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 (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. ERRATAJournal of Lipid ResearchVol. 51Issue 4PreviewThe authors of “Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies” (J. Lipid Res. 50: 2245–2257) have advised the Journal that the name of an oligo stated in the Materials and Methods section of their manuscript is incorrect. The mouse immunoglobulin kappa chain variable region (VL) was amplified by PCR using VK20, not MKV20. Full-Text PDF Open Access Sphingosine-1-phospate (S1P) is a bioactive lysophospholipid signaling molecule that serves important roles in normal development and physiological processes, including modulating the immune, cardiovascular, and central nervous systems (1Gardell S.E. Dubin A.E. Chun J. Emerging medicinal roles for lysophospholipid signaling.Trends Mol. Med. 2006; 12: 65-75Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 2Murph M. Mills G.B. Targeting the lipids LPA and S1P and their signalling pathways to inhibit tumour progression.Expert Rev. Mol. Med. 2007; 9: 1-18Crossref PubMed Scopus (52) Google Scholar, 3Skoura A. Sanchez T. Claffey K. Mandala S.M. Proia R.L. Hla T. Essential role of sphingosine 1-phosphate receptor 2 in pathological angiogenesis of the mouse retina.J. Clin. Invest. 2007; 117: 2506-2516Crossref PubMed Scopus (187) Google Scholar, 4Wymann M.P. Schneiter R. Lipid signalling in disease.Nat. Rev. Mol. Cell Biol. 2008; 9: 162-176Crossref PubMed Scopus (960) Google Scholar). S1P is a key player in the sphingolipid signaling cascade and is produced from ceramide and sphingosine the of sphingosine and are of S1P has in from from S1P as a key of multiple and pathways Targeting a novel for cancer 2006; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). The of S1P are by the binding of the bioactive lipid to a of receptors to the S1P receptor J. receptors as potential in and 2006; 12: PubMed Scopus Google Scholar). The levels S1P a that a is the is from of by PubMed Scopus Google Scholar, 1-phosphate signalling in J. PubMed Scopus Google Scholar, A. J. of 1-phosphate involved in the of to in human Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The key of is the S1P for and for and by the S1P receptors on the of and other several of human of and of of human sphingosine Res. Google Scholar, of sphingosine 1 in normal and PubMed Scopus Google with a of S1P production and progression with of with roles of sphingosine in of PubMed Scopus Google Scholar, Targeting for cancer J. 2006; PubMed Scopus Google Scholar). We that effective of of cancer a to the of sphingolipid This therapeutic of the S1P, and levels of the and several for a therapeutic that for of the of are involved in the sphingolipid signaling the of is molecule of not the production of S1P also the sphingolipid in of and an and effect that by S1P levels M. J. The role of sphingosine 1 in or 2008; PubMed Scopus Google Scholar). and that be their therapeutic is the S1P receptors are to be of potential therapeutic J. receptors as potential in and 2006; 12: PubMed Scopus Google with as the S1P is in clinical trials for multiple T. A. an sphingolipid of a novel clinical in multiple 2007; PubMed Scopus Google Scholar, J. T. for multiple J. Med. 2006; PubMed Scopus Google Scholar). is by the of receptors that have and are by and for the of cancer and other diseases the administration of monoclonal antibodies to as “molecular to or their in the pro-angiogenic and or the cytokine, We that in diseases associated with S1P as an anti-S1P antibody the S1P in the to and signaling S1P receptors on the cancer The production of an antibody directed against a lipid as S1P is a and have been successful to antibodies to bioactive lipids with the high affinity and specificity for and therapeutic In a for the generation of antibodies against bioactive as S1P, a that lipid as a key of the for both the immunization and the antibody and We and the monoclonal directed against bioactive S1P. This murine antibody also the of an that shows as a to S1P in as We report that LT1002 has been humanized and by region by This humanized LT1009, is a therapeutic for of with cancer or that both LT1002 and LT1009 can the S1P-mediated release of from human ovarian cancer and that both anti-angiogenic activity in the choroidal neovascularization of age-related macular as as of lymphocyte with other of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in and angiogenesis in multiple 2006; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. A. J. A. R. monoclonal antibodies inhibit angiogenesis and in a murine of choroidal Res. 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Med. 2006; 12: 65-75Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 2Murph M. Mills G.B. Targeting the lipids LPA and S1P and their signalling pathways to inhibit tumour progression.Expert Rev. Mol. Med. 2007; 9: 1-18Crossref PubMed Scopus (52) Google Scholar, and 3Skoura A. Sanchez T. Claffey K. Mandala S.M. Proia R.L. Hla T. Essential role of sphingosine 1-phosphate receptor 2 in pathological angiogenesis of the mouse retina.J. Clin. 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