Inhibition of NF-Kb Cell Signaling and Induction of the Metastasis Suppressor and Immune Surveillance Cancer Gene RKIP Expression in B-NHL by the Novel Humanized BM-Ca Anti-CD20 Monoclonal Antibody: Implication in Chemo and Immune-Sensitization
Authors: Vega Mario I , Martinez-Paniagua Melisa , Huerta-Yepez Sara , Bonavida Benjamin 
Published: April 2020

Abstract
Treatment of patients with relapsed or refractory low-grade follicular B-NHL lymphoma with rituximab has resulted in ~50% response rate. The mechanism underlying the failure of rituximab to affect the remaining 50% of the patients is not clear, though their tumors express CD20. The in vivo effector functions of rituximab include ADCC, CDC, and seldom apoptosis. In addition, we have reported that rituximab signals the cells and inhibits several intracellular cell survival pathways that are responsible for the immune and chemo-sensitizing effects of rituximab on resistant B-NHL cell lines (see review Jazirehi and Bonavida, Oncogene24:2121, 2005). The objective of this study was to develop novel and fully humanized anti-CD20 monoclonal antibodies with enhanced effector functions and molecular signaling that may potentiate their therapeutic efficacy. Novel humanized anti-CD20 monoclonal antibodies were derived from a chimerized form of murine anti-CD20 1K11791, shown to exert more important ADCC, CDC, and apoptotic activities as compared to rituximab (Nishida et al., Int J Oncol., 31:29, 2007). A representative humanized monoclonal antibody, BM-ca (Biomedics Inc., Tokyo, Japan) was used to examine its molecular cell signaling on a representative Ramos B-NHL cell line. The studies were also performed in parallel with rituximab treatment. Ramos cells were treated with various concentrations of BM-ca monoclonal antibody and it was found to inhibit cell proliferation in a concentration-dependent manner. The optimal concentration of BM-ca mAb (20–40 μg/ml) was used for further molecular analyses. Following treatment of Ramos with BM-ca mAb for 24 hours, cell lysates were assessed for the expression of various gene products by Western. Compared to untreated cells, treatment with BM-ca inhibited constitutively activated NF-kB as assessed by inhibition of phospho-p65, phospho-IkB-α, but not unphosphorylated forms. In addition, the NF-kB upstream kinase phospho-p38 MAPK was also inhibited. Inhibition of NF-kB and phospho-p38 MAPK resulted in downstream inhibition of the anti-apoptotic gene product Mcl-1 and induction of the proapototic gene products Bax and Bak. BM-ca significantly induced the expression of the metastasis suppressor and immune surveillance cancer gene product, Raf-1 kinase inhibitor protein (RKIP). RKIP has been shown to inhibition NF-kB activity and inhibition of NF-kB results downstream in the inhibition of the metastasis-inducer gene product Snail. Snail is a transcription factor that has been shown recently to negatively regulate the expression of RKIP (Beach et al, Oncogene27:2243, 2008). BM-ca inhibited Snail expression in Ramos cells. In comparison with rituximab, BM-ca showed qualitative and quantitative differences in the regulation of gene expression. These findings demonstrate that BM-ca triggers CD20-expressing B-NHL cells and results in significant alteration of several gene products that regulate chemo and immune-resistance and metastasis.