Isolated antigen binding proteins and uses thereof

Abstract

An isolated antigen binding protein capable of specifically binding to an LILRB4 protein. The invention also relates to a polypeptide, a fusion protein and a drug molecule comprising the isolated antigen-binding protein, a nucleic acid encoding the isolated antigen-binding protein, a vector comprising the isolated antigen-binding protein, a cell comprising the nucleic acid or the vector, and a method for preparing the antigen-binding protein. The present invention relates to isolated antigen binding proteins, methods of making the isolated antigen binding proteins and uses of the isolated antigen binding proteins.

Description

Isolated antigen-binding proteins and their uses Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an antigen binding protein targeting LILRB4 and its use. Background Art

[0002] Leukocyte immunoglobulin-like receptor B4 (LILRB4) is a member of the leukocyte Ig-like receptors (LILRs). Under physiological conditions, LILRB4 plays a crucial role in immune system function through its expression on various immune cells, such as T cells and plasma cells. Under pathological conditions, LILRB4 influences the progression of various diseases, such as the transformation and infiltration of tumors and leukemia, through multiple signaling pathways. Differential expression of LILRB4 is found in a variety of immune system diseases, such as Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis. Recent studies have suggested that LILRB4 also plays a role in psychiatric disorders. Given its important role in the immune system and its differential expression in a variety of diseases, LILRB4 has become a potential therapeutic target for a variety of diseases.

[0003] Based on the therapeutic potential of LILRB4, a variety of antibodies that can specifically bind to LILRB4 still need to be developed.

[0004] Summary of the Invention

[0005] In one aspect, the present application provides an isolated antigen-binding protein having one or more of the following properties: 1) being able to specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) being able to -7 M or lower K D The invention discloses a novel novel anti-ILR-4 inhibitor that can bind to LILRB4 protein; 3) can block the activity of LILRB4 / APOE binding; 4) can stimulate the activation of CD8+ T cells; 5) can inhibit the migration of human monocytic leukemia cells THP-1; 6) can inhibit the growth and / or metastasis of tumor cells; 7) can block the binding of LILRB4 to Fibronectin.

[0006] In certain embodiments, the LILRB4 is human LILRB4.

[0007] In certain embodiments, the isolated antigen-binding protein comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33 and SEQ ID NO: 22.

[0008] In certain embodiments, the isolated antigen-binding protein comprises a HCDR3 comprising the amino acid sequence of SEQ ID NO: 83 or SEQ ID NO: 29.

[0009] In certain embodiments, the isolated antigen-binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20, and SEQ ID NO: 29.

[0010] In certain embodiments, the isolated antigen-binding protein comprises a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 28.

[0011] In certain embodiments, the isolated antigen-binding protein comprises HCDR1, which comprises the amino acid sequence shown in SEQ ID NO: 69 (X1X2WMX3, wherein X1 is D or S; X2 is A or Y; and X3 is D or H).

[0012] In certain embodiments, the isolated antigen-binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27.

[0013] In certain embodiments, the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from the group consisting of:

[0014] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0015] (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and

[0016] (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29.

[0017] In certain embodiments, the isolated antigen-binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:73.

[0018] In certain embodiments, the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:17, SEQ ID NO:30 and SEQ ID NO:41.

[0019] In certain embodiments, the isolated antigen-binding protein comprises H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:74.

[0020] In certain embodiments, the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:31, SEQ ID NO:42 and SEQ ID NO:53.

[0021] In certain embodiments, the isolated antigen-binding protein comprises H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:75.

[0022] In certain embodiments, the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:54 and SEQ ID NO:57.

[0023] In certain embodiments, the isolated antigen-binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:76.

[0024] In certain embodiments, the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 44.

[0025] In certain embodiments, the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from the group consisting of:

[0026] (1) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0027] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7;

[0028] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:21, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7;

[0029] (4) the H-FR1 comprises the amino acid sequence of SEQ ID NO: 30, the H-FR2 comprises the amino acid sequence of SEQ ID NO: 31, the H-FR3 comprises the amino acid sequence of SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 7;

[0030] (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:42, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:43, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44;

[0031] (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:54, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44;

[0032] (7) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:57, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:44.

[0033] In certain embodiments, the isolated antigen-binding protein comprises a VH comprising the amino acid sequence of any one of SEQ ID NO:81, SEQ ID NO:33, and SEQ ID NO:22.

[0034] In certain embodiments, the VH of the isolated antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:55 and SEQ ID NO:58.

[0035] In certain embodiments, the isolated antigen binding protein comprises an antibody heavy chain constant region that is derived from an IgG heavy chain constant region.

[0036] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0037] In certain embodiments, the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

[0038] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:63.

[0039] In certain embodiments, the isolated antigen-binding protein comprises an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in any one of SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:91.

[0040] In certain embodiments, the isolated antigen-binding protein comprises at least one CDR in the light chain variable region VL of an antibody, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40.

[0041] In certain embodiments, the isolated antigen-binding protein comprises LCDR3, which comprises the amino acid sequence shown in SEQ ID NO:72 (QX1X2X3X4X5PX6T, X1 is H or Q; X2 is G or S; X3 is D, N or W; X4 is E or T; X5 is I or L; X6 is P or R).

[0042] In certain embodiments, the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:11, SEQ ID NO:24 and SEQ ID NO:36.

[0043] In certain embodiments, the isolated antigen binding protein comprises LCDR2, which comprises the amino acid sequence shown in SEQ ID NO:71.

[0044] In certain embodiments, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:35.

[0045] In certain embodiments, the isolated antigen binding protein comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:70 or SEQ ID NO:34.

[0046] In certain embodiments, the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:23 and SEQ ID NO:34.

[0047] In certain embodiments, the isolated antigen binding protein comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of:

[0048] (1) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11;

[0049] (2) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; and

[0050] (3) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0051] In certain embodiments, the isolated antigen-binding protein comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:77.

[0052] In certain embodiments, the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 37 and SEQ ID NO: 47.

[0053] In certain embodiments, the isolated antigen-binding protein comprises L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:78.

[0054] In certain embodiments, the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 38, and SEQ ID NO: 48.

[0055] In certain embodiments, the isolated antigen-binding protein comprises L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:79.

[0056] In certain embodiments, the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 14, SEQ ID NO: 39, SEQ ID NO: 49 and SEQ ID NO: 60.

[0057] In certain embodiments, the isolated antigen-binding protein comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:80.

[0058] In certain embodiments, the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 50.

[0059] In certain embodiments, the isolated antigen-binding protein comprises L-FR1, L-FR2, L-FR3 and L-FR4, wherein L-FR1, L-FR2, L-FR3 and L-FR4 comprise an amino acid sequence selected from the group consisting of:

[0060] (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 12, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0061] (2) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 25, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0062] (3) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 37, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 38, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0063] (4) the L-FR1 comprises the amino acid sequence of SEQ ID NO:47, the L-FR2 comprises the amino acid sequence of SEQ ID NO:48, the L-FR3 comprises the amino acid sequence of SEQ ID NO:49, and the L-FR4 comprises the amino acid sequence of SEQ ID NO:50; and

[0064] (5) The L-FR1 comprises the amino acid sequence shown in SEQ ID NO:47, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:48, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:60, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

[0065] In certain embodiments, the isolated antigen binding protein comprises a VL comprising the amino acid sequence shown in SEQ ID NO:82 or SEQ ID NO:40.

[0066] In certain embodiments, the VL comprises the amino acid sequence shown in any one of SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:51 and SEQ ID NO:61.

[0067] In certain embodiments, the isolated antigen binding protein comprises an antibody light chain constant region, and the antibody light chain constant region comprises a human Igκ constant region.

[0068] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:64.

[0069] In certain embodiments, the isolated antigen-binding protein comprises an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NO:52, SEQ ID NO:62, SEQ ID NO:87, SEQ ID NO:90 and SEQ ID NO:92.

[0070] In certain embodiments, the isolated antigen binding protein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from the group consisting of:

[0071] (1) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0072] (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; and

[0073] (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0074] In certain embodiments, the isolated antigen-binding protein comprises a VH and a VL, wherein the VH and VL comprise an amino acid sequence selected from the group consisting of:

[0075] (1) the VH comprises the amino acid sequence shown in SEQ ID NO: 8, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16;

[0076] (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 16;

[0077] (3) the VH comprises the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26;

[0078] (4) the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40;

[0079] (5) the VH comprises the amino acid sequence shown in SEQ ID NO:45, and the VL comprises the amino acid sequence shown in SEQ ID NO:51;

[0080] (6) the VH comprises the amino acid sequence shown in SEQ ID NO: 55, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51;

[0081] (7) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51; and

[0082] (8) The VH comprises the amino acid sequence shown in SEQ ID NO:45, and the VL comprises the amino acid sequence shown in SEQ ID NO:61.

[0083] In certain embodiments, the isolated antigen-binding protein antibody heavy chain and antibody light chain comprise an amino acid sequence selected from the group consisting of:

[0084] (1) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87;

[0085] (2) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87;

[0086] (3) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 90;

[0087] (4) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 92;

[0088] (5) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52;

[0089] (6) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52;

[0090] (7) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 59, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; and

[0091] (8) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:62.

[0092] In certain embodiments, the isolated antigen binding protein comprises an antibody or an antigen binding fragment thereof.

[0093] In certain embodiments, the antibody is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

[0094] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, di-scFv, and dAb fragments.

[0095] In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein.

[0096] In another aspect, the present application provides a fusion protein comprising the isolated antigen-binding protein.

[0097] In another aspect, the present application provides a drug molecule comprising the isolated antigen-binding protein.

[0098] In another aspect, the present application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding protein.

[0099] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0100] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0101] On the other hand, the present application provides a pharmaceutical composition comprising the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0102] In certain embodiments, the pharmaceutical composition further comprises one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), Clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, ensidipine, Idhifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibody, PD-L1 antibody, anti-CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0103] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein, which comprises culturing the cell under conditions such that the isolated antigen-binding protein is expressed.

[0104] On the other hand, the present application provides the use of the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating diseases and / or conditions, including diseases or conditions related to LILRB4 signaling.

[0105] In certain embodiments, the disease and / or condition comprises a tumor.

[0106] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0107] In certain embodiments, the drug is used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, emcitabine, Idhifa, IDO inhibitors agents, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0108] In certain embodiments, the disease or disorder associated with LILRB4 signaling comprises an immune system disease and / or a hematological malignancy.

[0109] In certain embodiments, the immune system disease is selected from one or more of the following groups: Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0110] In certain embodiments, the solid tumor is selected from one or more of the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0111] In certain embodiments, the hematological malignancy is selected from one or more of the following groups: myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia, or acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, acute myeloblastic leukemia, and polycythemia vera.

[0112] On the other hand, the present application provides the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition, which are used to prevent and / or treat diseases and / or conditions, including diseases or conditions related to LILRB4 signaling.

[0113] In certain embodiments, the disease and / or condition comprises a tumor.

[0114] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0115] In certain embodiments, the isolated antigen binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition is used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors agents, IDH2 inhibitors, ensidipine, Idhifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0116] In certain embodiments, the disease or disorder associated with LILRB4 signaling comprises an immune system disease and / or a hematological malignancy.

[0117] In certain embodiments, the immune system disease is selected from one or more of the following groups: Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0118] In certain embodiments, the solid tumor is selected from one or more of the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0119] In certain embodiments, the hematological malignancy is selected from one or more of the following groups: myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia, or acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, acute myeloblastic leukemia, and polycythemia vera.

[0120] On the other hand, the present application provides a method for preventing and / or treating diseases and / or conditions, which comprises administering an effective amount of the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition to a subject in need thereof, wherein the diseases and / or conditions include diseases or conditions associated with LILRB4 signaling.

[0121] In certain embodiments, the disease and / or condition comprises a tumor.

[0122] In certain embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0123] In certain embodiments, in the method, the isolated antigen binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition is used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH 1 inhibitors, IDH2 inhibitors, enzidipine, Idhifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0124] In certain embodiments, the disease or disorder associated with LILRB4 signaling comprises an immune system disease and / or a hematological malignancy.

[0125] In certain embodiments, the immune system disease is selected from one or more of the following groups: Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0126] In certain embodiments, the solid tumor is selected from one or more of the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0127] In certain embodiments, the hematological malignancy is selected from one or more of the following groups: myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia, or acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, acute myeloblastic leukemia, and polycythemia vera.

[0128] On the other hand, the present application provides a method for diagnosing a disease or condition associated with the expression of LILRB4 protein in a subject, the method comprising: contacting a sample derived from the subject with the isolated antigen-binding protein, and determining the presence and / or amount of a substance in the sample that can specifically bind to the isolated antigen-binding protein.

[0129] In another aspect, the present application provides a method for detecting LILRB4 in a sample, comprising administering the isolated antigen-binding protein.

[0130] In certain embodiments, the method is an in vitro and / or ex vivo method.

[0131] In another aspect, the present application provides a detection kit comprising the isolated antigen-binding protein, wherein the detection kit is used to detect the presence and / or content of LILRB4 protein in a sample or a subject.

[0132] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0134] FIG1 shows the binding assay of the isolated antigen binding protein described herein to human LILRB4.

[0135] FIG2 shows the results of specific binding detection between the isolated antigen-binding protein described in the present application and the LILRB family protein.

[0136] FIG3A and FIG3B show the activity detection of the isolated antigen binding protein described in the present application in blocking LILRB4 / APOE binding.

[0137] FIG4 shows the detection of the effect of the isolated antigen binding protein described in the present application on activating CD8+ T cells.

[0138] FIG5 shows the inhibitory effect of the isolated antigen-binding protein described in the present application on THP-1 cell migration.

[0139] FIG6 is a graph showing the growth trend of tumor volume in mice after drug administration.

[0140] FIG7 shows the proportion of hCD45+ cells in different tissues and organs after administration of the isolated antigen-binding protein described herein.

[0141] FIG8 shows a binding assay of the isolated antigen binding proteins described herein to LILRB4.

[0142] FIG9 shows a binding assay of the isolated antigen binding protein described herein to THP-1 cells.

[0143] FIG10 shows the inhibitory effect of the isolated antigen-binding protein described in the present application on THP-1 cell migration.

[0144] FIG11 shows the activity detection of the isolated antigen binding protein described in the present application in blocking LILRB4 / APOE binding.

[0145] FIG12 shows the ADCC activity of the isolated antigen-binding protein described herein on 293T-hLILRB4 cells.

[0146] Figure 13 shows the detection of the effect of the isolated antigen binding protein activating CD8+ T cells described in the present application.

[0147] FIG14 is a graph showing the growth trend of tumor volume in mice after drug administration.

[0148] FIG15 shows the fluorescence images of the changes in tumor volume after drug administration in mice. DETAILED DESCRIPTION

[0149] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0150] Definition of terms

[0151] The present application is further described below. Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those commonly used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0152] In this application, the term "isolated" generally refers to a substance obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been altered, or that the substance has been separated from its natural environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal and has not been separated is considered isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0153] In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained from a natural state by artificial means. The "isolated antigen-binding protein" may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding portion to the antigen. The antigen-binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or an artificial scaffold having transplanted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Additionally, peptide antibody mimetics ("PAMs") as well as antibody mimetic-based scaffolds utilizing fibronectin components can be used as scaffolds.

[0154] In this application, the term "KD" or "K D” is often referred to as “affinity constant” or “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (kd) by the association rate constant (ka). The association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) are used to express the binding affinity of a binding protein (e.g., an isolated antigen-binding protein described herein) for an antigen (e.g., LILRB4 protein). Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples in physiological buffers at equilibrium. For example, the KD can be determined by Octet The KD value can also be determined using other experimental approaches and instruments such as BIAcore (biomolecular interaction analysis) (e.g., an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). In addition, the KD value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho), or using a surface plasmon resonance (SPR) instrument to determine the KD value.

[0155] In this application, the term "LILRB4 protein" generally refers to leukocyte immunoglobulin-like receptor B4 (LILRB4). In this application, the term covers the entire LILRB4 and its functionally active fragments, variants, homologues, analogs and derivatives.

[0156] In this application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which can be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) is one that binds to that target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In one embodiment, the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). For example, in this application, the isolated antigen binding protein is capable of binding to an unrelated target at a rate of <2x10 -7 The specific binding may include but is not limited to exclusive binding.

[0157] In this application, the term "variable domain" generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains can be referred to as "VH" and "VL," respectively (or "VH" and "VL," respectively). These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0158] In this application, the term "variable" generally refers to the fact that certain segments of the variable domains differ greatly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three CDRs, which form a loop connection and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR region, and the CDRs from the other chain together contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0159] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function (e.g., specific binding to LILRB4). Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites. IgA antibodies, on the other hand, consist of two to five basic four-chain units that can combine with the J chain to form multivalent combinations. For IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are interconnected by one or more disulfide bonds that depend on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. The VL corresponds to the VH, and the CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be divided into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of its heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively.The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl, and IgKl.

[0160] In this application, the term "CDR" generally refers to the region of the antibody variable domain, whose sequence is highly variable and / or forms a structural definition loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In natural antibodies, HCDR3 and LCDR3 show most of the diversity of the six CDRs, and HCDR3 in particular is considered to play a unique role in conferring fine specificity on antibodies. See, for example, Xu et al, Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains function normally and stably in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

[0161] In the present application, the term "FR" generally refers to the more highly conserved part of the antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each include four FR regions, i.e., four (H-FR1, H-FR2, H-FR3, and H-FR4) in VH, and four (L-FR1, L-FR2, L-FR3, and L-FR4) in VL. For example, the VL of the antigen-binding proteins of the separation described herein can include framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the antigen-binding proteins of the separation described herein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0162] As used herein, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind to an antigen (e.g., LILRB4 protein). As used herein, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb.

[0163] In this application, the term "monoclonal antibody" or "monoclonal antibody" or "monoclonal antibody composition" generally refers to an antibody molecule preparation of a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.

[0164] In this application, the term "single-chain antibody" generally refers to a molecule comprising an antibody heavy chain variable region and a light chain variable region. For example, the single-chain antibody can be formed by connecting the antibody heavy chain variable region and the light chain variable region through a linker (e.g., a connecting peptide).

[0165] In this application, the term "human antibody" generally refers to antibodies whose variable region framework and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from human germline immunoglobulin sequences. The human antibodies of the present application may contain amino acid residues that are not encoded by human germline immunoglobulin sequences, such as mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo. However, the term "human antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into human framework sequences.

[0166] In this application, the term "murine antibody" generally refers to antibodies whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences. The murine antibody of the present application may contain amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by in vitro random mutations or point mutations or by in vivo somatic mutations. However, the term "murine antibody" does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse framework sequence.

[0167] In this application, the term "chimeric antibody" generally refers to an antibody that is derived from a combination of non-human genetic material and human genetic material. Or more generally, a chimeric antibody refers to an antibody that combines genetic material from one species with genetic material from another species.

[0168] In this application, the term "multispecific antibody" generally refers to an antibody molecule that can simultaneously recognize two or more antigens or epitopes. The multispecific antibody can be obtained in a eukaryotic expression system or a prokaryotic expression system by chemical coupling, hybridization-hybridoma method, genetic engineering antibody preparation method, etc.

[0169] In this application, the term "humanized antibody" generally refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to naturally occurring antibodies in humans.

[0170] In this application, the term "fully human antibody" generally refers to a fully human antibody, i.e., an antibody whose constant and variable regions are both derived from humans. Such fully human antibodies can be achieved through phage antibody library technology, transgenic mouse production of humanized antibodies, ribosome display technology, EBV-transformed B cell cloning technology, single B cell cloning, and other technologies.

[0171] In the present application, the term "directly connected" is contrasted with the term "indirectly connected", and the term "directly connected" generally refers to direct connection. For example, the direct connection can be a situation where the substances are directly connected without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly connected" generally refers to a situation where the substances are not directly connected. For example, the indirect connection can be a situation where the substances are connected through a spacer. For example, in the isolated antigen-binding protein described in the present application, the C-terminus of the L-FR1 and the N-terminus of the LCDR1 can be directly or indirectly connected.

[0172] In this application, the terms "polypeptide," "peptide," and "protein" are used interchangeably and generally refer to a polymer of amino acid residues. The term may be used to refer to an amino acid polymer in which one or more amino acid residues is a synthetic chemical mimetic of a corresponding naturally occurring amino acid, and may also be used to refer to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

[0173] In this application, the term "fusion protein" generally refers to a protein composed of two or more polypeptides, which, although not normally bound in nature, are bound together by peptide bonds at their respective amino and carboxyl termini to form a continuous polypeptide. It should be understood that the two or more polypeptide components can be bound directly or indirectly through a peptide linker / spacer.

[0174] In this application, the term "drug molecule" generally refers to a molecule with a desired biological effect. A drug can be prophylactic or therapeutic. Drug molecules may include, but are not limited to: protein molecules, including but not limited to peptides, polypeptides, proteins, including post-translationally modified proteins, fusion proteins, antibodies, etc.; small molecules, including inorganic or organic compounds; nucleic acid molecules, including but not limited to double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA (such as antisense (molecules), RNAi, etc.), intron sequences, triple-helical nucleic acid molecules and aptamers; or vaccines.

[0175] As used herein, the term "isolated nucleic acid molecule" generally refers to isolated forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, separated from their natural environment or artificially synthesized.

[0176] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein expressed. A vector can be used to transform, transduce, or transfect host cells, allowing the genetic material elements it carries to be expressed in host cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector may contain multiple elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain a replication initiation site. Vectors may also include components that assist in their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0177] In this application, the term "cell" generally refers to a single cell, cell line or cell culture that may be or has been a recipient of a subject's plasmid or vector, including nucleic acid molecules of the present invention or vectors of the present invention. Cells may include the offspring of a single cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical to the original parent cell (in the form of the total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described herein. The cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NSO cell, a human non-small cell lung cancer A549 cell, a human skin squamous cell carcinoma A431 cell, a renal clear cell adenocarcinoma 786-0 cell, a human pancreatic cancer MIA PaCa-2 cell, an erythroleukemia K562 cell, an acute T-cell leukemia Jurkat cell, a human breast cancer MCF-7 cell, a human breast cancer MDA-MB-231 cell, a human breast cancer MDA-MB-468 cell, a human breast cancer SKBR3 cell, a human ovarian cancer SKOV3 cell, a lymphoma U-937 cell, a lymphoma Raji cell, a human myeloma U266 cell, or a human multiple myeloma RPMI8226 cell. In certain embodiments, the cell is a mammalian cell.

[0178] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for application to a patient, preferably a human patient. For example, the pharmaceutical composition described herein may include the antigen-binding proteins of the separation described herein, the immunoconjugates described herein, the nucleic acid molecules described herein, the carrier described herein and / or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably nontoxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0179] As used herein, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0180] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0181] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0182] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0183] Detailed Description of the Invention

[0184] Isolated antigen binding protein

[0185] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR. For example, the CDRs of the isolated antigen-binding proteins described in the sequence listing of the present application can be determined using Kabat.

[0186] On the one hand, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR in the VH as shown in any one of SEQ ID NO:81, SEQ ID NO:33 and SEQ ID NO:22; and / or comprise at least one CDR in the VL as shown in SEQ ID NO:82 or SEQ ID NO:40.

[0187] X1VQLX2QX3GAEX4X5KPGASVKX6SCKASGYTFTSYWMHWVX7QX8PGQGLEWX9GEINPSNGRTNYNEKFKSX 10 X 11 TX 12 TX 13 DX 14 SX 15 STX 16 YX 17 X 18 LX 19 SLX 20 SEDX 21 AVYYCARDRYDEGNAMDYWGQGTX22 VTVSS (SEQ ID NO: 81). wherein X1 can be E or Q; X2 can be Q or V; X3 can be P or S; X4 can be L or V; X5 can be K or V; X6 can be L or V; X7 can be K or R; X8 can be A or R; X9 can be I or M; X 10 Can be K or R; X 11 Can be A or V; X 12 Can be L or M; X 13 Can be R or V; X 14 Can be K or T; X 15 Can be S or T; X 16 Can be A or V; X 17 Can be M or V; X 18 Can be E or Q; X 19 Can be N or S; X 20 Can be R or T; X 21 Can be S or T; X 22 Can be S or T.

[0188] DIX1MTX2X3X4SSLSASX5GDRVTIX6CRASQDIX7NYLNWYQQKPX8X9X 10 X 11 KLLIYYT SRLHSGVPSRFSGSGSGTX 12 YX 13 X 14 TISX 15 LX 16 X 17 X 18 DX 19 ATYX 20 CQQGX 21 TLPX 22 TFGX 23 GTX 24 LEIK (SEQ ID NO: 82). wherein X1 may be Q or V; X2 may be H or Q; X3 may be S or T; X4 may be P or T; X5 may be L or V; X6 may be S or T; X7 may be S or T; X8 may be D or G; X9 may be G or K; X 10 Can be A or T; X 11 Can be P or V; X 12 Can be D or E; X 13 Can be S or T; X 14 Can be F or L; X 15 Can be N or S; X 16 Can be E or Q; X 17 Can be P or Q; X18 Can be D or E; X 19 Can be F or I; X 20 Can be F or Y; X 21 Can be D or N; X 22 Can be P or R; X 23 Can be G or Q; X 24 Can be K or R.

[0189] In the present application, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:55 and SEQ ID NO:58.

[0190] In the present application, the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 40, SEQ ID NO: 51 and SEQ ID NO: 61.

[0191] In the present application, the HCDR of the isolated antigen-binding protein can be divided in any form. As long as the VH and / or VL are identical to the amino acid sequences of VH and / or VL shown in any of the items in the present application, the HCDR obtained by any division can fall within the scope of protection of the present application.

[0192] For example, the methods of dividing VH (SEQ ID NO: 8) and / or VL (SEQ ID NO: 16) using different CDR labeling methods can be summarized in the following table.

[0193] For example, the methods of dividing VH (SEQ ID NO: 19) and / or VL (SEQ ID NO: 16) using different CDR labeling methods can be summarized in the following table.

[0194] For example, the methods of dividing VH (SEQ ID NO: 22) and / or VL (SEQ ID NO: 26) using different CDR labeling methods can be summarized in the following table.

[0195] For example, the methods of dividing VH (SEQ ID NO: 33) and / or VL (SEQ ID NO: 40) using different CDR labeling methods can be summarized in the following table.

[0196] Among them, Laa-Lbb may refer to the amino acid sequence starting from the N-terminus of the antibody light chain variable region and from positions aa to bb; Haa-Hbb may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain variable region and from positions aa to bb. For example, L24-L34 may refer to the amino acid sequence starting from the N-terminus of the antibody light chain and from positions 24 to 34; H26-H35 may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and from positions 26 to 35.

[0197] As one of the cases, in the sequence listing of the present application, the CDR sequences and framework region (FR) sequences of the isolated antigen-binding protein are determined by the Kabat numbering system.

[0198] In the present application, the VH of the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3.

[0199] In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 69.

[0200] X1X2WMX3 (SEQ ID NO: 69), wherein X1 can be D or S; X2 can be A or Y; and X3 can be D or H.

[0201] For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27.

[0202] In the present application, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28.

[0203] In the present application, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 83 or SEQ ID NO: 29.

[0204] [-G][DI][RY]Y[DG][EY][DG][NY]AMDY (SEQ ID NO: 83). wherein X1 may be absent or G; X2 may be D or I; X3 may be R or Y; X4 may be D or G; X5 may be E or Y; X6 may be D or G; and X7 may be N or Y.

[0205] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 29.

[0206] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

[0207] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 20.

[0208] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0209] In the present application, the VL of the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3.

[0210] In the present application, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 34.

[0211] RASQDIX1NYLN (SEQ ID NO: 70), wherein X1 can be S or T.

[0212] For example, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:23 and SEQ ID NO:34.

[0213] In the present application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 71.

[0214] YX1SX2LX3S (SEQ ID NO: 71), wherein X1 can be A or T, X2 can be N or R, and X3 can be H or Q.

[0215] For example, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35.

[0216] In the present application, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 72.

[0217] QX1X2X3X4X5PX6T (SEQ ID NO: 72). For example, X1 can be H or Q; X2 can be G or S; X3 can be D, N or W; X4 can be E or T; X5 can be I or L; and X6 can be P or R.

[0218] For example, the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36.

[0219] In the present application, the LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:70 or SEQ ID NO:34, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:71, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:72 (QX1X2X3X4X5PX6T, X1 is H or Q; X2 is G or S; X3 is D, N or W; X4 is E or T; X5 is I or L; X6 is P or R).

[0220] In the present application, the LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:9, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:11.

[0221] In the present application, the LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0222] In the present application, the LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0223] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0224] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 29, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36.

[0225] In the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the isolated antigen-binding protein may comprise an amino acid sequence selected from the group consisting of:

[0226] (1) The HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 11;

[0227] (2) the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 20, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 24; and

[0228] (3) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0229] In the present application, the VH of the isolated antigen-binding protein may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0230] In the present application, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:73.

[0231] X1VX2LX3X4X5GX6X7X8X9X 10 PGX 11 SX 12 KX 13 SCX 14 ASGX 15 TFX 16 (SEQ ID NO: 73). X1 can be E or Q; X2 can be K or Q; X3 can be E, V or Q; X4 can be E or Q; X5 can be P or S; X6 can be A or G; X7 can be E or G; X8 can be L or V; X9 can be K or V; X 10 Can be K or Q; X 11 Can be A or G; X 12 Can be M or V; X 13 Can be L or V; X 14 Can be A or K; X 15 Can be F or Y; X 16 Can be S or T.

[0232] For example, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 30 and SEQ ID NO: 41.

[0233] In the present application, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 74.

[0234] WVX1QX2PX3X4GLEWX5X6 (SEQ ID NO: 74), wherein X1 can be K or R; X2 can be A, R or S; X3 can be E or G; X4 can be K or Q; X5 can be I, M or V; and X6 can be A or G.

[0235] For example, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 42 and SEQ ID NO: 53.

[0236] In the present application, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 75.

[0237] X1X2TX3X4X5DX6SX7SX8X9YX 10 X 11 X 12 X 13 SLX 14 X 15 EDX 16 X 17 X 18 YYCX 19 R (SEQ ID NO: 75). Wherein, X1 can be K or R; X2 can be A, F or V; X3 can be I, L or M; X4 can be I, S or T; X5 can be R or V; X6 can be D, K or T; X7 can be K, S or T; X8 can be S or T; X9 can be A or V; X 10 Can be L, M or V; X 11 Can be E or Q; X 12 Can be L or M; X 13 Can be N or S; X 14 Can be R or T; X 15 Can be A or S; X 16 Can be S or T; X 17 Can be A or G; X 18 Can be I or V; X19 Can be A or T.

[0238] For example, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:54 and SEQ ID NO:57.

[0239] In the present application, the N-terminus of the H-FR4 may be connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 76.

[0240] GQGTX1VTVSS (SEQ ID NO: 76), wherein X1 can be S or T.

[0241] For example, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 44.

[0242] For example, H-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO:73, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:74, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:75, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:76.

[0243] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequences shown in the following group:

[0244] (1) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0245] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7;

[0246] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:21, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7;

[0247] (4) the H-FR1 comprises the amino acid sequence of SEQ ID NO: 30, the H-FR2 comprises the amino acid sequence of SEQ ID NO: 31, the H-FR3 comprises the amino acid sequence of SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 7;

[0248] (5) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:42, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:43, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:44;

[0249] (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:54, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44;

[0250] (7) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:57, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:44.

[0251] In the present application, the VL of the isolated antigen-binding protein may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0252] In the present application, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 77.

[0253] DIX1MTX2X3X4X5SLX6X7SX8GX9RX 10 TIX 11C (SEQ ID NO: 77). wherein X1 may be Q or V; X2 may be H or Q; X3 may be S or T; X4 may be P or T; X5 may be A or S; X6 may be A or S; X7 may be A or V; X8 may be L or V; X9 may be D or Q; X 10 Can be A or V; X 11 Can be S or T.

[0254] For example, the L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 37 and SEQ ID NO: 47.

[0255] In the present application, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 78.

[0256] WYQQKPX1X2X3X4KLLIX5 (SEQ ID NO: 78), wherein X1 can be D or G; X2 can be G, K or Q; X3 can be A, P or T; X4 can be P or V; and X5 can be K or Y.

[0257] For example, the L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 38, and SEQ ID NO: 48.

[0258] In the present application, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 79.

[0259] GVPX1RFSGSGSGTX2X3X4X5X6IX7X8X9X 10 X 11 X 12 DX 13 ATYX 14 C (SEQ ID NO: 79). wherein X1 may be A or S; X2 may be D or E; X3 may be F or Y; X4 may be S or T; X5 may be F or L; X6 may be N or T; X7 may be H or S; X8 may be N, P or S; X9 may be L or V; X 10 Can be E or Q; X 11 Can be E, P or Q; X 12 Can be D or E; X 13 Can be F, I or S; X 14 Can be F or Y.

[0260] For example, the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 14, SEQ ID NO: 39, SEQ ID NO: 49 and SEQ ID NO: 60.

[0261] In the present application, the N-terminus of the L-FR4 may be connected to the C-terminus of the HCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 80.

[0262] FGX1GTX2LEIK (SEQ ID NO: 80), wherein X1 can be G or Q; and X2 can be K or R.

[0263] For example, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 50.

[0264] For example, L-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 77, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 78, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 79, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 80.

[0265] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein described in the present application may comprise the amino acid sequences shown in the following group:

[0266] (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 12, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0267] (2) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 25, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0268] (3) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 37, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 38, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0269] (4) the L-FR1 comprises the amino acid sequence of SEQ ID NO:47, the L-FR2 comprises the amino acid sequence of SEQ ID NO:48, the L-FR3 comprises the amino acid sequence of SEQ ID NO:49, and the L-FR4 comprises the amino acid sequence of SEQ ID NO:50; and

[0270] (5) The L-FR1 comprises the amino acid sequence shown in SEQ ID NO:47, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:48, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:60, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:50.

[0271] The isolated antigen binding proteins described herein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0272] For example, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 11.

[0273] For example, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 24.

[0274] For example, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0275] The isolated antigen-binding protein described herein may comprise an antibody light chain variable region VH and an antibody heavy chain variable region VL.

[0276] In the present application, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33 and SEQ ID NO: 22. For example, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 45, SEQ ID NO: 55 and SEQ ID NO: 58.

[0277] In the present application, the VL may comprise the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40. For example, the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 40, SEQ ID NO: 51 and SEQ ID NO: 61.

[0278] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0279] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 19, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0280] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 22, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0281] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 33, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 40.

[0282] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:45, and the VL may comprise the amino acid sequence shown in SEQ ID NO:51.

[0283] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 55, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 51.

[0284] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 58, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 51.

[0285] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:45, and the VL may comprise the amino acid sequence shown in SEQ ID NO:61.

[0286] In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.

[0287] In certain embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In other embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG4 heavy chain constant region.

[0288] For example, the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 63.

[0289] In the present application, the isolated antigen-binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Igκ constant region. For example, the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO: 64.

[0290] In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence shown in any one of SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:91.

[0291] In the present application, the isolated antigen-binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence shown in any one of SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 87, SEQ ID NO: 90 and SEQ ID NO: 92.

[0292] The isolated antigen-binding protein described herein may comprise an antibody heavy chain and an antibody light chain.

[0293] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 87.

[0294] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 87.

[0295] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 90.

[0296] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:91, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:92.

[0297] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:52.

[0298] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 56, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 52.

[0299] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 59, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 52.

[0300] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:62.

[0301] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy chain and / or light chain sequences that have one or more conservative sequence modifications with the antigen-binding proteins described herein. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not abolish antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissrov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0302] The proteins, polypeptides and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0303] In the present application, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., the isolated antigen-binding protein described in the present application). For example, the variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., the ability to specifically bind to the LILRB4 protein) of the protein or polypeptide before the change.

[0304] In the present application, the homolog can be a protein or polypeptide having at least about 80% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (for example, the antibody or antigen-binding fragment thereof described in the present application).

[0305] In the present application, described homology generally refers to the similarity between two or more sequences or the degree of association.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0306] In the present application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding protein described herein may include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a polyclonal antibody, an Fv fragment, a scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelized single-domain antibody.

[0307] In the present application, the antibody may be a humanized antibody. In other words, the isolated antigen-binding protein described herein may be an antibody or a variant, derivative, analog or fragment thereof that immunospecifically binds to a relevant antigen (e.g., human LILRB4) and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Here, "substantially" in the case of CDRs means that the amino acid sequence of the CDR is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the CDR of a non-human antibody. The humanized antibody may comprise substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., an antibody) and all or substantially all framework regions are framework regions having a consensus sequence of a human immunoglobulin. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically a constant region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domains of a light chain and a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only a humanized variable domain of a light chain and / or a humanized heavy chain.

[0308] In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0309] In the present application, the isolated antigen-binding protein may have one or more of the following properties: 1) being able to specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) being able to -7 M or lower K D The invention discloses a novel novel anti-ILR-4 inhibitor that can bind to LILRB4 protein; 3) can block the activity of LILRB4 / APOE binding; 4) can stimulate the activation of CD8+ T cells; 5) can inhibit the migration of human monocytic leukemia cells THP-1; 6) can inhibit the growth and / or metastasis of tumor cells; 7) can block the binding of LILRB4 to Fibronectin.

[0310] In the present application, the isolated antigen binding protein can be expressed as 2×10 -7 M or lower K D Binds to LILRB4 protein. For example, the isolated antigen binding protein described herein binds to the K of human LILRB4 protein.D The value can be ≤2×10 - 7 M, ≤1.5×10 -7 M, ≤1×10 -7 M, ≤9×10 -8 M, ≤8×10 -8 M, ≤7×10 -8 M, ≤6×10 -8 M, ≤5×10 -8 M, ≤4×10 -8 M, ≤3×10 -8 M, ≤2×10 -8 M, ≤1.5×10 -8 M, ≤1.2×10 -8 M, ≤1.15×10 -8 M, ≤1.1×10 -8 M, ≤1.05×10 -8 M, ≤1×10 -8 M, ≤5×10 -9 M or ≤1×10 -9 M.

[0311] In the present application, the KD value can also be determined by ELISA, competitive ELISA, BIACORE or KINEXA.

[0312] In the present application, the isolated antigen-binding protein is capable of specifically binding to human LILRB4 protein. The specific binding can be determined by FACS.

[0313] In the present application, the LILRB4 protein may be human LILRB4 protein (GenBank accession number: CAG46845.1).

[0314] The LILRB4 protein may include variants of the LILRB4 protein. For example, the variant may be: 1) a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the LILRB4 protein; and 2) a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to the LILRB4 protein.

[0315] The LILRB4 protein may also include a functionally active fragment of the LILRB4 protein.

[0316] Polypeptides, fusion proteins, drug molecules, nucleic acid molecules, vectors, cells and pharmaceutical compositions

[0317] In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein.

[0318] In another aspect, the present application provides a fusion protein comprising the isolated antigen-binding protein.

[0319] In another aspect, the present application provides a drug molecule comprising the isolated antigen-binding protein.

[0320] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the isolated antigen-binding protein. The one or more isolated nucleic acid molecules described herein can be nucleotides, deoxyribonucleotides or ribonucleotides, in an isolated form of any length, or analogs isolated from their natural environment or artificially synthesized, but can encode the isolated antigen-binding protein described herein.

[0321] On the other hand, the present application also provides vectors, which may contain the nucleic acid molecules described herein. The vectors can be transformed, transduced, or transfected into host cells, so that the genetic material elements they carry are expressed in the host cells. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and animal viruses. Types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). For another example, the vectors may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vectors may also contain a replication initiation site. Furthermore, the vectors may also include components that facilitate entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0322] On the other hand, the application also provides cells, which can comprise nucleic acid molecules described herein or vectors described herein. The cells can include the offspring of individual cells. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical with the original mother cell (in the form of total DNA complement or on the genome). In some embodiments, the cells can also include cells transfected in vitro with the vectors described in the present invention. In some embodiments, the cells can be bacterial cells (e.g., Escherichia coli), yeast cells or other eukaryotic cells.

[0323] On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described herein, the polypeptide described herein, the fusion protein described herein, the nucleic acid molecule described herein, the vector described herein and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0324] In certain embodiments, the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or suitable formulations of preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes but is not limited to liquid, frozen and lyophilized compositions.

[0325] In certain embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0326] In certain embodiments, the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and / or intranasal administration or direct injection into tissue.For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered uninterruptedly. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015 / 036583.

[0327] In the present application, the pharmaceutical composition may further include one or more drugs selected from the following groups: anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, emcitabine, Idhifa, IDO inhibitors Preparations, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibody, PD-L1 antibody, anti-CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0328] Preparation method and use

[0329] On the other hand, the present application also provides a method for preparing the isolated antigen-binding protein described herein, which may include culturing the cells described herein under conditions that allow the isolated antigen-binding protein described herein to be expressed.

[0330] On the other hand, the present application provides the use of the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition in the preparation of a medicament for preventing and / or treating a disease and / or condition.

[0331] On the other hand, the present application provides the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition, which are used to prevent and / or treat diseases and / or disorders.

[0332] On the other hand, the present application provides a method for preventing and / or treating a disease and / or condition, comprising administering an effective amount of the isolated antigen-binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition to a subject in need.

[0333] In the present application, the administration can be performed by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0334] In the present application, the disease and / or disorder may include a disease or disorder associated with LILRB4 signaling.

[0335] In the present application, the disease and / or disorder may include a tumor.

[0336] In the present application, the diseases and / or disorders may include LILRB4-positive diseases and / or disorders.

[0337] In the present application, the tumor may include solid tumors and / or hematological tumors.

[0338] In the present application, the disease or disorder associated with LILRB4 signaling may include immune system diseases and / or hematological malignancies.

[0339] In the present application, the immune system disease may be selected from one or more of the following groups: Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0340] In the present application, the solid tumor may be selected from one or more of the following groups: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer and pancreatic cancer.

[0341] In the present application, the malignant blood disease can be selected from one or more of the following groups: myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia, or acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, acute myeloblastic leukemia, and polycythemia vera.

[0342] In the present application, the isolated antigen binding protein, the polypeptide, the fusion protein, the drug molecule, the nucleic acid molecule, the vector, the cell, the pharmaceutical composition and / or the drug can be used in combination with one or more drugs selected from the following group: anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, ensidipine, Idhifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibody, PD-L1 antibody, anti-CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0343] In another aspect, the present application provides use of the antibody or antigen-binding fragment thereof for preparing a diagnostic agent, wherein the diagnostic agent is used to diagnose a disease or condition associated with the expression of the LILRB4 protein.

[0344] In another aspect, the present application provides an isolated antigen-binding protein according to the method, which is used for diagnosing a disease or condition associated with the expression of the LILRB4 protein.

[0345] In this application, the diagnostic agent can be used alone or in combination with an instrument, apparatus, device, or system. In the prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation, and prediction of genetic diseases, the diagnostic agent can be used to perform in vitro detection on human samples (e.g., various body fluids, cells, tissue samples, etc.). The diagnostic agent can be selected from the following group: reagents, kits, calibrators, and quality control products.

[0346] The in vitro detection method can be selected from the group consisting of Western Blot, ELISA, and immunohistochemistry. For example, the reagent can include a reagent capable of measuring the expression level of the LILRB4 protein. For example, the reagent can be selected from the group consisting of a reagent for performing Western Blot, a reagent for performing ELISA, and a reagent for performing immunohistochemistry.

[0347] In another aspect, the present application provides a detection kit comprising the isolated antigen-binding protein, for detecting the presence and / or amount of LILRB4 protein in a sample. In particular, the present application relates to an immunoassay kit for use using the antibodies of the present application in conjunction with immunoassay methods such as ELISA, immunohistochemistry, Western blotting, and flow cytometry.

[0348] The kit includes the antibody of the present application for detecting LILRB4-related cancer cells. Specifically, the immunodetection kit contains the antibody of the present application as a first antibody that binds to LILRB4, and optional immunodetection reagents in a suitable container component.

[0349] In certain embodiments, the antibodies may be pre-bound to a solid support, such as a column matrix and / or a microtiter plate well.

[0350] The immunodetection reagents in the kit can be in any of a variety of forms, including those detectable labels that are bound or linked to a given antibody. Detectable labels that are bound or linked to a secondary binding ligand can also be included. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[0351] Other immunodetection reagents suitable for use in the present kits include two-component reagents comprising a secondary antibody having binding affinity for the first antibody, and a third antibody having binding affinity for the second antibody, the third antibody being linked to a detectable label. As described above, a variety of exemplary labels are known in the art and all such labels can be used in conjunction with the present disclosure.

[0352] The kit may further comprise an appropriate aliquot of a LILRB4 composition, either labeled or unlabeled, which can be used to prepare a standard curve for the assay. The kit may contain the antibody-label conjugate in fully conjugated form, in the form of an intermediate, or with individual portions conjugated by the user of the kit. The components of the kit may be packaged in an aqueous medium or in lyophilized form.

[0353] The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the antibody or preferably an appropriate aliquot of the antibody can be placed. The kits of the present disclosure will also typically include means for containing the antibody, antigen, and any other reagent containers, which are in a sealed form for commercial sale. Such containers may include injection-molded or blow-molded plastic containers in which the vials described above are retained.

[0354] On the other hand, the present application provides a method for diagnosing a disease or condition associated with the expression of LILRB4 protein in a subject, the method comprising: contacting a sample derived from the subject with the isolated antigen-binding protein, and determining the presence and / or amount of a substance in the sample that can specifically bind to the isolated antigen-binding protein.

[0355] In another aspect, the present application provides a method for detecting LILRB4 in a sample or subject, the method comprising administering the isolated antigen-binding protein. In the present application, the administration can be performed by various routes, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

[0356] Without intending to be bound by any theory, the following examples are merely intended to illustrate the antigen-binding proteins, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0357] Example

[0358] Example 1 Screening of Monoclonal Antibodies Using Hybridoma Technology

[0359] Balb / c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were routinely immunized with a human LILRB4 protein extracellular domain-human IgG1 Fc fusion protein (named LILRB4-Fc, where the sequence information of human LILRB4 is referenced to Q8NHJ6, the sequence of the human IgG1 Fc fragment is referenced to AXN93652.1, the amino acid sequence of the human LILRB4 protein extracellular domain is shown in SEQ ID NO: 84; the amino acid sequence of LILRB4-Fc is shown in SEQ ID NO: 85).

[0360] On days 0, 14, and 28, Balb / c mice were subcutaneously injected with 100 μg of LILRB4-Fc in the presence of Freund's complete adjuvant (first injection) or Freund's incomplete adjuvant (second and third injections). Splenocytes from the mice were fused with mouse myeloma cells SP2 / 0 (ATCC) using conventional hybridoma technology protocols (Salhi et al., Biochem. J. 2004). The cells were cultured in plates containing HAT medium (10 cells per well). 5Cells were used for hybridoma selection. After 12 days, the supernatant was harvested and clones with binding specificity to LILRB4 (positive clones) were screened by direct enzyme-linked immunosorbent assay (ELISA). Through screening, a series of mouse monoclonal antibodies targeting the extracellular domain (ECD) of human LILRB4 were obtained.

[0361] The variable region genes of the obtained mouse monoclonal antibodies were cloned into the eukaryotic expression vector pCMV-163 containing the human IgG1 constant region gene to construct antibody expression vectors, which were transfected into CHO-S cells for expression. The chimeric antibodies were then affinity purified using conventional Protein A and named 1A2-7, 3A2-18, 3G9-1, and 5E11-2. The amino acid sequences of the chimeric antibodies are shown in the table below, where the CDR sequences were divided using the Kabat method.

[0362] Example 2 Binding of Chimeric Antibodies to Human LILRB4

[0363] ELISA plates were coated with LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag, Sino Biological) and incubated at 37°C for 2 h. After washing with PBST, 10% fetal bovine serum was added and the plates were blocked at 37°C for 1 h. Chimeric antibodies and positive antibody C84 at different concentrations were added and reacted at 37°C for 1 h. After washing with PBST, goat anti-human IgG Fc secondary antibody labeled with horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37°C for 30 min. The plates were washed five times with PBST. 100 μL of TMB (eBioscience) was added to each well and incubated at room temperature (20±5°C) in the dark for 2–3 min. 100 μL of 2N The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm on a microplate reader to analyze the binding ability of the chimeric antibody to LILRB4.

[0364] As shown in FIG1 , the chimeric antibodies 1A2-7, 3A2-18, 3G9-1, and 5E11-2 had binding abilities to LILRB4 similar to that of the positive antibody C84.

[0365] Example 3 Detection of binding specificity of chimeric antibodies to human LILRB family proteins

[0366] The LILRB family proteins LILRB1 (Human LILRB1 / CD85 / ILT2 / ILR1 Protein (ECD, His Tag), Sino Biological), LILRB2 (Human LILRB2 / ILT4 / LIR-2 Protein (ECD, His Tag), Sino Biological), LILRB3 (Human LILRB3 / LIR3 / ILT5 / CD85a Protein (ECD, His Tag), Sino Biological), LILRB4 (Human LILRB4 / CD85k / ILT3 protein (His Tag), Sino Biological), LILRB5 (Human LILRB5 / CD85c / LIR-8 Protein (ECD, His Tag), Sino ELISA plates were coated with 10% fetal bovine serum (FBS) proteins at 4°C overnight. After washing with PBST, 10% fetal bovine serum was added and the plates were blocked at 37°C for 1 hour. 10 μg / mL of chimeric antibody was added to each well and reacted at 37°C for 1 hour. After washing with PBST, goat anti-human IgG Fc antibody (horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37°C for 30 minutes. The plates were washed five times with PBST. 100 μL of TMB (eBioscience) was added to each well and the plates were incubated at room temperature (20±5°C) in the dark for 1–2 minutes. Subsequently, 100 μL of 2N H2SO4 stop solution was added to each well to terminate the substrate reaction. The OD values ​​were read at 450 nm on a microplate reader to analyze the nonspecific binding ability of the chimeric antibody to LILRB1, LILRB2, LILRB3, and LILRB5.

[0367] The results are shown in Figure 2 , and none of the chimeric antibodies non-specifically bind to LILRB1, LILRB2, LILRB3, and LILRB5.

[0368] Example 4 Detection of Chimeric Antibody Blocking Activity of LILRB4 / APOE Binding

[0369] The activity of chimeric antibodies in blocking LILRB4 / APOE binding was detected using the Jurkat-LILRB4-CHO-CD3-APOE reporter cell line, and 128-3 (Oncoimmune antibody 128-3) was used as a positive control.

[0370] APOE / TCR activator / CHO cells in logarithmic growth were seeded into 96-well plates and cultured overnight at 37°C. The next day, the supernatant was discarded, the cells were washed twice with PBS, and the antibody sample diluted in 1640 medium was added. LILRB4 effector reporter cells in logarithmic growth were then added and cultured in a 37°C incubator. After 5 hours, the cells were removed, Bright-Glo luciferase was added, and the luciferase readings were read on a microplate reader. The values ​​for each well were analyzed using Prism GraphPad software.

[0371] The results are shown in Figures 3A and 3B , showing that chimeric antibodies 1A2-7 and 3A2-18 were able to block the binding of LILRB4 to APOE, wherein the blocking activity of 1A2-7 was superior to that of the positive control 128-3.

[0372] Example 5 Chimeric Antibody Stimulation of CD8 + T cell activation

[0373] PBMCs were isolated from whole blood of healthy donors and CD8 + T cell isolation kit (CD8 + CD8 T Cell Isolation Kit, Miltenyi) was isolated + T cells, 5×10 4 Cells / well were added to a 96-well plate (96-well Clear Round Bottom TC-treated Microplate, Corning); T cell activation magnetic beads ( Human T-Activator CD3 / CD28 / CD137, GIBCO) was placed in a 37°C 5% CO2 incubator for 2 days; chimeric antibodies with a final concentration of 200 μg / mL, positive control antibody C84, and isotype control were added, and then 5×10 4 Human monocytic leukemia THP-1 cells were added to each well and incubated in a 37°C 5% CO2 incubator for 5 days. The supernatant was collected and the IFN-γ level in the supernatant was detected using Human IFN-γ Elisa Kit (ExCell Bio) to evaluate the activity of the chimeric antibody in THP-1 / CD8 + Activated CD8 T lymphocytes in co-culture experiments + T cell capacity.

[0374] The results are shown in Figure 4. Chimeric antibodies 1A2-7, 3A2-18, 3G9-1, and 5E11-2 were expressed in THP-1 / CD8 + Activated CD8 T lymphocytes in co-culture +The T cell potency was similar to that of the positive control antibody C84.

[0375] Example 6 Inhibitory Effect of Chimeric Antibodies on THP-1 Cell Migration

[0376] Human monocytic leukemia THP-1 cells were serum-starved for 24 h and added to a 24-well cell culture plate. Chimeric antibodies and positive control antibody C84 were added at a final concentration of 10 μg / mL and incubated in a 37°C 5% CO2 incubator for 1 h. 100 μL of the cell-antibody mixture was added to a Transwell (6.5 mm The cells were placed in the upper chamber of a 96-well plate (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning) and 700 μL of complete culture medium was added to the lower chamber. The cells were placed in a 37°C 5% CO2 incubator and incubated for 24 h. 100 μL of the solution was taken from the lower chamber and added to a 96-well plate (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning). 100 μL of cell viability assay reagent (CellTiter-LumiNano) was added. TM Luminescence cell viability detection kit (Biyuntian) was used. After incubation at room temperature in the dark for 10 min, the relative fluorescence intensity of chemiluminescence was detected using a multifunctional microplate reader. A standard curve was drawn using the gradient dilution of THP-1 cells and their corresponding relative fluorescence intensity values. The number of cells in the lower chamber was calculated, and the inhibitory effect of the chimeric antibody on THP-1 cell migration was analyzed.

[0377] The results are shown in FIG5 . The chimeric antibodies 1A2-7, 3A2-18, and 3G9-1 have stronger abilities to inhibit THP-1 cell migration than the positive control antibody C84, and the inhibitory ability of 5E11-2 is similar to that of the positive control antibody C84.

[0378] Example 7 Chimeric Antibodies Inhibit the Growth and Metastasis of THP-1 in NSG Mice

[0379] 5×10 THP1-Luc human monocytic leukemia cells resuspended in PBS 6 The mice were injected into the tail vein at a volume of 0.2 mL / mouse. The mice were divided into groups according to the imaging signal (average imaging signal 1.9x10 7p / s) and body weight (average body weight 22.4g) were randomly divided into 5 experimental groups, with 5 mice in each group, namely G1 (PBS), G2 (1A2-7, 10mg / kg), G3 (3A2-18, 10mg / kg), and G5 (C84, 10m / kg). Drugs were administered on the day of grouping (day 0) and day 3 after grouping, and all mice were administered intraperitoneally. After cell inoculation (i.e., before grouping and drug administration, day 0), on days 3, 7, 14, 21, and 25 after drug administration, and before the end of the experiment (day 26), mice were imaged using a small animal in vivo imaging device to obtain imaging signal images and signal intensity, and the growth of tumors in vivo was observed. After the observation was completed, the experiment was terminated. During the drug administration and observation period, the body weight of the animals was measured twice a week, and the measurement values ​​were recorded.

[0380] At the end of the experiment, the tumor imaging signal intensity of the control group (PBS) was 1.43x10 9 ±1.32x10 8 p / s; tumor imaging signal intensity in group 1A2-7 was 1.27x10 6 ±2.35x10 5 p / s, TGITV was 101.3%; the tumor imaging signal intensity of group 3A2-18 was 1.18x10 6 ±2.79x10 5 p / s, TGITV was 101.3%; the tumor imaging signal intensity of group C84 was 3.74x10 7 ±1.92x10 7 p / s, TGITV was 98.7%. Compared with the G1 group, the P values ​​of each group were all <0.001, indicating that the test products 1A2-7, 3A2-18, and C84 at a dose of 10 mg / kg had a significant inhibitory effect on the growth of tumors in B-NDG mice transplanted with THP1-LUC cells.

[0381] The results are shown in FIG6 . Chimeric antibodies 1A2-7 and 3A2-18 can effectively inhibit tumor growth, and the inhibitory effect is significantly better than that of the positive control antibody C84.

[0382] At the end of the above experiment, the mice were weighed and imaged, then euthanized and the survival curve of the experimental animals was drawn. The hCD45 expression in the liver, lung and bone marrow of each group of mice was analyzed by flow cytometry. + The ratio of cells (live / dead, hCD45+, mCD45+).

[0383] Before the tumor model experiment, flow cytometry analysis showed that THP1-Luc cells were all hCD45 +Before administration, hCD45 was detected in the lungs, bone marrow (BM) and liver of the B-NDG mice, the model mice constructed in this experiment. + cell.

[0384] The results of flow cytometry after administration showed that compared with the G1 control group, the levels of hCD45 in the liver, lung and bone marrow of mice in each group were significantly higher than those in the G1 control group. + The cell proportions were significantly reduced, indicating that the test products C84, 1A2-7 and 3A2-18 at a dose of 10 mg / kg can significantly reduce the amount of tumor cells in the liver, lungs and bone marrow of mice and inhibit tumor cell migration. The results are shown in Figure 7.

[0385] Example 8 Antibody Humanization

[0386] The antibodies were humanized using standard CDR grafting methods. Human heavy and light chain variable sequences were BLAST searched, and three or four sequences from each sequence were selected as humanized acceptor frameworks. The heavy and light chain CDR1, CDR2, and CDR3 were cloned into three different heavy chain frameworks (H1-H4) and two different light chain frameworks (L1-L2). Four different antibody combinations (see Table 1) were then tested for expression levels and antigen binding ability in CHO-S cells.

[0387] Example 9 Binding of Humanized Antibodies to Human LILRB4

[0388] ELISA plates were coated with LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag, Sino Biological) and incubated at 37°C for 2 h. After washing with PBST, 10% fetal bovine serum was added and the plates were blocked at 37°C for 1 h. Different concentrations of humanized antibodies and positive antibody 128-3 were added and reacted at 37°C for 1 h. After washing with PBST, goat anti-human IgG Fc secondary antibody labeled with horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37°C for 30 min. The plates were washed five times with PBST. 100 μL of TMB (eBioscience) was added to each well and incubated at room temperature (20±5°C) in the dark for 2–3 min. 100 μL of 2N The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm on a microplate reader to analyze the binding ability of the humanized antibody to LILRB4.

[0389] The results are shown in FIG8 . The binding abilities of humanized antibodies RB4-G1-018, RB4-G1-019, RB4-G1-020, and RB4-G1-022 to LILRB4 were similar to those of the positive control antibody 128-3.

[0390] Example 10 Humanized Antibody Affinity Determination

[0391] Antibody affinity was analyzed using a BIACORE 8K biomacromolecular interaction instrument (GE). The test antibodies were captured using a Series S Sensor Chip Protein A chip (GE). The test antibody concentration was set at 15 μg / mL, and the sample was injected at 10 μl / min until RU = 500. LILRB4 (Beijing Biopsies Technology Co., Ltd., Catalog No. LI4-H52H7, 6× His tag, theoretical molecular weight 28.2 kDa) was used as the mobile phase. A six-step concentration gradient (ranging from 0.0977 nM to 200 nM) was used. The association time was 180 seconds, and the dissociation time was 300 seconds. The results of the antibody affinity tests are shown below.

[0392] Example 11 Determination of Binding Ability of Humanized Antibodies to LILRB4-Positive Cells

[0393] Human monocytic leukemia THP-1 cells were collected and 5×10 cells were added to each tube. 5 Cells were added to 1.5 mL EP tubes; different concentrations of humanized antibodies and positive control antibody 128-3 were added and incubated on ice in the dark for 30 minutes; after washing with FACS buffer, PE fluorescent-labeled goat anti-human IgG Fc secondary antibody (Invitrogen) was added and incubated on ice in the dark for 30 minutes; washed twice with FACS buffer; 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells, mixed well, and the relative fluorescence intensity of PE fluorescence was measured on a microscope to analyze the binding ability of the humanized antibody to LILRB4-positive THP-1 cells.

[0394] The results are shown in FIG9 . The humanized antibody had a similar binding activity to LILRB4-positive THP-1 cells and was stronger than that of the positive antibody 128-3.

[0395] Example 12 Humanized Antibodies Inhibit LILRB4+ Tumor Migration

[0396] 5×10 THP1-Luc human monocytic leukemia cells resuspended in PBS 6 / 0.2mL / mouse was injected into the tail vein of B-NDG mice. On the day of inoculation, the imaging signal of mice (average imaging signal 3.6x10 7 Based on the p / s and mouse body weight (average weight 23 g), mice were randomly assigned to six experimental groups, with five mice in each group: G1 (PBS), G2 (C84, 10 mg / kg), G3 (RB4-G1-018, 10 mg / kg), G4 (RB4-G1-019, 10 mg / kg), G5 (RB4-G1-020, 10 mg / kg), and G6 (RB4-G1-022, 10 mg / kg). All mice were intraperitoneally injected with the drug on the day of grouping (day 0) and day 3 after grouping. Mice were imaged using a small animal in vivo imaging system before grouping and dosing (day 0), and on days 7, 14, 21, and 25 after dosing and at the end of the experiment (day 28). Imaging signal patterns and signal intensities were obtained to observe tumor growth in vivo. Animal body weights were measured twice weekly during the dosing and observation period, and the measured values ​​were recorded.

[0397] At the end of the experiment, the mice were weighed and imaged, then euthanized and the survival curve of the experimental animals was drawn. The hCD45 expression in the liver, lung and bone marrow of each group of mice was analyzed by flow cytometry. + The ratio of cells (live / dead, hCD45 + , mCD45 + ).

[0398] The results of flow cytometry are shown in Figure 10. Compared with the G1 control group, the expression of hCD45 in the liver, lung and bone marrow of mice in each group was significantly higher than that in the control group. + The cell proportions were significantly reduced, indicating that the test products C84, RB4-G1-018, RB4-G1-019, RB4-G1-020 and RB4-G1-022 at a dose of 10 mg / kg could significantly reduce the amount of tumor cells in the liver, lungs and bone marrow of mice.

[0399] Example 13 Detection of the Activity of LILRB4 Humanized Antibodies to Block LILRB4 / APOE Binding Using Jurkat-LILRB4-CHO-CD3-APOE Reporter Cell Line

[0400] APOE / TCR activator / CHO cells in the logarithmic growth phase were seeded into 96-well plates and cultured overnight at 37°C. The next day, the supernatant was discarded, the cells were washed twice with PBS, and 50 μl of antibody sample diluted in 1640 medium was added to each well. LILRB4 effector reporter cells in the logarithmic growth phase were then added and cultured in a 37°C incubator. After 5 hours, the cells were removed, Bright-Glo luciferase was added, and the values ​​were read on a microplate reader. The values ​​for each well were analyzed using Prism GraphPad software.

[0401] The results are shown in Figure 11. All humanized antibodies can block the binding of APOE to LILRB4, and the blocking ability is stronger than that of the positive control antibodies 128-3 and C84.

[0402] Example 14 ADCC activity of humanized antibodies on 293T-hLILRB4 cells

[0403] 293T-hLILRB4 cells overexpressing the human LILRB4 gene were collected and added to a white transparent bottom 96-well plate (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning); different concentrations of humanized antibodies and positive antibody 128-3 were added; then effector cells Jurkat-hFcγRⅢa-NFAT overexpressing the human FcγRIIIa gene and the NFAT fluorescent reporter gene were added, and the 96-well plate was placed in a 37°C 5% CO2 incubator for 6 hours; 100 μL of luciferase detection reagent (Bright-Lumi TM Firefly luciferase reporter gene assay kit (Biyuntian) was used. After incubation at room temperature in the dark for 10 min, the relative fluorescence intensity of chemiluminescence was measured by a multifunctional microplate reader to analyze the ADCC activity mediated by the humanized antibody on 293T-hLILRB4 cells.

[0404] The results are shown in Figure 12. The ADCC activities of humanized antibodies RB4-G1-018, RB4-G1-019, and RB4-G1-022 were similar and stronger than the positive control antibody 128-3. The ADCC activity of RB4-G1-020 was similar to that of the positive control antibody 128-3.

[0405] Example 15 Humanized Antibodies Stimulate CD8+ T Cell Activation

[0406] PBMCs were isolated from whole blood of healthy donors and CD8 +CD8 T cell isolation kit (CD8+ T Cell Isolation Kit, Miltenyi) was used to isolate CD8 + T cells, 5×10 4 Cells / well were added to a 96-well plate (96-well Clear Round Bottom TC-treated Microplate, Corning); T cell activation magnetic beads ( Human T-Activator CD3 / CD28 / CD137, GIBCO) was placed in a 37°C 5% CO2 incubator for 2 days; humanized antibodies with a final concentration of 200 μg / mL, positive antibody 128-3, and isotype control were added, and 5×10 4 Human monocytic leukemia THP-1 cells were added to each well and incubated in a 37°C 5% CO2 incubator for 5 days. The supernatant was collected and the level of TNF-α in the supernatant was detected using Human TNF-α Elisa Kit (ExCell Bio) to evaluate the effect of humanized antibodies on THP-1 / CD8 + Activated CD8 T lymphocytes in co-culture experiments + T cell capacity.

[0407] The results are shown in Figure 13. Humanized antibodies RB4-G1-018 and RB4-G1-020 were expressed in THP-1 / CD8 + Activated CD8 T lymphocytes in co-culture + The ability of T cells is stronger than that of positive antibody 128-3.

[0408] Example 16 LILRB4 humanized antibody can inhibit tumor growth in B-NDG mice transplanted with THP1-LUC cells

[0409] 5×10 THP1-Luc human monocytic leukemia cells resuspended in PBS 6 / 0.2mL / mouse was injected into the tail vein of B-NDG mice. On the day of inoculation, the imaging signal of mice (average imaging signal 3.6x10 7Based on the p / s and mouse body weight (average weight 23 g), mice were randomly assigned to six experimental groups, with 5 mice in each group: G1 (PBS), G2 (C84, 10 mg / kg), G3 (RB4-G1-018, 10 mg / kg), G4 (RB4-G1-019, 10 mg / kg), G5 (RB4-G1-020, 10 mg / kg), and G6 (RB4-G1-022, 10 mg / kg). All mice were intraperitoneally injected with the drug on the day of grouping (day 0) and day 3 after grouping. Mice were imaged using a small animal in vivo imaging device before grouping and dosing (day 0), on days 7, 14, 21, and 25 after dosing, and on the end of the experiment (day 28). Imaging signal patterns and signal intensity were obtained to observe tumor growth in vivo. During the dosing and observation period, animal body weights were measured twice a week and the measured values ​​were recorded.

[0410] The results showed that on the 28th day after grouping, the tumor imaging signal intensity in group G1 was 2.54x10 9 ±3.87x10 8 p / s; tumor imaging signal intensity in group G2 was 1.06x10 7 ±4.50x10 6 p / s, TGITV was 101.0%; the tumor imaging signal intensity in group G3 was 4.24x10 6 ±1.71x10 6 p / s, TGITV was 101.3%; the tumor imaging signal intensity in group G4 was 1.29x10 6 ±2.79x10 5 p / s, TGITV was 101.4%; the tumor imaging signal intensity in the G5 group was 5.82x10 6 ±2.35x10 6 p / s, TGITV was 101.2%; the tumor imaging signal intensity in the G6 group was 4.08x10 6 ±2.63x10 6 p / s, TGITV is 101.3%.

[0411] The results are shown in Figures 14 and 15 . Compared with the G1 control group, the P values ​​for G2-G6 were all 0.000, indicating that the test products C84, RB4-G1-018, RB4-G1-019, RB4-G1-020, and RB4-G1-022 significantly inhibited THP1-LUC tumor growth at a dose of 10 mg / kg. RB4-G1-018, RB4-G1-019, RB4-G1-020, and RB4-G1-022 were more effective than C84.

Claims

1. An isolated antigen-binding protein capable of binding to leukocyte immunoglobulin-like receptor B4 (LILRB4), wherein the isolated antigen-binding protein comprises at least one CDR in the antibody heavy chain variable region VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33 and SEQ ID NO:

22.

2. The isolated antigen-binding protein according to claim 1, comprising a HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 83 or SEQ ID NO:

29.

3. The isolated antigen-binding protein according to claim 2, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO:

29.

4. The isolated antigen-binding protein according to any one of claims 1 to 3, comprising a HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

28.

5. The isolated antigen-binding protein according to any one of claims 1 to 4, comprising HCDR1 comprising SEQ ID NO: 69(X1X2WMX3, wherein X1 is D or S; X2 is A or Y; X3 is D or H). The isolated antigen-binding protein according to claim 5 , wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO:

27.

7. The isolated antigen-binding protein according to any one of claims 1 to 6, comprising HCDR1, HCDR2 and HCDR3, wherein the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from the group consisting of: (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

29.

8. The isolated antigen-binding protein according to any one of claims 1 to 7, comprising H-FR1, wherein the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:

73.

9. The isolated antigen-binding protein according to claim 8, wherein the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 30 and SEQ ID NO:

41.

10. The isolated antigen-binding protein according to any one of claims 1 to 9, comprising H-FR2, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:

74.

11. The isolated antigen-binding protein according to claim 10, wherein the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 42 and SEQ ID NO:

53.

12. The isolated antigen-binding protein according to any one of claims 1 to 11, comprising H-FR3, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:

75.

13. The isolated antigen-binding protein of claim 12, wherein the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 32, SEQ ID NO: 43, SEQ ID NO: 54 and SEQ ID NO:

57.

14. The isolated antigen-binding protein according to any one of claims 1 to 13, comprising H-FR4, wherein the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

76.

15. The isolated antigen-binding protein of claim 14, wherein the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO:

44.

16. The isolated antigen-binding protein according to any one of claims 1 to 15, comprising H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1, H-FR2, H-FR3 and H-FR4 comprise an amino acid sequence selected from the group consisting of: (1) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7; (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7; (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:21, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7; (4) the H-FR1 comprises the amino acid sequence of SEQ ID NO: 30, the H-FR2 comprises the amino acid sequence of SEQ ID NO: 31, the H-FR3 comprises the amino acid sequence of SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 7; (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:42, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:43, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44; (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:54, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44; (7) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:57, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:

44.

17. The isolated antigen-binding protein according to any one of claims 1 to 16, comprising a VH comprising the amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33 and SEQ ID NO:

22.

18. The isolated antigen-binding protein of any one of claims 1-17, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 45, SEQ ID NO: 55, and SEQ ID NO:

58.

19. The isolated antigen binding protein of any one of claims 1-18, comprising an antibody heavy chain constant region derived from an IgG heavy chain constant region.

20. The isolated antigen binding protein of claim 19, wherein the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

21. The isolated antigen binding protein of any one of claims 19-20, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

22. The isolated antigen-binding protein of any one of claims 19-21, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

63.

23. The isolated antigen-binding protein according to any one of claims 1 to 22, comprising an antibody heavy chain comprising the amino acid sequence shown in any one of SEQ ID NO:46, SEQ ID NO:56, and SEQ ID NO:59, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:

91.

24. The isolated antigen-binding protein according to any one of claims 1 to 23, comprising at least one CDR in an antibody light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO:

40.

25. The isolated antigen-binding protein according to any one of claims 1 to 24, comprising a LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 72 (QX1X2X3X4X5PX6T, X1 is H or Q; X2 is G or S; X3 is D, N or W; X4 is E or T; X5 is I or L; X6 is P or R).

26. The isolated antigen-binding protein of claim 25, wherein the LCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO:

36.

27. The isolated antigen binding protein of any one of claims 1-26, comprising a LCDR2 comprising the amino acid sequence shown in SEQ ID NO:

71.

28. The isolated antigen-binding protein of claim 27, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO:

35.

29. The isolated antigen binding protein of any one of claims 1-28, comprising LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 70 or SEQ ID NO:

34.

30. The isolated antigen-binding protein of claim 29, wherein the LCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO:

34.

31. The isolated antigen binding protein of any one of claims 1-30, comprising LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of: (1) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 11; (2) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24; and (3) The LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

36.

32. The isolated antigen-binding protein according to any one of claims 1 to 31, comprising L-FR1, wherein the C-terminus of said L-FR1 is directly or indirectly linked to the N-terminus of said LCDR1, and said L-FR1 comprises the amino acid sequence shown in SEQ ID NO:

77.

33. The isolated antigen-binding protein of claim 32, wherein the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 37 and SEQ ID NO:

47.

34. The isolated antigen-binding protein of any one of claims 1-33, comprising L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:

78.

35. The isolated antigen-binding protein of claim 34, wherein the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 38 and SEQ ID NO:

48.

36. The isolated antigen-binding protein of any one of claims 1-35, comprising L-FR3, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:

79.

37. The isolated antigen-binding protein of claim 36, wherein the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 14, SEQ ID NO: 39, SEQ ID NO: 49 and SEQ ID NO:

60.

38. The isolated antigen-binding protein according to any one of claims 1 to 37, comprising L-FR4, wherein the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

80.

39. The isolated antigen-binding protein of claim 38, wherein the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO:

50.

40. The isolated antigen-binding protein of any one of claims 1-39, comprising L-FR1, L-FR2, L-FR3 and L-FR4, wherein the L-FR1, L-FR2, L-FR3 and L-FR4 comprise an amino acid sequence selected from the group consisting of: (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 12, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15; (2) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 25, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15; (3) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 37, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 38, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15; (4) the L-FR1 comprises the amino acid sequence of SEQ ID NO:47, the L-FR2 comprises the amino acid sequence of SEQ ID NO:48, the L-FR3 comprises the amino acid sequence of SEQ ID NO:49, and the L-FR4 comprises the amino acid sequence of SEQ ID NO:50; and (5) The L-FR1 comprises the amino acid sequence shown in SEQ ID NO:47, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:48, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:60, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:

50.

41. The isolated antigen binding protein of any one of claims 1-40, comprising a VL comprising the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO:

40.

42. The isolated antigen-binding protein of claims 24-41, wherein the VL comprises the amino acid sequence of any one of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 40, SEQ ID NO: 51 and SEQ ID NO:

61.

43. The isolated antigen binding protein of any one of claims 1-42, comprising an antibody light chain constant region, wherein the antibody light chain constant region comprises a human Ig kappa constant region.

44. The isolated antigen-binding protein of claim 43, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

64.

45. The isolated antigen-binding protein according to any one of claims 43-44, comprising an antibody light chain, wherein the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 87, SEQ ID NO: 90 and SEQ ID NO:

92.

46. ​​The isolated antigen binding protein of any one of claims 1-45, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from the group consisting of: (1) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 11; (2) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 23, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24; and (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

36.

47. The isolated antigen-binding protein of any one of claims 1-46, comprising a VH and a VL comprising an amino acid sequence selected from the group consisting of: (1) the VH comprises the amino acid sequence shown in SEQ ID NO: 8, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16; (2) the VH comprises the amino acid sequence set forth in SEQ ID NO: 19, and the VL comprises the amino acid sequence set forth in SEQ ID NO: 16; (3) the VH comprises the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26; (4) the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40; (5) The VH comprises the amino acid sequence shown in SEQ ID NO: 45, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51 The amino acid sequence shown; (6) the VH comprises the amino acid sequence shown in SEQ ID NO: 55, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51; (7) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51; and (8) The VH comprises the amino acid sequence shown in SEQ ID NO:45, and the VL comprises the amino acid sequence shown in SEQ ID NO:

61.

48. The isolated antigen-binding protein of any one of claims 1-47, comprising an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise an amino acid sequence selected from the group consisting of: (1) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87; (2) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87; (3) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 90; (4) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 92; (5) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; (6) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; (7) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 59, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; and (8) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

62.

49. The isolated antigen binding protein of any one of claims 1-48, which comprises an antibody or antigen-binding fragment thereof.

50. The isolated antigen binding protein of claim 49, wherein the antibody is selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a multispecific antibody, a humanized antibody, and a fully human antibody.

51. The isolated antigen binding protein of any one of claims 49-50, wherein the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv, F(ab')2, scFv, di-scFv and dAb fragments.

52. The isolated antigen binding protein of any one of claims 1-51, having one or more of the following properties: 1) Able to specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) Ability to bind to LILRB4 protein with a KD value of 2×10-7M or lower; 3) can block the activity of LILRB4 / APOE binding; 4) can stimulate the activation of CD8+ T cells; 5) It can inhibit the migration of human monocytic leukemia cells THP-1; 6) Ability to inhibit the growth and / or metastasis of tumor cells; 7) It can block the binding of LILRB4 to Fibronectin.

53. The isolated antigen binding protein of claim 52, wherein the LILRB4 is human LILRB4.

54. A polypeptide comprising the isolated antigen binding protein of any one of claims 1-53.

55. A fusion protein comprising the isolated antigen binding protein of any one of claims 1-53.

56. A drug molecule comprising the isolated antigen binding protein of any one of claims 1-53.

57. An isolated nucleic acid molecule or molecules encoding the isolated antigen binding protein of any one of claims 1-53.

58. A vector comprising the nucleic acid molecule of claim 57.

59. A cell comprising the nucleic acid molecule of claim 57 or the vector of claim 58.

60. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of claims 1-53, the polypeptide of claim 54, the fusion protein of claim 55, the drug molecule of claim 56, the nucleic acid molecule of claim 57, the vector of claim 58 and / or the cell of claim 59, and optionally a pharmaceutically acceptable carrier.

61. The pharmaceutical composition of claim 60, further comprising one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin-2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), cytochrome P60 inhibitors, ... g), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, ensidipine, Idhifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, TIGIT antibodies, TIM3 antibodies, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

62. A method of making the isolated antigen binding protein of any one of claims 1-53, the method comprising culturing the cell of claim 59 under conditions such that the isolated antigen binding protein of any one of claims 1-53 is expressed.

63. Use of the isolated antigen binding protein of any one of claims 1-53, the polypeptide of claim 54, the fusion protein of claim 55, the drug molecule of claim 56, the nucleic acid molecule of claim 57, the vector of claim 58, the cell of claim 59, and / or the pharmaceutical composition of any one of claims 60-61 in the preparation of a medicament for preventing and / or treating diseases and / or conditions, including diseases or conditions associated with LILRB4 signaling.

64. Use according to claim 63, wherein the disease and / or disorder comprises a tumor.

65. The use according to claim 64, wherein the tumor comprises a solid tumor and / or a hematological tumor.

66. The method of claim 63, wherein the drug is used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, emcitabine, Idh ifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor ibrutinib, PD-1 antibody, PD-L1 antibody, anti-CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to cell or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

67. The use according to any one of claims 63-66, wherein the disease or disorder associated with LILRB4 signaling comprises an immune system disease and / or a hematological malignancy.

68. The use according to claim 67, wherein the immune system disease is selected from one or more of the following groups: Kawasaki disease, systemic lupus erythematosus (SLE), sepsis.

69. The use according to any one of claims 65-68, wherein the solid tumor is selected from one or more of the following groups: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer and pancreatic cancer.

70. The method of any one of claims 67-69, wherein the hematological malignancy is selected from one or more of the following groups: myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia (CMML), chronic myeloid leukemia, or acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, acute myeloblastic leukemia, and polycythemia vera.

71. A method of diagnosing a disease or condition associated with expression of LILRB4 protein in a subject, the method comprising: The sample derived from the subject is contacted with the isolated antigen-binding protein according to any one of claims 1 to 53, and the presence and / or amount of a substance capable of specifically binding to the isolated antigen-binding protein in the sample is determined.

72. A method for detecting LILRB4 protein in a sample, the method comprising administering the isolated antigen binding protein of any one of claims 1-53.

73. The detection method according to claim 72, which is an in vitro and / or ex vivo method.

74. A detection kit comprising the isolated antigen binding protein of any one of claims 1-53, for detecting the presence and / or amount of LILRB4 protein in a sample or a subject.

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