Tumor-localized ligation of CD3 and CD28 with systemic regulatory T-cell depletion induces potent innate and adaptive antitumor responses

Clin Cancer Res. 2009 Apr 15;15(8):2756-66. doi: 10.1158/1078-0432.CCR-08-2311. Epub 2009 Mar 24.


Purpose: Tumor-localized activation of immune cells by membrane-tethered anti-CD3 antibodies (CD3L) is under investigation to treat poorly immunogenic tumors. Here we sought to elucidate the mechanism of antitumor immunity elicited by CD3L.

Experimental design: CD3L and CD86 were expressed on poorly immunogenic B16 melanoma cells (B16/3L86 cells) and the effect of various lymphocytes, including CD4(+) and CD8(+) T cells, natural killer T (NKT) cells, and regulatory T cells, on antitumor activity was investigated.

Results: B16/3L86 cells activated naïve T cells; suppressed tumor growth in subcutaneous, peritoneal, and metastasis models; and protected mice from rechallenge with B16 melanoma cells. However, in vivo antitumor activity against primary B16/3L86 tumors unexpectedly depended on NKT cells rather than CD4(+) or CD8(+) T cells. Treatment of mice with low-dose cyclophosphamide or anti-CD25 antibody to deplete regulatory T cells unmasked latent T-cell antitumor activity; the number of activated CD8(+) T cells in tumors increased and B16/3L86 tumors were completely rejected in a CD8(+) and CD4(+) T-cell-dependent fashion. Furthermore, fibroblasts expressing CD3L and CD86 suppressed the growth of neighboring B16 cancer cells in vivo, and direct intratumoral injection of adenoviral vectors expressing CD3L and CD86 or CD3L and a membrane-tethered anti-CD28 antibody significantly suppressed the growth of subcutaneous tumors.

Conclusions: Tumor-located ligation of CD3 and CD28 can activate both innate (NKT cells) and adaptive (CD4(+) and CD8(+) T cells) responses to create a tumor-destructive environment to control tumor growth, but modulation of regulatory T cells is necessary to unmask local adaptive antitumor responses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents, Alkylating / pharmacology
  • B7-2 Antigen / immunology
  • B7-2 Antigen / metabolism
  • CD28 Antigens / immunology*
  • CD3 Complex / immunology*
  • Cell Line, Tumor
  • Cyclophosphamide / pharmacology
  • Female
  • Genetic Vectors / immunology
  • Genetic Vectors / metabolism
  • Immunity, Active
  • Immunity, Innate
  • Interferon-gamma / immunology
  • Interferon-gamma / metabolism
  • Lymphocyte Depletion*
  • Melanoma, Experimental / therapy*
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Knockout
  • T-Lymphocytes, Regulatory / drug effects
  • T-Lymphocytes, Regulatory / immunology*
  • Transfection


  • Antineoplastic Agents, Alkylating
  • B7-2 Antigen
  • CD28 Antigens
  • CD3 Complex
  • Interferon-gamma
  • Cyclophosphamide