Unbiased electrothermal flow-enhanced identification of antigen-specific T cells in lung cancer

项目摘要/摘要 尽管免疫疗法取得了成功，但它们未能在抗原依赖的基础上调动T细胞，而是选择了 根据T细胞的位置或表型重新激活未知特异性的广谱T细胞。这样的标记 由于PD-1已被认为是描绘抗肿瘤T细胞，但也表达在那些识别非 肿瘤抗原。同样，肿瘤驻留的T细胞被推测表现出肿瘤抗原识别，尽管它们 我们和其他人已经证明也识别病毒和细菌抗原(即。旁观者T细胞)。MHC 多聚体为识别抗肿瘤T细胞提供了一种有效的替代方案，但受到前向需求的限制 鉴定感兴趣的抗原(S)。由于缺乏对抗原的了解，这种局限性变得更加严重。 免疫原性和识别它们的T细胞。肿瘤突变影响的最新发现 负担(TMB)加强了我们对肿瘤抗原来源的把握，尽管TMB未能始终如一地 预测临床结果。这一点最好的缩影可能是令人沮丧的抗原确认率徘徊在 ~1%。因此，我们了解哪些T细胞在实体瘤中显示出治疗抗肿瘤的潜力 仍然是有问题的。因此，迫切需要能够识别反歧视的不偏不倚的方法 肿瘤T细胞反应的缺乏将严重阻碍实体肿瘤免疫治疗的进展。 我们之前开发了ATTACH(评估与I/II类抗原组织相容性相关的T细胞)，a 强大的微流控检测技术，可利用人类白细胞抗原/多肽结合直接分离抗肿瘤T细胞 T细胞受体(TCR)对相匹配的肿瘤细胞的亲和力实际上是“四聚体池”。在这里，我们建议 微流控装置附着器(电热流强化回收附着器)的研制 能够简化这一检测，同时提高敏感性、特异性和抗原特异性T细胞回收。 我们的中心假设是，识别多种肿瘤抗原的T细胞可以在体外用 一种T细胞附着物，并且这些细胞与原生T细胞相比具有更强的抗肿瘤细胞毒活性 细胞。我们根据对依恋能力的初步研究提出了这一假说 增加肿瘤浸润性淋巴细胞(TIL)的细胞毒活性。建议的理由是 研究表明，通过开发T细胞附着器，将电热流应用于附着物将促进T细胞 细胞/肿瘤细胞接触，并允许T细胞扫描多个MHC/抗原复合体，以遇到其 同源抗原。在目标1中，我们将设计和建造T细胞附着器，并评估其恢复能力 在20种不同的人类抗原和不同亲和力的TCR上形成的抗原特异性T细胞 我们的实验室。在目标2中，我们将鉴定使用T细胞贴壁器分离的TIL的表型和 从功能上使用配对的人患者来源的异种移植和来自ICON的肿瘤浸润性淋巴细胞 一群人。我们预计，我们的工作将使我们能够展示T细胞附着者的多功能性及其能力 提高体外抗肿瘤反应。总体而言，我们的工作将允许直接、准确和不偏不倚地识别 在几小时内将抗肿瘤T细胞转移到实体肿瘤中，而不是目前所要求的几个月，奠定了 为后续研究这种方法在实体肿瘤中的预测和治疗潜力奠定了基础。

Project Abstract/Summary Despite their success, immunotherapies fail to engage T cells on an antigen-dependent basis rather opting to reactivate a broad spectrum of T cells of unknown specificity based on their location or phenotype. Markers such as PD-1 have been posited to delineate anti-tumor T cells but are also expressed on those recognizing non- tumor antigens. Likewise, tumor-resident T cells are surmised to exhibit tumor antigen recognition though they have been shown by us and others to also recognize viral and bacterial antigens (ie. bystander T cells). MHC multimers present a potent alternative to identify anti-tumor T cells but are limited by a need for anterior identification of the antigen(s) of interest. This limitation is compounded by a lack of understanding of the antigens that are immunogenic and the T cells that recognize them. Recent discoveries into the impact of tumor mutational burden (TMB) have bolstered our grasp of the origins of tumor antigens, despite TMB failing to consistently predict clinical outcomes. This is perhaps best epitomized by the dismal antigen validation rates which hover at ~1%. Hence, our understanding of which T cells exhibit therapeutic anti-tumor potential in solid tumors remains problematic. Therefore, there is a critical need for unbiased approaches capable of identifying anti- tumor T cell responses, a lack of which will substantially impede the progress of immunotherapies in solid tumors. We previously developed ATTACH (Assessment of T cells Tethered to Antigen Class I/II Histocompatibility), a powerful microfluidics assay which allows direct isolation of anti-tumor T cells by leveraging HLA/peptide binding avidity of T cell receptors (TCR) to matched tumor cells serving as de facto “tetramer pools”. Here, we propose the development of an ATTACHER (ATTACH via Electrothermal flow-enhanced Recovery), a microfluidic device capable of streamlining this assay while increasing sensitivity, specificity, and antigen-specific T cell recovery. Our central hypothesis is that T cells recognizing multiple tumor antigens can be directly isolated in vitro using a T cell ATTACHER, and that these cells harbor increased anti-tumor cytotoxic potential compared to bulk T cells. We have formulated this hypothesis on the basis of preliminary studies outlining the ability of ATTACH to enrich for tumor-infiltrating lymphocytes (TILs) with increased cytotoxic potential. The rationale for the proposed research is that applying electrothermal flow to ATTACH via development of a T cell ATTACHER will promote T cell/tumor cell contact and allow T cells to scan multiple MHC/antigen complexes in order to encounter their cognate antigen. In Aim 1, we will design and construct the T cell ATTACHER and evaluate its ability to recover antigen-specific T cells across a set of 20 different human antigens and TCRs of different affinities developed in our lab. In Aim 2, we will characterize TILs isolated using the T cell ATTACHER both phenotypically and functionally using paired human patient-derived xenografts and tumor-infiltrating lymphocytes from the ICON cohort. We anticipate our work will allow us to demonstrate the versatility of the T cell ATTACHER and its ability to improve anti-tumor responses in vitro. Overall, our work will allow direct, accurate and unbiased identification of anti-tumor T cells in solid tumors within hours rather than months as is currently required, laying the foundation for subsequent studies into the predictive and therapeutic potential of this method in solid tumors.