Sorafenib Affects Membrane Complement Inhibitors and Improves Antitumor Activity of Rituximab,
Authors: Golab Jakub , Bil Jacek , Bojarczuk Kamil , Nowis Dominika , Okroj Marcin , Basak Grzegorz , Stoklosa Tomasz , Winiarska Magdalena 
Published: October 2019

Abstract
Abstract 3723
Monoclonal antibodies (mAbs) targeting CD20 antigen are now routinely used in the treatment of various types of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). Their antitumor action results from the ability to trigger complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, direct cell death can be induced upon crosslinking with secondary antibodies. Previous studies have demonstrated a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC but not ADCC. Next to CD20 expression level also other potential mechanisms of anti-CD20-mediated cytotoxicity have been observed. Among them an increased expression of complement regulatory proteins (CRP), such as CD46, CD55 or CD59 has been shown to contribute to resistance to mAb-mediated CDC. Antibodies blocking CRP or siRNA that knocks-down CRP expression facilitate rituximab-mediated cytotoxicity. Also in xenograft in vivo models it was shown that antibodies blocking the activity of CD55 and CD59 or a recombinant adenoviral fiber knob protein that cross-links CD46 molecules enhance therapeutic effects of rituximab. Fludarabine, the nucleoside analogue clinically active against CLL and indolent NHL has been shown to act synergistically with rituximab and to down-regulate the membrane expression of CD55 without significantly altering CD20 levels.
The aim of this study was to investigate the influence of sorafenib, a multikinase inhibitor on the expression of CRPs in human CD20+ B-cell lymphomas and to evaluate its ability to trigger CDC in combination with rituximab. A 48-h incubation of four different human non-Hodgkin lymphoma cells DoHH2 (Fig. 1A), Daudi, Raji and Ramos with sorafenib resulted in a marked concentration-dependent drop in the surface expression of CD46, CD55 and CD59, but did not affect the levels of CD20. Analysis of primary cells isolated from 5 consecutive patients with CD20+ CLL also revealed a significant drop in the levels of CD46 (5 of 5 patients), CD55 (4 of 5 patients), and CD55 (5 of 5 patients) after a 48 h incubation of tumor cells with sorafenib. Accordingly, with previous reports, sorafenib significantly decreased phosphorylation of STAT3 transcription factors that are involved in the regulation of CRP expression. Incubation of tumor cells with sorafenib lowered the levels of mRNA for all three CRPs, but not that of CD20 indicating that sorafenib-mediated downregulation of CRPs occurs at transcriptional level. Pre-incubation of all four lymphoma cell lines cells with sorafenib at concentrations that down-modulate CRPs levels also significantly sensitized tumor cells to rituximab-mediated CDC (Fig. 1B). Moreover, sorafenib led to increased incorporation of membrane-attack complex (C5b-9) into the membranes of DoHH2 cells. On the other hand, sorafenib did not affect rituximab-mediated ADCC nor apoptosis in DoHH2 cells. Sunitinib, another multi-kinase inhibitor, did not affect rituximab-mediated complement-dependent cytolysis.Figure 1.(A) DoHH2 cells were incubated with either diluent or sorafenib at 1.25, 2.5 and 3.75 μM concentration for 48h. Then, cells (1 × 105/100 μl) were stained for CD20, CD46, CD55 and CD59 and analyzed in a FACSCalibur using Cell-Quest Pro software version 5.2. (B) DoHH2, Daudi, Raji or Ramos cells were incubated with either diluent or sorafenib at appropriate concentrations for 48h. Then, equal numbers of cells (1 × 105/well) were incubated for 1h with serial dilutions (from 1 to 100 μg/ml) of rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured with a MTT assay. The survival of cells is presented as percentage of corresponding diluent- or sorafenib-pretreated cells without rituximab. *P<0.05 (Student's t -test).Figure 1. (A) DoHH2 cells were incubated with either diluent or sorafenib at 1.25, 2.5 and 3.75 μM concentration for 48h. Then, cells (1 × 105/100 μl) were stained for CD20, CD46, CD55 and CD59 and analyzed in a FACSCalibur using Cell-Quest Pro software version 5.2. (B) DoHH2, Daudi, Raji or Ramos cells were incubated with either diluent or sorafenib at appropriate concentrations for 48h. Then, equal numbers of cells (1 × 105/well) were incubated for 1h with serial dilutions (from 1 to 100 μg/ml) of rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured with a MTT assay. The survival of cells is presented as percentage of corresponding diluent- or sorafenib-pretreated cells without rituximab. *P<0.05 (Student's t -test).
Altogether, these results indicate that sorafenib, which undergoes clinical trials in patients with NHL and B-CLL might be effectively used in combination with anti-CD20 mAbs.

Disclosures:
No relevant conflicts of interest to declare.