Direct Growth Inhibition of Both Mouse and Human B Cell Lymphomas by CpG Oligodeoxynucleotides

Abstract 2854

Immunostimulatory CpG oligodeoxynucleotides (ODN) are potent activators of T cell immunity and antibody-dependent cellular cytotoxicity (ADCC), and under study as immunotherapeutic agents for a variety of cancers, including B cell lymphomas. Recently, anti-CD20 antibody-CpG conjugates have been shown to eradicate rituximab-resistant B cell lymphoma in a syngeneic murine lymphoma model (D. Betting et al, ASH 2009). CpG is known to strongly stimulate the proliferation of normal B cells. Paradoxically, CpG has been reported to markedly inhibit the in vitro growth of the murine B cell lymphoma A20 (J. Li et al, J. Immunol. 2007), thereby prompting us to investigate the direct effects of CpGs on the growth of human B cell lymphomas. We first demonstrated that CpGs, especially those of the B class, potently inhibited proliferation of the A20 mouse B cell line in vitro by up to 81.5% (class A 58.7% and class C 52.7%). Moreover, in non-tumor bearing mice intratumoral injections of CpG activated normal B cells, while mice bearing subcutaneous A20 tumors showed suppressed tumor growth after CpG injections. Similarly, in humans, CpGs strongly stimulated the proliferation of normal peripheral blood B cells (stimulation index for class B 27.5 at 5 μg/ml). A panel of 12 human lymphoma cell lines (DLBCL, Burkitt's, mantle cell) were cultured in the presence or absence of varying concentrations of CpGs of A, B, or C classes (50, 10, or 2 μg/ml) or control ODN. Proliferation was measured by [³H]-thymidine incorporation in quadruplicate 72 hour cultures, and apoptosis measured by Annexin-V and PI flow cytometry. In contrast to the stimulation observed with normal human B cells, the proliferation of all 12 lymphoma lines were inhibited by CpGs. The strongest inhibitory effects were seen with CpG 7909, a class B CpG under clinical development for cancer therapy (Pfizer, PF-3512676). Raji cells were inhibited by 77.9%, 40.7%, and 8.8% at CpG concentrations of 50, 10, and 2 μg/ml, respectively (p≤0.01 for all comparisons vs. media alone). Among the 12 tested cell lines, the percentage growth inhibition using 50 μg/ml CpG 7909 was 61.2–80.4% for germinal center-type DLBCL (SUDHL-4, SUDHL-6, OCI-Ly19), 50–59.5% for activated B cell-type DLBCL (SUDHL-2, OCI-Ly3, OCI-Ly10), 56.4–79.3% for Burkitt's lymphomas (Raji, Ramos, Daudi, BJAB), and 69.6–69.9% for mantle cell lymphomas (Jeko-1, Granta-519). Interestingly, although all of the human cell lines expressed TLR9 by semi-quantitative RT-PCR, inhibition in the proliferation levels did not correlate with TLR9 expression levels. CpG 7909 also induced significant levels of apoptosis in Raji and Jeko-1 cells, 10.1% and 27.6% respectively at 50 μg/ml. In conclusion, we have demonstrated that CpGs have divergent effects on normal versus malignant B cells in both mouse and human systems. Delivery of CpG to mouse lymphoma cells inhibited their growth in vivo, while normal mouse B cells were activated. Furthermore, CpGs directly inhibit the proliferation of a large panel of human B cell lymphomas representing the majority of aggressive histologies. These results provide a novel mechanism of action for CpGs as therapeutic agents for B cell lymphomas.