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Utilising assay systems relevant to IL-6 mechanisms in Rheumatoid Arthritis to demonstrate activity of a novel human anti-IL-6 antibody, CAT6001 Purpose: IL-6 was first identified as a B-cell differentiating factor. Recent clinical data with tocilizumab have shown IL-6 to have multiple effects in RA (Smolen et al, 2008; Genovese et al, 2008). Using a panel of in vitro and in vivo assays, each designed to reflect an aspect of RA pathology with IL-6 involvement, we describe the activity of a novel anti human IL-6 neutralizing antibody (CAT6001), engineered for very high affinity to IL-6.
Method: The IL-6-dependent proliferation of TF1 and B9 cells was measured using 3H-thymidine incorporation. IL-6-dependent IgM release from a B cell line (SKW6.4), was measured by ELISA. Primary fibroblast-like synoviocytes from RA patients were stimulated with IL-1β and IL-6Rα for 48hrs to produce IL-6 and VEGF, which were detected by ELISA. In a model of murine acute phase protein production, mice were treated with 12μg/kg huIL-6 i.p. daily for 7 days; CAT6001 (8 to 467 μg/kg s.c.) or isotype control was given in a single administration. Animals were sacrificed on day 7 and plasma haptaglobin levels measured.
Results: CAT6001 (<1 pM affinity for IL-6) potently blocked IL-6 driven cell proliferation of the haematopoietic cell line TF-1 (IC50 1.61pM). IL-6-induced production of IgM from a B cell line (SKW6.4) and IL-6 induced proliferation of a B cell hybridoma cell line (B9), were potently inhibited by CAT6001 (IC50 2.63Pm and Kb 0.3pM respectively). Primary fibroblast-like synoviocytes from RA patients dose dependently expressed IL-6 upon stimulation with IL-1β. The addition of exogenous soluble IL-6Rα resulted in the release of VEGF, a key angiogenic factor. CAT6001 blocked this IL-1β-stimulated VEGF release (IC50 340pM). Thus IL-6 plays a key role in mediating the effects of other pro-inflammatory cytokines on angiogenic factor release from activated RA synovial fibroblasts. In a murine in vivo model, recombinant human IL-6 induced an increase in serum levels of the acute phase protein haptoglobin. The production of this acute phase protein was inhibited in a dose dependent manner by administration of CAT6001.
Conclusions: These assay systems were used to exemplify the classical role of IL-6 in RA, such as IgM production from B cells, proliferative effects on multiple cell lineages, activation of synovial fibroblasts and production of angiogenic factors exvivo, and systemic release of acute phase proteins in vivo. In all these models, CAT6001 potently inhibited these IL-6 driven mechanisms which supports the continued development of this molecule as a possible therapeutic agent for patients with RA.
Smolen JS et al. (2008) Lancet 371, 987-997 |
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