GENE-MODIFIED DENDRITIC CELLS FOR TUMOR IMMUNOTHERAPY

描述：（申请人摘要）人乳头瘤病毒感染 (HPV)已经成为一个主要的公共卫生问题，因为他们的高 患病率、发病率增加和致癌性。 HPV介导的 致癌性已定位于E6和E7开放阅读框， 与90%以上的宫颈癌有关。 虽然E6和 从HPV转化细胞表达的E7可以作为有效的靶点， 动物模型中的细胞毒性反应，对HPV的天然细胞介导的免疫 在人类身上的表现出奇的差。 最近，树突状细胞（DC）已被 被鉴定为有效的专业APC，其刺激T细胞识别 并对特定抗原作出反应。 肽脉冲DC已被证明 在动物模型和一个人中产生有效的抗肿瘤细胞毒性反应 审判 树突状细胞与HLA联合提呈肽表位 分子，不同的HLA等位基因与不同的肽相关联。 然而，鉴定具有免疫原性表位的肽， 与HLA等位基因的大量排列相关， 人口是一项艰巨的任务。 因此，引入编码 将感兴趣的免疫原导入DC中，并随后选择最佳免疫原。 表位通过DC T细胞相互作用对于产生免疫应答是有吸引力的。 免疫力 然而，通过转染或逆转录病毒将基因转移到人DC中 由于它们的非增殖状态，转导已被证明是困难的。 腺相关病毒（AAV）载体最近已被鉴定为是高度非特异性的。 有希望的基因治疗工具，因为它们的宿主范围广，缺乏 细胞致病性，稳定整合， 非增殖细胞靶点。 申请人已证明持续 和有效转导原代人单核细胞-巨噬细胞，也就是APC 在功能和个体发育上与DC密切相关。 在这项研究中，她将 测试以下HLA限制性、抗原特异性CTL的诱导 腺相关病毒载体介导人乳头瘤病毒E7转化基因转染树突状细胞 转导 同基因转导的小鼠DC的体内移植 将测试对肿瘤攻击的保护作用和对先前的 现有的肿瘤以及用于诱导记忆反应。 最后， 来自HPV 16+宫颈癌患者的DC将用 AAV-E7并测试它们产生CTL的能力，所述CTL裂解自体免疫细胞。 肿瘤细胞 这些研究将1）测试肿瘤特异性 在AAV载体转导编码免疫原性的基因后， 免疫原转化为DC，2）潜在地简化和促进开发 分子疫苗，3）提供重要的临床前信息 关于AAV载体用于肿瘤免疫治疗的用途，并可能形成 为发展基于基因的免疫策略奠定了基础， 癌

DESCRIPTION: (Applicant's Abstract) Infection with human papilloma viruses (HPV) has become a major public health concern because of their high prevalence, increasing incidence, and oncogenicity. HPV-mediated oncogenicity has been mapped to the E6 and E7 open reading frames and has been associated with more than 90% of cervical carcinomas. Although E6 and E7 expressed from HPV-transformed cells can serve as effective targets for cytotoxic responses in animal models, natural cell mediated immunity to HPV in humans is surprisingly poor. Recently, dendritic cells (DC) have been identified as potent, professional APC which stimulate T cells to recognize and respond to specific antigens. Peptide pulsed DC have been shown to generate potent antitumor cytotoxic responses in animal models and one human trial. Peptide epitopes are presented by DC in association with HLA molecules, with different HLA alleles associating with different peptides. However, the identification of peptides with immunogenic epitopes which can be presented in association with the vast array of HLA alleles in the population is an arduous task. Thus the introduction of genes encoding the immunogen of interest into DC and the subsequent selection of optimal epitopes by DC T cell interactions is attractive for the generation of immunity. However gene transfer into human DC by transfection or retroviral transduction has proven difficult due to their non-proliferative status. Adeno-associated virus (AAV) vectors have recently been identified as highly promising vehicles for gene therapy because of their wide host range, lack of cytopathogenicity, stable integration, and ability to transduce nonproliferating cellular targets. The applicant has demonstrated sustained and efficient transduction of primary human monocyte-macrophages, also APCs closely related to DCs in function and ontogeny. In this study she will test the induction of HLA-restricted, antigen-specific CTL following introduction of the HPV E7 transforming gene into DC by AAV vector transduction. In vivo transplantation of syngeneic transduced murine DC will be tested for protection from tumor challenge and effects on previously existing tumors as well as for the induction of memory responses. Lastly, DCs from patients with HPV16+ cervical cancers will be transduced with AAV-E7 and tested for their ability to generate CTL which lyse autologous tumor cells. These studies will 1) test the generation of tumor-specific immunity following AAV vector transduction of the gene encoding the immunogen into DCs, 2) potentially simplify and facilitate the development of molecular vaccines, 3) provide important preclinical information regarding the use of AAV vectors for tumor immunotherapy and may form the groundwork for the development of gene-based immune strategies against cancer.