Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.
OBJECTIVE
Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA.


METHODS
The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA.


RESULTS
The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity.


CONCLUSIONS
We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.
Swearingen
C A
CA
Musculoskeletal Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Carpenter
J W
JW
Siegel
R
R
Brittain
I J
IJ
Dotzlaf
J
J
Durham
T B
TB
Toth
J L
JL
Laska
D A
DA
Marimuthu
J
J
Liu
C
C
Brown
D P
DP
Carter
Q L
QL
Wiley
M R
MR
Duffin
K L
KL
Mitchell
P G
PG
Thirunavukkarasu
K
K
D051722:ADAM Proteins / Q000032:analysis*; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D002358:Cartilage, Articular / Q000201:enzymology* / Q000276:immunology; D003404:Creatinine / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D020370:Osteoarthritis, Knee / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D011348:Procollagen N-Endopeptidase / Q000032:analysis*; D013582:Synovial Fluid / Q000201:enzymology
D053674:Aggrecans; D000911:Antibodies, Monoclonal; D015415:Biomarkers; D010446:Peptide Fragments; D003404:Creatinine; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein