ADALIMUMAB, ETANERCEPT, AND INFLIXIMAB BIND TO SOLUBLE AND TRANSMEMBRANE TNF WITH SIMILAR AFFINITIES

Background: Adalimumab (Ada), etanercept (Eta), and infliximab (Inf), and have distinct clinical efficacy profiles. It is unclear whether differences in their TNF-binding properties are relevant, because commonly held beliefs, such as "weaker" binding by Eta, are poorly documented, and because direct comparisons of the 3 with current technologies have not been published.Objectives: To compare the binding properties of Eta, Inf, and Ada with: 1) soluble TNF (sTNF) and 2) transmembrane TNF (mTNF), and 3) to determine the range of concentration ratios at which Ada and TNF form complexes that exhibit enhanced binding to Fc receptors (FcR) on cells.Methods: A BIAcore 3000 instrument measured the binding of sTNF to anti-TNFs captured on a biosensor chip by antibodies to the constant region of IgG. On-rate was measured over 10 minutes, and off-rate over 30 minutes. Sensorgrams were analyzed using the global fit program. Binding to mTNF was assessed with I-labeled anti-TNFs and cells transfected with a TNF deletion mutant that expresses a stable form of mTNF not cleaved by TACE. Binding isotherms were generated to determine binding affinities. Binding of TNF-Ada complexes to FcR was studied by incubating THP-1 cells with mixtures of TNF and I-Ada. Each mixture used Ada at a molar equivalence to TNF or in molar excess (up to 33-fold).Results: Measurements showed similar affinities of TNF antagonists for sTNF, but differences in their on-rate constants. (Table. Results for Apparent on-rate are x 10 Ms, for Apparent off-rate are x 10s, and for Intrinsic affinity are x 10 M).Measurements of I-Eta, I-Inf, and I-Ada binding to mTNF on transfected cells showed that the TNF antagonists had similar affinities for mTNF (4.45, 4.68, and 4.83 x 10 M, respectively).Binding of I-Ada to FcR-bearing THP-1 cells was enhanced by complexing with TNF when the Ada-TNF complexes were formed at close to molar equivalence, but almost no effect was seen at Ada:TNF ratios ≥10:1. Binding of TNF antagonists to sTNF Eta Inf Ada Apparent on-rate 5.65 2.40 1.69 Apparent off-rate 6.66 6.56 4.71 Intrinsic affinity 1.18 2.73 3.04 Conclusion: Ada, Inf and Eta have overall similar affinities for sTNF, but Eta has a faster on-rate. The 3 anti-TNF agents bind with similar affinities to cells expressing mTNF, and these affinities are lower than those for sTNF. Binding to and cross-linking of FcRs by Ada-TNF complexes is unlikely to occur in vivo, where Ada concentrations greatly exceed those of sTNF.Citation: Ann Rheum Dis, volume 65, supplement II, year 2006, page 458Session: Cytokines and inflammatory mediators