Anti-lilrb1 antibody 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 the contents disclosed in the documents of the Korean patent applications are included as part of this specification.

[0002] The present invention relates to an anti - LILRB1 antibody and its uses, specifically, to an anti - LILRB1 antibody or its antigen - binding fragment, and its use for cancer treatment.

Background Art

[0003] Leukocyte immunoglobulin - like receptor subfamily B member 1 (LILRB1, or 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 for immune evasion. When the binding of LILRB1 to MHC class I is inhibited, it is expected to restore the activity of suppressed immune cells and exhibit an anti - cancer effect.

[0005] Therefore, the development of new drugs that bind to LILRB1 and inhibit the binding of LILRB1 to MHC class I and / or inhibit the interaction between LILRB1 and MHC class I is required.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Antibodies that bind to LILRB1, act on immune cells expressing LILRB1, regulate the activity of immune cells, and exhibit anti-cancer efficacy, and their uses in cancer treatment are provided.

[0007] One embodiment provides an anti-LILRB1 antibody or an antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or its antigen-binding fragment may have an activity of inhibiting the binding of LILRB1 to MHC class I and / or an activity of inhibiting the interaction between LILRB1 and MHC class I. The anti-LILRB1 antibody or its antigen-binding fragment may have an activity of inhibiting immune escape of cancer cells. Further, the anti-LILRB1 antibody or its antigen-binding fragment may have excellent anti-cancer activity. The cancer may be one that expresses or overexpresses MHC class I on its surface.

[0008] Another embodiment provides a pharmaceutical composition for the treatment and / or prevention of cancer, comprising the anti-LILRB1 antibody or its antigen-binding fragment.

[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 its antigen-binding fragment.

[0010] Another embodiment provides a pharmaceutical composition for inhibiting immune escape of cancer cells, comprising the anti-LILRB1 antibody or its antigen-binding fragment.

Means for Solving the Problems

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

[0012] The anti-LILRB1 antibody or its antigen-binding fragment may include 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 including 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 including 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 including 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 including 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, A 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 comprises 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, or (2) Based on the CDR definition by IMGT numbering (http: / / www.imgt.org / ), a 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, a 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, a 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, a 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, a 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 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 the 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 may be as described above, comprising a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3, and a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3.

[0015] In an embodiment, the anti-LILRB1 antibody or antigen-binding fragment thereof comprises a light chain variable region having an 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 a heavy chain variable region having an 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] The combinations of the light chain variable region and heavy chain variable region that can be included in the anti-LILRB1 antibody or antigen-binding fragment 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 an antigen-binding fragment thereof (e.g., a CDR, variable region, or heavy / light chain) “comprises or consists of or is represented by a specific amino acid sequence” can mean all cases where the amino acid sequence is necessarily included, and cases where meaningless mutations (e.g., substitutions, deletions, and / or additions of amino acid residues) that do not affect antibody activity are introduced into the amino acid sequence.

[0018] The anti-LILRB1 antibodies or antigen-binding fragments thereof provided herein have a binding affinity (K for LILRB1 (e.g., human LILRB1) D) can 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, for example, that measured by surface plasmon resonance (SPR), and can be, for example, from 0.001 nM to 10 mM, from 0.005 nM to 10 mM, from 0.01 nM to 10 mM, from 0.05 nM to 10 mM, from 0.1 nM to 10 mM, from 0.5 nM to 10 mM, from 1 nM to 10 mM, from 0.001 nM to 5 mM, from 0.005 nM to 5 mM, from 0.01 nM to 5 mM, from 0.05 nM to 5 mM, from 0.1 nM to 5 mM, from 0.5 nM to 5 mM, from 1 nM to 5 mM, from 0.001 nM to 1 mM, from 0.005 nM to 1 mM, from 0.01 nM to 1 mM, from 0.05 nM to 1 mM, from 0.1 nM to 1 mM, from 0.5 nM to 1 mM, from 1 nM to 1 mM, from 0.001 nM to 0.5 mM, from 0.005 nM to 0.5 mM, from 0.01 nM to 0.5 mM, from 0.05 nM to 0.5 mM, from 0.1 nM to 0.5 mM, from 0.5 nM to 0.5 mM, from 1 nM to 0.5 mM, from 0.001 nM to 0.2 mM, from 0.005 nM to 0.2 mM, from 0.01 nM to 0.2 mM, from 0.05 nM to 0.2 mM, from 0.1 nM to 0.2 mM, from 0.5 nM to 0.2 mM, from 1 nM to 0.2 mM, from 0.001 nM to 0.15 mM, from 0.005 nM to 0.15 mM, from 0.01 nM to 0.15 mM, from 0.05 nM to 0.15 mM, from 0.1 nM to 0.15 mM, from 0.5 nM to 0.15 mM, or from 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 can be a pharmaceutical composition for the treatment and / or prevention of cancer. The pharmaceutical composition can have an activity of inhibiting the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I. The cancer can be a cancer associated with the interaction between LILRB1 and MHC class I. In one example, the pharmaceutical composition can have an activity of inhibiting the immune escape of cancer cells. The cancer cells can be those expressing or overexpressing MHC class I on the 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 example 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 pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal including a human) in need of treatment and / or prevention of cancer.

[0023] Another example provides a method for inhibiting the binding of LILRB1 to MHC class I and / or a method for inhibiting the interaction between LILRB1 and MHC class I, comprising administering (orally or parenterally) a pharmaceutically effective amount of the anti-LILRB1 antibody or an antigen-binding fragment thereof 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 between LILRB1 and 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 an 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 comprise, prior to the administering step, identifying a subject in need of treatment and / or prevention of cancer, a subject in need of inhibiting the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I, and / or a subject in need of inhibiting immune evasion of cancer cells.

[0026] Other examples provide 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] Other examples provide a recombinant vector comprising the nucleic acid molecule. In one example, the recombinant vector may 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 may comprise them together (e.g., in one vector). The recombinant vector can be used as an expression vector.

[0028] Other examples provide a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0029] Other examples provide a method for producing an anti-LILRB1 antibody or an antigen-binding fragment thereof, which comprises the step of expressing the nucleic acid molecule in a cell. The step of expressing the nucleic acid molecule may include the step of culturing the recombinant cell.

[0030] The antigen-binding fragment of the anti-LILRB1 antibody described in this specification means a fragment derived from the anti-LILRB1 antibody and possessing the binding ability to the antigen (LILRB1), and can be any polypeptide containing the 6 CDRs of the anti-LILRB1 antibody, for example, scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-Cλ (lambda constant region), (scFv)2, Fab, Fab’ or F(ab’)2, but is not limited thereto. In one example, the antigen-binding fragment can be scFv, or a fusion polypeptide (scFv-Fc) in which scFv is fused with the Fc site of an immunoglobulin (for example, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) or a fusion polypeptide (scFv-Ck or scFv-Cλ) in which scFv is fused with a light chain constant region (for example, kappa or lambda).

[0031] The anti-LILRB1 antibody or its antigen-binding fragment can be one that has a regulatory effect on the LILRB1 protein, for example, antagonism or agonism. The anti-LILRB1 antibody or its antigen-binding fragment can be one that has the activity of inhibiting the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I. Further, the anti-LILRB1 antibody or its antigen-binding fragment can be one that has the activity of inhibiting the immune escape of cancer cells. Further, the anti-LILRB1 antibody or its antigen-binding fragment can be one that has 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, 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.), but is not limited thereto.

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

[0034] As used herein, the term "antibody" generally refers to a protein that specifically binds to a particular antigen, and can be a protein produced by antigen stimulation in the immune system or a protein produced by recombinant or chemical synthesis, and its type is not particularly limited. The antibody can be non-naturally produced, for example, recombinantly or synthetically produced. The antibody can be an animal antibody (e.g., a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody can be a monoclonal antibody or a polyclonal antibody.

[0035] The anti-LILRB1 antibodies or antigen-binding fragments thereof provided herein, excluding the heavy-chain CDR and light-chain CDR sites, or the remaining sites excluding the heavy-chain variable region and the light-chain variable region, as defined above, can be derived from all subtypes of immunoglobulins (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), for example, they can be derived from the framework regions of all the above subtypes of immunoglobulins, and / or the light-chain constant region and / or the heavy-chain constant region. In one example, the anti-LILRB1 antibodies provided herein can be human IgG-type antibodies, for example, antibodies in the form of IgG1, IgG2, IgG3, or IgG4, but are 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 the heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy-chain constant region and a light-chain constant region. The heavy-chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, and has subclasses such as gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), or alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

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

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

[0039] In the present specification, 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, unless otherwise specifically mentioned.

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

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

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

[0043] The F(ab’)2 antibody is generated while the cysteine residues in the hinge region of Fab’ form disulfide bonds. Fv is the smallest antibody fragment having only the heavy-chain variable region and the light-chain variable region, and the recombinant techniques for generating Fv fragments are widely known in the art.

[0044] In two-chain Fv, the heavy-chain variable region and the light-chain variable region are linked by non-covalent bonds, and in single-chain Fv, generally, the variable region of the heavy chain and the variable region of the light chain are covalently linked via a peptide linker or directly linked at the C-terminus. Thus, like two-chain Fv, it can form a structure such as a dimer.

[0045] The antigen-binding fragment can be obtained using proteolytic enzymes (for example, Fab can be obtained by limited cleavage of the whole antibody with papain, and F(ab’)2 fragment can be obtained by cleavage with pepsin.), and is made using recombinant techniques.

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

[0047] The anti-LILRB1 antibody can be a monoclonal antibody. Monoclonal antibodies can be produced by methods known in the art. For example, they can be produced using phage display techniques. Alternatively, the anti-LILRB1 antibody can be produced as a mouse-derived monoclonal antibody by ordinary methods.

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

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

[0050] The effective amount of the pharmaceutical composition, or the antibody or antigen-binding fragment thereof, can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, intranasal administration, intratracheal administration, rectal administration, or local administration to the lesion site. When administered orally, since proteins or peptides are digested, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. Further, the composition can be administered by any device that enables the active substance to reach the target cells (e.g., cancer cells).

[0051] The anti-LILRB1 antibody or its antigen-binding fragment is contained in the pharmaceutical composition in a pharmaceutically effective amount or can be administered to a patient. As used herein, "pharmaceutically effective amount" means the amount of the active ingredient (anti-LILRB1 antibody or its antigen-binding fragment) that can exert the 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, gender, pathological condition, diet, excretion rate, responsiveness, formulation method, administration time, administration interval, administration route, administration mode, etc. For example, the daily dose of the anti-LILRB1 antibody or its antigen-binding fragment can be in the range of 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 250 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, but is not limited thereto. The daily dose can be formulated in a single formulation in unit volume form, or appropriately dispensed and formulated, or manufactured 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 forms such as extracts, powders, powders, granules, tablets or capsules, and may further contain a dispersant or stabilizer for formulation.

[0053] The patients to whom the present invention is applicable can be mammals including primates such as humans and monkeys, and rodents such as mice and rats.

[0054] The cancer may be a solid cancer or a blood cancer, and is not limited thereto, but may be one or more selected from the group consisting of lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, etc.), peritoneal cancer, skin cancer, cutaneous or uveal melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, 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 have MHC class I expressed or overexpressed on its surface, and may be, for example, colon adenocarcinoma, small cell lung carcinoma, breast cancer, pancreatic cancer, malignant melanoma, bone osteosarcoma, renal cell carcinoma, gastric cancer. The overexpression of MHC class I means that it is overexpressed in the cancer cells to which the antibody is applied as compared with normal cells or cancer cells in which no anti-cancer effect appears (non-responsive or resistant to the immunotherapy), for example, T-cell (e.g., cytotoxic T-cell)-mediated immunotherapy.

[0055] In this specification, the treatment of cancer means all anti-cancer effects that prevent, relieve or improve the deterioration of cancer symptoms such as suppression of cancer cell proliferation, cancer cell death, metastasis suppression, or eliminate the cancer partially or completely.

[0056] The anti-LILRB1 antibodies or antigen-binding fragments thereof provided in this specification can be used in combination with one or more other drugs selected from the group consisting of, for example, commonly used immunotherapeutic agents, anti-cancer agents, cytotoxic agents, and the like. Thus, as an example, there is provided a pharmaceutical composition for combination administration for the prevention and / or treatment of cancer, comprising (1) an anti-LILRB1 antibody or an antigen-binding fragment thereof, and (2) one or more drugs selected from the group consisting of immunotherapeutic agents, anti-cancer agents, cytotoxic agents, and the like. Another example provides a method for the prevention and / or treatment of cancer, which includes the step of administering to a patient in need of prevention and / or treatment of cancer (1) an anti-LILRB1 antibody or an antigen-binding fragment thereof, and (2) one or more drugs selected from the group consisting of immunotherapeutic agents, anti-cancer agents, cytotoxic agents, and the like. The immunotherapeutic agents, anti-cancer agents, and cytotoxic agents generally include all drugs used in cancer treatment and / or having cytotoxic activity, and are selected from one or more of proteins such as antibodies, nucleic acid molecules such as siRNA, and / or small molecule compounds such as paclitaxel and docetaxel, but are not limited thereto.

[0057] As another example, there is provided a polypeptide molecule comprising the heavy chain complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, or a combination thereof), light chain complementarity determining regions (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 in antibody production as a precursor of an antibody, but also included as a component of a protein scaffold (such as 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 part in a target cell therapeutic agent such as CAR-T, a secreted antibody, a cell therapeutic agent prepared to secrete the anti-LILRB1 antibody, and the like.

[0058] As another example, provided is 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.

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

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

[0061] As another example, provided is a recombinant cell containing the nucleic acid molecule or the recombinant vector.

[0062] The term "vector" means an expression means for expressing a target gene in a host cell. For example, it includes 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 frequently used plasmids in the art (such as pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (such as λgt4λB, λ-Charon, λΔz1, and M13, etc.), or viruses (such as SV40, etc.).

[0063] In the recombinant vector, the nucleic acid molecule can be operatively linked to a promoter. The term "operatively linked" means a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. By being "operatively linked", the regulatory sequence can 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 use those 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 a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector used is an expression vector and a prokaryotic cell is used as the host, it generally contains a strong promoter for promoting transcription (e.g., pL λ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for the initiation of translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as the host, the origins of replication that function in eukaryotic cells contained in the vector include, but are not limited to, f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adenovirus origin of replication, AAV origin of replication, and BBV origin of replication. Also, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter), or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, etc.) is used, and it generally has a polyadenylation sequence as a transcription termination sequence.

[0066] The recombinant cell can be obtained by introducing the recombinant vector into an appropriate host cell. The host cell can be any cell known in the art that can stably and continuously clone or express the recombinant vector. As prokaryotic cells, for example, Escherichia coli such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis and other Bacillus strains, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. When transforming eukaryotic cells, host cells such as yeast (Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, for example, Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, CHO S, CHO DXB11, CHO GS-KO, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell lines, etc. can be used, but are not limited thereto.

[0067] Introduction (transfection) of the nucleic acid molecule or the recombinant vector containing the same into the host cell can use delivery methods widely known in the art. For the transfection method, for example, when the host cell is a prokaryotic cell, the CaCl2 method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, the liposome-mediated transfection method and the gene bombardment method can be used, but are not limited thereto.

[0068] The method for screening 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 screened by culturing the transformant in a medium containing the antibiotic.

[0069] As another example, provided is a method for producing an anti-LILRB1 antibody or an antigen-binding fragment thereof, which includes the step of expressing the nucleic acid molecule or a recombinant vector containing the same in a host cell. The step of expressing can be carried out by culturing a recombinant cell containing the nucleic acid molecule (for example, contained in a recombinant vector) under conditions that permit the expression of the nucleic acid molecule. The production method may include, after the step of expressing or culturing, a step of separating and / or purifying the antibody or antigen-binding fragment from the culture medium.

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 escape mechanism of cancer cells and enabling it to be well exerted without inhibiting the anti-cancer efficacy of immune cells.

Brief Description of the Drawings

[0071]

Figure 1

Figure 2

Figure 3

Figure 4a

Figure 4b

Figure 4c

Figure 5

Figure 6

Figure 7a

Figure 7b

Figure 7c

Figure 7d

Figure 8a

Figure 8b

Figure 8c

Figure 8d

Figure 9

Figure 10

Figure 11

Figure 12

Modes for Carrying Out the Invention

[0072] Hereinafter, the present invention will be described more specifically with reference to examples, which are merely illustrative and not intended to limit the scope of the present invention. It will be apparent 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, phage display selection was performed using a library of human scFv antibodies. As antigens, human LILRB1-His (Cat.No.8989-T2) and human LILRB1-Fc (Cat.No.2017-T2) manufactured by RnD systems were used respectively. Also, 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 antigens (LILRB1-His, LILRB1-Fc, LILRB1-His-Biotin, LILRB1-Fc-Biotin) by solid-phase and solution-phase selection methods. Additional selection was performed by methods such as gradually reducing the antigen concentration used, eluting competitively using a control antibody against LILRB1, or performing negative selection against Fc when LILRB1-Fc was used as the antigen. The selected products were confirmed for binding to the antigen by multi-clone phage ELISA.

[0075] 1.2. Single clone soluble scFv screening and analysis The gene encoding the scFv whose binding to the antigen was confirmed in Example 1.1 above was amplified by PCR to prepare an expression vector. For screening for each selection group, a certain number of transformants were transferred to a 96 well culture plate. After expressing the antibody in scFv form using Autoinduction media (Studier,F.W.(2005)Protein Expression and Purification 41,207-34), DELFIA immune assay (PerkinElmer) was performed to confirm binding to the antigen. Also, after coating a certain amount of scFv antibody on the surface, DELFIA against the antigen was performed to determine the ranking of the antigen-antibody binding force.

[0076] 1.3. Conversion of the Selected scFv Antibodies into IgG Antibodies A total of 376 clones were selected from among the clones in which binding to the antigen was confirmed in Example 1.2 above, and the nucleic acid sequences of the genes encoding the selected scFvs were analyzed by a general DNA sequence analysis method to remove overlapping clones. Also, a total of 93 clones were selected based on the ranking for the antigen-antibody binding force determined in Example 1.2 above. Sequences corresponding to the respective heavy chain variable regions (VH) and light chain variable regions (VL) were amplified by PCR from the genes encoding the selected scFvs, and an expression vector (pTRIOZ-hIgG4; InvivoGen, which also has a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1), and either one of the vectors containing the constant region sequences of the heavy chain of human IgG4 and the constant region of kappa or lambda light chain can be used) prepared to encode an IgG4-form human antibody (IgG4 Fc: SEQ ID NO: 341, Kappa constant region: SEQ ID NO: 342, Lambda constant region: SEQ ID NO: 343) was inserted. The DNA sequence of the expression vector was confirmed by sequencing.

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

[0078] Specifically, the vector constructed in Example 1.3 above was transfected into ExpiCHO-S TM cells (Gibco) (1.5×10 8 cells / Culture Volume 25 mL) with ExpiFectamine TM80 μL of CHO reagent (Thermo Fisher) was added for transfection. One day after transfection, ExpiCHO TM Enhancer (Thermo Fisher) 150 μL and ExpiCHO TM Feed (Thermo Fisher) 4 mL were added. On the 5th day, 4 mL of ExpiCHO TM Feed was added. The transfected cells were cultured for a total of 7 - 11 days under the conditions of 32°C and 5% CO₂.

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

[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 solution was purified using AKTA Pure L (GE healthcare). An Hitrap MabSelectSure 1 mL column (GE healthcare) was attached to the AKTA Pure L, and the culture solution was flowed at a flow rate of 1 mL / min and then washed with 1X PBS at 20 column volume (CV). The elution buffer (0.1 M sodium citrate pH 3.4 buffer) was flowed to elute the target protein. 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 prepare a concentration of approximately 1 mg / mL. After mixing 10 μL of Reducing Loading Buffer (3X) or Non-reducing Loading Buffer (3X) with 20 μL of the purified antibody sample, the mixture was left standing in a 95°C heating bath for 2 minutes, then taken out and cooled. After installing an SDS-PAGE Gradient Gel (4~20% or 4~12%) in the electrophoresis apparatus, 10 μg of the sample was injected per well and the gel was developed. Precision Plus Protein TM Dual Color Standards (BIO-RAD) were injected into a separate well. The gel was stained with Coomassie staining solution and destained, and then a gel photo was taken.

[0082] Among these 93 antibodies, the gel electrophoresis photos of antibodies A10, B3, E3, G1, G9, and H2 are shown in Figure 1 as representatives. As shown in Figure 1, it was possible to confirm the production of antibodies having disulfide bonds.

[0083] 1.5. Analysis of the binding affinity of the selected antibodies The affinity of the 93 antibodies selected in Example 1.3 for the LILRB1 antigen was 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 flowed 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) and immobilized at approximately 5000 - 7000 RU. The antigen, human LILRB1 protein (LILRB1-His, RnD systems Cat. No. 8989-T2), was injected at a rate of 30 μL / min at 4 - 9 different concentrations within a concentration range from 3.13 nM to 1600 nM, and k a and k d values were determined, and from these the K D value was calculated.

[0084] Among the 93 antibodies, 20 antibodies showing excellent binding force (K D value) were selected, and the results are shown in Table 3 below. Among them, the SPR sensorgrams of B3, which showed a binding force of approximately 99.8 nM for the LILRB1 antigen, and E3, which showed a binding force of approximately 101.2 nM for the LILRB1 antigen, are shown in Figure 2 and Figure 3 respectively (Figure 2: SPR sensorgram for B3, Figure 3: SPR sensorgram for E3).

Table 3

[0085] 1.6. Sequence analysis of the selected antibodies Based on the CDR definition according to the Kabat numbering of the 20 antibodies whose binding ability to the antigen was confirmed in Example 1.5 above, the CDRs defined thereby, the amino acid sequences of the light chain variable region, heavy chain variable region, light chain, and heavy chain, and the nucleic acid sequences of the coding genes of the light chain variable region and heavy chain variable region were analyzed by general amino acid sequence analysis and DNA sequence analysis methods and organized 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 the selected antibodies 2.1. NK cell surface binding assay To confirm whether the 93 antibodies selected in Example 1.4 also bind to LILRB1 expressed on the surface of immune cells, an NK cell surface binding assay was performed. KHYG-1 cells (JCRB), which are human NK cells, were cultured in RPMI 1640 medium (Gibco) containing 10% (w / v) FBS (Gibco) and 100 U / mL interleukin-2 (Novartis). KHYG-1 cells were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at a density of 5×10 4 cells / well. The test antibody with a final concentration of 50 μg / mL per well was added and left to stand at 4°C for 1 hour.

[0087] For specific binding to LILRB1, a human IgG4 isotype control antibody (Biolegend) was similarly processed. 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 suspension was analyzed using an iQue screener (Sartorius).

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

Table 24

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

[0090] 2.2. Analysis of the ability of the 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 analysis of the ability to inhibit LILRB1 / HLA - G binding was performed.

[0091] Therefore, 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 dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at a density of 5×10 4 cells / 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 added to the cells and then left standing on ice for 2 hours. As a positive control group, an anti-LILRB1 antibody (clone HP-F1, Abcam) and, as a negative control group, an anti-lysozyme IgG4 antibody (clone D1.3) were prepared by the same treatment as above. After washing twice with FACS buffer, a PE-anti-huIgG-Fc antibody (Biolegend, 10 μg / mL) was added to each well and left standing on ice for 1 hour. After washing twice with FACS buffer, the cells were suspended in 100 μL of the same buffer and analyzed with an iQue screener (Sartorius).

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

[0093] 2.3. Analysis of the ability of NK cells to kill cancer cells To confirm whether the selected antibodies increase the ability of NK cells to kill cancer cells, the killing rate of HLA-G overexpressing HEK293 cells by NK cells KHYG-1 was analyzed. KHYG-1 cells (JCRB) were adjusted to 2×104 cells / well (4×10 4 cells / mL, total volume 50 μL) was dispensed into a 96-well tissue culture plate (BD Falcon). The test antibody (Table 25) was added so that the final concentration per well was 20 μg / mL, and the plate was left standing at 37°C for 1 hour.

[0094] As a negative control group, the 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 lentivirus prepared to express HLA-G1 to produce an HLA-G overexpressing cell line) were stained using IncuCyte CytoLight Rapid Red Reagent (Sartorius) according to the manufacturer's instructions. After 1 hour, the HLA-G overexpressing HEK293 cells were added to the plate at 1×10 4 cells / well (2×10 4 cells / mL, total volume 50 μL). The plate was placed in an IncuCyte S3 (Sartorius) provided in a 37°C, 5% CO2 incubator, images were taken for 72 hours, and the Red area confluence indicating the density of living HLA-G overexpressing HEK293 was measured to determine the cell viability. The cell viability is shown in Table 25 below (expressed as a relative value with the control antibody set to 1):

Number

Table 25

[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: Analysis of the in vivo biological activity of the selected antibodies Among the antibodies selected in Example 1.5 above, the need to improve the anti-cancer efficacy of two test antibodies (E3, B3) was confirmed in vivo. For this purpose, the Bioware Brite Cell Line HCT116 Red-Fluc colorectal cancer cells (PerkinElmer) and THP-1 derived macrophages were used, and the in vivo effect of administering the two antibodies on reducing the tumor size was confirmed in a xenograft colorectal cancer mouse animal model administered with the antibodies. As a negative control group, a xenograft colorectal cancer mouse animal model administered with a human IgG1 isotype control antibody (BioXcell, Cat. No. BP0297) in the same manner as above was prepared. The above process will be described more specifically below:

[0098] Preparation of THP-1 derived macrophages

[0099] The THP-1 derived macrophages used above were prepared by differentiating 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] Analysis of anti-cancer efficacy in a mouse animal model Five-week-old female CIEA NOG Mouse [NOG immunodeficient mouse] (Experimental Animal Central Institute, Incorporated Administrative Agency) was inoculated with 3×10 6Individual HCT116 Red-Fluc colorectal cancer cells and 3×10 6 individual THP-1-derived macrophages, and a mixture of each of two test antibodies (E3 or B3 antibody; administered at 20 μg per animal) were used by subcutaneous injection. After tumor transplantation, starting from the 4th day, the antibody was administered twice a week by intraperitoneal injection at a concentration of 5 mg / kg, and the size of the transplanted tumor (mm 3 ) was 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 colorectal cancer and THP-1-derived macrophages, especially the E3 antibody, showed a clear tumor growth inhibitory effect at a statistically significant level.

[0101] Example 4: Preparation 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 showed particularly excellent effects in Example 3 above, was PCR amplified, and after PCR amplifying the nucleic acid sequence corresponding to Ser1 to Leu110 of the light chain variable region (VL) (SEQ ID NO: 221) of the E3 antibody, it was ligated with the lambda constant region (Lambda CL.1, SEQ ID NO: 344) nucleic acid sequence to PCR amplify the lambda light chain region coding nucleic acid sequence. The amplified sequence was inserted into an expression vector (pTRIOZ-hIgG4; InvivoGen, and alternatively, any vector having a CMV promoter, or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and containing sequences encoding the heavy chain constant region of human IgG4 and the constant region of lambda light chain can be used) prepared to code for a human antibody in IgG4 form. The DNA sequence of the expression vector was confirmed by sequencing.

[0102] Using the constructed expression vector, an antibody (E3.1) was prepared 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 A 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 alternatively, any vector having a CMV promoter or a CMV / CHO beta-actin fusion promoter (KR10-1038126B1) and containing the constant region sequence of the heavy chain of human IgG4 and the constant region sequence of lambda light chain) prepared to encode the sequence. The sequence of the expression vector was confirmed by sequencing. The constructed vector was transfected into CHO cells to prepare 11 stable cell lines that overexpress each LILR protein on the cell surface.

Table 27-1

Table 27-2

Table 27-3

Table 27-4

[0104] Example 6: EC for the Binding of the Selected Antibodies to the Surface of LILRB1-Overexpressing Cells 50 Measurement To measure the EC value of the antibodies prepared in Example 1 and Example 4 for binding to the human LILRB1-overexpressing cell line 50 a cell surface binding assay was performed. As representatives of the prepared antibodies, the EC values of E3.1 and H11 antibodies 50The value was measured. CHO cells overexpressing LILRB1 prepared in Example 5 on the cell surface were dispensed into a U-bottom 96-well tissue culture plate (BD Falcon) at 1×10 5 cells / well. After serially diluting E3.1 from 600 μg / mL and H11 from 27 μg / mL by one-third each for each well, the cells were treated and left standing at 4°C for 60 minutes. After washing with FACS buffer, the cells were treated with an anti-human Fc-biotin antibody (Invitrogen) and left standing at 4°C for 30 minutes. After washing with FACS buffer, the cells were treated with streptavidin labeled with PE fluorescence (BD Pharmigen) and left standing 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 formula of GraphPad Prism software, and the obtained results are shown in Table 28: [Table 28]

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

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

[0107] Example 8: Measurement of Granzyme B and Perforin Secretion by the Enzyme-Linked Immune Absorbent Spot (ELISPOT) To confirm whether E3.1 and H11 antibodies increase the cytotoxic performance of NK cells, an enzyme-linked immunosorbent spot (ELISPOT) assay was performed. The cytotoxic performance was confirmed by the expression levels of granzyme B and perforin, which are cytotoxic substances in NK cells.

[0108] 5×10 3 cells of the KHYG-1 cell line (JCRB) expressing LILRB1 were co-cultured with 5×10 3 cells of K562 cells (American Type Culture Collection) overexpressing HLA-G (K562 cells were transduced with lentivirus prepared to express HLA-G to generate an HLA-G overexpressing cell line). At this time, E3.1, H11, and human IgG4 isotype control antibodies were added so that the final concentration per well was 50 μg / mL, and the cells were cultured at 37°C for 30 minutes. The cells co-cultured were transferred to a 96-well plate for ELISPOT (Immunospot, Cat. HGZBPFN-2M) (PVDF membrane) coated with anti-perforin antibody and anti-granzyme B antibody, and further cultured at 37°C for 8 hours. After washing the PVDF membrane with a washing solution (0.05% tween 20 in PBS), anti-granzyme B-HRP and anti-perforin-biotin were added sequentially, and then the detection process was carried out according to the manufacturer's manual. After drying the PVDF membrane at room temperature for 24 hours, the number of granzyme B and perforin spots was measured using an ELISPOT analyzer manufactured by Immunospot.

[0109] The above-mentioned obtained results are shown in Figure 9 (granzyme b; Gzmb) and Figure 10 (perforin; Prf), respectively (the Y-axis indicates the total number of spots). As shown in Figures 9 and 10, in the case of granzyme B and perforin, it can be confirmed that the secretion amount was statistically significantly increased during the treatment with E3.1 or H11 antibody compared to the treatment with human IgG4 isotype control antibody. The statistical analysis was performed by unpaired T-test, and all experiments were conducted 3 times under the same conditions for the reliability of the experiment, and the results were represented as average values.

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

[0111] Specifically, after analyzing the amino acid sequence of the GHI / 75 antibody by Peptide mapping, a vector was prepared by substituting the nucleic acid sequence corresponding to the variable region (VH, VL domain) of the mouse GHI / 75 antibody with the nucleic acid sequence of the variable region (VH, VL domain) of the human IgG4 antibody. The portion corresponding to the upper hinge of human IgG4 was substituted with the amino acid sequence of the human IgG1 upper hinge (EPKSCDKTHT; SEQ ID NO: 359). The antibody obtained by expressing and purifying the vector through the same process as in Example 1.4 was used as a comparative antibody in the following tests.

[0112] Example 10: Measurement of the Ability of the Selected Antibody to Inhibit LILRB1 Signaling Using IL-2 Promoter Luciferase Assay To confirm whether the antibodies prepared in Example 1 and Example 4 inhibit signal transduction by LILRB1, a luciferase reporter assay was performed. Among the antibodies prepared in Example 1 and Example 4, typically, tests were conducted on E3.1 and H11 antibodies, 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 the Jurkat cell line (American Type Culture Collection) and then transducing with a lentivirus prepared to express LILRB1) and a K562 cell line overexpressing HLA-G were used. After adding anti-CD3 antibody (Biolegend) to a 96-well plate, it was coated overnight at 4°C. The next day, 1×10 5 cells / well of Jurkat cells expressing LILRB1 and IL-2 promoter luciferase were added to a U-bottom 96-well plate, and E3.1, H11, chimeric GHI / 75, and human IgG4 isotype (control group) antibodies were each treated to a final concentration of 20 μg / mL and then left standing at 37°C for 1 hour. Then, 1×10 5 K562 cells overexpressing HLA-G were added to the plate and left standing at 37°C for 30 minutes. Then, the suspension was transferred to the plate coated with anti-CD3, and anti-CD28 antibody (Biolegend) was added to a final concentration of 10 μg / mL. After leaving the plate standing at 37°C for 6 hours, Steady-Glo® (Promega) solution was added to each well, and then analyzed using a luminometer (Envision, PerkinElmer).

[0113] The results obtained above are shown in Fig. 11. As shown in Fig. 11, the E3.1 antibody and the 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 comparison antibody.

[0114] Example 11: Analysis of the anti-cancer efficacy of the selected antibodies in a mouse animal model For the analysis of the anti-cancer efficacy of the selected antibodies, 5-week-old female CIEA NOG Mice (NOG immunodeficient mice, Central Institute for Experimental Animals, Incorporated Administrative Agency) were subcutaneously injected with 3×10 6 cells of HCT116 Red-Fluc colorectal cancer cells, 3×10 6 cells of THP-1-derived macrophages, and two test antibodies (administered at 20 μg per mouse) to prepare a mouse animal model. The test antibodies used were E3.1 and H11, and a human IgG4 isotype control antibody was used for comparison. After tumor cell transplantation, the antibodies were intraperitoneally injected twice a week at a volume of 5 mg / kg starting from the 4th day, and the tumor volume was measured. The results are shown in Fig. 12. As shown in Fig. 12, both the E3.1 and H11 antibodies showed excellent tumor growth inhibitory efficacy against the control antibody in the mouse animal model transplanted with HCT116 colorectal cancer cells and THP-1-derived macrophages.

Claims

Claim 1 An anti-LILRB1 antibody or an antigen-binding fragment thereof, comprising the following complementarity determining regions (CDRs): 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 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 (The CDRs are defined based on Kabat numbering.) Claim 2 The anti-LILRB1 antibody or an antigen-binding fragment thereof according to claim 1, comprising: (1) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 2, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 6; (2) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 9, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 11, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 12; (3) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 15, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18; (4) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 19, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 20, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 21, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 22, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 24; (5) 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; (6) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 31, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 32, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; (7) 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; (8) 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; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 52, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 53, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 54; 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; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 61, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 62, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 68, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 69, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 71, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 73, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 74, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 75, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 76, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 77, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 78; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 79, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 80, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 81, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 82, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 83, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 84; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 85, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 86, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 87, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 88, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 89, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 90; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 91, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 92, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 93, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 94, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 95, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 96; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 97, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 98, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 99, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 100, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 101, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 102; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 103, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 104, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 105, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 106, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 107, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 108; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 109, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 110, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 111, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 112, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 113, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 114; or CDR-L1 comprising the amino acid sequence of SEQ ID NO: 115, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 116, CDR-L3 comprising the amino acid sequence of SEQ ID NO: 117, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 118, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 119, and CDR-H3 comprising the amino acid sequence of SEQ ID NO:

120.

3. 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 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: 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258 or 260.

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

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

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

7. The pharmaceutical composition according to claim 6, wherein the cancer has the property of overexpressing MHC class I.

8. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

9. A recombinant vector comprising the nucleic acid molecule according to claim 8.

10. A recombinant cell comprising the nucleic acid molecule according to claim 8 or a recombinant vector comprising the same.

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

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