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

Introduction:

Clinical studies of 2^nd generation CD19 Chimeric Antigen Receptor (CAR) T-cells have delivered unprecedented clinical responses in patients with B-ALL. Increasing demand for CAR T-cells places huge pressure on the small number of laboratories with expertise in GMP CAR T-cell manufacture. Automation of CAR T-cell production using the CliniMACS Prodigy (Miltenyi Biotec) has enormous potential to simplify manufacture and to enhance CAR T-cell accessibility to patients.

We have optimized and successfully validated GMP-grade CD19CAR T-cell manufacture on the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cells in 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-cell manufacture and to assess the engraftment and safety profile of allogeneic CD19CAR T-cells generated using the CliniMACS Prodigy.

Methods:

To validate automated CAR T-cell manufacture on the CliniMACS Prodigy, large-scale experiments using cryopreserved or fresh healthy donor leukapheresis were performed and assessed for T-cell expansion, transduction efficiency, viability, purity, immunophenotype and functionality. T-cells were activated with TransAct, a polymeric nanomatrix incorporating CD3/CD28-specific antibodies, transduced with a SIN lentiviral vector encoding a second generation CD19 CAR (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 cells at intervals of no shorter than 8 weeks, titrated to clinical response. Cell doses are based on CD3+ T-cell numbers rather than on absolute CD19CAR T-cell numbers (dose 1 = 1x10⁶/kg CD3+ T-cells; dose 2 = 3x10⁶/kg CD3+ T-cells and 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-cell manufacturing process with healthy donor leucapheresate confirmed equivalence of CAR T-cell specification and yield when compared to products manufactured using established WAVE-Bioreactor-based GMP T-cell processes in our facility (Qasim et al, Cytotherapy, 2016). Based on this data, automated CliniMACS Prodigy-based CD19CAR T-cell manufacture 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-cell doses 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 cell dose, with evidence of engraftment and expansion despite the absence of preconditioning.

Conclusions:

We have optimized and successfully validated GMP-grade CD19CAR T-cell manufacture using the CliniMACS Prodigy and have delivered the first in man Prodigy-manufactured CD19CAR T-cell products to adult patients with CD19+ disease relapse following allo-SCT. Preliminary results of CAR T-cell manufacture and clinical outcomes in initial patients will be presented.