Original article

Ahead-of-Print

Advantages of high cell concentration prior to cryopreservation of initial leukapheresis in CAR-T cell therapy

Authors

Key words: chimeric antigen receptor T cells, protocol, leukapheresis, high concentration, cryopreservation

Abstract

Background - Chimeric antigen receptor (CAR) T-cell therapy is increasingly used in patients affected by B-cell lymphoma and acute lymphoblastic leukemia. For logistical reasons, initial apheresis products may be cryopreserved for shipment to manufacturing centers. Due to the characteristics of these patients, cells are often collected in large volumes, meaning more bags must be cryopreserved. This requires increased storage, time and monetary costs. In this context, we aimed to evaluate a high cell concentration cryopreservation protocol by centrifugation to standardize the initial CAR-T manufacturing procedure.
Materials and methods - Sixty-eight processes of leukapheresis of 57 patients affected by refractory/relapsed B cell lymphoma and 9 patients affected by acute lymphoblastic leukemia who were eligible for anti-CD19 CAR-T cell treatment performed between June 2019 and October 2022 were analyzed. Whole blood count, percentage and number of T cells were assessed on the apheresis final product. The apheresis product, which was alternatively stored overnight at 4°C, was centrifuged, adjusting the volume to approximately 40 mL. The product was immediately cryopreserved to achieve a final cell concentration of 50-200×106 cells/ml for cryopreservation.
Results - Leukapheresis volume was reduced by almost fivefold (median: 185 to 40 mL), resulting in a higher product concentration in one bag. In addition, the number of non-target cells (monocytes, platelets and erythrocytes) was also reduced during the development of CAR T cell therapy, thereby maintaining T lymphocyte levels and providing a purer starting material.
Discussion - The advantages of the protocol include reducing economic costs, saving storage space, simplifying the manufacturing process, and facilitating shipping logistics. In conclusion, we present a validated, simple, and cost-effective cell enrichment processing protocol that provides high-quality cryopreserved products as starting material for the CAR-T cell manufacturing process.

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Authors

Diego Carbonell - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain https://orcid.org/0000-0003-2960-0301

Silvia Monsalvo - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain

Eva Catalá - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain

Ana Pérez-Corral - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain

Carolina Villegas - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain

Carmen Falero - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain

Gloria Ruano - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain

Monica Martinez - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain

Mi Kwon - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain

Cristina Muñoz-Martínez - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain

José Luis Díez-Martín - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain; Department of Medicine, School of Medicine, Complutense University of Madrid, Madrid, Spain

Jorge Gayoso - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain

Javier Anguita - Department of Hematology, Gregorio Marañón General University Hospital, Madrid, Spain; Gregorio Marañón Health Research Institute (IiSGM), Madrid, Spain

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