基因工程抗体的研究

本文综述了本实验室近年来在基因工程抗体方面所做的工作：①从杂交瘤细胞基因组中筛选和鉴定出抗乙型脑炎病毒单克隆抗体的重链、轻链可变区基因，构建成人-鼠嵌舍基因并在骨髓瘤细胞中表达出抗乙型脑炎病毒的人-鼠嵌合抗体；②根据文献中抗CD3单抗的序列，进行了分子设计，设计出改形抗体的分子序列．用合成和PCR补齐的方法构建了改形的单域抗体（Vn)表达载体，并在大肠杆菌中表达；④构建了外分泌型-附着型的表达单链抗体的表达载体；④克隆和测序了抗人肺腺癌．膀胱癌、CD3的单抗重,轻链可变区基因，并正在构建鼠的抗体库。     

抗人胃癌3H11人-鼠嵌合抗体的构建及表达

为了降低抗人胃癌鼠单抗3H11的免疫原性,以利于该单抗在临床中的应用。我们构建并表达了3H11的人-鼠嵌合抗体,将3H11的轻、重链可变区基因分别插入到含有人k链及IgGl重链恒定区基因的真核细胞表达载体中,构建了3H11人-鼠嵌合抗体轻、重链表达载体。应用Lipofectin方法先将嵌合轻链表达载体转染到Sp2/0细胞中,经用含霉酚酸的选择培养基筛选及克隆化培养,获得稳定分泌3H11人-鼠嵌合轻链的转染细胞株。再将嵌合重链表达载体转染该细胞系,用含有组氨醇的选择培养基筛选,获得组氨醇抗性细胞株,经亚克隆后得到可稳定分泌人k链和人IgGl的转染细胞系,经ELISA检测该细胞系所分泌的上清含有可与人胃癌细胞系803结合的人IgG抗体活性,RT-PCR结果显示该细胞株有人-鼠嵌合抗体mRNA的转录,证明已获得分泌3H11人-鼠嵌合抗体的细胞系。     

抗人CD3人/鼠嵌合抗体的表达及特性

将从HIT3a基因库分离克隆的功能性抗CD3轻重链可变区基因插入含有人k轻链及γ1重链恒定区基因的哺乳动物表达载体中，构建了抗CD3嵌合抗体基因,用Lipofectin将抗CD3人／鼠嵌合轻重链基因共转染SP2／0细胞表达，获得稳定传代和稳定表达抗CD3嵌合抗体的转染杂交瘤细胞株C-HIT3a，ELISA，免疫荧光和PCR实验证实嵌合抗体与原鼠CD3单抗有相同的特异性和亲和性。体外生物活性的初步分析表明嵌合抗体与鼠CD3单抗对人的PBMC细胞的增殖有相同类型的作用，高剂量抗体抑制人T细胞的增殖，低剂量显示明显的激活增殖作用，当与IL-2联合应用其激活增殖作用增加20倍。细胞毒实验表明嵌合CD3抗体或由该抗体介导的免疫活性细胞(CD3-AK)对多种肿瘤细胞有明显的杀伤活性，较单用IL-2或单用LAK细胞其杀伤活性增加25倍。     

抗CD3人／鼠嵌合抗体的研究

已有的研究表明抗人T细胞CD3抗原的单抗具有激活和抑制人T细胞的双向功能。该单抗在抗肿瘤、抗多种器官移植排斥反应及再生障碍贫血的治疗中已显示出广阔的应用前景。但目前所用的CD3单抗都是鼠源的，大大地限制了它在临床治疗中的应用。为解决这一难题，本研究应用基因工程技术将鼠源CD3单机进行“人化”改造，在国内首次成功地构建并在其核细胞表达了抗CD3人／鼠嵌合抗体。这里综合报道研究结果如下：一、CD3单抗可变区基因cDNA的克隆和鉴定从分泌抗CD3单抗的小鼠杂交瘤HIT3a提取分离RNA，用小鼠Ig轻重涟恒定区保守序列为引物，…     

抗人CD3人/鼠嵌合抗体基因的构建

为了降低鼠源抗人CD3单抗的免疫原性．增加其在人体内的生物活性及治疗作用，使该抗体能更广泛更有效地长期多次用于人体治疗肿瘤、器官移植排斥反应及自身免疫性疾病。本文采用PCR技术从分泌抗CD3单抗的杂交瘤细胞HIT3a的mRNA中分离克隆了抗体的轻重链可变区基因的cDNA。以此轻重链可变区cDNA为特异探针从HIT3a基因文库中分离带有调控序列的功能性轻重链可变区基因，并将其插入到含有人k轻链及人71重链恒定区基因的哺乳动物表达载体中成功地构建了抗人CD3人／鼠轻重链嵌合抗体基因，为研制人抗CD3入／鼠嵌合抗体完成了关键性的第一步。     

抗人血管内皮生长因子165嵌合抗体的构建及其真核表达

目的 在真核细胞中表达抗人血管内皮生长因子１６５（ｖａｓｃｕｌａｒ ｅｎｄｏｔｈｅｌｉａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ １６５，ＶＥＧＦ１６５）人／鼠嵌合抗体。方法 将抗ＶＥＧＦ１６５鼠单抗ＶｍＤ１１的轻，重甸可变区基因克隆入基因工程抗体真核表达载体中，转染二氢叶酸还原酶缺陷型中国仓鼠卵巢（ｄｉｈｙｄｒｏｆｏｌａｔｅ ｒｅｄｕｃｔａｓｅ －ｄｅｆｉｃｉｅｎｔ Ｃｈｉｎｅｓｅ ｈａｍｓｔｅｒ ｏｖａ     

188Re标记CEA嵌合人源化抗体在荷人结肠癌裸鼠的放射免疫显像研究

背景与目的：癌胚抗原（carcinoembbryonic antigen,CEA）在多种肿瘤,尤其是结肠癌中高表达,抗CEA抗体在人结肠癌的诊断治疗中具有良好的应用前景。本研究采用^188Re标记CEA嵌合抗体,研究其在荷人结肠癌裸鼠体内的生物分布及放射免疫显像。方法：用氯化亚锡还原法标记CEA嵌合抗体及其亲本鼠单抗C50,薄层层析法鉴定标记抗本的标记率、放化纯度及体外稳定性,ELISA鉴定标记后的免疫活性,研究^188Re-CEA嵌合抗体在荷人结肠癌裸鼠体内的分布及放射免疫显像效果,并与C50鼠单抗的显像效果进行比较。结果：^188Re-CEA嵌合抗体的放化纯度大于95%;比活为36MBq/mg;免疫活性为61%;薄层层析示其在体外稳定。^188Re-CEA嵌合抗体的生物学分布显示：注射后24h,肿瘤与肾脏、血液的放射性比值分别为0.75、0.99,与其余各脏器的放射性比值均大于1.78;48h,肿瘤与肾脏、血液的放射性比值分别为1.02、1.12,与其余各脏器的放射性比值均大于2.08。20h后,肿瘤显影清晰。CEA嵌合抗体的放射生物学特性及显像效果与C50鼠单抗基本相同。结论：^188Re-CEA嵌合抗体在裸鼠体内放射免疫显像显示出良好的肿瘤特异性,且显像速度较快,可显示的肿瘤最小可达0.5cm。CEA嵌合抗体降低了免疫原性,在人体内显像更优于其亲本鼠单抗C50。     

抗人血管内皮生长因子嵌合抗体在真核细胞中的高效表达

目的：在中国仓鼠卵巢（Chinese hamster ovary,CHO)细胞中高效表达有活性的抗人血管内皮生长因子（vascular endothelial growth factor,VEGF)嵌合抗体,并探索获得最佳表达的途径。方法：采用一种新型的基因工程抗体真核高效表达载体系统,将抗VEGF嵌合抗体轻,将抗VEGF嵌合抗体轻、重链基因导入二氢叶酸还原酶缺陷型CHO细胞,筛选表达抗VEGF嵌合抗体的克隆,再进行递增逍度的氨甲喋呤（methotrexate,MTX)加压扩增表达,采用ELISA检测所表达的嵌合抗体的生物学特性和产量。结果：采用三种不同的筛选加压扩增表达方法获得的结果有差异,其中采用每轮加压扩增表达后进行亚克隆的方法获得了高表达产量的克隆,产量可达28ug/ml。ELISA结果证实所表达的抗体为特异地与VEGF结合的、含人抗体恒定区的抗VEGF嵌合抗体。结论：成功地在真核细胞中高效表达了有活性的抗VEGF嵌合抗体,为下一步该嵌合抗体的临床试用奠定了重要的基础。并探索出该表达系统最佳的筛选加压扩增表达方法。     

抗膀胱癌单抗Fab段基因的克隆及表达

目的：克隆抗膀胱癌单抗BDI的Fab段基因并在大肠杆菌中表达。方法：用逆转录－聚合酶链反应技术（RT－PCR），从分泌抗人膀胱癌的鼠单抗杂交瘤细胞系中克隆k链和Fd段基因，克隆到Fab表达载体中，在大肠杆菌表达噬菌体抗体和可溶Fab；运用PCR介导的定位点突变改造VH氨基端序列；用ELISA、免疫组化法等进行特异性鉴定。结果：从分泌抗膀胱癌的鼠单抗杂交瘤细胞系中克隆了重链Fd段和k链基因，在大肠杆菌中获得有抗原结合活性的噬菌体抗体和可溶性Fab的表达，但活性很弱，将VH氨基端序列矫正为亲本单抗原始序列后，明显改善了其活性，通过ELISA、免疫组化及模拟抗体库筛选证实了所获抗体片段的特异性结合及在抗体库技术中的可用性。结论：获得了功能性抗膀胱癌小分子抗体，并再次提示抗体氨基端序列对抗体活性的影响的重要性。     

嵌合轻链为模板导向筛选人源性抗肝癌抗体重链Fd片段

目的:利用噬菌体抗体库和导向选择技术,以抗HAb18G嵌合抗体轻链为模板,筛选人源性抗肝癌抗体Fd片段.方法:利用RT-PCR自肝癌患者外周血淋巴细胞中扩增全套人抗体重链Fd基因片段,克隆入含嵌合L链的展示载体pComb3X,建立人-鼠杂合Fab噬菌体抗体库.然后,利用大肠杆菌表达的非融合HAb18GE为抗原进行亲和筛选,利用pⅢ融合抗体的形式进行克隆鉴定,并对筛选出的杂合抗体进行初步的功能检测.结果:构建成功库容量为2×107PFU的杂合人-鼠噬菌体抗体库,利用非融合表达的HAb18GE进行6轮筛选.ELISA及流式细胞仪检测,其中7个克隆呈特异性阳性反应.其中克隆AP6-2和AP6-7亲和力较高,测序显示其基因序列相同,且与亲本抗体恒定区序列相同,属IgG2亚类,可变区属VH3家族.结论:通过本实验,利用嵌合轻链为模板,进行Fd片段替换,成功筛选到人源性抗体Fd片段.     

Construction and expression of a human-mouse chimeric antibody against human bladder cancer

Human bladder cancer is one of the most common malignant diseases in urogenital system. Traditional therapies are far from successful, especially in advanced cases[1,2]. Targeted therapy with specific antibody is considered a promising strategy for the treatment of diseases as carcinoma, immune disorders and infectious diseases, and so on[3-5]. BDI-1 is an anti-human bladder cancer monoclonal antibody produced through hybridoma technique. It has undergone a series of trials with good results…     

Cloning of the genes for T84.66, an antibody that has a high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human/mouse T84.66 genes in myeloma and Chinese hamster ovary cells

Carcinoembryonic antigen (CEA) is one of the best characterized tumor-associated antigens and is extensively used in the in vitro immunodiagnosis of human colon adenocarcinomas. Among a number of anti-CEA monoclonal antibodies, the murine monoclonal antibody T84.66 shows the highest specificity and affinity for CEA and has been used successfully for in vivo tumor imaging in mice and humans. We report here the cloning and sequencing of the genes coding for monoclonal antibody T84.66 and the amino acid sequence of the variable regions for the heavy and light chains. We also report the construction of mouse/human chimeric IgG1 antibody genes using T84.66 variable region genes and human constant region genes. The resulting chimeric gene constructs were transfected into murine myeloma cells (Sp2/0) by electroporation and into Chinese hamster ovary cells by lipofection. The chimeric antibodies obtained exhibited the same specificity and affinity for CEA as that of the T84.66 immunoglobulin produced by the murine hybridoma cell line. Antibody concentrations in culture medium supernatants were clonally variable but similar (15-480 ng/ml) for both Sp2/0 and Chinese hamster ovary transfectants; the average production by Chinese hamster ovary transfectants was only 3-5-fold less than Sp2/0 transfectants. Ascites production of Sp2/0 transfectants is sufficiently high (900 micrograms/ml) for initial in vivo studies with humanized T84.66.     

Establishment and Characterization of Mouse-Human Chimeric Monoclonal Antibody to erbB-2 Product

A mouse-human chimeric antibody for erbB -2 product was established by a new procedure using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The E401 hybridoma secreted anti- erbB -2 product monoclonal antibody (MoAb) (IgG1, k ). The gene of the mouse variable regions of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the E401 hybridoma RNA. The variable region of heavy chain was joined with the expression vector, which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells E401-12, which produced only murine immunoglobulin light chains. A chimeric monoclonal antibody CH401 retained full binding reactivity to erbB -2 product, compared with murine E401 parental antibody. Furthermore, the chimeric MoAb CH401 was much more efficient in supporting antibody dependent cell-mediated cytotoxicity activity against erbB -2-bearing human adenocarcinoma cells than murine MoAb E401. These suggest that a chimeric monoclonal antibody CH401 may be a potent reagent for therapy of human adenocarcinomas.     

A chimeric antibody to varicella-zoster virus glycoprotein e

Varicella-Zoster virus (VZV) immune globulin (VZIG) derived from pooled human serum is currently used in immunotherapy of VZV-associated complications of chickenpox and shingles. We developed a mouse-human chimeric antibody against a VZV glycoprotein E (gE) epitope as a safer replacement for VZIG. Variable (V) heavy- and V kappa light-chain exons, derived from an anti-VZV gE antibody secreting mouse hybridoma cell line, were cloned into expression vectors containing an immunoglobulin promoter and enhancer, and human IgG1 or kappa constant (C) region genes. The expression vectors were cotransfected into mouse myeloma cell line (NSO), generating transformants that secreted chimeric human-mouse IgGs. The chimeric and the parent mouse antibody were indistinguishable in their antigen binding specificity. VZV gE chimeric antibody may prove to be a prophylactic antibody that could provide significant advantages over VZIG in having defined specificity, lessened possibility of contamination with viral pathogens, and consistent availability.     

Expression of an ovarian cancer anti-idiotype antibody (6B11VLVHCH3) in Chinese hamster ovary (CHO) cells with improved immunoactivity and stability over proteins expressed in prokaryotic cells

The 6B11VLVHCH3 is an anti-idiotype antibody to ovarian cancer. It can mimic the ovarian cancer-associated antigen OC166-9. We have previously expressed it in Escherichia coli. We now express the 6B11VLVHCH3 in a Chinese hamster ovary (CHO) cell system. The 6B11VLVHhCH3 sequence was amplified from the prokaryotic vector and cloned into the eukaryotic expression vector pSecTag2. The construct was transfected into CHO cells. Then the transfected monoclonal CHO cells were sequentially adapted to growth in serum-free medium. Functional differences between the antibody produced in E. coli or CHO cells were assessed. The antibody produced by CHO cells was able to both mimic ovarian cancer antigen and retain FC region functionality. Importantly, the antigen mimicry activity of 6B11VLVHhCH3 produced by CHO cells is approximately 20 times higher than antibody expressed by E. coli. This method of expression provides a safer and higher quality antibody for clinical drug development.     

抗VEGF嵌合抗体的生物学特性分析

目的：分析抗人血管内皮生长因子（VEGF）嵌合抗体VcD11的各种生物学特性,确定其人源性,特异性,中和VEGF的中和活性以及体内抑制肿瘤生长转移的抗肿瘤活性。方法：采用纯化的VcD11进行ELISA和Westerm blot实验,分析其人源性、特异性;采用抑制由VEGF刺激引起的内皮细胞增殖实验分析VcD11的中和活性;采用近交系小鼠T739的小鼠肺腺癌LA795实验动物肿瘤模型分析VcD11的抗肿瘤活性。结果VcD11在ELISA和Western blot实验中均呈阳性结果,并可抑制由VcD11的刺激引起的内皮细胞增殖,虽未能明显抑制小鼠肺腺癌LA795原发瘤的生长,但可显著抑制其肺转移灶的形成和工。结论,抗VEGF嵌合抗体VcD11具有人抗体的恒定区,可与人VEGF特异地结合,并具有良好的中和活性和抗肿瘤转移的活性。它可能在临床治疗肿瘤中有着重要、广泛的应用潜力。     

Generation of a recombinant mouse-human chimaeric monoclonal antibody directed against human carcinoembryonic antigen

A procedure was devised for the identification and specific cloning of functionally rearranged variable region immunoglobulin (Ig) gene segments from genomic DNA of a murine hybridoma cell line which produces a high-affinity monoclonal antibody (MAb) directed against human carcinoembryonic antigen (CEA). The cloned, functionally-rearranged murine Ig H-chain and L-chain variable region gene segments were incorporated into plasmid vectors capable of directing the expression of a chimaeric mouse-human antibody molecule with human (gamma 4, kappa) constant region sequences. Expression plasmids were transfected into a mouse myeloma cell line by electroporation and transfectomas secreting functional chimaeric antibody selected. Chimaeric antibody generated by transfectomas was analysed and shown to compete effectively with its murine counterpart for binding to the CEA epitope, and to have an equivalent antigen-binding affinity. This anti-CEA recombinant antibody should find application in in vivo diagnosis by immunoscintigraphy of human colonic carcinoma, and possibly also in therapy of the disease, overcoming some of the difficulties associated with the repeated use of non-human immunoglobulins in human patients.     

A Human/Mouse Chimeric Monoclonal Antibody against Intercellular Adhesion Molecule-1 for Tumor Radioimmunoimaging

A mouse-human chimeric antibody for intercellular adhesion molecule-1 (ICAM-1) was established by using heavy chain loss mouse mutant hybridoma and human immunoglobulin expression vector. The HA58 hybridoma secreted anti-ICAM-1 monoclonal antibody (MoAb) (IgG1, k ). The gene of the mouse variable region of heavy chain was amplified and cloned by the polymerase chain reaction technique directly from the HA58 hybridoma RNA. The variable region of heavy chain was joined with an expression vector which contains human γ1 constant gene. The expression vector was transfected into heavy chain loss mutant cells HA58-7, which produced only murine immunoglobulin light chains. The resultant chimeric MoAb HA58, chHA58, retained full-binding reactivity to ICAM-1 compared with murine HA58 parental antibody. The chimeric MoAb chHA58 showed little antibody dependent cell-mediated cytotoxic activity against cultured tumor cells. Biodistribution studies with ^99mTc-labeled chHA58 in nude mice bearing human gastric carcinoma JRST cells demonstrated that the tumor-blood ratio was 1.55 at 18 h after injection, when the tumors were clearly visible in gamma scintigraphy. These data suggest that chHA58 may be of practical use for radioimmunoimaging of a wide variety of tumors.