Construction and preclinical evaluation of an anti-CD19 chimeric antigen receptor.
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-gamma 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.
Kochenderfer
James N
JN
Surgery Branch of the National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. kochendj@mail.nih.gov
Feldman
Steven A
SA
Zhao
Yangbing
Y
Xu
Hui
H
Black
Mary A
MA
Morgan
Richard A
RA
Wilson
Wyndham H
WH
Rosenberg
Steven A
SA
D018941:Antigens, CD19 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D004353:Drug Evaluation, Preclinical; D004797:Enzyme-Linked Immunosorbent Assay; D017988:Gammaretrovirus / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D020014:K562 Cells; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology / Q000473:pathology / Q000628:therapy; D016853:Muromonab-CD3 / Q000276:immunology; D011946:Receptors, Antigen / Q000235:genetics / Q000276:immunology*; D017475:Receptors, Nerve Growth Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D014161:Transduction, Genetic
D018941:Antigens, CD19; D007376:Interleukin-2; D016853:Muromonab-CD3; D011946:Receptors, Antigen; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma