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Updated: Jan 19, 2026
An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells
Engineered triple inhibitory receptor resistance improves anti-tumor CAR-T cell performance via CD56
Fan Zou1,2,3,4, Lijuan Lu1,2,3, Jun Liu1,2,3
1Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, China.
Abstract:
The inhibitory receptors PD-1, Tim-3, and Lag-3 are highly expressed on tumor-infiltrating lymphocytes and compromise their antitumor activity. For efficient cancer immunotherapy, it is important to prevent chimeric antigen receptor T (CAR-T)-cell exhaustion. Here we downregulate these three checkpoint receptors simultaneously on CAR-T cells and that show the resulting PTL-CAR-T cells undergo epigenetic modifications and better control tumor growth. Furthermore, we unexpectedly find increased tumor infiltration by PTL-CAR-T cells and their clustering between the living and necrotic tumor tissue. Mechanistically, PTL-CAR-T cells upregulate CD56 (NCAM), which is essential for their effector function. The homophilic interaction between intercellular CD56 molecules correlates with enhanced infiltration of CAR-T cells, increased secretion of interferon-γ, and the prolonged survival of CAR-T cells. Ectopically expressed CD56 promotes CAR-T cell survival and antitumor response. Our findings demonstrate that genetic blockade of three checkpoint inhibitory receptors and the resulting high expression of CD56 on CAR-T cells enhances the inhibition of tumor growth.
Insights
Simultaneously blocking PD-1, Tim-3, and Lag-3 on CAR-T cells enhances their antitumor activity. Upregulating CD56 on these modified CAR-T cells improves their survival and tumor control.
Area of Science:
- Immunology
- Oncology
- Cell Biology
Background:
- Inhibitory receptors programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), and Lymphocyte-activation gene 3 (Lag-3) are highly expressed on tumor-infiltrating lymphocytes, impairing their anti-tumor functions.
- Chimeric antigen receptor T (CAR-T)-cell exhaustion is a significant hurdle in effective cancer immunotherapy.
Purpose of the Study:
- To investigate the simultaneous downregulation of PD-1, Tim-3, and Lag-3 on CAR-T cells to enhance anti-tumor activity.
- To explore the epigenetic modifications, tumor infiltration, and effector functions of these modified CAR-T cells.
Main Methods:
- Simultaneous genetic blockade of PD-1, Tim-3, and Lag-3 on CAR-T cells, creating PTL-CAR-T cells.
- Analysis of epigenetic modifications in PTL-CAR-T cells.
- Assessment of tumor infiltration, CD56 (NCAM) expression, interferon-gamma secretion, and CAR-T cell survival in vivo.
- Evaluation of the homophilic interaction of CD56 molecules.
Main Results:
- PTL-CAR-T cells exhibited epigenetic modifications and improved control over tumor growth.
- Unexpectedly, PTL-CAR-T cells showed increased tumor infiltration and clustered at the interface of living and necrotic tumor tissue.
- PTL-CAR-T cells upregulated CD56 (NCAM), which was crucial for their effector functions.
- Homophilic CD56 interactions correlated with enhanced CAR-T cell infiltration, increased interferon-gamma secretion, and prolonged CAR-T cell survival.
Conclusions:
- Genetic blockade of PD-1, Tim-3, and Lag-3, coupled with high CD56 expression, significantly enhances CAR-T cell survival and anti-tumor responses.
- This dual strategy offers a promising approach to overcome CAR-T cell exhaustion and improve cancer immunotherapy efficacy.
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