Precision quality check of immunotherapeutics via single-cell cytokine mapping

已经出现了经工程化以表达嵌合抗原受体（汽车）的自体T细胞的连续转移 作为恶性血液病患者的一种有前途的免疫疗法，例如白血病中的CD 19汽车 和淋巴瘤。然而，在制造一致性和功能概况方面仍然存在挑战 由于输注的细胞作为“活药物”，可以被激活并释放多种药物， 细胞因子对特异性抗原识别的影响。这些分泌的细胞因子不仅可以产生治疗功效， 而且还有危及生命的免疫毒性，例如细胞因子释放综合征和神经毒性。必须 癌症患者在输注前具有CAR-T细胞产物的全谱效力和毒性特征。 我们已经开发了单细胞条形码芯片（SCBC）技术，该技术使得（a）能够测定30+分泌的 每个活细胞的蛋白质，（B）涵盖功效和安全性的广泛免疫细胞功能，（c） 设计为适合各种类型的免疫细胞，（d）是每个细胞的这些蛋白质的第一个定量度量，以及（e） 所需的样本量总计仅为5000个细胞，所有这些都是基于流式细胞术平台的关键飞跃。 通过与两家领先的CAR-T制药公司的合作，IsoPlexis的单细胞深度分析已经 首次揭示了输注前与输注后患者反应的相关性。SCBC分析20 淋巴瘤患者表现出CAR-T细胞的多功能强度的显著相关性， 对CAR-T细胞治疗有客观应答（完全或部分）（p = 0.012），表明这是一个强有力的指标， 在质量评估中，其他黄金标准技术没有检测到任何显著的相关性。 根据临床数据和SBIR第一阶段资助的自动化进展，我们提出以下建议 在这个为期两年的第二阶段申请的具体目标：目标1：开发自动化的“流动池”消耗品 隔室，它捕获32重单细胞细胞因子反应，以准备所需的全自动化 大型CAR-T试验（1-10个月）; AIM 2：生产全自动检测盒分析和工作流程系统， 允许10个样本同时进行分析，用户交互最少，便于引入临床核心 实验室（1-15个月）;目标3：制作一个全面的信息学套件，并全面测试我们的自动化系统， 在两项30例患者队列试验中的信息学，为输注前CAR-T提供稳健和预测性生物标志物 质量.建立MSKCC和UCLA beta研究中心，用于II期后过渡（7-24个月）。 通过此次II期申报，我们将提供首个有效的CAR-T输注前质量检查检测，以预测 患者的客观反应，在一个易于使用的自动化系统，可用于所有细胞 免疫治疗试验

Adoptive transfer of autologous T cells engineered to express chimeric antigen receptors (CARs) has emerged as a promising immunotherapy for patients with hematologic malignancies, such as CD19 CARs in leukemias and lymphomas. However, challenges remain in terms of manufacturing consistency and the functional profile of the CAR-T cell product, since infused cells, as “living drugs”, can be activated and release a variety of cytokines upon specific antigen recognition. These secreted cytokines may result in not only therapeutic efficacy, but also life-threatening immunotoxicity, such as cytokine release syndrome and neurologic toxicity. It is essential for cancer patients to have a full spectrum potency and toxicity profiling of CAR-T cell products before infusion. We have developed a single-cell barcode chip (SCBC) technology, which enables (a) assaying 30+ secreted proteins per individual live cell, (b) a broad range of immune cell functions covering efficacy and safety, (c) is designed to fit various types of immune cells, (d) is the first quantitative metric of these proteins per cell, and (e) requires samples sizes totaling only 5000 cells, all critical leaps over flow cytometry based platforms. With the collaboration of two leading CAR-T pharmaceutical companies, IsoPlexis’ single-cell deep profiling has revealed, for the first time, a pre-infusion correlate to post-infusion patient response. The SCBC analysis of 20 patients with lymphoma demonstrates a significant correlation of the polyfunctional strength of the CAR-T cells with objective response (complete or partial) to the CAR-T cell therapy (p = 0.012), indicating a powerful metric of quality assessment, where the other gold standard technologies did not detect any significant correlations. Based on the clinical data and the automation progress in our SBIR Phase I grant, we are proposing the following specific aims in this two-year Phase II application: AIM 1: Develop automated “flow cell” consumable compartment, which captures 32-plex single-cell cytokine response, to prepare for full automation required by large CAR-T trial (months 1-10); AIM 2: Produce a fully automated cartridge analysis and workflow system, to allow 10 samples assayed in parallel with minimal user interaction, allowing easy introduction into clinical core labs (months 1-15); AIM 3: Produce a comprehensive informatics suite, and fully test our automated system and informatics in two 30 patient cohort trials, providing robust and predictive biomarkers for pre-infusion CAR-T quality. Establish MSKCC and UCLA beta sites for post-phase II transition (months 7-24). With this Phase II submission, we will deliver the first effective CAR-T pre-infusion quality check assay to predict objective response in patients, in an easy-to-use, automated system that can be used throughout all cellular immunotherapy trials.