HLA-G specific antibodies and their uses

Abstract

The present invention relates to an antibody that specifically binds to HLA-G and its use in the treatment of cancer. The antibody can bind to both HLA-G monomers and multimers and prevent HLA-G from binding to its receptor, and therefore can be useful in the treatment of cancer.

Description

[Technical field]

[0001] The present invention relates to an antibody that specifically binds to HLA-G and its use in cancer treatment. [Background technology]

[0002] Despite intensive research into cancer over the past several decades, cancer remains the leading cause of death worldwide. Many cancer treatment methods have been developed, and methods such as surgery, chemotherapy, and radiation therapy have been used from ancient times to the present. However, most of these existing cancer treatment methods are only effective in the early stages of cancer before it has metastasized, and there is a problem that in cases where cancer has already metastasized, there is a high possibility of recurrence even after surgery. Therefore, in order to provide more effective cancer treatment, research has recently been ongoing into immune anticancer agents that utilize immune responses.

[0003] Immunotherapy is a treatment that prevents cancer cells from evading the body's immune system, allowing immune cells to better recognize and attack cancer cells. Specifically, immunotherapy can induce the death of cancer cells through the activation of non-specific immune cells, or can induce an immune response against tumors in the cancer patient's immune cells by using tumor-specific antigens.

[0004] On the other hand, human major histocompatibility complex antigens class I and G, also known as human leukocyte antigen-G (HLA-G), are immune barrier factors that suppress the activity and division of T cells, NK cells, cytotoxic T cells, etc. ILT2 (Ig-like transcript 2), ILT4, and KIR2DL4 (Killer Cell Immunoglobulin Like Receptor, Two Ig Domains And Long cytoplasmic tail 4) are known as immune cell receptors for HLA-G, and all of these receptors have an ITIM (immunoreceptor tyrosine-based inhibitory motif). When HLA-G binds to the receptor, this motif activates a signaling system that suppresses immune cells.

[0005] It has been reported that HLA-G expression is limited in general cells, but is expressed in many types of cancer cells (Lin A, Yan WH.Human Leukocyte Antigen-G (HLA-G) Expression in Cancers; Roles in Immune Evasion, Metastasis and Target for Therapy. Mol. Med. 21(1), 782-791 (2015)). It has also been reported that the expression level of HLA-G in cancer cells of many cancer patients is correlated with the stage of cancer disease, and that patients with HLA-G expression have a low survival rate (Ye, Sr., Yang, H., Li, K. et al.Human leukocyte antigen G expression: as a significant prognostic indicator for patients with colorectal cancer. Mod. Pathol. 20, 375-383 (2007)). Summary of the Invention [Problem to be solved by the invention]

[0006] In the above circumstances, the present inventors have endeavored to develop an HLA-G specific antibody to enhance the targeting ability of immune cells against cancer cells by suppressing the function of HLA-G, and as a result, have developed an HLA-G specific antibody capable of binding to both HLA-G monomers and dimers, thus completing the present invention.

[0007] It is therefore an object of the present invention to provide an HLA-G specific antibody or an antigen-binding fragment thereof.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises an HLA-G specific antibody or an antigen-binding fragment thereof as an active ingredient. [Means for solving the problem]

[0009] To achieve the above object, one aspect of the present invention provides an anti-HLA-G antibody or antigen-binding fragment thereof, comprising: (a) a heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or (d) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:31.

[0010] In the present invention, the anti-HLA-G antibody or antigen-binding fragment thereof may comprise the following sequence: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 4, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO: 8; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 16; (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO: 24; or (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:28 or an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO:28, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:32 or an amino acid sequence having 80% or more homology to the amino acid sequence of SEQ ID NO:32.

[0011] More specifically, in one embodiment of the present invention, the anti-HLA-G antibody or antigen-binding fragment thereof may comprise the following sequence: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 12 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 20 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 24; or (d) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 32.

[0012] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor to specifically recognize an antigen, including an immunoglobulin molecule that is immunologically reactive with a particular antigen. The antibody includes monoclonal antibodies, polyclonal antibodies, mixtures of monoclonal and / or polyclonal antibodies, full-length antibodies, and antibody fragments. Full-length antibody or intact antibody are used interchangeably to refer to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain that contains an Fc region as defined herein.

[0013] Antibodies can also include bivalent or bispecific molecules (e.g., bispecific antibodies).Antibodies can also be human, humanized or chimeric, depending on the origin of the sequence.

[0014] In the present invention, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such a monoclonal antibody exhibits a single binding property and affinity for a specific epitope, unlike a polyclonal antibody that can bind to multiple epitopes. In the present invention, the term "full-length antibody" refers to a structure having two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond. The constant regions of the heavy chains have gamma, mu, alpha, delta, and epsilon types, with subclasses of gamma 1, gamma 2, gamma 3, gamma 4, alpha 1, and alpha 2. The constant regions of the light chains have kappa and lambda types. IgG has subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0015] In the present invention, the term "heavy chain" may include a full-length heavy chain or a fragment thereof, which includes a variable region VH and three constant regions CH1, CH2, and CH3, each of which has an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, and the term "light chain" in the present invention may include a full-length light chain or a fragment thereof, which includes a variable region VL and a constant region CL, each of which has an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0016] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remaining portion of the heavy and / or light chain is derived from a different source or species. For example, there is a chimeric antibody in which the variable region of a mouse antibody and the constant region of a human antibody are combined, and the immune response is greatly improved compared to that of a mouse antibody.

[0017] In the present invention, the term "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to resemble that of an antibody naturally produced by humans. For example, the humanized antibody can be produced by recombining mouse-derived CDR with human antibody-derived FR (framework region) to produce a humanized variable region, which can then be recombined with a constant region of a preferred human antibody to produce a humanized antibody. However, since the affinity of the humanized antibody decreases when only CDR grafting is performed, the degree of substitution can be increased to the same level as that of the original mouse antibody by making some important FR amino acid residues that are expected to affect the three-dimensional structure of the CDR compatible with those of a mouse antibody.

[0018] The present invention also provides an antigen-binding fragment of the anti-HLA-G antibody, which may be selected from the group consisting of Fab (fragment antigen binding), Fab', F(ab')2, Fv (variable fragment), dsFv (disulfide-stabilized Fv fragments), scFv (single chain Fv), diabody, triabody, and tetrabody.

[0019] As used herein, the terms "fragment," "antibody fragment," "antigen-binding fragment," or "antigen-binding domain" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv.

[0020] The Fab has a structure including light and heavy chain variable regions, a light chain constant region and the first constant region of the heavy chain (CH1 domain), and has one antigen-binding site. Fab' is different from Fab in that it has a hinge region including one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced by disulfide bonds formed between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are disclosed in PCT International Patent Publications WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086 and WO88 / 09344. In a two-chain Fv, the variable region of the heavy chain is linked to the variable region of the light chain by a non-covalent bond, and in a single-chain Fv, the variable region of the heavy chain is linked to the variable region of the light chain by a covalent bond or directly at the C-terminus via a peptide linker, so that the two-chain Fv can form a dimer-like structure. Such antibody fragments can be obtained using protease (for example, Fab can be obtained by restrictive cleavage of a whole antibody with papain, and F(ab')2 fragment can be obtained by cleavage with pepsin), and can be preferably produced through gene recombination technology.

[0021] The diabody is a small bivalent antibody composed of two heavy chain variable and two light chain variable domains, each of which is linked to a heavy chain variable domain by a short linker. When the light chain variable domain is linked to the heavy chain variable domain by an even shorter linker, a triabody or tetrabody can be formed.

[0022] Furthermore, the antibody or antigen-binding fragment thereof of the present invention may include not only the sequences of the anti-HLA-G antibodies described herein, but also biological equivalents thereof, so long as it can exhibit binding ability to HLA-G. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of residues in the amino acid sequence of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutes reveals that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine have similar sizes; and phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on this, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.

[0023] Another aspect of the invention is an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof, wherein the antibody and antigen-binding fragment thereof are as described above.

[0024] The term "nucleic acid" as used herein is intended to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and the basic building block of a nucleic acid molecule, a nucleotide, includes not only natural nucleotides but also analogues in which the sugar or base moiety is modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews (1990) 90:543-584). The sequences of the nucleic acid molecules encoding the variable regions of the heavy and light chains of the present invention may be modified, and such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0025] In the present invention, the isolated nucleic acid may comprise the following sequence: (a) the nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO: 37 and the nucleotide sequence of SEQ ID NO: 38 or a nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO: 38; (b) the nucleotide sequence of SEQ ID NO: 39 or a nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO: 39 and the nucleotide sequence of SEQ ID NO: 40 or a nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO: 40; (c) the nucleotide sequence of SEQ ID NO: 41 or a nucleotide sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 42 or a nucleotide sequence having 80% or more homology to the nucleotide sequence of SEQ ID NO: 42; or

[0026] (d) the nucleotide sequence of SEQ ID NO: 43 or a nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO: 43 and the nucleotide sequence of SEQ ID NO: 44 or a nucleotide sequence having 80% or more identity to the nucleotide sequence of SEQ ID NO: 44.

[0027] More specifically, in one embodiment of the invention, the isolated nucleic acid may comprise the following sequence: (a) the nucleotide sequence of SEQ ID NO:37 and the nucleotide sequence of SEQ ID NO:38; (b) the nucleotide sequence of SEQ ID NO:39 and the nucleotide sequence of SEQ ID NO:40; (c) the nucleotide sequence of SEQ ID NO: 41 and the nucleotide sequence of SEQ ID NO: 42; or (d) the nucleotide sequence of SEQ ID NO:43 and the nucleotide sequence of SEQ ID NO:44.

[0028] Another aspect of the present invention is a recombinant vector comprising the nucleic acid molecule.

[0029] The term "vector" as used herein refers to a means for expressing a gene of interest in a host cell, and includes plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and is preferably, but not limited to, a plasmid vector.

[0030] According to one embodiment of the present invention, a recombinant vector comprising a nucleic acid molecule of the present invention may comprise a nucleic acid molecule encoding a sequence comprising a heavy and / or light chain variable region comprising or comprising the following CDRs: (a) a heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or (d) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:31.

[0031] In the vector of the present invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region may be operatively linked to a promoter.

[0032] As used herein, the term "operably linked" refers to a functional association between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0033] The recombinant vector system of the present invention can be constructed through a variety of methods known in the relevant art, specific methods for which are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0034] The vector of the present invention can typically be constructed as a vector for cloning or a vector for expression. The recombinant vector of the present invention is preferably an expression vector. The vector of the present invention can also be constructed as a prokaryotic or eukaryotic host.

[0035] For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, it generally contains a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for the initiation of decoding, and a transcription / decoding termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., (1984) 158:1018-1024) and the left-directed promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14:399-445) can be used as a regulatory site. When Bacillus bacteria are used as the host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64: 3932-3938; Mol. Gen. Genet. (1996) 250: 734-741) or any promoter capable of being expressed in Bacillus bacteria can be used as the regulatory region.

[0036] Meanwhile, the recombinant vector of the present invention may be prepared by manipulating a plasmid (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., λgt4·λB, λ-Charon, λΔz1, M13, etc.) or a virus (e.g., SV40, etc.) frequently used in the related technical field.

[0037] On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and respiratory syncytial virus (RSV) promoter) may be used, which generally has a polyadenylation sequence as a transcription termination sequence. Specifically, the recombinant vector of the present invention may include a CMV promoter.

[0038] The recombinant vector of the present invention may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Examples of sequences to be fused include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6xHis (hexahistidine; Quiagen, USA). In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like even without an additional sequence for purification.

[0039] Meanwhile, the recombinant vector of the present invention may contain an antibiotic resistance gene commonly used in the related art as a selection marker, for example, resistance genes against ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline. According to one embodiment of the present invention, the anti-HLA-G expression vector may contain an ampicillin resistance gene.

[0040] The vector expressing the antibody of the present invention may be either a vector system in which the light chain and the heavy chain are simultaneously expressed in one vector or a system in which the light chain and the heavy chain are each expressed in a separate vector. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation. Co-transformation is a method in which vector DNAs encoding the light chain and the heavy chain are simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing the light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing the heavy chain (or light chain), and finally cells expressing both the light chain and the heavy chain are selected.

[0041] Nucleic acids encoding the heavy and light chains of the antibody of the invention are inserted into an expression vector encoding the heavy and light chain constant regions of human IgG1, IgG2, and IgG4. The light and heavy chains may be cloned in the same or different expression vectors. The DNA sequences encoding the immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences. Expression vectors are capable of replicating in the host organism either as an integral part of the host chromosomal DNA or as episomes.

[0042] If the heavy and light chains of an antibody are each cloned into separate vectors, the expression vectors encoding the heavy and light chains can be co-transfected into a host cell for expression of the two chains, which can be assembled to form intact antibodies in vivo or in vitro.

[0043] Alternatively, an expression vector encoding the heavy chain and an expression vector encoding the light chain can be introduced into different host cells for expression of the heavy and light chains, respectively, which can then be purified and assembled to form intact antibodies in vitro. The antibodies or fragments thereof as described herein can be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, insect and mammalian cells. Although it is not necessary for the recombinant antibodies of the invention to be glycosylated or expressed in eukaryotic cells, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are human embryonic kidney cell lines (HEK293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells / - or +DHFR (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells) and human stem cells (Hep G2 cells).

[0044] Expression vectors for such cells can include expression control sequences such as an origin of replication, a promoter and an enhancer, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, etc. Vectors containing plasmid sequences producing antibodies can be transferred into host cells by widely known methods that can vary depending on the type of host cell.

[0045] Another aspect of the present invention is a host cell comprising the recombinant vector. Preferably, the host cell of the present invention is a host cell transformed with a recombinant vector.

[0046] Host cells in which the vectors of the present invention can be stably and continuously cloned and expressed may be any host cell known in the relevant art, including, but not limited to, prokaryotic host cells such as Escherichia coli, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus).

[0047] Suitable eukaryotic host cells transformed with the vectors may include, but are not limited to, fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, and Neurospora crassa, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells of plant or mammalian origin.

[0048] Specifically, the host cells may be monkey kidney cells 7 (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, Mardin Darby canine kidney (MDCK) cells, myeloma cell lines, HuT78 cells, or 293 cells.

[0049] Mammalian host cell culture is preferred for expressing and producing anti-HLA-G antibodies or antigen-binding fragments thereof, since a number of suitable host cell lines capable of secreting intact immunoglobulin have been developed in the art.

[0050] In the present invention, "transformation" and / or "transfection" of a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextrin sulfate, lipofectamine, and desiccation / repression-mediated transformation methods.

[0051] Another aspect of the present invention is a method for producing an anti-HLA-G antibody or an antigen-binding fragment thereof, comprising the step of culturing a transformed host cell of the present invention. Preferably, the method for producing an anti-HLA-G antibody or an antigen-binding fragment thereof of the present invention may further comprise the step of expressing the anti-HLA-G antibody or an antigen-binding fragment thereof in the cultured transformed host cell.

[0052] In the method for producing the antibody or antigen-binding fragment thereof, the transformed cells may be cultured by suitable culture and culture conditions known in the relevant technical field. Such a culture process can be easily adjusted and used by a person of ordinary skill in the art depending on the selected bacterial strain. Such various culture methods are disclosed in various literature (e.g., James M. Lee. Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture is classified into suspension culture and adherent culture according to the cell growth method, and into batch, fed-batch and continuous culture methods according to the culture method. The medium used for culture should appropriately satisfy the requirements of the specific bacterial strain.

[0053] In the culture of animal cells, the medium contains various carbon sources, nitrogen sources and trace element components. Examples of usable carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these carbon sources can be used alone or in combination.

[0054] The nitrogen sources that can be used in the present invention can include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steeping liquor (CSL) and soybean meal, and inorganic nitrogen sources such as iodine, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, which can be used alone or in combination. The medium can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and corresponding sodium-containing salts as phosphorus sources. Metal salts such as magnesium sulfate or ferrous sulfate can also be included. Others can include amino acids, vitamins and appropriate precursors.

[0055] During the culture, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner. During the culture, the generation of bubbles can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. In order to maintain the culture in an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture may be usually 20°C to 45°C, preferably 25°C to 40°C, but is not limited thereto.

[0056] The antibody obtained by culturing the transformed host cells may be used without purification, or may be further purified to a high purity using various conventional methods, such as dialysis, salt precipitation, chromatography, etc. Among these, the method using chromatography is most commonly used, and the type and order of columns may be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc., depending on the characteristics of the antibody, the culture method, etc.

[0057] The anti-HLA-G antibody according to the present invention is prepared using the monomeric structure of HLA-G as an antigen, and is characterized by its ability to bind to both HLA-G monomers and multimers. Since HLA-G is known to suppress the activation of immune cells and to be overexpressed in cancer cells, the present inventors have confirmed the anti-cancer ability of the anti-HLA-G antibody.

[0058] As a result, it was confirmed that anti-HLA-G antibodies inhibited the binding of HLA-G to CD8a (Table 4). In addition, when PBMCs (potency cells) and target cells treated with anti-HLA-G antibodies were mixed and co-cultured with cancer cells, the cancer cells were killed (Figure 3) and tumor growth was inhibited in tumor-induced mice (Table 6).

[0059] Thus, another aspect of the present invention provides a pharmaceutical composition for use in the prevention or treatment of cancer, comprising the antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0060] In the present invention, the pharmaceutical composition may be used for the treatment of cancers that overexpress HLA-G, but is not limited thereto, which may be selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer, and melanoma.

[0061] The blood cancer may be Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), Chronic Myelogenous Leukemia (CML), Multiple Myeloma (MM), or Lymphoma.

[0062] As used herein, the term "prevention" refers to any action of suppressing or slowing the progression of a disease by administering a composition of the present invention, and "treatment" refers to suppressing, reducing or eliminating the development of a disease.

[0063] The pharmaceutical composition of the present invention may further include a pharma- ceutically acceptable carrier, which is a carrier that is commonly used in formulation, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition for preventing or treating cancer of the present invention may further include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharma- ceutically acceptable carriers and formulations are described in detail in the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)].

[0064] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. During oral administration, proteins or peptides are digested, so oral compositions are formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be administered by any device that can transport the active agent to target cells.

[0065] The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, and a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention may be 0.1-100 mg / kg, preferably 0.1-10 mg / kg, more preferably 0.1-2 mg / kg. As used herein, the term "pharmaceutical effective amount" means an amount sufficient for treating, preventing and diagnosing a disease.

[0066] The pharmaceutical composition of the present invention may be prepared in a unit dose form or in a multi-capacity container by formulating it using pharma- ceutical acceptable carriers and / or excipients in a manner that can be easily carried out by a person having ordinary skill in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

[0067] The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, either sequentially or simultaneously with conventional therapeutic agents.

[0068] The antibody or antigen-binding fragment thereof of the present invention can be administered into the body in the form of an antibody-therapeutic agent (functional molecule) or bispecific antibody-therapeutic agent (functional molecule) conjugate to be used for the treatment of cancer. Various suitable and preferred conditions for targeting a drug to a specific target site are reported in, for example, Trouet et al., Plenum Press, New York and London, (1982) 19-30.

[0069] The antibody or antigen-binding fragment thereof of the present invention may be used for the prevention, treatment, and diagnosis of diseases associated with HLA-G overexpression by additionally binding or coadministering a functional molecule. The functional molecule may include chemical substances, radionuclides, immunotherapeutic agents, cytokines, chemokines, toxins, biological agents, and enzyme inhibitors.

[0070] Another aspect of the present invention is a method for diagnosing, preventing or treating a disease associated with HLA-G overexpression, such as cancer, comprising administering a pharma- ceutical effective amount of an antibody or antigen-binding fragment thereof, or the pharmaceutical composition of the present invention to a subject, such as a human or a non-human mammal. The present invention also provides a method for diagnosing a disease associated with HLA-G overexpression, such as cancer, comprising administering an antibody or antigen-binding fragment thereof of the present invention to an individual in need thereof, such as a human or a non-human mammal.

[0071] In the present invention, the term "individual" refers to any animal, including monkeys, cows, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs, including humans, that have or may develop an HLA-G overexpression associated disease.

[0072] Meanwhile, the inventors of the present invention confirmed the binding site between the antibody and HLA-G and confirmed that the amino acid regions 339-344 (DYEATL), 390-402 (VVVPSGEEQRYTC), and 323-338 (QRADPPKTHVTHHPVF) of the amino acid sequence (sequence number 33) of the antigen used for antibody screening were epitopes.

[0073] Thus, another aspect of the present invention provides an HLA-G epitope consisting of a sequence selected from the group consisting of SEQ ID NOs:34-36.

[0074] In the present invention, the term "epitope" refers to a localized site on an antigen to which an antibody or a fragment thereof can specifically bind. Epitopes are often composed of surface groups of molecules such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to conformational epitopes, but not to non-conformational epitopes, is lost in the presence of denaturing solvents. Epitopes can include amino acid residues that are directly involved in binding (also called immunodominant components of the epitope) and other amino acid residues that are not directly involved in binding, for example, amino acid residues that are effectively blocked by a specific antigen-binding peptide (i.e., amino acid residues that are present within the footprint of a specific antigen-binding peptide).

[0075] The present invention also provides an antibody that specifically binds to the HLA-G epitope. Specific examples of the antibody are as described above, but are not limited thereto. Effect of the Invention

[0076] The anti-HLA-G antibody according to the present invention can bind to both HLA-G monomers and polymers, and can therefore effectively block the binding of HLA-G to its receptor. [Brief description of the drawings]

[0077] [Figure 1] FIG. 1 shows a schematic diagram for producing b2m-HLA-G antigen according to one embodiment of the present invention. [Diagram 2] The vector structure used for producing anti-HLA-G antibody is shown. [Diagram 3]HLA-G-overexpressing target cells treated with anti-HLA-G antibody were mixed with PBMCs, and the mixed cells were co-cultured with SK-OV-3 cells or MDA-MB-231 cells, after which the extent of cancer cell death was confirmed. [Figure 4] Tumors were induced by transplanting 4T-1 cells that overexpress human HLA-G into mice, and after administering anti-HLA-G antibodies, the tumors were isolated and the numbers of cytotoxic T cells / regulatory T cells were confirmed. [Diagram 5] These are the results of confirming the epitope sequence to which anti-HLA-G antibodies and HLA-G bind. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] Hereinafter, one or more embodiments will be described in more detail with reference to examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0079] Example 1: Antigen design In order to discover HLA-G specific antibodies and inhibit the mechanism of action of HLA-G, an antigen similar to the b2m-HLA-G multimer structure, which is an activated form of HLA-G, was produced. A protein sequence was designed in which a signal peptide-b2m-HLA-G-6X His was linked to easily produce the antigen. A DNA sequence was designed using this protein sequence (Figure 1). Then, the DNA sequence was introduced into animal cells, and the protein was expressed and purified. Specifically, the cell culture solution containing the signal peptide-b2m-HLA-G-6X His antigen was purified in the first step using an NI-NTA column. Then, the monomer and dimer were separated from the result of the first purification using size exclusion chromatography. The production of the antigen is not limited to the animal cells, and it can also be expressed and purified in, for example, E. coli.

[0080] SEQ ID NO: 33: Antigen sequence MSRSVALAVLALLSLSGLEARIIPRHLQLGCGGSGGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFT PTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNT KAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWGSGLNDIFEAQKIEWHEHHHHHH

[0081] Example 2: Antibody Production 2-1. Library screening The phage / scFv library was screened using Immunotubes (15501, Piece) coated with HLA-G antigen. Panning was performed four times to increase the antigen binding specificity of the phage / scFv library. For recovery and amplification of the selected phages, exponentially growing E. coli cultures were infected with the phage suspension eluted after each round of panning. After completion of panning, phage-ELISA was performed using HLA-G antigen to evaluate the antigen reactivity of the selected phages. After the fourth panning, the antigen reaction signal of the phage increased to three times the background. According to phage display technology, such an increase in signal indicates that the selected phages are enriched in HLA-G antigen-specific binders. Phagemid DNA of the selected phages was extracted from the library after the fourth panning. 96 clones were isolated and cultured on 24-well plates. The supernatant containing the phage particles was tested by phage-ELISA method, and the DNA of the clones showing HLA-G specific binding was isolated and sequenced.

[0082] 2-2. Antibody maturation To mature HLA-G specific antibodies, mutations were introduced into CDR1, 2, and 3 of the heavy and light chains to obtain various heavy and light chain products. To select HLA-G specific antibodies by combining the products, HLA-G specific mature antibodies were obtained through ELISA analysis using HLA-A, HLA-B, and HLA-C.

[0083] 2-3. Recombinant production of antibodies In this example, an antibody was produced using the vector structure of Figure 2. The vector contains a human cytomegalovirus promoter (CMV promoter) for expression in animal cells, a Kozak sequence for enhanced expression, a signal sequence for extracellular transport of the expressed protein, a cloning site for the variable region, a coding sequence for the constant region of the light or heavy chain of IgG1, Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), an ampicillin resistance gene, and pUC for amplifying the plasmid in E. coli. The cloning site for the variable region contains HindIII and EcoR1 restriction enzymes, and after treating the expression vector with the restriction enzymes, the PCR product of the variable region of the heavy or light chain was inserted into the vector using infusion enzyme (TAKARA). Heavy chain constant domains of IgG2, IgG3, and IgG4 may also be used.

[0084] The light chain expression vector and the heavy chain expression vector were transduced into Expi293 cells using a transduction kit (Thermo), and the cells were then cultured. The cell culture medium was harvested after 4 to 6 days, and the cells were removed to collect only the supernatant. The collected supernatant was filtered through a 0.45 um filter, and the filtered culture medium was purified using a Mabselect (Cytiva) purification column on an AKTA purification device to obtain an antibody that specifically binds to human HLA-G.

[0085] 2-4. Antibody analysis The obtained antibodies were analyzed, and the CDR and variable region sequences are summarized in Tables 1 and 2 below.

[0086] [Table 1]

[0087] [Table 2(1)] [Table 2(2)]

[0088] Example 3: Evaluation of antigen-binding ability of antibodies The anti-HLA-G specific antibodies obtained in Example 2 were screened for their binding properties to the HLA-G antigen prepared in Example 1. The analysis results are summarized in Table 3 below.

[0089] [Table 3]

[0090] Surface magnetic resonance (SPR) and octet analysis confirmed that anti-HLA-G specific antibodies have high affinity for antigens, as they showed nanomolar KD values ​​for HLA-G multimers. In addition, anti-HLA-G specific antibodies obtained from HLA-G multimers showed higher affinity for multimers than monomers.

[0091] <Example 4: Confirmation of the ability to inhibit binding of HLA-G specific antibodies> Since anti-HLA-G specific antibodies have high affinity for HLA-G multimers, we examined whether the antibodies could inhibit the binding between ILT2 (Ig-like transcript 2) and HLA-G multimers.

[0092] 4-1. Receptor binding inhibition 1: Inhibition of ILT2 binding to HLA-G by anti-HLA-G antibodies a.ILT2 Block ELISA For analysis, ILT-2-Fc was diluted in 1xPBS to a concentration of 1.5μg / mL. 150ng / 100uL of diluted ILT-2 protein was dispensed into each well of a 96-well immune plate. The immune plate was stored at 4℃ overnight to allow ILT-2 protein to be immobilized on the surface of the wells. The next day, the ILT-2 protein solution was completely removed and each well was washed with 200uL of 1xPBS-T. 200ul of blocking solution was dispensed into each well and incubated at 37℃ for 1 hour to block non-specific protein binding.

[0093] The antigen prepared in Example 1 was diluted in the blocking solution to 1,200 nM, which is twice the concentration to be treated. In addition, the anti-HLA-G monoclonal antibody was diluted in the blocking solution to 2,000 nM, which is twice the concentration to be treated, and serially diluted 3-fold to 0.914 nM.

[0094] The prepared antigen of Example 1 and anti-HLA-G monoclonal antibody diluted according to concentration were mixed at a ratio of 1:1 and dispensed in 120 ul portions into each well, followed by pre-incubation for 30 minutes at 37° C. After completely removing the blocking solution from each well, the wells were washed with PBS-T.

[0095] A 1:1 mixture of the antigen of Example 1 and anti-HLA-G monoclonal antibody was added to each well in an amount of 100 ul, and reacted at 37° C. for 1 hour. Anti-His-HRP, a detection antibody for the antigen of Example 1, was diluted at a ratio of 1:10,000 and dispensed in an amount of 100 ul into each well, and reacted at 37° C. for 1 hour. Each well was washed three times with 200 ul of 1x PBS-T, and the absorbance was measured at 450 nm using a Multi-mode Microplate Reader.

[0096] The measurement results are shown in Table 4. Table 4 below shows the results of ELISA analysis to determine the antibody concentration that inhibits HLA-G:ILT2 interaction by 50% or more when different antibody concentrations are applied to unblocked HLA-G:ILT2 interaction. The antibody obtained in Example 2 was shown to inhibit HLA-G:ILT2 binding by 50% or more at the 150 nM level.

[0097] [Table 4]

[0098] b.ILT-2 Block Cell On day 0 (D0), HLA-G expressing cells were cultured in a 96-well microplate at a concentration of 3.0.E+05 cells / well. On day 1, the cell supernatant was removed. Anti-HLA-G monoclonal antibody was diluted in blocking solution to 200 nM, twice the concentration to be treated, and serially diluted 6-fold to 0.714 pM. This dilution was dispensed into the wells from which the supernatant had been removed and reacted for 30 minutes. ILT2-mFc protein was diluted to 100 ug / mL and then dispensed into the antibody-cell reaction solution and reacted for 1 hour. When the reaction was completed, the supernatant was diluted and dispensed with anti-mouse IgG-FITC, which can detect ILT2-mFC, as described above. After reacting for 2 hours, the remaining amount of ILT2-mFc was confirmed using a cell-based analyzer.

[0099] As a result, the IgG1-treated group, which was the negative control group, did not inhibit the binding between HLA-G and ILT2 even at high concentrations, but the antibody obtained in Example 2 inhibited the binding between HLA-G and ILT2 in a concentration-dependent manner. In addition, the antibody concentration (EC50) at which the binding between HLA-G expressing cells and ILT2 was reduced by 50% or less was confirmed, and it was shown that the obtained antibody effectively inhibited the binding between HLA-G on the cell surface and ILT2 at the nanomolar level (Table 4).

[0100] 4-2. Receptor binding inhibition 2: Inhibition of CD8a binding to HLA-G by anti-HLA-G antibodies When HLA-G antigen binds to CD8a expressed in immune cells, it suppresses immune cells, so inhibiting the binding of HLA-G and CD8a is important for activating immune cells. Therefore, the CD8a blocking effect of the antibody obtained in Example 2 was confirmed as follows.

[0101] The dimeric antigen prepared in Example 1 was diluted in PBS solution to a concentration of 5 ug / mL. The diluted antigen was dispensed in 100 ul portions into a 96-well microplate, and the antigen was coated on the wells for 24 hours.

[0102] 100 μL of blocking solution was dispensed into each well of the streptavidin 96-well plate to block the plate. The blocking solution was prepared by dissolving skim milk at a concentration of 4% in PBS containing 1% Tween-20. Each well of the streptavidin 96-well plate was washed three times with 100 μL of PBS containing 0.1% Tween-20.

[0103] Then, anti-HLA-G antibody was diluted in blocking solution to a final concentration of 0.391 nM. The diluted anti-HLA-G antibody was dispensed into 96 wells and reacted for 1 hour. CD8a-His diluted in blocking solution at a concentration of 100 nM was dispensed into each well and reacted for 1 hour. When the reaction was completed, each well was washed with PBS-T. Anti-CD8a-HRP, a CD8a detection antibody, was diluted in blocking solution and dispensed into each well. After reacting for 1 hour, each well was washed with PBS-T, and TMB solution was added to each well to develop color. 1N sulfuric acid was added to terminate the reaction, and the absorbance was measured at 450 nM using a microplate reader.

[0104] The results showed that the antibody of the present invention blocks the binding of HLA-G to CD8a at a level similar to or higher than that of conventional HLA-G (Table 4). These results suggest that anti-HLA-G antibodies can activate immune cells by suppressing the binding of HLA-G to CD8a.

[0105] <Example 5: Confirmation of cancer cell death using PBMC (peripheral blood mononuclear cells)> SK-OV-3 cells or MDA-MB-231 cells overexpressing HLA-G (target cells) were cultured for 24 hours in an incubator at 37° C. After culture, the HLA-G overexpressing cells (target cells) were pretreated with 100 nM anti-HLA-G antibody for 1 hour at 37° C. PBMCs (potency cells) and antibody-treated target cells were mixed at a ratio of 5:1 and co-cultured with SK-OV-3 cells or MDA-MB-231 cells for 48 hours.

[0106] After the culture was completed, the cell culture medium was collected. The LDH (lactate dehydrogenase) level was measured from the collected cell culture medium using a cytotoxicity assay kit (cytotox96 non-radioactive cytotoxicity assay, Promega) according to the manufacturer's protocol. Since LDH is released when cells die, the degree of cell death can be determined by measuring the LDH level.

[0107] The measurement results confirmed that anti-HLA-G antibody 2, antibody 1, and antibody 3 enhanced the cancer cell killing ability of PBMCs compared to the negative control group (pretreated with IgG1) that does not specifically bind to HLA-G (Figure 3).

[0108] Example 6: Confirmation of tumor growth inhibitory effect of HLA-G antibody 4T-1 cells overexpressing human HLA-G were transplanted into the right abdominal cavity of Balb / c mice. 7 to 14 days after transplantation, tumor growth was observed and tumors were classified into groups according to the tumor size (>100 mm). 3 Mice were grown until tumor size reached the desired size. Once tumor size reached the desired size, tumor-implanted mice were randomly grouped according to the test groups in Table 5.

[0109] [Table 5]

[0110] Anti-HLA-G antibody was intraperitoneally administered to each test group at a dose of 1 mg / kg (mg / kg=mpk) or 10 mg / kg twice weekly (BIW) for 2 weeks, a total of 4 times. On the 12th day after the start of the experiment, the volume of the subcutaneous tumor visible in the mice was measured, and the tumor growth inhibition rate was calculated according to the following formula 1.

[0111] [Formula 1] Tumor growth inhibition rate (TGI) (%) = (tumor volume of negative control group (IgG) - tumor volume of test group) / tumor volume of negative control group

[0112] The calculated tumor growth inhibition rates are shown in Table 6 below.

[0113] [Table 6]

[0114] The IgG1-treated group, which was the negative control group, did not suppress tumor growth, while all of the anti-HLA-G antibody-treated groups suppressed tumor growth. Antibody 4 showed a 24.9% tumor growth inhibition effect when administered at 10 mpk, and Antibody 1, Antibody 2, and Antibody 3 showed a tumor growth inhibition effect of 20% or more even when administered at 1 mpk (Table 6).

[0115] In addition, tumor tissue was isolated from mice treated with anti-HLA-G antibodies, and the immune cells that had infiltrated into the tumor were analyzed. The action of the immune anticancer drug changes the pool of immune cells in the tumor tissue, and cancer cells are killed by activating the immune cells. Cytotoxic T cells are a representative immune cell that has the function of killing cancer cells, and regulatory T (Treg) cells are a representative immune suppressor.

[0116] When the numbers of cytotoxic T cells / regulatory T cells in the tumor tissue were checked, it was found that the numbers of cytotoxic T cells / regulatory T cells were higher in all experimental groups that received anti-HLA-G antibodies compared to the negative control group (mock) (Figure 5). This result means that anti-HLA-G antibodies increase the function of cytotoxic T cells.

[0117] <Example 7: Confirmation of the binding site of antibody and HLA-G> Through preliminary experiments, it was confirmed that the highest total sequence coverage of 95.5% was obtained when the antigen was treated with a quenching buffer and pepsin solution (1:1 w / w) at low temperature for 5 minutes. Therefore, epitope mapping experiments were carried out under these conditions. The quenching buffer contained 100 mM potassium phosphate, 1 M urea, and 500 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) (pH 2.5).

[0118] Epitope mapping was performed using hydrogen deuterium exchange mass spectrometry (HDX-MS) technology, with β2M-HLA-G as the antigen and anti-HLA-G antibody 1 or 2 as the antibody.

[0119] The antigen alone and antigen-antibody complex samples were each labeled with D2O buffer (three samples were used). Mass spectrometry was then performed at five time points: 0 min, 0.33 min (20 sec), 10 min, 60 min, and 240 min. The molecular weight values ​​obtained through the spectrum were compared to identify the area where deuterium uptake of the complex was reduced compared to the antigen alone.

[0120] As a result, it was confirmed that when the antigen bound to each of the two antibodies, the amino acid sequence 339-344 (DYEATL) and 390-402 (VVVPSGEEQRYTC) of the antigen acted as the same epitope. In addition, for antibody 1, the 323-338 (QRADPPKTHVTHHPVF) portion was also found to be an epitope adjacent to 339-344 (Figure 5).

Claims

1. An anti-HLA-G antibody or antigen-binding fragment thereof, comprising: (a) a heavy chain complementarity determining region 1 (CDR1) comprising the amino acid sequence of SEQ ID NO:1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:3; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:7; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and A light chain CDR1 comprising the amino acid sequence of SEQ ID NO:21, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:23; or (d) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:27; and a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:30, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

31.

2. The anti-HLA-G antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8; (b) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 12 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 16; (c) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 20 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 24; or (d) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 28 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO:

32. The anti-HLA-G antibody or antigen-binding fragment thereof according to claim 1,

3. The anti-HLA-G antibody or its antigen-binding fragment according to claim 1, wherein the anti-HLA-G antibody is a recombinant antibody, a monoclonal antibody, a polyclonal antibody, a mixture of monoclonal antibodies and / or polyclonal antibodies, a human antibody, a humanized antibody or a chimeric antibody.

4. The anti-HLA-G antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody-binding fragment is selected from the group consisting of Fab (fragment antigen binding), Fab', F(ab')2, Fv (variable fragment), dsFv (disulfide-stabilized Fv fragments), scFv (single chain Fv), diabody, triabody, and tetrabody.

5. An isolated nucleic acid comprising a nucleotide sequence encoding the anti-HLA-G antibody or antigen-binding fragment thereof of claim 1.

6. A vector comprising the isolated nucleic acid of claim 5.

7. A host cell comprising the vector according to claim 6.

8. A pharmaceutical composition for use in the prevention or treatment of cancer, comprising one or more active ingredients selected from the group consisting of: (a) the anti-HLA-G binding antibody or antigen-binding fragment thereof according to claim 1; (b) the isolated nucleic acid of claim 5; (c) the vector according to claim 6; or (d) A cell according to claim 7.

9. The pharmaceutical composition of claim 8, wherein the cancer is selected from the group consisting of pancreatic cancer, breast cancer, ovarian cancer, glioma, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, colon cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, blood cancer and melanoma.

10. A method for preventing or treating cancer, comprising administering the anti-HLA-G binding antibody or antigen-binding fragment thereof described in claim 1, or the pharmaceutical composition described in claim 8, to a subject in need thereof.

11. An HLA-G epitope, characterized in that it consists of a sequence selected from the group consisting of SEQ ID NOs: 34 to 36.

12. An antibody, characterized in that it binds to an HLA-G epitope consisting of a sequence selected from the group consisting of SEQ ID NOs: 34 to 36.

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