Galiximab (anti-CD80 mAb)-Induced Cell Triggering Results in the Inhibition of Constitutive NF-Kb Activity in Raji: Contrasting Roles of Snail and RKIP in the Regulation of Drug/Immune Resistance

The CD80 antigen, also called B7.1, is the natural ligand for the T cell receptor CD28 and which maintains T cell and B cell adhesion. Galiximab (anti-CD80 mAb) is a primatized (human IgG1 constant regions and Cynomolgous macaque variable regions) mAb that binds CD80 on lymphoma cells and has been shown in vitro to inhibit tumor cell proliferation, upregulate apoptosis and induce ADCC. A phase I/II trial as single agent Galiximab with dose escalation demonstrated that it is well tolerated and produced modest clinical activity. Also, a phase I/II trial evaluated the combination of Rituximab and Galiximab in patients with relapse refractory follicular NHL. The combination produced an overall response rate of 66% with a median PFS of 12.4 months. We have reported that Galiximab sensitized Raji and IM-9 cells to drug-induced apoptosis. The present study extends these findings and examines the underlying molecular mechanism by which Galiximab sensitizes NHL cells to apoptosis by cytotoxic drugs. We hypothesized that Galiximab inhibits intracellularly cell survival anti-apoptotic pathways such as constitutively activated NF-kB, leading to sensitization to drug-induced apoptosis. We have used CD80-expressing Raji cells as a model for our studies. We demonstrate that following treatment of Raji with Galiximab (25–50 μg/ml) for 24 hours, cell lysates were assessed for various gene products of the NF-kB pathway by Western. There were significant downregulation of both p65 and phospho-p65, both IkB-α and phospho-IkB-α and downstream inhibition of Bcl-2 and Bcl_XL and induction of Bak. In addition, there was a strong induction of the metastasis suppressor and immune surveillance cancer gene product Raf-1 kinase inhibitor protein (RKIP) and downregulation of the inactive and phosphorylated form of RKIP. The induction of RKIP by Galiximab was, in part, the result of NF-kB-induced inhibition downstream of the metastasis inducer and RKIP transcription repressor Snail. Galiximab also inhibited downstream both the Fas and DR5 transcription repressor Yin-Yang 1 (YY1) concomitantly with upregulation of Fas and DR5. These findings establish a molecular mechanism by which Galiximab sensitizes tumor cells to drug/immune-induced apoptosis via inhibition of NF-kB and Snail and induction of RKIP expression. We have previously reported that Rituximab modifies intracellular pathways including NF-κB and sensitizes B-NHL to apoptosis (