CART4-34 Manufacturing
Improving lentiviral transduction for a selective CAR T cell therapy, with reagent screening and follow-up cell expansion.
2024–2026 · Published in Science Translational Medicine
CART4-34 is an experimental CAR T cell approach that recognizes IGHV4-34, a variable region of the B cell receptor enriched in certain B cell cancers and autoimmune disease. The project aimed to target these pathogenic cells more selectively than CD19-directed therapies, which also deplete much of the healthy B cell population. At Penn’s Center for Cellular Immunotherapies, I worked on a practical part of that effort: producing enough engineered T cells for the study.
The broader study demonstrated selective killing of IGHV4-34-positive malignant cells, antitumor activity in mouse models, and targeting of patient-derived lupus B cells outside the body. Shorter CAR hinges improved the contact interface between T cells and their targets. CART4-34 largely preserved the IGHV4-34-negative healthy B cell population; it also depleted the small IGHV4-34-positive subset found in healthy donors. These were preclinical findings.
My contribution
My work focused on lentiviral transduction, the step that introduces the CAR gene into T cells. Too few cells were expressing the receptor, so I looked for ways to improve that step. I screened transduction-enhancing reagents, measured reporter expression by flow cytometry, and followed cell growth under the selected conditions.
The screen compared Poloxamer 407 (P407), beta-mercaptoethanol, Polybrene, and LentiBOOST, each with and without RetroNectin. RetroNectin brings viral particles and target cells into proximity. The CAR construct included a truncated EGFR reporter, allowing the fraction of EGFR-positive cells to serve as a readout of successful gene delivery and expression.
Screening result
RetroNectin plus P407 reached 35.6% EGFR positivity, the highest value reported among the tested enhancer conditions. The no-enhancer control without RetroNectin was 13.6%, the RetroNectin-only control was 18.7%, and P407 without RetroNectin reached 23.2%. The combination outperformed either reagent alone in this screen.
P407 is a nonionic surfactant. The poster proposed that reduced repulsion between viral particles and cells might complement RetroNectin’s proximity effect, but the experiment did not establish that mechanism.
Following the cells over time
A subsequent expansion using RetroNectin and P407 showed continued cell growth through day 16, with EGFR positivity tracked on days 5–11. That gave us a useful condition to carry into later experiments.
I presented this work at Penn’s CURF Fall Research Expo in September 2024. It became part of the broader collaborative study published in 2026.
Tools and methods: CAR T cell culture, lentiviral transduction, reagent screening, flow cytometry, reporter-based expression analysis, and cell expansion.