In vivo activity of rituximab-CpG oligodeoxynucleotide conjugate against rituximab-resistant human CD20+ B-cell lymphoma

Abstract

8529 Background: The anti-CD20 monoclonal antibody rituximab (R) is a mainstay in the treatment of B cell non-Hodgkin's lymphoma (NHL), exerting anti-tumor effects via antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and apoptosis induction. Toll-like receptor 9 agonist CpG oligodeoxynucleotides (CpG) are potent activators of ADCC and T cell immunity, and have been studied for anti-NHL effects when administered by systemic or intratumoral routes. We sought to optimize the delivery of CpG to sites of NHL to improve R efficacy. Methods: We utilized an aggressive syngeneic human CD20-expressing murine B cell lymphoma (38C13-huCD20) to study the in vivo augmentation of R efficacy by CpG. Established tumors in immunocompetent mice were treated with R plus CpG given systemically, intratumorally, or chemically linked to antibody using maleimide-sulfhydryl chemistry (D. Betting et al, J. Immunol. 2008; 181: 4131). Results: 5- to 7-day established tumors were completely resistant to single agent R. Combining intratumoral, but not systemic administration of CpG with R resulted in tumor eradication from up to 42% of mice (p < 0.0003 vs. R alone, CpG alone, R + systemic CpG). Mechanistic studies indicated that both natural killer cells and complement participated in the cure of tumors by intratumoral CpG + R, by increasing tumor cell sensitivity to complement and ADCC lysis, and by augmenting the cytotoxicity of ADCC effectors. To overcome the need for repeated direct intratumoral injections and allow precise targeting of CpG to tumor cells, we chemically linked CpG to R using a cleavable linker. A single i.v. injection of this R-CpG conjugate achieved eradication of established tumors from 100% of mice. In contrast, equivalent doses of unlinked i.v. R + CpG, CpG alone, or R alone cured only 8% of mice. Thus, combining CpG with R was most effective using direct conjugation to the antibody. Conclusions: In conclusion, enhancement of R efficacy required sustained intratumoral delivery of CpG to maximize anti-tumor responses. R-CpG conjugate efficiently eradicated an established B cell lymphoma that is fully resistant to single-agent R. Clinical testing of anti-CD20-CpG conjugates against B cell NHL is thus warranted. [Table: see text]