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bioRxiv · 10.1101/2020.09.18.296913

The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

Muller, Y.·Nguyen, D.·Ferreira, L. M. R.·Ho, P.·Raffin, C.·Beltran Valencia, R.·Congrave-Wilson, Z.·Roth, T.·Eyquem, J.·Van Gool, F.·Marson, A. M. R.·Wells, J. A.·Bluestone, J. A.·Tang, Q.
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Abstract

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge (HD) and transmembrane (TMD) domains between the extracellular antigen-targeting and the intracellular signaling modalities of CARs has not been systemically studied. Here, a series of CD19-CARs differing only by their HD (CD8/CD28/IgG4) and TMD (CD8/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 to anti-CD28 stimulation. This dimerization depended on polar amino-acids in the CD28-TMD. CD28-CAR heterodimerization was more efficient in CARs containing a CD8-HD or CD28-HD as compared to an IgG4-HD. CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but led to a significant reduction of CD28 cell-surface expression. These data unveil a new property of the CD28-TMD and suggest that TMDs can modulate CAR T-cell activities by engaging endogenous partners. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/296913v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@bcd17aorg.highwire.dtl.DTLVardef@5cbca9org.highwire.dtl.DTLVardef@660eecorg.highwire.dtl.DTLVardef@5db163_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Muller, Y., Nguyen, D., Ferreira, L. M. R., Ho, P., Raffin, C., Beltran Valencia, R., Congrave-Wilson, Z., Roth, T., Eyquem, J., Van Gool, F., Marson, A. M. R., Wells, J. A., Bluestone, J. A., Tang, Q.. 2020-09-19. The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28. https://doi.org/10.1101/2020.09.18.296913

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