基于EGFR“二聚体界面”靶向策略的单克隆抗体与多肽疫苗研究

Epidermal growth factor recepor (EGFR) was the first receptor to be proposed as the target for cancer therapy, however,the targeting strategies were defective,which resulted in a low resoponse rate of anti-EGFR therapy clinically.Dimerizaiotn plays a key role in the activation of receptor of EGFR family, so targeting the conservative dimer interface may be the key for improving the responses of anti-EGFR. A B-cell epitope peptide"EGFR237-267" from the interface domain was found, by our laboratory recently, to be of immunogenicity and potentiality clinically in the cancer therapy. This study will focus on the feasibility of targeting dimer interface in anti-EGFR therapy, furthermore, a novel monoclonal antibody (mAb) and a therapeutic peptide vaccine will be developed based on the strategy and "EGFR 237-267". The feasibility of targeting dimer interface of EGFR will be evaluated by a series of in vitro experiments with the novel mAb， epithelioma cell lines of distinct subtypes of EGFR and A431 exnografts grown in nude mice.A series of antigen peptide based on EGFR237-267 will be constructed with Neisseria meningitidis P64k (P64k) and MVF, a "promiscuous Th cell epitope "from the measles virus fusion protein, as the imunnopotentiating protein or peptide, and screened with C57BL6 mice as the" poor antibody responder (PAR)" animal. Futhermore,the efficacy of pepetide vaccine will be evaluated with Lewis lung cancer (LLC) exnografts grown in C57BL6 mice.We hope that our work on the strategy for targeting EGFR should be helpful to tap the potential of anti-EGFR therapy for cancer.

表皮生长因子受体（EGFR）是恶性肿瘤靶向治疗理想的靶分子，但现有靶向策略存在缺陷，导致靶向药物临床反应率偏低。"二聚化"是EGFR及其突变体活化所需的关键步骤，靶向此过程有可能提高药物的抗瘤谱和临床反应率。我们曾发现一个来自EGFR"二聚体界面"区的全新B细胞表位，以其所获多克隆抗体可有效抑制EGFR过表达肿瘤细胞的生长。本研究拟以该B细胞表位为基础，构建一类EGFR"二聚体界面"靶向的单克隆抗体和基于新型载体蛋白"p64K"与通用Th表位"MVF"的多肽疫苗，并以EGFR代表性表型的细胞模型、人上皮癌细胞A431裸鼠移植模型和小鼠Lewis肺癌移植模型来系统研究单克隆抗体和多肽疫苗的抑瘤效果与作用机制，以期研制一类具有自主知识产权的、可针对各种EGFR表型恶性肿瘤的单克隆抗和治疗性多肽疫苗，并系统探讨EGFR"二聚体界面"靶向策略的可行性，为"抗EGFR"治疗提供新的策略与手段。

表皮生长因子受体（EGFR）是恶性肿瘤靶向治疗理想的靶分子，但现有靶向策略存在缺陷，导致该类药物临床反应率偏低。“二聚化”是EGFR家族受体活化的关键步骤，“二聚体界面区”是高度保守的序列，靶向该区域有可能提高临床反应率和疗效。本课题提出EGFR"二聚化界面"靶向策略，并拟构建一类基于此策略的治疗性单克隆抗体与多肽疫苗。 我们从人源EGFR的二聚化界面区分离了一个全新B细胞表位肽——EGFR237-267，该抗原肽已获中国发明专利授权（ZL201010618851.4）；以EGFR237-267为基础，通过与麻疹病毒融合蛋白的通用Th表位肽MVF或脑膜炎球菌外膜蛋白p64K的融合或偶联，构建了5种形式的抗原肽，并基于DNA重组技术建立了相应抗原肽的高效制备方法；以C57BL6小鼠免疫模型和Lewis肺癌（LLC）移植瘤模型评价了该几种形式抗原肽的免疫原性和体内抑瘤效果，确定了最佳抗原肽构建形式p64K* EGFR237-267，并初步观察到该类抗原肽免疫可有效抑制小鼠LLC移植瘤的生长；获得了4株高亲和力的抗EGFR237-267单克隆抗体，系列体内、外实验结果表明，该类抗体可特异性结合于细胞表面EGFR，并且属于EGFR二聚体界面靶向，可有效抑制EGFR过表达肿瘤细胞的生长；发表了2篇SCI论文和1篇核心期刊论文，已获1项中国发明专利，正准备申请1项中国发明专利，培养了4名硕士研究生和1名在职博士研究生。.本课题为一年期项目，希望进一步获得国家自然科学基金的资助，以本项目构建的抗EGFR237-267单抗和抗原肽p64K* EGFR237-267为基础，系统、深入研究EGFR“二聚体界面”靶向策略的可行性，同时对抗EGFR237-267单抗进行人源化改造，并进行系统的体内外抗瘤效果与机制研究，以期研制一类具有自主知识产权的治疗性人源化抗体。.