T cells can be engineered to express the genes of chimeric antigen receptors (CARs) that recognize tumor-associated antigens. We constructed and compared 2 CARs that contained a single chain variable region moiety that recognized CD19. One CAR contained the signaling moiety of the 4-1BB molecule and the other did not. We selected the CAR that did not contain the 4-1BB moiety for further preclinical development. We demonstrated that gammaretroviruses encoding this receptor could transduce human T cells. Anti-CD19-CAR-transduced CD8+ and CD4+ T cells produced interferon-γ and interleukin-2 specifically in response to CD19+ target cells. The transduced T cells specifically killed primary chronic lymphocytic leukemia (CLL) cells. We transduced T cells from CLL patients that had been previously treated with chemotherapy. We induced these T cells to proliferate sufficiently to provide enough cells for clinical adoptive T cell transfer with a protocol consisting of an initial stimulation with an anti-CD3 monoclonal antibody (OKT3) before transduction followed by a second OKT3 stimulation 7 days after transduction. This protocol was successfully adapted for use in CLL patients with high peripheral blood leukemia cell counts by depleting CD19+ cells before the initial OKT3 stimulation. In preparation for a clinical trial that will enroll patients with advanced B cell malignancies, we generated a producer cell clone that produces retroviruses encoding the anti-CD19 CAR, and we produced sufficient retroviral supernatant for the proposed clinical trial under good manufacturing practice conditions.
*Surgery Branch of the National Cancer Institute
†Metabolism Branch of the National Cancer Institute, National Institutes of Health, Bethesda, MD
This work was supported by intramural funding of the Center for Cancer Research, National Cancer Institute, NIH.
Reprints: James N. Kochenderfer, Surgery Branch of the National Cancer Institute, NIH 10 Center Drive CRC Room 3-3888 Bethesda, MD 20892 (e-mail: firstname.lastname@example.org).
Received for publication January 6, 2009
accepted April 21, 2009
Financial Disclosure: All authors have declared there are no financial conflicts of interest in regards to this work.