ABSTRACT
Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.
Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Complement C5/immunology , Animals , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antibodies, Monoclonal, Humanized/pharmacology , Antigen-Antibody Reactions , Binding Sites , Complement Activation/drug effects , Complement C5/chemistry , Complement C5/genetics , Genetic Variation , Half-Life , Hemolysis/drug effects , Humans , Inhibitory Concentration 50 , Macaca fascicularis , Mice , Protein Structure, QuaternaryABSTRACT
PURPOSE: To characterize molecular weight fractions of bovine corneal endothelial cell conditioned medium (CM) supporting retinal pigment epithelium (RPE) cell survival on aged and age-related macular degeneration (AMD) Bruch's membrane. METHODS: CM was subject to size separation using centrifugal filters. Retentate and filtrate fractions were tested for bioactivity by analyzing RPE survival on submacular Bruch's membrane of aged and AMD donor eyes and behavior on collagen I-coated tissue culture wells. Protein and peptide composition of active fractions was determined by mass spectrometry. RESULTS: Two bioactive fractions, 3-kDa filtrate and a 10-50-kDa fraction, were necessary for RPE survival on aged and AMD Bruch's membrane. The 3-kDa filtrate, but not the 10-50-kDa fraction, supported RPE growth on collagen 1-coated tissue culture plates. Mass spectrometry of the 10-50-kDa fraction identified 175 extracellular proteins, including growth factors and extracellular matrix molecules. Transforming growth factor (TGF)ß-2 was identified as unique to active CM. Peptides representing 29 unique proteins were identified in the 3-KDa filtrate. CONCLUSIONS: These results indicate there is a minimum of two bioactive molecules in CM, one found in the 3-kDa filtrate and one in the 10-50-kDa fraction, and that bioactive molecules in both fractions must be present to ensure RPE survival on Bruch's membrane. Mass spectrometry analysis suggested proteins to test in future studies to identify proteins that may contribute to CM bioactivity. TRANSLATIONAL RELEVANCE: Results of this study are the first steps in development of an adjunct to cell-based therapy to ensure cell transplant survival and functionality in AMD patients.
