Development of Therapeutic CD59 inhibitor for treating B-cell malignancies

描述（由申请方提供）：利妥昔单抗（RTX）和奥法木单抗（OFA）在癌症治疗中的疗效部分取决于补体依赖性细胞毒性（CDC）的诱导。人CD 59（hCD 59）是一种关键的补体调节蛋白，其限制膜攻击复合物（MAC）的形成，从而抑制CDC的诱导。hCD 59在B细胞恶性肿瘤如非霍奇金淋巴瘤（NHL）和慢性淋巴细胞白血病（CLL）中高度表达，并且hCD 59表达的上调是那些癌细胞对RTX治疗的敏感性的重要决定因素。因此，我们迫切需要开发一种能够消除癌细胞中CD 59功能的分子，从而促进抗体介导的癌症治疗。然而，来自对hCD 59具有特异性的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。最近，我们开发了一种特异性和有效的hCD 59抑制剂，rILYd 4（114 aa），并证明了rILYd 4在体外和体内增强CDC，从而使RTX抗性淋巴瘤细胞和原发性CLL对RTX和OFA治疗敏感。通过定义rILYd 4在小鼠中的PK/PD特征，我们表明rILYd 4本身不会不利地介导表达hCD 59的红细胞的体内溶血。我们证明了RTX抗性细胞中hCD 59表达水平的增加是由于对预先存在的克隆的选择，而不是hCD 59的从头诱导。我们还证明了OFA或RTX介导的CDC效应对RTX抗性B细胞恶性细胞系和CLL细胞的敏感性与细胞表面上的CD 59水平呈负相关。综上所述，我们的结果合理使用rILYd 4作为RTX和OFA治疗RTX耐药NHL和CLL的治疗性佐剂。基于这些结果，我们建议优化rILYd 4并开发免疫原性低或无免疫原性、有效且稳定的rILYd 4衍生肽。我们假设优化的rILYd 4将改善亲本rILYd 4的物理化学性质，消除或显著降低免疫原性，增加代谢稳定性和循环中的半衰期，并增强治疗功效，从而导致有效的rILYd 4相关治疗剂，用于增强在先前基于抗体的治疗后复发的B细胞恶性肿瘤中作为抗癌药物如OFA和RTX的佐剂。我们计划1）通过噬菌体展示和合成方法鉴定rILYd 4中负责其抗hCD 59活性的序列，并使用它们开发小的、有效的和非免疫原性的rILYd 4衍生肽（衍生物）（目的1）和2）研究其在人HLA转基因小鼠中的免疫应答，并表征其在人源化hCD 59转基因小鼠中的PK/PD、功效和毒性特征。(Aim 2）。这项研究的成功结果将产生一种用于临床试验的新型治疗性rILYd 4衍生剂，该临床试验将测试其作为基于RTX或OFA的B细胞恶性肿瘤治疗的佐剂的功效，并提供急需的手段来克服毁灭性的RTX或OFA抗性B细胞恶性肿瘤。

DESCRIPTION (provided by applicant): Rituximab (RTX) and Ofatumumab (OFA) efficacies in cancer therapy depend in part on the induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of membrane attack complex (MAC), thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell malignancies such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) and up-regulation of hCD59 expression is an important determinant of the sensitivity of those cancer cells to RTX treatment. Therefore, it is imperative for us to develop a molecule capable of abrogating CD59 function in cancer cells and thus facilitate antibody- mediated cancer therapy. However, targeted toxicity effect from antibody specific against hCD59 and less efficacy of C8 or C9 peptides limit them for therapeutic purposes. Recently, we developed a specific and potent hCD59 inhibitor, rILYd4 (114aa), and demonstrated that rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing RTX resistant lymphoma cells and primary CLL to RTX and OFA treatment. By defining PK/PD profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. We demonstrated that increasing expression level of hCD59 in RTX-resistant cells occurs due to selection of pre-existing clones, rather than de novo induction of hCD59. We also demonstrated that the sensitivity to CDC effects mediated by OFA or RTX on RTX-resistant B-cell malignant cell lines and CLL cells negatively correlated with the level of CD59 on the cell surface. Taken together, our results rationalize the use of rILYd4 as a therapeutic adjuvant for RTX and OFA treatment of RTX-resistant NHL and CLL. Based on these results, we propose to optimize rILYd4 and develop less or non immunogenic, potent and stable rILYd4-derived peptide. We hypothesize that the optimized rILYd4 will improve the physicochemical properties of parental rILYd4, eliminate or reduce markedly immunogenicity, increase metabolic stability and half life in circulation, and enhance therapeutic efficacy, thereb leading to effective rILYd4-related therapeutics for enhancing as a adjuvant to anti-cancer drugs such as OFA and RTX in B-cell malignancies that have relapsed after prior antibody-based therapies. We propose to 1) to identify the sequences in rILYd4 responsible for its anti-hCD59 activity by phage display and synthetic approaches, and use them to develop a small, potent and non-immunogenic rILYd4-derived peptide (derivative) (Aim 1) and 2) to study its immune responses in human HLA transgenic mice and characterize its PK/PD, efficacy and toxicity profiles in humanized hCD59 transgenic mice. (Aim 2). Successful outcome of this research will generate a novel therapeutic rILYd4-derived agent for clinical trials that will test its efficacy a an adjuvant for RTX- or OFA-based B-cell malignancy therapy and provide the much needed means to overcome the devastating RTX- or OFA-resistant B-cell malignancies.