Antibody-mediated Gene Therapy for the Treatment of Cancer

描述(由申请人提供)：拟议的研究重点是开发和表征新的高靶向性基因治疗方法，用于系统治疗侵袭性B细胞淋巴瘤，重点是套细胞淋巴瘤(MCL)。淋巴瘤分为霍奇金淋巴瘤(HL)和非霍奇金淋巴瘤(NHL)，在美国，超过85%的淋巴瘤是NHL。MCL是一种侵袭性B细胞非霍奇金淋巴瘤，预后差。MCL占NHL病例的5%-10%，中位生存期约4年，长期生存率不到15%，在过去20年中没有明显变化。目前，对于MCL的治疗没有公认的治疗标准，这种疾病被认为是不治之症。因此，迫切需要新的治疗方法。在本申请中描述的策略中，MCL靶向将通过两种机制发生：1)通过靶向恶性B细胞表面的肿瘤相关抗原(TAA)；2)通过使用细胞特异性启动子选择性表达有毒基因。癌细胞表面的TAA是免疫治疗的极佳靶点。因此，我的第一级靶向策略将通过使用转铁蛋白受体(TFR)特异性的鼠/人嵌合抗体-亲和素融合蛋白来实现。这种受体由于其在癌细胞表面的高表达、内化的能力以及在癌症细胞病理中的中心作用而成为癌症治疗的一个有吸引力的靶点。然而，TFR在一些正常细胞上有不同程度的表达。为了进一步提高恶性细胞的靶向性，我的靶向策略的第二个层面专注于通过使用免疫球蛋白启动子来限制有毒基因对恶性细胞的表达。本研究的中心假设是MCL表面TFR的过度表达可以作为TFR介导的基因传递的有效靶点，从而使转基因受到转录上的限制。由于肿瘤靶向将在两个水平上发生，我还假设这种策略将在体内消除恶性B细胞，而不会出现限制大多数癌症治疗效果的严重副作用。针对TFR的抗体-亲和素融合蛋白是一种独特的药物，因为它是各种生物素化试剂的通用递送系统。 抗体-亲和素融合蛋白将连接到生物素标记的DNA或生物素化的慢病毒载体上，通过受体介导的内吞作用将有毒基因传递到恶性B细胞。在本申请中提出了两种独立的和非排他性的基因治疗策略。第一个基因编码Saporin毒素，这是一种来自植物防风的核糖体失活蛋白。皂苷是一种单链毒素，由于缺乏细胞结合域而不能自行进入细胞。皂苷是一种剧毒物质，一旦进入细胞，它就会通过N-糖苷酶活性抑制蛋白质合成，导致28S核糖体亚单位失活。第二个基因将编码一种嵌合酵母酶(FCU1)，它由胞嘧啶脱氨酶(CD)和尿嘧啶磷酸核糖转移酶(UPRT)组成。这种酶将前体药物5-氟胞嘧啶转化为毒性代谢产物5-氟尿嘧啶(5-FU)和5-氟尿嘧啶5‘-单磷酸(5-FUMP)，因此是一种抗体导向的酶前药疗法(ADEPT)。前药将在肿瘤微环境中转化为有毒代谢物。预计将会出现与熟练治疗相关的旁观者效应，因为有毒代谢物可以从靶细胞中释放出来，并被肿瘤环境中的非靶向恶性细胞以及支持恶性细胞生长的基质细胞吸收。重要的是，这两种策略可以在未来结合使用，以最大限度地发挥其抗肿瘤作用。与单靶向药物相比，使用这种使用任何一种有毒基因的双重靶向策略有望增加抗肿瘤活性，并消除治疗的潜在全身毒性。为了执行这个项目，我提出了三个具体目标： 目的1：报告基因载体的构建及体外基因传递的优化。目的：构建毒性基因载体并体外评价其靶向抗癌活性。目的3：在动物模型中评价其毒性、药代动力学和抗肿瘤活性。 这个项目不仅有望在癌症基因治疗和MCL治疗领域取得重要进展，而且还将在我的职业发展方面取得重要进展。事实上，我将获得许多新的技能，这些技能将增加我的知识和研究经验。这一培训将有助于未来准备拨款提案，使我成为一家领先机构的学术教员职位的更好候选人，这是我的长期目标。我的目标是成为一名独立的研究员，更好地了解癌细胞生物学，以便开发新的治疗方法，有助于减轻癌症患者遇到的痛苦和痛苦。加州大学洛杉矶分校出色的研究环境，我经验丰富的导师的指导，以及这个奖项将极大地促进我成功地实现我的目标，并将开启我职业发展的新维度。

DESCRIPTION (provided by applicant): The proposed studies focus on the development and characterization of new highly targeted gene therapy approaches for the systemic treatment of aggressive B-cell lymphomas with an emphasis on mantle cell lymphoma (MCL). Lymphomas are subdivided into Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), of which in the United States more than 85% are NHL. MCL is an aggressive form of B-cell NHL with a very poor prognosis. MCL comprises 5-10% of NHL cases, has a median survival of about 4 years, and a long- term survival rate of less than 15%, which has not significantly changed in the past 20 years. Currently, there is no accepted standard of care for the treatment of MCL and the disease is considered incurable. Therefore, novel therapeutic approaches are urgently needed. In the strategies described in this application, MCL targeting will occur via two mechanisms: 1) through the targeting of a tumor-associated antigen (TAA) on the surface of malignant B cells and 2) through the selective expression of toxic genes using a cell-specific promoter. TAA on the surface of cancer cells serve as excellent targets for immunotherapy. Therefore, the first level of my targeted strategy will occur through the use of a mouse/human chimeric antibody-avidin fusion protein specific for the transferrin receptor (TfR). This receptor is an attractive target for cancer therapy due to its elevated expression on the surface of cancer cells, its ability to internalize, and its central role in the cellular pathology of cancer. However, the TfR is expressed on some normal cells at various levels. In order to further improve malignant cell targeting, the second level of my targeted strategy focuses on limiting the expression of toxic genes to malignant cells by using the immunoglobulin promoter. The central hypothesis of the present proposal is that TfR overexpression on the surface of MCL can be used as an effective target for TfR- mediated gene delivery, for which the transgene will be transcriptionally restricted. Since tumor targeting will occur on two levels, I also hypothesize that this strategy will be extremely effective in eliminating malignant B cells in vivo without the severe side effects that limit the efficacy of most cancer therapeutics. The antibody-avidin fusion protein that targets the TfR is a unique drug since it serves as a universal delivery system for a wide variety of biotinylated agents. The antibody-avidin fusion protein will be conjugated to either biotinylated DNA or biotinylated lentiviral vectors in order to deliver a toxic gene into malignant B cells by receptor-mediated endocytosis. The use of two independent and non-exclusive gene therapy strategies is proposed in this application. The first gene encodes the toxin saporin, a ribosomal inactivating protein that is derived from the plant Saponaria officinialis. Saporin is a single chain toxin that cannot enter cells by itself due to the lack of a cell-binding domain. Saporin is a highly toxic and once inside the cell it inhibits protein synthesis through its N-glycosidase activity that leads to the inactivation of the 28S ribosomal subunit. The second gene that will be used encodes a chimeric yeast enzyme (FCU1) that consists of cytosine deaminse (CD) and uracil phosphoribosyltransferase (UPRT). This enzyme converts the prodrug 5-fluorocytosine to the toxic metabolites 5-fluorouracil (5-FU) and 5-fluorouridine 5'monophosphate (5-FUMP) and thus is an antibody- directed enzyme prodrug therapy (ADEPT) approach. The prodrug will be converted to its toxic metabolites within the tumor microenvironment. It is expected there will be a bystander effect associated with ADEPT therapy since the toxic metabolites can be released from targeted cells and taken up by non-targeted malignant cells in the tumor environment as well as stromal cells that support the growth of the malignant cells. Importantly, these two strategies can be used in the future in combination to maximize their anti-tumor effects. The use of this dual targeting strategy using either toxic gene is expected to increase the anti-tumor activity compared to singularly targeted agents, as well as eliminate the potential systemic toxicity of the treatment. To execute this project I propose three specific aims: Aim 1: Reporter gene vector construction and in vitro optimization of gene delivery. Aim 2: Toxic gene vector construction and in vitro evaluation of targeted anti-cancer activity. Aim 3: Evaluation of toxicity, pharmacokinetics, and anti-tumor activity in animal models. This project is expected to result in important advances not only in the fields of cancer gene therapy and treatment of MCL, but also in my career development. In fact, many new skills will be acquired that will increase my knowledge and research experience. This training will aid in the future preparation of grant proposals that will allow me to become a better candidate for an academic faculty position at a leading institution, which is my long-term goal. It is my goal to become an independent investigator to better understand cancer cell biology in order to develop new therapeutics that will help reduce the pain and suffering encountered by cancer patients. The outstanding research environment at UCLA, the guidance from my experienced mentors, and this award will greatly facilitate my success in reaching my goals and will open a new dimension in my professional development.