Biochemical and Structural Characterization of Lysophosphatidic Acid Binding by a Humanized Monoclonal Antibody

Abstract

Lysophosphatidic acid (LPA) is a common product of glycerophospholipid metabolism and an important mediator of signal transduction. Aberrantly high LPA concentrations accompany multiple disease states. One potential approach for treatment of these diseases, therefore, is the therapeutic application of antibodies that recognize and bind LPA as their antigen. We have determined the x-ray crystal structure of an anti-LPA antibody (LT3015) Fab fragment in its antigen-free form to 2.15 Å resolution and in complex with two LPA isotypes (14:0 and 18:2) to resolutions of 1.98 and 2.51 Å, respectively. The variable complementarity determining region (CDR) loops at the antigen binding site adopt nearly identical conformations in the free and antigen-bound crystal structures. The crystallographic models reveal that the LT3015 antibody employs both heavy and light chain CDR loops to create a network of eight hydrogen bonds with the glycerophosphate head group of its LPA antigen. The head group is almost completely excluded from contact with solvent, while the hydrocarbon tail is partially solvent exposed. In general, mutation of amino acid residues at the antigen binding site disrupts LPA binding. However, the introduction of particular mutations chosen strategically based upon the structures can positively influence LPA binding affinity. Finally, these structures elucidate the exquisite specificity demonstrated by an anti-lipid antibody for binding a structurally simple and seemingly unconstrained target molecule.

A novel approach for measuring sphingosine-1-phosphate and lysophosphatidic acid binding to carrier proteins using monoclonal antibodies and the Kinetic Exclusion Assay

“…1A). Based on the LT3015 fragment antigen-binding LPA(14:0) and fragment antigen-binding LPA(18:2) crystal structures, the LPA epitope consists of the glycerolphosphate group plus the juxtaposed C1-C8 carbon atoms of the acyl chain (29). As visualized in both cocrystal structures, this portion of the bound LPA hydrocarbon chain is fully extended and forms extensive intermolecular hydrophobic contacts with the antibody.…”

“…1B). The production and characterization of the two humanized IgG1k mAbs, LT1009 and LT3015, 3 which specifically recognize S1P and LPA, respectively, and the structural basis for lipid recognition are described elsewhere (28,29). These antibodies directly compete with carrier proteins for binding target lipids in vitro; the equilibrium binding curve for LT3015 binding LPA shifts toward weaker apparent affinity as the concentration of fatty acid-free (FAF)-BSA is increased (Fig.…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…113–115 Similarly, conventional antibodies tend to accommodate short linear peptides and nucleic acid polymers within grooves formed from both heavy- and light-chain CDRs. 104 Four studies have reported structures of camelid V H Hs in complex with haptens and peptides (Table 2); the recognition mechanism of all but one (a methotrexate-specific V H H with a non-canonical binding site involving framework region (FR)3 residues located below CDR1 113 ) was basically similar to that of conventional antibodies, with the hapten-binding pocket formed from two or more CDRs and extending in some instances into the former V L interface.…”

Drug development against the hippo pathway in mesothelioma

“…Conversely, activating Gα s coupled receptors by adrenaline, glucagon and dihydrexidine turns on the cAMP-dependent protein kinase A (PKA) signaling pathway and promotes LAST1/2 activity, resulted in YAP suppression (24,25). Therapeutic approaches targeting GPCR to attenuate YAP/Hippo pathway in cancers have been evaluated in clinic trials, including S1P-blocking antibody Sphingomab and Phosphatase-resistant LPA analog (26,27). In pre-clinical studies, G protein-coupled β-adrenergic receptor agonist dobutamine has been proposed as anti-cancer treatments (28).…”