Anti-CD248 Recombinant Antibody (Ontuxizumab) (CAT#: TAB-773)

Figure 1 TEM-1 Expression: High and Null Expressing Sarcoma Lines.

FACS analysis using Ontuxizumab antibody and FITC-labeled anti-human secondary antibody to determine TEM1 expression. RD-ES (Ewing's sarcoma) and FUJI (synovial sarcoma) show high TEM-1 expression. LUPI, a Ewing's sarcoma line, and SYO-1, a synovial sarcoma line, display null to low expression of TEM-1, respectively.

Lange, S. E., Zheleznyak, A., Studer, M., O'Shannessy, D. J., Lapi, S. E., & Van Tine, B. A. (2016). Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications. Oncotarget, 7(11), 13082.

Figure 2 TEM-1 Expression: High and Null Expressing Sarcoma Lines.

Semi-quantitative immunofluorescence of TEM-1 expression. Cell lines RD-ES and FUJI display strong Ontuxizumab binding while LUPI and SYO-1 display null binding. Images were captured using an exposure time of 25 msec through a 60x objective keeping time constant across all images.

Lange, S. E., Zheleznyak, A., Studer, M., O'Shannessy, D. J., Lapi, S. E., & Van Tine, B. A. (2016). Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications. Oncotarget, 7(11), 13082.

Figure 3 Binding Study and Uptake.

A. 89Zr-Df-Bz-NCS-Ab binding to selected sarcoma cell lines. Radiolabeled antibody was allowed to bind to selected cell lines for 35 min as described in the Methods section. RD-ES and FUJI cells demonstrated good binding of 89Zr-Df-Bz-NCS-Ab, while SYO-1 and LUPI showed no appreciable binding. B. Immunoreactivity of 89Zr-Df-Bz-NCS-Ab determined using 89Zr-Df-Bz-NCS-Ab binding to immobilized TEM-1 in the presence of increasing amounts of Df-Bz-NCS-Ab (conjugated) or Ab (unconjugated). Df-Bz-NCS-Ab IC50 = 13.1 nM, Ab IC50 = 14.5 nM. Assay performed as described in the Methods section.

Lange, S. E., Zheleznyak, A., Studer, M., O'Shannessy, D. J., Lapi, S. E., & Van Tine, B. A. (2016). Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications. Oncotarget, 7(11), 13082.

Figure 4 TEM-1 Expression: High and Null Expressing Sarcoma Lines.

NATIVE-PAGE Western blot showing high-expressing TEM-1 lines RD-ES and FUJI, with null-expressing lines LUPI and SYO-1.

Lange, S. E., Zheleznyak, A., Studer, M., O'Shannessy, D. J., Lapi, S. E., & Van Tine, B. A. (2016). Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications. Oncotarget, 7(11), 13082.

Figure 5 Fc-TEM1 binds to ECM proteins.

(A) ELISA plates precoated with different ECM proteins were blocked with ELISA buffer before the addition of purified Fc-TEM1 protein at the indicated concentrations. After 2 h of incubation, the plates were washed, and HRP-conjugated goat anti-mouse antibody was added to detect bound Fc-TEM1. FN, fibronectin; Col I/IV, collagen I/IV; VN, vitronectin; LN, laminin; Gel, gelatin. (B) An ELISA plate was precoated overnight with the following antigens: Staphylococcus Enterotoxin B (STEB), ovalbumin (OVA), bovine γ-globulin (BGG), tumor-associated 90-kDa glycoprotein antigen expressed on most melanoma cells (TA90), hen egg lysozyme (HEL), tetanus toxoid (TT), 1% BSA, human mesothelin, human GM-CSF, goat IgG, and mouse IgG. Fc-TEM1 was added at increasing amounts (5, 10, and 50 μg/ml) and allowed to adhere for 2 h. Plates were then washed, and HRP-conjugated goat anti-mouse antibody was added to detect bound Fc-TEM1.

Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., ... & Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences, 104(46), 17965-17970.

Figure 6 MORAb-004 Inhibits Fc-TEM1 binding to FN and Col I.

First, 1.25 μg/ml Fc-TEM1 was preincubated for 1 h at 4°C with increasing concentrations of MORAb-004 or human IgG. The protein–antibody complex was then added to an ELISA plate precoated with FN (A) or Col I (B). After 2 h of incubation at room temperature, the plate was washed, and HRP-conjugated goat anti-mouse antibody was added to detect bound Fc-TEM1.

Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., ... & Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences, 104(46), 17965-17970.

Figure 7 Endosialin/TEM1 expressed in CHO cells interacts with FN.

For immunoprecipitation (IP), polyclonal antibodies against FN were absorbed to protein G Sepharose beads. We subjected 107 cells to overnight IP at 4°C, followed by Western blot analysis with either the anti-FN (Upper) or anti-endosialin/TEM1 (Lower) antibodies.

Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., ... & Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences, 104(46), 17965-17970.

Figure 8 Expression of endosialin/TEM1 changes cell morphology on matrigel.

We seeded 8 × 104 cells of either CHO-K1 (Upper) or CHO-TEM1 (Lower) onto a 96-well plate coated with matrigel and incubated them at 37°C. After overnight incubation, the wells were photographed for macroscopic examination of tubule formation.

Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., ... & Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences, 104(46), 17965-17970.

Figure 9 Expression of endosialin/TEM1 enhances FN-dependent cell adhesion.

(A) Cells were added to a precoated 96-well plate containing various ECM proteins. The cells were allowed to adhere for 1 h at 37°C, and wells were washed extensively to remove any loosely bound cells. The number of attached cells was determined by using the CellTiter-Glo Luminescent Cell Viability Assay. Col, collagen; FN, fibronectin; LN, laminin; TN, tenascin; VN, vitronectin; Neg, BSA. (B) To determine whether MORAb-004 blocks endosialin/TEM1-mediated cell adhesion to FN, cells (1.5 × 105) were preincubated for 1 h at 4°C with 100 μg/ml MORAb-004 or 100 μg/ml IgG isotype control antibody. After pretreatment, cells were added to a 96-well plate coated with FN and incubated for 1 h at 37°C. (C) The region within FN that mediates adhesion of endosialin/TEM1-positive cells was determined by precoating 96-well plates with equimolar amounts of protein fragments derived from whole FN, and the number of attached cells was determined as described before. Matrigel served as a positive control.

Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., ... & Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences, 104(46), 17965-17970.

Figure 10 Expression of endosialin/TEM1 enhances MMP 9 activity.

Supernatants from serum-starved CHO-TEM1 or CHO-K1 cells were subjected to gelatin zymography to assess the activation state of MMPs. Equal amounts of protein supernatant collected after 48 h were loaded onto a gelatin-containing gel and subjected to electrophoresis under nonreducing/nondenaturing conditions. The positive MMP 2/9 marker (right lane) served as a control for MMP 2/9 migration and activity. Arrows indicate pro and active forms of MMP 2/9 and protease bands of unknown origin.

Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., ... & Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences, 104(46), 17965-17970.

Figure 1 Anti-CD248 Recombinant Antibody (TAB-773) in SDS-PAGE

SDS-PAGE analysis of TAB-773 in non-reduced (Lane 1) and reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result, different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively.

The purity of TAB-773 was greater than 95% as determined by SDS-PAGE.

Figure 2 Anti-CD248 Recombinant Antibody (TAB-773) in HPLC

The purity of TAB-773 was greater than 95% as determined by SEC-HPLC.