Immunoconjugate and use thereof

Abstract

The present application relates to an immunoconjugate, comprising an antigen-binding protein targeting LILRB4, an optional linker moiety, and a drug molecule. The present application further provides a pharmaceutical composition comprising the immunoconjugate, a pharmaceutical combination comprising the immunoconjugate, a preparation method for the immunoconjugate, and use of the immunoconjugate in the preparation of a drug.

Description

An immunoconjugate and its use Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to an immunoconjugate comprising an antigen binding protein targeting LILRB4 and a drug molecule. Background Art

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

[0003] Based on the therapeutic potential of LILRB4, it is necessary to develop more diverse drug forms based on the LILRB4 target to fully utilize the characteristics of the target and improve the therapeutic effect. Summary of the Invention

[0004] The present application provides an immunoconjugate comprising an antigen binding protein targeting LILRB4 and a drug molecule. The immunoconjugate of the present invention can specifically kill target cells that highly express LILRB4, achieving a targeted therapeutic effect. In the present application, the antigen binding protein targeting LILRB4 has one or more of the following properties: 1) can specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) can be used to treat leukocytes with a specific expression of 2×10 -7 M or lower K D The invention discloses a novel novel anti-ILR-4 inhibitor that can bind to LILRB4 protein; 3) can block the activity of LILRB4 / APOE binding; 4) can stimulate the activation of CD8+ T cells; 5) can inhibit the migration of human monocytic leukemia cells THP-1; 6) can inhibit the growth and / or metastasis of tumor cells; and 7) can block the binding of LILRB4 to Fibronectin.

[0005] In certain embodiments, the immunoconjugate has the following general formula: Ab-(Q) n (Formula 1)

[0006] Wherein, Ab represents the antigen binding protein targeting LILRB4, Q represents the portion comprising the drug molecule connected to Ab, n is an integer from 1 to 10, and each Q may be the same or different.

[0007] In certain embodiments, the LILRB4 is human LILRB4.

[0008] In certain embodiments, the antigen binding protein targeting LILRB4 comprises at least one CDR in the antibody heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO:81, SEQ ID NO:33 and SEQ ID NO:22.

[0009] In certain embodiments, the antigen binding protein targeting LILRB4 comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69 (X1X2WMX3, wherein X1 is D or S; X2 is A or Y; X3 is D or H), the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:83 or SEQ ID NO:29.

[0010] In certain embodiments, the antigen binding protein targeting LILRB4 comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:28, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:20 and SEQ ID NO:29.

[0011] In certain embodiments, the antigen binding protein targeting LILRB4 comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from the group consisting of:

[0012] (1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3;

[0013] (2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 20; and

[0014] (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29.

[0015] In certain embodiments, the antigen binding protein targeting LILRB4 comprises a VH comprising the amino acid sequence shown in any one of SEQ ID NO:81, SEQ ID NO:33, and SEQ ID NO:22.

[0016] In certain embodiments, the antigen binding protein targeting LILRB4 comprises a VH comprising 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.

[0017] In certain embodiments, the antigen binding protein targeting LILRB4 comprises at least one CDR in the antibody light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40.

[0018] In certain embodiments, the antigen binding protein targeting LILRB4 comprises LCDR1, LCDR2 and LCDR3, wherein the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:70 or SEQ ID NO:34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:71, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:72 (QX1X2X3X4X5PX6T, X1 is H or Q; X2 is G or S; X3 is D, N or W; X4 is E or T; X5 is I or L; X6 is P or R).

[0019] In certain embodiments, the antigen binding protein targeting LILRB4 comprises LCDR1, LCDR2 and LCDR3, 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, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:10 or SEQ ID NO:35, and 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.

[0020] In certain embodiments, the antigen binding protein targeting LILRB4 comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise an amino acid sequence selected from the group consisting of:

[0021] (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;

[0022] (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

[0023] (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.

[0024] In certain embodiments, the antigen binding protein targeting LILRB4 comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40.

[0025] In certain embodiments, the antigen binding protein targeting LILRB4 comprises a VL comprising 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.

[0026] In certain embodiments, the antigen binding protein targeting LILRB4 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from any one of the following groups:

[0027] (1) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 9, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 11;

[0028] (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

[0029] (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 36.

[0030] In certain embodiments, the antigen binding protein targeting LILRB4 comprises VH and VL, wherein the VH and VL comprise an amino acid sequence selected from any one of the following groups:

[0031] (1) the VH comprises the amino acid sequence shown in SEQ ID NO: 8, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16;

[0032] (2) the VH comprises the amino acid sequence shown in SEQ ID NO: 19, and the VL comprises the amino acid sequence shown in SEQ ID NO: 16;

[0033] (3) the VH comprises the amino acid sequence shown in SEQ ID NO: 22, and the VL comprises the amino acid sequence shown in SEQ ID NO: 26;

[0034] (4) the VH comprises the amino acid sequence shown in SEQ ID NO: 33, and the VL comprises the amino acid sequence shown in SEQ ID NO: 40;

[0035] (5) the VH comprises the amino acid sequence shown in SEQ ID NO:45, and the VL comprises the amino acid sequence shown in SEQ ID NO:51;

[0036] (6) the VH comprises the amino acid sequence shown in SEQ ID NO: 55, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51;

[0037] (7) the VH comprises the amino acid sequence shown in SEQ ID NO: 58, and the VL comprises the amino acid sequence shown in SEQ ID NO: 51; and

[0038] (8) The VH comprises the amino acid sequence shown in SEQ ID NO:45, and the VL comprises the amino acid sequence shown in SEQ ID NO:61.

[0039] In certain embodiments, the antigen binding protein targeting LILRB4 comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from an IgG constant region.

[0040] In certain embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:63.

[0041] In certain embodiments, the antigen binding protein targeting LILRB4 comprises an antibody light chain constant region, and the antibody light chain constant region is derived from a human Igκ constant region.

[0042] In certain embodiments, the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:64.

[0043] In certain embodiments, the antigen binding protein targeting LILRB4 comprises an antibody heavy chain and an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise an amino acid sequence selected from the group consisting of:

[0044] (1) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87;

[0045] (2) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87;

[0046] (3) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 90;

[0047] (4) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 92;

[0048] (5) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52;

[0049] (6) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52;

[0050] (7) the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 59, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; and

[0051] (8) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:62.

[0052] In certain embodiments, the drug molecules in the immunoconjugate include macromolecular drugs and / or small molecule drugs.

[0053] In certain embodiments, the macromolecular drugs include other antibody drugs, nucleic acid drugs, molecular glues and protacs. The other antibody drugs include antibodies that bind to tumor treatment-related antigens, and the nucleic acid drugs include sgRNA, mRNA, siRNA, shRNA and antisense RNA.

[0054] In certain embodiments, the drug molecule in the immunoconjugate is selected from a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent.

[0055] In certain embodiments, the radioactive element comprises 177 Lu, 68 Ga, 89 Zr, 225 Ac, 111 In, 90 Y.213 Bi, 131 I. 125 I. 99 mTc, 3 H. 14 C. 15 N. 35 S. 18 F and 64 One or more of Cu.

[0056] In certain embodiments, the drug molecule is selected from the group consisting of a V-ATPase inhibitor, a Bcl2 inhibitor, an MCL1 inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a maytansinoid, a MetAP (methionine aminopeptidase), a nuclear export inhibitor of protein CRM1, a DPPIV inhibitor, a proteasome inhibitor, an inhibitor of phosphotransferase in mitochondria, a protein synthesis inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, a DHFR inhibitor, a nucleoside analog, an HDAC inhibitor, an anthracycline, a NAMPT inhibitor, SN-38 or a derivative thereof, an etoposide phosphate, a nitrogen mustard, a proteosome inhibitor, a cytokine, a Tubulysin B analog, and a Toll-like receptor agonist.

[0057] In certain embodiments, the drug molecule is selected from antibiotics, microtubule-disrupting drugs, DNA-damaging drugs, apoptosis inducers, tylanstatin and its analogs, amatoxin, nicotinamide phosphoribosyltransferase, and carmacin.

[0058] In certain embodiments, the microtubule-disrupting drug is selected from: calendula, maytansine derivatives, tubulysin, cryptocolistin, and anti-mitotic EG5 inhibitors, wherein, preferably, the calendula comprises MMAE and / or MMAF, and the maytansine derivatives comprise DM1, DM2, DM3 and / or DM4.

[0059] In certain embodiments, the DNA damaging drug is selected from the group consisting of: pyrrolebenzazepines, indolechlorobenzazepines, dukamycin, camptothecin and its derivatives, and calicheamicin, wherein, preferably, the pyrrolebenzazepines comprise pyrrolo[2,1-c][1,4]benzodiazepine (PBD), the camptothecin and its derivatives comprise SN-38 and / or DXd / DX8951, and the apoptosis inhibitor comprises a Bcl-xL inhibitor.

[0060] In certain embodiments, the immunoconjugate further comprises a linker, which is capable of linking the antigen binding protein targeting LILRB4 and the drug molecule.

[0061] In certain embodiments, the linker is selected from the group consisting of a hydrophilic linker, a hydrophobic linker, a charged linker, an uncharged linker, and a dicarboxylic acid-based linker.

[0062] In certain embodiments, the linker is selected from the group consisting of a non-cleavable linker and a cleavable linker.

[0063] In certain embodiments, the cleavable linker comprises a chemically active linker, an acid-cleavable linker, a reducing condition linker, and / or an enzyme-cleavable linker.

[0064] In certain embodiments, the linker is linked to the LILRB4 antigen binding protein via a sulfhydryl group, an azide group, or an amide group on the LILRB4 antigen binding protein.

[0065] In certain embodiments, the linker is selected from one or more of the following groups: Acetyl Butyrate, MC-Val-Cit-PABC, SMCC, maleimide, CL2A, MC, MC-VC-PAB, PY-VC-PAB, PEG8-VA-PABC, MC-GGFG, MC-Val-Ala-PAB, MC-Val-Lys(Ac)-PAB, MC-Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, a disulfide bond and an acidolytic hydrazone bond.

[0066] In certain embodiments, the immunoconjugate comprises the structure shown in Formula 2: Ab-(L1) a -(L2) b -D (Formula 2),

[0067] Wherein Ab represents the antigen binding protein targeting LILRB4;

[0068] L1 represents the linker connected to Ab, L2 represents the linker connected to D,

[0069] a, b are each independently selected from 0-10,

[0070] D represents the drug molecule,

[0071] Wherein, preferably, L1 is a group or molecule that can react with a thiol group, an azide group or an amide group;

[0072] L2 comprises a combination of one or more of C1-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, C6-C24 aryl, C6-C15 heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, polyethylene glycol, O, S, alkylamino, carbonyl, carboxyl, sulfonic acid, Val-Cit-PABC, VA-PABC, GGFG, Val-Lys(Ac)-PAB, Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB and Ala-PAB.

[0073] In certain embodiments, Q in the general formula 1 of the immunoconjugate is selected from one or more combinations of the following groups:

[0074] Wherein, * is the connection site between Q and the antibody molecule.

[0075] In certain embodiments, Q in Formula 1 is selected from the group consisting of -MC-MK4827, -MC-MK4827&MMAF, -MC-MMAF, -MC-MMAF, -MC-MMAF&SN38, -MC-SN38, -MC-2MMAF, -MC-MC-VC-PAB-MMAE, and -PY-VC-PAB-MMAE.

[0076] In certain embodiments, the immunoconjugate has the following formula:

[0077] wherein n is selected from 1-8.

[0078] In another aspect, the present application provides a pharmaceutical composition comprising the immunoconjugate, and optionally a pharmaceutically acceptable carrier.

[0079] In another aspect, the present application provides a drug combination and an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor comprises PD-1, PD-L1, GITR, and / or CTLA-4. Preferably, the immune checkpoint inhibitor comprises one or more selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, and a GITR antibody.

[0080] On the other hand, the present application provides a method for preparing the immunoconjugate, which comprises linking the antigen binding protein targeting LILRB4 and the drug molecule under suitable conditions.

[0081] In another aspect, the present application provides a method for preventing, diagnosing and / or treating a disease and / or condition, comprising administering the immunoconjugate, the pharmaceutical composition or the pharmaceutical combination to a subject in need thereof.

[0082] In another aspect, the present application provides use of the immunoconjugate, the pharmaceutical composition or the pharmaceutical combination in the preparation of a medicament for preventing, diagnosing and / or treating a disease and / or condition.

[0083] In another aspect, the present application provides the immunoconjugate, the pharmaceutical composition or the pharmaceutical combination for use in preventing, diagnosing and / or treating diseases and / or disorders.

[0084] In certain embodiments, the disease and / or condition comprises a disease or condition associated with LILRB4 signaling and / or LILRB4 expression. In certain embodiments, the disease and / or condition comprises a tumor, and in certain embodiments, the disease and / or condition is selected from a hematological tumor and / or a solid tumor.

[0085] In certain embodiments, the solid tumor is selected from one or more of the following groups: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, head and neck cancer, gastric cancer, pancreatic cancer, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; wherein the hematological tumor is selected from multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, myelodysplastic syndrome and myeloproliferative neoplasms.

[0086] The leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia (AML), myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia, acute promyelocytic leukemia (APL) or M3AML, acute myelomonocytic leukemia or M4AML, acute monocytic leukemia or M5AML, wherein preferably, the acute myeloid leukemia is M4 or M5 type acute myeloid leukemia; preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia. The lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, T cell lymphoma, and Waldenstrom's macroglobulinemia. The sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0087] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0089] Figure 1 shows the binding of LILRB4 hybridoma antibodies to LILRB4-overexpressing cells.

[0090] FIG2 shows the binding of LILRB4 hybridoma antibodies to LILRB family proteins.

[0091] FIG3A shows the internalization activity of LILRB4 hybridoma antibodies on LILRB4-positive cells.

[0092] FIG3B shows the inhibitory effect of LILRB4 hybridoma antibody on THP-1 cell migration.

[0093] FIG4 shows the binding of the 3A2-18A humanized antibody to the human LILRB4 recombinant protein.

[0094] FIG5 shows the binding of the 3A2-18A humanized antibody to LILRB family proteins.

[0095] FIG6 shows the internalization of the 3A2-18A humanized antibody on LILRB4-positive cells.

[0096] FIG7 shows the inhibitory effect of LILRB4 humanized antibody on THP-1 cell migration.

[0097] FIG8 shows the activity detection of LILRB4 humanized antibody blocking LILRB4 / APOE binding.

[0098] FIG9 shows the detection of the effect of LILRB4 humanized antibody on activating CD8+ T cells.

[0099] FIG10A shows the MicroPET / CT scanning data results of the test antibodies 89Zr-SG2919 and 89Zr-HEL-G1.

[0100] Figure 10B shows the SUVmean data of 89Zr-SG2919 and 89Zr-HEL-G1 in different tissues of LILRB4 humanized MC38-LILRB4 tumor-bearing mice and non-tumor-bearing mice at 48 h.

[0101] FIG11 shows the killing effect of SG2919-ADC conjugated with different toxins on THP-1 cells.

[0102] FIG12A shows the flow cytometry detection results of LILRB4 expression abundance on the surface of different tumor cells.

[0103] FIG12B shows the killing of THP-1 cells by SG2918-ADC.

[0104] Figure 12C shows the killing of MOLM-13 cells by SG2918-ADC.

[0105] Figure 12D shows the killing of MOLM-16 cells by SG2918-ADC.

[0106] FIG13A shows the flow cytometry results of the expression abundance of LILRB4 on the surface of different multiple myeloma MM cells.

[0107] FIG13B shows the killing of U-266 cells by SG2918-PYAE.

[0108] FIG13C shows the killing of MM.1S cells by SG2918-PYAE.

[0109] FIG13D shows the killing effect of SG2918-PYAE on ALMC-1 cells.

[0110] Figure 14A shows the in vivo efficacy and survival data of SG2918-ADC in the THP-1 system tumor model.

[0111] Figure 14B shows the in vivo efficacy and fluorescence intensity data of SG2918-ADC in the THP-1 system tumor model.

[0112] FIG15 shows the in vivo efficacy of SG2918-PYAE in the OPM-2 multiple myeloma model.

[0113] FIG16 shows the efficacy curve of SG2918-PYAE in the LLC1 model.

[0114] FIG17 shows the trend of tumor volume changes after drug administration. DETAILED DESCRIPTION

[0115] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0116] Definition of terms

[0117] In this application, the term "LILRB4 protein" generally refers to leukocyte immunoglobulin-like receptor B4 (LILRB4). In this application, the term covers the entire LILRB4 and its functionally active fragments, variants, homologues, analogs and derivatives.

[0118] In this application, the term "antigen binding protein" generally refers to a protein with antigen binding ability. The "antigen binding protein" may comprise a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes the binding of the antigen binding portion to the antigen. The antigen binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold having a transplanted CDR or CDR derivative. Such scaffolds include, but are not limited to, antibody-derived scaffolds that contain mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein and fully synthetic scaffolds that contain, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). In addition, peptide antibody mimics ("PAMs") and scaffolds based on antibody mimics using a fibronectin component can be used as scaffolds.

[0119] In this application, the term "antibody" generally refers to an immunoglobulin or its fragment or derivatives thereof, encompassing any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybridized, mutated and transplanted antibodies. Unless otherwise modified by the term "complete", as in "complete antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other antibody fragments that retain antigen binding function. Typically, such fragments should include an antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies are composed of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites. IgA antibodies, on the other hand, consist of two to five basic four-chain units that can combine with the J chain to form multivalent combinations. For IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are interconnected by one or more disulfide bonds that depend on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. The VL corresponds to the VH, and the CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from any vertebrate species can be divided into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Depending on the amino acid sequence of its heavy chain (CH) constant domain, immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively.The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl, and IgKl.

[0120] In this application, the term "CDR" generally refers to the region of the antibody variable domain, whose sequence is highly variable and / or forms a structural definition loop. Typically, an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3). In natural antibodies, HCDR3 and LCDR3 show most of the diversity of the six CDRs, and HCDR3 in particular is considered to play a unique role in conferring fine specificity on antibodies. See, for example, Xu et al, Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting only of heavy chains function normally and stably in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

[0121] In the present application, the term "FR" generally refers to the more highly conserved part of the antibody variable domain, which is referred to as the framework region. Typically, the variable domains of native heavy and light chains each include four FR regions, i.e., four (H-FR1, H-FR2, H-FR3, and H-FR4) in VH, and four (L-FR1, L-FR2, L-FR3, and L-FR4) in VL. For example, the VL of the antigen-binding proteins of the separation described herein can include framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the antigen-binding proteins of the separation described herein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0122] In the art, antibody CDRs can be divided by a variety of methods, such as 1) the Kabat definition rule based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991), 2) the Chothia definition rule based on the position of the structural loop region (Al-Lazikani et al., J Mol Biol 273:927-48, 1997), 3) the AbM definition rule using Oxford Molecular's AbM antibody model software that balances the above two rules, and 4) the Contact definition rule based on analysis of the resulting complex crystal structure.

[0123] In this application, the term "antigen-binding fragment" generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The antigen-binding function of an antibody can also be achieved by a heavy chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2, or a light chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab')2. (1) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH domains; (2) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (3) Fd fragments consisting of the VH and CH domains; (4) Fv fragments consisting of the VL and VH domains of a single antibody arm; (5) dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341: 544-546); (6) isolated complementarity determining regions (CDRs) and (7) combinations of two or more isolated CDRs, optionally linked by a linker. Furthermore, monovalent single-chain molecules Fv (scFv) formed by pairing VL and VH can also be included (see Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883). The "antigen binding portion" may also include a fusion protein comprising an immunoglobulin. For example, the fusion protein may comprise a binding domain selected from the group consisting of: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and / or (3) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region.

[0124] In this application, the term "K D " is used interchangeably with "KD" and generally refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, expressed in M ​​(mol / L). KD can be calculated using the concentrations of substance AB and the dissociated substances A and B: KD = c(A) * c(B) / c(AB). As can be seen from this formula, a larger KD value indicates greater dissociation, meaning a weaker affinity between substances A and B; conversely, a smaller KD value indicates less dissociation, meaning a stronger affinity between substances A and B.

[0125] In this application, the term "tumor" generally refers to a physiological condition typically characterized by dysregulated cell proliferation or survival. The tumor can include all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and all stages and grades of cancer, including pre-metastatic and post-metastatic cancers. The tumor can also include one or more tumor cells.

[0126] In this application, the term "monoclonal antibody" generally refers to a group of substantially homologous antibodies, i.e., the individual antibodies contained in the group are identical except for possible naturally occurring mutations present in trace amounts. Monoclonal antibodies can be highly specific, being directed against a single antigenic site. The monoclonal antibodies can be prepared by hybridoma technology or by using recombinant DNA methods to produce monoclonal antibodies in bacteria, eukaryotic animals or plant cells. Monoclonal antibodies can also be obtained from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., Mol. Biol., 222: 581-597 (1991).

[0127] In this application, the term "single-chain antibody" (scFv) generally refers to a molecule comprising an antibody heavy chain variable region and a light chain variable region. For example, the scFv can be formed by connecting the antibody heavy chain variable region and the light chain variable region through a linker (e.g., a connecting peptide).

[0128] In this application, the term "chimeric antibody" generally refers to an antibody in which a portion of the heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species or belongs to a certain category, while the other parts of the chain are homologous to the corresponding sequence in another species. For example, the variable regions of the light and heavy chains can all be derived from the variable regions of antibodies from one animal species (such as mice, rats, etc.), while the constant portion is homologous to the antibody sequence from another species (such as humans). For example, to obtain chimeric antibodies, non-human B cells or hybridoma cells can be used to produce variable regions, and the constant regions combined therewith are from humans. Since the constant regions of chimeric antibodies can be derived from humans, the possibility of chimeric antibodies triggering an immune response upon injection will be lower than that of antibodies using non-human constant regions.

[0129] In this application, the term "humanized antibody" generally refers to an antibody that contains fewer sequences from non-human immunoglobulins, thereby reducing the immunogenicity of xenogeneic antibodies when introduced into humans. For example, CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof can be used; including "reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, et al., 1988 Nature 332:323-337; Tempest, et al., Bio / Technol 1991 9:266-271), "hyperchimerization", (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol The binding domain of non-human origin is humanized using techniques such as veneering (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies." In: Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994: 291-312), resurfacing (U.S. Pat. No. 5,639,641). If other regions, such as hinge regions and constant region domains, are also derived from non-human sources, these regions can also be humanized.

[0130] In this application, the term "fully human antibody" generally refers to a fully human antibody, i.e., an antibody whose constant and variable regions are both derived from humans. Such fully human antibodies can be achieved through phage antibody library technology, transgenic mouse production of humanized antibodies, ribosome display technology, EBV-transformed B cell cloning technology, single B cell cloning, and other technologies.

[0131] In this application, the term "multispecific antibody" generally refers to an antibody molecule that can simultaneously recognize two or more antigens or epitopes. The multispecific antibody can be obtained in a eukaryotic expression system or a prokaryotic expression system by chemical coupling, hybridization-hybridoma method, genetic engineering antibody preparation method, etc.

[0132] In the present application, the term "immunoconjugate" generally refers to a conjugate formed by connecting the other agent / drug molecule (e.g., a chemotherapeutic agent, a radioactive element, a cell growth inhibitor, and a cytotoxic agent) to the antibody or its antigen-binding fragment. The connection can be through a covalent bond, or a non-covalent interaction, and can include chelation. The conjugate can deliver the other agent to the target cell (e.g., a tumor cell) through the specific binding of the antibody or its antigen-binding fragment to the antigen on the target cell. The immunoconjugate is then internalized and eventually enters the interior of the target cell (e.g., into a vesicle such as a lysosome), at which point the connecting molecule in the immunoconjugate can be cleaved, releasing the other agent to exert its cytotoxic effect. In addition, the antigen can also be secreted by the target cell and located in the gap outside the target cell. A variety of connecting molecules known in the art can be used to form immunoconjugates. In the present application, the term "linker" generally refers to a functional molecule that connects or links two molecules. For example, the connecting molecule can connect one molecule to another (e.g., one molecule is a protein molecule and the other molecule is also a protein molecule, or can be a small molecule drug). The linker molecule can be used in the construction of the immunoconjugate. In the immunoconjugate, the linker molecule can have two functional characteristics: 1. It has circulatory system stability, and the immunoconjugate cannot be cleaved in the circulatory system to release the other agents before reaching the target cell, thereby avoiding toxic effects; 2. After entering the target cell, the linker molecule needs to be quickly and effectively broken so that the other agents can be effectively released to exert their due pharmacological activity. The linker molecule can be composed of polar or non-polar amino acids. The linker molecule can also be a carbon chain containing heteroatoms (such as nitrogen atoms, sulfur atoms, etc.). The length of the linker molecule can be 2 to 100 atoms, for example, between 2 and 50 atoms, or 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 atoms; for example, the length of the linker can be 20 to 26 (20, 21, 22, 23, 24, 25 or 26) atoms. The linker molecule may include a substituent selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocycle, aromatic heterocycle, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, sulfhydryl, and urea. In addition, the linker molecule may be selected from the group consisting of pH-sensitive linkers, protease-cleavable linkers, nuclease-sensitive linkers, lipase-sensitive linkers, glycosidase-sensitive linkers, anoxic linkers, photocleavable linkers, heat-labile linkers, ultrasound-sensitive linkers, and peptide linkers.Exemplary linker molecules can include, but are not limited to, 1,4-bis(maleimido)butane, (1,4-bismaleimido-2,3-dihydroxybutane), bis(maleimido)hexane, bis(maleimido)ethane, 1,4-bis-[3′-(2′-pyridyldithio)propionamido]butane, 1,6-hexane-bis-vinylsulfone, bismaleimidoethane dithioate, 1,8-bismaleimido-diethylene glycol, and 1,11-bismaleimido-triethylene glycol. Common peptide linkers are well known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

[0133] In this application, the term "covalent" generally refers to a covalent bond, that is, two or more atoms share a pair of electrons, reaching a state of electron saturation to form a relatively stable chemical structure. The formation of a covalent bond is that electrons with opposite spin directions between two adjacent atoms pair with each other. At this time, the atomic orbitals overlap with each other, and the electron cloud density between the two nuclei increases relatively, thereby increasing the attraction to the two nuclei. Covalent bonds can have saturation and directionality. Covalent bonds can be divided into non-polar covalent bonds, polar covalent bonds and coordinate bonds. Compounds containing only covalent bonds can be called covalent compounds.

[0134] In this application, the term "drug molecule" generally refers to an active agent molecule attached to an antigen binding protein. The drug molecule can be various types of drug molecules. For example, the drug molecule can be a macromolecular drug or a small molecule drug. The macromolecular drug can be other antibody drugs, nucleic acid drugs, molecular glues and protacs. The other antibody drugs can be antibodies other than LILRB4 antibodies that bind to tumor therapy-related antigens, for example, PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, 4-1BB antibodies, TIGIT antibodies, TIM3 antibodies, CD47 antibodies, antibodies that bind to T lymphocyte surface markers, antibodies that bind to tumor stromal cells, antibodies that bind to myeloid-derived suppressor cells or NK cell surface markers. The nucleic acid drug antigens include but are not limited to sgRNA, mRNA, siRNA, shRNA and antisense RNA. PROTAC and molecular glue are two main modes of targeted protein degradation technology based on the ubiquitin proteasome system. Among them, PROTAC recruits E3 ubiquitin ligases to induce the target protein to approach the E3 ubiquitin ligase, resulting in ubiquitination and degradation of the target protein. Molecular glues modify the surface of ubiquitin ligases, promoting or inducing protein-protein interactions (PPIs) between E3 ubiquitin ligases and target proteins, leading to ubiquitination and subsequent degradation of target proteins. Molecular glues include, but are not limited to, immunomodulators (IMiDs; thalidomide, lenalidomide, pomalidomide), CFT-7455, BAY-2666605, DKY-709, ICP-490, CC-9009, CC-92480, CC-99282, BTX-1188, and MRT-2359.

[0135] The small molecule drug can be a chemotherapeutic agent, a radioactive element, a cell growth inhibitor, and / or a cytotoxic agent. In this application, when referring to a specific type of drug molecule, its meaning includes not only the drug molecule itself, but also other forms such as its analogs, derivatives, variants, etc. In some cases, the drug molecule can have therapeutic activity. In some cases, the drug molecule can have cytotoxicity. In this application, the term "chemotherapeutic agent" generally refers to a chemotherapy agent that can inhibit tumor and / or tumor cell proliferation. The chemotherapeutic agent can be selected from the following groups: mitotic inhibitors, kinase inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, histone deacetylase inhibitors, antisurvival agents, biological response regulators, anti-hormones such as anti-androgens and anti-angiogenic agents. For example, the chemotherapeutic agent can be selected from the group consisting of capecitabine, daunorubicin, daunorubicin, dactinomycin D, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytarabine, bischloroethyl nitrosourea, busulfan, mitomycin C, dactinomycin D, plicamycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentomethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclobutanone ... Hexylnitrosourea, nitrogen mustard, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, and / or diethylstilbestrol (DES).

[0136] In this application, the term "radioactive element" generally refers to an element used in radiotherapy that can inhibit the proliferation of tumors and / or tumor cells. The radioactive element can be selected from the following group: 177 Lu, 68 Ga, 89 Zr, 225 Ac, 90 Y. 213 Bi, 99 mTc, 18 F. 64 Cu, 3 H. 14 C. 15 N. 35 S. 99 Tc, 111 In, 125 I and / or 131 I.

[0137] In this application, the term "cytostatic" generally refers to an agent that inhibits tumors by inhibiting growth factors that promote the growth and replication of tumor cells. Growth factors activate intracellular signaling pathways after binding to receptors on the cell surface. Complex pathways may cause cells to grow out of control, resulting in excessive cell division and development into tumors. The cytostatic can inhibit the effects of these growth factors. The cytostatic can be selected from the following group: angiogenesis inhibitors, deacetylase (HDAC) inhibitors, Hedgehog signaling pathway blockers, mTOR inhibitors, p53 / mdm2 inhibitors, PARP inhibitors, proteasome inhibitors and / or tyrosine kinase inhibitors.

[0138] In the present application, the term "cytotoxic agent" generally refers to an agent that inhibits the proliferation of tumors and / or tumor cells by producing toxins to the cells on which it acts. The cytotoxic agent can be selected from the following groups: alkylating agents, such as busulfan, hexamethylmelamine, thiotepa, cyclophosphamide, mechlorethamine, uracil, melphalan, chlorambucil, carmustine, streptozotocin, dacarbazine, temozolomide, ifosfamide, etc.; antitumor agents, such as mitomycin C, etc.; antimetabolites, such as methotrexate, azathioprine, mercaptopurine, fludarabine, 5-fluorouracil, etc.; platinum-containing anticancer agents, such as cisplatin, carboplatin, etc.; anthracyclines, such as daunorubicin, doxorubicin, epirubicin, etc. Rubicin, idarubicin, mitoxantrone, etc.; plant alkaloids and terpenoids, such as vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, docetaxel, etc.; topoisomerase inhibitors, such as irinotecan, amsacrine, topotecan, etoposide, teniposide, etc.; antibodies, such as rituximab, trastuzumab, bevacizumab, erlotinib, dactinomycin, etc.; finasteride; aromatase inhibitors; tamoxifen; goserelin; paclitaxel and / or imatinib mesylate. The cytotoxic agent can be administered orally, by injection, etc.

[0139] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, preferably a human patient. For example, the pharmaceutical composition described herein may comprise the immunoconjugate described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical composition of the present invention includes, but is not limited to, liquid, frozen and lyophilized compositions.

[0140] As used herein, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration and are generally safe, non-toxic, and neither biologically nor otherwise undesirable.

[0141] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0142] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0143] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "to be", "to be composed of..."

[0144] Detailed Description of the Invention

[0145] Antigen binding proteins targeting LILRB4

[0146] The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or antigenic epitope. Antibody CDRs can be determined using a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art, and for details, see, for example, http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region based on the sequence and structure of the antibody. Using different coding systems, there may be differences in the CDR region. In this application, the CDR covers CDR sequences obtained by any CDR division method; it also covers variants thereof, wherein the variant includes the amino acid sequence of the CDR being substituted, deleted, and / or having one or more amino acids added. For example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and / or insertions; homologs thereof are also encompassed, and the homologs can be amino acid sequences having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology to the amino acid sequence of the CDR. For example, the CDRs of the isolated antigen-binding proteins described in the sequence listing of the present application can be determined using Kabat.

[0147] On the one hand, the present application provides an isolated antigen-binding protein, which may comprise at least one CDR in the VH as shown in any one of SEQ ID NO:81, SEQ ID NO:33 and SEQ ID NO:22; and / or comprise at least one CDR in the VL as shown in SEQ ID NO:82 or SEQ ID NO:40.

[0148] X1VQLX2QX3GAEX4X5KPGASVKX6SCKASGYTFTSYWMHWVX7QX8PGQGLEWX9GEINPSNGRTNYNEKFKSX 10 X 11 TX 12 TX 13 DX 14 SX 15 STX 16 YX 17 X 18 LX 19 SLX 20 SEDX 21 AVYYCARDRYDEGNAMDYWGQGTX22 VTVSS (SEQ ID NO: 81). wherein X1 can be E or Q; X2 can be Q or V; X3 can be P or S; X4 can be L or V; X5 can be K or V; X6 can be L or V; X7 can be K or R; X8 can be A or R; X9 can be I or M; X 10 Can be K or R; X 11 Can be A or V; X 12 Can be L or M; X 13 Can be R or V; X 14 Can be K or T; X 15 Can be S or T; X 16 Can be A or V; X 17 Can be M or V; X 18 Can be E or Q; X 19 Can be N or S; X 20 Can be R or T; X 21 Can be S or T; X 22 Can be S or T.

[0149] DIX1MTX2X3X4SSLSASX5GDRVTIX6CRASQDIX7NYLNWYQQKPX8X9X 10 X 11 KLLIYYTSRLHSGVPSRFSGSGSGTX 12 YX 13 X 14 TISX 15 LX 16 X 17 X 18 DX 19 ATYX 20 CQQGX 21 TLPX 22 TFGX 23 GTX 24 LEIK (SEQ ID NO: 82). wherein X1 may be Q or V; X2 may be H or Q; X3 may be S or T; X4 may be P or T; X5 may be L or V; X6 may be S or T; X7 may be S or T; X8 may be D or G; X9 may be G or K; X 10 Can be A or T; X 11 Can be P or V; X 12 Can be D or E; X 13 Can be S or T; X 14 Can be F or L; X 15 Can be N or S; X 16 Can be E or Q; X 17 Can be P or Q; X18 Can be D or E; X 19 Can be F or I; X 20 Can be F or Y; X 21 Can be D or N; X 22 Can be P or R; X 23 Can be G or Q; X 24 Can be K or R.

[0150] In the present application, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:55 and SEQ ID NO:58.

[0151] In the present application, the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 40, SEQ ID NO: 51 and SEQ ID NO: 61.

[0152] In the present application, the HCDR of the isolated antigen-binding protein can be divided in any form. As long as the VH and / or VL are identical to the amino acid sequences of VH and / or VL shown in any of the items in the present application, the HCDR obtained by any division can fall within the scope of protection of the present application.

[0153] For example, the methods of dividing VH (SEQ ID NO: 8) and / or VL (SEQ ID NO: 16) using different CDR labeling methods can be summarized in the following table.

[0154] For example, the methods of dividing VH (SEQ ID NO: 19) and / or VL (SEQ ID NO: 16) using different CDR labeling methods can be summarized in the following table.

[0155] For example, the methods of dividing VH (SEQ ID NO: 22) and / or VL (SEQ ID NO: 26) using different CDR labeling methods can be summarized in the following table.

[0156] For example, the methods of dividing VH (SEQ ID NO: 33) and / or VL (SEQ ID NO: 40) using different CDR labeling methods can be summarized in the following table.

[0157] Among them, Laa-Lbb may refer to the amino acid sequence starting from the N-terminus of the antibody light chain variable region and from positions aa to bb; Haa-Hbb may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain variable region and from positions aa to bb. For example, L24-L34 may refer to the amino acid sequence starting from the N-terminus of the antibody light chain and from positions 24 to 34; H26-H35 may refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and from positions 26 to 35.

[0158] As one of the cases, in the sequence listing of the present application, the CDR sequence and framework region (FR) sequence of the isolated antigen-binding protein are determined by the Kabat numbering method.

[0159] In the present application, the VH of the isolated antigen-binding protein may comprise HCDR1, HCDR2 and HCDR3.

[0160] In the present application, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 69.

[0161] X1X2WMX3 (SEQ ID NO: 69), wherein X1 can be D or S; X2 can be A or Y; and X3 can be D or H.

[0162] For example, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27.

[0163] In the present application, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28.

[0164] In the present application, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 83 or SEQ ID NO: 29.

[0165] [-G][DI][RY]Y[DG][EY][DG][NY]AMDY (SEQ ID NO: 83). wherein X1 may be absent or G; X2 may be D or I; X3 may be R or Y; X4 may be D or G; X5 may be E or Y; X6 may be D or G; and X7 may be N or Y.

[0166] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 29.

[0167] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.

[0168] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 20.

[0169] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0170] In the present application, the VL of the isolated antigen binding protein may comprise LCDR1, LCDR2 and LCDR3.

[0171] In the present application, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 70 or SEQ ID NO: 34.

[0172] RASQDIX1NYLN (SEQ ID NO: 70), wherein X1 can be S or T.

[0173] For example, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:9, SEQ ID NO:23 and SEQ ID NO:34.

[0174] In the present application, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 71.

[0175] YX1SX2LX3S (SEQ ID NO: 71), wherein X1 can be A or T, X2 can be N or R, and X3 can be H or Q.

[0176] For example, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35.

[0177] In the present application, the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 72.

[0178] QX1X2X3X4X5PX6T (SEQ ID NO: 72). For example, X1 can be H or Q; X2 can be G or S; X3 can be D, N or W; X4 can be E or T; X5 can be I or L; and X6 can be P or R.

[0179] For example, the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36.

[0180] In the present application, the LCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:70 or SEQ ID NO:34, LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:71, and LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:72 (QX1X2X3X4X5PX6T, X1 is H or Q; X2 is G or S; X3 is D, N or W; X4 is E or T; X5 is I or L; X6 is P or R).

[0181] In the present application, the isolated antigen binding protein may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.

[0182] In the present application, the HCDR1 of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 28, the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 20 and SEQ ID NO: 29, the LCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 23 and SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 11, SEQ ID NO: 24 and SEQ ID NO: 36.

[0183] In the present application, the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the isolated antigen-binding protein may comprise an amino acid sequence selected from any one of the following groups:

[0184] (1) The HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 3, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 9, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 11;

[0185] (2) the HCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 2, the HCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 20, the LCDR1 may comprise the amino acid sequence set forth in SEQ ID NO: 23, the LCDR2 may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the LCDR3 may comprise the amino acid sequence set forth in SEQ ID NO: 24; and

[0186] (3) The HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 34, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 35, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0187] In the present application, the VH of the isolated antigen-binding protein may comprise framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0188] In the present application, the C-terminus of the H-FR1 may be directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:73.

[0189] X1VX2LX3X4X5GX6X7X8X9X 10 PGX 11 SX 12 KX 13 SCX 14 ASGX 15 TFX 16 (SEQ ID NO: 73). X1 can be E or Q; X2 can be K or Q; X3 can be E, V or Q; X4 can be E or Q; X5 can be P or S; X6 can be A or G; X7 can be E or G; X8 can be L or V; X9 can be K or V; X 10 Can be K or Q; X 11 Can be A or G; X 12 Can be M or V; X 13 Can be L or V; X 14 Can be A or K; X 15 Can be F or Y; X 16 Can be S or T.

[0190] For example, the H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 4, SEQ ID NO: 17, SEQ ID NO: 30 and SEQ ID NO: 41.

[0191] In the present application, the H-FR2 may be located between the HCDR1 and the HCDR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 74.

[0192] WVX1QX2PX3X4GLEWX5X6 (SEQ ID NO: 74), wherein X1 can be K or R; X2 can be A, R or S; X3 can be E or G; X4 can be K or Q; X5 can be I, M or V; and X6 can be A or G.

[0193] For example, the H-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO: 42 and SEQ ID NO: 53.

[0194] In the present application, the H-FR3 may be located between the HCDR2 and the HCDR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 75.

[0195] X1X2TX3X4X5DX6SX7SX8X9YX 10 X 11 X 12 X 13 SLX 14 X 15 EDX 16 X 17 X 18 YYCX 19 R (SEQ ID NO: 75). Wherein, X1 can be K or R; X2 can be A, F or V; X3 can be I, L or M; X4 can be I, S or T; X5 can be R or V; X6 can be D, K or T; X7 can be K, S or T; X8 can be S or T; X9 can be A or V; X 10 Can be L, M or V; X 11 Can be E or Q; X 12 Can be L or M; X 13 Can be N or S; X 14 Can be R or T; X 15 Can be A or S; X 16 Can be S or T; X 17 Can be A or G; X 18 Can be I or V; X 19 Can be A or T.

[0196] For example, the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:32, SEQ ID NO:43, SEQ ID NO:54 and SEQ ID NO:57.

[0197] In the present application, the N-terminus of the H-FR4 may be connected to the C-terminus of the HCDR3, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 76.

[0198] GQGTX1VTVSS (SEQ ID NO: 76), wherein X1 can be S or T.

[0199] For example, the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 44.

[0200] For example, H-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO:73, H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:74, H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:75, and H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:76.

[0201] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein described in the present application may comprise an amino acid sequence as shown in any one of the following groups:

[0202] (1) the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 4, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 6, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 7;

[0203] (2) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO: 17, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO: 18, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO: 7;

[0204] (3) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:4, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:5, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:21, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:7;

[0205] (4) the H-FR1 comprises the amino acid sequence of SEQ ID NO: 30, the H-FR2 comprises the amino acid sequence of SEQ ID NO: 31, the H-FR3 comprises the amino acid sequence of SEQ ID NO: 32, and the H-FR4 comprises the amino acid sequence of SEQ ID NO: 7;

[0206] (5) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:42, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:43, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44;

[0207] (6) the H-FR1 comprises the amino acid sequence set forth in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:54, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:44;

[0208] (7) The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:41, the H-FR2 comprises the amino acid sequence shown in SEQ ID NO:53, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:57, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:44.

[0209] In the present application, the VL of the isolated antigen-binding protein may comprise framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0210] In the present application, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 77.

[0211] DIX1MTX2X3X4X5SLX6X7SX8GX9RX 10 TIX 11 C (SEQ ID NO: 77). wherein X1 may be Q or V; X2 may be H or Q; X3 may be S or T; X4 may be P or T; X5 may be A or S; X6 may be A or S; X7 may be A or V; X8 may be L or V; X9 may be D or Q; X 10 Can be A or V; X 11 Can be S or T.

[0212] For example, the L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 12, SEQ ID NO: 25, SEQ ID NO: 37 and SEQ ID NO: 47.

[0213] In the present application, the L-FR2 may be located between the LCDR1 and the LCDR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 78.

[0214] WYQQKPX1X2X3X4KLLIX5 (SEQ ID NO: 78), wherein X1 can be D or G; X2 can be G, K or Q; X3 can be A, P or T; X4 can be P or V; and X5 can be K or Y.

[0215] For example, the L-FR2 may comprise the amino acid sequence shown in any one of SEQ ID NO: 13, SEQ ID NO: 38, and SEQ ID NO: 48.

[0216] In the present application, the L-FR3 may be located between the LCDR2 and the LCDR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 79.

[0217] GVPX1RFSGSGSGTX2X3X4X5X6IX7X8X9X 10 X 11 X 12 DX 13 ATYX 14 C (SEQ ID NO: 79). wherein X1 may be A or S; X2 may be D or E; X3 may be F or Y; X4 may be S or T; X5 may be F or L; X6 may be N or T; X7 may be H or S; X8 may be N, P or S; X9 may be L or V; X 10 Can be E or Q; X 11 Can be E, P or Q; X 12 Can be D or E; X 13 Can be F, I or S; X 14 Can be F or Y.

[0218] For example, the L-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 14, SEQ ID NO: 39, SEQ ID NO: 49 and SEQ ID NO: 60.

[0219] In the present application, the N-terminus of the L-FR4 may be connected to the C-terminus of the HCDR3, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 80.

[0220] FGX1GTX2LEIK (SEQ ID NO: 80), wherein X1 can be G or Q; and X2 can be K or R.

[0221] For example, the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 50.

[0222] For example, L-FR1 of the isolated antigen-binding protein described herein may comprise the amino acid sequence shown in SEQ ID NO: 77, L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 78, L-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 79, and L-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 80.

[0223] For example, H-FR1, H-FR2, H-FR3 and H-FR4 of the isolated antigen-binding protein described in the present application may comprise an amino acid sequence as shown in any one of the following groups:

[0224] (1) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 12, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0225] (2) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 25, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 13, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 14, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0226] (3) the L-FR1 comprises the amino acid sequence of SEQ ID NO: 37, the L-FR2 comprises the amino acid sequence of SEQ ID NO: 38, the L-FR3 comprises the amino acid sequence of SEQ ID NO: 39, and the L-FR4 comprises the amino acid sequence of SEQ ID NO: 15;

[0227] (4) the L-FR1 comprises the amino acid sequence of SEQ ID NO:47, the L-FR2 comprises the amino acid sequence of SEQ ID NO:48, the L-FR3 comprises the amino acid sequence of SEQ ID NO:49, and the L-FR4 comprises the amino acid sequence of SEQ ID NO:50; and

[0228] (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.

[0229] The isolated antigen-binding protein described herein may comprise an antibody light chain variable region VH and an antibody heavy chain variable region VL.

[0230] In the present application, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 81, SEQ ID NO: 33 and SEQ ID NO: 22. For example, the VH may comprise the amino acid sequence shown in any one of SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 33, SEQ ID NO: 45, SEQ ID NO: 55 and SEQ ID NO: 58.

[0231] In the present application, the VL may comprise the amino acid sequence shown in SEQ ID NO: 82 or SEQ ID NO: 40. For example, the VL may comprise the amino acid sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 40, SEQ ID NO: 51 and SEQ ID NO: 61.

[0232] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 8, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0233] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 19, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0234] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 22, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0235] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 33, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 40.

[0236] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:45, and the VL may comprise the amino acid sequence shown in SEQ ID NO:51.

[0237] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 55, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 51.

[0238] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 58, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 51.

[0239] For example, the VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:45, and the VL may comprise the amino acid sequence shown in SEQ ID NO:61.

[0240] In the present application, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.

[0241] In certain embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region. In other embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG4 heavy chain constant region. The isolated antigen-binding protein may include a natural Fc and an Fc mutant, and the Fc mutant includes but is not limited to S239D / I332E and S239D / I332E / A330L that significantly promote binding and ADCC effects, and S239D / I332E that promotes B cell depletion. Fc variants include the S239D / I332E mutation that enhances ADCC / ADCP effects, variants such as S298A / E333A / K334A and F243L / R292P / Y300L / V305I / P396L that promote FcγRIIIa binding and enhance ADCC effects, IgG1 antibodies with the N297 terminal carbohydrate fucose removed that can significantly improve ADCC effects, L234A / L235A, D265A and N297A that reduce ADCC effects, S267E / H268F / S324T that can increase C1q affinity and promote CDC effects, G236A / I332E that improves poor ADCC effects, M252Y / S254T / T256E (Y / T / E) that increase the affinity of IgG for FcRn, and M428L / N434S.

[0242] For example, the antibody heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 63.

[0243] In the present application, the isolated antigen-binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Igκ constant region. For example, the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO: 64.

[0244] In the present application, the isolated antigen-binding protein may comprise an antibody heavy chain HC, and the HC may comprise the amino acid sequence shown in any one of SEQ ID NO:46, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:89 and SEQ ID NO:91.

[0245] In the present application, the isolated antigen-binding protein may comprise an antibody light chain LC, and the LC may comprise the amino acid sequence shown in any one of SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 87, SEQ ID NO: 90 and SEQ ID NO: 92.

[0246] The isolated antigen-binding protein described herein may comprise an antibody heavy chain and an antibody light chain.

[0247] The isolated antigen-binding protein described herein may comprise an antibody heavy chain and an antibody light chain.

[0248] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 87.

[0249] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 87.

[0250] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 90.

[0251] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:91, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:92.

[0252] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:52.

[0253] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 56, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 52.

[0254] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 59, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 52.

[0255] For example, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO:46, and the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO:62.

[0256] In addition, it should be noted that the isolated antigen-binding proteins described herein may include heavy chain and / or light chain sequences that have one or more conservative sequence modifications with the antigen-binding proteins described herein. The so-called "conservative sequence modifications" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the isolated antigen-binding proteins described herein by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR region of the isolated antigen-binding protein described herein can be replaced with other amino acid residues from the same side chain group. Those skilled in the art will appreciate that some conservative sequence modifications will not abolish antigen binding. For details, see, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissrov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0257] The proteins, polypeptides and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0258] In the present application, the variant may be a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., the isolated antigen-binding protein described in the present application). For example, the variant may comprise a protein or polypeptide having an amino acid change by at least 1, for example, 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., the ability to specifically bind to the LILRB4 protein) of the protein or polypeptide before the change.

[0259] In the present application, the homolog can be a protein or polypeptide having at least about 80% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (for example, the antibody or antigen-binding fragment thereof described in the present application).

[0260] In the present application, described homology generally refers to the similarity between two or more sequences or the degree of association.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0261] In the present application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding protein described herein may include, but is not limited to, a recombinant antibody, a monoclonal antibody, a human antibody, a murine antibody, a humanized antibody, a chimeric antibody, a single-chain antibody, a polyclonal antibody, an Fv fragment, a scFv fragment, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a camelized single-domain antibody.

[0262] In the present application, the antibody may be a humanized antibody. In other words, the isolated antigen-binding protein described herein may be an antibody or a variant, derivative, analog or fragment thereof that immunospecifically binds to a relevant antigen (e.g., human LILRB4) and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Here, "substantially" in the case of CDRs means that the amino acid sequence of the CDR is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of the CDR of a non-human antibody. The humanized antibody may comprise substantially all of at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all CDR regions correspond to the CDR regions of a non-human immunoglobulin (i.e., an antibody) and all or substantially all framework regions are framework regions having a consensus sequence of a human immunoglobulin. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically a constant region of a human immunoglobulin. In some embodiments, the humanized antibody contains at least the variable domains of a light chain and a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only a humanized heavy chain. In specific embodiments, the humanized antibody contains only a humanized variable domain of a light chain and / or a humanized heavy chain.

[0263] In the present application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0264] In the present application, the isolated antigen-binding protein may have one or more of the following properties: 1) being able to specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); 2) being able to -7 M or lower K D The invention discloses a novel novel anti-ILR-4 inhibitor that can bind to LILRB4 protein; 3) can block the activity of LILRB4 / APOE binding; 4) can stimulate the activation of CD8+ T cells; 5) can inhibit the migration of human monocytic leukemia cells THP-1; 6) can inhibit the growth and / or metastasis of tumor cells; 7) can block the binding of LILRB4 to Fibronectin.

[0265] In the present application, the isolated antigen binding protein can be expressed as 2×10 -7 M or lower K D Binds to LILRB4 protein. For example, the isolated antigen binding protein described herein binds to the K of human LILRB4 protein.D The value can be ≤2×10 -7 M, ≤1.5×10 -7 M, ≤1×10 -7 M, ≤9×10 -8 M, ≤8×10 -8 M, ≤7×10 -8 M, ≤6×10 -8 M, ≤5×10 -8 M, ≤4×10 -8 M, ≤3×10 -8 M, ≤2×10 -8 M, ≤1.5×10 -8 M, ≤1.2×10 -8 M, ≤1.15×10 -8 M, ≤1.1×10 -8 M, ≤1.05×10 -8 M, ≤1×10 -8 M, ≤5×10 -9 M or ≤1×10 -9 M.

[0266] In the present application, the KD value can also be determined by ELISA, competitive ELISA, BIACORE or KINEXA.

[0267] In the present application, the isolated antigen-binding protein is capable of specifically binding to human LILRB4 protein. The specific binding can be determined by FACS.

[0268] In the present application, the LILRB4 protein may be human LILRB4 protein (GenBank accession number: CAG46845.1).

[0269] The LILRB4 protein may include variants of the LILRB4 protein. For example, the variant may be: 1) a protein or polypeptide in which one or more amino acids are substituted, deleted, or added in the amino acid sequence of the LILRB4 protein; and 2) a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity to the LILRB4 protein.

[0270] The LILRB4 protein may also include a functionally active fragment of the LILRB4 protein.

[0271] Linker

[0272] The linkers used in immunoconjugates can be divided into two types: cleavable and non-cleavable. Cleavable linkers release the drug at the target cell under specific conditions. Cleavable linkers can be divided into chemically cleavable or enzymatically cleavable. There are three main types of chemically induced cleavable linkers: acid cleavable, cleavable under reducing conditions (disulfide, etc.) and linkers that can be cleaved by exogenous stimuli.

[0273] Acid-cleavable linkers need to maintain circulation stability under physiological conditions of pH ≈ 7.4, enter the acidic environment of endosomes (pH approximately 5.5-6.2) and lysosomes (pH approximately 4.5-5.0), and be removed by acid-catalyzed hydrolysis.

[0274] Acid-cleavable linkers can also include carbonate linkers. In some cases, simple carbonates have limited stability in serum, and the half-life can be improved by adding a p-aminobenzyl (PAB) group. For example, carbonate structures can hydrolyze under acidic conditions to release the SN-38 drug and carbon dioxide.

[0275] Specific enzymes exist in both the intracellular lysosomes and the extracellular tumor microenvironment, which can selectively cleave the corresponding substrates. Therefore, the linker used can be an enzymatically cleavable linker.

[0276] For example, cathepsins can cleave dipeptides, and dipeptide-based linkers can be used. For example, the dipeptide can be a Val-Cit dipeptide, a Val-Ala dipeptide, or a Phe-Lys dipeptide. Additionally, a self-degradable spacer can be introduced to facilitate enzyme access. For example, the self-degradable spacer can be PABC. In some cases, a tetrapeptide linker can also be used. For example, the tetrapeptide linker can be Gly-Gly-Phe-Gly. For example, when a tetrapeptide linker is used, the self-degradable spacer can be semi-aminated.

[0277] Like cathepsins, pyrophosphatases and phosphatases are hydrolases that are selectively expressed in lysosomes. For example, cathepsin B-sensitive Val-Cit-PABA can be coupled using linkers containing phosphate and pyrophosphate.

[0278] β-Glucuronidases are a class of glycosidases that catalyze the hydrolysis of β-glucuronic acid residues and are highly expressed in lysosomes and tumor stroma. In certain cases, glucuronic acid-containing linkers can be used. Glucuronic acid can be attached to a self-degradable spacer. Glucuronic acid-containing linkers can also be applied to other amine-containing payloads, such as camptothecin analogs, SN38, dulcamycin, and matrine, via an additional dimethylethylenediamine (DMED) self-degradable spacer.

[0279] In certain embodiments, the linker may further comprise a β-galactosidase cleavable linker. The β-galactosidase cleavable linker may comprise a PEG10 spacer. The spacer may be substituted with a nitro group to increase the self-degradation rate.

[0280] In certain embodiments, the linker comprises a sulfatase-cleavable linker.

[0281] In the present application, the linker may include a charged linker or an uncharged linker.

[0282] In the present application, the linker may comprise a dicarboxylic acid-based linker.

[0283] In the present application, the linker may include a hydrophilic linker (e.g., PEG4Mal and sulfo-SPDB) and a hydrophobic linker. For example, the hydrophilic linker may reduce the extent to which the antibody drug conjugate can be pumped out of resistant cancer cells via MDR (multidrug resistance) or functionally similar transporters.

[0284] In the present application, the linker can also function to directly or indirectly inhibit cell growth and / or cell proliferation. For example, the linker can function as an intercalator in the cleavage. For example, the linker can inhibit macromolecular biosynthesis.

[0285] In the present application, the linker can facilitate the entry of the antibody drug conjugate into cells (eg, can facilitate "internalization"), and can also be designed to improve the stability of the antibody drug conjugate.

[0286] For example, the linker can have a functional group capable of reacting with a free cysteine ​​present on the antibody to form a bond (e.g., a covalent bond). For example, the functional group can include: maleimide, haloacetamides, α-haloacetyl, active esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and / or isothiocyanates.

[0287] In some cases, the linker can include a functional group that is capable of reacting with an electrophilic group present on the antibody. For example, the functional group can include: an aldehyde, a ketocarbonyl, a hydrazine, an oxime, an amino, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and / or an arylhydrazide.

[0288] In the present application, the linker can be linked to the LILRB4 antigen binding protein via a sulfhydryl group, an azide group, or an amide group on the LILRB4-targeting antigen binding protein, for example, via a sulfhydryl group.

[0289] In the present application, the linker can be selected from one or more of the following groups: Acetyl Butyrate, MC-Val-Cit-PABC, SMCC, maleimide, CL2A, MC, MC-VC-PAB, PY-VC-PAB, PEG8-VA-PABC, MC-GGFG, MC-Val-Ala-PAB, MC-Val-Lys(Ac)-PAB, MC-Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, disulfide bond and acidolytic hydrazone bond.

[0290] In this application, the types of linkers include, but are not limited to, those currently known and disclosed in patent documents such as CN107921030A, WO2023237050, and US20050238649A1. The linkers disclosed in these documents can be used alone or in combination. For example, an immunoconjugate molecule may have one linker or two or more linkers.

[0291] Drug molecules

[0292] In the present application, the immunoconjugate comprises a drug molecule. In the present application, the drug molecule can comprise any known molecule with pharmaceutical activity. For example, the drug molecule can be any type of molecule. For example, the drug molecule can be at least one other agent selected from the group consisting of a chemotherapeutic agent, a radioactive element, a cytostatic agent, and a cytotoxic agent.

[0293] In the present application, the radioactive element may be selected from 177 Lu, 68 Ga, 89 Zr, 225 Ac, 111 In, 90 Y. 213 Bi, 131 I. 99 mTc, 3 H. 14 C. 15 N. 35 S. 18 F and 64 One or more of Cu.

[0294] In the present application, the drug molecules may include macromolecular drugs and / or small molecule drugs. The macromolecular drugs may be other antibody drugs, nucleic acid drugs, molecular glues and protacs. The other antibody drugs may be antibodies other than LILRB4 antibodies that bind to tumor therapy-related antigens, for example, PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, 4-1BB antibodies, TIGIT antibodies, TIM3 antibodies, CD47 antibodies, antibodies that bind to T lymphocyte surface markers, antibodies that bind to tumor stromal cells, antibodies that bind to myeloid-derived suppressor cells or NK cell surface markers. The nucleic acid drug antigens include but are not limited to sgRNA, mRNA, siRNA, shRNA and antisense RNA.

[0295] PROTAC and molecular glue are two main modes of targeted protein degradation technology based on the ubiquitin proteasome system. Among them, PROTAC recruits E3 ubiquitin ligases to induce the target protein to approach the E3 ubiquitin ligase, resulting in ubiquitination and degradation of the target protein. Molecular glues modify the surface of ubiquitin ligases to promote or induce protein-protein interactions (PPIs) between E3 ubiquitin ligases and target proteins, leading to ubiquitination and degradation of target proteins. Molecular glues include but are not limited to immunomodulators (IMiDs; thalidomide, lenalidomide, pomalidomide), CFT-7455, BAY-2666605, DKY-709, ICP-490, CC-9009, CC-92480, CC-99282, BTX-1188, MRT-2359, etc.

[0296] The small molecule drug can be a chemotherapeutic agent, a radioactive element, a cytostatic agent, and / or a cytotoxic agent. In this application, when referring to a specific type of drug molecule, its meaning includes not only the drug molecule itself, but also other forms such as its analogs, derivatives, and variants. In some cases, the drug molecule can have therapeutic activity. In some cases, the drug molecule can have cytotoxicity.

[0297] In the present application, the drug molecule can be selected from the following group: V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, maytansinoids, MetAP (methionine aminopeptidase), nuclear export inhibitors of protein CRM1, DPPIV inhibitors, proteasome inhibitors, mitochondrial phosphorylation reaction inhibitors, protein synthesis inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HD AC inhibitors, anthracyclines, NAMPT inhibitors, SN-38 glucuronide, etoposide phosphate, nitrogen mustard, proteosome inhibitors, cytokines, Tubulysin B analogs and Toll-like receptor agonists.

[0298] In the present application, the drug molecule may be a DNA topoisomerase I inhibitor. In the present application, the drug molecule may be a camptothecin derivative.

[0299] In the present application, Tubulysin B can be a microtubule lysin with cytotoxic activity, which can inhibit tubulin polymerization and cause cell cycle arrest and apoptosis. The CAS number of Tubulysin B is 205304-87-6. In the present application, MMAE, namely Monomethyl auristatin E, is a synthetic derivative of dolastatin 10, which can effectively inhibit mitosis by inhibiting tubulin polymerization. The CAS number of MMAE is 474645-27-7. In the present application, SN38 is an active metabolite of the topoisomerase I inhibitor irinotecan. Its CAS number is 119577-28-5. In the present application, the drug can be SN38 or MMAE.

[0300] In the present application, the drug molecule can be selected from microtubule-damaging drugs, DNA-damaging drugs, apoptosis inducers, tylanstatin and its analogs, amatoxin, nicotinamide phosphoribosyltransferase, and carmamycin. For example, the microtubule-damaging drug can be calendula, maytansine derivatives, tubulysin, cryptocolistin and / or anti-mitotic EG5 inhibitors. For example, the calendula can include MMAE and / or MMAF. For example, the maytansine derivative can include DM1, DM2, DM3 and / or DM4. For example, the DNA-damaging drug can be selected from: pyrrole benzazepines, indolechlorobenzazepines, dukamycin, camptothecin and its derivatives, calicheamicin. For example, the pyrrole benzazepines can include pyrrolo[2,1-c][1,4]benzodiazepine (PBD). For example, the camptothecin and its derivatives include SN-38, and / or DXd / DX8951. For example, the apoptosis inhibitor may comprise a Bcl-xL inhibitor.

[0301] In the present application, the drug molecules may include analogs and derivatives of the drug molecules. The analogs and derivatives of the drug molecules can still retain the functions of the drug molecules.

[0302] Immunoconjugates

[0303] In another aspect, the present application provides an immunoconjugate comprising an antigen binding protein targeting LILRB4 and a drug molecule. In the present application, the antigen binding protein targeting LILRB4 and the drug molecule may be linked via a linker.

[0304] In the present application, the immunoconjugate may comprise a structure shown in Formula 1: Ab-(Q) n (Formula 1), wherein Ab represents the antigen binding protein targeting LILRB4, Q represents a portion comprising the drug molecule linked to Ab, and n is an integer from 1 to 10.

[0305] For example, in Formula 1, Q may be selected from one or more of the following groups:

[0306] Wherein, * is the connection site between Q and the antibody molecule.

[0307] In certain specific embodiments, the Q in Formula 1 can be selected from the following group: -MC-MK4827, -MC-MK4827&MMAF, -MC-MMAF, -MC-MMAF, -MC-MMAF&SN38, -MC-SN38, -MC-2MMAF, -MC-MC-VC-PAB-MMAE and -PY-VC-PAB-MMAE.

[0308] In the present application, the antigen binding protein targeting LILRB4 in the immunoconjugate can be directly or indirectly linked to the drug molecule, for example, via a linker as described herein.

[0309] In the present application, the immunoconjugate may comprise a structure shown in Formula 2: Ab-(L1) a -(L2) b -D (Formula 2), wherein Ab represents the antigen-binding protein targeting LILRB4 described herein; L1 represents a linker connected to Ab; L2 represents a linker connected to D; a and b are each independently selected from 0 to 10; and D represents a drug molecule. For example, L1 can be a group or molecule reactive with a thiol, an azide, or an amide group. For example, L2 can include a combination of one or more of C1-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, C6-C24 aryl, C6-C15 heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclyl, polyethylene glycol, O, S, alkylamino, carbonyl, carboxyl, sulfonic acid, Val-Cit-PABC, VA-PABC, GGFG, Val-Lys(Ac)-PAB, Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB and Ala-PAB.

[0310] In the present application, the Ab can be a complete antibody structure (e.g., a complete IgG antibody structure, comprising two identical light chains and two identical heavy chains). In the present application, the light chain constant region, the CH1 of the heavy chain constant region, and / or the hinge region of the Ab can comprise a connection site capable of connecting to the L1. For example, the light chain constant region, the CH1 of the heavy chain constant region, and the hinge region of the Ab can comprise a connection site capable of connecting to the L1.

[0311] In the present application, the attachment site may comprise a cysteine. In the present application, the attachment site may comprise a sulfhydryl group in a ligatable state. In the present application, the number of the attachment sites may be 8. In the present application, the attachment sites may include: 2 attachment sites located in the light chain constant region, 2 attachment sites located in CH1 of the heavy chain constant region, and 4 attachment sites located in the hinge region.

[0312] In the present application, the linking site may comprise a group that can be linked to the L1 after deglycosylation modification. In the present application, the linking site may comprise a group that can be linked to the L1 after glycosylation modification.

[0313] In the present application, a may be an integer selected from 0-10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0314] In the present application, b may be an integer selected from 0-10, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0315] In some cases, the linker in the immunoconjugate can, for example, maintain the specific binding properties of the antigen binding protein targeting LILRB4; participate in the delivery of the drug molecule, and / or maintain the therapeutic effect of the drug (e.g., cytotoxic effect).

[0316] In the present application, the immunoconjugate may comprise a structure shown in Formula 1: Ab-(Q) n (Formula 1), wherein Ab represents the antigen binding protein targeting LILRB4, Q represents a portion comprising the drug molecule linked to Ab, and n is an integer from 1 to 10.

[0317] For example, in Formula 1, Q may be selected from one or more of the following groups:

[0318] Wherein, * is the connection site between Q and the antibody molecule.

[0319] In certain specific embodiments, the Q in Formula 1 can be selected from the following group: -MC-MK4827, -MC-MK4827&MMAF, -MC-MMAF, -MC-MMAF, -MC-MMAF&SN38, -MC-SN38, -MC-2MMAF, -MC-MC-VC-PAB-MMAE and -PY-VC-PAB-MMAE.

[0320] In certain embodiments, the immunoconjugate may have the following formula:

[0321] wherein n is selected from 1-8, preferably 1-6, for example, n is 1, 2, 3, 4, 5, 6, 7 or 8.

[0322] Pharmaceutical composition and use

[0323] In another aspect, the present application provides a pharmaceutical composition comprising the immunoconjugate, and optionally a pharmaceutically acceptable carrier.

[0324] In the present application, the pharmaceutically acceptable carrier may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counter ion, a metal complex and / or a nonionic surfactant, etc.

[0325] In the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or administration via a subcutaneous reservoir. For example, for an injectable formulation, the pharmaceutical composition can be prepared as an ampoule, such as a single-dose dosage form, or a unit dosage form, such as a multi-dose container. The pharmaceutical composition can also be prepared as a solution, a suspension, a tablet, a pill, a capsule, and a long-acting formulation.

[0326] In the present application, the pharmaceutical composition may further include one or more drugs selected from the following group: anthracycline topoisomerase inhibitors, daunorubicin, cytarabine nucleoside metabolic inhibitors, combined daunorubicin, cytarabine, daunorubicin and cytarabine liposome injection, Vyxeos, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine hydrochloride, histamine dihydrochloride (Ceplene), interleukin-2, gemtuzumab ozogamicin (Mylotarg), clofarabine, farnesyl transferase inhibitors, decitabine, IDH1 inhibitors, IDH2 inhibitors, ensidipine, Idhifa, IDO inhibitors, epacadostat, platinum complex derivatives, oxaliplatin, kinase inhibitors agents, tyrosine kinase inhibitors, PI3 kinase inhibitors, BTK inhibitors, antibodies that bind to tumor antigens, such as PD-1 antibodies, PD-L1 antibodies, anti-CTLA-4 antibodies, LAG3 antibodies, ICOS antibodies, 4-1BB antibodies, TIGIT antibodies, TIM3 antibodies and CD47 antibodies, neovascularization inhibitors, antibodies that bind to T lymphocyte surface markers, antibodies that bind to tumor stromal cells, myeloid-derived suppressor cells or NK cell surface markers, alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, alkaloids from plants, topoisomerase inhibitors, microtubule inhibitors, hormone therapy drugs, hormone antagonists, aromatase inhibitors, P-glycoprotein inhibitors, radiotherapy drugs, and nucleic acid drugs.

[0327] In another aspect, the present application provides a drug combination comprising the immunoconjugate and an immune checkpoint inhibitor. The immune checkpoint inhibitor comprises PD-1, PD-L1, GITR and / or CTLA-4. The immune checkpoint inhibitor comprises one or more selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, and a GITR antibody.

[0328] In another aspect, the present application provides the immunoconjugate and / or the pharmaceutical composition for use in preventing and / or treating a disease and / or disorder.

[0329] In another aspect, the present application provides a method for preventing, diagnosing and / or treating a disease and / or condition, comprising administering an effective amount of the immunoconjugate and / or the pharmaceutical composition to a subject in need thereof.

[0330] In the present application, the administration can be performed by different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

[0331] In the present application, the disease and / or disorder may include a disease or disorder associated with LILRB4 signaling and / or LILRB4 expression.

[0332] In the present application, the disease and / or disorder may include a tumor.

[0333] In the present application, the diseases and / or disorders may include LILRB4-positive diseases and / or disorders.

[0334] In the present application, the tumor may include solid tumors and / or hematological tumors.

[0335] In the present application, the solid tumor can be selected from one or more of the following groups: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, head and neck cancer, gastric cancer, pancreatic cancer, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; wherein the blood tumor is selected from multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, myelodysplastic syndrome and myeloproliferative neoplasms.

[0336] The leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia (AML), myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia, acute promyelocytic leukemia (APL) or M3AML, acute myelomonocytic leukemia or M4AML, acute monocytic leukemia or M5AML, wherein preferably, the acute myeloid leukemia is M4 or M5 type acute myeloid leukemia; preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia. The lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B cell lymphoma, T cell lymphoma, and Waldenstrom's macroglobulinemia. The sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

[0337] Without intending to be bound by any theory, the following examples are merely intended to illustrate the immunoconjugates, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0338] Example

[0339] Example 1 Evaluation of LILRB4 hybridoma antibody binding activity

[0340] ELISA plates were coated with LILRB4 recombinant protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag, Sino Biological) at 4°C overnight. After washing with PBST, the plates were blocked with 10% fetal bovine serum in blocking buffer at 37°C for 1 hour. Six anti-LILRB4 hybridoma antibodies were added at different dilutions at a maximum concentration of 20 μg / mL and reacted at 37°C for 1 hour. After washing with PBST, goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody (HRP, Invitrogen) was added and reacted at 37°C for 30 minutes. The plates were washed five times with PBST. 100 μL of TMB (eBioscience) was added to each well and incubated at room temperature (20±5°C) in the dark for 2–3 minutes. 100 μL of 2N The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm on a microplate reader to analyze the binding ability of the anti-LILRB4 hybridoma antibody to the LILRB4 recombinant protein.

[0341] The results are shown in Figure 1. Figure 1 shows that among the four anti-LILRB4 hybridoma antibodies, 1A2-7 and 3A2-18 had stronger binding abilities to LILRB4 than the other antibodies.

[0342] The amino acid sequences of 1A2-7, 3A2-18, 3G9-1, and 5E11-2 are shown in the table below, where the CDR sequences are divided using the Kabat method.

[0343] Example 2 Detection of cross-binding activity of LILRB4 family proteins

[0344] The leukocyte Ig-like receptor subfamily B inhibitory proteins, such as LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag), Sino Biological), LILRB1 protein (LILRB1 Protein, Human, Recombinant (ECD, His Tag), Sino Biological), LILRB2 protein (ILT4 Protein, Human, Recombinant (ECD, His Tag), Sino Biological), LILRB3 protein (LILRB3 Protein, Human, Recombinant (ECD, His Tag), Sino Biological), and LILRB5 protein (LILRB5 / CD85c Protein, Human, Recombinant (ECD, His Tag), Sino Biological) were used to express the inhibitory leukocyte Ig-like receptor subfamily B ELISA plates were coated with LILRB) at 4°C overnight; after washing with PBST, 10% fetal bovine serum was added and the plates were blocked at 37°C for 1 hour; 100 μL of anti-LILRB4 antibody at a concentration of 10 μg / mL was added to each well and reacted at 37°C for 1 hour; after washing with PBST, horseradish peroxidase-labeled goat anti-human IgG secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP, Invitrogen) was added and reacted at 37°C for 30 minutes; the plates were washed five times with PBST; 100 μL of TMB (eBioscience) was added to each well and the plates were incubated at room temperature (20±5°C) in the dark for 2-3 minutes; 100 μL of 2N H2SO4 stop solution was then added to each well to terminate the substrate reaction, and the OD values ​​were read at 450 nm on a microplate reader to analyze the binding ability of the anti-LILRB4 antibody to LILRB family proteins other than the specific antigen.

[0345] The results are shown in Figure 2. Figure 2 shows that the four antibodies tested did not bind to other proteins in the LILRB family.

[0346] Example 3 Evaluation of biological activity of LILRB4 antibody

[0347] Example 3-1 Evaluation of LILRB4 Antibody Internalization Activity

[0348] Human monocytic leukemia THP-1 cells were collected and 1×10 6Cells were added to a 1.5 mL EP tube; different concentrations of hybridoma-derived anti-LILRB4 antibody and isotype control antibody HG1k were added and incubated on ice in the dark for 30 min; after washing with FACS buffer, PE fluorescent-labeled goat anti-human IgG Fc secondary antibody (Invitrogen) was added and incubated on ice in the dark for 30 min; washed twice with FACS buffer; each tube of cells was resuspended in complete culture medium, divided equally into two groups, and incubated at 4°C and 37°C for 2 h, respectively, with mixing every 30 min. After washing twice with FACS buffer, 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells. After mixing, the mean fluorescence intensity (MFI) of PE fluorescence in each group was measured on a microscope. The endocytosis efficiency was calculated according to the formula: endocytosis efficiency (%) = [total surface MFI (4°C) - total surface MFI (37°C)] / total surface MFI (4°C) × 100%. The internalization efficiency of the antibody after binding to LILRB4-positive tumor THP-1 cells was analyzed.

[0349] The results are shown in Figure 3A. Figure 3A shows that among the hybridoma-derived anti-LILRB4 antibodies, 3G9-1 (i.e., 3G9-1A in the figure), 1A2-7, and 3A2-18 (i.e., 3A2-18A in the figure) had high internalization efficiency after binding to LILRB4-positive tumor cells THP-1.

[0350] Example 3-2 Effect of LILRB4 Antibody on THP-1 Cell Migration

[0351] Human monocytic leukemia THP-1 cells were serum-starved for 24 h and added to a 24-well cell culture plate. LILRB4 antibody and positive control antibody C84 (disclosed in patent WO2016144728A3) were added at a final concentration of 10 μg / mL and incubated in a 37°C 5% CO2 incubator for 1 h. 100 μL of the mixture of cells and antibodies was added to a Transwell (6.5 mm The cells were placed in the upper chamber of a 96-well plate (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning) and 700 μL of complete culture medium was added to the lower chamber. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h. 100 μL of the solution was taken from the lower chamber and added to a 96-well plate (96-well Black / Clear and White / Clear Bottom Polystyrene Microplates, Corning). 100 μL of cell viability assay reagent (CellTiter-LumiNano) was added. TMLuminescence cell viability detection kit (Biyuntian) was used. After incubation at room temperature in the dark for 10 min, the relative fluorescence intensity of chemiluminescence was detected using a multifunctional microplate reader. A standard curve was drawn using the gradient dilution of THP-1 cells and their corresponding relative fluorescence intensity values. The number of cells in the lower chamber was calculated to analyze the inhibitory effect of LILRB4 antibody on THP-1 cell migration.

[0352] The results are shown in Figure 3B . The ability of 1A2-7, 3A2-18, and 3G9-1 to inhibit THP-1 cell migration was stronger than that of the positive control antibody C84, and the inhibitory ability of 5E11-2 was similar to that of the positive control antibody C84.

[0353] Example 4-1 Antibody Humanization

[0354] The antibodies were humanized using standard CDR grafting methods. Human heavy and light chain variable sequences were BLAST searched, and three or four sequences from each were selected as humanized acceptor frameworks. The heavy and light chain CDR1, CDR2, and CDR3 were cloned into three different heavy chain frameworks (H1-H4) and two different light chain frameworks (L1-L2). The four different antibody combinations were then tested for expression levels and antigen binding ability in CHO-S cells.

[0355] Example 4-2 Evaluation of humanized antibody binding activity (ELISA)

[0356] ELISA plates were coated with LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag, Sino Biological) at 4°C overnight. After washing with PBST, 10% fetal bovine serum was added and the plates were blocked at 37°C for 1 hour. Different concentrations of 3A2-18A humanized antibody (corresponding to 3A2-18) and positive antibody 128-3 were added and reacted at 37°C for 1 hour. After washing with PBST, horseradish peroxidase-labeled goat anti-human IgG secondary antibody (H+L Cross-Adsorbed Secondary Antibody, Invitrogen) was added and reacted at 37°C for 30 minutes. The plates were washed five times with PBST. 100 μL of TMB (eBioscience) was added to each well and incubated at room temperature (20±5°C) in the dark for 2-3 minutes. Subsequently, 100 μL of 2N The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm on a microplate reader to analyze the binding ability of the 3A2-18A humanized antibody to LILRB4.

[0357] The results are shown in Figure 4. Figure 4 shows that the binding abilities of the four humanized antibodies 3A2-18A to LILRB4 are similar and similar to that of the positive antibody 128-3 (heavy chain amino acid sequence shown in SEQ ID NO: 65, light chain amino acid sequence shown in SEQ ID NO: 66).

[0358] Example 5 Determination of Binding Affinity between Humanized Antibodies and LILRB4 Protein

[0359] Take 100μl NHS and 100 microliters EDC, mix them, and centrifuge at 12000rpm for 5 minutes; take a certain volume of antibody, dilute it according to the pre-binding ratio, 150μl is required after dilution, and take 100μl ethanolamine HCL. The above reagents are balanced to room temperature of about 25 degrees Celsius, and centrifuged at high speed for five minutes; put the above three reagents on the sample rack, set the sample flow rate to 10μl / min, and inject in the injection mode, NHS / EDC, sample, ethanolamine-HCl are loaded at 100, 100, and 60μl respectively. Dilute the antigen to 7 concentrations with a suitable buffer, load the sample after centrifugation, and observe the antigen-antibody binding process. After testing, the affinity of 3A2-18A humanized antibody to human LILRB4 is 10 -9 M level, with a slightly decreased affinity compared to the parent antibody and similar affinity to the positive control antibody 128-3. The results are shown in Table 1.

[0360] Table 1. Binding affinity of 3A2-18A humanized antibody to LILRB4 protein determined by Biacore T200

[0361] Example 6 Detection of cross-binding activity of humanized antibody family proteins

[0362] The leukocyte Ig-like receptor subfamily B inhibitory proteins, such as LILRB4 protein (Human LILRB4 / CD85k / ILT3 Protein (ECD, His Tag), Sino Biological), LILRB1 protein (LILRB1 Protein, Human, Recombinant (ECD, His Tag), Sino Biological), LILRB2 protein (ILT4 Protein, Human, Recombinant (ECD, His Tag), Sino Biological), LILRB3 protein (LILRB3 Protein, Human, Recombinant (ECD, His Tag), Sino Biological), and LILRB5 protein (LILRB5 / CD85c Protein, Human, Recombinant (ECD, His Tag), Sino Biological) were used to express the inhibitory leukocyte Ig-like receptor subfamily B ELISA plates were coated with LILRB) at 4°C overnight; after washing with PBST, PBS containing 10% fetal bovine serum was added and the plates were blocked at 37°C for 1 hour; 100 μL of anti-LILRB4 antibody at a concentration of 10 μg / mL was added to each well (the LILRB4 antigen group was coated with antibody concentrations of 1 μg / mL and 0.1 μg / mL in test wells) and reacted at 37°C for 1 hour; after washing with PBST, horseradish peroxidase-labeled goat anti-human IgG secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP, Invitrogen) was added and reacted at 37°C for 30 minutes; the plates were washed with PBST five times; 100 μL of TMB (eBioscience) was added to each well and the plates were incubated at room temperature (20±5°C) in the dark for 2-3 minutes; then 100 μL of 2N The substrate reaction was terminated with H2SO4 stop solution, and the OD value was read at 450 nm on a microplate reader to analyze the binding ability of the anti-LILRB4 antibody to LILRB subfamily proteins other than the specific antigen.

[0363] The results are shown in Figure 5. Figure 5 shows that the four tested anti-LILRB4 antibodies, 3A2-18A humanized antibodies, only bind to LILRB4 protein among LILRB subfamily proteins and do not bind to other proteins in the same subfamily.

[0364] Example 7 Evaluation of internalization activity of humanized antibodies

[0365] Human monocytic leukemia THP-1 cells were collected and 1×10 6 Cells were added to a 1.5 mL EP tube; different concentrations of hybridoma-derived anti-LILRB4 antibody and isotype control antibody HG1k were added and incubated on ice in the dark for 30 min; after washing with FACS buffer, PE fluorescent-labeled goat anti-human IgG Fc secondary antibody (Invitrogen) was added and incubated on ice in the dark for 30 min; washed twice with FACS buffer; each tube of cells was resuspended in complete culture medium, divided equally into two groups, and incubated at 4°C and 37°C for 2 h, respectively, with mixing every 30 min. After washing twice with FACS buffer, 400 μL of 1% paraformaldehyde fixative (Solarbio) was added to each tube to fix the cells. After mixing, the mean fluorescence intensity (MFI) of PE fluorescence in each group was measured on a microscope. The endocytosis efficiency was calculated according to the formula: Endocytosis efficiency (%) = [total surface MFI (4°C) - total surface MFI (37°C)] / total surface MFI (4°C) × 100%. The internalization efficiency of the antibody after binding to LILRB4-positive tumor THP-1 cells was analyzed.

[0366] The results are shown in Figure 6. Figure 6 shows that the humanized antibody 3A2-18A can be internalized after binding to LILRB4-positive tumor cells THP-1, and the internalization efficiency is close to that of the control positive antibody 128-3.

[0367] Example 8 Humanized Antibodies Inhibit LILRB4+ Tumor Migration

[0368] 5×10 THP1-Luc human monocytic leukemia cells resuspended in PBS 6 / 0.2mL / mouse was injected into the tail vein of B-NDG mice. On the day of inoculation, the imaging signal of mice (average imaging signal 3.6x10 7Based on the p / s and mouse body weight (average weight 23 g), mice were randomly assigned to six experimental groups, with 5 mice in each group: G1 (PBS), G2 (C84, 10 mg / kg), G3 (RB4-G1-018, 10 mg / kg), G4 (RB4-G1-019, 10 mg / kg), G5 (RB4-G1-020, 10 mg / kg), and G6 (RB4-G1-022, 10 mg / kg). All mice were intraperitoneally injected with the drug on the day of grouping (day 0) and day 3 after grouping. Mice were imaged using a small animal in vivo imaging device before grouping and dosing (day 0), on days 7, 14, 21, and 25 after dosing, and on the end of the experiment (day 28). Imaging signal patterns and signal intensity were obtained to observe tumor growth in vivo. During the dosing and observation period, animal body weights were measured twice a week and the measured values ​​were recorded.

[0369] At the end of the experiment, the mice were weighed and imaged, then euthanized and the survival curve of the experimental animals was drawn. The hCD45 expression in the liver, lung and bone marrow of each group of mice was analyzed by flow cytometry. + The ratio of cells (live / dead, hCD45 + , mCD45 + ).

[0370] The results of flow cytometry are shown in Figure 7. Compared with the G1 control group, the expression of hCD45 in the liver, lung and bone marrow of mice in each group was significantly higher than that in the control group. + The cell proportions were significantly reduced, indicating that the test products C84, RB4-G1-018, RB4-G1-019, RB4-G1-020 and RB4-G1-022 at a dose of 10 mg / kg could significantly reduce the amount of tumor cells in the liver, lungs and bone marrow of mice.

[0371] Example 9: Detection of LILRB4 Humanized Antibody Blocking LILRB4 / APOE Binding Using Jurkat-LILRB4-CHO-CD3-APOE Reporter Cell Line

[0372] APOE / TCR activator / CHO cells in the logarithmic growth phase were seeded into 96-well plates and cultured overnight at 37°C. The next day, the supernatant was discarded, the cells were washed twice with PBS, and 50 μl of antibody sample diluted in 1640 medium was added to each well. LILRB4 effector reporter cells in the logarithmic growth phase were then added and cultured in a 37°C incubator. After 5 hours, the cells were removed, Bright-Glo luciferase was added, and the values ​​were read on a microplate reader. The values ​​for each well were analyzed using Prism GraphPad software.

[0373] The results are shown in Figure 8 . All humanized antibodies can block the binding of APOE to LILRB4, and their blocking abilities are stronger than those of the positive control antibodies 128-3 and C84.

[0374] Example 10 Humanized Antibodies Stimulate CD8+ T Cell Activation

[0375] PBMCs were isolated from whole blood of healthy donors and CD8 + CD8 T cell isolation kit (CD8+ T Cell Isolation Kit, Miltenyi) was used to isolate CD8 + T cells, 5×10 4 Cells / well were added to a 96-well plate (96-well Clear Round Bottom TC-treated Microplate, Corning); T cell activation magnetic beads ( Human T-Activator CD3 / CD28 / CD137, GIBCO) was placed in a 37°C 5% CO2 incubator for 2 days; humanized antibodies with a final concentration of 200 μg / mL, positive antibody 128-3, and isotype control were added, and then 5×10 4 Human monocytic leukemia THP-1 cells were added to each well and incubated in a 37°C 5% CO2 incubator for 5 days. The supernatant was collected and the level of TNF-α in the supernatant was detected using Human TNF-α Elisa Kit (ExCell Bio) to evaluate the effect of humanized antibodies on THP-1 / CD8 + Activated CD8 T lymphocytes in co-culture experiments + T cell capacity.

[0376] The results are shown in Figure 9. The humanized antibodies RB4-G1-018 and RB4-G1-020 were expressed in THP-1 / CD8 + Activated CD8 T lymphocytes in co-culture + The ability of T cells is stronger than that of positive antibody 128-3.

[0377] Example 11 Tissue distribution of SG2919 in LILRB4 humanized MC38-LILRB4 tumor-bearing mice

[0378] Humanized antibodies were labeled with 89Zr. After a single tail vein injection of 89Zr-SG2919 antibody (corresponding to RB4-G1-019 humanized antibody) and 89Zr-control antibody, in vivo scanning was performed using small animal PET / CT at different time points to observe the tissue distribution of SG2919 antibody and control antibody in humanized tumor-bearing mice and humanized mice. Group G1-1: After the 89Zr-SG2919 antibody was injected into the tail vein of 3 humanized mice + positive tumor cell models, static Micro PET / CT scans were performed at 24h, 48h, 96h, and 168h; Group G1-2: After the 89Zr-control antibody was injected into the tail vein of 3 humanized mice + positive tumor cell models, static Micro PET / CT scans were performed at 24h, 48h, 96h, and 168h; Group G2-1: After the 89Zr-SG2919 antibody was injected into the tail vein of 3 humanized mouse models, static Micro PET / CT scans were performed at 24h, 48h, 96h, and 168h; Group G2-2: After the 89Zr-control antibody was injected into the tail vein of 3 humanized mouse models, static Micro PET / CT scans were performed at 24h, 48h, 96h, and 168h. As can be seen from the results of Figures 10A and 10B, the radioactive concentration of the test antibody 89Zr-SG2919 in the tumors of humanized mice + positive tumor cell-bearing mice is higher than the radioactive concentration of the control antibody 89Zr-HEL in the tumors of humanized mice + positive tumor cell-bearing mice. The peak radioactive concentration of the test antibody 89Zr-SG2919 and the control antibody 89Zr-HEL in the tumors of humanized mice + positive tumor cell-bearing mice is at 48 hours, and then the radioactive concentration in the tumor gradually decreases with time. This result shows that the radiolabeled (e.g., 89Zr-labeled) antibody of the present application can be used for the diagnosis of LILRB4-positive tumors.

[0379] Example 12 Preparation of SG2919-ADC Conjugated with Different Toxins

[0380] The humanized antibody RB4-G1-019, designated SG2919, was conjugated to various small molecule compounds using conventional antibody-drug conjugate conjugate techniques (e.g., the method disclosed in CN107921030A) to prepare antibody-drug conjugates: SG2919-MC-MK4827 & MMAF, SG2919-MC-MMAF & SN38, SG2919-MC-2MMAF, SG2919-MC-MC-VC-PAB-MMAE, and SG2919-PY-VC-PAB-MMAE. SG2919-MC-MK4827 & MMAF is a conjugate in which both MK4827 and MMAF are conjugated to the SG2919 antibody, while SG2919-MC-MMAF & SN38 is a conjugate in which both MMAF and SN38 are conjugated to the antibody. The sample parts are named as follows: SG2919 antibody intermediate (i.e. naked antibody), SG2919 antibody drug conjugate: SG2919-MC-MK4827&MMAF (abbreviated as SG2919-MC4827AF), SG2919-MC-MMAF&SN38 (abbreviated as SG2919-MC38AF), SG2919-MC-2MMAF (abbreviated as SG2919-MC2AF), SG2919-MC-MC-VC-PAB-MMAE (abbreviated as SG2919-MACE), SG2919-PY-VC-PAB-MMAE (abbreviated as SG2919-PYAE)).

[0381] The humanized antibody RB4-G1-018, designated SG2918, was conjugated to various small molecule compounds using conventional antibody-drug conjugate conjugate techniques (e.g., the method disclosed in CN107921030A) to prepare the following antibody-drug conjugates: SG2918-MC-MK4827 & MMAF, SG2918-MC-MMAF & SN38, SG2918-MC-2MMAF, SG2918-MC-MC-VC-PAB-MMAE, SG2918-MC-VC-PAB-MMAE, and SG2918-PY-VC-PAB-MMAE. SG2918-MC-MK4827 & MMAF is a conjugate in which both MK4827 and MMAF are conjugated to the SG2918 antibody, while SG2918-MC-MMAF & SN38 is a conjugate in which both MMAF and SN38 are conjugated to the antibody. The sample parts are named as follows: SG2918 antibody intermediate (i.e. naked antibody), SG2918 antibody-drug conjugate: SG2918-MC-MK4827&MMAF (abbreviated as SG2918-MC4827AF), SG2918-MC-MMAF&SN38 (abbreviated as SG2918-MC38AF), SG2918-MC-2MMAF (abbreviated as SG2918-MC2AF), SG2918-MC-MC-VC-PAB-MMAE (abbreviated as SG2918-MACE), SG2918-PY-VC-PAB-MMAE (abbreviated as SG2918-PYAE)).

[0382] Example 13 Killing of THP-1 cells by SG2919-ADC coupled with different toxins (AML model)

[0383] THP-1 cells in the logarithmic growth phase were collected and counted at 1.5×10 4 Cells were plated per well in a transparent 96-well plate (Nunc™ Edge™ 96-well, Thermo). Different concentrations of SG2919-ADC conjugated with different toxins were added to the cells. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 3 days. CCK-8 detection reagent (Cell Counting Kit-8, Dojindo) was added to each well at a final concentration of 10% (v / v). The cells were incubated at 37°C for 1-2 hours, and the absorbance at 450 nm / 600 nm was measured using a multi-function microplate reader. The cytotoxic activity of SG2919-ADC conjugated with different toxins on THP-1 cells was analyzed.

[0384] The results are shown in Figure 11. Figure 11 shows that SG2919-ADCs conjugated with different toxins all have significant dose-dependent killing activity on THP-1 cells. SG2919-MC4827AF (i.e., SG2919-MC-MK4827 & MMAF), SG2919-MC38AF (i.e., SG2919-MC-MMAF & SN38), and SG2919-MC2AF (i.e., SG2919-MC-2MMAF) have the strongest killing activity against THP-1 cells; SG2919-MCAE (i.e., SG2919-MC-MC-VC-PAB-MMAE) and SG2919-PYAE (i.e., SG2919-PY-VC-PAB-MMAE) have relatively strong activity. Naked anti-RB4-G1-019, which is not conjugated with a toxin, has no killing activity.

[0385] Example 14: Killing of AML cells with different LILRB4 expression abundances by SG2918-ADC coupled with different toxins

[0386] THP-1, MOLM-13, and MOLM-16 cells in the logarithmic growth phase were collected and cultured at a rate of 1.5 × 10 4 Cells were added to a clear 96-well plate (Nunc™ Edge™ 96-well, Thermo). Different concentrations of SG2918-ADC conjugated with different toxins were added to the cells. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 3 days. CCK-8 detection reagent (Cell Counting Kit-8, Dojindo) was added to each well at a final concentration of 10% (v / v). The cells were incubated at 37°C for 1-2 hours, and the absorbance at 450 nm / 600 nm was measured using a multi-function microplate reader. The cytotoxicity of SG2918-ADC conjugated with different toxins was analyzed against AML cells with varying LILRB4 expression levels (THP-1 > MOLM-13 > MOLM-16, Figure 12A).

[0387] The results are shown in Figures 12B-12D. Figures 12B, C, and D show that SG2918-ADCs conjugated with different toxins exhibited significant dose-dependent cytotoxic activity on LILRB4-expressing THP-1 and MOLM-13 cells. SG2918-MC2AF (SG2918-MC-2MMAF) and SG2918-38AF (SG2918-MC-MMAF&SN38) showed the strongest and similar cytotoxic activities, while SG2918-MCAE (SG2918-MC-VC-PAB-MMAE) and SG2918-PYAE (SG2918-PY-VC-PAB-MMAE) showed relatively strong and similar cytotoxic activities. On MOLM-16 cells, which do not express LILRB4, SG2918-ADCs conjugated with different toxins exhibited cytotoxic activity at higher concentrations (>10ug / ml), with the effective concentration being significantly higher than that on THP-1 and MOLM-13 cells. Unconjugated naked anti-RB4-G1-018 showed no cytotoxic activity on any of the three cell types.

[0388] Example 15 SG2918--PYAE kills multiple myeloma (MM) cells with different LILRB4 expression abundances

[0389] U-266, MM.1S, and ALMC-1 cells in the logarithmic growth phase were collected and cultured at a rate of 1.5 × 10 4 Cells were plated per well in a clear 96-well plate (Nunc™ Edge™ 96-well, Thermo). SG2918-PYAE was added to the cells at varying concentrations. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 5 days. CCK-8 assay reagent (Cell Counting Kit-8, Dojindo) was added to each well at a final concentration of 10% (v / v). The cells were incubated at 37°C for 1-2 hours, and the absorbance at 450 nm / 600 nm was measured using a multi-function microplate reader. The cytotoxic activity of SG2918-PYAE was analyzed against MM cells with varying LILRB4 expression levels (ALMC-1, U-266, and MM.1S, Figure 13A).

[0390] The results, as shown in Figures 13B, C, and D, show that SG2918-PYAE exhibited significant dose-dependent cytotoxic activity against LILRB4-expressing U-266, MM.1S, and ALMC-1 cells. In ALMC-1 cells, SG2918-PYAE exhibited stronger cytotoxic activity than Blenrep, a marketed ADC targeting BCMA.

[0391] Example 16 In vivo efficacy of SG2918-ADC coupled with different toxins (THP-1 model)

[0392] Human monocytic leukemia cells THP-1-Luc labeled with luciferase were inoculated into female NCG mice via the tail vein. 1×10 7 Mice were randomly divided into four groups (6 animals per group) 14 days after inoculation based on in vivo imaging signals: a PBS (iv, QW x 3) control group, an SG2918 (4 mg / kg, iv, QW x 3) group, an SG2918-MCAE (4 mg / kg, iv, QW x 3) group, and an SG2918-PYAE (4 mg / kg, iv, QW x 3) group. Mouse body weights were measured weekly, and tumor bioluminescence signals were observed using a small animal in vivo imaging device. Bioluminescence values ​​and T / C (%) were calculated for each tumor-bearing mouse group. Mouse survival was recorded, and the median survival time and survival extension rate of each tumor-bearing mouse group were calculated and statistically analyzed. The results are shown in Figures 14A and 14B.

[0393] The SG2918-MCAE and SG2918-PYAE treatment groups showed significant inhibition of tumor growth early after administration. On PG-D7 (i.e., 21 days after tumor inoculation), the bioluminescence signal (p / s / cm2 / sr) of the PBS control group was 5.72×10 6 ±8.80×10 5 The bioluminescence signals (p / s / cm2 / sr) of the SG2918 group, SG2918-MCAE group, and SG2918-PYAE group were 3.89×10 6 ±4.61×10 5 , 8.24×10 5 ±1.57×10 5 and 4.50×10 5 ±8.64×10 4 The relative tumor proliferation T / C (%) of each treatment group compared to the PBS control group was 68%, 14%, and 8%, respectively. The bioluminescence signal intensities of the SG2918-MCAE and SG2918-PYAE treatment groups were significantly lower than those of the PBS control group (p<0.05). Compared with the SG2918 group, the bioluminescence signal intensities of the SG2918-MCAE and SG2918-PYAE treatment groups were significantly lower than those of the SG2918 group (p<0.05), while no statistical difference was observed between the SG2918-MCAE and SG2918-PYAE treatment groups (p>0.05).

[0394] At the end of the observation period (PG-D42), all mice in the PBS control, SG2918, SG2918-MCAE, and SG2918-PYAE groups became ill and died, with median survival times of 14, 15, 35, and 40 days, respectively. The survival extension rates for each treatment group were 7%, 150%, and 186%, respectively. Compared with the PBS control and SG2918 groups, the survival times of the SG2918-MCAE and SG2918-PYAE groups were significantly prolonged (p<0.05). Furthermore, the survival time of the SG2918-PYAE group was significantly longer than that of the SG2918-MCAE group (p<0.05).

[0395] Example 17 In vivo efficacy of SG2918-PYAE on multiple myeloma (OPM-2 model)

[0396] Human myeloma OPM-2 cells were inoculated subcutaneously in the right shoulder of female NCG immunodeficient mice. 3 At the same time, appropriate mice were randomly selected based on their tumor volume and body weight, with a total of 2 groups, each with 6 mice, namely G1: vehicle and G2: SG2918-PYAE. The dosage of SG2918-PYAE was 5 mg / kg, and it was intravenously injected twice a week for a total of 4 doses. The body weight of the mice was measured twice a week during the dosing and observation period, and the experiment was terminated on the 20th day after grouping. The results are shown in Figure 15. At the end of the experiment, the average tumor volume of the vehicle control group was 2503.3±199.9mm 3 The average tumor volume of the SG2918-PYAE group was 5.1±0.7 mm 3 The tumor inhibition rate TGI% was 102% (p<0.01), showing a very significant tumor inhibition effect.

[0397] The body weight of the SG2918-PYAE group continued to increase throughout the experimental period without any abnormalities, indicating that the animal tumor model showed good tolerance to the test drugs.

[0398] Example 18 In vivo efficacy of SG2918-PYAE in solid tumors (LLC1 model)

[0399] LLC1 cells resuspended in PBS were plated at 2×10 5 The concentration of 0.1 mL was used to subcutaneously inoculate 18 B-Tg (hLILRB1-hLILRB4) mice. When the tumor volume grew to 80 mm 3At the time of the experiment, 12 suitable animals were randomly selected based on tumor volume and animal body weight, with 6 animals in each group, for a total of 2 groups, namely G1: PBS and G2: SG2918-PYAE (administered with SG2918-Py-VC-PAB-MMAE). Each group was administered via the tail vein twice a week for a total of 3 doses. The experiment ended on the 9th day after grouping. The body weight and tumor volume of the mice were measured twice a week during the administration and observation period, and the measured values ​​were recorded and the tumor volume growth inhibition rate (TGITV) was calculated. At the end of the experiment, the tumors and spleens of the animals in the G1-G2 groups were collected for FACS analysis.

[0400] The results are shown in Figure 16. During the experiment, the mice in each group showed no obvious clinical symptoms. Similar to the G1 control group, the weight of the animals in the G2 test article group gradually increased, indicating that the animals had good tolerance to the test article. On the 9th day of grouping, the average tumor volume of the control group G1 was 1776±232mm 3 The average tumor volume of the G2 group was 1137±78mm 3 The corresponding TGITV is 37.7%.

[0401] Example 19 In vivo efficacy of SG2918-PYAE combined with PD-1 antibody in the treatment of solid tumors

[0402] LLC1 cells resuspended in PBS were plated at 5×10 5 34 B-Tg (hLILRB1-hLILRB4) mice were inoculated subcutaneously on the right side of the body at a volume of 0.1 mL / mouse. When the average tumor volume reached 79 mm 3 At the same time, appropriate mice were selected according to the tumor volume and body weight of the mice and randomly assigned to 4 experimental groups, with 6 mice in each group, namely: G1: SG641 (negative control), G2: SG2918-PYAE (administered with SG2918-Py-VC-PAB-MMAE), G3: SG2918-PYAE+Anti-mouse PD-1 and G4: Anti-mouse PD-1. The dosage, administration method, administration frequency and number of administrations for each group were carried out in accordance with 6.2. The experiment was terminated on the 17th day after grouping. The body weight and tumor volume of the mice were measured twice a week during the administration and observation period. At the end of the experiment, the mice were euthanized, the tumors were removed, weighed, photographed, and the tumor growth inhibition rate was calculated.

[0403] During the experiment, mice in each group showed no obvious clinical symptoms. Similar to the G1 control group, the weight of animals in the G2 test product group gradually increased, indicating that the animals had good tolerance to the test product. On the 14th day after grouping, the average tumor volume in the G4 group was 3868±284mm 3 The average tumor volumes of G1-G3 groups were 3474±257mm 3、2961±326mm 3 and 2573±243mm 3 The corresponding tumor volume growth inhibition rates (TGITV) were 10.4%, 23.9%, and 34.2%, respectively. The results are shown in Figure 17. Under these experimental conditions, compared with anti-mouse PD-1, the combined administration of SG2918-PYAE and anti-mouse PD-1 significantly inhibited tumor growth in LLC1 cell-transplanted B-Tg (hLILRB1-hLILRB4) mice (p < 0.05). The animals tolerated the test products well.

Claims

1. An immunoconjugate comprising an antigen-binding protein targeting LILRB4 and a drug molecule, the immunoconjugate having the following general formula: Ab-(Q) n (Formula 1) Among them, Ab represents the antigen-binding protein targeting LILRB4, Q represents the part containing the drug molecule linked to Ab, n is an integer from 1 to 10, and each Q is the same or different; The antigen-binding protein targeting LILRB4 has one or more of the following properties: (1) It can specifically recognize leukocyte immunoglobulin-like receptor B4 (LILRB4); (2) capable of binding to the LILRB4 protein with a KD value of 2×10 -7 M or lower; (3) It can block the activity of LILRB4 / APOE binding; (4) capable of stimulating 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) The activity of internalization can occur after binding to the LILRB4 target; and (8) It can block the binding of LILRB4 to Fibronectin.

2. The immunoconjugate according to claim 1, wherein the LILRB4 is human LILRB4.

3. The immunoconjugate according to any one of claims 1-2, wherein the antigen-binding protein targeting LILRB4 comprises at least one CDR in the heavy chain variable region VH of the antibody, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:81, SEQ ID NO:33, and SEQ ID NO:

22.

4. The immunoconjugate according to any one of claims 1-3, wherein the antigen-binding protein targeting LILRB4 comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises SEQ ID NO:69 (X1X2WMX3, wherein, The amino acid sequence shown in (where X1 is D or S; X2 is A or Y; X3 is D or H), the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:83 or SEQ ID NO:

29.

5. The immunoconjugate according to any one of claims 1-4, wherein the antigen-binding protein targeting LILRB4 comprises HCDR1, HCDR2, and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:28, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:3, SEQ ID NO:20, and SEQ ID NO:

29.

6. The immunoconjugate according to any one of claims 1-5, wherein the antigen-binding protein targeting LILRB4 comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 comprise amino acid sequences selected from the following group: (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:20; and (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

29.

7. The immunoconjugate according to any one of claims 1-6, wherein the antigen-binding protein targeting LILRB4 comprises a VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:81, SEQ ID NO:33, and SEQ ID NO:

22.

8. The immunoconjugate according to any one of claims 1-7, wherein the antigen-binding protein targeting LILRB4 comprises a VH, and the VH comprises the amino acid sequence shown in any one of SEQ ID NO:8, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:55, and SEQ ID NO:

58.

9. The immunoconjugate according to any one of claims 1-8, wherein the antigen-binding protein targeting LILRB4 comprises at least one CDR in the variable light chain VL of the antibody, and the VL comprises the amino acid sequence shown in SEQ ID NO:82 or SEQ ID NO:

40.

10. The immunoconjugate according to any one of claims 1-9, wherein the antigen-binding protein targeting LILRB4 comprises LCDR1, LCDR2, and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:70 or SEQ ID NO:34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:71, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:72 (QX1X2X3X4X5PX6T, X1 is H or Q; X2 is G or S; X3 is D, N or W; X4 is E or T; X5 is I or L; X6 is P or R).

11. The immunoconjugate according to any one of claims 1-10, wherein the antigen-binding protein targeting LILRB4 comprises LCDR1, LCDR2 and LCDR3, 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, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:35, and 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.

12. The immunoconjugate according to any one of claims 1-11, wherein the antigen-binding protein targeting LILRB4 comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from the group consisting of: (1) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11; (2) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:23, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:24; and (3) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

36.

13. The immunoconjugate according to any one of claims 1-12, wherein the antigen-binding protein targeting LILRB4 comprises VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:82 or SEQ ID NO:

40.

14. The immunoconjugate according to any one of claims 1-13, wherein the antigen-binding protein targeting LILRB4 comprises VL, and 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.

15. The immunoconjugate according to any one of claims 1-14, wherein the antigen-binding protein targeting LILRB4 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from the group consisting of: (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, 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; (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:28, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:29, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:34, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:35, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:

36.

16. The immunoconjugate according to any one of claims 1-15, wherein the antigen-binding protein targeting LILRB4 comprises VH and VL, and the VH and VL comprise amino acid sequences selected from any one of the following groups: (1) The VH comprises the amino acid sequence shown in SEQ ID NO:8, and the VL comprises the amino acid sequence shown in SEQ ID NO:16; (2) The VH comprises the amino acid sequence shown in SEQ ID NO:19, and the VL comprises the amino acid sequence shown in SEQ ID NO:16; (3) The VH comprises the amino acid sequence shown in SEQ ID NO:22, and the VL comprises the amino acid sequence shown in SEQ ID NO:26; (4) The VH comprises the amino acid sequence shown in SEQ ID NO:33, and the VL comprises the amino acid sequence shown in SEQ ID NO:40; (5) The VH comprises the amino acid sequence shown in SEQ ID NO:45, and the VL comprises the amino acid sequence shown in SEQ ID NO:51; (6) The VH comprises the amino acid sequence shown in SEQ ID NO:55, and the VL comprises the amino acid sequence shown in SEQ ID NO:51; (7) The VH 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.

17. The immunoconjugate according to any one of claims 1-16, wherein the antigen-binding protein targeting LILRB4 comprises an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from an IgG constant region.

18. The immunoconjugate according to claim 17, wherein the antibody heavy chain constant region comprises an IgG1 constant region, an IgG4 constant region, and their respective mutants.

19. The immunoconjugate according to any one of claims 17-18, wherein the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

63.

20. The immunoconjugate according to any one of claims 1-19, wherein the antigen-binding protein targeting LILRB4 comprises an antibody light chain constant region, and the antibody light chain constant region is derived from a human Igκ constant region.

21. The immunoconjugate according to claim 20, wherein the antibody light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

64.

22. The immunoconjugate according to any one of claims 1-21, wherein the antigen-binding protein targeting LILRB4 comprises an antibody heavy chain and an antibody light chain, and the antibody heavy chain and the antibody light chain comprise an amino acid sequence selected from any one of the following groups: (1) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 86, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87; (2) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 88, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 87; (3) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 89, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 90; (4) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 91, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 92; (5) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 46, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; (6) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; (7) The antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 59, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 52; and (8) The heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 46, and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

62.

23. The immunoconjugate according to any one of claims 1-22, wherein the drug molecule comprises a macromolecular drug and / or a small molecule drug.

24. The immunoconjugate according to claim 23, wherein the macromolecular drug comprises other antibody drugs, nucleic acid drugs, molecular glues, and PROTACs, the other antibody drugs comprise antibodies that bind to tumor treatment-related antigens, and the nucleic acid drugs comprise sgRNA, mRNA, siRNA, shRNA, and antisense RNA.

25. The immunoconjugate according to any one of claims 1-24, wherein the drug molecule is selected from chemotherapeutic agents, radioactive elements, cell growth inhibitors, and cytotoxic agents.

26. The immunoconjugate according to claim 25, wherein the radioactive element comprises 177 Lu, 68 Ga, 89 Zr, 225 Ac, 111 In, 90 Y, 213 Bi, 131 I, 125 I, 99 mTc, 3 H, 14 C, 15 N, 35 S, 18 F and 64 one or more of Cu.

27. The immunoconjugate according to any one of claims 1-26, wherein the drug molecule is selected from the group consisting of: V-ATPase inhibitors, Bcl2 inhibitors, MCL1 inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, dolastatin, maytansine alkaloids, MetAP (methionyl aminopeptidase), nuclear export inhibitors of protein CRM1, DPPIV inhibitors, proteasome inhibitors, inhibitors of phosphotransfer reactions in mitochondria, protein synthesis inhibitors, CDK2 inhibitors, CDK9 inhibitors, kinesin inhibitors, HDAC inhibitors, DNA-damaging agents, DNA alkylating agents, DNA intercalating agents, DNA minor groove binders, DHFR inhibitors, nucleoside analogs, HDAC inhibitors, anthracyclines, NAMPT inhibitors, SN-38 or its derivatives, etoposide phosphate, nitrogen mustard, proteasome inhibitors, cytokines, Tubulysin B analogs, and Toll-like receptor agonists.

28. The immunoconjugate according to any one of claims 1-27, wherein the drug molecule is selected from antibiotics, microtubule-damaging drugs, DNA-damaging drugs, apoptosis inducers, talystatin and its analogs, amanitin, nicotinamide phosphoribosyltransferase, and calicheamicin.

29. The immunoconjugate according to claim 28, wherein the microtubule-disrupting agent is selected from: calceolarioside A, maytansine derivatives, laulimalide, cryptophycin, anti-mitotic EG5 inhibitors, wherein, Preferably, the calicheamicin comprises MMAE and / or MMAF, and the maytansine derivative comprises DM1, DM2, DM3, and / or DM4.

30. The immunoconjugate according to any one of claims 28-29, wherein the DNA damaging agent is selected from: pyrrolobenzodiazepines, indolobenzodiazepines, duocarmycin, camptothecin and its derivatives, and calicheamicin, wherein, Preferably, the pyrrolobenzodiazepine drug comprises pyrrolo[2,1-c][1,4]benzodiazepine (PBD), the camptothecin and its derivatives comprise SN-38 and / or DXd / DX8951, and the apoptosis inhibitor comprises a Bcl-xL inhibitor.

31. The immunoconjugate according to any one of claims 1-30, further comprising a linker that can link the antigen-binding protein targeting LILRB4 and the drug molecule.

32. The immunoconjugate according to claim 31, wherein the linker is selected from: non-cleavable linkers and cleavable linkers, preferably the cleavable linker comprises a chemically active linker, an acid-cleavable linker, a linker cleavable under reducing conditions, and / or an enzyme-cleavable linker.

33. The immunoconjugate according to any one of claims 31-32, wherein the linker is linked to the LILRB4 antigen-binding protein through a thiol group, an azide group or an amide group on the antigen-binding protein targeting LILRB4.

34. The immunoconjugate according to any one of claims 31-33, wherein the linker comprises a combination of one or more selected from the group consisting of: Acetyl Butyrate, MC-Val-Cit-PABC, SMCC, maleimide, CL2A, MC, MC-VC-PAB, PY-VC-PAB, PEG8-VA-PABC, MC-GGFG, MC-Val-Ala-PAB, MC-Val-Lys(Ac)-PAB, MC-Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, Ala-PAB, disulfide bond and acid-labile hydrazone bond.

35. The immunoconjugate according to any one of claims 1-34, which comprises the structure shown in formula 2: Ab-(L1) a -(L2) b -(D) n (Formula 2) Wherein Ab represents an antigen-binding protein targeting LILRB4; L1 represents a linker linked to Ab, and L2 represents a linker linked to D, a and b are each independently selected from 0-10, D represents a drug molecule, n is selected from 1-10, Among them, Preferably, L1 is a group or molecule capable of reacting with a thiol group, an azide group or an amide group, preferably Py-MAA; L2 comprises C1-C9 alkyl, C2-C9 alkenyl, C2-C9 alkynyl, C6-C24 aryl, C6-C15 heteroaryl, C3-C9 cycloalkyl, C3-C9 heterocyclic group, polyethylene glycol, O, S, alkylamino, carbonyl, carboxyl, sulfonic acid group, Val-Cit-PAB, VA-PAB, GGFG, Val-Lys(Ac)-PAB, Phe-Lys-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB and Ala-PAB in a combination of one or more.

36. The immunoconjugate according to any one of claims 1-35, wherein Q in Formula 1 is selected from one or more combinations of the following groups: Among them, * is the linking site of Q to the antibody molecule.

37. The immunoconjugate according to any one of claims 1-36, wherein Q in formula 1 is selected from the group consisting of: -MC-MK4827, -MC-MK4827&MMAF, -MC-MMAF, -MC-MMAF, -MC-MMAF&SN38, -MC-SN38, -MC-2MMAF, -MC-MC-VC-PAB-MMAE and -PY-VC-PAB-MMAE.

38. The immunoconjugate according to any one of claims 1-37, which has the following molecular formula: Wherein n is selected from 1-8.

39. A pharmaceutical composition comprising the immunoconjugate according to any one of claims 1-38, and optionally a pharmaceutically acceptable carrier.

40. A pharmaceutical combination comprising the immunoconjugate according to any one of claims 1-38, and an immune checkpoint inhibitor, preferably the immune checkpoint comprises PD-1, PD-L1, GITR and / or CTLA-4; more preferably, the immune checkpoint inhibitor comprises one or more selected from the group consisting of: a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody and a GITR antibody.

41. A method for preparing the immunoconjugate according to any one of claims 1-38, which comprises linking the antigen-binding protein targeting LILRB4 according to any one of claims 1-38 and the drug molecule according to any one of claims 1-38 under suitable conditions.

42. A method for preventing, diagnosing and / or treating a disease and / or disorder, which comprises administering to a subject in need thereof the immunoconjugate according to any one of claims 1-38, the pharmaceutical composition according to claim 39 or the pharmaceutical combination according to claim 40.

43. The method according to claim 42, wherein the disease and / or disorder comprises a disease or disorder related to LILRB4 signaling and / or LILRB4 expression.

44. The method according to any one of claims 42-43, wherein the disease and / or disorder comprises a tumor, preferably a hematological tumor and / or a solid tumor.

45. The method according to claim 44, wherein the solid tumor is selected from one or more of the following group: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, head and neck cancer, gastric cancer, pancreatic cancer, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma.

46. The method according to claim 44, wherein the hematological tumor is selected from multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, myelodysplasia syndrome and myeloproliferative neoplasm.

47. The method according to claim 46, wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia (AML), myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia, acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, wherein preferably, the acute myeloid leukemia is M4 or M5 type acute myeloid leukemia; preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia; the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia.

48. The method according to claim 45, wherein the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

49. Use of the immunoconjugate according to any one of claims 1-38, or the pharmaceutical composition according to claim 39, or the pharmaceutical combination according to claim 40, in the manufacture of a medicament for the prevention, diagnosis, and / or treatment of a disease and / or disorder.

50. The use according to claim 49, wherein the disease and / or disorder includes a tumor, preferably selected from hematological tumors and / or solid tumors.

51. The use according to claim 50, wherein the solid tumor is selected from one or more of the following: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, head and neck cancer, gastric cancer, pancreatic cancer, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma, preferably, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

52. The use according to claim 49, wherein the disease and / or disorder includes a disease or disorder associated with LILRB4 signaling and / or LILRB4 expression.

53. The use according to claim 50, wherein the hematological tumor is selected from multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, myelodysplasia syndrome, and myeloproliferative neoplasm.

54. The use according to claim 53, wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia (AML), myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia, acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, wherein preferably, the acute myeloid leukemia is M4 or M5 acute myeloid leukemia; preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia.

55. The use according to claim 50, wherein the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia.

56. The immunoconjugate according to any one of claims 1-38, or the pharmaceutical composition according to claim 39, or the pharmaceutical combination according to claim 40, for the prevention, diagnosis, and / or treatment of diseases and / or disorders.

57. The immunoconjugate or pharmaceutical composition or pharmaceutical combination according to claim 56, wherein the diseases and / or disorders include diseases or disorders associated with LILRB4 signaling and / or LILRB4 expression.

58. The immunoconjugate or pharmaceutical composition or pharmaceutical combination according to claim 56, wherein the diseases and / or disorders include tumors, preferably selected from hematological tumors and / or solid tumors.

59. The immunoconjugate or pharmaceutical composition or pharmaceutical combination according to claim 58, wherein the solid tumor is selected from one or more of the following: breast cancer, melanoma, colon cancer, lung cancer, kidney cancer, head and neck cancer, gastric cancer, pancreatic cancer, lymphoma, non-small cell lung cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma; wherein the hematological tumor is selected from multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, myelodysplasia syndrome, and myeloproliferative neoplasm.

60. The immunoconjugate or pharmaceutical composition or combination according to claim 59, wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia (AML), myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myeloid leukemia, acute promyelocytic leukemia (APL) or M3 AML, acute myelomonocytic leukemia or M4 AML, acute monocytic leukemia or M5 AML, and preferably, the acute myeloid leukemia is M4 or M5 acute myeloid leukemia; preferably, the chronic myeloid leukemia is chronic myelomonocytic leukemia.

61. The immunoconjugate or pharmaceutical composition or combination according to claim 59, wherein the lymphoma is selected from Hodgkin lymphoma and non-Hodgkin lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and Waldenström macroglobulinemia.

62. The immunoconjugate or pharmaceutical composition or combination according to claim 59, wherein the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

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