Human Anti-S1P Recombinant Antibody (clone LT1009) (CAT#: PABL-701)

Figure 1 Comparison of the in vivo effi cacy 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 fi ve 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).

O'Brien, N. , Jones, S. T. , Williams, D. G. , Cunningham, H. B. , Moreno, K. , & Visentin, B. , et al. (2009). Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies. Journal of Lipid Research, 50(11), 2245.

Figure 2 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 M S1P in the presence or absence of increasing concentrations of either LT1009 or LT1002 (2–2,500 ug/ml). The addition of increasing concentrations of either LT1009 or LT1002 was able to reduce cytokine release in the cell conditioned media in a dosedependent fashion. IC 50 values ( ug/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).

O'Brien, N. , Jones, S. T. , Williams, D. G. , Cunningham, H. B. , Moreno, K. , & Visentin, B. , et al. (2009). Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies. Journal of Lipid Research, 50(11), 2245.

Figure 3 Thermostability comparison of the humanized antibody variants.

The thermostability of the variants was determined by measuring its S1P-binding affi nity by direct binding ELISA. Each antibody (25 ug/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.

O'Brien, N. , Jones, S. T. , Williams, D. G. , Cunningham, H. B. , Moreno, K. , & Visentin, B. , et al. (2009). Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies. Journal of Lipid Research, 50(11), 2245.

Figure 4 Antibody designation and binding affinity.

All antibody variants are described in figure.

O'Brien, N. , Jones, S. T. , Williams, D. G. , Cunningham, H. B. , Moreno, K. , & Visentin, B. , et al. (2009). Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies. Journal of Lipid Research, 50(11), 2245.

Figure 5 Schematic representations of S1P and LT1009.

(A) The structure of S1P in its uncharged form with stereochemistry and numbering of the carbon atoms indicated. (B) The primary amino acid sequence of the CDRs from the LT1009 heavy and light chains is shown in single letter code. Numbering is according to Kabat and Wu convention with letters A–D below indicating insertions. For comparison, the CDR sequences from the anti-CD4 Q425 antibody are aligned and identical and homologous residues are indicated by black and light gray boxes, respectively. Residues directly involved in S1P binding are indicated by the following letters above the sequence: M-binds Ca2+; H-hydrogen bonds through a side chain; h-hydrogen bonds through peptide backbone atoms; Φ-mediates hydrophobic interactions. Amino acids that were observed to contact Ca2+ in the Q425 Fab x-ray structure are indicated by the letter "m" below.

Wojciak, J. M. , Zhu, N. , Schuerenberg, K. T. , Moreno, K. , Shestowsky, W. S. , & Hiraiwa, M. , et al. (2009). The crystal structure of sphingosine-1-phosphate in complex with a fab fragment reveals metal bridging of an antibody and its antigen. Proceedings of the National Academy of Sciences, 106(42), 17717-17722.

Figure 6 Bridging Ca2+ ions are required for S1P binding by LT1009.

SPR sensorgrams of titrated LT1009 Fab binding to surface-bound S1P in the absence (Top) and presence (Bottom) of 50 μM CaCl2. Raw measurements are plotted in black while curves that best fit the data are overlayed in orange.

Wojciak, J. M. , Zhu, N. , Schuerenberg, K. T. , Moreno, K. , Shestowsky, W. S. , & Hiraiwa, M. , et al. (2009). The crystal structure of sphingosine-1-phosphate in complex with a fab fragment reveals metal bridging of an antibody and its antigen. Proceedings of the National Academy of Sciences, 106(42), 17717-17722.