抗人CD154人—鼠嵌合抗体Fab片段的制备及其结合活性的研究
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摘要
CD154是一种分子量为39kD的Ⅱ型跨膜糖蛋白，其基因定位于Xq26.3-Xq27.1，属于肿瘤坏死因子(TNF)超家族，主要表达在活化的CD4~+T细胞和肥大细胞表面。越来越多的研究表明，CD40／CD154在特异性免疫应答中发挥重要的作用，通过CD40／CD154途径：(1)促使抗原递呈细胞的有效活化，进而介导其成熟、分化，启动免疫应答的产生；(2)活化T淋巴细胞，介导细胞免疫应答，参与记忆性T淋巴细胞的形成；(3)促使B细胞的活化、增殖、分化，分泌Ig及类别转换，对生发中心和记忆性B细胞的形成及维持起重要作用；(4)促进免疫活性细胞分泌多种细胞因子(IL-1、IL-8、IL-12、TNF-α等)，参与免疫应答的调节。
业有报道，运用抗CD154单克隆抗体阻断CD40／CD154途径，能有效地抑制特异性免疫应答，减少特异性抗体和CTL的产生，在自身免疫、移植排斥、炎症等病理免疫反应中发挥重要作用。同时，在皮肤、心脏、肾脏等小鼠移植模型中，运用抗CD154单克隆抗体能显著延长移植物的存活期，为移植排斥的防止和移植耐受的诱导提供了新方法。该单抗在基础及临床免疫学中的应用前景已受到广泛关注。
但是，鼠源性单抗作为异源蛋白，容易引起人体的人抗鼠反应(HAMA)，且抗体分子量大，在体内很难快速到达靶器官，从而限制了其临床应用，随着基因工程技术的发展，基因工程抗体为解决以上问题提供了一条有效途径。人-鼠嵌合抗体Fab片段的稳定性好，分子量不到完整抗体的1／3，使其比完整抗体更易接近靶组织。
本研究利用本实验室特有的抗人CD154单克隆抗体4F1，运用RT-PCR技术，从4F1杂交瘤细胞中克隆出抗人CD154单抗的可变区基因，并与人免疫球蛋白IgG1的恒定区基因重组成抗人CD154人-鼠嵌合抗体Fab融合基因，构建嵌合Fab共表达载体pTXB1-Fab，利用大肠杆菌表达简单，高效，成本低廉的特点，对重组抗体进行原核表达。实验表明联合应用各种表达优化条件，包括降低温度，减少IPTG浓度，延长诱导时间可获得可溶性蛋白表达，且蛋白表达动力学分析表明：在0.05mmol／L
抗人CD巧4人一鼠嵌合抗体Fab片段的制备及其结合活性的研究
摘要
IPTG，1%蔗糖，18℃，诱导16h可获得可溶性蛋白表达。以羊抗人IgG(H+L)多
抗为一抗，兔抗羊IgG为二抗，Western一blot检测，结果显示两条特异性条带，分子
量大小约为25kD、27kD，与重、轻链蛋白理论值基本一致，且目的条带深浅一致，
说明重轻链表达基本平衡，进而表明构建的共表达载体在原核细胞中得到了较好的
表达。为了研究其活性和抗原结合能力，用竞争抑制实验检测Fab结合的特异性，
选取高表达人CD154的CD154一L929转基因细胞(CD154一TC)与reFab反应，竞争
性抑制鼠单抗4FI与CD154一TC的结合，阳性对照为CD154-TC加4FI，阴性对照只
加荧光二抗。FACS结果显示，rcFab能部分竞争性抑制4FI与CD154L一TC的结合，
说明rcFab能识别结合4FI的抗原位点，与CD 1 54一TC能特异性结合，部分抑制鼠源
亲本抗体的结合。但是，尽管使用较大量的rcFab，考虑到重组嵌合抗体抗原结合力
较天然抗体大为降低，且由于rcFab为单价，而4FI为二价，故人源化嵌合Fab抗体
的亲和力较鼠源单抗有所降低，不能完全将其阻断。
总之，本实验成功克隆获得阻断型抗人CD154 mAb(4FI)单克隆抗体的轻链、
重链可变区基因及人免疫球蛋白IgGI恒定区基因，并且拼接完成了完整的嵌合重链
和嵌合轻链基因，成功构建pTXBI一Fab共表达载体，在大肠杆菌中实现可溶性表达，
为抗体的分离纯化带来了便捷，同时也为本所其它鼠源性单抗的人源化嵌合Fab小
分子抗体的制备奠定了技术基础。表达产物在大肠杆菌质周腔中能形成正确的空间
折叠，装配成具有生物学活性的小分子抗体片段，为该分子的进一步研究和临床应
用奠定了基础。
CD 154 (CD40L, gp39) is a 39kD glycoprotein expressed as a type II intergrel membrane protein on the surface of activated CD4+ T cells. CD 154, encoded by a gene located at Xq26.3-Xq27.1, belongs to tumor necrosis factor (TNF) superfamily.
The contribution of CD40/CD154 interactions to the process of T cell priming, differentiation, and effector functions has been extensively reviewed. CD40/CD154 interactions play a critical role in T cell priming and have been suggested to prohibit tolerance induction. Therefore, interference with CD 154 mAbs has been extensively investigated to prolong the survival of experimentally transplanted organs. The treatment with anti-CD 154 monoclonal antibody prolonged survival of experimentally transplanted organs in some murine models including allografts of heart, skin, aorta, and pancreatic islets. It is important that in rhesus monkey model of kidney transplantation, exciting therapeutic effects of anti-CD 154 mAb treatment have been observed. Although mAbs have held great promise for the treatment of human disease such as cancer, viral infection and autoimmune disorders, the most important impediments in murine mAb have been the immune response against murine immunoglobulins and insufficient activation of hu
man effector function. These problems could be overcome using genetic engineering techniques to produce humanized antibodies.
In our study, an expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli.
The single-strand cDNA was prepared by AMV reverse transcription from poly A+ RNA extracted from hybridoma cells (4F1) secreting mouse anti-human CD 154 mAb. Amplification of murine variable regions (mVH & mVL) was achieved by PCR using a pair of specific consensus primers. At the same time, constant regions of human
immunoglobulin IgG1 (huFc, huCk and huCH1) were obtained from human spleen cells by RT-PCR using corresponding primers. By TP-PCR, the variable regions are fused directly to corresponding domains of the constant regions without any mutation or endonuclese sites to constitute a mouse and human chimeric H and L chains. For antibody expression, a novel co-expression vector was constructed containing a pelB signal sequence and at the C terminus of CHI there is an 11 amino acid c-myc peptide tag which could be recognized by a polyclonal anti-c-myc antibody and aids detection of the expressed Fab during purification. The pelB signal sequence directs chimeric heavy or light chains into the bacterial periplasm, where protein folding as well as heterodimer association occurred correctly to form functional Fab. Thus, the assembly pathway for the Fab fragment of CD154 mAb is similar to that of a whole antibody in the eukaryotic cell. The Fab fragment of CD 154 mAb was purified to homogeneity with affinity chromatograph
y in a single step. The antigen-binding ability of the recombinant Fab fragment was demonstrated by competitive inhibitory assay showing that the affinity constant of the Fab fragment is a little lower than that of the parental murine antibody CD154mAb(4Fl).
In conclusion, our studies indicated that mAbs from murine hybridomas may be simply and rapidly converted to chimeric Fab which can be expressed in bacteria and purified in reasonable quantities. This expression system should facilitate future protein engineering experiments on murine monoclonal antibodies. Besides, cloned antibody fragments also have the additional advantages in that they may be manipulated to alter affinity or to make fusion proteins with enzymes or toxins.
业有报道，运用抗CD154单克隆抗体阻断CD40／CD154途径，能有效地抑制特异性免疫应答，减少特异性抗体和CTL的产生，在自身免疫、移植排斥、炎症等病理免疫反应中发挥重要作用。同时，在皮肤、心脏、肾脏等小鼠移植模型中，运用抗CD154单克隆抗体能显著延长移植物的存活期，为移植排斥的防止和移植耐受的诱导提供了新方法。该单抗在基础及临床免疫学中的应用前景已受到广泛关注。
但是，鼠源性单抗作为异源蛋白，容易引起人体的人抗鼠反应(HAMA)，且抗体分子量大，在体内很难快速到达靶器官，从而限制了其临床应用，随着基因工程技术的发展，基因工程抗体为解决以上问题提供了一条有效途径。人-鼠嵌合抗体Fab片段的稳定性好，分子量不到完整抗体的1／3，使其比完整抗体更易接近靶组织。
本研究利用本实验室特有的抗人CD154单克隆抗体4F1，运用RT-PCR技术，从4F1杂交瘤细胞中克隆出抗人CD154单抗的可变区基因，并与人免疫球蛋白IgG1的恒定区基因重组成抗人CD154人-鼠嵌合抗体Fab融合基因，构建嵌合Fab共表达载体pTXB1-Fab，利用大肠杆菌表达简单，高效，成本低廉的特点，对重组抗体进行原核表达。实验表明联合应用各种表达优化条件，包括降低温度，减少IPTG浓度，延长诱导时间可获得可溶性蛋白表达，且蛋白表达动力学分析表明：在0.05mmol／L
抗人CD巧4人一鼠嵌合抗体Fab片段的制备及其结合活性的研究
摘要
IPTG，1%蔗糖，18℃，诱导16h可获得可溶性蛋白表达。以羊抗人IgG(H+L)多
抗为一抗，兔抗羊IgG为二抗，Western一blot检测，结果显示两条特异性条带，分子
量大小约为25kD、27kD，与重、轻链蛋白理论值基本一致，且目的条带深浅一致，
说明重轻链表达基本平衡，进而表明构建的共表达载体在原核细胞中得到了较好的
表达。为了研究其活性和抗原结合能力，用竞争抑制实验检测Fab结合的特异性，
选取高表达人CD154的CD154一L929转基因细胞(CD154一TC)与reFab反应，竞争
性抑制鼠单抗4FI与CD154一TC的结合，阳性对照为CD154-TC加4FI，阴性对照只
加荧光二抗。FACS结果显示，rcFab能部分竞争性抑制4FI与CD154L一TC的结合，
说明rcFab能识别结合4FI的抗原位点，与CD 1 54一TC能特异性结合，部分抑制鼠源
亲本抗体的结合。但是，尽管使用较大量的rcFab，考虑到重组嵌合抗体抗原结合力
较天然抗体大为降低，且由于rcFab为单价，而4FI为二价，故人源化嵌合Fab抗体
的亲和力较鼠源单抗有所降低，不能完全将其阻断。
总之，本实验成功克隆获得阻断型抗人CD154 mAb(4FI)单克隆抗体的轻链、
重链可变区基因及人免疫球蛋白IgGI恒定区基因，并且拼接完成了完整的嵌合重链
和嵌合轻链基因，成功构建pTXBI一Fab共表达载体，在大肠杆菌中实现可溶性表达，
为抗体的分离纯化带来了便捷，同时也为本所其它鼠源性单抗的人源化嵌合Fab小
分子抗体的制备奠定了技术基础。表达产物在大肠杆菌质周腔中能形成正确的空间
折叠，装配成具有生物学活性的小分子抗体片段，为该分子的进一步研究和临床应
用奠定了基础。
CD 154 (CD40L, gp39) is a 39kD glycoprotein expressed as a type II intergrel membrane protein on the surface of activated CD4+ T cells. CD 154, encoded by a gene located at Xq26.3-Xq27.1, belongs to tumor necrosis factor (TNF) superfamily.
The contribution of CD40/CD154 interactions to the process of T cell priming, differentiation, and effector functions has been extensively reviewed. CD40/CD154 interactions play a critical role in T cell priming and have been suggested to prohibit tolerance induction. Therefore, interference with CD 154 mAbs has been extensively investigated to prolong the survival of experimentally transplanted organs. The treatment with anti-CD 154 monoclonal antibody prolonged survival of experimentally transplanted organs in some murine models including allografts of heart, skin, aorta, and pancreatic islets. It is important that in rhesus monkey model of kidney transplantation, exciting therapeutic effects of anti-CD 154 mAb treatment have been observed. Although mAbs have held great promise for the treatment of human disease such as cancer, viral infection and autoimmune disorders, the most important impediments in murine mAb have been the immune response against murine immunoglobulins and insufficient activation of hu
man effector function. These problems could be overcome using genetic engineering techniques to produce humanized antibodies.
In our study, an expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli.
The single-strand cDNA was prepared by AMV reverse transcription from poly A+ RNA extracted from hybridoma cells (4F1) secreting mouse anti-human CD 154 mAb. Amplification of murine variable regions (mVH & mVL) was achieved by PCR using a pair of specific consensus primers. At the same time, constant regions of human
immunoglobulin IgG1 (huFc, huCk and huCH1) were obtained from human spleen cells by RT-PCR using corresponding primers. By TP-PCR, the variable regions are fused directly to corresponding domains of the constant regions without any mutation or endonuclese sites to constitute a mouse and human chimeric H and L chains. For antibody expression, a novel co-expression vector was constructed containing a pelB signal sequence and at the C terminus of CHI there is an 11 amino acid c-myc peptide tag which could be recognized by a polyclonal anti-c-myc antibody and aids detection of the expressed Fab during purification. The pelB signal sequence directs chimeric heavy or light chains into the bacterial periplasm, where protein folding as well as heterodimer association occurred correctly to form functional Fab. Thus, the assembly pathway for the Fab fragment of CD154 mAb is similar to that of a whole antibody in the eukaryotic cell. The Fab fragment of CD 154 mAb was purified to homogeneity with affinity chromatograph
y in a single step. The antigen-binding ability of the recombinant Fab fragment was demonstrated by competitive inhibitory assay showing that the affinity constant of the Fab fragment is a little lower than that of the parental murine antibody CD154mAb(4Fl).
In conclusion, our studies indicated that mAbs from murine hybridomas may be simply and rapidly converted to chimeric Fab which can be expressed in bacteria and purified in reasonable quantities. This expression system should facilitate future protein engineering experiments on murine monoclonal antibodies. Besides, cloned antibody fragments also have the additional advantages in that they may be manipulated to alter affinity or to make fusion proteins with enzymes or toxins.
引文
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[2] Flamand V, Donckier V, Demoor F, et al. Cutting Edge: CD40 Ligation Prevents Neonatal Induction of Transplantation Tolerance. The Journal of Immunology. 1998; 160:4666—4669
[3] Pankhurst T and Adu D. Antibodies in the prevention of renal allograft rejection. Expert Opin Biol Ther. 2004 Feb; 4(2):243-252
[4] Villa A, Notarangelo LD, Di Santo JP, et al. Organizaiton of the human CD40L gene: implications for molecular defects in X-chromosome-linked hyper-IgM syndrome and prenatal diagnosis. Proc. Natl. Acad. Sci. USA. 1994; 91:2110-2114
[5] Hollenbaugh D, Grosmaire LS, Kullas CD, et al. The human antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 1992; 11,4313-4321
[6] Spriggs MK, Armitage RJ, Strockbine L, et al. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J. Exp. Med. 1992; 176:1543-1550
[7] Karpusas M, Hsu YM, Wang JH, et al. 2A crystal structure of an extracelular fragment of human CD40L. Structure. 1995; 3:1031-1039
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[34] Kim MS, Kim NS, Sung YH, et al. Biphasic culture strategy based on hyperosmotic pressure for improved humanized antibody production in Chinese hamster ovary cell culture. In Vitro Cell Dev Biol Anim. 2002 Jun; 38(6):314-319
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[36] Pankhurst T, Adu D. Antibodies in the prevention of renal alloraft rejection. Eur J Immuno. 2004 Mar; 34(3):850-8
[37] Allann D, Kirk L, Linda C, et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates. Nature Medicine. 1999; 5:686-693
[38] Xu H, Tadaki DK, Elster EA, et al. Humanized anti-CD154 antibody therapy for the treatment of allograft rejection in nonhuman primates. Transplantatin. 2002 Oct; 74(7):940-943
[39] Schuler W, Bigaud M, Brinkmann V, et al. Efficacy and safety of ABI93, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation. Transplantation. 2004 Mar; 77(5):717-26
[2] Flamand V, Donckier V, Demoor F, et al. Cutting Edge: CD40 Ligation Prevents Neonatal Induction of Transplantation Tolerance. The Journal of Immunology. 1998; 160:4666—4669
[3] Pankhurst T and Adu D. Antibodies in the prevention of renal allograft rejection. Expert Opin Biol Ther. 2004 Feb; 4(2):243-252
[4] Villa A, Notarangelo LD, Di Santo JP, et al. Organizaiton of the human CD40L gene: implications for molecular defects in X-chromosome-linked hyper-IgM syndrome and prenatal diagnosis. Proc. Natl. Acad. Sci. USA. 1994; 91:2110-2114
[5] Hollenbaugh D, Grosmaire LS, Kullas CD, et al. The human antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity. EMBO J. 1992; 11,4313-4321
[6] Spriggs MK, Armitage RJ, Strockbine L, et al. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion. J. Exp. Med. 1992; 176:1543-1550
[7] Karpusas M, Hsu YM, Wang JH, et al. 2A crystal structure of an extracelular fragment of human CD40L. Structure. 1995; 3:1031-1039
[8] Hermann P, Kooten van C, Gaillard C, et al. CD40 Iigand -positive CDS+ T cell clones allow b cell growth and differentiation. Eur. J. Immunol. 1995; 25:2972-2977
[9] Math F, Schonbeck U, Sukhova GK, et al. Functional CD40 Iigand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 Iigand signaling in atherosclerosis. Proc. Natl. Aead. Sci. USA. 1997; 94:1931-1936
[10] Essen D, Kikutani H. and Gray D. CD40 ligand-transduces co-stimulation of T cells in the development of helper function. Nature. 1995; 378:620-623
[11] Flores-Romol, Bjorek P, Duvert B, et al. CD40 ligation on human cord blood CD34+
hematopoietic Progenitors induces their Proliferation and differentiation into. functional dendritic cells. J. Exp. Med. 1997; 185:341-349
[12] Romani N, Lenz A, Glassl H, et al. Clutured human Langerhans cells resemble hymphoid dendritic cells in phynotye and function. J. Invest. Dermatol. 1989; 93:600-609
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