Using microfluidics to realize patient-specific anti-cancer immunotherapies

利用微流控实现患者特异性抗癌免疫疗法

• 批准号: 10702214
• 负责人: Polly Morrell Fordyce
• 金额: $ 108.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702214
• 关键词: Address; Affinity; Agonist; Alleles; Amino Acids; Antigen-Presenting Cells; Binding; Biomechanics; Cancerous; Cell surface; Cells; Clone Cells; Complex; Dangerousness; Data; Disease; Encapsulated; Engineering; Hour; Immune response; Immunologic Surveillance; Immunotherapy; In Vitro; Malignant Neoplasms; Methods; Microfluidics; Patients; Peptide Receptor; Peptide Vaccines; Peptides; Physiological; Play; Proteins; Qualifying; Recombinants; Role; Sensitivity and Specificity; Sorting; Surface; T cell response; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; TCR Activation; Techniques; Technology; Testing; Training; Transfusion; Tumor Antigens; Work; antigen-specific T cells; cancer immunotherapy; cancer therapy; cost; cytotoxic; engineered T cells; fighting; improved; in vivo; invention; microfluidic technology; neoantigens; new technology; next generation sequencing; novel; receptor binding; screening; side effect; tool

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).

项目摘要 T细胞在免疫响应中起着核心作用，检测到MHC分子中的抗原petides （PMHC）通过特定的与细胞表面T细胞接收器的特定相互作用显示在解剖细胞的表面上 （TCR）。这种识别触发了下游T细胞激活和细胞毒性杀伤。在体内，T细胞是 精心敏感和特异 细胞。免疫疗法试图利用这种敏感性和特异性，以消除两种 输血的T细胞患者设计，以显示针对肿瘤相关抗原的TCRS （新抗原）或注射肽“疫苗”以刺激新抗原特异性T细胞克隆的扩张。 预测哪种新抗原/TCR组合将激活患者的潜在T细胞反应 强大的挑战。有大量潜在的PMHC/TCR复合物：MHC分子是 由23,000 HLA等位基因编码，每个MHC显示一个〜9个氨基酸肽（209个可能性），每个氨基酸肽 患者可以表达> 1020可能的TCR。虽然许多技术利用下一代测序 屏幕数百万的PMHC/TCR组合用于高亲和力粘合剂，这些屏幕只能测试一个小的 可能组合的一部分。 此外，PMHC/TCR结合的强度无法预测激活： 许多高亲和力的宠物没有激活T细胞，许多潜在的激动剂仅与中等亲和力结合。 T细胞在PMHC/TCR复合物上在抗原呈递细胞上爬行时会产生PN的PN，并且 新兴的证据已经确定，这些生物力学力对于敏感和特异性至关重要 TCR-PMHC识别：PMHC/TCR络合物驱动电势激活形式“捕获”键的强度 在有效的情况下，虽然那些不形成“滑动”键的人更有可能破裂。这是发展的 免疫疗法需要能够测试大量候选PMHC/TCR的新技术 相互作用的相互作用能够形成捕获键并在物理力下激活T细胞的能力。 我的实验室具有独特的资格来满足这一关键需求。在先前的工作中，我们开发了一个微流体平台 使重组无细胞表达，纯化和定量在体外表征> 1,500 蛋白质以低成本为单位。在这里，我们将应用这项强大的技术系统调查 哪种PMHC/TCR组合形成了预测激活的“捕获”键（平台1）和哪些新抗原 由1000秒的不同MHC序列（通过可变HLA等位基因（平台2））进行有效显示。 为了进一步测试候选PMHC/TCR在其细胞环境中的组合，我们将应用一种新型基于液滴 我们发明的技术是为了共同填充了100千万个T细胞/APC对的技术，并根据激活对它们进行排序 （平台3）。