Related Experiment Video
Updated: Oct 17, 2025
Generation of Human Chimeric Antigen Receptor Regulatory T Cells
Published on: January 3, 2025
PD-1 and TIGIT downregulation distinctly affect the effector and early memory phenotypes of CD19-targeting CAR
Young-Ho Lee1, Hyeong Ji Lee1, Hyung Cheol Kim2
1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, Republic of Korea; Curocell Inc., Daejeon 34109, Republic of Korea.
Abstract:
CD19-targeting chimeric antigen receptor (CAR) T cells have become an important therapeutic option for patients with relapsed and refractory B cell malignancies. However, a significant portion of patients still do not benefit from the therapy owing to various resistance mechanisms, including high expression of multiple inhibitory immune checkpoint receptors. Here, we report a lentiviral two-in-one CAR T approach in which two checkpoint receptors are downregulated simultaneously by a dual short hairpin RNA cassette integrated into a CAR vector. Using this system, we evaluated CD19-targeting CAR T cells in the context of four different checkpoint combinations-PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT-and found that CAR T cells with PD-1/TIGIT downregulation uniquely exerted synergistic antitumor effects. Importantly, functional and phenotypic analyses suggested that downregulation of PD-1 enhances short-term effector function, whereas downregulation of TIGIT is primarily responsible for maintaining a less differentiated/exhausted state, providing a potential mechanism for the observed synergy. The PD-1/TIGIT-downregulated CAR T cells generated from diffuse large B cell lymphoma patient-derived T cells also showed robust antitumor activity and significantly improved persistence in vivo. The efficacy and safety of PD-1/TIGIT-downregulated CD19-targeting CAR T cells are currently being evaluated in adult patients with relapsed or refractory large B cell lymphoma (ClinicalTrials.gov: NCT04836507).
Insights
Chimeric antigen receptor (CAR) T-cell therapy shows promise for B cell malignancies. Combining PD-1 and TIGIT downregulation in CAR T-cells enhances synergistic antitumor effects and persistence, offering a new therapeutic strategy.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- CD19-targeting chimeric antigen receptor (CAR) T-cell therapy is a key treatment for relapsed/refractory B cell malignancies.
- Therapeutic resistance, often due to inhibitory immune checkpoint receptors, limits CAR T-cell efficacy.
- Overcoming resistance mechanisms is crucial for improving patient outcomes.
Purpose of the Study:
- To develop a novel CAR T-cell therapy that simultaneously downregulates multiple immune checkpoint receptors.
- To investigate the synergistic antitumor effects of combining PD-1 and TIGIT downregulation in CD19-targeting CAR T-cells.
- To explore the mechanisms underlying the enhanced efficacy and persistence of these modified CAR T-cells.
Main Methods:
- A lentiviral two-in-one CAR T approach was engineered using a dual short hairpin RNA cassette to downregulate two checkpoint receptors.
- CD19-targeting CAR T-cells were evaluated with four checkpoint combinations: PD-1/TIM-3, PD-1/LAG-3, PD-1/CTLA-4, and PD-1/TIGIT.
- Functional, phenotypic, and in vivo analyses were performed using patient-derived T-cells and lymphoma models.
Main Results:
- CAR T-cells with combined PD-1 and TIGIT downregulation demonstrated unique synergistic antitumor effects.
- PD-1 downregulation enhanced short-term effector function, while TIGIT downregulation maintained a less differentiated/exhausted state.
- PD-1/TIGIT-downregulated CAR T-cells from diffuse large B cell lymphoma patients showed robust antitumor activity and improved in vivo persistence.
Conclusions:
- Simultaneous downregulation of PD-1 and TIGIT in CD19-targeting CAR T-cells offers a promising strategy to overcome resistance mechanisms.
- This approach enhances both the immediate antitumor response and the long-term persistence of CAR T-cells.
- Clinical trials are underway to evaluate the safety and efficacy of PD-1/TIGIT-downregulated CAR T-cells in patients with relapsed or refractory large B cell lymphoma.
