Development of Therapeutic CD59 inhibitor for treating B-cell malignancies

描述(由申请人提供)：利妥昔单抗(RTX)和奥法单抗(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CD59(HCD59)是一种关键的补体调节因子，可抑制膜攻击复合体(MAC)的形成，从而抑制CDC的诱导。HCD59在非霍奇金淋巴瘤(NHL)和慢性淋巴细胞白血病(CLL)等B细胞恶性肿瘤中高表达，hCD59表达上调是决定这些肿瘤细胞对RTX治疗敏感性的重要因素。因此，开发一种能够抑制肿瘤细胞hCD59功能从而促进肿瘤免疫治疗的分子势在必行。然而，针对hCD59的抗体的靶向性毒性作用以及C8或C9多肽的疗效较差限制了它们的治疗目的。最近，我们开发了一种特异而有效的hCD59抑制剂ILYd4(114AA)，并证明：1)ILYd4增强CDC，从而使RTX耐药的NHL细胞和原代CLL对RTX或OFA治疗敏感；2)ILYd4本身并不负面地介导表达hCD59的红细胞在体内的溶血；3)RTX或OFA介导的CDC效应的敏感性与细胞表面CD59的水平负相关；以及4)过表达hCD59的NHL细胞直接与RTX介导的CDC耐药有关。这些结果使ILYd4作为一种主要的生物候选者的使用合理化，以开发一种潜在的RTX治疗RTX耐药的NHL和CLL的治疗佐剂。基于这些结果，我们建议开发边缘或非免疫原性、有效和稳定的ILYd4衍生多肽。我们推测，优化的ILYd4衍生多肽将改善ILYd4衍生多肽的物理化学性质，使父母能够给药，显著消除或降低免疫原性，增加代谢稳定性和循环中的半衰期，并增强治疗效果，从而导致有效的ILYd4相关治疗将作为佐剂发挥作用 对于先前用基于抗体的疗法治疗后复发的B细胞恶性肿瘤，如OFA和RTX等抗癌药物。此外，hCD59和ILY晶体结构的可用性使我们能够进行计算对接，以确定ILYd4和hCD59之间的潜在界面，并预测非功能表位和免疫原性表位。最近，我们通过去除N-末端具有免疫原性的21个氨基酸残基(ILYd4del21AA)产生了一个短型ILYd4，发现ILYd4del21AA具有与亲本ILYd4相当的抗hCD59活性。因此，我们建议研制具有活性的和灭活的ILYd4del21AA，并聚乙二醇化去免疫的ILYd4del21AA，并研究其免疫原性、PK/PD、体内疗效和毒性。这项研究的成功成果将产生 一种用于临床试验的新型治疗性ILYd4衍生物，将测试其作为基于抗体的癌症治疗(如基于RTX或OFA的B细胞恶性肿瘤治疗)的有效性，并提供急需的手段来克服毁灭性的RTX或OFA耐药B细胞肿瘤。

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 regulator 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 to develop a molecule capable of inhibiting hCD59 function in cancer cells and thus facilitate cancer immune 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 have developed a specific and potent hCD59 inhibitor, ILYd4 (114AA), and demonstrated that 1) ILYd4 enhances CDC, thereby sensitizing RTX-resistant NHL cells and primary CLL to RTX or OFA treatment; 2) by itself ILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes; 3) the sensitivity to CDC effects mediated by RTX or OFA on RTX-resistant NHL cell lines and CLL cells negatively correlated with the level of CD59 on the cell surface; and 4) NHL cells overexpressing hCD59 were directly responsible for the resistance to RTX-mediated CDC. These results rationalize the use of ILYd4 as a lead biological candidate to develop a potential therapeutic adjuvant for RTX treatment of RTX-resistant NHL and CLL. Based on these results, we propose to develop marginally- or non- immunogenic, potent and stable ILYd4-derived peptides. We hypothesize that the optimized ILYd4-derived peptides will improve the physicochemical properties to enable parental administration, eliminate or reduce markedly immunogenicity, increase metabolic stability and half-life in circulation, and enhance therapeutic efficacy, thereby leading to effective ILYd4-related therapeutics that will function as an adjuvant to anti-cancer drugs such as OFA and RTX in B-cell malignancies that have relapsed after prior treatment with Ab-based therapies. Supportively, the availability of crystal structures of hCD59 and ILY enabled us to perform computational docking to identify potential interfaces between ILYd4 and hCD59 and predict nonfunctional and immunogenic epitopes. Recently, we generated a short form of ILYd4 by removing an immunogenic 21 AA residues in N-terminal region (ILYd4del21AA) and found that ILYd4del21AA had an anti-hCD59 activity at the level comparable to parental ILYd4. Therefore, we propose to develop active and deimmunized ILYd4del21AA and pegylate the deimmunized ILYd4del21AA, and to study the immunogenicity, the PK/PD, efficacy and toxicity profile in vivo. Successful outcome of this research will generate a novel therapeutic ILYd4 derivative for clinical trials that will test its efficacy as an adjuvantfor antibody-based cancer therapy such as 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.