Nano and biomolecular engineered technologies for neoantigen-specific T cell capture and characterization

项目总结 许多癌症免疫治疗都集中在改造或激活肿瘤抗原特异性CD8T细胞上， 在较小程度上，CD4T细胞。特别是，新抗原特异性T细胞之所以吸引人，是因为它们可以杀死 肿瘤细胞具有高度的特异性。一般的方法是从识别新抗原的T细胞开始。 在肿瘤内广泛表达，分离T细胞并确定其T细胞受体(TCR)序列。 然后，这些TCR可以被导入患者的T细胞，或许可以通过额外的基因工程来促进 更持久的抗肿瘤作用，并扩展为用于患者治疗的输液产品。3事实上，这是 方法最近进入临床，一项试验(NCT03970382)借鉴了NCI的发明- 由PPI牵头的CCNE U54基金的这项建议。4、5然而，仍然有一些基本的和 与推进新抗原特异性TCR工程疗法相关的技术挑战。首先， 新抗原特异性TCR的发现依赖于来自算法的指导，例如Net MHCpan4.1，以预测 抗原/MHC呈递(基于结合和其他考虑)，以及产生于 主干突变，如突变的KRAS或突变的TP53，被预测为不太可能，但已有报道称 临床有效靶点。6，7，8秒，新抗原特异性的CD4T细胞及其II类限制性新抗原， 虽然9，10在免疫治疗诱导的抗肿瘤反应中很重要，但在很大程度上仍未被开发 治疗资源，对于II类抗原/MHC结合的预测算法11，12开发得较少。三分之一 挑战在于，对患者血液中新抗原特异性T细胞的分析通常需要超过20M 外周血单核细胞(PBMC)，因此并不是特别有效。在此，我们提出了三个具体目标 旨在解决这些悬而未决的问题。技术解决方案的核心是组合 设计用于高效和选择性的工程纳米颗粒(NPs)和生物分子工程结构 捕获、分析和验证躯干新抗原特异性的CD4和CD8T细胞群。意义重大 提供了所有3个AIM的初步数据，其中一些使用了我们工作生成的新冠肺炎患者数据 在当前的大流行期间。13，14这项工作的结果将是为最小偏见的人设计的强大的工具集 寻找抗主干肿瘤抗原的CD4和CD8 T细胞群(独立于患者的人类白细胞抗原 单倍型)，为快速验证和表征这些新抗原特异性T细胞而设计的工具包 克隆类型，以及I类和II类干新抗原和T细胞受体基因特异性的公共数据库 对这些新抗原的反应。

Project summary Much of cancer immunotherapy is focused on engineering or activating tumor-antigen specific CD8+ T cells and, to a lesser extent, CD4+ T cells. In particular, neoantigen-specific T cells are attractive because they can kill cancer cells with high specificity. 1 A general approach starts with identifying T cells that recognize neoantigens broadly expressed within the tumor, isolating the T cells and determining their T cell receptor (TCR) sequences. These TCRs can then be transfected into patient T cells, perhaps with additional genetic engineering2 to promote more durable anti-tumor effects, and expanded into an infusion product for patient treatment. 3 In fact, this approach has recently entered the clinic, with one trial (NCT03970382) drawing from inventions from an NCI- funded CCNE U54 grant led by the PI of this proposal. 4,5 However, there are still a number of fundamental and technological challenges associated with advancing neoantigen-specific TCR-engineered therapies. First, the discovery of neoantigen-specific TCRs relies on guidance from algorithms, such as NET MHCpan 4.1, to predict antigen/MHC presentation (based upon binding and other considerations), and many neoantigens arising from truncal mutations, such as mutant KRAS or mutant TP53, are predicted as unlikely, yet have been reported as clinically effective targets. 6,7,8 Second, neoantigen-specific CD4+ T cells and their class II restricted neoantigens, while identified as important for immunotherapy-induced anti-tumor responses, 9,10 remain a largely untapped therapeutic resource, with prediction algorithms 11,12 for Class II antigen/MHC binding less developed. A third challenge is that analysis of a patient blood for neoantigen-specific T cells typically requires upwards of 20M peripheral blood mononuclear cells (PBMCs), and so isn't particularly efficient. Here we propose 3 specific Aims designed to address these outstanding issues. At the heart of the technology solutions are combinations of engineered nanoparticles (NPs) and biomolecular engineered constructs designed for efficient and selective capture, analysis, and validation of truncal neoantigen-specific CD4+ and CD8+ T cell populations. Significant preliminary data is presented for all 3 Aims, some of which uses COVID-19 patient data generated by our work during the current pandemic. 13,14 The result of this work will be a powerful toolset designed for a minimally-biased search for CD4+ and CD8+ T cell populations against truncal neoantigens (independent of patient HLA haplotype), a toolset designed for the rapid validation and characterization of those neoantigen-specific T cell clonotypes, and a public data base of Class I and Class II truncal neoantigens and T cell receptor genes specific to those neoantigens.