靶向肿瘤AGR2微环境信号和PD1免疫检查点的双特异抗体对肺部AGR2阳性肿瘤的靶向富集、药理活性及分子机制研究

Programmed cell death protein 1(PD-1) is one of the inhibitory immune-checkpoint receptors that plays an important role in down-regulating the immune system to prevent auto immune attack. Activation of the PD-1 pathway by its ligand (PD-L1) expressed on the surface of tumor cells causing immune escape in cancer. Currently, antibody therapies against PD-1 are one of the most successful strategies against cancer. However, serious side effects are also associated with the inherited problems of anti-PD-1 therapies caused by the widespread locations of the PD-1 positive immune cells. Therefore, PD-1 blocking therapeutic antibodies often cause auto-immune attacks against skin, joint, red blood cells and organs such as thymus and heart in patients. Localizing and enriching these antibodies to tumor tissue through a tumor-specific guiding target in the tumor itself will greatly limit the side effect of the immune-checkpoint antibodies. We have showed that as a paracrine signal for tumor microenvironment formation, secreted AGR2 is highly concentrated in interstitial fluid of AGR2 positive tumors. Preliminary data showed the anti-AGR2 antibody agtuzumab not only promoted tumor apoptosis and necrosis but the antibody was also effectively concentrated in the tumor tissues. In this proof–of–concept proposal, we will construct various forms of bi-specific antibodies targeting AGR2 and PD-1 in order to verify whether such AGR2/PD1 antibody will be guided by the high AGR2 concentration to be enriched in tumor tissue; whether AGR2/PD-1 bi-specific antibody will reduce auto-immune and increase anti-tumor effects compared with anti-PD-1 antibody alone, anti-AGR2 alone and in combination; we will also explore the underlying mechanism of the possible different outcome between AGR2/PD-1 bi-specific antibody and the combination with antiAGR2 and anti-PD1 antibodies. We will perform this investigation at molecular cellular levels, as well as at the animal level using xenograft animal models. We believe that this work will provide bases to validate the concept that a tumor specific localized paracrine signal gradient in tumor microenvironment can effectively guide immune-checkpoint inhibiting antibodies in a bi-specific form to home in on and localize to the target tumor mass to reduce therapeutic side effect in other tissues, and hereby, greatly facilitate the development a series of next-generation immune-checkpoint bi-specific or multi-specific therapeutic antibodies.

抑制性免疫检查点蛋白存在于免疫系统细胞中抑制自身免疫反应而肿瘤细胞通过结合免疫细胞的检查点蛋白造成免疫逃逸。用抗体阻断PD-1免疫检查点蛋白是阻止肿瘤逃逸免疫系统的抗癌新星，但由于PD-1抗体靶向体内免疫细胞而不是肿瘤细胞缺乏特异性，会造成多种自身免疫反应。使PD-1抗体富集于肿瘤组织内可减少其副作用。分泌型AGR2是肿瘤微环境形成信号，在很多肿瘤组织如肺癌中高表达。阻断AGR2的人源化艾克舒单抗，富集在肿瘤组织中，促进AGR2阳性肿瘤细胞凋亡与坏死。本课题通过检验同时靶向AGR2和PD-1的双特异抗体，验证其能否获得肿瘤靶向性，富集于AGR2阳性的肺部肿瘤组织内同时保持PD-1阻断活性，从而减少PD-1抗体的副作用，提高AGR2抗体的活性。项目通过揭示双特异抗体靶向肿瘤微环境AGR2与免疫检查点PD-1之间的相互影响，为靶向肿瘤微环境的新型双特异抗体药物的研发提供概念验证和分子理论基础。

研究背景：靶向抑制PD-1免疫检查点蛋白的抗体治疗是治疗晚期肿瘤的有效方法。 但由于免疫检查点蛋白普遍存在于免疫系统多种细胞表面，分布于多种重要组织器官，针对免疫检查点的抗体治疗也会产生免疫毒性。使免疫检查点治疗抗体靶向富集于肿瘤组织内发挥作用对提高疗效、减少副作用具有科学和应用价值。分泌型AGR2是肿瘤微环境形成的重要信号，我们针对AGR2在卵巢癌、肺癌等多肿瘤组织高表达的特点，开发了具有国际专利的人源化抗体18A4HU，并在本项目提出构建PD1/AGR2双特异抗体的研究。.研究内容：1构建靶向AGR2肿瘤微环境信号和PD1免疫检查点的双特异抗体。2在体外细胞分子水平研究验证阻断AGR2信号网络和PD1免疫检查点蛋白网络之间的相互作用和影响及相关机制。3在整体动物水平研究验证PD1/AGR2双特异抗体能否富集于表达AGR2的肿瘤内并具有抗PD1的免疫激活功能和更高的药理活性，减少PD-1抗体的副作用。 .重要结果： 1构建的靶向AGR2和PD-1的双特异抗体通过质粒测序和免疫印迹，确认了抗体结构的完整性2通过western确认双特异抗体对AGR和PD1的识别 3通过对高表达细胞克隆的选择进行实验室工艺表达纯化，制备纯度达到90%以上。 4在体外细胞分子水平研究验证阻断AGR2信号和PD-1免疫检查点之间的相互作用和影响，发现被双特异抗体抑制后PD-L1 表达下降并促进CD8 表达。5对照单独抗体组合，细胞实验说明抗体阻断AGR表达抑制了Catenin肿瘤信号通道。 6裸鼠模型验证双特异抗体的靶向性和双特异抗体的富集对AGR2肿瘤的特异性。AGR2阳性移植瘤比阴性瘤富集更多的双特异抗体。 7对肺癌组织切片的荧光染色表明AGR2强阳性的肺癌PD1表达也多呈阳性。以上结果奠定了双特异抗体的实验室工艺， 揭示了双特异抗体的作用，确认了其治疗AGR/PD1双阳性肺癌的可行性。

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