Development of Oligonucleotide-based CTLA-4 Inhibitors

描述(申请人提供)：CD28是主要的共刺激分子，在大多数情况下是有效激活抗原刺激的T细胞所必需的。CTLA-4是第二个共刺激分子，与CD28有相当大的同源性，但与CD28不同的是，它的功能是抑制抗原激活的T细胞的增殖。与CTLA-4的作用一致的是，用抗CTLA-4的单抗在体内瞬时阻断CTLA-4的功能，当与免疫方案结合使用时，可以增强小鼠的抗肿瘤免疫。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治疗的有用辅助手段，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子组成的新型CTLA-4抑制剂。适配子是一种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的体外选择技术SELEX来分离。重要的是，这些适配子中的大多数已经被证明能够抑制它们结合的蛋白质的功能。这项拨款申请的目标是分离与人和小鼠CTLA-4结合并抑制的适体，但不包括CD28。该建议的具体目的是：a)分离与人CTLA-4特异结合但不与CD28结合并与小鼠CTLA-4发生交叉反应的耐核酸酶适配子；b)确定CTLA-4特异的适配子是否能够在体内外(小鼠体内)抑制CTLA-4的功能以及给小鼠注射CTLA-4适配子是否会产生不良反应；c)探索进一步提高CTLA-4适配子的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联合使用CTLA-4结合适体和免疫疗法的治疗益处。

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.