Development of an in vivo screening technology for cancer vaccine immunogens

癌症疫苗免疫原体内筛选技术的开发

• 批准号: 8508685
• 负责人: Steven L. Zeichner
• 金额: $ 23.02万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-19 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8508685
• 关键词: Address; Animals; Antibody Formation; Antigens; Bacteria; Bar Codes; Cancer Vaccines; Cell Extracts; Cells; Chimeric Proteins; Cloning; Computer software; DNA; DNA biosynthesis; Development; Devices; Discipline; Doctor of Philosophy; Ecology; Escherichia coli; Feces; Gastrointestinal tract structure; Genes; Genetic Engineering; Genomics; Gram-Negative Bacteria; Immune; Immune response; Immune system; Institution; Knowledge; Libraries; Life; Malignant Neoplasms; Microarray Analysis; Modeling; Monitor; Mucosal Immune Responses; Mus; Peptide Library; Peptide Vaccines; Peptides; Plasmids; Principal Investigator; Procedures; Production; Proteins; Recombinants; Relative (related person); Research Personnel; Salmonella; Salmonella Vaccines; Serum; Site; Study of serum; Surface; System; Techniques; Technology; Testing; Theoretical model; Therapeutic; Time; Tumor Antigens; Tumor Cell Derivative Vaccine; Vaccines; base; cancer immunotherapy; cancer therapy; comparative efficacy; design; experience; expression vector; feeding; gastrointestinal; genetic analysis; high risk; high throughput screening; immunogenic; immunogenicity; improved; in vivo; innovation; insight; member; microbial; microbiome; mouse model; new technology; novel; novel vaccines; oncology; programs; rRNA Genes; research study; response; screening; survivin; synthetic biology; technology development; tumor; vaccine candidate

DESCRIPTION (provided by applicant): This application addresses the mandates of the RFA by proposing to develop a highly novel and previously untested technology to identify, assess, and compare immunogens for cancer vaccines, employing an in vivo immune system in a high throughput screening mode. The development of the new technology will bring together multiple investigators experienced in a wide variety of complementary techniques, from different institutions and disciplines. If successful, the new technology will have great impact on oncology by providing investigators with an important new technology to evaluate and compare immunogens for cancer vaccines. We will develop and then employ this new technology in a test case to identify immunogens that elicit immune responses to the survivin tumor-associated antigen, an attractive and widely studied tumor vaccine candidate useful for this developmental proof-of-principle study due to its demonstrated activity in cancer vaccine models and relatively small size. The technology exploits an intact GI mucosal immune system as a massively parallel in vivo screening device. To develop this new screening technology we will: 1) Create a library of bacteria transformed with DNA bar-coded plasmids that include expression cassettes that place large amounts of chimeric protein, which will include sequences encoding an exhaustive, overlapping library of peptides derived from survivin, on the Gram-negative bacterial surface, 2) Feed the library to mice, 3) Use PhyloChip microarray technologies (or high throughput sequencing) to identify, via the barcodes, members of the library showing decreased relative abundance in the mouse feces over time, which we would take as evidence of the induction of a mucosal immune response against the chimeric proteins encoded by plasmids showing decreased abundance. We will rescreen the clones to confirm their ability to induce an immune response and evaluate sera and cells from animals inoculated with bacteria expressing the proteins and animals immunized with the proteins identified in the screen to determine if they induce anti-survivin humoral and cellular immune responses. Clones and the peptides encoded by the clones able to induce an anti-survivin response will be studied in an in vivo tumor vaccine model and compared to peptides already known to induce an anti-tumor immune response in the model. The study proposes to develop an innovative, rapid, high-throughput approach to the identification of potential immunogens useful as cancer vaccine employing the survivin tumor associated antigen as a model. If successfully developed, the technology also would be useful in the search for other immunogens for cancer vaccines and for comparing the immunogenicity of different candidate proteins/peptides. Since the proteins are expressed in Gram-negative bacteria, including vaccine strains of Salmonella, the technology could be used to rapidly and cheaply produce a candidate vaccine. Although the project is risky, the project could therefore potentially have a very large impact on cancer immunotherapy.

描述（由申请人提供）：本申请通过提议开发一种高度新颖且之前未经测试的技术来识别、评估和比较癌症疫苗的免疫原，并在高通量筛选模式下采用体内免疫系统，从而解决了 RFA 的要求。新技术的开发将汇集来自不同机构和学科的、在各种互补技术方面经验丰富的多名研究人员。如果成功，这项新技术将为研究人员提供评估和比较癌症疫苗免疫原的重要新技术，从而对肿瘤学产生巨大影响。我们将开发并在测试用例中使用这项新技术，以识别可引发对存活蛋白肿瘤相关抗原的免疫反应的免疫原，这是一种有吸引力且经过广泛研究的肿瘤疫苗候选物，可用于这项开发原理验证研究，因为它已被证明癌症疫苗模型中的活性和相对较小的尺寸。该技术利用完整的胃肠道粘膜免疫系统作为大规模并行的体内筛查装置。为了开发这种新的筛选技术，我们将：1) 创建一个用 DNA 条形码质粒转化的细菌文库，其中包括放置大量嵌合蛋白的表达盒，其中包括编码源自生存素的详尽重叠肽文库的序列，在革兰氏阴性细菌表面，2) 将文库喂给小鼠，3) 使用 PhyloChip 微阵列技术（或高通量测序）通过条形码识别小鼠粪便中相对丰度下降的文库成员随着时间的推移，我们将其作为诱导针对由丰度下降的质粒编码的嵌合蛋白的粘膜免疫反应的证据。我们将重新筛选克隆，以确认它们诱导免疫反应的能力，并评估接种表达蛋白质的细菌的动物和接种筛选中鉴定的蛋白质的动物的血清和细胞，以确定它们是否诱导抗生存素体液和细胞免疫反应。将在体内肿瘤疫苗模型中研究能够诱导抗生存素应答的克隆和由克隆编码的肽，并与已知在模型中诱导抗肿瘤免疫应答的肽进行比较。该研究拟开发一种创新、快速、高通量的方法，以生存素肿瘤相关抗原为模型，鉴定可用作癌症疫苗的潜在免疫原。如果成功开发，该技术还将可用于寻找癌症疫苗的其他免疫原以及比较不同候选蛋白质/肽的免疫原性。由于这些蛋白质在革兰氏阴性细菌（包括沙门氏菌疫苗株）中表达，因此该技术可用于快速且廉价地生产候选疫苗。尽管该项目存在风险，但该项目可能因此对癌症免疫治疗产生非常大的影响。