WO2022121239A1 - 一种双特异性抗体及其应用 - Google Patents

一种双特异性抗体及其应用 Download PDF

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WO2022121239A1
WO2022121239A1 PCT/CN2021/094672 CN2021094672W WO2022121239A1 WO 2022121239 A1 WO2022121239 A1 WO 2022121239A1 CN 2021094672 W CN2021094672 W CN 2021094672W WO 2022121239 A1 WO2022121239 A1 WO 2022121239A1
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antibody
seq
cancer
il15rα
bispecific antibody
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French (fr)
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屈向东
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启愈生物技术(上海)有限公司
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Priority to EP21901953.6A priority Critical patent/EP4261231A1/en
Priority to US18/256,679 priority patent/US20240043567A1/en
Priority to JP2023535529A priority patent/JP2023552851A/ja
Publication of WO2022121239A1 publication Critical patent/WO2022121239A1/zh

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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
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    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
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    • 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
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    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • 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
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the invention belongs to the field of biomedicine, relates to a bispecific antibody, and also relates to the application of the bispecific antibody.
  • Bispecific antibodies also known as bifunctional antibodies, can simultaneously recognize and bind two different antigens and epitopes, and block two different signaling pathways to exert their effects. Compared with ordinary antibodies, BsAb adds a specific antigen-binding site, which shows the following advantages in treatment:
  • Bispecific antibodies have two antigen-binding arms, one of which binds to the target antigen, and the other binds to effector cells. Tag antigen binding, which activates effector cells to target and kill tumor cells.
  • the two bispecific antibody products that have been approved for marketing belong to this category.
  • the catumaxomab developed by Trion Pharma can target the tumor surface antigen EpCAM and the T cell surface receptor CD3, while the blinatumomab developed by Micromet and Amgen can simultaneously bind CD19 and CD3. Both of them achieve the purpose of treating tumors by activating and recruiting killer T cells.
  • Dual-target signal blocking, exerting unique or overlapping functions, effectively preventing drug resistance: simultaneously combining dual targets and blocking dual signaling pathways is another important mechanism of action of bispecific antibodies.
  • Receptor tyrosine kinases RTKs
  • RTKs are the largest class of enzyme-linked receptors and play an important regulatory role in the process of cell proliferation, such as the Her family.
  • RTKs are abnormally highly expressed on the surface of tumor cells, leading to malignant proliferation of tumor cells, so they are also important targets for tumor therapy.
  • Single-target monoclonal antibodies against RTKs have been widely used in tumor therapy, however, tumor cells can activate intracellular activation by switching signaling pathways or by homologous or heterodimerization of HER family members themselves or between different members Signal for immune escape. Therefore, using bispecific antibody drugs to block two or more RTKs or their ligands at the same time can reduce tumor cell escape and improve the therapeutic effect.
  • bispecific antibodies can bind to different antigens. Binding specificity and targeting of cancer cells, reducing side effects such as off-target.
  • BiTE Effectively reduce the cost of treatment: Taking BiTE as an example, compared with traditional antibodies, it has strong competitiveness in terms of tissue penetration rate, tumor cell killing efficiency, off-target rate and clinical indications, and has significant clinical advantages. Especially in terms of dosage, because its therapeutic effect can reach 100-1000 times that of ordinary antibodies, the minimum dosage can be 1/2000 of the original, which significantly reduces the cost of drug treatment. Compared to combination therapy, the cost of bispecific antibodies is also much lower than that of two single-agent combination therapy.
  • the European Union approved the first therapeutic bispecific antibody, Catumaxomab (targeting CD3 and EpCAM) from TrionPharma, for the treatment of cancerous ascites.
  • the FDA quickly approved Amgen based on The bispecific antibody drug Blinatumomab (targeting CD3 and CD19) developed by the technology is used for the treatment of acute B lymphocytic leukemia, and it is also the first approved drug targeting CD19.
  • Amgen has more than a dozen in clinical development across a range of hematological malignancies and solid tumors. molecular.
  • AMG420 which targets BCMA/CD3, has been granted fast track status by the FDA.
  • PSMA prostate-specific membrane antigen
  • Bispecific antibodies themselves have many advantages and can be used in a variety of therapeutic areas, such as cancer, chronic inflammatory diseases, autoimmune diseases, and infections.
  • the main technical difficulty in producing bispecific antibodies is to obtain correctly paired bispecific antibodies, especially for asymmetric bispecific antibodies containing an Fc region (IgG-like), which are faced with both HC/HC and LC/HC mismatches.
  • KiH, ART-Ig and BiMab technologies were subsequently developed to reduce HC/HC mismatches.
  • the CrossMAb technology in which a pair of heavy chain and light chain variable domains VL and VH are replaced with each other, and the constant domains CL and CH1 are also replaced with each other
  • YBODY technology one side uses traditional Fab to form a target antigen A Monovalent unit Fab-Fc, the other side uses single-chain antibody to form a single-chain unit targeting antigen B (ScFv-Fc), WuXiBody (TCR constant region replaces heavy chain constant region CH1 and light chain constant region CL) and common light chain technology Used to reduce LC/HC mismatches.
  • ScFv-Fc single-chain unit targeting antigen B
  • WuXiBody TCR constant region replaces heavy chain constant region CH1 and light chain constant region CL
  • common light chain technology Used to reduce LC/HC mismatches.
  • ErbB family of receptor tyrosine kinases are important mediators of cell growth, differentiation and survival.
  • This receptor family includes four unique members, including epidermal growth factor receptor (EGFR or ErbB1), Her2 (ErbB2), Her3 (ErbB3) and Her4 (ErbB4 or tyro2).
  • EGFR epidermal growth factor receptor
  • Her2 ErbB2
  • Her3 Her3
  • Her4 ErbB4 or tyro2
  • Her2 is a transmembrane, surface-bound receptor tyrosine kinase and is normally involved in signal transduction pathways leading to cell growth and differentiation.
  • Her2 overexpression may lead to normal cellular dysfunction and is often closely associated with tumor initiation and progression. Homologous or heterologous polymerization of Her2 can lead to phosphorylation of receptor tyrosine residues and initiate numerous signaling pathways leading to cell proliferation and tumorigenesis. As a prognostic and predictive biomarker, amplification or overexpression of the Her2 gene occurs in approximately 15-30% of breast cancers and 10-30% of gastric/esophageal cancers. Her2 overexpression has also been observed in other tumors such as ovarian, endometrial, bladder, lung, colon and head and neck tumors.
  • trastuzumab recognizes the juxtamembrane epitope of Her2 extracellular domain IV. Specifically, an epitope consisting of 3 loops (557-561, 570-573 and 593-603) at the C-terminus of the IV portion of the Her2 ectodomain. Because the epitope can be near or interact directly with the binding domain of its dimerization partner, binding of trastuzumab to the epitope can induce steric hindrance that inhibits the dimerization process. In addition, the binding of trastuzumab also protects the extracellular domain of the Her2 receptor from protease attack and hydrolysis.
  • Trastuzumab is currently used as a first-line drug for the treatment of breast cancer, and it is effective in the treatment of metastatic breast cancer with overexpression of Her2, and the objective response rate of single-agent first-line therapy is 30-50%; however, in the treatment of patients with low Her2 expression Metastatic breast cancer is less effective, and resistance develops in many patients where the antibody is initially effective within 1 year.
  • Her2, along with other members of the family (Her1, Her3, and Her4), can form ligand-dependent or ligand-independent heterodimers that activate downstream pathways that then lead to tumor cell proliferation, whereas trastuzumab cannot Inhibits heterodimer formation, so this may be one of the reasons for the development of drug resistance.
  • Pertuzumab is a humanized monoclonal antibody specifically designed to prevent the HER2 receptor from pairing (dimerization) with other HER receptors (EGFR/HER1, HER3 and HER4) on the cell surface ), a process believed to play a role in tumor growth and survival.
  • Pertuzumab has a certain therapeutic effect on advanced prostate cancer, non-small cell lung cancer, ovarian cancer and breast cancer, but its therapeutic effect also depends on the expression level of Her2.
  • Pertuzumab recognizes a key site for heterodimerization of Her2 extracellular domain II, and the epitope thus recognized is located in the segment 245-311 in the center of the II subregion, and the key residues are H245, V286, S288, L295, H296 and K311.
  • L295 and H296 are the key sites mediating the heterodimerization of Her2 and Her3, and the L295A/H296A double mutation can completely block the heterodimerization of Her2/Her3 (Franklin, M.C. et al., Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex. Cancer Cell, 2004.5(4):pp.317-28). Therefore, Pertuzumab can be used to effectively inhibit the formation of Her2/Her3 heterodimers, but it does not show a significant inhibitory effect on the formation of EGFR/Her2 heterodimers.
  • IL-15 is a 14–15kDa long cytokine that is important for NK cell, NKT cell and memory CD8 + T cell function.
  • the content of IL-15 in the body is very small, but it is transduced and transported to the target cells by combining with its receptor IL-15R ⁇ to produce a complex IL-15 superagonist (IL-15SA) with extremely high biological potency.
  • IL-15SA strongly activates IL-15-responsive cells, especially NK cells, thereby promoting antitumor and antiviral functions.
  • IL-15 acts differently from other cytokines.
  • IL-15 receptor alpha is expressed on IL-15-producing cells (such as macrophages and dendritic cells) and forms IL-15SA with IL-15 to deliver signals to express IL-15R ⁇ (also known as IL-2R ⁇ ) and common ⁇ chain (shared with IL-2, IL-4, IL-7, IL-9 and IL-21) NK, NKT and memory CD8 + T cells, very It is possible that this unique presentation confers IL-15 the ability to mediate its unique functions.
  • Mouse IL-15 shares 70% amino acid sequence homology with human IL-15, and human IL-15 and mouse IL-15 also have similar trans-expression patterns, signaling pathways and biological activities.
  • IL-15 is expressed in many cell types and tissues, including monocytes, macrophages, DCs, keratinocytes, fibroblasts, muscle cells, and neural cells. IL-15 plays an important role in innate and adaptive immunity as a pleiotropic cytokine.
  • Trans-expressed IL-15/IL-15R ⁇ signaling induces the recruitment and activation of JAK1 and JAK3 in response to the ⁇ and ⁇ chains expressed on cells, and the activated JAK1 and JAK3 further phosphorylate STAT3 and STAT5.
  • STAT3 and STAT5 are phosphorylated to form homodimers that translocate to the nucleus and promote transcription of target genes.
  • IL-15 signaling stimulates a series of downstream responses that induce cell growth, reduce apoptosis, and enhance activation and metastasis of immune cells.
  • IL-15R ⁇ moderate-affinity
  • IL-15R ⁇ ⁇ receptor complex
  • Lck tyrosine kinases
  • IL-15 Since IL-15 has similar immunological properties to IL-2: it induces the proliferation and survival of T cells, promotes the proliferation and differentiation of NK cells, and induces the production of cytotoxic T lymphocytes. But unlike IL-2, IL-15 has no obvious effect on Treg cells and does not cause capillary leak syndrome in mice or non-human primates (NHP), so compared with IL-2, IL-15 is a better choice for tumor immunotherapy.
  • Rhesus monkey IL-15 (rIL-15) was the first form of IL-15 used in in vivo experiments, and researchers believe that rIL-15 can preferentially bind to cell surface IL-15R ⁇ .
  • Heterodimeric IL-15/IL-15R ⁇ is the native form of IL-15 cleaved from cells and can respond independently of cell-stimulatory interactions, and Novartis is currently developing this form of molecule in solid tumors (NIZ985 ) clinical trials.
  • the RLI developed by Cytune is a fusion protein composed of IL-15 linked to the Sushi domain of IL-15R ⁇ , which acts as a soluble IL-15 agonist.
  • the IL-15/IL-15R ⁇ -Fc complex produced by mixing a commercially available IL-15R ⁇ -Fc chimeric fusion protein with rIL-15, has been used extensively in preclinical studies.
  • cytokines have the disadvantage of poor targeting of single-drug administration. Only high-concentration administration can achieve anti-tumor effects, while high-concentration administration will produce immunosuppressive effects and high toxicity. Moreover, the activation of the immune system by non-targeted cytokines is systemic, and the immune system is widely activated with lethal side effects. In addition, because cytokines are small molecular weight proteins and do not have the protective mechanism of antibodies in vivo, simple cytokines often have a short half-life and require repeated high-dose administration in a short period of time. At present, most clinical research drugs use PEGylation or Fc fusion to improve the half-life of cytokines. Although the half-life is prolonged, it still cannot solve the problem of poor targeting of cytokines.
  • a bispecific antibody comprising: a) a light chain and a heavy chain of a first antibody that specifically binds to a first antigen; and b) a first antibody that specifically binds to a second antigen
  • the light chain and heavy chain of the secondary antibody; IL15 and IL15R ⁇ are introduced into the two chains of the primary antibody respectively (for example, IL15 and IL15R ⁇ replace CL1 and CH1, respectively), and the high affinity of IL15 and IL15R ⁇ is used to form an IL15/IL15R ⁇ complex, thereby realizing Correct pairing of the primary antibody light/heavy chain to resolve bispecific antibody light/heavy chain mismatches.
  • the primary antibody was further enhanced by adding one or more pairs of disulfide bonds between VH1 and VL1 and between IL15 and IL15R ⁇ by mutating the amino acid sequences of the variable domains of the primary antibody VH1, VL1, IL15 and IL15R ⁇ .
  • the light chain/heavy chain binding activity can effectively overcome the bispecific antibody light chain/heavy chain mismatch problem.
  • a bispecific antibody constructed by the present invention comprises: a) a light chain and a heavy chain of a first antibody that specifically binds to a first antigen; and b) a light chain and a second antibody that specifically binds to a second antigen and Heavy chain; wherein the two chains in the primary antibody comprise IL15 and IL15R ⁇ , respectively, and are capable of forming an IL15/IL15R ⁇ complex.
  • the IL15 comprises a mutant capable of binding IL15R ⁇
  • the IL15R ⁇ comprises a mutant capable of binding IL15.
  • the first antibody variable domains VH1 and VL1 are linked or polymerized, and the IL15 and IL15R ⁇ are linked or polymerized.
  • the IL15 and IL15R ⁇ replace the constant domains CH1 and CL of the first antibody.
  • variable domains VL1 and VH1 of the first antibody are linked to the N-termini of IL15 and IL15R ⁇ .
  • variable domains VL1 and VH1 of the first antibody are linked to the C-termini of IL15 and IL15R ⁇ .
  • variable domains VL1 and VH1 of the first antibody exchange positions.
  • the light chain of the first antibody (from the N-terminus to the C-terminus) and the heavy chain of the first antibody (from the N-terminus to the C-terminus) are polymerized in the same direction.
  • the light chain of the first antibody (from the C-terminus to the N-terminus) and the heavy chain of the first antibody (from the N-terminus to the C-terminus) are reversely polymerized.
  • variable domains VL1 and VH1 of the first antibody are linked to IL15 and IL15R ⁇ through amino acid linker sequences with low immunogenicity.
  • the IL15 comprises the following mutations, and the counting method starts from the first amino acid of IL15 shown in SEQ ID No. 1 and counts it as the first position.
  • the IL15 and IL15R ⁇ comprise the combination of mutations shown below, and the counting method starts from the first amino acid of IL15 shown in SEQ ID No.1 as the first position; IL15R ⁇ shown in SEQ ID No.3 is the first An amino acid starts at position 1.
  • variable domains VH1 and VL1 of the first antibody have one or more pairs of disulfide bonds.
  • VH1 and VL1 comprise the following mutant combinations, counted according to EU.
  • the first and second antibody heavy chains respectively comprise A and B chains with different mutations in the Fc segment, and the A and B chains have the following combinations of mutations, according to EU counts.
  • the Fc segment comprises Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc and mutants thereof.
  • one chain in the A chain and the B chain of the Fc segment, one chain can bind protein A, and the other chain is a mutant that cannot bind protein A, and the mutation comprises H435R or H435R/Y436F, according to EU count.
  • the first antigen is CD3, CD20, CD19, CD30, CD33, CD38, CD40, CD52, slamf7, GD2, CD24, CD47, CD133, CD217, CD239, CD274, CD276, PD-1 , CEA, Epcam, Trop2, TAG72, MUC1, MUC16, mesothelin, folr1, CLDN18.2, PDL1, EGFR, EGFR VIII, C-MET, HER2, FGFR2, FGFR3, PSMA, PSCA, EphA2, ADAM17, 17-A1, NKG2D ligands, MCSP, LGR5, SSEA3, SLC34A2, BCMA, GPNMB, IL-6R, IL-2R, CCR4, VEGFR-2, CD6, CTLA-4, integrin ⁇ 4, DNA/histone complex, PDGFR ⁇ , NeuGcGM3, IL- Any one of 4R ⁇ and IL-6R ⁇ , and the second antigen is a different epitop
  • the first/second antibody is a chimeric, humanized or fully human antibody.
  • the bispecific antibody has the structure shown in formula I:
  • VL1 or VH1 is linked to the N-terminus or C-terminus of IL15 or IL15R ⁇ through L1;
  • Chain 2 arranged from N-terminal to C, VH1 or VL1, L2, IL15R ⁇ or IL15, L3, Fc;
  • Chain 3 The secondary antibody heavy chain is arranged from N-terminus to C-terminus, VH2-CH1-Fc;
  • the secondary antibody light chain is arranged from N-terminus to C-terminus, VL2-CL;
  • L1, L2 and L3 are each independently a bond or linker sequence
  • the bispecific antibody has the structure shown in formula II:
  • Chain 1 IL15 or IL15R ⁇ is linked to the N-terminus or C-terminus of VL1 or VH1 through L1;
  • Chain 2 arranged from N-terminal to C, IL15R ⁇ or IL15, L2, VH1 or VL1, L3, Fc;
  • Chain 3 The secondary antibody heavy chain is arranged from N-terminus to C-terminus, VH2-CH1-Fc;
  • the secondary antibody light chain is arranged from N-terminus to C-terminus, VL2-CL;
  • L1, L2 and L3 are each independently a bond or linker sequence.
  • chain 1 and chain 2 include the following combinations:
  • the bispecific antibody has the structure shown in formula III:
  • Chain 1 Arranged from N-terminus to C-terminus, VL1-L1-IL15, L1 is a low immunogenic amino acid linker sequence;
  • Chain 2 arranged from N-terminal to C-terminal, VH1-L2-IL15R ⁇ -L3-Fc, L2, L3 are low immunogenic amino acid linker sequences;
  • Chain 3 arranged from N-terminal to C-terminal, VH2-CH1-Fc;
  • Chain 4 Arranged from N-terminal to C-terminal, VL2-CL;
  • the L1, L2 and L3 comprise glycine (G) and serine (S) residues.
  • the L1, L2 and L3 comprise one or more GGGGS repeats.
  • the IL15 sequence is shown in SEQ ID No. 1 or SEQ ID No. 2; the IL15R ⁇ sequence is shown in SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 2 ID No.6, SEQ ID No.7, SEQ ID No.8 or SEQ ID No.9.
  • the Fc sequence is shown in SEQ ID No.18 or SEQ ID No.19.
  • the first antigen and the second antigen are antigens that bind to two different epitopes of Her2, respectively.
  • the bispecific antibody is obtained by fusing the sequences of SEQ ID No.10, SEQ ID No.11, SEQ ID No.13, and SEQ ID No.12.
  • the first antigen and the second antigen are CS1 antigen and CD38 antigen, respectively.
  • the bispecific antibody is obtained by fusing the sequences of SEQ ID No.14, SEQ ID No.15, SEQ ID No.17 and SEQ ID No.16.
  • a second aspect of the present invention provides a pharmaceutical composition comprising:
  • the pharmaceutical composition further contains other drugs for treating cancer (or tumor), such as chemotherapy drugs.
  • the third aspect of the present invention provides the application of the bispecific antibody of the first aspect of the present invention and the pharmaceutical composition of the sixth aspect of the present invention in the preparation of a drug for the treatment of cancer (or tumor), infection or immune regulation disease .
  • the fourth aspect of the present invention provides the application of the bispecific antibody according to the first aspect of the present invention in preparing a drug for inhibiting tumor growth.
  • the cancer or tumor comprises cancer or tumor from the following sites: colorectal, breast, ovary, pancreas, stomach, prostate, kidney, cervix, thyroid, endometrium, uterus, bladder, neuroendocrine , head and neck, liver, nasopharynx, testes.
  • the cancer comprises: bone marrow cancer, lymphoma, leukemia, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, carina Dermatofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • the fifth aspect of the present invention provides the use of the bispecific antibody binding to the Her bi-epitope according to the first aspect of the present invention in preparing a reagent or kit for diagnosing HER2 positive tumors (eg breast cancer, gastric cancer).
  • HER2 positive tumors eg breast cancer, gastric cancer.
  • a bispecific antibody that introduces IL15 and IL15R ⁇ into the two chains of the primary antibody (for example, IL15 and IL15R ⁇ replace CL1 and CH1, respectively), utilizes IL15 and IL15R ⁇ , respectively.
  • the high affinity of IL15R ⁇ forms the IL15/IL15R ⁇ complex, thereby realizing the correct pairing of the light chain/heavy chain of the primary antibody, and solving the problem of mismatching the light chain/heavy chain of the bispecific antibody.
  • the primary antibody was further enhanced by adding one or more pairs of disulfide bonds between VH1 and VL1 and between IL15 and IL15R ⁇ by mutating the amino acid sequences of the variable domains of the primary antibody VH1, VL1, IL15 and IL15R ⁇ .
  • the binding activity between light chain/heavy chain can effectively overcome the challenges of light chain/heavy chain mismatch, high by-products, poor stability and other challenges in the preparation of bispecific antibodies, and finally prepare correctly paired bispecific targeting cytokines multifunctional antibody.
  • the development cycle of bispecific antibodies is shortened and the production cost is reduced.
  • the bispecific antibody constructed in the present invention has IL-15/IL-15R ⁇ activity while overcoming the problem of light chain/heavy chain mismatch, can target cytokines to tumor sites, and can specifically target tumors in tumors.
  • the site expands and activates T cells and NK cells in PMBC, and increases the number of immune cells and the release of killer cytokines, killing tumor cells more effectively and reducing the dosage.
  • the present invention successfully constructs correctly paired Trastuzumab/Pertuzumab/IL15 bispecific antibodies, utilizes the targeting of Her2 bispecific antibodies to target IL15 to tumor tissues, stimulates immune responses, and kills Her2 positive tumors with multiple mechanisms.
  • the present invention has successfully constructed a correctly paired Elotuzumab/Daratumumab/IL15 bispecific antibody that binds to the CS1/CD38 antigen, has the CS1 antigen binding capacity equivalent to the CS1 monoclonal antibody, and has the CD38 antigen equivalent to the CD38 monoclonal antibody. Combined with the binding ability, it has great potential in the treatment of hematological tumors.
  • the “antibody” in the present invention includes not only complete antibodies, but also fragments, polypeptide sequences, derivatives and analogs thereof with antigen-binding activity.
  • the antigen-binding fragment refers to one or more portions of a full-length antibody that retain the ability to bind an antigen (eg, HER2), competing with the intact antibody for specific binding to the antigen.
  • an antigen eg, HER2
  • Recombinant DNA techniques Or by enzymatic or chemical cleavage of intact antibody to produce antigen-binding portion.
  • antigen-binding portion includes Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, Single-chain antibodies (eg, scFv), chimeric antibodies, comprising at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • CDR complementarity determining region
  • Fd fragment means an antibody fragment consisting of VH and CH1 domains
  • Fv fragment means an antibody fragment consisting of the VL and VH domains of a one-armed antibody
  • dAb fragment means an antibody fragment consisting of Antibody fragments consisting of VH domains (Ward et al., Nature 341:544-546 (1989))
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F(ab ')2 fragment means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • epitope refers to a site on an antigen that is specifically bound by an immunoglobulin or an antibody.
  • Epitopes are also known in the art as “antigenic determinants”. Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear” “or conformational”. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.
  • the "IL-15” of the present invention can be any IL-15 or a mutant capable of binding IL15R ⁇ , such as human IL-15 or non-human mammalian or non-mammalian IL-15.
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, etc.; preferably human IL-15.
  • the term "mutant capable of binding to IL15R ⁇ ” refers to an increased or decreased affinity for IL-15 and its receptor obtained by one or more amino acid substitutions, additions or deletions mutations, or an increase or decrease in the activity of stimulating T cells or NK cells. mutant molecules.
  • the "IL-15" of the present invention is preferably its variant form, more preferably the amino acid sequence is SEQ ID No.1 or SEQ ID No.2.
  • the "IL-15R ⁇ ” described in the present invention can be any species of IL-15R ⁇ or a mutant capable of binding IL15R ⁇ , such as human IL-15R ⁇ or non-human mammalian IL-15R ⁇ or non-mammalian IL-15R ⁇ .
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens, and the like.
  • human IL-15R ⁇ more preferably human IL-15R ⁇ extracellular domain fragment, abbreviated as IL-15R ⁇ ECD (see database UniProtKB, accession number Q13261, 31-205aa).
  • mutant capable of binding IL15R ⁇ refers to a functional mutant formed by one or more amino acid deletion, insertion or substitution mutations in IL-15R ⁇ , which has the ability to bind to its ligand molecule such as IL-15, preferably human IL
  • the -15R ⁇ molecule is more preferably a shortened form of the human IL-15R ⁇ extracellular domain fragment, that is, a molecule with human IL-15 receptor ⁇ activity obtained by one or more amino acid deletion mutations from the C-terminal of the extracellular domain fragment, preferably retaining 65 - Deletion mutant forms of 178 amino acids, such as IL-15R ⁇ (SEQ ID NOs: 3-9).
  • the "Fc segment” in the present invention refers to the C-terminal region of immunoglobulins, which has no antigen-binding activity, is the site where antibody molecules interact with effector molecules and cells, and is an antibody heavy chain Fc region polypeptide comprising two disulfide linkages. of dimer molecules. Fc regions can be produced by papain digestion or IdeS digestion into trypsinization of intact (full length) antibodies or can be produced recombinantly.
  • the "Fc portion” preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of IgG.
  • Fc heterodimer mutation refers to a change in Fc structure or function by the presence of one or more amino acid substitution, insertion or deletion mutations at appropriate sites in the Fc. Space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, etc. can be formed between the mutant-designed Fc variants. Interaction between Fc mutants contributes to the formation of stable heterodimers. Preferred mutagenesis designs are those in the "Knob-in-hole" format.
  • the Fc of the present invention may also have other mutations that lead to changes in its function, such as glycosylation mutations, Fc ⁇ R binding region mutations (to adjust ADCC activity), and amino acid mutations to improve antibody stability.
  • Fc includes Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc and mutations thereof, wherein one chain can bind proteinA, and the other chain is a mutant that cannot bind proteinA, including mutation H435R or H435R/Y436F , according to EU counts.
  • the "heterodimer” of the present invention is preferably the product of gene co-expression.
  • gene co-expression refers to the co-expression of multiple genes in a cell and the simultaneous appearance of their products. These genes can be co-existing and individually or jointly controlled expression.
  • co-expression in one eukaryotic cell is preferred.
  • the gene expression product obtained by co-expression is favorable for the efficient and simple formation of complexes; in the present invention, it is favorable for the formation of heterodimers.
  • the mutation design technology of Fc variants has been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion proteins.
  • the representative is the "Knob-in-Hole" form proposed by Cater et al. (Protein Engineering vol. 9no. 7pp617-621, 1996); Amgen company technicians use electrostatic steering to form an Fc-containing heterodimeric form (US2010286374A1); The heterodimeric form (SEED bodies) formed by IgG/IgA chain exchange proposed by Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.
  • the Knob-in-Hole structure on the Fc variant fragment of the present invention refers to the mutation of the two Fc fragments, which can be combined in the form of "Knob-in-Hole" after the mutation.
  • the "Knob-in-Hole" model of Cater et al. is preferably used for site mutation engineering in the Fc region, so that the resulting first and second Fc variants can be in the form of "Knob-in-Hole" bind together to form heterodimers.
  • the selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is within the purview of those skilled in the art.
  • the Fc regions of human antibodies IgG1, IgG2, IgG3, and IgG4 are preferred, and the Fc regions of human antibodies IgG1 and IgG4 are more preferred.
  • One of the first Fc variant or the second Fc variant is randomly selected to mutate the knob and the other to mutate the hole.
  • the first Fc variant is mutated with knob; the second Fc variant is mutated with hole.
  • linker sequence refers to insertion into an immunoglobulin domain of one or more amino acid residues that provide sufficient mobility for the domains of the light and heavy chains to fold into an exchange dual variable region immunoglobulin. It is used in the present invention to link IL-15 or IL-15R ⁇ to the corresponding light or heavy chain to ensure proper protein folding and peptide stability.
  • the "linker peptide” of the present invention is preferably a low immunogenic amino acid disability, preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5 or more, preferably n is 1-5.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Figure 1 is the structural form one of the bispecific antibody.
  • Figure 2 is a structural version two of the bispecific antibody.
  • Figure 3 is structural form three of the bispecific antibody.
  • Figure 4 is the structural version four of the bispecific antibody.
  • Figure 5 is structural version five of the bispecific antibody.
  • Figure 6 is structural version six of the bispecific antibody.
  • Figure 7 is structural version seven of the bispecific antibody.
  • Figure 8 is structural version eight of the bispecific antibody.
  • Figure 9 shows the reduced and non-reduced bands of QP34563457 protein analyzed by SDS-PAGE electrophoresis.
  • FIG. 10 is HPLC-SEC analysis of QP34563457 protein purity.
  • Figure 11 is a peptide map analysis of QP34563457 protein
  • Figure 11a shows 36% sequence coverage of QP34563457 protein for Trastuzumab VL-IL15 chain
  • Figure 11b shows 56% sequence coverage of QP34563457 protein for Trastuzumab VH-IL15Ra-Fc chain
  • Figure 11c The QP34563457 protein shows 64% sequence coverage for the pertuzumab VL-CL chain
  • Figure 11d shows the QP34563457 protein 56% sequence coverage for the pertuzumab VH-CH1-Fc chain.
  • Figure 12 is an ELISA detection of QP34563457 and other molecules bound to Her2-Fc fusion protein.
  • Figure 13 is an ELISA detection of QP34563457 and other molecules bound to Her2M2-Fc fusion protein.
  • Figure 14 is an ELISA detection of QP34623463 and other molecules bound to CD38 protein.
  • Figure 15 is an ELISA detection of QP34623463 and other molecules binding to CS1 protein.
  • Figure 16 is the FACS detection of QP34563457 and other molecules bound to SK-BR-3 cells.
  • Figure 17 is the FACS detection of QP34563457 and other molecules bound to BT-474 cells.
  • Figure 18 is the FACS detection of QP34563457 and other molecules bound to SK-OV-3 cells.
  • Figure 19 is the proliferation inhibition curve of QP34563457 and other molecules on human breast cancer cell SK-BR-3.
  • Figure 20 is the proliferation inhibition curve of QP34563457 and other molecules on human breast cancer cell BT-474.
  • Figure 21 is the proliferation inhibition curve of QP34563457 and other molecules on human ovarian cancer cell SK-OV-3.
  • Figure 22 shows the killing of human breast cancer cells SK-BR-3 cells by PBMC mediated by QP34563457 and the like.
  • Figure 23 shows the killing of human breast cancer cells BT-474 cells by PBMC mediated by QP34563457 and the like.
  • Figure 24 shows the killing of human ovarian cancer cells SK-OV-3 cells by PBMC mediated by QP34563457 and the like.
  • Figure 25 is an experiment to detect the proliferation of Mo7e cells by molecules such as QP34563457.
  • Figure 26 is the tumor growth curve of the in vivo pharmacodynamic model of SK-OV-3.
  • the protein sequence is as follows:
  • VH-IL15Ra-Fc (Knob) SEQ ID NO: 11
  • the clone design is shown in Table 1: construct vectors encoding anti Her2(Tmab)VL-IL15(SEQ ID NO:10), anti Her2(Tmab)VH-IL15Ra-Fc(Knob)(SEQ ID NO:11), anti Her2(Tmab)VH-IL15Ra-Fc(Knob)(SEQ ID NO:11), respectively.
  • CS1 VL-IL15 SEQ ID NO: 14
  • anti CS1 VH-IL15Ra-Fc Knob
  • the plasmid contains DHFR as a screening marker, which can be used for stable strain screening; the construction vector encodes the light chain sequence (sequence) of anti-tumor-specific antigen antibody and the heavy chain sequence of anti-tumor-specific antigen antibody (where Fc contains the Hole mutation (T366S).
  • the plasmid contains GS as a screening marker, which can be used for stable strain screening.
  • the structural schematic diagram of the protein molecule is shown in the figure 6. In other embodiments, the protein molecule may be in other structural forms, as shown in Figures 2 to 8, respectively.
  • control antibodies Trastuzumab, Pertuzumab, Elotuzumab and Daratumumab were designed and constructed.
  • Expi CHO-S cells were seeded into Forti CHO medium (Gibco, A1148301) with an additional addition of 8 mM GlutaMax and incubated at 37°C, 120 rpm, 8% CO 2 .
  • the density of Expi CHO-S cells was adjusted to 3*10E6/mL, placed in a shaker, 37°C, 120 rpm, 8% CO 2 and cultured; on the day of transfection, samples were taken, counted, and the cell density was diluted to 6*10E6/ml, 40ml per bottle, placed in a 125ml shake flask; mix 20ug of the corresponding plasmid with 4.8mL of Opti MEM, add 120ul of Polyplus-FectoPRO transfection reagent, mix the DNA and transfection reagent evenly, set At room temperature for 10 min, the mixture was slowly placed in the cells, and after mixing evenly, it was placed on a shaker for incubation.
  • each bottle was supplemented with 2 mL of Feed PFF05 (OPM, F81279-001) and 1 m of 30% glucose solution on the 1st, 4th, 6th, and 8th days, respectively.
  • Feed PFF05 OPM, F81279-001
  • glucose solution 3 mL
  • the temperature was lowered to 32°C and the CO2 concentration was lowered to 5%.
  • samples were collected, centrifuged at 8000 rpm for 20 min, and the supernatant was taken for purification.
  • PAC-EP
  • This example uses LC-MS to identify the amino acid sequence of QP34563457 protein. After denaturation and reduction of QP34563457 protein, trypsin was added for enzymatic digestion and then analyzed by LC-MS/MS (LC instrument Agilent 1290 Infinity II, column Agilent Peptide Plus column. MS instrument Agilent 6545Q-TOF), the obtained data was searched by Peaks Library, Peaks search results were filtered by strict card value to obtain credible peptides, which achieved a sequence coverage of 36% for the Trastuzumab VL-IL15 chain of the QP34563457 protein (Fig.
  • Her2 variant protein that only binds Pertuzumab.
  • Matthew C. Franklin published the complex structure of Pertuzumab Fab and Her2 extracellular structure in Cancer cell. The team also used alanine scanning to study which key amino acids of Her2 affect the binding to the Pertuzumab Fab.
  • the Her2 variant protein that binds only pertuzumab but not trastuzumab was designated as Her2M2 (protein number QP3732).
  • the amino acid sequences of Her2 wt (protein number QP3731) and Her2M2 (protein number QP3732) are as follows:
  • Blocking solution Weigh 5g of milk into PBS. The blocking solution needs to be prepared and used immediately. Stop solution (1mol/LH2SO4): Take 109mL 98% concentrated H2SO4 and slowly add it dropwise to 2000mL ddH2O. TMB developed color at 37°C for 10min, placed in a shaker (120rpm), 100 ⁇ l/well;
  • Her2 and its variant proteins QP3731 and QP3732 were coated on plates, 1ug/ml, 4 degrees overnight; washed with PBS for 3 times. Add blocking solution 5% milk at 200 ⁇ L/well and incubate at 37°C for 1 h. After blocking, wash 3 times with PBS, incubate the samples, dilute 5 times according to 20ug/ml, and dilute 7 gradients in total.
  • the last hole is diluted 100 times, and mix well with 100 ⁇ l/well, incubate for 1 h, and wash 3 times with PBST; add enzyme Standard antibody: Incubate HRP-anti human Fab antibody according to the dilution ratio of 1:5000, 100 ⁇ l/well, mix well, incubate for 1 h, and wash 6 times with PBST.
  • Add substrate chromogenic solution add substrate chromogenic solution TMB at a dosage of 100 ⁇ L/well, place in a shaker, 200 rpm, 35 °C for color development for 10 min in the dark.
  • Termination After the color development is completed, 100 ⁇ L/well of stop solution is added quickly to terminate the reaction. Detection: On a microplate reader, measure the OD value of A450nm, and use graphpad prism software to analyze the results.
  • trastuzumab can bind Her2wt well, but hardly bind Her2M2; QP34563457 and Pertuzumab can bind Her2wt and Her2M2 well (results are shown in Figure 12, Figure 13).
  • Example 5 ELISA detection of QP34623463 binding to CD38 and CS1 activity
  • Blocking solution Weigh 5g of milk into PBS. The blocking solution needs to be prepared and used immediately. Stop solution (1mol/LH2SO4): Take 109mL 98% concentrated H2SO4 and slowly add it dropwise to 2000mL ddH2O. TMB developed color at 37°C for 10min, placed in a shaker (120rpm), 100 ⁇ l/well;
  • CD38-Fc and CS1-Fc fusion proteins were coated on plates, respectively, at 1 ug/ml, overnight at 4 degrees; washed three times with PBS. Add blocking solution 5% milk at 200 ⁇ L/well and incubate at 37°C for 1 h.
  • BT474 cells human breast cancer cell line
  • SK-BR-3 cells human breast cancer cell line
  • SK-OV-3 cells human ovarian cancer cell line
  • BT474 cells use RPMI1640 (Gibco) medium containing 10% FBS, 0.11g/L sodium pyruvate and 2.5g/L glucose
  • SK-BR-3 cells use DMEM medium (Gibco) containing 10% FBS
  • SK-OV-3 cells use McCoy's 5a medium (Gibco) containing 10% FBS.
  • BT474 cells human breast cancer cell line
  • SK-BR-3 cells human breast cancer cell line
  • SK-OV-3 cells human ovarian cancer cell line
  • CCK-8 Cell Proliferation and Toxicity Detection Kit
  • BT474 cells use RPMI1640 (Gibco) medium containing 10% FBS, 0.11g/L sodium pyruvate and 2.5g/L glucose
  • SK-BR-3 cells use DMEM medium (Gibco) containing 10% FBS
  • SK-OV-3 cells use McCoy's 5a medium (Gibco) containing 10% FBS.
  • QP34563457 showed a significant proliferation inhibitory effect on HER2-positive tumor cells BT474, SK-BR-3, and SK-OV-3, which was significantly better than Trastuzumab or Pertuzumab monoclonal antibody and their combination (with synergy) ( as shown in Figure 19-21).
  • Example 8 HER2 bispecific antibody-mediated ADCC effect
  • PBMC peripheral blood mononuclear cells
  • BT474 cells human breast cancer cell line
  • SK-BR-3 cells human breast cancer cell line
  • SK-OV-3 cells human ovarian cancer cell line
  • Cytotox96 non-radioactive cytotoxicity assay kit was purchased from Promega (G1780).
  • mice resuscitate PBMC and collect cells for use on the next day.
  • target cells SK-BR-3, BT-474, SK-OV-3 were trypsinized, 1000rpm for 5min. After washing twice with PBS, a 96-well plate was plated, 20,000 cells per well, 50 ⁇ l per well, and incubated at 37°C with 5% CO 2 for 2 h.
  • LDH detection For the maximum release group and the volume correction group, add 10 ⁇ l of lysate 45 minutes in advance, and continue to place in the incubator for cultivation.
  • Mo7e cells human giant cell leukemia cell line
  • CCK-8 Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences, Cell Proliferation and Toxicity Detection Kit (CCK-8), purchased from Meilun Biotech, Cat. No. MA0218
  • recombinant Human GM-CSF purchased from perprotech, Cat. No. 300-03
  • human IgG from Sigma, Cat. No. I4506; other antibodies were prepared in-house.
  • Mo7e cells were cultured in RPMI1640 containing 10% FBS, 2mM L-glutamine and 8ng/ml GM-CSF based on 37°C, 5% CO2 incubator; Mo7e cells were collected, centrifuged at 800rpm for 5 minutes and discarded In the supernatant, the cells were washed twice with RPMI1640 medium without GM-CSF; the cells were resuspended in RPMI1640 medium without GM-CSF and counted, and the cells were seeded in 96-well plates at 2 ⁇ 104 cells, 80 ⁇ l per well.
  • the inhibitory effect of QP34563457 on tumor growth was evaluated in a human ovarian cancer cell SK-OV-3 model with high expression of HER2.
  • SK-OV-3 cells were cultured in McCoy's 5A+10% FBS medium containing 10% fetal bovine serum. SK-OV-3 cells in exponential growth phase were collected and resuspended in PBS to an appropriate concentration for inoculation. Each BALB/C-nude mouse was subcutaneously inoculated with 1 ⁇ 10 6 SK-OV- 3 cells on the right back, and the tumor growth was observed regularly. Group administration (administration volume is 10ul/g body weight), and the day of the first administration is defined as D0.
  • the dosing regimen is as follows:

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Abstract

本发明提供一种双特异性抗体及其应用，在本发明的双特异性抗体的第一抗体的两条链当中分别引入IL15和IL15Rα(例如IL15和IL15Rα分别替换CL1和CH1)，利用IL15和IL15Rα的高亲和力形成IL15/IL15Rα复合物，从而实现第一抗体轻链/重链的正确配对，解决双特异性抗体轻链/重链错配问题。同时，通过第一抗体可变结构域VH1、VL1、IL15和IL15Rα氨基酸序列突变，在VH1和VL1之间，以及IL15和IL15Rα之间添加一对或多对二硫键，进一步地增强第一抗体轻链/重链间的结合活性，从而有效克服双特异性抗体制备过程中轻链/重链错配、副产物高、稳定性差等挑战，最终制备得到正确配对的双特异性靶向细胞因子的多功能抗体。同时，缩短双特异性抗体开发周期，降低生产成本。

Description

[根据细则26改正07.06.2021]　一种双特异性抗体及其应用 技术领域
本发明属于生物医药领域，涉及一种双特异抗体，本发明还涉及这种双特异性抗体的应用。
背景技术
双特异性抗体(BsAb)又称双功能抗体，可同时识别和结合两种不同的抗原和表位，并阻断两种不同的信号通路以发挥其作用。BsAb与普通抗体相比增加了一个特异性抗原结合位点，在治疗方面表现出了以下优势：
介导免疫细胞对肿瘤的杀伤：双特异性抗体的一个重要作用机制是介导免疫细胞杀伤，双特异性抗体有两条抗原结合臂，其中一条与靶抗原结合，另一条与效应细胞上的标记抗原结合，后者可以激活效应细胞，使其靶向杀伤肿瘤细胞。目前已经批准上市的2个双特异性抗体产品都属于这个类别，Trion Pharma公司开发的catumaxomab能够靶向肿瘤表面抗原EpCAM和T细胞表面受体CD3，而Micromet公司和Amgen公司开发的Blinatumomab可以同时结合CD19和CD3。两者都是通过激活并召集杀伤性T细胞，从而达到治疗肿瘤的目的。
双靶点信号阻断，发挥独特的或重叠的功能，有效防止耐药：同时结合双靶点，阻断双信号通路是双特异性抗体的另一个重要作用机制。受体络氨酸激酶(receptor tyrosine kinase，RTKs)是最大的一类酶联受体，在细胞增殖过程中发挥重要的调节作用，如Her家族等。RTKs在肿瘤细胞表面异常高表达，导致肿瘤细胞恶性增生，因此也是肿瘤治疗的重要靶点。针对RTKs的单靶点单克隆抗体已在肿瘤治疗中得到广泛应用，但是，肿瘤细胞可以通过转换信号通路或通过HER家族成员自身或不同成员之间的同源或异源二聚体激活细胞内信号进行免疫逃逸。因此采用双特异性抗体药物同时阻断两个或多个RTKs或其配体，可以减少肿瘤细胞逃逸，提高治疗效果。
具备更强特异性、靶向性和降低脱靶毒性：利用双特异性抗体两个抗原结合臂可以结合不同抗原的特点，两个抗原结合臂分别结合癌细胞表面2种抗原，可以有效增强抗体对癌细胞的结合特异性和靶向性，降低脱靶等副作用。
有效降低治疗成本：以BiTE为例，与传统抗体相比在组织渗透率、杀伤肿瘤细胞效率、脱靶率和临床适应症等指标方面都具有较强的竞争力，临床优势显著。特别在使用剂量方面，由于其治疗效果可以达到普通抗体的100-1000倍，使用剂量最低可将为原来的1/2000，显著降低药物治疗成本。相对于组合疗法，双特异性抗体的成本也远远低于两个单药联合治疗。
目前，全球有三款双特异性抗体药物获得上市批准：
2009年，欧盟批准了第一个治疗性双特异性抗体—TrionPharma公司的Catumaxomab(靶向CD3和EpCAM)，用于治疗癌性腹水。2014年，FDA快速批准Amgen公司基于
Figure PCTCN2021094672-appb-000001
技术开发的双特异性抗体药物Blinatumomab(靶向CD3和CD19)用于治疗急性B淋巴细胞白血病，它也是第一个获批的靶点为CD19的药 物。Amgen一系列的血液恶性肿瘤和实体瘤的临床开发中就拥有十几种
Figure PCTCN2021094672-appb-000002
分子。其中靶向BCMA/CD3的AMG420已获得FDA授权快速审批通道地位。在实体肿瘤的治疗中，靶向前列腺特异膜抗原(PSMA)的
Figure PCTCN2021094672-appb-000003
分子AMG212(Pasotuxizumab)的临床试验结果也展露锋芒。2017年11月，FDA快速批准了罗氏的双特异性抗体Emicizumab(靶向凝血因子X和因子IXa)，用于治疗血友病。2018年12月在中国获批上市，是首个在国内获批的双特异性抗体。
双特异性抗体本身具有诸多优势，能够应用于多个治疗领域，比如：癌症、慢性炎症性疾病、自身免疫疾病以及感染等。但生产双特异性抗体的主要技术难点在于获得正确配对的双特异性抗体，尤其对于包含Fc区(IgG样)的不对称双特异性抗体，同时面临HC/HC和LC/HC错配问题。后续开发出KiH、ART-Ig和BiMab技术来减少HC/HC的错配。而CrossMAb技术(对其中一对重链和轻链可变结构域VL和VH相互替换，同时恒定结构域CL和CH1也相互替换)、YBODY技术(一侧采用传统的Fab构成靶向抗原A的单价单元Fab-Fc，另一侧采用单链抗体构成靶向抗原B的单链单元ScFv-Fc)、WuXiBody(TCR恒定区取代重链恒定区CH1和轻链恒定区CL)以及共同轻链技术被用来减少LC/HC的错配。但是对双特异抗体领域而言，依然有必要进一步开发新的技术解决LC/HC的错配问题。
1.Her2
受体酪氨酸激酶的ErbB家族的成员是细胞生长，分化和存活的重要介导物。该受体家族包括四个独特成员，包括表皮生长因子受体(EGFR或ErbB1)，Her2(ErbB2)，Her3(ErbB3)和Her4(ErbB4或tyro2)。Her2是一种跨膜的表面结合的受体酪氨酸激酶，而且正常情况下牵涉导致细胞生长和分化的信号转导途径。
Her2过度表达可能导致细胞正常功能紊乱，并且通常与肿瘤的发生和发展密切相关。Her2的同源或异源聚合可导致受体酪氨酸残基磷酸化，并且引发许多信号途径并引起细胞增殖和肿瘤发生。作为预后和预测的生物标志物，Her2基因的扩增或过度表达在约15-30％的乳腺癌和10-30％的胃癌/食道癌中发生。在其它肿瘤诸如卵巢、子宫内膜、膀胱、肺、结肠和头颈肿瘤中也可观察到Her2过度表达。
曲妥珠单抗(Trastuzumab)识别Her2胞外结构域IV近膜表位。具体为Her2胞外结构域IV部分的C端处由3个环(557-561、570-573和593-603)组成的表位。因为表位可以靠近其二聚化伴侣的结合结构域或直接与之相互作用，所以曲妥珠单抗与表位的结合可以诱导抑制二聚化过程的空间位阻。另外，曲妥珠单抗的结合还可以保护Her2受体的细胞外结构域免受蛋白酶的攻击而水解。
目前将曲妥珠单抗用作治疗乳腺癌的一线药物，有效治疗Her2过度表达的转移性乳腺癌，并且单药一线治疗的客观反映率为30-50％；但是在治疗Her2表达较低的转移性乳腺癌方面效果不理想，并且抗体最初在1年内有效的许多患者中发展出耐药性。Her2连同该家族的其它成员(Her1、Her3和Her4)可以形成配体依赖性或配体非依赖性异源二聚体，从而激活下游途径，然后导致肿瘤细胞增殖，而曲妥珠单抗无法 抑制异源二聚体形成，所以这可能是耐药性发展的原因之一。
帕妥珠单抗(Pertuzumab)是一种人源化单克隆抗体，其特异性设计成阻止HER2受体与细胞表面上的其它HER受体(EGFR/HER1，HER3和HER4)配对(二聚化)，认为该过程在肿瘤生长和存活中发挥作用。帕妥珠单抗对晚期前列腺癌、非小细胞肺癌、卵巢癌和乳腺癌有一定治疗效果，但其治疗效果还取决于Her2表达水平。帕妥珠单抗识别Her2细胞外结构域II异源二聚化的关键位点，由此识别的表位位于II亚区中心的区段245-311，关键残基为H245、V286、S288、L295、H296和K311。其中，L295、H296是介导Her2和Her3异源二聚化的关键位点，并且L295A/H296A双重突变可完全阻断Her2/Her3的异源二聚化(Franklin，M.C.等人，Insights into ErbB signaling from the structure of the ErbB2-pertuzumab complex.Cancer Cell，2004.5(4):第317-28页)。因此，帕妥珠单抗可用于有效抑制Her2/Her3异源二聚体的形成，但对EGFR/Her2异源二聚体的形成并未显示出明显的抑制作用。
2.IL15/IL15Ra
IL-15是一种长14–15kDa的细胞因子，对于NK细胞、NKT细胞和memory CD8 +T细胞功能非常重要。IL-15在体内含量很少，但通过与其受体IL-15Rα结合产生具有极高生物效价的复合物IL-15超激动剂(IL-15SA)后一起被转导转运至靶细胞。IL-15SA强烈激活响应IL-15的细胞，特别是NK细胞，从而促进抗肿瘤和抗病毒功能。
研究人员在1994年首次将IL-15鉴定为T淋巴细胞生长因子，它与IL-2具有大约19％的同源序列，许多生物学特性也非常类似。IL-15三维结构与IL-2类似，由四个“上-下-下-下”螺旋束组成，IL-4，IL-7和IL-9等其他细胞因子也包含这种构象。IL-15与其他细胞因子作用方式不同，IL-15受体α表达于产生IL-15的细胞(例如巨噬细胞和树突状细胞)，与IL-15形成IL-15SA将信号递送至表达IL-15Rβ(也称为IL-2Rβ)和共同γ链(与IL-2，IL-4，IL-7，IL-9和IL-21共享)的NK、NKT和memory CD8 +T细胞，很有可能正是由于这一独特的呈递方式赋予了IL-15介导其独特功能的能力。小鼠IL-15与人IL-15具有70％的氨基酸序列同源性，人IL-15和小鼠IL-15也具有相似的反式表达模式，信号通路以及生物学活性。IL-15在许多细胞类型和组织中表达，包括单核细胞、巨噬细胞、DC、角质形成细胞、成纤维细胞、肌细胞和神经细胞。IL-15作为多效细胞因子在先天性和适应性免疫中起重要作用。
反式表达的IL-15/IL-15Rα信号通过响应细胞上表达的β和γ链，诱导JAK1和JAK3的募集和激活，活化的JAK1和JAK3进一步磷酸化STAT3和STAT5。STAT3和STAT5磷酸化后形成同源二聚体，易位至细胞核并促进靶基因的转录。IL-15信号传导刺激一系列下游反应，诱导细胞生长、减少凋亡以及增强免疫细胞的激活和转移。在没有高亲和力IL-15Rα的情况下，IL-15也可以单独与中等亲和力(Ka＝1.10 9/M)的β、γ受体复合物(IL-15Rβγ)结合，诱导其他酪氨酸激酶(如Lck、Fyn、Lyn、Syk)磷酸化激活，并与PI3K、MAPK途径发生作用。有研究表明，代谢检查 点激酶mTOR也可以被高浓度的IL-15激活，这与NK细胞的增殖和激活增强有关：选择性敲除mTOR会导致骨髓NK细胞成熟受阻。IL-15促进NK细胞增殖的能力部分是由于IL-15介导的有氧糖酵解作用引起的，在没有IL-15的情况下NK细胞的基础代谢很低，但通过提高IL-15浓度可以明显增强这一生理活动。
由于IL-15具有与IL-2类似的免疫学特性：诱导T细胞的增殖和存活，促进NK细胞的增殖和分化，并诱导细胞毒性T淋巴细胞的产生。但不同于IL-2，IL-15对Treg细胞没有明显作用，并且不会在小鼠或非人类灵长类动物(NHP)中引起毛细血管渗漏综合征，因此相较于IL-2，IL-15是肿瘤免疫疗法的更优选择。猕猴IL-15(rIL-15)是第一种用于体内实验的IL-15形式，研究人员认为rIL-15可以优先结合细胞表面IL-15Rα。异二聚体IL-15/IL-15Rα是从细胞上裂解下来的IL-15的天然形式，可以独立于细胞刺激相互作用的应答，目前诺华公司正在实体瘤中开展这种形式的分子(NIZ985)的临床试验。Cytune开发的RLI是一种融合蛋白，由与IL-15Rα的Sushi结构域相连的IL-15组成，可以作为可溶性的IL-15激动剂。IL-15/IL-15Rα-Fc复合物通过将市售的IL-15Rα-Fc嵌合融合蛋白与rIL-15混合产生的，已在临床前研究中广泛使用。
然而，细胞因子在临床上的使用存在着单药给药靶向性差的缺点，只有高浓度给药才可以达到抗肿瘤作用，而高浓度给药会产生免疫抑制作用和高毒性。并且，非靶向性细胞因子对于免疫系统的激活是系统性的，免疫系统被广泛的激活，具有致命的副作用。此外，由于细胞因子属于小分子量蛋白，不具备抗体的体内循环保护机制，单纯的细胞因子往往半衰期较短，需要短时间重复高剂量给药。目前临床研究药物多采用PEG化或者Fc融合来提高细胞因子的半衰期，虽然半衰期得以延长，但仍无法解决细胞因子的靶向性差的问题。
发明内容
在本发明的第一方面，提供了一种双特异性抗体，其包含：a)特异性结合第一抗原的第一抗体的轻链和重链；和b)特异性结合第二抗原的第二抗体的轻链和重链；其中第一抗体中两条链分别引入IL15和IL15Rα(例如IL15和IL15Rα分别替换CL1和CH1)，利用IL15和IL15Rα的高亲和力形成IL15/IL15Rα复合物，从而实现第一抗体轻链/重链的正确配对，解决双特异性抗体轻链/重链错配问题。同时，通过第一抗体可变结构域VH1、VL1、IL15和IL15Rα氨基酸序列突变，在VH1和VL1之间，以及IL15和IL15Rα之间添加一对或多对二硫键，进一步地增强第一抗体轻链/重链间的结合活性，从而有效克服双特异性抗体轻链/重链错配问题。
本发明构建的一种双特异性抗体，其包含：a)特异性结合第一抗原的第一抗体的轻链和重链；和b)特异性结合第二抗原的第二抗体的轻链和重链；其中第一抗 体中两条链分别包含IL15和IL15Rα，且能够形成IL15/IL15Rα复合物。
在另一优选例中，所述IL15包含能够结合IL15Rα的突变体，所述IL15Rα包含能够结合IL15的突变体。
在另一优选例中，所述第一抗体可变结构域VH1和VL1连接或聚合，所述IL15和IL15Rα连接或聚合。
在另一优选例中，所述IL15和IL15Rα取代第一抗体的恒定结构域CH1和CL。
在另一优选例中，所述第一抗体的可变结构域VL1和VH1连接在IL15和IL15Rα的N端。
在另一优选例中，所述第一抗体的可变结构域VL1和VH1连接在IL15和IL15Rα的C端。
在另一优选例中，所述第一抗体的可变结构域VL1和VH1互换位置。
在另一优选例中，所述第一抗体轻链(从N端到C端)与第一抗体重链(从N端到C端)同向聚合。
在另一优选例中，所述第一抗体轻链(从C端到N端)与第一抗体重链(从N端到C端)反向聚合。
在另一优选例中，所述第一抗体可变结构域VL1和VH1通过低免疫原性的氨基酸接头序列与IL15和IL15Rα连接。
在另一优选例中，所述IL15和IL15Rα之间具有一对或多对二硫键。
在另一优选例中，所述IL15包含以下所示突变，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位。
Figure PCTCN2021094672-appb-000004
Figure PCTCN2021094672-appb-000005
在另一优选例中，所述IL15和IL15Rα包含以下所示突变组合，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位；SEQ ID No.3所示IL15Rα第一个氨基酸开始算为第1位。
组合 IL15 IL15Ra
1 wt D96
2 wt D96/P97
3 wt D96/P97/A98
4 E87C D96/C97
5 E87C D96/P97/C98
6 E87C D96/C97/A98
7 V49C S40C
8 L52C S40C
9 E89C K34C
10 Q48C G38C
11 E53C L42C
12 C42S A37C
13 L45C G38C
14 L45C A37C
在另一优选例中，所述第一抗体的可变结构域VH1和VL1之间具有一对或多对二硫键。
在另一优选例中，所述VH1和VL1包含以下突变组合形式，根据EU计数。
Figure PCTCN2021094672-appb-000006
Figure PCTCN2021094672-appb-000007
在另一优选例中，所述第一、第二抗体重链分别包含Fc段不同突变的A、B链，所述A、B链具有以下突变组合形式，根据EU计数。
Figure PCTCN2021094672-appb-000008
在另一优选例中，所述Fc段包含Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc及其突变体。
在另一优选例中，所述Fc段的A链和B链，其中一条链能够结合protein A，另一条链为不能够结合protein A的突变体,所述突变包含H435R或H435R/Y436F，根据EU计数。
在另一优选例中，所述第一抗原为CD3、CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、CD217、CD239、CD274、CD276、PD-1、CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB、IL-6R、IL-2R、CCR4、VEGFR-2、CD6、CTLA-4、整合素α4、DNA/histone complex、PDGFRα、NeuGcGM3、IL-4Rα、IL-6Rα之任一，所述第二抗原为第一抗原的不同表位或上述另一抗原。
在另一优选例中，所述第一/第二抗体为嵌合、人源化或全人源抗体。
在另一优选例中，所述双特异性抗体具有式I所示结构：
Figure PCTCN2021094672-appb-000009
其中：
链1：VL1或VH1通过L1连接在IL15或IL15Rα的N端或C端；
链2：从N端到C排列，VH1或VL1、L2、IL15Rα或IL15、L3、Fc；
链3：第二抗体重链从N端到C端排列，VH2-CH1-Fc；
链4：第二抗体轻链从N端到C端排列，VL2-CL；
“-”代表肽键；
L1、L2和L3各自独立地为键或接头序列；
在另一优选例中，所述双特异性抗体具有式II所示的结构：
Figure PCTCN2021094672-appb-000010
Figure PCTCN2021094672-appb-000011
其中：
链1：IL15或IL15Rα通过L1连接在VL1或VH1的N端或C端；
链2：从N端到C排列，IL15Rα或IL15、L2、VH1或VL1、L3、Fc；
链3：第二抗体重链从N端到C端排列，VH2-CH1-Fc；
链4：第二抗体轻链从N端到C端排列，VL2-CL；
“-”代表肽键；
L1、L2和L3各自独立地为键或接头序列。
在另一优选例中，上述链1及链2包含下表组合形式：
Figure PCTCN2021094672-appb-000012
Figure PCTCN2021094672-appb-000013
在另一优选例中，所述的双特异性抗体具有式III所示的结构：
Figure PCTCN2021094672-appb-000014
其中:
链1：从N端到C端排列，VL1-L1-IL15，L1为低免疫源性氨基酸接头序列；
链2：从N端到C端排列，VH1-L2-IL15Rα-L3-Fc，L2、L3为低免疫源性氨基酸接头序列；
链3：从N端到C端排列，VH2-CH1-Fc；
链4：从N端到C端排列，VL2-CL；
“-”代表肽键。
在另一优选例中，所述L1、L2和L3包含甘氨酸(G)和丝氨酸(S)残基。
在另一优选例中，所述L1、L2和L3包含一个或多个GGGGS重复。
在另一优选例中，所述IL15序列如SEQ ID No.1或SEQ ID No.2所示；所述IL15Rα序列如SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ ID No.9所示。
在另一优选例中，所述Fc序列如SEQ ID No.18或SEQ ID No.19所示。
在另一优选例中，所述第一抗原和第二抗原分别为结合Her2两个不同表位的抗原。
在另一优选例中，所述双特异抗体由SEQ ID No.10、SEQ ID No.11、SEQ ID No.13、SEQ ID No.12序列融合得到。
在另一优选例中，所述第一抗原和第二抗原分别为CS1抗原和CD38抗原。
在另一优选例中，所述双特异抗体由SEQ ID No.14、SEQ ID No.15、SEQ ID No.17、SEQ ID No.16序列融合得到。
本发明的第二方面提供了一种药物组合物含有：
(a)本发明第一方面所述的双特异性抗体；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物中还含有治疗癌症(或肿瘤)的其他药物，如化疗药物。
本发明的第三方面，提供了本发明第一方面所述的双特异抗体以及本发明第六方面所述药物组合物在制备治疗癌症(或肿瘤)、感染或免疫调节疾病的药物中的应用。
本发明的第四方面，提供了本发明第一方面所述的双特异抗体，在制备抑制肿瘤生长的药物中的应用。
在另一优选例中，所述癌症或肿瘤包含来自如下部位的癌症或肿瘤：结直肠、乳腺、卵巢、胰腺、胃、前列腺、肾、宫颈、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸。
在另一优选例中，所述癌症(或肿瘤)包含：骨髓癌、淋巴癌、白血病、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症。
本发明的第五方面，提供了本发明第一方面所述的结合Her双表位的双特异抗体，在制备诊断HER2阳性肿瘤(例如乳腺癌、胃癌)的试剂或试剂盒中的用途。
发明的有益效果
本发明的发明人通过实验，令人惊奇地发现，一种双特异性抗体，在第一抗体的两条链当中分别引入IL15和IL15Rα(例如IL15和IL15Rα分别替换CL1和CH1)，利用IL15和IL15Rα的高亲和力形成IL15/IL15Rα复合物，从而实现第一抗体轻链/重链的正确配对，解决双特异性抗体轻链/重链错配问题。同时，通过第一抗体可变结构域VH1、VL1、IL15和IL15Rα氨基酸序列突变，在VH1和VL1之间，以及IL15和IL15Rα之间添加一对或多对二硫键，进一步地增强第一抗体轻链/重链间的结合活性，从而有效克服双特异性抗体制备过程中轻链/重链错配、副产物高、稳定性差等挑战，最终制备得到正确配对的双特异性靶向细胞因子的多功能抗体。同时，缩短双特异性抗体开发周期，降低生产成本。
另一方面，本发明构建的双特异性抗体，在克服轻链/重链错配问题的同时，具有IL-15/IL-15Rα活性，能够靶向细胞因子到肿瘤部位，可以特异的在肿瘤部位扩增和活化PMBC中的T细胞及NK细胞，并使得免疫细胞数目以及杀伤性细胞因子的释放增加，更加有效地杀伤肿瘤细胞，并能降低给药剂量。
第三方面，本发明成功构建正确配对的Trastuzumab/Pertuzumab/IL15双特异抗 体，利用Her2双特异性抗体的靶向性将IL15靶向到肿瘤组织，刺激免疫反应，用多重机制杀伤Her2阳性肿瘤。
第四方面，本发明成功构建正确配对的结合CS1/CD38抗原的Elotuzumab/Daratumumab/IL15双特异抗体，具有与CS1单抗相当的CS1抗原结合结合能力，同时具有与CD38单抗具有相当的CD38抗原结合结合能力，在血液瘤治疗当中具有极大潜力。
为了更容易解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件的它处另有明确定义，否则本文中使用的所有其他技术和科学术语都具有本发明所属技术领域的一般技术人员通常理解的含义。
术语
本发明所用氨基酸三字母代码和单字母代码如J.Boil.Chem.,243,p3558(1968)中所述。
本发明所述“抗体”，不仅包括完整的抗体，还包括具有抗原结合活性的片段、多肽序列、及其衍生物和类似物。
所述抗原结合片段是指全长抗体的一个或多个部分，所述部分保持结合抗原(例如，HER2)的能力，与完整抗体竞争对抗原的特异性结合。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗原结合部分。在一些情况下，抗原结合部分包括Fab、Fab'、F(ab')2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如单克隆抗体2E12)获得抗体的抗原结合部分(例如，上述抗体片段)，并且以与对于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合部分。术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab')2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明所述“表位”或“抗原表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。
本发明所述的“IL-15”可以是任何IL-15或能结合IL15Rα的突变体，如人IL-15或非人哺乳动物或非哺乳动物的IL-15。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等；优选人的IL-15。术语“能结合IL15Rα的突变体”指通过一个或多个氨基酸替换、增加或者缺失突变获得的对IL-15与其受体间亲和力提高或者降低，或其刺激T细胞或者NK细胞活性增加或者降低的突变体分子。本发明所述“IL-15”优选其变体形式，更优选氨基酸序列为SEQ ID No.1或SEQ ID No.2。
本发明中所述的“IL-15Rα”可以是任何物种的IL-15Rα或者能结合IL15Rα的突变体，如人IL-15Rα或非人哺乳动物IL-15Rα或非哺乳动物IL-15Rα。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。优选人的IL-15Rα，更优选人IL-15Rα胞外域片段，简称IL-15RαECD(见数据库UniProtKB，登录号Q13261，31-205aa)。术语“能结合IL15Rα的突变体”指在IL-15Rα上通过一个或者多个氨基酸 缺失、插入或替换突变形成的具有与其配体分子如IL-15结合能力的功能性突变体，优选人的IL-15Rα分子更优选人的IL-15Rα胞外域片段的缩短形式，即从胞外域片段C端开始通过一个或多个氨基酸缺失突变所得的具有人IL-15受体α活性的分子，优选保留65-178个氨基酸的缺失突变形式，比如IL-15Rα(SEQ ID NO:3-9)。
本发明所述“Fc段”指免疫球蛋白的C端区域，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位，是包含两个二硫化物连接的抗体重链Fc区多肽的二聚体分子。Fc区可以通过木瓜蛋白酶消化或IdeS消化成胰蛋白酶消化完整(全长)抗体来产生或可以重组产生。“Fc部分”优选包括至少一个免疫球蛋白铰链区，以及IgG的CH2和CH3区。
Fc异源二聚体突变是指通过在Fc的合适位点处存在一个或多个氨基酸替换、插入或缺失突变引起Fc结构或功能的变化。经突变设计的Fc变异体之间可以形成空间填充效应、静电转向、氢键作用、疏水作用等。Fc突变体间相互作用有助于形成稳定的异源二聚体。优选的突变设计为“Knob-in-hole”形式的突变设计。此外，本发明所述Fc上还可以存在其他导致其功能变化的突变，例如糖基化改造突变、FcγR结合区域突变(以调整ADCC活性)和改善抗体稳定性的氨基酸突变等。本发明中Fc包含Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc及其突变，其中一条链能够结合proteinA，另一条链为不能够结合proteinA的突变体，包含突变H435R或H435R/Y436F，根据EU计数。
本发明所述的“异源二聚体”优选为基因共表达的产物。如在原核细胞在大肠杆菌中共表达；或在真核细胞，如293、CHO中共表达。所述“共表达”指在一个细胞中多个基因一起表达，同时出现它们的产物。这些基因可以是同时存在而分别或共同地受控表达。在本发明中，优选在一个真核细胞中共表达。共表达得到的基因表达产物有利于高效、简单地形成复合物；在本发明中，有利于形成异源二聚体。
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(ProteinEngineeringvol.9no.7pp617-621,1996)提出的“Knob-in-Hole”形式；Amgen公司技术人员利用静电转向形成含Fc的异源二聚体形式(US2010286374A1)；Jonathan H.Davis等人(ProteinEngineering,Design&Selectionpp.1–8,2010)提出的通过IgG/IgA链交换形成的异源二聚体形式(SEED bodies)；Genmab公司DuoBody (Science,2007.317(5844))平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(mAbs3:6,546-557；November/December2011)；苏州康宁杰瑞公司的基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本发明所述的Fc变体片段上的Knob-in-Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-in-Hole”形式进行结合。优选用Cater等人的“Knob-in-Hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“Knob-in-Hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1和IgG4的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。在实施例中，所述的第一Fc变体做knob的突变；所述的第二Fc变体做hole的突变。
术语“接头序列”是指插入免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。在本发明中用于将IL-15或IL-15Rα连接至相应的轻链或重链中，以保证蛋白的正确折叠和肽稳定性。本发明的“连接肽”优选为低免疫原性氨基酸残疾，优选(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多，优选n为1-5。
本发明抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
附图说明
图1是双特异抗体的结构形式一。
图2是双特异抗体的结构形式二。
图3是双特异抗体的结构形式三。
图4是双特异抗体的结构形式四。
图5是双特异抗体的结构形式五。
图6是双特异抗体的结构形式六。
图7是双特异抗体的结构形式七。
图8是双特异抗体的结构形式八。
图9是SDS-PAGE电泳分析QP34563457蛋白还原与非还原条带。
图10是HPLC-SEC分析QP34563457蛋白纯度。
图11是QP34563457蛋白的肽图分析，图11a显示QP34563457蛋白对于Trastuzumab VL-IL15链36％的序列覆盖率，图11b显示QP34563457蛋白对于Trastuzumab VH-IL15Ra-Fc链56％的序列覆盖率，图11c显示QP34563457蛋白对于pertuzumab VL-CL链64％的序列覆盖率，图11d显示QP34563457蛋白对于pertuzumab VH-CH1-Fc链56％的序列覆盖率。
图12是ELISA检测QP34563457等分子结合Her2-Fc融合蛋白。
图13是ELISA检测QP34563457等分子结合Her2M2-Fc融合蛋白。
图14是ELISA检测QP34623463等分子结合CD38蛋白。
图15是ELISA检测QP34623463等分子结合CS1蛋白。
图16是FACS检测QP34563457等分子结合SK-BR-3细胞。
图17是FACS检测QP34563457等分子结合BT-474细胞。
图18是FACS检测QP34563457等分子结合SK-OV-3细胞。
图19是QP34563457等分子对人乳腺癌细胞SK-BR-3的增殖抑制曲线。
图20是QP34563457等分子对人乳腺癌细胞BT-474的增殖抑制曲线。
图21是QP34563457等分子对人卵巢癌细胞SK-OV-3的增殖抑制曲线。
图22是QP34563457等介导的PBMC杀伤人乳腺癌细胞SK-BR-3细胞。
图23是QP34563457等介导的PBMC杀伤人乳腺癌细胞BT-474细胞。
图24是QP34563457等介导的PBMC杀伤人卵巢癌细胞SK-OV-3细胞。
图25是检测QP34563457等分子对Mo7e细胞增殖实验。
图26是SK-OV-3体内药效模型肿瘤生长曲线。
具体实施方式
以下实施方式中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件和方法进行。如分子克隆，实验室手册，冷泉港实验室，当代分子生物学方法，细胞生物学等等。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：融合蛋白的克隆和表达
蛋白序列如下：
anti Her2(Her2胞外域IV区)VL-IL15SEQ ID NO:10
Figure PCTCN2021094672-appb-000015
anti Her2(Her2胞外域IV区)VH-IL15Ra-Fc(Knob)SEQ ID NO:11
Figure PCTCN2021094672-appb-000016
Figure PCTCN2021094672-appb-000017
anti Her2′(Her2胞外域II区)VL-CL SEQ ID NO:12
Figure PCTCN2021094672-appb-000018
anti Her2′(Her2胞外域II区)VH-CH1-CH2-CH3(Hole)SEQ ID NO:13
Figure PCTCN2021094672-appb-000019
anti CS1 VL-IL15SEQ ID NO:14
Figure PCTCN2021094672-appb-000020
anti CS1 VH-IL15Ra-Fc(Knob)SEQ ID NO:15
Figure PCTCN2021094672-appb-000021
anti CD38 VL-CL SEQ ID NO:16
Figure PCTCN2021094672-appb-000022
Figure PCTCN2021094672-appb-000023
anti CD38 VH-CH1-CH2-CH3(Hole)SEQ ID NO:17
Figure PCTCN2021094672-appb-000024
克隆构建方法：
克隆设计如表1所示：分别构建载体编码anti Her2(Tmab)VL-IL15(SEQ ID NO:10)、anti Her2(Tmab)VH-IL15Ra-Fc(Knob)(SEQ ID NO:11)、anti CS1 VL-IL15(SEQ ID NO:14)、anti CS1 VH-IL15Ra-Fc(Knob)(SEQ ID NO:15)。质粒含有DHFR作为筛选标记,可以用于稳定株筛选；构建载体编码抗肿瘤特异性抗原抗体的轻链序列(序列)及抗肿瘤特异性抗原抗体的重链序列(其中Fc含有Hole突变即(T366S,L368A,Y407V)anti Her2′VL(Pmab)-CL(SEQ ID NO:12)、anti Her2′(Pmab)VH-CH1-CH2-CH3(Hole)(SEQ ID NO:13)、anti CD38 VL-CL(SEQ ID NO:16)、anti CD38 VH-CH1-CH2-CH3(Hole)(SEQ ID NO:17)，质粒含有GS作为筛选标记,可以用于稳定株筛选。蛋白分子的结构示意图如图6所示。在其它的实施方式中，蛋白分子可以为其它结构形式，分别如图2至图8所示。
表6.克隆设计构建
Figure PCTCN2021094672-appb-000025
按照表7所示，设计构建对照抗体Trastuzumab，Pertuzumab，Elotuzumab，Daratumumab。
表7.对照蛋白序列及编号
Figure PCTCN2021094672-appb-000026
Figure PCTCN2021094672-appb-000027
Figure PCTCN2021094672-appb-000028
瞬转表达目标分子
接种Expi CHO-S细胞到Forti CHO培养基中(Gibco，A1148301)额外加入8mM GlutaMax，37℃，120rpm，8％CO 2培养。转染前一天，将Expi CHO-S细胞密度调整为3*10E6/mL，置于摇床中，37℃，120rpm，8％CO 2培养；转染当天，取样，计数，将细胞密度稀释为6*10E6/ml，每瓶40ml，置于125ml的摇瓶中；取对应质粒各20ug与4.8mL Opti MEM混合，加入120ul Polyplus-FectoPRO转染试剂，将DNA和转染试剂混合均匀后，置于室温10min，将混合物缓慢置于细胞中，混合均匀后，置于摇床培养。培养过程中，分别在第1，4，6，8天每瓶补加2mL Feed PFF05(OPM，F81279-001)及1m的30％葡萄糖溶液。转染第一天，将温度降为32℃，CO2浓度降为5％。13天收样，8000rpm离心20min，取上清，待纯化。
实施例2：融合蛋白的纯化
Protein A亲和层析纯化
用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
CH1-XL亲和层析
将Protein A处理后样品8000rpm×15min离心，取上清；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后上清经上样环过柱，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至10mAU时开始收集洗脱峰(PAC-EP)，UV280下降至10mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将CH1-EP调至中性。
PNGase F酶切
取39.5μl样品，加入1.98μl 10％SDS和1.58μl 1M DTT，混合均匀后100℃煮沸10分钟；待样品冷却，加入4.8μl 10％NP-40和1.0μl PNGase F，混匀后4℃水浴过夜；将过夜后样品75℃加热10分钟热失活，样品进行SDS-PAGE电泳及SEC，检测蛋白纯度。
SDS-PAGE电泳分析及HPLC-SEC分析
酶切前和酶切后样品各取5μg，用1×PBS将体积定容至48μl，再加入12μl 5×Protein Loading Dye，95℃加热10分钟。在电泳槽中倒入pH为8.3的Tris-HCl缓冲液(含0.1％SDS)，用移液枪向样品槽中加入样品，每个样品槽加样60μl。将电压设置为140V，时间设置为100分钟即可开始电泳。电泳结束后取出凝胶，浸泡在染色液中30分钟，再用脱色液脱色，直至蛋白质区条带清晰。图9是SDS-PAGE电泳分析QP34563457蛋白还原与非还原条带。图10是HPLC-SEC分析QP34563457蛋白纯度。
实施例3：蛋白QP34563457肽图分析
本实施例利用LC-MS来鉴定QP34563457蛋白的氨基酸序列。QP34563457蛋白变性还原后，加入胰蛋白酶酶解后用LC-MS/MS分析，(LC仪器Agilent 1290 Infinity II，柱子Agilent Peptide Plus column。MS仪器Agilent 6545Q-TOF)，将得到的数据采用Peaks进行搜库，Peaks搜库结果经严格卡值过滤后得到可信肽段，实现了对于QP34563457蛋白Trastuzumab VL-IL15链36％的序列覆盖率(图11a，灰色为鉴定到的氨基酸序列)，Trastuzumab VH-IL15Ra-Fc链56％的序列覆盖率(图11b，灰色为鉴定到的氨基酸序列)，pertuzumab VL-CL链64％的序列覆盖率(图11c，灰色为鉴定到的氨基酸序列)，pertuzumab VH-CH1-Fc链56％的序列覆盖率(图11d，灰色为鉴定到的氨基酸序列)。通过LC-MS进行肽图分析，分别发现QP34563457 分子中各个片段，包括，Trastuzumab VL、IL15、Trastuzumab VH、IL15Ra、Fc、pertuzumab VL、CL、pertuzumab VH、CH1、Fc等。
实施例4：ELISA检测QP34563457结合Her2及其突变体活性
只结合帕托珠单抗Pertuzumab的Her2变体蛋白的设计.Matthew C.Franklin在Cancer cell上发表了帕托珠单抗Fab与Her2胞外结构的复合物结构。该团队同时用丙氨酸扫描的方法研究了Her2哪些关键氨基酸会影响与帕托珠单抗Fab的结合。将只结合帕托珠单抗Pertuzumab而不结合曲妥珠单抗trastuzumab的Her2变体蛋白命名为Her2M2(蛋白编号QP3732)。Her2 wt(蛋白编号QP3731)及Her2M2(蛋白编号QP3732)的氨基酸序列分别如下：
QP3731:(Her2 ECD-Fc)
Figure PCTCN2021094672-appb-000029
QP3732(Her2M2-Fc):
Figure PCTCN2021094672-appb-000030
Figure PCTCN2021094672-appb-000031
实验材料：milk(BD,232100)，PBS(生工，B548117-0500)；HRP-anti human IgG(H+L)(jackson，109-035-088)；TMB(洛阳佰奥通实验材料中心，C060201)；Elisa板(costa,9018)1×PBS缓冲液：称取NaCl 8.00g、KCl 0.20g、Na2HPO4·12H202.9g、KH2PO4 0.2g至800mL ddH2O中溶解，充分溶解后定容至1L，调节pH至7.4，高温灭菌备用。或购买商品化10×、20×的PBS溶液稀释至1×PBS缓冲液使用。封闭液：称取5g的milk至PBS中,封闭液需现配现用。终止液(1mol/LH2SO4)：取109mL 98％的浓H2SO4缓慢滴加至2000mL ddH2O中。TMB 37℃显色10min,置于摇床中(120rpm)，100μl/well；
实验过程：Her2及其变体蛋白QP3731及QP3732包板，1ug/ml，4度过夜；PBS洗3遍。按200μL/孔加入封闭液5％milk，37度孵育1h。封闭结束后，PBS洗3遍，孵育样品，按照20ug/ml 5倍稀释，共稀释7个梯度最后一孔稀释100倍,100μl/well充分混匀后，孵育1h,PBST洗3遍；加酶标抗体：孵育HRP-anti human Fab抗体，按照1:5000稀释比例,100μl/孔，充分混匀后，孵育1h，PBST洗6遍。加底物显色液：按100μL/孔用量加底物显色液TMB，置于摇床中，200rpm,35℃避光显色10min。
终止：显色完成后，按100μL/孔用量快速加入终止液终止反应。检测：在酶标仪上，测其A450nm的OD值，使用graphpad prism软件分析结果。
实验结果：表明曲妥珠单抗trastuzumab能很好的结合Her2wt，而基本不结合Her2M2；QP34563457和帕托珠单抗Pertuzumab均能很好的结合Her2wt和Her2M2(结果如图12，图13)。
实施例5：ELISA检测QP34623463结合CD38及CS1活性
实验材料：milk(BD,232100)，PBS(生工，B548117-0500)；HRP-anti human IgG(H+L)(jackson，109-035-088)；TMB(洛阳佰奥通实验材料中心，C060201)；Elisa板(costa,9018)1×PBS缓冲液：称取NaCl 8.00g、KCl 0.20g、Na2HPO4·12H20 2.9g、KH2PO4 0.2g至800mL ddH2O中溶解，充分溶解后定容至1L，调节pH至7.4，高温灭菌备用。或购买商品化10×、20×的PBS溶液稀释至1×PBS缓冲液使用。封闭液：称取5g的milk至PBS中,封闭液需现配现用。终止液(1mol/LH2SO4)：取109mL 98％的浓H2SO4缓慢滴加至2000mL ddH2O中。TMB 37℃显色10min,置于摇床中(120rpm)，100μl/well；
实验过程：CD38-Fc和CS1-Fc融合蛋白分别包板，1ug/ml，4度过夜；PBS洗3遍。按200μL/孔加入封闭液5％milk，37度孵育1h。封闭结束后，PBS洗3遍，孵育样品，按照20ug/ml 5倍稀释，共稀释7个梯度最后一孔稀释100倍，100μl/well充分混匀后，孵育1h,PBST洗3遍；加酶标抗体：孵育HRP-anti human Fab抗体， 按照1:5000稀释比例，100μl/孔，充分混匀后，孵育1h，PBST洗6遍。加底物显色液：按100μL/孔用量加底物显色液TMB，置于摇床中，200rpm,35℃避光显色10min。终止：显色完成后，按100μL/孔用量快速加入终止液终止反应。检测：在酶标仪上，测其A450nm的OD值，使用graphpad prism软件分析结果。
实验结果：QP34623463既能够结合CD38抗原又能结合CS1抗原(如图14和图15所示)。
实施例6：FACS检测QP34563457结合Her2高表达细胞
实验材料：BT474细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-BR-3细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-OV-3细胞(人卵巢癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。
实验方法：
实验过程：BT474细胞使用含有10％FBS，0.11g/L丙酮酸钠和2.5g/L葡萄糖的RPMI1640(Gibco)培养基，SK-BR-3细胞使用含有10％FBS的DMEM培养基(Gibco)，SK-OV-3细胞使用含有10％FBS的McCoy′s 5a培养基(Gibco)。于37℃5％CO2培养箱中培养，胰酶消化收集细胞，按100000个细胞每孔接种于96孔板中，2％FBS/PBS冰上封闭1h，孵育不同浓度蛋白QP34563457及对照蛋白，冰上1h，PBS洗3遍，孵育PE-anti human Fc(1：200)稀释，PBS洗3遍，200微升PBS重悬，FACS读取平均荧光值，使用graphpad prism软件分析结果。
实验结果：QP34563457能很好的结合BT474、SK-BR-3和SK-OV-3这些Her2高表达细胞，结合能力与阳性对照单抗Trastuzumab和pertuzumab接近，而阴性对照抗体不能结合Her2高表达细胞(如图16-18所示)。
实施例7：Her2双特异抗体介导的增殖抑制
实验目的：确定Her2双特异抗体对肿瘤细胞的增殖抑制作用
实验材料：BT474细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-BR-3细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-OV-3细胞(人卵巢癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。细胞增殖及毒性检测试剂盒(CCK-8)，购自美仑生物，货号MA0218；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验过程：BT474细胞使用含有10％FBS，0.11g/L丙酮酸钠和2.5g/L葡萄糖的RPMI1640(Gibco)培养基，SK-BR-3细胞使用含有10％FBS的DMEM培养基(Gibco)，SK-OV-3细胞使用含有10％FBS的McCoy′s 5a培养基(Gibco)。于37℃，5％CO 2培养箱中培养，胰酶消化收集细胞，离心后用含有1％FBS的培养基重悬，按10000个细胞，50μl每孔接种于96孔板中，37℃贴壁培养3小时；将各个待测抗体用培养基按3倍梯度稀释后，50μl每孔与细胞悬液混合均匀，37℃，5％CO 2培养箱中培养3天；将CCK-8试剂按10μl每孔加入待测的96孔板中，37℃，5％CO 2培养箱中孵育2小时；将96孔板取出，于酶标仪中检测450nm波长的吸光值，计算细胞活力值，与样品浓度对数作图，使用graphpad prism软件分析结果。通过四参数拟合出细胞增殖抑制曲线。
实验结果：QP34563457对HER2阳性肿瘤细胞BT474、SK-BR-3、SK-OV-3均显示出显著的增殖抑制效果，明显优于Trastuzumab或Pertuzumab单抗及两者联用(具有协同性)(如图19-21所示)。
实施例8：HER2双特异抗体介导的ADCC作用
实验目的：确定HER2双特异抗体的ADCC作用
实验材料：PBMC购买自上海赛笠生物科技有限公司，BT474细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-BR-3细胞(人乳腺癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心。SK-OV-3细胞(人卵巢癌细胞株)，购于中国医学科学院基础医学研究所细胞资源中心，Cytotox96 non-radioactive cytotoxicity assay检测试剂盒购自Promega(G1780)。
实验过程：复苏PBMC，收集细胞待第二天备用。准备靶细胞:SK-BR-3，BT-474，SK-OV-3用胰酶消化，1000rpm 5min。PBS清洗2次后，铺96孔板，20000个每孔，50μl每孔，37℃5％CO 2孵育2h。准备抗体:用培养基(RPMI1640含10％low-IgG FBS)1:4梯度稀释抗体(80ug/ml—0.000512ug/ml,0ug/ml)10个浓度。加入上面稀释各浓度的抗体50μl每孔，37℃孵育15min。准备PBMC:将第一天复苏的PBMC离心，用培养基(RPMI1640含10％low-IgG FBS)重悬，计数。按PBMC:Target cell＝30:1，加入上面的孔板中50μl每孔，37度孵育4小时。LDH检测:对于最大释放组和体积矫正组，提前45min加入10μl裂解液,继续放置培养箱培养。培养4h后，吸取50μl上清至酶标板中,按照Cytotox96non-radioactive cytotoxicity assay检测试剂盒(Promega，G1780)说明书，加入50μl reagent。室温，避光反应30min后，加入50μl stop solution.490nm吸光值读数(加入stop solution后1h内完成读数)。计算：杀伤百分比＝(样品释放-靶细胞自发-效应细胞自发释放)/(靶细胞最大释放-靶细胞自发释放)*100。将自发释放(对应于在没有抗体的情况下与效应细胞一起温育的靶细胞)定义为0％细胞毒性，并将最大释放(用1％Triton X-100裂解的靶细胞)定义为100％细胞毒性。
实验结果：表明QP34563457对于BT474、SK-BR-3、SK-OV-3肿瘤细胞均保持了相对于两个HER2单抗相当的ADCC活性(如图22-24所示)。
实施例9：Mo7e细胞增殖实验
实验材料：Mo7e细胞(人巨细胞白血病细胞株)，购于中国医学科学院基础医学研究所细胞资源中心,细胞增殖及毒性检测试剂盒(CCK-8)，购自美仑生物，货号MA0218；重组人GM-CSF，购自perprotech，货号300-03；人IgG，购自Sigma，货号I4506；其他抗体来自内部制备。
实验过程：Mo7e细胞使用含有10％FBS，2mM L-谷氨酰胺和8ng/ml GM-CSF的RPMI1640培养基于37℃，5％CO 2培养箱中培养；收集Mo7e细胞，800rpm离心5分钟倒掉上清，细胞用不含有GM-CSF的RPMI1640培养基洗涤2遍；用无GM-CSF的RPMI1640培养基重悬细胞并计数，将细胞按2×104个，80μl每孔接种于96孔板中，37℃，5％CO 2培养箱中培养1小时；将各个待测抗体用培养基按4倍梯度稀释后，20μl每孔与细胞悬液混合均匀，37℃，5％CO 2培养箱中培养3天；将CCK-8试剂按10μl每孔加入待测的96孔板中，37℃，5％CO 2培养箱中孵育4小时；将96 孔板取出，于酶标仪中检测450nm波长的吸光值。
实验结果：Mo7e细胞增殖实验验证其具有合适的IL15活性(如图25所示)。
实施例10：动物药效实验
在高表达HER2的人卵巢癌细胞SK-OV-3模型中评价QP34563457对肿瘤生长的抑制作用。
实验过程：SK-OV-3细胞培养在含10％胎牛血清的McCoy’s 5A+10％FBS培养液中。收集指数生长期的SK-OV-3细胞，PBS重悬至适合浓度接种。每只BALB/C-nude小鼠于右侧背部皮下接种1×10 6SK-OV-3细胞，定期观察肿瘤生长情况，待肿瘤生长至平均体积约50mm 3时根据肿瘤大小和小鼠体重随机分组给药(给药体积为10ul/g体重)，第一次给药当天定义为D0。给药方案如下：
表3：给药方案
Figure PCTCN2021094672-appb-000032
每次给药观察、记录小鼠体重和肿瘤体积，用游标卡尺测量肿瘤长短径，并按公式肿瘤体积(mm 3)＝0.5×(a×b 2)计算、记录肿瘤生长情况，绘制肿瘤生长曲线。最后一次给药后观察6天，结束实验，并记录实验数据。
实验结果：结果显示，Tastuzumab、Pertuzumab、Trastuzumab+Pertuzumab及QP34563457均能明显抑制SK-OV-3肿瘤生长，6次给药后肿瘤发生消退(TGItv＞100％)，证明QP34563457在更低用药量(单抗用药量的10％)的前提下依然在huPBMC-graft NCG小鼠模型中具有显著的肿瘤生长抑制活性(如图26和表4所示)。
表4：SK-OV-3体内药效数据及分析
Figure PCTCN2021094672-appb-000033
Figure PCTCN2021094672-appb-000034
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (21)

  1. 一种双特异性抗体，其包含：
    a)特异性结合第一抗原的第一抗体的轻链和重链；和
    b)特异性结合第二抗原的第二抗体的轻链和重链；
    其中第一抗体中两条链分别包含IL15和IL15Rα，且能够形成IL15/IL15Rα复合物；
    所述IL15包含能够结合IL15Rα的突变体，所述IL15Rα包含能够结合IL15的突变体。
  2. 如权利要求1所述的双特异性抗体，其中所述IL15和IL15Rα之间具有一对或多对二硫键；
    所述IL15包含以下所示突变，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位；
    组合 IL15突变 1 N1D 2 N4D 3 D8N 4 D30N 5 D61N 6 E64Q 7 N65D 8 Q108E 9 N1D/D61N 10 N1D/E64Q 11 N4D/D61N 12 N4D/E64Q 13 D8N/D61N 14 D8N/E64Q 15 D61N/E64Q 16 E64Q/Q108E 17 N1D/N4D/D8N 18 D61N/E64Q/N65D 19 N1D/D61N/E64Q/Q108E 20 N4D/D61N/E64Q/Q108E
    或所述IL15和IL15Rα包含以下所示突变组合，计数方式根据SEQ ID No.1所示IL15第一个氨基酸开始算为第1位；SEQ ID No.3所示IL15Rα第一个氨基酸开始算为第1位。
    Figure PCTCN2021094672-appb-100001
    Figure PCTCN2021094672-appb-100002
  3. 如权利要求1所述的双特异性抗体，其中所述第一抗体的可变结构域VH1和VL1之间具有一对或多对二硫键，所述VH1和VL1包含以下突变组合形式，根据EU计数。
    Figure PCTCN2021094672-appb-100003
  4. 如权利要求1所述的双特异性抗体，其中所述第一、第二抗体重链分别包含Fc段；所述Fc段分别包含不同突变的A、B链；所述A、B链具有以下突变组合形式，根据EU计数。
    Figure PCTCN2021094672-appb-100004
  5. 如权利要求4所述的双特异性抗体，其中所述Fc段包含Human IgG1 Fc、Human IgG2 Fc、Human IgG3 Fc、Human IgG4 Fc及其突变体；所述Fc段的A链和B链，其中一条链能够结合protein A，另一条链为不能够结合protein A的突变体,所述突变包含H435R或H435R/Y436F，根据EU计数。
  6. 如权利要求1所述的双特异性抗体，其中所述第一抗原为CD3、CD20、CD19、CD30、CD33、CD38、CD40、CD52、slamf7、GD2、CD24、CD47、CD133、 CD217、CD239、CD274、CD276、PD-1、CEA、Epcam、Trop2、TAG72、MUC1、MUC16、mesothelin、folr1、CLDN18.2、PDL1、EGFR、EGFR VIII、C-MET、HER2、FGFR2、FGFR3、PSMA、PSCA、EphA2、ADAM17、17-A1、NKG2D ligands、MCSP、LGR5、SSEA3、SLC34A2、BCMA、GPNMB、IL-6R、IL-2R、CCR4、VEGFR-2、CD6、CTLA-4、整合素α4、DNA/histone complex、PDGFRα、NeuGcGM3、IL-4Rα、IL-6Rα之任一，所述第二抗原为第一抗原的不同表位或上述另一抗原。
  7. 如权利要求1所述的双特异性抗体，其中所述第一抗体和/或第二抗体为嵌合、人源化或全人源抗体。
  8. 如权利要求1-7任一项所述的双特异抗体，其具有式I或式II所示的结构：
    Figure PCTCN2021094672-appb-100005
    其中：
    链1：VL1或VH1通过L1连接在IL15或IL15Rα的N端或C端；
    链2：从N端到C排列，VH1或VL1、L2、IL15Rα或IL15、L3、Fc；
    链3：第二抗体重链从N端到C端排列，VH2-CH1-Fc；
    链4：第二抗体轻链从N端到C端排列，VL2-CL；
    VH1、VL1代表第一抗体的可变结构域；
    VH2、VL2代表第二抗体的可变结构域；
    CH1、CL代表第二抗体的恒定结构域；
    “-”代表肽键；
    L1、L2和L3各自独立地为键或接头序列；
    Figure PCTCN2021094672-appb-100006
    其中：
    链1：IL15或IL15Rα通过L1连接在VL1或VH1的N端或C端；
    链2：从N端到C排列，IL15Rα或IL15、L2、VH1或VL1、L3、Fc；
    链3：第二抗体重链从N端到C端排列，VH2-CH1-Fc；
    链4：第二抗体轻链从N端到C端排列，VL2-CL；
    VH1、VL1代表第一抗体的可变结构域；
    VH2、VL2代表第二抗体的可变结构域；
    CH1、CL代表第二抗体的恒定结构域；
    “-”代表肽键；
    L1、L2和L3各自独立地为键或接头序列。
  9. 如权利要求8所述双特异性抗体，其中所述的链1及链2包含下表组合形式。
    Figure PCTCN2021094672-appb-100007
    Figure PCTCN2021094672-appb-100008
  10. 如权利要求8所述的双特异性抗体，其从N端到C端排列具有式III所示的结构：
    Figure PCTCN2021094672-appb-100009
    其中:
    VH1、VL1代表第一抗体的可变结构域；
    VH2、VL2代表第二抗体的可变结构域；
    CH1、CL代表第二抗体的恒定结构域；
    “-”代表肽键；
    L1、L2和L3各自独立地为低免疫源性氨基酸接头序列
  11. 如权利要求8-10任一项所述的双特异性抗体，其中所述IL15序列如SEQ ID No.1或SEQ ID No.2所示；所述IL15Rα序列如SEQ ID No.3、SEQ ID No.4、SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ ID No.9所示。
  12. 如权利要求8-10任一项所述的双特异性抗体，其中所述Fc序列如SEQ ID No.18或SEQ ID No.19所示。
  13. 如权利要求1所述的双特异性抗体，其中所述第一抗原和第二抗原分别为结合Her2两个不同表位的抗原。
  14. 如权利要求12所述的双特异性抗体，其中所述双特异抗体由SEQ ID No.10、SEQ ID No.11、SEQ ID No.13、SEQ ID No.12序列融合得到。
  15. 如权利要求1所述的双特异性抗体，其中所述第一抗原和第二抗原分别为CS1抗原和CD38抗原。
  16. 如权利要求14所述的双特异性抗体，其中所述双特异抗体由SEQ ID  No.14、SEQ ID No.15、SEQ ID No.17、SEQ ID No.16序列融合得到。
  17. 一种药物组合物，其中所述药物组合物含有：
    (a)如权利要求1-16任一项所述的双特异性抗体；和
    (b)药学上可接受的载体。
  18. 如权利要求1-16任一项所述的双特异性抗体在制备用于治疗癌症(或肿瘤)、感染和免疫调节疾病的药物中的应用。
  19. 如权利要求1-16中任意一项所述的双特异性抗体在制备抑制肿瘤生长的药物中的应用。
  20. 如权利要求18或19任一项所述双特异性抗体，其中所述癌症(或肿瘤)包含：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、前列腺癌、肾癌、宫颈癌、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头部颈部癌、肝癌、鼻咽癌、睾丸癌、骨髓癌、淋巴癌、白血病、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症。
  21. 如权利要求14所述的双特异性抗体在制备诊断Her2阳性肿瘤(例如乳腺癌、胃癌)的试剂或试剂盒中的用途。
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