ABSTRACT
Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.
Subject(s)
Antigens, CD19/genetics , Batch Cell Culture Techniques , Cell Engineering , Gene Editing , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Alleles , Animals , Dependovirus/genetics , Disease Models, Animal , Gene Expression , Gene Knockout Techniques , Gene Order , Genetic Loci , Genetic Vectors/genetics , Humans , Immunotherapy, Adoptive , Lymphoma/genetics , Lymphoma/immunology , Lymphoma/therapy , Mice , Neoplasms , Transduction, GeneticABSTRACT
Medulloblastoma (MB) is the most common pediatric brain tumor with few reports of successful immunologic targeting. We have recently demonstrated the immune tumor microenvironment as well as response to immune checkpoint blockade differ across subtypes of murine MB.
ABSTRACT
PURPOSE: Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma, the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and group 3 medulloblastoma for preclinical evaluation in immunocompetent C57BL/6 mice. EXPERIMENTAL DESIGN AND RESULTS: Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid-derived suppressor cells, and tumor-associated macrophages in murine SHH model tumors compared with group 3 tumors. However, murine group 3 tumors had higher percentages of CD8(+) PD-1(+) T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial group 3 tumors compared with SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1(+) peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3(+) T cells within the tumor microenvironment. CONCLUSIONS: This is the first immunologic characterization of preclinical models of molecular subtypes of medulloblastoma and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment.
Subject(s)
Antineoplastic Agents/pharmacology , Medulloblastoma/immunology , Medulloblastoma/pathology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Animals , Biomarkers , Disease Models, Animal , Immunophenotyping , Lymphocyte Count , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Lymphocytes, Tumor-Infiltrating/pathology , Medulloblastoma/drug therapy , Medulloblastoma/genetics , Medulloblastoma/mortality , Mice , Mice, Knockout , Myeloid Cells/immunology , Myeloid Cells/metabolism , Myeloid Cells/pathology , Patched Receptors , Phenotype , Receptors, Cell Surface/deficiency , Receptors, Cell Surface/genetics , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Tumor Microenvironment/geneticsABSTRACT
Many attempts to use genetically modified T cells to halt tumor progression have been met with disappointment and significant challenges in the successful application within human patients. Porter et al., however, describe the use of genetically modified lymphocytes bearing a chimeric antigen receptor that bypasses many of the common limitations of adoptive lymphocyte therapy. Through incorporation of a costimulatory domain within the chimeric antigen receptor, the investigators engineered lymphocytes with significantly higher tumor rejection activity and demonstrated significant expansion and prolonged survival after in vivo transfer to a single patient who showed a complete regression of refractory chronic lymphoid leukemia. This recent success in using genetically modified T cells to kill chronic lymphoid leukemia tumor cells is an encouraging advancement in the development of specific and targeted immune-based therapies against cancer.
