Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Targeting properties of an anti-CD16/anti-CD30 bispecific antibody in an in vivo system

Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkin's lymphoma and Hodgkin's lymphoma. We have previously performed two clinical trials in patients with Hodgkin's disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either ¹²⁵I or ¹¹¹In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. ¹¹¹In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30⁺ tumors which did not differ significantly between the two (maximum uptake 16.5% ± 4.2% vs. 18.4% ± 3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkin's disease. Received: 26 October 2000 / Accepted: 15 December 2000     

端粒酶活性调节的分子机制

人端粒酶由RNA亚基、hTERT催化亚基和hTEP1调节蛋白等组成。端粒酶对端粒结构的稳定起着重要的作用,而端粒结构和端粒结合蛋白也影响着端粒酶活性。某些化疗药物通过破坏端粒结构下调端粒酶活性。端粒酶的激活需要hTERT基因的从头转录和各个蛋白亚基正确装配为端粒酶全酶。端粒酶活性调节的分子机制包括：（1）TERT基因的表达和转录是决定端粒酶活性的重要环节,受多种因素调控;（2）蛋白激酶Cα和蛋白激酶B磷酸化端粒酶蛋白而激活端粒酶,蛋白磷酸酯酶2A（PP2A）可逆转这一过程,下调端粒酶活性;（3）多种癌基因和抑癌基因及其编码的蛋白质也直接或间接与端粒蛋白、端粒酶蛋白反应,参与端粒酶活性的调控。     

原位杂交技术在检测染色体畸变中的研究

实验运用原位杂交技术检测肿瘤组织的染色体畸变。用3、7、8、9、12号染色体着丝粒特异性DNA探针,分别对外周静脉血的淋巴细胞、新鲜实体瘤组织、瘤组织石蜡切片进行染色体检测。二个染色体杂交点代表一对正常的染色体、三个染色体杂交点显示该细胞有染色体三体。整个实验过程简单、快速、准确、易重复。此方法和传统的细胞遗传学方法相结合,对探讨肿瘤的发生、发展有很大的意义。     

Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast

The BRCA1 gene, mutations of which contribute significantly to hereditary breast cancer, was not identified in the existing YAC and BAC libraries. The gene is now available only as a set of overlapping fragments that form a contig. In this work we describe direct isolation of a genomic copy of BRCA1 from human DNA by transformation-associated recombination (TAR) cloning. Despite the presence of multiple repeats, most of the primary BRCA1 YAC isolates did not contain detectable deletions and could be stably propagated in a host strain with conditional RAD52. Similar to other circular YACs, 90 kb BRCA1 YACs were efficiently and accurately retrofitted into bacterial artificial chromosomes (BACs) with the Neo^R mammalian selectable marker and transferred as circular BAC/YACs in E. coli cells. The BRCA1 BAC/YAC DNAs were isolated from bacterial cells and were used to transfect mouse cells using the Neo^R gene as selectable marker. Western blot analysis of transfectants showed that BRCA1 YACs isolated by a TAR cloning contained a functional gene. The advantage of this expression vector is that the expression of BRCA1 is generated from its own regulatory elements and does not require additional promoter elements that may result in overexpression of the protein. In contrast to the results with cDNA expression vectors, the level of BRCA1 expression from this TAR vector is stable, does not induce cell death, maintains serum regulation, and approximates the level of endogenously expressed BRCA1 in human cells. The entire isolation procedure of BRCA1 described in this paper can be accomplished in approximately 10 days and can be applied to isolation of gene from clinical material. We propose that the opportunity to directly isolate normal and mutant forms of BRCA1 will greatly facilitate analysis of the gene and its contribution to breast cancer.     

增殖诱导配体(APRIL)研究进展

增殖诱导配体 (aproliferation inducingligand ,APRIL)是肿瘤坏死因子 (TNF)家族的新成员 ,在多种肿瘤组织中有高表达 ,能促进肿瘤细胞增殖 ,防止肿瘤细胞受CD95L、FasL等诱导的凋亡 ;调节体液免疫 ;并在T、B淋巴细胞的成熟和活化中起一定作用。因此 ,APRIL与肿瘤的发生、发展以及免疫系统的调节有密切关系。     

癌蛋白D52家族研究进展

肿瘤蛋白D52家族近年来引起了人们的研究兴趣。D52最早是从人乳腺癌中发现的。该家族成员分子中都含有一个叫做Coli-Coli基序的高度保守结构域,这个结构域在低等生物到哺乳动物或者同种生物的不同成员间也是高度保守的。已有的研究表明该家族成员可以通过选择性剪接的方式产生功能不同的拼接体。D52家族基因在多种癌症中广泛扩增,蛋白表达水平升高。目前认为,他们的基因功能可能与包括癌症在内的人类疾病相关,关于这个家族成员发挥作用的分子机制还有待于进一步的探讨。