FRI0367 ADALIMUMAB IS MORE EFFECTIVE THAN ETANERCEPT AT PREVENTING TNF-ENHANCED OSTEOCLAST DEVELOPMENT THROUGH DOWNREGULATION OF PRO-OSTEOCLASTOGENIC FACTORS ICAM-1 AND IGFBP2 AND UPREGULATION OF ANTI-OSTEOCLASTOGENIC FACTOR FABP4
Authors: B. Harvey, L. Jin, J. Maull, C. Hu, Y. Tian, Z. Kaymakcalan
Journal: Annals of the Rheumatic Diseases (2020)

Background: TNF has been shown to contribute to osteoclastogenesis independently and in conjunction with M-CSF and RANKL. We have previously demonstrated that TNF enhances the kinetics of RANKL-induced human osteoclastogenesis and that its effects are mitigated more effectively by the anti-TNF biologic adalimumab (ADA) as compared to etanercept (ETN). Objectives: To determine the mechanism responsible for the difference in effectiveness between the two biologics, a label-free quantitative proteomics study was conducted on TNF-activated human osteoclasts (OC) upon biologic treatment. Methods: Human bone marrow-derived OC precursors (OCP, 3 donors) were exposed for 5 days to M-CSF, M-CSF+RANKL (RANKL) alone or in combination with 100 ng/mL TNF +/- 5 ug/mL ADA, ETN or human IgG1 (IGG) as pre-formed complexes. OC differentiation was confirmed by measuring TRAP5b activity. Shotgun proteomics was performed on peptides generated from detergent based cell extracts subjected to methanol/chloroform precipitation and trypsin/Lys-C digestion. Data acquisition was performed with Orbitrap Q Exactive™ HF-X mass spectrometer. MaxQuant was utilized to quantify proteins based on MS1 peak intensities. Data matrix was normalized, imputed and subjected to differential expression analysis by limma. DAVID pathway analysis (DPA) was used to identify pathways impacted by various treatment conditions based on proteins exhibiting significant (p&lt;0.05) 1.2-fold change in expression as compared to RANKL. Protein levels within culture supernatant or cell lysate were verified by ELISA or MSD. Results: Principle component analysis (PCA) of the proteomic profiling data for the 3 donors indicated that exposure of OCP to TNF induced a distinct profile from that of M-CSF and RANKL. The addition of ADA:TNF complexes restored the profile to that of RANKL, whereas those exposed to ETN:TNF complexes exhibited an intermediate profile matching differences observed in TRAP 5b activity levels. DPA identified 3 pathways most associated with osteoclastogenesis: receptor-mediated endocytosis (e.g. CD163, IGFBP2), oxidation-reduction process (e.g. FABP4), and cell adhesion (e.g. ICAM-1, TGFBI) that were significantly impacted by TNF with ADA:TNF being more effective than ETN:TNF in restoring most pathway-associated proteins to RANKL levels. Based on ELISA, two pro-osteoclastogenic factors IGFBP2 and ICAM-1 were increased 2-fold in OCP culture supernatants in response to TNF with only ADA:TNF complexes reducing these levels of both to RANKL. TNF-induced reduction of intracellular levels of CD163, an M2 macrophage polarization marker, and TGFBI were not only restored to RANKL levels by ADA:TNF but brought to a level closer to M-CSF alone, unlike ETN:TNF. One anti-osteoclastogenic factor FABP4 was found to be increased intracellularly above that of RANKL only following exposure to ADA:TNF complexes. Conclusion: Shotgun proteomic profiling of human OCP differentiated in vitro with TNF revealed at least 3 novel pathways by which TNF exerts its pro-osteoclastogenic effects. Only with the addition of ADA:TNF, not with ETN:TNF, were most pathway-associated proteins significantly restored to RANKL levels including pro-osteoclastogenic factors ICAM-1 and IGFBP2. Moreover, ADA:TNF complexes enhanced the intracellular levels of CD163 and the anti-osteoclastogenic factor FABP4, suggesting these complexes are exerting an effect on OCP beyond simple TNF neutralization. Additional in vitro and in vivo studies need to be performed to verify our findings. Acknowledgments: The authors thank Drs. Jochen Salfeld, Yonghao Cao and Dhaval Nanavati for their critical review of our data, and Yucheng Zhang for her technical support. Disclosure of Interests: Bohdan Harvey Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc., Liang Jin Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc., John Maull Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc., Chenqi Hu Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc., Yu Tian Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc., Zehra Kaymakcalan Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc.