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WO2024121380A1 - Composition vaccinale et adjuvant - Google Patents

Composition vaccinale et adjuvant Download PDF

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Publication number
WO2024121380A1
WO2024121380A1 PCT/EP2023/084879 EP2023084879W WO2024121380A1 WO 2024121380 A1 WO2024121380 A1 WO 2024121380A1 EP 2023084879 W EP2023084879 W EP 2023084879W WO 2024121380 A1 WO2024121380 A1 WO 2024121380A1
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Prior art keywords
antigen
antibody
cancer
vista
cell
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Inventor
Christine Libon
Pierre FERRE
Isabelle VANDENBERGHE
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Pierre Fabre Medicament SA
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Pierre Fabre Medicament SA
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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • VISTA V-Domain Ig Suppressor of T Cell Activation
  • VISTA is a negative checkpoint control protein that regulates T cell activation and immune responses.
  • VISTA is a member of the B7 family which comprises several immune checkpoint proteins such as PD-L1.
  • VISTA comprises a single unusually large Ig- like V-type domain.
  • VISTA cytoplasmic tail domain contains several docketing sites for effector proteins, suggesting that VISTA could potentially function as both a receptor and a ligand.
  • VISTA Human VISTA has two confirmed binding partners with immunosuppressive functions, PSGL-1 and VSIG3.
  • VISTA interacts with VSIG3 at physiological pH, but at acidic pH VISTA- expressing cells can bind to PSGL-1 on T cells (Wang et al. Immunology. 156(1 ): 74-85 (2019); Johnston et al. Nature. 574(7779): 565-570 (2019)). Both interactions result in inhibition of T cell function.
  • Other receptors including VSIG8 (WO 2016/090347A1 ) and LRIG1 (WO 2015/187359), have also been reported.
  • VISTA exerts a regulatory function on the immune system at several levels, particularly by modulating T cells activation. More recently, VISTA was identified as the earliest checkpoint regulator of peripheral T cell tolerance, particularly in the maintenance of naive T cell quiescence. In the context of cancer, VISTA is upregulated on immunosuppressive tumour infiltrating leukocytes such as inhibitory regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The presence of VISTA in the tumour microenvironment hinders effective T cell responses and has been implicated in a number of human cancers including prostate, colon, skin, pancreatic, and lung.
  • Tregs inhibitory regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • Vaccination is considered one of the greatest achievements of modern medicine. In particular, the impact of vaccination to prevent infectious diseases has been outstanding. Vaccination approaches also show promises in other fields, such as e.g., cancer, with the hope that vaccine strategies will enable patients to develop long-lasting anti-tumour immunity (see e.g., Morse et al. Target Oncol. 16(2) : 121 -152 (2021 )).
  • An adjuvant is a substance that is added to a vaccine to stimulate and enhance the quality, magnitude, and durability of the specific immune response.
  • Combined therapy with immune checkpoint inhibitors and cancer vaccines lead notably to an increase in the activation of the T cells, thereby promoting increased immunogenicity and circumventing immunosuppressive activity in the tumour microenvironment.
  • the present disclosure provides improved vaccinal compositions, compositions, kits and systems incorporating such constructs, and methods for preparing and using such constructs.
  • the present disclosure provides a combination comprising:
  • an anti-VISTA antibody or an antigen-binding fragment thereof, wherein the anti- VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO:3-5 and three light chain CDRs of SEQ ID NO:6-8.
  • the anti-VISTA antibody or antigen-binding fragment thereof is a monoclonal antibody, more preferably a humanised antibody.
  • the anti-VISTA antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:9 and a VL of SEQ ID N0:10.
  • the anti-VISTA antibody or antigen-binding fragment thereof comprises a human lgG1 constant region.
  • the anti- VISTA antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO:11 and a light chain of SEQ ID NO: 12.
  • the antigen-binding fragment is preferably selected in the group consisting of Fab, Fab', (Fab')2, Fv, scFv (sc for single chain), Bis-scFv, scFv-Fc fragments, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies, and nanobodies.
  • the antigen is a tumour antigen, a bacterial antigen, a parasite antigen, a viral antigen, or a fungal antigen.
  • the tumour antigen is preferably selected from: 19.9; oncofoetal protein 5T4; antigen 4.2; A33; adenosine deaminase-binding protein (ADAbp), adenomatous polyposis coli protein (APC), AFP; ALCAM; ALK, AML1 , BAGE; BCL-1 /lgH, BCL-2/lgH, BCL-6, BCR/ABL, BN, brain glycogen phosphorylase, E-cadherin, CAGE 3, CAP-1 , CAP-2, o-catenin, B-catenin, .y- catenin, CA125; Carboxypeptidase M; B1 ;CD5; CD19; CD20; CD22; CD23; CD25; CD27; CD30; CD33; CD36; CD46; CD52; CD79a/CD79b; CD123; CD317; CDK4, CEA, including include CD66a, CD66b, CD66c, CD66d,
  • the viral antigen is preferably selected from the antigens of papilloma viruses, for example, human papilloma virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, dengue fever virus, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T- cell leukaemia virus (HTLV-I), hairy cell leukaemia virus (HTLV-II), California encephalitis virus, Hanta virus (haemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus,
  • the parasite antigen is preferably selected from the antigens of Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, and Wuchereria bancrofti.
  • the bacterial antigen is preferably an antigen of a bacterium selected from: an anthrax bacterium, an antibiotic resistant bacterium, a disease-causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium.
  • the bacterium can be a mycobacterium, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile, and Mycobacterium tuberculosis.
  • the fungal antigen is preferably an antigen of a fungus selected from Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.
  • a fungus selected from Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocysti
  • the polynucleotide is DNA or RNA.
  • the polynucleotide comprises a modified DNA or RNA sequence, wherein the codon usage in the at least one open reading frame of the modified DNA or RNA sequence is adapted to the human codon usage.
  • the polynucleotide is an mRNA, even more preferably an mRNA produced by in vitro transcription
  • the mRNA comprises one or more of the following:
  • the polynucleotide is complexed or associated with at least one carrier selected from (a) one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins including protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids; and/or
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the combination described therein and a pharmaceutically acceptable excipient, preferably a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an adjuvant.
  • the pharmaceutical composition is a vaccine.
  • the pharmaceutical composition is formulated for simultaneous, separate or sequential administration.
  • the combination disclosed herein have a therapeutical activity.
  • the present disclosure relates to the use of the combination or the pharmaceutical composition disclosed herein as a medicament.
  • the disclosure also relates to the combination or the pharmaceutical composition for use as a medicament.
  • the disclosure relates to the use of the combination or the pharmaceutical composition as a vaccine.
  • the disclosure also relates to the combination or the pharmaceutical composition for use as a vaccine.
  • the disclosure provides a method of treatment of a cancer, an infectious disease, an autoimmune disorder or an inflammatory disorder comprising the administration of the combination or the pharmaceutical composition disclosed therein to a subject in need thereof.
  • the disclosure provides the combination or the pharmaceutical composition disclosed therein for use in the treatment of a cancer, an infectious disease, an autoimmune disorder or an inflammatory disorder.
  • the at least one epitope of an antigen or a polynucleotide encoding this at least epitope; and the anti-VISTA antibody, or antigen-binding fragment thereof, are administered simultaneously, separately or sequentially.
  • the treatment is therapeutic or prophylactic.
  • the treatment comprises the generation of an immune response against the antigen. More preferably, the generation of an immune response comprises the generation of a humoral immune response. In another more preferred embodiment, the generation of a humoral immune response involves the production of antibodies against the antigen.
  • the cancer is selected in the group consisting of Acute Lymphoblastic Leukaemia, Adult; Acute Lymphoblastic Leukaemia, Childhood; Acute Myeloid Leukaemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumour, Adult; Brain Tumour, Brain Stem Glioma, Childhood; Brain Tumour, Cerebellar Astrocytoma, Childhood; Brain Tumour, Cerebellar Astrocytoma, Childhood; Brain Tumour, Cerebral Astrocytoma/Malignant
  • the infectious disease is the infectious disease is selected in the group consisting of Botulism, Bubonic plague, Calicivirus infection (Norovirus and Sapovirus), Chickenpox, Chlamydia, Cholera, Clostridium difficile infection, Common cold (Acute viral rhinopharyngitis; Acute coryza), COVID-19 (coronavirus disease 2019), Creutzfeldt- Jakob disease (CJD), Dengue fever, Diphtheria, Ebola haemorrhagic fever, Gonorrhoea, Hand, foot and mouth disease (HFMD), Helicobacter pylori infection, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, human immunodeficiency virus (HIV), Human papillomavirus (HPV) infection, Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza (flu), Legionellosis (L)
  • the disclosure relates to the use of an anti-VISTA antibody as an adjuvant in a vaccinal composition, wherein the anti-VISTA antibody comprises three heavychain CDRs of SEQ ID NO: 1 -3 and three light chain CDRs of SEQ ID NO:4-6.
  • the anti-VISTA antibody or antigen-binding fragment thereof is a monoclonal antibody, more preferably a humanised antibody.
  • the anti-VISTA antibody or antigen-binding fragment thereof comprises a VH of SEQ ID NO:7 and a VL of SEQ ID NO:8.
  • the anti-VISTA antibody or antigen-binding fragment thereof comprises a human lgG1 constant region.
  • the anti- VISTA antibody or antigen-binding fragment thereof comprises a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO: 10.
  • the antigen-binding fragment is preferably selected in the group consisting of Fab, Fab', (Fab')2, Fv, scFv (sc for single chain), Bis-scFv, scFv-Fc fragments, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies, and nanobodies.
  • the antigen is a tumour antigen, a bacterial antigen, a parasite antigen, a viral antigen, or a fungal antigen.
  • the tumour antigen is preferably selected from: 19.9; oncofoetal protein 5T4; antigen 4.2; A33; adenosine deaminase-binding protein (ADAbp), adenomatous polyposis coli protein (APC), AFP; ALCAM; ALK, AML1 , BAGE; BCL-1 /lgH, BCL-2/lgH, BCL-6, BCR/ABL, BN, brain glycogen phosphorylase, E-cadherin, CAGE 3, CAP-1 , CAP-2, o-catenin, B-catenin, .y- catenin, CA125; Carboxypeptidase M; B1 ;CD5; CD19; CD20; CD22; CD23; CD25; CD27; CD30; CD33; CD36; CD46; CD52; CD79a/CD79b; CD123; CD317; CDK4, CEA, including include CD66a, CD66b, CD66c, CD66d,
  • the viral antigen is preferably selected from the antigens of papilloma viruses, for example, human papilloma virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, dengue fever virus, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T- cell leukaemia virus (HTLV-I), hairy cell leukaemia virus (HTLV-II), California encephalitis virus, Hanta virus (haemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus,
  • the parasite antigen is preferably selected from the antigens of Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, and Wuchereria bancrofti.
  • the bacterial antigen is preferably an antigen of a bacterium selected from: an anthrax bacterium, an antibiotic resistant bacterium, a disease-causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium.
  • the bacterium can be a mycobacterium, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile, and Mycobacterium tuberculosis.
  • the fungal antigen is preferably an antigen of a fungus selected from Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.
  • a fungus selected from Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocysti
  • the polynucleotide is DNA or RNA.
  • the polynucleotide comprises a modified DNA or RNA sequence, wherein the codon usage in the at least one open reading frame of the modified DNA or RNA sequence is adapted to the human codon usage.
  • the polynucleotide is an mRNA, even more preferably an mRNA produced by in vitro transcription
  • the mRNA comprises one or more of the following:
  • polynucleotide is complexed or associated with at least one carrier selected from
  • cationic or polycationic compounds preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins including protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids; and/or
  • Figure 1 Sampling schedule and experimental design.
  • FIG. 2 IFNy specific T cell responses of animals were obtained by ELISPOT after in vitro stimulation of PBMCs with SIV Gag peptide pool. Signal detected after stimulation with medium was subtracted.
  • FIG. 3 RM9 (A) and GAG (B) specific T cell response before the 3 rd immunization (BL) and at the peak of response following the 3 rd immunization and treatment. Signal detected after stimulation with medium was subtracted.
  • Figure 4 Frequency of proliferation marker KI67 expressed by CD8+ T cells before the treatment (BL) and at D14 post treatment (Peak).
  • Figure 5 Frequency of proliferation marker KI67 expressed by B cells before the treatment (BL) and at D14 post treatment (Peak).
  • an “adjuvant” as used herein is a substance or a composition of matter which is 1 ) not in itself capable of mounting a specific immune response against the immunogen of the vaccine, but which is 2) nevertheless capable of enhancing the immune response against the immunogen.
  • vaccination with the adjuvant alone does not provide an immune response against the immunogen
  • vaccination with the immunogen may or may not give rise to an immune response against the immunogen, but the combined vaccination with immunogen and adjuvant induces an immune response against the immunogen which is stronger than that induced by the immunogen alone.
  • Adjuvants can include a suspension of minerals (alum, aluminium salts, aluminium hydroxide, or phosphate) on which antigen is adsorbed; or water -in-oil emulsion in which antigen solution is emulsified in mineral oil (for example, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity.
  • Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants (for example, see U.S. Patent Nos.
  • Adjuvants also include biological molecules, such as lipids and costimulatory molecules.
  • biological adjuvants include AS04 (Didierlaurent, A.M. et al, J. Immunol., 2009, 183: 6186-6197), I L-2, RANTES, G M-CSF, TNF-a, IFN-y, G-CSF, LFA-3, CD72, B7-1 , B7-2, OX-40L and 41 BBL.
  • administer refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. , an anti-VISTA antibody provided herein) into a patient, such as by oral, mucosal (e.g., sublingual, buccal, rectal, vaginal, nasal, etc.), subcutaneous, cutaneous, intradermal, intravenous, intramuscular, intrathecal, ocular, otic, inhalation, nebulisation, cutaneous, or transdermal delivery and/or any other method of physical delivery described herein or known in the art.
  • administration of the substance typically occurs after the onset of the disease or symptoms thereof.
  • administration of the substance typically occurs before the onset of the disease or symptoms thereof.
  • antibody and “immunoglobulin” or “Ig” are used interchangeably herein. These terms are used herein in the broadest sense and specifically cover monoclonal antibodies (including full length monoclonal antibodies) of any isotype such as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies, chimeric antibodies, and antibody fragments, provided that said fragments retain the desired biological function.
  • polypeptide product of B cells within the immunoglobulin class of polypeptides that is capable of binding to a specific molecular antigen and is composed of two identical pairs of polypeptide chains inter-connected by disulfide bonds, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed., Oxford University Press.; Kuby (1997) Immunology, Third Ed., W.H. Freeman and Company, New York).
  • Each variable region of each heavy and light chain is composed of three complementarity-determining regions (CDRs), which are also known as hypervariable regions and four frameworks (FRs), the more highly conserved portions of variable domains, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. , effector cells) and the first component (C1q) of the classical complement system.
  • the specific molecular antigen can be bound by an antibody provided herein includes the target VISTA polypeptide, fragment or epitope.
  • An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunising an animal with the antigen or an antigen-encoding nucleic acid.
  • Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanised antibodies, camelised antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-ld) antibodies, and functional fragments of any of the above, which refers a portion of an antibody heavy or light chain polypeptide that retains some or all of the biological function of the antibody from which the fragment was derived.
  • the antibodies provided herein can be of any type (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g.
  • lgG1 lgG2, lgG3, lgG4, lgA1 and lgA2
  • any subclass e.g. , lgG2a and lgG2b of immunoglobulin molecule.
  • anti-VISTA antibodies refer to antibodies that bind to a VISTA polypeptide, such as a VISTA antigen or epitope.
  • Such antibodies include polyclonal and monoclonal antibodies, including chimeric, humanised, and human antibodies.
  • An antibody that binds to a VISTA antigen may be cross -reactive with related antigens.
  • an antibody that binds to VISTA does not crossreact with other antigens such as e.g. , other peptides or polypeptides belonging to the B7 superfamily.
  • An antibody that binds to VISTA can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
  • An antibody binds to VISTA, for example, when it binds to VISTA with higher affinity than to any cross -reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs), for example, an antibody that specifically binds to VISTA.
  • a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background.
  • an antibody “which binds” an antigen of interest is one that binds the antigen with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins.
  • the extent of binding of the antibody to a “non-target” protein will be less than about 10% of the binding of the antibody to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIPA).
  • the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction.
  • Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.
  • specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labelled target. In this case, specific binding is indicated if the binding of the labelled target to a probe is competitively inhibited by excess unlabelled target.
  • telomere binding or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10' 4 M, alternatively at least about 10' 5 M, alternatively at least about 10' 6 M, alternatively at least about 10' 7 M, alternatively at least about 10' 8 M, alternatively at least about 10' 9 M, alternatively at least about 1O' 10 M, alternatively at least about 10' 11 M, alternatively at least about 10' 12 M, or greater.
  • the term "specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • an antibody that binds to VISTA has a dissociation constant (KD) of ⁇ 1pM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1nM, or ⁇ 0.1 nM.
  • an “antigen” is a substance, which is capable of being recognised (typically via its epitope(s)) by the immune system, preferably by the adaptive immune system, and which is capable of eliciting an antigen-specific immune response, e.g., by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • An antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound.
  • an antigen may be or may comprise a peptide or protein, which may be presented to (antigen-specific) T-cells on MHC surface molecules by antigen- presenting cells.
  • an antigen may be the product of translation of a provided nucleic acid molecule, e.g., an epitope-encoding DNA or an epitope-encoding RNA.
  • an antigen such as a peptide or a protein
  • fragments or variants of an antigen comprising at least one epitope are understood as antigens.
  • antigen binding fragment refers to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g. , the complementarity determining regions (CDRs)).
  • CDRs complementarity determining regions
  • an antibody By the expression “antigen-binding fragment” of an antibody, it is intended to indicate any peptide, polypeptide, or protein retaining the ability to bind to the target (also generally referred to as antigen) of the said antibody, generally the same epitope, and comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, or at least 200 contiguous amino acid residues, of the amino
  • the said antigen-binding fragment comprises at least one CDR of the antibody from which it is derived. Still in a preferred embodiment, the said antigen binding fragment comprises 2, 3, 4 or 5 CDRs, more preferably the 6 CDRs of the antibody from which it is derived.
  • the “antigen-binding fragments” can be selected, without limitation, in the group consisting of Fab, Fab', (Fab )?, Fv, scFv (sc for single chain), Bis-scFv, scFv-Fc fragments, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies, and nanobodies, and fusion proteins with disordered peptides such as XTEN (extended recombinant polypeptide) or PAS motifs, and any fragment of which the half-life time would be increased by chemical modification, such as the addition of poly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation”) (pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab’)z-PEG or Fab’- PEG) (“PEG” for Poly(Ethylene) Glycol), or by incorporation in a liposome, said
  • Fab has a structure including variable regions of light chain and heavy chain, a constant region of a light chain, and the first constant region of a heavy chain (CH1 ), and it has one antigen binding site.
  • Fab' is different from Fab in that it has a hinge region including one or more cysteine residues at C terminus of heavy chain CH1 domain.
  • F(ab')? antibody is generated as the cysteine residues of the hinge region of Fab' form a disulfide bond.
  • Fv is a minimum antibody fragment which has only a heavy chain variable region and a light chain variable region, and a recombination technique for producing the Fv fragment is described in International Publication WO 88/10649 or the like.
  • double chain Fv the heavy chain variable region and light chain variable region are linked to each other via a disulfide bond
  • scFv single chain Fv
  • the heavy chain variable region and light chain variable region are covalently linked to each other via a peptide linker in general.
  • Those antibody fragments can be obtained by using a proteinase (e.g. , Fab can be obtained by restriction digestion of whole antibody with papain, and F(ab')? fragment can be obtained by restriction digestion with pepsin), and it can be preferably produced by genetic engineering techniques.
  • said “antigen-binding fragments” will be constituted or will comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same specificity of binding as the antibody from which it is descended and a sufficient affinity, preferably at least equal to 1 /100, in a more preferred manner to at least 1 /10, of the affinity of the antibody from which it is descended, with respect to the target.
  • antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al.
  • binding refers to an interaction between molecules to form a complex which, under physiologic conditions, is relatively stable. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as VISTA, is the affinity of the antibody or functional fragment for that epitope.
  • the ratio of association (ki) to dissociation (k-i) of an antibody to a monovalent antigen (ki/ k-i) is the association constant K, which is a measure of affinity.
  • K is a measure of affinity.
  • the value of K varies for different complexes of antibody and antigen and depends on both ki and k-i.
  • the association constant K for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art.
  • the affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen.
  • the avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.
  • said antibody, or antigen-binding fragment thereof binds to VISTA with an affinity that is at least two-fold greater than its affinity for binding to a non-specific molecule such as BSA or casein.
  • said antibody, or antigen-binding fragment thereof binds only to VISTA.
  • biological sample refers to a sample that has been obtained from a biological source, such as a patient or subject.
  • a “biological sample” as used herein refers notably to a whole organism or a subset of its tissues, cells or component parts (e.g., blood vessel, including artery, vein and capillary, body fluids, including but not limited to blood, serum, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen).
  • Bio sample further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
  • biological sample refers to a medium, such as a nutrient broth or gel in which an organism has been propagated, which contains cellular components, such as proteins or nucleic acid molecules.
  • cell proliferative disorder and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation.
  • the cell proliferative disorder is a tumour or cancer.
  • Tuour refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer, “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumour” are not mutually exclusive as referred to herein.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterised by unregulated cell growth.
  • a “cancer” as used herein is any malignant neoplasm resulting from the undesired growth, the invasion, and under certain conditions metastasis of impaired cells in an organism.
  • the cells giving rise to cancer are genetically impaired and have usually lost their ability to control cell division, cell migration behaviour, differentiation status and/or cell death machinery. Most cancers form a tumour but some hematopoietic cancers, such as leukaemia, do not.
  • a “cancer” as used herein may include both benign and malignant cancers.
  • the term “cancer” as used herein refers in particular to any cancer that can be treated by the human antibody of the present disclosure without any limitation.
  • cancer examples include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukaemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, multiple myeloma
  • the cancer is a haematological cancer, which refers to cancer that begins in blood-forming tissue, such as the bone marrow, or in the cells of the immune system.
  • haematologic cancer are leukaemia (e.g., acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myelogenous leukaemia (CML), chronic lymphocytic leukaemia (CLL), or acute monocytic leukaemia (AMoL)), lymphoma (Hodgkin lymphoma or non-Hodgkin lymphoma), and myeloma (multiple myeloma, plasmacytoma, localised myeloma or extramedullary myeloma).
  • AML acute myeloid leukaemia
  • ALL acute lymphoblastic leukaemia
  • CML chronic myelogenous leukaemia
  • CLL chronic lymphocytic leukaemia
  • AoL acute mono
  • CDR or “complementary-determining region” refers to one of three hypervariable regions (H1 , H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH B-sheet framework, or one of three hypervariable regions (L1 , L2 or L3) within the non-framework region of the antibody VL B-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al. (1977) J. Biol. Chem.
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modelling software.
  • the “contact” hypervariable regions are based on an analysis of the available complex crystal structures.
  • IMGT ImMunoGeneTics
  • the IMGT universal numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P. (1997) Immunol. Today 18: 509; Lefranc M.-P.
  • the IMGT universal numbering provides a standardised delimitation of the framework regions (FR1 -IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1 -IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, e.g. [8.8.13]) become crucial information.
  • the IMGT universal numbering is used in 2D graphical representations, designated as IMGT Colliers de Perles [Ruiz, M.
  • Hypervariable regions may comprise "extended hypervariable regions” as follows: 24- 36 or 24-34 (L1 ), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26-35A (H1 ), 50-65 or 49-65 (H2) and 93-102, 94-1 02, or 95-102 (H3) in the VH.
  • the variable domain residues are 25 numbered according to Kabat et al. , supra, for each of these definitions.
  • the terms “HVR” and “CDR” are used interchangeably.
  • cellular immune response refers typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.
  • the cellular immune response is not based on antibodies, but on the activation of cells of the immune system.
  • a cellular immune response may be characterised e.g. , by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g. , specific immune cells like dendritic cells or other cells, displaying epitopes of foreign antigens on their surface.
  • Such cells may be virus-infected or infected with intracellular bacteria, or cancer cells displaying tumour antigens. Further characteristics may be activation of macrophages and natural killer cells, enabling them to destroy pathogens and stimulation of cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses
  • constant region or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor.
  • the terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
  • the constant domain contains the CH1 , CH2 and CH3 domains of the heavy chain and the CL domain of the light chain.
  • the term “decreased”, as used herein, refers to the activity of a protein, e.g., VISTA, at least 1 -fold (e.g. , 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) lower than its reference value. “Decreased”, as it refers to the activity of a protein, e.g. , VISTA, of a subject, signifies also at least 5% lower (e.g.
  • the term “decreased”, as used herein, also refers to the level of a biomarker, e.g. , VISTA, of a subject at least 1 -fold (e.g. , 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000- fold or more) lower than its reference value.
  • a biomarker e.g. , VISTA
  • a biomarker e.g. VISTA
  • derivative refers to a polypeptide that comprises an amino acid sequence of a VISTA polypeptide, a fragment of a VISTA polypeptide, or an antibody that binds to a VISTA polypeptide which has been altered by the introduction of amino acid residue substitutions, deletions or additions.
  • derivative also refers to a VISTA polypeptide, a fragment of a VISTA polypeptide, or an antibody that binds to a VISTA polypeptide which has been chemically modified, e.g. , by the covalent attachment of any type of molecule to the polypeptide.
  • a VISTA polypeptide, a fragment of a VISTA polypeptide, or a VISTA antibody may be chemically modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, derivatisation by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • the derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide.
  • a derivative of a VISTA polypeptide, a fragment of a VISTA polypeptide, or a VISTA antibody may be chemically modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a VISTA polypeptide, a fragment of a VISTA polypeptide, or a VISTA antibody may contain one or more non-classical amino acids.
  • a polypeptide derivative possesses a similar or identical function as a VISTA polypeptide, a fragment of a VISTA polypeptide, or a VISTA antibody described herein.
  • an “effective amount” or “therapeutically effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to elicit the desired biological response in a subject. Such response includes alleviation of the symptoms of the disease or disorder being treated, prevention, inhibition or a delay in the recurrence of symptom of the disease or of the disease itself, an increase in the longevity of the subject compared with the absence of the treatment, or prevention, inhibition or delay in the progression of symptom of the disease or of the disease itself.
  • An “effective amount” is in particular the amount of the agent effective to achieve the desired therapeutic or prophylactic result. More specifically, an “effective amount” as used herein is an amount of the agent that confers a therapeutic benefit. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.
  • encode or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof.
  • the antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.
  • epitope typically refers to the part of an antigen which is recognised by the adaptive immune system.
  • epitope in particular refers to a part or fragment of an antigen presented on a MHC surface molecule.
  • Such a fragment comprising or consisting of an epitope as used herein may typically comprise from about 5 to about 20 amino acids.
  • Epitopes can be distinguished in T cell epitopes and B cell epitopes.
  • T cell epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g.
  • fragments as processed and presented by MHC class I molecules preferably having a length of about 8 to about 10 amino acids, e.g., 8, 9, or 10, (or even 11 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g., 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence.
  • These fragments are typically recognised by T cells in form of a complex consisting of the peptide fragment and an MHC surface molecule, i.e., the fragments are typically not recognised in their native form.
  • a B cell epitope refers to the region of an antigen to which an antibody binds.
  • a B cell epitope as used herein is a localised region on the surface of an antigen that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g. , a human), that is capable of eliciting a humoral immune response.
  • a B cell epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal.
  • a B cell epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, for example, by an immunoassay. Antigenic B cell epitopes need not necessarily be immunogenic.
  • B cell epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three- dimensional structural characteristics as well as specific charge characteristics.
  • a B cell epitope can be formed by contiguous residues or by non-contiguous residues brought into close proximity by the folding of an antigenic protein.
  • B cell epitopes formed by contiguous amino acids are typically retained on exposure to denaturing solvents, whereas B cell epitopes formed by non-contiguous amino acids are typically lost under said exposure.
  • an antigen has several or many different epitopes, including several or many different T cell epitopes and/or B cell epitopes.
  • an antigen has generally several or many different B cell epitopes and reacts with many different antibodies.
  • excipient refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilising agent for drugs which imparts a beneficial physical property to a formulation, such as increased protein stability, increased protein solubility, and decreased viscosity.
  • excipients include, but are not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, non-ionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, which is hereby incorporated by reference in its entirety.
  • proteins e.g., serum albumin, etc.
  • amino acids e.g., aspartic acid, glutamic acid, lysine, arginine
  • FR residues refers to those variable domain residues other than the hypervariable region residues herein defined. FR residues are those variable domain residues flanking the CDRs. FR residues are present, e.g., in chimeric, Humanised, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies.
  • the term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region.
  • the constant region can be one of five distinct types, referred to as alpha (a), delta (6), epsilon (E), gamma (y) and mu (p), based on the amino acid sequence of the heavy chain constant region.
  • the distinct heavy chains differ in size: a, 6 and y contain approximately 450 amino acids, while p and s contain approximately 550 amino acids.
  • a heavy chain can be a human heavy chain.
  • hinge region refers herein to a flexible amino acid stretch in the central part of the heavy chains of the IgG and IgA immunoglobulin classes, which links these 2 chains by disulfide bonds.
  • the hinge region is generally defined as stretching from Glu216 to Pro230 of human lgG1 (Burton, Mol Immunol, 22: 161 -206, 1985).
  • Hinge regions of other IgG isotypes may be aligned with the lgG1 sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.
  • the "CH2 domain” of a human IgG Fc portion usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilise the CH2 domain (Burton, Mol Immunol, 22: 161 -206, 1985).
  • the "CH3 domain” comprises the stretch of residues C- terminal to a CH2 domain in an Fc portion (i.e. , from about amino acid residue 341 to about amino acid residue 447 of an IgG).
  • host refers to an animal, such as a mammal (e.g. , a human).
  • host cell refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.
  • a “humanised” antibody refers to a chimeric antibody that contains minimal sequence derived from non-human immunoglobulin.
  • a humanised antibody is a human immunoglobulin (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • some of the skeleton segment residues (called FR for framework) can be modified to preserve binding affinity, according to techniques known by a man skilled in the art (Jones et al. , Nature, 321 :522-525, 1986).
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of TJ a human antibody.
  • a humanised antibody optionally may comprise at least a portion of an antibody constant region (Fc), typically that of a human immunoglobulin.
  • Fc antibody constant region
  • the goal of humanisation is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody.
  • a xenogenic antibody such as a murine antibody
  • the goal of humanisation is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody.
  • a xenogenic antibody such as a murine antibody
  • Humoral immune response refers typically to antibody production and optionally to accessory processes accompanying antibody production.
  • a humoral immune response may be typically characterised, e.g., by Th2 activation and cytokine production, germinal centre formation and isotype switching, affinity maturation and memory cell generation.
  • Humoral immune response also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralisation, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
  • immune response means the activation of a host's immune system, e.g., that of a mammal, in response to the introduction of antigen.
  • the immune response can be in the form of a cellular or humoral response, or both.
  • the immune response can be T cell-mediated and/or B cell-mediated immune responses that are influenced by modulation of T cell costimulation.
  • the term “increased”, as used herein, refers to the activity of a protein, e.g. , VISTA, at least 1 -fold (e.g. , 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) greater than its reference value. “Increased”, as it refers to the activity of a protein, e.g. , VISTA, of a subject, signifies also at least 5% greater (e.g.
  • the term “increased”, as used herein, also refers to the level of a biomarker, e.g. , VISTA, of a subject at least 1 -fold (e.g. 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) greater than its reference value.
  • a biomarker e.g. , VISTA
  • a biomarker e.g., VISTA
  • inhibitor when used in the context of an antibody refers to an antibody that suppresses, restrains or decreases a biological activity of the antigen to which the antibody binds.
  • the inhibitory effect of an antibody can be one which results in a measurable change in the antigen’s biological activity.
  • inhibitor or “block” refers to an antibody that prevents or stops a biological activity of the antigen to which the antibody binds.
  • a blocking antibody includes an antibody that combines with an antigen without eliciting a reaction, but that blocks another protein from later combining or complexing with that antigen.
  • an anti-VISTA antibody described herein blocks the ability of VISTA to bind VSIG3, which can result in inhibiting or blocking suppressive signals of VISTA.
  • Certain anti-VISTA antibodies described herein inhibit or block suppressive signals of VISTA on VISTA-expressing cells, including by about 98% to about 100% as compared to the appropriate control (e.g. , the control being cells not treated with the antibody being tested).
  • the anti-VISTA antibody described herein blocks the binding of the extracellular domain VISTA to VSIG3 and/or blocks the binding of a VISTA-expressing cell to a VSIG3-expressing cell.
  • an anti-VISTA antibody described herein blocks the ability of VISTA to bind PSGL-1 , preferably at acidic pH (pH between 5.9 and 6.5), which can result in inhibiting or blocking suppressive signals of VISTA.
  • Certain anti- VISTA antibodies described herein inhibit or block suppressive signals of VISTA on VISTA- expressing cells, including by about 98% to about 100% as compared to the appropriate control (e.g. , the control being cells not treated with the antibody being tested).
  • the anti-VISTA antibody described herein blocks, preferably at acidic pH (pH between 5.9 and 6.5), the binding of the extracellular domain VISTA to PSGL-1 and/or blocks, preferably at acidic pH (pH between 5.9 and 6.5), the binding of a VISTA-expressing cell to a PSGL-1 -expressing cell.
  • the term “in combination” in the context of the administration of other therapies refers to the use of more than one therapy (e.g. , an anti-VISTA antibody and at least one epitope of an antigen or a polynucleotide encoding this at least one epitope).
  • the use of the term “in combination” does not restrict the order or the time in which therapies are administered to a subject (e.g. , one therapy before, concurrent with, or after another therapy).
  • a first therapy can be administered before (e.g.
  • any additional therapy can be administered in any order or time with the other additional therapies (e.g. , an anti-VISTA antibody and at least one epitope of an antigen or a polynucleotide encoding this at least one epitope).
  • the antibodies can be administered in combination with one or more therapies (e.g. , therapies that are not the antibodies that are currently administered to prevent, treat, manage, and/or ameliorate a disease, disorder or condition).
  • Non-limiting examples of therapies that can be administered in combination with an antibody include an antagonist to a co-inhibitory molecule, an agonist to a co-stimulatory molecule, a chemotherapeutic agent, radiation, analgesic agents, anaesthetic agents, antibiotics, or immunomodulatory agents or any other agent listed in the U.S. Pharmacopoeia and/or Physician’s Desk Reference.
  • an “isolated” antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g. , ion exchange or reverse phase HPLC).
  • electrophoresis e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g. , ion exchange or reverse phase HPLC.
  • nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • KD used herein means a dissociation constant of a specific antibody-antigen interaction and is used as an indicator for measuring the affinity of an antibody for an antigen.
  • Lower KD means higher affinity of an antibody for an antigen.
  • light chain when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region.
  • the approximate length of a light chain is 211 to 217 amino acids.
  • K kappa
  • A lambda
  • Light chain amino acid sequences are well known in the art.
  • a light chain can be a human light chain.
  • the term “monoclonal antibody” designates an antibody arising from a nearly homogeneous antibody population, wherein population comprises identical antibodies except for a few possible naturally-occurring mutations which can be found in minimal proportions.
  • a monoclonal antibody arises from the growth of a single cell clone, such as a hybridoma, and is characterised by heavy chains of one class and subclass, and light chains of one type.
  • a monoclonal antibody shows specific binding to a single antigenic site (i.e., single epitope) when the antibody is presented to it.
  • the monoclonal antibody can be produced by various methods that are well known in the corresponding technical area.
  • nucleic acid molecules when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not manipulated by a human being.
  • PEGylation means a processing method for increasing the retention time of an antibody in blood by introducing polyethylene glycol to the aforementioned monoclonal antibody or an antigen -binding fragment thereof.
  • hydrophilicity on a nanoparticle surface is enhanced, and, accordingly, fast degradation in living body can be prevented due to so-called stealth effect which prevents recognition by immune activity including macrophage in a human body to cause phagocytosis and digestion of pathogens, waste products, and foreign materials introduced from an outside.
  • the retention time of an antibody in blood can be increased by PEGylation.
  • the PEGylation employed in the present disclosure can be carried out by a method by which an amide group is formed based on a bond between the carboxyl group of hyaluronic acid and the amine group of polyethylene glycol, but it is not limited thereto, and the PEGylation can be carried out by various methods.
  • the polyethylene glycol to be used polyethylene glycol having molecular weight of 100 to 1 ,000 and a linear or branched structure is preferably used, although it is not particularly limited thereto.
  • the “percentage identity” or “% identity” between two sequences of nucleic acids or amino acids refers to the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length.
  • the comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an “alignment window”. Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of methods known by a man skilled in the art.
  • amino acid sequence exhibiting at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a reference amino acid sequence
  • preferred examples include those containing the reference sequence, certain modifications, notably a deletion, addition or substitution of at least one amino acid, truncation or extension.
  • substitutions are preferred in which the substituted amino acids are replaced by “equivalent” amino acids.
  • Equivalent amino acids is meant to indicate any amino acids likely to be substituted for one of the structural amino acids without however modifying the biological activities of the corresponding antibodies and of those specific examples defined below. Equivalent amino acids can be determined either on their structural homology with the amino acids for which they are substituted or on the results of comparative tests of biological activity between the various antibodies likely to be generated.
  • Table 1 summarises the possible substitutions likely to be carried out without resulting in a significant modification of the biological activity of the corresponding modified antigen binding protein; inverse substitutions are naturally possible under the same conditions.
  • pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognised Pharmacopeia for use in animals, and more particularly in humans. More specifically, when referring to a carrier, the expression “pharmaceutically acceptable” means that the carrier(s) is compatible with the other ingredient(s) of the composition and is not deleterious to the recipient thereof. Accordingly, as used herein, the expression “pharmaceutically acceptable carrier” refers to a carrier or a diluent which does not inhibit the biological activity and characteristics of a compound for administration without stimulating a living organism. The type of carrier can be selected based upon the intended route of administration.
  • each carrier used may vary within ranges conventional in the art.
  • a pharmaceutically acceptable carrier in the composition which is prepared as a liquid solution physiological saline, sterilised water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and a mixture of one or more of them can be used as a sterilised carrier suitable for a living organism. If necessary, common additives like anti-oxidant, buffer solution, and bacteriostat may be added.
  • the composition can be prepared as a formulation for injection like aqueous solution, suspension, and emulsion, a pill, a capsule, a granule, or a tablet.
  • polyclonal antibody refers to an antibody which was produced among or in the presence of one or more other, non-identical antibodies.
  • polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes producing non-identical antibodies.
  • polyclonal antibodies are obtained directly from an immunised animal.
  • nucleic acid molecule As used herein, the terms “polynucleotide,” “nucleotide,” nucleic acid” “nucleic acid molecule” and other similar terms are used interchangeable and include DNA, RNA, mRNA and the like.
  • prevention refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition (e.g., cancer).
  • prevention is assessed on a population basis such that an agent is considered to “prevent” a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition.
  • signal peptide refers to a peptide capable of enhancing the processing through the endoplasmic reticulum (ER)-and/or secretion of a polypeptide when present at its N-terminus.
  • signal peptides usually comprise, consist essentially of or consist of 15 to 35 essentially hydrophobic amino acids, are inserted at the N-terminus of the polypeptide downstream of the codon for initiation of translation, initiate its passage into the endoplasmic reticulum (ER) and are then removed by a specific ER-located endopeptidase to give the mature polypeptide.
  • ER endoplasmic reticulum
  • Appropriate signal peptides are known in the art.
  • cellular or viral polypeptides such as those of immunoglobulins, tissue plasminogen activator, insulin, rabies glycoprotein (see e.g; W099/03885 or W02008/ 138649), the HIV virus envelope glycoprotein or the measles virus F protein or may be synthetic.
  • stimulation of the immune system means that a substance or composition of matter exhibits a general, non-specific immunostimulatory effect.
  • a number of adjuvants and putative adjuvants (such as certain cytokines) share the ability to stimulate the immune system.
  • the result of using an immunostimulating agent is an increased “alertness” of the immune system meaning that simultaneous or subsequent immunisation with an immunogen induces a significantly more effective immune response compared to isolated use of the immunogen.
  • a “subject” which may be subjected to the methodology described herein may be any of mammalian animals including human, dog, cat, cattle, goat, pig, swine, sheep and monkey.
  • a human subject can be known as a patient.
  • “subject” or “subject in need” refers to a mammal that is suffering from a disease, disorder or condition, such as e.g. , cancer or is suspected of suffering from cancer or has been diagnosed with a disease, disorder or condition, such as e.g. , cancer.
  • a “cancer-suffering subject” as used herein refers to a mammal that is suffering from cancer or has been diagnosed with cancer.
  • a “control subject” refers to a mammal that is not suffering from a disease, disorder or condition, such as e.g. , cancer, and is not suspected of suffering from cancer.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • “substantially” refers to refers to at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • a therapeutic agent refers to any agent that can be used in treating, preventing or alleviating a disease, disorder or condition, including in the treatment, prevention or alleviation of one or more symptoms of a Disease, disorder, or condition and/or a symptom related thereto.
  • a therapeutic agent refers to an anti-VISTA antibody provided herein.
  • a therapeutic agent refers to an agent other than an anti-VISTA antibody provided herein.
  • a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment, prevention or alleviation of one or more symptoms of a disease, disorder, condition, and/or a symptom related thereto.
  • the combination of therapies can be more effective than the additive effects of any two or more single therapies.
  • a synergistic effect of a combination of therapeutic agents permits the use of lower dosages of one or more of the agents and/or less frequent administration of the agents to a subject with a Disease, disorder or condition and/or a symptom related thereto.
  • the ability to utilise lower dosages of therapeutic therapies and/or to administer the therapies less frequently reduces the toxicity associated with the administration of the therapies to a subject without reducing the efficacy of the therapies in the prevention, treatment or alleviation of one or more symptoms of a disease, disorder or condition and/or a symptom related thereto.
  • synergistic effect can result in improved efficacy of therapies in the prevention, treatment or alleviation of one or more symptoms of a disease, disorder or condition and/or a symptom related thereto.
  • synergistic effect of a combination of therapies e.g., therapeutic agents
  • terapéuticaally effective amount refers to the amount of a therapeutic agent (e.g., an anti-VISTA antibody or any other therapeutic agent, including as described herein, including, for example, an epitope of an antigen or a polypeptide encoding an epitope of an antigen) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition and/or a symptom related thereto.
  • a therapeutic agent e.g., an anti-VISTA antibody or any other therapeutic agent, including as described herein, including, for example, an epitope of an antigen or a polypeptide encoding an epitope of an antigen
  • a therapeutically effective amount of a therapeutic agent can be an amount necessary for the reduction or amelioration of the advancement or progression of a given disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given disease, disorder or condition and/or to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an anti-VISTA antibody, including as described herein).
  • another therapy e.g., a therapy other than the administration of an anti-VISTA antibody, including as described herein.
  • the term “therapy” refers to any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of a disease, disorder or condition.
  • the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the treatment, prevention and/or amelioration of a disease, disorder or condition known to one of skill in the art such as medical personnel.
  • “treating” a disease in a subject or “treating” a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that the extent of the disease is decreased or prevented.
  • treating results in the reduction of at least one sign or symptom of the disease or condition.
  • Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, and may be performed either prophylactically, or subsequent to the initiation of a pathologic event. Treatment can require administration of an agent and/or treatment more than once.
  • such terms refer to the reduction or amelioration of the progression, severity, and/or duration of a disease, that is responsive to immune modulation, such modulation resulting from increasing T cell activation.
  • the term “vaccination” or “vaccinating” refers to, but is not limited to, a process to elicit an immune response in a subject against a particular antigen.
  • Vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and/or to the development of one or more symptoms, and in some embodiments, before, during, and/or shortly after exposure to the agent.
  • vaccination may be therapeutic or prophylactic.
  • therapeutic vaccination is meant the administration of a vaccine to a subject already suffering from a disease (e.g., a tumour), typically for the purpose of heightening or broadening the immune response to thereby halt, impede, or reverse the progression of the disease.
  • Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous or intramuscular. Vaccines may be administered with an adjuvant to boost the immune response. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccinating composition.
  • the term "vaccine composition” is intended to mean a composition which can be administered to humans or to animals in order to induce an immune system response; this immune system response can result in the activation of certain cells, in particular APCs, T lymphocytes and B lymphocytes.
  • variable domain refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen.
  • the variable domains differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable domain are referred to as framework regions (FR).
  • FR framework regions
  • Each variable region comprises three CDRs which are connected to four FR.
  • the CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen.
  • the FR determines the folding of the molecules and thus the amount of CDR that is presented on the surface of the variable region for interaction with the antigen.
  • the variable region is a human variable region.
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • variable domain of the heavy chain may be referred to as “VH”.
  • variable domain of the light chain may be referred to as “VL”. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
  • variant when used in relation to VISTA or to an anti-VISTA antibody refers to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence.
  • a VISTA variant may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of native VISTA.
  • a variant of an anti- anti-VISTA antibody may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified anti- anti-VISTA antibody.
  • a variant of an anti-VISTA antibody may result from one change to an amino acid sequence of a native or previously unmodified anti- anti-VISTA antibody.
  • the VISTA variant or anti-VISTA antibody variant at least retains VISTA or anti-VISTA antibody functional activity, respectively.
  • an anti-VISTA antibody variant does not undergo deamidation in the CDRs.
  • an anti-VISTA antibody variant binds VISTA and/or is antagonistic to VISTA activity. In some embodiments, an anti-VISTA antibody variant does not undergo deamidation in the CDRs, binds VISTA and/or is antagonistic to VISTA activity.
  • Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed.
  • the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes VISTA or anti-VISTA antibody VH or VL regions or subregions. Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding the variants.
  • SNP single nucleotide polymorphism
  • vector refers to a substance that is used to introduce a nucleic acid molecule into a host cell.
  • a “vector,” as used herein, is a nucleic acid molecule capable of propagating another nucleic acid molecule to which it is linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • the term “vector” thus includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced.
  • Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome.
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retrovirus, EBV-derived episomes, and all the other vectors that the skilled man will know to be convenient for ensuring the expression of the heavy and/or light chains of the antibody of interest (e.g. , an anti-VISTA antibody).
  • the polynucleotides encoding the heavy and the light chains can be cloned into different vectors or in the same vector.
  • the vectors can include one or more selectable marker genes and appropriate expression control sequences.
  • Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media.
  • Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art.
  • the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter.
  • the introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product.
  • PCR polymerase chain reaction
  • the nucleic acid molecule is expressed in a sufficient amount to produce the desired product (e.g. an anti-VISTA antibody provided herein), and it is further understood that expression levels can be optimised to obtain sufficient expression using methods well known in the art.
  • VISTA or “VISTA polypeptide” and similar terms refers to the polypeptide (“polypeptide,” “peptide” and “protein” are used interchangeably herein) encoded by the human Chromosome 10 Open Reading Frame 54 (VISTA) gene, which is also known in the art as B7-H5, platelet receptor Gi24, GI24, Stress Induced Secreted Proteinl , SISP1 , and PP2135, for example, comprising the amino acid sequence of:
  • the VISTA polypeptide has been shown to or is predicted to comprise several distinct regions within the amino acid sequence including: a signal sequence (residues 1 -32; see Zhang et al. , Protein Sci. 13:2819- 2824 (2004)); an immunoglobulin domain - IgV-like (residues 33-162); and a transmembrane region (residues 195-215).
  • the mature VISTA protein includes amino acid residues 33-311 of SEQ ID NO: 1 .
  • the extracellular domain of the VISTA protein includes amino acid residues 33-194 of SEQ ID NO: 1.
  • Related polypeptides include allelic variants (e.g. , SNP variants); splice variants; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, preferably, which retain VISTA activity and/or are sufficient to generate an anti-VISTA immune response.
  • VISTA can exist in a native or denatured form.
  • the VISTA polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • a “native sequence VISTA polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding VISTA polypeptide derived from nature. Such native sequence VISTA polypeptides can be isolated from nature or can be produced by recombinant or synthetic means.
  • the term “native sequence VISTA polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific VISTA polypeptide (e.g. , an extracellular domain sequence), naturally-occurring variant forms (e.g. , alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide.
  • a cDNA nucleic acid sequence encoding the VISTA polypeptide for example, comprises:
  • VISTA is predominantly expressed on the myeloid cell population, particularly myeloid- derived suppressor cells (MDSCs), neutrophils, monocytes, macrophages, and dendritic cells. VISTA can also be expressed on regulatory T cells and CD4 + naive T lymphocytes. As described herein, VISTA is an immunomodulator, that is a negative checkpoint regulator of immune responses (e.g. , inhibits or suppresses immune responses). VISTA has been identified as a negative checkpoint regulator of T cell function and is known to suppress autoimmune responses in a variety of human and mouse models of autoimmunity.
  • MDSCs myeloid- derived suppressor cells
  • VISTA can also be expressed on regulatory T cells and CD4 + naive T lymphocytes.
  • VISTA is an immunomodulator, that is a negative checkpoint regulator of immune responses (e.g. , inhibits or suppresses immune responses).
  • VISTA has been identified as a negative checkpoint regulator of T cell function and is
  • VISTA has in particular been shown to promote tumourigenesis, block T cell function, and modulate the activity of macrophages and immunosuppressive myeloid-derived suppressor cells (MDSCs).
  • VISTA is upregulated on immunosuppressive tumour infiltrating leukocytes such as inhibitory regulatory T cells (Tregs) and MDSCs.
  • Tregs inhibitory regulatory T cells
  • Orthologs to the VISTA polypeptide are also well known in the art.
  • the mouse ortholog to the VISTA polypeptide is V-region Immunoglobulin-containing Suppressor of T cell Activation (VISTA) (also known as PD-L3, PD-1 H, PD-XL, Pro1412 and UNQ730), which shares approximately 70% sequence identity to the human polypeptide.
  • VISTA V-region Immunoglobulin-containing Suppressor of T cell Activation
  • Orthologs of VISTA can also be found in additional organisms including chimpanzee, cow, rat and zebrafish.
  • VISTA is a type-l transmembrane protein belonging to the B7-related immunoglobulin superfamily which is highly expressed in the haematopoietic compartment. VISTA acts both as a ligand and a receptor and negatively regulates T-cell activation through inhibiting CD4 + and CD8 + T-cell proliferation and proinflammatory cytokines (e.g., IFNy, TNFo, or IL-2) production.
  • cytokines e.g., IFNy, TNFo, or IL-2
  • Antibodies inhibiting VISTA function are particularly useful for treating cancer.
  • the present inventors have previously described antibodies directed against VISTA which induce strong tumor growth inhibition (see e.g., WO 2014/197849, WO 2016/094837, and WO 2022/229469).
  • Other anti-VISTA antibodies with anti-cancer properties have also been described in the art (see e.g., WO 2014/039983A1 , WO 2015/145360A1 , WO 2015/097536, WO 2017/137830, WO 2017/181139).
  • an anti-VISTA antibody is a powerful immune adjuvant when administered with a vaccine.
  • the anti- VISTA antibody disclosed herein reverses VISTA-mediated suppression of T-cell activation however, surprisingly, this anti-VISTA antibody is also capable of inducing a humoral response characterised by a significant boost in antibody production.
  • an anti-VISTA antibody inhibits B-cell proliferation in vitro (WO 2015/191881 ; Green et al. J Virol. 89(18):9693-9698 (2015)).
  • the present inventors have presently shown that stimulation of B-cell proliferation is not predictive of an increased antibody production. It is therefore all the more surprising that administration of the present antibody with a vaccine leads both to increased B-cell proliferation and to enhanced antibody production.
  • the present disclosure relates to a vaccine that can be used to increase or enhance an immune response, i.e., create a more effective immune response, by combining an isolated antigen with an anti-VISTA antibody.
  • the present disclosure thus provides a combination of:
  • anti-VISTA antibodies may be polyclonal (“anti-VISTA PAbs”) or monoclonal (“anti-VISTA MAbs”).
  • a polyclonal antibody is an antibody which was produced among or in the presence of one or more other, non-identical antibodies.
  • polyclonal antibodies are produced from a B-lymphocyte in the presence of several other B-lymphocytes producing non-identical antibodies.
  • polyclonal antibodies are obtained directly from an immunised animal.
  • a monoclonal antibody is an antibody arising from a nearly homogeneous antibody population, wherein population comprises identical antibodies except for a few possible naturally-occurring mutations which can be found in minimal proportions.
  • a monoclonal antibody arises from the growth of a single cell clone, such as a hybridoma, and is characterised by heavy chains of one class and subclass, and light chains of one type.
  • the anti-VISTA antibody disclosed herein is a monoclonal antibody, such as any of the monoclonal antibodies disclosed in WO 2014/197849, WO 2016/094837, and WO 2022/229469.
  • a monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies, i.e. , the individual antibodies of the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. Such monoclonal antibody may be produced by a single clone of B cells or hybridoma. Monoclonal antibodies may also be recombinant, i.e., produced by protein engineering. Monoclonal antibodies may also be isolated from phage antibody libraries. In addition, in contrast with preparations of polyclonal antibodies which typically include various antibodies directed against various determinants, or epitopes, each monoclonal antibody is directed against a single epitope of the antigen. The disclosure relates to an antibody isolated or obtained by purification from natural sources or obtained by genetic recombination or chemical synthesis.
  • the monoclonal anti-VISTA antibody disclosed herein is the antibody 26A described in WO 2016/094837 and WO 2022/229469.
  • This antibody is capable of overriding VISTA- mediated inhibition of T-cell responses, notably through blockade of the interaction between VISTA and each of its two binding partners, PSG-L1 and VSIG3.
  • the antibody 26A inhibits tumour proliferation in vivo, indicating that the antibody 26A is capable of generating anti-tumour immunity. Effector functions are required for antibody 26A suppression of VISTA-mediated inhibition of T-cell activation and for its consequent anti-tumour activity.
  • the monoclonal anti-VISTA antibody 26A disclosed herein comprises three heavy-chain CDRS and three light-chain CDRs.
  • the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the three heavy-chain CDRs and the light chain comprises the three light-chain CDRs.
  • the antibody 26A disclosed herein comprises three heavy-chain CDRs and three light-chain CDRs, wherein the sequence of each CDR is selected in the group consisting of sequences set forth in SEQ ID NOS:3-8.
  • the anti-VISTA antibody 26A disclosed herein comprises three heavychain CDRs comprising sequences set forth as SEQ ID NO:3-5. In an embodiment, the anti-VISTA antibody 26A disclosed herein comprises three lightchain CDRs comprising sequences set forth as SEQ ID NO:6-8.
  • the anti-VISTA antibody 26A disclosed herein comprises a heavy chain comprising 3 CDRs comprising sequences set forth as SEQ ID NO:3-5 and a light chain comprising 3 CDRs comprising sequences set forth as SEQ ID NO:6-8.
  • the present disclosure thus provides a combination of:
  • an anti-VISTA antibody or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO. 3-5 and three light chain CDRs of SEQ ID NO. 6-8.
  • the anti-VISTA monoclonal antibodies described herein can be in the form of full-length antibodies, multiple chain or single chain antibodies, fragments of such antibodies that selectively bind to VISTA (including but not limited to Fab, Fab', (Fab )?, Fv, and scFv), surrobodies (including surrogate light chain construct), single domain antibodies, humanised antibodies, camelised antibodies and the like. They also can be of, or derived from, any isotype, including, for example, IgA (e.g. , lgA1 or lgA2), IgD, IgE, IgG (e.g.
  • the anti-VISTA antibody is an IgG (e.g. , lgG1 , lgG2, lgG3 or lgG4).
  • the antibody further comprises a human constant region.
  • the human constant region is selected from the group consisting of lgG1 , lgG2, lgG2, lgG3and lgG4.
  • the human constant region is IgG 1 .
  • the heavy chain constant region has gamma (y), mu (p), alpha (a), delta (6) and epsilon (E) types, and, as a subclass, it has gammal (y1 ), gamma2 (y2), gamma3 (y3), gamma4 (y4), alphal (o1 ) and alpha2 (o2).
  • the light chain constant region has kappa (K) and lambda (A) types.
  • the monoclonal anti-VISTA antibody 26A disclosed herein includes murine, chimeric and humanised antibodies, such as described after.
  • the anti-VISTA antibody 26A disclosed herein is a murine (m) antibody, or any antigen binding fragments, wherein the antibody also comprises light-chain and heavy-chain constant regions derived from an antibody of a species heterologous with the mouse, notably man.
  • the anti-VISTA antibody 26A disclosed herein is a chimeric antibody.
  • a chimeric antibody is an antibody containing a natural variable region (light chain and heavy chain) derived from an antibody of a given species in combination with constant regions of the light chain and the heavy chain of an antibody of a species heterologous to said given species.
  • the antibodies, or chimeric fragments of same can be prepared by using the techniques of recombinant genetics.
  • the chimeric antibody could be produced by cloning recombinant DNA containing a promoter and a sequence coding for the variable region of a nonhuman monoclonal antibody of the invention, notably murine, and a sequence coding for the human antibody constant region.
  • a chimeric antibody according to the invention coded by one such recombinant gene could be, for example, a mouse-human chimera, the specificity of this antibody being determined by the variable region derived from the murine DNA and its isotype determined by the constant region derived from human DNA.
  • the chimeric antibodies 26A herein described can be also characterised by the constant domain and, more particularly, these chimeric antibodies 26A can be selected or designed such as, without limitation, lgG1 , lgG2, lgG3, lgG4, IgM, IgA, IgD or IgE. More preferably, in the context of the present disclosure, said the chimeric antibodies 26A are lgG1 , lgG2, lgG2, lgG3 or lgG4. Even more preferably, the chimeric antibodies disclosed herein are IgG 1 .
  • the anti-VISTA antibody 26A is a humanised antibody.
  • a humanised antibody is an antibody that contains CDR regions derived from an antibody of nonhuman origin, the other parts of the antibody molecule being derived from one (or several) human antibodies.
  • some of the skeleton segment residues (called FR) can be modified to preserve binding affinity.
  • the humanised antibody 26A thus has the same CDRs as the murine antibody 26A, but differs in that the other parts of the antibody molecule are derived from one (or several) human antibodies.
  • the humanised antibody 26A comprises a heavy chain comprising 3 CDRs comprising sequences set forth as SEQ ID NO:3-5 and a light chain comprising 3 CDRs comprising sequences set forth as SEQ ID NO:6-8.
  • the humanised antibody 26A comprises a heavy chain variable domain of sequence SEQ ID NO:9 or any sequence exhibiting at least 80% identity with SEQ ID N0:9 and a light chain comprising three light-chain CDRs of sequences SEQ ID NOs:6, 7 and 8.
  • the humanised antibody 26A comprises a heavy chain comprising three light-chain CDRs of sequences SEQ ID NOs:3, 4 and 5, and a light chain variable domain of sequence SEQ ID NO: 10 or any sequence exhibiting at least 80% identity with SEQ ID NO: 10.
  • the humanised antibody 26A comprises a heavy chain variable domain of sequence SEQ ID NO:9 and a light chain variable domain of sequence SEQ ID N0:10.
  • the humanised antibodies 26A herein described can be also characterised by the constant domain and, more particularly, these humanised antibodies 26A can be selected or designed such as, without limitation, lgG1 , lgG2, lgG3, lgG4, IgM, IgA, IgD or IgE. More preferably, in the context of the present disclosure, the humanised antibodies 26A are lgG1 , lgG2, lgG2, lgG3 or lgG4. Even more preferably, the humanised antibodies 26A disclosed herein are lgG1 .
  • the complete heavy chain of humanised antibody 26A preferably has the sequence represented by SEQ ID NO:11.
  • the complete light chain of humanised antibody 26A has the sequence represented by SEQ ID NO: 12.
  • the humanised antibody 26A disclosed herein has a complete heavy chain that has the sequence represented by SEQ ID NO:11 , and the complete light chain that has the sequence represented by SEQ ID NO:12.
  • the anti-VISTA antibody 26A, or an antigen-binding fragment thereof, which is particularly useful in the present disclosure is represented in Table 2.
  • Table 2 Sequences of the 26ADMD anti-VISTA antibody.
  • humanised antibodies disclosed therein or fragments thereof can be prepared by any technique known to the person skilled in the art. Such humanised antibodies are preferred for their use in methods involving in vitro diagnoses or preventive and/or therapeutic treatment in vivo.
  • Other humanisation techniques also known to a person skilled in the art, such as, for example, “CDR grafting” described by PDL in patents EP 0 451 216, EP 0 682 040, EP 0 939 127, EP 0 566 647 or US 5,530,101 , US 6,180,370, US 5,585,089 and US 5,693,761.
  • US patents 5,639,641 or 6,054,297, 5,886,152 and 5,877,293 can also be cited.
  • Anti-VISTA antibodies include labelled antibodies, useful in diagnostic applications.
  • the antibodies can be used diagnostically, for example, to detect expression of a target of interest in specific cells, tissues, or serum; or to monitor the development or progression of an immunologic response as part of a clinical testing procedure to, e.g. , determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance or “label.”
  • a label can be conjugated directly or indirectly to an anti-VISTA antibody of the disclosure.
  • the label can itself be detectable (e.g.
  • radioisotope labels, isotopic labels, or fluorescent labels can catalyse chemical alteration of a substrate compound or composition which is detectable.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
  • the detectable substance can be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, B- galactosidase, acetylcholinesterase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • luciferases e.g., firefly luciferas
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, dimethylamine-1 -napthalenesulfonyl chloride, or phycoerythrin and the like;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable isotopic materials include 13 C, 15 N, and deuterium; and
  • suitable radioactive material include 125 l, 131 l, 111 ln or "Tc.
  • anti-VISTA antibodies of the present invention can be further modified to contain additional non -proteinaceous moieties that are known in the art and readily available.
  • anti-VISTA monoclonal antibodies which are derivatised, covalently modified, or conjugated to other molecules, for use in diagnostic and therapeutic applications.
  • derivatised antibodies include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, derivatisation by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids.
  • the monoclonal antibody of the present invention or an antigen-binding fragment thereof may be subjected to derivatisation as described above, notably by e.g., glycosylation and/or PEGylation, in order to enhance the residence time in a living body to which the antibody is administered.
  • glycosylation and/or PEGylation various patterns of glycosylation and/or PEGylation can be modified by a method well known in the art, as long as the function of the antibody of the present invention is maintained, and included in the antibody of the present invention are a variant monoclonal antibody in which various patterns of glycosylation and/or PEGylation are modified, or an antigen-binding fragment thereof.
  • the moieties suitable for derivatisation of the antibody are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 , 3- dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n- vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatisation can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • the anti-VISTA antibodies of the present disclosure can be attached to Poly(ethyleneglycol) (PEG) moieties.
  • the antibody is an antibody fragment and the PEG moieties are attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group.
  • Such amino acids can occur naturally in the antibody fragment or can be engineered into the fragment using recombinant DNA methods. See, for example U.S. Patent No. 5,219,996. Multiple sites can be used to attach two or more PEG molecules.
  • PEG moieties can be covalently linked through a thiol group of at least one cysteine residue located in the antibody fragment. Where a thiol group is used as the point of attachment, appropriately activated effector moieties, for example thiol selective derivatives such as maleimides and cysteine derivatives, can be used.
  • an anti-VISTA antibody conjugate is a modified Fab' fragment which is PEGylated, i.e. , has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g. , according to the method disclosed in EP0948544.
  • PEG poly(ethyleneglycol)
  • PEG can be attached to a cysteine in the hinge region.
  • a PEG-modified Fab' fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region.
  • a lysine residue can be covalently linked to the maleimide group and to each of the amine groups on the lysine residue can be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000 Da.
  • the total molecular weight of the PEG attached to the Fab' fragment can therefore be approximately 40,000 Da.
  • conjugates of an antibody and non -proteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the non- proteinaceous moiety to a temperature at which cells proximal to the antibody-non- proteinaceous moiety are killed.
  • the present disclosure encompasses polynucleotides encoding immunoglobulin light and heavy chain genes for antibodies, notably anti-VISTA antibodies, vectors comprising such nucleic acids, and host cells capable of producing the antibodies of the disclosure. Also provided herein are polynucleotides that hybridise under high stringency, intermediate or lower stringency hybridisation conditions, e.g. , as defined supra, to polynucleotides that encode an antibody or modified antibody provided herein.
  • the present disclosure relates to one or more polynucleotides encoding an antibody, notably an antibody capable of binding specifically to VISTA, or a fragment thereof, as described above.
  • the present disclosure notably provides a polynucleotide encoding the heavy chain and/or the light chain of the anti-VISTA antibody disclosed herein.
  • nucleic acid molecules provided herein comprise or consist of a nucleic acid sequence encoding the heavy chain variable region and light chain variable region disclosed herein, or any combination thereof (e.g. , as a nucleotide sequence encoding an antibody provided herein, such as e.g. , a full-length antibody, heavy and/or light chain of an antibody, or a single chain antibody provided herein).
  • the polynucleotide encodes three heavy-chain CDRs of the anti-VISTA antibody described herein.
  • the polynucleotide encodes three light-chain CDRs of the anti-VISTA antibody described herein.
  • the polynucleotide encodes three heavy-chain CDRs and three light-chain CDRs of the anti-VISTA antibody described herein.
  • Another example provides a couple of polynucleotides, wherein the first polynucleotide encodes three heavy-chain CDRs of the anti-VISTA antibody described herein; and the second polynucleotide encodes three light-chain CDRs of the same anti-VISTA antibody described herein.
  • the polynucleotide encodes the heavy-chain variable region of the anti-VISTA antibody described herein.
  • the polynucleotide encodes the lightchain variable region of the anti-VISTA antibody described herein.
  • the polynucleotide encodes the heavy-chain variable region and the light-chain variable region of the anti-VISTA antibody described herein.
  • Another instance provides a couple of polynucleotides, wherein the first polynucleotide encodes the heavy-chain variable region of the anti-VISTA antibody described herein; and the second polynucleotide encodes the light-chain variable region of the same anti-VISTA antibody described herein.
  • the polynucleotide encodes the heavy-chain of the anti-VISTA antibody described herein. In an embodiment, the polynucleotide encodes the light-chain of the anti-VISTA antibody described herein. In an embodiment, the polynucleotide encodes the heavy-chain and the light-chain of the anti-VISTA antibody described herein. Another embodiment provides a couple of polynucleotides, wherein the first polynucleotide encodes the heavy-chain of the anti-VISTA antibody described herein; and the second polynucleotide encodes the light-chain of the same anti-VISTA antibody described herein.
  • the polynucleotide encodes the heavy chain of the anti-VISTA antibody described above is provided.
  • the heavy chain comprises three heavychain CDRs of sequence SEQ ID NOS: 13-15. More preferably, the heavy chain comprises a heavy chain comprising the variable region of sequence SEQ ID NO: 19. Even more preferably, the heavy chain has the sequence represented by SEQ ID NO:21 .
  • the polynucleotide encodes the light chain of an anti-VISTA antibody described above.
  • said light chain comprises three light-chain CDRs of sequence SEQ ID NOS: 16-18. More preferably, said light chain comprises a light chain comprising the variable region of sequence SEQ ID N0:20. Even more preferably, the light chain has the sequence represented by SEQ ID NO:22.
  • the polynucleotide encoding the light chain and heavy chain of the monoclonal antibody of the present invention or an antigen-binding fragment thereof can have various variations in the coding region within a range in which the amino acid sequence of the light chain and heavy chain of an antibody expressed from the coding region is not changed, and, even in a region other than the coding region, various changes or modifications can be made within a range in which the gene expression is not affected by them.
  • the skilled person will easily understand that those variant genes also fall within the scope of the present invention.
  • nucleic acid bases can be changed by substitution, deletion, insertion, or a combination thereof, and those also fall within the scope of the present invention.
  • Sequence of the polynucleotide may be either a single chain or a double chain, and it may be either a DNA molecule or an RNA (mRNA) molecule.
  • expression systems may be used to express the antibody of the invention.
  • such expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transiently transfected with the appropriate nucleotide coding sequences, express an IgG antibody in situ.
  • the disclosure provides vectors comprising the polynucleotides described above.
  • the vector contains a polynucleotide encoding a heavy chain of the anti-VISTA antibody of interest.
  • the polynucleotide encodes the light chain of the anti-VISTA antibody of interest.
  • the polynucleotide encodes the heavy chain and the light chain of the anti-VISTA antibody of interest.
  • a couple of polynucleotides are provided, wherein the first polynucleotide encodes the heavy chain of the anti-VISTA antibody of interest, and the second polynucleotide encodes the light chain of the same anti-VISTA antibody of interest.
  • the disclosure also provides vectors comprising polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments, and probes thereof.
  • the polynucleotides encoding said heavy and/or light chains are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational sequences.
  • these polynucleotides are cloned into two vectors.
  • “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • expression control sequence refers to polynucleotide sequences which are necessary to affect the expression and processing of coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilise cytoplasmic mRNA; sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • polynucleotides of the invention and vectors comprising these molecules can be used for the transformation of a suitable host cell.
  • host cell is intended to refer to a cell into which a recombinant expression vector has been introduced in order to express the anti-VISTA antibody of interest. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • Transformation can be performed by any known method for introducing polynucleotides into a cell host. Such methods are well known of the man skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, biolistic injection and direct microinjection of DNA into nuclei.
  • the host cell may be co-transfected with one or more expression vectors.
  • a host cell can be transfected with a vector encoding both the heavy chain and the light chain of the anti-VISTA antibody of interest, as described above.
  • the host cell can be transformed with a first vector encoding the heavy chain of the anti-VISTA antibody of interest, and with a second vector encoding the light chain of said antibody.
  • Mammalian cells are commonly used for the expression of a recombinant therapeutic immunoglobulins, especially for the expression of whole recombinant antibodies.
  • mammalian cells such as HEK293 or CHO cells, in conjunction with a vector, containing the expression signal such as one carrying the major intermediate early gene promoter element from human cytomegalovirus, are an effective system for expressing the humanised anti-VISTA antibody of the invention (Foecking et al., 1986, Gene 45:101 ; Cockett et al., 1990, Bio /Technology 8: 2).
  • a host cell may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing of protein products may be important for the function of the protein.
  • Different host cells have features and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems are chosen to ensure the correct modification and processing of the expressed antibody of interest.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product may be used.
  • Such mammalian host cells include, but are not limited to, CHO, COS, HEK293, NS/0, BHK, Y2/0, 3T3 or myeloma cells (all these cell lines are available from public depositories such as the Collection Nationale des Cultures de Microorganismes, Paris, France, or the American Type Culture Collection, Manassas, VA, U.S.A.).
  • cell lines which stably express the antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells are transformed with DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences known to the person skilled in art, and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for one to two days in an enriched media, and then are moved to a selective media.
  • appropriate expression regulatory elements including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences known to the person skilled in art, and a selectable marker.
  • the selectable marker on the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and be expanded into a cell line.
  • Other methods for constructing stable cell lines are known in the art.
  • methods for site-specific integration have been developed. According to these methods, the transformed DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences is integrated in the host cell genome at a specific target site which has previously been cleaved (Moele et al., Proc. Natl. Acad. Sci. U.S.A., 104(9): 3055-3060; US 5,792,632; US 5,830,729; US 6,238,924; WO 2009/054985; WO 03/025183; WO 2004/067753).
  • a number of selection systems may be used according to the invention, including but not limited to the Herpes simplex virus thymidine kinase (Wigler et al., Cell 11 :223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48: 202, 1992), glutamate synthase selection in the presence of methionine sulfoximide (Adv Drug Del Rev, 58: 671 , 2006, and website or litreature of Lonza Group Ltd.) and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817, 1980) genes in tk, hgprt or aprt cells, respectively.
  • Herpes simplex virus thymidine kinase Wigler et al., Cell 11 :223, 1977
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77: 357, 1980); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78: 2072, 1981 ); neo, which confers resistance to the aminoglycoside, G-418 (Wu et al., Biotherapy 3: 87, 1991 ); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147, 1984).
  • a modified zinc finger protein can be engineered that is capable of binding the expression regulatory elements upstream of the gene of the invention; expression of the said engineered zinc finger protein (ZFN) in the host cell of the invention leads to increases in protein production (see e.g. Reik et al., Biotechnol. Bioeng., 97(5): 1180-1189, 2006).
  • ZFN can stimulate the integration of a DNA into a predetermined genomic location, resulting in high-efficiency site-specific gene addition (Moehle et al, Proc Natl Acad Sci USA, 104: 3055, 2007).
  • the anti-VISTA antibody of interest may be prepared by growing a culture of the transformed host cells under culture conditions necessary to express the desired antibody.
  • the resulting expressed antibody may then be purified from the culture medium or cell extracts. Soluble forms of the anti-VISTA antibody of interest can be recovered from the culture supernatant. It may then be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by Protein A affinity for Fc, and so on), centrifugation, differential solubility or by any other standard technique for the purification of proteins. Suitable methods of purification will be apparent to a person of ordinary skills in the art.
  • Another aspect of the invention thus relates to a method for the production of an antibody (e.g., an anti-VISTA antibody) described herein, said method comprising the steps of: a) growing the above-described host cell in a culture medium under suitable culture conditions; and b) recovering the antibody (e.g., an anti-VISTA antibody), from the culture medium or from said cultured cells.
  • an antibody e.g., an anti-VISTA antibody
  • the antibody obtained by culturing the transformant can be used in a non-purified state. Impurities can be removed by additional various commons methods like centrifuge or ultrafiltration, and the resultant may be subjected to dialysis, salt precipitation, chromatography or the like, in which the method may be used either singly or in combination thereof. Among them, affinity chromatography is most widely used, including ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, and the like.
  • the present combinations can comprise an isolated antigen or a polynucleotide encoding at least one epitope of this antigen.
  • the antigen can be anything that induces an immune response in a subject. Some antigens can induce a strong immune response. Other antigens can induce a weak immune response. The antigen can elicit a greater immune response when combined with the VISTA antibody 26A described above.
  • the antigen disclosed herein can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or any other naturally occurring or synthetic compound.
  • the antigen is a nucleic acid sequence, an amino acid sequence, or a combination thereof.
  • the nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof.
  • the nucleic acid sequence can also include additional sequences that encode linker or tag sequences that are linked to the antigen by a peptide bond.
  • the amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof.
  • the antigen can be contained in a protein, a nucleic acid, or a fragment thereof, or a variant thereof, or a combination thereof from any number of organisms, for example, a virus, a parasite, a bacterium, a fungus, or a mammal.
  • the antigen can be associated with a cancer, an infectious disease, an autoimmune disorder, or an inflammatory disorder.
  • the antigen can be associated with a cancer or an infectious disease.
  • the antigen of the combinations disclosed herein can be a “full-length” antigen, i.e., an antigen that substantially comprises the entire amino acid sequence of the naturally occurring (wild-type) antigen.
  • a naturally occurring (wild-type) antigen may be encoded by a naturally occurring (wild-type) nucleic acid sequence or by a nucleic acid sequence “variant” (because of the degeneracy of the genetic code).
  • the polynucleotide encoding at least one epitope of this antigen of the combinations disclosed herein comprises a wild-type nucleic acid sequence or a nucleic acid sequence "variant" encoding a full-length, wild-type antigen as described above.
  • the combinations disclosed herein can also comprise a variant of an antigen, e.g., a variant of naturally occurring (wild type) antigen.
  • a variant of an antigen e.g., a variant of naturally occurring (wild type) antigen.
  • sequence of an antigen “variant” or “sequence variant” differs in at least one amino acid residue from the amino acid sequence of the naturally occurring (wild-type) antigen serving as a reference (or “parent”) sequence.
  • variant antigens thus preferably comprise at least one amino acid mutation, substitution, insertion or deletion as compared to their respective reference sequence.
  • the term “variant” as used herein comprises any homolog, isoform or transcript variant of a protein antigen as defined herein, wherein the homolog, isoform or transcript variant is preferably characterised by a degree of identity or homology, respectively, as defined herein.
  • an antigen “variant” disclosed herein may comprise at least one amino acid substitution as compared to the wild-type (naturally occurring) antigen amino acid sequence.
  • the substitution may be selected from a conservative or non-conservative substitution.
  • these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can form hydrogen bridges, e.g. , side chains which have a hydroxyl function.
  • an amino acid having a polar side chain may be replaced by another amino acid having a corresponding polar side chain, or, for example, an amino acid characterised by a hydrophobic side chain may be substituted by another amino acid having a corresponding hydrophobic side chain (e.g. , serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)).
  • a “variant” of an antigen may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring (wild-type) antigen.
  • the combinations disclosed herein comprise a fragment of an antigen (or a variant thereof).
  • This fragment can be of any length, provided that it preferably comprises at least one functional epitope.
  • a “fragment” of an antigen (or a variant thereof) may comprise a sequence of an antigen (or a variant thereof) as defined above, which is, with regard to its amino acid sequence (or its encoding nucleic acid sequence), N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the naturally occurring antigen or a variant thereof (or its encoding nucleic acid sequence).
  • Such truncation may thus occur either at the amino acid level or at the nucleic acid level, respectively.
  • a sequence identity with respect to such a fragment as defined herein therefore preferably refers to the entire antigen (or a variant thereof) as defined herein or to the entire (coding) nucleic acid sequence of such an antigen (or a variant thereof).
  • a “fragment” of antigen (or a variant thereof) may comprise or consist of an amino acid sequence of said antigen (or a variant thereof) as defined herein, having a length of about 5 to about 20 or even more amino acids and which is preferably processed and presented by an MHC complex.
  • a fragment of an antigen (or a variant thereof) may comprise or consist of an amino acid sequence of said antigen (or a variant thereof) as defined herein, which has a length of about 6 to about 20 or even more amino acids, e.g. a fragment as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g.
  • fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein the fragment may be selected from any part of the amino acid sequence.
  • These fragments are typically recognized by T-cells in the form of a complex consisting of the peptide fragment and an MHC molecule, i.e. , the fragments are typically not recognized in their “native” or “free” form, but rather in MHC-bound form.
  • a “fragment” of an antigen (or a variant thereof) encoded by the at least one coding sequence of the epitope-encoding RNA of the inventive combination may typically comprise or consist of an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective full-length wildtype antigen (or variant thereof).
  • the at least one antigen “fragment” encoded by the epitope-encoding RNA may be a (N-terminally, C-terminally and/or intrasequentially) truncated fragment of (a) a wild-type antigen or (b) an antigen variant as defined herein.
  • fragment may however also include “fragment variants” comprising or consisting of an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97% to a fragment of (a) a wild-type antigen or (b) an antigen variant as defined herein.
  • the combinations disclosed herein thus comprise an isolated antigen, a variant thereof, or a fragment thereof, or a polynucleotide encoding at least one epitope of this antigen, or variant thereof, or fragment thereof.
  • the combinations disclosed herein comprise an isolated antigen, a variant thereof, or a fragment thereof, wherein the isolated antigen, a variant thereof, or a fragment thereof is a peptide.
  • Peptide-based vaccines are useful for eliciting immune responses in diseases such as cancer, infectious diseases, autoimmune disorders, and inflammatory disorders.
  • diseases such as cancer, infectious diseases, autoimmune disorders, and inflammatory disorders.
  • small peptides are poor immunogens because they act as haptens that lack the necessary Th-cell epitopes and/or that are captured with low efficiency by antigen presenting cells (APC).
  • APC antigen presenting cells
  • a peptide immunogen can be linked to a suitable carrier to help elicit an immune response.
  • suitable carriers include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from other pathogenic bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an attenuated toxin derivative.
  • cytokines such as IL-1o, IL-1 B, IL-2, INF-y, IL-10, GM-CSF, and chemokines, such as M1 P1 a and B and RANTES.
  • Immunogenic agents can also be linked to peptides that enhance transport across tissues, as described in WO 97/17613 and WO 97/17614.
  • Immunogenic agents can be linked to carriers by chemical crosslinking.
  • Techniques for linking an immunogen to a carrier include the formation of disulfide linkages using N- succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl-4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) (if the peptide lacks a sulfhydryl group, this can be provided by addition of a cysteine residue).
  • SPDP N- succinimidyl-3-(2-pyridyl-thio) propionate
  • SMCC succinimidyl-4-(N- maleimidomethyl)cyclohexane-1 -carboxylate
  • reagents create a disulfide linkage between themselves and peptide cysteine resides on one protein and an amide linkage through the epsilon-amino on a lysine, or other free amino group in other amino acids.
  • disulfide/amide-forming agents are described by Immun. Rev. 62, 185 (1982).
  • Other bifunctional coupling agents form a thioether rather than a disulfide linkage.
  • Many of these thio-ether-forming agents are commercially available and include reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid, 4- (N-maleimido-methyl)cyclohexane-l -carboxylic acid.
  • the carboxyl groups can be activated by combining them with succinimide or 1 -hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
  • Immunogenic peptides can also be expressed as fusion proteins with carriers.
  • the immunogenic peptide can be linked at the amino terminus, the carboxyl terminus, or at a site anywhere within the peptide (internally) to the carrier. In some instances, multiple repeats of the immunogenic peptide can be present in the fusion protein, as detailed above.
  • the antigen disclosed herein is a tumour antigen, a bacterial antigen, a parasite antigen, a viral antigen, or a fungal antigen.
  • the antigen comprised in the combinations disclosed herein is a tumour antigen.
  • the combinations disclosed herein preferably any isolated tumour antigen or polypeptides comprising one or more epitopes of this tumour antigen.
  • Tumour antigens as disclosed herein can be any that are known in the art and comprise notably tumour-associated antigens (TAA) and a tumour-specific antigens (TSA).
  • TAA tumour-associated antigens
  • TSA tumour-specific antigens
  • a “tumour- associated antigen” is a protein or other molecule that is found on cancer cells whilst a “tumour-specific antigen” is a protein or other molecule that is found on cancer cells and not on normal cells.
  • Tumour-specific antigens are known in the art (Smith et al. Nat Rev Cancer 19, 465-478 (2019)).
  • the tumour is a malignant tumour.
  • the tumour antigen is preferably an antigen associated with a cancer disease.
  • tumour antigen as used herein is typically derived from a tumour cell, preferably a mammalian tumour cell.
  • a tumour antigen is preferably located in or on the surface of a tumour cell derived from a mammalian, preferably from a human, tumour, such as a systemic or a solid tumour.
  • Tumour antigens can be classified in a variety of ways. Tumour antigens include antigens encoded by genes that have undergone chromosomal alteration. Many of these antigens are found in lymphoma and leukaemia. Even within this classification, antigens can be characterised as those that involve activation of quiescent genes.
  • BCL-1 and IgH Mantel cell lymphoma
  • BCL-2 and IgH Follicular lymphoma
  • BCL-6 Diffuse large B-cell lymphoma
  • TAL-1 and TCR delta or SIL T-cell acute lymphoblastic leukaemia
  • c-MYC and IgH or IgL Burkitt lymphoma
  • MUN/IRF4 and IgH Myeloma
  • PAX-5 (BSAP) (Immunocytoma).
  • tumour antigens that involve chromosomal alteration and thereby create a novel fusion gene and/or protein include RARoa, PML, PLZF, NPMor NuM4 (Acute promyelocytic leukaemia), BCR and ABL (Chronic myeloid/acute lymphoblastic leukaemia), MLL (HRX) (Acute leukaemia), E2A and PBXor HLF (B-cell acute lymphoblastic leukaemia), NPM, ALK (Anaplastic large cell leukaemia), and NPM, MLF-1 (Myelodysplastic syndrome/acute myeloid leukaemia).
  • tumour antigens are specific to a tissue or cell lineage. These include cell surface proteins such as CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, Chronic lymphocytic leukaemia (CLL)), CD52 (B-cell CLL), CD33 (Acute myelogenous leukaemia (AML)), CD 10 (gp100) (Common (pre-B) acute lymphocytic leukaemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukaemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukaemia), CD26 (Epithelial and lymphoid malignancies), Human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (Lymphoid malignancies), RCAS1 (Gynaecological carcinomas, biliary adenocarcinomas and
  • Tissue- or lineage-specific tumour antigens also include epidermal growth factor receptors (high expression) such as EGFR (HER1 or erbB1 ) and EGFRvlll (Brain, lung, breast, prostate and stomach cancer), erbB2 (HER2 or HER2/neu) (Breast cancer and gastric cancer), erbB3 (HER3) (Adenocarcinoma), and erbB4 (HER4) (Breast cancer).
  • epidermal growth factor receptors high expression
  • EGFR HER1 or erbB1
  • EGFRvlll Brain, lung, breast, prostate and stomach cancer
  • erbB2 HER2 or HER2/neu
  • HER3 HER3
  • HER4 erbB4
  • Tissue- or lineage-specific tumour antigens also include cell-associated proteins such as Tyrosinase, Melan-A/MART-1 , tyrosinase related protein (TRP)-1 /gp75 (Malignant melanoma), Polymorphic epithelial mucin (PEM) (Breast tumours), and Human epithelial mucin (MUC1 ) (Breast, ovarian, colon and lung cancers).
  • TRP tyrosinase related protein
  • PEM Polymorphic epithelial mucin
  • MUC1 Human epithelial mucin
  • Tissue- or lineage-specific tumour antigens also include secreted proteins such as Monoclonal immunoglobulin (Multiple myeloma and plasmacytoma), Immunoglobulin light chains (Multiple Myeloma), alpha-fetoprotein (Liver carcinoma), Kallikreins 6 and 10 (Ovarian cancer), Gastrin-releasing peptide/bombesin (Lung carcinoma), and Prostate specific antigen (Prostate cancer).
  • Monoclonal immunoglobulin Multiple myeloma and plasmacytoma
  • Immunoglobulin light chains Multiple Myeloma
  • alpha-fetoprotein Liver carcinoma
  • Kallikreins 6 and 10 Ovarian cancer
  • Gastrin-releasing peptide/bombesin Lung carcinoma
  • Prostate specific antigen Prostate cancer
  • tumour antigens are cancer testis (CT) antigens that are expressed in some normal tissues such as testis and in some cases placenta. Their expression is common in tumours of diverse lineages and as a group the antigens form targets for immunotherapy.
  • CT antigens include MAGE-A1 , -A3, -A6, -A12, BAGE, GAGE, HAGE, LAGE-1 , NY-ESO-1 , RAGE, SSX-1 , -2, -3, -4, -5, -6, -7, -8, -9, HOM-TES-14/SCP-1 , HOM- TES-85 and PRAME.
  • CT antigens and the cancers in which they are expressed include SSX-2, and -4 (Neuroblastoma), SSX-2 (HOM-MEL-40), MAGE, GAGE, BAGE and PRAME (Malignant melanoma), HOM-TES-14/SCP-1 (Meningioma), SSX-4 (Oligodendroglioma), HOM-TES-14/SCP-1 , MAGE-3 and SSX-4 (Astrocytoma), SSX member (Head and neck cancer, ovarian cancer, lymphoid tumours, colorectal cancer and breast cancer), RAGE-1 , -2, -4, GAGE-1 -2, -3, -4, -5, -6, -7 and -8 (Head and neck squamous cell carcinoma (HNSCC)), HOM-TES14/SCP-1 , PRAME, SSX-1 and CT-7 (Non-Hodgkin's lymphoma), and PRAME (Acute lymph
  • tumour antigens are not specific to a particular tissue or cell lineage. These include members of the carcinoembryonic antigen (CEA) family: CD66a, CD66b, CD66c, CD66d and CD66e. These antigens can be expressed in many different malignant tumours and can be targeted by immunotherapy.
  • CEA carcinoembryonic antigen
  • tumour antigens are viral proteins and these include Human papilloma virus protein (cervical cancer), and EBV-encoded nuclear antigen (EBNA)-1 (lymphomas of the neck and oral cancer).
  • EBNA EBV-encoded nuclear antigen
  • tumour antigens are mutated or aberrantly expressed molecules such as but not limited to CDK4 and beta-catenin (melanoma).
  • the tumour antigen is selected from the group consisting of 19.9; oncofoetal protein 5T4; antigen 4.2; A33; adenosine deaminase-binding protein (ADAbp), adenomatous polyposis coli protein (APC), AFP; ALCAM; ALK, AML1 , BAGE; BCL-1 /lgH, BCL-2/lgH, BCL-6, BCR/ABL, BN, brain glycogen phosphorylase, E-cadherin, CAGE 3, CAP-1 , CAP-2, o-catenin, B-catenin, .y-catenin, CA125; Carboxypeptidase M; B1 ;CD5; CD19; CD20; CD22; CD23; CD25; CD27; CD30; CD33; CD36; CD46; CD52; CD79a/CD79b; CD123; CD317; CDK4, CEA, including include CD66a, CD66b, CD66c
  • Cancer or tumour antigens can also be classified according to the cancer or tumour they are associated with (i.e. , expressed by). Cancers or tumours associated with tumour antigens include acute lymphoblastic leukaemia (etv6; am11 ; cyclophilin b), B cell lymphoma (Ig-idiotype); Burkitt's (Non-Hodgkin's) lymphoma (CD20); glioma (E-cadherin; o- catenin; B-catenin; y-catenin; p120ctn), bladder cancer (p21 ras), biliary cancer (p21 ras), breast cancer (MUC family; HER2/neu; c-erbB-2), cervical carcinoma (p53; p21 ras), colon carcinoma (p21 ras; HER2/neu; c-erbB-2; MUC family), colorectal cancer (Colorectal associated antigen (CRC)-0017-1A/GA73
  • antigens of interest for the present disclosure relate to infectious diseases.
  • antigens include notably viral antigens, parasite antigens, bacterial antigens, and fungal antigens.
  • the antigen can be a viral antigen, or fragment thereof, or variant thereof.
  • the viral antigen can be from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae.
  • the viral antigen can be from papilloma viruses, for example, human papilloma virus (HPV), coronaviruses, such as SARS-CoV1 , SARS-CoV2, and MERS-CoV, human immunodeficiency virus (HIV), polio virus, hepatitis viruses, for example, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV), smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, dengue fever virus, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T- cell leukaemia virus (HTLV-I), hairy cell leukaemia virus (HTLV-II), California encephalitis virus, Hanta virus (haemorrhagic fever
  • the antigen can be a parasite antigen or fragment or variant thereof.
  • the parasite can be a protozoa, helminth, or ectoparasite.
  • the helminth i.e. , worm
  • the ectoparasite can be lice, fleas, ticks, and mites.
  • the parasite can be any parasite causing the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloid
  • the parasite can be Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.
  • the antigen can be a bacterial antigen or fragment or variant thereof.
  • the bacterium can be from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.
  • the bacterium can be a gram-positive bacterium or a gram-negative bacterium.
  • the bacterium can be an aerobic bacterium or an anaerobic bacterium.
  • the bacterium can be an autotrophic bacterium or a heterotrophic bacterium.
  • the bacterium can be a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, a psychrophile, an halophile, or an osmophile.
  • the bacterium can be an anthrax bacterium, an antibiotic resistant bacterium, a disease-causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium.
  • the bacterium can be a mycobacterium, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile, or Mycobacterium tuberculosis.
  • the antigen can be a fungal antigen or fragment or variant thereof.
  • the fungus can be Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.
  • the combinations disclosed herein comprise at least one polynucleotide encoding at least one epitope of the antigen, or variant thereof, or fragment thereof.
  • an immune response is against the antigen is induced in a subject when somatic cells of the patient are brought to express the at least one polynucleotide encoding at least one epitope of the antigen, or variant thereof, or fragment thereof.
  • the polynucleotide disclosed herein encodes one epitope of the antigen. In other embodiments, the polynucleotide disclosed herein encodes several epitopes of the antigen. For example, the polynucleotide of the present combinations can encode at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 epitopes of the antigen.
  • the epitope-encoding polynucleotide is mono-, bi-, or multici stronic.
  • Mono, bi- or multicistronic polynucleotides typically comprise one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORF).
  • An open reading frame in this context is a sequence of codons that is translatable into a peptide or protein.
  • the polynucleotide of the combinations disclosed herein can comprise one, two, or more ORFs.
  • the polynucleotide disclosed herein comprises a single ORF.
  • the polynucleotide disclosed herein comprises 2 ORFs.
  • the polynucleotide disclosed herein comprises more than 2 ORFs.
  • the polynucleotide of the present combinations can comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 ORFs.
  • the coding sequences in a bi- or multicistronic epitope-encoding polynucleotide preferably encode distinct epitopes (or antigens or variants or fragments thereof comprising said epitopes) as defined herein.
  • Bi- or even multicistronic epitopeencoding polynucleotide may encode, for example, at least two, three, four, five, six or more (preferably different) epitopes (or antigens or variants or fragments thereof comprising said epitopes) as defined herein.
  • the several epitopes of the antigen are carried by several nucleic acid molecules.
  • Each ORF can be present on the same or on separate polynucleotides; however, it is to be understood that one or more ORFs can be present on a single polynucleotide, and these coding regions can be under the control of a single or multiple promoters.
  • the polynucleotide can encode a separate peptide expression product for each encoded epitope or that the expression vector can encode a plurality of peptide expression products, where at least some exhibit(s) several encoded epitopes, of which at least some optionally are separated by peptide linkers.
  • this polynucleotide may encode a plurality of separate proteinaceous expression products or as few as 2 or even one single expression product - in this context it is only relevant whether the encoded epitopes are satisfactorily presented to the immune system and the choice of their presence in separate on combined expression products is therefore of minor relevance.
  • the codon usage of the at least one ORF of the polynucleotide is optimised to the human codon usage.
  • codon optimisation it is referred to the alterations to the coding sequences for the antigen which improve the sequences for codon usage in the cell of the subject, e.g., a human cell.
  • Many parasites, bacteria, viruses, or fungi use a large number of codons which are not so frequently used in e.g., humans. By changing these to correspond to commonly used human codons, increased expression of the at least one epitope of the antigen in the cells of the subject can be achieved. Codon usage tables are known in the art for human cells, as well as for a variety of other organisms.
  • the ORF described herein may additionally or alternatively encode a secretory signal peptide.
  • signal peptides are sequences, typically of about 15 to 30 amino acids, preferably located at the N-terminus of the encoded peptide, which allow the transport of the epitope into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non-classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201 ), signal sequences of cytokines or immunoglobulins, signal sequences of the invariant chain of immunoglobulins or antibodies, signal sequences of Lampl , Tapasin, Erp57, Calretikulin, Calnexin, PLAT, EPO or albumin and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.
  • MHC-molecules e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201
  • signal sequences of cytokines or immunoglobulins e.g. signal sequences of MHC I and II molecules,
  • signal sequences are derived from (human) HLA-A2; (human) PLAT; (human) sEPO; (human) ALB; (human) IgE-leader; (human) CD5; (human) IL2; (human) CTRB2; (human) IgG-HC; (human) Ig-HC; (human) Ig-LC; GpLuc; (human) Ig kappa or a fragment or variant of any of the aforementioned proteins, in particular HLA-A2; HsPLAT; sHsEPO; HsALB; HsPLAT(aa1 -21 ); HsPLAT(aa1 -22); IgE-leader; HsCD5(aa1 -24); HslL2(aa1 -20); HsCTRB2(aa1 - 18); lgG-HC(aa1 -19); lg-HC(aa1 -19); lg-LC(aa1 -19); GpLuc(1
  • the polynucleotide is provided in a complexed form, i.e., complexed or associated with one or more (poly-)cationic compounds, preferably (poly- )cationic lipids.
  • the polynucleotide is present in essentially stable combination with one or more (poly-)cationic compounds, with cationic or polycationic polymers, cationic or polycationic peptides or proteins including protamine, cationic or polycationic polysaccharides and/or cationic or (poly-)cationic lipids, into larger complexes or assemblies without covalent binding.
  • the polynucleotide described herein is complexed or associated with lipids, notably cationic lipids, to form one or more liposomes, lipoplexes, and/or lipid nanoparticles comprising the polynucleotide.
  • lipids notably cationic lipids
  • the polynucleotide is complexed or associated with (a) one or more cationic or polycationic compounds, preferably with cationic or polycationic polymers, cationic or polycationic peptides or proteins including protamine, cationic or polycationic polysaccharides and/or cationic or polycationic lipids; and/or
  • the cationic lipid is a cationic fatty acid, a cationic glycerolipid, a cationic glycerophospholipid, a cationic sphingolipid, a cationic sterol lipid, a cationic prenol lipid, a cationic saccharolipid, or a cationic polyketide.
  • the cationic lipid comprises two fatty acyl chains, each chain of which is independently saturated or unsaturated.
  • the cationic lipid is a diglyceride.
  • the cationic lipid is DOTAP (l,2-dioleoyl-3-trimethylammonium-propane), or a derivative thereof.
  • the cationic lipid is DOTMA (l,2-di-0-octadecenyl-3- trimethylammonium propane), or a derivative thereof.
  • the polynucleotide can be DNA, including cDNA; RNA, e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA); a variant thereof; a fragment thereof; or a combination thereof, e.g., a combination of RNA with DNA and/or cDNA.
  • RNA e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA)
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • mRNA messenger RNA
  • a variant thereof e.g., a fragment thereof
  • a combination thereof e.g., a combination of RNA with DNA and/or cDNA.
  • the polynucleotide may be a DNA or an RNA, notably a mRNA, in particular a mRNA produced by in vitro transcription.
  • the polynucleotide is DNA, e.g., cDNA.
  • the DNA molecule encodes at least one epitope of the antigen.
  • the vector can have a nucleic acid sequence containing an origin of replication.
  • the vector can be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
  • the vector can be either a self-replication extra chromosomal vector, or a vector which integrates into a host genome.
  • the one or more vectors can be an expression construct, which is generally a plasmid that is used to introduce a specific gene into a target cell. Once the expression vector is inside the cell, the protein that is encoded by the gene is produced by the cellular- transcription and translation machinery ribosomal complexes.
  • the plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.
  • the vectors of the present invention express large amounts of stable messenger RNA, and therefore proteins.
  • the vectors may have expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
  • expression signals such as a strong promoter, a strong termination codon, adjustment of the distance between the promoter and the cloned gene, and the insertion of a transcription termination sequence and a PTIS (portable translation initiation sequence).
  • the vector can be a circular plasmid or a linear nucleic acid.
  • the circular plasmid and linear nucleic acid are capable of directing expression of a particular nucleotide sequence in an appropriate subject cell.
  • the vector can have a promoter operably linked to the antigen-encoding nucleotide sequence, or the adjuvant-encoding nucleotide sequence, which may be operably linked to termination signals.
  • the vector can also contain sequences required for proper translation of the nucleotide sequence.
  • the vector comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue or organ or stage of development.
  • the vector may be circular plasmid, which may transform a target cell by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).
  • Numerous plasmids known in the art may be used for the production of nucleic acid vaccines. Plasmids suitable for expressing an epitope of an antigen as disclosed herein include plasmids VR1012 (Vical Inc., San Diego Calif.), pCMVI.UBF3/2 (S.
  • linear nucleic acid vaccine or linear expression cassette (“LEC”), that is capable of being efficiently delivered to a subject via electroporation and expressing one or more desired epitopes of the antigen.
  • the LEC may be any linear DNA devoid of any phosphate backbone.
  • the DNA may encode one or more epitopes of the antigen.
  • the LEC may contain a promoter, an intron, a stop codon, and/or a polyadenylation signal.
  • the expression of the epitope(s) may be controlled by the promoter.
  • the LEC may not contain any antibiotic resistance genes and/or a phosphate backbone.
  • the LEC may not contain other nucleic acid sequences unrelated to the desired epitope gene expression.
  • the LEC may be derived from any plasmid capable of being linearised.
  • the plasmid may be capable of expressing the desired epitope(s) of the antigen.
  • the plasmid can be pNP (Puerto Rico/34) or pM2 (New Caledonia/99).
  • the plasmid may be WLV009, pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA encoding the epitope(s) of the antigen, and enabling a cell to translate the sequence to one or more epitopes of an antigen that is recognised by the immune system.
  • viral vectors are provided herein which are capable of delivering a DNA molecule as disclosed herein to a cell.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001 ), and in Ausubel et al. (1997), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • a promoter sequence for example, WO 01 /96584; WO 01 /29058; and U.S. Pat. No. 6,326,193
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno- associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • the vector may have a promoter.
  • a promoter may be any promoter that is capable of driving gene expression and regulating expression of the isolated DNA. Such a promoter is a cis-acting sequence element required for transcription via a DNA dependent RNA polymerase, which transcribes the sequence of the epitopes of the antigen described herein. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter may be positioned about the same distance from the transcription start in the vector as it is from the transcription start site in its natural setting. However, variation in this distance may be accommodated without loss of promoter function.
  • the promoter may be operably linked to the nucleic acid sequence encoding the antigen and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the promoter may be operably linked to the nucleic acid sequence encoding the adjuvant and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination.
  • the promoter may be a CMV promoter, SV40 early promoter, SV40 later promoter, metallo thionein promoter, murine mammary tumour virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or another promoter shown effective for expression in eukaryotic cells.
  • a strong viral promoter other than CMV-IE that may be usefully employed is the early/late promoter of the SV40 virus or the LTR promoter of the Rous sarcoma virus.
  • a strong cellular promoter that may be usefully employed is the promoter of a gene of the cytoskeleton, such as e.g. , the desmin promoter or the actin promoter.
  • the vector may include an enhancer and an intron with functional splice donor and acceptor sites.
  • the vector may contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the vector advantageously comprises a polyadenylation sequence.
  • the polyadenylation signal (poly A) of the bovine growth hormone (bGH) gene, or the poly(A) signal of the rabbit B-globin gene or the poly(A) signal of the SV40 virus can be used advantageously.
  • the vector disclosed herein advantageously contain immunostimulatory sequences (ISS).
  • ISS immunostimulatory sequences
  • These sequences in the vaccine vector are used to enhance T-cell response towards encoded epitopes, in particular Th1 cell responses, which are elicited by adjuvants that incorporate agonists of the toll-like receptors TLR3, TLR7-TLR8, and TLR9. and/or cytosolic RNA receptors such as, but not limited to, RIG- 1 , MDA5 and LGP2 (Desmet et al. Nat. Rev. Imm. 12(7): 479-491 , 2012)
  • the present vectors may further comprise additional elements for proper initiation, regulation and/or termination of transcription (e.g. a transcription termination sequences), mRNA transport (e.g. nuclear localisation signal sequences, polyadenylations sequences), processing (e.g. splicing signals, selfcleaving peptides like T2A, P2A, E2A, F2A, linkers), stability (e.g. introns, like 16S/19S or chimeric human B globin/IgG, and non-coding 5' and 3' sequences), translation (e.g.
  • transcription termination sequences e.g. nuclear localisation signal sequences, polyadenylations sequences
  • processing e.g. splicing signals, selfcleaving peptides like T2A, P2A, E2A, F2A, linkers
  • stability e.g. introns, like 16S/19S or chimeric human B globin/IgG, and non-coding 5' and 3' sequences
  • an initiator Met tripartite leader sequences, IRES ribosome binding sites, signal peptides, etc.
  • targeting sequences e.g. linkers composed of flexible residues like glycine and serine
  • linkers e.g. linkers composed of flexible residues like glycine and serine
  • transport sequences secretion signal
  • sequences involved in replication or integration Such sequences have been reported in the literature and can be readily obtained by those skilled in the art.
  • the polynucleotide encoding at least one epitope disclosed herein is an RNA.
  • the RNA molecules comprise tRNA, rRNA, mRNA, or a combination thereof.
  • the RNA is total RNA isolated from a cell.
  • the RNA is total RNA isolated from a diseased cell, such as, for example, a tumour cell or a cancer cell.
  • mRNA is in vitro transcribed mRNA.
  • the mRNA molecules are produced by in vitro transcription (IVT). Suitable techniques of carrying out IVT are known in the art.
  • an IVT kit is employed. The kit can comprise one or more IVT reaction reagents.
  • the term “in vitro transcription (IVT) reaction reagent” refers to any molecule, compound, factor, or salt, which functions in an IVT reaction.
  • the kit may comprise prokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts to synthesise proteins from exogenous DNA templates.
  • the RNA is in vitro transcribed mRNA, wherein the in vitro transcription template is cDNA made from RNA extracted from a tumour cell.
  • the present RNA can be advantageously modified by the addition of a “5' cap” structure.
  • a 5’ cap is a modified nucleotide whose presence in 5’ of an RNA, notably an mRNA, results in increased stability, in particular in vivo.
  • m7GpppN is the 5'-cap structure, which naturally occurs in mRNA transcribed by RNA polymerase II and is therefore preferably not considered as modification comprised in a modified mRNA in this context.
  • an epitope-encoding RNA described herein preferably comprises a m7GpppN as 5'-cap. Additionally an epitopeencoding RNA described herein typically comprises at least one further modification as defined herein.
  • the epitope-encoding RNA described herein contains a poly(A) sequence or poly(A) tail.
  • a poly(A) sequence also called poly(A) tail or 3'-poly(A) tail, is typically understood to be a sequence of adenosine nucleotides, e.g., of up to about 400 adenosine nucleotides, e.g., from about 20 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides.
  • a poly(A) sequence may also comprise about 10 to 200 adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides, more preferably about 40 to 80 adenosine nucleotides or even more preferably about 50 to 70 adenosine nucleotides.
  • a poly(A) sequence is typically located at the 3' end of an RNA, in particular a mRNA.
  • the present RNA may contain a poly(C) tail on the 3' terminus of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70 cytosine nucleotides or even more preferably about 20 to 60 or even 10 to 40 cytosine nucleotides.
  • the epitope-encoding RNA disclosed herein comprises at least one 5 - or 3 -UTR element.
  • an UTR element comprises or consists of a nucleic acid sequence, which is derived from the 5 - or 3 -UTR of any naturally occurring gene or which is derived from a fragment, a homolog or a variant of the 5 - or 3 - UTR of a gene.
  • the 5 - or 3 -UTR element used according to the present invention is heterologous to the at least one coding sequence of said epitope-encoding RNA (or other nucleic acid, in particular RNA). Even if 5 - or 3 -UTR elements derived from naturally occurring genes are preferred, also synthetically engineered UTR elements may be used in the context of the present invention.
  • the RNA described herein comprises at least one 5'-untranslated region element (5' UTR element).
  • 5 -UTR refers to a part of a nucleic acid molecule, which is located 5' (i.e. , “upstream”) of an open reading frame and which is not translated into protein.
  • the 5 -UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame.
  • the 5 -UTR may comprise elements for controlling gene expression, also called regulatory elements, including e.g., ribosomal binding sites.
  • the 5 -UTR may be post- transcriptionally modified, for example by addition of a 5'-Cap.
  • a 5 -UTR corresponds to the sequence of a mature mRNA, which is located between the 5'-Cap and the start codon.
  • the RNA described herein comprises a 3’ UTR.
  • the term “3 -UTR” refers to a part of a nucleic acid molecule, which is located 3' (i.e., “downstream”) of an open reading frame and which is not translated into protein.
  • a 3 -UTR corresponds to a sequence which is located between the stop codon of the protein coding sequence, preferably immediately 3' to the stop codon of the protein coding sequence, and the poly(A) sequence of the epitope-encoding RNA described herein.
  • the at least one 3'UTR element comprises or consists of a nucleic acid sequence derived from the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 3'UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.
  • the RNA described herein comprises a histone stem-loop sequence/structure.
  • a histone stem-loop is an RNA element bound by the stem-loop binding protein (SBLP).
  • SBLP stem-loop binding protein
  • the protein is essential for efficient 3 - end processing of histone pre-mRNA by the U7 snRNP.
  • SLBP remains associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm.
  • the histone stem-loop structure is thus involved in nucleocytoplasmic transport of mRNAs and in the regulation of stability and of translation efficiency in the cytoplasm.
  • Such histone stem-loop sequences are preferably selected from histone stem-loop sequences as disclosed in WO 2012/019780.
  • the combination of a poly(A) sequence and at least one histone- stern loop act synergistically to increase the protein expression beyond the level observed with either of the individual elements.
  • the mRNA comprises one or more of the following:
  • compositions comprising the anti-VISTA antibody described herein and/or the antigen or a polypeptide encoding at least one epitope of the antigen, as described herein.
  • the composition comprises a combination of:
  • an anti-VISTA antibody or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO:3-5 and three light chain CDRs of SEQ ID NO:6-8.
  • the present immunogenic composition can be provided as a kit-of-parts, as described below.
  • the anti-VISTA antibody and the antigen or a polypeptide encoding at least one epitope of the antigen can be administered simultaneously, separately, or sequentially.
  • compositions disclosed herein are pharmaceutical compositions. More preferably, the pharmaceutical compositions comprise the combination disclosed herein and a pharmaceutically acceptable excipient, preferably a pharmaceutically acceptable carrier, and optionally an adjuvant.
  • the pharmaceutically acceptable excipient can be functional molecules such as vehicles, adjuvants other than the VISTA antibody described herein, carriers, or diluents.
  • Pharmaceutically acceptable carriers preferably have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a subject to be treated.
  • compositions can exhibit different functional roles and include, without limitation, diluents, fillers, bulking agents, carriers, disintegrants, binders, lubricants, glidants, coatings, solvents and co-solvents, buffering agents, preservatives, adjuvants, anti-oxidants, wetting agents, anti-foaming agents, thickening agents, sweetening agents, flavouring agents and humectants.
  • Suitable pharmaceutically acceptable carriers are typically chosen based on the formulation of the pharmaceutical composition.
  • the pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analogue including monophosphoryl lipid A, muramyl peptides, quinone analogues, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.
  • surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analogue including monophosphoryl lipid A, muramyl peptides, quinone analogues, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticle
  • the pharmaceutically acceptable excipient can be an adjuvant in addition to the VISTA antibody disclosed herein. Accordingly, in this embodiment, the pharmaceutical composition further comprises at least an additional adjuvant.
  • Adjuvants come in many different forms, for example, mineral salts, oil emulsions, sterile constituents of bacteria or other pathogens, cytokines, and so forth.
  • Adjuvants commonly used in are well known to the skilled person and include, inter alia, mineral salts such as aluminium salts; emulsions, e.g., complete and incomplete Freund’s adjuvants, MF59, AS03; microparticles, including virus-like particles (VLPs), virosomes, and PLA/PLGA; immune potentiators, such as e.g., L-pampo, MALP-2, Pam2CSK4 and Pam3CSK4, Poly(l:C) (polyinosinic:polycytidylic acid)Poly-ICLC, monophosphoryl lipid A (MPL), flagellin, imiquimod (R837; 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine) and res
  • the additional adjuvant can in particular be other genes that are expressed in an alternative plasmid or are delivered as proteins in combination with the antigen or the polynucleotide described above in the vaccine.
  • the adjuvant may be selected from the group consisting of: a-interferon (IFN- a), B-interferon (IFN-B), y-interferon, platelet derived growth factor (PDGF), TNFa, TNPB, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE.
  • IFN- a a-interferon
  • IFN-B B-interferon
  • PDGF platelet derived growth factor
  • the adjuvant can be IL-12, IL- 15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFo, TNFB, GM-CSF, epidermal growth factor (EGF), IL-1 , IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL- 18, or a combination thereof.
  • genes that can be useful as adjuvants in addition to the VISTA antibody include those encoding: MCP-1 , MIP-la, MIP-lp, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1 , MadCAM-1 , LFA-1 , VLA-1 , Mac-1 , pl50.95, PECAM, ICAM-1 , ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G- CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1 , p55, WSL-1 , DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase
  • the immunogenic compositions will include one or more inactive agents such as a sterile, biocompatible carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution.
  • a sterile, biocompatible carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution.
  • the composition can contain any of a variety of additives, such as stabilisers, buffers, excipients ⁇ e.g., sugars, amino acids, etc.), or preservatives.
  • Pharmaceutically acceptable carriers used in particular embodiments include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition are sterile, and the formulation suits the mode of administration.
  • an immunogenic composition contains minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • a pharmaceutical composition is a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used.
  • the medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
  • Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil.
  • the pharmaceutical composition can be formulated according to the mode of administration to be used.
  • an immunogenic composition is formulated for intradermal injection, intranasal administration or intramuscular injection.
  • injectables are prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • injection solutions and suspensions are prepared from sterile powders, granules, etc. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington's Pharmaceutical Sciences, 19 th ed., Mack Publishing Co., Easton, PA, 1995; incorporated herein by reference.
  • An injectable pharmaceutical composition can be sterile, pyrogen free and particulate free.
  • An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose.
  • the vaccine can comprise a vasoconstriction agent.
  • the isotonic solutions can include phosphate buffered saline.
  • Pharmaceutical compositions can further comprise stabilisers including gelatine and albumin. The stabilisers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.
  • composition of the present disclosure can be suitable for administration by any acceptable route, including parenteral and subcutaneous.
  • Other routes include intravenous, intradermal, intramuscular, intraperitoneal, intranodal and intrasplenic, for example.
  • the composition is systemically administered via parenteral administration.
  • the composition is administered via injection or infusion.
  • the composition is administered subcutaneously or intravenously or intramuscularly.
  • the composition is administered intravenously.
  • compositions in accordance with the invention are administered using a device that delivers a metered dosage of composition ⁇ e.g., of the present combination).
  • Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent No. 4,886,499, U.S. Patent No. 5,190,521 , U.S. Patent No. 5,328,483, U.S. Patent No. 5,527,288, U.S. Patent No. 4,270,537, U.S. Patent No. 5,015,235, U.S. Patent No. 5,141 ,496, U.S. Patent No. 5,417,662 (all of which are incorporated herein by reference).
  • Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO 99/34850, incorporated herein by reference, and functional equivalents thereof.
  • jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Patent No. 5,480,381 , U.S. Patent No. 5,599,302, U.S. Patent No. 5,334,144, U.S. Patent No. 5,993,412, U.S. Patent No. 5,649,912, U.S. Patent No. 5,569,189, U.S.
  • Patent No. 5,704,911 U.S. Patent No. 5,383,851 , U.S. Patent No. 5,893,397, U.S. Patent No. 5,466,220, U.S. Patent No. 5,339,163, U.S. Pat. No. 5,312,335, U.S. Pat. No. 5,503,627, U.S. Pat. No. 5,064,413, U.S. Patent No. 5,520,639, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941 ,880, U.S. Patent No.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti- oxidants, chelating agents, and inert gases and the like.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid
  • compositions are in a form intended for administration to a subject, it can be made to be isotonic with the intended site of administration.
  • the composition typically is sterile. In certain embodiments, this may be accomplished by filtration through sterile filtration membranes.
  • parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag, or vial having a stopper pierceable by a hypodermic injection needle, or a prefilled syringe.
  • the composition may be stored either in a ready-to-use form or in a form (e.g., lyophilised) that is reconstituted or diluted prior to administration.
  • composition disclosed herein can advantageously be formulated for simultaneous, separate or sequential administration of (1 ) the antigen or polynucleotide encoding at least one epitope of the antigen and (2) the anti -VISTA antibody.
  • the pharmaceutical composition disclosed herein is a vaccine.
  • a “vaccine” as used herein is a prophylactic or therapeutic material providing at least one epitope of an antigen.
  • Vaccines are used to stimulate an immune response in an individual to provide protection against and/or treatment for a particular disease. Some vaccines include an antigen to induce the immune response. Some antigens elicit a strong immune response while other antigens elicit a weak immune response. A weak immune response to an antigen can be strengthened by including an adjuvant in the vaccine.
  • the present disclosure thus relates to a vaccine comprising a combination of:
  • an anti-VISTA antibody or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO. 1 -3 and three light chain CDRs of SEQ ID NO. 4-6.
  • the present vaccine can be provided as a kit-of-parts, as described below.
  • the present vaccine can increase antigen presentation and the overall immune response to the antigen in a subject.
  • the combination of antigen and anti-VISTA antibody induces the immune system more efficiently than a vaccine comprising the antigen alone, notably by inducing a humoral response leading to the production of antibodies by the subject.
  • This more efficient immune response provides increased efficacy in the treatment and/or prevention of any disease, including cancer and infectious diseases.
  • the anti-VISTA antibody is thus a powerful adjuvant in the compositions disclosed here, notably in the vaccinal compositions. Accordingly, the disclosure also relates to the use of the present anti-VISTA antibody as an adjuvant in a vaccinal composition.
  • the vaccinal composition can induce an immune response in the subject administered with the composition.
  • the induced immune response can be specific for the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the present disclosure provides a composition for inducing an immune response against an antigen, wherein the composition comprises (a) at least one epitope of the isolated antigen or a polypeptide encoding at least one epitope of that antigen, in combination with (b) an anti-VISTA antibody, or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO. 1 -3 and three light chain CDRs of SEQ ID NO. 4-6.
  • the vaccinal composition can induce a humoral immune response in the subject administered the immunogenic composition.
  • the induced humoral immune response can be specific for the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the humoral immune response can be induced in the subject administered the vaccinal composition by about 1.5-fold to about 16-fold, about 2-fold to about 12- fold, or about 3-fold to about 10-fold.
  • the humoral immune response can be induced in the subject administered the vaccinal composition by at least about 1 .5 -fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5- fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5- fold, or at least about 16.0- fold as
  • the humoral immune response induced by the immunogenic composition can include an increased level of IgG antibodies associated with the subject administered the vaccinal composition as compared to a subject not administered the vaccinal composition.
  • IgG antibodies can be specific for the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • These IgG antibodies can be reactive with at least the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the level of IgG antibody associated with the subject administered the vaccinal composition can be increased by about 1.5-fold to about 16- fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold as compared to the subject not administered the immunogenic composition.
  • the level of IgG antibody associated with the subject administered the vaccinal composition can be increased by at least about 1 .5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5- fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11 .0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0- fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0-
  • the immunogenic composition can induce a cellular immune response in the subject administered the immunogenic composition.
  • the induced cellular immune response can be specific for the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the induced cellular immune response can be reactive for the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the induced cellular immune response can include eliciting a CD8+ T cell response.
  • the elicited CD8+ T cell response can be reactive with the antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the elicited CD8 + T cell response can be polyfunctional.
  • the induced cellular immune response can include eliciting a CD8 + T cell response, in which the CD8 + T cells produce interferongamma (IFN-y).
  • the induced cellular immune response can include an increased CD8 + T cell response associated with the subject administered the vaccinal composition as compared to the subject not administered the vaccinal composition.
  • the CD8 + T cell response associated with the subject administered the vaccinal composition can be increased by about 2-fold to about 30-fold, about 3 -fold to about 25 -fold, or about 4-fold to about 20-fold as compared to the subject not administered the vaccinal composition.
  • the CD8 + T cell response associated with the subject administered the vaccinal composition can be increased by at least about 1.5- fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11 .0- fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5- fold, at least about 13.0-fold, at least about 13.5 -fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21 .0-fold, at least
  • the cellular immune response induced by the immunogenic composition can include eliciting a CD4 + T cell response.
  • the elicited CD4 + T cell response can be reactive with antigen comprised in the combination or encoded by the polynucleotide comprised in the combination.
  • the elicited CD4 + T cell response can be polyfunctional.
  • the induced cellular immune response can include an increased CD4 + T cell response associated with the subject administered the vaccinal composition as compared to the subject not administered the vaccinal composition.
  • the CD4 + T cell response associated with the subject administered the vaccinal composition can be increased by about 2-fold to about 30-fold, about 3 -fold to about 25 -fold, or about 4-fold to about 20-fold as compared to the subject not administered the vaccinal composition.
  • the CD4 + T cell response associated with the subject administered the vaccinal composition can be increased by at least about 1.5- fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0- fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5- fold, at least about 13.0-fold, at least about 13.5 -fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21 .0-fold, at least about 22
  • the present invention relates to a kit or kit-of-parts comprising the inventive combination, pharmaceutical composition or vaccine, in other words, the kit-of- parts typically comprises (i) the antigen or polynucleotide encoding at least one epitope of the antigen as defined herein and (ii) the anti-VISTA antibody disclosed herein as its components.
  • kits of parts comprising:
  • a first component comprising at least one epitope of an antigen or a polynucleotide encoding this at least epitope
  • a second component comprising an anti-VISTA antibody, or an antigen-binding fragment thereof, as described herein.
  • the aforementioned components may each be provided in the form of a pharmaceutical composition in the kit-of-parts.
  • the first component comprises at least one epitope of an antigen or a polynucleotide encoding this at least epitope and a pharmaceutically acceptable excipient.
  • the second component comprises the anti-VISTA antibody described above and a pharmaceutically acceptable excipient.
  • both first and second components comprise a pharmaceutically acceptable excipient.
  • the (i) the antigen or polynucleotide encoding at least one epitope of the antigen and (ii) the anti-VISTA antibody may be provided -independently from each other- in lyophilised or liquid form, optionally together with one or more pharmaceutically acceptable carrier(s), excipients, adjuvants or further agents as described above in the context of the pharmaceutical composition.
  • the disclosure also relates to a kit-of-parts as described above, wherein the first component and/or the second component further comprises a pharmaceutically acceptable excipient, preferably a pharmaceutically acceptable carrier, and optionally an adjuvant.
  • the kit-of-parts may comprise at least one further agent as defined herein in the context of the pharmaceutical composition, antimicrobial agents, RNAse inhibitors, solubilising agents or the like.
  • the kit-of-parts may be a kit of two or more parts and typically comprises the components in suitable containers.
  • each container may be in the form of vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes or pouches, tubes or blister packages or any other suitable form provided the container is configured so as to prevent premature mixing of components.
  • Each of the different components may be provided separately, or some of the different components may be provided together (i.e. , in the same container).
  • a container may also be a compartment or a chamber within a vial, a tube, a jar, or an envelope, or a sleeve, or a blister package or a bottle, provided that the contents of one compartment are not able to associate physically with the contents of another compartment prior to their deliberate mixing by a pharmacist or physician.
  • the kit-of-parts may furthermore contain technical instructions with information on the administration and dosage of any of its components.
  • the present disclosure also relates to a kit of parts comprising:
  • a first component comprising at least one epitope of an antigen or a polynucleotide encoding this at least epitope and a pharmaceutically acceptable excipient
  • a second component comprising an anti-VISTA antibody, or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO:3-5 and three light chain CDRs of SEQ ID NO:6-8, and a pharmaceutically acceptable excipient, for the use disclosed herein, wherein component A and component B are administered jointly or independently at the same time or separately within time intervals.
  • Administration regimen comprising:
  • the present combination, pharmaceutical composition, vaccine, or kit-of-parts and the respective components may be administered to a subject in need thereof several times a day, daily, every other day, weekly, or monthly.
  • the components may be administered simultaneously (i.e., at the same time via the same or different administrations routes) or separately (i.e., sequentially at different time points and/or via different administrations routes).
  • a sequential administration scheme is also referred to as “time-staggered” administration.
  • Time-staggered administration of the several components of the present combination, pharmaceutical composition, vaccine or kit-of- parts may ensure that the separate mechanisms elicited by said components do not negatively influence each other.
  • Time-staggered administration may for instance mean that the antigen or polynucleotide encoding at least one epitope of the antigen is administrated e.g., prior, concurrent or subsequent to the anti-VISTA antibody.
  • This procedure preferably allows immune cells such as antigen-presenting cells and T cells to encounter the epitope (either directly or after translation of the epitope-encoding polynucleotide), before the immune system is stimulated by inhibition of the VISTA pathway, even though a concurrent administration or an administration, wherein the anti-VISTA antibody is administered prior to the antigen or polynucleotide encoding at least one epitope of the antigen, may lead to the same or at least comparable results.
  • the components of the inventive combination, pharmaceutical composition, vaccine or kit-of-parts i.e., the anti-VISTA antibody and the antigen or polynucleotide encoding at least one epitope of the antigen
  • are administered simultaneously i.e., at the same time via the same or different administrations routes.
  • the components of the inventive combination, pharmaceutical composition, vaccine or kit-of-parts i.e., the anti- VISTA antibody and the antigen or polynucleotide encoding at least one epitope of the antigen
  • are administered separately i.e., sequentially at different time points and/or via different administrations routes).
  • the present disclosure provides methods comprising administering present combination, pharmaceutical composition, vaccine, or kit-of-parts to a subject in need thereof.
  • the present combination, pharmaceutical composition, vaccine or kit-of-parts and the respective components are preferably administered to the subject in need thereof in a “pharmaceutically effective” amount.
  • a “pharmaceutically effective amount” is typically understood as an amount that is sufficient to induce a desired pharmaceutical effect, such as an immune response.
  • the administered amount of the present combination, pharmaceutical composition, vaccine or kit-of-parts is also sufficient for the alleviation of the symptoms of the disease or condition being treated and/or for prophylaxis of the symptoms of the disease or condition being prevented.
  • a “therapeutically effective amount” is preferably small enough to avoid serious side-effects, i.e., to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
  • a “therapeutically effective amount” of the present combination, pharmaceutical composition, vaccine or kit-of-parts will furthermore vary in connection with the particular disease or condition to be treated, characteristics of the patient (including age, physical condition, body weight, sex and diet), concurrent treatments, pharmacokinetic properties of the active agent(s), treatment regime and the desired effect (amelioration vs. complete remission), etc.
  • the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
  • the “pharmaceutically effective amount” of the present combination, pharmaceutical composition, vaccine or kit-of- parts disclosed herein or their components can be determined by routine experiments, e.g., by using cell cultures and/or animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models.
  • therapeutic efficacy and toxicity of the present combination, pharmaceutical composition, vaccine or kit-of-parts or the respective components can be determined by e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Combinations, pharmaceutical composition, vaccine or kit-of-parts which exhibit large therapeutic indices are generally preferred.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • therapeutically effective doses of the inventive combination, pharmaceutical composition, vaccine or kit- of-parts or the respective components described herein may range from about 0.001 mg to 10 mg, preferably from about 0.01 mg to 5 mg, more preferably from about 0.1 mg to 2 mg per dosage unit or from about 0.01 nmol to 1 mmol per dosage unit, in particular from 1 nmol to 1 mmol per dosage unit, preferably from 1 pmol to 1 mmol per dosage unit.
  • the therapeutically effective dose of the inventive combination, pharmaceutical composition, vaccine or kit-of-parts may range (per kg body weight) from about 0.01 mg/kg to 10 g/kg, preferably from about 0.05 mg/kg to 5 g/kg, more preferably from about 0.1 mg/kg to 2.5 g/kg.
  • the present disclosure also relates to the combination, pharmaceutical composition, vaccine, or kit-of-parts disclosed herein disclosed herein for use as medicament.
  • the present combination, pharmaceutical composition, vaccine, or kit-of-parts are notably useful for treatment of cancer, infectious diseases, autoimmune disorders or inflammatory disorders.
  • the present disclosure provides a combination of: at least one epitope of an isolated antigen or at least one polypeptide encoding at least one epitope of that antigen; and an anti-VISTA antibody, or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO. 3-5 and three light chain CDRs of SEQ ID NO. 6-8; for use in treatment of cancer, infectious diseases, autoimmune disorders or inflammatory disorders.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • an anti-VISTA antibody or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO. 3-5 and three light chain CDRs of SEQ ID NO. 6-8; for use in treatment of cancer, infectious diseases, autoimmune disorders or inflammatory disorders.
  • the present disclosure also provides a vaccine comprising:
  • an anti-VISTA antibody or an antigen-binding fragment thereof, wherein the anti-VISTA antibody comprises three heavy-chain CDRs of SEQ ID NO. 3-5 and three light chain CDRs of SEQ ID NO. 6-8; for use in treatment of cancer, infectious diseases, autoimmune disorders or inflammatory disorders.
  • kit-of-parts comprising:
  • a first component comprising at least one epitope of an isolated antigen or at least one polypeptide encoding at least one epitope of that antigen
  • the treatment comprises the joint or independent administration of the anti- VISTA antibody with at least one epitope of an antigen or a polynucleotide encoding this at least epitope.
  • (1 ) the anti-VISTA antibody and (2) the antigen or polynucleotide encoding at least one epitope of the antigen are administered jointly or independently at the same time or separately within time intervals.
  • (1 ) the anti-VISTA antibody and (2) the antigen or polynucleotide encoding at least one epitope of the antigen are administered simultaneously, separately or sequentially.
  • treatment it is herein referred to both prophylactic and therapeutic treatment. Accordingly, the combination, pharmaceutical composition, vaccine, or kit-of-parts have a positive effect on humoral response leading to the production of antibodies, not only in patients afflicted with one of the above conditions, but also in healthy subjects. In the former case, the antibodies generated through the humoral response help cure the condition afflicting the patient, whereas in the latter case, these antibodies help prevent the subject from becoming afflicted with one of these conditions.
  • the present disclosure relates to the combination, pharmaceutical composition, vaccine, or kit-of-parts and the respective components disclosed herein for use in the treatment of a cancer, an infectious disease, an autoimmune disorder or an inflammatory disorder, wherein the use comprises the simultaneous, separate or sequential administration of (1 ) the antigen or polynucleotide encoding at least one epitope of the antigen and (2) the anti-VISTA antibody.
  • the present disclosure relates to the combination, pharmaceutical composition, vaccine, or kit-of-parts for use in the treatment of a cancer, an infectious disease, an autoimmune disorder or an inflammatory disorder, wherein the treatment comprises the generation of an immune response against the antigen.
  • the generation of an immune response comprises the generation of a humoral immune response. More preferably, the generation of a humoral immune response involves the production of antibodies against the antigen.
  • the disclosure also relates to the anti-VISTA antibody, or an antigenbinding fragment thereof, as disclosed herein, for use in the treatment of a cancer, an infectious disease, an autoimmune disorder, or an inflammatory disorder, wherein the treatment comprises further administering at least one epitope of an antigen or a polynucleotide encoding this at least epitope, as described above.
  • the treatment comprises the joint or independent administration of the anti- VISTA antibody with at least one epitope of an antigen or a polynucleotide encoding this at least epitope.
  • (1 ) the anti-VISTA antibody and (2) the antigen or polynucleotide encoding at least one epitope of the antigen are administered jointly or independently at the same time or separately within time intervals.
  • (1 ) the anti-VISTA antibody and (2) the antigen or polynucleotide encoding at least one epitope of the antigen are administered simultaneously, separately or sequentially.
  • the treatment comprises the generation of an immune response against the antigen.
  • Cancers or tumours associated with tumour antigens which can thus be treated by the combination disclosed herein or to the composition disclosed herein, including the vaccine, include Acute Lymphoblastic Leukaemia, Adult; Acute Lymphoblastic Leukaemia, Childhood; Acute Myeloid Leukaemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumour, Adult; Brain Tumour, Brain Stem Glioma, Childhood; Brain Tumour,
  • the combination disclosed herein or the composition disclosed herein, notably the vaccine can be used to treat an infectious disease.
  • the infectious disease is selected in the group consisting of Botulism, Bubonic plague, Calicivirus infection (Norovirus and Sapovirus), Chickenpox, Chlamydia, Cholera, Clostridium difficile infection, Common cold (Acute viral rhinopharyngitis; Acute coryza), COVID-19 (coronavirus disease 2019), Creutzfeldt- Jakob disease (CJD), Dengue fever, Diphtheria, Ebola haemorrhagic fever, Gonorrhoea, Hand, foot and mouth disease (HFMD), Helicobacter pylori infection, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, Hepatitis E, Herpes simplex, human immunodeficiency virus (HIV), Human papillomavirus (HPV) infection, Epstein-Barr
  • the objective of this study is to use the Multi-SIV GTU-DNA vaccine model to reveal the activity of immune-checkpoints inhibitors.
  • Electroporation was performed immediately following vaccine injection using the Nepa GeneTM CUY21 EDIT In Vivo Electroporator with CUY650P10 Tweezers w/Variable Gap 2 Round Plate Electrodes (7 mm diameter, 10 mm diameter):
  • pulse-1 10 ms. pulse-1 90 ms interval 6 pulses.
  • Vehicle solution or Pembrolizumab solution were injected by the IV route (Bolus) at the appropriate dose as described in Table 2.
  • the dose volume for each animal was based on the most recent body weight measurement (W14 post prime).
  • the anti-VISTA antibody solution was diluted at final concentration of 10 mg/mL with NaCl 0.9% buffer, filtered with 0.22 pm strainer and administered at 10 mL/kg to the appropriate animals by intravenous infusion via an appropriate peripheral vein over a targeted period of 1 hour.
  • the dose volume for each animal was based on the most recent body weight measurement (W14 post prime).
  • Each infused dose was administered using a catheter inserted in a peripheral vein and an injection set or butterfly connected to an infusion pump. Each dose was followed by a flush of saline.
  • T cell subsets phenotyping and tetramer assays were performed on fresh whole blood collected into EDTA tubes. Three steps were performed during this assay:
  • Cell subsets phenotyping was performed on fresh whole blood collected on EDTA tubes. Surface markers were added to identify myeloid subsets and their activation state.
  • PBMC peripheral blood mononuclear cells
  • Monkey IFN-y ELISpot PRO kit (Mabtech Monkey IFN-y ELISPOT pro, #3421M-2APT) was used following manufacturer’s instructions.
  • Comparison of ELISPOT responses among the 3 experimental groups was performed using Kruskal-Wallis and Mann-Whitney tests. Comparison of flow cytometry parameters among the 3 experimental groups was performed using Mann-Whitney test and Kruskal-Wallis. Analyses were performed using GraphPad Prism V.8 software. The threshold for statistical significance was fixed at p ⁇ 0.05.
  • Anti-SIV antibodies in monkey EDTA-K3 plasma were detected by direct ELISA. Briefly, the anti-SIV antibodies can bind the recombinant SIV-2 gag protein pre-coated on a plate. Then, the bound anti-SIV antibodies are detected using the A/G-HPR protein. Finally, the addition of TMB allows the formation of a coloured complex for which the intensity of the optical density (read at 450 nm) is directly related to the quantity of anti-SIV antibodies bound.
  • Recombinant SIV-1 gag protein (Mac239) [His] (reference Vang-LSX0495 - supplied by Creative Biolabs / Interchim) was coated onto a 96-well plate by adding 100 pL/well of a 0.125 pg/mL solution of protein in PBS and incubating overnight. After three washes in PBS + Tween200.05%, the plate was saturated in PBS + BSA 2% for one hour at room temperature. After washing the plate 3 times in PBS + Tween 20 0.05%, 500 pL of each individual plasma sample diluted 1 /25 in PBS were added to each well and incubated for two hours at room temperature.
  • the commercial analytical kit reference ab190549 supplied by Abeam was used for the quantification of the total IgG in monkey K3-EDTA plasma samples.
  • the 6 cytokines (IFN-y, IL-1 B, IL-2, IL-6, IL-8, and IL-10) were quantified in monkey K3- EDTA plasma samples using the commercial analytical Proinflammatory Panel 1 - NHP V-plex kit reference K15056D-2 supplied by MSD.
  • each group was treated either with the anti -VISTA antibody, pembrolizumab (i.e. , an anti-PD-1 antibody), or a vehicle.
  • the monkeys were divided 3 groups of 3 individuals and immunised with SIV. A day after the last immunisation, each group was treated either with the anti-VISTA antibody, pembrolizumab (i.e., an anti-PD-1 antibody), or a vehicle. Animals were longitudinally followed for clinical signs, blood haematology and antigen-specific T cell responses.
  • Anti-RM9 and anti-GAG specific T-cell responses were measured by y ELISPOT. This assay measures antigen-specific T cell responses by quantifying the number of IFNy- producing T cells in response to stimulation by the antigen (RM9 or GAG in the present case).
  • IFNy ELISPOT assay was performed on PBMCs in the presence of Nef RM 9 peptide. The secretion of IFNy was then monitored over time. The general time course of RM 9 IFNy ELISPOT response to in vivo SIV immunisation showed that a specific immune response to SIV was evidenced in all treatment groups (Fig. 2).

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Abstract

L'invention concerne des compositions vaccinales, des compositions, des kits et des systèmes incorporant de telles constructions améliorés, et des méthodes de préparation et d'utilisation de telles constructions.
PCT/EP2023/084879 2022-12-08 2023-12-08 Composition vaccinale et adjuvant Ceased WO2024121380A1 (fr)

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