Lack of Increased Clinical Efficacy When Interleukin-12 Is Added to Rituximab in B-Cell Lymphoma Patients Is Related to Inadequate Delivery of the Cytokine to the Sites of Lymphoma.

Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B-lymphocytes. Binding of the Fab domain to B-cells directly induces apoptosis, while the Fc domain recruits immune effector functions to mediate cell lysis. Because rituximab therapy alone does not result in durable responses for all patients, combination therapies have been explored. We have previously shown in a phase I study that interleukin-12 (IL-12), which facilitates cytolytic T-cell responses and enhances the lytic activity of NK cells, can be safely combined with rituximab and that IL-12 significantly upregulated gamma interferon, CXCL10 (inducible protein-10) and NK cell activity in the peripheral blood. To confirm whether IL-12 could augment the immune mediated cell lysis induced by rituximab, a subsequent randomized phase II study of the combination was performed in patients with B-cell lymphoma. While the combination of IL-12 and rituximab was well tolerated with acceptable toxicity, only moderate disease activity was seen and the response rate to the combination was similar to that seen with rituximab alone. Additionally, the sequential administration of IL-12 at the time of disease progression after treatment with rituximab did not result in any clinical responses. This study was therefore performed to determine potential biologic reasons for the lack of increased clinical efficacy when IL-12 was added to rituximab therapy in patients with B-cell non-Hodgkin lymphoma. Of the 52 patients treated on the phase II study, 8 patients had matched tumor biopsies and peripheral blood specimens obtained prior to therapy and again 2 weeks after treatment was started. Six of the patients were receiving IL-12 plus rituximab at the time the specimens were obtained while 2 were receiving rituximab alone. Gene expression array analysis using the Affymetrix U133 plus chip was performed on RNA isolated from cells from involved lymph nodes and from peripheral blood mononuclear cells. Specimens from the peripheral blood of patients who received IL-12 in combination with rituximab showed a greater than 5-fold increase in the expression of multiple genes known to be upregulated by IL-12 signaling including interferon gamma, CXCL10, IFIT2 and 4 (interferon-induced protein with tetratricopeptide repeats 2 and 4), IL-8 and CXCL2 (macrophage inflammatory protein-2). These increases in gene expression were not seen in the peripheral blood of patients who received rituximab alone. Furthermore, the significant changes seen in cells obtained from the peripheral blood were not seen in cells obtained from lymph nodes involved by lymphoma, despite the samples being obtained from the same patient on the same day. In the tumor specimens, the changes in gene expression involved none of the genes downstream of IL-12 signaling. Instead, many of the upregulated genes were associated with cell cycle and spindle checkpoint proteins suggesting ongoing tumor cell proliferation. In conclusion, while IL-12 significantly upregulated gene expression in the peripheral blood, the same changes were not seen in the tumor. This would suggest that systemically administered IL-12 may not be effectively delivered to the site of tumor involvement. This finding may explain the lack of additional clinical benefit when IL-12 was added to rituximab as therapy for patients with non-Hodgkin lymphoma.