Automated Manufacture of CAR19 Donor Lymphocytes Incorporated into an Escalated Dose DLI Protocol: Preliminary Results from the CARD Study

Introduction: Clinical studies of 2nd generation CD19 Chimeric Antigen Receptor(CAR) T-cellshave delivered unprecedented clinical responses in patients with B-ALL. Increasing demand for CAR T-cellsplaces huge pressure on the small number of laboratories with expertise in GMP CAR T-cellmanufacture. Automation of CAR T-cellproduction using the CliniMACS Prodigy (Miltenyi Biotec) has enormous potential to simplify manufacture and to enhance CAR T-cellaccessibility to patients. We have optimized and successfully validated GMP-grade CD19CAR T-cellmanufacture on the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cellsin the Phase I CARD study (NCT02893189). Recruitment to the CARD study is open to adult patients with relapsed CD19+ disease following allogeneic stem cell transplantation (allo-SCT). The study is designed to assess the feasibility of automated CD19CAR T-cellmanufacture and to assess the engraftment and safety profile of allogeneic CD19CAR T-cellsgenerated using the CliniMACS Prodigy. Methods: To validate automated CAR T-cellmanufacture on the CliniMACS Prodigy, large-scale experiments using cryopreserved or fresh healthy donor leukapheresiswere performed and assessed for T-cellexpansion, transduction efficiency, viability, purity, immunophenotype and functionality. T-cellswere activated with TransAct, a polymeric nanomatrix incorporating CD3/CD28-specific antibodies, transduced with a SIN lentiviral vector encoding a second generation CD19CAR (4G7-41BBz CAR) and underwent expansion in a closed system with automated feeding and media exchange prior to cryopreservation. Small-scale controls with TransAct / TexMACS or Dynabeads/ X-vivo were run in parallel. The CARD study is designed to mirror escalated dose DLI protocols, allowing up to 3 consecutive doses of CD19CAR T cellsat intervals of no shorter than 8 weeks, titrated to clinical response. Cell doses are based on CD3+ T-cellnumbers rather than on absolute CD19CAR T-cellnumbers (dose 1 = 1x106/kg CD3+ T-cells; dose 2 = 3x106/kg CD3+ T-cellsand dose 3 = 1x107/kg CD3+ T-cells). A minimum transduction efficiency of 10% is stipulated as a release criterion for all trial products. Results: Preclinical scale up studies of the automated CAR T-cellmanufacturing process with healthy donor leucapheresate confirmed equivalence of CAR T-cellspecification and yield when compared to products manufactured using established WAVE-Bioreactor-based GMP T-cellprocesses in our facility (Qasim et al, Cytotherapy, 2016). Based on this data, automated CliniMACS Prodigy-based CD19CAR T-cellmanufacture was subsequently approved by the MHRA, the UK regulatory body, for use in the CARD clinical trial. To date, we have enrolled 3 patients on study. Transduction efficiency was on average 46.6% (range 40.7 - 55.3%), with successful manufacture of 3 CD19CAR T-celldoses per patient. All products met sterility release criteria. GMP capacity, staffing and quality assurance systems permit manufacture of one CliniMACS Prodigy product per month using a team of 2 GMP staff with the possibility for further upscaling. Two patients have been treated at the initial T celldose, with evidence of engraftment and expansion despite the absence of preconditioning. Conclusions: We have optimized and successfully validated GMP-grade CD19CAR T-cellmanufacture using the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cellproducts to adult patients with CD19+ disease relapse following allo-SCT. Preliminary results of CAR T-cellmanufacture and clinical outcomes in initial patients will be presented. Disclosures Nickolay: Miltenyi Biotec GmbH: Research Funding. Mock: Miltenyi Biotec GmbH: Research Funding. Himoudi: Autolus: Research Funding. Thrasher: Orchard Therapeutics: Consultancy; Torus Therapeutics, Inc: Other: Advisory Board; Rocket Pharmaceuticals Ltd: Consultancy; 4bio Ventures Management Ltd: Other: Advisory Board. Pule: UCL: Patents & Royalties: UCL Business; Autolus Ltd: Employment, Equity Ownership, Research Funding. Qasim: Miltenyi: Research Funding; Servier: Research Funding; NIHR: Research Funding; Autolus Ltd: Consultancy, Equity Ownership; Bellicum: Research Funding; Cellectis: Research Funding; Orchard Therapeutics: Consultancy, Equity Ownership.