Antibody-mediated Gene Therapy for the Treatment of Cancer

抗体介导的癌症基因疗法

• 批准号: 8519364
• 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/8519364
• 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; tumor microenvironment; 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（由申请方提供）：拟定研究的重点是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））的新型高靶向基因治疗方法。淋巴瘤被细分为霍奇金淋巴瘤（HL）和非霍奇金淋巴瘤（NHL），其中在美国超过85%是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%，在过去20年中没有显著变化。目前，MCL的治疗没有公认的标准护理，并且该疾病被认为是不可治愈的。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细胞表面上的肿瘤相关抗原（TAA）和2）通过使用细胞特异性启动子选择性表达毒性基因。癌细胞表面的TAA是免疫治疗的极好靶点。因此，我的靶向策略的第一个层面将通过使用转铁蛋白受体（TfR）特异性的小鼠/人嵌合抗体-抗生物素蛋白融合蛋白来实现。该受体是癌症治疗的有吸引力的靶标，因为其在癌细胞表面上的表达升高、其内化能力以及其在癌症的细胞病理学中的中心作用。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，我的靶向策略的第二个层次集中在通过使用免疫球蛋白启动子限制恶性细胞的毒性基因的表达。本建议的中心假设是MCL表面上的TfR过表达可用作TfR介导的基因递送的有效靶标，为此转基因将受到转录限制。由于肿瘤靶向将发生在两个层面上，我还假设这种策略将在消除体内恶性B细胞方面非常有效，而没有限制大多数癌症治疗效果的严重副作用。靶向TfR的抗体-抗生物素蛋白融合蛋白是一种独特的药物，因为它作为多种生物素化试剂的通用递送系统。 抗体-抗生物素蛋白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。在本申请中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，这是一种核糖体失活蛋白，来源于植物皂草。皂草素是一种单链毒素，由于缺乏细胞结合结构域，其本身不能进入细胞。皂草素是一种高毒性物质，一旦进入细胞，它通过其N-糖苷酶活性抑制蛋白质合成，导致28 S核糖体亚基失活。将使用的第二个基因编码由胞嘧啶脱氨酶（CD）和尿嘧啶磷酸核糖基转移酶（UPRT）组成的嵌合酵母酶（FCU 1）。该酶将前药5-氟胞嘧啶转化为毒性代谢物5-氟尿嘧啶（5-FU）和5-氟尿嘧啶苷5 '单磷酸（5-FUMP），因此是抗体导向的酶前药治疗（ADEPT）方法。前药将在肿瘤微环境中转化为其毒性代谢物。预期将存在与ADEPT治疗相关的旁观者效应，因为毒性代谢物可从靶细胞释放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摄取。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。与单一靶向药物相比，使用任一毒性基因的这种双重靶向策略的使用预期会增加抗肿瘤活性，并消除治疗的潜在全身毒性。为了执行这个项目，我提出三个具体目标： 目的1：报告基因载体的构建及体外基因递送的优化。目的2：构建毒力基因载体并进行靶向抗癌活性的体外评价。目的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，而且有望在我的职业发展中取得重要进展。事实上，许多新的技能将获得，这将增加我的知识和研究经验。这项培训将有助于未来准备赠款提案，使我能够成为领先机构学术教师职位的更好候选人，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，以便开发新的治疗方法，帮助减轻癌症患者遇到的疼痛和痛苦。加州大学洛杉矶分校出色的研究环境，来自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实现我的目标，并将在我的专业发展中开辟一个新的层面。