Anti-LILRB1 antibodies and uses thereof

Abstract

To provide: an anti-LILRB1 antibody having increased specificity for LILRB1, and uses thereof; and specifically, an anti-LILRB1 antibody or antigen-binding fragment thereof, and uses thereof in treating cancer.SOLUTION: This disclosure provides: antibodies which bind to LILRB1, act on LILRB1-expressing immune cells, regulate activity of the immune cells and exhibit anti-cancer effect; and uses thereof in treating cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority based on Korean Patent Application No. 10-2019-0173414 filed on December 23, 2019, and Korean Patent Application No. 10-2020-0061907 filed on May 22, 2020, and all contents disclosed in the documents of said Korean patent applications are incorporated herein by reference.

[0002] The present invention relates to an anti-LILRB1 antibody and uses thereof, specifically, to an anti-LILRB1 antibody or an antigen-binding fragment thereof, and uses thereof in cancer treatment. [Background technology]

[0003] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1, also known as ILT2, CD85j, LIR-1) is an inhibitory receptor expressed on B cells, T cells, NK cells, dendritic cells, macrophages, and other immune cells. LILRB1 binds to classical and non-classical MHC class-I and is involved in the signal transduction mechanism that suppresses the activity of immune cells.

[0004] On the other hand, it is known that various cancer cells overexpress MHC class I, such as HLA-G, to evade the immune system. Inhibiting the binding of LILRB1 to MHC class I is expected to restore the activity of suppressed immune cells and show anti-cancer effects.

[0005] Therefore, there is a need to develop new drugs that bind to LILRB1 and inhibit the binding of LILRB1 to MHC class I and / or inhibit the interaction of LILRB1 with MHC class I. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides an antibody that binds to LILRB1, acts on immune cells expressing LILRB1, and regulates the activity of the immune cells to exhibit anti-cancer efficacy, and uses thereof in cancer treatment.

[0007] One embodiment provides an anti-LILRB1 antibody or antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or antigen-binding fragment thereof may have activity of inhibiting binding of LILRB1 to MHC class I and / or activity of inhibiting interaction of LILRB1 with MHC class I. The anti-LILRB1 antibody or antigen-binding fragment thereof may have activity of inhibiting immune evasion of cancer cells. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have excellent anti-cancer activity. The cancer may express or overexpress MHC class I on its surface.

[0008] Another embodiment provides a pharmaceutical composition for treating and / or preventing cancer, comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof.

[0009] Another embodiment provides a pharmaceutical composition for inhibiting the binding of LILRB1 to MHC class I and / or a pharmaceutical composition for inhibiting the interaction between LILRB1 and MHC class I, comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof.

[0010] Another embodiment provides a pharmaceutical composition for inhibiting immune evasion of cancer cells, comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof. [Means for solving the problem]

[0011] One embodiment of the present invention provides an anti-LILRB1 antibody or antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or antigen-binding fragment thereof may have activity of inhibiting the binding of LILRB1 to MHC class I and / or inhibiting the interaction of LILRB1 with MHC class I. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have activity of inhibiting immune evasion of cancer cells. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have excellent anti-cancer activity.

[0012] The anti-LILRB1 antibody or antigen-binding fragment thereof may comprise the following complementarity determining regions (CDRs): (1) Based on the CDR definition by Kabat numbering (Kabat, EA, Wu, TT, Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242; http: / / www.abysis.org / ), CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79, 85, 91, 97, 103, 109 or 115; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, 8, 14, 20, 26, 32, 38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110 or 116; CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111 or 117; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 100, 106, 112 or 118; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77, 83, 89, 95, 101, 107, 113 or 119, and or comprising a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, or 120; (2) Based on the CDR definition by IMGT numbering (http: / / www.imgt.org / ), CDR-L1 comprising the amino acid sequence of SEQ ID NO: 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191, 196, 201, 206, 211 or 216; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 122, 127, 132, 137, 142, 147, 152, 157, 162, 167, 172, 177, 182, 187, 192, 197, 202, 207, 212 or 217; CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75, 81, 87, 93, 99, 105, 111 or 117; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 123, 128, 133, 138, 143, 148, 153, 158, 163, 168, 173, 178, 183, 188, 193, 198, 203, 208, 213 or 218; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 124, 129, 134, 139, 144, 149, 154, 159, 164, 169, 174, 179, 184, 189, 194, 199, 204, 209, 214 or 219, and It may comprise a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215 or 220.

[0013] In one embodiment, the combinations of six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) that can be included in the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein are shown in Table 1 below: [Table 1-1] [Table 1-2] [Table 1-3]

[0014] In one example, the anti-LILRB1 antibody or antigen-binding fragment thereof is a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3; and It may comprise a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3.

[0015] In embodiments, the anti-LILRB1 antibody or antigen-binding fragment thereof is a light chain variable region comprising the amino acid sequence of SEQ ID NO: 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, or 345; and The heavy chain variable region may comprise the amino acid sequence of SEQ ID NO: 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258 or 260.

[0016] Combinations of light chain variable regions and heavy chain variable regions that can be included in the anti-LILRB1 antibodies or antigen-binding fragments thereof provided herein are shown in Table 2 below: [Table 2-1] [Table 2-2] [Table 2-3]

[0017] As used herein, an antibody or antigen-binding fragment thereof (e.g., CDR, variable region, or heavy chain / light chain) "comprises, consists of, or is expressed by a specific amino acid sequence" can mean both cases where the amino acid sequence is necessarily contained, and cases where meaningless mutations (e.g., substitution, deletion, and / or addition of amino acid residues) that do not affect antibody activity have been introduced into the amino acid sequence.

[0018] The anti-LILRB1 antibodies or antigen-binding fragments thereof provided herein have a binding affinity (K D) may be, for example, 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM, or 0.15 mM or less based on the value measured by surface plasmon resonance (SPR), and may be, for example, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM M~10mM, 1nM~10mM, 0.001nM~5mM, 0.005nM~5mM, 0.01nM~5mM, 0.05nM~5mM, 0.1nM~5mM, 0 .5nM~5mM, 1nM~5mM, 0.001nM~1mM, 0.005nM~1mM, 0.01nM~1mM, 0.05nM~1mM, 0.1nM~1mM, 0 .5nM~1mM, 1nM~1mM, 0.001nM~0.5mM, 0.005nM~0.5mM, 0.01nM~0.5mM, 0.05nM~0.5mM, 0. 1nM~0.5mM, 0.5nM~0.5mM, 1nM~0.5mM, 0.001nM~0.2mM, 0.005nM~0.2mM, 0.01nM~0.2mM, 0 It can be 0.05nM to 0.2mM, 0.1nM to 0.2mM, 0.5nM to 0.2mM, 1nM to 0.2mM, 0.001nM to 0.15mM, 0.005nM to 0.15mM, 0.01nM to 0.15mM, 0.05nM to 0.15mM, 0.1nM to 0.15mM, 0.5nM to 0.15mM, or 1nM to 0.15.

[0019] Another example provides a pharmaceutical composition comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient. For example, the pharmaceutical composition may be a pharmaceutical composition for treating and / or preventing cancer. The pharmaceutical composition may have activity of inhibiting the binding of LILRB1 to MHC class I and / or the interaction of LILRB1 with MHC class I. The cancer may be a cancer associated with the interaction of LILRB1 with MHC class I. In one example, the pharmaceutical composition may have activity of inhibiting immune evasion of cancer cells. The cancer cells may express or overexpress MHC class I on their surface.

[0020] Another example provides a composition for inhibiting the binding of LILRB1 to MHC class I and / or a composition for inhibiting the interaction between LILRB1 and MHC class I, comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0021] Another embodiment provides a composition for inhibiting immune evasion of cancer cells, comprising the anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0022] Another example provides a method for treating and / or preventing cancer, comprising administering (orally or parenterally) a pharma- tically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject (e.g., a mammal, including a human) in need of such treatment and / or prevention.

[0023] Another example provides a method for inhibiting the binding of LILRB1 to MHC class I and / or the interaction of LILRB1 with MHC class I, comprising administering (orally or parenterally) a pharma- ceutical effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject (e.g., a mammal, including a human) in need of inhibition of the binding of LILRB1 to MHC class I and / or the interaction of LILRB1 with MHC class I.

[0024] Another example provides a method for inhibiting immune evasion of cancer cells, comprising administering (orally or parenterally) a pharma- tically effective amount of the anti-LILRB1 antibody or antigen-binding fragment thereof to a subject (e.g., a mammal, including a human) in need of inhibiting immune evasion of cancer cells.

[0025] The methods provided herein may further include, prior to the administering step, a step of identifying a subject in need of treatment and / or prevention of cancer, a subject in need of inhibition of binding of LILRB1 to MHC class I and / or inhibition of the interaction of LILRB1 with MHC class I, and / or a subject in need of inhibition of immune evasion of cancer cells.

[0026] Another example provides a nucleic acid molecule (polynucleotide) encoding one or more polypeptides selected from the group consisting of the CDRs of the anti-LILRB1 antibody (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, or a combination of CDR-L1, CDR-L2, and CDR-L3, or a combination of CDR-H1, CDR-H2, and CDR-H3); a light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3; a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3; a light chain comprising the light chain variable region; and a heavy chain comprising the heavy chain variable region.

[0027] Another example provides a recombinant vector comprising the nucleic acid molecule. In one example, the recombinant vector can comprise the light chain variable region or light chain encoding nucleic acid molecule and the heavy chain variable region or heavy chain encoding nucleic acid molecule (e.g., in two vectors) respectively or together (e.g., in one vector). The recombinant vector can be used as an expression vector.

[0028] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0029] Another example provides a method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising expressing the nucleic acid molecule in a cell. Expressing the nucleic acid molecule can include culturing the recombinant cell.

[0030] The antigen-binding fragment of the anti-LILRB1 antibody described herein refers to a fragment derived from the anti-LILRB1 antibody and retains the binding ability to the antigen (LILRB1), and may be any polypeptide containing the six CDRs of the anti-LILRB1 antibody, such as, but not limited to, scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-Cλ (lambda constant region), (scFv)2, Fab, Fab', or F(ab')2. In one example, the antigen-binding fragment may be an scFv, or a fusion polypeptide (scFv-Fc) in which the scFv is fused to the Fc portion of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) or a fusion polypeptide (scFv-Ck or scFv-Cλ) in which the scFv is fused to a light chain constant region (e.g., kappa or lambda).

[0031] The anti-LILRB1 antibody or antigen-binding fragment thereof may have a regulatory effect, for example, antagonism or agonism, on LILRB1 protein. The anti-LILRB1 antibody or antigen-binding fragment thereof may have activity of inhibiting binding of LILRB1 to MHC class I and / or interaction of LILRB1 with MHC class I. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have activity of inhibiting immune evasion of cancer cells. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have excellent anti-cancer activity.

[0032] The LILRB1 that acts as an antigen for the antibodies or antigen-binding fragments provided herein can be of mammalian origin, for example, but is not limited to, human-derived LILRB1 (e.g., GenBank accession numbers AAH15731.1 (SEQ ID NO: 348), NP_001265328.2, NP_001265327.2, NP_001075108.2, NP_001075107.2, NP_001075106.2, NP_006660.4, NM_001081637.2, NM_001081638.3, NM_001081639.3, NM_001278398.2, NM_001278399.2, etc.).

[0033] The MHC class I described herein is a classification of major histocompatibility complex (MHC) molecules. In one example, the MHC class I may be of human origin and may be one or more selected from the group consisting of, but not limited to, human leukocyte antigen (HLA)-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0034] As used herein, the term "antibody" is a general term for a protein that specifically binds to a specific antigen, and may be a protein produced by antigen stimulation in the immune system or a protein produced by recombinant or chemical synthesis thereof, with no particular limitation on the type. The antibody may be a non-naturally produced antibody, for example, a recombinant or synthetically produced antibody. The antibody may be an animal antibody (e.g., a mouse antibody), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.

[0035] The heavy chain CDR and light chain CDR regions, or the remaining regions excluding the heavy chain variable region and the light chain variable region, defined above in the anti-LILRB1 antibody or antigen-binding fragment thereof provided herein may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), for example, from the framework regions, and / or the light chain constant region and / or the heavy chain constant region of any of the above subtypes of immunoglobulin. In one example, the anti-LILRB1 antibody provided herein may be a human IgG type antibody, for example, an IgG1, IgG2, IgG3, or IgG4 type antibody, but is not limited thereto.

[0036] A complete antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) type, and has a gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) or alpha 2 (α2) subclass. The light chain constant region has a kappa (κ) and lambda (λ) type.

[0037] The term "heavy chain" refers to a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three constant region domains C H1 , C H2 and C H3 The term "light chain" is intended to include a full-length heavy chain, including a hinge, and a fragment thereof. The term "light chain" is intended to include a variable region domain V that contains an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and constant region domain C L The term "light chain" is intended to include all full-length light chains and fragments thereof.

[0038] The term "CDR (complementarity determining region)" refers to a site in the variable site of an antibody that confers binding specificity to an antigen, and refers to the amino acid sequence of the hypervariable region of an immunoglobulin heavy chain and light chain. Each of the heavy chain and light chain may contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide the main contact residues for an antibody to bind to an antigen or epitope. Meanwhile, in this specification, the terms "specifically bind" and "specifically recognize" have the same meaning as commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact with each other to react immunologically.

[0039] In this specification, unless otherwise specified, an antibody can be understood to include not only a complete antibody but also an antigen-binding fragment of an antibody having antigen-binding ability.

[0040] The term "antigen-binding fragment" refers to any form of polypeptide that includes a portion capable of binding to an antigen (e.g., six CDRs as defined herein). For example, it may be, but is not limited to, an scFv, (scFv)2, scFvFc, Fab, Fab', or F(ab')2 of an antibody. As described above, the antigen-binding fragment may be an scFv, or a fusion polypeptide in which an scFv is fused to the Fc portion or light chain constant region (e.g., kappa or lambda) of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).

[0041] Among the antigen-binding fragments, Fab contains the variable regions of the light chain and the heavy chain, the constant region of the light chain, and the first constant region of the heavy chain (C H1 ) structure.

[0042] Fab' is the heavy chain C H1It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain.

[0043] F(ab')2 antibodies are produced by forming disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv is the minimum antibody fragment that contains only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art.

[0044] In a two-chain Fv, the heavy-chain variable domain and the light-chain variable domain are linked by a non-covalent bond, whereas in a single-chain Fv, the heavy-chain variable domain and the light-chain variable domain are generally linked by a covalent bond via a peptide linker or directly at the C-terminus, and thus can form a dimer-like structure similar to the two-chain Fv.

[0045] The antigen-binding fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be subjected to limited cleavage with papain to obtain Fab fragments, or pepsin to obtain F(ab')2 fragments) or can be produced using recombinant gene techniques.

[0046] The term "hinge region" refers to a region contained in an antibody heavy chain, which is located between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site in the antibody.

[0047] The anti-LILRB1 antibody may be a monoclonal antibody. The monoclonal antibody may be produced by a method known in the art. For example, the monoclonal antibody may be produced by using a phage display technique. Alternatively, the anti-LILRB1 antibody may be produced as a mouse-derived monoclonal antibody by a conventional method.

[0048] Meanwhile, individual monoclonal antibodies can be screened based on their binding ability to LILRB1 using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format. The binding bodies can be assayed for inhibitory activity by functional assays such as competitive ELISA to assay molecular interactions or cell-based assays. Then, the monoclonal antibody members selected based on their strong inhibitory activity can be assayed for their respective affinities (Kd values) for LILRB1.

[0049] The pharmaceutical composition provided herein may further comprise a pharma- ceutically acceptable carrier in addition to the active ingredient (anti-LILRB1 antibody or its antigen-binding fragment). The pharma- ceutically acceptable carrier is one that is commonly used in formulations, and may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. The pharmaceutical composition may also further comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like that are commonly used in the manufacture of pharmaceutical compositions.

[0050] The pharmaceutical composition or an effective amount of the antibody or antigen-binding fragment thereof can be administered orally or parenterally. Parenteral administration can be by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, or local administration to the lesion site. Since proteins or peptides are digested during oral administration, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. The composition can also be administered by any device that allows the active agent to be transferred to the target cells (e.g., cancer cells).

[0051] The anti-LILRB1 antibody or antigen-binding fragment thereof may be contained in the pharmaceutical composition or administered to a patient in a pharma- ceutical effective amount. As used herein, the term "pharma-ceutical effective amount" refers to the amount of the active ingredient (anti-LILRB1 antibody or antigen-binding fragment thereof) that can exert the intended effect (e.g., anti-cancer effect). The pharma-ceutical effective amount may be formulated in various ways depending on factors such as the patient's age, weight, sex, pathological condition, diet, excretion rate, reaction sensitivity, formulation method, administration time, administration interval, administration route, and administration method. For example, the daily dosage of the anti-LILRB1 antibody or antigen-binding fragment thereof may be 0.005 ug / kg to 1000 mg / kg, 0.005 ug / kg to 500 mg / kg, 0.005 ug / kg to 250 mg / kg, 0.005 ug / kg to 100 mg / kg, 0.005 ug / kg to 75 mg / kg, 0.005 ug / kg to 50 mg / kg, 0.01 ug / kg to 1000 mg / kg, 0.01 ug / kg to 500 mg / kg, 0.01 ug / kg to 2 The daily dose may be in the range of, but is not limited to, 50 mg / kg, 0.01 ug / kg to 100 mg / kg, 0.01 ug / kg to 75 mg / kg, 0.01 ug / kg to 50 mg / kg, 0.05 ug / kg to 1000 mg / kg, 0.05 ug / kg to 500 mg / kg, 0.05 ug / kg to 250 mg / kg, 0.05 ug / kg to 100 mg / kg, 0.05 ug / kg to 75 mg / kg, or 0.05 ug / kg to 50 mg / kg. The daily dose may be formulated in a single formulation in a unit dose form, or may be appropriately divided and formulated, or may be prepared in a multi-dose container.

[0052] The pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated into the form of an extract, powder, powder, granule, tablet or capsule, etc., and may further contain a dispersing agent or stabilizer for formulation.

[0053] The subject patient of the present invention may be a mammal, including humans, primates including monkeys, and rodents including mice and rats.

[0054] The cancer may be a solid cancer or a blood cancer, including, but not limited to, one or more selected from the group consisting of lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), peritoneal cancer, skin cancer, skin or intraocular melanoma, colon cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, leukemia (e.g., chronic or acute leukemia), lymphoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, renal cell carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, osteosarcoma, etc. The cancer may be a primary cancer or a metastatic cancer. The cancer may be one in which MHC class I is expressed or overexpressed on the surface, such as colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma, bone osteosarcoma, renal cell carcinoma, and gastric cancer. Overexpression of MHC class I refers to overexpression in cancer cells to which the antibody is applied, compared to normal cells or cancer cells that do not show anti-cancer effects in immunotherapy, such as T-cell (e.g., cytotoxic T-cell)-mediated immunotherapy (non-responsive or resistant to the immunotherapy).

[0055] As used herein, the treatment of cancer refers to any anti-cancer effect that prevents, alleviates or improves the worsening of symptoms of cancer, such as inhibition of cancer cell proliferation, annihilation of cancer cells, and inhibition of metastasis, or that partially or completely eradicates cancer.

[0056] The anti-LILRB1 antibody or antigen-binding fragment thereof provided herein can be used in combination with other drugs, for example, one or more selected from the group consisting of commonly available immunotherapeutic agents, anticancer agents, cytotoxic agents, etc. Thus, as one example, there is provided a pharmaceutical composition for combined administration for preventing and / or treating cancer, comprising (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) one or more drugs selected from the group consisting of immunotherapeutic agents, anticancer agents, cytotoxic agents, etc. As another example, there is provided a method for preventing and / or treating cancer, comprising the step of administering (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) one or more drugs selected from the group consisting of immunotherapeutic agents, anticancer agents, cytotoxic agents, etc. to a patient in need of cancer prevention and / or treatment. The immunotherapeutic agents, anticancer agents and cytotoxic agents are generally used in cancer treatment and / or include any drug with cytotoxic activity, and may be selected from one or more of proteins such as antibodies, nucleic acid molecules such as siRNA, and / or small molecule compounds such as paclitaxel, docetaxel, etc., but are not limited to these.

[0057] As another example, a polypeptide molecule is provided that includes the heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), the light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof) of the anti-LILRB1 antibody described above, or a combination thereof; or the heavy chain variable region, the light chain variable region, or a combination thereof. The polypeptide molecule is not only used as a precursor of an antibody for antibody production, but is also included as a component of a protein scaffold (e.g., a peptibody) having a structure similar to that of an antibody, a bispecific antibody, or a multispecific antibody. As yet another example, the polypeptide molecule can be used as a target (antigen) recognition portion in a targeted cell therapy such as CAR-T, a secreted antibody, or a cell therapy produced to secrete the anti-LILRB1 antibody.

[0058] As another example, a nucleic acid molecule encoding a heavy chain complementarity determining region (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), a heavy chain variable region, or a heavy chain of an anti-LILRB1 antibody is provided.

[0059] As another example, a nucleic acid molecule encoding the light chain complementarity determining region (CDR-L1, CDR-L2, CDR-L3, or a combination thereof), the light chain variable region, or the light chain of an anti-LILRB1 antibody is provided.

[0060] As another example, there is provided a recombinant vector that contains a nucleic acid molecule encoding the heavy chain complementarity determining region, heavy chain variable region or heavy chain of the anti-LILRB1 antibody and a nucleic acid molecule encoding the light chain complementarity determining region, light chain variable region or light chain of the anti-LILRB1 antibody together in one vector or each in a separate vector.

[0061] In another embodiment, a recombinant cell comprising the nucleic acid molecule or recombinant vector is provided.

[0062] The term "vector" refers to an expression means for expressing a gene of interest in a host cell. Examples include plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, lentivirus vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. The vectors that can be used for the recombinant vector can be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.) or viruses (e.g., SV40, etc.) that are frequently used in the art.

[0063] In the recombinant vector, the nucleic acid molecule can be operatively linked to a promoter. The term "operatively linked" refers to a functional connection between a nucleotide expression control sequence (e.g., a promoter sequence) and another nucleotide sequence. The control sequence can be "operatively linked" to control the transcription and / or translation of the other nucleotide sequence.

[0064] The recombinant vector can typically be constructed as a vector for cloning or a vector for expression. The expression vector can be a vector commonly used in the art for expressing foreign proteins in plants, animals, or microorganisms. The recombinant vector can be constructed by various methods known in the art.

[0065] The recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter (e.g., pL λ Generally, the vector contains a ribosome binding site for initiation of translation and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the replication origin contained in the vector that operates in a eukaryotic cell includes, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, and a BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, a tk promoter of HSV, etc.) is used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0066] The recombinant cell may be obtained by introducing the recombinant vector into a suitable host cell. The host cell may be any host cell known in the art that stably and continuously clones or expresses the recombinant vector. Examples of prokaryotic cells include Escherichia coli such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, and E. coli W3110, Bacillus sp. strains such as Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In the case of transformation into eukaryotic cells, examples of host cells include yeast (Saccharomyces cerevisiae), insect cells, plant cells, and animal cells such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO Cell lines that may be used include, but are not limited to, GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines.

[0067] The nucleic acid molecule or a recombinant vector containing the same can be introduced (transfected) into a host cell by a delivery method well known in the art, for example, when the host cell is a prokaryotic cell, the CaCl2 method or the electroporation method can be used, and when the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome-mediated transfection method, the gene bombardment method, etc. can be used, but are not limited to these.

[0068] The method of selecting the transformed host cell can be easily carried out by a method widely known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0069] As another example, there is provided a method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising expressing the nucleic acid molecule or a recombinant vector comprising the same in a host cell. The expressing step can be performed by culturing a recombinant cell comprising the nucleic acid molecule (e.g., contained in a recombinant vector) under conditions that permit expression of the nucleic acid molecule. The method can include isolating and / or purifying the antibody or antigen-binding fragment from the culture medium after the expressing or culturing step. Effect of the Invention

[0070] The anti-LILRB1 antibody or antigen-binding fragment thereof provided by the present invention can exhibit excellent anti-cancer activity by inhibiting the immune evasion mechanism of cancer cells and allowing immune cells to exert their anti-cancer efficacy well without inhibition. [Brief description of the drawings]

[0071] [Figure 1] 1 is an electrophoretic photograph showing the results of SDS-PAGE gel analysis of purified anti-LILRB1 antibody in one embodiment. [Diagram 2] FIG. 1 shows a surface plasmon resonance (SPR) sensorgram result for anti-LILRB1 antibody B3 according to one embodiment. [Diagram 3] FIG. 1 shows SPR sensorgram results for anti-LILRB1 antibody E3 according to one embodiment. [Figure 4a] 1 is a graph showing the binding ability of anti-LILRB1 antibody A10 according to one embodiment to KHYG-1 cells, which are human NK cells. [Figure 4b] 1 is a graph showing the binding ability of anti-LILRB1 antibody E3 according to one embodiment to KHYG-1 cells, which are human NK cells. [Figure 4c]Fig. 1 is a graph showing the binding affinity of the human IgG4 isotype control antibody to KHYG-1 cells, which are human NK cells. [Diagram 5] 1 is a graph showing the results of analyzing the degree of binding of recombinant LILRB1-Fc protein to the surface of HLA-G-overexpressing cells when treated with an anti-LILRB1 antibody according to one embodiment and a human IgG4 isotype control antibody using iQue screener. [Figure 6] 1 is a graph showing the in vivo anti-tumor effect of anti-LILRB1 antibodies E3 and B3 according to one embodiment. [Figure 7a] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody E3.1 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 7b] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody E3.1 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 7c] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody E3.1 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 7d] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody E3.1 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 8a] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody H11 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 8b] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody H11 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 8c]1 is a flow cytometry diagram showing that anti-LILRB1 antibody H11 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 8d] 1 is a flow cytometry diagram showing that anti-LILRB1 antibody H11 according to one embodiment binds to various human LILR family overexpressing cells. [Figure 9] 1 is a graph showing the amount of granzyme B secreted in KHYG-1 cells, which are human NK cells, when treated with anti-LILRB1 antibodies E3.1 or H11 according to one embodiment, in comparison with a control antibody (human IgG4 isotype). [Figure 10] 1 is a graph showing the amount of perforin secretion in human NK cells, KHYG-1 cells, upon treatment with anti-LILRB1 antibodies E3.1 or H11 according to one embodiment, in comparison with a control antibody (human IgG4 isotype). [Figure 11] 1 is a graph showing the results of a luciferase reporter assay for evaluating the ability of anti-LILRB1 antibodies E3.1 or H11 according to one embodiment to inhibit LILRB1 signaling. [Figure 12] 1 is a graph showing the in vivo anti-tumor effect of anti-LILRB1 antibody E3.1 or H11 according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It will be obvious to those skilled in the art that the examples described below can be modified without departing from the essential gist of the invention.

[0073] Example 1: Production of human antibodies against LILRB1 1.1. Selection of human antibodies against LILRB1 using phage display To select antibodies that specifically recognize human LILRB1, a phage display selection method was performed using a library of human scFv antibodies. The antigens used were human LILRB1-His (Cat. No. 8989-T2) and human LILRB1-Fc (Cat. No. 2017-T2) manufactured by RnD systems. Each antigen was conjugated with biotin using the EZ-Link Sulfo-NHS-Biotin kit (ThermoFisher Scientific).

[0074] Phage display selection was performed using a total of four forms of LILRB1 antigen (LILRB1-His, LILRB1-Fc, LILRB1-His-Biotin, LILRB1-Fc-Biotin) using solid-phase and solution-phase selection methods. Additional selection was performed by gradually decreasing the antigen concentration used, competitive elution using a control antibody against LILRB1, or negative selection against Fc when LILRB1-Fc was used as the antigen. The selected products were confirmed to bind to the antigen by polyclonal phage ELISA.

[0075] 1.2. Monoclonal soluble scFv screening and analysis Genes encoding scFvs confirmed to bind to antigens in Example 1.1 were amplified by PCR to prepare expression vectors. For screening of each selection group, a certain number of transformants were transferred to a 96-well culture plate. Antibodies in the form of scFvs were expressed using autoinduction media (Studier, FW (2005) Protein Expression and Purification 41, 207-34), and then DELFIA immune assay (PerkinElmer) was performed to confirm the binding to the antigen. In addition, a certain amount of scFv antibodies was coated on the surface, and DELFIA was performed on the antigens to determine the ranking of antigen-antibody binding strength.

[0076] 1.3. Conversion of selected scFv antibodies to IgG antibodies A total of 376 clones were selected from the clones confirmed to bind to the antigen in Example 1.2, and the nucleic acid sequences of the genes encoding the selected scFvs were analyzed by a general DNA sequence analysis method to remove duplicated clones. In addition, a total of 93 clones were selected based on the ranking of antigen-antibody binding strength determined in Example 1.2. From the gene encoding the selected scFv, sequences corresponding to the heavy chain variable region (VH) and light chain variable region (VL) were amplified by PCR and inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen, or any vector having a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and containing the heavy chain constant region of human IgG4 and the constant region sequence of kappa or lambda light chain) constructed to encode a human antibody in the form of IgG4 (IgG4 Fc: SEQ ID NO: 341, Kappa constant region: SEQ ID NO: 342, Lambda constant region: SEQ ID NO: 343). The DNA sequence of the expression vector was confirmed by sequencing.

[0077] 1.4. Production of Selected Antibodies The vector constructed in Example 1.3 was purified using a Plasmid Plus Maxi kit (Qiagen). The vector thus purified was then transformed into ExpiCHO-S TM Cells or Expi293 TM The cells were used for antibody expression.

[0078] Specifically, the vector constructed in Example 1.3 was used as ExpiCHO-S TM Cells (Gibco) (1.5×10 8 cells / Culture Volume 25mL) with ExpiFectamine TMCHO reagent (Thermo Fisher) (80 ul) was added and transfection was performed. One day after transfection, ExpiCHO TM Enhancer (Thermo Fisher) 150ul and ExpiCHO TM Feed (Thermo Fisher) 4 mL was added. On the fifth day, ExpiCHO TM Feed (4 mL) was added. The transfected cells were cultured at 32°C and 5% CO2 for a total of 7 to 11 days.

[0079] In addition, the vector constructed in Example 1.3 was used as Expi293F TM Cells (Gibco) (3×10 8 cells / Culture Volume 100mL) with ExpiFectamine TM 320 ul of 293 Reagent (Gibco) was added and transfection was performed according to the manufacturer's instructions. One day after transfection, ExpiFectamine TM 0.6 mL of 293 Enhancer1 (Thermo Fisher) per 100 mL of culture volume, ExpiFectamine TM Enhancer2 (Thermo Fisher) was added at 6 mL per 100 mL culture volume, and glucose was added at 3.6 g per liter. The transfected cells were cultured at 36.5°C and 5% CO2 for a total of 5 days.

[0080] The two cultured cells were centrifuged at 4000 rpm for 20 minutes at 4°C, and then filtered using a 0.22um bottle-top filter system (Corning). The collected culture fluid was purified using AKTA Pure L (GE Healthcare). A Hitrap MabSelectSure 1mL column (GE Healthcare) was attached to the AKTA Pure L, and the culture fluid was passed through the column at a flow rate of 1mL / min, followed by washing with 20 column volumes (CV) of 1X PBS. The target protein was eluted by passing an elution buffer (0.1M sodium citrate pH3.4 buffer). The eluate was concentrated using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, and then buffer exchange was performed with 1xPBS buffer solution.

[0081] The purified antibody sample was diluted with 1X PBS to approximately 1mg / mL. 20ul of the purified antibody sample was mixed with 10ul of Reducing Loading Buffer (3X) or Non-reducing Loading Buffer (3X), and then placed in a 95℃ heating bath for 2 minutes, after which it was removed and cooled. SDS-PAGE Gradient Gel (4-20% or 4-12%) was placed in an electrophoresis apparatus, and 10μg of sample was injected per well and the gel was developed. Precision Plus Protein Ion Imaging was used to analyze the molecular weight of the sample. TM Dual Color Standards (BIO-RAD) were poured into separate wells. The gel was stained with Coomassie staining solution, and after destaining, the gel was photographed.

[0082] Representative gel electrophoretic photographs of the A10, B3, E3, G1, G9 and H2 antibodies among these 93 antibodies are shown in Figure 1. As shown in Figure 1, the production of antibodies having disulfide bonds was confirmed.

[0083] 1.5. Analysis of binding affinity of selected antibodies The affinity of the 93 antibodies selected in Example 1.3 to the LILRB1 antigen was measured using Biacore T200 (GE healthcare). Anti-human IgG (Fc) antibody (GE healthcare, Cat. No. BR-1008-39, final concentration 25ug / mL) was injected at 5ul / min for 360 seconds on a Series S Sensor Chip CM5 (GE healthcare, Cat. No. BR-1005-30) using an Amine Coupling Kit (GE healthcare, Cat. No. BR-1000-508) to immobilize about 5000-7000RU. Human LILRB1 protein (LILRB1-His, RnD systems Cat. No. 8989-T2), which is an antigen, was injected at a rate of 30ul / min at 4 to 9 different concentrations within the concentration range from 3.13nM to 1600nM, and the k values ​​were measured as shown in the table below. a and k d From this, we calculate K D values ​​were calculated.

[0084] Out of the 93 antibodies, the binding strength (K D Twenty antibodies that showed binding affinity of approximately 99.8 nM to the LILRB1 antigen were selected, and the results are shown in Table 3 below. Among them, the SPR sensorgrams of B3, which showed a binding affinity of approximately 99.8 nM to the LILRB1 antigen, and E3, which showed a binding affinity of approximately 101.2 nM to the LILRB1 antigen, are shown in Figures 2 and 3, respectively (Figure 2: SPR sensorgram for B3, Figure 3: SPR sensorgram for E3). [Table 3]

[0085] 1.6. Sequence analysis of selected antibodies The CDRs defined by the CDR definition based on Kabat numbering, the amino acid sequences of the light chain variable region, the heavy chain variable region, the light chain, and the heavy chain of the 20 antibodies whose antigen-binding ability was confirmed in Example 1.5, and the nucleic acid sequences of the genes encoding the light chain variable region and the heavy chain variable region were analyzed by general amino acid sequence analysis and DNA sequence analysis methods, and are summarized in the following Tables 4 to 23. [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21] [Table 22] [Table 23]

[0086] Example 2: In vitro biological activity analysis of selected antibodies 2.1.NK cell surface binding assay To confirm whether the 93 antibodies selected in Example 1.4 bind to LILRB1 expressed on the surface of immune cells, a NK cell surface binding assay was performed. Human NK cells, KHYG-1 cells (JCRB), were cultured in RPMI 1640 medium (Gibco) containing 10% (w / v) FBS (Gibco) and 100 U / mL interleukin-2 (Novartis). KHYG-1 cells were cultured at 5×10 4 The plates were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 100 cells / well. A test antibody was added to each well at a final concentration of 50 μg / mL, and the plates were left to stand at 4° C. for 1 hour.

[0087] For specific binding to LILRB1, human IgG4 isotype control antibody (Biolegend) was treated in the same manner. After washing with FACS buffer, anti-human Fc-biotin antibody (life technologies) was added to each well and incubated at 4°C for 1 hour. After washing with FACS buffer, streptavidin PE (BD Pharmigen) was added to each well and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were suspended and analyzed with iQue screener (Sartorius).

[0088] Among the obtained results, the results for A10, E3, E4, F12, G1, G9, G11, H2, and H11 are representatively shown in Table 24 in comparison with human IgG4 isotype (control), and flow cytometry diagrams for A10, E3, and human IgG4 isotype (control) are shown in Figure 4a (A10), Figure 4b (E3), and Figure 4c (isotypeIg G4), respectively: [Table 24]

[0089] As shown in Table 24 and Figures 4a to 4c, the test antibodies showed a higher degree of binding to human NK cells (surface) than the human IgG4 isotype control antibody.

[0090] 2.2. Analysis of LILRB1 / HLA-G binding inhibition ability of selected antibodies In order to confirm whether the antibodies selected in Example 1.5 inhibit the binding of LILRB1 to its ligand HLA-G, an LILRB1 / HLA-G binding inhibition assay was performed.

[0091] For this purpose, JEG-3 (ATCC cat# HTB-36), which is known to overexpress HLA-G, was used. JEG-3 was cultured in MEM medium (Gibco) containing 10% (v / v) FBS (Gibco) and 1% (v / v) pen-strep (Gibco). JEG-3 cells were cultured at 5 × 10 4 The cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 100 μg / mL / well. The wall plate was washed with 1X PBS buffer. The test antibodies (A10, E3, F12, G1, G9, H2, H11) selected in Example 1.5 and LILRB1-Fc (RnD systems) were mixed in FACS buffer (1X PBS + 1% BSA + 1 mM EDTA) to a final concentration of 10 μg / mL and 5 μg / mL, respectively, and 100 μl per well was treated with the cells, and then the cells were left on ice for 2 hours. Anti-LILRB1 antibody (clone HP-F1, Abcam) was used as a positive control, and anti-lysozyme IgG4 antibody (clone D1.3) was used as a negative control, and the treatment was performed in the same manner as above. After washing twice with FACS buffer, PE-anti-huIgG-Fc antibody (Biolegend, 10 μg / mL) was added to each well and the wells were left to stand on ice for 1 hour. After washing twice with FACS buffer, the wells were suspended in 100 μl of the same buffer and analyzed with an iQue screener (Sartorius).

[0092] The results are shown in Figure 5. As can be seen from Figure 5, the test antibodies A10, E3, F12, G1, G9, H2, and H11 all effectively inhibited the binding of LILRB1-Fc to HLA-G overexpressing cell lines.

[0093] 2.3. Analysis of cancer cell killing ability by NK cells To confirm whether the selected antibodies increase the ability of NK cells to kill cancer cells, we analyzed the killing rate of HLA-G-overexpressing HEK293 cells by NK cells KHYG-1. KHYG-1 cells (JCRB) were cultured at 2 × 104 cells / well (4×10 4 The plate was dispensed into a 96-well tissue culture plate (BD Falcon) at a final concentration of 20 μg / mL per well. The test antibody (Table 25) was added to each well at a final concentration of 20 μg / mL, and the plate was left to stand at 37° C. for 1 hour.

[0094] As a negative control, a human IgG4 isotype control antibody (Biolegend) was treated in the same manner.

[0095] HLA-G overexpressing HEK293 cells (HEK293 cells (American Type Culture Collection) were transduced with a lentivirus produced to express HLA-G1 to produce an HLA-G overexpressing cell line) were stained with IncuCyte CytoLight Rapid Red Reagent (Sartorius) according to the manufacturer's instructions. After 1 hour, 1 × 10 HLA-G overexpressing HEK293 cells were stained with IncuCyte CytoLight Rapid Red Reagent (Sartorius) according to the manufacturer's instructions. 4 Cells / well (2×10 4 The plate was then added to a total volume of 50 ul (cells / mL). The plate was placed in an IncuCyte S3 (Sartorius) in a 37°C, 5% CO2 incubator and images were taken for 72 hours. Red area confluence, which indicates the density of live HLA-G-overexpressing HEK293 cells, was measured to determine cell viability. The cell viability is shown in Table 25 below (expressed as a relative value with the control antibody set to 1):

number

[0096] As can be seen from Table 25, the test antibodies A10, B9, D3, E1, E3, F12, G1, G6, G9, G11, H2, and H11 increased the killing of HLA-G-overexpressing HEK293 cells by KHYG-1 compared to the human IgG4 isotype control.

[0097] Example 3: In vivo biological activity analysis of selected antibodies Among the antibodies selected in Example 1.5, the anti-cancer efficacy of two test antibodies (E3, B3) was examined in vivo to see if it was possible to improve the efficacy. To this end, Bioware Brite Cell Line HCT116 Red-Fluc colon cancer cells (PerkinElmer) and THP-1 derived macrophages, as well as a xenografted mouse animal model of colon cancer administered with the antibodies, were used to examine whether administration of the two antibodies reduced tumor size in vivo. As a negative control, a xenografted mouse animal model of colon cancer was prepared by administering human IgG1 isotype control antibody (BioXcell, Cat. No. BP0297) in the same manner as above. The above process will be described in more detail below:

[0098] Preparation of THP-1 derived macrophages

[0099] The THP-1-derived macrophages used above were prepared by differentiation of THP-1 cells (ATCC) by adding 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma), 20 ng / mL interferon gamma (Peprotech), and 10 pg / mL lipopolysaccharide (LPS, Sigma).

[0100] Anti-cancer efficacy analysis in mouse animal models Five-week-old female CIEA NOG mice (Central Institute for Experimental Animals, Public Interest Foundation) were treated with 3 × 10 6HCT116 Red-Fluc colon cancer cells and 3 × 10 6 A mixture of THP-1-derived macrophages and two test antibodies (E3 or B3 antibody; 20 μg per mouse) was subcutaneously injected. From day 4 after tumor implantation, the antibody was administered twice a week at a concentration of 5 mg / kg by intraperitoneal injection, and the size of the implanted tumor (mm 3 ) were measured, and the results are shown in Figure 6. As shown in Figure 6, all the antibodies tested in the mouse animal model transplanted with HCT116 colon cancer and THP-1-derived macrophages, especially the E3 antibody, showed clear tumor growth inhibitory efficacy at a statistically significant level.

[0101] Example 4: Generation of anti-LILRB1 antibody (E3.1) The entire nucleic acid sequence corresponding to the heavy chain (SEQ ID NO: 302) of the E3 antibody, which was confirmed to have a particularly excellent effect in Example 3, was PCR amplified, and the nucleic acid sequence corresponding to Ser1 to Leu110 of the light chain variable region (VL) (SEQ ID NO: 221) of the E3 antibody was PCR amplified, and then ligated with the nucleic acid sequence of the Lambda constant region (Lambda CL.1, SEQ ID NO: 344) to PCR amplify the nucleic acid sequence encoding the Lambda light chain region. The amplified sequence was inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen, or any vector having a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and a sequence encoding the heavy chain constant region of human IgG4 and the constant region of lambda light chain) prepared to encode a human antibody in the IgG4 form. The DNA sequence of the expression vector was confirmed by sequencing.

[0102] The constructed expression vector was used to produce an antibody (E3.1) with reference to Example 1.4, and the antibody sequence was analyzed with reference to Example 1.6, and the results are shown in Table 26: [Table 26]

[0103] Example 5: Preparation of cell lines overexpressing human LILR The nucleic acid sequence encoding the entire sequence of the human LILR family protein (see Table 27 below) was amplified by PCR, and the amplified sequence was inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen, or any vector having a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and containing the heavy chain constant region and lambda light chain constant region sequences of human IgG4) that was constructed to code for the nucleic acid sequence. The sequence of the expression vector was confirmed by sequencing. The constructed vectors were transfected into CHO cells, and 11 stable cell lines overexpressing each LILR protein on the cell surface were generated. [Table 27-1] [Table 27-2] [Table 27-3] [Table 27-4]

[0104] Example 6: EC of selected antibodies binding to LILRB1-overexpressing cell surface 50 measurement EC values ​​of the antibodies prepared in Examples 1 and 4 for binding to human LILRB1-overexpressing cell lines 50 To measure the EC value, a cell surface binding assay was performed. The EC values ​​of E3.1 and H11 antibodies were used as representatives of the antibodies prepared above. 50The values ​​were measured. The CHO cells in which LILRB1 was overexpressed on the cell surface prepared in Example 5 were used at 1 × 10 5 The cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) so that the final concentration per well was 1 / 3 for E3.1 and 1 / 3 for H11, which were serially diluted from 600 ug / mL and 27 ug / mL, respectively, and then treated with the dilution and incubated at 4°C for 60 minutes. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (Invitrogen) and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin labeled with PE fluorescence (BD Pharmigen) and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were suspended and analyzed with an iQue screener (Sartorius). EC 50 was calculated using the nonlinear regression equation of GraphPad Prism software, and the results are shown in Table 28: [Table 28]

[0105] Example 7: Cross-reactivity evaluation of selected antibodies against human LILR family overexpressing cell lines To confirm whether the selected antibodies bind to other human LILR family members besides LILRB1, a cell surface binding assay was performed. The various LILR proteins prepared in Example 5 were overexpressed on the cell surface of CHO cells at 1 × 10 5The cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 1000 cells / well. The antibody was treated with a final concentration of 20ug / mL and incubated at 4℃ for 60 minutes. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (Invitrogen) and incubated at 4℃ for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin labeled with PE or FITC fluorescence (BD Pharmigen) and incubated at 4℃ for 30 minutes. After washing with FACS buffer, the cells were suspended and analyzed with an iQue screener (Sartorius). Cells treated with antibodies specific to each LILR protein (see Table 27) were used as positive controls, and cells treated with human IgG4 isotype control antibody (Biolegend) were used as negative controls.

[0106] The results obtained using the E3.1 antibody are shown in Figures 7a to 7d (E3.1: red; LILR-specific antibody: blue; isotype (hIgG4) control: gray), and the results obtained using the H11 antibody are shown in Figures 8a to 8d (H11: red; LILR-specific antibody: blue; isotype (hIgG4) control: gray). As shown in Figures 7a to 7d and 8a to 8d, it was confirmed that all of the E3.1 and H11 antibodies tested did not bind at all or barely bound to other LILRs other than LILRB1. These results indicate that the antibodies provided in this embodiment have specific binding ability to LILRB1.

[0107] Example 8: Measurement of Granzyme B and Perforin secretion by enzyme-linked immune absorbent spot (ELISPOT) To determine whether E3.1 and H11 antibodies increase the cytotoxicity of NK cells, we performed enzyme-linked immunosorbent assay (ELISPOT), which was performed by measuring the expression levels of granzyme B and perforin, which are cytotoxic substances in NK cells.

[0108] KHYG-1 cell line expressing LILRB1 (JCRB) 5 × 10 3 5 × 10 cells in a U-bottom 96-well tissue culture plate 3 The cells were co-cultured with K562 cells overexpressing HLA-G (K562 cells (American Type Culture Collection) were transduced with lentivirus produced to express HLA-G to produce an HLA-G overexpressing cell line). E3.1, H11, and human IgG4 isotype control antibodies were added to each well at a final concentration of 50ug / mL, and the cells were cultured at 37℃ for 30 minutes. The co-cultured cells were transferred to a 96-well plate (Immunospot, Cat. HGZBPFN-2M) (PVDF membrane) for ELISPOT coated with anti-perforin antibody and anti-granzyme B antibody, respectively, and further cultured at 37℃ for 8 hours. After washing the PVDF membrane with washing solution (0.05% Tween 20 in PBS), anti-granzyme B-HRP and anti-perforin-biotin were added in sequence, and the detection process was carried out according to the manufacturer's manual. After drying the PVDF membrane at room temperature for 24 hours, the numbers of granzyme B and perforin spots were measured using an ELISPOT analyzer manufactured by Immunospot.

[0109] The results are shown in FIG. 9 (granzyme b; Gzmb) and FIG. 10 (perforin; Prf), respectively (Y axis indicates total number of spots). As shown in FIG. 9 and FIG. 10, it can be confirmed that the secretion amount of both granzyme B and perforin was statistically significantly increased by treatment with E3.1 or H11 antibody compared to treatment with human IgG4 isotype control antibody. Statistical analysis was performed by unpaired T-test, and all experiments were performed three times under the same conditions to ensure experimental reliability, and the results were expressed as average values.

[0110] Example 9: Production of Chimeric GHI / 75 antibody In order to confirm the superior efficacy of the antibody provided in this example over existing antibodies, chimeric GHI / 75 was constructed having the variable region of the mouse-derived anti-human LILRB1 antibody GHI / 75 antibody (Biolegend, cat#333721) and the constant region of a human-derived antibody.

[0111] Specifically, the amino acid sequence of the GHI / 75 antibody was analyzed by peptide mapping, and then a vector was prepared in which the nucleic acid sequence corresponding to the variable region (VH, VL domain) of the mouse GHI / 75 antibody was replaced with the nucleic acid sequence of the variable region (VH, VL domain) of a human IgG4 antibody. The portion corresponding to the upper hinge of human IgG4 was replaced with the amino acid sequence of the human IgG1 upper hinge (EPKSCDKTHT; SEQ ID NO: 359). The vector was expressed and purified in the same manner as in Example 1.4, and the obtained antibody was used as a comparative antibody in the following tests.

[0112] Example 10: Measurement of LILRB1 signaling inhibitory ability of selected antibodies using IL-2 promoter luciferase assay A luciferase reporter assay was performed to confirm whether the antibodies prepared in Examples 1 and 4 inhibited signal transduction by LILRB1. Among the antibodies prepared in Examples 1 and 4, the E3.1 and H11 antibodies were representatively tested, and the chimeric GHI / 75 antibody prepared in Example 9 was used as a comparative antibody. A Jurkat cell line expressing LILRB1 and interleukin 2 (IL-2) promoter luciferase (prepared by inserting an IL-2 promoter luciferase vector (Promega) into a Jurkat cell line (American Type Culture Collection) and then transducing a lentivirus prepared to express LILRB1) and a K562 cell line overexpressing HLA-G were used. Anti-CD3 antibody (Biolegend) was added to a 96-well plate and coated overnight at 4°C. The next day, 1 x 10 Jurkat cells expressing LILRB1 and IL-2 promoter luciferase were added to a U-bottom 96-well plate. 5 The plate was then treated with E3.1, H11, chimeric GHI / 75, and human IgG4 isotype (control) antibodies at a final concentration of 20 μg / mL, and then incubated at 37° C. for 1 hour. 5 K562 cells overexpressing HLA-G were added and left to stand at 37°C for 30 minutes. The suspension was then transferred to a plate coated with anti-CD3, and anti-CD28 antibody (Biolegend) was added to a final concentration of 10ug / mL. The plate was left to stand at 37°C for 6 hours, after which Steady-Glo (registered trademark) (Promega) solution was added to each well and analyzed using a luminometer (Envision, PerkinElmer).

[0113] The results obtained above are shown in Figure 11. As shown in Figure 11, the E3.1 antibody and H11 antibody according to this example showed significantly increased LILRB1 signaling inhibitory activity compared to the human IgG4 isotype control antibody and the chimeric GHI / 75 comparative antibody.

[0114] Example 11: Anti-cancer efficacy analysis of selected antibodies in mouse animal models To analyze the anti-cancer efficacy of the selected antibodies, 5-week-old female CIEA NOG mice (immunodeficient mice, Central Institute for Experimental Animals, Public Interest Foundation) were incubated with 3×10 6 HCT116 Red-Fluc colon cancer cells and 3 × 10 6 Mouse animal models were prepared by subcutaneously injecting 10 THP-1-derived macrophages and two types of test antibodies (20 μg per mouse). The test antibodies used were E3.1 and H11, and a human IgG4 isotype control antibody was used for comparison. Starting from day 4 after tumor cell transplantation, the antibodies were administered twice a week via intraperitoneal injection at a dose of 5 mg / kg each, and the tumor volume was measured, and the results are shown in FIG. 12. As shown in FIG. 12, in a mouse animal model transplanted with HCT116 colon cancer cells and THP-1-derived macrophages, both the E3.1 and H11 antibodies showed superior tumor growth inhibitory efficacy compared to the control antibody.

Claims

1. An anti-LILRB1 antibody or antigen-binding fragment thereof, comprising the following complementarity determining regions (CDRs): CDR-L1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 38, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:42 (The CDRs are defined according to Kabat numbering.)

2. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 231; and The anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

232.

3. The anti-LILRB1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is a human IgG1 or IgG4 antibody.

4. The antigen-binding fragment may be an scFv, (scFv) ２ , Fab, Fab', F(ab') ２ 2. The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1, which is a fusion polypeptide in which the scFv is fused with the Fc of an immunoglobulin, or a fusion polypeptide in which the scFv is fused with a constant region of a light chain.

5. A pharmaceutical composition for preventing or treating cancer, comprising the anti-LILRB1 antibody or its antigen-binding fragment according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. The pharmaceutical composition of claim 5 , wherein the cancer is characterized by overexpression of MHC class I.

7. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

8. A recombinant vector comprising the nucleic acid molecule of claim 7.

9. A recombinant cell comprising the nucleic acid molecule of claim 7 or a recombinant vector comprising the same.

10. A method for producing an anti-LILRB1 antibody or antigen-binding fragment thereof, comprising culturing the recombinant cell of claim 9.

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