Microfluidic CAR-T Cell Processing Device

摘要 这个快速通道STTR项目的目标是开发一个确定性横向位移（DLD） 微流控装置可以在1小时内从典型的白细胞去除单元富集白色血细胞（WBC）， 用于制造癌症细胞免疫疗法。嵌合抗原受体T细胞（CAR-T）疗法 已建议FDA批准用于治疗复发性或难治性儿童和年轻成人患者， B细胞急性淋巴细胞白血病迫切需要具有成本效益的自动化方法来改善 用于制造CAR-T和其他细胞的WBC的大规模富集的效率和产率 治疗 GPB是开发新型DLD微芯片以处理血细胞进行细胞分析的先驱（19，26）。GPB现在 提出开发、评估和商业化一种紧凑的装置，其中整个白细胞去除单元（up 高达300 ml中的5x1010个WBC）可以在一次性使用多通道的"Leuko-stack"中进行处理 DLD芯片，以在1小时内产生富含WBC和耗尽红血的洗涤细胞悬浮液 细胞（RBC）和血小板（PLT）。 在第一阶段，目标1是通过当前的原型芯片增加细胞吞吐量：1）优化DLD芯片 设计和操作，以增加流速; 2）通过堆叠塑料碎片并运行它们来增加吞吐量 并行（"Leuko-stacks"）;以及3）将芯片生产转化为大批量制造材料， 环烯烃聚合物（COP）。进入第二阶段的第一阶段的最后里程碑是：1）最终的芯片设计， 通过单个芯片的流速至少为25 mL/hr，活WBC和免疫表型的回收率至少为70%- 确定的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个芯片的白细胞堆叠 并行运行，具有与#1相同的输出; 3）经由#1和#2的吞吐量的组合增加足以 在1小时内处理300 ml白细胞分离单元; 4）确认可以从COP生产芯片。 在第二阶段，目标2是建立最终的原型COP塑料芯片为基础的微流控装置，能够处理一个 目标3是测试目标2中原型的性能，其中白细胞去除法为300 mL/hr 等分试样，然后是全尺寸的人白细胞分离样本。这个项目的最后一个里程碑是制作一个 一套商业原型Leuko-stacks，可在1小时内处理整个300 ml白细胞分离装置 WBC和T淋巴细胞回收率至少为70%，RBC耗竭率至少为90%， 血小板耗竭，T细胞扩增能力恢复至少70%（与输入相比 在2个地点检测的样品中，显著超过50%的样品中含有这些化合物。 GPB Leuko-stack平台将保留DLD微流体细胞处理相对于当前 方法，同时大规模提高吞吐率和细胞处理能力，从而从 从分析到验证规模的WBC富集，用于随后的CAR-T和其他细胞疗法的生产。

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.