Background/Purpose: TNF-alpha (TNFa) has been shown to contribute to osteoclastogenesis independently and in conjunction with M-CSF or RANKL, two key cytokines involved in osteoclast development.  Both TNFa and RANKL have been concomitantly detected in the synovial fluid of RA patients.  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 as compared to etanercept.  Based on these findings, we sought to determine the mechanism that was responsible for this difference and to identify the TNF-receptor that is predominantly involved in TNF-enhanced osteoclastogenesis. 

Methods: Primary human osteoclast precursors (OCP) were exposed to various combinations of M-CSF, RANKL and TNFa (100 ng/mL) +/- increasing equimolar concentrations of adalimumab (ADA) [whole, F(ab’)2 or Fab], etanercept (ETN) [whole or Fc-deficient (pepsin digested) molecule] or certolizumab pegol (CZP) for up to 6 days.  Prior to adding to the cells, the biologics were pre-incubated with the TNFa for 30 min.  To artificially generate large immune complexes (IC), polyclonal anti-human IgG Fc-specific F(ab’)2 antibody (20 µg/mL) was added to the preformed TNF:biologic complexes.  To determine contribution of individual TNF receptors to TNF-mediated osteoclast development, OCP were treated with 50 µg/mL anti-human TNF-RI or -RII blocking antibodies prior to the addition of TNF.   Osteoclast differentiation was determined by the presence of large multinucleated cells positive for tartrate-resistant acid phosphatase (TRAP) and by TRAP5b activity.  Resorptive activity was assessed by measuring the release of cross-linked C-telopeptide of type I collagen (CTX-I) from human bone.  

Results: Each of the biologics as whole molecule was able to reduce TNF-enhanced osteoclast development; however, both CZP and ADA were more effective at lower concentrations as compared to ETN (9.6, 14.4 and >130 nM, respectively).  The F(ab’)2 of ADA inhibited osteoclast activity to the same level as the whole IgG demonstrating that the Fc domain was not contributing to the inhibitory effects of the biologic. Following the cross-linking of the biologics with anti-human IgG to generate artificially large IC, the inhibitory effect of ADA improved two-fold, whereas etanercept suppressed osteoclast development to levels comparable to non-cross-linked ADA, suggesting that IC formation by ADA contributes to its ability to block TNF-enhanced osteoclastogenesis.  As to which receptor mediates the effect of TNF on human OCP, blocking TNF-RI curtailed TNF-dependent development demonstrating that the effective anti-TNF biologics are restricting TNF access to TNF-RI. 

Conclusion: The mechanistic feature that distinguishes ADA from ETN in its ability to more effectively inhibit TNF-enhanced osteoclastogenesis is its TNF binding mode.  Surprisingly, the Fc domain of ADA does not contribute to its suppressive function.  Overall, our results may provide a mechanistic explanation for the sustained potency of adalimumab in preventing bone erosion due to chronic TNF exposure.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-complex-formation-by-adalimumab-contributes-significantly-to-its-inhibitory-effect-on-tnf-enhanced-human-osteoclast-development-even-in-the-absence-of-fc-receptor-binding/