In Vivo Eradication of a Rituximab-Resistant Human CD20⁺ B Cell Lymphoma by Rituximab-CpG Oligodeoxynucleotide Conjugate Is Mediated by Natural Killer Cells and Complement.

Abstract 723

The anti-CD20 monoclonal antibody rituximab exerts its anti-tumor effects against B cell non-Hodgkin's lymphoma (NHL) via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG ODN) are potent activators of ADCC and T cell immunity, and have been studied for anti-NHL effects when administered by systemic or intratumoral (i.t.) routes. We have used an aggressive human CD20-expressing syngeneic murine B cell lymphoma (38C13-huCD20) that is resistant to single agent rituximab to study techniques for improving antibody efficacy. We recently reported that combining rituximab with i.t. (but not systemic) CpG promotes eradication of 7-day established rituximab-resistant 38C13-huCD20 lymphoma from 42% of mice (D. Betting et al, J. Immunotherapy 2009). Six doses of i.t. CpG (50 μg days 7, 8, and 9 and 25 μg days 11, 13, and 15) were required to achieve tumor eradication in this setting. Both natural killer cells and complement participated in the cure of tumors by rituximab plus i.t CpG, by increasing tumor cell sensitivity to ADCC and complement lysis, and by augmenting the cytotoxicity of ADCC effectors. To overcome the need for repeated i.t. injections, eliminate the need for an accessible tumor site, and allow for more direct targeting of CpG to tumor cells, we chemically linked CpG to rituximab using a cleavable linker. The conjugate retained both antibody binding activity to human CD20 and potent CpG biologic activity, demonstrated via upregulation of costimulatory molecules on cultured lymphoma cells and activation of bone marrow-derived dendritic cells. Just 2 injections of rituximab-CpG conjugate (containing 50 μg CpG, days 7 and 9) achieved eradication of 7-day established tumors from 100% of mice, while injection of rituximab-control ODN or trastuzumab-CpG conjugates had no therapeutic effects. Treatment with equivalent doses of rituximab plus i.v. or i.t. CpG prolonged survival slightly, but did not result in tumor eradication. In vivo depletion of NK cells or complement nearly eliminated tumor eradication effects. In contrast, in vivo depletion of macrophages, or CD4⁺ or CD8⁺ T cells had no significant effects on tumor eradication by rituximab-CpG, indicating that adaptive T cell immunity did not contribute to elimination of tumor during rituximab-CpG conjugate therapy. As CpGs can stimulate leukocytes to secrete alpha interferon (αaIFN), which can inhibit the growth of B cell lymphomas, we sought to determine whether αaIFN played a significant role in tumor control after rituximab-CpG therapy. 38C13-huCD20 tumor cells rendered deficient in αaIFN receptor (IFNAR) by lentiviral transduction with shRNA were nearly as sensitive to rituximab-CpG as wild-type IFNAR⁺ 38C13-huCD20 cells, indicating no role for direct αaIFN tumor cytotoxicity in eradication of 38C13-huCD20. In conclusion, natural killer cells and complement, but not T cells, macrophages, or αaIFN, are critical to the efficacy of rituximab-CpG conjugate in efficiently eradicating an established human CD20⁺ B cell lymphoma that is fully resistant to single-agent rituximab. Further pre-clinical studies of anti-CD20-CpG conjugates against B cell lymphomas are thus warranted. Anti-CD20-CpG conjugates may represent a novel treatment modality for human NHL.