Anti-LILRB1 antibodies and uses thereof

Abstract

The present invention relates to anti-LILRB1 antibodies and uses thereof. In particular, the present invention relates to anti-LILRB1 antibodies with increased specificity for LILRB1 and uses thereof. Specifically, there is provided an anti-LILRB1 antibody, or an antigen-binding fragment thereof, and its use in the treatment of cancer.

Description

[0001] This application is a divisional application of the application with international application date of December 22, 2020, international application number PCT / KR2020 / 018931, entering the Chinese national phase on June 16, 2022, application number 202080087379.8, and invention name “Anti-LILRB1 antibodies and their uses”. Technical Field

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of KR 10-2019-0173414, filed on December 23, 2019, and KR 10-2020-0061907, filed on May 22, 2020, in the Korean Intellectual Property Office, the disclosures of which are incorporated herein by reference in their entirety.

[0004] The present disclosure relates to an anti-LILRB1 antibody and its use. More specifically, an anti-LILRB1 antibody or an antigen-binding fragment thereof and its use for cancer treatment are provided. Background Art

[0005] Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1; also known as ILT2, CD85j or LIR-1) is an inhibitory receptor expressed in cells such as B cells, T cells, NK cells, dendritic cells, macrophages and other immune cells. LILRB1 participates in signal transduction mechanisms that inhibit immune cell activity by binding to classical and nonclassical MHC class I.

[0006] Meanwhile, various cancer cells have been reported to overexpress MHC class I such as HLA-G for immune escape. Blocking the binding of LILRB1 to MHC class I is expected to allow the restoration of suppressed immune cell activity, thereby exhibiting anticancer effects.

[0007] Therefore, there is a need to develop novel agents that bind to LILRB1 and block the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I. Summary of the invention

[0008] Technical issues

[0009] The present disclosure provides antibodies that bind to LILRB1, act on immune cells expressing LILRB1, regulate the activity of immune cells and exhibit anti-cancer effects, and uses thereof for cancer treatment.

[0010] One embodiment provides an anti-LILRB1 antibody or an antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or an antigen-binding fragment thereof may have the activity of blocking the binding of LILRB1 to MHC class I and / or blocking the interaction between LILRB1 and MHC class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have the activity of inhibiting the immune escape of cancer cells. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect. The anti-cancer effect may be directed against cancer cells that express or overexpress MHC class I on the cell surface.

[0011] Another embodiment provides a pharmaceutical composition for treating and / or preventing cancer, the composition comprising an anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0012] Another embodiment provides a pharmaceutical composition for inhibiting the binding of LILRB1 to MHC class I and / or blocking the interaction between LILRB1 and MHC class I, the composition comprising an anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0013] Another embodiment provides a pharmaceutical composition for inhibiting the immune escape of cancer cells, the composition comprising an anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0014]

Technical Solution

[0015] One embodiment provides an anti-LILRB1 antibody or an antigen-binding fragment thereof that binds to LILRB1. The anti-LILRB1 antibody or an antigen-binding fragment thereof may have the activity of blocking the binding of LILRB1 to MHC class I and / or blocking the interaction between LILRB1 and MHC class I. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have the activity of inhibiting the immune escape of cancer cells. In addition, the anti-LILRB1 antibody or an antigen-binding fragment thereof may have an anti-cancer effect.

[0016] The anti-LILRB1 antibody or antigen-binding fragment thereof may comprise the following complementarity determining regions (CDRs):

[0017] (1) CDR definitions based on Kabat numbering (Kabat, EA, Wu, TT, Perry, H., Gottesman, K., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242; http: / / www.abysis.org / ),

[0018] CDR-L1 comprises 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,

[0019] CDR-L2 comprises 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,

[0020] CDR-L3 comprises 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,

[0021] CDR-H1 comprises 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,

[0022] 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

[0023] 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

[0024] (2) based on the definition of CDRs according to IMGT numbering (http: / / www.imgt.org / ), CDR-L1 comprising an 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,

[0025] CDR-L2 comprises 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,

[0026] CDR-L3 comprises 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,

[0027] CDR-H1 comprises 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,

[0028] 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

[0029] 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.

[0030] In a specific embodiment, the combination of 6 CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3) that may be included in the anti-LILRB1 antibodies or antigen-binding fragments thereof provided in the present disclosure is shown in Table 1:

[0031]

Table 1

[0032]

[0033]

[0034]

[0035]

[0036] In one embodiment, the anti-LILRB1 antibody or antigen-binding fragment thereof may comprise:

[0037] a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3, and

[0038] a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3,

[0039] wherein the CDRs are as described above.

[0040] More specifically, the anti-LILRB1 antibody or antigen-binding fragment thereof may comprise:

[0041] 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

[0042] 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.

[0043] In a specific embodiment, the combinations of light chain variable regions and heavy chain variable regions that may be included in the anti-LILRB1 antibodies or antigen-binding fragments thereof provided in the present disclosure are shown in Table 2:

[0044]

Table 2

[0045]

[0046]

[0047]

[0048] In the present disclosure, the expression "an antibody or antigen-binding fragment (e.g., CDR, variable region or heavy / light chain) comprising, consisting of or represented by a specific amino acid sequence" may refer to an antigen-binding fragment consisting essentially of: (1) the specific amino acid sequence or (2) an amino acid sequence in which non-significant mutations (e.g., substitution, deletion and / or addition of amino acid residues; resulting in no effect on antibody activity) are introduced into the amino acid sequence (1).

[0049] When measured by surface plasmon resonance (SPR), the anti-LILRB1 antibodies or antigen-binding fragments thereof provided in the present disclosure can have a molecular weight of 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 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. ...6 nM to 10 mM, 0.17 nM to 10 mM, 0.26 nM to 15 mM, 0.28 nM to 15 mM, 0.29 nM to 16 mM, 0.30 nM to 17 mM, 0.31 nM to 18 mM, 0.32 nM to 19 mM, 0.33 nM to 19 mM, 0.34 nM to 19 mM, 0.35 nM to 19 mM, 0.36 nM to 19 mM, 0.37 nM to 19 mM, 0.38 nM to 19 mM 0.5nM to 10mM, 1nM to 10mM, 0.001nM to 5mM, 0.005nM to 5mM, 0.01nM to 5mM, 0.05nM to 5mM, 0.1nM to 5mM, 0.5nM to 5mM, 1nM to 5mM, 0.001nM to 1mM, 0.005nM to 1mM, 0.01nM to 1mM, 0.05nM to 1mM, 0.1nM to 1mM, 0.5nM to 1mM, 1nM to 1mM, 0.001nM to 0.5mM, 0.005nM to 0.5mM, 0.01nM to 0.5mM, 0.05nM to 0.5mM, 0.1nM to 0.5mM, 0.5nM to 0.5mM, 1nM to 0.5mM, 0.001nM to 0.2mM, 0.005nM to 0.2mM, 0.01nM to 0.2mM, 0.05nM to 0.2mM, 0.05nM to 0.2mM, 0.01nM to 0.5mM, 0.05nM to 0.5mM, 0.1nM to 0.5mM, 0.5nM to 0.5mM, 1nM to 0.5mM, 0.001nM to 0.2mM, 0.005nM to 0.2mM, 0.01nM to 0.2mM, 0.05nM to 0.2mM, 0 The binding affinity (K) of LILRB1 (e.g., human LILRB1) to the target protein is preferably between 0.1 nM and 0.2 mM, 0.5 nM and 0.2 mM, 1 nM and 0.2 mM, 0.001 nM and 0.15 mM, 0.005 nM and 0.15 mM, 0.01 nM and 0.15 mM, 0.05 nM and 0.15 mM, 0.1 nM and 0.15 mM, 0.5 nM and 0.15 mM or 1 nM to 0.15 mM. D ).

[0050] Another embodiment provides a pharmaceutical composition comprising an anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient. For example, the pharmaceutical composition may be a pharmaceutical composition for treating and / or preventing cancer. The pharmaceutical composition may have the activity of inhibiting the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I. The cancer may be a cancer associated with the interaction between LILRB1 and MHC class I. In one embodiment, the pharmaceutical composition may have the activity of inhibiting the immune escape of cancer cells. The cancer cell may be a cell expressing or overexpressing MHC class I on the cell surface.

[0051] Another embodiment provides a composition for blocking the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I, the composition comprising an anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0052] Another embodiment provides a composition for inhibiting cancer cell immune escape, comprising an anti-LILRB1 antibody or an antigen-binding fragment thereof as an active ingredient.

[0053] Another embodiment provides a method for treating and / or preventing cancer, comprising administering (orally or parenterally) a pharmaceutically effective amount of an anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal, including a human) in need of treating and / or preventing cancer.

[0054] Another embodiment provides a method for blocking the binding of LILRB1 to MHC class I and / or a method for blocking the interaction between LILRB1 and MHC class I, the method comprising administering (orally or parenterally) a pharmaceutically effective amount of an anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal, including a human) in need of inhibition of the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I.

[0055] Another embodiment provides a method for inhibiting cancer cell immune escape, comprising administering (orally or parenterally) a pharmaceutically effective amount of an anti-LILRB1 antibody or an antigen-binding fragment thereof to a subject (e.g., a mammal, including a human) in need of inhibiting cancer cell immune escape.

[0056] The methods provided in the present disclosure may further include, prior to the administering step, the step of identifying a subject in need of treating and / or preventing cancer, inhibiting the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I, and / or inhibiting cancer cell immune escape.

[0057] Another embodiment provides a nucleic acid molecule (polynucleotide) encoding at least one polypeptide, wherein the polypeptide is selected from the CDR of the above-mentioned anti-LILRB1 antibody (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, CDR-H3, 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.

[0058] Another embodiment provides a recombinant vector comprising the nucleic acid molecule. In one embodiment, the recombinant vector can, separately (e.g., in two separate vectors) or all together (e.g., in one vector), comprise a nucleic acid molecule encoding a light chain variable region or a light chain and a nucleic acid molecule encoding a heavy chain variable region or a heavy chain. The recombinant vector can be used as an expression vector.

[0059] Another embodiment provides a recombinant cell comprising the nucleic acid molecule or the recombinant vector.

[0060] Another embodiment provides a method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, the method comprising expressing a nucleic acid molecule in a cell. The step of expressing the nucleic acid molecule may comprise culturing the recombinant cell.

[0061] As described herein, the antigen-binding fragment of an anti-LILRB1 antibody may refer to a fragment derived from an anti-LILRB1 antibody and retaining the antigen (LILRB1) binding affinity of the antibody. In one embodiment, the antigen-binding fragment may be a polypeptide comprising the six CDRs of the anti-LILRB1 antibody as described above, and may be, for example, scFv, scFv-Fc, scFv-Ck (κ constant region), scFv-Cλ (λ constant region), (scFv) 2 , Fab, Fab' or F(ab') 2 , but not limited thereto. In one embodiment, the antigen binding fragment may be scFv; a fusion polypeptide (scFv-Fc) in which scFv is fused to the Fc region 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 scFv is fused to the constant region of a light chain (e.g., κ or λ).

[0062] The anti-LILRB1 antibody or antigen-binding fragment thereof may have a regulatory activity, such as an antagonistic or agonistic activity, on the LILRB1 protein. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of blocking the binding of LILRB1 to MHC class I and / or the interaction between LILRB1 and MHC class I. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an activity of inhibiting the immune escape of cancer cells. In addition, the anti-LILRB1 antibody or antigen-binding fragment thereof may have an anti-cancer effect.

[0063] The protein LILRB1 used as an antigen for the anti-LILRB1 antibody or antigen-binding fragment thereof provided in the present disclosure may be derived from a mammal. For example, the LILRB1 used as an antigen may be human LILRB1 (e.g., GenBank Accession No. 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.

[0064] MHC class I may be a class of major histocompatibility complex (MHC) molecules. In one embodiment, the MHC class I may be human MHC class I and may be at least one selected from HLA (human leukocyte antigen)-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G, but is not limited thereto.

[0065] As described herein, the term "antibody" may refer to a protein that specifically binds to a specific antigen, and may be a protein produced by antigen stimulation in the immune system, or a protein produced by chemical synthesis or recombinant production, without specific limitations. The antibody may be non-naturally occurring, for example, produced by recombinant or synthetic production. The antibody may be an animal antibody (e.g., a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal or polyclonal antibody.

[0066] In the anti-LILRB1 antibodies or antigen-binding fragments thereof provided herein, the portion other than the heavy chain CDR and light chain CDR portions or the heavy chain variable region and light chain variable region as defined above may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.), and, for example, derived from a framework portion and / or a light chain constant region and / or a heavy chain constant region. In one embodiment, the anti-LILRB1 antibodies provided in the present disclosure may be antibodies in the form of human IgG, such as IgG1, IgG2, IgG3, or IgG4, but are not limited thereto.

[0067] A complete antibody (e.g., IgG type) has a structure containing two full-length light chains and two full-length heavy chains, wherein each light chain is connected to a corresponding heavy chain via a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region is of γ, μ, α, δ, or ε type, and has γ1, γ2, γ3, γ4, α1, or α2 as its subclass. The light chain constant region has a κ or λ type.

[0068] As used herein, the term "heavy chain" may be intended to encompass full-length heavy chains and fragments thereof, wherein the full-length heavy chain may include a variable region VH (including an amino acid sequence sufficient to provide specificity for an antigen), three constant regions CH1, CH2 and CH3, and a hinge. The term "light chain" may be intended to encompass full-length light chains and fragments thereof, wherein the full-length light chain may include a variable region VL (including an amino acid sequence sufficient to provide specificity for an antigen), and a constant region CL.

[0069] The term "complementarity determining region (CDR)" may refer to a portion of the variable region of an antibody that confers antigen binding specificity, and may refer to an amino acid sequence found in a hypervariable region of a heavy or light chain of an immunoglobulin. The heavy chain and light chain may include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3), respectively. The CDRs may provide contact residues that play an important role in the binding of an antibody to its antigen or antigenic epitope. As used herein, the terms "specific binding" and "specific recognition" may have the same general meaning as known to those of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to cause an immune response.

[0070] In the present disclosure, unless otherwise specified, the term "antibody" may encompass not only intact antibodies but also antigen-binding fragments of antibodies having antigen-binding ability.

[0071] The term "antigen-binding fragment" as used herein may refer to any type of polypeptide that comprises a portion capable of binding to an antigen (e.g., the 6 CDRs described herein), and may be, for example, scFv, (scFv)2 , scFv-Fc, Fab, Fab′ or F(ab′) 2 , but not limited thereto. In addition, as described above, the antigen binding fragment may be a scFv, a fusion polypeptide in which the scFv is fused to the Fc region or constant region (e.g., κ or λ) of an immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).

[0072] Among antigen-binding fragments, Fab includes light and heavy chain variable regions, a light chain constant region and the first heavy chain constant region, CH1.

[0073] Fab' differs from Fab in that Fab' comprises a hinge region having at least one cysteine ​​residue at the C-terminus of CH1.

[0074] F(ab') 2 Antibodies are formed by disulfide bridges of cysteine ​​residues in the Fab' hinge region.

[0075] Fv is the smallest antibody fragment consisting of only the heavy chain variable region and the light chain variable region. Recombinant techniques for producing Fv fragments are well known in the art.

[0076] Double-chain Fv comprises a heavy chain variable region and a light chain variable region connected to each other by a non-covalent bond. Single-chain Fv usually comprises a heavy chain variable region and a light chain variable region, which are connected to each other by a covalent bond or directly connected at the C-terminus via a peptide linker to have a dimer structure similar to a double-chain Fv.

[0077] Antigen-binding fragments can be obtained using proteases (e.g., Fab can be obtained by restrictive cleavage of whole antibodies with papain, and F(ab') 2 The fragment can be obtained by cleavage with pepsin), or can be prepared by using gene recombination technology.

[0078] The term "hinge region" may refer to the region between the CH1 and CH2 domains within an antibody heavy chain, which functions to provide flexibility to the antigen binding site in the antibody.

[0079] The anti-LILRB1 antibody may be a monoclonal antibody or a polyclonal antibody, and is, for example, a monoclonal antibody. The monoclonal antibody may be prepared using methods well known in the art, such as phage display technology. Alternatively, the anti-LILRB1 antibody may be constructed in the form of a mouse-derived monoclonal antibody by conventional methods.

[0080] Meanwhile, based on the binding potential to LILRB1, individual monoclonal antibodies can be screened using a typical ELISA (enzyme-linked immunosorbent assay) format. The inhibitory activity can be verified by functional assays (e.g., competitive ELISA for verifying the molecular interaction of the binding assembly) or functional assays (e.g., cell-based assays). Then, for the monoclonal antibody members selected based on their strong inhibitory activity, their affinity (Kd value) to LILRB1 can be verified individually.

[0081] In addition to the active ingredient (anti-LILRB1 antibody or antigen-binding fragment thereof), the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be selected from any carrier commonly used for antibody preparations. For example, a pharmaceutically acceptable carrier may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. One or more, but not limited thereto. The pharmaceutical composition may also include one or more selected from diluents, excipients, lubricants, wetting agents, sweeteners, flavor enhancers, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions.

[0082] The pharmaceutical composition or the antibody or its Fab can be administered orally or parenterally in a pharmaceutically effective amount. Parenteral administration can be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, intranasal administration, intrapulmonary administration, rectal administration or topical administration in a lesion. Since proteins or peptides are digested when administered orally, the active ingredient in the composition for oral administration can be coated or formulated to prevent digestion in the stomach. In addition, the antibody or the composition can be administered using an optional device that can deliver the active ingredient to target cells (e.g., cancer cells).

[0083] The anti-LILRB1 antibody or antigen-binding fragment thereof can be contained in a pharmaceutical composition or administered to a subject in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" may refer to the amount of active ingredient that enables the active ingredient (antibody or fragment thereof) to exert a desired effect (e.g., anti-cancer effect). The pharmaceutically effective amount can be specified in a variety of ways, depending on a variety of factors, such as the subject's age, weight, sex, pathological condition, diet, excretion rate and / or reaction sensitivity, formulation type, administration time, administration interval, administration route, administration method, etc. For example, the anti-LILRB1 antibody or antigen-binding fragment thereof can be administered daily at 0.005 ug / kg to 1000 mg / kg, 0.005 ug / kg to 500 mg / kg, 0.005 ug / kg to 250 mg / kg, 0.005 ug / kg to 100 mg / kg, 0.005 ug / kg to 75 mg / kg, 0.005 ug / kg to 50 mg / kg, 0.01 ug / kg to 1000 mg / kg, 0.01 ug / kg to 500 mg / kg, 0.01 ...250 mg / kg, 0.005 ug / kg to 1000 mg / kg, 0.01 ug / kg to 500 mg / kg, 0.01 ug / kg to 250 mg / kg, 0.005 ug / kg to 250 mg / kg, 0.005 ug / kg to 1000 mg / kg, 0.01 ug / kg to 500 mg / kg, 0.01 ug / kg to 250 mg / kg, 0.005 ug / kg to 250 mg / kg, 0.005 ug / kg to 250 mg / kg, 0.005 ug / kg to 1000 mg / kg, 0 The amount of 0.01 ug / kg to 250 mg / kg, 0.01 ug / kg to 100 mg / kg, 0.01 ug / kg to 75 mg / kg, 0.01 ug / kg to 50 mg / kg, 0.05 ug / kg to 1000 mg / kg, 0.05 ug / kg to 500 mg / kg, 0.05 ug / kg to 250 mg / kg, 0.05 ug / kg to 100 mg / kg, 0.05 ug / kg to 75 mg / kg or 0.05 ug / kg to 50 mg / kg is used, but is not limited thereto. The daily dose can be formulated into a single preparation of a unit dosage form or formulated into a suitable separate dosage form, or it can be manufactured to be contained in a multi-dose container.

[0084] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup, emulsified solution, extract, powder, granule, tablet or capsule in oil or aqueous medium, and may further contain a dispersant or stabilizer for formulation.

[0085] The subject to which the antibodies, pharmaceutical compositions or methods provided in the present disclosure are applied can be selected from mammals, including primates such as humans and monkeys, rodents such as rats and mice, and the like.

[0086] Cancer can be solid cancer or blood cancer.Cancer can be but not limited to be selected from lung cancer (for example, squamous cell carcinoma of the lung, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma), peritoneal cancer, skin cancer, squamous cell carcinoma, skin or eyeball melanoma, rectal cancer, cancer near anus, esophageal cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, leukemia (for example chronic or acute leukemia), lymphocytic lymphoma, liver cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland tumor, renal cell carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, biliary tract cancer, gallbladder cancer, one or more of bone osteosarcoma, etc.Cancer can be primary cancer or metastatic cancer. The cancer may be a cancer characterized by expression or overexpression of MHC class I on the surface of cancer cells, and may be, for example, colon adenocarcinoma, small cell lung cancer, breast cancer, pancreatic cancer, malignant melanoma, skeletal osteosarcoma, renal cell carcinoma, or gastric cancer. Overexpression of MHC class I may refer to overexpression compared to normal cells or cancer cells that are unresponsive or resistant to immunotherapy, such as T cell (e.g., cytotoxic T cell)-mediated immunotherapy.

[0087] As used herein, the term "cancer treatment" may refer to all anti-cancer effects that prevent, alleviate or improve cancer symptoms or partially or completely eliminate cancer, such as cancer cell death, inhibition of cancer cell proliferation, inhibition of cancer metastasis, etc.

[0088] The anti-LILRB1 antibody or antigen-binding fragment thereof provided in the present disclosure may be co-administered with another drug, the other drug being, for example, at least one selected from conventionally used immunotherapeutic agents, anticancer agents, cytotoxic agents, and the like. Therefore, one embodiment provides a pharmaceutical composition for combined administration for treating and / or preventing cancer, comprising (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) at least one selected from immunotherapeutic agents, anticancer agents, cytotoxic agents, and the like. Another embodiment provides a method for treating and / or preventing cancer, the method comprising administering (1) an anti-LILRB1 antibody or antigen-binding fragment thereof, and (2) at least one selected from immunotherapeutic agents, anticancer agents, cytotoxic agents, and the like to a subject in need of treating and / or preventing cancer. The immunotherapeutic agent, anticancer agent, and cytotoxic agent may include any drug conventionally used for cancer treatment and / or having cytotoxic activity, and for example, they may be at least one selected from proteins such as antibodies, nucleic acid molecules such as siRNA, and / or small molecule chemicals such as paclitaxel, docetaxel, and the like, but are not limited thereto.

[0089] Another embodiment provides a polypeptide molecule comprising a heavy chain complementary determining region (CDR-H1, CDR-H2, CDR-H3 or a combination thereof), a light chain complementary determining region (CDR-L1, CDR-L2, CDR-L3 or a combination thereof), a combination thereof, or a heavy chain variable region, a light chain variable region or a combination thereof of an anti-LILRB1 antibody as described above. The polypeptide molecule can be used as an antibody precursor for the preparation of an antibody, or as a component contained in a protein scaffold (e.g., a peptibody), a bispecific antibody or a multispecific antibody having an antibody-like structure. In another embodiment, the polypeptide molecule can be used as a target (antigen) recognition domain or secreted antibody in a cell therapeutic agent such as CAR-T for targeted therapy. In another embodiment, the polypeptide molecule can be used to construct an anti-LILRB1 antibody-secreting cell as a cell therapeutic agent.

[0090] Another embodiment provides 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.

[0091] Another embodiment provides a nucleic acid molecule encoding a light chain complementary 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.

[0092] Another embodiment provides a recombinant vector comprising nucleic acid molecules encoding the heavy chain variable region or heavy chain of an anti-LILRB1 antibody and the light chain variable region or light chain of an anti-LILRB1 antibody in two separate vectors or all together in one vector.

[0093] Another embodiment provides a recombinant cell comprising a nucleic acid molecule or a recombinant vector.

[0094] The term "vector" refers to a tool for expressing a target gene in a host cell, such as a plasmid vector, a cosmid vector, and a viral vector such as a phage vector, a lentiviral vector, an adenoviral vector, a retroviral vector, and an adeno-associated viral vector. The recombinant vector can be constructed by or by manipulating a plasmid (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, M13, etc.) or a viral vector (e.g., SV40, etc.), which is commonly used in the art.

[0095] In a recombinant vector, a nucleic acid molecule can be operably linked to a promoter. The term "operably linked" is intended to refer to a functional connection between a target nucleotide sequence and an expression control sequence (e.g., a promoter sequence). When "operably linked", a regulatory element can control transcription and / or translation of a target polynucleotide.

[0096] Recombinant vectors can be constructed as cloning vectors or expression vectors. For recombinant expression vectors, vectors commonly available in the relevant art for expressing foreign proteins in plants, animals or microbial cells can be used. Various methods well known in the art can be used to construct recombinant vectors.

[0097] For use in a host such as a prokaryotic or eukaryotic cell, a recombinant vector can be constructed accordingly. For example, when constructing a vector as an expression vector for a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., pL λ Promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation and a transcription / translation termination sequence. On the other hand, the expression vector for eukaryotic hosts includes a replication origin that can be operated in eukaryotic cells, such as f1 replication origin, SV40 replication origin, pMB1 replication origin, adenovirus replication origin, AAV replication origin and BBV replication origin, but is not limited thereto. In addition, the expression vector generally includes a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, etc.), and a polyadenylation sequence as a transcription termination sequence.

[0098] Recombinant cells can be prepared by introducing the recombinant vector into a suitable host cell. Any host cell known in the art can be used in the present disclosure as long as it allows the recombinant vector to be continuously cloned and expressed in a stable manner. Examples of prokaryotic host cells that can be used in the present disclosure can be selected from Escherichia coli (E.coli) such as Escherichia coli JM109, Escherichia coli BL21, Escherichia coli RR1, Escherichia coli LE392, Escherichia coli B, Escherichia coli X 1776, Escherichia coli W3110, Bacillus species such as Bacillus subtilis and Bacillus thuringiensis, and Enterobacteriaceae strains, such as Salmonella typhimurium, Serratia marcescens and various Pseudomonas species. Eukaryotic host cells that can be used for transformation can be selected from, but are not limited to, Saccharomyces cerevisiae, insect cells, and animal cells, such as 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, etc.

[0099] Nucleic acid molecules or recombinant vectors carrying nucleic acid molecules can be introduced (transfected) into host cells using methods well known in the relevant art. For example, when the host cell is a prokaryotic cell, CaCl can be used. 2 For eukaryotic host cells, gene introduction can be achieved using, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle bombardment.

[0100] In order to select transformed host cells, the phenotype associated with the selection marker can be utilized according to methods well known in the art. For example, when the selection marker is a gene that confers resistance to certain antibiotics, host cells can be grown in culture medium in the presence of antibiotics to select target transformants.

[0101] Another embodiment provides a method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, the method comprising expressing a nucleic acid molecule or a recombinant vector in a host cell. The expression step can be performed by culturing a recombinant cell comprising a nucleic acid molecule (e.g., in a recombinant vector) under conditions that allow expression of the nucleic acid molecule. The method can also include isolating and / or purifying the antibody or fragment thereof from the cell culture after the expression or culturing step.

[0102] Beneficial Effects

[0103] The anti-LILRB1 antibodies or antigen-binding fragments thereof provided in the present disclosure may have a high anti-cancer effect by inhibiting the immune escape mechanism of cancer cells so that immune cells can exhibit their anti-cancer effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 This is an electrophoretic diagram showing the results of SDS-PAGE gel analysis of the anti-LILRB1 antibody purified in the Example.

[0105] Figure 2 is a sensorgram showing the result of SPR (surface plasmon resonance) measurement of the anti-LILRB1 antibody B3 according to one example.

[0106] Figure 3 is a sensorgram showing the SPR measurement result of the anti-LILRB1 antibody E3 according to one embodiment.

[0107] Figure 4a is a graph showing the binding ability of the anti-LILRB1 antibody A10 according to one embodiment to the human natural killer cell KHYG-1; Figure 4b is a graph showing the binding ability of the anti-LILRB1 antibody E3 according to one embodiment to human natural killer cells KHYG-1; and Figure 4c This is a graph showing the ability of a human IgG4 isotype control antibody to bind to human natural killer cells KHYG-1.

[0108] Figure 5 is a graph showing the binding level of recombinant LILRB1-Fc protein to the surface of HLA-G overexpressing cells measured by the iQue screener when treated with an anti-LILRB1 antibody according to one embodiment and a human IgG4 isotype control antibody, respectively.

[0109] Figure 6 is a graph showing the in vivo anti-tumor effects of anti-LILRB1 antibodies E3 and B3 according to one embodiment.

[0110] Figures 7a to 7d is a flow cytometric graph of the binding of the anti-LILRB1 antibody E3.1 to cells expressing various members of the human LILR family according to one embodiment.

[0111] Figures 8a to 8d is a flow cytometric graph of the binding of the anti-LILRB1 antibody H11 to cells expressing various members of the human LILR family according to one embodiment.

[0112] Fig. 9is a graph showing the release level of granzyme B in human natural killer cells KHYG-1 when treated with the anti-LILRB1 antibody E3.1 or H11 according to one embodiment, compared with the release level of granzyme B in cells treated with a control antibody (human IgG4 isotype).

[0113] Fig.10 is a graph showing the release level of perforin in human natural killer cells KHYG-1 when treated with the anti-LILRB1 antibody E3.1 or H11 according to one embodiment, compared with the release level of perforin in cells treated with a control antibody (human IgG4 isotype).

[0114] Fig.11 is a graph showing the results of a luciferase reporter assay for evaluating the ability of the anti-LILRB1 antibody E3.1 or H11 to block the LILRB1 signaling pathway according to one embodiment.

[0115] Fig.12 is a graph showing the in vivo anti-tumor effects of the anti-LILRB1 antibodies E3.1 and H11 according to one embodiment. DETAILED DESCRIPTION

[0116] Hereinafter, the present invention is described in detail by way of examples.

[0117] The following examples are intended only to illustrate the present invention and are not to be construed as limiting the present invention.

[0118] Example 1: Preparation of human antibodies against LILRB1

[0119] 1.1. Selection of human antibodies against LILRB1 using phage display

[0120] In order to select antibodies that specifically recognize human LILRB1, phage display screening was performed using a library consisting of human scFv antibodies. As antigens, human LILRB1-His (Catalog No. 8989-T2) and human LILRB1-Fc (Catalog No. 2017-T2) (RnD systems) were used, respectively. Each antigen was conjugated to biotin by the EZ-Link Sulfo-NHS-Biotin Kit (ThermoFisherScientific) for use.

[0121] Phage display screening was performed using a total of 4 types of LILRB1 antigens (LILRB1-His, LILRB1-Fc, LILRB1-His-biotin, and LILRB1-Fc-biotin) by solid phase screening and solution phase screening. Additional screening was performed by gradually decreasing the concentration of the antigen used, competitive elution against LILRB1 with a control antibody, negative selection for Fc when LILRB1-Fc was used as an antigen, etc. The binding of the selected products to the antigen was confirmed by polyclonal phage ELISA.

[0122] 1.2. Screening and analysis of monoclonal soluble scFv

[0123] In Example 1.1, it is confirmed that the gene of the coding scFv combined with the antigen is amplified by PCR to prepare an expression vector. For each selection, a certain number of transformants are transferred to 96-well culture plates for screening. The antibody in the form of scFv is expressed using autoinduction medium (Studier, FW (2005) Protein Expression and Purification 41, 207-34), and then the combination of them and antigen is analyzed by performing DELFIA immunoassay (PerkinElmer). In addition, after capturing a certain amount of each scFv antibody on the surface, DELFIA is performed to the antigen to determine the ranking of antigen-antibody binding affinity.

[0124] 1.3. Converting the screened scFv into IgG antibodies

[0125] Among the clones confirmed to bind to the antigen in Example 1.2, a total of 376 clones were selected, and the DNA sequence of the gene encoding the selected scFv was analyzed by universal DNA sequencing to remove duplicate clones. In addition, based on the ranking of antigen-antibody binding affinity determined in Example 1.2, a total of 93 clones were selected. The genes encoding the heavy chain variable region (VH) and the light chain variable region (VL) were amplified by PCR from each gene encoding the selected scFv, and inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of the vectors containing CMV promoter or CMV / CHOβ-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and κ or λ light chain constant region can be used), wherein the expression vector is designed to encode human IgG4 antibody (IgG4 Fc: SEQ ID NO: 341, κ constant region: SEQ ID NO: 342, λ constant region: SEQ ID NO: 343). The DNA sequence of the expression vector was confirmed by sequencing.

[0126] 1.4. Preparation of selected antibodies

[0127] The vector constructed in Example 1.3 was purified using the Plasmid Plus Maxi Kit (Qiagen). The purified vector was used to express TM Cell or Expi293 TM Antibodies are expressed in cells.

[0128] Specifically, by adding 80 μL of ExpiFectamine TM CHO reagent (Thermo Fisher) was used to transfect the vector constructed in Example 1.3 into ExpiCHO-S TM Cells (Gibco) (1.5×10 8 One day after transfection, 150 μL of ExpiCHO TM Enhancer (Thermo Fisher) and 4mL ExpiCHO TM Feed (ThermoFisher) was added to the culture. On day 5, 4 mL of ExpiCHO TM Feed was added to the culture. Transfected cells were incubated at 32°C and 5% CO 2 The culture conditions were maintained for a total of 7-11 days.

[0129] Additionally, according to the manufacturer's protocol, 320 μL of ExpiFectamine TM 293 reagent (Gibco), the vector constructed in Example 1.3 was transfected into Expi293F TM Cells (Gibco) (3×10 8 One day after transfection, add ExpiFectamine TM 293Enhancer 1 (Thermo Fisher), ExpiFectamine TM Enhancer 2 (Thermo Fisher) and glucose were used in an amount of 0.6 mL per 100 mL culture volume, 6 mL per 100 mL culture volume, and 3.6 g per 1 liter, respectively. The transfected cells were incubated at 36.5°C and 5% CO 2 The culture conditions were maintained for a total of 5 days.

[0130] The two types of cultured cells were centrifuged at 4000rpm for 20 minutes at 4 ° C, and then filtered using a 0.22um bottle top filter system (Corning). The culture supernatant was collected and purified using AKTA Pure L (GE Healthcare). The culture supernatant was loaded into an AKTA Pure L equipped with a Hitrap MabSelectSure 1mL column (GE Healthcare) at a flow rate of 1mL / min, and the column was washed with 20 column volumes (CV) of 1X PBS. Then, elution buffer (0.1M sodium citrate pH3.4 buffer) was loaded into the column to elute the target protein. The eluate was concentrated using Amicon Ultra Filter Device (MWCO10K, Merck), centrifuged and buffer exchanged with 1xPBS buffer.

[0131] Dilute the purified antibody sample with 1X PBS to a final concentration of about 1 mg / mL. Mix 10 μL of reducing loading buffer (3X) or non-reducing loading buffer (3X) and 20 μL of purified antibody sample and place in a 95°C heating bath for 2 minutes, then remove and cool. Inject the sample at 10 μg per well into an SDS-PAGE gradient gel (4-20% or 4-12%) equipped on an electrophoresis device and develop on the gel. To analyze the molecular weight of the sample, place the Precision Plus Protein TM A dual color standard (BIO-RAD) was injected into another separate well. The gel was stained with Coomassie staining solution and destained to obtain the gel image.

[0132] Among the 93 antibodies, the gel electrophoresis images of antibodies A10, B3, E3, G1, G9, and H2 are representatively shown in Figure 1 In. Figure 1 As shown, it was confirmed that an antibody having a disulfide bond was produced.

[0133] 1.5. Analysis of Binding Affinity of Selected Antibodies

[0134] The binding affinity of the 93 antibodies selected in Example 1.3 to the antigen LILRB1 was measured using Biacore T200 (GE Healthcare). Anti-human IgG (Fc) antibody (GE Healthcare, catalog number BR-1008-39, final concentration 25 μg / mL) was flowed at a flow rate of 5 μL / min for 360 seconds to be immobilized on S series sensor chip CM5 (GE healthcare, catalog number BR-1005-30) at 5000-7000 RU using an amine coupling kit (GE healthcare, catalog number BR-1000-508). The antigen human LILRB1 protein (LILRB1-His, RnD system catalog number 8989-T2) was injected at 4 to 9 different concentrations ranging from 3.13 nM to 1600 nM at a flow rate of 30 μL / min to determine the k as shown in Table 3. a and k d value and calculate K from it D value.

[0135] Among the 93 antibodies, 20 that showed excellent binding affinity (K D The antibodies with the highest affinity (K) for LILRB1 were analyzed and summarized in Table 3. D ) was about 99.8 nM for antibody B3 and showed LILRB1 binding affinity (K D ) is about 101.2 nM, and the SPR sensorgrams of antibody E3 are shown in Figure 2 and Figure 3 middle( Figure 2 : SPR sensorgram of B3, Figure 3 :SPR sensorgram of E3):

[0136]

Table 3

[0137] Antigen binding affinity of anti-LILRB1 antibodies to human LILRB1 (K D )

[0138] Clone Name <![CDATA[k a (x 10 5 )(1 / Ms)]]> <![CDATA[k d (x 10 -4 )(1 / s)]]> <![CDATA[K D (nM)]]> A10 0.504 76.5 152 A11 0.001801 9.814 5448 B3 0.149 14.87 99.8 B9 0.09324 6.16 66.1 B12 1.84 14.42 7.84 D1 1.165 57.44 49.33 D3 0.0311 5.58 180 E3 0.3460 35.00 101.2 E4 0.1065 7.73 72.55 E6 0.2679 16.27 60.73 E9 0.105 10.48 99.86 E12 2.331 102.6 44.01 F11 2.72 6.15 2.26 F12 2.811 9.731 3.462 G1 4.33 14.19 3.28 G6 2.58 152.4 59.06 G9 1.43 4.36 3.05 G11 0.454 20.53 45.23 H2 5.865 95 16.20 H11 2.962 22.57 7.621

[0139] 1.6. Sequence analysis of selected antibodies

[0140] Among the 20 antibodies analyzed for antigen binding affinity in Example 1.5, the CDRs, light chain variable regions, heavy chain variable regions, amino acid sequences of light chains and heavy chains defined according to Kabat numbering, and nucleic acid sequences encoding light chain variable regions and heavy chain variable regions were analyzed by general amino acid sequencing and DNA sequencing methods, and are summarized in Tables 4 to 23:

[0141]

Table 4

[0142] Antibody Clone E3

[0143]

[0144]

Table 5

[0145] Antibody Clone B3

[0146]

[0147]

Table 6

[0148] Antibody clone A10

[0149]

[0150]

Table 7

[0151] Antibody Clone G1

[0152]

[0153]

Table 8

[0154] Antibody Clone G9

[0155]

[0156]

Table 9

[0157] Antibody Clone H2

[0158]

[0159]

Table 10

[0160] Antibody Clone H11

[0161]

[0162]

Table 11

[0163] Antibody clone F12

[0164]

[0165]

Table 12

[0166] Antibody Clone B9

[0167]

[0168]

Table 13

[0169] Antibody Clone G11

[0170]

[0171]

Table 14

[0172] Antibody Clone G6

[0173]

[0174]

Table 15

[0175] Antibody clone F11

[0176]

[0177]

Table 16

[0178] Antibody Clone D3

[0179]

[0180]

Table 17

[0181] Antibody Clone B12

[0182]

[0183]

Table 18

[0184] Antibody Clone E4

[0185]

[0186]

Table 19

[0187] Antibody clone E12

[0188]

[0189]

Table 20

[0190] Antibody Clone D1

[0191]

[0192]

Table 21

[0193] Antibody Clone E6

[0194]

[0195]

Table 22

[0196] Antibody Clone E9

[0197]

[0198]

Table 23

[0199] Antibody Clone A11

[0200]

[0201] Example 2: In vitro biological activity assay of selected antibodies 2.1. Natural killer cell (NK cell) surface binding assay

[0202] To test whether the 93 antibodies selected in Example 1.4 bind to LILRB1 expressed on the surface of immune cells, a natural killer cell (NK cell) surface binding assay was performed. Human NK cells, KHYG-1 cells (JCRB), were cultured in RPMI 1640 medium (Gibco) supplemented with 10% (w / v) FBS (Gibco) and 100 U / mL interleukin-2 (Novartis). KHYG-1 cells were cultured at 5×10 4 Each selected antibody was added to the wells to a final concentration of 50 μg / mL per well and incubated at 4°C for 1 hour.

[0203] To understand the level of LILRB1-specific binding of the selected antibodies, a human IgG4 isotype control antibody (Biolegend) was treated in the same manner. After washing with FACS buffer, the cells were treated with anti-human Fc-biotin antibody (life technologies) and incubated at 4°C for 1 hour. After washing with FACS buffer, the cells were treated with streptavidin PE (BD Pharmigen) and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were resuspended and analyzed using an iQue screener (Sartorius).

[0204] Among the results obtained, the results of antibodies A10, E3, E4, F12, G1, G9, G11, H2 and H11 were representatively compared with the results of human IgG4 isotype (control), which are shown in Table 24. The flow cytometry graphs of A10, E3 and human IgG4 isotype (control) are shown in Figure 4a (A10), Figure 4b (E3) and Figure 4c (isotype IgG4):

[0205]

Table 24

[0206] Mean fluorescence intensity % of Group 2 Human IgG4 Isotype Control 142917.2 2.95 A10 222660.2 28.68 E3 268702.2 40.22 E4 272295.5 43.25 F12 262012.7 38.02 G1 321051.7 56.23 G9 263079.3 41.32 G11 262771.9 40.11 H2 238570.9 29.00 H11 244818.2 32.59

[0207] As shown in Table 24 and Figures 4a to 4c As shown in , the antibodies tested showed higher levels of binding to human NK cells (surface) compared to a human IgG4 isotype control antibody.

[0208] 2.2. Analysis of the inhibitory effect of selected antibodies on the binding of LILRB1 to HLA-G

[0209] In order to test whether the antibodies selected in Example 1.5 exert an inhibitory effect on the binding of LILRB1 to its ligand HLA-G, the degree of blocking of the selected antibodies was analyzed.

[0210] For this purpose, JEG-3 cells (ATCC catalog number HTB-36) showing high HLA-G expression levels were used. JEG-3 cells were cultured in MEM medium (Gibco) supplemented with 10% (v / v) FBS (Gibco) and 1% (v / v) penicillin-streptomycin (Gibco). JEG-3 cells were cultured at 5×10 4 The amount of cells / well was added to a U-shaped bottom 96-well tissue culture plate (BD Falcon). The well plate was washed with 1X PBS buffer. Each antibody selected in Example 1.5 (A10, E3, F12, G1, G9, H2 and H11) and LILRB1-Fc (RnD systems) were mixed in FACS buffer (1X PBS + 1% BSA + 1mM EDTA) to a final concentration of 10μg / mL and 5μg / mL, respectively. The cells were treated with 100μL of the mixture solution per well and incubated on ice for 2 hours. The anti-LILRB1 antibody (clone HP-F1, Abcam) as a positive control and the anti-lysozyme IgG4 antibody (clone D1.3) as a negative control were treated in the same manner. After washing twice with FACS buffer, the cells were treated with PE-anti-huIgG-Fc antibody (Biolegend, 10μg / mL) and incubated on ice for one hour. After washing twice with FACS buffer, cells were resuspended in 100 μL of the same buffer and analyzed using the iQue screener (Sartorius).

[0211] The results are shown in Figure 5 In. Figure 5 As shown, all tested antibodies A10, E3, F12, G1, G9, H2 and H11 effectively inhibited the binding of LILRB1-Fc to HLA-G overexpressing cell lines.

[0212] 2.3. Determination of cancer cell lysis induced by NK cells

[0213] To test whether the selected antibodies increased the extent of cancer cell lysis induced by NK cells, the cell death rate of HLA-G overexpressing HEK293 cells induced by NK cell KHYG-1 was analyzed. KHYG-1 cells (JCRB) were cultured at 2×10 4 cells / well (4×10 4Cells were added to a 96-well tissue culture plate (BD Falcon) in an amount of 50 μL (cells / mL, total volume 50 μL). Cells were treated with each antibody (Table 25) to a final concentration of 20 μg / mL per well and placed at 37° C. for one hour.

[0214] As a negative control, human IgG4 isotype control antibody (Biolegend) was treated in the same manner.

[0215] HLA-G overexpressing HEK293 cells (prepared by transducing HEK293 cells (American Typo Culture Collection) with a lentivirus constructed to express HLA-G) were stained with IncuCyte CytoLight Rapid Red reagent (Sartorius) according to the manufacturer's protocol. One hour later, HLA-G overexpressing HEK293 cells were plated at 1×10 4 cells / well (2×10 4 cells / mL, total volume 50 μL) was added to the plate. Incubate at 37°C and 5% CO 2 The plate was placed in an IncuCyte S3 (Sartorius) equipped in an incubator under the conditions of , and images were taken for 72 hours. The red area confluence indicating the density of HLA-G overexpressing live HEK293 cells was measured, and cell viability was calculated. The obtained cell viability is shown in Table 25 (wherein cell viability is shown as a relative value relative to the control antibody (cell viability of the IgG4 isotype treated well = 1)):

[0216]

[0217]

Table 25

[0218]

[0219]

[0220] As shown in Table 25, all tested antibodies including A10, B9, D3, E1, E3, F12, G1, G6, G9, G11, H2 and H11 increased cell death of HLA-G overexpressing HEK293 cells caused by KHYG-1 compared to the human IgG4 isotype control antibody.

[0221] Example 3: In vivo biological activity determination of selected antibodies

[0222] Among the antibodies selected in Example 1.5, the in vivo anticancer efficacy of two antibodies (E3 and B3) was tested. For this purpose, it was tested whether the administration of the two antibodies would reduce tumor size, wherein the tumor was produced by transplanting human colorectal cancer cells (Bioware Brite Cell Line HCT116 Red-Fluc colorectal cancer cells (PerkinElmer)) and THP-1 derived macrophages to mice. As a negative control, human colon cancer xenograft mice prepared as above were treated with human IgG1 isotype control antibodies (BioXcell, catalog number BP0297). Hereinafter, the process is described in detail:

[0223] Preparation of THP-1 derived macrophages

[0224] The THP-1-derived macrophages used above were prepared by differentiating THP-1 cells (ATCC) with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma), 20 ng / mL interferon γ (Peprotech), and 10 pg / mL lipopolysaccharide (LPS, Sigma).

[0225] Measuring anticancer efficacy in mouse models

[0226] 3 × 10 6 HCT116 Red-Fluc colorectal cancer cells, 3×10 6 A mixture of THP-1 derived macrophages of 100 cells and one of the two test antibodies (E3 or B3 antibody; 20 μg per mouse) was administered to the mouse model at a dose of 5 mg / kg by intraperitoneal injection twice a week starting from day 4 after tumor implantation. Then, the size of the transplanted tumor (mm 3 ) and displayed in Figure 6 In. Figure 6 As shown, all tested antibodies, especially antibody E3, exhibited statistically significant inhibitory effects on tumor growth in a mouse model transplanted with HCT116 colon cancer cells and THP-1-derived macrophages.

[0227] Example 4: Preparation of anti-LILRB1 antibody (E3.1)

[0228] The nucleic acid sequence encoding the full-length heavy chain (SEQ ID NO: 302) of antibody E3 (which was confirmed to have a particularly significant effect in Example 3) was amplified by PCR. The nucleic acid sequence encoding the Ser1 to Leu110 region of the light chain variable region (VL) (SEQ ID NO: 221) of antibody E3 was amplified by PCR and connected to the nucleic acid sequence encoding the λ constant region (λCL.1, SEQ ID NO: 344) to amplify the nucleic acid sequence encoding the λ light chain by PCR. The amplified sequence was inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any one of the vectors containing a CMV promoter or a CMV / CHO β-actin fusion promoter (KR10-1038126B1) and genes encoding the human IgG4 heavy chain constant region and the λ light chain constant region can be used), wherein the expression vector is designed to encode a human IgG4 antibody. The DNA sequence of the expression vector was confirmed by sequencing.

[0229] The antibody (E3.1) was prepared using the constructed expression vector according to Example 1.4, and the sequence of the antibody was analyzed according to Example 1.6 and summarized in Table 26:

[0230]

Table 26

[0231] Antibody clone E3.1

[0232]

[0233] Example 5: Generation of human LILR overexpressing cell lines

[0234] Nucleic acid sequences encoding full-length human LILR family proteins (Table 27) were amplified by PCR, and each amplified sequence was inserted into an expression vector (pTRIOZ-hIgG4, InvivoGen; alternatively, any of the vectors containing a CMV promoter or a CMV / CHO β-actin fusion promoter (KR10-1038126B1) and genes encoding human IgG4 heavy chain constant region and λ light chain constant region can be used). The DNA sequence of the expression vector was confirmed by sequencing. The constructed vector was transfected into CHO cells to generate 11 stable cell lines that overexpressed each LILR protein on the surface.

[0235]

Table 27

[0236]

[0237]

[0238]

[0239]

[0240] Example 6: Determination of ECs for Binding of Selected Antibodies to the Surface of LILRB1-Overexpressing Cells 50

[0241] In order to determine the EC binding of the antibodies prepared in Examples 1 and 4 to the human LILRB1 overexpressing cell lines 50 The EC values ​​of E3.1 and H11 antibodies were measured. 50 The CHO cells overexpressing LILRB1 on the surface prepared in Example 5 were cultured at 1×10 5 Cells / well were added to a U-bottom 96-well tissue culture plate (BD Falcon). Three-fold serial dilutions of E3.1 and H11 antibodies were prepared starting from final concentrations of 600ug / mL and 27ug / mL, respectively. Cells were treated with each diluted antibody 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 resuspended and analyzed using an iQue filter (Sartorius). EC was calculated using the nonlinear regression formula of GraphPad Prism software. 50 The values ​​are shown in Table 28:

[0242]

Table 28

[0243] E3.1 H11 <![CDATA[EC 50 (nM)]]> 7.154 0.376

[0244] Example 7: Evaluation of cross-reactivity of selected antibodies against human LILR family overexpressing cell lines

[0245] In order to confirm whether the selected antibodies bind to human LILR family proteins other than LILRB1, a cell surface binding assay was performed. CHO cells expressing one of the various LILR family proteins on their surfaces (prepared in Example 5) were incubated at 1×10 5The amount of cells / well was added to a U-shaped bottom 96-well tissue culture plate (BD Falcon). The cells in each well were treated with the selected antibody at a final concentration of 20ug / 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 fluorescently labeled streptavidin (BD Pharmigen) and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were resuspended and analyzed using an iQue filter (Sartorius). Cells treated with each LILR protein-specific antibody (Table 27) were used as positive controls, and cells treated with human IgG4 isotype control antibody (Biolegend) were used as negative controls.

[0246] The results obtained for antibody E3.1 are shown in Figures 7a to 7d (E3.1: red; LILR-specific antibody: blue; isotype (hIgG4) control: grey), and the results obtained for antibody H11 are shown in Figures 8a to 8d (H11: red; LILR-specific antibody: blue; isotype (hIgG4) control: gray). Figures 7a to 7d and Figures 8a to 8d As shown, E3.1 and H11 antibodies do not bind at all or hardly bind to LILRs other than LILRB1. These results indicate that the antibodies provided in the Examples have the ability to specifically bind to LILRB1.

[0247] Example 8: Measurement of Granzyme B and Perforin Release by ELISPOT Assay

[0248] To confirm whether E3.1 and H11 antibodies increase the cytotoxicity level of NK cells, an enzyme-linked immunosorbent spot (ELISPOT) assay was performed. The cytotoxicity level was determined by the release of cytotoxic granules, granzyme B, and perforin in NK cells.

[0249] 5×10 3 5×10 cells of the KHYG-1 cell line expressing LILRB1 (JCRB) and 5×10 3HLA-G overexpressing K562 cells (prepared by transducing K562 cells (American Type Culture Collection) with a lentivirus constructed to express HLA-G) were co-cultured in a U-bottom 96-well tissue culture plate. Each antibody (E3.1, H11 or human IgG4 isotype control antibody) was added thereto at a final concentration of 50 ug / mL per well and incubated at 37°C for 30 minutes. The co-cultured cells were transferred to a 96-well plate for ELISPOT (Immunospot, catalog HGZBPFN-2M) (PVDF membrane), which was coated with anti-perforin antibody and anti-granzyme B antibody, respectively, and further incubated at 37°C for 8 hours. The PVDF membrane was washed with a washing solution (0.05% tween 20 in PBS) and then treated with anti-granzyme B-HRP and anti-perforin-biotin antibodies. Then, the detection process was performed according to the manufacturer's protocol. The PVDF membrane was dried at room temperature for 24 hours, and the number of spots of granzyme B and perforin was counted by ELISPOT analyzer (Immunospot).

[0250] The results are shown in Fig. 9 (granzyme b; Gzmb) and Fig.10 (perforin; Prf) (the Y axis represents the total number of spots). Fig. 9 and Fig.10 As shown, the release levels of granzyme B and perforin in the E3.1 or H11 antibody-treated group were significantly increased compared with the human IgG4 isotype control antibody-treated group. Unpaired T tests were performed, and all experiments were performed three times under the same conditions to ensure the reliability of the experiments, and the results are shown as average values.

[0251] Example 9: Preparation of chimeric GHI / 75 antibodies

[0252] In order to understand whether the antibodies provided in the examples show higher efficacy than previously existing antibodies, a chimeric GHI / 75 antibody comprising the variable region of GHI / 75 antibody (Biolegend, catalog number 333721, which is a mouse-derived anti-human LILRB1 antibody) and the constant region of a human antibody was prepared.

[0253] More specifically, the amino acid sequence of the GHI / 75 antibody was analyzed by peptide mapping, and a vector was prepared in which the nucleic acid sequence corresponding to the variable region (VH and VL domains) of the human IgG4 antibody was replaced with the nucleic acid sequence corresponding to the variable region (VH and VL domains) of the mouse GHI / 75 antibody. In the vector, the region corresponding to the upper hinge of human IgG4 was replaced with the nucleic acid sequence corresponding to the amino acid sequence of the upper hinge of human IgG1 (EPKSCDKTHT; SEQ ID NO: 359). The vector was expressed as described in Example 1.4, and the obtained antibody was purified and used as a comparative antibody in the following examples.

[0254] Example 10: Measuring the inhibitory effects of selected antibodies on LILRB1 signaling using an IL-2 promoter luciferase assay

[0255] In order to confirm whether the antibodies prepared in Examples 1 and 4 inhibit the signaling of LILRB1, a luciferase reporter assay was performed. Among the antibodies prepared in Examples 1 and 4, E3.1 and H11 antibodies were representatively assayed, and the chimeric GHI / 75 antibody prepared in Example 9 was used for comparison. Jurkat cells expressing LILRB1 and interleukin 2 (IL-2) promoter luciferase (prepared by inserting an IL-2 promoter luciferase vector (Promega) into a Jurkat cell line (American Type Culture Collection), followed by transduction with a lentivirus constructed to express LILRB1) and HLA-G overexpressing K562 cells were used. A 96-well plate was coated with an anti-CD3 antibody (Biolegend) by incubating with the antibody overnight at 4°C. The next day, Jurkat cells expressing LILRB1 and IL-2 promoter luciferase were plated at 1×10 5 1×10 cells / well were added to a U-bottom 96-well plate and the plate was treated with each antibody (E3.1, H11, chimeric GHI / 75, or human IgG4 isotype (control)) to a final concentration of 20 ug / mL and incubated at 37°C for one hour. 5 Cells / well) were added to the plate and incubated at 37°C for 30 minutes. The resulting suspension was transferred to a plate coated with anti-CD3 antibody, and anti-CD28 antibody (Biolegend) was added thereto to a final concentration of 10 ug / mL. The plate was incubated at 37°C for 6 hours. Steady- Solution (Promega) was added to each well, and the luminescence intensity was recorded using a luminometer (Envision, PerkinElmer).

[0256] The results are shown in Fig.11 In. Fig.11As shown, the E3.1 and H11 antibodies provided in the Examples exhibited significantly increased LILRB1 signaling inhibitory activity compared to a human IgG4 isotype control antibody and a chimeric GHI / 75 control antibody.

[0257] Example 11: Analysis of the anti-cancer effects of selected antibodies in mouse models

[0258] To analyze the anticancer effects of the selected antibodies, referring to Example 3, 3×10 6 HCT116 Red-Fluc colorectal cancer cells, 3×10 6 A mixture of THP-1 derived macrophages of 10 cells and antibodies (20 μg / mouse) was added. The antibodies used were E3.1 or H11 antibodies, and human IgG4 isotype was used as a control antibody for comparison. Starting from the 4th day after tumor cell transplantation, the antibodies were administered to the mouse model at a dose of 5 mg / kg by intraperitoneal injection twice a week, and the tumor volume was measured and shown in Fig.12 In. Fig.12 As shown, E3.1 and H11 antibodies exhibited significant inhibitory effects on tumor growth in a mouse model transplanted with HCT116 colon cancer cells and THP-1-derived macrophages compared with a control antibody.

Claims

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

2. The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1, comprising: A light chain variable region, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 231, and The heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

232. 3 . The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is a human IgG1 or IgG4 antibody.

4. The anti-LILRB1 antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is scFv, (scFv)2, Fab, Fab', F(ab')2, a fusion polypeptide comprising scFv fused to immunoglobulin Fc, or a fusion polypeptide comprising scFv fused to a light chain constant region. 5 . A pharmaceutical composition comprising the anti-LILRB1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier. 6 . A nucleic acid molecule encoding the anti-LILRB1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 . A recombinant vector comprising the nucleic acid molecule according to claim 6 .

8. A recombinant cell, comprising the nucleic acid molecule according to claim 6 or the recombinant vector according to claim 7. 9 . A method for preparing an anti-LILRB1 antibody or an antigen-binding fragment thereof, the method comprising culturing the recombinant cell according to claim 8 .

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