HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis

Abstract

Background: Incorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin's lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule.

Design and methods: By retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells.

Results: We show that HuMab-7D8 efficiently killed CD20(low) cells that are not susceptible to rituximab-induced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20(low) cells survived.

Conclusions: Cells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic.

Figure 1.

Resistance to rituximab-mediated killing is not explained by ineffective translocation into lipid rafts. (A) FACS-analysis of HuMab-7D8 (light gray) and rituximab (dark gray) binding to CEM-CD20 cells at saturating concentrations. The black line displays transduced cells that were stained with FITC conjugated human IgG. (B) Individual CEM-CD20 clones were incubated with 10 μg/ml rituximab or HuMab-7D8 and CDC was induced by adding 20% normal human serum as source of complement. The expression of CD20 in number of molecules per cell was plotted against the extent of CDC (expressed as % PI positive cells). The open dots represent rituximab-mediated CDC and the filled dots HuMab-7D8. (C) CEM-CD20 clones (A-F) with increasing CD20 expression (see Y-axis) were incubated with 10 μg/mL of CD20 mAb. One half of the cells were directly stained with anti-IgG1-FITC (open bars). The other half was first treated with Triton X-100 prior to staining with anti-IgG1-FITC (filled bars). (D) The open bars show the percentage CDC of the clones in the presence of rituximab and normal human serum, the filled bars show the HuMab-7D8-induced CDC. All experiments were performed in duplicate and repeated once with similar results.

Figure 2.

Preparation of luciferase expressing CEM-CD20 cells. (A) Schematic representation of CD20- and LucR-encoding retroviral vectors. (B) FACS analysis of transduced and FACS-sorted LucR-tagged CEM-CD20 cell pool. (C) In vitro luciferase activity of LucR-tagged CEM-CD20 cells and control CEM-CD20 cells. (D) In vitro CDC assay of the LucR-CEM-CD20 cell pool prior to in vivo use; cell kill is expressed as % PI positive cells.

Figure 3.

In vivo bioluminescence imaging. (A) In vivo imaging at day 20 and day 38 of mice inoculated with LucR-CEM-CD20 cells as described in the Design and Methods section. Mice were treated either with PBS, rituximab or HuMab-7D8. (B) After the last bioluminescence (on day 38) the mice were euthanized. Luciferase activity in relative light units was plotted against time. (C) Survival curve of mice inoculated with LucR-CEM-CD20 cells treated with PBS (control), rituximab or HuMab-7D8.

Figure 4.

HuMab-7D8 eradicates CD20^low-expressing CEM cells in vivo. FACS-analyses of CEM-CD20 cells harvested from bone marrow of treated mice and cultured for two weeks. Each graph represents cells from an individual mouse except graph A (see below). The gray plots represent the non-transduced CEM cells. (A) CD20 expression of the pool of retrovirally CD20-transduced CEM cells that was injected into the mice and kept in culture during the experiment. (B) CD20 expression of CEM cells retrieved from the PBS-treated mice. (C) CD20 expression of CEM cells retrieved from rituximab-treated mice and (D) CD20 expression of CEM cells retrieved from the HuMab-7D8-treated mice.

Figure 5.

Rituximab-resistant cells are lysed by HuMab-7D8. (A) CD20 mAb-induced CDC of a CEM-CD20 clone with low CD20-expression (MFI of 325) by rituximab and HuMab-7D8. Cells were incubated with human serum only or with either rituximab or HuMab-7D8 and human serum. After 24 h the cells were washed and a sample was taken and analyzed for CDC. Results are expressed as % PI positive cells. The bars indicate the mean ± SD of triplicate measurements. (B) The cells that survived the one day initial rituximab- or Humab-7D8-mediated killing were cultured for an additional two weeks. Rituximab treated cells were subjected to a second exposure of CDC induction using either rituximab or HuMab-7D8. Conversely, the HuMab-7D8-treated cells from the first experiment were subjected to CDC induction using either rituximab or HuMab-7D8. Background lysis was assessed by adding normal human serum only.