T Cell Modulation Combined with Intratumoral CpG Cures Lymphoma without the Need for Chemotherapy.

Introduction: We have previously shown that intratumoral injection of CpG oligodeoxynucleotide plus systemic chemotherapy can induce T cell immunity against lymphoma and serve as a therapeutic vaccine to cure tumors in a murine lymphoma model (Li et al., J Immunol. 2007). CpG is a Toll-like receptor 9 agonist capable of activating tumor-infiltrating dendritic cells and/or tumor B cells to force tumor antigen presentation in situ when injected intratumorally. Here, we demonstrate that antibody-mediated modulation of T cells enhances the efficacy of CpG vaccination, thereby eliminating the need for chemotherapy.

Materials and Methods: Mice were inoculated s.c. with A20 lymphoma tumor cells at two sites (right and left abdomen). Only one site was injected with CpG allowing us to evaluate the systemic anti-tumor response at the distant site. Treatment started when tumors became palpable. CpG was administered intratumorally. T cell modulation was accomplished using systemic (i.p.) administration of monoclonal antibodies against T cell targets:

• - Regulatory T cells were depleted using anti-Folate Receptor 4 (FR4) antibody or functionally blocked using anti-GITR antibody,

• - Effector T cells were stimulated using anti-OX40 antibody (to trigger their costimulatory molecule) or anti-CTLA4 antibody (to block inhibitory signals).

Results: Treatment with intratumoral injection of CpG alone did not cure any mice. Treatment with CpG and a single antibody (anti-OX40, anti-CTLA4, anti-FR4, or anti-GITR) cured 20–30% of mice. Interestingly, some combinations of antibodies (anti-OX40+anti-CTLA4, anti-OX40+anti-FR4, anti-CTLA4+anti-GITR) potentiated T cell modulation and further enhanced the efficacy of CpG vaccination. In particular, the combination of anti-OX40 and anti-CTLA4 appeared to be especially potent when combined with intratumoral CpG. Indeed, this combination (CpG+anti-OX40+anti-CTLA4) induced anti-tumor T cells capable of secreting IFN-γ in response to overnight culture with A20 tumor cells; it cured more than 80% of mice bearing large and systemic lymphoma tumors without the need for chemotherapy (effective therapy required both CD4 and CD8 T cells); finally, this therapy produced high numbers of anti-tumor memory T cells and provided long-lasting immunity against tumor re-challenge.

Conclusions: Our results show that antibody-mediated T cell modulation greatly enhances the therapeutic efficacy of intratumoral vaccination with CpG. Importantly, we show that T cell modulation can be potentiated by appropriate combinations of antibodies against T cell targets. As these reagents (CpG, anti-OX40 and anti-CTLA4 notably) are becoming available for human clinical trials, the combination of intratumoral CpG and immunomodulatory T cell antibodies holds promise for therapeutic vaccination against lymphoma.