WO2021057451A1 - 一种抗体融合蛋白及其制法和在抗肿瘤中的应用 - Google Patents

一种抗体融合蛋白及其制法和在抗肿瘤中的应用 Download PDF

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WO2021057451A1
WO2021057451A1 PCT/CN2020/113556 CN2020113556W WO2021057451A1 WO 2021057451 A1 WO2021057451 A1 WO 2021057451A1 CN 2020113556 W CN2020113556 W CN 2020113556W WO 2021057451 A1 WO2021057451 A1 WO 2021057451A1
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fusion protein
antibody fusion
seq
acid sequence
amino acid
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French (fr)
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朱祯平
黄浩旻
顾昌玲
祝海霞
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三生国健药业（上海）股份有限公司
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the invention belongs to the field of tumor treatment and biotechnology, and relates to an antibody fusion protein composed of an anti-HER2 monoclonal antibody IgG and the D2 domain of VEGFR1, and a preparation method and application thereof.
  • HER2 human epidermal growth factor receptor2
  • receptor tyrosine protein kinase activity is a member of the human epidermal growth factor receptor family, and is only expressed at low levels in a few normal tissues of adults.
  • HER2 is overexpressed in a variety of tumors. For example, there is overexpression in about 30% of breast cancer patients and 16% of gastric cancer patients. Overexpression of HER2 in tumors can significantly promote tumor growth and enhance The ability of tumor invasion and metastasis is an important indicator of poor prognosis for such patients. Therefore, as early as 1998, Herceptin, the first monoclonal antibody drug targeting HER2, was approved by the FDA for the treatment of breast and gastric cancers overexpressing HER2.
  • VEGF vascular endothelial growth factors
  • VEGF-A165 VEGF-A165
  • VEGF vascular endothelial growth factors
  • VEGFR1-D2 competes for binding to VEGF and blocks the binding of VEGFR2 to VEGF, thereby blocking the signal pathway, inhibiting endothelial cell proliferation and angiogenesis, thereby inhibiting the rapid proliferation and metastasis of tumors.
  • the present invention introduces an antibody fusion protein that can simultaneously block the HER2 and VEGFR2 signaling pathways.
  • the antibody fusion protein can bind to the HER2 antigen on the surface of tumor cells to inhibit tumor proliferation; on the other hand, it can compete to bind to VEGF and inhibit endothelial cells. Proliferation and angiogenesis. This mechanism of action occurs in the tumor microenvironment and can effectively inhibit the formation of blood vessels inside the tumor, thereby inhibiting tumor growth.
  • the antibody fusion protein has better tumor proliferation than HER2 monoclonal antibody and HER2 monoclonal antibody + FcD2, and has broad application prospects in the treatment of tumor diseases.
  • the purpose of the present invention is to provide an antibody fusion protein capable of simultaneously blocking the HER2 and VEGFR2 signaling pathways, to provide a nucleotide molecule encoding the antibody fusion protein; to provide an expression vector containing the nucleotide molecule; to provide the The host cell of the expression vector; the preparation method of the antibody fusion protein is provided; the pharmaceutical composition containing the antibody fusion protein is provided; the antibody fusion protein is provided in the production
  • the present invention provides an antibody fusion protein capable of simultaneously blocking the HER2 and VEGFR2 signaling pathways, which comprises an anti-HER2 monoclonal antibody IgG and the D2 domain of VEGFR1, and the D2 domain of VEGFR1 is connected to the IgG heavy chain via a peptide linker L C terminal.
  • the "antibody fusion protein" of the present invention is a recombinantly produced antigen binding molecule in which an antibody or antibody fragment is linked to another protein or peptide. It contains the anti-HER2 monoclonal antibody IgG and the D2 domain of VEGFR1, and the D2 domain of VEGFR1 is connected to the C-terminus of the IgG heavy chain through a peptide linker L.
  • the "anti-HER2 monoclonal antibody IgG" of the present invention is a molecule of about 150kDa, which is composed of four peptide chains, containing two identical heavy chains of about 50kDa and two identical light chains of about 25kDa, thus having a tetrameric Body four-level structure.
  • the two heavy chains are connected to each other by disulfide bonds, and each is connected to a light chain.
  • the resulting tetramer has the same two halves, which form a fork or Y-like shape, and each end of the fork contains the same antigen binding site.
  • IgG antibodies can be classified into multiple subclasses (for example, IgG1, 2, 3, 4) based on small differences in the amino acid sequence of the constant region of the heavy chain.
  • the heavy chain of the IgG includes the 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: 2. As shown in SEQ ID NO: 3;
  • the light chain of the IgG includes the 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 shown.
  • the binding region of an antibody usually contains one light chain variable region and one heavy chain variable region, and each variable region contains three CDRs and three domains.
  • the CDR domains of the heavy chain and light chain of an antibody are called HCDR and LCDR, respectively. Therefore, a conventional antibody antigen binding site contains six CDRs, including a set of CDRs from the V regions of the heavy and light chains, respectively.
  • amino acid sequence of the heavy chain variable region of the IgG is shown in SEQ ID NO: 7
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • amino acid sequence of the peptide linker L is shown in SEQ ID NO: 9.
  • the heavy chain amino acid sequence of the antibody fusion protein is shown in SEQ ID NO: 10
  • the light chain amino acid sequence of the antibody fusion protein is shown in SEQ ID NO: 11.
  • the heavy chain amino acid sequence of the antibody fusion protein is shown in SEQ ID NO: 15, and the light chain amino acid sequence is shown in SEQ ID NO: 11.
  • the problems related to the chemical and physical stability of the antibody fusion protein are also solved, such as expressing physically stable molecules, increasing heat and salt-dependent stability, reducing aggregation, and increasing Solubility at high concentrations and maintaining affinity for HER2 and VEGF respectively.
  • Another aspect of the present invention provides a nucleotide molecule that encodes any one of the above-mentioned antibody fusion proteins.
  • nucleotide sequence encoding the heavy chain of the antibody fusion protein is shown in SEQ ID NO: 12, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 13.
  • nucleotide sequence of the heavy chain of the nucleotide molecule encoding the antibody fusion protein is shown in SEQ ID NO: 16
  • nucleotide sequence of the light chain encoding thereof is shown in SEQ ID NO: 13.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the field, and preferably includes the following preparation method: obtain the nucleotide molecule encoding the above-mentioned antibody fusion protein by gene cloning technology such as PCR method, or by The synthetic method of artificial whole sequence obtains the nucleotide molecule encoding the above-mentioned antibody fusion protein.
  • nucleotide sequence encoding the amino acid sequence of the above-mentioned antibody fusion protein can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homolog.
  • the polynucleotide homologs of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the antibody fusion protein within the scope of maintaining the activity of the antibody.
  • Another aspect of the present invention provides an expression vector containing any of the above-mentioned nucleotide molecules.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate regulatory sequences.
  • the expression vector may be a virus or a plasmid, such as a suitable phage or phagemid.
  • a suitable phage or phagemid for more technical details, please refer to, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.
  • the expression vector of the present invention is preferably pDR1, pcDNA3.4(+), pDHFR or pTT5.
  • the present invention additionally provides a host cell containing the above-mentioned expression vector.
  • the host cell of the present invention is a variety of conventional host cells in the field, as long as the above-mentioned recombinant expression vector can 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), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3) Or TG1, more preferably E. coli TG1, BL21 (DE3) cells (expressing single-chain antibodies or Fab antibodies) or CHO-K1 cells (expressing full-length IgG antibodies).
  • the aforementioned expression vector is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention.
  • the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
  • the host cells are eukaryotic cells, preferably CHO cells and 293E cells.
  • Another aspect of the present invention provides a method for preparing the above-mentioned antibody fusion protein, and the preparation method includes the following steps:
  • the method for culturing the host cell and the method for separation and purification of the antibody of the present invention are conventional methods in the art.
  • the specific operation method please refer to the corresponding cell culture technical manual and the antibody separation and purification technical manual.
  • the recombinant protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.
  • the antibody fusion protein disclosed in the present invention can be separated and purified by the method of affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, changing the pH, etc. can be used to eluate the binding in the affinity The antibody fusion protein on the column.
  • the inventors of the present invention have conducted detection experiments on the obtained antibody fusion protein, and the experimental results show that the antibody fusion protein can well bind to target cells and antigens and has a high affinity.
  • compositions comprising the above-mentioned antibody fusion protein and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the antibody fusion protein provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect.
  • These preparations can ensure the conformational integrity of the amino acid core sequence of the antibody fusion protein disclosed by the present invention.
  • it also protects the protein's multifunctional groups to prevent its degradation (including but not limited to aggregation, deamination or oxidation).
  • it can be stored at 2°C-8°C for at least one year, and for freeze-dried preparations, it can be kept stable at 30°C for at least six months.
  • the antibody fusion protein preparation can be suspension, water injection, freeze-drying 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 .
  • the surfactant preferably includes but is not limited to: non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (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 added should minimize the tendency of the antibody fusion protein to granulate.
  • non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Plur
  • the solution stabilizer preferably includes, but is 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 enable the final formulation to maintain a stable state within a period of time considered by those skilled in the art to be stable.
  • the isotonicity adjusting agent preferably includes, but is 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 application of the above-mentioned antibody fusion protein or the above-mentioned pharmaceutical composition in the preparation of medicines for the treatment of tumors.
  • the drugs used in the present invention for the treatment of tumors refer to drugs capable of inhibiting and/or treating tumors, which may include delays in the development of tumor-related symptoms and/or reduction in the severity of these symptoms, and further include existing tumor-associated drugs.
  • the reduction of symptoms and the prevention of other symptoms including the reduction or prevention of tumor metastasis, etc.
  • the tumors targeted by the drug of the present invention preferably include, but are not limited to: breast cancer, lung cancer, bone cancer, gastric cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, testicular cancer, uterine cancer, fallopian tube Cancer, endometrial cancer, cervical cancer, vagina cancer, vulvar cancer, rectal cancer, colon cancer, anal area cancer, esophagus 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 cancer, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, Hodgkin's disease, non-Hodgkin's lymphoma , Soft tissue sarcoma, childhood solid tumor, lymphocytic lymphoma, central nervous system (CNS) tumor, primary central nervous system lymphoma,
  • the dosage of administration varies with the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. You can refer to the animal experiment Results and various circumstances, the total dose can not exceed a certain range. Specifically, the dose of intravenous injection is 1-1800 mg/day.
  • the antibody fusion protein and its composition disclosed in the present invention can also be administered in combination with other anti-tumor drugs to achieve the purpose of more effective treatment of tumors.
  • anti-tumor drugs include but are not limited to: 1.
  • Cytotoxic drugs 1) Effect Drugs based on the chemical structure of nucleic acids: alkylating agents such as nitrogen mustards, nitrosoureas, and methylsulfonates; platinum compounds such as cisplatin, carboplatin and oxaliplatin; Antibiotics such as Adriamycin/Doxorubicin, Dactinomycin D, Daunorubicin, Epirubicin, Mithramycin, etc.; 2) Affect nucleic acid metabolism Drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and pemetrexed (Pemetrexed); thymidine synthase inhibitors such as fluorouracil (5-fluorouracil, capecitabine), etc.;
  • Hormonal drugs anti-estrogens Hormones such as Tamoxifen, Droloxifene, Exemestane, etc.; aromatase inhibitors such as Aminoglutethimide, Formestane, Letra Letrozle, Anastrozole, etc.; Antiandrogens: Flutamine RH-LH agonist/antagonist: Norride, Enantone, etc.; 3.
  • Biological response modifier drugs this type of drug Mainly adjust the body's immune function to achieve anti-tumor effects, such as interferon (Interferon); interleukin-2 (Interleukin-2); thymosin (Thymosins), etc.; 4.
  • Monoclonal antibody drugs trastux Trastuzumab (Trastuzumab), Rituximab (Rituximab), Cetuximab (Cetuximab), Bevacizumab (Bevacizumab), etc.; 5.
  • Other anti-tumor drugs including some current mechanisms that are unclear and need to be Drugs for further research, etc.
  • the antibody fusion protein 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 reagents and raw materials used in the present invention are all commercially available.
  • the antibody fusion protein provided by the present invention can simultaneously block the signaling pathways of HER2 and VEGFR2.
  • In vitro activity test results show that: at the molecular level, the affinity of the antibody fusion protein of the present invention to HER2 antigen is equivalent to that of monoclonal antibodies; the affinity to VEGF is equal to that of Fc-D2; at the cellular level, the antibody fusion protein of the present invention can inhibit human umbilical vein endothelium
  • the proliferation and biological activity of HUVEC cells are equivalent to that of Fc-D2; it can inhibit the proliferation of HER2-positive tumor cells, and the inhibitory effect on NCI-N87, SK-OV3, and SK-BR3 is better than HER2 mAb and HER2 mAb+ FcD2.
  • the antibody fusion protein provided by the present invention has strong stability, provides a candidate drug with better therapeutic effect for anti-tumor therapy, and has great application prospects in the treatment of tumor diseases.
  • Figure 1 Schematic diagram of the structure of antibody fusion protein HD2.
  • FIG. 2A HPLC-SEC detection pattern of antibody fusion protein HD2.
  • Figure 2B Polyacrylamide gel electrophoresis image of antibody fusion protein HD2.
  • FIG. 3A ELISA detects the binding of antibody fusion proteins HD2 and HD2-2aa to HER2.
  • Figure 3B ELISA detects the binding of antibody fusion proteins HD2 and HD2-2aa to VEGF.
  • FIG. 4 ELISA detection antibody fusion protein HD2 blocks the binding of VEGF and VEGFR2.
  • Figure 5 FACS detects the binding of antibody fusion protein HD2 to BT474 cells.
  • Figure 6A FACS detects the binding of anti-VEGFR2 antibody to NCI-N87 cells.
  • Figure 6B FACS detects the binding of anti-VEGFR2 antibody to SK-OV3 cells.
  • Figure 6C FACS detects the binding of anti-VEGFR2 antibody to SK-BR3 cells.
  • Figure 6D FACS detects the binding of anti-VEGFR2 antibody to BT474 cells.
  • Figure 6E Proliferation inhibition curve of antibody fusion protein HD2 on NCI-N87 tumor cells.
  • Figure 6F The proliferation inhibition curve of the antibody fusion protein HD2 on SK-OV3 tumor cells.
  • Figure 6G The proliferation inhibition curve of the antibody fusion protein HD2 on SK-BR3 tumor cells.
  • Figure 6H Proliferation inhibition curve of antibody fusion protein HD2 on BT474 tumor cells.
  • Figure 7 Antibody fusion protein HD2 and HD2-2aa's proliferation inhibition curve of HUVEC.
  • Figure 8A The results of the pharmacokinetic parameters of the antibody fusion protein HD2 calculated by HER2 coated ELISA in rats.
  • Figure 8B The pharmacokinetic parameters of the antibody fusion protein HD2 calculated by proteinA-coated ELISA in rats.
  • Figure 9 Antibody fusion protein HD2 inhibiting tumor proliferation curve on mouse tumor model.
  • Figure 10A DSC curve of antibody fusion protein HD2.
  • FIG. 10B Thermal stability HPLC-SEC of antibody fusion protein HD2.
  • 293E cells from NRC Biotechnology Research Institute.
  • HUVEC Human umbilical vein endothelial cells
  • Human breast cancer cell BT474 from the Cell Bank of Chinese Academy of Sciences.
  • Human ovarian cancer cell SK-OV3 from the Cell Bank of Chinese Academy of Sciences.
  • Human breast cancer cell SK-BR3 from the Cell Bank of Chinese Academy of Sciences.
  • Human gastric cancer cell line NCI-N87 purchased from the American Type Culture Collection (ATCC).
  • Protein A chip label No: 29139131-AA; lot: 10261132.
  • SD rats purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd., production license SCXK (Zhejiang) 2018-0001.
  • BALB/c nude mice purchased from Shanghai Lingchang Biological Technology Co., Ltd.
  • VEGF-A165 Annotated as VEGF in the text, self-made according to the sequence of UniProt's sequence number p15692.
  • VEGFR2 purchased from R&D, item number 357-KD.
  • Biotinylated VEGF antibody purchased from R&D, catalog number BAF293.
  • HRP-labeled mouse anti-human Fab antibody purchased from sigma, catalog number A0293.
  • Streptavidin HRP purchased from BD Biosciences, catalog number 554066.
  • Goat anti-human IgG-FITC purchased from sigma, item number F4143.
  • PBS purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117.
  • BSA purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number A60332.
  • TMB purchased from BD company, article number 555214.
  • FBS purchased from Gibco, item number 10099.
  • HBS-EP working fluid purchased from Lifescience, BR-1006-69.
  • HiTrap MabSelectSuRe column purchased from GE Company.
  • Beckman Coulter CytoFLEX flow cytometer purchased from Beckman.
  • SpectraMax i3x microplate reader purchased from Molecular Devices company.
  • SpectraMaxM5 microplate reader purchased from Molecular Devices company.
  • the HER2 monoclonal antibodies described in the embodiments of the present invention all refer to human-mouse chimeric monoclonal antibodies obtained by Sansheng Guojian Pharmaceutical in accordance with the Herceptin amino acid sequence, expressed by the CHO cell expression system, and independently developed by the cell culture production process. Antibody.
  • the anti-HER2 monoclonal antibody IgG and the D2 domain of VEGFR1 are connected in series to construct the antibody fusion protein HD2.
  • the D2 domain (SEQ ID NO: 14) of VEGFR1 and the heavy chain (SEQ ID NO: 7) of the anti-HER2 monoclonal antibody are connected through the peptide linker Linker (SEQ ID NO: 9) to obtain the heavy chain of the fusion protein ( SEQ ID NO: 10).
  • the light chain (SEQ ID NO: 11) of the HER2 monoclonal antibody remains unchanged.
  • Jin Weizhi was entrusted to optimize the codons of the nucleic acid sequence of the HD2 molecule.
  • the optimization mainly considers factors such as codon preference, GC content, mRNA secondary structure, repetitive sequence, etc., and then commissioned Jinweizhi Company to synthesize.
  • the HD2 heavy chain nucleic acid sequence is SEQ ID NO: 12
  • the light chain nucleic acid sequence is SEQ ID NO: 13. See the appendix for the sequence.
  • the structure of HD2 is shown in Figure 1.
  • the anti-HER2 monoclonal antibody IgG and the domain shown in SEQ ID NO: 14 above were truncated in series with two amino acids at the end to construct the antibody fusion protein HD2-2aa, the weight of HD2-2aa
  • the chain amino acid sequence is SEQ ID NO: 15
  • the HD2-2aa heavy chain nucleic acid sequence is SEQ ID NO: 16
  • the light chain of HD2-2aa is the same as HD2.
  • the DNA fragments of HD2 heavy chain and light chain were cloned into pTT5 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells and/or 293E cells. After the cells are cultured for 5-7 days, the culture solution is filtered by high-speed centrifugation and vacuum filtration through a microporous membrane, and then loaded onto a HiTrap MabSelect SuRe column. The protein is eluted in one step with an eluent containing 100 mM citric acid and pH 3.5 to recover the target The samples were dialyzed to pH 7.4 PBS. The purified protein was detected by HPLC, and the HPLC-SEC detection patterns of HD2 were shown in Figure 2A. The antibody molecules were in uniform state and the monomer purity reached more than 98%.
  • the antibody fusion protein HD2-2aa was expressed and purified.
  • Example 3 Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of HD2 and HD2-2aa to HER2 antigen and VEGF
  • the HER2-ECD-His protein made by Sansheng Guojian was diluted to 250ng/ml with pH7.4 PBS buffer, and then 100 ⁇ l/well was added to the ELISA plate , Incubate overnight at 4°C. The plate was washed twice with PBST the next day, PBST + 1% BSA was added to each well for blocking, blocked at 37°C for 1 h, and the plate was washed twice with PBST. Then add the antibody fusion protein HD2, HD2-2aa, and anti-HER2 monoclonal antibody diluted with PBS+1% BSA as a positive control.
  • the initial concentration is 100 nM, and 12 gradients of 3-fold dilution are gradually added.
  • VEGF was diluted to 500ng/ml with pH7.4 PBS, 100 ⁇ l/well was added to the microtiter plate, and coated overnight at 4°C. The plate was washed twice with PBST, 200 ⁇ l/well was blocked by adding PBS+2% BSA, placed at 37°C for 1 hour and then washed with PBST once for use. Then, the antibody fusion proteins HD2, HD2-2aa, and Fc-D2, which were gradually diluted with PBS + 1% BSA, were added as a positive control, the initial concentration was 200 nM, and 12 gradients were gradually diluted 3 times.
  • Enzyme-linked immunosorbent assay measures HD2 blocking the binding of VEGF and VEGFR2
  • VEGFR2 Dilute VEGFR2 with pH 7.4 PBS to 400 ng/mL, add 100 ⁇ L/well to the microtiter plate, and coat overnight at 4°C. The plate was washed twice with PBST, 200 ⁇ l/well was blocked by adding PBS+2% BSA. After standing at 37°C for 1 hour, the plate was washed twice with PBST for use. Dilute VEGF to 4nM with PBS containing 1% BSA, and then use 4nM with 1% BSA. Dilute the sample to be tested with %BSA in PBS with an initial concentration of 200nM, and 12 gradients of three-fold dilution step by step.
  • the human breast cancer cell BT474 with high expression of HER2 on the cell surface was used as the target cell.
  • the cells were washed three times with PBS containing 0.5% BSA, centrifuged at 300g for 5 minutes each time, the supernatant was discarded, and the cells were resuspended in 0.5% BSA in PBS.
  • the obtained data was fitted and analyzed by GraphPad Prism7 software.
  • the experimental results are shown in Figure 5.
  • the EC 50 of the antibody fusion protein HD2 and the positive control HER2 monoclonal antibody bound to BT474 cells were 1.238nM and 1.054nM, respectively, and the HD2 and positive control HER2 monoclonal antibodies It has the same affinity with BT474.
  • Example 6 In vitro proliferation inhibition and synergistic effect of HD2 on HER2-positive tumor cells
  • NCI-N87, SK-OV3, SK-BR3, BT474 tumor cells all have HER2 antigen expression on the surface, and the addition of anti-HER2 antibody can inhibit their proliferation.
  • HD2 synergistic mechanism Some tumor cell culture supernatants secrete VEGF, which may play a role in tumor cell proliferation. And some tumor cells have the receptor VEGFR2 expression on the surface. HD2 can compete with VEGFR2 to bind to VEGF, and tumor cell proliferation may be inhibited to a certain extent.
  • ELISA to detect tumor cell VEGF secretion Take the adherent cultured NCI-N87, BT474 cell culture supernatant for three days for detection.
  • the ELISA procedure is: HER2-His antigen 50ng/well is coated with an enzyme-labeled plate, incubated overnight at 4°C, and washed with PBST twice. Dilute HD2 with PBS plus 1% BSA, add it to an enzyme-labeled plate, 200ng/well, incubate at 37°C for 1 hour, and then wash the plate twice with PBST.
  • FACS detection of tumor cell surface receptor VEGFR2 Adherent cultured logarithmic growth phase cells NCI-N87, SK-OV3, SK-BR3, BT474 were trypsinized, centrifuged at 300g for 5 minutes to discard the supernatant, and resuspended in PBS. 10 6 cells / mL, 100 ⁇ L / hole plated in 96-well cell culture plate. Add anti-VEGFR2 antibody (made by our company), start at 600nM, and dilute three times. After incubating for 1 hour at 4°C, the cells were washed twice with PBS.
  • NCI-N87, SK-OV3, SK-BR3, BT474 were trypsinized, resuspended and counted, and the cell density was adjusted with medium containing 1% FBS , Pave a 96-well cell culture plate, 100 ⁇ L/well.
  • NCI-N87 is 10,000 pieces/hole
  • BT474, SK-OV3, and SK-BR3 are respectively 5000 pieces/hole.
  • HD2 had better inhibitory effects on the proliferation of SK-BR3, SK-OV3, and NCI-N87 than HER2 mAb and HER2 mAb + FcD2, while FcD2 had no inhibitory effect on tumor cells.
  • the HD2 fusion protein has a synergistic effect. While HD2 binds to HER2-positive tumor cells, the VEGFR1-D2 domain of HD2 competes with tumor cell surface receptor VEGFR2 to bind to VEGF, and tumor cell proliferation may be inhibited to a certain extent. . For BT474 cells where VEGFR2 expression is almost undetectable, HD2 has a stronger inhibitory effect on tumor cells than HER2 monoclonal antibody.
  • VEGF can stimulate HUVEC (Human umbilical vein endothelial cell) proliferation.
  • HD2 inhibits the proliferation of HUVEC cells in vitro by binding to VEGF.
  • ECM basal medium containing 0.5% FBS to dilute VEGF to 60ng/mL, and use this solution to prepare samples to be tested, HD2, HD2-2aa, Fc-D2, negative control IgG1 starting concentration 400nM, step by step three Dilute by 10 gradients, add to a 96-well plate with HUVEC in the middle, culture in a 37°C, 5% CO 2 incubator for three days, aspirate the supernatant, add 10 ⁇ L/well of CCK-8 to develop color, and continue to culture for 4-8 After hours, the microplate reader uses 650nm as the reference wavelength to measure the OD value at 450nm. The data obtained was analyzed by GraphPad Prism7 software. The experimental results are shown in Figure 7.
  • the IC 50 of HD2, HD2-2aa and the positive control Fc-D2 are 0.42nM, 0.51nM and 0.61nM, respectively.
  • the inhibition rates of the three are equivalent, and the negative control IgG1
  • the antibody has no inhibitory effect on the proliferation of HUVEC.
  • the proteinA capture method was used to determine the kinetic parameters of the binding and dissociation of HD2 and antigen HER2-ECD-his.
  • HD2 at a concentration of 5 ⁇ g/ml was bound to the Protein A chip, and the antigen HER2-ECD-his was diluted with 1 ⁇ HBS working solution , Set up 6 concentration gradients to bind to the antibody and dissociate in the HBS working solution.
  • the proteinA capture method was used to determine the kinetic parameters of the binding and dissociation of HD2 and antigen VEGF-A165.
  • HD2 at a concentration of 5 ⁇ g/ml was bound to the Protein A chip, and the antigen VEGF was diluted with 1 ⁇ HBS working solution, with 6 concentration gradients. Combine with antibody and dissociate in HBS working solution.
  • KD is the affinity constant
  • kon is the association rate constant
  • kdis is the dissociation rate constant
  • the serum drug concentration of HD2 is detected by the following methods:
  • HER2-His coated ELISA plate 50ng/well, coated overnight at 4°C, washed the plate twice with PBST the next day, and then blocked with PBS+2% BSA at 37°C for 2 hours.
  • PBS+2% BSA PBS+2% BSA at 37°C for 2 hours.
  • Dilute the serum sample 2000 times add the above sample to the blocked ELISA plate, incubate at 37°C for one hour, then wash the plate twice with PBST, add HRP-labeled mouse anti-human Fab antibody, diluted 1:3000, 100 ⁇ L/well. Incubate at 37°C for 40 min.
  • ProteinA coats the ELISA plate, detects the Fab fragment of the antibody, the proteinA coating amount is 100ng/well, overnight at 4°C, the plate is washed twice with PBST the next day, and then blocked with PBS+2% BSA at 37°C for 2 hours. The plate was washed twice with PBST, the HD2 standard substance was started from 1000ng/mL, and 12 gradients were gradually diluted twice.
  • the rat serum samples were diluted 2000 times, the above two groups of samples were added to the blocked ELISA plate, incubated for 1 hour, washed with PBST twice, then added HRP-labeled mouse anti-human Fab antibody, placed at 37°C for 30 minutes, washed with PBST 3 times After that, pat dry the remaining droplets on absorbent paper as much as possible, add 100 ⁇ l of TMB to each well, and place 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. Read the OD value at 450nm on the scaler.
  • Rat HL_Lambda_z(hr) Cmax(ug/mL) 1 207.19207 86.5 2 220.68823 73.5 3 148.78511 68.5 4 157.4815 68.5 average 183h
  • Rat HL_Lambda_z(hr) Cmax(ug/mL) 1 159.76559 74 2 159.58479 73 3 261.44609 59 4 230.41973 53 average 203h
  • Example 10 The anti-tumor effect of HD2 on the NCI-N87 xenograft tumor model
  • the human gastric cancer cell line NCI-N87 expresses HER2 antigen on the cell surface, and HER2 antibody binds to it, which can block cell signaling pathways and inhibit tumor proliferation.
  • 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 cell suspension in nude mice under sterile conditions Subcutaneously on the back, use a vernier caliper to measure the length and width of the transplanted tumor to calculate the tumor volume. After the tumor grows to 100-200 mm 3, the animals are randomly divided into groups.
  • the dosage of HD2 of the sample to be tested is divided into two groups, 17mg/kg, 1.7mg/kg, and the dose of the positive control drug HER2 monoclonal antibody is 15mg/kg, which is equimolar with HD2.
  • the control group was given the same volume of PBS, the administration method was intraperitoneal administration, the administration volume was 0.2mL/mouse (20g), the administration was administered twice a week for three consecutive weeks, and the tumor volume was measured twice a week. .
  • the experimental results are shown in Figure 9.
  • the antibody fusion protein HD2 exhibits anti-tumor activity in vivo, and there is a dose-dependent relationship.
  • the anti-tumor effect is better than that of HER2 monoclonal antibody, indicating that the D2 domain of the second extramembrane region of VEGFR1 of HD2 exerts a synergistic anti-tumor effect.
  • the experiment uses MicroCal VP-Capillary DSC, filters the sample and its buffer with a 0.22um filter membrane, respectively take 400 ⁇ l of the sample and its matching buffer and place it in a 96-well plate.
  • the sample is scanned at 25°C-100°C, scanning rate At 120°C per hour, HD2 is stored in pH 7.4 PBS.
  • the Tm value of HD2 detected by DSC is shown in Table 4, and the map is shown in Fig. 10A. It can be seen that the antibody fusion protein HD2 is relatively stable, which is also verified by the subsequent stability experiment results at 37°C.
  • the antibody fusion protein provided by the present invention has an affinity for antigen and target cells equivalent to that of monoclonal antibodies; at the same time, it has good biological activity and can inhibit the proliferation of HER2-positive tumor cells.
  • SK-OV3 tumor cells showed better inhibitory effects than HER2 monoclonal antibody and HER2 monoclonal antibody + FcD2; at the same time, it can inhibit the proliferation of human umbilical vein endothelial cells HUVEC.
  • the detection results on the mouse tumor model show that the antibody fusion protein of the present invention at the same molar concentration has a better tumor proliferation inhibitory effect than the HER2 monoclonal antibody; and the antibody fusion protein has strong stability and broad application prospects.

Abstract

一种抗体融合蛋白及其制法和在抗肿瘤中的应用，其包含抗HER2单克隆抗体IgG和VEGFR1的D2结构域，VEGFR1的D2结构域通过肽接头L连接至IgG重链的C末端。该抗体融合蛋白能同时阻断HER2和VEGFR2信号通路，具有优于单克隆抗体的抑制肿瘤增殖的作用，为抗肿瘤治疗提供了一种治疗效果更佳的候选药物，在肿瘤疾病的治疗上具有广阔的应用前景。

Description

一种抗体融合蛋白及其制法和在抗肿瘤中的应用 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单克隆抗体IgG和VEGFR1的D2结构域组成的抗体融合蛋白及其制备方法和用途。
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。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被FDA批准上市，并用于HER2过表达的乳腺癌和胃癌的治疗。
肿瘤的生长有两个阶段，从无血管的缓慢生长期到有血管的快速增殖期。如果肿瘤内部没有血管的生成，则原发肿瘤生长缓慢，转移无法实现。因此抑制肿瘤血管生成被认为是当前具有前途的肿瘤治疗方法之一。血管内皮生长因子(VEGFs)家族中，VEGF-A165(以下简称VEGF)是最丰富活跃的亚型。VEGF通过与II型受体VEGFR2结合，激活信号通路发生一系列级联反应，促进新生血管形成并维持其完整性。但I型受体VEGFR1与VEGF结合的能力远大于VEGFR2，发生作用部位主要是VEGFR1的胞外区D2结构域。VEGFR1-D2通过竞争结合VEGF，阻断VEGFR2与VEGF结合，从而阻断信号通路，抑制内皮细胞增殖与血管生成，从而抑制肿瘤的快速增殖与转移。
本发明介绍了一种能同时阻断HER2和VEGFR2信号通路的抗体融合蛋白，该抗体融合蛋白一方面可以结合肿瘤细胞表面HER2抗原，抑制肿瘤增殖；另一方面，可以竞争结合VEGF，抑制内皮细胞增殖与血管生成。这个作用机制发生在肿瘤微环境中，可以有效地抑制肿瘤内部血管的生成，从而抑制肿瘤生长。该抗体融合蛋白具有优于HER2单抗及HER2单抗+FcD2的抑制肿瘤增殖的作用，在肿瘤疾病的治疗上具有广阔的应用前景。
发明内容
本发明的目的在于提供一种能同时阻断HER2和VEGFR2信号通路的抗体融合蛋白，提供编码所述抗体融合蛋白的核苷酸分子；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；提供所述抗体融合蛋白的制备方法；提供包含所述抗体融合蛋白的药物组合物；提供所述抗体融合蛋白在制
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：
本发明提供了一种能同时阻断HER2和VEGFR2信号通路的抗体融合蛋白，其包含抗 HER2单克隆抗体IgG和VEGFR1的D2结构域，VEGFR1的D2结构域通过肽接头L连接至IgG重链的C末端。
本发明“抗体融合蛋白”是重组产生的抗原结合分子，其中抗体或抗体片段连接至另一种蛋白质或肽。其包含抗HER2单克隆抗体IgG和VEGFR1的D2结构域，VEGFR1的D2结构域通过肽接头L连接至IgG重链的C末端。
本发明“抗HER2单克隆抗体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IgG的重链包含互补决定区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IgG的轻链包含互补决定区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。
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三个结构域。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，所述IgG的重链可变区的氨基酸序列如SEQ ID NO：7所示，轻链可变区的氨基酸序列如SEQ ID NO：8所示。
作为优选的方案，所述肽接头L的氨基酸序列如SEQ ID NO：9所示。
作为优选的方案，所述抗体融合蛋白的重链氨基酸序列如SEQ ID NO：10所示，其轻链氨基酸序列如SEQ ID NO：11所示。
作为优选的方案，所述抗体融合蛋白的重链氨基酸序列如SEQ ID NO：15所述，其轻链氨基酸序列如SEQ ID NO：11所示。
在构建本发明的抗体融合蛋白时，与该抗体融合蛋白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HER2和VEGF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任一所述的抗体融合蛋白。
作为优选的方案，所述核苷酸分子编码抗体融合蛋白的重链的核苷酸序列如SEQ ID NO：12所示，编码其轻链的核苷酸序列如SEQ ID NO：13所示。
作为优选的方案，所述核苷酸分子编码抗体融合蛋白的重链的核苷酸序列如SEQ ID NO：16所示，编码其轻链的核苷酸序列如SEQ ID NO：13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，获得编码上述抗体融合蛋白的核苷酸分子，或者通过人工全序列合成的方法得到编码上述抗体融合蛋白的核苷酸分子。
本领域技术人员知晓，编码上述抗体融合蛋白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抗体融合蛋白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任一所述的核苷酸分子。
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，pcDNA3.4(+)，pDHFR或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，优选自CHO细胞和293E细胞。
本发明另一方面提供了上述抗体融合蛋白的制备方法，所述制备方法包括以下步骤：
a)在表达条件下，培养如上述任一所述的宿主细胞，从而表达能能同时阻断HER2和VEGFR2信号通路的抗体融合蛋白；
b)分离并纯化步骤a)所述的抗体融合蛋白。
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。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体融合蛋白进行分离纯化，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体融合蛋白。本发明的发明人对所得抗体融合蛋白进行了检测实验，实验结果表明该抗体融合蛋白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的抗体融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明提供的抗体融合蛋白，可以和药学上可以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公开的抗体融合蛋白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。所述抗体融合蛋白制剂可为制药领域常用的混悬、水针、冻干等制剂。
对于本发明公开的抗体融合蛋白的水针或冻干制剂，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，其加入量应使抗体融合蛋白的颗粒化趋势最小。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抗体融合蛋白、或上述药物组合物在制备药物中的应用，所述药物用于治疗肿瘤。
本发明所称的用于治疗肿瘤的药物，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：乳腺癌、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、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。
本发明中抗体融合蛋白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。
本发明公开的抗体融合蛋白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。本发明公开的抗体融合蛋白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。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
其一、本发明提供的抗体融合蛋白能同时阻断HER2和VEGFR2的信号通路。体外活性检测结果表明：分子水平上，本发明抗体融合蛋白对HER2抗原亲和力与单克隆抗体相当；对VEGF的亲和力与Fc-D2相当；细胞水平上，本发明抗体融合蛋白能抑制人脐静脉内皮细胞HUVEC的增殖，生物学活性均与Fc-D2相当；能抑制HER2阳性肿瘤细胞的增殖，其中对NCI-N87，SK-OV3，SK-BR3的抑制效果优于HER2单抗以及HER2单抗+FcD2。
其二、动物实验表明，本发明抗体融合蛋白的抑瘤效果，明显优于同等摩尔浓度的抗HER2单抗，说明本发明抗体融合蛋白的VEGFR1-D2结构域发挥了协同抗肿瘤作用。
其三、本发明提供的抗体融合蛋白稳定性强，为抗肿瘤治疗提供了一种治疗效果更佳的候选药物，在肿瘤疾病的治疗上具有巨大的应用前景。
附图说明
图1：抗体融合蛋白HD2结构示意图。
图2A：抗体融合蛋白HD2的HPLC-SEC检测图谱。
图2B：抗体融合蛋白HD2的聚丙烯酰胺凝胶电泳图。
图3A：ELISA检测抗体融合蛋白HD2、HD2-2aa与HER2的结合。
图3B：ELISA检测抗体融合蛋白HD2、HD2-2aa与VEGF的结合。
图4：ELISA检测抗体融合蛋白HD2阻断VEGF与VEGFR2的结合。
图5：FACS检测抗体融合蛋白HD2对BT474细胞的结合。
图6A：FACS检测抗VEGFR2抗体对NCI-N87细胞的结合。
图6B：FACS检测抗VEGFR2抗体对SK-OV3细胞的结合。
图6C：FACS检测抗VEGFR2抗体对SK-BR3细胞的结合。
图6D：FACS检测抗VEGFR2抗体对BT474细胞的结合。
图6E：抗体融合蛋白HD2对NCI-N87肿瘤细胞的增殖抑制曲线。
图6F：抗体融合蛋白HD2对SK-OV3肿瘤细胞的增殖抑制曲线。
图6G：抗体融合蛋白HD2对SK-BR3肿瘤细胞的增殖抑制曲线。
图6H：抗体融合蛋白HD2对BT474肿瘤细胞的增殖抑制曲线。
图7：抗体融合蛋白HD2、HD2-2aa对HUVEC的增殖抑制曲线。
图8A：HER2包被ELISA计算所得的抗体融合蛋白HD2在大鼠体内药代动力学参数结果。
图8B：proteinA包被ELISA计算所得的抗体融合蛋白HD2在大鼠体内药代动力学参数结果。
图9：小鼠肿瘤模型上抗体融合蛋白HD2抑制肿瘤增殖的曲线。
图10A：抗体融合蛋白HD2的DSC曲线。
图10B：抗体融合蛋白HD2的热稳定性HPLC-SEC。
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,2 nd 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：
293E细胞：来自NRC biotechnology Research Institute。
人脐静脉内皮细胞HUVEC：购自Sciencell。
人乳腺癌细胞BT474：来自中科院细胞库。
人卵巢癌细胞SK-OV3：来自中科院细胞库。
人乳腺癌细胞SK-BR3：来自中科院细胞库。
人胃癌细胞株NCI-N87：购自美国典型培养物保藏中心(ATCC)。
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。
BALB/c裸小鼠：购自上海灵畅生物科技有限公司。
实验试剂：
VEGF-A165：正文中标注为VEGF，根据UniProt的序列号p15692的序列自制。
VEGFR2：购自R&D，货号357-KD。
生物素化的VEGF抗体：购自R&D，货号BAF293。
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。
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。
FBS：购自Gibco，货号10099。
HBS-EP工作液：购自Life science，BR-1006-69。
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。
本发明实施例中所述的HER2单克隆抗体均是指三生国健药业按照Herceptin的氨基酸序列、用CHO细胞表达系统进行表达、自主研发的细胞培养生产工艺得到的人鼠嵌合单克隆抗体。
实施例1.抗体融合蛋白HD2和HD2-2aa的分子构建
本发明采用了抗HER2单克隆抗体IgG和VEGFR1的D2结构域串联的方式，构建了抗体融合蛋白HD2。将VEGFR1的D2结构域(SEQ ID NO：14)和抗HER2单克隆抗体的重链(SEQ ID NO：7)通过肽接头Linker(SEQ ID NO：9)连接起来，得到融合蛋白的重链(SEQ ID NO：10)。HER2单抗的轻链(SEQ ID NO：11)则保持不变。为了提高该分子在293E细胞中的表达效率，委托金唯智公司对HD2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。拼接后HD2重链核酸序列为SEQ ID NO：12，轻链核酸序列为SEQ ID NO：13。序列见附录。HD2结构如图1所示。
采用上述相同的方法，将抗HER2单克隆抗体IgG和上述SEQ ID NO：14所示结构域末端截掉了两个氨基酸的结构域串联，构建了抗体融合蛋白HD2-2aa，HD2-2aa的重链氨基 酸序列为SEQ ID NO：15，HD2-2aa重链核酸序列为SEQ ID NO：16，HD2-2aa的轻链与HD2相同。
实施例2.抗体融合蛋白HD2和HD2-2aa的表达与纯化
将HD2的重链和轻链的DNA片段分别克隆到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，HD2的HPLC-SEC检测图谱分别如图2A所示，抗体分子状态均一，单体纯度达到98％以上。
取纯化后的抗体融合蛋白HD2分别加入非还原电泳缓冲液，SDS-聚丙烯酰胺凝胶电泳检测，取纯化后的抗体融合蛋白HD2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2B所示，抗体融合蛋白HD2理论分子量为169KD。
采用上述相同的方法，表达与纯化抗体融合蛋白HD2-2aa。
实施例3.酶联免疫吸附法(ELISA)测定HD2和HD2-2aa对HER2抗原以及VEGF的亲和力
为了检测HD2和HD2-2aa抗体融合蛋白与HER2抗原的亲和力，用pH7.4的PBS缓冲液将三生国健自制HER2-ECD-His蛋白稀释至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融合蛋白HD2、HD2-2aa,抗HER2单克隆抗体作为阳性对照，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50μl的2MH 2SO 4终止底物反应，酶标仪450nm处读取OD值。GraphPad Prism7进行数据分析，作图并计算EC 50，实验结果如图3A所示，HD2，HD2-2aa和阳性对照HER2单抗，与HER2结合的EC 50分别为0.15nM，0.16nM，0.11nM,三者亲和力相当。
为了检测HD2和HD2-2aa与VEGF的结合能力，将VEGF用pH7.4的PBS稀释至500ng/ml，100μl/孔加入酶标板，4℃包被过夜。PBST洗板2次，200μl/孔加入PBS+2％BSA进行封闭，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融合蛋白HD2、HD2-2aa，Fc-D2作为阳性对照，起始浓度为200nM，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Fc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50μl的2MH 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。GraphPad Prism7进行数据分析，作图并计算EC 50。实验结果如图3B所示，抗体融合蛋白HD2，HD2-2aa和阳性对照Fc-D2与VEGF结合的EC 50分 别为0.22nM，0.22nM和0.19nM，三者亲和力相当。
实施例4.酶联免疫吸附法(ELISA)测定HD2阻断VEGF与VEGFR2结合
由于VEGF通过结合到VEGFR2，是调节血管内皮细胞增殖迁移的关键步骤，而VEGF与VEGFR1结合能力强于VEGFR2。因此本实验检测了HD2阻断VEGF与VEGFR2结合的能力。
将VEGFR2用pH7.4的PBS稀释至400ng/mL，100μL/孔加入酶标板，4℃包被过夜。PBST洗板2次，200μl/孔加入PBS+2％BSA进行封闭，37℃放置1小时后PBST洗板2次待用，用含1％BSA的PBS稀释VEGF至4nM，再用含4nM，1％BSA的PBS稀释待检样品，起始浓度200nM，逐级三倍稀释12个梯度。100μl/孔加入封闭后的酶标板，37℃放置1小时，PBST洗板2次，用PBS+1％BSA稀释生物素化的VEGF抗体至0.2μg/mL，100μl/孔加入酶标板，37℃放置1小时后PBST洗板2次。加入HRP标记的链霉亲和素(SA)，37℃孵育30min，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50μl的2MH 2SO 4终止底物反应，酶标仪450nm处读取OD值。GraphPad Prism7进行数据分析，作图并计算IC 50，实验结果如图4所示，抗体融合蛋白HD2和阳性对照Fc-D2阻断VEGF与VEGFR2结合的IC 50，分别为1.587nM和1.466nM，两者阻断能力相当。
实施例5.检测HD2对靶细胞BT474的结合力
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，将抗体融合蛋白HD2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1:1000稀释的羊抗人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HD2对该细胞的结合亲和力。所得数据通过GraphPad Prism7软件拟合分析，实验结果如图5所示，抗体融合蛋白HD2和阳性对照HER2单抗与BT474细胞结合的EC 50分别为1.238nM和1.054nM，HD2和阳性对照HER2单抗与BT474亲和力相当。
实施例6.HD2对HER2阳性肿瘤细胞的体外增殖抑制以及协同作用
NCI-N87，SK-OV3，SK-BR3，BT474肿瘤细胞表面均有HER2抗原表达，加入抗HER2抗体可以抑制其增殖。
HD2协同作用机制：有些肿瘤细胞培养上清中分泌有VEGF，可能对肿瘤细胞的增殖起一定作用。并且有些肿瘤细胞表面有受体VEGFR2表达。HD2可以与VEGFR2竞争结合VEGF，肿瘤细胞的增殖可能由此受到一定程度的抑制作用。
ELISA检测肿瘤细胞VEGF分泌：取贴壁培养的NCI-N87，BT474细胞培养三天的上清，进行检测。ELISA步骤为：HER2-His抗原50ng/孔包酶标板，4℃孵育过夜后PBST洗板2次。用PBS加1％BSA稀释HD2，加入酶标板，200ng/孔，37℃孵育1小时后PBST洗板2次。加待检样品，空白培养基，NCI-N87培养上清，BT474培养上清，VEGF-A165标准品(200nM起始，逐级三倍稀释11个梯度)，37℃孵育1小时后PBST洗板2次。加Bio-anti-hVEGF，按1:500稀释，37℃孵育1小时后PBST洗板2次。加HRP标记的链霉亲和素，按1:5000稀释，37℃孵育1小时后PBST洗板3次，在吸水纸上尽量拍干残留液滴。每孔加入100μl TMB，室温(20±5℃)避光放置5分钟，每孔加入50μl的2MH 2SO 4终止底物反应，酶标仪450nm处读取OD值。根据VEGF-A165标准曲线，标定NCI-N87，BT474细胞培养上清中，VEGF分泌约为1-1.5ng/mL。
FACS检测肿瘤细胞表面受体VEGFR2:将贴壁培养的对数生长期细胞NCI-N87，SK-OV3，SK-BR3，BT474用胰酶消化，300g离心5分钟弃上清，用PBS重悬为10 6cells/mL，100μL/孔铺96孔细胞培养板。加入抗VEGFR2抗体(本公司自制)，600nM起始，三倍稀释。4℃孵育1小时后PBS洗涤细胞两次。将细胞与1:1000稀释的羊抗人IgG-FITC于4℃孵育30分钟后，PBS洗涤细胞两次，最后将细胞重悬在200μl PBS中，通过Beckman Coulter CytoFLEX流式细胞仪测定抗VEGFR2抗体对该细胞的结合亲和力。所得数据通过GraphPad Prism7软件拟合分析，实验结果如图6A，6B，6C，6D所示。数据显示NCI-N87，SK-OV3，SK-BR3这三株细胞表面有VEGFR2表达，其表达量高低为：NCI-N87>SK-OV3>SK-BR3。BT474细胞几乎检测不到VEGFR2表达。
细胞增殖抑制检测：将贴壁培养的对数生长期细胞NCI-N87，SK-OV3，SK-BR3，BT474用胰酶消化，重悬后进行计数，用含1％FBS的培养基调整细胞密度，铺96孔细胞培养板，100μL/孔。其中NCI-N87为10000个/孔，BT474，SK-OV3，SK-BR3分别为5000个/孔。周圈加入200μL/孔的培养基或PBS封边，放置37℃，5％CO 2培养箱中培养过夜。次日加入待检抗体。将HD2，HER2单抗，HER2单抗+FcD2三组样品，用含1％FBS的培养基稀释配制为300nM溶液，再逐级3倍稀释，共计10个梯度。另设一板1500nM起始浓度的FcD2的抑制实验作为对照。将稀释好的样品，100μL/孔加入到对应的96孔板细胞中，置于37℃、5％CO 2的培养箱内继续培养6天。孵育6天的细胞培养板，加入10μL/孔的CCK-8显色，放入CO 2培养箱中继续孵育2～5h，酶标仪以650nm为参比波长，450nm下测定OD值。所得数据通过GraphPad Prism7软件分析，实验结果如图6E，6F，6G，6H所示。
结果显示，HD2对SK-BR3，SK-OV3，NCI-N87的增殖抑制作用均优于HER2单抗及HER2单抗+FcD2，而FcD2对肿瘤细胞没有抑制作用。这说明HD2融合蛋白发挥了协同作用，HD2与HER2阳性肿瘤细胞结合的同时，HD2的VEGFR1-D2结构域与肿瘤细胞表面受体VEGFR2竞争结合VEGF，肿瘤细胞的增殖可能由此受到一定程度的抑制。对于几乎检测不到VEGFR2表达的BT474细胞，则检测不到HD2比HER2单抗对肿瘤细胞更强的抑制效果。
实施例7.HD2和HD2-2aa对细胞HUVEC体外增殖抑制作用
VEGF可以刺激HUVEC(Human umbilical vein endothelial cell)增殖。HD2通过结合VEGF，抑制细胞HUVEC体外增殖。
将培养的HUVEC用胰酶消化，重悬后进行细胞计数，细胞活率在95％以上，用无菌PBS洗一次，再用含0.5％FBS的ECM基础培养基重悬至3×10 4细胞/mL，100μL/孔加入96孔细胞培养板的中间60孔，其余用培养基补齐，放置37℃，5％CO 2培养箱培养过夜。次日，用含0.5％FBS的ECM基础培养基，稀释VEGF至60ng/mL，用此溶液配制待检样品，HD2，HD2-2aa，Fc-D2，阴性对照IgG1起始浓度400nM，逐级三倍稀释10个梯度，加入中间铺有HUVEC的96孔板，37℃，5％CO 2培养箱培养三天后，吸去上清，加入10μL/孔的CCK-8显色，继续培养4-8小时，酶标仪以650nm为参比波长，450nm下测定OD值。所得数据通过GraphPad Prism7软件分析，实验结果如图7所示，HD2，HD2-2aa和阳性对照Fc-D2的IC 50分别为0.42nM,0.51nM和0.61nM，三者抑制率相当，阴性对照IgG1抗体对HUVEC的增殖没有抑制作用。
实施例8.Octet测定HD2对抗原的亲和解离常数KD
使用proteinA捕获法测定HD2和抗原HER2-ECD-his结合解离的动力学参数，将浓度为5μg/ml的HD2结合在Protein A芯片上，将抗原HER2-ECD-his用1×HBS工作液稀释，设6个浓度梯度与抗体结合，于HBS工作液中解离。
使用proteinA捕获法测定HD2和抗原VEGF-A165结合解离的动力学参数，将浓度为5μg/ml的HD2结合在Protein A芯片上，将抗原VEGF用1×HBS工作液稀释，设6个浓度梯度与抗体结合，于HBS工作液中解离。
HD2与两组抗原的亲和解离常数见下表，结果表明，HD2与抗原HER2和VEGF有良好的亲和力。
表1
抗原 样品 kon(1/Ms) kdis(1/s) KD(M)
VEGF-A165 HD2 2.26E+06 9.35E-05 4.13E-11
HER2-ECD-His HD2 3.72E+05 2.05E-04 5.51E-10
KD为亲和力常数；kon为结合速率常数；kdis为解离速率常数。
实施例9.抗体融合蛋白HD2的药代动力学研究
取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的抗体融合蛋白HD2。分别在给药后的间隔时间眼眶取血，血液自然凝固后8000rpm/min离心取血清。HD2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，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1000ng/mL的HD2标准品，逐级两倍稀释12个梯度。将血清样品稀释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，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)proteinA包被ELISA板，检测抗体Fab段，proteinA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，HD2标准品从1000ng/mL起始，逐级两倍稀释12个梯度。大鼠血清样品稀释2000倍，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，药代动力学参数见下表，实验结果如图8A，8B所示，用两种ELISA方法检测的大鼠体内半衰期分别为183h和203h，两种ELISA方法计算得到的半衰期结果相差不大，说明数据可靠。
用HER2检测，计算HD2的半衰期见下表。
表2
大鼠 HL_Lambda_z(hr) Cmax(ug/mL)
1 207.19207 86.5
2 220.68823 73.5
3 148.78511 68.5
4 157.4815 68.5
平均 183h  
用proteinA检测，计算HD2的半衰期见下表：
表3
大鼠 HL_Lambda_z(hr) Cmax(ug/mL)
1 159.76559 74
2 159.58479 73
3 261.44609 59
4 230.41973 53
平均 203h  
实施例10.HD2在NCI-N87移植瘤模型上的抗肿瘤作用
人胃癌细胞株NCI-N87，细胞表面表达HER2抗原，HER2抗体与其结合，可以阻断细 胞信号通路，抑制肿瘤增殖。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。待检样品HD2的用药剂量分为两组，17mg/kg，1.7mg/kg，阳性对照药HER2单抗单药的剂量为15mg/kg，与HD2等摩尔量。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)，每周给药两次，连续给药三周，每周测量2次移植瘤体积。实验结果如图9所示，在NCI-N87裸小鼠移植瘤模型上，抗体融合蛋白HD2体现体内抗肿瘤活性，且存在剂量依赖关系，HD2融合蛋白与相同摩尔浓度的HER2单抗相比，抑瘤效果优于HER2单抗，说明HD2的VEGFR1的第二膜外区D2结构域发挥了协同抗肿瘤作用。
实施例11.HD2的热稳定性研究
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℃，HD2保存在pH7.4的PBS中。DSC检测HD2的Tm值见表4，图谱见图10A，由此可知，抗体融合蛋白HD2较稳定，后续的37℃稳定性实验结果也验证了这一点。
表4：HD2的DSC数值
样品号 Tm Onset Tm1 Tm2
HD2 67.2 72.2 82.2
Fc-D2 63.77 71.3 82.1
37℃稳定性实验：将HD2融合蛋白透析到Ph7.4的PBS缓冲液中，调整浓度为2mg/mL，放于37℃温箱中，间隔时间取样，HPLC-SEC检测纯度。结果在14天时，HPLC-SEC纯度几乎没有变化，为97％，28天的HPLC-SEC检测结果，HPLC-SEC纯度稍有下降，为95％，说明HD2融合蛋白较稳定。HPLC-SEC图谱见图10B。
采用上述相同的方法，测定抗体融合蛋白HD2-2aa阻断VEGF与VEGFR2结合的能力、对靶细胞的结合力、对肿瘤细胞的体外增殖抑制以及协同作用及热稳定性，实验结论与上述相同。
由上述实验可知，本发明提供的抗体融合蛋白对抗原和靶细胞的亲和力与单克隆抗体相当；同时具有良好的生物学活性，能抑制HER2阳性肿瘤细胞的增殖，在NCI-N87，SK-BR3，SK-OV3肿瘤细胞上表现出优于HER2单抗及HER2单抗+FcD2的抑制作用；同时能抑制人脐静脉内皮细胞HUVEC的增殖。小鼠肿瘤模型上的检测结果表明，相同摩尔浓度的本发明的抗体融合蛋白具有优于HER2单抗的抑制肿瘤增殖的效应；并且该抗体融合蛋白稳定性强，具有广阔应用前景。

Claims (16)

  1. 一种能同时阻断HER2和VEGFR信号通路的抗体融合蛋白，其特征在于，其包含抗HER2单克隆抗体IgG和VEGFR1的D2结构域，VEGFR1的D2结构域通过肽接头L连接至IgG重链的C末端。
  2. 根据权利要求1所述的抗体融合蛋白，其特征在于，所述IgG的重链包含互补决定区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IgG的轻链包含互补决定区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。
  3. 根据权利要求1所述的抗体融合蛋白，其特征在于，所述IgG的重链可变区的氨基酸序列如SEQ ID NO：7所示，轻链可变区的氨基酸序列如SEQ ID NO：8所示。
  4. 根据权利要求1所述的抗体融合蛋白，其特征在于，所述肽接头L的氨基酸序列如SEQ ID NO：9所示。
  5. 根据权利要求1所述的抗体融合蛋白，其特征在于，所述抗体融合蛋白的重链氨基酸序列如SEQ ID NO：10所述，其轻链氨基酸序列如SEQ ID NO：11所示。
  6. 根据权利要求1所述的抗体融合蛋白，其特征在于，所述抗体融合蛋白的重链氨基酸序列如SEQ ID NO：15所述，其轻链氨基酸序列如SEQ ID NO：11所示。
  7. 一种核苷酸分子，所述核苷酸分子编码如权利要求1-6任一所述的抗体融合蛋白。
  8. 如权利要求7所述的核苷酸分子，其特征在于，所述核苷酸分子编码抗体融合蛋白的重链的核苷酸序列如SEQ ID NO：12所示，编码其轻链的核苷酸序列如SEQ ID NO：13所示。
  9. 如权利要求7所述的核苷酸分子，其特征在于，所述核苷酸分子编码抗体融合蛋白的重链的核苷酸序列如SEQ ID NO：16所示，编码其轻链的核苷酸序列如SEQ ID NO：13所示。
  10. 一种表达载体，其特征在于，所述表达载体含有权利要求7-9任一所述的核苷酸分子。
  11. 根据权利要求10所述的表达载体，其特征在于，所述表达载体选自pDR1，pcDNA3.4(+)，pDHFR或pTT5。
  12. 一种宿主细胞，所述宿主细胞含有权利要求10所述的表达载体。
  13. 根据权利要求12所述的宿主细胞，其特征在于，所述宿主细胞是真核细胞，优选自CHO细胞和293E细胞。
  14. 一种如权利要求1-6任一项所述的抗体融合蛋白的制备方法，其特征在于，所述制备方法包括以下步骤：
    a)在表达条件下，培养如权利要求12-13任一项所述的宿主细胞，从而表达能能同时阻断 HER2和VEGFR信号通路的抗体融合蛋白；
    b)分离并纯化步骤a)所述的抗体融合蛋白。
  15. 一种组合物，所述组合物包含权利要求1-6任一项所述的抗体融合蛋白和一种或多种药学上可接受的载体、稀释剂或赋形剂。
  16. 权利要求1-6任一项所述的抗体融合蛋白、或权利要求15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肿瘤。
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