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Unveiling the influence of CAR-negative T-cells: enhancing efficacy and ensuring safety in CAR-T therapies
Belén Sierro-Martínez1, Beatriz Guijarro-Albaladejo1, Ricardo Fernández-Cisnal2
1Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain.
Background:
Despite the promising results of CAR-T cell therapy, still a significant proportion of patients experience treatment failure due to absence of response, progression or treatment-related toxicities. Our research aims to investigate how the CAR-T cell product characteristics and particularly the percentage of CAR-negative cells present in the infusion product affects its efficacy and safety.
Methods:
CAR-positive (CAR-pos) and CAR-negative (CAR-neg) cells were analyzed during in vitro expansion with IL-2 or IL-7/IL-15. FACS-sorted populations were assessed for cytotoxic effect and secretion of cytokines. Transcriptomic analyses were conducted for CAR-pos and CAR-neg populations.
Results:
A significant reduction in the percentage of transduced (CAR-pos) T cells was observed throughout the in vitro cell culture, where CAR-neg T cells displayed greater expansion along the procedure compared to CAR-pos T cells, using either IL-2 or IL-7/IL-15. CAR-pos T cells exhibited increased expression of activation and exhaustion markers. Comparative analyses of CAR-pos and CAR-neg showed an increased cytotoxic effect in purified samples with 100% CAR-pos T cells compared to non-purified product. Quantification of cytokine levels revealed a significant decrease in IL-6 in purified compared to non-purified products. RNA sequencing analysis revealed significant differential gene expression profile between CAR-pos and CAR-neg cells, the prior exhibiting an upregulation of genes associated with immune checkpoint regulation, cytokine-cytokine receptor interaction and Th1/Th2 and Th17 cell differentiation pathways.
Conclusion:
CAR-neg T cells exhibit a significantly greater expansion compared to CAR-pos T cells alongside CAR-T cell production. The percentage of CAR-neg cells influences the specific cytotoxicity of the product and on the cytokine pattern, which might influence both efficacy and toxicity and, therefore, the number of CAR-neg T cells within the infusion product must be considered in the clinical setting. Purification of CAR-pos T cells could potentially optimize product characteristics.
Insights
CAR-negative cells expand more than CAR-positive cells during CAR-T therapy production. The percentage of CAR-negative cells impacts product efficacy and safety, suggesting purification may optimize CAR-T therapy outcomes.
Area of Science:
- Immunotherapy
- Cellular Therapy
- Oncology
Background:
- CAR-T cell therapy shows promise but faces challenges with treatment failure.
- Patient outcomes are affected by response, progression, and toxicities.
- Understanding CAR-T product characteristics is crucial for improving efficacy and safety.
Purpose of the Study:
- To investigate the impact of CAR-T cell product characteristics on therapy efficacy and safety.
- To analyze the role of CAR-negative cells in CAR-T cell production and function.
- To determine if purification of CAR-positive cells can optimize CAR-T product quality.
Main Methods:
- In vitro expansion of CAR-positive (CAR-pos) and CAR-negative (CAR-neg) T cells with IL-2 or IL-7/IL-15.
- Assessment of cytotoxic effects and cytokine secretion in FACS-sorted populations.
- Transcriptomic analysis of CAR-pos and CAR-neg cells.
Main Results:
- CAR-neg T cells exhibited significantly greater expansion than CAR-pos T cells during in vitro culture.
- CAR-pos T cells showed increased activation and exhaustion markers, with higher cytotoxicity in purified samples.
- Differential gene expression revealed distinct pathways, including immune checkpoint regulation, in CAR-pos cells.
Conclusions:
- CAR-neg T cells outcompete CAR-pos cells during CAR-T production, influencing product efficacy and safety.
- The percentage of CAR-neg cells affects cytotoxicity and cytokine profiles, necessitating consideration in clinical settings.
- Purification of CAR-pos T cells presents a potential strategy to enhance CAR-T product characteristics.
