Using microfluidics to realize patient-specific anti-cancer immunotherapies

项目摘要 T细胞在免疫反应中起着核心作用，检测MHC分子内的抗原肽 通过与细胞表面T细胞受体的特异性相互作用， （TCR）。这种识别触发下游T细胞活化和细胞毒性杀伤。在体内，T细胞是 它非常敏感和特异，能够被患病的人展示的单一抗原肽激活。 cell.免疫疗法试图利用这种敏感性和特异性来消除癌细胞， 使用经工程改造的T细胞来展示对肿瘤相关抗原特异性的TCR的患者 新抗原（neoantigens）或注射肽“疫苗”以刺激新抗原特异性T细胞克隆的扩增。 预测哪种新抗原/TCR组合将激活患者中的有效T细胞应答仍然是一个挑战。 艰巨的挑战存在大量潜在的pMHC/TCR复合物： 由23，000个HLA等位基因编码，每个MHC展示约9个氨基酸的肽（209种可能性），并且每个 患者可以表达>1020个可能的TCR。虽然许多技术利用下一代测序， 筛选数以百万计的pMHC/TCR组合的高亲和力结合剂，这些筛选只能测试一小部分 可能的组合的一部分。 此外，pMHC/TCR结合的强度不能预测活化： 许多高亲和力肽不能激活T细胞，许多有效的激动剂仅以中等亲和力结合。 当T细胞在抗原呈递细胞上爬行时，它们在pMHC/TCR复合物上产生pN至nN力， 新出现的证据表明，这些生物力学力量对于敏感和特异性的 TCR-pMHC识别：驱动有效激活的pMHC/TCR复合物形成“捕获”键， 而那些没有形成“滑动”键的更有可能断裂。因此，开发改进 免疫治疗需要能够测试大量候选pMHC/TCR的新技术 它们之间的相互作用是因为它们在生理力下形成捕获键并激活T细胞的能力。 我的实验室是唯一有资格满足这一关键需求。在之前的工作中，我们开发了一个微流体平台， 能够实现重组无细胞表达、纯化和体外定量表征， 蛋白质在几个小时内和低成本。在这里，我们将应用这一强大的技术， 哪些pMHC/TCR组合形成预测激活的“捕获”键（平台1），以及哪些新抗原 通过由可变HLA等位基因编码的1000个不同MHC序列有效地展示（平台2）。 为了进一步测试候选pMHC/TCR组合在其细胞环境中的作用，我们将应用一种新的基于液滴的方法。 我们发明了一种技术，可以共同封装成千上万的T细胞/APC对，并根据激活情况对它们进行分类， （3号站台）。

Project Summary T cells play a central role in the immune response, detecting antigenic peptides cradled within MHC molecules (pMHCs) displayed on the surface of diseased cells via specific interactions with cell-surface T cell receptors (TCRs). This recognition triggers downstream T cell activation and cytotoxic killing. In vivo, T cells are exquisitely sensitive and specific, able to be activated by a single antigenic peptide displayed by a diseased cell. Immunotherapies attempt to harness this sensitivity and specificity to eliminate cancerous cells by either transfusing patients with T cells engineered to display TCRs specific for tumor-associated antigens (neoantigens) or injecting peptide ‘vaccines’ to stimulate expansion of neoantigen-specific T cell clones. Predicting which neoantigen/TCR combinations will activate a potent T cell response in a patient remains a formidable challenge. There are a vast number of potential pMHC/TCR complexes: MHC molecules are encoded by 23,000 HLA alleles, each MHC displays a ~9 amino acid peptide (209 possibilities), and each patient can express >1020 possible TCRs. While many techniques leverage next-generation sequencing to screen millions of pMHC/TCR combinations for high-affinity binders, these screens can test only a small fraction of possible combinations. Moreover, the strength of pMHC/TCR binding does not predict activation: many high-affinity peptides do not activate T cells, and many potent agonists bind with only moderate affinities. T cells generate pN to nN forces on pMHC/TCR complexes as they crawl over antigen-presenting cells, and emerging evidence has established that these biomechanical forces are essential for sensitive and specific TCR-pMHC recognition: pMHC/TCR complexes that drive potent activation form ‘catch’ bonds that strengthen under force, while those that do not form ‘slip’ bonds more likely to break. Thus, developing improved immunotherapies requires new technologies capable of testing large numbers of candidate pMHC/TCR interactions for their ability to form catch bonds and activate T cells under physiological forces. My lab is uniquely qualified to address this critical need. In prior work, we developed a microfluidic platform that enables recombinant cell-free expression, purification, and quantitative in vitro characterization of >1,500 proteins in hours and at low cost. Here, we will apply this powerful technology to systematically investigate which pMHC/TCR combinations form ‘catch’ bonds that predict activation (Platform 1) and which neoantigens are efficiently displayed by 1000s of different MHC sequences encoded by variable HLA alleles (Platform 2). To further test candidate pMHC/TCR combinations in their cellular context, we will apply a novel droplet-based technology we invented to co-encapsulate 10s of millions of T cell/APC pairs and sort them based on activation (Platform 3).