Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 10 ⁸ cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.
Competing Interests: B.J.S., S.B., F.F., L.H.W., and R.S.P. are or were employed by and have an equity interest in Indee Labs. L.M.R.F., J.B., L.L., F.L.P. and J.E. are or were consultants to Indee Labs. R.S.P. is an investor in and a venture partner at both Pioneer Fund and Axial, which have a financial interest in Indee Labs. The Ferreira Lab received support from Indee Labs as a subaward from the National Institute of Diabetes and Digestive and Kidney Diseases (Grant No. 1R43DK133029-01). Indee Labs has a commercial interest in developing patents related to Hydropore™ (WO2016109864A1 & WO2019084624A1). M.F. and R.B. are or were employed by and have an equity interest in CellChorus. CellChorus received support from the National Center for Translational Sciences (R44TR005137) and the National Institute of General Medical Sciences (R44GM149106) of the National Institutes of Health and the National Science Foundation (NSF2229323). CellChorus has a commercial interest in developing the TIMING assay. J.E. is a compensated co-founder at Mnemo Therapeutics; owns stocks in Mnemo Therapeutics and Cytovia Therapeutics; is a compensated scientific advisor for Enterome, Treefrog Therapeutics and Resolution Therapeutics; and is a holder of patents pertaining to but not resulting from this work. The Eyquem Lab received research support from Cytovia Therapeutic, Mnemo Therapeutics, Takeda and Indee Labs as a subaward from the National Cancer Institute (Contract No. 7591022C00053).