Antibody-mediated Gene Therapy for the Treatment of Cancer

抗体介导的癌症基因疗法

• 批准号: 8319671
• 负责人: Tracy Ruth Daniels-Wells
• 金额: $ 14.27万
• 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/8319671
• 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 Line; Cells; Cellular biology; Chimeric Proteins; Cytosine; DNA; Development; Dimensions; Disease; Drug Kinetics; Environment; Enzymes; Evaluation; Faculty; Flucytosine; Fluorouracil; Future; Gene Delivery; Gene Expression; Genes; Goals; Growth; Health; 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; Mus; N glycosidase; Non-Hodgkin' s Lymphoma; Normal Cell; Oncogenes; Pain; Pharmaceutical Preparations; Plants; Positioning Attribute; Preparation; Prodrugs; Property; Protein Biosynthesis; Proteins; Rattus; Receptors, Antigen, B-Cell; Reporter Genes; Research; Research Personnel; Role; SCID Beige Mouse; Saponaria; Species Specificity; Sprague-Dawley Rats; Stromal Cells; Surface; Survival Rate; System; Testing; Therapeutic; Toxic effect; Toxin; Training; Transferrin Receptor; Transgenes; 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; novel therapeutic intervention; novel therapeutics; outcome forecast; overexpression; promoter; 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.

描述（由申请人提供）：拟议的研究着重于用于全身治疗侵袭性B细胞淋巴瘤的新型高度靶向基因治疗方法的开发和表征，重点是地幔细胞淋巴瘤（MCL）。将淋巴瘤细分为霍奇金的淋巴瘤（HL）和非霍奇金淋巴瘤（NHL），在美国，NHL超过85％。 MCL是B细胞NHL的一种侵略性形式，预后较差。 MCL占NHL病例的5-10％，中位存活率约为4年，长期存活率少于15％，在过去20年中没有显着变化。当前，尚无接受MCL治疗的公认护理标准，并且该疾病被认为无法治愈。因此，迫切需要新颖的治疗方法。在本应用中描述的策略中，MCL靶向将通过两种机制进行：1）通过使用细胞特异性启动子选择性表达有毒基因，通过在恶性B细胞表面靶向肿瘤相关的抗原（TAA）和2）。癌细胞表面的TAA是免疫疗法的极好靶标。因此，我的靶向策略的第一级将通过使用针对转铁蛋白受体（TFR）的小鼠/人嵌合抗体 - 阿维丁蛋白融合蛋白发生。由于其在癌细胞表面的表达升高，其内在化的能力以及其在癌症的细胞病理学中的核心作用，因此该受体是癌症治疗的有吸引力的靶标。但是，TFR在某些正常细胞上以各种水平表达。为了进一步改善恶性细胞靶向，我的靶向策略的第二级集中于通过使用免疫球蛋白启动子将有毒基因的表达限制为恶性细胞。本提案的中心假设是MCL表面上的TFR过表达可以用作TFR介导的基因输送的有效靶标，该基因递送将在该基因上进行转录限制。由于靶向肿瘤将在两个层面上发生，因此我还假设该策略将在消除体内恶性B细胞中非常有效，而没有严重的副作用，从而限制了大多数癌症治疗剂的功效。靶向TFR的抗体 - 阿维丁融合蛋白是一种独特的药物，因为它是多种生物素化剂的通用递送系统。 为了通过受体介导的内吞作用，将抗体 - 阿维丁蛋白融合蛋白偶联与生物素化的DNA或生物素化的慢病毒载体。在本应用中提出了两种独立和非排他性基因治疗策略的使用。第一个基因编码毒素糖蛋白，这是一种核糖体灭活蛋白，源自植物saponaria officinialis。 Saporin是一种单链毒素，由于缺乏细胞结合域而无法单独进入细胞。 Saporin是一种剧毒，一旦在细胞内，它就会通过其N-糖苷酶活性抑制蛋白质的合成，从而导致28S核糖体亚基的失活。第二种将使用的基因编码由胞嘧啶二氨基（CD）和尿嘧啶磷酸糖基转移酶（UPRT）组成的嵌合酵母酶（FCU1）。该酶将前药5-氟环肽转化为有毒的代谢物5-氟尿嘧啶（5-FU）（5-FU）和5-氟尿嘧啶5'Monophophate（5-fump），因此是一种抗体 - 定向的酶Proprug Therapy（Adept）方法。前药将在肿瘤微环境中转化为其有毒代谢产物。预计可以从靶向细胞中释放出毒性代谢产物，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释放出来的旁观者效应。重要的是，将来可以使用这两种策略来最大程度地发挥其抗肿瘤作用。与单个靶向药物相比，使用这种双重靶向策略的使用有望增加抗肿瘤活性，并消除了治疗的潜在全身毒性。要执行该项目，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：有毒基因载体的构建和靶向抗癌活性的体外评估。目标3：在动物模型中评估毒性，药代动力学和抗肿瘤活性。 预计该项目将不仅在癌症基因疗法和MCL治疗领域都带来重要进展，而且还会在我的职业发展中取得重要的进步。实际上，将获得许多新技能，以增加我的知识和研究经验。这项培训将有助于将来准备赠款提案，这将使我成为领先机构的学术职位的更好候选人，这是我的长期目标。我的目标是成为一名独立研究者，以更好地了解癌细胞生物学，以开发新的治疗剂，以减轻癌症患者遇到的疼痛和痛苦。加州大学洛杉矶分校（UCLA）的杰出研究环境，我经验丰富的导师的指导以及该奖项将极大地促进我成功实现目标的成功，并将在我的专业发展中开辟新的方面。