Transmembrane Proteins Involved in Human Tumor Expansion

描述（由申请人提供）：为了鉴定在功能上有助于人类肿瘤细胞转移的特定蛋白抗原，我们的实验室启动了减法免疫方法，以产生抑制人类肿瘤传播的独特单克隆抗体（mab）。对生成的单克隆抗体进行了筛选，以确定其抑制人类肿瘤在鸡胚胎和小鼠转移模型中的扩散的能力。通过这种方法，我们产生了独特的抗体。其中之一，mAb 1A5，识别四蛋白家族的一个特定成员CD151，并相应地抑制> 90%的转移。另一种减法免疫抗体mAb 41-2可识别肿瘤细胞跨膜蛋白CDCP1/SIMA-135。mAb 41-2可抑制整体转移60-80%，但mAb 41-2及其靶抗原的作用机制尚不清楚。因此，本研究的目标是阐明mAb 41-2的转移抑制机制，并对其同源抗原CDCP1进行结构-功能分析，以确定CDCP1在癌症传播中的作用。定时向动物中添加mAb 41-2，然后定量测量肿瘤细胞血管内阻滞、凋亡、存活、外渗和继发灶的建立，将确定mAb 41-2何时、何地以及如何阻断恶性功能。这些方法将在肿瘤播散的小鼠模型中得到扩展和证实，包括原位植入方法。mAb 41-2的抗原CDCP1作为存活因子的功能将通过CDCP1细胞质结构域和CDCP1胞外CUB结构域的突变分析进行测试。组合文库将用于鉴定未知的CDCP1天然配体，并产生新的CDCP1特异性抗体。这项更新的另一个目的是表征在肿瘤形成能力上相似但在转移特性上有很大差异的其他同源人类肿瘤变异对，并在新的减法免疫方法中使用这些细胞对来产生新的功能阻断抗体。在我们建立的模型中，将筛选单克隆抗体的差异反应性和功能阻断特性。我们还建议启动潜在的翻译方法，并产生新的转移阻断，单链可变片段（sc Fv）人源化抗体，这些抗体在癌症类型特异性的原位小鼠模型中有效，并可能代表可能的靶向肿瘤治疗的独特试剂。

DESCRIPTION (provided by applicant): To identify specific protein antigens that functionally contribute to human tumor cell metastasis, our laboratory initiated subtractive immunization approaches to raise unique monoclonal antibodies (mAbs) that inhibit human tumor dissemination. Panels of generated mAbs are screened for their ability to inhibit human tumor dissemination in chick embryo and mouse models of metastasis. Using this approach, we have generated unique antibodies. One of them, mAb 1A5, recognizes a specific member of the tetraspanin family, CD151, and correspondingly inhibits metastasis by > 90%. Another subtractive immunization antibody, mAb 41-2, recognizes a tumor cell transmembrane protein, CDCP1/SIMA-135. This mAb 41-2 inhibits overall metastasis by 60-80%, however the mechanism of mAb 41-2 and that of its target antigen are unknown. Thus, a proposed goal of this renewal is to elucidate the metastasis-inhibition mechanism of mAb 41-2 and to conduct structure- function analysis of its cognate antigen, CDCP1, in order to determine the contributory role of CDCP1 in cancer dissemination. Timed additions of mAb 41-2 into animals followed by quantitative measurements of tumor cell intravascular arrest, apoptosis, survival, extravasation and establishment of secondary foci will determine when, where and how mAb 41-2 blocks malignant function. These approaches will be expanded and confirmed in mouse models of tumor dissemination, including orthotopic implantation approaches. That mAb 41-2's antigen, CDCP1, functions as a survival factor will be tested with mutational analyses of CDCP1's cytoplasmic domain and CDCP1's extracellular CUB domains. The use of combinatorial libraries will be employed to identify unknown natural ligand(s) for CDCP1 and to generate new distinctive antibodies to CDCP1. Another aim of this renewal is to characterize additional pairs of congenic human tumor variants that are similar in tumor-forming ability but differ substantially in their metastasis properties, and to use these cell pairs in new subtractive immunization approaches to generate novel function-blocking antibodies. Panels of mAbs will be screened for differential reactivity and function-blocking properties in our established models. We also propose to initiate potential translational approaches and generate new metastasis-blocking, single chain variable fragment (sc Fv) humanized antibodies that are effective in cancer type-specific orthotopic mouse models and could represent unique reagents for possible targeted tumor therapy.