抗体-白细胞介素2融合蛋白18382scFv-Interleukin2的表达及活性检测

目的在哺乳动物细胞内表达抗体细胞因子融合蛋白18382scFv-Interleukin2，为卵巢癌提供一种新的治疗方法。方法通过基因工程的方法将两段基因IL-2和18382scFv开放读框的编码序列克隆在一起，在CHO-K1细胞内人巨细胞病毒启动子的作用下表达可溶性融合蛋白，并检测其对肿瘤细胞的靶向性。结果采用哺乳动物细胞表达，系统表达的这种小分子融合蛋白，既保留了与卵巢癌OC18382抗原结合的特性，又保持了IL-2的生物学活性。融合蛋白在细胞培养上清中保持稳定，更重要的是融合蛋白将IL-2靶向表达OC18382抗原的卵巢癌细胞表面化的同时，又能刺激IL-2依赖细胞株的增殖，从而诱发局部有效的抗肿瘤反应。既提高了融合蛋白的穿透组织能力和肿瘤组织部位的浓聚，又避免了大剂量全身应用细胞因子引起的副作用。结论真核表达系统表达的融合蛋白具有很好的生物学活性。     

抗卵巢癌单链免疫毒素183B2ScFvPE38融合蛋白的高效表达及活性测定

目的　制备抗人卵巢癌单链免疫毒素融合蛋白 183B2 ScFvPE38,并对其活性进行测定 ,为卵巢癌导向治疗打下基础。　方法　在异丙基硫代 β D 半乳糖苷 (IPTG)的诱导下 ,高效表达免疫毒素 183B2 ScFvPE38,并采用直接ELISA及细胞毒性实验对抗体及毒素部分的活性进行检查。　结果　表达蛋白 183B2 ScFvPE38以包涵体形式为主高效表达 ,并有少量可溶性表达。该蛋白具有较好的抗体活性及毒素活性。　结论　抗卵巢癌单链免疫毒素183B2 ScFvPE38具有良好的免疫学活性及生物学活性 ,有良好的应用前景     

超抗原葡萄球菌肠毒素与抗人大肠癌单链抗体ND-1 scFv融合基因的构建、表达及活性分析

目的:构建并表达超抗原葡萄(SEA)和鼠抗人大肠癌单链抗体ND-1scFv的融合蛋白,以提高SEA的靶向杀伤作用。方法:构建超抗原SEA和鼠抗人大肠癌单链抗ND-1scFv的融合基因ND-1 scFv/SEA的表达载体,转化到大肠杆菌E.coli M15中进行诱导表达。Ni-NTA亲和层析对表达产物进行分离、纯化。间接免疫荧光法检测融合蛋白的靶向结合活性,MTT法检测靶向杀伤效率。结果:成功构建了融合基因ND-1scFv/SEA,实现功能性表达,纯化的ND-1scFv/SEA融合蛋白与表达有ND-1相应抗原的大肠癌细胞CCL-187有高度亲和活性,通过激活外周血单核细胞,可特异性杀伤靶细胞,在4μg/mL浓度下对CCL-187的杀伤率达到91%,明显优于SEA的杀伤活性。结论:融合蛋白ND-1 scFv/SEA对大肠癌细胞CCL-187具有靶向结合和杀伤活性,为SEA用于靶向性的大肠癌治疗奠定了基础。     

抗HER2单链抗体融合凋亡蛋白对SKOV3细胞的靶向杀伤

目的：探讨分泌表达的抗HER2单链抗体与重构型人caspase—3融合蛋白对HER2抗原阳性肿瘤细胞的靶向杀伤作用。方法：将重构型人caspase—3基因亚克隆人pCMV—eDscFv—PEII—PEIII的相应位点，构建重组真核表达载体pCMV—e23scFv—PEII—revcasP3，并转染人T淋巴瘤细胞系Jurkat，筛选并建系。用ELISA检测培养上清中融合蛋白的分泌表达。通过共培养实验观察含有该融合蛋白的培养上清对人卵巢癌细胞SKOV3生长的抑制作用。结果：融合蛋白基因可在Jurkat细胞中，分泌表达并杀伤SKOV3细胞。结论：分泌表达的抗erbB2单链抗体与重构型人caspase3融合蛋白能够靶向诱导SKOV3细胞死亡。     

人抗HER2单链抗体/精氨酸九聚体融合蛋白的基因构建、表达及鉴定

目的:构建人源性抗HER2单链抗体(scFv)/精氨酸九聚体(9R)融合蛋白基因,在大肠杆菌里表达纯化并检测该融合蛋白的活性。方法:采用PCR的方法,扩增融合基因scFv-9R,将获得的基因克隆入原核表达载体pQE30,在大肠杆菌M15中表达,表达产物经SDS-PAGE和Western blot鉴定,通过Ni-NTA螯合层析纯化,透析复性,超滤浓缩。ELISA分析scFv-9R融合蛋白抗原亲和活性,凝胶迁移实验检测scFv-9R融合蛋白与siRNA结合活性。结果:成功构建了人源性抗HER2 scFv-9R融合基因,经IPTG诱导后在M15中以包涵体形式表达。表达的目的蛋白scFv-9R能与HER2抗原结合,同时具有siRNA结合能力。结论:scFv-9R具有结合抗原与siRNA双重活性,为靶向递送siRNA的研究奠定了基础。     

来自噬菌体抗体库识别KG1a细胞的scFv5C1的高效表达、纯化及其生物学特性

目的为深入研究源于噬菌体抗体库特定单链抗体（scFv）的功能和其识别抗原的分子特征,将识别KG1a细胞的单链抗体5C1scFv高效表达、纯化;用scFv测定未知抗原的相对分子质量（Mr）;并研究5C1scFv对KG1a部分细胞生物学特性的影响。方法基因重组构建表达5C1scFv的载体pSTE-5C1,在大肠杆菌中诱导表达,金属离子螯和亲和层析法纯化,获得高纯度的活性5C1scFv;借助生物素-链亲和素的高亲和力和高灵敏度的显色系统,用Westernblot分析其识别的KG1a细胞膜蛋白的Mr;用聚集实验分析5C1scFv对KG1a细胞同型聚集的影响。结果5C1scFv在大肠杆菌中获高效表达,纯化后活性蛋白产量可达每升培养物50～60mg,纯度大于95%;成功地测定了其识别抗原的Mr为（85.0/124.5）×103;并确认5C1scFv以浓度依赖方式抑制KG1a细胞的同型聚集。结论高效表达纯化的5C1scFv特异性识别Mr为（85.0/124.5）×103的KG1a细胞膜表面分子,此分子可能是一种在KG1a细胞表面表达的参与细胞同型聚集的分子或受体。这些结果为进一步深入研究抗原本质及克隆抗原分子基因奠定了良好的基础。     

具有中和活性的抗人IL-1β单链抗体的构建

目的构建抗人IL-1β单链抗体(anti-hIL-1βscFv)的原核表达载体,并对其表达的蛋白进行生物学活性检测。方法使用PCR的方法从含有抗人IL-1β抗体基因的质粒中获得抗人IL-1β的单链抗体基因,将其插入原核表达载体pET-27b中,构建重组表达载体pET-27b-hIL-1βscFv。将该载体转化大肠杆菌Rosetta(DE3)后诱导表达,经凝胶过滤色谱柱上复性得到可溶的抗人IL-1β的scFv蛋白。使用ELISA方法鉴定scFv抗体的特异性结合活性,使用Real time-PCR的方法检测scFv抗体的中和活性。结果成功地对抗人IL-1β单链抗体进行诱导、表达和复性,蛋白相对分子质量约为28 000。ELISA结果证实scFv蛋白对hIL-1β具有特异性结合能力。Real time-PCR实验结果证实该scFv抗体可以有效阻断人IL-1β刺激人T细胞表达细胞因子IL-18及IL-1β的作用。结论通过原核表达系统得到的抗人IL-1β单链抗体经复性后,与人IL-1β蛋白有特异性结合能力,并且表现出中和活性,为进一步研究人IL-1β相关疾病及其治疗性抗体奠定了基础。     

IL-10在系统性红斑狼疮发病机制中的角色

系统性红斑狼疮的B细胞过度活化 ,细胞介导的免疫应答受损。而IL 10既是有效的B细胞刺激因子 ,又能抑制T细胞和抗原递呈细胞的功能 ,在SLE发病机制的免疫调节紊乱中扮演重要角色 :IL 10的基因与SLE易感性有关 ,在SLE病人的健康亲属体内IL 10也呈高表达 ,该因子还参与疾病状态下的细胞因子谱偏移及细胞调亡异常。IL 10分泌增加与狼疮环境的形成密切相关     

抗TfR单链抗体-AP融合蛋白的构建、表达与鉴定

目的构建、表达和鉴定抗转铁蛋白受体(TfR)单链抗体———碱性磷酸酶(AP)融合蛋白。方法用SfiⅠ和NotⅠ分别酶切抗转铁蛋白受体scFv表达质粒pUC19/119,获得scFv基因,直接亚克隆到表达载体pDAP2中,在TG1菌中表达scFvAP融合蛋白,SDSPAGE分析scFvAP的分子量,酶免疫测定鉴定其抗体和酶活性。结果scFvAP融合蛋白表达载体经scFv特异性引物PCR扩增,扩增产物经琼脂糖凝胶电泳可见约700bp条带,符合scFv基因理论值大小,IPTG诱导产物经SDSPAGE分析可见约为75ku的蛋白条带,符合scFvAP融合蛋白分子量的理论值大小,直接细胞ELISA测定证明表达产物scFvAP融合蛋白具有结合人TfR和AP活性的双重功效。结论抗人scFvAP融合蛋白表达载体构建成功,表达产物具有结合人TfR和AP活性的双重功效,为其临床检测应用奠定了基础。     

抗SEB单克隆抗体可变区基因克隆及单链抗体的基因构建和表达

目的 从分泌抗SEB的单克隆抗体(FMU-SEB-No.1)杂交瘤细胞中,克隆出FMU-SEB-No.1重链和轻链可变区(VH和VL)基因,构建FMU-SEB-No.1单链抗体(scFv)的原核表达载体,并进行scFv基因的蛋白表达.方法 从FMU-SEB-No.1杂交瘤细胞中提取总RNA,采用RT-PCR扩增出VH和VL基因;通过在引物上设计linker序列,拼接VH和VL为完整FMU-SEB-No.1的scFv基因(FMU-SEB-scFv).将测序正确的scFv基因克隆入PGEX4T-1载体,转化入E.coli BL21(DE3)进行蛋白表达.通过SDS-PAGE,Western blot法分析其表达水平和特异性,酶联免疫吸附试验(ELISA)鉴定其抗原结合活性.结果 测序结果显示,本实验成功克隆出FMU-SEB-No.1重链及轻链可变区基因,并成功构建FMU-SEB-scFv基因,所得的基因全长为750 bp,编码250个氨基酸.SDS-PAGE和Western blot分析表明,PGEX4T1-FMU-SEB-scFv在E.coli BL21(DE3)可表达为Mr约54 000的可溶型scFv/GST融合蛋白.间接ELISA检测结果表明,可溶型scFv/GST融合蛋白与SEB具有较高的抗原结合活性.结论 制备并鉴定了针对SEB的基因工程抗体,为开发出针对SEB的治疗性抗体奠定了实验基础.     

Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis.

Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2/neu protooncogene product and the human FcgammaRIII (CD16). To address these obstacles, we have constructed and characterized a fully human gene-fused bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD16. The human anti-CD16 scFv component, NM3E2, was isolated from a human scFv phage display library. As binding of NM3E2 to human neutrophil-associated CD16 decreased in the presence of plasma IgG, we have concluded that NM3E2 recognizes an epitope in the vicinity of the Fc binding pocket. Furthermore, the NM3E2 scFv was found by surface plasmon resonance-based epitope mapping to share an overlapping epitope with the Leu-11c mAb. The human anti-HER2/neu scFv component, C6.5, which was previously isolated from a human scFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv, peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies performed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of 125I-labeled C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h following injection. These results indicated that both scFv components of the bs-scFv retained their function in the fusion protein. This bsAb should overcome some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv offers promise as a platform for multifunctional binding proteins with potential clinical applications as a result of its human origin, lack of an Fc domain, ease of production, high level of in vitro tumor cell cytotoxicity and highly selective tumor targeting.     

Expression of a humanized single-chain variable fragment antibody targeting chronic myeloid leukemia cells in Escherichia coli and its characterization

Chronic myeloid leukemia (CML) is a malignant blood disease originating from hematopoietic stem cells. Drug resistance and tumor recurrence have become major problems for the treatment of this disease. Therefore, new therapeutic methods need to be developed. Antigens expressed on the surface of cancer cells are potential targets for antibody-mediated drug delivery. In our study, an anti-CML cell single-chain variable fragment (scFv) antibody has been produced and characterized because it is the first step towards the construction of a novel cancer-targeted agent for cancer diagnosis and treatment. Here, a 46?kDa antibody derivative was produced by genetic fusion of a humanized scFv antibody against a CML cell surface antigen with the 6xHis-tag, which can specifically bind to CML cells. The recombinant scFv against CML cells was expressed as a fusion protein containing the 6xHis-tag at its N-termini, and purified by Ni2+-NTA column chromatography. The recombinant scFv, which was soluble, was expressed and produced in bacteria, and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays. Finally, its cell-binding activity and immunoactivity were demonstrated by enzyme-linked immunosorbent assay (ELISA). Furthermore, flow cytometry analysis demonstrated that this scFv specifically targeted CML cells expressing the associated antigen (47.9 and 34.4%) other than non-expressing tumor cells (1.25%) in?vitro. The results presented in this study illustrate that the humanized anti-CML cell scFv antibody may function as a novel therapeutic tool for CML.     

Generation and characterization of a single-gene encoded single-chain immunoglobulin-interleukin-2 fusion protein

Background: Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancer; however, the high doses of IL-2 required to mediate patients' immune responses can cause considerable systemic toxicity. The murine monoclonal antibody (MAb) CC49, which reacts with tumor-associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials. Objectives: Development and characterization of a single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein which, by delivering IL-2 selectively to the tumor site, can serve as an effective reagent for CC49/IL-2 combination therapy. Study design: A single-gene encoding the SCIg-IL-2 fusion protein derived from the chimeric (c) CC49 was designed, generated and inserted in an expression vector. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently joined through a (GGGGS)3 linker peptide. The carboxyl end of the variable domain of the light chain was linked to the amino terminus of the human γl Fc through the hinge region, and the carboxyl end of the CH3 domain was linked to the amino terminus of the human IL-2 through a GGGSGGG linker peptide. The SCIg-IL-2, expressed from the murine myeloma cells transfected with the expression construct, was characterized for its antigen-binding specifity, antibody effector functions and IL-2 biological activity. Results and conclusion: Transfection of murine myeloma cells with the single-gene expression construct SCIg-IL-2 expressed a single-chain protein of approximately 70 kD, which was secreted into tissue culture fluid as a homodimer of approximately 140 kD. SCIg-IL-2 competed completely with cCC49 for binding to the TAG-72 antigen, but approximately three- to four-fold more of the SCIg-IL-2 was required to achieve levels of competition similar to those observed with the murine or chimeric CC49. With human effector cells, the fusion protein mediated lysis of TAG-72 positive human carcinoma cells. Prior treatment of human effector cells with 100 U/ml of human IL-2 enhanced the fusion protein-mediated cytolysis from 32 to 65%. At doses of a 1 ng/ml, the stimulatory effect of SCIg-IL-2 on IL-2 dependent murine HT-2 cell proliferation was comparable to that of the recombinant human IL-2. The single-gene construct may also facilitate inoculation of the gene in animal tissue for in vivo expression of the fusion protein.     

A NOD/SCID tumor model for human ovarian cancer that allows tracking of tumor progression through the biomarker Sp17

No experimental animal model employing a primary human ovarian carcinoma (OC) cell line is presently available that tracks the progression of this cell line with an identifiable marker. This hinders investigations related to developing new approaches for treating OC. Here, we describe the development of a tumor model in NOD/SCID mice for human OC that makes use of the endogenously expressed tumor specific sperm protein 17 (Sp17) cancer testis antigen. In this model, human SKOV-3 OC cell lines were intra-peritoneally seeded. Subsequently viable SKOV-3 cells were recovered from primary organ cell cultures from the liver ovaries, abdomen, and ascitic fluid, and their presence was confirmed by the detection of Sp17 mRNA by RT-PCR and Sp17 protein by immunocytochemistry and FACS analysis. When SKOV-3 tumor cells were administered intravenously the mice developed primarily lung tumor foci. This model makes it possible to evaluate new immunotherapeutic strategies for the treatment of human OC based on the biomarker Sp17.     

Monocyte chemotaxis mediated by formyl-methionyl-leucyl-phenylalanine conjugated with monoclonal antibodies against human ovarian carcinoma

Availability of a chemically defined chemoattractant (fMLP) and of appropriate monoclonal antibodies may permit local manipulation of the inflammatory response to human tumors. fMLP has been conjugated with two monoclonal antibodies (OC125 and OC133) which react with human ovarian carcinomas. Conjugates retained the ability to bind to a human ovarian carcinoma line (OVCA433) judged by indirect immunofluorescence and by radioimmunoassay. The fMLP conjugate was maximally chemotactic for human blood monocytes and human peritoneal macrophages at protein concentrations of 300-900 micrograms/ml. Conjugates stimulated chemotaxis rather than chemokinesis. After incubation with an fMLP-antibody conjugate, antigen positive OVCA433 cells released chemotactic activity and attracted monocytes in vitro, whereas an antigen-negative ovarian cell line failed to do so. As monocytes can be important effectors of antibody dependent cell mediated cytoxicity, fMLP conjugates might increase monocyte concentrations at tumor sites and potentiate serotherapy for certain human neoplasms.     

Generation of human monoclonal antibodies to cancer-associated antigens using limited numbers of patient lymphocytes

A limiting dilution method for the efficient transformation by Epstein-Barr virus (EBV) of human B lymphocytes has been applied to the production of human monoclonal antibodies to ovarian cancer-associated antigens. Limited numbers (e.g., 2 X 10(5)) of EBV-infected B lymphocytes from ovarian cancer patient spleen, lymph node, tumor, ascites and blood were successfully transformed using this method. An immunofiltration assay system was employed to identify EBV transformants secreting IgM antibody which reacted selectively with ovarian cancer patient ascites tumor cells, but not with a mixture of normal cell types. A miniature Western blot assay was utilized to screen for IgG reactivity to protein species in detergent extracts of ovarian cancer tumor cells. EBV-transformed cells selected after screening were then fused with heteromyeloma fusion partner SHM-D33 resulting in efficient recovery of hybridomas secreting MAb of the desired specificity. Human MAbs which selectively react with antigens associated with ovarian cancer tumor cells were obtained.     

Implication of IL-17 producing ɑβT and γδT cells in patients with ovarian cancer

ObjectiveTo investigate the expression and cellular source of IL-17A in human ovarian cancer (OC), benign ovarian tumor (BOT) and borderline ovarian tumor (OBT).MethodsRT-PCR and immunohistochemistry were used to measure the expression level of IL-17A in human OC tissues. Find concrete source of the elevated IL-17A levels in OC tissues by flow cytometry.ResultsWe found that IL-17A is expressed at higher levels in OC tissues than in BOT or OBT tissues at both the mRNA and protein levels. Moreover, high tumor IL-17A expression was significantly associated with poor tumor differentiation and positive lymph node status. Flow cytometric analysis demonstrated that significantly higher proportions of tumor-infiltrating IL-17A-producing CD4⁺ T cells (Th17), CD8⁺ T cells (Tc17), and γδT cells (IL-17⁺ γδT) were present in OC tissue compared with BOT tissue. Of these, tumor-infiltrating γδT cells were the predominant source of IL-17A in OC and BOT patients. Finally, we found that the abundance of tumor-infiltrating IL-17⁺ γδT cells, but not Th17 or Tc17 cells, was positively correlated with larger tumor size and lymph node metastasis in patients with advanced OC. These data suggest that increased tumor-infiltrating IL-17⁺ γδ T cells may be associated with cancer progression in OC patients.     

抗肝癌单链抗体融合TNFα导向作用的初步实验研究

目的 获得有效果的抗肝癌基因工程单链免疫细胞因子（TNFα）。方法 将抗肝癌单克隆抗体（mAb)HAb25的单链抗体（scFv25)与人TNFα基因偶联,构建抗肝癌免疫细胞因子（scFv25-TNFα,亚克隆人原核GST融合表达载体pGEX4T-1中,在大杆菌中进行表达。对肝癌细胞SMMC-7721涂片进行间接免疫荧光染色,测定纯化后目的蛋白的活性,通过对荷肝癌（SMMC-7721）裸鼠进行的初步抑瘤实验,检测scFv25-TNFα的导向治疗效果。结果scFv25-TNFα具有与亲本抗体HAb25类似的抗原结合特异性。scFv25-TNFα对荷肝癌裸鼠体内直径2mm左右的肿瘤具有明确的导向杀伤作用,达到3/3完全缓解,明显强于TNFα对照组。该组有交待得只有2/3,且无完全缓解。结论 scFv250-TNFα为具有一定效果的抗肝癌双功能抗体。     

从噬菌体抗体库筛选人源性抗白细胞介素8抗体

目的: 从噬菌体抗体库中筛选人源性抗IL- 8单链抗体(scFV)。方法: 采用原核表达载体pRSET- IL- 8在大肠杆菌BL21(DE3)中表达IL- 8 His融合蛋白, 并通过亲和层析纯化。以该融合蛋白为固相抗原, 对噬菌体抗体库进行 3轮淘筛, 并对所获阳性克隆进行抗原结合活性测定和DNA序列测定。结果: 获得 2株特异性抗IL- 8噬菌体抗体。对其DNA序列测定结果表明, 其VH基因均属于人IgGVH3亚群, Vλ基因分别属于人VλDPL5和VλDPL2亚群。结论: 利用噬菌体抗体库技术可不经免疫制备人源性抗IL- 8抗体, 为银屑病及相关疾病的临床应用提供了条件。