WO2021219127A1 - 一种靶向her2和pd-1的双特异性抗体及其应用 - Google Patents

一种靶向her2和pd-1的双特异性抗体及其应用 Download PDF

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WO2021219127A1
WO2021219127A1 PCT/CN2021/091441 CN2021091441W WO2021219127A1 WO 2021219127 A1 WO2021219127 A1 WO 2021219127A1 CN 2021091441 W CN2021091441 W CN 2021091441W WO 2021219127 A1 WO2021219127 A1 WO 2021219127A1
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antibody
her2
bispecific antibody
chain
cells
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French (fr)
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王春河
耿美玉
谢作权
王艳菲
许慧
陈艺丽
黄承浩
罗文新
夏宁邵
丁健
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中国科学院上海药物研究所
厦门大学
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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
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/2818—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 immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—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)

Definitions

  • the invention relates to the field of tumor immunotherapy. Specifically, the present invention relates to a fusion protein containing a targeted PD-1 molecule. More specifically, it relates to bispecific antibodies targeting HER2 and PD-1 and their application as anti-tumor drugs.
  • Bispecific antibodies are defined as artificial antibodies that contain two specific antigen binding sites, which are characterized by connecting two specific antibodies, which can produce functions that the parent monoclonal antibody does not have. Now the number of bispecific antibodies entering clinical trials is increasing, and most of them are concentrated in the field of tumor treatment. According to the therapeutic mechanism of bispecific antibodies, they can be divided into three types: (1) Recruitment and activation of immune cells. Redirect specific immune cells (T cells or NK cells) to tumor cells and enhance the lethality of tumors; (2) Co-stimulation or inhibition of receptors. It can block two different pathways at the same time and exert unique or overlapping functions; (3) Promote the formation of protein complexes.
  • PD-1 Programmed death receptor 1
  • B cells B cells, monocyte dendritic cells, etc.
  • PD-L1 and PD-L2 are expressed in macrophages, epithelial cells, etc. Cell surface.
  • the combination of PD-1/PD-L1 inhibits the transcription and translation of genes and cytokines required for T cell activation, and plays a negative role in regulating T cell activity.
  • tumor cells use the PD-1/PD-L1 pathway to inhibit the immune activity of T cells, thereby generating immune escape and continuing to grow.
  • Antibodies targeting PD-1 can recognize the PD-1 antigen and block the PD-1/PD-L1 pathway, activate T cells, and produce anti-tumor effects.
  • Anti-PD-1 antibodies that target immune checkpoints have attracted much attention because of their wide indications and the improvement of the tumor immune environment. Compared with other immune enhancement methods such as CTLA-4 inhibitors, PD-1 inhibitors have a better objective response rate and fewer immune-related adverse reactions, but there are still many patients with ineffective drug treatment.
  • HER2 Human epidermal growth factor receptor 2
  • trastuzumab Herceptin, US005821337A
  • HER2 antibody targeting HER2 has significantly improved the clinical outcome of the indication, its drug resistance is also very obvious.
  • the combined administration of HER2 antibody and PD-1 antibody is effective.
  • blocking the PD-1/PD-L1 pathway on the basis of inhibiting the HER2 signal can activate T cells, enhance immunity, more effectively kill HER2-positive cells and reduce the probability of drug resistance.
  • bispecific antibodies targeting HER2 and PD-1 have been published, such as CN109021110 and WO2019153200A1.
  • the heterodimer form of asymmetric structure used in both is a monovalent antibody for a single-target bispecific antibody, and the step of reassembling the bispecific antibody during the production process will reduce the yield. In actual production and clinical applications, it is still necessary to further develop products with superior performance.
  • the purpose of the present invention is to provide a bispecific antibody targeting HER2 and PD-1 with stable structure, good specificity, and excellent anti-tumor effect.
  • the invention provides a tetravalent bispecific antibody with a symmetric configuration and its application.
  • a bispecific antibody comprising:
  • the scFv of the anti-HER2 antibody and the anti-PD-1 antibody are connected by a linker sequence.
  • the ScFv of the anti-PD-1 antibody is connected to a region of the anti-HER2 antibody selected from the group consisting of a heavy chain variable region, a heavy chain constant region, or a combination thereof.
  • the ScFv of the anti-PD-1 antibody is connected to the end of the heavy chain constant region of the anti-HER2 antibody.
  • the anti-HER2 antibody is humanized.
  • the ScFv of the PD-1 antibody is humanized.
  • the bispecific antibody is a homodimer.
  • the bispecific antibody is a tetravalent antibody.
  • the bispecific antibody is formed by the fusion of anti-HER2 antibody and anti-PD-1 antibody ScFv, and has two pairs of symmetrical peptide chains, and each pair of peptide chains contains light chain, L chain and The heavy chain H chain, all peptide chains are connected by disulfide bonds, and any pair of peptide chains has the structure of the H chain and the L chain shown in formula I from the N-terminus to the C-terminus:
  • VH stands for the variable region of the heavy chain of the anti-HER2 antibody
  • VL stands for the variable region of the light chain of the HER2 antibody
  • CH1, CH2 and CH3 represent the heavy chain constant regions CH1, CH2 and CH3 of the anti-HER2 antibody, respectively;
  • CL represents the constant region of the light chain of the anti-HER2 antibody
  • ScFv stands for ScFv of anti-PD-1 antibody
  • L is the linker sequence
  • the bispecific antibody has the activity of simultaneously binding to HER2 and binding to PD-1.
  • the variant of (G 4 S) n includes: a variant of the (G 4 S) n linker sequence obtained by substituting amino acids with similar or similar properties in the sequence, such as One or more S are respectively mutated to T; or 1-3 amino acids are inserted into the sequence.
  • amino acid sequence of the linker sequence is shown in SEQ ID NO: 50.
  • amino acid sequence of the linker sequence may be another flexible sequence, and the amino acid sequence is shown in SEQ ID NO: 51 or 52.
  • the ScFv of the anti-PD-1 antibody includes an anti-PD-1 heavy chain variable region and an anti-PD-1 light chain variable region.
  • the ScFv of the anti-PD-1 antibody further includes a heavy chain variable region of the anti-PD-1 antibody and a light chain variable region of the anti-PD-1 antibody for connecting the The connecting peptide of the variable region of the heavy chain and the variable region of the light chain.
  • amino acid sequence of the connecting peptide is shown in SEQ ID NO: 19.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody is as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17, or the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17 has ⁇ 85% (preferably 90%, more preferably 95%) sequence identity; and the amino acid sequence of the light chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18. Or it has ⁇ 85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16 or 18.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 3, 9 or 17, or is The sequence shown in SEQ ID NO: 3, 9 or 17 has a sequence identity of ⁇ 85% (preferably 90%, more preferably 95%); and the amino acid of the light chain variable region of the anti-PD-1 antibody
  • the sequence is shown in SEQ ID NO: 4, 10 or 18, or has a sequence identity of ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 4, 10 or 18.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody has an amino acid sequence selected from the group consisting of SEQ ID NO: 9 Mutations: the 44th amino acid residue is mutated to Cys, the 89th amino acid residue is mutated to Thr, the 100th amino acid residue is mutated to Cys, the 108th amino acid residue is mutated to Thr, or a combination thereof.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody has the following mutation based on SEQ ID NO: 10: 100th The amino acid residue at position mutated to Cys.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody has the following mutations based on SEQ ID NO: 3: No. 44 The amino acid residue at position was mutated to Cys.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody has an amino acid sequence selected from the group consisting of SEQ ID NO: 4 Mutation: The 9th amino acid residue is mutated to Ala, the 10th amino acid residue is mutated to Phe, the 100th amino acid residue is mutated to Cys, or a combination thereof.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody has the following mutation based on SEQ ID NO: 17: No. 44 The amino acid residue at position was mutated to Cys.
  • the amino acid sequence of the heavy chain variable region of the anti-PD-1 antibody has an amino acid sequence selected from the group consisting of SEQ ID NO: 18 Mutation: The 9th amino acid residue is mutated to Ala, the 10th amino acid residue is mutated to Phe, the 100th amino acid residue is mutated to Cys, or a combination thereof.
  • amino acid sequence of the ScFv of the anti-PD-1 antibody is as shown in SEQ ID NO: 20-28, or has ⁇ 85% (preferably 90%) with the sequence of SEQ ID NO: 20-28. , More preferably 95%) sequence identity.
  • amino acid sequence of the ScFv of the anti-PD-1 antibody is shown in SEQ ID NO:21.
  • the heavy chain constant regions CH1, CH2, and CH3, and the light chain constant region CL of the anti-HER2 antibody are all derived from human IgG1 or IgG4, preferably human IgG4.
  • amino acid sequence of the VH-CH1-CH2-CH3 segment in the formula I is shown in SEQ ID NO: 29, or has ⁇ 85% (preferably, the sequence of SEQ ID NO: 29) 90%, more preferably 95%) sequence identity.
  • amino acid sequence of the VL-CL segment in Formula I is shown in SEQ ID NO: 30, or has ⁇ 85% (preferably 90%, more preferably 90%) with the sequence of SEQ ID NO: 30. Preferably 95%) sequence identity.
  • the H chain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38 or 39; and the bispecific antibody
  • the L chain of a sex antibody has an amino acid sequence as shown in SEQ ID NO: 30.
  • the bispecific antibody further contains (preferably coupled with) a detectable label, a targeting label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the bispecific antibody is conjugated with a tumor targeting marker conjugate.
  • the bispecific antibody of the present invention further includes the active fragment and/or derivative of the antibody, and the derivative contains the active fragment and/or the derivative of the bispecific antibody 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% of anti-HER2 and/or anti-PD-1 activity are retained.
  • the polynucleotide has an H chain encoding the bispecific antibody as shown in SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48 or 49 Sequence; and has the sequence encoding the L chain of the bispecific antibody as shown in SEQ ID NO: 40.
  • the ratio of the polynucleotide encoding the H chain to the polynucleotide encoding the L chain is 1:2 to 3:1, preferably 2:1.
  • a vector which contains the polynucleotide according to the second aspect of the present invention.
  • the vector contains all the polynucleotides in the polynucleotide of the second aspect of the present invention at the same time.
  • the vector contains the polynucleotide encoding the H chain and the polynucleotide encoding the L chain as described in the second aspect of the present invention, respectively.
  • the vector is an expression vector.
  • the vector includes a plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • a genetically engineered host cell contains the vector as described in the third aspect of the present invention or the genome integrates the polynucleotide according to the second aspect of the present invention .
  • step (ii) Purifying and/or separating the mixture obtained in step (i) to obtain the antibody according to the first aspect of the present invention.
  • the purification can be purified and separated by a protein A affinity column to obtain the target antibody.
  • the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
  • a pharmaceutical composition which contains:
  • the pharmaceutical composition also contains an anti-tumor agent.
  • the pharmaceutical composition is in a unit dosage form.
  • the anti-tumor agent comprises paclitaxel, doxorubicin, cyclophosphamide, axitinib, levatinib, or pembrolizumab, camptothecin derivatives or Auristatin E and its analog.
  • the anti-tumor agent and the bispecific antibody may be separately present in a separate package, or the anti-tumor agent may be coupled to the bispecific antibody.
  • the dosage form of the pharmaceutical composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.
  • the parenteral administration dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
  • the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce Detection of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (for example, cisplatin) or any form of nanoparticles, or other active substances.
  • fluorescent or luminescent markers for example, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce Detection of enzymes, radionuclides, biotoxins, cytok
  • the use of the bispecific antibody according to the first aspect of the present invention or the immunoconjugate according to the seventh aspect of the present invention is provided for preparing a pharmaceutical composition for treating tumors.
  • the tumor includes solid tumors, lymphomas and/or leukemias.
  • the solid tumor includes a malignant tumor.
  • the tumor or solid tumor is selected from the group consisting of ovarian cancer, colorectal cancer, malignant melanoma, lung cancer, gastric cancer, liver cancer, kidney cancer, head and neck squamous cell carcinoma, bladder cancer, pancreatic cancer, breast cancer , Or a combination thereof.
  • a method for treating tumors comprising the steps of: administering to a subject in need a safe and effective amount of the antibody as described in the first aspect of the present invention, or the antibody as described in the sixth aspect of the present invention A pharmaceutical composition, or an immunoconjugate according to the seventh aspect of the present invention.
  • Figure 1 shows the effect of anti-PD-1 monoclonal antibody on blocking PD-1/PD-L1 detected by ELISA method.
  • Figure 2 shows the binding of anti-PD-1 monoclonal antibodies to PD-1 antigen detected by ELISA.
  • FIG. 3 shows the affinity of anti-PD-1 monoclonal antibody T16# detected by the BLI method.
  • Figure 4 shows the binding of anti-PD-1 monoclonal antibody T16# to PD-1 antigen before and after light detected by ELISA.
  • Figure 5 shows a schematic diagram of the structure of the bispecific antibody.
  • Figure A is a schematic diagram of the structure of the anti-PD-1/HER2 bispecific antibody
  • Figure B is a schematic diagram of the structure of the anti-HER2/PD-1 bispecific antibody.
  • Figure 6 shows the binding of bispecific antibodies to cells.
  • Picture A is the binding of bispecific antibody to CHO/PD-1 cells
  • Picture B is the binding of bispecific antibody to NCI-N87 cells.
  • Figure 7 shows the ADCC effect of anti-HER2/PD-1 bispecific antibodies on T cells detected by the BATDA method.
  • Figure 8 shows the purity of the anti-HER2/PD-1 bispecific antibody determined by SE-HPLC.
  • Figure 9 shows the thermal stability of the anti-HER2/PD-1 bispecific antibody determined by the DSF method.
  • Figure 10 shows the binding of the anti-HER2/PD-1 bispecific antibody to the antigen determined by the ELISA method.
  • Picture A is the binding of anti-HER2/PD-1 bispecific antibody to antigen PD-1
  • Picture B is the binding of anti-HER2/PD-1 bispecific antibody to antigen HER2.
  • Figure 11 shows the binding of anti-HER2/PD-1 bispecific antibodies to cells determined by flow cytometry.
  • Picture A is the binding of anti-HER2/PD-1 bispecific antibody to CHO/PD-1 cells
  • Picture B is the binding of anti-HER2/PD-1 bispecific antibody to HER2-positive tumor cells.
  • Figure 12 shows that the anti-HER2/PD-1 bispecific antibody simultaneously binds to the antigen PD-1 and HER2 as determined by the ELISA method.
  • Figure 13 shows the approach of the anti-HER2/PD-1 bispecific antibody to CHO/PD-1 cells and NCI-N87 cells determined by flow cytometry.
  • Figure 14 shows the blocking of the PD-1/PD-L1 pathway by the anti-HER2/PD-1 bispecific antibody determined by HTRF.
  • FIG. 15 shows the activation of T cells by anti-HER2/PD-1 bispecific antibodies detected by MLR.
  • Figure 16 shows the inhibition of NCI-N87 cells by anti-HER2/PD-1 bispecific antibodies detected by CCK8 method.
  • Figure 17 shows the efficacy of the anti-HER2/PD-1 bispecific antibody on a nude mouse model of gastric cancer NCI-N87 tumor transplanted subcutaneously.
  • Figure 18 shows the efficacy of the anti-HER2/PD-1 bispecific antibody on the human PD-1 knock-in mouse model of EMT-6/HER2 tumor transplanted subcutaneously.
  • Figure 19 shows the efficacy of the anti-HER2/PD-1 bispecific antibody on a mouse model of gastric cancer NCI-N87 tumor NCG transplanted subcutaneously.
  • Figure 20 shows the survival curve in the mouse model experiment in Example 13.
  • the bispecific antibody of the present invention is a homodimer.
  • the experimental results show that the bispecific antibody of the present invention has good stability, high affinity to the antigens PD-1 and HER2, can effectively block the PD-1/PD-L1 pathway, and can effectively activate T cells. Thereby effectively inhibiting tumors. Therefore, the bispecific antibody of the present invention can be developed as an antitumor drug with superior curative effect.
  • the present invention has been completed on this basis.
  • antibody is also called “immunoglobulin", which can be a natural or conventional antibody, in which two heavy chains are connected to each other by disulfide bonds and each heavy chain and light chain are connected by disulfide bonds.
  • light chains There are two types of light chains, ⁇ (l) and ⁇ (k).
  • heavy chains or isotypes that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains.
  • the light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable region of the heavy chain (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH).
  • the variable regions of the light chain (VL) and heavy chain (VH) both determine the binding recognition and specificity of the antigen.
  • the constant domain (CL) of the light chain and the constant domain (CH) of the heavy chain confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the immunoglobulin Fab fragment and consists of the variable part of one light chain and one heavy chain.
  • the specificity of an antibody depends on the structural complementarity between the antibody binding site and the epitope.
  • the antibody binding site is composed of residues mainly derived from the hypervariable region or complementarity determining region (CDR). Occasionally, residues from non-hypervariable or framework regions (FR) affect the overall domain structure and thus the binding site.
  • Complementarity determining region or CDR refers to an amino acid sequence that collectively defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site.
  • the light chain and the heavy chain of an immunoglobulin each have three CDRs, which are referred to as CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, and CDR3-H.
  • Conventional antibody antigen binding sites therefore include six CDRs, including a set of CDRs from each of the heavy and light chain v regions.
  • variable means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a b-sheet configuration and are connected by three CDRs forming a connecting loop, which can form a partial b-sheet structure in some cases.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIHPubl. No. 91-3242, Vol. 1, pp. 647-669 ( 1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • FR framework region
  • the light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%. , 97%, 98% or 99%.
  • human framework region is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .
  • monoclonal antibody refers to an antibody molecule having a single amino acid composition against a specific antigen, and should not be understood as requiring the production of the antibody by any specific method.
  • Monoclonal antibodies can be produced by a single clone of B cells or hybridomas, but can also be recombinant, that is, produced by protein engineering.
  • the term "antigen" or "target antigen” refers to a molecule or part of a molecule that can be bound by an antibody or antibody-like binding protein.
  • the term further refers to a molecule or part of a molecule that can be used in an animal to produce an antibody capable of binding to an epitope of the antigen.
  • the target antigen can have one or more epitopes.
  • the antibody-like binding protein can compete with a complete antibody that recognizes the target antigen.
  • linker As used herein, the terms “linker”, “connecting peptide”, and “linker” are used interchangeably and refer to insertion into the immunoglobulin domain to provide sufficient mobility for the light and heavy chain domains to fold into Exchange one or more amino acid residues of a dual variable region immunoglobulin.
  • suitable linkers include monoglycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements that need to be implemented in the linker.
  • bispecific antibody As used herein, the terms “bispecific antibody”, “bifunctional antibody”, “antibody of the present invention”, “biantibody of the present invention”, “biantibody” and “bifunctional fusion antibody” are used interchangeably and refer to the present invention.
  • the anti-HER2/PD-1 bispecific antibody described in the first aspect can simultaneously target HER2 and PD-1.
  • the bispecific antibody is formed by the fusion of scFv of an anti-HER2 antibody and an anti-PD-1 antibody, and has two pairs of symmetrical peptide chains, each pair of peptide chains contains a light chain L chain As with the heavy chain H chain, all peptide chains are connected by disulfide bonds, and any pair of peptide chains has the structure of the H chain and the L chain shown in formula I from the N-terminus to the C-terminus:
  • VH stands for the variable region of the heavy chain of the anti-HER2 antibody
  • VL stands for the variable region of the light chain of the HER2 antibody
  • CH1 and CH2 distinguish between CH1 and CH2 representing the heavy chain constant regions of the anti-HER2 antibody
  • CL represents the constant region of the light chain of the anti-HER2 antibody
  • ScFv stands for ScFv of anti-PD-1 antibody
  • L is no or linker sequence
  • the bispecific antibody has the activity of simultaneously binding to HER2 and binding to PD-1.
  • the H chain of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38 or 39; and the L of the bispecific antibody The chain has an amino acid sequence as shown in SEQ ID NO:30.
  • the double antibody of the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or proprotein sequence, or with Fusion protein formed by 6His tag
  • the double antibody of the present invention refers to an antibody having anti-HER2 and anti-PD-1 activities and including two of the above-mentioned structures of formula I.
  • the term also includes variant forms of the antibody having the same function as the double antibody of the present invention, including two of the above-mentioned structures of formula I. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the dual antibodies of the present invention.
  • the variant forms of the double antibody include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions
  • the encoded protein and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • “conservative variants of the double antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, compared with the amino acid sequence of the double antibody of the present invention. Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions 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 Preferred substitution Ala(
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • nucleic acid (and nucleic acid combination) of the present invention can be used to produce the recombinant antibody of the present invention in a suitable expression system.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Fortunately, hybridization occurs when more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • One feasible method is to use artificial synthesis to synthesize relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli; Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the double antibody of the present invention can be used alone, or can be combined or coupled with a detectable marker (for diagnostic purposes), a therapeutic agent, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nano magnetic particles; 8. Tumor therapeutics (for example, cisplatin) or any form of anti-tumor drugs, etc.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavity injection.
  • the pharmaceutical composition of the present invention can be directly used to bind HER2 or PD-1 antigen molecules, and thus can be used to treat tumors.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned single domain antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient.
  • a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the present invention optimizes the stability of bispecific antibodies.
  • the bispecific antibody provided by the present invention has a high affinity for both the antigen PD-1 and HER2.
  • the bispecific antibody provided by the present invention can effectively block the PD-1/PD-L1 pathway.
  • the bispecific antibody provided by the present invention can effectively activate T cells.
  • the present invention can specifically narrow the distance between HER2-positive tumor cells and activated T cells.
  • the bispecific antibody provided by the present invention can inhibit the growth of HER2-positive tumors more effectively.
  • variable region (SEQ ID NO: 1 and 2) of the anti-PD-1 human-mouse chimeric antibody is derived from hybridoma screening, and the constant region is derived from human antibody IgG.
  • the chimeric antibody needs to be humanized, that is, the mouse CDR sequence is transplanted into the human antibody variable region framework, the purpose is to effectively reduce the immunogenicity of the antibody in clinical use.
  • the CDR region primers are synthesized according to the sequence and combined with the various variable region frameworks of human antibodies to form VH and VL. Using PCR and DNA recombination technology to construct on the vector with CH1 or Ck, electrotransfer to Escherichia coli to construct a humanized Fab phage library.
  • the antigen human PD-1 protein is used for multiple rounds of adsorption-elution-amplification, and the specific phage antibodies are enriched.
  • Select clones for ELISA to detect the binding of antigen PD-1 and determine the variable region sequence select 8 antigen-binding epitopes with higher activity, namely T1#, T4#, T8#, T16#, T68#, T77 #, T79# and T82#, the sequence of the variable region is SEQ ID NO: 2-18, respectively.
  • variable region sequence of the humanized PD-1 antibody was connected with the human IgG constant region to construct a plasmid, and the cell was transiently transfected to express the protein, and the purity was checked by high performance liquid chromatography (SE-HPLC).
  • SE-HPLC high performance liquid chromatography
  • the molecular level compares the binding of 8 anti-PD-1 antibodies to the antigen PD-1 and their ability to block PD-1 and PD-L1.
  • ELISA method is used to determine the blocking activity of the antibody.
  • the operation is: coat the antigen PD-1 protein at the bottom of the 96-well plate, add gradually diluted anti-PD-1 antibody to it, and then add biotinylated PD-L1, as HRP- After streptavidin is combined with biotin, the color is developed with TMB, and finally the inhibition rate of the antibody is calculated (Figure 1).
  • ELISA method is used to determine the binding activity of antibodies.
  • the operation is to coat protein A on the bottom of a 96-well plate, add anti-PD-1 antibody to it, and then add the diluted biotinylated antigen PD-1 protein, as HRP-Streptavidin After combining with biotin, the color was developed with TMB, and finally the EC50 was calculated (Figure 2).
  • the BLI method is used to determine the binding activity of the antibody.
  • the operation is: fix the anti-PD-1 antibody on the protein A probe, firstly bind to the gradiently diluted antigen PD-1, then immerse in the blank buffer for dissociation, and use the software to calculate the equilibrium
  • the dissociation constant KD value (Figure 3), the smaller the KD value, the better the binding activity of the antibody.
  • the light stability experiment is to expose the antibody to light of no less than 1.2 million lux and near-ultraviolet energy no less than 200 Wh/m2. The comparison of the effect of light on the activity of the sample is shown in Figure 4.
  • T1#, T16# and T82# have better purity, activity and stability.
  • Two bispecific antibodies targeting HER2 and PD-1 were constructed in the IgG-scFv configuration (Figure 5).
  • One is to connect an IgG antibody targeting PD-1 and a single-chain antibody targeting HER2 with a linker (SEQ ID NO: 19) to form an anti-PD-1/HER2 bispecific antibody.
  • the other is to connect the IgG antibody targeting HER2 and the single-chain antibody targeting PD-1 with a linker (SEQ ID NO: 10) to form an anti-HER2/PD-1 bispecific antibody.
  • the single-chain antibody scFv is formed by connecting the VH and VL of the parent antibody (such as anti-PD-1 antibody or anti-HER2 antibody) with (G 4 S) 4 chains. Construct plasmid and express purified antibody.
  • variable region of the anti-PD-1 antibody was selected to construct a single-chain antibody, which was linked to the C-terminus of the anti-HER2 antibody with reference to the IgG-scFv configuration (as shown in Figure 5B) to construct an anti-HER2/PD-1 bispecific antibody. It is considered that although the IgG1 subtype can increase the killing of antibodies against HER2-positive tumors, the damage to T cells expressing PD-1 should also be considered.
  • Two anti-HER2/PD-1 bispecific antibodies, IgG1 and IgG4 were expressed, and the antibody-dependent cell-mediated cytotoxicity was detected by the BATD method. The experimental results are shown in Figure 7.
  • the anti-HER2/PD-1 bispecific antibody IgG4 is more conducive to the safety of the drug.
  • scFv makes the antibody unstable and prone to aggregation.
  • VH44 and VL100 of scFv are mutated to cysteine to form scFv interchain disulfide bonds; after the DS software simulates the spatial structure of scFv, it predicts and mutates the amino acid sites with a tendency to aggregate; compares the variable regions of anti-PD-1 antibodies Sequence search for optimization sites.
  • the heavy and light chains are transfected into mammalian cells such as HEK293 by transient transfection or stable transfection. After high-speed centrifugation of the cell culture medium, the protein A can be combined with the Fc of the anti-HER2/PD-1 bispecific antibody to purify the protein using the affinity filler protein A.
  • the purified protein was placed in an ultrafiltration concentration tube (standard molecular weight cut-off is 50KDa), and the solution was replaced with PBS, pH 7.2.
  • High performance liquid chromatography SE-HPLC was used to detect the purity. After diluting the protein solution to 1 mg/ml, load 20 ⁇ L of the sample and use a high-performance liquid exclusion liquid chromatography column MAbPac TM SEC-1 (7.8 mm ⁇ 300 mm, Thermo Fisher Scientific) for detection.
  • the mobile phase is 20 mM phosphate buffer pH 7.0, containing 150 mM sodium chloride.
  • the flow rate is 0.6ml/min, and a 280nm UV detector is used to obtain the signal value. As shown in Figure 8, the main peak is the monomer of the bispecific antibody.
  • the purity values of AF are 86.38%, 92.82%, 91.92%, 92.49%, respectively , 92.11%, 84.04%, 90.03%, 84.10% and 90.00%.
  • Differential Scanning Fluorescence is to detect the change curve of the binding amount of protein and fluorescent dye SYPRO orange as the temperature gradually increases.
  • Use a fluorescent quantitative PCR instrument to program the temperature and measure the fluorescence value to determine the structural changes of the protein.
  • the midpoint temperature from the natural state to the deformed state is the melting temperature (Tm value), which quantifies the stability of the protein under certain conditions.
  • Tm value melting temperature
  • the specific operation is as follows: dilute the protein sample with PBS to a final concentration of 2 ⁇ M and 250 x gem orange stain, and mix the volume according to 24:1. Placed in a qPCR instrument to program the temperature from 25°C to 95°C, and the Tm value obtained after data processing is shown in Figure 9.
  • the Tm values of AF are 62.70°C, 63.90°C, 63.75°C, 63.90°C, 63.90°C, 62.70 °C, 60.30°C, 64.65°C, 60.60°C.
  • the stability of the antibody is better when the scFv increases the interchain disulfide bond; the 9th and 10th positions of the scFv light chain are mutated to Ala and Phe for better stability, such as HER2/PD-1 T1# AF and HER2 /PD-1 T82# AF.
  • Enzyme-linked immunosorbent immunoassay detects the binding of antibodies to antigens.
  • the detection process is as follows: Dilute antigen recombinant human HER2 protein (Nearshore protein, Item No. CP69) or recombinant human PD-1 protein (Nearshore protein, Item No. CX91) to 50 nM in PBS with pH 7.2, 50 ⁇ L/well and coat 96 wells Incubate the plate at 37°C for 1.5 hours. Wash with PBS 3 times. Block the 96-well plate with 200 ⁇ L/well of blocking solution and place it at 37°C for 1.5 hours. Wash with PBS 3 times.
  • FCM detection antibodies bind to stable CHO/PD-1 cells and cancer cells with high HER2 expression. 1 ⁇ 10 6 cells were collected and washed twice with PBS. Add 1 ⁇ g of antibody to 500 ⁇ L of the final system and incubate for 30 minutes. Wash with PBS twice. Add 1 ⁇ g PE anti-human IgG Fc Antibody to 500 ⁇ L of the final system, and incubate for 15 minutes in the dark. The fluorescence intensity is read by the flow cytometer. As shown in Figure 11, the anti-HER2/PD-1 bispecific antibody binds to CHO/PD-1 cells expressing PD-1 antigen and NCI-N87 and SK-BR-3 expressing HER2 antigen.
  • Enzyme-linked immunosorbent immunoassay detects that the antibody binds to two antigens at the same time. Dilute the antigen recombinant human HER2 protein to 1 ⁇ g/mL in pH7.2 PBS, coat a 96-well plate with 50 ⁇ L/well, and incubate at 37°C for 1.5 hours. Wash with PBS 3 times. Block the 96-well plate with 200 ⁇ L/well of blocking solution and place it at 37°C for 1.5 hours. Wash with PBS 3 times. Add antibodies that are serially diluted with blocking solution, including bispecific antibodies and control antibodies, and incubate at 50 ⁇ L/well for 1 hour at room temperature. Wash with PBST 3 times.
  • ELISA Enzyme-linked immunosorbent immunoassay
  • the anti-HER2/PD-1 bispecific antibody can bind to the antigens HER2 and PD-1 at the same time, while the anti-PD-1 monoclonal antibody and the anti-HER2 monoclonal antibody cannot bind at the same time.
  • FCM Flow cytometry detects the simultaneous binding of anti-HER2/PD-1 bispecific antibodies to CHO/PD-1 stable transgenic cells and antigens on the surface of NCI-N87 cells with high HER2 expression.
  • CFSE dye labels NCI-N87 cells.
  • the method refers to the instructions as follows: resuspend NCI-N87 cells in PBS, and adjust the cell density to 1 ⁇ 10 6 cells/mL after washing twice in PBS. Add CFSE dye to a final concentration of 1 ⁇ M, mix and incubate at 37°C for 10 minutes in the dark. Centrifuge at 1000 rpm for 5 minutes to collect the cells. Resuspend the cells in pre-chilled complete medium (containing 10% FBS) and incubate on ice for 5 minutes.
  • Proliferation 670 dye labels CHO/PD-1 cells The method refers to the instructions, as follows: CHO/PD-1 cells were resuspended in PBS, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL after washing twice with PBS. Add Proliferation 670 dye to a final concentration of 5 ⁇ M, mix and incubate at 37°C for 10 minutes in the dark. Centrifuge at 1000 rpm for 5 minutes to collect the cells. Resuspend the cells in pre-chilled complete medium (containing 10% FBS) and incubate on ice for 5 minutes. The complete medium was washed 3 times.
  • the two groups of cells were washed twice with pre-cooled PBS (containing 1% FBS) to a cell density of 5 ⁇ 10 6 cells/mL.
  • the anti-HER2/PD-1 bispecific antibody brought the stable CHO/PD-1 cell line and the NCI-N87 cell with high HER2 expression closer together.
  • Example 8 Blocking of PD-1/PD-L1 pathway by bispecific antibody
  • HTRF Homogeneous time-resolved fluorescence technology detects the blocking effect of anti-HER2/PD-1 bispecific antibodies on the PD-1/PD-L1 pathway. 4 ⁇ L each of Tag1-PD-L1 and Tag2-PD-1 proteins with different tags, and add 2 ⁇ L of serially diluted antibodies. After incubating at room temperature for 15 minutes, add 10 ⁇ L of tag antibody solution, where anti-Tag1-Eu3+ and anti-Tag2-XL665 are mixed in a 1:1 ratio. After incubating for 2 hours at room temperature, the fluorescence values at the wavelengths of 665nm and 620nm were read. Calculate the data using 665nm/620nm ⁇ 10 4. As shown in Figure 14, the anti-PD-1 scFv part of the anti-HER2/PD-1 bispecific antibody can still block the PD-1/PD-L1 pathway better.
  • the mixed lymphocyte reaction detects the activation of T cells by the anti-HER2/PD-1 bispecific antibody.
  • Human dendritic cells DC were obtained: PBMC cells were sorted using CD14 magnetic beads, and the sorted CD14+ monocytes were stimulated with 500ng/mL IL-4 and 500ng/mL GM-CSF for 5 days. Change the medium in half every 2 days and replenish cytokines. Add 30ng/mL TNF- ⁇ , 300ng/mL IL-1 ⁇ , 300ng/mL IL-6, 3ug/mL PGE2, and co-stimulate for 2 days to induce DC maturation.
  • CD4 magnetic beads sort PBMC cells, which are of different origin from the PBMC cells obtained from DC.
  • Example 10 Bispecific antibodies inhibit the growth of HER2-positive tumor cells
  • Example 11 The curative effect of the bispecific antibody on the nude mouse model of gastric cancer NCI-N87 tumor transplanted subcutaneously.
  • the nude mice used in the experiment were female BALB/c (Charles river). Each mouse was subcutaneously inoculated with 5 ⁇ 10 6 HER2 high-expressing NCI-N87 cells, divided into negative control group, positive control group (trastuzumab) and sample group (anti-HER2/PD-1 double antibody) ), 10 mice per group.
  • the administration concentration is 50 nmol/kg, that is, the administration concentration of the control group is 7.5 mg/kg and the double antibody is 10 mg/kg, and the administration method is intraperitoneal administration.
  • the drug was administered twice a week and the tumor volume was measured.
  • the anti-HER2/PD-1 bispecific antibody can significantly inhibit the growth of HER2-positive tumors and is comparable to trastuzumab.
  • Example 12 The curative effect of bispecific antibody on human PD-1 knock-in mouse model of subcutaneously transplanted tumor
  • the human-derived HER2 sequence lentivirus was transformed into EMT-6 cells to obtain EMT-6/HER2 stable transfected cell line.
  • Flow cytometry confirmed that EMT-6/HER2 cells overexpress HER2.
  • trastuzumab targeting HER2 cannot inhibit tumor growth, that is, this model cannot evaluate anti-HER2 antibodies.
  • the overexpression of HER2 is beneficial to increase the immunogenicity of tumors.
  • mice used in the experiment were female PD-1 knock-in BALB/c (Jichui Yaokang). Each mouse was subcutaneously inoculated with 5 ⁇ 10 5 EMT-6/HER2 cells under the armpit, divided into negative control group, positive control group (pembrolizumab) and sample group (anti-HER2/PD-1 double antibody). Group 7 mice.
  • the administration concentration is 50 nmol/kg, that is, the administration concentration of the control group is 7.5 mg/kg and the double antibody is 10 mg/kg, and the administration method is intraperitoneal administration. It is administered twice a week. Weigh and measure the tumor volume.
  • the anti-HER2/PD-1 bispecific antibody can inhibit the growth of EMT-6/HER2 tumors and is comparable to pambrolizumab.
  • Example 13 Efficacy of bispecific antibody on mouse model of gastric cancer NCI-N87 tumor NCG transplanted subcutaneously
  • NCG mouse Jichui Yaokang
  • the NCG mouse used in the experiment was female, 6-8 weeks old. Each mouse was subcutaneously inoculated with 5 ⁇ 10 6 NCI-N87 cells under the armpit and divided into 8 mice/groups. On the seventh day, 5 ⁇ 10 5 cells of human PBMC (Miaotong Biology) were injected at the same location to achieve immune humanization. Then intraperitoneally administered at a concentration of 50 nmol/kg, that is, the concentration of the control antibody is 7.5 mg/kg, the double antibody is 10 mg/kg, and the combination of the two drugs is 7.5 mg/kg for each antibody, for a total of 6 Second-rate.
  • tumor volume (mm 3 ) long side (mm) ⁇ short side (mm) 2 /2.
  • tumor volume (mm 3 ) long side (mm) ⁇ short side (mm) 2 /2.
  • the combination of anti-HER2/PD-1 bispecific antibody and anti-HER2 monoclonal antibody and anti-PD-1 monoclonal antibody has comparable tumor inhibition effect, and the anti-HER2/PD-1 double antibody is safe Sex may be better than the combination of two antibodies.

Abstract

提供一种能够同时与人PD-1抗原以及HER2抗原相结合的双功能抗体，能够抑制PD-1和HER2与其相应的受体的结合，可以将T细胞特异性的靶向到HER2高表达的肿瘤组织中、激活T细胞，并产生抗肿瘤效应，可应用于HER2阳性肿瘤的治疗。

Description

一种靶向HER2和PD-1的双特异性抗体及其应用 技术领域
本发明涉及肿瘤免疫药物治疗领域。具体而言，本发明涉及到含有靶向PD-1分子的融合蛋白。更具体的涉及靶向HER2和PD-1的双特异性抗体以及其作为抗肿瘤药物的应用。
背景技术
双特异性抗体被定义为含有两个特异性抗原结合位点的人工抗体，其特点是将两个特异性抗体连接，这可以产生亲本单抗不具备的功能。现在进入临床实验中的双特异性抗体增多，且大部分集中在肿瘤治疗领域。根据双特异性抗体的治疗机制可以将其分为3种类型：(1)免疫细胞的招募和激活。将特定的免疫细胞(T细胞或NK细胞)重定向至肿瘤细胞并增强对肿瘤的杀伤力；(2)受体的共刺激或抑制。可以同时阻断两种不同通路而发挥独特的或重叠的功能；(3)促进蛋白复合物的形成。
程序性死亡受体1(PD-1)表达在成熟的T细胞、B细胞、单核细胞树突细胞等，其相应配体PD-L1、PD-L2则表达与巨噬细胞、上皮细胞等细胞表面。正常情况下，PD-1/PD-L1结合后抑制T细胞活化所需基因及细胞因子的转录和翻译，发挥负向调控T细胞活性的作用。在肿瘤微环境中肿瘤细胞利用PD-1/PD-L1通路抑制T细胞免疫活性，从而产生免疫逃逸并持续生长。靶向PD-1的抗体可以识别PD-1抗原并阻断PD-1/PD-L1通路，激活T细胞，并产生抗肿瘤效果。靶向免疫检查点的抗PD-1抗体因其具有适应症广泛且改善肿瘤免疫环境的特点而备受关注。比起其他的免疫增强方法如CTLA-4抑制剂，PD-1抑制剂有更好的客观反应率和更少的免疫相关不良反应，但是仍有许多患者药物治疗无效。
人表皮生长因子受体2(HER2)是重要肿瘤相关抗原，在乳腺癌和胃癌等癌症中过表达。靶向HER2的曲妥珠单抗(赫赛汀，US005821337A)虽然明显改善了适应症的临床结果，但是耐药性也十分明显。在一项针对HER2抗体耐药的乳腺癌患者的I/II期临床试验中，HER2抗体与PD-1抗体联合给药效果明显。
因此，在抑制HER2信号的基础上阻断PD-1/PD-L1通路，可以激活T细胞，增强免疫，更有效的杀伤HER2阳性细胞并且降低耐药性出现几率。
目前已有靶向HER2和PD-1的双特异性抗体公开，如CN109021110和WO2019153200A1。但是都采用的非对称结构的异源二聚体形式，对于单靶点双特异性抗体为单价抗体，且在生产过程中有重新组装的双特异性抗体的步骤将降低收率。在实际的生产和临床应用上仍需要进一步开发具有更优越性能的产品。
因此，本领域迫切需要开发一种结构稳定、特异性佳，并且具有优异的抗肿瘤效果的靶向HER2和PD-1的双特异性抗体。
发明内容
本发明的目的就是提供一种结构稳定、特异性佳，并且具有优异的抗肿瘤效果的靶向HER2和PD-1的双特异性抗体。
本发明提供了一种对称构型的四价双特异性抗体及其应用。
在本发明的第一方面，提供了一种双特异性抗体，所述双特异性抗体包括：
(a)抗HER2抗体；和
(b)与所述抗HER2抗体相连接的抗PD-1抗体的单链可变区(single-chain FV,ScFv)。
在另一优选例中，所述抗HER2抗体和所述抗PD-1抗体的ScFv通过接头序列相连。
在另一优选例中，所述抗PD-1抗体的ScFv连接至所述抗HER2抗体的选自下组的区域：重链可变区、重链恒定区，或其组合。
在另一优选例中，所述抗PD-1抗体的ScFv连接至所述抗HER2抗体的重链恒定区的末端。
在另一优选例中，所述抗HER2抗体是人源化的。
在另一优选例中，所述PD-1抗体的ScFv是人源化的。
在另一优选例中，所述的双特异性抗体是同源二聚体。
在另一优选例中，所述的双特异性抗体是四价抗体。
在另一优选例中，所述的双特异性抗体由抗HER2抗体和抗PD-1抗体的ScFv融合而成，且具有相互对称的两对肽链，每对肽链含有轻链L链和重链H链，所有的肽链均由二硫键相连，其中任意一对肽链从N端到C端具有式I所示的H链和L链的结构：
Figure PCTCN2021091441-appb-000001
其中，
VH代表抗HER2抗体的重链可变区；
VL代表HER2抗体的轻链可变区；
CH1、CH2和CH3分别代表抗HER2抗体的重链恒定区CH1、CH2和CH3；
CL代表抗HER2抗体的轻链恒定区；
ScFv代表抗PD-1抗体的ScFv；
L为接头序列；
“～”代表二硫键；
“-”代表肽键；
其中，所述双特异性抗体具有同时结合HER2和结合PD-1的活性。
在另一优选例中，所述接头序列的氨基酸序列为(G 4S) n或其变体，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝4。
在另一优选例中，所述(G 4S) n的变体包括：将所述序列中性能相近或相似的氨基酸进行取代所获得的(G 4S)n接头序列的变体，如将一个或多个S分别突变为T；或者在所述序列中插入1-3个氨基酸。
在另一优选例中，所述接头序列的氨基酸序列如SEQ ID NO:50所示。
在另一优选例中，所述接头序列的氨基酸序列可以是其他柔性序列，其氨基酸序列如SEQ ID NO:51或52所示。
在另一优选例中，所述接头序列的氨基酸序列还可以是刚性序列，例如(EA 3K) n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝4。
在另一优选例中，所述抗PD-1抗体的ScFv包括抗PD-1的重链可变区和抗PD-1的轻链可变区。
在另一优选例中，所述抗PD-1抗体的ScFv还包括位于抗PD-1抗体的重链可变区和抗PD-1抗体的轻链可变区之间的用于连接所述重链可变区和轻链可变区的连接肽。
在另一优选例中，所述连接肽的氨基酸序列为(G 4S) n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n＝4。
在另一优选例中，所述连接肽的氨基酸序列如SEQ ID NO:19所示。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列如SEQ ID NO:1、3、5、7、9、11、13、15或17所示，或与SEQ ID NO:1、3、5、7、9、11、13、15或17所示的序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述抗PD-1抗体的轻链可变区的氨基酸序列如SEQ ID NO:2、4、6、8、10、12、14、16或18所示，或与SEQ ID NO:2、4、6、8、10、12、14、16或18所示的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列如SEQ ID NO:3、9或17所示，或与SEQ ID NO:3、9或17所示的序列具有≥85％(优选地90％，更优选地95％)的序列同一性；并且所述抗PD-1抗体的轻链可变区的氨基酸序列如SEQ ID NO:4、10或18所示，或与SEQ ID NO:4、10或18所示的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列中，具有基于SEQ ID NO:9的选自下组的突变：第44位氨基酸残基被突变为Cys、第89位氨基酸残基突变为Thr、第100位氨基酸残基突变为Cys、第108位氨基酸残基突变为Thr，或其组合。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列中，具有基于SEQ ID NO:10的以下突变：第100位氨基酸残基突变为Cys。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列中，具有基于SEQ ID NO:3的以下突变：第44位氨基酸残基被突变为Cys。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列中，具有基于SEQ ID NO:4的选自下组的突变：第9位氨基酸残基被突变为Ala、第10位氨基酸残基被突变为Phe、第100位氨基酸残基突变为Cys，或其组合。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列中，具有基于SEQ ID NO:17的以下突变：第44位氨基酸残基被突变为Cys。
在另一优选例中，所述抗PD-1抗体的ScFv中，所述的抗PD-1抗体的重链可变区的氨基酸序列中，具有基于SEQ ID NO:18的选自下组的突变：第9位氨基酸 残基被突变为Ala、第10位氨基酸残基被突变为Phe、第100位氨基酸残基突变为Cys，或其组合。
在另一优选例中，所述抗PD-1抗体的ScFv的氨基酸序列如SEQ ID NO:20-28所示，或与SEQ ID NO:20-28的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗PD-1抗体的ScFv的氨基酸序列如SEQ ID NO:21所示。
在另一优选例中，所述抗HER2抗体的重链恒定区CH1、CH2和CH3、以及轻链恒定区CL均来源于人IgG1或IgG4，优选为人IgG4。
在另一优选例中，所述式I中的VH-CH1-CH2-CH3区段的氨基酸序列如SEQ ID NO:29所示，或与SEQ ID NO:29的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述式I中的VL-CL区段的氨基酸序列如SEQ ID NO:30所示，或与SEQ ID NO:30的序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述双特异性抗体的H链具有如SEQ ID NO:31、32、33、34、35、36、37、38或39所示的氨基酸序列；并且所述双特异性抗体的L链具有如SEQ ID NO:30所示的氨基酸序列。
在另一优选例中，所述双特异性抗体还含有(优选偶联有)可检测标记物、靶向标记、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述双特异性抗体偶联有肿瘤靶向标记偶联物。
在另一优选例中，本发明所述双特异性抗体还包括所述抗体的活性片段和/或衍生物，所述衍生物含有所述双特异性抗体的活性片段和/或所述衍生物保留了70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、100％的抗HER2和/或抗PD-1活性。
在本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第一方面所述的双特异性抗体。
在另一优选例中，所述的多核苷酸具有如SEQ ID NO:41、42、43、44、45、46、47、48或49所示的编码所述双特异性抗体的H链的序列；并且具有如SEQ ID NO:40所示的编码所述双特异性抗体的L链的序列。
在另一优选例中，所述的多核苷酸中，编码H链的多核苷酸和编码L链的多 核苷酸的比例为1:2至3:1，优选地为2:1。
在本发明的第三方面，提供了一种载体，所述载体含有如本发明第二方面所述的多核苷酸。
在另一优选例中，所述载体同时含有如本发明第二方面所述多核苷酸中的所有多核苷酸。
在另一优选例中，所述载体分别含有如本发明第二方面所述的编码H链的多核苷酸和编码L链的多核苷酸。
在另一优选例中，所述载体为表达载体。
在另一优选例中，所述载体包括质粒、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。
在本发明的第四方面，提供了一种遗传工程化的宿主细胞，所述宿主细胞含有如本发明第三方面所述的载体或基因组中整合有本发明第二方面所述的多核苷酸。
在本发明的第五方面，提供了一种制备如本发明第一方面所述的双特异性抗体的方法，包括步骤：
(i)在合适的条件下，培养如本发明第四方面所述的宿主细胞，获得含有如本发明第一方面所述双特异性抗体的混合物；
(ii)对步骤(i)中得到的混合物进行纯化和/或分离，从而获得如本发明第一方面所述的抗体。
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。
在本发明的第六方面，提供了一种药物组合物，所述药物组合物含有：
(I)如本发明第一方面所述的双特异性抗体；和
(II)药学上可接受的载体。
在另一优选例中，所述药物组合物中还含有抗肿瘤剂。
在另一优选例中，所述药物组合物为单元剂型。
在另一优选例中，所述抗肿瘤剂包含紫杉醇、多柔比星、环磷酰胺、阿西替尼、乐伐替尼、或派姆单抗，喜树碱衍生物或Auristatin E及其类似物。
在另一优选例中，所述的抗肿瘤剂可以与所述双特异性抗体单独存在于独立的包装内，或所述抗肿瘤剂可以与所述双特异性抗体偶联。
在另一优选例中，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型。
在另一优选例中，所述的胃肠外给药剂型包括静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射、或腔内注射。
在本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物包括：
(a)如本发明第一方面所述的双特异性抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联物部分选自：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、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒，或其它活性物质。
在本发明的第八方面，提供了如本发明第一方面所述的双特异性抗体或如本发明第七方面所述的免疫偶联物的用途，用于制备治疗肿瘤的药物组合物。
在另一优选例中，所述肿瘤包括实体肿瘤、淋巴瘤和/或白血病。
在另一优选例中，所述实体瘤包括恶性肿瘤。
在另一优选例中，所述肿瘤或实体瘤选自下组：卵巢癌、结直肠癌、恶性黑色素瘤、肺癌、胃癌、肝癌、肾癌、头颈鳞癌、膀胱癌、胰腺癌、乳腺癌、或其组合。
在本发明的第九方面，提供了一种治疗肿瘤的方法，包括步骤：向需要的对象施用安全有效量的如本发明第一方面所述的抗体、或如本发明第六方面所述的药物组合物、或如本发明第七方面所述的免疫偶联物。
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ELISA法检测的抗PD-1单抗阻断PD-1/PD-L1的效果。
图2显示了ELISA法检测的抗PD-1单抗与PD-1抗原的结合。
图3显示了BLI法检测的抗PD-1单抗T16#的亲和力。
图4显示了ELISA法检测的抗PD-1单抗T16#光照前后与PD-1抗原的结合。
图5显示了双特异性抗体的结构示意图。其中，A图为抗PD-1/HER2双特异性抗体结构示意图；B图为抗HER2/PD-1双特异性抗体结构示意图。
图6显示了双特异性抗体与细胞的结合。其中，A图为双特异性抗体与CHO/PD-1细胞的结合；B图为双特异性抗体与NCI-N87细胞的结合。
图7显示了BATDA法检测的抗HER2/PD-1双特异性抗体对T细胞的ADCC作用。
图8显示了SE-HPLC测定的抗HER2/PD-1双特异性抗体的纯度。
图9显示了DSF法测定的抗HER2/PD-1双特异性抗体的热稳定性。
图10显示了ELISA法测定的抗HER2/PD-1双特异性抗体与抗原的结合。其中，A图为抗HER2/PD-1双特异性抗体与抗原PD-1的结合；B图为抗HER2/PD-1双特异性抗体与抗原HER2的结合。
图11显示了流式细胞术测定的抗HER2/PD-1双特异性抗体与细胞的结合。其中，A图为抗HER2/PD-1双特异性抗体与CHO/PD-1细胞的结合；B图为抗HER2/PD-1双特异性抗体与HER2阳性肿瘤细胞的结合。
图12显示了ELISA法测定的抗HER2/PD-1双特异性抗体同时结合抗原PD-1和HER2结合。
图13显示了流式细胞术测定的抗HER2/PD-1双特异性抗体对CHO/PD-1细胞和NCI-N87细胞的拉近。
图14显示了HTRF测定的抗HER2/PD-1双特异性抗体对PD-1/PD-L1通路的阻断。
图15显示了MLR检测的抗HER2/PD-1双特异性抗体对T细胞的活化。
图16显示了CCK8法检测的抗HER2/PD-1双特异性抗体对NCI-N87细胞的抑制。
图17显示了抗HER2/PD-1双特异性抗体对皮下移植胃癌NCI-N87肿瘤的裸鼠模型的疗效。
图18显示了抗HER2/PD-1双特异性抗体对皮下移植EMT-6/HER2肿瘤的人源PD-1敲入小鼠模型的疗效。
图19显示了抗HER2/PD-1双特异性抗体对皮下移植胃癌NCI-N87肿瘤NCG的小鼠模型的疗效。
图20显示了实施例13中小鼠模型实验中的生存曲线。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种能够同时靶向HER2和PD-1的双特异性抗体，其由抗HER2抗体和抗PD-1抗体的ScFv串联而成。本发明的双特异性抗体属于同源二聚体。实验结果表明，本发明的双特异性抗体的稳定性好、对抗原PD-1和HER2均具有较高的亲和力、能够有效地阻断PD-1/PD-L1通路、能够有效活化T细胞，从而有效地抑制肿瘤。因此，本发明的双特异性抗体可以被开发为一种疗效优越的抗肿瘤药物。在此基础上完成了本发明。
术语
通常，“抗体”也称为“免疫球蛋白“其可以是天然或常规的抗体，其中两条重链通过二硫键彼此连接且每条重链与轻链通过二硫键连接。存在两种类型的轻链，λ(l)和κ(k)。存在五种主要的重链种类(或同型)，其决定抗体分子的功能活性：IgM、IgD、IgG、IgA和IgE。每种链包含不同的序列结构域。轻链包括两个结构域或区，可变结构域(VL)和恒定结构域(CL)。重链包括四个结构域，重链可变区(VH)和三个恒定区(CH1、CH2和CH3，统称为CH)。轻链(VL)和重链(VH)的可变区都决定对抗原的结合识别和特异性。轻链的恒定结构域(CL)和重链的恒定区(CH)赋予重要的生物性质如抗体链结合、分泌、经胎盘的移动性、补体结合和与Fc受体(FcR)的结合。Fv片段是免疫球蛋白Fab片段的N-末端部分且由一条轻链和一条重链的可变部分组成。抗体的特异性取决于抗体结合位点和抗原决定区间的结构互补。抗体结合位点由主要来自高度可变 区或互补决定区(CDR)的残基组成。偶尔，来自非高度可变或框架区(FR)的残基影响整体结构域结构且进而影响结合位点。互补决定区或CDR指共同限定结合亲和力和天然免疫球蛋白结合位点天然Fv区的特异性的氨基酸序列。免疫球蛋白的轻链和重链各具有三个CDR，分另称为CDR1-L、CDR2-L、CDR3-L和CDR1-H、CDR2-H、CDR3-H。常规抗体抗原结合位点因此包括六个CDR，包含来自每个重链和轻链v区的CDR集合。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈b-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b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(参见Kabat等，NIHPubl.No.91-3242，卷I，647-669页(1991))。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“框架区(FR)”指插入CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“单克隆抗体”或“mAb”指针对特定抗原的具有单一氨基酸组成的抗体分子，且不应理解为需要通过任何特定方法产生该抗体。单 克隆抗体可由B细胞或杂交瘤的单一克隆产生，但还可为重组的，即通过蛋白工程产生。
如本文所用，术语“抗原”或“靶抗原”指能够由抗体或抗体样结合蛋白所结合的分子或分子的部分。该术语进一步指能够用于动物以产生能够与该抗原的表位结合的抗体的分子或分子的部分。靶抗原可具有一个或多个表位。对于每种由抗体或由抗体样结合蛋白识别的靶抗原，抗体样结合蛋白能够与识别靶抗原的完整抗体竞争。
如本文所用，术语“接头”、“连接肽”、“衔接物”可互换使用，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。合适的接头实例包括单甘氨酸(Gly)、或丝氨酸(Ser)残基，接头中氨基酸残基的标识和序列可随着接头中需要实现的次级结构要素的类型而变化。
双特异性抗体
如本文所用，术语“双特异性抗体”、“双功能抗体”“本发明抗体”、“本发明双抗”、“双抗”、“双功能融合抗体”可互换使用，是指本发明第一方面所述的可同时靶向HER2和PD-1的抗HER2/PD-1双特异性抗体。
在一优选的实施方式中，所述的双特异性抗体由抗HER2抗体和抗PD-1抗体的ScFv融合而成，且具有相互对称的两对肽链，每对肽链含有轻链L链和重链H链，所有的肽链均由二硫键相连，其中任意一对肽链从N端到C端具有式I所示的H链和L链的结构：
Figure PCTCN2021091441-appb-000002
其中，
VH代表抗HER2抗体的重链可变区；
VL代表HER2抗体的轻链可变区；
CH1、CH2分辨代表抗HER2抗体的重链恒定区CH1和CH2；
CL代表抗HER2抗体的轻链恒定区；
ScFv代表抗PD-1抗体的ScFv；
L为无或接头序列；
“～”代表二硫键；
“-”代表肽键；
其中，所述双特异性抗体具有同时结合HER2和结合PD-1的活性。
优选地，所述双特异性抗体的H链具有如SEQ ID NO:31、32、33、34、35、36、37、38或39所示的氨基酸序列；并且所述双特异性抗体的L链具有如SEQ ID NO:30所示的氨基酸序列。
本发明双抗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。
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。
本发明双抗指具有抗HER2以及抗PD-1活性的、包括两条上述式I结构的抗体。该术语还包括具有与本发明双抗相同功能的、包括两条上述式I结构的抗体的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明双抗的活性片段和活性衍生物。
该双抗的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
在本发明中，“本发明双抗的保守性变异体”指与本发明双抗的氨基酸序列相比，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
编码核酸和表达载体
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还 包括附加编码和/或非编码序列的多核苷酸。
本发明的核酸(以及核酸组合)可用于在合适的表达系统产生本发明的重组抗体。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC、0.1％SDS、60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺、0.1％小牛血清/0.1％Ficoll、42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是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。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
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。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。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。此外，还可通过化学合成将突变引入本发明蛋白序列中。
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。代表性例子有：大肠杆菌；链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转 化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的双抗可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、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。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.肿瘤治疗剂(例如，顺铂)或任何形式的抗肿瘤药物等。
药物组合物
本发明还提供了一种组合物。优选地，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，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)：静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。
本发明的药物组合物可直接用于结合HER2或PD-1抗原分子，因而可用于治疗肿瘤。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％)的本发明上述的单域抗体(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10微克/千克体重-约50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，是将安全有效量的免疫偶联物施用于哺乳动物，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10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。
本发明的主要优点包括：
1)本发明优化了双特异性抗体的稳定性。
2)本发明提供的双特异性抗体对抗原PD-1和HER2均具有较高的亲和力。
3)本发明提供的双特异性抗体能够有效地阻断PD-1/PD-L1通路。
4)本发明提供的双特异性抗体能够有效活化T细胞。
5)本发明可以特异性的拉近HER2阳性肿瘤细胞与活化T细胞的距离。
6)本发明提供的双特异性抗体与单独的PD-1或HER2抗体相比可以更有效地抑制HER2阳性肿瘤生长。
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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：抗PD-1抗体的人源化
抗PD-1人鼠嵌合抗体，其可变区(SEQ ID NO:1和2)来自于杂交瘤筛选获得， 恒定区来自于人源抗体IgG。嵌合抗体需要进行人源化，也就是将小鼠的CDR序列移植到人的抗体可变区框架，目的是有效降低抗体在临床使用中的免疫源性。根据序列合成CDR区引物，与人源抗体的多样的可变区框架组合成VH和VL。应用PCR和DNA重组技术构建到带有CH1或者Ck载体上，电转到大肠杆菌中构建人源化Fab噬菌体库。测定噬菌体库滴度后，用抗原人源PD-1蛋白进行多轮的吸附-洗脱-扩增，特异性噬菌体抗体被富集。挑选克隆进行ELISA检测与抗原PD-1的结合并测定可变区序列，选择其中8个活性较高的抗原结合表位，分别是T1#、T4#、T8#、T16#、T68#、T77#、T79#和T82#，可变区的序列分别为SEQ ID NO:2-18。
实施例2：比较抗PD-1抗体
将人源化PD-1抗体的可变区序列与人源IgG恒定区连接并构建质粒，经过瞬时转染细胞表达蛋白，使用高效液相排阻液相色谱(SE-HPLC)检测纯度。分子水平比较8个抗PD-1抗体与抗原PD-1的结合与阻断PD-1和PD-L1的能力。ELISA法测定抗体的阻断活性，操作为：在96孔板底部包被抗原PD-1蛋白，向其中加入梯度稀释的抗PD-1抗体，再加入生物素化的PD-L1，当HRP-Streptavidin结合生物素后用TMB显色，最后计算抗体的抑制率(如图1)。ELISA法测定抗体的结合活性，操作为：在96孔板底部包被protein A蛋白，向其中加入抗PD-1抗体，然后加入度稀释的生物素化的抗原PD-1蛋白，当HRP-Streptavidin结合生物素后用TMB显色，最后计算EC50(如图2)。BLI法测定抗体的结合活性，操作为：将抗PD-1抗体固定在protein A探针上，先是与梯度稀释的抗原PD-1结合，然后浸入到空白缓冲液中解离，用软件计算平衡解离常数KD值(如图3)，KD值越小抗体的结合活性越好。光稳定性实验是将抗体暴露在光下不低于120万lux和近紫外线能量不低于200瓦时/平方米的光线下，比较光照对样品活性影响如图4所示。这8个抗PD-1抗体中T1#、T16#和T82#的纯度、活性以及稳定性较好。
实施例3：双特异性抗体的克隆和表达
以IgG-scFv构型构建2个靶向HER2和PD-1的双特异性抗体(如图5)。一个是将靶向PD-1的IgG抗体和靶向HER2单链抗体用衔接物(SEQ ID NO:19)连接，构成抗PD-1/HER2双特异性抗体。另一个是将靶向HER2的IgG抗体和靶向PD-1单链抗体用衔接物(SEQ ID NO:10)连接，构成抗HER2/PD-1双特异性抗体。单链抗体scFv是将亲本抗体(如抗PD-1抗体或者抗HER2抗体)的VH和VL用(G 4S) 4链 连接形成。构建质粒并表达纯化抗体。如图6所示，流式细胞术检测到抗HER2/PD-1双特异性抗体中靶向PD-1的scFv的对抗原的亲和力明显降低；抗PD-1/HER2双特异性抗体中靶向HER2的scFv的对抗原几乎不结合。
选取抗PD-1抗体可变区构建单链抗体，链接在抗HER2抗体的C端参照IgG-scFv构型(如图5B)构建抗HER2/PD-1双特异性抗体。考虑到虽然IgG1亚型可以增加抗体对HER2阳性肿瘤的杀伤，但是也要考虑到对表达PD-1的T细胞损伤。表达IgG1和IgG4两种抗HER2/PD-1双特异性抗体，用BATD法检测抗体依赖细胞介导的细胞毒作用，实验结果如图7所示。抗HER2/PD-1双特异性抗体IgG4更有利于药物的安全性。
因为附加scFv使得抗体不稳定，且容易产生聚集。现有多种方法进行scFv序列的优化，目的是提高双特异性抗体的稳定性。例如，scFv的VH44和VL100突变为半胱氨酸形成scFv链间二硫键；DS软件模拟scFv空间结构后，预测有聚集趋势的氨基酸位点并进行突变；对比抗PD-1抗体可变区序列寻找优化位点。利用同源重组技术将合成的scFv序列与靶向HER2的IgG序列(SEQ ID NO:29)连接为双特异性抗体重链，与轻链(SEQ ID NO:30)共转染HEK293蛋白表达系统，获得如表1所示抗体。
表1.HER2/PD-1双特异性抗体
Figure PCTCN2021091441-appb-000003
实施例4：双特异性抗体的纯化
将重链和轻链采用瞬时转染或者稳定转染的方案转染哺乳动物细胞如 HEK293。细胞培养液高速离心后，利用亲和填料protein A可与抗HER2/PD-1双特异性抗体的Fc结合的特性纯化蛋白。纯化后的蛋白置于超滤浓缩管中(标准截留分子量为50KDa)，将溶液置换为PBS，pH7.2。
高效液相排阻液相色谱(SE-HPLC)检测纯度。将蛋白溶液稀释至1mg/ml后，上样20μL，使用高效液相排阻液相色谱柱MAbPac TM SEC-1(7.8mm×300mm，Thermo Fisher Scientific)检测。流动相为pH7.0的20mM磷酸盐缓冲液，含有150mM氯化钠。流速为0.6ml/min，并使用280nm紫外检测器获得信号值。如图8所示，主峰为双特异性抗体的单体。
另外，经检测，HER2/PD-1 T16#、HER2/PD-1 T16# s-s、HER2/PD-1 T16# 89T、HER2/PD-1 T16# 108T、HER2/PD-1 T16# 89&108T、HER2/PD-1 T1# s-s、HER2/PD-1 T82# s-s、HER2/PD-1 T1# AF、HER2/PD-1 T82# AF的纯度值分别为86.38％、92.82％、91.92％、92.49％、92.11％、84.04％、90.03％、84.10％和90.00％。
实施例5：比较双特异性抗体的稳定性
差式扫描荧光(DSF)是检测在温度逐渐升高时蛋白与荧光染料宝石橙蛋白染色剂(SYPRO orange)结合量的变化曲线。利用荧光定量PCR仪程序性升温并测定荧光值，从而判断蛋白的结构变化。从天然状态到变形状态的中点温度为熔化温度(Tm值)，Tm值在一定条件下将蛋白质的稳定性进行量化。具体操作为：将蛋白样品用PBS稀释到终浓度为2μM与250×宝石橙蛋白染色剂，体积按照24:1混合。放置在qPCR仪中从25℃至95℃程序性升温，数据处理后获得Tm值如图9所示。HER2/PD-1 T16#、HER2/PD-1 T16# s-s、HER2/PD-1 T16# 89T、HER2/PD-1 T16# 108T、HER2/PD-1 T16# 89&108T、HER2/PD-1 T1# s-s、HER2/PD-1 T82# s-s、HER2/PD-1 T1# AF、HER2/PD-1 T82# AF的Tm值分别为62.70℃、63.90℃、63.75℃、63.90℃、63.90℃、62.70℃、60.30℃、64.65℃、60.60℃。
本次加速试验是考察抗体在高温下的纯度和聚集体变化。放置条件为60℃条件加热1小时，使用SE-HPLC测定加热后纯度变化，以及使用ANS染料判断加热后的聚集体变化情况。
从结果可知，scFv增加链间二硫键后抗体的稳定性更好；scFv的轻链9位和10位突变为Ala和Phe其稳定性更好，如HER2/PD-1 T1# AF和HER2/PD-1 T82# AF。
体外活性评价
实施例6：双特异性抗体与靶点结合
酶联吸附免疫(ELISA)检测抗体与抗原结合。检测流程如下：pH7.2的PBS稀释抗原重组人HER2蛋白(近岸蛋白，货号：CP69)或者重组人PD-1蛋白(近岸蛋白，货号：CX91)至50nM，50μL/孔包被96孔板，37℃孵育1.5小时。PBS清洗3次。封闭液200μL/孔封闭96孔板，37℃放置1.5小时。PBS清洗3次。加入用封闭液梯度稀释的抗体，包括双特异性抗体和对照抗体，50μL/孔室温孵育1小时。PBST清洗3次。加入用封闭液1:2000稀释的HRP Goat Anti Human IgG，50μL/孔室温孵育1小时。PBST清洗3次。加入TMB溶液50μL/孔，室温孵育5分钟后加入50μL/孔的2M硫酸终止反应。酶标仪读取450nm波长的吸光度值。如图10所示，抗HER2/PD-1双特异性抗体对抗原PD-1(A)和HER2(B)有较高的亲和力。
流式细胞术(FCM)检测抗体分别与CHO/PD-1稳转株细胞和HER2高表达的癌症细胞结合。1×10 6个细胞被收集，PBS清洗2次。加入1μg抗体至终体系500μL，孵育30分钟。PBS清洗2次。加入1μg PE anti-human IgG Fc Antibody至终体系500μL，避光孵育15分钟。流式细胞仪读取荧光强度。如图11所示，抗HER2/PD-1双特异性抗体与表达PD-1抗原的CHO/PD-1细胞以及表达HER2抗原的NCI-N87和SK-BR-3有结合。
实施例7：双特异性抗体与靶点同时结合
酶联吸附免疫(ELISA)检测抗体与2个抗原同时结合。pH7.2的PBS稀释抗原重组人HER2蛋白至1μg/mL，50μL/孔包被96孔板，37℃孵育1.5小时。PBS清洗3次。封闭液200μL/孔封闭96孔板，37℃放置1.5小时。PBS清洗3次。加入用封闭液梯度稀释的抗体，包括双特异性抗体和对照抗体，50μL/孔室温孵育1小时。PBST清洗3次。加入被生物素标记的PD-1蛋白，用封闭液稀释至0.5μg/mL，50μL/孔室温孵育1小时。PBST清洗3次。加入用封闭液1:2000稀释的streptavidin HRP，50μL/孔室温孵育1小时。PBST清洗3次。加入TMB溶液50μL/孔，室温孵育5分钟后加入50μL/孔的2M硫酸终止反应。酶标仪读取450nm波长的吸光度值。如图12所示，抗HER2/PD-1双特异性抗体可同时结合抗原HER2和PD-1，而抗PD-1单抗和抗HER2单抗不能同时结合。
流式细胞术(FCM)检测抗HER2/PD-1双特异性抗体与CHO/PD-1稳转株细胞和HER2高表达的NCI-N87细胞表面的抗原同时结合。CFSE染料标记NCI-N87细胞。方法参考说明书，如下：PBS重悬NCI-N87细胞，在PBS清洗2次后将细胞密度调 整为1×10 6个/mL。加入CFSE染料至终浓度1μM，混匀后37℃避光孵育10分钟。1000rpm离心5分钟，收集细胞。预冷的完全培养基(含有10％的FBS)重悬细胞，冰上孵育5分钟。完全培养基清洗3次。Proliferation 670染料标记CHO/PD-1细胞。方法参考说明书，如下：PBS重悬CHO/PD-1细胞，在PBS清洗2次后将细胞密度调整为1×10 6个/mL。加入Proliferation 670染料至终浓度5μM，混匀后37℃避光孵育10分钟。1000rpm离心5分钟，收集细胞。预冷的完全培养基(含有10％的FBS)重悬细胞，冰上孵育5分钟。完全培养基清洗3次。标记后，两组细胞分别用预冷的PBS(含有1％FBS)清洗细胞2次，至细胞密度为5×10 6个/mL。将NCI-N87细胞和CHO/PD-1细胞按1:1混合。取混合后细胞100μl并加入抗体50nM，用PBS(含有1％FBS)补足终体积至200μL。4℃混合孵育30分钟。预冷的PBS(含有1％FBS)清洗细胞2次后，用500μL预冷的PBS(含有1％FBS)重悬细胞，用流式细胞仪检测荧光强度。如图13所示，抗HER2/PD-1双特异性抗体将CHO/PD-1稳转株细胞和HER2高表达的NCI-N87细胞拉近。
实施例8：双特异性抗体对PD-1/PD-L1通路的阻断
均相时间分辨荧光技术(HTRF)检测抗HER2/PD-1双特异性抗体对PD-1/PD-L1通路的阻断作用。带有不同标签的Tag1-PD-L1和Tag2-PD-1蛋白各4μL，并加入2μL梯度稀释的抗体。室温孵育15分钟后加入10μL标签抗体溶液，其中anti-Tag1-Eu3+和anti-Tag2-XL665按照1:1混合后。室温孵育2小时后，读取665nm和620nm波长处荧光值。采用665nm/620nm×10 4计算数据。如图14所示，抗HER2/PD-1双特异性抗体中抗PD-1的scFv部分仍能较好的阻断PD-1/PD-L1通路。
实施例9：双特异性抗体对T细胞的活化
淋巴细胞混合反应(MLR)检测抗HER2/PD-1双特异性抗体对T细胞的活化。人树突状细胞(DC)得获得：使用CD14磁珠分选PBMC细胞，分选获得的CD14+单核细胞经过500ng/mL IL-4和500ng/mL GM-CSF刺激5天。每2天半量换液一次，并补足细胞因子。加入30ng/mL TNF-α、300ng/mL IL-1β、300ng/mL IL-6、3ug/mL PGE2，共刺激2天，诱导DC成熟。T细胞的获取：CD4磁珠分选PBMC细胞，此PBMC细胞与DC获取的PBMC细胞来源不同。将1×10 5个CD4+T细胞与1×10 4个DC细胞接种于96孔板中，分组设置对照及加药组，加入抗体后混合培养5天。此模型中细胞 因子IFN-γ的释放代表T细胞的活化，使用ELISA试剂盒测试上清液中IFN-γ含量。如图15所示，抗HER2/PD-1双特异性抗体显示与阳性抗体相当的IFN-γ释放，证明其可活化T细胞。
实施例10：双特异性抗体抑制HER2阳性肿瘤细胞生长
CCK8检测加入抗体对肿瘤细胞的生长抑制。取生长状态良好的对数生长期细胞，铺板中置于细胞培养箱中培养，待细胞贴壁后加入梯度稀释的抗体培养72小时。更换新鲜培养基每孔加入20μL的CCK8检测液，置于培养箱中继续培养2小时，酶标仪测定450nm处的吸光度值。按公式计算生长抑制率＝[(对照组-实验组)/对照组]×100％，以浓度为横坐标，生长抑制率为纵坐标，绘制细胞生长抑制率柱状图。结果如图16所示。
体内药效评价
实施例11：双特异性抗体对皮下移植胃癌NCI-N87肿瘤的裸鼠模型的疗效。
实验所用裸鼠为雌性BALB/c(Charles river)。每只小鼠腋下皮下接种5×10 6个HER2高表达的NCI-N87细胞，分为阴性对照组，阳性对照组(曲妥珠单抗)和样品组(抗HER2/PD-1双抗)，每组10只老鼠。给药浓度为50nmol/kg，即对照组给药浓度为7.5mg/kg和双抗为10mg/kg，给药方式为腹腔给药。每周给药2次并测量瘤体积。肿瘤体积的计算公式为：肿瘤体积(mm 3)＝长边(mm)×短边(mm) 2/2。如图17所示，抗HER2/PD-1双特异性抗体可明显抑制HER2阳性肿瘤生长，且与曲妥珠单抗相当。
实施例12：双特异性抗体对皮下移植肿瘤的人源PD-1敲入小鼠模型的疗效
将人源HER2序列慢病毒转入EMT-6细胞中，获得EMT-6/HER2稳转细胞株。流式细胞术确定EMT-6/HER2细胞过表达HER2。在EMT-6/HER2细胞移植瘤小鼠模型中，靶向HER2曲妥珠单抗是无法抑制肿瘤生长的，即此模型不可评价抗HER2抗体。但HER2的过表达利于增加肿瘤的免疫原性。
实验所用小鼠为雌性PD-1敲入的BALB/c(集萃药康)。每只小鼠腋下皮下接种5×10 5个EMT-6/HER2细胞，分为阴性对照组，阳性对照组(派姆单抗)和样品组(抗HER2/PD-1双抗)，每组7只老鼠。给药浓度为50nmol/kg，即对照组给药浓度为7.5mg/kg和双抗为10mg/kg，给药方式为腹腔给药。每周给药2次。称重并测量瘤 体积。肿瘤体积的计算公式为：肿瘤体积(mm 3)＝长边(mm)×短边(mm) 2/2。如图18所示，抗HER2/PD-1双特异性抗体的可抑制EMT-6/HER2肿瘤生长，且与帕姆单抗相当。
实施例13：双特异性抗体对皮下移植胃癌NCI-N87肿瘤NCG的小鼠模型的疗效
实验所用NCG鼠(集萃药康)为雌性，6-8周。每只小鼠腋下皮下接种5×10 6个NCI-N87细胞，分为8只/组。第七天在同一位置注射人PBMC(妙通生物)5×10 5个细胞，实现免疫人源化。然后腹腔给药，给药浓度为50nmol/kg，即对照抗体给药浓度为7.5mg/kg、双抗为10mg/kg以及两药联用为每个抗体各7.5mg/kg，共给药6次。称重并测量瘤体积，肿瘤体积的计算公式为：肿瘤体积(mm 3)＝长边(mm)×短边(mm) 2/2。如图19、图20所示，抗HER2/PD-1双特异性抗体与抗HER2单抗和抗PD-1单抗药物联用相当的抑制肿瘤的效果，抗HER2/PD-1双抗安全性可能优于两抗体联用。
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. 一种双特异性抗体，其特征在于，所述双特异性抗体包括：
    (a)抗HER2抗体；和
    (b)与所述抗HER2抗体相连接的抗PD-1抗体的单链可变区(single-chain FV,ScFv)。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述的双特异性抗体由抗HER2抗体和抗PD-1抗体的ScFv融合而成，且具有相互对称的两对肽链，每对肽链含有轻链L链和重链H链，所有的肽链均由二硫键相连，其中任意一对肽链从N端到C端具有式I所示的H链和L链的结构：
    Figure PCTCN2021091441-appb-100001
    其中，
    VH代表抗HER2抗体的重链可变区；
    VL代表HER2抗体的轻链可变区；
    CH1、CH2和CH3分别代表抗HER2抗体的重链恒定区CH1、CH2和CH3；
    CL代表抗HER2抗体的轻链恒定区；
    ScFv代表抗PD-1抗体的ScFv；
    L为接头序列；
    “～”代表二硫键；
    “-”代表肽键；
    其中，所述双特异性抗体具有同时结合HER2和结合PD-1的活性。
  3. 如权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体的H链具有如SEQ ID NO:31、32、33、34、35、36、37、38或39所示的氨基酸序列；并且所述双特异性抗体的L链具有如SEQ ID NO:30所示的氨基酸序列。
  4. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1所述的双特异性抗体。
  5. 一种载体，其特征在于，所述载体含有如权利要求4所述的多核苷酸。
  6. 一种遗传工程化的宿主细胞，其特征在于，所述宿主细胞含有如权利要求5所述的载体或基因组中整合有权利要求4所述的多核苷酸。
  7. 一种制备如权利要求1所述的双特异性抗体的方法，其特征在于，包括步骤：
    (i)在合适的条件下，培养如权利要求6所述的宿主细胞，获得含有如权利要求1所述双特异性抗体的混合物；
    (ii)对步骤(i)中得到的混合物进行纯化和/或分离，从而获得如权利要求1所述的抗体。
  8. 一种药物组合物，其特征在于，所述药物组合物含有：
    (I)如权利要求1所述的双特异性抗体；和
    (II)药学上可接受的载体。
  9. 一种免疫偶联物，其特征在于，所述免疫偶联物包括：
    (a)如权利要求1所述的双特异性抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  10. 如权利要求1所述的双特异性抗体或如权利要求9所述的免疫偶联物的用途，其特征在于，用于制备治疗肿瘤的药物组合物。
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