Targeting gp130 to prevent inflammation and promote insulin action.

Obesity and type 2 diabetes are now the most prevalent metabolic diseases in the Western world and the development of new strategies to treat these metabolic diseases is most warranted. Obesity results in a state of chronic low-grade inflammation in metabolically active tissues such as the liver, adipose tissue, brain and skeletal muscle. Work in our laboratory has focussed on the role of the cytokine interleukin-6 (IL)-6 and other IL-6-like cytokines that signal through the gp130 receptor complex. We have focussed on the role of blocking IL-6 trans-signalling to prevent inflammation on the one hand, and activating membrane-bound signalling to promote insulin sensitivity on the other hand. Since the cloning of the IL-6 gene nearly 30 years ago, a pattern has emerged associating IL-6 with a number of diseases associated with inflammation including rheumatoid arthritis (RA), Crohn's disease and several cancers. Accordingly, tocilizumab, an IL-6 receptor-inhibiting monoclonal antibody, is now useful for the treatment of RA. However, this may not be the most optimal strategy to block inflammation associated with IL-6 and may result in unwanted side effects that, paradoxically, could actually promote metabolic disease.

Potential treatment of cardiac hypertrophy and heart failure by inhibiting the sarcolemmal binding of phospholipase Cbeta1b.

Heart failure, the common end-point of many cardiac diseases, is a major contributor to mortality and morbidity and contributes considerably to health care costs. Current treatment regimens include beta-adrenergic antagonists, angiotensin converting enzyme inhibitors, and inotropic agents are used by some patients. Studies in experimental animals have demonstrated that inhibition of signaling pathways downstream of the heterotrimeric G protein Gq reduce ventricular hypertrophy and protects from the development of heart failure. However, targets identified, to date, have been limited by a lack of tissue specificity. In cardiomyocytes, Gq activates only one splice variant of one subtype of phospholipase Cbeta, specifically phospholipase Cbeta1b (PLCbeta1b) and PLCbeta1b is responsible for Gq mediated hypertrophic and apoptotic responses. PLCbeta1b targets to the sarcolemma via its unique C-terminal sequence and its activation can be inhibited by expressing the C-terminal sequence to compete for sarcolemmal binding. Inhibition of PLCbeta1b by the C-terminal peptide reduces hypertrophic responses in cardiomyocytes. We present the evidence that inhibition of the sarcolemmal association of PLCbeta1b provides a cardiac-specific target for the development of drugs to reduce pathological cardiac hypertrophy and thereby to reduce the burden of heart failure.

Orientation of antigen binding sites in dimeric and trimeric single chain Fv antibody fragments.

Electron microscopy of dimeric and trimeric single chain antibody Fv fragments (scFvs) complexed with anti-idiotype Fab fragments was used to reveal the orientation of antigen binding sites. This is the first structural analysis that discloses the multivalent binding orientation of scFv trimers (triabodies). Three different scFv molecules were used for the imaging analysis; NC10 scFv-5 and scFv-0, with five- and zero-residue linkers respectively between the VH and VL domains, were complexed with 3-2G12 anti-idiotype Fab fragments and 11-1G10 scFv-0 was complexed with NC41 anti-idiotype Fab fragments. The scFv-5 molecules formed bivalent dimers (diabodies) and the zero-linker scFv-0 molecules formed trivalent trimers (triabodies). The images of the NC10 diabody-Fab complex appear as boomerangs, not as a linear molecule, with a variable angle between the two Fab arms and the triabody-Fab complexes appear as tripods.

Single-chain Fv of anti-idiotype 11-1G10 antibody interacts with antibody NC41 single-chain Fv with a higher affinity than the affinity for the interaction of the parent Fab fragments.

A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 microg/ml) was stable for 7 days at 21 degrees C and 30 days at 4 degrees C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomerdimer equilibrium mixture after 30 days at 4 degrees C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of Mr approximately 156,000. Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-IG10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.

Nonspecific amine immobilization of ligand can Be a potential source of error in BIAcore binding experiments and may reduce binding affinities.

The interaction of monovalent forms of NC41, an anti-viral neuraminidase antibody, and the antiidiotype antibody 11-1G10 has been used as a model system for BIAcore analysis to demonstrate the potential problems resulting from the nonspecific amine coupling procedure. To avoid complications due to antibody bivalency, monovalent Fab fragments and monomeric recombinant scFvs were used. When immobilized by amine coupling, the 11-1G10 anti-idiotype fragments were found to have an artificially reduced affinity for NC41 compared to the results obtained using site-directed immobilization via C-terminal thiol residue and from solution equilibrium measurements. The NC41 antibody fragments, on the other hand, were able to retain their 11-1G10 binding affinity when immobilized nonspecifically through free amine groups. These data, in combination with the known sequences of the two antibodies, suggested that nonspecific immobilization through one or more lysine residues close to or within the CDR2 region of the 11-1G10 VH domain was responsible for the reduced strength of the interaction with NC41. These results emphasize the need to use site-specific immobilization strategies when accurate kinetic measurements are required.

Triabodies: single chain Fv fragments without a linker form trivalent trimers.

A single chain Fv fragment (scFv) of the murine monoclonal antibody 11-1G10 was constructed by directly joining the C-terminal residue of the V(H) domain to the N-terminal residue of V(L). 11-1G10 is an anti-idiotype and competes with the antigen, influenza virus neuraminidase (NA), for binding to the NC41 antibody. The scFv formed stable trimers with three active antigen combining sites for NC41 Fab fragments. We propose that trimeric scFvs may be the preferred conformation for directly linked V(H)-V(L) molecules, which contrasts the formation of scFv dimers (diabodies) when the V(H) and V(L) domains are joined by short flexible linkers of between 5-10 residues. BIAcore biosensor binding experiments showed that the trimeric scFv showed an expected increase in binding affinity, due to avidity, compared to the monomeric 15-residue linked scFv. The increase in avidity of scFv trimers offers advantages for imaging and immunotherapy.