Ovarian cancer gene therapy using HPV pseudovirion

描述（由申请人提供）：在美国，晚期卵巢癌在女性生殖系统癌症患者中死亡率最高。现有的卵巢癌治疗方法，如手术和化疗，有显着的副作用，很少导致局部晚期或转移性疾病患者的长期治愈。缺乏治愈性治疗和高比例的患者被诊断患有晚期疾病，强调迫切需要开发创新和靶向治疗来控制晚期卵巢癌。在目前的建议中，我们的目标是克服目前的治疗方法的主要局限性，采用人乳头瘤病毒（HPV）的假病毒（psVs）提供一个治疗性的DNA构建体的卵巢肿瘤细胞，以控制感染和未感染的卵巢肿瘤细胞通过细胞介导的和体液免疫反应。最近，我们证明了HPV psV能够优先感染体内卵巢肿瘤。此外，我们已经证明，含有肿瘤归巢分子NKG 2D的嵌合蛋白与免疫球蛋白G（IgG 2a）的Fc片段融合，能够包被肿瘤细胞并产生抗肿瘤作用（见初步数据）。肿瘤归巢分子NKG 2D以高亲和力与NKG 2D配体结合，与正常细胞相比，NKG 2D配体在大多数卵巢肿瘤细胞上过表达。与Fc片段融合的NKG 2D（NKG 2D-Fc）能够使表现出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，以诱导抗体依赖性和补体依赖性细胞毒性。我们建议进一步融合NKG 2D-Fc，与流感病毒的MHC I类限制性免疫原性CTL表位，由弗林蛋白酶切割位点分开。这将通过利用预先存在的细胞毒性T淋巴细胞（CTL）对抗在绝大多数个体中发现的常见外来病毒表位来绕过免疫耐受。我们以前已经证明，腹腔内注射一个类似的嵌合蛋白含有模型免疫原性CTL抗原肽，OVA，侧翼弗林蛋白酶切割位点，导致有针对性的交付的嵌合蛋白和介绍的OVA CTL肽的MHC I类分子的肿瘤细胞在荷瘤小鼠。因此，我们推断，从嵌合蛋白（NKG 2D-Fc-RM），NKG 2D部分将使嵌合蛋白特异性结合HPV psV感染和未感染的卵巢癌细胞，其中免疫原性CTL表位将通过弗林蛋白酶切割释放并包被肿瘤细胞，以被预先存在的CTL识别。此外，Fc的存在使得结合的肿瘤细胞易于受到体液免疫的攻击。总的来说，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、在肿瘤部位特异性弗林蛋白酶切割以及通过现有CTL免疫和Fc介导的杀伤的有效肿瘤靶向杀伤的优点，代表了具有高翻译价值的新策略。这项研究的成功实施将成为一种创新策略，可用于治疗其他类型的晚期癌症。

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.