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Updated: Jul 15, 2025
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Published on: November 12, 2019
Targeting and cytotoxicity of chimeric antigen receptor T cells grafted with PD1 extramembrane domain
Ang Zhang1,2, Shenyu Wang1,3, Yao Sun3
1Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, 100850, PR China.
Background:
Immunosuppression induced by programmed cell death protein 1 (PD1) presents a significant constraint on the effectiveness of chimeric antigen receptor (CAR)-T therapy. The potential of combining PD1/PDL1 (Programmed cell death 1 ligand 1) axis blockade with CAR-T cell therapy is promising. However, developing a highly efficient and minimally toxic approach requires further exploration. Our attempt to devise a novel CAR structure capable of recognizing both tumor antigens and PDL1 encountered challenges since direct targeting of PDL1 resulted in systemic adverse effects.
Methods:
In this research, we innovatively engineered novel CARs by grafting the PD1 domain into a conventional second-generation (2G) CAR specifically targeting CD19. These CARs exist in two distinct forms: one with PD1 extramembrane domain (EMD) directly linked to a transmembrane domain (TMD), referred to as PE CAR, and the other with PD1 EMD connected to a TMD via a CD8 hinge domain (HD), known as PE8HT CAR. To evaluate their efficacy, we conducted comprehensive assessments of their cytotoxicity, cytokine release, and potential off-target effects both in vitro and in vivo using tumor models that overexpress CD19/PDL1.
Results:
The findings of our study indicate that PE CAR demonstrates enhanced cytotoxicity and reduced cytokine release specifically towards CD19 + PDL1 + tumor cells, without off-target effects to CD19-PDL1 + tumor cells, in contrast to 2G CAR-T cells. Additionally, PE CAR showed ameliorative differentiation, exhaustion, and apoptosis phenotypes as assessed by flow cytometry, RNA-sequencing, and metabolic parameter analysis, after encountering CD19 + PDL1 + tumor cells.
Conclusion:
Our results revealed that CAR grafted with PD1 exhibits enhanced antitumor activity with lower cytokine release and no PD1-related off-target toxicity in tumor models that overexpress CD19 and PDL1. These findings suggest that our CAR design holds the potential for effectively addressing the PD1 signal.
Insights
Engineered chimeric antigen receptor (CAR)-T cells targeting CD19 and Programmed cell death 1 ligand 1 (PDL1) show improved antitumor activity. This novel CAR design effectively overcomes PD1-mediated immunosuppression with reduced toxicity.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Programmed cell death protein 1 (PD1) mediated immunosuppression limits chimeric antigen receptor (CAR)-T cell therapy efficacy.
- Combining PD1/PDL1 axis blockade with CAR-T therapy is promising but requires optimized, low-toxicity approaches.
- Directly targeting PDL1 can cause systemic adverse effects, necessitating alternative strategies.
Purpose of the Study:
- To engineer novel CAR structures that recognize both tumor antigens and PDL1.
- To evaluate the efficacy and safety of CARs engineered with PD1 domains.
- To assess the potential of these novel CARs in overcoming PD1-mediated immunosuppression in CD19-positive, PDL1-positive tumors.
Main Methods:
- Engineered novel CARs by grafting the PD1 domain into a CD19-targeting second-generation (2G) CAR.
- Developed two CAR variants: PE CAR (PD1 extramembrane domain directly linked to transmembrane domain) and PE8HT CAR (PD1 EMD linked via CD8 hinge domain).
- Assessed CAR efficacy, including cytotoxicity, cytokine release, and off-target effects, in vitro and in vivo using CD19/PDL1-overexpressing tumor models.
Main Results:
- PE CAR demonstrated enhanced cytotoxicity and reduced cytokine release against CD19+/PDL1+ tumor cells compared to 2G CAR-T cells.
- PE CAR showed no off-target effects on CD19-/PDL1+ tumor cells.
- Flow cytometry, RNA-sequencing, and metabolic analysis revealed improved differentiation, reduced exhaustion, and apoptosis phenotypes in PE CAR-T cells after encountering target cells.
Conclusions:
- CARs grafted with PD1 exhibit enhanced antitumor activity with reduced cytokine release and no PD1-related off-target toxicity.
- The novel CAR design effectively addresses PD1-mediated immunosuppression in CD19 and PDL1 co-overexpressing tumor models.
- This strategy holds potential for improving CAR-T cell therapy outcomes in relevant cancers.
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