Chimeric bacterial toxins and cancer therapy

描述（由申请人提供）：重组毒素，由细菌毒素和生长因子或重组单克隆抗体的一部分组成的杂合蛋白，在癌症治疗中受到了极大的关注。本申请提出了一种独特的研究，以开发和评估基于人消化酶和细菌细胞毒素之间的相似性的重组毒素。人DNA酶I已被用作治疗剂，用于治疗创伤和溃疡、支气管炎、炎性病症、疱疹感染以及最显著的囊性纤维化。细胞致死性膨胀毒素（Cdt）是一种由牙周致病菌伴放线放线杆菌产生的遗传毒素。Cdt含有亚基蛋白CdtB，其已被证明是与哺乳动物I型DNA酶在遗传和功能上相关的核酸酶。人DNA酶I具有CdtB的100至1000倍的比活性。相反，CdtB具有内置的细胞递送系统、将蛋白质靶向细胞核的功能结构域和导致快速增殖的上皮样细胞的生长停滞或凋亡的DNA损伤活性。我们的假设是，人DNA酶I和细菌CdtB的嵌合体可以通过遗传构建来结合联合收割机有效的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癌细胞试剂的功效。本研究的具体目的是：（1）构建人DNase 1/CdtB嵌合基因及其位点特异性突变体，并分离突变基因产物。(2)比较嵌合基因产物和突变嵌合基因产物的体外生物学活性。(3)评价嵌合体和突变体嵌合体构建体在细胞培养和转染系统中的功效。将使用一组头颈部鳞状细胞癌（HNSCC）细胞系评价证明能够进入细胞并诱导细胞周期停滞的突变嵌合蛋白。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鳞状癌细胞和适当小鼠模型中肿瘤的影响上。

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 DNA-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.