WO2011103701A1 - 全人源抗TNF-α单克隆抗体、其制备方法及用途 - Google Patents

全人源抗TNF-α单克隆抗体、其制备方法及用途 Download PDF

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WO2011103701A1
WO2011103701A1 PCT/CN2010/000512 CN2010000512W WO2011103701A1 WO 2011103701 A1 WO2011103701 A1 WO 2011103701A1 CN 2010000512 W CN2010000512 W CN 2010000512W WO 2011103701 A1 WO2011103701 A1 WO 2011103701A1
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tnf
antibody
seq
human anti
monoclonal antibody
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郭怀祖
李川
仝昕
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BIOMAB PHARMACEUTICALS Ltd
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BIOMAB PHARMACEUTICALS Ltd
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Priority to JP2012554186A priority Critical patent/JP2013520181A/ja
Priority to CA2790013A priority patent/CA2790013C/en
Priority to BR112012021329-6A priority patent/BR112012021329B1/pt
Priority to US13/579,208 priority patent/US8597648B2/en
Priority to EP10846325.8A priority patent/EP2540743B1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to the field of biotechnology, and more particularly, to a fully human monoclonal antibody, a process for its preparation and use. Background technique
  • TNF- ⁇ is a multifunctional immunomodulatory molecule in the body that binds to receptors on the cell membrane and acts to cause death of target cells (its name is derived from this) or to attract immune effector cells locally. Gather. TNF-a is a soluble homotrimeric subunit with a molecular weight of 17 kD (Smith, et al., J. Biol. Chem. 262:6951-6954, 1987). A transmembrane-bound precursor, TNF- ⁇ , was also found with a molecular weight of 26 kD (Kreegler, et al. Cell 53:45-53, 1988). Mononuclear macrophages, when stimulated by endotoxin and other stimuli, secrete TNF- ⁇ and TNF- ⁇ , and other cells secrete TNF-a.
  • TOF-a plays a key role in the pathogenesis of rheumatoid arthritis, bacterial or viral infections, chronic inflammation, autoimmune diseases such as AIDS (AIDS:), malignant tumors and/or neurodegenerative diseases.
  • the TNF-a monoclonal antibody neutralizes TNF-a and plays a negative regulatory role in TNF-a activity in vivo.
  • TNF-a is also the main mediator of septic shock syndrome. Elevated levels of TNF-a in the serum of patients with septic shock syndrome predict an increase in mortality or disability.
  • Clinical application of TNF-a antibody or its receptor has a certain effect on septic shock syndrome.
  • TNF-a is one of the main mediators for promoting the asymptomatic infection of HIV into AIDS, while monoclonal antibodies against ⁇ - ⁇ are able to neutralize the activity of TNF-a and negatively regulate the activity of TNF-a in vivo. It can remove the incentive for patients to enter AIDS from asymptomatic infection status and achieve certain AIDS treatment goals.
  • monoclonal antibodies to TNF-a in combination with other AIDS drugs to antagonize side effects caused by excessive TNF-a will significantly improve efficacy.
  • a murine anti-TNF-a monoclonal antibody was used to neutralize the treatment of TNF-a.
  • murine monoclonal antibody has many defects as a therapeutic drug. This is because murine monoclonal antibody is used in humans with strong immunogenicity, rapid elimination in vivo, and short half-life, resulting in limited clinical efficacy and side effects.
  • humanized monoclonal antibody technology the defects of anti-TNF-a murine monoclonal antibody were overcome.
  • the human-mouse chimeric anti-TNF-o monoclonal antibody ⁇ Infliximab, Remicade®) is based on genetic engineering upstream construction technology.
  • variable region obtained from the preparation was still taken from the murine TNF- ⁇ monoclonal antibody, retaining the specificity and affinity (Ka K ⁇ JVT 1 ) binding to the tumor necrosis factor soluble fragment and the transmembrane region, and the constant region was human. Substituted by the constant region, the half-life in vivo is greatly extended.
  • TNF- ⁇ inhibitors that have been approved for marketing abroad include tumor necrosis factor receptor-antibody fusion protein (Enbrel, Amgen) and anti-tumor necrosis factor a all-human monoclonal antibody (Humim, Abbott).
  • the present invention constructs a large-capacity natural human phage antibody library, and a whole human anti-TNF-ot antibody 4H16 was obtained by screening.
  • the present invention provides a fully human anti-TNP-a antibody, wherein the heavy chain variable region amino acid sequence is represented by SEQ ID NO: 6, and the light chain variable region amino acid sequence is represented by SEQ ID NO: ;
  • the whole human anti-TNF-a antibody of the present invention has a heavy chain amino acid sequence of SEQ ID NO: 10 and a light chain amino acid sequence of SEQ ID NO: 12;
  • the present invention also provides an isolated nucleotide encoding the above-described fully human anti-TNF-a antibody; the nucleotide of the present invention, wherein the core encoding the heavy chain variable region of the fully human anti-TNF-a antibody
  • the nucleotide sequence of SEQ ID NO: 5 the nucleotide sequence encoding the light chain variable region of the fully human anti-TNF-a antibody is set forth in SEQ ID NO: 7.
  • nucleotide of the present invention wherein the nucleotide sequence encoding the heavy chain of the fully human anti-TNF-a antibody is represented by SEQ ID NO: 9, and the nucleotide encoding the light chain of the whole human anti-TNF-a antibody The sequence is shown in SEQ ID NO:11;
  • the present invention also provides an expression vector comprising the above nucleotide, which is pcDNA3.1/ZEO(+) or pcDNA3.1 (+);
  • the present invention also provides a host cell transformed with the above expression vector, which is a CHO-K1 cell; the present invention further provides a method for preparing the above fully human antibody, which comprises screening a phage human antibody library to obtain a high affinity full human anti-TNF-a single Chain antibody; fully human anti-TNF-a intact antibody molecule Construction of eukaryotic expression vector; expression of fully human anti-TNF-ot intact antibody molecule in CHO cells; purification of fully human anti-TNF- ⁇ intact antibody molecule. way.
  • autoimmune diseases are rheumatoid arthritis, ankylosing spondylitis or 4 scurf diseases.
  • the invention utilizes the obtained antibody to carry out a series of experiments, and the experimental results show that: with D2E7 (adalimumab monoclonal antibody, Abbott company), Chinese invention patent application number 200310108440.0 application date November 6, 2003, the invention name is "full human tumor necrosis” In comparison with 7B3 disclosed in Factor Antibody, Its Preparation Method, and Pharmaceutical Composition, the antibody obtained by the present invention has higher antibody affinity and stronger TNF-oc neutralizing ability.
  • Anti-TNF- ⁇ antibody 4H16 blocks the binding of TNF- ⁇ to soluble P75 receptor;
  • Anti-TNF-a antibody 4H16 blocks the binding of TNF-a to U-937 cell surface receptors. ;
  • SEQ ID NO: 1 and SEQ ID NO: 2 respectively show the nucleotide sequence and amino acid sequence of the heavy chain constant region ((3 ⁇ 4).
  • SEQ ID NO: 3 and SEQ ID NO: 4 respectively show the light chain constant region ( Nucleotide sequence and amino acid sequence of C L )
  • the correct clones in this example are designated as PGEM-T/CH and pGEM-T/C L .
  • RNA of cells was extracted from isolated human peripheral blood lymphocytes using Ti'izol reagent (Invitrogen).
  • the cDNA was reverse transcribed using a cDNA reverse transcription kit (Shanghai Shenneng Gaming Biotechnology Co., Ltd.). The above steps are carried out according to the instructions provided by the manufacturer.
  • V H Back V H For
  • VjJBack Vi of the human heavy chain variable region (V H ) and light chain variable region (VL) genes Or primer.
  • V H Back V H For
  • V L Back V L For 'J see Immunotechnology, 1998, 3: 271-278.
  • a phage single-chain antibody library was constructed using a recombinant Phage antibody system kit (Amersham Biosciences), and then the library was panned with a specific antigen.
  • Antibody library construction and panning methods were performed with reference to the recombinant Phage antibody system kit, and the specific antigen "recombinant human TNF-a (rhTNP-a)" for panning was purchased from R&D.
  • An anti-human TNP-single-chain antibody 4H16ScFv was obtained after multiple panning of the antibody library, and the gene sequence was obtained after sequencing.
  • SEQ ID NO: 5 and SEQ ID NO: 6 show the nucleotide and amino acid sequences V H of the heavy chain variable region 4H16 ScFv.
  • SEQ ID NO: 7 and SEQ ID NO: 8 show the nucleotide sequence and amino acid sequence of the 4H16 ScFv light chain variable region VL ⁇ , respectively.
  • the full human antibody heavy chain gene was synthesized by overlapping PCR under the following conditions: 95 V for 15 minutes; 94 °C for 50 seconds, 58 °C for 50 seconds, 72 °C for 50 seconds, 30 cycles; 72 ° C for 10 minutes.
  • the 5' end of the full human antibody heavy chain gene contains the restriction enzyme site Hindlll and the signal peptide gene sequence, and the 3' end contains the translation stop codon TAA and restriction enzyme sites.
  • the PCR amplification product is separated by agarose gel electrophoresis, and recovered.
  • the target band was cloned into the pGEM-T vector (Promega product), and positive clones were screened for sequencing.
  • the clones with the correct sequencing were digested with Hindlll and EcoR I, and the whole human antibody heavy chain fragment 4H16V H CH was purified by agarose gel electrophoresis, and the plasmid pcDNA3.1(+) digested with Hindlll and EcoR I was used.
  • the 4H16ScFv gene and PGEM-T/CL vector were used as templates to synthesize the fully humanized antibody light chain gene by overlapping PCR.
  • the reaction conditions were: 95 ° C for 15 minutes; 94 ° C for 50 seconds, 58 ° C for 50 seconds, 72 ° C. 50 seconds, 30 cycles; 72 ° C for 10 minutes, the PCR product was obtained, the 5' end contains the restriction enzyme site Hindlll and the signal peptide gene sequence, and the 3' end contains the translation stop codon TAA and the restriction enzyme site EcoR I.
  • the signal peptide gene sequence is
  • Plasmid 1 ( ⁇ g (plasmid pcDNA3.1(+)(4H16V H CH)4 g, plasmid pcDNA3.1/ZEO) (+) (4H16V L C L )6 g) and 20 ⁇ 1 Lipofectamine2000 Reagent (Invitrogen) were transfected according to the instructions of Lipofectamine2000 Reagent kit. After transfection for 24h, the cells were replaced with 60 ( ⁇ g/ml G418 (Invitrogen) And resistant clones were screened in DMEM medium of 250 ⁇ / ⁇ 1 Zeocin (Invitrogen).
  • TNFa antibodies were detected by surface plasmon resonance (SPR) using a Biacore T100 system (Biacore AB, Uppsala, Sweden).
  • Recombinant human TNFa product of R&D was covalently bound to the CM5 biosensor chip (Biacore) by amino group, and 1 full human antibody 4H16; 2 full human antibody adalimumab (Humim, D2E7, commercially available product); Positive control whole human anti-TNFct antibody 7B3; 4 negative control antibody Tmstuzumab (with PBS/0.05% T EEN-20 (ICI Americas) (detergent) with solution, formulated into different concentrations (2 times diluted concentration) The chip was passed through the chip at a flow rate of 50 ⁇ l/ ⁇ .
  • ⁇ of the ⁇ 75 receptor-Fc fusion protein (in accordance with the Chinese patent application No. 01132074.5 application date October 31, 2001, the invention name is "recombinant gene of tumor necrosis factor receptor soluble part, and its fusion gene and product"
  • the method disclosed in the preparation of the coated plate was carried out at 37 ° C for 2 hours; 3% of BSA blocked the plate well and reacted at 4 ° C overnight.
  • PBS biotinylated TNF- ⁇ obtained for R&D product 210-TA-050, obtained using Pierce's EZ-Link Sulfo-NHS-Biotinylation Kit 21425
  • Anti-TNF- ⁇ monoclonal antibody 4H16 (antibody of the invention), D2E7 (adalimumab mAb, Abbott), 7B3 and negative control
  • the body Trastuzumab (Genentech) was diluted to 10 g / ml, and diluted 2 times, the diluted sample and the control were added to the washed ELISA plate, ⁇ / well, reacted at 37 ° C for 1 hour; Plate, PBS diluted 1:1000 HRP-avidin (Zymed), ⁇ / well added to the microplate, reaction at 37 ° C for 1 hour; wash the enzyme plate, HRP substrate TMB A and B (Jingmei biological), etc.
  • U937 cells (ATCC CRL1593) were cultured in RPMI-1640 medium (GIBCO) containing 10% fetal calf serum (RHH), and the surface of the cells expressed TNF-ot receptor. After counting the cells in logarithmic growth phase, centrifuge at 200g for 5 minutes, discard the supernatant, resuspend the cell pellet in PBS containing 1% fetal bovine serum, adjust the cell density to l x10 6 /ml, and divide the cells into flow cytometry. In the tube, ⁇ /tube.
  • PBS fluorescein isothiocyanate (FITC, Amresco)-labeled TNF-a obtained as R&D product 210-TA-050, obtained by dialysis labeling
  • FITC fluorescein isothiocyanate
  • TNF-a monoclonal antibody 4H16 (antibody of the invention), D2E7 (adalimumab mAb, Abbott), 7B3 and negative control antibody Trastuzumab (Genentech) diluted to 100 g / ml, and diluted 2 times, will be diluted
  • the sample and the control were added to the flow tube, ⁇ /tube, and reacted at 4 ° C for 1 hour in the dark; the cells were washed twice with PBS containing 1% fetal bovine serum, centrifuged at 200 g for 5 minutes each time, the supernatant was discarded, and the cell pellet was heavy.
  • U937 cell surface receptors have the lowest IC50 for binding, and therefore have the highest affinity for TNF-oc.
  • L929 cells (ATCC CCL-1) cells were cultured in RPMI-1640 medium (GIBCO) containing 10% fetal bovine serum (JRH). After counting the cells in the logarithmic growth phase, centrifuge at 200 g for 5 minutes, discard the supernatant, resuspend the cell pellet with the above culture solution, adjust the cell density to lxl0 5 /ml, and add the cells to a 96-well culture plate, ⁇ /well, Incubate overnight at 37 ° C in a 5% CO 2 incubator.
  • RPMI-1640 medium containing 10% fetal bovine serum (JRH). After counting the cells in the logarithmic growth phase, centrifuge at 200 g for 5 minutes, discard the supernatant, resuspend the cell pellet with the above culture solution, adjust the cell density to lxl0 5 /ml, and add the cells to a 96-well culture plate, ⁇ /well, Incubate overnight at 37 ° C in
  • the culture solution was added to actinomycin D (Huamei) to a concentration of 2 ( ⁇ g/ml, TNF-a (R&D product 210-TA-050) to a concentration of 4 ng/ml.
  • actinomycin D Human anti-TNP-a monoclonal antibody 4H16 (antibody of the invention), D2E7 (adalimumab mAb, Abbott), 7B3 and negative control antibody Trastuzumab (Genentech).
  • Lg recombinant human TN-a (R&D product 210-TA-050) and 20mg D-galactosamine (Amresco) can induce 80-90% C57BL6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to die. .
  • first an amount of each antibody administered intraperitoneally 30 minutes before administration of 1 ⁇ ⁇ Recombinant human TNF- ⁇ and 20 mg D galactosamine were intraperitoneally injected to observe the protective effects of various antibodies.
  • the injection dose and results of each group are shown in Table 5 below.
  • Recombinant human TNF-a was injected intravenously into New Zealand white rabbits to induce a thermal response. 5 g/kg recombination
  • Human TNF-a-induced fever response in rabbits was approximately 0.5 °C.
  • New Zealand white rabbits (source?) were injected intravenously with 5 g/kg of recombinant human TNF-a and various doses of various antibodies, and the body temperature of the test animals was monitored before injection and 60 minutes after injection to evaluate The ability of various antibodies to neutralize the biological effects of recombinant human TNF-a.
  • the injection dose and results of each group are shown in Table 6 below.

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Description

全人源抗 TNF-α单克隆抗体、 其制备方法及用途 技术领域
本发明涉及生物技术领域， 更具体地， 本发明公开了一种全人源单克隆 抗体、 其制备方法及用途。 背景技术
TNF-α是体内的一种多功能免疫调节分子， 它可以和细胞膜上的受体结合 · 发挥作用， 往往引起靶细胞的死亡（它的名称即来源于此）或招引免疫效应细 胞在局部的聚集。 TNF-a是一种可溶性的同源三聚体亚基， 分子量为 17kD ( Smith, et al., J. Biol. Chem. 262:6951-6954, 1987 )。 也发现有跨膜结合的前体 TNF-α, 分子量为 26kD ( Kriegler, et al. Cell 53:45-53, 1988 )。 单核巨噬细胞在 受到内毒素和其他一些刺激物刺激后， 能够分泌 TNF-α和 TNF-β, 另外其他一 些细胞也能分泌 TNF-a。
TOF-a在类风湿关节炎、 细菌或病毒感染、 慢性炎症、 自身免疫病如艾滋 病 （AIDS：)、 恶性肿瘤和 /或神经退行性疾病等的病理进程中起十分关键的作 用。 TNF-a单克隆抗体可以中和 TNF-a,在体内对 TNF-a的活性起到了负调节 的作用。而且有大量研究表明， TNF-a还是引起脓毒性休克综合症的主要介质。 脓毒性休克综合症患者血清中 TNF-a水平升高预示着死亡率或致残率升高。临 床上应用 TNF-a抗体或者其受体对脓毒性休克综合症有一定疗效。
此外， TNF-a是促进 HIV无症状感染状态进入 AIDS的主要介质之一， 而针 对 ΝΡ-α的单克隆抗体能够中和 TNF- a的活性， 在体内对 TNF-a的活性进行负 调节， 可去除患者从无症状感染状态进入 AIDS的诱因， 达到一定的 AIDS治疗 目的。将 TNF-a的单克隆抗体与其他 AIDS药物联合应用，拮抗由 TNF-a过多所 引起的副作用， 将会明显提高疗效。
最初科学家们制备获得的是鼠源抗 TNF-a单克隆抗体， 用于中和 TNF-a的 治疗。但研究发现鼠源单抗作为治疗药物存在许多缺陷， 这是因为鼠源单抗用 于人体具有强烈的免疫原性， 体内消除快， 半衰期短， 导致临床疗效有限， 副 作用大。 随着人源化单抗技术的发展， 克服了抗 TNF-a鼠源单抗的缺陷。 其中 人鼠嵌合抗 TNF-o单抗 { Infliximab, Remicade® )是利用基因工程上游构建技 术制备获得的， 其可变区仍取自鼠源 TNF-α单抗， 保留了与肿瘤坏死因子可溶 性片段和跨膜区结合的特异性和亲和力（Ka K^JVT1 ), 恒定区为人 的恒 定区所取代， 体内半衰期大大延长。 另外还有已经在国外批准上市的 TNF-α抑 制剂有肿瘤坏死因子受体一抗体融合蛋白 （Enbrel, Amgen公司）和抗肿瘤坏 死因子 a全人单抗（Humim, Abbott公司 ) 。
从作用的靶点和作用特异性的角度来说，上述几种药物的作用机理几乎是 完全相同的。但上述抗体或融合蛋白都存在不同程度的免疫原性高、特异性低 和 /或稳定性不够等问题， 因此， 本领域迫切需要建立既能够保持或提高抗体 的亲和力和特异性， 又能够降低或基本消除抗体免疫原性的抗 NF-a抗体， 从 而进一步提高临床应用的安全性和有效性。 发明内容
本发明构建了大容量的天然人源噬菌体抗体库， 从中筛选获得了一株全人 源抗 TNF-ot抗体 4H16。
更具体地，本发明提供一种全人源抗 TNP-a抗体，其重链可变区氨基酸序 列为 SEQ ID NO:6所示， 轻链可变区氨基酸序列为 SEQ ID NO:8所示；
本发明所述的全人源抗 TNF-a抗体， 其重链氨基酸序列为 SEQ ID NO:10 所示， 轻链氨基酸序列为 SEQ ID NO: 12所示；
本发明还提供一种分离的核苷酸， 编码上述的全人源抗 TNF-a抗体； 本发明所述的核苷酸，其中编码全人源抗 TNF-a抗体重链可变区的核苷酸 序列为 SEQ ID NO:5所示， 编码全人源抗 TNF-a抗体轻链可变区的核苷酸序 列为 SEQ ID NO:7所示；
本发明所述的核苷酸，其中编码全人源抗 TNF-a抗体重链的核苷酸序列为 SEQ ID NO:9所示， 编码全人源抗 TNF-a抗体轻链的核苷酸序列为 SEQ ID NO: 11所示；
本发明还提供一种表达载体， 含有上述的核苷酸， 为 pcDNA3.1/ZEO(+) 或 pcDNA3.1 (+);
本发明还提供上述表达载体转化的宿主细胞， 为 CHO-K1 细胞； 本发明进一步提供上述全人源抗体的制备方法，包括从噬菌体人源抗体库 筛选获得高亲和力全人源抗 TNF-a单链抗体； 全人源抗 TNF-a完整抗体分子 真核表达载体的构建； 全人源抗 TNF-ot完整抗体分子在 CHO细胞中的表达； 全人源抗 TNF-α完整抗体分子的纯化。 途。 其中自身免疫性疾病为类风湿性关节炎、 强直性脊柱炎或 4艮屑病。
本发明利用得到的抗体进行了一系列实验， 实验结果表明： 与 D2E7 ( adalimumab单抗， Abbott公司 )、 中国发明专利申请号 200310108440.0申请 日 2003年 11月 6 日发明名称为 《全人源肿瘤坏死因子抗体，其制备方法以及 药物组合物》 中公开的 7B3 比较起来， 本发明得到的抗体具有更高的抗体亲 和力和更强的 TNF-oc中和能力。 附图说明
图 1. 抗 TNF-α抗体 4H16阻断 TNF-α与可溶性 P75受体的结合实验结果； 图 2. 抗 TNF-a抗体 4H16阻断 TNF-a与 U-937细胞表面受体的结合实验 结果；
图 3. 抗 TNF-a抗体 4H16拮抗 TNF-a介导的 L929细胞的杀伤效应实验结 果； 具体实施方式
以下实施例、 实验例仅仅对本发明进行进一步的说明， 不应理解为对本发 明的限制。
实施例 抗体的制备
( 1 )人抗体轻、 重链恒定区基因的克隆
用淋巴细胞分离液（鼎国生物技术发展公司产品）分离健康人淋巴细胞， 用 Trizol试剂（Invitrogen公司产品)提取总 R A, 根据文献（Cell,1980,22: 197-207 )和文献（Nucleic Acids Research, 1982， 10: 4071-4079 )报道的序 列分别设计引物采用 RT-PCR反应扩增抗体重链和轻链恒定区基因。 PCR产物 经琼脂糖凝胶电泳纯化回收并克隆到 pGEM-T载体（Promega公司产品） 中， 测序验证后确认获得了正确的克隆。 SEQ ID ΝΟ:1和 SEQ ID NO:2分别显示了 重链恒定区（(¾ )的核苷酸序列和氨基酸序列。 SEQ ID NO:3和 SEQ ID NO:4 分别显示了轻链恒定区 (CL)的核苷酸序列和氨基酸序列。 本例中的正确克隆记 作 PGEM-T/CH和 pGEM-T/CL。 (2) cDNA的制备
收集 50健康人的外周血各 20ml， 混合， 用淋巴细胞分离液 (医科院天津 血研所生产)分离单个核细胞。 用 Ti'izol试剂 （Invitrogen公司）从分离的人外 周血淋巴细胞中提取细胞的总 RNA。 用 cDNA反转录试剂盒（上海申能博彩 生物科技有限公司）反转录出 cDNA。 以上步骤按照厂家提供的说明书进行。
(3)引物设计
参考文献（ Immunotechnology, 1998,3 :271 -278 )设计并合成克隆人抗体重 链可变区 (VH)和轻链可变区 (VL)基因的 VHBack, VHFor, VjJBack和 Vi or引 物 。 VHBack , VHFor , VLBack 和 VLFor 的 序 歹' J 见 Immunotechnology, 1998,3 :271-278。 其中在 VHBack引物的 5，端加上含有 Sfi I 位 、的序歹 'J atg gcc cag ccg gcc atg gcc, 在 VHFor引物 5，端力口上序歹1 J gcc aga acc acc gcc gcc gga gcc acc acc gcc,在 VLBack引物 ό 5，端力口上序歹1 J tec ggc ggc ggt ggt tct ggc gga ggc gga tct, 在 VLFor引物々 5，端力口上含有 ot I位 、 ό 序歹 'J atg egg ccg c。
( 4 ) .噬菌体抗体库的构建及筛选
采用（2)中的 cDNA和 (3)中的引物，利用 recombinant Phage antibody system 试剂盒（Amersham Biosciences公司）构建噬菌体单链抗体库， 然后用特异性 抗原对文库进行淘选。 抗体库构建和淘选方法参照 recombinant Phage antibody system试剂盒说明书进行， 用于淘选的特异性抗原"重组人 TNF-a(rhTNP-a)" 购自 R&D 公司。 经多次淘选抗体库后获得了一株抗人 TNP- 单链抗体 4H16ScFv， 测序后获得其基因序列。 SEQ ID NO:5和 SEQ ID NO:6分别显示 了 4H16 ScFv重链可变区 VH的核苷酸序列和氨基酸序列。 SEQ ID NO:7和 SEQ ID NO:8分别显示了 4H16 ScFv轻链可变区 VL^的核苷酸序列和氨基酸序列。
( 5 )全人源抗体在真核细胞中的表达
以 4H16ScFv基因和 PGEM-T/CH为模版， 通过重叠 PCR合成全人源抗体重 链基因， 反应条件为： 95 V 15分钟； 94 °C 50秒， 58°C50秒， 72°C 50秒， 30 个循环； 72°C 10分钟。 并使此全人源抗体重链基因的 5'端含有限制酶位点 Hindlll和信号肽基因序列， 3 '端含有翻译终止密码 TAA和限制酶位点
EcoR l 。 信 号 肽 基 因 序 列 为
GTCATAATATCCAGAGGA )。 最后琼脂糖凝胶电泳分离 PCR扩增产物， 回收 目的条带并克隆到 pGEM-T载体（Promega公司产品）中， 筛选阳性克隆测序。 挑选测序正确的克隆用 Hindlll和 EcoR I酶切， 经琼脂糖凝胶电泳纯化回收全 人源抗体重链片段 4H16VHCH, 与用 Hindlll和 EcoR I酶切的质粒 pcDNA3.1(+)
( Invitrogen公司）进行连接， 构建成全人源重链真核表达载体 pcDNA3.1 (+) (4H16VHCH)。
以 4H16ScFv基因和 PGEM-T/CL载体为模板，通过重叠 PCR合成全人源 化抗体轻链基因， 反应条件为： 95°C 15分钟； 94°C 50秒， 58°C50秒， 72°C 50秒， 30个循环； 72 °C 10分钟，得到 PCR产物，其 5'端含有限制酶位点 Hindlll 和信号肽基因序列， ，3 '端含有翻译终止密码 TAA和限制酶位点 EcoR I。 信 号肽基因序列为
GTCATAATATCCAGAGGA )。挑选测序正确的克隆用 Hindlll和 EcoR I酶切， 经琼脂糖凝胶电泳纯化回收全人源抗体轻链片段 4Η16ν! ^， 与用 Hindlll和 EcoR l酶切的盾粒 pcDNA3.1/ZEO(+) ( Invitrogen公司）载体进行连接， 构建 成全人源轻链真核表达载体 pcDNA3.1/ZEO(+) (4Η16ν]^Ο 。
于 3.5cm组织培养 i中接种 3 x 105 CHO-K1
细胞（ATCC CRL-9618 ) , 细胞培养至 90%-95%融合时进行转染： 取质粒 l(^g( 质 粒 pcDNA3.1(+)(4H16VHCH)4 g , 质 粒 pcDNA3.1/ZEO(+) (4H16VLCL)6 g)和 20μ1 Lipofectamine2000 Reagent ( Invitrogen公司产品）按 Lipofectamine2000 Reagent试剂盒说明书进行转染。转染进行 24h后细胞换含 60(^g/ml G418 ( Invitrogen公司产品 )和 250μ /ιη1 Zeocin ( Invitrogen公司 产品）的 DMEM培养基筛选抗性克隆。取细胞培养上清用 ELISA检测筛选高 表达克隆： 羊抗人 IgG (Fc) ( KPL公司） 包被于 ELISA板， 4。C过夜， 用 2 %BSA-PBS于 37。C封闭 2h, 加入待测的抗性克隆培养上清或标准品
Human myeloma IgGl,K ( Sigma), 37°C 温育 2h, 加入 HRP -羊抗人 IgG(K) ( (Southern Biotechnology Associates公司）进行结合反应， 37。C 温育 lh， 力口 入 TMB显色液于 37°C作用 5min, 最后用 H2S04终止反应， 测 A450值。 将筛 选得到的高表达克隆用无血清培养基扩大培养， 用 Protein A亲和柱（ GE公司 产品）分离纯化全人源抗体 4H16。 将纯化抗体用 PBS进行透析， 最后以紫外 重链核苷酸序列和氛基酸序列。 SEQ ID NO: 11和 SEQ ID NO: 12分别显示了全 人源抗体 4H16的轻链核苷酸序列和氨基酸序列。
实验例
7B3 : 按照中国专利申请号 200310108440.0, 申请日 2003年 11月 6 曰， 发明名称 《全人源肿瘤坏死因子抗体，其制备方法以及药物组合物》 中公开的 方法制备得到。
实验例 1.抗 TNFa抗体的亲和力检测
运用 Biacore T100系统 (Biacore AB, Uppsala, Sweden)通过表面等离子体 激元共振（ SPR )检测 TNFa抗体的亲和力常数。 将重组人 TNFa(R&D公司产 品）通过氨基共价结合与 CM5 生物传感芯片（Biacore)上， 将①全人源抗体 4H16; ②全人源抗体 adalimumab ( Humim, D2E7 , 市售产品）； ③阳性对照 全人源 抗 TNFct 抗体 7B3 ； ④ 阴 性对照 抗体 Tmstuzumab( 以 PBS/0.05%T EEN-20(ICI Americas) (去污剂） 配溶液， 配成不同的浓度 (2倍 比浓度稀释)， 以 50μ1/πώι的流速通过芯片。 每次检查之后， 用 5μ1 50mM盐 酸水溶液以 3μ1/ηώι的流速洗涤， 从而将残留的抗体从固定化的配体上洗脱下 来。 用 BIAevalution软件 （T100 evalution 2.0版， Biacore ), 通过非线性回归 法分析结合曲线。 结果如表 1所示， 全人源抗体 4H16的 KD值显著低于全人 源抗体 adalimumab和全人源 TN a抗体 7B3 , 说明全人源抗体 4H16对 ΤΝΡα 的亲和力高于 adalimumab和全人源抗体 TNFa抗体 7Β3 , 实验结果见表 1 表 1亲和力实验结果
抗体 Kon ( M-lS-1/105 ) Koff ( 105S-1 ) KD ( nM )
4H16 1.58 5.7 0.36 adalimumab 1.35 8.5 0.63
7B3 1.32 9.5 0.72
Trastuzumab ND ND ND 实验例 2.抗 TNF-a抗体 4H16阻断 TNF-ot与可溶性 F75受体的结合实验
ΙΟμ^πύ的 Ρ75受体 -Fc融合蛋白（按照中国专利申请号 01132074.5申请日 2001年 10月 31 日发明名称为 《肿瘤坏死因子受体可溶部分的重组基因，及其 融合基因与产物》中公开的方法制备得到）包被酶标板， 37°C反应 2小时； 3% 的 BSA封闭板孔， 4°C反应过夜。 PBS将生物素标记的 TNF-α (为 R&D公司 产品 210-TA-050 , 使用 Pierce公司 EZ-Link Sulfo-NHS-Biotinylation Kit 21425 标记获得）稀释至 10ng/ml, 用该稀释液将全人源抗 TNF-α单克隆抗体 4H16 (本发明抗体）、 D2E7 ( adalimumab单抗， Abbott公司）、 7B3及阴性对照抗 体 Trastuzumab ( Genentech )稀释至 lO g/ml, 并连续 2倍比稀释， 将稀释好 的样品及对照品加入洗涤后的酶标板中， ΙΟΟμΙ/孔， 37°C反应 1小时； 洗涤酶 标板， PBS按照 1 :1000稀释 HRP-avidin ( Zymed ), ΙΟΟμΙ/孔加入酶标板， 37°C 反应 1小时； 洗涤酶标板， HRP底物 TMB的 A液和 B液（晶美生物）等体 积混合后， ΙΟΟμΙ/孔加入酶标板， 室温避光反应 10分钟； 每孔加入 ΙΟΟμΙ浓度 为 0.5Μ的硫酸终止反应， 酶标仪读取 490nm的光吸收值。 以样品浓度为横坐 标， 光吸收值为纵坐标， 结果见表 2和图 1。
表 2
IC50 ( /ml )
实验编码 4H16 7B3 D2E7
1 0.25 1.19 2.30
2 0.31 1.42 2.15
3 0.27 1.06 2.59 平均 0.28±0.03 1.22±0.18 2.35±0.22 实验结果显示， 本发明全人源抗 TNF-α单克隆抗体 4H16阻断 TNF-a与 P75 受体的结合的 IC50最小， 因此其与 TNF-a的亲和力最高。
实验例 3.抗 TNF-a抗体 4H16阻断 TNF-a与 U-937细胞表面受体的结合实验
U937细胞 ( ATCC CRL1593 ) 细胞培养于含 10%胎牛血清 （ JRH ) 的 RPMI- 1640培养液（GIBCO ) 中， 细胞表面表达 TNF-ot受体。 对数生长期细胞 计数后， 200g离心 5分钟， 弃上清， 细胞沉淀用含 1%胎牛血清的 PBS重悬， 调 整细胞密度至 l xl06/ml, 将细胞分至流式细胞术检测管中， ΙΟΟμΙ/管。 PBS将异 硫氰酸荧光素（ FITC, Amresco )标记的 TNF-a (为 R&D公司产品 210-TA-050, 使用透析法标记获得）稀释至 100ng/ml， 用该稀释液将全人源抗 TNF-a单克隆 抗体 4H16 (本发明抗体）、 D2E7 ( adalimumab单抗， Abbott公司）、 7B3及阴性 对照抗体 Trastuzumab ( Genentech )稀释至 lOO g/ml, 并连续 2倍比稀释， 将稀 释好的样品及对照品加入流式管中， ΙΟΟμΙ/管， 4°C避光反应 1小时； 含 1%胎牛 血清的 PBS洗涤细胞 2次， 每次 200g离心 5分钟， 弃上清， 细胞沉淀重悬于 300μ1 含 1%胎牛血清的 PBS, 流式细胞仪检测每管的荧光强度。 以样品浓度为横坐 标， 收值为纵坐标， 结果见表 3和图 2。
IC50 ( μg /ml )
实验编码 4H16 7B3 D2E7
1 0.89 3.42 6.62
2 1.03 3.06 8.03 3 0.97 4.15 7.44
^ 0.96士 0.07 3.54±0.56 7.36士 0·71 实验结果显示， 本发明全人源抗 TNF-α单克隆抗体 4H16阻断 TNF-α与
U937细胞表面受体的结合的 IC50最小， 因此其与 TNF-oc的亲和力最高。
实验例 4.抗 TNF-OC抗体 4H16拮抗 TNF-a介导的 L929细胞的杀伤效应实验
L929细胞（ ATCC CCL-1 )细胞培养于含 10%胎牛血清（ JRH )的 RPMI-1640 培养液（GIBCO )中。 对数生长期细胞消化计数后， 200g离心 5分钟， 弃上清， 细胞沉淀用前述培养液重悬， 调整细胞密度至 lxl05/ml, 将细胞加至 96孔培养 板中， ΙΟΟμΙ/孔， 置 37°C , 5%C02培养箱培养过夜。 次日， 取培养液加入放线 菌素 D (华美生物）至浓度为 2(^g/ml, TNF-a ( R&D公司产品 210-TA-050 )至 浓度为 4ng/ml, 用含放线菌素 D和 TNF-a的培养液将全人源抗 TNP-a单克隆抗 体 4H16 (本发明抗体）、 D2E7 ( adalimumab单抗， Abbott公司）、 7B3及阴性对 照抗体 Trastuzumab ( Genentech )稀释至 l g/ml, 并连续 2倍比稀释， 将稀释好 的样品及对照品加入接种 L929细胞的 96孔培养板内， ΙΟΟμΙ/孔， 设置复孔， 37°C , 5%C02培养箱培养 20小时； 新鲜配制的非同位素细胞增殖检测试剂 ( Promega ), 即 MTS和 PMS按 20:1比例混合后， 20μ1/孔加入 96孔培养板中， 培 养箱内继续培养 3小时， 酶标仪检测 490nm的光吸收， 630nm为参比波长。 以样 品浓 横坐标， 光吸收值为纵坐标， 结果见表 4和图 3。
IC50 ( ng/ml )
实验编码 4H16 7B3 D2E7
1 59.2 224.3 296.3
2 45.9 247.1 335.2
3 66.4 210.2 317.8 平均 57.17±10.40 227.20±18.62 316.43±19.49 实验结果显示，本发明全人源抗 TNF-α单克隆抗体 4H 16拮抗 TNF-a介导的 L929细胞杀伤作用的 IC50最小， 因此其与 TNF-a的亲和力最高， 中和 TNF-oc的 能力最强。
实验例 5.抗 TNF-a抗体 4H16拮抗 TNF-a诱导的小鼠死亡实验
D半乳糖胺致敏的小鼠注射重组人 TNF-oc可诱导小鼠死亡。 l g重组人 TN -a ( R&D公司产品 210-TA-050 )及 20mg D半乳糖胺（ Amresco )腹腔注 射， 可诱导 80-90% C57BL6小鼠（北京维通利华实验动物技术有限公司） 死 亡。在本实验例中，先给予一定量的各种抗体腹腔注射， 30分钟后，再给予 1μ§ 重组人 TNF-α及 20mgD半乳糖胺腹腔注射， 观察各种抗体的保护效果。 各组 注射剂量及结果见下表 5。
表 5
小鼠存活情况
注射剂量 4H16 7B3 D2E7 Trastuzumab
10/10 ( 100% ) 8/10 (80%) 7/10 ( 70% ) 0/10 ( 0% )
8μΒ 10/10 ( 100%) 7/10 (70%) 6/10 (60%) 0/10 ( 0% )
9/10 (90 o%) 7/10 (70%) 5/10 (50%) 1/10 ( 10%)
8/10 (80% Ό) 6/10 (60%) 4/10 (40%) 0/10 ( 0% )
8/10 (80%) 5/10 (50%) 4/10 (40%) 1/10 ( 10%)
7/10 (70%) 5/10 (50%) 2/10 (20%) 0/10 ( 0% )
0 0/10 ( 0% ) 1/10 ( 10%) 1/10 ( 10%) 0/10 (0%) lO g (无 TNF-α) 10/10 ( 100%) 10/1 o0 ( 100%) 10/10 ( 100%) 10/10 ( 100%) 以上实验结果显示， 本发明全人源抗 TNF-α单克隆抗体 4H16拮抗 TNF-a 诱导的小鼠死亡的效果最佳， 因此该抗体在小鼠体内中和 TNF-a的能力最高。 实验例 6.抗 TNF-a抗体 4H16抑制 TNF-a诱导的家兔发热反应实验
重组人 TNF-a静脉注射新西兰大白兔， 可诱导发 o热反应。 5 g/kg的重组
P
人 TNF-a诱导的家兔发热反应约为 0.5°C。本实验例中，新西兰大白兔（来源？ ） 静脉注射 5 g/kg的重组人 TNF-a和各种抗体的不同剂量的混合物 , 注射前及 注射后 60分钟监测受试动物的体温， 以评价各种抗体中和重组人 TNF-a生物 效应的能力。 各组注射剂量及结果见下表 6。
表 6
兔体温上升情况
注射剂量 4ΗΪ6 7B3 D2E7 Trastuzumab
Figure imgf000010_0001
500 g/kg (无 TNF-a )
以上实验结果显示， 本发明全人源抗 ΤΝΡ-α单克隆抗体 4H16抑制 TNF-a 诱导的家兔发热反应的效果最佳， 因此该抗体在家兔体内中和 TNF-a的能力最 优。

Claims

权利要求书
1. 一种全人源抗 Τ Ρ-α单克隆抗体， 其具有如 SEQ ID NO:6所示的重链 可变区氨基酸序列， 以及如 SEQ ID NO:8所示的轻链可变区氨基酸。
2. 权利要求 1 所述的全人源抗 TNF-α单克隆抗体， 其具有如 SEQ ID NO: 10所示的重链氨基酸序列，以及如 SEQ ID NO:12所示的轻链氨基酸序列。
3. 编码权利要求 1所述的全人源抗 TNF-a单克隆抗体的核苷酸， 其具有 如 SEQ ID NO:5所示的重链可变区的核苷酸序列， 以及如 SEQ ID NO:7所示 的轻链可变区的核苷酸序列。
4. 权利要求 3所述的核苷酸， 其具有如 SEQ ID NO:9所示的重链核苷酸 序列， 以及如 SEQ ID NO:l l所示的轻链核苷酸序列。
5. 含有权利要求 3或 4所述的核苷酸的表达载体， 为 pcDNA3.1/ZEO(+) 和 pcDNA3.1 (+)。
6. 含有权利要求 5所述的表达载体的宿主细胞， 为 CHO-K1 细胞。
7. 权利要求 1所述的全人源抗 TNF-cx单克隆抗体的制备方法， 包括从噬 菌体人源抗体库筛选获得高亲和力全人源抗 TNF-oc单链抗体、全人源抗 TNF-a 完整抗体分子真核表达载体的构建、 全人源抗 TNF-oc完整抗体分子在 CHO细 胞中的表达和全人源抗 TNF-a完整抗体分子的纯化四个步骤。
8. 权利要求 1或 2所述的全人源抗 TNF-a单克隆抗体在制备治疗自身免 疫性疾病药物中的用途。
9. 权利要求 8所述的用途， 其中自身免疫性疾病为类风湿性关节炎、 强 直性脊柱炎或银屑病。
PCT/CN2010/000512 2010-02-25 2010-04-16 全人源抗TNF-α单克隆抗体、其制备方法及用途 Ceased WO2011103701A1 (zh)

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