The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28

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.

Precision Engineering of an Anti-HLA-A2 Chimeric Antigen Receptor in Regulatory T Cells for Transplant Immune Tolerance

“…A previous study has shown that site-specific integration of a CD19-CAR into the TCR alpha constant region ( TRAC ) of T cells results in a more uniform distribution and TCR-like regulation of CAR surface expression, thereby mitigating T-cell exhaustion and enhancing anti-tumor activity ( 21 ). In addition, we recently observed that CAR hi human T effector cells exhibited a surprisingly robust proliferative response to anti-CD28 stimulation alone, independent of CAR or TCR engagement, whereas CAR lo T effector cells did not ( 22 ). Thus, lentivirally engineered Tregs may result in heterogeneous CAR expression and unexpected properties of the engineered cells.…”

Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

“…A previous study has shown that site-specific integration of a CD19-CAR into the TCR alpha constant region ( TRAC ) of T cells results in a more uniform distribution and TCR-like regulation of CAR surface expression, thereby mitigating T-cell exhaustion and enhancing anti-tumor activity (13). In addition, we recently observed that CAR hi human T cells exhibited a surprisingly robust proliferative response to anti-CD28 stimulation alone, independent of CAR or TCR engagement, whereas CAR lo T cells did not (14). Thus, lentivirally engineered Tregs may result in heterogeneous CAR expression and unexpected properties of the engineered cells.…”

CAR T-Cell Therapy: Is CD28-CAR Heterodimerization Its Achilles’ Heel?

“…Crystal Mackall and co-workers demonstrated that swapping the CD8-TMD/HD in a CD19 4-1BB-ζ CAR for a CD28-TMD-HD lowered the antigen density threshold for CAR T-cell activation ( 14 ). Finally, we have recently demonstrated that CD28 TMD-containing CARs can recruit and dimerize with endogenous CD28, which normally exists as a homodimer on the cell surface, via a four amino acid motif in the TMD ( 15 , 16 ). Consistent with this, in-depth analysis of the CAR interactome and signalosome revealed that the top interacting partner of a CAR bearing a CD28-TMD/HD is endogenous CD28, and CAR mediated-signaling is associated with phosphorylation of endogenous CD28 ( 9 , 17 ).…”

“…Consistent with this, in-depth analysis of the CAR interactome and signalosome revealed that the top interacting partner of a CAR bearing a CD28-TMD/HD is endogenous CD28, and CAR mediated-signaling is associated with phosphorylation of endogenous CD28 ( 9 , 17 ). This association, through heterodimerization of the CAR with endogenous CD28 receptor via the CD28-TMD ( 15 ), may result in stronger signal transduction, facilitating CAR T-cell activation in the context of low levels of CAR antigen, such as in low-CD19 mural cells. It is interesting to note that CD28-CAR heterodimerizes inefficiently if the CAR is built with an IgG4-HD.…”

“…It is interesting to note that CD28-CAR heterodimerizes inefficiently if the CAR is built with an IgG4-HD. In silico modeling of the hinge-hinge interactions suggested that the membrane proximity of the IgG4 hinge is too short to form CAR-CD28 inter-molecular disulfide bonds for stabilizing the CAR-CD28 heterodimerization, leading to preferential CAR-homodimerization ( 15 ). This observation may explain why JCAR-14/-17, engineered with a CD28-TMD and IgG4-HD, caused less ICANS than KTE-C19/KTE-X19 or JCAR-15 ( Figures 1B, C ).…”