Application of a novel inhibitor of human CD59 for the enhancement of complement-dependent cytolysis on cancer cells

Abstract

Many monoclonal antibodies (mAbs) have been extensively used in the clinic, such as rituximab to treat lymphoma. However, resistance and non-responsiveness to mAb treatment have been challenging for this line of therapy. Complement is one of the main mediators of antibody-based cancer therapy via the complement-dependent cytolysis (CDC) effect. CD59 plays a critical role in resistance to mAbs through the CDC effect. In this paper, we attempted to investigate whether the novel CD59 inhibitor, recombinant ILYd4, was effective in enhancing the rituximab-mediated CDC effect on rituximab-sensitive RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells. Meanwhile, the CDC effects, which were mediated by rituximab and anti-CD24 mAb, on the refractory multiple myeloma (MM) cell line ARH-77 and the solid tumor osteosarcoma cell line Saos-2, were respectively investigated. We found that rILYd4 rendered the refractory cells sensitive to the mAb-mediated CDC effect and that rILYd4 exhibited a synergistic effect with the mAb that resulted in tumor cells lysis. This effect on tumor cell lysis was apparent on both hematological tumors and solid tumors. Therefore, rILYd4 may serve as an adjuvant for mAb mediated-tumor immunotherapy.

Figure 1

rILYd4 sensitized the original RL-7 lymphoma cells and rituximab-induced resistant RR51.2 cells to the rituximab-mediated CDC effect. (a, b) RL-7 cells and RR51.2 cells expressed CD20 and CD59 on the cell surface. Shaded histogram: isotype-matched Abs+FITC secondary Ab staining (negative control); open histogram: anti-hCD59 Ab or anti-CD20 Ab (0.2 µg/ml)+FITC secondary Ab. (c, d) rILYd4 dose dependently enhanced the rituximab (1 or 10 µg/ml)-mediated CDC effect on RL-7 lymphoma cells or RR51.2 cells. Triton X-100 was utilized as a positive control, and all treated cells were 100% cytolysis-positive. Statistical significance is indicated by an asterisk (P<0.01 versus the group treated with rituximab alone). (e, f) Cells were treated with either 376 or 320 nM of rILYd4 to neutralize the hCD59 on lymphoma RL-7 and RR51.2 cells. Statistical significance is indicated by an asterisk (P<0.01 for the rILYd4 group versus the PBS group). Results are expressed as mean±SEM, and the results are derived from three independent experiments. Ab, antibody; CDC, complement-dependent cytolysis; MFI, mean fluorescence intensity; NHS, normal human serum; PBS, phosphate-buffered saline.

Figure 2

rILYd4 senstized MM ARH-77 cells to rituximab-mediated CDC effect. (a) CD20 and CD59 are expressed on the surface of MM ARH-77 cells. Shaded histogram: isotype-matched Abs+FITC secondary Ab staining (negative control); open histogram: anti-hCD59 Ab or anti-CD20 Ab (0.2 µg/ml)+FITC secondary Ab. (b) rILYd4 dose dependently enhanced the rituximab (20 µg/ml)-mediated CDC effect in the ARH-77 cells. Triton X-100 was utilized as a positive control, and all treated cells were 100% cytolysis positive. Statistical significance is indicated by an asterisk (P<0.01 versus the group treated with rituximab alone). (c) rILYd4 (520 nM) sensitized the MM ARH-77 cells to the rituximab-mediated CDC effect. Statistical significance is indicated by an asterisk (P<0.05 for the rILYd4 group versus the PBS group). Results are expressed as mean±SEM, and the results are derived from three independent experiments. Ab, antibody; CDC, complement-dependent cytolysis; MM, multiple myeloma; NHS, normal human serum; PBS, phosphate-buffered saline.

Figure 3

The resistance of the ARH-77 cells to the rituximab-mediated CDC effect is associated with CD59 and other mCRPs levels. (a) CD46 and CD55 are expressed on the surface of RL-7, RR51.2 and ARH-77 cells. Shaded histogram: isotype-matched Abs+FITC secondary Ab staining (negative control); Open histogram: anti-hCD46 Ab or anti-CD55 Ab (0.2 µg/ml)+FITC secondary Abs. (b) Comparison of CD20 or mCRPs levels on the RL-7, RR51.2 and ARH-77 cells in response to the CDC effect in either the presence or the absence of rILYd4. (c) The dose of rILYd4 required for mediating 50% of the rituximab-mediated cytolysis in RL-7, RR51.2 and ARH-77 cells. Results are expressed as mean±SEM, and the results are derived from three independent experiments. (d) A positive linear correlation was observed between the IC₅₀ of rILYd4 and the expression level of CD59. The positive linear correlation between the IC₅₀ of rILYd4 and the expression level of CD59 was calculated with SPSS 16.0 statistics software. P<0.01 (F=7.803E4). Ab, antibody; CDC, complement-dependent cytolysis; mCRPs, membrane complement regulatory regulators; MFI, mean fluorescence intensity; Rit, rituximab.

Figure 4

Recombinant rILYd4 enhanced the anti-CD24 monoclonal Ab (clone SN3)-mediated CDC effect on osteosarcoma Saos-2 cells. (a) CD24 and CD59 are expressed on the surface of Saos-2 cells. Shaded histogram: isotype-matched Ab+FITC secondary Ab staining (negative control); open histogram: anti-hCD59 Ab or anti-CD24 Ab (0.2 µg/ml)+FITC secondary Ab. (b) The Saos-2 cells were effectively sensitized with the 20 µg/ml anti-CD24 monoclonal Ab-mediated CDC effect with 215 and 430 nM of rILYd4. Triton X-100 was utilized as a positive control, and all treated cells were 100% cytolysis-positive. Statistical significance is indicated by an asterisk (P<0.01 versus anti-CD24 Abs alone group). Results are expressed as mean±SEM, and derived from three independent experiments conducted in triplicate. Ab, antibody; MFI, mean fluorescence intensity.