Pharmacogenomics and translational simulations to bridge indications for an anti-interferon-α receptor antibody.

A type I interferon (IFN) gene signature shared by systemic lupus erythematous (SLE) and systemic sclerosis (SSc) was used to evaluate an anti-type I IFN-α receptor (IFN-αR) monoclonal antibody, MEDI-546, in a phase I trial in SSc. MEDI-546 suppressed IFN signature in blood and skin of SSc patients in a dose-dependent manner. To bridge clinical indications to SLE, we developed a model incorporating (i) pharmacokinetics (PK) and pharmacodynamics (PD) in SSc patients, (ii) internalization kinetics of MEDI-546/IFN-αR complex, and (iii) the different IFN signatures between SSc and SLE. Simulations predicted that i.v. administration of MEDI-546 at 300- or 1,000-mg monthly doses could suppress IFN signature in blood to levels of healthy subjects in 53 and 68% of SLE patients, respectively. An innovative approach utilizing a novel biomarker characterized the PD of MEDI-546 by modeling and simulation and allowed rapid progression of MEDI-546 from a phase I study in SSc to a randomized, multiple-dose phase II trial.

Protein engineering and preclinical development of a GM-CSF receptor antibody for the treatment of rheumatoid arthritis.

BACKGROUND AND PURPOSE: For antibody therapies against receptor targets, in vivo outcomes can be difficult to predict because of target-mediated clearance or antigen 'sink' effects. The purpose of this work was to engineer an antibody to the GM-CSF receptor α (GM-CSFRα) with pharmacological properties optimized for chronic, s.c. treatment of rheumatoid arthritis (RA) patients. EXPERIMENTAL APPROACH: We used an in silico model of receptor occupancy to guide the target affinity and a combinatorial phage display approach for affinity maturation. Mechanism of action and internalization assays were performed on the optimized antibody in vitro before refining the modelling predictions of the eventual dosing in man. Finally, in vivo pharmacology studies in cynomolgus monkeys were carried out to inform the predictions and support future clinical development. KEY RESULTS: Antibody potency was improved 8600-fold, and the target affinity was reached. The refined model predicted pharmacodynamic effects at doses as low as 1 mg kg(-1) and a study in cynomolgus monkeys confirmed in vivo efficacy at 1 mg kg(-1) dosing. CONCLUSIONS AND IMPLICATIONS: This rational approach to antibody drug discovery enabled the isolation of a potent molecule compatible with chronic, s.c. self-administration by RA patients. We believe this general approach enables the development of optimal biopharmaceuticals.

The insulin receptor substrate (IRS)-1 pleckstrin homology domain functions in downstream signaling.

The pleckstrin homology (PH) domain of the insulin receptor substrate-1 (IRS-1) plays a role in directing this molecule to the insulin receptor, thereby regulating its tyrosine phosphorylation. In this work, the role of the PH domain in subsequent signaling was studied by constructing constitutively active forms of IRS-1 in which the inter-SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase was fused to portions of the IRS-1 molecule. Chimeric molecules containing the PH domain were found to activate the downstream response of stimulating the Ser/Thr kinase Akt. A chimera containing point mutations in the PH domain that abolished the ability of this domain to bind phosphatidylinositol 4,5-bisphosphate prevented these molecules from activating Akt. These mutations also decreased by about 70% the amount of the constructs present in a particulate fraction of the cells. These results indicate that the PH domain of IRS-1, in addition to directing this protein to the receptor for tyrosine phosphorylation, functions in the ability of this molecule to stimulate subsequent responses. Thus, compromising the function of the PH domain, e.g. in insulin-resistant states, could decrease both the ability of IRS-1 to be tyrosine phosphorylated by the insulin receptor and to link to subsequent downstream targets.

Peptide rescue of an N-terminal truncation of the Stoffel fragment of taq DNA polymerase.

Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5' to 3' exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N-terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the Stof delta 12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the Stof delta 47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33-amino acid synthetic peptide, based on this critical region (i.e., residues 303-335 inclusive), was able to restore 85% of the Stof delta 12 activity when added back to the truncated Stof delta 47 protein as well as return the temperature optimum to that of the Stof delta 12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302-336 of the enzyme form a three-stranded beta-sheet structure that interacts with the remainder of the protein. CD analysis of the 33-amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% beta-sheet content. These data suggest that this 33-amino acid peptide constitutes a stable beta-sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well-documented complementation of Ribonuclease S protein by the 15-residue, alpha-helical, S peptide.

[Correlation of electric potentials of the hippocampus, amygdala and neocortex during instrumental conditioned reflexes in the dog]. / Sootnosheniia élektricheskikh potentsialov gippokampa, mindaliny i neokorteksa pri instrumental'nykh usloviiakh refleksakh u sobak.

Coherence and spectral functions of biopotentials of the hippocampus, amygdala, frontal and sensorimotor cortical areas were estimated in dogs during instrumental alimentary behaviour and during self-stimulation of emotionally positive brain areas. Formation of functional systems: frontal cortex--amygdala, frontal cortex--hippocampus, and sensorimotor cortex--amygdala, synchronously acting in delta- and theta bands of electrical activity, was observed at the stage of a stabilized motor habit. THe hippocampus and amaygdala poorly co-operated, probably reflecting their different roles in the organization of behaviour.

[Effect of hippocampal damage on emotional stress in rats]. / Vliianie povrezhdeniia gippokampa na émotsional'noe napriazhenie u krys.

Motor reactions and ECGs were recorded in rats with lesioned hippocampus and in intact animals during elaboration of conditioned switching over of heterogeneous conditioned reflexes (alimentary and defensive). In hippocampectomized rats, in contrast to intact ones, the formation of this complex conditioned activity is not accompanied by pronounced vegetative reactions and changes in the ECG. The data obtained point to an active involvement of the hippocampus in formation of emotional stress and confirm the concept of the hippocampus as a brain structure, providing for responses to signals of events with low probability.

[Behavioral, electrophysiologic and vegetative correlates of self stimulation in dogs]. / Povedencheskie, élektrofiziologicheskie i vegetativnye korreliaty samostimuliatsii u sobak

Summary electrical activity of different brain structures (chiefly the hippocampal theta-rhythm) and cardiac and respiratory rhythms were recorded during self-stimulation (SS) in dogs. Emotional-motivational excitation in dogs, preceding SS, is attended with a moderate increase in theta-activity in the hippocampus. The SS period is characterized by desynchronization of the electrical activity, the appearance of high-frequency rhythmics and diminished theta-rhythm. After withdrawal of the pedal, hypersynchronization of the theta-rhythm sets in in most of the structures studied. SS is accompanied by considerable shifts of the cardiac and respiratory rhythms. The dynamics of behavioral, electrophysiological and vegetative shifts during SS in dogs points to a successive involvment of the brain mechanisms of search, positive reinforcement and emotional-negative interruption of the pedal pressing series. Complex interaction of the three mechanisms underlies the external phenomenology of the SS instrumental conditioned reflex.