THU0002 THERAPEUTIC ANTI-TNF BIOLOGIC AGENTS EXHIBIT FUNCTIONAL DIFFERENCES IN BLOCKING TNF-INDUCED EFFECTS ON HUMAN MONOCYTES IN VITRO

Abstract

Background: Therapeutic anti-TNF biologic agents can be distinct in their structure, such as etanercept, a human TNFRII- Fc fusion protein, as compared to adalimumab, a human IgG molecule, and/or in their binding to TNF as shown by crystal structures of adalimumab as compared to infliximab in the presence of TNF. Whether these differences can affect the functional properties of these biologics in direct comparison to each other has not been thoroughly investigated. Objectives: To determine the equivalency of all anti-TNF biologic agents currently approved for the treatment of RA in preventing a variety of TNF-induced effects on human monocytes in vitro. Methods: Human monocytic U937 NF-kB luciferase reporter cell line was incubated with 100 ng/mL TNF +/- increasing conc. (0.15-338 nM) of adalimumab (ADA), etanercept (ETN), infliximab (IFX), golimumab (GOL) or certolizumab pegol (CZP) as pre-formed complexes generated for 1 h. at 37°C. Surface TNF-RI and –RII levels were monitored by flow cytometry following 1 h. incubation. Luciferase activity was measured in cell lysate after 4 h. to assess NF-kB activation. After 24 h., U937 cells were analyzed by flow cytometry for surface levels of ICAM-1, an NF-kB-induced adhesion molecule shown to contribute to monocyte migration and arthritis. Apoptosis was assessed by time-lapsed microscopy and flow cytometry using caspase 3/7 fluorescent substrate. Alpha-2,6 sialylation (Sia), a glycosylation modification shown to regulate TNF-RI internalization and apoptosis induction, was evaluated by flow cytometry using FITC-labeled Sambucus nigra lectin (SNA). Human PBMC were incubated with TNF +/- pre-formed complex with anti-TNF biologics for 24 h. and then stained with CD14 and ICAM-1 Abs for flow cytometry. Results: Surface levels of TNF-RI and –RII on U937 cells were both reduced by 2.4-fold in presence of TNF. TNF-RI was maintained at baseline levels by 16.7 nM ADA or CZP as pre-formed complexes with TNF; however, those complexes with ETN, IFX or GOL could only preserve a fraction of this receptor on the surface (43%, 52% and 62%, respectively). All anti-TNF biologics were equally effective in preventing loss of surface TNF-RII. TNF stimulation of U937 NF-kB reporter cells led to a 122-fold increase in luciferase activity which was reduced to baseline by only ADA or CZP with largest conc. range tested. Partial reduction by 3, 7 and 11-fold was observed with ETN, IFX or GOL, respectively. TNF-enhanced ICAM-1 surface expression (3-fold increase) on U937 cells was reduced to baseline by 16.7 nM ADA or CZP, whereas ETN, IFX or GOL were only partially effective (48%, 61% and 59% reduction, respectively). Exposure of CD14+ primary monocytes to ADA:TNF or CZP:TNF complexes not only prevented TNF induction of ICAM-1 but significantly reduced its level below that of baseline, whereas those with GOL or IFX brought ICAM-1 levels to baseline and those with ETN were only 46% effective. Both ADA:TNF and CZP:TNF complexes also completely inhibited TNF-induced apoptosis in a dose dependent manner unlike ETN, IFX and GOL, which were less effective (42%, 32% & 42% reduction, respectively). According to SNA staining, alpha-2,6 Sia surface levels dropped in presence of TNF specifically on the subset of cells undergoing apoptosis, and this subset was reduced proportionately to the inhibitory properties of anti-TNF biologics on apoptosis. Conclusion: For each of the conditions tested in vitro, many resembling features associated with RA pathogenesis, the pre-formed complexes of ADA:TNF and CZP:TNF were significantly more effective in preventing the TNF-induced effects (decrease of surface TNF-RI expression and alpha-2,6 Sia; increase in NF-kB activation, ICAM-1 surface expression, and apoptosis) on human monocytes than those complexes of TNF with ETN, IFX or GOL. Additional in vitro and in vivo studies need to be done to further elucidate the mechanism responsible for these differences. Disclosure of Interests: Bohdan Harvey Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc., Zehra Kaymakcalan Shareholder of: AbbVie, Inc., Employee of: AbbVie, Inc.