PF436 GMP MANUFACTURING OF ALLOGENEIC CD19 CHIMERIC ANTIGEN RECEPTOR (CAR) CYTOKINE INDUCED KILLER (CIK) CELLS WITH SLEEPING BEAUTY (SB) TRANSPOSON FOR ADOPTIVE IMMUNOTHERAPY
Authors: C.F. Magnani, G. Gaipa, D. Belotti, G. Matera, S. Tettamanti, B. Cabiati, S. Cesana, V. Colombo, G. Cazzaniga, G. Fazio, C. Buracchi, S. Rigamonti, A. Rovelli, A. Balduzzi, S. Napolitano, E. Montini, G.M. Borleri, G. Gritti, F. Lussana, M. Introna, A. Rambaldi, G. Dastoli, A. Biondi
Journal: HemaSphere (2019)

Background: Immunotherapy using patient-derived T cells engineered to express a chimeric antigen receptor (CAR) by viral vectors has achieved complete remission and durable response in highly refractory populations. Unmodified allogeneic Cytokine Induced Killer (CIK) cells (CD3+CD56+ T cells) have clearly demonstrated a high profile of safety in ALL patients. Aims: Here, we demonstrate the feasibility and reproducibility of a good manufacturing practices (GMP)-compliant culture of allogeneic CIK cells modified by non-viral Sleeping Beauty (SB) transposon to obtain CD19CAR T cells for the clinical application. Preliminary analysis of CARCIK-CD19 cellular kinetic in 6 patients are also reported. Methods: PBMCs were electro-transferred with the SB GMP-grade CD19.CAR/pTMNDU3 plasmid and pCMV-SB11 plasmid (kindly provided by L. Cooper, Houston). CIK cells were then generated according to the method enclosed in the filed patent EP20140192371. The manufacturing process were performed in a academic cell factory authorized by Agenzia Italiana del Farmaco (AIFA). CARCIK-CD19 were infused in pediatric and adult B-ALL patients relapsed post transplantation after standard lymphodepletion. Results: We manufactured ten batches by seeding a median of 103.16x106 allogeneic PBMCs derived from 50 ml of PB. After 20–28 days of culture (median 22) we harvested a median of 3.6x109 nucleated cells (range 1.40 – 15.75x109). At the end of expansion, cell viability was 97.24% (range 91.99%>98.96%), manufactured cells were mostly CD3+ lymphocytes (mean 98.73% ± SD 1.55%). Of these, 46.17% ± 17.92% were CAR+ and 43.89% ± 10.13% were CD56+, while median fold increase was 176.6 (37.0–1350) and had a median vector copy number (VCN) of 3.5 VCN/cells. In all the ten batches, CARCIK-CD19 cells demonstrated potent and specific in vitro cytotoxicity towards the CD19+ REH target cell line (mean 80.68%, range 61.89%>97.72%). Cell products appear to be highly polyclonal and no signs of genotoxicity by transposon insertions could be observed by integration site (IS) analysis performed using Sonication Linker Mediated (SLiM)-PCR. All the batches were released after about 10 days after the end of production. The quality requirements for batch release were met in all 10 productions. CARCIK-CD19 achieved robust expansion in the majority of the patients as defined by detectable CAR T-cell detection by VCN (range 4645–343403 transgene copies/ug) and flow (range 0.5–30%) in the blood. The median time to peak engraftment in peripheral blood was 14 days. The magnitude of expansion in peripheral blood correlate with the disease burden at the time of product infusion in the marrow. Summary/Conclusion: Overall, these results demonstrate that clinical-grade SB transduction of allogeneic CIK cells with CD19 CAR is feasible and allows efficient expansion of CARCIK-CD19 cells starting from easily available small amounts of PB, with important implications for non-viral technology. A clinical trial investigating allogeneic CARCIK-CD19 in r/r pediatric and adult ALL post HSCT is currently ongoing and demonstrates expansion of CARCIK-CD19 post infusion (NCT03389035).