WO2022143743A1 - 人lifr抗原结合蛋白及其制备方法和应用 - Google Patents
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- 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
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Abstract
Description
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 |
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 | - |
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 |
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 |
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 |
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 |
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 |
Claims (31)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 包含权利要求10所述的核酸分子的载体。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
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JP2023540785A JP2024501739A (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 |
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 |
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- 2021-12-29 CN CN202180088318.8A patent/CN117043189A/zh active Pending
- 2021-12-29 KR KR1020237025486A patent/KR20230125037A/ko active Search and Examination
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