Abstract
Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge domain (HD) and the transmembrane domain (TMD) between the extracellular antigen-targeting CARs and the intracellular signaling modalities of CARs has not been systemically studied. In this study, a series of 19-CARs differing only by their HD (CD8, CD28, or IgG4) and TMD (CD8 or CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation of anti-CD28 stimulation. This dimerization was dependent on polar amino acids in the CD28-TMD and was more efficient with CARs containing CD28 or CD8 HD than IgG4-HD. The CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but had a significantly reduced CD28 cell-surface expression. These data unveiled a fundamental difference between CD28-TMD and CD8-TMD and indicated that CD28-TMD can modulate CAR T-cell activities by engaging endogenous partners.
Highlights
Chimeric antigen receptor (CAR)-engineered T cells are emerging as promising therapies for otherwise untreatable diseases [1]
To investigate the role of CAR transmembrane domain (TMD), we first generated a panel of 19-CARs differing only by their hinge domain (HD) (CD8, CD28, or IgG4) and their TMD (CD8 vs. CD28), all of which have been used to engineer CAR T cells for clinical applications (Figure 1A, Supplementary Figure 1)
We discovered that the CD28-TMD mediates CAR and CD28 heterodimerization via a core of up to four polar amino acids
Summary
Chimeric antigen receptor (CAR)-engineered T cells are emerging as promising therapies for otherwise untreatable diseases [1]. A third CAR-T product, lisocabtagene maraleucel (JCAR-17, LISO-CEL, Bristol-Myers Squibb), is currently under review by the FDA for adults with relapsed/refractory large B-cell lymphoma. The success of these CAR-T products can be attributed to their antigen specificity, all conferred by the single-chain variable fragment (scFv) of the anti-CD19 antibody clone FMC63. Majzner et al [12] demonstrated that replacing a CD8HD/TMD with a CD28-HD/TMD lowers the threshold for CAR activation to CD19 in an ICD-independent fashion These results corroborate the findings reported by Kochenderfer et al and show that T cells with CD28-HD/TMD-containing CARs secrete higher levels of interferon-γ upon CAR stimulation [13, 14]
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