Anti-CTLA-4 monoclonal antibodies and uses thereof

Abstract

The present invention relates to anti-CTLA-4 monoclonal antibodies and uses thereof, more particularly to anti-CTLA-4 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. 10-2022-0111320, filed on September 2, 2022, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to anti-CTLA-4 monoclonal antibodies and uses thereof, more particularly to anti-CTLA-4 monoclonal antibodies and functional fragments thereof, and their use in the prevention or treatment of cancer. [Background technology]

[0003] CTLA-4 (cytotoxic T lymphocyte-associated antigen-4, CD152) is a membrane glycoprotein expressed by activated effector T cells. It binds to B7 ligands (CD80 / CD86) on antigen-presenting cells and is a co-inhibitory signaling receptor involved in suppressing T cell proliferation, cell cycle progression, and cytokine (IL-2, IFN-γ) production.

[0004] On the other hand, cancer cells, which are the targets of T cells, have a mechanism to suppress T cell activity and avoid attack by T cells in the body by stimulating CTLA-4 on T cells using B7 ligands. Focusing on this, third-generation anticancer drugs have been developed as immune anticancer drugs (cancer immunotherapy).

[0005] 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 CTLA-4, the binding site between cancer cells and T cells, and block immune evasion signals from co-inhibitory signal receptors, allowing T cells that are not hindered by the cancer cells' immune evasion mechanism 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, and have the effect of improving the quality of life of cancer patients and significantly extending their survival time with few side effects.

[0006] Antibody targeting of CTLA-4 is used as a therapeutic approach for various human malignancies, with the aim of blocking the inhibitory effect of CTLA-4 on T cells. Currently approved anti-CTLA-4 immunomodulatory monoclonal antibody therapeutics in Korea are ipilimumab and tremelimumab, which are used alone or in combination with other chemotherapy agents, vaccines, or other antibodies for the treatment of melanoma, non-small cell lung cancer, breast cancer, prostate cancer, pancreatic cancer, hepatocellular carcinoma, and mesothelioma.

[0007] This has led to an increasing need for anti-CTLA-4 antibodies that have significant CTLA-4 binding ability and can be used as effective immunological anti-cancer agents in anti-cancer therapy as third-generation anti-cancer drugs. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the inventors have been conducting research to develop an antibody that has specific binding ability to human CTLA-4 protein, and have confirmed that the antibody containing the unique CDR sequence provided in the present invention has excellent binding neutralization ability to B7-1 (CD80) and B7-2 (CD86), has strong ADCC (antibody-dependent cellular cytotoxicity) efficacy, and activates suppressed immune functions, thereby showing remarkable effects when applied as a cancer therapeutic agent, thereby completing the present invention.

[0009] Therefore, the object of the present invention is to i) an antibody heavy chain variable region (V) comprising a complementary-determining region (CDR) H1 containing the amino acid sequence represented by SEQ ID NO: 1, a complementary-determining region (CDR) H2 containing the amino acid sequence represented by SEQ ID NO: 2, and a complementary-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 ) an antibody comprising; and 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 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; The present invention provides an antibody or fragment thereof that binds to human CTLA-4 protein selected from the group consisting of:

[0010] 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.

[0011] 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.

[0012] Another object of the present invention is to provide use of said antibody or a fragment thereof for producing a pharmaceutical composition for treating cancer.

[0013] 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. [Means for solving the problem]

[0014] 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 ) an antibody comprising; and 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 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; The present invention provides an antibody or fragment thereof that binds to a human CTLA-4 protein selected from the group consisting of:

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] The present invention will be described in detail below.

[0020] CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) has a similar structure to CD28, a T cell surface molecule with similar functional characteristics. Like CD28, CTLA-4 is located on human chromosome 2, band q33-q34, and encodes a 223-amino acid protein containing one variable domain flanked by two hydrophobic regions. The chromosomal sequence identity between CD28 and CTLA-4 is approximately 20%, but the amino acid sequence identity is 30% in humans.

[0021] CTLA-4, the membrane receptor for cytotoxic T cells, contains B7-1 (CD80) and B7-2 (CD86), and shares the same B7 ligand as CD28, adversely affecting T cell activation. After T cell receptor (TCR) activation, CTLA-4 is upregulated and binds to B7 with higher avidity than the T lymphocyte receptor CD28, reducing T cell proliferation and cytokine secretion. CTLA-4 also induces B7-mediated reverse signaling, inducing indoleamine-2,3-dioxygenase (IDO), leading to tryptophan catabolism and consequent suppression of T cell proliferation. Furthermore, CTLA-4 is known to negatively regulate T cell activation by promoting the expression of casitas-B-lineage lymphoma (Cbl)-b protein or suppressing the formation of 70 kDa zeta-associated protein (ZAP 70). Recent studies have shown that CTLA-4 induces the PI3K / Akt pathway, cyclin D3, cyclin-dependent kinase (cdk4 / cdk6), and suppression of nuclear transcription factor (NF-κB).

[0022] Therefore, in the early stages of tumorigenesis, CTLA-4 reduces T cell activation by generating inhibitory signals that weaken the immune response against tumors, allowing cancer cells to evade T cell attack. Therefore, CTLA-4 plays an important role in regulating the immune response against tumors and is considered a potential target for immunotherapy.

[0023] Thus, the present invention provides antibodies or fragments thereof that bind to human CTLA-4 protein.

[0024] 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.

[0025] 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.

[0026] The variable region of an antibody, which contains the antigen-binding site, is subdivided into framework regions (FRs), which have little sequence variability, and complementary-determining regions (CDRs), which are hypervariable regions with 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. Within the variable region of an antibody, the CDRs, which have the highest sequence variability, are the sites that directly bind to the antigen and are therefore most important for the antigen specificity of the antibody.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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 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. LThe antibody or fragment thereof that binds to human CTLA-4 protein may be selected from the group consisting of: an antibody comprising

[0031] In another aspect of the present invention, the antibody or fragment thereof according to the present invention may be an antibody or fragment thereof that binds to human CTLA-4 protein, characterized in that it is an antibody selected from the group consisting of: i) an antibody comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 7 as a heavy chain variable region and the amino acid sequence of positions 1 to 110 of SEQ ID NO: 8 as a light chain variable region; ii) an antibody comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 15 as a heavy chain variable region and the amino acid sequence of positions 1 to 109 of SEQ ID NO: 16 as a light chain variable region; iii) an antibody comprising the amino acid sequence of positions 1 to 121 of SEQ ID NO: 23 as a heavy chain variable region and the amino acid sequence of positions 1 to 110 of SEQ ID NO: 24 as a light chain variable region; and iv) an antibody comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 31 as a heavy chain variable region and the amino acid sequence of positions 1 to 110 of SEQ ID NO: 32 as a light chain variable region.

[0032] In another aspect of the present invention, the antibody or fragment thereof according to the present invention may be an antibody or fragment thereof that binds to human CTLA-4 protein, more preferably characterized by being 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; and iv) an antibody comprising a heavy chain consisting of SEQ ID NO: 31 and a light chain consisting of SEQ ID NO: 32.

[0033] 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.

[0034] 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 said 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 may be a CDR configuration specific to human CTLA-4 protein, or a V H and V L It means a base sequence encoding an antibody consisting of heavy and light chains having the following structure:

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] According to one example of the present invention, it was confirmed that the antibody of the present invention has excellent binding neutralizing ability against B7-1 (CD80) and B7-2 (CD86) that bind to CLTA-4.

[0052] In another embodiment of the present invention, the antibody of the present invention was confirmed to bind to a HEK293 cell line expressing CTLA-4 and to exert antibody-dependent cell-mediated cytotoxicity (ADCC) via NK cells, demonstrating a strong concentration-dependent ADCC effect.

[0053] 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.

[0054] These results suggest that the antibodies of the present invention specifically bind to CTLA-4, thereby activating immune cells in the body and 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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. As long as the pharmaceutical composition of the present invention exhibits the effects of the present invention, there are no particular limitations on its formulation, administration route, or administration method.

[0062] Furthermore, the present invention provides use of the antibody or a fragment thereof for producing a pharmaceutical composition for treating cancer.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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]

[0067] Therefore, the present invention provides an antibody or fragment thereof that binds to human CTLA-4 protein. The antibody of the present invention binds to CTLA-4 on T cells and inhibits CTLA-4 stimulation by cancer cells, thereby preventing T cells from being inactivated and allowing T cells to attack cancer cells. At the same time, the antibody can exert a strong ADCC effect via NK cells, making it a highly effective cancer therapeutic agent. [Brief explanation of the drawings]

[0068] [Figure 1] FIG. 1 shows that the top four clones among the scFv sequences obtained via phage display were converted into full IgG1 form, and then the clones were expressed and purified through SDS-PAGE. [Figure 2] FIG. 2 shows the results of flow cytometry analysis of the binding ability of anti-CTLA-4 antibodies using the HEK293 cell line. [Figure 3] FIG. 3 shows the results of Western blotting using an HEK293 cell line overexpressing human CTLA-4 protein and an anti-CTLA-4 antibody. [Figure 4]FIG. 4 shows the results of immunoprecipitation (IP) performed using an HEK293 cell line overexpressing human CTLA-4 protein and an anti-CTLA-4 antibody. [Figure 5] FIG. 5 shows the results of competitive ELISA confirming the binding neutralizing ability of anti-CTLA-4 antibodies to B7-1 (CD80) and B7-2 (CD86). [Figure 6] FIG. 6 shows the results of an ADCC assay performed on the HEK293 cell line overexpressing human CTLA-4 protein using the NK92MI-CD16 cell line. [Figure 7] FIG. 7 shows the results of ELISA confirming immune activation by anti-CTLA-4 antibody after stimulation of human peripheral blood mononuclear cells with Staphylococcal enterotoxin B (SEB). [Figure 8] FIG. 8 shows the results of mixed lymphocyte reaction using human peripheral blood mononuclear cells from different donors, followed by ELISA to confirm immune activation by anti-CTLA-4 antibodies. [Figure 9] Figure 9 shows the results of confirming the in vivo anti-cancer efficacy of anti-CTLA-4 antibodies using a mouse model introduced with human CTLA-4 protein. DETAILED DESCRIPTION OF THE INVENTION

[0069] 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.

[0070] Example 1. Screening for scFv that specifically binds to human CTLA-4 protein

[0071] 1-1. Selection of antigen and scFv phage The antigen, CTLA-4, was provided in the form of a recombinant protein (hCTLA4-Fc, RND systems, Cat. No. 325-CT) during the screening process. Phage library display was used to screen for antibodies that specifically bind to CTLA-4. 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: 1 mL of the antigen (hCTLA4-Fc, RND systems, Cat. No. 325-CT) was placed in an immunotube at a concentration of 10 μg / mL and coated at 200 rpm and 4°C for at least 12 hours. The coated immunotubes were washed three times with 0.05% PBS-T and blocked for 2 hours at room temperature with 1 mL of 3% BSA blocking buffer. After 2 hours, the tubes were washed three times with 0.05% PBS-T. The blocked scFv library stock was added to the antigen-coated immunotubes and allowed to bind at 200 rpm at 37°C for 1 hour. Unbound scFv library stock was discarded and the tubes were washed three times with 0.05% PBS-T. To allow elution of specifically bound scFv-phage, the tubes were incubated with 100 mM TEA (triethylamine) for 5 minutes at room temperature and neutralized with Tris, pH 8.5, to prepare scFv-antigen conjugates. The prepared scFv-antigen conjugate was added to E. coli ER2537 cells for infection, and then incubated overnight at 37°C on LB / ampicillin / glucose agar medium. The E. coli ER2537 cells were transferred to SB / ampicillin medium and cultured until the OD600 value reached 0.5. Afterwards, 1 x 10 11 ~1×1012 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 CTLA-4 antigen.

[0072] 1-2. Selection of scFv antibodies that specifically bind to CTLA-4 To select scFvs with superior binding affinity from the scFv candidate group obtained in Example 1-1, ELISA analysis was performed on CTLA-4-expressing cell lines. CTLA-4-expressing cell lines were prepared by transfecting HEK293 cells with a human CTLA-4 expression vector and then treating the cells with 50 μg / ml hygromycin B to select transformants. Each library stock from each panning step in Example 1-1 was cultured 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 cultured again at 30°C overnight. After the culture was completed, 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 inoculated into a 1x10 CTLA-4 cell culture. 5 After incubation at room temperature for 1 hour, a secondary antibody (anti-HA HRP, Santa Cruz, Cat. No. sc-7392) was added and incubated for 40 minutes. After the secondary antibody incubation was complete, TMB was added to develop the color reaction, and the results were analyzed using an ELISA reader (450 nm). The ELISA values ​​were compared, and the top four scFvs (3E4, 4B6, 4F2, and 4G1) were selected. The amino acid sequences of the selected scFvs were analyzed, and the results are shown in Table 1 below.

[0073] [Table 1]

[0074] Example 2. Conversion of scFv antibody to IgG, expression, and purification

[0075] 2-1. Construction of a complete 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 each obtained by PCR, using the primers shown in Table 2 below. The light chain variable region sequence was cloned into pcDNA 3.3 (Invitrogen), an expression vector into which a light chain constant region sequence had been inserted, and the heavy chain variable region sequence was cloned into pOptiVEC (Invitrogen), an expression vector into which a heavy chain constant region sequence had been inserted. 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.

[0076] [Table 2]

[0077] 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 pCHO1.0 vector, transfection reagent (FreeStyle TM CHO-S cells were transduced with 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 first selection consisted of 10 μg / ml puromycin and 100 nM MTX, or 20 μg / ml puromycin and 200 nM MTX. Once cell viability met the criteria, the second selection process was initiated. The second selection consisted of 30 μg / ml puromycin and 500 nM MTX, or 50 μg / ml puromycin and 1000 nM MTX. Once cell viability met the criteria, the second selection process was terminated and high-expression populations were selected using Simple Fed Batch (SFB).

[0078] 2-3. Production and purification of whole IgG antibodies Each antibody-producing cell line prepared in Example 2-2 was designated 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. Protein A resin (Mabselect SuRe, 11-0026-01 AD, GE Healthcare Life Sciences) was used for purification, 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) were used. TM The column was purified using an avant column 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).

[0079] The IgG antibody proteins produced under reducing and non-reducing conditions were analyzed using standard SDS-PAGE techniques, and it was confirmed that 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.

[0080] Example 3. Evaluation of the binding specificity and avidity of the antibody according to the present invention to CTLA-4

[0081] 3-1. Generation of CTLA-4 / HEK293 cell line To confirm the antigen specificity of the antibody prepared in Example 2-3, the presence or absence of binding to human CTLA-4 protein was examined. The CTLA-4 gene was cloned into pCMV3-C-FLAG (Sino Biological) and transfected into HEK293 cells using Fugene HD (E231A, Promega) transfection reagent. Hygromycin B-resistant cell lines were then selected.

[0082] 3-2. Evaluation of the binding ability of the antibody of the present invention to the CTLA-4 / HEK293 cell line The cross-reactivity of the antibody prepared in Example 2-3 with the CTLA-4 / HEK293 cell line prepared in Example 3-1 was confirmed. Native HEK293 cells were used as a negative control. First, single cells were dissociated using cell dissociation buffer (Gibco, 13151-014), and then 2.5 x 10 cells were collected. 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 concentration 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 CTLA-4.

[0083] 3-3. Confirmation of CTLA-4 specific binding - Western blot The ability of the antibodies prepared in Examples 2-3 to recognize the denatured CTLA-4 antigen was confirmed using the CTLA-4 / HEK293 cell line prepared in Example 3-1. Native HEK293 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. The resulting mixture was then loaded on SDS-PAGE, separated by size, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at 4°C for 1 hour. The antibody prepared in Examples 2-3 was used as the primary antibody and attached to the PVDF membrane overnight. 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 does not recognize the denatured CTLA-4 antigen.

[0084] 3-4. Confirmation of CTLA-4 specific binding - immunoprecipitation In Example 3-1, the ability of the antibodies prepared in Examples 2-3 to recognize the native CTLA-4 antigen was confirmed for the CTLA-4 / HEK293 cell line. Native HEK293 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 TLA-4 antibody (Cell signaling technology) to confirm that the antibody recognized the intact CTLA-4 antigen. As a result, as shown in FIG. 4, it was confirmed that the antibody specifically binds to the intact CTLA-4 antigen.

[0085] Example 4. Confirmation of binding neutralizing ability to B7-1 (CD80) and B7-2 (CD86)

[0086] To confirm the binding neutralization ability of the antibodies prepared in Examples 2-3, the binding neutralization ability of CTLA4-B7-1 (CD80) and CTLA4-B7-2 (CD86) was measured by ELISA. 250 ng of human CTLA-4 protein (hCTLA4-Fc, RND Systems, Cat. No. 325-CT) 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. The blocked 96-well plate was washed three times with 0.1% PBS-T. Anti-CTLA-4 antibody, diluted from 60 μg / mL to a 1 / 3 concentration, was added. B7-1 (CD80)-biotin and B7-2 (CD86)-biotin (Sino Biological) were added and allowed to bind for 2 hours at room temperature. An equal amount of ipilimumab (BMS) was used as a positive control. 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, and 50 μl of stop solution was added to each plate. The wavelength at 450 nm was measured using an ELISA reader. As a result, as shown in FIG. 5, it was confirmed that the antibody inhibited the binding of CTLA4-B7-1 (CD80) and CTLA4-B7-2 (CD86).

[0087] Example 5. Confirmation of antibody-dependent cellular cytotoxicity

[0088] The antibody-dependent cellular cytotoxicity of the antibody prepared in Example 2-3 was confirmed against the CTLA-4 / HEK293 cell line prepared in Example 3-1. The CTLA-4 / HEK293 cell line was used as the target cells, and a cell line obtained by transfecting NK92MI cell line with human CD16 was used as the effector cells. The target cells were separated into single cells using a separation buffer, and then placed in a 96-well plate at 2.0 × 10 4The effector cells were divided into 1.0x10 cells and cultured overnight. 5 Anti-CTLA-4 antibodies were diluted from 1 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. 6, it was confirmed that the antibody concentration-dependently killed cells expressing CTLA-4 through antibody-dependent cellular cytotoxicity mediated by NK cells.

[0089] Example 6. Confirmation of immune activation ability

[0090] 6-1. Immune activation by anti-CTLA-4 antibody (IL-2) 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. Peripheral blood concentrated in an LRS chamber was diluted with 2% FBS / PBS. A 50 mL tube was filled with Histopaque. (R) 25 mL of Sigma-Aldrich HCl (A1077) 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 PBMC were then washed three times with 2% FBS / PBS washing buffer at 300 g for 8 minutes to obtain the PBMC. The isolated human PBMC were then plated at 2.0 x 10 5 The cells were cultured in a 100 ng / mL culture medium containing SEB (Abion) and anti-CTLA-4 antibody diluted to one-third of the original concentration of 30 μg / mL. The cells were then incubated at 37°C for 3 days. After 3 days, 100 μl of the supernatant was used for further analysis. IL-2 secretion due to immune activation was measured by ELISA (Human IL-2 DuoSet ELISA, R&D Systems, Cat. No. DY 202). As a result, as shown in FIG. 7, it was confirmed that the antibody maximized IL-2 secretion from human PBMCs through immune activation.

[0091] 6-2. Confirmation of immune activation by anti-CTLA-4 antibody 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 6-1, 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-CTLA-4 antibody diluted from 30 μg / ml to a concentration of 1 / 3 and then incubated at 37°C for 5 days. After 5 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. DY285 B). As a result, as shown in FIG. 8, it was confirmed that the antibody increased IFN-γ secretion in a concentration-dependent manner.

[0092] Example 7. Confirmation of in vivo anti-cancer efficacy of antibodies To confirm the in vivo anti-cancer efficacy of the anti-CTLA-4 antibody, the anti-CTLA-4 antibody prepared above was injected into cancer-induced human CTLA-4 transfected mice, and changes in tumor size were observed. First, 5x10 MC38 cells, a mouse colon cancer cell line, were added to human CTLA-4 protein-transfected mice. 5 The tumors were injected subcutaneously. The average tumor size was 150 mm. 3 When tumor size reached 100%, five animals 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 4G1 antibody at a dose of 20 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 mm3 was calculated. As a result, as can be seen in FIG. 9, the tumor size was statistically significantly reduced in the 4G1 antibody administration group compared to the control group (t-test, p value=0.04). [Industrial Applicability]

[0093] As described above, the present invention provides an antibody or fragment thereof that binds to human CTLA-4 protein. The antibody of the present invention binds to CTLA-4 on T cells and inhibits CTLA-4 stimulation by cancer cells, thereby preventing T cell inactivation and allowing T cells to attack cancer cells. At the same time, the antibody can exert a strong ADCC effect via NK cells, demonstrating significant efficacy as a cancer therapeutic agent, making it highly industrially applicable.

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 An antibody or fragment thereof that binds to human CTLA-4 protein selected from the group consisting of:

2. The antibody i) an antibody comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 7 in the heavy chain variable region and the amino acid sequence of positions 1 to 110 of SEQ ID NO: 8 in the light chain variable region; ii) an antibody comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 15 in the heavy chain variable region and the amino acid sequence of positions 1 to 109 of SEQ ID NO: 16 in the light chain variable region; iii) an antibody comprising the amino acid sequence of positions 1 to 121 of SEQ ID NO: 23 in the heavy chain variable region and the amino acid sequence of positions 1 to 110 of SEQ ID NO: 24 in the light chain variable region; and iv) an antibody comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 31 in its heavy chain variable region and the amino acid sequence of positions 1 to 110 of SEQ ID NO: 32 in its light chain variable region; An antibody or fragment thereof that binds to the human CTLA-4 protein according to 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; and iv) an antibody comprising a heavy chain consisting of SEQ ID NO: 31 and a light chain consisting of SEQ ID NO: 32; An antibody or fragment thereof that binds to the human CTLA-4 protein according to 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. A method for preparing an antibody or fragment thereof that binds to human CTLA-4 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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