Melina Grasmeier from the Luppa Group (Klinikum rechts der Isar) successfully defended her PhD Thesis-Congratulations!

Biosensor-based monitoring of TNF antagonists and anti-drug antibodies in inflammatory bowel disease

Summary

Tumor necrosis factor (TNF) antagonists, a class of therapeutic proteins, have significantly
improved the treatment of inflammatory bowel diseases (IBD), such as Crohn’s disease
and ulcerative colitis. The pro-inflammatory cytokine TNF is a key player in IBD-related
intestinal inflammation. Pharmacological blockade of TNF using TNF antagonists in IBD
has been shown to be very effective and in some cases safer compared with application of
traditional small-molecule drugs. The chimeric therapeutic antibody infliximab (IFX) was
the first TNF antagonist to receive drug approval and remains widely prescribed. Yet, up
to 65 % of IFX-treated patients develop anti-drug antibodies. Anti-drug antibodies can
emerge with parenteral application of biologics. They can neutralize drug function and
enhance its clearance from the blood stream, which may lead to treatment failure.
Measuring serum levels of biologic drug and corresponding anti-drug antibodies, termed
therapeutic drug monitoring (TDM), is an essential tool to optimize effectiveness and
safety of biologic therapies. However, current TDM assays suffer from the analytic complexity
of anti-drug antibody assessment and poor harmonization of available assays. This
hampers the establishment of evidence-based consensus strategies for anti-drug antibody
management. In addition to the mainly (semi-)quantitative data provided by state of the
art TDM assays, qualitative data on patient-individual anti-drug antibodies may be of
importance for therapeutic decision-making. Though, the research literature regarding
qualitative analysis of anti-drug antibodies is very limited.
The aim of this dissertation was to develop surface plasmon resonance (SPR) spectroscopybased
biosensor assays for the TDM of the exemplary TNF antagonist IFX and to evaluate
the feasibility of SPR biosensors for routine TDM. With SPR, analytes can both
be quantified and characterized regarding their binding properties. Thus, it was additionally
investigated whether knowledge about the binding stability of anti-infliximab
antibody(ADA):IFX complexes adds diagnostic value in IFX therapy management. Using
a Biacore X100 SPR instrument, two biosensor assays were developed: “IFXmon”
for the quantification of IFX and “ADAmon” for the quantification and characterization
of ADA. Both IFXmon and ADAmon were compared to in vitro diagnostics-approved
enzyme-linked immunosorbent assays (ELISA) by analysis of sera from IFX-treated IBD
patients.
Method validation showed that IFXmon comprised acceptable biosensor stability, analytic
sensitivity, precision and accuracy. Analysis of 84 patient sera by IFXmon and
ELISA demonstrated method interchangeability. While IFXmon utilized diluted serum as
sample material, a pre-analytic affinity purification protocol with magnetic beads was developed
for the ADAmon assay. This additional step was necessary to allow IFX-tolerant
ADA quantification, that is, even in the presence of typical IFX serum concentrations.
Overall, ADAmon achieved similar performance parameters as ELISA with respect to
biosensor stability, analytic sensitivity, precision and accuracy. However, the results of
ADA quantification by ADAmon and ELISA in patient sera differed substantially and did
not correlate. This suggests that ADAmon and ELISA assess different, potentially ADA
quality-dependent ADA populations.
In order to characterize patient-individual ADA:IFX binding stabilities, the dissociation
ratio “DissR” was introduced. DissR is an easily accessible index that describes the dissociation
velocity of ADA:drug complexes. After successful validation of its robustness, DissR
was retrospectively calculated for ADAmon-positive patients in the so far largest study to
analyze ADA:IFX binding stability. It was shown that high ADA:IFX binding stability
was significantly correlated with the development of therapy failure and undetectable IFX
concentrations in the future, suggesting a prognostic value of ADA:IFX binding properties.
The assay principles of IFXmon and ADAmon, in particular also DissR, were
successfully transferred with minimal adaptations to adalimumab as another widely used
TNF antagonist. This implies that the established assays can be rapidly adapted.
In conclusion, the presented findings suggest that SPR-based biosensor assays suffice the
bioanalytic requirements for routine TDM. Particularly the assessment of ADA binding
stability may enable earlier therapeutic decision-making in IBD treatment. This dissertation
substantiates the practical and economic suitability of SPR for laboratory medical
analysis and highlights its potential for proactive TDM in the context of personalized
medicine.