全新肿瘤免疫治疗药物CTLA-4阻断型纳米抗体的抗肿瘤作用研究

Immunotherapy is a new cancer treatment modality that has the potential to treat all types of cancer. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a key negative regulator of T-cell activation. CTLA-4 blockade using anti—CTLA-4 monoclonal antibody therapy has great appeal and a fully human monoclonal antibody against CTLA-4 was recently approved by (FDA) for the treatment of metastatic melanoma. Nanobodies, derived from heavy chain only fragment in camelid animals, are now proving their clinical values for antibody drug development. In our research group, we have developed a platform for rapid identification of Nanobody by phage display immunized library techniques. Our team has initiated several nanobody projects, which are involved in tumor and auto-immune diseases. In our preliminary experiments, we have immunized two camels with CTLA-4 antigens individually. After 3 rounds biopanning, 22 nanobodies with different CDR3 sequences have been screened from phage display library. 1 out of 22 showed great potential to inhibit CTLA-4/CD80/CD86 interaction, named Nb1408. In this proposal, we will continue to humanize the blocking nanobody and to understand the epitope of Nb1408 against CTLA-4. By using immunodeficient mice model, we will test the in vivo anti-tumor activity of Nb1408 to treat A549 cancer cells. Moreover, we will systemically investigate the mechanisms of blocking Nb1408 against tumor by studying the effect of Nb1408 on the activation of T cells and inhibition of Treg cells. Overall, this proposal will lay the good foundation to develop a humanized CTLA-4 blocking therapeutic innovative nanobody with proprietary intellectual property rights.

肿瘤免疫治疗是肿瘤治疗的一个重要突破口。细胞毒性T淋巴细胞相关抗原（CTLA-4）作为免疫治疗靶点开发的抗体药物在肿瘤治疗中取得了重大突破。骆驼科动物体内存在的一种天然缺失轻链的纳米抗体（Nanobody），逐渐成为新一代抗体治疗的新兴力量。申请人成功搭建了噬菌体展示纳米抗体快速筛选平台，在国内率先开展了多项肿瘤与自身免疫性疾病治疗性纳米抗体的研究。我们前期免疫了两只新疆双峰驼，筛选出22株CDR3区序列完全不同的CTLA-4高亲和力纳米抗体。其中1株Nb1408能有效阻断CTLA-4与CD80/CD86相互作用。本项目将继续进行阻断型CTLA-4纳米抗体人源化、抗原表位的研究。小鼠肿瘤模型验证CTLA-4纳米抗体在体内对肿瘤的抑制效果。系统地研究纳米抗体对T细胞的激活及对调节性T细胞的抑制并揭示其作用机制，为开发具有自主知识产权的CTLA-4全人源化纳米抗体创新药物奠定基础。

肿瘤免疫治疗是肿瘤治疗的一个重要突破口。细胞毒性T淋巴细胞相关抗原（CTLA-4）作为免疫治疗靶点开发的抗体药物在肿瘤治疗中取得了重大突破。骆驼科动物体内存在的一种天然缺失轻链的纳米抗体（Nanobody），逐渐成为新一代抗体治疗的新兴力量。申请人成功搭建了噬菌体展示纳米抗体快速筛选平台，在国内率先开展了多项肿瘤与自身免疫性疾病治疗性纳米抗体的研究。我们前期免疫了两只新疆双峰驼，筛选出多个CDR3区序列完全不同的CTLA-4高亲和力纳米抗体，其中Nb6能有效阻断CTLA-4与CD80/CD86相互作用。人源化后的抗体能够识别人和猴子CTLA-4，不与其他种属及家族相关蛋白发生交叉反应。该抗体具有良好的亲和力，能够有效激活T细胞，同时表现出较好的肿瘤杀伤活性。此外，四价人源化纳米抗体具有良好的稳定性及较高的表达产量，具有很好的成药性。由此，该项目的进行为开发具有自主知识产权的CTLA-4全人源化纳米抗体创新药物奠定基础。