This project is a combination of a clinical trial of infusions of autologous anti-CD19 chimeric-antigen-receptor-transduced T cells and laboratory experiments performed on cells obtained from patients that received infustions of the chimeric-antigen-receptor-transduced T cells. During 2012 this project resulted in a Plenary Paper published in Blood entitled """"""""B-cell Depletion and Remissions of Malignancy Along With Cytokine-associated Toxicity in a Clinical Trial of Anti-CD19 Chimeric-antigen-receptor-transduced T cells"""""""". The project also resulted in an abstract that was presented as an oral presentation at the most important hematology meeting in the world, the Annual Meeting of the American Society of Hematology. The rest of this section summarizes the project. New therapies are needed for chemotherapy-resistant B-cell malignancies. Adoptive transfer of T cells genetically-engineered to express chimeric antigen receptors (CARs) that specifically recognize the B-cell antigen CD19 is a promising new approach for treating B-cell malignancies. We are conducting a clinical trial in which patients receive infusions of autologous T cells that are transduced with gamma-retroviruses encoding an anti-CD19 CAR. The CAR is made up of the variable regions of an anti-CD19 antibody, a portion of the CD28 molecule, and a portion of the CD3-zeta molecule. Our clinical protocol consists of cyclophosphamide plus fludarabine chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of high-dose IL-2. We have treated 11 patients on this clinical trial. Five of the patients had chronic lymphocytic leukemia (CLL), and 6 patients had B-cell lymphoma. Anti-CD19-CAR-transduced T cells that specifically recognized CD19-expressing target cells were produced for all patients. The total number of cells administered to each patient ranged from 0.25x10e7 to 5.5x10e7 cells per kg of bodyweight. Of the patients traeted, 4 have obtained complete remissions, 3 of which are ongoing for greater than 6 months. Four patients achieved partial remissions, two of which are ongoing for more than one year. One patient obtained stable disease. One patient died of influenza, and another patient died of progressive lymphoma. A striking depletion of CD19+ B-lineage cells occurred in 6 of 8 evaluable patients. This B-cell depletion lasted for up to 18 months. Because of the long duration of B-cell depletion, it cannot be attributed to the chemotherapy that the patients received. For example, a patient with follicular lymphoma had a normal level of polyclonal blood B cells before treatment on our protocol. Six months after treatment, he had a blood B cell count of 1 per microliter (normal range 61-321 B cells per microliter). A patient with CLL had a regression of adenopathy in the first 32 days after chemotherapy and CAR-transduced T cell administration. CAR-transduced cells were detected in the blood of all 8 patients by quantitative PCR. The percentage of peripheral blood mononuclear cells (PBMC) containing the CAR gene varied widely, but in 2 patients the CAR gene was detected in greater than 0.1 percent of PBMC more than 90 days after infusion. At early time-points after infusion, CAR-expressing T cells constituted up to 20 percent of all blood T cells. Patients had significant toxicity during the first 10 days after CAR-transduced T cell infusion. The most prominent toxicity was hypotension. CAR-transduced cells were detected in the blood of all 8 patients by quantitative PCR. The percentage of peripheral blood mononuclear cells (PBMC) containing the CAR gene varied widely, but in 3 patients the CAR gene was detected in greater than 0.1 percent of PBMC more than 90 days after infusion. At early time-points after infusion, CAR-expressing T cells constituted up to 50 percent of all blood T cells. Patients had significant toxicity during the first 10 days after CAR-transduced T cell infusion. The most prominent toxicity was hypotension. These results demonstrate that CAR-expressing T cells can specifically eliminate targeted cells and cause significant cytokine-mediated toxicity in humans.
