Peptide-scFv antigen recognition domains effectively confer CAR T cell multiantigen specificity

Summary

The emergence of immune escape is a significant roadblock to developing effective chimeric antigen receptor (CAR) T cell therapies against hematological malignancies, including acute myeloid leukemia (AML). Here, we demonstrate feasibility of targeting two antigens simultaneously by combining a GRP78-specific peptide antigen recognition domain with a CD123-specific scFv to generate a peptide-scFv bispecific antigen recognition domain (78.123). To achieve this, we test linkers with varying length and flexibility and perform immunophenotypic and functional characterization. We demonstrate that bispecific CAR T cells successfully recognize and kill tumor cells that express GRP78, CD123, or both antigens and have improved antitumor activity compared to their monospecific counterparts when both antigens are expressed. Protein structure prediction suggests that linker length and compactness influence the functionality of the generated bispecific CARs. Thus, we present a bispecific CAR design strategy to prevent immune escape in AML that can be extended to other peptide-scFv combinations.

Graphical abstract

Highlights

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  The most effective strategy to target multiple antigens remains elusive

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  Peptide-scFv antigen recognition domains allow for effective dual-antigen targeting

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  Structural configuration impacts antigen accessibility and CAR effector function

Abstract

Zoine et al. engineer a peptide-scFv antigen recognition domain as part of a bispecific chimeric antigen receptor (CAR). They utilize a systematic approach to design and test combinations by performing structural and functional evaluation. Their bispecific CAR demonstrates antitumor activity in vitro and in vivo while maintaining dual-antigen specificity.