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Updated: Oct 13, 2025
Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy
Published on: December 17, 2019
NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity
Rebecca M Richards1, Feifei Zhao2,3,4, Katherine A Freitas5
1Department of Pediatrics, Stanford University School of Medicine, Stanford, California.
Abstract:
Chimeric antigen receptor (CAR) T cells hold promise for the treatment of acute myeloid leukemia (AML), but optimal targets remain to be defined. We demonstrate that CD93 CAR T cells engineered from a novel humanized CD93-specific binder potently kill AML in vitro and in vivo but spare hematopoietic stem and progenitor cells (HSPC). No toxicity is seen in murine models, but CD93 is expressed on human endothelial cells, and CD93 CAR T cells recognize and kill endothelial cell lines. We identify other AML CAR T-cell targets with overlapping expression on endothelial cells, especially in the context of proinflammatory cytokines. To address the challenge of endothelial-specific cross-reactivity, we provide proof of concept for NOT-gated CD93 CAR T cells that circumvent endothelial cell toxicity in a relevant model system. We also identify candidates for combinatorial targeting by profiling the transcriptome of AML and endothelial cells at baseline and after exposure to proinflammatory cytokines.
Significance:
CD93 CAR T cells eliminate AML and spare HSPCs but exert on-target, off-tumor toxicity to endothelial cells. We show coexpression of other AML targets on endothelial cells, introduce a novel NOT-gated strategy to mitigate endothelial toxicity, and demonstrate use of high-dimensional transcriptomic profiling for rational design of combinatorial immunotherapies.See related commentary by Velasquez and Gottschalk, p. 559. This article is highlighted in the In This Issue feature, p. 549.
Insights
Chimeric antigen receptor (CAR) T cells targeting CD93 show promise for acute myeloid leukemia (AML) treatment, effectively killing cancer cells while sparing healthy stem cells. However, they can harm endothelial cells, leading to toxicity. A NOT-gated strategy mitigates this risk.
Area of Science:
- Immunotherapy
- Hematologic Malignancies
- Cellular Therapy
Background:
- Chimeric antigen receptor (CAR) T cells are a promising therapy for acute myeloid leukemia (AML).
- Identifying optimal and safe CAR T cell targets for AML is crucial.
- CD93 is a potential target for CAR T cell therapy in AML.
Purpose of the Study:
- To evaluate CD93 CAR T cells as a potential therapy for AML.
- To assess the safety and efficacy of CD93 CAR T cells, including potential off-target toxicities.
- To develop strategies to mitigate CAR T cell-associated toxicities.
Main Methods:
- Engineered novel humanized CD93-specific CAR T cells.
- Assessed CAR T cell efficacy against AML cell lines and in vivo models.
- Investigated CD93 expression on normal human cells, particularly endothelial cells.
- Utilized transcriptomic profiling to identify combinatorial targets and assess cytokine effects.
Main Results:
- CD93 CAR T cells demonstrated potent killing of AML cells in vitro and in vivo.
- CD93 CAR T cells spared hematopoietic stem and progenitor cells (HSPCs).
- On-target, off-tumor toxicity was observed in human endothelial cells due to CD93 expression.
- A NOT-gated CD93 CAR T cell strategy effectively reduced endothelial cell toxicity.
Conclusions:
- CD93 CAR T cells are effective against AML and spare HSPCs but cause endothelial toxicity.
- Other AML targets also show co-expression on endothelial cells, necessitating careful target selection.
- A novel NOT-gated CAR T cell approach can mitigate endothelial toxicity.
- Transcriptomic profiling aids in designing combinatorial immunotherapies for AML.
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