CAR Linker Length Modulates CD22-Targeted CAR T-cell Efficacy in ALL

A variety of chimeric antigen receptor (CAR) T-cell therapies have been engineered and are undergoing thorough studies, but potential effects of the linker that joins the V_L and V_H regions of the antigen-recognition domain have not been extensively explored. After comparing the disappointing efficacy seen in two clinical trials of CD22-directed CAR T cells in which the CAR had a 20–amino acid residue linker (with four repeats of GGGGS) in patients with acute lymphoblastic leukemia (ALL) with the promising results observed with a CAR that differed only in that it contained a five–amino acid residue linker (with only one GGGGS sequence) in patients with ALL, Singh, Frey, Engels, and colleagues investigated the possible impacts of these linkers. In vitro experiments revealed that linker length had minimal effects on CAR affinity for CD22, ruling out target-antigen affinity as a cause for the difference in efficacy. Further probing revealed that the CAR with a long linker existed as a monomer, whereas the CAR with a short linker formed homodimers, leading to clustering on the cell surface. This clustering had functional relevance: Upon activation, clustered CARs triggered greater phosphorylation of T-cell signaling proteins of the PI3K and MAPK pathways along with higher p53 activation than non-clustered CARs. These findings held true for CARs directed toward CD33 but not those directed toward CD19. Indicating that they promote strong immune synapse formation and downstream receptor activation, CD22-directed CAR T cells with short linkers exhibited greater effector function, as evidenced by increases in perforin accumulation at immune synapses along with greater phosphorylation of the T-cell receptor subunit CD3ζ and the downstream signaling protein ZAP70. In vitro coculture experiments and in vivo analyses revealed that CD22-directed CAR T cells with short linkers were more cytotoxic toward leukemia cells and improved disease control (leading to extended survival), respectively, relative to CD22-targeted CAR T cells with long linkers. Collectively, these findings reveal unforeseen impacts of seemingly minor modifications to CAR structure, highlighting how much remains to be learned about this type of therapy.

Singh N, Frey NV, Engels B, Barrett DM, Shestova O, Ravikumar P, et al. Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells. Nat Med 2021;27:842–50.

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