WO2019091436A1 - 4-1bb抗体及其制备方法和应用 - Google Patents

4-1bb抗体及其制备方法和应用 Download PDF

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WO2019091436A1
WO2019091436A1 PCT/CN2018/114641 CN2018114641W WO2019091436A1 WO 2019091436 A1 WO2019091436 A1 WO 2019091436A1 CN 2018114641 W CN2018114641 W CN 2018114641W WO 2019091436 A1 WO2019091436 A1 WO 2019091436A1
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antibody
seq
variable region
chain variable
heavy chain
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PCT/CN2018/114641
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English (en)
French (fr)
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潘恒
刘浩涵
高营营
胡少平
李勇
潘明明
杨达志
段清
刘礼乐
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上海开拓者生物医药有限公司
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Priority to US16/762,871 priority Critical patent/US11555077B2/en
Priority to EP18876555.6A priority patent/EP3708582A4/en
Priority to JP2020544094A priority patent/JP7215759B2/ja
Publication of WO2019091436A1 publication Critical patent/WO2019091436A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to the field of biomedicine, and more particularly to 4-1BB antibodies and methods for their preparation and use.
  • 4-1BB also known as CD137, or TNFRF9, is a member of the TNF receptor family.
  • 4-1BB is a type I transmembrane protein with a reading frame of 255 amino acids (NCBI: NP_001552), consisting of a 17-amino acid N-terminal signal peptide, a 169 amino acid extracellular region, a 27 amino acid transmembrane region and The 42-amino acid C-terminal intracellular domain is composed.
  • 4-1BB molecules are mainly expressed in activated T cells, NK cells, regulatory T cells, dendritic cells, monocytes, neutrophils and eosinophils. Endothelial cells of tumor blood vessels have also been reported to express 4-1BB. .
  • the 4-1BB molecule provides a costimulatory signal during the activation of T cells.
  • the T cell receptor contacts the antigen, it will increase the expression of 4-1BB.
  • 4-1BB binds to the ligand, it will activate the NF ⁇ B signaling pathway, which leads to the activation and proliferation of T cells, and 4-1BB can also inhibit. Activate apoptosis.
  • Animal models and in vitro experiments confirmed that anti-4-1BB agonistic monoclonal antibodies have antitumor activity. It can selectively induce the proliferation of CD8+ T cells, up-regulate the expression of the pro-inflammatory cytokine IFN- ⁇ , and enhance the killing effect of antigen-specific effector T cells, thereby promoting tumor clearance.
  • Anti-4-1BB agonistic monoclonal antibodies also cause expansion of NK cells and can increase the cytotoxic activity of CD8+ T cells.
  • Anti-4-1BB agonistic antibodies can also cause endothelial cells of tumor cells to up-regulate the expression of adhesion molecules and promote the infiltration of activated T lymphocytes into tumor tissues.
  • anti-4-1BB activating antibodies can also alleviate autoimmune diseases such as autoimmune encephalomyelitis, lupus-like syndrome and collagen-induced arthritis.
  • the existing 4-1BB antibody in the art has many shortcomings in antigen binding activity, activation of 4-1BB molecular downstream signal, etc., and there is no widely used 4-1BB antibody drug product, and 4-1BB agonistic antibody is developed. It is an urgent need to treat cancer as well as autoimmune diseases.
  • the object of the present invention is to provide a high affinity 4-1BB antibody and a preparation method and application thereof.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes 1 amino acid and is capable of retaining 4-1BB binding affinity.
  • the heavy chain variable region comprises the following three complementarity determining region CDRs:
  • any one of the above amino acid sequences further comprises a derivative sequence which optionally adds, deletes, modifies and/or substitutes 1 amino acid and is capable of retaining 4-1BB binding affinity.
  • the amino acid sequence of CDR3 comprises a derivative sequence that optionally adds, deletes, modifies, and/or substitutes 1 amino acid and is capable of retaining 4-1BB binding affinity.
  • the heavy chain variable region further comprises a FR region of a human source or a FR region of a murine source.
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO.: 1 or SEQ ID NO.: 9.
  • a heavy chain of an antibody having a heavy chain variable region according to the first aspect of the invention.
  • the heavy chain of the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region is of human, murine or rabbit origin.
  • a light chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • CDR3' represented by SEQ ID NO.: 8 or SEQ ID NO.: 16.
  • the light chain variable region comprises the following three complementarity determining region CDRs:
  • the light chain variable region further comprises a human FR region or a murine FR region.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO.: 5 or SEQ ID NO.: 13.
  • a light chain of an antibody having a light chain variable region according to the third aspect of the invention.
  • the light chain of the antibody further comprises a light chain constant region.
  • the light chain constant region is of human, murine or rabbit origin.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
  • the antibody affinity for the NF ⁇ B transcription factor EC 50 is 0.1-5nM.
  • the antibody affinity for the postcrosslinking of NF ⁇ B transcription factor EC 50 is 0.4-1nM
  • the NF ⁇ B transcription factor is located downstream of the human 4-1BB protein.
  • the antibody is selected from the group consisting of an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.
  • the antibody is a diabody, or a single chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a partially or fully humanized monoclonal antibody.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO.: 1; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 5.
  • the heavy chain variable region sequence of the antibody is set forth in SEQ ID NO.: 9; and the light chain variable region sequence of the antibody is set forth in SEQ ID NO.: 13.
  • the antibody is in the form of a drug conjugate.
  • a recombinant protein having:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein comprises a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • a CAR construct wherein the scFV segment of the monoclonal antibody antigen-binding region of the CAR construct is a binding region that specifically binds to 4-1BB, and
  • the scFv has a heavy chain variable region according to the first aspect of the invention and a light chain variable region according to the third aspect of the invention.
  • a recombinant immune cell characterized in that said immune cell expresses an exogenous CAR construct according to the seventh aspect of the invention.
  • the immune cells are selected from the group consisting of NK cells, T cells.
  • the immune cells are from a human or non-human mammal (e.g., a mouse).
  • the invention provides an antibody drug conjugate, characterized in that the antibody drug conjugate comprises:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody moiety is coupled to the coupling moiety via a chemical bond or linker.
  • an active ingredient selected from the group consisting of the heavy chain variable region according to the first aspect of the invention, the weight according to the second aspect of the invention a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, the recombinant according to the sixth aspect of the invention A protein, an immune cell according to the eighth aspect of the invention, the antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent or A kit; and/or (b) a medicament for the prevention and/or treatment of a disease associated with 4-1BB.
  • the 4-1BB-related disease is selected from the group consisting of cancer, autoimmune disease, viral infection, graft versus host disease, inflammatory disease, immune disease, or a combination thereof.
  • the cancer comprises a solid tumor, a blood cancer.
  • the solid tumor is selected from the group consisting of bladder cancer, biliary tract cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer. Cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary cancer, gastric cancer, thymic epithelial cancer, and thyroid cancer, or a combination thereof.
  • the autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, or a combination thereof.
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is used to diagnose a 4-1BB related disease.
  • the detection reagent or kit is used to detect 4-1BB in a sample.
  • the detection reagent is a test piece.
  • a pharmaceutical composition comprising:
  • an active ingredient the active ingredient being selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, according to the third aspect of the invention a light chain variable region, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, according to the eighth aspect of the invention Immunocyte, an antibody drug conjugate according to the ninth aspect of the invention, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:
  • nucleic acid encoding the heavy chain variable region is set forth in SEQ ID NO.: 106 and/or the nucleic acid encoding the light chain variable region is set forth in SEQ ID NO.: 107.
  • nucleic acid encoding the heavy chain variable region is as set forth in SEQ ID NO.: 108 and/or the nucleic acid encoding the light chain variable region is set forth in SEQ ID NO.: 109 Show.
  • the invention provides a vector comprising the polynucleotide of the twelfth aspect of the invention.
  • the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
  • a genetically engineered host cell comprising the vector or genome according to the thirteenth aspect of the present invention, which is integrated with the twelfth aspect of the present invention, is provided Polynucleotide.
  • a method of in vitro detection (including diagnostic or non-diagnostic) of 4-1BB in a sample comprising the steps of:
  • a test panel comprising: a substrate (support plate) and a test strip, the test strip comprising the antibody according to the fifth aspect of the invention or The immunoconjugate of the ninth aspect of the invention.
  • a kit of the present invention characterized in that the kit comprises:
  • a first container comprising the antibody of the fifth aspect of the invention.
  • the kit contains the test plate according to the sixteenth aspect of the invention.
  • a method for preparing a recombinant polypeptide comprising:
  • a nineteenth aspect of the invention provides a method of treating a disease associated with 4-1BB, characterized in that the method comprises: administering to a subject in need thereof the antibody according to the fifth aspect of the invention, the antibody An antibody-drug conjugate, or a CAR-T cell expressing the antibody, or a combination thereof.
  • a method for detecting a cell overexpressing a 4-1BB protein comprising the steps of: contacting the antibody according to the fifth aspect of the present invention with a sample to be tested, detecting the antibody and The combination of samples to be tested.
  • composition for detecting a cell overexpressing a 4-1BB protein comprising the antibody of the fifth aspect of the invention as an active ingredient.
  • the technical problem to be solved by the present invention is to overcome the shortcomings of the current lack of 4-1BB antibody, especially the humanized 4-1BB antibody, and to provide a 4-1BB antibody with high specificity and high biological activity and a preparation method thereof.
  • the 4-1BB antibody has high affinity with human and monkey-derived 4-1BB proteins, and can activate downstream signals of 4-1BB molecules, and can significantly increase IFN- ⁇ in human mixed lymphocyte or T lymphocyte reaction. And the expression level of IL-2.
  • the present invention first prepares human 4-1BB as an immunogen, and adopts a human antibody transgenic mouse technique to prepare a fully human antibody, and obtains a lead antibody of the 4-1BB antibody. Through the preliminary production, purification and verification of the lead antibody, it can effectively stimulate the 4-1BB receptor and activate its downstream signal, resulting in an excellent increase in the expression levels of IFN- ⁇ and IL-2 in the human T lymphocyte reaction.
  • Biological level antibodies The amino acid sequences of the heavy chain variable region and the light chain variable region of the 4-1BB antibody were then sequenced by molecular biological methods.
  • Figure 1 shows the binding activity of the 4-1BB-hFc protein to the fusion protein his-muCD8a-4-1BBL.
  • Figure 2 shows FACS screening assay of 4-1BB protein transfected HEK293 cells.
  • Figure 3 shows the serum antibody titer of Harbour transgenic mice after ELISA for 4-1BB immunization.
  • Figures 4A-4H show ELISA assays for binding of individual 4-1BB fully human antibodies to human 4-1BB-hFc protein, respectively.
  • Figures 5A and 5B show the ELISA assay for the binding of each 4-1BB fully human antibody to the monkey 4-1BB-hFc protein, respectively.
  • Figure 6A and Figure 6B show the binding reaction of each 4-1BB fully human antibody to the monkey 4-1BB-hFc protein by ELISA, respectively.
  • Figure 7 shows the ELISA assay for the binding of 4-1BB fully human antibody to other immunological checkpoint proteins.
  • Figures 8A-8E show FACS detection of the binding reaction of each 4-1BB fully human antibody to CHOk1-h4-1BB, respectively.
  • Figures 9A-9D show FACS detection of the binding reaction of each 4-1BB fully human antibody to CHOk1-c4-1BB, respectively.
  • Figure 10A and Figure 10B show reporter gene experiments for each 4-1BB full human antibody activating the NF- ⁇ B downstream promoter, respectively.
  • Figure 11 shows that the 4-1BB fully human antibody blocked the binding reaction of the 4-1BB protein to its ligand 4-1BBL.
  • Figure 12 shows the effect of 4-1BB fully human antibody on IFN- ⁇ secretion in a T lymphocyte stimulation assay.
  • Figure 13 shows the effect of 4-1BB fully human antibody on IL-2 secretion in a T lymphocyte stimulation assay.
  • Figure 14 shows the results of a comparative test using Pfizer's Utomilumab antibody as a comparative antibody.
  • Figure 15 shows the results of an experiment in which 4-1BB antibody grows on mouse MC38 tumors.
  • Figure 16 shows the tumor volume of individual mice in different antibody treatment groups.
  • the present inventors have extensively and intensively studied, and for the first time, unexpectedly discovered a 4-1BB antibody having high affinity and specificity, and based on the antibody, a fully humanized antibody was obtained.
  • the antibody of the invention has high affinity to the 4-1BB protein, can effectively activate the downstream signal of 4-1BB, and significantly increases the expression levels of IFN- ⁇ and IL-2 in human mixed lymphocytes or T lymphocytes, and can be used for treating cancer. And autoimmune diseases.
  • the present invention has been completed on this basis.
  • conjugate refers to a soluble receptor or fragment thereof or analog thereof, or an antibody or fragment thereof, or an analog thereof, that is capable of binding to a target.
  • 4-1BB conjugate As used herein, the terms "4-1BB conjugate”, “4-1BB antibody”, “anti-4-1BB antibody”, “antibody of the invention” have the same meaning, and are capable of specifically recognizing 4-1BB and with 4- 1BB-bound antibody or fragment thereof or analog thereof.
  • administering and “treating” mean that the exogenous drug, therapeutic, diagnostic, or composition is applied to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering and “treatment” can refer to treatment, pharmacokinetics, diagnostics, research, and experimental methods.
  • the treatment of the cells includes contact of the reagents with the cells, contact of the reagents with the fluid, and contact of the fluid with the cells.
  • administering and “treatment” also mean in vitro and ex vivo treatment by reagents, diagnostics, binding compositions or by another cell.
  • Treatment when applied to a human, animal or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnosis; includes 4-1BB conjugates with humans or animals, subjects, cells, tissues, Contact with physiological compartments or physiological fluids.
  • treating refers to the administration of a therapeutic agent for internal or external use to a patient, comprising any of the 4-1BB conjugates of the invention, and compositions thereof, which have one or more symptoms of the disease, and are known Therapeutic agents have a therapeutic effect on these symptoms.
  • the patient is administered an amount (a therapeutically effective amount) of a therapeutic agent effective to relieve one or more of the symptoms of the disease.
  • antibody refers to an immunoglobulin that is a tetrapeptide chain structure formed by the joining of two identical heavy chains and two identical light chains by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or different types of immunoglobulins, namely, IgM, IgD, IgG, IgA, and IgE, and the heavy chain constant regions corresponding to different classes of immunoglobulins are respectively referred to as ⁇ . , ⁇ , ⁇ , ⁇ , and ⁇ .
  • IgG stands for the most important class of immunoglobulins and can be divided into four subclasses due to differences in chemical structure and biological function: IgG1, IgG2, IgG3 and IgG4.
  • Light chains are divided into kappa or lambda chains by the constant region.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved FR regions (FR). The amino acid sequences of the four FRs are relatively conservative and are not directly involved in the binding reaction.
  • the three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and 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 i.e., the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2, and LCDR3;
  • the three CDR regions of the heavy chain, the heavy chain hypervariable region (HCDR) refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention conform to known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or in accordance with the kabat and chothia numbering rules (HCDR1) ).
  • the four FR regions in the native heavy and light chain variable regions are generally in a beta-sheet configuration, joined by three CDRs forming a linker, and in some cases may form a partial beta sheet structure.
  • the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody.
  • the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
  • the term "antigen-binding fragment” refers to a Fab fragment having an antigen-binding activity, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment.
  • the Fv antibody contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • antigenic determinant refers to a three-dimensional spatial site that is discrete on an antigen and that is recognized by an antibody of the invention or an antigen-binding fragment.
  • the present invention encompasses not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using recombinant DNA techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody that is secreted from a clone of a single cell source. Monoclonal antibodies are highly specific and target a single epitope.
  • the cell may be a eukaryotic, prokaryotic or phage clonal cell line.
  • chimeric antibody is obtained by splicing a V region gene of a murine antibody with a C region gene of a human antibody into a chimeric gene, and then inserting the vector into an antibody molecule expressed by the host cell. It not only retains the high specificity and affinity of the parental mouse antibody, but also enables its human Fc segment to effectively mediate biological effects.
  • humanized antibody is a variable region engineered form of a murine antibody of the invention having a CDR region derived from (or substantially derived from) a non-human antibody, preferably a mouse monoclonal antibody. And FR regions and constant regions derived substantially from human antibody sequences; the CDR region sequences of the murine antibodies are grafted onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing an expression vector.
  • the antibody may be monospecific, bispecific, trispecific, or more multiple specificity.
  • the antibody of the present invention further includes a conservative variant thereof, which means that there are up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of the antibody of the present invention. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.
  • substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala
  • 4-1BB also known as CD137, or TNFRF9, is a member of the TNF receptor family.
  • 4-1BB is a type I transmembrane protein with a reading frame of 255 amino acids (NCBI: NP_001552), consisting of a 17-amino acid N-terminal signal peptide, a 169 amino acid extracellular region, a 27 amino acid transmembrane region and The C-terminal intracellular region of 42 amino acids is composed, and the specific sequence can be seen in SEQ ID NO: 22 of the present patent.
  • 4-1BB generally refers to natural or recombinant human 4-1BB, as well as non-human homologs of human 4-1BB.
  • the molar concentration of 4-1BB was calculated using the molecular weight of the homodimer of 4-1BB unless otherwise indicated.
  • human 4-1BB includes the mature form of human 4-1BB protein and its natural variants and polymorphisms.
  • the present invention provides a high affinity antibody against 4-1BB comprising a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) amino acid sequence, the light chain comprising a light chain variable region ( VL) amino acid sequence.
  • VH heavy chain variable region
  • VL light chain variable region
  • the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the group consisting of:
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 90%, more preferably at least 95. %, optimally at least 99% of the amino acid sequence.
  • the number in Table 1 is the sequence number "SEQ ID NO.”
  • the amino acid sequence of the heavy chain protein variable region of 57B3D10 is as shown in SEQ ID NO.: 1
  • the heavy chain protein of 113F6C6 is variable.
  • the amino acid sequence of the CDR1 domain in the region is as shown in SEQ ID NO.: 2.
  • the inventors also targeted 4-1BB of the clones: 11H10C9, 15G10D4, 23G3B8, 52F4G2, 118F3A2, 170D7F2, 172E3E3, 178D10D11, 182A5B3, 100F3C4, 119B6G5, 258F5A8, 259G10E11, 263A11E3, 289B6G6 during antibody screening.
  • relevant experiments were carried out, and since the respective performances were lower than those of the clones 57B3D10 and 113F6C6, no further experiments were carried out, and the results of the detailed experiments are shown in the examples.
  • the antibody of the present invention may be an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody fragment (scFv), a single-domain antibody (sdAb). And one or more of a single-domain antibody (Signle-domain antibody), and a monoclonal antibody or a polyclonal antibody produced by the above antibody.
  • the monoclonal antibodies can be developed by a variety of pathways and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc.
  • the mainstream is the preparation of monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.
  • the full-length antibody protein is a conventional full-length antibody of the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
  • the heavy chain variable region and the light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full human antibody full length protein.
  • the full length protein of the antibody is IgG1, IgG2, IgG3 or IgG4.
  • the single-chain antibody is a conventional single-chain antibody in the art, which comprises a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids.
  • the antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment of the art comprising a light chain variable region, a light chain constant region, and an Fd segment of a heavy chain constant region.
  • the antigen-antibody binding domain protein fragments are Fab and F(ab').
  • the single domain antibodies are conventional single domain antibodies in the art which include heavy chain variable regions and heavy chain constant regions.
  • the single region antibodies are conventional single region antibodies of the art which include only heavy chain variable regions.
  • the antibody of the present invention may be a double-stranded or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, more preferably a humanized antibody, a human-animal chimeric antibody, and more preferably For fully humanized antibodies.
  • the antibody derivative of the present invention may be a single chain antibody, and/or an antibody fragment such as Fab, Fab', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, IgE. Any one or more of IgG and IgM antibodies or antibodies of other subtypes.
  • the animal is preferably a mammal, such as a mouse.
  • the antibody of the invention may be a murine antibody, chimeric antibody, humanized antibody, CDR grafted and/or modified antibody that targets human 4-1BB.
  • the above SEQ ID NO.: 2, 3 and 4, or any one or more of SEQ ID NO.: 6, 7 and 8, or they are added, deleted, A sequence having 4-1BB binding affinity for modifying and/or substituting at least one amino acid, located in the CDR region of the heavy chain variable region (VH).
  • VH heavy chain variable region
  • the above SEQ ID NO.: 10, 11 and 12, or any one or more of SEQ ID NO.: 14, 15 and 16, or they are added, deleted, A sequence having 4-1BB binding affinity for modifying and/or substituting at least one amino acid, located in the CDR region of the light chain variable region (VL).
  • VL light chain variable region
  • the number of amino acids added, deleted, modified and/or substituted is preferably not more than 30%, more preferably not more than 20%, more preferably 1-15% of the total amino acid number of the initial amino acid sequence. More preferably, it is 1-10%.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2. The best is 1.
  • the invention also provides a nucleic acid encoding the above polypeptide.
  • the nucleic acid encoding the heavy chain variable region is represented by SEQ ID NO.: 106 of the Sequence Listing or SEQ ID NO.: 108 of the Sequence Listing; and/or nucleic acid encoding the variable region of the light chain
  • the nucleotide sequence is shown in SEQ ID NO.: 107 of the Sequence Listing or SEQ ID NO.: 109 of the Sequence Listing.
  • nucleic acid encoding the heavy chain variable region is set forth in SEQ ID NO.: 106, and the nucleic acid encoding the light chain variable region is set forth in SEQ ID NO.: 107;
  • the nucleic acid of the heavy chain variable region is set forth in SEQ ID NO.: 108 of the Sequence Listing, and the nucleic acid encoding the variable region of the light chain is set forth in SEQ ID NO.: 109 of the Sequence Listing.
  • the number in Table 2 is the sequence number "SEQ ID NO.”
  • the nucleotide sequence of the heavy chain protein variable region encoding 57B3D10 is SEQ ID NO.: 106 of the Sequence Listing.
  • nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 57B3D10 is the 91st to 105th positions in SEQ ID NO.:106 of the Sequence Listing;
  • the nucleotide sequence encoding the CDR2 domain of the heavy chain protein variable region of 57B3D10 is from positions 148 to 195 of SEQ ID NO.: 106;
  • nucleotide sequence of the CDR3 domain of the heavy chain protein variable region encoding 57B3D10 is position 292 to 342 of SEQ ID NO.: 106 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain of the light chain protein variable region encoding 57B3D10 is from position 67 to position 102 of SEQ ID NO.: 107;
  • the nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 57B3D10 is from positions 148 to 168 of SEQ ID NO.: 107;
  • nucleotide sequence of the CDR3 domain of the light chain protein variable region encoding 57B3D10 is position 265 to 288 of SEQ ID NO.: 107 of the Sequence Listing;
  • the nucleotide sequence of the CDR1 domain in the heavy chain protein variable region encoding 113F6C6 is position 91 to 105 in SEQ ID NO.: 108 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain in the heavy chain protein variable region encoding 113F6C6 is 148th to 198th in the sequence listing SEQ ID NO.:108;
  • the nucleotide sequence of the CDR3 domain in the heavy chain protein variable region encoding 113F6C6 is from positions 295 to 351 of SEQ ID NO.: 108;
  • the nucleotide sequence of the CDR1 domain in the light chain protein variable region encoding 113F6C6 is the 70th to 102nd positions in SEQ ID NO.: 109 of the Sequence Listing;
  • the nucleotide sequence of the CDR2 domain of the light chain protein variable region encoding 113F6C6 is from positions 148 to 168 of SEQ ID NO.: 109 of the Sequence Listing;
  • the nucleotide sequence of the CDR3 domain in the light chain protein variable region encoding 113F6C6 is from position 265 to position 291 in SEQ ID NO.: 109 of the Sequence Listing;
  • the preparation method of the nucleic acid is a preparation method conventional in the art, and preferably includes the steps of obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial total sequence synthesis. .
  • the base sequence encoding the amino acid sequence of the above protein may be appropriately introduced with a substitution, deletion, alteration, insertion or addition to provide a homologue of a polynucleotide.
  • a homologue of a polynucleotide of the invention can be made by replacing, deleting or increasing one or more bases encoding a gene of the protein sequence within the range of activity of the antibody.
  • any method suitable for the production of monoclonal antibodies can be used to produce the anti-4-1BB antibodies of the invention.
  • an animal can be immunized with a 4-1BB homodimer or a fragment thereof that is ligated or naturally occurring.
  • Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • any suitable form of 4-1BB can be used as an immunogen (antigen) for the production of non-human antibodies specific for 4-1BB, and the biological activity of the antibodies is screened.
  • the stimulating immunogen can be a full length mature human 4-1BB, including a native homodimer, or a peptide containing a single/multiple epitope.
  • the immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art.
  • the immunogen can be purified from natural sources or produced in genetically modified cells.
  • the DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in its source.
  • the DNA encoding the immunogen can be expressed using a suitable genetic vector including, but not limited to, an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a material, and a non-viral vector.
  • a suitable genetic vector including, but not limited to, an adenoviral vector, an adeno-associated viral vector, a baculovirus vector, a material, and a non-viral vector.
  • Example 1 An exemplary method of producing an anti-human 4-1BB antibody of the invention is described in Example 1.
  • the fully humanized antibody can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA, and IgE.
  • the antibody is an IgG antibody, and an IgG4 subtype is used. Optimization of the necessary constant domain sequences is readily achieved by screening antibodies using the biological assays described in the Examples below to produce the desired biological activity.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.
  • Example 3 An exemplary method of humanizing an anti-human 4-1BB antibody of the invention is described in Example 3.
  • sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell is a variety of host cells conventional in the art, as long as it satisfies the stable self-replication of the above recombinant expression vector, and the nucleic acid carried can be efficiently expressed.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Preferred animal cells include, but are not limited to, CHO-S, CHO-K1, HEK-293 cells.
  • Preferred host cells include E. coli TG1 or BL21 cells (expressing single-chain antibodies or Fab antibodies), or CHO-K1 cells (expressing full-length IgG antibodies)
  • the step of transforming a host cell with recombinant DNA as described in the present invention can be carried out by techniques well known in the art.
  • the obtained transformant can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.
  • the resulting host cells are cultured under conditions suitable for expression of the antibody of the invention.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
  • the antibodies of the present invention are purified by conventional separation and purification means well known to those skilled in the art.
  • the resulting monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the invention provides the use of an antibody of the invention, for example, for the preparation of a diagnostic preparation, or for the preparation of a medicament for the prevention and/or treatment of a 4-1BB related disease.
  • the 4-1BB related diseases include cancer, autoimmune diseases, viral infections, graft versus host diseases, inflammatory diseases, immune diseases, or a combination thereof.
  • the cancer comprises solid tumor, blood cancer, and the solid tumor is selected from the group consisting of bladder cancer, biliary cancer, brain cancer, breast cancer, colon cancer, esophageal cancer, gastric cancer, glioma, head cancer.
  • Immune diseases include: systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis, or a combination thereof.
  • the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof or an ADC thereof or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the antibody of the present invention may also be a cell therapy for expression of a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used for binding to a 4-1BB protein molecule, and thus can be used for the prevention and treatment of diseases associated with 4-1BB.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 100 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the pharmaceutical composition is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight.
  • the dose is from about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the antibodies of the invention can be used in detection applications, for example for detecting samples, to provide diagnostic information.
  • the sample (sample) used includes cells, tissue samples, and biopsy specimens.
  • biopsy shall include all types of biopsies known to those skilled in the art.
  • the biopsy used in the present invention may include a tissue sample prepared, for example, by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.
  • the antibody of the invention may be immobilized on a test plate.
  • the present invention also provides a method for detecting cells overexpressing a 4-1BB protein, comprising the steps of: contacting the above-mentioned protein with a sample to be tested in vitro, and detecting the binding of the protein to the sample to be tested.
  • overexpression is conventional in the art and refers to the overexpression of RNA or protein of the 4-1BB protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and protein degradation), and Local overexpression and increased functional activity due to changes in protein transport patterns (increased nuclear localization) (as in the case of increased enzymatic hydrolysis of the substrate).
  • the detection mode of the combination is a conventional detection method in the art, preferably FACS detection.
  • the present invention provides a composition for detecting a cell overexpressing a 4-1BB protein, which comprises the above-described protein as an active ingredient.
  • a composition for detecting a cell overexpressing a 4-1BB protein which comprises the above-described protein as an active ingredient.
  • it further comprises a compound consisting of functional fragments of the above proteins as an active ingredient.
  • the reagents and starting materials used in the present invention are commercially available.
  • the protein of the present invention is a fully human 4-1BB antibody having high affinity with the 4-1BB protein, capable of binding to the extracellular region of the 4-1BB protein receptor, and capable of activating 4-1BB molecules at the cellular level. Downstream signal.
  • the mixed lymphocyte assay and the T cell stimulation assay demonstrated that the 4-1BB antibody has good biological activity, and it significantly increases the expression levels of IFN- ⁇ and IL-2 in human mixed lymphocytes or T lymphocytes.
  • the 4-1BB antibody has excellent properties including binding to human 4-1BB protein and regulation of human lymphocyte immune response.
  • the room temperature described in the examples is room temperature conventional in the art, and is generally from 10 to 30 °C.
  • the PBS described in the examples was a PBS phosphate buffer, pH 7.2.
  • the human 4-1BB protein extracellular domain amino acid sequence Leu24-Gln186 (shown in SEQ ID NO.: 21) was cloned into the pCpC vector carrying the human IgG Fc fragment (hFc) (purchased from Invitrogen, V044-50) Plasmids were prepared according to established standard molecular biology methods. In HEK293 cells (purchased from Invitrogen) for transient transfection (PEI, Polysciences), using FreeStyle TM 293 (Invitrogen) were expanded at 37 °C. After 4 days, the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing the extracellular region of the 4-1BB protein.
  • hFc human IgG Fc fragment
  • the culture supernatant was applied to a Protein A affinity chromatography column (Mabselect Sure, available from GE Healthcare) while monitoring the change in ultraviolet absorption value (A280 nm) with an ultraviolet (UV) detector.
  • the protein A affinity column was washed with PBS phosphate buffer (pH 7.2) until the UV absorbance value returned to the baseline, and then eluted with 0.1 M glycine hydrochloride (pH 2.5) to collect affinity from protein A.
  • the hFc-tagged 4-1BB protein (4-1BB-hFc) eluted on the column was dialyzed against a PBS phosphate buffer (pH 7.2) at 4 ° C overnight.
  • the dialyzed protein was sterile-filtered at 0.22 ⁇ m and stored at -80 ° C to obtain purified immunogen A.
  • Immunogen A requires a series of quality control tests before use, such as detection of protein concentration, purity, molecular weight and biological activity, and the results are shown in Figure 1 and Table 3.
  • Table 3 illustrates that the binding of 4-1BB to its ligand protein 4-1BBL at the protein level varies with the concentration of 4-1BBL, where the control protein is a non-4-1BB fusion protein and the data in the table is the OD 450 nm value.
  • the immunogenic A biological activity is detected by ELISA, specifically:
  • the hFc-tagged 4-1BB extracellular domain protein (4-1BB-hFc) was diluted to 0.5 ⁇ g/mL with PBS, added to an ELISA microplate at 100 ⁇ l/well, and incubated overnight at 4 °C. After blocking with ELISA blocking solution (containing 1% BSA, PBS phosphate buffer pH 7.4, the percentage is mass%) at 37 ° C for two hours, add gradient dilution of his-muCD8a-4-1BBL ECD fusion protein 37 ° C Incubate for 1 hour.
  • ELISA blocking solution containing 1% BSA, PBS phosphate buffer pH 7.4, the percentage is mass
  • His-muCD8a-4-1BBL ECD is fused by the extracellular domain of 4-1BB ligand 4-1BBL (Arg71-Glu254 of NCBI sequence NP_003802.1) and the extracellular domain of mouse CD8a (Lys28-Asp194 of NCBI sequence NP_001074579.1) Composition, his tag is at the N end.
  • Biotinylated rat anti-mouse CD8a antibody was then added and incubated for 1 hour at 37 °C.
  • Streptavidin-labeled horseradish peroxidase purchased from Sigma trade number S2438, was added, and after incubation for 30 minutes at room temperature, 100 ⁇ l/well of TMB color developing solution was added. After incubation for 15 minutes at room temperature, the color reaction was stopped by the addition of 50 ⁇ l of 1N hydrochloric acid, and the OD 450 nm reading was read using an ELISA plate reader.
  • the human 4-1BB full-length amino acid sequence (shown in SEQ ID NO.: 23 of the Sequence Listing) was cloned into the pIRES vector (purchased from Clontech) and plasmids were prepared. Plasmid transfection of HEK293 cell line and CHOK1 cell line (both purchased from Invitrogen) (transfection using X-treme GENE HP DNA Transfection Reagent, available from Roche, Cat. No.
  • the cells were selectively cultured in DMEM medium containing 0.5 ⁇ g/mL of 10% (w/w) FBS for 2 weeks, subcloned in a 96-well culture plate by limiting dilution, and placed at 37 ° C, 5%. (v/v) CO 2 culture. After approximately 2 weeks, a portion of the monoclonal wells were selected for amplification into 6-well plates. The amplified clones were subjected to screening by flow cytometry using a known 4-1BB antibody (purchased from BD).
  • the cell line with good growth and high fluorescence intensity is selected, and the monoclonal cell line continues to expand and the liquid nitrogen is frozen, that is, the immunogen B is obtained.
  • the specific selection results are shown in Table 4 and Figure 2.
  • the positive cells (%) in Table 4 refer to the percentage of positive cells in the total number of cells. Table 4 illustrates that a series of HEK293 cell lines with positive expression of 4-1BB have been made.
  • the Harbour transgenic mouse introduced the human immunoglobulin variable region gene and the rat immunoglobulin constant region gene, while the mouse Ig expression was silenced.
  • the transgenic mouse is immunized with an antigen to produce an immune response and antibody titer comparable to that of a normal mouse (e.g., Balb/c).
  • Immunogen A immunization 6 to 8 weeks old Harbour human antibody transgenic mice (purchased from Beijing Vital Lihua Co., Ltd.) were used, and the mice were raised under SPF conditions.
  • the immunogen (1) protein was emulsified with Freund's complete adjuvant and intraperitoneally injected with 0.25 ml, that is, 100 ⁇ g of immunogen A protein was injected per mouse.
  • the immunogen A protein was emulsified with Freund's incomplete adjuvant and intraperitoneally injected with 0.25 ml, that is, 50 ⁇ g of immunogen A protein was injected per mouse.
  • the cells were washed twice with DMEM basal medium, and after cell counting, the cells were diluted with phosphate buffer (pH 7.2) to 2 ⁇ 10 7 cells per ml. Each mouse was intraperitoneally injected with 0.5 ml of cell suspension per immunization. The first and second immunizations were separated by 2 weeks, and each subsequent immunization interval was 3 weeks. In addition to the first immunization, blood was collected 1 week after each immunization, and antibody titer and specificity in serum were measured by FACS. After secondary booster immunization, the serum antibody titer of FACS is usually above 1:1000.
  • the FACS titer of most mice after 3 immunizations can reach 1:1000 or more.
  • each mouse selected was intraperitoneally injected with 100 ⁇ g of purified 4-1BB-hFc (mouse against immunogen A and immunogen C) or human source 4 -1BB HEK293-h4-1BB stable cell line (mouse that immunoreacted against immunogen B), mice were sacrificed 5 days later, and spleen cells were collected. NH 4 OH was added to a final concentration of 1% (w/w), and the erythrocytes mixed in the spleen cells were lysed to obtain a spleen cell suspension.
  • the cells were washed three times with DMEM basal medium at 1000 rpm, then mixed with mouse myeloma cells SP2/0 (purchased from ATCC) at a ratio of 5:1 viable cells, and cell fusion was performed by high-efficiency electrofusion or PEG method. .
  • the fused cells were diluted into DMEM medium containing 20% fetal calf serum, 1 ⁇ HAT, and the percentage was a mass percentage. Then, 1 ⁇ 10 5 / 20 ⁇ l per well was added to a 96-well cell culture plate and placed in a 5% CO 2 , 37 ° C incubator, the percentage being a volume percentage.
  • the cell fusion plates were screened by ELISA and Acumen (microplate assay), and positive clones with OD 450nm >1.0 in ELISA and MFI values >100 in Acumen were amplified into 24-well plates at 10% (in 10%).
  • w/w) fetal bovine serum in DMEM (Invitrogen) medium was expanded at 37 ° C under 5% (v/v) CO 2 conditions. After culturing for 3 days, the culture medium expanded in a 24-well plate was centrifuged, and the supernatant was collected, and the supernatant was subjected to antibody subtype analysis.
  • the binding activity of 4-1BB protein and 4-1BB positive cells was determined by ELISA and FACS (for the detection method of binding activity, please refer to Example 5A and Example 5B, respectively), and then determined by NF- ⁇ B luciferase reporter gene assay.
  • the activity of the antibody sample on 4-1BB receptor activation see Example 5 for the detection method).
  • OD 450nm >1.0 in the ELISA experiment, MFI value >50 in the FACS experiment, and the hybridoma cell culture supernatant in the NF- ⁇ B luciferase reporter gene experiment will be 4-1BB compared to the control IgG group.
  • Hybridoma cells with receptor activation above 1.0-fold were eligible positive clones.
  • Selected hybridoma cells were subcloned in a 96-well plate by limiting dilution in DMEM medium containing 10% (w/w) FBS (purchased from Invitrogen) at 37 ° C, 5% (v/v) CO Culture under 2 conditions.
  • the positive clones were expanded in DMEM (purchased from Invitrogen) medium containing 10% (w/w) FBS, and cultured at 37 ° C under 5% (v/v) CO 2 .
  • the cells were suspended in a cryopreservation solution [DMEM containing 20% (w/w) FBS and 10% (w/w) DMSO], and the hybridoma cells of the present invention were obtained by routine freezing of liquid nitrogen, and can be used for subsequent Antibody sequencing.
  • RNA isolation The supernatant obtained by subcloning culture of Example 1 was tested for antigen binding (that is, after the assays and activity assays of Examples 3 to 6), and partial antibodies were selected (see Tables 6 and 7 for sequencing). .
  • 5 ⁇ 10 7 hybridoma cells were collected by centrifugation, mixed with 1 mL of Trizol, and transferred to a 1.5 mL centrifuge tube, and allowed to stand at room temperature for 5 minutes. 0.2 mL of chloroform was added, shaken for 15 seconds, allowed to stand for 2 minutes, centrifuged at 12000 g for 5 minutes at 4 ° C, and the supernatant was transferred to a new 1.5 mL centrifuge tube.
  • RNA RNA was added, and the liquid in the tube was gently mixed. After standing at room temperature for 10 minutes, it was centrifuged at 12,000 g for 15 minutes at 4 ° C, and the supernatant was discarded. Add 1 mL of 75% ethanol (the percentage is a percentage by volume), gently wash the precipitate, centrifuge at 12000g for 5 minutes at 4 ° C, discard the supernatant, dry the precipitate, and add DEPC-treated H 2 O to dissolve (55 ° C water bath Promote dissolution for 10 minutes) to obtain total RNA.
  • 75% ethanol the percentage is a percentage by volume
  • Reverse transcription and PCR 1 ⁇ g of total RNA was taken, 20 ⁇ l of the system was placed, reverse transcriptase was added, and the reaction was carried out at 42 ° C for 60 minutes, and the reaction was terminated at 7 ° C for 10 minutes.
  • a 50 [mu]l PCR system was configured, including 1 [mu]l cDNA, 25 pmol of each primer, 1 [mu]l DNA polymerase, and a matching buffer system, 250 [mu]mol dNTPs.
  • the PCR program was set up, pre-denatured at 95 ° C for 3 minutes, denatured at 95 ° C for 30 seconds, annealed at 55 ° C for 30 seconds, extended at 72 ° C for 35 seconds, and 35 cycles and then extended at 72 ° C for 5 minutes to obtain a PCR product.
  • the kit for reverse transcription was PrimeScript RT Master Mix, purchased from Takara, catalog number RR036; the kit used for PCR was Q5 super-fidelity enzyme, purchased from NEB, catalog number M0492.
  • the ligation reaction was carried out: 50 ng of sample, 50 ng of T vector, 0.5 ⁇ l of ligase, 1 ⁇ l of buffer, 10 ⁇ l of reaction system, and reacted at 16 ° C for half an hour to obtain a ligation product, wherein the ligated kit was T4 DNA ligase, purchased from NEB, article number M0402 5 ⁇ l of the ligation product was added to 100 ⁇ l of competent cells (Ecos 101competent cells, available from Yeastern, Cat. No. FYE607), and ice bathed for 5 minutes.
  • T4 DNA ligase purchased from NEB
  • article number M0402 5 ⁇ l of the ligation product was added to 100 ⁇ l of competent cells (Ecos 101competent cells, available from Yeastern, Cat. No. FYE607), and ice bathed for 5 minutes.
  • the strain was preserved on an LB solid culture dish. After the end of the PCR reaction, 5 ⁇ l was taken for agarose gel electrophoresis detection, and the positive samples were sequenced. Among them, the steps of sequencing are described in Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991).
  • the number in Table 6 is the sequence number "SEQ ID NO.”
  • the amino acid sequence of the heavy chain protein variable region of 57B3D10 is SEQ ID NO.: 1
  • the heavy chain protein variable region of 57B3D10 The amino acid sequence of the middle CDR1 domain is SEQ ID NO.: 2 of the Sequence Listing.
  • the number in Table 7 is the sequence number "SEQ ID NO.”
  • the nucleotide sequence of the heavy chain protein variable region encoding 57B3D10 is SEQ ID NO.: 106 of the Sequence Listing.
  • Example 2 A purified 4-1BB antibody was obtained from the culture supernatant of hybridoma cells, and the heavy chain of the 4-1BB antibody was confirmed according to the sequencing result of Example 1. Region and light chain variable region sequences. Recombination of the heavy chain variable region sequence of the 4-1BB antibody into an expression vector comprising the signal peptide and the human IgG1 constant region of the human heavy chain antibody (wherein the expression vector was purchased from Invitrogen, and the recombination step was also performed by Shanghai Ruizhi Chemical), 4 The light chain variable region sequence of the -1BB antibody was recombined into an expression vector containing the signal peptide and the human antibody light chain kappa constant region, and the recombinant plasmid was obtained and verified by sequencing (sequencing method was the same as the sequencing method in Example 7).
  • the recombinant plasmid having a purity increased by mass spectrometry using an alkaline lysis kit was used at a mass of 500 ⁇ g or more, and filtered through a 0.22 ⁇ m filter (purchased from Millopore) for transfection.
  • 293E cells purchased from Invitrogen
  • medium Freestyle 293 expression medium purchased from Invitrogen.
  • the shaker was set to 37 ° C, 130 RPM, 8% CO 2 (v/v) concentration.
  • Freestyle 293 expression medium was added with 10% (v/v) F68 (purchased from Invitrogen) to a final concentration of 0.1% (v/v) at transfection to obtain Freestyle 293 expression medium containing 0.1% (v/v) F68. , medium A.
  • Peptone was added the next day to a final concentration of 0.5% (w/v).
  • the antibody titer of the culture solution was measured.
  • the supernatant was collected by centrifugation (3500 RPM, 30 minutes), and filtered through a 0.22 ⁇ m filter to obtain a filtered cell supernatant for purification.
  • the obtained human 4-1BB antibody was characterized (the same as Examples 4-8), and the results are shown in Figures 4-13 and 8-18, respectively.
  • Figures 4-13 and Tables 8-17 illustrate that the fully human 4-1BB antibody produced by whole human IgG transformation can activate the downstream signaling pathway of the 4-1BB molecule.
  • A enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibodies to 4-1BB protein
  • the purified whole human 4-1BB antibody obtained in Example 2 was subjected to the human 4-1BB-hFc protein, the monkey 4-1BB-his, and the other immunodetection point proteins OX40, GITR of the family of the 4-1BB protein, respectively. Perform a cross reaction.
  • the purified immunogen A (4-1BB-hFc), monkey 4-1BB-his, and mouse 4-1BB-his obtained in Example 1 were prepared.
  • the preparation of the immunogen A in the step (1) of Example 1 was prepared.
  • the monkey 4-1BB protein extracellular domain purchased from ACRO biosystems
  • mouse 4-1BB protein extracellular domain purchased from sino biological
  • other immunodetection point proteins were diluted with PBS to a final concentration of 1.0 ⁇ g/mL. Then, 100 ⁇ l per well was added to a 96-well ELISA plate.
  • the pIRES plasmid containing the nucleotide sequence encoding the full-length amino acid sequence of human 4-1BB as described in the step (2) of Example 1 was transfected into the CHOK1 cell line to obtain a CHOK1 stably transfected cell line containing human 4-1BB (here)
  • the CHOk1-h4-1BB stable cell line, the pIRES plasmid carrying the full-length gene of the monkey-derived PDL1 carry the human IgG Fc fragment.
  • the pCpC vector of (hFc) was prepared in the same manner, in which the database accession number of the amino acid sequence of the 4-1BB protein extracellular region (Phe19-Thr239) was G7PSE7).
  • the transfected CHOK1 cell line was stably transformed with the monkey 4-1BB.
  • Cell line (herein referred to as CHOK1-c4-1BB stable cell line).
  • the CHOK1-h4-1BB stable cell line and the CHOK1-c4-1BB stable cell line were expanded to a 90% confluence in a T-75 cell culture flask, and the medium was exhausted, and the HBSS buffer (Hanks Balanced Salt Solution, purchased) was used.
  • the cells were washed twice from Invitrogen and then treated and collected with an enzyme-free cell dissociation solution (Versene solution, purchased from Life Technology).
  • the cells were washed twice with HBSS buffer, and after cell counting, the cells were diluted with HBSS buffer to 2 ⁇ 10 6 cells per ml, and 1% goat serum blocking solution was added, the percentage being mass percentage.
  • the collected cells were suspended in FACS buffer (HBSS containing 1% BSA, the percentage is mass%) to 2 ⁇ 10 6 cells/mL, and added to a 96-well FACS reaction plate at 100 ⁇ L per well, and the addition was carried out.
  • the purified 4-1BB antibody obtained in Example 2 was sampled at 100 ⁇ L per well and incubated on ice for 2 hours.
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of a fluorescent (Alexa 488)-labeled secondary antibody (purchased from Invitrogen) per well was added and incubated on ice for 1 hour.
  • the cells were washed three times with FACS buffer, and 100 ⁇ l of the fixing solution [4% (v/v) paraformaldehyde] per well was used to suspend the cells, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD).
  • the results are shown in Figures 8-9 and 12-13.
  • the results indicate that the antibody to be tested binds to the 4-1BB protein on the cell surface.
  • the IgG control is human IgG, and the data in the table is the average fluorescence intensity value of the cell population measured by MFI.
  • the human 4-1BB protein-positive HEK293 stable cell line (see Example 1 for preparation) was further transfected with the NF ⁇ B luciferase reporter gene plasmid to produce a human 4-1BB protein-positive stable cell with the NF ⁇ B luciferase reporter gene.
  • Strain. The antibody to be detected is cross-linked with anti-human or rat Fc F(ab') 2 , and then the antibody which has been cross-linked or not cross-linked is added to the stable cell strain for cell culture. After 5 hours, the luciferase assay reagent was added and the fluorescence value was read. The results are shown in Figures 10A-10B and Table 14.
  • Table 14 shows that the fully human 4-1BB antibody has a strong activation of the NF ⁇ B signaling pathway downstream of the 4-1BB protein, and the antibody activation ability is further enhanced after antibody cross-linking (EC50). The value is lowered). This indicates that when the 4-1BB antibody is used in vivo, it can be cross-linked by FcR-expressing cells after reaching a specific region, such as tumor tissue, thereby enhancing antibody activity and safety.
  • Table 14 shows the fluorescence value multiples and EC50 values of the whole human 4-1BB antibody group relative to the control group (human IgG).
  • Detection of 4-1BB antibody by CHO cells expressing the 4-1BB protein blocked the binding of the 4-1BB protein to its receptor 4-1BBL.
  • the CHOK1-h4-1BB cell line was partially treated according to Example 4B to obtain 2 ⁇ 10 6 cells/mL of single suspension cells, and 100 ⁇ L per well was added to a 96-well FACS reaction plate, and the purified 4 obtained in Example 3 was added.
  • -1 BB antibody test sample 50 ⁇ l per well, then add his-muCD8a-4-1BBL ECD (see Example 1 for the preparation method) 50 ⁇ l of the fusion protein and incubate for 2 hours on ice.
  • the cells were washed twice with FACS buffer, and 100 ⁇ l of anti-mouse CD8a fluorescent (Alexa 488)-labeled secondary antibody per well was added and incubated on ice for 1 hour.
  • the cells were washed three times with FACS buffer, and 100 ⁇ l of fixative [4% (v/v) paraformaldehyde] suspension cells were added to each well, and after 10 minutes, the cells were washed twice with FACS buffer.
  • the cells were suspended in 100 ⁇ l of FACS buffer, and the results were detected and analyzed by FACS (FACS Calibur, available from BD). The results are shown in Figure 11 and Table 15.
  • Table 15 shows that the antibody to be tested inhibits the binding of the 4-1BB ligand protein to 4-1BB.
  • the IgG control was human IgG, and the data in the table is the inhibition rate of ligand binding to the control (human IgG) whole human 4-1BB antibody.
  • the freshly obtained whole blood was diluted with a phosphate buffered saline PBS in a volume ratio of 1:1 to obtain diluted whole blood, and the diluted whole blood was gently flattened on a Ficoll liquid surface (purchased from GE Healthcare) using a sterile pipette.
  • the volume ratio of Ficoll to diluted whole blood is 3:4, avoiding shaking and mixing, and centrifuging at 400g for 20 minutes at room temperature 20°C.
  • the centrifuge tube after centrifugation is divided into three layers, the upper layer is plasma, and the middle layer is milky white. It is a mononuclear lymphocyte.
  • the intermediate layer cells were gently aspirated with a sterile pipette, collected into a new centrifuge tube, diluted to three volumes with PBS phosphate buffer, centrifuged at 100 g for 10 minutes at room temperature, and the supernatant was discarded.
  • the lymphocytes were resuspended to 10 mL with PBS phosphate buffer, and the platelets were removed by repeating the previous procedure.
  • the lymphocytes were resuspended to 10 mL of multi-component RPMI 1640 medium (purchased from Invitrogen) containing 10% fetal calf serum for use as peripheral blood mononuclear lymphocyte PBMC, the percentage being mass percentage.
  • the above plasmid pIRES-OS8 was transfected into CHOK1 cells, and the stably passaged cell line CHOK1-OS8 was prepared by the plasmid transfected cells described in Example 1, and used as a T lymphocyte stimulating factor. At the same time, an equal volume ratio of the purified 4-1BB antibody obtained in Example 2 to be diluted was prepared to obtain a sample solution to be tested.
  • Human CD3 positive T cells were purified from human peripheral blood mononuclear lymphocytes obtained in the step (I) of Example 7 using a T cell purification kit (purchased from Stemcell) according to the method provided by the manufacturer.
  • the CD3-positive T cells and the cell stimulating factor CHOK1-OS8 were added to a 96-well cell culture plate, and the antibody to be detected which was cross-linked with anti-human or rat Fc F(ab') 2 was added , and finally each reaction well was The final volume of 200 microliters.
  • the supernatant was collected after co-cultivation for 72 hours at 37 ° C in a 5% CO 2 incubator, and the obtained cell supernatant was frozen at -20 ° C, and the percentage was a volume percentage.
  • C cytokine gamma interferon (IFN- ⁇ ) or interleukin IL-2 enzyme-linked immunosorbent assay in cell supernatant.
  • IFN- ⁇ cytokine gamma interferon
  • IFN-2 interleukin IL-2 enzyme-linked immunosorbent assay in cell supernatant.
  • Enzyme-linked immunosorbent assay for the determination of cytokine gamma interferon (IFN- ⁇ ) or interleukin IL-2 in cell supernatants using a double-antibody sandwich ELISA kit purchased from R&D Systems, IFN- ⁇ Cat#DIF50 and IL-2Cat) #S2050.
  • the experimental procedure is strictly in accordance with the instructions of the kit, and all test reagents are provided by the kit.
  • the specific experiment is briefly described as follows: IFN- ⁇ or IL-2 polyclonal antibody is coated on an ELISA microplate, and the cell supernatant obtained in the step (2) of the step 5 is used as a sample to be tested, and the standard product is added. The samples were incubated for 2 hours at room temperature.
  • Table 17 and Figure 13 demonstrate that the fully humanized 4-1BB antibody is capable of stimulating IL-2 secretion in a T cell stimulation assay.
  • the IgG control was human IgG (hIgG) and the data in the table was IL-2 value (pg/mL).
  • the affinity constant was determined using a Biacore T200 instrument (purchased from). Specific procedures and methods Root instrument manuals and detailed methods provided by the manufacturer. Five different gradients of the his-tagged human 4-1BB protein antigen were tested for dissociation with an anti-human Fc-labeled sensor in combination with anti-4-1BB full human antibody. Then, the dissociation constant and the binding constant were obtained by software fitting. The affinity constant is the ratio of the dissociation constant to the binding constant. The results of the affinity test are shown in Table 18.
  • mice The anti-tumor activity of the antibody in mice was evaluated using a human 4-1BB knock-in C57BL/6 mouse using the MC38 syngeneic mouse model.
  • the experimental design is as follows:
  • C57BL/6 mice knocked into human 4-1BB gene were selected and divided into 4 groups, 6 in each group, Utomilumab, Urellumab and homoantibody hIgG4 were used as controls, and the sample was administered as 113F6C6 antibody.
  • the route of administration was intraperitoneal injection at a dose of 3 mg/kg (hIgG control), 1 mg/kg (Utomilumab, Uruleum and 113F6C6), and intraperitoneal injection at 0, 4, 7, 11, 14, 18 days twice a week.
  • the antibody of the present invention (113F6C6) was able to significantly inhibit tumor growth, and the inhibitory effect was remarkably superior to the control antibodies Utomilumab and Urumemb.
  • a Pfizer Utomilumab antibody (PF-05082566) identical to the antibody target of the present application was used as a comparative antibody, and a comparative test was conducted in the same manner as in Example 7.
  • the 4-1BB antibody of the present application can stimulate T cells to secrete more IFN- ⁇ cytokines and activate T cells more than Utomilumab.

Abstract

本发明提供了一种4-1BB抗体及其制备方法和应用。具体地，本发明提供了一种4-1BB抗体，所述抗体对4-1BB蛋白具有高亲和力，能有效激活4-1BB下游信号，并且在人混合淋巴细胞或T淋巴细胞中显著增加IFN-γ和IL-2的表达量，可以用于治疗癌症以及自身免疫性疾病。

Description

4-1BB抗体及其制备方法和应用 技术领域
本发明涉及生物医药领域，更具体地涉及4-1BB抗体及其制备方法和应用。
背景技术
4-1BB又名CD137，或TNFRF9，是TNF受体家族的成员之一。4-1BB是一个阅读框含有255个氨基酸(NCBI：NP_001552)的I型跨膜蛋白，由含有17个氨基酸的N端信号肽，169个氨基酸的胞外区，27个氨基酸的跨膜区和42个氨基酸的C端胞内区组成。4-1BB分子主要表达在活化的T细胞，NK细胞，调节性T细胞，树突状细胞，单核细胞，中性粒细胞和嗜酸性细胞中，肿瘤血管的内皮细胞也有报道表达4-1BB。
在T细胞的活化过程中，4-1BB分子能为其提供共刺激信号。当T细胞受体接触到抗原后，会增加4-1BB的表达量，4-1BB与配体结合后会引起NFκB信号通路的激活，从而导致T细胞的活化增殖，并且4-1BB还能抑制活化细胞凋亡。动物模型和体外实验证实，抗4-1BB激动性单抗具有抗肿瘤活性。它能够选择性地引起CD8+T细胞的增殖，上调促炎性细胞因子IFN-γ的表达，并且能够增强抗原特异性效应型T细胞的杀伤作用,从而促进肿瘤的清除。抗4-1BB激动性单抗也能引起NK细胞的扩增，并能通过其增加CD8+T细胞的细胞毒活性。抗4-1BB的激动性抗体还能够引起肿瘤细胞的血管的内皮细胞上调粘附分子的表达，促进活化的T淋巴细胞浸润肿瘤组织。
除此之外，在动物模型中，抗4-1BB激活性抗体还能减轻自身免疫性疾病，如自身免疫性脑脊髓炎，狼疮样综合征和胶原诱导型关节炎。
目前，本领域现有的4-1BB抗体在抗原结合活性、激活4-1BB分子下游信号等方面还有许多的不足，没有广泛应用的4-1BB抗体药物产品，开发4-1BB的激动性抗体是治疗癌症以及自身免疫性疾病的迫切需求。
发明内容
本发明的目的在于提供一种高亲和力的4-1BB抗体及其制备方法和应用。
本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:2或SEQ ID NO.:10所示的CDR1，
SEQ ID NO.:3或SEQ ID NO.:11所示的CDR2，和
SEQ ID NO.:4或SEQ ID NO.:12所示的CDR3，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代1个氨基酸并能够保留4-1BB结合亲和力的衍生序列。
在另一优选例中，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:2所示的CDR1，
SEQ ID NO.:3所示的CDR2，和
SEQ ID NO.:4所示的CDR3；
或者，
SEQ ID NO.:10所示的CDR1，
SEQ ID NO.:11所示的CDR2，和
SEQ ID NO.:12所示的CDR3，
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代1个氨基酸并能够保留4-1BB结合亲和力的衍生序列。
在另一优选例中，所述的CDR3的氨基酸序列包括任选地经过添加、缺失、修饰和/或取代1个氨基酸并能够保留4-1BB结合亲和力的衍生序列。
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述重链可变区具有SEQ ID NO.:1或SEQ ID NO.:9所示的氨基酸序列。
本发明的第二方面，提供了一种抗体的重链，所述的重链具有如本发明第一方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。
本发明的第三方面，提供了一种抗体的轻链可变区，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:6或SEQ ID NO.:14所示的CDR1’，
SEQ ID NO.:7或SEQ ID NO.:15所示的CDR2’，和
SEQ ID NO.:8或SEQ ID NO.:16所示的CDR3’。
在另一优选例中，所述的轻链可变区包括以下三个互补决定区CDR：
SEQ ID NO.:6所示的CDR1’，
SEQ ID NO.:7所示的CDR2’，和
SEQ ID NO.:8所示的CDR3’；
或者，
SEQ ID NO.:14所示的CDR1’，
SEQ ID NO.:15所示的CDR2’，和
SEQ ID NO.:16所示的CDR3’。
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。
在另一优选例中，所述轻链可变区具有SEQ ID NO.:5或SEQ ID NO.:13所示的氨基酸序列。
本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有如本发明第三方面所述的轻链可变区。
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。
本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区；
或者，所述抗体具有：如本发明第二方面所述的重链；和/或如本发明第四方面所述的轻链。
在另一优选例中，所述抗体对NFκB转录因子的亲和力的EC 50为0.1-5nM。
在另一优选例中，所述抗体交联后对NFκB转录因子的亲和力的EC 50为0.4-1nM
在另一优选例中，所述交联是指抗体可通过其本身的Fc段与其他介质结合富集，所述的其他介质包括特异性结合抗体的Fc段，包括但不限于抗Fc抗体，细胞表面Fc受体。
在另一优选例中，所述的NFκB转录因子位于人4-1BB蛋白下游。
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:1所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:5所示。
在另一优选例中，所述抗体的重链可变区序列如SEQ ID NO.:9所示；并且所述的抗体的轻链可变区序列如SEQ ID NO.:13所示。
在另一优选例中，所述的抗体为药物偶联物形式。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
本发明的第七方面，提供了一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于4-1BB的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。
本发明的第八方面，提供了一种重组的免疫细胞，其特征在于，所述的免疫细胞表达外源的如本发明第七方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明的第九方面，提供了一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第八方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、或其组合，其特征在于，所述活性成分用于(a)制备检测试剂或试剂盒；和/或(b)制备预防和/或治 疗4-1BB相关疾病的药物。
在另一优选例中，所述4-1BB相关疾病选自下组：癌症、自身免疫疾病、病毒感染、移植物抗宿主病、炎症性疾病、免疫性疾病、或其组合。
在另一优选例中，所述的癌症包括实体瘤、血液癌。
在另一优选例中，所述的实体瘤选自下组：膀胱癌、胆道癌、脑癌、乳腺癌、结肠癌、食管癌、胃癌、神经胶质瘤、头癌、白血病、肝癌、肺癌癌症、淋巴瘤、骨髓瘤、颈癌、卵巢癌、黑素瘤、胰腺癌、肾癌、唾液癌、胃癌、胸腺上皮癌和甲状腺癌、或其组合。
在另一优选例中，所述的自身免疫疾病选自下组：系统性红斑狼疮、类风湿关节炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症、或其组合。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的检测试剂或试剂盒用于诊断4-1BB相关疾病。
在另一优选例中，所述检测试剂或试剂盒用于检测样品中4-1BB。
在另一优选例中，所述的检测试剂为检测片。
本发明的第十一方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第八方面所述的免疫细胞、如本发明第九方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
本发明的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；
(3)如本发明第七方面所述的CAR构建物。
在另一优选例中，编码所述重链可变区的核酸如SEQ ID NO.：106所示和/或编码所述轻链可变区的核酸如SEQ ID NO.：107所示。
在另一优选例中，编码所述重链可变区的核酸如SEQ ID NO.：108所示和/ 或且编码所述轻链可变区的核酸如序列表SEQ ID NO.：109所示。
本发明的第十三方面，提供了一种载体，所述的载体含有如本发明第十二方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明第十三方面所述的载体或基因组中整合有如本发明第十二方面所述的多核苷酸。
本发明的第十五方面，提供了一种体外检测(包括诊断性或非诊断性)样品中4-1BB的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在4-1BB。
本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第九方面所述的免疫偶联物。
本发明的第十七方面，提供了一种试剂盒，其特征在于，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明第十六方面所述的检测板。
本发明的第十八方面，提供了一种重组多肽的制备方法，其特征在于，所述方法包括：
(a)在适合表达的条件下，培养如本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白。
本发明的第十九方面，提供了一种治疗4-1BB相关疾病的方法，其特征在于，所述方法包括：给需要的对象施用如本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
在本发明的另一方面，提供了一种检测过表达4-1BB蛋白的细胞的方法，包括步骤：如本发明第五方面所述的抗体与待检样品在体外接触，检测所述抗 体与待检样品的结合。
在本发明的另一方面，提供了一种检测过表达4-1BB蛋白的细胞的组合物，包括本发明第五方面所述的抗体作为活性成分。
本发明所要解决的技术问题是为了克服目前缺少4-1BB抗体，尤其是人源化4-1BB抗体的不足，提供一种特异性强，生物学活性高的4-1BB抗体及其制备方法。所述的4-1BB抗体与人源和猴源的4-1BB蛋白均具有高度亲和力，能够激活4-1BB分子的下游信号，能在人混合淋巴细胞或T淋巴细胞反应中显著增加IFN-γ和IL-2的表达水平。
本发明先制备人源4-1BB作为免疫原，采用人源抗体转基因小鼠技术进行全人源抗体的制备，获得4-1BB抗体的先导抗体。再通过对先导抗体的初步生产、纯化和检定，获得能有效刺激4-1BB受体，激活其下游信号，导致在人T淋巴细胞反应中显著增加IFN-γ和IL-2的表达水平的优异生物水平的抗体。然后通过分子生物学方法测序获知4-1BB抗体的重链可变区和轻链可变区的氨基酸序列。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了4-1BB-hFc蛋白与融合蛋白his-muCD8a-4-1BBL的结合活性。
图2显示了4-1BB蛋白转染的HEK293细胞FACS筛选检测。
图3显示了ELISA检测4-1BB免疫后Harbour转基因小鼠血清抗体效价情况。
图4A-图4H分别显示了ELISA检测各个4-1BB全人源抗体与人4-1BB-hFc蛋白的结合反应。
图5A和图5B分别显示了ELISA检测各4-1BB全人源抗体与猴4-1BB-hFc蛋白的结合反应。
图6A和图6B分别显示了ELISA检测各4-1BB全人源抗体与猴4-1BB-hFc蛋白的结合反应。
图7显示了ELISA检测4-1BB全人源抗体与其他免疫检查点蛋白的结合反应。
图8A-图8E分别显示了FACS检测各4-1BB全人源抗体与CHOk1-h4-1BB的结合反应。
图9A-图9D分别显示了FACS检测各4-1BB全人源抗体与CHOk1-c4-1BB的结合反应。
图10A和图10B分别显示了各4-1BB全人源抗体激活NF-κB下游启动子的报告基因实验。
图11显示了4-1BB全人源抗体阻断4-1BB蛋白与其配体4-1BBL的结合反应。
图12显示了4-1BB全人源抗体在T淋巴细胞刺激试验中对IFN-γ分泌的影响。
图13显示了4-1BB全人源抗体在T淋巴细胞刺激试验中对IL-2分泌的影响。
图14显示了以辉瑞公司的Utomilumab抗体作为对比抗体进行对比试验的结果。
图15显示了4-1BB抗体对小鼠MC38肿瘤生长的实验结果。
图16显示了不同抗体治疗组的小鼠个体的肿瘤体积
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种具有高亲和力和特异性的4-1BB抗体，并基于该抗体而获得了全人源化抗体。本发明抗体对4-1BB蛋白具有高亲和力，能有效激活4-1BB下游信号，并且在人混合淋巴细胞或T淋巴细胞中显著增加IFN-γ和IL-2的表达量，可以用于治疗癌症以及自身免疫性疾病。在此基础上完成了本发明。
术语
如本文所用，术语“结合物”是指能够与靶点结合的可溶性受体或其片段或其类似物，或抗体或其片段或其类似物。
如本文所用，术语“4-1BB结合物”、“4-1BB抗体”、“抗4-1BB抗体”、“本发明抗体”具有相同含义，是指能特异性识别4-1BB并与4-1BB结合的抗体或其片段或其类似物。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括4-1BB结合物与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的任何一种4-1BB结合物及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症 状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
抗体
如本文所用，术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的不同种型，即IgM、IgD、IgG、IgA和IgE，对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。IgG代表免疫球蛋白中最重要的一类，由于化学结构和生物功能差异，它又可以分为4个子类：IgG1、IgG2、IgG3和IgG4。轻链通过恒定区的不同分为κ或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的FR区(FR)。4个FR的氨基酸序列相对比较保守，不直接参与结合反应。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到羧基端依次排列的顺序为FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区，即轻链高变区(LCDR),指LCDR1、LCDR2和LCDR3；重链的3个CDR区，即重链高变区(HCDR),指HCDR1、HCDR2和HCDR3。发明所述的抗体或抗原结合片段的LCVR和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。天然重链和轻链可变区中的四个FR区大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，或单一Fv片段。Fv抗体含有抗体重链可变区、轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般的，Fv抗体还包含VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗 原结合片段识别的三维空间位点。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
如本文所用，术语“嵌合抗体”是由鼠源性抗体的V区基因与人抗体的C区基因拼接为嵌合基因，然后插入载体，转染宿主细胞表达的抗体分子。既保留了亲本鼠抗体的高特异性和亲和力，又使其人源Fc段能有效介导生物学效应功能。
如本文所用，术语“人源化抗体”，是本发明鼠抗的一种可变区改造形式，具有源自(或基本上源自)非人类抗体(优选小鼠单克隆抗体)的CDR区，和基本源自人源抗体序列的FR区和恒定区；即将鼠抗的CDR区序列嫁接到不同类型的人种系抗体构架序列上。因为CDR序列负责大部分的抗体-抗原相互作用，所以可以通过构建表达载体来表达模拟特定天然存在的抗体性质的重组抗体。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗4-1BB抗体
4-1BB又名CD137，或TNFRF9，是TNF受体家族的成员之一。4-1BB是一个阅读框含有255个氨基酸(NCBI：NP_001552)的I型跨膜蛋白，由含有17个氨基酸的N端信号肽，169个氨基酸的胞外区，27个氨基酸的跨膜区和42个氨基酸的C端胞内区组成，具体序列可见本专利中SEQ ID NO：22。
如本文所用，术语“4-1BB”一般是指天然的或重组的人4-1BB，以及人4-1BB的非人同源物。除非另有指示，否则使用4-1BB的同源二聚体的分子量计算4-1BB的摩尔浓度。
如本文所用，术语“人4-1BB”包括人4-1BB蛋白的成熟形式及其天然变体和多态性。
本发明提供一种针对4-1BB的高亲和力的抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
优选地，重链可变区(VH)氨基酸序列和轻链可变区(VL)氨基酸序列的各自CDR选自下组：
a1)SEQ ID NO.:2或SEQ ID NO.:10；
a2)SEQ ID NO.:3或SEQ ID NO.:11；
a3)SEQ ID NO.:4或SEQ ID NO.:12；
a4)SEQ ID NO.:6或SEQ ID NO.:14；
a5)SEQ ID NO.:7或SEQ ID NO.:15；
a6)SEQ ID NO.:8或SEQ ID NO.:16；
a7)上述氨基酸序列中任意一种氨基酸序列经过添加、缺失、修饰和/或取代至少一个(如1-5、1-3个，较佳地1-2个，更佳地1个)氨基酸的具有4-1BB结 合亲和力的序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少90％，更佳地至少为95％，最佳地至少99％的氨基酸序列。
优选地，上述氨基酸序列的编号如表1所示。
表1 4-1BB抗体蛋白序列编号
Figure PCTCN2018114641-appb-000001
其中，表1中的数字即为序列表“SEQ ID NO.”编号，如57B3D10的重链蛋白可变区的氨基酸序列为如SEQ ID NO.:1所示，而113F6C6的重链蛋白可变区中CDR1域的氨基酸序列为如SEQ ID NO.:2所示。
事实上，发明人在抗体筛选过程中，还针对克隆号为：11H10C9、15G10D4、23G3B8、52F4G2、118F3A2、170D7F2、172E3E3、178D10D11、182A5B3、100F3C4、119B6G5、258F5A8、259G10E11、263A11E3、289B6G6的4-1BB抗体，进行了相关实验，由于其各项性能低于克隆号57B3D10和113F6C6的抗体，未进行进一步实验，详细实验结果参见实施例。
本发明抗体可以是抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single-domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15～20个氨基酸的短肽。
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定 区。
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。
具体地，本发明的抗体可以是双链或单链抗体，并且可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab’、(Fab’) 2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
其中，所述动物优选为哺乳动物，如鼠。
本发明抗体可以是靶向人4-1BB的鼠源抗体、嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。
在本发明的一种优选实施例中，上述SEQ ID NO.:2、3和4，或者SEQ ID NO.:6、7和8中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有4-1BB结合亲和力的序列，位于重链可变区(VH)的CDR区。
在本发明的一种优选实施例中，上述SEQ ID NO.:10、11和12，或者SEQ ID NO.:14、15和16中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有4-1BB结合亲和力的序列，位于轻链可变区(VL)的CDR区。
在本发明的一种更优选实施例中，VH CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:2、3和4，或者SEQ ID NO.:6、7和8中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有4-1BB结合亲和力的序列；VL CDR1、CDR2、CDR3分别独立地选自SEQ ID NO.:10、11和12，或者SEQ ID NO.:14、15和16中任意一种或几种序列、或它们经过添加、缺失、修饰和/或取代至少一个氨基酸的具有4-1BB结合亲和力的序列。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的30％，更优选为不超过20％，更优选为1-15％，更优选为1-10％。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
多核苷酸
本发明还提供一种核酸，其编码上述多肽。
较佳地，编码所述重链可变区的核酸如序列表SEQ ID NO.：106或序列表SEQ ID NO.：108所示；和/或，编码所述轻链可变区的核酸的核苷酸序列如 序列表SEQ ID NO.：107或序列表SEQ ID NO.：109所示。
更佳地，编码所述重链可变区的核酸如序列表SEQ ID NO.：106所示,且编码所述轻链可变区的核酸如序列表SEQ ID NO.：107所示；编码所述重链可变区的核酸如序列表SEQ ID NO.：108所示,且编码所述轻链可变区的核酸如序列表SEQ ID NO.：109所示。
上述核苷酸序列的编号如表2所示：
表2 4-1BB抗体基因序列编号
克隆号 重链蛋白可变区 轻链蛋白可变区
57B3D10 106 107
113F6C6 108 109
其中，表2中的数字即为序列表“SEQ ID NO.”编号，如编码57B3D10的重链蛋白可变区的核苷酸序列为序列表SEQ ID NO.：106。
其中，编码57B3D10的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO.：106中的第91位至第105位；
编码57B3D10的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO.：106中的第148位至第195位；
编码57B3D10的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO.：106中的第292位至第342位；
编码57B3D10的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO.：107中的第67位至第102位；
编码57B3D10的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO.：107中的第148位至第168位；
编码57B3D10的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO.：107中的第265位至第288位；
编码113F6C6的重链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO.：108中的第位91至第105位；
编码113F6C6的重链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO.：108中的第148位至第198位；
编码113F6C6的重链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID NO.：108中的第295位至第351位；
编码113F6C6的轻链蛋白可变区中CDR1域的核苷酸序列为序列表SEQ ID NO.：109中的第70位至第102位；
编码113F6C6的轻链蛋白可变区中CDR2域的核苷酸序列为序列表SEQ ID NO.：109中的第148位至第168位；
编码113F6C6的轻链蛋白可变区中CDR3域的核苷酸序列为序列表SEQ ID  NO.：109中的第265位至第291位；
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本发明的抗4-1BB抗体。例如，可以用连接或天然存在的4-1BB同源二聚体或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的4-1BB都可以作为免疫原(抗原)，用于产生对4-1BB特异的非人抗体，筛选所述抗体的生物学活性。激发免疫原可以是全长的成熟人4-1BB，包括天然的同源二聚体，或含单个/多个表位的肽。免疫原可以单独使用，或与本领域已知的一种或多种免疫原性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、腺相关病毒载体、杆状病毒载体、质料和非病毒载体。
生产本发明的抗人4-1BB抗体的示例性方法描述于实施例1。
全人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。在本发明中，抗体是IgG抗体，使用IgG4亚型。通过用下文实施例中描述的生物学测定筛选抗体易于实现必需恒定结构域序列的最优化，以产生所需生物学活性。
同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本发明的化合物和方法中是可以用的。
人源化本发明的抗人4-1BB抗体的示例性方法描述于实施例3。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中 分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。具体地，宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、CHO-K1、HEK-293细胞。
优选的宿主细胞包括E.coli TG1或BL21细胞(表达单链抗体或Fab抗体)，或者CHO-K1细胞(表达全长IgG抗体)
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
应用
本发明提供了本发明抗体的用途，例如用于制备诊断制剂、或制备用于预防和/或治疗4-1BB相关的疾病的药物。所述4-1BB相关的疾病包括癌症、自身免疫疾病、病毒感染、移植物抗宿主病、炎症性疾病、免疫性疾病、或其组合。其中，所述的癌症包括实体瘤、血液癌，所述的实体瘤选自下组：膀胱癌、胆道癌、脑癌、乳腺癌、结肠癌、食管癌、胃癌、神经胶质瘤、头癌、白血病、肝癌、肺癌癌症、淋巴瘤、骨髓瘤、颈癌、卵巢癌、黑素瘤、胰腺癌、肾癌、唾液癌、胃癌、胸腺上皮癌和甲状腺癌、或其组合；所述的自身免疫疾病包括：系统性红斑狼疮、类风湿关节炎、溃疡性结肠炎、I型糖尿病、银屑病、多发性硬化症、或其组合。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合4-1BB蛋白分子，因而可用于预防和治疗4-1BB相关的疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约100毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
本发明还提供一种检测过表达4-1BB蛋白的细胞的方法，包括如下的步骤：上述的蛋白质与待检样品在体外接触，检测上述的蛋白质与所述待检样品的结合即可。
所述的过表达的含义为本领域常规，指4-1BB蛋白在待检样品中的RNA或蛋白质的过表达(由于转录增加、转录后加工、翻译、翻译后加工以及蛋白质降解改变)，以及由于蛋白质运送模式改变(核定位增加)而导致的局部过表达和功能活性提高(如在底物的酶水解作用增加的情况下)。
所述结合的检测方式是本领域常规的检测方式，较佳地为FACS检测。
本发明提供一种检测过表达4-1BB蛋白的细胞的组合物，其包括上述的蛋白质作为活性成分。较佳地，其还包括上述的蛋白质的功能片段组成的化合物作为活性成分。
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。
本发明的主要优点包括：
本发明所述的蛋白质是一种全人源4-1BB抗体，其与4-1BB蛋白具有高亲和力，能够结合4-1BB蛋白受体的胞外区，并能够在细胞水平激活4-1BB分子下游信号。混合淋巴细胞实验和T细胞刺激实验证明，所述的4-1BB抗体具有良好的生物活性，其在人混合淋巴细胞或T淋巴细胞中会显著增加IFN-γ和IL-2的表达量。由此可见，所述的4-1BB抗体除了为全人源序列外，还具有包括结合人4-1BB蛋白的高度特异性、能够调节人淋巴细胞免疫反应等优异的特性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例中所述的室温为本领域常规的室温，一般为10～30℃。
若无特别说明，实施例中所述的PBS为PBS磷酸缓冲液，pH7.2。
实施例1
4-1BB抗体的制备
(一)、免疫原A的制备
将人源4-1BB蛋白胞外区氨基酸序列Leu24-Gln186(如序列表SEQ ID NO.：21所示)克隆到带有人IgG Fc片段(hFc)的pCpC载体(购自Invitrogen，V044-50) 并按已建立的标准分子生物学方法制备质粒。对HEK293细胞(购自Invitrogen)进行瞬时转染(PEI，Polysciences)并使用FreeStyle  TM 293(Invitrogen)在37℃下进行扩大培养。4天后收集细胞培养液，离心去除细胞成分，得含4-1BB蛋白胞外区的培养上清液。将培养上清液上样到蛋白A亲和层析柱(Mabselect Sure，购自GE Healthcare)，同时用紫外(UV)检测仪监测紫外吸收值(A280nm)的变化。上样后用PBS磷酸盐缓冲液(pH7.2)清洗蛋白A亲和层析柱直到紫外吸收值回到基线，然后用0.1M甘氨酸盐酸(pH2.5)洗脱，收集从蛋白A亲和层析柱上洗脱下来的带hFc标签的4-1BB蛋白(4-1BB-hFc)，用PBS磷酸盐缓冲液(pH7.2)在4℃冰箱透析过夜。透析后的蛋白经0.22微米无菌过滤后分装于-80℃保存，即获得纯化的免疫原A。免疫原A在使用前需要进行一系列质控检测，如检测其蛋白浓度、纯度、分子量和生物活性等，结果如图1和表3所示。表3说明4-1BB与其配体蛋白4-1BBL在蛋白水平的结合随着4-1BBL的浓度变化而变化，其中对照蛋白为非4-1BB融合蛋白，表中的数据为OD 450nm值。
其中，免疫原A生物活性采用ELISA检测，具体为：
将带hFc标签的4-1BB胞外区蛋白(4-1BB-hFc)用PBS稀释至0.5μg/mL，以100μl/孔加入ELISA微孔板，4℃孵育过夜。用ELISA封闭液(含1％BSA，pH7.4的PBS磷酸缓冲液，所述百分比为质量百分比)37℃封闭两小时后，再加入梯度稀释的his-muCD8a-4-1BBL ECD融合蛋白37℃温育1小时。his-muCD8a-4-1BBL ECD由4-1BB配体4-1BBL胞外区(NCBI序列NP_003802.1的Arg71-Glu254)和小鼠CD8a胞外区(NCBI序列NP_001074579.1的Lys28-Asp194)融合组成，his标签在N端。his-muCD8a-4-1BBL ECD蛋白的表达方法如免疫源A，由镍柱亲和层析法制备纯化。
接着再加入生物素标记的大鼠抗小鼠CD8a抗体，37℃温育1小时。加入链霉亲和素标记的辣根过氧化物酶(购自Sigma商品号S2438)，室温孵育30分钟后加入100微升/孔TMB显色液。室温孵育15分钟后，加入50微升1N盐酸终止显色反应，用ELISA读板机读取OD 450nm读数。
表3 4-1BB-hFc蛋白与受体4-1BBL的结合活性
Figure PCTCN2018114641-appb-000002
(二)、免疫原B的制备
人源4-1BB全长氨基酸序列(如序列表SEQ ID NO.：23所示)被克隆到pIRES载体(购自Clontech)并制备质粒。对HEK293细胞系和CHOK1细胞系(均购自Invitrogen)进行质粒转染(转染使用X-treme GENE HP DNA Transfection Reagent，购自Roche公司，货号Cat#06 366 236 001，并按说明书操作)后，在含0.5μg/mL的含10％(w/w)FBS的DMEM培养基中选择性培养2周，用有限稀释法在96孔培养板中进行亚克隆，并置于37℃，5％(v/v)CO 2培养。大约2周后选择部分单克隆孔扩增到6孔板中。对扩增后的克隆用已知的4-1BB抗体(购自BD公司)经流式细胞分析法进行筛选。选择长势较好、荧光强度较高、单克隆的细胞系继续扩大培养并液氮冻存，即获得免疫原B。具体选择结果如表4和图2所示，表4中阳性细胞(％)指阳性细胞占总细胞数目的百分比。表4说明，已经制得一系列4-1BB阳性表达的HEK293细胞系。
表4 4-1BB蛋白转染的HEK293细胞FACS筛选检测结果
Figure PCTCN2018114641-appb-000003
Figure PCTCN2018114641-appb-000004
(三)、杂交瘤细胞的制备和抗体筛选
Harbour转基因小鼠引入了人免疫球蛋白可变区基因和大鼠免疫球蛋白恒定区基因，而小鼠本身的Ig表达则被沉默。该转基因小鼠经抗原免疫后能产生与正常小鼠(如Balb/c)相当的免疫反应和抗体效价。
A、免疫原A免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。初次免疫时，免疫原(1)蛋白用弗氏完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射100微克免疫原A蛋白。加强免疫时，免疫原A蛋白用弗氏不完全佐剂乳化后腹腔注射0.25毫升，即每只小鼠注射50微克免疫原A蛋白。初次免疫与第一次加强免疫之间间隔2周，以后每次加强免疫之间间隔3周。每次加强免疫1周后采血，用ELISA和FACS检测血清中免疫原(1)的抗体效价和特异性，结果如图3和表5所示。表5说明，经4-1BB-hFc免疫的小鼠的免疫后血清对免疫原均有不同程度的结合，呈现抗原抗体反应，其中最高稀释度在一百万左右。其中空白对照为1％(w/w)BSA，其中批次指第三次加强免疫后第七天的小鼠血清，表中的数据为OD 450nm值
表5 ELISA检测4-1BB蛋白免疫后Harbour转基因小鼠血清抗体效价
Figure PCTCN2018114641-appb-000005
B、免疫原B免疫采用6～8周龄Harbour人源抗体转基因小鼠(购自北京维通利华公司)，小鼠在SPF条件下饲养。将按实施例1步骤(二)中的得到的含人源4-1BB的HEK293-h4-1BB稳定细胞系在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基。用DMEM基础培养基(购自Invitrogen)洗涤2次，然后用无酶细胞解离液(购自Invitrogen)37℃处理直至细胞从培养皿壁上可脱落，收集细胞。用DMEM基础培养基洗涤2次，进行细胞计数后将细胞用磷酸盐缓冲液(pH7.2)稀释至2╳10 7细胞每毫升。每只小鼠每次免疫时腹腔注射0.5毫升细胞悬液。第一次与第二次免疫之间间隔2周，以后每次免疫间隔3周。除第一次免疫以外，每次免疫1周后采血，用FACS检测血清中抗体效价和特异性。在二次加强免疫后，FACS检测血清抗体效价通常达到1:1000以上。
通常以免疫原A～B进行免疫，大部分小鼠经3次免疫后FACS效价可达到1:1000以上。
A～B步骤完成前，将所选择的每只小鼠最后一次免疫腹腔注射100微克纯化的4-1BB-hFc(针对免疫原A和免疫原C进行免疫反应的小鼠)或含人源4-1BB的HEK293-h4-1BB稳定细胞系(针对免疫原B进行免疫反应的小鼠)，5天后处死小鼠，收集脾细胞。加入NH 4OH至终浓度1％(w/w)，裂解脾细胞中参杂的红细胞，获得脾细胞悬液。用DMEM基础培养基1000转每分钟离心清洗细胞3次，然后按活细胞数目5:1比率与小鼠骨髓瘤细胞SP2/0混合(购自ATCC)，采用高效电融合或PEG方法进行细胞融合。融合后的细胞稀释到含20％胎牛血清、1╳HAT的DMEM培养基中，所述百分比为质量百分比。然后按1╳10 5/20微升每孔加入到96孔细胞培养板中，放入5％CO 2、37℃培养箱中，所述百分比为体积百分比。14天后用ELISA和Acumen(微孔板细胞检测法)筛选细胞融合板上清，将ELISA中OD 450nm>1.0和Acumen中MFI值>100的阳性克隆扩增到24孔板，在含10％(w/w)胎牛血清的DMEM(Invitrogen)的培养基中，在37℃，5％(v/v)CO 2条件下扩大培养。培养3天后取24孔板中扩大培养的培养液进行离心，收集上清液，对上清液进行抗体亚型分析。用ELISA、FACS确定对4-1BB蛋白和4-1BB阳性细胞的结合活性(结合活性的检测方法请分别参见实施例5A和实施例5B)，再通过NF-κB荧光素酶报告基因实验来确定抗体样品对4-1BB受体激活的活性(检测方法请参见实施例5)。
根据24孔板筛选结果，挑选ELISA实验中OD 450nm>1.0、FACS实验中MFI值>50，和NF-κB荧光素酶报告基因实验中杂交瘤细胞培养上清相对于对照IgG组将4-1BB受体激活在1.0倍以上的杂交瘤细胞为符合条件的阳性克隆。选择符合条件的杂交瘤细胞用有限稀释法在96孔板进行亚克隆，在含10％(w/w)FBS的DMEM培养基中(购自Invitrogen)37℃，5％(v/v)CO 2条件下培养。亚克隆后10天用ELISA和Acumen进行初步筛选，挑选阳性单克隆扩增到24孔板继续培养。3天后用FACS确定抗原结合阳性并用4-1BB受体NF-κB荧光素酶报告基因实验评估生物活性(评估标准为ELISA实验中OD 450nm>1.0、FACS实验中MFI值>5，和NF-κB荧光素酶报告基因实验中杂交瘤细胞培养上清相对于对照IgG组将4-1BB受体激活在1.0倍以上)。
根据24孔板样品检测结果，阳性克隆在含10％(w/w)FBS的DMEM(购自Invitrogen)培养基中，在37℃，5％(v/v)CO 2条件下进行扩大培养，细胞悬浮于冻存液[含有20％(w/w)FBS和10％(w/w)DMSO的DMEM]中，按常规方法液氮冻存即得本发明杂交瘤细胞，并可用于后续的抗体测序。
实施例2
轻重链可变区氨基酸序列测定
总RNA分离：将实施例1亚克隆培养所得的上清液检验过抗原结合后(即经过实施例3～6的检定和活性测定后)，选择部分抗体(详见表6、7)进行测序。通过离心搜集5×10 7个杂交瘤细胞，加入1mL Trizol混匀并转移到1.5mL离心管中，室温静置5分钟。加0.2mL氯仿，振荡15秒，静置2分钟后于4℃，12000g离心5分钟，取上清转移到新的1.5mL离心管中。加入0.5mL异丙醇，将管中液体轻轻混匀，室温静置10分钟后于4℃，12000g离心15分钟，弃上清。加入1mL 75％乙醇(所述百分比为体积百分比)，轻轻洗涤沉淀，4℃，12000g离心5分钟后弃上清，将沉淀物晾干，加入DEPC处理过的H 2O溶解(55℃水浴促溶10分钟)，即得总RNA。
逆转录与PCR：取1μg总RNA，配置20μl体系，加入逆转录酶后于42℃反应60分钟，于7℃反应10分钟终止反应。配置50μl PCR体系，包括1μl cDNA、每种引物25pmol、1μl DNA聚合酶以及相配的缓冲体系、250μmol dNTPs。设置PCR程序，预变性95℃3分钟，变性95℃30秒，退火55℃30秒，延伸72℃35秒，35个循环后再额外于72℃延伸5分钟，得PCR产物。其中逆转录所用的试剂盒为PrimeScript RT Master Mix，购自Takara，货号RR036；PCR所用的试剂盒为Q5超保真酶，购自NEB，货号M0492。
克隆与测序：取5μl PCR产物进行琼脂糖凝胶电泳检测，将检测阳性样品使用柱回收试剂盒纯化，其中回收试剂盒为
Figure PCTCN2018114641-appb-000006
Gel&PCR Clean-up，购自MACHEREY-NAGEL，货号740609。进行连接反应：样品50ng，T载体50ng，连接酶0.5μl，缓冲液1μl，反应体系10μl，于16℃反应半小时得连接产物，其中连接的试剂盒为T4DNA连接酶，购自NEB，货号M0402；取5μl连接产物加入100μl的感受态细胞(Ecos 101competent cells，购自Yeastern，货号FYE607)中，冰浴5分钟。而后于42℃水浴热激1分钟，放回冰上1分钟后加入650μl无抗生素SOC培养基，于37℃摇床上以200RPM的速度复苏30分钟，取出200μl涂布于含抗生素的LB固体培养基上于37℃孵箱过夜培养。次日，使用T载体上引物M13F和M13R配置30μl PCR体系，进行菌落PCR，用移液器枪头蘸取菌落于PCR反应体系中吹吸，并吸出0.5μl点于另一块含100nM氨苄青霉素的LB固体培养皿上以保存菌株。PCR反应结束后，取出5μl进行琼脂糖凝胶电泳检测，将阳性样品进行测序。其中，测序的步骤参见Kabat，Sequences of Proteins of Immunological Interest，National Institutes of Health,Bethesda,Md.(1991)。
测序结果如表6～7所示。
表6 4-1BB抗体的氨基酸序列的序列表编号
Figure PCTCN2018114641-appb-000007
Figure PCTCN2018114641-appb-000008
其中，表6中的数字即为序列表“SEQ ID NO.”编号，如57B3D10的重链蛋白可变区的氨基酸序列为序列表SEQ ID NO.：1，而57B3D10的重链蛋白可变区中CDR1域的氨基酸序列为序列表SEQ ID NO.：2。
表7 4-1BB抗体的核苷酸序列的序列表编号
Figure PCTCN2018114641-appb-000009
Figure PCTCN2018114641-appb-000010
其中，表7中的数字即为序列表“SEQ ID NO.”编号，如编码57B3D10的重链蛋白可变区的核苷酸序列为序列表SEQ ID NO.：106。
实施例3
全人抗体IgG转化和制备
(1)质粒构建与准备：实施例2已从杂交瘤细胞的培养上清液中获得了纯化的4-1BB抗体，并根据实施例1的测序结果明确了4-1BB抗体的重链可变区和轻链可变区序列。将4-1BB抗体的重链可变区序列重组到包含信号肽和人源重链抗体IgG1恒定区的表达载体(其中表达载体购买自Invitrogen，重组步骤也由上海睿智化学完成)中，将4-1BB抗体的轻链可变区序列重组到包含信号肽和人源抗体轻链kappa恒定区的表达载体当中，得重组质粒并经测序验证(测序方法与实施例7中测序方法相同)。使用碱裂解法试剂盒(购自MACHEREY-NAGEL)中量抽提高纯度的重组质粒，质量为500μg以上，经0.22μm滤膜(购自Millopore)过滤，供转染使用。
(2)细胞转染：
在培养基Freestyle 293expression medium(购自Invitrogen)培养293E细胞(购自Invitrogen)。摇床设置为37℃、130RPM，8％CO 2(v/v)浓度。
Freestyle 293expression medium在转染时添加10％(v/v)F68(购自Invitrogen)至F68终浓度为0.1％(v/v)，得含0.1％(v/v)F68的Freestyle 293表达培养基，即培养基A。
取5mL培养基A和200μg/mL PEI(购自Sigma)混匀，得培养基B。取5mL培养基A和100μg/mL步骤(1)所得的重组质粒混匀，得培养基C。5分钟后将培养基B和培养基C合并混匀，静置15分钟，得混合液D。将10mL混合液D缓缓加入100mL含293E细胞的培养基Freestyle 293expression medium中至293E的细胞密度为1.5×10 6/mL，边加边振荡，避免PEI过度集中，放入摇床培养。第二天加入蛋白胨至终浓度为0.5％(w/v)。第5～7天，测培养液抗体效价。第6～7天，离心(3500RPM，30分钟)收集上清，经0.22μm滤膜过滤，得滤好的细胞上清液，以供纯化。
(3)抗体纯化：对于连续生产的无内毒素的层析柱和Protein A填料(购自GE)，使用0.1M NaOH处理30分钟或者5个柱体积的0.5M NaOH冲洗。对于长期未 使用的柱料和层析柱至少使用1M NaOH浸泡1h，用无内毒的水冲洗至中性，用10倍柱体积的1％(v/v)Triton×100对柱料清洗。使用5个柱体积的PBS(PBS磷酸缓冲液，pH7.2)进行平衡，将步骤(2)所得过滤好的细胞上清液上柱，必要时收集流穿液。上柱完成后，使用5倍柱体积的PBS清洗。用5倍柱体积的0.1M pH3.0的Glycine-HCl进行洗脱，收集洗脱液，并用0.5倍柱体积洗脱液的pH8.5的1MTris-HCl(1.5M NaCl)中和，收获全人4-1BB抗体。上述所用溶液均需要新配置。收获全人4-1BB抗体后，在1╳PBS中透析4小时，避免内毒素污染。透析结束后，使用分光光度或试剂盒测定浓度，使用HPLC-SEC测定抗体纯度，使用内毒素检测试剂盒(购自Lonza)检测抗体内毒素含量。并对所获全人4-1BB抗体进行特性鉴定(同实施例4-8)，检测结果分别如图4-13和表8-18所示。图4～13和表8～17说明，经过全人IgG转化并制备所得全人4-1BB抗体能够激活4-1BB分子下游信号通路。
实施例4
先导抗体的检定
A、酶联免疫吸附实验(ELISA)检测抗体与4-1BB蛋白的结合
对实施例2所得的纯化的全人源4-1BB抗体进行与人4-1BB-hFc蛋白、与猴4-1BB-his和与4-1BB蛋白所在家族的其他免疫检测点蛋白OX40，GITR分别进行交叉反应。
将实施例1获得的纯化的免疫原A(4-1BB-hFc)、猴4-1BB-his、小鼠4-1BB-his[其制备方法参见实施例1步骤(一)免疫原A的制备，其中猴源4-1BB蛋白胞外区(购自ACRO biosystems)，小鼠4-1BB蛋白胞外区(购自sino biological)或其他免疫检测点蛋白分别用PBS稀释到终浓度1.0μg/mL，然后以100μl每孔加到96孔ELISA板。用塑料膜封好4℃孵育过夜，第二天用洗板液[含有0.01％(v/v)Tween20的PBS]洗板2次，加入封闭液[含有0.01％(v/v)Tween20和1％(v/v)BSA的PBS]室温封闭2小时。倒掉封闭液，加入实施例2所得的纯化的4-1BB抗体100μl每孔。37℃孵育2小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入HRP(辣根过氧化物酶)标记的二抗(购自Sigma)，37℃孵育2小时后，用洗板液[含有0.01％(v/v)Tween20的PBS]洗板3次。加入TMB底物100μl每孔，室温孵育30分钟后，加入终止液(1.0N HCl)100μl每孔。用ELISA读板机(SpectraMax 384plus,Molecular Device)读取A450nm数值，结果如图4～7和表8～11所示，表8～11说明，纯化后的抗体与4-1BB重组蛋白在ELISA水平特异性结合。其中空白为人IgG，CTLA4-Fc作为阴性对照(negative control，NC)，表中的数据为OD 450nm值。
表8 ELISA检测全人源4-1BB抗体与人4-1BB-hFc蛋白的结合反应
Figure PCTCN2018114641-appb-000011
表9 ELISA检测4-1BB抗体与猴4-1BB-his蛋白的结合反应
Figure PCTCN2018114641-appb-000012
表10 ELISA检测4-1BB抗体与小鼠4-1BB-hFc蛋白的结合反应
Figure PCTCN2018114641-appb-000013
Figure PCTCN2018114641-appb-000014
表11 ELISA检测4-1BB抗体与其他免疫检查点蛋白的结合反应
Figure PCTCN2018114641-appb-000015
B、流式细胞实验(FACS)检测抗体与4-1BB表达细胞的结合
将实施例1步骤(二)中所述含有编码人源4-1BB全长氨基酸序列的核苷酸序列的pIRES质粒转染CHOK1细胞株得含人4-1BB的CHOK1稳转细胞株(此处称为CHOk1-h4-1BB稳定细胞株)，将带有猴源PDL1全长基因的pIRES质粒(其制备方法与实施例1步骤(一)“免疫原A的制备”中带有人源IgG Fc片段(hFc)的pCpC载体的制备方法相同，其中猴源4-1BB蛋白胞外区(Phe19-Thr239)氨基酸序列的数据库登录号为G7PSE7)转染CHOK1细胞株得含猴4-1BB的CHOK1稳转细胞株(此处称为CHOK1-c4-1BB稳定细胞株)。将CHOK1-h4-1BB稳定细胞株和CHOK1-c4-1BB稳定细胞株在T-75细胞培养瓶中扩大培养至90％汇合度，吸尽培养基，用HBSS缓冲液(Hanks Balanced Salt Solution，购自Invitrogen)洗涤2次，然后用无酶细胞解离液(Versene solution，购自Life technology公司)处理和收集细胞。用HBSS缓冲液洗涤细胞2次，进行细胞计数后将细胞用HBSS缓冲液稀释至2╳10 6细胞每毫升，加入1％山羊血清封闭液，所述百分比为质量百分比。冰上孵育30分钟，然后用HBSS缓冲液离心洗涤2次。将收集的细胞用FACS缓冲液(含有1％BSA的HBSS，所述百分比为质量百分比)悬浮至2╳10 6细胞/mL，按每孔100微升加入到96孔FACS反应板中，加入实施例2所得的纯化的4-1BB抗体待测样品每孔100微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升荧光(Alexa 488)标记的二抗(购自Invitrogen)，冰上孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛] 悬浮细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图8-9和表12-13所示，结果说明，待测抗体可结合细胞表面的4-1BB蛋白。其中IgG对照为人IgG，表中的数据为MFI所测细胞群的平均荧光强度值。
表12 FACS检测4-1BB抗体与CHOK1-h4-1BB的结合反应
Figure PCTCN2018114641-appb-000016
表13 FACS检测4-1BB抗体与CHOK1-c4-1BB的结合反应
Figure PCTCN2018114641-appb-000017
Figure PCTCN2018114641-appb-000018
实施例5
在NFκB荧光素酶报告基因实验中检测4-1BB抗体活性
在人4-1BB蛋白阳性的HEK293稳定细胞株(制备方法见实施例1)中进一步转染NFκB荧光素酶报告基因质粒，制成人4-1BB蛋白阳性的带NFκB荧光素酶报告基因的稳定细胞株。用抗人或大鼠Fc F(ab’) 2对待检测抗体进行交联，然后将交联好或没有进行交联的抗体加入此稳定细胞株中进行细胞培养。5小时后加入荧光素酶检测试剂，读取荧光值。结果如图10A-10B和表14所示，表14说明，全人4-1BB抗体对4-1BB蛋白下游NFκB信号通路有很强的激活，且在抗体交联后抗体激活能力进一步增强(EC50值降低)。这表明4-1BB抗体在体内应用时，可在达到特定区域，如肿瘤组织后，通过表达FcR的细胞介导进行交联，从而增强抗体活性和安全性。表14所示为全人4-1BB抗体组相对对照组(人IgG)的荧光值倍数和EC50值。
表14 全人源4-1BB抗体对4-1BB蛋白下游NFκB转录因子的激活
Figure PCTCN2018114641-appb-000019
实施例6
检测4-1BB抗体阻断4-1BB蛋白与其受体4-1BBL的结合
用表达4-1BB蛋白的CHO细胞检测4-1BB抗体阻断4-1BB蛋白与其受体4-1BBL的结合。
按照实施例4B部分处理CHOK1-h4-1BB细胞株得到2╳10 6细胞/mL的单悬细胞，按每孔100微升加入到96孔FACS反应板中，加入实施例3所得的纯化的4-1BB抗体待测样品每孔50微升，再加入his-muCD8a-4-1BBL ECD(制作方法见实施例1)融合蛋白50微升，冰上孵育2小时。用FACS缓冲液离心洗涤2次，加入每孔100微升抗小鼠CD8a的荧光(Alexa 488)标记的二抗，冰上孵育1小时。用FACS缓冲液离心洗涤3次，加入每孔100微升固定液[4％(v/v)多聚甲醛]悬浮细胞，10分钟后用FACS缓冲液离心洗涤2次。用100微升FACS缓冲液悬浮细胞，用FACS(FACS Calibur，购自BD公司)检测和分析结果。结果如图11和表15所示，表15说明，待测抗体可抑制4-1BB配体蛋白与4-1BB的结合。其中IgG对照为人IgG，表中的数据为相对于对照组(人IgG)全人4-1BB抗体对配体结合的抑制率。
表15 全人源4-1BB抗体对4-1BB蛋白与其受体4-1BBL的结合的抑制
Figure PCTCN2018114641-appb-000020
实施例7
T淋巴细胞刺激实验
(一)Ficoll分离全血获取外周血单核淋巴细胞PBMC。
将新鲜获取的全血用磷酸缓冲液PBS以1:1的体积比例稀释得稀释后的全血，用无菌吸管轻轻将稀释后的全血铺平在Ficoll液面(购自GE Healthcare)，Ficoll与稀释后的全血的体积比为3:4，避免震荡混匀，以400g转速室温20℃梯度离心30分钟，离心后的离心管分为三层，上层为血浆，中间乳白色分层即为单核淋巴细胞。用无菌吸管轻轻吸取中间层细胞，收集至新的离心管，用PBS磷酸缓冲液稀释至三倍体积，100g转速室温离心10分钟，弃上清。将淋巴细胞用PBS磷酸缓冲液重悬至10mL，重复前面步骤取出血小板。最后将淋巴细胞重悬至10mL含有10％胎牛血清的多组份RPMI1640培养基(购自Invitrogen)备用，即为外周血单核淋巴细胞PBMC，所述百分比为质量百分比。
(二)T淋巴细胞刺激实验
构建anti-CD3单链抗体在细胞膜上表达的CHOK1细胞株：为使anti-CD3(OKT3)(参见Kipriyanov et al.1997,PEDS 10:445-453)嵌合的ScFv能锚定在细胞膜上，将该ScFv与mouse CD8a(NCBI Accession No:NP —001074579.1)的C端113-220氨基酸序列连接，构建成质粒pIRES-OS8。质粒pIRES-OS8的制备方法参见Joe Sambrook，Molecular Cloning:A Laboratory Manual。将上述质粒pIRES-OS8转染CHOK1细胞，按实施例1中所述的质粒转染细胞的方法制备稳定传代细胞株CHOK1-OS8，并将其作为T淋巴细胞刺激因子。同时配制等体积比稀释的待测实施例2所得的纯化的4-1BB抗体，得待测样品溶液。
用T细胞提纯试剂盒(购自Stemcell)按照厂家提供的方法从将实施例7步骤(一)获得的人外周血单核淋巴细胞中纯化人CD3阳性T细胞。将此CD3阳性的T细胞与细胞刺激因子CHOK1-OS8加入96孔细胞培养板，同时加入用抗人或大鼠Fc F(ab’) 2交联好的待检测抗体，最终每个反应孔有200微升的终体积。于37℃、5％CO 2培养箱共培养72小时后收集上清，将得到的细胞上清于-20℃冻存，上所述百分比为体积百分比。
(三)细胞上清中细胞因子伽马干扰素(IFN-γ)或白介素IL-2酶联免疫吸附检测。
细胞上清中细胞因子伽马干扰素(IFN-γ)或白介素IL-2酶联免疫吸附检测使用R&D system相关检测试剂盒Human IFN-gamma Quantikin(DIF50)和Quantikine ELISA human IL-2(S2050)，并按照说明书操作。除检测抗体外的所有检测试剂均由检测试剂盒提供。
测定细胞上清中细胞因子伽马干扰素(IFN-γ)或白介素IL-2含量的酶联免疫吸附检测采用双抗夹心ELISA试剂盒(购自R&D Systems，IFN-γCat#DIF50和IL-2Cat#S2050)。实验操作严格按照试剂盒说明书要求，所有检测试剂均由试剂盒提供。具体实验简述如下：将IFN-γ或IL-2多克隆抗体包被于ELISA微孔板上，将实施例5步骤(二)获得的细胞上清液作为待测样品，加入标准品和待测样品室温孵育2小时。每孔加入400微升洗液，重复洗板4次；再加入抗人IFN-γ或IL-2的辣根过氧化物酶标抗体，室温孵育2小时，与微孔板上的IFN-γ或IL-2形成免疫复合物，清洗微孔；加入底物显色，避光室温30分钟，最终加入终止液，用酶标仪测定A450nm吸光度。
检测抗体在实施例7步骤(二)所述T细胞刺激实验中对IFN-γ分泌的影响。结果如图12，和表16所示，表16说明，在PBMC淋巴细胞刺激试验中待测抗体可使PBMC的IFN-γ分泌增强。其中hIgG对照为人IgG，表中的数据为IFN-g值(pg/mL)。
表16 4-1BB全人源抗体在T细胞刺激试验中对IFN-γ分泌的影响
Figure PCTCN2018114641-appb-000021
检测抗体在实施例7步骤(二)所述T细胞刺激实验中对IL-2分泌的影响。表17和图13说明，全人源化4-1BB抗体在T细胞刺激试验中能够刺激IL-2分泌。其中IgG对照为人IgG(hIgG)，表中数据为IL-2值(pg/mL)。
表17 4-1BB全人源抗体在T细胞试验中对IL-2分泌的影响
Figure PCTCN2018114641-appb-000022
实施例8
抗4-1BB抗体亲和力的分析测定
使用Biacore T200仪器(购自)进行亲和常数的测定。具体操作和方法根仪器说明书和厂家提供的详细方法。用抗人Fc标记的传感器结合抗4-1BB全人源抗体，检测五个不同梯度的带his标签的人4-1BB蛋白抗原与之结合解离情况。然后用软件拟合得到解离常数与结合常数，亲和力常数为解离常数与结合常数的比值。亲和力检测结果如表18所示。
表18 4-1BB抗体对人4-1BB的亲和常数
Figure PCTCN2018114641-appb-000023
实施例9 抗4-1BB抗体小鼠体内抗肿瘤活性评价
采用MC38同系小鼠模型，使用人4-1BB基因敲入的C57BL/6小鼠评价抗体在小鼠体内的抗肿瘤活性。实验设计如下：
选择人4-1BB基因敲入的C57BL/6小鼠，分为4组，每组6只，使用Utomilumab,Urelumab和同型抗体hIgG4作为对照，给药样品为113F6C6抗体。给药途径为腹腔注射，给药剂量为3mg/kg(hIgG对照)、1mg/kg(Utomilumab、Urelumab和113F6C6)，在0、4、7、11、14、18天腹腔注射，每周两次测量， 测量肿瘤体积，小鼠体重及小鼠存活率。肿瘤体积(Tumor volume)计算公式为1/2*L 长*L 短 2。
结果如表20和图15，16所示，本发明的抗体(113F6C6)能够显著抑制肿瘤生长，抑制效果显著优于对照抗体Utomilumab和Urelumab。
表20 4-1BB抗体对小鼠MC38肿瘤的生长抑制总结
Figure PCTCN2018114641-appb-000024
对比例
以与本申请的抗体靶点相同的辉瑞公司的Utomilumab抗体(PF-05082566)作为对比抗体，进行对比试验，试验方法与实施例7完全一致。
结果如图14和表19所示，本申请的4-1BB抗体相对于Utomilumab，能刺激T细胞分泌更多的IFN-γ细胞因子，对T细胞的激活更强。
表19 抗体在T细胞刺激试验中对IFN-γ分泌的影响
Figure PCTCN2018114641-appb-000025
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.:10或SEQ ID NO.:2所示的CDR1，
    SEQ ID NO.:11或SEQ ID NO.:3所示的CDR2，和
    SEQ ID NO.:12或SEQ ID NO.:4所示的CDR3，
    其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代1个氨基酸并能够保留4-1BB结合亲和力的衍生序列。
  2. 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。
  3. 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：
    SEQ ID NO.:14或SEQ ID NO.:6所示的CDR1’，
    SEQ ID NO.:15或SEQ ID NO.:7所示的CDR2’，和
    SEQ ID NO.:16或SEQ ID NO.:8所示的CDR3’。
  4. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。
  5. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的重链可变区；和/或
    (2)如权利要求3所述的轻链可变区；
    或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。
  6. 如权利要求5所述的抗体，其特征在于，所述抗体的重链可变区序列如SEQ ID NO.:9所示；和/或所述的抗体的轻链可变区序列如SEQ ID NO.:13所示。
  7. 如权利要求5所述的抗体，其特征在于，所述抗体的重链可变区序列如SEQ ID NO.:1所示；和/或所述的抗体的轻链可变区序列如SEQ ID NO.:5所示。
  8. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  9. 一种CAR构建物，其特征在于，所述的CAR构建物的单克隆抗体抗原结合区域的scFV段为特异性结合于4-1BB的结合区，并且所述scFv具有如权利要求1所述的重链可变区和权利要求3所述的轻链可变区。
  10. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、如权利要求8所述的重组蛋白、或其组合，其特征在于，所述活性成分用于(a)制备预防和/或治疗4-1BB相关疾病的药物；和/或(b)制备检测试剂或试剂盒。
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