BCL-2 Inhibition During CAR-T Manufacturing Improves CD19-Targeted Antitumor Function in Cell and Mouse Studies
Background: Targeting cell-death regulation during manufacturing
A preclinical study reports that treating cells with venetoclax during ex vivo culture improves the antitumor function of CD19-targeted chimeric antigen receptor T cells (CAR-T). The researchers observed functional changes in CAR-T cells made from healthy donors and patients who had received cancer treatment, and found delayed tumor growth and improved survival in leukemia and lymphoma xenograft mice. These findings should be distinguished from clinical trial results in patients treated using this manufacturing method.
The study focused on BCL-2, a protein that inhibits cell death, and venetoclax, a drug that blocks its function. BCL-2 family proteins contribute to T-cell survival, signaling, and metabolism. The researchers investigated how CAR-T cells adapt when BCL-2 is inhibited during manufacturing and whether those changes improve tumor-killing function.
Key findings: Greater killing activity observed without drug
In a representative manufacturing experiment, researchers isolated and activated T cells, introduced the CAR, and treated the cells daily with venetoclax or vehicle from days 2–13. Final cell numbers tended to decrease with increasing drug dose, but the difference was not statistically significant. CAR expression, the proportions of CD4 and CD8 cells, and the memory T-cell composition immediately after manufacturing also did not differ significantly. These results alone, however, cannot establish equivalence in commercial manufacturing yield.
CAR-T cells from the treated group eliminated OCI-Ly8 lymphoma cells and NALM6 leukemia cells more effectively than vehicle-treated controls. A representative killing assay co-cultured CAR-T cells and tumor cells at a 1:5 ratio for 48 hours. Venetoclax was absent from the culture medium, separating the result from any direct drug effect on tumor cells. The CAR-T cells did not kill target cells from which CD19 had been removed, supporting antigen specificity.
After tumor stimulation, treated cells showed increased expression of cytotoxic proteins and cytokines, while markers associated with cell death and exhaustion were lower. Expressing a BCL-2 variant resistant to venetoclax binding weakened the functional improvement, supporting target dependence. Phosphorylation of the signaling proteins STAT5 and AKT increased, and inhibiting either reduced the enhanced killing activity in the treated group. However, the molecular links from BCL-2 inhibition to these signaling changes have not all been established.
Metabolic measurements showed increases in basal oxygen consumption rate, maximal respiration, and spare respiratory capacity, which are associated with oxidative phosphorylation (OXPHOS). Basal glycolysis did not differ, while glycolytic capacity and glycolytic reserve increased. The observed changes should therefore be interpreted as altered metabolic response capacity rather than reduced energy consumption.
Meaning and outlook: Potential in patient-derived cells, with limits to durability
In two xenograft models using CAR-T cells from healthy donors, the treated group showed slower tumor growth and improved mouse survival. CAR-T cells manufactured from cells of patients who had received cancer treatment also showed improved killing activity in vitro and efficacy in mouse studies. Observing improvements in samples with different starting-cell compositions and exhaustion states provides a basis for further manufacturing research.
The researchers noted, however, that improvements in CAR-T function in vivo and tumor clearance did not appear to persist long term. The limitations of immunodeficient mouse models also mean these results cannot be extrapolated to improved long-term remission or cure rates in patients. Further clinical research and durability testing are needed to determine whether drug treatment during manufacturing translates into better patient outcomes and how long any improvement lasts.
This account is based on the provided open-access full text of the Signal Transduction and Targeted Therapy paper.
Targeting the BCL-2 family of proteins, key regulators of cellular apoptosis, with BH3-mimetics has been a major therapeutic goal to overcome cancer cell death resistance. However, beyond their canonical roles in apoptosis, BCL-2 proteins also play vital roles in cellular metabolism, signaling, and, increasingly, immune cell regulation. T cells in particular depend heavily on BCL-2 family proteins during ontogeny and maintenance, yet the broader consequences of pharmacologically inhibiting anti-apoptotic BCL-2 proteins in T cells remain underexplored. Our group has previously demonstrated that BCL-2 inhibition with the BH3-mimetic venetoclax induces gene expression and plasticity changes in murine T cells. Building upon this, we here investigate whether venetoclax-driven T cell reprogramming can be leveraged to enhance adoptive cell therapies, specifically using chimeric antigen receptor (CAR) T cells targeting CD19 as a model system. Our findings reveal that venetoclax treatment during ex vivo expansion of CART cells, prepared from T cells from healthy donors or chemotherapeutically pretreated patients, potently augments antitumor efficacy in a BCL-2-dependent manner. Transcriptomic and functional analyses demonstrate that venetoclax reprograms CART cells by regulating signaling pathways (e.g., IL-2/STAT5 and PI3K/AKT) and metabolic programs (e.g., OXPHOS and glycolysis), yielding CART cells with superior fitness and effector functionalities. These results highlight a novel therapeutic approach using anti-apoptotic drugging to enhance the performance of adoptive T cell therapies and support further examination of venetoclax and other BH3-mimetics as immune modulators.
These results point to the possibility of improving cell function by adjusting the ex vivo expansion environment for CD19 CAR-T cells. One practical scenario during manufacturing process development would be to split the same starting sample into drug-treated and control groups, then compare cell yield, antigen-specific killing, and maintenance of function after repeated stimulation. Improvements were also observed in cells from patients who had received cancer treatment, providing a basis for further evaluation, but results from the tested samples cannot be generalized to all patients. Durability of the in vivo effect, as well as safety and efficacy in patients, still require validation. Findings from an ex vivo manufacturing step should not be interpreted as a recommendation to administer venetoclax alongside CAR-T therapy in patients.