HIgh efficiency protocol for lymphocytes gene modification by lentiviral vector within an automated closed system

Abstract

Background & Aim Gene therapy with genetically modified lymphocytes has been progressively entering in the clinical settings mainly as clinical trials and recently also with approved CAR-T cells. This therapeutic approach holds great promises for cancer therapy but is also limited by the current manufacturing methods. Most protocols rely on transduction with viral vectors followed by cell expansion; this method requires lengthy process optimization on the vector and on the transduction conditions and is often limited by the cell number that needs to be achieved for a clinical dose. In addition, variability of the cell product from batch to batch and manufacturing costs need to be taken into account. Our group isolated TCRs targeting viral antigens and is in the process of isolating TCRs targeting cancer neoantigens; we intend to develop a process for transduction of T-cells that will guarantee us a high and robust level of exogenous TCRs expression coupled with a significant cell yield. TCR-T cells manufactured with our new method will be used for adoptive cell therapy trials in cancer patients and patients affected by chronic viral hepatitis. Methods, Results & Conclusion PBMC were obtained from healthy donor buffy coat, CD4+ and CD8+ cells were enriched, activated and expanded in the CliniMacs Prodigy (Miltenyi) system. The TCT process provided by Miltenyi has been modified to increase its overall efficiency with our model lentiviral vector (pSSLSIN.cppt.MSCV-GFP). Parameters like cells number, percentage of transduced cells and cell phenotype were used to identify the best protocol. CD3/CD4 and CD3/CD8 enrichment was on average 1.5 fold compared to the initial starting material. The optimal time for lentiviral transduction was identified at 24h after cell activation with TransAct (Miltenyi). After 7 days, the gene modification efficiency was evaluated by flow cytometry and was not less 60% with high viability (96%) of the transduced cells and the expected phenotypes (CD3/CD4/CD8). We subsequently focused on cell yield, and starting from 100 × 106 cells we established protocol that allowed us to generate 2,3 × 109 cells in 10 days and with more than 95% of viability. Based on this optimized protocol, we are now transducing T-cells with a vector encoding TCRs for hepatitis C virus antigens, specifically the non-structural 3 and 5 antigens (NS3/NS5). We here report an optimized GMP-compliant protocol to generate high number of TCR-modified T cells that can be used for adoptive cell immunotherapy.