ABSTRACT
Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies, as well being developed for 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, like lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies, such as CAR-T. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7 to 2x 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 demonstrated scaled-up hydroporation can process 5 × 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T cell manufacturing with the potential for improved therapeutic outcomes.
Competing Interest Statement
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 HydroporeTM (WO2016109864A1 & WO2019084624A1). M.F. and R.B. are or were employed by and have an equity interest in CellChorus Inc.. 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).
Footnotes
↵* Joint first authors
ABBREVIATIONS
- 4-1BB
- Tumor Necrosis Factor Ligand Superfamily Member 9
- AAV
- Adeno-Associated Virus
- AICD
- Activation Induced Cell Death
- BLI
- Bioluminescence Imaging
- CAR-T
- Chimeric Antigen Receptor T-cell
- Cas9
- CRISPR-Associated Protein 9
- CCL1
- Chemokine Ligand 1
- CCL5
- Chemokine Ligand 5
- ccRCC
- Clear Cell Renal Cell Carcinoma
- CD3
- Cluster of Differentiation 3
- CD3ζ
- Cluster of Differentiation 3 Zeta Chain
- CD4
- Cluster of Differentiation 4
- CD8
- Cluster of Differentiation 8
- CD19
- Cluster of Differentiation 19
- CD28
- Cluster of Differentiation 28
- CD45RA
- Cluster of Differentiation 45 RA
- CD62L
- Cluster of Differentiation 62 L-selectin
- CD70
- Cluster of Differentiation 70
- cGMP
- Current Good Manufacturing Practices
- CM
- Central Memory Cells
- CRISPR
- Clustered Regularly Interspaced Short Palindromic Repeats
- CRS
- Cytokine Release Syndrome
- CT
- Cell Therapy
- CXCR1
- Cysteine-Amino Acid-Cysteine Motif Chemokine Receptor 1
- CXCR2
- Cysteine-Amino Acid-Cysteine Motif Chemokine Receptor 2
- DIC
- Disseminated Intravascular Coagulation
- DNA
- Deoxyribonucleic Acid
- DSB
- Double Stranded Break
- EGFRt
- Truncated Epidermal Growth Factor Receptor
- ELISA
- Enzyme Linked Immunosorbent Assay
- EM
- Effector Memory Cells
- EMRA
- Terminal Effector Memory Cells
- E:T
- Effector-to-Target
- FDA
- Food and Drug Administration
- G4S
- 4-O-Sulfo-Beta-D-Galactopyranose
- HDR
- Homology Directed Repair
- HDRT
- Homology Directed Repair Template
- HLH
- Hemophagocytic Lymphohistiocytosis
- Hy-CAR-Ts
- Hydroporated Chimeric Antigen Receptor T-cells
- ICANS
- Immune Effector Cell-Associated Neurotoxicity Syndrome
- IL-2
- Interleukin 2
- IL-6
- Interleukin 6
- IL-7
- Interleukin 7
- IL-8
- Interleukin 8
- IL-10
- Interleukin 10
- IL-15
- Interleukin 15
- IL-17A
- Interleukin 17A
- IL-22
- Interleukin 22
- IFNɣ
- Interferon Gamma
- KI
- Knock-in
- KO
- Knockout
- Log2fc
- Logarithmic 2 Fold Change
- MHC
- Major Histocompatibility Complex
- mRNA
- Messenger Ribonucleic Acid
- NIR
- Near Infrared
- NSG
- NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
- Nuc-CAR-Ts
- Nucleofected Chimeric Antigen Receptor T-cells
- P2A
- Porcine Teschovirus-1 2A
- PBMC
- Peripheral Blood Mononuclear Cell
- PERC
- Peptide-enabled RNP Delivery for CRISPR
- RNP
- Ribonucleoprotein
- ROS
- Reactive Oxygen Species
- RUO
- Research Use Only
- scFv
- Single-chain Fragment Variable
- sgRNA
- Single Guide Ribonucleic Acid
- TCR (α/β)
- T-cell Receptor (Alpha/Beta Chains)
- TIMING
- Time-lapse Imaging Microscopy in Nanowell Grids
- TME
- Tumor Microenvironment
- TNFɑ
- Tumor Necrosis Factor Alpha
- TRAC
- T-cell Receptor Alpha Constant
- Tregs
- Regulatory T-cells
- WT
- Wild Type
- µVS
- Microfluidic Vortex Shedding
