Microfluidic CAR-T Cell Processing Device

摘要 该快速通道STTR项目的目标是开发一种确定性横向位移(DLD) 可以在1小时内从典型的白细胞分离单元中浓缩白细胞(WBC)的微流控设备， 用于制造癌症细胞免疫疗法。嵌合抗原受体T细胞(CAR-T)治疗 已被推荐FDA批准用于治疗复发或难治性儿童和青壮年患者 B细胞急性淋巴细胞性白血病。迫切需要具有成本效益的自动化方法来改进 用于制造CAR-T和其他蜂窝的WBC的大规模浓缩的效率和产量 治疗。 GPB是开发新型DLD微芯片以处理用于细胞分析的血细胞的先驱(19，26)。GPB Now 提议开发、评估一种紧凑型设备并将其商业化，其中整个白细胞分离单元(UP 高达300毫升的5x1010个WBC)可在一次性使用多通道的“白血球堆”中处理 DLD芯片在1小时内产生富含WBC并耗尽红血球的洗涤细胞悬浮液 细胞(红细胞)和血小板(PLT)。 在第一阶段，目标1是通过当前的原型芯片来提高小区吞吐量：1)优化DLD芯片 设计和运行以增加流量；2)通过堆叠塑料芯片和运行它们来增加吞吐量 并行(“Leuko-Stack”)；以及3)将芯片生产转变为大批量制造材料，如 环烯烃聚合物(COP)。进入第二阶段的最终第一阶段里程碑是：1)最终芯片设计，具有 通过单芯片的流速至少为25毫升/小时，活性白细胞和免疫表型的回收率至少为70%- 定义的T淋巴细胞，以及处理细胞1小时而不堵塞的能力；2)至少6个芯片的白血球堆叠 并行运行，输出与#1相同；3)通过#1和#2增加的吞吐量合计足以 在1小时内处理一个300毫升的白细胞分离装置；4)确认芯片可以从COP中生产。 在第二阶段，目标2是建立最终原型，基于铜塑芯片的微流控装置能够处理 以300毫升/小时的速度采集白细胞样本。目标3是用白细胞分离法测试目标2的原型的性能 等分，然后是全尺寸的人类白细胞分离样本。这个项目的最后一个里程碑是制作一个 一套商业原型Leuko-Stack，可在1小时内处理整个300毫升白细胞分离单元 至少70%的WBC和T淋巴细胞恢复，至少90%的红细胞耗尽，至少80% PLT耗尽，T细胞扩增能力至少恢复70%(与输入相比 样本)，在两个地点测试的样本中，明显超过50%。 GPB Leuko-Stack平台将保留DLD微流控电池处理相对于当前技术的优势 方法，同时大幅提高吞吐量和信元处理能力，从而从 分析性-制备规模的WBC浓缩，用于后续CAR-T和其他细胞疗法的制造。

ABSTRACT The goal of this Fast-Track STTR project is to develop a Deterministic Lateral Displacement (DLD) microfluidic device that can enrich white blood cells (WBCs) from a typical leukapheresis unit in 1 hr, for use in manufacturing cancer cellular immunotherapy. Chimeric antigen receptor T cell (CAR-T) therapy has been recommended for FDA approval to treat relapsed or refractory pediatric and young adult patients with B-cell acute lymphoblastic leukemia. There is a critical need for cost-effective automated methods to improve the efficiency and yield of large-scale enrichment of WBCs for use in manufacturing CAR-T and other cellular therapies. GPB is a pioneer in developing novel DLD microchips to process blood cells for cell analysis (19,26). GPB now proposes to develop, evaluate and commercialize a compact device in which an entire leukapheresis unit (up to 5x1010 WBCs in up to 300 ml) can be processed in a “Leuko-stack” of disposable single-use multi-channel DLD chips to produce in 1 hr a washed cell suspension that is enriched in WBCs and depleted of red blood cells (RBCs) and platelets (PLT). In Phase I, Aim 1 is to increase cell throughput through the current prototype chips by: 1) optimizing DLD chip design and operation to increase flow rate; 2) increasing throughput by stacking plastic chips and running them in parallel (“Leuko-stacks”); and 3) translating chip production to high-volume manufacturing material such as Cyclic Olefin Polymer (COP). Final Phase I milestones to proceed to Phase II are: 1) final chip design with a flow rate of at least 25 mL/hr via a single chip, at least 70% recovery of viable WBCs and immunophenotype- defined T-lymphocytes, and ability to process cells for 1 hr without clogging; 2) Leuko-stack of at least 6 chips run in parallel, with the same output as in #1; 3) combined increases in throughput via #1 and #2 sufficient to process a 300 ml leukapheresis unit in 1 hr; 4) confirmation that the chips can be produced from COP. In Phase II, Aim 2 is to build final prototype COP plastic chip-based microfluidic device capable of processing a leukapheresis sample at 300 mL/hr. Aim 3 is to test performance of prototypes from Aim 2 with leukapheresis aliquots and then full-size human leukapheresis samples. The final milestone of this project is to produce a set of commercial prototype Leuko-stacks that can process an entire 300-ml leukapheresis unit in 1 hr with at least 70% WBC and T-lymphocyte recovery, at least 90% depletion of RBCs, at least 80% depletion of PLTs, and at least 70% recovery of T-cell expansion capacity (as compared with the input samples) in significantly more than 50% of samples tested at 2 sites. The GPB Leuko-stack platform will preserve the advantages of DLD microfluidic cell processing over current methods, while massively increasing throughput rate and cell processing capacity, thus transitioning from analytic- to preparative-scale WBC enrichment for subsequent manufacture of CAR-T and other cell therapies.