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Updated: Jan 19, 2026
A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Published on: November 12, 2019
Multispecific Targeting with Synthetic Ankyrin Repeat Motif Chimeric Antigen Receptors
Ashwini Balakrishnan1, Anusha Rajan1, Alexander I Salter1,2
1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.
Purpose:
The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition.
Experimental Design:
A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo.
Results:
The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen.
Conclusions:
DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.
Insights
Engineered T cells using Designed Ankyrin Repeat Proteins (DARPins) in chimeric antigen receptors (CARs) overcome tumor escape. This multispecific CAR approach enhances efficacy against heterogeneous cancers by targeting multiple antigens, reducing antigen-negative variant outgrowth.
Area of Science:
- Immunotherapy
- Cancer Biology
- Molecular Engineering
Background:
- Adoptive T cell therapy faces challenges from antigen-negative tumor escape variants.
- Chimeric antigen receptors (CARs) typically use single-chain variable fragments (scFvs) for antigen recognition.
Purpose of the Study:
- To design and evaluate CARs utilizing Designed Ankyrin Repeat Proteins (DARPins) for enhanced tumor recognition and overcoming antigen escape.
- To compare DARPin-based CARs with traditional scFv-based CARs.
Main Methods:
- A monospecific CAR with an anti-EGFR DARPin (E01) was created and compared to an anti-EGFR scFv CAR.
- Multispecific CARs were engineered by linking DARPins targeting EGFR, EpCAM, and HER2.
- In vivo efficacy of DARPin CAR-T cells against heterogeneous tumors was assessed.
Main Results:
- The monospecific anti-EGFR DARPin CAR demonstrated potent tumor regression, comparable to the scFv CAR.
- Linking three DARPins into a single CAR was feasible and effectively targeted heterogeneous tumor cells.
- The multispecific DARPin CAR exhibited synergistic activity when tumor cells expressed multiple target antigens.
Conclusions:
- DARPins are effective high-affinity recognition motifs for CAR design.
- The modularity of DARPins allows for the creation of multispecific CARs, enhancing functional synergy and reducing tumor antigen escape.
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