Development and validation of immunoassays to quantify the half-antibody exchange of an IgG4 antibody, natalizumab (Tysabri®) with endogenous IgG4.

Natalizumab is a humanized IgG4 monoclonal antibody which binds human α4 integrin and is approved for treatment of multiple sclerosis and Crohn's disease. Assessment of the in vivo disposition of natalizumab presents a unique assay development challenge due to the ability of human IgG4 antibodies to undergo half-antibody exchange in vivo. Such exchange generates IgG4 molecules of mixed specificity comprising a natalizumab heavy-light chain pair coupled to an IgG4 heavy-light chain pair of unknown specificity.

Resolution of disulfide heterogeneity in Nogo receptor I fusion proteins by molecular engineering.

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively).

Suppression of sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample preparation artifacts for analysis of IgG4 half-antibody.

Human IgG4 subtype antibodies have often been reported to have a significant portion (5-50%) of a heavy chain-light chain dimer ("half-antibody") on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the heavy chain is not covalently linked through the hinge disulfides to another heavy chain. We demonstrate here that there can be artifactual sources of half-antibody. One occurred during SDS-PAGE sample preparation where rapid disulfide scrambling was initiated by preexisting free sulfhydryls in the monoclonal antibody (mAb) and by free sulfhydryl produced by destruction of disulfide bonds during heating.

The contribution of Fc effector mechanisms in the efficacy of anti-CD154 immunotherapy depends on the nature of the immune challenge.

Blockade of the CD154-CD40 co-stimulatory pathway with anti-CD154 mAbs has shown impressive efficacy in models of autoimmunity and allotransplantation. Clinical benefit was also demonstrated in systemic lupus erythematosus (SLE) and idiopathic thrombocytopenia patients with the humanized anti-CD154 mAb, 5C8 (hu5C8). However, thromboembolic complications that occurred during the course of the hu5C8 clinical trials have proven to be a major setback to the field and safe alternative therapeutics targeting the CD154-CD40 pathway are of great interest.

FcR interactions do not play a major role in inhibition of experimental autoimmune encephalomyelitis by anti-CD154 monoclonal antibodies.

It has been demonstrated that anti-CD154 mAb treatment effectively inhibits the development of experimental autoimmune encephalomyelitis (EAE). However, although it appears to prevent the induction of Th1 cells and reactivation of encephalitogenic T cells within the CNS, little information is available regarding the involvement of alternative mechanisms, nor has the contribution of Fc effector mechanisms in this context been addressed. By contrast, efficacy of anti-CD154 mAbs in models of allotransplantation has been reported to involve long-term unresponsiveness, potentially via activation of T regulatory cells, and recently was reported to depend on Fc-dependent functions, such as activated T cell depletion through FcgammaR or complement.

A monoclonal antibody to alpha1beta1 blocks antigen-induced airway responses in sheep.

The integrin alpha1beta1 (very late antigen-1; CD49a/CD29) is a major adhesion receptor for collagen I, IV, and VI, and its induced expression on activated monocytes and lymphocytes plays a central role in their retention and activation at inflammatory sites in autoimmune pathologies. However, the role of alpha1beta1 in allergic settings has not been explored. In this study, we show that a single 45-mg dose of aerosolized monoclonal antibody AQC2 to the alpha1 chain of human and sheep very late antigen-1, given 30 minutes before challenge, blocks both the allergen-induced late response and the associated airway hyperresponsiveness, functional indicators of allergen-induced inflammation, in sheep.

Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications.

We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of Sonic hedgehog and its receptor, Patched, in adult nervous tissue suggests possible applications for the protein in the treatment of neurodegenerative disease and injury.

Crystal structure of the alpha1beta1 integrin I domain in complex with an antibody Fab fragment.

The alpha1beta1 (VLA-1) integrin is a cell-surface receptor for collagen and laminin and has been implicated in biological pathways involved in several pathological processes. These processes may be inhibited by the monoclonal antibody AQC2, which binds with high affinity to human alpha1beta1 integrin. To understand the structural basis of the inhibition we determined the crystal structure of the complex of a chimeric rat/human I domain of the alpha1beta1 integrin and the Fab fragment of humanized AQC2 antibody.

Long-acting forms of Sonic hedgehog with improved pharmacokinetic and pharmacodynamic properties are efficacious in a nerve injury model.

The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment.