Novel anti-VISTA antibody formulations

Abstract

The present invention provides anti-VISTA antibodies suitable for pharmaceutical development, pharmaceutical compositions comprising the antibodies, and methods of treating VISTA mediated diseases.

Description

Background Art

[0001] Developing therapeutic antibodies is a complex process. Various physicochemical and functional liabilities may impair the production or therapeutic efficacy of antibodies. The present disclosure provides anti-VISTA antibodies suitable for drug development, pharmaceutical compositions comprising the antibodies, and methods for treating VISTA-mediated diseases.

[0002] Immunotherapy has become a game changer in the field of cancer treatment. In order to ensure that once the tumor antigen stimulates the reaction, the immune inflammatory response will not be continuously activated, multiple controls or "checkpoints" are in place or activated. VISTA (V-domain Ig suppressor of T cell activation) is a negative checkpoint-controlled protein that regulates the activation and immune response of T cells. VISAT is a member of the B7 family, which contains several immune checkpoint proteins such as PD-L1. However, unlike the members of this family, VISTA contains a single unusually large Ig-like V-type domain. In addition, the VISTA cytoplasmic tail domain contains multiple effector protein docking sites, indicating that VISTA may act as both a receptor and a ligand.

[0003] From a physiological perspective, VISTA exerts regulatory functions on the immune system at multiple levels, particularly by regulating T cell activation. Recently, VISTA has been identified as the earliest checkpoint regulator of peripheral T cell tolerance, particularly in maintaining naive T cell quiescence. In the context of cancer, VISTA is upregulated in immunosuppressive tumor-infiltrating leukocytes such as suppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). The presence of VISTA in the tumor microenvironment impedes the effective response of T cells and is involved in a variety of human cancers, including prostate cancer, colon cancer, skin cancer, pancreatic cancer, and lung cancer.

[0004] Several antagonistic anti-VISTA antibodies that can be used to treat cancer have been described (ElTanbouly et al., Clin Exp Immunol. 200(2):120-130 (2020); Mehta et al., Sci Rep. 10(1):1 5171 (2020); Yuan et al., Trends Immunol. 42(3):209-227 (2021); Tagliamento et al., Immunotargets Ther. 10:185-200 (2021); Thakkar et al., J Immunother Cancer. 10(2):e003382 (2022); WO 2015 / 097536; WO 2016 / 094837; WO 2017 / 181139; WO 2019 / 183040). Specifically, WO 2016 / 094837 discloses an antibody capable of inhibiting VISTA's inhibition of anti-tumor immune response, thereby conferring protective anti-tumor immunity. However, the antibody contains several Asn residues that are potentially susceptible to deamidation, thereby affecting drug efficacy and clinical and manufacturing development.

[0005] In addition, antibody drugs are unstable due to their large molecular weight, complex structure, easy degradation, polymerization or undesirable chemical modification. In order to make antibodies suitable for administration and maintain stability during storage and subsequent use, research on stable formulations of antibody drugs is particularly important.

[0006] Therefore, there is a need to develop pharmaceutical compositions (formulations) comprising homogeneous, safe and effective anti-VISTA antibodies that are suitable for administration. Summary of the invention

[0007] This article provides the following aspects:

[0008] In the first aspect, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antigen-binding fragment thereof that specifically binds to VISTA; and a pharmaceutically acceptable carrier and / or excipient. The antibody has a heavy chain and a light chain provided herein, for example, a heavy chain of a sequence represented by SEQ ID NO: 21 and a light chain of a sequence represented by SEQ ID NO: 22. Specifically, the anti-VISTA antibody of the present disclosure has aspartic acid at position 55. The anti-VISTA antibody disclosed herein is preferably not susceptible to deamidation.

[0009] Preferably, the antibody is a monoclonal antibody, more preferably a humanized antibody.

[0010] In one example, the concentration of the monoclonal anti-VISTA antibody is between 5 and 100 mg / mL, preferably between 10 and 70 mg / mL, more preferably between 15 and 50 mg / mL, and most preferably between 20 and 30 mg / mL. In a preferred example, the concentration of the monoclonal anti-VISTA antibody is 20 mg / mL.

[0011] In another aspect, the present disclosure provides a pharmaceutical composition comprising an anti-VISTA antibody and a buffer, preferably a citrate buffer, a phosphate buffer or a histidine buffer, more preferably a histidine buffer.

[0012] In one example, the buffer is a histidine buffer with a concentration between 5 and 45 mM, preferably between 10 and 40 mM, more preferably between 15 and 35 mM, and most preferably between 20 and 30 mM. In a preferred example, the buffer is a histidine buffer with a concentration of 25 mM.

[0013] In another aspect, the present disclosure provides a pharmaceutical composition comprising an anti-VISTA antibody and a tension regulator. Preferably, the tension regulator is selected from polyols (e.g., trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol), salts, and amino acids; more preferably, the salt is selected from sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, and calcium chloride; even more preferably NaCl, MgCl2 and / or CaCl2, most preferably NaCl.

[0014] In one example, the tonicity adjusting agent is present in the formulation at a concentration between 0 mM and 300 mM, preferably between 50 mM and 250 mM, more preferably between 100 mM and 200 mM.

[0015] In one example, the tonicity modifier is NaCl, and the concentration is between 0mM and 300mM, preferably between 50mM and 250mM, and more preferably between 100mM and 200mM. In a preferred example, the pharmaceutical composition comprises NaCl at a concentration of 150mM NaCl.

[0016] In another aspect, the present disclosure provides a pharmaceutical composition comprising a nonionic surfactant, preferably a polysorbate, such as polysorbate 20 or polysorbate 80.

[0017] In one example, the nonionic surfactant is polysorbate 80 at a concentration of about 0.001% to about 0.5% (v / v), about 0.002% to about 0.2% (v / v), about 0.003% to about 0.1% (v / v), about 0.004% to about 0.009% (v / v), about 0.005% to about 0.008% (v / v), or about 0.006% to about 0.007% (v / v). In a preferred example, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.006% (v / v).

[0018] In another aspect, the present disclosure provides a pharmaceutical composition having a pH value between about 3.0 and about 9.0, between about 4.0 and about 8.0, between about 5.0 and about 8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0, between about 5.5 and about 7.0, or between about 5.5 and about 6.5. In one example, the pH value of the pharmaceutical composition is about 6.5.

[0019] Preferably, in addition to the monoclonal anti-VISTA antibody disclosed herein, the pharmaceutical composition further comprises 25 mM histidine, 150 mM NaCl, 0.006% polysorbate 80 (v / v), pH 6.5.

[0020] In another aspect, the monoclonal anti-VISTA antibody or immunoconjugate or pharmaceutical composition disclosed herein is used to treat a VISTA-mediated disease in a patient, especially cancer. Preferably, the use includes inducing an immune response in a patient. Preferably, the immune response includes inducing CD4+T cell proliferation, inducing CD8+T cell proliferation, inducing CD4+T cells to produce cytokines, and inducing CD8+T cells to produce cytokines. Preferably, the use disclosed herein includes activation of the effector function of the antibody.

[0021] In a preferred aspect, the therapeutic use disclosed herein comprises administering a second therapeutic agent. The second therapeutic agent is advantageously an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0022] Preferably, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer (such as leukemia, lymphoma or myeloma), laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1: Identification of charge variants by cation exchange chromatography, pH gradient. Left: Several peaks are visible for product 1 containing Ab3, indicating that product 1 has been degraded, especially deamidation at amino acid residue 55. Right: A single peak is visible for product 2 containing Ab1, indicating that it is no longer sensitive to deamidation at amino acid residue 55 and is stable.

[0024] Figure 2 : Graphical representation of average data obtained in three experiments (N=3) in direct rhVISTA ELISA using the third series of antibodies. Solid squares: Ab1, diamonds: Ab3 batch 1, inverted triangles: Ab3 batch 2, triangles: IgG1 anti-VISTA (positive control), open circles: c9G4 (negative control), open circles and dashed lines: anti-hVISTA polyclonal antibody (positive control).

[0025] Figure 3 : Graphical representation of average data obtained in three experiments (N=3) in the indirect rhVISTA ELISA using the third series of antibodies. Solid squares: Ab1, diamonds: Ab3 batch 1, inverted triangles: Ab3 batch 2, triangles: IgG1 anti-VISTA (positive control), open circles: c9G4 (negative control), open circles and dotted lines: anti-hVISTA polyclonal antibody (positive control).

[0026] Figure 4 : Evaluation of T cell activation and cytokine release in CHO-VISTA and PBMC co-culture: Schematic representation of the experiment.

[0027] Figure 5 : Evaluation of T cell activation and cytokine release in CHO-VISTA and PBMC co-culture: Anti-VISTA Ab1 effectively induced T cell activation and cytokine release in CHO-VISTA and PBMC co-culture (Donor 119). Ab1 silencing: Ab1 variant with N298A mutation.

[0028] Figure 6 : Binding of rhVISTA-Fc and rhVISTA-TagHis on rhVSIG3-Fc = f([anti-VISTA Ab1]).

[0029] Figure 7 : In vivo activity of potent anti-VISTA Ab1 in the MC38 xenograft model.

[0030] Figure 8 : In vivo activity of silent anti-VISTA Ab1 (N298A variant) in the MC38 xenograft model.

[0031] Fig. 9 : Derivative plot type and melting temperature (Tm) obtained in step 1 at T0 for all conditions.

[0032] Fig.10 : MODDE analysis of TSA results at T0 in step 1. The values ​​in the squares represent the weights when the factors are significant (intervals do not cross zero).

[0033] Fig.11 : Analysis of mAb 1 remaining in step 1. Values ​​in squares represent weights when the factor is significant (interval does not cross zero).

[0034] Fig.12 : Figure 4 : Analysis of monomer content in step 1. The values ​​in the squares represent the weights when the factors are significant (intervals do not cross zero). DETAILED DESCRIPTION

[0035] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, wherein suitable methods and materials are described herein. Unless otherwise indicated, the practice of the present invention adopts conventional techniques or protein chemistry, molecular virology, microbiology, recombinant DNA technology and pharmacology within the technical scope of the art. Such technology is fully described in the literature (see, for example, Ausubel et al., Short Protocols in Molecular Biology, Current Protocols; 5th edition, 2002; Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co. Easton, Pa., 1985; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd edition, 2001). The nomenclature and laboratory procedures and techniques used in combination with molecular and cell biology, protein biochemistry, enzymology, and medical chemistry and pharmaceutical chemistry described herein are well known in the art and commonly used. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Unless otherwise specified, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

[0036] The present invention will be more fully understood from the detailed description given herein and the accompanying drawings, which are given by way of illustration only and do not limit the intended scope of the present invention.

[0037] definition

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the fields of chemistry, biochemistry, cell biology, molecular biology and medical sciences.

[0039] The term "about" or "approximately" refers to the normal error range of a given value or range known to those skilled in the art. It usually means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range.

[0040] As used herein, the expression "antibody-dependent cell-mediated cytotoxicity," "antibody-dependent cellular cytotoxicity," or "ADCC" refers to a form of cytotoxicity in which immunoglobulins bind to Fc receptors (FcRs) present on certain cytotoxic effector cells, enabling these cytotoxic effector cells to specifically bind to target cells bearing the antigen and subsequently kill the target cells with cytotoxins. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement. Cell destruction can occur, for example, by lysis or phagocytosis. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model, such as that disclosed in Clynes et al., PNAS (USA) 95: 652-656 (1998).

[0041] As used herein, "antibody-dependent phagocytosis" or "ADCP" or "opsonization" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcyR recognize bound antibody on a target cell, subsequently resulting in phagocytosis of the target cell.

[0042] As used herein, "administer" or "administration" refers to the act of physically delivering a substance present in vitro (e.g., an anti-VISTA antibody provided herein) to a patient by injection or otherwise, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is typically administered before the onset of the disease or its symptoms.

[0043] As used herein, "antagonist" or "inhibitor" refers to a molecule that can inhibit or otherwise reduce one or more biological activities of a target protein (such as VISTA). In some embodiments, an antagonist of VISTA (e.g., an antagonist antibody provided herein) can, for example, act by inhibiting or otherwise reducing the activation and / or cell signaling pathways of cells expressing VISTA (e.g., tumor cells, regulatory T cells, myeloid-derived suppressor cells, or dendritic suppressor cells carrying VISTA), thereby inhibiting the biological activity of the cell relative to the biological activity in the absence of an antagonist. For example, an antagonist of VISTA can inhibit the inhibitory effect of VISTA on T cell immunity (CD4+ and / or CD8+T cell immunity) and / or proinflammatory cytokine expression. More specifically, an antagonist of VISTA can block or reduce the interaction of VISTA with at least one of its ligands (including VSIG3, PSG-L1, VSIG8, and LRIG1). Even more specifically, VISTA's antagonists can block or reduce the interaction between VISTA and any of VSIG3 or PSG-L1. Preferably, VISTA's antagonists can block or reduce the interaction between VISTA and PSG-L1 at acidic pH (i.e., pH between 5.9 and 6.5). In some embodiments, the antibodies provided herein are antagonistic anti-VISTA-1 antibodies. Certain antagonistic antibodies substantially or completely inhibit one or more biological activities of the antigen. For example, antagonistic anti-VISTA can inhibit the inhibitory effect of VISTA on T cell immunity (CD4+ and / or CD8+T cell immunity) and / or proinflammatory cytokine expression. More specifically, antagonistic anti-VISTA antibodies can block or reduce the interaction between VISTA and at least one of its ligands (including VSIG3, PSG-L1, VSIG8 and LRIG1). Even more specifically, VISTA's antagonists can block or reduce the interaction between VISTA and VSIG3 or PSG-L1. Preferably, the antagonist anti-VISTA antibody can block or reduce the interaction of VISTA with PSG-L1 at acidic pH (i.e., at a pH between 5.9 and 6.5).

[0044] The terms "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein. These terms are used in the broadest sense herein, specifically covering monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies and antibody fragments of any isotype such as IgG, IgM, IgA, IgD and IgE, provided that the fragment retains the desired biological function. These terms are intended to include polypeptide products of B cells in the immunoglobulin class of polypeptides, which are capable of binding to specific molecular antigens and are composed of two pairs of identical polypeptide chains interconnected by disulfide bonds, wherein each pair has a heavy chain (about 50-70 kDa) and a light chain (about 25 kDa), each amino terminal portion of each chain contains a variable region of about 100 to about 130 or more amino acids, and each carboxyl terminal portion of each chain contains a constant region (see Borrebaeck (ed.) (1995) AntibodyEngineering , 2nd edition, Oxford University Press.; Kuby (1997) Immunology , 3rd edition, WH Freeman and Company, New York). Each variable region of each heavy chain and light chain consists of three complementary determining regions (CDRs, also called hypervariable regions) and four frameworks (FRs, more highly conserved parts of the variable domains), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate immunoglobulin binding 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. In some embodiments, the antibodies provided herein can bind to specific molecular antigens, including target VISTA polypeptides, fragments or epitopes. Antibodies that can react with specific antigens can be produced by recombinant methods, such as selecting a recombinant antibody library in a phage or similar vector or by immunizing an animal with the antigen or a nucleic acid encoding the antigen.

[0045] Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above (referring to a portion of an antibody heavy chain or light chain polypeptide that retains some or all of the biological functions of the antibody from which the fragment is derived). The antibodies provided herein can be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0046] The terms "anti-VISTA antibody", "antibody binding to VISTA", "antibody binding to VISTA epitope" and similar terms are used interchangeably herein and refer to antibodies that bind to VISTA polypeptides (such as VISTA antigens or epitopes). Such antibodies include polyclonal and monoclonal antibodies, including chimeric antibodies, humanized antibodies and human antibodies. Antibodies that bind to VISTA antigens may have cross-reactivity with related antigens. In some embodiments, antibodies that bind to VISTA do not cross-react with other antigens (e.g., other peptides or polypeptides belonging to the B7 superfamily). Antibodies that bind to VISTA can be identified by, for example, immunoassays, BIAcore, or other techniques known to those skilled in the art. For example, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), when the affinity of the antibody binding to VISTA is higher than the affinity of the antibody binding to any cross-reactive antigen, the antibody that binds to VISTA, for example, can be identified as an antibody that specifically binds to VISTA. Typically, a specific or selective reaction will be at least twice the background signal or noise, and can be more than 10 times the background. For a discussion of antibody specificity, see, e.g., Paul, ed., 1989, Fundamental Immunology 2nd Edition, Raven Press, New York, pp. 332-336. In some embodiments, the antibody "binding" to an antigen of interest is an antibody that binds to the antigen with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent for cells or tissues that target the expression of the antigen, and does not significantly cross-react with other proteins. In such embodiments, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIPA), the degree of binding of the antibody to "non-target" protein will be less than about 10% of the binding of the antibody to its specific target protein. With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specific binding to" or "specific to" an epitope on a specific polypeptide or specific polypeptide target refers to a measurable binding different from nonspecific interactions. For example, specific binding can be measured by determining the binding of a molecule compared with the binding of a control molecule, and a control molecule is typically a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competing with a control molecule similar to a target (e.g., an excessive amount of unlabeled target). In this case, if excess unlabeled target competitively inhibits the binding of the labeled target to the probe, specific binding is indicated. As used herein, the term "specifically binds" or "binds specifically to" or "specific for" with respect to a particular polypeptide or epitope on a particular polypeptide target may mean, for example, that the molecule has a K of D : At least about 10 -4 M, or at least about 10 -5 M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 - 9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 M, or greater. In some embodiments, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, an antibody that binds to VISTA has a dissociation constant (K) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. D ).

[0047] An "antigen" is a predetermined antigen to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide, including, for example, a VISTA polypeptide.

[0048] The terms "antigen binding fragment," "antigen binding domain," "antigen binding region" and similar terms refer to the portion of an antibody comprising the amino acid residues that interact with an antigen and confer upon the binding agent specificity and affinity for the antigen (e.g., the complementarity determining region (CDR)).

[0049] The terms "antigen binding fragment", "antigen binding domain", "antigen binding region" and similar terms refer to the portion of an antibody that comprises the amino acid residues that interact with the antigen and impart specificity and affinity to the binding agent for the antigen (e.g., complementary determining region (CDR)). By the expression "antigen binding fragment" of an antibody, it is intended to mean any peptide, polypeptide or protein that retains the ability of the antibody to bind to a target (also commonly referred to as an antigen), typically to bind to the same epitope, and comprises the following amino acid sequence: at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, or at least 200 consecutive amino acid residues in the amino acid sequence of the antibody. In a specific embodiment, the antigen-binding fragment comprises at least one CDR of the antibody from which it is derived. Still in a preferred embodiment, the antigen-binding fragment comprises 2, 3, 4 or 5 CDRs of the antibody from which it is derived, more preferably 6 CDRs.

[0050] "Antigen binding fragments" may be selected from, but are not limited to: Fab, Fab', (Fab')2, Fv, scFv (sc stands for single chain), Bis-scFv, scFv-Fc fragments, Fab2, Fab3, miniantibodies, diabodies, triabodies, tetrabodies and nanobodies, and fusion proteins with disordered peptides (such as XTEN (extended recombinant polypeptide) or PAS motifs) and any fragments that will increase the half-life by chemical modification, such as adding poly (alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments are called Fv-PEG, scFv-PEG, Fab-PEG, F (ab')2-PEG or Fab'-PEG) ("PEG" means polyethylene glycol), or any fragments that increase the half-life by incorporation into liposomes, the fragments having at least one characteristic CDR of an antibody according to the present invention. Among antibody fragments, Fab has a structure including light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1), and has one antigen binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminal end of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region, and the recombinant technology for producing Fv fragments is described in International Publication WO 88 / 10649, etc. In double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are mutually connected by a disulfide bond, while in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are usually covalently connected to each other by a peptide linker. These antibody fragments can be obtained by using proteases (for example, Fab can be obtained by restrictive digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by restrictive digestion with pepsin), and these antibody fragments can preferably be produced by genetic engineering technology. Preferably, the "antigen-binding fragment" will be composed of or contain a partial sequence of the heavy chain or light chain variable chain of the antibody from which it is derived, which is sufficient to maintain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least equal to 1 / 100 of the affinity of the antibody from which it is derived for the target, and more preferably at least equal to 1 / 10. Antibodies: ALaboratory 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., Cell Biophysics, 22: 189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and in Day, ED, Advanced Immunochemistry, A description of such antibody fragments is found in Antibody, 2nd Edition, Wiley-Liss, Inc., New York, NY (1990).

[0051] The term "antigen presenting cell" or "APC" refers to a heterogeneous group of immune cells that mediate cellular immune responses by processing and presenting antigens recognized by certain lymphocytes (such as T cells). APCs include, but are not limited to, dendritic cells, macrophages, Langerhans cells, and B cells.

[0052] As used herein, the term "bind" or "binding" refers to the interaction between molecules to form a complex that is relatively stable under physiological conditions. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. The 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 interaction 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 binding (k1) and dissociation (k2) of an antibody to a monovalent antigen are related to the binding (k3) and dissociation (k4) of an antibody to a monovalent antigen. -1 ) ratio (k1 / k -1 ) is the binding constant K, which is a measure of affinity. The value of K varies for different antibody and antigen complexes and depends on k1 and k -1. Any method provided herein or any other method well known to those skilled in the art can be used to determine the binding constant K of the antibody provided herein. The affinity of a binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeated antigenic determinants (such as multivalent VISTA) contacts an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site will increase the possibility of a reaction at a second site. The strength of this multiple interaction between a multivalent antibody and an antigen is called avidity. The avidity of an antibody can better measure its binding ability than the affinity of its single binding site. For example, high avidity can make up for the low affinity of pentamer IgM antibodies that are sometimes found, and pentamer IgM antibodies have a lower affinity than IgG, but the multivalence of IgM leads to high avidity, enabling it to effectively bind to antigens. Methods for determining whether two molecules are bound are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, and the like. In a specific embodiment, the antibody or its antigen-binding fragment binds to VISTA with an affinity that is at least twice as strong as its affinity for binding to non-specific molecules (such as BSA or casein). In a more specific embodiment, the antibody or its antigen-binding fragment binds only to VISTA.

[0053] As used herein, the term "biological sample" or "sample" refers to a sample that has been obtained from a biological source (such as a patient or subject). As used herein, "biological sample" refers in particular to a whole organism or a subset of its tissues, cells or components (e.g., blood vessels (including arteries, veins and capillaries), body fluids (including but not limited to blood, serum, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" further refers to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or components, or a fraction or part thereof. Finally, "biological sample" refers to a culture medium containing cellular components (such as proteins or nucleic acid molecules), such as a nutrient broth or gel in which an organism is propagated.

[0054] "Buffer" refers to a buffer that is resistant to changes in pH due to its conjugate acid-base components. Examples of buffers that control pH within an appropriate range include acetate buffers, succinate buffers, gluconate buffers, histidine buffers, oxalate buffers, lactate buffers, phosphate buffers, citrate buffers, tartrate buffers, fumarate buffers, glycylglycine and other organic acid buffers.

[0055] Histidine buffer refers to a buffer containing histidine ions. Examples of histidine buffers include histidine-hydrochloride buffer, histidine-acetate buffer, histidine-phosphate buffer, histidine-sulfate buffer, etc., preferably histidine-hydrochloride buffer. Histidine-hydrochloride buffer is prepared from histidine and hydrochloric acid or from histidine and histidine hydrochloride.

[0056] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a tumor or cancer. As used herein, "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer", "cancer (of)", "cell proliferative disorder", "proliferative disorder" and "tumor" as mentioned herein are not mutually exclusive. The terms "cancer" and "cancer (of)" refer to or describe a physiological condition in mammals, which is usually characterized by unregulated cell growth. As used herein, "cancer" is any malignant tumor caused by the undesirable growth, invasion and, under certain conditions, metastasis of damaged cells in an organism. The cells that cause cancer are genetically damaged and have generally lost the ability to control cell division, cell migration behavior, differentiation state and / or cell death mechanisms. Most cancers form tumors, but some hematopoietic cancers (such as leukemias) do not form tumors. Therefore, "cancer" as used herein can include benign and malignant cancers. As used herein, the term "cancer" refers in particular to any cancer that can be treated by the human antibodies of the present disclosure, without any limitation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penis cancer, melanoma, multiple myeloma and B cell lymphoma, brain cancer and head and neck cancer, and related metastasis. In some embodiments, the cancer is a blood cancer, which refers to a cancer that begins in cells of the blood-forming tissues (e.g., bone marrow) or the immune system. Examples of blood cancers are leukemias (e.g., acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), or acute monocytic leukemia (AMoL)), lymphomas (Hodgkin's lymphoma or non-Hodgkin's lymphoma), and myelomas (multiple myeloma, plasmacytoma, limited myeloma, or extramedullary myeloma).

[0057] A "chemotherapeutic agent" is a chemical or biological agent (e.g., an agent including a small molecule drug or a biological agent such as an antibody or cell) that can be used to treat cancer, regardless of the mechanism of action. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy. Chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, or immunomodulators.

[0058] As used herein, "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody) or one of the three hypervariable regions (L1, L2 or L3) within the non-framework region of the VL β-sheet framework of an antibody. Thus, CDRs are variable region sequences interspersed among framework region sequences. CDR regions are well known to those skilled in the art and have been defined, for example, by Kabat as the most hypervariable region within an antibody variable (V) domain (Kabat et al. (1977) J. Biol. Chem. 252: 6609-6616; Kabat (1978) Adv. Prot. Chem. 32: 1-75). Kabat CDRs are based on sequence variability and are the most commonly used (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., Public Health Servic, National Institutes of Health, Bethesda, MD). Chothia refers to the position of the structural loop (Chothia and Lesk, (1987) J Mol. Biol. 196: 901-917). CDR region sequences are also structurally defined by Chothia as those residues that are not part of the conserved β-sheet framework and are therefore able to adopt different conformations (Chothia and Lesk (1987), J. Mol. Biol. 196: 901-917). When numbered according to the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). Both terms are well known in the art. CDR region sequences are also defined as AbM, Contact, and IMGT. The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on analysis of available complex crystal structures. A universal numbering system, ImMunoGeneTics (IMGT), has recently been developed and widely adopted. (Lafranc et al., (2003) Dev. Comp. Immunol. 27(1):55-77). Regardless of the antigen receptor, chain type or species, the IMGT universal numbering has been defined to compare variable domains [Lefranc M.-P. (1997) Immunol. Today 18:509; Lefranc M.-P. (1999) The Immunologist 7:132-136]. In the IMGT universal numbering, conserved amino acids always have the same position, such as cysteine ​​23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine ​​104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT universal numbering provides standardized delimitations of 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 complementarity determining regions (CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65, and CDR3-IMGT: 105 to 117). Since gaps represent unoccupied positions, the CDR-IMGT length (shown between brackets and separated by dots, e.g., [8.8.13]) becomes key information. The IMGT universal numbering is used for 2D graphical representations, named IMGTColliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002); Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2:21-30 (2007)], and for 3D structures in IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32:D208-D210 (2004). The positions of CDRs in typical antibody variable domains have been determined by comparing multiple structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Since the number of residues in the hypervariable region varies among different antibodies, additional residues associated with typical positions are usually numbered with a, b, c, etc. next to the residue numbers in the typical variable domain numbering scheme (Al-Lazikani et al., supra (1997)).Such nomenclature is also well known to those skilled in the art.

[0059] The hypervariable region may comprise an "extended hypervariable region" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered 25 according to Kabat et al. (supra). As used herein, the terms "HVR" and "CDR" can be used interchangeably.

[0060] As used herein, "checkpoint inhibitors" refer to molecules that target immune checkpoints and block the immune checkpoint function, such as small molecules, soluble receptors, or antibodies. More specifically, as used herein, "checkpoint inhibitors" are molecules that can inhibit or otherwise reduce one or more biological activities of immune checkpoints, such as small molecules, soluble receptors, or antibodies. In some embodiments, inhibitors of immune checkpoint proteins (e.g., antagonistic anti-VISTA antibodies provided herein) can, for example, inhibit or otherwise reduce the activation and / or cell signaling pathways of cells (e.g., T cells) expressing the immune checkpoint proteins, thereby inhibiting the biological activity of the cell relative to the biological activity in the absence of an antagonist. Examples of immune checkpoint inhibitors include small molecule drugs, soluble receptors, and antibodies.

[0061] As used herein, the term "complement-dependent cytotoxicity" or "CDC" refers to the process of antibody-mediated complement activation that leads to cell lysis following binding of the antibody to its antigen on the cell, according to the mechanism outlined above. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santaro et al., J. Immunol. Methods, 202: 163 (1996). In the art, normal human serum is used as a source of complement.

[0062] The term "constant region" or "constant domain" refers to the carboxyl terminal portion of the light chain and the heavy chain, which is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as interaction with Fc receptors. The term refers to a portion of the immunoglobulin molecule that has a more conserved amino acid sequence relative to another portion of the immunoglobulin (i.e., 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.

[0063] As described herein, "cytotoxic agent" refers to an agent that treats or prevents the development of cell proliferation, preferably cancer development in a subject, by inhibiting or preventing cell function and / or causing cell death when applied to a subject. The cytotoxic agent that can be used in the antibody drug conjugate of the present invention includes any agent, part or residue that has a cytotoxic effect or inhibitory effect on cell proliferation. Examples of such agents include (i) chemotherapeutic agents that can act as tubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors or DNA chelators; (ii) protein toxins with enzymatic functions; and (iii) radioactive isotopes (radionuclides). Cytotoxic agents can be conjugated with antibodies (such as, for example, anti-VISTA antibodies) to form immunoconjugates. Preferably, the cytotoxic agent is released from the antibody under specific conditions (e.g., under acidic conditions), so as to affect the target cell therapeutically, for example, by preventing the proliferation of target cells or by showing cytotoxic effects.

[0064] As used herein, the term "reduced" refers to an activity of a protein (e.g., VISTA) that is at least 1 times (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more) lower than its reference value. "Reduced" refers to the activity of a subject's protein (e.g., VISTA), and also refers to an activity of a protein of a reference sample or a reference value of the protein that is at least 5% lower (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%). As used herein, the term "reduction" also refers to a level of a subject's biomarker (e.g., VISTA) that is at least 1 times lower than its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more). When "reduction" refers to the level of a subject's biomarker (e.g., VISTA), it also means that it is at least 5% lower than the level of a reference sample or relative to the reference value of the marker (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%).

[0065] As used herein, the term "detecting" encompasses quantitative or qualitative detection.

[0066] As used herein, the term "detectable probe" refers to a composition that provides a detectable signal. The term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment that provides a detectable signal through its activity.

[0067] In the context of polypeptides, as used herein, the term "derivative" refers to a polypeptide comprising an amino acid sequence of a VISTA polypeptide that is changed by introducing amino acid residue substitutions, deletions or additions, a fragment of a VISTA polypeptide, or an antibody bound to a VISTA polypeptide. As used herein, the term "derivative" also refers to a VISTA polypeptide that has been chemically modified, a fragment of a VISTA polypeptide, or an antibody bound to a VISTA polypeptide, wherein the chemical modification, for example, covalently connects any type of molecule to a polypeptide. For example, but not limited to, a VISTA polypeptide, a fragment of a VISTA polypeptide, or a VISTA antibody can be chemically modified (e.g., by glycosylation, acetylation, pegylation, phosphorylation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cell ligand or other protein, etc.). Derivatives are modified in a manner different from naturally occurring or starting peptides or polypeptides (regardless of the type or position of the molecule connected). Derivatives also include the absence of one or more chemical groups naturally occurring on a peptide or polypeptide. The derivatives of VISTA polypeptides, fragments of VISTA polypeptides or VISTA antibodies can be chemically modified by chemical modification using techniques known to those skilled in the art (including but not limited to specific chemical cleavage, acetylation, formulations, metabolic synthesis of tunicamycin, etc.). In addition, the derivatives of VISTA polypeptides, fragments of VISTA polypeptides or VISTA antibodies may contain one or more non-classical amino acids. The polypeptide derivatives have similar or identical functions to the VISTA polypeptides, fragments of VISTA polypeptides or VISTA antibodies described herein.

[0068] The term "diagnostic agent" refers to a substance administered to a subject that aids in disease diagnosis. Such substances can be used to reveal, identify, and / or define the location of pathogenic processes. In some embodiments, the diagnostic agent includes a substance conjugated to an antibody provided herein, which, when administered to a subject or contacted with a subject's sample, aids in the diagnosis of cancer, tumor formation, or any other VISTA-mediated disease, illness, or condition.

[0069] The term "detectable agent" refers to a substance that can be used to determine the presence or existence of a desired molecule (e.g., an antibody provided herein) in a sample or subject. A detectable agent can be a substance that can be visualized or can be otherwise determined and / or measured (e.g., by quantification).

[0070] As used herein, the term "detecting" encompasses quantitative or qualitative detection.

[0071] An "effective amount" or "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to induce a desired biological response in a subject at the necessary dosage and time period. Such responses include: alleviating the symptoms of the disease or disorder being treated, preventing, inhibiting or delaying the symptoms of the disease or the recurrence of the disease itself, increasing the life span of the subject compared to the absence of treatment, or preventing, inhibiting or delaying the symptoms of the disease or the progression of the disease itself. An "effective amount" is particularly an amount of an agent that is effective to achieve the desired therapeutic or preventive result. More specifically, an "effective amount" as used herein is an amount of an agent that confers a therapeutic benefit. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of an agent outweigh any toxic or deleterious effects.

[0072] The effective amount can be applied in one or more administrations, applications or dosages.Such delivery depends on many variables, including the time period of the single dosage unit to be used, the bioavailability of the reagent, the route of administration, etc. In some embodiments, the effective amount also refers to the amount of the antibody (e.g., anti-VISTA antibody) provided herein that achieves a specific result (e.g., suppressing the biological activity of immune checkpoints, such as regulating T cell activation). In some embodiments, the term refers to the amount of therapy (e.g., immune checkpoint inhibitors, such as, for example, anti-VISTA antibodies) sufficient to reduce and / or improve the severity and / or duration of a given disease, illness or condition and / or symptoms associated therewith. The term also encompasses the amount required for the following: reduce or improve the development (advancement) or progress (progression) of a given disease, illness or condition, reduce or improve the recurrence, development or onset of a given disease, illness or condition, and / or improve or enhance the prevention or therapeutic effect of another therapy (e.g., therapy other than the immune checkpoint inhibitor). In the context of cancer treatment, a therapeutic benefit refers to, for example, any improvement in cancer, including any one or a combination of the following: stopping or slowing cancer progression (e.g., from one stage of cancer to the next), stopping or delaying aggravation or deterioration of symptoms or signs of cancer, reducing the severity of cancer, inducing remission of cancer, inhibiting tumor cell proliferation, tumor size or number of tumors, or reducing the level of a biomarker indicative of cancer. In some embodiments, an effective amount of an antibody is from about 0.1 mg / kg (mg antibody / kg subject body weight) to about 100 mg / kg. In some embodiments, the effective amount of an antibody provided herein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg (or ranges therein).

[0073] As used herein, the term "effector function" refers to the biological function carried by the Fc domain of an immunoglobulin (e.g., an anti-VISTA antibody described herein). The activity mediated by these Fc domains is mediated by immune effector cells, such as killer cells, natural killer cells, and activated macrophages or various complement components. These effector functions involve activating receptors on the surface of the effector cells by binding of the Fc domain of an antibody to the receptor or complement components. "Effector function" as used herein covers activities such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), etc.

[0074] As used herein, "effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. Cells express at least FcγRI, FCγRII, FcγRIII and / or FcγRIV and perform ADCC effector functions. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.

[0075] The term "encoding" or its grammatical equivalents when used to refer to a nucleic acid molecule refers to a nucleic acid molecule that can be transcribed to produce mRNA when in its native state or when manipulated by methods well known to those skilled in the art, which is then translated into a polypeptide and / or fragments thereof. The antisense strand is the complementary strand of such a nucleic acid molecule, from which the coding sequence can be deduced.

[0076] As used herein, the term "epitope" refers to the region of an antigen (such as a VISTA polypeptide or a VISTA polypeptide fragment) to which an antibody binds. Preferably, as used herein, an epitope is a local region on the surface of an antigen (such as a VISTA polypeptide or a VISTA polypeptide fragment) that can bind to one or more antigen binding regions of an antibody and has antigenicity or immunogenic activity that can induce an immune response in an animal (such as a mammal, such as a human). An epitope with immunogenic activity is a part of a polypeptide that triggers an antibody response in an animal. An epitope with antigenic activity is a part of a polypeptide that an antibody binds to, and the binding is determined by any method well known in the art, such as by an immunoassay. Antigenic epitopes do not necessarily have to be immunogenic. Epitopes are generally composed of chemically active surface groups (e.g., amino acids or sugar side chains) of molecules and have specific three-dimensional structural features and specific charge characteristics. Epitopes can be formed by continuous residues or non-continuous residues that are close to each other by folding of antigenic proteins. Epitopes formed by continuous amino acids are usually retained when exposed to a denaturing solvent, while epitopes formed by non-continuous amino acids are usually lost under the exposure. In some embodiments, the VISTA epitope is a three-dimensional surface feature of the VISTA polypeptide. In other embodiments, the VISTA epitope is a linear feature of the VISTA polypeptide. Typically, an antigen has several or many different epitopes and reacts with many different antibodies.

[0077] The term "excipient" as used herein refers to an inert substance commonly used as a diluent, carrier, preservative, binder or stabilizer for a drug that imparts beneficial physical properties to the formulation, such as increased protein stability, increased protein solubility and reduced viscosity. Examples of 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, octanoates, etc.), surfactants (e.g., SDS, polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See also Remington' sPharmaceutical Sciences, (1990), Mack Publishing Co. Easton, PA, the entire contents of which are incorporated herein by reference.

[0078] The term "framework" or "FR" residues refers to those variable domain residues other than the hypervariable region residues as defined herein. FR residues are those variable domain residues that flank the CDRs. FR residues are found, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies.

[0079] When used to refer to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino terminal portion contains a variable region of about 120 to 130 or more amino acids, and the carboxyl terminal portion contains a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types, called alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ). Different heavy chains are of different sizes: α, δ and γ contain about 450 amino acids, while μ and ε contain about 550 amino acids. When combined with a light chain, five well-known classes of antibodies are produced by these different types of heavy chains, namely IgA, IgD, IgE, IgG and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. The heavy chain can be a human heavy chain.

[0080] The term "hinge region" refers herein to a flexible amino acid segment in the central portion of the heavy chain of the IgG and IgA immunoglobulin classes, which connects the two chains by a disulfide bond. The hinge region is generally defined as extending from Glu216 of human IgG1 to Pro230 (Burton, Mol Immunol, 22: 161-206, 1985). By placing the first and last cysteine ​​residues that form the inter-heavy chain SS bond in the same position, the hinge region of other IgG isotypes can be compared with the IgG1 sequence. The "CH2 domain" (also referred to as the "Cγ2" domain) of the human IgG Fc portion generally extends from about amino acid 231 to about amino acid 340. The uniqueness of the CH2 domain is that it is not tightly paired with another domain. Instead, two N-connected branched carbohydrate chains are inserted between the two CH2 domains of a complete natural IgG molecule. It is speculated that carbohydrates can provide substitutions for domain-domain pairing and contribute to stabilizing the CH2 domain (Burton, Mol Immunol, 22: 161-206, 1985). The "CH3 domain" includes the stretch of residues from the C-terminal end to the CH2 domain in the Fc portion (ie, from about amino acid residue 341 to about amino acid residue 447 of IgG).

[0081] As used herein, the term "host" refers to an animal, such as a mammal (eg, a human).

[0082] As used herein, the term "host cell" refers to a specific subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may be different from the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations or the nucleic acid molecule is integrated into the host cell genome.

[0083] "Humanized antibody" refers to a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor CDR are replaced with residues from the CDR of a non-human species (donor antibody) (such as mouse, rat, rabbit or non-human primate) with desired specificity, affinity and / or ability. In some cases, according to techniques known to those skilled in the art (Jones et al., Nature, 321:522-525, 1986), some skeleton segment residues (referred to as FR, representing framework) can be modified to maintain binding affinity. In some embodiments, the FR residues of human immunoglobulin are replaced with corresponding non-human residues. In certain embodiments, a humanized antibody will substantially comprise at least one, typically all of two variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody optionally may comprise at least a portion of an antibody constant region (Fc), typically at least a portion of a human immunoglobulin. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. The goal of humanization is to reduce the immunogenicity of a xenogeneic antibody (such as a murine antibody) for introduction into humans while maintaining the full antigen binding affinity and specificity of the antibody. For more details, see, e.g., Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0084] As used herein, "identifying" when referring to a subject having a disorder, refers to the process of evaluating a subject and determining that the subject has the disorder (eg, has cancer).

[0085] As used herein, the term "immune checkpoint" or "immune checkpoint protein" refers to certain proteins produced by certain types of immune system cells (such as T cells) and certain cancer cells. Such proteins regulate T cell function in the immune system. Notably, they help monitor immune responses and can prevent T cells from killing cancer cells. The immune checkpoint proteins achieve this result by interacting with specific ligands that send signals into T cells, essentially shutting down or inhibiting T cell function. Inhibiting these proteins results in restoration of T cell function and immune response to cancer cells. Examples of checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIGIT, TIM3, GAL9, LAG3, VSIG4, KIR, 2B4 (belonging to the CD2 family of molecules, expressed in all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, IDO1, A2aR, and various B7 family ligands.

[0086] As used herein, the term "increase" refers to an activity of a protein (e.g., VISTA) that is at least 1 times (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more) of its reference value. When "increase" refers to the activity of a subject's protein (e.g., VISTA), it also means that the activity of the protein of the reference sample or relative to the reference value of the protein is at least 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%) higher. As used herein, the term "increase" also refers to a subject's biomarker (e.g., VISTA) level that is at least 1 times its reference value (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000 times or more). When "increase" refers to a subject's biomarker (e.g., VISTA) level, it also means at least 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%) higher than the level of a reference sample or relative to the reference value of the marker.

[0087] When used in the context of antibodies, the term "inhibit" or "block" or its grammatical equivalent refers to an antibody that inhibits, limits or reduces the biological activity of an antigen bound to the antibody. The inhibitory effect of an antibody can be one that causes a measurable change in the biological activity of the antigen. In certain cases, "inhibition" or "blocking" refers to an antibody that prevents or terminates the biological activity of the antigen to which the antibody binds. Blocking antibodies include antibodies that bind to an antigen without causing a reaction but block another protein from subsequently binding or compounding with the antigen. The blocking effect of an antibody can be an effect that causes a measurable change in the biological activity of an antigen. In some embodiments, the anti-VISTA antibodies described herein block the ability of VISTA to bind to VSIG3, which can result in inhibition or blocking of VISTA's inhibitory signal. Certain anti-VISTA antibodies described herein inhibit or block the inhibitory signal of VISTA on cells expressing VISTA, including inhibition or blocking of about 98% to about 100% compared to an appropriate control (e.g., a control is a cell that has not been treated with the antibody to be tested). In some embodiments, the anti-VISTA antibodies described herein block the binding of the extracellular domain VISTA to VSIG3 and / or block the binding of cells expressing VISTA to cells expressing VSIG3. In some embodiments, the anti-VISTA antibodies described herein block the ability of VISTA to bind to PSGL-1, preferably at an acidic pH (pH between 5.9 and 6.5), which can result in inhibition or blocking of VISTA's inhibitory signal. Certain anti-VISTA antibodies described herein inhibit or block the inhibitory signal of VISTA on cells expressing VISTA, including inhibition or blocking of about 98% to about 100% compared to an appropriate control (e.g., a control is a cell treated with an antibody to be tested). In some embodiments, the anti-VISTA antibodies described herein block, preferably at an acidic pH (pH between 5.9 and 6.5) the binding and / or blocking of the extracellular domain VISTA to PSGL-1, preferably at an acidic pH (pH between 5.9 and 6.5) the binding of cells expressing VISTA to cells expressing PSGL-1.

[0088] The term "immune infiltrate" or "tumor immune cell" refers to cells that infiltrate the tumor microenvironment, including, but not limited to, lymphocytes (e.g., T cells, B cells, natural killer (NK) cells), dendritic cells, mast cells, and macrophages.

[0089] As used herein, the term "combination" in the context of administering other therapies refers to the use of more than one therapy (e.g., an anti-VISTA antibody and an immune checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody). The use of the term "combination" does not limit the order or timing of administering the therapy to a subject (e.g., one therapy before, simultaneously with, or after another therapy). The first therapy is administered before (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) the second therapy is administered to a subject who has suffered from, has suffered from or is susceptible to a VISTA-mediated disease, illness or condition, simultaneously or afterwards (e.g., 1 minute, 45 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks). Any additional therapy can be administered in any order or time with other additional therapies (e.g., anti-VISTA antibodies and immune checkpoint inhibitors, such as anti-PD-1 antibodies or anti-PD-L1 antibodies). In some embodiments, the antibody can be administered in combination with one or more therapies (e.g., the therapy is not an antibody currently administered to prevent, treat, control and / or ameliorate a VISTA-mediated disease, disorder or condition). Non-limiting examples of therapies that can be administered in combination with the antibody include antagonists of co-inhibitory molecules, agonists of co-stimulatory molecules, chemotherapeutic agents, radiation, analgesics, anesthetics, antibiotics or immunomodulators or in combination with USPharmacopoeia and / or P hysician'sDeskReference Any other reagents listed.

[0090] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0091] An "isolated" 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.

[0092] The term "K D” refers to the dissociation constant of a specific antibody-antigen interaction and is used as a measure of the affinity of the antibody for the antigen. A lower K D This means that the antibody has a higher affinity for the antigen.

[0093] As used herein, the "level" of a biomarker (e.g., VISTA) is composed of the quantitative value of the biomarker in a sample (e.g., in a sample collected from a patient with cancer). In some embodiments, the quantitative value is not composed of the absolute value of the actual measurement, but is composed of the final value produced after considering the signal-to-noise ratio occurring in the assay form used, and / or considering the calibration reference value for improving the repeatability of the cancer marker level measurement value between assays. In some embodiments, the "level" of a biomarker (e.g., VISTA) is expressed as an arbitrary unit, because it is important that between (i) assays, or (ii) between a patient with cancer and other patients, or (iii) between assays performed on the same patient at different time periods, or (iv) between the biomarker level measured in a patient sample and a predetermined reference value (also referred to herein as a "cutoff" value), the same type of arbitrary unit is compared.

[0094] When used to refer to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion comprises a variable region of about 100 to about 110 or more amino acids, and the carboxyl terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two different types, called kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0095] As used herein, the term "monoclonal antibody" refers to an antibody derived from an almost homogeneous antibody population, wherein the population contains identical antibodies except for some possible naturally occurring mutations that can be found in minimal proportions. Monoclonal antibodies are derived from the growth of a single cell clone (such as a hybridoma) and are characterized by a class and subclass of heavy chains and a type of light chain. As used herein, when a monoclonal antibody is presented to a single antigenic site (i.e., a single epitope), the antibody exhibits specific binding to the site. Monoclonal antibodies can be produced by various methods known in the corresponding technical field.

[0096] The term "native" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, etc., refers to those found in nature and have not been manipulated by humans.

[0097] As used herein, "percent identity" or "% identity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acid residues between the two sequences to be compared after optimal alignment, which is purely statistical, and the differences between the two sequences are randomly distributed along their lengths. The comparison of two nucleic acid or amino acid sequences is traditionally performed by comparing the sequences after optimal alignment, which can be performed by segments or by using a "comparison window". In addition to manual comparison, the sequences to be compared can also be optimally aligned by methods known to those skilled in the art.

[0098] For amino acid sequences that show 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 to a reference amino acid sequence, preferred examples include those containing the reference sequence, certain modifications, especially deletions, additions or substitutions, truncations or extensions of at least one amino acid. In the case of substitution of one or more consecutive or non-consecutive amino acids, preferred substitutions are those in which the substituted amino acid is replaced with an "equivalent" amino acid. Here, the expression "equivalent amino acid" refers to any amino acid that may replace one of the structural amino acids without changing the biological activity of the corresponding antibody and those specific embodiments defined below. Equivalent amino acids can be determined based on the structural homology of the equivalent amino acid with the amino acid it replaces or based on the results of comparative biological activity tests between various antibodies that may be generated.

[0099] As non-limiting examples, Table 1 below summarizes possible substitutions that can be made without resulting in a significant change in the biological activity of the corresponding modified antigen binding protein; reverse substitutions can naturally be made under the same conditions.

[0100] Table 1

[0101] original residue replace Ala(A) Val, Gly, Pro Arg(R) Lys、His Asn(N) Gln Asp(D) Glu Cys(C) Ser Gln(Q) Asn Glu(E) Asp Gly(G) Ala His(H) Arg Ile(I) Leu Leu(L) Ile, Val, Met Lys(K) Arg Met(M) Leu Phe(F) Tyr Pro(P) Ala Ser(S) Thr、Cys Thr(T) Ser Trp(W) Tyr Tyr(Y) Phe、Trp Val(V) Leu, Ala

[0102] As used herein, the term "pharmaceutically acceptable" refers to approved by a regulatory agency of a federal or state government, or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia or other recognized pharmacopoeias for use in animals, more particularly for humans. More specifically, when referring to a carrier, the expression "pharmaceutically acceptable" refers to the carrier being compatible with the other components of the composition and being harmless to its recipient. Therefore, as used herein, the expression "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism, which does not inhibit the biological activity and characteristics of the compound used for administration. The type of carrier can be selected based on the intended route of administration. The amount of each carrier used can vary within the conventional range of the art. As a pharmaceutically acceptable carrier in a composition prepared as a liquid solution, physiological saline, sterile water, buffered saline, albumin injection, glucose solution, maltodextrin solution, glycerol and a mixture of one or more thereof can be used as a sterile carrier suitable for living organisms. If necessary, common additives such as antioxidants, buffers and antibacterial agents can be added. Furthermore, by further adding a diluent, a dispersant, a surfactant, a binder or a lubricant, the composition can be prepared into an injection preparation such as an aqueous solution, a suspension, and an emulsion, a pill, a capsule, a granule or a tablet.

[0103] As used herein, the term "polyclonal antibody" refers to an antibody produced in the presence of or among one or more other different antibodies. Polyclonal antibodies are usually produced by B lymphocytes, while there are several other different antibodies produced by non-B lymphocytes. Polyclonal antibodies are usually obtained directly from immunized animals.

[0104] As used herein, the terms "polynucleotide," "nucleotide," "nucleic acid," "nucleic acid molecule" and other similar terms are used interchangeably and include DNA, RNA, mRNA, and the like.

[0105] When used in a therapeutic context, the term "radiation" refers to a type of treatment that uses intense energy beams to kill target cells (e.g., cancer cells). Radiation therapy includes the use of X-rays, protons, or other forms of energy that are administered through external beams. Radiation therapy also includes radiation therapy placed inside the patient's body (e.g., brachytherapy), in which small containers of radioactive material are implanted directly into or near a tumor.

[0106] As used herein, the term "reference value" refers to the expression level of the biomarker (e.g., VISTA) considered in the reference sample. As used herein, "reference sample" refers to a sample obtained from a subject known to be free of disease, preferably two or more subjects, alternatively from the general population. The suitable reference expression level of the biomarker can be determined by measuring the expression level of the biomarker in several suitable subjects, and such reference levels can be adjusted for a specific subject population. Reference values ​​or reference levels can be absolute values; relative values; values ​​with upper or lower limits; a range of values; average values; median values, mean values, or values ​​compared to a specific control or baseline value. Reference values ​​can be based on the value of a single sample, such as from a sample from a tested subject, but at an earlier time point. Reference values ​​can be based on a large number of samples, such as from a subject population of an actual age-matched group, or based on a sample pool containing or not containing a sample to be tested.

[0107] As used herein, the term "side effect" encompasses an undesirable and adverse effect of a therapy (e.g., a prophylactic or therapeutic agent). An undesirable effect is not necessarily adverse. An adverse effect of a therapy (e.g., a prophylactic or therapeutic agent) may be harmful, uncomfortable, or risky. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, hair loss, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rash or swelling at the site of administration, flu-like symptoms (such as fever, chills, and tiredness), digestive problems, and allergic reactions. Other undesirable effects experienced by patients are numerous and known in the art. Many are described in Physician's Desk Reference (67th edition, 2013).

[0108] As used herein, in the context of a liquid formulation comprising an antibody (including an antibody fragment thereof) that specifically binds an antigen of interest (e.g., VISTA), the terms "stability" and "stable" refer to the resistance of the antibody (including its antibody fragment) in the formulation to aggregation, degradation or fragmentation under given manufacturing, preparation, transportation and storage conditions. The "stable" formulation of the present disclosure retains biological activity under given manufacturing, preparation, transportation and storage conditions. The stability of the antibody (including its antibody fragment) can be assessed by the degree of aggregation, degradation or fragmentation compared to a reference formulation, such as by HPSEC, reverse phase chromatography, static light scattering (SLS), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea unfolding technology, intrinsic tryptophan fluorescence, differential scanning calorimetry and / or ANS binding technology. For example, the reference preparation can be a reference standard frozen at -70°C, which consists of 20 mg / ml antibody (including antibody fragments thereof) (for example, but not limited to an antibody comprising the heavy chain sequence of SEQ ID NO: 21 and the light chain sequence of SEQ ID NO: 22) in 25 mM histidine (pH 6.5), containing 150 mM NaCl and 0.006% polysorbate 80, (v / v), which typically gives a single monomer peak (for example, ≥ 97% area) by HPSEC. The overall stability of the preparation comprising the antibody (including antibody fragments thereof) can be assessed by various immunological assays, including, for example, ELISA and radioimmunoassay using isolated antigen molecules.

[0109] "Subjects" that can be subjected to the methods described herein can be any mammal, including humans, dogs, cats, cows, goats, pigs, swine, sheep, and monkeys. Human subjects can be referred to as patients. In one embodiment, a "subject" or "subject in need" refers to a mammal that has cancer or is suspected of having cancer or has been diagnosed with cancer. As used herein, a "subject with cancer" refers to a mammal that has cancer or has been diagnosed with cancer. A "control subject" refers to a mammal that does not have cancer and is not suspected of having cancer.

[0110] As used herein, "substantially all" means 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%.

[0111] As used herein, the term "therapeutic agent" refers to any agent that can be used to treat, prevent or alleviate a disease, illness or condition, including any agent used to treat, prevent or alleviate one or more symptoms and / or symptoms associated with VISTA-mediated diseases, illnesses or conditions. In some embodiments, a therapeutic agent refers to an anti-VISTA antibody provided herein. In some embodiments, a therapeutic agent refers to an agent other than an anti-VISTA antibody provided herein. In some embodiments, a therapeutic agent is a reagent known to be used or has been used or is currently used to treat, prevent or alleviate one or more symptoms and / or symptoms associated with VISTA-mediated diseases, illnesses, conditions.

[0112] The combination of therapies (e.g., using multiple therapeutic agents) can be more effective than the cumulative effect of any two or more monotherapies. For example, the synergistic effect of the therapeutic agent combination allows the use of one or more agents at lower doses and / or the agent is administered to a subject suffering from a disease, illness or condition mediated by VISTA and / or symptoms associated therewith at a lower frequency. The ability of the therapy using a lower dose of the therapeutic agent and / or the ability of the therapy administered at a lower frequency reduces the toxicity associated with the administration of therapy to the subject without reducing the efficacy of the therapy in preventing, treating or alleviating one or more symptoms in a disease, illness or condition mediated by VISTA and / or symptoms associated therewith. In addition, in terms of preventing, treating or alleviating one or more symptoms in a disease, illness or condition mediated by VISTA and / or symptoms associated therewith, synergistic effect can improve the therapeutic efficacy. Finally, the synergistic effect of the therapy (e.g., therapeutic agent) combination can avoid or reduce the adverse or undesirable side effects associated with the use of any monotherapy.

[0113] As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent (e.g., an anti-VISTA antibody or any other therapeutic agent, including those described herein, including, for example, immune checkpoint inhibitors, such as, for example, anti-PD-1 antibodies or anti-PD-L1 antibodies), sufficient to reduce and / or improve the severity and / or duration of a given disease, illness or condition and / or symptoms associated therewith. A therapeutically effective amount of a therapeutic agent can be an amount necessary to reduce or improve the development or progression of a given disease, illness or condition, an amount necessary to reduce or improve the recurrence, progression or onset of a given disease, illness or condition, and / or an amount necessary to improve or enhance the preventive or therapeutic effect of another therapy (e.g., a therapy other than administering an anti-VISTA antibody, including a therapy described herein).

[0114] As used herein, the term "therapy" refers to any regimen, method and / or agent that can be used to prevent, manage, treat and / or improve a VISTA-mediated disease, illness or condition. In some embodiments, the terms "therapies" and "therapy" refer to biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (e.g., medical personnel) that can be used to treat, prevent and / or improve a VISTA-mediated disease, illness or condition.

[0115] As used herein, "treating" a disease in a subject or "treating" a subject with a disease refers to subjecting the subject to drug therapy (e.g., administration of a drug) to reduce or prevent the extent of the disease. For example, treatment results in at least one sign of a disease or condition or reduces symptoms. Treatment includes, but is not limited to, administration of a composition (e.g., a pharmaceutical composition), and may be performed prophylactically or after the onset of a pathological event. Treatment may require more than one administration of an agent and / or treatment. In some embodiments, such terms refer to a reduction or improvement in the course, severity, and / or duration of a disease that responds to immunomodulation, which is caused by increasing T cell activation.

[0116] The term "tumor microenvironment" refers to the cellular environment in which a tumor exists. The tumor microenvironment may include surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix.

[0117] The term "variable domain" or "variable region" refers to a part of the light chain or heavy chain of an antibody, which is usually located at the amino terminal of the light chain 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 is used in the binding and specificity of each specific antibody to its specific antigen. The sequence of the variable domain varies greatly between different antibodies. The variability of the sequence is concentrated in the CDR, and the part with lower variability in the variable domain is called the framework region (FR). Each variable region includes three CDRs connected to four FRs. The CDRs of the light chain and heavy chain are mainly responsible for the interaction of the antibody with the antigen. Although not directly involved in antigen binding, FR determines the folding of the molecule, thereby determining the amount of CDRs that interact with the antigen presented on the surface of the variable region. In some embodiments, the variable region is a human variable region.

[0118] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of an antibody heavy or light chain. The variable domain of the heavy chain may be referred to as the "V H The variable domain of the light chain may be referred to as the "V L ”. These domains are usually the most variable parts of antibodies and contain the antigen binding site.

[0119] When used in conjunction with VISTA or anti-VISTA antibodies, the term "variant" refers to a peptide or polypeptide comprising one or more (e.g., such as 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 compared to a native or unmodified sequence. For example, a VISTA variant may be produced by 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 the amino acid sequence of a natural VISTA. For another example, a variant of an anti-VISTA antibody may be produced by 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 the amino acid sequence of a natural or previously unmodified anti-VISTA antibody. Preferably, a variant of an anti-VISTA antibody may be produced by a change to the amino acid sequence of a natural or previously unmodified anti-VISTA antibody. In some embodiments, VISTA variants or anti-VISTA antibody variants retain at least the functional activity of VISTA or anti-VISTA antibodies, respectively. In some embodiments, anti-VISTA antibody variants do not undergo deamidation in CDR. In some embodiments, anti-VISTA antibody variants bind VISTA and / or antagonize VISTA activity. In some embodiments, anti-VISTA antibody variants do not undergo deamidation in CDR, bind VISTA and / or antagonize VISTA activity. Variants may be naturally occurring (such as alleles or splice variants), or may be artificially constructed. In some embodiments, variants are encoded by single nucleotide polymorphism (SNP) variants of nucleic acid molecules encoding VISTA or anti-VISTA antibody VH or VL regions or subregions. Polypeptide variants can be prepared from the corresponding nucleic acid molecules encoding variants.

[0120] The term "vector" refers to a substance used to introduce a nucleic acid molecule into a host cell. In particular, as used herein, a "vector" is a nucleic acid molecule that can propagate another nucleic acid molecule connected thereto. An example of a vector is a "plasmid", which refers to a circular double-stranded DNA loop in which an additional DNA segment can be connected. Another example of a vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-free mammalian vectors) can be integrated into the genome of the host cell, thereby replicating with the host genome. Therefore, the term "vector" includes vectors as self-replicating nucleic acid structures and vectors incorporated into the host cell genome into which they are introduced. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include operable sequences or markers selected to be stably integrated into the host cell chromosome.

[0121] Certain vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, the present invention is intended to include such forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retroviruses, EBV-derived episomes, and all other vectors known to technicians to be conveniently used to ensure the expression of heavy chains and / or light chains of antibodies of interest (e.g., anti-VISTA antibodies). Technicians will recognize that polynucleotides encoding heavy and light chains can be cloned into different vectors or the same vector.

[0122] The vector can include one or more selective marker genes and appropriate expression control sequences. The selective marker genes that can be included, for example, provide resistance to antibiotics or toxins, supplement nutritional deficiencies or provide key nutrients that are not available in the culture medium. The expression control sequence can include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc. that are well known in the art. When two or more nucleic acid molecules (for example, both antibody heavy chains and light chains) are to be co-expressed, the two nucleic acid molecules can be inserted, for example, in a single expression vector or in a separate expression vector. For single vector expression, the encoding nucleic acid can be operably connected to a common expression control sequence, or connected to different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm that nucleic acid molecules are introduced into host cells. Such methods include, for example, nucleic acid analysis, such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequence or its corresponding gene product. Those skilled in the art understand that nucleic acid molecules are expressed in amounts sufficient to produce a desired product (e.g., an anti-VISTA antibody provided herein), and further understand that expression levels can be optimized using methods well known in the art to obtain sufficient expression.

[0123] The term "VISTA" or "VISTA polypeptide" and similar terms refer to a polypeptide encoded by the open reading frame 54 (VISTA) gene of human chromosome 10 ("polypeptide", "peptide" and "protein" are used interchangeably herein), also known in the art as B7-H5, platelet receptor Gi24, GI24, stress-induced secreted protein 1, SISP1 and PP2135, for example, comprising the following amino acid sequence:

[0124]

[0125] And related polypeptides, including SNP variants thereof. It has been shown or predicted that the VISTA polypeptide contains several different 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; homologs between species, preferably, which retain VISTA activity and / or are sufficient to produce an immune response against VISTA. VISTA can exist in a native or denatured form. The VISTA polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or another source, or prepared by recombinant or synthetic methods. "Native sequence VISTA polypeptides" include polypeptides having the same amino acid sequence as the corresponding VISTA polypeptides 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 (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternative splicing forms) and allelic variants of naturally occurring polypeptides of specific VISTA polypeptides.

[0126] A cDNA nucleic acid sequence encoding a VISTA polypeptide, for example comprising

[0127]

[0128] VISTA is primarily expressed on myeloid cell populations, particularly myeloid-derived suppressor cells (MDSCs), neutrophils, monocytes, macrophages, and dendritic cells. VISTA can also be expressed on regulatory T cells and CD4 +It is expressed on initial T lymphocytes. As described herein, VISTA is an immunomodulator, which is a negative checkpoint regulator of the immune response (e.g., inhibiting or suppressing the immune response). VISTA has been identified as a negative checkpoint regulator of T cell function, known to inhibit autoimmune responses in a variety of human and mouse autoimmune models. VISTA has been shown to be particularly capable of promoting tumorigenesis, blocking T cell function, regulating the activity of macrophages and immunosuppressive myeloid-derived suppressor cells (MDSC). VISTA is upregulated in immunosuppressive tumor-infiltrating leukocytes (such as suppressive regulatory T cells (Treg) and MDSC). The presence of VISTA in the tumor microenvironment impedes effective T cell responses and is associated with many human cancers, including prostate, colon, skin, pancreatic and lung cancers (ElTanbouly et al., Clin Exp Immunol. 200(2):120-130 (2020); Mehta et al., Sci Rep. 10(1):1 5171 (2020); Yuan et al., Trends Immunol. 42(3):209-227 (2021); Tagliamento et al., Immunotargets Ther. 10:185-200 (2021); Thakkar et al., J Immunother Cancer. 10(2):e003382 (2022); WO 2015 / 097536; WO 2016 / 094837; WO 2017 / 181139; WO 2019 / 183040).

[0129] Orthologs of VISTA polypeptides are also well known in the art. For example, a mouse ortholog of VISTA polypeptide is an inhibitor of T cell activation containing a V region immunoglobulin ( V -region I mmunoglobulin-containing S uppressor of Tcell A Activation, VISTA) (also known as PD-L3, PD-1H, PD-XL, Pro1412 and UNQ730), has about 70% sequence identity with the human polypeptide. Orthologs of VISTA can also be found in other organisms, including chimpanzees, cows, rats and zebrafish.

[0130] "VISTA-expressing cells", "VISTA-expressing cells" or their grammatical equivalents refer to cells that express endogenous or transfected VISTA on the cell surface. VISTA-expressing cells include tumor cells carrying VISTA, regulatory T cells (e.g., CD4 +Foxp3 + regulatory T cells), myeloid-derived suppressor cells (e.g., CD11b + CD11b 高 Myeloid-derived suppressor cells) and / or suppressor dendritic cells (e.g., CD11b + CD11b 高 Dendritic cells). Cells expressing VISTA produce sufficient levels of VISTA on their surface so that anti-VISTA antibodies can bind to it and / or PSGL-1 or cells expressing PSGL-1 can bind to it. In some aspects, inhibiting or blocking this binding can have a therapeutic effect. Cells that "overexpress" VISTA are cells with significantly higher levels of VISTA on their cell surfaces compared to cells of the same tissue type known to express VISTA. This overexpression may be due to gene amplification or by increasing transcription or translation. By assessing the increased level of VISTA protein present on the cell surface, VISTA overexpression can be determined in a diagnostic or prognostic assay (e.g., by immunohistochemical assay; FACS analysis). Alternatively or additionally, the level of nucleic acid encoding VISTA or mRNA encoding VISTA in the cell can be measured, for example, by fluorescence in situ hybridization; (FISH; see WO98 / 45479 published in October 1998), Southern blotting, Northern blotting or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). In addition to the above assays, technicians can also perform various in vivo assays. For example, cells in a patient's body can be exposed to an antibody optionally labeled with a detectable agent, and the binding of the antibody to cells in the patient's body can be assessed, for example, by external scanning of radioactivity or by analyzing a biopsy obtained from a patient previously exposed to the antibody. Tumor cells expressing VISTA include, but are not limited to, acute myeloid leukemia (AML) tumor cells.

[0131] "VISTA-mediated disease", "VISTA-mediated illness" and "VISTA-mediated condition" can be used interchangeably and refer to any disease, illness or condition caused or caused in whole or in part by VISTA. Such diseases, illnesses or conditions include those caused by or otherwise associated with VISTA, including those caused by or associated with cells expressing VISTA (e.g., tumor cells, myeloid-derived suppressor cells (MDSC), inhibitory dendritic cells (inhibitory DC), and / or regulatory T cells (T-reg)). In some embodiments, VISTA is abnormally (e.g., highly) expressed on the cell surface. In some embodiments, VISTA can be abnormally upregulated on a specific cell type. In other embodiments, normal, abnormal or excessive cell signaling is caused by VISTA in combination with a VISTA receptor (e.g., PSGL-1, VSIG3, VSIG8 or LRIG1), which can bind to or otherwise interact with VISTA. Preferably, as used herein, "VISTA-mediated disease" refers to a tumor whose proliferation is associated with VISTA activity (i.e., a "VISTA-mediated tumor"). For example, expression of VISTA in cells present in the tumor microenvironment (e.g., MDSC) can lead to suppression of the immune response against the tumor. In certain cases, expression of VISTA in cells present in the tumor microenvironment (e.g., MDSC) can suppress T cell immunity (CD4 + and CD8 + T cell immunity) and / or prevent the expression of pro-inflammatory cytokines. In particular, CD4 + and / or CD8 + The proliferation of T cells can be inhibited. The expression of cytokines such as IFNγ, IL-2 or TNFα can be prevented. In a specific aspect, these effects are mediated by the expression of VISTA on cells (e.g., MDSC) present in the tumor microenvironment, which interact with receptors (e.g., PSG-L1, VSIG3, VSIG8 or LRIG1) expressed on immune cells (e.g., T cells or tumor cells).

[0132] Antibody preparations

[0133] The present disclosure provides a stable pharmaceutical composition (formulation) comprising an anti-VISTA antibody or an antigen-binding fragment thereof, such as any anti-VISTA antibody described herein.

[0134] These compositions can be used, for example, to stimulate an immune response in a subject. The antibody that specifically binds to VISTA of the present invention induces T cell activation by binding to a VISTA protein that inhibits T cell activation, and thus the antibody can stimulate an immune response.

[0135] The compositions described herein can also be used to treat cancer. Protective anti-tumor immunity can be established by administering such compositions comprising an anti-VISTA antibody, an antigen-binding fragment thereof, or a conjugate thereof disclosed herein.

[0136] Immune checkpoints play a crucial role in maintaining self-tolerance and limiting immune-mediated tissue damage under physiological conditions. VISTA is a type I transmembrane protein that belongs to the B7-related immunoglobulin superfamily and is highly expressed in the hematopoietic compartment. VISTA acts as both a ligand and a receptor, inhibiting CD4 + and CD8 + T cell activation is negatively regulated by T cell proliferation and pro-inflammatory cytokine (e.g., IFNγ, TNFα, or IL-2) production.

[0137] WO 2016 / 094837 describes a monoclonal antibody Ab3 that can inhibit VISTA immunosuppression and thereby enhance anti-tumor immune response. The CDRs of the antibody are represented by SEQ ID NOs: 3-8, V H and V L They are represented by SEQ ID NO: 9 and SEQ ID NO: 10 respectively. The complete heavy chain of Ab3 has the sequence represented by SEQ ID NO: 11, and the complete light chain of Ab3 has the sequence represented by SEQ ID NO: 12.

[0138] The complete sequence of antibody Ab3 shows that it contains 11 potential deamidation sites. Although all of these sites are predicted to be effective deamidation sites, the inventors found that only one of them actually undergoes deamidation, namely the Asn residue at position 55 in the heavy chain CDR2. Surprisingly, replacing the Asn with an Asp residue does not affect Ab3 binding to its target, which is completely different from the teachings of the prior art, in which similar mutations in CDRs result in a 400-fold decrease in affinity for the corresponding CD52 antigen (Liu et al., 2022). In addition, the mutated antibody retains the ability to inhibit the immunosuppressive activity of VISTA, because the mutated antibody is able to block the interaction between VISTA and each of its two binding partners, PSG-L1 and VSIG3, which results in the inhibition of T cell function. Therefore, the mutated antibody inhibits tumor proliferation in vivo.

[0139] The present disclosure relates to a stable liquid formulation of an antibody or fragment thereof that specifically binds to VISTA, which is not easily deamidated while retaining its ability to inhibit VISTA immunosuppressive activity. In certain embodiments, a stable liquid formulation of an anti-human VISTA antibody or fragment thereof is suitable for parenteral administration to a human subject. In a specific embodiment, the stable liquid formulation of the present disclosure is suitable for subcutaneous administration to a human subject.

[0140] The present disclosure relates to a sterile, stable aqueous formulation comprising an antibody or fragment thereof that specifically binds to human VISTA, and is more homogeneous and can stimulate anti-cancer immunity. The present disclosure provides a preparation of an anti-VISTA antibody described in WO 2022 / 229469. In a particular example, the preparation of the present disclosure comprises an anti-VISTA antibody comprising an Asp residue at position 55 in the CDR2 of the heavy chain. In another embodiment, the preparation of the present disclosure comprises an anti-VISTA antibody comprising a heavy chain sequence of SEQ ID NO: 21 and a light chain sequence of SEQ ID NO: 22. In one example, the preparation of the present disclosure is provided in a prefilled syringe.

[0141] In one example, the liquid formulation of the present disclosure is an aqueous formulation. Preferably, the liquid formulation of the present disclosure is an aqueous formulation, wherein the aqueous carrier is distilled water.

[0142] Advantageously, the compositions of the present disclosure are sterile.

[0143] Advantageously, the compositions of the present disclosure are homogeneous.

[0144] Advantageously, the compositions of the present disclosure are isotonic.

[0145] The present disclosure encompasses stable liquid formulations comprising a single antibody of interest or an antibody fragment thereof, e.g., an antibody that specifically binds to VISTA, in particular the antibodies described in WO 2022 / 229469. Optionally, the formulations disclosed herein may comprise one or more additional therapeutic agents, such as immune checkpoint inhibitors described below.

[0146] In one example, the formulations disclosed herein contain at least about 1 mg / mL, at least about 5 mg / mL, at least about 10 mg / mL, at least about 15 mg / mL, at least about 20 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, at least about 80 mg / mL, at least about 90 mg / mL, or at least about 100 mg / mL of an anti-VISTA antibody or fragment thereof, such as the antibodies described in WO 2022 / 229469. In another example, the formulation disclosed herein comprises less than 100 mg / mL, less than 90 mg / mL, less than 80 mg / mL, less than 70 mg / mL, less than 60 mg / mL, less than 50 mg / mL, less than 40 mg / mL, less than 30 mg / mL, less than 20 mg / mL, less than 15 mg / mL, less than 10 mg / mL or less than 5 mg / mL of an anti-VISTA antibody or fragment thereof, such as the antibody described in WO 2022 / 229469. In another example, the formulation disclosed herein comprises an anti-VISTA antibody or fragment thereof between 5 and 100 mg / mL, between 10 and 70 mg / mL, between 15 and 50 mg / mL or between 20 and 30 mg / mL, such as the antibody described in WO 2022 / 229469. Preferably, the formulation disclosed herein comprises 20 mg / mL of an anti-VISTA antibody or fragment thereof, such as the antibody described in WO 2022 / 229469.

[0147] Optionally, the preparation of the present disclosure may also include common excipients and / or additives, such as buffers, saccharides, salts and surfactants. Additionally or alternatively, the preparation of the present disclosure may also include common excipients and / or additives, such as but not limited to solubilizers, diluents, adhesives, stabilizers, salts, lipophilic solvents, amino acids, chelating agents, preservatives, etc.

[0148] In some examples, the buffer is selected from histidine, citrate, phosphate, glycine and acetate. In other examples, the carbohydrate excipient is selected from trehalose, sucrose, mannitol, maltose and raffinose. In other examples, the surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 80 and Pluronic F68. In other embodiments, the salt is selected from NaCl, KCl, MgCl2 and CaCl2.

[0149] The formulations of the present invention include buffers or pH adjusting agents to provide improved pH control, thereby maintaining the pH within a desired range. For example, the formulations disclosed herein have a pH between about 3.0 to about 9.0, between about 4.0 to about 8.0, between about 5.0 to about 8.0, between about 5.0 to about 7.0, between about 5.0 to about 6.5, between about 5.5 to about 8.0, between about 5.5 to about 7.0, or between about 5.5 to about 6.5. In further embodiments, the compositions of the present disclosure have a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, the compositions of the present disclosure have a pH of about 6.5.

[0150] Buffers suitable for use in the present invention include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, etc. Preferably, the buffer is selected from citrate buffer, phosphate buffer and histidine buffer. More preferably, the buffer is a histidine buffer.

[0151] The buffer, e.g., histidine, can be present in a concentration of about 2 mM to about 50 mM. Preferably, the buffer is present in a concentration of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, or 45 mM. Preferably, the buffer is present in a concentration of less than 45, 40, 35, 30, 25, 20, 15, 10, 5, or 2 mM. More preferably, the concentration of the buffer is between 5 and 45 mM, between 10 and 40 mM, between 15 and 35 mM, between 20 and 30 mM. Even more preferably, the concentration of the buffer is about 25 mM.

[0152] More preferably, the buffer is a histidine buffer and is present at a concentration of 25 mM.

[0153] Technicians will appreciate that preparations disclosed herein can be isotonic with human blood, that is, the preparation has substantially the same osmotic pressure as human blood. Preferably, the osmotic pressure range of preparations of the present invention is from about 100mOSm to about 1200mOSm, or from about 200mOSm to about 1000mOSm, or from about 200mOSm to about 800mOSm, or from about 200mOSm to about 600mOSm, or from about 250mOSm to about 500mOSm, or from about 250mOSm to about 400mOSm, or from about 250mOSm to about 350mOSm. Preparations of the present invention will more preferably have an osmotic pressure from about 250mOSm to about 350mOSm. Isotonicity can be measured, for example, using vapor pressure or a frozen type osmometer. The tension of preparation is regulated by using a tension regulator. "Tonicity adjusters" refer to pharmaceutically acceptable inert substances that can be added to the formulation to ensure the isotonicity of the liquid formulations of the present disclosure, including polyols, such as tri- or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol, salts and amino acids.

[0154] In certain embodiments, the formulations of the present disclosure have an osmotic pressure of from about 100 mOSm to about 1200 mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800 mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or from about 250 mOSm to about 350 mOSm.

[0155] In certain embodiments, the formulations of the present disclosure have an osmotic pressure of from 100 mOSm to 1200 mOSm, or from 200 mOSm to 1000 mOSm, or from 200 mOSm to 800 mOSm, or from 200 mOSm to 600 mOSm, or from 250 mOSm to 500 mOSm, or from 250 mOSm to 400 mOSm, or from 250 mOSm to 350 mOSm.

[0156] The concentration of any one or any combination of the different components of the formulations described herein is adjusted to achieve the desired tension of the final formulation. Amino acids that are pharmaceutically acceptable and suitable for use as tension regulators in the present disclosure include, but are not limited to proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine, serine, lysine and histidine. The desired isotonicity of the final formulation can be achieved, in particular, by adjusting the salt concentration of the formulation. Salts that are pharmaceutically acceptable and suitable for use as tension regulators in the present disclosure include, but are not limited to sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate and calcium chloride. Advantageously, the formulations of the present invention include NaCl, MgCl2 and / or CaCl2. Preferably, the formulations of the present invention include NaCl.

[0157] In one example, the tonicity adjusting agent is present in the formulation at a concentration between 0 mM and 300 mM, preferably between 50 mM and 250 mM, more preferably between 100 mM and 200 mM.

[0158] In one example, the formulations disclosed herein include at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, at least about 80 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, or at least about 300 mM NaCl. In another example, the formulations described herein include between 10 mM and 300 mM, between 10 mM and 200 mM, between 10 mM and 175 mM, between 10 mM and 150 mM, between 25 mM and 300 mM, between 25 mM and 200 mM, between 25 mM and 175 mM, between 25 mM and 150 mM, between 50 mM and 300 mM, between 50 mM and 200 mM. In another example, the formulation disclosed herein comprises NaCl between 0mM and 300mM, between 50mM and 175mM, between 50mM and 150mM, between 75mM and 300mM, between 75mM and 200mM, between 75mM and 175mM, between 75mM and 150mM, between 100mM and 300mM, between 100mM and 200mM, between 100mM and 175mM, or between 100mM and 150mM. In another example, the formulation disclosed herein comprises NaCl between 0mM and 300mM, between 50mM and 250mM, between or between 100mM and 200mM. Preferably, the formulation disclosed herein comprises 150mM NaCl.

[0159] Preservatives may be added to retard microbial growth, and the amount added ranges from 0.2% to 1% (w / v). Preservatives suitable for use in the present disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chlorides, bromides, and iodides), hexamethylammonium chloride, and alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Stabilizers refer to a broad class of excipients, ranging in function from fillers to additives that make therapeutic agents (i.e., anti-VISTA antibodies, antigen-binding fragments thereof, or conjugates thereof) soluble or that help prevent denaturation or adhesion to the container wall. Typical stabilizers can be polyols (listed above); amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol (myoinisitol), galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; amino acids Polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose and trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in a range of 0.1 to 10,000 weight per weight portion of active protein (e.g., anti-VISTA antibodies or antigen-binding fragments comprising such antibodies). Preferably, the formulations described herein include at least one stabilizer selected from arginine and sucrose. Arginine may, for example, be present at a concentration between 0 and 50 mM. In another example, the concentration range of sucrose may be 0 to 6%.

[0160] Nonionic surfactants or detergents (also called "wetting agents") may be added to help make the anti-VISTA antibody soluble, as well as to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxyheteroisomeric (184, 188, etc.), pluronic polyols, polyoxyethylene sorbitan monoethers ( -20, -80, etc.). The nonionic surfactant may be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, for example, about 0.07 mg / ml to about 0.2 mg / ml. Preferably, the formulations described herein contain a nonionic surfactant, which is a polysorbate, such as polysorbate 20 or polysorbate 80. Preferably, the nonionic surfactant in the formulations disclosed herein is polysorbate 80. In one example, the formulations described herein contain at least about 0.001%, at least about 0.002%, at least about 0.003%, at least about 0.004%, at least about 0.005%, at least about 0.006%, at least about 0.007%, at least about 0.008%, at least about 0.009%, at least about 0.01%, at least about 0.02%, at least about 0.05%, at least about 0.1%, at least about 0.2% or at least about 0.5% polysorbate 80 (v / v). In another example, the formulations described herein include between about 0.001% to about 0.5% (v / v), between about 0.002% to about 0.2% (v / v), between about 0.003% to about 0.1% (v / v), between about 0.004% to about 0.009% (v / v), between about 0.005% to about 0.008% (v / v), or between about 0.006% to about 0.007% polysorbate 80 (v / v). In another example, the formulations disclosed herein include about 0.006% polysorbate 80 (v / v).

[0161] Other miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and solubilizing agents.

[0162] Preferably, the formulation disclosed herein comprises histidine buffer, NaCl, polysorbate 80, and an anti-VISTA antibody or fragment thereof disclosed in WO 2022 / 229469. Optionally, the formulation disclosed herein comprises sucrose.

[0163] Advantageously, the formulations disclosed herein comprise between 10 mM and 50 mM histidine, preferably 25 mM; between 0 and 150 mM NaCl, preferably 150 mM; between 0 and 0.5% polysorbate 80 (v / v), preferably 0.006% (v / v); and between 0 and 6% sucrose, preferably 0%; and a pH between 5.5 and 7.0, preferably 6.5.

[0164] Preferably, the formulation disclosed herein comprises 25 mM histidine, 150 mM NaCl, 0.006% polysorbate 80 (w / w), pH 6.5. More preferably, the pharmaceutical composition comprises 20 mg / mL of an anti-VISTA antibody or a fragment thereof disclosed in WO 2022 / 229469.

[0165] The formulation is typically non-pyrogenic, for example containing <1 Eu (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The formulation is preferably gluten-free.

[0166] As described below, the anti-VISTA antibody or antigen-binding fragment thereof or conjugate thereof may be mixed with a second therapeutic agent in the present formulation.

[0167] The preparation is generally provided as part of a sterile pharmaceutical composition, which generally comprises a pharmaceutically acceptable carrier and / or excipient. Therefore, in another aspect, the present disclosure provides a pharmaceutical composition comprising an anti-VISTA antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier and / or excipient.

[0168] The composition can be in any suitable form (depending on the desired method of applying it to the patient). The composition used in the method described herein can be applied, for example, as follows: intravitreal (e.g., by intravitreal injection), by eye drops, intramuscular, intravenous, intradermal, percutaneous, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, local, intratumoral, transperitoneal, subcutaneous, subconjunctival, intravesicular, transmucosal, pericardial, umbilical, intraocular, intraorbital, oral, local, transdermal, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local perfusion to directly bathe target cells, by catheter, by lavage, in cream or in lipid composition. The composition utilized in the method described herein can also be applied systemically or topically. The method of application can vary depending on various factors (e.g., the compound or composition applied and the severity of the illness, disease or disorder treated). In any given example, the most suitable route of administration will depend on the specific antibody, subject, and the nature and severity of the disease and the physical condition of the subject. For example, anti-VISTA antibodies or their antigen-binding fragments can be formulated into an aqueous solution and administered by subcutaneous injection. Preferably, anti-VISTA is formulated into an aqueous solution and administered by infusion.

[0169] The formulation of the present invention can be conveniently present in a unit dosage form, each dose containing a predetermined amount of anti-VISTA or its antigen-binding fragment. Such units may contain, for example, but not limited to, 5 mg to 5 g, for example, 10 mg to 1 g or 20 to 50 mg. The pharmaceutically acceptable carrier for the present disclosure may take a variety of forms, depending on, for example, the condition to be treated or the route of administration.

[0170] The formulation disclosed herein can be prepared by mixing the antibody with the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer commonly used in the art (all of which are referred to herein as "carriers", e.g., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants and other various additives) for storage as a lyophilized formulation or an aqueous solution. See Remington's Pharmaceutical Sciences, 16th edition (Osol, 1980). Such additives must be nontoxic to the receptor at the dosage and concentration used. Preferably, the formulation disclosed herein is a liquid formulation. More preferably, the liquid formulation disclosed herein is an aqueous formulation. Still more preferably, the liquid formulation disclosed herein is an aqueous composition, wherein the aqueous carrier is distilled water.

[0171] Stability of formulation

[0172] In one aspect, the formulation disclosed herein stabilizes an anti-VISTA antibody or a fragment thereof. Preferably, the formulation disclosed herein prevents aggregation of an anti-VISTA antibody or a fragment thereof. In a specific example, the formulation disclosed herein comprises an anti-VISTA antibody comprising a heavy chain sequence of SEQ ID NO: 21 and a light chain sequence of SEQ ID NO: 22, such as an anti-VISTA antibody or a fragment thereof disclosed in WO 2022 / 229469.

[0173] The present disclosure provides a stable liquid formulation comprising an anti-VISTA antibody disclosed herein. The stability of the antibody can be assessed by the degree of aggregation, degradation or fragmentation compared with the reference preparation comprising a reference antibody, such as by HPSEC, reverse phase chromatography, static light scattering (SLS), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea unfolding technology, intrinsic tryptophan fluorescence, differential scanning calorimetry and / or ANS binding technology. The overall stability of the preparation comprising an antibody (including its antibody fragment) can also be assessed by various immunological assays, including, for example, ELISA and radioimmunoassay using separated antigen molecules. In addition, the stability of the preparation comprising an antibody can also be measured using various assays intended to measure antibody functional characteristics, for example, intended to measure antigen binding affinity, in vitro ADCC activity, in vivo consumption activity, in vitro CDC activity.

[0174] Preferably, the formulations disclosed herein are stable when stored at about 5° C. for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least 48 months. More preferably, the formulations disclosed herein are stable when stored at about 5° C. for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.

[0175] In one embodiment, a formulation disclosed herein comprises an anti-VISTA antibody having a VISTA binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the VISTA binding activity of a reference antibody, wherein the formulation is stored at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least 48 months. More preferably, the formulations disclosed herein comprise an anti-VISTA antibody having a VISTA binding activity that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the VISTA binding activity of the reference antibody, wherein the formulation is stored at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.

[0176] In one embodiment, the formulation disclosed herein comprises an anti-VISTA antibody, wherein at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least 48 months are stored at about 5 ° C. After being determined by SEC, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, or less than 10% of the antibodies form aggregates. More preferably, the formulation disclosed herein comprises an anti-VISTA antibody, wherein after being stored at about 5 ° C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, or less than 10% of the antibodies form aggregates by SEC.

[0177] In one embodiment, the formulation disclosed herein comprises an anti-VISTA antibody, wherein the antibody fragmentation is less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, or less than 10% after storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least 48 months. More preferably, the formulation disclosed herein comprises an anti-VISTA antibody, wherein the antibody fragmentation is less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, or less than 10% after storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years, by SEC determination.

[0178] In one embodiment, a formulation disclosed herein comprises an anti-VISTA antibody, wherein less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, or less than 10% of the antibody is not the full-length antibody (H2L2) as determined by CE-SDS after storage at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least 48 months. More preferably, the formulations disclosed herein comprise an anti-VISTA antibody, wherein less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 7%, or less than 10% of the antibody is not the full-length antibody (H2L2) as determined by CE-SDS after storage at about 5°C for at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 6 years, at least about 7 years, at least about 8 years, at least about 9 years, at least about 10 years, at least about 11 years, or at least about 12 years.

[0179] Preparations with high antibody concentrations may have a shorter shelf life, and the formulated antibodies may lose biological activity due to chemical and physical instabilities during storage. Among them, aggregation, deamidation and oxidation are the most common causes of known antibody degradation. Specifically, aggregation can lead to enhanced immune responses in patients, thereby causing safety issues. Therefore, aggregation must be minimized or prevented.

[0180] Preferably, the formulations of the present disclosure maintain enhanced aggregation properties upon storage, e.g., storage at 5°C for extended periods of time (e.g., but not limited to, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years).

[0181] A variety of methods are known in the art that can be used to determine the degree of aggregation and / or the type and / or size of aggregates present in a protein formulation (e.g., an antibody formulation of the present disclosure), including but not limited to size exclusion chromatography (SEC), high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), urea-induced protein unfolding techniques, endogenous tryptophan fluorescence, differential scanning calorimetry, and 1-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques. For example, size exclusion chromatography (SEC) can be performed to separate molecules according to molecular size, passing the molecules through a column filled with an appropriate resin, and larger molecules (e.g., aggregates) will elute before smaller molecules (e.g., monomers). The molecules are typically detected by UV absorbance at 280 nm, and the molecules can be collected for further characterization. High pressure liquid chromatography columns are typically used for SEC analysis (HP-SEC). Specific SEC methods are described in detail in the examples herein. Alternatively, analytical ultracentrifugation (AUC) can be used. AUC is an orthogonal technique for determining the sedimentation coefficient of macromolecules in liquid samples (reported in Svedberg, S). Like SEC, AUC is able to separate and detect antibody fragments / aggregates from monomers and further provide information about the molecular mass. Protein aggregation in the formulation can also be characterized by a particle counter using a Coulter counter or turbidity measurement using a nephelometer. Turbidity is a measure of the amount of light scattered by particles in a solution and can therefore be used as a general indicator of protein aggregation. In addition, non-reducing polyacrylamide gel electrophoresis (PAGE) or capillary gel electrophoresis (CGE) can be used to characterize the aggregation and / or fragmentation state of antibodies or fragments thereof in the formulations of the present disclosure.

[0182] Anti-VISTA Antibodies Used in Formulations

[0183] Immune checkpoints play a crucial role in maintaining self-tolerance and limiting immune-mediated tissue damage under physiological conditions. VISTA is a type I transmembrane protein that belongs to the B7-related immunoglobulin superfamily and is highly expressed in the hematopoietic compartment. VISTA acts as both a ligand and a receptor, inhibiting CD4 + and CD8 + T cell activation is negatively regulated by T cell proliferation and pro-inflammatory cytokine (e.g., IFNγ, TNFα, or IL-2) production.

[0184] WO 2016 / 094837 describes a monoclonal antibody Ab3 that can inhibit VISTA immunosuppression and thereby enhance anti-tumor immune response. The CDRs of the antibody are represented by SEQ ID NOs: 3-8, V H and V LThey are represented by SEQ ID NO: 9 and SEQ ID NO: 10 respectively. The complete heavy chain of Ab3 has the sequence represented by SEQ ID NO: 11, and the complete light chain of Ab3 has the sequence represented by SEQ ID NO: 12.

[0185] The complete sequence of antibody Ab3 shows that it contains 11 potential deamidation sites. Although all of these sites are predicted to be effective deamidation sites, the inventors found that only one of them actually undergoes deamidation, namely the Asn residue at position 55 in the heavy chain CDR2. Surprisingly, replacing the Asn with an Asp residue does not affect Ab3 binding to its target, which is in sharp contrast to the teachings of the prior art, in which similar mutations in CDRs result in a 400-fold decrease in affinity for the corresponding CD52 antigen (Liu et al., 2022). In addition, the mutated antibody retains the ability to inhibit the immunosuppressive activity of VISTA, because the mutated antibody is able to block the interaction between VISTA and each of its two binding partners, PSG-L1 and VSIG3, which results in the inhibition of T cell function. Therefore, the mutated antibody inhibits tumor proliferation in vivo.

[0186] In a first aspect, the present disclosure provides a novel anti-VISTA antibody, wherein Asp is substituted for Asn in the heavy chain CDR2 comprised by the antibody.

[0187] As used herein, anti-VISTA monoclonal antibodies include, but are not limited to: synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above. Anti-VISTA monoclonal antibodies may be human or non-human. Examples of non-human anti-VISTA antibodies include, but are not limited to, those antibodies of mammalian origin (e.g., monkeys, rodents, goats, and rabbits). Since each structure of a human antibody is derived from humans, it is less likely to have an immune response than conventional humanized antibodies or mouse antibodies, so it has the advantage of not causing any undesirable immune response when administered to humans. Therefore, it can be used very advantageously as a therapeutic antibody. Therefore, the anti-VISTA monoclonal antibodies for human therapeutic use are preferably humanized or fully human. More preferably, they are humanized.

[0188] The antibodies disclosed herein are antibodies that have substantially the same affinity for antigens as antibody Ab3. The term "affinity" refers to the property of specifically recognizing and binding to a specific antigenic site, and the specificity and high affinity of an antibody for an antigen are important factors in an immune response. In the present case, the affinity of the antibodies disclosed in the present invention can be determined by competitive ELISA. In addition to this method, various methods for measuring affinity for antigens can be used, and surface plasmon resonance technology is an example of these methods.

[0189] To the extent that VISTA can be specifically recognized, the monoclonal antibodies disclosed herein may include not only the sequences of the anti-VISTA antibodies of the present invention described in this specification, but also their biological equivalents, wherein the biological equivalents exhibit improved binding affinity and / or other biological properties of the antibodies. For example, in order to further improve the binding affinity and / or other biological properties of the antibodies, additional changes may be made to the amino acid sequence of the antibodies. For example, deletions, insertions and / or substitutions of the antibody amino acid sequences are included in these modifications. Those modifications of amino acids are based on the relative similarity (e.g., hydrophobicity, hydrophilicity, charge, size, etc.) between the amino acid side chain substituents. Based on the analysis of the size, shape and type of the amino acid side chain substituents, it was found that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine are similar in size; phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, it can be said that biologically, arginine, lysine and histidine; alanine, glycine and serine; phenylalanine, tryptophan and tyrosine are functional equivalents.

[0190] In one embodiment, the anti-VISTA monoclonal antibodies described herein may be in the form of full-length antibodies, multi-chain or single-chain antibodies, fragments of such antibodies that selectively bind to VISTA (including but not limited to Fab, Fab', (Fab')2, Fv and scFv), surrobody (including surrogate light chain constructs), single domain antibodies, humanized antibodies, camelized antibodies, etc. They may also be or be derived from any isotype, including, for example, IgA (eg, Ig11 or IgA2), IgD, IgE, IgG (eg, IgG1, IgG2, IgG3 or IgG4) or IgM. In some embodiments, the anti-VISTA antibody is IgG (eg, IgG1, IgG2, IgG3 or IgG4). In one embodiment, the antibody further comprises a human constant region. In a further embodiment, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, IgG4. In a further specific embodiment, the human constant region is IgG1. Furthermore, the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and it has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2) as subclasses. The light chain constant region has kappa (κ) and lambda (λ) types.

[0191] In the context of the present disclosure, antibodies comprising a human IgG1 constant region are particularly preferred. They not only bind to VISTA with the same affinity as antibody Ab3, but are also able to suppress the immunosuppressive effects of VISTA. Surprisingly, this activity requires the effector function of the antibody, which has never been recorded for anti-VISTA antibody Ab3.

[0192] Preferably, the anti-VISTA antibody disclosed herein comprises a heavy chain of sequence SEQ ID NO: 21 and a light chain of sequence SEQ ID NO: 22.

[0193] The antibody is more stable and more homogeneous than antibody Ab3 because it contains Asp at position 55 and therefore does not undergo deamidation. In addition, the antibody has the same affinity as antibody Ab3, suppressing the immunosuppressive activity of VISTA. In particular, this inhibition is the result of the destruction of the interaction between VISTA and each of its binding partners (PSG-L1 and VSIG-3). On the contrary, there is no sign that antibody Ab3 interferes with these interactions. In addition, the inhibition of VISTA immunosuppression surprisingly requires the effector function of the antibody disclosed herein.

[0194] The sequences of Ab1 and Ab3 are shown in Table 2 below.

[0195] Table 2

[0196]

[0197]

[0198] Preferably, the antibody of the formulation of the present invention is an anti-VISTA antibody or a fragment thereof disclosed in WO 2022 / 229469.

[0199] Nucleic Acids and Expression Systems

[0200] The present disclosure encompasses polynucleotides encoding immunoglobulin light and heavy chain genes of antibodies (especially anti-VISTA antibodies), vectors comprising such nucleic acids, and host cells capable of producing antibodies of the present disclosure. Also provided herein are polynucleotides hybridized with polynucleotides encoding antibodies or modified antibodies provided herein under highly stringent, moderately or less stringent hybridization conditions (e.g., as defined above).

[0201] In the first aspect, as described above, the present disclosure relates to one or more polynucleotides encoding antibodies (especially antibodies or fragments thereof that can specifically bind to VISTA). The present disclosure particularly provides polynucleotides encoding the heavy chain and / or light chain of the anti-VISTA antibodies disclosed herein. More specifically, in certain embodiments, the nucleic acid molecules provided herein include nucleic acid sequences 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 a full-length antibody, an antibody heavy chain and / or light chain or a single-chain antibody provided herein), or consisting thereof.

[0202] For example, the polynucleotide encodes three heavy chain CDRs of the anti-VISTA antibodies described herein. For example, the polynucleotide encodes three light chain CDRs of the anti-VISTA antibodies described herein. For example, the polynucleotide encodes three heavy chain CDRs and three light chain CDRs of the anti-VISTA antibodies described herein. Another example provides a pair of polynucleotides, wherein the first polynucleotide encodes three heavy chain CDRs of the anti-VISTA antibodies described herein; and the second polynucleotide encodes three light chain CDRs of the same anti-VISTA antibodies described herein.

[0203] In another case, the polynucleotide encodes the heavy chain variable region of the anti-VISTA antibody described herein. For example, the polynucleotide encodes the light chain variable region of the anti-VISTA antibody described herein. For example, the polynucleotide encodes the heavy chain variable region and the light chain variable region of the anti-VISTA antibody described herein. Another example provides a pair of polynucleotides, wherein the first polynucleotide encodes the heavy chain variable region of the anti-VISTA antibody described herein; the second polynucleotide encodes the light chain variable region of the same anti-VISTA antibody described herein.

[0204] In one embodiment, the polynucleotide encodes the heavy chain of an anti-VISTA antibody described herein. In one embodiment, the polynucleotide encodes the light chain of an anti-VISTA antibody described herein. In one embodiment, the polynucleotide encodes the heavy chain and light chain of an anti-VISTA antibody described herein. Another embodiment provides a pair of polynucleotides, wherein the first polynucleotide encodes the heavy chain of an anti-VISTA antibody described herein; the second polynucleotide encodes the light chain of the same anti-VISTA antibody described herein.

[0205] In one embodiment, a polynucleotide encoding the heavy chain of the above-mentioned anti-VISTA antibody is provided. Preferably, the heavy chain comprises three heavy chain CDRs of sequence SEQ ID NO: 13-15. More preferably, the heavy chain comprises a heavy chain containing a variable region of sequence SEQ ID NO: 19. Even more preferably, the heavy chain has a sequence represented by SEQ ID NO: 21.

[0206] In another embodiment, the polynucleotide encodes a light chain of an anti-VISTA antibody described herein. Preferably, the light chain comprises three light chain CDRs of sequences 16-18. More preferably, the light chain comprises a light chain containing a variable region of sequence SEQ ID NO: 20. Even more preferably, the light chain has a sequence represented by SEQ ID NO: 222.

[0207] Due to codon degeneracy or in consideration of preferred codons in an organism in which the light and heavy chains of human antibodies or fragments thereof are to be expressed, the polynucleotides encoding the light and heavy chains of the monoclonal antibodies of the present invention or their antigen-binding fragments may have various variations in the coding region without changing the amino acid sequence of the light and heavy chains of the antibodies expressed from the coding region, and may be variously changed or modified in regions outside the coding region without affecting gene expression. It will be readily understood by those skilled in the art that those variant genes also fall within the scope of the present invention. That is, as long as the polynucleotides of the present invention encode proteins with equivalent activity, one or more nucleic acid bases may be changed by substitution, deletion, insertion or a combination thereof, which also fall within the scope of the present invention. The sequence of the polynucleotide may be single-stranded or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.

[0208] According to the present invention, multiple expression systems can be used to express antibody of the present invention.On the one hand, such expression system representative is carrier (it can produce target encoding sequence and purify subsequently), and another representative is the cell that can in situ express IgG antibody when using suitable nucleotide encoding sequence transient transfection.

[0209] The present disclosure provides a vector comprising the above-mentioned polynucleotides. In one embodiment, the vector contains a polynucleotide encoding an anti-VISTA antibody heavy chain of interest. In another embodiment, the polynucleotide encodes an anti-VISTA antibody light chain of interest. In another embodiment, the polynucleotide encodes the heavy chain and light chain of an anti-VISTA antibody of interest. In yet another embodiment, a pair of polynucleotides is provided, wherein the first polynucleotide encodes the heavy chain of an anti-VISTA antibody of interest, and the second polynucleotide encodes the light chain of the same anti-VISTA antibody of interest.

[0210] The present disclosure also provides vectors comprising polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments and probes thereof.

[0211] In order to express the heavy chain and / or light chain of the anti-VISTA antibody of interest, the polynucleotide encoding the heavy chain and / or light chain is inserted into an expression vector so that the gene is operably linked to the transcription and translation sequence. In a preferred embodiment, these polynucleotides are cloned into two vectors.

[0212] "Operably linked" sequences include expression control sequences that are continuous with the gene of interest, and expression control sequences that control the gene of interest in trans or at a distance. As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary for achieving expression and processing of the coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when necessary. The nature of such control sequences varies depending on the host organism: in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0213] The polynucleotides of the present invention and vectors comprising these molecules can be used to transform suitable host cells. As used herein, the term "host cell" is intended to refer to cells into which a recombinant expression vector has been introduced to express an anti-VISTA antibody of interest. It should be understood that such terms are intended not only to refer to specific test cells, but also to the offspring of such cells. Because some modifications may occur in offspring due to mutations or environmental influences, such offspring may actually be different from parental cells, but are still included in the scope of the term "host cell" as used herein.

[0214] Transformation can be carried out by any known method for introducing a polynucleotide into a cellular host. Such methods are well known to those skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, gene gun injection, and direct microinjection of DNA into the nucleus.

[0215] Host cells can be co-transfected with one or more expression vectors. For example, host cells can be transfected with vectors encoding both the heavy chain and the light chain of the anti-VISTA antibody of interest as described above. Alternatively, host cells can be transformed with a first vector encoding the heavy chain of the anti-VISTA antibody of interest and a second vector encoding the light chain of the antibody. Mammalian cells are commonly used to express recombinant therapeutic immunoglobulins, particularly for expressing fully recombinant antibodies. For example, mammalian cells (such as HEK293 or CHO cells) are combined with vectors containing expression signals (such as expression signals carrying the major intermediate early gene promoter elements from human cytomegalovirus), which are effective systems for expressing humanized anti-VISTA antibodies of the present invention (Foecking et al., 1986, Gene 45: 101; Cockett et al., 1990, Bio / Technology 8: 2).

[0216] In addition, the host cell that can select to regulate the expression of insertion sequence or modify and process gene product in the ad hoc manner of expectation.Such modification (such as glycosylation) and processing of protein product may be very important to the function of protein.Different host cells have the post-translational processing and the modified characteristics and specific mechanisms of protein and gene product.Select appropriate cell line or host system to ensure the correct modification and processing of the antibody of interest expressed.Therefore, eukaryotic host cells with cellular mechanisms for appropriately processing primary transcript and glycosylated gene product can 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 can be obtained from public deposits, such as the National Microbiological Culture Collection in Paris, France or the American Type Culture Collection in Manassas, Virginia, USA).

[0217] In order to produce recombinant proteins for a long time and in high yield, stable expression is preferred. In one embodiment of the present invention, a cell line of stably expressed antibodies can be engineered. The host cells are transformed with DNA and selectable markers under the control of suitable expression regulatory elements (including promoters, enhancers, transcription terminators, polyadenylation sites and other sequences known to those skilled in the art), without using expression vectors containing viral replication origins. After introducing foreign DNA, engineered cells can be allowed to grow in enriched medium for one to two days and then moved to selective medium. The selectable markers on the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into the chromosome and amplify into a cell line. Other methods for constructing stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, the transforming DNA under the control of appropriate expression regulatory elements (including promoters, enhancers, transcription terminators, polyadenylation sites and other appropriate sequences) is integrated into the host cell genome at a specific target site that has been previously cut (Moele et al., Proc. Natl. Acad. Sci. USA, 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).

[0218] Many selection systems can be used according to the present invention, including but not limited to 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 in the presence of methionine sulfoximine (Adv Drug Del Rev, 58:671, 2006, and the website or literature of Lonza Group Ltd.), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes in tk, hgprt or aprt cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr (Wigler et al., Proc Natl Acad Sci USA 77:357, 1980), which confers resistance to methotrexate; gpt (Mulligan et al., Proc Natl Acad Sci USA 78:2072, 1981), which confers resistance to mycophenolic acid; neo (Wu et al., Biotherapy 3:87, 1991), which confers resistance to the aminoglycoside G-418; and hygro (Santerre et al., Gene 30:147, 1984), which confers resistance to hygromycin. Methods known in the art of recombinant DNA technology can be routinely applied to select desired recombinant clones, such methods being described, for example, in Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons (1993). Expression levels of antibodies can be increased by vector amplification. When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture will increase the number of copies of the marker gene. Since the amplified region is associated with the gene encoding the IgG antibody of the present invention, the production of the antibody will also increase (Crouse et al., Mol Cell Biol 3:257, 1983). Alternative methods for expressing the genes of the present invention exist, which are known to those skilled in the art. For example, a modified zinc finger protein capable of binding to an expression control element upstream of the gene of the present invention can be engineered; expressing the engineered zinc finger protein (ZFN) in the host cell of the present invention results in increased protein production (see, e.g., Reik et al., Biotechnol. Bioeng., 97 (5): 1180-1189, 2006). In addition, ZFN can stimulate DNA integration into a predetermined genomic location, resulting in efficient site-specific gene addition (Moehle et al., Proc Natl Acad Sci USA, 104:3055, 2007).

[0219] The anti-VISTA antibody of interest can be prepared by growing the culture of the transformed host cells under the culture conditions necessary for expressing the desired antibody. The expressed antibody can then be purified from the culture medium or cell extract. The soluble form of the anti-VISTA antibody of interest can be recovered from the culture supernatant. The antibody can then be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly by affinity of protein A to Fc, etc.), centrifugation, solubility differences, or by any other standard technique for purifying protein. Suitable purification methods will be apparent to those of ordinary skill in the art.

[0220] Therefore, another aspect of the present invention relates to a method for producing an antibody described herein (e.g., an anti-VISTA antibody), the method comprising the following steps:

[0221] a) growing the above host cell in a culture medium under suitable culture conditions; and

[0222] b) recovering the antibody (e.g., anti-VISTA antibody) from the culture medium or from the cultured cells.

[0223] The antibodies obtained by culturing the transformants can be used in an unpurified state. Impurities can be removed by various other common methods such as centrifugation or ultrafiltration, and the resulting product can be dialyzed, salt precipitated, chromatographed, etc., wherein the methods can be used alone or in combination. Among them, affinity chromatography is the most widely used, including ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc.

[0224] Effective amount

[0225] Anti-VISTA antibodies and conjugates thereof, optionally in combination with immune checkpoint inhibitors, will generally be used in an effective amount to achieve the desired result, such as in an amount effective to treat cancer in a subject in need thereof. Pharmaceutical compositions comprising anti-VISTA antibodies (or their conjugates) and / or immune checkpoint inhibitors can be administered to a patient (e.g., a human subject) in a therapeutically effective dose.

[0226] Determining the effective amount is entirely within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. The toxicity and therapeutic efficacy of the compound or conjugate can be determined by standard pharmaceutical procedures in cell cultures and experimental animals. The effective amount of this combination or other therapeutic agent to be administered to a subject will depend on the stage, category and state of the disease (e.g., cancer), and the characteristics of the subject such as general health, age, sex, body weight and drug tolerance. The effective amount of the therapeutic agent of the present invention to be administered or the combination will also depend on the route of administration and dosage form. Dosage and interval can be adjusted individually to provide a plasma level of the active compound sufficient to maintain the desired therapeutic effect.

[0227] The amount of anti-VISTA antibody or antigen-binding fragment administered will depend on a variety of factors, including the nature and staging of the disease (e.g., cancer) being treated, the form, route and site of administration, the treatment regimen (e.g., whether the therapeutic agent is used in combination with an immune checkpoint inhibitor), the age and condition of the specific subject being treated, and the sensitivity of the patient treated with the antibody or conjugate. A person skilled in the art can easily determine a suitable dose. Finally, the doctor will determine a suitable dosage for use. The dose can be repeated at a suitable frequency. If side effects occur, the amount and / or frequency of the dose can be changed or reduced according to common clinical practice. Appropriate dosage and treatment regimens can be established by monitoring the progress of treatment using conventional techniques known to those skilled in the art.

[0228] The effective dose can be preliminarily estimated by in vitro assays. For example, an initial dose for an animal can be formulated to reach a circulating blood or serum concentration of an anti-VISTA antibody, the concentration being equal to or higher than the binding affinity of the antibody to VISTA measured in vitro. Considering the bioavailability of a particular antibody, the circulating dose reaching such circulating blood or serum concentrations is completely within the capabilities of the technician. For guidance, the reader is referred to Fingl & Woodbury, "General Principles" in Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, latest edition, Pagamonon Press and references cited therein. The initial dose can be estimated from in vivo data (such as animal models). Animal models that can be used to test the efficacy of compounds for treating specific diseases (such as cancer) are generally well known in the art. Ordinary technicians can routinely adjust such information to determine a dose suitable for administration to humans.

[0229] The effective dose of an anti-VISTA antibody as described herein can be within the range of about 0.001 to about 75 mg / kg per single (e.g., bolus) administration, multiple administrations, or continuous administration, or reach a serum concentration of 0.01-5000 μg / ml serum concentration per single (e.g., bolus) administration, multiple administrations, or continuous administration, or depending on the disorder being treated, the route of administration, and the age, weight, and disorder of the subject, any effective range or value therein. In a certain embodiment, the range of each dose can be about 0.5 μg to about 50 μg per kilogram of body weight, such as about 3 μg to about 30 μg per kilogram of body weight.

[0230] The amount, frequency and duration of administration will depend on various factors, such as the patient's age, weight and disease condition. The treatment regimen for administration can last for 2 weeks to indefinitely, 2 weeks to 6 months, 3 months to 5 years, 6 months to 1 or 2 years, 9 months to 18 months, etc. Optionally, the treatment regimen provides repeated administration, for example, once a day, twice a day, every two days, three days, five days, one week, two weeks, three weeks or once a month. Repeated administration can be performed with the same dose or different doses. It can be repeatedly administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. The anti-VISTA antibody or its antigen-binding fragment (optionally combined with an immune checkpoint inhibitor) of a therapeutically effective amount can be administered in a single dose or during the course of the treatment regimen, for example, in a week, two weeks, three weeks, one month, three months, six months, one year or more.

[0231] Methods of administering antibody preparations

[0232] The present disclosure provides a method for preventing, treating and / or managing a disease (e.g., a disease or disease mediated by abnormal expression and / or activity of VISTA) by administering an effective amount of a liquid preparation of the present disclosure to a subject. Various delivery systems are known and can be used to administer the liquid preparation disclosed herein. The method of administering the antibody liquid preparation of the present invention includes, but is not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, topical administration and mucosal administration (e.g., but not limited to intranasal and oral routes). In a specific example, the liquid preparation of the present invention is administered intramuscularly, intravenously or subcutaneously. In one example, the liquid preparation disclosed herein is administered subcutaneously. The preparation can be administered by any convenient route, for example, by infusion or bolus injection, by absorption of epithelial or mucosal lining (lining) (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents. Administration can be systemic or local.

[0233] The present disclosure also provides packaging of the liquid preparation of the present disclosure in a sealed container, such as an ampoule or a pouch, wherein the quantity of the antibody (including its antibody fragment) is indicated. In one embodiment, the liquid preparation of the present disclosure is in a sealed container, wherein the quantity and concentration of the antibody (including its antibody fragment) is indicated. In one embodiment, a liquid formulation of the present disclosure is provided in a sealed container, comprising about 1 mg / mL, about 5 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL or about 100 mg / mL of an anti-VISTA antibody or fragment thereof, such as the antibody described in WO 2022 / 229469, in an amount of about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 15 ml or about 20 ml. Specifically, the liquid formulation disclosed herein can be provided in a sealed container, comprising at least about 1 mg / mL, at least about 5 mg / mL, at least about 10 mg / mL, at least about 15 mg / mL, at least about 20 mg / mL, at least about 25 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, at least about 80 mg / mL, at least about 90 mg / mL or at least about 100 mg / mL of an anti-VISTA antibody or a fragment thereof, such as WO 2022 / 229469 for intravenous injection, and at least about 1 mg / mL, at least about 5 mg / mL, at least about 10 mg / mL, at least about 15 mg / mL, at least about 20 mg / mL, at least about 25 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, at least about 80 mg / mL, at least about 90 mg / mL or at least about 100 mg / mL of an anti-VISTA antibody or fragment thereof, such as an antibody described in WO 2022 / 229469, for repeated subcutaneous administration.

[0234] The amount of the liquid preparation of the present invention that is effective in preventing, treating and / or managing diseases or patients characterized by abnormal expression and / or activity of VISTA can be determined by standard clinical techniques well known in the art or described herein. The precise dosage used in the preparation also depends on the severity of the route of administration and VISTA-mediated diseases, and should be determined according to the judgment of the clinician and the situation of each patient. The effective dose can be inferred from the dose-response curve obtained from the in vitro or animal model test system as described above.

[0235] It will be appreciated that the anti-VISTA antibodies or antigen-binding fragments thereof of the present invention may advantageously be administered with a second therapeutic agent (e.g., an immune checkpoint inhibitor as described below).

[0236] The present disclosure further relates to a pharmaceutical composition comprising at least:

[0237] i) an anti-VISTA antibody, an antigen-binding fragment thereof, or a conjugate thereof as disclosed herein, and

[0238] ii) a second therapeutic agent, such as an immune checkpoint inhibitor as described below,

[0239] As a combination product for simultaneous, separate or sequential use.

[0240] As used herein, "simultaneous use" means that the two compounds in the composition of the present invention are administered in a single and identical pharmaceutical form.

[0241] As used herein, "separate use" means that the two compounds in the composition of the present invention are administered simultaneously in different pharmaceutical forms.

[0242] As used herein, "sequential use" means that the two compounds of the combination according to the present invention are administered consecutively, each in a different pharmaceutical form.

[0243] Compositions of anti-VISTA antibodies (or their antigen-binding fragments or their conjugates) and second therapeutic agents (such as, for example, immune checkpoint inhibitors) can be administered alone, as a mixture of one or more anti-VISTA antibodies (or their antigen-binding fragments or their conjugates) and / or one or more second therapeutic agents (such as immune checkpoint inhibitors as described below), mixed with other agents that can be used to treat cancer or other adjuvant therapies for cancer, or administered in combination. Examples of suitable combinations and adjuvant therapies are provided below.

[0244] The present disclosure encompasses pharmaceutical kits containing anti-VISTA antibodies (or antigen-binding fragments thereof or conjugates thereof) and are described herein. A pharmaceutical kit is a package comprising an anti-VISTA antibody (e.g., in lyophilized form or as an aqueous solution) and one or more of the following:

[0245] A second therapeutic agent, e.g., an immune checkpoint inhibitor as described below;

[0246] a device for administering the anti-VISTA antibody, such as a pen, needle, and / or syringe; and

[0247] (If the inhibitor is in the form of an antibody) Pharmaceutical grade water or buffer for resuspending the antibody.

[0248] Each unit dose of an anti-VISTA antibody (or its antigen-binding fragment or its conjugate) can be packaged separately, and the kit can contain one or more unit doses (e.g., two unit doses, three unit doses, four unit doses, five unit doses, eight unit doses, ten unit doses or more). In a specific embodiment, the one or more unit doses are each contained in a syringe or pen.

[0249] Treatment

[0250] The anti-VISTA antibodies or their antigen-binding fragments described herein can promote T cell activation, including T cell proliferation and cytokine production, especially by the activation of the effector function of the antibody. Therefore, the preparation of the present invention comprising the anti-VISTA antibodies or their antigen-binding fragments described herein can be used in a method of inducing an immune response, wherein the method comprises administering to a patient in need thereof a preparation comprising an effective amount of an anti-VISTA antibody or its antigen-binding fragment disclosed herein. Specifically, the preparation of the present invention comprising an anti-VISTA antibody or its antigen-binding fragment is used to induce an immune response. The present disclosure also relates to the use of the preparation of the present invention comprising an anti-VISTA antibody or its antigen-binding fragment for the preparation of a drug for inducing an immune response. In a specific embodiment of the method described herein, the induction of an immune response requires the effector function of the activated antibody.

[0251] The preparations of the present invention comprising an anti-VISTA antibody or an antigen-binding fragment thereof as described herein can be used in a method of inducing an immune response, wherein the induction of the immune response includes suppressing VISTA-mediated immunosuppression, wherein the method comprises administering an effective amount of a preparation comprising an anti-VISTA antibody or an antigen-binding fragment thereof to a patient in need thereof. Therefore, preferably, a preparation comprising an anti-VISTA antibody or an antigen-binding fragment thereof as described herein can be used in a method of inducing an immune response, wherein the induction of the immune response includes promoting T cell activation, and wherein the method comprises administering an effective amount of a preparation comprising an anti-VISTA antibody or an antigen-binding fragment thereof to a patient in need thereof. T cell activation can specifically include stimulating T cell proliferation (e.g., CD4 + T cell proliferation and / or CD8 + T cell proliferation) and / or cytokine production, particularly pro-inflammatory cytokines, such as INF-γ, IL-2 and / or TNF-α.

[0252] The ability of the anti-VISTA antibodies of the invention to induce an immune response (e.g., by promoting T cell activation, particularly by inducing CD4 + T cell proliferation, CD8 + T cell proliferation, CD4 + T cells produce cytokines and / or CD8 +T cells produce cytokines), thereby inhibiting VISTA-mediated immunosuppression, making it useful for treating a variety of VISTA-mediated conditions, including cancer. Therefore, therapeutic intervention of the VISTA inhibition pathway represents a promising method for regulating inflammation and T cell-mediated immunity to treat a variety of VISTA-mediated diseases, especially cancer. In fact, the antibodies disclosed herein inhibit tumor growth in vivo.

[0253] Therefore, the preparation comprising the anti-VISTA antibody or its antigen binding fragment as described herein can be used for treating VISTA-mediated diseases, especially cancer methods, wherein the method comprises administering an effective amount of a preparation comprising an anti-VISTA antibody or its antigen binding fragment to a patient in need thereof. Therefore, the preparation comprising the anti-VISTA antibody or its antigen binding fragment as described herein can be used for treating VISTA-mediated diseases, especially cancer methods, wherein the treatment includes suppressing VISTA-mediated immunosuppression, and wherein the method comprises administering an effective amount of a preparation comprising an anti-VISTA antibody or its antigen binding fragment to a patient in need thereof. Therefore, preferably, the preparation comprising the anti-VISTA antibody or its antigen binding fragment as described herein can be used for treating VISTA-mediated diseases, especially cancer methods, wherein the treatment includes promoting T cell activation, and wherein the method comprises administering an effective amount of a preparation comprising an anti-VISTA antibody or its antigen binding fragment to a patient in need thereof.

[0254] Therefore, the preparations comprising the anti-VISTA antibodies or antigen-binding fragments thereof described herein can be used in the following methods: for treating VISTA-mediated diseases (especially cancer), inducing CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and / or induce CD8 + T cells produce cytokines, wherein the method comprises administering to a patient in need thereof an effective amount of a formulation comprising an anti-VISTA antibody or an antigen-binding fragment thereof. Preferably, therefore, the formulation comprising an anti-VISTA antibody or an antigen-binding fragment thereof as described herein can be used in a method for treating a VISTA-mediated disease (especially cancer), wherein the treatment comprises inducing CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and / or induce CD8 + T cells produce cytokines, and wherein the method comprises administering to a patient in need thereof an effective amount of a formulation comprising an anti-VISTA antibody or an antigen-binding fragment thereof.

[0255] Surprisingly, the antibodies of the invention require effector function for T cell activation. In contrast, a version of the humanized IgG1 anti-VISTA mAb was engineered to avoid binding to human Fcγ receptors via the N298A mutation (Herbs et al., Nature 515(7528):563–567) and therefore lacks any effector function and cannot induce CD4 + Proliferation, CD8 + Proliferation, unable to induce CD4 + T cell cytokines and CD8 + T cell cytokines. Therefore, the variants of the anti-VISTA antibodies described herein are unable to inhibit tumor proliferation in vivo.

[0256] More preferably, the formulations comprising the anti-VISTA antibodies or antigen-binding fragments thereof described herein can be used in a method for treating VISTA-mediated diseases, especially cancer, wherein the treatment comprises promoting T cell activation by activating the effector function of the antibody, and wherein the method comprises administering an effective amount of a formulation comprising an anti-VISTA antibody or antigen-binding fragment thereof to a patient in need thereof. Even more preferably, the formulations comprising the anti-VISTA antibodies or antigen-binding fragments thereof described herein can be used in a method for treating VISTA-mediated diseases, especially cancer, wherein the treatment comprises inducing CD4 by activating the effector function of the antibody. + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and / or induce CD8 + T cells produce cytokines, wherein the method includes administering an effective amount of a preparation comprising an anti-VISTA antibody or its antigen-binding fragment to a patient in need thereof. The methods of treatment described herein may include administering a preparation comprising an antibody or its antigen-binding fragment specifically binding to VISTA as described herein to a patient in need thereof. Therefore, the VISTA antibodies disclosed herein and their conjugates are used to regulate immunity (especially T cell immunity) to treat VISTA-mediated diseases, especially cancer.

[0257] Therefore, one aspect of the present disclosure relates to a preparation comprising an anti-VISTA antibody or an antigen-binding fragment thereof disclosed herein for treating a VISTA-mediated disease in a patient, especially cancer. Also provided herein is a method for treating a VISTA-mediated disease, especially cancer, in a patient in need thereof, the method comprising administering to the patient a preparation comprising an anti-VISTA antibody or an antigen-binding fragment thereof disclosed herein. The present disclosure also relates to the use of a preparation of an anti-VISTA antibody or an antigen-binding fragment thereof in the preparation of a medicament for treating cancer.

[0258] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an anti-VISTA antibody or an antigen-binding fragment thereof disclosed herein, for treating a VISTA-mediated disease in a patient, especially cancer. Also provided herein is a method for treating a VISTA-mediated disease, especially cancer, in a patient in need thereof, the method comprising administering to the patient a pharmaceutical composition comprising an anti-VISTA antibody or an antigen-binding fragment thereof disclosed herein. The present disclosure also relates to the use of a pharmaceutical composition comprising an anti-VISTA antibody or an antigen-binding fragment thereof disclosed herein in the preparation of a medicament for treating a VISTA-mediated disease, especially cancer.

[0259] Cancers that can be treated with a formulation or pharmaceutical composition comprising an antibody disclosed herein can include any malignant or benign tumor of any organ or body system. Examples include, but are not limited to, breast cancer, digestive / gastrointestinal cancer, endocrine cancer, neuroendocrine cancer, eye cancer, genitourinary cancer, germ cell cancer, gynecological cancer, head and neck cancer, hematologic / blood cancer, musculoskeletal cancer, neural cancer, respiratory / thoracic cancer, bladder cancer, colon cancer, rectal cancer, lung cancer, endometrial cancer, kidney cancer, pancreatic cancer, salivary gland cancer, liver cancer, gastric cancer, peritoneal cancer, testicular cancer, esophageal cancer, prostate cancer, brain cancer, cervical cancer, ovarian cancer, and thyroid cancer. Other cancers can include melanoma, mesothelioma, sarcoma, glioblastoma, blood cancers such as leukemia, myeloma, and lymphoma, as well as any cancer described herein. In some embodiments, solid tumors are infiltrated by bone marrow cells and / or T cells. In some embodiments, cancer is leukemia, lymphoma, myelodysplastic syndrome, mesothelioma, and / or myeloma. In some embodiments, cancer can be any kind or type of leukemia, including lymphocytic leukemia or myeloid leukemia, such as, for example, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid (myeloid) leukemia (AML), chronic granulocytic leukemia (CML), hairy cell leukemia, T cell prolymphocytic leukemia, large granular lymphocytic leukemia or adult T cell leukemia. In some embodiments, lymphoma is histiocytic lymphoma, follicular lymphoma or Hodgkin's lymphoma, and in some embodiments, cancer is multiple myeloma. In some embodiments, cancer is a solid tumor, such as melanoma or bladder cancer. In a specific embodiment, cancer is lung cancer, such as non-small cell lung cancer (NSCLC). The present invention also provides a method for regulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, the malignant disease including but not limited to at least one of the following: leukemia, acute leukemia, acute lymphocytic leukemia (ALL), B cells, T cells or FAB ALL, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal cancer, pancreatic cancer, nasopharyngeal carcinoma, malignant histiocytosis, malignant paraneoplastic syndrome / hypercalcemia, solid tumors, adenocarcinomas, sarcomas, malignant melanomas, hemangiomas, metastatic diseases, cancer-related bone resorption, cancer-related bone pain, etc. In some embodiments, the solid tumor is infiltrated by bone marrow cells and / or T cells. In a specific embodiment, the solid tumor is lung cancer, such as non-small cell lung cancer (NSCLC). In another embodiment, the solid tumor is mesothelioma.

[0260] Preferably, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer (e.g., leukemia, lymphoma or myeloma), laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer.

[0261] The antibody of the present invention is particularly useful because it can induce an immune response in patients (e.g., cancer patients) suffering from VISTA-mediated diseases, as described in detail above. Therefore, in one embodiment, a preparation comprising an anti-VISTA antibody or its antigen-binding fragment is used to treat a VISTA-mediated disease in a patient, especially cancer, wherein the use includes inducing an immune response in the patient. Also provided herein is a method for treating a VISTA-mediated disease, especially cancer, in a patient in need thereof, the method comprising administering to the patient a preparation comprising an anti-VISTA antibody or its antigen-binding fragment disclosed herein, and inducing an immune response in the patient. The present disclosure also relates to the use of a preparation comprising an anti-VISTA antibody or its antigen-binding fragment in the preparation of a medicament for treating a VISTA-mediated disease, especially cancer, wherein the treatment includes inducing an immune response in the patient.

[0262] In one embodiment, the present disclosure relates to a pharmaceutical composition as disclosed herein, wherein the pharmaceutical composition comprises an anti-VISTA antibody or an antigen-binding fragment thereof of the present invention, for use in treating a VISTA-mediated disease (especially cancer) of a patient, wherein the use includes inducing an immune response in the patient. Also provided herein is a method for treating a VISTA-mediated disease, especially cancer, in a patient in need thereof, the method comprising administering to the patient a pharmaceutical composition comprising an anti-VISTA antibody or an antigen-binding fragment thereof disclosed herein and inducing an immune response in the patient. The present disclosure also relates to the use of a pharmaceutical composition disclosed herein, wherein the pharmaceutical composition comprises an anti-VISTA antibody or an antigen-binding fragment thereof of the present invention, for the preparation of a medicament for treating a VISTA-mediated disease (especially cancer), wherein the treatment includes inducing an immune response in a patient.

[0263] One embodiment provides a preparation comprising an anti-VISTA antibody or its antigen-binding fragment, which is used to induce an immune response in a patient (e.g., a cancer patient) suffering from a VISTA-mediated disease. Also provided herein is a method of inducing an immune response in a patient (e.g., a cancer patient) suffering from a VISTA-mediated disease in need thereof, the method comprising administering to the patient a preparation comprising an anti-VISTA antibody or its antigen-binding fragment disclosed herein. The disclosure also relates to the use of a preparation comprising an anti-VISTA antibody or its antigen-binding fragment in the preparation of a drug for inducing an immune response in a patient (e.g., a cancer patient) suffering from a VISTA-mediated disease.

[0264] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an anti-VISTA antibody disclosed herein or a conjugate thereof, for use in inducing an immune response in a patient (e.g., a cancer patient) suffering from a VISTA-mediated disease. Also provided herein is a method for inducing an immune response in a patient (e.g., a cancer patient) suffering from a VISTA-mediated disease in need thereof, the method comprising administering a pharmaceutical composition comprising an anti-VISTA antibody disclosed herein or a conjugate thereof to the patient. The present disclosure also relates to the use of a pharmaceutical composition comprising an anti-VISTA antibody disclosed herein or a conjugate thereof in the preparation of a drug for inducing an immune response in a patient (e.g., a cancer patient) suffering from a VISTA-mediated disease.

[0265] Thus, the immune response generated by the antibodies disclosed herein includes, but is not limited to: induction of CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and induce CD8 + T cells produce cytokines. Preferably, the antibodies disclosed herein produce effector functions required for immune response including, but not limited to: inducing CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and induce CD8 + T cells produce cytokines.

[0266] In the formulations of the present invention, the anti-VISTA antibody or antigen-binding fragment thereof, or a conjugate thereof, may be mixed with a second therapeutic agent.

[0267] "Therapeutic agent" encompasses biological agents, such as antibodies, peptides, proteins, enzymes, and chemotherapeutic agents. Therapeutic agents also encompass immunoconjugates of cell-binding agents (CBAs) and chemical compounds, such as antibody-drug conjugates (ADCs). The drug in the conjugate can be a cytotoxic agent, such as a cytotoxic agent described herein.

[0268] As used herein, if an anti-VISTA antibody or its antigen-binding fragment or its conjugate and other therapeutic agents are administered to a patient on the same day (e.g., during the same patient visit), they are referred to as continuous administration. Continuous administration can be performed at intervals of 1, 2, 3, 4, 5, 6, 7, or 8 hours. On the contrary, if the anti-VISTA antibody or its antigen-binding fragment or its conjugate of the present disclosure and other therapeutic agents are administered to patients on different days, they are considered to be administered separately, for example, the anti-VISTA antibody or its antigen-binding fragment or its conjugate of the present disclosure and other therapeutic agents can be administered at intervals of 1 day, 2 days or 3 days, one week, 2 weeks, or monthly. In the method disclosed herein, the administration of the anti-VISTA antibody or its antigen-binding fragment or conjugate of the present disclosure can be before or after the administration of other therapeutic agents.

[0269] As a non-limiting example, an anti-VISTA antibody or antigen-binding fragment thereof or conjugate thereof and the other therapeutic agent can be administered simultaneously for a period of time, and then the anti-VISTA antibody or antigen-binding fragment thereof or conjugate thereof of the present disclosure and the other therapeutic agent are administered alternately for a second period of time.

[0270] In the case where the anti-VISTA antibody or its antigen-binding fragment or its conjugate or other therapeutic agent is not sufficient to produce a therapeutic effect when administered in a single amount, the combination therapy disclosed herein can produce a greater than additive or synergistic effect, providing a therapeutic benefit. Therefore, such agents can be administered in lower amounts, reducing the likelihood and / or severity of adverse reactions.

[0271] In a preferred embodiment, the other therapeutic agent is a chemotherapeutic agent. The chemotherapeutic agent is preferably an alkylating agent, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, a chromatin function inhibitor, an antiangiogenic agent, an antiestrogen, an antiandrogen or an immunomodulator.

[0272] As used herein, the term "alkylating agent" refers to any substance that can crosslink or alkylate any molecule, preferably nucleic acid (e.g., DNA) within a cell. Examples of alkylating agents include nitrogen mustards such as dichloromethyldiethylamine, chlorambucil, melphalan, loratadine, pipebutium bromide, prednimustine, disodium phosphate or estramustine; oxazolines such as cyclophosphamide, hexamethylmelamine, trofosfamide, sulfonphosphamide or ifosfamide; aziridine or imine-ethylenes such as thiotepa, triethylamine or azoxystrobin; nitrosoureas such as carmustine, streptozocin, fotemustine or lomustine; alkyl sulfonates such as busulfan, trithiodan or isoprosulfan; triazenes such as dacarbazine; or platinum complexes such as cisplatin, oxaliplatin and carboplatin.

[0273] As used herein, the expression "antimetabolite" refers to a substance that blocks cell growth and / or metabolism by interfering with certain activities, usually DNA synthesis. Examples of antimetabolites include methotrexate, 5-fluorouracil, floxuridine, 5-fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytarabine, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), chlorodeoxyadenosine, 5-azacytidine, gemcitabine, cladribine, deoxycoformycin, and pentostatin.

[0274] As used herein, "antitumor antibiotics" are compounds that can prevent or inhibit DNA, RNA and / or protein synthesis. Examples of antitumor antibiotics include doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin and procarbazine.

[0275] As used herein, "mitotic inhibitors" impede the normal progression of the cell cycle and mitosis. In general, microtubule inhibitors or taxanes (such as paclitaxel and docetaxel) can inhibit mitosis. Vinca alkaloids such as vinblastine, vincristine, vindesine and vinorelbine can also inhibit mitosis.

[0276] As used herein, the term "chromatin function inhibitor" or "topoisomerase inhibitor" refers to a substance that inhibits the normal function of a chromatin modeling protein such as topoisomerase I or topoisomerase II. Examples of chromatin function inhibitors include camptothecin and its derivatives (such as topotecan or irinotecan) for topoisomerase I, and etoposide, etoposide phosphate, and teniposide for topoisomerase II.

[0277] As used herein, the term "anti-angiogenic agent" refers to any drug, compound, substance or agent that inhibits blood vessel growth. Exemplary anti-angiogenic agents include, but are not limited to, razoxin, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, halofuginon, COL-3, neovastat, BMS-275291, thalidomide, CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferon-α, EMD121974, interleukin-12, IM862, angiostatin and vitaxin.

[0278] As used herein, the term "anti-estrogen" or "anti-estrogen agent" refers to any substance that reduces, antagonizes or inhibits the effects of estrogen. Examples of anti-estrogen agents are tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, anastrozole, letrozole and exemestane.

[0279] As used herein, the term "anti-androgen" or "anti-androgen agent" refers to any substance that reduces, antagonizes or inhibits the action of androgens. Examples of anti-androgens are flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride and cimetidine.

[0280] As used herein, an "immunomodulator" is a substance that stimulates the immune system.

[0281] Examples of immunomodulators include interferons, interleukins (such as aldesleukin, OCT-43, denileukin, and interleukin-2), tumor necrosis factors (such as tasonermine), or other immunomodulators such as lentinan, sizofiran, roquinimex, pidotimod, pegademase, thymopentin, poly I:C, or levamisole combined with 5-fluorouracil.

[0282] For more details, those skilled in the art can refer to the Handbook entitled "Traité de chimie thérapeutique, vol. 6, Traité de chimie thérapeutique, vol.

[0283] Mention may also be made of chemical or cytotoxic agents, all kinase inhibitors, such as gefitinib or erlotinib.

[0284] More generally, examples of suitable chemotherapeutic agents include, but are not limited to, 1-dehydrotestosterone, 5-fluorouracil dicarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, doxorubicin, aldesleukin, alkylating agents, allopurinol sodium, hexamethylmelamine, amifostine, anastrozole, anthramycin (AMC), antimitotic agents, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, cupric copper, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), chlorambucil, cisplatin, cladribine, colchicine, conjugated estrogens, cyclophosphamide, cyclophosphamide, cytarabine, cytarabine, cytochalasin B, cyclophosphamide, dacarbazine, actinomycin, actinomycin (formerly actinomycin), daunorubicin hydrochloride, daunorubicin citrate, denidone Albumin, dexrazoxane, dibromomannitol, dihydroxyanthracenedione, docetaxel, dolasetron mesylate, doxorubicin hydrochloride, dronabinol, Escherichia coli L-asparaginase, emetine, epoetin-α, Erwinia L-asparaginase, esterified estrogen, estradiol, estramustine sodium phosphate, ethidium bromide, ethinyl estradiol, etidronic acid, etoposide citrate factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine hydrochloride, glucocorticoids, gosere Acetate, Gramicidin D, Granisetron Hydrochloride, Hydroxyurea, Idarubicin Hydrochloride, Ifosfamide, Interferon α-2b, Irinotecan Hydrochloride, Letrozole, Calcium Folinate, Leuprorelin Acetate, Levamisole Hydrochloride, Lidocaine, Lomustine, Maytansinoids, Nigrometholone Hydrochloride, Medroxyprogesterone Acetate, Megestrol Acetate, Melphalan HCL, Thiocarbine, Mesna, Methotrexate, Methyltestosterone, Mithramycin, Mitomycin C, Mitotane, Mitoxantrone, Nilutamide, Octreotide Acetate, Ondansetron Hydrochloride, Oxaliplatin, Purple Taxol, pamidronate disodium, pentostatin, pilocarpine hydrochloride, prinomycin, polyphenprosan 20 with carmustine implant, porfimer sodium, procaine, procarbazine hydrochloride, propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, paclitaxel, tegafur, teniposide, tenoposide, testolactone, tetracaine, thioguanine, thiotepa, topotecan hydrochloride, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

[0285] Anti-VISTA antibodies disclosed herein or their antigen-binding fragments thereof or their conjugates can be administered to patients who need to receive cancer treatment with a combination of chemotherapeutic agents. Exemplary combinations of chemotherapeutic agents include a combination of 5-fluorouracil (5FU) and folinic acid (leucovorin or LV); a combination of capecitabine with uracil (UFT) and folinic acid; a combination of tegafur with uracil (UFT) and folinic acid; an oxaliplatin with 5FU, or an oxaliplatin with capecitabine; an irinotecan with capecitabine, a combination of mitomycin C with 5FU, irinotecan or capecitabine. Other combinations using chemotherapeutic agents disclosed herein are also possible.

[0286] Anti-VISTA antibodies or their antigen-binding fragments or conjugates can also be combined with other therapeutic antibodies. Therefore, therapies based on the anti-VISTA antibodies or their antigen-binding fragments or conjugates disclosed herein can be combined or adjuvanted with different monoclonal antibodies, such as, for example but not limited to, by means of anti-EGFR (EGF receptor) monoclonal antibodies or anti-VEGF monoclonal antibodies. Specific examples of anti-EGFR antibodies include cetuximab and panitumumab. A specific example of an anti-VEGF antibody is bevacizumab.

[0287] In particular, the methods of treatment described herein may include administering an immune checkpoint inhibitor together with an anti-VISTA antibody, or an antigen-binding fragment thereof, or a conjugate thereof. The immune checkpoint inhibitor and the anti-VISTA antibody, or an antigen-binding fragment thereof, or a conjugate thereof may be administered simultaneously, separately, or sequentially.

[0288] As used herein, "checkpoint inhibitors" refers to molecules that target immune checkpoints and block the function of the immune checkpoints, such as, for example, small molecules, soluble receptors, or antibodies. More specifically, as used herein, "checkpoint inhibitors" are molecules (such as, for example, small molecules, soluble receptors, or antibodies) that block certain proteins made by certain types of immune system cells (such as T cells and certain cancer cells).

[0289] In a first embodiment, the immune checkpoint inhibitor is any one of the following inhibitors: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIGIT, TIM3, GAL9, LAG3, PSG-L1, VSIG4, KIR, 2B4 (belonging to the CD2 family of molecules, expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, IDO1, A2aR and various B-7 family ligands.

[0290] Exemplary immune checkpoint inhibitors include: anti-CTLA-4 antibodies (e.g., ipilimumab), anti-LAG-3 antibodies (e.g., BMS-986016), anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-Tim3 antibodies (e.g., TSR-022, MBG453), anti-BTLA antibodies, anti-KIR antibodies, anti-A2aR antibodies, anti-CD200 antibodies, anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, cemiplimab, pilizumab), anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab, BMS936559), anti-TIGIT antibodies (e.g., tiragolumab, vibostolimab), anti-VSIG4 antibodies, anti- CD28 antibody, anti-CD80 or anti-CD86 antibody, anti-B7RP1 antibody, anti-B7-H3 antibody, anti-HVEM antibody, anti-CD137 antibody (e.g., Urecitabine), anti-CD137L antibody, anti-OX40 (e.g., 9B12, PF-04518600, MEDI6469), anti-OX40L antibody, anti-CD40 or anti-CD40L antibody, anti-GAL9 antibody, anti-IL-10 antibody, fusion protein of PD-1 ligand (e.g., PDL-1 or PD-L2) extracellular domain and IgG1 (e.g., AMP-224), fusion protein of OX40 ligand (e.g., OX40L) extracellular domain and IgG1 (e.g., MEDI6383), IDO1 drug (e.g., icaridostat) and A2aR drug. Many immune checkpoint inhibitors have been approved or are undergoing clinical trials. Such inhibitors include ipilimumab, pembrolizumab, nivolumab, cemiplimab, pilizumab, atezolizumab, avelumab, durvalumab, tiragliflozin, vebrilumab, BMS 936559, usrelumab, 9B12, PF-04518600, BMS-986016, TSR-022, MBG453, MEDI6469, MEDI6383, and icadolizumab.

[0291] Examples of immune checkpoint inhibitors are listed in, e.g., Marin-Acevedo et al., Journal of Haematology & Oncology 11:8, 2018; Kavecansky and Pavlick, AJHO 13(2):9-20, 2017; Wei et al., Cancer Discov 8(9):1069–86, 2018.

[0292] Preferably, the immune checkpoint inhibitor is an inhibitor of CTLA-4, LAG-3, Tim3, PD-1, PD-L1, PSG-L1, VSIG4, CD137, OX40 or IDO1. More preferably, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1. Even more preferably, the immune checkpoint inhibitor is an antibody that inhibits PD-1 or an antibody that inhibits PD-L1.

[0293] Therefore, the present disclosure preferably relates to a combination therapy of an anti-VISTA antibody or its antigen-binding fragment or its conjugate with an anti-PD-1 antibody or an anti-PD-L1 antibody for the treatment of VISTA-mediated diseases, especially cancer. In the first aspect, the anti-VISTA antibody or its antigen-binding fragment or its conjugate of the present invention is used to treat VISTA-mediated diseases, especially cancer, wherein the treatment further includes the administration of anti-PD-1 or anti-PD-L1 antibodies. The present disclosure also relates to a method for treating VISTA-mediated diseases, especially cancer, comprising administering an effective amount of an anti-VISTA antibody or its antigen-binding fragment or its conjugate of the present invention and an effective amount of anti-PD-1 or anti-PD-L1 antibodies to a subject in need thereof. In another aspect, the present disclosure also relates to the use of an anti-VISTA antibody or its antigen-binding fragment or its conjugate disclosed herein for the preparation of a medicament for treating a VISTA-mediated disease, especially cancer, wherein the treatment includes the administration of an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0294] The anti-VISTA antibody or antigen-binding fragment thereof or its conjugate and the anti-PD-1 or anti-PD-L1 antibody can be administered simultaneously, separately or sequentially.

[0295] The present invention is described in detail below based on examples. However, the following examples are given only for illustrating the present invention, and it is obvious that the present invention is not limited to the following examples.

[0296] Example

[0297] Example 1: Identification of the deamidation site in Ab3

[0298] Monoclonal antibody Ab3 was initially disclosed in WO 2016 / 94837. Ab3 comprises a heavy chain of SEQ ID NO: 11 and a light chain of SEQ ID NO: 12. Bioinformatics analysis predicts that there are 2 potential deamidation sites in the light chain and 9 potential deamidation sites in the heavy chain of Ab3.

[0299] To investigate whether deamidation indeed occurs at either of these sites, Ab3 was subjected to cation exchange chromatography (CEX) as described by Goyon et al. (J Chromatogr B Analyt Technol Biomed Life Sci. 1065-1066:119-128 (2017)).

[0300] method: CEX (pH gradient)

[0301] Material:

[0302] Column MabPac SCX-10 4×250 mm, 10 μm (Thermo, reference number: 74625)

[0303] Eluent:

[0304] Buffer A: CX-1 pH gradient buffer A pH 5.6 (Thermo, reference number: 85349)

[0305] Buffer B: CX-1 pH gradient buffer B pH 10.2 (Thermo, reference number: 85349)

[0306] · Buffer was diluted 1 / 10 with MilliQ water and filtered / 0.22μm (for temporary use)

[0307] Sample preparation & methods:

[0308] Dilute the sample to 1 mg / mL with MilliQ water

[0309] Inject 20 μL of diluted sample

[0310] Gradient: See Table 3.

[0311] Table 3: Gradients used

[0312]

[0313] result

[0314] like Figure 1As shown, Ab3 is a highly heterogeneous mixture of 3 major charge variants, with heavy chain N55 / N55 accounting for 40%, N55 and D55 deamidation variants accounting for 33%, and all D55 deamidation variants accounting for 8%. This result was confirmed by structural evaluation (LC-MS). Forced degradation studies (pH 9, 40°C, 3 days; vs. pembrolizumab) came to the same conclusion: when Asn residues in the VL and VH chains of Ab3 were examined under these conditions, only N55 in the heavy chain was observed to be degraded, and other Asn residues seemed unaffected. Therefore, under all these different experimental conditions, position N55 in the heavy chain was identified as the major (if not the only) deamidation site in Ab3.

[0315] Example 2: Generation and characterization of Ab1

[0316] Based on the results of Example 1, a variant of Ab3 was created by mutating Asn to Asp at position 55 in the heavy chain. This variant was designated Abl.

[0317] Ab1 is a humanized monoclonal antibody against VISTA based on the framework of human immunoglobulin G1 (IgG1k; G1m3 (R215) allotype). The recombinant antibody is produced in Chinese hamster ovary (CHO) cells and consists of two heavy chains (HC) of 448 amino acid residues each and two kappa light chains (LC) of 213 amino acid residues each, with typical IgG1 inter-chain and intra-chain disulfide bonds.

[0318] The structural, physicochemical, immunological and biological properties of the anti-VISTA antibody Ab1 were established using a comprehensive approach:

[0319] Molecular weight :147213Da (G0F / G0F, pE / pE, 16 disulfide bridges)

[0320] Molecular formula :C 6410 H 9904 N 1686 O 2009 S 50

[0321] N-glycosylation site: 298,298"

[0322] The disulfide bridges are located :

[0323] ○ Intrachain (light chain): Cys(23) to Cys(87); Cys(133) to Cys(193)

[0324] ○ Intrachain (heavy chain): Cys(22) to Cys(96); Cys(145) to Cys(201);

[0325] Cys(262) to Cys(322); Cys(368) to Cys(426)

[0326] ○ Interchain (light and heavy chains): Cys(213)LC to Cys(221)HC; Cys(227)HC to Cys(227)HC; Cys(230)HC to Cys(230)HC

[0327] The expected average molar masses of full-length IgG, deglycosylated IgG, IdeS digested and reduced IgG were confirmed. The N-terminal residue of the heavy chain was encoded as glutamine, but was mainly present as pyroglutamate. There was one N-glycosylation site (Asn298) on the heavy chain, which was mainly occupied by a typical core fucosylated biantennary glycan with 0, 1 or 2 terminal galactose residues, as expected for recombinant IgG produced by CHO. Most of the C-terminal lysine residues in the heavy chain were cleaved off.

[0328] The molecular weights are shown in Table 4 below:

[0329] Table 4: Determination of molecular weight of Ab1

[0330]

[0331] Example 3: Determination of the binding of Ab1 to VISTA

[0332] Mutations in CDRs are known to affect the binding efficacy of antibodies. For example, when Asn33 in CRDL1 of the anti-CD52a monoclonal antibody was replaced by Asp, a 400-fold decrease in antigen binding affinity was observed (Qiu et al., mAbs. 11(7): 1266-1275 (2019)).

[0333] The binding of Ab1 to the recombinant human (rh)VISTA-His protein VISTA was studied by direct and indirect ELISA. In the direct ELISA, the rhVISTA-His protein was directly immobilized on the plate, while in the indirect ELISA, the rhVISTA-His protein was captured using an immobilized anti-His antibody.

[0334] The antibodies tested are provided in Table 5.

[0335] Table 5: Antibodies tested by ELISA

[0336]

[0337]

[0338] The anti-VISTA antibody Ab1 with Asp at position 55 was compared with two different batches of Ab3 antibody (with Asn at position 55), as well as IgG1 anti-VISTA and anti-hVISTA rabbit polyclonal antibodies (positive control) and an unrelated c9G4 antibody (negative control).

[0339] Direct ELISA

[0340] method

[0341] 100 μl of rhVISTA (0.3 μg / ml in 1× D-PBS) was coated on the wells overnight at 4°C.

[0342] After incubation, the coating solution was removed and the plates were blocked by adding 250 μl of blocking buffer (0.5% gelatin in 1×PBS) for at least 1 hour at 37°C.

[0343] After blocking, primary antibodies (antibodies listed in Table 3) were serially diluted 1:3 from an initial concentration of 5 μg / mL in dilution buffer (1× PBS + 0.1% gelatin + 0.05% Tween 20) to a final volume of 100 μL per well and incubated at 37°C for 1 hour.

[0344] Wash the wells three times with 300 μl of 1× PBS

[0345] 100 μl of secondary antibody (AffiniPure goat anti-rabbit specific IgG (H+L) HRP (Immuno Research Jackson, reference number 111-035-003)) or AffiniPure goat anti-human specific IgG (Fc fragment) HRP (Immuno Research Jackson, #109-035-098) diluted 1:5000 in dilution buffer was added to the wells and incubated for 1 hour at 37°C.

[0346] Wash the wells three times with 300 μl of 1× PBS

[0347] Add 100 μL of TMB to each well and incubate the plate at room temperature for 5 minutes. Add 100 μL of 1M H2SO4 to each well to terminate the reaction and read the absorbance at 450 nm using a microplate reader.

[0348] result

[0349] Although several IgG1 monoclonal antibodies have been reported to lose activity due to deamidation, the binding affinity of the Ab1 anti-VISTA antibody was unexpectedly maintained. In fact, Ab1 had very similar characteristics compared to the unmutated antibody Ab3 (see Figure 2).EC 50 The value is about 8.83×10 -10 M (CV 21%) (Table 6), in contrast to the values ​​observed previously. As expected, the c9G4 negative control antibody showed no binding.

[0350] Table 6: EC obtained for the tested antibodies in each of three experiments (n1, n2 and n3) in the rhVISTA direct ELISA 50 value.

[0351]

[0352]

[0353] Indirect ELISA

[0354] method

[0355] Wells were coated with 100 μl of 2 μg / ml anti-6×histidine mouse monoclonal IgG1, clone AD1.1.10 (RD Systems Cat. No. MAB050) in 1×D-PBS overnight at 4° C. (indirect ELISA).

[0356] After incubation, the coating solution was removed and the plates were blocked by adding 250 μl of blocking buffer (0.5% gelatin in 1×PBS) for at least 1 hour at 37°C.

[0357] After blocking, 0.3 μg / ml rhVISTA in 100 μL of dilution buffer (1×PBS + 0.1% gelatin + 0.05% Tween20) was added to each well and incubated at 37°C for 1 hour.

[0358] The wells were washed three times with 300 μl of 1× PBS.

[0359] Primary antibodies (antibodies listed in Table 3) were serially diluted in dilution buffer from an initial concentration of 5 μg / mL to a final volume of 100 μL per well and incubated at 37°C for 1 hour.

[0360] The wells were washed three times with 300 μl of 1× PBS.

[0361] 100 μl of secondary antibody (AffiniPure goat anti-rabbit specific IgG (H+L) HRP (Immuno Research Jackson reference number 111-035-003) or AffiniPure goat anti-human specific IgG (Fc fragment) HRP (Immuno Research Jackson #109-035-098) diluted 1:5000 in dilution buffer) was added to the wells and incubated for 1 hour at 37°C.

[0362] The wells were washed three times with 300 μl of 1× PBS.

[0363] Add 100 μL of TMB to each well and incubate the plate at room temperature for 5 minutes. Add 100 μL of 1M H2SO4 to each well to stop the reaction and read the absorbance at 450 nm using a microplate reader.

[0364] result

[0365] While several IgG1 monoclonal antibodies have been reported to have reduced affinity due to deamidation, the affinity of the Ab1 anti-VISTA antibody was unexpectedly maintained. In fact, Ab1 had very similar characteristics compared to the unmutated antibody Ab3 (see Figure 3 ).EC 50 The value is about 4.20×10 -11 M (CV 10%) (Table 7). As expected, the c9G4 negative control antibody showed no binding.

[0366] Table 7: EC obtained for the tested antibodies in each of the three experiments (n1, n2 and n3) in the rhVISTA indirect ELISA 50 value

[0367]

[0368]

[0369] Example 4: Evaluation of T cell activation and cytokine release in CHO-VISTA and PBMC co-culture

[0370] VISTA is known to be an immune checkpoint protein that strictly regulates immune responses. Since Ab1 binds VISTA with the same affinity as the original antibody Ab3, we investigated whether Ab1 could reverse immunosuppression like Ab3.

[0371] The schematic diagram of the experiment is as follows Figure 4 shown.

[0372] Chinese hamster ovary (CHO) cells WT or transfected to express human VISTA protein were irradiated with 90 Gy using a Faxitron X-ray machine to reduce their proliferation and metabolism.

[0373] Then, 20,000 CHO cells were cultured together with 200,000 PBMC cells in a 96-well plate (ratio of CHO:PBMC = 1:10).

[0374] Then, the mixture was incubated with anti-CD3 / CD28 beads (ratio: 1 bead to 32 cells) in the presence of Ab1 or hIgG1 negative control 10 μg / mL total 200 μl / 96 wells at 37°C and 5% CO2.

[0375] On day 3, the supernatant was recovered and analyzed for cytokine release by MSD (Meso Scale Discovery), and the expression of CD25 T cell activation marker on CD4 and CD8 T cells was analyzed by flow cytometry (FACS).

[0376] As expected, in the presence of CHO-VISTA, CD4 + and CD8 + The proliferation of T cells was inhibited. Adding antibody Ab1 reversed this inhibition. In the presence of Ab1, CD4 + and CD8 + Strong proliferation of T cells. However, no such stimulation was detected with the negative control hIgG1 antibody. Similarly, the addition of Ab1 to the mixture of PBMC and CHO-VISTA triggered a large production of IFNγ, IL-2, and TNFα, confirming the activation of CD4 + and CD8 + T cell activation ( Figure 5 ). The hIgG1 negative control again showed no effect. Therefore, these results demonstrate that Ab1 retains activity in inhibiting VISTA-mediated immunosuppression.

[0377] In order to understand the mechanism of this inhibition, a mutation (N298A) was introduced at position 298 of the Fc domain of Ab1. Antibodies with this mutation are known to be unable to activate effector functions, such as ADCC, CDC, and ADCP, because this mutation eliminates their ability to bind to human Fcγ receptors (see, e.g., Liu et al., Antibodies (Basel), 9(4): 64. (2020); Herbst et al., Nature. Immunol. 515(7528): 563-567 (2014)).

[0378] Surprisingly, this mutation completely abolished Ab1-induced CD4 + and CD8 + The ability of T cells to proliferate. Similarly, when Asn298 was replaced by alanine, no cytokine release could be detected. Therefore, these results indicate that the interaction of Ab1 with Fcγ receptors and the resulting effector function of Ab1 are essential for Ab1 to reverse the immunosuppression of VISTA ( Figure 5 ).

[0379] The negative control was not affected by the introduction of the same mutations in its Fc.

[0380] Ab1 blocks VISTA and reverses immunosuppression. This activity requires the effector function of the antibody (ADCC and / or CDC and / or ADCP).

[0381] Example 5: Ab1 inhibited the binding of VISTA to PSG-L1 and VSIG3.

[0382] Several VISTA ligands have been described. Specifically, VSIG3 has been identified as the main ligand of VISTA, showing specific binding and functional in vitro inhibition of T cell activation (Wang et al., Immunology.156(1):74-85(2019)). In addition, pH-dependent binding of VISTA to P-selectin glycoprotein ligand-1 (PSGL-1) has been described; blocking this interaction in an acidic environment is sufficient to reverse VISTA-mediated immunosuppression in vivo (Johnston et al., Nature.574(7779):565-570.(2019)).

[0383] Therefore, it was investigated whether Ab1 could block the interaction between VISTA and VSIG3 and / or the interaction between VISTA and PSG-L1 in an acidic pH environment (pH = 6).

[0384] Evaluation of rhVISTA-His (monomer) or rhVISTA-Fc (dimer) with implantation in CM5 sensor chip Binding of rhVSIG3-Fc on (2200RU).

[0385] Interactions were measured by Biacore at pH = 7.4

[0386] 700 nM of rhVISTA-His (monomer) and rhVISTA-Fc (dimer) were tested in the presence of a range of concentrations (0 to 1200 nM) of anti-VISTA Ab1.

[0387] Figure 6Shown is a dose response curve for VISTA-VSIG3 binding in the presence of Ab 1 compared to VISTA-VSIG3 binding in the absence of Ab 1 (100% binding). Ab 1 disrupted the VISTA-VSIG3 interaction in a dose-dependent manner.

[0388] Evaluation of the interaction between VISTA-Fc-d2 and PSGL1-His by anti-VISTA antibodies.

[0389] Determination principle:

[0390] HTRF (Homogeneous Time-Resolved Fluorescence) technology is an assay developed to study interactions between biomolecules. The detection system is based on fluorescence resonance energy transfer (FRET). The interaction between d2-labeled hVISTA-Fc (VISTA-Fc-d2) and His-tagged hPSGL-1 (PSGL1-His) with terbium-labeled anti-His mAb (anti-His-Tb / Cisbio) allows for HTRF signaling.

[0391] The antibodies tested in the experiments were:

[0392] - Anti-VISTA Ab1

[0393] -Anti-VISTA R&D Systems#71261

[0394] - Human IgG1 control isotype.

[0395] Different concentrations (0 to 20 μg / mL) of antibodies were incubated with VISTA-Fc-d2 and anti-His-Tb indirectly labeled PSGL1-His at room temperature and pH=6 for 4 hours.

[0396] No signal reduction was observed using the negative control. On the other hand, the addition of the positive control, a commercial antibody (R&D System #71261) that prevents VISTA / PSGL-1 interaction, resulted in a decrease in the measured HTRF signal, as expected. The IC 50 The IC value of Ab1 in the VISTA / PSGL-1 HTRF interaction assay was 333 nM (Table 8). Importantly, Ab1 also triggered a decrease in the signal, indicating that this antibody blocked the interaction between VISTA and PSG-L1 under acidic pH conditions (see Table 9). 50 It was 2.3 nM (Table 7).

[0397] Table 8: Anti-VISTA Antibody IC in VISTA / PSGL-1 HTRF Interaction Assay 50

[0398] <![CDATA[IC 50 (nM)]]> Anti-VISTA(R&D Sytems) 333 Anti-VISTA Ab1 2.3

[0399] Table 9: Average values ​​of 3 experiments expressed as percentage of specific HTRF signal

[0400]

[0401] Example 6: In vivo evaluation of anti-VISTA mAb1 antibody in the MC38 mouse colon tumor model

[0402] Materials and methods

[0403] For each experiment, a vial of frozen MC38 cells was thawed and grown in DMEM / F12 containing 10% serum. After 2 days in culture, cells were harvested using trypsin and plated at 5 × 10 5 The cells were resuspended in DMEM / F12 at a concentration of 10 cells / ml and 100 μl was injected into each mouse.

[0404] Female C57Bl / 6hVISTA mice aged 8-10 weeks were purchased from Genoway (Lyon, France). After arrival, mice were allowed to adapt for 7 days, and then the hair on the right flank was shaved. 100 μl of MC38 cell suspension (50,000 cells) was injected subcutaneously (sc) into the shaved flank of mice.

[0405] When the tumor reaches about 6 mm in diameter (about 80 mm in volume) 3 ), the tumor is considered to have formed. Once established, treatment begins. The murinized anti-VISTA antibody (mAb1) (corresponding to the CDR of Ab1 with mouse Fc) or the corresponding isotype control antibody mIgG2a was administered intraperitoneally at 30 mg / kg (formulated in histidine 25mM, NaCl 150mM, 0.1% polysorbate 80 [v / v], pH 6.5), injected once every 3 to 4 days, for a total of 4 injections. Tumor growth was evaluated three times a week during treatment until the end of the experiment, and three dimensions were measured using an electronic caliper: length (L), width (W) at a 90° angle to the first measurement, and final height (H).

[0406] Tumor volume was calculated as follows: Volume = 0.52 × (L × W × H)

[0407] Since Ab1 activation of T cells was shown to be dependent on effector functions in vitro, the role of these activities in any anti-tumor activity of the antibody was investigated. A variant of mAb1 was created in which the Asn (equivalent residue to N298A in Ab1) that interacts with FcγR was replaced by an Ala residue, thereby eliminating any effector mechanism. The mIgG1 antibody was used as a negative control (Chen et al., Front Immunol. 10:292 (2019)).

[0408] result

[0409] Under the implantation conditions and administration regimen tested, the active form of mAb1 induced 47% tumor growth inhibition at day 21 ( Figure 7 The silenced form of mAb1 did not induce tumor growth inhibition ( Figure 8 ).

[0410] Example 7: Design of Ab1 formulation

[0411] Formulation development is an important aspect of product development and is often a critical path to successful clinical manufacturing and stability studies, which are critical for investigational new drug (IND) applications. Due to their large size, antibodies are usually administered by infusion. Due to their complex three-dimensional structure, antibodies often aggregate in solution, shortening their shelf life and availability.

[0412] Based on the experimental design, a two-step approach was used to perform pre-formulation studies and four antibody formulations were selected. The aim of this study was to define four antibody formulations using a two-step approach based on the experimental design. The first step aimed to identify important factors and the second step aimed to define the four formulations.

[0413] From D0 to 4 weeks, samples were stored at +5 ± 3 °C, +40 °C or after 3 freeze-thaw cycles for different time periods and analyzed by UV280 nm, size exclusion chromatography (SEC), field-flow fractionation (A4F), thermal shift assay (TSA) or differential scanning calorimetry (DSC), cation exchange chromatography (CEX) and microflow imaging (MFI) to determine the particle amount.

[0414] 7.1 Materials and methods

[0415] Experimental design

[0416] Step 1 Design

[0417] The following factors were selected for this step:

[0418] Buffer: 25 mM citrate (C) or 25 mM histidine (H) or 25 mM phosphate (P)

[0419] pH: 5.5 or 6 or 6.5

[0420] Sucrose (Suc): 0-6% (mass / volume) (m / v)

[0421] Arginine (Arg): 0–500 mM

[0422] NaCl: 0–150 mM

[0423] Surfactant: No polysorbate (PS), polysorbate 80 0.01%, or polysorbate 20 0.01% (volume of surfactant / volume of mAb solution) (v / v)

[0424] The mAb concentration was fixed at 20 mg / mL.

[0425] The experimental design was set up using MODDE (Umetrics) software in screening mode, which allows finding significant factors as the first stage of the project using linear and interaction models.

[0426] Step 2 Design

[0427] The following factors were selected for this step:

[0428] Buffer: 25 mM histidine

[0429] pH: 5.5-6.5

[0430] Sucrose: 0-6% (m / v)

[0431] NaCl: 0-150mM

[0432] Polysorbate 80: 0-0.01% (v / v)

[0433] The mAb concentration was fixed at 20 mg / mL.

[0434] The experimental design was set up using MODDE software in optimization mode (Response Surface Model, RSM), which allows detailed modeling and optimization using quadratic and cubic models. Central point analysis was performed in triplicate to assess experimental variability.

[0435] Operational Scheme

[0436] In two-step experiment, Ab1 is configured in suitable buffer by overnight dialysis at +5 ± 3 ℃ with 20mg / mL. The solution after dialysis is centrifuged for 2 minutes at 2000g, and the concentration of mAb is measured by UV280 method subsequently. The concentration of final preparation is adjusted to 20mg / mL by using corresponding buffer dilution. When needed, polysorbate 20 or polysorbate 80 (polysorbate volume / mAb solution volume) is added. Through aseptic filtration (0.22 μm filter), 0.3mL of each preparation is loaded in sterile 2mL Eppendorf tube, and before analysis, store as follows. In addition, in step 2, 2.5mL of each preparation is loaded in sterile 15mL Falcon tube, and stored for 4 weeks at +40 ℃.

[0437] Table 10

[0438]

[0439] Statistical analysis

[0440] The experiments were analyzed using MODDE dedicated software.

[0441] Material

[0442] equipment

[0443] NanoDrop One Micro-Volume UV-Vis Spectrophotometer (Thermo Scientific)

[0444] MicroCal VP-Capillary DSC (Malvern Instruments) - Software: MicroCalVP-CapillaryDSC Software 2.0

[0445] HPLC system:

[0446] -Shimadzu LC10-AD - Software LC Solution Version 1.25

[0447] -Agilent 1260 thermostat, UV detector 1260 (DAD) (Agilent)

[0448] -Waters 2695 (Waters) - Software Empower 2

[0449] Eclipse Dualtec A4F Split System (Wyatt)

[0450] A4F spacer short channel (SC) 490μm W, regenerated cellulose membrane 10kDa

[0451] Multi-angle light scattering (MALS) detector (Wyatt Dawn HELEOS II)

[0452] RI detector Optilab T-rEX (Wyatt)

[0453] A4F acquisition and processing software: Open Lab C.01.06 and Astra 6.1.6.5

[0454] Reagents

[0455] L-Histidine (Sigma Aldrich-H8000)

[0456] L-Histidine monohydrochloride monohydrate (Sigma Aldrich-H8125)

[0457] ·NaCl(Merck-Millipore–106400)

[0458] Sodium azide NaN3 (Sigma Aldrich-S2002)

[0459] BSA (Pierce–23029)

[0460] CAPDSC 96-well plate (Malvern Instruments–WEL190010-10)

[0461] CAPDSC 96-well plate cover (Malvern Instruments–WEL190020-010)

[0462] ·ProPac WCX-10 column (4x250mm) (ThermoScientific–054993)

[0463] CX-1 pH Gradient Buffer A pH 5.6 10x (Thermo Scientific–085346)

[0464] CX-1 pH Gradient Buffer B pH 10.2 10x (Thermo Scientific–085348)

[0465] Carbonic anhydrase (Sigma Aldrich—C7025) was prepared at 2 mg / mL in 25 mM His / His-HCl, 150 mM NaCl buffer (pH 6.5).

[0466] Ab1

[0467] Analysis Methods

[0468] Table 11: Methods used in the study

[0469] test method mAb concentration UV280 Aggregates, polymers, degradation products SEC Aggregates, polymers, degradation products A4F Melting temperature Tm DSF / TSA Melting temperature Tm DSC Charge variant CEX Particles >10μm and >25μm MFI

[0470] Protein concentration was determined by UV280 protein assay on a NanoDrop One spectrophotometer.

[0471] Aggregation and fragmentation were assessed by asymmetric flow field flow fractionation (A4F). For each sample, 40 μg was injected into a 490 μm W short channel. IgG was eluted using 25 mM histidine, 150 mM sodium chloride, pH 6.5 and 0.02% NaN3. Samples were analyzed by size exclusion chromatography "SEC" on a Superdex 200 Increase 10 / 300 GL column connected to a 2695 Waters HPLC system.

[0472] The melting temperatures (Tm) of the samples were determined by differential scanning fluorimetry (DSF) using a standard protocol.

[0473] The melting temperature (Tm) of the samples was also determined by differential scanning calorimetry (DSC). Each sample was diluted in its own dialysis buffer to reach a protein concentration of 1 mg / mL. 400 μL of these 1 mg / mL solutions and 400 μL of each dialysis buffer (run as a blank control) were dispensed into a 96-well plate. DSC analysis was performed in an automated microcalorimeter with a temperature range of 40 to 100°C and a scan rate of 60°C / h.

[0474] Charge variants were analyzed using cation exchange chromatography (CEX) on a ProPac WCX-10 column connected to a Shimadzu LC10-AD HPLC system. 20 μg of each sample was injected onto the column. 5 μg of carbonic anhydrase and 20 μg of hz1022C3 L1 / H13 (G1) were also injected as calibrants at the beginning of the batch.

[0475] Running conditions: CX-1 pH gradient buffer A (pH 5.6, 1x) and CX-1 pH buffer B (pH 10.2, 1x) - flow rate: 1 mL / min.

[0476] The results were analyzed using MicroCal VP-Capillary DSC automatic analysis software.

[0477] 7.2 Results

[0478] Results of step 1

[0479] Thermal shift measurement at T0

[0480] TSA analysis has been performed under 22 conditions. The spectrum of the derivative plot as well as the Tm value varies depending on the buffer properties ( Fig. 9 ). The three replicates of sample 11 showed good reproducibility, and the standard deviation showed that a difference of 1°C was significant. The TSA results were statistically reviewed ( Fig.10). Only 3 factors were identified as significant for each Tm. 25 mM citrate buffer (pH 5) and 500 mM arginine had negative effects on both Tms. 25 mM phosphate buffer (pH 6.5) and 6% sucrose had positive effects on Tm1 and Tm2, ​​respectively.

[0481] Analysis of remaining mAbs

[0482] The concentration of Ab1 was determined at each time point. The amount of mAb remaining was expressed as a percentage of the initial amount. The results were statistically reviewed ( Fig.11 25 mM phosphate buffer (pH 6.5) and 500 mM arginine had a negative effect on the remaining mAb after 10 days at 40°C, as did 25 mM citrate buffer (pH 5.5) after freeze-thaw cycles.

[0483] Monomer analysis

[0484] SEC analysis was performed for 22 conditions at each time point, and the monomer content is expressed as a percentage. At T0, the monomer content of each condition was comparable. Three replicates of sample 11 showed good reproducibility, and the standard deviation indicated that a difference of 1°C was significant. The results were statistically reviewed ( Fig.12 ). Similarly, 25 mM phosphate buffer (pH 6.5) had a negative effect on monomer after 10 days at 40°C and after freeze-thaw cycles. In addition, the absence of detergent had a negative effect after 10 days at 40°C. 500 mM arginine had a positive effect on monomer content after 10 days at 40°C.

[0485] Step 1 Results Overview

[0486] Table 12 below summarizes the weights of each research factor in Step 1 calculated by MODDE.

[0487] Table 12:

[0488]

[0489] All factors showing negative effects were not considered further in the next step.

[0490] The first step identified 25 mM citrate buffer (pH 5 or pH 5.5), 25 mM phosphate buffer (pH 6 or pH 6.5), and 500 mM arginine as factors that negatively affected melting temperature, residual mAb, and monomer content.

[0491] The selection factors that need further study are

[0492] Histidine buffer pH 5.5 to 6.5

[0493] NaCl: 0 -150mM

[0494] Sucrose: 0-6% (m / v)

[0495] Polysorbate 80: 0–0.01% (v / v)

[0496] Result of step 2

[0497] Ab1 analysis at T0

[0498] Ab1 was analyzed before being placed under stress conditions. The concentration of mAb was uniform between samples. The average percentage of monomer (SEC UV and A4F) and the percentage of the main peak of charge variants were similar for all preparations. Therefore, at T0, the effect of buffer on monomer and charge variants was negligible. However, the amount of particles at T0 varied with the buffer, indicating a potential effect of the buffer on this parameter.

[0499] DSC analysis showed different patterns depending on the nature of the buffer, as well as changes in Tm values.

[0500] Experimental design results

[0501] Results obtained after stress of freeze-thaw cycles, after two weeks (T2weeks) and four weeks (T4weeks) of storage at +5±3°C, or after 2 weeks of stress at 40°C were not used because stress did not produce sufficiently significant changes in the responses studied (percentage of mAb remaining, percentage of monomer measured by SEC UV or AF4, and percentage of major charge variant peak).

[0502] The results obtained for the particles were not analyzed because they appeared inconsistent.

[0503] Review of the complete results for the responses using MODDE showed good reproducibility across triplicates for each studied response. Analysis of all responses obtained versus those predicted by the model showed good regression, indicating that the model developed was a good model.

[0504] Response Surface

[0505] Surface response analysis allows a detailed examination of the behavior of each response. It is thus observed that:

[0506] • pH 6.5 increases the melting temperature but decreases the monomer level and the percentage of the main charge variant peak.

[0507] High concentration of NaCl (150 mM) is not conducive to achieving the optimal melting temperature and high levels of monomers.

[0508] • In general, NaCl concentrations greater than 80 mM were beneficial in maintaining a good percentage of the main peak of the charge variants.

[0509] Sucrose and polysorbate 80 had little effect on melting temperature and monomer levels.

[0510] • However, 0.25% polysorbate 80 (mass / mass) had a positive effect on the proportion of the main peaks of the charge variants.

[0511] Best Points

[0512] The optimal points defined by the MODDE software are summarized in Table 13 below.

[0513] Table 13: Optimal points for step 2

[0514]

[0515] In the second step of the study, the study of the optimal point showed that

[0516] The most favorable pH range is between 5.9 and 6.5

[0517] • The most favorable NaCl concentration varies depending on the response being studied. For melting temperature and monomer content (based on SECUV), low concentrations are required, but for combined analysis of charge variants and SEC UC_charge variants, high concentrations are required.

[0518] • The most favorable sucrose concentration varies depending on the response studied. For the combined analysis of melting temperature, A4F-based monomer content, and A4F_charge variants, high concentrations are required, but for the combined analysis of SEC UV, charge variants, and SEC UC_charge variants, low concentrations are required.

[0519] The most beneficial concentration of polysorbate 80 is between 0.006 and 0.01% (v / v).

[0520] Based on these results, four formulations were selected for stability studies:

[0521] →A: 25 mM histidine, 1% sucrose, 0.006% polysorbate 80 (v / v), pH 6;

[0522] →B: 25 mM histidine, 150 mM NaCl, 0.006% polysorbate 80 (v / v), pH 6.5;

[0523] →C: 25 mM histidine, 150 mM NaCl, 3% sucrose, 0.006% polysorbate 80

[0524] (v / v), pH 6.5; and

[0525] →D: 25 mM histidine, 15 mM NaCl, 5% sucrose, 0.01% polysorbate 80 (v / v), pH 6.5.

[0526] Example 8: Stability study of Ab1 formulation

[0527] Stability studies were performed using the four formulations selected in Example 7. The formulations were stored at -66°C, +5°C and +40°C for 1.5 months and 2 months + 3 weeks. The following tests were performed: appearance, opalescence, pH, protein content by UV, SEC-HPLC, antibody purity by CE-SDS (non-reducing), charge profile by CEX, DSC, MFI and target binding by ELISA.

[0528] 8.1 Materials and methods

[0529] Preparation of formulations

[0530] Batches in each formulation (A, B, C, and D) were reconstituted by running separate tangential flow filtration (TFF). Each TFF used Repligen's Sius Hystream LS 30 kDa 0.01 m 2 The product was processed at 300LMH without back pressure. Finally, the product concentration was adjusted to 20g / L.

[0531] Finally, polysorbate 80 (5% v / v polysorbate 80) was added using a concentrated solution, followed immediately by 0.22 μm filtration on a PES membrane.

[0532] The antibody solution was then dispensed into Pharmatainer 50 mL tubes (Cellon PET reference 1000PF-05), filling each tube with 4 or 7 mL.

[0533] pH

[0534] The test was performed using a pH meter according to Chapter 2.2.3 of the European Pharmacopoeia.

[0535] EC by ELISA 50 VISTA binding shown as a ratio (expressed as a percentage of the reference)

[0536] This test evaluates direct binding between Ab1 solution and VISTA receptors coated on a 96-well plate. After blocking the plate, serial dilutions of Ab1 solution were made across the plate to allow for binding to the receptor. The complex was detected with goat anti-human IgG conjugated to horseradish peroxidase (HRP) and quantified using a colorimetric substrate. Data analysis was performed using a four-parameter logistic fit to yield the EC values ​​for Ab1 solution. 50 value, and compared with the reference EC 50 The values ​​are compared.

[0537] Charge spectrum of CEX (%)

[0538] The heterogeneity of the charge variants of Ab1 solutions was investigated by a method based on liquid ion exchange chromatography developed according to Chapter 2.2.29 of the European Pharmacopoeia. The samples were desalted, diluted with water to approximately 5 mg / mL, and analyzed as follows:

[0539] Table 14

[0540]

[0541] Quantification was achieved by normalization. The results for the acidic variant peak, main peak, and basic variant peak were expressed as a percentage relative to the total area.

[0542] Size variants determined by SEC (%)

[0543] To investigate the size variants of Ab1 solutions, a method based on liquid chromatography (Ph. Eur. Chapter 2.2.29) was developed according to the following conditions:

[0544] Table 15

[0545]

[0546] Samples were diluted to approximately 1.5 mg / mL in mobile phase. Quantification was achieved by normalization. Results for aggregate, monomer, and fragment peaks were reported as relative percentages of the total area.

[0547] Antibody purity determined by CE-SDS (percentage of H2L2)

[0548] SDS gel capillary electrophoresis (CE-SDS) was used to evaluate the purity and integrity of Ab1 solution samples. Measurements were performed using a Beckman-Coulter ProteomeLab PA800 equipped with a photodiode array detector (220 nm). For non-reducing conditions, the samples were diluted with SDS-MW sample buffer. Internal standard and iodoacetamide (alkylating agent) were added to the solution and heated at 70°C for 10 minutes. For reducing conditions, the samples were diluted with SDS-MW (denaturing agent) sample buffer. Internal standard and 2-mercaptoethanol (reducing agent) were added to the solution and heated at 70°C for 10 minutes. Quantification (%) was achieved by normalization method.

[0549] Determination of subvisible particles by light obscuration (European Pharmacopoeia Chapter 2.9.19)

[0550] The test complies with European Pharmacopoeia Chapter 2.9.19. Method 1 (Light Obscuration Particle Count Test).

[0551] Protein content determined by UV (mg / mL)

[0552] The protein concentration in the Ab1 solution samples was determined by UV spectrophotometry at 280 nm. The samples were diluted with water and the working range was 0.054 mg / mL to 0.705 mg / mL. The protein concentration was calculated by dividing the absorbance reading by the determined specific absorbance coefficient of the Ab1 solution (1.45 mL.mg -1 .cm -1 This value is then corrected for the dilution factor.

[0553] Bacterial endotoxin (IU / mg)

[0554] The test follows the kinetic chromogenic method of Chapter 2.6.14 of the European Pharmacopoeia.

[0555] Sterility

[0556] The test follows Chapter 2.6.1 of the European Pharmacopoeia.

[0557] 8.2 Results

[0558] After 2 months and 3 weeks of stability studies, analytical criteria for antibody quality were not discriminatory.

[0559] The appearance, opalescence and pH of the formulation remained stable during the study period.

[0560] Regardless of temperature (<-66°C, +5°C or +40°C) and formulation, no significant changes in protein content were observed by UV. The concentration was maintained within ±10% of the initial concentration.

[0561] The SEC profiles were indeed stable and similar in the 4 frozen formulations (<-66°C). At +5°C, slight changes were observed over time, but no significant differences between the 4 buffers. Under accelerated conditions at +40°C, some fragments appeared, so the monomer level decreased after 2 months and 3 weeks of storage. However, all formulations progressed in the same way. In conclusion, the SEC profile cannot be a distinguishing factor between the 4 formulations, but it helps to choose the storage temperature (+5°C seems to be more favorable for SEC profile stability).

[0562] Since antibodies are sensitive to oxidation during production, the CE-SDS profiles were studied in detail. At +5°C, full-length IgG (H2L2) levels were completely stable for 2 months and 3 weeks. When frozen at <-66°C, a decrease in full-length IgG levels was observed in all formulations (87-88% at T0 and 84-86% at 3 months (T3 month). The same trend was observed under accelerated conditions at +40°C. In summary, the CE-SDS profile cannot be used as a discriminatory factor between the 4 formulations, but it can distinguish between storage temperatures: freezing at <-66°C should be avoided. These trends can be explained by the lack of control of the freezing and thawing process (static process).

[0563] The CEX profiles remained constant over time regardless of storage temperature and did not differ between formulations.

[0564] There were no significant differences in the amount of particles over time as measured by ELISA binding and by MFI among the four formulations.

[0565] Since the analytical criteria for antibody quality failed to reflect the ability to discriminate, the osmotic pressure of the buffer was also considered.

[0566] Buffer A is hypotonic, whereas buffer D is hypertonic. These two formulations were then eliminated. Between formulation B and formulation C, formulation B (i.e., 25 mM histidine, 150 mM NaCl, 0.006% polysorbate 80 (v / v), pH 6.5) was chosen to limit the amount of raw materials in the composition (especially to avoid sucrose, which may increase endotoxin levels).

[0567] 8.3 Results of 48-month stability study of formulation B

[0568] The stability of the formulation buffer was verified by physicochemical parameters (appearance (including opalescence), pH and osmotic pressure) measured under long-term conditions (+5°C ± 3°C). The stability data of formulation B over 48 months are shown in Table 16.

[0569] Study conclusion: The properties of the formulation buffer remain stable for at least 48 months at +5°C.

[0570]

[0571]

Claims

1. A pharmaceutical composition comprising: Monoclonal anti-VISTA antibody comprising o the heavy chain of the sequence represented by SEQ ID NO: 21, and o a light chain of the sequence represented by SEQ ID NO: 22, and Pharmaceutically acceptable carriers and / or excipients.

2. A pharmaceutical composition according to claim 1, wherein the concentration of the monoclonal anti-VISTA antibody is between 5 and 100 mg / mL, preferably between 10 and 70 mg / mL, more preferably between 15 and 50 mg / mL, and most preferably between 20 and 30 mg / mL.

3. A pharmaceutical composition according to any one of claim 1 or claim 2, wherein the concentration of the monoclonal anti-VISTA antibody is 20 mg / mL.

4. The pharmaceutical composition according to any one of claims 1 to 3, comprising a buffer, preferably a citrate buffer, a phosphate buffer or a histidine buffer, more preferably a histidine buffer.

5. The pharmaceutical composition according to claim 4, wherein the buffer is a histidine buffer having a concentration between 5 and 45 mM, preferably between 10 and 40 mM, more preferably between 15 and 35 mM, most preferably between 20 and 30 mM.

6. The pharmaceutical composition according to any one of claim 4 or claim 5, wherein the buffer is a histidine buffer at a concentration of 25 mM.

7. A pharmaceutical composition according to any one of claims 1 to claim 6, comprising a tension regulator, which is preferably selected from polyols (e.g., trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol), salts and amino acids; more preferably, the salt is selected from sodium chloride, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate and calcium chloride; even more preferably NaCl, MgCl2 and / or CaCl2, most preferably NaCl.

8. The pharmaceutical composition according to claim 4, wherein the tonicity modifier is NaCl, and its concentration is between 0 mM and 300 mM, preferably between 50 mM and 250 mM, more preferably between 100 mM and 200 mM.

9. The pharmaceutical composition according to any one of claims 7 to 8, wherein the tonicity modifier is NaCl at a concentration of 150 mM.

10. The pharmaceutical composition according to any one of claims 1 to claim 9, comprising a nonionic surfactant, preferably a polysorbate, such as polysorbate 20 or polysorbate 80, more preferably polysorbate 80.

11. The pharmaceutical composition of claim 10, wherein the nonionic surfactant is polysorbate 80 at a concentration between about 0.001% to about 0.5% (v / v), between about 0.002% to about 0.2% (v / v), between about 0.003% to about 0.1% (v / v), between about 0.004% to about 0.009% (v / v), between about 0.005% to about 0.008% (v / v), or between about 0.006% to about 0.007% (v / v).

12. The pharmaceutical composition according to any one of claims 10 to 11, wherein the non-ionic surfactant is polysorbate 80 at a concentration of 0.006% (v / v).

13. A pharmaceutical composition according to any one of claims 1 to claim 12, wherein the pH of the pharmaceutical composition is between about 3.0 to about 9.0, between about 4.0 to about 8.0, between about 5.0 to about 8.0, between about 5.0 to about 7.0, between about 5.0 to about 6.5, between about 5.5 to about 8.0, between about 5.5 to about 7.0, or between about 5.5 to about 6.

5.

14. The pharmaceutical composition of claim 13, wherein the pH of the pharmaceutical composition is about 6.

5.

15. The pharmaceutical composition according to any one of claims 1 to 14, comprising 25 mM histidine, 150 mM NaCl, 0.006% polysorbate 80 (v / v), pH 6.

5.

16. A pharmaceutical composition according to any one of claims 1 to 15 for use in treating cancer in a patient.

17. The pharmaceutical composition of any one of claims 1 to 15 for use according to claim 16, wherein the use comprises inducing an immune response in the patient.

18. The pharmaceutical composition of any one of claims 1 to 15 for use according to claim 17, wherein the immune response comprises inducing CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and induce CD8 + T cells produce cytokines.

19. The pharmaceutical composition of any one of claims 1 to 15 for use as described in any one of claims 16 to 18, wherein the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer (e.g., leukemia, lymphoma or myeloma), laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma and prostate cancer.

20. The pharmaceutical composition of any one of claims 1 to 15 for use according to any one of claims 16 to 19, wherein the use comprises activating the effector function of the antibody.

21. The pharmaceutical composition of any one of claims 1 to 15 for use as claimed in any one of claims 16 to 20, further comprising administering a second therapeutic agent.

22. The pharmaceutical composition of any one of claims 1 to 15 for use according to claim 21, wherein the second therapeutic agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

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