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Exploring the Effects of Chimeric Antigen Receptor Sequence on CAR-T Cell Function by Rational Protein Design


Adoptive T-cell therapy has emerged as one of the most promising treatment modalities for cancer. CD19 chimeric antigen receptor (CAR)-T cell therapy has shown remarkable success in treating hematological malignancies, but robust clinical efficacy has not yet been observed with the vast majority of CARs targeting other tumor-associated antigens. Several recent reports suggest that antigen-independent, tonic signaling by CARs may be detrimental to T-cell function, and that the CD19 CAR’s unique potency may be attributed to its lack of tonic signaling. Our goal is to study the impact of tonic signaling on CAR-T cell function and investigate protein engineering strategies to optimize non-CD19 CAR design. Here, we report that robust CAR-T cell function in vivo can be achieved by applying rational protein engineering to achieve a non-zero, calibrated level of tonic signaling. We also discovered a generalizable strategy to fine-tune CAR signaling by imposing structural changes through the insertion of alanine residues between the transmembrane and cytoplasmic domains of the CAR. The insertion of two alanines yielded superior CAR variants from a rituximab-based anti-CD20 CAR and a 14g2a-based GD2 CAR. In addition, we found that CAR tonic signaling could be tuned through sequence hybridization of single-chain variable fragment (scFv) domains. A hybrid CAR incorporating a hybridized scFv sequence derived from the anti-CD20 antibodies rituximab and leu16 demonstrated superior anti-tumor function compared to both of the parental CARs, resulting in the first example of a CD20 CAR that outperformed a CD19 CAR in the Raji xenograft model to our knowledge. Through transcriptomic, epigenetic and metabolomic studies, we observed that CAR tonic signaling was associated with an upregulation of metabolic activity, which likely potentiates rapid effector function upon antigen stimulation, but could also lead to early exhaustion. Tuning of tonic-signaling intensity through alanine insertion and scFv hybridization yielded CARs that enable T cells to both quickly eliminate established tumors and maintain long-term tumor clearance in vivo. RNA-seq and ATAC-seq analysis further indicated that the two protein-engineering strategies explored in our study could yield synergistic effects on improving CAR-T cell function. Our study reveals the importance of tonic-signaling intensity tuning as a previously unknown feature of CAR-T cell biology, and provides generalizable strategies for novel, next-generation CAR design.

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