Development of Oligonucleotide-based CTLA-4 Inhibitors

描述（由申请人提供）：CD28是主要的共刺激分子，在大多数情况下需要有效激活抗原刺激的T细胞。CTLA-4是第二种共刺激分子，与CD28有相当大的同源性，但与CD28不同的是，它的功能是减弱抗原活化T细胞的扩增。与CTLA-4的作用一致，当与免疫方案结合使用时，抗CTLA-4单克隆抗体在体内短暂阻断CTLA-4功能可增强小鼠的抗肿瘤免疫。总的来说，这些研究表明CTLA-4阻断可以作为抗原特异性免疫治疗的有用辅助，以增强疫苗产生的抗肿瘤反应。在这项拨款申请中，我们建议开发一类由短寡核苷酸适配体组成的新型CTLA-4抑制剂。适配体是高亲和力的单链核酸配体，可以通过组合化学过程使用迭代体外选择技术（SELEX）进行分离。重要的是，这些适体中的大多数已被证明能够抑制它们所结合的蛋白质的功能。这项拨款申请的目标是分离出结合和抑制人类和小鼠CTLA-4但不抑制CD28的适体。该建议的具体目标是：a)隔离nuclease-resistant寡核苷酸适配子专门结合人类CTLA-4但不是CD28和交叉与小鼠CTLA-4反应,b)确定CTLA-4特定寡核苷酸适配子可以抑制CTLA-4体外和体内的功能(老鼠)和管理是否CTLA-4寡核苷酸适配子老鼠与不利影响和c)探索方法进一步提高亲和力和体内生物利用度的CTLA-4寡核苷酸适配子。拟议研究的成功完成将为临床试验奠定基础，以测试将CTLA-4结合适体与癌症患者的免疫治疗相结合的治疗效果。

DESCRIPTION (provided by applicant): CD28 is the major costimulatory molecule which in most circumstances is required for the efficient activation of antigen stimulated T cells. CTLA-4 is a second costimulatory molecule which shares considerable homology with CD28 but unlike CD28 it functions to attenuate the expansion of antigen activated T cells. Consistent with the role of CTLA-4, transient blockade of CTLA-4 function in vivo with anti-CTLA-4 monoclonal antibodies led to enhanced antitumor immunity in mice when used in conjunction with immunization protocols. Overall these studies have shown that CTLA-4 blockade could serve as a useful adjunct to antigen-specific immunotherapy to potentiate vaccine generated antitumor responses. In this grant application we propose to develop a new class of CTLA-4 inhibitors composed of short oligonucleotide aptamers. Aptamers are high affinity single stranded nucleic acid ligands which can be isolated trough a combinatorial chemistry process using iterative in vitro selection techniques termed SELEX. Importantly, most of these aptamers have been shown to be capable of inhibiting the function of the protein to which they bind. The goal of this grant application is to isolate aptamers that bind and inhibit human as well as murine CTLA-4 but not CD28. The specific aims of the proposal are: a) to isolate nuclease-resistant aptamers that specifically bind human CTLA-4 but not CD28 and which cross react with murine CTLA-4, b) to determine whether the CTLA-4 specific aptamers are capable of inhibiting the function of CTLA-4 in vitro and in vivo (in mice) and whether administration of the CTLA-4 aptamers to mice is associated with adverse effects and c) to explore methods to further improve the affinity and the in vivo bioavailability of the CTLA-4 aptamers. Successful completion of the proposed studies will set the stage for clinical trials to test the therapeutic benefit of using CTLA-4 binding aptamers in conjunction with immunotherapy in cancer patients.