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. 2021 Aug 14;3(4):100207.
doi: 10.1016/j.ocarto.2021.100207. eCollection 2021 Dec.

A serological type II collagen neoepitope biomarker reflects cartilage breakdown in patients with osteoarthritis

Solveig Skovlund Groen  1   2 , Dovile Sinkeviciute  1   3 , Anne-Christine Bay-Jensen  1 , Christian S Thudium  1 , Morten A Karsdal  1 , Simon Francis Thomsen  4 , Sven Lindemann  5 , Daniela Werkmann  5 , Joseph Blair  1 , Line Mærsk Staunstrup  1 , Patrik Önnerfjord  3 , Lars Arendt-Nielsen  6 , Signe Holm Nielsen  1   7
Affiliations

A serological type II collagen neoepitope biomarker reflects cartilage breakdown in patients with osteoarthritis

Solveig Skovlund Groen et al. Osteoarthr Cartil Open. .

Abstract

Objectives: There is an unmet medical need for biomarkers in OA which can be applied in clinical drug development trials. The present study describes the development of a specific and robust assay measuring type II collagen degradation (T2CM) and discusses its potential as a noninvasive translational biomarker.

Methods: A type II collagen specific neoepitope (T2CM) was identified by mass spectrometry and monoclonal antibodies were raised towards the epitope, employed in a chemiluminescence immunoassay. T2CM was assessed in bovine cartilage explants with or without MMP-13 inhibitor, and explant supernatants were analyzed by Western blot. T2CM was measured in plasma samples from one study (n ​= ​48 patients) where OA patients were referred to total knee replacement (TKR). Additionally, T2CM was quantified in serum from OA patients receiving salmon calcitonin treatment (sCT) (n ​= ​50) compared to placebo (n ​= ​57).

Results: The T2CM assay was technically robust (13/4 ​% inter/intra-variation) and specific for the type II collagen fragment cleaved by MMP-1 and -13. The MMP-13 inhibitor reduced the T2CM release from bovine cartilage explants receiving catabolic treatment. These results were confirmed by Western blot. In human end-stage OA patients (scheduled for TKR), the T2CM levels were elevated compared to moderate OA (p<0.004). The OA patients receiving sCT had lower levels of T2CM compared to placebo group after 1, 6, and 24 months of treatment (p ​= ​0.0285, p ​= ​0.0484, p ​= ​0.0035).

Conclusions: To our knowledge, T2CM is the first technically robust serological biomarker assay which has shown biological relevance in ex vivo models and OA cohorts. This suggests that T2CM may have potential as a translational biomarker for cartilage degradation.

Keywords: Biomarker; Cartilage; Extracellular matrix; T2CM; Type II collagen.

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Conflict of interest statement

The study was supported by The Danish Council for Technology and Innovation and The Danish National Advanced Technology Foundation. Morten A. Karsdal, Anne-Christine Bay-Jensen, Christian S. Thudium and Signe Holm Nielsen, Joseph Blair, and Line Mærsk are employees of Nordic Bioscience. Morten A. Karsdal holds stock in Nordic Bioscience. Sven Lindemann and Daniela Werkmann are employees of Merck. Solveig Skovlund Groen, Simon Francis Thomsen, Dovile Sinkeviciute, Patrik Önnerfjord, and Lars Arendt-Nielsen have no competing interest.

Figures

Fig. 1
Fig. 1
Sequence alignment of the targeted T2CM sequence in human, mouse, bovine, and rat species (black box). The sequences were aligned using Clustal Omega algorithm.
Fig. 2
Fig. 2
A-C. Standard curve and native reactivity to various matrices. Standard curves and native reactivity against A) human serum, B) human plasma (EDTA, Citrate and Heparin), and C) animal samples (rat serum, mouse serum and BEX supernatant), diluted from 1:1, 1:2, 1:4 and 1:8 for the T2CM assay. Signals are shown as relative luminescence units (all wavelengths) per second, as a function of standard peptide concentration.
Fig. 3
Fig. 3
A-B. A) High specificity of the T2CM assay. Reactivity towards the standard peptide (MSAFAGLGPR), truncated peptide (SAFAGLGPR), elongated peptide (DMSAFAGLGPR). No background signal was detected when using a non-sense screening (coating) peptide (HDFSSDLENVK-Biotin). Signals are shown as relative luminescence units (RLU) per second, as a function of standard peptide concentration. B) MMP-1 and -13 primarily mediate the T2CM neoepitope. Generation of T2CM fragment by MMP-mediated cleavage of human articular cartilage obtained from an OA patient undergoing total knee replacement surgery. Results are shown as bars with as mean ​± ​SD.
Fig. 4
Fig. 4
A-D. A-B) T2CM was increased during prolonged stimulation with OSM ​+ ​TNF in BEX supernatant. T2CM levels were measured in supernatant from the BEX model harvested on day 0, 7, 14, and 21. Explants were either without treatment (w/o) or treated with different concentrations of OSM ​+ ​TNF-α (O ​+ ​T). Group comparisons were done using two-way ANOVA with Tukey's multiple comparison at each timepoint, n ​= ​6. Error bars are shown with mean ​± ​SD. C) Western blot results confirmed the BEX results. T2CM fragments were visualized in explants conditioned media from day 7, 14, and 21, with strongest bands at day 21 which confirms the BEX results. 1) without treatment, 2) 20/20 ​ng/mL OSM ​+ ​TNF-α, 3) 20/40 ​ng/mL OSM ​+ ​TNF-α. D) MMP-13 inhibitor decreased the release of T2CM. T2CM release measured in BEX supernatant treated with MMP-13 inhibitor in combination with OSM ​+ ​TNF-α (O ​+ ​T). Supernatants were harvested on day 0, 7, 14, and 21. Group comparisons were done using two-way ANOVA with Tukey's multiple comparison, n ​= ​6. Error bars are shown with mean ​± ​SD.
Fig. 5
Fig. 5
A-B. A) T2CM were increased in patients undergoing total knee replacement. In EDTA plasma levels of T2CM were assessed in patients with moderate OA (n ​= ​54) and patients referred to total knee replacement (TKR) (n ​= ​48). Data were analyzed by Mann-Whitney test, and data are presented as a Tukey boxplot. B) T2CM was decreased in OA patients treated with oral salmon calcitonin compared to placebo. Serum levels of T2CM were assessed in oral salmon calcitonin treated (sCT) OA patients (n ​= ​50) compared to placebo (n ​= ​57). Data were analyzed by one-way analysis of covariance (ANCOVA) with Bonferroni post hoc test corrected for age, gender and BMI and presented as percentage (%) difference from baseline. All values are presented as mean ​± ​SD. Significance difference is given by ∗ p=0.0285, ∗’ p ​= ​0.0484, ∗∗p=0.0035. M: Month.
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