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Systematic optimization of PD-L1 and CLDN18.2 CAR-T designs identifies a bicistronic dual-target, double-CD3ζ
Xiang Zhang1,2,3,4, Yao-Jie Kong1, Jing-Yao Li1
1School of Medicine, Shanghai University, Shanghai, China.
Background:
Gastric cancer is a leading cause of cancer-related mortality, with limited efficacy of immunotherapies due to intratumoral heterogeneity, an immunosuppressive microenvironment, and antigen-loss escape. The optimization of chimeric antigen receptor (CAR) T-cell therapy targeting PD-L1 and Claudin18.2 (CLDN18.2) is crucial for improving the antitumor response.
Methods:
We performed a stepwise optimization of CAR designs targeting PD-L1 and CLDN18.2, focusing on the costimulatory domains and intracellular signaling architecture. We engineered monospecific PD-L1 and CLDN18.2 CAR-T cells using CD28 or 4-1BB costimulatory domains and assessed their CAR expression, activation phenotype, subset composition, and cytotoxicity. We then engineered three bicistronic dual-target CARs with distinct CD3ζ configurations and evaluated their functionality through in vitro assays and in vivo NUGC4-Luc gastric cancer xenograft models.
Results:
The single-target CLDN18.2-CD28 CAR-T cells exhibited the strongest cytotoxicity in vitro. Among the bicistronic designs, the double-CD3ζ CAR-T cells showed superior expansion, differentiation, and cytotoxicity compared to alternative bicistronic constructs and single-target CAR-T cells. Bulk RNA sequencing revealed enhanced immune activation and reduced exhaustion markers in the double-CD3ζ CAR-T cells. In the NUGC4-Luc xenograft model, the double-CD3ζ CAR-T cells achieved a tumor growth inhibition of 63.23%, outperforming the 48.06% inhibition observed with single CLDN18.2 CAR-T cells.
Conclusions:
The study identifies a bicistronic dual-target, double-CD3ζ CAR-T design that enhances T-cell functional fitness and antitumor efficacy in gastric cancer models. This approach offers a promising strategy for addressing antigen heterogeneity and improving the durability of CAR-T therapies in solid tumors.
Insights
Optimized chimeric antigen receptor (CAR) T-cell therapy targeting PD-L1 and Claudin18.2 (CLDN18.2) shows improved efficacy in gastric cancer. A novel dual-target CAR with double CD3ζ signaling enhances T-cell function and antitumor response.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Gastric cancer poses a significant mortality risk, with current immunotherapies facing challenges from tumor heterogeneity and immunosuppression.
- Chimeric antigen receptor (CAR) T-cell therapy optimization is vital for overcoming these limitations in gastric cancer treatment.
Purpose of the Study:
- To engineer and optimize CAR T-cell designs targeting PD-L1 and Claudin18.2 (CLDN18.2) for enhanced gastric cancer treatment.
- To evaluate the efficacy of monospecific and bicistronic dual-target CAR T-cells in preclinical gastric cancer models.
Main Methods:
- Stepwise optimization of CAR designs, focusing on costimulatory domains (CD28, 4-1BB) and intracellular signaling (CD3ζ).
- Engineering and assessment of monospecific PD-L1 and CLDN18.2 CAR-T cells, followed by development of bicistronic dual-target CARs.
- In vitro functional assays and in vivo evaluation using NUGC4-Luc gastric cancer xenograft models.
Main Results:
- Single-target CLDN18.2-CD28 CAR-T cells demonstrated potent in vitro cytotoxicity.
- Bicistronic dual-target CAR-T cells with double CD3ζ signaling exhibited superior expansion, differentiation, and cytotoxicity compared to single-target CARs.
- In vivo studies showed the double CD3ζ CAR-T cells achieved 63.23% tumor growth inhibition, outperforming single-target CARs.
Conclusions:
- A bicistronic dual-target, double-CD3ζ CAR-T design significantly enhances T-cell functional fitness and antitumor efficacy in gastric cancer models.
- This optimized CAR T-cell strategy holds promise for overcoming antigen heterogeneity and improving the durability of immunotherapy for solid tumors.
