Selection of internalizing human antibodies targeting pancreatic tumor cells in s

描述(申请人提供)：这项建议的目标是鉴定原位靶向胰腺肿瘤细胞的内化人单链抗体(ScFv)，重点是(1)早期肿瘤(0/I期)，内化单链抗体可用于非侵入性成像以早期发现，(2)晚期肿瘤(III/IV期)，内化单链抗体可用于基于细胞内递送策略的靶向治疗。目前，针对胰腺肿瘤的人类单抗很少，更少的是专门检测早期胰腺肿瘤以实现无症状肿瘤的非侵入性成像，而无症状肿瘤是一个对治疗方案和结果有巨大影响的领域。因此，我们提议的研究将解决这一领域的一个关键需求。我们假设，像其他肿瘤一样，胰腺肿瘤具有独特的细胞表面表位特征，以区别于肿瘤和非肿瘤组织。这些表位中的一些可能出现在肿瘤进展的早期，使无症状肿瘤的早期发现成为可能。我们进一步假设，这些肿瘤细胞表面表位的一个子集正在内化，因此可以用来将有效载荷运送到胰腺肿瘤细胞内。由于肿瘤细胞表面的表位空间由复杂的抗原决定簇组成，包括翻译后修饰，我们采取了基于抗体的方法来研究肿瘤细胞表面。我们已经开发和利用了一个包含5亿个成员的天然噬菌体抗体库来识别针对肿瘤相关内化表位的人单链抗体(ScFv)，促进了基于细胞内递送策略的靶向治疗的进一步发展。认识到肿瘤细胞株通常表达与原位肿瘤细胞不同的细胞表面抗原特征，我们最近开发了一种新的方法，利用激光捕获显微切割(LCM)来原位选择肿瘤细胞上的噬菌体抗体库，从而产生针对临床代表的肿瘤抗原的抗体，而不是与体外培养的细胞系相关的可能的人工制品。通过使用这些技术，我们能够识别针对临床标本中原位表达的肿瘤抗原的人单抗，并具有新的治疗有用的功能，如靶向细胞内有效载荷递送。LCM对疾病细胞的精确获取使处于不同阶段的肿瘤细胞成为噬菌体抗体选择的靶点，从而提供了识别可能用于早期肿瘤检测的阶段相关抗体的机会。我们建议应用这一策略来选择内在化的人单链抗体，这些单链抗体在不同阶段原位靶向胰腺肿瘤细胞。这些新的抗体与人类的序列相同，未来可以用于开发靶向治疗和基于非侵入性成像的早期肿瘤检测。公共卫生相关性：该项目旨在通过激光捕获显微切割识别原位靶向胰腺肿瘤细胞的内在化人类单链抗体。这些内化单链抗体可用于开发非侵入性成像策略，从而实现灵敏而准确的早期肿瘤检测，并基于细胞内递送策略进行靶向治疗。

DESCRIPTION (provided by applicant): The objective of this proposal is to identify internalizing human single chain antibodies (scFvs) that target pancreatic tumor cells in situ, with emphasis on (1) early stage tumors (stages 0/I), for which internalizing scFvs can be used in non-invasive imaging for early detection, and (2) late stage tumors (stages III/IV), for which internalizing scFvs can be used to develop targeted therapy based on intracellular delivery strategies. Currently, there are very few human monoclonal antibodies that target pancreatic tumors, and even fewer that specifically detect early stage pancreatic tumor to allow non-invasive imaging of asymptomatic neoplasia, an area that has a huge impact on treatment options and outcomes. Thus our proposed research would address a critical need of this field. We hypothesize that like other tumors, pancreatic tumors possess unique cell surface epitope profiles that distinguish tumors from non-neoplastic tissues. Some of these epitopes may be present early in tumor progression, allowing early detection of asymptomatic neoplasia. We further hypothesize that a subset of these tumor cell surface epitopes are internalizing, and thus can be utilized to deliver payloads intracellularly to pancreatic tumor cells. Because the tumor cell surface epitope space is composed of complex antigenic determinants including posttranslational modifications, we have taken an antibody-based approach to study the tumor cell surface. We have previously developed and utilized a naive phage antibody library containing 500 million members to identify human single chain antibodies (scFvs) targeting tumor associated internalizing epitopes, facilitating further development of targeted therapy based on intracellular delivery strategies. Recognizing that tumor cell lines often express a different cell surface antigenic profile than that of tumor cells in situ, we have recently developed a novel method that utilizes laser capture microdissection (LCM) to select phage antibody libraries on tumor cells in situ, thereby generating antibodies against clinically represented tumor antigens as opposed to possible artifacts associated with cell lines cultured in vitro. By using these techniques we are able to identify human monoclonal antibodies that target tumor antigens expressed in situ in clinical specimens, and possess novel, therapeutically useful functions such as targeted intracellular payload delivery. The precise procurement of disease cells by LCM allows tumor cells at different stages to be targeted for phage antibody selection, offering opportunities to identify stage-associated antibodies that may be used in early tumor detection. We propose to apply this strategy to select internalizing human scFvs that target pancreatic tumor cells in situ at different stages. These novel antibodies, which are human in sequence, can in the future be used to develop targeted therapeutics and non-invasive imaging-based early tumor detection. PUBLIC HEALTH RELEVANCE: This project aims to identify by laser capture microdissection internalizing human single chain antibodies that target pancreatic tumor cells in situ. These internalizing scFvs can be used to develop non-invasive imaging strategies that allow sensitive and accurate early tumor detection, and targeted therapies based on intracellular delivery strategies.