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WO2023186111A1 - 一种靶向cd40的抗原结合蛋白及其制备和应用 - Google Patents

一种靶向cd40的抗原结合蛋白及其制备和应用 Download PDF

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WO2023186111A1
WO2023186111A1 PCT/CN2023/085538 CN2023085538W WO2023186111A1 WO 2023186111 A1 WO2023186111 A1 WO 2023186111A1 CN 2023085538 W CN2023085538 W CN 2023085538W WO 2023186111 A1 WO2023186111 A1 WO 2023186111A1
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amino acid
antibody
acid sequence
seq
binding protein
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PCT/CN2023/085538
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English (en)
French (fr)
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罗海山
李鹤
邓刚
王永强
王远东
陈飞
戎一平
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和铂医药(上海)有限责任公司
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Priority to EP23778475.6A priority Critical patent/EP4506364A1/en
Priority to CN202380030974.1A priority patent/CN118922450A/zh
Priority to US18/853,251 priority patent/US20250215100A1/en
Publication of WO2023186111A1 publication Critical patent/WO2023186111A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular to an antigen-binding protein targeting CD40 and its preparation and application.
  • CD40 is a glycosylated type I transmembrane protein, a member of the tumor necrosis factor receptor superfamily (TNFRSF), also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5).
  • TNFRSF tumor necrosis factor receptor superfamily
  • APCs antigen-presenting cells
  • DCs dendritic cells
  • B cells B cells
  • macrophages ligand CD40L
  • NK natural killer cells
  • CD40 downstream signaling pathways requires the formation of cross-linking between CD40 in the form of trimers and multimers of CD40L. They interact on the cell surface, causing CD40 to redistribute to membrane lipid rafts and undergo conformational changes. CD40 recruits TNFR-associated factors (TRAF) in the cytoplasm through the intracellular domain to promote intracellular signaling, thereby activating different signaling pathways.
  • TNF TNFR-associated factors
  • apoptosis apoptosis, cell cycle progression, cytokine production, and expression of cell surface immune regulators. Therefore, activating CD40 can increase antigen presentation, promote cytokine secretion, activate lymphocytes, and at the same time stimulate and activate the human innate immune system and acquired immune system to produce a synergistic effect and resist the occurrence and development of cancer.
  • PI3K phosphatidylinositol-3 kinase
  • phospholipase C ⁇ pathway etc.
  • CD40 is also widely expressed in tumor cells, and is expressed in almost all B-cell malignancies as well as a wide range of solid tumors, including melanoma, lung cancer, breast cancer, colon cancer, prostate cancer, pancreatic cancer, renal cancer, ovarian cancer, and Head and neck cancer, etc.
  • CD40 expressed on the surface of tumor cells can mediate tumor cell death.
  • CD40 expressed on the surface of various tumor cells via CD40L cross-linking will mediate direct cytotoxicity.
  • cross-linking CD40 via CD40L has been shown to induce tumor cell apoptosis and inhibit the growth of solid tumor cells and B malignant tumor cells.
  • activation of CD40 also mediates tumor suppressive effects.
  • Evidence shows that in immunodeficient mice, interfering with CD40 on the surface of tumor cells through CD40L can inhibit the growth of breast cancer cell transplant tumors or B lymphocyte transplant tumors even in the absence of lymphocyte activation.
  • CD40-mediated tumor cell death can be dual, that is, stimulating the immune system to kill tumor cells and direct tumor cytotoxicity, which can synergize against tumors.
  • Agonistic anti-CD40 antibodies similar to CD40L, can cross-link and activate CD40 on the surface of immune cells and tumor cells, exerting significant anti-tumor effects. This anti-tumor effect has been demonstrated in preclinical animal models and clinical trials with tumor patients, and can be combined with chemotherapy drugs, such as gemcitabine and paclitaxel, or with immunomodulatory drugs, such as PD-1 antibodies and CTLA-4 antibodies produce synergistic anti-tumor effects.
  • CD40 antibody drugs there are more than 20 CD40 antibody drugs in clinical trials, but the earliest products are only in the second phase of clinical trials and there are no marketed products yet. Therefore, the development of other CD40 antibodies will provide the possibility for the treatment of various tumors and immune system-related diseases. Very high market value.
  • CD40 antibodies In terms of anti-tumor, the main problems faced by CD40 antibodies in clinical practice include low objective response rate, significant toxic side effects and low tolerated dose. The possible reason for these results is the weak activity of agonists, such as Celldex's product CDX-1140. In its phase I clinical trial, no complete or partial response occurred among 42 patients treated with single drug; in combined treatment with rhFLT3L, only 1 patient out of 20 patients experienced partial response. This product has weak activation of DC cells in vitro, and in the case of cross-linking, the agonist activity cannot be enhanced.
  • the present invention provides an antigen-binding protein targeting CD40 and its preparation and application.
  • the CD40-targeting antigen-binding protein has the characteristics of high affinity to CD40 and strong agonistic activity on the signaling pathway.
  • the agonistic activity is enhanced after cross-linking, making the CD40-targeting antigen-binding protein of the present invention more effective. It has a large therapeutic window and good safety, and is expected to bring new opportunities for the treatment of various tumors.
  • an antigen-binding protein targeting CD40 includes a light chain variable region and a heavy chain variable region.
  • the light chain variable region (VL) includes LCDR1, LCDR2 and LCDR3, and the heavy chain variable region (VH) includes HCDR1, HCDR2 and HCDR3; wherein:
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:33 or variant 1 with 3, 2 or 1 amino acid mutations on SEQ ID NO:33
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:41
  • Amino acid sequence or variant 2 having 3, 2 or 1 amino acid mutations on SEQ ID NO:41
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:49 or having 3, 2 or 1 amino acid mutations on SEQ ID NO:49 Variant 3 with 2 or 1 amino acid mutations
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:7 or variant 4 with 3, 2 or 1 amino acid mutations on SEQ ID NO:7
  • HCDR2 includes the amino acid sequence as shown in SEQ ID NO:15 or variant 5 with 3, 2 or 1 amino acid mutations in SEQ ID NO:15
  • the HCDR3 includes the amino acid sequence as shown in SEQ ID NO:23 Or variant 6 with 3, 2 or 1 amino acid mutations on SEQ ID NO:23.
  • the variant 3 includes an amino acid sequence with a PTM site mutation on the amino acid sequence shown in SEQ ID NO:49, preferably at position 4 and/or at position 4 of the amino acid sequence shown in SEQ ID NO:49.
  • the amino acid mutation is preferably an amino acid substitution, and more preferably is a conservative substitution of an amino acid.
  • the variant 3 is the amino acid sequence shown in any one of SEQ ID NO: 50-73.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO:33
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO:41
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO:49 -Amino group represented by any one of -73 Acid sequence
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO:7
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO:15
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO:23
  • the specific CDR combination is shown in Table 1-1 below.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO:85 or is at least 85%, 90%, 92%, 94%, 95%, 96%, or similar to SEQ ID NO:85.
  • the light chain variable region includes the amino acid sequence shown in any one of SEQ ID NO:85-109
  • the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:81, specifically The combinations of light chain variable regions and heavy chain variable regions are shown in Table 1-2 below.
  • the CD40-targeting antigen-binding protein satisfies at least one of the following three items:
  • the CD40-targeting antigen-binding protein is a full-length antibody, Fab, Fab', F(ab') 2 or Fv, and the Fv is preferably scFv;
  • the antigen-binding protein targeting CD40 is a monospecific antibody, a bispecific antibody or a multispecific antibody;
  • the CD40-targeting antigen-binding protein is a monoclonal antibody or a polyclonal antibody.
  • the CD40-targeting antigen-binding protein is a full-length antibody, and the full-length antibody includes a light chain and a heavy chain;
  • the light chain includes a light chain constant region (CL), and the light chain constant region is preferably It is a human antibody light chain constant region;
  • the heavy chain includes a heavy chain constant region (CH), and the heavy chain constant region is preferably a human antibody heavy chain constant region, more preferably a heavy chain of hIgG1, hIgG2, hIgG3 or hIgG4 subtypes.
  • the chain constant region is further preferably a heavy chain constant region of hIgG1 subtype.
  • the CD40-targeting antigen-binding protein is a full-length antibody
  • the full-length antibody includes a light chain and a heavy chain
  • the heavy chain includes as shown in SEQ ID NO: 114 or 115 Amino acid sequence
  • the light chain includes the amino acid sequence shown in any one of SEQ ID NO: 119-143.
  • the heavy chain includes the amino acid sequence shown in SEQ ID NO: 114, the light chain includes the amino acid sequence shown in SEQ ID NO: 119; or, the heavy chain includes the amino acid sequence shown in SEQ ID NO:
  • variant 1, variant 2, variant 3, variant 4, variant 5 and variant 6 are only used to distinguish different variants and do not represent actual meanings.
  • the PTM site is a site where chemical modification is sometimes introduced after the protein or polypeptide amino acid chain is translated and synthesized in cells, which is called a post-translational modification (PTM) site.
  • PTM post-translational modification
  • amino acid sequence of the CDR in the present invention is determined according to Chothia definition rules.
  • a chimeric antigen receptor which contains the antigen-binding protein targeting CD40 as described in the present invention.
  • another technical solution provided by the present invention is: an isolated nucleic acid encoding the CD40-targeting antigen-binding protein according to the present invention or the chimeric antigen receptor according to the present invention.
  • a recombinant expression vector which contains the The isolated nucleic acid described above.
  • the recombinant expression vector includes a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
  • a transformant which contains the isolated nucleic acid according to the present invention or the recombinant expression vector according to the present invention.
  • the host cell of the transformant is a prokaryotic cell and/or a eukaryotic cell
  • the prokaryotic cell is preferably an E.coli cell such as TG1, BL21
  • the eukaryotic cell is preferably a HEK293 cell or CHO cell.
  • another technical solution provided by the present invention is: a method for preparing a CD40-targeting antigen-binding protein according to the present invention, which includes the following steps: cultivating the transformant according to the present invention, The CD40-targeting antigen-binding protein is obtained from culture.
  • an antibody drug conjugate (Antibody-drug conjugate, ADC), which contains the antigen-binding protein targeting CD40 as described in the present invention and a cytotoxicity agent. agent or label.
  • the cytotoxic agent is MMAF or MMAE
  • the label is a fluorescent agent.
  • a genetically modified cell expressing the chimeric antigen receptor according to the present invention is: a genetically modified cell expressing the chimeric antigen receptor according to the present invention.
  • the genetically modified cells are eukaryotic cells, preferably isolated human cells, more preferably immune cells such as T cells, or NK cells.
  • a pharmaceutical composition comprising the CD40-targeting antigen-binding protein according to the present invention, the isolated nucleic acid according to the present invention, and the The recombinant expression vector of the invention, the genetically modified cells of the invention and/or the antibody drug conjugate of the invention, and pharmaceutically acceptable carriers and/or pharmaceutically acceptable adjuvants .
  • the pharmaceutical composition also includes other anti-tumor antibodies as active ingredients.
  • a detection reagent which contains the antigen-binding protein targeting CD40 according to the present invention and/or the antibody-drug conjugate according to the present invention.
  • the detection reagent is in liquid dosage form, gas dosage form, solid dosage form or semi-solid dosage form.
  • the detection reagent further includes a secondary antibody, CD40 or a derivative thereof, such as an anti-human IgG antibody coupled to horseradish peroxidase and an anti-human IgG antibody coupled to biotin.
  • a pharmaceutical kit which includes a pharmaceutical kit A, and the pharmaceutical kit A contains the antigen-binding protein targeting CD40 as described in the present invention, as described in the present invention.
  • the pharmaceutical composition according to the invention, the detection reagent according to the invention, the genetically modified cells according to the invention and the antibody drug conjugate according to the invention is: a pharmaceutical kit, which includes a pharmaceutical kit A, and the pharmaceutical kit A contains the antigen-binding protein targeting CD40 as described in the present invention, as described in the present invention.
  • the kit also includes kit B, which contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations or Various.
  • kit B which contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations or Various.
  • the antigen-binding protein targeting CD40 according to the present invention is: the pharmaceutical composition according to the present invention, the detection reagent according to the present invention, such as One or more of the kit of the present invention, the genetically modified cells of the present invention and the antibody-drug conjugate of the present invention can be used to diagnose, prevent and/or treat tumors and other diseases. uses in medicines.
  • the tumors include solid tumors and hematological tumors.
  • the tumors include B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, Breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer and colon cancer.
  • NHL chronic lymphocytic leukemia
  • HCL hairy cell leukemia
  • Hodgkin's disease multiple myeloma
  • bladder cancer eloma
  • kidney cancer ovarian cancer
  • cervical cancer ovarian cancer
  • cervical cancer ovarian cancer
  • lung cancer nasopharyngeal cancer
  • malignant melanoma pancreatic cancer and colon cancer.
  • a method for detecting CD40 in a sample which includes using the antigen-binding protein targeting CD40 according to the present invention and the detection reagent according to the present invention. and the step of contacting one or more of the antibody drug conjugates of the present invention with the sample.
  • samples are, for example, blood samples (eg, whole blood samples and serum samples) and CD40-containing reagents.
  • the method is for non-diagnostic and/or therapeutic purposes, such as detecting the concentration of CD40 standards and whether other reagents are contaminated by CD40 in scientific research.
  • another technical solution provided by the present invention is: a method for diagnosing, treating and/or preventing tumors, which method includes administering a therapeutically effective amount of a target as described in the present invention to a patient in need.
  • the tumors include solid tumors and hematological tumors.
  • the tumors include B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, Breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer and colon cancer.
  • NHL chronic lymphocytic leukemia
  • HCL hairy cell leukemia
  • Hodgkin's disease multiple myeloma
  • bladder cancer eloma
  • kidney cancer ovarian cancer
  • cervical cancer ovarian cancer
  • cervical cancer ovarian cancer
  • lung cancer nasopharyngeal cancer
  • malignant melanoma pancreatic cancer and colon cancer.
  • the antigen-binding protein targeting CD40 as described in the present invention is: the antigen-binding protein targeting CD40 as described in the present invention, the isolated nucleic acid as described in the present invention, the recombinant expression vector as described in the present invention, Pharmaceutical composition according to the present invention, detection reagent according to the present invention, pharmaceutical kit according to the present invention, genetically modified cells according to the present invention and/or antibody drug according to the present invention Use of one or more conjugates in the preparation of medicaments for diagnosing, preventing or treating tumors.
  • the tumors include solid tumors and hematological tumors.
  • the tumors include B-lineage NHL, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), Hodgkin's disease, multiple myeloma, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, Breast cancer, lung cancer, nasopharyngeal cancer, malignant melanoma, pancreatic cancer and colon cancer.
  • NHL chronic lymphocytic leukemia
  • HCL hairy cell leukemia
  • Hodgkin's disease multiple myeloma
  • bladder cancer eloma
  • kidney cancer ovarian cancer
  • cervical cancer ovarian cancer
  • cervical cancer ovarian cancer
  • lung cancer nasopharyngeal cancer
  • malignant melanoma pancreatic cancer and colon cancer.
  • “having 3, 2 or 1 amino acid mutations” means including insertion, deletion or substitution of amino acids based on the original amino acid sequence.
  • the mutation of CDR can include the mutation of 3, 2 or 1 amino acid, and the same or different number of amino acid residues can optionally be selected for mutation between these CDRs, for example, it can be 1 mutation of CDR1. Amino acid mutations are carried out, and no amino acid mutations are carried out for CDR2 and CDR3.
  • PTM site mutation means that compared with the original amino acid sequence, the variant sequence has an amino acid mutation at the PTM site.
  • mutation design methods There are different mutation design methods. One method is to replace "hotspot" amino acids (such as N or S in the NS pattern) with amino acids with similar physical and chemical properties (such as mutating N to Q). If the PTM sequence pattern is derived from somatic high-frequency mutations and does not exist in the germline gene sequence, then another approach can be to replace the sequence pattern with the corresponding germline gene sequence.
  • the VH, VL or the full-length antibody may include mutations based on a defined sequence.
  • the mutation is the deletion, substitution or addition of one or more amino acid residues in the defined amino acid sequence, and the mutated amino acid sequence has at least 85% sequence identity with the defined amino acid sequence and maintains or The binding activity of the antigen-binding protein comprising the mutated amino acid sequence is improved; the at least 85% sequence identity is preferably at least 90% sequence identity; more preferably at least 95% sequence identity; most preferably at least 99% sequence identity Identity.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the CD40-targeting antigen-binding protein has the characteristics of high affinity to CD40 and strong agonistic activity on the signaling pathway.
  • the agonistic activity is enhanced after cross-linking, making the CD40-targeting antigen-binding protein of the present invention more effective.
  • the large therapeutic window provides a good basis for significantly improving patient response rates in clinical trials under safe and tolerable dosage conditions.
  • Figure 1A, Figure 1B, Figure 1C and Figure 1D respectively show the binding levels of some CD40 antibodies to CHO-K1 cells that highly express human CD40.
  • Figure 2A, Figure 2B, Figure 2C, Figure 2D and Figure 2E respectively show the binding levels of some CD40 antibodies to Raji cells that highly express human CD40.
  • Figure 3A, Figure 3B, Figure 3C and Figure 3D respectively show the binding levels of some CD40 antibodies to CHO-K1 cells that highly express cynomolgus monkey CD40.
  • Figure 4A, Figure 4B, Figure 4C and Figure 4D respectively show the effects of the CD40 antibody PR003379 to be tested and three control antibodies on HEK293-hCD40-NFkB fluorescent reporter cells with or without CHO-K1/hCD32B cell-mediated cross-linking. the level of activation.
  • Figure 5A shows the fluorescence intensity enhancement fold of the variant molecule of CD40 antibody PR003379 in activating HEK293-hCD40-NFkB cells under the condition of CHO-K1/hCD32B cell-mediated cross-linking.
  • Figure 5B shows the variant molecule of CD40 antibody PR003379. Fold enhancement in fluorescence intensity upon activation of HEK293-hCD40-NFkB cells in the absence of CHO-K1/hCD32B cell-mediated cross-linking.
  • Figure 6A, Figure 6B, Figure 6C and Figure 6D respectively show the activation effect of the tested CD40 antibody PR003379 and three control antibodies on human DC cells (IL12p40 release) with or without CHO-K1/hCD32B cell-mediated cross-linking. level).
  • FIG. 7 shows that CD40 antibodies inhibit the binding levels of CD40 and CD40L.
  • Figure 8 A shows the tumor volume changes of the anti-tumor effect of CD40 antibodies in the MC38-hPD-L1/CD40 humanized mouse model.
  • Figure 8B shows the changes in tumor volume of the CD40 antibodies in the MC38-hPD-L1/CD40 humanized mouse model. Anti-tumor effect on body weight changes in mice.
  • binding protein or "antigen-binding protein” generally refers to a protein that contains a moiety that binds an antigen, and optionally a scaffold or backbone that allows the antigen-binding moiety to adopt a conformation that promotes binding of the antigen-binding protein to the antigen. part.
  • An antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both may typically be included.
  • VH and VL regions can be further distinguished as Hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3 and FR-4 .
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the three CDRs of VH are represented as HCDR1, HCDR2 and HCDR3 respectively, and can also be represented as VH CDR1, VH CDR2 and VH CDR3;
  • the three CDRs of VL are represented as LCDR1, LCDR2 and LCDR3 respectively, and can also be represented as VL CDR1 and VL CDR2 and VL CDR3.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugates Antibodies, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they display the required antigen-binding activity.
  • Fab antigen-binding fragments
  • Fv fragments F(ab') 2
  • scFv fragments
  • di-scFv and/or dAb immunoconjugates
  • Antibodies multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they display the required antigen-binding activity.
  • CDRs of antibodies can be defined in a variety of ways, such as Kabat's definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th ed., U.S. National Institute of Health Research, Bethesda, MD (1991)) and Chothia definition rules based on the position of structural loop regions (see J Mol Biol 273:927-48, 1997).
  • the combined definition rules including Kabat definition and Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the Combined definition rule is to combine the scope defined by Kabat and Chothia. Based on this, a larger scope is taken. See Table 1-4 below for details. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass the above-mentioned as described by the present invention. Complementary determination zones defined by any of the known schemes. Although the scope of protection claimed in the present invention is the sequence shown based on the Chothia definition rules, the corresponding amino acid sequences according to other CDR definition rules should also fall within the scope of protection of the present invention.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rule) to position bb (Chothia coding rule);
  • Haa-Hbb can refer to starting from the N-terminus of the antibody heavy chain.
  • L24-L34 can refer to the amino acid sequence from position 24 to position 34 starting from the N-terminus of the antibody light chain and following the Chothia coding rules;
  • H26-H32 can refer to starting from the N-terminus of the antibody heavy chain and following the Chothia coding rules. Amino acid sequence from position 26 to position 32.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of antibodies that is essentially homogenous, i.e., the individual antibodies in the population are identical except for the possible presence of small amounts of natural mutations.
  • Monoclonal antibodies are usually highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations, which often have different antibodies directed against different determinants, each monoclonal antibody is directed against against a single determinant on an antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination from other immunoglobulins.
  • monoclonal indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies used according to the present invention can be produced in hybridoma cells or can be produced by recombinant DNA methods.
  • Fully human antibody generally refers to an antibody in which all human genes encoding antibodies are transferred into genetically engineered animals lacking antibody genes, allowing the animals to express them. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects of heterologous antibodies on the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, etc.
  • the terms “specific binding” and “targeting” generally mean that an antibody binds to an epitope through its antigen-binding domain, and that binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” an antigen when it binds to an epitope more readily through its antigen-binding domain than it would to a random, unrelated epitope.
  • Epitope refers to a specific atomic group (eg, sugar side chain, phosphoryl group, sulfonyl group) or amino acid on an antigen that binds to an antigen-binding protein (eg, an antibody).
  • Fab generally refers to the portion of a conventional antibody (eg, IgG) that binds to an antigen, including the heavy chain variable region VH, light chain variable region VL, and heavy chain constant region domain CH1, as well as the light chain variable region CH1.
  • Chain constant region CL In conventional antibodies, the C-terminus of VH is linked to the N-terminus of CH1 to form the heavy chain Fd fragment, the C-terminus of VL is linked to the N-terminus of CL to form a light chain, and the C-terminus of CH1 is further linked to the hinge region and other constants of the heavy chain. The domains join together to form a heavy chain.
  • Fab also refers to variant structures of Fab.
  • the C-terminus of VH is linked to the N-terminus of CL to form a polypeptide chain
  • the C-terminus of VL is linked to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH/VL) structure.
  • CH1 of Fab is not connected to the hinge region, but the C-terminal of CL is connected to the hinge region of the heavy chain, forming a Fab (cross Fd/LC) structure.
  • VH generally refers to the heavy chain variable region VH domain of an antibody, that is, it can be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals, or it can also be a camelid, etc.
  • the heavy chain variable region VHH of an animal's heavy chain antibody (HCAb structure) can also be the heavy chain variable region VH of a fully human heavy chain antibody (HCAb structure) produced using Harbor HCAb transgenic mice.
  • CD40 generally refers to the tumor necrosis factor receptor superfamily member 5 protein, functional variants and/or functional fragments thereof, also known as TNFRSF5.
  • CD40 sequences are known in the art. For example, an exemplary human CD40 protein amino acid sequence can be found under UniProt accession number P25942; an exemplary cynomolgus monkey CD40 protein sequence can be found under Uniprot accession number G7PG38; an exemplary mouse CD40 protein sequence can be found under Uniprot accession number Found under number P27512.
  • CD40L is a natural trimer ligand molecule of CD40.
  • the present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples.
  • the examples do not include a detailed description of traditional methods, such as those used to construct vectors and plasmids, insert genes encoding proteins into such vectors and plasmids, or introduce plasmids into host cells. Such a method would be useful to those with ordinary skill in the art Members are well known and described in many publications. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.
  • mice that carry a human immunoglobulin immune repertoire and are depleted of the endogenous murine immune repertoire.
  • the antibodies produced by this transgenic mouse have fully human sequences, so there is no need for further humanization, which greatly improves the efficiency of therapeutic antibody development.
  • Harbor H2L2 mice (Harbour Antibodies BV) are transgenic mice that carry a human immunoglobulin immune repertoire and produce antibodies with complete human antibody variable domains and rat constant domains.
  • mice were immunized for multiple rounds with soluble recombinant human CD40 extracellular segment fusion protein (Acrobiosystems, #CD0-H5253).
  • the antigenic protein is mixed with an immune adjuvant to form an immunogenic reagent, which is then injected subcutaneously through the groin or intraperitoneally.
  • the total injection dose received by each mouse was 100 ⁇ L.
  • each mouse was immunized with an immunogenic reagent prepared by mixing 50 ⁇ g of antigen protein and complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1.
  • each mouse was immunized with an immunogenic reagent formulated with 25 ⁇ g of antigenic protein mixed with Sigma Adjuvant System adjuvant (Sigma, #S6322).
  • the interval between each round of boosters is at least two weeks and usually no more than five rounds of boosters.
  • the immunization times were days 0, 14, 28, 42, 56, and 70; and on days 49 and 77, the mouse serum antibody titers were detected.
  • Three days before cell fusion, a final booster immunization was performed with a dose of 25 ⁇ g of antigen protein per mouse.
  • the spleen cells of the mouse are removed and fused with myeloma cell lines to obtain hybridoma cells; after multiple rounds of screening and cloning, the hybridoma cells are isolated Hybridomas expressing CD40 monoclonal antibody molecules were produced.
  • the isolated hybridomas express antibody molecules with intact human variable domains and rat constant domains of heavy and light chains.
  • the above monoclonal antibodies were further identified, and several hybridoma clones were selected for sequencing based on parameters such as their binding ability to human CD40, cynomolgus monkey CD40, and ability to activate CD40 downstream signaling pathways.
  • VH antibody heavy chain variable domain sequence
  • the antibody light chain variable domain sequence (VL) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig ⁇ light chain constant domain sequence to encode the full-length light chain of the resulting antibody.
  • VL antibody light chain variable domain sequence
  • PR006239 is generated through gene synthesis.
  • the nucleic acid encoding the heavy chain variable region (VH) of PR003379 is connected to the nucleic acid encoding the antibody heavy chain constant domain sequence of the IgG1 subtype containing L234A, L235A, and G237A mutations, and cloned into mammals.
  • the full-length heavy chain of IgG1 (L234A, L235A, G237A) is encoded.
  • the nucleic acid encoding the antibody light chain variable domain sequence (VL) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig ⁇ light chain constant domain sequence to encode the full-length light chain for the production of the antibody.
  • the two plasmids were co-transfected into mammalian cells, and after expression, production and purification, the fully human anti-CD40 recombinant IgG1 (L234A, L235A, G237A) antibody PR006239 was obtained. See Example 2 for specific antibody expression and purification methods.
  • This example introduces a general method for preparing antibodies using mammalian host cells (for example, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and their derivatives), transient transfection expression, and affinity capture isolation.
  • mammalian host cells for example, human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and their derivatives
  • transient transfection expression and affinity capture isolation.
  • This method is applicable to target antibodies containing Fc regions; the target antibodies can be composed of one or more protein polypeptide chains; and can be derived from one or more expression plasmids.
  • the amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence through codon optimization method; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with the host cell. Plasmids encoding antibody polypeptide chains are simultaneously transfected into mammalian host cells at a specific ratio, and conventional recombinant protein expression and purification techniques are used to obtain recombinant antibodies with correct folding and polypeptide chain assembly. Specifically, FreeStyle TM 293-F cells (Thermo, #R79007) were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504).
  • the cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • the heavy chain variable domain sequence of the antibody is derived from events such as gene rearrangements and somatic high-frequency mutations of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome; the light chain variable domain sequence is derived from Events such as gene rearrangement of germline gene V and J gene segments of the light chain gene group and somatic high-frequency mutations. Gene rearrangements and high-frequency somatic mutations are major factors that increase antibody diversity. Antibodies derived from the same germline V gene fragment may also produce different sequences, but overall the similarity is high.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • PTMs post-translational modifications
  • the amino acid sequence pattern of PTMs can be disrupted through amino acid mutations, thereby reducing or eliminating the formation of specific PTMs.
  • mutation design methods There are different mutation design methods.
  • One method is to replace "hotspot" amino acids (such as N or S in the NS pattern) with amino acids with similar physical and chemical properties (such as mutating N to Q).
  • hotspot amino acids such as N or S in the NS pattern
  • amino acids with similar physical and chemical properties such as mutating N to Q.
  • Another approach can be to replace the sequence pattern with the corresponding germline gene sequence.
  • multiple mutation design methods may be used for the same PTM sequence pattern.
  • Table 3 lists new antibody molecules obtained by amino acid mutation of antibody PR003379.
  • CD40 antibody in the present application and the amino acid sequence of the CDR defined according to the Chothia definition rules.
  • These CD40 antibodies include positive control antibodies, CD40 antibody PR003379 of the present invention and mutant molecules thereof.
  • Table 4 Sequence numbering list of CD40 antibody sequences and CDR sequences (Chothia) in the application of the present invention
  • This example is to study the binding activity of CD40-targeting antibodies to cells expressing CD40.
  • the highest final concentration of the antigen-binding protein is 300nM, a total of 8 concentrations, 5-fold concentration gradient dilution, and hIgG (Crownbio, #C0002) is used as a negative control.
  • the positive control molecules are the molecules in Table 1-5. The cells were placed at 4°C and incubated in the dark for 1 hour.
  • the software GraphPad Prism 8 was used for data processing and graph analysis. Through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cells and the EC50 value were obtained.
  • Figures 1A, 1B, 1C and 1D and Table 5 show the binding activity of CD40 antibody PR003379 and its variant molecules to CHO-K1/hCD40.
  • the results show that PR003379 and its variant molecules have good binding activity to human CD40; moreover, the binding activity of PR003379 is stronger than that of the control molecules.
  • the EC50 is better than Selicrelumab and CDX-1140, while the maximum MFI value is higher than APX005 and CDX-1140.
  • Figure 2A, Figure 2B, Figure 2C, Figure 2D and Figure 2E and Table 6 show the binding activity of CD40 antibody PR003379 and its variant molecules to Raji cells.
  • the results show that PR003379 and its variant molecules can bind strongly to Raji cells; moreover, the binding activity of PR003379 is stronger than that of the control molecule.
  • Figures 3A, 3B, 3C and 3D and Table 7 show the activity of CD40 antibody PR003379 and its variant molecules in binding to cynomolgus monkey CD40 cells CHO-K1/cyCD40.
  • the results show that PR003379 and its variant molecules have good binding activity to cynomolgus monkey CD40; moreover, the binding activity of PR003379 is stronger than that of the control molecule.
  • the sensor rotation speed is 1000 rpm.
  • PR003379 has a strong affinity for binding to human CD40 protein (approximately 138pM), and the affinity is stronger than the positive control antibody.
  • This example uses a fluorescent reporter gene experiment to study the activation effect of CD40 antibodies on CD40 with and without Fc cross-linking.
  • Cells that highly express human CD32B were used to conduct Fc-mediated cross-linking of the antibody to study whether cross-linking could further enhance the ability to activate CD40.
  • CHO-K1 cells expressing human CD32B CHO-K1/hCD32B (Genscript, #M00600) and CHO-K1 cells were spread into a 96 -well plate at 1 ⁇ 10 4 /well, 100 ⁇ L/well ( PerkinElmer, #6005181), incubate overnight at 37°C in 5% CO2 . The supernatant was removed, and HEK293-hCD40-NFkB reporter cells (BPS bioscience, #60626) expressing human CD40 and NFkB-luciferase reporter genes were collected and added to 96 wells at 5 ⁇ 10 4 /well and 50 ⁇ L /well. board.
  • PR003379 and the control antibody APX005M have a very obvious "cross-linking enhancement" effect, that is, under the conditions of CD32B-mediated antibody cross-linking, PR003379 and APX005M It can significantly amplify the signal of the fluorescent reporter gene, indicating that it significantly enhances the activation of downstream signaling molecules of the CD40 molecule.
  • Figures 5A and 5B and Table 10 show the fluorescence intensity enhancement fold of PR003379 variant molecules activating reporter cells under conditions with or without CD32B cell-mediated cross-linking.
  • the results show that the variant molecule of PR003379 can significantly enhance the activation of CD40 molecules in the case of cross-linking, as shown in the fact that its EC50 value is significantly reduced in the case of cross-linking compared with the case of non-cross-linking (reflected as " Multiple (uncross-linked EC50/cross-linked EC50)”>1).
  • the RLU multiples/folds represent the difference in the activation potential of the antibody in the two situations, or the therapeutic window, which reflects that the multiples of PR003379 and its derived antibodies are better than existing antibodies and have a larger therapeutic window. .
  • CD40 antibody activates the activity of HEK293-CD40-NFkB reporter cells
  • PR003379 variants activate reporter gene cellular activity under cross-linked and non-cross-linked conditions
  • This example uses cells that highly express human CD32B to conduct Fc-mediated cross-linking of antibodies to study whether cross-linking can further enhance the ability of CD40 antibodies to activate DC cells.
  • CHO-K1 cells expressing human CD32B, CHO-K1/hCD32B and CHO-K1 cells were spread into 96-well plates at 1 ⁇ 10 4 /well and 50 ⁇ L/well respectively (Corning, #3599).
  • 50 ⁇ L/well of the antibody protein dilution to be tested with a starting concentration of 100 nM and 5-fold dilution, for a total of 8 concentrations.
  • PR003379 has a very obvious "cross-linking enhancement" effect, that is, in the absence of CD32B-mediated antibody cross-linking, PR003379 activates DC cell release
  • the level of IL-12p40 is limited; however, in the presence of CD32B-mediated antibody cross-linking, PR003379 can significantly enhance the activation of DC cells, and the maximum value of IL-12p40 can reach 1243.0pg/mL, far exceeding other control antibodies.
  • CD40 antibody activates DC cells to release the cytokine IL-12p40
  • CHO-K1/hCD40 cells were plated into a 96-well plate (Corning, #3799) at 2 ⁇ 10 5 /well, 100 ⁇ L/well, centrifuged to take the supernatant, and added 50 ⁇ L/well of dilution solution of the antigen-binding protein to be tested, with a starting concentration of 500 nM and 5-fold dilution, for a total of 8 concentrations.
  • the software GraphPad Prism 8 was used for data processing and graph analysis, and parameters such as the antibody concentration-dependent inhibition rate curve and IC50 value were obtained through four-parameter nonlinear fitting.
  • PR003379 exhibited partial inhibitory activity on the binding of CD40 and CD40L, with a maximum inhibition rate of 41.9%.
  • CD40 antibodies inhibit the binding of CD40 and CD40L
  • the Octet Red 96e (Fortebio) molecular interaction analyzer was used to perform epitope competition experiments on the CD40 antibodies Selicrelumab, APX005M, and PR003379 through BLI technology.
  • the first step is to obtain 100% signal of the antibody: capture CD40 protein (Sino Biological, #10774-H08H) with a HIS1K sensor (Fortebio, #18-5120) with a capture height of 0.2nm.
  • the sensor was equilibrated in the buffer for 120 seconds and then immersed in each antibody diluted to 140 nM for 220 seconds.
  • the final signal of the antibody binding to CD40 was recorded as 100% signal of the antibody.
  • the second step, epitope competition use HIS1K sensor to capture CD40 protein with a capture height of 0.2nm. Immerse the sensor in the first antibody (concentration is 140nM) for 220 seconds, and then immerse the HIS1K sensor in the mixture of the first antibody and the second antibody (the final concentration of both antibodies is 140nM), for 220 seconds, and immerse the sensor The signal difference after immersion in the antibody mixture was recorded as the signal for that antibody as the secondary antibody.
  • A 100% signal of a certain antibody (obtained from the first step)
  • B signal of this antibody as a second antibody (obtained from the second step).
  • the inhibition rate obtained is greater than 80%, it means that the epitopes of the two antibodies almost completely overlap; if the inhibition rate is greater than 40% and less than 80%, it means that the epitopes bound by the two antibodies do not completely overlap or the epitopes are very small. Similar; if the inhibition rate is less than 40%, it means that the epitopes of the two antibodies are different.
  • MC38 mouse colon cancer cells overexpressing human PD-L1 (MC38/hPD-L1, provided by Shanghai Southern Model Biology Company) were cultured at 37 degrees with 5% CO2 .
  • the culture medium is DMEM medium containing 10% inactivated fetal calf serum. The cells are subcultured every 3 to 4 days when they are full grown.
  • mice with moderate individual tumor volumes are selected and enrolled into the group. The mice are randomly assigned to each experimental group according to tumor volume, with 6 mice in each group. Administration begins on the day of grouping.
  • the average tumor volume of IgG1 (3 mg/kg) in the control group was 1409.41 ⁇ 205.02 mm 3 .
  • Selicrelumab (3mg/kg) group, CDX-1104 (3mg/kg) group, PR003379 (3mg/kg) group The average tumor volumes were 460.96 ⁇ 68.61mm 3 , 1345.22 ⁇ 162.94mm 3 , and 447.89 ⁇ 50.74mm 3 respectively, and the tumor inhibition rates were 73.22%, 4.92%, and 74.24% respectively.
  • mice were 12.24%, 7.31%, 7.56%, and 1.86%, respectively. The mice were in normal condition.

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Abstract

本发明公开了一种靶向CD40的抗原结合蛋白及其制备和应用。所述靶向CD40的抗原结合蛋白具有与CD40的高亲和力和对信号通路的激动活性强的特性，尤其是交联后激动活性增强，使得本发明所述的靶向CD40的抗原结合蛋白具有更大的治疗窗口，有望为多种肿瘤的治疗带来新的机遇。

Description

一种靶向CD40的抗原结合蛋白及其制备和应用
本申请要求申请日为2022/4/2的中国专利申请2022103511889的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物制药领域，尤其涉及一种靶向CD40的抗原结合蛋白及其制备和应用。
背景技术
CD40是一种糖基化的I型跨膜蛋白，属于肿瘤坏死因子受体超家族(TNFRSF)成员，又称为肿瘤坏死因子受体超家族成员5(TNFRSF5)。CD40表达在一系列抗原呈递细胞(APC)表面，包括单核细胞、树突状细胞(DC)、B细胞和巨噬细胞。其配体CD40L，主要表达在淋巴细胞表面，包括T细胞、B细胞、自然杀伤细胞(NK)，通常以三聚体以及多聚体形式存在。CD40和CD40L是一对共刺激分子，CD40下游信号通路的激活，需要三聚体以及多聚体形式的CD40L对CD40形成交联。它们在细胞表面相互作用，引起CD40重新分布到膜脂筏，并发生构象变化，CD40通过胞内端结构域在细胞质中募集TNFR相关因子(TRAF)促进细胞内信号传导，从而激活不同的信号通路，例如经典和非经典核因子κB通路、p38丝裂原活化蛋白激酶、磷脂酰肌醇-3激酶(PI3K)和磷脂酶Cγ通路等，并通过这些信号通路靶向的基因，进一步调控细胞凋亡、细胞周期进程、细胞因子生产以及细胞表面免疫调节器的表达。由此，激活CD40可以增加抗原呈递、促进细胞因子分泌、激活淋巴细胞，同时刺激活化人先天性免疫系统和获得性免疫系统，产生协同效应，抵抗癌症的发生和发展。
CD40在肿瘤细胞中也有广泛表达，其在几乎所有的B细胞恶性肿瘤以及广泛的实体瘤中表达，包括黑色素瘤、肺癌、乳腺癌、结肠癌、前列腺癌、胰腺癌、肾癌、卵巢癌和头颈部癌等。表达在肿瘤细胞表面的CD40能介导的肿瘤细胞死亡。在没有免疫辅助细胞的情况下，通过CD40L交联表达在多种肿瘤细胞表面的CD40将介导直接的细胞毒作用。在体外，通过CD40L交联CD40被证明能诱导肿瘤细胞凋亡，抑制实体瘤细胞和B恶性肿瘤细胞的生长。在体内，激活CD40也介导肿瘤抑制作用。证据表明，在免疫缺陷型小鼠中，通过CD40L干预肿瘤细胞表面的CD40，即使没有淋巴细胞激活，也会抑制乳腺癌细胞移植瘤或者B淋巴细胞移植瘤的生长。
因此，CD40介导的肿瘤细胞死亡的机制可以是双重的，即刺激免疫系统对肿瘤细胞的杀伤和直接的肿瘤细胞毒作用，可以协同抗肿瘤。激动型的抗CD40抗体，类似CD40L，可以交联和激活免疫细胞及肿瘤细胞表面的CD40，起到显著的抗肿瘤效果。这种抗肿瘤效果已经在临床前动物模型和临床肿瘤患者的试验中得到证明，并且可以与化疗药物联用，比如吉西他滨和紫杉醇，也可以与免疫调节类药物联用，比如PD-1抗体和CTLA-4抗体，产生协同的抗肿瘤效果。
目前，处于临床试验阶段的CD40抗体药物已有20余项，但最早产品仅处于临床二期，尚未有任何上市产品。因此，开发其它CD40抗体将为多种肿瘤和免疫系统相关疾病的治疗提供可能，具有 很高的市场价值。
在抗肿瘤方面，CD40抗体在临床上面临的最主要问题包括，偏低的客观缓解率、显著的毒副作用和较低的耐受剂量等。导致这些结果的可能原因是激动剂活性偏弱，比如Celldex公司的产品CDX-1140。在其一期临床试验中，单药治疗的42位患者中，没有出现完全或者部分缓解；在联合rhFLT3L的治疗中，20位患者中，也仅1例患者出现部分缓解。该产品体外对DC细胞的激活较弱，且在交联的情况下，激动剂活性得不到加强。罗氏公司的CD40抗体Selicrelumab，临床试验中，用药剂量超过0.2mg/kg的情况下，即在部分受试患者体内引起显著的细胞因子释放综合症和肝脏毒性等毒副作用；Apexigen公司的CD40抗体APX005M，临床试验中，用药剂量超过0.3mg/kg的情况下，即在部分受试患者体内引起显著的嗜中性粒细胞减少症，以及进一步的败血症和败血性休克。另外，人鼠嵌合序列的CD40单抗ChiLob7/4，由于在一期临床实验中遭遇人抗嵌合抗体效应(Human Anti-Chimeric Antibody，HACA)而失败。综上所言，目前临床上的CD40抗体面临着有效性和安全性之间治疗窗口过小的问题和挑战。
发明内容
针对现有技术中CD40抗体存在的有效性和安全性之间治疗窗口小的技术缺陷，本发明提供了一种靶向CD40的抗原结合蛋白及其制备和应用。所述靶向CD40的抗原结合蛋白具有与CD40的高亲和力和对信号通路的激动活性强的特性，尤其是交联后激动活性增强，使得本发明所述的靶向CD40的抗原结合蛋白具有更大的治疗窗口，安全性好，有望为多种肿瘤的治疗带来新的机遇。
为解决上述技术问题，本发明提供的一个技术方案为：一种靶向CD40的抗原结合蛋白，所述抗原结合蛋白包含轻链可变区和重链可变区，所述轻链可变区(VL)包含LCDR1、LCDR2和LCDR3，所述重链可变区(VH)包含HCDR1、HCDR2和HCDR3；其中：
所述LCDR1包括如SEQ ID NO:33所示的氨基酸序列或在SEQ ID NO:33上具有3、2或1个氨基酸突变的变体1，所述LCDR2包括如SEQ ID NO:41所示的氨基酸序列或在SEQ ID NO:41上具有3、2或1个氨基酸突变的变体2，所述LCDR3包括如SEQ ID NO:49所示的氨基酸序列或在SEQ ID NO:49上具有3、2或1个氨基酸突变的变体3；所述HCDR1包括如SEQ ID NO:7所示的氨基酸序列或在SEQ ID NO:7上具有3、2或1个氨基酸突变的变体4，所述HCDR2包括如SEQ ID NO:15所示的氨基酸序列或在SEQ ID NO:15上具有3、2或1个氨基酸突变的变体5，所述HCDR3包括如SEQ ID NO:23所示的氨基酸序列或在SEQ ID NO:23上具有3、2或1个氨基酸突变的变体6。
较佳地，所述变体3包括在SEQ ID NO:49所示氨基酸序列上具有PTM位点突变的氨基酸序列，优选包括在SEQ ID NO:49所示氨基酸序列的第4位和/或第5位上发生氨基酸突变的氨基酸序列，所述氨基酸突变优选为氨基酸替代，更优选为氨基酸的保守替代。
在本发明一些技术方案中，所述变体3为如SEQ ID NO:50-73任一项所示的氨基酸序列。
在本发明一些技术方案中，所述LCDR1包括如SEQ ID NO:33所示的氨基酸序列，所述LCDR2包括如SEQ ID NO:41所示的氨基酸序列，所述LCDR3包括如SEQ ID NO:49-73任一项所示的氨基 酸序列；所述HCDR1包括如SEQ ID NO:7所示的氨基酸序列，所述HCDR2包括如SEQ ID NO:15所示的氨基酸序列，所述HCDR3包括如SEQ ID NO:23所示的氨基酸序列，具体CDR组合见下表1-1。
表1-1：具体CDR组合
较佳地，所述轻链可变区包括如SEQ ID NO:85所示的氨基酸序列或与SEQ ID NO:85有至少85％、90％、92％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述重链可变区包括如SEQ ID NO:81所示的氨基酸序列或与SEQ ID NO:81有至少85％、90％、92％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列。更佳地，所述轻链可变区包括如SEQ ID NO:85-109任一项所示的氨基酸序列，所述重链可变区包括如SEQ ID NO:81所示的氨基酸序列，具体轻链可变区和重链可变区组合如下表1-2所示。
表1-2：具体轻链可变区和重链可变区组合

较佳地，所述靶向CD40的抗原结合蛋白满足以下三项中至少一项：
(1)所述靶向CD40的抗原结合蛋白为全长抗体、Fab、Fab’、F(ab’)2或Fv，所述Fv优选为scFv；
(2)所述靶向CD40的抗原结合蛋白为单特异性抗体、双特异性抗体或多特异性抗体；
(3)所述靶向CD40的抗原结合蛋白为单克隆抗体或多克隆抗体。
较佳地，所述靶向CD40的抗原结合蛋白为全长抗体，所述全长抗体包含轻链和重链；所述轻链包括轻链恒定区(CL)，所述轻链恒定区优选为人源抗体轻链恒定区；所述重链包括重链恒定区(CH)，所述重链恒定区优选为人源抗体重链恒定区，更优选为hIgG1、hIgG2、hIgG3或hIgG4亚型的重链恒定区，进一步优选为hIgG1亚型的重链恒定区。
在本发明一些技术方案中，所述靶向CD40的抗原结合蛋白为全长抗体，所述全长抗体包含轻链和重链，所述重链包括如SEQ ID NO:114或115所示的氨基酸序列，所述轻链包括如SEQ ID NO:119-143任一项所示的氨基酸序列。较佳地，所述重链包括如SEQ ID NO:114所示的氨基酸序列，所述轻链包括如SEQ ID NO:119所示的氨基酸序列；或，所述重链包括如SEQ ID NO:115所示的氨基酸序列，所述轻链包括如SEQ ID NO:119-143任一项所示的氨基酸序列，具体轻链和重链组合见下表 1-3。
表1-3：具体轻链和重链组合
本发明中，所述变体1、变体2、变体3、变体4、变体5和变体6中的数字仅用以区分不同变体，并不表示实际含义。
本发明中，所述PTM位点为蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰的位点，称为翻译后修饰(PTM)位点。
本发明中CDR的氨基酸序列按照Chothia定义规则确定。
为解决上述技术问题，本发明提供的另一个技术方案为：一种嵌合抗原受体(CAR)，其包含如本发明所述的靶向CD40的抗原结合蛋白。
为解决上述技术问题，本发明提供的另一个技术方案为：一种分离的核酸，其编码如本发明所述的靶向CD40的抗原结合蛋白或如本发明所述的嵌合抗原受体。
为解决上述技术问题，本发明提供的另一个技术方案为：一种重组表达载体，其包含如本发明所 述的分离的核酸。较佳地，所述重组表达载体包含真核细胞表达载体和/或原核细胞表达载体。
为解决上述技术问题，本发明提供的另一个技术方案为：一种转化体，其包含如本发明所述的分离的核酸或如本发明所述的重组表达载体。较佳地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选为E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
为解决上述技术问题，本发明提供的另一个技术方案为：一种如本发明所述的靶向CD40的抗原结合蛋白的制备方法，其包括以下步骤：培养如本发明所述的转化体，从培养物中获得所述靶向CD40的抗原结合蛋白。
为解决上述技术问题，本发明提供的另一个技术方案为：一种抗体药物偶联物(Antibody-drug conjugate,ADC)，其包含如本发明所述的靶向CD40的抗原结合蛋白和细胞毒性剂或标签。较佳地，所述细胞毒性剂为MMAF或MMAE，所述标签为荧光剂。
为解决上述技术问题，本发明提供的另一个技术方案为：一种基因修饰的细胞，其表达如本发明所述的嵌合抗原受体。较佳地，所述基因修饰的细胞为真核细胞，优选分离的人细胞，更优选免疫细胞如T细胞，或NK细胞。
为解决上述技术问题，本发明提供的另一个技术方案为：一种药物组合物，其包含如本发明所述的靶向CD40的抗原结合蛋白、如本发明所述的分离的核酸、如本发明所述的重组表达载体、如本发明所述的基因修饰的细胞和/或如本发明所述的抗体药物偶联物，以及药学上可接受的载体和/或药学上可接受的佐剂。较佳地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分。
为解决上述技术问题，本发明提供的另一个技术方案为：一种检测试剂，其包含如本发明所述的靶向CD40的抗原结合蛋白和/或如本发明所述的抗体药物偶联物。较佳地，所述检测试剂为液体剂型、气体剂型、固体剂型和半固体剂型。更佳地，所述检测试剂还包括二抗、CD40或其衍生物，所述二抗例如抗人IgG的抗体偶联辣根过氧化物酶和抗人IgG的抗体偶联生物素蛋白。
为解决上述技术问题，本发明提供的另一个技术方案为：一种套装药盒，其包含药盒A，所述药盒A含有如本发明所述的靶向CD40的抗原结合蛋白、如本发明所述的药物组合物、如本发明所述的检测试剂、如本发明所述的基因修饰的细胞和如本发明所述的抗体药物偶联物中的一种或多种。
较佳地，所述套装药盒还包括药盒B，所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
为解决上述技术问题，本发明提供的另一个技术方案为：如本发明所述的靶向CD40的抗原结合蛋白、如本发明所述的药物组合物、如本发明所述的检测试剂、如本发明所述的套装药盒、如本发明所述的基因修饰的细胞和如本发明所述的抗体药物偶联物中的一种或多种在制备诊断、预防和/或治疗肿瘤等疾病的药物中的用途。较佳地，所述肿瘤包括实体瘤和血液瘤。更佳地，所述肿瘤包括B系NHL、慢性淋巴细胞性白血病(CLL)、多毛细胞白血病(HCL)、霍奇金病、多发性骨髓瘤、膀胱癌、肾癌、卵巢癌、宫颈癌、乳腺癌、肺癌、鼻咽癌、恶性黑色素瘤、胰腺癌和结肠癌。
为解决上述技术问题，本发明提供的另一个技术方案为：一种检测样品中CD40的方法，其包括使用如本发明所述的靶向CD40的抗原结合蛋白、如本发明所述的检测试剂和如本发明所述的抗体药物偶联物中的一种或多种与所述样品接触的步骤。所述样品例如血液样本(例如全血样本和血清样本)和包含CD40的试剂。较佳地，所述方法为非诊断和/或治疗目的，例如科学研究中检测CD40标准品的浓度、其他试剂是否被CD40污染等。
为解决上述技术问题，本发明提供的另一个技术方案为：一种诊断、治疗和/或预防肿瘤的方法，所述方法包括向有需要的患者施用治疗有效量的如本发明所述的靶向CD40的抗原结合蛋白、如本发明所述的分离的核酸、如本发明所述的重组表达载体、如本发明所述的药物组合物、如本发明所述的检测试剂、如本发明所述的基因修饰的细胞和如本发明所述的抗体药物偶联物中的一种或多种，或者使用如本发明所述的套装药盒诊断或治疗有需要的患者。较佳地，所述肿瘤包括实体瘤和血液瘤。更佳地，所述肿瘤包括B系NHL、慢性淋巴细胞性白血病(CLL)、多毛细胞白血病(HCL)、霍奇金病、多发性骨髓瘤、膀胱癌、肾癌、卵巢癌、宫颈癌、乳腺癌、肺癌、鼻咽癌、恶性黑色素瘤、胰腺癌和结肠癌。
为解决上述技术问题，本发明提供的另一个技术方案为：如本发明所述的靶向CD40的抗原结合蛋白、如本发明所述的分离的核酸、如本发明所述的重组表达载体、如本发明所述的药物组合物、如本发明所述的检测试剂、如本发明所述的套装药盒、如本发明所述的基因修饰的细胞和/或如本发明所述的抗体药物偶联物的一种或多种在制备诊断、预防或治疗肿瘤的药物中的用途。较佳地，所述肿瘤包括实体瘤和血液瘤。更佳地，所述肿瘤包括B系NHL、慢性淋巴细胞性白血病(CLL)、多毛细胞白血病(HCL)、霍奇金病、多发性骨髓瘤、膀胱癌、肾癌、卵巢癌、宫颈癌、乳腺癌、肺癌、鼻咽癌、恶性黑色素瘤、胰腺癌和结肠癌。
本发明中，“具有3、2或1个氨基酸突变”是指包括在原氨基酸序列的基础上发生氨基酸的插入、缺失或替换。示例性的解释是对CDR的突变可以包含3个、2个或1个氨基酸的突变，这些CDR之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如可以是对CDR1进行1个氨基酸的突变，对CDR2和CDR3不进行氨基酸突变。
本发明中，“PTM位点突变”是指相较于原氨基酸序列而言，变体的序列存在PTM位点上氨基酸的突变。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。
本发明中，所述的VH、VL或所述的全长抗体均可包含在所限定的序列的基础上进行突变的情形。所述突变为所限定的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所限定的氨基酸序列具有至少85％序列同一性，并保持或改善了包含该突变的氨基酸序列的抗原结合蛋白的结合活性；所述至少85％序列同一性优选为至少90％序列同一性；更优选为至少95％序列同一性；最优选为至少99％序列同一性。
本发明中，“包括”、“包含”和“为”在某些具体的实施例中表示一样的意思。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
所述靶向CD40的抗原结合蛋白具有与CD40的高亲和力和对信号通路的激动活性强的特性，尤其是交联后激动活性增强，使得本发明所述的靶向CD40的抗原结合蛋白具有更大的治疗窗口，为在临床试验中，在安全耐受的剂量条件下，显著提高患者响应率提供良好基础。
附图说明
图1A、图1B、图1C和图1D分别展示部分CD40抗体结合高表达人CD40的CHO-K1细胞的水平。
图2A、图2B、图2C、图2D和图2E分别展示部分CD40抗体结合高表达人CD40的Raji细胞的水平。
图3A、图3B、图3C和图3D分别展示部分CD40抗体结合高表达食蟹猴CD40的CHO-K1细胞的水平。
图4A、图4B、图4C和图4D分别展示待测CD40抗体PR003379和三个对照抗体在有或者没有CHO-K1/hCD32B细胞介导交联的条件下对HEK293-hCD40-NFkB荧光报告基因细胞的激活作用的水平。
图5A为CD40抗体PR003379的变体分子在有CHO-K1/hCD32B细胞介导交联的条件下对HEK293-hCD40-NFkB细胞的激活的荧光强度增强倍数，图5B为CD40抗体PR003379的变体分子在没有CHO-K1/hCD32B细胞介导交联的条件下对HEK293-hCD40-NFkB细胞的激活的荧光强度增强倍数。
图6A、图6B、图6C和图6D分别展示待测CD40抗体PR003379和三个对照抗体在有或者没有CHO-K1/hCD32B细胞介导交联的条件下对人DC细胞的激活作用(IL12p40释放水平)。
图7显示了CD40抗体抑制CD40和CD40L的结合水平。
图8 A为CD40抗体在MC38-hPD-L1/CD40人源化小鼠模型中的抗肿瘤效果的肿瘤体积变化，图8B为CD40抗体在MC38-hPD-L1/CD40人源化小鼠模型中的抗肿瘤效果的小鼠体重变化。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“结合蛋白”或者“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为 称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR-1、CDR1、FR-2、CDR2、FR-3、CDR3和FR-4。重链和轻链的可变区含有与抗原相互作用的结合结构域。VH的三个CDR分别表示为HCDR1、HCDR2和HCDR3，也可表示为VH CDR1、VH CDR2和VH CDR3；VL的三个CDR分别表示为LCDR1、LCDR2和LCDR3，也可表示为VL CDR1、VL CDR2和VL CDR3。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab)2，Fv片段，F(ab’)2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，所述CDR的氨基酸序列均是按照Chothia定义规则所示出的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。在本发明的技术方案中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则来确定可变结构域序列中的氨基酸残基。其中Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见下表1-4。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
表1-4：本申请抗体CDR定义方法
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。本领域技术人员应当知晓的是，在用Chothia编码CDR时，有些位置会有插入位点的情况(可参见http://bioinf.org.uk/abs/)。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针 对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本发明使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术等。
在本申请中，术语“特异性结合”、“靶向”通常是指抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“Fab”通常指常规抗体(例如IgG)中与抗原结合的部分，包括抗体的重链可变区VH、轻链可变区VL和重链恒定区结构域CH1以及轻链恒定区CL。在常规抗体中，VH的C端与CH1的N端联结形成重链Fd片段，VL的C端与CL的N端联结形成轻链，CH1的C端又进一步与重链的铰链区和其他恒定区结构域联结形成重链。在一些实施例中，“Fab”也指Fab的变体结构。例如，在某些实施例中，VH的C端与CL的N端联结形成一个多肽链，VL的C端与CH1的N端联结形成另一个多肽链，形成Fab(cross VH/VL)的结构；在某些实施例中，Fab的CH1不与铰链区联结，而是CL的C端与重链的铰链区联结，形成Fab(cross Fd/LC)的结构。
在本申请中，术语“VH”通常指抗体的重链可变区VH结构域，即可以是人或者其他动物的常规抗体(H2L2结构)的重链可变区VH，也可以是骆驼科等动物的重链抗体(HCAb结构)的重链可变区VHH，还可以是利用Harbour HCAb转基因小鼠产生的全人源重链抗体(HCAb结构)的重链可变区VH。
在本申请中，术语“CD40”通常是指肿瘤坏死因子受体超家族成员5蛋白、其功能变体和/或其功能片段，也称为TNFRSF5。CD40序列是本领域已知的。例如，示例性的人CD40蛋白的氨基酸序列可在UniProt登录号P25942下找到；示例性的食蟹猴CD40蛋白序列可在Uniprot登录号G7PG38下找到；示例性的小鼠CD40蛋白序列可在Uniprot登录号P27512下找到。CD40L是CD40的天然三聚体配体分子。
实施例
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人 员是众所周知的，并且在许多出版物中都有所描述。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1 CD40抗体的制备
转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
用可溶的重组人CD40胞外段融合蛋白(Acrobiosystems,#CD0-H5253)对Harbour H2L2小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100μL。在首轮免疫中，每只小鼠接受用50μg抗原蛋白与完全弗氏佐剂(Sigma，#F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25μg抗原蛋白与Sigma Adjuvant System佐剂(Sigma，#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行细胞融合前3天，以每只小鼠25μg抗原蛋白的剂量进行最后一次增强免疫。
当检测小鼠血清中CD40特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，分离出表达CD40单克隆抗体分子的杂交瘤。分离的杂交瘤表达具有完整的人可变结构域和大鼠恒定结构域的重链和轻链的抗体分子。对上述单克隆抗体进行进一步的鉴定，根据其对人CD40的结合能力、食蟹猴CD40的结合能力、激活CD40下游信号通路的能力等参数，选出若干个杂交瘤克隆进行测序。在筛选过程中，使用了如表1-5所列阳性对照抗体(相应序列编号见表4)。利用常规的杂交瘤测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中，从免疫的Harbour H2L2小鼠得到的CD40单克隆抗体分子可变结构域的序列是人源抗体序列。抗体可变结构域的CDR序列可以通过Kabat或者Chothia或者其他CDR定义规则(表1-5)进行分析。
表1-5：CD40阳性对照抗体
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG2抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG2抗体的全长重链。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。在本实施例中，得到全人源的抗CD40重组IgG2抗体。
PR006239是通过基因合成，将编码PR003379的重链可变区(VH)的核酸连接到编码含有L234A,L235A,G237A突变的IgG1亚型的抗体重链恒定结构域序列的核酸上，并克隆到哺乳动物细胞表达质粒中，编码产生IgG1(L234A,L235A,G237A)全长重链。编码抗体轻链可变结构域序列(VL)的核酸通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。将两个质粒共转到哺乳动物细胞中，表达生产以及纯化后，即得到全人源的抗CD40重组IgG1(L234A,L235A,G237A)抗体PR006239。具体的抗体表达和纯化方法参见实施例2。
实施例2抗体的瞬时转染表达和纯化
本实施例介绍了利用哺乳动物宿主细胞(例如，人胚肾细胞HEK293或中国仓鼠卵巢细胞CHO及其衍生细胞)、瞬时转染表达和亲和捕获分离等技术来制备抗体的一般方法。本方法适用于含有Fc区的目标抗体；目标抗体可以由一条或多条蛋白质多肽链组成；可以来源于一个或多个表达质粒。
将抗体多肽链的氨基酸序列通过密码子优化方法转换成核苷酸序列；合成编码的核苷酸序列并克隆到与宿主细胞兼容的表达载体上。将编码抗体多肽链的质粒按照特定比例同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到具有正确折叠和多肽链组装的重组抗体。具体地，将FreeStyleTM 293-F细胞(Thermo,#R79007)在FreeStyleTM F17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6–8×105细胞/ml，于37℃8％CO2摇床中培养24小时，细胞浓度在1.2×106细胞/ml。准备30ml培养的细胞。将编码抗体多肽链的质粒(pTT5，NRC)按照一定比例混合共计30μg质粒(质粒与细胞的比例为1μg:1ml)溶解于1.5ml Opti-MEM减血清培养基(Thermo,#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS pH7.4缓冲液平衡含有MabSelectTM(GE Healthcare,#71-5020-91)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS缓冲液冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，随后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,#UFC901024)浓缩换液至PBS缓冲液或者含有其他成分的缓冲液，得到纯化的重组抗体溶液。最后用NanoDrop(Thermo,NanoDropTM One)测定浓度，分装、存储备用。
实施例3抗体的序列分析和优化
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如 IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。将实施例1得到的CD40抗体序列进行分析，其重链可变结构域(VH)和轻链可变结构域(VL)的胚系基因V基因片段列于表2。
蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰，称为翻译后修饰(PTM)。如果在抗体的可变结构域尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM是和氨基酸序列的组成尤其是相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。表2列出了实施例1的抗体的可变结构域VH和VL的预测的PTM(NS可能是脱酰胺位点)。
可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
表3列出了对抗体PR003379进行氨基酸突变得到的新的抗体分子。
表4列出了本发明申请中CD40抗体的序列和根据Chothia定义规则定义的CDR的氨基酸序列。这些CD40抗体包括阳性对照抗体、本发明的CD40抗体PR003379及其突变体分子。
表2：CD40抗体的胚系基因分析和PTM位点分析
表3：CD40抗体的PR003379的突变位点设计

表4：本发明申请中CD40抗体的序列和CDR序列(Chothia)的序列编号表

实施例4结合表达CD40的细胞
本实施例是为了研究靶向CD40的抗体结合表达有CD40的细胞的结合活性。
利用流式细胞术FACS检测CD40抗体与高表达人CD40的CHO-K1/hCD40(北京康源博创构建和高表达食蟹猴CD40的CHO-K1/cyCD40(北京康源博创构建)以及高表达人CD40的淋巴瘤细胞Raji(ATCC,#CCL-86)等细胞的结合能力。具体地，消化细胞并用完全培养基(CHO-K1细胞用F-12K；Raji细胞用RPMI-1640)重悬；将细胞密度调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的待测抗原结合蛋白，混合均匀。其中抗原结合蛋白最高终浓度为300nM，共8个浓度，5倍浓度梯度稀释，并以hIgG(Crownbio,#C0002)作为阴性对照。阳性对照分子为表1-5中的分子。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g，4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(山羊抗人IgG(H+L)第二抗体,Alexa488 conjugate,Invitrogen，#A11013,1:1000稀释)，4℃，避光孵育30分钟。用200μL/孔预冷PBS洗涤细胞两次，于500g，4℃下离心5分钟，弃上清。最后，用200μL/孔预冷PBS重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
实施例4.1结合到人CD40细胞CHO-K1/hCD40
图1A、图1B、图1C和图1D以及表5显示了CD40抗体PR003379及其变体分子结合CHO-K1/hCD40的活性。结果显示PR003379及其变体分子具有很好的人CD40的结合活性；而且，PR003379的结合活性与对照分子相比更强，例如，EC50优于Selicrelumab和CDX-1140，而MFI最大值高于APX005和CDX-1140。
表5：PR003379及其变体分子结合CHO-K1/hCD40(对应图1A-图1D)

实施例4.2结合到人CD40细胞Raji
图2A、图2B、图2C、图2D和图2E以及表6显示了CD40抗体PR003379及其变体分子结合Raji细胞的活性。结果显示PR003379及其变体分子能够很强地结合Raji细胞；而且，PR003379的结合活性与对照分子相比更强。
表6：PR003379及其变体分子结合Raji细胞(对应图2A-图2E)

实施例4.3结合到食蟹猴CD40细胞CHO-K1/cyCD40
图3A、图3B、图3C和图3D以及表7显示了CD40抗体PR003379及其变体分子结合食蟹猴CD40细胞CHO-K1/cyCD40的活性。结果显示PR003379及其变体分子具有很好的食蟹猴CD40的结合活性；而且，PR003379的结合活性与对照分子相比更强。
表7：PR003379及其变体分子结合CHO-K1/cyCD40(对应图3A-图3D)
实施例5与CD40蛋白的亲和力
为了检测CD40抗体与抗原如人CD40蛋白的亲和力，通过生物膜干涉(BLI)技术，使用Octet Red 96e(Fortebio)分子相互作用分析仪来进行抗原抗体之间的结合动力学分析。
先将10×动力学缓冲液(ForteBio，#18-1105)稀释至1×，用于亲和力测试以及抗原、抗体的稀释。测定抗原和抗体的亲和力时，传感器转动速度为1000转/分钟。先将置于一列的AHC传感器(Fortebio，#18-5060)在上述稀释至1×的测试缓冲液中平衡10分钟，然后用AHC传感器捕获CD40抗体，捕获高度0.85nm；将AHC传感器在缓冲液中平衡120秒后与2倍梯度稀释的人CD40蛋白(与PR003379、CDX-1140结合的CD40浓度范围为20-5nM以及0nM；与APX005M、Selicrelumab结合的CD40浓度范围为120-30nM以及0nM结合180秒，再解离500秒。最后将AHC传感器浸入10mM甘氨酸-盐酸pH 1.5溶液进行再生，以洗脱结合在传感器上的蛋白。
使用Octet Data Analysis软件(Fortebio，版本11.0)进行数据分析时，以0nM为参照孔，扣除参照信号(reference subtraction)，选择“1:1Global fitting”方法进行数据拟合，计算出抗原与CD40抗体结合的动力学参数，得到kon(1/Ms)值、kdis(1/s)值和KD(M)值。
亲和力测试结果如表8所示，PR003379结合人CD40蛋白有很强的亲和力(约138pM)，亲和力强于阳性对照抗体。
表8：CD40抗体结合人CD40蛋白的亲和力
实施例6在荧光报告基因实验中的活性
本实施例利用荧光报告基因实验研究了CD40抗体在Fc交联以及不交联情况下对CD40的活化影响。利用高表达人CD32B的细胞对抗体进行Fc介导的交联，研究交联是否能进一步增强对CD40激活的能力。
将表达人CD32B的CHO-K1细胞CHO-K1/hCD32B(Genscript，#M00600)以及CHO-K1细胞以1×104/孔以1×104/孔，100μL/孔铺到96孔板中(PerkinElmer，#6005181)，37℃在5％CO2环境下孵育过夜。去除上清液，将表达人CD40和NFkB-荧光素酶报告基因的HEK293-hCD40-NFkB报告基因细胞(BPS bioscience,#60626)收集后以5×104/孔，50μL/孔加入到96孔板中。加入50μL/孔的待测抗体蛋白稀释液，起始浓度为100nM，5倍浓度稀释，共8个浓度。37℃在5％CO2环境下培养6小时。加入ONE-GloTM荧光素酶试剂(Promega,#E6110)，室温孵育5分钟，酶标仪(PerkinElmer EnSpire)检测发光值。应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体浓度依赖的相对荧光信号单位(RLU)的曲线及EC50值等参数。CD40抗体与CHO-K1/hCD32B细胞孵育的样品称为“交联”(crosslinked)；CD40抗体与CHO-K1细胞孵育的样品没有交联。
如图4A、图4B、图4C和图4D和表9所示，PR003379和对照抗体APX005M有非常明显的“交联增强”作用，即，在CD32B介导抗体交联的条件下，PR003379和APX005M能够显著地放大荧光报告基因的信号，说明显著增强了对CD40分子的下游信号分子的活化作用。
如图5A和图5B及表10显示了PR003379的变体分子在有或者没有CD32B细胞介导交联的条件下对报告基因细胞的激活的荧光强度增强倍数。结果说明，PR003379的变体分子在交联的情况下能显著增强对CD40分子的活化作用，表现在其EC50值在交联的情况下相比未交联的情况下显著减小(体现为“倍数(未交联EC50/交联EC50)”>1)。
从表9和表10中RLU倍数/倍数均代表了抗体在两种情况下的激活潜能的差异，或者治疗窗口，反应了PR003379及其衍生抗体的倍数较现有抗体更佳，治疗窗口更大。
表9：CD40抗体激活HEK293-CD40-NFkB报告基因细胞的活性
表10：PR003379变体在交联和未交联条件下激活报告基因细胞活性
实施例7在DC细胞激活实验中的活性
本实施例利用高表达人CD32B的细胞对抗体进行Fc介导的交联，研究交联是否能进一步增强CD40抗体激活DC细胞的能力。
将表达人CD32B的CHO-K1细胞CHO-K1/hCD32B以及CHO-K1细胞分别以1×104/孔，50μL/孔铺到96孔板中(Corning，#3599)。加入50μL/孔的待测抗体蛋白稀释液，起始浓度为100nM，5倍浓度稀释，共8个浓度。加入从人PBMC分离得到的诱导成熟的DC细胞，2×104/孔，100μL/孔，于37℃，5％CO2培养箱孵育2天。收集第二天的上清液，采用人IL-12p40ELISA试剂盒(R&D system，#DY240)检测上清中IL-12p40的含量。应用软件GraphPad Prism8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体浓度依赖的IL12p40释放水平的曲线及EC50值等参数。CD40抗体与CHO- K1/hCD32B细胞孵育的样品称为“交联”(crosslinked)；CD40抗体与CHO-K1细胞孵育的样品没有交联。
结果如图6A、图6B、图6C和图6D和表11所示，PR003379有非常明显的“交联增强”作用，即，在没有CD32B介导抗体交联的条件下，PR003379激活DC细胞释放IL-12p40水平有限；但是，在有CD32B介导抗体交联的条件下，PR003379能显著增强DC细胞的活化，IL-12p40的最大值可以达到1243.0pg/mL，远超过其他对照抗体。
表11：CD40抗体激活DC细胞释放细胞因子IL-12p40
实施例8阻断CD40和CD40L的结合
为了检测CD40抗体对CD40和CD40L结合的影响，将CHO-K1/hCD40细胞以2×105/孔，100μL/孔铺到96孔板中(Corning，#3799)，离心取上清液，加入50μL/孔的待测抗原结合蛋白稀释液，起始浓度为500nM，5倍浓度稀释，共8个浓度。加入20ug/mL人CD40L-his蛋白(Sino biological，#10239-H08E)，50μL/孔，4度孵育1小时后，用FACS缓冲液(1xPBS with 2％FBS)洗3次，加入1：400稀释的抗组氨酸标签的二抗(GenScript,#A01800)，4度孵育1小时后用FACS缓冲液洗2次，用预冷PBS重悬细胞。使用BD FACS CANTOII流式细胞仪或ACEA NovoCyte流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体浓度依赖的抑制率曲线及IC50值等参数。
如图7和表12所示，PR003379呈现出部分抑制CD40和CD40L结合的活性，最大抑制率为41.9％。
表12：CD40抗体抑制CD40和CD40L的结合

实施例9利用BLI方法测定抗体结合CD40的表位竞争
为了检测CD40抗体的结合表位，使用Octet Red 96e(Fortebio)分子相互作用分析仪通过BLI技术对CD40抗体Selicrelumab、APX005M、PR003379进行表位竞争实验。第一步，获取抗体的100％信号：用HIS1K传感器(Fortebio，#18-5120)捕获CD40蛋白(Sino Biological，#10774-H08H)，捕获高度为0.2nm。将传感器在缓冲液中平衡120秒后浸入稀释至140nM的各抗体中，时间220秒，将抗体与CD40结合的最终信号记录为该抗体的100％信号。第二步，表位竞争：用HIS1K传感器捕获CD40蛋白，捕获高度为0.2nm。将传感器浸入第一抗体中(浓度为140nM)，时间220秒，再将HIS1K传感器浸入第一抗体和第二抗体的混合物中(两种抗体的终浓度均为140nM)，时间220秒，将传感器浸入抗体混合物后的信号差，记录为该抗体作为第二抗体的信号。抑制率通过下式计算，
抑制率(％)＝(A–B)/A*100
A：某抗体的100％信号(从第一步中获得)，B：该抗体作为第二抗体的信号(从第二步获得)。
若得到的抑制率大于80％，则意味着两种抗体的表位几乎完全重叠；若抑制率大于40％且小于80％，则意味着两种抗体结合的表位不完全重叠或者表位很相近；若抑制率小于40％，则意味着两种抗体的表位不同。
结果如表13所示，PR003379和Selicrelumab的结合表位几乎一致，但是和APX005M的表位不同。
表13：BLI表位竞争实验
实施例10在MC38-hPD-L1/hCD40-C57BL/6J小鼠模型中的抗肿瘤活性和对小鼠体重的影响
为检测CD40抗体在体内的抗肿瘤功能，将过表达人PD-L1的MC38小鼠结肠癌细胞(MC38/hPD-L1，由上海南方模式生物公司提供)培养在37度，5％CO2的培养箱中，培养基为含10％灭活胎牛血清的DMEM培养基，细胞每隔3至4天长满即分皿传代。取对数生长期MC38/hPD-L1，重悬于PBS中，进行细胞计数，调整细胞浓度到1.0×107/mL；用1mL注射器将细胞悬液接种于CD40人源化转基因小鼠(hCD40-C57BL/6J，由上海南方模式生物公司提供)的右侧胁肋部皮下，100μL/只，每只接种约1.0×106个肿瘤细胞。当平均肿瘤体积达到114mm3时，挑选个体肿瘤体积适中的小鼠入组，将小鼠按肿瘤体积随机分配到各实验组中，每组6只，分组当天开始给药。
结果如图8A和图8B所示，给药开始后第10天，对照组IgG1(3mg/kg)平均肿瘤体积为1409.41±205.02mm3。Selicrelumab(3mg/kg)组、CDX-1104(3mg/kg)组、PR003379(3mg/kg)组的平 均肿瘤体积分别为460.96±68.61mm3、1345.22±162.94mm3、447.89±50.74mm3，肿瘤抑制率分别为73.22％、4.92％、74.24％。整个实验过程中，IgG1对照组、Selicrelumab、CDX-1104、PR003379小鼠的体重增长率分别为12.24％、7.31％、7.56％、1.86％。小鼠状态正常。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (18)

  1. 一种靶向CD40的抗原结合蛋白，所述抗原结合蛋白包含轻链可变区和重链可变区，所述轻链可变区(VL)包含LCDR1、LCDR2和LCDR3，所述重链可变区(VH)包含HCDR1、HCDR2和HCDR3；其中：
    所述LCDR1包括如SEQ ID NO:33所示的氨基酸序列，所述LCDR2包括如SEQ ID NO:41所示的氨基酸序列，所述LCDR3包括如SEQ ID NO:49所示的氨基酸序列或在SEQ ID NO:49上具有3、2或1个氨基酸突变的变体3；所述HCDR1包括如SEQ ID NO:7所示的氨基酸序列，所述HCDR2包括如SEQ ID NO:15所示的氨基酸序列，所述HCDR3包括如SEQ ID NO:23所示的氨基酸序列；
    较佳地，所述变体3包括在SEQ ID NO:49所示氨基酸序列上具有PTM位点突变的氨基酸序列，优选包括在SEQ ID NO:49所示氨基酸序列的第4位和/或第5位上发生氨基酸突变的氨基酸序列，所述氨基酸突变优选为氨基酸替代，更优选为氨基酸的保守替代。
  2. 如权利要求1所述的靶向CD40的抗原结合蛋白，其特征在于，所述变体3为如SEQ ID NO:50-73任一项所示的氨基酸序列；
    较佳地，所述LCDR1包括如SEQ ID NO:33所示的氨基酸序列，所述LCDR2包括如SEQ ID NO:41所示的氨基酸序列，所述LCDR3包括如SEQ ID NO:49-73任一项所示的氨基酸序列；所述HCDR1包括如SEQ ID NO:7所示的氨基酸序列，所述HCDR2包括如SEQ ID NO:15所示的氨基酸序列，所述HCDR3包括如SEQ ID NO:23所示的氨基酸序列。
  3. 如权利要求1或2所述的靶向CD40的抗原结合蛋白，其特征在于，所述轻链可变区包括如SEQ ID NO:85所示的氨基酸序列或与SEQ ID NO:85有至少85％、90％、92％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，所述重链可变区包括如SEQ ID NO:81所示的氨基酸序列或与SEQ ID NO:81有至少85％、90％、92％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；较佳地，所述轻链可变区包括如SEQ ID NO:85-109任一项所示的氨基酸序列，所述重链可变区包括如SEQ ID NO:81所示的氨基酸序列。
  4. 如权利要求1-3任一项所述的靶向CD40的抗原结合蛋白，其特征在于，所述靶向CD40的抗原结合蛋白满足以下三项中至少一项：
    (1)所述靶向CD40的抗原结合蛋白为全长抗体、Fab、Fab’、F(ab’)2或Fv，所述Fv优选为scFv；
    (2)所述靶向CD40的抗原结合蛋白为单特异性抗体、双特异性抗体或多特异性抗体；
    (3)所述靶向CD40的抗原结合蛋白为单克隆抗体或多克隆抗体；
    较佳地，所述靶向CD40的抗原结合蛋白为全长抗体，所述全长抗体包含轻链和重链；所述轻链包括轻链恒定区(CL)，所述轻链恒定区优选为人源抗体轻链恒定区；所述重链包括重链恒定区(CH)，所述重链恒定区优选为人源抗体重链恒定区，更优选为hIgG1、hIgG2、hIgG3或hIgG4亚型的重链恒定区，进一步优选为hIgG1亚型的重链恒定区。
  5. 如权利要求1-4任一项所述的靶向CD40的抗原结合蛋白，其特征在于，所述靶向CD40的抗 原结合蛋白为全长抗体，所述全长抗体包含轻链和重链，所述重链包括如SEQ ID NO:114或115所示的氨基酸序列，所述轻链包括如SEQ ID NO:119-143任一项所示的氨基酸序列；较佳地，所述重链包括如SEQ ID NO:114所示的氨基酸序列，所述轻链包括如SEQ ID NO:119所示的氨基酸序列；或，所述重链包括如SEQ ID NO:115所示的氨基酸序列，所述轻链包括如SEQ ID NO:119-143任一项所示的氨基酸序列。
  6. 一种嵌合抗原受体，其特征在于，所述嵌合抗原受体包含如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白。
  7. 一种分离的核酸，其特征在于，所述分离的核酸编码如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白或如权利要求6所述的嵌合抗原受体。
  8. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求7所述的分离的核酸；较佳地，所述重组表达载体包含真核细胞表达载体和/或原核细胞表达载体。
  9. 一种转化体，其特征在于，所述转化体包含如权利要求7所述的分离的核酸或如权利要求8所述的重组表达载体；较佳地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选为E.coli细胞如TG1、BL21，所述真核细胞优选HEK293细胞或CHO细胞。
  10. 一种如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白的制备方法，其包括以下步骤：培养如权利要求9所述的转化体，从培养物中获得所述靶向CD40的抗原结合蛋白。
  11. 一种抗体药物偶联物，其特征在于，所述抗体药物偶联物包含如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白和细胞毒性剂或标签；较佳地，所述细胞毒性剂为MMAF或MMAE，所述标签为荧光剂。
  12. 一种基因修饰的细胞，其特征在于，所述基因修饰的细胞表达如权利要求6所述的嵌合抗原受体；较佳地，所述基因修饰的细胞为真核细胞，优选分离的人细胞，更优选免疫细胞如T细胞，或NK细胞。
  13. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白、如权利要求7所述的分离的核酸、如权利要求8所述的重组表达载体，如权利要求11所述的抗体药物偶联物和如权利要求12所述的基因修饰的细胞中的一种或多种，以及药学上可接受的载体和/或药学上可接受的佐剂；较佳地，所述药物组合物还包括其他抗肿瘤抗体作为活性成分。
  14. 一种检测试剂，其特征在于，所述检测试剂包含如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白和/或如权利要求11所述的抗体药物偶联物；较佳地，所述检测试剂为液体剂型、气体剂型、固体剂型和半固体剂型；更佳地，所述检测试剂还包括二抗、CD40或其衍生物，所述二抗例如抗人IgG的抗体偶联辣根过氧化物酶和抗人IgG的抗体偶联生物素蛋白。
  15. 一种套装药盒，其特征在于，所述套装药盒包含药盒A，所述药盒A含有如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白、如权利要求13所述的药物组合物、如权利要求14所述的检测试剂、如权利要求11所述的抗体药物偶联物和如权利要求12所述的基因修饰的细胞中的一种或多种；
    较佳地，所述套装药盒还包括药盒B，所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  16. 如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白、如权利要求7所述的分离的核酸、如权利要求8所述的重组表达载体，如权利要求13所述的药物组合物、如权利要求14所述的检测试剂、如权利要求15所述的套装药盒、如权利要求11所述的抗体药物偶联物和如权利要求12所述的基因修饰的细胞中的一种或多种在制备诊断、预防和/或治疗肿瘤的药物中的用途；较佳地，所述肿瘤包括实体瘤和血液瘤；更佳地，所述肿瘤包括B系NHL、慢性淋巴细胞性白血病(CLL)、多毛细胞白血病(HCL)、霍奇金病、多发性骨髓瘤、膀胱癌、肾癌、卵巢癌、宫颈癌、乳腺癌、肺癌、鼻咽癌、恶性黑色素瘤、胰腺癌和结肠癌。
  17. 一种检测样品中CD40的方法，其特征在于，所述方法包括使用如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白、如权利要求14所述的检测试剂和/或如权利要求11所述的抗体药物偶联物与所述样品接触的步骤；较佳地，所述方法为非诊断和/或治疗目的。
  18. 一种诊断、治疗和/或预防肿瘤的方法，所述方法包括向有需要的患者施用治疗有效量的如权利要求1-5任一项所述的靶向CD40的抗原结合蛋白、如权利要求7所述的分离的核酸、如权利要求8所述的重组表达载体，如权利要求13所述的药物组合物、如权利要求14所述的检测试剂、如权利要求15所述的套装药盒、如权利要求11所述的抗体药物偶联物和如权利要求12所述的基因修饰的细胞中的一种或多种，或者使用如权利要求15所示的套装药盒诊断或治疗有需要的患者。较佳地，所述肿瘤包括实体瘤和血液瘤。更佳地，所述肿瘤包括B系NHL、慢性淋巴细胞性白血病(CLL)、多毛细胞白血病(HCL)、霍奇金病、多发性骨髓瘤、膀胱癌、肾癌、卵巢癌、宫颈癌、乳腺癌、肺癌、鼻咽癌、恶性黑色素瘤、胰腺癌和结肠癌。
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