WO2019185163A1 - Molécules de liaison à l'antigène vista - Google Patents

Molécules de liaison à l'antigène vista Download PDF

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Publication number
WO2019185163A1
WO2019185163A1 PCT/EP2018/058258 EP2018058258W WO2019185163A1 WO 2019185163 A1 WO2019185163 A1 WO 2019185163A1 EP 2018058258 W EP2018058258 W EP 2018058258W WO 2019185163 A1 WO2019185163 A1 WO 2019185163A1
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WIPO (PCT)
Prior art keywords
amino acid
acid sequence
seq
antigen
binding molecule
Prior art date
Application number
PCT/EP2018/058258
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English (en)
Inventor
Jerome Douglas BOYD-KIRKUP
Piers INGRAM
Dipti THAKKAR
Zhihao WU
Konrad PASZKIEWICZ
Original Assignee
Hummingbird Bioscience Holdings Pte. Ltd.
CLEGG, Richard Ian
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Application filed by Hummingbird Bioscience Holdings Pte. Ltd., CLEGG, Richard Ian filed Critical Hummingbird Bioscience Holdings Pte. Ltd.
Priority to PCT/EP2018/058258 priority Critical patent/WO2019185163A1/fr
Priority to US16/180,949 priority patent/US20190300610A1/en
Priority to PL19714654T priority patent/PL3645570T3/pl
Priority to DK19714654.1T priority patent/DK3645570T3/da
Priority to EP19714654.1A priority patent/EP3645570B1/fr
Priority to KR1020247002424A priority patent/KR20240015156A/ko
Priority to CA3095417A priority patent/CA3095417A1/fr
Priority to SG11202009515RA priority patent/SG11202009515RA/en
Priority to ES19714654T priority patent/ES2869549T3/es
Priority to CN202210352325.0A priority patent/CN114507285A/zh
Priority to JP2020552406A priority patent/JP7118332B2/ja
Priority to CN201980036745.4A priority patent/CN112513080B/zh
Priority to AU2019240906A priority patent/AU2019240906A1/en
Priority to PCT/EP2019/058036 priority patent/WO2019185879A1/fr
Priority to TW108111311A priority patent/TWI828673B/zh
Priority to KR1020207031017A priority patent/KR102629403B1/ko
Priority to PT197146541T priority patent/PT3645570T/pt
Priority to US17/042,855 priority patent/US20210380697A1/en
Priority to EP21151388.2A priority patent/EP3868786A1/fr
Publication of WO2019185163A1 publication Critical patent/WO2019185163A1/fr
Priority to JP2022076578A priority patent/JP2022128604A/ja
Priority to US18/518,192 priority patent/US20240158538A1/en

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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
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the fields of molecular biology, more specifically antibody technology.
  • the present invention also relates to 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-b and S100A8/A9 which promote neovascularisation, invasion, proliferation and metastasis.
  • soluble factors such as matrix metalloproteinases, VEGF, bFGF, TGF-b and S100A8/A9 which promote neovascularisation, invasion, proliferation and metastasis.
  • V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), an immune checkpoint inhibitor expressed primarily on MDSCs, is an attractive therapeutic strategy for removing MDSC-mediated suppression of effector immune cell function.
  • the present invention provides an antigen-binding molecule, optionally isolated, which is capable of binding to VISTA in the Ig-like V-type domain.
  • the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:6.
  • the antigen-binding molecule is capable of binding to a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:31.
  • VH heavy chain variable
  • 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:246;
  • 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 NO:245
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
  • VH heavy chain variable
  • 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;
  • 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 NO:42
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
  • VH heavy chain variable
  • 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;
  • 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 NO:67
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
  • VH heavy chain variable
  • 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;
  • 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 NO:58
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43.
  • VH heavy chain variable
  • 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;
  • VL light chain variable
  • the antigen-binding molecule comprises:
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:88
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:89
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:90;
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:98.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:88
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:89
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:90;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 137
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 138
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 139.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 107
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 108,
  • VL light chain variable
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 1 15.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 120
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 121
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 122;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 127
  • the antigen-binding molecule comprises:
  • VH heavy chain variable
  • 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;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 151
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 152
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 153.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 158
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 159
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 160;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 165
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 152
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 153.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 169 HC-CDR2 having the amino acid sequence of SEQ ID NO: 170 HC-CDR3 having the amino acid sequence of SEQ ID NO: 171 ;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 177
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 179.
  • VH heavy chain variable
  • 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;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO:247
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190.
  • the antigen-binding molecule comprises: (i) a heavy chain variable (VH) region incorporating the following CDRs:
  • 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;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 189
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190.
  • VH heavy chain variable
  • 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;
  • VL light chain variable
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 197
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:190.
  • VH heavy chain variable
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 184
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:200;
  • VL light chain variable
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190.
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:32;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:40;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:52;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:57;
  • VH region comprising an amino acid sequence having at least 70% 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 to the amino acid sequence of SEQ ID NO:66;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:48;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:50;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:87;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:95;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 106;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 1 13;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 143;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 150;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 157;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 164;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:71 ;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:79;
  • (x) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 102;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 104;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 126; or
  • VL region comprising an amino acid sequence having at least 70% 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 to the amino acid sequence of SEQ ID NO: 196;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 199;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:202;
  • (xv) a VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 133;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 136;
  • VH region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 168;
  • VL region comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:176.
  • the antigen-binding molecule is capable of binding to human VISTA and one or more of: mouse VISTA and cynomolgus macaque VISTA.
  • an antigen-binding molecule optionally isolated, comprising (i) an antigen-binding molecule according to the present invention, and (ii) an antigen-binding molecule capable of binding to an antigen other than VISTA.
  • the antigen-binding molecule is capable of binding to cells expressing VISTA at the cell surface.
  • the antigen-binding molecule is capable of inhibiting interaction between VISTA and a binding partner for VISTA.
  • the antigen-binding molecule is capable of inhibiting VISTA-mediated signalling.
  • a chimeric antigen receptor (CAR) comprising an antigen-binding molecule according to the present invention.
  • nucleic acid or a plurality of nucleic acids, optionally isolated, encoding an antigenbinding molecule or a CAR according to the present invention.
  • an expression vector or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to the present invention.
  • Also provided is a cell comprising an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors according to the present invention.
  • Also provided is a method comprising culturing a cell comprising a nucleic acid or a plurality of nucleic acids, or an expression vector or a plurality of expression vectors according to the invention, under conditions suitable for expression of the antigen-binding molecule or CAR from the nucleic acid(s) or expression vector(s).
  • composition comprising an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, expression vector or plurality of expression vectors, or a cell according to the present invention.
  • the composition additionally comprises an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor other than VISTA, optionally wherein the immune checkpoint inhibitor other than VISTA is selected from PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT and BTLA.
  • an antigen-binding molecule CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors, cell, or composition according to the invention for use in a method of medical treatment or prophylaxis.
  • an antigen-binding molecule CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors, cell, or composition of the invention for use in a method of treatment or prevention of a cancer or an infectious disease.
  • an antigen-binding molecule CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors, cell, or composition of the invention in the manufacture of a medicament for use in a method of treatment or prevention of a cancer or an infectious disease.
  • Also provided is a method of treating or preventing a cancer or an infectious disease comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors, cell, or composition of the invention.
  • the cancer is selected from: colorectal cancer, pancreatic cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC) and a solid tumor.
  • an antigen-binding molecule CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors, cell, or composition of the invention for use in a method of treatment or prevention of a disease in which myeloid-derived suppressor cells (MDSCs) are pathologically implicated.
  • MDSCs myeloid-derived suppressor cells
  • an antigen-binding molecule, CAR, nucleic acid or a plurality of nucleic acids, expression vector or a plurality of expression vectors, cell, or composition of the invention in the manufacture of a medicament for use in a method of treatment or prevention of a disease in which myeloid-derived suppressor cells (MDSCs) are pathologically implicated.
  • MDSCs myeloid-derived suppressor cells
  • MDSCs myeloid-derived suppressor cells
  • the methods additionally comprise administration of an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor other than VISTA, optionally wherein the immune checkpoint inhibitor other than VISTA is selected from PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA.
  • MDSCs myeloid-derived suppressor cells
  • an in vitro complex optionally isolated, comprising an antigen-binding molecule according to the invention bound to VISTA.
  • Also provided is a method comprising contacting a sample containing, or suspected to contain, VISTA with an antigen-binding molecule according to the invention, and detecting the formation of a complex of the antigen-binding molecule with VISTA.
  • an antigen-binding molecule in a method for detecting, localizing or imaging a cancer, optionally wherein the cancer is selected from: colorectal cancer, pancreatic cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC) and a solid tumor.
  • the cancer is selected from: colorectal cancer, pancreatic cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC) and a solid tumor.
  • NSCLC non-small cell lung cancer
  • the present invention relates to novel VISTA-binding molecules having improved properties as compared to known anti-VISTA antibodies.
  • the inventors generated antigen-binding molecules which bind to particular regions of interest in the extracellular region of VISTA.
  • the VISTA-binding molecules of the present invention are provided with combinations of desirable biophysical and functional properties as compared to VISTA-binding antigenbinding molecules disclosed in the prior art.
  • 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 sequence 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 (i.e. after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO:2.
  • Positions 33 to 194 of SEQ ID NO:1 form the extracellular domain (SEQ ID NO:3)
  • positions 195 to 215 form 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 sequence identity to the amino acid sequence 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 VSIG-3.
  • 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
  • VISTA has been shown to be a co-inhibitory receptor on CD4+ T cells or a co-inhibitory ligand for T cells.
  • VISTA _/ 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 VSIG-3 (IGSF1 1 ) - see e.g. Wang et al., J Immunol (2017),
  • VSIG-3 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 NO:7 at positions 1 to 17; and isoform 3 (UniProt: Q5DX21-3; SEQ ID NO:9), which comprises a different sequence to SEQ ID NO:7 at positions 1 to 17, and which also comprises a different sequence to SEQ ID NO:7 at positions 21 1-235.
  • the N-terminal 22 amino acids of SEQ ID NOs:7, 8 and 9 constitute a signal peptide, and so the mature form of VSIG-3 isoforms 1 , 2 and 3 (i.e. after processing to remove the signal peptide) have the amino acid sequences shown in SEQ ID NOs:10, 1 1 and 12, respectively. Positions 23 to 241 of SEQ ID NOs:7, and 8 form the extracellular domain of VSIG-3 isoforms 1 and 2 (SEQ ID NO:13), and positions 23 to 216 of SEQ ID NO:9 form the extracellular domain of VSIG-3 isoform 3 (SEQ ID NO: 14).
  • the transmembrane domain of VSIG-3 is 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 VSIG-3 isoforms 1 and 2 additionally comprise an Ig-like C2-type domain (shown in SEQ ID NO: 18).
  • VSIG-3 refers to VSIG-3 from any species and includes VSIG-3 isoforms, fragments, variants (including mutants) or homologues from any species.
  • a fragment of VSIG-3 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 VSIG-3 is VSIG-3 from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and/or a rodent (e.g. rat or murine) VSIG-3).
  • Isoforms, fragments, variants or homologues of VSIG-3 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 VSIG-3 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 VSIG-3, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of VSIG-3 may e.g. display association with VISTA.
  • the VSIG-3 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to one of SEQ ID NOs:7 to 12.
  • a fragment of VSIG-3 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%,
  • VISTA has also been proposed to interact with VSIG-8 - see e.g. WO/2017/090347 A1.
  • VSIG-8 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 VSIG-8 (i.e. after processing to remove the signal peptide) has the amino acid sequence shown in SEQ ID NO:20.
  • Positions 22 to 263 of SEQ ID NO: 19 form the extracellular domain of VSIG-8 (SEQ ID NO:21 ).
  • the transmembrane domain of VSIG-8 is shown in SEQ ID NO:22, 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).
  • VSIG-8 refers to VSIG-8 from any species and includes VSIG-8 isoforms, fragments, variants (including mutants) or homologues from any species.
  • a fragment of VSIG-8 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 VSIG-8 is VSIG-8 from a mammal (e.g. a primate (rhesus, cynomolgous, nonhuman primate or human) and/or a rodent (e.g. rat or murine) VSIG-8).
  • Isoforms, fragments, variants or homologues of VSIG-8 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 VSIG-8 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 VSIG-8, as determined by analysis by a suitable assay for the functional property/activity.
  • an isoform, fragment, variant or homologue of VSIG-8 may e.g. display association with VISTA.
  • the VSIG-8 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 VSIG-8 comprises, or consists of, an amino acid sequence having at least 70%, preferably one of 80%, 85%,
  • the antigen-binding molecules of the present invention 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 invention may be defined by reference to the region of VISTA which they bind to.
  • the antigen-binding molecules of the present invention 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 (i.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 invention binds to VISTA. In some embodiments, the antigen-binding molecule binds to the extracellular region of VISTA (e.g. the region shown in SEQ ID NO:3). In some embodiments, the antigen-binding molecule binds to the Ig-like V-type domain of VISTA (e.g. the region shown in SEQ ID NO:6). In some embodiments, the antigen-binding molecule binds to VISTA 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: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 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 antibody- antigen 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 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, 2M1-B12, 2M1-D2, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A11 or 9M2-C12 described herein.
  • 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 invention is capable of binding to a polypeptide comprising, or consisting of, the amino acid sequence of one of SEQ ID NOs:1 , 2, 3, 6 or 31.
  • the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:26. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:27. In some embodiments, the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:28. In some embodiments, the antigen-binding molecule is capable of binding to a
  • the antigen-binding molecule is capable of binding to a peptide/polypeptide comprising, or consisting of, the amino acid sequence of SEQ ID NO:30.
  • 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 (i.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, 2M1-B12, 2M1-D2, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A1 1 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 (i.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/EBRb and STAT3; decreased expression of IRF8; and increased production of S100A8/9 proteins.
  • NO reactive oxygen species
  • c/EBRb and STAT3 decreased expression of IRF8; and increased production of S100A8/9 proteins.
  • 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 CD1 1 b + Gr1 + cells.
  • Gr-1 hi cells are mostly PMN-MDSCs
  • Gr-1 '° cells are mostly M-MDSCs.
  • M-MDSCs are CD1 1 b + Ly6C hi Ly6G
  • PMN-MDSCs are CD1 1 b + Ly6C'°Ly6G +
  • MDSCs are identified in the mononuclear fraction.
  • PMN-MDSCs are CD14-CD1 1 b + CD33 + CD15 + or CD66b + cells
  • M-MDSCs are CD14 + HLA-DR-/'° 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
  • effector immune cell function e.g. effector T cell and NK cell function
  • VEGF vascular endothelial growth factor
  • bFGF vascular endothelial growth factor
  • 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 (i.e. VISTA may be expressed in or at the cell membrane).
  • the present invention provides antigen-binding molecules capable of binding to VISTA.
  • An“antigen-binding molecule” refers to a molecule which is capable of binding to a target antigen, and encompasses monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab ⁇ , diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as they display binding to the relevant target molecule(s).
  • monospecific and multispecific antibodies e.g., bispecific antibodies
  • antibody fragments e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab ⁇ , diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.
  • the antigen-binding molecule of the present invention 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): 181- 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 (i.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 (i.e. a single-domain
  • the antigen-binding molecules of the present invention 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.
  • polypeptide e.g. 2, 3, 4, 6, or 8 polypeptides
  • IgG-like antigen-binding molecule comprising two heavy chain polypeptides and two light chain polypeptides.
  • the antigen-binding molecules of the present invention 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 antigen-binding molecule comprises the CDRs of an antigen-binding molecule which is capable of binding to VISTA.
  • 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 (i.e. anti- VISTA antibody clones 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1 , V4H2, 2M1-B12, 2M1-D2, 1 M2-D2, 13D5p, 13D5-1 , 13D5-13, 5M1-A1 1 or 9M2-C12).
  • a VISTA-binding antibody clone described herein i.e. anti- VISTA antibody clones 4M2-C12, 4M2-B4, 4M2-C9, 4M2-D9, 4M2-D5, 4M2-A8, V4H1 , V4H2, 2M1-B12, 2M1-D2, 1 M2-D2, 13D
  • the antigen-binding molecule comprises a VH region according to one of (1 ) to (14) below:
  • 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:246,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • 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,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • 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,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • 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,
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:88
  • HC-CDR2 having the amino acid sequence of SEQ ID NO:89
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:90,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:33
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 107
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 108,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 120
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 121
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 122,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • 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,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 158
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 159
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 160,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO: 169
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 170
  • HC-CDR3 having the amino acid sequence of SEQ ID NO: 171 ,
  • 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,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • 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,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • 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,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • HC-CDR1 having the amino acid sequence of SEQ ID NO:72
  • HC-CDR2 having the amino acid sequence of SEQ ID NO: 184
  • HC-CDR3 having the amino acid sequence of SEQ ID NO:200,
  • HC-CDR1 HC- CDR2
  • HC-CDR3 HC-CDR3
  • the antigen-binding molecule comprises a VH region according to one of (15) to (29) below:
  • HC-FR1 having the amino acid sequence of SEQ ID NO:36
  • HC-FR2 having the amino acid sequence of SEQ ID NO:37
  • HC-FR3 having the amino acid sequence of SEQ ID NO:38
  • HC-FR4 having the amino acid sequence of SEQ ID NO:39,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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.
  • V4H1 a VH region incorporating the following FRs:
  • 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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 (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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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: 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 NO: 109
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 1 10
  • HC-FR3 having the amino acid sequence of SEQ ID NO: 1 1 1
  • HC-FR4 having the amino acid sequence of SEQ ID NO: 1 12,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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: 123
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 124
  • HC-FR3 having the amino acid sequence of SEQ ID NO: 125
  • HC-FR4 having the amino acid sequence of SEQ ID NO:78,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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,
  • 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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: 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,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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: 103
  • HC-FR2 having the amino acid sequence of SEQ ID NO: 186
  • HC-FR3 having the amino acid sequence of SEQ ID NO:201
  • HC-FR4 having the amino acid sequence of SEQ ID NO:86,
  • HC-FR1 HC-FR2
  • HC-FR3 HC-FR4
  • HC-FR4 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 (14) above, and the FRs according to one of (15) to (29) above.
  • the antigen-binding molecule comprises a VH region according to one of (30) to (47) below:
  • VH region comprising the CDRs according to (1 1 ) and the FRs according to (28) or (29).
  • the antigen-binding molecule comprises a VH region according to one of (48) to (63) 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:32.
  • 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.
  • 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.
  • 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.
  • 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: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 NO: 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 NO: 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: 1 19.
  • 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.
  • 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.
  • 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.
  • 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 (64) to (79) below:
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43;
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:43;
  • 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-CDR3 having the amino acid sequence of SEQ ID NO:98;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 127
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 129;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 137
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 138
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 139;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 151
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 152
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 153;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 165
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 152
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:153; or a 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-CDR1 having the amino acid sequence of SEQ ID NO: 177
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 179;
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO:190;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 189
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;
  • LC-CDR1 having the amino acid sequence of SEQ ID NO: 197
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;
  • LC-CDR2 having the amino acid sequence of SEQ ID NO: 178
  • LC-CDR3 having the amino acid sequence of SEQ ID NO: 190;
  • the antigen-binding molecule comprises a VL region according to one of (80) to (95) below:
  • LC-FR1 having the amino acid sequence of SEQ ID NO:44
  • LC-FR2 having the amino acid sequence of SEQ ID NO:45
  • LC-FR3 having the amino acid sequence of SEQ ID NO: 101
  • LC-FR4 having the amino acid sequence of SEQ ID NO:86
  • LC-FR1 having the amino acid sequence of SEQ ID NO: 105
  • LC-FR1 having the amino acid sequence of SEQ ID NO: 130
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 131
  • LC-FR3 having the amino acid sequence of SEQ ID NO: 132
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 141
  • LC-FR3 having the amino acid sequence of SEQ ID NO: 142
  • LC-FR1 having the amino acid sequence of SEQ ID NO: 154
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 155
  • LC-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: 166
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 155
  • LC-FR3 having the amino acid sequence of SEQ ID NO: 167
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 192
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 198
  • LC-FR2 having the amino acid sequence of SEQ ID NO: 192
  • the antigen-binding molecule comprises a VL region comprising the CDRs according to one of (64) to (79) above, and the FRs according to one of (80) to (95) above.
  • the antigen-binding molecule comprises a VL region according to one of (96) to (1 13) below:
  • (1 1 1 ) a VL region comprising the CDRs according to (77) and the FRs according to (93).
  • the antigen-binding molecule comprises a VL region according to one of (1 14) to (129) below:
  • 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.
  • 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 NO: 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: 1 13.
  • 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%,
  • 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%,
  • 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%,
  • 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%,
  • 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%,
  • 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%,
  • the antigen-binding molecule comprises a VH region according to any one of (1 ) to (63) above, and a VL region according to any one of (64) to (129) above.
  • substitutions may 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 invention 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 invention 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 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 1 10 of SEQ ID NO:205 form a hinge region between CH1 and CH2 regions (SEQ ID NO:207). Positions 1 1 1 to 223 of SEQ ID NO:205 form the CH2 region (SEQ ID NO:208). Positions 224 to 330 of SEQ ID NO:205 form the CH3 region (SEQ ID NO:209).
  • 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 CH 1 region comprises or consists of the sequence of SEQ ID NO:206, or a sequence having at least 60%, preferably one of 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%,
  • a CH1-CH2 hinge region comprises or consists of the sequence of SEQ ID NO: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 NO:207.
  • a CH2 region comprises or consists of the sequence of SEQ ID NO: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 NO:208.
  • a CH3 region comprises or consists of the sequence of SEQ ID NO: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 NO:209 or 210.
  • the antigen-binding molecule of the present invention 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:21 1 ).
  • the immunoglobulin light chain constant sequence is a human immunoglobulin lambda constant (IGLC; CK), 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%,
  • the VL and light chain constant (CL) region, and the VH region and heavy chain constant 1 (CH1 ) region of an antigen-binding region of an antibody together constitute the Fab region.
  • 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 invention 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).
  • Ig immunoglobulin
  • 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.
  • IgG e.g. lgG1 , lgG2, lgG3, lgG4
  • IgA e.g. lgA1 , lgA2
  • IgD IgE
  • IgM which binds to VISTA.
  • the antigen-binding molecule of the present invention 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 antigenbinding molecule is e.g. bivalent, trivalent or tetravalent for VISTA. Aspects of the present invention 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. In some
  • the antigen-binding molecule comprises at least two different antigen-binding domains (i.e. at least two antigen-binding domains, e.g. comprising non-identical 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 invention 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 invention 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 invention 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 (i.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 (i.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 invention is on the external surface of the immune cell (i.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 inhibitor (e.g. PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA) or a ligand thereof.
  • Multispecific antigen-binding molecules according to the invention 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. lgG ⁇ , F(ab’)2 or CovX-Body; IgG or IgG-like molecules, e.g. IgG, chimeric IgG, kl-body common HC; CH1/CL fusion proteins, e.g.
  • scFv2-CH1/CL, VHH2-CH1/CL;‘variable domain only’ bispecific antigen-binding 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-Fab3, DNL-Fab2-scFv, DNL- Fab2-lgG-cytokine2; asymmetric IgG or IgG-like molecules, e.g.
  • IgGs appended and Fc-modified IgGs, e.g. lgG(kih)-Fv, IgG HA-TF-Fv, lgG(kih)scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-lg, DVI-lg (four-in-one), CrossMab-Fab; modified Fc and CH3 fusion proteins, e.g.
  • modified IgGs e.g. DAF (two-in one-lgG), DutaMab, Mab 2 ; and non-lg fusions, e.g. DNL-Fab 4 -lgG.
  • 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:IV:2.13:2.13.1—2.13.16, which is hereby incorporated by reference in its entirety.
  • 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) ⁇ 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 according to the present invention 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:
  • a DNA construct encoding the light and heavy chain variable domains for the two antigen-binding fragments i.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
  • 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 invention comprise an Fc region.
  • An Fc region is 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.
  • the antigen-binding molecule of the present invention 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. Immnol (2016) 7:394, which is hereby incorporated by reference in its entirety.
  • the antigen antigen-binding molecule of the present invention 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.
  • one of the CH3 regions of the Fc region of the antigen-binding molecule of the present invention 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.
  • 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 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 invention comprises an Fc region modified as described in Labrijn et al., Proc Natl Acad Sci U S A. (2013) 1 10(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 invention 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
  • 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 invention comprises an Fc region comprising the“SEED” modification as described in Davis et al., Protein Eng Des Sel (2010) 23(4):195- 202, in which b-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 (201 1 ) 3(6):546-57).
  • one of the CH3 regions comprises the substitutions T350V, L351 Y, 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 present invention 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 invention 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 invention comprises, or consists of, a VH as described herein. In some embodiments a polypeptide according to the present invention 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). In some embodiments the polypeptide comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments the polypeptide comprises a CH 1 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 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; Q347
  • the CH2 and/or CH3 regions of the polypeptide comprise one or more amino acid substitutions for promoting association of the polypeptide with another polypeptide comprising a CH2 and/or CH3 region.
  • 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.
  • polypeptide according to the present invention comprises a structure from N- to C-terminus according to one of the following:
  • antigen-binding molecules composed of the polypeptides of the present invention.
  • the antigen-binding molecule of the present invention 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- CH 1-CH2-CH3, and two polypeptides comprising the structure VL-CL.
  • the antigen-binding molecule of the present invention comprises one of the following combinations of polypeptides:
  • VH (anti-VISTA) refers to the VH of an antigen-binding molecule capable of binding to VISTA as described herein, e.g. as defined in one of (1 ) to (63);“VL (anti-VISTA)” refers to the VL of an antigenbinding molecule capable of binding to VISTA as described herein, e.g. as defined in one of (64) to (129).
  • 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.
  • the antigen-binding molecules and polypeptides of the present invention comprise a hinge region.
  • a hinge region is provided between a CH 1 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 invention 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 invention 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
  • the antigen-binding molecule/polypeptide comprises a detectable moiety, e.g. a fluorescent, lunminescent, immuno-detectable, radio, chemical, nucleic acid or enzymatic label.
  • 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 invention 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
  • 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., 201 1 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176).
  • SignalP Protein et al., 201 1 Nature Methods 8: 785-786
  • Signal-BLAST Frank and Sippl, 2008 Bioinformatics 24: 2172- 2176.
  • the antigen-binding molecules of the present invention 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.
  • 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” 171 , Indium 111 , lndiurn 113m , Gallium 67 , Gallium 68 , Ruthenium 95 , Ruthenium 97 , Ruthenium 103 , Ruthenium 105 , Mercury 207 , Mercury 203 , Rhenium” 111 , Rhenium 101 , Rhenium 105 , Scandium 47 , Tellurium 121111 , Tellurium 122111 , Tellurium 125111 , Thulium 165 , Thuliuml 167 , Thulium 168 , Copper 67 , Fluorine 18 , Yttrium", Palladium 100 , Bismuth 217 and Antimony 211 .
  • 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 invention 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 sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:212; and
  • 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:214;
  • 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 NO:220;
  • 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: (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:224; and
  • 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 NO: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: (i) two polypeptides comprising, or consisting of, an amino acid sequence having at least 70%, preferably one of 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO:234; and
  • 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 NO:240;
  • 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 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:
  • VISTA e.g. human, murine and/or cynomolgus macague VISTA
  • VISTA inhibits interaction between VISTA and a binding partner for VISTA (e.g. VSIG-3 or VSIG-8); inhibits VISTA-mediated signalling;
  • a binding partner for VISTA e.g. VSIG-3 or VSIG-8
  • the antigen-binding molecules described herein preferably 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 a target molecule preferably binds the target 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) 907:41 1-442), 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 (i.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.
  • the antigen-binding molecule displays binding to human VISTA, murine (e.g. mouse) VISTA and/or cynomolgus macaque ( Macaca fascicularis) VISTA. That is, in some embodiments the antigen-binding molecule is cross-reactive for human VISTA and murine VISTA and/or cynomolgus macaque VISTA. In some embodiments the antigen-binding molecule of the present invention 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.
  • 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 (i.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 and/or HHLA2.
  • PD-L1 e.g. human PD-L1
  • HER3 e.g. human HER3
  • the antigen-binding molecule does not display specific binding to (i.e. does not cross-react with) another member of the B7 family of proteins.
  • the antigen-binding molecule does not display specific
  • the antigen-binding molecule binds to human VISTA, murine VISTA and/or cynomolgus macaque VISTA; and does not bind to PD-L1 (e.g. human PD-L1 ).
  • the antigen-binding molecule described herein binds to VISTA (e.g. human VISTA) with a KD of 10 mM or less, preferably one of ⁇ 5 mM, ⁇ 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
  • VISTA e.g. human 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.
  • KD ⁇ 500 pM, ⁇ 100 pM, ⁇ 90 pM, ⁇ 80 pM, ⁇ 70 pM or ⁇ 60 pM, ⁇ 50 pM, ⁇ 40 pM, ⁇ 30 pM, ⁇ 20 pM, ⁇ 10 pM, ⁇ 9 pM, ⁇ 8 pM, ⁇ 7 pM or ⁇ 6 pM, ⁇ 5 pM, ⁇ 4 pM, ⁇ 3 pM, ⁇ 2 pM or ⁇ 1 pM.
  • the antigen-binding molecules of the present invention 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 (i.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 invention 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 antigen-binding 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:26. In some embodiments the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:27.
  • the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:28. In some embodiments the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:29. In some embodiments the antigen-binding molecule is capable of binding to a peptide or polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO:30.
  • 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 ,
  • 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 antibody- antigen 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 invention binds to VISTA in a region which is accessible to an antigen-binding molecule (i.e., an extracellular antigen-binding molecule) when VISTA is expressed at the cell surface (i.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 invention may be an antagonist of VISTA.
  • the antigen-binding molecule is capable of inhibiting a function or process (e.g.
  • inhibitor refers to a reduction, decrease or lessening relative to a control condition.
  • the antigen-binding molecule of the present invention is capable of inhibiting interaction between VISTA and a binding partner for VISTA (e.g. VSIG-3, VSIG-8). In some embodiments the antigen-binding molecule of the present invention is capable of inhibiting interaction between VISTA and VSIG-3.
  • 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 a binding partner for VISTA may include VISTA-mediated signalling.
  • the ability of an antigen-binding molecule to inhibit interaction of VISTA and a binding partner for VISTA may be determined by analysis of production of IL-2, IFN-g and/or IL-17 in an MLR assay.
  • the antigen-binding molecule of the present invention is capable of inhibiting interaction between VISTA and a binding partner for VISTA (e.g. VSIG-3, VSIG-8) to less than less than 1 times, e.g.
  • the antigen-binding molecule inhibits VISTA-mediated signalling.
  • 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-g and/or IL-17.
  • the antigen-binding molecule of the present invention 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.
  • antigen-binding molecule comprises an Fc region the antigen-binding molecule may increasing 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 invention 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 invention 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.
  • the antigen-binding molecule of the present invention 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 invention 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.
  • 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 by analysis of production of IL-2, IFN-g and/or IL-17.
  • the antigen-binding molecule of the present invention 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 invention 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).
  • the antigen-binding molecule of the present invention 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 Sura. 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 invention 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 invention 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 invention is capable of increasing T cell proliferation, IL-2 production, IFN-g production and/or IL-17 production in a Mixed Lymphocyte Reaction (MLR) assay.
  • MLR assays may be performed as described in Bromelow et al J. Immunol Methods, 2001 Jan 1 ;247(1 -2): 1 -8, (hereby incorporated by reference in its entirety), or as described in the experimental examples herein.
  • 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 invention is capable of increasing T cell proliferation, IL-2 production, IFN-g production and/or IL-17 production in an MLR assay 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 invention 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
  • 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 invention 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 or a B16 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.
  • Cancers in which MDSCs are‘pathologically implicated’ include cancers in which MDSCs, or an increased number/proportion of MDSCs, is positively associated with onset, development or progression of the cancer, and/or severity of one or more symptoms of the cancer, or a cancer for which MDSCs, or an increased number/proportion of MDSCs, is a risk factor for the onset, development or progression of the cancer.
  • the cancer may comprise MDSCs in an organ/tissue which is affected by the disease (e.g. an organ/tissue in which the symptoms of the disease/condition manifest) or in a tumor.
  • administration of an antigen-binding molecule according to the present invention may cause one or more of: inhibition of the development/progression of the cancer, a delay to/prevention of onset of the cancer, a reduction in/delay to/prevention of tumor growth, a reduction in/delay to/prevention of metastasis, a reduction in the severity of the symptoms of the cancer, a reduction in the number of cancer cells, a reduction in tumour size/volume, and/or an increase in survival (e.g. progression free survival), e.g. as determined in an CT26 cell line and/or 4T-1 cell line-derived xenograft model.
  • survival e.g. progression free survival
  • the present invention also provides Chimeric Antigen Receptors (CARs) comprising the antigen-binding molecules or polypeptides of the present invention.
  • CARs Chimeric Antigen Receptors
  • CARs are recombinant receptors that provide both antigen-binding and T cell activating functions.
  • CAR structure and engineering is reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1 ), hereby incorporated by reference in its entirety.
  • CARs comprise an antigen-binding region linked to a cell membrane anchor region and a signalling region.
  • An optional hinge region may provide separation between the antigen-binding region and cell membrane anchor region, and may act as a flexible linker.
  • the CAR of the present invention comprises an antigen-binding region which comprises or consists of the antigen-binding molecule of the present invention, or which comprises or consists of a polypeptide according to the invention.
  • the cell membrane anchor region is provided between the antigen-binding region and the signalling region of the CAR and provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen-binding region in the extracellular space, and signalling region inside the cell.
  • the CAR comprises a cell membrane anchor region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the transmembrane region amino acid sequence for one of CDS-z, CD4, CD8 or CD28.
  • a region which is‘derived from’ a reference amino acid sequence comprises an amino acid sequence having at least 60%, e.g. one of at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the reference sequence.
  • the signalling region of a CAR allows for activation of the T cell.
  • the CAR signalling regions may comprise the amino acid sequence of the intracellular domain of CDS-z, which provides immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation and activation of the CAR-expressing T cell.
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Signalling regions comprising sequences of other ITAM-containing proteins such as FcyRI have also been employed in CARs (Haynes et al., 2001 J Immunol 166(1 ): 182-187).
  • Signalling regions of CARs may also comprise co-stimulatory sequences derived from the signalling region of co-stimulatory molecules, to facilitate activation of CAR-expressing T cells upon binding to the target protein.
  • Suitable co-stimulatory molecules include CD28, 0X40, 4-1 BB, ICOS and CD27.
  • CARs are engineered to provide for co-stimulation of different intracellular signalling pathways.
  • signalling associated with CD28 costimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, whereas the 4-1 BB-mediated signalling is through TNF receptor associated factor (TRAF) adaptor proteins.
  • TNF receptor associated factor (TRAF) adaptor proteins TNF receptor associated factor
  • the CAR of the present invention comprises one or more co-stimulatory sequences comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the intracellular domain of one or more of CD28, 0X40, 4-1 BB, ICOS and CD27.
  • an optional hinge region may provide separation between the antigen-binding domain and the transmembrane domain, and may act as a flexible linker. Hinge regions may be derived from IgG 1.
  • the CAR of the present invention comprises a hinge region comprising or consisting of an amino acid sequence which comprises, consists of, or is derived from, the amino acid sequence of the hinge region of lgG1 .
  • a cell comprising a CAR according to the invention.
  • the CAR according to the present invention may be used to generate CAR-expressing immune cells, e.g. CAR-T or CAR-NK cells.
  • the antigen-binding region of the CAR of the present invention may be provided with any suitable format, e.g. scFv, scFab, etc.
  • the present invention provides a nucleic acid, or a plurality of nucleic acids, encoding an antigen-binding molecule, polypeptide or CAR according to the present invention.
  • the nucleic acid is purified or isolated, e.g. from other nucleic acid, or naturally- occurring biological material.
  • the nucleic acid(s) comprise or consist of DNA and/or RNA.
  • the present invention also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present invention.
  • the nucleotide sequence may be contained in a vector, e.g. an expression vector.
  • A“vector” as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.
  • the vector may be a vector for expression of the nucleic acid in the cell.
  • Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the sequence to be expressed.
  • a vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the invention.
  • operably linked may include the situation where a selected nucleic acid sequence and regulatory nucleic acid sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of nucleic acid sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette).
  • a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence.
  • the resulting transcript(s) may then be translated into a desired
  • Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g.
  • gammaretroviral vectors e.g. murine Leukemia virus (MLV)-derived vectors
  • lentiviral vectors e.g. murine Leukemia virus (MLV)-derived vectors
  • lentiviral vectors e.g. lentiviral vectors
  • adenovirus vectors e.g. lentiviral vectors
  • adeno-associated virus vectors e.g. vaccinia virus vectors and herpesvirus vectors
  • transposon-based vectors e.g. yeast artificial chromosomes
  • the vector may be a eukaryotic vector, e.g. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell.
  • the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.
  • CMV cytomegalovirus
  • Constituent polypeptides of an antigen-binding molecule according to the present invention may be encoded by different nucleic acids of the plurality of nucleic acids, or by different vectors of the plurality of vectors.
  • the present invention also provides a cell comprising or expressing an antigen-binding molecule, polypeptide or CAR according to the present invention. Also provided is a cell comprising or expressing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the invention.
  • the cell may be a eukaryotic cell, e.g. a mammalian cell.
  • the mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Eguidae), donkey, and non-human primate).
  • a primate rhesus, cynomolgous, non-human primate or human
  • a non-human mammal e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e
  • the present invention also provides a method for producing a cell comprising a nucleic acid(s) or vector(s) according to the present invention, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present invention into a cell.
  • introducing an isolated nucleic acid(s) or vector(s) according to the invention into a cell comprises transformation, transfection, electroporation or transduction (e.g. retroviral transduction).
  • the present invention also provides a method for producing a cell expressing/comprising an antigenbinding molecule, polypeptide or CAR according to the present invention, comprising introducing a nucleic acid, a plurality of nucleic acids, a vector or a plurality of vectors according to the present invention in a cell.
  • the methods additionally comprise culturing the cell under conditions suitable for expression of the nucleic acid(s) or vector(s) by the cell.
  • the methods are performed in vitro.
  • the present invention also provides cells obtained or obtainable by the methods according to the present invention. Producing the antigen-binding molecules and polypeptides
  • Antigen-binding molecules and polypeptides according to the invention may be prepared according to methods for the production of polypeptides known to the skilled person.
  • Polypeptides may be prepared by chemical synthesis, e.g. liquid or solid phase synthesis.
  • peptides/polypeptides can by synthesised using the methods described in, for example, Chandrudu et al., Molecules (2013), 18: 4373-4388, which is hereby incorporated by reference in its entirety.
  • antigen-binding molecules and polypeptides may be produced by recombinant expression.
  • Molecular biology techniques suitable for recombinant production of polypeptides are well known in the art, such as those set out in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Press, 2012, and in Nat Methods. (2008); 5(2): 135-146 both of which are hereby incorporated by reference in their entirety.
  • Methods for the recombinant production of antigen-binding molecules are also described in Frenzel et al., Front Immunol. (2013); 4: 217 and Kunert and Reinhart, Appl Microbiol Biotechnol. (2016) 100: 3451-3461 , both of which are hereby incorporated by reference in their entirety.
  • the antigen-binding molecule of the present invention are comprised of more than one polypeptide chain.
  • production of the antigen-binding molecules may comprise transcription and translation of more than one polypeptide, and subsequent association of the polypeptide chains to form the antigen-binding molecule.
  • the cell may be a prokaryote or eukaryote.
  • the cell is a prokaryotic cell, such as a cell of archaea or bacteria.
  • the bacteria may be Gramnegative bacteria such as bacteria of the family Enterobacteriaceae, for example Escherichia coli.
  • the cell is a eukaryotic cell such as a yeast cell, a plant cell, insect cell or a mammalian cell, e.g. CHO, HEK (e.g. HEK293), HeLa or COS cells.
  • the cell is a CHO cell that transiently or stably expresses the polypeptides.
  • the cell is not a prokaryotic cell because some prokaryotic cells do not allow for the same folding or post-translational modifications as eukaryotic cells.
  • very high expression levels are possible in eukaryotes and proteins can be easier to purify from eukaryotes using appropriate tags.
  • Specific plasmids may also be utilised which enhance secretion of the protein into the media.
  • polypeptides may be prepared by cell-free-protein synthesis (CFPS), e.g.
  • Production may involve culture or fermentation of a eukaryotic cell modified to express the polypeptide(s) of interest.
  • the culture or fermentation may be performed in a bioreactor provided with an appropriate supply of nutrients, air/oxygen and/or growth factors.
  • Secreted proteins can be collected by partitioning culture media/fermentation broth from the cells, extracting the protein content, and separating individual proteins to isolate secreted polypeptide(s).
  • Culture, fermentation and separation techniques are well known to those of skill in the art, and are described, for example, in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th Edition; incorporated by reference herein above).
  • Bioreactors include one or more vessels in which cells may be cultured. Culture in the bioreactor may occur continuously, with a continuous flow of reactants into, and a continuous flow of cultured cells from, the reactor. Alternatively, the culture may occur in batches.
  • the bioreactor monitors and controls environmental conditions such as pH, oxygen, flow rates into and out of, and agitation within the vessel such that optimum conditions are provided for the cells being cultured.
  • the polypeptide(s) of interest may be isolated. Any suitable method for separating proteins from cells known in the art may be used. In order to isolate the polypeptide it may be necessary to separate the cells from nutrient medium. If the polypeptide(s) are secreted from the cells, the cells may be separated by centrifugation from the culture media that contains the secreted polypeptide(s) of interest. If the polypeptide(s) of interest collect within the cell, protein isolation may comprise centrifugation to separate cells from cell culture medium, treatment of the cell pellet with a lysis buffer, and cell disruption e.g. by sonification, rapid freeze-thaw or osmotic lysis.
  • polypeptide(s) of interest may be isolated from the supernatant or culture medium, which may contain other protein and non-protein components.
  • a common approach to separating protein components from a supernatant or culture medium is by precipitation. Proteins of different solubilities are precipitated at different concentrations of precipitating agent such as ammonium sulfate. For example, at low concentrations of precipitating agent, water soluble proteins are extracted. Thus, by adding different increasing concentrations of precipitating agent, proteins of different solubilities may be distinguished. Dialysis may be subsequently used to remove ammonium sulfate from the separated proteins.
  • precipitating agent such as ammonium sulfate
  • polypeptide(s) of interest may be desired or necessary to concentrate the polypeptide(s).
  • a number of methods for concentrating proteins are known in the art, such as ultrafiltration or lyophilisation.
  • the present invention also provides compositions comprising the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein.
  • the antigen-binding molecules, polypeptides, CARs, nucleic acids, expression vectors and cells described herein may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the composition may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration which may include injection or infusion.
  • Suitable formulations may comprise the antigen-binding molecule in a sterile or isotonic medium.
  • Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.
  • composition is formulated for injection or infusion, e.g. into a blood vessel or tumor.
  • such methods of production may comprise one or more steps selected from: producing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; isolating an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein; and/or mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • a further aspect the invention described herein relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease/condition (e.g. a cancer), the method comprising formulating a pharmaceutical composition or medicament by mixing an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof) or cell described herein with a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.
  • a disease/condition e.g. a cancer
  • antigen-binding molecules polypeptides, CARs, nucleic acids, expression vectors, cells and compositions described herein find use in therapeutic and prophylactic methods.
  • the present invention provides an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is the use of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition.
  • Also provided is a method of treating or preventing a disease or condition comprising administering to a subject a therapeutically or prophylactically effective amount of an antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition described herein.
  • the methods may be effective to reduce the development or progression of a disease/condition, alleviation of the symptoms of a disease/condition or reduction in the pathology of a disease/condition.
  • the methods may be effective to prevent progression of the disease/condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease/condition.
  • the methods may lead to an improvement in the disease/condition, e.g. a reduction in the symptoms of the
  • the methods may prevent development of the disease/condition a later stage (e.g. a chronic stage or metastasis).
  • the articles of the present invention may be used for the treatment/prevention of any disease/condition that would derive therapeutic or prophylactic benefit from a reduction in the number and/or activity of cells expressing VISTA (e.g. MDSCs).
  • VISTA cells expressing VISTA
  • the therapeutic and prophylactic utility of the present invention extends to essentially any disease/condition which would benefit from a reduction in the number or activity of MDSCs and/or other cells expressing VISTA, e.g. tumor-associated macrophages (TAMs) and neutrophils.
  • TAMs tumor-associated macrophages
  • Antagonism of VISTA effectively releases effector immune cells from suppression by MDSCs and/or other cells expressing VISTA.
  • the disease/condition may be a disease/condition in which cells expressing VISTA (e.g. MDSCs) are pathologically implicated, e.g. a disease/condition in which an increased number/proportion of cells expressing VISTA (e.g. MDSCs) is positively associated with the onset, development or progression of the disease/condition, and/or severity of one or more symptoms of the disease/condition, or for which an increased number/proportion of cells expressing VISTA (e.g. MDSCs), is a risk factor for the onset, development or progression of the disease/condition.
  • VISTA e.g. MDSCs
  • the disease/condition to be treated/prevented in accordance with the present invention is a disease/condition characterised by an increase in the number/proportion/activity of cells expressing VISTA (e.g. MDSCs), e.g. as compared to the number/proportion/activity of cells expressing VISTA (e.g. MDSCs) in the absence of the disease/condition.
  • VISTA e.g. MDSCs
  • a subject may be selected for treatment described herein based on the detection of an increase in the number/proportion/activity of cells expressing VISTA (e.g. MDSCs), e.g. in the periphery, or in an organ/tissue which is affected by the disease/condition (e.g. an organ/tissue in which the symptoms of the disease/condition manifest), or by the presence of cells expressing VISTA (e.g. MDSCs or tumor-associated macrophages) in a tumor.
  • the disease/condition may affect any tissue or organ or organ system. In some embodiments the disease/condition may affect several
  • a subject may be selected for therapy/prophylaxis in accordance with the present invention based on determination that the subject has an increase in the number/proportion/activity of cells expressing VISTA (e.g. MDSCs) in the periphery or in an organ/tissue relative to the number/proportion/activity of such cells in a healthy subject, or based on determination that the subject has a tumor comprising cells expressing VISTA (e.g. MDSCs).
  • VISTA e.g. MDSCs
  • the disease/condition to be treated/prevented is a cancer.
  • antigen-binding molecules are useful for the treatment of cancers in general, because antigen-binding molecules of the present invention are useful to release effector immune cells from MDSC-mediated suppression or suppression by cells expressing VISTA, and thereby enhance the anticancer immune response.
  • the cancer may be any unwanted cell proliferation (or any disease manifesting itself by unwanted cell proliferation), neoplasm or tumor.
  • the cancer may be benign or malignant and may be primary or secondary (metastatic).
  • a neoplasm or tumor may be any abnormal growth or proliferation of cells and may be located in any tissue.
  • the cancer may be of tissues/cells derived from e.g. the adrenal gland, adrenal medulla, anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum, central nervous system (including or excluding the brain) cerebellum, cervix, colon, duodenum, endometrium, epithelial cells (e.g.
  • kidney oesophagus
  • glial cells heart, ileum, jejunum, kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node, lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx, omentum, oral cavity, ovary, pancreas, parotid gland, peripheral nervous system, peritoneum, pleura, prostate, salivary gland, sigmoid colon, skin, small intestine, soft tissues, spleen, stomach, testis, thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, and/or white blood cells.
  • Tumors to be treated may be nervous or non-nervous system tumors.
  • Nervous system tumors may originate either in the central or peripheral nervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma.
  • Non-nervous system cancers/tumors may originate in any other non-nervous tissue, examples include melanoma, mesothelioma, lymphoma, myeloma, leukemia, Non-Hodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL), chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer, lung cancer , colon cancer, ovarian cancer, pancreatic cancer, thymic carcinoma, NSCLC, hematologic cancer and sarcoma.
  • NHL Non-Hodgkin’s lymphoma
  • CML chronic myelogenous leukemia
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • MDSCs are elevated in advanced colorectal cancer (Toor et al, Front Immunol. 2016; 7:560). MDSCs are also observed in breast cancer, and the percentage of MDSCs in the peripheral blood is increased in patients with later stage breast cancer (Markowitz et al, Breast Cancer Res Treat. 2013 Jul; 140(1 ): 13- 21 ). MDSC abundance is also correlated with poor prognosis in solid tumors (Charoentong et al, Cell Rep. 2017 Jan 3; 18(1 ):248-262), and MDSCs are enriched in liver cancer models (Connolly et al., J Leukoc Biol. (2010) 87(4):713-25).
  • VISTA has also been reported to be a target for the treatment of ovarian cancer (see e.g. US 9,631 ,018 B2) and lymphoma (see e.g. WO 2017/023749 A1 ).
  • the cancer is colorectal cancer, pancreatic cancer, breast cancer, liver cancer, prostate cancer, ovarian cancer, head and neck cancer, lymphoma, melanoma, thymoma, lung cancer, non-small cell lung cancer (NSCLC) and/or a solid tumor.
  • NSCLC non-small cell lung cancer
  • the treatment/prevention may be aimed at one or more of: delaying/preventing the onset/progression of symptoms of the cancer, reducing the severity of symptoms of the cancer, reducing the
  • the cancer to be treated/prevented comprises cells expressing VISTA.
  • the cells expressing VISTA are MDSCs.
  • the cancer comprises a tumor comprising cells expressing VISTA (e.g. MDSCs).
  • a subject may be selected for treatment described herein based on the detection of a cancer comprising cells expressing VISTA (e.g. MDSCs), or detection of a tumor comprising cells expressing VISTA (e.g. MDSCs), e.g. in a sample obtained from the subject.
  • the disease/condition in which the VISTA-expressing cells are pathologically implicated is an infectious disease, e.g. bacterial, viral, fungal, or parasitic infection.
  • infectious disease e.g. bacterial, viral, fungal, or parasitic infection.
  • T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections (including viral, bacterial and parasitic), as well as in cancer (Wherry Nature Immunology Vol.12, No.6, p492-499, June 201 1 ).
  • bacterial infections examples include infection by Bacillus spp., Bordetella pertussis, Clostridium spp., Corynebacterium spp., Vibrio chloerae, Staphylococcus spp., Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina, Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g. Mycobacterium tuberculosis) and Pseudomonas aeruginosa.
  • the bacterial infection may be sepsis or tuberculosis.
  • viral infections examples include infection by influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), Herpes simplex virus and human papilloma virus (HPV).
  • fungal infections examples include infection by Alternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungal infection may be fungal sepsis or histoplasmosis.
  • parasitic infections examples include infection by Plasmodium species (e.g.
  • the parasitic infection may be a disease such as malaria, leishmaniasis and toxoplasmosis.
  • Administration of the articles of the present invention is preferably in a "therapeutically effective” or “prophylactically effective” amount, this being sufficient to show therapeutic or prophylactic benefit to the subject.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease/condition and the particular article administered. Prescription of treatment, e.g.
  • Administration may be alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the antigen-binding molecule or composition described herein and a therapeutic agent may be administered simultaneously or sequentially.
  • the methods comprise additional therapeutic or prophylactic intervention, e.g. for the treatment/prevention of a cancer.
  • the therapeutic or prophylactic intervention is selected from chemotherapy, immunotherapy, radiotherapy, surgery, vaccination and/or hormone therapy.
  • the therapeutic or prophylactic intervention comprises leukapheresis.
  • the therapeutic or prophylactic intervention comprises a stem cell transplant.
  • the antigen-binding molecule is administered in combination with an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor other than VISTA.
  • the immune checkpoint inhibitor is e.g. PD-1 , CTLA-4, LAG-3, TIM-3, TIGIT or BTLA.
  • the antigen-binding molecule is administered in combination with an agent capable of promoting signalling mediated by a costimulatory receptor.
  • the costimulatory receptor is e.g. CD28, CD80, CD40L, CD86, 0X40, 4-1 BB, CD27 or ICOS.
  • compositions comprising an article according to the present invention (e.g. an antigen-binding molecule according to the invention) and an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor other than VISTA. Also provided are compositions comprising the articles of the present invention and an agent capable of promoting signalling mediated by a costimulatory receptor. Also provided is the use of such compositions in methods of medical treatment and prophylaxis of diseases/conditions described herein.
  • Agents capable of inhibiting signalling mediated by an immune checkpoint inhibitors are known in the art, and include e.g.
  • agents capable of binding to immune checkpoint inhibitors or their ligands, and inhibiting signalling mediated by the immune checkpoint inhibitor include agents capable of reducing gene/protein expression of the immune checkpoint inhibitor or a ligand for the immune checkpoint inhibitor (e.g.
  • RNA encoding the immune checkpoint inhibitor/ligand through inhibiting transcription of the gene(s) encoding the immune checkpoint inhibitor/ligand, inhibiting post-transcriptional processing of RNA encoding the immune checkpoint inhibitor/ligand, reducing stability of RNA encoding the immune checkpoint inhibitor/ligand, promoting degradation of RNA encoding the immune checkpoint inhibitor/ligand, inhibiting post-translational processing of the immune checkpoint inhibitor/ligand, reducing stability the immune checkpoint inhibitor/ligand, or promoting degradation of the immune checkpoint inhibitor/ligand), and small molecule inhibitors.
  • Agents capable of promoting signalling mediated by costimulatory receptors include e.g. agonist antibodies capable of binding to costimulatory receptors and triggering or increasing signalling mediated by the costimulatory receptor.
  • Other agents capable of promoting signalling mediated by costimulatory receptors include agents capable of increasing gene/protein expression of the costimulatory receptor or a ligand for the costimulatory receptor (e.g.
  • RNA encoding the costimulatory receptor/ligand through promoting transcription of the gene(s) encoding the costimulatory receptor/ligand, promoting post-transcriptional processing of RNA encoding the costimulatory receptor/ligand, increasing stability of RNA encoding the costimulatory receptor/ligand, inhibiting degradation of RNA encoding the costimulatory receptor/ligand, promoting post- translational processing of the costimulatory receptor/ligand, increasing stability the costimulatory receptor/ligand, or inhibiting degradation of the costimulatory receptor/ligand), and small molecule agonists.
  • VISTA may be a compensatory inhibitory pathway in prostate tumors after ipilimumab (i.e. anti-CTLA-4 antibody) therapy.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by PD-1.
  • the agent capable of inhibiting signalling mediated by PD-1 may be a PD-1- or PD-L1 -targeted agent.
  • the agent capable of inhibiting signalling mediated by PD-1 may e.g. be an antibody capable of binding to PD-1 or PD-L1 and inhibiting PD-1-mediated signalling.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by CTLA-4.
  • the agent capable of inhibiting signalling mediated by CTLA-4 may be a CTLA-4-targeted agent, or an agent targeted against a ligand for CTLA-4 such as CD80 or CD86.
  • the agent capable of inhibiting signalling mediated by CTLA-4 may e.g. be an antibody capable of binding to CTLA-4, CD80 or CD86 and inhibiting CTLA-4-mediated signalling.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by LAG-3.
  • the agent capable of inhibiting signalling mediated by LAG-3 may be a LAG-3-targeted agent, or an agent targeted against a ligand for LAG-3 such as MHC class II.
  • the agent capable of inhibiting signalling mediated by LAG-3 may e.g. be an antibody capable of binding to LAG-3 or MHC class II and inhibiting LAG-3-mediated signalling.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by TIM-3.
  • the agent capable of inhibiting signalling mediated by TIM-3 may be a TIM-3-targeted agent, or an agent targeted against a ligand for TIM-3 such as Galectin 9.
  • the agent capable of inhibiting signalling mediated by TIM-3 may e.g. be an antibody capable of binding to TIM-3 or Galectin 9 and inhibiting TIM- 3-mediated signalling.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by TIGIT.
  • the agent capable of inhibiting signalling mediated by TIGIT may be a TIG IT-targeted agent, or an agent targeted against a ligand for TIGIT such as CD113, CD112 or CD155.
  • the agent capable of inhibiting signalling mediated by TIGIT may e.g. be an antibody capable of binding to TIGIT, CD113, CD112 or CD155 and inhibiting TIGIT-mediated signalling.
  • the antigen-binding molecule of the present invention is administered in combination with an agent capable of inhibiting signalling mediated by BTLA.
  • the agent capable of inhibiting signalling mediated by BTLA may be a BTLA-targeted agent, or an agent targeted against a ligand for BTLA such as HVEM.
  • the agent capable of inhibiting signalling mediated by BTLA may e.g. be an antibody capable of binding to BTLA or HVEM and inhibiting BTLA - mediated signalling.
  • methods employing a combination of an antigen-binding molecule of the present invention and an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor provide an improved treatment effect as compared to the effect observed when either agent is used as a monotherapy.
  • an immune checkpoint inhibitor e.g. PD-1
  • the combination of an antigen-binding molecule of the present invention and an agent capable of inhibiting signalling mediated by an immune checkpoint inhibitor e.g. PD-1
  • Simultaneous administration refers to administration of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition and therapeutic agent together, for example as a pharmaceutical composition containing both agents (combined preparation), or immediately after each other and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel.
  • Sequential administration refers to administration of one of the antigen-binding molecule/composition or therapeutic agent followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments.
  • the time interval may be any time interval.
  • Chemotherapy and radiotherapy respectively refer to treatment of a cancer with a drug or with ionising radiation (e.g. radiotherapy using X-rays or g-rays).
  • the drug may be a chemical entity, e.g. small molecule pharmaceutical, antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor), or a biological agent, e.g. antibody, antibody fragment, aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, or protein.
  • the drug may be formulated as a pharmaceutical composition or medicament.
  • the formulation may comprise one or more drugs (e.g. one or more active agents) together with one or more pharmaceutically acceptable diluents, excipients or carriers.
  • a treatment may involve administration of more than one drug.
  • a drug may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • the chemotherapy may be a co-therapy involving administration of two drugs, one or more of which may be intended to treat the cancer.
  • the chemotherapy may be administered by one or more routes of administration, e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
  • routes of administration e.g. parenteral, intravenous injection, oral, subcutaneous, intradermal or intratumoral.
  • the chemotherapy may be administered according to a treatment regime.
  • the treatment regime may be a pre-determined timetable, plan, scheme or schedule of chemotherapy administration which may be prepared by a physician or medical practitioner and may be tailored to suit the patient requiring treatment.
  • the treatment regime may indicate one or more of: the type of chemotherapy to administer to the patient; the dose of each drug or radiation; the time interval between administrations; the length of each treatment; the number and nature of any treatment holidays, if any etc.
  • a single treatment regime may be provided which indicates how each drug is to be administered.
  • Chemotherapeutic drugs may be selected from: Abemaciclib, Abiraterone Acetate, Abitrexate
  • CARBOPLATIN-TAXOL Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant,
  • COPDAC COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio
  • Etoposide Phosphate Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate),
  • Fludarabine Phosphate Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE- OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), G
  • Liposome Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah
  • Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg
  • Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF,
  • Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-lntron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab,
  • Procarbazine Hydrochloride Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), [No Entries], Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine,
  • Recombinant Human Papillomavirus HPV
  • HPV Human Papillomavirus
  • HPV Nonavalent Vaccine
  • HPV Human Papillomavirus
  • HPV Quadrivalent Vaccine
  • Recombinant Interferon Alfa-2b Regorafenib
  • Relistor Metalnaltrexone Bromide
  • Revlimid Lidomide
  • Rheumatrex Metalhotrexate
  • Ribociclib R-ICE
  • Rituxan Rituximab
  • Rituxan Hycela Rituximab and Hyaluronidase Human
  • Rituximab Rituximab
  • Rituximab and
  • Multiple doses of the antigen-binding molecule, polypeptide, CAR, nucleic acid (or plurality thereof), expression vector (or plurality thereof), cell or composition may be provided.
  • One or more, or each, of the doses may be accompanied by simultaneous or sequential administration of another therapeutic agent.
  • Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.
  • doses may be given once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).
  • the invention also provides the articles of the present invention for use in methods for detecting, localizing or imaging VISTA, or cells expressing VISTA (e.g. MDSCs).
  • the antigen-binding molecules described herein may be used in methods that involve the antigen-binding molecule to VISTA. Such methods may involve detection of the bound complex of the antigen-binding molecule and VISTA.
  • detection of VISTA may be useful in methods of diagnosing/prognosing a disease/condition in which cells expressing VISTA (e.g. MDSCs) are pathologically implicated, identifying subjects at risk of developing such diseases/conditions, and/or may be useful in methods of predicting a subject’s response to a therapeutic intervention.
  • VISTA e.g. MDSCs
  • a method comprising contacting a sample containing, or suspected to contain, VISTA with an antigen-binding molecule as described herein, and detecting the formation of a complex of the antigen-binding molecule and VISTA. Also provided is a method comprising contacting a sample containing, or suspected to contain, a cell expressing VISTA with an antigen-binding molecule as described herein and detecting the formation of a complex of the antigen-binding molecule and a cell expressing VISTA.
  • a sample may be taken from any tissue or bodily fluid.
  • the sample may comprise or may be derived from: a quantity of blood; a quantity of serum derived from the individual’s blood which may comprise the fluid portion of the blood obtained after removal of the fibrin clot and blood cells; a tissue sample or biopsy; pleural fluid; cerebrospinal fluid (CSF); or cells isolated from said individual.
  • CSF cerebrospinal fluid
  • the sample may be obtained or derived from a tissue or tissues which are affected by the disease/condition (e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition).
  • a tissue or tissues which are affected by the disease/condition e.g. tissue or tissues in which symptoms of the disease manifest, or which are involved in the pathogenesis of the disease/condition.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un suppresseur contenant un domaine d'immunoglobuline de type V de molécules de liaison à l'antigène d'activation de lymphocytes T (VISTA). L'invention concerne également des acides nucléiques et des vecteurs d'expression codant, des compositions comprenant, et des procédés utilisant, les molécules de liaison à l'antigène VISTA.
PCT/EP2018/058258 2018-03-29 2018-03-29 Molécules de liaison à l'antigène vista WO2019185163A1 (fr)

Priority Applications (21)

Application Number Priority Date Filing Date Title
PCT/EP2018/058258 WO2019185163A1 (fr) 2018-03-29 2018-03-29 Molécules de liaison à l'antigène vista
US16/180,949 US20190300610A1 (en) 2018-03-29 2018-11-05 Vista antigen-binding molecules
CN202210352325.0A CN114507285A (zh) 2018-03-29 2019-03-29 Vista抗原结合分子
CN201980036745.4A CN112513080B (zh) 2018-03-29 2019-03-29 Vista抗原结合分子
EP19714654.1A EP3645570B1 (fr) 2018-03-29 2019-03-29 Molécules de liaison à l'antigène vista
KR1020247002424A KR20240015156A (ko) 2018-03-29 2019-03-29 Vista 항원 결합 분자
CA3095417A CA3095417A1 (fr) 2018-03-29 2019-03-29 Molecules de liaison a l'antigene vista
SG11202009515RA SG11202009515RA (en) 2018-03-29 2019-03-29 Vista antigen-binding molecules
ES19714654T ES2869549T3 (es) 2018-03-29 2019-03-29 Moléculas de unión a antígeno de VISTA
PL19714654T PL3645570T3 (pl) 2018-03-29 2019-03-29 Cząsteczki wiążące antygen VISTA
JP2020552406A JP7118332B2 (ja) 2018-03-29 2019-03-29 Ｖｉｓｔａ抗原結合性分子
DK19714654.1T DK3645570T3 (da) 2018-03-29 2019-03-29 VISTA-antigenbindende molekyler
AU2019240906A AU2019240906A1 (en) 2018-03-29 2019-03-29 VISTA antigen-binding molecules
PCT/EP2019/058036 WO2019185879A1 (fr) 2018-03-29 2019-03-29 Molécules de liaison à l'antigène vista
TW108111311A TWI828673B (zh) 2018-03-29 2019-03-29 Vista抗原結合分子
KR1020207031017A KR102629403B1 (ko) 2018-03-29 2019-03-29 Vista 항원 결합 분자
PT197146541T PT3645570T (pt) 2018-03-29 2019-03-29 Moléculas de ligação ao antigénio de vista
US17/042,855 US20210380697A1 (en) 2018-03-29 2019-03-29 Vista antigen-binding molecules
EP21151388.2A EP3868786A1 (fr) 2018-03-29 2019-03-29 Molécules de liaison à l'antigène vista
JP2022076578A JP2022128604A (ja) 2018-03-29 2022-05-06 Ｖｉｓｔａ抗原結合性分子
US18/518,192 US20240158538A1 (en) 2018-03-29 2023-11-22 Vista antigen-binding molecules

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PCT/EP2018/058258 WO2019185163A1 (fr) 2018-03-29 2018-03-29 Molécules de liaison à l'antigène vista

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US20200308308A1 (en) * 2018-09-07 2020-10-01 Hummingbird Bioscience Holdings Pte. Ltd. Vista antigen-binding molecules
US11306150B2 (en) 2017-01-11 2022-04-19 Bristol-Myers Squibb Company Method of identifying a P-selectin glycoprotein ligand-1 (PSGL-1) antagonist
WO2022263388A1 (fr) * 2021-06-14 2022-12-22 Hummingbird Bioscience Pte. Ltd. Cellules exprimant des molécules de liaison à l'antigène vista
US11603406B2 (en) 2017-03-14 2023-03-14 Five Prime Therapeutics, Inc. Antibodies binding to VISTA at acidic pH

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11306150B2 (en) 2017-01-11 2022-04-19 Bristol-Myers Squibb Company Method of identifying a P-selectin glycoprotein ligand-1 (PSGL-1) antagonist
US11603406B2 (en) 2017-03-14 2023-03-14 Five Prime Therapeutics, Inc. Antibodies binding to VISTA at acidic pH
US20200308308A1 (en) * 2018-09-07 2020-10-01 Hummingbird Bioscience Holdings Pte. Ltd. Vista antigen-binding molecules
US11873346B2 (en) * 2018-09-07 2024-01-16 Hummingbird Bioscience Pte. Ltd. VISTA antigen-binding molecules
WO2022263388A1 (fr) * 2021-06-14 2022-12-22 Hummingbird Bioscience Pte. Ltd. Cellules exprimant des molécules de liaison à l'antigène vista

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