Assessment of Eculizumab and C5 Levels in PNH Treatment: Molar Ratio of Eculizumab to C5 Level Effectively Predicts Ongoing Dosage Changes in a Cohort of 50 Patients Treated with Eculizumab

Introduction

Eculizumab is highly effective in preventing the intravascular haemolysis and long-term complications of PNH. However a significant proportion (>20%) of patients in the Leeds cohort require a dose in excess of the standard 900mg Q2W to achieve and maintain efficacy. It is, therefore, important to determine the circulating concentration of both the drug and its target, complement component C5, throughout treatment to accurately inform dosing and to minimise the risk of a potentially serious breakthrough event.

Methods

Serum samples were collected prior to scheduled treatment and were analysed for trough levels of eculizumab by bridge ELISA (Cambridge Biomedical, MA), and subsequently for both eculizumab levels and C5 levels by mass spectrometry and immunonephelometry, respectively (Mayo Clinic, MN). Eculizumab levels determined by the two techniques were compared for a total of 54 patient samples, and C5 levels were determined in these sample, as well as in 20 normal controls. For 7 patients multiple samples collected over the treatment course were analysed to determine the concentration of drug, target and the molar ratio between the two over a period of up to 24 months. Clinical data maintained by the UK PNH National Service (Leeds, UK) was correlated with these findings.

Results

ELISA measurements for eculizumab levels identified 35/50 (70%) patients below the target trough level of 100ug/ml; mass spectrometry indicated that only 7/50 (14%) patients were below this level. All 7 patients below 100ug/ml by mass spectrometry were also reported as below this limit by ELISA. There was some correlation between the two tests with respect to overall position within the range of values reported (r² = 0.65), but absolute values differed significantly. This is expected, the C5 ELISA assay is only able to detect free eculizumab while the mass spectrometry assay can detect total eculizumab.

The median C5 level in healthy controls was 205ug/ml (range:128-305ug/ml) and fell within the expected normal range (106-263ug/ml); C5 levels for PNH patients receiving eculizumab were elevated in all patients, with a median of 403ug/ml (range:241-655ug/ml).

For the 7 patients with multiple samples: 5/7 had increasing circulating C5 over time whereas 2/7 had decreasing C5 (though only one was reduced to the high end of the normal range: 241ug/ml)

The eculizumab levels of 3/4 four patients on standard dose decreased over time (all falling below the clinically significant cut-off of 100ug/ml), 1 was unchanged. All patients receiving a higher dose (1200mg) showed an increased eculizumab level in excess of 100ug/ml.

A molar ratio of C5:eculizumab < 2 is required for adequate protection from intravascular haemolysis, as there are two C5 binding sites per eculizumab molecule. Four patients with a ratio >2.0 on the standard dose (900mg) had the ratio reduced to 2.0 or less following an increase to 1200mg and have remained on this dose. One patient had a ratio that decreased from 3.7 to 2.6 following dose increase (to 1200mg), and remains on this dose. Two patients on standard dose across the sampling time showed ratios of 4.0 and 2.4 and were subsequently given increased doses (1200mg).

Conclusion

The two methods used here for measuring eculizumab and C5 levels returned values in line with previous studies highlighting the increase in C5 levels associated with eculizumab treatment. Whilst eculizumab levels alone are a reasonable predictor of efficacy, the molar ratio between eculizumab and C5 correlated well with the clinical decision to increase dosage and should be tested in larger, prospective studies to assess its potential to inform clinical decisions regarding dose escalation.

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