WO2022143743A1 - 人lifr抗原结合蛋白及其制备方法和应用 - Google Patents

人lifr抗原结合蛋白及其制备方法和应用 Download PDF

Info

Publication number
WO2022143743A1
WO2022143743A1 PCT/CN2021/142404 CN2021142404W WO2022143743A1 WO 2022143743 A1 WO2022143743 A1 WO 2022143743A1 CN 2021142404 W CN2021142404 W CN 2021142404W WO 2022143743 A1 WO2022143743 A1 WO 2022143743A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antigen
amino acid
antibody
lifr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/142404
Other languages
English (en)
French (fr)
Inventor
丛艳妮
张领兵
邓长静
朱广倍
周亮
马瑞鹏
陈银
刘礼乐
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Harbour Biomed Suzhou Co Ltd
Yinuoke Ltd
Original Assignee
Harbour Biomed Suzhou Co Ltd
Yinuoke Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Harbour Biomed Suzhou Co Ltd, Yinuoke Ltd filed Critical Harbour Biomed Suzhou Co Ltd
Priority to CA3203886A priority Critical patent/CA3203886A1/en
Priority to KR1020237025486A priority patent/KR20230125037A/ko
Priority to EP21914475.5A priority patent/EP4253413A4/en
Priority to AU2021414160A priority patent/AU2021414160B2/en
Priority to US18/259,986 priority patent/US20250136702A1/en
Priority to CN202180088318.8A priority patent/CN117043189A/zh
Priority to JP2023540785A priority patent/JP7691668B2/ja
Publication of WO2022143743A1 publication Critical patent/WO2022143743A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001119—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of biomedicine, and particularly relates to a human LIFR antigen binding protein and a preparation method and application thereof.
  • Leukemia inhibitory factor is a cytokine with multiple biological functions, named for its ability to induce M1 myeloid leukemia cells to differentiate into normal cells.
  • LIF belongs to the interleukin-6 (IL-6) family, which also includes IL-11, IL-27, ciliary neurotrophic factor (CNTF), myocardin-1 (CT-1), and oncostatin M ( OSM), etc., these cytokines share the gp130 receptor with LIF, therefore, LIF has overlapping and unique biological roles with other family members.
  • IL-6 interleukin-6
  • CNTF ciliary neurotrophic factor
  • CT-1 myocardin-1
  • OSM oncostatin M
  • the LIF protein in mammals consists of 180 amino acid residues and is highly conserved.
  • the human LIF gene is encoded on chromosome 22q12, while the mouse LIF gene is located on chromosome 11.
  • the mouse and human LIF genes are homologous > 75%.
  • Mature LIF protein is a highly glycosylated secreted protein, which can be secreted by a variety of adult cells such as oocytes and human embryonic extraembryonic cells, endometrial cells, fibroblasts, hepatocytes, and mononuclear macrophages.
  • LIF protein glycosylation Different tissue sources of LIF protein glycosylation are different, of which the most studied is a secreted, variable glycosylation protein (34-63kDa), which has induced autocrine and extensive paracrine effects, which may Contradictory due to different cell types and backgrounds, such as proliferation and differentiation, survival and apoptosis.
  • variable glycosylation protein 34-63kDa
  • LIF mainly induces downstream JAK/STAT3, PI3K/AKT, ERK1/2 by binding to its specific LIF receptor (LIF receptor, LIFR) and then recruiting the gp130 receptor subunit to form a high-affinity heterodimeric receptor complex , MAPK and other signaling pathways are activated to exert various cellular functions and regulate cell proliferation and survival.
  • LIF receptor LIF receptor
  • LIFR LIF receptor is abundantly expressed in the central nervous system, kidney, liver, bone, uterus and other tissues and organs. The wide distribution of LIF and LIFR and several LIF signaling pathways determine the complex diversity of LIF functions.
  • the LIF pathway is a promising new target in the field of immunology. It has been reported that the LIF signaling pathway plays an important role in the occurrence and progression of tumors, which can enhance the migration and infiltration of tumor cells and promote tumor metastasis; at the same time, it can regulate a variety of immune cells in the tumor microenvironment, including effector T cells (T cells). -eff), regulatory T cells (T-reg), Th17 and myeloid cells, etc. There are also studies showing that the LIF signaling pathway can promote the activity of tumor-initiating cells (CICs) and enhance resistance to anti-tumor treatments (chemotherapy and radiotherapy). In a variety of cancers, high expression of LIF is associated with poor prognosis.
  • CICs tumor-initiating cells
  • Patent CN111378036A discloses a preparation method and application of an anti-human leukemia inhibitory factor (LIF) monoclonal antibody, which uses mouse hybridoma cell technology to prepare an anti-human leukemia inhibitory factor monoclonal antibody. and lower yields.
  • Patent CN111868086A discloses a series of anti-LIF antibodies and their applications. The LIF antibodies can bind to the unique epitopes of LIF, thereby inhibiting the biological activity of LIF.
  • MSC-1 antibody is a humanized monoclonal antibody (IgG1) that binds the immunosuppressive human cytokine LIF for the treatment of adult patients with advanced solid tumors.
  • IgG1 humanized monoclonal antibody
  • the MSC-1 antibody can only bind to the specific epitope of LIF, so it cannot completely compete for the binding of LIF to the 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).
  • the present invention provides for the first time a fully human LIFR antigen-binding protein and a preparation method and application thereof.
  • the present invention adopts human LIFR-ECD protein to immunize H2L2 human antibody transgenic mice, and utilizes single B cell cloning technology (SBC) to obtain a series of human LIFR antibodies with biological functions, and the method greatly improves the efficiency of antibody drug discovery
  • SBC single B cell cloning technology
  • the obtained human LIFR antibody has good affinity and can block the binding of huLIF protein to huLIFR/gp130 cells, thereby inhibiting signaling pathways such as STAT3, inhibiting tumor growth, and achieving the purpose of tumor prevention and treatment.
  • the present invention provides a human LIFR antigen binding protein.
  • the human LIFR antigen-binding protein comprises the following complementarity determining regions:
  • the heavy chain complementarity determining region 1HCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4 or a variant sequence thereof;
  • the light chain complementarity determining region 2LCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO:33-SEQ ID NO:40 or its variant sequence;
  • the variant sequence is a CDR sequence having one or several amino acid substitutions, deletions or additions compared to the CDR from which it is derived; the substitutions are conservative substitutions.
  • the human LIFR antigen-binding protein comprises the following complementarity determining regions:
  • And/or (5) comprising the amino acid sequence shown in GX 13 SSRAT, the amino acid sequence shown in KASX 14 LEX 15 , the amino acid sequence shown in SEQ ID NO: 35: AASNRAT, and the amino acid sequence shown in SEQ ID NO :36:
  • And/or (6) comprise the amino acid sequence whose sequence is as shown in SEQ ID NO: 41: QQYGSSPFT, the amino acid sequence whose sequence is as shown in SEQ ID NO: 42: QQSYSTPLT, and the amino acid whose sequence is as shown in SEQ ID NO: 43: QQYKSFSPGGLT
  • described human LIFR antigen binding protein comprises:
  • the heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO:46-SEQ ID NO:59; and/or comprising any one of SEQ ID NO:60-SEQ ID NO:69
  • VH having one or several amino acid substitutions, deletions or additions, or any combination thereof, compared to any of the VHs in (1); and/or, compared to any of the VLs in (1)
  • described human LIFR antigen binding protein comprises:
  • a heavy chain HC comprising the amino acid sequence shown in any one of SEQ ID NO:70-SEQ ID NO:83; and/or comprising the amino acid shown in any one of SEQ ID NO:84-SEQ ID NO:93 the light chain LC of the sequence;
  • the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared to the heavy chain and light chain in (1) , 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.
  • described human LIFR antigen binding protein comprises:
  • HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:5, HCDR3 as shown in SEQ ID NO:16, and/or LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:33, LCDR3 as shown in SEQ ID NO:41;
  • HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO:17, and/or LCDR1 as shown in SEQ ID NO:27, LCDR2 as shown in SEQ ID NO:34, LCDR3 as shown in SEQ ID NO:41;
  • HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:18, and/or LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:33, LCDR3 as shown in SEQ ID NO:41;
  • HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:19, and/or LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:33, LCDR3 as shown in SEQ ID NO:41;
  • HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:8, HCDR3 as shown in SEQ ID NO:19, and/or LCDR1 as shown in SEQ ID NO:28, LCDR2 as shown in SEQ ID NO:35, LCDR3 as shown in SEQ ID NO:41;
  • HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:9, HCDR3 as shown in SEQ ID NO:19, and/or LCDR1 as shown in SEQ ID NO:26, LCDR2 as shown in SEQ ID NO:35, LCDR3 as shown in SEQ ID NO:41;
  • HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:20, and/or LCDR1 as shown in SEQ ID NO:29, LCDR2 as shown in SEQ ID NO:36, LCDR3 as shown in SEQ ID NO:42;
  • HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:21, and/or LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:37, LCDR3 as shown in SEQ ID NO:43;
  • HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:22, and/or LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:38, LCDR3 as shown in SEQ ID NO:44;
  • HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:22, and/or LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:39, LCDR3 as shown in SEQ ID NO:44;
  • HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:12, HCDR3 as shown in SEQ ID NO:23, and/or LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:38, LCDR3 as shown in SEQ ID NO:44;
  • HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:22, and/or LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:38, LCDR3 as shown in SEQ ID NO:44;
  • HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:13, HCDR3 as shown in SEQ ID NO:22, and/or LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:38, LCDR3 as shown in SEQ ID NO:44;
  • HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO:24, and/or LCDR1 as shown in SEQ ID NO:31, LCDR2 as shown in SEQ ID NO:37, LCDR3 as shown in SEQ ID NO:43;
  • HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:15, HCDR3 as shown in SEQ ID NO:25, and/or LCDR1 as shown in SEQ ID NO:32, LCDR2 as shown in SEQ ID NO:40, LCDR3 as shown in SEQ ID NO:45.
  • the human LIFR antigen-binding protein adopts Mega software to perform sequence similarity analysis, including the following five groups:
  • Group 1 the antigen-binding protein of (1)
  • Group 2 the antigen-binding proteins described in (2), (3), and (4);
  • Group three the antigen-binding proteins described in (5) and (6);
  • Group 4 the antigen-binding proteins described in (7), (8), (9), (10), (11), (12), and (13);
  • Group 5 the antigen-binding proteins described in (14) and (15).
  • described human LIFR antigen binding protein comprises:
  • VH as shown in SEQ ID NO:46, and/or VL as shown in SEQ ID NO:60;
  • VH as shown in SEQ ID NO:48, and/or VL as shown in SEQ ID NO:62;
  • VH as shown in SEQ ID NO:50, and/or VL as shown in SEQ ID NO:63;
  • VH as shown in SEQ ID NO:58, and/or VL as shown in SEQ ID NO:66;
  • At least 70% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, A VH of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or, 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% identical in sequence Sexual VL;
  • Any VL is compared to a VL having one or several amino acid substitutions, deletions or additions, or any combination thereof; the substitutions are conservative substitutions.
  • described human LIFR antigen binding protein comprises:
  • the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 90%, 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.
  • human LIFR antigen-binding proteins include chimeric antibodies, humanized antibodies or fully human antibodies.
  • the above-mentioned human LIFR antigen-binding proteins include full-length antibodies, Fab, Fab', F(ab')2, Fv, scFv, di-scFv, bispecific antibodies, multispecific antibodies, heavy chain antibodies and/or Or single-domain antibodies, or monoclonal and/or polyclonal antibodies prepared from the above-mentioned antibodies.
  • the present invention provides a series of nucleic acid molecules encoding the human LIFR antigen binding proteins.
  • the nucleic acid molecule comprises one or more codon-optimized nucleic acid molecules.
  • the present invention provides a series of vectors comprising one or more nucleic acid molecules described herein.
  • the vectors include but are not limited to plasmids, viruses, and bacteriophages.
  • the present invention provides a series of host cells comprising the above nucleic acid molecule or the above vector.
  • the host cells include, but are not limited to, microorganisms, plant or animal cells, and the vectors of the present invention can be introduced into the host cells by methods known to those skilled in the art, such as electroporation, lipofectine transfection , lipofectamin transfection and other methods.
  • the present invention provides a chimeric antigen receptor comprising the above-mentioned human LIFR antigen binding protein.
  • the present invention provides an immune cell comprising the above-mentioned chimeric antigen receptor.
  • the present invention provides an antigen-binding protein derivative comprising the above-mentioned human LIFR antigen-binding protein and a detectable marker molecule.
  • the detectable marker molecule is an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance) or biotin.
  • an enzyme eg, horseradish peroxidase
  • a radionuclide e.g., a radionuclide
  • a fluorescent dye e.g., a fluorescent dye
  • a luminescent substance eg, a chemiluminescent substance
  • biotin biotin
  • the present invention provides a multispecific antibody comprising the above-mentioned human LIFR antigen binding protein, and another antibody or a fragment or antibody analog thereof.
  • the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • the present invention provides an antibody-drug conjugate
  • the antibody-drug conjugate comprises an antibody part and a coupling part
  • the antibody part comprises the above-mentioned human LIFR antigen-binding protein
  • the coupling part comprises But not limited to detectable labels, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof, the antibody moieties and conjugation moieties are coupled via chemical bonds or linkers.
  • the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the above-mentioned human LIFR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen Binding protein derivatives, multispecific antibodies and/or antibody drug conjugates.
  • the pharmaceutical composition further comprises an optional pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers include but are not limited to: diluents, excipients, fillers, wetting agents, disintegrating agents, flavoring agents and binders.
  • the pharmaceutical composition further comprises a combined therapeutic agent, and the combined therapeutic agent includes but is not limited to chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, and cytotoxic drugs.
  • the present invention provides a method for producing the above-mentioned human LIFR antigen-binding protein, the method comprising culturing the above-mentioned host cell under the condition that the antigen-binding protein is expressed.
  • the method includes the following steps:
  • step (3) recovering the heavy chain and light chain sequences of the antibody from the single B cell screened in step (2);
  • step (3) Recombining the antibody heavy chain and light chain recovered in step (3) with human Fc, introducing it into host cells, and preparing the human LIFR antigen-binding protein through cell culture and purification.
  • the mouse described in step (1) is a humanized mouse.
  • the humanized mouse is a Harbor H2L2 mouse
  • the Harbor H2L2 transgenic mouse strain is capable of producing traditional two-heavy-chain two-light-chain immunoglobulin antibodies with fully human variable regions.
  • the method for screening antigen-specific B cells secreting antibodies described in step (2) is: using the plasma cell (plasma cell) discovery workflow of the Beacon photoelectric system for screening.
  • the method for recovering the heavy chain and light chain sequences of the antibody described in step (3) is single B cell sequencing.
  • the single B cell sequencing steps are: RNA purification from 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, uniqueness and cluster analysis of the obtained sequences, and finally the paired heavy and light chain DNA sequences were subjected to plasmid synthesis.
  • the present invention provides the 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 use of a pharmaceutical composition in the preparation of a drug, kit and/or medical device for LIF and/or LIFR blocking.
  • the LIFR blocking drugs, kits and/or devices are mainly used in diseases with increased expression of LIF and/or LIFR.
  • the present invention provides the 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 the use of the pharmaceutical composition in the preparation of a medicament, a kit and/or a drug delivery device for the prevention and/or treatment of LIFR positive diseases.
  • the present invention provides the use of the human LIFR antigen-binding protein and antigen-binding protein derivatives in the preparation of LIFR detection reagents or kits.
  • the present invention provides a LIFR detection method, which utilizes the above-mentioned human LIFR antigen-binding protein qualitative or quantitative analysis to detect LIFR.
  • the detection method is used for non-disease diagnosis or treatment purposes.
  • the present invention provides a method for treating LIFR-positive related diseases, which comprises administering to a subject in need thereof an effective amount of the above-mentioned human LIFR antigen-binding protein, immune cells, antigen-binding protein derivatives, polynucleotides, etc.
  • a method for treating LIFR-positive related diseases which comprises administering to a subject in need thereof an effective amount of the above-mentioned human LIFR antigen-binding protein, immune cells, antigen-binding protein derivatives, polynucleotides, etc.
  • Specific antibodies, antibody drug conjugates and/or pharmaceutical compositions are examples of the above-mentioned human LIFR antigen-binding protein, immune cells, antigen-binding protein derivatives, polynucleotides, etc.
  • the LIFR-positive disease is a tumor
  • the tumor includes but is not limited to cholangiocarcinoma, colorectal cancer, glioma, prostate cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, breast cancer, bladder cancer, pancreas cancer cancer, non-small cell lung cancer.
  • the present invention provides a kit comprising the human LIFR antigen-binding protein, nucleic acid molecule, vector, host cell, chimeric antigen receptor, immune cell, antigen-binding protein derivative , multispecific antibodies, antibody drug conjugates and/or pharmaceutical compositions, and optionally, instructions.
  • the present invention provides a drug delivery device comprising: (1) an infusion module for administering the pharmaceutical composition to a subject in need thereof, and (2) Optional efficacy monitoring module.
  • the present invention provides the use of LIFR antigen-binding protein in the preparation of a drug for the treatment of cancer
  • 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.
  • the LIFR antigen-binding protein includes any one of the above-mentioned LIFR antigen-binding proteins.
  • the present invention provides a human LIFR antibody for the first time.
  • the human LIFR antibody is prepared by immunizing human antibody transgenic mice (such as Harbour H2L2 mice), and has fully human antibody amino acid and gene sequences, thereby ensuring that Lowest possible immunogenicity for human use, with no toxic side effects.
  • the present invention adopts single B cell cloning technology (SBC) to screen positive clones, which can greatly improve the efficiency and yield of antibody discovery compared with traditional monoclonal antibody screening technology.
  • SBC single B cell cloning technology
  • the human LIFR antibody prepared by the present invention has high specificity, high affinity and good anti-tumor activity.
  • the antibody of the present invention is a fully human antibody, so that it can be safely administered to a human subject without causing an immunogenic reaction, and has great clinical value.
  • Figure 1 is a graph showing the detection of the binding ability of the human LIFR antibody of the present invention to LIFR-ECD at the protein level, wherein A is the detection result of the binding ability of the human LIFR antibody of the present invention to human LIFR-ECD, and B is the detection result of the binding ability of the human LIFR antibody of the present invention to human LIFR-ECD The detection result of the binding ability of the human LIFR antibody to the monkey LIFR-ECD, C is the detection result of the binding ability of the human LIFR antibody of the present invention to the mouse LIFR-ECD.
  • Figure 2 is a graph showing the detection of the binding ability of the antibody of the present invention to LIFR and LIFR/gp130 at the cellular level, wherein A is the detection result of the binding ability of the antibody of the present invention to LIFR, and B is the binding of the antibody of the present invention to LIFR/gp130 Ability test results.
  • Figure 3 is a graph showing the detection of the ability of the antibody of the present invention to block the binding of LIF to LIFR/gp130.
  • Fig. 4 is a graph showing the inhibition effect of the antibody of the present invention on the STAT3 signaling pathway.
  • Fig. 5 is a graph showing the in vivo PK detection of the antibody of the present invention in Balb/c mice.
  • Figure 6 is the PK detection chart of the antibody of the present invention in Sprague Dawley rats, wherein, A is the PK detection chart of the PR300516 antibody in the rat, B is the PK detection chart of the PR301168 antibody in the rat, and C is the PR300516 The average detection chart of PK of antibody and PR301168 antibody in rats.
  • Figure 7 is the PK detection chart of the antibody of the present invention in monkeys, wherein, A is the detection result of a single dose of 10 mg/mL, and B is the detection result of a single dose of 100 mg/mL.
  • Fig. 8 is a graph showing the detection of the pharmacodynamics of the antibody of the present invention in the Capan-2 mouse model.
  • Fig. 9 is a graph showing the detection of the pharmacodynamics of the antibody of the present invention in the Capan-1 mouse model.
  • Fig. 10 is a graph showing the detection of the pharmacodynamics of the antibody of the present invention in the NCI-H292 model mouse model.
  • Antigen-binding protein as used herein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone portion that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen.
  • An antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both may typically be included.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FR or FWR).
  • CDRs complementarity determining regions
  • FR or FWR framework regions
  • antigen binding proteins include, but are not limited to, antibodies, antigen binding fragments (Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv and/or dAbs), immunoconjugates, multispecific antibodies (eg 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.
  • the "antibody” in the present invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , ⁇ chain, ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL or LCVR) and heavy chain variable region (VH or HCVR) consists of 3 CDR regions and 4 FR regions, the order from the amino terminus to the carboxy terminus is: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • CDRs can be determined according to various numbering systems known in the art, such as the Kabat, Chothia or, AbM or IMGT numbering systems.
  • the "Kabat numbering convention” (see Kabat et al. (1991)) is used in the present invention to determine the amino acid sequence boundaries of CDRs.
  • framework region or "FR” residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variant antibodies, the individual antibodies comprising the population are identical and/or bind to the same epitope. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants, each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. therefore.
  • “Monoclonal” refers to the properties of an antibody obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the manufacture of the antibody by any particular method.
  • the monoclonal antibodies of the present invention can be prepared by various techniques known to those skilled in the art, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and transgenic methods.
  • humanized monoclonal antibody refers to an antibody produced by grafting murine CDR sequences into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence, which can overcome the large number of murine antibodies carried by chimeric antibodies. protein components, thereby inducing a heterologous response.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation (or back mutation) to maintain activity.
  • murine CDR regions can be inserted into human framework sequences using methods known in the art (see US Patent Nos. 5,225,539 to Winter; US Patent Nos.
  • transgenic animals can also be utilized that are capable of producing no endogenous immunoglobulins following immunization, and are capable of producing fully human antibody repertoires (see, eg, Jakobovits et al., 1993, Proc. Natl. Acad. Sci.
  • transgenic animals include, but are not limited to, H2L2 humanized mice from HARBOURBIOMED.
  • 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 against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed in terms of the dissociation equilibrium constant (KD) of the interaction.
  • KD dissociation equilibrium constant
  • the term “KD” refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe 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.
  • the antibody binds with an affinity (KD) of about less than 10-8 M, eg, about less than 10-9 M, 10-10 M, 10-11 M or less.
  • identity generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to residues in a second reference polypeptide sequence or part thereof after alignment of the sequences, introducing gaps (GAPS) where necessary to Maximum percent sequence identity was achieved and any conservative substitutions were not considered as part of the sequence identity.
  • Alignment for determining percent amino acid/nucleotide sequence identity can be accomplished by various means known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR). Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Percent identity can be determined over the length of the entire defined polypeptide/polynucleotide sequence, or can be determined over shorter lengths, such as the length of fragments obtained from larger defined polypeptide/polynucleotide sequences . It is to be understood that in the Tables, Figures or Sequence Listing, any fragment length supported by the sequences shown herein can be used as a length for which percent identity can be measured. A sequence with "% identity" retains important biological activities, such as antibody binding specificity, of the sequence to which it is compared or from which it is derived.
  • a nucleotide sequence having "% identity" or a nucleotide sequence that differs by no more than 3, 6, 15, 30 or 45 nucleotides is capable of performing a similar function to the nucleotide sequence from which it is compared or derived, as described
  • the expressed proteins can all specifically bind to the same antigen or molecule.
  • conservative substitution refers to amino acid substitutions that do not adversely affect or alter the intended properties of the protein/polypeptide comprising the amino acid sequence.
  • 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 include substitutions of amino acid residues with amino acid residues that have similar side chains, e.g., that are physically or functionally similar to the corresponding amino acid residues (e.g., have similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.) Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.
  • alanine, valine, leucine, isoleucine amino acid, proline, phenylalanine, methionine), beta branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of amino acids are well known in the art (see, eg, 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. Set USA 94:412-417 (1997), which is incorporated herein by reference).
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it 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) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” generally refers to an individual cell, cell line or cell culture that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or capable of expressing an antibody or antigen-binding fragment thereof described herein .
  • the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells can be obtained by transfecting cells in vitro with the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be mammalian cells.
  • the mammalian cells can be CHOK1 cells.
  • pharmaceutically acceptable carrier refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient and is well 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, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • pH adjusting agents 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 agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate salts and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (eg, buffered saline), alcohols and polyols (eg, glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate) and the like.
  • sugars such as sorbitol, mannitol, starch, sucrose , lactose, glucan, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dry whey, albumin or casein
  • degradation products such as lactalbumin hydrolyzate
  • composition generally refers to a formulation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to whom the composition is to be administered.
  • the composition is sterile.
  • subject refers to a mammal, eg, a primate, eg, a non-human primate, or a human.
  • the subject eg, human
  • has cancer including but not limited to bile duct cancer, colorectal cancer, glioma, prostate cancer, ovarian cancer, stomach cancer, nasopharyngeal cancer, breast cancer
  • bladder cancer pancreatic cancer, non-small cell lung cancer
  • an effective amount refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a disease-prophylactically (eg, LIFR-positive associated) effective amount refers to an amount sufficient to prevent, prevent, or delay the onset of a disease (eg, LIFR-positive associated disease);
  • a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent The amount of disease and its complications in patients who already have the disease. Determining such effective amounts is well within the ability of those skilled in the art.
  • an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other concurrently administered treatments and many more.
  • chimeric antigen receptor refers to a chimeric antigen receptor T cell coupled to a chimeric protein in vitro by the antigen-binding site of an antibody that recognizes a tumor antigen and the intracellular portion of the CD3-delta chain or Fc ⁇ RI ⁇ , the patient's T cells are transfected by gene transduction to express chimeric antigen receptors, so that 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 added to the patient's body. This chimeric antigen receptor can specifically track and recognize and guide T cells to kill tumor cells like GPS.
  • chimeric antigen receptors consist of an extracellular antigen-binding region (consisting of light and heavy chains derived from monoclonal antibodies, linked by a flexible hinge region to form a single-chain antibody), a transmembrane region, and an intracellular signal transduction region. composition of the guide area.
  • the CAR structure was obtained by genetically recombining the scFv that recognizes tumor-associated antigens and the intracellular signaling domain "immunoreceptor tyrosine activation motif" in vitro.
  • immune cell includes cells of hematopoietic origin and that play a role in the immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils Red blood cells, mast cells, basophils and granulocytes.
  • lymphocytes such as B cells and T cells
  • natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils Red blood cells, mast cells, basophils and granulocytes.
  • CDRs Complementarity Determining Regions in Immunoglobulin Variable Regions.
  • VH antibody heavy chain variable region.
  • VL antibody light chain variable region.
  • HC antibody heavy chain
  • LC antibody light chain
  • IgG Immunoglobulin G.
  • AbM CDR definition method comes from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272), this definition method integrates Partial definitions of both Kabat and Chothia.
  • Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia An immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917 ; Chothia et al. (1989) Nature 342:878-883).
  • IMGT Based on The international ImMunoGeneTics information system initiated by Lefranc et al. (IMGT)), see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
  • mAb Monoclonal antibody.
  • EC50 Concentration producing 50% efficacy or binding.
  • IC50 Concentration producing 50% inhibition.
  • HRP Horseradish peroxidase.
  • LIFR leukemia inhibitory factor receptor
  • IL7R ⁇ Interleukin 7 receptor alpha subunit.
  • TARC Thymus Activation Regulated Chemokine.
  • hFc Human IgG antibody Fc fragment.
  • HCDR1 Complementarity Determining Region 1 in the variable region of an immunoglobulin heavy chain.
  • HCDR2 Complementarity Determining Region 2 in the variable region of an immunoglobulin heavy chain.
  • HCDR3 Complementarity determining region 3 in the variable region of immunoglobulin heavy chains.
  • LCDR1 Complementarity determining region 1 in the variable region of immunoglobulin light chains.
  • LCDR2 Complementarity determining region 2 in the variable region of immunoglobulin light chains.
  • LCDR3 Complementarity determining region 3 in the variable region of immunoglobulin light chains.
  • the present invention prepares a series of fully human monoclonal antibodies.
  • These anti-human LIFR antibodies are prepared by immunizing fully human antibody transgenic mice, molecular biology and antibody engineering techniques, and have fully human antibody amino acid and gene sequences to ensure the lowest possible immunogenicity when used in humans.
  • Human antibody transgenic mouse technology was first developed by Abgenix (XenoMouse) and Madarex (HuMab mouse) and used for the preparation 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.
  • the early transgenic mice produced fully human immunoglobulin (Ig), that is, the variable and constant regions of the antibody were all human sequences. Because 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 is very low, which is manifested in the low efficiency of monoclonal antibody preparation. In recent years, the technology has undergone major innovations and improvements, mainly in the use of the same and similar constant region sequences as the host immunoglobulin (Ig) in the variable regions of the heavy and light chains.
  • the present invention adopts the transgenic mouse (Harbour H2L2) developed by Harbour BioMed Company to successfully prepare LIFR fully human antibody.
  • Harbour transgenic mice introduced human immunoglobulin variable region genes and rat immunoglobulin constant region genes, and the mouse's own Ig expression was inactivated.
  • the transgenic mice can produce immune responses and antibody titers equivalent to normal mice (eg Balb/c) after being immunized with the antigen.
  • the antigen binding proteins provided by the invention include the following complementarity determining regions:
  • the heavy chain complementarity determining region 1HCDR1 comprising the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 4 or a variant sequence thereof;
  • the light chain complementarity determining region 2LCDR2 comprising the amino acid sequence shown in any one of SEQ ID NO:33-SEQ ID NO:40 or its variant sequence;
  • the variant sequence is a CDR sequence with one or several amino acid substitutions, deletions or additions compared to the CDR from which it was derived.
  • the human LIFR antigen-binding proteins provided by the invention comprise the following complementarity determining regions:
  • And/or (5) comprising the amino acid sequence shown in GX 13 SSRAT, the amino acid sequence shown in KASX 14 LEX 15 , the amino acid sequence shown in SEQ ID NO: 35: AASNRAT, and the amino acid sequence shown in SEQ ID NO :36:
  • And/or (6) comprise the amino acid sequence whose sequence is as shown in SEQ ID NO: 41: QQYGSSPFT, the amino acid sequence whose sequence is as shown in SEQ ID NO: 42: QQSYSTPLT, and the amino acid whose sequence is as shown in SEQ ID NO: 43: QQYKSFSPGGLT
  • the antigen binding proteins provided by the present invention comprise the following heavy chain variable region VH and light chain variable region VL:
  • the heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO:46-SEQ ID NO:59; and/or comprising any one of SEQ ID NO:60-SEQ ID NO:69
  • VH having one or several amino acid substitutions, deletions or additions, or any combination thereof, compared to any of the VHs in (1); and/or, compared to any of the VLs in (1)
  • the antigen binding proteins provided by the invention include the following heavy chain HC and light chain LC:
  • a heavy chain HC comprising the amino acid sequence shown in any one of SEQ ID NO:70-SEQ ID NO:83; and/or comprising the amino acid shown in any one of SEQ ID NO:84-SEQ ID NO:93 the light chain LC of the sequence;
  • the heavy chain has at least 80%, at least 85%, at least 90%, at least 91%, at least 92% compared to the heavy chain and light chain in (1) , 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.
  • the human LIFR antibody prepared by the present invention comprises the combination of heavy chain and light chain described in Table 1 below.
  • the human LIFR antibodies shown in Table 1 were subjected to sequence similarity analysis by Mega software and can be divided into the following five groups:
  • Group 4 PR301153, PR301177, PR301195, PR301196, PR301211, PR301215, PR301218;
  • PR300516, PR301127, PR301152, PR301168, and PR301211 are selected from five histone antibodies for experiments.
  • Antigen-binding proteins provided herein as described in the above section include antibodies or antigen-binding fragments, which can be derivatized (eg, linked to another molecule, eg, another polypeptide or protein).
  • derivatization eg, labeling
  • the antibodies or antigen-binding fragments thereof of the invention are also intended to include such derivatized forms.
  • an antibody or antigen-binding fragment thereof of the invention can be linked to one or more other molecular moieties to form bispecific antibodies, detection reagents, pharmaceutical reagents, and/or capable of mediating the interaction of the antibody or antigen-binding fragment with another A protein or polypeptide (eg, avidin or polyhistidine tag) to which a molecule binds.
  • a protein or polypeptide eg, avidin or polyhistidine tag
  • One type of derivatized antibody is a labeled antibody.
  • an antibody or antigen-binding fragment thereof of the invention can be linked to a detectable label.
  • the detectable label of the present invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • Such labels are well known in the art, examples of which include, but are not limited to, enzymes (eg, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides Fluorescein (eg, 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas red, rhodamine, quantum dots or cyanine derivatives (eg Cy7, Alexa 750)), acridine esters, magnetic beads (eg, ), calorimetric labels such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and modified avidins (eg, streptavidin) for
  • Patents teaching the use of this marker include, but are not limited to, US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference).
  • Detectable labels as described above can be detected by methods known in the art. For example, radiolabels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.
  • such labels can be suitable for use in immunological detection (eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescence immunoassays, etc.).
  • a detectable label as described above can be attached to an antibody or antigen-binding fragment thereof of the invention via linkers of various lengths to reduce potential steric hindrance.
  • the antibodies or antigen-binding fragments thereof of the present invention can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
  • PEG polyethylene glycol
  • methyl or ethyl methyl or ethyl
  • glycosyl groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
  • the present invention provides a multispecific antibody comprising a first antibody or fragment thereof, and an additional antibody or fragment thereof, or an antibody analog, wherein the first antibody or fragments thereof, additional antibodies or fragments thereof, or antibody analogs retain their original binding specificity.
  • the first antibody or fragment thereof is any of the TSLP-binding (monoclonal) antibodies or antigen-binding fragments thereof of the invention.
  • antibody mimetic refers to the same specific binding to an antigen as an antibody, but without the antibody structure. They are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa, such as ankyrin repeat proteins (DARPins) and fynomers.
  • DARPins ankyrin repeat proteins
  • Designed ankyrin repeat proteins can be linked to IgG antibodies, scFv-Fc antibody fragments, or a combination thereof, as described in CN104341529A.
  • Anti-IL-17a fynomers were fused to anti-IL-6R antibodies to produce bispecific fusion polypeptides as described in WO2015141862A1.
  • the multispecific antibody is formed by conjugation of a first antibody, or antigen-binding fragment thereof, and other antibodies, or antigen-binding fragments or antibody analogs thereof, and wherein each antibody, or antigen-binding fragment or antibody thereof The analog retains its original binding specificity, and the first antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of the present invention.
  • the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • Antibody drug conjugates include antigen binding protein moieties and conjugate moieties provided by the present invention.
  • the antigen-binding protein portion includes the "antigen-binding protein" as described above, as well as antigen-binding fragments obtained on the basis of the "antigen-binding protein". Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', Fv fragments, F(ab')2, scFv, di-scFv and/or dAb, and the like.
  • the conjugation moiety may comprise at least one loaded drug.
  • the loaded drug may comprise a pharmaceutical active ingredient and/or the aforementioned marker molecule.
  • Loaded drug is a kind of small molecule compound or toxin or other drug molecule form with pharmacological activity, which can be but not limited to small molecule compound, toxin molecule, oligonucleotide, protein degradation targeting chimera (PROTAC), affinity Ligands, fluorophores, nuclide groups, etc.
  • a variety of drug payloads are known in the art.
  • small molecule compounds usually refer to a class of substances with strong cytotoxicity.
  • Exemplary small molecule compounds can exert such cytotoxic and cytostatic effects by mechanisms including, but not limited to, tubulin binding, DNA binding, inhibition of RNA polymerase, protein synthesis, or inhibition of topoisomerase.
  • the small molecule compound can be a tubulin inhibitor; for example, the tubulin inhibitor can be maytansine (eg, DM1 or DM4) and auristatin (eg, MMAE or MMAF), among others.
  • the small molecule compound may be a DNA damaging agent; for example, the DNA damaging agent may be Calicheamicins, pyrrolobenzodiazepines (PBD), and the like.
  • PPD pyrrolobenzodiazepines
  • PROTACs protein degradation targeting chimeras
  • PROTACs are a class of compounds capable of causing degradation of a target protein by inducing polyubiquitination of the target protein; for example, the PROTAC can be a BET protein degrader.
  • the drug conjugate may also comprise at least one linker.
  • the linker may comprise a cleavable linker or a non-cleavable linker.
  • the linker is used to link one or more drug payloads to the antigen binding protein.
  • a cleavable linker may be a "cleavable" linker that facilitates release of the drug.
  • cleavable linkers can include, but are not limited to, acid-sensitive linkers, protease-sensitive linkers, light-sensitive linkers, or disulfide-containing linkers.
  • the linker may comprise one or more linker members, various linker members are known in the art, for example, maleimidocaproyl (MC, maleimidocaproyl), maleimidopropionyl (MP), Val-citrulline (val-cit or vc, valine-citrulline), p-aminobenzyloxycarbonyl (PAB, paminobenzyloxycarbonyl).
  • MC maleimidocaproyl
  • MP maleimidopropionyl
  • Val-citrulline val-cit or vc, valine-citrulline
  • PAB paminobenzyloxycarbonyl
  • the antigen-binding protein of the present invention can be prepared by various methods known in the art, for example, by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the invention are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present invention.
  • the present invention provides isolated nucleic acid molecules comprising an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region and/or a light chain variable region thereof, or one or Nucleotide sequences of multiple CDRs.
  • the nucleotide sequences are replaceable according to codon degeneracy as known in the art.
  • the nucleotide sequence is codon-optimized.
  • the present invention provides cloning vectors or expression vectors comprising the isolated nucleic acid molecules of the present invention.
  • the vector is, for example, a plasmid, cosmid, phage, lentivirus, and the like.
  • the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject (eg, a mammal, eg, a human).
  • the present invention provides host cells comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention.
  • Host cells can be eukaryotic cells (eg, mammalian cells, insect cells, yeast cells) or prokaryotic cells (eg, E.
  • Suitable eukaryotic cells include, but are not limited to, NSO 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.
  • the host cells of the invention are mammalian cells, such as CHO (eg, CHO-K1, CHO-S, CHO DXB11, CHO DG44).
  • the present invention provides methods of making an antibody or antigen-binding fragment thereof of the present invention, comprising, culturing a host cell of the present invention under conditions that permit expression of the antibody or antigen-binding fragment thereof, and The antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • the present invention provides the human LIFR antigen-binding protein, antigen-binding protein derivatives, multispecific antibodies, immune cells, and antibody-drug conjugates in the preparation of LIF and/or LIFR blocking drugs, preparation for prevention and /or use in a medicament for the treatment of LIF and/or LIFR-positive disease.
  • the present invention provides the human LIFR antigen binding protein, antigen binding protein derivatives, multispecific antibodies, immune cells, antibody drug conjugates for blocking LIF and/or LIFR, for prevention and/or Or methods of treating LIF and/or LIFR positive disease.
  • the present invention provides a pharmaceutical composition for use in therapy, said pharmaceutical composition comprising said human LIFR antigen binding protein, nucleic acid molecule, vector, host cell, immune cell, antigen binding protein derivative drug, multispecific antibody and/or antibody drug conjugate and optionally a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include, but are not limited to, diluents, excipients, fillers, wetting agents, disintegrating agents, flavoring agents and binders.
  • the pharmaceutical composition further comprises a combination therapeutic agent, including but not limited to chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, and cytotoxic drugs.
  • a combination therapeutic agent including but not limited to chemotherapeutic agents, radiotherapeutic agents, immunosuppressive agents, and cytotoxic drugs.
  • the antigen binding protein of the invention and the combination therapeutic agent are provided as separate components or as components of the same composition.
  • the antigen binding protein of the present invention and the combination therapeutic agent may be administered in combination or separately, simultaneously or sequentially.
  • Anti-LIFR antibodies or antigen-binding fragments thereof can be administered alone or in combination with other therapeutic agents.
  • the anti-LIFR antibody or antigen-binding fragment thereof and the one or more other therapeutic agents can be administered separately, simultaneously or sequentially.
  • the pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient) and may be administered by a variety of routes (eg, oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular,
  • routes eg, oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular
  • the human LIFR antibodies of the invention are administered to a patient topically, intrathecally, and intracerebroventricularly.
  • the most appropriate route of administration in any given situation will depend on the particular antibody, the nature and severity of the individual and disease, and the individual's physical condition.
  • the present invention also provides a drug delivery device for administering the antigen binding proteins, antigen binding protein derivatives, multispecific antibodies, immune cells, antibody drug conjugates, and pharmaceutical compositions comprising the foregoing components , the device includes:
  • an infusion module for administering to a subject a pharmaceutical composition comprising an active ingredient
  • compositions for infusion containing an active ingredient selected from the group consisting of antigen binding proteins, antigen binding protein derivatives, multispecific antibodies, immune cells, Antibody drug conjugates or combinations thereof; and
  • the administration includes parenteral administration and parenteral administration.
  • the parenteral administration includes injection administration, and the route of injection used includes: intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, Tracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and boluses.
  • the parenteral administration includes topical, epidermal or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical application.
  • the infusion module is a needle-free hypodermic injection device, a microinfusion pump, a transdermal drug delivery device, a bolus injection device, or an osmotic device.
  • the antibodies or antigen-binding fragments thereof of the present invention are capable of binding LIFR and thus can be used to detect the presence or level of LIFR in a sample.
  • the present invention provides a kit comprising the antigen binding protein of the present invention.
  • the antigen binding proteins of the invention are detectably labeled.
  • the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the present invention.
  • the second antibody further comprises a detectable label.
  • the detectable label may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. It is particularly preferred that such labels be suitable for use in immunological detection (eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescence immunoassays, etc.).
  • immunological detection eg, enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescence immunoassays, etc.
  • detection of LIFR expression comprising contacting a biological sample (individual cells, tissues or body fluids) with one or more human LIFR antibodies of the invention (optionally conjugated to a detectable moiety), and The sample is tested for positive LIFR expression, or whether the sample has altered (eg, decreased or increased) expression compared to a control sample.
  • the tissue or body fluid is peripheral blood, peripheral blood leukocytes, biopsy tissue (eg, lung or skin biopsy), and tissue.
  • the methods can be used for diagnostic purposes, or non-diagnostic purposes (eg, for LIF/LIFR pathway studies, drug screening, histochemical analysis, etc.).
  • the sample used for non-diagnostic purposes is a cell sample, such as a cell line or an ex vivo cell culture.
  • the present invention provides a method of detecting the presence or level of LIFR in a sample, the method comprising under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof of the present invention and LIFR , contacting the sample with the antibody or antigen-binding fragment thereof, and detecting the formation of the complex.
  • the present invention provides a method for diagnosing a LIFR-positive disease in a subject, comprising combining the antibody or antigen-binding fragment thereof, multispecific antibody, or antibody-drug conjugate of the present invention with an antibody from the subject
  • the subject's sample is contacted under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and LIFR, and the formation of the complex is detected, wherein elevated levels of LIFR compared to healthy controls are indicative of the presence of cancer.
  • the subject is a mammal, including non-human mammals and humans.
  • the subject is a human.
  • 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.
  • 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 comprises a method for describing its use Instructions for use of the method.
  • Immunogen The amino acid sequence of recombinant human LIFR protein (GenBank accession number: NP_001121143.1) 1-833 of the extracellular region was cloned into the pcDNA3.1 vector containing his tag and the recombinant expression plasmid was prepared. Transient transfection and expanded culture of HEK293 cells, collect cell culture medium after 4 days, and purify the culture supernatant to obtain high-purity huLIFR-ECD protein (>90%), which has biological activity, and is frozen and stored in - 80°C.
  • huLIFR-ECD protein was used to immunize Harbor H2L2 transgenic mice for 6-8 weeks. All transgenic mice were housed in SPF grade environment. In the primary immunization, 50 ⁇ g of huLIFR-ECD protein was emulsified with Freund's complete adjuvant (Sigma, F5881) and injected into mice intraperitoneally. For subsequent booster immunizations, 25 ⁇ g of huLIFR-ECD protein was mixed with RIBI adjuvant (Sigma, S6322) and injected into mice intraperitoneally. 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 titers were selected for subsequent single B cell screening experiments.
  • Example 2 is based on Single B cell screening with Optofluidic system
  • the system uses photo-electric localization (OEP TM ) technology to perform the movement of individual cells.
  • OEP TM photo-electric localization
  • the Beacon optoelectronic system is an automated biological instrument that allows simultaneous biological function tests, experimental analysis, positive clone selection and other operations under cell culture conditions.
  • the Beacon platform can perform these tasks in a massively parallel, automated fashion on thousands of cells.
  • This application uses the plasma cell discovery workflow, and can screen up to 14k single plasma cells in each experiment for the selection of antigen-specific plasma cells secreted by antibodies. These specific antibody-secreting plasma cells were then individually exported into 96-well plates containing cell lysates for subsequent single B cell sequencing to identify the heavy and light chains of antibodies produced by single B cells (monoclonal). chain sequence.
  • the present invention adopts the single B cell sequencing method to obtain antibody heavy chain and light chain sequences from a single plasma cell.
  • Single B cell sequencing has become a powerful tool for obtaining antibody sequences.
  • General procedures include purification of RNA from single plasma cell lysate, reverse transcription synthesis of cDNA, amplification and purification of cDNA, amplification of heavy and light chains, cloning and transfection, and Sanger sequencing.
  • the resulting sequences were subjected to uniqueness and cluster analysis, and the paired heavy and light chain DNA sequences were then subjected to plasmid synthesis.
  • VH antibody heavy chain variable domain sequences
  • Antibody light chain variable domain sequences were genetically synthesized and cloned into mammalian cell expression plasmid vectors encoding human antibody Ig ⁇ light chain constant domain sequences to encode full-length light chains for antibody production.
  • the sequence of the variable domain of the anti-LIFR monoclonal antibody molecule obtained from the immunized Harbour H2L2 mice is a human antibody sequence, this example also obtained a fully human anti-LIFR recombinant IgG1 antibody.
  • the human LIFR antibody heavy chain and light chain complementary region determining region (CDR) sequences obtained in the present invention, heavy chain and light chain variable region (V), heavy chain and light chain sequences are shown in Table 1 above.
  • the DNA nucleotide sequence of the PR300516 heavy chain variable region is shown in SEQ ID NO: 94, and the DNA nucleotide sequence of the PR300516 light chain variable region is shown in SEQ ID NO: 95; PR301127
  • the DNA nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 96, the DNA nucleotide sequence of the light chain variable region of PR301127 is shown in SEQ ID NO: 97; the DNA core of the heavy chain variable region of PR301152
  • the nucleotide sequence is as shown in SEQ ID NO:98, the DNA nucleotide sequence of the variable region of the PR301152 light chain is as shown in SEQ ID NO:99; the DNA nucleotide sequence of the variable region of the PR301168 heavy chain is as shown in SEQ ID NO:
  • As shown in 100 the DNA nucleotide sequence of the variable region of the PR301168 light chain is shown in SEQ ID NO: 101; the DNA nucleotide sequence of the
  • 293F cells were used for antibody expression. Adjust the cell density to 1 ⁇ 10e6 cells/mL. Add the heavy chain and light chain antibody plasmids into the tube at a ratio of 1:1.5, then add the transfection reagent PEI, the ratio of PEI to DNA is 4:1, and incubate at room temperature for 15 minutes after mixing. The above mixture was added to the cells, and the transfected cells were incubated in a 37°C, 5% CO2 incubator with shaking at 125rpm. 24h after transfection, OPM-CHO PFF05 nutrition (Opmel, Cat. No. FB1279-001) was supplemented to a final concentration of 3%. After the cells were cultured for 5 days, the supernatant was collected when the cell viability dropped below 70%. Protein purification was performed on the transfected supernatant using a protein G column.
  • the five antibodies PR300516, PR301127, PR301152, PR301168 and PR301211 prepared according to the methods of Examples 1-4 of the present invention were tested by the method of Biolayer Interferometry (BLI) (Fortebio octet RED 96e). Binding kinetics of ECD proteins.
  • the antibody to be tested was diluted to a final concentration of 6 ⁇ g/mL, directly immobilized on the AHC biosensor for kinetic measurement, and the antigen protein (Human LIF) was diluted with 0.02% PBST20 to 3 concentrations of 100nM, 50nM, and 25nM, and injected for 70s. , the binding time was 300 s, the dissociation time was 300 s, and the regeneration was 15 s with 10 mM glycine-HCl (pH 1.5).
  • the binding ability of the 15 antibodies of the present invention prepared by Examples 1-4 to LIFR-ECD protein was detected based on ELISA assay.
  • the binding capacity of different antibodies was determined by comparing their binding curves to LIFR-ECD protein.
  • the specific experimental process is as follows: first, human LIFR-ECD, monkey LIFR-ECD and mouse LIFR-ECD proteins were sequentially diluted to a concentration of 1 ⁇ g/mL, added to a 96-well plate, 100 ⁇ L per well, and placed at 4°C overnight. After the 96-well plate was washed three times with PBST solution, 2% BSA in PBS solution was added and placed at 37° C. for 1 h.
  • the antibody to be tested was subjected to concentration gradient dilution (100nM, 10-fold-dilution), added to a 96-well plate, and incubated at 37°C for 1h. After washing three times with PBST solution, add anti-human IgG Fc-HRP secondary antibody (diluted at 5000x) and incubate at 37°C for 30-60min. After washing three times with PBST solution, add TMB color development solution for 5-15min, and then add stop solution to stop color development.
  • test results are shown in Table 3 and Figure 1.
  • PR300516, PR301127, PR301172, PR301152, PR301153, PR301160, PR301164, PR301168, PR301176, PR301177, PR301195, PR301196, PR301211, PR301215 and PR30 The ability of the monkey LIFR-ECD protein to bind, but only the PR301127 and PR301172 antibodies cross-bound to the mouse LIFR-ECD protein.
  • the five antibodies PR300516, PR301127, PR301152, PR301168, and PR301211 of the present invention prepared in Examples 1-4 were detected based on flow cytometry assay to be different from the human LIFR receptor or human LIFR/gp130 receptor expressed on the surface of 293T cells
  • the binding capacity of dimers was determined by comparing their binding curves to human LIFR receptor or human LIFR/gp130 receptor heterodimer expressed on the surface of 293T cells.
  • the specific experimental process is as follows: (1) Infect HEK293T cells with lenti-huLIFR and lenti-LIFR/gp130 lentiviruses, and screen the infected cells with puromycin to obtain stable cell pools: HEK293T-huLIFR and HEK293T-huLIFR/gp130; (2) ) Perform subclonal screening on the stable cell pool to obtain monoclonal cell lines with high expression of the target gene; (3) Incubate the monoclonal cell lines with different concentrations of the antibody to be tested at 4°C for 30 min, wash three times with PBS, and then use FITC The labeled goat anti-human secondary antibody was incubated with it for 30 min at 4°C, washed with PBS for three times, and then resuspended the cells with 100 ⁇ L of FACS buffer; (4) The median fluorescence value of the first channel was measured by flow cytometer. Take the logarithm of the antibody concentration as the base 10 as the ab
  • the ability of the 15 antibodies prepared in Examples 1-4 to block the binding of huLIF protein to the human LIFR/gp130 receptor heterodimer expressed on the surface of 293T cells was tested based on flow cytometry.
  • the blocking ability of different antibodies was determined by comparing the curves of different antibodies blocking the binding of huLIF protein to HEK293T-huLIFR/gp130 cells.
  • the specific experimental process is as follows: (1) First, determine the EC80 concentration of huLIF protein binding to HEK293T-huLIFR/gp130 cells.
  • Biotin labeling huLIF protein to obtain huLIF-biotin protein (2) using different concentrations of the test antibody and huLIF protein (the final concentration is the value of EC80) and HEK29ET-huLIFR/gp130 cells incubated at 4°C For 1 h, after washing three times with PBS, use APC-labeled Strepavidin secondary antibody to incubate with it at 4°C for 30 min. After washing three times with PBS, resuspend the cells with 100 ⁇ L FACS buffer; (3) Use flow cytometry to measure the four-channel median fluorescence degree value. Take the logarithm of the antibody concentration as the base 10 as the abscissa and the four-channel median fluorescence value as the ordinate to compare the IC50 and the peak value of the curve.
  • Antibody PR300516 PR301127 PR301172 PR301152 PR301153 hIgG1 IC50 0.562 NA NA 2.018 7.382 0.001774
  • Antibody PR301160 PR301164 PR301168 PR301176 PR301177 - IC50 2.404 1.129 0.991 1.436 0.496 -
  • Antibody PR301195 PR301196 PR301211 PR301215 PR301218 - IC50 0.753 0.799 0.572 0.533 0.628 -
  • test results are shown in Table 5 and Figure 3.
  • PR300516, PR301152, PR301153, PR301160, PR301164, PR301168, PR301176, PR301177, PR301195, PR301196, PR301211, PR301215 and PR301218 have obvious blocking huLIF Function of binding to HEK293T-huLIFR/gp130.
  • the inhibitory effect of LIF-induced STAT3 tyrosine 705 phosphorylation was detected based on Western blot, including Capan-2 pancreatic cancer cells, MDA-MB-231 breast cancer cells, A549 and NCI-H292 non-small cell lung cancer cells.
  • the specific experimental process is as follows: firstly, the tumor cells are plated on a 6-well plate, 1 ⁇ 10 6 cells per well, and starved for 24 hours (the medium does not add serum). Then a certain concentration of huLIF protein and the corresponding antibody were co-incubated for 1 h in a 37°C incubator, and the mixture of huLIF protein and antibody was added to the tumor cells, and treated in a 37°C incubator for 30 min.
  • the purpose of this study was to determine the pharmacokinetic parameters of the antibody following intravenous injection in female Balb/c mice.
  • the specific experimental process is as follows: before the antibody is injected into the tail vein of Balb/c mice, blood samples are taken first. Then according to the dose of 5mg/kg antibody after tail vein injection, blood was collected at 15min, 5h, 24h, day2, day4, day7, day14 and day21 to detect the antibody concentration in serum.
  • the PK experimental design is shown in Table 6 below.
  • the purpose of this study was to determine the pharmacokinetic parameters of the antibody following intravenous injection in male Sprague Dawley rats.
  • the specific experimental process is as follows: before the antibody is injected into the tail vein of Sprague Dawley rats, blood samples are taken first. Then, after tail vein injection at a dose of 5mg/kg antibody, blood was collected at time points 10min, 2h, 8h, 24h, day4, day7, day10, day14, day21 and day28 before administration to detect the antibody concentration in serum. Each group of PR300516 antibody and PR301168 antibody arranged 3 rats for the experiment.
  • the PK experimental design is shown in Table 8 below.
  • the aim of this study was to determine the pharmacokinetic parameters of antibodies following intravenous injection in male and female monkeys.
  • the specific experimental process is as follows: before the antibody is injected into the tail vein of male and female monkeys, blood samples are taken first. Then, after tail vein injection of low-dose 10mg/kg antibody and high-dose 100mg/kg antibody, blood was collected at the time points of 10min, 2h, 8h, 24h, day4, day7, day10, and day14 in the 10 mg/kg group before administration. , 100mg/kg measurement group in the measurement group before administration, 10min, 2h, 8h, 24h, day4, day7 blood was collected to detect the antibody concentration in serum.
  • the PK experimental design is shown in Table 10 below.
  • Capan-2 mouse tumor model was used to study the anti-tumor activity of anti-human LIFR antibody.
  • the pharmacodynamics of PR300516 antibody administered alone (5 mg/kg and 15 mg/kg) in the Capan-2 mouse model was investigated.
  • Human pancreatic cancer Capan-2 cells (ATCC cell bank) 5x10e6 cells were inoculated subcutaneously into female Balb/c nude mice (Viton Lever).
  • the treatment methods of the antibody group were as follows: mice in each group were intraperitoneally injected with corresponding antibodies on the 5th day, the 9th day, the 12th day, the 16th day, the 19th day and the 23rd day after tumor cell inoculation.
  • the treatment methods of the small molecule inhibitor group were: day 5, day 7, day 9, day 12, day 14, day 16, day 19, day 21 and day 23 after tumor cell inoculation.
  • the anti-human LIFR antibody PR300516 of the present application has a certain inhibitory effect on tumor growth when used alone.
  • the anti-human LIFR antibody described in this application has stronger inhibitory effect on tumor growth. In all groups of mice in this study, there was no significant change in the body weight of mice 37 days after inoculation.
  • Capan-1 mouse tumor model was used to study the anti-tumor activity of anti-human LIFR antibody.
  • the pharmacodynamics of PR300516 and PR301168 antibodies administered alone (15 mg/kg) in the Capan-1 mouse model were investigated.
  • Human pancreatic cancer Capan-1 cells (ATCC cell bank) 5x10e6 cells were subcutaneously inoculated into female Balb/c nude mice (Viton Lever).
  • the treatment methods of the antibody group were as follows: mice in each group were intraperitoneally injected with corresponding antibodies on the 5th, 8th, 12th, 15th, 18th and 21st days after tumor cell inoculation.
  • NCI-H292 mouse tumor model was used to study the anti-tumor activity of anti-human LIFR antibodies.
  • the pharmacodynamics of PR300516 and PR301168 antibodies administered alone (15 mg/kg) in the NCI-H292 mouse model were investigated.
  • Human pancreatic cancer NCI-H292 cells (ATCC cell bank) 5x10e6 cells were subcutaneously inoculated into female Balb/c nude mice (Viton Lever).
  • the treatment methods of the antibody group were as follows: mice in each group were intraperitoneally injected with corresponding antibodies on the 5th, 8th, 12th, 15th, 18th and 21st days after tumor cell inoculation.
  • the results are shown in Table 17 and Figure 10.
  • the anti-human LIFR antibodies PR300516 and PR301168 of the present application have a certain inhibitory effect on tumor growth when used alone.
  • the anti-human LIFR antibody described in this application has a weaker inhibitory effect on tumor growth. In all groups of mice in this study, the body weight of mice decreased 25 days after inoculation.
  • TGI(%) 1 Isotype 5mg/kg 0 2 EC359, 5mg/kg 40.99 3 EC359, 15mg/kg 47.85 4 PR300516, 5mg/kg 54.92 5 PR300516, 15mg/kg 55.32 6 MSC-1, 5mg/kg 48.93 7 MSC-1, 15mg/kg 49.34
  • TGI(%) 1 Isotype 15mg/kg 0 2 MSC-1, 15mg/kg 53.96 3 PR300516, 15mg/kg 59.32 4 PR301168, 15mg/kg 59.89
  • TGI(%) 1 Isotype 15mg/kg 0 2 MSC-1, 15mg/kg 39.99 3 PR300516, 15mg/kg 36.35 4 PR301168, 15mg/kg 15.58

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

提供了一种人LIFR抗原结合蛋白及其制备方法和应用。采用人LIFR-ECD蛋白免疫H2L2人源抗体转基因小鼠，利用单个B细胞克隆技术(SBC)，及单B细胞测序分析，Fc重组得到了一系列具有生物学功能的人LIFR抗体，该方法大大提高了抗体药物发现的效率和产率，且获得的人LIFR抗体具有很好的亲和力，可阻断huLIF蛋白与huLIFR/gp130细胞的结合，从而抑制STAT3等信号通路，进一步抑制肿瘤的生长，达到肿瘤预防和治疗的目的。

Description

人LIFR抗原结合蛋白及其制备方法和应用 技术领域
本发明属于生物医药领域，具体涉及人LIFR抗原结合蛋白及其制备方法和应用。
背景技术
白血病抑制因子(leukemia inhibitory factor，LIF)是一种具有多种生物学功能的细胞因子，因其能够诱导M1髓系白血病细胞向正常细胞分化而被命名。LIF属于白细胞介素-6(IL-6)家族，后者还包括IL-11、IL-27、睫状神经营养因子(CNTF)、心肌素-1(CT-1)和制瘤素M(OSM)等，这些细胞因子与LIF共享gp130受体，因此，LIF具有与其他家族成员重叠而独特的生物学作用。
哺乳动物中LIF蛋白由180个氨基酸残基组成，具有高度保守性，人的LIF基因编码在染色体22q12上，而小鼠LIF基因定位于11号染色体上，小鼠和人类LIF基因同源性＞75％。成熟LIF蛋白是一种高度糖基化分泌蛋白，可由卵母细胞和人胚胎的胚外细胞、子宫内膜细胞、成纤维细胞、肝细胞、单核巨噬等多种成体细胞分泌。不同组织来源的LIF蛋白糖基化不同，其中研究最多的是一种分泌型、可变异的糖基化蛋白(34-63kDa)，它具有诱导自分泌和广泛的旁分泌效应，这种作用可能因细胞类型和背景不同而相互矛盾，如增殖与分化、存活和凋亡等。
LIF主要通过结合其特异性LIF受体(LIF receptor，LIFR)，然后招募gp130受体亚基形成高亲和力异源二聚体受体复合物，诱导下游JAK/STAT3，PI3K/AKT，ERK1/2，MAPK等信号通路的活化从而发挥各种细胞功能，调节细胞的增殖和存活。且LIFR丰富表达于中枢神经系统、肾脏、肝脏、骨骼、子宫等多种组织器官。LIF和LIFR的广泛分布和数条LIF信号通路决定了LIF功能的复杂多样性。
LIF通路在免疫学领域是一个很有前途的新靶点。有研究报道，LIF信号通路在肿瘤的发生和进展中发挥了重要作用，能够增强肿瘤细胞的迁移和浸润，促进肿瘤转移；同时可以调节肿瘤微环境中多种免疫细胞，包括效应T细胞(T-eff)，调节T细胞(T-reg)，Th17以及髓系细胞等。还有研究表明，LIF信号通路可以促进肿瘤起始细胞(CIC)的活性，增强抗肿瘤治疗(化疗和放疗)的抗性。在多种癌症中，LIF的高表达与不良预后相关。
专利CN111378036A公开了一种抗人白血病抑制因子(LIF)单克隆抗体的制备方法及其应用，其采用小鼠杂交瘤细胞技术制备得到抗人白血病抑制因子单克隆抗体，但该方法抗体筛选的效率和产率较低。专利CN111868086A公开了一系列抗LIF抗体及其应用，其LIF抗体可以结合LIF独特的表位，从而抑制LIF的生物活性。
目前临床上针对LIF靶点的抗体有Northern biologics的MSC-1抗体，该抗体于2019年完成了I期临床研究，目前，正计划开展多项临床疗效试验，将MSC-1用于治疗胰腺癌及肺癌等一系列实体肿瘤。MSC-1抗体是一个人源化单克隆抗体(IgG1)，可以结合免疫抑制人细胞因子LIF，用于治疗成年晚期实体瘤患者。然而，MSC-1抗体只能够结合LIF的特异性表位，因此不能完全竞争掉LIF与受体的结合。EC359是靶向LIFR的小分子抑制剂(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)。
技术问题
现有技术中尚未有针对LIFR治疗性抗体的报道，因此，亟需开发针对LIF受体(LIFR)的高效特异性的阻断抗原结合蛋白，用于LIFR相关疾病的治疗。
技术解决方案
针对上述不足，本发明首次提供了一种全人LIFR抗原结合蛋白及其制备方法和应用。本发明采用人LIFR-ECD蛋白免疫H2L2人源抗体转基因小鼠，利用单个B细胞克隆技术(SBC)，得到了一系列具有生物学功能的人LIFR抗体，该方法大大提高了抗体药物发现的效率和产率，且得到的人LIFR抗体具有很好的亲和力，可阻断huLIF蛋白与huLIFR/gp130细胞的结合，从而抑制STAT3等信号通路，抑制肿瘤的生长，达到肿瘤预防和治疗的目的。
为了实现上述发明目的，本发明的技术方案如下：
一方面，本发明提供了一种人LIFR抗原结合蛋白。
具体地，所述的人LIFR抗原结合蛋白包含以下互补决定区：
(1)包含SEQ ID NO:1-SEQ ID NO:4中任一个所示的氨基酸序列或其变体序列的重链互补决定区1HCDR1；
(2)包含SEQ ID NO:5-SEQ ID NO:15中任一个所示的氨基酸序列或其变体序列的重链互补决定区2HCDR2；
(3)包含SEQ ID NO:16-SEQ ID NO:25中任一个所示的氨基酸序列或其变体序列的重链互补决定区3HCDR3；
(4)包含SEQ ID NO:26-SEQ ID NO:32中任一个所示的氨基酸序列或其变体序列的轻链互补决定区1LCDR1；
(5)包含SEQ ID NO:33-SEQ ID NO:40中任一个所示的氨基酸序列或其变体序列的轻链互补决定区2LCDR2；
(6)包含SEQ ID NO:41-SEQ ID NO:45中任一个所示的氨基酸序列或其变体序列的轻链互补决定区3LCDR3；
优选地，所述变体序列为与其来源的CDR相比具有一个或几个氨基酸的置换、缺失或添加的CDR序列；所述的置换为保守置换。
进一步具体地，所述的人LIFR抗原结合蛋白包含如下互补决定区：
(1)包含序列如GFTFSSYGMX 1所示的氨基酸序列或序列如SEQ ID NO:4：GFTFSNYAMT所示的氨基酸序列的重链互补决定区1HCDR1，其中X 1＝H、D或N；
和/或(2)包含序列如VIWYDGX 2NKYYX 3DSVKG所示的氨基酸序列、序列如VIWFDGSX 4KYYADSVKG所示的氨基酸序列、序列如SEQ ID NO:7：VIWYDGSNKFYADSVRG所示的氨基酸序列、序列如SEQ ID NO:14：TISGSGAFTYYADAVKG所示的氨基酸序列或序列如SEQ ID NO:15：VISGSGFLTYYADAVKG所示的氨基酸序列的重链互补决定区2HCDR2，其中X 2＝N或S，X 3＝E、A或T，X 4＝N、I、L或V；
和/或(3)包含序列如SEQ ID NO:16：DGESSMVRGLLNWFDP所示的氨基酸序列、序列如SEQ ID NO:17：ELRYFDWLLSPFDY所示的氨基酸序列、序列如SEQ ID NO:21：GERTLDL所示的氨基酸序列、序列如ELWFGELLSPX 5DF所示的氨基酸序列、序列如GQLVX 6DX 7所示的氨基酸序列或序列如GGILTGFDX 8所示的氨基酸序列的重链互补决定区3HCDR3，其中X 5＝L或F，X 6＝G或Q，X 7＝Y、F或L，X 8＝N或Y；
和/或(4)包含序列如RASQSX 9SSSX 10LA所示的氨基酸序列、序列如RASQSISSX 11LX 12所示的氨基酸序列或序列如SEQ ID NO:32：RASQNLNSNLA所示的 氨基酸序列的轻链互补决定区1LCDR1，其中X 9＝V或I，X 10＝Y或F，X 11＝Y、W或N，X 12＝N或A；
和/或(5)包含序列如GX 13SSRAT所示的氨基酸序列、序列如KASX 14LEX 15所示的氨基酸序列、序列如SEQ ID NO:35：AASNRAT所示的氨基酸序列、序列如SEQ ID NO:36：AASSLQS所示的氨基酸序列或序列如SEQ ID NO:40：GASTRAP所示的氨基酸序列的轻链互补决定区2LCDR2，其中X 13＝A或T，X 14＝S或N，X 15＝S或N；
和/或(6)包含序列如SEQ ID NO:41：QQYGSSPFT所示的氨基酸序列、序列如SEQ ID NO:42：QQSYSTPLT所示的氨基酸序列、序列如SEQ ID NO:43：QQYKSFSPGGLT所示的氨基酸序列、序列如SEQ ID NO:44：QQYKSNPLT所示的氨基酸序列或序列如SEQ ID NO:45：QQYNNWPRT所示的氨基酸序列的轻链互补决定区3LCDR3。
进一步具体地，所述的人LIFR抗原结合蛋白包含：
(1)、包含SEQ ID NO:46-SEQ ID NO:59中任一个所示的氨基酸序列的重链可变区VH；和/或包含SEQ ID NO:60-SEQ ID NO:69中任一个所示的氨基酸序列的轻链可变区VL；
或者(2)、与(1)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(1)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；
或者(3)、与(1)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与(1)中的任一VL相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VL；所述的置换是保守置换。
进一步具体地，所述的人LIFR抗原结合蛋白包含：
(1)包含SEQ ID NO:70-SEQ ID NO:83中任一个所示的氨基酸序列的重链HC；和/或包含SEQ ID NO:84-SEQ ID NO:93中任一个所示的氨基酸序列的轻链LC；
或者(2)重链和轻链，其中，与(1)中的重链和轻链相比，所述重链具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，所述轻链具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
进一步具体地，所述的人LIFR抗原结合蛋白包含：
(1)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:5所示的HCDR2，如SEQ ID NO:16所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:33所示的LCDR2，如SEQ ID NO:41所示的LCDR3；
(2)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:6所示的HCDR2，如SEQ ID NO:17所示的HCDR3，和/或如SEQ ID NO:27所示的LCDR1，如SEQ ID NO:34所示的LCDR2，如SEQ ID NO:41所示的LCDR3；
(3)如SEQ ID NO:2所示的HCDR1，如SEQ ID NO:7所示的HCDR2，如SEQ ID NO:18所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:33所示的LCDR2，如SEQ ID NO:41所示的LCDR3；
(4)如SEQ ID NO:2所示的HCDR1，如SEQ ID NO:7所示的HCDR2，如SEQ ID NO:19所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:33所示的LCDR2，如SEQ ID NO:41所示的LCDR3；
(5)如SEQ ID NO:2所示的HCDR1，如SEQ ID NO:8所示的HCDR2，如SEQ ID NO:19所示的HCDR3，和/或如SEQ ID NO:28所示的LCDR1，如SEQ ID NO:35所示的LCDR2，如SEQ ID NO:41所示的LCDR3；
(6)如SEQ ID NO:2所示的HCDR1，如SEQ ID NO:9所示的HCDR2，如SEQ ID NO:19所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:35所示的LCDR2，如SEQ ID NO:41所示的LCDR3；
(7)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:10所示的HCDR2，如SEQ ID NO:20所示的HCDR3，和/或如SEQ ID NO:29所示的LCDR1，如SEQ ID NO:36所示的LCDR2，如SEQ ID NO:42所示的LCDR3；
(8)如SEQ ID NO:3所示的HCDR1，如SEQ ID NO:10所示的HCDR2，如SEQ ID NO:21所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:37所示的LCDR2，如SEQ ID NO:43所示的LCDR3；
(9)如SEQ ID NO:3所示的HCDR1，如SEQ ID NO:11所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；
(10)如SEQ ID NO:3所示的HCDR1，如SEQ ID NO:11所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:39所示的LCDR2，如SEQ ID NO:44所示的LCDR3；
(11)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:12所示的HCDR2，如SEQ ID NO:23所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；
(12)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:11所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；
(13)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:13所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；
(14)如SEQ ID NO:4所示的HCDR1，如SEQ ID NO:14所示的HCDR2，如SEQ ID NO:24所示的HCDR3，和/或如SEQ ID NO:31所示的LCDR1，如SEQ ID NO:37所示的LCDR2，如SEQ ID NO:43所示的LCDR3；
(15)如SEQ ID NO:4所示的HCDR1，如SEQ ID NO:15所示的HCDR2，如SEQ ID NO:25所示的HCDR3，和/或如SEQ ID NO:32所示的LCDR1，如SEQ ID NO:40所示的LCDR2，如SEQ ID NO:45所示的LCDR3。
优选地，所述的人LIFR抗原结合蛋白采用Mega软件进行序列相似性分析，包含以下五组：
组一：(1)所述的抗原结合蛋白；
组二：(2)、(3)、(4)所述的抗原结合蛋白；
组三：(5)、(6)所述的抗原结合蛋白；
组四：(7)、(8)、(9)、(10)、(11)、(12)、(13)所述的抗原结合蛋白；
组五：(14)、(15)所述的抗原结合蛋白。
进一步具体地，所述的人LIFR抗原结合蛋白包含：
(1)如SEQ ID NO:46所示的VH，和/或如SEQ ID NO:60所示的VL；
(2)如SEQ ID NO:47所示的VH，和/或如SEQ ID NO:61所示的VL；
(3)如SEQ ID NO:48所示的VH，和/或如SEQ ID NO:62所示的VL；
(4)如SEQ ID NO:49所示的VH，和/或如SEQ ID NO:62所示的VL；
(5)如SEQ ID NO:50所示的VH，和/或如SEQ ID NO:63所示的VL；
(6)如SEQ ID NO:51所示的VH，和/或如SEQ ID NO:64所示的VL；
(7)如SEQ ID NO:52所示的VH，和/或如SEQ ID NO:65所示的VL；
(8)如SEQ ID NO:53所示的VH，和/或如SEQ ID NO:66所示的VL；
(9)如SEQ ID NO:54所示的VH，和/或如SEQ ID NO:67所示的VL；
(10)如SEQ ID NO:54所示的VH，和/或如SEQ ID NO:68所示的VL；
(11)如SEQ ID NO:55所示的VH，和/或如SEQ ID NO:67所示的VL；
(12)如SEQ ID NO:56所示的VH，和/或如SEQ ID NO:67所示的VL；
(13)如SEQ ID NO:57所示的VH，和/或如SEQ ID NO:67所示的VL；
(14)如SEQ ID NO:58所示的VH，和/或如SEQ ID NO:66所示的VL；
(15)如SEQ ID NO:59所示的VH，和/或如SEQ ID NO:69所示的VL；
或者，与(1)-(15)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(1)-(15)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；
或者，与(1)-(15)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与(1)-(15)中的任一VL相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VL；所述的置换是保守置换。
进一步具体地，所述的人LIFR抗原结合蛋白包含：
(1)如SEQ ID NO:70所示的HC，和/或如SEQ ID NO:84所示的LC；
(2)如SEQ ID NO:71所示的HC，和/或如SEQ ID NO:85所示的LC；
(3)如SEQ ID NO:72所示的HC，和/或如SEQ ID NO:86所示的LC；
(4)如SEQ ID NO:73所示的HC，和/或如SEQ ID NO:86所示的LC；
(5)如SEQ ID NO:74所示的HC，和/或如SEQ ID NO:87所示的LC；
(6)如SEQ ID NO:75所示的HC，和/或如SEQ ID NO:88所示的LC；
(7)如SEQ ID NO:76所示的HC，和/或如SEQ ID NO:89所示的LC；
(8)如SEQ ID NO:77所示的HC，和/或如SEQ ID NO:90所示的LC；
(9)如SEQ ID NO:78所示的HC，和/或如SEQ ID NO:91所示的LC；
(10)如SEQ ID NO:78所示的HC，和/或如SEQ ID NO:92所示的LC；
(11)如SEQ ID NO:79所示的HC，和/或如SEQ ID NO:91所示的LC；
(12)如SEQ ID NO:80所示的HC，和/或如SEQ ID NO:91所示的LC；
(13)如SEQ ID NO:81所示的HC，和/或如SEQ ID NO:91所示的LC；
(14)如SEQ ID NO:82所示的HC，和/或如SEQ ID NO:90所示的LC；
(15)如SEQ ID NO:83所示的HC，和/或如SEQ ID NO:93所示的LC；
或者，与(1)-(15)中的重链和轻链相比，所述重链具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，所述轻链具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
进一步具体地，上述人LIFR抗原结合蛋白包括嵌合抗体、人源化抗体或全人源抗体。
进一步具体地，上述人LIFR抗原结合蛋白包括全长抗体、Fab、Fab’、F(ab’)2、Fv、scFv、di-scFv、双特异性抗体、多特异性抗体、重链抗体和/或单域抗体，或由上述抗体制得的单克隆抗体和/或多克隆抗体。
另一方面，本发明提供了一系列编码所述人LIFR抗原结合蛋白的核酸分子。
具体地，所述的核酸分子包含一种或多种经密码子优化的核酸分子。
又一方面，本发明提供了一系列载体，所述的载体包含本申请所述的一个或多个核酸分子。
具体地，所述的载体包括但不限于质粒、病毒、噬菌体。
又一方面，本发明提供了一系列包含上述核酸分子或上述载体的宿主细胞。
具体地，所述的宿主细胞包括但不限于微生物、植物或动物细胞，可通过本领域技术人员已知的方法将本发明所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等方法。
又一方面，本发明提供了一种嵌合抗原受体，所述的嵌合抗原受体包含上述人LIFR抗原结合蛋白。
又一方面，本发明提供了一种免疫细胞，所述的免疫细胞包含上述嵌合抗原受体。
又一方面，本发明提供了一种抗原结合蛋白衍生物，所述的抗原结合蛋白衍生物包含上述人LIFR抗原结合蛋白和可检测的标记分子。
具体地，所述的可检测的标记分子为酶(例如辣根过氧化物酶)、放射性核素、荧光染料、发光物质(如化学发光物质)或生物素。
又一方面，本发明提供了一种多特异性抗体，所述的多特异性抗体包含上述人LIFR抗原结合蛋白，和另外的抗体或其片段或抗体类似物。
具体地，所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。
又一方面，本发明提供了一种抗体药物偶联物，所述的抗体药物偶联物包括抗体部分和偶联部分，所述抗体部分包含上述人LIFR抗原结合蛋白，所述偶联部分包括但不限于可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合，所述抗体部分和偶联部分通过化学键或接头进行偶联。
又一方面，本发明提供了一种药物组合物，其特征在于，所述的药物组合物包含上述人LIFR抗原结合蛋白、核酸分子、载体、宿主细胞、嵌合抗原受体、免疫细胞、抗原结合蛋白衍生物、多特异性抗体和/或抗体药物偶联物。
具体地，所述的药物组合物还包含任选的药学上可接受的载体。
进一步具体地，所述的药学上可接受的载体包括但不限于：稀释剂、赋形剂、填充剂、润湿剂、崩解剂、矫味剂和粘合剂。
具体地，所述的药物组合物还包含组合治疗剂，所述的组合治疗剂包括但不限于化学治疗剂、放射治疗剂、免疫抑制剂、细胞毒性药物。
又一方面，本发明提供了上述人LIFR抗原结合蛋白的生产方法，所述的方法包括在使得所述抗原结合蛋白表达的情况下，培养上述宿主细胞。
具体地，所述的方法包括以下步骤：
(1)采用LIFR-ECD蛋白作为抗原免疫小鼠；
(2)筛选分泌抗体的抗原特异性B细胞；
(3)从步骤(2)筛选出的单个B细胞中恢复抗体的重链和轻链序列；
(4)将步骤(3)恢复的抗体重链和轻链与人Fc重组，引入宿主细胞，经细胞培养、纯化制备得到人LIFR抗原结合蛋白。
进一步具体地，步骤(1)中所述的小鼠为人源化小鼠。
优选地，所述的人源化小鼠为Harbour H2L2小鼠，所述的Harbour H2L2转基因小鼠品系能够产生具有全人源可变区的传统的两重链两轻链免疫球蛋白抗体。
进一步具体地，步骤(2)中所述的筛选分泌抗体的抗原特异性B细胞的方法为：使用Beacon光电系统的浆细胞(plasma cell)发现工作流程进行筛选。
进一步具体地，步骤(3)中所述的恢复抗体的重链和轻链序列的方法为单B细胞测序。
进一步具体地，所述的单B细胞测序的步骤为：从单B细胞裂解液中纯化RNA，cDNA的逆转录合成，cDNA的扩增纯化，重链和轻链的扩增，克隆和转染，Sanger测序，对得到的序列进行唯一性和聚类分析，最后将配对的重链和轻链DNA序列进行质粒合成。
又一方面，本发明提供了所述的人LIFR抗原结合蛋白、核酸分子、载体、宿主细胞、嵌合抗原受体、免疫细胞、抗原结合蛋白衍生物、多特异性抗体、抗体药物偶联物和/或药物组合物在制备用于LIF和/或LIFR阻断药物、试剂盒和/或医疗装置中的应用。
具体地，所述的LIFR阻断药物、试剂盒和/或装置主要应用于LIF和/或LIFR表达量增高的疾病中。
又一方面，本发明提供了所述的人LIFR抗原结合蛋白、核酸分子、载体、宿主细胞、嵌合抗原受体、免疫细胞、抗原结合蛋白衍生物、多特异性抗体、抗 体药物偶联物和/或药物组合物在制备用于预防和/或治疗LIFR阳性疾病的药物、试剂盒和/或给药装置中的应用。
又一方面，本发明提供了所述的人LIFR抗原结合蛋白、抗原结合蛋白衍生物在制备LIFR检测试剂或试剂盒中的用途。
又一方面，本发明提供了LIFR检测方法，利用上述人LIFR抗原结合蛋白定性或定量分析检测LIFR，优选地，所述的检测方法用于非疾病诊断或治疗目的。
又一方面，本发明提供了LIFR阳性相关疾病的治疗方法，所述的治疗方法为向有需要的受试者施用有效量的上述人LIFR抗原结合蛋白、免疫细胞、抗原结合蛋白衍生物、多特异性抗体，抗体药物偶联物和/或药物组合物。
具体地，所述的LIFR阳性疾病为肿瘤，所述肿瘤包括但不限于胆管癌、结直肠癌、神经胶质瘤、前列腺癌、卵巢癌、胃癌、鼻咽癌、乳腺癌、膀胱癌、胰腺癌、非小细胞肺癌。
又一方面，本发明提供了一种试剂盒，所述的试剂盒包括所述的人LIFR抗原结合蛋白、核酸分子、载体、宿主细胞、嵌合抗原受体、免疫细胞、抗原结合蛋白衍生物、多特异性抗体、抗体药物偶联物和/或药物组合物，及任选地，说明书。
又一方面，本发明提供了一种给药装置，所述的给药装置包含：(1)用于对有需要的受试者施用所述的药物组合物的输注模块，以及(2)任选的药效监控模块。
又一方面，本发明提供了LIFR抗原结合蛋白在制备癌症治疗药物中的用途，所述癌症选自胆管癌、结直肠癌、神经胶质瘤、前列腺癌、卵巢癌、胃癌、鼻咽癌、乳腺癌、膀胱癌、胰腺癌、非小细胞肺癌。
具体地，所述的LIFR抗原结合蛋白包含上述任意一项LIFR抗原结合蛋白。
有益效果
与现有技术相比，本发明的积极和有益效果在于：
(1)本发明首次提供了一种人LIFR抗体，所述的人LIFR抗体通过免疫人源抗体转基因小鼠(如Harbour H2L2小鼠)制备得到，具有全人源抗体氨基酸和基因序列，从而确保人体使用时的最低可能的免疫原性，去除了毒副作用。
(2)本发明采用单个B细胞克隆技术(SBC)筛选阳性克隆，与传统的单克隆抗体筛选技术相比，可大大提高抗体发现的效率和产率。
(3)本发明制备的人LIFR抗体特异性高，亲和力高，抗肿瘤活性好。
(4)本发明的抗体为全人抗体，从而可安全地施用给人受试者，而不引发免疫原性反应，具有重大的临床价值。
附图说明
图1为本发明所述人LIFR抗体在蛋白水平与LIFR-ECD的结合能力检测图，其中A为本发明所述人LIFR抗体与人LIFR-ECD的结合能力检测结果，B为本发明所述人LIFR抗体与猴LIFR-ECD的结合能力检测结果，C为本发明所述人LIFR抗体与小鼠LIFR-ECD的结合能力检测结果。
图2为本发明所述抗体在细胞水平与LIFR和LIFR/gp130结合能力检测图，其中，A为本发明所述抗体与LIFR结合能力检测结果，B为本发明所述抗体与LIFR/gp130结合能力检测结果。
图3为本发明所述抗体阻断LIF与LIFR/gp130结合能力检测图。
图4为本发明所述抗体对STAT3信号通路的抑制作用检测图。
图5为本发明所述抗体在Balb/c小鼠体内PK检测图。
图6为本发明所述抗体在Sprague Dawley大鼠体内的PK检测图，其中，A为PR300516抗体在大鼠体内的PK检测图，B为PR301168抗体在大鼠体内的PK检测图，C为PR300516抗体和PR301168抗体在大鼠体内的PK平均值检测图。
图7为本发明所述抗体在猴子体内的PK检测图，其中，A为10mg/mL单次给药检测结果图，B为100mg/mL单次给药检测结果图。
图8为本发明所述抗体在Capan-2小鼠模型体内的药效学实验检测图。
图9为本发明所述抗体在Capan-1小鼠模型体内的药效学实验检测图。
图10为本发明所述抗体在NCI-H292模型小鼠模型体内的药效学实验检测图。
本发明的实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
术语定义
为了更容易理解本发明，以下具体定义了某些技术和科学用语。除非在本文中另有明确定义，本文使用的所有的其他技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968,IUPAC-IUB委员会)中所述。
本发明所述的“抗原结合蛋白”通常是指包含结合抗原部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR或FWR)的更保守的区域中。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物、嵌合抗原受体或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键链接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA、IgE，其相应的重链分别为μ链、δ链、γ链、α链、ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可以分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补决定区(CDR)。每条轻链可变区(VL或LCVR)和重链可变区(VH或HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3，重链的3个CDR区指HCDR1、HCDR2和HCDR3。
术语“互补决定区”(CDR)指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。CDR可根据本领域已知的各种编号系统确定，例如Kabat、Chothia或，AbM或IMGT编号系统。本发明中使用“Kabat编号规则”(参见Kabat等(1991))确定CDR的氨基酸序列边界。
术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“单克隆抗体”、“单抗”、“mAb”是指从基本上均质抗体的群里获得的抗体，即除可能的变体抗体外，构成所述群体的个别抗体相同和/或结合相同的表位。与通常包含针对不同决定簇的不同抗体的多克隆抗体制备物不同，单克隆抗体制备物的每个单克隆抗体是针对抗原上的单一决定簇的。因此。“单克隆”是指从基本上均质抗体群体获得的抗体的特性，且不应解释为需要通过任何特定方法来制造抗体。本发明所述的单克隆抗体可通过各种本领域技术人员已知的技术制备，所述技术包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法及转基因方法等。
术语“人源化单克隆抗体”是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体，可以克服由于嵌合抗体携带大量鼠蛋白成分，从而诱导的异源性反应。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变(或回复突变)，以保持活性。为制备人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539；Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370；以及Lo,Benny,K.C.,editor,in Antibody Engineering:Methods and Protocols,volume 248,Humana Press,New Jersey,2004)。或者，还可以利用转基因动物，其能够在免疫后不产生内源性免疫球蛋白、并且能够产生完整人抗体库(参见例如，Jakobovits等，1993，Proc.Natl.Acad.Sci.USA 90：2551；Jakobovits等，1993，Nature 362：255-258；Bruggermann等，1993，Year in Immunology 7：33；和Duchosal等，1992，Nature 355：258；Lonberg等人(1994)Nature 368(6474):856-859；WO02/43478)。其他 抗体人源化改造的方法还包括噬菌体展示技术(Hoogenboom等，1991，J.Mol.Biol.227：381；Marks等，J.Mol.Biol.1991，222：581-597；Vaughan等，1996，Nature Biotech 14：309)。其中，“转基因动物”的实例包括但不限于HARBOURBIOMED的H2L2人源化小鼠。术语“特异性结合”、“选择性结合”是指两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以用相互作用的解离平衡常数(KD)表示。在本文中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。解离平衡常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语“同一性”通常是指在比对序列后，查询序列中氨基酸残基或核苷酸与第二参考多肽序列或其部分中相同的残基所占百分比，必要时引入空位(GAPS)以实现最大序列同一性百分比，并且不考虑任何保守取代作为序列同一性的一部分。用于确定氨基酸/核苷酸序列同一性百分比的比对可以通过本领域已知的各种方式来实现，例如，使用可公开获得的计算机软件，例如BLAST，BLAST-2，ALIGN，NEEDLE或Megalign(DNASTAR)。本领域技术人员可以确定用于测量比对的合适参数，包括在所比较的序列的全长上实现最大比对所需的任何算法。同一性百分比可以在整个确定的多肽/多核苷酸序列的长度上测定，或者可以在较短的长度上，例如在从较大的确定的多肽/多核苷酸序列中获得的片段的长度上测定。应当理解，在表，附图或序列表中，本文所示的序列所支持的任何片段长度可用于作为可测量同一性百分比的长度。具有“％同一性”的序列保留与其比较或来源的序列的重要生物活性，如抗体结合特异性。具有一个或几个氨基酸的置换、缺失或添加或其任意组合的序列保留与其比较或来源的序列的重要生物活性，如抗体结合特异性。具有“％同一性”的核苷酸序列或相差不超过3、6、15、30或45个核苷酸的核苷酸序列能够实现与其比较或来源的核苷酸序列近似的功能，如所表达的蛋白都能特异地结合同一抗原或分子。
术语“保守置换”指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残 基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
术语“宿主细胞”，通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通 过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHOK1细胞。
术语“药学上可接受的载体”是指在药理学和/或生理学上与受试者和活性成分相容的载体，是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。所述组合物是无菌的。
术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如非人灵长类哺乳动物或人。在某些实施方式中，所述受试者(例如人)患有癌症(包括但不限于胆管癌、结直肠癌、神经胶质瘤、前列腺癌、卵巢癌、胃癌、鼻咽癌、乳腺癌、膀胱癌、胰腺癌、非小细胞肺癌)，或者，具有患有上述疾病的风险。
术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防 疾病(例如，LIFR阳性相关)有效量是指，足以预防，阻止，或延迟疾病(例如，LIFR阳性相关疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
术语“嵌合抗原受体”指的是嵌合抗原受体T细胞通过识别某种肿瘤抗原的抗体的抗原结合部位与CD3-δ链或FcεRIγ的胞内部分在体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体，使得患者的T细胞被“重编码”后，能够生成大量肿瘤特异性的CAR-T细胞。把重新编码后的嵌合抗原受体T细胞加进患者身体里，这种嵌合抗原受体就像GPS一样可以特异性地追踪和识别并引导T细胞杀伤肿瘤细胞。大多数嵌合抗原受体由胞外抗原结合区(由来源于单克隆抗体的轻链和重链组成，中间由带韧性的铰链区连接形成单链抗体)、跨膜区域和胞内信号转导区组成。通过将识别肿瘤相关抗原的scFv和胞内信号域“免疫受体酪氨酸活化基序”在体外进行基因重组获得CAR结构。
术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞，例如淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
缩略词
CDR：免疫球蛋白可变区中的互补决定区。
VH：抗体重链可变区。
VL：抗体轻链可变区。
HC：抗体重链。
LC：抗体轻链。
IgG：免疫球蛋白G。
AbM：AbM CDR定义方式来源于Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272)，此定义方法整合了Kabat及Chothia两者的部分定义。
Kabat：由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
Chothia：由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia & Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
IMGT：基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information
Figure PCTCN2021142404-appb-000001
(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
mAb：单克隆抗体。
EC50：产生50％功效或结合的浓度。
IC50：产生50％抑制的浓度。
ELISA：酶联免疫吸附测定。
PCR：聚合酶链式反应。
HRP：辣根过氧化物酶。
LIFR：白血病抑制因子受体。
IL7Rα：白介素7受体alpha亚基。
TARC：胸腺激活调节趋化因子。
hFc：人IgG抗体Fc段。
KD：解离平衡常数。
HCDR1：免疫球蛋白重链可变区中的互补决定区1。
HCDR2：免疫球蛋白重链可变区中的互补决定区2。
HCDR3：免疫球蛋白重链可变区中的互补决定区3。
LCDR1：免疫球蛋白轻链可变区中的互补决定区1。
LCDR2：免疫球蛋白轻链可变区中的互补决定区2。
LCDR3：免疫球蛋白轻链可变区中的互补决定区3。
发明详述
抗原结合蛋白
本发明制备了一系列全人源单克隆抗体。这些抗人LIFR抗体是通过免疫全人源抗体转基因小鼠、分子生物学和抗体工程技术制备，具有全人源抗体氨基酸和基因序列，从而确保人体使用时的最低可能的免疫原性。人源抗体转基因小鼠技术最早由Abgenix(XenoMouse)和Madarex(HuMab mouse)开发及用于全人源抗体的制备(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)。早期的转基因小鼠产生全人免疫球蛋白(Ig)，即抗体的可变区和恒定区全为人源序列，因人Ig Fc与小鼠宿主Fc受体不匹配，因而导致免疫反应很弱，抗原免疫后血清中抗体效价很低，表现在低下的单抗制备效率。近年来该技术经过了重大革新和改进，主要表现在重链和轻链可变区采用与宿主免疫球蛋白(Ig)相同和相似的恒定区序列。本发明采用Harbour BioMed公司研发的转基因小鼠(Harbour H2L2)成功地制备了LIFR全人源抗体。Harbour转基因小鼠引入了人免疫球蛋白可变区基因和大鼠免疫球蛋白恒定区基因，小鼠本身的Ig表达则被灭活。该转基因小鼠经抗原免疫后能产生与正常小鼠(如Balb/c)相当的免疫反应和抗体效价。
在某些实施方式中，本发明提供的抗原结合蛋白包括以下互补决定区：
(1)包含SEQ ID NO:1-SEQ ID NO:4中任一个所示的氨基酸序列或其变体序列的重链互补决定区1HCDR1；
(2)包含SEQ ID NO:5-SEQ ID NO:15中任一个所示的氨基酸序列或其变体序列的重链互补决定区2HCDR2；
(3)包含SEQ ID NO:16-SEQ ID NO:25中任一个所示的氨基酸序列或其变体序列的重链互补决定区3HCDR3；
(4)包含SEQ ID NO:26-SEQ ID NO:32中任一个所示的氨基酸序列或其变体序列的轻链互补决定区1LCDR1；
(5)包含SEQ ID NO:33-SEQ ID NO:40中任一个所示的氨基酸序列或其变体序列的轻链互补决定区2LCDR2；
(6)包含SEQ ID NO:41-SEQ ID NO:45中任一个所示的氨基酸序列或其变体序列的轻链互补决定区3LCDR3；
所述变体序列为与其来源的CDR相比具有一个或几个氨基酸的置换、缺失或添加的CDR序列。
在某些实施方式中，本发明提供的人LIFR抗原结合蛋白包含如下互补决定区：
(1)包含序列如GFTFSSYGMX 1所示的氨基酸序列或序列如SEQ ID NO:4：GFTFSNYAMT所示的氨基酸序列的重链互补决定区1HCDR1，其中X 1＝H、D或N；
和/或(2)包含序列如VIWYDGX 2NKYYX 3DSVKG所示的氨基酸序列、序列如VIWFDGSX 4KYYADSVKG所示的氨基酸序列、序列如SEQ ID NO:7：VIWYDGSNKFYADSVRG所示的氨基酸序列、序列如SEQ ID NO:14：TISGSGAFTYYADAVKG所示的氨基酸序列或序列如SEQ ID NO:15：VISGSGFLTYYADAVKG所示的氨基酸序列的重链互补决定区2HCDR2，其中X 2＝N或S，X 3＝E、A或T，X 4＝N、I、L或V；
和/或(3)包含序列如SEQ ID NO:16：DGESSMVRGLLNWFDP所示的氨基酸序列、序列如SEQ ID NO:17：ELRYFDWLLSPFDY所示的氨基酸序列、序列如SEQ ID NO:21：GERTLDL所示的氨基酸序列、序列如ELWFGELLSPX 5DF所示的氨基酸序列、序列如GQLVX 6DX 7所示的氨基酸序列或序列如GGILTGFDX 8所示的氨基酸序列的重链互补决定区3HCDR3，其中X 5＝L或F，X 6＝G或Q，X 7＝Y、F或L，X 8＝N或Y；
和/或(4)包含序列如RASQSX 9SSSX 10LA所示的氨基酸序列、序列如RASQSISSX 11LX 12所示的氨基酸序列或序列如SEQ ID NO:32：RASQNLNSNLA所示的氨基酸序列的轻链互补决定区1LCDR1，其中X 9＝V或I，X 10＝Y或F，X 11＝Y、W或N，X 12＝N或A；
和/或(5)包含序列如GX 13SSRAT所示的氨基酸序列、序列如KASX 14LEX 15所示的氨基酸序列、序列如SEQ ID NO:35：AASNRAT所示的氨基酸序列、序列如SEQ ID NO:36：AASSLQS所示的氨基酸序列或序列如SEQ ID NO:40：GASTRAP所示的氨基酸序列的轻链互补决定区2LCDR2，其中X 13＝A或T，X 14＝S或N，X 15＝S或N；
和/或(6)包含序列如SEQ ID NO:41：QQYGSSPFT所示的氨基酸序列、序列如SEQ ID NO:42：QQSYSTPLT所示的氨基酸序列、序列如SEQ ID NO:43：QQYKSFSPGGLT所示的氨基酸序列、序列如SEQ ID NO:44：QQYKSNPLT所示的氨 基酸序列或序列如SEQ ID NO:45：QQYNNWPRT所示的氨基酸序列的轻链互补决定区3LCDR3。
在某些实施方式中，本发明提供的抗原结合蛋白包含如下重链可变区VH和轻链可变区VL：
(1)、包含SEQ ID NO:46-SEQ ID NO:59中任一个所示的氨基酸序列的重链可变区VH；和/或包含SEQ ID NO:60-SEQ ID NO:69中任一个所示的氨基酸序列的轻链可变区VL；
或者(2)、与(1)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(1)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；
或者(3)、与(1)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与(1)中的任一VL相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VL；所述的置换是保守置换。
在某些实施方式中，本发明提供的抗原结合蛋白包括如下重链HC和轻链LC：
(1)包含SEQ ID NO:70-SEQ ID NO:83中任一个所示的氨基酸序列的重链HC；和/或包含SEQ ID NO:84-SEQ ID NO:93中任一个所示的氨基酸序列的轻链LC；
或者(2)重链和轻链，其中，与(1)中的重链和轻链相比，所述重链具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，所述轻链具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在某些具体的实施方式中，本发明制备获得的人LIFR抗体包括下表1所述的重链及轻链的组合。
表1 结合LIFR蛋白抗体的序列特征
Figure PCTCN2021142404-appb-000002
Figure PCTCN2021142404-appb-000003
Figure PCTCN2021142404-appb-000004
表1中所示的人LIFR抗体通过Mega软件进行序列相似性分析，可分为以下五组：
组一：PR300516；
组二：PR301164、PR301168、PR301176；
组三：PR301152、PR301160；
组四：PR301153、PR301177、PR301195、PR301196、PR301211、PR301215、PR301218；
组五：PR301127、PR301172。
在某些实施例中，从五组蛋白抗体中分别选取PR300516、PR301127、PR301152、PR301168、PR301211进行实验。
抗体衍生物和多特异性抗体
如上部分所述的本发明提供的抗原结合蛋白包括抗体或抗原结合片段，其可进行衍生化(例如被连接至另一个分子，例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会对其与LIFR(特别是人LIFR)的结合产生不利影响。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段连接于一个或多个其它分子基团，形成双特异性抗体，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
一种类型的衍生化抗体是标记的抗体。例如，可以将本发明的抗体或其抗原结合片段连接至可检测的标记。本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，
Figure PCTCN2021142404-appb-000005
)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。如上所述的可检测的标记可通过本领域已知的方法检测。例 如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
此外，本发明的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清半衰期。
作为另一种抗体的衍生物，本发明提供一种多特异性抗体，所述多特异性抗体包含第一抗体或其片段，和另外的抗体或其片段，或抗体类似物，其中第一抗体或其片段，另外的抗体或其片段，或抗体类似物保留其原始结合特异性。所述第一抗体或其片段是本发明的任一结合TSLP的(单克隆)抗体或其抗原结合片段。如本文中所使用的，“抗体类似物(antibody mimetic)”，指与抗体一样特异性结合抗原，但却没有抗体结构。它们通常是人工肽或蛋白质，摩尔质量约为3至20kDa，例如锚蛋白重复蛋白(DARPin)和fynomer。经设计的锚蛋白重复蛋白(DARPin)可以与IgG抗体、scFv-Fc抗体片段或其组合相连，如CN104341529A所述。抗IL-17a的fynomer与抗IL-6R抗体融合产生双特异性融合多肽，如WO2015141862A1所述。
在某些实施方案中，所述多特异性抗体由第一抗体或其抗原结合片段与其他抗体或其抗原结合片段或抗体类似物通过偶联形成，并且其中各抗体或其抗原结合片段或抗体类似物保持其原结合特异性，所述第一抗体或其抗原结合片段为本发明所述的抗体或其抗原结合片段。某些实施方案中，所述多特异性抗体为双特异性抗体或三特异性抗体或四特异性抗体。
抗体药物偶联物
抗体药物偶联物包括本发明提供的抗原结合蛋白部分以及偶联部分。抗原结合蛋白部分包括如上所述的“抗原结合蛋白”，以及在“抗原结合蛋白”基础上 获得的抗原结合片段。抗原结合片段的实例包括但不限于Fab，Fab’，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb等。偶联部分可以包含至少一个载荷药物。所述载荷药物可以包含药物活性物成分和/或前述的标记分子。载荷药物是一类具有药学活性的小分子化合物或者毒素或其他药物分子形式，可以是但不局限于小分子化合物、毒素分子、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、荧光基团、核素基团等。本领域已知多种载荷药物。例如，小分子化合物通常是指具有较强细胞毒性的一类物质。示例性的小分子化合物可通过包括但不限于微管蛋白结合、DNA结合、抑制RNA聚合酶、蛋白质合成或抑制拓扑异构酶等机制来发挥此类细胞毒性和细胞抑制性效应。例如，小分子化合物可以是微管蛋白抑制剂；例如，所述微管蛋白抑制剂可以是美登素(例如，DM1或DM4)和奥瑞司他汀(例如，MMAE或MMAF)等等。例如，所述小分子化合物可以是DNA损伤剂；例如，所述DNA损伤剂可以是卡奇霉素(Calicheamicins)、吡咯并苯并二氮杂卓(PBD，pyrrolobenzodiazepines)等等。例如，蛋白降解靶向嵌合体(PROTAC)是一类能够通过诱导靶蛋白的多聚泛素化而导致靶蛋白降解的化合物；例如，所述PROTAC可以是BET蛋白降解剂。所述药物偶联物还可以包含至少一个连接子。例如，所述连接子可包含可断裂连接子或非可断裂连接子。所述连接子用于将一个或多个载荷药物连接到抗原结合蛋白。在本申请中，可断链的连接子可以是便于释放药物的“可切割”连接子。例如，可切割连接子可以包括但不局限于酸敏感连接子，蛋白酶敏感连接子，光敏感连接子，或含二硫化物连接子。连接子可包含一种或多种连接子构件，本领域已知多种连接子构件，例如，马来酰亚胺基己酰基(MC，maleimidocaproyl)，马来酰亚胺基丙酰基(MP)，缬氨酸-瓜氨酸(val-cit或vc，valine-citrulline)，对氨基苄氧羰基(PAB，paminobenzyloxycarbonyl)。
核酸、载体、宿主细胞及抗体的制备
本发明的抗原结合蛋白可通过本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
在某些具体的实施方式中，本发明提供了分离的核酸分子，包含编码本发明的抗体或其抗原结合片段、或其重链可变区和/或轻链可变区、或其一个或多个CDR的核苷酸序列。根据本领域已知的密码子简并性，在某些实施方案中，所述核苷酸序列是可以根据密码子简并性进行替换的。在某些实施方案中，所述核苷酸序列是密码子最优化的。
在某些实施方式中，本发明提供了包含本发明的分离的核酸分子的克隆载体或表达载体。在某些实施方案中，所述的载体是例如质粒，粘粒，噬菌体，慢病毒等。在某些实施方案中，所述的载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。在某些实施方式中，本发明提供了包含本发明的分离的核酸分子或本发明的载体的宿主细胞。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本发明的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、CHO DG44)。
在某些实施方式中，本发明提供了制备本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
制药用途、治疗方法、药物组合物和给药装置
本发明提供了所述的人LIFR抗原结合蛋白、抗原结合蛋白衍生物、多特异性抗体、免疫细胞、抗体药物偶联物在制备用于LIF和/或LIFR阻断药物、制备用于预防和/或治疗LIF和/或LIFR阳性疾病的药物中的用途。
相应地，本发明提供了所述的人LIFR抗原结合蛋白、抗原结合蛋白衍生物、多特异性抗体、免疫细胞、抗体药物偶联物用于阻断LIF和/或LIFR、用于预防和/或治疗LIF和/或LIFR阳性疾病方法。
在某些实施方式中，本发明提供了用于治疗的药物组合物，所述的药物组合物包含所述的人LIFR抗原结合蛋白、核酸分子、载体、宿主细胞、免疫细胞、抗原结合蛋白衍生物、多特异性抗体和/或抗体药物偶联物及任选地药学上可接 受的载体。“药学上可接受的载体”包括但不限于：稀释剂、赋形剂、填充剂、润湿剂、崩解剂、矫味剂和粘合剂。
在某些实施方式中，所述的药物组合物还包含组合治疗剂，所述的组合治疗剂包括但不限于化学治疗剂、放射治疗剂、免疫抑制剂、细胞毒性药物。
在某些实施方式中，在所述药物组合物中，本发明的抗原结合蛋白与所述组合治疗剂作为分离的组分或作为同一组合物的组分提供。因此，本发明的抗原结合蛋白与所述组合治疗剂可以联合或分开，同时或相继施用。抗LIFR的抗体或其抗原结合片段可单独或与其它治疗剂组合给药。抗LIFR的抗体或其抗原结合片段和一种或多种其它治疗剂可分开、同时或相继给药。
药物组合物可呈任何适合形式(视将其投予患者的所需方法而定)，可通过多种途径(例如经口、经皮、皮下、鼻内、静脉内、肌肉内、眼内、局部、鞘内和脑室内)向患者投予本发明的人LIFR抗体。在任何指定情况下最适于投药的途径将视特定抗体、个体和疾病的性质和严重度以及个体的身体状况而定。
在某些实施方式中，本发明还提供了施用所述抗原结合蛋白、抗原结合蛋白衍生物、多特异性抗体、免疫细胞、抗体药物偶联物及包含上述成分的药物组合物的给药装置，所述装置包括：
(i)输注模块，所述输注模块用于对受试者施用包括具有一活性成分的药物组合物；
(ii)用于输注的药物组合物，所述药物组合物中含有一活性成分，所述活性成分选自下组：抗原结合蛋白、抗原结合蛋白衍生物、多特异性抗体、免疫细胞、抗体药物偶联物或其组合；以及
(iii)任选的药效监控模块。
在另一优选例中，所述的施用包括肠道外施用和非肠道外施用。在另一优选例中，所述肠道外施用包括注射施用，所用的注射的路径包括：静脉内、肌内、动脉内、膜内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬脑膜上和胸骨内注射和推注。在另一优选例中，所述非肠道外施用包括外用、表皮施用或粘膜施用，例如鼻内、经口、阴道、直肠、舌下、或局部外用。在另一优选例中，所述的输注模块为无针皮下注射设备、微量输液泵、经皮给药设备、推注设备，或渗透设备。
检测方法、试剂盒
本发明的抗体或其抗原结合片段能够结合LIFR，从而可用于检测LIFR在样品中的存在或其水平。
在一方面，本发明提供了一种试剂盒，其包括本发明的抗原结合蛋白。在某些实施方案中，本发明的抗原结合蛋白带有可检测的标记。在一个优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。
在本发明中，所述可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。特别优选的是，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。
在本发明中，提供了对LIFR表达的检测，其包含使用一种或一种以上本发明的人LIFR抗体(任选接合于可检测部分)接触生物样品(个体细胞、组织或体液)，和检测样品关于LIFR表达是否呈阳性，或检测样品相较于对照样品是否具有改变(例如减少或增加)的表达。在某些实施例中，组织或体液为周边血液、周边血液白血球、活组织检查组织(例如肺或皮肤活组织检查)以及组织。所述方法可以用于诊断目的，或者非诊断目的(例如，用于LIF/LIFR途径研究、药物筛选、组化分析等)。在某些实施方案中，用于非诊断目的的样品是细胞样品，例如细胞系或离体细胞培养物。
在一个实施方案中，本发明提供了一种检测样品中LIFR的存在或其水平的方法，所述方法包括在允许本发明所述抗体或其抗原结合片段与LIFR之间形成复合物的条件下，使所述样品与所述抗体或其抗原结合片段接触，并且检测所述复合物的形成。
在另一个方面，本发明提供了一种诊断受试者中LIFR阳性疾病的方法，包括将本发明所述的抗体或其抗原结合片段、多特异性抗体、或抗体药物偶联物与来自受试者的样品在允许抗体或其抗原结合片段与LIFR之间形成复合物的条件下接触，检测所述复合物的形成，其中与健康对照相比，LIFR水平升高指示存在癌症。
可选地，所述受试者是哺乳动物，包括非人哺乳动物和人。优选地，所述受试者是人。
优选地，所述癌症为胆管癌、结直肠癌、神经胶质瘤、前列腺癌、卵巢癌、胃癌、鼻咽癌、乳腺癌、膀胱癌、胰腺癌、非小细胞肺癌。
在另一个方面，本发明提供了检测试剂盒，其包含本发明所述的人LIFR抗原结合蛋白、抗原结合蛋白衍生物等，在优选的实施例中，所述试剂盒包含用于描述其使用方法的使用说明书。
实施例1Harbour转基因小鼠免疫
1.免疫原：重组人LIFR蛋白氨基酸序列(GenBank登录号：NP_001121143.1)胞外区1-833位被克隆到含有his标签的pcDNA3.1载体并制备重组表达质粒。对HEK293细胞进行瞬时转染并扩大培养，4天后收集细胞培养液，对培养上清进行纯化，得到高纯度的huLIFR-ECD蛋白(>90％)，具有生物活性，分装后冻存于-80℃。
2.Harbour H2L2小鼠免疫：huLIFR-ECD蛋白用于免疫6-8周Harbour H2L2转基因小鼠。所有的转基因小鼠饲养于SPF级环境中。初次免疫中，50μg huLIFR-ECD蛋白与弗式完全佐剂(Sigma，F5881)乳化后，腹腔注射到小鼠体内。对于后续的加强免疫，25μg huLIFR-ECD蛋白与RIBI佐剂(Sigma，S6322)混匀后，腹腔注射到小鼠体内。每次免疫之间的间隔为2周。在免疫后7天取血用ELISA检测血清抗体滴度，选择血清滴度高的小鼠用于后续单个B细胞筛选实验。
实施例2基于
Figure PCTCN2021142404-appb-000006
Optofluidic system的单个B细胞筛选
Figure PCTCN2021142404-appb-000007
system使用光-电定位(OEP TM)技术来进行单个细胞的移动。Beacon光电系统是一个自动化的生物仪器设备，能够允许在细胞培养条件下同时进行生物功能试验、实验分析、阳性克隆挑选等多种操作。Beacon平台可以在数千个细胞上以大规模并行、自动化的方式执行这些任务。
本申请使用浆细胞(plasma cell)发现工作流程，每次实验中最多可以筛选14k个单个plasma细胞，用于挑选抗体分泌的抗原特异性的浆细胞。然后，将 这些分泌特异性抗体的浆细胞分别导出到含细胞裂解液的96孔板中，用于后续的单个B细胞测序，以鉴定单个B细胞(单克隆)产生的抗体的重链和轻链序列。
实施例3单B细胞测序和全人源抗体的制备
本发明采用单B细胞测序方法从单个浆细胞中获得抗体重链和轻链序列。单B细胞测序已经成为获得抗体序列的强有力工具。一般程序包括从单浆细胞裂解液中纯化RNA，cDNA的逆转录合成，cDNA的扩增纯化，重链和轻链的扩增，克隆和转染，Sanger测序。对得到的序列进行唯一性和聚类分析，然后将配对的重链和轻链DNA序列进行质粒合成。
具体的在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG1抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG1抗体的全长重链。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。在本实施例中，由于从免疫的Harbour H2L2小鼠得到的抗LIFR单克隆抗体分子可变结构域的序列是人源抗体序列，因而本实施例也得到全人源的抗LIFR重组IgG1抗体。其中，本发明获得的所述人LIFR抗体重链、轻链互补区决定区(CDR)序列，重链、轻链可变区(V)，重链、轻链序列如上表1所示。
通过Mega软件对表1中的15个抗体进行序列相似性分析，发现上述抗体可分为5组：(1)组一：PR300516；(2)组二：PR301164、PR301168、PR301176；(3)组三：PR301152、PR301160；(4)组四：PR301153、PR301177、PR301195、PR301196、PR301211、PR301215、PR301218；(5)组五：PR301127、PR301172。所有抗体均进行了实施例4.2和4.4的试验。从5组中分别挑选了代表性抗体PR300516、PR301127、PR301152、PR301211、PR301168进行了实施例4.1，4.3，实施例5的试验。代表性抗体PR300516和PR301168进行了体内PK和药效学试验。
5个代表性抗体中，PR300516重链可变区的DNA核苷酸序列如SEQ ID NO:94所示，PR300516轻链可变区的DNA核苷酸序列如SEQ ID NO:95所示；PR301127重链可变区的DNA核苷酸序列如SEQ ID NO:96所示，PR301127轻链可变区的DNA核苷酸序列如SEQ ID NO:97所示；PR301152重链可变区的DNA核苷酸序列如SEQ ID NO:98所示，PR301152轻链可变区的DNA核苷酸序列如SEQ ID NO:99所示；PR301168重链可变区的DNA核苷酸序列如SEQ ID NO:100所示，PR301168轻链可变区的DNA核苷酸序列如SEQ ID NO:101所示；PR301211重链可变区的DNA核苷酸序列如SEQ ID NO:102所示，PR301211轻链可变区的DNA核苷酸序列如SEQ ID NO:103所示。
实施例4重组抗体的表达和纯化
293F细胞用于抗体的表达。调整细胞密度为1×10e6cells/mL。将重链和轻链抗体的质粒按照1：1.5的比例加入管中，然后加入转染试剂PEI，PEI与DNA的比例为4：1，混匀后室温孵育15min。将上述混合物加入细胞中，转染细胞于37℃，5％CO 2孵箱中培养，125rpm振荡。转染后24h补充OPM-CHO PFF05营养(奥浦迈，货号FB1279-001)至终浓度为3％。细胞培养5天后，待细胞活率降为70％以下，收集上清。对转染上清用protein G柱子进行蛋白纯化。
4.1抗体亲和力检测实验
将按照本发明实施例1-4的方法制备得到的5个抗体PR300516、PR301127、PR301152、PR301168、PR301211通过生物膜干涉(Biolayerinterferometry，BLI)的方法(Fortebio octet RED 96e)，检测其对于人LIFR-ECD蛋白的结合动力学。
将待测抗体稀释至终浓度6μg/mL，直接固化到AHC biosensor上，进行动力学测量，将抗原蛋白(Human LIF)用0.02％PBST20分别稀释至100nM、50nM、25nM 3个浓度，进样70s，结合时间为300s，解离时间为300s，10mM glycine-HCl(pH1.5)再生15s。使用简单一对一Languir结合模型(Octet Red96数据分析软件)，计算结合速率(kon)和解离速率(kdis)，平衡解离常数(kD)以比率kdis/kon计算。抗体亲和力检测结果如下表2所示。
表2 抗体亲和力检测结果
抗体 PR301127 PR301152 PR301168 PR301211 PR300516 MSC-1
KD - 1.57E-08 4.92E-10 4.21E-10 3.04E-10 1.25E-09
由表2可知，人LIFR抗体的亲和力高于LIF抗体MSC-1的亲和力。
4.2抗体在蛋白水平与LIFR-ECD的结合能力检测
基于ELISA测定法检测本发明通过实施例1-4制备得到的15个抗体与LIFR-ECD蛋白的结合能力。通过比较不同抗体与LIFR-ECD蛋白的结合曲线来测定其结合能力。具体实验过程为：先将人LIFR-ECD，猴LIFR-ECD和小鼠LIFR-ECD蛋白依次稀释成1μg/mL浓度，加入96孔板中，每孔100μL，放置于4℃过夜。96孔板用PBST溶液洗三遍后，加入2％BSA的PBS溶液于37℃放置1h。将待测抗体进行浓度梯度稀释后(100nM，10-fold-dilution)，加入96孔板中，37℃孵育1h。PBST溶液洗三遍后，加入anti-human IgG Fc-HRP二抗(5000x稀释使用)于37℃孵育30-60min。PBST溶液洗三遍后，加入TMB显色液5-15min，然后加入终止液终止显色。
表3 抗体在蛋白水平与LIFR-ECD的结合能力检测结果
Figure PCTCN2021142404-appb-000008
Figure PCTCN2021142404-appb-000009
检测结果如表3和图1所示，PR300516、PR301127、PR301172、PR301152、PR301153、PR301160、PR301164、PR301168、PR301176、PR301177、PR301195、PR301196、PR301211、PR301215和PR301218抗体都具有和人LIFR-ECD蛋白和猴LIFR-ECD蛋白结合的能力，但只有PR301127和PR301172抗体可与小鼠LIFR-ECD蛋白交叉结合。
4.3抗体在细胞水平与LIFR和LIFR/gp130结合能力检测
基于流式细胞术测定法检测本发明通过实施例1-4制备得到的5个抗体PR300516、PR301127、PR301152、PR301168、PR301211与表达于293T细胞表面的人LIFR受体或者人LIFR/gp130受体异二聚体的结合能力。通过比较不同抗体与表达于293T细胞表面的人LIFR受体或人LIFR/gp130受体异二聚体的结合曲线来测定其结合能力。具体实验过程为：(1)使用lenti-huLIFR和lenti-LIFR/gp130慢病毒感染HEK293T细胞，用嘌呤霉素筛选感染后的细胞得到稳定细胞池：HEK293T-huLIFR和HEK293T-huLIFR/gp130；(2)对稳定的细胞池进行亚克隆筛选，得到高表达目的基因的单克隆细胞系；(3)使用不同浓度的待测抗体与单克隆细胞系于4℃孵育30min，PBS清洗三次后，使用FITC标记的羊抗人二抗与之4℃孵育30min，PBS再次清洗三次后，使用l00μL FACS缓冲液重悬细胞；(4)使用流式细胞仪测定其一通道中位荧光度值。以抗体浓度以10为底的对数为横坐标，以一通道中位荧光度值为纵坐标，比较其EC50及曲线峰值。
表4 抗体在细胞水平与LIFR和LIFR/gp130结合能力检测结果
Figure PCTCN2021142404-appb-000010
检测结果如表4和图2所示，人LIFR抗体在同等浓度条件下，与HEK293T-huLIFR/gp130细胞的结合能力要强于与HEK293T-huLIFR细胞的结合。
4.4抗体阻断LIF与LIFR/gp130结合的能力检测
基于流式细胞术测定法检测本发明通过实施例1-4制备得到的15个抗体阻断huLIF蛋白与表达于293T细胞表面的人LIFR/gp130受体异二聚体的结合的能力。通过比较不同抗体阻断huLIF蛋白与HEK293T-huLIFR/gp130细胞结合的曲线来测定其阻断能力。具体实验过程为：(1)首先确定huLIF蛋白与HEK293T-huLIFR/gp130细胞结合的EC80浓度。对huLIF蛋白进行生物素标记(biotin labeling)，得到huLIF-biotin蛋白；(2)使用不同浓度的待测抗体和huLIF蛋白(终浓度为EC80的值)与HEK29ET-huLIFR/gp130细胞于4℃孵育1h，PBS清洗三次后，使用APC标记的Strepavidin二抗与之4℃孵育30min，PBS再次清洗三次后，使用l00μL FACS buffer重悬细胞；(3)使用流式细胞仪测定其四通道中位荧光度值。以抗体浓度以10为底的对数为横坐标，以四通道中位荧光度值为纵坐标作图，比较其IC50及曲线峰值。
表5 抗体阻断LIF与LIFR/gp130结合的能力检测结果
抗体 PR300516 PR301127 PR301172 PR301152 PR301153 hIgG1
IC50 0.562 NA NA 2.018 7.382 0.001774
抗体 PR301160 PR301164 PR301168 PR301176 PR301177 -
IC50 2.404 1.129 0.991 1.436 0.496 -
抗体 PR301195 PR301196 PR301211 PR301215 PR301218 -
IC50 0.753 0.799 0.572 0.533 0.628 -
检测结果如表5和图3所示，与阴性对照hIgG1相比，PR300516、PR301152、PR301153、PR301160、PR301164、PR301168、PR301176、PR301177、PR301195、PR301196、PR301211、PR301215和PR301218都有明显的阻断huLIF与HEK293T-huLIFR/gp130结合的功能。
实施例5抗体对STAT3信号通路的抑制作用检测
基于Western blot检测LIF诱导的STAT3酪氨酸705位点磷酸化的抑制作用，包括Capan-2胰腺癌细胞，MDA-MB-231乳腺癌细胞，A549和NCI-H292非小细胞肺癌细胞。具体实验过程为：先将肿瘤细胞铺于6孔板，每孔1x10 6个细胞，饥饿处理24小时(培养基不加血清)。再将一定浓度的huLIF蛋白与对应的抗体于37℃培养箱中共孵育1h，将huLIF蛋白和抗体混合物加入到肿瘤细胞中，于 37℃培养箱中处理30min。弃掉培养基，用预冷的PBS洗两次。每孔加入200μL预冷的裂解buffer，加入一定量的蛋白酶抑制剂(100×稀释)和磷酸酶抑制剂(50×稀释)。冰上孵育30min后，12000转离心10min。将上清转移到新的EP管中，取25μL上清蛋白进行BCA定量，余下的加入5×SDS buffer，95℃金属浴处理5min。对蛋白样品跑SDS-PAGE胶，转膜后，加block buffer室温孵育1h。一抗Stat3(124H6)Mouse mAb(Cell Signaling Technology，#9139)、Phospho-Stat3(Tyr705)Antibody(Cell Signaling Technology，#9131)4℃过夜。PBST溶液洗三次，每次10min。加二抗Anti-Mouse IgG(Fc Specific)-Peroxidase(Sigma-Aldrich，A0168)、Peroxidase AffiniPure Goat Anti-Rabbit IgG(H+L)(Jackson immune research，111-035-045)室温孵育1h。PBST洗三次后，加入A+B显色液，ChemiDoc Imaging System曝光拍照。
检测结果如图4所示，人LIFR抗体在Capan-2胰腺癌细胞，MDA-MB-231乳腺癌细胞，A549和NCI-H292非小细胞肺癌细胞四种肿瘤细胞中均可以抑制由LIF诱导的STAT3信号通路的激活。
实施例6抗体在体内的PK检测
6.1.Balb/c小鼠体内PK
本研究的目的是在雌性Balb/c小鼠静脉注射抗体后确定其药代动力学参数。具体实验过程为：Balb/c小鼠尾静脉注射抗体前，先取血留样。然后按照5mg/kg抗体的剂量尾静脉注射后，在15min，5h，24h，day2，day4，day7，day14和day21时间点取血，检测血清中的抗体浓度。PK实验设计如下表6。
表6 PK实验设计
Figure PCTCN2021142404-appb-000011
表7 PK参数统计
抗体 PR300516 MSC-1
T1/2(hr) 178.25 212.84
Vz(mL/kg) 114.09 81.57
AUCall(hr*μg/mL) 9521.05 16003.13
Cl(mL/hr/kg) 0.45 0.27
C0(μg/mL) 92.90 134.97
由表7和图5可知，人LIFR抗体PR300516在小鼠中的半衰期为178.25小时。
6.2.Sprague Dawley大鼠体内PK
本研究的目的是在雄性Sprague Dawley大鼠静脉注射抗体后确定其药代动力学参数。具体实验过程为：Sprague Dawley大鼠尾静脉注射抗体前，先取血留样。然后按照5mg/kg抗体的剂量尾静脉注射后，在给药前，10min，2h，8h，24h，day4，day7，day10，day14，day21和day28时间点取血，检测血清中的抗体浓度。PR300516抗体和PR301168抗体每组各安排3只大鼠进行实验。PK实验设计如下表8。
表8 PK实验设计
Figure PCTCN2021142404-appb-000012
表9 大鼠PK参数统计
抗体 PR300516 PR301168
T1/2(hr) 219 242
Vz(mL/kg) 122 177
AUCall(hr*μg/mL) 11056.83 8173.60
Cl(mL/hr/kg) 0.40 0.53
C0(μg/mL) 92.78 65.32
由表9和图6可知，人LIFR抗体PR300516和PR301168在大鼠中的半衰期分别为219小时和242小时。
6.3.猴子体内PK实验
本研究的目的是在雄性和雌性猴子静脉注射抗体后确定其药代动力学参数。具体实验过程为：雄性和雌性猴子尾静脉注射抗体前，先取血留样。然后按照低剂量10mg/kg和高剂量100mg/kg抗体的剂量尾静脉注射后，10mg/kg计量组在给药前，10min，2h，8h，24h，day4，day7，day10，day14时间点取血，100mg/kg计量组在计量组在给药前，10min，2h，8h，24h，day4，day7时间点取血，检测血清中的抗体浓度。PK实验设计如下表10。
表10 PK实验设计
Figure PCTCN2021142404-appb-000013
表11 猴PK参数统计
Figure PCTCN2021142404-appb-000014
由表11和图7可知，低剂量人LIFR抗体PR300516在猴子中的半衰期分别为173.7小时和182.9小时，高剂量人LIFR抗体PR300516在猴子中的半衰期分别为239.3小时和128.2小时。
实施例7抗体在体内的药效学实验
7.1.Capan-2模型
本实验利用Capan-2小鼠肿瘤模型研究抗人LIFR抗体的抗肿瘤活性。研究了PR300516抗体单独给药(5mg/kg和15mg/kg)在Capan-2小鼠模型中的药效学。人胰腺癌Capan-2细胞(ATCC细胞库)5x10e6细胞数皮下接种到雌性Balb/c nude小鼠(维通利华)。抗体组处理方法为：肿瘤细胞接种后的第5天，第9天，第12天，第16天，第19天和第23天给各组小鼠腹腔注射相应的抗体。小分子抑制剂组处理方法为：肿瘤细胞接种后的第5天，第7天，第9天，第12天，第14天，第16天，第19天，第21天和第23天对小鼠口服EC359小分子抑制剂。肿瘤细胞接种后的第5天，第9天，第12天，第16天，第19天和第23天测量肿瘤的体积，之后对小鼠实施安乐死。结果如表15和图8所示，本申请的抗人LIFR抗体PR300516与阴性对照相比，在单独使用时对肿瘤生长有一定的抑制作用。与EC359小分子抑制剂和MSC-1阳性对照抗体相比，本申请所述的抗人LIFR抗体对肿瘤生长的抑制作用更强。本研究中所有组小鼠在接种后37天，小鼠体重均无明显变化。
7.2.Capan-1模型
本实验利用Capan-1小鼠肿瘤模型研究抗人LIFR抗体的抗肿瘤活性。研究了PR300516和PR301168抗体单独给药(15mg/kg)在Capan-1小鼠模型中的药效学。人胰腺癌Capan-1细胞(ATCC细胞库)5x10e6细胞数皮下接种到雌性Balb/c nude小鼠(维通利华)。抗体组处理方法为：肿瘤细胞接种后的第5天，第8天，第12天，第15天，第18天和第21天给各组小鼠腹腔注射相应的抗体。肿瘤细胞接种后的第5天，第8天，第12天，第15天，第18天,第21天和测量肿瘤的体积，之后对小鼠实施安乐死。结果如表16和图9所示，本申请的抗人LIFR抗体PR300516和PR301168与阴性对照相比，在单独使用时对肿瘤生长有一定的抑制作用。和MSC-1阳性对照抗体相比，本申请所述的抗人LIFR抗体对肿瘤生长的抑制作用更强。本研究中所有组小鼠在接种后25天，小鼠体重均无明显变化。
7.3.NCI-H292模型
本实验利用NCI-H292小鼠肿瘤模型研究抗人LIFR抗体的抗肿瘤活性。研究了PR300516和PR301168抗体单独给药(15mg/kg)在NCI-H292小鼠模型中的药效学。人胰腺癌NCI-H292细胞(ATCC细胞库)5x10e6细胞数皮下接种到雌性 Balb/c nude小鼠(维通利华)。抗体组处理方法为：肿瘤细胞接种后的第5天，第8天，第12天，第15天，第18天和第21天给各组小鼠腹腔注射相应的抗体。肿瘤细胞接种后的第5天，第8天，第12天，第15天，第18天,第21天和测量肿瘤的体积，之后对小鼠实施安乐死。结果如表17和图10所示，本申请的抗人LIFR抗体PR300516和PR301168与阴性对照相比，在单独使用时对肿瘤生长有一定的抑制作用。和MSC-1阳性对照抗体相比，本申请所述的抗人LIFR抗体对肿瘤生长的抑制作用弱一些。本研究中所有组小鼠在接种后25天，小鼠体重均有所下降。
表12 抗体体内药效学实验设计(Capan-2)
Figure PCTCN2021142404-appb-000015
表13 抗体体内药效学实验设计(Capan-1)
Figure PCTCN2021142404-appb-000016
表14 抗体体内药效学实验设计(NCI-H292)
Figure PCTCN2021142404-appb-000017
Figure PCTCN2021142404-appb-000018
表15 Capan-2模型中肿瘤生长抑制率统计
组别 给药 TGI(％)
1 Isotype，5mg/kg 0
2 EC359，5mg/kg 40.99
3 EC359，15mg/kg 47.85
4 PR300516，5mg/kg 54.92
5 PR300516，15mg/kg 55.32
6 MSC-1，5mg/kg 48.93
7 MSC-1，15mg/kg 49.34
表16 Capan-1模型中肿瘤生长抑制率统计
组别 给药 TGI(％)
1 Isotype，15mg/kg 0
2 MSC-1，15mg/kg 53.96
3 PR300516，15mg/kg 59.32
4 PR301168，15mg/kg 59.89
表17 NCI-H292模型中肿瘤生长抑制率统计
组别 给药 TGI(％)
1 Isotype，15mg/kg 0
2 MSC-1，15mg/kg 39.99
3 PR300516，15mg/kg 36.35
4 PR301168，15mg/kg 15.58
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为 准。

Claims (31)

  1. 一种人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含以下互补决定区：
    (1)包含SEQ ID NO:1-SEQ ID NO:4中任一个所示的氨基酸序列或其变体序列的重链互补决定区1HCDR1；
    (2)包含SEQ ID NO:5-SEQ ID NO:15中任一个所示的氨基酸序列或其变体序列的重链互补决定区2HCDR2；
    (3)包含SEQ ID NO:16-SEQ ID NO:25中任一个所示的氨基酸序列或其变体序列的重链互补决定区3HCDR3；
    (4)包含SEQ ID NO:26-SEQ ID NO:32中任一个所示的氨基酸序列或其变体序列的轻链互补决定区1LCDR1；
    (5)包含SEQ ID NO:33-SEQ ID NO:40中任一个所示的氨基酸序列或其变体序列的轻链互补决定区2LCDR2；
    (6)包含SEQ ID NO:41-SEQ ID NO:45中任一个所示的氨基酸序列或其变体序列的轻链互补决定区3LCDR3；
    所述变体序列为与其来源的CDR相比具有一个或几个氨基酸的置换、缺失或添加的CDR序列。
  2. 根据权利要求1所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含如下互补决定区：
    (1)包含序列如GFTFSSYGMX 1所示的氨基酸序列或序列如SEQ ID NO:4：GFTFSNYAMT所示的氨基酸序列的重链互补决定区1 HCDR1，其中X 1＝H、D或N；
    和/或(2)包含序列如VIWYDGX 2NKYYX 3DSVKG所示的氨基酸序列、序列如VIWFDGSX 4KYYADSVKG所示的氨基酸序列、序列如SEQ ID NO:7：VIWYDGSNKFYADSVRG所示的氨基酸序列、序列如SEQ ID NO:14：TISGSGAFTYYADAVKG所示的氨基酸序列或序列如SEQ ID NO:15：VISGSGFLTYYADAVKG所示的氨基酸序列的重链互补决定区2 HCDR2，其中X 2＝N或S，X 3＝E、A或T，X 4＝N、I、L或V；
    和/或(3)包含序列如SEQ ID NO:16：DGESSMVRGLLNWFDP所示的氨基酸序列、序列如SEQ ID NO:17：ELRYFDWLLSPFDY所示的氨基酸序列、序列如SEQ IDNO:21：GERTLDL所示的氨基酸序列、序列如ELWFGELLSPX 5DF所示的氨基酸序列、序列如GQLVX 6DX 7所示的氨基酸序列或序列如GGILTGFDX 8所示的氨基酸序列的重链互补决定区3HCDR3，其中X 5＝L或F，X 6＝G或Q，X 7＝Y、F或L，X 8＝N或Y；
    和/或(4)包含序列如RASQSX 9SSSX 10LA所示的氨基酸序列、序列如RASQSISSX 11LX 12所示的氨基酸序列或序列如SEQ ID NO:32：RASQNLNSNLA所示的氨基酸序列的轻链互补决定区1LCDR1，其中X 9＝V或I，X 10＝Y或F，X 11＝Y、W或N，X 12＝N或A；
    和/或(5)包含序列如GX 13SSRAT所示的氨基酸序列、序列如KASX 14LEX 15所示的氨基酸序列、序列如SEQ ID NO:35：AASNRAT所示的氨基酸序列、序列如SEQ ID NO:36：AASSLQS所示的氨基酸序列或序列如SEQ ID NO:40：GASTRAP所示的氨基酸序列的轻链互补决定区2LCDR2，其中X 13＝A或T，X 14＝S或N，X 15＝S或N；
    和/或(6)包含序列如SEQ ID NO:41：QQYGSSPFT所示的氨基酸序列、序列如SEQ ID NO:42：QQSYSTPLT所示的氨基酸序列、序列如SEQ ID NO:43：QQYKSFSPGGLT所示的氨基酸序列、序列如SEQ ID NO:44：QQYKSNPLT所示的氨基酸序列或序列如SEQ ID NO:45：QQYNNWPRT所示的氨基酸序列的轻链互补决定区3 LCDR3。
  3. 根据权利要求2所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含：
    (1)、包含SEQ ID NO:46-SEQ ID NO:59中任一个所示的氨基酸序列的重链可变区VH；和/或包含SEQ ID NO:60-SEQ ID NO:69中任一个所示的氨基酸序列的轻链可变区VL；
    或者(2)、与(1)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(1)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；
    或者(3)、与(1)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添 加或其任意组合的VH；和/或，与(1)中的任一VL相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VL；所述的置换是保守置换。
  4. 根据权利要求3所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含：
    (1)包含SEQ ID NO:70-SEQ ID NO:83中任一个所示的氨基酸序列的重链HC；和/或包含SEQ ID NO:84-SEQ ID NO:93中任一个所示的氨基酸序列的轻链LC；
    或者(2)重链和轻链，其中，与(1)中的重链和轻链相比，所述重链具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，所述轻链具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
  5. 根据权利要求1-4任意一项所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含：
    (1)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:5所示的HCDR2，如SEQ ID NO:16所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:33所示的LCDR2，如SEQ ID NO:41所示的LCDR3；和/或
    (2)如SEQ ID NO:2所示的HCDR1，如SEQ ID NO:7所示的HCDR2，如SEQ ID NO:18所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:33所示的LCDR2，如SEQ ID NO:41所示的LCDR3；和/或
    (3)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:6所示的HCDR2，如SEQ ID NO:17所示的HCDR3，和/或如SEQ ID NO:27所示的LCDR1，如SEQ ID NO:34所示的LCDR2，如SEQ ID NO:41所示的LCDR3；和/或
    (4)如SEQ ID NO:2所示的HCDR1，如SEQ ID NO:7所示的HCDR2，如SEQ ID NO:19所示的HCDR3，和/或如SEQ ID NO:26所示的LCDR1，如SEQ ID NO:33所示的LCDR2，如SEQ ID NO:41所示的LCDR3；和/或
    (5)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:12所示的HCDR2，如SEQ ID NO:23所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；和/或
    (6)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:10所示的HCDR2，如SEQ ID NO:20所示的HCDR3，和/或如SEQ ID NO:29所示的LCDR1，如SEQ ID NO:36所示的LCDR2，如SEQ ID NO:42所示的LCDR3；和/或
    (7)如SEQ ID NO:3所示的HCDR1，如SEQ ID NO:10所示的HCDR2，如SEQ ID NO:21所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:37所示的LCDR2，如SEQ ID NO:43所示的LCDR3；和/或
    (8)如SEQ ID NO:3所示的HCDR1，如SEQ ID NO:11所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；和/或
    (9)如SEQ ID NO:3所示的HCDR1，如SEQ ID NO:11所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:39所示的LCDR2，如SEQ ID NO:44所示的LCDR3；和/或
    (10)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:11所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3；和/或
    (11)如SEQ ID NO:1所示的HCDR1，如SEQ ID NO:13所示的HCDR2，如SEQ ID NO:22所示的HCDR3，和/或如SEQ ID NO:30所示的LCDR1，如SEQ ID NO:38所示的LCDR2，如SEQ ID NO:44所示的LCDR3。
  6. 根据权利要求5所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含：
    (1)如SEQ ID NO:46所示的VH，和/或如SEQ ID NO:60所示的VL；
    (2)如SEQ ID NO:48所示的VH，和/或如SEQ ID NO:62所示的VL；
    (3)如SEQ ID NO:47所示的VH，和/或如SEQ ID NO:61所示的VL；
    (4)如SEQ ID NO:49所示的VH，和/或如SEQ ID NO:62所示的VL；
    (5)如SEQ ID NO:55所示的VH，和/或如SEQ ID NO:67所示的VL；
    (6)如SEQ ID NO:52所示的VH，和/或如SEQ ID NO:65所示的VL；
    (7)如SEQ ID NO:53所示的VH，和/或如SEQ ID NO:66所示的VL；
    (8)如SEQ ID NO:54所示的VH，和/或如SEQ ID NO:67所示的VL；
    (9)如SEQ ID NO:54所示的VH，和/或如SEQ ID NO:68所示的VL；
    (10)如SEQ ID NO:56所示的VH，和/或如SEQ ID NO:67所示的VL；
    (11)如SEQ ID NO:57所示的VH，和/或如SEQ ID NO:67所示的VL；
    或者，与(1)-(11)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(1)-(11)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；
    或者，与(1)-(11)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与(1)-(11)中的任一VL相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合的VL；所述的置换是保守置换。
  7. 根据权利要求6所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包含：
    (1)如SEQ ID NO:70所示的HC，和/或如SEQ ID NO:84所示的LC；
    (2)如SEQ ID NO:72所示的HC，和/或如SEQ ID NO:86所示的LC；
    (3)如SEQ ID NO:71所示的HC，和/或如SEQ ID NO:85所示的LC；
    (4)如SEQ ID NO:73所示的HC，和/或如SEQ ID NO:86所示的LC；
    (5)如SEQ ID NO:79所示的HC，和/或如SEQ ID NO:91所示的LC；
    (6)如SEQ ID NO:76所示的HC，和/或如SEQ ID NO:89所示的LC；
    (7)如SEQ ID NO:77所示的HC，和/或如SEQ ID NO:90所示的LC；
    (8)如SEQ ID NO:78所示的HC，和/或如SEQ ID NO:91所示的LC；
    (9)如SEQ ID NO:78所示的HC，和/或如SEQ ID NO:92所示的LC；
    (10)如SEQ ID NO:80所示的HC，和/或如SEQ ID NO:91所示的LC；
    (11)如SEQ ID NO:81所示的HC，和/或如SEQ ID NO:91所示的LC；
    或者，与(1)-(11)中的重链和轻链相比，所述重链具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，所述轻链具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
  8. 根据权利要求1-7任意一项所述的人LIFR抗原结合蛋白，其特征在于，所述 的人LIFR抗原结合蛋白包括嵌合抗体、人源化抗体或全人源抗体。
  9. 根据权利要求1-8任意一项所述的人LIFR抗原结合蛋白，其特征在于，所述的人LIFR抗原结合蛋白包括全长抗体、Fab、Fab’、F(ab’)2、Fv、scFv、di-scFv、双特异性抗体、多特异性抗体、重链抗体和/或单域抗体，或由上述抗体制得的单克隆抗体和/或多克隆抗体。
  10. 编码权利要求1-9任意一项所述的人LIFR抗原结合蛋白的核酸分子。
  11. 包含权利要求10所述的核酸分子的载体。
  12. 包含权利要求10所述的核酸分子或权利要求11所述的载体的宿主细胞。
  13. 一种嵌合抗原受体，其特征在于，所述的嵌合抗原受体包含权利要求1-9中任意一项所述的人LIFR抗原结合蛋白。
  14. 一种免疫细胞，其特征在于，所述的免疫细胞包含权利要求13所述的嵌合抗原受体。
  15. 一种抗原结合蛋白衍生物，其特征在于，所述的抗原结合蛋白衍生物包含权利要求1-9任意一项所述的人LIFR抗原结合蛋白和可检测的标记分子，所述的可检测的标记分子为酶、放射性核素、荧光染料、发光物质或生物素。
  16. 一种多特异性抗体，其特征在于，所述的多特异性抗体包含权利要求1-9任一项所述的人LIFR抗原结合蛋白，和另外的抗体或其片段或抗体类似物；所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。
  17. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物包括抗体部分和偶联部分，所述抗体部分包含权利要求1-9任意一项所述的人LIFR抗原结合蛋白，所述偶联部分包括但不限于可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合，所述抗体部分和偶联部分通过化学键或接头进行偶联。
  18. 一种药物组合物，其特征在于，所述的药物组合物包含权利要求1-9任意一项所述的人LIFR抗原结合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞、权利要求14所述的免疫细胞、权利要求15所述的抗原结合蛋白衍生物、权利要求16所述的多特异性抗体和/或权利要求17所述的抗体药物偶联物及任选地药学上可接受的载体。
  19. 根据权利要求18所述的药物组合物，其特征在于，所述的药物组合物还包含组合治疗剂，所述的组合治疗剂包括但不限于化学治疗剂、放射治疗剂、免疫抑制剂、细胞毒性药物。
  20. 权利要求1-9任意一项所述的人LIFR抗原结合蛋白的生产方法，其特征在于，所述的方法包括在使得权利要求1-9任一项所述的抗原结合蛋白表达的情况下，培养权利要求12所述的宿主细胞。
  21. 根据权利要求20所述的人LIFR抗原结合蛋白的方法，其特征在于，所述的方法包括以下步骤：
    (1)采用LIFR-ECD蛋白作为抗原免疫小鼠；
    (2)筛选分泌抗体的抗原特异性B细胞；
    (3)从步骤(2)筛选出的单个B细胞中恢复抗体的重链和轻链序列；
    (4)将步骤(3)恢复的抗体重链和轻链引入宿主细胞，经细胞培养、纯化制备得到人LIFR抗原结合蛋白。
  22. 权利要求1-9任意一项所述的人LIFR抗原结合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞、权利要求13所述的嵌合抗原受体、权利要求14所述的免疫细胞、权利要求15所述的抗原结合蛋白衍生物、权利要求16所述的多特异性抗体、权利要求17所述的抗体药物偶联物和/或权利要求18-19所述的药物组合物在制备用于LIF和/或LIFR阻断药物、试剂盒和/或医疗装置中的应用。
  23. 权利要求1-9任意一项所述的人LIFR抗原结合蛋白、权利要求11所述的核酸分子、权利要求12所述的载体、权利要求13所述的宿主细胞、权利要求14所述的嵌合抗原受体、权利要求15所述的免疫细胞、权利要求16所述的抗原结合蛋白衍生物、权利要求17所述的多特异性抗体、权利要求18所述的抗体药物偶联物和/或权利要求19-20所述的药物组合物在制备用于预防和/或治疗LIFR阳性疾病的药物、试剂盒和/或给药装置中的应用。
  24. 权利要求1-9任意一项所述的人LIFR抗原结合蛋白、权利要求15所述的抗原结合蛋白衍生物在制备LIFR检测试剂或试剂盒中的用途。
  25. LIFR检测方法，其特征在于，利用权利要求1-9任意一项所述的人LIFR抗原结合蛋白定性或定量分析检测LIFR，所述的检测方法用于非疾病诊断或治疗目的。
  26. LIFR阳性相关疾病的治疗方法，其特征在于，所述的方法为向有需要的受试者施用有效量的权利要求1-9任意一项所述的人LIFR抗原结合蛋白、权利要求14所述的免疫细胞、权利要求15所述的抗原结合蛋白衍生物、权利要求16所述的多特异性抗体，权利要求17所述的抗体药物偶联物和/或权利要求18-19所述的药物组合物。
  27. 根据权利要求23所述的应用或权利要求26所述的方法，其特征在于，所述LIFR阳性疾病为肿瘤，所述肿瘤包括但不限于胆管癌、结直肠癌、神经胶质瘤、前列腺癌、卵巢癌、胃癌、鼻咽癌、乳腺癌、膀胱癌、胰腺癌和非小细胞肺癌。
  28. 试剂盒，其特征在于，所述的试剂盒包括权利要求1-9任意一项所述的人LIFR抗原结合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞、权利要求13所述的嵌合抗原受体、权利要求14所述的免疫细胞、权利要求15所述的抗原结合蛋白衍生物、权利要求16所述的多特异性抗体、权利要求17所述的抗体药物偶联物和/或权利要求18-19所述的药物组合物，及任选地，说明书。
  29. 给药装置，其特征在于，所述的给药装置包含：(1)用于对有需要的受试者施用权利要求18或19所述的药物组合物的输注模块，以及(2)任选的药效监控模块。
  30. LIFR抗原结合蛋白在制备癌症治疗药物中的用途，其特征在于，所述癌症选自胆管癌、结直肠癌、神经胶质瘤、前列腺癌、卵巢癌、胃癌、鼻咽癌、乳腺癌、膀胱癌、胰腺癌和非小细胞肺癌。
  31. 根据权利要求30所述的用途，其特征在于，所述LIFR抗原结合蛋白如权利要求1-9任一项所述。
PCT/CN2021/142404 2020-12-31 2021-12-29 人lifr抗原结合蛋白及其制备方法和应用 Ceased WO2022143743A1 (zh)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CA3203886A CA3203886A1 (en) 2020-12-31 2021-12-29 Human lifr antigen binding protein, preparation method therefor, and application thereof
KR1020237025486A KR20230125037A (ko) 2020-12-31 2021-12-29 인간 lifr 항원 결합 단백질, 이의 제조 방법, 및 이의응용
EP21914475.5A EP4253413A4 (en) 2020-12-31 2021-12-29 HUMAN LIFR ANTIGEN-BINDING PROTEIN, PRODUCTION PROCESS AND USE THEREOF
AU2021414160A AU2021414160B2 (en) 2020-12-31 2021-12-29 Human lifr antigen binding protein, preparation method therefor, and application thereof
US18/259,986 US20250136702A1 (en) 2020-12-31 2021-12-29 Human lifr antigen binding protein, preparation method therefor, and application thereof
CN202180088318.8A CN117043189A (zh) 2020-12-31 2021-12-29 人lifr抗原结合蛋白及其制备方法和应用
JP2023540785A JP7691668B2 (ja) 2020-12-31 2021-12-29 ヒトｌｉｆｒ抗原結合タンパク質、その製造方法及び応用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011619032.1 2020-12-31
CN202011619032 2020-12-31

Publications (1)

Publication Number Publication Date
WO2022143743A1 true WO2022143743A1 (zh) 2022-07-07

Family

ID=82260235

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/142404 Ceased WO2022143743A1 (zh) 2020-12-31 2021-12-29 人lifr抗原结合蛋白及其制备方法和应用

Country Status (9)

Country Link
US (1) US20250136702A1 (zh)
EP (1) EP4253413A4 (zh)
JP (1) JP7691668B2 (zh)
KR (1) KR20230125037A (zh)
CN (1) CN117043189A (zh)
AU (1) AU2021414160B2 (zh)
CA (1) CA3203886A1 (zh)
TW (1) TWI904315B (zh)
WO (1) WO2022143743A1 (zh)

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3939350A (en) 1974-04-29 1976-02-17 Board Of Trustees Of The Leland Stanford Junior University Fluorescent immunoassay employing total reflection for activation
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4277437A (en) 1978-04-05 1981-07-07 Syva Company Kit for carrying out chemically induced fluorescence immunoassay
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
WO2002043478A2 (en) 2000-11-30 2002-06-06 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
CN104341529A (zh) 2013-07-26 2015-02-11 三星电子株式会社 带有DARPin的双特异性嵌合蛋白
WO2015141862A1 (en) 2014-03-17 2015-09-24 Mitsubishi Tanabe Pharma Corporation Antibody-fynomer conjugates
CN111378036A (zh) 2020-01-09 2020-07-07 湖北医药学院 一种抗人白血病抑制因子单克隆抗体的制备方法及其应用
CN111868086A (zh) 2016-12-19 2020-10-30 马赛克生物医学一人有限责任公司 抗lif抗体及其用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2097291A1 (en) * 1990-12-13 1992-06-14 David P. Gearing Leukemia inhibitory factor receptors
GB2556632A (en) * 2016-11-18 2018-06-06 Owen Mumford Ltd Medicament delivery device

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3850752A (en) 1970-11-10 1974-11-26 Akzona Inc Process for the demonstration and determination of low molecular compounds and of proteins capable of binding these compounds specifically
US3817837A (en) 1971-05-14 1974-06-18 Syva Corp Enzyme amplification assay
US3939350A (en) 1974-04-29 1976-02-17 Board Of Trustees Of The Leland Stanford Junior University Fluorescent immunoassay employing total reflection for activation
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4277437A (en) 1978-04-05 1981-07-07 Syva Company Kit for carrying out chemically induced fluorescence immunoassay
US4275149A (en) 1978-11-24 1981-06-23 Syva Company Macromolecular environment control in specific receptor assays
US4366241A (en) 1980-08-07 1982-12-28 Syva Company Concentrating zone method in heterogeneous immunoassays
US4366241B1 (zh) 1980-08-07 1988-10-18
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5693762A (en) 1988-12-28 1997-12-02 Protein Design Labs, Inc. Humanized immunoglobulins
US6180370B1 (en) 1988-12-28 2001-01-30 Protein Design Labs, Inc. Humanized immunoglobulins and methods of making the same
WO2002043478A2 (en) 2000-11-30 2002-06-06 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
CN104341529A (zh) 2013-07-26 2015-02-11 三星电子株式会社 带有DARPin的双特异性嵌合蛋白
WO2015141862A1 (en) 2014-03-17 2015-09-24 Mitsubishi Tanabe Pharma Corporation Antibody-fynomer conjugates
CN111868086A (zh) 2016-12-19 2020-10-30 马赛克生物医学一人有限责任公司 抗lif抗体及其用途
CN111378036A (zh) 2020-01-09 2020-07-07 湖北医药学院 一种抗人白血病抑制因子单克隆抗体的制备方法及其应用

Non-Patent Citations (34)

* Cited by examiner, † Cited by third party
Title
"Antibody Engineering: Methods and Protocols", vol. 248, 2004, HUMANA PRESS
"GenBank", Database accession no. NP_001121143.1
BRUGGERMANN ET AL., YEAR IN IMMUNOLOGY, vol. 7, 1993, pages 33
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC. NATL ACAD. SET USA, vol. 94, 1997, pages 412 - 417
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 878 - 883
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
DATABASE Protein 1 December 2020 (2020-12-01), "Chain L, Antibody Fab fragment light chain", XP055950799, retrieved from NCBI Database accession no. 6MVL_L *
DATABASE Protein 1 December 2020 (2020-12-01), "IGH c2032_heavy_IGHV3-33_IGHD3-10_IGHJ4, partial [Homo sapiens] - - ", XP055950909, retrieved from NCBI Database accession no. QEP16307 *
DATABASE Protein 18 February 1997 (1997-02-18), "immunoglobulin heavy chain variable region, partial [Homo sapiens] - - ", XP055950829, retrieved from NCBI Database accession no. AAC51020 *
DATABASE Protein 18 February 1997 (1997-02-18), "immunoglobulin heavy chain variable region, partial [Homo sapiens] ", XP055950844, retrieved from NCBI Database accession no. AAC51023 *
DATABASE Protein 19 October 2015 (2015-10-19), "1-732 antibody light chain variable region, partial [synthetic constru ]", XP055950839, retrieved from NCBI Database accession no. ALJ30113 *
DATABASE Protein 24 July 2016 (2016-07-24), "immunoglobulin kappa light chain, partial [Homo sapiens] ", XP055950798, retrieved from NCBI Database accession no. BAH04687 *
DATABASE Protein 25 July 2016 (2016-07-25), "immunoglobulin heavy chain variable region, partial [Homo sapiens] ", XP055950842, retrieved from NCBI Database accession no. AAC18328 *
DATABASE Protein 26 July 2016 (2016-07-26), "immunoglobulin heavy chain variable region EM2-PPS-4-VH3-4, partial [H ", XP055950816, retrieved from NCBI Database accession no. ABC66902 *
DATABASE Protein 26 July 2016 (2016-07-26), "immunoglobulin heavy chain, partial [Homo sapiens]", XP055950913, retrieved from NCBI Database accession no. AAS68225 *
DATABASE Protein 26 July 2016 (2016-07-26), "immunoglobulin kappa light chain variable region, partial [Homo sapien", XP055950837, retrieved from NCBI Database accession no. AAZ09096 *
DUCHOSAL ET AL., NATURE, vol. 355, 1992, pages 258
HARDING, F.LONBERG, N., ANN. NYACAD. SCI., vol. 764, 1995, pages 536 - 546
HOOGENBOOM ET AL., J. MOL. BIOL., vol. 222, 1991, pages 581 - 597
HUSZAR, D., INTERN. REV. IMMUNOL., vol. 13, 1995, pages 65 - 93
ILJPAC-ILJB COMMITTEE, J. BIOOL. CHEM, vol. 243, 1968, pages 3558
JAKOBOVITS ET AL., NATURE, vol. 362, 1993, pages 255 - 258
JAKOBOVITS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 2551
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
KOBAYASHI ET AL., PROTEIN ENG, vol. 12, no. 10, 1999, pages 879 - 884
LEFRANC ET AL., DEV. COMPARAT. IMMUNOL., vol. 27, 2003, pages 55 - 77
LONBERG ET AL., NATURE, vol. 368, no. 6474, 1994, pages 856 - 859
MARTIN ACRCHEETHAM JCREES AR: "Modelling antibody hypervariable loops: A combined algorithm", PROC NATL ACAD SCI USA, vol. 86, 1989, pages 9268 - 9272, XP000165667, DOI: 10.1073/pnas.86.23.9268
See also references of EP4253413A4
VAUGHAN ET AL., NATURE BIOTECH, vol. 14, 1996, pages 309
VISWANADHAPALLI SURYAVATHI, LUO YILIAO, SAREDDY GANGADHARA R., SANTHAMMA BINDU, ZHOU MEI, LI MENGXING, MA SHIHONG, SONAVANE RAJNI,: "EC359: A First-in-Class Small-Molecule Inhibitor for Targeting Oncogenic LIFR Signaling in Triple-Negative Breast Cancer", MOLECULAR CANCER THERAPEUTICS, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 18, no. 8, 1 August 2019 (2019-08-01), US , pages 1341 - 1354, XP055950580, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-18-1258 *
WU, H. X. ET AL.: "LIFR promotes tumor angiogenesis by up-regulating IL-8 levels in colorectal cancer.", BIOCHIMICA ET BIOPHYSICA ACTA (BBA)-MOLECULAR BASIS OF DISEASE., vol. 1864, no. 9, 9 May 2018 (2018-05-09), XP085428762, DOI: 10.1016/j.bbadis.2018.05.004 *
ZHAO FENGQING, CHEN SHUAI, XING DAN: "Expressions of LIFR and MMP-12 in Gallbladder Carcinoma and Its Clinical Significance", JOURNAL OF CLINICAL AND PATHOLOGICAL RESEARCH, 314000, vol. 38, no. 8, 31 December 2018 (2018-12-31), pages 1601 - 1607, XP055950600, ISSN: 2095-6959, DOI: 10.3978/j.issn.2095-6959.2018.08.002 *

Also Published As

Publication number Publication date
JP2024501739A (ja) 2024-01-15
TWI904315B (zh) 2025-11-11
AU2021414160A9 (en) 2024-05-09
US20250136702A1 (en) 2025-05-01
AU2021414160A1 (en) 2023-07-20
TW202227502A (zh) 2022-07-16
EP4253413A4 (en) 2024-11-06
JP7691668B2 (ja) 2025-06-12
EP4253413A1 (en) 2023-10-04
CN117043189A (zh) 2023-11-10
AU2021414160B2 (en) 2025-08-07
KR20230125037A (ko) 2023-08-28
CA3203886A1 (en) 2022-07-07

Similar Documents

Publication Publication Date Title
CN112912396B (zh) 抗cldn18.2抗体及其用途
US6924360B2 (en) Antibodies against the MUC18 antigen
US7067131B2 (en) Methods for using anti-MUC18 antibodies
ES2639056T3 (es) Anticuerpos anti-EGFR y sus usos
JP2005538682A (ja) カルボキシックアンヒドラーゼｉｘ（ｃａｉｘ）腫瘍抗原に対する抗体
CN112175085B (zh) 抗gpc3抗体及其用途
JP7827890B2 (ja) Ｐｒｌｒ抗原結合タンパク質、その製造方法及び使用
WO2022068809A1 (zh) 抗cd3抗体以及其用途
CN116323676A (zh) 抗Claudin18.2和CD3的双特异性抗体以及其用途
US12534541B2 (en) Anti-PTK7 antibody and uses thereof
KR20250042908A (ko) 항-메소텔린 항체 및 이의 용도
TWI904315B (zh) 人lifr抗原結合蛋白及其製備方法和應用
TW202604963A (zh) 人lifr抗原結合蛋白及其製備方法和應用
WO2024131835A1 (en) Ligand-cytotoxicity drug conjugates and pharmaceutical uses thereof
WO2024131846A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
WO2025201493A1 (zh) Dll3结合分子及其用途
KR20250160321A (ko) 항-메소텔린 항체 및 이의 용도
KR20250126941A (ko) 항-메소텔린 인간화 항체 및 이의 용도
CN118715243A (zh) Cd39抗原结合蛋白及其应用
CN121181702A (zh) 一种抗epha2的抗体及其相关应用
TW202140551A (zh) 抗cldn18.2抗體及其用途
CN113321730A (zh) Cldn18.2抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21914475

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202180088318.8

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 3203886

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2023540785

Country of ref document: JP

ENP Entry into the national phase

Ref document number: 2021914475

Country of ref document: EP

Effective date: 20230630

ENP Entry into the national phase

Ref document number: 2021414160

Country of ref document: AU

Date of ref document: 20211229

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20237025486

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWP Wipo information: published in national office

Ref document number: 18259986

Country of ref document: US