Precision Quality Check of Immunotherapeutics via Single Cell Cytokine Mapping

尽管靶向CD19的CAR-T免疫治疗剂的益处已得到证实，但仍存在两个挑战， 将治疗药物推向市场。第一个关键挑战是可重复地制造治疗， 细胞的细胞因子介导的功能在制造后以可预测的一致方式起作用。第二 一个关键的挑战是控制细胞的副作用（免疫毒性），即细胞因子释放综合征 (CRS)IL-6和其他炎性细胞因子，同时还确保对CD19靶点的功效。一个更 不仅要解决确保一致和安全运行有效的输注前质量控制测试 这两个关键的挑战，但帮助制药公司满足FDA在这两个领域的关注。等 测试将对确保制药公司帮助这些疗法进入市场产生深远的影响， 需要治疗的无法治愈的B细胞恶性肿瘤患者可以获得这些革命性的治疗方法。一个更 可靠的体外CAR-T功能测试将允许医生去除或修改不一致或不安全的 在注射前进行细胞治疗，显著降低患者的风险，并提高治疗的可能性。 成功然而，目前领先的输注前测试并不能解决最重要的临床问题。 要求.这样的测试的第一个要求是评估CAR功能性细胞的细胞因子活化， 单细胞多功能细胞亚群，与免疫应答质量相关，还监测 这些细胞亚群之间的不良功能反应。这需要多达18个非重叠的细胞因子， cell.第二个要求是以"离体"方式测量细胞的真实分泌物，而不是固定细胞的分泌物。 细胞和操纵它们的真正功能。IsoPlexis的微芯片技术满足了这两个需求， 时间它测量多功能细胞因子标志物（抗肿瘤、刺激性、趋化性）的功效范围， 同时测量CRS相关的炎性细胞因子以及来自这些亚群的调节性细胞因子（多达42 细胞因子/细胞）。它在离体平台中这样做，该平台不仅测量针对CD19靶标的真实细胞分泌， 而且还可以直接在设备上与目标细胞相互作用并对其作出响应。IsoPlexis计划利用其核心 技术和学习，以创建一个体外评估测试，以测量一致的功能范围， CAR-T细胞疗法，并监测安全性和有效性的标志物。我们建议采取以下具体措施 目的：（1）开发一种可重复的测定方法，用于测量与CD19 + CAR细胞相互作用后的CD19 + CAR细胞。 ⑶ 19（固定的）以确保对CAR靶标的一致功能。(2)执行上述测定， 转染的CAR-T供体样品，并证明了临床上有用的工作流程。第一阶段奖 支持，我们希望在未来使用它作为一个伴随诊断工具，以监测病人的反应 并确保在每位患者中对这些个性化的活体药物进行适当的管理， 免疫肿瘤学中的微型设备。

Despite the demonstrated benefit of CD19 targeted CAR-T immunotherapeutics, two challenges remain to bringing the therapeutics to market. The first key challenge is to reproducibly manufacture the therapy so that the cells' cytokine mediated function acts in a predictably consistent fashion post manufacturing. The second key challenge is to manage the cell's adverse effect (immuno-toxicity), namely cytokine release syndrome (CRS) of IL-6 and other inflammatory cytokines, while also ensuring efficacy against the CD19 target. A more effective pre-infusion quality control test to ensure consistent and safe functioning would be not only address these two critical challenges, but help pharmaceutical firms satisfy FDA concerns in both of these areas. Such a test would have deep impact on ensuring the pharmas help these therapies to market, and in making sure that patients in need with incurable B-cell malignancies have access to these revolutionary therapies. A more reliable in vitro CAR-T functional test would allow physicians to remove or modify the inconsistent or unsafe cell therapies prior to injection, significantly reducing risk to the patient, and improving odds of therapeutic success. However, the current leading pre-infusion tests do not address the most important clinical requirements. The first requirement of such a test is to evaluate the CAR functional cell cytokine activation in the single-cell poly-functional cell subsets, a correlate of quality immune response, and also monitor for adverse functional reactions amongst these cell subsets. That requires up to 18 non-overlapping cytokines per cell. A second requirement is to measure the cells' true secretions in an “ex vivo” manner, rather than fixing the cells and manipulating their true function. IsoPlexis' microchip technology meets these two needs for the first time. It measures the range of efficacy polyfunctional cytokine markers (anti-tumor, stimulatory, chemotactic), while measuring the CRS related inflammatory and also regulatory cytokines from those subsets (up to 42 cytokines per cell). It does so in an ex vivo platform that not only measures true cell secretion to CD19 target, but can also interact with and respond to the target cell directly on device. IsoPlexis plans to use its core technology and learning to create an in vitro assessment test to measure for consistent range of function for in CAR-T cell therapies and to monitor for markers of safety and efficacy. We propose to do the following specific aims: (1) Develop a panel and reproducible assay for measurement of CD19+ CAR cells upon interaction with CD19 (immobilized) to ensure consistent function to CAR target. (2) Implement the assay above with transfected CAR-T donor samples, and demonstrate a clinically useful workflow. With the Phase I award support, we expect in the future to use it as a companion diagnostics tool to monitor the response of patients and ensure proper management of these personalized living drugs in every patient, justifying the broad impact of the proposed microdevice in immune-oncology.