A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation

Abstract

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

A low cartilage formation and repair endotype predicts radiographic progression of symptomatic knee osteoarthritis

“…Type II collagen is nearly exclusively localized to cartilage, being the major structural component of this tissue. Recently, immunoassays suitable for measuring serological collagen type IIB N-propeptide fragment (PIIBNP or PRO-C2) were developed and proposed for the estimation of cartilage formation [14].…”

“…This suggests that PRO-C2 could serve as a biomarker not only for prediction of OA progression but also for treatment response. We recently demonstrated that FGF-18 induced PRO-C2 in both human and animal chondrocytes [14,32]. In addition, in the phase II FORWARD (FGF-18 Osteoarthritis Randomized Trial with Administration of Repeated Doses) study, intra-articular FGF-18 demonstrated a dose-dependent increase in the cartilage thickness on Magnetic Resonance Imaging (MRI) [33,34].…”

“…Serum or plasma PRO-C2 was assessed at baseline and 24 months in blood using a high sensitivity (hs) PRO-C2 electro-chemiluminescence ImmunoAssay (ECLIA) (Nordic Bioscience, Herlev, Denmark). It is a competitive assay based on a monoclonal antibody speci c to the epitope (QDVRQPGPKG) derived from the N-terminal propeptide region of the type IIB procollagen α1-chain [14]. We have previously developed the PRO-C2 enzyme-linked immunosorbent assay (ELISA), which, had some sensitivity limitations.…”

“…We have previously developed the PRO-C2 enzyme-linked immunosorbent assay (ELISA), which, had some sensitivity limitations. Therefore, we converted the assay to the ECLIA platform, resulting in a seven-fold increased sensitivity [14]. PRO-C2 was measured in the NYU cohort (non-fasting plasma) and the placebo (PLB) and sCT arms of the SMC subcohort (fasting serum) at baseline and 24 months.…”

“…We recently demonstrated that PRO-C2 concentrations were lower in OA patients compared to healthy controls and could be induced by potential cartilage anabolic therapy [14].…”