Transmembrane Proteins Involved in Human Tumor Expansion

描述(由申请人提供)：为了确定在功能上有助于人类肿瘤细胞转移的特定蛋白质抗原，我们实验室启动了消减免疫方法，以产生独特的单抗(MAbs)来抑制人类肿瘤的扩散。对所产生的mAb板进行筛选，以确定它们在鸡胚胎和小鼠转移模型中抑制人类肿瘤扩散的能力。使用这种方法，我们已经产生了独特的抗体。其中，单抗1A5识别Tetraspanin家族的一个特定成员CD151，并相应地抑制90%的转移。另一种消减免疫抗体mAb41-2识别肿瘤细胞跨膜蛋白CDCP1/SIMA-135。该单抗41-2可抑制60-80%的肿瘤整体转移，但其作用机制及其靶抗原机制尚不清楚。因此，这次更新的目标之一是阐明mAb41-2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在肿瘤转移中的贡献。将mAb41-2定时添加到动物体内，然后对肿瘤细胞血管内停滞、细胞凋亡、存活、渗出和继发病灶的建立进行定量测量，将决定mAb41-2何时、何地和如何阻断恶性功能。这些方法将在肿瘤扩散的小鼠模型中得到扩展和确认，包括原位种植方法。41-2‘S抗原CDCP1作为生存因子，将通过CDCP1的S胞内结构域和CDCP1的S胞外区的突变分析来验证。将利用组合文库来鉴定CDCP1的未知天然配体(S)，并产生新的独特的抗体。这次更新的另一个目的是鉴定另外一对在成瘤能力上相似但在转移特性上有很大差异的同源人类肿瘤变体，并将这些细胞对用于新的消减免疫方法，以产生新的功能阻断抗体。在我们已建立的模型中，将对单抗的面板进行筛选，以确定不同的反应性和功能阻断属性。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的原位小鼠模型中有效，并可代表可能的靶向肿瘤治疗的独特试剂。公共卫生相关性：癌症死亡的主要原因是肿瘤细胞从原发肿瘤部位扩散到其他重要器官和组织。为了让肿瘤细胞逃离原发肿瘤并转移到远处的器官，它们会产生不同水平的功能蛋白。这项拟议的研究的目标是使用一种改进的单抗产生系统，以便识别有助于肿瘤细胞扩散的相关蛋白质。肿瘤学家可以针对这些关键的蛋白质分子，也可能使用特定的抗体作为治疗剂。

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. PUBLIC HEALTH RELEVANCE: Deaths from cancer occur mainly because tumor cells spread from the primary tumor site to other vital organs and tissues. In order for tumor cells to escape from the primary tumor and travel to distant organs, they produce different levels of functioning proteins. The goal of the proposed research is to use a modified system of monoclonal antibody generation in order to identify the relevant proteins that contribute to tumor cell spread. Oncologists could target those critical protein molecules and might also employ the specific antibodies as therapeutic agents.