Anti-S1P Antibody as a Novel Therapeutic Strategy for VEGFR TKI-Resistant Renal Cancer.

PURPOSE: VEGFR2 tyrosine kinase inhibition (TKI) is a valuable treatment approach for patients with metastatic renal cell carcinoma (RCC). However, resistance to treatment is inevitable. Identification of novel targets could lead to better treatment for patients with TKI-naïve or -resistant RCC. EXPERIMENTAL DESIGN: In this study, we performed transcriptome analysis of VEGFR TKI-resistant tumors in a murine model and discovered that the SPHK-S1P pathway is upregulated at the time of resistance. We tested sphingosine-1-phosphate (S1P) pathway inhibition using an anti-S1P mAb (sphingomab), in two mouse xenograft models of RCC, and assessed tumor SPHK expression and S1P plasma levels in patients with metastatic RCC. RESULTS: Resistant tumors expressed several hypoxia-regulated genes. The SPHK1 pathway was among the most highly upregulated pathways that accompanied resistance to VEGFR TKI therapy. SPHK1 was expressed in human RCC, and the product of SPHK1 activity, S1P, was elevated in patients with metastatic RCC, suggesting that human RCC behavior could, in part, be due to overproduction of S1P. Sphingomab neutralization of extracellular S1P slowed tumor growth in both mouse models. Mice bearing tumors that had developed resistance to sunitinib treatment also exhibited tumor growth suppression with sphingomab. Sphingomab treatment led to a reduction in tumor blood flow as measured by MRI. CONCLUSIONS: Our findings suggest that S1P inhibition may be a novel therapeutic strategy in patients with treatment-naïve RCC and also in the setting of resistance to VEGFR TKI therapy.

Blockage of lysophosphatidic acid signaling improves spinal cord injury outcomes.

Evidence suggests a proinflammatory role of lysophosphatidic acid (LPA) in various pathologic abnormalities, including in the central nervous system. Herein, we describe LPA as an important mediator of inflammation after spinal cord injury (SCI) in zebrafish and mice. Furthermore, we describe a novel monoclonal blocking antibody raised against LPA that potently inhibits LPA's effect in vitro and in vivo. This antibody, B3, specifically binds LPA, prevents it from interacting with its complement of receptors, and blocks LPA's effects on the neuronal differentiation of human neural stem/progenitor cells, demonstrating its specificity toward LPA signaling. When administered systemically to mice subjected to SCI, B3 substantially reduced glial inflammation and neuronal death. B3-treated animals demonstrated significantly more neuronal survival upstream of the lesion site, with some functional improvement. This study describes the use of anti-LPA monoclonal antibody as a novel therapeutic approach for the treatment of SCI.

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

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 A 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.

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.

Sphingosine-1-phosphate and sphingosine kinase are critical for transforming growth factor-beta-stimulated collagen production by cardiac fibroblasts.

AIMS: Following injury, fibroblasts transform into myofibroblasts and produce extracellular matrix (ECM). Excess production of ECM associated with cardiac fibrosis severely inhibits cardiac function. Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, regulates the function of numerous cell types. In this study, we determined the role of S1P in promoting pro-fibrotic actions of cardiac fibroblasts (CFs). METHODS AND RESULTS: S1P-mediated effects on myofibroblast transformation, collagen production, and cross-talk with transforming growth factor-beta (TGF-beta) using mouse CF were examined. S1P increased alpha-smooth muscle actin (a myofibroblast marker) and collagen expression in a S1P2 receptor- and Rho kinase-dependent manner. TGF-beta increased sphingosine kinase 1 (SphK1; the enzyme responsible for S1P production) expression and activity. TGF-beta-stimulated collagen production was inhibited by SphK1 or S1P2 siRNA, a SphK inhibitor, and an anti-S1P monoclonal antibody. CONCLUSION: These findings suggest that TGF-beta-stimulated collagen production in CF involves 'inside-out' S1P signalling whereby S1P produced intracellularly by SphK1 can be released and act in an autocrine/paracrine fashion to activate S1P2 and increase collagen production.

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.

Sphingosine-1-phosphate (S1P) is a novel fibrotic mediator in the eye.

Sphingosine-1-phosphate (S1P) is a pleiotropic lysolipid that has recently been implicated in the regulation of tissue fibrosis. However, the fibrogenic potential of S1P in the eye has not previously been investigated. In the current study, we evaluated cells from the anterior and posterior segments of the eye for the presence of S1P and their potential ability to produce and respond to S1P. In addition, we investigated the regulatory role of S1P as a mediator of proliferation, cellular transformation and pro-fibrotic protein expression in human retinal pigmented epithelial cells. Expression of S1P receptors and sphingosine kinases (the enzymes that produce S1P) was examined using RT-PCR, and intracellular localization of S1P was examined using immunoblotting, immunohistochemistry and ELISA in primary human retinal pigmented epithelial (RPE) cells, primary human conjunctival fibroblasts (ConF), and primary human corneal fibroblasts (CF). RPE cell proliferation was determined using an MTT-based cell proliferation assay, and RPE myofibroblast transformation, collagen type I production and profibrotic protein expression were assessed using immunofluorescence, ELISA and immunoblot. S1P(1-3, 5) receptors and sphingosine kinases 1 and 2 were expressed and intracellular pools of S1P were detected in RPE cells, ConF and CF. S1P stimulated RPE cell proliferation in a dose- and time-dependent manner. S1P induced myofibroblast transformation of RPE cells, as indicated by increased alpha-smooth muscle actin (alpha-SMA) expression and its incorporation into prominent stress fibers, and promoted collagen type I production. S1P stimulated the expression of plasminogen activator inhibitor-1 (PAI-1) and heat shock protein 47 (HSP47), two proteins that are linked to increased tissue fibrosis. Combined, these data demonstrate that RPE cells, ConF and CF from the human eye not only have the molecular ability to produce and respond to S1P, but also contain S1P. Furthermore, S1P promotes proliferation, myofibroblast transformation, collagen production and pro-fibrotic protein expression by human RPE cells. These data suggest that S1P is a previously unrecognized mediator of profibrotic cellular function and signaling in the eye.

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.