Ovarian cancer gene therapy using HPV pseudovirion

描述(申请人提供)：晚期卵巢癌是美国女性生殖系统癌症患者中死亡率最高的。现有的卵巢癌治疗方法，如手术和化疗，都有显著的副作用，很少能为局部晚期或转移性疾病的患者带来长期治愈。缺乏根治疗法和高比例的晚期疾病患者强调了迫切需要开发创新的和有针对性的治疗方法来控制晚期卵巢癌。在目前的方案中，我们的目标是通过使用人乳头瘤病毒(HPV)假病毒(PSV)将治疗性DNA载体传递到卵巢肿瘤细胞，以通过细胞介导和体液免疫反应控制感染和未感染的卵巢肿瘤细胞，从而克服现有治疗方法的主要局限性。最近，我们证明了HPV PSV能够在体内优先感染卵巢肿瘤。此外，我们已经证明，含有肿瘤归巢分子NKG2D的嵌合蛋白融合到免疫球蛋白G(IgG2a)的Fc片段上，能够包裹肿瘤细胞并产生抗肿瘤效应(见初步数据)。肿瘤归巢分子NKG2D与NKG2D配体具有高亲和力，与正常细胞相比，NKG2D配体在大多数卵巢肿瘤细胞上过度表达。NKG2D与Fc片段的融合(NKG2D-Fc)能够使具有Fc受体的效应细胞(如巨噬细胞和自然杀伤细胞)与肿瘤细胞结合，从而诱导抗体依赖和补体依赖的细胞毒作用。我们建议进一步将NKG2D-Fc与流感病毒MHC-I类限制性免疫原性CTL表位融合，由Furin裂解位点分隔。这将通过利用先前存在的针对一种常见的外来病毒表位的细胞毒性T淋巴细胞(CTL)来绕过免疫耐受，这种表位在绝大多数人中都存在。我们先前已经证明，在荷瘤小鼠体内注射含有模型免疫原性CTL抗原肽OVA的类似嵌合蛋白，侧翼有Furin裂解位点，导致嵌合蛋白的靶向递送和OVA CTL肽在荷瘤小鼠肿瘤细胞的MHC-I类分子上的呈递。因此，我们推测，从嵌合蛋白(NKG2D-Fc-RM)中，NKG2D部分将使嵌合蛋白与HPV PSV感染和未感染的卵巢癌细胞特异性结合，在那里免疫原性CTL表位将通过Furin切割释放，并覆盖肿瘤细胞以供预先存在的CTL识别。此外，Fc的存在使结合的肿瘤细胞容易受到体液免疫的攻击。总体而言，我们提出的由HPV PSV递送的治疗性嵌合蛋白具有靶向肿瘤递送、肿瘤部位特异的Furin切割以及通过现有的CTL免疫和Fc介导的杀伤而有效的肿瘤靶向杀伤的优势，代表了一种具有很高翻译价值的新策略。这项拟议研究的成功实施将成为一项创新战略，可应用于其他类型的晚期癌症的治疗。

DESCRIPTION (provided by applicant): Advanced ovarian cancer has the highest mortality rate among patients with cancers of the female reproductive system in the United States. Existing therapies for ovarian cancer, such as surgery and chemotherapy, have significant side effects and rarely result in long-term cures for patients with locally advanced or metastatic disease. The lack of curative treatments and high proportion of patients diagnosed with advanced disease underscores the urgent need to develop innovative and targeted therapies to control advanced stage ovarian cancer. In the current proposal, we aim to overcome major limitations of current therapies by employing human papillomavirus (HPV) pseudovirions (psVs) to deliver a therapeutic DNA construct to ovarian tumor cells in order to control infected and uninfected ovarian tumor cells through cell-mediated and humoral immune responses. Recently, we demonstrated that HPV psV is able to preferentially infect ovarian tumors in vivo. Furthermore, we have shown that a chimeric protein containing a tumor-homing molecule, NKG2D, fused to the Fc fragment of immunoglobulin G (IgG2a) was capable of coating tumor cells and generating antitumor effects (see preliminary data). The tumor-homing molecule, NKG2D, binds with high affinity to NKG2D ligand, which is overexpressed on the majority of ovarian tumor cells, compared to normal cells. NKG2D fused to the Fc fragment (NKG2D-Fc) enables the binding of effector cells exhibiting Fc receptor, such as macrophages and natural killer cells, to the tumor cells for the induction of antibody-dependent and complement-dependent cytotoxicity. We propose to further fuse NKG2D-Fc, with an MHC class I-restricted immunogenic CTL epitope for influenza virus, separated by a furin cleavage site. This will bypass immune tolerance by exploiting pre-existing cytotoxic T lymphocytes (CTLs) against a common foreign viral epitope, which is found in the vast majority of individuals. We have previously demonstrated that intraperitoneal injection of a similar chimeric protein containing model immunogenic CTL antigenic peptide, OVA, flanked with a furin cleavage site, led to the targeted delivery of the chimeric protein and the presentation of OVA CTL peptide on MHC class I molecules of tumor cells in tumor-bearing mice. Therefore, we reason that from the chimeric protein (NKG2D-Fc-RM), the NKG2D portion will make the chimeric protein bind specifically to HPV psV infected and uninfected ovarian cancer cells, where the immunogenic CTL epitopes will be released through cleavage by furin and coat the tumor cells for recognition by pre-existing CTLs. In addition, the presence of Fc renders the bound tumor cell susceptible to attack by humoral immunity. Overall, our proposed therapeutic chimeric protein delivered by HPV psV has the advantages of targeted tumor delivery, specific furin cleavage at the tumor site, and potent tumor-targeted killing through existing CTL immunity and Fc-mediated killing, representing a novel strategy with high translational value. The successful implementation of the proposed study would serve as an innovative strategy that may be applied to the treatment of other types of late stage cancers.