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Systemic Targeting of CpG-ODN to the Tumor Microenvironment with Anti–neu-CpG Hybrid Molecule and T Regulatory Cell Depletion Induces Memory Responses in BALB-neuT Tolerant Mice

¹ Mayo Clinic College of Medicine, Department of Immunology, Mayo Clinic Arizona, Scottsdale, Arizona; ² Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Torino, Torino, Italy; and ³ Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

Requests for reprints: Joseph Lustgarten, Cancer Center Scottsdale, Mayo Clinic Arizona, 13400 East Shea Boulevard, Scottsdale, AZ 85259. Phone: 480-301-6662; Fax: 480-301-9162; E-mail: lustgarten.joseph{at}mayo.edu.

Key Words: tolerance • TLR ligands • tumor immunology • T regulatory cells • immunotherapy • hybrid molecules

We have shown that neu transgenic mice are immunotolerant and that immunizations with dendritic cells (DC) pulsed with neu-derived antigens were not able to control tumor growth in these animals. We tested whether, by modulating the tumor microenvironment with Toll-like receptor ligands, it could be possible to induce the activation of antitumor responses in neu mice. Our results indicate that only intratumoral (i.t.) injections of CpG-ODN induce an antitumor response in neu mice. To target the CpG-ODN to the tumor site anywhere within the body, we chemically conjugated an anti–Her-2/neu monoclonal antibody (mAb) with CpG-ODN. The anti–neu-CpG hybrid molecule retained its ability to bind to Her-2/neu⁺ tumors, activate DCs, and induce antitumor responses. Our results indicated that injections of anti–neu-CpG induced the rejection of primary tumors in 100% of BALB/c mice and only in 30% of BALB-neuT mice. After challenging the BALB/c and BALB-neuT mice, we observed that BALB/c mice developed a protective memory response; in contrast, BALB-neuT mice succumbed to the challenge. After injections of anti–neu-CpG, T regulatory cells (T-reg) were drastically reduced at the tumor site, but a large number were still present in the lymphoid organs. When BALB-neuT mice were treated with anti–neu-CpG plus anti-GITR mAb, but not with anti-CD25 mAb, 100% of the BALB-neuT mice rejected the primary tumor and developed a protective memory response indicating the critical role of T-regs in regulating the repertoire against self antigens. Taken together, these results indicate that CpG-ODN–targeted therapy and depletion of T-regs optimally activate a primary response and generate a protective memory response against self-tumor antigens. [Cancer Res 2008;68(18):7530–40]