Abstract
Drug discovery opportunities where loss-of-function alleles of a target gene link to a disease-relevant phenotype often require an agonism approach to up-regulate or re-establish the activity of the target gene. Antibody therapy is increasingly recognized as a favored drug modality due to multiple desirable pharmacological properties. However, agonistic antibodies that enhance the activities of the target enzymes are rarely developed because the discovery of agonistic antibodies remains elusive. Here we report an innovative scheme of discovery and characterization of human antibodies capable of binding to and agonizing a circulating enzyme lecithin cholesterol acyltransferase (LCAT). Utilizing a modified human LCAT protein with enhanced enzymatic activity as an immunogen, we generated fully human monoclonal antibodies using the XenoMouseTM platform. One of the resultant agonistic antibodies, 27C3, binds to and substantially enhances the activity of LCAT from humans and cynomolgus macaques. X-ray crystallographic analysis of the 2.45 Å LCAT-27C3 complex shows that 27C3 binding does not induce notable structural changes in LCAT. A single administration of 27C3 to cynomolgus monkeys led to a rapid increase of plasma LCAT enzymatic activity and a 35% increase of the high density lipoprotein cholesterol that was observed up to 32 days after 27C3 administration. Thus, this novel scheme of immunization in conjunction with high throughput screening may represent an effective strategy for discovering agonistic antibodies against other enzyme targets. 27C3 and other agonistic human anti-human LCAT monoclonal antibodies described herein hold potential for therapeutic development for the treatment of dyslipidemia and cardiovascular disease.
Highlights
lecithin cholesterol acyltransferase (LCAT) Activation and high density lipoprotein (HDL) Modulation by Agonistic Antibodies prevents loss of the lipid-free apoA-I and small HDL particles via kidney filtration
Studies performed in rodent species that lack cholesteryl ester transfer protein (CETP) showed inconsistent results with LCAT intervention, presumably because CETP plays a role in the reverse cholesterol transport (RCT) pathway at a step immediately downstream of LCAT action to transfer cholesteryl ester (CE) from HDL to the apoB-containing lipoproteins toward the liver portal [13]
CVD risk has been reported in both types of LCAT-deficient patients [19], but in several cases, clinical CVD manifestations are not readily apparent and are perhaps complicated by reduced low density lipoprotein (LDL)-cholesterol levels associated with LCAT deficiency in these cases
Summary
Generation of Recombinant Human and Cynomolgous Monkey LCAT Proteins—Full-length human LCAT-coding region was cloned from liver and brain cDNAs (BioChain Institute, Inc., Hayward, CA) and subcloned into the expression vector pDSRa25. Primary activity screening for identification of human antihuman LCAT-agonistic antibodies was performed using a scintillation proximity assay (SPA) to first filter out LCAT antagonistic antibodies This SPA used 0.5 l of hybridoma culture supernatant samples to incubate with rLCAT(C31Y) at a concentration of 0.1 g/ml protein in the reaction mixture containing 1% human serum albumin (fatty acid-free), 7 mM Tris (pH 7.4), 100 mM NaCl, and 2 mM -mercaptoethanol. LCAT Activation Assays—The non-antagonistic antibodies selected by the SPA assay were examined using another LCAT activity assay with thin layer chromatography (TLC) to quantify the rate of cholesterol esterification in reconstituted HDL particles [34] For this assay, 10 l of hybridoma exhaust supernatants were incubated with purified wild type rLCAT protein at a concentration of 0.8 g/ml protein in the presence of 1% human serum albumin, 7 mM Tris (pH 7.4), 4 mM EDTA, 100 mM NaCl, and 2 mM -mercaptoethanol. Comparisons were deemed statistically significant if the p value was Ͻ0.05
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