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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M900048-JLR200 on June 9, 2009

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Journal of Lipid Research, Vol. 50, 2245-2257, November 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies1

Nicole O'Brien*,{dagger}, S. Tarran Jones§, David G. Williams§, H. Brad Cunningham*, Kelli Moreno*, Barbara Visentin*,{dagger}, Angela Gentile*, John Vekich*,{dagger}, William Shestowsky*, Masao Hiraiwa*, Rosalia Matteo*, Amy Cavalli*, Douglas Grotjahn**, Maria Grant{dagger}{dagger}, Geneviève Hansen*, Mary-Ann Campbell* and Roger Sabbadini2,*,{dagger}

* Lpath Inc., San Diego, CA 92121
{dagger} Department of Biology, San Diego State University, San Diego, CA 92182
** Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182
§ MRC Technology, London NW7 1AD, United Kingdom
{dagger}{dagger} University of Florida, Gainsville, FL

2 To whom correspondence should be addressed. e-mail: rsabbadini{at}lpath.com

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.

Supplementary key words sphingosine-1-phosphate • monoclonal antibodies • angiogenesis • cancer • bioactive lipids • humanization • age-related macular degeneration • choroidal neovascularization • complementarity determining region • lymphocyte trafficking

Abbreviations: AMD, age-related macular degeneration; ATCC, American Type Culture Collections; CDR, complementarity determining region; CER, ceramide; CNV, choroidal neovascularization; EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride; FR, framework region; GPCR, G protein-coupled receptor; IL-8, interleukin-8; IOA, iodoacetyl aminobenzoate; KLH, keyhole limpet hemocyanin; LPA, lysophosphatidic acid; mAb, monoclonal antibody; NHP, nonhuman primate; OD, optical density; S1P, sphingosine-1-phosphate; PS, phosphatidylserine; SMCC, sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate crosslinker; SPH, sphingosine; SPHK, sphingosine kinase; SPR, surface plasmon resonance; sulfo-MBS, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester


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