Variations of post-translational modifications are important for stability and behavior of

Variations of post-translational modifications are important for stability and behavior of therapeutic antibodies. benzoylecgonine and cocaethylene by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1 5 and 20?nM for cocaethylene cocaine and benzoylecgonine respectively. Tryptophan fluorescence quenching (emission at 330?nm) was measured after either excitation of tyrosine and tryptophan (280?nm) or selective excitation of tryptophan alone (295?nm). More accurate binding constants are Isorhamnetin-3-O-neohespeidoside obtained using tryptophan selective excitation at 295?nm likely due to interfering absorption of cocaine and metabolites at 280?nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody’s variable region. ramifications. Common post-translational modifications which introduce heterogeneity in monoclonal antibodies have recently been reviewed 1 and include charge variants (e.g. variations in the removal of heavy chain C-terminal lysine N-terminal pyroglutamate formation asparagine deamidation) oxidation variants (e.g. methionine and tryptophan) disulfide bond scrambled/mispaired variants glycosylation variants (e.g. immature glycosylation and the presence or absence of fucose and sialic acid on the glycan chains) and differential glycation variants. Although antibody glycosylation commonly increases solubility and stability while decreasing aggregation 3 Fc glycosylation is also functionally relevant due to modulation of antibody binding to Fcγ receptors with increased binding leading to increased antibody-dependent cell-mediated cytotoxicity (ADCC).4 Immature glycosylation (high mannose glycan chains) can result in shorter plasma half-lives for the antibodies.5 Asparagine deamidation and aspartic acid isomerization can influence storage stability and shelf life of monoclonal antibodies.6 Tryptophan is commonly found in the antibody complementarity determining regions (CDRs) and its oxidation can result in an attenuation or loss of antigen affinity.7 In addition aggregation of monoclonal antibodies can occur during production and Isorhamnetin-3-O-neohespeidoside purification and is therapeutically relevant affecting their efficacy and safety since aggregates tend to increase plasma clearance Isorhamnetin-3-O-neohespeidoside systemic side effects and the likelihood of undesired host immunogenic responses. A chimeric murine/human anti-cocaine monoclonal antibody (2E2) has been generated 8 using technology described Isorhamnetin-3-O-neohespeidoside by Lonberg.9 The Isorhamnetin-3-O-neohespeidoside murine/human chimeric anti-cocaine 2E2 monoclonal antibody was previously shown to inhibit the distribution of cocaine to the brain in mice.10 A re-engineered version of the 2E2 antibody incorporating a humanized light chain (h2E2) was recently shown to have binding properties and behavior in rats Isorhamnetin-3-O-neohespeidoside consistent with its likely utility as a therapy for human cocaine abuse.11 The affinity for cocaine of the recombinant h2E2 antibody was shown to be 3.9?nM by radioligand binding and the desirable relative selectivity of the re-engineered antibody for cocaine over the major inactive metabolites of cocaine Mouse monoclonal to GSK3B was not adversely affected by the humanization process.11 Thus the h2E2 monoclonal antibody is a lead candidate for advancement to clinical trials as a therapeutic agent for cocaine abuse. For some non-clinical studies and clinical uses of therapeutic monoclonal antibodies it may be desirable to generate Fab fragments which bind antigens but lack the constant region and therefore antibody effector functions. The Fab fragment is smaller and has better tissue and tumor penetration than intact monoclonal antibodies. In the case of an anti-cocaine antibody it is not unreasonable to assume that the Fab fragment of the h2E2 monoclonal antibody may be preferable to the intact mAb for treatment of acute cocaine overdose since the Fab fragment has no Fcγ binding region resulting in much reduced chances for ADCC and as shown in this study the Fab fragment has identical affinity for cocaine as the intact h2E2 antibody. The h2E2 Fab fragment is also very likely to have a substantially shorter plasma half-life.