Agonistic Human Antibodies Binding to Lecithin-Cholesterol Acyltransferase Modulate High Density Lipoprotein Metabolism

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.

A retractable lid in lecithin:cholesterol acyltransferase provides a structural mechanism for activation by apolipoprotein A-I

“…Electron density is observed starting at residue 20 and ending at 399, similar to that observed in previous structural determinations ( Fig. 1A) (17,26,27). The enzyme crystallized with four copies in the asymmetric unit, with an average root mean square deviation (r.m.s.d.)…”

“…The overall domain architecture is conserved with LPLA2 and previously reported LCAT structures, with an ␣/␤ hydrolase domain positioning the active site at the center of the enzyme, in a cavity flanked by the cap and membrane-binding domains ( Fig. 1A) (17,26,27). Surprisingly, residues 226 -246 in the cap domain of LCAT (referred to as the "lid"), which includes the dynamic "lid loop" of LPLA2 (residues 226 -236 in LCAT), adopt a different conformation from all prior structures of LCAT and LPLA2 and is almost fully ordered ( Fig.…”

“…1C and supplemental Fig. S1A) (26,27). In the first, LCAT was crystallized in complex with a Fab fragment that recognizes the membrane-binding domain.…”

“…We used an IDFP-bound LPLA2 crystal structure to model possible paths for acyl chains in the open-2Fab structure and to understand how protection from HDX might occur (Fig. 6, C and D) (17,27). Certain regions on the front side of LCAT are more likely protected directly by IDFP, as they form the active site where IDFP binds.…”

“…Retraction of the lid could therefore represent an aspect of interfacial activation, a phenomenon wherein the presence of a lipid/solvent interface stimulates enzyme activity on soluble substrates (20,24,25). Recently, higher resolution structures of human LCAT in complex with one (26) or two (27) Fab fragments have been reported and confirm the overall domain structure of LCAT, but, unlike LPLA2, they contain largely disordered but conformationally distinct lid regions. The presence of bound Fab and a crystal contact formed in the active site also complicates interpretation of how lid conformation correlates with activity in these structures.…”