Abrogating human CD59 activity for antibody-based cancer therapy

描述（由申请人提供）：CD 59是一种关键调节因子，通过与补体成分C8和C9结合并阻止C9在细胞膜中掺入和聚合，限制补体的膜攻击复合物（MAC）的形成。补体是基于抗体的癌症治疗的关键介质，例如补体依赖性细胞毒性（CDC）和抗体依赖性细胞介导的细胞毒性（ADCC）。CD 59在所有细胞中普遍表达，并且在包括B细胞淋巴瘤在内的多种癌细胞中高度表达。CD 59似乎是在保护肿瘤细胞免受抗体介导的CDC方面最有效的膜补体调节剂。广泛的研究表明，CD 59的上调是淋巴瘤对利妥昔单抗治疗耐药的原因。因此，我们必须开发一种能够消除癌细胞中CD 59功能并促进抗体介导的癌症治疗的分子。然而，特异性针对hCD 59的微型抗体的靶向毒性作用和C8或C9肽的较低功效限制了它们用于治疗目的。中间溶素（ILY）是一种由中间链球菌分泌的细胞溶解性成孔毒素，由于其对人CD 59（hCD 59）的受体特异性，仅溶解人细胞。最近，我们使用hCD 59转基因小鼠来证实ILY在体内仅与hCD 59结合的发现。ILY的结构域4与hCD 59中的AA 42 -58结合，其也参与与C8和C9的结合。因此，我们假设仅呈递结构域4的截短的ILY将特异性地消除hCD 59功能并促进抗体介导的和补体依赖性的癌症细胞溶解。因此，我们的初步结果已经证明来源于ILY结构域4（rILYd 4）的重组蛋白（114 AA）特异性地阻断hCD 59离体功能。此外，我们证明了将rILYd 4（体外IC 50 = 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具有抗性的B淋巴瘤细胞系（拉莫斯）（所谓的RRR细胞），使它们对利妥昔单抗介导的CDC在淋巴瘤中敏感，而没有脱靶毒性效应。我们建议使用利妥昔单抗作为一种治疗性抗体在淋巴瘤异种移植无胸腺裸鼠表达转基因hCD 59来测试这一假设。具体而言，我们将确定rILY 4的功效和特异性（具体目标1）并表征rILYd 4免疫原性（目标2）。该应用的成功将产生一种新鉴定的CD 59抑制剂，ILY的结构域4，不仅可作为基于利妥昔单抗的淋巴瘤治疗的辅助药物，还可用于其他基于抗体的癌症治疗。 公共卫生相关性：在本申请中，我们将评估天然产物rILYd 4用于抗体（利妥昔单抗）介导的癌症（B淋巴瘤）治疗的体内功效。这项工作的成功将促进我们开发新的抗癌试剂，抗人CD 59抑制剂rILYd 4，其可以显著促进抗体介导的癌症治疗，不仅在淋巴瘤中，而且在其他癌症中。

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.