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

By Yannick D. Muller, Duy P. Nguyen, Leonardo Ferreira, Patrick Ho, Caroline Raffin, Roxxana Beltran Valencia, Zion Congrave-Wilson, Theodore Roth, Justin Eyquem, Frederic Van Gool, Alex Marson, James A Wells, Jeffrey A. Bluestone, Qizhi Tang

Posted 19 Sep 2020
bioRxiv DOI: 10.1101/2020.09.18.296913

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. ![Figure][1]</img> ### Competing Interest Statement Conflict of interest. A provisional patent has been submitted. JAB and QT are co-founders of Sonoma Biotherapeutics. AM and TR are co-founders of Arsenal Biosciences. AM is also a co-founder of Spotlight Therapeutics.. JAB and AM have served as advisors to Juno Therapeutics. AM was a member of the scientific advisory board at PACT Pharma and was an advisor to Trizell. QT, JAB, and AM have received sponsored research support from Juno Therapeutics. AM has received research support from Epinomics, Sanofi, GlaxoSmithKline, and gifts from Gilead and Anthem. JAW is co-Founder of Soteria Biotherapeutics developing small molecule switchable biologics, on the SAB of Spotlight, and recipient of sponsored research from Bristol Myers Squibb. JE is an advisor for Mnemo Therapeutics and Cytovia and received research support from Cytovia. The authors declare no other relevant conflict of interest. * CAR : Chimeric Antigen Receptor HD : Hinge domain ICD : Intracellular signaling domain scFv : Single chain variable fragment TMD : Transmembrane domain, [1]: pending:yes

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