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Tuning the Antigen Density Requirement for CAR T-cell Activity
Robbie G Majzner1,2, Skyler P Rietberg2, Elena Sotillo2
1Department of Pediatrics, Stanford University School of Medicine, Stanford, California.
Abstract:
Insufficient reactivity against cells with low antigen density has emerged as an important cause of chimeric antigen receptor (CAR) T-cell resistance. Little is known about factors that modulate the threshold for antigen recognition. We demonstrate that CD19 CAR activity is dependent upon antigen density and that the CAR construct in axicabtagene ciloleucel (CD19-CD28ζ) outperforms that in tisagenlecleucel (CD19-4-1BBζ) against antigen-low tumors. Enhancing signal strength by including additional immunoreceptor tyrosine-based activation motifs (ITAM) in the CAR enables recognition of low-antigen-density cells, whereas ITAM deletions blunt signal and increase the antigen density threshold. Furthermore, replacement of the CD8 hinge-transmembrane (H/T) region of a 4-1BBζ CAR with a CD28-H/T lowers the threshold for CAR reactivity despite identical signaling molecules. CARs incorporating a CD28-H/T demonstrate a more stable and efficient immunologic synapse. Precise design of CARs can tune the threshold for antigen recognition and endow 4-1BBζ-CARs with enhanced capacity to recognize antigen-low targets while retaining a superior capacity for persistence. SIGNIFICANCE: Optimal CAR T-cell activity is dependent on antigen density, which is variable in many cancers, including lymphoma and solid tumors. CD28ζ-CARs outperform 4-1BBζ-CARs when antigen density is low. However, 4-1BBζ-CARs can be reengineered to enhance activity against low-antigen-density tumors while maintaining their unique capacity for persistence.This article is highlighted in the In This Issue feature, p. 627.
Insights
Chimeric antigen receptor (CAR) T-cell therapy faces challenges with low-antigen-density tumors. Optimizing CAR construct design, particularly the CD28 hinge-transmembrane region, can improve T-cell recognition and persistence against these difficult targets.
Area of Science:
- Immunology
- Cancer Biology
- Biotechnology
Background:
- Chimeric antigen receptor (CAR) T-cell therapy shows promise but faces resistance due to insufficient reactivity against cancer cells with low antigen density.
- Understanding the factors that influence the antigen recognition threshold for CAR T-cells is crucial for improving therapeutic efficacy.
Purpose of the Study:
- To investigate how CAR construct design modulates antigen recognition thresholds.
- To compare the efficacy of different CAR constructs against tumors with varying antigen densities.
- To explore strategies for re-engineering CAR T-cells to overcome resistance in low-antigen-density tumors.
Main Methods:
- Comparative analysis of CD19 CAR constructs (axicabtagene ciloleucel vs. tisagenlecleucel) against antigen-low tumor models.
- Modulation of CAR signaling strength by altering immunoreceptor tyrosine-based activation motifs (ITAMs).
- Investigating the impact of hinge-transmembrane (H/T) regions (CD8 vs. CD28) on CAR T-cell function and immunologic synapse formation.
Main Results:
- CAR T-cell activity is significantly dependent on target antigen density.
- Axicabtagene ciloleucel (CD19-CD28ζ) demonstrated superior activity against antigen-low tumors compared to tisagenlecleucel (CD19-4-1BBζ).
- Enhancing ITAMs increased CAR T-cell recognition of low-antigen-density cells, while ITAM deletions increased the antigen threshold. Replacing the CD8 H/T region with CD28 H/T in a 4-1BBζ CAR lowered the reactivity threshold and improved synapse stability.
Conclusions:
- CAR T-cell efficacy against tumors is critically influenced by antigen density.
- CAR construct engineering, specifically the hinge-transmembrane region and ITAM content, can precisely tune antigen recognition thresholds.
- Strategies exist to enhance 4-1BBζ-based CAR T-cells for improved recognition of low-antigen-density tumors while preserving their persistence advantage.
