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Chimeric antigen receptors (CARs) incorporating mutations in the IgG4 Fc spacer region to eliminate Fc receptor recognition results in improved CAR T cell persistence and anti-tumor efficacy

Adoptive immunotherapy using T cells genetically redirected via expression of chimeric antigen receptors (CARs) is a promising approach for cancer treatment. However, this immunotherapy is dependent in part on the optimal molecular design of the CAR, which involves an extracellular ligand-binding domain connected to an intracellular signaling domain by spacer and/or transmembrane sequences. CAR designs frequently incorporate extracellular linker regions based on the immunoglobulin constant regions of either IgG1 or IgG4. In this study we evaluated the potential for the IgG4-Fc linker to result in off-target interactions between the CAR and Fc gamma receptors (FcγRs). As proof of principle, we have focused on a CD19-specific CD19scFv-IgG4-CD28-zeta CAR, and indeed found that CAR+ T cells bound to soluble FcγRs, and did not engraft in NSG mice compared to CAR-negative T cells that only expressed an EGFRt tracking marker. We hypothesized that mutations to avoid FcγR interactions would improve CAR+ T cell persistence and anti-tumor efficacy. To this end, we generated a CD19-specific CAR that has been mutated at two sites within the CH2 region (L235E; N297Q) of the IgG4 Fc spacer, here called CD19R(EQ), as well as a CD19-specific CAR that has a CH2 deletion in its IgG4 Fc spacer (CD19Rch2Δ). These mutations/deletion do not alter the functional ability of the CAR, when expressed by T cells, to mediate antigen-specific lysis of tumor cells. However, compared to T cells that express a non-mutated CAR, T cells expressing the CD19R(EQ) and CD19Rch2Δ exhibit impaired binding to recombinant soluble FcγRs. These CD19R(EQ) and CD19Rch2Δ T cells also exhibit improved engraftment in NSG mice. Indeed the engraftment levels seen with the mutated CAR were similar to that seen with CAR-negative T cells that only expressed the EGFRt tracking marker. Importantly, elimination of CAR/FcγR interactions also significantly improves CD19-specific CAR+ T cell anti-lymphoma efficacy in NSG mice. These studies provide evidence that optimal CAR function necessitates the elimination of cellular FcγR interactions in order to improve T cell persistence and anti-tumor responses.

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Correspondence to Mahesh Jonnalagadda.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Chimeric Antigen Receptor
  • Adoptive Immunotherapy
  • Impaired Binding
  • Transmembrane Sequence
  • Intracellular Signaling Domain