Development of an in vivo screening technology for cancer vaccine immunogens

描述(由申请人提供)：该申请旨在解决RFA的任务，建议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，在高通量筛选模式下使用体内免疫系统。这项新技术的开发将汇集来自不同机构和学科、在各种互补技术方面经验丰富的多名调查人员。如果成功，这项新技术将为研究人员提供一项重要的新技术，以评估和比较癌症疫苗的免疫原，从而对肿瘤学产生重大影响。我们将开发这项新技术，然后在测试案例中应用这项新技术来识别对Survivin肿瘤相关抗原产生免疫反应的免疫原，Survivin肿瘤相关抗原是一种有吸引力且被广泛研究的肿瘤疫苗候选，由于其在癌症疫苗模型中显示出活性且相对较小，因此适用于这项发展中的原则证明研究。这项技术利用完整的胃肠道粘膜免疫系统作为大规模平行的活体筛选设备。为了开发这项新的筛选技术，我们将：1)创建用DNA条码质粒转化的细菌库，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，其中将包括编码来自Survivin的详尽、重叠的多肽文库的序列；2)将文库喂给小鼠；3)使用PhyloChip微阵列技术(或高通量测序)来通过条形码鉴定小鼠粪便中相对丰度随时间降低的文库成员，我们将此视为针对由显示丰度降低的质粒编码的嵌合蛋白而诱导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，并评估接种了表达这些蛋白的细菌的动物的血清和细胞，以及用筛选中确定的蛋白免疫的动物，以确定它们是否诱导抗Survivin体液和细胞免疫反应。克隆和克隆编码的能够诱导抗Survivin反应的多肽将在体内肿瘤疫苗模型中进行研究，并与模型中已知的诱导抗肿瘤免疫反应的多肽进行比较。这项研究建议以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可用作癌症疫苗的潜在免疫原。如果开发成功，这项技术也将有助于为癌症疫苗寻找其他免疫原，并比较不同候选蛋白质/多肽的免疫原性。由于这些蛋白质是在包括沙门氏菌疫苗株在内的革兰氏阴性细菌中表达的，这项技术可以用来快速、廉价地生产候选疫苗。尽管该项目风险很大，但该项目可能会对癌症免疫疗法产生非常大的影响。

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.