WO2019196531A1 - 特异性诱导肿瘤细胞凋亡的重组人Fc抗体及其制备方法、用途 - Google Patents

特异性诱导肿瘤细胞凋亡的重组人Fc抗体及其制备方法、用途 Download PDF

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WO2019196531A1
WO2019196531A1 PCT/CN2019/071725 CN2019071725W WO2019196531A1 WO 2019196531 A1 WO2019196531 A1 WO 2019196531A1 CN 2019071725 W CN2019071725 W CN 2019071725W WO 2019196531 A1 WO2019196531 A1 WO 2019196531A1
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antibody
recombinant human
protein
solution
fragment
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赵洪礼
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赵洪礼
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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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    • C07K2319/00—Fusion polypeptide
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Definitions

  • the present invention belongs to antibody pharmacy and its application in malignant tumors, in particular to a recombinant human Fc antibody for specifically inducing tumor cell apoptosis, a preparation method thereof, and a preparation obtained by the preparation method for treating malignant tumors .
  • the human immune system is an important organ for the prevention and development of various diseases in the body. It is responsible for four major immune functions: immune defense, immune surveillance, immune tolerance and immune regulation. Antibodies are one of the most important molecules for performing these four functions.
  • the antibody molecule is an immunoglobulin produced by the body after being stimulated by an antigenic substance. It can specifically bind to the corresponding antigen and remove the antigen by other immune functions, and plays an important role in disease prevention.
  • the antibody molecule consists of 4 peptide chains and consists of two functional regions, that is, the amino terminus of the specific binding antigen, called the F(ab') segment. The molecular structure of the F(ab') segment is different because of different antigens.
  • variable region V region
  • Fc fragment carboxy terminus responsible for immunoregulatory function
  • C region the constant region
  • the antibody is divided into five isotypes, namely IgM, IgG, IgA, IgD and IgE; even the molecular structure of the same type of Ig is different, according to which the same type of Ig can be divided into different sub- Classes such as human IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • the antibody Fc fragment does not specifically bind to the antigen, it is indispensable for exerting immune function.
  • ADCC antibody-dependent dell-mediated cytotoxicity
  • Fc antibodies antigen-binding Fc proteins
  • the anti-tumor antibodies currently used in clinical practice are mostly directed against tumor cell surface dominant antigens, such as CD antigen series, rather than tumor cell-specific antigens, because there are few tumor-specific antigens that can be proved at present, which is to a large extent. It affects the therapeutic effect of the antibody and increases the toxic side effects of the antibody.
  • tumor cell surface dominant antigens such as CD antigen series
  • anti-tumor antibodies are always in shortcomings.
  • the current antibody development pays more attention to the role of the V region, while ignoring the function of the C region.
  • the clinically available Fcabs-like monoclonal antibodies are antigen-blocking.
  • Enbrel for the treatment of rheumatoid arthritis is a therapeutic effect by blocking TNF receptors. It acts to stabilize the protein and increase the half-life without further exerting its immunomodulatory function.
  • VP3 has a specific killing effect on various human tumor cells and abnormally transformed cells, and is named as a tumor-specific apoptin (Apoptin), which is hopeful to be a novel biological preparation for effectively treating cancer.
  • VP3 induces tumor cell apoptosis, which is not dependent on the tumor suppressor gene-53 pathway, nor is it inhibited by overexpression of the inhibitory gene (Bcl-2), but since VP3 is not a structural protein of chicken anemia virus, It is very unstable in vitro and restricts large-scale production.
  • VP3 does not have its associated protein receptor on the surface of human cell membrane.
  • VP3 cannot bind to human cells and enter tumor cells, causing tumor cell apoptosis.
  • the principle of VP3-induced apoptosis is to enter cells and activate apoptosis-related enzymes (mainly caspase) to induce apoptosis.
  • apoptosis-related enzymes mainly caspase
  • the human Fc antibody not only increases its half-life in vivo, but also utilizes the function of the Fc fragment to bind to the Fc receptor and its induced DACC action and the function of crossing cells, the function of activating complement and the function of VP3-specific induction of tumor cell apoptosis. It has triple biological functions of anti-tumor and can be scaled up and applied.
  • the object of the present invention is to provide a preparation method of recombinant human Fc antibody which is highly expressed in eukaryotic cells, is stable, has high purity, and can specifically induce apoptosis of tumor cells, and the preparation obtained by the method is anti-tumor treatment.
  • the recombinant human Fc antibody provided by the invention can bind to tumor cells and specifically induce tumor cell apoptosis, so as to meet the purpose of clinically treating tumors.
  • the technical scheme of the present invention is: based on the construction of the Fc-Linker-VP3 fusion protein (recombinant human Fc antibody) expression vector pPIC9K vector, further establishing a technical method for purifying recombinant human Fc antibody, so that it is easily dissolved in vitro, Stable, high purity; able to bind to tumor cells and induce apoptosis, becoming a novel recombinant human Fc antibody.
  • novel recombinant human Fc antibody gene provided by the present invention has the nucleotide sequence shown in SEQ1 of the Sequence Listing;
  • novel recombinant human Fc antibody amino acid provided by the present invention has the amino acid sequence shown in SEQ5 of the Sequence Listing;
  • the preparation method of the recombinant human Fc antibody provided by the invention comprises the following steps:
  • Recombinant human Fc antibody induces apoptosis of tumor cells.
  • Buffer A 1 M sorbitol, 10 mM bicine (N-bis(hydroxyethyl)glycine) pH 8.35, 3% (v/v) ethylene glycol;
  • Buffer B 40% (w / v) polyethylene glycol 1000, 0.2 M N-bis (hydroxyethyl) glycine pH 8.35;
  • Buffer C 0.15 M NaCl, 10 mM N-bis(hydroxyethyl)glycine pH 8.35;
  • the untransfected cells and empty vector cells were used as negative controls, and cultured in serum-free 1640 medium.
  • the supernatant was collected every 24 h in the experimental group, filtered through a 0.45 ⁇ m filter, and the supernatant was stored at -20 ° C; the cell culture supernatant was collected for purification, and the CHO cell line was highly expressed.
  • the above culture supernatant was slowly added to an equal volume of saturated ammonium sulfate, mixed and allowed to stand at 4 ° C for 4 hours or more, and centrifuged at 5000 g for 20 minutes at 4 ° C to collect the precipitate.
  • the precipitate was dissolved with 100 ml of the balance solution, and dialyzed against the balance solution 2000 for 12 hours.
  • the middle is changed once a night; the sample is then stored on an anion column equilibrated with an equilibrium solution or stored at -20 °C, and equilibrated with 0.1 mol/L, 0.2 mol/L, 0.5 mol/L and 1 mol/L NaCl, respectively.
  • the solution was eluted with liquid, and the eluted peak was collected and identified by SDS electrophoresis.
  • the protein fraction containing molecular weight of 39 kD was taken; the Separose 12 column was equilibrated with the equilibration solution; the concentrated 39 kD protein fraction was loaded at a ratio of 3% of the bed volume, and the flow rate was 2 ml/ Min; step-by-step collection; after SDS electrophoresis identification, the protein fraction with high molecular weight of 39kD is recombinant human Fc antibody; stored at -20 °C to be identified.
  • YPD or YPED Yeast Extract Peptone Dextrose Medium (1L) 1% Yeast Extract 2% Peptone 2% Dextrose (glucose)
  • YPD-genemycin plate 1% yeast extract, 2% peptone 2% glucose 2% different amounts of geneticin 100 mg/ml geneticin: 30 ml 100 mg/ml geneticin stock solution was prepared with sterile water, filtered Sterilization, stored at -20 °C. Used to prepare geneticin plates containing different final concentrations.
  • MGY and MGYH minimal glycerol medium ⁇ histidine 1L: 1.34% YNB 1% glycerol 4 ⁇ 10-5% biotin ⁇ 0.004% histidine
  • Saturated ammonium sulfate It is prepared in the following ratio in units of 1000 ml.
  • the culture and purification steps of the recombinant human Fc antibody CHO cell line described in (4) are: expression of a highly expressed CHO cell line, cultured in serum-free 1640 medium for 24 hours, and the supernatant is collected and filtered with a 0.45 ⁇ m filter. Slowly add an equal volume of saturated ammonium sulfate, mix and leave at 4 ° C for 4 hours or more, and centrifuge at 5000 g for 20 min at 4 ° C to collect the precipitate. The precipitate is dissolved with 100 ml of equilibration solution, and dialyzed against the balance solution 2000 for 12 hours or more.
  • the sample is then stored on an anion column equilibrated with an equilibration solution or stored at -20 ° C, and eluted with an equilibration solution containing 0.1 mol/L, 0.2 mol/L, 0.5 mol/L, and 1 mol/L NaCl, respectively.
  • the eluted peaks were collected and identified by SDS electrophoresis, and the protein fraction containing molecular weight of 39 kD was taken; the Separose 12 column was equilibrated with the equilibration solution; the concentrated 39 kD protein fraction was loaded at a ratio of 3% of the bed volume, and the flow rate was 2 ml/min; After collection by SDS electrophoresis, a protein fraction having a high molecular weight of 39 kD was collected as a recombinant human Fc antibody.
  • Saturated ammonium sulfate It is prepared in the following ratio in units of 1000 ml.
  • the above recombinant human Fc antibody preparation is used for inducing apoptosis of tumor cells, and the cells are cultured in vitro as an example: the tumor cells are inoculated into a 96-well cell culture plate and cultured overnight; and different concentrations of recombinant human Fc antibody are added to continue. After 24 hours of culture, apoptosis was measured by the Annexin V method, and the test was set to a normal cell control. Results 6g/L recombinant single-chain antibody can cause apoptosis in more than 31.45% of tumor cells.
  • mice are subcutaneously inoculated with tumor cells, and when the tumor length is about 150 mm 3 , the mice are randomly grouped and subcutaneously injected, respectively.
  • Two different doses of recombinant single-chain antibody and normal saline (control group) were injected every other day for 20 consecutive days. The results showed that the recombinant single-chain antibody had obvious anti-tumor effect on tumor cells, compared with the control group. Proliferation is markedly slow.
  • Figure 1 shows the construction of a synthetic Fc-Linker-VP3 fragment, which was cloned into the pPIC9K vector EcoRI and NotI restriction site expression vector.
  • Figure 2 shows the expression and purification results of recombinant human Fc antibody.
  • 1, 2, 3, 4, and 5 are purified recombinant human Fc antibodies; 6 is the molecular weight standard, which is 97.0, 66.2, 43.0, 31.0, in descending order. 14.3 kDa; 7 is a recombinant human Fc antibody protein expressed by fermentation.
  • Figure 3 shows the expression and purification results of recombinant human Fc antibody.
  • 1 is the molecular weight standard, which is 97.0, 66.2, 43.0, 31.0, 14.3 kDa from large to small; 2, 3 are purified recombinant human Fc antibody; 4 is CHO Recombinant human Fc antibody protein expressed by cells
  • Figure 4 shows the inhibition of proliferation of breast cancer MCF-7 cells by recombinant human Fc antibody in vitro, indicated in the figure: the ordinate is the cell proliferation inhibition rate; the abscissa is the concentration gradient of the recombinant human Fc antibody.
  • Figure 5 shows the effect of recombinant human Fc antibody on apoptosis induction of breast cancer MCF-7 cells.
  • concentrations of recombinant human Fc antibodies indicated as follows: 1, 2, and 3 are: 0g/L 3g/L and 6g/L, respectively.
  • Figure 6 is a therapeutic effect of recombinant human Fc antibody on breast cancer in nude mice.
  • the figures in the figure are: the above picture is the tumor volume line drawing, and the lower picture is the physical picture of the tumor.
  • yeast system GS115 strain competent cells add 50ug of DNA in 20ul water, add the DNA directly into the competent cell tube still in the frozen state, mix in water bath for 5min at 37°C for 2min; remove the transformation tube and add 1.5ml Buffer B, completely Mix well, boil at 30 ° C for 1 hour; centrifuge at 1500 g for 10 min at room temperature, remove the supernatant, resuspend the cells with 1.5 ml Buffer C, centrifuge, resuspend the cells with 0.2 ml Buffer C and apply them all to the selected growth plates. Incubate for 3 to 4 days at 30 ° C and screen for genetic resistant high resistant clones (Figure 1).
  • Experimental material YPD-plate MGY and MGYH medium, saturated ammonium sulfate, equilibration solution: 20 mmol PB buffer (pH 7.2), anion chromatography column, Separose 12 chromatography column.
  • the above culture supernatant was slowly added to an equal volume of saturated ammonium sulfate, mixed and allowed to stand at 4 ° C for 4 hours or more, and centrifuged at 5000 g for 20 minutes at 4 ° C to collect the precipitate.
  • the precipitate was dissolved with 100 ml of the balance solution, and dialyzed against the balance solution 2000 for 12 hours.
  • the middle is changed once a night; the sample is then stored on an anion column equilibrated with an equilibrium solution or stored at -20 °C, and equilibrated with 0.1 mol/L, 0.2 mol/L, 0.5 mol/L and 1 mol/L NaCl, respectively.
  • the solution was eluted by liquid, and the eluted peak was collected and identified by SDS electrophoresis.
  • the protein fraction containing molecular weight of 39 kD was taken; the Separose 12 column was equilibrated with the equilibration solution; the concentrated 39 kD protein fraction was loaded at a ratio of 3% of the bed volume, and the flow rate was 2 ml/ Min; step-by-step collection; after SDS electrophoresis identification, the protein fraction with high molecular weight of 39kD is recombinant human Fc antibody; stored at -20 °C to be identified.
  • the supernatant was collected every 24 h in each experimental group, filtered through a 0.45 ⁇ m filter, and the supernatant was stored at -20 ° C.
  • the supernatant of the IgG1-VP3 fusion protein was purified as follows; the culture supernatant was slowly added to an equal volume of saturated ammonium sulfate. After mixing, the mixture was allowed to stand at 4 ° C for 4 hours or more, and the precipitate was collected by centrifugation at 5000 g for 20 min at 4 ° C.
  • the precipitate was dissolved with 100 ml of the equilibrium solution, and dialyzed against the balance solution 2000 for 12 hours or more, and the middle was replaced by the night;
  • the well-balanced anion column was stored at -20 °C, and eluted with an equilibrium solution containing 0.1 mol/L, 0.2 mol/L, 0.5 mol/L and 1 mol/L NaCl, and the eluted peaks were collected and identified by SDS electrophoresis.
  • cell inhibition rate (%) (Ac-As) / (Ac-Ab) ⁇ 100%.
  • IC 50 value The half inhibition concentration (IC 50 value) was calculated using Graphpad Prism software.
  • the treatment group nude mice were injected subcutaneously with recombinant human Fc antibody to treat the 30 ⁇ g/ml and 50 ⁇ g/ml (0.1 mL)/only, and the normal saline group was injected with an equal volume of physiological saline.
  • Each group of nude mice was injected once every other day; the general living conditions of the mice were observed daily, the tumor size was measured, and the body weight was measured every other day.
  • the cervical vertebrae were sacrificed 3 weeks after inoculation, and the tumor volume was separated and the tumor volume was measured.
  • mice in each group were fed normally, and the general physiological conditions within 1 wk of inoculation did not change significantly.
  • the mice in the saline group had reduced eating, weight loss, weight loss, tar loss, and tumor masses significantly increased; while the treatment group mice were generally in good condition.
  • the mice were killed 32 days later, and the tumor volume was measured by tumor tissue.
  • the tumor inhibition rates of the treatment group were 30.6% and 64.6% in the 30 ⁇ g/ml and 50 ⁇ g/ml groups, respectively. The tumor inhibition rate was significantly different (p ⁇ 0.01) (attached table). (See Figure 6).
  • the invention has the beneficial effects that the recombinant human Fc antibody can be highly expressed in eukaryotic cells, has good stability, high purity and high activity, and has a remarkable therapeutic effect, in particular, the invention has no special requirements for production equipment, and is suitable for large-scale production. And industrialization is of great significance for humans to overcome tumors and prolong human life.

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Abstract

提供一种特异性诱导肿瘤细胞凋亡的重组人Fc抗体及其制备方法、用途，其要点是：通过人工合成方法直接合成人IgG抗体的Fc片段的HC2和HC3区基因片段，同时合成能特异性诱导肿瘤细胞凋亡的VP3基因，构建含Fc-linker-VP3单链抗体基因；将上述基因片段亚克隆到pPIC9K载体并转染毕赤酵母系统GS115菌株，或亚克隆到哺乳细胞表达载体及在哺乳细胞中表达重组的人Fc抗体蛋白；此重组人Fc抗体具有激活补体活性，引起ADCC作用，同时有很强的特异性诱导肿瘤细胞凋亡作用，可用于恶性肿瘤的治疗。

Description

特异性诱导肿瘤细胞凋亡的重组人Fc抗体及其制备方法、用途 技术领域
本发明属抗体制药及其在恶性肿瘤中的应用，尤其是一种特异性诱导肿瘤细胞凋亡的重组人Fc抗体及其制备方法、及用该制备方法获得的制品在恶性肿瘤治疗中的应用。
背景技术
人体免疫系统是防御体内各种疾病发生发展的重要器官，它担负着免疫防御、免疫监视、免疫耐受和免疫调节4大免疫功能。抗体是执行这4大功能的最重要分子之一。抗体分子是机体受到抗原性物质刺激后产生的免疫球蛋白，它可与相应抗原特异性结合并借助其它免疫功能将抗原清除掉，起到防病的重要作用。抗体分子由4条肽链组成，有2个功能区构成，即特异性结合抗原的氨基端，称为F(ab＇)段，因抗原不同F(ab＇)段的分子结构不同，又称为可变区(V区)；和担负免疫调节功能的羧基端，称为Fc片段，同种生物的Fc片段是相对保守的，又称为恒定区(C区)。根据恒定区的抗原性不同将抗体分为5个同种型，即IgM、IgG、IgA、IgD和IgE；即使是同一类Ig的分子结构也有不同，据此又可将同类Ig分为不同亚类，如人IgG可分为IgG1、IgG2、IgG3和IgG4。尽管抗体Fc片段不与抗原发生特异性结合，但在发挥免疫功能方面是不可或缺的。当抗体V区与抗原发生结合后，必须借助C区的作用，在体外可发生各种抗原抗体结合反应，有力于抗原或抗体的检测和功能的判定，在体内可中和毒素、阻断病原入侵、清楚病原微生物等。Fc片段主要功能有：补体激活功能、结合Fc受体功能和穿过胎盘和粘膜功能。抗体通过与Fc受体结合进一步发挥免疫调理功能和抗体依赖的细胞介导的细胞毒作用(antibody-dependent dell-mediated cytotoxicity,ADCC),ADCC作用是机体清楚病原体和肿瘤细胞的重要功能之一。人体内的Fc受体分为3钟，广泛的分布于体内各种免疫细胞和体细胞的表面，发挥着重要的免疫调节功能，同时抗体在体内的半衰期起着重要作用。
近年来，随着单克隆抗体技术和基因工程技术的快速发展，涌现出大量的人源化抗体、基因工程抗体和抗原结合的Fc融合蛋白，俗称Fc抗体，(antigen-binding Fc proteins.Fcabs,)。这些抗体的研制成功为各种疑难杂症提供了有力的精确诊断和精准治疗的手段，已有350多种抗体获批应用于临 床，每年获得数百亿元的经济效益。特别是出现了大量的针对肿瘤细胞的单克隆抗体或人源化抗体。但是目前临床上所应用的抗肿瘤抗体多为针对肿瘤细胞表面优势抗原的，如CD抗原系列，而非肿瘤细胞特异性抗原，因为目前能证明的肿瘤特异性抗原寥寥无几，这在很大程度上影响了抗体的治疗效果，同时增加了抗体的毒副作用。目前在缺乏肿瘤特异性抗原的前提下，制备的抗肿瘤抗体总是有不足之处。另外目前抗体研制多注重V区的作用，而忽略了C区的功能。已上临床的Fcabs类单抗制品均为抗原阻断型，如用于治疗风湿性关节炎的依那西普(Enbrel),就是阻断TNF受体而发挥治疗作用，其中的抗体Fc段只起到稳定蛋白和增加半衰期的作用，而没有进一步发挥其免疫调节功能。
众所周知癌症治疗是世界性难题，目前常规的手术、放疗、化疗等方法还不能治愈癌症，寻找更有效的治疗手段一直是国际上重点研究领域之一。所以研发具有治疗作用的新型生物制剂具有重要的社会意义和经济意义。
国内外研究证明鸡贫血病毒VP3蛋白对人的各种肿瘤细胞和异常转化细胞具有特异性杀伤作用，被命名为肿瘤特异性凋亡因子(Apoptin)，有希望成为有效治疗癌症的新型生物制剂。VP3诱导肿瘤细胞凋亡的特点是不依赖于抑癌基因-53作用途径，也不被抑制细胞凋亡基因(Bcl-2)过量表达所抑制，但是由于VP3不是鸡贫血病毒的结构蛋白质，在体外非常不稳定，限制规模化生产；另外由于种属间的差异，VP3在人体细胞膜表面没有其相关的蛋白受体，所以，VP3不能与人的细胞结合，进入肿瘤细胞，引起肿瘤细胞凋亡。而VP3诱导细胞凋亡的原理是进入细胞，激活与细胞凋亡相关的酶(主要为caspase)引起细胞凋亡。上述两个技术难题严重限制了VP3的产业化和临床应用。为解决上述理论问题，我们曾经将VP3与GST融合表达，获得了体外稳定批量获得VP3蛋白的技术(专利号：ZL201010107675.8)，解决了体外大量获得VP3蛋白的技术难题；同时我们将叶酸通过化学方法联结到VP3蛋白上，使其能自主进入肿瘤细胞，解决了种属间差异造成的VP3不能自主进入人肿瘤细胞的理论问题(专利号：ZL201010107682.8)。在解决这两个技术难题的基础上，我们又对VP3进行了进一步的改进，将其与人抗体IgG1的Fc片段融合，研制出能特异性诱导肿瘤细胞凋亡的重组人Fc抗体，此重组人Fc抗体不仅增加了其体内半衰期，而且利用了Fc片段结合Fc受体的功能及其诱发的DACC作用和穿越细胞的作用、激活补体的功能和VP3特异性诱导肿瘤细胞凋亡的功能，使其具备了三重抗肿瘤的生物学功能，且能规模化生产与应用。
发明内容
本发明的目的在于提供一种在真核细胞中高效表达、稳定好、纯度高、且能特异性诱导肿瘤细胞凋亡的重组人Fc抗体的制备方法，及该方法获得的制品在抗肿瘤治疗中的应用。本发明提供的重组人Fc抗体能与肿瘤细胞发生结合，并特异性诱导肿瘤细胞凋亡，以达到满足临床治疗肿瘤的目的。
本发明的技术方案是：在构建Fc-Linker-VP3融合蛋白(重组人Fc抗体)表达载体pPIC9K载体的基础上，进一步建立一种纯化重组人Fc抗体的技术方法，使其在体外易溶解、稳定、纯度高；能够与肿瘤细胞结合并诱导其凋亡，成为一种新型的重组人Fc抗体。
本发明提供的新型重组人Fc抗体基因具有序列表中SEQ1所示的核苷酸序列；
本发明提供的新型重组人Fc抗体氨基酸具有序列表中SEQ5所示的氨基酸序列；
本发明提供的重组人Fc抗体的制备方法，包括以下步骤：
(1)通过人工合成的方法直接获得重组人Fc抗体的基因片段，构建Fc-Linker-VP3基因，亚克隆到pPIC9K表达载体；用酵母系统GS115菌株表达蛋白，用载体信号肽作分泌表达。
(2)通过人工合成的方法直接获得重组人Fc抗体的基因片段，构建Fc-Linker-VP3基因，亚克隆到哺乳细胞表达载体pATX2载体，脂质体法转染哺乳细胞CHO细胞株表达，筛选高效表达细胞株，表达重组人Fc抗体蛋白；
(3)重组人Fc抗体酵母菌株的发酵,高效表达及纯化；
(4)重组人Fc抗体CHO细胞株的发酵,高效表达及纯化；
(5)重组人Fc抗体诱导肿瘤细胞凋亡作用。
(1)中所说的构建重组人Fc抗体pPIC9K载体及工程菌株的建立的方法:直接人工合成Fc-Linker-VP3片段，定向亚克隆到pPIC9K载体EcoRI和NotI酶切位点，如图1所示；常规制备酵母系统GS115菌株感受态细胞；在20ul水中加入50ug DNA,将DNA直接加入仍处于冰冻状态的感受态细胞管中，37℃水浴5min，其间混匀2次；取出转化管，加入1.5ml Buffer B，完全混匀，30℃水浴1小时；室温下，1500g离心样品10min，去净上清，用1.5ml Buffer C重悬细胞，再次离心，用0.2ml Buffer C重悬细胞，将管中内容物全部涂在选择生长平板上，30℃孵育3～4天，筛选遗传霉素高抗性克隆。
溶液准备：
Buffer A:1M山梨醇，10mM bicine(N－二(羟乙基)甘氨酸)pH8.35，3％(v/v)乙二醇；
Buffer B:40％(w/v)聚乙二醇1000，0.2M N－二(羟乙基)甘氨酸pH8.35；
Buffer C:0.15M NaCl,10mM N－二(羟乙基)甘氨酸pH8.35；
过滤除菌，存于-20℃。新鲜的试剂级DMSO，未打开或刚配制，存于-70℃直至使用。
(2)中所说的构建重组人Fc抗体哺乳细胞表达载体pATX2载体及CHO细胞转染的方法:直接人工合成Fc-Linker-VP3片段，定向亚克隆到表达质粒pATX2EcoRI和NotI酶切位点；CHO细胞的转染方法：弃去CHO细胞原来的培养基，加入约5mL新鲜培养基，再加入DNA/DOTAP混合物150止，轻轻摇晃培养瓶，使之在培养基中分布均匀，置37℃细胞培养箱培养3～10h后换新鲜完全1640培养基，48h后加G418(800μg/mL)筛选，实验同时设未转染细胞和空载体细胞为阴性对照，以无血清1640培养基培养，各实验组每24h收集上清液，用0.45μm滤器过滤，上清液-20℃保存；收集细胞培养上清液，用于纯化，筛选高效表达CHO细胞株。
(3)中所说重组人Fc抗体的菌株发酵,高效表达及纯化步骤是：取单个高效表达的克隆菌株，接种于含25mlMGY，BMG或BMGY的250ml摇瓶中，28～30℃，250～300rpm培养到OD 600＝2～6(约12～20小时)；将上述25ml培养基转钟到含1L MGY，BMG或BMGY的5L的摇瓶中，28～30℃度高速振荡(250～300rpm)，培养到对数生长期(OD 600＝2～5)；用灭菌离心管，室温1500～3000g离心5min收集细胞；用MM，BMM或BMMY重悬细胞至OD 600＝1.0(2～6L)进行诱导；分装培养基至N个5L隔板摇瓶，1L/瓶，用8层灭菌纱布盖住瓶口，放入摇床28～30℃继续培养；每24小时加甲醇1次，终浓度为0.5％，直到达最佳诱导时间；室温，1500～3000g离心5min，保留上清，置4℃预冷，为蛋白纯化备用。将上述培养上清缓慢加入等体积的饱和硫酸铵，混匀后4℃放置4小时以上，4℃下5000g离心20min收集沉淀，沉淀用100ml平衡液溶解，并对平衡液2000平衡液透析12小时以上，中间换夜一次；样品再上用平衡液平衡好的阴离子层析柱或－20℃保存备用，分别用含0.1mol/L、0.2mol/L、0.5mol/L和1mol/LNaCl的平衡液洗脱，收集洗脱峰，SDS电泳鉴定，取含分子量39kD蛋白质部分；用平衡液平衡Separose12层析柱；将上述浓缩的39kD蛋白质部分按床体积的3％比例上样， 流速为2ml/min；分步收集；SDS电泳鉴定后，取分子量为39kD纯度高的蛋白质部分即为重组人Fc抗体；－20℃保存待鉴定。
溶液准备：
YPD或YPED：Yeast Extract Peptone Dextrose Medium(1L)1％Yeast Extract 2％Peptone 2％Dextrose(glucose)
1溶解10gYE,20gPEP于900ml水中，如制平板加入20g琼脂粉
2高压20min
3加入100ml 10×D
YPD-遗传霉素平板：1％酵母浸出物，2％蛋白胨2％葡萄糖2％不同量的遗传霉素100mg/ml遗传霉素：用无菌水制备30ml 100mg/ml遗传霉素贮存液，过滤除菌，-20℃保存。用来制备含不同终浓度遗传霉素平板。
MGY及MGYH(minimal glycerol medium±histidine)1L：1.34％YNB 1％甘油4×10-5％生物素±0.004％组氨酸
1在800ml水中加入100ml 10×YNB,，2ml 500×B，100ml 10×GY
2培养his4菌株，需加入组氨酸(称为MGYH)，加入10ml 100×H贮存液，4℃保存，
饱和硫酸铵：以1000ml为单位按下列比例配制。
硫酸铵     1000g以上
平衡液：20mmol PB缓冲液(pH7.2)，以1000ml为单位按下列比例配制。
磷酸二氢钠.2H 2O      3.12g
磷酸氢二钠.12H 2O     7.16g
EDTA                 0.37g
(4)中所说的重组人Fc抗体CHO细胞株的培养及纯化步骤是：高效表达的CHO细胞株表达，以无血清1640培养基培养24h，收集上清液，用0.45μm滤器过滤，上清缓慢加入等体积的饱和硫酸铵，混匀后4℃放置4小时以上，4℃下5000g离心20min收集沉淀，沉淀用100ml平衡液溶解，并对平衡液2000平衡液透析12小时以上，中间换夜一次；样品再上用平衡液平衡好的阴离子层析柱或-20℃保存备用，分别用含0.1mol/L、0.2mol/L、0.5mol/L和1mol/LNaCl的平衡液洗脱，收集洗脱峰，SDS电泳鉴定，取含分子量39kD蛋白质部分；用平衡液平衡Separose12层析柱；将上述浓缩的39kD蛋白质部分按床体积的3％比例上样，流速为2ml/min；分步收集；SDS电泳鉴定后，收集分子量为39kD 纯度高的蛋白质部分即为重组人Fc抗体。
溶液准备：
无血清1640培养基。
饱和硫酸铵：以1000ml为单位按下列比例配制。
硫酸铵     1000g以上
平衡液：20mmol PB缓冲液(pH7.2)，以1000ml为单位按下列比例配制。
磷酸二氢钠.2H 2O      3.12g
磷酸氢二钠.12H 2O     7.16g
EDTA                 0.37g
(5)将上述重组人Fc抗体制品用于诱导肿瘤细胞凋亡，以体外培养细胞为例：将肿瘤细胞接种到96孔细胞培养板内，培养过夜；加入不同浓度的重组人Fc抗体，继续培养24小时，用AnnexinV法测定细胞凋亡，试验设正常细胞对照。结果6g/L重组单链抗体可使31.45％以上的肿瘤细胞发生凋亡。
(5)将上述重组人Fc抗体制品用于抗肿瘤治疗,以动物实验为例:将裸鼠皮下接种肿瘤细胞，待肿瘤长至150mm 3左右时，将小鼠随机分组，分别皮下注射高、低二种不同计量的重组单链抗体及生理盐水(对照组)，隔日注射，连续20天，实验结果表明重组单链抗体对肿瘤细胞具有明显的抑瘤作用，与对照组比治疗组肿瘤体积增殖明显缓慢。
附图说明
下面结合附图进一步说明本发明。
图1为将人工合成Fc-Linker-VP3片段，定向克隆到pPIC9K载体EcoRI和NotI酶切位点表达载体构建方案。
图2为重组人Fc抗体表达及纯化结果,图中1 2、3、4、5，为纯化的重组人Fc抗体；6为分子量标准，从大到小依次为97.0，66.2，43.0，31.0，14.3kDa；7为发酵表达的重组人Fc抗体蛋白。
图3为重组人Fc抗体表达及纯化结果,图中1为分子量标准，从大到小依次为97.0，66.2，43.0，31.0，14.3kDa；2、3为纯化的重组人Fc抗体；4为CHO细胞表达的重组人Fc抗体蛋白
图4重组人Fc抗体体外对乳腺癌MCF-7细胞的增殖抑制，图中标示分别为：纵坐标为细胞增殖抑制率；横坐标为重组人Fc抗体的浓度梯度。
图5为重组人Fc抗体对乳腺癌MCF-7细胞凋亡诱导作用，图中标示分别为： 1、2、3的重组人Fc抗体浓度分别为：0g/L 3g/L和 6g/L。
图6为重组人Fc抗体对裸鼠体内乳腺癌的治疗作用。图中标示分别为：上图为肿瘤体积线条图，下图是肿瘤的实体照片。
具体实施方式
(1)通过直接人工合成的方法直接获得重组人Fc抗体的基因片段；将Fc-Linker-VP3片段，定向克隆到pPIC9K载体EcoRI和NotI酶切位点，如图1所示；常规制备酵母系统GS115菌株感受态细胞，在20ul水中加入50ug DNA,将DNA直接加入仍处于冰冻状态的感受态细胞管中，37℃水浴5min，其间混匀2次；取出转化管，加入1.5ml Buffer B，完全混匀，30℃水浴1小时；室温下，1500g离心样品10min，去净上清，用1.5ml Buffer C重悬细胞，离心，用0.2ml Buffer C重悬细胞并全部涂在选择生长平板上，30℃孵育3～4天，筛选遗传霉素高抗性克隆(图1)。
(2)重组人Fc抗体的发酵，高效表达及纯化，具体是：
(2.1)实验材料YPD-平板：MGY及MGYH培养基，饱和硫酸铵，平衡液：20mmol PB缓冲液(pH7.2)，阴离子层析柱，Separose12层析柱。
(2.2)实验方法取单个高效表达的克隆菌株，接种于含25mlMGY，BMG或BMGY的250ml摇瓶中，28～30℃，250～300rpm培养到OD 600＝2～6(约12～20小时)；将上述25ml培养基转钟到含1L MGY，BMG或BMGY的5L的摇瓶中，28～30℃度高速振荡(250～300rpm)，培养到对数生长期(OD 600＝2～5)；用灭菌离心管，室温1500～3000g离心5min收集细胞；用MM，BMM或BMMY重悬细胞至OD 600＝1.0(2～6L)进行诱导；分装培养基至N个5L隔板摇瓶，1L/瓶，用8层灭菌纱布盖住瓶口，放入摇床28～30℃继续培养；每24小时加甲醇1次，终浓度为0.5％，直到达最佳诱导时间；室温，1500～3000g离心5min，保留上清，置4℃预冷，为蛋白纯化备用。将上述培养上清缓慢加入等体积的饱和硫酸铵，混匀后4℃放置4小时以上，4℃下5000g离心20min收集沉淀，沉淀用100ml平衡液溶解，并对平衡液2000平衡液透析12小时以上，中间换夜一次；样品再上用平衡液平衡好的阴离子层析柱或－20℃保存备用，分别用含0.1mol/L、0.2mol/L、0.5mol/L和1mol/LNaCl的平衡液洗脱，收集洗脱峰，SDS电泳鉴定，取含分子量39kD蛋白质部分；用平衡液平衡Separose12层析柱；将上述浓缩的39kD蛋白质部分按床体积的3％比例上样，流速为2ml/min；分步收集；SDS电泳鉴定后，取分子量为39kD纯度高的蛋白质部分即为重组人Fc抗体；－20℃ 保存待鉴定。
(2.3)实验结果采用SDS电泳法或高效液相法测定蛋白质纯度，纯度在95％以上,分子量为39kD，采用紫外分光光度计测定蛋白质含量，约为0.5mg/ml。(图2)
(3)重组人Fc抗体哺乳细胞株的构建、培养及纯化
(3.1)实验材料：中国仓鼠卵巢细胞即CHO细胞，脂质体DOTAP，完全1640培养基。
(3.2)试验方法将人工合成的基因片段“Fc-linker-VP3”亚克隆到哺乳细胞表达载体pATX2载体，脂质体法转染哺乳细胞CHO。CHO细胞的转染方法：弃去CHO细胞原来的培养基，加入约5m新鲜培养基L，再加入DNA/DOTAP混合物150止，轻轻摇晃培养瓶，使之在培养基中分布均匀，置37℃细胞培养箱培养3～10h后换新鲜完全1640培养基，48h后加G418(800μg/mL)筛选，实验同时设未转染细胞和空载体细胞为阴性对照，以无血清1640培养基培养，各实验组每24h收集上清液，用0.45μm滤器过滤，上清液-20℃保存；上清液中IgG1-VP3融合蛋白的纯化如下；将上述培养上清缓慢加入等体积的饱和硫酸铵，混匀后4℃放置4小时以上，4℃下5000g离心20min收集沉淀，沉淀用100ml平衡液溶解，并对平衡液2000平衡液透析12小时以上，中间换夜一次；样品再上用平衡液平衡好的阴离子层析柱或－20℃保存备用，分别用含0.1mol/L、0.2mol/L、0.5mol/L和1mol/LNaCl的平衡液洗脱，收集洗脱峰，SDS电泳鉴定，取含分子量39kD蛋白质部分；用平衡液平衡Separose12层析柱；将上述浓缩的39kD蛋白质部分按床体积的3％比例上样，流速为2ml/min；分步收集；SDS电泳鉴定后，取分子量为39kD纯度高的蛋白质部分即为重组人Fc抗体；－20℃保存待鉴定。
(3.3)实验结果采用SDS电泳法或高效液相法测定蛋白质纯度，纯度在95％以上,分子量为39kD，采用紫外分光光度计测定蛋白质含量，约为0.5mg/ml。(图3)
(4)重组人Fc抗体体外细胞培养及体外增殖-毒性实验
(4.1)实验材料人乳腺癌MCF-7细胞购买于中国科学院典型培养物保藏委员会细胞库，在含10％胎牛血清的DMEM/F12培养基中培养，37℃、5％CO 2饱和湿度的条件下贴壁生长。
(4.2)实验方法制备乳腺癌MCF-7细胞单细胞悬液，将悬液加入96孔板 内，调节每孔细胞数为3×10 4个/ml。96孔板内培养细胞24h后加入重组人Fc抗体，使其终浓度为2、4、6、8、10、12、14、16、18、20g/L，终体积为200μl，每个浓度为3孔，并设置调零孔(只加培养基)、对照组(加入终浓度为0.1％的DMSO)。培养24h后加10ul的CCK-8试剂和100ul培养基，混匀后孵育24h后用酶标仪检测450nm下的各孔吸光度OD值，以每个浓度的3个复孔OD值的平均值为各浓度的平均OD值，计算不同药物浓度的细胞抑制率(IR)：细胞抑制率(％)＝(Ac-As)/(Ac-Ab)×100％。As：实验孔，含细胞+培养基+CCK-8+毒性物质；Ac：对照孔，含细胞+培养基+CCK-8；Ab：空白孔，含培养基+CCK-8。使用Graphpad Prism软件计算半数抑制浓度(IC 50值)。
(4.3)实验结果终浓度为2、4、6、8、10、12、14、16、18、20g/L重组人Fc抗体处理乳腺癌MCF-7细胞24h，对细胞的抑制率分别：13.4％、32.2％、49.7％、63.6％、80.1％、89.7％、91.2％、93.1％、93.9％、94.1％。与对照组相比，重组人Fc抗体能明显抑制乳腺癌MCF-7细胞的增殖(P<0.05),并存在一定的浓度依赖性，24h的IC 50为6.192g/L。以上实验数据均来自三次不同实验。(图4)。
(5)重组人Fc抗体体外肿瘤细胞培养-细胞凋亡实验
(5.1)实验材料人乳腺癌MCF-7细胞购买于中国科学院典型培养物保藏委员会细胞库，在含10％胎牛血清的DMEM/F12培养基中培养，37℃、5％CO 2饱和湿度的条件下贴壁生长。
(5.2)实验方法乳腺癌MCF-7细胞接种培养瓶中，实验组参考CCK-8试验结果，加入重组人Fc抗体的浓度分别为3g/L和6g/L。24h后终止培养，胰酶消化细胞，制成单细胞悬液，调整细胞浓度为1×10 6个/ml，取1ml细胞，1000r/min 4℃离心5min，弃上清后加入1ml冷的PBS，轻轻震荡使细胞悬浮，重复离心2次。将细胞重悬于400ul结合缓冲液(Binding Buffer)，加入异硫氰酸荧光素(F1TC)标记的AnnexinV 5ul，避光4℃反应15min，再加入10ul PI后轻轻混匀后4℃孵育5min，然后用流式细胞仪检测细胞凋亡。
(5.3)实验结果结果显示实验组细胞的早期和晚期凋亡率和坏死率均明显大于对照组。0、3、6g/L重组人Fc抗体实验组早期凋亡率分别为2.42％、17.14％、31.45％，晚期凋亡和细胞坏死率为1.30％、2.73％、9.26％(图5)。
(6)重组人Fc抗体体内抗肿瘤作用：实验委托日本大阪癌症化学治疗研究所完成。
(6.1)实验材料：裸鼠雄性(体重18～20g)，人癌乳腺癌(H185株)均为该 研究所饲养和保存。
(6.2)实验方法将人乳腺癌H185株组织块5mm 3接种于裸鼠右侧腋部皮下,所有动物常规饲养，待肿瘤包快长到150mm 3后，随机分为重组人Fc抗体治疗30μg/ml组、重组人Fc抗体治疗50μg/ml组和生理盐水组，7只/组。治疗组：裸鼠右侧腋部皮下注射重组人Fc抗体治疗30μg/ml和50μg/ml(0.1mL)/只，生理盐水组分别注射等体积的生理盐水。每组裸鼠隔日注射1次；每日观察小鼠一般生活状况，肿瘤大小，隔日测量体重，于接种后3wk脱颈椎处死，分离肿瘤组织测量瘤体积。
(6.3)实验结果实验裸鼠的一般状况，接种瘤细胞后,各组小鼠正常喂养，接种1wk内一般生理状况没有明显变化。1wk后，与治疗组相比较，生理盐水组小鼠进食减少、消瘦、体重下降、皮毛失去光泽,肿瘤包块明显增大；而治疗组小鼠一般状况良好。32天后活杀小鼠，取肿瘤组织测量瘤体积，治疗组30μg/ml和50μg/ml组的抑瘤率分别为61.6％和64.6％；抑瘤率差异显著(p<0.01)(附表)(参见图6)。
附表：重组人Fc抗体对裸鼠体内乳腺癌的瘤体体积和重量的影响
Figure PCTCN2019071725-appb-000001
注：**：p<0.01
本发明的有益效果是：重组人Fc抗体可在真核细胞中高效表达、稳定好、纯度高且有高活性，治疗效果显著，特别是实施本发明对生产设备没有特殊的要求，适合规模化和产业化，对于人类攻克肿瘤、延长人类寿命具有重要意义。

Claims (5)

  1. 一种特异性诱导肿瘤细胞凋亡的重组人Fc抗体，其特征是：
    (1)通过人工合成的方法直接获得重组人Fc抗体基因片段，其完整核酸序列为SEQ1，SEQ1序列由下列三个基因片段构成：人IgG H链Fc片段的基因片段、序列为SEQ2；链接片段基因、序列为SEQ2；VP3基因片段、序列为SQE4；
    (2)此重组人Fc抗体的完整氨基酸序列为序列SEQ5，该序列由下列3个氨基酸片段构成：IgG H链Fc片段的氨基酸片段、序列为SEQ6；链接片段氨基酸、序列为SEQ7；VP3氨基酸片段、序列为SQE8。
  2. 按照权利要求1所述的特异性诱导肿瘤细胞凋亡的重组人Fc抗体，其特征是：所述重组人Fc抗体基因片段亚克隆到酵母表达载体及在酵母细胞中表达；或亚克隆到哺乳细胞表达载体及在哺乳细胞中表达。
  3. 制备权利要求2所述的特异性诱导肿瘤细胞凋亡的重组人Fc抗体的方法，其特征是：
    (1)重组人Fc抗体酵母工程菌的构建、发酵及回收，将人工合成的基因片段“Fc-linker-VP3”亚克隆到酵母表达载体pPIC9K载体，转化毕赤酵母系统GS115菌株，筛选高效表达细胞株，表达重组人Fc抗体蛋白；
    (2)重组人Fc抗体蛋白的纯化取发酵培养上清缓慢加入等体积的饱和硫酸铵，混匀后4℃放置4小时以上，4℃下5000g离心20min收集沉淀，沉淀用100ml平衡液溶解，并对平衡液2000平衡液透析12小时以上，中间换夜一次；样品再上用平衡液平衡好的阴离子层析柱或-20℃保存备用，分别用含0.1mol/L、0.2mol/L、0.5mol/L和1mol/LNaCl的平衡液洗脱，收集洗脱峰，SDS电泳鉴定，取含分子量39kD蛋白质部分；用平衡液平衡Separose12层析柱；将上述浓缩的39kD蛋白质部分按床体积的3％比例上样，流速为2ml/min；分步收集；SDS电泳鉴定后，收集分子量为39kD纯度高的蛋白质部分即为重组人Fc抗体。
    (3)纯化的重组人Fc抗体鉴定采用SDS电泳法或高效液相法测定蛋白质纯度，纯度在95％以上,分子量为39kD，采用紫外分光光度计测定蛋白质含量，约为0.5mg/ml。
  4. 制备权利要求2所述的特异性诱导肿瘤细胞凋亡的重组人Fc抗体的方法，其特征是：
    (1)重组人Fc抗体哺乳细胞株的构建、发酵及回收，将人工合成的基因片 段“Fc-linker-VP3”亚克隆到哺乳细胞表达载体pATX2载体，脂质体法转染哺乳细胞CHO细胞株表达，筛选高效表达细胞株，表达重组人Fc抗体蛋白；
    (2)重组人Fc抗体蛋白的纯化取CHO细胞培养上清，缓慢加入等体积的饱和硫酸铵，混匀后4℃放置4小时以上，4℃下5000g离心20min收集沉淀，沉淀用100ml平衡液溶解，并对平衡液2000平衡液透析12小时以上，中间换夜一次；样品再上用平衡液平衡好的阴离子层析柱或-20℃保存备用，分别用含0.1mol/L、0.2mol/L、0.5mol/L和1mol/LNaCl的平衡液洗脱，收集洗脱峰，SDS电泳鉴定，取含分子量39kD蛋白质部分；用平衡液平衡Separose12层析柱；将上述浓缩的39kD蛋白质部分按床体积的3％比例上样，流速为2ml/min；分步收集；SDS电泳鉴定后，收集分子量为39kD纯度高的蛋白质部分即为重组人Fc抗体。
    (3)纯化的重组人Fc抗体鉴定采用SDS电泳法或高效液相法测定蛋白质纯度，纯度在96％以上,分子量为39kD，采用紫外分光光度计测定蛋白质含量，约为0.5mg/ml。
  5. 按照权利要求3或4所述的一种特异性诱导肿瘤细胞凋亡的重组人Fc抗体的制备方法获得的制品，用于抗恶性肿瘤治疗。
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