Development of Oligonucleotide-based CTLA-4 Inhibitors

描述（由申请人提供）：CD 28是主要的共刺激分子，在大多数情况下，有效激活抗原刺激的T细胞需要CD 28。CTLA-4是第二种共刺激分子，其与CD 28具有相当大的同源性，但与CD 28不同，其功能是减弱抗原活化的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，用抗CTLA-4单克隆抗体在体内瞬时阻断CTLA-4功能导致小鼠中增强的抗肿瘤免疫力。总体而言，这些研究表明，CTLA-4阻断可以作为抗原特异性免疫疗法的有用辅助，以增强疫苗产生的抗肿瘤应答。在这项授权申请中，我们提出开发一类由短寡核苷酸适体组成的新的CTLA-4抑制剂。适体是高亲和力的单链核酸配体，其可以通过使用称为SELEX的迭代体外选择技术的组合化学过程分离。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。该授权申请的目标是分离结合并抑制人以及鼠CTLA-4但不抑制CD 28的适体。该提案的具体目标是：a）分离特异性结合人CTLA-4但不结合CD 28且与鼠CTLA-4交叉反应的核酸酶抗性适体，B）确定所述CTLA-4特异性适体是否能够在体外和体内（在小鼠中）抑制CTLA-4的功能，以及向小鼠施用所述CTLA-4适体是否与副作用相关，以及探索进一步提高CTLA-4适体亲和力和体内生物利用度的方法。拟议研究的成功完成将为临床试验奠定基础，以测试在癌症患者中使用CTLA-4结合适体与免疫疗法结合的治疗益处。

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.