WO2023046979A1 - Traitement et prévention du cancer à l'aide de molécules de liaison à l'antigène vista - Google Patents

Traitement et prévention du cancer à l'aide de molécules de liaison à l'antigène vista Download PDF

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WO2023046979A1
WO2023046979A1 PCT/EP2022/076793 EP2022076793W WO2023046979A1 WO 2023046979 A1 WO2023046979 A1 WO 2023046979A1 EP 2022076793 W EP2022076793 W EP 2022076793W WO 2023046979 A1 WO2023046979 A1 WO 2023046979A1
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amino acid
acid sequence
seq
antigen
vista
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Piers INGRAM
Jerome Douglas BOYD-KIRKUP
Dipti THAKKAR
Shalini PALIWAL
Bhushan DHARMADHIKARI
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Hummingbird Bioscience Pte. Ltd.
CLEGG, Richard Ian
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Publication of WO2023046979A1 publication Critical patent/WO2023046979A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present disclosure relates to the fields of molecular biology, more specifically antibody technology and methods of medical treatment and prophylaxis.
  • MDSC Myeloid Derived Suppressor Cell
  • MDSCs exert suppression over T cells through multiple mechanisms, including the production of reactive oxygen species, nitric oxide, and arginase. These ultimately lead to suppression of DC, NK and T cell activity and increased tumor burden (Umansky et al., Vaccines (Basel) (2016) 4(4):36). MDSCs also contribute to the tumor development and metastasis through the production of soluble factors such as matrix metalloproteinases, VEGF, bFGF, TGF-p and S100A8/A9 which promote neovascularisation, invasion, proliferation and metastasis.
  • soluble factors such as matrix metalloproteinases, VEGF, bFGF, TGF-p and S100A8/A9 which promote neovascularisation, invasion, proliferation and metastasis.
  • V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), an immune checkpoint molecule expressed primarily on MDSCs, is an attractive therapeutic strategy for removing MDSC-mediated suppression of effector immune cell function.
  • WO 2017/137830 A1 discloses anti-VISTA antibody VSTB174, which is disclosed at e.g. paragraph [00221] to comprise the variable regions of anti-VISTA antibody VSTB112.
  • Paragraph [00362] discloses that VSTB123 comprises the variable regions of VSTB174.
  • Example 25 of WO 2017/137830 A1 at paragraph [0417] and Figure 42A disclose that mlgG2a antibody VSTB123 was able to inhibit tumor growth in a MB49 tumor model.
  • Paragraph [0418] and Figure 42A disclose that by contrast VSTB124 - which is the same antibody provided in lgG2a LALA format; see paragraph [0408] - did not inhibit tumor growth.
  • Example 25 concludes at paragraph [0419] that efficacy with anti-VISTA antibody treatment might require active Fc. Accordingly, the proposed mechanism of action for the anti- VISTA antibody represented schematically at Figure 47 (see the legend to Figure 47 at paragraph [0053]) involves Fc-mediated engagement of FcyRIII expressed by NK cells.
  • Hamster monoclonal anti-VISTA antibody mAb13F3 is disclosed in Le Mercier et al. Cancer Res. (2014) 74(7):1933-44 to inhibit tumor growth in B16OVA and B16-BL6 melanoma models. Page 1942, paragraph spanning left and right columns teaches that immunogenicity and the FcR binding activity of the VISTA mAb might be critical limiting factors for achieving optimal target neutralization and therapeutic efficacy.
  • VISTA-binding antibodies are also disclosed e.g. in WO 2019/185879 A1 .
  • the present disclosure provides an antigen-binding molecule that binds to VISTA for use in a method of treating or preventing a cancer in a subject, wherein the cancer is characterised by the presence of cells expressing an interaction partner for VISTA (e.g. an interaction partner for VISTA that binds to the C-C’ region of VISTA, e.g. LRIG1 or VSIG3), wherein the antigen-binding molecule inhibits interaction between VISTA and the interaction partner for VISTA.
  • an interaction partner for VISTA e.g. an interaction partner for VISTA that binds to the C-C’ region of VISTA, e.g. LRIG1 or VSIG3
  • an antigen-binding molecule that binds to VISTA in the manufacture of a medicament for treating or preventing a cancer in a subject, wherein the cancer is characterised by the presence of cells expressing an interaction partner for VISTA (e.g. an interaction partner for VISTA that binds to the C-C’ region of VISTA, e.g. LRIG1 or VSIG3), wherein the antigen-binding molecule inhibits interaction between VISTA and the interaction partner for VISTA.
  • an interaction partner for VISTA e.g. an interaction partner for VISTA that binds to the C-C’ region of VISTA, e.g. LRIG1 or VSIG3
  • Also provided is a method of treating or preventing a cancer in a subject comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule that binds to VISTA, wherein the cancer is characterised by the presence of cells expressing an interaction partner for VISTA (e.g. an interaction partner for VISTA that binds to the C-C’ region of VISTA, e.g. LRIG1 or VSIG3), wherein the antigen-binding molecule inhibits interaction between VISTA and the interaction partner for VISTA.
  • an interaction partner for VISTA e.g. an interaction partner for VISTA that binds to the C-C’ region of VISTA, e.g. LRIG1 or VSIG3
  • an antigen-binding molecule that binds to VISTA for use in a method of treating or preventing a cancer in a subject, wherein the cancer is characterised by the presence of: (i) cells expressing VISTA, and (ii) cells expressing an interaction partner for VISTA that binds to the C-C’ region of VISTA; and wherein the antigen-binding molecule inhibits interaction between VISTA and the interaction partner for VISTA.
  • an antigen-binding molecule that binds to VISTA in the manufacture of a medicament for treating or preventing a cancer in a subject, wherein the cancer is characterised by the presence of: (i) cells expressing VISTA, and (ii) cells expressing an interaction partner for VISTA that binds to the C-C’ region of VISTA; and wherein the antigen-binding molecule inhibits interaction between VISTA and the interaction partner for VISTA.
  • the method comprises administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule that binds to VISTA, wherein the cancer is characterised by the presence of: (i) cells expressing VISTA, and (ii) cells expressing an interaction partner for VISTA that binds to the C-C’ region of VISTA; and wherein the antigen-binding molecule inhibits interaction between VISTA and the interaction partner for VISTA.
  • the cancer comprises a tumor comprising: (i) cells expressing VISTA, and (ii) cells expressing an interaction partner for VISTA.
  • step (b) selecting a subject for treatment with an antigen-binding molecule that inhibits interaction between VISTA and an interaction partner for VISTA that binds to the C-C’ region of VISTA where the subject’s cancer is determined in step (a) to comprise cells expressing VISTA, and cells expressing an interaction partner for VISTA that binds to the C-C’ region of VISTA.
  • the method further comprises:
  • step (c) administering an antigen-binding molecule that inhibits interaction between VISTA and an interaction partner for VISTA that binds to the C-C’ region of VISTA to a subject selected for treatment in step (b).
  • the interaction partner for VISTA that binds to the C-C’ region of VISTA is selected from LRIG1 and VSIG3.
  • the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:340.
  • the antigen-binding molecule comprises:
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:305
  • HC-CDR2 having the amino acid sequence of SEQ ID NQ:306
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:307;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:41
  • LC-CDR2 having the amino acid sequence of SEQ ID NQ:308
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
  • the antigen-binding molecule comprises:
  • HC-CDR1 having the amino acid sequence of SEQ ID NQ:290
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:291
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278; and (ii) a VL region incorporating 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.
  • 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.
  • the antigen-binding molecule comprises: a VH region incorporating 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.
  • FRs framework regions
  • the antigen-binding molecule comprises: a VL region incorporating 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.
  • the antigen-binding molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:331 .
  • the antigen-binding molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO:317.
  • the cancer is selected from: a hematological cancer, leukemia, acute myeloid leukemia, lymphoma, B cell lymphoma, T cell lymphoma, multiple myeloma, mesothelioma, a solid tumor, lung cancer, non-small cell lung carcinoma, gastric cancer, gastric carcinoma, colorectal cancer, colorectal carcinoma, colorectal adenocarcinoma, uterine cancer, uterine corpus endometrial carcinoma, breast cancer, triple negative breast cancer, triple negative breast invasive 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 carcinoma, ovarian serous
  • the cancer is selected from: colorectal cancer, pancreatic cancer, breast cancer, triple-negative breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, leukemia, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC) and a solid tumor.
  • NSCLC non-small cell lung cancer
  • the present disclosure relates to VISTA-binding molecules that bind to particular regions of interest in the extracellular domain of VISTA.
  • aspects and embodiments of the present disclosure are concerned in particular with VISTA-binding antigen-binding molecules that bind to VISTA and inhibit interaction with an interaction partner for VISTA, in particular interaction partners for VISTA that bind to the C-C’ region of VISTA (e.g. LRIG1 and VSIG3).
  • Such antigen-binding molecules are useful for inhibiting downstream signalling by complexes comprising VISTA and such interaction partners, and are therefore particularly useful for the treatment/prevention of cancers characterised by the presence of cells expressing VISTA and cells expressing such interaction partners.
  • V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA; also known e.g. as B7- H5, SISP1 , PD-1 H) is the protein identified by UniProt Q9H7M9, having the amino acid seguence shown in SEQ ID NO:1 (Q9H7M9-1 , v3).
  • the structure and function of VISTA is described e.g. in Lines et al., Cancer Res. (2014) 74(7): 1924-1932, which is hereby incorporated by reference in its entirety.
  • VISTA is a ⁇ 50 kDa single-pass type I transmembrane that functions as an immune checkpoint and is encoded by the C10orf54 gene.
  • the extracellular domain of VISTA is homologous to PD-L1.
  • the N-terminal 32 amino acids of SEQ ID NO:1 constitutes a signal peptide, and so the mature form of VISTA (/.e. after processing to remove the signal peptide) has the amino acid seguence 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 a transmembrane domain (SEQ ID NO:4)
  • positions 216 to 311 form the cytoplasmic domain (SEQ ID NO:5).
  • the extracellular domain comprises an Ig-like V-type domain (positions 33 to 168 of SEQ ID NO:1 , shown in SEQ ID NO:6).
  • VISTA refers to VISTA from any species and includes VISTA isoforms, fragments, variants (including mutants) or homologues from any species.
  • a “fragment”, “variant” or “homologue” of a protein may optionally be characterised 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 seguence identity to the amino acid seguence of the reference protein (e.g. a reference isoform).
  • fragments, variants, isoforms and homologues of a reference protein may be characterised by ability to perform a function performed by the reference protein.
  • a “fragment” generally refers to a fraction of the reference protein.
  • a “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 the reference protein, but retaining a considerable degree of sequence identity (e.g. at least 60%) to the amino acid sequence of the reference protein.
  • An “isoform” generally refers to a variant of the reference protein expressed by the same species as the species of the reference protein.
  • a “homologue” generally refers to a variant of the reference protein produced by a different species as compared to the species of the reference protein. Homologues include orthologues.
  • a “fragment” may be of any length (by number of amino acids), although may optionally be at least 20% of the length of the reference protein (that is, the protein from which the fragment is derived) and may 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 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250 or 300 amino acids.
  • the VISTA is VISTA from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and/or a rodent (e.g. rat or murine) VISTA).
  • Isoforms, fragments, variants or homologues of VISTA may optionally be characterised 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.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference VISTA, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of VISTA may e.g. display association with LRIG1 , VSIG3, PSGL-1 and/or VSIG8.
  • the 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.
  • 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 NOs:2, 3 or 6.
  • VISTA is a member of the B7 family of proteins, and is primarily expressed by leukocytes, and in particular CD14+ monocytes (including monocyte-derived suppressor cells (MDSCs)) and CD33+ myeloid cells. VISTA is also expressed by CD56+ NK cells, dendritic cells, and to a lesser extent on CD4+ and CD8+ T cells. VISTA is highly expressed on MDSCs, in particular tumor-infiltrating MDSCs, and also on tumor-infiltrating myeloid DCs (Le Mercier et al, Cancer Res. (2014) 74(7):1933-44), as well as on tumor- associated macrophages (TAMs) and neutrophils.
  • MDSCs including monocyte-derived suppressor cells (MDSCs)
  • VISTA tumor-associated macrophages
  • VISTA can act as both a ligand and a receptor on T cells to inhibit T cell effector function and maintain peripheral tolerance; tumors engineered to overexpress VISTA evade immune control and grow faster than tumors which do not overexpress VISTA (Wang et al., Journal of Experimental Medicine. (201 1) 208 (3): 577-92; Lines et al., Cancer Res. (2014) 74(7): 1924-1932).
  • VISTA has been shown to be a co-inhibitory receptor on CD4+ T cells or a co-inhibitory ligand for T cells.
  • VISTA -7 CD4+ T cells have been reported to display stronger antigen-specific proliferation and cytokine production than wildtype CD4+ T cells, suggesting that VISTA functions as an inhibitory receptor on CD4+ T cells.
  • Blocking VISTA function using monoclonal anti-VISTA antibody has been shown to enhance infiltration, proliferation and effector function of tumor-reactive T cells within the tumor microenvironment (Le Mercier et al, Cancer Res. (2014) 74(7): 1933-4).
  • VISTA has been proposed to interact with VSIG3 (IGSF11) - see e.g. Wang et al., J Immunol (2017), 198 (1 Supplement) 154.1 , which is hereby incorporated by reference in its entirety. Engagement of VSIG3 through VISTA on activated T cells inhibits T cell proliferation, and reduces production of cytokines and chemokines such as IFN-y, IL-2, IL-17, CCL5/RANTES, CCL3/MIP-1 a, and CXCL11/l-TAC.
  • cytokines and chemokines such as IFN-y, IL-2, IL-17, CCL5/RANTES, CCL3/MIP-1 a, and CXCL11/l-TAC.
  • VSIG3 is the protein identified by UniProt Q5DX21 .
  • Alternative splicing of mRNA encoded by the human IGSF1 1 gene yields three different isoforms: isoform 1 (UniProt: Q5DX21-1 , v3; SEQ ID NO:7); isoform 2 (UniProt: Q5DX21-2; SEQ ID NO:8), which comprises a different sequence to SEQ ID NOT at positions 1 to 17; and isoform 3 (UniProt: Q5DX21-3; SEQ ID NO:9), which comprises a different sequence to SEQ ID NOT at positions 1 to 17, and which also comprises a different sequence to SEQ ID NOT at positions 211-235.
  • the N-terminal 22 amino acids of SEQ ID NOsT, 8 and 9 constitute a signal peptide, and so the mature form of VSIG3 isoforms 1 , 2 and 3 (/.e. after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs:10, 11 and 12, respectively.
  • Positions 23 to 241 of SEQ ID NOsT, and 8 form the extracellular domain of VSIG3 isoforms 1 and 2 (SEQ ID NO:13), and positions 23 to 216 of SEQ ID NO:9 form the extracellular domain of VSIG3 isoform 3 (SEQ ID NO:14).
  • the transmembrane domain of VSIG3 is shown in SEQ ID NO:15, and the cytoplasmic domain is shown in SEQ ID NO:16.
  • the extracellular domain comprises an Ig-like V-type domain (shown in SEQ ID NO:17), and the extracellular domains of VSIG3 isoforms 1 and 2 additionally comprise an Ig-like C2-type domain (shown in SEQ ID NO:18).
  • VSIG3 refers to VSIG3 from any species and includes VSIG3 isoforms, fragments, variants (including mutants) or homologues from any species.
  • a fragment of VSIG3 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids.
  • the VSIG3 is VSIG3 from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and/or a rodent (e.g. rat or murine) VSIG3).
  • Isoforms, fragments, variants or homologues of VSIG3 may optionally be characterised 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 VSIG3 isoform from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference VSIG3, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of VSIG3 may e.g. display association with VISTA.
  • the VSIG3 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.
  • a fragment of VSIG3 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 NQs:10 to 14, 17 or 18.
  • VISTA has also been proposed to interact with VSIG8 - see e.g. WO/2017/090347 A1 .
  • VSIG8 is the protein identified by UniProt P0DPA2 (SEQ ID NO:19).
  • the N-terminal 21 amino acids of SEQ ID NO:19 constitutes a signal peptide, and so the mature form of VSIG8 (/.e. after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NQ:20.
  • Positions 22 to 263 of SEQ ID NO:19 form the extracellular domain of VSIG8 (SEQ ID NO:21).
  • the transmembrane domain of VSIG8 is shown in SEQ ID NO:22, and the cytoplasmic domain is shown in SEQ ID NO:23.
  • the extracellular domain comprises an Ig-like V-type domain 1 (shown in SEQ ID NO:24), and an Ig-like V-type domain 2 (shown in SEQ ID NO:25).
  • VSIG8 refers to VSIG8 from any species and includes VSIG8 isoforms, fragments, variants (including mutants) or homologues from any species.
  • a fragment of VSIG8 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids.
  • the VSIG8 is VSIG8 from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and/or a rodent (e.g. rat or murine) VSIG8).
  • Isoforms, fragments, variants or homologues of VSIG8 may optionally be characterised 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 VSIG8 isoform from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference VSIG8, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of VSIG8 may e.g. display association with VISTA.
  • the VSIG8 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:19 or 20.
  • a fragment of VSIG8 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:20, 21 , 24 or 25.
  • VISTA has also been proposed to interact with PSGL-1 - see e.g. WO 2018/132476 A1 .
  • Johnston et al. Nature (2019) 574: 565-570 discloses that PSGL-1 associates with VISTA via interaction involving positions Y46, Y48, Y51 , E56 and T57 of PSGL-1 , and positions H98, H100, H153, H154 and H155 of VISTA.
  • PSGL-1 isoform 1 is the protein identified by UniProt Q14242-1 (SEQ ID NO:323).
  • PSGL-1 isoform 2 is the protein identified by UniProt Q14242-2 (SEQ ID NO:324), and differs from PSGL-1 isoform 1 in that it comprises an additional 16 amino acids after position 1 of SEQ ID NO:323.
  • SEQ ID NO:323 The N-terminal 17 amino acids of SEQ ID NO:323 constitutes a signal peptide, and so the mature form of PSGL-1 (/.e. after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO:325. Positions 18 to 320 of SEQ ID NO:323 form the extracellular domain of PSGL-1 (SEQ ID NO:326).
  • the transmembrane domain of PSGL-1 is shown in SEQ ID NO:327, and the cytoplasmic domain is shown in SEQ ID NO:328.
  • the extracellular domain comprises 12, 10 amino acid tandem repeats; the repeat region is shown in SEQ ID NO:329.
  • PSGL-1 refers to PSGL-1 from any species and includes PSGL-1 isoforms, fragments, variants (including mutants) or homologues from any species.
  • a fragment of PSGL-1 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350 or 400 amino acids.
  • the PSGL-1 is PSGL-1 from a mammal (e.g. a primate (rhesus, cynomolgous, non-human primate or human) and/or a rodent (e.g. rat or murine) PSGL-1).
  • Isoforms, fragments, variants or homologues of PSGL-1 may optionally be characterised 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.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference PSGL-1 , as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of PSGL-1 may e.g. display association with VISTA.
  • the PSGL-1 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:323 or 324.
  • a fragment of PSGL-1 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:325, 326 or 329.
  • VISTA has also been proposed to interact with LRIG1 - see e.g. WO/2019/165233 A1 .
  • WO/2019/165233 A1 discloses that LRIG1 associates with VISTA via interaction involving positions 245 to 260 of LRIG1 , and positions 68 to 92 of VISTA.
  • LRIG1 isoform 1 is the protein identified by UniProt Q96JA1-1 (SEQ ID NO:332).
  • LRIG1 isoform 2 is the protein identified by UniProt Q96JA1-2 (SEQ ID NO:334), and differs from LRIG1 isoform 1 in that it comprises an additional 14 amino acids after position 387 of SEQ ID NO:332, and in that positions 644 to 691 of SEQ ID NO:332 are instead Q.
  • the N-terminal 34 amino acids of SEQ ID NO:332 constitutes a signal peptide, and so the mature form of LRIG1 isoforms 1 and 2 (/.e. after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs:333 and 335, respectively.
  • the extracellular domain of LRIG1 isoform 1 is shown in SEQ ID NO:336, and the extracellular domain of LRIG1 isoform 2 is shown in SEQ ID NO:337.
  • the transmembrane domain of LRIG1 is shown in SEQ ID NO:338, and the cytoplasmic domain is shown in SEQ ID NO:339.
  • the extracellular domain comprises 15, leucine-rich repeats, followed by three Ig-like domains proximal to the transmembrane domain (see e.g. Xu et al. J Mol Biol. (2015) 427(10): 1934-1948).
  • LRIG1 refers to LRIG1 from any species and includes LRIG1 isoforms, fragments, variants (including mutants) or homologues from any species.
  • a fragment of LRIG1 may have a minimum length of one of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600 or 700 amino acids, and may have a maximum length of one of 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600 or 700 amino acids.
  • the LRIG1 is LRIG1 from a mammal (e.g. a primate (rhesus, cynomolgus, nonhuman primate or human) and/or a rodent (e.g. rat or murine) LRIG1).
  • Isoforms, fragments, variants or homologues of LRIG1 may optionally be characterised 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 LRIG1 isoform from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference LRIG1 , as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of LRIG1 may e.g. display association with VISTA.
  • the LRIG1 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:332, 333, 334 or 335.
  • a fragment of LRIG1 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:336 or 337.
  • VSIG3 and LRIG1 are both thought to bind to VISTA via interaction with the C-C’ loop region of VISTA, the amino acid sequence of which is shown in SEQ ID NO:344.
  • the antigen-binding molecules of the present disclosure were specifically designed to target regions of VISTA of particular interest.
  • VISTA regions to be targeted were selected following analysis for predicted antigenicity, function and safety.
  • Antibodies specific for the target regions of VISTA were then prepared using peptides corresponding to the target regions as immunogens to raise specific monoclonal antibodies, and subsequent screening to identify antibodies capable of binding to VISTA in the native state. This approach provides extraordinar control over the antibody epitope.
  • the antigen-binding molecules of the present disclosure may be defined by reference to the region of VISTA which they bind to.
  • the antigen-binding molecules of the present disclosure may bind to a particular region of interest of VISTA.
  • the antigen-binding molecule may bind to a linear epitope of VISTA, consisting of a contiguous sequence of amino acids (/.e. an amino acid primary sequence).
  • the antigen-binding molecule may bind to a conformational epitope of VISTA, consisting of a discontinuous sequence of amino acids of the amino acid sequence.
  • the antigen-binding molecule of the present disclosure binds to VISTA. In some embodiments, the antigen-binding molecule binds to the extracellular region of VISTA (e.g. the region shown in SEQ ID NO:3). In some embodiments, the antigen-binding molecule binds to the Ig-like V-type domain of VISTA (e.g. the region shown in SEQ ID NO:6). In some embodiments, the antigen-binding molecule binds to VISTA in the region corresponding to positions 61 to 162 of SEQ ID NO:1 (shown in SEQ ID NO:31).
  • the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:322. In some embodiments, the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:26. In some embodiments, the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:27. In some embodiments, the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:28. In some embodiments, the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:29. In some embodiments, the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:30.
  • the antigen-binding molecule does not bind to the region of VISTA shown in SEQ ID NO:271. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA shown in SEQ ID NO:272. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA shown in SEQ ID NO:273. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA shown in SEQ ID NO:274. In some embodiments, the antigen-binding molecule does not bind to the region of VISTA shown in SEQ ID NO:275.
  • the region of a peptide/polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibodyantigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety.
  • the region of a peptide/polypeptide to which an antigen-binding molecule according the present disclosure binds is evaluated by hydrogen-deuterium exchange mass spectrometry (HDXMS) analysis, e.g. as described in the experimental examples of the present disclosure.
  • HDXMS hydrogen-deuterium exchange mass spectrometry
  • the antigen-binding molecule binds to the C-C’ region of VISTA. In some embodiments, the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:344. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:344. In some embodiments, the antigen-binding molecule contacts the region of VISTA shown in SEQ ID NO:344. In some embodiments, the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:344. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:344.
  • the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NQ:340. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NQ:340. In some embodiments, the antigenbinding molecule contacts the region of VISTA shown in SEQ ID NQ:340. In some embodiments, the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NQ:340. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NQ:340.
  • the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:341. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:341. In some embodiments, the antigenbinding molecule contacts the region of VISTA shown in SEQ ID NO:341 . In some embodiments, the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:341. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:341 .
  • the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:342. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:342. In some embodiments, the antigenbinding molecule contacts the region of VISTA shown in SEQ ID NO:342. In some embodiments, the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:342. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:342.
  • the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:341 , and/or binds to the region of VISTA shown in SEQ ID NO:342. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:341 and/or binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:342. In some embodiments, the antigen-binding molecule contacts the region of VISTA shown in SEQ ID NO:341 and/or contacts the region of VISTA shown in SEQ ID NO:342.
  • the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:341 and/or binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:342.
  • the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:341 , and/or the amino acid sequence shown in SEQ ID NO:342.
  • the antigen-binding molecule binds to the region of VISTA bound by an interaction partner for VISTA that binds to C-C’ region of VISTA (e.g. LRIG1 or VSIG3). In some embodiments, the antigen-binding molecule binds to the region of VISTA bound by LRIG1 . In some embodiments, the antigen-binding molecule binds to the region of VISTA bound by VSIG3.
  • the antigen-binding molecule binds to the region of VISTA shown in SEQ ID NO:343. In some embodiments, the antigen-binding molecule binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:343. In some embodiments, the antigenbinding molecule contacts the region of VISTA shown in SEQ ID NO:343. In some embodiments, the antigen-binding molecule binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:343. In some embodiments, the epitope of the antigen-binding molecule comprises or consists of the amino acid sequence shown in SEQ ID NO:343.
  • the antigen-binding molecule is capable of binding the same region of VISTA, or an overlapping region of VISTA, to the region of VISTA which is bound by an antibody comprising the VH and VL sequences of one of 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, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A11 or 9M2-C12 described herein.
  • the antigen-binding molecule is capable of binding the same region of VISTA, or an overlapping region of VISTA, to the region of VISTA which is bound by an antibody comprising the VH and VL sequences of one of antibody clones 4M2-C12, V4H1 , V4H2, V4-C1 , V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4- C30, V4-C31.
  • the antigen-binding molecule is capable of binding the same region of VISTA, or an overlapping region of VISTA, to the region of VISTA which is bound by an antibody comprising the VH and VL sequences of V4-C26.
  • a “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds.
  • a peptide typically has a length in the region of about 2 to 50 amino acids.
  • a “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.
  • the antigen-binding molecule of the present disclosure is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of one of SEQ ID NOs:1 , 2, 3, 6 or 31 .
  • 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.
  • 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 NQ:30.
  • the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:271. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:272. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:273. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:274. In some embodiments, the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence of SEQ ID NO:275.
  • an antigen-binding molecule to bind to a given peptide/polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442) or Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507).
  • ELISA immunoblot
  • SPR Surface Plasmon Resonance
  • Bio-Layer Interferometry see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507.
  • the peptide/polypeptide may comprise one or more additional amino acids at one or both ends of the reference amino acid sequence.
  • the peptide/polypeptide comprises e.g. 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, IQ- 20, 10-30, 10-40, 10-50, 20-30, 20-40 or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.
  • the additional amino acid(s) provided at one or both ends (/.e. the N-terminal and C-terminal ends) of the reference sequence correspond to the positions at the ends of the reference sequence in the context of the amino acid sequence of VISTA.
  • the additional two amino acids may be arginine and asparagine, corresponding to positions 90 and 91 of SEQ ID NO:1.
  • the antigen-binding molecule is capable of binding to a peptide/polypeptide which is bound by an antibody comprising the VH and VL sequences of one of 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, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A11 or 9M2-C12 described herein.
  • MDSCs Myeloid-Derived Suppressor Cells
  • MDSCs Myeloid-Derived Suppressor Cells
  • MDSC are characterised by a number of biochemical and genomic features that distinguish these cells from mature myeloid cells (/.e. macrophages, dendritic cells and neutrophils) such as: increased expression of NADPH oxidase (Nox2), increased production of reactive oxygen species (ROS) (such as superoxide anion (O 2 ‘), hydrogen peroxide (H2O2), and peroxynitrite (PNT; ONOO’)); increased expression of arginase 1 and nitric oxide synthase 2 (nos2), and increased production of nitric oxide (NO); increased expression of c/EBPp and STAT3; decreased expression of IRF8; and increased production of S100A8/9 proteins.
  • ROS reactive oxygen species
  • MDSC polymorphonuclear MDSCs
  • PMN-MDSCs polymorphonuclear MDSCs
  • M-MDSCs monocytic MDSCs
  • the morphologic and phenotypic characteristics of MDSCs are described e.g. in Marvel and Gabrilovich J Clin Invest. 2015 Sep 1 ; 125(9): 3356-3364, which is hereby incorporated by reference in its entirety.
  • MDSCs are broadly identified as CD11 b + Gr1 + cells.
  • Gr-1 hi cells are mostly PMN-MDSCs
  • Gr-1 10 cells are mostly M-MDSCs.
  • M-MDSCs are CD11 b + Ly6C hi Ly6G
  • PMN-MDSCs are CD11 b + Ly6C l0 Ly6G +
  • MDSCs are identified in the mononuclear fraction.
  • PMN-MDSCs are CD14 _ CD11 b + CD33 + CD15 + or CD66b + cells
  • M-MDSCs are CD14 + HLA-DR7 10 cells.
  • Populations of Lin _ HLA-DR _ CD33 + MDSCs represent a mixed group of cells enriched for myeloid progenitors.
  • M-MDSCs and PMN-MDSCs employ different mechanisms of immune suppression.
  • M-MDSCs suppress both antigen-specific and non-specific T cell responses through production of NO and cytokines, and are more strongly immunosuppressive than PMN-MDSCs.
  • PMN-MDSCs suppress immune responses in an antigen-specific manner through production of ROS.
  • MDSCs are pathologically implicated in the development and progression of cancer and infectious disease.
  • MDSCs are abundant in tumor tissues, and contribute to the development and progression of cancer through multiple mechanisms, reviewed e.g. in Umansky et al., Vaccines (Basel) (2016) 4(4):36. MDSCs are recruited to the tumor site through chemokine expression, and proinflammatory factors in the tumor microenvironment result in significant upregulation of immunosuppressive function by MDSCs. MDSCs contribute to tumor development, neovascularization and metastasis through suppression of effector immune cell function (e.g. effector T cell and NK cell function), promotion of regulatory T cell production/activity, production of growth factors such as VEGF and bFGF, and production of ECM- modifying factors such as matrix metalloproteinases.
  • effector immune cell function e.g. effector T cell and NK cell function
  • ECM- modifying factors such as matrix metalloproteinases.
  • MDSCs may be characterised by reference to expression of VISTA.
  • the MDSCs may be “VISTA-expressing MDSCs” or “VISTA+ MDSCs”.
  • the MDSCs may express VISTA at the cell surface (/.e. VISTA may be expressed in or at the cell membrane).
  • the present disclosure relates to the therapeutic and prophylactic use of antigen-binding molecules which bind to VISTA.
  • an “antigen-binding molecule” refers to a molecule which is capable of binding to a target antigen.
  • Antigen-binding molecules include e.g. 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, minibodies, single domain antibodies (e.g. VhH), etc.), as long as they display binding to the relevant target molecule(s).
  • Antigen-binding molecules also include antibody-derived molecules, e.g. molecules comprising an antigen-binding region/domain derived from an antibody.
  • Antibody-derived antigen-binding molecules may comprise an antigen-binding region/domain that comprises, or consists of, the antigen-binding region of an antibody (e.g. an antigen-binding fragment of an antibody).
  • the antigen-binding region/domain of an antibody-derived antigen-binding molecule may be or comprise the Fv (e.g. provided as an scFv) or the Fab region of an antibody, or the whole antibody.
  • antigen-binding molecules according to the present disclosure include antibody-drug conjugates (ADCs) comprising a (cytotoxic) drug moiety (e.g. as described hereinbelow).
  • ADCs antibody-drug conjugates
  • Antigen-binding molecules according to the present disclosure also include multispecific antigen-binding molecules such as immune cell engager molecules comprising a domain for recruiting (effector) immune cells (reviewed e.g. in Goebeler and Bargou, Nat. Rev. Clin. Oncol. (2020) 17: 418-434 and Ellerman, Methods (2019) 154:102-117, both of which are hereby incorporated by reference in their entirety), including BiTEs, BiKEs and TriKEs.
  • Antigen-binding molecules according to the present disclosure also include chimeric antigen receptors (CARs), which are recombinant receptors providing both antigen-binding and T cell activating functions (CAR structure, function and engineering is reviewed e.g. in Dotti et al., Immunol Rev (2014) 257(1), which is hereby incorporated by reference in its entirety).
  • CARs chimeric antigen receptors
  • the antigen-binding molecule of the present disclosure comprises a moiety capable of binding to a target antigen(s).
  • the moiety capable of binding to a target antigen comprises an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) of an antibody capable of specific binding to the target antigen.
  • the moiety capable of binding to a target antigen comprises or consists of an aptamer capable of binding to the target antigen, e.g. a nucleic acid aptamer (reviewed, for example, in Zhou and Rossi Nat Rev Drug Discov. 2017 16(3):1 SI- 202).
  • the moiety capable of binding to a target antigen comprises or consists of a antigen-binding peptide/polypeptide, e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (/.e. a single-domain antibody (sdAb)) affilin, armadillo repeat protein (ArmRP), OBody or fibronectin - reviewed e.g. in Reverdatto et al., Curr Top Med Chem.
  • a antigen-binding peptide/polypeptide e.g. a peptide aptamer, thioredoxin, monobody, anticalin, Kunitz domain, avimer, knottin, fynomer, atrimer, DARPin, affibody, nanobody (/.e. a single-domain
  • the antigen-binding molecules of the present disclosure generally comprise an antigen-binding domain comprising a VH and a VL of an antibody capable of specific binding to the target antigen.
  • the antigenbinding domain formed by a VH and a VL may also be referred to herein as an Fv region.
  • An antigen-binding molecule may be, or may comprise, an antigen-binding polypeptide, or an antigenbinding polypeptide complex.
  • An antigen-binding molecule may comprise more than one polypeptide which together form an antigen-binding domain.
  • the polypeptides may associate covalently or non- covalently.
  • the polypeptides form part of a larger polypeptide comprising the polypeptides (e.g. in the case of scFv comprising VH and VL, or in the case of scFab comprising VH-CH1 and VL-CL).
  • An antigen-binding molecule may refer to a non-covalent or covalent complex of more than one polypeptide (e.g. 2, 3, 4, 6, or 8 polypeptides), e.g. an IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.
  • the antigen-binding molecules of the present disclosure may be designed and prepared using the sequences of monoclonal antibodies (mAbs) capable of binding to VISTA.
  • mAbs monoclonal antibodies
  • Antigen-binding regions of antibodies, such as single chain variable fragment (scFv), Fab and F(ab’)2 fragments may also be used/provided.
  • An “antigen-binding region” is any fragment of an antibody which is capable of binding to the target for which the given antibody is specific.
  • Antibodies generally comprise 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.
  • the six CDRs together define the paratope of the antibody, which is the part of the antibody which binds to the target antigen.
  • VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs.
  • FRs framework regions
  • VH regions comprise the following structure: N term-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]- [LC-CDR3]-[LC-FR4]-C term.
  • the CDRs and FRs of the VH regions and VL regions of the antibody clones described herein were defined according to the international IMGT (ImMunoGeneTics) information system (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.
  • the antigen-binding molecule comprises the CDRs of an antigen-binding molecule which is capable of binding to VISTA. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule which is capable of binding to VISTA. In some embodiments, the antigen-binding molecule comprises the CDRs and the FRs of an antigen-binding molecule which is capable of binding to VISTA. That is, in some embodiments, the antigen-binding molecule comprises the VH region and the VL region of an antigen-binding molecule which is capable of binding to VISTA.
  • the antigen-binding molecule comprises a VH region and a VL region which is, or which is derived from, the VH/VL region of a VISTA-binding antibody clone described herein (/.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, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A11 or 9M2-C12).
  • the antigen-binding molecule comprises a VH region according to one of (1) to (18) below: (1) (4M2-C12 derived consensus) a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:305
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:306
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:307, or a variant thereof in which 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.
  • V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NQ:290
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:291
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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.
  • V4-C1 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:277
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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.
  • V4-C9 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:286
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:244
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:34
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:35, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:34
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:35, or a variant thereof in which 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.
  • V4H1 a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:53
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:34
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:35, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:73
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:74, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:88
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:89
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:90, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NQ:107
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:108, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NQ:120
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:121
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:122, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:144
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:145
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:146, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:158
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:159
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:160, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:169
  • HC-CDR2 having the amino acid sequence of SEQ ID NQ:170
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:171 , or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:184
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:246, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:184
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:185, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:184
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:195, or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:184
  • the antigen-binding molecule comprises a VH region according to one of (19) to (35) below:
  • V4-C24, V4-C26, V4-C27, V4-C28, V4-C30, V4-C31 a VH region incorporating the following 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 , or a variant thereof in which 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.
  • V4-C1 , V4-C9 a VH region incorporating the following FRs:
  • HC-FR1 having the amino acid sequence of SEQ ID NO:63
  • HC-FR2 having the amino acid sequence of SEQ ID NO:279
  • HC-FR3 having the amino acid sequence of SEQ ID NQ:280
  • HC-FR4 having the amino acid sequence of SEQ ID NO:281 , or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:36
  • HC-FR2 having the amino acid sequence of SEQ ID NO:37
  • HC-FR3 having the amino acid sequence of SEQ ID NO:38
  • HC-FR4 having the amino acid sequence of SEQ ID NO:39, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:49
  • HC-FR2 having the amino acid sequence of SEQ ID NO:37
  • HC-FR3 having the amino acid sequence of SEQ ID NO:38
  • HC-FR4 having the amino acid sequence of SEQ ID NO:39, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:54
  • HC-FR2 having the amino acid sequence of SEQ ID NO:55
  • HC-FR3 having the amino acid sequence of SEQ ID NO:56
  • HC-FR4 having the amino acid sequence of SEQ ID NO:39, or a variant thereof in which 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.
  • V4H2 a VH region incorporating the following FRs:
  • HC-FR1 having the amino acid sequence of SEQ ID NO:63
  • HC-FR2 having the amino acid sequence of SEQ ID NO:64
  • HC-FR3 having the amino acid sequence of SEQ ID NO:65
  • HC-FR4 having the amino acid sequence of SEQ ID NO:39, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:75
  • HC-FR2 having the amino acid sequence of SEQ ID NO:76
  • HC-FR3 having the amino acid sequence of SEQ ID NO:77
  • HC-FR4 having the amino acid sequence of SEQ ID NO:78, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:91
  • HC-FR2 having the amino acid sequence of SEQ ID NO:92
  • HC-FR3 having the amino acid sequence of SEQ ID NO:93
  • HC-FR4 having the amino acid sequence of SEQ ID NO:94, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NQ:103
  • HC-FR2 having the amino acid sequence of SEQ ID NO:76
  • HC-FR3 having the amino acid sequence of SEQ ID NO:77
  • HC-FR4 having the amino acid sequence of SEQ ID NO:78, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NQ:109
  • HC-FR2 having the amino acid sequence of SEQ ID NQ:110
  • HC-FR3 having the amino acid sequence of SEQ ID NO:111
  • HC-FR4 having the amino acid sequence of SEQ ID NO:1 12, or a variant thereof in which 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.
  • HC-FR2 having the amino acid sequence of SEQ ID NO:124
  • HC-FR3 having the amino acid sequence of SEQ ID NO:125
  • HC-FR4 having the amino acid sequence of SEQ ID NO:78, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:134
  • HC-FR2 having the amino acid sequence of SEQ ID NO:92
  • HC-FR3 having the amino acid sequence of SEQ ID NO:93
  • HC-FR4 having the amino acid sequence of SEQ ID NO:135, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:147
  • HC-FR2 having the amino acid sequence of SEQ ID NO:148
  • HC-FR3 having the amino acid sequence of SEQ ID NO:149
  • HC-FR4 having the amino acid sequence of SEQ ID NO:135, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:161
  • HC-FR2 having the amino acid sequence of SEQ ID NO:162
  • HC-FR3 having the amino acid sequence of SEQ ID NO:163
  • HC-FR4 having the amino acid sequence of SEQ ID NO:135, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NO:172
  • HC-FR2 having the amino acid sequence of SEQ ID NO:173
  • HC-FR3 having the amino acid sequence of SEQ ID NO:174
  • HC-FR4 having the amino acid sequence of SEQ ID NO:175, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NQ:103
  • HC-FR2 having the amino acid sequence of SEQ ID NO:186
  • HC-FR3 having the amino acid sequence of SEQ ID NO:187
  • HC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • HC-FR1 having the amino acid sequence of SEQ ID NQ:103
  • HC-FR2 having the amino acid sequence of SEQ ID NO:186
  • HC-FR3 having the amino acid sequence of SEQ ID NQ:201
  • HC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • the antigen-binding molecule comprises a VH region comprising the CDRs according to one of (1) to (18) above, and the FRs according to one of (19) to (35) above.
  • the antigen-binding molecule comprises a VH region according to one of (36) to
  • (40) a VH region comprising the CDRs according to (5) and the FRs according to (21), (22), (23) or (24).
  • VH region comprising the CDRs according to (10) and the FRs according to (28).
  • VH region comprising the CDRs according to (15) and the FRs according to (34) or (35).
  • the antigen-binding molecule comprises a VH region according to one of (58) to (76) below:
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:276.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:285.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:289.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:32.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:48.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:52.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:62.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:71 .
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:87.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:102.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:106.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:119.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:133.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:143.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:157.
  • a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:168.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:183.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:194.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:199.
  • the antigen-binding molecule comprises a VL region according to one of (77) to (96) below:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • V4-C27, V4-C30, V4-C31 a VL region incorporating 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 NQ:300
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • V4H2 a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:82; or a variant thereof in which 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.
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:97
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:98; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:115, or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:129; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:139; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:153; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:153; or a variant thereof in which 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.
  • LC-CDR2 having the amino acid sequence of SEQ ID NO:178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:179; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NQ:190; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NQ:190; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NQ:190; or a variant thereof in which 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.
  • LC-CDR3 having the amino acid sequence of SEQ ID NQ:190; or a variant thereof in which 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.
  • the antigen-binding molecule comprises a VL region according to one of (97) to (120) below:
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • V4-C9 a VL region incorporating the following FRs:
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • V4-C30 a VL region incorporating the following FRs:
  • LC-FR1 having the amino acid sequence of SEQ ID NO:288
  • LC-FR2 having the amino acid sequence of SEQ ID NO:283
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • V4-C31 a VL region incorporating the following FRs:
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • V4H1 a VL region incorporating the following FRs:
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR3 having the amino acid sequence of SEQ ID NQ:70
  • LC-FR4 having the amino acid sequence of SEQ ID NO:47, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR1 having the amino acid sequence of SEQ ID NO:154
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • (119) (13D5-1) a VL region incorporating the following FRs:
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86, or a variant thereof in which 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.
  • the antigen-binding molecule comprises a VL region comprising the CDRs according to one of (77) to (96) above, and the FRs according to one of (97) to (120) above.
  • the antigen-binding molecule comprises a VL region according to one of (121) to (148) below:
  • (121) a VL region comprising the CDRs according to (77) and the FRs according to (97), (98), (99), (100), (101), (102), (103), (104), (105), (106), (107) or (108).
  • VL region comprising the CDRs according to (82) and the FRs according to (102).
  • (131) a VL region comprising the CDRs according to (81) and the FRs according to (105), (106), (107) or (108).
  • the antigen-binding molecule comprises a VL region according to one of (149) to (173) below: (149) a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:310.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:282.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:287.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:294.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:297.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:299.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:301 .
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:302.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:303.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:40.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:50.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:57.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:66.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:79.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:95.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:104.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:113.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:126.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:136.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:150.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:164.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:176.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:188.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:196.
  • VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:202.
  • the antigen-binding molecule comprises a VH region according to any one of (1) to (76) above, and a VL region according to any one of (77) to (173) above.
  • the antigen-binding molecule comprises the CDRs of, or comprises the VH and VL of, a VISTA-binding antibody clone selected from: 4M2-C12, V4H1 , V4H2, V4-C1 , V4-C9, V4-C24, V4-C26, V4-C27, V4-C28, V4-C30 or V4-C31.
  • the antigen-binding molecule comprises the CDRs of, or comprises the VH and VL of, V4-C26.
  • the antigen-binding molecule comprises:
  • HC-CDR1 having the amino acid sequence of SEQ ID NQ:305
  • HC-CDR2 having the amino acid sequence of SEQ ID NQ:306
  • HC-CDR3 having the amino acid sequence of SEQ ID NQ:307, or a variant thereof in which 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; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • B a VH region incorporating the following CDRs:
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:290
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:291
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:53
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:34
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:35, or a variant thereof in which 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; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:34
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:35, or a variant thereof in which 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; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:277
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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
  • VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:286
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NQ:290
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:291
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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; and a VL region incorporating the following CDRs:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NQ:290
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:291
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:278, or a variant thereof in which 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; and a VL region incorporating 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:42
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:34
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:35, or a variant thereof in which 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
  • VL region incorporating 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:42
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43; or a variant thereof in which 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.
  • the antigen-binding molecule comprises:
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:297.
  • (L) a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:62; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:66.
  • (M) a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:276; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:282.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:285; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:287.
  • VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:294.
  • (P) a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:299.
  • (Q) a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:301 .
  • (R) a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:289; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NQ:302.
  • (S) a VH region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:32; and a VL region comprising an amino acid sequence having at least 70% sequence identity more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequence identity to the amino acid sequence of SEQ ID NO:40.
  • the antigen-binding molecule comprises, or consists of:
  • 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 ;
  • 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.
  • substitutions may be conservative substitutions, for example according to the following Table.
  • amino acids in the same block in the middle column are substituted.
  • amino acids in the same line in the rightmost column are substituted:
  • substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target binding) of the antigen-binding molecule comprising the substitution as compared to the equivalent unsubstituted molecule.
  • the VH and VL region of an antigen-binding region of an antibody together constitute the Fv region.
  • the antigen-binding molecule according to the present disclosure comprises, or consists of, an Fv region which binds to VISTA.
  • the VH and VL regions of the Fv are provided as single polypeptide joined by a linker region, i.e. a single chain Fv (scFv).
  • the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin heavy chain constant sequence.
  • the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE or IgM.
  • the immunoglobulin heavy chain constant sequence is, or is derived from, the heavy chain constant sequence of lgG4.
  • the immunoglobulin heavy chain constant sequence is human immunoglobulin G 1 constant (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 NQ:205 form a hinge region between CH1 and CH2 regions (SEQ ID NQ:207). Positions 111 to 223 of SEQ ID NQ:205 form the CH2 region (SEQ ID NQ:208). Positions 224 to 330 of SEQ ID NQ:205 form the CH3 region (SEQ ID NQ:209).
  • the exemplified antigen-binding molecules may be prepared using pFUSE-CHIg-hG1 , which comprises the substitutions D356E, L358M (positions numbered according to EU numbering) in the CH3 region.
  • the amino acid sequence of the CH3 region encoded by pFUSE-CHIg-hG1 is shown in SEQ ID NO:210. It will be appreciated that CH3 regions may be provided with further substitutions in accordance with modification to an Fc region of the antigen-binding molecule as described herein.
  • a CH1 region comprises or consists of the sequence of SEQ ID NQ:206, or a sequence 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 SEQ ID NQ:206.
  • a CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID NQ:207, or a sequence 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 SEQ ID NQ:207.
  • a CH2 region comprises or consists of the sequence of SEQ ID NQ:208, or a sequence 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 SEQ ID NQ:208.
  • a CH3 region comprises or consists of the sequence of SEQ ID NQ:209 or 210, or a sequence 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 SEQ ID NQ:209 or 210.
  • the antigen-binding molecule of the present disclosure comprises the sequence of SEQ ID NO:345, or a sequence 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 SEQ ID NO:345.
  • the antigen-binding molecule of the present disclosure comprises the sequence of SEQ ID NO:346, or a sequence 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 SEQ ID NO:346.
  • the antigen-binding molecule of the present disclosure comprises one or more regions of an immunoglobulin light chain constant sequence.
  • the immunoglobulin light chain constant sequence is human immunoglobulin kappa constant (IGKC; CK; UniProt: P01834-1 , v2; SEQ ID NO:211).
  • the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CA), e.g. IGLC1 , IGLC2, IGLC3, IGLC6 or IGLC7.
  • a CL region comprises or consists of the sequence of SEQ ID NO:211 , or a sequence 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 SEQ ID NO:211.
  • the antigen-binding molecule comprises a Fab region comprising a VH, a CH1 , a VL and a CL (e.g. CK or CA).
  • the Fab region comprises a polypeptide comprising a VH and a CH1 (e.g. a VH-CH1 fusion polypeptide), and a polypeptide comprising a VL and a CL (e.g. a VL-CL fusion polypeptide).
  • the Fab region comprises a polypeptide comprising a VH and a CL (e.g. a VH-CL fusion polypeptide) and a polypeptide comprising a VL and a CH (e.g. a VL-CH1 fusion polypeptide); that is, in some embodiments, the Fab region is a CrossFab region.
  • the VH, CH1 , VL and CL regions of the Fab or CrossFab are provided as single polypeptide joined by linker regions, i.e. as a single chain Fab (scFab) or a single chain CrossFab (scCrossFab).
  • the antigen-binding molecule of the present disclosure comprises, or consists of, a Fab region which binds to VISTA.
  • the antigen-binding molecule described herein comprises, or consists of, a whole antibody which binds to VISTA.
  • whole antibody refers to an antibody having a structure which is substantially similar to the structure of an immunoglobulin (Ig). Different kinds of immunoglobulins and their structures are described e.g. in Schroeder and Cavacini J Allergy Clin Immunol. (2010) 125(202): S41-S52, which is hereby incorporated by reference in its entirety.
  • Immunoglobulins of type G are ⁇ 150 kDa glycoproteins comprising two heavy chains and two light chains. From N- to C-terminus, the heavy chains comprise a VH followed by a heavy chain constant region comprising three constant domains (CH1 , CH2, and CH3), and similarly the light chain comprise a VL followed by a CL.
  • immunoglobulins may be classed as IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM.
  • the light chain may be kappa (K) or lambda (A).
  • the antigen-binding molecule described herein comprises, or consists of, an IgG (e.g. lgG1 , lgG2, lgG3, lgG4), IgA (e.g. lgA1 , lgA2), IgD, IgE, or IgM which binds to VISTA.
  • the antigen-binding molecule is an lgG4.
  • the antigen-binding molecule of the present disclosure is at least monovalent binding for VISTA.
  • Binding valency refers to the number of binding sites in an antigen-binding molecule for a given antigenic determinant. Accordingly, in some embodiments, the antigen-binding molecule comprises at least one binding site for VISTA.
  • the antigen-binding molecule comprises more than one binding site for VISTA, e.g. 2, 3 or 4 binding sites. The binding sites may be the same or different.
  • the antigen-binding molecule is e.g. bivalent, trivalent or tetravalent for VISTA. Aspects of the present disclosure relate to multispecific antigen-binding molecules. By “multispecific” it is meant that the antigen-binding molecule displays specific binding to more than one target.
  • the antigen-binding molecule is a bispecific antigen-binding molecule.
  • the antigen-binding molecule comprises at least two different antigen-binding domains (/.e. at least two antigen-binding domains, e.g. comprising non-identical VHs and VLs).
  • the antigen-binding molecule binds to VISTA and another target (e.g. an antigen other than VISTA), and so is at least bispecific.
  • another target e.g. an antigen other than VISTA
  • bispecific means that the antigen-binding molecule is able to bind specifically to at least two distinct antigenic determinants.
  • an antigen-binding molecule according to the present disclosure may comprise antigen-binding molecules capable of binding to the targets for which the antigen-binding molecule is specific.
  • an antigen-binding molecule which is capable of binding to VISTA and an antigen other than VISTA may comprise: (i) an antigenbinding molecule which is capable of binding to VISTA, and (ii) an antigen-binding molecule which is capable of binding to an antigen other than VISTA.
  • an antigen-binding molecule according to the present disclosure may comprise antigen-binding polypeptides or antigen-binding polypeptide complexes capable of binding to the targets for which the antigen-binding molecule is specific.
  • an antigen-binding molecule according to the present disclosure may comprise e.g.
  • an antigen-binding polypeptide complex capable of binding to VISTA comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3)
  • an antigen-binding polypeptide complex capable of binding to an antigen other than VISTA comprising a light chain polypeptide (comprising the structure VL-CL) and a heavy chain polypeptide (comprising the structure VH-CH1-CH2-CH3).
  • a component antigen-binding molecule of a larger antigen-binding molecule may be referred to e.g. as an “antigen-binding domain” or “antigen-binding region” of the larger antigen-binding molecule.
  • the antigen-binding molecule comprises an antigen-binding molecule capable of binding to VISTA, and an antigen-binding molecule capable of binding to an antigen other than VISTA.
  • the antigen other than VISTA is an immune cell surface molecule.
  • the antigen other than VISTA is a cancer cell antigen.
  • the antigen other than VISTA is a receptor molecule, e.g. a cell surface receptor.
  • the antigen other than VISTA is a cell signalling molecule, e.g. a cytokine, chemokine, interferon, interleukin or lymphokine.
  • the antigen other than VISTA is a growth factor or a hormone.
  • a cancer cell antigen is an antigen which is expressed or over-expressed by a cancer cell.
  • a cancer cell antigen may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof.
  • a cancer cell antigen’s expression may be associated with a cancer.
  • a cancer cell antigen may be abnormally expressed by a cancer cell (e.g. the cancer cell antigen may be expressed with abnormal localisation), or may be expressed with an abnormal structure by a cancer cell.
  • a cancer cell antigen may be capable of eliciting an immune response.
  • the antigen is expressed at the cell surface of the cancer cell (/.e. the cancer cell antigen is a cancer cell surface antigen).
  • the part of the antigen which is bound by the antigen-binding molecule described herein is displayed on the external surface of the cancer cell (/.e. is extracellular).
  • the cancer cell antigen may be a cancer-associated antigen.
  • the cancer cell antigen is an antigen whose expression is associated with the development, progression or severity of symptoms of a cancer.
  • the cancer-associated antigen may be associated with the cause or pathology of the cancer, or may be expressed abnormally as a consequence of the cancer.
  • the cancer cell antigen is an antigen whose expression is upregulated (e.g. at the RNA and/or protein level) by cells of a cancer, e.g.
  • the cancer-associated antigen may be preferentially expressed by cancerous cells, and not expressed by comparable non-cancerous cells (e.g. non-cancerous cells derived from the same tissue/cell type).
  • the cancer- associated antigen may be the product of a mutated oncogene or mutated tumor suppressor gene.
  • the cancer-associated antigen may be the product of an overexpressed cellular protein, a cancer antigen produced by an oncogenic virus, an oncofetal antigen, or a cell surface glycolipid or glycoprotein.
  • An immune cell surface molecule may be any peptide/polypeptide, glycoprotein, lipoprotein, glycan, glycolipid, lipid, or fragment thereof expressed at or on the cell surface of an immune cell.
  • the part of the immune cell surface molecule which is bound by the antigen-binding molecule of the present disclosure is on the external surface of the immune cell (/.e. is extracellular).
  • the immune cell surface molecule may be expressed at the cell surface of any immune cell.
  • the immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte.
  • the lymphocyte may be e.g.
  • the immune cell surface molecule may be a costimulatory molecule (e.g. CD28, 0X40, 4-1 BB, ICOS or CD27) or a ligand thereof.
  • the immune cell surface molecule may be a checkpoint molecule (e.g. PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA) or a ligand thereof.
  • Multispecific antigen-binding molecules may be provided in any suitable format, such as those formats described in described in Brinkmann and Kontermann MAbs (2017) 9(2): 182-212, which is hereby incorporated by reference in its entirety.
  • Suitable formats include those shown in Figure 2 of Brinkmann and Kontermann MAbs (2017) 9(2): 182-212: antibody conjugates, e.g. lgG2, F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, KA-body common HC; CH1/CL fusion proteins, e.g.
  • scFv2-CH1/CL, VHH2-CH1/CL ‘variable domain only’ bispecific antigenbinding molecules, e.g. tandem scFv (taFV), triplebodies, diabodies (Db), dsDb, Db(kih), DART, scDB, dsFv-dsFv, tandAbs, triple heads, tandem dAb/VHH, tertravalent dAb.VHH;
  • Non-lg fusion proteins e.g.
  • scFv2-albumin scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab2, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2, ImmTAC (TCR-scFv); modified Fc and CH3 fusion proteins, e.g.
  • Fab-scFv (bibody), Fab-scFv2 (tribody), Fab- Fv, Fab-dsFv, Fab-VHH, orthogonal Fab-Fab; non-lg fusion proteins, e.g. DNL-Fabs, DNL-Fab2-scFv, DNL-Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g.
  • DAF two-in one-IgG
  • bispecific antigen-binding molecules The skilled person is able to design and prepare bispecific antigen-binding molecules.
  • Methods for producing bispecific antigen-binding molecules include chemically crosslinking of antigen-binding molecules or antibody fragments, e.g. with reducible disulphide or non-reducible thioether bonds, for example as described in Segal and Bast, 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14:l V:2.13:2.13.1 — 2.13.16, which is hereby incorporated by reference in its entirety.
  • SPDP A/-succinimidyl-3-(-2-pyridyldithio)-propionate
  • SPDP can be used to chemically crosslink e.g. Fab fragments via hinge region SH- groups, to create disulfide-linked bispecific F(ab)2 heterodimers.
  • bispecific antigen-binding molecules include fusing antibody-producing hybridomas e.g. with polyethylene glycol, to produce a quadroma cell capable of secreting bispecific antibody, for example as described in D. M. and Bast, B. J. 2001. Production of Bispecific Antigen-binding molecules. Current Protocols in Immunology. 14: IV:2.13:2.13.1 — 2.13.16.
  • Bispecific antigen-binding molecules can also be produced recombinantly, by expression from e.g. a nucleic acid construct encoding polypeptides for the antigenbinding molecules, for example as described in Antibody Engineering: Methods and Protocols, Second Edition (Humana Press, 2012), at Chapter 40: Production of Bispecific Antigen-binding molecules: Diabodies and Tandem scFv (Hornig and Farber-Schwarz), or French, How to make bispecific antigenbinding molecules, Methods Mol. Med. 2000; 40:333-339, the entire contents of both of which are hereby incorporated by reference.
  • a DNA construct encoding the light and heavy chain variable domains for the two antigen-binding fragments (/.e. the light and heavy chain variable domains for the antigen-binding fragment capable of binding VISTA, and the light and heavy chain variable domains for the antigen-binding fragment capable of binding to another target protein), and including sequences encoding a suitable linker or dimerization domain between the antigen-binding fragments can be prepared by molecular cloning techniques.
  • Recombinant bispecific antibody can thereafter be produced by expression (e.g. in vitro) of the construct in a suitable host cell (e.g. a mammalian host cell), and expressed recombinant bispecific antibody can then optionally be purified.
  • the antigen-binding molecules of the present disclosure comprise an Fc region.
  • Fc regions are composed of CH2 and CH3 regions from one polypeptide, and CH2 and CH3 regions from another polypeptide. The CH2 and CH3 regions from the two polypeptides together form the Fc region.
  • Fc regions In IgM and IgE isotypes the Fc regions contain three constant domains (CH2, CH3 and CH4), and CH2 to CH4 from the two polypeptides together form the Fc region.
  • Fc regions provide for interaction with Fc receptors and other molecules of the immune system to bring about functional effects.
  • IgG Fc-mediated effector functions are reviewed e.g. in Jefferis et al., Immunol Rev 1998 163:59-76 (hereby incorporated by reference in its entirety), and are brought about through Fc- mediated recruitment and activation of immune cells (e.g. macrophages, dendritic cells, NK cells and T cells) through interaction between the Fc region and Fc receptors expressed by the immune cells, recruitment of complement pathway components through binding of the Fc region to complement protein C1q, and consequent activation of the complement cascade.
  • immune cells e.g. macrophages, dendritic cells, NK cells and T cells
  • Fc-mediated functions include Fc receptor binding, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), formation of the membrane attack complex (MAC), cell degranulation, cytokine and/or chemokine production, and antigen processing and presentation.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • MAC membrane attack complex
  • cell degranulation cell degranulation
  • cytokine and/or chemokine production and antigen processing and presentation.
  • Fc region/CH2/CH3 is described as comprising modification(s) “corresponding to” reference substitution(s), equivalent substitution(s) in the homologous Fc/CH2/CH3 are contemplated.
  • L234A/L235A substitutions in human lgG1 correspond to L to A substitutions at positions 117 and 118 of the mouse Ig gamma-2A chain C region, A allele, numbered according to SEQ ID NO:256.
  • an Fc region is described as comprising a modification
  • the modification may be present in one or both of the polypeptide chains which together form the Fc region.
  • the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and/or CH3 regions.
  • the Fc region comprises modification to increase an Fc-mediated function. In some embodiments, the Fc region comprises modification to increase ADCC. In some embodiments, the Fc region comprises modification to increase ADCP. In some embodiments, the Fc region comprises modification to increase CDC.
  • An antigen-binding molecule comprising an Fc region comprising modification to increase an Fc-mediated function induces an increased level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.
  • the Fc region comprises modification to increase binding to an Fc receptor. In some embodiments, the Fc region comprises modification to increase binding to an Fey receptor. In some embodiments, the Fc region comprises modification to increase binding to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRI Ila and FcyRlllb. In some embodiments, the Fc region comprises modification to increase binding to FcyRI Ila. In some embodiments, the Fc region comprises modification to increase binding to FcyRlla. In some embodiments, the Fc region comprises modification to increase binding to FcyRllb. In some embodiments, the Fc region comprises modification to increase binding to FcRn.
  • the Fc region comprises modification to increase binding to a complement protein. In some embodiments, the Fc region comprises modification to increase binding to C1q. In some embodiments, the Fc region comprises modification to promote hexamerisation of the antigen-binding molecule. In some embodiments, the Fc region comprises modification to increase antigen-binding molecule half-life. In some embodiments, the Fc region comprises modification to increase coengagement.
  • the Fc region comprises modification corresponding to the combination of substitutions F243L/R292P/Y300L/V305I/P396L as described in Stavenhagen et al. Cancer Res. (2007) 67:8882-8890. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions 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 modification corresponding to the combination of substitutions S298A/E333A/K334A as described in Shields et al., J Biol Chem.
  • the Fc region comprises modification to one of heavy chain polypeptides corresponding to the combination of substitutions L234Y/L235Q/G236W/S239M/H268D/D270E/S298A, and modification to the other heavy chain polypeptide corresponding to the combination of substitutions D270E/K326D/A330M/K334E, as described in Mimoto et al., MAbs. (2013): 5:229-236.
  • the Fc region comprises modification corresponding to the combination of substitutions G236A/S239D/I332E as described in Richards et al., Mol Cancer Ther. (2008) 7:2517-2527.
  • the Fc region comprises modification corresponding to the combination of substitutions K326W/E333S as described in Idusogie et al. J Immunol. (2001) 166(4):2571-5. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions S267E/H268F/S324T as described in Moore et al. MAbs. (2010) 2(2):181-9. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions described in Natsume et al., Cancer Res. (2008) 68(10):3863-72. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions E345R/E430G/S440Y as described in Diebolder et al. Science (2014) 343(6176):1260-3.
  • the Fc region comprises modification corresponding to the combination of substitutions M252Y/S254T/T256E as described in Dall’Acqua et al. J Immunol. (2002) 169:5171-5180.
  • the Fc region comprises modification corresponding to the combination of substitutions M428L/N434S as described in Zalevsky et al. Nat Biotechnol. (2010) 28:157-159.
  • the Fc region comprises modification corresponding to the combination of substitutions S267E/L328F as described in Chu et al., Mol Immunol. (2008) 45:3926-3933. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions N325S/L328F as described in Shang et al. Biol Chem. (2014) 289:15309-15318.
  • the Fc region comprises modification to reduce/prevent an Fc-mediated function. In some embodiments, the Fc region comprises modification to reduce/prevent ADCC. In some embodiments, the Fc region comprises modification to reduce/prevent ADCP. In some embodiments, the Fc region comprises modification to reduce/prevent CDC.
  • An antigen-binding molecule comprising an Fc region comprising modification to reduce/prevent an Fc-mediated function induces an reduced level of the relevant effector function as compared to an antigen-binding molecule comprising the corresponding unmodified Fc region.
  • the Fc region comprises modification to reduce/prevent binding to an Fc receptor. In some embodiments, the Fc region comprises modification to reduce/prevent binding to an Fey receptor. In some embodiments, the Fc region comprises modification to reduce/prevent binding to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the Fc region comprises modification to reduce/prevent binding to FcyRllla. In some embodiments, the Fc region comprises modification to reduce/prevent binding to FcyRlla. In some embodiments, the Fc region comprises modification to reduce/prevent binding to FcyRllb.
  • the Fc region comprises modification to reduce/prevent binding to a complement protein. In some embodiments, the Fc region comprises modification to reduce/prevent binding to C1q. In some embodiments, the Fc region comprises modification to reduce/prevent glycosylation of the amino acid residue corresponding to N297. In some embodiments, the Fc region is not able to induce one or more Fc-mediated functions (/.e. lacks the ability to elicit the relevant Fc-mediated function(s)). Accordingly, antigen-binding molecules comprising such Fc regions also lack the ability to induce the relevant function(s). Such antigen-binding molecules may be described as being devoid of the relevant function(s).
  • the Fc region is not able to induce ADCC. In some embodiments, the Fc region is not able to induce ADCP. In some embodiments, the Fc region is not able to induce CDC. In some embodiments, the Fc region is not able to induce ADCC and/or is not able to induce ADCP and/or is not able to induce CDC.
  • the Fc region is not able to bind to an Fc receptor. In some embodiments, the Fc region is not able to bind to an Fey receptor. In some embodiments, the Fc region is not able to bind to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the Fc region is not able to bind to FcyRllla. In some embodiments, the Fc region is not able to bind to FcyRlla. In some embodiments, the Fc region is not able to bind to FcyRllb.
  • the Fc region is not able to bind to FcRn. In some embodiments, the Fc region is not able to bind to a complement protein. In some embodiments, the Fc region is not able to bind to C1q. In some embodiments, the Fc region is not glycosylated at the amino acid residue corresponding to N297.
  • the Fc region comprises modification corresponding to N297A or N297Q or N297G as described in Leabman et al., MAbs. (2013) 5:896-903.
  • the Fc region comprises modification corresponding to L235E as described in Alegre et al., J Immunol. (1992) 148:3461-3468.
  • the Fc region comprises modification corresponding to the combination of substitutions L234A/L235A or F234A/L235A as described in Xu et al., Cell Immunol. (2000) 200:16-26.
  • the Fc region comprises 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 modification corresponding to the combination of substitutions L234A/L235A/P329G as described in Lo et al. J. Biol. Chem (2017) 292(9):3900-3908. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions described in Rother et al., Nat Biotechnol. (2007) 25:1256-1264.
  • the Fc region comprises modification corresponding to the combination of substitutions S228P/L235E as described in Newman et al., Clin. Immunol. (2001) 98:164-174. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions H268Q/V309L/A330S/P331S as described in An et al., MAbs. (2009) 1 :572-579. In some embodiments, the Fc region comprises modification corresponding to the combination of substitutions
  • the Fc region comprises modification corresponding to the combination of substitutions L234A/L235E/G237A/A330S/P331S as described in US 2015/0044231 A1.
  • substitutions “L234A/L235A” and corresponding substitutions are known to disrupt binding of Fc to Fey receptors and inhibit ADCC, ADCP, and also to reduce C1q binding and thus CDC (Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466, hereby incorporated by reference in entirety).
  • substitutions “P329G” and “P329A” reduce C1q binding (and thereby CDC).
  • the Fc region comprises modification corresponding to the substitution S228P as described in Silva et al., J Biol Chem. (2015) 290(9):5462-5469.
  • the substitution S228P in lgG4 Fc reduces Fab-arm exchange (Fab arm exchange can be undesirable).
  • the Fc region comprises modification corresponding to corresponding to the combination of substitutions L234A/L235A. In some embodiments, the Fc region comprises modification corresponding to corresponding to the substitution P329G. In some embodiments, the Fc region comprises modification corresponding to corresponding to the substitution N297Q.
  • the Fc region comprises modification corresponding to corresponding to the combination of substitutions L234A/L235A/P329G.
  • the Fc region comprises modification corresponding to corresponding to the combination of substitutions L234A/L235A/P329G/N297Q.
  • the Fc region comprises modification corresponding to corresponding to the combination of substitutions L234A/L235E/G237A/A330S/P331S.
  • the Fc region comprises modification corresponding to corresponding to the substitution S228P, e.g. in lgG4.
  • the antigen-binding molecule of the present disclosure comprises an Fc region comprising modification in one or more of the CH2 and CH3 regions promoting association of the Fc region.
  • Recombinant co-expression of constituent polypeptides of an antigen-binding molecule and subsequent association leads to several possible combinations.
  • modification(s) promoting association of the desired combination of heavy chain polypeptides.
  • Modifications may promote e.g. hydrophobic and/or electrostatic interaction between CH2 and/or CH3 regions of different polypeptide chains. Suitable modifications are described e.g. in Ha et al., Front.
  • the antigen antigen-binding molecule of the present disclosure comprises an Fc region comprising paired substitutions in the CH3 regions of the Fc region according to one of the following formats, as shown in Table 1 of Ha et al., Front. Immnol (2016) 7:394: KiH, KiH s s , HA-TF, ZW1 , 7.8.60, DD-KK, EW-RVT, EW-RVTs-s, SEED or A107.
  • the Fc region comprises the “knob-into-hole” or “KiH” modification, e.g. as described e.g. in US 7,695,936 and Carter, J Immunol Meth 248, 7-15 (2001).
  • one of the CH3 regions of the Fc region comprises a “knob” modification
  • the other CH3 region comprises a “hole” modification.
  • the “knob” and “hole” modifications are positioned within the respective CH3 regions so that the “knob” can be positioned in the “hole” in order to promote heterodimerisation (and inhibit homodimerisation) of the polypeptides and/or stabilise heterodimers.
  • Knobs are constructed by substituting amino acids having small chains with those having larger side chains (e.g. tyrosine or tryptophan). Holes are created by substituting amino acids having large side chains with those having smaller side chains (e.g. alanine or threonine).
  • one of the CH3 regions of the Fc region of the antigen-binding molecule of the present disclosure comprises the substitution (numbering of positions/substitutions in the Fc, CH2 and CH3 regions herein is according to the EU numbering system as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) T366W, and the other CH3 region of the Fc region comprises the substitution Y407V.
  • one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W
  • the other CH3 region of the Fc region comprises the substitutions T366S and L368A.
  • one of the CH3 regions of the Fc region of the antigen-binding molecule comprises the substitution T366W, and the other CH3 region of the Fc region comprises the substitutions Y407V, T366S and L368A.
  • the Fc region comprises the “DD-KK” modification as described e.g. in WO 2014/131694 A1 .
  • one of the CH3 regions comprises the substitutions K392D and K409D, and the other CH3 region of the Fc region comprises the substitutions E356K and D399K. The modifications promote electrostatic interaction between the CH3 regions.
  • the antigen-binding molecule of the present disclosure comprises an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci U S A. (2013) 110(13):5145-50, referred to as ‘Duobody’ format.
  • one of the CH3 regions comprises the substitution K409R
  • the other CH3 region of the Fc region comprises the substitution K405L.
  • the antigen-binding molecule of the present disclosure comprises an Fc region comprising the “EEE-RRR” modification as described in Strop et al., J Mol Biol. (2012) 420(3):204-19.
  • one of the CH3 regions comprises the substitutions D221 E, P228E and L368E, and the other CH3 region of the Fc region comprises the substitutions D221 R, P228R and K409R.
  • 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.
  • one of the CH3 regions comprises the substitutions K360E and K409W
  • the other CH3 region of the Fc region comprises the substitutions Q347R, D399V and F405T.
  • one of the CH3 regions comprises the substitution S354C
  • the other CH3 region of the Fc region comprises the substitution Y349C.
  • Introduction of these cysteine residues results in formation of a disulphide bridge between the two CH3 regions of the Fc region, further stabilizing the heterodimer (Carter (2001), J Immunol Methods 248, 7-15).
  • the Fc region comprises the “KiHs-s” modification.
  • one of the CH3 regions comprises the substitutions T366W and S354C, and the other CH3 region of the Fc region comprises the substitutions T366S, L368A, Y407V and Y349C.
  • the antigen-binding molecule of the present disclosure comprises an Fc region comprising the “SEED” modification as described in Davis et al., Protein Eng Des Sei (2010) 23(4):195- 202, in which p-strand segments of human lgG1 CH3 and IgA CH3 are exchanged.
  • one of the CH3 regions comprises the substitutions S364H and F405A
  • the other CH3 region of the Fc region comprises the substitutions Y349T and T394F (see e.g. Moore et al., MAbs (2011) 3(6):546-57).
  • one of the CH3 regions comprises the substitutions T350V, L351Y, F405A and Y407V
  • the other CH3 region of the Fc region comprises the substitutions T350V, T366L, K392L and T394W (see e.g. Von Kreudenstein et al., MAbs (2013) 5(5):646-54).
  • one of the CH3 regions comprises the substitutions K360D, D399M and Y407A
  • the other CH3 region of the Fc region comprises the substitutions E345R, Q347R, T366V and K409V (see e.g. Leaver-Fay et al., Structure (2016) 24(4):641-51).
  • one of the CH3 regions comprises the substitutions K370E and K409W
  • the other CH3 region of the Fc region comprises the substitutions E357N, D399V and F405T (see e.g. Choi et al., PLoS One (2015) 10(12):e0145349).
  • the antigen-binding molecule of the present disclosure comprises an Fc region which does not bind to an Fc y receptor. In some embodiments, the antigen-binding molecule comprises an Fc region which does not bind to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the antigen-binding molecule comprises an Fc region which does not bind to one or more of FcyRlla, FcyRllb and FcyRllla.
  • the antigen-binding molecule comprises an Fc region which does not bind to one or both of FcyRlla and FcyRllb.
  • a reference protein e.g. an Fc receptor
  • ELISA ELISA
  • immunoblot immunoprecipitation
  • SPR Surface Plasmon Resonance
  • BLI Bio-Layer Interferometry
  • an Fc region “which does not bind to” a reference protein may display substantially no binding to the reference protein, e.g. as determined by ELISA, immunoblot (e.g. western blot), immunoprecipitation, SPR or BLI).
  • “Substantially no binding” may be a level of interaction that is not significantly greater than the level of interaction determined for proteins that do not bind to one another in a given assay.
  • “Substantially no binding” may be a level of interaction which is ⁇ 5 times, e.g. ⁇ 4 times, ⁇ 3 times, ⁇ 2.5 times, ⁇ 2 times or ⁇ 1 .5 times the level of interaction determined for proteins that do not bind to one another, in a given assay.
  • the antigen-binding molecule comprises an Fc region which binds to FcRn.
  • the antigen-binding molecule comprises an Fc region which binds to FcRn, and which does not bind to one or more of FcyRlla, FcyRllb and FcyRllla. In some embodiments, the antigenbinding molecule comprises an Fc region which binds to FcRn, and which does not bind to one or both of FcyRlla and FcyRllb.
  • the antigen-binding molecule of the present disclosure comprises an Fc region which does not induce ADCC. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region which does not induce ADCP. In some embodiments, the antigenbinding molecule of the present disclosure comprises an Fc region which does not induce CDC. In some embodiments, the antigen-binding molecule of the present disclosure comprises an Fc region which does not induce ADCC, ADCP or CDC.
  • an Fc region/antigen-binding molecule which does not induce (/.e. is not able to induce) ADCC/ADCP/CDC elicits substantially no ADCC/ADCP/CDC activity, e.g. as determined by analysis in an appropriate assay for the relevant activity.
  • substantially no ADCC/ADCP/CDC activity refers to a level of ADCC/ADCP/CDC that is not significantly greater than ADCC/ADCP/CDC determined for an appropriate negative control molecule in a given assay (e.g. an antigen-binding molecule lacking an Fc region, or an antigen-binding molecule comprising a ‘silent’ Fc region (e.g.
  • “Substantially no activity” may be a level of the relevant activity which is ⁇ 5 times, e.g. ⁇ 4 times, ⁇ 3 times, ⁇ 2.5 times, ⁇ 2 times or ⁇ 1 .5 times the level of activity determined for an appropriate negative control molecule in a given assay.
  • the ability of an Fc region, or an antigen-binding molecule comprising an Fc region, to induce ADCC can be analysed e.g. according to the method described in Yamashita et al., Scientific Reports (2016) 6:19772 (hereby incorporated by reference in its entirety), or by 51 Cr release assay as described e.g. in Jedema et al., Blood (2004) 103: 2677-82 (hereby incorporated 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 analysed e.g. according to the method described in Kamen et al., J Immunol (2017) 198 (1 Supplement) 157.17 (hereby incorporated by reference in its entirety).
  • an Fc region, or an antigen-binding molecule comprising an Fc region, to induce CDC can be analysed e.g. using a C1q binding assay, e.g. as described in Schlothauer et al., Protein Engineering, Design and Selection (2016), 29(10):457-466 (incorporated by reference hereinabove).
  • the antigen-binding molecule comprises an Fc region comprising a polypeptide having 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:254.
  • the antigen-binding molecule comprises an Fc region comprising a polypeptide having 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:257.
  • the antigen-binding molecule comprises an Fc region comprising a polypeptide having 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:259.
  • the antigen-binding molecule comprises an Fc region comprising a polypeptide having 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 NQ:260.
  • the antigen-binding molecule comprises an Fc region comprising a polypeptide having 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:347.
  • the antigen-binding molecules of the present disclosure lack an Fc region.
  • Fc receptors are polypeptides which bind to the Fc region of immunoglobulins. Fc receptor structure and function is reviewed e.g. in Masuda et al., Inflamm Allergy Drug Targets (2009) 8(1): 80-86, and Bruhns, Blood (2012) 119:5640-5649, both of which are hereby incorporated by reference in their entirety.
  • Fc receptors are expressed at surface of hematopoietic cells including macrophages, neutrophils, dendritic cells, eosinophils, basophils, mast cells, and NK cells. They include the IgG-binding Fc y receptors, the high-affinity receptor for IgE (FcsRI), the IgA receptor, and the polymeric Ig receptor for IgA and IgM.
  • the neonatal Fc receptor (FcRn) is a further Fc receptor for IgG, and is involved in IgG transport across epithelial barriers (transcytosis), protecting IgG from degradation, and antigen presentation.
  • FcyRI mFcyRI
  • FcyRlla mFcyRIII
  • FcyRllb mFcyRllb
  • FcyRllc FcyRllla
  • FcyRlllb FcyRlllb
  • FCYRI, FcyRlla, FcyRllc and FcyRIHa comprise immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains, and ligation by Fc leads to activation of cells expressing the receptors.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • FcyRllb comprises immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its intracellular domain, and negatively regulates cell activation and degranulation, cell proliferation, endocytosis, and phagocytosis upon ligation by Fc.
  • ITIMs immunoreceptor tyrosine-based inhibitory motifs
  • an “Fey receptor” may be from any species, and includes isoforms, fragments, variants (including mutants) or homologues from any species.
  • “FcyRI”, “FcyRlla”, “FcyRllb”, “FcyRllc”, “FcyRllla” and “FcyRlllb” refer respectively to FcYRI/FcYRIIa/FcYRIIb/FcYRIIc/FcYRIIIa/FcyRlllb from any species, and include isoforms, fragments, variants (including mutants) or homologues from any species.
  • the Fc y receptor e.g. FcYRI/FcYRIIa/FcYRIIb/FcYRIIc/FcYRIIIa/FcYRIIIb
  • a mammal e.g. a primate (rhesus, cynomolgous, non-human primate or human) and/or a rodent (e.g. rat or mouse).
  • Isoforms, fragments, variants or homologues may optionally be characterised 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 Fc y receptor (e.g. FcYRI/FcYRIIa/FcYRIIb/FcYRIIc/FcYRIIIa/FcYRIIIb) from a given species, e.g. human.
  • FcYRI/FcYRIIa/FcYRIIb/FcYRIIc/FcYRIIIa/FcYRIIIb e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference Fc y receptor, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of FCYRI may e ⁇ 7 display association with human lgG1 Fc.
  • an “FcRn receptor” may be from any species, and includes isoforms, fragments, variants (including mutants) or homologues from any species.
  • the FcRn receptor is from a mammal (e.g. a primate (rhesus, cynomolgous, non- human primate or human) and/or a rodent (e.g. rat or mouse).
  • Isoforms, fragments, variants or homologues may optionally be characterised 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 FcRn receptor from a given species, e.g. human.
  • Isoforms, fragments, variants or homologues may optionally be functional isoforms, fragments, variants or homologues, e.g. having a functional property/activity of the reference FcRn, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of FcRn may e.g. display association with human lgG1 Fc.
  • the present disclosure also provides polypeptide constituents of antigen-binding molecules.
  • the polypeptides may be provided in isolated or substantially purified form.
  • the antigen-binding molecule of the present disclosure may be, or may comprise, a complex of polypeptides.
  • a polypeptide comprises more than one domain or region
  • the plural domains/regions are preferably present in the same polypeptide chain. That is, the polypeptide comprises more than one domain or region is a fusion polypeptide comprising the domains/regions.
  • a polypeptide according to the present disclosure comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present disclosure comprises, or consists of, a VL as described herein.
  • the polypeptide additionally comprises one or more antibody heavy chain constant regions (CH). In some embodiments, the polypeptide additionally comprises one or more antibody light chain constant regions (CL). In some embodiments, the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).
  • CH antibody heavy chain constant regions
  • CL antibody light chain constant regions
  • the polypeptide comprises a CH1 , CH2 region and/or a CH3 region of an immunoglobulin (Ig).
  • 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.
  • the polypeptide comprises a CH2 and/or CH3 region comprising any one 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/H268F/S324T; E345R/E430G/S440Y;
  • the polypeptide comprises a CH3 region comprising any one of the following amino acid substitutions/combinations of amino acid substitutions (shown e.g. in Table 1 of Ha et al., Front. Immnol (2016) 7:394, incorporated by reference hereinabove): 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; Q3
  • 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.
  • 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.
  • polypeptide according to the present disclosure comprises a structure from N- to C-terminus according to one of the following:
  • antigen-binding molecules composed of the polypeptides of the present disclosure.
  • the antigen-binding molecule of the present disclosure comprises one of the following combinations of polypeptides:
  • the antigen-binding molecule comprises more than one of a polypeptide of the combinations shown in (A) to (I) above.
  • the antigen-binding molecule comprises two polypeptides comprising the structure VH- CH1-CH2-CH3, and two polypeptides comprising the structure VL-CL.
  • the antigen-binding molecule of the present disclosure comprises one of the following combinations of polypeptides:
  • VH anti-VISTA
  • VL anti-VISTA
  • 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.
  • the antigen-binding molecules and polypeptides of the present disclosure comprise a hinge region.
  • a hinge region is provided between a CH1 region and a CH2 region.
  • a hinge region is provided between a CL region and a CH2 region.
  • the hinge region 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 SEQ ID NO:207.
  • the antigen-binding molecules and polypeptides of the present disclosure comprise one or more linker sequences between amino acid sequences.
  • a linker sequence may be provided at one or both ends of one or more of a VH, VL, CH1-CH2 hinge region, CH2 region and a CH3 region of the antigen-binding molecule/polypeptide.
  • Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety.
  • a linker sequence may be a flexible linker sequence.
  • Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence.
  • Flexible linkers are known to the skilled person, and several are identified in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and/or serine residues.
  • the linker sequence comprises at least one glycine residue and/or at least one serine residue. In some embodiments, the linker sequence consists of glycine and serine residues. In some embodiments, the linker sequence has a length of 1 -2, 1-3, 1-4, 1 -5 or 1 -10 amino acids.
  • the antigen-binding molecules and polypeptides of the present disclosure may additionally comprise further amino acids or sequences of amino acids.
  • the antigen-binding molecules and polypeptides may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection of the antigen-binding molecule/polypeptide.
  • the antigen-binding molecule/polypeptide may comprise a sequence encoding a His, (e.g. 6XHis), Myc, GST, MBP, FLAG, HA, E, or Biotin tag, optionally at the N- or C- terminus of the antigen-binding molecule/polypeptide.
  • the antigen-binding molecule/polypeptide comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.
  • the antigen-binding molecules and polypeptides of the present disclosure may additionally comprise a signal peptide (also known as a leader sequence or signal sequence).
  • Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides.
  • the signal peptide may be present at the N-terminus of the antigen-binding molecule/polypeptide, and may be present in the newly synthesised antigen-binding molecule/polypeptide.
  • the signal peptide provides for efficient trafficking and secretion of the antigen-binding molecule/polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature antigen-binding molecule/polypeptide secreted from the cell expressing the antigen-binding molecule/polypeptide.
  • 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 e.g. 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).
  • SignalP Protein et al., 2011 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176.
  • the antigen-binding molecules of the present disclosure additionally comprise a detectable moiety.
  • the antigen-binding molecule comprises a detectable moiety, e.g. a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label.
  • the antigen-binding molecule may be covalently or non- covalently labelled with the detectable moiety.
  • Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP) chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5.
  • GFP green fluorescent protein
  • Radiolabels include radioisotopes such as Iodine 123 , Iodine 125 , Iodine 126 , Iodine 131 , Iodine 133 , Bromine 77 , Technetium 99m , Indium 111 , lndium 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 , Thuliuml 167 , Thulium 168 , Copper 67 , Fluorine 18 , Yttrium 90 , Palladium 100 , Bismuth 217 and Antimony 211 .
  • radioisotopes such as Iodine
  • Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels.
  • Immuno- detectable labels include haptens, peptides/polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin.
  • Nucleic acid labels include aptamers.
  • Enzymatic labels include e.g. peroxidase, alkaline phosphatase, glucose oxidase, beta-galactosidase and luciferase.
  • the antigen-binding molecules of the present disclosure are conjugated to a chemical moiety.
  • the chemical moiety may be a moiety for providing a therapeutic effect.
  • Antibody-drug conjugates are reviewed e.g. in Parslow et al., Biomedicines. 2016 Sep; 4(3):14.
  • the chemical moiety may be a drug moiety (e.g. a cytotoxic agent).
  • the drug moiety may be a chemotherapeutic agent.
  • the drug moiety is selected from calicheamicin, DM1 , DM4, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), SN-38, doxorubicin, duocarmycin, D6.5 and PBD.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid seguence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid seguence identity to the amino acid seguence of SEQ ID NO:212; and
  • polypeptides comprising, or consisting of, an amino acid seguence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid seguence identity to the amino acid seguence of SEQ ID NO:213.
  • the antigen-binding molecule comprises, or consists of:
  • polypeptides comprising, or consisting of, an amino acid seguence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid seguence identity to the amino acid seguence of SEQ ID NO:214; and (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.
  • the antigen-binding molecule comprises, or consists of:
  • 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
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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 NQ:220; and
  • 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 .
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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 (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.
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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 NQ:230; and
  • 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 .
  • the antigen-binding molecule comprises, or consists of:
  • 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
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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 (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.
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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 NQ:240; and
  • 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 .
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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.
  • the antigen-binding molecule comprises, or consists of:
  • 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 (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.
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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 NQ:250.
  • the antigen-binding molecule comprises, or consists of:
  • 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
  • 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 NQ:250.
  • the antigen-binding molecule comprises, or consists of:
  • 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;
  • 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 NQ:250.
  • the antigen-binding molecule comprises, or consists of:
  • 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 NQ:330; and
  • 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.
  • the antigen-binding molecules described herein may be characterised by reference to certain functional properties.
  • the antigen-binding molecule described herein may possess one or more of the following properties: binds to VISTA (e.g. human, murine and/or cynomolgus macaque VISTA); does not bind to PD-L1 and/or HER3; does not bind to an Fey receptor; does not bind to C1q; does not induce ADCC; does not induce ADCP; does not induce CDC; binds to an FcRn receptor; binds to VISTA with similar affinity at pH from 5.5 to pH 7.5; binds to VISTA-expressing cells; inhibits interaction between VISTA and an interaction partner for VISTA (e.g.
  • LRIG1 PSGL-1 , VSIG3 or VSIG8; inhibits VISTA-mediated signalling; inhibits VISTA-mediated signalling independently of Fc-mediated function; increases killing of VISTA-expressing cells; does not induce/increase killing of VISTA-expressing cells; reduces the number/proportion of VISTA-expressing cells; does not reduce the number/proportion of VISTA-expressing cells; increases effector immune cell number/activity; reduces suppressor immune cell number/activity; reduces suppressor immune cell proliferation; decreases immune suppression mediated by VISTA-expressing cells; increases antigen presentation by antigen-presenting cells; increases production of IL-6 by immune cells; increases production of IFN-y, IL-2 and/or IL-17 in a mixed lymphocyte reaction (MLR) assay; increases T cell proliferation, IFN-y production, TNFa production and/or T cell-mediated lysis of cancer cells; inhibits the development and/or progression of cancer in vivo; and does not induce cytokine release syndrome in vivo.
  • a given antigen-binding molecule may display more than one of the properties recited in the preceding paragraph.
  • a given antigen-binding molecule may be evaluated for the properties recited in the preceding paragraph using suitable assays.
  • the assays may be e.g. in vitro assays, which may be cell-free or cell-based assays.
  • the assays may be e.g. in vivo assays, i.e. performed in non-human animals.
  • assays are cell-based assays, they may comprise contacting cells with a given antigen-binding molecule in order to determine whether the antigen-binding molecule displays one or more of the recited properties.
  • Assays may employ species labelled with detectable entities in order to facilitate their detection.
  • Assays may comprise evaluating the recited properties following treatment of cells separately with a range of quantities/concentrations of antigen-binding molecule (e.g. a dilution series). It will be appreciated that the cells are preferably cells that express VISTA, e.g. MDSCs. Analysis of the results of such assays may comprise determining the concentration at which 50% of the maximal level of the relevant activity is attained.
  • the concentration of antigen-binding molecule at which 50% of the maximal level of the relevant activity is attained may be referred to as the ‘half-maximal effective concentration’ of the antigen-binding molecule in relation to the relevant activity, which may also be referred to as the ‘ECso’.
  • the ECso of a given antigen-binding molecule for binding to VISTA may be the concentration at which 50% of the maximal level of binding to the relevant species is achieved.
  • the ECso may also be referred to as the ‘half-maximal inhibitory concentration’ or ‘ICso’, this being the concentration of antigen-binding molecule at which 50% of the maximal level of inhibition of a given property is observed.
  • the IC50 of a given antigen-binding molecule for inhibiting interaction between VISTA and an interaction partner for VISTA e.g. LRIG1 , PSGL-1 , VSIG3 or VSIG8
  • LRIG1 LRIG1 , PSGL-1 , VSIG3 or VSIG8
  • the antigen-binding molecules described herein bind to VISTA.
  • the antigenbinding molecules display specific binding to VISTA.
  • specific binding refers to binding which is selective for the antigen, and which can be discriminated from non-specific binding to non-target antigen.
  • An antigen-binding molecule that specifically binds to VISTA preferably binds to VISTA with greater affinity, and/or with greater duration than it binds to other, non-target molecules.
  • the ability of a given polypeptide to bind specifically 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 e.g. Hearty et al., Methods Mol Biol (2012) 907:411-442), Bio-Layer Interferometry (see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay.
  • SPR Surface Plasmon Resonance
  • RIA radiolabeled antigen-binding assay
  • the extent of binding of the antigen-binding molecule to an non-target molecule is less than about 10% of the binding of the antibody to the target molecule as measured, e.g. by ELISA, SPR, Bio-Layer Interferometry or by RIA.
  • binding specificity may be reflected in terms of binding affinity where the antigen-binding molecule binds with a dissociation constant (KD) that is at least 0.1 order of magnitude (/.e. 0.1 x 10 n , where n is an integer representing the order of magnitude) greater than the KD of the antigen-binding molecule towards a non-target molecule.
  • KD dissociation constant
  • This may optionally be one of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, or 2.0.
  • an antigen-binding molecule binds to VISTA with a KD of 10 pM or less, preferably one of ⁇ 5 pM, ⁇ 2 pM, ⁇ 1 pM, ⁇ 500 nM, ⁇ 100 nM, ⁇ 75 nM, ⁇ 50 nM, ⁇ 40 nM, ⁇ 30 nM, ⁇ 20 nM, ⁇ 15 nM, ⁇ 12.5 nM, ⁇ 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM, ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM ⁇ 3 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 500 pM, ⁇ 400 pM, ⁇ 300 pM, ⁇ 200 pM, ⁇ 100 pM, ⁇ 50 pM, ⁇ 40 pM, ⁇ 30 pM, ⁇ 20
  • an antigen-binding molecule according to the present disclosure binds to VISTA with a KD (e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure) of ⁇ 1 nM (e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM, ⁇ 500 pM, ⁇ 400 pM, ⁇ 300 pM).
  • a KD e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure
  • ⁇ 1 nM e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM, ⁇ 500 pM, ⁇ 400 pM, ⁇ 300 pM.
  • an antigen-binding molecule according to the present disclosure binds to human VISTA with a KD (e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure) of ⁇ 1 nM (e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM, ⁇ 500 pM).
  • a KD e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure
  • ⁇ 1 nM e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM, ⁇ 500 pM.
  • an antigen-binding molecule according to the present disclosure binds to cynomolgus macaque VISTA with a KD (e.g. as determined by SPR (Biocore) analysis, e
  • an antigen-binding molecule according to the present disclosure binds to rat VISTA with a KD (e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure) of ⁇ 1 nM (e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM, ⁇ 500 pM, ⁇ 400 pM).
  • a KD e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure
  • ⁇ 1 nM e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM, ⁇ 500 pM, ⁇ 400 pM.
  • an antigen-binding molecule according to the present disclosure binds to mouse VISTA with a KD (e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure) of ⁇ 1 nM (e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM).
  • KD e.g. as determined by SPR (Biocore) analysis, e.g. SPR analysis as described in the Examples of the present disclosure
  • ⁇ 1 nM e.g. one of ⁇ 900 pM, ⁇ 800 pM, ⁇ 700 pM, ⁇ 600 pM.
  • an antigen-binding molecule according to the present disclosure binds to VISTA with an ECso (e.g. as determined by ELISA, e.g. an ELISA as described in the Examples of the present disclosure) of 1 pM or less, e.g.
  • the antigen-binding molecule displays binding to human VISTA, murine (e.g. mouse) VISTA, rat VISTA, and/or cynomolgus macaque (Macaca fascicuiaris) VISTA. In some embodiments, the antigen-binding molecule binds to human VISTA and mouse VISTA and rat VISTA and cynomolgus macaque VISTA. In some embodiments, the antigen-binding molecule is cross-reactive for human VISTA and mouse VISTA and rat VISTA and cynomolgus macaque VISTA.
  • the antigen-binding molecule of the present disclosure displays cross-reactivity with VISTA of a non-human primate. Cross-reactivity to VISTA in model species allows in vivo exploration of efficacy in syngeneic models without relying on surrogate molecules.
  • an antigen-binding molecule according to the present disclosure binds to human VISTA with an ECso (e.g. as determined by ELISA, e.g. an ELISA as described in the Examples of the present disclosure) of ⁇ 20 pM (e.g. one of ⁇ 15 pM, ⁇ 12.5 pM, ⁇ 10 pM, ⁇ 7.5 pM).
  • an antigen-binding molecule according to the present disclosure binds to cynomolgus macaque VISTA with an ECso (e.g. as determined by ELISA, e.g. an ELISA as described in the Examples of the present disclosure) of ⁇ 50 pM (e.g. one of ⁇ 25 pM, ⁇ 20 pM, ⁇ 15 pM).
  • an antigen-binding molecule according to the present disclosure binds to rat VISTA with an ECso (e.g. as determined by ELISA, e.g. an ELISA as described in the Examples of the present disclosure) of ⁇ 20 pM (e.g.
  • an antigen-binding molecule according to the present disclosure binds to mouse VISTA with an ECso (e.g. as determined by ELISA, e.g. an ELISA as described in the Examples of the present disclosure) of ⁇ 20 pM (e.g. one of ⁇ 15 pM, ⁇ 12.5 pM, ⁇ 10 pM, ⁇ 7.5 pM, ⁇ 5 pM).
  • an ECso e.g. as determined by ELISA, e.g. an ELISA as described in the Examples of the present disclosure
  • ⁇ 20 pM e.g. one of ⁇ 15 pM, ⁇ 12.5 pM, ⁇ 10 pM, ⁇ 7.5 pM, ⁇ 5 pM.
  • an antigen-binding molecule according to the present disclosure binds to VISTA (e.g. human VISTA) with similar affinity at pH from 5.5 to pH 7.5.
  • VISTA e.g. human VISTA
  • the antigen-binding molecule displays similar affinity for VISTA at pH 5.5 as the affinity for VISTA at pH 7.5.
  • a binding affinity which is ‘similar’ to a reference binding affinity means a binding affinity which is within 50%, e.g. within one of 40%, 45%, 30%, 25%, 20% 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the reference binding affinity, as determined under comparable conditions.
  • the KD for binding to VISTA may be similar at pH from 5.5 to pH 7.5.
  • the ECso for binding to VISTA e.g. human VISTA
  • the KD for binding to VISTA may be similar at pH from 5.5 to pH 7.5.
  • a ‘similar’ KD or ECso value to a reference value may be > 0.5 times and ⁇ 2 times, e.g. one of > 0.7 times and ⁇ 1 .5 times, > 0.75 times and ⁇ 1 .25 times, > 0.8 times and ⁇ 1 .2 times, > 0.85 times and ⁇ 1.15 times, > 0.9 times and ⁇ 1 .1 times, > 0.91 times and ⁇ 1 .09 times, > 0.92 times and ⁇ 1 .08 times, > 0.93 times and ⁇ 1 .07 times, > 0.94 times and ⁇ 1 .06 times, > 0.95 times and ⁇ 1 .05 times, > 0.96 times and ⁇ 1 .04 times, > 0.97 times and ⁇ 1 .03 times, > 0.98 times and ⁇ 1 .02 times, or > 0.99 times and ⁇ 1 .01 times the reference value.
  • the antigen-binding molecule does not display specific binding to PD-L1 (e.g. human PD-L1). In some embodiments, the antigen-binding molecule does not display specific binding to HER3 (e.g. human HER3). In some embodiments, the antigen-binding molecule does not display specific binding to (/.e. does not cross-react with) another member of the B7 family of proteins. In some embodiments, the antigen-binding molecule does not display specific binding to PD-L1 , PD-L2 CD80, CD86, ICOSLG, CD276, VTCN1 , NCR3LG1 , HHLA2 and/or CTLA4.
  • PD-L1 e.g. human PD-L1
  • HER3 e.g. human HER3
  • the antigen-binding molecule does not display specific binding to (/.e. does not cross-react with) another member of the B7 family of proteins.
  • the antigen-binding molecule does
  • the antigen-binding molecule does not display specific binding to PD-1 , PD-L1 , B7H3, VTCN1 (B7H4), NCR3LG1 (B7H6), HHLA2 (B7H7) and/or CTLA4.
  • the antigen-binding molecule is not able to induce one or more Fc-mediated functions (/.e. lacks the ability to elicit the relevant Fc-mediated function(s)).
  • Such antigen-binding molecules may be described as being devoid of the relevant function(s).
  • an Fc region/antigen-binding molecule which does not induce (/.e. is not able to induce) ADCC/ADCP/CDC elicits substantially no ADCC/ADCP/CDC activity, e.g. as determined by analysis in an appropriate assay for the relevant activity.
  • an antigen-binding molecule “which does not bind to” a reference protein e.g. a given Fc receptor or complement protein
  • the antigen-binding molecule does not induce ADCC. In some embodiments, the antigen-binding molecule does not induce ADCP. In some embodiments, the antigen-binding molecule does not induce CDC. In some embodiments, the antigen-binding molecule does not induce ADCC and/or does not induce ADCP and/or does not induce CDC.
  • the antigen-binding molecule does not bind to an Fc receptor. In some embodiments, the antigen-binding molecule does not bind to an Fey receptor. In some embodiments, the antigen-binding molecule does not bind to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb. In some embodiments, the antigen-binding molecule does not bind to FcyRIII (e.g. FcyRllla and/or FcyRlllb). In some embodiments, the antigen-binding molecule does not bind to FcyRllla.
  • the antigen-binding molecule does not bind to FcyRlla. In some embodiments, the antigenbinding molecule does not bind to FcyRllb. In some embodiments, the antigen-binding molecule binds to FcRn. In some embodiments, the antigen-binding molecule does not bind to a complement protein. 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.
  • 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).
  • CTLA4 e.g. human PD-L1/PD- 1/B7H3/VTCN1/NCR3LG1/HHLA2/CTLA4
  • the antigen-binding molecule described herein binds to VISTA (e.g. human VISTA, mouse VISTA) with a KD of 10 pM or less, preferably one of ⁇ 5 pM, ⁇ 2 pM, ⁇ 1 pM, ⁇ 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.
  • VISTA e.g. human VISTA, mouse VISTA
  • VISTA e.g. human VISTA, mouse VISTA
  • KD 10 nM, ⁇ 9 nM, ⁇ 8 nM, ⁇ 7 nM or ⁇ 6 nM, ⁇ 5 nM, ⁇ 4 nM, ⁇ 3 nM, ⁇ 2 nM or ⁇ 1 nM.
  • the antigen-binding molecule binds to VISTA (e.g.
  • the antigen-binding molecules of the present disclosure may bind to a particular region of interest of VISTA.
  • the antigen-binding region of an antigen-binding molecule according to the present domain may bind to a linear epitope of VISTA, consisting of a contiguous sequence of amino acids (/.e. an amino acid primary sequence).
  • the antigen-binding region molecule may bind to a conformational epitope of VISTA, consisting of a discontinuous sequence of amino acids of the amino acid sequence.
  • the antigen-binding molecule of the present disclosure is capable of binding to VISTA. In some embodiments, the antigen-binding molecule is capable of binding to VISTA in an extracellular region of VISTA. In some embodiments, the antigen-binding molecule is capable of binding to VISTA in the Ig-like V-type domain (e.g. the region shown in SEQ ID NO:6). In some embodiments, the antigen-binding molecule is capable of binding to VISTA in the region shown in SEQ ID NO:31 .
  • the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:6. In some embodiments, the antigenbinding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:31. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:322. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:26.
  • the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:27. In some embodiments, the antigenbinding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:28. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:29. In some embodiments, the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NQ:30.
  • the antigen-binding molecule does not bind to the region of VISTA bound by IGN175A (described e.g. in WO 2014/197849 A2). In some embodiments, the antigen-binding molecule does not bind to the region of VISTA bound by an antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:267 and a polypeptide consisting of the sequence of SEQ ID NO:268.
  • the antigen-binding molecule does not compete with IGN175A (described e.g. in WO 2014/197849 A2) for binding to VISTA. In some embodiments, the antigen-binding molecule does not compete with an antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:267 and a polypeptide consisting of the sequence of SEQ ID NO:268 for binding to VISTA.
  • the ability of a given antigen-binding molecule to compete with IGN175A or the antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:267 and a polypeptide consisting of the sequence of SEQ ID NO:268 for binding to VISTA can be analysed e.g. by competition ELISA, or by epitope binning as described in Abdiche et al., J Immunol Methods (2012) 382(— 2):101 -116 (hereby incorporated by reference in its entirety). Epitope binning can be performed e.g. by BLI analysis, e.g. as described in Example 8 of the present application.
  • the antigen-binding molecule is not capable of binding to a peptide consisting of the amino acid sequence shown in SEQ ID NO:275.
  • a “peptide” refers to a chain of two or more amino acid monomers linked by peptide bonds.
  • a peptide typically has a length in the region of about 2 to 50 amino acids.
  • a “polypeptide” is a polymer chain of two or more peptides. Polypeptides typically have a length greater than about 50 amino acids.
  • an antigen-binding molecule to bind to a given peptide/polypeptide can be analysed by methods well known to the skilled person, including analysis by ELISA, immunoblot (e.g. western blot), immunoprecipitation, surface plasmon resonance and biolayer interferometry.
  • the antigen-binding molecule is capable of binding the same region of VISTA, or an overlapping region of VISTA, to the region of VISTA which is bound by an antibody comprising the VH and VL sequences of one of 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, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A11 or 9M2-C12.
  • the antigen-binding molecule is capable of binding to a region of VISTA which is different to the region of VISTA bound by IGN175A (described e.g. in WO 2014/197849 A2). In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA which is different to the region of VISTA bound by an antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:267 and a polypeptide consisting of the sequence of SEQ ID NO:268.
  • the antigen-binding molecule is capable of binding to a region of VISTA which does not overlap the region of VISTA bound by IGN175A (described e.g. in WO 2014/197849 A2). In some embodiments, the antigen-binding molecule is capable of binding to a region of VISTA which does not overlap with the region of VISTA bound by an antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:267 and a polypeptide consisting of the sequence of SEQ ID NO:268.
  • the antigen-binding molecule binds to VISTA through contact with residues of VISTA which are non-identical to the residues of VISTA which are contacted by VSTB112 (described e.g. in WO 2015/097536 A2). In some embodiments, the antigen-binding molecule binds to VISTA through contact with residues of VISTA which are non-identical to the residues of VISTA which are contacted by an antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:269 and a polypeptide consisting of the sequence of SEQ ID NQ:270.
  • the epitope for the antigen-binding molecule is non-identical to the epitope for VSTB112. In some embodiments, the epitope for the antigen-binding molecule is non-identical to the epitope for an antigen-binding molecule comprised of a polypeptide consisting of the sequence of SEQ ID NO:269 and a polypeptide consisting of the sequence of SEQ ID NO:270.
  • the region of a peptide/polypeptide to which an antibody binds can be determined by the skilled person using various methods well known in the art, including X-ray co-crystallography analysis of antibodyantigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competition ELISA and proteolysis-based ‘protection’ methods. Such methods are described, for example, in Gershoni et al., BioDrugs, 2007, 21 (3):145-156, which is hereby incorporated by reference in its entirety.
  • the antigen-binding molecule of the present disclosure binds to VISTA in a region which is accessible to an antigen-binding molecule (/.e., an extracellular antigen-binding molecule) when VISTA is expressed at the cell surface (/.e. in or at the cell membrane).
  • the antigen-binding molecule is capable of binding to VISTA expressed at the cell surface of a cell expressing VISTA.
  • the antigen-binding molecule is capable of binding to VISTA-expressing cells (e.g. CD14+ monocytes (such as monocyte-derived suppressor cells (MDSCs)) and/or CD33+ myeloid cells, tumor associated macrophages (TAMs), and neutrophils).
  • the ability of an antigen-binding molecule to bind to a given cell type can be analysed by contacting cells with the antigen-binding molecule, and detecting antigen-binding molecule bound to the cells, e.g. after a washing step to remove unbound antigen-binding molecule.
  • the ability of an antigen-binding molecule to bind to immune cell surface molecule-expressing cells and/or cancer cell antigen-expressing cells can be analysed by methods such as flow cytometry and immunofluorescence microscopy.
  • the antigen-binding molecule of the present disclosure may be an antagonist of VISTA.
  • the antigen-binding molecule is capable of inhibiting a function or process (e.g. interaction, signalling or other activity) mediated by VISTA and/or an interaction partner for VISTA (e.g. LRIG1 , VSIG3, PSGL-1 , VSIG8).
  • a function or process e.g. interaction, signalling or other activity
  • an interaction partner for VISTA e.g. LRIG1 , VSIG3, PSGL-1 , VSIG8
  • ‘inhibition’ refers to a reduction, decrease or lessening relative to a control condition.
  • An antigen-binding molecule which inhibits a given interaction/activity/process may be referred to as inhibitor or antagonist of the interaction/activity/process, and may be said to ‘block’ or ‘neutralise’ the interaction/activity/process.
  • VISTA-binding antigen-binding molecules described herein are able to inhibit VISTA-mediated functions/processes by a mechanism not requiring Fc-mediated functions such as ADCC, ADCP and CDC. That is, VISTA-binding antigen-binding molecules described herein are able to inhibit the immunosuppressive activity of VISTA-expressing cells without the need to elicit ADCC, ADCP and/or CDC.
  • VISTA-binding antigen-binding molecules described herein are able to inhibit VISTA via a mechanism not requiring binding to Fey receptors and/or binding to C1q.
  • the antigen-binding molecule of the present disclosure is capable of inhibiting interaction between VISTA and an interaction partner for VISTA (e.g. LRIG1 , VSIG3, PSGL-1 , VSIG8).
  • the antigen-binding molecule is capable of inhibiting interaction between VISTA and an interaction partner for VISTA that binds to the C-C’ region of VISTA.
  • an interaction partner for VISTA that binds to the C-C’ region of VISTA binds to the region of VISTA shown in SEQ ID NO:344. In some embodiments, an interaction partner for VISTA that binds to the C-C’ region of VISTA binds to a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:344. In some embodiments, an interaction partner for VISTA that binds to the C-C’ region of VISTA contacts the region of VISTA shown in SEQ ID NO:344. In some embodiments, an interaction partner for VISTA that binds to the C-C’ region of VISTA binds to VISTA via contact with one or more amino acids of the region shown in SEQ ID NO:344.
  • an interaction partner for VISTA that binds to the C-C’ region of VISTA is selected from LRIG1 and VSIG3. In some embodiments, an interaction partner for VISTA is LRIG1. In some embodiments, an interaction partner for VISTA is VSIG3.
  • the antigen-binding molecule of the present disclosure is capable of inhibiting interaction between VISTA and LRIG1. In some embodiments, the antigen-binding molecule of the present disclosure is capable of inhibiting interaction between VISTA and PSGL-1 . In some embodiments, the antigen-binding molecule of the present disclosure is capable of inhibiting interaction between VISTA and VSIG3.
  • an antigen-binding molecule to inhibit interaction between two factors can be determined for example by analysis of interaction in the presence of, or following incubation of one or both of the interaction partners with, the antibody/fragment.
  • Assays for determining whether a given antigen-binding molecule is capable of inhibiting interaction between two interaction partners include competition ELISA assays and analysis by SPR.
  • An antigen-binding molecule which is capable of inhibiting a given interaction is identified by the observation of a reduction/decrease in the level of interaction between the interaction partners in the presence of - or following incubation of one or both of the interaction partners with - the antigen-binding molecule, 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).
  • Suitable analysis can be performed in vitro, e.g. using recombinant interaction partners or using cells expressing the interaction partners. Cells expressing interaction partners may do so endogenously, or may do so from nucleic acid introduced into the cell.
  • one or both of the interaction partners and/or the antigen-binding molecule may be labelled or used in conjunction with a detectable entity for the purposes of detecting and/or measuring the level of interaction.
  • the ability of an antigen-binding molecule to inhibit interaction between two binding partners can also be determined by analysis of the downstream functional consequences of such interaction.
  • downstream functional consequences of interaction between VISTA and an interaction partner for VISTA may include VISTA-mediated signalling.
  • the ability of an antigen-binding molecule to inhibit interaction of VISTA and an interaction partner for VISTA may be determined by analysis of production of IL-2, IFN-y and/or IL-17 in an MLR assay.
  • the antigen-binding molecule of the present disclosure is capable of inhibiting interaction between VISTA and an interaction partner for VISTA (e.g. LRIG1 , VSIG3, PSGL-1 , VSIG8) to less than less than 1 times, e.g.
  • the antigen-binding molecule inhibits VISTA-mediated signalling.
  • VISTA-mediated signalling may be signalling mediated by a polypeptide complex comprising VISTA.
  • VISTA-mediated signalling may be signalling mediated by a polypeptide complex comprising VISTA and an interaction partner for VISTA (e.g. LRIG1 , VSIG3, PSGL- 1 , VSIG8).
  • VISTA-mediated signalling may be signalling mediated by a polypeptide complex comprising VISTA and an interaction partner for VISTA that binds to the C-C’ region of VISTA (e.g. LRIG1 or VSIG3).
  • VISTA-mediated signalling may be signalling mediated by a polypeptide complex comprising VISTA and LRIG1. In some embodiments, VISTA- mediated signalling may be signalling mediated by a polypeptide complex comprising VISTA and VSIG3.
  • VISTA-mediated signalling can be analysed e.g. using an assay of effector immune cell number/activity, such as an MLR assay as described in the experimental examples herein. Inhibition of VISTA-mediated signalling can be identified by detection of an increase in the number and/or activity of effector immune cells, as determined e.g. by an increase in production of IL-2, IFN-y and/or IL-17.
  • the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring or involving Fc-mediated function. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling independently of Fc-mediated function. That is, in some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling in an Fc region-independent manner.
  • an antigen-binding molecule to inhibit VISTA-mediated signalling by a mechanism not requiring/involving Fc-mediated function can be evaluated e.g. by analysing the ability of the antigenbinding molecule provided in a format lacking a functional Fc region to inhibit VISTA-mediated signalling.
  • the effect on VISTA-mediated signalling can be investigated using an antigen-binding molecule comprising a ‘silent’ Fc region (e.g. comprising LALA PG substitutions), or using an antigenbinding molecule provided in a format lacking an Fc region (e.g. scFv, Fab etc.).
  • the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not involving ADCC. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not involving ADCP. In some embodiments, the antigenbinding molecule is able to inhibit VISTA-mediated signalling by a mechanism not involving CDC.
  • the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding of the antigen-binding molecule to an Fc receptor. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding of the antigen-binding molecule to an Fey receptor. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding of the antigen-binding molecule to one or more of FcyRI, FcyRlla, FcyRllb, FcyRllc, FcyRllla and FcyRlllb.
  • the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding to FcyRllla. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding to FcyRlla. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding to FcyRllb. In some embodiments, the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding to a complement protein.
  • the antigen-binding molecule is able to inhibit VISTA-mediated signalling by a mechanism not requiring binding to C1q. In some embodiments, the antigen-binding molecule is able to inhibit VISTA- mediated signalling by a mechanism not requiring N297 glycosylation.
  • the antigen-binding molecule of the present disclosure is capable of increasing killing of VISTA-expressing cells. Killing of VISTA-expressing cells may be increased through an effector function of the antigen-binding molecule. In embodiments wherein antigen-binding molecule comprises an Fc region the antigen-binding molecule may increase killing of VISTA-expressing cells through one or more of complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • An antigen-binding molecule which is capable of increasing killing of VISTA-expressing cells can be identified by observation of an increased level of killing of VISTA-expressing cells in the presence of - or following incubation of the VISTA-expressing cells with - the antigen-binding molecule, as compared to the level of cell killing detected in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule), in an appropriate assay. Assays of CDC, ADCC and ADCP are well known the skilled person. The level of killing of VISTA-expressing cells can also be determined by measuring the number/proportion of viable and/or non-viable VISTA-expressing cells following exposure to different treatment conditions.
  • the antigen-binding molecule of the present disclosure is capable of increasing killing of VISTA-expressing cells (e.g. VISTA-expressing MDSCs) to more than 1 times, e.g. >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 the level of killing observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • VISTA-expressing cells e.g. VISTA-expressing MDSCs
  • the antigen-binding molecule of the present disclosure is capable of reducing the number of VISTA-expressing cells (e.g. VISTA-expressing MDSCs) to less than less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times, ⁇ 0.05 times, or ⁇ 0.01 times the number of VISTA-expressing cells (e.g. VISTA- expressing MDSCs, TAMs, neutrophils) detected following incubation in the absence of the antigenbinding molecule (or following incubation in the presence of an appropriate control antigen-binding molecule), in a comparable assay.
  • VISTA-expressing cells
  • the antigen-binding molecule is a non-depleting antigen-binding molecule. That is, in some embodiments, the antigen-binding molecule does not cause substantial depletion of VISTA- expressing cells. In some embodiments, the antigen-binding molecule does not elicit/increase ADCC, ADCP and/or CDC against VISTA-expressing cells.
  • the antigen-binding molecule of the present disclosure does not induce/increase killing of VISTA-expressing cells, e.g. in embodiments wherein the antigen-binding molecule lacks an Fc region, or embodiments wherein the antigen-binding molecule comprises an Fc region which is not able to induce an Fc-mediated antibody effector function. In some embodiments, the antigen-binding molecule of the present disclosure does not reduce the number/proportion of VISTA-expressing cells.
  • the antigen-binding molecule of the present disclosure (i) inhibits VISTA-mediated signalling, and (ii) does not induce/increase killing of VISTA-expressing cells. In some embodiments, the antigen-binding molecule of the present disclosure (i) inhibits VISTA-mediated signalling, and (ii) does not reduce the number/proportion of VISTA-expressing cells.
  • VISTA is expressed by cells that it is not desirable to deplete.
  • VISTA is expressed at low levels by immune cells (e.g. certain types of T cells and dendritic cells) that it is not desirable to kill or reduce the number/proportion of.
  • the antigen-binding molecule of the present disclosure is capable of increasing the number and/or activity of effector immune cells relative to a negative control condition, e.g. in an appropriate in vitro assay, or in vivo.
  • the antigen-binding molecules of the present disclosure may be capable of releasing effector immune cells from MDSC-mediated suppression of effector immune cell proliferation and function.
  • the effector immune cells may be e.g. CD8+ T cells, CD8+ cytotoxic T lymphocytes (CD8+ CTLs), CD4+ T cells, CD4+ T helper cells, NK cells, IFNy-producing cells, memory T cells, central memory T cells, antigen-experienced T cells or CD45RO+ T cells.
  • the antigen-binding molecule of the present disclosure is capable of increasing the number of an effector immune cell type to more than 1 times, e.g. >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 the number observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • the antigen-binding molecule of the present disclosure is capable of increasing the level of a correlate of effector immune cell activity to more than 1 times, e.g. >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 the level observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • Cell numbers and proportions can be determined e.g. by flow cytometry analysis using antibodies allowing detection of cell types.
  • Cell division can be analysed, for example, by in vitro analysis of incorporation of 3 H-thymidine or by CFSE dilution assay, e.g. as described in Fulcher and Wong, Immunol Cell Biol (1999) 77(6): 559-564, hereby incorporated by reference in entirety.
  • Effector immune cell activity can be analysed by measuring a correlate of such activity.
  • effector immune cell activity can be determined e.g. by analysis of production of IL-2, IFN-y and/or IL-17.
  • the antigen-binding molecule of the present disclosure is capable of decreasing the level of immune suppression mediated by VISTA-expressing cells.
  • a change in the level of immune suppression may be determined using methods to measure the expression of arginase 1 and/or the production of reactive oxygen species (ROS) by VISTA-expressing cells, for example as described in Ochoa et al., Ann Surg. 2001 Mar; 233(3): 393-399 and Dikalov and Harrison Antioxid Redox Signal. 2014 Jan 10; 20(2): 372-382.
  • ROS reactive oxygen species
  • the antigen-binding molecule of the present disclosure is capable of increasing antigen presentation by antigen-presenting cells, e.g. as determined using a suitable assay of antigen presentation.
  • the antigen-binding molecule of the present disclosure is capable of increasing phagocytosis by phagocytic cells (e.g. neutrophils, monocytes, macrophages, mast cells, and/or dendritic cells), e.g. as determined using a suitable assay of the level of phagocytosis.
  • phagocytic cells e.g. neutrophils, monocytes, macrophages, mast cells, and/or dendritic cells
  • the antigen-binding molecule of the present disclosure is capable of increasing the number and/or activity of antigen-presenting cells (e.g. CD11 b+ MHCII+ cells) relative to a negative control condition, e.g. in an appropriate in vitro assay, or in vivo (e.g. in a tumor).
  • the antigen-binding molecule is capable of increasing the number and/or activity of macrophages (e.g. CD11 b+ F4/80+ cells) relative to a negative control condition, e.g. in an appropriate in vitro assay, or in vivo (e.g. in a tumor).
  • the antigen-binding molecule is capable of increasing the number and/or activity of dendritic cells (e.g. CD11c+ cells) relative to a negative control condition, e.g. in an appropriate in vitro assay, or in vivo (e.g. in a tumor).
  • the antigen-binding molecule of the present disclosure is capable of increasing the number of a cell type recited in the preceding paragraph to more than 1 times, e.g.
  • the antigenbinding molecule of the present disclosure is capable of increasing the level of a correlate of activity of a cell type recited in the preceding paragraph to more than 1 times, e.g. >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 the level observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • the antigen-binding molecule of the present disclosure is capable of increasing production of IL-6 by immune cells.
  • the immune cells may be e.g. PBMCs, lymphocytes, T cells, B cells, NK cells, or monocytes.
  • the immune cells are monocytes.
  • the antigen-binding molecule is capable of increasing production of IL-6 by immune cells following stimulation, e.g. with LPS.
  • the ability of an antigen-binding molecule to increase production of IL-6 by immune cells can be analysed in an in vitro assay e.g. as described in Example 10 herein.
  • Such methods may comprise stimulating monocytes (e.g. THP1 cells) with LPS, and incubating the stimulated cells with the antigen-binding molecule.
  • the antigen-binding molecule of the present disclosure is capable of increasing IL- 6 production by immune cells (e.g. LPS-stimulated THP1 cells) to more than 1 times, e.g. >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 the level observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • immune cells e.g. LPS-stimulated THP1 cells
  • the antigen-binding molecule of the present disclosure is capable of increasing the number and/or activity of Th1/Th17 cells. In some embodiments, the antigen-binding molecule is capable of upregulating the Th1/Th17 response. In some embodiments, the antigen-binding molecule favours the Th1/Th17 response over the Th2 response. In some embodiments, the antigen-binding molecule of the present disclosure is capable of increasing T cell proliferation, IL-2 production, IFN-y production, TNFa production and/or IL-17A production in a Mixed Lymphocyte Reaction (MLR) assay. MLR assays may be performed as described in Bromelow et al J.
  • IL-2, IFNy and/or IL-17 production may be analysed e.g. by antibody-based methods well known to the skilled person, such as western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or by reporter-based methods.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell (e.g. Th1/Th17 cell) proliferation, IL-2 production, IFN-y production and/or IL-17 production in an MLR assay to more than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell (e.g. Th1/Th17 cell) proliferation, IFN-y production and/or TNFa production, e.g. in the presence of VISTA/VISTA expressing cells.
  • Antigen-binding molecules may be evaluated for such properties e.g. in in vitro assays as described in the experimental examples herein.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell (e.g. Th1/Th17 cell) proliferation, IFN-y production and/or TNFa production (e.g. in the presence of VISTA/VISTA expressing cells) to more than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell (e.g. CD4+ T cell and/or CD8+ T cell, e.g. Th1/Th17 cell) proliferation to a greater extent than a VISTA-binding antibody disclosed in the prior art (e.g. VSTB112, described e.g. in WO 2015/097536 A2).
  • T cell proliferation may be evaluated in an in vitro assay e.g. as described in Example 9 herein, and may involve stimulating T cell proliferation by culture in the presence of agonist anti-CD3 antibody.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell proliferation in such an assay to more than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell-mediated lysis of cancer cells to more than 1 times, e.g. >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 the level observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell-mediated lysis of cancer cells, e.g. in the presence of VISTA/VISTA expressing cells.
  • Antigen-binding molecules may be evaluated for such properties e.g. in in vitro assays as described in the experimental examples herein.
  • the antigen-binding molecule of the present disclosure is capable of increasing T cell-mediated lysis (e.g. in the presence of VISTA/VISTA expressing cells) to more than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure is capable of increasing IL- 6 production by THP1 cells to a greater extent than a VISTA-binding antibody disclosed in the prior art (e.g. VSTB112, described e.g. in WO 2015/097536 A2).
  • IL-6 production by THP1 cells may be evaluated in an in vitro assay e.g. as described in Example 10 herein, and may involve stimulating THP1 cells with LPS.
  • the antigen-binding molecule of the present disclosure is capable of increasing IL-6 production in such an assay to more than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure is capable of: reducing the number and/or activity of suppressor immune cells, inhibiting proliferation of suppressor immune cells, and/or reducing the proportion of suppressor immune cells within a population of cells (e.g. CD45+ cells, e.g. CD45+ cells obtained from a tumor) relative to control condition, e.g. as determined in an appropriate in vitro assay, or in vivo.
  • a population of cells e.g. CD45+ cells, e.g. CD45+ cells obtained from a tumor
  • control condition e.g. as determined in an appropriate in vitro assay, or in vivo.
  • the suppressor immune cells may be e.g. VISTA-expressing cells, Arg 1 -expressing cells, MDSCs, granulocytic MDSCs (g-MDSCs) or monocytic MDSCs (m-MDSCs).
  • the suppressor immune cells are CD11 b+ GR1 + MHCII- cells.
  • the reduction in the number/activity/proliferation/proportion is to less than 1 times, e.g. ⁇ 0.99 times, ⁇ 0.95 times, ⁇ 0.9 times, ⁇ 0.85 times, ⁇ 0.8 times, ⁇ 0.75 times, ⁇ 0.7 times, ⁇ 0.65 times, ⁇ 0.6 times, ⁇ 0.55 times, ⁇ 0.5 times, ⁇ 0.45 times, ⁇ 0.4 times, ⁇ 0.35 times, ⁇ 0.3 times, ⁇ 0.25 times, ⁇ 0.2 times, ⁇ 0.15 times, ⁇ 0.1 times, ⁇ 0.05 times, or ⁇ 0.01 times the number/activity/proliferation/proportion observed in the absence of the antigen-binding molecule (or in the presence of an appropriate control antigen-binding molecule).
  • the antigen-binding molecule is able to reduce the number/activity/proliferation/proportion of suppressor immune cells by a mechanism not involving emediated function. In some embodiments, the antigen-binding molecule is able to reduce the number/activity/proliferation/proportion of suppressor immune cells independently of Fc-mediated function (/.e. in an Fc region-independent manner). In some embodiments, the antigen-binding molecule is able to reduce the number/activity/proliferation/proportion of suppressor immune cells by a mechanism not involving ADCC, ADCP and/or CDC. In some embodiments, the antigen-binding molecule is able to reduce the number/activity/proliferation/proportion of suppressor immune cells by a mechanism not involving depletion of VISTA-expressing cells.
  • the antigen-binding molecule of the present disclosure inhibits the development and/or progression of cancer in vivo.
  • the antigen-binding molecule causes an increase in the killing of cancer cells, e.g. by effector immune cells. In some embodiments, the antigen-binding molecule causes a reduction in the number of cancer cells in vivo, e.g. as compared to an appropriate control condition. In some embodiments, the antigen-binding molecule inhibits tumor growth, e.g. as determined by measuring tumor size/volume over time.
  • the antigen-binding molecule of the present disclosure is capable of increasing serum levels of IFN-y and/or IL-23 in mice treated with the antigen-binding molecule. Serum levels of IFN- y and/or IL-23 can be analysed e.g. by ELISA of serum derived from blood samples obtained from the mice. In some embodiments, administration of the antigen-binding molecule of the present disclosure increases serum level of IFN-y and/or IL-23 to more than 1 times, e.g.
  • the antigen-binding molecule of the present disclosure may be analysed for the ability to inhibit development and/or progression of cancer in an appropriate in vivo model, e.g. cell line-derived xenograft model such as CT26 cell-derived model, a 4T-1 cell-derived model, an LL2 cell-derived model, a B16 cell- derived model, or an EL4 cell-derived model.
  • the cancer may be a cancer in which VISTA-expressing cells and/or MDSCs (e.g. VISTA-expressing MDSCs, TAMs, neutrophils) are pathologically implicated.

Abstract

La présente divulgation concerne des molécules de liaison à l'antigène qui se lient à VISTA pour une utilisation dans une méthode de traitement ou de prévention d'un cancer, des compositions comprenant lesdites molécules, des méthodes thérapeutiques et prophylactiques utilisant lesdites molécules, et des méthodes de sélection d'un sujet pour un traitement.
PCT/EP2022/076793 2021-09-27 2022-09-27 Traitement et prévention du cancer à l'aide de molécules de liaison à l'antigène vista WO2023046979A1 (fr)

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