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。     

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CEA人鼠嵌合F(ah’)_2在昆虫细胞中的表达

癌胚抗原(CEA)是一种非特异性肿瘤标记(TW).在结肠癌,胃癌,肺癌,乳腺癌,宫颈癌等许多恶性肿瘤中均有较高表达.利用抗CEA单克隆抗体进行放免显像研究近年来发展较快,但都存在着放射性本底较高的问题,影响了显像结果.F(ab’)_2.由于分子量小,没有Fc段,从血液中清除较快,可减少非特异结合,而因显像效果较好,但仍然受HAMA反应的影响.为此,本研究利用昆虫杆状病毒表达系统在SF9细胞中表达了抗CEA人鼠嵌合F(ah’)_2,以利于临床肿瘤的放免显像.提取一株表达完整抗CEA嵌合抗体的CHO细胞的总RNA,分别用与人轻链k恒区基因及α1重链CH2基因互补的3’引物进行反转录,再以CDNA为模板,5’引物相同,都与鼠重链前导肽互补,3’引物与反转录相同分别进行PCR扩增,得到含有鼠重链前导肽,轻链可变区,完整的k恒区的680bp的轻链基因和含有鼠重链前导肽,重链可变区,     

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.     

基因工程改造鼠源性抗人脑胶质瘤单克隆抗体的初步报告

为了提高人脑胶质瘤导向诊治中单抗载体的临床实用价值，必须把鼠源性大分子单抗改造成人源化、小分子单抗。采用ＲＴ－ＰＣＲ法，从人脑胶质瘤单抗杂交瘤细胞系ＳＺ３９中克隆出３４８ｂｐ的重链可变区（ＶＨ）和３１８ｂｐ的轻链可变区（ＶＬ）ｃＤＮＡ片断。又采用重组ＤＮＡ技术，将ＶＨ和ＶＬ与人免疫球蛋白ＩｇＧ１重链ＣＨ１和轻链κ恒定区基因拼接，或将ＶＨ和ＶＬ以通用Ｌｉｎｋｅｒ直接连接，分别构建表达载体ｐＨＥＮ１－ＳＺ３９Ｆａｂ／Ｈｕ（嵌合抗体）和ｐＨＥＮ１－ＳＺ３９ＳｃＦｖ（单链抗体）。而后将此二载体转染至非抑制性大肠杆菌ＨＢ２１５１中表达。结果表明，两者的表达产物均为可溶性，以ＥＬＩＳＡ和Ｗｅｓｔ－ｅｒｎｂｌｏｔ法检测，均与人脑胶质瘤体外细胞系ＳＨＧ４４细胞膜表面抗原发生特异结合，与亲本抗体ＳＺ３９特异识别１８００００膜抗原的条带相一致。该结果显示，基因工程改造的人源化、小分子单抗为今后的临床应用展示了美好的前景。     

抗CEA抗体可变区基因的克隆及其嵌合抗体的表达

目的 在真核细胞中表达抗癌胚抗原（carcinoembryonic antigen,CEA)人－鼠嵌合抗体。方法 从分泌抗CEA鼠单抗C50的杂交瘤细胞扩增、克隆可变区基因。测序鉴定后连入真核表达载体,导入二氢叶酸还原酶缺陷型中国仓鼠卵巢（dihydrofolate reductase-deficient Chinese hamster ovary CHO-dhfr）细胞进行表达。采用间接和竞争抑制ELISA检测表达产物的人源性和抗原结合特异性。结果 序列测定初步确定所克隆的是功能性抗体可变区基因。在转染细胞上清中测到抗CEA嵌合抗体的表达。ELISA试验证实嵌合抗体具有人的恒定区,并与原鼠单抗C50有相同的抗原结合特异性。结论 成功地在真核细胞中表达了抗CEA人－鼠嵌合抗体。     

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     

分泌抗CD71人-鼠嵌合抗体的转染瘤细胞分泌抗体的特异性与稳定性的研究

 目的　研究在液氮中冻存 2年的分泌抗CD71人 鼠嵌合抗体的转染瘤细胞分泌抗体的特异性、稳定性及抗体产量。方法　利用人IgG和鼠IgG作为浓度标准 ,绘制浓度曲线 ,比较研究转染瘤细胞培养上清的抗体分泌量。腹腔接种转染瘤细胞诱导裸鼠产生腹水抗体 ,经离子交换法纯化 ,电泳鉴定纯化嵌合抗体。结果　 1× 10 ⁵个 5ml转染瘤细胞培养 5天的上清中 ,嵌合抗体分泌量为 (0 .5～ 5 ) μg ml。Balb c裸鼠腹腔接种转染瘤细胞后 ,每只裸鼠腹水量在 (3～ 5 )ml,腹水抗体经纯化 ,抗体产量约为 (1～ 2 )mg (ml腹水 )。经离子交换法纯化 ,电泳鉴定 ,在分子量 5 5kDa和 2 5kDa处 ,可见有抗体IgG蛋白质的重链和轻链的染色条带。结论　由我们制备的在液氮中冻存 2年的分泌抗CD71人 鼠嵌合抗体 (D2C)的转染瘤细胞体外生长良好 ,抗体分泌稳定 ,产量高 ,特异性强。     

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…     

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.     

Recombinant human-mouse chimeric monoclonal antibody specific for common acute lymphocytic leukemia antigen

A human-mouse chimeric antibody constructed in the present study was specific for a human tumor-associated antigen, common acute lymphocytic leukemia antigen. The antibody consisted of human heavy and light chain constant domains (gamma 1 and kappa type) and mouse heavy and light chain variable domains, which were derived from human plasma cell leukemia line (ARH77) and mouse hybridoma cells (NL-1) specific for common acute lymphocytic leukemia antigen, respectively. The artificially fused immunoglobulin molecules were produced in mouse myeloma cells, X63Ag8.653 which were transformed with the chimeric heavy and light chain genes formed by joining the corresponding gene segments in vitro at the J-C introns. The human heavy chain enhancer element was ligated tot he chimeric heavy and light chain genes, and this enhancer appeared to be obligatory for the efficient production of the chimeric antibody molecules. The stably transformed cells secreted the chimeric antibody, which specifically bound a common acute lymphocytic leukemia antigen expressing cell line. The amount of the chimeric antibody produced (10-30 micrograms/ml in the serum-free medium) was comparable to that made by murine hybridoma line, NL-1. The molecular weight of the chimeric heavy chain molecules was reduced from 54,000 to 50,000 upon treatment with tunicamycin, suggesting that the peptide was normally glycosylated in the transformants. The chimeric antibody exhibited complement-dependent cytotoxicity, in which glycosylation is thought to be indispensable. The antibody also mediated antibody-dependent cell-mediated cytotoxicity to the human target cells. The antibody-dependent cell-mediated cytotoxicity activity of the chimeric antibody was twice that of the murine NL-1 monoclonal antibody when human peripheral blood mononuclear cells were used as effectors.     

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.     

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.     

Epitope mapping and use of anti-idiotypic antibodies to the L6 monoclonal anticarcinoma antibody.

Mouse monoclonal anti-idiotypic antibodies (anti-ids) were raised against L6, a murine IgG2a monoclonal antibody specific for a cell surface antigen expressed by many human carcinomas. Ten distinct anti-ids were generated. Eight anti-ids were shown to inhibit the binding of L6 to its target antigen and were characterized in detail. The heavy and light chain variable region gene segments of the monoclonal antibody L6 linked to human constant regions (chimeric L6) were expressed separately or together, to map the epitopes recognized by the anti-ids. Individual anti-ids were shown to recognize heavy chain, light chain, or combinatorial variable region determinants. Defining these specificities enabled us to select particular anti-ids for assays to monitor the pharmacokinetics of either murine or chimeric L6 antibodies in the circulation of human patients. A quantitative enzyme-linked immunosorbent assay developed with two anti-ids accurately detects less than 5 ng/ml. Anti-ids specific for light chain variable region-encoded determinants were capable of recognizing L6 antigen-binding fragments bound to the surface of human carcinoma cells. These anti-ids can be used to study the binding of chimeric L6 antibody at the surface of tumor cells in histological sections of tumor biopsies.     

Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells

Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies.     

Effect of plasmid-mediated RNA interference targeting telomerase reverse transcriptase on lung cancer cells

In the present study, a plasmid-mediated siRNA interference vector targeting the hTERT gene was constructed and stably transfected into H1299 lung cancer cells. Using real-time quantitative fluorescent PCR technology, western blotting and flow cytometry-based cell cycle profiling, the silencing effect of this vector and its inhibitory effect on proliferation in lung cancer cells were explored. Based upon the results of our previous study, a pair of siRNA sequences was selected, and a DNA template primer was designed and synthesized. After cloning of the template primer into the promoter of the pGenesil-1.1 expression vector, the constructed interference vector was validated using enzyme digestion and gene sequencing. The recombinant interference vector and empty vector were separately transfected into H1299 lung cancer cells with cationic liposomes, and stable monoclonally transfected cells were obtained after selection with G418. After stable transfection, hTERT mRNA and protein expression levels were detected using real-time RT-PCR technology and western blotting. Using the MTT method and a colony formation assay, the growth and proliferation of the stably transfected lung cancer cells were determined. Changes in the cell cycle profile of the stably transfected lung cancer cells were detected using flow cytometry. An interference vector targeting the hTERT gene (pGenesil.1-hTERT) was successfully constructed. Enzyme digestion and gene sequencing confirmed that the sequence insertion met the criteria of the design. After transfection of H1299 cells with pGenesil.1-hTERT or an empty vector, the stably transfected monoclonal cell lines H1299-pGenesil.1-hTERT and H1299-pGenesil.1 were obtained. Compared to the control cells transfected with the empty vector, the H1299-pGenesil.1-hTERT cells had significantly lower mRNA expression of hTERT (93.97±0.83% inhibition, with P<0.001). The protein expression of hTERT in H1299-pGenesil.1-hTERT cells was significantly lower compared to that in H1299-pGenesil.1 cells. The rate of proliferation of H1299-pGenesil.1-hTERT cells was lower compared to that of H1299-pGenesil.1 lung cancer cells. In H1299-pGenesil.1-hTERT cells, the number of cells in the G1 phase increased by 18.3% (P<0.05) compared to the control group; the number of cells in the S and G2 phases decreased by 10.4 and 7.9%, respectively (P<0.05). A recombinant plasmid that interfered with the expression of the hTERT target gene was successfully constructed. Upon transfection of the recombinant interference plasmid into H1299 lung cancer cells, hTERT mRNA and protein expression were down-regulated effectively, telomerase activity and cell proliferation were inhibited, and the cell cycle profile was altered.     

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。