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

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 naive 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.