HLA-G specific antibodies and their applications

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 Art

[0002] Despite intensive research on cancer over the past few decades, cancer remains a major cause of death worldwide. A number of cancer treatment methods have been developed, and methods such as surgical operations, chemotherapy, and radiotherapy have been continuously used from the past to the present. However, most of these existing cancer treatment methods are effective only in the initial stage where cancer has not metastasized. In a state where metastasis has already progressed, for example, even if a surgical operation is performed, there is a problem that recurrence is likely to occur subsequently. Therefore, for more effective cancer treatment, recently, research on immune anticancer agents that utilize the immune response has been continuing.

[0003] An immune anticancer agent is a therapeutic agent that enables immune cells to better recognize and attack cancer cells by preventing cancer cells from evading the human immune system. Specifically, an immune anticancer agent can induce the death of cancer cells through the activation of non-specific immune cells or induce an immune response against tumors in the immune cells of cancer patients using tumor-specific antigens.

[0004] On the other hand, human major histocompatibility complex antigens (HLA-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, and cytotoxic T cells. Known immune cell receptors for HLA-G include ILT2 (Ig-like transcript 2), ILT4, and KIR2DL4 (Killer Cell Immunoglobulin Like Receptor, Two Ig Domains And Long cytoplasmic tail 4), all of which possess an ITIM (immunoreceptor tyrosine-based inhibitory motif). When HLA-G binds to its receptor, this motif activates a signaling system that suppresses immune cells.

[0005] While HLA-G expression is restrictive in normal cells, it has been reported that HLA-G 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)). Furthermore, there have been reports of a correlation between HLA-G expression levels and the stage of cancer in cancer cells of many cancer patients, and some reports indicate that patients with HLA-G expression have lower survival rates (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)). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Under the circumstances described above, the inventors strived 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. As a result, they developed an HLA-G specific antibody that can bind to both HLA-G monomers and dimers, thus completing the present invention.

[0007] Therefore, an object of the present invention is to provide an HLA-G specific antibody or its antigen-binding fragment.

[0008] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, which provides 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 objective, one aspect of the present invention provides an anti-HLA-G antibody or its antigen-binding fragment comprising the following: (a) heavy chain complementarity determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO: 1, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3; and Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 5, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 6, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 7; (b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 9, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 10, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 11; and Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 13, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 14, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 15; (c) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 17, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 18, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 19; and Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 21, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 22, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 23; or (d) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 25, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 26, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 27; and Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 29, light chain CDR2 containing the amino acid sequence of SEQ ID NO: 30, and light chain CDR3 containing the amino acid sequence of SEQ ID NO: 31.

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

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

[0013] Furthermore, the antibody may be bivalent or a bispecific molecule (e.g., a bispecific antibody). Depending on the origin of its sequence, the antibody may also be a human antibody, a humanized antibody, or a chimeric antibody.

[0014] In this invention, the term "monoclonal antibody" refers to an antibody molecule with a single molecular composition obtained from a substantially identical antibody population. Unlike polyclonal antibodies, which can bind to multiple epitopes, such monoclonal antibodies exhibit single binding and affinity to a specific epitope. In this invention, the term "full-length antibody" refers to a structure having two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The invariant regions of the heavy chains have γ, μ, α, δ, and ε types, and have subclasses γ1, γ2, γ3, γ4, α1, and α2. The invariant regions of the light chains have κ and λ types. IgG is a subtype and includes IgG1, IgG2, IgG3, and IgG4.

[0015] In this invention, the term "heavy chain" can include both the full-length heavy chain and its fragments, which include a variable region VH containing an amino acid sequence having a sufficiently variable region sequence for conferring specificity to an antigen, and three invariant regions CH1, CH2, and CH3. Furthermore, in this invention, the term "light chain" can include all of the full-length light chain and its fragments, which include a variable region VL containing an amino acid sequence having a sufficiently variable region sequence for conferring specificity to an antigen, and an invariant region CL.

[0016] In this invention, the term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain originates from a specific source or species, while the remaining portion of the heavy chain and / or light chain originates from a different source or species. For example, there are chimeric antibodies in which the variable region of a mouse antibody and the invariant region of a human antibody are recombinant, and these antibodies exhibit a significantly improved immune response compared to mouse antibodies.

[0017] In this invention, the term "humanized antibody" refers to an antibody whose protein sequence, derived from a non-human species, has been modified to resemble that of an antibody naturally produced by humans. As an example, the humanized antibody can be produced by creating a humanized variable region by recombining a mouse-derived CDR with a human antibody-derived FR (framework region), and then recombining this with the invariant region of a preferred human antibody. However, simply performing CDR grafting alone reduces the affinity of the humanized antibody. Therefore, by making several important FR amino acid residues, which are expected to affect the three-dimensional structure of the CDR, compatible with those of the mouse antibody, the degree of substitution can be raised to the same level as that of the original mouse antibody.

[0018] Furthermore, the present invention provides antigen-binding fragments of the anti-HLA-G antibody. The antibody-binding fragments can 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] In this invention, the terms “fragment,” “antibody fragment,” “antigen-binding fragment,” or “antigen-binding domain” are interchangeable to refer to any fragment of the antibody of this invention that possesses 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 having the variable regions of the light and heavy chains and the constant region of the light chain and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. Fab’ is different from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. The F(ab’)2 antibody is produced while the cysteine residues in the hinge region of Fab’ form disulfide bonds. Fv is the smallest antibody fragment having only the variable regions of the heavy and light chains, and the recombinant technology for generating Fv fragments is disclosed in PCT International Patent Application Publications WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344, etc. The two-chain Fv has the variable regions of the heavy and light chains linked by non-covalent bonds, and the single-chain Fv generally has the variable regions of the heavy and light chains linked by covalent bonds through a peptide linker or immediately linked at the C-terminus, and thus can form a structure like a dimer together with the two-chain Fv. Such antibody fragments can be obtained by using proteolytic enzymes (for example, when the whole antibody is restrictively cleaved with papain, Fab can be obtained, and when cleaved with pepsin, an F(ab’)2 fragment can be obtained), and preferably can be produced through gene recombination technology.

[0021] The diabody is a small bivalent antibody composed of two heavy chain variables and two light chain variable domains, and each light chain variable domain is linked to the 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 a tetrabody can be formed.

[0022] In addition, the antibody or antigen-binding fragment thereof of the present invention can include not only the sequences of the anti-HLA-G antibodies described herein but also their biological equivalents, as long as they can exhibit the ability to bind to HLA-G. For example, additional changes can be made to the amino acid sequence of the antibody in order to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substituents 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 point, 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 present invention is an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof. The antibody and its antigen-binding fragment are as described above.

[0024] As used herein, the term "nucleic acid" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is the basic structural unit in the nucleic acid molecule, includes not only natural nucleotides but also analogues with modified sugar or base moieties (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 can be modified, and such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.

[0025] In the present invention, the isolated nucleic acid can include the following sequences: (a) The nucleotide sequence of SEQ ID NO: 37 or a nucleotide sequence having 80% or more homology 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 homology 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 homology 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 homology 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 homology 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 homology to the nucleotide sequence of SEQ ID NO: 44.

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

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

[0029] As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, and includes, but is not limited to, plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Plasmid vectors are preferred, but are not limited thereto.

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

[0031] The nucleic acid molecules that encode the variable region of the light chain and the nucleic acid molecules that encode the variable region of the heavy chain in the vector of the present invention may be operatively linked to a promoter.

[0032] As used herein, the term “operatably linked” means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, thereby the regulatory sequence modulates the transcription and / or decoding 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 thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is inserted herein by reference.

[0034] The vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. The recombinant vectors of the present invention are preferably expression vectors. Furthermore, the vectors of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.

[0035] For example, if the vector of the present invention is an expression vector and the host is a prokaryotic cell, it typically includes a potent promoter capable of driving 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, and T7 promoter), a ribosome binding site for initiating sequencing, and a transcription / sequencing termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthesis 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 regulatory sites. When Bacillus bacteria are used as host cells, the promoter of the Bacillus thuringiensis toxin protein gene (Appl. Environ. Microbiol. (1998) 64:3932-3938; Mol. Gen. Genet. (1996) 250:734-741) or any promoter expressible in Bacillus bacteria can be used as the regulatory site.

[0036] On the other hand, the recombinant vectors of the present invention can be produced by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.) that are frequently used in the relevant art.

[0037] On the other hand, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, promoters derived from the genome of mammalian cells (e.g., metallothione promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., late adenovirus 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, and generally have a polyadenylated sequence as the transcription termination sequence. Specifically, the recombinant vector of the present invention may contain a CMV promoter.

[0038] The recombinant vector of the present invention can be fused with other sequences to facilitate the purification of the antibody expressed therefrom. Examples of sequences that can be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine; Quiagen, USA). Furthermore, 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 without the need for additional sequences for purification.

[0039] On the other hand, the recombinant vector of the present invention may include antibiotic resistance genes commonly used in the relevant art as select labels, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline. According to one specific example of the present invention, an anti-HLA-G expression vector may include an ampicillin resistance gene.

[0040] The vector expressing the antibody of the present invention can be either a vector system in which the light chain and heavy chain are expressed simultaneously in a single vector, or a system in which the light chain and heavy chain are expressed in separate vectors. In the latter case, the two vectors are introduced into host cells through co-transformation and targeted transformation. Co-transformation is a method of simultaneously introducing the respective vector DNAs encoding the light chain and heavy chain into host cells, and then selecting cells that express both the light chain and heavy chain. Targeted transformation is a method of selecting cells transformed with a vector containing the light chain (or heavy chain), and then transforming the selected cells that express the light chain back into a vector containing the heavy chain (or light chain) to finally select cells that express both the light chain and heavy chain.

[0041] The nucleic acids encoding the heavy and light chains of the antibody of the present invention are inserted into an expression vector encoding the invariant regions of the heavy and light chains of human IgG1, IgG2, and IgG4. The light and heavy chains can be cloned in the same or different expression vectors. The DNA sequence encoding the immunoglobulin chain is operably ligated to a control sequence in an expression vector that ensures the expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences. The expression vector can be replicated in the host organism as an essential portion of host chromosomal DNA or as an episome.

[0042] When the heavy and light chains of an antibody are cloned into separate vectors, the expression vectors encoding the heavy and light chains can be assembled to form an antibody that is not damaged in vivo or in vitro, and can be co-transfected into a single host cell for the expression of both chains.

[0043] Alternatively, expression vectors encoding the heavy chain and light chain may be introduced into different host cells for the expression of the heavy and light chains, respectively, which can then be purified and assembled in vitro to form indestructible antibodies. Antibodies or fragments thereof as described herein may be produced in prokaryotic or eukaryotic expression systems such as bacteria, yeast, filamentous fungi, insects, and mammalian cells. The recombinant antibodies of the present invention do not necessarily need to be glycosylated or expressed in eukaryotic cells, but expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines include human embryonic kidney cell line (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] Such cell expression vectors may include replication bases, expression regulatory sequences such as promoters and enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Preferred expression regulatory sequences are promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papillomaviruses, giant cell viruses, etc. Vectors containing antibody-producing plasmid sequences can be transmitted into host cells by widely known methods, which may vary depending on the host cell type.

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

[0046] The host cells capable of stably and continuously cloning and expressing the vector of the present invention may be any host cells known in the relevant art, including, but not limited to, Bacillus strains such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, and prokaryotic host cells such as Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylococcus carnosus).

[0047] The host cells of the compatible eukaryotic cells transformed with the vector may be, 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, cells of higher eukaryotes such as insect-derived cells, and cells derived from plants or mammals.

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

[0049] Mammalian host cell cultures are preferred for expressing and producing anti-HLA-G antibodies or their antigen-binding fragments, because a number of suitable host cell lines capable of secreting undamaged immunoglobulins have been developed in the industry.

[0050] In the present invention, “transformation” and / or “phenoinfection” of host cells include any method of introducing nucleic acids into an organism, cell, tissue, or organ, and can be carried out by selecting a standard technique suitable for the host cell, as is known in the art. Such methods include, but are not limited to, electroporation, plasmofusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextrin sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

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

[0052] The culture of transformed cells using the method for producing the antibody or its antigen-binding fragment can be carried out by appropriate culture and culture conditions known in the relevant art. Such culture processes can be easily adjusted and used by a skilled technician depending on the selected bacterial strain. Such diverse culture methods are disclosed in various publications (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture is classified into suspension culture and adhesion culture depending on the cell growth method, and into batch, fed-batch, and continuous culture methods depending on the culture method. The culture medium used must appropriately satisfy the requirements of the specific bacterial strain.

[0053] In the culture of animal cells, the culture medium contains a variety of carbon sources, nitrogen sources, and trace element components. Examples of usable carbon sources may 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. These carbon sources may be used individually or in combination.

[0054] Nitrogen sources usable in the present invention may include, for example, organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn saturation solution (CSL), and soybean meal, as well as inorganic nitrogen sources such as iodine, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, which may be used alone or in combination. The culture medium may contain, as a phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt. It may also contain metal salts such as magnesium sulfate or iron sulfate. Other possible materials include amino acids, vitamins, and suitable precursors.

[0055] During cultivation, 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. Furthermore, during cultivation, the formation of bubbles can be suppressed using antifoaming agents such as fatty acid polyglycol esters. In addition, oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain an aerobic state. The culture temperature is usually 20°C to 45°C, preferably 25°C to 40°C, but is not limited to this range.

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

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

[0058] As a result, we confirmed that the anti-HLA-G antibody inhibits the binding of HLA-G to CD8a (Table 4). Furthermore, when PBMCs (effective cells) and target cells treated with the anti-HLA-G antibody were mixed and co-cultured with cancer cells, the cancer cells were killed (Figure 3), and tumor growth was suppressed in tumor-inducing mice (Table 6).

[0059] Therefore, yet 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, but is not limited to, be used to treat cancers that overexpress HLA-G. The cancer 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, hematological 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” means all actions that suppress or delay the progression of a disease by administering the compositions of the present invention, and “treatment” means the suppression, mitigation, or elimination of the development of a disease.

[0063] The pharmaceutical composition of the present invention may further contain pharmaceutically acceptable carriers, which are those commonly used in formulation and include, but are not limited to, lactose, dextrose, straw, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The cancer prevention or treatment composition of the present invention may further contain, in addition to the above components, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in the literature [Remington's Pharmaceutical Sciences (19th ed., 1995)].

[0064] The pharmaceutical compositions of the present invention can be administered orally or parenterally. In the case of parenteral administration, they can be administered by intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial infusion, local infusion, intranasal infusion, intrapulmonary infusion, and intrarectal infusion. When administered orally, proteins or peptides are digested, so oral compositions are formulated to coat the active agent or protect it from digestion in the stomach, and the compositions of the present invention can be administered by any device that allows the active substance to move to target cells.

[0065] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity. Usually, a skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to one embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention may be 0.1-100 mg / kg, preferably 0.1-10 mg / kg, and more preferably 0.1-2 mg / kg. In this specification, the term "pharmaceutical effective dose" means a sufficient amount to treat, prevent, and diagnose a disease.

[0066] The pharmaceutical compositions of the present invention may be manufactured in unit volume form by being formulated using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains, or by being contained in a multi-volume container. 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 additionally contain a dispersant or stabilizer.

[0067] The composition of the present invention may be administered as a standalone therapeutic agent, in combination with other therapeutic agents, or sequentially or simultaneously with conventional therapeutic agents.

[0068] The antibody or antigen-binding fragment of the present invention is used for cancer treatment by introducing it into the body in the form of antibody-therapeutic agent (functional molecule) and bispecific antibody-therapeutic agent (functional molecule) conjugates. Various suitable and preferred conditions for targeting the drug to a specific target site have been reported, for example, in the literature [Trouet et al., Plenum Press, New York and London, (1982) 19-30].

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

[0070] Another aspect of the present invention is a method for diagnosing, preventing, or treating an HLA-G overexpression-related disease, such as cancer, comprising administering a pharmaceutically effective amount of the antibody or antigen-binding fragment thereof, or the pharmaceutically effective composition thereof, to a target organism, such as a human or a non-human mammal. The present invention also provides a method for diagnosing an HLA-G overexpression-related disease, such as cancer, comprising the step of administering the antibody or antigen-binding fragment thereof to an individual requiring it, such as a human or a non-human mammal.

[0071] In this invention, the term "individual" means all animals, including humans who have developed or may develop HLA-G overexpression-related diseases, as well as monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quail, cats, dogs, mice, rats, rabbits, or guinea pigs.

[0072] On the other hand, the inventors of the present invention confirmed the binding sites of the antibody and HLA-G, and found that amino acids 339-344 (DYEATL), 390-402 (VVVPSGEEQRYTC), and 323-338 (QRADPPKTHVTHHPVF) of the amino acid sequence (SEQ ID NO: 33) of the antigen used for antibody screening were epitopes.

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

[0074] In this invention, the term "epitope" refers to a localized site on an antigen to which an antibody or fragment thereof can specifically bind. Epitopes typically consist of surface groups of molecules, such as amino acid or sugar side chains, and generally possess specific three-dimensional structural and specific charge properties. Stereomorphic and non-stereomorphic epitopes are distinguished in that binding to stereomorphic epitopes is lost in the presence of a denaturing solvent, while binding to non-stereomorphic epitopes is not. Epitopes may include amino acid residues directly involved in binding (also called the immunodominant component of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides (i.e., amino acid residues located within the footprint of the specific antigen-binding peptide).

[0075] Furthermore, the present invention provides antibodies that specifically bind to the HLA-G epitope, and specific examples of antibodies are as described above, but are not limited thereto. [Effects of the Invention]

[0076] Since the anti-HLA-G antibody according to the present invention can bind to both monomers and polymers of HLA-G, it can effectively block the binding of HLA-G to its receptor. [Brief explanation of the drawing]

[0077] [Figure 1] A schematic diagram for producing a b2m-HLA-G antigen according to an example of the present invention is shown. [Figure 2] The vector structure used for the production of anti-HLA-G antibodies is shown. [Figure 3]This study involved mixing HLA-G overexpressing target cells treated with anti-HLA-G antibodies with PBMCs, and then co-culturing these mixed cells with SK-OV-3 cells or MDA-MB-231 cells to confirm the degree of cancer cell death. [Figure 4] This study involved transplanting 4T-1 cells overexpressing human HLA-G into mice to induce tumors, then administering anti-HLA-G antibodies. The tumors were isolated, and the levels of cytotoxic T cells and regulatory T cells were examined. [Figure 5] This is the result of confirming the epitope sequence to which anti-HLA-G antibodies and HLA-G bind. [Modes for carrying out the invention]

[0078] The following describes one or more specific examples in more detail through embodiments. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited by these embodiments.

[0079] <Example 1: Antigen Design> To identify HLA-G-specific antibodies and suppress the mechanism of action of HLA-G, an antigen similar to the b2m-HLA-G multimer structure, which is the activated form of HLA-G, was constructed. To facilitate antigen production, a protein sequence linked to the signal peptide-b2m-HLA-G-6XHis was designed. A DNA sequence was then designed using this protein sequence (Figure 1). Subsequently, the DNA sequence was introduced into animal cells, and the protein was expressed and then purified. Specifically, the cell culture medium containing the signal peptide-b2m-HLA-G-6XHis antigen was purified in a primary process using an NI-NTA column. Then, the monomer and dimer were separated from the results of the primary purification using size exclusion chromatography. Antigen production is not limited to the aforementioned animal cells; for example, expression and purification can also be performed using E. coli.

[0080] Sequence ID 33: Antigen sequence MSRSVALAVLALLSLSGLEARIIPRHLQLGCGGSGGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFT PTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNT KAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWGSGLNDIFEAQKIEWHEHHHHHH

[0081] <Example 2: Antibody Production> 2-1. Library Screening Phage / scFv libraries were screened using HLA-G antigen-coated immunotubes (15501, Piece). Panning was performed four times to increase the antigen-binding specificity of the phage / scFv libraries. For the recovery and amplification of selected phages, geometrically growing E. coli cultures were infected with eluted phage suspensions after each panning. After completion of panning, phage-ELISA was performed using HLA-G antigen to evaluate the antigenic reactivity of the selected phages. After the fourth panning, the antigenic reaction signal of the phages increased to three times the background level. According to phage display techniques, such an increase in signal indicates that the selected phages are rich in HLA-G antigen-specific conjugates. Phagemid DNA was extracted from the phages selected from the library after the fourth panning. 96 clones were isolated and cultured on 24-well plates. The supernatant containing phage particles was tested using the phage-ELISA method, and the DNA of 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 the CDR1, 2, and 3 of the heavy and light chains to obtain diverse heavy and light chain products. These products were then combined to select HLA-G specific antibodies, and mature HLA-G specific antibodies were secured through ELISA analysis using HLA-A, HLA-B, and HLA-C.

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

[0084] The light chain expression vector and 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 4-6 days, the cells were removed, and only the upper layer was collected. The collected upper layer was filtered through a 0.45 μm filter, and the filtered culture medium was purified using an AKTA purifier with a Mabselect (Cytiva) purification column to obtain antibodies that specifically bind to human HLA-G.

[0085] 2-4. Antibody Analysis The obtained antibodies were analyzed, and the CDR and variable region sequences were compiled 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 binding properties of the anti-HLA-G specific antibody obtained in Example 2 to the HLA-G antigen prepared in Example 1 were screened. The analytical results are summarized in Table 3 below.

[0089] [Table 3]

[0090] Surface magnetic resonance (SPR) and octet analysis revealed that the anti-HLA-G specific antibody exhibited nanomolar-level KD values ​​against HLA-G polymers, confirming its high affinity for the antigen. Furthermore, the anti-HLA-G specific antibody, when obtained from HLA-G polymers, showed higher affinity for polymers than for monomers.

[0091] <Example 4: Confirmation of the binding inhibitory ability of HLA-G specific antibodies> Since anti-HLA-G specific antibodies have high affinity for HLA-G polymers, we investigated whether these antibodies can suppress the binding between ILT2 (Ig-like transcript 2) and HLA-G polymers.

[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 1x PBS to a concentration of 1.5 μg / mL. The diluted ILT-2 protein was dispensed at 150 ng / 100 μL into each well of a 96-well immunoplate. The immunoplate was stored overnight at 4°C to allow the ILT-2 protein to fix to the well surface. The following day, the ILT-2 protein solution was completely removed, and each well was washed with 200 μL of 1x PBS-T. A blocking solution was dispensed at 200 μL into each well, and the wells were incubated at 37°C for 1 hour to block nonspecific protein binding.

[0093] The antigen prepared in Example 1 was diluted in the blocking solution to a concentration of 1,200 nM, which is twice the concentration intended for treatment. The anti-HLA-G monoantibody was also diluted in the blocking solution to a concentration of 2,000 nM, which is twice the concentration intended for treatment, and then sequentially diluted threefold to 0.914 nM.

[0094] The antigen prepared in Example 1 and the anti-HLA-G monoantibody diluted to different concentrations were mixed in a 1:1 ratio, and 120 μl was dispensed into each well. The mixture was then pre-incubated at 37°C for 30 minutes. After completely removing the blocking solution from each well, it was washed with PBS-T.

[0095] A 1:1 mixture of the antigen from Example 1 and the anti-HLA-G monoantibody was added to each well at a rate of 100 µl, and the mixture was reacted at 37°C for 1 hour. Anti-His-HRP, the detection antibody against the antigen from Example 1, was diluted at a ratio of 1:10,000 and dispensed into each well at a rate of 100 µl, and the mixture was reacted at 37°C for 1 hour. Each well was washed three times with 200 µl 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 confirming the antibody concentration that suppressed the HLA-G:ILT2 interaction by 50% or more when treated with different antibody concentrations against the unblocked HLA-G:ILT2 interaction. The antibody obtained in Example 2 was shown to suppress HLA-G:ILT2 binding by 50% or more at a 150 nM level.

[0097] [Table 4]

[0098] b. ILT-2 Block Cell HLA-G expressing cells were cultured in a 96-well microplate at a concentration of 3.0.E+05 cells / well on day 0 (D0) of the experiment. The cell supernatant was removed on day 1 of the experiment. The anti-HLA-G monoantibody was diluted in a blocking solution to 200 nM, which is twice the concentration intended for treatment, and then serially diluted sixfold to 0.714 pM. This diluted solution was dispensed into the wells from which the supernatant had been removed and allowed to react for 30 minutes. The ILT2-mFc protein was diluted to 100 ug / mL and then dispensed into the antibody-cell reaction mixture and allowed to react for 1 hour. After the reaction was complete, the supernatant was removed as described above, and diluted anti-mouse IgG-FITC, which can detect ILT2-mFCs, was dispensed. After reacting for 2 hours, the residual amount of ILT2-mFc was confirmed using a cell analyzer.

[0099] Confirmation revealed that the IgG1-treated group, which was the negative control group, did not suppress the binding of HLA-G to ILT2 even at high concentrations. However, the antibody obtained in Example 2 suppressed the binding of HLA-G to ILT2 in a concentration-dependent manner. Furthermore, the antibody concentration (EC50) at which the binding of HLA-G-expressing cells to ILT2 was reduced to 50% or less was identified, demonstrating that the obtained antibody effectively suppressed the binding of HLA-G to ILT2 on the cell surface 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 antigens bind to CD8a expressed on immune cells, suppression of immune cells occurs; therefore, inhibiting the binding of HLA-G to CD8a is important for the activation of immune cells. Accordingly, the CD8a blocking effect of the antibody obtained in Example 2 was confirmed as follows.

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

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

[0103] Next, the anti-HLA-G antibody was diluted in the blocking solution to a final concentration of 0.391 nM. The diluted anti-HLA-G antibody was dispensed into 96 wells and allowed to react for 1 hour. A 100 nM concentration of CD8a-His, diluted in the blocking solution, was dispensed into each well and allowed to react for 1 hour. After the reaction was complete, each well was washed with PBS-T. Anti-CD8a-HRP, a CD8a detection antibody, was diluted in the 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] Measurement results showed that the antibody of the present invention blocked 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 cell)> SK-OV-3 cells or MDA-MB-231 cells overexpressing HLA-G (target cells) were cultured in a 37°C incubator for 24 hours. After culturing, the HLA-G overexpressing cells (target cells) were pre-treated with a 100 nM anti-HLA-G antibody at 37°C for 1 hour. PBMCs (effective cells) and the antibody-treated target cells were mixed in a 5:1 ratio 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 in the collected cell culture medium was measured 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 antibodies 2, 1, and 3 enhanced the cancer cell killing ability of PBMCs compared to a negative control group (IgG1 pretreatment) that did not specifically bind to HLA-G (Figure 3).

[0108] <Example 6: Confirmation of the tumor growth inhibitory effect of HLA-G antibody> 4T-1 cells overexpressing human HLA-G were transplanted into the right peritoneal cavity of Balb / c mice. Tumor growth was observed 7-14 days after transplantation, and when the tumor size exceeded a certain level (100 mm), it was considered a success. 3 Mice were grown until the tumors reached the desired size. Once the tumor size reached the desired size, the tumor-transplanted mice were randomly grouped into the test groups shown in Table 5.

[0109] [Table 5]

[0110] Each test group received intraperitoneal administration of anti-HLA-G antibody at a dose of 1 mg / kg (mg / kg = mpk) or 10 mg / kg twice weekly (BIW) for a total of four doses over two weeks. On day 12 of the experiment, the volume of tumors visible subcutaneously in the mice was measured, and the tumor growth inhibition rate was calculated using the following formula 1.

[0111] [Formula 1] Tumor Growth Inhibition (TGI) (%) = (Tumor volume of negative control group (IgG) - Tumor volume of test group) / Tumor volume of negative control group

[0112] The results of the calculation of the tumor growth inhibition rate are shown in Table 6 below.

[0113] [Table 6]

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

[0115] Furthermore, tumor tissue was isolated from mice treated with anti-HLA-G antibodies, and immune cells that had infiltrated the tumor were analyzed. The action of immunosuppressants alters the pool of immune cells within tumor tissue, and cancer cells are killed by the activation of these immune cells. Cytotoxic T cells are representative immune cells with cancer cell death function, and regulatory T (Treg) cells are representative immunosuppressive factors.

[0116] Analysis of cytotoxic T cell / regulatory T cell levels in tumor tissue revealed that all experimental groups administered anti-HLA-G antibodies showed higher levels of cytotoxic T cells / regulatory T cells compared to the negative control group (mock) (Figure 5). This result suggests 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> Preliminary experiments confirmed that the highest overall sequence coverage of 95.5% was obtained when the antigen was treated with a quenching buffer and a pepsin solution (1:1 w / w) at low temperature for 5 minutes. Based on this, 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] Samples in the antigen-only state and the antigen-antibody complex state were labeled with D2O buffer (3 repeated samples were used). Mass spectrometry was then performed at five time points: 0 minutes, 0.33 minutes (20 seconds), 10 minutes, 60 minutes, and 240 minutes. The molecular weight values ​​obtained through Spectra were compared to identify the portion where the deuterium uptake of the complex decreased compared to the antigen alone.

[0120] As a result, we were able to confirm that when the antigen bound to the two types of antibodies, the amino acid sequences 339-344 (DYEATL) and 390-402 (VVVPSGEEQRYTC) of the antigen acted as the same epitope. Furthermore, for antibody 1, it was found that the 323-338 (QRADPPKTHVTHHPVF) portion was also an epitope adjacent to 339-344 (Figure 5).

Claims

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

31.

2. The anti-HLA-G antibody or its antigen-binding fragment is (a) Variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 4 and variable region of the light chain consisting of the amino acid sequence of SEQ ID NO: 8; (b) Variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 12 and variable region of the light chain consisting of the amino acid sequence of SEQ ID NO: 16; (c) A variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 20 and a variable region of the light chain consisting of the amino acid sequence of SEQ ID NO: 24; or (d) A variable region of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 28 and a variable region of the light chain 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, characterized in that it comprises these elements.

3. The anti-HLA-G antibody or its antigen-binding fragment according to claim 1, characterized in that 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 its antigen-binding fragment according to claim 1, characterized in that 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 characterized by comprising a nucleotide sequence encoding the anti-HLA-G antibody or its antigen-binding fragment as described in claim 1.

6. A vector characterized by comprising the isolated nucleic acid described in claim 5.

7. A host cell characterized by containing the vector described in claim 6.

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

9. The pharmaceutical composition according to claim 8, characterized in that 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, colorectal cancer, nasopharyngeal cancer, oral cancer, thyroid cancer, prostate cancer, kidney cancer, gallbladder cancer, bile duct cancer, hematological cancer, and melanoma.

10. A method for preventing or treating cancer (excluding medical acts on humans) characterized by administering the pharmaceutical composition described in Claim 8 to a subject that requires it.

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