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. 2009 Nov;50(11):2245-57.
doi: 10.1194/jlr.M900048-JLR200. Epub 2009 Jun 9.

Production and Characterization of Monoclonal anti-sphingosine-1-phosphate Antibodies

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Free PMC article

Production and Characterization of Monoclonal anti-sphingosine-1-phosphate Antibodies

Nicole O'Brien et al. J Lipid Res. .
Free PMC article

Abstract

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.

Figures

Fig. 1.
Fig. 1.
A: Direct ELISA for measurement of the murine mAb, LT1002, binding to S1P. The S1P binding affinity of the murine mAb, LT1002, and a commercially available murine IgM antibody were compared by direct binding ELISA. Each antibody was measured at six concentrations ranging from 400–12.5 ng/ml on a 96-well plate coated with S1P-SMCC-BSA and detected using HRP-conjugated anti-mouse IgG and IgM secondary antibodies. The binding activity is expressed as OD at 450 nm. B: Direct ELISA for measurement of the murine mAb, LT1002, binding to S1P versus binding to cross-linker or carrier proteins. The specific binding of the murine mAb, LT1002, to S1P versus binding to cross-linkers IOA or SMCC and to carrier proteins KLH or BSA was assessed using a direct binding ELISA. LT1002 antibody was measured at six concentrations ranging from 400–12.5 ng/ml on a 96-well plate coated with S1P-SMCC-BSA (positive control), C1P-IOA-BSA, LPA-SMCC-BSA, and PAF-KLH. Primary antibody was then detected using HRP-conjugated anti-mouse IgG secondary antibody. The binding activity is expressed as OD at 450 nm.
Fig. 2.
Fig. 2.
Thermostability comparison of the humanized antibody variants. The thermostability of the variants was determined by measuring its S1P-binding affinity by direct binding ELISA. Each antibody (25 μg/ml in PBS) was incubated at 60, 62, 64, 66, 68, 70, and 72, up to 78°C for 10 min and then removed insoluble material by centrifugation for 1 min at 13,000 rpm. The binding activity of the supernatant was submitted to direct S1P-binding ELISA. The TM of the variants was determined after thermal challenge at the indicated temperature (60, 62, 64, 66, 68, 70, and 72, up to 78°C). The binding activity to S1P was then measured by ELISA and is expressed as OD at 450 nm.
Fig. 3.
Fig. 3.
Comparison of the in vivo efficacy of the anti-S1P mAb variants. Animals were treated with vehicle, a murine NS Ab (NS IgG), and all the anti-S1P antibodies 1 day prior to laser treatment and 6 days after laser treatment. CNV lesion volumes were measured 14 days after laser photocoagulation treatment (three burns/eye, one eye per animal, and five mice per treatment group). The areas for each burn were converted to a volume, and the volumes were averaged to produce a single CNV wound volume for each animal. All antibody treatment groups were statistically different than the saline treatment group (ANOVA followed by Bonferroni's post test: *P < 0.05 and **P < 0.001).
Fig. 4.
Fig. 4.
Antibody inhibition of IL-8 release in response to S1P. Cell conditioned media from SKOV3 human ovarian cancer cells were tested for CXCL8/IL-8 levels after 18 h of incubation with 5 μM S1P in the presence or absence of increasing concentrations of either LT1009 or LT1002 (2–2,500 μg/ml). The addition of increasing concentrations of either LT1009 or LT1002 was able to reduce cytokine release in the cell conditioned media in a dose-dependent fashion. IC50 values (μg/ml; mean ± SD, n = 3) were calculated for both LT1009 (374 ± 52) and LT1002 (513 ± 57) curves using a “nonlin fit log dose versus response” equation (GraphPad software).
Fig. 5.
Fig. 5.
Comparison of the ability of murine and human anti-S1P to alter lymphocyte trafficking. Peripheral blood lymphocytes were measured in mice (five/group) pre and 24 h after treatment with a single dose of antibody at 50 mg/kg. Murine (LT1002) and humanized (LT1009) anti-S1P antibodies caused a statistically significant decline in peripheral lymphocyte count when compared with pretreatment values; human nonspecific IgG (NS IgG) or PBS had no effect (two-way repeated measures ANOVA with Bonferron's post test: *P < 0.05).

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