B-Cell Depletion, Remissions of Malignancy, and Cytokine-Associated Toxicity in a Clinical Trial of T Cells Genetically-Engineered to Express An Anti-CD19 Chimeric Antigen Receptor

Abstract 167^FN2

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 8 patients on this clinical trial. Four of the patients had chronic lymphocytic leukemia (CLL), and 4 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 0.5×10⁷ to 5.5×10⁷ cells per kg of bodyweight. A mean of 54% of the administered cells expressed the anti-CD19 CAR. One patient with CLL obtained a complete remission that is ongoing 15 months after treatment. Five patients obtained partial remissions that are ongoing in 3 patients. One patient with CLL had stable disease. One patient with lymphoma died of culture-proven influenza A pneumonia and is not evaluable for lymphoma response. A striking depletion of CD19⁺ B-lineage cells occurred in 4 of 8 patients. This B-cell depletion lasted for up to 15 months, and it is ongoing in 3 of 4 patients. 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/microliter (normal range 61–321 B cells/microliter). A patient with CLL had a regression of adenopathy in the first 32 days after chemotherapy and CAR-transduced T cell administration. Interestingly, this adenopathy continued to substantially regress between 33 and 133 days after chemotherapy ended. 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% of PBMC more than 90 days after infusion. At early time-points after infusion, CAR-expressing T cells constituted up to 20% 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. Sharp increases in serum interferon gamma (IFN) and tumor necrosis factor (TNF) occurred in 4 of 8 patients during the first 10 days after cell infusion. In the 4 patients with prominent elevations in inflammatory cytokines, peak serum IFN levels ranged from 865 to 2305 pg/mL, and peak TNF levels ranged from 49 to 118 pg/mL. As measured by an objective sequencial organ failure assessment score, the 4 patients with prominent elevations in IFN and TNF had more severe clinical toxicity during the first 10 days after cell infusion than the other 4 patients who did not have prominent elevations of IFN and TNF. We analyzed PBMC of three patients with elevations of serum IFN and TNF by using an ex vivo assay consisting of a 6-hour incubation of PBMC with target cells followed by intracellular cytokine staining. In all three patients, we detected T cells that produced IFN and TNF in a CD19-specific manner in PBMC samples collected after anti-CD19-CAR-transduced T cell infusions but not in PBMC samples collected before CAR-transduced T cell infusions. This indicates that anti-CD19-CAR-transduced T cells were a source of the elevated serum cytokines. In multiple patients receiving infusions of anti-CD19-CAR-transduced T cells, we have demonstrated elimination of CD19⁺ B-lineage cells, regressions of malignancy, elevated levels of serum inflammatory cytokines, and CD19-specific T cells that produce inflammatory cytokines ex vivo. These results demonstrate that CAR-expressing T cells can specifically eliminate targeted cells and cause significant cytokine-mediated toxicity in humans.