Safety and Clinical Activity of MEDI1873, a Novel GITR Agonist, in Advanced Solid Tumors.

PURPOSE: The safety and preliminary efficacy of MEDI1873, an agonistic IgG1 fusion protein targeting glucocorticoid-induced TNF receptor-related protein (GITR), were evaluated in an open-label, first-in-human, phase I, dose escalation study in previously treated patients with advanced solid tumors. PATIENTS AND METHODS: Two single-patient cohorts at 1.5 and 3 mg i.v. were followed by 3+3 dose escalation in six cohorts at 7.5, 25, 75, 250, 500, and 750 mg, all every 2 weeks, for up to 52 weeks. Primary endpoints were safety and tolerability, dose-limiting toxicities (DLT), and MTD. Secondary endpoints included antitumor activity, pharmacokinetics, immunogenicity, and pharmacodynamics. RESULTS: Forty patients received MEDI1873. Three experienced DLTs: grade 3 worsening tumor pain (250 mg); grade 3 nausea, vomiting, and headache (500 mg); and grade 3 non-ST segment elevation myocardial infarction (750 mg). An MTD was not reached and treatment was well tolerated up to 500 mg. Most common treatment-related adverse events were headache (25%), infusion-related reaction (17.5%), and decreased appetite (17.5%). MEDI1873 exposure was dose proportional. Antidrug-antibody incidence was low. MEDI1873 increased peripheral CD4+ effector memory T-cell proliferation as well as cytokines associated with effector T-cell activation at dose levels ≥75 mg. The best response was stable disease (SD) in 17 patients (42.5%), including 1 unconfirmed partial response. Eight patients (20.0%) had SD ≥24 weeks. CONCLUSIONS: MEDI1873 showed acceptable safety up to 500 mg i.v. every 2 weeks with pharmacodynamics activity, and prolonged SD in some patients. However, further development is not planned because of lack of demonstrated tumor response.

Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.

Late intervention with a myeloperoxidase inhibitor stops progression of experimental chronic obstructive pulmonary disease.

RATIONALE: Inflammation and oxidative stress are linked to the deleterious effects of cigarette smoke in producing chronic obstructive pulmonary disease (COPD). Myeloperoxidase (MPO), a neutrophil and macrophage product, is important in bacterial killing, but also drives inflammatory reactions and tissue oxidation. OBJECTIVES: To determine the role of MPO in COPD. METHODS: We treated guinea pigs with a 2-thioxanthine MPO inhibitor, AZ1, in a 6-month cigarette smoke exposure model, with one group receiving compound from Smoking Day 1 and another group treated after 3 months of smoke exposure. RESULTS: At 6 months both treatments abolished smoke-induced increases in lavage inflammatory cells, largely ameliorated physiological changes, and prevented or stopped progression of morphologic emphysema and small airway remodeling. Cigarette smoke caused a marked increase in immunohistochemical staining for the myeloperoxidase-generated protein oxidation marker dityrosine, and this effect was considerably decreased with both treatment arms. Serum 8-isoprostane, another marker of oxidative stress, showed similar trends. Both treatments also prevented muscularization of the small intrapulmonary arteries, but only partially ameliorated smoke-induced pulmonary hypertension. Acutely, AZ1 prevented smoke-induced increases in expression of cytokine mediators and nuclear factor-κB binding. CONCLUSIONS: We conclude that an MPO inhibitor is able to stop progression of emphysema and small airway remodeling and to partially protect against pulmonary hypertension, even when treatment starts relatively late in the course of long-term smoke exposure, suggesting that inhibition of MPO may be a novel and useful therapeutic treatment for COPD. Protection appears to relate to inhibition of oxidative damage and down-regulation of the smoke-induced inflammatory response.

Whole-molecule antibody engineering: generation of a high-affinity anti-IL-6 antibody with extended pharmacokinetics.

The differentiation of therapeutic monoclonal antibodies in an increasingly competitive landscape requires optimization of clinical efficacy combined with increased patient convenience. We describe here the generation of MEDI5117, a human anti-interleukin (IL)-6 antibody generated by variable domain engineering, to achieve subpicomolar affinity for IL-6, combined with Fc (fragment crystallizable) engineering to enhance pharmacokinetic half-life. MEDI5117 was shown to be highly potent in disease-relevant cellular assays. The pharmacokinetics of MEDI5117 were evaluated and compared to those of its progenitor, CAT6001, in a single-dose study in cynomolgus monkeys. The antibodies were administered, either subcutaneously or intravenously, as a single dose of 5 mg/kg. The half-life of MEDI5117 was extended by approximately 3-fold, and clearance was reduced by approximately 4-fold when compared to CAT6001. MEDI5117 therefore represents a potential 'next-generation' antibody; future studies are planned to determine the potential for affinity-driven efficacy and/or less frequent administration.

Identification of iNOS inhibitors using InteraX.

Inducible nitric oxide synthase (iNOS) is active as a homodimer. A cell-based assay suitable for high-throughput screening (HTS) was generated to identify inhibitors of iNOS dimerization using the InteraX enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of beta-galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in beta-galactosidase activity. Surprisingly, the assay was also able to identify compounds that have the same profile as known inhibitors of fully formed dimeric iNOS by causing an increase in beta-galactosidase activity. The iNOS InteraX assay was used to screen approximately 800,000 compounds in a 384-well format. After hit confirmation, 3359 compounds were taken forward for full IC50 determination in InteraX and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraX compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit beta-galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in beta-galactosidase activity. Many of these were known inhibitors of iNOS. After IC50 determination in InteraX and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS.

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase.

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.