Background 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. 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.
Objectives To determine the mechanism that is responsible for the difference in effectiveness between adalimumab and etanercept and to identify the TNF-receptor that is predominantly involved in TNF-enhanced osteoclast function.
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 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. Artificially large complexes were generated using polyclonal anti-human IgG Fc-specific F(ab')2 antibody (20 μg/mL) to bind the preformed TNF:biologic complexes. To determine contribution of individual TNF receptors to TNF-mediated osteoclast function, 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 multi-nucleated 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 function; 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, the inhibitory effect of ADA improved two-fold, whereas etanercept suppressed osteoclast function to levels comparable to non-cross-linked ADA, suggesting that complex formation by ADA with TNF contributes to its inhibitory properties. As to the receptor that mediates the effect of TNF on OCP function, blocking TNF-RI curtailed TNF-dependent OCP functions demonstrating that the effective anti-TNF biologics are restricting TNF access to TNF-RI.
Conclusions The mechanistic feature that distinguishes ADA from ETN in its ability to more effectively inhibit TNF-enhanced osteoclast function is its ability as an antibody to form complexes with TNF unlike that of the TNF receptor:Fc fusion protein ETN. 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.
Acknowledgements The authors would like to acknowledge Wendy Waegell, Dr. Jochen Salfeld and Dr. Anna Yarilina for their input and critical review of the science.
Disclosure of Interest B. Harvey Shareholder of: AbbVie Inc., Employee of: AbbVie Inc., Z. Kaymakcalan Shareholder of: AbbVie Inc., Employee of: AbbVie Inc.