Physiological (TCR-like) regulated lentiviral vectors: a platform for the generation of improved CAR-T cells

M Tristán-Manzano(1) N Maldonado-Pérez(2) P L Justicia-Lirio(1,2) C Barbero-Jiménez(2) P Muñoz(2) M Cortijo-Gutiérrez(2) K Pavlovic(2,3) A Aguilar-González(2,4) A Hinckley(1) C Blanco-Benítez(1) M Castella(5) M Juan(5) C Marañon(2) J A Marchal(6) K Benabdellah(2) C Herrera(3) F Martín(2,7)

1:LentiStem Biotech.; 2:Department of Genomic Medicine, GENYO.; 3:IMIBIC, Cellular Therapy Unit, Reina Sofia University Hospital, University of Córdoba.; 4:Department of Medicinal and Organic Chemistry, University of Granada.; 5:Department of Hematology, ICMHO, Hospital Clínic de Barcelona.; 6:Department of Human Anatomy and Embryology, University of Granada.; 7:Departament of Biochemistry, Molecular Biology and Immunology III, University of Granada.

Anti-CD19-CAR-T cells for B malignancies have provide impressive clinical responses. However, important limitations still remain due to severe adverse events and relapse of ~50% of the treated patients. Several studies indicate that expressing the CAR following the natural behaviour of T cell receptor (TCR-like) can improve CAR-T cell’s fitness and their antitumor efficacy. However, different levels of CAR basal expression are required depending on the antigen’s density of target cells or CAR’s affinity. Here, we propose to use different TCR-like lentiviral vectors (LVs) to drive diverse expression levels as a platform to express different CARs. First, using a synthetic WAS-gene promoter, we generated LVs for driven low/moderate transgene expression, following TCR-like pattern and which were applied to the ARI-0001 academic CAR to create AWARI-CAR-T cells. AWARI-CAR-T cells exhibited lower tonic signalling, higher proportion of stem/memory TSCM, less exhausted phenotype and milder secretion of TNF-α and IFN-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo. Moreover, we also showed their improved efficacy using an in vitro CD19+ pancreatic tumour model. We next designed a panel of novel LVs for transgene expression through different chimeric promoters (CD4, B2M and LCK loci) with different strengths. All the generated-LVs mimicked the TCR/CD3 pattern after stimulation at different levels, although the B2M-LV was selected for its strong basal expression and quicker downregulation upon T cell activation. In summary, we present here two different platforms for CAR expression on T cells at different levels and with a TCR-like behaviour.