Local Manufacture of CD19 CAR-T Cells Using an Automated Closed-System: Robust Manufacturing and High Clinical Efficacy with Low Toxicities
Authors: Molostova Olga , Shelikhova Larisa , Schneider Dina , Khismatullina Rimma , Muzalevsky Yakov , Kazachenok Alexey , Preussner Liane , Rauser Georg , Abugova Julia , Kurnikova Elena , Pershin Dmitriy , Zubachenko Viktoria , Popov Alexander , Illarionova Olga , Miakova Natalia , Litvinov Dmitry , Novichkova Galina , Maschan Alexey A. , Orentas Rimas , Dropulic Boro , Maschan Michael 
Published: November 2019

Introduction CD19 CAR-T cell products were recently approved as therapy for B-lineage malignancies. We initiated an IIT trial where manufacture of CAR-T cells was performed locally using a unique CD19 CAR with potent anti-leukemic effects.
Patients and methods
A total of 37 pts with relapsed/refractory B-acute lymphoblastic leukemia (12 female, 25 male, median age 10 y) were screened, 27 pts were enrolled for a trial, 10 were eligible for compassionate use of CD19 CAR-T cell therapy. Sixteen patients had relapsed B-ALL after haploidentical HSCT, 19 pts refractory relapse, 2 induction failure, 13 patients had previous blinatumomab infusion. Eighteen patients had >20% blast cells, median bone marrow leukemia burden for patients with full blown disease was 89%, 19 pts had minimal residual disease (MRD) >0.1% in BM, 3 had skeletal involvement with multiple mass lesions, one had CNS involvement.
The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells. The automated production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct and transduced with lentiviral vector expressing the CD19CAR gene (second generation CD19.4-1BB zeta with alternate transmembrane domain derived from the TNF superfamily) (Lentigen, Miltenyi Biotec company). T cells were expansion over 10 days in the presence of serum-free TexMACS GMP Medium supplemented with MACS GMP IL-7 and IL-15. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab at 8mg/kg before CAR-T cell infusion. Patients did not receive HSCT as consolidation after CAR-T therapy.
Results Thirty-five manufacturing cycles were successful. Median transduction efficacy was 60% (20-80). Median expansion of T cells was x 46 (18-51). CD4:CD8 ratio in the final product was 0.73. The cell products were administered at a dose of 3*106/kg of CAR-T cells in 4 pts, 1*106/kg in 9 pts, 0.5*106/kg in 14 pts, 0.1*106/kg in 8 pts. Two patients received 0.1*106/kg of CAR-T cells produced from haploidentical donors.
The cytokine release syndrome (CRS) occurred in 22 (59%) pts and was mostly mild and moderate: grade I - 15 pts, grade II- 4 pts, grade III - 2 pt, grade IV - 1 pt. CAR-T cell related encephalopathy occurred in 15 (40%). Grade I-II neurotoxicity developed in 10 pts, grade III - in 2 pt, grade IV - 1 pt, grade V - 2 pt. In one patient with grade V neurotoxicity concomitant K. pneumonia encephalitis was documented. Severe (grade 3-5) CRS and neurotoxicity were associated exclusively with large leukemia burden (>20% in the bone marrow) at enrollment, p=0,002.
Thirty-one patient was evaluable for response at day 28. Four pts had persistent leukemia. In 27 (87%) cases Flow MRD-negative remission was achieved. Disease relapse after initial response was registered in 9 (33%) cases (7 patients had CD19 negative, 2 had CD19 positive relapse). At the moment of reporting, 10 patients have died (3 due to sepsis, 1 due to brain edema, 1 due to brain edema and K. pneumonia encephalitis, 5 due to progression of disease or relapse). Twenty-seven pts are alive, 19 in complete remission with a median follow up of 223 days (41-516 days).
Conclusion CliniMACS Prodigy TCT process is a robust CAR-T cell manufacturing platform that enables rapid and flexible provision of CAR-T cells to patients in need. Significant toxicity of CD19 CAR-T cells was associated exclusively with high leukemia burden at enrollment. In the absence of HSCT consolidation relapse rate exceeds 30%.

Disclosures
Schneider: Lentigen Technology, A Miltenyi Biotec Company: Employment. Preussner:Miltenyi Biotec: Employment. Rauser:Miltenyi Biotec: Employment. Orentas:Lentigen Technology Inc., a Miltenyi Biotec Company: . Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Maschan:Miltenyi Biotec: Other: lecture fee.