单链抗体在肿瘤诊断、治疗中的应用研究

单链抗体是由免疫球蛋白的重链可变区(V_H)和轻链可变区(V_L)通过一段连接肽连接而成的重组蛋白,它是具有完全抗原结合位点的最小抗体片段。它分子小,容易进入肿瘤内部,免疫原性低,可用基因工程大量生产,克服了单克隆抗体在人体内应用的许多不足。在单链抗体C末端引入毒素、细胞因子、前体药物降解酶等构建双功能单链抗体,或者构建双特异抗体,在肿瘤诊断和治疗方面具有潜在的巨大的临床应用价值,目前国外的一些抗肿瘤单链抗体已进入临床Ⅰ期试验阶段。本文主要介绍了单链抗体在肿瘤诊断和治疗方面研究的新进展。     

肿瘤血管特异结合抗体ScFvH1的靶向性和抑瘤性

目的：检测从肿瘤特异单链抗体库中筛选到的肿瘤血管特异结合单链抗体ScFvH1的靶向性和抑瘤性，探讨该抗体在癌症诊断和治疗中应用的可能性。方法：将该单链抗体基因插入到含绿色荧光蛋白（EGFP）基因及带有硫氧还原蛋白（Trx）基因的原核表达载体pET-28a（＋）／EGFP及pTIG—Trx中，在大肠杆菌中进行表达，并经镍柱（Ni—NTA）纯化。建立人宫颈癌HeLa细胞裸鼠皮下移植瘤模型，尾静脉注射纯化的单链抗体-EGFP融合蛋白，通过荧光显微镜观察肿瘤部位及其他器官中EGFP信号，考察该单链抗体的靶向性；同时在裸鼠致瘤部位注射纯化的单链抗体蛋白，观察该单链抗体对肿瘤生长的抑制性。结果：在大肠杆菌中表达了该单链抗体基因片段，并使单链抗体-EGFP融合蛋白得到了很好的表达，经镍柱纯化后得到了电泳级的单一条带。靶向性实验结果显示，单链抗体-EGFP融合蛋白在肿瘤部位得到了富集，而只注射EGFP蛋白的肿瘤组织中荧光很少，并且在裸鼠肺部组织中没有观察到EGFP荧光信号。抑瘤性实验发现，单链抗体处理组移植瘤生长速率与PBS组类似。结论：从肿瘤特异单链抗体库中筛选到的肿瘤血管特异抗体ScFvH1具有较好的肿瘤血管靶向性，而对肿瘤生长的抑制作用不明显，为进一步研究抗体在肿瘤诊断和治疗中的应用奠定了基础。     

肿瘤基因工程抗体研究进展

综述单链抗体、抗体融合蛋白、抗独特型抗体、双特异性抗体的研究进展，显示它们在临床肿瘤治疗方面的良好应用前景。     

肿瘤基因工程抗体研究进展

综述单链抗体、抗体融合蛋白、抗独特型抗体、双特异性抗体的研究进展，显示它们在临床肿瘤治疗方面的良好应用前景。     

小鼠结肠癌RNA转染mIL12修饰的树突状细胞诱发抗肿瘤活性

目的:构建肿瘤组织特异的噬菌体呈现型单链抗体库,用噬菌体体内筛选与肿瘤血管特异性结合的单链抗体,为癌症的诊断和治疗奠定基础。方法:取食道癌、胃癌、脑癌、肺癌、脊髓瘤患者肿瘤组织,提取各肿瘤组织膜蛋白混合后免疫BALB/c小鼠,取鼠脾脏淋巴细胞提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区基因(VH和VL),经Linker连接形成ScFv基因片段,将ScFv基因片段与噬菌粒载体pCANTAB5E的连接产物转化大肠杆菌TG1,在辅助噬菌体M13KO7的作用下,获得重组噬菌体单链抗体库。以人宫颈癌HeLa细胞致瘤裸鼠为实验模型,用所构建的单链抗体库进行了4轮特异抗体的体内筛选。挑取了24个PCR鉴定为阳性的单链抗体做免疫组化分析,将在HeLa致瘤的组织切片上呈阳性染色、而在对照的肾组织切片上没有阳性染色的单链抗体进行序列测定。结果:选用7株不同的肿瘤细胞系建立裸鼠致瘤模型,以HeLa细胞致瘤裸鼠成瘤效果最好。用所构建的库容量为1.6×106的单链抗体库对肿瘤血管特异结合抗体进行体内筛选,得到1株单链抗体ScFv(VH-linker-VL),命名为ScFvH1。结论:成功构建肿瘤特异单链抗体库,并用此抗体库筛选到与肿瘤血管结合特异性较好的单链抗体,为肿瘤的早期诊断和治疗提供了一条新的技术路线。     

基因工程抗体在肿瘤诊治中的应用

基因工程抗体(GEA)发展十分迅速,利用其特异性的靶向作用,在肿瘤诊断、治疗及预后等方面已初步显示良好效果和广阔应用前景.随着研究的不断深入,基因工程抗体必将发挥更重要的作用.现综述基因工程抗体在肿瘤应用中的最新进展.     

单链抗体及其在肿瘤免疫上的应用

随着分子生物学的进一步发展,抗体技术也逐步由细胞工程抗体向基因工程抗体演替,特别是单链抗体(ScFv)在理论和应用方面的研究令人尤为关注。ScFv应用范围的不断扩大和研究的不断深入,它在肿瘤的研究中也发挥了越来越大的作用。现综述ScFv的构建、表达及其在肿瘤免疫诊断和免疫治疗中的研究应用。     

单链抗体在肿瘤诊治中的应用及改进

单链抗体(scFv)是一种新型小分子抗体,其基本结构为VH-Linker-VL或VL-Linker- VH,具有分子量小、免疫原性低、组织穿透力强、保持了亲本抗体的抗原亲和力和特异性等优点,可将细胞因子、毒素、药物、放射性核素等导向肿瘤,对肿瘤实现靶向性诊断和治疗。     

核酸适配子及其在肿瘤诊断与治疗中的应用

核酸适配子是采用指数富集配体的系统进化(SELEX)技术从随机单链寡核苷酸库中筛选出的能与靶物质高特异性、高亲和力结合的配体,其特性在很多方面优于抗体,在分子识别研究中具有重要价值,可用于靶物质的测定、阻断靶物质的生物活性,在肿瘤的诊断和治疗中有着广阔的应用前景。     

单链抗体及其在肿瘤免疫上的应用

随着分子生物学的进一步发展，抗体技术也逐步由细胞工程抗体向基因工程抗体演替，特别是单链抗体（ScFv）在理论和应用方面的研究令人尤为关注。ScFv应用范围的不断扩大和研究的不断深入，它在肿瘤的研究中也发挥了越来越大的作用。现综述ScFv的构建、表达及其在肿瘤免疫诊断和免疫治疗中的研究应用。     

Monoclonal antibodies: potential role in radiation therapy and oncology

Specificity, which is a hallmark of the immune system, will be used in radiation oncology in both diagnosis and therapy through the application of radiolabelled monoclonal and polyclonal antibodies. Antigenic specificities, antibody preparations, and the tumor as a target for radiolabelled antibody is reviewed. Several clinical situations, i.e. single tumor cell suspensions, intraperitoneal single cells and masses, and solid tumors are reviewed in regard to both immune antibody targeting and specific differences between tumors in these regions. The concentration of tumor associated antigens is introductory to radiolabelled antibodies in diagnosis. In the radiation therapy of solid tumors, data regarding tumor dose, tumor effective half-life, varied antibody preparations, and the use of radiolabelled antibody as a method of tumor implantation is discussed using antiferritin 131I-IgG as a model as a model in hepatoma. The theoretical applications of monoclonal antibody integrated in cancer therapy are then presented as a new goal for future development.     

Intratumoral phenotypic diversity of cloned human lung tumor cell lines and consequences for analyses with monoclonal antibodies

Cloned cell lines and a number of subclones from these lines were established in vitro from biopsies of small cell lung carcinomas and squamous cell lung carcinomas. The cloned cultures, including the cloned subclones, were analyzed in respect to morphology, karyotype, growth rates, clonogenicity in semisolid agar medium, and tumorigenicity in nude mice. A remarkable biologic diversity was found in respect to most of these biologic features. In addition, four murine monoclonal antibodies with high specificity for lung tumor cells were generated. Their reactivity pattern to clonogenic cells was for some clones different as compared to the nonclonogenic cells. Subclones of tumor cells not binding the antibody were identified for each monoclonal antibody. It is concluded that intratumoral phenotypic diversity may have a severe negative impact on the use of monoclonal antibodies in cancer diagnosis/therapy. The work also indicates that a mixture of antibodies may be more useful in tumor diagnosis than individual antibodies and perhaps even therapy, particularly if they bind to the clonogenic part of a cell population.     

Clinical application of monoclonal antibodies to human malignant melanoma-associated antigens

Monoclonal antibodies to human malignant melanoma-associated antigens were reviewed from the viewpoint of the target structure recognized by them. With regard to clinical application of monoclonal antibodies for diagnosis, immunohistochemistry, serological diagnosis and tumor imaging were described, together with the possibility of therapy involving monoclonal antibody infusion.     

Tumor markers in hepatoma, gallbladder-biliary tract and pancreas cancer

Possibilities for the early diagnosis of hepatoma, gallbladder-biliary tract cancer and pancreas cancer became getting higher by using method of monoclonal antibody. AFP, gamma-GTP, hepatoma specific gamma-GTP, P-III-P, PIVKA-II and 5'-NPD-V are sensitive and useful markers for diagnosis of hepatoma, CA 19-9 and CEA for gallbladder-biliary tract cancer and pancreas cancer, and DUPAN-2 and POA for pancreas cancer. Screening of sera of the patients with these tumor markers combined with image diagnosis are necessary for early and accurate diagnosis of the cancers. Everlasting effort to get the more sensitive and specific tumor markers are necessary. And missile therapy binding anti-cancer drugs to monoclonal antibodies are expected.     

Reactivity of tumor cells in malignant effusions with a panel of monoclonal and polyclonal antibodies

A panel of 13 mouse monoclonal antibodies (mAb) and 1 rabbit polyclonal antibody was tested for reactivity with tumor cells in 26 effusions obtained from patients with carcinoma of ovary, breast, or mesothelioma, using an immunoperoxidase staining reaction. Specific staining of tumor cells but not reactive mesothelial cells was demonstrated with some of the mAb in the panel. In 4 of 26 effusions no evidence of malignancy was obtained after routine cytological staining, but this was reversed on the basis of immunoperoxidase staining of tumor cells with the mAb in the panel. Serial effusions were evaluated in 4 patients during the course of chemotherapy, allowing an assessment of effect of therapy on the antigenic characteristics of the tumor cells. In another 4 patients, the results of immunoperoxidase staining of effusions were compared with those obtained by applying the same antibody panel to solid tumor nodules. There was a tendency to develop changes in the pattern of reactivity during therapy, and the pattern of reactivity was more restricted in tumor nodules than in effusions. One of the mAb in our panel (2G3) was consistently shown to produce strong staining of a high proportion of tumor cells in effusions and tumor nodules from patients with ovarian or breast cancer and may be of value in immunocytochemical diagnosis and therapy of epithelial malignancies.     

Production and characterization of a high-affinity nanobody against human endoglin

Abstract Antibodies or antibody fragments are almost exclusively applied in human therapy and diagnosis. The high affinity and specificity of antibodies makes them suitable for these applications. Nanobody, the variable domain of Camelidae heavy chain antibodies, have superior properties compared with conventional antibodies in that they are small, non-immunogenic, very stable, highly soluble, and easy to produce in large quantities. In the present study, we report the isolation and characterization of a high-affinity binder against human endoglin retrieved from camels' nanobody gene library. Endoglin (CD105), an accessory protein of the transforming growth factor beta receptor complex, has become an attractive molecule for the targeting of the tumor vasculature. Upregulation of endoglin on proliferating endothelial cells is associated with tumor neovascularization. Here, we generated two nanobody gene libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the recombinant extracellular domain of human endoglin. The other selected anti-endoglin nanobody (AR1-86) showed strong binding to human endoglin expressing endothelial cells (HUVECs), while no binding was observed with the endoglin-negative cell line (HEK293). This high-affinity single-domain antibody could be a good candidate for the generation of vascular or tumor targeting agents in cancer therapy.     

Tumor localization by combinations of monoclonal antibodies in a new human colon carcinoma cell line (LIM1899)

One of the problems of in vivo diagnosis and therapy of tumors with monoclonal antibodies is their heterogeneity with respect to antigen expression, with some cells expressing no antigen and others being weakly or strongly positive. Selected mixtures of antibodies to different antigens are therefore likely to react with more cells than single antibodies and be more effective for imaging and therapy. With this in mind, we have examined a new human colon cancer cell line (LIM1899) which has a heterogeneous expression of several cell surface molecules: by flow cytometry 38% were carcinoembryonic antigen positive; 64%, human milk fat globule positive, and 73%, CD46 positive; 87% of tumor cells bound a mixture of all three antibodies in vitro. Some blocking of the binding of anti-human milk fat globule antibody by the anti-CD46 antibody was noted. LIM1899 was established as a xenograft in nude mice and in vivo biodistribution studies performed using antibodies alone or in combination. Mixtures of antibodies clearly showed a higher percentage of injected dose of antibody in the tumor than did single antibodies: one antibody gave 10%; two together, 17 to 21%; and all three together gave 29% of the injected dose in the tumor. Tumor:blood ratios were also superior for combinations of antibodies, provided that low doses of the antibodies were used; at higher doses the effect was lost. The study demonstrates that combinations of antibodies are better than single antibodies for localization, provided that the dose used is carefully selected.     

Clinical application of anti-idiotypic antibody

The concept of idiotype networks are supposed to play important roles in regulation of the immune response, and anti-idiotypic (anti-Id) antibodies have enabled to analyze the pathogenesis of autoimmune diseases and structural relations of receptors and ligands in clinical fields. We applied anti-idiotypic monoclonal antibodies (MoAb) to tumor immunology to analyze idiotype network system of tumor associated antigen (TAA) and to investigate the fine structure of TAAs and their corresponding antibodies. New assays were established to detect circulating TAAs, anti-TAA antibodies and their immune complex regarding YH 206 antigen and carcinoembryonic antigen (CEA) systems. The measurement of anti-TAA antibodies and immune complex was found to be of value for the cancer diagnosis. The results about immune response with use of anti-Id monoclonal antibodies suggest that some of the anti-Id antibodies might be useful for cancer therapy, since anti-Id antibody could induce anti-anti-Id antibodies reactive with tumor antigen. In this review, the usefulness of anti-Id MoAb to serodiagnosis and immunotherapy for cancer was discussed.     

Immunotargeting of tumors: state of the art and prospects in 2000

Following 15 years of experimental studies, tumor immunotargeting using monoclonal antibodies directed against tumor associated antigens shows now important monoclonal antibodies directed against tumor associated antigens shows now important clinical developments. This is mainly due to encouraging therapeutic results which have obtained using humanized antibodies such as the anti-CD20 rituximab in follicular B lymphomas and the anti-DrbB2 herceptin in breast carcinomas. Thanks to genetic engineering it is possible to graft variable or hypervariable regions from murine antibodies to human IgG, and even to obtain fully human antibodies by using either transgenic mice containing a large part of the human repertoire of human IgG, or selection of human antibody fragments expressed by phages. Radiolabeling of antibodies played a major role to demonstrate the tumor immunotargeting specificity and remains attractive for the diagnosis by immunoscintigraphy as well as for the treatment by radioimmunotherapy of some cancers. In this review, the current results and the prospects of diagnostic and therapeutic uses of anti-tumor antibodies and their fragments will be described. Concerning diagnosis, 123-iodine or 99m-technetium labeled Fab fragments allowed very demonstrative tumor images but this technique has a limited effect upon the therapeutic attitude. Immuno-PET (positron emission tomography) could enhance the sensitivity of this imaging method. Radio-immunoguided surgery and immunophotodetection are attractive techniques still under evaluation. Concerning therapy, 131-iodine labeled anti-CD20 antibodies gave spectacular results in non-Hodgkin's B lymphomas. In solid tumors which as less radiosensitive, radioimmunotherapy could concern small tumors and need the use of two-steps targeting and/or alpha emitters radioisotopes. Some other strategies will be described such as bispecific antibodies directed against tumors and immune effector cells, some antibody fragments expressed on T cells called T-bodies or some biological studies using intrabodies. Published data and works in progress demonstrate that immunotargeting of tumors will have a growing place in the treatments of cancer patients.