ABSTRACT
Clinical success of autologous CD19-directed chimeric antigen receptor T cells (CAR Ts) in acute lymphoblastic leukemia and non-Hodgkin lymphoma suggests that CAR Ts may be a promising therapy for hematological malignancies, including multiple myeloma. However, autologous CAR T therapies have limitations that may impact clinical use, including lengthy vein-to-vein time and manufacturing constraints. Allogeneic CAR T (AlloCAR T) therapies may overcome these innate limitations of autologous CAR T therapies. Unlike autologous cell therapies, AlloCAR T therapies employ healthy donor T cells that are isolated in a manufacturing facility, engineered to express CARs with specificity for a tumor-associated antigen, and modified using gene-editing technology to limit T cell receptor (TCR)-mediated immune responses. Here, transcription activator-like effector nuclease (TALEN) gene editing of B cell maturation antigen (BCMA) CAR Ts was used to confer lymphodepletion resistance and reduced graft-versus-host disease (GvHD) potential. The safety profile of allogeneic BCMA CAR Ts was further enhanced by incorporating a CD20 mimotope-based intra-CAR off switch enabling effective CAR T elimination in the presence of rituximab. Allogeneic BCMA CAR Ts induced sustained antitumor responses in mice supplemented with human cytokines, and, most importantly, maintained their phenotype and potency after scale-up manufacturing. This novel off-the-shelf allogeneic BCMA CAR T product is a promising candidate for clinical evaluation.
Subject(s)
B-Cell Maturation Antigen/immunology , Cell Transplantation/methods , Immunotherapy, Adoptive/methods , Multiple Myeloma/therapy , Receptors, Chimeric Antigen/immunology , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Animals , Antineoplastic Agents, Immunological/therapeutic use , B-Cell Maturation Antigen/genetics , Blood Donors , Cell Line, Tumor , Cell Transplantation/adverse effects , Cytotoxicity, Immunologic/genetics , Gene Editing , Genetic Vectors , Graft vs Host Disease/therapy , Humans , Immunotherapy, Adoptive/adverse effects , Mice , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/pathology , Progression-Free Survival , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Rituximab/therapeutic use , T-Lymphocytes/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Transduction, Genetic , Transplantation, Homologous/methodsABSTRACT
TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.
