Transposon-based CAR-T cells for lymphoma treatment

B Díez(1) C Calviño(2) M Fernández-García(1) S I Inoges(2,3) A López Díaz de Cerio(2,3) M Hernando-Rodríguez(1) L Álvarez(1) M L Lozano(1) C Ceballos(4) L López-Corral(5) A Navarro(5) A Alfonso(2) J Rifon(2) J Serrano-López(6) P Llamas(6) M Redondo(4) M C Viguria(4) M Juan(7) J M Moraleda(8) F Sánchez-Guijo(5) J R Rodríguez-Madoz(9) R M Yáñez(1) J A Bueren(1) F Prosper(2,9)

1:Division of Hematopoietic Innovative Therapies, Biomedical Innovation Unit, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain; 2:Hematology and Cell Therapy Department, Clínica Universidad de Navarra, IdiSNA, Pamplona, Spain; 3:Immunology and Inmunotherapy Department, Clínica Universidad de Navarra, Pamplona, Spain; 4:Servicio de Hematología, Hospital Universitario de Navarra, IdiSNA, Pamplona, Spain; 5:Hematology Department, University Hospital of Salamanca (HUS/IBSAL), CIBERONC and Center for Cancer Research-IBMCC (USALCSIC), Salamanca, Spain; 6:Division of Hematology and Hemotherapy, IIS Fundación Jiménez Díaz, Madrid, Spain; 7:Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Department of Immunology and Advanced Therapies Unit, Hospital Clínic de Barcelona, Barcelona, Spain; 8:Deparment of Hematology, Stem Cell Transplant and Cell Therapy Unit, IMIB-Arrixaca, Virgen de la Arrixaca University Hospital, University of Murcia, Murcia, Spain; 9:Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, CIBERONC, Pamplona, Spain

TranspoCART is a project conceived to develop an innovative CART medicinal product based on a viral vector-free gene-transfer strategy, which is based on the use of transposons. A Sleeping Beauty transposon minicircle (SBmC) carrying the 4-1BB CAR endodomain, an anti-CD19 ScFv and a truncated EGFR -as a safety switch- was designed for the treatment of lymphoma patients. Our optimized manufacturing protocol reached transposition efficacies up to 44% of CAR⁺ cells using PB cells from healthy donors and 50% from patients’ samples. Extensive in vitro characterization of TranspoCART19 cells showed an enriched stem-cell memory/central memory phenotype, no signs of cell exhaustion and high level of specific cytotoxicity activity against CD19⁺ cells. Dilution of SBmC and hyperactive form of transposase (SB100x) during cell manufacturing was confirmed, with no detectable levels of both components at the end of the expansion period. The in vivo antitumoral efficacy of TranspoCART19 cells was evaluated in NALM6 xenograft models using immunodeficient NSG mice. Treated animals showed improved survival compared to untreated mice, showing a similar efficacy compared to lentivirus-produced CART cells. Biodistribution and toxicity studies showed a safe integration profile of the transposon. Additionally, the efficacy of the safety switch construct was confirmed in vivo using the same xenograft model. Finally, scale-up and GMP validations of TranspoCART19 cell manufacturing were carried out in two independent GMP facilities, which demonstrated that the protocol is reproducible and highly efficient. These encouraging results will allow us to initiate a clinical trial with the TransproCART19 cell product.