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 December 23, 2019, and Korean Patent Application No. 10-2020-0061907, filed 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, and more particularly 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, or 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 receptors and is involved in signal transduction mechanisms that suppress immune cell activity.

[0004] On the other hand, various cancer cells are known to overexpress MHC class I, such as HLA-G, in order 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 exhibit 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 that express LILRB1, and regulates the activity of the immune cells, thereby exhibiting 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 the activity of inhibiting the binding of LILRB1 to MHC class I and / or the activity of inhibiting the interaction of LILRB1 with MHC class I. The anti-LILRB1 antibody or antigen-binding fragment thereof may have the activity of inhibiting immune evasion of cancer cells. Furthermore, 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 antigen-binding fragment thereof.

[0009] Another embodiment provides a pharmaceutical composition for inhibiting the binding of LILRB1 to MHC class I and / or 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 the activity of inhibiting the binding of LILRB1 to MHC class I and / or inhibiting the interaction of LILRB1 with MHC class I. The anti-LILRB1 antibody or antigen-binding fragment thereof may also have the activity of inhibiting immune evasion by cancer cells. The anti-LILRB1 antibody or antigen-binding fragment thereof may also 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, E.A., Wu, T.T., 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 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 as a specific amino acid sequence" can mean both cases where the antibody necessarily contains the amino acid sequence, 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), 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 to 10 mM, 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 0.05 nM to 0.2 mM, 0.1 nM to 0.2 mM, 0.5 nM to 0.2 mM, 1 nM to 0.2 mM, 0.001 nM to 0.15 mM, 0.005 nM to 0.15 mM, 0.01 nM to 0.15 mM, 0.05 nM to 0.15 mM, 0.1 nM to 0.15 mM, 0.5 nM to 0.15 mM, or 1 nM to 0.15.

[0019] Another example provides a pharmaceutical composition comprising the anti-LILRB1 antibody or its antigen-binding fragment as an active ingredient. For example, the pharmaceutical composition may be for treating and / or preventing cancer. The pharmaceutical composition may have the 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 the 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, which comprises the anti-LILRB1 antibody or its antigen-binding fragment as an active ingredient.

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

[0022] Another example provides a method for treating and / or preventing cancer, comprising administering (orally or parenterally) a pharmaceutically 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 the step of administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or its antigen-binding fragment to a subject (e.g., a mammal, including a human) in need of inhibiting 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 pharmaceutically 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 inhibition of immune evasion of cancer cells.

[0025] The methods provided herein may further include, prior to the administering step, identifying a subject in need of treatment and / or prevention of cancer, a subject in need of inhibition of the 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 containing the nucleic acid molecule. In one example, the recombinant vector can contain 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 can contain both (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 that retains binding ability to the antigen (LILRB1), and may be any polypeptide comprising 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 an 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 an 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 on the LILRB1 protein, for example, antagonism or agonism. The anti-LILRB1 antibody or antigen-binding fragment thereof may have the activity of inhibiting the binding of LILRB1 to MHC class I and / or the interaction of LILRB1 with MHC class I. Furthermore, the anti-LILRB1 antibody or antigen-binding fragment thereof may have the 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 serves as the 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. It may be a protein produced by antigen stimulation in the immune system or a protein produced by recombinant or chemical synthesis, and the type of antibody is not particularly limited. The antibody may be a non-naturally occurring 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 regions, of the anti-LILRB1 antibodies or antigen-binding fragments thereof provided herein as defined above may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), and may be derived from, for example, framework regions and / or light chain constant regions and / or heavy chain constant regions of any of the above subtypes of immunoglobulin. In one example, the anti-LILRB1 antibodies provided herein may be human IgG-type antibodies, for example, IgG1, IgG2, IgG3, or IgG4 antibodies, but are not limited thereto.

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

[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 the heavy chain and hinge, and a fragment thereof. The term "light chain" also refers to 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 interpreted to mean a full-length light chain including the above chain and fragments thereof.

[0038] The term "CDR (complementarity determining region)" refers to the portion of an antibody variable region that confers antigen-binding specificity, and refers to the amino acid sequence of the hypervariable region of an immunoglobulin heavy chain or light chain. Each 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 antibody binding to an antigen or epitope. Meanwhile, as used herein, the terms "specifically bind" and "specifically recognize" have the same meaning as commonly known to those skilled in the art, meaning that an antigen and an antibody specifically interact and immunologically react with each other.

[0039] In this specification, unless otherwise specified, the term "antibody" can be understood to include not only complete antibodies but also antigen-binding fragments of antibodies that have antigen-binding ability.

[0040] The term "antigen-binding fragment" refers to any form of polypeptide comprising a portion capable of binding to an antigen (e.g., six CDRs as defined herein). For example, the fragment 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 with 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 variable regions of the light chain and heavy chain, a constant region of the light chain, and the first constant region of the heavy chain (C H1 ) is a structure having the following 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 disulfide bonding between cysteine ​​residues in the hinge region of Fab'. Fv is the minimum antibody fragment containing 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 region and the light-chain variable region are linked non-covalently, while in a single-chain Fv, the heavy-chain variable region and the light-chain variable region are generally linked covalently via a peptide linker or directly at the C-terminus, and thus can form a dimer-like structure similar to a two-chain Fv.

[0045] The antigen-binding fragments can be obtained using proteolytic enzymes (for example, whole antibodies can be digested with papain to obtain Fab fragments, or with pepsin to obtain F(ab')2 fragments), or can be produced using recombinant DNA 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 within the antibody.

[0047] The anti-LILRB1 antibody may be a monoclonal antibody. Monoclonal antibodies can be produced by methods known in the art, such as phage display technology. Alternatively, anti-LILRB1 antibodies can be produced as mouse-derived monoclonal antibodies by conventional methods.

[0048] Alternatively, individual monoclonal antibodies can be screened based on their binding ability to LILRB1 using a typical enzyme-linked immunosorbent assay (ELISA) format. The binding partners can then be assayed for inhibitory activity using functional assays such as competitive ELISA or cell-based assays to test for molecular interactions. Subsequently, the affinities (Kd values) of monoclonal antibodies selected based on their strong inhibitory activity for LILRB1 can be assayed.

[0049] The pharmaceutical compositions provided herein may further comprise a pharmaceutically acceptable carrier in addition to the active ingredient (anti-LILRB1 antibody or antigen-binding fragment thereof). The pharmaceutically acceptable carrier is one commonly used in pharmaceutical 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 to these. 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 commonly used in the manufacture of pharmaceutical compositions.

[0050] An effective amount of the pharmaceutical composition or 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. Because 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. Furthermore, the composition can be administered by any device that allows the active agent to be delivered to target cells (e.g., cancer cells).

[0051] The anti-LILRB1 antibody or its antigen-binding fragment can be contained in the pharmaceutical composition or administered to a patient in a pharmaceutically effective amount. As used herein, "pharmaceutically effective amount" refers to the amount of the active ingredient (anti-LILRB1 antibody or its antigen-binding fragment) that is capable of exerting its intended effect (e.g., anti-cancer effect). The pharmaceutically effective amount can be formulated in various ways depending on factors such as the patient's age, weight, sex, pathological condition, diet, excretion rate, responsiveness, formulation method, administration time, administration interval, administration route, and administration method. For example, the daily dose of the anti-LILRB1 antibody or antigen-binding fragment thereof may be 0.005 μg / kg to 1000 mg / kg, 0.005 μg / kg to 500 mg / kg, 0.005 μg / kg to 250 mg / kg, 0.005 μg / kg to 100 mg / kg, 0.005 μg / kg to 75 mg / kg, 0.005 μg / kg to 50 mg / kg, 0.01 μg / kg to 1000 mg / kg, 0.01 μg / kg to 500 mg / kg, 0.01 μg / kg to 2 The daily dose may be in the range of, but is not limited to, 50 mg / kg, 0.01 μg / kg to 100 mg / kg, 0.01 μg / kg to 75 mg / kg, 0.01 μg / kg to 50 mg / kg, 0.05 μg / kg to 1000 mg / kg, 0.05 μg / kg to 500 mg / kg, 0.05 μg / kg to 250 mg / kg, 0.05 μg / kg to 100 mg / kg, 0.05 μg / kg to 75 mg / kg, or 0.05 μg / kg to 50 mg / kg. The daily dose may be formulated in a unit dosage form in a single formulation, or may be appropriately divided into dosage forms, or may be packaged 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, granule, tablet or capsule, etc., and may further contain a dispersant 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, and may be one or more types selected from the group consisting of, but not limited to, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, colon cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal gland 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, or 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, e.g., T-cell (e.g., cytotoxic T-cell)-mediated immunotherapy (i.e., are unresponsive 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 progression of cancer symptoms, such as the inhibition of cancer cell proliferation, the death of cancer cells, and the inhibition of metastasis, or that partially or completely eliminates cancer.

[0056] The anti-LILRB1 antibodies or antigen-binding fragments 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 the prevention and / or treatment of 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. Another example is provided a method for the prevention and / or treatment of cancer, comprising the step of administering to a patient in need of cancer prevention and / or treatment (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. The immunotherapeutic agents, anticancer agents and cytotoxic agents include all drugs that are typically used in cancer treatment and / or have cytotoxic activity, and may be selected from, but are not limited to, one or more of proteins such as antibodies, nucleic acid molecules such as siRNA, and / or small molecule compounds such as paclitaxel and docetaxel.

[0057] As another example, there is provided a polypeptide molecule comprising 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), or a combination thereof of the anti-LILRB1 antibody described above; or a heavy chain variable region, a light chain variable region, or a combination thereof. The polypeptide molecule is not only used as an antibody precursor for antibody production, but is also included as a component of protein scaffolds (e.g., peptibodies) with a structure similar to that of antibodies, bispecific antibodies, and multispecific antibodies. As yet another example, the polypeptide molecule can be used as a target (antigen) recognition moiety in targeted cell therapies such as CAR-T, as a secreted antibody, or as a cell therapy engineered to secrete the anti-LILRB1 antibody.

[0058] As another example, nucleic acid molecules encoding the heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), heavy chain variable regions, or heavy chains of anti-LILRB1 antibodies are provided.

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

[0060] As another example, the present invention provides recombinant vectors that contain a nucleic acid molecule encoding a heavy chain complementarity determining region, a heavy chain variable region, or the heavy chain of the anti-LILRB1 antibody, and a nucleic acid molecule encoding a light chain complementarity determining region, a light chain variable region, or the light chain of the anti-LILRB1 antibody, either together in a single vector or separately in separate vectors.

[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, lentiviral vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors. Vectors that can be used for the recombinant vector can be prepared 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), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40) commonly used in the art.

[0063] In the recombinant vector, the nucleic acid molecule can be operably linked to a promoter. The term "operably linked" refers to a functional connection between a nucleotide expression control sequence (e.g., a promoter sequence) and another nucleotide sequence. The control sequence is "operably linked" so as to regulate 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 the host, a strong promoter (e.g., pL λ Typically, the vector contains a promoter (e.g., CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. When a eukaryotic cell is used as the host, the vector may contain an origin of replication that functions in eukaryotic cells, including, but not limited to, the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adenovirus origin of replication, the AAV origin of replication, and the BBV origin of replication. Furthermore, promoters derived from the genome of mammalian cells (e.g., the metallothionine promoter) or promoters derived from mammalian viruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the cytomegalovirus promoter, the HSV tk promoter, etc.) are used, and generally contain 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 can be any host cell known in the art that can stably and continuously clone or express 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 strains such as Bacillus subtilis and Bacillus thuringiensis; and Enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When transforming 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 can be used include, but are not limited to, GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK.

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

[0068] The transformed host cells can be easily selected by a method well 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 transformants can be easily selected by culturing them 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 containing the same in a host cell. The expression step can be carried out by culturing a recombinant cell containing the nucleic acid molecule (e.g., contained in a recombinant vector) under conditions that allow expression of the nucleic acid molecule. The production method can also include a step of isolating and / or purifying the antibody or antigen-binding fragment from the culture medium after the expression or culturing step. [Effects 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 fully exert their anti-cancer efficacy without inhibiting it. [Brief explanation of the drawings]

[0071] [Figure 1] 1 is an electrophoresis photograph showing the results of SDS-PAGE gel analysis of purified anti-LILRB1 antibodies according to one embodiment. [Figure 2] FIG. 1 shows the results of an SPR (surface plasmon resonance) sensorgram for anti-LILRB1 antibody B3 according to one embodiment. [Figure 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 ability of a human IgG4 isotype control antibody to KHYG-1 cells, which are human NK cells. [Figure 5] 1 is a graph showing the results of analyzing, using iQue screener, 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. [Figure 6] 1 is a graph showing the in vivo anti-tumor effects 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 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 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 INVENTION

[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 within the scope of 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, we performed phage display screening 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) from 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 four forms of LILRB1 antigen (LILRB1-His, LILRB1-Fc, LILRB1-His-Biotin, and LILRB1-Fc-Biotin) using solid-phase and solution-phase selection methods. Additional selections were performed by gradually decreasing the antigen concentration, competitive elution with a control antibody against LILRB1, or negative selection against Fc when LILRB1-Fc was used as the antigen. The selected products were confirmed for antigen binding by polyclonal phage ELISA.

[0075] 1.2. Monoclonal soluble scFv screening and analysis Genes encoding scFvs confirmed to bind to the antigen 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 a DELFIA immune assay (PerkinElmer) was performed to confirm antigen binding. Furthermore, a certain amount of scFv antibody was coated on the surface, and then DELFIA was performed on the antigen 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 using a standard DNA sequence analysis method to remove duplicate clones. Furthermore, a total of 93 clones were selected based on the ranking of antigen-antibody binding ability determined in Example 1.2. Sequences corresponding to the heavy chain variable region (VH) and light chain variable region (VL) from the genes encoding the selected scFvs were amplified by PCR and inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen; alternatively, a vector containing a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and containing the human IgG4 heavy chain constant region and the kappa or lambda light chain constant region sequence may be used) constructed to encode an IgG4-type human antibody (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 purified vector 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 TM80 μl of CHO reagent (Thermo Fisher) was added and transfection was performed. One day after transfection, ExpiCHO TM Enhancer (Thermo Fisher) 150ul and ExpiCHO TM 4 mL of ExpiCHO feed (Thermo Fisher) was added. TM Four milliliters of feed was added, and the transfected cells were cultured at 32°C in 5% CO 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 μl of 293 Reagent (Gibco) was added and transfection was performed according to the manufacturer's instructions. One day after transfection, ExpiFectamine TM 293 Enhancer1 (Thermo Fisher) 0.6 mL per 100 mL culture volume, ExpiFectamine TM Enhancer 2 (Thermo Fisher) was added at 6 mL per 100 mL of culture volume, and glucose was added at 3.6 g per liter. The transfected cells were cultured at 36.5°C in 5% CO 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 0.22 μm bottle-top filter system (Corning). The collected culture medium was purified using an AKTA Pure L (GE Healthcare). A Hitrap MabSelectSure 1 mL column (GE Healthcare) was attached to the AKTA Pure L, and the culture medium was passed through at a flow rate of 1 mL / min. The column was then washed with 20 column volumes (CV) of 1X PBS. The target protein was eluted with elution buffer (0.1 M sodium citrate, pH 3.4). The eluate was concentrated using an Amicon Ultra Filter Device (MWCO 10K, Merck) and a centrifuge, followed by buffer exchange with 1X PBS.

[0081] The purified antibody sample was diluted with 1X PBS to approximately 1 mg / mL. 20 μL of purified antibody sample was mixed with 10 μL of Reducing Loading Buffer (3X) or Non-reducing Loading Buffer (3X), and then placed in a 95°C heating bath for 2 minutes. The mixture was then removed and allowed to cool. SDS-PAGE Gradient Gels (4-20% or 4-12%) were placed in an electrophoresis apparatus, and 10 μg of sample was injected per well. The gel was then developed using Precision Plus Protein Ion Imaging (PIM) for molecular weight analysis of the sample. TM Dual color standards (BIO-RAD) were injected into separate wells. The gel was stained with Coomassie staining solution, and after destaining, the gel was photographed.

[0082] Representative gel electrophoresis images of the A10, B3, E3, G1, G9, and H2 antibodies from these 93 antibodies are shown in Figure 1. As shown in Figure 1, the production of antibodies with disulfide bonds was confirmed.

[0083] 1.5. Analysis of binding affinity of selected antibodies The affinities of the 93 antibodies selected in Example 1.3 for the LILRB1 antigen were measured using a Biacore T200 (GE Healthcare). Anti-human IgG (Fc) antibody (GE Healthcare, Cat. No. BR-1008-39, final concentration 25 μg / mL) was injected at 5 μL / min for 360 seconds onto 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), resulting in immobilization at approximately 5,000 to 7,000 RU. The antigen, human LILRB1 protein (LILRB1-His, RnD Systems, Cat. No. 8989-T2), was injected at a rate of 30 μL / min at four to nine different concentrations ranging from 3.13 nM to 1,600 nM. The k values ​​were measured as shown in the table below. a and k d From this, K D values ​​were calculated.

[0084] Among the 93 antibodies, the one with the highest binding strength (K D Twenty antibodies showing binding affinity (value) 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, heavy chain variable region, light chain, and 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 heavy chain variable region were analyzed by conventional amino acid sequence analysis and DNA sequence analysis methods, and are summarized in Tables 4 to 23 below. [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 screened in Example 1.4 also bind to LILRB1 expressed on the surface of immune cells, an 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). 5 × 10 KHYG-1 cells were cultured at 10 °C. 4 The cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 100 cells / well. Test antibodies were added to each well at a final concentration of 50 μg / mL, and the plate was left to stand at 4°C for 1 hour.

[0087] For specific binding to LILRB1, a human IgG4 isotype control antibody (Biolegend) was also treated in the same manner. After washing with FACS buffer, an 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 using an iQue screener (Sartorius).

[0088] Among the results obtained, the results for representative antibodies A10, E3, E4, F12, G1, G9, G11, H2, and H11 are shown in Table 24 in comparison with the human IgG4 isotype (control). Flow cytometry diagrams for A10, E3, and the human IgG4 isotype (control) are shown in Figure 4a (A10), Figure 4b (E3), and Figure 4c (isotype IgG 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 the ability of selected antibodies to inhibit LILRB1 / HLA-G binding To confirm whether the antibodies selected in Example 1.5 inhibit the binding of LILRB1 to its ligand, HLA-G, an assay for inhibiting LILRB1 / HLA-G binding was performed.

[0091] JEG-3 (ATCC cat# HTB-36), known to overexpress HLA-G, was used for this study. JEG-3 cells were cultured in MEM medium (Gibco) containing 10% (v / v) FBS (Gibco) and 1% (v / v) pen-strep (Gibco). 5 × 10 JEG-3 cells were cultured at 4 °C. 4 The cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 100 μg / mL of cells / well. The wall plate was washed with 1X PBS buffer. The test antibodies (A10, E3, F12, G1, G9, H2, and H11) selected in Example 1.5 and LILRB1-Fc (RnD Systems) were mixed in FACS buffer (1X PBS + 1% BSA + 1 mM EDTA) to final concentrations of 10 μg / mL and 5 μg / mL, respectively. 100 μl of this mixture was applied to the cells and then placed on ice for 2 hours. An anti-LILRB1 antibody (clone HP-F1, Abcam) was used as a positive control, and an anti-lysozyme IgG4 antibody (clone D1.3) was used as a negative control, treated 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 cells were allowed to stand on ice for 1 hour. After washing twice with FACS buffer, the cells were suspended in 100 μl of the same buffer and analyzed using 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 enhance 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 (cells / mL, total volume 50 μl). Test antibodies (Table 25) were added to each well to 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, human IgG4 isotype control antibody (Biolegend) was also treated in the same manner.

[0095] HLA-G-overexpressing HEK293 cells (HEK293 cells (American Type Culture Collection) were transduced with a lentivirus engineered to express HLA-G1 to create 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. 4 cells / well (2 × 10 4 The plate was then added to a total volume of 50 μl (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. The 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 enhanced 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, two test antibodies (E3 and B3) were tested in vivo to determine whether they had improved anti-cancer efficacy. To this end, we tested whether tumor size was reduced in vivo by administering these two antibodies to Bioware Brite Cell Line HCT116 Red-Fluc colon cancer cells (PerkinElmer) and THP-1 derived macrophages, as well as a mouse xenograft colon cancer model administered with the antibodies. As a negative control, a mouse xenograft colon cancer model administered with a human IgG1 isotype control antibody (BioXcell, Cat. No. BP0297) in the same manner as above was also prepared. The above process is 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) with 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) 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. Starting from day 4 after tumor implantation, the antibody was administered twice a week at a concentration of 5 mg / kg via intraperitoneal injection. The size of the implanted tumor (mm 3 ) were measured, and the results are shown in Figure 6. As shown in Figure 6, all antibodies tested in a 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, whose particularly excellent effects were confirmed 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. This was then ligated with the nucleic acid sequence of the Lambda constant region (Lambda CL.1, SEQ ID NO: 344) to PCR-amplified the nucleic acid sequence encoding the Lambda light chain region. The amplified sequence was inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen) designed to encode a human IgG4 antibody (although any vector containing a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and encoding the human IgG4 heavy chain constant region and the lambda light chain constant region may also be used). 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: Construction of a cell line 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 inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen, or any vector containing a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and the heavy chain constant region and lambda light chain constant region sequences of human IgG4) encoding the amplified 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 the E3.1 and H11 antibodies were used as representatives of the antibodies prepared above. 50The values ​​were measured. 1 × 10 CHO cells overexpressing LILRB1 on the cell surface, prepared in Example 5, were used. 5 The cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 100 cells / well. The final concentrations per well were serially diluted from 600 μg / mL for E3.1 and 27 μg / mL for H11, and then treated with the diluted solutions 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 PE fluorescently labeled streptavidin (BD Pharmigen) and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were suspended and analyzed using an iQue screener (Sartorius). EC 50 was calculated using the nonlinear regression formula in GraphPad Prism software and the results obtained 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. Various LILR family 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 100 cells / well. Each well was treated with antibodies at a final concentration of 20 μg / mL 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 PE- or FITC-labeled streptavidin (BD Pharmigen) and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were suspended and analyzed using an iQue screener (Sartorius). Cells treated with antibodies specific to each LILR protein (see Table 27) served as positive controls, and cells treated with a human IgG4 isotype control antibody (Biolegend) served 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 to any LILRs other than LILRB1. These results demonstrate 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) An enzyme-linked immunosorbent assay (ELISPOT) was performed to determine whether E3.1 and H11 antibodies increased the cytotoxic activity of NK cells. The cytotoxic activity was determined by measuring the expression levels of granzyme B and perforin, which are cytotoxic substances in NK cells.

[0108] 5 × 10 KHYG-1 cell line (JCRB) expressing LILRB1 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 a lentivirus engineered to express HLA-G to generate 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 50 μg / mL and incubated at 37°C for 30 minutes. The co-cultured cells were transferred to a 96-well ELISPOT plate (Immunospot, Cat. HGZBPFN-2M) (PVDF membrane) coated with anti-perforin antibody and anti-granzyme B antibody, respectively, and incubated at 37°C for an additional 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 sequentially. The detection process was then carried out according to the manufacturer's instructions. The PVDF membrane was dried at room temperature for 24 hours, and the number of granzyme B and perforin spots was then measured using an ELISPOT analyzer manufactured by Immunospot.

[0109] The results are shown in Figure 9 (granzyme B; Gzmb) and Figure 10 (perforin; Prf), respectively (the Y axis represents the total number of spots). As shown in Figures 9 and 10, it can be seen that the secretion levels of both granzyme B and perforin were statistically significantly increased following treatment with E3.1 or H11 antibody compared to treatment with the human IgG4 isotype control antibody. Statistical analysis was performed using an unpaired t-test. To ensure experimental reliability, all experiments were performed three times under the same conditions, and the results are expressed as averages.

[0110] Example 9: Production of Chimeric GHI / 75 antibody To confirm the superior efficacy of the antibody provided in this example over existing antibodies, chimeric GHI / 75 was constructed, which contains the variable region of the mouse-derived anti-human LILRB1 antibody GHI / 75 (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 constructed in which the nucleic acid sequence corresponding to the variable regions (VH and VL domains) of the mouse GHI / 75 antibody was replaced with the nucleic acid sequence of the variable regions (VH and VL domains) of a human IgG4 antibody. The portion corresponding to the upper hinge of human IgG4 was replaced with the amino acid sequence of the upper hinge of human IgG1 (EPKSCDKTHT; SEQ ID NO: 359). The vector was expressed and purified using the same process as in Example 1.4, and the resulting antibody was used as a comparative antibody in the following tests.

[0112] Example 10: Measurement of the ability of selected antibodies to inhibit LILRB1 signaling using an IL-2 promoter luciferase assay A luciferase reporter assay was performed to confirm whether the antibodies prepared in Examples 1 and 4 inhibited LILRB1-mediated signal transduction. Representative antibodies prepared in Examples 1 and 4, E3.1 and H11, were tested, with the chimeric GHI / 75 antibody prepared in Example 9 used as a comparative antibody. A Jurkat cell line expressing LILRB1 and interleukin 2 (IL-2) promoter luciferase (generated by inserting an IL-2 promoter luciferase vector (Promega) into a Jurkat cell line (American Type Culture Collection) and then transducing a lentivirus engineered 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 plates were 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 1000 HLA-G were added and incubated at 37°C for 30 minutes. The suspension was then transferred to an anti-CD3-coated plate, and anti-CD28 antibody (Biolegend) was added to a final concentration of 10 μg / mL. The plate was incubated at 37°C for 6 hours, after which Steady-Glo® (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 of this example exhibited 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, 3 × 10 mice were cultured in 5-week-old female CIEA NOG mice (NOG immunodeficient mice, Central Institute for Experimental Animals, Public Interest Foundation) with reference to Example 3. 6 HCT116 Red-Fluc colon cancer cells and 3 × 10 6 Mouse animal models were prepared by subcutaneously injecting 100 THP-1-derived macrophages and two 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 four days after tumor cell inoculation, the antibodies were administered intraperitoneally at a dose of 5 mg / kg twice a week, and tumor volume was measured. The results are shown in Figure 12. As shown in Figure 12, both the E3.1 and H11 antibodies demonstrated superior tumor growth inhibitory efficacy compared to the control antibody in a mouse animal model inoculated with HCT116 colon cancer cells and THP-1-derived macrophages.

Claims

1. An anti-LILRB1 antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs): (1) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 25; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 26; CDR-L3 comprising the amino acid sequence of SEQ ID NO: 27; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; (2) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44; CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 46; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 47, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 48; or (3) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55; CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56; CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57; CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58; CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60, (The CDRs are defined according to Kabat numbering.)

2. (1) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 233, and comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 234; (2) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 237, and comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 238; or (3) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 239, and The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

240.

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 pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to 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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