Development of an in vivo screening technology for cancer vaccine immunogens

描述（由申请人提供）：本申请通过提出开发一种高度新颖且以前未经测试的技术来识别、评估和比较癌症疫苗的免疫原，从而解决了RFA的任务，采用高通量筛选模式的体内免疫系统。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一种评估和比较癌症疫苗免疫原的重要新技术，对肿瘤学产生重大影响。我们将开发这项新技术，然后在一个测试案例中使用这项新技术来识别免疫原，这些免疫原会引发对存活肿瘤相关抗原的免疫反应，这是一种有吸引力的、被广泛研究的肿瘤疫苗候选物，由于其在癌症疫苗模型中已被证明具有活性，而且体积相对较小，因此对这项发展性原理验证研究很有用。该技术利用完整的胃肠道黏膜免疫系统作为大规模平行体内筛选装置。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，其中包括在革兰氏阴性细菌表面放置大量嵌合蛋白的表达盒，其中包括编码来自survivin的完整重叠肽库的序列，2)将文库喂养小鼠，3)使用PhyloChip微阵列技术（或高通量测序）通过条形码识别，随着时间的推移，文库成员在小鼠粪便中显示相对丰度下降，我们将以此作为诱导粘膜免疫反应的证据，以对抗丰度下降的质粒编码的嵌合蛋白。我们将重新筛选克隆以确认其诱导免疫反应的能力，并评估用表达该蛋白的细菌接种动物的血清和细胞，以及用筛选中鉴定的蛋白质免疫动物的血清和细胞，以确定它们是否诱导抗survivin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反应的克隆和克隆编码的肽，并将其与模型中已知的诱导抗肿瘤免疫反应的肽进行比较。本研究提出以survivin肿瘤相关抗原为模型，开发一种创新、快速、高通量的方法来鉴定潜在的可作为癌症疫苗的免疫原。如果开发成功，该技术还可用于寻找用于癌症疫苗的其他免疫原以及比较不同候选蛋白/肽的免疫原性。由于这些蛋白在革兰氏阴性细菌中表达，包括沙门氏菌的疫苗菌株，因此该技术可用于快速而廉价地生产候选疫苗。尽管该项目有风险，但该项目可能因此对癌症免疫治疗产生非常大的影响。

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.