The kinetic and physical properties of the allosteric enzyme, aspartate transcarbamoylase from Escherichia coli, have been analyzed according to the two-state model (Monod, J., Wyman, J., and Changeux, J.-P. (1965), J . Mol. Biol. 12, 88). An internally consistent set of calculated parameters accounted quantitatively for the results from diverse experiments including: (a) enzyme kinetics as a function of the concentration of aspartate in the presence of saturating carbamoyl phosphate; (b) gross conformational changes of the enzyme, revealed by the decrease in the sedimentation coefficient and the increase in the reactivity of the sulfhydryl groups of the regulatory subunits, as a function of the extent of saturation of the active sites by the bisubstrate analogue, N-(phosphonacety1)-L-aspartate; (c) stimulation of enzymic activity at low concentrations of the substrate analogue, succinate; and (d) effects of the inhibitor, CTP, and the activator, ATP, on the kinetic and physical properties of the enzyme. The data were interpreted in terms of an equilibrium between a constrained or low-affinity (T) state and a relaxed or highaffinity (R) form of the enzyme and the perturbation of the equilibrium by the addition of various ligands. In the absence

SUMMARYThe type 2 helper T cell (T H 2) cytokine interleukin (IL)-4 is thought to play a central role in the early stages of asthma. In an effort to develop an antibody treatment for asthma that neutralizes the effects of IL-4, a murine monoclonal antibody, 3B9, was generated with specificity for human IL-4. In vitro studies demonstrated that 3B9 inhibited IL-4-dependent events including IL-5 synthesis, T H 2 cell activation and up-regulation of immunoglobulin E expression. 3B9 was then humanized (pascolizumab, SB 240683) to reduce immunogenicity in humans. SB 240683 demonstrated species specificity for both monkey and human IL-4 with no reactivity to mouse, rat, cow, goat or horse IL-4. Pascolizumab inhibited the response of human and monkey T cells to monkey IL-4 and effectively neutralized IL-4 bioactivity when tested against several IL-4-responsive human cell lines. Affinity studies demonstrated rapid IL-4 binding by pascolizumab with a slow dissociation rate. In vivo pharmacokinetic and chronic safety testing in cynomolgus monkeys demonstrated that pascolizumab was well tolerated, and no adverse clinical responses occurred after up to 9 months of treatment. Three monkeys developed an anti-idiotypic response that resulted in rapid pascolizumab clearance. However, in the chronic dosing study the antibody response was transient and not associated with clinical events. In conclusion, pascolizumab is a humanized anti-IL-4 monoclonal antibody that can inhibit upstream and downstream events associated with asthma, including T H 2 cell activation and immunoglobulin E production. Clinical trials are under way to test the clinical efficacy of pascolizumab for asthma.

Abstract-A murine antihuman factor IX monoclonal antibody (BC2) has been generated and evaluated for its capacity to prolong the activated partial thromboplastin time (aPTT) in vitro and ex vivo and to prevent arterial thrombosis in a rat model in vivo. BC2 extended aPTT to a maximum of 60 to 80 seconds at 100 to 1000 nmol/L in vitro (rat and human plasma, respectively) and ex vivo (rat) after dosing of rats up to 6 mg/kg in vivo. BC2, administered as bolus (1 to 6 mg/kg) followed by infusion (0.3 to 2 mg ⅐ kg Ϫ1 ⅐ h Ϫ1 ), dose-dependently prevented thrombosis of an injured rat carotid artery (FeCl 3 -patch model), increased time to artery occlusion, and reduced incidence of vessel occlusion. BC2 efficacy in preventing arterial thrombosis exceeded that of heparin (bolus 15 to 120 U/kg followed by infusion 0.5 to 4.0 U ⅐ kg Ϫ1 ⅐ min Ϫ1 ), whereas the latter rendered the blood incoagulable (aPTTϾ1000 seconds). BC2 demonstrated complete antithrombotic efficacy also as a single bolus given either as prevessel or postvessel injury as evidenced by reduction of thrombus mass (from 4.18Ϯ0.49 to 1.80Ϯ0.3 mg, PϽ0.001), increasing vessel patency time (from 14.9Ϯ0.9 minutes to 58.3Ϯ1.7 minutes, PϽ0.001) and decreasing incidence of vessel occlusion from 100% to 0% in vehicle-versus BC2-treated rats, respectively. BC2 (3 mg/kg, IV) administered in a single bolus resulted in 50% reduction in thrombus mass (PϽ0.01), extended vessel patency time (PϽ0.001), extended aPTT only 4-fold, and had no effect on blood loss via a tail surgical wound; heparin, at doses that reduced thrombus mass to a similar extent, extended aPTT beyond 1000 seconds (over 500-fold) and increased blood loss from 1.8Ϯ0.7 to 3.3Ϯ0.6 mL (PϽ0.001). These data suggest that BC2 may provide enhanced therapeutic efficacy in humans at lesser interference with blood hemostasis than heparin.

Upon treatment with denaturing agents, vitronectin has been observed to exhibit conformational alterations which are similar to the structural changes detected when vitronectin binds the thrombin-antithrombin complex or associates with the terminal attack complex of complement. Denaturation and renaturation of vitronectin isolated from human plasma were characterized by changes in intrinsic fluorescence. Unfolding by chemical denaturants was irreversible and accompanied by self-association of the protein to form vitronectin multimers. Self-association was evaluated by equilibrium analytical ultracentrifugation which demonstrated that multimers form only during the refolding process after removal of denaturant, that multimeric vitronectin dissociates to constituent subunits readily upon treatment with chemical denaturant, and that intermolecular disulfide cross-linking occurs primarily at the dimer level among a subset of constituent vitronectin subunits within the multimer. The monomeric form of vitronectin isolated from human plasma partially unfolds at intermediate concentrations of denaturant to an altered conformation with a high propensity to associate into multimers. Folding of vitronectin in vivo appears to be regulated by partitioning of folding intermediates toward either of two conformations, one that exists as a stable monomer and another that associates into a multimeric form.