Isolated antigen-binding protein and its use

Abstract

This application relates to an isolated antigen-binding protein that can specifically bind to the LILRB4 protein. This application relates to a polypeptide, a fusion protein, a drug molecule containing the isolated antigen-binding protein, a nucleic acid encoding the isolated antigen-binding protein, a vector containing the isolated antigen-binding protein, a cell containing the nucleic acid or the vector, a method for preparing the isolated antigen-binding protein, and the use of the isolated antigen-binding protein.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceuticals, 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 receptor (LILR). Under physiological conditions, LILRB4 plays a very important role in the function of the immune system through its expression in various immune cells such as T cells and plasma cells. Under pathological conditions, LILRB4 affects the progression of various diseases such as tumor and leukemia transformation and invasion through various signaling pathways. Differential expression of LILRB4 exists in various immune system diseases such as Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis, and recent studies have shown that LILRB4 also plays a role in mental diseases. In view of the important role of LILRB4 in the immune system and its differential expression in various diseases, LILRB4 is a potential therapeutic target for various diseases.

[0003] Based on the therapeutic potential of LILRB4, it is necessary to develop various antibodies that can specifically bind to LILRB4.

Summary of the Invention

[0004] In one aspect, the present application provides an isolated antigen-binding protein having one or more of the following characteristics: 1) capable of specifically identifying leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) capable of binding to LILRB4 protein with a K -7 value of 2×10 D M or less; 3) having the activity to block the binding of LILRB4 / APOE; 4) capable of stimulating the activation of CD8+ T cells; 5) capable of inhibiting the migration of human monocytic leukemia cells THP-1; 6) capable of inhibiting the growth and / or metastasis of tumor cells; 7) capable of blocking the binding of LILRB4 to fibronectin.

[0005] In some embodiments, the LILRB4 is human LILRB4.

[0006] In some embodiments, the isolated antigen-binding protein comprises at least one CDR in the variable region VH of the antibody heavy chain, and the VH comprises an amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33, and SEQ ID NO: 22.

[0007] In some embodiments, the isolated antigen-binding protein comprises HCDR3, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO: 83 or SEQ ID NO: 29.

[0008] In some embodiments, the isolated antigen-binding protein comprises HCDR3, and the HCDR3 comprises an amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20, and SEQ ID NO: 29.

[0009] In some embodiments, the isolated antigen-binding protein comprises HCDR2, and the HCDR2 comprises an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28.

[0010] In some embodiments, the isolated antigen-binding protein comprises HCDR1, and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO: 69 (X1X2WMX3, where X1 is D or S, X2 is A or Y, and X3 is D or H).

[0011] In some embodiments, the isolated antigen-binding protein comprises HCDR1, and the HCDR1 comprises an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27.

[0012] In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2, and HCDR3, The HCDR1, the HCDR2, and the HCDR3 are (1) The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 3. (2) The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 20, and (3) The HCDR1 includes the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29. It includes an amino acid sequence selected from the group consisting of.

[0013] In some embodiments, the isolated antigen-binding protein includes 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 includes the amino acid sequence shown in SEQ ID NO: 73.

[0014] In some embodiments, the H-FR1 includes 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.

[0015] In some embodiments, the isolated antigen-binding protein includes H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 74.

[0016] In some embodiments, the H-FR2 includes 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.

[0017] In some embodiments, the isolated antigen-binding protein includes H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 75.

[0018] In some embodiments, the H-FR3 comprises an 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.

[0019] In some 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 an amino acid sequence shown in SEQ ID NO: 76.

[0020] In some embodiments, the H-FR4 comprises an amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 44.

[0021] In some embodiments, the isolated antigen-binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein the H-FR1, H-FR2, H-FR3, and H-FR4 (1) the H-FR1 comprises an amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises an amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises an amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises an amino acid sequence shown in SEQ ID NO: 7; (2) the H-FR1 comprises an amino acid sequence shown in SEQ ID NO: 17, the H-FR2 comprises an amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises an amino acid sequence shown in SEQ ID NO: 18, and the H-FR4 comprises an amino acid sequence shown in SEQ ID NO: 7; (3) the H-FR1 comprises an amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises an amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises an amino acid sequence shown in SEQ ID NO: 21, and the H-FR4 comprises an amino acid sequence shown in SEQ ID NO: 7; (4) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 30, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 31, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO: 7. (5) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 42, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 43, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO: 44. (6) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 53, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 54, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO: 44, and (7) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 53, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 57, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO: 44. It contains an amino acid sequence selected from the group consisting of.

[0022] In some embodiments, the isolated antigen-binding protein contains VH, and the VH contains the amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33, and SEQ ID NO: 22.

[0023] In some embodiments, the isolated antigen-binding protein contains VH, and the VH contains 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.

[0024] In some embodiments, the isolated antigen-binding protein contains an antibody heavy chain constant region derived from the IgG heavy chain constant region.

[0025] In some embodiments, the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.

[0026] In some 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.

[0027] In some embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 63.

[0028] In some 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.

[0029] In some embodiments, the isolated antigen-binding protein comprises at least one CDR in the variable region VL of the antibody light chain, and the VL comprises the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40.

[0030] In some embodiments, the isolated antigen-binding protein comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 72 (QX1X2X3X4X5PX6T, where 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, and X6 is P or R).

[0031] In some 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.

[0032] In some embodiments, the isolated antigen-binding protein comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 71.

[0033] In some embodiments, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35.

[0034] In some embodiments, the isolated antigen-binding protein comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 34.

[0035] In some 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.

[0036] In some embodiments, the isolated antigen-binding protein comprises LCDR1, LCDR2, and LCDR3, the LCDR1, the LCDR2, and the LCDR3 are (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. and comprises an amino acid sequence selected from the group consisting of

[0037] In some embodiments, the isolated antigen-binding protein comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 77.

[0038] In some 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.

[0039] In some embodiments, the isolated antigen-binding protein comprises L-FR2, 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.

[0040] In some 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.

[0041] In some embodiments, the isolated antigen-binding protein comprises L-FR3, 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.

[0042] In some 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.

[0043] In some embodiments, the isolated antigen-binding protein comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 80.

[0044] In some embodiments, the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 50.

[0045] In some embodiments, the isolated antigen-binding protein comprises L-FR1, L-FR2, L-FR3, and L-FR4, the L-FR1, the L-FR2, the L-FR3, and the L-FR4 are (1) the L-FR1 comprises the amino acid sequence shown in SEQ ID NO: 12, the L-FR2 comprises the amino acid sequence shown in SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence shown in SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15; (2) The L-FR1 contains the amino acid sequence shown in SEQ ID NO: 25, the L-FR2 contains the amino acid sequence shown in SEQ ID NO: 13, the L-FR3 contains the amino acid sequence shown in SEQ ID NO: 14, and the L-FR4 contains the amino acid sequence shown in SEQ ID NO: 15. (3) The L-FR1 contains the amino acid sequence shown in SEQ ID NO: 37, the L-FR2 contains the amino acid sequence shown in SEQ ID NO: 38, the L-FR3 contains the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 contains the amino acid sequence shown in SEQ ID NO: 15. (4) The L-FR1 contains the amino acid sequence shown in SEQ ID NO: 47, the L-FR2 contains the amino acid sequence shown in SEQ ID NO: 48, the L-FR3 contains the amino acid sequence shown in SEQ ID NO: 49, and the L-FR4 contains the amino acid sequence shown in SEQ ID NO: 50, and (5) The L-FR1 contains the amino acid sequence shown in SEQ ID NO: 47, the L-FR2 contains the amino acid sequence shown in SEQ ID NO: 48, the L-FR3 contains the amino acid sequence shown in SEQ ID NO: 60, and the L-FR4 contains the amino acid sequence shown in SEQ ID NO: 50. It contains an amino acid sequence selected from the group consisting of.

[0046] In some embodiments, the isolated antigen-binding protein contains VL, and the VL contains the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40.

[0047] In some embodiments, the VL contains 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.

[0048] In some embodiments, the isolated antigen-binding protein contains an antibody light chain constant region containing a human Igκ constant region.

[0049] In some embodiments, the antibody light chain constant region contains the amino acid sequence shown in SEQ ID NO: 64.

[0050] In some embodiments, the isolated antigen-binding protein comprises an antibody light chain, and the antibody light chain comprises an amino acid sequence set forth 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.

[0051] In some embodiments, the isolated antigen-binding protein comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 (1) 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: 3, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 9, 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: 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 set forth in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 28, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 36. The antigen-binding protein comprises an amino acid sequence selected from the group consisting of

[0052] In some embodiments, the isolated antigen-binding protein comprises VH and VL, The VH and the VL are (1) The VH contains the amino acid sequence shown in SEQ ID NO: 8, and the VL contains the amino acid sequence shown in SEQ ID NO: 16. (2) The VH contains the amino acid sequence shown in SEQ ID NO: 19, and the VL contains the amino acid sequence shown in SEQ ID NO: 16. (3) The VH contains the amino acid sequence shown in SEQ ID NO: 22, and the VL contains the amino acid sequence shown in SEQ ID NO: 26. (4) The VH contains the amino acid sequence shown in SEQ ID NO: 33, and the VL contains the amino acid sequence shown in SEQ ID NO: 40. (5) The VH contains the amino acid sequence shown in SEQ ID NO: 45, and the VL contains the amino acid sequence shown in SEQ ID NO: 51. (6) The VH contains the amino acid sequence shown in SEQ ID NO: 55, and the VL contains the amino acid sequence shown in SEQ ID NO: 51. (7) The VH contains the amino acid sequence shown in SEQ ID NO: 58, and the VL contains the amino acid sequence shown in SEQ ID NO: 51, and (8) The VH contains the amino acid sequence shown in SEQ ID NO: 45, and the VL contains the amino acid sequence shown in SEQ ID NO: 61. comprises an amino acid sequence selected from the group consisting of

[0053] In some embodiments, the isolated antigen-binding protein comprises an antibody heavy chain and an antibody light chain, the antibody heavy chain and the antibody light chain are (1) The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain contains the amino acid sequence shown in SEQ ID NO: 87. (2) The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain contains the amino acid sequence shown in SEQ ID NO: 87. (3) The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain contains the amino acid sequence shown in SEQ ID NO: 90 (4) The heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 91, and the light chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 92. (5) The heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 46, and the light chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 52. (6) The heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 56, and the light chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 52. (7) The heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 59, and the light chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 52, and (8) The heavy chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 46, and the light chain of the antibody contains the amino acid sequence shown in SEQ ID NO: 62. It contains an amino acid sequence selected from the group consisting of. In some embodiments, the isolated antigen-binding protein comprises an antibody or an antigen-binding fragment thereof.

[0054] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, single-chain antibodies, chimeric antibodies, multispecific antibodies, humanized antibodies, and fully human antibodies.

[0055] In some 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.

[0056] In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein.

[0057] In another aspect, the present application provides a fusion protein comprising the isolated antigen-binding protein.

[0058] In another aspect, the present application provides a drug molecule comprising the isolated antigen-binding protein.

[0059] In another aspect, the present application provides one or more nucleic acid molecules encoding the isolated antigen-binding protein.

[0060] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0061] In another aspect, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0062] In another aspect, there is provided 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.

[0063] In some embodiments, the pharmaceutical composition further comprises one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, daunorubicin-cytarabine combined 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, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0064] In another aspect, the present application provides a method for preparing the isolated antigen-binding protein, comprising culturing the cells under conditions that express the isolated antigen-binding protein.

[0065] In another aspect, 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 drug for preventing and / or treating a disease and / or a symptom comprising a disease or a symptom associated with LILRB4 signaling.

[0066] In some embodiments, the disease and / or symptom comprises a tumor.

[0067] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0068] In some embodiments, the drug is used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin-cytarabine combined 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, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors.

[0069] In some embodiments, the diseases or conditions associated with LILRB4 signaling include immune system diseases and / or hematological malignancies.

[0070] In some embodiments, the immune system disease is one or more selected from the group consisting of Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0071] In some embodiments, the solid tumor is one or more selected from the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0072] In some embodiments, the malignant blood disease is one or more selected from the group consisting of myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia or acute myelogenous 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.

[0073] In another aspect, 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 for preventing and / or treating a disease and / or a symptom including a disease or a symptom related to LILRB4 signaling.

[0074] In some embodiments, the disease and / or the symptom includes a tumor.

[0075] In some embodiments, the tumor includes a solid tumor and / or a hematological tumor.

[0076] In some 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 combined with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin-cytarabine combined 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, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors for use.

[0077] In some embodiments, the diseases or conditions associated with LILRB4 signaling include immune system diseases and / or hematologic malignancies.

[0078] In some embodiments, the immune system disease is one or more selected from the group consisting of Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0079] In some embodiments, the solid tumor is one or more selected from the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0080] In some embodiments, the malignant blood disease is one or more selected from the group consisting of myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia or acute myelogenous 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.

[0081] In another aspect, the present application provides a method for preventing and / or treating a disease and / or condition including a disease or condition related to LILRB4 signaling, the method including 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.

[0082] In some embodiments, the disease and / or condition includes a tumor.

[0083] In some embodiments, the tumor includes a solid tumor and / or a hematological tumor.

[0084] In some 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 combined with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin-cytarabine combined 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, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, oxaliplatin, kinase inhibitors, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitor Ibrutinib, PD-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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors for use.

[0085] In some embodiments, the diseases or conditions associated with LILRB4 signaling include immune system diseases and / or hematological malignancies.

[0086] In some embodiments, the immune system disease is one or more selected from the group consisting of Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0087] In some embodiments, the solid tumor is one or more selected from the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0088] In some embodiments, the malignant blood disease is one or more selected from the group consisting of myelodysplastic syndrome, myeloproliferative tumor, chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia or acute myelogenous 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.

[0089] In another aspect, the present application provides a method for diagnosing a disease or condition related to the expression of LILRB4 protein in a subject, comprising contacting a sample derived from the subject with the isolated antigen-binding protein, and determining the presence and / or content of a substance that can specifically bind to the isolated antigen-binding protein in the sample.

[0090] In another aspect, the present application provides a method for detecting LILRB4 in a sample, comprising administering the isolated antigen-binding protein.

[0091] In some embodiments, the method is an in vitro method and / or an ex vivo method.

[0092] In another aspect, the present application provides a detection kit comprising the isolated antigen-binding protein, which is used for detecting the presence and / or content of LILRB4 protein in a sample or a subject.

[0093] Those skilled in the art can easily understand other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art understand, based on the content of the present application, those skilled in the art can modify the disclosed specific embodiments without departing from the spirit and scope of the invention according to the present application. Correspondingly, the descriptions of the drawings and the specification of the present application are only exemplary and not intended to be limiting.

Brief Description of the Drawings

[0094] The specific features of the invention according to this application are described in the appended claims. By referring to the exemplary embodiments and drawings described in detail below, the features and advantages of the invention according to this application can be better understood. A brief description of the drawings is as follows:

[0095]

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Modes for Carrying Out the Invention

[0096] The embodiments of the invention of the present application will be described below with specific specific examples. However, those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0097] Definition of Terms The present application will be further described below. In the present invention, unless otherwise specified, the scientific terms and technical terms used in this specification have the meanings generally understood by those skilled in the art. In addition, the terms and experimental procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this specification are terms and ordinary procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and interpretations of related terms are provided below.

[0098] In this application, the term "isolated" generally refers to being obtained by artificial means from its natural state. When an "isolated" substance or component occurs in nature, the natural environment in which it exists may have changed, or the substance may have been separated from the natural environment, or both. For example, if an unisolated polynucleotide or polypeptide naturally exists in the body of a living animal, a highly purified same polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the mixing of artificial or synthetic substances, nor the presence of other impurity substances that do not affect the activity of the substance.

[0099] In this application, the term "isolated antigen-binding protein" generally refers to a protein having antigen-binding ability obtained by artificial means from its natural state. The "isolated antigen-binding protein" can include an antigen-binding portion and, optionally, a scaffold portion 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 can include, for example, a protein scaffold derived from an antibody, or an alternative protein scaffold having a transplanted CDR or CDR derivative, or an artificial scaffold. Such scaffolds include, but are not limited to, an antibody-derived scaffold containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and a fully synthetic scaffold containing, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003) and Roque et al., Biotechnol. Prog. 20:639-654 (2004). Also, peptide antibody mimetics ("PAMs") and scaffolds based on antibody mimetics utilizing fibronectin components can be used as scaffolds.

[0100] In this application, "KD" or "K DThe term " " usually refers to the "affinity constant" or "equilibrium dissociation constant", which refers to the value obtained at equilibrium in a titration measurement or the value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka). The binding affinity of a binding protein (e.g., the isolated antigen-binding protein described herein) to an antigen (e.g., LILRB4 protein) is represented by the association rate constant (ka), the dissociation rate constant (kd), and the equilibrium dissociation constant (KD). Methods for determining the association rate constant and the dissociation rate constant are well known in the art. By using fluorescence-based techniques, high sensitivity and the ability to examine samples at equilibrium in physiological buffer are provided. For example, the KD value can be measured by Octet, and the KD value can also be measured using other experimental approaches and instruments such as BIAcore (biomolecular interaction analysis) (e.g., an instrument available for purchase from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Also, the KD value can be measured using KinExA (Kinetic Exclusion Assay) available for purchase from Sapidyne Instruments (Boise, Idaho), or the KD value can be measured using a surface plasmon resonance apparatus (SPR).

[0101] In this application, the term "LILRB4 protein" usually refers to leukocyte immunoglobulin-like receptor B4 (LILRB4). In this application, the term includes full-length LILRB4 and its functionally active fragments, variants, homologs, analogs, and derivatives.

[0102] In the present application, the terms "specific binding" or "specific" generally refer to a measurable and reproducible interaction, such as the binding of a target and an antibody, that can determine the presence of a target when a heterogeneous population of molecules (including biomolecules) is present. For example, an antibody that specifically binds to a target (which may be an epitope) binds to that target with a higher affinity, avidity, more readily, and / or for a longer duration than when binding to other targets. In one embodiment, the degree to which the antibody binds to an irrelevant 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 the present application, the isolated antigen-binding protein can bind to the LILRB4 protein with a dissociation constant (KD) of <2x10 -7 M or less. In some embodiments, specific binding can include, but does not necessarily require, exclusive binding.

[0103] In the present application, the term "variable domain" generally refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively (or are referred to as "VH" and "VL", respectively). These domains are generally the most variable parts of the antibody (relative to other antibodies of the same type) and include the antigen-binding site.

[0104] In the present application, the term "variable" generally refers to the fact that the sequences of segments within the variable domain vary widely between antibodies. The V domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments called the hypervariable regions (CDRs or HVRs) in the variable domains of the light and heavy chains. The more highly conserved portions of the variable domains are called the framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, predominantly adopt a β-sheet structure, and are connected by three CDRs, forming loop connections and in some cases forming part of the β-sheet structure. The CDRs of each chain are held in close proximity through the FR regions, and the CDRs from the other chain together promote 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 do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as the involvement of the antibody in antibody-dependent cell cytotoxicity.

[0105] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, nonspecific antibodies, humanized antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, and grafted antibodies. Unless otherwise modified by the term "intact" as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes Fab, F(ab’)2, Fv, scFv, Fd, dAb, and other antibody fragments that maintain antigen-binding function (e.g., specifically bind to LILRB4). Usually, such fragments should contain an antigen-binding domain. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L chains) and two identical heavy chains (H chains). IgM antibodies are composed of five basic heterotetrameric units and another polypeptide called the J chain, and contain ten antigen-binding sites, while IgA antibodies contain two to five basic four-chain units that can bind to the J chain and polymerize to form a multivalent aggregate. In the case of IgG, the four-chain unit generally weighs about 150,000 daltons. Each L chain is connected to the H chain by a disulfide covalent bond, while the two H chains are connected to each other by one or more disulfide bonds depending on the heavy-chain isotype. Each H chain and L chain also have regularly spaced intra-chain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) in the case of each of the α and γ chains, and four CH domains in the case of the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other terminus. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain. VH and VL pair to form a single antigen-binding site.For the structures and properties of different types 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 chains from any vertebrate species may be divided into one of two clearly different types called κ and λ based on the amino acid sequence of their constant domains. Immunoglobulins may be divided into different classes or isotypes according to the amino acid sequence of the heavy chain (CH) constant domains. There are five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, which have heavy chains named α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in the sequence and function of CH. For example, humans express subclasses of IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0106] In the present application, the term "CDR" generally refers to the regions of the variable domains of an antibody, the sequences of which are highly variable and / or form structurally defined loops. Typically, an antibody contains six CDRs, with three CDRs (HCDR1, HCDR2, HCDR3) in VH and three CDRs (LCDR1, LCDR2, LCDR3) in VL. In natural antibodies, HCDR3 and LCDR3 exhibit most of the diversity among the six CDRs, and in particular, HCDR3 is thought to play a unique role in conferring fine specificity to the antibody. 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, N.J., 2003). In fact, natural camel antibodies composed of only heavy chains function properly and stably even without light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993), Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

[0107] In the present application, the term "FR" generally refers to the more highly conserved portions of the variable domains of an antibody and is referred to as the framework region. Typically, the variable domains of natural heavy and light chains each contain four FR regions, i.e., there are four FR regions (H-FR1, H-FR2, H-FR3, and H-FR4) in VH and four FR regions (L-FR1, L-FR2, L-FR3, and L-FR4) in VL. For example, the VL of the isolated antigen-binding protein described herein can include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen-binding protein described herein can include the framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0108] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind to an antigen (e.g., LILRB4 protein). In the present application, the antigen-binding fragment can include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0109] In the present application, the term "monoclonal antibody" or "single antibody" or "monoclonal antibody composition" generally refers to an antibody molecule product composed of a single molecule. Monoclonal antibodies are constructed to exhibit a single binding specificity and affinity for a specific epitope.

[0110] In the present application, the term "single-chain antibody" generally refers to a molecule containing the variable region of the heavy chain and the variable region of the light chain of an antibody. For example, the single-chain antibody may be one in which the variable region of the heavy chain and the variable region of the light chain of the antibody are connected by a linker molecule (e.g., a linker peptide).

[0111] In the present application, the term "human antibody" generally refers to an antibody in which the variable region framework and the CDR region are derived from human germline immunoglobulin sequences. Also, when the antibody contains a constant region, that is also derived from human germline immunoglobulin sequences. The human antibodies of the present application can include amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced by in vitro random mutagenesis or point mutagenesis, or somatic mutations in vivo. However, the term "human antibody" does not include antibodies in which CDR sequences from other mammalian species are inserted into the human framework sequence.

[0112] In this application, the term "mouse antibody" generally refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. Also, when the antibody contains a constant region, that too is derived from mouse germline immunoglobulin sequences. The mouse antibodies of this application can contain amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by in vitro random mutagenesis or point mutagenesis, or somatic mutations in vivo. However, the term "mouse antibody" does not include antibodies in which CDR sequences from other mammalian species are inserted into the mouse framework sequence.

[0113] In this application, the term "chimeric antibody" generally refers to an antibody that combines non-human genetic material and human genetic material. Or, more generally, a chimeric antibody refers to an antibody that combines genetic material of one species with genetic material of another species.

[0114] 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 methods such as chemical coupling methods, hybridization-hybridoma methods, and genetic engineering antibody preparation methods.

[0115] In this application, the term "humanized antibody" generally refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to enhance its similarity to naturally occurring human antibodies.

[0116] In this application, the term "fully human antibody" generally refers to a complete human antibody, i.e., an antibody in which both the constant region and the variable region are derived from humans. The fully human antibody may be realized by techniques such as phage antibody library technology, transgenic mice for human antibody preparation, ribosome display technology, EBV-transformed B cell cloning technology, and single B cell cloning.

[0117] In the present application, the term "direct connection" is relative to the term "indirect connection". The term "direct connection" usually means direct connection. For example, direct connection may mean that substances are directly connected without a spacer therebetween. The spacer may be a linker. For example, it may be the peptide linker. The term "indirect connection" usually means that substances are not directly connected. For example, the indirect connection may be connection via a spacer. For example, in the isolated antigen-binding protein described in the present application, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1.

[0118] In the present application, the terms "polypeptide", "peptide" and "protein" may be used interchangeably and usually refer to a polymer of amino acid residues. This term may be used to refer to an amino acid polymer in which one or more amino acid residues are artificially synthesized chemical mimics of the corresponding natural amino acids, or may be used to refer to natural amino acid polymers, amino acid polymers containing modified residues, and non-natural amino acid polymers.

[0119] In the present application, the term "fusion protein" refers to a protein composed of two or more polypeptides that are not usually bound in nature but whose respective amino and carboxy termini are joined by peptide bonds to form one continuous polypeptide. It should be understood that the two or more polypeptide components may be directly bound or indirectly bound via a peptide linker / spacer.

[0120] In this application, the term "drug molecule" generally refers to a molecule having a desired biological effect. The drug may be prophylactic or therapeutic. Drug molecules include, but are not limited to, protein molecules such as peptides, polypeptides, proteins including post-translationally modified proteins, fusion proteins, antibodies, etc., small molecules including inorganic or organic compounds, and nucleic acid molecules that can include, but are not limited to, double-stranded or single-stranded DNA, or double-stranded or single-stranded RNA (antisense (molecules), RNAi, etc.), intron sequences, triple helix nucleic acid molecules, or aptamers, or vaccines, among others.

[0121] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized.

[0122] In this application, the term "vector" generally refers to a nucleic acid delivery tool that can insert a polynucleotide encoding a certain protein and express that protein. A vector can express the genetic material elements contained therein within a host cell by transforming, transducing, or transfecting the host cell. For example, vectors include artificial chromosomes such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as λ phage or M13 bacteriophage, and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain an origin of replication site. Vectors may contain components that assist in entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these.

[0123] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that can be or has been a recipient of a plasmid or vector from a subject, including the nucleic acid molecules described in the present invention or the vectors described in the present invention. A cell can include the progeny of a single cell. Due to natural mutations, accidental mutations, or intentional mutations, the progeny need not be exactly identical to the original parental cell (either in the form of the entire DNA complement or the genome). A cell can include cells transfected in vitro using the vectors described in this application. The cells can be bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, human non-small cell lung cancer A549 cells, human cutaneous squamous cell carcinoma A431 cells, clear cell renal cell carcinoma 786-O cells, human pancreatic cancer MIA PaCa-2 cells, erythroleukemia K562 cells, acute T cell leukemia Jurkat cells, human breast cancer MCF-7 cells, human breast cancer MDA-MB-231 cells, human breast cancer MDA-MB-468 cells, human breast cancer SKBR3 cells, human ovarian cancer SKOV3 cells, lymphoma U-937 cells, lymphoma Raji cells, human myeloma U266 cells, or human multiple myeloma RPMI8226 cells. In some embodiments, the cell is a mammalian cell.

[0124] In the present application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, preferably a human patient. For example, the pharmaceutical compositions described in the present application can include the isolated antigen-binding proteins described in the present application, the immune complexes described in the present application, the nucleic acid molecules described in the present application, the vectors and / or cells described in the present application, and optionally a pharmaceutically acceptable adjuvant. Further, the pharmaceutical composition can further include a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions and lyophilized compositions.

[0125] In the present application, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coating agents, isotonic agents, absorption delaying agents, etc. that are compatible with the administration of a drug, and are generally safe, non-toxic and not biologically or otherwise undesirable.

[0126] In the present application, the term "subject" generally refers to a human or a non-human animal including, but not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

[0127] In the present application, the term "comprising" generally means including the explicitly specified features, but not excluding other elements.

[0128] In the present application, the term "about" generally refers to a variation within the range of ±0.5% to 10% of the specified value, for example, a variation within the 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% or 10% of the specified value.

[0129] Detailed Description of the Invention Isolated Antigen-Binding Protein The CDRs of an antibody, called complementarity-determining regions, are part of the variable region. The amino acid residues in this region can contact an antigen or an antigen epitope. The CDRs of an antibody may be determined by various coding systems such as CCG, Kabat, Chothia, IMGT, AbM, and combinations of Kabat / Chothia. These coding systems are known in the art, and specifically, for example, reference can be made to http: / / www.bioinf.org.uk / abs / index.html#kabatnum. A person skilled in the art can determine the CDR region using different coding systems based on the sequence and structure of the antibody. When using different coding systems, there may be differences in the CDR region. In the present application, the CDR includes a CDR sequence classified according to any CDR classification method, and also includes variants thereof. The variants include those in which one or more amino acids are substituted, deleted, and / or added to the amino acid sequence of the CDR. For example, 1 to 30, 1 to 20, or 1 to 10 amino acids are substituted, deleted, and / or inserted, and also include homologs thereof. The homologs may be amino acid sequences having at least about 85% (for example, at least 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 protein described in the sequence listing of the present application may be determined using Kabat.

[0130] In one aspect, the present application provides an isolated antigen-binding protein that can include at least one CDR in VH shown in any one of SEQ ID NO: 81, SEQ ID NO: 33, and SEQ ID NO: 22, and / or at least one CDR in VL shown in SEQ ID NO: 82 or SEQ ID NO: 40.

[0131] X1VQLX2QX3GAEX4X5KPGASVKX6SCKASGYTFTSYWMHWVX7QX8PGQGLEWX9GEINPSNGRTNYNEKFKSX 10 X11 TX 12 TX 13 DX 14 SX 15 STX 16 YX 17 X 18 LX 19 SLX 20 SEDX 21 AVYYCARDRYDEGNAMDYWGQGTX 22 In VTVSS (SEQ ID NO: 81), X1 may be E or Q, X2 may be Q or V, X3 may be P or S, X4 may be L or V, X5 may be K or V, X6 may be L or V, X7 may be K or R, X8 may be A or R, X9 may be I or M, X 10 may be K or R, X 11 may be A or V, X 12 may be L or M, X 13 may be R or V, X 14 may be K or T, X 15 may be S or T, X 16 may be A or V, X 17 may be M or V, X 18 may be E or Q, X 19 may be N or S, X 20 may be R or T, X 21 may be S or T, X 22 may be S or T.

[0132] DIX1MTX2X3X4SSLSASX5GDRVTIX6CRASQDIX7NYLNWYQQKPX8X9X 10 X 11 KLLIYYTSRLHSGVPSRFSGSGSGTX 12 YX 13 X 14 TISX 15 LX 16 X 17 X 18 DX 19 ATYX 20 CQQGX 21TLPX 22 TFGX 23 GTX 24 In LEIK (SEQ ID NO: 82), 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 may be A or T, X 11 may be P or V, X 12 may be D or E, X 13 may be S or T, X 14 may be F or L, X 15 may be N or S, X 16 may be E or Q, X 17 may be P or Q, X 18 may be D or E, X 19 may be F or I, X 20 may be F or Y, X 21 may be D or N, X 22 may be P or R, X 23 may be G or Q, X 24 may be K or R.

[0133] In the present application, the VH can include 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.

[0134] In the present application, the VL can include 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.

[0135] In the present application, the HCDRs of the isolated antigen-binding protein may be divided in any form, and as long as the VH and / or VL have the same amino acid sequence as the VH and / or VL shown in any one of the claims of the present application, the HCDRs divided in any form can be included within the protection scope of the present application.

[0136] For example, the separation of VH (SEQ ID NO: 8) and / or VL (SEQ ID NO: 16) using different CDR marking methods can be summarized in the following table. [Table 1]

[0137] For example, the separation of VH (SEQ ID NO: 19) and / or VL (SEQ ID NO: 16) using different CDR marking methods can be summarized in the following table. [Table 2]

[0138] For example, the separation of VH (SEQ ID NO: 22) and / or VL (SEQ ID NO: 26) using different CDR marking methods can be summarized in the following table. [Table 3]

[0139] For example, the separation of VH (SEQ ID NO: 33) and / or VL (SEQ ID NO: 40) using different CDR marking methods can be summarized in the following table. [Table 4]

[0140] Here, Laa-Lbb can refer to the amino acid sequence from position aa to position bb counting from the N-terminus of the variable region of the antibody light chain, and Haa-Hbb can refer to the amino acid sequence from position aa to position bb counting from the N-terminus of the variable region of the antibody heavy chain. For example, L24~L34 can refer to the amino acid sequence from position 24 to position 34 counting from the N-terminus of the light chain of the antibody, and H26~H35 can refer to the amino acid sequence from position 26 to position 35 counting from the N-terminus of the heavy chain of the antibody.

[0141] In one of these 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.

[0142] In the present application, the VH of the isolated antigen-binding protein can include HCDR1, HCDR2, and HCDR3.

[0143] In the present application, the HCDR1 can include the amino acid sequence shown in SEQ ID NO: 69.

[0144] In X1X2WMX3 (SEQ ID NO: 69), X1 can be D or S, X2 can be A or Y, and X3 can be D or H.

[0145] For example, the HCDR1 can include the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27.

[0146] In the present application, the HCDR2 can include the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28.

[0147] In the present application, the HCDR3 can include the amino acid sequence shown in SEQ ID NO: 83 or SEQ ID NO: 29.

[0148] [-G][DI][RY]Y[DG][EY][DG][NY]AMDY (SEQ ID NO: 83), X1 may not be present or may be 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.

[0149] In the present application, the HCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27, the HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28, and the HCDR3 may contain the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20, and SEQ ID NO: 29.

[0150] In the present application, the HCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 3.

[0151] In the present application, the HCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 20.

[0152] In the present application, the HCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may contain the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 may contain the amino acid sequence shown in SEQ ID NO: 29.

[0153] In the present application, the VL of the isolated antigen-binding protein may contain LCDR1, HCDR2, and LCDR3.

[0154] In the present application, the LCDR1 can include the amino acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 34.

[0155] In RASQDIX1NYLN (SEQ ID NO: 70), X1 may be S or T.

[0156] For example, the LCDR1 can include the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 23, and SEQ ID NO: 34.

[0157] In the present application, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 71.

[0158] In YX1SX2LX3S (SEQ ID NO: 71), X1 may be A or T, X2 may be N or R, and X3 may be H or Q.

[0159] For example, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35.

[0160] In the present application, the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 72.

[0161] In QX1X2X3X4X5PX6T (SEQ ID NO: 72), for example, X1 may be H or Q, X2 may be G or S, X3 may be D, N or W, X4 may be E or T, X5 may be I or L, and X6 may be P or R.

[0162] For example, the LCDR3 can include the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24, and SEQ ID NO: 36.

[0163] In the present application, the LCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 34, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 71, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 72 (wherein in 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, and X6 is P or R).

[0164] In the present application, the LCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 11.

[0165] In the present application, the LCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 24.

[0166] In the present application, the LCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 36.

[0167] In the present application, the isolated antigen-binding protein can include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0168] In the present application, the HCDR1 of the isolated antigen-binding protein can comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28, the HCDR3 can 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 can 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 can comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35, and the LCDR3 can comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36.

[0169] In the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the isolated antigen-binding protein are (1) the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 3, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 11; (2) the HCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 can comprise the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 can comprise the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 can comprise the amino acid sequence shown in SEQ ID NO: 24; and (3) The HCDR1 can include the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 can include the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 can include the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 can include the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 36. It can include an amino acid sequence selected from the group consisting of.

[0170] In the present application, the VH of the isolated antigen-binding protein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0171] 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 can include the amino acid sequence shown in SEQ ID NO: 73.

[0172] X1VX2LX3X4X5GX6X7X8X9X 10 PGX 11 SX 12 KX 13 SCX 14 ASGX 15 TFX 16 (In SEQ ID NO: 73), X1 may be E or Q, X2 may be K or Q, X3 may be E, V or Q, X4 may be E or Q, X5 may be P or S, X6 may be A or G, X7 may be E or G, X8 may be L or V, X9 may be K or V, X 10 may be K or Q, X 11 may be A or G, X 12 may be M or V, X 13 may be L or V, X 14 may be A or K, X 15 may be F or Y, X 16 may be S or T.

[0173] For example, the H-FR1 can include an 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.

[0174] In the present application, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 can include an amino acid sequence shown in SEQ ID NO: 74.

[0175] In WVX1QX2PX3X4GLEWX5X6 (SEQ ID NO: 74), X1 may be K or R, X2 may be A, R, or S, X3 may be E or G, X4 may be K or Q, X5 may be I, M, or V, and X6 may be A or G.

[0176] For example, the H-FR2 can include an 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.

[0177] In the present application, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 can include an amino acid sequence shown in SEQ ID NO: 75.

[0178] X1X2TX3X4X5DX6SX7SX8X9YX 10 X 11 X 12 X 13 SLX14X 15 EDX 16 X 17 X 18 YYCX 19 R (SEQ ID NO: 75), X1 may be K or R, X2 may be A, F, or V, X3 may be I, L, or M, X4 may be I, S, or T, X5 may be R or V, X6 may be D, K, or T, X7 may be K, S, or T, X8 may be S or T, X9 may be A or V, X 10 may be L, M, or V, and X11 may be E or Q, and X 12 may be L or M, and X 13 may be N or S, and X 14 may be R or T, and X 15 may be A or S, and X 16 may be S or T, and X 17 may be A or G, and X 18 may be I or V, and X 19 may be A or T.

[0179] For example, the H-FR3 may include 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.

[0180] 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 include the amino acid sequence shown in SEQ ID NO: 76.

[0181] In GQGTX1VTVSS (SEQ ID NO: 76), X1 may be S or T.

[0182] For example, the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 44.

[0183] For example, the H-FR1 of the isolated antigen-binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 73, the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 74, the H-FR3 may include the amino acid sequence shown in SEQ ID NO: 75, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 76.

[0184] For example, the H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen-binding protein described in the present application are (1) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7. (2) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 17, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 18, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7. (3) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 21, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7. (4) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 30, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 31, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 7. (5) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 42, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 43, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 44. (6) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 53, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 54, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 44, and (7) The H-FR1 includes the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 53, the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 57, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 44, can include the amino acid sequences shown in the group consisting of.

[0185] In the present application, the VL of the isolated antigen-binding protein can include framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0186] 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 can include the amino acid sequence shown in SEQ ID NO: 77.

[0187] DIX1MTX2X3X4X5SLX6X7SX8GX9RX 10 TIX 11 In C (SEQ ID NO: 77), 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 may be A or V, and X 11 may be S or T.

[0188] For example, the L-FR1 can include 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.

[0189] In the present application, the L-FR2 can be located between the LCDR1 and the LCDR2, and the L-FR2 can include the amino acid sequence shown in SEQ ID NO: 78.

[0190] In WYQQKPX1X2X3X4KLLIX5 (SEQ ID NO: 78), X1 may be D or G, X2 may be G, K, or Q, X3 may be A, P, or T, X4 may be P or V, and X5 may be K or Y.

[0191] For example, the L-FR2 can include the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 38, and SEQ ID NO: 48.

[0192] In the present application, the L-FR3 can be located between the LCDR2 and the LCDR3, and the L-FR3 can include the amino acid sequence shown in SEQ ID NO: 79.

[0193] GVPX1RFSGSGSGTX2X3X4X5X6IX7X8X9X 10 X11X 12 DX 13 ATYX 14 In C (SEQ ID NO: 79), 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 may be E or Q, X 11 may be E, P or Q, X 12 may be D or E, X 13 may be F, I or S, X 14 may be F or Y.

[0194] For example, the L-FR3 can include 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.

[0195] 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 can include the amino acid sequence shown in SEQ ID NO: 80.

[0196] In FGX1GTX2LEIK (SEQ ID NO: 80), X1 may be G or Q, and X2 may be K or R.

[0197] For example, the L-FR4 can include the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 50.

[0198] For example, the L-FR1 of the isolated antigen-binding protein described in the present application can include the amino acid sequence shown in SEQ ID NO: 77, the L-FR2 can include the amino acid sequence shown in SEQ ID NO: 78, the L-FR3 can include the amino acid sequence shown in SEQ ID NO: 79, and the L-FR4 can include the amino acid sequence shown in SEQ ID NO: 80.

[0199] For example, the H-FR1, H-FR2, H-FR3, and H-FR4 of the isolated antigen-binding protein described in the present application (1) the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 12, the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 13, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 14, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 15, (2) the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 25, the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 13, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 14, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 15, (3) the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 37, the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 38, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 39, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 15, (4) the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 47, the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 48, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 49, the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 50, and (5) the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 47, the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 48, the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 60, the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 50, and can include the amino acid sequence shown in the group consisting of

[0200] The isolated antigen-binding protein described in the present application can include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0201] In the present application, the HCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 can include the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 can include the amino acid sequence shown in SEQ ID NO: 3, the LCDR1 can include the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 11.

[0202] In the present application, the HCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 can include the amino acid sequence shown in SEQ ID NO: 2, the HCDR3 can include the amino acid sequence shown in SEQ ID NO: 20, the LCDR1 can include the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 24.

[0203] In the present application, the HCDR1 of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 can include the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 can include the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 can include the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 can include the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 can include the amino acid sequence shown in SEQ ID NO: 36.

[0204] The isolated antigen-binding protein described in the present application can include an antibody light chain variable region VH and an antibody heavy chain variable region VL.

[0205] In the present application, the VH can include 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 can include 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.

[0206] In the present application, the VL can include the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40. For example, the VL can include 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.

[0207] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 8, and the VL can include the amino acid sequence shown in SEQ ID NO: 16.

[0208] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 19, and the VL can include the amino acid sequence shown in SEQ ID NO: 16.

[0209] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 22, and the VL can include the amino acid sequence shown in SEQ ID NO: 26.

[0210] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 33, and the VL can include the amino acid sequence shown in SEQ ID NO: 40.

[0211] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 45, and the VL can include the amino acid sequence shown in SEQ ID NO: 51.

[0212] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 55, and the VL can include the amino acid sequence shown in SEQ ID NO: 51.

[0213] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 58, and the VL can include the amino acid sequence shown in SEQ ID NO: 51.

[0214] For example, the VH of the isolated antigen-binding protein can include the amino acid sequence shown in SEQ ID NO: 45, and the VL can include the amino acid sequence shown in SEQ ID NO: 61.

[0215] In the present application, the isolated antigen-binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG heavy chain constant region.

[0216] In some embodiments, the isolated antigen-binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG1 heavy chain constant region. In some other embodiments, the isolated antigen-binding protein can include an antibody heavy chain constant region, and the antibody heavy chain constant region can be derived from a human IgG4 heavy chain constant region.

[0217] For example, the antibody heavy chain constant region can include the amino acid sequence shown in SEQ ID NO: 63.

[0218] In the present application, the isolated antigen-binding protein can include an antibody light chain constant region, and the antibody light chain constant region can be derived from a human Igκ constant region. For example, the antibody light chain constant region can include the amino acid sequence shown in SEQ ID NO: 64.

[0219] In this application, the isolated antigen-binding protein can include an antibody heavy chain HC, and the HC can include an 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.

[0220] In this application, the isolated antigen-binding protein can include an antibody light chain LC, and the LC can include an 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.

[0221] The isolated antigen-binding protein described in this application can include an antibody heavy chain and an antibody light chain.

[0222] For example, the antibody heavy chain can include the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain can include the amino acid sequence shown in SEQ ID NO: 87.

[0223] For example, the antibody heavy chain can include the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain can include the amino acid sequence shown in SEQ ID NO: 87.

[0224] For example, the antibody heavy chain can include the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain can include the amino acid sequence shown in SEQ ID NO: 90.

[0225] For example, the antibody heavy chain can include the amino acid sequence shown in SEQ ID NO: 91, and the antibody light chain can include the amino acid sequence shown in SEQ ID NO: 92.

[0226] For example, the antibody heavy chain can include the amino acid sequence shown in SEQ ID NO: 46, and the antibody light chain can include the amino acid sequence shown in SEQ ID NO: 52.

[0227] For example, the heavy chain of the antibody can include the amino acid sequence shown in SEQ ID NO: 56, and the light chain of the antibody can include the amino acid sequence shown in SEQ ID NO: 52.

[0228] For example, the heavy chain of the antibody can include the amino acid sequence shown in SEQ ID NO: 59, and the light chain of the antibody can include the amino acid sequence shown in SEQ ID NO: 52.

[0229] For example, the heavy chain of the antibody can include the amino acid sequence shown in SEQ ID NO: 46, and the light chain of the antibody can include the amino acid sequence shown in SEQ ID NO: 62.

[0230] Also, to be described, the isolated antigen-binding protein described in the present application can include a heavy chain sequence and / or a light chain sequence modified with one or more conservative sequences with the antigen-binding protein described in the present application. "Conservative sequence modification" refers 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. The modifications can be introduced into the isolated antigen-binding protein described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitution is the substitution of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR regions of the isolated antigen-binding protein described in the present application may be substituted with other amino acid residues of the same side chain group.Those skilled in the art know that some conservative array modifications do not eliminate the binding properties of antigens. Specifically, for example, reference can be made to Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot.Eng.10:835-41; Komissarov 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.

[0231] The protein, polypeptide and / or amino acid sequence according to the present application should be understood to at least include the scope of variants or homologues having the same or similar functions as the protein or polypeptide.

[0232] In the present application, the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added to the amino acid sequence of the protein and / or the polypeptide (for example, the isolated antigen-binding protein described in the present application). For example, the variant may include a protein or polypeptide in which the amino acids are changed by substitution, deletion and / or insertion of at least one, for example 1 to 30, 1 to 20 or 1 to 10, for example 1, 2, 3, 4 or 5 amino acids. The functional variant can basically maintain the biological properties of the protein or polypeptide before the change (for example, substitution, deletion or addition). For example, the functional variant can maintain at least 60%, 70%, 80%, 90% or 100% of the biological activity of the protein or polypeptide before the change (for example, the ability to specifically bind to the LILRB4 protein).

[0233] In the present application, the homolog may be a protein or polypeptide having at least about 80% (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 protein and / or the polypeptide (e.g., the antibody or antigen-binding fragment thereof described in the present application).

[0234] In the present application, the homology generally refers to the similarity or relatedness between two or more sequences. The "percent sequence homology" can be calculated as follows: Two sequences to be aligned are compared in a comparison window, and the number of positions where the same nucleobase (e.g., A, T, C, G, I, etc.) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exists in the two sequences is determined to obtain the matching positions. The number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to generate the percent sequence homology. The alignment for determining the percent sequence homology may be achieved in various ways known in the art, for example, by using publicly available computer software such as LAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment within the full-length sequences being compared or within the target sequence region. The homology may be measured by FASTA and BLAST. For an explanation of the FASTA algorithm, reference can be made to "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman, Proceedings of the National Academy of Sciences, 85:2444-2448, 1988, and "Rapid and Sensitive Protein Similarity Searches" by D.J. Lipman and W.R. Pearson, Science, 227:1435-1441, 1989. For an explanation of the BLAST algorithm, reference can be made to "Basic Local Alignment Search Tool" by S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, Journal of Molecular Biology, 215:403-410, 1990.

[0235] In the present application, the isolated antigen-binding protein can comprise an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding protein described in the present application includes, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, mouse antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, multi-specific antibodies, Fv fragments, scFv fragments, Fab fragments, Fab’ fragments, F(ab’)2 fragments, and camelized single-domain antibodies.

[0236] In the present application, the antibody may be a humanized antibody. In other words, the isolated antigen-binding protein described in the present application may be an antibody or a variant, derivative, analog, or fragment thereof that immunospecifically binds to a relevant antigen (e.g., human LILRB4) and has a framework (FR) region having basically the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having basically the amino acid sequence of a non-human antibody. Here, “basically” means that in the case of the CDR, 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% the same as the amino acid sequence of the CDR of the non-human antibody. The humanized antibody basically includes all of at least one and usually two variable domains (Fab, Fab’, F(ab’)2, FabC, Fv), and all or basically all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., antibody), and all or basically all of the framework regions are framework regions having a human immunoglobulin consensus sequence. Preferably, the humanized antibody includes a constant region of an immunoglobulin (e.g., Fc), usually at least a part of the constant region of a human immunoglobulin. In some embodiments, the humanized antibody includes at least the variable domains of the light chain and the heavy chain. The antibody can further include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody includes only the humanized light chain. In some embodiments, the humanized antibody includes only the humanized heavy chain. In certain embodiments, the humanized antibody includes only the humanized variable domains of the light chain and / or the humanized heavy chain.

[0237] In the present application, the antigen-binding fragment can include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0238] In the present application, the isolated antigen-binding protein can include one or more of the following characteristics: 1) it can specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) it can bind to the LILRB4 protein at a K -7 value of 2×10 D M or less; 3) it has the activity to block the binding of LILRB4 / APOE; 4) it can stimulate the activation of CD8+ T cells; 5) it can inhibit the migration of human monocytic leukemia cells THP-1; 6) it can inhibit the growth and / or metastasis of tumor cells; 7) it can block the binding of LILRB4 to fibronectin.

[0239] In the present application, the isolated antigen-binding protein can bind to the LILRB4 protein at a K -7 value of 2×10 D M or less. For example, the K D value of the isolated antigen-binding protein described in the present application binding to the human LILRB4 protein is ≦ 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 -9It may also be M.

[0240] In the present application, the KD value may be measured by ELISA, competitive ELISA, BIACORE or KINEXA.

[0241] In the present application, the isolated antigen-binding protein can specifically bind to the human LILRB4 protein. The specific binding may be measured by FACS.

[0242] In the present application, the LILRB4 protein may be the human LILRB4 protein (GenBank accession number: CAG46845.1).

[0243] The LILRB4 protein can 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 to 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 homology to the LILRB4 protein.

[0244] The LILRB4 protein can further include a functionally active fragment of the LILRB4 protein.

[0245] Polypeptide, fusion protein, drug molecule, nucleic acid molecule, vector, cell and pharmaceutical composition In another aspect, the present application provides a polypeptide comprising the isolated antigen-binding protein.

[0246] In another aspect, the present application provides a fusion protein comprising the isolated antigen-binding protein.

[0247] In another aspect, the present application provides a drug molecule comprising the isolated antigen-binding protein.

[0248] In another aspect, the present application provides one or more nucleic acid molecules encoding the isolated antigen-binding protein. The one or more isolated nucleic acid molecules described in the present application may be isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized, but can encode the isolated antigen-binding protein described in the present application.

[0249] In another aspect, the present application further provides a vector that can contain the nucleic acid molecule described in the present application. By transforming, transducing or transfecting a host cell, the genetic material element contained therein can be expressed in the host cell. For example, the vector can include artificial chromosomes such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as λ phage or M13 bacteriophage, and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomavirus vacuolating viruses (such as SV40). Also, for example, the vector may contain various elements for controlling expression, including a promoter sequence, a transcription start sequence, an enhancer sequence, a selection element, a reporter gene. Further, the vector can further include an origin of replication site. Further, the vector can further include components that assist in entry into cells, such as virus particles, liposomes, protein shells, but are not limited thereto.

[0250] In another aspect, the present application further provides a cell that can contain the nucleic acid molecule described in the present application or the vector described in the present application. The cell can contain the progeny of a single cell. Due to natural mutations, accidental mutations or intentional mutations, the progeny do not necessarily have to be exactly the same as the original parental cell (either in the form of the full DNA complement or the genome). In some embodiments, the cell can also include a cell transfected in vitro using the vector described in the present invention. In some embodiments, the cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell or other eukaryotic cells.

[0251] In another aspect, the present application further provides a pharmaceutical composition that can contain the isolated antigen-binding protein described in the present application, the polypeptide described in the present application, the fusion protein described in the present application, the nucleic acid molecule of the present application, the vector of the present application and / or the cell described in the present application, and optionally a pharmaceutically acceptable carrier.

[0252] In some embodiments, the pharmaceutical composition can further contain an appropriate formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosages and concentrations used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions and lyophilized compositions.

[0253] In some embodiments, the "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coating agents, isotonic agents and absorption delaying agents that are compatible with the administration of the drug, and are usually safe, non-toxic and not undesirable biologically or otherwise.

[0254] In some embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intraluminally, intraarterially, intrathecally and / or intranasally, or may be directly injected into a tissue. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways such as intravenous, intraperitoneal administration, subcutaneous administration, intramuscular administration, topical administration or intradermal administration. In some embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be achieved by a small pump system worn by the patient for measuring the therapeutic agent flowing into the patient's body, as described in WO2015 / 036583.

[0255] In the present application, the pharmaceutical composition may further comprise one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin-cytarabine combined 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, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0256] Preparation method and use In another aspect, the present application further provides a method for preparing the isolated antigen-binding protein described in the present application, which can include the step of culturing the cells described in the present application under conditions for expressing the isolated antigen-binding protein described in the present application.

[0257] In another aspect, 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 described in the present application in the preparation of a drug for preventing and / or treating a disease and / or a symptom.

[0258] In another aspect, 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 described in the present application for preventing and / or treating a disease and / or a symptom.

[0259] In another aspect, the present application provides a method for preventing and / or treating a disease and / or a symptom, which includes the step of 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 described in the present application to a subject in need thereof.

[0260] In the present application, the administration may be performed in various ways such as intravenous administration, intratumoral administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, topical administration or intradermal administration.

[0261] In the present application, the disease and / or the symptom may include a disease or a symptom related to LILRB4 signal transduction.

[0262] In the present application, the disease and / or the symptom may include a tumor.

[0263] In the present application, the disease and / or the symptom may include a LILRB4-positive disease and / or a symptom.

[0264] In the present application, the tumor can include a solid tumor and / or a hematological tumor.

[0265] In the present application, the disease or condition associated with LILRB4 signaling can include an immune system disease and / or a malignant hematological disease.

[0266] In the present application, the immune system disease may be one selected from the group consisting of Kawasaki disease, systemic lupus erythematosus (SLE), and sepsis.

[0267] In the present application, the solid tumor may be one or more selected from the group consisting of breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, and pancreatic cancer.

[0268] In the present application, the malignant hematological disease may be one or more selected from the group consisting of myelodysplastic syndrome, myeloproliferative tumor, chronic myelomonocytic leukemia (CMML), chronic myelogenous leukemia or acute myelogenous leukemia (AML), acute promyelocytic leukemia or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, acute myeloblastic leukemia, and polycythemia vera.

[0269] 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 may be used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin-cytarabine combined 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, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors.

[0270] In another aspect, the present application provides the use of an antibody or an antigen-binding fragment thereof in the preparation of a diagnostic agent for diagnosing a disease or condition associated with the expression of the LILRB4 protein.

[0271] In another aspect, the present application provides the isolated antigen-binding protein for diagnosing a disease or condition associated with the expression of the LILRB4 protein.

[0272] In the present application, the diagnostic agent may be used alone or in combination with an apparatus, instrument, device or system. In the processes of disease prevention, diagnosis, treatment monitoring, prognosis observation, health status evaluation and prediction of genetic diseases, the diagnostic agent can be used to detect human samples (such as various body fluids, cells, tissue samples, etc.) in vitro. The diagnostic agent may be selected from the group consisting of a reagent, a kit, a calibrator and a control.

[0273] The in vitro detection method may be selected from the group consisting of Western blot, ELISA and immunohistochemistry. For example, the reagent may include a reagent capable of measuring the expression level of the LILRB4 protein. For example, the reagent may be selected from the group consisting of a reagent for performing Western blot, a reagent for performing ELISA and a reagent for performing immunohistochemistry.

[0274] In another aspect, the present application provides a detection kit comprising the isolated antigen-binding protein for detecting the presence and / or content of LILRB4 protein in a sample. In particular, the present application relates to an immunoassay kit using the antibody of the present application in combination with immunological detection methods such as ELISA, immunohistochemistry, Western blot, flow cytometry technology and the like.

[0275] The kit includes the antibody of the present application for detecting LILRB4-related cancer cells. Specifically, the immunoassay kit includes the antibody of the present application as a first antibody that binds to LILRB4 and any immunoassay reagent in a suitable container member.

[0276] In some embodiments, the antibody may be pre-bound to a solid vector such as a column matrix and / or a well of a microtiter plate.

[0277] The immunoassay reagents in the kit can assume any one of various forms, including a detectable marker bound or conjugated to a specific antibody. It can also include a detectable marker bound or conjugated to a secondary binding ligand. An exemplary secondary ligand is a secondary antibody having binding affinity for a first antibody.

[0278] Other immunoassay reagents applicable to the kit of the present application can include a two-component reagent comprising a secondary antibody having binding affinity for a first antibody and a tertiary antibody having binding affinity for a second antibody, wherein the third antibody is connected to a detectable marker. As described above, various exemplary markers are known in the art, and all such markers can be used in combination with the present disclosure.

[0279] The kit can further include an appropriate aliquot of the LILRB4 composition that can be used to create a standard curve for detection and analysis, regardless of marking. The kit can include the fully conjugated form, an intermediate form of the antibody-marker conjugate, or separate parts where the conjugation is completed by the user of the kit. The components of the kit can be encapsulated in an aqueous medium or in lyophilized form.

[0280] The container member of the kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container member capable of holding an antibody or preferably an appropriate aliquot of an antibody. The kit of the present disclosure usually further includes a member for holding antibody, antigen, and other reagent containers in a sealed form for commercial sale. Such containers can include injection-molded or blow-molded plastic containers in which the above vials are held.

[0281] In another aspect, the present application provides a method for diagnosing a disease or condition related to 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 content of a substance that can specifically bind to the isolated antigen-binding protein in the sample.

[0282] In another aspect, the present application provides a method for detecting LILRB4 in a sample or a subject, the method comprising administering the isolated antigen-binding protein. In the present application, the administration may be performed in various ways such as intravenous administration, intratumoral administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, local administration or intradermal administration.

[0283] Without intending to be limited by any theory, the following examples are only for explaining the antigen-binding protein, preparation method and use of the present application, but not for limiting the scope of the invention of the present application.

[0284] Examples Example 1 Screening of Monoclonal Antibodies by Hybridoma Technology Using the extracellular domain of human LILRB4 protein-human IgG1 Fc fusion protein (named LILRB4-Fc), referring to Q8NHJ6 for the sequence information of human LILRB4, referring to AXN93652.1 for the sequence of human IgG1 Fc fragment, the amino acid sequence of the extracellular domain of human LILRB4 protein is shown in SEQ ID NO: 84, the amino acid sequence of LILRB4-Fc is shown in SEQ ID NO: 85, and conventional immunization of Balb / c mice is performed (Beijing Vital River Laboratory Animal Technology Co., Ltd.).

[0285] On days 0, 14, and 28, 100 μg of LILRB4-Fc was subcutaneously injected into Balb / c mice in the presence of complete Freund's adjuvant (first injection) or incomplete Freund's adjuvant (second and third injections). Mouse-derived spleen cells were fused with mouse myeloma cells SP2 / 0 (ATCC) using a conventional hybridoma technology scheme (Salhi et al., Biochem. J. 2004). To select hybridomas, the cells were cultured in plates containing HAT medium (10 5 cells per well). After 12 days, the supernatants were collected and clones with binding specificity to LILRB4 (positive clones) were screened directly by enzyme-linked immunosorbent assay (ELISA). Through screening, a series of mouse monoclonal antibodies targeting the extracellular domain (ECD) of human LILRB4 were obtained.

[0286] 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 affinity purified using conventional protein A and named 1A2-7, 3A2-18, 3G9-1, 5E11-2. The amino acid sequences of the chimeric antibodies are shown in the following table, and the CDR sequences were divided by Kabat.

Table 5

[0287] Example 2 Binding of Chimeric Antibodies to Human LILRB4 Coat the ELISA plate with LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag), Sino Biological), leave it at 37 °C for 2 h, wash it with PBST, then add 10% fetal bovine serum, seal it at 37 °C for 1 h, add chimeric antibodies and positive antibody C84 at different concentrations, react at 37 °C for 1 h, wash it with PBST, then add goat anti-human IgG Fc secondary antibody (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) marked with horseradish peroxidase, react at 37 °C for 30 min, wash it 5 times with PBST, add 100 μL of TMB (eBioscience) to each well, leave it in the dark at room temperature (20 ± 5 °C) for 2 - 3 min, then add 100 μL of 2N H2SO4 reaction stop solution to each well to stop the substrate reaction, read the OD value at 450 nm of the microplate reader, and analyze the binding ability of the chimeric antibody to LILRB4.

[0288] The results are shown in Figure 1, and the binding abilities of chimeric antibodies 1A2 - 7, 3A2 - 18, 3G9 - 1, and 5E11 - 2 to LILRB4 are the same as those of the positive antibody C84.

[0289] Example 3 Detection of the binding specificity of chimeric antibodies to human LILRB family proteins Coat ELISA plates with proteins of the LILRB family, namely 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), and LILRB5 (Human LILRB5 / CD85c / LIR-8 Protein (ECD, His Tag), Sino Biological), respectively. Leave them overnight at 4°C, wash with PBST, then add 10% fetal bovine serum and incubate for 1 hour at 37°C. Add 10 μg / mL of the chimeric antibody to each well, react for 1 hour at 37°C, wash with PBST, then add a goat anti-human IgG Fc secondary antibody conjugated with horseradish peroxidase (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen), react for 30 minutes at 37°C, wash 5 times with PBST, add 100 μL of TMB (eBioscience) to each well, leave in the dark at room temperature (20 ± 5°C) for 1 - 2 minutes, then add 100 μL of 2N H2SO4 reaction stop solution to each well to stop the substrate reaction. Read the OD value at 450 nm on a microplate reader to analyze the non-specific binding ability of the chimeric antibody to LILRB1, LILRB2, LILRB3, and LILRB5.

[0290] The results are shown in Figure 2, and none of the chimeric antibodies non-specifically bind to LILRB1, LILRB2, LILRB3, or LILRB5.

[0291] Example 4, Detection of the Activity of Blocking the Binding of Chimeric Antibody LILRB4 / APOE The jurkat-LILRB4-CHO-CD3-APOE reporter gene cell line was used to detect the activity of blocking the binding of chimeric antibody LILRB4 / APOE, and 128-3 (Oncoimmune antibody 128-3) was used as a positive control antibody.

[0292] APOE / TCR activator / CHO cells in the logarithmic growth phase were taken and inoculated into a 96-well plate, and cultured overnight at 37°C. The next day, the supernatant was discarded, and after washing twice with PBS, the antibody sample diluted in 1640 medium was added, and then LILRB4 effector reporter cells in the logarithmic growth phase were added, and they were left in an incubator at 37°C for culture. After 5 hours, the cells were taken out, bright-Glo luciferase was added, the value of the microplate reader was read, and analysis was performed using prism Graphpad software based on the value of each well.

[0293] The results are shown in Figures 3A and 3B. The chimeric antibodies 1A2-7 and 3A2-18 can block the binding of LILRB4 and APOE, and the blocking activity of 1A2-7 is superior to that of the positive control antibody 128-3.

[0294] Example 5 Chimeric Antibody Stimulates the Activation of CD8 + T Cells PBMC was extracted from the whole blood of healthy donors, and CD8 + T cell isolation kit (CD8 + T Cell Isolation Kit, Miltenyi) was used to isolate CD8 + T cells, and 5×10 were placed in a 96-well plate (96-well Clear Round Bottom TC-treated Microplate, Corning). 4Add to cells / wells, add T cell activation magnetic beads (Dynabeads(R) Human T-Activator CD3 / CD28 / CD137, GIBCO), leave in a 5% CO2 incubator at 37°C and incubate for 2 days, add chimeric antibody at a final concentration of 200 μg / mL, positive control antibody C84 and isotype control antibody, and add human monocytic leukemia cells THP-1 at 5×10 4 Add to cells / wells, continue to incubate in a 5% CO2 incubator at 37°C for 5 days, then collect the supernatant, detect the level of IFN-γ in the supernatant using a Human IFN-γ Elisa Kit (ExCell Bio), and the THP-1 / CD8 of the chimeric antibody + In the CD8 + ability to activate T cells was evaluated.

[0295] The results are shown in Figure 4. The ability of the chimeric antibodies 1A2-7, 3A2-18, 3G9-1 and 5E11-2 to activate CD8 + T cells in THP-1 / CD8+ T lymphocyte co-culture is similar to that of the positive control antibody C84.

[0296] Example 6 Inhibitory effect of chimeric antibody on migration of THP-1 cells Human monocytic leukemia cells THP-1 in a 24-hour serum-starved state were collected and added to a 24-well cell culture plate. A chimeric antibody at a final concentration of 10 μg / mL and a positive control antibody C84 were added, and the plate was left in a 5% CO2 incubator at 37°C for 1 hour of incubation. 100 μL of the cell-antibody mixture was added to the upper chamber of a Transwell (6.5 mm Transwell(R) with 8.0 μm Pore Polycarbonate Membrane Insert, Corning), 700 μL of complete medium was added to the lower chamber, and the plate was left in a 5% CO2 incubator at 37°C for 24 hours of incubation. 100 μL of the solution was taken out from the lower chamber and added to a 96-well plate with a white and transparent bottom (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning). 100 μL of a cell viability detection reagent (CellTiter-Lumi TM Luminescent Cell Viability Detection Kit, Beyotime) was added, and after incubation in the dark at room temperature for 10 minutes, a multifunctional microplate reader was used to detect the relative fluorescence intensity value of chemiluminescence. A standard curve was drawn using THP-1 cells diluted in a gradient 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 the migration of THP-1 cells was analyzed.

[0297] The results are shown in Figure 5. The inhibitory abilities of chimeric antibodies 1A2-7, 3A2-18, and 3G9-1 on the migration of THP-1 cells were stronger than that of the positive control antibody C84, and the inhibitory ability of 5E11-2 was the same as that of the positive control antibody C84.

[0298] Example 7: Chimeric antibody inhibits the growth and metastasis of THP-1 in NSG mice THP1-Luc human monocytic leukemia cells resuspended in PBS were injected into the tail vein of LB-NDG mice at 5×10 6 cells / 0.2 mL, 0.2 mL per mouse. On the day of grouping, the imaging signal of the mice (average imaging signal 1.9x10 7Based on the body surface area (p / s) and body weight (average body weight 22.4 g), the mice were randomly assigned to five experimental groups: G1 (PBS), G2 (1A2-7, 10 mg / kg), G3 (3A2-18, 10 mg / kg), and G5 (C84, 10 mg / kg), with 5 mice assigned to each group. Administration was performed on the day of grouping (day0) and on the third day after grouping, and all mice were administered intraperitoneally. After inoculating the cells (i.e., before grouping and administration, day0), on the third, seventh, fourteenth, twenty-first, twenty-fifth days after administration, and before the end of the experiment (day26), the mice were imaged using a small animal live imager to obtain an imaging signal map and signal intensity, observe the growth of tumors in the body. After the observation was completed, the experiment was terminated. During the administration and observation period, the body weight of the animals was measured twice a week and the measured values were recorded.

[0299] When the experiment ended, the tumor imaging signal intensity of the control group (PBS) was 1.43x10 9 ±1.32x10 8 p / s, the tumor imaging signal intensity of the 1A2-7 group was 1.27x10 6 ±2.35x10 5 p / s, the TGITV was 101.3%, the tumor imaging signal intensity of the 3A2-18 group was 1.18×10 6 ±2.79×10 5 p / s, the TGITV was 101.3%, the tumor imaging signal intensity of the C84 group was 3.74x10 7 ±1.92x10 7 p / s, the TGITV was 98.7%. Compared with the G1 group, the P value of each group was <0.001, indicating that the test products 1A2-7, 3A2-18, and C84 at a dose of 10 mg / kg had an obvious inhibitory effect on the tumor growth of B-NDG mice transplanted with THP1-LUC cells.

[0300] The results are shown in Figure 6. The chimeric antibodies 1A2-7 and 3A2-18 can effectively inhibit the growth of tumors, and their inhibitory effect is significantly better than that of the positive control antibody C84.

[0301] When the above experiment was completed, after measuring and imaging the body weight of the mice, the mice were euthanized, the survival curve of the experimental animals was drawn, and hCD45 in the liver, lung and bone marrow of the mice in each group was detected by flow cytometry + The ratios of cells (live / dead, hCD45+, mCD45+) were analyzed.

[0302] Prior to the tumor model construction experiment, the results detected and analyzed by flow cytometry showed that all THP1-Luc cells were hCD45 + cells and there was no expression of mCD45 in the cells. Before administration, hCD45 + cells were detected in the lungs, bone marrow (BM) and liver of B-NDG mice, which were the model mice constructed in this experiment.

[0303] The results detected and analyzed by flow cytometry after administration showed that, compared with the G1 control group, the ratios of hCD45 + cells in the liver, lung and bone marrow of the mice in each group were significantly decreased, indicating that the test products C84, 1A2-7 and 3A2-18 at a dose of 10 mg / kg could significantly reduce the amount of tumor cells in the liver, lung and bone marrow of mice and inhibit the migration of tumor cells. The results are shown in Figure 7.

[0304] Example 8 Humanization of Antibodies When humanizing the antibody, it was humanized using a standard CDR grafting method. BLAST searches were performed on the variable sequences of the human heavy and light chains, and three or four sequences were selected from each sequence as the acceptor frameworks for humanization. CDR1, CDR2 and CDR3 of the heavy and light chains were cloned into three different heavy chain frameworks (H1-H4) and two different light chain frameworks (L1-L2). Next, the expression levels and antigen-binding abilities of four different combinations of antibodies (see Table 1 for details) in CHO-S cells were detected.

Table 6

[0305] Example 9 Binding of Humanized Antibodies to Human LILRB4 The ELISA plate was coated with LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag), Sino Biological), left at 37°C for 2 h, washed with PBST, then 10% fetal bovine serum was added and sealed at 37°C for 1 h. Different concentrations of humanized antibodies and the positive antibody 128-3 were added and reacted at 37°C for 1 h. After washing with PBST, a goat anti-human IgG Fc secondary antibody marked with horseradish peroxidase (Goat anti-human IgG Fc antibody, horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37°C for 30 min, washed 5 times with PBST, 100 μL of TMB (eBioscience) was added to each well, left at room temperature (20 ± 5°C) in the dark for 2 - 3 min, then 100 μL of 2N H2SO4 reaction stop solution was added to each well to stop the substrate reaction, and the OD value at 450 nm of the microplate reader was read to analyze the binding ability of the humanized antibody to LILRB4.

[0306] The results are shown in Figure 8. The binding abilities of the humanized antibodies RB4-G1-018, RB4-G1-019, RB4-G1-020, and RB4-G1-022 to LILRB4 are the same as those of the positive control antibody 128-3.

[0307] Example 10 Measurement of the Affinity of Humanized Antibodies The affinity of the antibody was analyzed using a BIACORE 8K biomacromolecule interaction apparatus (manufactured by GE). The antibody to be measured was captured using a Series S Sensor Chip Protein A chip (manufactured by GE). The concentration of the antibody to be measured was set at 15 μg / mL and injected at 10 μl / min until RU = 500. LILRB4 (Beijing Protein Innovation Co., Ltd., product number LI4-H52H7, 6×His tag, theoretical molecular weight 28.2 Kda) was used as the mobile phase, and six concentration gradients (0.0977 nM to 200 nM) were used. The binding time was 180 seconds and the dissociation time was 300 seconds. The results of the affinity of the antibody to be measured are shown below.

Table 7

[0308] Example 11 Measurement of the binding ability of the humanized antibody to LILRB4-positive cells Human monocytic leukemia cells THP-1 were collected and added to 1.5 mL EP tubes at 1×10 5 cells per tube. Different concentrations of the humanized antibody and the positive control antibody 128-3 were added, and incubated on ice in the dark for 30 min. After washing with FACS wash buffer, a goat anti-human IgG Fc secondary antibody (Goat Anti-Human IgG Fc Secondary Antibody, Invitrogen) labeled with PE fluorescence was added, and incubated on ice in the dark for 30 min. After washing twice with FACS wash buffer, 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells. After uniform mixing, the relative fluorescence intensity of PE fluorescence was detected with an instrument to analyze the binding ability of the humanized antibody to LILRB4-positive cells THP-1.

[0309] The results are shown in Figure 9. The binding activity of the humanized antibody to LILRB4-positive cells THP-1 is the same as or stronger than the binding activity of the positive antibody 128-3.

[0310] Example 12 The humanized antibody inhibits the migration of LILRB4+ tumors THP1-Luc human monocytic leukemia cells resuspended in PBS were injected into the tail vein of B-NDG mice at 5×106 cells / 0.2 mL, 0.2 mL / mouse. On the day of inoculation, based on the imaging signal of the mice (average imaging signal 3.6x10 7 p / s) and the body weight of the mice (average body weight 23 g), the mice were randomly assigned to six experimental groups: 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 m / kg), and G6 (RB4-G1-022, 10 mg / kg), with 5 mice assigned to each group. On the day of grouping (day0) and on the 3rd day after grouping, all mice were administered intraperitoneally. Before grouping and administration (day0), on the 7th, 14th, 21st, 25th days after drug administration, and on the day when the experiment ended (day28), the mice were imaged using a small animal live imager to obtain an imaging signal map and signal intensity, and the growth of tumors in the body was observed. During the administration and observation period, the body weight of the animals was measured twice a week and the measured values were recorded.

[0311] When the experiment ended, after measuring the body weight and imaging the mice, the mice were euthanized, the survival curve of the experimental animals was drawn, and the proportion of hCD45 + cells (live / dead, hCD45 + , mCD45 + ) in the liver, lung, and bone marrow of the mice in each group was detected by flow cytometry and analyzed.

[0312] The flow cytometry detection results are shown in Figure 10. Compared with the G1 control group, the proportion of hCD45 + cells in the liver, lung, and bone marrow of the mice in each group was significantly decreased, 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 can significantly reduce the amount of tumor cells in the liver, lung, and bone marrow of mice.

[0313] Using the Jurkat-LILRB4-CHO-CD3-APOE reporter cell line, detect the activity of blocking the LILRB4 / APOE binding of the humanized LILRB4 antibody APOE / TCR activator / CHO cells in the logarithmic growth phase were inoculated into 96-well plates and cultured overnight at 37°C. The next day, the supernatant was discarded, and after washing twice with PBS, 50 μl of the antibody sample diluted in 1640 medium was added to each well. Next, LLILRB4 effector reporter cells in the logarithmic growth phase were added, and the cells were placed in an incubator at 37°C for culture. After 5 hours, the cells were taken out, bright-Glo luciferase was added, the value of the microplate reader was read, and analysis was performed using prism Graphpad software based on the value of each well.

[0314] The results are shown in Figure 11. All humanized antibodies can block the binding of APOE to LILRB4, and their blocking ability is stronger than that of the positive control antibodies 128-3 and C84.

[0315] Example 14 ADCC activity of humanized antibodies in 293T-hLILRB4 cells Collect 293T-hLILRB4 cells that overexpress the human LILRB4 gene, add them to 96-well plates with a white and transparent bottom (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning), add different concentrations of humanized antibodies and the positive antibody 128-3, and further add effector cells Jurkat-hFcγRIIIa-NFAT that overexpress the human FcγRIIIa gene and the NFAT fluorescent reporter gene. Leave the 96-well plates in a 5% CO2 incubator at 37°C for 6 h of culture, and add 100 μL of luciferase detection reagent (Bright-Lumi TMThe Firefly Luciferase Reporter Gene Assay Kit (Beyotime) was added to each well and incubated at room temperature for 10 min in the dark. Then, a multifunctional microplate reader was used to detect the relative fluorescence intensity value of chemiluminescence, and the ADCC activity mediated by the humanized antibody in 293T-hLILRB4 cells was analyzed.

[0316] The results are shown in Figure 12. The ADCC activities of the humanized antibodies RB4-G1-018, RB4-G1-019, and RB4-G1-022 were similar to and stronger than that of the positive control antibody 128-3.

[0317] Example 15 Humanized antibody stimulates the activation of CD8+ T cells PBMCs were extracted from the whole blood of healthy donors, and CD8 + T cells were isolated using a CD8+ T Cell Isolation Kit (Miltenyi) and added to a 96-well plate (96-well Clear Round Bottom TC-treated Microplate, Corning) at 5×10 + cells / well. T cell activation magnetic beads (Dynabeads(R) Human T-Activator CD3 / CD28 / CD137, GIBCO) were added, and the cells were incubated in a 5% CO2 incubator at 37°C for 2 days. Then, humanized antibodies, positive antibody 128-3, and isotype control antibodies at a final concentration of 200 μg / mL were added, and human monocytic leukemia cells THP-1 were added at 5×10 4 cells / well. The cells were continuously incubated in a 5% CO2 incubator at 37°C for 5 days. Then, the supernatant was collected, and the level of IFN-γ in the supernatant was detected using a Human TNF-α Elisa Kit (ExCell Bio) to evaluate the ability of the humanized antibody to activate CD8 4 T lymphocytes in the THP-1 / CD8 + T lymphocyte co-culture experiment. + The ability to activate CD8

[0318] The results are shown in Figure 13, and the ability of the humanized antibodies RB4-G1-018 and RB4-G1-020 to activate CD8 + T lymphocytes in the THP-1 / CD8 + T cells is stronger than that of the positive antibody 128-3.

[0319] Example 16 The humanized LILRB4 antibody can inhibit the growth of tumors in B-NDG mice transplanted with THP1-LUC cells THP1-Luc human monocytic leukemia cells resuspended in PBS were injected into the tail vein of B-NDG mice at 5×10 6 cells / 0.2 mL, 0.2 mL / mouse. On the day of inoculation, based on the imaging signal of the mice (average imaging signal 3.6x10 7 p / s) and the body weight of the mice (average body weight 23 g), the mice were randomly assigned to six experimental groups: 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 m / kg), and G6 (RB4-G1-022, 10 mg / kg), with 5 mice assigned to each group. On the day of grouping (day0) and on the 3rd day after grouping, all mice were administered intraperitoneally. Before administration (day0), on the 7th, 14th, 21st, 25th, and the last day of the experiment (day28), the mice were imaged using a small animal live imager to obtain an imaging signal map and signal intensity, and the growth of tumors in the body was observed. During the administration and observation period, the body weight of the animals was measured twice a week and the measured values were recorded.

[0320] The results showed that on the 28th day after grouping, the tumor imaging signal intensity of the G1 group was 2.54x10 9 ±3.87x10 8 p / s, the tumor imaging signal intensity of the G2 group was 1.06x10 7 ±4.50x10 6 p / s, and the TGITV was 101.0%. The tumor imaging signal intensity of the G3 group was 4.24x10 6 ±1.71x106 p / s, with TGITV being 101.3%, and the tumor imaging signal intensity of group G4 being 1.29x10 6 ±2.79x10 5 p / s, with TGITV being 101.4%, and the tumor imaging signal intensity of group G5 being 5.82x10 6 ±2.35x10 6 p / s, with TGITV being 101.2%, and the tumor imaging signal intensity of group G6 being 4.08×10 6 ±2.63×10 6 p / s, indicating that TGITV is 101.3%.

[0321] The results are shown in Figures 14 and 15. Compared with the G1 control group, the P-values of G2 - G6 are all 0.000, 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 have an obvious inhibitory effect on THP1 - LUC tumor growth. Also, the effects of RB4 - G1 - 018, RB4 - G1 - 019, RB4 - G1 - 020, and RB4 - G1 - 022 are superior to those of 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 variable region VH of the antibody heavy chain, and the VH comprises an 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 HCDR3, wherein the HCDR3 comprises an 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 an 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 HCDR2, wherein the HCDR2 comprises an amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

28.

5. Comprising HCDR1, said HCDR1 having the amino acid sequence set forth in SEQ ID NO: 69 (X １ X ２ WMX ３ , where X １ is D or S, X ２ is A or Y, and X ３ is D or H), the isolated antigen-binding protein according to any one of claims 1 to 4.

6. The isolated antigen-binding protein according to claim 5, wherein the HCDR1 comprises an 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, the HCDR2, and the HCDR3 (1) the HCDR1 comprises an amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO: 3; (2) the HCDR1 comprises an amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO: 20; and (3) the HCDR1 comprises an amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises an amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises an amino acid sequence shown in SEQ ID NO: 29 and comprises an amino acid sequence selected from the group consisting of.

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 an amino acid sequence shown in SEQ ID NO:

73.

9. The isolated antigen-binding protein according to claim 8, wherein the H-FR1 comprises an 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. An 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 an amino acid sequence shown in SEQ ID NO:

74.

11. The isolated antigen-binding protein according to claim 10, wherein the H-FR2 comprises an 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. An 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 an amino acid sequence shown in SEQ ID NO:

75.

13. The isolated antigen-binding protein according to claim 12, wherein the H-FR3 comprises an 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. An 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 an amino acid sequence shown in SEQ ID NO:

76.

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

44.

16. An 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, the H-FR2, the H-FR3, and the H-FR4 are (1) the H-FR1 comprises an amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises an amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises an amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises an amino acid sequence shown in SEQ ID NO:

7. (2) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 17, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 18, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO:

7. (3) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 21, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO:

7. (4) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 30, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 31, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 32, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO:

7. (5) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 42, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 43, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO:

44. (6) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 53, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 54, the H-FR4 contains the amino acid sequence shown in SEQ ID NO: 44, and (7) The H-FR1 contains the amino acid sequence shown in SEQ ID NO: 41, the H-FR2 contains the amino acid sequence shown in SEQ ID NO: 53, the H-FR3 contains the amino acid sequence shown in SEQ ID NO: 57, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO:

44. An isolated antigen-binding protein comprising an amino acid sequence selected from the group consisting of.

17. The isolated antigen-binding protein according to any one of claims 1 to 16, comprising VH, wherein the VH contains 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 according to any one of claims 1 to 17, wherein the VH contains 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. An isolated antigen-binding protein according to any one of claims 1 to 18, comprising an antibody heavy chain constant region derived from an IgG heavy chain constant region.

20. The isolated antigen-binding protein according to 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 according to any one of claims 19 to 20, wherein the antibody heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG2 heavy chain constant region.

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

63.

23. An isolated antigen-binding protein according to any one of claims 1 to 22, comprising an antibody heavy chain, wherein 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.

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

40.

25. Including LCDR3, wherein the LCDR3 has the amino acid sequence shown in SEQ ID NO: 72 (QX １ X ２ X ３ X ４ X ５ PX ６ T, X １ is H or Q, X ２ is G or S, X ３ is D, N or W, X ４ is E or T, X ５ is I or L, X ６ is P or R), the isolated antigen-binding protein according to any one of claims 1 to 24.

26. The isolated antigen-binding protein according to 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. An isolated antigen-binding protein according to any one of claims 1 to 26, comprising LCDR2, wherein the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:

71.

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

35.

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

34.

30. The isolated antigen-binding protein according to 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. An isolated antigen-binding protein according to any one of claims 1 to 30, comprising LCDR1, LCDR2, and LCDR3, The LCDR1, the LCDR2, and the LCDR3 are, (1) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 9, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 11; (2) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 23, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 10, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 24; and (3) The LCDR1 includes the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:

36. An isolated antigen-binding protein comprising an amino acid sequence selected from the group consisting of.

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

77.

33. The isolated antigen-binding protein according to claim 32, wherein the L-FR1 includes 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. An isolated antigen-binding protein according to any one of claims 1 to 33, comprising L-FR2, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO:

78.

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

48.

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

79.

37. The isolated antigen-binding protein according to claim 36, wherein the L-FR3 includes 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. An 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 connected 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 according to claim 38, wherein the L-FR4 comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO:

50.

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

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

40.

42. An isolated antigen-binding protein according to any one of claims 24 to 41, wherein 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.

43. An isolated antigen-binding protein according to any one of claims 1 to 42, comprising an antibody light chain constant region containing a human Igκ constant region.

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

64.

45. An isolated antigen-binding protein according to any one of claims 43 to 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. An isolated antigen-binding protein according to any one of claims 1 to 45, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are (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, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, 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 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, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20, 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 HCDR1 contains the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 contains the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 contains the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 contains the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 contains the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 contains the amino acid sequence shown in SEQ ID NO:

36. An isolated antigen-binding protein comprising an amino acid sequence selected from the group consisting of.

47. The isolated antigen-binding protein according to any one of claims 1 to 46, comprising VH and VL, wherein the VH and the VL are (1) The VH contains the amino acid sequence shown in amino acid sequence 8, and the VL contains the amino acid sequence shown in SEQ ID NO:

16. (2) The VH contains the amino acid sequence shown in amino acid sequence 19, and the VL contains the amino acid sequence shown in SEQ ID NO:

16. (3) The VH contains the amino acid sequence shown in amino acid sequence 22, and the VL contains the amino acid sequence shown in SEQ ID NO:

26. (4) The VH contains the amino acid sequence shown in amino acid sequence 33, and the VL contains the amino acid sequence shown in SEQ ID NO:

40. (5) The VH contains the amino acid sequence shown in amino acid sequence 45, and the VL contains the amino acid sequence shown in SEQ ID NO:

51. (6) The VH contains the amino acid sequence shown in amino acid sequence 55, and the VL contains the amino acid sequence shown in SEQ ID NO:

51. (7) The VH contains the amino acid sequence shown in amino acid sequence 58, and the VL contains the amino acid sequence shown in SEQ ID NO: 51, and (8) The VH contains the amino acid sequence shown in amino acid sequence 45, and the VL contains the amino acid sequence shown in SEQ ID NO:

61. An isolated antigen-binding protein comprising an amino acid sequence selected from the group consisting of.

48. The isolated antigen-binding protein according to any one of claims 1 to 47, comprising an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain and the antibody light chain are (1) The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain contains the amino acid sequence shown in SEQ ID NO:

87. (2) The antibody heavy chain contains the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain contains the amino acid sequence shown in SEQ ID NO:

87. (3) The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 89, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:

90. (4) The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 91, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:

92. (5) The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 46, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:

52. (6) The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 56, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:

52. (7) The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 59, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 52, and (8) The heavy chain of the antibody includes the amino acid sequence shown in SEQ ID NO: 46, and the light chain of the antibody includes the amino acid sequence shown in SEQ ID NO:

62. An isolated antigen-binding protein comprising an amino acid sequence selected from the group consisting of:

49. An isolated antigen-binding protein according to any one of claims 1 to 48, comprising an antibody or an antigen-binding fragment thereof.

50. The isolated antigen-binding protein according to 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 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, and is the isolated antigen-binding protein according to any one of claims 49 to 50.

52. An isolated antigen-binding protein according to any one of claims 1 to 51, having one or more of the following characteristics: 1) It can specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4). 2) It can bind to the LILRB4 protein with a KD value of 2×10−7 M or less. 3) It has the activity to block the binding of LILRB4 / APOE. 4) It can stimulate the activation of CD8+ T cells. 5) It can inhibit the migration of human monocytic leukemia cell THP-1. 6) It can 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 according to claim 52, wherein the LILRB4 is human LILRB4.

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

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

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

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

58. A vector comprising the nucleic acid molecule according to claim 57.

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

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

61. Anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin-cytarabine combined liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceple ne), interleukin-2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyltransferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant-derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P-glycoprotein inhibitors, one or more drugs selected from the group consisting of, further comprising the pharmaceutical composition according to claim 60.

62. A method for preparing an antigen-binding protein according to any one of claims 1 to 53, comprising culturing the cell according to claim 59 under conditions for expressing the isolated antigen-binding protein according to any one of claims 1 to 53.

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

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

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

66. The drug is used in combination with one or more drugs selected from the group consisting of anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolism inhibitors, daunorubicin - cytarabine combination liposome injection, Vyxeos, all - trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceple ne), interleukin - 2, interleukin 2 (Proleukin), gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyltransferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, enasidenib, Idhifa, IDO inhibitors, epacadostat, derivatives of platinum complexes, 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 surface markers of T cells, antibodies that bind to surface markers of cells or NK cells, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant - derived alkaloids, topoisomerase inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors and P - glycoprotein inhibitors, the use according to any one of claims 63 to 65.

67. The disease or condition related to LILRB4 signaling includes immune system diseases and / or malignant blood diseases, the use according to any one of claims 63 to 66.

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

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

70. The use according to any one of claims 67 to 69, wherein the malignant blood disease is one or more selected from the group consisting of myelodysplastic syndrome, myeloproliferative neoplasm, 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 for diagnosing a disease or condition associated with the expression of LILRB4 protein in a subject, comprising: contacting a sample derived from the subject with the isolated antigen-binding protein according to any one of claims 1 to 53; and determining the presence and / or content of a substance capable of specifically binding to the isolated antigen-binding protein in the sample.

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

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

74. A detection kit comprising the isolated antigen-binding protein according to any one of claims 1 to 53, wherein the detection kit is used to detect the presence and / or content of LILRB4 protein in the sample or subject.

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