Construction and Preclinical Evaluation of an anti-CD19 Chimeric Antigen Receptor

J Immunother. 2009 Sep;32(7):689-702. doi: 10.1097/CJI.0b013e3181ac6138.

Abstract

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.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Antigens, CD19 / genetics
  • Antigens, CD19 / immunology*
  • Antigens, CD19 / metabolism
  • CD4-Positive T-Lymphocytes / cytology
  • CD4-Positive T-Lymphocytes / immunology
  • CD4-Positive T-Lymphocytes / metabolism
  • CD8-Positive T-Lymphocytes / cytology
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism
  • Cell Line, Tumor
  • Cytotoxicity Tests, Immunologic
  • Cytotoxicity, Immunologic / immunology*
  • Drug Evaluation, Preclinical
  • Enzyme-Linked Immunosorbent Assay
  • Gammaretrovirus / genetics
  • Genetic Vectors / genetics
  • Humans
  • Immunotherapy / methods
  • Interferon-gamma / metabolism
  • Interleukin-2 / metabolism
  • K562 Cells
  • Leukemia, Lymphocytic, Chronic, B-Cell / immunology
  • Leukemia, Lymphocytic, Chronic, B-Cell / pathology
  • Leukemia, Lymphocytic, Chronic, B-Cell / therapy
  • Muromonab-CD3 / immunology
  • Receptors, Antigen / genetics
  • Receptors, Antigen / immunology*
  • Receptors, Nerve Growth Factor / genetics
  • Receptors, Nerve Growth Factor / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / metabolism
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism
  • Transduction, Genetic

Substances

  • Antigens, CD19
  • Interleukin-2
  • Muromonab-CD3
  • Receptors, Antigen
  • Receptors, Nerve Growth Factor
  • Recombinant Fusion Proteins
  • Interferon-gamma