Anti-VISTA monoclonal antibodies and uses thereof

Abstract

The present invention relates to anti-VISTA monoclonal antibodies and uses thereof, more particularly to anti-VISTA monoclonal antibodies and functional fragments thereof, and their use in the prevention or treatment of cancer.

Description

[Technical Field]

[0001] This application claims priority from Korean Patent Application No. 10-2022-0112412, filed on September 5, 2022, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to anti-VISTA monoclonal antibodies and uses thereof, more particularly to anti-VISTA monoclonal antibodies and functional fragments thereof, and their use in the prevention or treatment of cancer. [Background technology]

[0003] Anticancer drugs are broadly divided into first-generation chemical anticancer drugs, second-generation targeted anticancer drugs, and third-generation immunotherapy drugs. Unlike the drawbacks of first- and second-generation anticancer drugs, third-generation immunotherapy drugs bind to the binding sites between cancer cells and T cells, blocking immune evasion signals from co-inhibitory signal receptors, allowing T cells that are not hindered by the cancer cells' immune evasion mechanisms to destroy the cancer cells. In other words, immunotherapy drugs have a new mechanism for strengthening the body's immune function that has been suppressed against cancer cells, thereby killing cancer cells. They have few side effects and are effective in improving the quality of life of cancer patients and significantly extending their survival time.

[0004] In the tumor microenvironment (TME), various immune cells (T cells, NK cells, Tregs, myeloid-derived suppressor cells, dendritic cells, M2 macrophages, M1 macrophages, etc.) and general cells form the tumor microenvironment (TME). In this environment, cancer creates an environment that suppresses the patient's innate and adaptive immunity, preventing the patient's immune system from attacking cancer cells. Therefore, methods to kill cancer cells by blocking negative immune checkpoints such as PD-1 are being studied as a way to eliminate cancer in the TME environment.

[0005] PD-1 (programmed death-1) and its ligands, PD-L1 / PD-L2, represent another immune negative checkpoint axis. The PD-1 pathway impairs T cell responses and inhibits Foxp3 in the periphery. + It promotes Treg induction and downregulates tumor-specific immune responses. On the other hand, VISTA (V-domain Ig suppressor of T cell activation) has homology to PD-L1 and shows a distinct expression pattern as a new negative checkpoint ligand that suppresses T cell activation. Therefore, there is a need to develop antibodies that specifically block VISTA as new immunological anticancer agents. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the inventors have been conducting research to develop antibodies with specific binding affinity to human VISTA protein, and have confirmed that antibodies containing the unique CDR sequence provided in the present invention have excellent binding neutralization ability against PSGL1 and VSIG3, which bind to VISTA, have strong ADCC efficacy, and activate suppressed immune functions, thereby showing significant effects when used as cancer therapeutic agents, thereby completing the present invention.

[0007] Therefore, the object of the present invention is to i) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 2, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 3; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 6. L ) antibodies; ii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 9, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 10, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 11; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 12, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 14. L ) antibodies; iii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 17, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 18, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 19; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 22. L ) antibodies; iv) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 25, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 26, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 27; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 28, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 30. L ) antibodies; v) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 33, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 34, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 35; H), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 36, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 38. L ) an antibody comprising; and vi) an antibody heavy chain variable region (V) comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 41, a complementarity-determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 42, and a complementarity-determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 43; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 44, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 46. L ) antibodies; The present invention provides an antibody or fragment thereof that binds to a human VISTA protein selected from the group consisting of:

[0008] Another object of the present invention is to provide a polynucleotide encoding the antibody or a fragment thereof, a recombinant vector containing the same, a transformed cell line containing the same, and a method for producing the antibody or a fragment thereof using the same.

[0009] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the antibody or a fragment thereof as an active ingredient. Furthermore, another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or a fragment thereof. Furthermore, another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which consists essentially of the antibody or a fragment thereof.

[0010] Another object of the present invention is to provide the use of an antibody or a fragment thereof for the manufacture of a pharmaceutical composition for treating cancer.

[0011] Another object of the present invention is to provide a method for treating cancer, which comprises administering an effective amount of a pharmaceutical composition containing the antibody or a fragment thereof as an active ingredient to an individual in need thereof.

[0012] In order to achieve the above object, the present invention provides: i) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 2, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 3; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 6. L ) antibodies; ii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 9, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 10, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 11; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 12, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 14. L ) antibodies; iii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 17, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 18, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 19; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 22. L) antibodies; iv) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 25, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 26, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 27; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 28, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 30. L ) antibodies; v) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 33, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 34, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 35; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 36, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 38. L ) an antibody comprising; and vi) an antibody heavy chain variable region (V) comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 41, a complementarity-determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 42, and a complementarity-determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 43; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 44, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 46. L ) antibodies; The present invention provides an antibody or fragment thereof that binds to a human VISTA protein selected from the group consisting of:

[0013] To achieve another object of the present invention, the present invention provides a polynucleotide encoding the antibody or a fragment thereof, a recombinant vector containing the same, a transformed cell line containing the same, and a method for producing the antibody or a fragment thereof using the same.

[0014] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the antibody or a fragment thereof as an active ingredient. Furthermore, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which comprises the antibody or a fragment thereof. Furthermore, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which consists essentially of the antibody or a fragment thereof.

[0015] Furthermore, to achieve another object of the present invention, the present invention provides use of the antibody or a fragment thereof for preparing a pharmaceutical composition for treating cancer.

[0016] Furthermore, in order to achieve another object of the present invention, the present invention provides a method for treating cancer, which comprises administering an effective amount of a pharmaceutical composition containing the antibody or a fragment thereof as an active ingredient to an individual in need thereof.

[0017] The present invention will be described in detail below.

[0018] VISTA (V-domain Ig suppressor of T cell activation) is a negative checkpoint ligand that shares homology with PD-L1 and inhibits T cell activation. It is expressed on myeloid cells and Foxp3. + CD4 + VISTA is highly expressed on tumor cells but not on tumor cells within the tumor microenvironment (TME). Within the hematopoietic compartment, VISTA is expressed in the CD11b highConstitutively highly expressed in myeloid cells and CD4 + and CD8 + Cells and Foxp3 + VISTA is expressed at lower levels on Treg cells. + and CD8 + Directly inhibits T cell proliferation and cytokine production. Therefore, VISTA is considered a novel immune negative checkpoint and a potential target for immune anticancer drugs.

[0019] Thus, the present invention provides antibodies or fragments thereof that bind to human VISTA protein.

[0020] In the present invention, "antibody" (also called immunoglobulin (Ig)) is a general term for proteins that selectively react with antigens and participate in biological immunity. Whole antibodies found in nature generally consist of two pairs of light chains (LC) and heavy chains (HC), which are polypeptides consisting of multiple domains, or have these two LC / HC pairs as their basic unit. There are five types of heavy chains that make up mammalian antibodies, represented by the Greek letters α, β, γ, δ, and μ, and different types of antibodies, such as IgA, IgD, IgE, IgG, and IgM, are made up depending on the type of heavy chain. There are two types of light chains that make up mammalian antibodies, represented by λ and κ.

[0021] Antibody heavy and light chains are structurally divided into variable and constant regions based on the variability of their amino acid sequences. The heavy chain constant region consists of three or four heavy chain constant regions, such as CH1, CH2, and CH4 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the antibody type. The light chain consists of a single constant region, the LC. The heavy and light chains are aligned side-by-side, with their respective variable and constant regions linked by a single covalent disulfide bond. They specifically bind to antigens via the light chain and the heavy and light chain variable regions. Because a whole antibody consists of two heavy and light chain pairs (HC / LC), a single antibody molecule has bivalent monospecificity, binding to the same two antigens via its two variable regions. The antibody variable region that binds to an antigen is called the antibody's antigen-binding site, and the portion of the antigen surface that is recognized by the antibody is called an epitope.

[0022] The variable region of an antibody, which contains the antigen-binding site, is subdivided into a framework region (FR), which has little sequence variability, and a hypervariable region called a complementary-determining region (CDR), which has high sequence variability. VH and VL each contain two CDRs and four FRs, arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. The CDRs, which have the highest sequence variability within the variable region of an antibody, are the sites that directly bind to the antigen and are therefore most important for the antigen specificity of the antibody.

[0023] The antibody or antibody fragment of the present invention is not limited in type as long as it has the above-described CDR, VH and VL, or light and heavy chain configuration. The antibody may be an IgG, IgA, IgM, IgE, or IgD antibody, with an IgG antibody being particularly preferred. The IgG subtypes include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. Furthermore, the antibody may be a monoclonal antibody derived from a single B cell or a polyclonal antibody derived from multiple B cells, but is preferably a monoclonal antibody, which is a population of antibodies whose heavy and light chain amino acid sequences are substantially identical. The antibody or antibody fragment of the present invention may also be conjugated to, but is not limited to, an enzyme, a fluorescent substance, a radioactive substance, a protein, or the like.

[0024] The antibodies of the present invention may be derived from any animal, including mammals including humans, birds, etc., and are preferably derived from humans. They may also be chimeric antibodies containing a portion of an antibody derived from a human and a portion of an antibody derived from another animal species.

[0025] Furthermore, in the present invention, an antibody fragment refers to a fragment that maintains the antigen-specific binding ability of the whole antibody, and specifically may be in the form of Fab, F(ab'), F(ab')2, Fv, scFv, diabody, dsFv, or the like. Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain from each of the heavy and light chains. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and consists of two Fabs linked by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to Fab separated by reducing the disulfide bond of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment consisting only of the variable regions of both the heavy and light chains. scFv (single chain variable fragment) is an antibody fragment consisting only of the heavy chain variable region (V H ) and the light chain variable region (V L ) are recombinant antibody fragments in which the V of scFv is linked with a flexible peptide linker. H and V L are connected by a very short linker and cannot bind to each other, and V of other scFvs of the same shape H and V L dsFv refers to a fragment in which V binds to form a dimer. H and V L The amino acid residue to be substituted with a cysteine ​​residue can be selected based on a predicted three-dimensional antibody structure according to known methods.

[0026] In one embodiment of the present invention, the antibody or fragment thereof according to the present invention comprises: i) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 2, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 3; H), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 6. L ii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 9, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 10, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 11; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 12, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 14. L iii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 17, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 18, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 19; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 22. L iv) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 25, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 26, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 27; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 28, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 30. Lv) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 33, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 34, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 35; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 36, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 38. L and vi) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 41, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 42, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 43. H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 44, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO: 46. L or a fragment thereof that binds to human VISTA protein selected from the group consisting of: an antibody comprising

[0027] In another embodiment of the present invention, the antibody or fragment thereof according to the present invention is preferably an antibody comprising: i) an antibody having a heavy chain variable region comprising the amino acid sequence of positions 1 to 117 of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of positions 1 to 108 of SEQ ID NO: 8; ii) an antibody having a heavy chain variable region comprising the amino acid sequence of positions 1 to 117 of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of positions 1 to 108 of SEQ ID NO: 16; iii) an antibody having a heavy chain variable region comprising the amino acid sequence of positions 1 to 118 of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of positions 1 to 107 of SEQ ID NO: 24; or iv) an antibody having a heavy chain variable region comprising: The antibody or fragment thereof that binds to human VISTA protein may be an antibody selected from the group consisting of: an antibody comprising the amino acid sequence of positions 1 to 118 of SEQ ID NO: 31 and having a light chain variable region comprising the amino acid sequence of positions 1 to 107 of SEQ ID NO: 32; v) an antibody comprising the amino acid sequence of positions 1 to 115 of SEQ ID NO: 39 and having a light chain variable region comprising the amino acid sequence of positions 1 to 106 of SEQ ID NO: 40; and vi) an antibody comprising the amino acid sequence of positions 1 to 119 of SEQ ID NO: 47 and having a light chain variable region comprising the amino acid sequence of positions 1 to 106 of SEQ ID NO: 48.

[0028] In another aspect of the present invention, the antibody or fragment thereof according to the present invention may more preferably be an antibody or fragment thereof that binds to human VISTA protein, characterized in that it is an antibody selected from the group consisting of: i) an antibody comprising a heavy chain consisting of SEQ ID NO: 7 and a light chain consisting of SEQ ID NO: 8; ii) an antibody comprising a heavy chain consisting of SEQ ID NO: 15 and a light chain consisting of SEQ ID NO: 16; iii) an antibody comprising a heavy chain consisting of SEQ ID NO: 23 and a light chain consisting of SEQ ID NO: 24; iv) an antibody comprising a heavy chain consisting of SEQ ID NO: 31 and a light chain consisting of SEQ ID NO: 32; v) an antibody comprising a heavy chain consisting of SEQ ID NO: 39 and a light chain consisting of SEQ ID NO: 40; and vi) an antibody comprising a heavy chain consisting of SEQ ID NO: 47 and a light chain consisting of SEQ ID NO: 48.

[0029] The present invention also provides polynucleotides encoding the antibodies or fragments thereof, recombinant vectors containing the same, transformed cell lines containing the same, and methods for producing the antibodies or fragments thereof using the same.

[0030] The "polynucleotide" of the present invention can be described as an oligonucleotide or a nucleic acid, and includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA obtained using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The polynucleotide may be single-stranded or double-stranded. The polynucleotide refers to a base sequence encoding an antibody consisting of a heavy chain and a light chain having a CDR configuration specific to the human CTLA-4 protein, or a VH and VL configuration.

[0031] Polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present invention can be obtained by methods well known in the art. For example, they can be synthesized using oligonucleotide synthesis techniques well known in the art, such as the polymerase chain reaction (PCR), based on DNA sequences encoding part or all of the heavy and light chains of the antibody or the amino acid sequences.

[0032] The "vector" of the present invention is used for replicating or expressing a polynucleotide of the present invention for recombinant production of an antibody or antigen-binding fragment thereof, and generally comprises one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The vector of the present invention may preferably be an expression vector, and more preferably may be a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence, such as a promoter.

[0033] A plasmid, a type of vector, refers to a linear or circular double-stranded DNA molecule to which external polynucleotide segments can be ligated. Another form of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors, including those of bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction, and thereby are replicated along with the host genome.

[0034] In the present invention, the term "vector" can be understood to have the same meaning as "expression vector," which is a form of vector capable of expressing the polynucleotide. A polynucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence. The regulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can exert its influence, for example, directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., the regulatory sequence and / or a polypeptide that binds to the nucleic acid). The regulatory sequence includes promoters, enhancers, and other expression control elements.

[0035] The cell line of the present invention is not particularly limited in type, as long as it can be used to express a polynucleotide encoding an antibody or fragment thereof contained in an expression vector of the present invention. Cells (host cells) transformed with an expression vector of the present invention can be prokaryotes (e.g., Escherichia coli), eukaryotes (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells), or hybridomas derived therefrom. Preferably, the cells are derived from mammals, including humans.

[0036] Suitable prokaryotes for this purpose include gram-negative or gram-positive organisms, such as bacteria from the family Enterobacteriaceae, for example, Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella; bacteria from the family Bacillus (e.g., B. subtilis and B. licheniformis); bacteria from Pseudomonas (e.g., P. aeruginosa); The cells of the present invention are not particularly limited as long as they are capable of expressing the vectors of the present invention, but are preferably Escherichia coli (E. coli).

[0037] As the eukaryote cell of the present invention, Saccharomyces cerevisiae is most commonly used. However, many other genera, species, and strains are compatible with the host, including, but not limited to, Schizosaccharomyces pombe, Kluyveromyces spp. hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, Yarrowia spp. (EP 402,226), Pichia pastoris (EP 183,070; Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger, can be used.

[0038] The term "transformation" refers to the modification of the genotype of a host cell by the introduction of an exogenous polynucleotide (a polynucleotide encoding the antibody or fragment thereof of the present invention), regardless of the method used for the transformation. The exogenous polynucleotide introduced into a host cell may be integrated into and maintained in the genome of the host cell, or may be maintained without integration, and the present invention encompasses both.

[0039] Recombinant vectors capable of expressing the antibodies of the present invention or fragments thereof can be introduced into and transformed into cells for producing the antibodies or fragments thereof by methods known in the art, including, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns, and other known methods for introducing nucleic acids into cells.

[0040] Furthermore, the cells of the present invention are cultured cells that can be transformed or transfected with the polynucleotides of the present invention or vectors containing them, which can then be expressed in the host cells. A recombinant cell refers to a cell that has been transformed or transfected with a polynucleotide to be expressed. A cell of the present invention can also be a cell that contains a polynucleotide of the present invention but does not express it at a desired level unless a regulatory sequence is introduced into the cell so that the polynucleotide is operably linked to it.

[0041] The cells of the present invention can be cultured in a variety of media. Commercially available media, such as Ham's FO (Sigma-Aldrich Co., St. Louis, MO), minimal essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich Co.), are suitable for culturing cells. The media can be supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or an equivalent energy source, as needed.

[0042] The present invention provides a method for producing an antibody or fragment thereof that binds to human CTLA-4 protein, comprising the steps of culturing the cells under conditions in which the polynucleotide is expressed to produce a polypeptide comprising light chain and heavy chain variable regions, and recovering the polypeptide from the cells or the culture medium in which they are cultured.

[0043] The cells used in the production method of the present invention are as described above and contain a polynucleotide encoding the antibody of the present invention. The polypeptide used in the production method may be the antibody of the present invention or a fragment thereof itself, or may be one to which an amino acid sequence other than the antibody of the present invention or a fragment thereof is further bound.

[0044] In this case, the antibody or fragment thereof of the present invention can be removed using a method well known to those skilled in the art. The culture medium composition and culture conditions may vary depending on the type of cell, and these can be appropriately selected and adjusted by those skilled in the art.

[0045] The antibody molecule can be accumulated in the cell cytoplasm, secreted from the cell, or targeted to the periplasm or extracellular medium by an appropriate signal sequence, preferably to the periplasm or extracellular medium. The produced antibody molecule is preferably refolded to a functional conformation using methods well known to those skilled in the art. Recovery of the polypeptide may vary depending on the characteristics of the produced polypeptide and the characteristics of the cell, and can be appropriately selected and adjusted by those skilled in the art.

[0046] The polypeptide can be produced intracellularly, in the surrounding cytoplasmic space, or directly secreted into the medium. If the polypeptide is produced intracellularly, the cells can be disrupted as a first step to release the protein. Particulate debris, host cells, or lysed fragments are removed, for example, by centrifugation or ultrafiltration. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, can be included in any previous step to prevent proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants. Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography; the antibodies of the present invention are preferably purified via affinity chromatography.

[0047] According to one example of the present invention, it was confirmed that the antibody of the present invention has excellent binding neutralizing ability to PSGL1 and VSIG3.

[0048] Furthermore, according to another embodiment of the present invention, the antibody of the present invention was tested for NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against the NIH / 3T3-VISTA cell line using the NK92MI-CD16 cell line, and it was confirmed that the antibody of the present invention has strong ADCC efficacy in a concentration-dependent manner.

[0049] Furthermore, according to another embodiment of the present invention, it was confirmed that the antibody of the present invention activates immunity in a concentration-dependent manner.

[0050] These results suggest that the antibodies of the present invention specifically bind to the VISTA protein, activating immune cells in the body and thereby killing cancer cells. Indeed, when the antibodies of the present invention were administered to mice induced with colon cancer, tumor size was significantly reduced compared to the control group administered IgG.

[0051] Therefore, the present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the antibody or a fragment thereof as an active ingredient.

[0052] In the present invention, the cancer may be a solid cancer or a non-solid cancer. A solid cancer refers to a cancer tumor that develops in an organ such as the liver, lung, breast, or skin. A non-solid cancer is a cancer that develops in the blood and is also called a blood cancer. The cancer may be a carcinoma, a sarcoma, a cancer derived from hematopoietic cells, a germ cell tumor, or a blastoma. The cancer may be selected from the group consisting of, for example, breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, stomach cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, brain and spinal cord tumors, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumors, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, Hodgkin's Disease, endocrine system cancer, and sarcoma.

[0053] The composition according to the present invention may contain only the antibody or fragment thereof according to the present invention, or may be formulated in an appropriate form together with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. The term "pharmaceutically acceptable" as used herein refers to a non-toxic composition that is physiologically acceptable and does not normally cause allergic or similar reactions, such as gastrointestinal disorders or dizziness, when administered to humans. The carrier includes any type of solvent, dispersion medium, oil-in-water or water-in-oil emulsion, aqueous composition, liposome, microbead, and microsome.

[0054] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Furthermore, various drug delivery materials used for oral administration of peptide formulations may also be included. Furthermore, carriers for parenteral administration may include water, a suitable oil, saline, aqueous glucose, glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above ingredients, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may be found in known literature.

[0055] The compositions of the present invention can be administered to mammals, including humans, by any method, for example, orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration.

[0056] The pharmaceutical composition of the present invention can be formulated into oral or parenteral preparations according to the administration route. For oral preparations, the composition of the present invention can be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., using methods known in the art. For example, oral preparations can be obtained by blending the active ingredient with a solid excipient, then pulverizing the mixture, and adding appropriate excipients to form a granular mixture to obtain tablets or sugar-coated tablets. Examples of suitable excipients include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol, and starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; gelatin; and fillers such as polyvinylpyrrolidone. In some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, sodium alginate, or the like may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further contain an anti-agglomerating agent, a lubricant, a humectant, a flavoring, an emulsifier, and a preservative. In the case of parenteral administration, the composition may be formulated in the form of an injection, a cream, a lotion, an ointment for external use, an oil, a moisturizer, a gel, an aerosol, or a nasal inhalant by a method known in the art. These formulations are described in all commonly known literature in medicinal chemistry.

[0057] The total effective amount of the composition of the present invention can be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. The content of the active ingredient in the pharmaceutical composition of the present invention can be varied depending on the severity of the disease. It can be approximately 0.01 μg to 10,000 mg per kg, most preferably 0.1 μg to 500 mg per kg. However, the effective dose of the pharmaceutical composition is determined by taking into account various factors, such as the formulation method, administration route, and frequency of treatment, as well as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate. Taking these factors into consideration, those skilled in the art will be able to determine the appropriate effective dose of the composition of the present invention. The pharmaceutical composition of the present invention is not particularly limited in its formulation, administration route, or administration method, as long as it exhibits the effects of the present invention.

[0058] Furthermore, the present invention provides use of the antibody or a fragment thereof for producing a pharmaceutical composition for treating cancer.

[0059] Furthermore, the present invention provides a method for treating cancer, which comprises administering an effective amount of a pharmaceutical composition containing the antibody or a fragment thereof as an active ingredient to an individual in need thereof.

[0060] The "effective amount" of the present invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, detecting, or diagnosing cancer or suppressing or reducing the disease, and the "individual" may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect.

[0061] The "treatment" of the present invention refers comprehensively to improving symptoms of cancer or the disease, including, but not limited to, alleviating, curing, or preventing one or most symptoms resulting from the disease, which may include curing, substantially preventing, or ameliorating the condition of the disease.

[0062] As used herein, the term "comprising" is used interchangeably with "including" or "characterized by" and does not exclude additional components or method steps, etc., not specifically mentioned in a composition or method according to the present invention. Additionally, the term "consisting of" means excluding additional elements, steps, or ingredients, etc., not otherwise recited. The term "essentially consisting of" means that a composition or method may include, in addition to the recited materials or steps, materials or steps that do not materially affect its basic properties. [Effects of the Invention]

[0063] Therefore, the present invention provides an antibody or fragment thereof that binds to human VISTA protein. The antibody of the present invention binds to VISTA protein in T cells and inhibits T cell inactivation by cancer cells, allowing T cells to attack cancer cells. At the same time, it can exert a strong ADCC effect through NK cells, demonstrating significant efficacy as a cancer therapeutic agent. [Brief explanation of the drawings]

[0064] [Figure 1] Figure 1 shows that the top six clones from the scFv sequences obtained by phage display were converted into full IgG1 form, and then the clones were expressed and purified by SDS-PAGE. [Figure 2] FIG. 2 shows the results of flow cytometry confirming the binding ability of anti-VISTA antibodies using the NIH / 3T3 cell line. [Figure 3] FIG. 3 shows the results of Western blotting using an NIH / 3T3 cell line overexpressing human VISTA protein and an anti-VISTA antibody. [Figure 4]FIG. 4 shows the results of immunoprecipitation (IP) performed using an NIH / 3T3 cell line overexpressing human VISTA protein and an anti-VISTA antibody. [Figure 5] FIG. 5 shows the results of confirming the human-mouse substitution reaction of anti-VISTA antibodies using ELISA. [Figure 6] FIG. 6 shows the results of flow cytometry of changes in the binding ability of anti-VISTA antibodies due to changes in pH. [Figure 7] FIG. 7 shows the results of competitive ELISA confirming the ability to neutralize binding of VSIG3, which binds to VISTA at neutral pH (pH 7.0). [Figure 8] FIG. 8 shows the results of competitive ELISA confirming the ability to neutralize binding of VISTA to PSGL1 at acidic pH (pH 6.0). [Figure 9] FIG. 9 shows the results of flow cytometry confirming the ability to neutralize the binding of PSGL1 to VISTA at an acidic pH (pH 6.0). [Figure 10] FIG. 10 shows the results of an ADCC assay performed on the NIH / 3T3 cell line in which human VISTA protein was overexpressed, using the NK92MI-CD16 cell line. [Figure 11] FIG. 11 shows the results of CD14 mononuclear cells and CD4 T cells isolated from human peripheral blood mononuclear cells, activated, and then co-cultured with anti-VISTA antibody. [Figure 12] Figure 12 shows the results of ELISA for anti-VISTA antibody and anti-PD-1 antibody after stimulation of human peripheral blood mononuclear cells with Staphylococcal enterotoxin B (SEB). [Figure 13] FIG. 13 shows the results of ELISA in which human peripheral blood mononuclear cells were stimulated with Staphylococcal enterotoxin B (SEB), treated with VISTA protein, and then treated with anti-VISTA antibody (INF-γ). [Figure 14]FIG. 14 shows the results of ELISA in which human peripheral blood mononuclear cells were stimulated with Staphylococcal enterotoxin B (SEB), treated with VISTA protein, and then treated with anti-VISTA antibody (TNF-α). [Figure 15] FIG. 15 shows the results of ELISA in which human peripheral blood mononuclear cells were stimulated with Staphylococcal enterotoxin B (SEB), treated with VISTA protein, and then treated with anti-VISTA antibody (IL-6). [Figure 16] FIG. 16 shows the results of ELISA in which mixed lymphocyte reactions were performed using human peripheral blood mononuclear cells from different donors and then treated with anti-VISTA antibody (IL-6). [Figure 17] Figure 17 shows the results of confirming the in vivo anti-cancer efficacy of anti-VISTA antibodies using a mouse model into which human VISTA protein was introduced. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention will be described in detail below. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0066] Example 1. Screening for scFv that specifically binds to human VISTA protein

[0067] 1-1. Selection of antigen and scFv phage The VISTA antigen was provided in the form of a recombinant protein (hVISTA-Fc, RND systems, Cat. No. 7126-B7) during the screening process. Phage library display was used to screen for antibodies that specifically bind to VISTA. A synthetic human scFv library was used, and specific information about the library is described in literature known in the art. The scFvs expressed in the scFv library were tagged with an HA tag, allowing them to be detected with an anti-HA FITC antibody (Genscript, A01621). Biopanning was performed using the scFv library as follows: The scFv library stock was blocked with 3% skim ml / PBS at room temperature. One mL of antigen (hVISTA-Fc, RND Systems, Cat. No. 7126-B7) was added to an immunotube at a concentration of 15 μg / mL and coated at 200 rpm for at least 12 hours at 4°C. The coated immunotube was washed three times with 0.05% PBS-T and blocked for 2 hours at room temperature with 1 mL of 6% Skim mlik blocking buffer. After 2 hours, the tube was washed three times with 0.05% PBS-T. The blocked scFv library stock was added to the antigen-coated immunotube and allowed to bind at 200 rpm for 1 hour at 37°C. Unbound scFv library stock was removed and then washed three times with 0.05% PBS-T. To elute only the specifically bound scFv-phage, the phage was incubated with 100 mM TEA (triethylamine) for 5 minutes at room temperature, neutralized with pH 8.5 Tris, and prepared in the form of an scFv-antigen conjugate. The prepared scFv-antigen conjugate was added to E. coli TG1 cells for infection, and then incubated overnight at 37°C on LB / ampicillin / glucose agar medium. E. coli TG1 cells were cultured in SB / The cells were transferred to ampicillin medium and cultured until the OD600 value reached 0.5. Then, 1 × 10 11 ~1×10 12 A helper phage was added and the cells were incubated again for 1 hour at 37°C. Kanamycin was then added and the cells were incubated again overnight. The overnight culture was centrifuged, and the supernatant was reacted with PEG solution at 4°C and centrifuged again to separate the pellet. The pellet was dissolved in PBS and centrifuged, and the resulting supernatant was used as the scFv library solution. This process was repeated four times to identify scFv candidates that specifically bind to the VISTA antigen.

[0068] 1-2. Selection of scFv antibodies that specifically bind to VISTA To select scFvs with superior binding affinity from the scFv candidate group obtained in Example 1-1, ELISA analysis was performed on VISTA-expressing cell lines. VISTA-expressing cell lines were prepared by transfecting NIH / 3T3 cells with a human VISTA expression vector and then treating the cells with 500 μg / ml hygromycin B to select transformants. Each panned library stock from Example 1-1 was incubated overnight on SB / ampicillin / glucose agar medium, and a single colony from each was inoculated into 200 μl of SB / ampicillin medium and cultured at 37°C for 3 hours. The mixture was then mixed to a 1 mM IPTG concentration and again incubated at 30°C overnight. Upon completion of the culture, the culture medium was centrifuged to separate the cells, which were then lysed in TES buffer to isolate the scFvs. The isolated scFvs were then extracted from VISTA-expressing cells at a concentration of 1x10. 5The plate was treated with aliquots of each antibody and incubated at room temperature for 1 hour. A secondary antibody (anti-HA HRP, Santa Cruz, Cat. No. sc-7392) was then added and incubated for 40 minutes. After the secondary antibody reaction was complete, TMB was added to develop the color reaction, and the results were analyzed using an ELISA reader (450 nm). The ELISA results were compared, and the top six scFvs (4D2, 4F2, 4G3, 4H2, 4A2, and 4A3) were selected. The amino acid sequences of the selected scFvs were analyzed, and the results are shown in Table 1 below.

[0069] [Table 1]

[0070] Example 2. Conversion of scFv antibody to IgG, expression, and purification

[0071] 2-1. Construction of whole IgG expression vector The scFv selected above was converted into the form of an IgG, a more commonly used antibody. An expression vector capable of expressing the entire IgG form was constructed based on the CDR region of the scFv. First, the light chain variable region and heavy chain variable region of the scFv were obtained by PCR, respectively, using the primers shown in Table 2 below. The light chain variable region sequence was cloned into pcDNA 3.3 (Invitrogen), an expression vector containing a light chain constant region sequence, and the heavy chain variable region sequence was cloned into pOptiVEC (Invitrogen), an expression vector containing a heavy chain constant region sequence. The light chain variable region and heavy chain constant region of the scFv cloned from the vector were expressed together with the light chain variable region and heavy chain variable region, resulting in the production of an entire IgG antibody containing the CDR region of the scFv.

[0072] [Table 2]

[0073] 2-2. Generation of whole IgG antibody expressing cell lines Using CHO-S cells (Life Technologies Inc.), IgG antibody-expressing cell lines were generated. The gene sequences encoding the heavy and light chains obtained in Example 2-1 were subjected to codon optimization in the Cricetulus griseus species, and these sequences were cloned into the Freedom (R) After cloning into pCHO 1.0 Vector, transfection reagent (FreeStyle TM CHO-S cells were transduced using MAX Reagent (Life Technologies Inc.). To select antibody-expressing cell lines after transduction, a two-stage selection process using puromycin and methotrexate (MTX) was performed. Specifically, the primary selection proceeded to 10 μg / ml puromycin and 100 nM MTX or 20 μg / ml puromycin and 200 nM MTX. Once cell viability met the criteria, the secondary selection process was initiated. The secondary selection proceeded to 30 μg / ml puromycin and 500 nM MTX or 50 μg / ml puromycin and 1000 nM MTX. Once the final cell viability criteria were met, secondary selection was terminated and high-expression populations were selected using Simple Fed Batch (SFB).

[0074] 2-3. Production and purification of whole IgG antibodies Each antibody-producing cell line prepared in Example 2-2 was used as CD FortiCHO TMThe cells were cultured in medium at 8% CO2, 37°C, and 100-120 rpm, with glucose added at 4g / L, 4g / L, and 6g / L on days 3, 5, and 7, respectively, for a total of 14 days. After completion of the culture, the culture medium was centrifuged at 6000xg in an ultracentrifuge, and the supernatant was filtered through a 0.2µm filter. Purification was performed using Protein A resin (Mabselect SuRe, 11-0026-01 AD, GE Healthcare Life Sciences) and equilibration buffer (20mM sodium phosphate, 150mM NaCl, pH 7.2), wash buffer (35mM sodium phosphate, 500mM NaCl, pH 7.2), and elution buffer (0.1M sodium citrate, pH 3.6). TM The column was purified using the avant filter with an equilibration buffer of 2 times the column volume, a wash buffer of 5 times the column volume, and an elution buffer of 5 times the column volume. A pH 8.0 Tris-HCl solution was added in 1 / 5 portions for neutralization during elution. The column was buffered twice with PBS using a filter membrane (CelluSep, 1430-45), and then concentrated using a centrifugal filter (Amicon Ultra-15, UFC905024, Merck). The IgG antibody proteins produced under reducing and non-reducing conditions were analyzed using standard SDS-PAGE techniques, and it was confirmed that both the light and heavy chains of each antibody were well expressed at the expected molecular weights. Figure 1 shows the SDS-PAGE results for each IgG antibody.

[0075] Example 3. Evaluation of binding specificity and binding strength of antibodies according to the present invention to VISTA

[0076] 3-1. Preparation of VISTA / NIH / 3T3 cell lines To confirm the antigen specificity of the antibody prepared in Example 2-3, the presence or absence of binding to human VISTA protein was examined. The VISTA gene was cloned into pCMV3-C-FLAG (Sino Biological), and each VISTA expression vector was transfected into NIH / 3T3 cells using Fugene HD (E231A, Promega) transfection reagent, followed by selection of resistant cell lines with hygromycin B.

[0077] 3-2. Evaluation of the binding ability of the antibody of the present invention to the VISTA / NIH / 3T3 cell line The cross-reactivity of the antibodies prepared in Example 2-3 was confirmed for the VISTA / NIH / 3T3 cell line prepared in Example 3-1. Original NIH / 3T3 cells were used as a negative control. First, the cells were dissociated into single cells using cell dissociation buffer (Gibco, 13151-014), and then 2.5 × 10 cells were cultured. 5 Each seeding was added to 10 μg / ml of each antibody and incubated on ice for 1 hour. After incubation, the cells were washed with 1% FBS / PBS and treated with the secondary antibody mouse anti-human IgG-PE (366904, Biolegend) at a 1:100 dilution and incubated on ice for 1 hour. After incubation, the cells were washed with 1% FBS / PBS and analyzed by flow cytometry using a BD FACS Lyrics. FIG. 2 shows the results of the flow cytometry, which allows comparative confirmation of the binding ability of the antibody to VISTA.

[0078] 3-3. Confirmation of VISTA specific binding - Western blot The ability of the antibodies prepared in Examples 2-3 to recognize denatured VISTA antigens was confirmed using the VISTA / NIH / 3T3 cell line prepared in Example 3-1. Native NIH / 3T3 cells were used as a negative control. First, cells were dissociated into single cells using cell dissociation buffer. Then, they were lysed by adding RIPA buffer (BIOSESANG), protease inhibitors, and phosphatase inhibitors at 200 rpm and 4°C for 1 hour. The supernatant was then collected by centrifugation at 15,000 rpm and 4°C for 15 minutes, and the dissolved protein was quantified using a BCA assay (ThermoFisher Scientific). The quantified protein was added to 5x sample loading buffer (BIOSESANG) containing DTT and boiled for 10 minutes to reduce all proteins. Equal amounts of protein were loaded on SDS-PAGE, separated according to size, and transferred to a PVDF membrane. The sections were then blocked with 5% skim milk at 4°C for 1 hour, and then attached overnight to a PVDF membrane using the antibody prepared in 2-3 as the primary antibody. After washing three times with 0.05% TBS-T, the sections were treated with a secondary antibody, anti-human Fc HRP antibody (Genscript), at a dilution of 1:2000 and incubated for 30 minutes. After washing three times with 0.05% TBS-T, ECL solution (Bio-Rad) was added and the sections were printed onto film in a darkroom. As a result, as shown in FIG. 3, it was confirmed that the antibody did not recognize the denatured VISTA antigen.

[0079] 3-4. Confirmation of VISTA specific binding - immunoprecipitation In Example 3-1, the ability of the antibodies prepared in Examples 2-3 to recognize intact (native) VISTA antigen was confirmed for the VISTA / NIH / 3T3 cell line. Native NIH / 3T3 cells were used as a negative control. Cells were detached with a scraper and then released into PBS containing protease inhibitors and phosphatase inhibitors. After physical cell disruption using a sonicator, protein was quantified using a BCA assay. The quantified protein was allowed to bind with the antibody for 1 hour. At the same time, Protein A beads (Sigma) were blocked with 5% skim milk for 1 hour. The protein and antibody solution was then allowed to bind to the blocked beads for 1 hour and washed three times with PBS. The beads were then incubated at 70°C for 10 minutes in 5x sample loading buffer containing DTT to separate and reduce both the protein and antibody bound to the Protein A beads. Western blots were performed using the samples and an anti-human VISTA antibody (Cell signaling technology) to confirm that the antibody recognized the intact VISTA antigen. As a result, as shown in FIG. 4, it was confirmed that the antibody specifically binds to the intact VISTA antigen.

[0080] Example 4. Confirmation of human-mouse cross-reactivity of anti-VISTA antibodies using ELISA

[0081] The human-mouse cross-reactivity of the antibodies prepared in Examples 2-3 was confirmed by ELISA. 100 ng each of mouse VISTA protein (mVISTA-Fc, R&D Systems, Cat. No. 7005-B7) and human VISTA protein (hVISTA-Fc, R&D Systems, Cat. No. 7126-B7) was added to a 96-well plate and coated overnight at 4°C. The plate was washed three times with 0.05% PBS-T and blocked with 300 μL of 5% skim milk at room temperature for 2 hours. After blocking, the plate was washed three times with 0.05% PBS-T. 100 ng of each antibody was added and allowed to bind for 1 hour at room temperature. Equal amounts of VSTB174 (J&J) and BMS767 (BMS) were used as positive controls. After binding, the plate was washed three times with 0.05% PBS-T, treated with anti-human Fc HRP antibody (Genscript) at a dilution of 1:2000, and incubated for 30 minutes. After incubation, the plate was washed three times with 0.05% PBS-T, and 100 μl of TMB solution (Surmodics) was added to each plate. The plate was incubated at room temperature for 20 minutes, and 50 μl of stop solution was added to each plate. The 450 nm wavelength reading was measured using an ELISA reader. As a result, as shown in FIG. 5, it was confirmed that the antibody was unable to recognize mouse VISTA and bound strongly only to human VISTA.

[0082] Example 5. Changes in binding ability of anti-VISTA antibodies depending on pH and confirmation of their ability to neutralize binding to VSIG3 and PSGL1

[0083] 5-1. Changes in binding ability of anti-VISTA antibodies depending on pH The VISTA / NIH / 3T3 cell line prepared in Example 3-1 was examined by flow cytometry to determine whether the antibody prepared in Example 2-3 recognized the VISTA antigen depending on the pH. First, single cells were separated using a cell separation buffer, and then 2.5 × 10 5The cells were seeded at 100 μg / ml each. Using a pH meter, the solution was titrated with HCl to prepare six 1% BSA / PBS buffer solutions ranging from pH 7.0 to 6.0. Each antibody was added at 10 μg / ml and incubated on ice for 1 hour. After incubation, the cells were washed with 1% BSA / PBS buffer at the appropriate pH, and treated with the secondary antibody goat anti-human IgG-FITC (109-095-098, Jackson Immunoresearch) at a 1:100 concentration. The cells were incubated on ice for 1 hour. After incubation, the cells were washed with 1% BSA / PBS buffer at the appropriate pH, and analyzed by flow cytometry using a BD FACS Lyrics. FIG. 6 shows the results of the flow cytometry, which allows comparative confirmation of the binding strength of the antibody to VISTA as a function of pH.

[0084] 5-2. Confirmation of the binding neutralizing ability of anti-VISTA antibodies to VSIG3 To confirm the binding neutralization ability of the antibodies prepared in Example 2-3, their VISTA-VSIG3 binding neutralization ability at pH 7.4 was assessed by ELISA. 250 ng of human VSIG3 protein (hVSIG3 Fc, R&D Systems, Cat. No. 9229-VS) was added to a 96-well plate and coated overnight at 4°C. The plate was washed three times with 0.1% PBS-T and blocked with 50 mg / mL BSA / PBS at room temperature for 2 hours. Meanwhile, 100 nM human VISTA-biotin protein (hVISTA-Avi-Biotin, R&D Systems, Cat. No. AVI9057) and an anti-VISTA antibody diluted from 60 μg / mL to a 1 / 3 concentration were allowed to bind at room temperature for 1 hour. The blocked 96-well plate was washed three times with 0.1% PBS-T, and 100 μL of VISTA-antibody binding solution was added to each well. The plate was then allowed to bind at room temperature for 2 hours. As positive controls, the same amount of VSTB174 (J&J) and BMS767 (BMS) were used. After binding, the plates were washed three times with 0.1% PBS-T, treated with Streptavidin-HRP (R&D Systems) at a dilution of 1:5000, and then incubated at room temperature for 1 hour. After incubation, the plates were washed three times with 0.1% PBS-T, and 100 μl of TMB solution (Surmodics) was added to each plate. The plate was incubated at room temperature for 20 minutes. 50 μl of stop solution was added to each plate, and the 450 nm wavelength reading was measured using an ELISA reader. As a result, as shown in FIG. 7, it was confirmed that the antibody inhibited the binding of VISTA-VSIG3 at neutral pH (pH 7.4).

[0085] 5-3. Confirmation of the binding neutralizing ability of anti-VISTA antibodies to PSGL1 - ELISA To confirm the binding neutralization ability of the antibodies prepared in Examples 2-3, their VISTA-PSGL1 binding neutralization ability at pH 6.0 was assessed by ELISA. 250 ng of human PSGL1 protein (hPSGL1 Fc, R&D Systems, Cat. No. 3345-PS) was added to a 96-well plate and coated overnight at 4°C. All subsequent steps were performed by titrating all buffers to pH 6.0 using HCl. The plate was washed three times with 0.1% PBS-T and blocked with 50 mg / mL BSA / PBS at room temperature for 2 hours. Meanwhile, 100 nM human VISTA-biotin protein (hVISTA-Avi-Biotin, R&D Systems, Cat. No. AVI9057) and anti-VISTA antibody were diluted from 60 μg / mL to 1 / 3 concentration and allowed to bind for 1 hour at room temperature. The blocked 96-well plate was washed three times with 0.1% PBS-T, and 100 μl of VISTA-antibody binding solution was added. Binding was allowed for 2 hours at room temperature. The same amount of VSTB174 (J&J) and BMS767 (BMS) were used as positive controls. After binding, the plate was washed three times with 0.1% PBS-T, treated with Streptavidin-HRP (R&D Systems) at a 1:5000 dilution, and then incubated for 1 hour at room temperature. After incubation, the plate was washed three times with 0.1% PBS-T, and 100 μl of TMB solution (Surmodics) was added. The plate was incubated for 20 minutes at room temperature. 50 μl of stop solution was added, and the 450 nm wavelength reading was measured using an ELISA reader. As a result, as shown in FIG. 8, it was confirmed that the antibody inhibited the binding of VISTA-PSGL1 at acidic pH (pH 6.0).

[0086] 5-4. Confirmation of the binding neutralizing ability of anti-VISTA antibodies to PSGL1 - Flow cytometry To confirm the binding neutralization ability of the antibodies prepared in Examples 2-3, the neutralization ability of VISTA-PSGL1 binding at pH 6.0 was examined by flow cytometry. Using a Jurkat cell line expressing PSGL1, the Jurkat cell line and VISTA protein (R&D Systems) were incubated with anti-VISTA antibody in 1% BSA / PBS buffer at pH 6.0 at 4°C for 30 minutes. After washing three times with the same buffer, the secondary antibody, goat anti-human IgG-FITC (Jackson Immunoresearch), was incubated at 4°C for 30 minutes. After washing three times with the same buffer, the cells were analyzed by flow cytometry using a BD FACS Lyrics. As a result, as shown in FIG. 9, it was confirmed that the antibody inhibited the binding of PSGL1 expressed in Jurkat cells to VISTA protein at an acidic pH (pH 6.0).

[0087] Example 6. Confirmation of antibody-dependent cellular cytotoxicity

[0088] The antibody-dependent cellular cytotoxicity of the antibody prepared in Example 2-3 was confirmed against the VISTA / NIH / 3T3 cell line prepared in Example 3-1. The target cells were the VISTA / NIH / 3T3 cell line, and the effector cells were a cell line obtained by transfecting NK92MI cells with human CD16. The target cells were separated into single cells using a separation buffer, and then placed in a 96-well plate at 2.0 × 10 4 The effector cells were divided into 1.0x10 cells and cultured overnight. 5 Anti-VISTA antibodies were diluted from 10 μg / ml to 1 / 10 concentration and added to the target cells. The cells were then incubated at 37°C for 4 hours. 50 μL of the supernatant was then used for the LDH assay (Promega, CytoTox 96). (R) ) was performed to confirm cytotoxicity. As a result, as shown in FIG. 10, it was confirmed that the antibody killed VISTA-expressing cells in a concentration-dependent manner through antibody-dependent cellular cytotoxicity mediated by NK cells.

[0089] Example 7. Confirmation of immune activation ability

[0090] 7-1. Immune activation by anti-VISTA antibody (IFN-γ + CD4 + ) To confirm the immunostimulatory activity of the antibody prepared in Example 2-3, flow cytometry analysis was performed using human immune cells. Peripheral blood concentrated in an LRS chamber was diluted with 2% FBS / PBS. Histopaque was placed in a 50 mL tube. (R) 25 mL of Sigma-Aldrich HCl (Ab-1077) was added, and 25 mL of diluted peripheral blood was slowly added. The mixture was then centrifuged at 1200 g for 10 minutes. After centrifugation, the supernatant was removed, and the human peripheral blood mononuclear cell (PBMC) layer was collected and transferred to a new 50 mL tube. The PBMCs were then washed three times with 2% FBS / PBS washing buffer at 300 g for 8 minutes to obtain PBMCs. Human CD4 T cells and human CD14 mononuclear cells were isolated from the PBMCs using CD4 MicroBeads (Miltenyi Biotec, Cat. No. 130-045-101) and CD14 MicroBeads (Miltenyi Biotec, Cat. No. 130-050-201). For T cell activation, a 96-well plate was coated with an anti-CD3 antibody (OKT3, Abcam) at a concentration of 2.5 μg / mL for 2 hours at room temperature, and 1.0 × 10 isolated human CD4 T cells were then transferred to the plate. 5 5.0 x 10 VISTA-expressing human CD14 mononuclear cells were placed on the plate. 5 The cells were incubated with 10 μg / ml of anti-VISTA antibody at 37°C for 8 hours. To confirm interferon-gamma expression in CD4 T cells, Brefeldin A (Biolegend) was added, followed by treatment with mouse anti-human CD4 APC antibody (Biolegend) and mouse anti-human IFN-γ FITC antibody (Biolegend) and analysis was performed using a BD FACS Lyrics flow cytometer. As a result, as shown in Figure 11, the antibody inhibited IFN-γ production by CD14 mononuclear cells. + CD4 + It was confirmed that T cells were restored.

[0091] 7-2. Immune activation by anti-VISTA antibody and anti-PD-1 antibody (IFN-γ) To confirm the immunostimulatory activity of the antibody prepared in Example 2-3, a Staphylococcus aureus enterotoxin B (SEB) activity assay was performed using human PBMCs. 2.0 × 10 human PBMCs isolated in Example 7-1 were plated onto a 96-well plate. 5 The cells were cultured in a single cell culture medium containing 100 ng / mL SEB (Abion), 10 μg / mL anti-VISTA antibody, and 10 μg / mL anti-PD-1 antibody (pembrolisumab) and incubated at 37°C for 3 days. After 3 days, 100 μl of the supernatant was used for further analysis. IFN-γ secretion due to immune activation was measured by ELISA (Human IFN-gamma DuoSet ELISA, R&D Systems, Cat. No. DY285B). As a result, as shown in Figure 12, it was confirmed that the antibody exhibited immune activation ability and maximized IFN-γ secretion when administered in combination with an anti-PD-1 antibody.

[0092] 7-3. Recovery of VISTA immunosuppression by anti-VISTA antibody (IFN-γ, TNF-α, and IL-6) To confirm the immunosuppression recovery ability of the antibody prepared in Example 2-3, a Staphylococcus aureus enterotoxin B (SEB) activity assay was performed using VISTA protein and human PBMCs. Human VISTA protein (hVISTA-Fc, RND Systems, Cat. No. 7126-B7) was coated onto a 96-well plate at a concentration of 5 μg / ml overnight at 4°C. The next day, after washing three times with PBS, the human PBMCs isolated in Example 7-1 were applied to the 96-well plate at 2.0 × 10 5Each cell was treated with 100 ng / mL of SEB (Abion) and anti-VISTA antibody diluted from 20 μg / mL to a quarter concentration and incubated at 37°C for 3 days. After 3 days, 100 μl of the supernatant was used for further analysis. The secretion of IFN-γ, TNF-α, and IL-6 in response to immune activation was measured using the Human IFN-gamma DuoSet ELISA (R&D systems, Cat. No. DY285B), Human TNF-alpha DuoSet ELISA (R&D systems, Cat. No. DY210), and Human IL-6 DuoSet ELISA (R&D systems, Cat. No. DY206), respectively. As a result, as shown in Figures 13 to 15, it was confirmed that the antibody concentration-dependently restored IFN-γ secretion (Figure 13), TNF-α (Figure 14), and IL-6 (Figure 15) suppressed by VISTA protein.

[0093] 7-4. Confirmation of immune activation by anti-VISTA antibodies using mixed lymphocyte reaction (MLR) assay To confirm the immune activation ability of the antibodies prepared in Example 2-3, a mixed lymphocyte reaction (MLR) was performed using PBMCs from different donors. Using the human PBMCs isolated in Example 7-1, the PBMCs used as stimulators were treated with mitomycin C to inhibit cell proliferation. PBMCs from other donors used as responders and PBMCs used as stimulators were plated at 1.0 x 10 s in a 96-well plate. 5 Each cell was treated with anti-VISTA antibody diluted from 20 μg / ml to a quarter concentration and then incubated at 37°C for 5 days. After 5 days, 100 μl of the supernatant was used for further analysis. IL-6 secretion due to immune activation was measured by ELISA (Human IL-6 DuoSet ELISA, R&D Systems, Cat. No. DY206). As a result, as shown in FIG. 16, it was confirmed that the antibody increased IL-6 secretion in a concentration-dependent manner.

[0094] Example 8. Confirmation of in vivo anti-cancer efficacy of antibodies

[0095] To confirm the in vivo anti-cancer efficacy of the anti-VISTA antibody, the prepared anti-VISTA antibody was injected into cancer-induced human VISTA protein transgenic mice, and changes in tumor size were observed. First, 5x10 MC38 cells, a mouse colon cancer cell line, were added to human VISTA protein-transfected mice. 5 The tumors were injected subcutaneously. The average tumor size was 140 mm. 3 When tumor size reached 100%, six mice per group were randomly assigned to a control group and an antibody-treated group. The control group received IgG, and the antibody-treated group received 4A2 antibody at a dose of 3 mg / kg, three times a week for a total of two weeks. After that, tumor size was measured. The tumor size was measured using calipers, and the long axis x (short axis) 2 mm 3 was calculated. As a result, as can be seen in FIG. 17, it was confirmed that the tumor size was statistically significantly reduced in the 4A2 antibody administration group compared to the control group (t-test, p value=0.04). [Industrial Applicability]

[0096] As described above, the present invention provides an antibody or fragment thereof that binds to human VISTA protein. The antibody of the present invention binds to VISTA on T cells and inhibits T cell inactivation by cancer cells, thereby allowing T cells to attack cancer cells. At the same time, it can exert a strong ADCC effect through NK cells, demonstrating significant efficacy as a cancer therapeutic agent and therefore having high industrial applicability.

Claims

1. i) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 1, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 2, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 3; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO:

6. Ｌ an antibody comprising ii) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 9, a complementarity determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 10, and a complementarity determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 11; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 12, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO:

14. Ｌ an antibody comprising iii) an antibody heavy chain variable region (V) comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 17, a complementarity-determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 18, and a complementarity-determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 19; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO:

22. Ｌ an antibody comprising iv) an antibody heavy chain variable region (V) comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 25, a complementarity-determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 26, and a complementarity-determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 27; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 28, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO:

30. Ｌ an antibody comprising v) an antibody heavy chain variable region (V) comprising a complementarity determining region (CDR) H1 comprising the amino acid sequence represented by SEQ ID NO: 33, a complementarity determining region (CDR) H2 comprising the amino acid sequence represented by SEQ ID NO: 34, and a complementarity determining region (CDR) H3 comprising the amino acid sequence represented by SEQ ID NO: 35; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 36, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO:

38. Ｌ an antibody comprising vi) an antibody heavy chain variable region (V) comprising a complementarity-determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 41, a complementarity-determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 42, and a complementarity-determining region (CDR) H3 containing the amino acid sequence represented by SEQ ID NO: 43; H ), and an antibody light chain variable region (V) comprising a complementarity determining region (CDR) L1 comprising the amino acid sequence represented by SEQ ID NO: 44, a complementarity determining region (CDR) L2 comprising the amino acid sequence represented by SEQ ID NO: 45, and a complementarity determining region (CDR) L3 comprising the amino acid sequence represented by SEQ ID NO:

46. Ｌ an antibody comprising An antibody or fragment thereof that binds to human VISTA protein selected from the group consisting of:

2. The antibody i) an antibody comprising the amino acid sequence of positions 1 to 117 of SEQ ID NO: 7 in its heavy chain variable region and the amino acid sequence of positions 1 to 108 of SEQ ID NO: 8 in its light chain variable region; ii) an antibody comprising the amino acid sequence of positions 1 to 117 of SEQ ID NO: 15 in its heavy chain variable region and the amino acid sequence of positions 1 to 108 of SEQ ID NO: 16 in its light chain variable region; iii) an antibody comprising the amino acid sequence of positions 1 to 118 of SEQ ID NO: 23 in its heavy chain variable region and the amino acid sequence of positions 1 to 107 of SEQ ID NO: 24 in its light chain variable region; iv) an antibody comprising the amino acid sequence of positions 1 to 118 of SEQ ID NO: 31 in its heavy chain variable region and the amino acid sequence of positions 1 to 107 of SEQ ID NO: 32 in its light chain variable region; v) an antibody comprising the amino acid sequence of positions 1 to 115 of SEQ ID NO: 39 in its heavy chain variable region and the amino acid sequence of positions 1 to 106 of SEQ ID NO: 40 in its light chain variable region; and vi) an antibody comprising the amino acid sequence of positions 1 to 119 of SEQ ID NO: 47 in its heavy chain variable region and the amino acid sequence of positions 1 to 106 of SEQ ID NO: 48 in its light chain variable region; An antibody or fragment thereof that binds to the human VISTA protein of claim 1, characterized in that it is an antibody selected from the group consisting of:

3. The antibody i) an antibody comprising a heavy chain consisting of SEQ ID NO: 7 and a light chain consisting of SEQ ID NO: 8; ii) an antibody comprising a heavy chain consisting of SEQ ID NO: 15 and a light chain consisting of SEQ ID NO: 16; iii) an antibody comprising a heavy chain consisting of SEQ ID NO: 23 and a light chain consisting of SEQ ID NO: 24; iv) an antibody comprising a heavy chain consisting of SEQ ID NO: 31 and a light chain consisting of SEQ ID NO: 32; v) an antibody comprising a heavy chain consisting of SEQ ID NO: 39 and a light chain consisting of SEQ ID NO: 40; and vi) an antibody comprising a heavy chain consisting of SEQ ID NO: 47 and a light chain consisting of SEQ ID NO: 48; An antibody or fragment thereof that binds to the human VISTA protein of claim 1, characterized in that it is an antibody selected from the group consisting of:

4. The antibody or fragment thereof according to claim 1, characterized in that the fragment is a fragment selected from the group consisting of diabody, Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.

5. A polynucleotide encoding the antibody or fragment thereof of claim 1.

6. A recombinant vector comprising the polynucleotide of claim 5.

7. A transformed cell line comprising the vector of claim 6.

8. An antibody or fragment thereof that binds to human VISTA protein, comprising the steps of: culturing the cell line described in claim 7 under conditions in which the polynucleotide is expressed to produce a polypeptide comprising light chain and heavy chain variable regions; and recovering the polypeptide from the cell line or the culture medium in which it is cultured.

9. A pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof according to claim 1 as an active ingredient.

10. 10. The pharmaceutical composition of claim 9, wherein the cancer is any one selected from the group consisting of breast cancer, colorectal cancer, head and neck cancer, colon cancer, skin cancer, pancreatic cancer, lung cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, liver cancer, kidney cancer, clear cell sarcoma, melanoma, brain and spinal cord tumor, brain cancer, thymoma, mesothelioma, esophageal cancer, biliary tract cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndromes (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's Disease, endocrine system cancer, and sarcoma.

11. Use of the antibody or fragment thereof according to claim 1 for preparing a pharmaceutical composition for treating cancer.

12. A method for treating cancer, comprising administering to an individual in need thereof an effective amount of a pharmaceutical composition comprising the antibody or fragment thereof according to claim 1 as an active ingredient.

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