WO2020103629A1 - 抗her2/pd1双特异性抗体 - Google Patents

抗her2/pd1双特异性抗体

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Publication number
WO2020103629A1
WO2020103629A1 PCT/CN2019/112467 CN2019112467W WO2020103629A1 WO 2020103629 A1 WO2020103629 A1 WO 2020103629A1 CN 2019112467 W CN2019112467 W CN 2019112467W WO 2020103629 A1 WO2020103629 A1 WO 2020103629A1
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Prior art keywords
seq
her2
acid sequence
amino acid
bispecific antibody
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PCT/CN2019/112467
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English (en)
French (fr)
Inventor
朱祯平
黄浩旻
顾昌玲
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Sunshine Guojian Pharmaceutical Shanghai Co Ltd
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Priority to US17/295,391 priority Critical patent/US12384853B2/en
Priority to EP19886519.8A priority patent/EP3885367A4/en
Priority to JP2021527066A priority patent/JP7165265B2/ja
Priority to CN201980068587.0A priority patent/CN113227151B/zh
Publication of WO2020103629A1 publication Critical patent/WO2020103629A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K2317/30—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
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of tumor therapy and biotechnology, and relates to a preparation method and application of anti-HER2 and PD1 bispecific antibody molecule.
  • HER2 human epidermal growth factor receptor 2
  • receptor tyrosine protein kinase activity is one of the members of the human epidermal growth factor receptor family and is only expressed at low levels in a small number of normal tissues in adults.
  • studies have shown that HER2 is overexpressed in a variety of tumors, such as overexpression in about 30% of breast cancer patients and 16% of gastric cancer patients.
  • Overexpression of HER2 in tumors can significantly promote tumor angiogenesis, The growth of tumors and the enhancement of tumor invasion and metastasis ability are important indicators of poor prognosis for these patients. Therefore, as early as 1998, the first monoclonal antibody drug Herceptin (Genentech / Roche) targeting HER2 was approved by the FDA for use in the treatment of breast cancer and gastric cancer with HER2 overexpression.
  • Human programmed cell death receptor-1 is a type I membrane protein composed of 288 amino acids, the extracellular segment is the Ig variable (V-type) domain responsible for binding ligands, and the intracellular segment is responsible for binding
  • the cytoplasmic tail of signal transduction molecules contains two tyrosine-based signal transduction motifs, namely ITIM (immunoreceptor tyrosine inhibitory motif) and ITSM (immunoreceptor tyrosine conversion motif).
  • ITIM immunoimmunoreceptor tyrosine inhibitory motif
  • ITSM immunommunoreceptor tyrosine conversion motif
  • PD-L1 programmed death receptor-ligand 1, programmed cell death-Ligand 1
  • PD-L2 programmed death receptor-ligand 2
  • Programmed cell death-Ligand 2 can inhibit the activity of T lymphocytes and related cellular immune responses in vivo.
  • a large number of studies have shown that the interaction of PD1 and PD-L1 not only maintains the balance of the immune system in the body, but also the main mechanism that causes tumor cells that express PD-L1 expression to evade immune surveillance.
  • the immune system By blocking the PD1 / PD-L1 signaling pathway, the immune system can be activated and the immune killing function of T cells can be restored.
  • (pembrolizumab) is the first humanized monoclonal antibody directed against PD1 that was approved by the FDA for the treatment of melanoma in September 2014.
  • the indications approved by 2018 include: melanoma, non-small cell lung cancer , Hodgkin's lymphoma, head and neck squamous cell carcinoma, bladder cancer, gastric cancer, and solid tumors with MSI-H or dMMR.
  • (nivolumab) is a PD1 monoclonal antibody from Bristol-Myers Squibb. It was approved by the FDA in December 2014.
  • anti-PD1 monoclonal antibody independently developed by Sansheng Guojian is a brand new anti-PD1 humanized monoclonal antibody.
  • In vivo and in vitro biological activity and anti-tumor activity studies have shown that the biological activity of anti-PD1 is between the positive control drugs Opdivo and Keytruda, which is slightly better than the positive control drug Opdivo in some aspects.
  • Bispecific antibody refers to an antibody molecule that can simultaneously bind two (or more) different epitopes.
  • bispecific antibodies have a unique mechanism of action: 1) Bispecific antibodies can bind two or more different antigen molecules or different epitopes of the same molecule at the same time. With this effect. 2) Mediate the interaction between cells.
  • Bispecific antibodies can bind to two kinds of antigens on effector cells and target cells respectively, build a bridge between effector cells and target cells, and promote the interaction between cells, such as mediating Immune cells kill tumor cells. Therefore, bispecific antibodies have unique advantages that traditional monoclonal antibodies do not have.
  • the present invention provides a new bispecific antibody that can specifically bind to HER2 and PD1, and also provides a preparation method and application of the bispecific antibody.
  • the object of the present invention is to provide a bispecific antibody that can specifically bind to HER2 and PD1; provide a nucleotide molecule encoding the bispecific antibody; provide an expression vector containing the nucleotide molecule; provide A host cell of the expression vector; providing a method for preparing the bispecific antibody; providing a pharmaceutical composition containing the bispecific antibody; providing an application of the bispecific antibody in the preparation of a drug.
  • One aspect of the present invention provides a bispecific antibody that specifically binds to HER2 and PD1, which comprises an immunoglobulin antibody IgG and two identical single-chain variable region fragments scFv, wherein each single-chain variable fragment scFv It contains a variable region VH and a variable region VL.
  • VH and VL are connected by a peptide linker L1, and each single-chain variable fragment scFv is connected in series with an immunoglobulin antibody IgG through a linker peptide L2.
  • the "bispecific antibody” in the present invention refers to a bispecific antibody that has two different antigen binding sites and can simultaneously bind HER2 and PD1, and contains two single-chain variable fragments scFv and conjugated thereto Immunoglobulin antibody IgG, each scFv is connected to each heavy chain of immunoglobulin antibody IgG via peptide linker L2 to form a heavy chain fusion protein of bispecific antibody, wherein each scFv contains variable region VH and variable region VL, VH and VL are connected by peptide linker L1.
  • the "single-chain variable region fragment scFv" of the present invention refers to a fusion protein comprising the immunoglobulin heavy chain VH and light chain VL variable regions, VH and VL are connected by a peptide linker, wherein the fusion protein remains intact Immunoglobulins have the same antigen specificity.
  • the "immunoglobulin antibody IgG" described in the present invention is a molecule of about 150 kDa, which is composed of four peptide chains, contains two identical ⁇ heavy chains of about 50 kDa, and two identical light chains of about 25 kDa, thereby having Tetramer quaternary structure.
  • the two heavy chains are connected to each other through a disulfide bond, and each is connected to a light chain.
  • the resulting tetramer has the same two halves, and the two form a fork shape or a Y-like shape, and each end of the fork contains an identical antigen binding site.
  • IgG antibodies can be divided into multiple subclasses (eg IgG1, 2, 3, 4) based on small differences in amino acid sequence in the constant region of the heavy chain.
  • the VH comprises a complementarity determining region HCDR1-3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3 shown;
  • the VL includes a complementarity determining region LCDR1-3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 6 ;
  • the heavy chain of the immunoglobulin antibody IgG includes a complementarity determining region HCDR4-6, wherein the amino acid sequence of HCDR4 is shown in SEQ ID NO: 7, and the amino acid sequence of HCDR5 is shown in SEQ ID NO: 8, wherein the amino acids of HCDR6 The sequence is shown in SEQ ID NO: 9;
  • the light chain of the immunoglobulin antibody IgG contains a complementarity determining region LCDR4-6, wherein the amino acid sequence of LCDR4 is shown in SEQ ID NO: 10, and the amino acid sequence of LCDR5 is shown in SEQ ID NO: 11, wherein the amino acid of LCDR6 The sequence is shown in SEQ ID NO: 12.
  • the binding regions of antibodies usually contain a light chain variable region and a heavy chain variable region, and each variable region contains 3 CDR domains.
  • the CDR domains of the heavy and light chains of antibodies are called HCDR and LCDR, respectively. Therefore, the conventional antibody antigen-binding site contains six CDRs, including a collection of CDRs from the heavy and light chain V regions, respectively.
  • amino acid sequence of the VH of scFv is shown in SEQ ID NO: 13
  • amino acid sequence of the VL is shown in SEQ ID NO: 14
  • amino acid sequence of the heavy chain variable region of the immunoglobulin antibody IgG is SEQ ID NO: 15
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • amino acid sequence of the peptide linker L1 is shown in SEQ ID NO: 17.
  • amino acid sequence of the peptide linker L2 is shown in SEQ ID NO: 18.
  • the molecular structure of the single-chain variable fragment scFv1 is VL-L1-VH, and the N-terminus of each scFv is connected to the C-terminus of the immunoglobulin antibody IgG heavy chain via a peptide linker L2.
  • amino acid sequence of the single-chain variable fragment scFv1 is shown in SEQ ID NO: 19.
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 20, and the light chain amino acid sequence is shown in SEQ ID NO: 21.
  • the molecular structure of the single-chain variable fragment scFv2 is VH-L1-VL, and the C-terminus of each scFv is connected to the N-terminus of the immunoglobulin antibody IgG heavy chain via a peptide linker L2.
  • amino acid sequence of the single-chain variable fragment scFv2 is shown in SEQ ID NO: 24.
  • the heavy chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 25, and the light chain amino acid sequence is shown in SEQ ID NO: 21.
  • bispecific antibody of the present invention problems related to the chemical and physical stability of the bispecific antibody are also solved, such as expressing physically stable molecules, increasing the stability of heat and salt dependence, reducing aggregation, Increase the solubility at high concentrations and maintain the affinity for the two antigens HER2 and PD1, respectively.
  • Another aspect of the invention provides a nucleotide molecule that encodes the bispecific antibody described above.
  • the nucleotide molecule encodes the heavy chain nucleotide sequence of the bispecific antibody that can specifically bind to HER2 and PD1 as shown in SEQ ID NO: 22, and encodes the light chain nucleotide sequence As shown in SEQ ID NO: 23; or the nucleotide sequence of the heavy chain encoding the bispecific antibody that can specifically bind to HER2 and PD1 is shown in SEQ ID NO: 26, encoding the light chain The nucleotide sequence is shown in SEQ ID NO: 23.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the art, and preferably includes the following preparation method: obtaining the nucleotide molecule encoding the above-mentioned monoclonal antibody through gene cloning technology such as PCR method, etc., or The method of artificial full sequence synthesis obtains the nucleotide molecule encoding the monoclonal antibody.
  • nucleotide sequence encoding the amino acid sequence of the above-mentioned bispecific antibody may appropriately introduce substitutions, deletions, changes, insertions or additions to provide a homologue of the polynucleotide.
  • the homologue of the polynucleotide in the present invention can be prepared by replacing, deleting, or adding one or more bases encoding the bispecific antibody gene within the range of maintaining antibody activity.
  • Another aspect of the present invention provides an expression vector, the expression vector containing the aforementioned nucleotide molecule.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequence, terminator sequence, polyadenylation sequence, enhancer sequence, marker gene and / or sequence and other appropriate Expression vector.
  • the expression vector may be a virus or a plasmid, such as a suitable bacteriophage or phagemid.
  • a suitable bacteriophage or phagemid for more technical details, please see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and solutions for nucleic acid manipulation can be found in Current Protocols, Molecular Biology, Second Edition, edited by Ausubel et al.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.1 (+), pcDNA3.1 / ZEO (+), pDHFR, pTT5, pDHFF, pGM-CSF or pCHO 1.0, more preferably pTT5.
  • the present invention further provides a host cell containing the above-mentioned expression vector.
  • the host cell described in the present invention is a variety of conventional host cells in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the nucleotides carried can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells, and the expression vectors preferably include: COS, CHO (Chinese Hamster Ovary, Chinese Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3) Or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single chain antibody or Fab antibody) or CHO-K1 cells (expressing full-length IgG antibody).
  • the conversion method is a conventional conversion method in the art, preferably a chemical conversion method, a heat shock method or an electric conversion method.
  • the host cell is a eukaryotic cell.
  • CHO cells or 293E cells are preferred.
  • Another aspect of the present invention provides a method for preparing the bispecific antibody that specifically binds to HER2 and PD1.
  • the method includes the following steps:
  • the above-mentioned host cells are cultured to express bispecific antibodies that can specifically bind to HER2 and PD1;
  • the method for cultivating host cells and the method for separating and purifying antibodies described in the present invention are conventional methods in the art.
  • For specific operation methods please refer to the corresponding cell culture technical manual and antibody separation and purification technical manual.
  • the preparation method of the anti-HER2 / PD1 bispecific antibody disclosed in the present invention includes: cultivating the above-mentioned host cells under expression conditions, thereby expressing the bispecific antibody that can specifically bind to HER2 and PD1; isolating and purifying the Anti-HER2 / PD1 bispecific antibody.
  • the recombinant protein can be purified to a substantially uniform substance, for example, a single band on SDS-PAGE electrophoresis.
  • the anti-HER2 / PD1 bispecific antibody disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer and pH change can be used to wash De-bound anti-HER2 / PD1 bispecific antibody on the affinity column.
  • the inventors of the present invention conducted a detection experiment on the obtained anti-HER2 / PD1 bispecific antibody, and the experimental results show that the anti-HER2 / PD1 bispecific antibody can bind well to target cells and antigens and has a high affinity.
  • compositions comprising the above-mentioned bispecific antibody capable of specifically binding to HER2 and PD1 and one or more pharmaceutically acceptable carriers, diluents or excipients Form agent.
  • the bispecific antibody provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to exert a more stable therapeutic effect, and these preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody of the present invention , While also protecting the protein's multifunctional groups from degradation (including but not limited to coagulation, deamination, or oxidation).
  • a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition to exert a more stable therapeutic effect
  • these preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody of the present invention , While also protecting the protein's multifunctional groups from degradation (including but not limited to coagulation, deamination, or oxidation).
  • it can be stored at 2 ° C-8 ° C for at least one year, and for lyophilized formulations, it is stable at 30 ° C for at least six months.
  • the bispecific antibody preparation may be suspension, water injection, lyophilization and other preparations commonly used in the pharmaceutical field.
  • pharmaceutically acceptable carriers preferably include, but are not limited to, one or a combination of surfactants, solution stabilizers, isotonic regulators and buffers .
  • Surfactants preferably include, but are not limited to: nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; MONAQUATTM, etc., the amount of which should be added to minimize the tendency of anti-HER2 / PD1 bispecific antibody granulation.
  • nonionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl s
  • Solution stabilizers preferably include but are not limited to one or a combination of the following: sugars, for example, reducing sugars and non-reducing sugars; amino acids, for example, monosodium glutamate or histidine; alcohols, For example: triols, higher sugar alcohols, propylene glycol, polyethylene glycol, etc., the amount of solution stabilizer added should be such that the final formulation maintains a stable state within the time that the person skilled in the art believes to reach stability.
  • Isotonicity adjusting agents preferably include, but are not limited to, one of sodium chloride, mannitol, or a combination thereof.
  • the buffer preferably includes, but is not limited to, one of Tris, histidine buffer, phosphate buffer, or a combination thereof.
  • Another aspect of the present invention provides the use of the above-mentioned bispecific antibody that specifically binds to HER2 and PD1, or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating cancer or tumor.
  • the medicine for treating cancer or tumor referred to in the present invention refers to a medicine for inhibiting and / or treating tumor, which may include the delay of the development of tumor-related symptoms and / or the reduction of the severity of these symptoms, further including the existing ones Tumors are accompanied by the reduction of symptoms and the prevention of other symptoms, including the reduction or prevention of tumor metastasis.
  • the tumors targeted by the drugs of the present invention preferably include, but are not limited to: lung cancer, bone cancer, stomach cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube cancer, uterus Endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, rectal cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer , Penile cancer, prostate cancer, pancreatic cancer, brain cancer, testicular cancer, lymphoma, transitional cell carcinoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma , Soft tissue sarcoma, pediatric solid tumor, lymphocytic lymphoma, central nervous system (CNS) tumor, primary central nervous system lymphom
  • the dosage varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Refer to animal experiments The results and various circumstances, the total dose should not exceed a certain range. Specifically, the dose for intravenous injection is 1-1800 mg / day.
  • the bispecific antibody and its composition of the present invention can also be co-administered with other anti-tumor drugs to achieve a more effective treatment of tumors.
  • anti-tumor drugs include but are not limited to: 1. Cytotoxic drugs: 1) Action Drugs specific to the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosourea, and methanesulfonates; platinum compounds such as cisplatin (Cisplatin), carboplatin (Carboplatin) and platinum oxalate (Oxaliplatin), etc .; Antibiotics such as Adriamycin / Doxorubicin, DactinomycinD, Daunorubicin, Epirubicin, Mithramycin, etc .; 2) Affect nucleic acid metabolism Of drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and pemetrexed (Pemetrexed), etc .; thymidine syntha
  • Hormone drugs anti-estrogen Hormones such as Tamoxifen, Droloxifene, Exemestane, etc .; Aromatase inhibitors such as Aminoglutethimide, Formestane, and Lytrax Letrozle, Anastrozole, etc .; anti-androgen: flutamide RH-LH agonist / antagonist: norrad, enatone, etc .; 3. biological response modifier drugs: such drugs Mainly by adjusting the body's immune function to achieve anti-tumor effects, such as Interferon (Interferon); Interleukin-2 (Interleukin-2); Thymosin (Thymosins), etc .; 4.
  • Interferon Interferon
  • Interleukin-2 Interleukin-2
  • Thymosins Thymosins
  • Monoclonal antibody drugs trastux Monoclonal antibody (Trastuzumab), rituximab (Rituximab), cetuximab (Cetuximab), bevacizumab (Bevacizumab), etc .; 5.
  • Other anti-tumor drugs including some current mechanisms are not yet clear Further study of drugs, etc.
  • the bispecific antibody and its composition disclosed in the present invention can be used in combination with one of the above-mentioned anti-tumor drugs or a combination thereof.
  • the invention provides a bispecific antibody which can target the tumor cell surface molecule HER2 and the T lymphocyte surface molecule PD1, which can maintain the activity of the antibodies at both ends and can simultaneously bind the PD1 and HER2 antigens; at the cellular level, it can inhibit HER2 positive Tumor cell proliferation can block the binding of PD-1 / PD-L1, and in the N87-PDL1 tumor cell model targeting two targets, it showed superiority to HER2 monoclonal antibody, HER2 monoclonal antibody combined with PD1 monoclonal antibody Used activity.
  • the double antibody of the present invention maintains the activity of both ends well, and can exert a synergistic antitumor effect.
  • the positive progress effect of the present invention lies in that this HER2 / PD1 bispecific antibody can simultaneously exert three aspects of effects and synergistically exert a tumor killing effect.
  • First block the PD-1 / PD-L1 signal path.
  • PD-L1 is expressed on tumor cells and some immunoregulatory cells, while PD-1 is expressed on T cells.
  • the combination of PD-1 and PD-L1 can inhibit the proliferation and activation of T cells. Blocking this pathway can restore the immune killing function of T cells.
  • the Fc segment of the anti-HER2 antibody of this double antibody can bind to the Fc receptor of NK and other cells, so that the immune effector cells of the Fc receptor exert ADCC effect, killing tumor cells, but not obviously killing T cells.
  • this double antibody can be combined with tumor cells that highly express HER2 antigen to inhibit tumor proliferation.
  • this HER2 / PD1 bispecific antibody can simultaneously bind PD1 and HER2 antigens, block signaling pathways, and activate immune effector cells.
  • the three aspects of efficacy simultaneously play a role in inhibiting the killing of tumor cells, and the bispecific antibody has good stability.
  • Figure 1A Schematic diagram of anti-HER2 / PD1 double antibody a
  • Figure 1B Schematic diagram of anti-HER2 / PD1 double antibody b
  • Figure 2A HPLC detection pattern of anti-HER2 / PD1 double antibody a
  • Figure 2B HPLC detection pattern of anti-HER2 / PD1 double antibody b
  • FIG. 2C SDS-PAGE detection results of anti-HER2 / PD1 double antibodies a and b
  • Figure 3A ELISA detection of anti-HER2 / PD1 double antibody a, b binding to HER2
  • Figure 3B ELISA detection of anti-HER2 / PD1 double antibody a, b binding to PD1-ECD
  • Figure 4A Binding of anti-HER2 / PD1 double antibody a, anti-HER2 / PD1 double antibody b to BT474 cells FACS
  • Figure 4B Binding of anti-HER2 / PD1 double antibody a to PD1 / CHO cells FACS
  • Figure 4C Binding of anti-HER2 / PD1 double antibody b to PD1 / CHO cells FACS
  • FIG. 6A Anti-HER2 / PD1 double antibody a blocks the activity of PD1 / PD-L1 binding at the cellular level
  • FIG. 6B Anti-HER2 / PD1 dual antibody b blocks PD1 / PD-L1 binding cell-level activity
  • Figure 7A Detection of the half-life of anti-HER2 antibodies against HER2 / PD1 double anti-a
  • Figure 7B Using biotinylated PD1 to detect the half-life of anti-HER2 / PD1 bi-anti-a antibody
  • FIG. 7C ProteinA was used to detect the half-life of anti-HER2 / PD1 anti-a antibody
  • Figure 7F Using proteinA to detect the half-life of anti-HER2 / PD1 anti-b antibody
  • Figure 8A Killing effect of NK on CD4 + T cells
  • Figure 8B NK ADCC effect on BT474 tumor cells
  • Figure 9A Synergistic killing effect of anti-HER2 / PD1 double antibody a on N87-PDL1 cells
  • Figure 11 Anti-tumor effect of anti-HER2 / PD1 double antibody a on humanized PD1 mouse MC38 transplanted tumor model
  • Figure 12A DSC chart of anti-HER2 / PD1 double antibody a
  • Figure 12B DSC chart of anti-HER2 / PD1 dual antibody b
  • Figure 12C Stability of anti-HER2 / PD1 double antibody a at 37 ° C, SEC-HPLC at 0 and day 24
  • Figure 12D Stability of anti-HER2 / PD1 double antibody b at 37 ° C, SEC-HPLC at 0 and day 24
  • CHO cells purchased from Thermo Fisher Company, catalog number A29133.
  • 293E cells from NRC biotechnology Research Institute.
  • Human breast cancer cell BT474 from the cell bank of the Chinese Academy of Sciences, catalog number TCHu143.
  • PD-L1aAPC / CHO-K1 cells purchased from Promega, catalog number J1252.
  • CD4 + T cells purchased from Allcells, Catalog No. LP180329.
  • NK cells purchased from Allcells, catalog number PB012-C.
  • Protein A chip label No: 29139131-AA; lot: 10261132.
  • SD rat purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd., production license SCXK (Zhejiang) 2018-0001.
  • Human gastric cancer cell line NCI-N87 purchased from the American Type Culture Collection (ATCC).
  • BALB / c nude mice purchased from Shanghai Lingchang Biological Technology Co., Ltd.
  • MC38 mouse colon cancer cell line Heyuan Biotechnology (Shanghai) Co., Ltd.
  • PBMC purchased from Saikai Biology, article number SLB-HP040A.
  • HRP-labeled mouse anti-human Fab antibody purchased from sigma, catalog number A0293.
  • Streptavidin HRP purchased from BD Biosciences, catalog number 554066.
  • Sheep anti-human IgG-FITC purchased from sigma, catalog number F4143.
  • Anti-CD28 antibody purchased from Abcam, catalog number ab213043.
  • IL-2 purchased from R & D, catalog number 202-IL.
  • PBS purchased from Biotechnology (Shanghai) Co., Ltd., article number B548117.
  • TMB purchased from BD Company, article number 555214.
  • Bio-Glo purchased from Promega, catalog number G7940.
  • FBS purchased from Gibco, catalog number 10099.
  • HBS-EP working solution purchased from Life Science, BR-1006-69.
  • CellTiter-Glo purchased from promega, catalog number G775B.
  • HiTrap MabSelectSuRe column purchased from GE.
  • Beckman Coulter CytoFLEX flow cytometer purchased from Beckman.
  • SpectraMax i3x microplate reader purchased from Molecular Devices.
  • SpectraMaxM5 microplate reader purchased from Molecular Devices.
  • the HER2 monoclonal antibody in the examples of the present invention refers to the human and mouse chimeric monoclonal antibody obtained by Sansheng Guojian Pharmaceutical according to the amino acid sequence of Herceptin and referring to the same expression and purification method as the double antibody in Example 2.
  • the PD1 monoclonal antibody described in the embodiments of the present invention refers to a brand-new anti-PD1 humanized monoclonal antibody independently developed by Sansheng Guojian Pharmaceutical Co., Ltd. disclosed in Chinese patent application CN201710054783.5.
  • the anti-HER2 / PD1 bispecific antibody a is constructed by using the scFv tandem of HER2 monoclonal antibody IgG and PD1 monoclonal antibody.
  • the light chain variable region VL (SEQ ID NO: 14) and the heavy chain variable region VH (SEQ ID NO: 13) of the anti-PD1 monoclonal antibody are connected through the peptide linker L1 (SEQ ID NO: 17) to obtain anti-PD1
  • the anti-HER2 / PD1 double antibody a heavy chain nucleic acid sequence is SEQ ID NO: 22, and the light chain nucleic acid sequence is SEQ ID NO: 23.
  • the structure of anti-HER2 / PD1 double antibody a is shown in Figure 1A, and the sequence is shown in the appendix sequence table.
  • anti-HER2 / PD1 double antibody b The molecular construction of anti-HER2 / PD1 double antibody b is as follows:
  • the anti-HER2 / PD1 double-antibody b heavy chain nucleic acid sequence is SEQ ID NO: 26, and the light chain nucleic acid sequence is SEQ ID NO: 23.
  • the structure of anti-HER2 / PD1 double antibody b is shown in Figure 1B, and the sequence is shown in the appendix sequence table.
  • the DNA fragments of the heavy chain and light chain of the double antibody were subcloned into the pTT5 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells and / or 293E cells. After 5-7 days of cell culture, the culture solution was filtered through high-speed centrifugation and microporous membrane vacuum filtration, and then loaded onto a HiTrap MabSelectSuRe column. The protein was eluted in one step with an eluent containing 100 mM citric acid at pH 3.5 to recover the target. Sample and dialyze to PBS pH 7.4. The purified protein was detected by HPLC.
  • the HPLC detection patterns of anti-HER2 / PD1 double antibodies a and b are shown in Figures 2A and 2B, respectively.
  • the antibody molecules are in a uniform state, and the purity of the monomer reaches more than 97%.
  • the electropherogram is shown in Figure 2C.
  • the full-length theoretical molecular weight of the double antibody is 199KD.
  • the HER2-ECD-His protein (manufactured by Sansheng Guojian) was diluted to 250ng / ml with PBS buffer pH7.4, then 100 ⁇ l / Add the wells to the ELISA plate; incubate at 4 ° C overnight; wash the plate twice with PBST the next day; add PBST + 1% BSA to each well for blocking, block at 37 ° C for 1 hour; wash the plate twice with PBST; The antibody to be tested in BSA gradient dilution, HER2 monoclonal antibody was used as a positive control, the initial concentration was 100nM, and 12 gradients were diluted by 3 times.
  • the binding capacity to PD1 the recombinant PD1-ECD-hFc protein (made by Sansheng Guojian) was diluted with PBS pH7.4 to 200g / ml, 100 ⁇ l / well was added Enzyme plate, coated overnight at 4 ° C. Wash the plate twice with PBST, add 200 ⁇ l / well blocking solution (PBS + 2% BSA), and leave it at 37 ° C for 1 hour after washing the plate once with PBST for use.
  • PBS + 2% BSA 200 ⁇ l / well blocking solution
  • human breast cancer cells BT474 with high expression of HER2 on the cell surface were used as target cells, washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 minutes each time, and the supernatant was discarded. Resuspend the cells in 0.5% BSA in PBS at a cell concentration of 1 ⁇ 10 6 cells / mL, and add 100 ⁇ L / well to a 96-well plate.
  • the anti-HER2 / PD1 double antibodies a, b and the positive control HER2 monoclonal antibody were diluted to 400 nM, 11 gradients were added in steps, 100 ⁇ L / well was added to 96-well plates, and BT474 cells were mixed evenly.
  • the experimental results are shown in FIG. 4A.
  • the EC 50 of the anti-HER2 / PD1 double antibodies a, b and the positive control HER2 monoclonal antibody binding to BT474 cells were 1.64, 5.669, and 1.556, respectively.
  • the anti-HER2 / PD1 double antibody a and the positive control HER2 monoclonal antibody have the same affinity, while the anti-HER2 / PD1 double antibody b has a slightly weaker affinity than the positive control HER2 monoclonal antibody.
  • the anti-HER2 / PD1 double antibody a and anti-HER2 / PD1 double antibody b were measured by flow cytometry, and the binding affinity of the cells was determined. The method is as described above, and the obtained data is fitted and analyzed by GraphPad Prism 6 software. The experimental results are shown in Figures 4B and 4C. Both anti-HER2 / PD1 double antibody a and anti-HER2 / PD1 double antibody b can specifically bind PD1 expressed on the cell surface.
  • the anti-HER2 / PD1 double antibody a and positive control PD1 monoclonal antibody EC 50 were 1.77 and 0.8981 respectively; the anti-HER2 / PD1 double antibody b and positive control anti PD1 monoclonal antibody EC 50 were 1.192 and 0.8891, respectively.
  • the three have the same affinity.
  • the human breast cancer cell line BT474 expresses HER2 antigen molecules on its cell surface. Due to the normal proliferation of BT474 cells in vitro, they depend in part on the growth signals transmitted by the HER2 receptor. Adding anti-HER2 antibody to the culture medium can inhibit the cell proliferation. Within a certain range, the concentration of antibody has a dose-effect relationship with the degree of cell proliferation inhibition. The degree of cell proliferation can be detected by CCK-8 (Cell Counting Kit-8) cell proliferation toxicity reagent. The dose-effect relationship curve is an inverse "S" curve.
  • the anti-HER2 / PD1 double antibodies a, b and the positive control HER2 monoclonal antibody samples were prepared as a 300 nM solution with complete medium as the diluent, and then diluted 3 times in stages, for a total of 11 gradients.
  • Double antibodies block the activity at the cellular level of PD1 / PD-L1 binding
  • PD1 effector cells with a density of 1.4-2 ⁇ 10 6 cells / mL and a cell viability rate of more than 95% are taken and pancreatin digested into a single cell suspension of 1.25 ⁇ 10 6 cells / ml.
  • a Anti-HER2 / PD1 double antibody a Anti-PD1 monoclonal antibody Bottom 2.707 -1.048 Top 101.1 95.61 lgIC 50 0.5161 -0.05017 HillSlope 1.048 0.8425 IC 50 3.282 0.8909
  • Anti-HER2 / PD1 b of the double antibody IC 50 top, bottom, hillslope other data. As shown in table 2:
  • the proteinA capture method was used to determine the kinetic parameters of double antibody binding to the antigen HER2-ECD-his. Bind the double antibody with a concentration of 1 ⁇ g / ml on the Protein A chip, dilute the antigen HER2-ECD-his 2 times from 50nM with 1 ⁇ HBS-EP working solution, and set up 6 concentration gradients to bind the antibody to HBS -Dissociation in EP working fluid.
  • the proteinA capture method was used to determine the kinetic parameters of the double antibody binding to the antigen PD1-ECD-his. Bind the double antibody with a concentration of 1 ⁇ g / ml on the Protein A chip, dilute the antigen PD1-ECD-his with 1 ⁇ HBS-EP working solution from 250nM down to 2 times and set up 5 concentration gradients to bind to the antibody. Dissociated in EP working fluid.
  • Example 8 Pharmacokinetic study of anti-HER2 / PD1 double antibody a, anti-HER2 / PD1 double antibody b
  • the drug concentration in the serum of anti-HER2 / PD1 double antibody a was detected by the following method:
  • HER2-His coated ELISA plate 50ng / well. There are two boards. Coated overnight at 4 ° C, washed twice with PBST the next day, and then blocked with PBS + 2% BSA at 37 ° C for 2 hours. Take the anti-HER-2 / PD1 double antibody a standard with an initial concentration of 0.5 ⁇ g / mL and dilute 12 gradients in two steps. Each serum sample was diluted 2000-fold. Add the above sample to the sealed ELISA plate. Incubate at 37 ° C for one hour. Then wash the plate twice with PBST.
  • Detection of anti-HER2 antibody one of the plates was added with HRP-labeled mouse anti-human Fab antibody, diluted 1: 3000, 100 ⁇ L / well. Incubate at 37 ° C for 40 min. Wash the plate 4 times with PBST and pat dry. Add 100 ⁇ l of TMB to each well and leave it at room temperature (20 ⁇ 5 ° C) in the dark for 5 minutes; add 50 ⁇ l of 2M H 2 SO 4 stop solution to each well to stop the substrate reaction, and read the OD value at 450 nm using a microplate reader.
  • Detection of anti-PD1 antibody take another plate to add biotinylated PD1-hFc, 7.5ng / well, and incubate for 1 hour. Wash the plate and add Streptavidin HRP, diluted 1: 1000. Leave at 37 ° C for 30 minutes; wash the plate 4 times with PBST and pat dry. Add 100 ⁇ l of TMB to each well and leave it at room temperature (20 ⁇ 5 ° C) in the dark for 5 minutes; add 50 ⁇ l of 2M H 2 SO 4 stop solution to each well to stop the substrate reaction, and read the OD value at 450 nm using a microplate reader.
  • ProteinA coated ELISA plate to detect antibody Fab segment Coated with proteinA, the coating amount was 100ng / well, overnight at 4 °C; the plate was washed twice with PBST the next day, and then blocked with PBS + 2% BSA at 37 °C for 2 hours. Wash the plate twice with PBST.
  • the anti-HER2 / PD1 double antibody a standard starts at 1000 ng / mL and is diluted twice in 12 steps. Rat serum samples were diluted 2000-fold.
  • the drug concentration in the serum of anti-HER2 / PD1 double antibody b was detected by the following method:
  • HER2-His coated ELISA plate 50ng / well. Coated overnight at 4 ° C, washed twice with PBST the next day, and then blocked with PBS + 2% BSA at 37 ° C for 2 hours. Take the anti-HER2 / PD1 double antibody b standard with a starting concentration of 0.5 ⁇ g / mL and dilute 12 gradients in two steps. Each serum sample was diluted 2000-fold and added to a closed ELISA plate. Incubate at 37 ° C for 1 hour. Then wash the plate twice with PBST. Add HRP-labeled mouse anti-human Fab antibody, 1: 3000 dilution, 100 ⁇ L / well.
  • Detection of anti-PD1 antibody PD1-ECD-hFc coated ELISA plate, 20ng / well. Coating, plate washing, and standard dilution method are the same as above. Dilute the serum sample 1000-2000 times and add it to the sealed ELISA plate. Incubate at 37 ° C for 1 hour. Then wash the plate twice with PBST. Add HRP-labeled mouse anti-human Fab antibody, 1: 3000 dilution, 100 ⁇ L / well. Incubate at 37 ° C for 40 min. Wash the plate 4 times with PBST and pat dry.
  • ProteinA coated ELISA plate to detect antibody Fab segment Coated with proteinA, the coating amount was 100ng / well, overnight at 4 °C; the plate was washed twice with PBST the next day, and then blocked with PBS + 2% BSA at 37 °C for 2 hours. Wash the plate twice with PBST.
  • the anti-HER2 / PD1 double antibody b standard starts at 1000 ng / mL and is diluted twice in 12 steps.
  • Rat serum samples were diluted 500-1000 times, added to the blocked ELISA plate, and incubated for 1 hour; after washing the plate twice with PBST, HRP-labeled mouse anti-human Fab antibody was added and left at 37 ° C for 30 minutes; washing plate with PBST 4 times, taking dry.
  • the Fc segment of the antibody can also bind to NK cells.
  • NK cells have a killing effect on CD4 + T cells: PD1 is expressed on activated T cells, and anti-HER2 / PD1 double anti-a antibody is added to bind to it, and the Fc segment of anti-HER2 / PD1 double anti-a and the effector cell NK Fc receptors are bound and added to NK cells to detect whether they kill T cells.
  • the experimental method is as follows:
  • CD4 + T cells D-PBS was used to prepare anti-CD3 antibody at a concentration of 5 ⁇ g / mL and coated with a 24-well cell culture plate at 4 ° C overnight. The next day, 5 ⁇ 10 5 CD4 + T cells were added to each well, and 2 ⁇ g / mL anti-CD28 antibody and 100 U / mL IL2 were added at the same time. Place at 37 ° C in a CO 2 incubator and activate for 72h.
  • T cells highly expressing PD1 were formulated into 2 ⁇ 10 5 cells / mL and 50 ⁇ L / well with 1640 medium containing 5% FBS. Add 96-well plate.
  • Dilution of anti-HER2 / PD1 double antibody a and negative control sample HER2 monoclonal antibody the anti-HER2 / PD1 double antibody a and HER2 monoclonal antibody were formulated to an initial concentration of 400 nM, after 5 times dilution, and added to 96 wells plated with T cells In the board. Place at 37 ° C in a CO 2 incubator and incubate for 15 minutes. During this period, NK cells were adjusted to 5 ⁇ 10 5 cells / mL in 1640 medium containing 5% FBS, 100 ⁇ L / well was added to the 96-well plate, and placed at 37 ° C. in a CO 2 incubator for 3 hours of killing.
  • NK cells have no significant killing effect on CD4 + T cells, and only have a weak killing effect at high concentrations of anti-HER2 / PD1 double antibody a.
  • the surface of BT474 cells expresses HER2 antigen, which can be combined with anti-HER2 / PD1 double antibody a.
  • the Fc segment of anti-HER2 / PD1 double antibody a binds to the Fc receptor of effector cell NK, and the addition of NK cells can detect whether it kills BT474 cells.
  • the experimental method is as follows:
  • BT474 cells were diluted with 1640 medium containing 5% FBS to 2 ⁇ 10 5 cells / mL, added to 96-well flat bottom plate, 50 ⁇ L / well. Place at 37 ° C in a 5% CO 2 incubator overnight.
  • HER2 monoclonal antibody Dilution of anti-HER2 / PD1 double antibody a and negative control sample HER2 monoclonal antibody: the anti-HER2 / PD1 double antibody a and HER2 monoclonal antibody were formulated to an initial concentration of 200nM, after 4 times dilution, added to 96 wells spread into BT474 cells In the board. Place in 37 ° C, 5% CO 2 incubator and incubate for 15 minutes. During this period, NK cells were adjusted to 5 ⁇ 10 5 cells / mL with 1640 medium containing 5% FBS, 100 ⁇ L / well was added to the 96-well plate, placed in a 37 ° C. CO 2 incubator, and incubated for 3 h.
  • HER2 antigen expression on tumor cells and tumor cell proliferation can be inhibited by HER2 antibody; while PD-L1 expression can It binds to PD-1 on T cells, so PD1 antibody is added to block PD-1 / PD-L1 binding, and T cell suppression is released, playing a role in killing tumors. Since no cell lines meeting these conditions were selected, the lentiviral transfection method was used to recombine the PD-L1 gene into human gastric cancer cell line NCI-N87. The constructed N87-PDL1 cells were detected by FACS with high expression of PD-L1 on the cell surface.
  • N87-PDL1 cells in logarithmic growth phase, trypsinize, dilute to 1 ⁇ 10 5 / mL with 1640 medium and 1% FBS, and transfer 100 ⁇ L / well to a white transparent bottom 96-well plate. Place at 37 ° C in a 5% CO 2 incubator overnight. The next day add the antibody to be tested and fresh PBMC cells, each 50 ⁇ L / well.
  • Antibodies were anti-HER2 / PD1 double antibody a, anti-HER2 monoclonal antibody, anti-HER2 monoclonal antibody plus anti-PD1 monoclonal antibody, anti-PD1 monoclonal antibody at a concentration of 4 nM.
  • PBMC peripheral blood mononuclear cells
  • Collect human gastric cancer cell line NCI-N87 cells cultured in vitro adjust the cell concentration to 5 ⁇ 10 7 cells / mL, resuspend in serum-free medium, and inoculate 100 ⁇ L of cell suspension in nude mice under sterile conditions Back skin.
  • the dosage of the test sample anti-HER2 / PD1 double antibody a is divided into two groups, 20mg / kg, 4mg / kg, that is 0.4mg / only, 0.08mg / only, the dose of the positive control drug HER2 monoclonal antibody is 15mg / kg, that is 0.3mg / piece.
  • the control group was given the same volume of PBS.
  • the mode of administration was intraperitoneal administration, the administration volume was 0.2 mL / mouse (20 g), administration was twice a week, and administration was continuous for three weeks.
  • the experimental results are shown in Figure 10.
  • the anti-HER2 / PD1 double antibody a is similar to the positive control HER2 monoclonal antibody.
  • the dose of anti-HER2 / PD1 double antibody a of the test sample was 13 mg / kg, the dose of the positive control anti-PD1 monoclonal antibody was set to 10 mg / kg, and the control group was given the same volume of normal saline.
  • the colon cancer cells of MC38 mice cultured in vitro were collected, and the concentration of the cell suspension was adjusted to 1 ⁇ 10 7 cells / ml. Under sterile conditions, 100 ⁇ l of cell suspension was inoculated subcutaneously on the right rib of humanized PD1 mice. Humanized PD1 mouse subcutaneously transplanted tumors were measured with vernier calipers. After the average tumor volume grew to 100-200 mm 3, the animals were randomly divided into groups.
  • Example 13 Stability study of anti-HER2 / PD1 double antibody a and anti-HER2 / PD1 double antibody b
  • thermodynamic parameters related to the interaction such as protein unfolding with the addition of excipients, so as to reveal the important mechanism information needed to develop the optimal formulation.
  • MicroCal VP-Capillary DSC was used to filter the sample and its buffer with a 0.22um filter membrane. 400 ⁇ l of the sample and its matching buffer were placed in a 96-well plate. The sample was scanned at 25 °C -100 °C with a scanning rate It is 120 ° C per hour.
  • Anti-HER2 / PD1 double antibody a, anti-HER2 / PD1 double antibody b were stored in PBS pH7.4. See Table 11 for Tm value of DSC detection of double antibody. The spectrum is shown in Figures 12A and 12B. It can be seen that this double antibody is relatively stable. Subsequent 37 °C long-term stability test results also verified this. The HPLC-SEC results are shown in Figures 12C and 12D.
  • the bispecific antibody provided by the present invention has a stable structure. Can simultaneously bind HER2 and PD1 antigens. Blocking the HER2 signaling pathway can inhibit the proliferation of tumor cells expressing the HER2 antigen; at the same time, it can block the PD-1 / PD-L1 pathway, which can restore the immune killing function of T cells and play a role in killing tumor cells. At the same time, the Fc segment of the double anti-HER2 antibody can bind to the Fc receptor of NK cells, exert ADCC effect, kill tumor cells, but have no obvious killing effect on T lymphocytes.

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Abstract

一种抗HER2/PD1双特异性抗体、制备方法和在抗肿瘤中的应用。具体地，单链可变片段scFv和免疫球蛋白抗体IgG通过肽接头连接获得双特异性抗体，该双特异性抗体能同时靶向肿瘤细胞表面分子HER2抗原和T淋巴细胞表面分子PD1。实验结果显示，提供的双特异性抗体，能够抑制HER2阳性肿瘤细胞的增殖，同时能够阻断PD-1/PD-L1的结合，解除T细胞的抑制状态，发挥抗肿瘤的作用。

Description

抗HER2/PD1双特异性抗体 技术领域
本发明属于肿瘤治疗和生物技术领域，涉及一种抗HER2和PD1的双特异性抗体分子制备方法和用途。
背景技术
HER2(human epidermal growth factor receptor2)，具有受体酪氨酸蛋白激酶活性，是人表皮生长因子受体家族成员之一，只在成年人的少数正常组织中呈低水平表达。但研究表明，HER2在多种肿瘤中过表达，如在约30％的乳腺癌患者和16％的胃癌患者中均存在过度表达情况，HER2在肿瘤中的过表达可以显著促进肿瘤血管的新生、肿瘤的生长，并增强肿瘤的侵袭和转移能力，是这类患者预后较差的重要指征。因此，早在1998年，第一个靶向于HER2的单克隆抗体药物Herceptin(Genentech/Roche)被FDA批准上市，并用于HER2过表达的乳腺癌和胃癌的治疗。
人程序性细胞死亡受体-1(PD1)是由288个氨基酸组成的I型膜蛋白，胞外段为负责结合配体的Ig可变型(V-型)结构域，胞内段为负责结合信号转导分子的胞质尾区。PD1胞质尾区含有两个基于酪氨酸的信号转导模体，分别为ITIM(免疫受体酪氨酸抑制作用模体)和ITSM(免疫受体酪氨酸转换作用模体)。PD1表达在已经激活的T淋巴细胞表面，它与配体PD-L1(程序性死亡受体-配体1，programmed cell death-Ligand 1)和PD-L2(程序性死亡受体-配体2，programmed cell death-Ligand 2)结合可以抑制T淋巴细胞的活性及相关的体内细胞免疫反应。大量研究表明，PD1和PD-L1的相互作用不仅维持了体内免疫系统的平衡，也是导致PD-L1表达阳性的肿瘤细胞规避免疫监视的主要机制。通过阻断PD1/PD-L1信号通路，能够激活免疫系统，恢复T细胞的免疫杀伤功能。
Figure PCTCN2019112467-appb-000001
(pembrolizumab)是第一个上市的针对PD1的人源化单克隆抗体，于2014年9月被FDA批准用于治疗黑色素瘤，至2018年获批的适应症包括：黑色素瘤、非小细胞肺癌、霍奇金淋巴瘤、头颈部鳞癌、膀胱癌、胃癌和带有MSI-H或dMMR的实体肿瘤。
Figure PCTCN2019112467-appb-000002
(nivolumab)是百时美施贵宝公司的一款PD1单克隆抗体，于2014年12月获FDA批准上市，适应症包括：黑色素瘤、非小细胞肺癌、肾细胞癌、经典霍奇金淋巴瘤、头颈部鳞癌、膀胱癌、结直肠癌和肝细胞癌。由三生国健自主研发的抗PD1单克隆抗体是一种全新的抗PD1人源化单克隆抗体。体内外生物学活性以及抗肿瘤活性研究显示，抗PD1的生物学活性介于阳性对照药Opdivo和Keytruda之间，在某些方面略优于阳性对照药Opdivo。
双特异性抗体(bispecific antibody，BsAb)是指能同时结合两个(或多个)不同抗原表位的抗体分子。与传统的单克隆抗体相比，双特异性抗体具有独特的作用机制：1)双特异性抗体可以同时结合2个或多个不同的抗原分子或相同分子的不同表位，而联合用药往往不具备这种效应。2)介导细胞间的相互作用，双特异性抗体可分别结合效应细胞和靶细胞上的 两种抗原上，在效应细胞和靶细胞之间架起桥梁，促进细胞间的相互作用，例如介导免疫细胞对肿瘤细胞的杀伤。因此双特异性抗体具有传统单克隆抗体不具备的独特优势。
发明内容
本发明提供了一种新的能与HER2和PD1特异结合的双特异性抗体，还提供了该双特异性抗体的制备方法和应用。
因此，本发明的目的在于提供一种能与HER2和PD1特异结合的双特异性抗体；提供编码所述双特异性抗体的核苷酸分子；提供包含所述核苷酸分子的表达载体；提供所述表达载体的宿主细胞；提供所述双特异性抗体的制备方法；提供包含所述双特异性抗体的药物组合物；提供所述双特异性抗体在制备药物中的应用。
为了实现上述目的，本发明采用了如下技术方案：
本发明一方面提供了一种能与HER2和PD1特异结合的双特异性抗体，其包含免疫球蛋白抗体IgG和两个相同的单链可变区片段scFv，其中每个单链可变片段scFv包含可变区VH和可变区VL，VH与VL通过肽接头L1连接，每个单链可变片段scFv通过接头肽L2与免疫球蛋白抗体IgG串联。
本发明所述的“双特异性抗体”是指拥有两个不同的抗原结合位点，能同时结合HER2和PD1的双特异性抗体，其包含两个单链可变片段scFv和与之缀合的免疫球蛋白抗体IgG，每个scFv经由肽接头L2连接至免疫球蛋白抗体IgG每条重链，形成双特异性抗体的重链融合蛋白，其中每个scFv包含可变区VH和可变区VL，VH与VL通过肽接头L1连接。
本发明所述的“单链可变区片段scFv”是指包含免疫球蛋白重链VH和轻链VL可变区的融合蛋白，VH与VL通过肽接头相连，其中所述融合蛋白保留了完整免疫球蛋白相同的抗原特异性。
本发明所述的“免疫球蛋白抗体IgG”是约150kDa的分子，它由四条肽链构成，含有两条相同的约50kDa的γ重链，和两条相同的约25kDa的轻链，从而具有四聚体四级结构。两条重链通过二硫键相互连接，并各自与一条轻链连接。所成的四聚体具有相同的两半，二者形成叉型或者类似Y的形状，叉的每一端含有一个相同的抗原结合位点。IgG抗体可以基于重链的恒定区中氨基酸序列的微小差异而分为多个亚类(例如IgG1、2、3、4)。
作为优选的方案，所述VH包含互补决定区HCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：1所示，HCDR2的氨基酸序列如SEQ ID NO：2所示，HCDR3的氨基酸序列如SEQ ID NO：3所示；
所述VL包含互补决定区LCDR1-3，其中LCDR1的氨基酸序列如SEQ ID NO：4所示，LCDR2的氨基酸序列如SEQ ID NO：5所示，LCDR3的氨基酸序列如SEQ ID NO：6所示；
所述免疫球蛋白抗体IgG的重链包含互补决定区HCDR4-6，其中HCDR4的氨基酸序列如SEQ ID NO：7所示，其中HCDR5的氨基酸序列如SEQ ID NO：8所示，其中HCDR6的氨基酸序列如SEQ ID NO：9所示；
所述免疫球蛋白抗体IgG的轻链包含互补决定区LCDR4-6，其中LCDR4的氨基酸序列 如SEQ ID NO：10所示，其中LCDR5的氨基酸序列如SEQ ID NO：11所示，其中LCDR6的氨基酸序列如SEQ ID NO：12所示。
本领域中，抗体的结合区通常均含有一条轻链可变区和一条重链可变区，每一个可变区均含有3个CDR结构域。抗体的重链和轻链的CDR结构域分别称为HCDR和LCDR。因此，常规抗体抗原结合位点包含六个CDR，包括分别来自重链和轻链V区的CDR集合。
作为优选的方案，scFv的VH的氨基酸序列如SEQ ID NO：13所示，VL的氨基酸序列如SEQ ID NO：14所示；所述免疫球蛋白抗体IgG的重链可变区的氨基酸序列如SEQ ID NO：15所示，轻链可变区的氨基酸序列如SEQ ID NO：16所示。
作为优选的方案，所述肽接头L1的氨基酸序列如SEQ ID NO：17所示。
作为优选的方案，所述肽接头L2的氨基酸序列如SEQ ID NO：18所示。
作为优选的方案，所述单链可变片段scFv1的分子结构形式为VL-L1-VH，每个scFv的N末端经由肽接头L2连接至免疫球蛋白抗体IgG重链的C末端。
作为优选的方案，所述单链可变片段scFv1的氨基酸序列如SEQ ID NO：19所示。
作为优选的方案，所述双特异性抗体的重链氨基酸序列如SEQ ID NO：20所示，其轻链氨基酸序列如SEQ ID NO：21所示。
作为优选的方案，所述单链可变片段scFv2的分子结构形式为VH-L1-VL，每个scFv的C末端经由肽接头L2连接至免疫球蛋白抗体IgG重链的N末端。
作为优选的方案，所述单链可变片段scFv2的氨基酸序列如SEQ ID NO：24所示。
作为优选的方案，所述双特异性抗体的重链氨基酸序列如SEQ ID NO：25所示，其轻链氨基酸序列如SEQ ID NO：21所示。
在构建本发明的双特异性抗体时，与该双特异性抗体的化学和物理稳定性相关的问题也得到了解决，诸如表达物理稳定的分子、增加热和盐依赖的稳定性、降低聚集、增加在高浓度下的溶解度以及维持分别针对两种抗原HER2和PD1的亲和力等。
本发明另一方面提供了一种核苷酸分子，所述核苷酸分子编码上述所述的双特异性抗体。
作为优选的方案，所述核苷酸分子编码能与HER2和PD1特异结合的双特异性抗体的重链的核苷酸序列如SEQ ID NO：22所示，编码其轻链的核苷酸序列如SEQ ID NO：23所示；或所述核苷酸分子编码能与HER2和PD1特异结合的双特异性抗体的重链的核苷酸序列如SEQ ID NO：26所示，编码其轻链的核苷酸序列如SEQ ID NO：23所示。
本发明所述核苷酸分子的制备方法为本领域常规的制备方法，较佳地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码上述单克隆抗体的核苷酸分子，或者通过人工全序列合成的方法得到编码上述单克隆抗体的核苷酸分子。
本领域技术人员知晓，编码上述双特异性抗体的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该双特异性抗体基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明另一方面提供了一种表达载体，所述表达载体含有上述的核苷酸分子。
其中所述表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。本发明所述表达载体较佳地为pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR，pTT5，pDHFF，pGM-CSF或pCHO 1.0，更佳地为pTT5。
本发明另外提供了一种宿主细胞，所述宿主细胞含有上述的表达载体。
本发明所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞，所述表达载体较佳地包括：COS、CHO(中国仓鼠卵巢，Chinese H amster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1，更佳地为E.coli TG1、BL21(DE3)细胞(表达单链抗体或Fab抗体)或者CHO-K1细胞(表达全长IgG抗体)。将前述表达载体转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。
作为优选的方案，所述宿主细胞是真核细胞。优选CHO细胞或293E细胞。
本发明另一方面提供了上述能与HER2和PD1特异结合的双特异性抗体的制备方法，所述制备方法包括以下步骤：
a)在表达条件下，培养上述的宿主细胞，从而表达能与HER2和PD1特异结合的双特异性抗体；
b)分离并纯化步骤a)所述的双特异性抗体。
本发明所述的宿主细胞的培养方法、所述抗体的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及抗体分离纯化技术手册。本发明中公开的抗HER2/PD1双特异性抗体的制备方法包括：在表达条件下，培养上述的宿主细胞，从而表达能与HER2和PD1特异结合的双特异性抗体；分离和纯化所述的抗HER2/PD1双特异性抗体。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
可以利用亲和层析的方法对本发明公开的抗HER2/PD1双特异性抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的抗HER2/PD1双特异性抗体。本发明的发明人对所得抗HER2/PD1双特异性抗体进行了检测实验，实验结果表明该抗HER2/PD1双特异性抗体能很好地与靶细胞和抗原结合，具有较高的亲和力。
本发明另一方面提供了一种组合物，所述组合物包含上述所述的能与HER2和PD1特异结合的双特异性抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明提供的双特异性抗体，可以和药学上可接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明的双特异性抗体的氨基酸核心序列的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。通常情况下，对于液体制剂，通常可以在2℃-8℃条件下保存至少稳定一年，对于冻干制剂，在30℃至少六个月保持稳定。所述双特异性抗体制剂可为制药领域常用的混悬、水针、冻干等制剂。
对于本发明的双特异性抗体的水针或冻干制剂，药学上可以接受的载体较佳地包括但不限于：表面活性剂、溶液稳定剂、等渗调节剂和缓冲液之一或其组合。其中表面活性剂较佳地包括但不限于：非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基、亚油基或十八烷基肌氨酸；Pluronics；MONAQUATTM等，其加入量应使抗HER2/PD1双特异性抗体的颗粒化趋势最小。溶液稳定剂较佳地包括但不限于以下列举之一或其组合：糖类，例如，还原性糖和非还原性糖；氨基酸类，例如，谷氨酸单钠或组氨酸；醇类，例如：三元醇、高级糖醇、丙二醇、聚乙二醇等，溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态。等渗调节剂较佳地包括但不限于氯化钠、甘露醇之一或其组合。缓冲液较佳地包括但不限于：Tris、组氨酸缓冲液、磷酸盐缓冲液之一或其组合。
本发明另一方面提供了上述能与HER2和PD1特异结合的双特异性抗体、或上述药物组合物在制备药物中的应用，所述药物用于治疗癌症或肿瘤。
本发明所称的用于治疗癌症或肿瘤的药物，指具有抑制和/或治疗肿瘤的药物，可以包括伴随肿瘤相关症状发展的延迟和/或这些症状严重程度的降低，进一步还包括已存在的肿瘤伴随症状的减轻并防止其他症状的出现，还包括减少或防止肿瘤的转移等。
本发明所述的药物所针对的肿瘤较佳地包括但不限于：肺癌、骨癌、胃癌、胰腺癌、皮肤癌、头颈癌、子宫癌、卵巢癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、子宫颈癌、阴道癌、外阴癌、直肠癌、结肠癌、肛门区癌、乳腺癌、食管癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、尿道癌、阴茎癌、前列腺癌、胰腺癌、脑癌、睾丸癌、淋巴癌、移行细胞癌、膀胱癌、肾癌或输尿管癌、肾细胞癌、肾盂癌、霍奇金病、非霍奇金淋巴瘤、软组织肉瘤、儿童实体瘤、淋巴细胞性淋巴瘤、中枢神经系统(CNS)肿瘤、原发性中枢神经系统淋巴瘤、肿瘤血管生成、脊柱肿瘤、脑干神经胶质瘤、垂体腺瘤、黑素瘤、卡波西肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、慢性或急性白血病和所述癌的组合。
本发明的双特异性抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。具体讲静脉注射的剂量是1-1800mg/天。
本发明的双特异性抗体及其组合物还可以和其他的抗肿瘤药联合给药以达到更加有效治疗肿瘤的目的，这些抗肿瘤药包括但不限于：1、细胞毒类药物：1)作用于核酸化学结构的药物：烷化剂如氮芥类、亚硝脲类、甲基磺酸酯类；铂类化合物如顺铂(Cisplatin)、卡铂 (Carboplatin)和草酸铂(Oxaliplatin)等；抗生素类如阿霉素(Adriamycin/Doxorubicin)、放线菌素D(DactinomycinD)、柔红霉素(Daunorubicin)、表阿霉素(Epirubicin)、光辉霉素(Mithramycin)等；2)影响核酸代谢的药物：二氢叶酸还原酶抑制剂如甲氨喋呤(MTX)和培美曲塞(Pemetrexed)等；胸腺核苷合成酶抑制剂如氟尿嘧啶类(5-氟尿嘧啶、卡培他滨)等；嘌呤核苷合成酶抑制剂如6-巯基嘌呤等；核苷酸还原酶抑制剂如羟基脲(Hydroxycarbamide)等；DNA多聚酶抑制剂如阿糖胞苷(Cytosinearabinoside)和吉西他滨(Gemcitabine)等；3)作用于微管蛋白的药物：多西他赛(Docetaxel)、长春花碱(Vincristine)、长春瑞滨(Vinorelbine)、鬼臼硷类、高三尖杉酯碱等；2、激素类药物：抗雌激素如他莫昔芬(Tamoxifen)、屈洛昔芬(Droloxifene)、依西美坦(Exemestane)等；芳香化酶抑制剂如氨鲁米特(Aminoglutethimide)、福美司坦(Formestane)、来曲唑(Letrozle)、阿那曲唑(Anastrozole)等；抗雄激素：氟它氨RH-LH激动剂/拮抗剂：诺雷德、依那通等；3、生物反应调节剂类药物：此类药物主要通过调节机体免疫功能以到抗肿瘤的效果，如干扰素类(Interferon)；白细胞介素-2(Interleukin-2)；胸腺肽类(Thymosins)等；4、单克隆抗体类药物：曲妥昔单抗(Trastuzumab)、利妥昔单抗(Rituximab)、西妥昔单抗(Cetuximab)、贝伐单抗(Bevacizumab)等；5、其他类抗肿瘤药物：包括一些目前机制尚不明确、有待进一步研究的药物等。本发明公开的双特异性抗体及其组合物可以和上述的抗肿瘤药物之一或其组合联合用药。
本发明提供了能靶向肿瘤细胞表面分子HER2和T淋巴细胞表面分子PD1的双特异性抗体，其能够保持两端抗体的活性，能同时结合PD1和HER2抗原；细胞水平上，能够抑制HER2阳性的肿瘤细胞增殖，能阻断PD-1/PD-L1的结合，并且在针对两个靶点的N87-PDL1肿瘤细胞模型上，表现出优于HER2单抗、HER2单抗与PD1单抗联合使用的活性。动物实验上，小鼠N87肿瘤模型的实验结果显示，该双特异性抗体HER2端能抑制肿瘤增殖；人源化PD1小鼠MC38移植瘤模型的实验结果显示，该双特异性抗体PD-1端能抑制肿瘤增殖。因此，本发明的双抗较好地保持了两端的活性，能够发挥协同抗肿瘤作用。
本发明的积极进步效果在于：此HER2/PD1双特异抗体，可以同时发挥三方面的功效，协同发挥杀伤肿瘤的作用。其一，阻断PD-1/PD-L1信号通路。PD-L1在肿瘤细胞和一些免疫调节细胞上表达，而T细胞上表达PD-1。PD-1与PD-L1结合能够抑制T细胞增殖活化。阻断此通路，可恢复T细胞的免疫杀伤功能。其二，此双抗的抗HER2抗体的Fc段可与NK等细胞的Fc受体结合，使Fc受体的免疫效应细胞发挥ADCC作用，杀伤肿瘤细胞，而对T细胞无明显杀伤。其三，此双抗的抗HER2活性，可以与高表达HER2抗原的肿瘤细胞结合，抑制肿瘤增殖。综上所述，此HER2/PD1双特异抗体可以同时结合PD1与HER2抗原，阻断信号通路，激活免疫效应细胞。三方面功效同时发挥作用抑制杀伤肿瘤细胞，并且该双特异性抗体具有良好的稳定性。
附图说明
图1A：抗HER2/PD1双抗a结构示意图
图1B：抗HER2/PD1双抗b结构示意图
图2A：抗HER2/PD1双抗a的HPLC检测图谱
图2B：抗HER2/PD1双抗b的HPLC检测图谱
图2C：抗HER2/PD1双抗a、b的SDS-PAGE检测结果
图3A：ELISA检测抗HER2/PD1双抗a，b与HER2的结合
图3B：ELISA检测抗HER2/PD1双抗a，b与PD1-ECD的结合
图4A：抗HER2/PD1双抗a、抗HER2/PD1双抗b与BT474细胞的结合FACS
图4B：抗HER2/PD1双抗a与PD1/CHO细胞的结合FACS
图4C：抗HER2/PD1双抗b与PD1/CHO细胞的结合FACS
图5：抗HER2/PD1双抗a、b对BT474细胞体外增殖抑制作用
图6A：抗HER2/PD1双抗a阻断PD1/PD-L1结合的细胞水平的活性
图6B：抗HER2/PD1双抗b阻断PD1/PD-L1结合的细胞水平的活性
图7A：检测抗HER2/PD1双抗a的抗HER2抗体的半衰期
图7B：用生物素化的PD1检测抗HER2/PD1双抗a抗体的半衰期
图7C：用proteinA检测抗HER2/PD1双抗a抗体的半衰期
图7D：检测抗HER2/PD1双抗b的抗HER2抗体的半衰期
图7E：检测抗HER2/PD1双抗b的抗PD1抗体的半衰期
图7F：用proteinA检测抗HER2/PD1双抗b抗体的半衰期
图8A：NK对CD4+T细胞的杀伤作用
图8B：NK对BT474肿瘤细胞的ADCC作用
图9A：抗HER2/PD1双抗a对N87-PDL1细胞的协同杀伤作用
图9B：PD1对照单抗对N87-PDL1细胞的作用
图10：抗HER2/PD1双抗a在NCI-N87移植瘤模型上的抗肿瘤作用
图11：抗HER2/PD1双抗a在人源化PD1小鼠MC38移植瘤模型上的抗肿瘤作用
图12A：抗HER2/PD1双抗a的DSC图
图12B：抗HER2/PD1双抗b的DSC图
图12C：抗HER2/PD1双抗a的37℃稳定性，0时与第24天的SEC-HPLC
图12D：抗HER2/PD1双抗b的37℃稳定性，0时与第24天的SEC-HPLC
具体实施方式
以下实施例、实验例是对本发明进行进一步的说明，不应理解为是对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J., Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:A LaboratoryManual,2nd edition,Cold spring Harbor Laboratory Press.
以下实施例中使用的实验材料和来源以及实验试剂的配制方法具体说明如下。
实验材料：
CHO细胞：购自Thermo fisher公司，货号A29133。
293E细胞：来自NRC biotechnology Research Institute。
人乳腺癌细胞BT474：来自中科院细胞库，目录号TCHu143。
PD-L1aAPC/CHO-K1细胞：购自Promega公司，货号J1252。
CD4+T细胞：购自Allcells，货号LP180329。
NK细胞:购自Allcells公司，货号PB012-C。
Protein A芯片：label No:29139131-AA；lot:10261132。
SD大鼠：购自浙江维通利华实验动物技术有限公司，生产许可证SCXK(浙)2018-0001。
人胃癌细胞株NCI-N87：购自美国典型培养物保藏中心(ATCC)。
BALB/c裸小鼠：购自上海灵畅生物科技有限公司。
MC38小鼠结肠癌细胞株：和元生物技术(上海)股份有限公司。
人源化PD1小鼠品系C57BJ/6J-PDCD1em1(Hpdcd1)/Smoc：产品编号：NM-KI-00015，购自上海南方模式生物科技股份有限公司。
PBMC：购自塞笠生物，货号SLB-HP040A。
实验试剂：
HRP标记的鼠抗人Fab抗体：购自sigma，货号A0293。
Streptavidin HRP：购自BD Biosciences，货号554066.
羊抗人IgG-FITC：购自sigma，货号F4143。
抗CD28抗体：购自Abcam,货号ab213043。
IL-2：购自R&D，货号202-IL。
PBS：购自生工生物工程(上海)股份有限公司，货号B548117。
PBST：PBS+0.05％Tween 20。
BSA：购自生工生物工程(上海)股份有限公司，货号A60332。
TMB：购自BD公司，货号555214。
Bio-Glo：购自Promega，货号G7940。
FBS：购自Gibco，货号10099。
HBS-EP工作液：购自Life science，BR-1006-69。
CellTiter-Glo：购自promega，货号G775B。
实验仪器：
HiTrap MabSelectSuRe柱：购自GE公司。
Beckman Coulter CytoFLEX流式细胞仪：购自Beckman公司。
SpectraMax i3x酶标仪：购自MolecularDevices公司。
SpectraMaxM5酶标仪：购自MolecularDevices公司。
微量热差式扫描量热仪MicroCal VP-Capillary DSC。
本发明实施例中所述的HER2单克隆抗体是指三生国健药业按照Herceptin的氨基酸序列，参照与实施例2中双抗相同的表达纯化方法得到的人鼠嵌合单克隆抗体。本发明实施例中所述的PD1单克隆抗体是指中国专利申请CN201710054783.5公开的由三生国健药业自主研发的全新的抗PD1人源化单克隆抗体。
实施例1.抗HER2/PD1双抗分子的构建
本发明采用了HER2单抗IgG和PD1单抗的scFv串联的方式构建了抗HER2/PD1双特异性抗体a。
将抗PD1单克隆抗体的轻链可变区VL(SEQ ID NO：14)和重链可变区VH(SEQ ID NO：13)通过肽接头L1(SEQ ID NO：17)连接起来，得到抗PD1的单链抗体片段VL-L1-VH，即抗PD1片段scFv1(SEQ ID NO：19)。利用L2(SEQ ID NO：18)将该单链抗体片段和抗HER2单克隆抗体的重链连接起来，从而得到双特异性抗体分子抗HER2/PD1双抗a的重链(SEQ ID NO：20)，HER2单抗的轻链(SEQ ID NO：21)则保持不变。为了提高抗体分子在CHO细胞中的表达效率，委托金唯智公司对抗HER2/PD1双抗a分子的核酸序列进行密码子优化。优化主要考虑密码子的偏好性、GC含量、mRNA二级结构、重复序列等因素，随后委托金唯智公司合成。抗HER2/PD1双抗a重链核酸序列为SEQ ID NO：22，轻链核酸序列为SEQ ID NO：23。抗HER2/PD1双抗a结构如图1A所示，序列见附录序列表。
抗HER2/PD1双抗b的分子构建如下所述：
将抗PD1单克隆抗体PD1单抗的轻链可变区VL(SEQ ID NO：14)和重链可变区VH(SEQ ID NO：13)通过肽接头L1(SEQ ID NO：17)连接起来，得到抗PD1的单链抗体片段VH-L1-VL，即抗PD1片段scFv2(SEQ ID NO：24)。利用L2(SEQ ID NO：18)将该单链抗体片段和抗HER2单克隆抗体的重链连接起来，从而得到双特异性抗体分子抗HER2/PD1双抗b的重链(SEQ ID NO：25)，HER2单抗的轻链(SEQ ID NO：21)则保持不变。为了提高抗体分子在CHO细胞中的表达效率，委托金唯智公司对抗HER2/PD1双抗b分子的核酸序列进行密码子优化。优化主要考虑密码子的偏好性、GC含量、mRNA二级结构、重复序列等因素，随后委托金唯智公司合成。抗HER2/PD1双抗b重链核酸序列为SEQ ID NO：26，轻链核酸序列为SEQ ID NO：23。抗HER2/PD1双抗b结构如图1B所示，序列见附录序列表。
实施例2.双抗的表达与纯化
将双抗的重链和轻链的DNA片段分别亚克隆到pTT5载体中，提取重组质粒共转染CHO细胞和/或293E细胞。细胞培养5-7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后，上样至HiTrap MabSelectSuRe柱，用含有100mM柠檬酸，pH3.5的洗脱液一步洗脱蛋白，回 收目标样品并透析至pH7.4的PBS。将纯化后的蛋白用HPLC检测，抗HER2/PD1双抗a、b的HPLC检测图谱分别如图2A、2B所示，抗体分子状态均一，单体纯度达到97％以上。取纯化后的抗HER2/PD1双抗a、b分别加入非还原电泳缓冲液，SDS-聚丙烯酰胺凝胶电泳检测；取纯化后的抗HER2/PD1双抗a、b分别加入还原电泳缓冲液并煮沸，SDS-聚丙烯酰胺凝胶电泳检测。电泳图见图2C。双抗全长理论分子量为199KD。
实施例3.酶联免疫吸附法(ELISA)测定双抗对抗原的亲和力
为了检测抗HER2/PD1双抗a和b，分别与HER2抗原的亲和力，用pH7.4的PBS缓冲液将HER2-ECD-His蛋白(三生国健自制)稀释至250ng/ml，然后100μl/孔加入ELISA板中；4℃孵育过夜；次日用PBST洗板两次；每孔加入PBST+1％BSA进行封闭，37℃封闭1h；用PBST洗板两次；然后加入用PBS+1％BSA梯度稀释的待检测抗体，HER2单抗作为阳性对照，起始浓度为100nM，逐级3倍稀释12个梯度。37℃孵育1h；PBST洗板两次，加入HRP标记的鼠抗人Fab抗体，37℃再孵育40min；PBST洗板三次并拍干，每孔加入100μl TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2MH 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。实验结果如图3A所示，抗HER2/PD1双抗a，b和阳性对照HER2单抗与HER2结合的EC 50分别为0.1975，0.2294和0.221，三者亲和力相当。
为了检测抗HER2/PD1双抗a和b，分别与PD1的结合能力，将重组PD1-ECD-hFc蛋白(三生国健自制)用pH7.4的PBS稀释至200g/ml，100μl/孔加入酶标版，4℃包被过夜。PBST洗板2次，加入200μl/孔封闭液(PBS+2％BSA)，37℃放置1小时后PBST洗板1次待用。然后加入用PBS+1％BSA梯度稀释的待检测抗体，PD1单抗作为阳性对照，起始浓度为100nM，逐级3倍稀释12个梯度。加入封闭后的酶标板，100μl/孔，37℃放置1小时。PBST洗板2次，加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟。PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2MH 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism6进行数据分析，作图并计算EC 50。实验结果如图3B所示，抗HER2/PD1双抗a，b和阳性对照PD1单抗与PD1结合的EC 50分别为0.1384，0.1525和0.1557。三者亲和力相当。
实施例4.检测双抗对靶细胞的结合亲和力
本实验以细胞表面HER2高表达的人乳腺癌细胞BT474作为靶细胞，用含有0.5％BSA的PBS洗涤三次，每次300g离心5分钟，弃上清。0.5％BSA的PBS重悬细胞，细胞浓度为1×10 6细胞/mL，100μL/孔加入96孔板。将抗HER2/PD1双抗a、b及阳性对照HER2单抗稀释为400nM，逐级稀释11个梯度，100μL/孔加入96孔板，将BT474细胞混合均匀。4℃孵育1h。PBS洗涤细胞两次以去除未结合的待检抗体。再将细胞与100μl的1g/ml羊抗人IgG-FITC于4℃孵育30分钟。300g离心5分钟，PBS洗涤细胞两次以去除未结合的二抗。 最后将细胞重悬在200μl PBS中，通过Beckman Coulter CytoFLEX流式细胞仪测定双抗对该细胞的结合亲和力。所得数据通过GraphPad Prism6软件拟合分析。实验结果如图4A所示，抗HER2/PD1双抗a，抗HER2/PD1双抗b都能特异性的结合细胞表面表达的HER2。实验结果如图4A所示，抗HER2/PD1双抗a，b和阳性对照HER2单抗与BT474细胞结合的EC 50分别为1.64，5.669，1.556。其中抗HER2/PD1双抗a和阳性对照HER2单抗亲和力相当，而抗HER2/PD1双抗b与阳性对照HER2单抗相比，亲和力稍弱。
同样的，以细胞表面表达PD1的CHO稳转细胞作为靶细胞，通过流式细胞仪测定抗HER2/PD1双抗a，抗HER2/PD1双抗b，对该细胞的结合亲和力。方法如前所述，所得数据通过GraphPad Prism 6软件拟合分析。实验结果如图4B，4C所示，抗HER2/PD1双抗a，抗HER2/PD1双抗b都可以特异性的结合细胞表面表达的PD1。抗HER2/PD1双抗a和阳性对照PD1单抗EC 50分别为1.777和0.8981；抗HER2/PD1双抗b和阳性对照抗PD1单抗EC 50分别为1.192和0.8891。三者亲和力相当。
实施例5.双抗对BT474细胞体外增殖抑制作用
人乳腺癌细胞系BT474表达HER2抗原分子在其细胞表面。由于BT474细胞在体外培养过程中，其正常的增殖，部分依赖HER2受体传递的生长信号。在培养基中加入抗HER2抗体，则可抑制该细胞增殖。在一定范围内，抗体的浓度与细胞增殖抑制的程度成量效关系。细胞增殖的程度可通过CCK-8(Cell Counting Kit-8)细胞增殖毒性试剂来检测。量效关系曲线为反”S”曲线。
将BT474细胞用胰酶消化，重悬后进行细胞计数，根据活细胞的密度，用完全培养基调整细胞密度至5×10 4细胞/mL，100μL/孔加入96孔细胞培养板的B～G行。A、H两行加入200μL/孔的培养基或PBS封边。放置37℃，5％CO 2培养箱中贴壁培养3～5小时。将抗HER2/PD1双抗a、b与阳性对照HER2单抗样品用完全培养基作为稀释液配制为300nM溶液，再逐级3倍稀释，共计11个梯度。将稀释好的样品，加入对应的96孔板细胞中。置于37℃、5％CO 2的培养箱内继续培养7天。孵育7天的细胞培养板，按照1：10的比例(样品稀释液：CCK-8)加入显色液，放入CO 2培养箱中继续孵育3～5h。酶标仪以650nm为参比波长，450nm下测定OD值。所得数据通过GraphPad Prism 6软件分析，实验结果如图5所示。抗HER2/PD1双抗a，抗HER2/PD1双抗b和阳性对照HER2单抗IC 50分别为0.4967、0.9427、0.5914。三者抑制率相当。
实施例6.双抗阻断PD1/PD-L1结合的细胞水平的活性
取对数期生长的PD-L1aAPC/CHO-K1，胰酶消化成单个细胞后转移到白色底透96孔板，100μL/孔，40000细胞/孔，置于37℃，5％CO 2，孵育过夜。取抗HER2/PD1双抗a、b、抗PD1单抗、同型阴性对照样品逐级3倍梯度稀释成2×工作液浓度：起始浓度为600nM。取密度在1.4-2×10 6细胞/mL，细胞活率在95％以上的PD1效应细胞，胰酶消化成1.25×10 6细胞 /ml的单细胞悬液。取前一天铺好的PD-L1aAPC/CHO-K1细胞，弃掉上清，加入40μl梯度稀释的双抗/PD1单抗工作液；再加入等体积的PD1效应细胞。置于37℃，5％CO 2，孵育6小时。每孔加入80μl检测试剂Bio-Glo。室温孵育10分钟后，用spectramax i3读取luminescence。
所有数据均为双复孔，所得信号值取平均值后用4-parameter法拟合，绘制曲线，如图6A，6B所示，获得抗HER2/PD1双抗a的IC 50，top，bottom，hillslope等数据。如表1所示：
表1
  抗HER2/PD1双抗a 抗PD1单抗
Bottom 2.707 -1.048
Top 101.1 95.61
lgIC 50 0.5161 -0.05017
HillSlope 1.048 0.8425
IC 50 3.282 0.8909
抗HER2/PD1双抗b的IC 50，top，bottom，hillslope等数据。如表2所示：
表2
  抗HER2/PD1双抗b 抗PD1单抗
Bottom 4.435 4.39
Top 121.3 102.8
lgIC 50 0.6467 0.03983
HillSlope 0.8171 0.7548
IC 50 4.433 1.096
实施例7.BiacoreTM 8K测定双抗对抗原的亲和力
使用proteinA捕获法测定双抗和抗原HER2-ECD-his结合的动力学参数。将浓度为1μg/ml的双抗结合在Protein A芯片上，将抗原HER2-ECD-his用1×HBS-EP工作液从50nM往下2倍稀释，设6个浓度梯度与抗体结合，于HBS-EP工作液中解离。
使用proteinA捕获法测定双抗和抗原PD1-ECD-his结合的动力学参数。将浓度为1μg/ml的双抗结合在Protein A芯片上，将抗原PD1-ECD-his用1×HBS-EP工作液从250nM往下2倍稀释设5个浓度梯度与抗体结合，于HBS-EP工作液中解离。
抗HER2/PD1双抗a和HER2-ECD-His、PD1-ECD-his结合的动力学参数见表3。结果表明，抗HER2/PD1双抗a与抗原PD1和HER2有良好的亲和力。
表3
Analyte Solution ka(1/Ms) kd(1/s) KD(M)
PD1-ECD-his 2.43E+04 8.57E-05 3.53E-09
HER2-ECD-his 5.88E+04 1.92E-04 3.27E-09
KD为亲和力常数；ka为抗原抗体结合速率；kd为抗原抗体解离速率；KD＝kd/ka。
抗体抗HER2/PD1双抗b和HER2-ECD-His、PD1-ECD-his结合的动力学参数见表4。结果表明，抗HER2/PD1双抗b与抗原PD1和HER2有良好的亲和力。
表4.抗HER2/PD1双抗b的动力学参数
Analyte Solution ka(1/Ms) kd(1/s) KD(M)
PD1-ECD-his 3.85E+04 1.86E-04 4.83E-09
HER2-ECD-his 2.16E+05 1.71E-04 7.92E-10
KD为亲和力常数；ka为抗原抗体结合速率；kd为抗原抗体解离速率；KD＝kd/ka。
实施例8.抗HER2/PD1双抗a，抗HER2/PD1双抗b的药代动力学研究
取每组4只SD大鼠，体重200g左右，每只大鼠通过尾静脉注射剂量为2mg的抗体。分别在给药后的特定时间眼眶取血，血液自然凝固后8000rpm/min离心取血清。
抗HER2/PD1双抗a的血清中药物浓度采用以下方法检测：
1)HER2-His包被ELISA板，50ng/孔。共两板。4℃包被过夜，次日PBST洗板两次，然后用PBS+2％BSA于37℃封闭2小时。取起始浓度为0.5μg/mL的抗HER-2/PD1双抗a标准品，逐级两倍稀释12个梯度。将每个血清样品稀释2000倍。将以上样品加入封闭好的ELISA板。37℃孵育一小时。然后PBST洗板两次。
检测抗HER2的抗体：取其中一板加入HRP标记的鼠抗人Fab抗体，1:3000稀释，100μL/孔。37℃孵育40min。PBST洗板4次，拍干。每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
检测抗PD1的抗体：取另一板加入生物素化的PD1-hFc，7.5ng/孔，孵育1小时。洗板并加入Streptavidin HRP，1:1000倍稀释。37℃放置30分钟；PBST洗板4次，拍干。每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl的2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
2)proteinA包被ELISA板，检测抗体Fab段。用proteinA包被，包被量为100ng/孔，4℃过夜；次日PBST洗板两次，然后用PBS+2％BSA于37℃封闭2小时。PBST洗板两次。抗HER2/PD1双抗a标准品从1000ng/mL起始，逐级两倍稀释12个梯度。大鼠血清样品2000倍稀释。以上两组样品加入封闭后的ELISA板，孵育1小时；PBST洗板两次后加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
用Phoenix软件计算抗体药物在大鼠体内的半衰期，用GraphPad Prism6进行数据整理分析和作图。药代动力学参数见表5、6、7，实验结果如图7A，7B，7C所示，用两种方法检测的大鼠体内半衰期分别为：方法一的抗HER2抗体的为273小时，抗PD1抗体的为333小时；方法二检测的半衰期是333小时。三组数据相差不大，可以推测抗HER2/PD1双抗a的半衰期在300小时左右。
检测抗HER2抗体的半衰期见表5：
表5
Figure PCTCN2019112467-appb-000003
Figure PCTCN2019112467-appb-000004
用生物素化的PD1检测抗HER2/PD1双抗a抗体的半衰期见表6：
表6
group HL_Lambda_z(hr)
1 382.77325
2 294.69571
3 302.13064
4 353.0152
平均 333
用proteinA检测抗HER2/PD1双抗a抗体的半衰期见表7：
表7
group HL_Lambda_z(hr)
1 346.75496
2 369.60234
3 306.45773
4 310.91707
平均 333
抗HER2/PD1双抗b的血清中药物浓度采用以下方法检测：
1)检测抗HER2的抗体：HER2-His包被ELISA板，50ng/孔。4℃包被过夜，次日PBST洗板两次，然后用PBS+2％BSA于37℃封闭2小时。取起始浓度为0.5μg/mL的抗HER2/PD1双抗b标准品，逐级两倍稀释12个梯度。将每个血清样品稀释2000倍，加入封闭好的ELISA板。37℃孵育1小时。然后PBST洗板两次。加入HRP标记的鼠抗人Fab抗体，1:3000稀释，100μL/孔。37℃孵育40min。PBST洗板4次，拍干。每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
2)检测抗PD1的抗体：PD1-ECD–hFc包被ELISA板，20ng/孔。包被，洗板，以及标准品稀释方法同上。将血清样品稀释1000-2000倍，加入封闭好的ELISA板。37℃孵育1小时。然后PBST洗板两次。加入HRP标记的鼠抗人Fab抗体，1:3000稀释，100μL/孔。37℃孵育40min。PBST洗板4次，拍干。每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
3)proteinA包被ELISA板，检测抗体Fab段。用proteinA包被，包被量为100ng/孔，4℃过夜；次日PBST洗板两次，然后用PBS+2％BSA于37℃封闭2小时。PBST洗板两次。抗HER2/PD1双抗b标准品从1000ng/mL起始，逐级两倍稀释12个梯度。大鼠血清样品500-1000 倍稀释，加入封闭后的ELISA板，孵育1小时；PBST洗板两次后加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟；PBST洗板4次，拍干。每孔加入100μl的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μl2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。
用Phoenix软件计算抗体药物在大鼠体内的半衰期，用GraphPad Prism6进行数据整理分析和作图。药代动力学参数见表8、9、10，实验结果如图7D，7E，7F所示。用三种方法检测的大鼠体内半衰期分别为：方法一为312小时；方法二为280小时。方法三为277小时。三组数据相差不大，可以推测抗HER2/PD1双抗b的半衰期在280小时左右。
检测抗HER2抗体的半衰期见表8：
表8
group HL_Lambda_z(hr)
1 249.10194
2 279.51118
3 366.19333
4 355.05384
平均 312
检测抗PD1抗体的半衰期见表9：
表9
group HL_Lambda_z(hr)
1 375.61812
2 216.88057
3 196.51091
4 331.40524
平均 280
用proteinA检测抗HER2/PD1双抗b抗体的半衰期见表10：
表10
group HL_Lambda_z(hr)
1 189.38172
2 333.14994
3 389.77667
4 196.6228
平均 277
实施例9.抗HER2/PD1双抗a的ADCC效应
由于HER2/PD1双抗不仅可以结合肿瘤细胞，结合表达PD-1的T细胞，抗体的Fc段还可以结合NK细胞。
本实验一方面检测NK细胞对抗体结合的CD4+T细胞是否有杀伤；另一方面检测NK细胞对抗体结合的BT474肿瘤细胞的杀伤。
1)NK细胞对CD4+T细胞是否有杀伤作用：活化的T细胞上表达PD1，加入抗HER2/PD1 双抗a抗体与其结合，而抗HER2/PD1双抗a的Fc段与效应细胞NK的Fc受体结合，加入NK细胞可检测对T细胞是否有杀伤。
实验方法如下：
CD4+T细胞的激活：用D-PBS配制抗CD3抗体，浓度为5μg/mL，包被24孔细胞培养板，4℃过夜。次日，每孔加入5×10 5个CD4+T细胞，同时加入2μg/mL的抗CD28抗体以及100U/mL的IL2。放于37℃，CO 2培养箱，激活72h。
收集活化的T细胞，FACS检测PD1的表达。然后将高表达PD1的T细胞，用含5％FBS的1640培养基，配制成2×10 5细胞/mL，50μL/孔。加入96孔板。
抗HER2/PD1双抗a以及阴性对照样品HER2单抗的稀释：将抗HER2/PD1双抗a以及HER2单抗配制为初始浓度400nM，逐级5倍稀释后，加入铺入T细胞的96孔板中。放于37℃，CO 2培养箱，孵育15min。期间，将NK细胞用含5％FBS的1640培养基调整为5×10 5细胞/mL，100μL/孔加入上述96孔板中，放于37℃，CO 2培养箱，杀伤3h。
将96孔板300g离心5min，转移上清再离心一次，转移100μL上清到另一96孔板。加入50μL/孔的LDH底物，孵育15min。在SpectraMaxM5酶标仪上，以450nm为检测波长，650nm为参比波长，进行读数，用GraphPad Prism6进行数据分析，作图。结果如图8A显示，NK细胞对CD4+T细胞无明显杀伤作用，仅在抗HER2/PD1双抗a高浓度下有微弱的杀伤。
2)NK对BT474肿瘤细胞ADCC效应
BT474细胞表面表达HER2抗原，加入抗HER2/PD1双抗a可与其结合。而抗HER2/PD1双抗a的Fc段与效应细胞NK的Fc受体结合，加入NK细胞可检测对BT474细胞是否有杀伤。
实验方法如下：
将BT474细胞，用含5％FBS的1640培养基稀释为2×10 5个细胞/mL，加入96孔平底板中，50μL/孔。放于37℃，5％CO 2培养箱过夜。
抗HER2/PD1双抗a以及阴性对照样品HER2单抗的稀释：将抗HER2/PD1双抗a以及HER2单抗配制为初始浓度200nM，逐级4倍稀释后，加入铺入BT474细胞的96孔板中。放于37℃，5％CO 2培养箱，孵育15min。期间，将NK细胞用含5％FBS的1640培养基调整为5×10 5细胞/mL，100μL/孔加入上述96孔板中，放于37℃，CO 2培养箱，孵育3h。
将96孔板300g离心5min，转移上清再离心一次，转移100μL上清到另一96孔板。加入50μL/孔的LDH底物，孵育15min。在SpectraMaxM5酶标仪上，以450nm为检测波长，650nm为参比波长，进行读数，用GraphPad Prism6进行数据分析，作图。结果如图8B显示，NK细胞对BT474肿瘤细胞有明显杀伤作用，结果与HER2单抗相似。
实施例10.细胞水平上检测抗HER2/PD1双抗a的协同作用
细胞水平上检测抗HER2/PD1双抗两个靶点的协同作用，需要满足以下条件：肿瘤细胞上有HER2抗原表达，并且肿瘤细胞的增殖能被HER2抗体抑制；同时有PD-L1表达，可以 与T细胞上的PD-1结合，因此加入PD1抗体后阻断PD-1/PD-L1结合，T细胞的抑制被解除，发挥杀伤肿瘤的作用。由于未筛选到满足这些条件的细胞株，因此采用慢病毒转染法，将PD-L1基因重组到人胃癌细胞株NCI-N87。构建的N87-PDL1细胞，FACS检测细胞表面高表达PD-L1。
取对数生长期的N87-PDL1细胞，胰酶消化后，用1640培养基加1％FBS稀释为1×10 5/mL，100μL/孔转移到白色透底96孔板。放于37℃，5％CO 2培养箱过夜。次日加待检抗体和新鲜的PBMC细胞，各50μL/孔。抗体为抗HER2/PD1双抗a，抗HER2单抗，抗HER2单抗加抗PD1单抗，抗PD1单抗，浓度为4nM。PBMC用1640培养基加1％FBS稀释，为10 5/孔。放于37℃，5％CO 2继续培养6天后，用PBS洗板三次，用培养基1:1稀释CellTiter-Glo，100μL/孔加入96孔板。用spectramax i3读取luminescence。用GraphPad Prism进行数据分析作图，见图9A，9B。数据显示，抗HER2/PD1双抗a杀伤肿瘤的效果优于HER2单抗，并且优于抗HER2单抗加PD1单抗，说明双抗发挥了协同抗肿瘤的作用。
实施例11.抗HER2/PD1双抗a在NCI-N87移植瘤模型上的抗肿瘤作用
收集体外培养的人胃癌细胞株NCI-N87细胞，将细胞浓度调整为5×10 7细胞/mL，重悬于无血清培养基中，在无菌条件下，接种100μL细胞悬液于裸小鼠背部皮下。用游标卡尺测量移植瘤长与宽，计算肿瘤体积，待肿瘤生长至100-200mm 3后将动物随机分组。
受试样品抗HER2/PD1双抗a的用药剂量分为两组，20mg/kg，4mg/kg，即0.4mg/只，0.08mg/只，阳性对照药HER2单抗单药的剂量为15mg/kg，即0.3mg/只。对照组给以相同体积的PBS。给药方式为腹腔给药，给药体积为0.2mL/鼠(20g)，每周两次给药，连续给药三周。
每周测量2次移植瘤体积，同时对小鼠称重，记录。肿瘤体积(tumor volume，TV)的计算公式为：TV＝1/2×长×宽 2。根据测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为：RTV＝Vt/V0。其中V0为分笼给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式为：T/C(％)＝(TRTV/CRTV)×100(TRTV：治疗组RTV；CRTV：阴性对照组RTV)；抑瘤率＝1-T/C(％)。疗效评价标准：T/C(％)>40％为无效；T/C(％)≤40，并经统计学处理p≤0.05为有效。实验结果如图10所示，抗HER2/PD1双抗a与阳性对照HER2单抗近似。
实施例12.抗HER2/PD1双抗a在人源化PD1小鼠MC38移植瘤模型上的抗肿瘤作用
受试样品抗HER2/PD1双抗a的剂量为13mg/kg，阳性对照抗PD1单抗的剂量设置为10mg/kg，对照组给以相同体积的生理盐水。收集体外培养的MC38小鼠结肠癌细胞，将细胞悬液浓度调整为1×10 7细胞/ml。在无菌条件下，接种100μl细胞悬液于人源化PD1小鼠右侧肋部皮下。人源化PD1小鼠皮下移植瘤用游标卡尺测量移植瘤直径，待平均肿瘤体积生长至100-200mm 3后将动物随机分组。抗PD1单抗、抗HER2/PD1双抗a按剂量给药、对照组给等量生理盐水，每周腹腔注射给药2次，连续给药3周。整个实验过程中，每周2次测量移 植瘤直径，同时称小鼠体重。肿瘤体积(tumorvolume，TV)的计算公式为：TV＝1/2×长×宽 2。根据测量的结果计算出相对肿瘤体积(relativetumorvolume，RTV)，计算公式为：RTV＝Vt/V0。其中V0为分组给药时(即d0)测量所得肿瘤体积，Vt为每一次测量时的肿瘤体积。抗肿瘤活性的评价指标为相对肿瘤增殖率T/C(％)，计算公式如下：T/C(％)＝(TRTV/CRTV)×100(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。疗效评价标准：T/C(％)>40％为无效；T/C(％)≤40，并经统计学处理p≤0.05为有效。实验重复两次。实验结果如图11所示。结果表明，在人源化PD1小鼠MC38移植瘤模型上，抗HER2/PD1双抗a可以通过阻断PD1抑制肿瘤生长，抑瘤效果与阳性对照PD1单抗近似。
实施例13.抗HER2/PD1双抗a和抗HER2/PD1双抗b的稳定性研究
本实验可用于评估与相互作用有关的热力学参数，如在辅料加入情况下蛋白质去折叠的情况等，从而揭示研发最优制剂所需的重要机理信息。
实验使用MicroCal VP-Capillary DSC，用0.22um滤膜将样品及其缓冲液过滤，分别取400μl样品及其匹配缓冲液置于96孔板中，样品在25℃-100℃条件下扫描，扫描速率为每小时120℃。
抗HER2/PD1双抗a，抗HER2/PD1双抗b保存在pH7.4的PBS中。DSC检测双抗的Tm值见表11。图谱见图12A，12B。由此可知，此双抗较稳定。后续的37℃长期稳定性实验结果也验证了这一点。HPLC-SEC结果见图12C，12D。
表11
样品号 Tm Onset Tm1 Tm2
抗HER2/PD1双抗a-CHO 50 58 83
抗HER2/PD1双抗a-293E 51 58 83
抗HER2/PD1双抗b-293E 53 62 81
由上述实验可知，本发明提供的双特异性抗体，结构稳定。能同时结合HER2和PD1抗原。阻断HER2信号通路，可抑制表达HER2抗原的肿瘤细胞增殖；同时能阻断PD-1/PD-L1通路，可恢复T细胞的免疫杀伤功能，发挥杀伤肿瘤细胞的作用。同时，此双抗HER2抗体的Fc段可以与NK细胞的Fc受体结合，发挥ADCC效应，杀伤肿瘤细胞，而对T淋巴细胞并无明显杀伤作用。

Claims (22)

  1. 一种能与HER2和PD1特异结合的双特异性抗体，其特征在于，其包含免疫球蛋白抗体IgG和两个相同的单链可变片段scFv，其中每个单链可变片段scFv包含可变区VH和可变区VL，VH与VL通过肽接头L1连接，每个单链可变片段scFv通过接头肽L2与所述免疫球蛋白抗体IgG串联。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，所述VH包含互补决定区HCDR1-3，其中HCDR1的氨基酸序列如SEQ ID NO：1所示，HCDR2的氨基酸序列如SEQ ID NO：2所示，HCDR3的氨基酸序列如SEQ ID NO：3所述；
    所述VL包含互补决定区LCDR1-3，其中LCDR1的氨基酸序列如SEQ ID NO：4所示，LCDR2的氨基酸序列如SEQ ID NO：5所示，LCDR3的氨基酸序列如SEQ ID NO：6所示；
    所述免疫球蛋白抗体IgG的重链包含互补决定区HCDR4-6，其中HCDR4的氨基酸序列如SEQ ID NO：7所示，HCDR5的氨基酸序列如SEQ ID NO：8所示，HCDR6的氨基酸序列如SEQ ID NO：9所示；
    所述免疫球蛋白抗体IgG的轻链包含互补决定区LCDR4-6，其中LCDR4的氨基酸序列如SEQ ID NO：10所示，LCDR5的氨基酸序列如SEQ ID NO：11所示，LCDR6的氨基酸序列如SEQ ID NO：12所示。
  3. 根据权利要求1所述的双特异性抗体，其特征在于，所述VH的氨基酸序列如SEQ ID NO：13所示，VL的氨基酸序列如SEQ ID NO：14所示；
    所述免疫球蛋白抗体IgG包含重链可变区和轻链可变区，其重链可变区的氨基酸序列如SEQ ID NO：15所示，轻链可变区的氨基酸序列如SEQ ID NO：16所示。
  4. 根据权利要求1所述的双特异性抗体，其特征在于，所述肽接头L1的氨基酸序列如SEQ ID NO：17所示。
  5. 根据权利要求1所述的双特异性抗体，其特征在于，所述肽接头L2的氨基酸序列如SEQ ID NO：18所示。
  6. 根据权利要求1所述的双特异性抗体，其特征在于，所述单链可变片段scFv的分子结构形式为VL-L1-VH，每个scFv的N末端经由肽接头L2连接至所述免疫球蛋白抗体IgG重链的C末端。
  7. 根据权利要求1所述的双特异性抗体，其特征在于，所述单链可变片段scFv的氨基酸序列如SEQ ID NO：19所示。
  8. 根据权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体的重链氨基酸序列如SEQ ID NO：20所示，其轻链氨基酸序列如SEQ ID NO：21所示。
  9. 一种核苷酸分子，所述核苷酸分子编码如权利要求1-8任一项所述的双特异性抗体。
  10. 如权利要求9所述的核苷酸分子，其特征在于，所述核苷酸分子编码能与HER2和PD1特异结合的双特异性抗体的重链的核苷酸序列如SEQ ID NO：22所示，编码其轻链的核苷酸序列如SEQ ID NO：23所示。
  11. 根据权利要求1所述的双特异性抗体，其特征在于，所述单链可变片段scFv的分子结构形式为VH-L1-VL，每个scFv的C末端经由肽接头L2连接至所述免疫球蛋白抗体IgG重链的N末端。
  12. 根据权利要求1所述的双特异性抗体，其特征在于，所述单链可变片段scFv的氨基酸序列如SEQ ID NO：24所示。
  13. 根据权利要求1所述的双特异性抗体，其特征在于，所述双特异性抗体的重链氨基酸序列如SEQ ID NO：25所示，其轻链氨基酸序列如SEQ ID NO：21所示。
  14. 一种核苷酸分子，其特征在于，所述核苷酸分子编码如权利要求11-13任一项所述的双特异性抗体。
  15. 如权利要求14所述的核苷酸分子，其特征在于，所述核苷酸分子编码能与HER2和PD1特异结合的双特异性抗体的重链的核苷酸序列如SEQ ID NO：26所示，编码其轻链的核苷酸序列如SEQ ID NO：23所示。
  16. 一种表达载体，所述表达载体含有权利要求9、10、14或15任一项所述的核苷酸分子。
  17. 根据权利要求16所述的表达载体，其特征在于，所述表达载体选自pDR1，pcDNA3.1(+)，pcDNA3.1/ZEO(+)，pDHFR和pTT5。
  18. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求16所述的表达载体。
  19. 根据权利要求18所述的宿主细胞，其特征在于，所述宿主细胞选自CHO细胞和293E细胞。
  20. 一种制备如权利要求1-8、11-13任一项所述的能与HER2和PD1特异结合的双特异性抗体的方法，所述方法包括以下步骤：
    a)在表达条件下，培养如权利要求18-19任一所述的宿主细胞，从而表达能与HER2和PD1特异结合的双特异性抗体；
    b)分离并纯化步骤a)所述的双特异性抗体。
  21. 一种组合物，所述组合物包含权利要求1-8、11-13任一所述的能与HER2和PD1特异结合的双特异性抗体和一种或多种药学上可接受的载体、稀释剂或赋形剂。
  22. 权利要求1-8、11-13任一项所述的能与EGFR和PD1特异结合的双特异性抗体、或权利要求21所述的药物组合物在制备药物中的应用，其特征在于所述药物用于治疗癌症或肿瘤。
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