抗人CD25人鼠嵌合抗体的构建、表达及其生物学活性的研究
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摘要
本课题旨在对本科室自行研制的抗人CD25鼠源单抗进行人鼠嵌合改造,然后在CHO细胞中进行稳定表达,以期获得稳定分泌特异性的抗人CD25人鼠嵌合抗体的细胞株,并对表达的嵌合抗体的生物学活性进行鉴定,为开发更加安全有效的治疗性抗体打下物质基础,同时也期在基因工程抗体研制方面取得进展。
首先提取CD25杂交瘤细胞总RNA,逆转录成cDNA后,设计多组针对鼠抗体重轻链可变区信号肽的简并引物及针对鼠抗体恒定区的特异性引物,通过PCR法钓取抗体可变区重轻链基因。以含人抗体恒定区基因的质粒PAG4622为模板钓取人抗体重轻链恒定区基因,测序鉴定正确后通过PCR法将鼠抗体可变区与人抗体恒定区基因进行拼接。将拼接后的重轻链人鼠嵌合基因与表达质粒pOptiVEC和pcDNA3.3分别进行连接。使用脂质体法将表达质粒共转染293T细胞进行抗体的瞬时表达;使用双抗夹心ELISA法对细胞上清中抗体进行含量测定;使用流式细胞术(FCM)对表达的嵌合抗体的抗原结合活性进行鉴定。结果表明,表达的嵌合抗体具有正确的抗原结合活性,同时含有人抗体恒定区序列,真核表达质粒构建成功。
然后以CHO-dhfr_细胞作为宿主细胞,采用脂质体法共转染表达质粒pOptiVEC-H和pcDNA3.3-L进行抗体的稳定表达。ELISA定量结果显示,在MTX 300nM时抗体表达量为103ng/ml;通过对培养上清纯化、定量,一共收获抗体220μg;SDS-PAGE蛋白纯度分析表明抗体纯度>97%,同时含有完整的抗体重轻链;WB结果显示嵌合抗体含有人抗体重轻链恒定区序列;Dot Blotting及WB结果显示纯化的抗体能特异性识别人CD25分子;竞争性分析结果表明嵌合抗体与亲本抗体之间识别相同表位,存在竞争关系;CCK结果显示,嵌合抗体对PHA刺激的T淋巴细胞增殖存在抑制作用;生物传感器法测得嵌合抗体的亲和常数为1.9×1010L/mol;1C1细胞株体外连续培养3个月抗体分泌保持稳定。
综上所述,本试验成功构建了抗人CD25人鼠嵌合抗体真核表达质粒,并在CHO细胞中稳定表达,表达的抗体具有特异抗原结合活性及相应生物学功能,为抗人CD25基因工程抗体的开发奠定了试验基础。
Basing on the anti-human CD25 hybridoma cell established by our laboratory, we constructed a human-mouse chimeric antibody expression plasmid against human CD25 and then expressed it in CHO cell. The aim was to establish a CHO cell line which stably expressed chimeric antibody against human CD25 and characterize its biological activity and lay foundation for the further development of safe and effective therapeutic antibody, and we also hoped to achieve progress in the study of genetically engineered antibody.
Firstly, total RNA was extracted from the murine anti-human CD25 hybridoma cell line, the VH and VL gene of the hybridoma cells were amplified by RT-PCR with the degenerate primers binding to signal peptide sequences of the heavy and light variable chain and primers binding to constant region sequences. Human antibody heavy and light constant region were cloned by PCR using plasmid PAG4622 as a template. The mouse variable region and human constant region were spliced by PCR method, the heavy and light human-mouse chimeric genes were ligated with plasmids pOptiVEC and pcDNA3.3 respectively and cotransfected 293T cells throμgh lipofectomine method.The antibody expression lever was determined by sandwich ELISA; the antigen binding activity of the chimeric antibody was characterized by FCM. The results showed that the chimeric antibody could bind to the antigen and had human antibody constant region. The eukaryotic expression plasmids of chimeric antibody were constructed succesffuly.
Secondly, plasmids pOptiVEC-H and pcDNA3.3-L were cotransfected CHO-dhfr- cells for stable expression.Quantitative ELISA result showed that the antibody expression lever was 103ng/ml when MTX concertration was 300nM;220μg antibody was harvested in total; the antibody purity was more than 97% analysed by SDS-PAGE; Western blotting result showed that the chimeric antibody had human heavy and light chain constant region; Dot Blotting and Western blotting results showed that the chimeric antibody could bind to CD25 molecule specifically; the competitive analysis showed that there was a competitive relationship between the chimeric and parental antibody and they may recognize the same epitope; CCK result showed that T lymphocytes proliferation could be inhibited by the chimeric antibody; biocore results showed that the affinity of chimeric antibody was about 1.9×1010l/mol; the cell line 1C1 could stably secret antibody for 3 months continuously.
In summary, we constructed and expressed a human-mouse chimeric antibody against human CD25 in CHO cells successfully, which laid the foundation for the development of genetically engineered antibody against human CD25.
首先提取CD25杂交瘤细胞总RNA,逆转录成cDNA后,设计多组针对鼠抗体重轻链可变区信号肽的简并引物及针对鼠抗体恒定区的特异性引物,通过PCR法钓取抗体可变区重轻链基因。以含人抗体恒定区基因的质粒PAG4622为模板钓取人抗体重轻链恒定区基因,测序鉴定正确后通过PCR法将鼠抗体可变区与人抗体恒定区基因进行拼接。将拼接后的重轻链人鼠嵌合基因与表达质粒pOptiVEC和pcDNA3.3分别进行连接。使用脂质体法将表达质粒共转染293T细胞进行抗体的瞬时表达;使用双抗夹心ELISA法对细胞上清中抗体进行含量测定;使用流式细胞术(FCM)对表达的嵌合抗体的抗原结合活性进行鉴定。结果表明,表达的嵌合抗体具有正确的抗原结合活性,同时含有人抗体恒定区序列,真核表达质粒构建成功。
然后以CHO-dhfr_细胞作为宿主细胞,采用脂质体法共转染表达质粒pOptiVEC-H和pcDNA3.3-L进行抗体的稳定表达。ELISA定量结果显示,在MTX 300nM时抗体表达量为103ng/ml;通过对培养上清纯化、定量,一共收获抗体220μg;SDS-PAGE蛋白纯度分析表明抗体纯度>97%,同时含有完整的抗体重轻链;WB结果显示嵌合抗体含有人抗体重轻链恒定区序列;Dot Blotting及WB结果显示纯化的抗体能特异性识别人CD25分子;竞争性分析结果表明嵌合抗体与亲本抗体之间识别相同表位,存在竞争关系;CCK结果显示,嵌合抗体对PHA刺激的T淋巴细胞增殖存在抑制作用;生物传感器法测得嵌合抗体的亲和常数为1.9×1010L/mol;1C1细胞株体外连续培养3个月抗体分泌保持稳定。
综上所述,本试验成功构建了抗人CD25人鼠嵌合抗体真核表达质粒,并在CHO细胞中稳定表达,表达的抗体具有特异抗原结合活性及相应生物学功能,为抗人CD25基因工程抗体的开发奠定了试验基础。
Basing on the anti-human CD25 hybridoma cell established by our laboratory, we constructed a human-mouse chimeric antibody expression plasmid against human CD25 and then expressed it in CHO cell. The aim was to establish a CHO cell line which stably expressed chimeric antibody against human CD25 and characterize its biological activity and lay foundation for the further development of safe and effective therapeutic antibody, and we also hoped to achieve progress in the study of genetically engineered antibody.
Firstly, total RNA was extracted from the murine anti-human CD25 hybridoma cell line, the VH and VL gene of the hybridoma cells were amplified by RT-PCR with the degenerate primers binding to signal peptide sequences of the heavy and light variable chain and primers binding to constant region sequences. Human antibody heavy and light constant region were cloned by PCR using plasmid PAG4622 as a template. The mouse variable region and human constant region were spliced by PCR method, the heavy and light human-mouse chimeric genes were ligated with plasmids pOptiVEC and pcDNA3.3 respectively and cotransfected 293T cells throμgh lipofectomine method.The antibody expression lever was determined by sandwich ELISA; the antigen binding activity of the chimeric antibody was characterized by FCM. The results showed that the chimeric antibody could bind to the antigen and had human antibody constant region. The eukaryotic expression plasmids of chimeric antibody were constructed succesffuly.
Secondly, plasmids pOptiVEC-H and pcDNA3.3-L were cotransfected CHO-dhfr- cells for stable expression.Quantitative ELISA result showed that the antibody expression lever was 103ng/ml when MTX concertration was 300nM;220μg antibody was harvested in total; the antibody purity was more than 97% analysed by SDS-PAGE; Western blotting result showed that the chimeric antibody had human heavy and light chain constant region; Dot Blotting and Western blotting results showed that the chimeric antibody could bind to CD25 molecule specifically; the competitive analysis showed that there was a competitive relationship between the chimeric and parental antibody and they may recognize the same epitope; CCK result showed that T lymphocytes proliferation could be inhibited by the chimeric antibody; biocore results showed that the affinity of chimeric antibody was about 1.9×1010l/mol; the cell line 1C1 could stably secret antibody for 3 months continuously.
In summary, we constructed and expressed a human-mouse chimeric antibody against human CD25 in CHO cells successfully, which laid the foundation for the development of genetically engineered antibody against human CD25.
引文
1.G.K?hler&C,Milstein.Continuous cultures of fused cells secreting antibody of predefined specificity.Nature 1975,256 (5517):495-497.
2.Miller RA,Maloney DG, Warnke R.Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody. N Engl J Med.1982,306(9):517-22.
3.Janice M Reichert.Therapeutic monoclonal antibodies: trends in development and approval in the US.Current Opinion in Molecular Therapeutics ,2002,4:110-118.
4.Hudson PJ,Souriau C,et al.Engineered Antibodies[J].Nuture Med,2003,9(1):129- 134.
5.Handschuh k,A datamonitor Report:Monoclonal antibody therapies[M].Product Code:DMHC2069.Published:2005.4.
6.Waldmann TA.The interleukin-2 receptor.J Biol Chem,1990,266(1):2681–2684.
7.Leonard WJ,Depper JM,Kronke M.et al.Structure of the human interleukin-2 receptor gene.Science,1985,230:633–9.
8.Josefina C, Hastings A, Letitia A ,et al.Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction.Journal of immunological Methods, 1992,152(1):89-104.
9.沈倍奋,陈志南,刘民培.重组抗体.北京科学出版社,2005.
10. Nashan B,Light S,Hardie IR,Lin A,Johnson JR. Reduction of acute renal allograft rejection by daclizumab. Daclizumab Double Therapy Study Group. Transplantation, 1999 67(1):110-5.
11.Clark M.Antibody humanization:a case of the“Emperor’s new clothes”.Immunol Today,2000,21(8):397-402.
12.Lin M,Ming A,Zhao M.Two-dose basiliximab compared with two-dose daclizumab in renal transplantation:a clinical study.Clin Transplant.2006,20(3):325-9.
13.Josefina C,Hastings A, Letitia A et al.Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. Journal of immunological Methods,1992,152(1):89-104.
14.王玉刚,冯健男,沈倍奋.用RLM.RACE法克隆抗CD20单克隆抗体可变区基因及其信号肽基因.细胞与分子免疫学杂志,2005,21(4):466-469.
15.Morea V,Lesk AM,Tramontano A.Antibody modeling:Implications for engineering and design[J]. Methods,2000,20:267-279.
16.Yoo EM,Chintalacharuvu KR,Penichet ML,et al.Myeloma expression systems.J Immunol Methods.2002,261(1-2):1-20.
17.SoulillouJP.Immunosuppression through inhibition of interleukin-2/interl- eukin-2 receptor interaction. Transplant Proc.1999,31(1-2A):49S-51S.
18.安怀杰,刘志刚,俞炜源.治疗性全抗体的表达及其研究进展.生物技术通讯, 2004, 15 (3):299-301.
19.申烨华,耿信笃.CHO细胞表达系统研究新进展.生物工程研究进展,2000,20(4):23-25.
20.Kingston RE, Kaufman RJ, Bebbington CR,et al.Amplification using CHO cell expression vectors. Curr Protoc Mol Biol.2002,9:(16-23).
21.Gorman,C,Bullock,C.Site-specific gene targeting for gene expression in eukaryotes. Curr.Opin.Biotechnol.2000,11:455-460.
22.Huang Y, Li Y, Wang YG.An efficient and targeted gene integration system for high-level antibody expression. J Immunol Methods.2007,322(1-2):28-39.
23.Chusainow J, Yang YS, Yeo JH. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng.2009,102(4):1182-96.
24.白银,王琰,张海荣等,通过载体DHFR基因的弱化提高抗体在CHO细胞中的表达.细胞与分子免疫学杂志2003,19(1):62-64.
25.Kim NS,Kim SJ, Lee GM.Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure. Biotechnol Bioeng.1998,60(6):679-88.
26.R.P.Junghans,T.A.Waldmann,N.F.Landolfi.Anti-Tac-H, a Humanized Antibody to the Interleukin 2 Receptor with New Features for Immunotherapy in Malignant and Immune Disorders. Cancer Research,1990,50:1495-1502.
27.Kircher B,Latzer K,Gastl G,et.al.In vitro immunosuppressive activity of new anti-CD25 antibodies.Bone Marrow Transplant,2002,29(Supple 2):S173.
28.Mahendra P Deonarain. Recombinant antibodies for cancer therapy,Expert Opin.Biol. Ther.2008,8(8):1123-1141.
29.Friguent B,Chaffatte AF,Lisa DO,et al.Measurement of the ture affinity constant in solution of antigen-antibody complex by enzyme-linked immunosorbent assay[J].Immunol Methods,1985,77(2):305.
30.朱勇,金伯泉,刘雪松.用生物传感器测定重组人α2a-干扰素单克隆抗体的亲和力及抗原识别表位.中国免疫学杂志,2000,(16):322-328.
1.BJ Brandhuber,T Boone,WC Kenney.Three-dimensional structure of interleukin-2. Science,1987,238(4834):1707-1709.
2.John C Morris,Thomas A Waldmann.Advances in interleukin 2 receptor targeted treatment. Ann Rheum Dis 2000,59(suppl I):i109–i114.
3.Taniguchi T, Minami Y.The IL-2/IL-2 receptor system: a current overview.Cell, 1993,73:5-8.
4.Lin JX,Leonard WJ.The role of Stat5a and Stat5b in signaling by IL-2 family cytokines.Oncogene.2000,19(21):2566-76.
5.Leonard WJ,Depper JM,Kronke M.et al.Structure of the human interleukin-2 receptor gene.Science,1985,230:633–9.
6.杨振林,秦玉峰,郑树森.IL-2受体单克隆抗体的研究进展,2004,2(3):11-13.
7.Waldmann TA.The interleukin-2 receptor.J Biol Chem,1991,266:2681–4.
8.Li XC,Roy-Chaudhury P,Hancock WW,et al. IL-2 and IL-4 double knockout mice reject islet allografts:a role for novel T cell growth factors in allograft rejection.J Immunol, 1998;161:890–6.
9.Soulillou JP.Immunosuppression through inhibition of interleukin-2/ interleukin-2 receptor interaction.TransplantProc, 1999,31(Suppl.):49–51S.
10.Strom TB,Kelley VR,Murphy JR,et al.Interleukin-2 receptor directed therapies. Ann Rev Med 1993,44:343–53.
11.A.C. Church .Clinical advances in therapies targeting the interleukin-2 receptor.Q J Med,2003,96: 91-102.
12.Queen C,Schneider WP,Selick HE,et al.Ahumanised antibody that binds to the interleukin-2 receptor.Proc Natl Acad Sci USA,1989,86:10029–33.
13.Kozak RW, Atcher RW, Gansow OA,et al.Bismuth-212-labeled anti-Tac monoclonal antibody: alpha-particle-emitting radionuclides as modalitiesfor radioimmuno -therapy. Proc Natl Acad Sci 1986,83:474–8.
14.Waldmann TA.The interleukin-2 receptor.Ann RevBiochem,1989,58:875–911.
15.Waldmann TA, White JD, Goldmann CK.The IL-2 receptor:atarget for monoclonalantibody therapy of HTLV-1 inducedadult T-cell leukaemia.Blood 1993,82:1701–12.
16.Waldmann TA, White JD, Carrasquillo JA,et al.Radioimmunotherapy of IL-2 receptor alpha-expressing Adult T-cell leukaemia with Yttrium-90labelled anti-Tac. Blood 1995,86:4063–75.
17.Nussenblatt RB, Fortin E, Schiffman R,et al.Treatment of non-infectious intermediate and posterioruveitis with the humanized anti-Tac monoclonal antibody: aphase I/II clinical trial. Proc Natl Acad Sci USA 1999,96:7462–6.
18.Guex-Crosier Y, Raber J, Chan CC,et,al.Benichou J.Humanized antibodies against the alpha-chain of the IL-2 receptor and against the beta-chain shared by the IL-2 and IL-15 receptors in a monkey uveitis model of autoimmune diseases.J Immunol 1997,158:452–8.
19.Anasetti C, Hansen JA, Waldmann TA.Treatment of acuteGraft-versus-host disease with humanized anti-Tac. Blood,1994,84:1320-7.
20.Matas AJ, Gillingham KJ, Payne WD,et al.The impactof an acute rejection episode on long term renal allograftsurvival.Transplantation, 1994,57:857–9.
21.Vincenti F, Lantz M, Bimbaum J,et al. Moμld D.A phase I trial of humanized anti-IL-2 receptor antibody in renal transplantation.Transplantation,1997, 63:33–8.
22.Vincenti F, Kirkman R, Light S, et al.IL-2 receptor blockade with Daclizumab to preventacute rejection in renal transplantation.N Engl J Med,1998,338:161–5.
23.尤平洪,卢一平.一种新型的IL-2R受体阻滞剂-抗CD25单克隆抗体.华西医学,2OO7,22(3) :659-660.
24.Vincenti,Flavio;Lantz,Marianne,A PhaseI Trial of Humanized Anti-Interleukin 2 Receptor Antibody in Renal Transplantation 1.Transplantation,1997,63(1):33-38.
25.Haririan A, Morawski K, Sillix DH.Induction therapy with basiliximab versus Thymoglobulin in African-American kidney transplant recipients. Transplantation.2005,79(6):716-21.
26.Nashan B.Antibody inductin therapy in renal transplant patients receiving calcineurin- inhibitor immunosuppressive regimens:a comparative review.BioDrμgs,2005:19(1):39-46.
27.C Queen,W P Schneider, H E Selick.A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci U S A.1989,86(24):10029–10033.
28.M Lin, A Ming, M Zhao.Two-dose basiliximab compared with two dose daclizumab in renal transplantation:a clinical study.Clin Transplant,2006,20:325–329.
1. Kellermann SA,Green LL.Antibody discovery:the use of transgenic mice to generate human monoclonal antibodies for therapeutics.Curr Opin Biotechnol, 2002, 13 (6):593-7.
2.K?hler & C.Milstein.Continuous cultures of fused cells secreting antibody of predefined specificity.Nature ,1975,256(5517):495-497.
3.Miller RA, Maloney DG,Warnke R.Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody.N Engl J Med.1982,306(9):517-22.
4.Khazaeli MB,Conry RM,LoBμglio AF.Human immune response to monoclonal antibodies.J Immunother Emphasis Tumor Immunol.1994,15(1):42-52.
5.WEINER LM.An overview of monoclonal antibody therapy of cancer[J].Semin Oncol,1999,26(4 Suppl 12):41-50.
6.Reichert JM.Therapeutic monoclonal antibodies :trends in development and approval in the US[J]. Curr Opin MoI Ther,2002,4(2):110-118.
7.Morrison SL,Johnson MJ,Herzenberg LA.Chimeric human antibody molecules:mouse antigen-binding domains with human constant region domains.PNAS November 1,1984, 81(21):6851-6855.
8.Josefina C,Hastings A,Letitia A ,et al.Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction.Journal of immunological Methods,1992,152(1):89-104.
9.Jones.Replacing the complementarity-determining regions in a human antibody with those from a mouse.Nature,1986:321-522.
10.葛彦.人源化抗体研制策略分析及应用研究.国外医学(免疫学分册),2004,27(5):271-273.
11.Morea V,Lesk AM,Tramontano A.Antibody modeling:implications for engineering and design. Methods.2000,20(3):267-79.
12.Schirrmann T,AI-Halabi L,Diibel S,et al.Production systems for recombinant antibodies [J].Front Biosei,2008,1(13):4576-4594.
13.Dall'Acqua WF, Damschroder MM, Zhang J.Antibody humanization by framework shuffling. Methods, 2005,36(1):43-60.
14.Hanes.J,Jermutus.L,Weber-Bornhauser.A Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.Proc Natl Acad Sci USA,1998,95:14130-14135.
15. Michael J.Mendez,Larry L.Green,et al.Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.Nature Genetics,1997,15:146-156.
16.Demarest SJ,Glaser SM. Antibody therapeutics,antibody engineering,and the merits of protein stability[J].Curr Opin Drug Discov Devel,2008,11(5):675-687.
17.李菁,林彤,宋帅.基因工程抗体研究进展.生物技术通报,2009,10:40-43.
18.Tomizuka K,Yoshida H,Uejima H.Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice.Nature Genetics,1997,16: 133–143.
19.朱振洪,余勤,万海同.基因工程小分子抗体的研究进展.生物技术通报,2008,5:59-62.
20. Kipriyanov SM.Generation and characterization of bispecific tandem diabodies for tumor therapy.Methods Mol Biol.2003,207:323-33.
21.Hamers-Casterman C,Atarhouch T,Muyldermans S et al.Nuturally occurring antibodies devoid of light chains.Nature,1993,363(3):446-448.
22. Wagner,V, Dullaart,A, Bock.The emerging nanomedicine landscape.Nat Biotech, 2006,24:1211-7.
23.Chong HS,Milenic DE,Garmestani K,et al.In vitro and in vivo evaluation of novel ligands for radioimmunotherapy. Nucl Med Bio,2006,33(4):459-467.
24.Chen ZN,Mi L,Xu J,et al.Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: Clinical Phase I/II trials.Int J Radiat Oncol Biol Phys.2006, Jun 1;65(2):435-444.
25.陈立慧,宋海峰,刘秀文.治疗性人源化单克隆抗体研究进展[J].中国新药杂志,2004,13(7):590-594.
26.Bonnet D,Schamlta AA,Feltes TF.Infecton by the respiratory syncytial virus in infans and young children at high risk[J].Cardiol Yong,2005,15(3):256-265.
27.倪健.抗体药物研究进展与展望.第15次全国干扰素及细胞因子学术会议论文,2006:29-34.
2.Miller RA,Maloney DG, Warnke R.Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody. N Engl J Med.1982,306(9):517-22.
3.Janice M Reichert.Therapeutic monoclonal antibodies: trends in development and approval in the US.Current Opinion in Molecular Therapeutics ,2002,4:110-118.
4.Hudson PJ,Souriau C,et al.Engineered Antibodies[J].Nuture Med,2003,9(1):129- 134.
5.Handschuh k,A datamonitor Report:Monoclonal antibody therapies[M].Product Code:DMHC2069.Published:2005.4.
6.Waldmann TA.The interleukin-2 receptor.J Biol Chem,1990,266(1):2681–2684.
7.Leonard WJ,Depper JM,Kronke M.et al.Structure of the human interleukin-2 receptor gene.Science,1985,230:633–9.
8.Josefina C, Hastings A, Letitia A ,et al.Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction.Journal of immunological Methods, 1992,152(1):89-104.
9.沈倍奋,陈志南,刘民培.重组抗体.北京科学出版社,2005.
10. Nashan B,Light S,Hardie IR,Lin A,Johnson JR. Reduction of acute renal allograft rejection by daclizumab. Daclizumab Double Therapy Study Group. Transplantation, 1999 67(1):110-5.
11.Clark M.Antibody humanization:a case of the“Emperor’s new clothes”.Immunol Today,2000,21(8):397-402.
12.Lin M,Ming A,Zhao M.Two-dose basiliximab compared with two-dose daclizumab in renal transplantation:a clinical study.Clin Transplant.2006,20(3):325-9.
13.Josefina C,Hastings A, Letitia A et al.Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction. Journal of immunological Methods,1992,152(1):89-104.
14.王玉刚,冯健男,沈倍奋.用RLM.RACE法克隆抗CD20单克隆抗体可变区基因及其信号肽基因.细胞与分子免疫学杂志,2005,21(4):466-469.
15.Morea V,Lesk AM,Tramontano A.Antibody modeling:Implications for engineering and design[J]. Methods,2000,20:267-279.
16.Yoo EM,Chintalacharuvu KR,Penichet ML,et al.Myeloma expression systems.J Immunol Methods.2002,261(1-2):1-20.
17.SoulillouJP.Immunosuppression through inhibition of interleukin-2/interl- eukin-2 receptor interaction. Transplant Proc.1999,31(1-2A):49S-51S.
18.安怀杰,刘志刚,俞炜源.治疗性全抗体的表达及其研究进展.生物技术通讯, 2004, 15 (3):299-301.
19.申烨华,耿信笃.CHO细胞表达系统研究新进展.生物工程研究进展,2000,20(4):23-25.
20.Kingston RE, Kaufman RJ, Bebbington CR,et al.Amplification using CHO cell expression vectors. Curr Protoc Mol Biol.2002,9:(16-23).
21.Gorman,C,Bullock,C.Site-specific gene targeting for gene expression in eukaryotes. Curr.Opin.Biotechnol.2000,11:455-460.
22.Huang Y, Li Y, Wang YG.An efficient and targeted gene integration system for high-level antibody expression. J Immunol Methods.2007,322(1-2):28-39.
23.Chusainow J, Yang YS, Yeo JH. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng.2009,102(4):1182-96.
24.白银,王琰,张海荣等,通过载体DHFR基因的弱化提高抗体在CHO细胞中的表达.细胞与分子免疫学杂志2003,19(1):62-64.
25.Kim NS,Kim SJ, Lee GM.Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure. Biotechnol Bioeng.1998,60(6):679-88.
26.R.P.Junghans,T.A.Waldmann,N.F.Landolfi.Anti-Tac-H, a Humanized Antibody to the Interleukin 2 Receptor with New Features for Immunotherapy in Malignant and Immune Disorders. Cancer Research,1990,50:1495-1502.
27.Kircher B,Latzer K,Gastl G,et.al.In vitro immunosuppressive activity of new anti-CD25 antibodies.Bone Marrow Transplant,2002,29(Supple 2):S173.
28.Mahendra P Deonarain. Recombinant antibodies for cancer therapy,Expert Opin.Biol. Ther.2008,8(8):1123-1141.
29.Friguent B,Chaffatte AF,Lisa DO,et al.Measurement of the ture affinity constant in solution of antigen-antibody complex by enzyme-linked immunosorbent assay[J].Immunol Methods,1985,77(2):305.
30.朱勇,金伯泉,刘雪松.用生物传感器测定重组人α2a-干扰素单克隆抗体的亲和力及抗原识别表位.中国免疫学杂志,2000,(16):322-328.
1.BJ Brandhuber,T Boone,WC Kenney.Three-dimensional structure of interleukin-2. Science,1987,238(4834):1707-1709.
2.John C Morris,Thomas A Waldmann.Advances in interleukin 2 receptor targeted treatment. Ann Rheum Dis 2000,59(suppl I):i109–i114.
3.Taniguchi T, Minami Y.The IL-2/IL-2 receptor system: a current overview.Cell, 1993,73:5-8.
4.Lin JX,Leonard WJ.The role of Stat5a and Stat5b in signaling by IL-2 family cytokines.Oncogene.2000,19(21):2566-76.
5.Leonard WJ,Depper JM,Kronke M.et al.Structure of the human interleukin-2 receptor gene.Science,1985,230:633–9.
6.杨振林,秦玉峰,郑树森.IL-2受体单克隆抗体的研究进展,2004,2(3):11-13.
7.Waldmann TA.The interleukin-2 receptor.J Biol Chem,1991,266:2681–4.
8.Li XC,Roy-Chaudhury P,Hancock WW,et al. IL-2 and IL-4 double knockout mice reject islet allografts:a role for novel T cell growth factors in allograft rejection.J Immunol, 1998;161:890–6.
9.Soulillou JP.Immunosuppression through inhibition of interleukin-2/ interleukin-2 receptor interaction.TransplantProc, 1999,31(Suppl.):49–51S.
10.Strom TB,Kelley VR,Murphy JR,et al.Interleukin-2 receptor directed therapies. Ann Rev Med 1993,44:343–53.
11.A.C. Church .Clinical advances in therapies targeting the interleukin-2 receptor.Q J Med,2003,96: 91-102.
12.Queen C,Schneider WP,Selick HE,et al.Ahumanised antibody that binds to the interleukin-2 receptor.Proc Natl Acad Sci USA,1989,86:10029–33.
13.Kozak RW, Atcher RW, Gansow OA,et al.Bismuth-212-labeled anti-Tac monoclonal antibody: alpha-particle-emitting radionuclides as modalitiesfor radioimmuno -therapy. Proc Natl Acad Sci 1986,83:474–8.
14.Waldmann TA.The interleukin-2 receptor.Ann RevBiochem,1989,58:875–911.
15.Waldmann TA, White JD, Goldmann CK.The IL-2 receptor:atarget for monoclonalantibody therapy of HTLV-1 inducedadult T-cell leukaemia.Blood 1993,82:1701–12.
16.Waldmann TA, White JD, Carrasquillo JA,et al.Radioimmunotherapy of IL-2 receptor alpha-expressing Adult T-cell leukaemia with Yttrium-90labelled anti-Tac. Blood 1995,86:4063–75.
17.Nussenblatt RB, Fortin E, Schiffman R,et al.Treatment of non-infectious intermediate and posterioruveitis with the humanized anti-Tac monoclonal antibody: aphase I/II clinical trial. Proc Natl Acad Sci USA 1999,96:7462–6.
18.Guex-Crosier Y, Raber J, Chan CC,et,al.Benichou J.Humanized antibodies against the alpha-chain of the IL-2 receptor and against the beta-chain shared by the IL-2 and IL-15 receptors in a monkey uveitis model of autoimmune diseases.J Immunol 1997,158:452–8.
19.Anasetti C, Hansen JA, Waldmann TA.Treatment of acuteGraft-versus-host disease with humanized anti-Tac. Blood,1994,84:1320-7.
20.Matas AJ, Gillingham KJ, Payne WD,et al.The impactof an acute rejection episode on long term renal allograftsurvival.Transplantation, 1994,57:857–9.
21.Vincenti F, Lantz M, Bimbaum J,et al. Moμld D.A phase I trial of humanized anti-IL-2 receptor antibody in renal transplantation.Transplantation,1997, 63:33–8.
22.Vincenti F, Kirkman R, Light S, et al.IL-2 receptor blockade with Daclizumab to preventacute rejection in renal transplantation.N Engl J Med,1998,338:161–5.
23.尤平洪,卢一平.一种新型的IL-2R受体阻滞剂-抗CD25单克隆抗体.华西医学,2OO7,22(3) :659-660.
24.Vincenti,Flavio;Lantz,Marianne,A PhaseI Trial of Humanized Anti-Interleukin 2 Receptor Antibody in Renal Transplantation 1.Transplantation,1997,63(1):33-38.
25.Haririan A, Morawski K, Sillix DH.Induction therapy with basiliximab versus Thymoglobulin in African-American kidney transplant recipients. Transplantation.2005,79(6):716-21.
26.Nashan B.Antibody inductin therapy in renal transplant patients receiving calcineurin- inhibitor immunosuppressive regimens:a comparative review.BioDrμgs,2005:19(1):39-46.
27.C Queen,W P Schneider, H E Selick.A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci U S A.1989,86(24):10029–10033.
28.M Lin, A Ming, M Zhao.Two-dose basiliximab compared with two dose daclizumab in renal transplantation:a clinical study.Clin Transplant,2006,20:325–329.
1. Kellermann SA,Green LL.Antibody discovery:the use of transgenic mice to generate human monoclonal antibodies for therapeutics.Curr Opin Biotechnol, 2002, 13 (6):593-7.
2.K?hler & C.Milstein.Continuous cultures of fused cells secreting antibody of predefined specificity.Nature ,1975,256(5517):495-497.
3.Miller RA, Maloney DG,Warnke R.Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody.N Engl J Med.1982,306(9):517-22.
4.Khazaeli MB,Conry RM,LoBμglio AF.Human immune response to monoclonal antibodies.J Immunother Emphasis Tumor Immunol.1994,15(1):42-52.
5.WEINER LM.An overview of monoclonal antibody therapy of cancer[J].Semin Oncol,1999,26(4 Suppl 12):41-50.
6.Reichert JM.Therapeutic monoclonal antibodies :trends in development and approval in the US[J]. Curr Opin MoI Ther,2002,4(2):110-118.
7.Morrison SL,Johnson MJ,Herzenberg LA.Chimeric human antibody molecules:mouse antigen-binding domains with human constant region domains.PNAS November 1,1984, 81(21):6851-6855.
8.Josefina C,Hastings A,Letitia A ,et al.Novel vectors for the expression of antibody molecules using variable regions generated by polymerase chain reaction.Journal of immunological Methods,1992,152(1):89-104.
9.Jones.Replacing the complementarity-determining regions in a human antibody with those from a mouse.Nature,1986:321-522.
10.葛彦.人源化抗体研制策略分析及应用研究.国外医学(免疫学分册),2004,27(5):271-273.
11.Morea V,Lesk AM,Tramontano A.Antibody modeling:implications for engineering and design. Methods.2000,20(3):267-79.
12.Schirrmann T,AI-Halabi L,Diibel S,et al.Production systems for recombinant antibodies [J].Front Biosei,2008,1(13):4576-4594.
13.Dall'Acqua WF, Damschroder MM, Zhang J.Antibody humanization by framework shuffling. Methods, 2005,36(1):43-60.
14.Hanes.J,Jermutus.L,Weber-Bornhauser.A Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries.Proc Natl Acad Sci USA,1998,95:14130-14135.
15. Michael J.Mendez,Larry L.Green,et al.Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.Nature Genetics,1997,15:146-156.
16.Demarest SJ,Glaser SM. Antibody therapeutics,antibody engineering,and the merits of protein stability[J].Curr Opin Drug Discov Devel,2008,11(5):675-687.
17.李菁,林彤,宋帅.基因工程抗体研究进展.生物技术通报,2009,10:40-43.
18.Tomizuka K,Yoshida H,Uejima H.Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice.Nature Genetics,1997,16: 133–143.
19.朱振洪,余勤,万海同.基因工程小分子抗体的研究进展.生物技术通报,2008,5:59-62.
20. Kipriyanov SM.Generation and characterization of bispecific tandem diabodies for tumor therapy.Methods Mol Biol.2003,207:323-33.
21.Hamers-Casterman C,Atarhouch T,Muyldermans S et al.Nuturally occurring antibodies devoid of light chains.Nature,1993,363(3):446-448.
22. Wagner,V, Dullaart,A, Bock.The emerging nanomedicine landscape.Nat Biotech, 2006,24:1211-7.
23.Chong HS,Milenic DE,Garmestani K,et al.In vitro and in vivo evaluation of novel ligands for radioimmunotherapy. Nucl Med Bio,2006,33(4):459-467.
24.Chen ZN,Mi L,Xu J,et al.Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: Clinical Phase I/II trials.Int J Radiat Oncol Biol Phys.2006, Jun 1;65(2):435-444.
25.陈立慧,宋海峰,刘秀文.治疗性人源化单克隆抗体研究进展[J].中国新药杂志,2004,13(7):590-594.
26.Bonnet D,Schamlta AA,Feltes TF.Infecton by the respiratory syncytial virus in infans and young children at high risk[J].Cardiol Yong,2005,15(3):256-265.
27.倪健.抗体药物研究进展与展望.第15次全国干扰素及细胞因子学术会议论文,2006:29-34.