Antibody-mediated Gene Therapy for the Treatment of Cancer

抗体介导的癌症基因疗法

• 批准号: 7941907
• 负责人: Tracy Ruth Daniels-Wells
• 金额: $ 13.95万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-28 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941907
• 关键词: 5 fluorouridine; Adverse effects; Affect; Animal Model; Antibodies; Antibody-Directed Enzyme Prodrug Therapy; Applications Grants; Avidin; Award; B lymphoid malignancy; B-Cell Lymphomas; B-Cell NonHodgkins Lymphoma; B-Lymphocytes; Binding; Bystander Effect; Cancer Patient; Cell Death; Cell Line; Cell Nucleus; Cells; Cellular biology; Cessation of life; Chimeric Proteins; Complex; Cytosine; DNA; Development; Dimensions; Disease; Drug Kinetics; Endocytosis; Endosomes; Environment; Enzymes; Evaluation; Faculty; Flucytosine; Fluorouracil; Future; Gene Delivery; Gene Expression; Genes; Goals; Growth; Hematopoietic; Hematopoietic stem cells; Hodgkin Disease; Human; IgG3; Immunoglobulins; Immunotherapy; In Vitro; Institution; Knowledge; Lentivirus Vector; Lymphoma; Magic; Malignant - descriptor; Malignant Neoplasms; Mantle Cell Lymphoma; Mediating; Mentors; Messenger RNA; Mus; N glycosidase; Non-Hodgkin' s Lymphoma; Normal Cell; Oncogenes; Pain; Pharmaceutical Preparations; Plants; Positioning Attribute; Preparation; Prodrugs; Property; Protein Biosynthesis; Proteins; Public Health; RNA chemical synthesis; Rattus; Receptors, Antigen, B-Cell; Reporter Genes; Research; Research Personnel; Ribosomes; Role; SCID Beige Mouse; Saponaria; Species Specificity; Sprague-Dawley Rats; Stromal Cells; Subfamily lentivirinae; Surface; Survival Rate; System; Testing; Therapeutic; Tissues; Toxic effect; Toxin; Training; Transferrin Receptor; Transgenes; Translating; Travel; Tumor Antigens; Ubiquitin; United States; Uracil phosphoribosyltransferase; Xenograft procedure; Yeasts; base; cancer cell; cancer therapy; career development; cellular pathology; chimeric antibody; cytotoxicity; economic cost; effective therapy; experience; gene therapy; improved; in vivo; neoplastic cell; novel therapeutic intervention; novel therapeutics; outcome forecast; overexpression; promoter; public health relevance; receptor; receptor mediated endocytosis; selective expression; skills; standard of care; success; targeted delivery; therapeutic gene; transgene expression; tumor; vector

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. PUBLIC HEALTH RELEVANCE: Mantle Cell Lymphoma (MCL) is an aggressive and incurable form of B-cell lymphoma that has a very poor prognosis. Since there is no effective therapy for this malignancy, I propose a new combination of antibody-based and gene therapy strategies in which the antibodies will act as "magic bullets" and preferentially deliver therapeutic genes that can only be activated inside the targeted cancer cells. This strategy is expected to destroy MCL cells without affecting normal cells, eliminating the side effects of the treatment and contributing to a decrease in the human and economic cost associated with MCL.

描述（由申请人提供）：拟议的研究重点是新的高度靶向基因治疗方法的开发和表征，用于全身治疗侵袭性 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），其中在美国85%以上为NHL。 MCL 是 B 细胞 NHL 的一种侵袭性形式，预后非常差。 MCL占NHL病例的5-10%，中位生存期约为4年，长期生存率低于15%，近20年来没有明显变化。目前，MCL 的治疗尚无公认的护理标准，并且该疾病被认为是无法治愈的。因此，迫切需要新的治疗方法。在本申请描述的策略中，MCL靶向将通过两种机制发生：1)通过将肿瘤相关抗原（TAA)靶向恶性B细胞表面，以及2)通过使用细胞选择性表达毒性基因-特异性启动子。癌细胞表面的 TAA 是免疫治疗的绝佳靶点。因此，我的目标策略的第一级将通过使用转铁蛋白受体（TfR）特异性的小鼠/人嵌合抗体-亲和素融合蛋白来实现。该受体是癌症治疗的一个有吸引力的靶标，因为它在癌细胞表面表达升高、内化能力及其在癌症细胞病理学中的核心作用。然而，TfR 在一些正常细胞上以不同水平表达。为了进一步提高恶性细胞靶向性，我的靶向策略的第二层重点是通过使用免疫球蛋白启动子来限制毒性基因对恶性细胞的表达。本提议的中心假设是，MCL 表面的 TfR 过表达可用作 TfR 介导的基因传递的有效靶标，为此转基因将受到转录限制。由于肿瘤靶向将在两个层面上发生，我还假设这种策略在消除体内恶性 B 细胞方面将非常有效，并且不会产生限制大多数癌症治疗效果的严重副作用。靶向 TfR 的抗体-亲和素融合蛋白是一种独特的药物，因为它可作为多种生物素化药物的通用递送系统。 抗体-亲和素融合蛋白将与生物素化的 DNA 或生物素化的慢病毒载体缀合，以便通过受体介导的内吞作用将毒性基因递送到恶性 B 细胞中。本申请提出使用两种独立且非排他性的基因治疗策略。第一个基因编码毒素皂草素，一种源自植物皂草的核糖体失活蛋白。肥皂草素是一种单链毒素，由于缺乏细胞结合结构域，其本身无法进入细胞。皂草素具有剧毒，一旦进入细胞，它就会通过其 N-糖苷酶活性抑制蛋白质合成，从而导致 28S 核糖体亚基失活。将使用的第二个基因编码嵌合酵母酶 (FCU1)，该酶由胞嘧啶脱氨酶 (CD) 和尿嘧啶磷酸核糖基转移酶 (UPRT) 组成。该酶将前药 5-氟胞嘧啶转化为有毒代谢物 5-氟尿嘧啶 (5-FU) 和 5-氟尿苷 5' 单磷酸 (5-FUMP)，因此是一种抗体导向的酶前药疗法 (ADEPT) 方法。前药将在肿瘤微环境中转化为其有毒代谢物。预计 ADEPT 治疗将产生旁观者效应，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，这两种策略将来可以结合使用，以最大限度地发挥其抗肿瘤作用。与单一靶向药物相比，使用任一有毒基因的双重靶向策略预计将增加抗肿瘤活性，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： 目标 1：报告基因载体构建和基因递送的体外优化。目标2：毒性基因载体构建及靶向抗癌活性的体外评价。目标 3：在动物模型中评价毒性、药代动力学和抗肿瘤活性。 该项目预计不仅会在癌症基因治疗和 MCL 治疗领域取得重要进展，而且还会在我的职业发展方面取得重要进展。事实上，我将获得许多新技能，这将增加我的知识和研究经验。这次培训将有助于未来准备资助提案，使我能够成为领先机构学术教职职位的更好候选人，这是我的长期目标。我的目标是成为一名独立研究者，更好地了解癌细胞生物学，以便开发新的疗法，帮助减轻癌症患者所遭受的痛苦和痛苦。加州大学洛杉矶分校出色的研究环境、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实现我的目标，并将开启我的专业发展的新维度。 公共卫生相关性：套细胞淋巴瘤 (MCL) 是一种侵袭性且无法治愈的 B 细胞淋巴瘤，预后非常差。由于这种恶性肿瘤没有有效的治疗方法，我提出了一种基于抗体和基因治疗策略的新组合，其中抗体将充当“魔法子弹”，并优先传递只能在目标癌细胞内激活的治疗基因。该策略有望在不影响正常细胞的情况下破坏 MCL 细胞，消除治疗的副作用，并有助于降低与 MCL 相关的人力和经济成本。