703. Development of RD-MolPack-trLNGFR/dCK.DM.S74E Stable Packaging Cells

Abstract

The efficacy of several clinical approaches of adoptive T cell therapy (ACT) can be compromised by the insurgence of severe unfavorable events as graft-versus host disease (GvHD) secondary to bone marrow transplantation unless ex-vivo manipulation endow transplantable T cells with safety system. Introduction of drug-induced suicide genes, as for example the herpes simplex virus thymidine kinase (HSV-TK) transgene in T cells during bone marrow transplantation for leukemia and lymphoma treatment, represents a valid approach to control gene-modified cell proliferation and survival overtime. Transplanted modified cells are selectively deleted in vivo by specific activation of an otherwise inactive prodrug in those cells. Despite the well-documented utility of this approach, some limitations arose in terms of poor prodrug activation kinetics, escape from drug selection and immunological rejection.Engineered variants targeting the active site of the human deoxyCytidine Kinase (dCK) suicide enzyme were described as a powerful alternative to current strategies being able to activate a large variety of nucleoside analogue-based prodrugs, having low immunogenicity and permitting in vivo imaging of genetically modified cells. Lentiviral vector (LV)-mediated delivery of the triple mutant dCK.R104M.D133A.S74E (dCK.DM.S74E) fused to the truncated low density nerve growth factor receptor (LNGFR) selection marker renders human cells highly sensitive to the prodrugs bromovinyl-deoxyuridine (brivudine or BVdU), l-deoxythymidine (LdT), and l-deoxyuridine (LdU) via induction of apoptosis. MolMed has developed the proprietary RD-MolPack technology for the stable production of both second and third generation LVs, pseudotyped with the RD114-TR envelope that, in contrast to inducible systems based on VSV-G envelope, permits the generation of a simple, constitutive, no-toxic LV packaging system. We thus exploited the RD-MolPack technology to obtain several colonies of RD-MolPack-trLNGFR/dCK.DM.S74E producer cells by stable transfection of a SIN transfer vector plasmid carrying the trLNGFR/dCK.DM.S74E transgene followed by zeocin selection. The average titer calculated on CEM A3.01 target cells of several colonies is 1.9 × 105 ± 8.15 × 104 TU/106 cells/day, in line with the previous results obtained with the previously established RD2-MolPack-Chim3 and RD3-MolPack-GFP producer cells. These preliminary data suggest that the human suicide gene trLNGFR/dCK.DM.S74E can be constitutively produced in LV stable packaging cells without toxicity and its use can be therefore considered a valid option in the scenario of prodrug convertase enzymes.