Antibody specifically binding to HLA-g, and use thereof

Abstract

The present invention relates to an antibody specifically binding to human leukocyte antigen G (HLA-G) or an antigen-binding fragment thereof, and use thereof. The antibody specifically binding to HLA-G or an antigen-binding fragment thereof can exhibit excellent anticancer effects such as that of inhibiting tumor growth by inhibiting the interaction between HLA-G and ILT-2 while specifically binding to HLA-G.

Description

Antibodies specifically binding to HLA-G and uses thereof

[0001] The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to HLA-G (human leukocyte antigen G), and uses thereof.

[0002] The precise origins of cancer are unknown, but it appears to have been a threat to humanity since ancient times, as evidenced by the discovery in the late 19th century of a papyrus document believed to date back to 1600 BCE. Since the founding of the American Cancer Society in 1913, humanity has made significant efforts to conquer cancer. However, even after more than 100 years, cancer remains a persistent threat.

[0003] Efforts to conquer cancer are evident in the development of anticancer drugs, beginning with the development of first-generation cytotoxic anticancer drugs in the 1960s and 1970s, and transitioning to second-generation targeted anticancer drugs in the early 2000s. This paradigm shift to third-generation immunotherapies began on March 28, 2011, with the US Food and Drug Administration's approval of ipilimumab, an immune checkpoint CTLA-4 inhibitor, for the treatment of late-stage melanoma.

[0004] Since then, in September and December of 2014, the PD-1 inhibitors pembrolizumab and nivolumab were approved by the U.S. Food and Drug Administration for use as melanoma treatment. To date, a total of eight immune checkpoint inhibitor antibodies, including the three mentioned above, the PD-1 inhibitors cemiplimab and dostarlimab, and the PD-L1 inhibitors atezolizumab, durvalumab, and avelumab, have been approved by the U.S. Food and Drug Administration for use as solid tumor treatment, and third-generation immunotherapy drugs are now leading the anticancer drug market in earnest.

[0005] However, the responsiveness to existing immunotherapy drugs varies greatly by cancer type and patient, and in the case of certain cancers such as non-small cell lung cancer and ovarian cancer, the proportion of patients showing resistance to existing immunosuppressive drugs that target PD-1 and PD-L1 is approaching 80% (Front. Immunol. 2020, 11:577869). Moreover, the proportion of patients showing acquired resistance, which is responsive to the above drugs in the initial treatment but does not respond to the second treatment, is very high. The causes of initial and acquired resistance are very diverse, including mutations in downstream signaling molecules and expression of alternative immune checkpoint molecules, and researchers report that there are likely many more causes that have not been identified (Front. Cell Dev. Biol. 2020, 8:672).

[0006] As the proportion of patients resistant to existing immunotherapy drugs is very high, and the mechanism of resistance is also very complex, there is an unmet medical need for the discovery of new immune checkpoints different from the targets of existing immunotherapy drugs and the development of inhibitors for these checkpoints.

[0007] HLA-G (human leukocyte antigen G) is known to be expressed in the placenta of pregnant women and acts as an immune checkpoint, protecting the fetus from maternal immunity (Front. Immunol. 2020, 11:1685). Recently, with increasing reports of a link between HLA-G and cancer, it is attracting attention as an anticancer target. HLA-G is a novel anticancer target immune checkpoint substance different from CTLA-4, PD-1, and PD-L1, which are the targets of existing immunotherapy drugs. In particular, it is overexpressed in cancer types such as non-small cell lung cancer, colon cancer, gastric cancer, ovarian cancer, and pancreatic cancer, which show low responsiveness to existing immunotherapy drugs. It has been reported that the expression of HLA-G in patients with the above cancers has a negative effect on the prognosis of the patients (Trends Immunol. 2008, 29(3):125; J. Int. Med. Res. 2020, 48(11):0300060520970758; Oncol. Lett. 2022, 24(1):226; Ann. Sug. Oncol. 2007, 14(10):2721; Hum. Immunol. 2018, 79(6):463; Cancer Sci. 2020, 111(8):3057).

[0008] This correlation between HLA-G expression and patient prognosis is related to the role of HLA-G in cancer. HLA-G overexpressed in cancer inhibits the antibody production ability of B cells through binding to ILT-2, etc. expressed in immune cells in the tumor microenvironment, inhibits the cancer cell killing ability of NK and T cells, and promotes the differentiation of Treg cells to increase the secretion of anti-inflammatory cytokines, thereby worsening the immune system of the tumor microenvironment, making it a very attractive anticancer target substance (Immunol. 2020, 11:1685).

[0009] Moreover, while PD-1 and PD-L1 are tumor-associated antigens (TAAs) that are expressed in normal tissues, HLA-G is not expressed in normal tissues except for the placenta of pregnant women, and is a tumor-specific antigen (TSA) that is overexpressed only in cancer. Therefore, the concern about targeting normal tissues by HLA-G-specific drugs is significantly lower, and thus its value as an anticancer target substance can be said to be even higher.

[0010] The present inventors have conducted extensive research to develop novel anticancer agents. As a result, they have developed antibodies or antigen-binding fragments thereof that specifically bind to HLA-G and inhibit the interaction between HLA-G and ILT-2. They have confirmed that these antibodies exhibit both high binding affinity for the HLA-G protein and excellent anticancer effects, thereby completing the present invention.

[0011] Each description and embodiment disclosed in this invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions described below.

[0012] Additionally, terms not specifically defined herein should be understood to have the meanings commonly used in the technical field to which the present invention pertains. Furthermore, unless specifically defined in context, the singular includes the plural, and vice versa.

[0013]

[0014] One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human leukocyte antigen G (HLA-G).

[0015] As used herein, the term "HLA-G (human leukocyte antigen G)" refers to a protein encoded by the HLA-G gene, also called histocompatibility antigen, class I, G. The amino acid sequence constituting it and the base sequence encoding it are known in existing databases (NCBI Reference Sequence: NP_002118, NP_001350496, NM_002127, NM_001363567, NM_001384280, NM_001384290, etc.).

[0016] As used herein, the term "anti-HLA-G antibody" means an antibody or antigen-binding fragment thereof that specifically binds to the HLA-G protein and neutralizes or inhibits the activity of the HLA-G protein, or that specifically binds to HLA-G and inhibits or inhibits the interaction between HLA-G and the ILT-2 receptor. Specifically, the anti-HLA-G antibody may be a human antibody that targets the human HLA-G protein while also being reactive to HLA-G proteins derived from a mouse or rat.

[0017] As used herein, the term "antibody" refers to an immunoglobulin molecule that immunologically reacts with a specific antigen or a protein molecule that acts as a receptor that specifically recognizes the antigen. Therefore, in the present invention, "antibody" is used in a broad sense and is interpreted to include all of polyclonal antibodies, monoclonal antibodies, whole antibodies (antibodies composed of at least two heavy chains and two light chains interconnected by disulfide bonds), and antibody fragments. The whole antibodies include IgA, IgD, IgE, IgM, and IgG. In addition, the IgG may include IgG1, IgG2, IgG3, and IgG4 as subtypes. The term "antibody fragment" refers to an "antigen-binding fragment" or "antibody analog" that retains at least some of the binding specificity of a parent antibody and includes a portion of the antigen-binding region (e.g., one or more CDRs) or variable region of the parent antibody. The antibody fragment may be, for example, a Fab, Fab', F(ab')2, an Fv fragment, a scFv, a unibody, a diabody, a linear antibody, a nanobody, a domain antibody or a multispecific antibody formed from an antibody fragment.

[0018] As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof, including a heavy chain variable region and a heavy chain constant region. Heavy chains include gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types.

[0019] As used herein, the term "light chain" refers to a full-length light chain and fragments thereof, including a light chain variable region and a light chain constant region. Light chains include kappa (κ) and lambda (λ) types.

[0020] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region, which forms the antigen-binding site, as part of the variable region of antibodies produced by B cells and T cells. The amino acid sequences of the heavy and light chains each contain three non-contiguously arranged CDRs, namely the heavy chain CDR H1 , CDR H2 , CDR H3 and light chain CDR L1 , CDR L2 , CDR L3 This is included. CDR is a region involved in antigen recognition, providing key contact residues for antibody binding to antigen or epitope, and plays a crucial role in the diversity of antigen specificity.

[0021] The antibody of the present invention may be a full-length antibody or an antibody fragment having antigen-binding ability, and the heavy chain may be any one of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) types, and the light chain may be the kappa (κ) or lambda (λ) types.

[0022] In one specific embodiment according to the present invention, the antibody or antigen-binding fragment thereof may comprise a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, 17, 33, 49, 64, 79, 94, 109, 125, 140, 156, 172 or 188; a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, 18, 34, 50, 65, 80, 95, 110, 126, 141, 157, 173 or 189; and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3, 19, 35, 51, 66, 81, 96, 111, 127, 142, 158, 174 or 190.

[0023] Additionally, the antibody or antigen-binding fragment thereof may comprise a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 4, 20, 36, 52, 67, 82, 97, 112, 128, 143, 159, 175 or 191; a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 5, 21, 37, 53, 68, 83, 98, 113, 129, 144, 160, 176 or 192; and a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 6, 22, 38, 54, 69, 84, 99, 114, 130, 145, 161, 177 or 193.

[0024] Additionally, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15, 31, 47, 123, 154, 170, 186 or 202.

[0025] Additionally, the antibody or antigen-binding fragment thereof may comprise a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16, 32, 48, 63, 78, 93, 108, 124, 139, 155, 171, 187 or 203.

[0026] The sequence disclosed in the present invention includes a sequence that exhibits substantial identity with the sequence described in the sequence listing. The term "substantial identity" refers to a sequence homology of at least 80%, 90%, or 95%, as determined by aligning two sequences to maximize their correspondence and analyzing them using an algorithm commonly used in the art.

[0027] In addition, the antibody or antigen-binding fragment thereof of the present invention may include the sequence of the antibody or antigen-binding fragment thereof described herein, a sequence substantially identical to the sequence, as well as a biological equivalent thereof, within the scope of being capable of specifically recognizing and binding to the HLA-G protein. For example, the antibody or antigen-binding fragment thereof may include additional mutations in the sequence to improve antibody binding affinity and / or biological properties, and may include additional mutations within the scope that does not alter the overall activity of the molecule.

[0028] Therefore, it should be interpreted that sequences having high homology with the sequences represented by SEQ ID NOs: 1 to 223 of the present invention, for example, sequences having high homology of 70% or more, specifically 80% or more, and more specifically 90% or more, are also included in the scope of the present invention.

[0029]

[0030] In one embodiment of the present invention, the antibody of the present invention or an antigen-binding fragment thereof can be combined with a drug to form an antibody-drug conjugate (ADC). The drug to which the antibody of the present invention or an antigen-binding fragment thereof binds in the antibody-drug conjugate is also referred to as a "payload." As long as the antibody of the present invention or an antigen-binding fragment thereof is used, the drug used in the antibody-drug conjugate may be, for example, one that has already been approved for sale or approved, or one that is currently under development, but is not limited thereto.

[0031]

[0032] In one embodiment of the present invention, the antibody of the present invention or an antigen-binding fragment thereof may be included in a fusion protein or may constitute a fusion protein. In another embodiment of the present invention, the fusion protein may comprise the antibody of the present invention or an antigen-binding fragment thereof.

[0033] As used herein, the term "fusion protein" refers to a protein comprising part or all of two or more different proteins combined. The fusion protein comprises the amino acid sequences of the proteins that make up the fusion protein, and may additionally include, but is not limited to, an amino acid sequence such as a signal peptide that allows the protein produced within a cell to be secreted outside the cell.

[0034] Specifically, the fusion protein may include a biologically active molecule, a linker, an IgG Fc region, an IgM Fc region, and a J chain, and may be represented by the following structural formula:

[0035] [(X) a -(Y) b -(IgG Fc region) c -(IgM Fc region)] m -(Z) n

[0036] At this time, in the above structural formula,

[0037] The above X is a biologically active molecule,

[0038] The above Y is a linker,

[0039] The above IgG Fc region is a monomer,

[0040] The above IgM Fc region is a monomer,

[0041] The above Z is a J chain,

[0042] The above a is 1 or 2, the above b is 1 or 2, the above c is 0 or 1, the above m is 5 or 6, and the above n is 0 or 1.

[0043] As used herein, the term "IgG Fc region" refers to the Fc region (fragment crystallizable region) of immunoglobulin G (IgG), and refers to the remaining portion of IgG excluding the Fab region (fragment, antigen binding region). The IgG Fc region can be directly linked to a linker and an IgM Fc region within a fusion protein. In addition, the IgG Fc region can be indirectly linked to a biologically active molecule through a linker within the fusion protein, wherein the IgG Fc region can serve as a linker, supporter, carrier, etc. of the biologically active molecule so as to include one or two or more of the biologically active molecules. In the present specification, the 'IgG Fc region' can be used interchangeably with 'IgG Fc fragment' to have the same meaning.

[0044] Specifically, the IgG Fc region monomer may be composed of two heavy chains. One heavy chain may include at least one selected from the group consisting of a CH2 domain and a CH3 domain. For example, one heavy chain may include a CH2 domain and a CH3 domain, or may be composed of a CH2 domain and a CH3 domain.

[0045] In the present invention, the IgG Fc region may be derived from IgG1, IgG2, IgG3 or IgG4.

[0046] Additionally, the IgG Fc region may be derived from wild-type IgG or a mutant IgG. In this case, the mutant IgG may be caused by a mutation that reduces antibody-dependent cell-mediated cytotoxicity (ADCC) and / or a mutation that reduces complement-dependent cytotoxicity (CDC).

[0047] As used herein, the term "IgM Fc region" refers to the Fc region (fragment crystallizable region) of immunoglobulin M (IgM), and refers to the remaining portion of IgM excluding the Fab region (fragment, antigen binding region). The IgM Fc region can be directly linked to an IgG Fc region in a fusion protein, and can also be directly linked to a linker if an IgG Fc region does not exist in the fusion protein. In addition, the IgM Fc region can be indirectly linked to a biologically active molecule through the IgG Fc region and a linker in the fusion protein, or can be indirectly linked to a biologically active molecule only through a linker in the fusion protein. Here, the IgM Fc region can serve as a linker, supporter, carrier, etc. of the biologically active molecule so as to include one or two or more of the biologically active molecules. In this specification, the 'IgM Fc region' may be used interchangeably with 'IgM Fc fragment' with the same meaning.

[0048] In the present invention, the IgM Fc region monomer can form a pentamer or a hexamer, and in this case, each monomer can be connected through a disulfide bond.

[0049] Specifically, the IgM Fc region monomer may be composed of two heavy chains. One heavy chain may include at least one selected from the group consisting of a Cμ2 domain, a Cμ3 domain, and a Cμ4 domain.

[0050] As used herein, the term "J chain (joining chain)" refers to a polypeptide that is a component of an IgM or IgA antibody and binds IgM or IgA monomers to form a multimer. The J chain may be present between IgM Fc region monomers in the fusion protein and may function to connect them. Specifically, the J chain may be included when IgM Fc region monomers form a pentamer, and may not be included when IgM Fc region monomers form a hexamer. More specifically, when IgM Fc region monomers form a pentamer, the J chain may be joined to the first IgM Fc region monomer and the fifth IgM Fc region monomer via a disulfide bond.

[0051] Specifically, the J chain may be derived from a wild-type J chain or a mutant J chain.

[0052] In the present invention, the J chain may be further bound to a protein molecule. At this time, the protein molecule may exhibit a synergistic effect on the desired effect of the fusion protein of the present invention. Specifically, the protein molecule may be at least one selected from the group consisting of antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, soluble proteins, membrane-bound proteins, ligands, receptors, virus-like particles, protein toxins, and enzymes, but is not limited thereto. More specifically, the protein molecule may be at least one selected from the group consisting of co-stimulatory receptors, cytokines, and cell engagers. At this time, the cell engager may be an antibody, and specifically, an antibody that binds to a cancer cell or immune cell-specific marker protein, for example, a T-cell engager. In addition, the immune cell may include lymphocytes such as neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and B cells and T cells.

[0053] The term "biologically active molecule" as used herein refers to a substance that can exhibit a specific action or effect within a living organism, for example, by activating or deactivating a specific signaling pathway, thereby promoting or inhibiting the expression of a specific gene, enhancing or inhibiting the function of a specific protein, or inducing or inhibiting cell death. Preferably, the biologically active molecule may exhibit immune regulation and / or anticancer activity.

[0054] Specifically, the biologically active molecule may comprise an antibody or an antigen-binding fragment thereof according to the present invention, or may be an antibody or an antigen-binding fragment thereof according to the present invention.

[0055] In the present invention, the biologically active molecule may be indirectly linked to an IgG Fc region within the fusion protein via a linker, or the biologically active molecule may be indirectly linked to an IgM Fc region within the fusion protein via a linker. Here, the biologically active molecule may serve as a functional group or active moiety that can cause the fusion protein to exhibit the desired effect.

[0056] Specifically, the biologically active molecule may be at least one selected from the group consisting of antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, soluble proteins, membrane-bound proteins, ligands, receptors, virus-like particles, protein toxins, chemokines, cytokines, and enzymes, but is not limited thereto. In this case, the receptor may be a cytokine receptor and / or an immune checkpoint receptor, but is not limited thereto. For example, the fusion protein according to the present invention may include two or more types of biologically active molecules depending on the desired purpose, target, or effect.

[0057] As a specific example, the antigen-binding fragment of the antibody may be at least one selected from the group consisting of Fab, F(ab')2, F(ab)2, Fab', Fab2, Fab3, Fv, scFv, Bis-scFv, Minibody, Triabody, Diabody, Tandem Diabody (TandAb), Nanobody, and Tetrabody, but is not limited thereto. In addition, the antigen-binding fragment of the antibody may bind to HLA-G. HLA-G is a type of type 1 HLA, expressed in the placenta of a pregnant woman and involved in the immune tolerance of the fetus. HLA-G binds to ILT2, ILT4, and KIR2DL4, which are inhibitory receptors of NK (natural killer) cells, to inhibit NK cells and regulatory T cells (T reg ) plays a role in promoting differentiation. In addition, when cancer cells overexpress HLA-G on their surface, the activity of cytotoxic T cells, helper T cells, and NK cells involved in cytotoxicity is inhibited, thereby reducing the effectiveness of immunotherapy for cancer treatment. Therefore, an antigen-binding fragment that selectively binds to HLA-G and inhibits HLA-G from binding to inhibitory receptors can be utilized as an anticancer therapeutic agent. For example, the antigen-binding fragment of the antibody may be Fab or scFv.

[0058] As another specific example, the receptor may be, but is not limited to, HLA (Human Leukocyte Antigen). The term 'HLA' refers to a protein encoded by the human major histocompatibility complex (MHC) gene complex, and is an important immunological molecule that exists mainly in a membrane-bound form and is responsible for regulating the immune system. HLA presents peptide fragments of foreign or self-proteins from inside the cell to the cell surface and activates cytotoxic T cells by recognizing and binding to the T-cell antigen receptor (TCR) of the cytotoxic T cell. HLA is largely divided into two types: type 1 HLA is present on the surface of all cells, while type 2 HLA is present only on specific antigen-presenting cells (APCs) such as NK cells, macrophages, and dendritic cells. The HLA gene has a highly polymorphic characteristic and expresses six different HLA protein α chains (two HLA-A, two HLA-B, and two HLA-C). Since the type 1 HLA can selectively activate cytotoxic T cells expressing TCR when a specific peptide fragment is loaded into it, recombinant type 1 HLA with the membrane-bound domain portion removed is also used in the treatment of cancer and infectious diseases. In addition, most type 1 HLAs are unstable when the groove is empty without a peptide loaded, unlike type 2 HLAs. Therefore, when producing recombinant type 1 HLAs, the target synthetic peptide is loaded or the recombinant HLA protein is manufactured in a form fused to the α chain. When expressing using a microorganism, there is a concern that refolding may not occur properly, so it is generally expressed using animal cells.In the art, HLA and MHC are used interchangeably with the same meaning, and in humans, HLA is used instead of MHC. In this specification, the terms 'HLA' and 'MHC' may be used interchangeably with the same meaning.

[0059] The above HLA may be type 1 HLA such as HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G; or type 2 HLA such as HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. The above type 1 HLA is composed of a β2m domain, an α1 domain, an α2 domain, and an α3 domain, and the above type 2 HLA is composed of an α1 domain, an α2 domain, a β1 domain, and a β2 domain. The HLA of the present invention may include a β2m domain, an α1 domain, an α2 domain, or an α3 domain, and may include an α1 domain, an α2 domain, a β1 domain, and a β2 domain.

[0060] In addition, the HLA may include a substance that specifically binds to the active site of the target T-cell. The substance may selectively activate the target T-cell expressing the TCR, and / or may increase the stability of the HLA. The term, 'target T-cell' may be a T-cell to which the fusion protein of the present invention binds, and may be a cytotoxic T-cell (killer T cell; Tc) or a helper T-cell (Helper T cell; Th). The cytotoxic T-cell is a cell that can kill cancer cells, cells infected with viruses, etc., or damaged cells, and is, for example, CD8 + It is a T cell. CD8 +CD8, a protein receptor on T cells, is located on the cell surface and binds to type 1 HLA. These helper T cells do not directly kill cells, but play a role in activating other immune cells, such as promoting antibody production by B cells and activation of cytotoxic T cells. CD4 + T cells are an example of this. CD4 + CD4, a protein receptor on T cells, is located on the cell surface and binds to type 2 HLA.

[0061] A specific example of a substance that specifically binds to the active site of the above target T cell may be, but is not limited to, a peptide. The peptide may be derived from a virus or an animal cell. In this case, the virus may be, but is not limited to, cytomegalovirus (CMV). CMV is one of the most potent immunogenic antigens for the human immune system, and when infected with a human, it produces a very strong CD8 + Stimulates T-cell response. CD8 + T-cell immune responses are primarily activated by CMV proteins pp65 and IE-1, and the frequency of CMV-specific T-cells to individual peptides is approximately 10% of total CD8 + It is known to be very high, with a frequency of approximately 1% to 2% of the T-cell repertoire.

[0062] As another specific example, the ligand may be, but is not limited to, EPO (erythropoietin) or an EPO analog. The term, 'EPO', is a hormone protein essential for red blood cell production, and acts on hematopoietic stem cells in the bone marrow to promote the differentiation and growth of red blood cells, thereby treating anemia, protecting nerves in hypoxic environments such as stroke, and participating in the wound healing process. It is also known to help improve memory and be effective in treating depression by participating in the hippocampal response, synaptic connections, and neuronal networks of the brain. The 'EPO analog' is a recombinant protein that performs the same function as EPO but has a different structure, and has two additional sugar chains in addition to the three sugar chains of human EPO, and has a blood half-life that is three times longer than that of existing human EPO. The EPO analog may be a ligand, antibody, or antibody fragment that can bind to and activate an EPO receptor, and a specific example may be, but is not limited to, NESP (Novel erythropoiesis stimulating protein).

[0063] As used herein, the term "linker" refers to an amino acid present between each domain of a protein. The linker may be present between a biologically active molecule and an IgG Fc region within a fusion protein, and may serve to directly link the biologically active molecule and the IgG Fc region. In addition, the linker may be present between a biologically active molecule and an IgM Fc region when an IgG Fc region is not present within the fusion protein, and may serve to directly link the biologically active molecule and the IgM Fc region.

[0064] As a specific example, the linker may be at least one selected from the group consisting of an IgD hinge region, an IgA hinge region, an IgG hinge region, and a GS linker.

[0065] The above "hinge region" refers to a region located in the central portion of the heavy chain of an antibody, connecting two regions of the heavy chain with a disulfide bond. The hinge region is located between the CH1 domain and the CH2 domain of the heavy chain, and is connected to them. Unlike immunoglobulins with short hinge regions that have a Y shape, IgD specifically has a long hinge region, which allows it to have great flexibility to allow the target binding sites on both sides to spread apart to form a T shape. This flexibility allows IgD to have binding activity that can bind to multimeric foreign antigens while barely binding to self-antigens. In the present specification, 'hinge region' may be used interchangeably with 'hinge domain' with the same meaning.

[0066] In the present invention, the hinge region may include part or all of the hinge region, part or all of the CH1 domain, part or all of the CH2 domain, or a combination thereof.

[0067] For example, the hinge region may include a part or all of an IgD hinge (hereinafter, “part or all” is referred to as “part / all”). Specifically, the hinge region may include a part / all of an IgD hinge and a part / all of an IgD CH1 domain. Specifically, the hinge region may include a part / all of an IgD hinge and a part / all of an IgD CH2 domain. Specifically, the hinge region may include a part / all of an IgD hinge, a part / all of an IgD CH1 domain, and a part / all of an IgD CH2 domain.

[0068] For another example, the hinge region may comprise part / all of an IgA hinge. Specifically, the hinge region may comprise part / all of an IgA hinge and part / all of an IgA CH1 domain. Specifically, the hinge region may comprise part / all of an IgA hinge and part / all of an IgA CH2 domain. Specifically, the hinge region may comprise part / all of an IgA hinge, part / all of an IgA CH1 domain, and part / all of an IgA CH2 domain.

[0069] For another example, the hinge region may comprise a part / entire portion of an IgG hinge. Specifically, the hinge region may comprise a part / entire portion of an IgG hinge and a part / entire portion of an IgG CH1 domain. Specifically, the hinge region may comprise a part / entire portion of an IgG hinge and a part / entire portion of an IgG CH2 domain. Specifically, the hinge region may comprise a part / entire portion of an IgG hinge, a part / entire portion of an IgG CH1 domain, and a part / entire portion of an IgG CH2 domain.

[0070] The above "GS Linker" is (GS) d , (SG) d , (GGGS) d , (GGGGS) d , GCGS(GGGS) d and GCGGS (GGGGS) d It may be one or more selected from the group consisting of, wherein d is an integer from 2 to 6. Specifically, it may include 4 to less than 35 amino acids composed of glycine (Gly; G) and serine (Ser; S), and may include 4 to less than 35 amino acids composed of glycine, serine, and cysteine ​​(Cys; C) to increase stability through disulfide bonds, but is not limited thereto.

[0071]

[0072] Another aspect of the present invention provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0073] In the nucleic acid according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0074] As used herein, the term "nucleic acid" has a comprehensive meaning that includes DNA (gDNA and cDNA) and RNA. Nucleotides, which form the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogue nucleotides with modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0075] A nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the present invention comprises a nucleotide sequence encoding an amino acid sequence constituting the antibody or antigen-binding fragment thereof, and is not limited to any particular nucleotide sequence.

[0076] The above nucleotide sequence includes a nucleotide sequence that includes functionally equivalent codons or codons encoding the same amino acid (for example, there are six codons for arginine or serine due to codon degeneracy), or codons encoding biologically equivalent amino acids, taking into account the degeneracy of codons.

[0077] Considering the variants having biologically equivalent activity described above, the nucleic acid molecule of the present invention encoding the antibody or antigen-binding fragment thereof comprises a sequence that exhibits substantial identity therewith. The term "substantial identity" refers to a sequence homology of at least 80%, 90%, or 95%, as determined by aligning two sequences to maximize their correspondence and analyzing them using an algorithm commonly used in the art.

[0078]

[0079] Another aspect of the present invention provides a vector comprising the nucleic acid and an isolated host cell transformed with the vector.

[0080] In the vector and transformed host cell according to the present invention, unless specifically stated otherwise, the related terms are understood to have the same meaning as the terms described above.

[0081] As used herein, the term "vector" refers to anything that can be inserted into a host cell to replicate genes. Such vectors include plasmids, linear nucleic acids, cosmids, RNA vectors, viral vectors, and the like, and viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. The recombinant vector system of the present invention can be constructed using various methods known in the art. In addition, the vector of the present invention can be constructed as a vector for cloning or expression, and can be constructed using prokaryotic or eukaryotic cells as a host. Specifically, the vector may be a vector for expressing an antibody or an antigen-binding fragment thereof according to the present invention.

[0082] As used herein, the term "host cell" refers to a cell that includes the vector and can stably and continuously clone and express the antibody or antigen-binding fragment thereof of the present invention. For example, a prokaryotic host cell such as a Bacillus strain such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus; The host cell may be, but is not limited to, a fungus such as an Aspergillus species, a eukaryotic host cell such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces or Neurospora crassa; a lower eukaryotic cell; a higher eukaryotic cell such as an insect-derived cell; a plant cell; or a cell derived from a mammal such as a COS7 cell (monkey kidney cell), NSO cell, SP2 / 0, Chinese hamster ovary (CHO) cell, W138, baby hamster kidney (BHK) cell, MDCK, a myeloma cell line, HuT 78 cell or 293 cell, and any host cell commonly used in the art may be used without limitation.

[0083]

[0084] Another aspect of the present invention provides a method for producing the antibody or antigen-binding fragment thereof, comprising the step of introducing the vector into a host cell.

[0085] In the method for producing an antibody or an antigen-binding fragment thereof according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0086] Specifically, the method for producing the antibody or antigen-binding fragment thereof of the present invention comprises:

[0087] (a) a step of introducing the vector of the present invention into a host cell;

[0088] (b) a step of culturing the host cell; and

[0089] (c) may include a step of obtaining an antibody or an antigen-binding fragment thereof from the host cell.

[0090] The step (a) of introducing the vector of the present invention into a host cell may be a step of producing a transformant including a vector for expressing the antibody or an antigen-binding fragment thereof, and the step of producing the transformant may be transforming a host cell.

[0091] The above term, "transformant", refers to an organism in which genetic changes have been artificially introduced into cells by introducing external DNA into the host, thereby making the DNA replicable as a chromosomal element or by completing chromosomal integration.

[0092] Transformation of the aforementioned host cells can be performed using any transformation method, and can be easily performed according to a conventional method in the art. For example, the method may include, but is not limited to, CaCl2 precipitation method, Hanahan method with increased efficiency by using dimethyl sulfoxide (DMSO) in the CaCl2 method, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, PEG-based transformation method, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation method, and any transformation or transfection method conventionally used in the art can be used without limitation.

[0093] The above step (b) of culturing the host cell of the present invention can be performed according to media and culture conditions known in the art. Those skilled in the art can easily adjust and use this culture process according to the selected strain. Depending on the cell growth pattern, suspension culture or attachment culture may be used; depending on the culture method, batch, fed-batch, or continuous culture methods may be used.

[0094] In animal cell culture, the medium may include various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, or cellulose; fats such as soybean oil, sunflower oil, castor oil, or coconut oil; fatty acids such as palmitic acid, stearic acid, or linoleic acid; alcohols such as glycerol or ethanol; or organic acids such as acetic acid, and these carbon sources may be used alone or in combination. Examples of nitrogen sources include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, or soybean meal; or inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, and these nitrogen sources may be used alone or in combination. Examples of trace elements include phosphorus sources such as potassium dihydrogen phosphate or dipotassium hydrogen phosphate; It may contain metal salts such as magnesium sulfate or iron sulfate. In addition, it may contain amino acids, vitamins, or suitable precursors.

[0095] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid or sulfuric acid can be added to the culture in an appropriate manner during cultivation to adjust the pH of the culture, an antifoaming agent such as fatty acid polyglycol ester can be used during cultivation to suppress bubble formation, and oxygen or an oxygen-containing gas (e.g., air) can be injected into the culture to maintain an aerobic state of the culture.

[0096] Additionally, the culture can be cultured by maintaining the temperature of the culture at 20°C to 45°C, specifically 25°C to 40°C.

[0097] The step (c) of expressing the antibody or antigen-binding fragment thereof in the host cell of the present invention may be a step of recovering, purifying, and concentrating the antibody or antigen-binding fragment thereof of the present invention from the culture solution obtained through the step (b).

[0098] The above antibody or antigen-binding fragment thereof may be used in an unpurified state, or may be further recovered, purified, and concentrated for use. Specifically, a method commonly used in the art (e.g., dialysis, salt precipitation, chromatography, etc.) may be used, through which recovery, purification, and concentration can be performed simultaneously. More specifically, the antibody or antigen-binding fragment thereof may be recovered, purified, and concentrated using chromatography (e.g., ion exchange chromatography, size exclusion chromatography, or affinity chromatography), and the type and order of columns used therefor may be appropriately selected depending on the characteristics of the antibody or antigen-binding fragment thereof, the culture method, etc.

[0099]

[0100] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof.

[0101] In the pharmaceutical composition according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0102] The term "treatment" as used in the present invention means all instances where cancer is improved, reversed, cured, etc. by administration of the composition according to the present invention. In addition, the term "prevention" means all instances where the occurrence or recurrence of cancer is suppressed, delayed, prevented, etc. by administration of the composition according to the present invention.

[0103] Specifically, the cancer may be one or more selected from the group consisting of lung cancer, non-small cell lung cancer, colon cancer, rectal cancer, anal cancer, colon cancer, small intestine cancer, breast cancer, uterine cancer, ovarian cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, head cancer, cervical cancer, thyroid cancer, parathyroid cancer, stomach cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, cutaneous or intraocular melanoma, Hodgkin's disease, esophageal cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, central nervous system lymphoma, spinal cord tumor, glioblastoma, brainstem glioma, and pituitary adenoma, but is not limited thereto.

[0104] Additionally, the cancer may exhibit anticancer drug resistance or refractory behavior. The term "anticancer drug resistance" here refers to a situation where anticancer drugs are not effective from the outset of treatment, or where the initial therapeutic effect is diminished or lost over time. Furthermore, the term "anticancer drug refractory" refers to a situation where anticancer drugs are not effective from the outset of treatment, or where the response to treatment is not sustained for an extended period of time.

[0105] The pharmaceutical composition of the present invention can be administered to a subject in need of prevention or treatment of cancer by containing an effective amount of the antibody or an antigen-binding fragment thereof.

[0106] As used herein, the term "administration" refers to physically introducing a composition into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Such administration may be, but is not limited to, oral administration, or intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration, such as injection or infusion. The frequency of administration may be, for example, single, multiple, or over one or more extended periods of time.

[0107] As used herein, the term "subject" includes a human or any non-human animal, which may be a vertebrate, such as a primate, dog, cow, horse, pig, rodent, such as a mouse, rat, guinea pig, etc. As used herein, the term "subject" is used interchangeably with "individual" and "patient."

[0108] Additionally, the effective amount may be a "therapeutically effective amount" or a "prophylactically effective amount." The term "therapeutically effective amount" refers to any amount of a drug or therapeutic agent, when used alone or in combination with other therapeutic agents, that can exhibit a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to disease affliction. The term "prophylactically effective amount" refers to any amount that inhibits the occurrence or recurrence of cancer in a subject. The level of the effective amount may be determined based on factors such as the severity of the disease, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0109] In addition, the pharmaceutical composition may vary depending on the age, sex, and weight of the subject, and specifically, depending on the symptoms of the subject, the composition of the present invention may be administered once or several times daily at 0.1 to 100 mg / kg, or at intervals of several days to several months. In addition, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, body weight, age, etc.

[0110] In addition, the pharmaceutical composition may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Examples of carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0111] Additionally, the pharmaceutical composition may be administered in combination with another therapeutic agent. In this case, the pharmaceutical composition of the present invention and the other therapeutic agent may be administered simultaneously, sequentially, or separately. The other therapeutic agent may be, but is not limited to, a drug such as a compound or protein that has cancer prevention, treatment, or improvement effects.

[0112] Additionally, the pharmaceutical composition may be formulated to be administered simultaneously, sequentially, or separately with other therapeutic agents. For example, the antibody or antigen-binding fragment thereof and the other therapeutic agent may be administered simultaneously in a single formulation, or may be administered simultaneously, sequentially, or separately in separate formulations. To be administered simultaneously, sequentially, or separately, the antibody or antigen-binding fragment thereof and the other therapeutic agent included in the pharmaceutical composition of the present invention may be formulated separately in separate containers, or may be formulated together in the same container. Additionally, the antibody or antigen-binding fragment thereof and the other therapeutic agent included in the pharmaceutical composition of the present invention may be the same or different from each other in terms of the pharmaceutically effective amount, administration time, administration interval, administration route, treatment period, etc.

[0113]

[0114] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering to a subject the antibody or an antigen-binding fragment thereof.

[0115] Another aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof in a method for preventing or treating cancer.

[0116] Another aspect of the present invention provides a use of the antibody or antigen-binding fragment thereof for the preparation of a composition, preferably a pharmaceutical composition, for preventing, improving or treating cancer.

[0117] In the preventive or therapeutic method or use according to the present invention, unless specifically stated otherwise, the related terms are understood to have the same meaning as the terms described above.

[0118] Additionally, in the method or use for preventing or treating cancer according to the present invention, the antibody or antigen-binding fragment thereof may be administered to a subject simultaneously, sequentially, or separately with another therapeutic agent.

[0119] The above "simultaneous" administration means administering the antibody or antigen-binding fragment thereof; and another therapeutic agent at one time as a single formulation, or the antibody or antigen-binding fragment thereof; and another therapeutic agent at one time as separate formulations, in which case the route of administration of the antibody or antigen-binding fragment thereof and the route of administration of the other therapeutic agent may be different from each other. In addition, the above "sequential" administration means administering the antibody or antigen-binding fragment thereof; and another therapeutic agent relatively continuously, allowing the minimum possible time between administrations. In addition, the above "separate" administration means administering the antibody or antigen-binding fragment thereof; and another therapeutic agent at regular intervals. The method of administration of the antibody or antigen-binding fragment thereof; and another therapeutic agent may be appropriately selected by a physician or expert in the art in consideration of the therapeutic efficacy, side effects, etc. of the subject.

[0120]

[0121] Another aspect of the present invention provides a composition for diagnosing cancer, comprising the antibody or an antigen-binding fragment thereof.

[0122] Another aspect of the present invention provides a kit for diagnosing cancer, comprising the antibody or an antigen-binding fragment thereof.

[0123] In the cancer diagnostic composition and kit according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0124] Specifically, cancer diagnosis according to the present invention can be performed by reacting the antibody or an antigen-binding fragment thereof with a biological sample to determine whether cancer has developed or has the possibility of developing cancer, and preferably, by contacting the antibody or an antigen-binding fragment thereof with a biological sample to determine whether an antigen-antibody complex has been formed.

[0125] As used herein, the term "biological sample" includes, but is not limited to, tissue, cells, blood, serum, plasma, and tissue autopsy samples (brain, skin, lymph nodes, spinal cord).

[0126] As used herein, the term "antigen-antibody complex" means a combination of an HLA-G protein antigen in a sample and an antibody of the present invention or an antigen-binding fragment thereof that recognizes the same.

[0127] The formation of such antigen-antibody complexes can be detected by any method, such as a colorimetric method, an electrochemical method, a fluorimetric method, a luminometric method, a particle counting method, a visual assessment, or a scintillation counting method. However, the present invention is not limited thereto, and various applications and adaptations are possible according to methods known in the art.

[0128] Specifically, the diagnostic composition or kit of the present invention can be manufactured to be suitable for various immunoassays or immunostaining. The immunoassays or immunostaining include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, radioimmunoassay (RIA), and protein chips.

[0129] Additionally, labels for qualitatively or quantitatively measuring the formation of antigen-antibody complexes include, but are not limited to, enzymes, fluorophores, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes.

[0130]

[0131] Another aspect of the present invention provides a method for providing information for diagnosing cancer, comprising the step of detecting HLA-G protein through an antigen-antibody reaction or measuring HLA-G protein expression level or activity in a biological sample isolated from a subject suspected of having cancer using the antibody or an antigen-binding fragment thereof.

[0132] In the method for providing information for cancer diagnosis according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0133] In the present invention, the method for providing information for cancer diagnosis can be performed by reacting the antibody or an antigen-binding fragment thereof with a biological sample to provide information on whether cancer has developed or is likely to develop, and preferably, by contacting the antibody or an antigen-binding fragment thereof with a biological sample to determine whether an antigen-antibody complex is formed.

[0134] In one specific example according to the present invention, the method for providing information for diagnosing cancer may be a method for diagnosing cancer, may be a method for providing information for selecting a patient with cancer resistant or refractory to anticancer drugs, and may also be a method for selecting a patient with cancer resistant or refractory to anticancer drugs.

[0135] Specifically, the method may include the steps of (a) treating a biological sample isolated from a subject suspected of having cancer with the antibody or an antigen-binding fragment thereof to detect HLA-G protein through an antigen-antibody reaction; and (b) comparing the expression level or activity of the HLA-G protein detected in step (a) with that of a control group, and determining the subject as a cancer patient if the expression level or activity of the HLA-G protein is higher than that of the control group.

[0136] At this time, the control group may be a biological sample isolated from a normal person who has not developed cancer or a person who has developed cancer and then been cured.

[0137]

[0138] Another aspect of the present invention provides a method for providing information for cancer treatment, comprising a step of detecting HLA-G protein through an antigen-antibody reaction or measuring HLA-G protein expression level or activity in a biological sample isolated from a cancer patient using the antibody or an antigen-binding fragment thereof.

[0139] In the method for providing information for cancer treatment according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0140] In the present invention, the method for providing information for cancer treatment can be performed by reacting the antibody or an antigen-binding fragment thereof with a biological sample to provide information on anticancer drug resistance or non-responsiveness, and preferably, by contacting the antibody or an antigen-binding fragment thereof with a biological sample to determine whether an antigen-antibody complex is formed.

[0141] In one specific example according to the present invention, the method for providing information for cancer treatment may be a method for providing information for selecting a cancer patient resistant to or refractory to an anticancer drug, and may also be a method for selecting a cancer patient resistant to or refractory to an anticancer drug.

[0142] In another specific embodiment according to the present invention, the method may include the steps of (a) treating a biological sample isolated from a cancer patient with the antibody or an antigen-binding fragment thereof to detect HLA-G protein through an antigen-antibody reaction; and (b) comparing the expression level or activity of the HLA-G protein detected in step (a) with that of a control group, and determining that the cancer or tumor is more likely to have resistance or refractoryness to an anticancer agent if the expression level or activity of the HLA-G protein is higher than that of the control group.

[0143] At this time, the control group may be a biological sample isolated from a normal person who has not developed cancer, a person who has been cured of cancer, or a cancer patient who has been determined not to be resistant or refractory to anticancer drugs.

[0144]

[0145] Another aspect of the present invention provides a method for screening for an anticancer agent, comprising the steps of: (a) treating cancer cells with an anticancer agent candidate; (b) measuring the level of HLA-G protein expression or activity in cancer cells treated with the anticancer agent candidate using the antibody or an antigen-binding fragment thereof; and (c) determining the anticancer agent candidate treated in step (a) as an anticancer agent when the level of HLA-G protein expression or activity in step (b) is lower than that of cancer cells not treated with the anticancer agent candidate.

[0146] In the anticancer agent screening method according to the present invention, unless specifically stated otherwise, related terms are understood to have the same meaning as the terms described above.

[0147] Specifically, step (a) above is a step of treating cancer cells with an anticancer agent candidate, and can be performed using methods known in the art. For example, the anticancer agent candidate can be treated by treating cancer cells with the anticancer agent candidate and culturing them together, or by administering the anticancer agent candidate into a living body containing cancer cells. However, this is not limited to these methods, and those skilled in the art will be able to use a method suitable for the purpose of the present invention.

[0148] The term "anticancer agent" as used herein refers to a substance that exhibits a preventive or therapeutic effect on cancer, specifically a substance capable of killing cancer cells or tumors, or inhibiting their growth. In this specification, the term "anticancer agent" may be used interchangeably with "drug," and the term "treatment" may also be used interchangeably with "addition" or "administration."

[0149] The term "anticancer drug candidate" as used herein means a substance expected to exhibit a preventive or therapeutic effect on cancer, and specifically means a substance expected to kill cancer cells or tumors, or inhibit their growth.

[0150] Preferably, the anticancer agent or anticancer agent candidate includes, without limitation, a compound, a protein, a fusion protein, a compound-protein complex, a drug-protein complex, an antibody, a compound-antibody complex, a drug-antibody complex, an amino acid, a peptide, a virus, a carbohydrate, a lipid, a nucleic acid, an extract, a fraction, and the like. For example, the anticancer agent or anticancer agent candidate may include, but is not limited to, a compound, a peptide, a peptide mimetic, a fusion protein, an antibody, an aptamer, an antibody-drug conjugate (ADC), and the like. Preferably, the anticancer agent or anticancer agent candidate may be an antibody or an immuno-oncology agent.

[0151] The above step (b) is a step for measuring the level of HLA-G protein expression or activity, and any method known to those skilled in the art can be used. Specific examples include, but are not limited to, Western blot, co-immunoprecipitation assay, ELISA (Enzyme Linked Immunosorbent Assay), tissue immunostaining, and flow cytometry analysis. Those skilled in the art will be able to use a method suitable for the purpose of the present invention.

[0152] The above step (c) is a step for determining whether the above anticancer agent candidate can be used as an anticancer agent.

[0153] An antibody or an antigen-binding fragment thereof that specifically binds to HLA-G (human leukocyte antigen G) according to the present invention can exhibit excellent anticancer effects such as suppressing tumor growth by specifically binding to HLA-G and inhibiting the interaction between HLA-G and ILT-2.

[0154] Figure 1A shows the results of ELISA confirming the concentration-dependent binding of anti-HLA-G antibodies to HLA-G. Figure 1B shows the results confirming the specific binding ability of anti-HLA-G antibodies to HLA-G.

[0155] Figure 2 shows the results of confirming the binding inhibition ability of anti-HLA-G antibodies to HLA-G and recombinant ILT-2 protein using ELISA.

[0156] Figure 3 shows the results of confirming the concentration-dependent binding of anti-HLA-G antibodies to cancer cell lines that naturally express HLA-G using FACS.

[0157] Figure 4 shows the results of FACS analysis confirming the cancer cell killing ability of anti-HLA antibodies by blocking the binding of ILT-2, which is naturally expressed in the SKOV-3 / HLA-G cell line that overexpresses HLA-G, and natural killer cells NK92MI.

[0158] Figure 5 shows the results of FACS analysis confirming that anti-HLA-G antibodies bind to HLA-G expressed in various cancer types and block binding to ILT-2 naturally expressed in natural killer cells NK92MI, thereby increasing cancer cell killing ability.

[0159] Figure 6 shows the results of confirming antibody-dependent phagocytosis of anti-HLA-G antibodies by macrophages using FACS.

[0160] Figure 7 shows the results of confirming the cancer cell killing ability of an anti-HLA-G antibody in an in vivo xenograft mouse model using pancreatic cancer cell lines and gastric cancer cell lines.

[0161] Figure 8 shows the results of an in vitro assay evaluating the antibody-dependent cytotoxicity of anti-HLA-G antibodies and afucosylated anti-HLA-G antibodies.

[0162] Figure 9 shows the results of ELISA to confirm the binding inhibition ability of a material produced based on an anti-HLA-G antibody and an antigen-binding fragment to HLA-G and ILT-2.

[0163] Figure 10 shows the results of HLA-subtype selectivity of anti-HLA-G antibodies.

[0164] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited by these examples.

[0165]

[0166] Example 1. Screening of anti-HLA-G antibodies

[0167] Example 1.1. Production of library phages through phage display

[0168] A diverse human synthetic Fab library was placed in 2XYT medium containing glucose and cultured in a shaking incubator at 37°C until the optical density (OD600) of the culture medium reached 0.5 to 0.7.

[0169] To remove glucose in the medium, centrifugation (5,000 rpm, 4℃, 20 min) was performed, the supernatant was discarded, and the cells were lysed with 2XYT medium, infected with helper phage, and cultured in a shaking incubator at 37℃ for about 1 hour. Kanamycin was added at a concentration of 50 ㎍ / ml and cultured in a shaking incubator (30℃, 120 rpm) for 16 hours. The cultured cells were centrifuged (8,000 rpm, 4℃, 20 min), and 4% PEG (Sigma, P5413) and 3% NaCl (Sigma, S9888) were added to the supernatant to dissolve them well, and then reacted on ice for about 1 hour. After centrifugation again (12,000 rpm, 4℃, 30 minutes), DPBS (Wellgene, LB001-02) was added to the pellet to dissolve it, and then centrifugation (10,000 rpm, 4℃, 10 minutes) was performed to obtain the supernatant, which was placed in a new tube, stored at 5±3℃, and then used for panning.

[0170]

[0171] Example 1.2. Phage panning

[0172] To select antibodies that bind to HLA-G, HLA-G antigen was diluted in PBS at a concentration of 10 μg / mL, added to an immunotube, and reacted overnight at 4°C. For negative selection to remove phage that binds only to B2M and HLA subtypes HLA-B, C, and E, B2M, HLA-B, HLA-C, and HLA-E were diluted in PBS at a concentration of 25 μg / mL and reacted with magnetic beads overnight. After that, to remove antigens that did not bind to the immunotube and magnetic beads, the supernatant was removed, and a 5% skim milk solution was added to the immunotube and magnetic beads to protect the surfaces not adsorbed by HLA-G and B2M, HLA-B, C, and E. The phage produced in Example 1.1 was diluted in a 5% skim milk solution and placed on magnetic beads, then bound to the B2M antigen and reacted at room temperature for 1 hour. The supernatant was obtained using a magnet, and the supernatant was placed in an immunoassay tube to which HLA-G was attached and reacted at room temperature for 1 hour. After 1 hour, the supernatant in the immunoassay tube was removed, and the non-specifically bound phage was washed 4 times with PBS-T (Phosphate buffered saline - 0.05% Tween 20) solution and 2 additional times with DPBS. The remaining antigen-specific phage antibodies were collected after adding a 0.1 M glycine HCl pH 2.7 solution and reacting at room temperature for 10 minutes. The recovered phage was neutralized with 2 M Tris-HCl (pH 10.05) and then infected into TG1 Escherichia coli for 1 hour in a shaking incubator at 37°C. After that, the supernatant was removed by centrifugation (4000 rpm, 4℃, 30 minutes), and the cell pellet was dissolved in 2XYT medium. Then, it was spread on 2XYT-carbenicillin, 2% glucose plates and cultured in a 37℃ incubator for approximately 16 hours.The next day, 6 ml of 2XYT medium was added to the plate, the colonies were suspended in the culture medium, and some of the suspension was added to 50 ml of 2XYT-carbenicillin culture medium and cultured in a 37℃ shaking incubator. When the absorbance (OD600) of the culture medium reached 0.5 to 0.6, 20 MOI (Mode of infection) of helper phage was added and cultured for 1 hour at 37℃ with gentle stirring. After that, 50 ㎍ / ml kanamycin was added and cultured for 16 to 20 hours in a 30℃ shaking incubator. The next day, the culture medium was centrifuged, and only the culture medium was taken, 4% PEG (Sigma, P5413) and 3% NaCl (Sigma, S9888) were added, and the culture medium was precipitated at 4℃ for 1 hour and then centrifuged. The supernatant was removed, and the precipitated phage was suspended in 5 ml of PBS, which was used as a library, to amplify / enrich antigen-specific clones by repeating the above panning process four times. At this time, the amount of HLA-G coated on the immunoassay tube was reduced by half for each round, and the number of PBS-T washes was increased by one for each round. The antigen for negative selection was changed to HLA-B for the second round, HLA-C for the third round, and HLA-E for the fourth round.

[0173]

[0174] Example 1.3. Monoclonal phage ELISA

[0175] In order to select clones that specifically bind to the antigen among the phages obtained through panning in the above Example 1.2, a monoclonal phage ELISA was performed. After the fourth round of panning, single clones were selected from the spread solid medium and inoculated into a 96-well deep well plate containing 1 ml of 2XYT-carbenicillin medium, and then cultured in a 37°C shaking incubator until the optical density (OD600) reached 0.5. After culture, helper phage was added and infected for 1 hour in a 37°C shaking incubator, followed by the addition of kanamycin and culture at 30°C for 20 hours. After centrifugation, the cells were precipitated, and the supernatant was collected and the Fab binding to the HLA-G antigen was confirmed using the ELISA technique. The antigen-bound Fab was detected using HRP (Horseradish peroxidase) / anti-M13 antibody and TMB substrate. At this time, clones with an absorbance (OD450) of 0.4 or higher for HLA-G were selected and the sequence of the corresponding gene was analyzed.

[0176] As a result, clones 2F1 (SEQ ID NOs: 15 and 16), T22A4 (SEQ ID NOs: 31 and 32), and T23D5 (SEQ ID NOs: 47 and 48) were selected, as shown in Table 1 below.

[0177]

[0178]

[0179] Example 1.4. Antibody Optimization

[0180] In order to secure various antibodies that bind to HLA-G and to optimize the efficacy of the antibodies, various types of shuffling libraries were produced as follows, and antibodies were screened using the same method as in Example 1.2.

[0181] To produce a 2F1 light chain shuffling library, the heavy chain of 2F1, a clone selected from the library of Example 1.1, was fixed, and then CDRL1, 2, and 3 from a human synthetic Fab library were introduced to produce a light chain shuffling library in the form of scFv. Thereafter, antibody screening was performed in the same manner as in Examples 1.2 and 1.3.

[0182] As a result, 2F1_LS#17 (SEQ ID NOs: 15 and 63), 2F1_LS#24 (SEQ ID NOs: 15 and 78), 2F1_LS#26 (SEQ ID NOs: 15 and 93), and 2F1_LS#39 (SEQ ID NOs: 15 and 108) were selected as shown in Table 2 below.

[0183]

[0184] Next, to prepare the 2F1_LS#17 CDRH1, 2, CDRL1, 2, 3 shuffling library, CDRH3 of 2F1_LS#17 was fixed, and CDRH1, 2 and CDRL1, 2, 3 were introduced from a human synthetic Fab library to prepare a scFv type light chain and heavy chain shuffling library. Thereafter, antibody screening was performed in the same manner as in Examples 1.1 to 1.3.

[0185] As a result, 2F1HS6LS#38 (SEQ ID NOs: 123 and 124) and 2F1HS6LS#46 (SEQ ID NOs: 123 and 139) were selected, as shown in Table 3 below.

[0186]

[0187] Next, in order to create 2F1 CDRH1, 2 shuffling, CDRH3 mutant, and CDRL1, 2, 3 shuffling libraries, CDRH3 of 2F1_LS#17 was mutated using the NNK method, and light and heavy chain shuffling libraries in the form of scFv were created by introducing CDRH1, 2 and CDRL1, 2, 3 from a human synthetic Fab library. Thereafter, antibody screening was performed in the same manner as in Examples 1.1 to 1.3. As a result, as shown in Table 4 below, 2F1CDRH3#10 (SEQ ID NOs: 154 and 155), 2F1CDRH3#16 (SEQ ID NOs: 170 and 171), 2F1CDRH3#17 (SEQ ID NOs: 186 and 187), and 2F1CDRH3#26 (SEQ ID NOs: 202 and 203) were selected.

[0188]

[0189]

[0190] Example 2. Preparation of anti-HLA-G antibody

[0191] To prepare an anti-HLA-G antibody, based on the sequences of the antibody variable regions obtained in Examples 1.2 to 1.4 and the positive control antibodies (Fab and scFv) described in Table 5, the heavy chain variable region was linked to the heavy chain constant region (SEQ ID NO: 220) described in Table 5 below, and the light chain variable region was linked to the light chain constant region (SEQ ID NO: 221) described in Table 5 below. The positive control antibody is an antibody that selectively binds to HLA-G, including the sequences of SEQ ID NOs: 238 and 306 disclosed in US 2020 / 0102390 A1. In the drawings attached to this specification, "Ref ab" refers to the positive control antibody.

[0192] To produce the 2F1.eP antibody, the heavy chain variable region (SEQ ID NO: 15) and IgD hinge + IgG1 + IgM (SEQ ID NO: 222) were linked.

[0193] 클론아미노산 서열서열번호양성대조군 항체CDRH1SSSTYWS204CDRH2GIAYSGSTYYNPSLKS205CDRH3GVRRAVPEDY206CDRL1QAVSSNYLA207CDRL2GASSRAT208CDRL3QQVVHSPYT209FRH1QLQLQESGPGLVKPSETLSLTCTVSGGSIS210FRH2WIRQPPGKGLEWIG211FRH3RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR212FRH4WGQGTLVTVSS213FRL1EIVLTQSPGTLSLSPGERATLSCQAS214FRL2WYQQKPGQAPRLLIY215FRL3GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC216FRL4FGGGTKVEIK217중쇄가변영역QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSTYWSWIRQPPGKGLEWIGGIAYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGVRRAVPEDYWGQGTLVTVSS218경쇄가변영역EIVLTQSPGTLSLSPGERATLSCQASQAVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQVVHSPYTFGGGTKVEIK219중쇄 불변 영역 (IgG1)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK220경쇄 불변영역RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC221IgD 힌지+IgG1+IgMASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNSRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY222J chain (R106W)QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDWNKCYTAVVPLVYGGETKMVETALTPDACYPD223

[0194] Afterwards, the anti-HLA-G antibody and 2F1.eP were produced using the pcDNA3.1 expression vector (Invitrogen) and the Expi293F animal cell line. The transfected Expi293F cells were cultured in suspension at 37°C and 8% CO2, and the medium was cultured using Expi293F expression medium. For overexpression, the heavy and light chains were mixed in a 1:1 ratio according to the manual of the ExpiFectamine 293 Transfection kit, and 2F1.eP was produced and purified by adding the J chain (SEQ ID NO: 223). After transfection, the supernatant was collected and used for purification after culturing for 5 days. For antibody purification, the antibody was purified using a Mabselect sure (Sartorious) column, and then buffer exchange was performed with PBS pH 7.4 using Amicon Ultra 15 (Millipore, UFC903024) and used for analysis of anti-HLA-G antibodies.

[0195]

[0196] Example 3. Binding affinity analysis of anti-HLA-G antibodies

[0197] The affinity of the anti-HLA-G antibodies prepared in Example 2 for the HLA-G antigen was measured using Octet (Sartorious). Anti-HLA-G antibodies were attached to an anti-human IgG Fc Capture biosensor at a concentration of 1.5 μg / ml, and HLA-G antigen was added after being diluted by half from 100 nM to 1.56 nM to induce an antigen / antibody reaction. Using the antigen / antibody reaction results, the association constant (k on) and dissociation constant (K off) were measured, and the affinity was calculated, and the results are shown in Table 6 below.

[0198] As a result, all of 2F1_LS#17, 2F1_LS#24, 2F1_LS#26, and 2F1_LS#39, which were selected from the light chain shuffling library of 2F1 and 2F1, showed reliable affinities (KD) of 0.5 to 1.4 nM, similar to the positive control antibody.

[0199] KD (M)KD Errorka (1 / Ms)ka Errorkdis (1 / s)kdis ErrorFull R 2 2F15.232X10 -10 4.911X10 -12 2.479X10 -5 5.335X10 -2 1.297X10 -4 1.185X10 -6 0.99272F1_LS#176.893X10 -10 4.733X10 -12 2.006X10 -5 3.314X10 -2 1.383X10 -4 9.213X10 -7 0.99582F1_LS#245.533X10 -10 4.343X10 -12 2.175X10 -5 3.611X10 -2 1.203X10 -4 9.229X10 -7 0.99572F1_LS#266.983X10 -10 3.596X10 -11 4.034X10 -4 1.237X10 -2 2.817X10 -5 1.448X10 -6 0.9942F1_LS#395.669X10 -10 4.749X10 -12 1.873X10 -5 2.929X10 -2 1.062X10 -4 8.737X10 -7 0.9964 positive control antibody 1.359X10 -9 1.402X10 -11 1.156X10 -53.293X10 -2 1.571X10 -4 1.557X10 -6 0.9875

[0200]

[0201] Example 4. Efficacy analysis of anti-HLA-G antibodies

[0202] Example 4.1. Binding ability analysis for recombinant HLA-G

[0203] The antigen binding affinity of the selected anti-HLA-G antibody prepared in Example 2 was confirmed using ELISA. The recombinant HLA-G antigen was diluted in 1X PBS to a concentration of 1 μg / mL, coated on an immunoplate (Thermo scientific, 442404) at 50 ng / 50 μl / well, and reacted overnight at 4°C. After washing three times, the plate was reacted with blocking buffer (3% skim milk in PBST) at 37°C for 1 hour. After washing three times, the anti-HLA-antibody was diluted 1 / 4 each from 20 nM and treated at 50 μl / well at 7 concentrations, and reacted at 37°C for 1 hour. After washing three times, the anti-human IgG HRP was diluted 1:5,000 in blocking buffer, treated at 50 μl / well, and reacted at 37°C for 1 hour. After washing three times, TMB (Sigma, T0440) was treated at 50 μl / well, and color development was confirmed for about 10 minutes. Then, 1N H2SO4 was added at 50 μl / well, and the absorbance was measured using a 450 nm wavelength of a microplate reader.

[0204] As a result, as shown in Fig. 1, all antibodies produced in Example 2 bound to HLA-G in a concentration-dependent manner at a level equivalent to that of the positive control antibody. In addition, it was confirmed that they did not bind to B2M, but specifically bound only to HLA-G.

[0205]

[0206] Example 4.2. Analysis of HLA-G and ILT-2 binding inhibition capacity

[0207] To confirm whether the anti-HLA-G antibodies prepared in Example 2 above bind to the HLA-G antigen and inhibit the binding to recombinant ILT-2 protein (R&D systems, 2017-T2), the binding inhibition ability of HLA-G and ILT-2 was measured using ELISA. Recombinant ILT-2_hFc (R&D systems) was diluted in 1X PBS to a concentration of 1 μg / mL, coated on an immunoplate (Thermo scientific, 442404) at 100 ng / 100 μl / well, and reacted overnight at 4°C. After washing three times, the plate was reacted with blocking buffer (3% skim milk in PBST) at 37°C for 1 hour. After washing three times, 50 ㎕ of the sample diluted to a concentration of 1.2 μM in 1X PBS and 50 ㎕ of the analysis antibody diluted 1 / 4 from 2000 μM were mixed and treated at 100 ㎕ / well and reacted at 37℃ for 1 hour. After washing three times, anti-his HRP was diluted 1:10,000 in blocking buffer and treated at 100 ㎕ / well and reacted at 37℃ for 1 hour. After washing three times, TMB (Sigma, T0440) was treated at 50 ㎕ / well and color development was confirmed for about 10 minutes. After adding 50 ㎕ / well of 1 N H2SO4, the absorbance was measured using a microplate reader at a wavelength of 450 nm.

[0208] As a result, as shown in Fig. 2, the antibody produced in Example 2 exhibited binding inhibition ability of recombinant HLA-G protein and recombinant ILT-2 protein at a level equivalent to that of the positive control antibody.

[0209]

[0210] Example 4.3. Cell surface binding assay

[0211] To confirm whether the antibodies produced in Example 2 above also bind to HLA-G naturally expressed on the cell surface, a cell surface binding assay was performed. JEG-3 (uterine choriocarcinoma) and SCH (gastric cancer) cell lines, which are cancer cell lines that naturally express HLA-G, were cultured in a chemically defined medium, and 2 x 10 5 Cells were dispensed into 1.7 ml microtubes at a concentration of 1.7 cells / tube. Each antibody was diluted to six concentrations and treated, and incubated at 4°C for 1 hour. To confirm specific binding to HLA-G on the cell surface, a human IgG1 isotype control antibody (BioXcell, BE2097) was also treated in the same manner. After washing with FACS buffer, each tube was treated with anti-human IgG PE (Biolegend) and treated at 4°C for 45 minutes, washed with FACS buffer, and then FACSLyric TM was analyzed using .

[0212] As a result, as shown in Fig. 3, among the antibodies produced in Example 2, 2F1 and 2F1_LS#17 showed cell surface antigen binding affinity similar to that of the positive control antibody, and antibodies 2F1HS6LS#38, 2F1HS6LS#46, 2F1CDRH3#10, 2F1CDRH3#16, 2F1CDRH3#17, and 2F1CDRH3#26 selected through antibody optimization showed HLA-G binding affinity at least 3 times higher than that of the positive control antibody.

[0213]

[0214] Example 4.4. Antibody-dependent cytotoxicity evaluation

[0215] To determine whether the antibodies selected in Example 2 above bind to the HLA-G overexpressing cell line SKOV-3 / HLAG and induce cytotoxicity through the Fc of the antibodies, an antibody-dependent cytotoxicity evaluation was performed using an ADCC reporter bioassay kit (Promega).

[0216] SKOV-3 / HLA-G cells were seeded at 5x10 in 96-well white plates. 3 After plating with cells / well, they were cultured for 20 hours in a 37℃, 5% CO2 incubator. After removing the culture medium from the plate where SKOV-3 / HLA-G cells were plated, 25 ㎕ of ADCC effect medium (RPMI 1640 + 4% low IgG FBS) was added. The antibody was diluted 1 / 3 from 20 nM in ADCC effect medium and added to the SK-OV3 / HLA-G plate at 25 ㎕ / well, then the effect medium lid was closed and left to stand on a clean bench. After obtaining the ADCC reporter cells included in the kit, 4x10 6 After suspension at 100 cells / ml, 25 μl / well was added to a plate containing SKOV-3 / HLA-G cells and antibodies, and cultured for 6 hours in a 37°C, 5% CO2 incubator. After removing the plate and leaving it at room temperature for 15 minutes, 75 μl / well of luciferase substrate was added, and the reaction was carried out for 15 minutes under light blocking, and the luminescence was measured using a luminometer.

[0217] As a result, as shown in Fig. 8, the antibodies selected in Example 2 exhibited antibody-dependent cytotoxicity efficacy in a concentration-dependent manner by binding to HLA-G on the cell surface, and 2F1HS6LS#38, 2F1HS6LS#46, 2F1CDRH3#10, 2F1CDRH3#16, 2F1CDRH3#17, and 2F1CDRH3#26 all exhibited antibody-dependent cytotoxicity efficacy equivalent to or greater than that of the positive control antibody.

[0218]

[0219] Example 4.5. Analysis of increased cancer cell killing capacity by natural killer cells

[0220] In order to confirm whether the anti-HLA-G antibodies selected in Example 2 above bind to HLA-G of SKOV-3 / HLAG and prevent the binding to ILT-2 expressed on natural killer cells NK92-MI, thereby increasing the apoptotic ability of cancer cells, an analysis of the apoptotic ability of HLA-G-overexpressing SKOV-3 cells by natural killer cells NK92-MI was performed. SKOV-3 / HLA-G cell lines treated with the manual method of the kit (Cell Trace violet cell proliferation, Thermofisher scientific) were seeded in 96-well plates at 10 4 After treatment with cells / well, anti-HLA-G antibody and human IgG1 isotype control antibody (negative control) were diluted 1 / 5 from 50 nM and treated with 50 μl / well, and reacted for 1 hour in a 37°C, 5% CO2 incubator. After 1 hour, NK92-MI cell line was added to the wells treated with SKOV-3 / HLA-G cells and antibodies at a density of 5x10 4 After dispensing cells / well, the cells were incubated at 37°C and 5% CO for 24 hours. Then, TrypLE TM Express (Gibco) was used to transfer cells in the well to a FACS tube, washed with FACS buffer (1,000 rpm, 3 min), and the supernatant was removed. 7-AAD staining solution was diluted 1:20 in FACS buffer, added at 100 ㎕ / tube, and stained for 20 min at 4°C with FACSLyric TMwas analyzed using . At this time, in order to gate only the target cells stained with CellTrace Violet among the sample where target cells and effector cells were mixed, the range of cells positive for BV420 was specified among the entire FSC / SSC distribution and gated. In order to confirm the ratio of dead cells among the gated target cells, only the distribution of cells positive for 7-AAD was gated, and then the ratio of cells positive for 7-AAD (P3) among target cells (P2) was calculated (% of Lysis = [P3 cell number] / [P2 cell number] x 100). Since the spontaneous lysis of each target cell was less than 5%, it was not included in the calculation formula when calculating the cytotoxicity result.

[0221] As a result, as can be seen in Fig. 4, the cancer cell killing ability of 2F1, 2F1_LS#17 showed a similar level to that of the positive control antibody, and 2F1HS6LS#38, 2F1HS6LS#46, 2F1CDRH3#10, 2F1CDRH3#16, 2F1CDRH3#17, 2F1CDRH3#26 showed excellent efficacy with the cancer cell killing ability increased by about 2 to 10 times compared to the positive control antibody.

[0222]

[0223] Example 4.6. Analysis of increased cancer cell killing capacity by natural killer cells in various indications.

[0224] We confirmed whether the selected anti-HLA-G antibody 2F1CDRH3#10 binds to HLA-G expressed in various cancers and prevents the binding to ILT-2 expressed on NK-92MI natural killer cells, thereby increasing the cancer cell killing ability of natural killer cells. Pancreatic cancer cells HuP-T3, gastric cancer cells SCH, bladder cancer cells, lung cancer cells, and triple-negative breast cancer cells naturally expressing HLA-G were dispensed into 96-well plates at a density of 10,000 cells per well, treated with 10 nM of anti-HLA-G antibody, and reacted for 1 hour in a 5% CO2 incubator at 37°C. NK-92MI cell line was added to the wells treated with bladder cancer cells and pancreatic cancer cells at a density of 2 x 10 4 cells / well, 5x10 in the wells treated with gastric cancer cells and lung cancer cells. 4 After dispensing 5,000 cells / well into each well treated with triple-negative breast cancer cells, the cells were reacted in a 37°C, 5% CO2 incubator for 24 hours. The process until calculating the percentage of dead cells was performed as in Example 4.5 above.

[0225] As a result, as can be seen in Figure 5, the natural killer cell induction effect of 2F1CDRH3#10 was confirmed in various cancer types through the results.

[0226]

[0227] Example 4.7. Analysis of antibody-dependent phagocytosis by macrophages

[0228] We confirmed whether the selected anti-HLA-G antibody 2F1CDRH3#10 binds to HLA-G expressed on HLA-G overexpressing cell lines and induces macrophage phagocytosis. THP-1 macrophages and HLA-G overexpressing target cells were stained using the Cell Trace violet cell proliferation (Thermofisher scientific) and Cell Trace CFSE cell proliferation (Thermofisher scientific) fluorescent staining kits, respectively. 10,000 cells per well of each cell were added to a 96-well plate and mixed. 2F1CDRH3#10 and Human IgG1 isotype control antibodies were diluted 1 / 10 from 10 nM and then treated in the 96-well plate where macrophages and target cells were co-cultured, and then cultured in a 5% CO2 incubator at 37°C for 24 hours. TrypLE TM After transferring the cells in the well to a FACS tube by treating with Express (Gibco), they were washed with FACS buffer (1,000 rpm, 3 minutes). FACSLyric TM The proportion of macrophages expressing violet and CFSE simultaneously was confirmed using .

[0229] As a result, as shown in Fig. 6, the antibody-dependent phagocytic efficacy of 2F1CDRH3#10 was confirmed.

[0230]

[0231] Example 5. Evaluation of in vivo biological activity of anti-HLA-G antibodies

[0232] An in vivo xenograft mouse model was created by injecting SCH gastric cancer cell line and HuP-T3 pancreatic cancer cell line into 6-week-old BALB / C nude mice. Tumor size 100 mm 3Below, antibodies were administered twice a week via intravenous injection at a dose of 30 mg / kg / time, and the changes in tumor volume according to antibody administration were measured and shown in Table 7 below.

[0233] As a result, as shown in Table 7 and Figure 7 below, 2F1HS6LS#38 exhibited tumor growth inhibition in both mouse models, and showed a statistically significant difference from the negative control group.

[0234] Mouse model tumor growth inhibition (TGI, %) pancreatic cancer cell line 75.38 gastric cancer cell line 75

[0235]

[0236] Example 6. Evaluation of antibody-dependent cytotoxicity of afucosylated antibodies

[0237] In order to induce afucosylation of antibodies, 2-FF (2-deoxy-2-fluro-L-Fucose), a fucose derivative, was added to the culture medium at a concentration of 400 μM during production of the antibodies selected in Example 1.4, and the cultures were purified to confirm the ADCC effect. Antibody-dependent cytotoxicity evaluation (ADCC) was performed in the same manner as in Example 5.3.

[0238] As a result, as shown in FIG. 8 and Table 8 below, the ADCC efficacy of anti-HLA-G antibodies increased by about 6 to 15 times as afucosylation progressed, and the efficacy of the six afucosylated ADCC antibodies selected and produced in Example 1.4 was 2 to 10 times higher than that of the afucosylated positive control antibody.

[0239] EC50 (nM) 2F1CDRH3 #10 2F1CDRH3 #16 2F1CDRH3 #17 2F1CDRH3 #26 2F1HS6LS #38 2F1HS6LS #46 Positive control antibody (Ref ab) IgG 0.08 0.17 0.07 0.10 0.93 0.15 1.59 Nonfucosylated 0.01 0.02 0.01 0.02 0.07 0.02 0.13

[0240] Example 7. Analysis of HLA-G and ILT-2 binding inhibition ability of 2F1.eP

[0241] To confirm the difference in the binding inhibition ability of 2F1 and 2F1.eP to HLA-G and ILT-2, the binding inhibition ability of anti-HLA-G was analyzed using recombinant HLA-G antigen and JEG-3 cell line. The binding inhibition ability analysis of recombinant antigen HLA-G and ILT-2 was performed in the same manner as in Example 5.1, and the binding inhibition ability analysis of HLA-G and ILT-2 using JEG-3 cell line was confirmed using FACS. JEG-3 cells 2x10 5 Cells / tubes / 50 ㎕ were suspended using stain buffer and blocked on ice for 30 minutes. Recombinant human ILT-2, mouse Fc-tagged protein was diluted to a 4X concentration of 1.5 ㎕ and treated with 25 ㎕ each, and pre-incubated on ice for 30 minutes. After that, 25 ㎕ each of 2F1 and 2F1.eP were treated using stain buffer and reacted on ice for 1 hour. After adding 1 mL of stain buffer, the washing process was repeated three times using centrifugation (1,000 rpm, 3 minutes). The cell pellet was resuspended in 100 μL stain buffer and incubated with goat anti-mouse IgG Fc cross-adsorbed secondary antibody, PE (1:100) on ice for 45 min. After washing twice with 1 mL stain buffer, the cells were analyzed using BD FACSLyric™.

[0242] As a result, as shown in Fig. 9, it was confirmed that not only 2F1 but also 2F1.eP showed significant ILT-2 binding inhibition ability.

[0243]

[0244] Example 8. Analysis of HLA-subtype selectivity of anti-HLA-G antibodies

[0245] The binding of the antibodies obtained in Example 2 to HLA subtype antigens (97 different types of antigens including alleles of HLA-A, B, and C) was confirmed using a kit (Labscreen single antigen HLA-class I-combi, One Lambda). 1000 nM of antibody was added to the beads, incubated for 45 minutes under light blocking, and washed twice (1300 g, room temperature, 5 minutes) with 1X wash buffer using centrifugation. Each sample was treated with anti-human IgG PE, incubated for 30 minutes at room temperature under light blocking, washed with 1X wash buffer, and analyzed using Luminex.

[0246] As a result, as shown in Fig. 10, all antibodies obtained in Example 2 were observed to have an MFI of 1000 or less, which is the cut-off standard value of the kit (Labscreen single antigen HLA-class 1-combi), confirming selectivity for HLA-G.

[0247]

[0248] Example 9. In vitro cytotoxicity evaluation of ADC

[0249] The cell proliferation inhibition activity of the antibody-drug-conjugates described in Table 9 below was measured in a pancreatic cancer cell line expressing HLA-G. After seeding 1,000 cells per well in a 96-well plate, the cancer cell lines were treated with the compounds at concentrations of 0.256 pM to 100 nM (5-fold serial dilution) 24 hours later, and the cancer cell lines were cultured for 144 hours, and the cell proliferation was measured using the CTG (cell titer glo, Promega) assay. As a result, the CC50 of the antibody-drug conjugate 2F1CDRH3#26-MMAE in the pancreatic cancer cell line was measured to be 0.83 nM.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to HLA-G (human leukocyte antigen G).

2. In paragraph 1, The above antibody or antigen-binding fragment thereof, A heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, 17, 33, 49, 64, 79, 94, 109, 125, 140, 156, 172 or 188; A heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, 18, 34, 50, 65, 80, 95, 110, 126, 141, 157, 173, or 189; and An antibody or antigen-binding fragment thereof comprising a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3, 19, 35, 51, 66, 81, 96, 111, 127, 142, 158, 174 or 190.

3. In paragraph 1, The above antibody or antigen-binding fragment thereof, A light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, 20, 36, 52, 67, 82, 97, 112, 128, 143, 159, 175 or 191; a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, 21, 37, 53, 68, 83, 98, 113, 129, 144, 160, 176 or 192; and An antibody or antigen-binding fragment thereof comprising a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6, 22, 38, 54, 69, 84, 99, 114, 130, 145, 161, 177 or 193.

4. In paragraph 1, The above antibody or antigen-binding fragment thereof, An antibody or antigen-binding fragment thereof comprising a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 15, 31, 47, 123, 154, 170, 186 or 202.

5. In paragraph 1, The above antibody or antigen-binding fragment thereof, An antibody or antigen-binding fragment thereof comprising a light chain variable region represented by the amino acid sequence of SEQ ID NO: 16, 32, 48, 63, 78, 93, 108, 124, 139, 155, 171, 187 or 203.

6. A nucleic acid encoding the antibody of paragraph 1 or an antigen-binding fragment thereof.

7. A vector containing the nucleic acid of paragraph 6.

8. An isolated host cell transformed with the vector of paragraph 7.

9. A method for producing the antibody or antigen-binding fragment thereof of claim 1, comprising the step of introducing the vector of claim 7 into a host cell.

10. Antibody-drug conjugate (ADC) comprising the antibody of paragraph 1 or an antigen-binding fragment thereof.

11. A fusion protein comprising the antibody of paragraph 1 or an antigen-binding fragment thereof.

12. A pharmaceutical composition for preventing or treating cancer, comprising the antibody of paragraph 1 or an antigen-binding fragment thereof.

13. A composition for diagnosing cancer, comprising the antibody of paragraph 1 or an antigen-binding fragment thereof.

14. A kit for diagnosing cancer, comprising the antibody of paragraph 1 or an antigen-binding fragment thereof.

15. A method for providing information for cancer diagnosis, comprising the following steps: A step of detecting HLA-G protein through an antigen-antibody reaction or measuring the level of HLA-G protein expression or activity in a biological sample isolated from a subject suspected of having cancer using the antibody of claim 1 or an antigen-binding fragment thereof.

16. A method of providing information for cancer treatment, comprising the following steps: A step of detecting HLA-G protein through an antigen-antibody reaction or measuring the level of HLA-G protein expression or activity in a biological sample isolated from a subject suspected of having cancer using the antibody of claim 1 or an antigen-binding fragment thereof.

17. A method for screening an anticancer agent, comprising the following steps: (a) a step of treating cancer cells with an anticancer drug candidate; (b) a step of measuring the level of HLA-G protein expression or activity in cancer cells treated with the anticancer agent candidate using the antibody of claim 1 or an antigen-binding fragment thereof; and (c) A step of determining that the anticancer drug candidate treated in step (a) is an anticancer drug if the expression level or activity of the HLA-G protein in step (b) is lower than that of cancer cells not treated with the anticancer drug candidate.

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