Chimeric bacterial toxins and cancer therapy

描述(申请人提供)：重组毒素，由细菌毒素和生长因子或重组单抗的一部分组成的杂交蛋白，在癌症治疗中受到了极大的关注。这项申请提出了一项独特的研究，以开发和评估基于人类消化酶和细菌细胞毒素之间的相似性的重组毒素。人类DNase I已被用作治疗伤口和溃疡、支气管炎、炎症条件、疱疹感染以及最显著的囊性纤维化的治疗剂。细胞致死性膨胀毒素(CDT)是一种遗传性毒素，由牙周病原体放线菌伴生放线杆菌产生。CDT含有一个亚基蛋白，CDtB，已被证明是一种核酸酶，在基因和功能上与哺乳动物I型DNA酶相关。人DNase I的比活力是CDtB的100到1000倍。相比之下，CDtB有一个内置的细胞递送系统，一个针对细胞核的蛋白质的功能域，以及导致快速增殖的上皮样细胞生长停滞或凋亡的DMA破坏活性。我们的假设是，人类DNase I和细菌CDtB的嵌合体可以在基因上构建，以结合强大的DNA损伤活性、细胞递送和核定位机制。本研究的目的是开发这些嵌合蛋白作为潜在的抗肿瘤细胞试剂的有效性。本研究的具体目的是：(1)构建人脱氧核糖核酸酶L/CDtB嵌合基因及嵌合体定点突变株，并分离突变基因产物。(2)比较嵌合和突变嵌合基因产物的体外生物学活性。(3)评价嵌合和突变型嵌合载体在细胞培养和转染系统中的应用效果。将使用一组头颈部鳞状细胞癌(HNSCC)细胞株来评估突变嵌合蛋白进入细胞并诱导细胞周期停滞的能力。如果这些研究成功，未来的应用将集中在这些突变嵌合体对癌症患者的鳞癌细胞和适当小鼠模型中的肿瘤的影响。

DESCRIPTION (provided by applicant): Recombinant toxins, hybrid proteins composed of a bacterial toxin and either a growth factor or a portion of a recombinant monoclonal antibody, have received significant attention in cancer therapeutics. This application proposes a unique study to develop and evaluate a recombinant toxin based on the similarities between a human digestive enzyme and a bacterial cytotoxin. Human DNase I has been used as a therapeutic agent for the treatment of wounds and ulcers, bronchitis, inflammatory conditions, herpes infection and most notably, cystic fibrosis. The cytolethal distending toxin (Cdt) is a genotoxin, produced by the periodontal pathogen Actinobacillus actinomycetemcomitans. The Cdt contains a subunit protein, CdtB, that has been shown to be a nuclease that is genetically and functionally related to mammalian type I DNases. Human DNase I has 100 to 1000 times the specific activity of CdtB. In contrast, CdtB has a built-in cell delivery system, a functional domain that targets the protein to the cell nucleus and a DMA-damaging activity that leads to the growth arrest or apoptosis of rapidly proliferating epithelial-like cells. Our hypothesis is that chimeras of human DNase I and bacterial CdtB can be genetically constructed to combine potent DNA- damaging activity, cell delivery and nuclear localization mechanisms. The objective of this study is to develop the efficacy of these chimeric proteins for use as the potential anti-cancer cell reagents. The specific aims of the study are: (1) to construct human DNase l/CdtB chimeric genes and site-specific mutants of the chimeras and to isolate the mutant gene products. (2) To compare the in vitro biological activities of the chimeric and mutated chimeric gene products. (3) To evaluate the efficacy of the chimeric and mutant chimeric constructs in cell culture and transfection systems. Mutant chimeric proteins that demonstrate the ability to enter cells and induce cell cycle arrest will be evaluated using a panel of head and neck squamous cell carcinoma (HNSCC) cell lines. If these studies are successful, future applications will be focused on the effects of these mutant chimeras on sqamous carcinoma cells from cancer patients and on tumors in appropriate mouse models.