WO2023169126A1 - 一种抗folr1/vegf的全人双特异性抗体及其筛选方法和应用 - Google Patents

一种抗folr1/vegf的全人双特异性抗体及其筛选方法和应用 Download PDF

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WO2023169126A1
WO2023169126A1 PCT/CN2023/075029 CN2023075029W WO2023169126A1 WO 2023169126 A1 WO2023169126 A1 WO 2023169126A1 CN 2023075029 W CN2023075029 W CN 2023075029W WO 2023169126 A1 WO2023169126 A1 WO 2023169126A1
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vegf
antibody
fully human
bispecific antibody
frα
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梁辉
苏冰
刘斗
倪啸天
崔丹丹
伍宁波
刘欣宇
程小涛
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苏州思萃免疫技术研究所有限公司
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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    • 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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    • 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
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    • 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)
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    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention belongs to the fields of immunology and biotechnology, and specifically relates to a fully human bispecific antibody against FOLR1/VEGF and its screening method and application.
  • Ovarian cancer is the cancer that causes the highest mortality rate in women. 65-75% of ovarian cancer patients are already in advanced stages (stage III and IV) when diagnosed. Ovarian cancer ascites is the main symptom of late-stage ovarian cancer, and ovarian cancer ascites is The manifestation of tumor metastasis. Studies have found that the ascites of ovarian cancer patients contains multiple types of immune cells. Ascites may be the tertiary lymphoid structures (TLSs) of ovarian cancer. However, there are currently no studies to clarify the role of immune cells in the ascites of ovarian cancer patients in ovarian cancer metastasis and ovarian cancer metastasis. role in deterioration.
  • TLSs tertiary lymphoid structures
  • FR ⁇ is encoded by FOLR1 and is a cell surface glycoprotein with a molecular weight of 38-40KD. FR ⁇ has been confirmed to be widely expressed in solid tumors, such as mesothelioma (72-100%), triple-negative breast cancer (35-68%), ovarian cancer (76-89%), non-small cell lung cancer (14- 74%); in non-malignant tissues, only the epithelial cells of the apical bronchi of the lungs have a certain proportion of expression. The specific expression of FR ⁇ in malignant tumors makes it a good target for anti-tumor drug development.
  • drugs such as small molecule drugs, antibody drugs, bispecific antibodies, CAR-T, ADC, and folic acid-cytotoxic drug conjugates are under development.
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • VEGF plays an important role in tumorigenesis and the occurrence of eye diseases such as wet macular degeneration.
  • Significant expression of VEGF and its receptors has been detected in most malignant tumors such as colon cancer, breast cancer, lung cancer, prostate cancer, kidney cancer, glioma, uterine cancer, esophageal cancer, gastric cancer, ovarian cancer and other tissues, and Its expression level is closely related to the occurrence and development of malignant tumors.
  • VEGF maintains the rapid growth of malignant tumors It is a key cytokine that grows long, promotes its dissemination and metastasis, and predicts the survival prognosis of patients. Bevacizumab targeting VEGF has shown efficacy in the clinical treatment of multiple malignant tumors and has also been widely used in the treatment of gynecological tumors.
  • bispecific antibodies Different from monoclonal antibodies, bispecific antibodies have the ability to target two different epitopes at the same time and can perform special biological functions, such as immune cell recruitment, receptor co-stimulation or co-inhibition, and multivalent virus neutralization. wait.
  • Bispecific antibodies are divided into symmetric structures and asymmetric structures according to the left-right symmetry of the structure. They are divided into quasi-intact antibodies and quasi-antibody fragments according to the integrity of the IgG molecules. They are also divided into bivalent, trivalent, and trivalent antibodies according to the number and configuration of the antigen-binding region. Tetravalent or more valent configurations, etc.
  • bispecific antibody designs have their own advantages and disadvantages, but the design of bispecific antibodies for clinical treatment purposes must solve the same problem: first, ensure the correct coupling of two (or more) pairs of different light chains and heavy chains or Pairing; secondly, maintain the independence of each monoclonal antibody's binding domain, and at the same time bind different epitopes without causing steric interference with each other; thirdly, the antibody molecules must be easy to express in mammalian cells , does not require complex protein modification processes, and has good drugability.
  • bispecific antibodies that can be used in clinical treatments
  • the following issues need to be solved: first, the correct pairing of light and heavy chains; second, the druggability of the molecule; third, each binding domain must not interfere with each other; and finally, We must also pay attention to immunogenicity, minimize mutation sites and additional peptide chains, and at the same time be able to use existing cells and purification processes for efficient large-scale production.
  • the more mainstream bispecific antibody platforms in the world include DVDIg (AbbVie), CrossMab (Roche), Duobody (GenMab), etc.
  • the purpose of the present invention is to provide a fully human bispecific antibody against FOLR1/VEGF, which has low immunogenicity and good targeting ability.
  • Another object of the present invention is to provide a screening method for fully human bispecific antibodies, which has simple steps and a high success rate.
  • Another object of the present invention is to provide the application of fully human bispecific antibodies against FOLR1/VEGF.
  • the present invention adopts the following technical solutions to achieve it:
  • a fully human bispecific antibody against FOLR1/VEGF is jointly constructed from an anti-human FRa antibody and an anti-human VEGF antibody.
  • the anti-human FRa antibody contains the heavy chain variable region sequence shown in SEQ ID No. 1 and the light chain variable region sequence shown in SEQ ID No. 2.
  • the anti-human FRa antibody is expressed in the form of a single chain antibody (scFv), that is, a heavy chain
  • scFv single chain antibody
  • the variable region and the light chain variable region are expressed in the same polypeptide chain, connected by (GGGGS) 3 in the middle.
  • the anti-human VEGF antibody contains the heavy chain variable region sequence shown in SEQ ID No. 6 and the light chain variable region sequence shown in SEQ ID No. 5.
  • VEGF antibody is expressed in the form of scFv, that is, the heavy chain variable region and the light chain variable region are expressed in the same polypeptide chain, connected by (GGGGS) 3 in the middle.
  • the anti-human FRa antibody heavy chain variable region is encoded by the nucleotide sequence shown in SEQ ID No. 3; the anti-human FRa antibody light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No. 4.
  • the nucleotide sequence code is encoded by the nucleotide sequence shown in SEQ ID No. 3.
  • anti-human VEGF antibody heavy chain variable region is encoded by the nucleotide sequence shown in SEQ ID No. 8; the anti-human VEGF antibody light chain variable region is encoded by the nucleotide sequence shown in SEQ ID No. 7 The nucleotide sequence code.
  • the fully human bispecific antibody also contains an Fc mutant fragment with the sequence shown in SEQ ID No. 12.
  • Fc mutant fragment is encoded by the nucleotide sequence shown in SEQ ID No. 17.
  • mutation sites of the Fc mutant fragment include S298A, T307A, E333A, K334A, E380A, and N430A.
  • the invention also provides a screening method for the fully human bispecific antibody.
  • the screening method includes the following steps:
  • the screening method includes the following steps:
  • VEGF antibodies have specific binding properties to antigens
  • FR ⁇ antibodies and VEGF antibodies to establish a bispecific antibody platform to obtain FR ⁇ antibodies and VEGF antibodies in scFv form, namely FR ⁇ scFv and VEGF scFv;
  • the FR ⁇ (scFv)-Fcmu-VEGF (scFv) protein includes FR ⁇ (scFv) with a sequence shown in SEQ ID No. 9, linker1 with a sequence shown in SEQ ID No. 10, and linker1 with a sequence shown in SEQ ID No. 10.
  • the Fcmu mutation sites include S298A, E333A, K334A, T307A, E380A, and N430A.
  • the invention also provides the use of the fully human bispecific antibody in the preparation of drugs for inhibiting angiogenesis.
  • the invention also provides the use of the fully human bispecific antibody in the preparation of anti-tumor drugs.
  • the tumors include lung cancer, prostate cancer, breast cancer, ovarian cancer, intestinal cancer, lymphoma, nasopharyngeal cancer, gastric cancer, liver cancer, kidney cancer, cervical cancer and endometrial cancer, and osteosarcoma.
  • the present invention also provides a pharmaceutical composition comprising a fully human bispecific antibody against FOLR1/VEGF.
  • the present invention has the following advantages and beneficial effects:
  • the current antibodies targeting ovarian cancer treatment are all mouse-derived or humanized monoclonal antibodies, which have strong immunogenicity and severe side effects.
  • the present invention obtains single antigen-specific B cells by sorting from ascitic fluid derived from ovarian cancer patients, and clones fully human FR ⁇ antibodies and VEGF antibodies.
  • a brand new Bispecific antibody is a fully human antibody derived from the human body. It has low immunogenicity and good targeting ability. It can target FOLR1 and VEGF at the same time.
  • the structure of the bispecific antibody constructed by the present invention is simple, easy to express and develop, and the constant region of the antibody has been specially designed. Tumor activity is stronger.
  • the antibody screening method of the present invention directly obtains fully human antibodies, the screening period is short, and the antibodies obtained are fully human antibodies. Compared with the current hybridoma screening, phage or yeast display screening methods, it has more technical advantages. Moreover, the dual antibodies have good tumor killing activity and can inhibit angiogenesis and tumor formation.
  • Figure 1 shows the ELISA results of FOLR1 antibody and VEFG antibody.
  • Figure 2 is a structural diagram of the ADCC enhanced bispecific antibody platform.
  • Figure 3 is a protein expression dot hybridization diagram of stable cell line screening with Bis-FR ⁇ -Fcmu-VEGF double antibody.
  • Figure 4 shows the SDS-PAGE electrophoresis results of Bis-FR ⁇ -Fcmu-VEGF double antibody after purification.
  • Figure 5 shows the affinity detection results of Bis-FR ⁇ -Fcmu-VEGF double antibodies, where A is the affinity detection result for FOLR1 antibody; B is the affinity detection result for VEFG antibody.
  • Figure 6 shows the results of the inhibitory activity of Bis-FR ⁇ -Fcmu-VEGF on the proliferation of different types of tumor cells.
  • the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.
  • the present invention obtains single antigen-specific B cells by sorting from ascites fluid derived from ovarian cancer patients, and clones fully human FR ⁇ antibodies and VEGF antibodies.
  • a new bispecific antibody is constructed.
  • the two specific antibody fragments were independently screened and adopted a new connection combination method.
  • the FcR affinity-enhanced Fc segment is used as a linker to improve the ability of the double antibody to recruit immune cells and kill.
  • Example 1 Screening and cloning of FRa and VEGF-specific B cells
  • CD27+ memory B cells are isolated from purified B cells by immunomagnetic positive selection according to the EasySep TM Human Memory B Cell Isolation Kit, STEMCELL. In short First, CD27+ memory B cells were labeled with magnetic beads conjugated with CD27 antibodies and separated using EasySep TM magnets. Purified CD27+ B cells were eluted and washed in PBS containing 2% (v/v) fetal bovine serum (FBS) and 1 mM EDTA. CD27+ B cells were counted using 0.4% (w/v) trypan blue stain and a Countess automated cell counter.
  • FBS fetal bovine serum
  • Biotinylated FRa and VEGF recombinant proteins were purchased from Sino Biological Inc. Fresh antigen/streptavidin M-280 Dynabeads (Thermofisher) complexes were prepared each time before B cell enrichment. Briefly, 100 ⁇ L of M-280 beads containing 6.5 ⁇ 10 beads was vortexed for 30 s and brought to room temperature before use. Beads were washed twice with 1 mL of 1 ⁇ PBS on a magnetic rack and eluted in 100 ⁇ L of 1 ⁇ PBS. Beads (100 ⁇ L) were mixed with 20 ⁇ g of biotinylated FRa and VEGF recombinant protein and incubated at room temperature for 30 min.
  • the complexes were washed 3 times using 500 ⁇ L of 1 ⁇ PBS on a magnetic stand. Washed complexes were eluted in 100 ⁇ L 1 ⁇ PBS and stored on ice until use. Prior to antigen enrichment, the complexes were equilibrated at room temperature. The amount of bead complex used was calculated based on a 1:1 ratio to the number of purified B cells. Add the FRa and VEGF magnetic bead complex directly to the B cell mixture, mix and incubate on a thermomixer at 4 °C for 30 min. After incubation, place the mixture on a magnetic stand and remove the supernatant. Wash the beads by mixing the magnet four times in total.
  • RNA from B cells was isolated using Trizol (Thermo), and reverse transcriptase (Invitrogen) was used to obtain cDNA. The above operations are all carried out in accordance with the manufacturer's instructions.
  • the amplification enzyme used is KOD plus (Thermo) to ensure that possible mutations are reduced during the amplification process.
  • the amplified enzyme digestion products are entrusted to a sequencing company for sequencing and confirmation.
  • the PCR obtains FR ⁇ and For the VEGF target gene fragment, FR ⁇ and VEGF scFv gene fragments were connected into the PCDNA3.1 vector through double enzyme digestion.
  • sequences of FR ⁇ and VEGF antibodies obtained by sequencing are as follows:
  • FR ⁇ (VH-VL) (abbreviated as: FR ⁇ scFv):
  • VL-VH Amino acid sequence of VEGF (VL-VH) (abbreviation: VEGF scFv):
  • Transient cell transfection 293 cell transfection was carried out according to the Lipofectamine TM 2000 operating instructions. Taking 1 well in a 6-well plate as an example, the steps are as follows: inoculate 4 ⁇ 10 5 cells into the well, and culture at 37°C and 5% CO 2 for 18 hours. Change the medium once and start transfection 12 hours later. The ideal confluence of adherent cells during transfection is 90-95%. Dilute 4 ⁇ g of plasmid DNA and 10 ⁇ l of Lipofectamine TM 2000 to 250 ⁇ l with antibiotic-free and serum-free medium respectively, mix gently, and let stand at room temperature for 15 minutes to form a liposome complex. After three days of continuous culture, the supernatant was collected and the indirect ELISA method was used to detect the specificity of the target protein.
  • Antigen coating (set blank control, negative control): Use coating diluent to dilute FR ⁇ or VEGF antigen to an appropriate 2ug/ml, add 100 ⁇ l of antigen to each well, 4°C, and coat overnight for 24 hours Be; discard the liquid in the well (to avoid evaporation, the plate should be covered or placed flat in a metal wet box with wet gauze at the bottom).
  • Washing method Absorb the reaction solution in the wells, fill the plate wells with the washing solution, leave it for 2 minutes and shake slightly, absorb the liquid in the wells, pour out the liquid and pat dry on absorbent paper, wash 3 times.
  • the structure of the ADCC enhanced bispecific antibody platform is shown in Figure 2.
  • the bispecific antibody FR ⁇ scFv-linker1-CH2-CH3-linker2-VEGF scFv was constructed, and its amino acid sequence is as follows:
  • the underlined areas are mutation sites, specifically S298A, T307A, E333A, K334A, E380A, and N430A.
  • nucleotide sequence of FR ⁇ scFv-linker1-CH2-CH3-linker2-VEGF scFv is:
  • FRa (scFv) and VEGF (scFv) screened in Example 1 were spliced with CH2-CH3 using overlap PCR to obtain FRa (scFv)-Fc mutant (Fcmu)-VEGF (scFv); where the Fc mutant ( Fcmu) uses the mutated sequence (i.e. CH2-CH3).
  • Fcmu uses the mutated sequence (i.e. CH2-CH3).
  • the mutations of S298A, E333A, and K334A in the Fc mutant can increase the affinity, and the mutations of T307A, E380A, and N430A can increase the half-life.
  • the mutated Fc fragment was selected as the linker between FR ⁇ (scFv) and VEGF (scFv), and FR ⁇ (scFv)-Fcmu-VEGF (scFv) was constructed into the PCDNA3.1 vector. After sequencing and double enzyme digestion, the obtained pFR132-FR ⁇ (scFv)-Fcmu-VEGF(scFv), the expression plasmid was extracted using an endotoxin-free plasmid extraction kit.
  • Bio-Rad electroporation system uses the Bio-Rad electroporation system to transfer pFR132-FR ⁇ (scFv)-Fcmu-VEGF(scFv) into host cells (CHO cells), use this cell to select the marker DHFR, and perform MTX pressure screening (final pressure is 200nM), label each The wells are: Bis-FR ⁇ -Fcmu-VEGF.
  • Use DOT Blotting to evaluate the expression level of the clone pool and gradually obtain high-expressing clone pool cells.
  • the Bis-FR ⁇ -Fcmu-VEGF-P2P3472 cell line with the highest expression amount was selected for amplification and transfection. Cultivate in a 200ml shake flask. When the cell growth density reaches 80%, collect the cells by centrifugation and transfer them to a 5L fermentation tank for continuous culture and fermentation.
  • the cell density was measured every day. When the cell density reached 90%, the jar was collected, centrifuged to remove the cell pellet, and the supernatant was purified.
  • the obtained fermentation broth was first captured using Protein A (alkali-resistant Protein A medium BXK26/20 column tube, Shanghai Borgron), and then subjected to cation medium purity (SP-5PW, TOSOH) to separate the acid and alkali peaks, and finally with WCX , SEC and SDS-PAGE were used to analyze the purity of the obtained samples.
  • the samples collected in stages during the purification process were analyzed by SDS-PAGE electrophoresis.
  • F1, F2, and F3 are the purified Bis-FR ⁇ -Fcmu-VEGF double antibodies.
  • the protein bands are clear and single, and the molecular weight is correct.
  • the molecular interaction instrument uses biofilm interference technology (Biolayer-interferometry, BLI) to perform kinetic measurements or concentration measurements. Visible light will form two reflection spectra at the two interfaces of the optical film at the end of the sensor, which are superimposed to form an interference spectrum. The combination of molecules causes the thickness of the film to change and causes the displacement of the original interference spectrum. By detecting and analyzing the interference The detection function is realized by using the shift value of the spectrum.
  • Bis-FR ⁇ -Fcmu-VEGF double antibody can bind to FR ⁇ and VEGF at the same time. By detecting the affinity of the antibody, the antibody performance of the sample to be tested can be evaluated.
  • the Bis-FR ⁇ -Fcmu-VEGF double antibody test sample is fixed to the Protein A sensor, and then combined and dissociated with FR ⁇ and VEGF proteins.
  • the software analyzes the data of the fitting curve to evaluate the performance of the test sample. Affinity.
  • the binding constants of Bis-FR ⁇ -Fcmu-VEGF double antibody to FR ⁇ and VEGF antigens are: 2.99 ⁇ 10 5 and 2.25 ⁇ 10 5 respectively, and the dissociation constants are: 2.45 ⁇ 10 -4 and 1.87 ⁇ 10 -4 .
  • Bis-FR ⁇ -Fcmu-VEGF double anti-antibody inhibits the growth of vascular endothelial cells and in vitro blood tube formation
  • Human breast cancer cells MDA-MB-231, lung cancer cells A549, human umbilical vein endothelial cells HUVEC and prostate cancer PC-3 were seeded in a 96-well plate at 5 ⁇ 10 6. After the cells adhered, 100 ⁇ g was added to the culture medium. /ml HAI-178, set up PBS control, and use MTT method to detect the effect of Bis-FR ⁇ -Fcmu-VEGF antibody on the growth of the above cells in vitro.
  • the Bis-FR ⁇ -Fcmu-VEGF double antibody can effectively inhibit the growth of HUVEC cells, with an inhibition rate of about 20%.
  • the growth of vascular endothelial cells is an important step in angiogenesis.
  • Bis-FR ⁇ -Fcmu-VEGF double anti-antibody can inhibit the growth of new blood vessels by inhibiting the growth of vascular endothelial cells.
  • the Matrigel vascular branch formation experiment was used to observe the effect of Bis-FR ⁇ -Fcmu-VEGF dual antibodies on the vascular formation of HUVEC cells in vitro. It was observed that the formation of vascular branches in HUVEC cells was significantly reduced under the action of Bis-FR ⁇ -Fcmu-VEGF double antibody, and its effect was equivalent to that of Angiostatin 2 as a positive control.
  • the present invention has successfully obtained a cell line that highly expresses Bis-FR ⁇ -Fcmu-VEGF double antibody, and obtained high-purity Bis-FR ⁇ -Fcmu-VEGF double antibody through purification, and biologically verified that it has high Good tumor killing activity, able to inhibit angiogenesis and tumor formation.

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Abstract

本发明公开了一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用。所述全人双特异性抗体由抗人FRα抗体和抗人VEGF抗体联合构建而成，其筛选方法简单，通过从卵巢癌患者来源的腹水中分选获得单个抗原特异性靶向的B细胞，最终构建了一个全新的双特异性抗体，所述抗体为来自人体的全人抗体，其免疫原性低，靶向性好，可以同时靶向FOLR1和VEGF，具有很好的肿瘤杀伤活性，能够抑制血管生成和肿瘤生成。

Description

一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用
相关申请交叉引用
本专利申请要求于2021年03月11日提交的、申请号为2022102420172、发明名称为“一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用”的中国专利申请的优先权，上述申请的全文以引用的方式并入本文中。
技术领域
本发明属于免疫学和生物技术领域，具体涉及一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用。
背景技术
卵巢癌是导致女性死亡率最高的一种癌症，65-75％的卵巢癌患者在确诊时已经处于晚期(III期和IV期)，卵巢癌腹水是卵巢癌晚期的主要症状，卵巢癌腹水是肿瘤发生转移的表现。研究发现卵巢癌患者的腹水中含有多种类型的免疫细胞，腹水有可能是卵巢癌的Tertiary lymphoid structures(TLSs)，但目前还没有研究来阐明卵巢癌患者腹水中的免疫细胞在卵巢癌转移和恶化中的作用。
FRα由FOLR1编码，是分子量38-40KD的细胞表面糖蛋白。FRα被证实在实体瘤中有广泛高表达，比如间皮瘤(72-100％)，三阴性乳腺癌(35-68％)，卵巢癌(76-89％)，非小细胞肺癌(14-74％)；而非恶性组织，只有肺部顶端支气管的上皮细胞，有一定比例的表达。FRα在恶性肿瘤的特异性表达，使之成为一个良好的抗肿瘤药物开发靶点。因而小分子药物、抗体药物、双特异性抗体、CAR-T、ADC、叶酸-细胞毒性药物偶联药物等不同形式的药物都在开发中。进展较快的如：Mirvetuximab soravtansine、Farletuzumab等均已经处于临床试验阶段。
血管内皮生长因子(vascular endothelial growth factor，VEGF)是最重要的促血管生成因子，可在体内诱导血管的新生。VEGF在肿瘤生成和湿性黄斑变性等眼科疾病发生中扮演重要的角色。在多数恶性肿瘤如结肠癌、乳腺癌、肺癌、前列腺癌、肾癌、神经胶质瘤、子宫癌、食管癌、胃癌、卵巢癌等组织中均检测到了VEGF及其受体的明显表达，且其表达水平与恶性肿瘤的发生、发展存在密切关系。VEGF是维持恶性肿瘤快速生 长、促进其播散转移、预测患者生存预后的关键细胞因子。靶向VEGF的贝伐珠单抗在多重恶性肿瘤的临床治疗显示疗效，在妇科肿瘤治疗方面也已广泛应用。
与单克隆抗体不同，双特异性抗体具有同时靶向2个不同表位的能力，并能起到特殊的生物学功能，例如免疫细胞召集、受体共刺激或共抑制、多价病毒中和等。双特异性抗体根据结构左右对称性分为对称结构和不对称结构，根据IgG分子完整性分为类完整抗体和类抗体片段，以及根据抗原结合区域的数量构型分为两价、三价、四价或更多价的构型等。不同的双特异性抗体设计各有利弊，但是以临床治疗为目的的双特异性抗体设计都要解决同样的问题：第一，保证两对(或以上)不同轻链与重链的正确偶合或配对；第二，保持每个单克隆抗体各自结合域的独立性，同时结合不同表位的时候互相之间不会产生空间位阻的干扰；第三，抗体分子要易于用哺乳动物细胞进行表达，不需要复杂的蛋白修饰工艺，有较好的成药性。
构建并开发可以应用于临床治疗的双特异性抗体需要亟需解决以下几个问题：一是轻重链的正确配对，二是分子的成药性，三是要做到各个结合域不能互相干扰，最后还要注意免疫原性，尽量减少突变位点和额外的肽链，同时又能够利用现有的细胞及纯化工艺进行高效规模化生产。现在国际上比较主流的双特异性抗体平台有DVDIg(艾伯维)、CrossMab(罗氏)、Duobody(GenMab)等。
发明内容
本发明的目的在于提供一种抗FOLR1/VEGF的全人双特异性抗体，所述全人双特异性抗体免疫原性低，靶向性好。
本发明的另一目的在于提供一种全人双特异性抗体的筛选方法，其步骤简单，成功率高。
本发明的另一目的在于提供抗FOLR1/VEGF的全人双特异性抗体的应用。
为实现上述发明目的，本发明采用以下技术方案予以实现：
一种抗FOLR1/VEGF的全人双特异性抗体，所述全人双特异性抗体由抗人FRα抗体和抗人VEGF抗体联合构建而成。
进一步的，所述抗人FRα抗体含有SEQ ID No.1所示的重链可变区序列和SEQ ID No.2所示的轻链可变区序列。
进一步的，所述抗人FRα抗体以单链抗体(scFv)形式表达，即重链 可变区和轻链可变区表达在同一条多肽链中，中间以(GGGGS)3连接。
进一步的，所述抗人VEGF抗体含有SEQ ID No.6所示的重链可变区序列和SEQ ID No.5所示的轻链可变区序列。
进一步的，所述VEGF抗体以scFv形式表达，即重链可变区和轻链可变区表达在同一条多肽链中，中间以(GGGGS)3连接。
进一步的，所述抗人FRα的抗体重链可变区由SEQ ID No.3所示的核苷酸序列编码；抗人FRα的抗体轻链可变区由SEQ ID No.4所示的核苷酸序列编码。
进一步的，所述抗人VEGF的抗体重链可变区由SEQ ID No.8所示的核苷酸序列编码；抗人VEGF的抗体轻链可变区由SEQ ID No.7所示的核苷酸序列编码。
进一步的，所述全人双特异性抗体中还含有序列如SEQ ID No.12所示的Fc突变体片段。
进一步的，所述Fc突变体片段由SEQ ID No.17所示的核苷酸序列编码。
进一步的，所述Fc突变体片段的突变位点包括S298A、T307A、E333A、K334A、E380A、N430A。
本发明还提供了所述的全人双特异性抗体的筛选方法，所述筛选方法包括以下步骤：
(1)利用卵巢癌腹水筛选、富集抗原特异性B细胞；
(2)从抗原特异性B细胞中，获取FRα抗体和VEGF抗体；
(3)利用FRα抗体和VEGF抗体建立双特异性抗体平台，得到scFv形式的FRα抗体和VEGF抗体；
(4)拼接scFv形式的FRα抗体和VEGF抗体，将Fc突变体片段Fcmu作为连接子，构建入载体，获取同时含有FRα(scFv)、Fcmu和VEGF(scFv)的质粒；
(5)将质粒转入宿主细胞，筛选得到高表达的细胞株；
(6)对高表达的细胞株培养纯化，得到同时含有FRα抗体、VEGF抗体和Fc突变体的蛋白，即为全人双特异性抗体。
具体的，所述筛选方法包括以下步骤：
(1)利采集卵巢癌患者术后腹水，经筛选、富集抗原特异性B细胞；
(2)提取抗原特异性B细胞RNA，反转录得到cDNA，经巢式PCR扩增获取FRα和VEGF目的基因片段，通过双酶切将FRα和VEGF scFv基因片段连接到PCDNA3.1载体中，获取FRα抗体和VEGF抗体；
(3)将FRα抗体和VEGF抗体进行活性鉴定，以确定FRα抗体和 VEGF抗体对抗原均具有特异性的结合特性；
(4)利用FRα抗体和VEGF抗体建立双特异性抗体平台，得到scFv形式的FRα抗体和VEGF抗体，即FRαscFv和VEGF scFv；
(4)利用overlap PCR的方法拼接FRαscFv和VEGF scFv，将Fc突变体片段Fcmu作为连接子，得到FRα(scFv)-Fcmu-VEGF(scFv)蛋白，构建入PCDNA3.1载体中，经测序和双酶切后，得到pFR132-FRα(scFv)-Fcmu-VEGF(scFv)质粒；
(5)将pFR132-FRα(scFv)-Fcmu-VEGF(scFv)质粒利用电穿孔系统转进宿主细胞，得到Bis-FRα-Fcmu-VEGF，筛选高表达的细胞株；
(6)扩增高表达的细胞株，经培养纯化得到Bis-FRα-Fcmu-VEGF蛋白，即为全人双特异性抗体Bis-FRα-Fcmu-VEGF，并同时获得了高表达全人双特异性抗体Bis-FRα-Fcmu-VEGF的细胞株。
进一步的，所述FRα(scFv)-Fcmu-VEGF(scFv)蛋白中包含序列如SEQ ID No.9所示的FRα(scFv)、序列如SEQ ID No.10所示的linker1、序列如SEQ ID No.11所示的linker2、序列如SEQ ID No.12所示的Fcmu、序列如SEQ ID No.13所示的VEGF(scFv)。
进一步的，所述Fcmu的突变位点包括S298A、E333A、K334A、T307A、E380A、N430A。
本发明还提供了所述的全人双特异性抗体在制备用于抑制血管生成的药物中应用。
本发明还提供了所述的全人双特异性抗体在制备用于抗肿瘤药物中的应用。
进一步的，所述肿瘤包括肺癌、前列腺癌、乳腺癌、卵巢癌、肠癌、淋巴瘤、鼻咽癌、胃癌、肝癌、肾癌、子宫颈癌和子宫内膜癌、骨肉瘤。
本发明还提供了一种药物组合物，所述药物组合物包含抗FOLR1/VEGF的全人双特异性抗体。
本发明与现有技术相比，具有以下优点和有益效果：
1、目前靶向卵巢癌治疗的抗体均为鼠源或者是人源化的单克隆抗体，免疫原性较强，副作用大。但本发明通过从卵巢癌患者来源的腹水中分选获得单个抗原特异性靶向的B细胞，克隆获得了全人的FRα抗体和VEGF的抗体，为了提高抗体治疗的效果，构建了一个全新的双特异性抗体，该抗体为来自人体的全人抗体，其免疫原性低，靶向性好，可以同时靶向FOLR1和VEGF。
2、本发明构建的双特异性抗体的结构与当前已有的双特异性结构的相比结构简单，易于表达和开发，并且对抗体的恒定区做了特别的设计，抗 肿瘤活性更强。
3、本发明中的抗体筛选方法由于是直接获得全人抗体，其筛选的周期短，获得的抗体为全人抗体。与目前由于的杂交瘤筛选，噬菌体或者是酵母展示筛选方法相比，更有技术优势。且所述双抗具有很好的肿瘤杀伤活性，能够抑制血管生成和肿瘤生成。
附图说明
图1为FOLR1抗体和VEFG抗体ELISA结果。
图2为ADCC增强型双特异性抗体平台的结构图。
图3为Bis-FRα-Fcmu-VEGF双抗的稳定细胞株筛选的蛋白表达点杂交图。
图4为Bis-FRα-Fcmu-VEGF双抗的纯化后SDS-PAGE电泳结果。
图5为Bis-FRα-Fcmu-VEGF双抗的亲和力检测结果，其中A为对FOLR1抗体亲和力检测结果；B为对VEFG抗体亲和力检测结果。
图6为Bis-FRα-Fcmu-VEGF对不同类型肿瘤细胞增殖的抑制活性结果。
具体实施方式
结合以下具体实例对本发明的技术方案作进一步详细的说明。
下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学试剂公司购买。
本发明通过从卵巢癌患者来源的腹水中分选获得单个抗原特异性靶向的B细胞，克隆获得了全人的FRα抗体和VEGF抗体，为了提高抗体治疗的效果，构建了一个全新的双特异性抗体，其中的两个特异性的抗体片段为自主筛选获得，采用了全新的连接组合方式，通过FcR亲和力增强型的Fc段为linker，提高双抗募集免疫细胞和杀伤的能力。
实施例1：FRα和VEGF的特异性B细胞的筛选和克隆
收集临床病例中已经确诊为卵巢高级别浆液性癌的患者术后腹水液体，取腹水800*g离心后收集细胞沉淀，采用RPMI-1640培养液重悬，离心后最终使用PBS缓冲液重悬细胞，此时细胞活力超过85％。采用如下步骤扩增抗原特异性B细胞。
1、CD27+记忆B细胞富集
根据EasySepTM人类记忆B细胞分离试剂盒，STEMCELL，通过免疫磁性阳性选择从纯化的B细胞中分离CD27+记忆B细胞。简而言 之，CD27+记忆B细胞用结合了CD27抗体的磁珠进行标记，并使用EasySepTM磁体进行分离。纯化的CD27+B细胞在含有2％(v/v)胎牛血清(FBS)和1mM EDTA的PBS中洗脱和洗涤。使用0.4％(w/v)台盼蓝染色剂和Countess自动细胞计数器对CD27+B细胞进行计数。
2、抗原结合B细胞富集
生物素化的FRα和VEGF重组蛋白购自Sino Biological Inc。每次在B细胞富集前制备新鲜抗原/链霉亲和素M-280Dynabeads(Thermofisher)复合物。简而言之，将含有6.5×107个珠子的100μL M-280珠子涡旋30秒，并在使用前置于室温。珠子在磁架上用1mL 1×PBS洗涤两次，并在100μL 1×PBS中洗脱。将珠子(100μL)与20μg生物素化FRα和VEGF重组蛋白混合，并在室温下孵育30分钟。孵育后，在磁性架上使用500μL 1×PBS将复合物洗涤3次。洗涤后的复合物在100μL 1×PBS中洗脱，并储存在冰上直至使用。在抗原富集之前，复合物在室温下平衡。使用的珠子复合物的数量是根据与纯化B细胞数量的1:1比例计算的。将FRα和VEGF磁珠复合物直接加入B细胞混合物中，混合并在4℃下在热混合器上孵育30分钟。孵育后，将混合物置于磁力架上，去除上清液。通过总共混合四次磁体来洗涤珠子。最终的抗原富集B细胞在含有2％(v/v)胎牛血清(FBS)和1mM EDTA的1×PBS中洗脱。使用0.4％(w/v)台盼蓝染色剂和Countess自动细胞计数器对富集的B细胞进行计数。
3、单B细胞抗体序列的克隆
用Trizol(Thermo)分离B细胞的总RNA，使用反转录酶(Invitrogen)以获取cDNA。上述操在均按照厂家说明书进行。用巢式PCR的方法，通过2次PCR反应，所用扩增酶为KOD plus(Thermo)确保扩增过程中减少可能的突变，扩增获得的酶切产物委托测序公司测序确认，PCR获得FRα和VEGF目的基因片段，通过双酶切将FRα和VEGF scFv基因片段连接到PCDNA3.1载体中。
PCR扩增体系：

PCR反应条件：
扩增不同类型的B细胞使用到的引物：
表1：第一轮PCR上游引物

表2：第一轮PCR下游引物
表3：第二轮PCR上游引物


表4：第二轮PCR下游引物

经过测序获得的FRα和VEGF抗体的序列如下：
FRα(VH-VL)(简称为：FRαscFv)的氨基酸序列：
FRα(VH-VL)的核苷酸序列：
VEGF(VL-VH)(简称为：VEGF scFv)的氨基酸序列：
VEGF(VL-VH)的核苷酸序列：
实施例2：FRα和VEGF抗体的表达和活性鉴定
1、FRα和VEGF抗体的瞬时表达
细胞瞬时转染：293细胞转染按LipofectamineTM 2000操作说明书进行，以6孔板中的1孔为例，步骤如下：向该孔接种4×105细胞，37℃、5％CO2培养18h后换液1次，12h后开始转染，贴壁细胞在转染时的理想汇合度为90-95％。将4μg质粒DNA和10μl LipofectamineTM 2000分别用无抗生素、无血清的培养基稀释至250μl，温和混匀，室温静置15min形成脂质体复合物。连续培养三天后收取上清采用间接ELISA方法检测目的蛋白的特异性。
2、FRα和VEGF抗体与抗原的特异性结合ELISA试验
(1)抗原包被(设置空白对照,、阴性对照)：将FRα或者VEGF抗原使用包被稀释液稀释到适当2ug/ml，每孔抗原加入100μl，4℃，24h过夜包 被；弃去孔中液体(为避免蒸发，板上应加盖或将板平放在底部有湿纱布的金属湿盒中)。
(2)封闭酶标反应孔：5％小牛血清置37℃封闭40min，封闭时将封闭液加满各反应孔，并去除各孔中的气泡，封闭结束后用洗涤液满孔洗涤3遍，每遍3min。
洗涤方法：吸干孔内反应液，将洗涤液注满板孔，放置2min略作摇动，吸干孔内液，倾去液体后在吸水纸上拍干，洗涤次数3次。
(3)加入FRα或者VEGF抗体(建立合适的浓度梯度)：采用1:100-1:100000的稀释度，将20μl稀释好的样品加入酶标反应孔中，每样品至少加双孔，每孔100μl，置于37℃，40-60min；用洗涤液满孔洗涤3遍，每遍3min。
(4)加入酶标抗体：加入HRP标记的羊抗人抗体，稀释比例为1：1000，37℃，孵育30-60min。
(5)加入底物液(现用现配)：每孔加入100μl TMB底物，置37℃避光放置3-5分钟，加入2ml硫酸终止液显色。
(6)结果判断：450nm波长来检测，与空白对照对确定抗体的特异性和效价。
从ELISA结果可知(图1)，FRα和VEGF抗体对抗原均有特异性的结合。
实施例3：ADCC增强型双特异性抗体平台的建立
ADCC增强型双特异性抗体平台的结构如图2所示。构建得到双特异性抗体FRαscFv-linker1-CH2-CH3-linker2-VEGF scFv，其氨基酸序列如下：
FRα(scFv)：
linker1：
linker2：
CH2-CH3：

其中下划线为突变位点，具体为S298A、T307A、E333A、K334A、E380A、N430A
VEGF(scFv)：
FRαscFv-linker1-CH2-CH3-linker2-VEGF scFv的核苷酸序列为：
FRα(scFv)：
linker 1：
linker 2：
CH2-CH3：
VEGF(scFv)：
将实施例1中筛选获得的FRα(scFv)和VEGF(scFv)与CH2-CH3采用overlap PCR的方法拼接获得FRα(scFv)-Fc突变体(Fcmu)-VEGF(scFv)；其中Fc突变体(Fcmu)采用突变后的序列(即CH2-CH3)，根据已知文献报道， Fc突变体中S298A、E333A、K334A的突变可以增加亲和力，T307A、E380A、N430A的突变可以提高半衰期。选用突变后的Fc片段做FRα(scFv)和VEGF(scFv)的连接子，将FRα(scFv)-Fcmu-VEGF(scFv)构建入PCDNA3.1载体中，经测序和双酶切后，确认获得pFR132-FRα(scFv)-Fcmu-VEGF(scFv)，采用无内毒素质粒大抽试剂盒抽提表达质粒。
实施例4：FRα和VEGF抗体双特异性抗体的制备和活性鉴定
1、Bis-FRα-Fcmu-VEGF双抗的电转和稳定细胞株的筛选
使用Bio-Rad电穿孔系统将pFR132-FRα(scFv)-Fcmu-VEGF(scFv)转进宿主细胞(CHO细胞)，利用本细胞筛选标记DHFR，进行MTX压力筛选(终压力为200nM)，标记每孔为：Bis-FRα-Fcmu-VEGF，以DOT Blotting评价克隆池的表达水平，逐步获得高表达的克隆池细胞。
结果如图3和表5所示，最终选择2株P2P261和P2P3472株细胞株进行发酵表达培养。
表5：Bis-FRα-Fcmu-VEGF双抗克隆池的表达水平检测
2、Bis-FRα-Fcmu-VEGF双抗的发酵和纯化
选用表达量最高的Bis-FRα-Fcmu-VEGF-P2P3472细胞株进行扩增，转 到200ml摇瓶中培养，带细胞生长密度达到80％时，离心收集细胞，转入5L的发酵罐中进行连续培养发酵。
每天检测细胞密度，待胞密度达到90％时，收罐，离心出去细胞陈沉淀，对上清进行纯化。
所获得的发酵液首先使用Protein A(耐碱型Protein A介质BXK26/20柱管，上海博格隆)进行捕获，然后进行阳离子中纯(SP-5PW，TOSOH)分离酸碱峰，最后以WCX、SEC以及SDS-PAGE对所获得的样品进行纯度分析，纯化过程中分段收集的样本采用SDS-PAGE电泳分析。
结果如图4所示，F1，F2，F3为纯化获得的Bis-FRα-Fcmu-VEGF双抗，蛋白的条带清晰单一，分子量正确。
实施例5：Bis-FRα-Fcmu-VEGF双抗的生物活性
1、Bis-FRα-Fcmu-VEGF双抗的亲和力检测
分子互作仪是利用生物膜干涉技术(Biolayer-interferometry，BLI)进行动力学测定或浓度测定。可见光在传感器末端的光学膜层的两个界面会形成两束反射光谱，叠加形成一束干涉光谱，而分子的结合导致膜层厚度变化，并导致原有干涉光谱的位移，通过检测和分析干涉光谱的位移值而实现检测功能。Bis-FRα-Fcmu-VEGF双抗可以与FRα和VEGF同时结合，通过检测抗体的亲和力，评价待测样品的抗体性能。
利用分子互作仪，将Bis-FRα-Fcmu-VEGF双抗待测样品固定至Protein A传感器，再与FRα和VEGF蛋白进行结合和解离，通过软件分析拟合曲线的数据，评价待测样品的亲和力。
如图5和表6所示，Bis-FRα-Fcmu-VEGF双抗对FRα和VEGF抗原的结合常数分别为：2.99×105和2.25×105，解离常数分别为：2.45×10-4和1.87×10-4。
表6：Bis-FRα-Fcmu-VEGF双抗的亲和力检测试验
2、Bis-FRα-Fcmu-VEGF双抗抗体抑制血管内皮细胞的生长和和体外血 管形成
将人乳腺癌细胞MDA-MB-231、肺癌细胞A549、人脐静脉内皮细胞HUVEC和前列腺癌PC-3以5×106种植于96孔板内，待细胞贴壁后，培养液内加入100μg/ml HAI-178，设置PBS对照，采用MTT方法，检测Bis-FRα-Fcmu-VEGF抗体对上述细胞体外培养生长情况的影响。
(1)Bis-FRα-Fcmu-VEGF抑制血管内皮细胞生长
如图6所示，Bis-FRα-Fcmu-VEGF双抗可以有效抑制HUVEC细胞的生长，抑制率达20％左右。血管内皮细胞的生长是血管新生的一个重要环节，Bis-FRα-Fcmu-VEGF双抗抗体可以通过抑制血管内皮细胞的生长起到抑制新生血管生长的作用。
另外，采用Matrigel血管枝形成实验，观察Bis-FRα-Fcmu-VEGF双抗对HUVEC细胞体外血管形成影响作用。观察发现HUVEC细胞在Bis-FRα-Fcmu-VEGF双抗的作用下，血管枝形成显著减少，其作用与作为阳性对照的血管抑素Angiostatin 2相当。
(2)Bis-FRα-Fcmu-VEGF对肿瘤细胞的杀伤活性
结果显示(如图6)，100μg/ml Bis-FRα-Fcmu-VEGF抗体能显著抑制肿瘤细胞与内皮细胞的增殖。作用48小时后，对MDA-MB-231细胞、A549细胞、PC3细胞和HUVEC细胞的抑制率分别为77.6％、52.1％、17.7％和18.4％；作用72h后，抑制率分别达到83.2％、53.1％、12.5％和20.7％，而对作为对照的CHO细胞无抑制作用。
综上，本发明成功获得了一株高表达Bis-FRα-Fcmu-VEGF双抗的细胞株，通过纯化获得了高纯度的Bis-FRα-Fcmu-VEGF双抗，并且生物学验证了其具有很好的肿瘤杀伤活性，能够抑制血管生成和肿瘤生成。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。

Claims (14)

  1. 一种抗FOLR1/VEGF的全人双特异性抗体，其特征在于，所述全人双特异性抗体由抗人FRα抗体和抗人VEGF抗体联合构建而成。
  2. 根据权利要求1所述的全人双特异性抗体，其特征在于，所述抗人FRα抗体含有SEQ ID No.1所示的重链可变区序列和SEQ ID No.2所示的轻链可变区序列。
  3. 根据权利要求1所述的全人双特异性抗体，其特征在于，所述抗人VEGF抗体含有SEQ ID No.6所示的重链可变区序列和SEQ ID No.5所示的轻链可变区序列。
  4. 根据权利要求1所述的全人双特异性抗体，其特征在于，所述抗人FRα抗体和所述抗人VEGF抗体均以scFv形式表达。
  5. 根据权利要求2所述的全人双特异性抗体，其特征在于，所述抗人FRα的抗体重链可变区由SEQ ID No.3所示的核苷酸序列编码；所述抗人FRα的抗体轻链可变区由SEQ ID No.4所示的核苷酸序列编码。
  6. 根据权利要求3所述的全人双特异性抗体，其特征在于，所述抗人VEGF的抗体重链可变区由SEQ ID No.8所示的核苷酸序列编码；所述抗人VEGF的抗体轻链可变区由SEQ ID No.7所示的核苷酸序列编码。
  7. 根据权利要求1所述的全人双特异性抗体，其特征在于，所述全人双特异性抗体中还含有序列如SEQ ID No.12所示的Fc突变体片段。
  8. 根据权利要求7所述的全人双特异性抗体，其特征在于，所述Fc突变体片段由SEQ ID No.17所示的核苷酸序列编码。
  9. 权利要求1-8任一项所述的全人双特异性抗体的筛选方法，其特征在于，所述筛选方法包括以下步骤：
    (1)利用卵巢癌腹水筛选、富集抗原特异性B细胞；
    (2)从抗原特异性B细胞中，获取FRα抗体和VEGF抗体；
    (3)利用FRα抗体和VEGF抗体建立双特异性抗体平台，得到scFv形式的FRα抗体和VEGF抗体；
    (4)拼接scFv形式的FRα抗体和VEGF抗体，将Fc突变体片段Fcmu作为连接子，构建入载体，获取同时含有FRα(scFv)、Fcmu和VEGF(scFv)的质粒；
    (5)将质粒转入宿主细胞，筛选得到高表达的细胞株；
    (6)对高表达的细胞株培养纯化，得到同时含有FRα抗体、VEGF抗体和Fc突变体的蛋白，即为全人双特异性抗体。
  10. 根据权利要求9所述的全人双特异性抗体的筛选方法，其特征在 于，所述Fc突变体片段的突变位点包括S298A、T307A、E333A、K334A、E380A、N430A。
  11. 权利要求1-8任一项所述的全人双特异性抗体在制备用于抑制血管生成的药物中应用。
  12. 权利要求1-8任一项所述的全人双特异性抗体在制备用于抗肿瘤药物中的应用。
  13. 根据权利要求12所述的应用，其特征在于，所述肿瘤包括肺癌、前列腺癌、乳腺癌、卵巢癌、肠癌、淋巴瘤、鼻咽癌、胃癌、肝癌、肾癌、子宫颈癌和子宫内膜癌、骨肉瘤。
  14. 一种药物组合物，其特征在于，所述药物组合物包含抗FOLR1/VEGF的全人双特异性抗体。
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