In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody

Abstract

BackgroundS1P3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtype-selective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist.Methodology/Principal FindingsSpecific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3-mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts.Conclusions/SignificanceWe have developed the first-reported monoclonal antibody that selectively recognizes a lipid-activated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsis and breast cancer, it also provides proof of concept for the generation of novel GPCR-specific therapeutic antibodies.

The Prevalence, Maintenance, and Relevance of G Protein–Coupled Receptor Oligomerization

“…Although technically challenging, not least because of wide-ranging concerns over the specificity of many of the available GPCR antibodies (Beermann et al, 2012) and the modest expression levels of many GPCRs, approaches such as this offer the opportunity to begin to examine potential colocalization of GPCRs without resorting to more traditional methods based on, e.g., immunoelectron microscopy . Interestingly, each of large-scale GPCR protein production for crystallography trials, novel approaches to GPCR antigen presentation (Larsson et al, 2011), and indications that anti-GPCRdirected "biologicals" may have clinical utility (Harris et al, 2012) hint at ways forward in providing a wider range of more specific and high-affinity immunologic reagents. Furthermore, despite some of the concerns noted earlier about antibody specificity, in relation to the identification of heteromers, a small number of heteromer-specific antibodies have been described (Gupta et al, 2010;Rozenfeld et al, 2011) that potentially identify such complexes directly.…”

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of <em>Caenorhabditis elegans</em>

“…Representative data (Figure 1) from several different genotypes and their paired controls are presented 8,24 ; data were specifically chosen that highlight differences in the assayed animals. The degree of effect at 10 min of exposure is considered the initial sensitivity of a strain, which is shown on the left axes in Figure 1B-G.…”

“…Here, we describe an assay that has been used to identify several molecular and environmental mediators of the acute behavioral response to ethanol 8,10,20,24,25 . This method allows the differentiation and simultaneous examination of two different ethanol response behavior phenotypes, initial sensitivity and the development of acute functional tolerance, that together model the composite phenotype of level of response in mammals.…”

“…Statistically significant comparisons (paired t-tests) are shown: *, p < 0.05; **, p < 0.01. 8,24 The data presented in this figure are modified from 8,24 .…”

“…This assay has been used extensively to study the genetic and environmental influences on the LR to ethanol 8,10,20,24,25 . The mammalian LR phenotype is a composite of at least two components, initial sensitivity to ethanol and acute functional tolerance to ethanol 26,27 .…”