules can increase the killing of cells expressing VISTA by one or more of complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP).

[1013] Can be by observing the presence of antigen binding molecules or after the cells expressing VISTA are hatched with antigen binding molecules, compared with the cell killing level detected in the absence of antigen binding molecules (or in the presence of appropriate control antigen binding molecules) in appropriate assays, the cell killing level of expressing VISTA increases, to identify the antigen binding molecules that can increase the cell killing of expressing VISTA. The determination of CDC, ADCC and ADCP is well known to technicians. The killing level of the cells expressing VISTA can also be determined by measuring the number / ratio of the cells expressing VISTA that survive and / or do not survive after being exposed to different treatment conditions.

[1014] In some embodiments, the antigen binding molecules of the invention are capable of increasing the killing of VISTA-expressing cells (e.g., VISTA-expressing MDSCs) by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the level of killing observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1015] In some embodiments, the antigen binding molecules of the present invention are capable of reducing the number of VISTA-expressing cells (e.g., VISTA-expressing MDSCs) by at least 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, compared to a comparable assay of the number of VISTA-expressing cells (e.g., VISTA-expressing MDSCs, TAMs, neutrophils) performed after incubation in the absence of the antigen binding molecule (or after incubation in the presence of an appropriate control antigen binding molecule).

[1016] In some embodiments, the antigen binding molecules are non-consumable antigen binding molecules. That is, in some embodiments, the antigen binding molecules do not cause substantial consumption of cells expressing VISTA. In some embodiments, the antigen binding molecules do not induce / increase ADCC, ADCP and / or CDC for cells expressing VISTA.

[1017] In some embodiments, the antigen binding molecules of the present invention do not induce / increase killing of cells expressing VISTA, for example, in embodiments in which the antigen binding molecules lack an Fc region, or in embodiments in which the antigen binding molecules comprise an Fc region that cannot induce Fc-mediated antibody effector functions. In some embodiments, the antigen binding molecules of the present invention do not reduce the number / ratio of cells expressing VISTA.

[1018] In some embodiments, the antigen binding molecules of the present invention (i) inhibit VISTA-mediated signal transduction, and (ii) do not induce / increase the killing of cells expressing VISTA. In some embodiments, the antigen binding molecules of the present invention (i) inhibit VISTA-mediated signal transduction, and (ii) do not reduce the number / ratio of cells expressing VISTA.

[1019] This may be particularly advantageous because VISTA is expressed by cells that are not depleted. For example, VISTA is expressed at low levels by immune cells (such as certain types of T cells and dendritic cells) that are not desired to be killed or have their number / ratio reduced.

[1020] In some embodiments, the antigen binding molecules of the present invention can increase the quantity and / or activity of effector immune cells relative to negative control conditions (such as performing appropriate analysis in vitro or in vivo).As an explanation, the antigen binding molecules of the present invention may be able to release effector immune cells from the effector immune cell proliferation and function suppression mediated by MDSC.In some embodiments, effector immune cells can be such as CD8+T cells, CD8+ cytotoxic T lymphocytes (CD8+CTL), CD4+T cells, CD4+T helper cells, NK cells, the cells producing IFN γ, memory T cells, central memory T cells, T cells or CD45RO+T cells exposed through antigen.

[1021] Cell numbers and proportions can be determined, for example, by flow cytometric analysis using antibodies to detect cell types. 3 In vitro assays for H-thymidine incorporation or analysis of cell division by CFSE dilution assays, as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, incorporated herein by reference in its entirety. Effector immune cell activity can be analyzed by measuring the correlation of such activity. In some embodiments, the activity of effector immune cells can be determined, for example, by analyzing the production of IL-2, IFN-γ, and / or IL-17.

[1022] In some embodiments, the antigen binding molecules of the invention are capable of increasing the number of effector immune cell types by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the number observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule). In some embodiments, the antigen binding molecules of the invention are capable of increasing the level of a factor associated with effector immune cell activity by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, relative to the level observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1023] In some embodiments, the antigen binding molecules of the present invention can reduce the level of immunosuppression mediated by cells expressing VISTA. The change in immunosuppression level can be determined using a method for measuring the expression of arginase 1 of cells expressing VISTA and / or the generation of reactive oxygen species (ROS), such as Ochoa et al., Ann Surg 2001 March; 233 (3): 393-399 and Dikalov and Harrison, Antioxidant Redox Signaling, January 10, 2014; 20 (2): described in 372–382.

[1024] In some embodiments, the antigen binding molecules of the present invention can increase the antigen presentation of antigen presenting cells, such as determined by a suitable assay for antigen presentation. In some embodiments, the antigen binding molecules of the present invention can increase the phagocytosis of phagocytes (such as neutrophils, monocytes, macrophages, mast cells and / or dendritic cells), such as determined by a suitable assay for phagocytosis levels.

[1025] In some embodiments, the antigen binding molecules of the present disclosure are capable of increasing the number and / or activity of antigen presenting cells (e.g., CD11b+MHCII+ cells) relative to negative control conditions, e.g., in an appropriate in vitro assay, or in vivo (e.g., in a tumor).

[1026] In some embodiments, the antigen binding molecules of the present invention can increase the number and / or activity of macrophages (e.g., CD11b+F4 / 80+ cells) relative to negative control conditions, for example, in an appropriate in vitro assay, or in vivo (e.g., in a tumor). In some embodiments, the antigen binding molecules of the present invention can increase the number and / or activity of dendritic cells (e.g., CD11c+ cells) relative to negative control conditions, for example, in an appropriate in vitro assay, or in vivo (e.g., in a tumor).

[1027] In some embodiments, the antigen binding molecules of the present disclosure are capable of increasing the number of cell types described in the preceding paragraph by more than 1-fold, for example, ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, relative to the number observed in the absence of the antigen binding molecules (or in the presence of an appropriate control antigen binding molecule). In some embodiments, the antigen binding molecules of the present disclosure are capable of increasing the activity-related levels of the cell types described in the preceding paragraph to more than 1-fold the level in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule), for example: ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold or ≥10-fold.

[1028] In some embodiments, the antigen binding molecules of the present invention can increase the ability of immune cells to produce IL-6. 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 can increase the IL-6 production of immune cells (for example, using LPS) after stimulation. The ability of antigen binding molecules to increase immune cell production of IL-6 can be analyzed in in vitro assays (as described in Example 10 herein). The method can include stimulating monocytes (for example, THP1 cells) with LPS, and incubating the stimulated cells with antigen binding molecules.

[1029] In some embodiments, the antigen binding molecules of the invention are capable of increasing IL-6 production by immune cells (e.g., LPS-stimulated THP1 cells) by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, relative to the level observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1030] In some embodiments, the antigen binding molecules of the present invention can increase the number and / or activity of Th1 / Th17 cells. In some embodiments, the antigen binding molecules can upregulate Th1 / Th17 reactions. In some embodiments, the antigen binding molecules tend to Th1 / Th17 reactions rather than Th2 reactions. In some embodiments, the antigen binding molecules of the present invention can increase T cell proliferation, IL-2 production, IFN-γ production, TNF-α production and / or IL-17A production in a mixed lymphocyte reaction (MLR) assay. The MLR assay can be performed as described in Bromelow et al., J. Immunol Methods, January 1, 2001; 247 (1-2): 1-8 (incorporated herein by reference in its entirety), or as described in the experimental examples herein. The production of IL-2, IFNγ and / or IL-17 can be analyzed by, for example, antibody-based methods well known to technicians, such as Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or by a reporter-based method.

[1031] In some embodiments, the antigen binding molecules of the present invention are capable of increasing T cell (e.g., Th1 / Th17 cell) proliferation, IL-2 production, IFN-γ production, TNF-α production, and / or IL-17A production in a mixed lymphocyte reaction (MLR) assay. The MLR assay can be performed as described in Bromelow et al., J. Immunol Methods, 2001 Jan 1; 247(1-2): 1-8 (incorporated herein by reference in its entirety), or as described in the experimental examples herein. The production of IL-2, IFNγ, and / or IL-17 can be analyzed by, for example, antibody-based methods well known to those skilled in the art, such as Western blotting, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or by reporter-based methods.

[1032] In some embodiments, the antigen binding molecules of the invention are capable of increasing T cell proliferation, IL-2 production, IFN-γ production and / or IL-17 production in an MLR assay by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, relative to the level observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1033] In some embodiments, the antigen binding molecules of the present invention can increase T cell proliferation, IFN-γ production and / or TNF α production, for example in the presence of VISTA / VISTA expressing cells. Such properties of antigen binding molecules can be assessed, for example, as described in the experimental examples herein in an in vitro assay.

[1034] In some embodiments, the antigen binding molecules of the invention are capable of increasing T cell (e.g., Th1 / Th17 cell) proliferation, IFN-γ production, and / or TNFα production (e.g., in the presence of VISTA / VISTA-expressing cells) by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the levels observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1035] In some embodiments, the antigen binding molecules of the present invention can increase the proliferation of T cells (e.g., CD4+ T cells and / or CD8+ T cells) to a greater extent than the VISTA binding antibodies disclosed in the prior art (e.g., VSTB112, as described in WO 2015 / 097536 A2). T cell proliferation can be assessed in an in vitro assay (e.g., as described in Example 9 herein) and can include stimulating T cell proliferation by culture in the presence of an agonist anti-CD3 antibody. In some embodiments, the antigen binding molecules of the invention are capable of increasing T cell proliferation in this assay by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the level of proliferation caused by prior art VISTA-binding antibodies (e.g., VSTB112).

[1036] In some embodiments, the antigen binding molecules of the present disclosure are capable of increasing T cell-mediated lysis of cancer cells by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the level observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1037] In some embodiments, the antigen binding molecules of the present disclosure are capable of increasing T cell-mediated lysis of cancer cells, for example in the presence of VISTA / VISTA-expressing cells. The properties of the antigen binding molecules can be assessed, for example, in in vitro assays described in the experimental examples herein.

[1038] In some embodiments, the antigen binding molecules of the present disclosure are capable of increasing T cell-mediated lysis (e.g., in the presence of VISTA / VISTA-expressing cells) by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the level observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1039] In some embodiments, the antigen binding molecules of the present invention can enhance IL-6 production by THP1 cells to a greater extent than VISTA binding antibodies disclosed in the prior art (e.g., VSTB112, e.g., described in WO2015 / 097536 A2). IL-6 production by THP1 cells can be assessed in an in vitro assay (e.g., as described in Example 10 herein) and can involve stimulating THP1 cells with LPS. In some embodiments, the antigen binding molecules of the invention are capable of increasing IL-6 production in this assay by more than 1-fold, e.g., ≥1.01-fold, ≥1.02-fold, ≥1.03-fold, ≥1.04-fold, ≥1.05-fold, ≥1.1-fold, ≥1.2-fold, ≥1.3-fold, ≥1.4-fold, ≥1.5-fold, ≥1.6-fold, ≥1.7-fold, ≥1.8-fold, ≥1.9-fold, ≥2-fold, ≥3-fold, ≥4-fold, ≥5-fold, ≥6-fold, ≥7-fold, ≥8-fold, ≥9-fold, or ≥10-fold, compared to the level of proliferation caused by prior art VISTA-binding antibodies (e.g., VSTB112).

[1040] In some embodiments, the antigen binding molecules of the invention are capable of: reducing the number and / or activity of suppressor immune cells, inhibiting the proliferation of suppressor immune cells, and / or reducing the proportion of suppressor immune cells in a cell population (e.g., CD45+ cells, such as CD45+ cells obtained from a tumor) relative to control conditions (e.g., as determined by an appropriate in vitro assay or in vivo).

[1041] The suppressive immune cells can be, for example, cells expressing VISTA, cells expressing Arg1, MDSCs, granulocytic MDSCs (g-MDSCs) or mononuclear MDSCs (m-MDSCs). In some embodiments, the suppressive immune cells are CD11b+GR1+MHCII- cells.

[1042] In some embodiments, the reduction in number / activity / proliferation / ratio is less than 1-fold, e.g., ≤0.99-fold, ≤0.95-fold, ≤0.9-fold, ≤0.85-fold, ≤0.8-fold, ≤0.75-fold, ≤0.7-fold, ≤0.65-fold, ≤0.6-fold, ≤0.55-fold, ≤0.5-fold, ≤0.45-fold, ≤0.4-fold, ≤0.35-fold, ≤0.3-fold, ≤0.25-fold, ≤0.2-fold, ≤0.15-fold, ≤0.1-fold, ≤0.05-fold, or ≤0.01-fold, of the number / activity / proliferation / ratio observed in the absence of the antigen binding molecule (or in the presence of an appropriate control antigen binding molecule).

[1043] In some embodiments, the antigen binding molecules can reduce the number / activity / proliferation / ratio of suppressive immune cells by the mechanism not involving the function of Fc mediation. In some embodiments, the antigen binding molecules can reduce the number / activity / proliferation / ratio of suppressive immune cells independently of the function of Fc mediation (i.e., in an Fc region independent manner). In some embodiments, the antigen binding molecules can reduce the number / activity / proliferation / ratio of suppressive immune cells by the mechanism not involving ADCC, ADCP and / or CDC. In some embodiments, the antigen binding molecules can reduce the number / activity / proliferation / ratio of suppressive immune cells by the mechanism not involving the cell exhaustion of expressing VISTA.

[1044] In some embodiments, the antigen binding molecules of the present invention inhibit the development and / or progression of cancer in vivo.

[1045] In some embodiments, the antigen binding molecules cause an increase in cancer cell killing, such as by effector immune cells. In some embodiments, the antigen binding molecules cause a decrease in the number of cancer cells in vivo, such as compared to appropriate control conditions. In some embodiments, the antigen binding molecules inhibit tumor growth, such as by measuring tumor size / volume over time.

[1046] In some embodiments, the antigen binding molecules of the present invention can increase serum IFN-γ and / or IL-23 levels in mice treated with antigen binding molecules.The serum levels of IFN-γ and / or IL-23 can be analyzed by obtaining from the ELISA of mouse blood sample from serum.In some embodiments, the administration of the antigen binding molecules of the present invention makes the serum levels of IFN-γ and / or IL-23 increase to more than 1 times of the level (or the level observed after administering appropriate control antigen binding molecules) observed when not administering antigen binding molecules, such as ≥1.01 times, ≥1.02 times, ≥1.03 times, ≥1.04 times, ≥1.05 times, ≥1.1 times, ≥1.2 times, ≥1.3 times, ≥1.4 times, ≥1.5 times, ≥1.6 times, ≥1.7 times, ≥1.8 times, ≥1.9 times, ≥2 times, ≥3 times, ≥4 times, ≥5 times, ≥6 times, ≥7 times, ≥8 times, ≥9 times or ≥10 times.

[1047] The antigen binding molecules of the present invention can be analyzed in suitable in vivo models (xenograft models of such as cell line origin, such as CT26 cell source model.4T-1 cell source model, LL2 cell source model, B16 cell source model or EL4 cell source model) suppress the ability of cancer development and / or progress. The cancer can be pathologically related to the cancer of the cell and / or MDSC (MDSC, TAM, neutrophil expressing VISTA) expressing VISTA. The cancer of " pathologically related to " MDSC includes that MDSC or MDSC quantity / ratio increase and the occurrence of cancer, development or progress and / or the severity of one or more cancer symptoms are positively correlated with cancer, or MDSC or MDSC quantity / ratio increase is the risk factor for cancer occurrence, development or progress. The cancer can include MDSC in the organ / tissue (such as showing the organ / tissue of disease / symptom) or tumor affected by disease.

[1048] In some embodiments, administration of antigen binding molecules according to the present invention (e.g., as determined in a xenograft model derived from CT26 cells, 4T-1 cells, LL2 cells, B16 cells, or EL4 cells) can result in one or more of the following: inhibition of cancer development / progression, delay / prevention of cancer onset, reduction / delay / prevention of tumor growth, reduction / delay / prevention of metastasis, reduction in the severity of cancer symptoms, reduction in the number of cancer cells, reduction in tumor size / volume, and / or improvement in survival rate (e.g., progression-free survival).

[1049] In some embodiments, administration of an antigen binding molecule of the invention is capable of inhibiting tumor growth by greater than 5%, e.g., ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75%, ≥80%, ≥85%, ≥90% or ≥95%, compared to tumor growth observed in the absence of the antigen binding molecule (or after administration of an appropriate control antigen binding molecule).

[1050] In some embodiments, compared to the tumor growth observed in the absence of antigen binding molecules (or after administration of appropriate control antigen binding molecules), in the CT26 cell-derived model of the experimental examples of the present disclosure, the antigen binding molecules are administered according to the dosage and cycle described in the experiment and can inhibit more than 5%, for example, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50%, ≥55%, ≥60%, ≥65%, ≥70%, ≥75% or ≥80% tumor growth. In some embodiments, compared to the tumor growth observed in the absence of antigen binding molecules (or after administration of appropriate control antigen binding molecules), in the 4T-1 cell-derived model of the experimental examples of the present disclosure, the antigen binding molecules are administered according to the dosage and cycle described in the experiment and can inhibit more than 5%, for example, ≥10%, ≥15%, ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, ≥50% tumor growth.

[1051] In some embodiments, the administration of antigen binding molecules according to the present disclosure is unrelated to cytokine release syndrome. In some embodiments, the administration of antigen binding molecules is unrelated to the systemic activation of leukocytes (such as B cells, T cells, NK cells, macrophages, dendritic cells and / or monocytes). In some embodiments, the administration of antigen binding molecules is unrelated to the systemic upregulation of leukocyte 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).

[1052] In some embodiments, treatment of a subject with an antigen binding molecule or other article (e.g., composition, nucleic acid, etc.) disclosed herein, e.g., where the antigen binding molecule / article is administered to the subject at a dose and / or according to a dose schedule described herein, can be associated with one or more of the following outcomes:

[1053] No adverse events (AEs);

[1054] No serious adverse events (SAEs);

[1055] Minimal or reduced frequency of adverse events (AEs), for example compared with other anti-VISTA therapeutic antibodies;

[1056] Minimal or reduced frequency of serious adverse events (SAEs), for example compared with other anti-VISTA therapeutic antibodies;

[1057] No dose-limiting toxicities (DLTs);

[1058] Minimal or reduced frequency of dose-limiting toxicities (DLTs), for example compared with other anti-VISTA therapeutic antibodies (such as W0180 and CA-170);

[1059] Reduction in circulating tumor markers (e.g., cell-free (cf) DNA altered allele fraction / tumor fraction, ctDNA) after treatment, e.g., compared to the same subject before treatment;

[1060] No, minimal, or reduced frequency of infusion-related reactions (e.g., cytokine release syndrome and associated cytokine increases), compared to, for example, other anti-VISTA therapeutic antibodies (e.g., W0180 and CA-170);

[1061] No, minimal, or reduced frequency of gastrointestinal reactions, for example compared to other anti-VISTA therapeutic antibodies (such as W0180 and CA-170);

[1062] No, minimal, or reduced frequency of rash or dermatitis, for example compared with other anti-VISTA therapeutic antibodies (such as W0180 and CA-170);

[1063] No, minimal, or reduced frequency of hematologic changes, for example compared with other anti-VISTA therapeutic antibodies (such as W0180 and CA-170);

[1064] No, minimal, or reduced frequency of cardiotoxicity, for example compared with other anti-VISTA therapeutic antibodies (such as W0180 and CA-170);

[1065] No, minimal, or reduced frequency of plasma albumin elevation, for example compared to other anti-VISTA therapeutic antibodies (such as W0180 and CA-170).

[1066] An adverse event (AE) can be defined as any unpleasant, unwanted, or unplanned medical occurrence in a patient taking an investigational medicinal product (IMP), a comparator product, or an approved drug. An AE can be a sign, symptom, disease, and / or laboratory or physiological observation that may or may not be related to the IMP or comparator. AEs include, but are not limited to, those listed below:

[1067] Clinically significant worsening of a previous disorder. This includes disorders that may have completely resolved and then become abnormal again.

[1068] Adverse reactions due to an overdose of IMP, whether accidental or intentional.

[1069] Adverse events that occur due to a lack of efficacy of the IMP, for example, if the investigator suspects that a batch of drug product is ineffective, or if the investigator suspects that the IMP promotes disease progression.

[1070] A serious adverse event (SAE) is any adverse event not related to dose, causality, or expectation:

[1071] resulting in death;

[1072] is life-threatening, meaning the patient faces a significant risk of death at the time of the adverse event or continued use of the device or other medical product may result in the patient's death;

[1073] Requires hospitalization or prolongs an existing hospitalization (some hospitalizations do not require SAE reporting, such as planned hospitalizations before the patient entered the trial; overnight stays for planned procedures such as blood transfusions);

[1074] Causes persistent or significant incapacity or disability;

[1075] Congenital anomaly or birth defect;

[1076] is any other medically significant event, that is, any event that may endanger the patient or may require intervention to prevent one of the outcomes listed above.

[1077] In some embodiments, the therapeutic response according to the present disclosure can be characterized by reference to the tumor / lesion response. In some embodiments, the tumor / lesion response is assessed according to the Response Evaluation Criteria in Solid Tumors (RECIST), such as the RECIST 1.1 criteria described in Eisenhauer et al., Eur J Cancer. 2009 January; 45(2): 228-47, which is hereby incorporated by reference in its entirety.

[1078] In some embodiments, treatment of a subject with an antigen binding molecule or article described herein, e.g., wherein the antigen binding molecule / article is administered to the subject at a dose and / or according to a dosing schedule described herein, may be associated with one or more of the following outcomes (e.g., assessed according to RECIST 1.1 criteria, as appropriate; see Example 19.9 for details and assessment methods), e.g., 12 and / or 24 months after initiation of treatment:

[1079] Anti-tumor response;

[1080] Complete response (CR). CR is the complete macroscopic disappearance of all target and / or non-target tumors. CR may involve normalization of tumor marker levels;

[1081] an increased likelihood of a complete remission (CR), for example, compared to the likelihood of a complete remission (CR) in the same subject who did not receive the antigen binding molecule, in a subject who did not receive the antigen binding molecule, or in a subject who received a different anti-VISTA antigen binding molecule;

[1082] an increased proportion of subjects who achieve a complete response (CR), for example, compared to the proportion of subjects who achieve a CR in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1083] Overall survival (OS). OS is defined as the time from the start of treatment to death from any cause;

[1084] The likelihood of increased overall survival (OS), for example, compared to the likelihood of overall survival (OS) in the same subject who did not receive the antigen binding molecule, in a subject who did not receive the antigen binding molecule, or in a subject who received a different anti-VISTA antigen binding molecule;

[1085] the proportion of subjects who demonstrate an increase in overall survival (OS), e.g., compared to the proportion of subjects who demonstrate OS in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1086] Progression-free survival (PFS). PFS is the time from the start of treatment to disease progression or death.

[1087] the likelihood of increased progression-free survival (PFS), for example, compared to the likelihood of PFS in the same subject who did not receive the antigen binding molecule, in a subject who did not receive the antigen binding molecule, or in a subject who received a different anti-VISTA antigen binding molecule;

[1088] the proportion of subjects who demonstrate an increase in progression-free survival (PFS), e.g., compared to the proportion of subjects who demonstrate PFS in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1089] 6-month progression-free survival (PFS6): PFS6 is the percentage of patients who are alive and progression-free 6 months (26 weeks) after the start of treatment.

[1090] the likelihood of increased progression-free survival at 6 months (PFS6), for example, compared to the likelihood of PFS6 in the same subject who did not receive the antigen binding molecule, in a subject who did not receive the antigen binding molecule, or in a subject who received a different anti-VISTA antigen binding molecule;

[1091] an increased proportion of subjects demonstrating 6-month progression-free survival (PFS6), for example, compared to the proportion of subjects demonstrating PFS6 in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1092] Partial response (PR). PR refers to a reduction of at least 30% in the sum of the diameters of all target tumors compared to the sum of the preoperative tumor diameters calculated before treatment;

[1093] an increased likelihood of a partial response (PR), for example, compared to the likelihood of a PR in the same subject who did not receive the antigen binding molecule, in a subject who did not receive the antigen binding molecule, or in a subject who received a different anti-VISTA antigen binding molecule;

[1094] an increased proportion of subjects who demonstrate a partial response (PR), for example, compared to the proportion of subjects who demonstrate a PR in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1095] Mixed response (MR): MR refers to one or more tumor lesions that meet PR criteria and other tumor lesions that meet criteria for progressive disease (an increase of at least 20% in the sum of the diameters of all tumors from the smallest tumor size and / or the appearance of new tumor lesions);

[1096] an increased likelihood of a mixed response (MR), for example, compared to the likelihood of a MR in the same subject who did not receive the antigen binding molecule, in a subject who did not receive the antigen binding molecule, or in a subject who received a different anti-VISTA antigen binding molecule;

[1097] an increased proportion of subjects showing mixed responses (MRs), for example, compared to the proportion of subjects showing MRs in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1098] Stable disease (SD). SD is defined as neither partial response nor progressive disease compared with the tumor burden at the start of treatment.

[1099] an increased likelihood of stable disease (SD), for example, compared to the likelihood of SD in the same subject not receiving the antigen binding molecule, a subject not receiving the antigen binding molecule, or a subject receiving a different anti-VISTA antigen binding molecule;

[1100] an increased proportion of subjects who demonstrate stable disease (SD), e.g., compared to the proportion of subjects who demonstrate SD in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1101] Increased duration of remission (DoR) or increased duration of CR, for example, compared to the duration in the same subject who did not receive the antigen binding molecule treatment, in a subject who did not receive the antigen binding molecule treatment, or in a subject who received a different anti-VISTA antigen binding molecule treatment. Duration of remission (DoR) is defined as the time from the first achievement of PR or CR measurement criteria to the first achievement of disease progression (PD) measurement criteria. Duration of CR (DoCR) is defined as the time from the satisfaction of CR measurement criteria to the first achievement of PD measurement criteria;

[1102] an increase in the proportion of subjects who demonstrate increased duration of response (DoR) or increased duration of CR, for example, compared to the proportion of subjects who demonstrate DoR or DoCR in subjects who have not received the antigen binding molecule treatment or who have received a different anti-VISTA antigen binding molecule treatment;

[1103] Overall response (OR). OR was defined as complete response (CR) or partial response (PR);

[1104] Overall response rate (ORR). ORR is defined as the proportion of patients achieving complete response (CR) or partial response (PR);

[1105] Increased ORR, e.g., compared to the proportion of subjects who experienced ORs who were not treated with the antigen binding molecule or who were treated with a different anti-VISTA antigen binding molecule.

[1106] The efficacy of the tumor can be assessed using appropriate imaging techniques, such as CT scans, MRI scans, and FDG-PET scans, depending on the tumor and its location. Suitable techniques will be familiar to the skilled artisan and are described in Eisenhauer et al., supra, and / or in Example 19.9 herein.

[1107] The subject can be a subject as defined herein, eg, having, or determined to have, cancer or a solid tumor, eg, according to the present disclosure.

[1108] Chimeric antigen receptors (CARs)

[1109] The present invention also provides chimeric antigen receptors (CARs) comprising the antigen-binding molecules or polypeptides of the present invention.

[1110] CAR is a recombinant receptor that provides antigen binding and T cell activation functions. For example, the structure and engineering of CAR are reviewed in Dotti et al., Immunol Rev (2014) 257 (1), which is incorporated herein by reference in its entirety. CAR includes an antigen binding region connected to a cell membrane anchoring region and a signaling region. An optional hinge region can provide separation between the antigen binding region and the cell membrane anchoring region and can act as a flexible joint.

[1111] The CAR of the present invention comprises an antigen-binding region, which comprises or consists of an antigen-binding molecule of the present invention, or comprises or consists of a polypeptide of the present invention.

[1112] The cell membrane anchoring region is located between the antigen binding region and the signal transduction region of the CAR, and is used to anchor the CAR to the cell membrane of the cell expressing the CAR, and its antigen binding region is located in the extracellular space, and the intracellular signal transduction region. In some embodiments, the CAR comprises a cell membrane anchoring region, and the cell membrane anchoring region comprises or consists of the following amino acid sequence: a transmembrane region amino acid sequence comprising one of CD3-ζ, CD4, CD8 or CD28, or consisting of or derived therefrom. As used herein, the region “derived from” a reference amino acid sequence includes an amino acid sequence having at least 60%, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference sequence.

[1113] The signaling region of CAR can activate T cells. The signaling region of CAR can include an amino acid sequence of the intracellular domain of CD3-ζ, which provides an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of T cells expressing CAR. Signaling regions containing sequences of other ITAM-containing proteins (e.g., FcγRI) have also been used in CAR (Haynes et al., 2001 J Immunol 166(1):182-187). The signaling region of CAR can also include a costimulatory sequence derived from the signaling region of a costimulatory molecule to promote the activation of T cells expressing CAR after binding to the target protein. Suitable costimulatory molecules include CD28, OX40, 4-1BB, ICOS, and CD27. In some cases, CAR is designed to provide co-stimulation of different intracellular signaling pathways. For example, signaling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, while 4-1BB-mediated signaling is through the TNF receptor-associated factor (TRAF) adapter protein. Therefore, the signaling region of CAR sometimes includes a costimulatory sequence derived from the signaling region of more than one costimulatory molecule. In some embodiments, the CAR of the present invention includes one or more costimulatory sequences comprising an amino acid sequence comprising one or more intracellular domains of CD28, OX40, 4-1BB, ICOS and CD27 or consisting of or derived from the amino acid sequence thereof, or consisting of.

[1114] The optional hinge region can provide separation between the antigen binding domain and the transmembrane domain and can act as a flexible joint. The hinge region can be derived from IgG1. In some embodiments, the CAR of the present invention comprises a hinge region comprising or consisting of the following amino acid sequence, the amino acid sequence comprising the amino acid sequence of the hinge region of IgG1 or consisting of or derived therefrom.

[1115] Also provided are cells comprising a CAR according to the present invention. The CAR according to the present invention can be used to generate immune cells expressing CAR, such as CAR-T or CAR-NK cells. CAR can be transformed into immune cells during in vitro culture.

[1116] The antigen binding region of the CAR of the present invention can be provided in any suitable form, such as scFv, scFab, etc.

[1117] Nucleic acids and vectors

[1118] The present invention provides a nucleic acid or multiple nucleic acids encoding an antigen binding molecule, polypeptide or CAR according to the present invention.

[1119] In some embodiments, the nucleic acid is purified or isolated, for example from other nucleic acids or naturally occurring biological materials. In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA.

[1120] The invention also provides one or more vectors comprising one or more nucleic acids according to the invention.

[1121] The nucleotide sequence can be contained in a vector, such as an expression vector. As used herein, a "vector" is a nucleic acid molecule used as a vehicle for transferring exogenous nucleic acid into a cell. The vector can be a vector for expressing nucleic acid in a cell. Such a vector can include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed. The vector can also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art can be used to express a peptide or polypeptide from a vector according to the present invention.

[1122] The term "operably linked" can include situations where a selected nucleotide sequence and a regulatory nucleotide sequence (e.g., a promoter and / or enhancer) are covalently linked (forming an expression cassette) in a manner that places the expression of the nucleotide sequence under the influence or control of the regulatory sequence. Thus, if the regulatory sequence is capable of influencing the transcription of the nucleic acid sequence, the regulatory sequence is operably linked to the selected nucleic acid sequence. The resulting transcript can then be translated into the desired peptide / polypeptide.

[1123] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., gammaretroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes).

[1124] In some embodiments, the vector can be a eukaryotic vector, for example, comprising elements necessary for expressing a protein from the vector in a eukaryotic cell. In some embodiments, the vector can be a mammalian vector, for example, comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[1125] The constituent polypeptides of the antigen-binding molecules according to the present invention may be encoded by different nucleic acids among multiple nucleic acids, or by different vectors among multiple vectors.

[1126] Cells containing / expressing antigen-binding molecules and polypeptides

[1127] The present invention also provides cells comprising or expressing antigen binding molecules, polypeptides or CARs according to the present invention. A cell is also provided, comprising or expressing a nucleic acid or multiple nucleic acids, a vector or multiple vectors according to the present invention.

[1128] The cell can be a eukaryotic cell, such as a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (e.g., rabbit, guinea pig, rat, mouse or other rodent (including any rodent), cat, dog, pig, sheep, goat, cattle (including cows, such as dairy cows, or any bovine), horse (including any equine), donkey and non-human primate).

[1129] The present invention also provides a method for producing a biological cell comprising one or more nucleic acids or vectors according to the present invention, comprising introducing into the cell one or more nucleic acids, one or more vectors according to the present invention. In some embodiments, introducing the isolated nucleic acid or vector according to the present invention into the cell comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[1130] The present invention also provides a method for producing cells expressing / comprising the antigen binding molecules, polypeptides or CARs of the present invention, comprising introducing a nucleic acid or multiple nucleic acids, a vector or multiple vectors according to the present invention into cells. In some embodiments, the method further comprises culturing cells under conditions suitable for cell expression of nucleic acids or vectors. In some embodiments, the method is performed in vitro.

[1131] The invention also provides a cell obtained or obtainable by a method according to the invention.

[1132] Production of antigen-binding molecules and peptides

[1133] The antigen-binding molecules and polypeptides according to the present invention can be prepared according to polypeptide production methods known to those skilled in the art.

[1134] Polypeptides can be prepared by chemical synthesis, such as liquid phase or solid phase synthesis. For example, peptides / polypeptides can be synthesized using methods such as those described in Chandrudu et al., Molecules (2013), 18: 4373-4388, which is incorporated herein by reference in its entirety.

[1135] Alternatively, antigen binding molecules and polypeptides can be produced by recombinant expression. Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as Green and Sambrook in Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5 (2): 135-146, both of which are incorporated herein by reference in their entirety. Methods for recombinant production of antigen binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451–3461, both of which are incorporated herein by reference in their entirety.

[1136] In some cases, the antigen binding molecules of the present invention are composed of more than one polypeptide chain. In this case, the production of the antigen binding molecules may include the transcription and translation of more than one polypeptide, and the subsequent association of polypeptide chains to form the antigen binding molecules.

[1137] For recombinant production according to the present invention, any cell suitable for expressing the polypeptide can be used. The cell can be a prokaryotic organism or a eukaryotic organism. In some embodiments, the cell is a prokaryotic cell, such as a cell of an archaebacterium or a bacterium. In some embodiments, the bacterium can be a gram-negative bacterium, such as a bacterium of the family Enterobacteriaceae, such as Escherichia coli. In some embodiments, the cell is a eukaryotic cell, such as a yeast cell, a plant cell, an insect cell or a mammalian cell, such as a CHO, HEK (e.g., HEK293), HeLa or COS cell. In some embodiments, the cell is a CHO cell transiently or stably expressing the polypeptide.

[1138] In some cases, the cells are not prokaryotes, as some prokaryotes do not allow the same folding or post-translational modifications as eukaryotic cells. Alternatively, very high expression levels are possible in eukaryotes, and proteins can be more easily purified from eukaryotes using appropriate tags. Specific plasmids can also be used to enhance secretion of proteins into the culture medium.

[1139] In some embodiments, the polypeptide can be produced by cell-free protein synthesis (CFPS), for example using the system described in Zemella et al., Chembiochem (2015) 16(17): 2420-2431, which is incorporated herein by reference in its entirety.

[1140] Production can involve the cultivation or fermentation of eukaryotic cells modified to express the polypeptide of interest. The cultivation or fermentation can be carried out in a bioreactor equipped with appropriate nutrients, air / oxygen and / or growth factors. The secreted protein can be collected by separating the culture medium / fermentation broth from the cells, extracting the protein content and isolating the individual proteins to isolate the secreted polypeptide. Cultivation, fermentation and separation techniques are well known to those skilled in the art and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th edition; incorporated herein by reference above).

[1141] A bioreactor consists of one or more containers in which cells can be cultured. Cultivation in a bioreactor can be continuous, with reactants flowing continuously into the reactor and cultured cells continuously flowing out. Alternatively, culturing can be performed in batches. Bioreactors monitor and control environmental conditions, such as pH, oxygen, flow rates in and out of the container, and agitation within the container, to provide optimal conditions for the cultured cells.

[1142] After culturing cells expressing the antigen binding molecules / polypeptides, the target polypeptide can be isolated. Any suitable method known in the art for isolating proteins from cells can be used. In order to isolate the polypeptide, it may be necessary to separate the cells from the nutrient medium. If the polypeptide is secreted from the cells, the cells can be separated from the culture medium containing the secreted target polypeptide by centrifugation. If the target polypeptide is aggregated within the cells, protein separation can include centrifugation to separate the cells from the cell culture medium, treating the cell pellet with a lysis buffer, and, for example, destroying the cells by ultrasonic treatment, rapid freeze-thaw, or osmotic lysis.

[1143] It may then be necessary to separate the target polypeptide from the supernatant or culture medium, which may contain other proteins and non-protein components. A common method for separating protein components from the supernatant or culture medium is precipitation. Proteins of different solubility will precipitate under different concentrations of precipitants (e.g., ammonium sulfate). For example, at low concentrations of precipitants, water-soluble proteins are extracted. Therefore, by adding increasing precipitants at different concentrations, proteins with different solubility can be distinguished. Dialysis can then be used to remove ammonium sulfate from the separated proteins.

[1144] Other methods for distinguishing different proteins are known in the art, such as ion exchange chromatography and size chromatography. These can be used as an alternative to precipitation, or can be performed after precipitation.

[1145] Once the polypeptide of interest is isolated from the culture, it may be desirable or necessary to concentrate the polypeptide. Many methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilization.

[1146] Composition

[1147] The present invention also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors, and cells described herein.

[1148] Antigen binding molecules as described herein, polypeptide, CAR, nucleic acid, expression vector and cell can be formulated into pharmaceutical compositions or medicines for clinical use, and can include pharmaceutically acceptable carriers, diluents, excipients or adjuvants. Compositions can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration, including injection or infusion.

[1149] Suitable formulations can include the antigen binding molecules in sterile or isotonic culture medium. Medicines and pharmaceutical compositions can be formulated into fluid forms including gels. Liquid formulations can be formulated to be administered to selected areas of the human or animal body by injection or infusion (e.g., via a catheter).

[1150] In some embodiments, the composition is formulated for injection or infusion, for example, into a blood vessel or a tumor.

[1151] According to the invention described herein, a method for producing a pharmaceutically useful composition is also provided, which production method may include one or more steps selected from the following steps: producing an antigen binding molecule, polypeptide, CAR, nucleic acid (or multiple thereof), expression vector (or multiple thereof) or cell as described herein; isolating the antigen binding molecule, polypeptide, CAR, nucleic acid (or multiple thereof), expression vector (or multiple thereof) or cell as described herein; and / or mixing the antigen binding molecule, polypeptide, CAR, nucleic acid (or multiple thereof), expression vector (or multiple thereof) or cell as described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[1152] For example, another aspect of the invention described herein relates to a method for formulating or producing a medicament or pharmaceutical composition for treating a disease / disorder (e.g., cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing the antigen binding molecules, polypeptides, CARs, nucleic acids (or multiples thereof), expression vectors (or multiples thereof) or cells described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

[1153] In aspects and embodiments of the present disclosure, the antigen binding molecules may be provided in compositions comprising specific chemical components at specific concentrations / ratios.

[1154] In some embodiments, the antigen binding molecules are provided by a buffer. As used herein, "buffer" refers to a buffer solution that resists pH changes through the action of its acid-base conjugate components. The buffer disclosed herein preferably has a pH in the range of about 4.5 to about 7.0, preferably a pH in the range of about 5.0 to about 6.5. Examples of buffers that control the pH value within this range include acetic acid, succinic acid, histidine, histidine arginine, histidine methionine and other organic acid buffers.

[1155] In some embodiments, the composition comprising the antigen binding molecule has a pH of 4.0 to 7.0, for example, one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.5, pH 4.8 to pH 6.3, or pH 5.0 to pH 6.3. In some embodiments, the composition has a pH of about 5.5. In some embodiments, the composition has a pH of about 5.8. In some embodiments, the composition has a pH of about 6.3.

[1156] In some embodiments, the antigen binding molecules are provided by an acetate buffer, i.e., a buffer comprising acetate ions. In some embodiments, the antigen binding molecules are provided by a composition comprising a final concentration of 2 mM to 200 mM acetic acid, such as one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise approximately 20 mM acetic acid. The buffer may contain sodium acetate.

[1157] In some embodiments, the antigen binding molecules are provided by a histidine buffer, i.e., a buffer comprising histidine ions. In some embodiments, the antigen binding molecules are provided by a composition comprising a final concentration of 2 mM to 200 mM histidine, such as one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise about 20 mM histidine.

[1158] In some embodiments, the antigen binding molecules are provided by a succinate buffer, i.e., a buffer comprising succinate ions. In some embodiments, the antigen binding molecules are provided by a composition comprising a final concentration of 2 mM to 200 mM succinic acid, such as one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise approximately 20 mM succinic acid. The buffer may contain sodium succinate.

[1159] In some embodiments, the antigen binding molecules are provided by sodium phosphate buffer, i.e., a buffer comprising sodium and phosphate ions. In some embodiments, the antigen binding molecules are provided by a composition comprising a final concentration of 2 mM to 200 mM sodium phosphate, such as one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise about 20 mM sodium phosphate.

[1160] In some embodiments, the antigen binding molecules are provided by sodium acetate buffer, i.e., a buffer comprising sodium and acetate ions. In some embodiments, the antigen binding molecules are provided by a composition comprising a final concentration of 2 mM to 200 mM sodium acetate, such as one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise approximately 20 mM sodium acetate.

[1161] In some embodiments, the antigen binding molecules are provided by an arginine buffer, i.e., a buffer comprising arginine ions. In some embodiments, the antigen binding molecules are provided by a composition comprising a final concentration of 1 mM to 250 mM arginine, such as one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM. In some embodiments, the composition may comprise about 20 mM arginine.

[1162] In some embodiments, antigen binding molecules are provided by histidine arginine buffer, i.e., a buffer comprising histidine and arginine ions. In some embodiments, antigen binding molecules are provided by a composition comprising a final concentration of 2mM to 200mM histidine, such as 5mM to 100mM, 10mM to 40mM, 12mM to 30mM, 15 to 25mM, or one of 18 to 22mM, and a composition with a final concentration of 1mM to 300mM arginine, such as 10mM to 250mM, 50mM to 200mM, 75mM to 200mM, 100 to 180mM, or one of 125 to 175mM. In some embodiments, compositions can include about 20mM histidine and about 150mM arginine.

[1163] In some embodiments, the composition comprising the antigen binding molecules includes an isotonic agent. An isotonic agent can be used to provide an isotonic formulation. Examples of isotonic agents include, for example, salts (e.g., sodium chloride, potassium chloride) and sugars (e.g., sucrose, glucose, trehalose).

[1164] In some embodiments, the antigen binding molecules are provided by a composition containing sodium chloride, i.e., sodium ions and chloride ions. The sodium chloride component of the composition can be provided by sodium chloride having a final concentration of 1 mM to 250 mM, such as 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 to 180 mM, or 125 to 175 mM. In some embodiments, the composition may include about 150 mM sodium chloride.

[1165] In some embodiments, the antigen binding molecule is provided by a composition containing methionine. The methionine component of the composition can be provided by methionine at a final concentration of 1 mM to 250 mM, for example, 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 to 180 mM, or 125 to 175 mM. In some embodiments, the composition may contain about 150 mM methionine.

[1166] In some embodiments, antigen binding molecules are provided by compositions containing sucrose. The sucrose component of the composition can be provided by 2% to 20% sucrose of the final concentration (by volume weight), for example, 2% to 15%, 3% to 12%, 4% to 10%. In some embodiments, the composition may include about 2% (w / v), about 4% (w / v), about 6% (w / v), or about 8% (w / v) sucrose. The sucrose component of the composition can be provided with a final concentration of 200 to 300 nM, for example, about 240 mM.

[1167] In some embodiments, the composition comprising the antigen binding molecule includes a surfactant. As used herein, a "surfactant" refers to an agent that reduces interfacial tension. The surfactant is preferably a nonionic surfactant. Examples of surfactants include polysorbates (polysorbate-20, polysorbate-80), poloxamers (poloxamer-188), and Triton X-100. The surfactant is preferably present in the composition in an amount ranging from about 0.001% (w / v) to about 0.5% (w / v).

[1168] In some embodiments, the antigen binding molecules are provided by a composition containing polysorbate 20. The polysorbate 20 component of the composition can be provided by a final concentration (by volume weight) of 0.001% to 0.1% polysorbate 20, such as 0.002% to 0.08%, 0.006% to 0.05%, or one of 0.008% to 0.04%. In some embodiments, the composition may include about 0.02% (w / v) polysorbate 20. In some embodiments, the composition may include about 0.05% (w / v) polysorbate 20.

[1169] In some embodiments, the antigen binding molecules are provided by a composition containing polysorbate 80. The polysorbate 80 component of the composition can be provided by a final concentration (by volume weight) of 0.001% to 0.1% polysorbate 80, such as 0.002% to 0.08%, 0.006% to 0.05%, or one of 0.008% to 0.04%. In some embodiments, the composition may include about 0.01% (w / v) polysorbate 80. In some embodiments, the composition may include about 0.02% (w / v) polysorbate 80.

[1170] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1171] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1172] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose;

[1173] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1174] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[1175] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1176] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1177] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose;

[1178] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1179] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 5.8.

[1180] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1181] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1182] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 4% (w / v) sucrose;

[1183] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1184] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 5.8.

[1185] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1186] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1187] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 2% (w / v) sucrose;

[1188] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1189] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 5.8.

[1190] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1191] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1192] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose;

[1193] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1194] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 6.3.

[1195] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1196] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1197] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose;

[1198] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1199] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 5.8.

[1200] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1201] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of acetic acid, more preferably about 20 mM acetic acid;

[1202] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose;

[1203] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1204] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[1205] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1206] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) succinic acid, more preferably about 20 mM succinic acid;

[1207] 2% to 20% (e.g., one of 2% to 15%, 3% to 12%, or 4% to 10%) sucrose (w / v), more preferably about 8% (w / v) sucrose;

[1208] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1209] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably about pH 5.5.

[1210] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1211] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1212] 0.001% to 0.1% (e.g., one of 0.002% to 0.08%, 0.006% to 0.05%, or 0.008% to 0.04%) polysorbate-80 (w / v), more preferably about 0.02% (w / v) polysorbate-80; and

[1213] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 5.8.

[1214] In some embodiments, the antigen binding molecule is provided by a composition comprising:

[1215] 2 mM to 200 mM (e.g., one of 5 mM to 100 mM, 10 mM to 40 mM, 12 mM to 30 mM, 15 to 25 mM, or 18 to 22 mM) of histidine, more preferably about 20 mM of histidine;

[1216] 1 mM to 250 mM (e.g., one of 10 mM to 250 mM, 50 mM to 200 mM, 75 mM to 200 mM, 100 mM to 180 mM, or 125 to 175 mM) of sodium chloride, more preferably about 150 mM sodium chloride; and

[1217] pH 4.0 to 7.0 (e.g., one of pH 4.5 to pH 6.8, pH 4.6 to pH 6.4, pH 4.8 to pH 6.2, or pH 5.0 to pH 6.2), more preferably pH about 5.8.

[1218] The composition may comprise from about 0.025 mg / mL to about 100 mg / mL of antigen binding molecule. The composition may comprise from about 0.05 mg / mL to about 80 mg / mL of antigen binding molecule. The composition may comprise from about 0.06 mg / mL to about 70 mg / mL of antigen binding molecule. The composition may comprise from about 0.07 mg / mL to about 60 mg / mL of antigen binding molecule. The composition may comprise from about 0.07 mg / mL to about 50 mg / mL of antigen binding molecule.

[1219] Antigen binding molecules can be prepared at a concentration of about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, or about 70 mg / mL, or any range therein, such as in a composition according to the present disclosure. Antigen binding molecules can be prepared at a concentration of about 50 mg / mL, such as in a composition according to the present disclosure.

[1220] As used herein, "about" refers to the specified concentration, as well as plus or minus 10% of that concentration. For example, a concentration of "about 50 mg / mL" refers to a concentration of 45 mg / mL to 55 mg / mL, including 50 mg / mL.

[1221] The antigen binding molecules can be formulated at a concentration of at least 0.05 mg / mL, at least 0.1 mg / mL, at least 0.15 mg / mL, at least 0.2 mg / mL, at least 0.25 mg / mL, at least 0.3 mg / mL, at least 0.35 mg / mL, at least 0.4 mg / mL, at least 0.5 mg / mL, at least 0.8 mg / mL, at least 1 mg / mL, at least 1.4 mg / mL, at least 2 mg / mL, at least 2.4 mg / mL, at least 4 mg / mL, at least 6 mg / mL, at least 8 mg / mL, at least 10 mg / mL, at least 12 mg / mL, at least 14 mg / mL, or at least 16 mg / mL, e.g., in a composition according to the present disclosure.

[1222] 50mg / mL antigen binding molecule solution can be used any suitable excipient dilution before administration.In some embodiments, 50mg / mL antigen binding molecule solution is diluted with 5% glucose for administration.In some embodiments, such as in the volume of 50mL, 50mg / mL antigen binding molecule solution is diluted to at least 0.07mg / mL, at least 0.14mg / mL, at least 0.21mg / mL, at least 0.35mg / mL, at least 0.42mg / mL, at least 0.8mg / mL, at least 1.4mg / mL, at least 2.4mg / mL concentration for administration.In some embodiments, such as in the volume of 100mL, 50mg / mL antigen binding molecule solution is diluted to at least 2.4mg / mL, at least 4mg / mL, at least 8mg / mL, at least 12mg / mL or at least 16mg / mL concentration for administration. In some embodiments, the diluted antigen binding molecule solution is then administered to the subject, e.g., intravenously, e.g., using a dosage / dosage regimen according to the present disclosure, e.g., to treat a disease / disorder according to the present disclosure. In some embodiments, the diluted antigen binding molecule solution is administered to the subject within 48 hours after the initial dilution step.

[1223] According to the antigen binding molecules of the present disclosure, for example, using the dosage and / or dosage regimen described herein, it is possible to co-administer with an agent capable of inhibiting PD-1-mediated signaling. The agent capable of inhibiting PD-1-mediated signaling may be a PD-1 or PD-L1 targeting agent. For example, the agent capable of inhibiting PD-1-mediated signaling may be an agent capable of binding to PD-1 or PD-L1 and inhibiting PD-1-mediated signaling. In some embodiments, the agent is an antagonist of an anti-PD-1 antibody. In certain embodiments, the agent is an antagonist. In some embodiments, the agent is pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab (Tecentriq), avelumab (Bavencio), or durvalumab (Imfinzi). These agents can be prepared and administered according to the drug preparation instructions provided with the agent.

[1224] Therapeutic and preventive applications

[1225] The antigen binding molecules, polypeptides, CARs, nucleic acids, expression vectors, cells and compositions described herein can be used in methods of treatment and prevention.

[1226] The present invention provides antigen binding molecules as described herein, polypeptide, CAR, nucleic acid (or its multiple), expression vector (or its multiple), cell or composition, for medical treatment or prevention method.Also provided are antigen binding molecules as described herein, polypeptide, CAR, nucleic acid (or its multiple), expression vector (or its multiple), cell or composition in the preparation of medicine for treating or preventing disease or illness.Also provided is a method for treating or preventing disease or illness, which includes applying to the experimental subject an antigen binding molecule as described herein, polypeptide, CAR, nucleic acid (or its multiple), expression vector (or its multiple), cell or composition for treating or preventing an effective amount.All treatments and prevention methods / purposes described herein can be used for experimental subjects.

[1227] The methods can effectively reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition, or reduce the pathology of a disease / condition. The methods can effectively prevent the progression of a disease / condition, such as preventing the disease / condition from worsening or slowing its progression. In some embodiments, the methods can result in an improvement in a disease / condition, such as alleviating the symptoms of a disease / condition or reducing other factors associated with the severity / activity of the disease / condition. In some embodiments, the methods can prevent the disease / condition from progressing to a later stage (e.g., a chronic stage or metastasis).

[1228] It should be understood that the articles of the present invention can be used to treat / prevent any disease / disorder that will benefit from a reduction in the number and / or activity of cells (e.g., MDSC) expressing VISTA. It will also be clear that the treatment and prevention uses of the present invention are substantially extended to diseases / disorders that will benefit from a reduction in the number or activity of MDSC and / or other cells expressing VISTA, such as tumor-associated macrophages (TAMs) and neutrophils. The antagonism of VISTA can effectively release effector immune cells from the inhibition of MDSC and / or other cells expressing VISTA.

[1229] For example, the disease / disorder can be a disease / disorder that pathologically involves cells expressing VISTA (e.g., MDSCs), such as a disease / disorder in which an increase in the number / ratio of cells expressing VISTA (e.g., MDSCs) is positively correlated with the onset, development or progression of the disease / disorder and / or the severity of one or more symptoms of the disease / disorder, or an increase in the number / ratio of cells expressing VISTA (e.g., MDSCs) is a risk factor for the onset, development or progression of the disease / disorder.

[1230] In some embodiments, the disease / disorder to be treated / prevented according to the present invention is a disease / disorder characterized by an increase in the number / proportion / activity of cells expressing VISTA (e.g., MDSCs), e.g., compared to the number / proportion / activity of cells expressing VISTA (e.g., MDSCs) in the absence of the disease / disorder.

[1231] In some embodiments, the test subjects described herein can be selected based on an increase in the number / ratio / activity of cells (e.g., MDSC) expressing VISTA, such as MDSCs, such as in a peripheral or organ / tissue affected by a disease / disorder (e.g., an organ / tissue exhibiting symptoms of a disease / disorder), or in a tumor where cells expressing VISTA (e.g., MDSCs or tumor-associated macrophages) are present. The disease / disorder may affect any tissue, organ, or organ system. In some embodiments, the disease / disorder can affect several tissues / organs / organ systems.

[1232] In some embodiments, a subject for treatment / prevention according to the present invention can be selected based on determining that the subject has an increase in the number / ratio / activity of cells expressing VISTA (e.g., MDSCs) in the periphery or in an organ / tissue (relative to the number / ratio / activity of such cells in a healthy subject), or based on determining that the subject has a tumor containing cells expressing VISTA (e.g., MDSCs).

[1233] In some embodiments, the disease / condition to be treated / prevented is cancer.

[1234] It will be appreciated that the antigen binding molecules may be used to treat cancer generally, as the antigen binding molecules of the invention may be used to release effector immune cells from MDSC-mediated inhibition or inhibition of VISTA-expressing cells and thereby enhance anti-cancer immune responses.

[1235] The cancer can be any unwanted cell proliferation (or any disease manifested by unwanted cell proliferation), neoplasm or tumor. The cancer can be benign or malignant, primary or secondary (metastatic). A neoplasm or tumor can be any abnormal growth or proliferation of cells and can be located in any tissue. Cancerous tissue / cells can be from, for example, the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain), cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g., renal epithelium), gall bladder, esophagus, glial cells, heart, ileum, jejunum, kidney, lacrimal gland, larynx, liver, lung, lymph, lymph node, lymphoblasts, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intesti...

Claims

1. An optionally isolated antigen-binding molecule, characterized in that The antigen binding molecule is able to bind to VISTA and inhibit VISTA-mediated signal transduction without relying on Fc-mediated function.

2. The antigen-binding molecule according to claim 1, 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 of SEQ ID NO: 290 HC-CDR2 having the amino acid sequence of SEQ ID NO: 291 an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; and (ii) a light chain variable (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 309 LC-CDR3 having the amino acid sequence of SEQ ID NO:

43.

3. The antigen-binding molecule according to claim 1 or 2, 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 of SEQ ID NO: 290 HC-CDR2 having the amino acid sequence of SEQ ID NO: 291 an HC-CDR3 having the amino acid sequence of SEQ ID NO: 278; and (ii) a light chain variable (VL) region comprising the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO:41 LC-CDR2 having the amino acid sequence of SEQ ID NO: 295 LC-CDR3 having the amino acid sequence of SEQ ID NO:

43.

4. The antigen-binding molecule according to any one of claims 1 to 3, characterized in that The antigen binding molecule comprises: A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 289; and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

310.

5. The antigen-binding molecule according to any one of claims 1 to 4, characterized in that The antigen binding molecule comprises: A VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 289; and A VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:

297.

6. The antigen-binding molecule according to any one of claims 1 to 5, characterized in that The antigen binding molecule comprises: A VH region comprising the following framework regions (FRs): HC-FR1 having the amino acid sequence of SEQ ID NO: 63 HC-FR2 having the amino acid sequence of SEQ ID NO: 292 HC-FR3 having the amino acid sequence of SEQ ID NO: 293 HC-FR4 having the amino acid sequence of SEQ ID NO:

281.

7. The antigen-binding molecule according to any one of claims 1 to 6, characterized in that The antigen binding molecule comprises: A VL region comprising the following framework regions (FRs): LC-FR1 having the amino acid sequence of SEQ ID NO: 288 LC-FR2 having the amino acid sequence of SEQ ID NO: 298 LC-FR3 having the amino acid sequence of SEQ ID NO: 284 LC-FR4 having the amino acid sequence of SEQ ID NO:

47.

8. The antigen-binding molecule according to any one of claims 1 to 7, characterized in that The antigen binding molecule includes a heavy chain comprising the amino acid sequence of SEQ ID NO:

331.

9. The antigen-binding molecule according to any one of claims 1 to 8, characterized in that The antigen binding molecule includes a light chain comprising the amino acid sequence of SEQ ID NO:

317. 10 . A composition comprising the antigen-binding molecule according to any one of claims 1 to 9 .

11. The composition according to claim 10, characterized in that The composition comprises: (i) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate-80, pH 4.0 to 7.0; or (ii) 2 mM to 200 mM histidine, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 20, pH 4.0 to 7.0; or (iii) 2 mM to 200 mM histidine, 1 mM to 250 mM sodium chloride, pH 4.0 to 7.0, optionally containing 0.001% to 0.1% (w / v) polysorbate 20 or polysorbate 80; or (iv) 2 mM to 200 mM histidine, 0.001% to 0.1% (w / v) polysorbate 20 or polysorbate 80, pH 4.0 to 7.0; or (v) 2 mM to 200 mM acetate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate-80, pH 4.0 to 7.0; or (vi) 2 mM to 200 mM acetate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate 20, pH 4.0 to 7.0; or (vii) 2 mM to 200 mM succinate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate-80, pH 4.0 to 7.0; or (viii) 2 mM to 200 mM succinate, 2% to 20% (w / v) sucrose, 0.001% to 0.1% (w / v) polysorbate-20, pH 4.0 to 7.

0.

12. The composition according to claim 10 or claim 11, characterized in that The composition comprises: (i) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and having a pH of 5.5; or (ii) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (iii) 20 mM histidine, 4% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (iv) 20 mM histidine, 2% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (v) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80, and having a pH of 6.3; or (vi) 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-20, and having a pH of 5.8; or (vii) 20 mM acetate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and having a pH of 5.5; or (viii) 20 mM succinate, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and having a pH of 5.5; or (ix) 20 mM histidine, 0.02% (w / v) polysorbate-80, and having a pH of 5.8; or (x) 20 mM histidine, 150 mM sodium chloride, and having a pH of 5.

8.

13. The composition according to any one of claims 10 to 12, characterized in that The composition comprises 20 mM histidine, 8% (w / v) sucrose; 0.02% (w / v) polysorbate-80 and has a pH of 5.

5.

14. The composition according to any one of claims 10 to 13, characterized in that The composition comprises about 50 mg / mL of the antigen binding molecule.

15. The antigen-binding molecule or composition according to any one of claims 1 to 14, for use as a medicament.

16. An antigen binding molecule or composition according to any one of claims 1 to 14, for use in a method for treating or preventing cancer in a subject.

17. Use of the antigen binding molecule or composition according to any one of claims 1 to 14 for preparing a medicament for treating or preventing cancer in a subject.

18. A method for treating or preventing cancer in a subject, characterized in that: The method comprises administering a therapeutically or prophylactically effective amount of the antigen binding molecule or composition according to any one of claims 1 to 14.

19. The use of the antigen binding molecule or composition according to claim 16, the use according to claim 17 or the method according to claim 18, characterized in that: The cancer is characterized by the presence of cells expressing VISTA and / or signal transduction mediated by a complex comprising VISTA.

20. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 19, characterized in that: The cancer is selected from the group consisting of hematological cancer, leukemia (e.g., T-cell leukemia), acute myeloid leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, multiple myeloma, mesothelioma, solid tumors, lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, gastric malignancy, colorectal cancer, colorectal tumors, colorectal adenocarcinoma, uterine cancer, endometrial tumors, breast cancer, triple negative breast cancer (TBNC), triple negative breast invasive carcinoma, invasive ductal carcinoma, 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, squamous cell carcinoma of the head and neck (SCCHN), ovarian cancer, ovarian cancer, ovarian serous cystadenocarcinoma, bladder cancer, prostate cancer and / or prostate adenocarcinoma.

21. The antigen binding molecule or composition, use or method according to any one of claims 16 to 20, characterized in that: The cancer is triple negative cancer (TBNC), non-small cell lung cancer (NSCLC) and / or a solid tumor.

22. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 21, characterized in that: The method includes the step of detecting the presence of cells expressing VISTA and / or by signal transduction mediated by a complex comprising VISTA.

23. The antigen binding molecule or composition, use or method according to claim 22, characterized in that: When the presence of cells expressing VISTA and / or signal transduction mediated by a complex comprising VISTA is detected, the subject is selected for treatment with the antigen binding molecule or composition.

24. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 23, characterized in that: The antigen binding molecules are administered weekly, every two weeks, or every three weeks.

25. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 24, characterized in that: The antigen binding molecule is administered once, twice or three times within a 21-day dosing cycle, and optionally the treatment comprises up to 35 dosing cycles.

26. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 25, characterized in that: The antigen binding molecule is administered on days 1, 8 and / or 15 within a 21 day dosing cycle, and optionally the treatment comprises up to 35 dosing cycles.

27. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 26, characterized in that: The treatment comprises administering 3.5 mg to 2200 mg of the antigen-binding molecule each time.

28. The antigen binding molecule or composition, use or method according to any one of claims 16 to 27, characterized in that: The treatment comprises administering at least one of: 3.5 mg, 7 mg, 10.5 mg, 17.5 mg, 20 mg, 21 mg, 40 mg, 60 mg, 72 mg, 120 mg, 180 mg, 240 mg, 360 mg, 400 mg, 800 mg, 1200 mg, 1600 mg, 1900 mg or 2200 mg of the antigen binding molecule per administration.

29. The antigen binding molecule or composition for use, use or method according to any one of claims 16 to 28, characterized in that: The treatment comprises administering up to 10.5 mg, up to 21 mg, up to 31.5 mg, up to 52.5 mg, up to 60 mg, up to 63 mg, up to 120 mg, up to 180 mg, up to 216 mg, up to 360 mg, up to 540 mg, up to 720 mg, up to 1080 mg, up to 1200 mg, up to 2400 mg, up to 3600 mg, up to 4800 mg, up to 5700 mg or up to 6600 mg of the antigen binding molecule per 21 day dosing cycle.

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