The crystal structure of sphingosine-1-phosphate in complex with a Fab fragment reveals metal bridging of an antibody and its antigen

Abstract

The pleiotropic signaling lipid sphingosine-1-phosphate (S1P) plays significant roles in angiogenesis, heart disease, and cancer. LT1009 (also known as sonepcizumab) is a humanized monoclonal antibody that binds S1P with high affinity and specificity. Because the antibody is currently in clinical trials, it is important to confirm by structural and biochemical analyses that it binds its target in a predictable manner. Therefore, we determined the structure of a complex between the LT1009 antibody Fab fragment and S1P refined to 1.90 Å resolution. The antibody employs unique and diverse strategies to recognize its antigen. Two metal ions bridge complementarity determining regions from the antibody light chain and S1P. The coordination geometry, inductively coupled plasma spectroscopy, surface plasmon resonance spectroscopy, and biochemical assays suggest that these are Ca 2+ . The amino alcohol head group of the sphingosine backbone is recognized through hydrogen bonding interactions from 1 aa side chain and polypeptide backbone atoms of the antibody light and heavy chains. The S1P hydrophobic tail is almost completely enclosed within a hydrophobic channel formed primarily by the heavy chain. Both treatment of the complex with metal chelators and mutation of amino acids in the light chain that coordinate the metal atoms or directly contact the polar head group abrogate binding, while mutations within the hydrophobic cavity also decrease S1P binding affinity. The structure suggests mechanistic details for recognition of a signaling lipid by a therapeutic antibody candidate. Moreover, this study provides direct structural evidence that antibodies are capable of using metals to bridge antigen:antibody complexes.

Local Inactivation of Sphingosine 1-Phosphate in Lymph Nodes Induces Lymphopenia

“…Another possibility would be that the S1P concentration in the local environment of pLNs is rather low and procures an equilibrium of S1P binding and dissociation. The dissociation constant of the Ab is lower than that of the S1P 1 receptor (34,35). The local presence of Sphingomab in pLN would snatch S1P away from the receptor.…”

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

“…Calcium was included in the running buffer because divalent metal ions bridge the antibody-S1P interface and are required for strong affinity binding (28). A series of 2-fold S1P dilutions were prepared in glass vials using a glass syringe using the running buffer above with 100 M FAF-BSA.…”

“…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.…”

Antibodies to Bioactive Lysophospholipids

“…This humanized, optimized antibody is referred to as sonepcizumab or LT1009. The crystal structure of the Fab' fragment of LT1009 was solved to 1.9 Angstrom resolution demonstrating that the hypervariable domains of the Fab' interact with the ligand, S1P, in a manner predicted by site-directed mutagenesis studies (14). Interestingly, single mutations in key light-chain CDRs such as changing AspL30, AspL32, or GluL50 individually to alanines completely disrupt the ability of the humanized mAb, LT1009, to bind S1P ( fig.…”

“…In both these methods, the thiolated S1P was fundamental for elucidating the interactions between the anti-S1P antibody and its bioactive lipid target. It is important to note the X-ray crystal structure of the LT1009 Fab bound to S1P shows two calcium atoms bridging the antibody-antigen interface, and these ions are essential for high-affinity binding (14). Normally, there is enough calcium present in the reagents one might use, particularly if plasma or serum is used.…”