Abrogating human CD59 activity for antibody-based cancer therapy

描述（由申请人提供）：CD59是限制补体膜攻击复合物（MAC）形成的关键调节剂，通过与补体组分C8和C9结合，阻止C9在细胞膜内掺入和聚合。补体是基于抗体的癌症治疗的关键介质，如补体依赖性细胞毒性（CDC）和抗体依赖性细胞毒性（ADCC）。CD59在所有细胞中普遍表达，并在包括b细胞淋巴瘤在内的多种癌细胞中高表达。CD59似乎是膜补体调节剂，在保护肿瘤细胞免受抗体介导的CDC中最有效。大量研究表明，CD59的上调是淋巴瘤对利妥昔单抗治疗耐药的原因。因此，我们迫切需要开发一种能够消除CD59在癌细胞中的功能并促进抗体介导的癌症治疗的分子。然而，针对hCD59的小型抗体的靶向毒性作用和C8或C9肽的低疗效限制了它们的治疗目的。中间溶素（ILY）是一种由中间链球菌分泌的细胞溶孔毒素，由于其对人CD59 （hCD59）的受体特异性，只能溶解人细胞。最近，我们利用hCD59转基因小鼠在体内证实了ILY仅与hCD59结合的发现。在hCD59中，ILY的结构域4与AA42-58结合，它们也参与与C8和C9的结合。因此，我们假设截断的仅呈递结构域4的ILY将特异性地剥夺hCD59的功能，并促进抗体介导和补体依赖性的癌细胞溶解。我们的初步结果一致地表明，来自ILY结构域4 （rILYd4）的重组蛋白（114AA）在体外特异性地阻断了hCD59的功能。此外，我们证明，将rILYd4（体外IC50 = 33 nM，体内有效剂量= 2.5 ug/g体重）应用于对利妥昔单抗介导的CDC（所谓的RRR细胞）耐药的B淋巴瘤细胞系（RAMOS），使它们对利妥昔单抗介导的淋巴瘤CDC增敏，而无脱靶毒性作用。我们建议在表达转基因hCD59的淋巴瘤异种移植胸腺裸小鼠中使用利妥昔单抗作为治疗性抗体来验证这一假设。具体来说，我们将确定rILY4的有效性和特异性（特异性目标1），并表征rILYd4的免疫原性（特异性目标2）。这项应用的成功将产生一种新发现的CD59抑制剂，即ILY的结构域4，不仅可以作为基于利妥昔单抗的淋巴瘤治疗的辅助药物，还可以作为其他基于抗体的癌症治疗的辅助药物。

DESCRIPTION (provided by applicant): CD59 is a key regulator for restricting the formation of the membrane attack complex (MAC) of complement by binding to complement components C8 and C9 and preventing C9 incorporation and polymerization in the cell membrane. Complement is a key mediator for antibody-based cancer therapy such as complement-dependent cytotoxicity (CDC) and antibody-dependent-cell mediated cytotoxicity (ADCC). CD59 is universally expressed in all cells and highly expressed in many kinds of cancer cells including B-cell lymphoma. CD59 appears to be the membrane complement regulator that is most effective at protecting tumor cells from antibody-mediated CDC. Extensive studies indicate that upregulation of CD59 is responsible for lymphoma resistance to rituximab treatment. Therefore, it is imperative for us to develop a molecule capable of abrogating CD59 function in cancer cells and facilitating antibody-mediated cancer therapy. However, targeted toxicity effect from miniantibody specific against hCD59 and less efficacy of C8 or C9 peptides limit them for therapeutic purposes. Intermedilysin (ILY), a cytolytic pore-forming toxin secreted by Streptococcus intermedius, lyses only human cells due to its receptor specificity for human CD59 (hCD59). Recently, we used hCD59 transgenic mice to confirm the finding that ILY binds only to hCD59 in vivo. Domain 4 of ILY binds to AA42-58 in hCD59, which also participate in the binding to C8 and C9. Thus, we hypothesize that the truncated ILY presenting only domain 4 will specifically abrogate hCD59 function and facilitate antibody- mediated and complement-dependant cancer cytolysis. Consistently, our preliminary results have demonstrated that the recombinant protein (114AA) derived from the ILY domain 4 (rILYd4) specifically blocks hCD59 function ex vivo. Furthermore, we demonstrated that application of rILYd4 (IC50 = 33 nM in vitro and effective dose = 2.5 ug/g body weight in vivo ) to B lymphoma cell lines (RAMOS), which are resistant to Rituximab-mediated CDC (so called RRR cells), sensitized them to Rituximab-mediated CDC in lymphoma without off target toxicity effects. We propose to use rituximab as a therapeutic antibody in lymphoma xenografted athymic nude mice that express transgenic hCD59 to test this hypothesis. Specifically, we will determine and the efficacy and specificity of rILY4 (specific aim 1) and characterize rILYd4 immunogenicity (Aim 2). Success in this application will yield a newly-identified CD59 inhibitor, domain 4 of ILY as an adjunct to not only rituximab-based lymphoma therapy, but also other antibody-based cancer therapy. PUBLIC HEALTH RELEVANCE: In this application, we will assess the in vivo efficacy of the nature product rILYd4 for antibody (rituximab)-mediated cancer (B-lymphoma) therapy. Success of this work will foster us to develop novel anti-cancer reagents, an anti-human CD59 inhibitor rILYd4, which can significantly facilitate antibody- mediated cancer therapy, not only in lymphoma, but also in other cancers.