Anti-vista antibody or antigen-binding fragment thereof and use thereof

Abstract

Provided are an anti-VISTA antibody or an antigen-binding fragment thereof and the use thereof. The antibody or the antigen-binding fragment thereof comprises complementarity determining region (CDR) sequences selected from SEQ ID NOs: 2-37, or comprises amino acid sequences respectively having at least 80% identity to the complementarity determining region sequences. The antibody or the antigen binding fragment thereof can bind to a human VISTA antigen, has a high binding activity, and can be endocytosed by a target cell.

Description

Anti-VISTA antibodies or antigen-binding fragments thereof and uses thereof Technical Field

[0001] The present application relates to the field of biopharmaceutical technology, and in particular to an anti-VISTA antibody or an antigen-binding fragment thereof and uses thereof.

[0002] Sequence listing submitted simultaneously

[0003] The entire contents of the following XML file are incorporated herein by reference in their entirety: Computer Readable Form (CRF) of the Sequence Listing (File Name: TFH01055PCT-sequencelisting.TXT, Date: 20240617, Size: 57.8 KB). Background Art

[0004] V-domain Ig inhibitor of T cell activation (VISTA) is a novel immunomodulatory receptor and a member of the B7 family that maintains homeostasis of T cells and myeloid cells. It shares high homology with PD-L1 but has a different mechanism of action. VISTA is widely expressed on myeloid and lymphocytes and is often considered an indicator of poor prognosis in various cancers. Importantly, VISTA's antibodies target both innate and adaptive immunity. VISTA's widespread expression gives it higher non-redundant activity than other immune checkpoints, making it a very promising target for tumor immunotherapy.

[0005] VISTA is an immunomodulatory receptor expressed on most hematopoietic cell subsets. Myeloid cells show the highest expression, with significantly increased levels in microglia and neutrophils. Other myeloid subsets, including monocytes, macrophages, basophils, and dendritic cells, are also expressed. Unlike other checkpoints, which are induced at different stages after activation, VISTA is expressed in a steady-state manner on these subsets.

[0006] On lymphocytes, VISTA is most highly expressed on naive CD4+ T cells and FoxP3+ T regulatory cells (Tregs). It is expressed at lower levels on CD8+ T cells, natural killer cell subsets, and thymocytes. Although VISTA is not expressed on B cells, it is significantly expressed on plasma cells. It is also worth noting that studies have found that reduced VISTA expression is detected under inflammatory conditions. In preclinical animal models, gene knockout or antibody blocking of VISTA can activate T cell responses to tumor cells.

[0007] Clear scientific evidence shows that VISTA is a ligand that can induce profound T cell suppression. Various groups, including Dartmouth College and Jannsen, have reported on a variety of antagonistic anti-human VISTA antibodies. Such antibodies can be used to treat conditions where it is desirable to inhibit the immunosuppressive effects of VISTA on T cell immunity, such as cancer.

[0008] Summary of the Invention

[0009] Based on the existing technology, this application uses a fully human antibody library to screen a new type of anti-human T cell activated V domain Ig inhibitory factor (VISTA) antibody, which can bind to the human VISTA antigen and exhibit high antibody affinity.

[0010] The specific technical solutions of this application are as follows:

[0011] 1. An anti-VISTA antibody or antigen-binding fragment thereof, comprising a complementary determining region (CDR) sequence selected from the following: SEQ ID NO: 2-37, or comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the complementary determining region sequence, respectively, wherein the complementary determining region is determined based on the Kabat numbering system.

[0012] 2. The anti-VISTA antibody or antigen-binding fragment thereof according to item 1, wherein the complementarity determining regions comprise a heavy chain complementarity determining region and a light chain complementarity determining region;

[0013] Wherein, the heavy chain complementarity determining region is:

[0014] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 2, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 3, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 4;

[0015] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO:8, a CDR2 having an amino acid sequence as set forth in SEQ ID NO:9, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO:10;

[0016] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 14, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 15, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 16;

[0017] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 20, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 21, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 22;

[0018] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 27, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 28; or

[0019] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 32, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 33, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 34; and

[0020] Wherein, the light chain complementary determining region is

[0021] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO:5, a CDR2 having an amino acid sequence as set forth in SEQ ID NO:6, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO:7;

[0022] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 11, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 12, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 13;

[0023] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 17, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 18, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 19;

[0024] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 23, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 24, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 25;

[0025] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 29, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 30, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 31; or

[0026] It comprises a CDR1 with the amino acid sequence shown in SEQ ID NO: 35, a CDR2 with the amino acid sequence shown in SEQ ID NO: 36, and a CDR3 with the amino acid sequence shown in SEQ ID NO: 37.

[0027] 3. The antibody or antigen-binding fragment thereof according to item 2, comprising the heavy chain complementarity determining regions and light chain complementarity determining regions shown below:

[0028] (1) the heavy chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 2, CDR2 with the amino acid sequence set forth in SEQ ID NO: 3, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 4, and the light chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 5, CDR2 with the amino acid sequence set forth in SEQ ID NO: 6, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 7;

[0029] (2) the heavy chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 8, CDR2 with the amino acid sequence set forth in SEQ ID NO: 9, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 10, and the light chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 11, CDR2 with the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 13;

[0030] (3) the heavy chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 14, CDR2 with the amino acid sequence set forth in SEQ ID NO: 15, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 16, and the light chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 17, CDR2 with the amino acid sequence set forth in SEQ ID NO: 18, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 19;

[0031] (4) the heavy chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 20, CDR2 with the amino acid sequence set forth in SEQ ID NO: 21, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 22, and the light chain complementary determining regions are CDR1 with the amino acid sequence set forth in SEQ ID NO: 23, CDR2 with the amino acid sequence set forth in SEQ ID NO: 24, and CDR3 with the amino acid sequence set forth in SEQ ID NO: 25;

[0032] (5) the heavy chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 26, CDR2 of the amino acid sequence set forth in SEQ ID NO: 27, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 28, and the light chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 29, CDR2 of the amino acid sequence set forth in SEQ ID NO: 30, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 31; or

[0033] (6) The heavy chain complementary determining regions are CDR1 with the amino acid sequence shown in SEQ ID NO: 32, CDR2 with the amino acid sequence shown in SEQ ID NO: 33, and CDR3 with the amino acid sequence shown in SEQ ID NO: 34, and the light chain complementary determining regions are CDR1 with the amino acid sequence shown in SEQ ID NO: 35, CDR2 with the amino acid sequence shown in SEQ ID NO: 36, and CDR3 with the amino acid sequence shown in SEQ ID NO: 37.

[0034] 4. The antibody or antigen-binding fragment thereof according to any one of items 1 to 3, which comprises a human framework region amino acid sequence, preferably, the human framework region amino acid sequence is a human consensus framework region amino acid sequence.

[0035] 5. An antibody or antigen-binding fragment thereof according to any one of items 1-4, comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence selected from the following: SEQ ID NO: 38-49, or comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence, respectively.

[0036] 6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, the amino acid sequence shown in SEQ ID NO: 40, the amino acid sequence shown in SEQ ID NO: 42, the amino acid sequence shown in SEQ ID NO: 44, the amino acid sequence shown in SEQ ID NO: 46, the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence, respectively; and / or

[0037] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, the amino acid sequence shown in SEQ ID NO:41, the amino acid sequence shown in SEQ ID NO:43, the amino acid sequence shown in SEQ ID NO:45, the amino acid sequence shown in SEQ ID NO:47, the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence, respectively.

[0038] 7. The antibody or antigen-binding fragment thereof according to item 6, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 38 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, respectively;

[0039] the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:41, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, respectively;

[0040] the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:42, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:43, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, respectively;

[0041] the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, respectively;

[0042] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 46, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to said amino acid sequence, respectively; or

[0043] The heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:48, and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:49, or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence, respectively.

[0044] 8. The antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof.

[0045] 9. The antibody or antigen-binding fragment thereof according to item 7, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.

[0046] 10. The antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises a constant domain of human IgG1, IgG2, IgG3 or IgG4.

[0047] 11. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of items 1 to 10.

[0048] 12. An expression vector comprising the polynucleotide according to item 11.

[0049] 13. A host cell comprising the expression vector according to item 12, preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

[0050] 14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of items 1 to 10, comprising:

[0051] (a) cultivating the host cell according to item 13; and

[0052] (b) recovering the antibody or antigen-binding fragment thereof from the cultured host cells.

[0053] 15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 10 and a pharmaceutically acceptable excipient.

[0054] 16. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

[0055] 17. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

[0056] 18. A method for treating a subject suffering from a VISTA-related disease, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof according to any one of items 1 to 10, or the pharmaceutical composition according to item 15, the conjugate according to claim 16, or the fusion protein according to claim 17.

[0057] 19. The method of claim 18, wherein the VISTA-mediated disease is leukemia, lymphoma, myelodysplasia, myeloma, or a combination thereof.

[0058] 20. Use of the antibody or antigen-binding fragment thereof according to any one of items 1 to 10, the pharmaceutical composition according to item 15, the conjugate according to item 16, or the fusion protein according to item 17 in the preparation of a medicament for treating VISTA-mediated related diseases.

[0059] 21. Use of the antibody or antigen-binding fragment thereof described in any one of items 1 to 10, the pharmaceutical composition described in item 15, the conjugate described in item 16, or the fusion protein described in item 17 in the treatment of VISTA-mediated related diseases.

[0060] 22. The use according to item 20 or 21, wherein the VISTA-mediated related disease is leukemia, lymphoma, myelodysplasia, myeloma or a combination thereof.

[0061] Effects of the Invention

[0062] The antibodies or antigen-binding fragments thereof described in the present application can bind to the human VISTA antigen with high binding activity and can be internalized by target cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1A is an SDS-PAGE electrophoresis purity diagram of anti-VISTA antibodies.

[0064] Figure 1B is a SEC-HPLC chromatographic purity graph.

[0065] Figure 2 is a diagram of the binding activity detection of anti-VISTA antibodies using the ELISA method.

[0066] Figure 3A is a flow cytometry fluorescence sorting detection diagram of HL-60 cells incubated with fluorescently labeled anti-VISTA antibodies for 1 hour.

[0067] Figure 3B is a flow cytometry fluorescence sorting detection diagram of fluorescently labeled anti-VISTA antibodies and HL-60 cells incubated for 24 hours.

[0068] Figure 4 is a fluorescence microscopy image of fluorescently labeled anti-VISTA antibodies and HL-60 cells incubated for 24 hours. DETAILED DESCRIPTION

[0069] The present application is described in detail below in conjunction with the described embodiments. Although specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0070] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of this application shall be as defined by the attached claims.

[0071] definition

[0072] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunoreactive against a specific antigen. Antibodies can include, for example, polyclonal, monoclonal, and genetically engineered antibodies and antigen-binding fragments thereof. Antibodies can be, for example, murine antibodies, chimeric antibodies, humanized antibodies, heterologous conjugate antibodies, bispecific antibodies, diabodies, triabodies, or tetrabodies.

[0073] As used herein, the term "antigen-binding fragment" refers to a region of a molecule that specifically binds to an antigen. An antigen-binding domain can be the antigen-binding portion of an antibody or an antigen-binding fragment thereof. An antigen-binding fragment can include, for example, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single domain antibody, VHH, VNAR, sdAb, or nanobody.

[0074] As used herein, the term "variable region" refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are kept close together by the FRs and, together with the CDRs from the other chains, contribute to the formation of the antigen binding site of the antibody.

[0075] As used herein, the term "identity" refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., identity = # of identical positions / # of total positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment, and the length of each gap. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0076] As used herein, the term "human consensus framework region" is a framework representing the amino acid residues that are most commonly present in a selection of human immunoglobulin VL or VH framework sequences. In general, the human immunoglobulin VL or VH sequence is selected from a subset of variable domain sequences. In general, the sequence subsets are those described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), Vols. 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described by Kabat et al. (supra). In one embodiment, for VH, the subgroup is subgroup III as described by Kabat et al. (supra).

[0077] As used herein, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may exist in trace amounts. Monoclonal antibodies are highly specific for a single antigenic site. In addition, unlike polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates that the antibody derives from a substantially homologous antibody population and should not be construed as requiring the production of the antibody by any ad hoc method.

[0078] As used herein, the term "chimeric antibody" refers to an antibody in which part or all of the variable region amino acid sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region amino acid sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0079] As used herein, the term "humanized" antibody refers to a form of non-human (e.g., murine) antibody that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof containing minimal sequence derived from a non-human immunoglobulin (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody). Preferably, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from one or more CDRs of the recipient are replaced by residues from one or more CDRs of a non-human species (such as mouse, rat, or rabbit) (donor antibody) having the desired specificity, affinity, and capacity.

[0080] As used herein, the term "multispecific antibody" refers to an antibody that binds to two or more different epitopes on at least two or more different targets, including bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, and the like.

[0081] As used herein, the term "conjugate" refers to a molecule conjugated to another molecule. In the present application, the conjugate is a conjugate obtained by conjugating the anti-VISTA antibody or antigen-binding fragment thereof described in the present application to another agent, such as a small molecule drug. In the present application, there is no limitation on small molecule drugs, and those skilled in the art can make routine selections as needed.

[0082] As used herein, the term "fusion protein" refers to a substance obtained by combining an antibody with a substance via a non-peptide linker or a peptide linker, or directly combining the antibody. In the present application, the fusion protein refers to a substance obtained by combining the anti-VISTA antibody or its antigen-binding fragment with a substance via a non-peptide linker or a peptide linker, or directly combining the antibody. In the present application, there is no limitation on a substance and a non-peptide linker, and those skilled in the art can make conventional selections as needed.

[0083] As used herein, "specifically binds" or is referred to as "preferentially binds" (used interchangeably herein) an antibody, conjugate or polypeptide to a target or antigen (e.g., VISTA protein) is a conventional technical term in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds to a particular cell or substance more often, more quickly, for a greater duration and / or with greater affinity than an alternative cell or substance. An antibody "specifically binds" or "preferentially binds" to a target or antigen if it binds with greater affinity, more readily and / or for a greater duration than other substances. For example, an antibody that specifically or preferentially binds to a VISTA epitope is an antibody that binds to the epitope with greater affinity, avidity, more readily and / or for a greater duration than it binds to other VISTA epitopes or non-VISTA epitopes.

[0084] As used herein, the term "operably linked" means a functional connection between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or binding site array for a transcriptional regulator) and another nucleic acid sequence, and enables the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0085] When prokaryotic cells are used as host cells, they generally contain a strong promoter capable of carrying out transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. In addition, for example, when eukaryotic cells are used as host cells, they contain a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter, a β-actin promoter, a human hemoglobin promoter, or a human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter), and they generally have a polyadenylation sequence as a transcription termination sequence.

[0086] Optionally, the vector can be fused to another sequence to purify the antibody expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6x His (six histidine; Qiagen, USA), etc. The vector includes antibiotic resistance genes commonly used as selective markers in the art, and the resistance genes include: ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0087] As used herein, the term "administering" refers to parenteral, intravenous, intraperitoneal, intramuscular, intratumoral, intralesional, intranasal or subcutaneous administration to a subject in need thereof, oral administration, administration by suppository, topical contact, intrathecal administration, or implantation of a sustained-release device, such as a mini-osmotic pump.

[0088] As used herein, "subject" refers to an individual who is at risk of or has a disease, disorder, or condition, and who can be treated or improved with the antibodies or pharmaceutical compositions described herein. In some embodiments, the antibodies or pharmaceutical compositions of the present application are administered to the subject to treat the disease.

[0089] As used herein, the terms "treatment," "treat," and "treating" refer to any intervention that results in any observable beneficial effect of treatment, or any statistically significant indicator of success in treating or ameliorating a disease or condition, such as improving the signs, symptoms, or progression of a disease or pathological condition. For example, a beneficial effect can be demonstrated by a decrease in disease in a subject, delaying the onset of disease, or lessening the severity of clinical symptoms of disease, reducing the frequency with which a subject experiences symptoms of disease, slowing the progression of disease, reducing the number of relapses of disease, improving the subject's overall health, or by other parameters specific to a particular disease.

[0090] As used herein, the term "prevention" is treatment administered to a subject who does not exhibit signs of a disease or who exhibits only early signs of the disease, with the goal of reducing the risk of developing the pathology or further progression of the early stage disease.

[0091] As used herein, the term "effective amount" or "effective dose" refers to an amount of an antibody or its pharmaceutical composition sufficient to achieve a desired (e.g., beneficial) effect in a subject receiving treatment with the antibody or its pharmaceutical composition, such as an amount sufficient to improve one or more symptoms of the disease being treated in a statistically significant manner, to slow the deterioration of a progressive disease in a statistically significant manner, or to prevent the onset of other related symptoms or diseases in a statistically significant manner, or any combination thereof. In some embodiments, an effective amount of an antibody or its pharmaceutical composition is an amount sufficient to inhibit or treat a disease, with minimal or no toxicity in the subject, excluding the presence of one or more adverse side effects. An effective amount or dose can be administered once or multiple times within a given time period. The effective amount or dose can depend on the purpose of the treatment, and one skilled in the art can determine it according to the needs of the subject. When referring to a single active ingredient administered alone, the effective amount or dose refers to that single ingredient. When referring to a combination, the effective amount or dose refers to the combined amount of the active ingredients that produce a therapeutic effect, whether administered continuously or simultaneously.

[0092] Antibodies or antigen-binding fragments thereof

[0093] The present application provides an anti-VISTA antibody or antigen-binding fragment thereof, comprising a complementary determining region (CDR) sequence selected from the following: SEQ ID NO: 2-37, or comprising an amino acid sequence having at least 80% identity with the complementary determining region (CDR) sequence, wherein the complementary determining region is determined based on the kabat numbering system. In some embodiments, it comprises a human framework region amino acid sequence, preferably, the human framework region amino acid sequence is a human consensus framework region amino acid sequence. In some embodiments, it comprises a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence selected from the following: SEQ ID NO: 38-49, or comprising an amino acid sequence having at least 80% identity with the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence, respectively.

[0094] The amino acid sequence of SEQ ID NO: 2 is as follows:

[0095] CDRH1:STGYSWS

[0096] The amino acid sequence of SEQ ID NO: 3 is as follows:

[0097] CDRH2:NIYHSGSTYYNPSLES

[0098] The amino acid sequence of SEQ ID NO:4 is as follows:

[0099] CDRH3:QGGSSSSWYYYYMDV

[0100] The amino acid sequence of SEQ ID NO:5 is as follows:

[0101] CDRL1:SGSSSNIGNNYVS

[0102] The amino acid sequence of SEQ ID NO:6 is as follows:

[0103] CDRL2:DNNKRPS

[0104] The amino acid sequence of SEQ ID NO:7 is as follows:

[0105] CDRL3:GTWDSSLSAVV

[0106] The amino acid sequence of SEQ ID NO:8 is as follows:

[0107] CDRH1:SYAIS

[0108] The amino acid sequence of SEQ ID NO:9 is as follows:

[0109] CDRH2:GIIPIFGTANYAQKFQG

[0110] The amino acid sequence of SEQ ID NO: 10 is as follows:

[0111] CDRH3:ELGSTGAYYYYGMDV

[0112] The amino acid sequence of SEQ ID NO: 11 is as follows:

[0113] CDRL1:SGSSSNIGSNTVN

[0114] The amino acid sequence of SEQ ID NO: 12 is as follows:

[0115] CDRL2:SNNQRPS

[0116] The amino acid sequence of SEQ ID NO: 13 is as follows:

[0117] CDRL3:AAWDDSLSGWV

[0118] The amino acid sequence of SEQ ID NO: 14 is as follows:

[0119] CDRH1:GSAMH

[0120] The amino acid sequence of SEQ ID NO: 15 is as follows:

[0121] CDRH2:RIRSKANSYATAYAASVKG

[0122] The amino acid sequence of SEQ ID NO: 16 is as follows:

[0123] CDRH3:VNSGYYGPDY

[0124] The amino acid sequence of SEQ ID NO: 17 is as follows:

[0125] CDRL1:TRNGGSIASNYVQ

[0126] The amino acid sequence of SEQ ID NO: 18 is as follows:

[0127] CDRL2:EDNQRPS

[0128] The amino acid sequence of SEQ ID NO: 19 is as follows:

[0129] CDRL3:QSYDSSNVV

[0130] The amino acid sequence of SEQ ID NO: 20 is as follows:

[0131] CDRH1:TYAIS

[0132] The amino acid sequence of SEQ ID NO:21 is as follows:

[0133] CDRH2:WITTYEGNTHYAPKFHG

[0134] The amino acid sequence of SEQ ID NO:22 is as follows:

[0135] CDRH3:HNYEMNWTFQDY

[0136] The amino acid sequence of SEQ ID NO:23 is as follows:

[0137] CDRL1:SGSSSNIGSNSVD

[0138] The amino acid sequence of SEQ ID NO:24 is as follows:

[0139] CDRL2:NNNQRPS

[0140] The amino acid sequence of SEQ ID NO:25 is as follows:

[0141] CDRL3:AAWDDSLNGLV

[0142] The amino acid sequence of SEQ ID NO:26 is as follows:

[0143] CDRH1:TSGVGVG

[0144] The amino acid sequence of SEQ ID NO:27 is as follows:

[0145] CDRH2:LIYWDDDKHYRPSLES

[0146] The amino acid sequence of SEQ ID NO:28 is as follows:

[0147] CDRH3:RLYYDSSGYYKTFDI

[0148] The amino acid sequence of SEQ ID NO: 29 is as follows:

[0149] CDRL1:TLSSEHSTYTIE

[0150] The amino acid sequence of SEQ ID NO:30 is as follows:

[0151] CDRL2:VKSDGSHIKGD

[0152] The amino acid sequence of SEQ ID NO:31 is as follows:

[0153] CDRL3:GESHTIDGQVGYV

[0154] The amino acid sequence of SEQ ID NO:32 is as follows:

[0155] CDRH1: SNSAAWN

[0156] The amino acid sequence of SEQ ID NO:33 is as follows:

[0157] CDRH2:RTYYRSKWYYDYAVSLKS

[0158] The amino acid sequence of SEQ ID NO:34 is as follows:

[0159] CDRH3:NYAFDF

[0160] The amino acid sequence of SEQ ID NO:35 is as follows:

[0161] CDRL1:SGSSSNIGSHYVF

[0162] The amino acid sequence of SEQ ID NO:36 is as follows:

[0163] CDRL2:SNDQRPS

[0164] The amino acid sequence of SEQ ID NO:37 is as follows:

[0165] CDRL3:VAWDGGLRNYV

[0166] The amino acid sequence of SEQ ID NO:38 is as follows:

[0167] The amino acid sequence of SEQ ID NO:39 is as follows:

[0168] The amino acid sequence of SEQ ID NO:40 is as follows:

[0169] The amino acid sequence of SEQ ID NO:41 is as follows:

[0170] The amino acid sequence of SEQ ID NO:42 is as follows:

[0171] The amino acid sequence of SEQ ID NO:43 is as follows:

[0172] The amino acid sequence of SEQ ID NO:44 is as follows:

[0173] The amino acid sequence of SEQ ID NO:45 is as follows:

[0174] The amino acid sequence of SEQ ID NO:46 is as follows:

[0175] The amino acid sequence of SEQ ID NO:47 is as follows:

[0176] The amino acid sequence of SEQ ID NO:48 is as follows:

[0177] The amino acid sequence of SEQ ID NO:49 is as follows:

[0178] For example, when the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80% identical to the CDR, respectively, it may comprise an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to the CDR, respectively;

[0179] When the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80% identical to the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence, respectively, it may comprise an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or 100% identical to the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence, respectively.

[0180] In some embodiments, the antibody is 2C12 antibody, 2G10 antibody, 2B7 antibody, 2A4 antibody, 2H2 antibody, or 1H11 antibody.

[0181] When the antibody comprises an amino acid sequence of SEQ ID NO: 2-7 in the complementarity determining region, it is a 2C12 antibody;

[0182] When the antibody comprises the amino acid sequence of SEQ ID NOs: 8-13 in the complementarity determining region, it is the 2G10 antibody;

[0183] When the antibody comprises the amino acid sequence of SEQ ID NOs: 14-19 in the complementarity determining region, it is a 2B7 antibody;

[0184] When the antibody comprises the amino acid sequence of SEQ ID NOs: 20-25 in the complementarity determining region, it is a 2A4 antibody;

[0185] When the antibody comprises the amino acid sequence of SEQ ID NOs: 26-31 in the complementarity determining region, it is a 2H2 antibody;

[0186] When the antibody comprises the amino acid sequence of SEQ ID NOs: 32-37 in the complementarity determining region, it is the 1H11 antibody;

[0187] When the antibody comprises a heavy chain variable region and a light chain variable region of SEQ ID NOs: 38-39, respectively, it is a 2C12 antibody;

[0188] When the antibody comprises a heavy chain variable region and a light chain variable region of SEQ ID NOs: 40-41, respectively, it is a 2G10 antibody;

[0189] When the antibody comprises a heavy chain variable region and a light chain variable region of SEQ ID NOs: 42-43, respectively, it is a 2B7 antibody;

[0190] When the antibody comprises a heavy chain variable region and a light chain variable region of SEQ ID NOs: 44-45, respectively, it is a 2A4 antibody;

[0191] When the antibody comprises a heavy chain variable region and a light chain variable region of SEQ ID NOs: 46-47, respectively, it is a 2H2 antibody;

[0192] When the antibody comprises a heavy chain variable region and a light chain variable region of SEQ ID NOs: 48-49, respectively, it is a 1H11 antibody.

[0193] In some embodiments, the heavy chain of the 2C12 antibody comprises the amino acid sequence shown in SEQ ID NO:50 or is SEQ ID NO:50 and the light chain of the 2C12 antibody comprises the amino acid sequence shown in SEQ ID NO:51 or is SEQ ID NO:51.

[0194] The amino acid sequence of SEQ ID NO:50 is as follows:

[0195] The amino acid sequence of SEQ ID NO:51 is as follows:

[0196] In some embodiments, the heavy chain of the 2G10 antibody comprises the amino acid sequence shown in SEQ ID NO: 52 or is SEQ ID NO: 52, and the light chain of the 2G10 antibody comprises the amino acid sequence shown in SEQ ID NO: 53 or is SEQ ID NO: 53.

[0197] The amino acid sequence of SEQ ID NO:52 is as follows:

[0198] The amino acid sequence of SEQ ID NO:53 is as follows:

[0199] In some embodiments, the heavy chain of the 2B7 antibody comprises the amino acid sequence shown in SEQ ID NO: 54 or is SEQ ID NO: 54, and the light chain of the 2B7 antibody comprises the amino acid sequence shown in SEQ ID NO: 55 or is SEQ ID NO: 55.

[0200] The amino acid sequence of SEQ ID NO:54 is as follows:

[0201] The amino acid sequence of SEQ ID NO:55 is as follows:

[0202] In some embodiments, the heavy chain of the 2A4 antibody comprises the amino acid sequence shown in SEQ ID NO:56 or is SEQ ID NO:56, and the light chain of the 2A4 antibody comprises the amino acid sequence shown in SEQ ID NO:57 or is SEQ ID NO:57.

[0203] The amino acid sequence of SEQ ID NO:56 is as follows:

[0204] The amino acid sequence of SEQ ID NO:57 is as follows:

[0205] In some embodiments, the heavy chain of the 2H2 antibody comprises the amino acid sequence shown in SEQ ID NO: 58 or is SEQ ID NO: 58, and the light chain of the 2H2 antibody comprises the amino acid sequence shown in SEQ ID NO: 59 or is SEQ ID NO: 59.

[0206] The amino acid sequence of SEQ ID NO:58 is as follows:

[0207] The amino acid sequence of SEQ ID NO:59 is as follows:

[0208] In some embodiments, the heavy chain of the 1H1 antibody comprises the amino acid sequence shown in SEQ ID NO: 60 or is SEQ ID NO: 60, and the light chain of the 1H1 antibody comprises the amino acid sequence shown in SEQ ID NO: 61 or is SEQ ID NO: 61.

[0209] The amino acid sequence of SEQ ID NO:60 is as follows:

[0210] The amino acid sequence of SEQ ID NO:61 is as follows:

[0211] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the V-domain Ig inhibitory factor of human T cell activation (VISTA), and preferably, the amino acid sequence of the VISTA antigen comprises the amino acid sequence shown in SEQ ID NO: 1 or is SEQ ID NO: 1.

[0212] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0213] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a scFv fragment. In some embodiments, the antibody or antigen-binding fragment thereof comprises the constant domain of human IgG1, IgG2, IgG3, or IgG4.

[0214] In some embodiments, the anti-VISTA antibody or antigen-binding fragment thereof comprises a complementarity determining region (CDR) sequence selected from the following: SEQ ID NO: 2-37, or comprises an amino acid sequence having at least 80% identity with each of the complementarity determining region sequences. In some embodiments, the complementarity determining region comprises a heavy chain complementarity determining region and a complementarity determining region;

[0215] The heavy chain complementarity determining regions are:

[0216] comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 2, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 3, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 4;

[0217] comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:8, a CDR2 having the amino acid sequence set forth in SEQ ID NO:9, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:10;

[0218] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 14, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 15, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 16;

[0219] comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 20, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 21, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 22;

[0220] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 27, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 28; or

[0221] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO:32, a CDR2 having an amino acid sequence as set forth in SEQ ID NO:33, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO:34;

[0222] The light chain complementarity determining region is

[0223] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO:5, a CDR2 having an amino acid sequence as set forth in SEQ ID NO:6, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO:7;

[0224] comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 13;

[0225] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 17, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 18, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 19;

[0226] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 23, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 24, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 25;

[0227] comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO:29, a CDR2 having an amino acid sequence as set forth in SEQ ID NO:30, and a CDR3 having an amino acid sequence as set forth in SEQ ID NO:31; or

[0228] comprising a CDR1 with the amino acid sequence set forth in SEQ ID NO: 35, a CDR2 with the amino acid sequence set forth in SEQ ID NO: 36, and a CDR3 with the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the heavy chain complementary determining region is the CDR1 with the amino acid sequence set forth in SEQ ID NO: 2, the CDR2 with the amino acid sequence set forth in SEQ ID NO: 3, and the CDR3 with the amino acid sequence set forth in SEQ ID NO: 4, and the light chain complementary determining region is the CDR1 with the amino acid sequence set forth in SEQ ID NO: 5, the CDR2 with the amino acid sequence set forth in SEQ ID NO: 6, and the CDR3 with the amino acid sequence set forth in SEQ ID NO: 7;

[0229] The heavy chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 8, CDR2 of the amino acid sequence set forth in SEQ ID NO: 9, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 10, and the light chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 11, CDR2 of the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 13;

[0230] The heavy chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 14, CDR2 of the amino acid sequence set forth in SEQ ID NO: 15, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 16, and the light chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 17, CDR2 of the amino acid sequence set forth in SEQ ID NO: 18, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 19;

[0231] The heavy chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 20, CDR2 of the amino acid sequence set forth in SEQ ID NO: 21, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 22, and the light chain complementary determining regions are CDR1 of the amino acid sequence set forth in SEQ ID NO: 23, CDR2 of the amino acid sequence set forth in SEQ ID NO: 24, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 25;

[0232] The heavy chain complementary determining regions are CDR1 of the amino acid sequence shown in SEQ ID NO: 26, CDR2 of the amino acid sequence shown in SEQ ID NO: 27, and CDR3 of the amino acid sequence shown in SEQ ID NO: 28, and the light chain complementary determining regions are CDR1 of the amino acid sequence shown in SEQ ID NO: 29, CDR2 of the amino acid sequence shown in SEQ ID NO: 30, and CDR3 of the amino acid sequence shown in SEQ ID NO: 31; or

[0233] The heavy chain complementary determining region is CDR1 of the amino acid sequence set forth in SEQ ID NO: 32, CDR2 of the amino acid sequence set forth in SEQ ID NO: 33, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 34, and the light chain complementary determining region is CDR1 of the amino acid sequence set forth in SEQ ID NO: 35, CDR2 of the amino acid sequence set forth in SEQ ID NO: 36, and CDR3 of the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human framework region amino acid sequence, preferably, the human framework region amino acid sequence is a human consensus framework region amino acid sequence. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence selected from the group consisting of SEQ ID NOs: 38-49, or comprises an amino acid sequence that is at least 80% identical to the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence, respectively. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38, the amino acid sequence shown in SEQ ID NO: 40, the amino acid sequence shown in SEQ ID NO: 42, the amino acid sequence shown in SEQ ID NO: 44, the amino acid sequence shown in SEQ ID NO: 46, or the amino acid sequence shown in SEQ ID NO: 48; and / or

[0234] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39, the amino acid sequence shown in SEQ ID NO: 41, the amino acid sequence shown in SEQ ID NO: 43, the amino acid sequence shown in SEQ ID NO: 45, the amino acid sequence shown in SEQ ID NO: 47, or the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39; or

[0235] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 40 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 41; or

[0236] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 42 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 43; or

[0237] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 44 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 45; or

[0238] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 46 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47; or

[0239] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies. In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or a scFv fragment. In some embodiments, the antibody or its antigen-binding fragment comprises the constant domain of human IgG1, IgG2, IgG3, or IgG4.

[0240] Polynucleotides, expression vectors and host cells

[0241] The present application provides a polynucleotide encoding the antibody or antigen-binding fragment thereof described above. The present application provides an expression vector comprising the polynucleotide described above. The present application provides a host cell comprising the expression vector described above. The present application provides a method for preparing the antibody or antigen-binding fragment thereof, comprising: culturing the host cell described above and recovering the antibody or antigen-binding fragment thereof from the cultured host cell.

[0242] The polynucleotides encompass DNA (gDNA and cDNA) and RNA molecules, as well as nucleotides, which are the basic building blocks of polynucleotides and include nucleotides of natural origin and analogs with modified sugar or base moieties. The sequences of the polynucleotides encoding the heavy and light chain variable regions of the present application can be altered. Such variations include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0243] DNA encoding the antibodies or antigen-binding fragments thereof can be readily isolated or synthesized using conventional procedures (eg, using oligonucleotide probes that are capable of binding specifically to DNA encoding the antibody heavy and light chains).

[0244] The expression vector refers to a tool for expressing a target gene in a host cell, and includes plasmid vectors, cosmid vectors, and viral vectors, such as phage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. The nucleic acid encoding the antibody or antigen-binding fragment thereof in the expression vector is operably linked to a promoter.

[0245] The host cell is a host cell used to produce the antibody or antigen-binding fragment thereof described in the present application. The host cell used to produce the antibody or antigen-binding fragment thereof can be a prokaryotic organism, yeast or higher eukaryotic cell, but is not limited thereto. Prokaryotic host cells that can be used include, for example, Escherichia coli; Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis; Streptomyces species; Pseudomonas species (e.g., Pseudomonas putida); Proteus mirabilis; and Staphylococcus species (e.g., Staphylococcus carnosus). Animal cells that can be used include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S and HT1080.

[0246] In the present application, host cells can be cultured in various culture media. Any commercially available culture medium can be used as a culture medium. All other necessary supplements well known to those skilled in the art can be added at appropriate concentrations. Culture conditions such as temperature and pH are all conventional culture conditions in the art, and those skilled in the art can make conventional selections as needed.

[0247] The antibody or antigen-binding fragment thereof can be recovered by, for example, centrifugation or ultrafiltration to remove impurities and purifying the resulting product using, for example, affinity chromatography. Other purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite (HA) chromatography, can also be used.

[0248] Pharmaceutical composition

[0249] The present application provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment thereof and a pharmaceutically acceptable excipient.

[0250] The antibodies or antigen-binding fragments thereof described herein can be formulated into pharmaceutical compositions by combining with suitable pharmaceutically acceptable carriers or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous form, for example, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols, etc.

[0251] In pharmaceutical dosage forms, the antibody or antigen-binding fragment thereof can be administered in combination with a pharmaceutically acceptable excipient, or can be used alone or in appropriate combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are not intended to limit the methods and excipients.

[0252] The antibody or antigen-binding fragment thereof can be formulated into an injectable preparation by dissolving, suspending or emulsifying in water or a non-aqueous solvent (e.g., vegetable oil or other similar oils, synthetic fatty acid glycerides, higher fatty acid esters or propylene glycol); if necessary, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives can also be added.

[0253] Pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof are prepared by mixing an antibody or antigen-binding fragment thereof of the desired purity with optional physiologically acceptable excipients (e.g., carriers, excipients, stabilizers, surfactants, buffers, and / or tonicity agents). Acceptable carriers, excipients, and / or stabilizers are non-toxic to the recipient at the doses and concentrations used. Acceptable carriers, excipients, and / or stabilizers include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (e.g., ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl parahydroxybenzoate, benzalkonium chloride, or a combination thereof); amino acids (e.g., arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, thiazolinone ... amino acids, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof); monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins (e.g., gelatin or serum albumin); chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbitol, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0254] The pharmaceutical composition may be in liquid form, in lyophilized form, or in liquid form reconstituted in a lyophilized form, wherein the lyophilized formulation is reconstituted with a sterile solution prior to administration.

[0255] Therapeutic applications

[0256] The present application provides a method for treating a subject suffering from a VISTA-mediated disease, the method comprising administering to the subject an effective amount of the above-described antibody or antigen-binding fragment thereof or the above-described pharmaceutical composition. In some embodiments, the VISTA-mediated disease is leukemia, lymphoma, myelodysplasia, myeloma, or a combination thereof.

[0257] The present application provides the use of the above-mentioned antibody or its antigen-binding fragment or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating a disease related to VISTA mediation. The present application also provides the use of the above-mentioned antibody or its antigen-binding fragment or the above-mentioned pharmaceutical composition for treating a disease related to VISTA mediation. In some embodiments, the disease related to VISTA mediation is leukemia, lymphoma, myelodysplasia, myeloma or a combination thereof.

[0258] The antibodies or antigen-binding fragments thereof and pharmaceutical compositions described herein can be used in a variety of different subjects, including but not limited to mammals, humans, non-human mammals, domesticated animals (e.g., laboratory animals, household pets or livestock), non-domesticated animals (e.g., wild animals), dogs, cats, rodents, mice, hamsters, cows, birds, chickens, fish, pigs, horses, goats, sheep, rabbits, and any combination thereof. In some embodiments, the subject is a human.

[0259] The antibody or its antigen-binding fragment and pharmaceutical composition of the present application can be used as a therapeutic agent, for example, a therapeutic product that can be applied to a subject in need. The therapeutic effect of the antibody or its antigen-binding fragment and pharmaceutical composition of the present application can be obtained in the subject by reducing, suppressing, alleviating or eradicating the disease state (including but not limited to its symptoms). The therapeutic effect in a subject suffering from a disease or susceptible to or just beginning to suffer from a disease or condition can be obtained by reducing, suppressing, preventing, alleviating or eradicating the condition or disease or before the condition or pre-disease state.

[0260] The antibody or its antigen-binding fragment or its pharmaceutical composition of the present application of an effective amount or dosage to the subject can be carried out by one or more routes, and can occur once or multiple times within a given time period. It will be understood by those of ordinary skill in the art that the amount, duration and frequency of application of the conjugate of the present application or its pharmaceutical composition to the subject in need depends on several factors, including, for example, the health status of the subject, the specific disease or condition of the subject, the grade or level of the specific disease or condition of the subject, the additional treatment that the subject is receiving or has received, etc. Exemplary routes of administration include systemic, cutaneous, subcutaneous, intravenous, intraarterial, subdural, intramuscular, intracranial, intrasternal, intratumoral, intraperitoneal. In addition, the conjugate of the present application or its pharmaceutical composition can be applied to the subject by other routes of administration, for example, by inhalation, or administered orally, cutaneously, intranasally or intrathecally.

[0261] The antibodies or antigen-binding fragments thereof and pharmaceutical compositions described herein can be administered to a subject in need in a first administration and subsequently administered to a subject in need in one or more additional administrations. One or more additional administrations can be administered to a subject in need a few minutes, hours, days, weeks, or months after the first administration. Any of the additional administrations can be administered to a subject in need less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days, or less than 1 day after the first administration. One or more administrations can occur more than once a day, more than once a week, or more than once a month. Administration can be weekly, biweekly / every two weeks, every three weeks, monthly, or every two months.

[0262] Example

[0263] This application provides general and / or specific descriptions of the materials and experimental methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight. All reagents or instruments used without manufacturer indication are commercially available conventional reagents.

[0264] Example 1 Preparation of Antibodies

[0265] The Human VISTA (M1-A194)-Fc sequence (Nishant Mehta1, et al., 2021.) was selected as the antibody screening antigen (abbreviated as hVISTA-Fc). First, the hVISTA-Fc sequence was gene synthesized and plasmid constructed, and then the ExpiCHO-S TM The antigen protein was transiently expressed in cells (manufacturer: ThermoFisher) and obtained through Protein A affinity chromatography.

[0266] Using hVISTA-Fc as the target antigen, two antibody libraries, scFv-κ190625 and scFv-λ190718, were screened. The two antibody libraries were subjected to a "bind-elute-amplification" process, and positive clones were screened through three rounds of solid-phase panning and monoclonal ELISA identification. The scFv sequences of the positive clones were transformed into human IgG1 format, and gene synthesis and vector construction were performed. The resulting plasmids expressed antibodies according to the transient transfection method provided by the CHO-S cell manufacturer. The cell culture supernatant was purified using Protein A affinity chromatography (manufacturer: Cytiva). The resulting samples were tested for purity by SDS-PAGE and SEC-HPLC. After passing the test, subsequent antibody evaluation experiments were carried out. The results are shown in Figures 1A and 1B, respectively.

[0267] As can be seen from FIG. 1A and FIG. 1B , the 2C12 antibody, 2G10 antibody, 2B7 antibody, 2A4 antibody, 2H2 antibody or 1H11 antibody obtained through screening were all correctly expressed, which was consistent with the expected theory.

[0268] Example 2 ELISA binding activity test of anti-VISTA antibodies and human VISTA antigen

[0269] Human hVISTA-Fc was diluted to 10 μg / ml with coating solution, and 50 μl / well was added to a 96-well plate. The plate was coated at 37°C for 2 h. After washing, 200 μl of 5% skim milk powder was added to each well and the plate was blocked at 4°C overnight.

[0270] The antibodies prepared in Example 1 were diluted to 100 μg / ml with PBS and then to 10 μg / ml with 5% skim milk powder as the starting concentration. Each antibody was then serially diluted 10-fold for a total of eight dilutions and blocked at 37°C for 0.5 h. After washing, the blocked antibodies were transferred to antigen-coated ELISA plates, with 50 μl / well incubated at 37°C for 1 h. After washing, 1 μl of secondary antibody (manufacturer: Jackson ImmunoResearch) was added to 5 ml of 5% skim milk powder at a 1:5000 dilution, mixed, and 50 μl of the diluted secondary antibody was added to each well. The plates were incubated at 37°C for 0.5 h. 50 μl of TMB colorimetric solution (manufacturer: KPL) was added to each well and color developed at room temperature for approximately 5 min. After the positive control developed significant color, 50 μl of stop solution was added to each well to stop color development. The readings were detected using a microplate reader at a wavelength of 450 nm. The binding activity results are shown in Figure 2.

[0271] As can be seen from Figure 2, the obtained 2C12 antibody and 2A4 antibody have good binding activity with the human VISTA antigen.

[0272] Example 3 Anti-VISTA antibody-mediated HL-60 cell endocytosis activity test

[0273] The antibody prepared in Example 1 was stained with Alexa Fluor TM HL-60 cells (manufacturer: ATCC) were plated in 96-well plates at a cell density of 1×10 6 cells / mL, the culture medium was 80% IMDM + 20% FBS + 1% antibiotics. Alexa Fluor was added 2 hours after plating. TM 647 fluorescently labeled anti-VISTA antibody was added to give a final antibody concentration of 4 μg / mL. Flow cytometry fluorescence sorting (FACS) detection was performed 1 h and 24 h after the addition of the antibody, respectively, with an excitation wavelength of 638 nm and a fluorescence detection channel of 660 nm. The detection results are shown in Figures 3A and 3B, wherein Figure 3A is a flow cytometry fluorescence sorting detection diagram of fluorescently labeled anti-VISTA antibody and HL-60 cells incubated for 1 h, and Figure 3B is a flow cytometry fluorescence sorting detection diagram of fluorescently labeled anti-VISTA antibody and HL-60 cells incubated for 24 h.

[0274] As can be seen from Figures 3A and 3B, at 1 hour, the 2C12 antibody had good endocytosis activity in HL-60 cells, with the highest intracellular mean fluorescence intensity. At 24 hours, the 2C12, 2A4, and 2H2 antibodies all showed good endocytosis activity.

[0275] Example 4 Fluorescence detection of anti-VISTA antibody-mediated endocytosis in HL-60 cells

[0276] The antibody prepared in Example 1 was stained with Alexa Fluor TM 594 (manufacturer: ThermoFisher) for fluorescent labeling. HL-60 cells were plated in 96-well plates at a cell density of 1×10 6 cells / mL, the culture medium was 80% IMDM + 20% FBS + 1% antibiotics. Alexa Fluor was added 2 hours after plating. TM 594 fluorescently labeled anti-VISTA antibody was added to a final concentration of 4 μg / mL. 24 h after adding the antibody, the internalization of the VISTA antibody into HL-60 cells was observed by fluorescence microscopy. The detection results are shown in Figure 4, where the arrows in Figure 4 represent red fluorescent signals.

[0277] As shown in Figure 4, after 24 hours of incubation of the antibodies with HL-60 cells, no significant red fluorescence signal was observed in cells treated with 1H11, indicating that it was unable to be internalized into the cells. However, after incubation of HL-60 cells with 2C12, 2A4, and 2H2, red fluorescence signals of varying intensities appeared, indicating that these antibodies could be internalized into the cells, with 2C12 exhibiting the best endocytic activity.

[0278] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application in any other manner. Any person skilled in the art may utilize the above disclosed technical content to modify or modify the present application into equivalent embodiments with equivalent variations. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present application and are based on the technical essence of the present application shall still fall within the scope of protection of the present application.

Claims

1. An anti-VISTA antibody or an antigen-binding fragment thereof, comprising a complementary determining region (CDR) sequence selected from the following: SEQ ID NO: 2-37, or an amino acid sequence having at least 80% identity with the complementary determining region sequence, respectively, wherein the complementary determining region is determined based on the Kabat numbering system.

2. The anti-VISTA antibody or antigen-binding fragment thereof according to claim 1, wherein The complementary determining regions include a heavy chain complementary determining region and a light chain complementary determining region; Wherein, the heavy chain complementary determining region is: comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:2, a CDR2 having an amino acid sequence as shown in SEQ ID NO:3, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:4; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:8, a CDR2 having an amino acid sequence as shown in SEQ ID NO:9, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:10; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 14, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 15, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 16; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:20, a CDR2 having an amino acid sequence as shown in SEQ ID NO:21, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:22; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:26, a CDR2 having an amino acid sequence as shown in SEQ ID NO:27, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:28; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:32, a CDR2 having an amino acid sequence as shown in SEQ ID NO:33, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:34; and Wherein, the light chain complementary determining region is comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:5, a CDR2 having an amino acid sequence as shown in SEQ ID NO:6, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:7; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 11, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 12, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 13; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 18, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 19; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:23, a CDR2 having an amino acid sequence as shown in SEQ ID NO:24, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:25; comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:29, a CDR2 having an amino acid sequence as shown in SEQ ID NO:30, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:31; or It comprises a CDR1 with the amino acid sequence shown in SEQ ID NO:35, a CDR2 with the amino acid sequence shown in SEQ ID NO:36, and a CDR3 with the amino acid sequence shown in SEQ ID NO:

37.

3. The antibody or antigen-binding fragment thereof according to claim 2, comprising a heavy chain complementary determining region and a light chain complementary determining region as shown below: (1) the heavy chain complementary determining region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:2, a CDR2 comprising the amino acid sequence of SEQ ID NO:3, and a CDR3 comprising the amino acid sequence of SEQ ID NO:4, and the light chain complementary determining region comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:5, a CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a CDR3 comprising the amino acid sequence of SEQ ID NO:7; (2) the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO:8, a CDR2 comprising the amino acid sequence of SEQ ID NO:9, and a CDR3 comprising the amino acid sequence of SEQ ID NO:10, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO:11, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:13; (3) the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO: 14, a CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 19; (4) the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO:20, a CDR2 comprising the amino acid sequence of SEQ ID NO:21, and a CDR3 comprising the amino acid sequence of SEQ ID NO:22, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO:23, a CDR2 comprising the amino acid sequence of SEQ ID NO:24, and a CDR3 comprising the amino acid sequence of SEQ ID NO:25; (5) the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

31. CDR3 of the amino acid sequence shown in NO:31; or (6) the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:32, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:33, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:34, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:35, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:36, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

37.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a human framework region amino acid sequence, preferably, the human framework region amino acid sequence is a human consensus framework region amino acid sequence.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a heavy chain variable region amino acid sequence and a light chain variable region amino acid sequence selected from the following: SEQ ID NO: 38-49, or an amino acid sequence that has at least 80% identity with the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38, the amino acid sequence shown in SEQ ID NO:40, the amino acid sequence shown in SEQ ID NO:42, the amino acid sequence shown in SEQ ID NO:44, the amino acid sequence shown in SEQ ID NO:46, the amino acid sequence shown in SEQ ID NO:48, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences; and / or The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, the amino acid sequence shown in SEQ ID NO:41, the amino acid sequence shown in SEQ ID NO:43, the amino acid sequence shown in SEQ ID NO:45, the amino acid sequence shown in SEQ ID NO:47, the amino acid sequence shown in SEQ ID NO:49, or an amino acid sequence that is at least 80% identical to the amino acid sequence, respectively.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39, or an amino acid sequence that is at least 80% identical to said amino acid sequence; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence that is at least 80% identical to said amino acid sequence; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:43, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:45, or an amino acid sequence that is at least 80% identical to said amino acid sequence; The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:46 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence that has at least 80% identity to each of said amino acid sequences; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 49, or an amino acid sequence that has at least 80% identity with each of the amino acid sequences.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antibody or antigen-binding fragment thereof is a monoclonal antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antigen-binding fragment thereof.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein The antigen binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein The antibody or antigen-binding fragment thereof comprises the constant domain of human IgG1, IgG2, IgG3 or IgG4.

11. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10. An expression vector comprising the polynucleotide of claim 11 .

13. A host cell comprising the expression vector according to claim 12, preferably, the host cell is a prokaryotic cell or a eukaryotic cell.

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising: (a) culturing the host cell according to claim 13; as well as (b) recovering the antibody or antigen-binding fragment thereof from the cultured host cells.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

16. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

17. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

18. A method for treating a subject suffering from a VISTA-mediated disease, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-10, the pharmaceutical composition of claim 15, the conjugate of claim 16, or the fusion protein of claim 17.

19. The method according to claim 18, wherein: The VISTA-mediated related diseases are leukemia, lymphoma, myelodysplasia, myeloma or a combination thereof.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the pharmaceutical composition according to claim 15, the conjugate according to claim 16, or the fusion protein according to claim 17 in the preparation of a drug for treating VISTA-mediated related diseases.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the pharmaceutical composition according to claim 14, the conjugate according to claim 16, or the fusion protein according to claim 17 for treating VISTA-mediated related diseases.

22. The use according to claim 20 or 21, wherein The VISTA-mediated related diseases are leukemia, lymphoma, myelodysplasia, myeloma or a combination thereof.

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