Anti-CTLA-4 monoclonal antibody and application thereof

Abstract

The present invention relates to an anti-CTLA-4 monoclonal antibody and a use thereof, and more particularly, to an anti-CTLA-4 monoclonal antibody and a functional fragment thereof, and a use thereof for preventing or treating cancer.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2022-0111320, filed on September 2, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to anti-CTLA-4 monoclonal antibodies and uses thereof, and more particularly to anti-CTLA-4 monoclonal antibodies and functional fragments thereof, and uses thereof for preventing or treating cancer. Background of the Invention

[0004] CTLA-4 (cytotoxic T lymphocyte-associated antigen-4, CD152) is a membrane glycoprotein expressed by activated effector T cells. It is a co-inhibitory signal receptor that participates in the inhibition of T cell proliferation, cell cycle progression and cytokine (IL-2, IFN-γ) production by binding to the B7 ligand (CD80 / CD86) of antigen-presenting cells.

[0005] At the same time, cancer cells targeted by T cells have a mechanism to escape attack by T cells in the body by stimulating CTLA-4 on T cells using the B7 ligand to suppress T cell activity. Based on this mechanism, cancer immunotherapy developed as a third-generation anticancer agent has been introduced.

[0006] Anticancer agents are roughly divided into first-generation chemical anticancer agents, second-generation targeted anticancer agents, and third-generation immune anticancer agents. Different from the drawbacks of the first and second generation anticancer agents, the immune anticancer agents belonging to the third generation bind CTLA-4 (which is a binding site between cancer cells and T cells) and block the immune escape signal mediated by co-inhibitory signal receptors. This mechanism allows T cells (which are not affected by the immune escape mechanism of cancer cells) to destroy cancer cells. In other words, immune anticancer agents have a new mechanism, which enhances the suppressed immune function of cancer cells in the body, thereby causing the destruction of cancer cells. They have fewer side effects, improve the quality of life of cancer patients, and significantly prolong the survival period.

[0007] Targeting CTLA-4 with antibodies is used as a therapeutic approach in a variety of human malignancies to block the inhibitory effects mediated by CTLA-4 in T cells. Currently, anti-CTLA-4 immunomodulatory monoclonal antibody therapies approved in South Korea include ipilimumab and tremelimumab, which are used alone or in combination with other chemotherapy, vaccines or other antibodies for melanoma, non-small cell lung cancer, breast cancer, prostate cancer, pancreatic cancer, hepatocellular carcinoma and mesothelioma.

[0008] Therefore, there is an increasing demand for anti-CTLA-4 antibodies with excellent CTLA-4 binding ability, which can be used as effective immunological anticancer agents for cancer treatment as third-generation anticancer agents.

[0009] Issues to be resolved

[0010] Therefore, the present inventors, while conducting studies to develop antibodies having specific binding affinity to human CTLA-4 protein, found that antibodies comprising the unique CDR sequences provided in the present invention exhibit excellent binding neutralization ability to B7-1 (CD80) and B7-2 (CD86), have strong ADCC (antibody-dependent cellular cytotoxicity) activity, and activate suppressed immune functions, thereby showing significant effects in their use as cancer therapeutic agents, and thus completed the present invention.

[0011] Therefore, an object of the present invention is to provide an antibody or a fragment thereof that binds to a human CTLA-4 protein, wherein the antibody or the fragment thereof is selected from the group consisting of:

[0012] i) an antibody comprising an antibody heavy chain variable region (the variable region in the heavy chain, V H ) and the antibody light chain variable region (the variable region in the light chain, V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 1, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 2, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 3, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 6;

[0013] ii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO:9, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO:10, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO:11, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO:12, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO:13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO:14;

[0014] iii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 17, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 19, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 22; and

[0015] iv) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region contains a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:25, a complementarity determining region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO:26, and a complementarity determining region (CDR) H3 comprising the amino acid sequence shown in SEQ ID NO:27, and the antibody light chain variable region contains a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:28, a complementarity determining region (CDR) L2 comprising the amino acid sequence shown in SEQ ID NO:29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence shown in SEQ ID NO:30.

[0016] Another object of the present invention is to provide a polynucleotide encoding the antibody or a fragment thereof, a recombinant vector comprising the polynucleotide, a transformed cell line comprising the recombinant vector, and a method for producing the antibody or a fragment thereof using the transformed cell line.

[0017] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.

[0018] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which consists of the antibody or a fragment thereof.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which consists essentially of the antibody or fragment thereof.

[0020] Another object of the present invention is to provide use of the antibody or fragment thereof in preparing a pharmaceutical composition for treating cancer.

[0021] 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 a subject in need thereof.

[0022] Means of solving the problem

[0023] In order to achieve the above objectives, the present invention provides an antibody or a fragment thereof that binds to human CTLA-4 protein, wherein the antibody or the fragment thereof is selected from the group consisting of:

[0024] i) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 1, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 2, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 3, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 6;

[0025] ii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO:9, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO:10, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO:11, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO:12, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO:13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO:14;

[0026] iii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 17, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 19, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 22; and

[0027] iv) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region contains a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:25, a complementarity determining region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO:26, and a complementarity determining region (CDR) H3 comprising the amino acid sequence shown in SEQ ID NO:27, and the antibody light chain variable region contains a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:28, a complementarity determining region (CDR) L2 comprising the amino acid sequence shown in SEQ ID NO:29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence shown in SEQ ID NO:30.

[0028] The present invention also provides a polynucleotide encoding the antibody or its fragment, a recombinant vector comprising the polynucleotide or its fragment, a transformed cell line comprising the polynucleotide or its fragment, and a method for producing the antibody or its fragment using the polynucleotide or its fragment.

[0029] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or fragment thereof as an active ingredient.

[0030] In addition, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which is composed of the above-mentioned antibody or a fragment thereof.

[0031] In addition, in order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer, which is mainly composed of the above-mentioned antibody or a fragment thereof.

[0032] In addition, to achieve another object of the present invention, the present invention provides use of the antibody or fragment thereof in preparing a pharmaceutical composition for treating cancer.

[0033] In addition, 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 comprising the antibody or a fragment thereof as an active ingredient to a subject in need thereof.

[0034] The present invention will now be described in detail.

[0035] CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) has a similar structure to the T cell surface molecule CD28 and has similar functional properties. Like CD28, CTLA-4 is located on the q33-q34 band of human chromosome 2 and encodes a 223 amino acid protein that contains a single variable domain adjacent to two hydrophobic regions. The chromosomal sequence homology between CD28 and CTLA-4 is approximately 20%, while the amino acid sequence homology in humans is 30%.

[0036] The membrane receptor CTLA-4 of cytotoxic T cells has the same B7 ligands as CD28, including B7-1 (CD80) and B7-2 (CD86), and has a negative impact on T cell activation. After T cell receptor (TCR) activation, CTLA-4 is upregulated and binds to B7 with a higher affinity than the T lymphocyte receptor CD28, reducing T cell proliferation and cytokine secretion. In addition, CTLA-4 induces reverse signaling through B7, leading to the induction of indoleamine-2,3-dioxygenase (IDO), which causes the catabolism of tryptophan, thereby inhibiting T cell proliferation. In addition, CTLA-4 is known to negatively regulate T cell activation by promoting the expression of casitas-B lineage lymphoma (Cbl)-b protein or inhibiting the formation of 70kDa zeta-associated protein (ZAP 70). Recent studies have shown that CTLA-4 induces inhibition of the PI3K / Akt pathway, cyclin D3, cyclin-dependent kinases (cdk4 / cdk6), and nuclear transcription factors (NF-κB).

[0037] Therefore, in the early stages of tumor formation, CTLA-4 generates inhibitory signals that weaken the immune response to the tumor, thereby reducing T cell activation and allowing cancer cells to evade T cell attack. Therefore, CTLA-4 plays a key role in regulating the immune response to tumors and is considered a potential target for immuno-oncology therapy.

[0038] Therefore, the present invention provides antibodies or fragments thereof that bind to human CTLA-4 protein.

[0039] In the present invention, the term "antibody" refers to a protein, also called immunoglobulin (Ig), that selectively acts on antigens and participates in immune responses in living organisms. Naturally occurring complete antibodies are usually composed of two pairs of polypeptides, light chains (LC) and heavy chains (HC), each chain consisting of several domains or basic units of these LC / HC pairs. The heavy chain types in mammalian antibodies are divided into five types, represented by the Greek letters α, β, γ, δ, and μ, which form different types of antibodies according to the heavy chain type, such as IgA, IgD, IgE, IgG, and IgM. The light chain types in mammalian antibodies are divided into two types, represented by λ and κ.

[0040] The heavy chain and light chain of an antibody are structurally divided into a variable region and a constant region according to the variability of its amino acid sequence. Depending on the type of antibody, the constant region of the heavy chain is composed of three or four heavy chain constant regions CH1, CH2 and CH3 (in IgA, IgD and IgG antibodies) or CH4 (in IgE and IgM antibodies), while the light chain is composed of a constant region LC. The heavy chain and the light chain are arranged side by side with their respective variable regions and constant regions and are connected by a single covalent disulfide bond. The variable regions of the heavy chain and the light chain can both bind specifically to the antigen, and the complete antibody is composed of two pairs of heavy chains and light chains (HC / LC), so that a single antibody molecule has a bivalent monospecificity, which binds to two identical antigens through its two variable regions. The variable region of an antibody that binds to an antigen is called an antigen binding site, and the part of the antigen that the antibody recognizes is called an epitope.

[0041] The variable region of an antibody, which contains the antigen binding site, is subdivided into framework regions (FRs) with low sequence variability and hypervariable regions (complementarity determining regions (CDRs)) with high sequence variability. H and V L Each is arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from N-terminus to C-terminus, and contains 2 CDRs and 4 FRs. Among the variable regions of antibodies, the CDR with the highest sequence variability is the region that directly binds to the antigen and is most important for the antigen specificity of the antibody.

[0042] The antibody or fragment thereof of the present invention has the above-mentioned CDR, V H and V L , or the configuration of light chain and heavy chain, then its type is not limited. The antibody can be in the form of IgG, IgA, IgM, IgE or IgD, and is particularly preferably an IgG antibody. IgG may include but is not limited to its subclasses, such as IgG1, IgG2, IgG3 and IgG4. In addition, it can 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 an antibody group with substantially the same amino acid sequence of heavy chain and light chain. In addition, the antibody of the present invention or its fragment can be conjugated with an enzyme, a fluorescent substance, a radioactive substance, a protein, etc., but is not limited thereto.

[0043] The antibody of the present invention may be derived from any animal, including mammals and birds, including humans, and preferably, it may be derived from humans, or may be a chimeric antibody comprising a portion of an antibody derived from humans and a portion of an antibody derived from another species.

[0044] In the present invention, an antibody fragment refers to a fragment that retains the antigen-specific binding ability of the entire antibody, and specifically can be in the form of Fab, F(ab'), F(ab')2, Fv, scFv, diabody or dsFv.

[0045] Fab (Fragment of antigen binding) is the antigen binding fragment of an antibody, consisting of one variable domain each of the heavy and light chains and one constant domain. F(ab')2 is a fragment produced after digestion of an antibody with pepsin, in which two Fabs are linked by disulfide bonds at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which the heavy chain hinge is added to the Fab separated by reducing the disulfide bonds of the F(ab')2 fragment. Fv (Fragment variable) is an antibody fragment consisting of only the variable regions of the heavy and light chains. scFv (Single chain Variable fragment) is a recombinant antibody fragment in which the variable region of the heavy chain (V H ) and light chain variable region (V L ) are connected by a flexible peptide linker. Diabodies are fragments in which the V H and V L They are connected by a very short linker to prevent them from binding to each other, while the V of another scFv of the same type H and V L dsFv refers to a polypeptide in which V H and V L One of the amino acid residues on each of the two is substituted with a cysteine ​​residue, and the two are bound via an SS bond mediated by the cysteine ​​residue. The amino acid residue to be substituted with a cysteine ​​residue can be selected based on the prediction of the three-dimensional structure of the antibody according to a known method.

[0046] In one embodiment of the present invention, the antibody or fragment thereof of the present invention may be an antibody selected from the group consisting of: i) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 1, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 2, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 3, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:9, a complementarity determining region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO:10, and a complementarity determining region (CDR) H3 comprising the amino acid sequence shown in SEQ ID NO:11, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:12, a complementarity determining region (CDR) L2 comprising the amino acid sequence shown in SEQ ID NO:13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence shown in SEQ ID NO:14; iii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 17, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 19, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 22; and iv) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region contains a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:25, a complementarity determining region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO:26, and a complementarity determining region (CDR) H3 comprising the amino acid sequence shown in SEQ ID NO:27, and the antibody light chain variable region contains a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:28, a complementarity determining region (CDR) L2 comprising the amino acid sequence shown in SEQ ID NO:29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence shown in SEQ ID NO:30.

[0047] In another embodiment of the present invention, the antibody or fragment thereof of the present invention may preferably be an antibody selected from the group consisting of: i) an antibody comprising a heavy chain variable region comprising the amino acid sequence from 1 to 116 of SEQ ID NO:7 and a light chain variable region comprising the amino acid sequence from 1 to 110 of SEQ ID NO:8; ii) an antibody comprising a heavy chain variable region comprising the amino acid sequence from 1 to 116 of SEQ ID NO:15 and a light chain variable region comprising the amino acid sequence from 1 to 109 of SEQ ID NO:16; iii) an antibody comprising a heavy chain variable region comprising the amino acid sequence from 1 to 121 of SEQ ID NO:23 and a light chain variable region comprising the amino acid sequence from 1 to 110 of SEQ ID NO:24; and iv) an antibody comprising a heavy chain variable region comprising the amino acid sequence from 1 to 116 of SEQ ID NO:31 and a light chain variable region comprising the amino acid sequence from 1 to 110 of SEQ ID NO:32.

[0048] In another embodiment of the present invention, the antibody or fragment thereof of the present invention may more preferably be 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.

[0049] The present invention also provides a polynucleotide encoding the antibody or its fragment, a recombinant vector comprising the polynucleotide or its fragment, a transformed cell line comprising the polynucleotide or its fragment, and a method for producing the antibody or its fragment using the polynucleotide.

[0050] In the present invention, the term "polynucleotide" 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 DNA or RNA produced 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 polypeptide encoding a CDR configuration specific to human CTLA-4 protein, or having a V H and V L The nucleotide sequences of the heavy and light chains of the antibody are shown in Figure 2.

[0051] The polynucleotides encoding the antibodies of the present invention or their antigen-binding fragments 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 polymerase chain reaction (PCR), based on partial or complete DNA sequences or corresponding amino acid sequences encoding antibody heavy and light chains.

[0052] The term "vector" in the present invention is used to clone or express the polynucleotide of the present invention for the purpose of recombinantly producing the antibody or antigen-binding fragment thereof of the present invention, and generally includes 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 a vector comprising a polynucleotide of the present invention operably linked to a regulatory sequence (e.g., a promoter).

[0053] Plasmid (which is a kind of vector) refers to a linear or circular double-stranded DNA molecule to which external polynucleotide fragments can be attached. Other forms of vectors include viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA fragments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in host cells (e.g., bacterial vectors, including bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction and replicated together with the host genome.

[0054] In the present invention, the term "vector" can be understood to have the same meaning as "expression vector", which is a type of vector capable of expressing polynucleotides. When a regulatory sequence affects the expression (e.g., the level, time or position of expression) of a polynucleotide sequence, the polynucleotide sequence is "operably connected" to a regulatory sequence. A regulatory sequence is a sequence that affects the expression (e.g., the level, time or position of expression) of a nucleic acid that is operably connected thereto. A regulatory sequence can directly exert its influence on the regulated nucleic acid or exert its influence through the effect of one or more other molecules (e.g., a polypeptide bound to a regulatory sequence and / or nucleic acid). Regulatory sequences include promoters, enhancers and other expression control elements.

[0055] The cell line of the present invention is not particularly limited, as long as it is a cell that can be used to express the polynucleotide encoding the antibody or its fragment included in the expression vector of the present invention. The host cell transformed by the expression vector of the present invention can be a prokaryotic organism (e.g., Escherichia coli), a eukaryotic organism (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, insect cells), or a hybridoma derived from these cells. Preferably, it can be a cell derived from a mammal (including humans).

[0056] Prokaryotes suitable for this purpose include Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens), and Shigella; Bacillus belonging to the class Bacillus (e.g., B. subtilis and B. licheniformis); Pseudomonas (e.g., P. aeruginosa); and Streptomyces. The cell of the present invention is not particularly limited as long as it can express the vector of the present invention, but Escherichia coli is preferred.

[0057] As the eukaryotic organism, the cell of the present invention is most commonly Saccharomyces cerevisiae. However, many other genera, species and strains may be used, including but not limited to: Schizosaccharomyces pombe, Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesei (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0058] The term "transformation" refers to changing the genotype of a host cell by introducing an exogenous polynucleotide (a polynucleotide encoding an antibody of the present invention or a fragment thereof), regardless of the method used for transformation, and means that the exogenous polynucleotide has been introduced into the host cell. The exogenous polynucleotide introduced into the host cell may remain integrated into the host cell genome, or may remain unintegrated, and the present invention includes both cases.

[0059] Recombinant vectors capable of expressing the antibodies or fragments thereof of the present invention can be introduced into cells to produce 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.

[0060] In addition, the cell of the present invention can be a cultured cell transformed or transfected with a polynucleotide of the present invention or a vector comprising the same, which can then be expressed in a host cell. A recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed. The cell of the present invention can also include a polynucleotide of the present invention, but may not express it at a desired level unless a regulatory sequence is operably linked to the polynucleotide in the cell.

[0061] The cells of the present invention can be cultured in a variety of culture media. Commercially available culture media, such as Ham's F10 (Sigma-Aldrich Co., St. Louis, MO), Minimum Essential Medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich Co.) are suitable for culturing cells. If necessary, the culture medium can be supplemented with hormones and / or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or an equivalent energy source.

[0062] The present invention provides a method for producing an antibody or a fragment thereof that binds to a human CTLA-4 protein, comprising the steps of culturing cells under conditions where a 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 the cells are cultured.

[0063] In the present invention, the cell used in the production method is as described above, and includes a polynucleotide encoding the antibody of the present invention. The polypeptide of the production method may be the antibody of the present invention or its fragment itself, or may be in the form of an antibody of the present invention or its fragment additionally bound to other amino acid sequences.

[0064] In this case, additional amino acid sequences can be removed from the antibody or fragment thereof of the present invention using methods well known to those skilled in the art. Culture conditions, including the composition of the culture medium and culture conditions, may vary depending on the type of cell, and these may be appropriately selected and adjusted by those skilled in the art.

[0065] The antibody molecule can be accumulated in the cytoplasm of the cell, can be secreted from the cell, or can be targeted to the periplasm or extracellular medium (supernatant) by an appropriate signal sequence, and it is preferably targeted to the periplasm or extracellular medium. In addition, it is preferred to refold the antibody molecule produced using methods well known to those skilled in the art to achieve a functional conformation. The recovery of the polypeptide varies according to the characteristics of the polypeptide produced and the cell, and this can be appropriately selected and adjusted by those skilled in the art.

[0066] Polypeptide can be produced in the cytoplasmic space around the cell, or directly secreted into the culture medium. If the polypeptide is produced in the cell, the first step is to destroy the cell to release the protein. For example, particle debris, host cells or lysate fragments can be removed by centrifugation or ultrafiltration. When the antibody is secreted into the culture medium, usually first a commercially available protein concentration filter (such as Amicon or Millipore Pellicon ultrafiltration unit) is used to concentrate the supernatant from this type of expression system. Protease inhibitors, such as PMSF, can be optionally included in the initial step to inhibit protein degradation, and antibiotics can be included to prevent the growth of unexpected pollutants. Hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography purification can be used to purify the antibody produced from the cell, and the antibody of the present invention is preferably purified by affinity chromatography.

[0067] According to one embodiment of the present invention, it has been demonstrated that the antibody of the present invention exhibits excellent binding neutralization ability against B7-1 (CD80) and B7-2 (CD86) bound to CTLA-4.

[0068] According to another embodiment of the present invention, it was demonstrated that the antibody of the present invention can bind to HEK293 cell line expressing CTLA-4 and exhibit concentration-dependent and strong antibody-dependent cellular cytotoxicity (ADCC) by NK cells.

[0069] According to still another embodiment of the present invention, it was confirmed that the antibody of the present invention activated immunity in a concentration-dependent manner.

[0070] These results suggest that the antibodies of the present invention specifically bind to CTLA-4, thereby activating immune cells in the body and ultimately killing cancer cells. In fact, when the antibodies of the present invention were administered to mice induced with colorectal cancer, the tumor size was significantly reduced compared to the control group administered with IgG.

[0071] Therefore, the present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the antibody or fragment thereof as an active ingredient.

[0072] In the present invention, cancer can be solid or non-solid cancer. Solid cancer refers to cancer tumors occurring in organs such as liver, lung, breast and skin. Non-solid cancer refers to cancer occurring in the blood, also known as blood cancer. Cancer can be carcinoma, sarcoma, cancer derived from hematopoietic cells, germ cell tumor or blastoma. The cancer can be 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 tumors, brain cancer, thymoma, mesothelioma, esophageal cancer, bile duct cancer, testicular cancer, germ cell tumor, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer and sarcoma.

[0073] The composition of the present invention may comprise only the antibody of the present invention or its fragment, or may be formulated into a suitable form together with a pharmaceutically acceptable carrier and may further comprise an excipient or diluent. The term "pharmaceutically acceptable" refers to a physiologically acceptable non-toxic composition and does not cause allergic or similar reactions, such as gastrointestinal disturbances or dizziness, when administered to humans. The carrier may include all types of solvents, dispersion media, water-in-oil or oil-in-water emulsions, aqueous compositions, liposomes, microbeads and microemulsions.

[0074] Pharmaceutically acceptable carriers may further include carriers such as for oral or parenteral administration. Oral carriers may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, it may include a variety of drug delivery materials for oral administration of peptide preparations. In addition, carriers for parenteral administration may include water, suitable oils, saline, aqueous glucose solutions, and ethylene glycol, and may also 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 parabens, and chlorobutanol. The pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and preparations can refer to known literature.

[0075] The compositions of the present invention can be administered to mammals, including humans, by any method. For example, they can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0076] The pharmaceutical composition of the present invention can be formulated into oral or parenteral preparations according to the above-mentioned administration routes. In the case of oral preparations, the composition of the present invention can be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquid preparations, gels, syrups, slurries, suspensions, etc. by methods known in the art. For example, oral preparations can be obtained by mixing the active ingredient with a solid excipient, grinding the mixture, adding a suitable adjuvant, and processing it into a granular mixture to produce tablets or sugar-coated tablets. Examples of suitable excipients include sugars, such as lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches, such as corn starch, wheat starch, rice starch, and potato starch; celluloses, such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and fillers, such as gelatin and polyvinyl pyrrolidone. In addition, disintegrants such as cross-linked polyvinyl pyrrolidone, agar, alginic acid, or sodium alginate can be added as needed. In addition, the pharmaceutical composition of the present invention may additionally include anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives. In the case of parenteral preparations, the composition can be formulated into forms such as injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants using methods known in the art. These preparations are described in the literature generally known in the field of pharmaceutical chemistry.

[0077] The total effective amount of the composition of the present invention can be administered to the patient as a single dose, or by a divided treatment regimen involving long-term multiple doses. The content of the active ingredient of the pharmaceutical composition of the present invention can be changed according to the severity of the disease. Preferably, the total daily dose of the pharmaceutical composition of the present invention is about 0.01 μg to 10,000 mg per kg of patient body weight, more preferably 0.1 μg to 500 mg. However, the dosage of the pharmaceutical composition is determined by considering various factors, such as formulation method, route of administration, frequency of treatment, and age, weight, health status, sex, severity of the disease, diet, excretion rate of the patient. Therefore, those skilled in the art can determine the appropriate effective dose of the composition of the present invention by considering these factors. The pharmaceutical composition of the present invention, its preparation, route of administration or method of administration are not particularly limited, as long as it exhibits the effect of the present invention.

[0078] In addition, the present invention provides use of the antibody or fragment thereof in preparing a pharmaceutical composition for treating cancer.

[0079] In addition, the present invention provides a method for treating cancer, which comprises administering an effective amount of a pharmaceutical composition comprising the antibody or a fragment thereof as an active ingredient to a subject in need thereof.

[0080] The term "effective amount" in the present invention refers to an amount that exhibits an effect, such as improvement, treatment, detection, diagnosis, inhibition or alleviation of cancer or disease, when administered to a subject. The term "subject" may include animals, preferably mammals, particularly humans, and may also include cells, tissues or organs derived from animals. The subject may be a patient in need of the aforementioned effect.

[0081] The term "treatment" in the present invention generally refers to improving the symptoms caused by cancer or disease, and may include curing the disease, substantially preventing the disease, or improving the condition. It may also include alleviating, curing or preventing one or more symptoms or most symptoms caused by the disease, but is not limited thereto.

[0082] As used herein, the term "comprising" is synonymous with "including" or "characterized by," and does not exclude additional components or steps not specifically mentioned in the compositions or methods of the invention. The term "consisting of" means excluding additional elements, steps or components not specifically described. The term "consisting essentially of" means that the composition or method may include substances or steps that do not substantially affect the basic characteristics of the substances or steps.

[0083] Effects of the Invention

[0084] Therefore, the present invention provides an antibody or a fragment thereof that binds to human CTLA-4 protein. The antibody of the present invention binds to CTLA-4 of T cells, thereby inhibiting the stimulation of CTLA-4 caused by cancer cells, preventing T cell inactivation, and enabling T cells to attack cancer cells. At the same time, it can exert a powerful ADCC effect through NK cells, thereby showing a significant effect as an anticancer agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 It was shown that the clones of the first 4 positions in the scFv sequence obtained by phage display were converted into a complete IgG1 format, and SDS-PAGE confirmed that the clones were expressed and purified.

[0087] Figure 2 The results of detecting the binding ability of anti-CTLA-4 antibody to HEK293 cell line using flow cytometry are shown.

[0088] Figure 3 Shown are the results of Western blotting using HEK293 cell lines overexpressing human CTLA-4 protein and anti-CTLA-4 antibodies.

[0089] Figure 4 Shown are the results of immunoprecipitation (IP) using HEK293 cell line overexpressing human CTLA-4 protein and anti-CTLA-4 antibody.

[0090] Figure 5 The results of competitive ELISA testing of the neutralizing ability of anti-CTLA-4 antibodies against B7-1 (CD80) and B7-2 (CD86) are shown.

[0091] Figure 6 Shown are the results of ADCC assays using the NK92MI-CD16 cell line against the HEK293 cell line overexpressing the human CTLA-4 protein.

[0092] Figure 7 Shown are the results of ELISA testing of immune activation by anti-CTLA-4 antibodies after stimulation of human peripheral blood mononuclear cells with Staphylococcal enterotoxin B (SEB).

[0093] Figure 8 Shown are the results of mixed lymphocyte reaction with human peripheral blood mononuclear cells from different donors followed by ELISA for immune activation by anti-CTLA-4 antibody.

[0094] Fig. 9 The results show the in vivo anticancer effect of anti-CTLA-4 antibodies using a mouse model into which human CTLA-4 protein has been introduced. DETAILED DESCRIPTION OF THE INVENTION

[0096] The present invention is described in detail below.

[0097] However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited to the following examples.

[0098] Example 1: Screening of scFvs that specifically bind to human CTLA-4 protein

[0099] 1-1. Antigen and scFv phage selection

[0100] CTLA-4 is provided as an antigen in the form of a recombinant protein (hCTLA4-Fc, RND system, Cat.No 325-CT) during the screening process. In order to screen antibodies that specifically bind to CTLA-4, a phage display method (phage library display) was used. The library used is a synthetic human scFv library, and specific information about the library is described in the publicly known literature in this field. The scFv expressed from the scFv library carries an HA tag to allow detection by an anti-HAFITC antibody (Genscript, A01621). Using the scFv library, bio-panning was performed as follows. The antigen hCTLA4-Fc, RND system, Cat.No 325-CT) was coated by adding 1 mL at a concentration of 10 μg / mL to the immunotube and placing it at 200 rpm, 4°C for more than 12 hours. The coated immunotube was washed three times with 0.05% PBS-T and blocked with 1 mL 3% BSA blocking buffer at room temperature for 2 hours. After 2 hours, wash three times with 0.05% PBS-T. Add the blocked scFv library stock solution to the antigen-coated immunotube and incubate at 200rpm, 37°C for 1 hour. Discard the unbound scFv library stock solution and wash the tube three times with 0.05% PBS-T. In order to elute the specifically bound scFv-phage, react it with 100mM TEA (triethylamine) at room temperature for 5 minutes, neutralize it with Tris at pH 8.5, and prepare it in the form of scFv-antigen conjugate. The prepared scFv-antigen conjugate is added to Escherichia coli ER2537 cells for infection and cultured overnight at 37°C in LB / ampicillin / glucose agar medium. Escherichia coli ER2537 cells are transferred to SB / ampicillin medium and cultured until the OD600 value reaches 0.5, and then 1x10 11 ~1x10 12 The scFv library was prepared by adding helper phages and culturing at 37°C for 1 hour. Kanamycin was then added and the culture was incubated overnight. The overnight culture was centrifuged and the supernatant was reacted with a PEG solution at 4°C and then centrifuged to separate the precipitate. The precipitate was dissolved in PBS, centrifuged, and the resulting supernatant was stored as a scFv library solution. This process was repeated four times to ensure that the scFv candidates specifically bound to the CTLA-4 antigen.

[0101] 1-2. Selection of scFv antibodies that specifically bind to CTLA-4

[0102] In order to select scFvs with excellent binding ability from the scFv candidates obtained in Example 1-1, ELISA analysis was performed on cell lines expressing CTLA-4. Cell lines expressing CTLA-4 were prepared by transfecting human CTLA-4 expression vectors into HEK293 cells and selecting transformants treated with 50 μg / mL hygromycin B. Each library stock at the end of each panning step in Example 1-1 was cultured overnight in SB / ampicillin / glucose agar medium, and each single colony was inoculated into 200 μL SB / ampicillin medium and cultured at 37°C for 3 hours. Subsequently, IPTG was added to a final concentration of 1 mM and cultured again overnight at 30°C. After the culture was completed, the culture medium was centrifuged to separate the cells, and then the cells were lysed using TES buffer to obtain scFv. The obtained scFv was treated on a plate on which CTLA-4 cells were plated at 1x10 per well. 5 The cells were dispersed and reacted at room temperature for 1 hour. Then, a secondary antibody (anti-HAHRP, Santa Cruz, Cat. NO. sc-7392) was added and reacted for 40 minutes. After the secondary antibody reaction was completed, TMB was added to induce a color reaction, and the results were analyzed using an ELISA reader (450 nm). By relatively comparing the ELISA analysis values, the top 4 excellent scFvs (3E4, 4B6, 4F2, 4G1) were selected.

[0103] The amino acid sequences of the selected scFvs are shown in Table 1 below.

[0104] [Table 1]

[0105]

[0106]

[0107] Example 2: Conversion, expression and purification of scFv antibodies into IgG

[0108] 2-1. Preparation of complete IgG expression vector

[0109] The selected scFv is converted into the form of IgG, which is a more commonly used antibody. Based on the CDR region of scFv, an expression vector capable of expressing the complete IgG form is prepared. First, the light chain variable region and heavy chain variable region of scFv are obtained by PCR using the primers shown in Table 2 below. The light chain variable region sequence is cloned from the expression vector pcDNA3.3 (Invitrogen), which contains the light chain constant region sequence, and the heavy chain variable region sequence is cloned from the expression vector pOptiVEC (Invitrogen), which contains the heavy chain constant region sequence. Together with the light chain and heavy chain constant regions cloned from the vector, the light chain variable region and heavy chain variable region of scFv are expressed to produce a complete IgG antibody containing the CDR region of scFv.

[0110] [Table 2]

[0111]

[0112]

[0113]

[0114] 2-2. Preparation of complete IgG antibody-expressing cell line

[0115] CHO-S cells (Life Technologies Inc.) were used to prepare a cell line expressing IgG antibodies. The gene sequences encoding the heavy and light chains obtained in Example 2-1 were codon-optimized for the gray hamster (Cricetulus griseus) species and cloned into pCHO1.0 vector, and then transfected using a transfection reagent (FreeStyle TM MAXReagent; Life Technologies Inc.) was transfected into CHO-S cells. In order to select the cell line expressing the antibody after transfection, a two-step selection process was performed using puromycin and MTX (methotrexate). Specifically, in the first selection, puromycin 10 μg / mL, MTX 100nM, or puromycin 20 μg / mL, MTX 200nM were used, and when the cell survival rate met the standard, the second process was performed. In the second selection, puromycin 30 μg / mL, MTX 500nM, or puromycin 50 μg / mL, MTX 1000nM were used, and when the final cell survival rate met the standard, the second selection was completed, and the group with a high expression level was selected by SFB (Simple Fed Batch).

[0116] 2-3. Production and purification of intact IgG antibodies

[0117] On CD FortiCHOTM In the culture medium, each antibody-producing cell line prepared in Example 2-2 was cultured under the conditions of 8% CO2, 37°C, and 100-120rpm, and 4g / L, 4g / L, and 6g / L of glucose were added on the 3rd, 5th, and 7th days, respectively, and cultured for 14 days. After the culture is completed, the culture medium is centrifuged at 6000g with an ultracentrifuge, and the supernatant is filtered with a 0.2μm filter. For purification, protein A resin (Mabselect SuRe, 11-0026-01AD, GE Healthcare LifeSciences) is used, and an equilibrium buffer (20mM sodium phosphate, 150mM NaCl, pH 7.2), a wash buffer (35mM sodium phosphate, 500mM NaCl, pH 7.2) and an elution buffer (0.1M sodium citrate, pH 3.6) are used. AKTA TM avant, 2 column volumes of equilibration buffer, 5 column volumes of washing buffer, and 5 column volumes of elution buffer were used for purification, and 1 / 5 volume of Tris-HCl solution at pH 8.0 was added for neutralization during elution. After two buffer exchanges with PBS using a filter membrane (CelluSep, 1430-45), the solution was concentrated using a centrifugal filter (Amicon Ultra-15, UFC905024, Merck).

[0118] IgG antibody proteins produced under reducing and non-reducing conditions were confirmed using conventional SDS-PAGE techniques and verified that the light and heavy chains of each antibody were correctly expressed at the expected molecular weight. Figure 1 The results of SDS-PAGE confirmation of each IgG antibody are shown.

[0119] Example 3: Evaluation of antibody binding specificity and binding strength to CTLA-4

[0120] 3-1. Preparation of CTLA-4 / HEK293 cell line

[0121] In order to confirm the antigen specificity of the antibody prepared in Example 2-3, binding to human CTLA-4 protein was verified.

[0122] The gene encoding CTLA-4 was cloned into pCMV3-C-FLAG (Sino Biological). Each of the resulting CTLA-4 expression vectors was transfected into HEK293 cells using Fugene HD (E231A, Promega) transfection reagent, and hygromycin B was used to select for resistant cell lines.

[0123] 3-2. Evaluation of the binding ability of the antibody of the present invention to the CTLA-4 / HEK293 cell line

[0124] For the CTLA-4 / HEK293 cell line generated in Example 3-1, the cross-reactivity of the antibodies prepared in Example 2-3 was confirmed. Naive HEK293 cells were used as a negative control. First, cells were dissociated into single cells using a cell dissociation buffer (Gibco, 13151-014) at 2.5×10 5 Cells / well were inoculated and reacted with 10 μg / ml of each antibody on ice for 1 hour. After the reaction, the cells were washed with 1% FBS / PBS, treated with secondary antibody mouse anti-human IgG-PE (366904, Biolegend) at a dilution of 1:100, and then reacted on ice for 1 hour. After the reaction, the cells were washed with 1% FBS / PBS and analyzed using a flow cytometer BD FACS Lyrics.

[0125] Figure 2 The results of flow cytometry analysis are shown, and comparison thereof confirmed the binding ability of the antibody to CTLA-4.

[0126] 3-3. Confirmation of CTLA-4 specific binding - Western blotting

[0127] For the CTLA-4 / HEK293 cell line produced in Example 3-1, the ability of the antibody prepared in Example 2-3 to recognize the denatured CTLA-4 antigen was confirmed. Naive HEK293 cells were used as negative controls. First, the cells were dissociated into single cells with a cell dissociation buffer, lysed with a RIPA buffer (BIOSESANG) containing protease inhibitors and phosphatase inhibitors at 200 rpm for 1 hour at 4°C, and centrifuged at 15,000 rpm for 15 minutes at 4°C to collect the supernatant. The dissolved protein was quantified using a BCA assay (ThermoFisher Scientific). The quantified protein was reduced by adding a 5X sample loading buffer (BIOSESANG) containing DTT and boiling for 10 minutes. Equal amounts of protein were loaded onto SDS-PAGE, separated by size, transferred to a PVDF membrane, and blocked with 5% skim milk at 4°C for 1 hour. The antibody prepared in Example 2-3 was used as the primary antibody and incubated with a PVDF membrane overnight. After washing three times with 0.05% TBS-T, the membrane was treated with secondary 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 membrane was developed on film in a dark room.

[0128] like Figure 3 As shown, it was confirmed that this antibody does not recognize the denatured CTLA-4 antigen.

[0129] 3-4. Confirmation of CTLA-4 specific binding - immunoprecipitation

[0130] For the CTLA-4 / HEK293 cell line produced in Example 3-1, the ability of the antibodies prepared in Example 2-3 to recognize the natural CTLA-4 antigen was confirmed. Naive HEK293 cells were used as negative controls. The cells were separated using a scraper and resuspended in PBS containing protease inhibitors and phosphatase inhibitors. The cells were physically lysed using an ultrasonic generator, and the protein was quantified using the BCA assay. The quantified protein was incubated with the antibody for 1 hour, while the Protein A beads (Sigma) were blocked with 5% skim milk for 1 hour. The protein-antibody solution was then incubated with the blocked beads for 1 hour and washed three times with PBS. The protein and antibody bound to the Protein A beads were separated and reduced by adding 5X sample loading buffer containing DTT and reacting at 70°C for 10 minutes. The samples were analyzed by Western blot using an anti-human CTLA-4 antibody (Cell Signaling Technology) to confirm whether the antibody recognizes the natural CTLA-4 antigen.

[0131] like Figure 4 As shown, it was confirmed that the antibody specifically binds to the native CTLA-4 antigen.

[0132] Example 4: Confirmation of neutralization ability of binding to B7-1 (CD80) and B7-2 (CD86)

[0133] In order to confirm the binding neutralization ability of the antibodies prepared in Example 2-3, the binding neutralization ability of CTLA4-B7-1 (CD80) and CTLA4-B7-2 (CD86) was evaluated using ELISA. Human CTLA-4 protein (hCTLA4-Fc, RND Systems, Cat. No 325-CT) was coated at 250 ng / well on a 96-well plate and incubated overnight at 4 ° C. The plate was washed 3 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, and the anti-CTLA-4 antibody was diluted from 60 μg / ml in a 1 / 3 dilution series and added. B7-1 (CD80)-biotin and B7-2 (CD86)-biotin (Sino Biological) were added and incubated at room temperature for 2 hours. The same amount of Ipilimumab (Ipilimumab) (BMS) was used as a positive control. After binding, the plate was washed three times with 0.1% PBS-T, treated with 1:5000 dilution of Streptavidin-HRP (R&D Systems), and incubated at room temperature for 1 hour. After incubation, the plate was washed three times with 0.1% PBS-T, 100 μL of TMB solution (Surmodics) was added to each well, and reacted at room temperature for 20 minutes. 50 μL of stop solution was added to each well, and the absorbance at 450 nm was measured using an ELISA reader.

[0134] like Figure 5 As shown, it was confirmed that the antibody inhibits the binding of CTLA4-B7-1 (CD80) and CTLA4-B7-2 (CD86).

[0135] Example 5: Confirmation of antibody-dependent cellular cytotoxicity

[0136] For the CTLA-4 / HEK293 cell line generated in Example 3-1, the antibody-dependent cellular cytotoxicity of the antibody prepared in Example 2-3 was confirmed. The CTLA-4 / HEK293 cell line was used as a target cell, and the cell line transfected with human CD16 into NK92MI cells was used as an effector cell. The target cells were dissociated into single cells using a dissociation buffer and the concentration was 2.0×10 4 Cells / well were seeded in a 96-well plate and incubated overnight. 5 Effector cells were added to each well, and anti-CTLA-4 antibody was diluted from 1 μg / ml in a 1 / 10 dilution series and added to target cells. The cells were incubated at 37°C for 4 hours. Then, 50 μL of the supernatant was used for LDH assay (Promega, CytoTox ) to confirm cytotoxicity.

[0137] like Figure 6 As shown, it was confirmed that the antibody induced concentration-dependent cell death of CTLA-4 expressing cells through NK cell-mediated antibody-dependent cellular cytotoxicity.

[0138] Example 6: Confirmation of immune activation ability

[0139] 6-1. Immune activation by anti-CTLA-4 antibody (IL-2)

[0140] In order to confirm the immune activation ability of the antibodies prepared in Example 2-3, a Staphylococcus enterotoxin B (SEB) activation assay was performed using human PBMC. Peripheral blood concentrated in the LRS chamber was diluted with 2% FBS / PBS. 25 mL -1077 (Sigma) was added to a 50 mL tube, and 25 mL of diluted peripheral blood was gently layered on top, and 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 washed three times at 300 g for 8 minutes with 2% FBS / PBS washing buffer to obtain PBMCs. The isolated human PBMCs were washed at 2.0 x 10 5 Cells / well were seeded in a 96-well plate, and 100 ng / mL of SEB (Abion) and anti-CTLA-4 antibody diluted from 30 μg / ml in a 1 / 3 dilution series were added to the cell culture medium and incubated at 37°C for 3 days. After 3 days, 100 μL of supernatant was taken for subsequent analysis. The amount of IL-2 secreted by immune activation was measured using ELISA (Human IL-2 DuoSet ELISA, R&D Systems, Cat. No. DY202).

[0141] like Figure 7 As shown, it was confirmed that this antibody maximizes the secretion of IL-2 from human PBMCs upon immune activation.

[0142] 6-2. Confirmation of immune activation by anti-CTLA-4 antibody by mixed lymphocyte reaction (MLR)

[0143] In order to confirm the immune activation ability of the antibodies prepared in Example 2-3, mixed lymphocyte reaction (MLR) assay was performed using PBMCs from different donors. The PBMCs to be used as stimulator cells were treated with mitomycin C to inhibit cell proliferation by human PBMCs isolated in Example 6-1. The PBMCs from another donor to be used as reactant cells and the PBMCs to be used as stimulator cells were each incubated at 1.0×10 5Cells / well were added to a 96-well plate, and anti-CTLA-4 antibody was serially diluted to a concentration of 30 μg / ml at 1 / 3 and treated. The plate was incubated at 37°C for 5 days. After 5 days, 100 μl of supernatant was collected for subsequent analysis. The amount of IFN-γ secreted by immune activation was measured using ELISA (Human IFN-γ DuoSet ELISA, R&D systems, Cat. No. DY285B).

[0144] Results, such as Figure 8 As shown, it was confirmed that the antibody increased the secretion of IFN-γ in a concentration-dependent manner.

[0145] Example 7: Confirmation of the Anticancer Efficacy of Antibodies in Vivo

[0146] In order to confirm the anti-cancer efficacy of the anti-CTLA-4 antibody in vivo, the prepared anti-CTLA-4 antibody was injected into cancer-induced mice into which human CTLA-4 was introduced, and the changes in tumor size were observed.

[0147] First, 5×10 cells of mouse colon cancer cell line MC38 were cultured. 5 The cells were injected subcutaneously into mice that had been transfected with human CTLA-4 protein. When the average tumor size reached 150 mm 3 At the time of the experiment, the mice were randomly divided into a control group and an antibody-treated group, with 5 mice in each group. The control group was administered with IgG, and the antibody-treated group was administered with 4G1 antibody at a dose of 20 mg / kg, three times a week for 2 weeks. Tumor size was measured using a caliper and calculated as long axis x (short axis) 2 mm 3 .

[0148] Results, such as Fig. 9 As shown, it was confirmed that the tumor size of the 4G1 antibody-treated group was statistically significantly reduced compared with the control group (t-test, p value = 0.04).

[0149] Industrial Applicability

[0150] As described above, the present invention provides an antibody or a fragment thereof that binds to human CTLA-4 protein. The antibody of the present invention binds to CTLA-4 of T cells, thereby inhibiting the stimulation of CTLA-4 caused by cancer cells, preventing T cells from being inactivated, and enabling T cells to attack cancer cells. At the same time, it can exert a powerful ADCC effect through NK cells, showing significant efficacy as an anticancer agent, and therefore has high industrial applicability.

Claims

1. An antibody or fragment thereof that binds to human CTLA-4 protein, selected from the group consisting of: i) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 1, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 2, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 3, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 4, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 5, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 6; ii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO:9, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO:10, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO:11, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO:12, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO:13, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO:14; iii) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region comprises a complementarity determining region (CDR) H1 comprising the amino acid sequence of SEQ ID NO: 17, a complementarity determining region (CDR) H2 comprising the amino acid sequence of SEQ ID NO: 18, and a complementarity determining region (CDR) H3 comprising the amino acid sequence of SEQ ID NO: 19, and the antibody light chain variable region comprises a complementarity determining region (CDR) L1 comprising the amino acid sequence of SEQ ID NO: 20, a complementarity determining region (CDR) L2 comprising the amino acid sequence of SEQ ID NO: 21, and a complementarity determining region (CDR) L3 comprising the amino acid sequence of SEQ ID NO: 22; and iv) an antibody comprising an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ), the antibody heavy chain variable region contains a complementarity determining region (CDR) H1 comprising the amino acid sequence shown in SEQ ID NO:25, a complementarity determining region (CDR) H2 comprising the amino acid sequence shown in SEQ ID NO:26, and a complementarity determining region (CDR) H3 comprising the amino acid sequence shown in SEQ ID NO:27, and the antibody light chain variable region contains a complementarity determining region (CDR) L1 comprising the amino acid sequence shown in SEQ ID NO:28, a complementarity determining region (CDR) L2 comprising the amino acid sequence shown in SEQ ID NO:29, and a complementarity determining region (CDR) L3 comprising the amino acid sequence shown in SEQ ID NO:

30.

2. The antibody or fragment thereof that binds to human CTLA-4 protein according to claim 1, wherein the antibody is selected from the group consisting of: i) an antibody comprising a heavy chain variable region comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of positions 1 to 110 of SEQ ID NO: 8; ii) an antibody comprising a heavy chain variable region comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO: 15 and a light chain variable region comprising the amino acid sequence of positions 1 to 109 of SEQ ID NO: 16; iii) an antibody comprising a heavy chain variable region comprising the amino acid sequence of positions 1 to 121 of SEQ ID NO: 23 and a light chain variable region comprising the amino acid sequence of positions 1 to 110 of SEQ ID NO: 24; and iv) an antibody comprising a heavy chain variable region comprising the amino acid sequence of positions 1 to 116 of SEQ ID NO:31 and a light chain variable region comprising the amino acid sequence of positions 1 to 110 of SEQ ID NO:

32.

3. The antibody or fragment thereof that binds to human CTLA-4 protein according to claim 1, wherein the antibody is 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.

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

5. A polynucleotide encoding the antibody or fragment thereof according to claim 1. A recombinant vector comprising the polynucleotide according to claim 5.

7. A transformed cell line comprising the vector according to claim 6.

8. A method for producing an antibody or fragment thereof that binds to a human CTLA-4 protein, comprising culturing the cell line of 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 the cell line is cultured.

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

10. The pharmaceutical composition of claim 9, wherein the cancer is selected from the group consisting of 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, bile duct cancer, testicular cancer, germ cell tumors, thyroid cancer, parathyroid cancer, cervical cancer, endometrial cancer, lymphoma, myelodysplastic syndrome (MDS), myelofibrosis, acute leukemia, chronic leukemia, multiple myeloma, Hodgkin's disease, endocrine cancer, and sarcoma.

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

12. A method for treating cancer, comprising administering to a subject 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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