重组免疫毒素CTLA4-ScFv-Mel的构建、制备及活性研究
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摘要
器官移植是治疗终末期器官功能衰竭的有效手段,免疫抑制剂的应用可以减轻移植排斥反应,但化学类免疫抑制剂毒副作用明显,影响了其临床应用。生物制剂类免疫抑制剂尤其是免疫毒素可以通过其特异性的结合作用,清除抗原特异性T细胞,且毒副作用较小,有更好的应用前景。
免疫毒素是具有导向能力的分子(载体)和具有细胞毒性的分子(毒素)偶联而成的具有特异性细胞杀伤能力的杂合分子,在治疗恶性肿瘤、移植排斥等方面有良好的应用前景。
CTLA-4是一种仅表达于活化T细胞而静止T细胞不表达的分子,因此CTLA-4的抗原结合分子是一种理想的免疫毒素导向分子,由之产生的免疫毒素只对活化的T细胞或肿瘤细胞有杀伤作用,避免了对正常静息T细胞的非特异性损伤。
蜂毒肽(Melittin, Mel)是蜜蜂蜂毒的主要成分,是一种重要的抗菌肽,具有抗菌、抗辐射、抗肿瘤等作用。它由26个氨基酸组成,分子量小,免疫原性低,不易产生过敏反应；具有膜活性,直接对细胞的磷脂膜起溶解作用,抑制细胞发育,是一种较理想毒素片段。
本研究以人源化抗CTLA-4单链抗体为靶向片段,特异性结合在表达CTLA-4的活化T细胞表面,以蜂毒肽类似物为毒素片段,特异性杀伤活化T细胞,设计构建出了高效低毒的免疫毒素分子,主要用于抗移植排斥反应。
利用重叠延伸PCR技术,通过两步PCR获得了重组免疫毒素全长基因,将其与表达载体pBV220连接,转化大肠杆菌,重组基因在大肠杆菌表达系统中以包涵体形式表达。包涵体用8M脲变性,采用稀释复性,然后用SP-Sepharose-Fast-Flow阳离子交换柱分离纯化,Sephacryl S-200凝胶排阻层析柱精制,制备得到目的蛋白CTLA4-ScFv-Mel.
在体外活性研究中,2μmo1/L的CTLA4-ScFv-Mel分别作用于非活化的T淋巴细胞、阴性对照细胞ECV-304、ConA激活的T淋巴细胞、CTLA-4阳性细胞Raji知Jurkat细胞。结果显示,给药24h后各组细胞存活率下降,其中前两组细胞的存活率分别为78.9%和77.3%；而后三组分别为31.2%、32.7%和30.8%。说明CTLA4-ScFv-Mel对CTLA-4阳性细胞的杀伤率高于正常细胞,表现出细胞杀伤的选择性和高效率。
体内活性研究以BEL-7402肝癌移植瘤模型和S180小鼠移植瘤模型为对象,分低、中、高三个剂量给药,比较试验组和阴性对照组移植瘤相对体积等药效学指标。结果显示,CTLA4-ScFv-Mel能够发挥促进两个试验组模型中移植瘤的存活和生长的作用,且呈现一定的剂量效应关系。推测该现象的出现与抑制T细胞免疫有关。
本研究成功制备了一种全新的免疫毒素融合蛋白,并进行了初步活性研究和药效学评价,为器官移植提供了潜在的选择药物,具有一定的科学价值和开发前景。
Organ transplantation has become the best treatment for end-stage organ diseases.However, rejection remains one of the major obstacles for the survival of allograft.Chemical immunosuppressant can suppress rejection, but associated with significantside effects, which jeopardized its clinical application. Biological immunosuppressiveagents, especially immunotoxin, can clear antigen-specific T cells through its specificbinding with less side effects and better prospect for clinical application.
An immunotoxin is a human-made protein that consists of a targeting portion (vector)linked to cytotoxic molecules (toxin). When the protein binds to that cell, it is taken inthrough endocytosis, and the toxin kills the cell. It has been reported in the treatment ofcancer and transplant rejection.
CTLA-4is a molecule that only expressed on the surface of activated T cells but noton stationary T cells. Therefore, CTLA-4antigen binding molecule is an idealimmunotoxin vector. Immunotoxins generated by these vectors only kill activated Tcells and tumor cell, avoiding non-specific damage to normal stationary T cells.
Melittin (Mel), the main component of bee venom, is an important antimicrobialpeptide with antibacterial, anti-radiation, anti-tumor effects. It is composed of26aminoacids. Melittin has low molecular weight, low immunogenicity, and low susceptibilityto allergic reactions. Its membrane activity can directly dissolute the phospholipidmembranes of cells, inhibit the development of cell. Melittin is an ideal toxin fragment.
In this study, we constructed a humanized anti-CTLA-4single-chain antibody as atargeting fragment, which can specifically bind to the activated T cell expressingCTLA-4. Melittin analogues were used as the toxin fragment to specifically killactivated T cells. The immunotoxin molecule prepared in our study displayed highefficiency and low toxicity that could be used for anti-transplant rejection.
Using the overlap extension PCR technique, recombinant immunotoxins full-lengthcDNA were obtained by two-step PCR. Then connected with the expression vectorpBV220, and transformed into Escherichia coli. The recombinant gene was expressed asinclusion bodies in Escherichia coli expression system. Inclusion bodies weredenaturized with8M urea, renatured by the dilution method, and then purified usingSP-Sepharose Fast-Flow cation exchange column. After chromatography purificationthrough Sephacryl S-200gel filtration column, the target protein CTLA4-ScFv-Mel wasprepared.
In the in vitro activity study, non-activated T lymphocytes, negative control ECV-304cells, T lymphocytes activated by ConA, and CTLA-4positive Raji cells and Jurkatcells were treated by2μmol/L of CTLA4-ScFv-Mel. The results showed that the cellsurvival decreased24h after administration of CTLA4-ScFv-Mel. The survival rates ofthe first two groups were78.9%and77.3%, respectively; while the survival rates of thelatter three groups were31.2%,32.7%and30.8%, respectively. The results suggestedthat CTLA4-ScFv-Mel has higher destruction rate in CTLA-4positive cells than that innormal cells, showing its selectivity and high efficiency in cell killing.
BEL-7402liver cancer xenograft and S180mouse xenograft tumor model were usedin the vivo activity study. CTLA4-ScFv-Mel were given in low, middle, and high dose.Pharmacodynamic indicators including tumor xenografts relative volume was comparedbetween the test group and negative control group. The results showed thatCTLA4-ScFv-Mel can promote the survival and growth of transplanted tumors in thetwo test groups, presenting a certain dose-effect relationship. The inhibition of T cellimmunity was suggested to be related with this phenomenon.
This study has successfully prepared a new immunotoxin fusion protein, andevaluated its initial activity and pharmacodynamic properties, providing a potential drugfor the treatment of organ transplant rejection with good scientific value anddevelopment prospects.
免疫毒素是具有导向能力的分子(载体)和具有细胞毒性的分子(毒素)偶联而成的具有特异性细胞杀伤能力的杂合分子,在治疗恶性肿瘤、移植排斥等方面有良好的应用前景。
CTLA-4是一种仅表达于活化T细胞而静止T细胞不表达的分子,因此CTLA-4的抗原结合分子是一种理想的免疫毒素导向分子,由之产生的免疫毒素只对活化的T细胞或肿瘤细胞有杀伤作用,避免了对正常静息T细胞的非特异性损伤。
蜂毒肽(Melittin, Mel)是蜜蜂蜂毒的主要成分,是一种重要的抗菌肽,具有抗菌、抗辐射、抗肿瘤等作用。它由26个氨基酸组成,分子量小,免疫原性低,不易产生过敏反应；具有膜活性,直接对细胞的磷脂膜起溶解作用,抑制细胞发育,是一种较理想毒素片段。
本研究以人源化抗CTLA-4单链抗体为靶向片段,特异性结合在表达CTLA-4的活化T细胞表面,以蜂毒肽类似物为毒素片段,特异性杀伤活化T细胞,设计构建出了高效低毒的免疫毒素分子,主要用于抗移植排斥反应。
利用重叠延伸PCR技术,通过两步PCR获得了重组免疫毒素全长基因,将其与表达载体pBV220连接,转化大肠杆菌,重组基因在大肠杆菌表达系统中以包涵体形式表达。包涵体用8M脲变性,采用稀释复性,然后用SP-Sepharose-Fast-Flow阳离子交换柱分离纯化,Sephacryl S-200凝胶排阻层析柱精制,制备得到目的蛋白CTLA4-ScFv-Mel.
在体外活性研究中,2μmo1/L的CTLA4-ScFv-Mel分别作用于非活化的T淋巴细胞、阴性对照细胞ECV-304、ConA激活的T淋巴细胞、CTLA-4阳性细胞Raji知Jurkat细胞。结果显示,给药24h后各组细胞存活率下降,其中前两组细胞的存活率分别为78.9%和77.3%；而后三组分别为31.2%、32.7%和30.8%。说明CTLA4-ScFv-Mel对CTLA-4阳性细胞的杀伤率高于正常细胞,表现出细胞杀伤的选择性和高效率。
体内活性研究以BEL-7402肝癌移植瘤模型和S180小鼠移植瘤模型为对象,分低、中、高三个剂量给药,比较试验组和阴性对照组移植瘤相对体积等药效学指标。结果显示,CTLA4-ScFv-Mel能够发挥促进两个试验组模型中移植瘤的存活和生长的作用,且呈现一定的剂量效应关系。推测该现象的出现与抑制T细胞免疫有关。
本研究成功制备了一种全新的免疫毒素融合蛋白,并进行了初步活性研究和药效学评价,为器官移植提供了潜在的选择药物,具有一定的科学价值和开发前景。
Organ transplantation has become the best treatment for end-stage organ diseases.However, rejection remains one of the major obstacles for the survival of allograft.Chemical immunosuppressant can suppress rejection, but associated with significantside effects, which jeopardized its clinical application. Biological immunosuppressiveagents, especially immunotoxin, can clear antigen-specific T cells through its specificbinding with less side effects and better prospect for clinical application.
An immunotoxin is a human-made protein that consists of a targeting portion (vector)linked to cytotoxic molecules (toxin). When the protein binds to that cell, it is taken inthrough endocytosis, and the toxin kills the cell. It has been reported in the treatment ofcancer and transplant rejection.
CTLA-4is a molecule that only expressed on the surface of activated T cells but noton stationary T cells. Therefore, CTLA-4antigen binding molecule is an idealimmunotoxin vector. Immunotoxins generated by these vectors only kill activated Tcells and tumor cell, avoiding non-specific damage to normal stationary T cells.
Melittin (Mel), the main component of bee venom, is an important antimicrobialpeptide with antibacterial, anti-radiation, anti-tumor effects. It is composed of26aminoacids. Melittin has low molecular weight, low immunogenicity, and low susceptibilityto allergic reactions. Its membrane activity can directly dissolute the phospholipidmembranes of cells, inhibit the development of cell. Melittin is an ideal toxin fragment.
In this study, we constructed a humanized anti-CTLA-4single-chain antibody as atargeting fragment, which can specifically bind to the activated T cell expressingCTLA-4. Melittin analogues were used as the toxin fragment to specifically killactivated T cells. The immunotoxin molecule prepared in our study displayed highefficiency and low toxicity that could be used for anti-transplant rejection.
Using the overlap extension PCR technique, recombinant immunotoxins full-lengthcDNA were obtained by two-step PCR. Then connected with the expression vectorpBV220, and transformed into Escherichia coli. The recombinant gene was expressed asinclusion bodies in Escherichia coli expression system. Inclusion bodies weredenaturized with8M urea, renatured by the dilution method, and then purified usingSP-Sepharose Fast-Flow cation exchange column. After chromatography purificationthrough Sephacryl S-200gel filtration column, the target protein CTLA4-ScFv-Mel wasprepared.
In the in vitro activity study, non-activated T lymphocytes, negative control ECV-304cells, T lymphocytes activated by ConA, and CTLA-4positive Raji cells and Jurkatcells were treated by2μmol/L of CTLA4-ScFv-Mel. The results showed that the cellsurvival decreased24h after administration of CTLA4-ScFv-Mel. The survival rates ofthe first two groups were78.9%and77.3%, respectively; while the survival rates of thelatter three groups were31.2%,32.7%and30.8%, respectively. The results suggestedthat CTLA4-ScFv-Mel has higher destruction rate in CTLA-4positive cells than that innormal cells, showing its selectivity and high efficiency in cell killing.
BEL-7402liver cancer xenograft and S180mouse xenograft tumor model were usedin the vivo activity study. CTLA4-ScFv-Mel were given in low, middle, and high dose.Pharmacodynamic indicators including tumor xenografts relative volume was comparedbetween the test group and negative control group. The results showed thatCTLA4-ScFv-Mel can promote the survival and growth of transplanted tumors in thetwo test groups, presenting a certain dose-effect relationship. The inhibition of T cellimmunity was suggested to be related with this phenomenon.
This study has successfully prepared a new immunotoxin fusion protein, andevaluated its initial activity and pharmacodynamic properties, providing a potential drugfor the treatment of organ transplant rejection with good scientific value anddevelopment prospects.
引文
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[1]郭怡,蔡常洁. CTLA4Ig诱导免疫耐受作用的研究进展[J].国际内科学杂志,2007,34(7):427-431.
[2]赵彦宗,郭喜桃,岳中瑾. CTLA-4与抑制免疫研究新进展[J].国外医学移植于血液净化分册,2004.2(2):31-33.
[3]王凤英.细胞毒性T淋巴细胞相关抗原4的研究进展[J].国际免疫学杂志,2007,30(1):43-47.
[4]翟树森,尉承泽.细胞毒T淋巴细胞抗原-4的研究进展[J].生物技术通讯,2007,18(3):493-496.
[5]袁岸龙,夏冰,黄梅芳. CTLA-4的免疫学作用与疾病[J].武汉大学学报（医学版）,2003,24(1):90-93.
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[8] Lenschow DJ, Walunas TL, Bluestone JA, et al. CD28/B7System of T CellCostimulation. Annual Review of Immunology.1996,14:233-258.
[9]罗良生,黄强.基因重组免疫毒素研究进展[J].国外医学肿瘤学分册,2000,27(5):269-273.
[10]卢丽琨,田利源,汪莉.人源化免疫毒素的研究进展[J].国际药学研究杂志,2009,36(6):426-430.
[11]Polito L, Bortolotti M, Farini V, et al. Saporin induces multiple death pathways inlymaphoma cells with different intensity and timing as compared to ricin[J]. TheInternaional Journal of Biochemistry&Cell Biology,2009,41(5):1055-1061.
[12]Tazzari PL, Polito L, Bolognesi A, et al. Immunotoxins Containing RecombinantAnti-CTLA-4Single-Chain Fragment Variable Antibodies and Saporin: In VitroResults and In Vivo Effects in an Acute Rejection Model[J]. J Immunol,2001,167(8):4222-4229.
[13]Zeng L, Wan L, Chen L, et al. Selective Depletion of Activated T Cells byRecombinant Immunotoxin Containing Anti-CTLA-4Single-Chain Fragment ofVariable Antibody and N-Terminal Fragment of Perforin[J]. Transplant Proc,2006,38(7):2151-2153.
[14]卢晓风,程惊秋,曾令宇,等.人源化CTLA-4单链抗体与人穿孔素通道形成肽P34的重组免疫毒素:中国, CN100443503C[P].2008-12-17.
[15]曾令宇.活化T细胞选择性清除分子hS83-P34的构建、制备及功能研究[D].成都:四川大学,2005.
[16]金永柱,张庆殷,谢蜀生. CTLA-4-FasL融合分子的构建及其生物学特性的鉴定[J].上海免疫学杂志,2003,23(3):173-176.
[17]金永柱,谢蜀生. CTLA-4-FasL双功能分子的构建及其体外免疫抑制作用的研究[J].北京大学学报（医学版）,2001,33(6):485.
[18]Orbach A, Rachmilewitz J, Parnas M, et al. CTLA-4FasL Induces Early Apoptosisof Activated T Cells by Interfering with Anti-Apoptotic Signals[J]. J Immunol,2007,179(11):7287-7294.
[1]郭怡,蔡常洁. CTLA4Ig诱导免疫耐受作用的研究进展[J].国际内科学杂志,2007,34(7):427-431.
[2] Vincent TH, Corey Cutler. Current and novel therapies in acute GVHD[J]. BestPractice&Research Clinical Haematology,2008,21(2):223-237.
[3] Mahmud N, Klipa D, Ahsan N. Antibody immunosuppressive therapy in solid-organtransplant[J]. Landes Bioscience,2010,2(2):148-156.
[4]李晓玉.免疫抑制剂的研究概述[J].药学服务与研究,2005,5(2):105-110.
[5]黄孝伦,黄洁夫.基因工程抗体在器官移植排斥反应中的应用前景[J].国外医学泌尿系统分册,2001,21(4):189-191.
[6]陈国江,黎燕.抗CD3单克隆抗体的作用机制及其在临床上的应用[J].国外医学药学分册,2005,32(1):46-49.
[7]屈浩. CD52抗原的研究进展及其抗体在临床中的应用[J].国外医学免疫学分册,2005,28(6):369-373.
[8]吴志贤.阿来佐单抗在肾移植中的应用研究进展[J].中华器官移植杂志,2007,28(7):443-444.
[9]马麟麟．抗体诱导疗法在器官移植领域的临床应用［J/CD］．中华移植杂志:电子版，2010，4(4):296-300．
[10]吴文林.器官移植排斥与耐受的研究进展[J].泉州师范学院学报,2003,21(4):77-82.
[11] Guillonneau C, Seveno C, Dugast AS, et al. Anti-CD28antibodies modify regulatorymechanisms and reinforce tolerance in CD40Ig-treated heart allograft recipients[J].J Immunol,2007,179(12):8164-8171.
[12]姜晓峰,张弘彬,郭大伟等.联合阻断共刺激信号对移植免疫耐受的影响[J].中国免疫学杂志,2009,25(7):634-637.
[13]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[J].华西医学,2007,22(3):659-660.
[14] Carella AM, Arena GD, Casavilla N. Monoclonal antibodies to treatGraft-Versus-Host Disease[J]. Mini-Reviews in Medicinal Chemistry,2011,11(6):473-479.
[15]孙凯,冯琦,金伯泉. Ig融合蛋白临床应用前景[J].中国肿瘤生物治疗杂志,2000,7(3):229-231.
[16]袁岸龙,夏冰,黄梅芳. CTLA-4的免疫学作用与疾病[J].武汉大学学报（医学版）,2003,24(1):90-93.
[17]王朝健,李元. CTLA-4基因工程药物研究进展[J].国外医学药学分册,2000,27(6):231-235.
[18] Weclawiak H, Kamar N, Ould-Mohamed A, et al. Biological agents in kidneytransplantation: belatacept is enetering the field[J]. Expert Opin Biol Ther,2010,10(10):1501-1508.
[19]郭亚军,候盛,寇庚等.一种免疫毒素及其制备方法和用途：中国, CN101062953A[P].2007-10-31.
[20] Tazzari PL, Polito L, Bolognesi A, et al. Immunotoxins Containing RecombinantAnti-CTLA-4Single-Chain Fragment Variable Antibodies and Saporin: In VitroResults and In Vivo Effects in an Acute Rejection Model[J]. J Immunol,2001,167(8):4222-4229.
[21] Zeng L, Wan L, Chen L, et al. Selective Depletion of Activated T Cells byRecombinant Immunotoxin Containing Anti-CTLA-4Single-Chain Fragment ofVariable Antibody and N-Terminal Fragment of Perforin[J]. Transplant Proc,2006,38(7):2151-2153.
[22]卢晓风,程惊秋,曾令宇等.人源化CTLA-4单链抗体与人穿孔素通道形成肽P34的重组免疫毒素:中国,CN100443503C[P].2008-12-17.
[23]曾令宇.活化T细胞选择性清除分子hS83-P34的构建、制备及功能研究[D].成都:四川大学,2005.
[24]张林,贾怡,李虹等.针对活化Th1细胞的重组免疫毒素Rantes-DT390的质粒及制备方法与应用:中国, CN1257975C.2006-5-31.
[25]张林,吕梅励,李虹等.针对活化Th1细胞的重组免疫毒素MIP-1α-DT390的质粒及制备方法与应用:中国, CN100348725C.2007-11-14.
[2]郭怡,蔡常洁. CTLA4Ig诱导免疫耐受作用的研究进展[J].国际内科学杂志,2007,34(7):427-431.
[3] Vincent TH, Corey Cutler. Current and novel therapies in acute GVHD[J]. BestPractice&Research Clinical Haematology,2008,21(2):223-237.
[4] Mahmud N, Klipa D, Ahsan N. Antibody immunosuppressive therapy in solid-organtransplant[J]. Landes Bioscience,2010,2(2):148-156.
[5]唐海淑，孙素荣.免疫毒素的研究及其应用[J].生物学通报,2008,43(11):7-10.
[6]赵彦宗,郭喜桃,岳中瑾. CTLA-4与移植免疫研究新进展[J].国外医学移植与血液净化分册,2004.2(2):31-33.
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[10] Tazzari PL, Polito L, Bolognesi A, et al. Immunotoxins Containing RecombinantAnti-CTLA-4Single-Chain Fragment Variable Antibodies and Saporin: In VitroResults and In Vivo Effects in an Acute Rejection Model[J]. J Immunol,2001,167(8):4222-4229.
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[12] Zeng L, Wan L, Chen L, et al. Selective Depletion of Activated T Cells byRecombinant Immunotoxin Containing Anti-CTLA-4Single-Chain Fragment ofVariable Antibody and N-Terminal Fragment of Perforin[J]. Transplant Proc,2006,38(7):2151-2153.
[13]卢晓风,程惊秋,曾令宇等.人源化CTLA-4单链抗体与人穿孔素通道形成肽P34的重组免疫毒素:中国,CN100443503C[P].2008-12-17.
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[15]卢丽琨,田利源,汪莉.人源化免疫毒素的研究进展[J].国际药学研究杂志,2009,36(6):426-430.
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[22]王凤英.细胞毒性T淋巴细胞相关抗原4的研究进展[J].国际免疫学杂志,2007,30(1):43-47.
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[24]袁岸龙,夏冰,黄梅芳. CTLA-4的免疫学作用与疾病[J].武汉大学学报（医学版）,2003,24(1):90-93.
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[1]郭怡,蔡常洁. CTLA4Ig诱导免疫耐受作用的研究进展[J].国际内科学杂志,2007,34(7):427-431.
[2]赵彦宗,郭喜桃,岳中瑾. CTLA-4与抑制免疫研究新进展[J].国外医学移植于血液净化分册,2004.2(2):31-33.
[3]王凤英.细胞毒性T淋巴细胞相关抗原4的研究进展[J].国际免疫学杂志,2007,30(1):43-47.
[4]翟树森,尉承泽.细胞毒T淋巴细胞抗原-4的研究进展[J].生物技术通讯,2007,18(3):493-496.
[5]袁岸龙,夏冰,黄梅芳. CTLA-4的免疫学作用与疾病[J].武汉大学学报（医学版）,2003,24(1):90-93.
[6]张旭东,郭树忠. CTLA4-Ig在异体移植方面的相关研究进展[J].第四军医大学学报,2005,26(18):1716-1718.
[7]王朝健,李元. CTLA-4基因工程药物研究进展[J].国外医学药学分册,2000,27(6):231-235.
[8] Lenschow DJ, Walunas TL, Bluestone JA, et al. CD28/B7System of T CellCostimulation. Annual Review of Immunology.1996,14:233-258.
[9]罗良生,黄强.基因重组免疫毒素研究进展[J].国外医学肿瘤学分册,2000,27(5):269-273.
[10]卢丽琨,田利源,汪莉.人源化免疫毒素的研究进展[J].国际药学研究杂志,2009,36(6):426-430.
[11]Polito L, Bortolotti M, Farini V, et al. Saporin induces multiple death pathways inlymaphoma cells with different intensity and timing as compared to ricin[J]. TheInternaional Journal of Biochemistry&Cell Biology,2009,41(5):1055-1061.
[12]Tazzari PL, Polito L, Bolognesi A, et al. Immunotoxins Containing RecombinantAnti-CTLA-4Single-Chain Fragment Variable Antibodies and Saporin: In VitroResults and In Vivo Effects in an Acute Rejection Model[J]. J Immunol,2001,167(8):4222-4229.
[13]Zeng L, Wan L, Chen L, et al. Selective Depletion of Activated T Cells byRecombinant Immunotoxin Containing Anti-CTLA-4Single-Chain Fragment ofVariable Antibody and N-Terminal Fragment of Perforin[J]. Transplant Proc,2006,38(7):2151-2153.
[14]卢晓风,程惊秋,曾令宇,等.人源化CTLA-4单链抗体与人穿孔素通道形成肽P34的重组免疫毒素:中国, CN100443503C[P].2008-12-17.
[15]曾令宇.活化T细胞选择性清除分子hS83-P34的构建、制备及功能研究[D].成都:四川大学,2005.
[16]金永柱,张庆殷,谢蜀生. CTLA-4-FasL融合分子的构建及其生物学特性的鉴定[J].上海免疫学杂志,2003,23(3):173-176.
[17]金永柱,谢蜀生. CTLA-4-FasL双功能分子的构建及其体外免疫抑制作用的研究[J].北京大学学报（医学版）,2001,33(6):485.
[18]Orbach A, Rachmilewitz J, Parnas M, et al. CTLA-4FasL Induces Early Apoptosisof Activated T Cells by Interfering with Anti-Apoptotic Signals[J]. J Immunol,2007,179(11):7287-7294.
[1]郭怡,蔡常洁. CTLA4Ig诱导免疫耐受作用的研究进展[J].国际内科学杂志,2007,34(7):427-431.
[2] Vincent TH, Corey Cutler. Current and novel therapies in acute GVHD[J]. BestPractice&Research Clinical Haematology,2008,21(2):223-237.
[3] Mahmud N, Klipa D, Ahsan N. Antibody immunosuppressive therapy in solid-organtransplant[J]. Landes Bioscience,2010,2(2):148-156.
[4]李晓玉.免疫抑制剂的研究概述[J].药学服务与研究,2005,5(2):105-110.
[5]黄孝伦,黄洁夫.基因工程抗体在器官移植排斥反应中的应用前景[J].国外医学泌尿系统分册,2001,21(4):189-191.
[6]陈国江,黎燕.抗CD3单克隆抗体的作用机制及其在临床上的应用[J].国外医学药学分册,2005,32(1):46-49.
[7]屈浩. CD52抗原的研究进展及其抗体在临床中的应用[J].国外医学免疫学分册,2005,28(6):369-373.
[8]吴志贤.阿来佐单抗在肾移植中的应用研究进展[J].中华器官移植杂志,2007,28(7):443-444.
[9]马麟麟．抗体诱导疗法在器官移植领域的临床应用［J/CD］．中华移植杂志:电子版，2010，4(4):296-300．
[10]吴文林.器官移植排斥与耐受的研究进展[J].泉州师范学院学报,2003,21(4):77-82.
[11] Guillonneau C, Seveno C, Dugast AS, et al. Anti-CD28antibodies modify regulatorymechanisms and reinforce tolerance in CD40Ig-treated heart allograft recipients[J].J Immunol,2007,179(12):8164-8171.
[12]姜晓峰,张弘彬,郭大伟等.联合阻断共刺激信号对移植免疫耐受的影响[J].中国免疫学杂志,2009,25(7):634-637.
[13]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[J].华西医学,2007,22(3):659-660.
[14] Carella AM, Arena GD, Casavilla N. Monoclonal antibodies to treatGraft-Versus-Host Disease[J]. Mini-Reviews in Medicinal Chemistry,2011,11(6):473-479.
[15]孙凯,冯琦,金伯泉. Ig融合蛋白临床应用前景[J].中国肿瘤生物治疗杂志,2000,7(3):229-231.
[16]袁岸龙,夏冰,黄梅芳. CTLA-4的免疫学作用与疾病[J].武汉大学学报（医学版）,2003,24(1):90-93.
[17]王朝健,李元. CTLA-4基因工程药物研究进展[J].国外医学药学分册,2000,27(6):231-235.
[18] Weclawiak H, Kamar N, Ould-Mohamed A, et al. Biological agents in kidneytransplantation: belatacept is enetering the field[J]. Expert Opin Biol Ther,2010,10(10):1501-1508.
[19]郭亚军,候盛,寇庚等.一种免疫毒素及其制备方法和用途：中国, CN101062953A[P].2007-10-31.
[20] Tazzari PL, Polito L, Bolognesi A, et al. Immunotoxins Containing RecombinantAnti-CTLA-4Single-Chain Fragment Variable Antibodies and Saporin: In VitroResults and In Vivo Effects in an Acute Rejection Model[J]. J Immunol,2001,167(8):4222-4229.
[21] Zeng L, Wan L, Chen L, et al. Selective Depletion of Activated T Cells byRecombinant Immunotoxin Containing Anti-CTLA-4Single-Chain Fragment ofVariable Antibody and N-Terminal Fragment of Perforin[J]. Transplant Proc,2006,38(7):2151-2153.
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