Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages.

S1P has been proposed to contribute to cancer progression by regulating tumor proliferation, invasion, and angiogenesis. We developed a biospecific monoclonal antibody to S1P to investigate its role in tumorigenesis. The anti-S1P mAb substantially reduced tumor progression and in some cases eliminated measurable tumors in murine xenograft and allograft models. Tumor growth inhibition was attributed to antiangiogenic and antitumorigenic effects of the antibody. The anti-S1P mAb blocked EC migration and resulting capillary formation, inhibited blood vessel formation induced by VEGF and bFGF, and arrested tumor-associated angiogenesis. The anti-S1P mAb also neutralized S1P-induced proliferation, release of proangiogenic cytokines, and the ability of S1P to protect tumor cells from apoptosis in several tumor cell lines, validating S1P as a target for therapy.

Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies.

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affinity and specificity for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, first-in-class therapeutic acting as a "molecular sponge" to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.

Anti-sphingosine-1-phosphate monoclonal antibodies inhibit angiogenesis and sub-retinal fibrosis in a murine model of laser-induced choroidal neovascularization.

The efficacy of novel monoclonal antibodies that neutralize the pro-angiogenic mediator, sphingosine-1-phosphate (S1P), were tested using in vitro and in vivo angiogenesis models, including choroidal neovascularization (CNV) induced by laser disruption of Bruch's membrane. S1P receptor levels in human brain choroid plexus endothelial cells (CPEC), human lung microvascular endothelial cells, human retinal vascular endothelial cells, and circulating endothelial progenitor cells were examined by semi-quantitative PCR. The ability of murine or humanized anti-S1P monoclonal antibodies (mAbs) to inhibit S1P-mediated microvessel tube formation by CPEC on Matrigel was evaluated and capillary density in subcutaneous growth factor-loaded Matrigel plugs was determined following anti-S1P treatment. S1P promoted in vitro capillary tube formation in CPEC consistent with the presence of cognate S1P(1-5) receptor expression by these cells and the S1P antibody induced a dose-dependent reduction in microvessel tube formation. In a murine model of laser-induced rupture of Bruch's membrane, S1P was detected in posterior cups of mice receiving laser injury, but not in uninjured controls. Intravitreous injection of anti-S1P mAbs dramatically inhibited CNV formation and sub-retinal collagen deposition in all treatment groups (p<0.05 compared to controls), thereby identifying S1P as a previously unrecognized mediator of angiogenesis and subretinal fibrosis in this model. These findings suggest that neutralizing S1P with anti-S1P mAbs may be a novel method of treating patients with exudative age-related macular degeneration by reducing angiogenesis and sub-retinal fibrosis, which are responsible for visual acuity loss in this disease.

Localization of sarcolemmal proteins to lipid rafts in the myocardium.

The localization of sarcolemmal proteins within the membrane can have a dramatic effect on excitation-contraction coupling. We examine the localization of the Na+-Ca2+ exchanger, the dihydropyridine receptor, and other proteins involved in excitation-contraction coupling in rat heart using biochemical and immunolocalization techniques. Specifically, we assess the distribution of proteins within the lipid raft fraction of the sarcolemma. We find that the distribution of proteins in lipid raft fractions is very dependent on the solubilization technique. A common technique using sodium carbonate/pH 11 to solubilize non-lipid raft proteins was inappropriate for use with sarcolemmal membranes. Use of Triton X-100 was more efficacious as a solubilization agent. A large majority of the Na+-Ca2+ exchanger, Na+/K+-ATPase, and plasma membrane Ca2+ pump are not present in lipid rafts. In contrast, most adenosine A1 receptors and dihydropyridine receptors were in lipid raft fractions. Most of the adenosine A1 receptors could be co-immunoprecipitated with caveolin indicating a localization to caveolae (a subclass of lipid rafts). In contrast, the dihydropyridine receptors could not be co-immunoprecipitated with caveolin. Most biochemical data were confirmed by high resolution immunolocalization studies. Using correlation analysis, only a small fraction of the Na+-Ca2+ exchangers colocalized with caveolin whereas a substantial fraction of dihydropyridine and adenosine A1 receptors did colocalize with caveolin. The most pertinent findings are that the Na+-Ca2+ exchanger and the dihydropyridine receptor are in separate sarcolemmal subcompartments. These spatial relationships may be relevant for understanding excitation-contraction coupling.

Expression and functional characterization of SCaMPER: a sphingolipid-modulated calcium channel of cardiomyocytes.

Calcium channels are important in a variety of cellular events including muscle contraction, signaling, proliferation, and apoptosis. Sphingolipids have been recognized as mediators of intracellular calcium release through their actions on a calcium channel, sphingolipid calcium release-mediating protein of the endoplasmic reticulum (SCaMPER). The current study investigates the expression and function of SCaMPER in cardiomyocytes. Northern analyses and RT-PCR cloning and sequencing revealed SCaMPER expression in both human and rat cardiac tissue. Immunofluorescence and Western blot analyses demonstrated that SCaMPER is abundant in cardiac tissue and is localized to the sarcotubular junction. This was confirmed by the colocalization of SCaMPER with dihydropyridine and ryanodine receptors by confocal microscopy. Purified T tubules were shown to contain SCaMPER and immunoelectron micrographs suggested that SCaMPER is located to the junctional T tubules, but a junctional SR localization cannot be ruled out. The sphingolipid ligand for SCaMPER, sphingosylphosphorylcholine (SPC), initiated calcium release from the cardiomyocyte SR. Importantly, antisense knockdown of SCaMPER mRNA produced a substantial reduction of sphingolipid-induced calcium release, suggesting that SCaMPER is a potentially important calcium channel of cardiomyocytes.