新型肿瘤靶向肽和免疫检查点阻断肽介导黑色素瘤免疫治疗

Cancer immunotherapy takes advantage of the body's immune system to fight cancer. One strategy is to use inhibitors that can specifically target immune checkpoints or their ligands to enhance the endogenous antitumor immune responses. One of the mostly studied immune checkpoint-ligand pairs is programmed cell death protein 1 (PD-1)/PD-1 ligand 1 (PD-L1). The use of monoclonal antibodies (mAbs) that can bind either PD-1 or PD-L1 to inhibit PD-1/PD-L1 interaction has achieved a series of clinical successes. However, with an increasing need for mAbs, this immunotherapy strategy faces two daunting challenges. First, the current manufacturing and purification processes lead to a high cost of mAbs. Second, the repeated high dose of mAbs may cause serious side effects in patients due to the lack of tumor-homing capability of the mAbs. Our group has successfully used a phage display biopanning method to discover peptides that can target materials, proteins, cells or tissues and employed these targeting peptides for disease diagnosis and therapy (Science 2004; Adv Funct Mater 2013, Nature Mater 2015; Acc Chem Res 2016; Angew Chem 2017; Chem Rev 2017; NPG Asia Mater 2017). In order to develop a novel PD-L1 inhibitor with a lower cost and minimum side effects, on the basis of these successes, we propose to use the phage display biopanning method to select a PD-L1 binding peptide, which can selectively bind PD-L1 to inhibit the PD-1/PD-L1 interaction, and a melanoma tumor-homing peptide, which can guide nanoparticles to home to a melanoma tumor. Then we will construct a pH responsive liposome by inserting the N-Isopropylacrylamide (NIPAM)-methacrylic acid (MAA)-octadecyl acrylate (ODA) copolymer into the liposome assembled from the egg phosphatidylcholine (EPC) and cholesterol, functionalize its surface with the melanoma tumor-homing peptide, and load the PD-L1 binding peptide into its core to form the dual tumor targeting anti-PD-L1 liposome (dtt-PDL1-lipo). The presence of the pH-responsive NIPAM-MAA-ODA copolymer in the dtt-PDL1-lipo will enable the lipid bilayer of the liposome to be broken open only in the unique acidic tumor microenvironment, leading to the controlled release of PD-L1 binding peptides site-specifically in the tumors. Finally, we will intravenously inject the dtt-PDL1-lipo into a melanoma tumor mouse model. The dtt-PDL1-lipo will selectively home to the tumor sites and then release the PD-L1 binding peptide upon the arrival at the tumor sites. The released PD-L1 binding peptide will then block the PD-1/PD-L1 recognition within the tumor sites and activate T cells there to inhibit tumor growth. The tumor-targeting capability of the dtt-PDL1-lipo will also prevent the activated T cells from attacking healthy tissue and thus reduce the side effects of the cancer immunotherapy. This project will open up a new multidisciplinary avenue to the development of cheap and safe PD-1/PD-L1 inhibitors to advance cancer immunotherapy.

PD-1/PD-L1免疫检查点阻断剂介导的肿瘤治疗是发展最快的免疫疗法，但现有的单抗阻断剂价格高，因缺乏肿瘤靶向性而副作用大。为解决这两大问题，在申请人使用噬菌体生物淘选筛选材料、蛋白、细胞和组织的特异性靶向肽用于疾病诊疗的基础上（Science 2004; Adv Funct Mater 2013, Nature Mater 2015; Acc Chem Res 2016; Angew Chem 2017; Chem Rev 2017; NPG Asia Mater 2017），本项目拟用生物淘选筛选出PD-L1亲和肽和黑色素瘤靶向肽, 构建基于靶向肽和pH响应的肿瘤双重靶向脂质体，将低成本化学合成的PD-L1亲和肽携带至肿瘤部位定点释放。亲和肽将在肿瘤处阻断免疫检查点，激活T细胞攻击肿瘤并抑制其生长，并降低T细胞对健康组织的攻击以减少副作用，为开发新型免疫检查点阻断剂治疗肿瘤拓展思路。

通过修改从随机肽库中筛选获得靶向肽的传统过程，我们首先发现了一种结合PD-L1蛋白胞外结构域的多肽和另一种靶向黑色素瘤并与黑色素瘤癌细胞结合的多肽。我们将PD-L1亲和肽和黑色素瘤靶向肽的相应基因插入侧壁蛋白（pVIII）和尖端蛋白（pIII）的基因中，将二者分别展示在fd噬菌体的侧壁和尖端。由此产生的双功能fd噬菌体具有生物相容性，可以有效结合黑色素瘤癌细胞，并阻断PD-1/PD-L1的相互识别，从而有效抑制肿瘤生长。噬菌体可以通过感染宿主细菌进行扩增，从而以低成本大量生产。且噬菌体侧壁或尖端的多肽可以独立地被替换为靶向其他癌细胞或信号通路的肽，因此噬菌体有望成为通用平台—一种有效的癌症靶向免疫治疗剂。