Stable Expression Of Chimeric Antigen Receptors (CARs) By Sleeping Beauty-Mediated Gene Transfer and Efficient Expansion Of Leukemia-Specific Cytokine-Induced Killer (CIK) Cells

Abstract

Gene-manipulation of effector T cells with CARs has recently turned into a powerful tool to redirect antigen specificity for adoptive immunotherapy of tumors. Although promising clinical efficacy has been demonstrated, critical issues concerning the profile of efficacy, safety and feasibility of cell manufacturing and gene therapy still remain partially unsolved. In order to rescue the concerns associated to viral vectors that limit so far their clinical applicability, we have explored here the use of the latest generation Sleeping Beauty Transposon-mediated gene transfer. Since current protocol of nucleofection associated with Transposons impaired subsequent expansion and vitality of modified cells, we generated and propagated CAR+ cytokine-induced killer (CIK) cells with the purpose of optimizing cell expansion. Actually, our experience with CIK cells clearly proved that the production of large numbers of unmanipulated allogeneic cytotoxic effector T cells is feasible under clinical-grade conditions, and repeated infusions in patients are safe and well tolerated (Introna et al., Haematologica 2007). Using an optimized stimulation protocol based on the addition of accessory cells, irradiated PBMCs, after nucleofection, we genetically modified CIK cells to express two distinct 3rd generation CARs (CD28/OX40/TCR zeta) specific for acute myelogenous leukemia (AML) CD123+ or acute lymphoblastic leukemia (ALL) CD19+ blasts. With this system, the average transfection at 24hours was 54.6% (±8.6, n=8) and mean survival percentage was 63.8% (±8.8, n=12). Nucleofection did not affect the phenotype of CIK cells, and, most importantly, the addition of accessory cells was effective in inducing T-cell expansion, with a fold increase of 39.4±9.8 within 3 weeks, sufficient to be translated into adoptive cell therapy clinical protocols. Transposed CIK cells displayed stable expression of CD123-CAR or CD19-CAR with a frequency of modified cells of 48.9%±3.3 (n=11) and 47%±6.4 (n=4), respectively. Efficient lysis of leukemic cell lines and primary blasts was observed and cytotoxic degranulation was associated to CAR expression, indicating a specific target recognition by the CAR. Interestingly, CAR triggering by the encounter with the specific antigen expressed by leukemic cells promoted specific cytokine secretion and proliferation, suggesting activation and selection of modified CIK cells upon encounter with cancer cells. Finally, preliminary insertion-site analysis by LAM-PCR confirmed the polyclonal profile of integrations in the genome of Sleeping Beauty system. These Results provide pre-clinical evidences of efficient transfection of CD123- and CD19- CARs using Sleeping Beauty-mediated gene transfer, specificity of action and improvements in Methods of expansion of cytotoxic effector T cells. The development of an adoptive cell therapy protocol based on a reproducible clinical-grade method of expansion and an innovative gene transfer process will be fundamental to envisage clinical protocols to control relapse in leukemic patients and to improve the range of applications of such novel therapeutic approaches. Disclosures:No relevant conflicts of interest to declare.