Title Point-of-Care Production of CD19 CAR-T Cells in an Automated Closed-System: Report of First Clinical Experience
Authors: Molostova Olga , Shelikhova Larisa , Muzalevsky Yakov , Kazachenok Alexey , Pershin Dmitriy , Kurnikova Elena , Popov Alexander , Dropulic Boro , Shneider Dina , Orentas Rimas , Alex Regina , Essl Mike , Miakova Natalia , Litvinov Dmitry , Karachunskiy Alexander , Novichkova Galina , Maschan Alexey A. , Maschan Michael 
Published: October 2019

Abstract
Introduction CD19 CAR-T cell products were recently approved as therapy for advanced B-cell lineage malignancies. The predominant manufacturing model for this type of therapy is a centralized industrial-type production process. An attractive model of CAR-T cell production and delivery to the patient is via a point-of care-manufacturing process. We report on first cases of implementation of this approach in a setting of compassionate use program.
Patients and methods Four patients with relapsed/refractory B-cell lineage malignancies were eligible for compassionate use of CD19 CAR-T cell therapy. Three patients, median age 15 years, had relapsed B-cell lineage acute lymphoblastic leukemia (B-ALL) after haploidentical hematopoietic stem cell transplantation (HSCT). Bone marrow disease burden at therapy start was 12%, 74% and 0,159. The patient with low minimal residual disease (MRD) in the bone marrow had skeletal involvement with multiple lymphomatous mass lesions. One patient had refractory primary mediastinal B-cell lymphoma (PMBCL). The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells from patient-derived leukapheresis product. Automatic production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct, transduction with lentiviral (second generation CD19.4-1BB zeta) vector (Lentigen, Miltenyi Biotec company) and expansion over 12 days in the presence of TexMACS GMP Medium supplemented with MACS GMP IL-7/IL-15 combination. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab 1 hour before CAR-T cell infusion at 8mg/kg.
Results All production cycles were successful. Median transduction efficacy achieved was 57%(52-63). Median expansion of T cells was x26(24-43). CD4/CD8 ratio in the final product was 0,750,22-6). All final products passed the in-process and quality controls. The cell products were administered at a dose of 1*106/kg of CAR-T cells. No immediate infusion reactions were reported. In 2 cases mild cytokine release syndrome (CRS) was diagnosed. In one case mild CAR-T cell related encephalopathy developed. In one case additional dose of tocilizumab and one dose of dexamethasone were administered to control CRS and encephalopathy. All patients survived to the point of response evaluation. In 3 cases CAR-T cell expansion was detected to a maximum 25%. MRD-negative responses detected by flow cytometry and PCR were achieved in 2 cases with bone marrow involvement. In two cases with prominent mass lesions an objective response was documented. In the patient with 74% blast in bone marrow at start of therapy, neither CAR-T cell expansion nor leukemia response were documented. Details of therapy and response are summarized in table 1.
Conclusion Production of CAR-T cells with the CliniMACS Prodigy TCT process is a feasible and an attractive option that provides a point-of-care manufacturing approach to enable rapid delivery of CAR-T cells to patients in need. Robustness and consistency of this approach provides a solid basis for multi-center academic trials in the field of adoptive cell therapy.
Table 1. Table 1.

Disclosures
Dropulic: Lentigen, A Miltenyi Biotec company: Employment. Shneider:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen, A Miltenyi Biotec company: Employment. Alex:Miltenyi Biotec: Employment. Essl:Miltenyi Biotec: Employment.