W157 - Fine Tuning CD19-CAR Sensitivity by Modulating the CD28 Transmembrane and Intracellular Domains

Erica Lana – Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Raphaël Porret – Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Laura Ermellino – Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Ana Alcaraz-Serna – Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Laurent Perez – Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland; Yannick Muller – Department of medicine – Service of Immunology and Allergy, Department of Medicine, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland

Abstract Text: CD19-CAR T cells are emerging as promising therapies, yet exposed to on- and off-target toxicities possibly resulting from the formation of CAR-CD28 heterodimer. Modulations and functional implications of CAR-CD28 interactions have not been thoroughly studied. We engineered 2nd generation CD19-CARs with either a CD28ζ or 4-1BBζ intracellular-domain (ICD) with selected mutations in the CD28 transmembrane-domain (TMD) targeting: (A) the CD28 evolutionary conserved YxxxxT motif (M1a=Y166L, M1b=T171L, M2=LxxxxL), (B) the CD28-core polar amino acids (CYSxxxT->LLLxxxL=M4), (C) the GxxxA dimerization motif (to YxxxL=M5). Control wild-type CD28-TMD-CAR (WT-CAR) was used. CARs were linked to an EGFRt reporter and inserted into the TRAC locus of primary human T cells to control genomic integration. Single-cell sorted clones with increasing CD19 levels were generated. Except for the M5 construct, all four mutants significantly reduced CAR-CD28 formation independently of the ICD assessed by the division index to anti-CD28 mAb stimulation and confirmed using a split fluorescent protein system. Mutating the threonine (M1b) strongly reduced cell-surface CAR expression and sensitivity against CD19 -low but not -high targets. CAR expression was progressively restored in the M2, and M4 CAR construct, respectively, although it remained inferior to WT-CAR and was ICD dependent. Interestingly, the M4-CAR had lower on-target sensitivity than WT-CAR only in the context of a 4-1BBz ICD. Our data suggest that the TMD conformation dictates CAR expression level and its interaction with endogenous CD28. CAR T cell sensitivity can be modulated using selected TMD-ICD pairs, opening the perspectives to engineer safer CAR-T cell products for non-oncological applications.