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。

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.