A Bioengineered Leukemia Chip for CAR T-Cell Immunotherapy Modeling and Screening

Introduction:: Adoptive CD19 CAR (Chimeric Antigen Receptor) T-cell transfer has emerged as a successful FDA-approved therapy for relapsed and refractory pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, with prolonged follow-up, a high relapse rate of 30~60% of CAR T-cell therapy remains a major issue that largely limits its clinical benefit (1). Several relapse mechanisms have been reported, such as failure of in vivo CAR T-cell expansion/persistence, targeted antigen modulation and leukemia lineage switch. The ability to pre-clinically assess the functionality of CAR T-cells and dissect the CAR T-cell immunotherapy relapse mechanisms will be of great significant but challenging with current animal models.

Materials and Methods:: To interrogate the CAR T-cell immunotherapy efficiency, we established a 3D microfluidics-based, organotypic ‘Leukemia-on-a-Chip’ to dissect and recapitulate the dynamic molecular and cellular interactions occurring between B-ALL and the bone marrow niche. The 3D microfluidics device is composed of three distinct functional regions (Fig. 1a,b): a central sinus region vascularized network by endothelial cells (HUVECs, Lonza), an inner ring region serves as a medullary cavity, an interface of multicellular interactions between leukemia cells (Reh, ATCC), bone marrow stromal cells (ECs and MSCs, Lonza), and bone marrow immune niche cells, and the outer ring channel as endosteal regions infused by osteoblasts and fibroblasts, connected with four media reservoirs, for cell culture media supplies and waste removal. ALL the cell types were embedded in a fibrin (Sigma) hydrogel to maintain 3D cell culture. The device was fabricated using standard soft lithography technique (2).

Results, Conclusions, and Discussions:: To assess CD19 CAR T-cell functionality, we first infused CAR T-cell into central sinus and monitored CAR T-cell dynamics during it extravasated out from the 3D vasculature and infiltrated into the leukemia niche (Fig. 1c). Importantly, we captured the whole eradication processes of a CD19+ tumor cell by a CAR T-cell, such as patrolling toward, recognizing, and killing of leukemia (Fig. 1d). We also characterized the CAR T-cell expansion and activation markers, granzyme B and perforin after interaction with leukemia blast sand cytokine secretion, which together induced remission (Fig. 1e-g). Next, we replicated CD19- relapse by spiking of CD19- B-ALL into CD19+ population at predefined percentages into our leukemia model. Following a chronological observation of leukemia burden under CD19 CAR T-cell therapy over 2 weeks, we found that the ex vivo leukemia system with addition of CD19- leukemia initially demonstrated partial remission then relapse accompanying with expansion of CD19- leukemia (Fig. 1h). In addition, we are currently expanding our assays to evaluate the functional performance of various CAR T cell products with different CAR designs and generations and as well as those derived from patients. We will also establish an integrative and analytical index to demarcate CAR T cell potency. Together, we have established a robust in vitro system for evaluating CAR T-cell therapy, which may translate to personalized therapies and improved clinical decision-making.

Acknowledgements (Optional): : This work was supported in part by the US National Institutes of Health (R35GM133646, R01CA243001) and the National Science Foundation (No.2103219). Chao Ma is the recipient of a Cancer Research Institute Irvington Postdoctoral Fellow-ship (CRI Award, CRI4018).

References (Optional): : [1] Schuster et al., Lancet Oncol. 2021;22(10):1403-1415;
[2] Ma C. et al. Science Advances. 6 (44), eaba5536;