Complement factor H-derived short consensus repeat 18-20 enhanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells

Abstract

The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.

Figure 1.

Production and heparin-binding activity of fH-derived proteins. (A) DNA fragments encoding hSCR18-20 and hSCR16-17 were cloned via EcoRI and XbaI restriction sites into pPICZα expression vector as indicated in the vector maps. (B) Culture supernatants were subjected to 6×His Ni-NTA affinity chromatography. Purified fractions were analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis and western blot. The specific bands of hSCR18-20 and hSCR16-17 in the culture supernatants (SN) appear in elution fraction 2 (E2) indicating the enrichment of the recombinant proteins mediated by the His-tag. (C) Heparin affinity chromatography was performed with hSCR18-20 and control domain hSCR16-17. Western blot analysis of flow-through (FT), wash (W1, W2) and elution (E1, E2) fractions was carried out. The distinct band of hSCR18-20 in E1 shows the heparin-binding activity of this domain, whereas hSCR16-17 was detected mainly in the FT indicating that the control SCR did not bind to heparin.

Figure 2.

Factor H-derived hSCR18-20 significantly enhanced ofatumumab(OFA)-induced CDC of primary CLL cells in vitro. (A) Twenty patients samples were treated in standard CDC assays using increasing concentrations of ofatumumab (1–100 μg/mL) in the absence or presence of hSCR18-20 (1200 μg/mL). Lysis of primary CLL cells was induced in a dose-dependent manner by ofatumumab (open circles) and was significantly enhanced by the addition of hSCR18-20 (closed circles, * P<0.05; ** P<0.01; *** P<0.001). In the presence of inactive complement (hiNHS) no reduction in the survival rates of CLL cells was observed after treatment either with ofatumumab alone (open circles, dashed line) or with ofatumumab and hSCR18-20 in combination (closed circles, dashed line). Circles: mean of 20 patients, bars: SEM. (B) Only 30% of all patient samples tested (6 out of 20) displayed a CDC non-responder phenotype showing less than 25% lysis at the highest ofatumumab concentration (open circles). Addition of hSCR18-20 rendered these cells more susceptible to ofatumumab-induced CDC and turned non-responder into responder samples at antibody concentrations ranging between 4 μg/mL and 100 μg/mL (closed circles). Circles: mean of six patients, bars: SEM, experiments performed in duplicate. (C) Fourteen out of 20 patients’ samples showed a dose-dependent response to ofatumumab (open circles) and were classified as CDC-responders according to the definition of >25% lysis at the highest antibody concentration. In the presence of hSCR18-20 ofatumumab-induced CDC was significantly increased at all antibody concentrations tested (closed circles). Circles: mean of 14 patients, bars: SEM.

Figure 3.

Complement-mediated effects of ofatumumab (OFA) and hSCR18-20 were specific to B cells. CDC assays were performed in a heterogeneous cell mixture of CLL patient PBMC and PBMC from healthy donors. Cells were stained with CD3 and CD19 antibodies and the survival rates were quantified for the individual populations. (A) Incubation of the cells in NHS only or in NHS and hSCR18-20 showed no effect on the PI-negative viable cell populations. Treatment with OFA (20 μg/mL) clearly reduced the viable CD19+ B-cell population (x axis) but not the CD3⁺ T-cell population. After addition of hSCR18-20 (1200 μg/mL) the viable B-cell fraction almost vanished, whereas the T-cell population was not affected. Results show one representative experiment. (B) Treatment with OFA or with OFA and hSCR18-20 significantly reduced the survival rates of the B-cell fraction (gray bars, 66% and 34%) in in vitro CDC assays, whereas no significant decrease in the survival rates of the T-cell population (black bars) was observed. The survival rates were calculated according to the hiNHS control, which defined 100% survival. Error bars: SEM, number of patients = 6.

Figure 4.

Western blot analysis of C3 fragments deposited on ofatumumab (OFA)-treated CLL cells in the presence or absence of hSCR18-20. CLL samples were incubated with NHS as a source of complement and with small amounts of OFA (2 μg/mL) to avoid complete lysis of the cells (lanes 3–10). SCR18-10 was added as indicated (samples 4, 6, 8, 10). C3 deposition was stopped at different time points and samples were subjected to western blot analysis. As a control, cells were incubated with hiNHS or NHS in the absence of OFA (lanes 1, 2), which allowed the detection of C3 α and β chains at 120 kDa and 75 kDa, respectively. Although the OFA concentration was suboptimal, the addition of hSCR18-20 resulted in the generation of active C3b, as indicated by the visible 110 kDa α′ chain (lanes 6, 8). In the absence of the SCR, no such activation product was detectable (lanes 3, 5, 7). After 60 min, more or less all C3 α chain has been consumed, as indicated by disappearance of the 120 kDa band (lane 10). As a loading control, samples were stained for β-actin.

Figure 5.

A trend toward higher CD20 expression was observed in CDC-responder patients. (A) A highly variable expression of CD20 was observed in 20 patients’ samples. CD20 MFI ranged from 4657 to 57575. (B) CD20 expression was considerably increased in the CDC-responder group (gray bar, 16096) as compared to the CDC non-responder group (black bar, 6427). However, statistical analysis (t test for unpaired data) did not show a statistically significant difference. Bars: SEM.

Figure 6.

The effects of blocking fH, CD55 or CD59 on ofatumumab (OFA)-induced CDC of primary CLL cells. (A) CLL cells were treated in standard CDC assays using OFA (20 μg/mL), hSCR16-17 (1200 μg/mL), hSCR18-20 (1200 μg/mL), blocking HD1A (10 μg/mL) or blocking MEM43 (10 μg/mL) either alone or in various combinations depending on the experimental design. The lysis induced by OFA (35.5%) was significantly (*P<0.05; **P<0.01; ***P<0.001) enhanced by additional treatment with hSCR18-20 (66.6%), HD1A (69.4%) or MEM43 (54.7%). The fH-derived control fragment hSCR16-17 did not affect survival rates (36.1%). Simultaneous blocking of fH and CD55 by hSCR18-20 and HD1A significantly improved the effects obtained with the individual agents. Additional blocking of CD59 by means of MEM43 showed minor effects. Survival rates were calculated with reference to the hiNHS control, which defined 100% survival. Bars: SEM, n = 20. (B) No induction of lysis was observed after treatment of primary CLL cells with hSCR18-20 (1200 μg/mL), HD1A (10 μg/mL) or MEM43 (10 μg/mL) under standard conditions in the absence of OFA. Survival rates were calculated according to the hiNHS control. Bars: SEM, n = 4.