Tumor-Localized Ligation of CD3 and CD28 with Systemic Regulatory T-Cell Depletion Induces Potent Innate and Adaptive Antitumor Responses

Abstract Purpose:Tumor-localized activationof immune cells bymembrane-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 B16melano-ma cells (B16/3L86 cells) and the effect of various lymphocytes, including CD4+ and CD8+ Tcells, natural killerT (NKT) cells, and regulatoryTcells, on antitumor activity was investigated. Results: B16/3L86 cells activated naI « veT 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 NKTcells rather than CD4+ or CD8+ T cells. Treatment of mice with low-dose cyclophosphamide or anti-CD25 antibody to deplete regulatoryTcells 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 o