Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Proteasome Inhibitors

Introduction: Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM). Despite the introduction of novel therapies more than 70% of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role on proliferation of MM and that its inhibition with small-molecule inhibitors sensitized MM cells to therapy. However, these small molecule inhibitors had low specificity to P-selectin and showed poor pharmacokinetics. In this study, we tested inhibition of P-selectin and PSGL-1 using functionally blocking monoclonal antibodies to sensitize MM cells to therapy.

Methods: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals. Endothelial cells, stromal cells derived from MM patients, and MM cell lines (MM1s, H929, OPM1 and RPMI8226) were used for in vitro expression, proliferation, apoptosis and immuno-blotting assays. The expression of P-selectin and PSGL-1 were tested by the interaction of SelG1 (5-20µg/mL) and SelK2 (5-20µg/mL) antibodies with endothelial cells, stromal cells and MM cells for 1hr, followed by addition of a secondary-FITC antibody and flow cytometry analysis. For adhesion assay, cells were treated with increasing concentrations of SelG1 and SelK2 for 1hr; pre-labeled MM cells were then applied to unlabeled endothelial or stromal cells for 1hr, non-adherent cells were washed, and adherent cells were analyzed by a fluorescent reader. For proliferation, MM1s-GFP+ cells cultured alone, with stroma, or with endothelial cells; and were treated with, or without, bortezomib and carfilzomib, in the presence, or absence, of SelG1 and SelK2 antibodies, and proliferation was determined by flow cytometry. Protein expression associated with survival, apoptosis and cell cycle signaling was analyzed by western blotting.

For in vivo, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumor progression followed for 4 weeks. Anti-mouse-P-selectin antibody was used to inhibit P-selectin in the mouse endothelial cells and stroma. Likewise, SelK2 and anti-mouse PSGL-1 were used to inhibit PSGL-1 on human MM cells in the mouse microenvironment, respectively. Mice were divided into 6 groups (1) vehicle treated control, (2) anti-mouse-P-selectin (5mg/kg) alone, (3) SelK2 (5mg/kg) and anti-mouse-PSGL-1 (5mg/kg) alone, (4) bortezomib (0.5mg/kg) alone, (5) a combination of anti-mouse-P-selectin and bortezomib, and (6) a combination of SelK2 (5mg/kg), anti-mouse-PSGL-1 (5mg/kg) and bortezomib. Anti-mouse-P-selectin, anti-mouse–PSGL-1, SelK2 and bortezomib were administered intraperitoneally twice a week.

Results:

The half-life of SelG1 in a Phase I clinical study was previously shown to be 363 hours while the half-life of SelK2 in a primate study was approximately 100 hours. P-selectin expression was detected on endothelial and stromal cells using the SelG1 antibody, while no expression was found on MM cells. PSGL-1 was highly expressed on MM cells as well as on endothelial cells and stromal cells as detected by SelK2 monoclonal antibody. Inhibition of P-selectin and PSGL-1 with SelG1 or SelK2, respectively, decreased MM cell adhesion to endothelial and stromal cells, and decreased the proliferation of MM cells induced by stromal and endothelial cells. Similarly, inhibition of the interaction between P-selectin and PSGL-1 sensitized MM cells to bortezomib and carfilzomib in vitro. In vivo results demonstrated that inhibition of the P-selectin or PSGL-1 as single agents delay tumor growth compared to non-treated mice and that it enhances the effect of bortezomib.

Conclusions: These results demonstrate that inhibition of P-selectin and PSGL-1 by SelG1 and SelK2 antibodies, respectively, disrupts the interaction between MM cells and BM microenvironment and decreases adhesion and proliferation of MM cells. Moreover, inhibition of P-selectin and PSGL-1 increased the sensitivity of MM cells to bortezomib and carfilzomib in vitro, and to bortezomib in vivo. These data provides a basis for future clinical trials for sensitization of refractory MM patients to therapy by inhibition of P-selectin and PSGL-1 using SelG1 and SelK2 monoclonal antibodies.