ABSTRACT
Patients with chemo-refractory acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T (CART) cell therapy has produced exciting results in CD19+ malignancies and may overcome many of the limitations of conventional leukemia therapies. We developed CART cells to target CD33 (CART33) using the anti-CD33 single chain variable fragment used in gemtuzumab ozogamicin (clone My96) and tested the activity and toxicity of these cells. CART33 exhibited significant effector functions in vitro and resulted in eradication of leukemia and prolonged survival in AML xenografts. CART33 also resulted in human lineage cytopenias and reduction of myeloid progenitors in xenograft models of hematopoietic toxicity, suggesting that permanently expressed CD33-specific CART cells would have unacceptable toxicity. To enhance the viability of CART33 as an option for AML, we designed a transiently expressed mRNA anti-CD33 CAR. Gene transfer was carried out by electroporation into T cells and resulted in high-level expression with potent but self-limited activity against AML. Thus our preclinical studies show potent activity of CART33 and indicate that transient expression of anti-CD33 CAR by RNA modification could be used in patients to avoid long-term myelosuppression. CART33 therapy could be used alone or as part of a preparative regimen prior to allogeneic transplantation in refractory AML.
Subject(s)
Blast Crisis/therapy , Cell- and Tissue-Based Therapy , Leukemia, Myeloid, Acute/therapy , Myelodysplastic Syndromes/therapy , Receptors, Antigen, T-Cell/immunology , Sialic Acid Binding Ig-like Lectin 3/immunology , Animals , Blast Crisis/immunology , Blast Crisis/metabolism , Blast Crisis/pathology , Blotting, Western , Cell Proliferation , Female , Flow Cytometry , Humans , Immunoenzyme Techniques , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Inbred NOD , Mice, SCID , Mice, Transgenic , Myelodysplastic Syndromes/immunology , Myelodysplastic Syndromes/metabolism , Myelodysplastic Syndromes/pathology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Antigen, T-Cell/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sialic Acid Binding Ig-like Lectin 3/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Alagille syndrome (AGS) is caused by heterozygous mutations in JAG1, and mutations have been previously reported in about 70% of patients who meet clinical diagnostic criteria. We studied a cohort of 247 clinically well-defined patients, and using an aggressive and sequential screening approach we identified JAG1 mutations in 94% of individuals. Mutations were found in 232 out of 247 patients studied and 83 of the mutations were novel. This increase in the mutation rate was accomplished by combining rigorous clinical phenotyping, with a combination of mutation detection techniques, including fluorescence in situ hybridization (FISH), genomic and cDNA sequencing, and quantitative PCR. This higher rate of mutation identification has implications for clinical practice, facilitating genetic counseling, prenatal diagnosis, and evaluation of living-related liver transplant donors. Our results suggest that more aggressive screening may similarly increase the rate of mutation detection in other dominant and recessive disorders.