Human LIFR antigen-binding protein, method for producing the same, and applications

Abstract

Provided is a human LIFR antigen binding protein and its preparation and application. Human LIFR-ECD protein is used to immunize H2L2 human antibody transgenic mice, and human LIFR antibody with a series of biological functions is obtained by Fc recombination using single B cell cloning technology (SBC) and single B cell sequencing analysis, which greatly improves the efficiency and yield of antibody drug development, and the obtained human LIFR antibody has good affinity, can block the binding of huLIF protein with huLIFR / gp130 cells, inhibit signal pathways such as STAT3, and further inhibit tumor growth, achieving the purpose of tumor prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and specifically relates to a human LIFR antigen-binding protein, a method for producing the same, and applications thereof.

Background Art

[0002] Leukemia inhibitory factor (LIF) is a cytokine with various biological functions and is named for inducing the differentiation of M1 myeloid leukemia cells into normal cells. LIF belongs to the interleukin-6 (IL-6) family, which further includes IL-11, IL-27, ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and Oncostatin M (OSM). Since these cytokines share the LIF and gp130 receptors, LIF has unique biological effects overlapping with other family members.

[0003] The LIF protein in mammals consists of 180 amino acid residues and has a high degree of conservation. The human LIF gene is encoded on chromosome 22q12, the mouse LIF gene is located on chromosome 11, and the homology between the mouse and human LIF genes is >75%. The mature LIF protein is a highly glycosylated secreted protein and is secreted from various adult cells such as oocytes, extraembryonic cells of human embryos, endometrial cells, fibroblasts, hepatocytes, and mononuclear macrophages. The glycosylation of LIF proteins derived from different tissues is different, and the most studied among them is the secreted variant glycosylated protein (34 - 63 kDa), which has autocrine induction and extensive paracrine effects, and such effects may conflict with each other due to differences in cell types and backgrounds, such as proliferation and differentiation, survival and apoptosis, etc.

[0004] LIF mainly binds to its specific LIF receptor (LIFR), then recruits the gp130 receptor subunit to form a high-affinity heterodimeric receptor complex, and induces the activation of downstream signaling pathways such as JAK / STAT3, PI3K / AKT, ERK1 / 2, and MAPK, thereby exerting various cell functions and regulating cell proliferation and survival. In addition, LIFR is abundantly expressed in various tissues and organs such as the central nervous system, kidney, liver, bone, and uterus. The wide distribution of LIF and LIFR and multiple LIF signaling pathways determine the complex diversity of LIF functions.

[0005] The LIF pathway is a very promising new target in the field of immunology. The LIF signaling pathway plays an important role in tumor occurrence and progression, can enhance tumor cell metastasis and invasion, promote tumor metastasis, and at the same time can regulate multiple immune cells including effector T cells (T-eff), regulatory T cells (T-reg), Th17, and myeloid cells in the tumor microenvironment. Studies have shown that the LIF signaling pathway can promote the activity of cancer-initiating cells (CIC) and enhance resistance to antitumor therapies (chemotherapy and radiotherapy). In multiple cancers, high expression of LIF is associated with poor prognosis.

[0006] Patent CN111378036A discloses a method for producing an anti-human leukemia inhibitory factor monoclonal antibody and its application by adopting mouse hybridoma cell technology, but the efficiency and yield of antibody screening in this method are low. Patent CN111868086A discloses a series of anti-LIF antibodies and their applications, and the LIF antibodies can bind to unique epitopes of LIF and inhibit the biological activity of LIF.

[0007] Currently, there is the MSC-1 antibody from Northern biologics as an antibody against the LIF target clinically. This antibody completed a Phase I clinical study in 2019 and currently plans to conduct multiple clinical therapeutic effect tests. MSC-1 is used in the treatment of a series of solid tumors such as pancreatic cancer and lung cancer. The MSC-1 antibody is a humanized monoclonal antibody (IgG1), binds to the immunosuppressive human cytokine LIF, and is used in the treatment of adult advanced solid tumor patients. However, since the MSC-1 antibody can only bind to the specific epitope of LIF, it cannot completely compete with the binding of LIF and its receptor. EC359 is a small molecule inhibitor targeting LIFR (EC359: A First-in-Class Small-Molecule Inhibitor for Targeting Oncogenic LIFR Signaling in Triple-Negative Breast Cancer, DOI: 10.1158 / 1535-7163.MCT-18-1258).

Summary of the Invention

Problems to be Solved by the Invention

[0008] Since there is no report on therapeutic antibodies against LIFR in the prior art, an efficient and specific antigen-blocking binding protein for the LIF receptor (LIFR) is needed for the treatment of LIFR-related diseases.

[0009] In response to the above drawbacks, the present invention provides a fully human LIFR antigen-binding protein, its production method and application for the first time. The present invention immunizes H2L2 human antibody transgenic mice with human LIFR-ECD protein and uses single B cell cloning technology (SBC) to obtain a series of human LIFR antibodies with biological functions. This method greatly improves the efficiency and yield of antibody drug development, and the obtained human LIFR antibodies have good affinity, can block the binding of huLIF protein and huLIFR / gp130 cells, suppress signal pathways such as STAT3, inhibit tumor growth, and achieve the purpose of tumor prevention and treatment.

[0010] To achieve the object of the present invention, the technical configuration of the present invention is as follows.

Means for Solving the Problems

[0011] In one aspect, the present invention provides a human LIFR antigen-binding protein. Specifically, the human LIFR antigen-binding protein (1) Heavy chain complementarity-determining region 1 HCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 1-SEQ ID NO: 4 or a variant sequence thereof, (2) Heavy chain complementarity-determining region 2 HCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 5-SEQ ID NO: 15 or a variant sequence thereof, (3) Heavy chain complementarity-determining region 3 HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 16-SEQ ID NO: 25 or a variant sequence thereof, (4) Light chain complementarity-determining region 1 LCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 26-SEQ ID NO: 32 or a variant sequence thereof, (5) Light chain complementarity-determining region 2 LCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO: 33-SEQ ID NO: 40 or a variant sequence thereof, (6) Light chain complementarity-determining region 3 LCDR3 comprising the amino acid sequence shown in any one of SEQ ID NO: 41-SEQ ID NO: 45 or a variant sequence thereof, including the complementarity-determining regions.

[0012] Preferably, the variant sequence is a CDR sequence having one or more amino acid substitutions, deletions or additions as compared to the CDR from which it is derived, and the substitution is a conservative substitution.

[0013] More specifically, the human LIFR antigen-binding protein (1) GFTFSSYGMX 1 is a heavy chain complementarity-determining region 1 HCDR1 comprising the amino acid sequence shown or the amino acid sequence shown in SEQ ID NO: 4: GFTFSNYAMT, wherein X 1 = H, D or N, and / or (2) VIWYDGX2 NKYYX 3 The amino acid sequence shown in DSVKG, VIWFDGSX 4 The heavy chain complementarity determining region 2 HCDR2 comprising the amino acid sequence shown in KYYADSVKG, SEQ ID NO: 7: the amino acid sequence shown in VIWYDGSNKFYADSVRG, SEQ ID NO: 14: the amino acid sequence shown in TISGSGAFTYYADAVKG or SEQ ID NO: 15: the amino acid sequence shown in VISGSGFLTYYADAVKG, wherein X 2 = N or S, X 3 = E, A or T, X 4 = N, I, L or V, and / or (3) the amino acid sequence shown in SEQ ID NO: 16: DGESSMVRGLLNWFDP, the amino acid sequence shown in SEQ ID NO: 17: ELRYFDWLLSPFDY, the amino acid sequence shown in SEQ ID NO: 21: GERTLDL, ELWFGELLSPX 5 The amino acid sequence shown in DF, GQLVX 6 DX 7 the amino acid sequence shown in or GGILTGFDX 8 The heavy chain complementarity determining region 3 HCDR3 comprising the amino acid sequence shown in or, wherein X 5 = L or F, X 6 = G or Q, X 7 = Y, F or L, X 8 = N or Y, and / or (4) RASQSX 9 SSSX 10 The amino acid sequence shown in LA, RASQSISSX 11 LX 12 The light chain complementarity determining region 1 LCDR1 comprising the amino acid sequence shown in or SEQ ID NO: 32: the amino acid sequence shown in RASQNLNSNLA, wherein X 9 = V or I, X 10 = Y or F, X 11 = Y, W or N, X 12 = N or A, and / or (5) GX 13 The amino acid sequence shown in SSRAT, KASX 14 LEX 15The light chain complementarity determining region 2 LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 35: AASNRAT, the amino acid sequence shown in SEQ ID NO: 36: AASSLQS, or the amino acid sequence shown in SEQ ID NO: 40: GASTRAP, wherein X 13 = A or T, X 14 = S or N, X 15 = S or N, and / or (6) the light chain complementarity determining region 3 LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 41: QQYGS SPFT, the amino acid sequence shown in SEQ ID NO: 42: QQSYS TPLT, the amino acid sequence shown in SEQ ID NO: 43: QQYKS FSPGGLT, the amino acid sequence shown in SEQ ID NO: 44: QQYKS NPLT, or the amino acid sequence shown in SEQ ID NO: 45: QQYNN WPRT, comprising the complementarity determining region as described above.

[0014] More specifically, the human LIFR antigen-binding protein is (1) the heavy chain variable region VH comprising the amino acid sequence shown in any of SEQ ID NOs: 46 - 59, and / or the light chain variable region VL comprising the amino acid sequence shown in any of SEQ ID NOs: 60 - 69, or (2) VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any of the VH in (1), and / or VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any of the VL in (1), or (3) VH having one or several amino acid substitutions, deletions or additions or any combination thereof compared to any of the VH in (1), and / or VL having one or several amino acid substitutions, deletions or additions or any combination thereof compared to any of the VL in (1), comprising wherein said substitution is a conservative substitution.

[0015] More specifically, the human LIFR antigen-binding protein is (1) a heavy chain HC comprising an amino acid sequence shown in any of SEQ ID NOs: 70 - 83, and / or a light chain LC comprising an amino acid sequence shown in any of SEQ ID NOs: 84 - 93, or (2) a heavy chain and a light chain, wherein, compared with the heavy chain and the light chain of (1), the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and / or the light chain has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and comprises.

[0016] More specifically, the human LIFR antigen-binding protein is (1) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 5, HCDR3 shown in SEQ ID NO: 16, and / or LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 33, LCDR3 shown in SEQ ID NO: 41, (2) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 17, and / or LCDR1 shown in SEQ ID NO: 27, LCDR2 shown in SEQ ID NO: 34, LCDR3 shown in SEQ ID NO: 41, (3) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 18, and / or LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 33, LCDR3 shown in SEQ ID NO: 41, (4)HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 19, and / or LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 33, LCDR3 shown in SEQ ID NO: 41, (5)HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 8, HCDR3 shown in SEQ ID NO: 19, and / or LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 35, LCDR3 shown in SEQ ID NO: 41, (6)HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 9, HCDR3 shown in SEQ ID NO: 19, and / or LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 35, LCDR3 shown in SEQ ID NO: 41, (7)HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 20, and / or LCDR1 shown in SEQ ID NO: 29, LCDR2 shown in SEQ ID NO: 36, LCDR3 shown in SEQ ID NO: 42, (8)HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 21, and / or LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 37, LCDR3 shown in SEQ ID NO: 43, (9)HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 22, and / or LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, LCDR3 shown in SEQ ID NO: 44, (10)HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 22, and / or LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 39, LCDR3 shown in SEQ ID NO: 44, (11)HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 23, and / or LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, LCDR3 shown in SEQ ID NO: 44, (12)HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 22, and / or LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, LCDR3 shown in SEQ ID NO: 44, (13) The HCDR1 shown in SEQ ID NO: 1, the HCDR2 shown in SEQ ID NO: 13, the HCDR3 shown in SEQ ID NO: 22, and / or the LCDR1 shown in SEQ ID NO: 30, the LCDR2 shown in SEQ ID NO: 38, the LCDR3 shown in SEQ ID NO: 44, (14) The HCDR1 shown in SEQ ID NO: 4, the HCDR2 shown in SEQ ID NO: 14, the HCDR3 shown in SEQ ID NO: 24, and / or the LCDR1 shown in SEQ ID NO: 31, the LCDR2 shown in SEQ ID NO: 37, the LCDR3 shown in SEQ ID NO: 43, (15) The HCDR1 shown in SEQ ID NO: 4, the HCDR2 shown in SEQ ID NO: 15, the HCDR3 shown in SEQ ID NO: 25, and / or the LCDR1 shown in SEQ ID NO: 32, the LCDR2 shown in SEQ ID NO: 40, the LCDR3 shown in SEQ ID NO: 45, comprising.

[0017] Preferably, the human LIFR antigen-binding protein performs sequence similarity analysis using Mega software and comprises the following five groups. Group 1: The antigen-binding protein described in (1). Group 2: The antigen-binding proteins described in (2), (3), (4). Group 3: The antigen-binding proteins described in (5), (6). Group 4: The antigen-binding proteins described in (7), (8), (9), (10), (11), (12), (13). Group 5: The antigen-binding proteins described in (14), (15).

[0018] More specifically, the human LIFR antigen-binding protein is (1) VH shown in SEQ ID NO: 46, and / or VL shown in SEQ ID NO: 60, (2) VH shown in SEQ ID NO: 47, and / or VL shown in SEQ ID NO: 61, (3) VH shown in SEQ ID NO: 48, and / or VL shown in SEQ ID NO: 62, (4) VH shown in SEQ ID NO: 49, and / or VL shown in SEQ ID NO: 62, (5) VH shown in SEQ ID NO: 50, and / or VL shown in SEQ ID NO: 63, (6) VH shown in SEQ ID NO: 51, and / or VL shown in SEQ ID NO: 64, (7)VH shown in SEQ ID NO: 52 and / or VL shown in SEQ ID NO: 65, (8)VH shown in SEQ ID NO: 53 and / or VL shown in SEQ ID NO: 66, (9)VH shown in SEQ ID NO: 54 and / or VL shown in SEQ ID NO: 67, (10)VH shown in SEQ ID NO: 54 and / or VL shown in SEQ ID NO: 68, (11)VH shown in SEQ ID NO: 55 and / or VL shown in SEQ ID NO: 67, (12)VH shown in SEQ ID NO: 56 and / or VL shown in SEQ ID NO: 67, (13)VH shown in SEQ ID NO: 57 and / or VL shown in SEQ ID NO: 67, (14)VH shown in SEQ ID NO: 58 and / or VL shown in SEQ ID NO: 66, (15)VH shown in SEQ ID NO: 59 and / or VL shown in SEQ ID NO: 69, or a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any of the VH in (1)-(15), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to any of the VL in (1)-(15), or a VH having one or several amino acid substitutions, deletions, additions, or any combination thereof compared to any of the VH in (1)-(15), and / or a VL having one or several amino acid substitutions, deletions, additions, or any combination thereof compared to any of the VL in (1)-(15), comprising, wherein said substitution is a conservative substitution.

[0019] More specifically, the human LIFR antigen-binding protein is (1) HC shown in SEQ ID NO: 70 and / or LC shown in SEQ ID NO: 84, (2) HC shown in SEQ ID NO: 71 and / or LC shown in SEQ ID NO: 85, (3) HC shown in SEQ ID NO: 72 and / or LC shown in SEQ ID NO: 86, (4) HC shown in SEQ ID NO: 73 and / or LC shown in SEQ ID NO: 86, (5) HC shown in SEQ ID NO: 74 and / or LC shown in SEQ ID NO: 87, (6) HC shown in SEQ ID NO: 75 and / or LC shown in SEQ ID NO: 88, (7) HC shown in SEQ ID NO: 76 and / or LC shown in SEQ ID NO: 89, (8) HC shown in SEQ ID NO: 77 and / or LC shown in SEQ ID NO: 90, (9) HC shown in SEQ ID NO: 78 and / or LC shown in SEQ ID NO: 91, (10) HC shown in SEQ ID NO: 78 and / or LC shown in SEQ ID NO: 92, (11) HC shown in SEQ ID NO: 79 and / or LC shown in SEQ ID NO: 91, (12) HC shown in SEQ ID NO: 80 and / or LC shown in SEQ ID NO: 91, (13) HC shown in SEQ ID NO: 81 and / or LC shown in SEQ ID NO: 91, (14) HC shown in SEQ ID NO: 82 and / or LC shown in SEQ ID NO: 90, (15) HC shown in SEQ ID NO: 83 and / or LC shown in SEQ ID NO: 93, Or, a heavy chain and a light chain, wherein compared with the heavy chain and light chain of (1)-(15), the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and / or the light chain has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. comprises

[0020] More specifically, the human LIFR antigen-binding protein comprises a chimeric antibody, a humanized antibody or a fully human antibody.

[0021] More specifically, the human LIFR antigen-binding protein comprises a full-length antibody, Fab, Fab’, F(ab’)2, Fv, scFv, di-scFv, bispecific antibody, multispecific antibody, heavy-chain antibody and / or single-domain antibody, or a monoclonal antibody and / or polyclonal antibody produced from the above antibodies.

[0022] In another aspect, the present invention provides a series of nucleic acid molecules encoding the human LIFR antigen-binding protein.

[0023] Specifically, the nucleic acid molecule comprises one or more codon-optimized nucleic acid molecules.

[0024] In another aspect, the present invention provides a series of vectors comprising one or more nucleic acid molecules described in the present invention.

[0025] Specifically, the vector includes, but is not limited to, a plasmid, a virus, and a phage.

[0026] In another aspect, the present invention provides a series of host cells comprising the nucleic acid molecule or the vector.

[0027] Specifically, the host cell includes, but is not limited to, a microorganism, a plant or an animal cell, and the vector described in the present invention can be introduced into the host cell by methods known to those skilled in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0028] In another aspect, the present invention provides a chimeric antigen receptor comprising the human LIFR antigen-binding protein.

[0029] In another aspect, the present invention provides an immune cell comprising the chimeric antigen receptor.

[0030] In another aspect, the present invention provides an antigen-binding protein derivative comprising the human LIFR antigen-binding protein and a detectable labeling molecule.

[0031] Specifically, the detectable labeling molecule is an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin.

[0032] In another embodiment, the present invention provides a multispecific antibody comprising the human LIFR antigen-binding protein and another antibody or a fragment thereof or an antibody mimetic.

[0033] Specifically, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetravalent antibody.

[0034] In another embodiment, the present invention provides an antibody-drug conjugate, the antibody-drug conjugate comprising an antibody portion and a conjugate portion, the antibody portion comprising the human LIFR antigen-binding protein, the conjugate portion comprising a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, but not limited thereto, and the antibody portion and the conjugate portion are conjugated by a chemical bond or a linker.

[0035] In another embodiment, the present invention provides a pharmaceutical composition comprising the human LIFR antigen-binding protein, a nucleic acid molecule, a vector, a host cell, a chimeric antigen receptor, an immune cell, an antigen-binding protein derivative, a multispecific antibody, and / or an antibody-drug conjugate.

[0036] Specifically, the pharmaceutical composition further comprises any pharmaceutically acceptable vector.

[0037] More specifically, the pharmaceutically acceptable vector includes, but is not limited to, diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents, and binders.

[0038] Specifically, the drug composition further includes a combination therapeutic agent, and the combination therapeutic agent includes, but is not limited to, chemotherapeutic agents, radiotherapy agents, immunosuppressive agents, and cytotoxic drugs.

[0039] In yet another aspect, the present invention provides a method for producing the human LIFR antigen-binding protein, and the method includes culturing the host cell in a state where the antigen-binding protein is expressed.

[0040] Specifically, the method (1) a step of immunizing a mouse with an LIFR-ECD protein as an antigen, (2) a step of screening for antigen-specific B cells that secrete antibodies, (3) a step of recovering the sequences of the antibody heavy chain and light chain from the single B cell screened in step (2), (4) a step of recombining the antibody heavy chain and light chain recovered in step (3) with human Fc, introducing them into a host cell, culturing the cells, and purifying to obtain a human LIFR antigen-binding protein, and includes.

[0041] More specifically, the mouse described in step (1) is a humanized mouse.

[0042] Preferably, the humanized mouse is a HarbourH2L2 mouse, and the HarbourH2L2 transgenic mouse can produce a conventional double-chain two-light-chain immunoglobulin antibody having a complete human variable region.

[0043] Even more specifically, the method for screening antigen-specific B cells that secrete the antibody described in step (2) is to screen using the plasma cell discovery workflow of the Beacon optoelectronic system.

[0044] More specifically, the method for restoring the sequences of the antibody heavy and light chains described in step (3) is single B cell sequencing.

[0045] Even more specifically, the steps of the single B cell sequencing are to purify RNA from a single B cell lysate, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of heavy and light chains, cloning and transfection, Sanger sequencing, perform uniqueness and clustering on the obtained sequences, and finally perform plasmid synthesis on the paired heavy and light chain DNA sequences.

[0046] In another aspect, the present invention provides the application of the above-mentioned human LIFR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, multispecific antibody, antibody-drug conjugate, and / or drug composition in the manufacture of LIF and / or LIFR blocking drugs, kits, and / or medical devices.

[0047] Specifically, the above-mentioned LIFR blocking drugs, kits and / or devices are mainly applied to diseases in which the expression levels of LIF and / or LIFR increase.

[0048] In another aspect, the present invention provides the application of the above-mentioned human LIFR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, multispecific antibody, antibody-drug conjugate, and / or drug composition in the manufacture of drugs, kits, and / or administration devices for preventing and / or treating LIFR-positive diseases.

[0049] In another aspect, the present invention provides the application of the above-mentioned human LIFR antigen-binding protein and antigen-binding protein derivative in the manufacture of LIFR detection reagents or kits.

[0050] In another aspect, the present invention provides a method for detecting LIFR by qualitatively or quantitatively analyzing and detecting LIFR using the human LIFR antigen-binding protein described above. Preferably, the detection method is used for non-disease diagnosis or non-therapeutic purposes.

[0051] In another aspect, the present invention provides a method for treating LIFR-positive related diseases, which comprises administering an effective amount of the above-mentioned human LIFR antigen-binding protein, immune cells, antigen-binding protein derivatives, bispecific antibodies, antibody-drug conjugates and / or pharmaceutical compositions to a subject in need thereof.

[0052] Specifically, the LIFR-positive disease is a tumor, and the tumor includes, but is not limited to, cholangiocarcinoma, colorectal cancer, glioma, prostate cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, breast cancer, bladder cancer, pancreatic cancer, non-small cell lung cancer.

[0053] In another aspect, the present invention provides a kit comprising the above-mentioned human LIFR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative, bispecific antibody, antibody-drug conjugate and / or pharmaceutical composition, as well as any instructions.

[0054] In another aspect, the present invention provides an administration device comprising (1) an infusion module for administering the pharmaceutical composition to a subject in need thereof, and (2) any drug efficacy monitoring module.

[0055] In another aspect, the present invention provides the application of the LIFR antigen-binding protein in the manufacture of an anti-cancer drug, and the cancer is selected from cholangiocarcinoma, colorectal cancer, glioma, prostate cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, breast cancer, bladder cancer, pancreatic cancer, non-small cell lung cancer.

[0056] Specifically, the LIFR antigen-binding protein includes any of the above-mentioned LIFR antigen-binding proteins.

Advantages of the Invention

[0057] Compared with the prior art, the present invention has the following advantageous effects. (1) The present invention provides a human LIFR antibody for the first time. The human LIFR antibody is produced by immunizing a human antibody transgenic mouse (for example, HarbourH2L2 mouse), and has a complete human antibody amino acid and gene sequence, thereby ensuring the lowest possible immunogenicity when used in the human body and removing toxic side effects. (2) The present invention uses single B cell cloning technology (SBC) to screen positive clones, and can significantly improve the efficiency and yield of antibody discovery compared with the conventional monoclonal antibody screening technology. (3) The human LIFR antibody produced by the present invention has high specificity, high affinity, and good antitumor activity. (4) Since the antibody of the present invention is a complete human antibody, it can be safely administered to human subjects without causing an immunogenic reaction, and has significant clinical value.

Brief Description of the Drawings

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Embodiments for Carrying out the Invention

[0059] Hereinafter, the present invention will be described in more detail with reference to specific examples. The following examples do not limit the present invention and are merely for explaining the present invention. The test methods used in the following examples are normal conditions unless otherwise specified in the examples when the specific conditions are not specified in the test methods, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0060] (Definition of Terms) To more easily understand the present invention, several technical terms and scientific terms are specifically defined below. Unless otherwise clearly defined in this specification, all other technical terms and scientific terms used in this specification have the meanings commonly understood by those skilled in the art to which this disclosure pertains.

[0061] The three-letter code and one-letter code of amino acids used in the present invention are described, for example, in J. biol. chem, 243, p3558 (1968, IUPAC-IUB Commission).

[0062] The "antigen-binding protein" described in the present invention generally refers to a protein containing a portion that binds to an antigen, and optionally employs a conformational stent or backbone portion that promotes the binding of the portion that binds to the antigen to the antigen. Typically, it may include an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions can be further distinguished as hypervariable regions called complementarity-determining regions (CDRs) dispersed in more conserved regions called framework regions (FR or FWR). The variable regions of the heavy and light chains contain binding domains that interact with the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab’, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb), immune complexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, chimeric antigen receptors or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

[0063] The "antibody" described in the present invention refers to an immunoglobulin, which has a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. Since the amino acid composition and sequence order of the heavy chain constant region of the immunoglobulin are different, their antigenicity is also different. Thus, immunoglobulins can be classified into five types, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig can be divided into different subclasses according to the differences in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain can be divided into κ chain or λ chain according to the difference in the constant region. Among the five types of Ig, each type of Ig may have a κ chain or a λ chain.

[0064] The sequences of approximately 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly and are the variable regions (Fv regions), while the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions. The variable region contains three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) consists of three CDR regions and four FR regions, and the order of arrangement from the amino terminus to the carboxy terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0065] The term "complementary determining region" (CDR) refers to one of six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The CDRs can be determined based on various numbering systems known in the art, such as, for example, the Kabat, Chothia, or AbM or IMGT numbering systems. In the present invention, the amino acid sequence boundaries of the CDRs are determined using the "Kabat numbering convention" (see Kabat et al. (1991)).

[0066] The term "framework region" or "FR" residues refers to amino acid residues in the antibody variable region other than the CDR residues defined above.

[0067] The terms "monoclonal antibody", "single clone antibody", "mAb" refer to an antibody obtained from a group of substantially homogeneous antibodies, i.e., except for possible variant antibodies, the individual antibodies constituting the population bind to the same and / or the same epitope. Usually, unlike polyclonal antibody preparations that contain different antibodies against different determinants, each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, "monoclonal" means the property of an antibody obtained from a substantially homogeneous antibody population and should not be construed as requiring any particular method for manufacturing the antibody. The monoclonal antibodies described in the present invention can be produced by various techniques known to those skilled in the art, including but not limited to the hybridoma method, recombinant DNA method, phage display method, and transgenic method.

[0068] The term "humanized monoclonal antibody" refers to an antibody produced by grafting mouse CDR sequences into the human antibody variable region framework, i.e., different types of human germline antibody framework sequences, which can overcome the heterologous reactions induced by chimeric antibodies carrying a large amount of mouse protein components. In order to avoid causing a decrease in activity along with the decrease in immunogenicity, minimal reverse mutations (or revertant mutations) can be made to the human antibody variable region framework sequence to retain the activity. To produce humanized antibodies, mouse CDR regions can be inserted into human-derived framework sequences using methods known in the art (see U.S. Patent No. 5,225,539 to Winter; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.; and Lo, Benny, K.C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be utilized, which do not produce endogenous immunoglobulins after immunization and can produce a fully human antibody library (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258; Lonberg et al., (1994) Nature 368(6474):856-859; WO02 / 43478). Another method for humanizing antibodies further includes phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).Here, examples of "transgenic animals" include, but are not limited to, HARBOUR BIOMED's H2L2 humanized mice. The terms "specific binding" and "selective binding" refer to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be represented by the dissociation equilibrium constant (KD) of the interaction. In this specification, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction for explaining the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Usually, an antibody binds with an affinity (KD) of less than about 10. -8 M, for example, about 10 -9 M, 10 -10 M, 10 -11 M or lower affinity (KD).

[0069] The term "identity" usually refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to those in a second reference polypeptide sequence or a portion thereof, after aligning the sequences, introducing gaps as necessary to achieve the maximum percent sequence identity, and not considering conservative substitutions as part of sequence identity. Alignment to determine the percent identity of amino acid / nucleotide sequences can be achieved by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR). One of ordinary skill in the art can determine appropriate parameters for measuring the comparison, including any algorithm necessary to achieve a maximum comparison over the entire length of the sequences being compared. The percent identity can be measured over the entire length of the determined polypeptide / polynucleotide sequence, or over a shorter length, for example, the length of a fragment obtained from a larger determined polypeptide / polynucleotide sequence. It should be understood that in a table, drawing, or sequence listing, the length of any fragment supported by the sequences shown herein can be used as the length for measuring the percent identity. Sequences having "% identity" retain important biological activities of the sequences to which they are compared or from which they are derived, such as antibody binding specificity. Sequences having one or more amino acid substitutions, deletions, or additions, or any combination thereof, retain important biological activities of the sequences to which they are compared or from which they are derived, such as antibody binding specificity. Nucleotide sequences having "% identity" or nucleotide sequences with differences of 3, 6, 15, 30, or 45 or fewer nucleotides can achieve functions similar to those of the nucleotide sequences to which they are compared or from which they are derived, for example, any of the expressed proteins can specifically bind to the same antigen or molecule.

[0070] The term "conservative substitution" refers to amino acid substitutions that do not adversely affect or change the desired properties of a protein / polypeptide containing an amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve using an amino acid residue with a similar side chain in place of an amino acid residue, e.g., substitution by a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Sci USA 94:412-417 (1997), which are incorporated herein by reference).

[0071] The term "vector" refers to a nucleic acid transport tool into which a polynucleotide can be inserted. When the protein encoded by the polynucleotide inserted with the vector is expressed, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, and the genetic material element carried thereon can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); phages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). Vectors can contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may contain an origin of replication site.

[0072] The term "host cell" generally refers to a somatic cell, cell line, or cell culture that may or does contain a plasmid or vector containing the nucleic acid molecule described in the present invention, or is capable of expressing the antibody or antigen-binding fragment thereof described in the present invention. Said cells can include the progeny of a single host cell. Due to natural, accidental, or deliberate mutations, the progeny cells and the original parent cells may not necessarily be exactly the same morphologically or genomically, but as long as they can express the antibody or antigen-binding fragment thereof described in the present application. Said cells can be obtained by transfecting cells in vitro using the vectors described in the present disclosure. Said cells may be prokaryotic cells (e.g., E. coli), or may be eukaryotic cells (e.g., yeast cells, e.g., COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells). In some cases, said cells may be mammalian cells. For example, said mammalian cells may be CHOK1 cells.

[0073] The term "pharmaceutically acceptable vector" refers to a vector that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is known in the art (see, e.g., Remington’s Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, reagents for maintaining osmotic pressure, reagents for delaying absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal reagents such as parabens, trichlorotertiary butanol, phenol, sorbic acid, etc. Reagents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl and its analogs. Reagents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyhydric alcohols (e.g., glycerin). Preservatives include, but are not limited to, various antibacterial and antifungal reagents such as thimerosal, 2-phenoxyethanol, parabens, trichlorotertiary butanol, phenol, sorbic acid, etc. Stabilizers have the meaning usually understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, and include, but are not limited to, sodium glutamate, gelatin, SPGA, saccharides (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin or casein) or their degradation products (e.g., lactalbumin hydrolysate).

[0074] The term "pharmaceutical composition" generally refers to a formulation that exists in a form that enables the biological activity of the active ingredient and does not contain additional ingredients that have unacceptable toxicity to the subject to whom the composition is administered. The composition is sterile.

[0075] The term "subject" refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human. In some embodiments, the subject (e.g., a human) has or is at risk of having cancer (including, but not limited to, cholangiocarcinoma, colorectal cancer, glioma, prostate cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, breast cancer, bladder cancer, pancreatic cancer, non-small cell lung cancer).

[0076] The term "effective amount" means an amount sufficient to obtain or at least partially obtain the desired effect. For example, a prophylactically effective amount of a disease (e.g., an LIFR-positive related disease) is an amount sufficient to prevent, inhibit or delay the occurrence of the disease (e.g., an LIFR-positive related disease), and a therapeutically effective amount of a disease is an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. Measuring such an effective amount is within the ability of those skilled in the art. For example, an amount effective for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously.

[0077] The term "chimeric antigen receptor" refers to a chimeric protein formed by in vitro binding of the intracellular portion of the CD3-δ chain or FcεRIγ to the antigen-binding site of an antibody that recognizes a tumor antigen by chimeric antigen receptor T cells, transfection of a patient's T cells by gene transfer methods to express the chimeric antigen receptor, and after the patient's T cells are "recoded", a large number of tumor-specific CAR-T cells can be generated. The recoded chimeric antigen receptor T cells are administered to the patient's body, and such chimeric antigen receptors can specifically track and identify T cells like a GPS and induce them to kill tumor cells. Most chimeric antigen receptors consist of an extracellular antigen-binding region (composed of a light chain and a heavy chain derived from a monoclonal antibody, with a middle tough hinge region connecting them to form a single-chain antibody), a transmembrane region, and an intracellular signaling region. The CAR structure is obtained by in vitro genetic recombination of a scFv that recognizes a tumor-associated antigen and an intracellular signaling domain "immunoreceptor tyrosine activation motif".

[0078] The term "immune cell" refers to cells having a hematopoietic origin and acting in the immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0079] Abbreviations CDR: Complementary determining region in the immunoglobulin variable region. VH: Variable region of the antibody heavy chain. VL: Variable region of the antibody light chain. HC: Antibody heavy chain. LC: Antibody light chain. IgG: Immunoglobulin G. AbM: The AbM CDR definition method is derived from the related research of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hyper variable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272). This definition method integrates the partial definitions of both Kabat and Chothia. Kabat: The immunoglobulin comparison and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md., 1991). Chothia: The immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the positions of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). IMGT: Based on the numbering system of the international ImMunoGeneTics information system (The international ImMunoGeneTics information system (registered trademark) (IMGT)) initiated by Lefranc et al., reference can be made to Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. mAb: Monoclonal antibody. EC50: The concentration at which 50% of the effect or binding occurs. IC50: The concentration at which 50% inhibition occurs. ELISA: Enzyme-linked immunosorbent assay. PCR: Polymerase chain reaction. HRP: Horseradish peroxidase. LIFR: Leukemia inhibitory factor receptor. IL7Rα: Interleukin 7 receptor alpha subunit. TARC: Thymus and activation regulated chemokine. hFc: Human IgG antibody Fc fragment. KD: Dissociation equilibrium constant. HCDR1: Complementary determining region 1 in the variable region of the immunoglobulin heavy chain. HCDR2: Complementary determining region 2 in the variable region of the immunoglobulin heavy chain. HCDR3: Complementary determining region 3 in the variable region of the immunoglobulin heavy chain. LCDR1: Complementary determining region 1 in the variable region of the immunoglobulin light chain. LCDR2: Complementary determining region 2 in the variable region of the immunoglobulin light chain. LCDR3: Complementary determining region 3 in the variable region of the immunoglobulin light chain.

[0080] Details of the Invention Antigen-binding protein The present invention has produced a series of fully human monoclonal antibodies. These anti-human LIFR antibodies are produced by immunizing fully human antibody transgenic mice, molecular biology, and antibody engineering techniques, and having fully human antibody amino acid and gene sequences, ensuring the lowest possible immunogenicity during human application. Human antibody transgenic mouse technology was first developed by Abgenix (Xeno Mouse) and Madarex (HuMab mouse) and is used for the production of fully human antibodies (Lonberg, et al. (1994) Nature. 368(6474):856-859, Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93, Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764:536-546). Early transgenic mice produced complete human immunoglobulins (Ig), that is, the variable and constant regions of the antibody were completely of human origin. Since the human Ig Fc did not match the mouse host Fc receptor, the immune response was very weak, the antibody titer in the serum after antigen immunization was very low, showing low monoclonal antibody production efficiency. In recent years, the technology has undergone significant innovations and improvements mainly by adopting the same and similar constant region sequences as the host immunoglobulin (Ig) in the heavy and light chain variable regions. In the present invention, transgenic mice (HarbourH2L2) developed by Harbour BioMed were used to successfully produce LIFR fully human antibodies. The Harbour transgenic mice were introduced with human immunoglobulin variable region genes and rat immunoglobulin constant region genes, and the Ig expression of the mice themselves was inactivated. After antigen immunization, the transgenic mice can produce an immune response and antibody titer corresponding to normal mice (e.g., Balb / c).

[0081] In some embodiments, the antigen-binding protein provided by the present invention comprises the following complementarity-determining regions. (1) Heavy chain complementarity-determining region 1 HCDR1 comprising the amino acid sequence shown in any of SEQ ID NO: 1-SEQ ID NO: 4 or a variant sequence thereof (2) A heavy chain complementarity-determining region 2 HCDR2 comprising an amino acid sequence shown in any of SEQ ID NO: 5 to SEQ ID NO: 15 or a variant sequence thereof, (3) A heavy chain complementarity-determining region 3 HCDR3 comprising an amino acid sequence shown in any of SEQ ID NO: 16 to SEQ ID NO: 25 or a variant sequence thereof, (4) A light chain complementarity-determining region 1 LCDR1 comprising an amino acid sequence shown in any of SEQ ID NO: 26 to SEQ ID NO: 32 or a variant sequence thereof, (5) A light chain complementarity-determining region 2 LCDR2 comprising an amino acid sequence shown in any of SEQ ID NO: 33 to SEQ ID NO: 40 or a variant sequence thereof, (6) A light chain complementarity-determining region 3 LCDR3 comprising an amino acid sequence shown in any of SEQ ID NO: 41 to SEQ ID NO: 45 or a variant sequence thereof.

[0082] The variant sequence is a CDR sequence having one or more amino acid substitutions, deletions or additions compared to the CDR from which it is derived.

[0083] In some embodiments, the human LIFR antigen-binding protein provided by the present invention comprises the following complementarity-determining regions. (1) A heavy chain complementarity-determining region 1 HCDR1 comprising an amino acid sequence shown in GFTFSSYGMX 1 or the amino acid sequence shown in SEQ ID NO: 4: GFTFSNYAMT, wherein X 1 = H, D or N, and / or (2) VIWYDGX 2 NKYYX 3 DSVKG, the amino acid sequence shown in VIWFDGSX 4 KYYADSVKG, the amino acid sequence shown in SEQ ID NO: 7: VIWYDGSNKFYADSVRG, the amino acid sequence shown in SEQ ID NO: 14: TISGSGAFTYYADAVKG or the amino acid sequence shown in SEQ ID NO: 15: VISGSGFLTYYADAVKG, a heavy chain complementarity-determining region 2 HCDR2, wherein X 2 = N or S, X 3 = E, A or T, X 4 = N, I, L or V, and / or (3) the amino acid sequence shown in SEQ ID NO: 16: DGESSMVRGLLNWFDP, the amino acid sequence shown in SEQ ID NO: 17: ELRYFDWLLSPFDY, the amino acid sequence shown in SEQ ID NO: 21: GERTLDL, ELWFGELLSPX 5 the amino acid sequence shown in DF, GQLVX 6 DX 7 the amino acid sequence shown in or GGILTGFDX 8 a heavy chain complementarity determining region 3 HCDR3 comprising the amino acid sequence shown in, where X 5 = L or F, X 6 = G or Q, X 7 = Y, F or L, X 8 = N or Y, and / or (4) RASQSX 9 SSSX 10 the amino acid sequence shown in LA, RASQSISSX 11 LX 12 a light chain complementarity determining region 1 LCDR1 comprising the amino acid sequence shown in or the amino acid sequence shown in SEQ ID NO: 32: RASQNLNSNLA, where X 9 = V or I, X 10 = Y or F, X 11 = Y, W or N, X 12 = N or A, and / or (5) GX 13 the amino acid sequence shown in SSRAT, KASX 14 LEX 15 a light chain complementarity determining region 2 LCDR2 comprising the amino acid sequence shown in, the amino acid sequence shown in SEQ ID NO: 35: AASNRAT, the amino acid sequence shown in SEQ ID NO: 36: AASSLQS or the amino acid sequence shown in SEQ ID NO: 40: GASTRAP, where X 13 = A or T, X 14 = S or N, X 15 = S or N, and / or (6) a light chain complementarity determining region 3 LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 41: QQYGS SPFT, the amino acid sequence shown in SEQ ID NO: 42: QQSYSTPLT, the amino acid sequence shown in SEQ ID NO: 43: QQYKSFSPGGLT, the amino acid sequence shown in SEQ ID NO: 44: QQYKSNPLT or the amino acid sequence shown in SEQ ID NO: 45: QQYNNWPRT.

[0084] In some embodiments, the antigen-binding protein provided by the present invention comprises the following heavy-chain variable region VH and light-chain variable region VL. (1) A heavy-chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NOs: 46-59, and / or a light-chain variable region VL comprising the amino acid sequence shown in any one of SEQ ID NOs: 60-69, or (2) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one of the VHs in (1), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to any one of the VLs in (1), or (3) a VH having one or several amino acid substitutions, deletions or additions or any combination thereof compared to any one of the VHs in (1), and / or a VL having one or several amino acid substitutions, deletions or additions or any combination thereof compared to any one of the VLs in (1). The above substitutions are conservative substitutions.

[0085] In some embodiments, the antigen-binding protein provided by the present invention comprises the following heavy chain HC and light chain LC. (1) A heavy chain HC comprising the amino acid sequence shown in any one of SEQ ID NOs: 70-83, and / or a light chain LC comprising the amino acid sequence shown in any one of SEQ ID NOs: 84-93, or (2) a heavy chain and a light chain, wherein the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to the heavy chain of (1), and / or the light chain has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, a heavy chain and a light chain.

[0086] In some specific embodiments, the human LIFR antibody produced by the present invention comprises the combinations of heavy and light chains described in Table 1 below.

[0087] [Table 1-1] [Table 1-2] [Table 1-3]

[0088] The human LIFR antibodies shown in Table 1 can be divided into the following five groups by performing sequence similarity analysis with Mega software. Group 1: PR300516, Group 2: PR301164, PR301168, PR301176, Group 3: PR301152, PR301160, Group 4: PR301153, PR301177, PR301195, PR301196, PR301211, PR301215, PR301218, Group 5: PR301127, PR301172.

[0089] In some embodiments, PR300516, PR301127, PR301152, PR301168, and PR301211 were each selected from five groups of protein antibodies for testing.

[0090] Antibody derivatives and bispecific antibodies The antigen-binding protein provided by the present invention described in the foregoing part includes an antibody or an antigen-binding fragment, which can be derivatized (for example, bound to another molecule, for example, another polypeptide or protein). Usually, derivatization (for example, labeling) of the antibody or its antigen-binding fragment does not adversely affect binding to LIFR (especially human LIFR). Therefore, the antibodies or antigen-binding fragments of the present invention are intended to include such forms of derivatization. For example, the antibodies or antigen-binding fragments of the present invention can be linked to one or more other molecular groups to form bispecific antibodies, detection reagents, pharmaceutical reagents, and / or proteins or polypeptides (for example, avidin or a multi-histidine tag) that can mediate the binding of the antibody or antigen-binding fragment to other molecules.

[0091] One type of derivatized antibody is a labeled antibody. For example, the antibody of the present invention or its antigen-binding fragment can be linked to a detectable label. The detectable label described in the present invention may be any substance detectable by fluorescence, spectral, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well-known in the art and are not particularly limited to specific examples. For example, enzymes (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (such as 3H, 125I, 35S, 14C or 32P, etc.), fluorescent dyes (such as fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa750)), acridinium ester compounds, magnetic beads (such as Dynabeads (registered trademark)), gold colloids or calorimetric markers such as colored glass or plastic (such as polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (such as streptavidin) modified with the above markers. Patents teaching the application of such markers include, but are not limited to, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The detectable markers as described above can be detected by known methods in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, fluorescent markers can be detected using a photodetector, and the emitted light can be detected. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product generated by the action of the enzyme on the substrate, and calorimetric markers are detected by simply visualizing the colored marker.In some embodiments, such labels can be applied to immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, linkers of different lengths can be used to connect the detectable labels as described above to the antibodies or antigen-binding fragments thereof of the present invention to reduce potential steric hindrance.

[0092] In addition, the antibodies or antigen-binding fragments thereof of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl group or ethyl group, or glycosyl group. These groups can be used to improve the biological properties of the antibody, for example, to increase the serum half-life.

[0093] As another derivative of the antibody, the present invention provides a multispecific antibody comprising a first antibody or a fragment thereof and another antibody or a fragment thereof, or an antibody mimetic, wherein the first antibody or a fragment thereof, another antibody or a fragment thereof, or an antibody mimetic retains the original binding specificity. The first antibody or a fragment thereof is a (monoclonal) antibody that binds to any of the TSLPs of the present invention or an antigen-binding fragment thereof. As used herein, "antibody mimetic" means that it specifically binds to an antigen in the same manner as an antibody but does not have an antibody structure. These are usually artificial peptides or proteins with a molar mass of about 3 - 20 kDa, for example, ankyrin repeat proteins (DARPins) and fynomers. The designed ankyrin repeat protein (DARPin) can be linked to an IgG antibody, an scFv-Fc antibody fragment or a combination thereof as described in CN104341529A. The fynomer against IL-17a can produce a bispecific fusion polypeptide by fusing with an anti-IL-6R antibody as described in WO2015141862A1.

[0094] In some embodiments, the multispecific antibody is formed by coupling a first antibody or an antigen-binding fragment thereof with another antibody or an antigen-binding fragment thereof or an antibody analog, each antibody or an antigen-binding fragment thereof or an antibody analog retains its original binding specificity, and the first antibody or an antigen-binding fragment thereof is the antibody or an antigen-binding fragment thereof described in the present invention. In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetravalent antibody.

[0095] Antibody-drug conjugate An antibody-drug conjugate comprises an antigen-binding protein portion and a conjugate portion provided by the present invention. The antigen-binding protein portion includes the above-mentioned "antigen-binding protein" and an antigen-binding fragment obtained based on the "antigen-binding protein". Examples of antigen-binding fragments include, but are not limited to, Fab, Fab’, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb, etc. The conjugate portion may include at least one payload. The payload can include a drug active substance component and / or the labeling molecule. The payload is a pharmaceutically active small molecule compound or toxin or other drug molecular form, and may be a small molecule compound, toxin molecule, oligonucleotide, proteolysis-targeting chimera (PROTAC), affinity ligand, fluorescent group, radionuclide group, etc., but is not limited thereto. Various payloads are known. For example, a small molecule compound usually refers to a substance having strong cytotoxicity. Exemplary small molecule compounds can exert such cytotoxic and cytostatic effects by including mechanisms such as tubulin binding, DNA binding, RNA polymerase inhibition, protein synthesis or topoisomerase inhibition, etc., but are not limited thereto. For example, the small molecule compound may be a tubulin inhibitor, for example, the tubulin inhibitor may be maytansine (e.g., DM1 or DM4) and auristatin (e.g., MMAE or MMAF), etc. For example, the small molecule compound may be a DNA damaging agent, for example, the DNA damaging agent may be Calicheamicins, pyrrolobenzodiazepines (PBD), etc. For example, a proteolysis-targeting chimera (PROTAC) is a compound that can cause the degradation of a target protein by inducing the polyubiquitination of the target protein. For example, the PROTAC may be a degrader of BET protein. The drug conjugate may further include at least one linker. For example, the linker may include a cleavable linker or a non-cleavable linker. The linker is used to link one or more payloads to the antigen-binding protein. In the present application, a cleavable linker may be a "cleavable" linker that is easy to release the drug.For example, the cleavable linker includes, but is not limited to, an acid-sensitive linker, a protease-sensitive linker, a photosensitive linker, or a disulfide-containing linker. The linker may include one or more linker members, and in this art, multiple types of linker members are known, for example, maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit or vc), p-aminobenzyloxycarbonyl (PAB).

[0096] Production of Nucleic Acids, Vectors, Host Cells, and Antibodies The antigen-binding protein of the present invention can be produced by various methods known in the art, such as genetic recombination technology. For example, DNA molecules encoding the heavy and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification. After inserting the obtained DNA molecule into an expression vector, it is transfected into a host cell. Then, the transfected host cell is cultured under specific conditions to express the antibody of the present invention.

[0097] In some specific embodiments, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof of the present invention, or a heavy chain variable region and / or a light chain variable region thereof, or one or more CDRs thereof. According to the degeneracy of codons known in the art, in some embodiments, the nucleotide sequence may be substituted according to the degeneracy of codons. In some embodiments, the nucleotide sequence is codon-optimized.

[0098] In some embodiments, the present invention provides a cloning vector or an expression vector comprising the isolated nucleic acid molecule of the present invention. In some embodiments, the vector is, for example, a plasmid, a cosmid, a phage, a lentivirus, etc. In some embodiments, the vector can express the antibody of the present invention or its antigen-binding fragment in a subject (e.g., a mammal such as a human). In some embodiments, the present invention provides a host cell comprising the isolated nucleic acid molecule of the present invention or the vector of the present invention. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In some embodiments, the host cell of the present invention is a mammalian cell such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0099] In some embodiments, the present invention provides a method for producing the antibody of the present invention or its antigen-binding fragment, comprising culturing the host cell of the present invention under conditions that permit the expression of the antibody or its antigen-binding fragment, and recovering the antibody or its antigen-binding fragment from the cultured host cell culture.

[0100] Pharmaceutical uses, treatment methods, drug compositions and administration devices The present invention provides the use of the human LIFR antigen-binding protein, antigen-binding protein derivative, multispecific antibody, immune cell, antibody-drug conjugate in the manufacture of a LIF and / or LIFR blocker, and in the manufacture of a drug for the prevention and / or treatment of LIF and / or LIFR positive diseases.

[0101] Correspondingly, the present invention provides a method for blocking LIF and / or LIFR of the human LIFR antigen-binding protein, antigen-binding protein derivative, multispecific antibody, immune cell, antibody-drug conjugate, and a method for preventing and / or treating LIF and / or LIFR positive diseases.

[0102] In some embodiments, the present invention provides a therapeutic drug composition comprising the human LIFR antigen-binding protein, nucleic acid molecule, vector, host cell, immune cell, antigen-binding protein derivative, multispecific antibody and / or antibody-drug conjugate, and any pharmaceutically acceptable vector. "Pharmaceutically acceptable vector" includes, but is not limited to, diluents, excipients, fillers, wetting agents, disintegrants, flavoring agents and binders.

[0103] In some embodiments, the drug composition further comprises a combination therapeutic agent, which includes, but is not limited to, chemotherapeutic agents, radiotherapy agents, immunosuppressive agents, and cytotoxic drugs.

[0104] In some embodiments, in the drug composition, the antigen-binding protein of the present invention and the combination therapeutic agent are provided as isolated components or as components of the same composition. Thus, the antigen-binding protein of the present invention and the combination therapeutic agent may be administered together or separately, simultaneously or sequentially. The anti-LIFR antibody or its antigen-binding fragment can be administered alone or in combination with other therapeutic agents. The anti-LIFR antibody or its antigen-binding fragment and one or more other therapeutic agents can be administered separately, simultaneously or sequentially.

[0105] The drug composition may have any suitable form (which may be determined according to the desired method of administration to the patient), and the human LIFR antibody of the present invention may be administered to the patient by various routes (for example, oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, topical, intrathecal and intracerebral). In any case, the optimal route of administration depends on the specific antibody, the nature and severity of the individual and the disease, and the physical condition of the individual.

[0106] In some embodiments, the present invention further provides an administration device for administering the antigen-binding protein, antigen-binding protein derivative, multispecific antibody, immune cell, antibody-drug conjugate and the drug composition comprising the components. The administration device includes the following. (i) An infusion module for administering a drug composition having one active ingredient to a subject, (ii) An infusion drug composition containing one active ingredient, wherein the active ingredient is selected from the group consisting of an antigen-binding protein, an antigen-binding protein derivative, a multispecific antibody, an immune cell, an antibody-drug conjugate, or a combination thereof, and (iii) Any drug efficacy monitoring module.

[0107] In another preferred example, the administration includes parenteral administration and non-parenteral administration. In another preferred example, the parenteral administration includes injection administration, and the routes of injection used include intravenous, intramuscular, intra-arterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and push injection. In another preferred example, the non-parenteral administration includes topical, epidermal, or mucosal administration, including, for example, intranasal, oral, vaginal, rectal, sublingual, or topical application. In another preferred example, the infusion module is a needleless subcutaneous injection device, a microinfusion pump, a transdermal administration device, an injection device, or an osmotic device.

[0108] Detection method, kit The antibody or antigen-binding fragment thereof of the present invention can bind to LIFR and can be used for detecting the presence or level of LIFR in a sample.

[0109] In one aspect, the present invention provides a kit containing the antigen-binding protein of the present invention. In some embodiments, the antigen-binding protein of the present invention has a detectable label. In a preferred embodiment, the kit further includes a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention. Preferably, the second antibody further includes a detectable label.

[0110] In the present invention, the detectable label may be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrochemistry, optics or chemical means. Particularly preferably, such a label is applicable to immunological detection (for example, enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.).

[0111] In the present invention, contacting a biological sample (somatic cells, tissues or body fluids) with one or more human LIFR antibodies of the present invention (optionally conjugated to a detectable moiety), and detecting whether the sample is positive with respect to LIFR expression, or detecting whether the sample has an expression that has changed (for example, decreased or increased) compared to a control sample, is provided for the detection of LIFR expression. In some embodiments, the tissue or body fluid is peripheral blood, peripheral blood leukocytes, a biopsy (for example, a lung or skin biopsy), and tissue. The method can be used for diagnostic purposes or non-diagnostic purposes (for example, LIF / LIFR pathway research, drug screening, histochemical analysis, etc.). In some embodiments, the sample used for non-diagnostic purposes is a cell sample such as a cell line or an in vitro cell culture.

[0112] In one embodiment, the present invention provides a method for detecting the presence or level of LIFR in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of the present invention under conditions capable of forming a complex between the antibody or antigen-binding fragment thereof and LIFR, and detecting the formation of the complex.

[0113] In another aspect, the present invention provides a method for diagnosing a LIFR-positive disease in a subject, comprising contacting an antibody or antigen-binding fragment thereof, a multispecific antibody, or an antibody-drug conjugate according to the present invention with a sample from the subject under conditions capable of forming a complex between the antibody or antigen-binding fragment thereof and LIFR, and detecting the formation of the complex, wherein an increase in LIFR level indicates the presence of cancer compared to a healthy control.

[0114] Preferably, the subject is a mammal, including non-human mammals and humans. Preferably, the subject is a human.

[0115] Preferably, the cancer is cholangiocarcinoma, colorectal cancer, glioma, prostate cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, breast cancer, bladder cancer, pancreatic cancer, non-small cell lung cancer.

[0116] In another aspect, the present invention provides a detection kit comprising the human LIFR antigen-binding protein, antigen-binding protein derivative, etc. described in the present invention. In a preferred embodiment, the kit includes an instruction manual for explaining its application method.

Example

[0117] Example 1 Immunization of Harbour transgenic mice 1. Immunogen: The extracellular region 1-833 of the recombinant human LIFR protein amino acid sequence (GenBank accession number: NP_001121143.1) was cloned into the pcDNA3.1 vector containing a his tag to produce a recombinant expression plasmid. HEK293 cells were transiently transfected and expanded in culture. Four days later, the cell culture medium was collected, and the culture supernatant was purified to obtain high-purity huLIFR-ECD protein (>90%), which had biological activity and was cryopreserved at -80°C after aliquoting. 2. Immunization of Harbour H2L2 mice: 6-8-week-old Harbour H2L2 transgenic mice were immunized with huLIFR-ECD protein. All transgenic mice were bred in a SPF-class environment. In the first immunization, 50 μg of huLIFR-ECD protein was emulsified with Freund's complete adjuvant (Sigma, F5881) and then intraperitoneally injected into the mice. For subsequent booster immunizations, 25 μg of huLIFR-ECD protein was uniformly mixed with RIBI adjuvant (Sigma, S6322) and then intraperitoneally injected into the mice. The interval between each immunization was 2 weeks. Blood was collected 7 days after immunization to detect serum antibody titers by ELISA, and mice with high serum antibody titers were selected for subsequent single B cell screening tests.

[0118] Example 2 Screening of Single B Cells Based on the Beacon (Registered Trademark) Optofluidic System The Beacon (Registered Trademark) Optofluidic System moves single cells using OptoElectro Positioning (OEP TM ) technology. The Beacon optoelectronic system is an automated biological instrument that can simultaneously perform various operations such as biological function tests, test analysis, and positive clone selection under cell culture conditions. The Beacon platform can automate these tasks in parallel on thousands of cells on a large scale. This application can screen up to 14k single plasma cells in each test using a plasma cell discovery workflow and is used to select antigen-specific plasma cells secreted by antibodies. Subsequently, the plasma cells secreting these specific antibodies are derived into 96-well plates containing cell lysates and used for subsequent single B cell sequencing to identify the sequences of the heavy and light chains of the antibodies produced by single B cells (monoclonal).

[0119] Example 3 Single B Cell Sequencing and Production of Fully Human Antibodies The present invention obtains the sequences of the heavy and light chains of antibodies from single plasma cells using a single B cell sequencing method. Single B cell sequencing has become a powerful tool for obtaining antibody sequences. General procedures include purifying RNA from single plasma cell lysates, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of heavy and light chains, cloning and transfection, and Sanger sequencing. Uniqueness and clustering are performed on the obtained sequences, and then plasmid synthesis is performed on the paired heavy and light chain DNA sequences. Specifically, after obtaining the light and heavy chain variable domain sequences encoding the antibody molecule, the light and heavy chain variable domain sequences and the corresponding light and heavy chain constant domain sequences of human antibodies can be fusion-expressed using ordinary recombinant DNA techniques to obtain recombinant antibody molecules. In this example, the antibody heavy chain variable domain sequence (VH) was cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence by gene synthesis, and encodes and produces the full-length heavy chain of the IgG1 antibody. The antibody light chain variable domain sequence (VL) was cloned into a mammalian cell expression plasmid vector encoding the human antibody Igκ light chain constant domain sequence by gene synthesis, and encodes and produces the full-length light chain of the antibody. In this example, since the sequence of the variable domain of the anti-LIFR monoclonal antibody molecule obtained from the immunized HarbourH2L2 mouse is a human antibody sequence, this example also obtained a fully human-derived anti-LIFR recombinant IgG1 antibody. Here, the human LIFR antibody heavy chain, light chain complementarity determining region (CDR) sequence, heavy chain, light chain variable region (V), heavy chain, and light chain sequences obtained in the present invention are as shown in Table 1 above. When sequence similarity analysis was performed on the 15 antibodies in Table 1 using Mega software, the antibodies could be divided into (1) Group 1: PR300516, (2) Group 2: PR301164, PR301168, PR301176, (3) Group 3: PR301152, PR301160, (4) Group 4: PR301153, PR301177, PR301195, PR301196, PR301211, PR301215, PR301218, (5) Group 5: PR301127, PR301172. Tests of Examples 4.2 and 4.4 were performed on all antibodies. Representative antibodies PR300516, PR301127, PR301152, PR301211, and PR301168 were selected from the 5 groups and tested in Examples 4.1, 4.3, and Example 5. In vivo PK and pharmacodynamics tests were performed on the representative antibodies PR300516 and PR301168. In five representative antibodies, the DNA nucleotide sequence of the PR300516 heavy chain variable region is represented by SEQ ID NO: 94, the DNA nucleotide sequence of the PR300516 light chain variable region is represented by SEQ ID NO: 95, the DNA nucleotide sequence of the PR301127 heavy chain variable region is represented by SEQ ID NO: 96, the DNA nucleotide sequence of the PR301127 light chain variable region is represented by SEQ ID NO: 97, the DNA nucleotide sequence of the PR301152 heavy chain variable region is represented by SEQ ID NO: 98, the DNA nucleotide sequence of the PR301152 light chain variable region is represented by SEQ ID NO: 99, the DNA nucleotide sequence of the PR301168 heavy chain variable region is represented by SEQ ID NO: 100, the DNA nucleotide sequence of the PR301168 light chain variable region is represented by SEQ ID NO: 101, the DNA nucleotide sequence of the PR301211 heavy chain variable region is represented by SEQ ID NO: 102, and the DNA nucleotide sequence of the PR301211 light chain variable region is shown in SEQ ID NO: 103.

[0120] Example 4 Expression and Purification of Recombinant Antibodies 293F cells are used for antibody expression. The cell density was adjusted to 1×10e6 cells / mL. Plasmids of the heavy and light chain antibodies were placed in a tube at a ratio of 1:1.5, and then the transfection reagent PEI was added. The ratio of PEI to DNA was 4:1. After uniform mixing, it was incubated at room temperature for 15 minutes. The above mixture was added to the cells to transfect the cells, and they were cultured in an incubator at 37°C and 5% CO 2 and shaken at 125 rpm. 24 hours after transfection, OPM-CHO PFF05 nutrient (OPM Biosciences, product number FB1279-001) was supplemented until the final concentration reached 3%. Five days after cell culture, when the cell viability decreased to 70% or less, the supernatant was collected. The transformed supernatant was subjected to protein purification using a protein G column.

[0121] 4.1 Antibody Affinity Detection Test Five antibodies, PR300516, PR301127, PR301152, PR301168, and PR301211, produced by the methods of Examples 1-4 of the present invention, were used to detect the binding kinetics to human LIFR-ECD protein by the method of Biolayer interferometry (BLI) (Fortebio octet RED 96e).

[0122] The test antibody was diluted to a final concentration of 6 μg / mL, immobilized directly on the AHC biosensor, and kinetic measurements were performed. The antigen protein (human LIF) was diluted to three concentrations of 100 nM, 50 nM, and 25 nM with 0.02% PBST20, injected in 70 s, with a binding time of 300 s, a dissociation time of 300 s, and regenerated with 10 mM glycine-HCl (pH 1.5) in 15 s. The association rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Langmuir binding model (Octet Red 96 data analysis software), and the equilibrium dissociation constant (kD) was calculated as the ratio kdis / kon. The measurement results of antibody affinity are shown in Table 2 below.

[0123]

Table 2

[0124] As can be seen from Table 2, the affinity of the human LIFR antibody is higher than that of the LIF antibody MSC-1.

[0125] 4.2 Detection of the binding ability of the antibody to LIFR-ECD at the protein level Based on the ELISA assay, the binding ability of the 15 antibodies produced in Examples 1-4 of the present invention to the LIFR-ECD protein was detected. By comparing the binding curves of different antibodies to the LIFR-ECD protein, their binding ability was measured. The specific test procedure was as follows: First, human LIFR-ECD, monkey LIFR-ECD, and mouse LIFR-ECD proteins were diluted to a concentration of 1 μg / mL in sequence, and 100 μL of each was added to a 96-well plate and left at 4°C overnight. After washing the 96-well plate three times with PBST solution, a PBS solution containing 2% BSA was added and left at 37°C for 1 hour. After serially diluting the antibody to be measured (100 nM, 10-fold dilution), it was added to the 96-well plate and incubated at 37°C for 1 h. After washing three times with PBST solution, an anti-human IgG Fc-HRP secondary antibody (used at a 5000-fold dilution) was added and incubated at 37°C for 30-60 minutes. After washing three times with PBST solution, TMB chromogenic solution was added for 5-15 minutes, and then the stop solution was added to terminate the color development.

[0126]

Table 3

[0127] As shown in Table 3 and Figure 1, the PR300516, PR301127, PR301172, PR301152, PR301153, PR301160, PR301164, PR301168, PR301176, PR301177, PR301195, PR301196, PR301211, PR301215, and PR301218 antibodies all have the ability to bind to human LIFR-ECD protein and monkey LIFR-ECD protein, but only the PR301127 and PR301172 antibodies can cross-bind to mouse LIFR-ECD protein.

[0128] 4.3 Detection of the binding ability of the antibody to LIFR and LIFR / gp130 at the cellular level Based on the flow cytometry measurement method, the binding ability of five antibodies PR300516, PR301127, PR301152, PR301168, and PR301211 produced in Examples 1-4 of the present invention to the human LIFR receptor or the human LIFR / gp130 receptor heterodimer expressed on the surface of 293T cells was detected. The binding ability was measured by comparing the binding curves of different antibodies to the human LIFR receptor or the human LIFR / gp130 receptor heterodimer expressed on the surface of 293T cells. The specific test procedure was as follows: (1) HEK293T cells were infected with lenti-huLIFR and lenti-LIFR / gp130 lentiviruses, and the infected cells were screened with puromycin to obtain stable cell pools: HEK293T-huLIFR and HEK293T-huLIFR / gp130; (2) Subcloning screening was performed on the stable cell pools to obtain monoclonal cell lines with high expression of the target gene; (3) Different concentrations of the antibody to be measured and the monoclonal cell line were incubated at 4°C for 30 minutes, washed three times with PBS, then incubated with an FITC-labeled goat anti-human secondary antibody at 4°C for 30 minutes, washed three times again with PBS, and then the cells were resuspended in 100 μL of FACS buffer; (4) The median fluorescence value of one channel was measured using a flow cytometer. The logarithm with base 10 of the antibody concentration was used as the horizontal axis, and the median fluorescence value of one channel was used as the vertical axis, and the EC50 and the curve peak value were compared.

[0129]

Table 4

[0130] As shown in Table 4 and Figure 2, the detection results show that the human LIFR antibody has a stronger binding ability to HEK293T-huLIFR / gp130 cells than to HEK293T-huLIFR cells under the same concentration conditions.

[0131] 4.4 Detection of the ability of the antibody to block the binding of LIF and LIFR / gp130 Based on the flow cytometry measurement method, the ability of 15 antibodies produced in Examples 1-4 of the present invention to block the binding of huLIF protein to the human LIFR / gp130 receptor heterodimer expressed on the surface of 293T cells was detected. By comparing the curves of different antibodies blocking the binding of huLIF protein to HEK293T-huLIFR / gp130 cells, the blocking ability was measured. The specific test procedure was as follows: (1) First, determine the EC80 concentration at which huLIF protein binds to HEK293T-huLIFR / gp130 cells. Biotin labeling was performed on huLIF protein to obtain huLIF-biotin protein; (2) Different concentrations of the antibody to be measured and huLIF protein (the final concentration was the EC80 value) and HEK29ET-huLIFR / gp130 cells were incubated at 4°C for 1 h, washed three times with PBS, then incubated with APC-labeled Strepavidin secondary antibody at 4°C for 30 min, washed three times again with PBS, and then the cells were resuspended with 100 μL of FACS buffer; (3) The central fluorescence value of the four channels was measured using a flow cytometer. The logarithm with base 10 of the antibody concentration was plotted on the x-axis, and the central fluorescence value of the four channels was plotted on the y-axis to compare the IC50 and the curve peak.

[0132]

Table 5

[0133] As shown in Table 5 and Figure 3, the detection results showed that compared with the negative control hIgG1, PR300516, PR301152, PR301153, PR301160, PR301164, PR301168, PR301176, PR301177, PR301195, PR301196, PR301211, PR301215, and PR301218 all had the function of clearly blocking the binding of huLIF to HEK293T-huLIFR / gp130.

[0134] Example 5 Detection of the inhibitory effect of the antibody on the STAT3 signaling pathway Based on Western blot, the inhibitory effect on the phosphorylation of STAT3 tyrosine 705 site in LIF induction was detected in Capan-2 pancreatic cancer cells, MDA-MB-231 breast cancer cells, A549 and NCI-H292 non-small cell lung cancer cells. The specific test procedure was as follows: First, tumor cells were seeded in 6-well plates, with 1x10 6 cells per well, and starved for 24 hours (serum was not added to the medium). Furthermore, a certain concentration of huLIF protein and the corresponding antibody were co-incubated in an incubator at 37°C for 1 h, the huLIF protein and antibody mixture was added to the tumor cells, and the cells were treated in an incubator at 37°C for 30 min. The medium was discarded and the cells were washed twice with pre-cooled PBS. 200 μL of pre-cooled buffer was added to each well, and a certain amount of protease inhibitor (100× dilution) and phosphatase inhibitor (50× dilution) were added. After incubating on ice for 30 min, the mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a new EP tube, 25 μL of the supernatant protein was taken for BCA quantification, and 5× SDS buffer was added to the rest, followed by treatment in a metal bath at 95°C for 5 min. After passing the protein sample through an SDS-PAGE gel and transferring the membrane, block buffer was added and incubated at room temperature for 1 h. The primary antibodies Stat 3 (124H6) Mouse mAb (Cell Signaling Technology, #9139), Phospho-Stat3 (Tyr705) Antibody (Cell Signaling Technology, #9131) were incubated overnight at 4°C. The membrane was washed 3 times with PBST solution, 10 min each time. The secondary antibodies Anti-Mouse IgG (Fc Specific)-Peroxidase (Sigma-Aldrich, A0168), Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson immune research, 111-035-045) were added and incubated at room temperature for 1 h. After washing 3 times with PBST, A+B developing solution and ChemiDoc Imaging System were added for exposure and photography.

[0135] As shown in Figure 4, the detection results indicate that the human LIFR antibody can inhibit the activation of the STAT3 signaling pathway induced by LIF in all four types of tumor cells, namely Capan-2 pancreatic cancer cells, MDA-MB-231 breast cancer cells, A549 and NCI-H292 non-small cell lung cancer cells.

[0136] Example 6 PK Detection of Antibody in Vivo 6.1. PK in Balb / c Mice The purpose of this study was to determine the pharmacokinetic parameters after intravenous injection of the antibody into female Balb / c mice. The specific test procedure was as follows: Before injecting the antibody into the tail vein of Balb / c mice, preliminary blood samples were first collected. Then, after intravenous injection at a dose of 5 mg / kg of the antibody, blood was collected at 15 minutes, 5 hours, 24 hours, on the 2nd day, 4th day, 7th day, 14th day, and 21st day, and the antibody concentration in the serum was detected. The PK test design was as shown in Table 6 below.

[0137]

Table 6

[0138]

Table 7

[0139] As can be seen from Table 7 and Figure 5, the half-life of the human LIFR antibody PR300516 in mice was 178.25 hours.

[0140] 6.2. PK in Sprague Dawley Rats The objective of this study was to determine the pharmacokinetic parameters after intravenous injection of the antibody in male Sprague Dawley rats. As a specific test procedure, before injecting the antibody into the tail vein of Sprague Dawley rats, first, preliminary blood samples were taken. Then, after intravenous injection at a dose of 5 mg / kg of the antibody, blood samples were collected at the time points before administration, 10 minutes later, 2 hours later, 8 hours later, 24 hours later, on the 4th day, 7th day, 10th day, 14th day, 21st day, and 28th day, and the antibody concentration in the serum was detected. Three rats were placed in each group of the PR300516 antibody and PR301168 antibody for the test. The PK test design is as shown in Table 8 below.

[0141]

Table 8

[0142]

Table 9

[0143] As can be seen from Table 9 and Figure 6, the half-lives of the human LIFR antibodies PR300516 and PR301168 in rats were 219 hours and 242 hours, respectively.

[0144] 6.3. In Vivo PK Test in Monkeys The objective of this study was to determine the pharmacokinetic parameters after intravenous injection of the antibody in male and female monkeys. The specific test procedure was that before intravenous injection of the antibody into the tails of male and female monkeys, first, blood samples were taken as samples. Then, after intravenous injection at doses of 10 mg / kg of the low dose and 100 mg / kg of the high dose of the antibody, for the 10 mg / kg dose group, blood samples were collected at the time points before administration, 10 minutes later, 2 hours later, 8 hours later, 24 hours later, on the 4th day, 7th day, 10th day, and 14th day, and for the 100 mg / kg dose group, blood samples were collected at the time points before administration, 10 minutes later, 2 hours later, 8 hours later, 24 hours later, on the 4th day, and 7th day, and the antibody concentration in the serum was detected. The PK test design is as shown in Table 10 below.

[0145]

Table 10

[0146]

Table 11

[0147] As can be seen from Table 11 and Figure 7, the half-lives of the low-dose human LIFR antibody PR300516 in monkeys are 173.7 hours and 182.9 hours respectively, and the half-lives of the high-dose human LIFR antibody PR300516 in monkeys are 239.3 hours and 128.2 hours respectively.

[0148] Example 7 Pharmacodynamic study of the antibody in vivo 7.1. Capan-2 model In this study, the anti-tumor activity of the anti-human LIFR antibody was studied using the Capan-2 mouse tumor model. The pharmacodynamics of the PR300516 antibody alone (5 mg / kg and 15 mg / kg) were studied in the Capan-2 mouse model. 5x10e6 cells of human pancreatic cancer Capan-2 cells (ATCC cell library) were subcutaneously inoculated into female Balb / c nude mice (Vitalriver). The treatment method for the antibody group was that on the 5th, 9th, 12th, 16th, 19th, and 23rd days after tumor cell inoculation, the corresponding antibodies were intraperitoneally injected into the mice of each group. The treatment method for the small molecule inhibitor group was that the mice were orally administered the EC359 small molecule inhibitor on the 5th, 7th, 9th, 12th, 14th, 16th, 19th, 21st, and 23rd days after tumor cell inoculation. On the 5th, 9th, 12th, 16th, 19th, and 23rd days after tumor cell inoculation, the tumor volume was measured and then the mice were euthanized. As shown in Table 15 and Figure 8, the anti-human LIFR antibody PR300516 of the present application has a certain inhibitory effect on tumor growth when used alone compared with the negative control. Compared with the EC359 small molecule inhibitor and the MSC-1 positive control antibody, the anti-human LIFR antibody described in the present application has a stronger inhibitory effect on tumor growth. In this study, 37 days after inoculation, the body weights of all groups of mice did not change significantly.

[0149] 7.2. Capan-1 Model In this study, the anti-tumor activity of anti-human LIFR antibodies was investigated using the Capan-1 mouse tumor model. The pharmacodynamics of the PR300516 and PR301168 antibodies administered alone (15 mg / kg) in the Capan-1 mouse model were studied. 5×10⁶ human pancreatic cancer Capan-1 cells (ATCC cell library) were subcutaneously inoculated into female Balb / c nude mice (Vitalriver). The treatment method for the antibody groups was that on the 5th, 8th, 12th, 15th, 18th, and 21st days after tumor cell inoculation, the corresponding antibodies were intraperitoneally injected into the mice in each group. On the 5th, 8th, 12th, 15th, 18th, and 21st days after tumor cell inoculation, the tumor volume was measured, and then the mice were euthanized. As shown in Table 16 and Figure 9, the anti-human LIFR antibodies PR300516 and PR301168 of this application have a certain inhibitory effect on tumor growth when used alone compared with the negative control. Compared with the MSC-1 positive control antibody, the anti-human LIFR antibody described in this application has a stronger inhibitory effect on tumor growth. In this study, for all groups of mice, there was no obvious change in the body weight of the mice 25 days after inoculation.

[0150] 7.3. NCI-H292 Model In this study, the antitumor activity of anti-human LIFR antibodies was investigated using the NCI-H292 mouse tumor model. The pharmacodynamics of single administration (15 mg / kg) of PR300516 and PR301168 antibodies in the NCI-H292 mouse model were studied. 5×10⁶ human pancreatic cancer NCI-H292 cells (ATCC cell library) were subcutaneously inoculated into female Balb / c nude mice (Vitalriver). The treatment method for the antibody group was that on the 5th, 8th, 12th, 15th, 18th, and 21st days after tumor cell inoculation, the corresponding antibodies were intraperitoneally injected into the mice in each group. On the 5th, 8th, 12th, 15th, 18th, and 21st days after tumor cell inoculation, the tumor volume was measured, and then the mice were euthanized. As shown in Table 17 and Figure 10, the anti-human LIFR antibodies PR300516 and PR301168 of this application have a certain inhibitory effect on tumor growth when used alone compared with the negative control. Compared with the MSC-1 positive control antibody, the anti-human LIFR antibody described in this application has a weak inhibitory effect on tumor growth. In this study, the body weight of the mice in all groups decreased 25 days after inoculation.

[0151]

Table 12

[0152]

Table 13

[0153]

Table 14

[0154]

Table 15

[0155]

Table 16

[0156]

Table 17

[0157] The above embodiments merely illustrate some embodiments of the present invention. Although the description is more specific and detailed, it does not limit the scope of the claims of the present invention. Those skilled in the art can make some modifications and improvements without departing from the idea of the present invention, and all of these belong to the protection scope of the present invention. Therefore, the protection scope of the present invention is based on the scope of the claims.

Claims

1. (1) Heavy chain complementarity-determining region (HCDR) 1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 5, HCDR3 shown in SEQ ID NO: 16, light chain complementarity-determining region (LCDR) 1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 33, LCDR3 shown in SEQ ID NO: 41, (2) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 33, LCDR3 shown in SEQ ID NO: 41, (3) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 6, HCDR3 shown in SEQ ID NO: 17, LCDR1 shown in SEQ ID NO: 27, LCDR2 shown in SEQ ID NO: 34, LCDR3 shown in SEQ ID NO: 41, (4) HCDR1 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 7, HCDR3 shown in SEQ ID NO: 19, LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 33, LCDR3 shown in SEQ ID NO: 41, (5) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 12, HCDR3 shown in SEQ ID NO: 23, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, LCDR3 shown in SEQ ID NO: 44, (6) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 20, LCDR1 shown in SEQ ID NO: 29, LCDR2 shown in SEQ ID NO: 36, LCDR3 shown in SEQ ID NO: 42, (7) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 21, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 37, LCDR3 shown in SEQ ID NO: 43, (8) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 22, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, LCDR3 shown in SEQ ID NO: 44, (9) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 22, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 39, LCDR3 shown in SEQ ID NO: 44, (10) HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 22, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, LCDR3 shown in SEQ ID NO: 44, or The anti-human LIFR antibody or antigen-binding fragment thereof comprising HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 13, HCDR3 shown in SEQ ID NO: 22, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 38, and LCDR3 shown in SEQ ID NO:

44. The anti-human LIFR antibody or antigen-binding fragment thereof comprising the above-mentioned HCDR and LCDR. **Claim 2** (1) A heavy chain variable region (VH) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 46, and a light chain variable region (VL) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

60. (2) A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 48, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

62. (3) A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 47, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

61. (4) A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 49, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

62. (5) A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 55, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:

67. A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 52, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 65 A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 53, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 66 A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 54, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 67 A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 54, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 68 A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 56, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 67, or A VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 57, and a VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 67 The anti-human LIFR antibody or antigen-binding fragment thereof according to claim 1, comprising VH and VL as described above.

3. The anti-human LIFR antibody is (1) a heavy chain (HC) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 70, and a light chain (LC) comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 84; (2) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 72, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 86; (3) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 71, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 85; (4) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 73, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 86; (5) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 79, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 91; (6) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 76, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 89 (7) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 77, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 90 (8) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 78, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 91 (9) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 78, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 92 (10) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 80, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 91, or (11) an HC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 81, and an LC comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 91 The anti-human LIFR antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising the HC and LC as described above.

4. The anti-human LIFR antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the anti-human LIFR antibody comprises a chimeric antibody, a humanized antibody or a fully human antibody.

5. The anti-human LIFR antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antigen-binding fragment comprises Fab, Fab’, F(ab’)2, Fv, scFv, and / or di-scFv.

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

7. A vector comprising the nucleic acid molecule according to claim 6.

8. A host cell comprising the nucleic acid molecule according to claim 6 or the vector according to claim 7.

9. A chimeric antigen receptor comprising the anti-human LIFR antibody or antigen-binding fragment thereof according to any one of claims 1-5.

10. An immune cell comprising the chimeric antigen receptor according to claim 9.

11. An antigen-binding protein derivative comprising the anti-human LIFR antibody or antigen-binding fragment thereof according to any one of claims 1-5 and a detectable label molecule which is an enzyme, a radionuclide, a fluorescent dye, a luminescent substance or biotin.

12. The anti-human LIFR antibody or antigen-binding fragment thereof according to any one of claims 1-5, and another antibody or fragment thereof or antibody analog, A multispecific antibody which is a bispecific antibody, a trispecific antibody or a tetravalent antibody.

13. An antibody-drug conjugate, wherein the antibody-drug conjugate comprises an antibody moiety and a conjugate moiety, the antibody moiety comprises the anti-human LIFR antibody or antigen-binding fragment thereof according to any one of claims 1-5, the conjugate moiety comprises a detectable marker, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof, but is not limited thereto, and the antibody moiety and the conjugate moiety are conjugated by a chemical bond or a linker.

14. A drug composition comprising an anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1-5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8, an immune cell according to claim 10, an antigen-binding protein derivative according to claim 11, a multispecific antibody according to claim 12 and / or an antibody-drug conjugate according to claim 13, and any pharmaceutically acceptable vector.

15. The drug composition further comprises a combination therapeutic agent, The combination therapeutic agent includes, but is not limited to, chemotherapeutic agents, radiotherapy agents, immunosuppressive agents, cytotoxic drugs, the drug composition according to claim 14.

16. A method for producing an anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1-5, A method comprising culturing the host cell according to claim 8 in a state where the anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1-5 is expressed.

17. Use of an anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1-5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8, a chimeric antigen receptor according to claim 9, an immune cell according to claim 10, an antigen-binding protein derivative according to claim 11, a multispecific antibody according to claim 12, an antibody-drug conjugate according to claim 13 and / or a drug composition according to claim 14 or 15 in the manufacture of an LIF and / or LIFR blocking drug, kit and / or medical device.

18. Use of an anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1-5, a nucleic acid molecule according to claim 6, a vector according to claim 7, a host cell according to claim 8, a chimeric antigen receptor according to claim 9, an immune cell according to claim 10, an antigen-binding protein derivative according to claim 11, a multispecific antibody according to claim 12, an antibody-drug conjugate according to claim 13 and / or a drug composition according to claim 14 or 15 in the manufacture of a drug, kit and / or administration device for preventing and / or treating LIFR-positive diseases.

19. The LIFR-positive disease is a tumor, and the tumor includes, but is not limited to, cholangiocarcinoma, colorectal cancer, glioma, prostate cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, breast cancer, bladder cancer, pancreatic cancer and non-small cell lung cancer, the use according to claim 18.

20. A method for detecting LIFR by qualitatively or quantitatively analyzing and detecting LIFR using the anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5.

21. A kit for use in detecting the expression of LIFR in a sample, comprising the anti-human LIFR antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, the antigen-binding protein derivative according to claim 11, and / or the multispecific antibody according to claim 12, and any instructions.

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