Abrogating human CD59 activity for antibody-based cancer therapy

描述(申请人提供)：CD59是一个关键的调节因子，通过与补体成分C8和C9结合来限制补体的膜攻击复合体(MAC)的形成，并阻止C9在细胞膜上的掺入和聚合。补体是以抗体为基础的肿瘤治疗的关键介质，如补体依赖细胞毒(CDC)和抗体依赖细胞介导的细胞毒(ADCC)。CD59在所有细胞中普遍表达，在包括B细胞淋巴瘤在内的多种癌细胞中高表达。CD59似乎是一种膜补体调节剂，在保护肿瘤细胞免受抗体介导的CDC的影响方面最有效。广泛的研究表明，CD59的上调是导致淋巴瘤对利妥昔单抗治疗耐药的原因。因此，开发一种能够消除肿瘤细胞中CD59功能并促进抗体介导的肿瘤治疗的分子势在必行。然而，针对hCD59的小抗体的靶向毒性作用以及C8或C9多肽的效果较差限制了它们的治疗目的。间质溶素(ILY)是一种由中间链球菌分泌的溶细胞致孔毒素，由于其与人CD59(HCD59)的受体特异性，它只裂解人类细胞。最近，我们用hCD59转基因小鼠证实了ILY在体内只与hCD59结合的发现。ILY的第4结构域与hCD59中的AA42-58结合，AA42-58也参与与C8和C9的结合。因此，我们假设截短的ILY只呈现结构域4将特异性地取消hCD59的功能，并促进抗体介导的和补体依赖的癌细胞溶解。我们的初步结果表明，来自ILY结构域4(RILYd4)的重组蛋白(114AA)在体外特异性地阻断hCD59的功能。此外，我们还证明了rILYd4(体外IC50=33 nM，体内有效剂量=2.5ug/g体重)应用于对利妥昔单抗介导的CDC耐药的B淋巴瘤细胞系(RAMOS)(即所谓的RRR细胞)，使其对利妥昔单抗介导的CDC增敏，而没有非靶向毒性作用。我们建议在表达转基因hCD59的淋巴瘤移植裸鼠中使用利妥昔单抗作为治疗性抗体来验证这一假设。具体地说，我们将确定rILY4的有效性和特异性(特异性目标1)，并鉴定rILYd4的免疫原性(目标2)。这一应用的成功将产生一种新发现的CD59抑制剂，ILY的第4结构域，不仅作为基于利妥昔单抗的淋巴瘤治疗的辅助，也作为其他基于抗体的癌症治疗的辅助。 公共卫生相关性：在这项应用中，我们将评估天然产品rILYd4对抗体(利妥昔单抗)介导的癌症(B淋巴瘤)治疗的体内疗效。这项工作的成功将促进我们开发新型抗癌试剂，一种抗人CD59抑制剂rILYd4，它可以显著促进抗体介导的癌症治疗，不仅适用于淋巴瘤，也适用于其他癌症。

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.