Anti-Human ENG Recombinant Antibody (Carotuximab) (CAT#: TAB-013ML)

Figure 1 Tube formation, proliferation and survival of CSC-TEC and TEC treated with TRC105.

(A and E) Representative micrograph of tubular structures formed by CSC-TEC (A) or TEC (E) at increasing levels of TRC105 (TRC40-TRC320, corresponding to TRC105 from 40 to 320 μg/ml). Original magnification x100. (B and F) Tube length of CSC-TEC (B) and TEC (F) at different doses (40-320 μg/ml) of TRC105. Data are represented as mean ± SD of at least three independent experiments normalized to untreated cells (CTL). (C and G) Proliferation levels of CSC-TEC (C) and TEC (G) at different doses (40-320 μg/ml) of TRC105. Data are represented as mean ± SD of at least three independent experiments normalized to CTL. (D and H) Percentage of apoptotic CSC-TEC (D) and TEC (H) treated with two different doses of TRC105 (80 and 160 μg/ml).

Brossa, A., Buono, L., & Bussolati, B. (2018). Effect of the monoclonal antibody TRC105 in combination with Sunitinib on renal tumor derived endothelial cells. Oncotarget, 9(32), 22680.

Figure 2 Tube formation, proliferation and survival of CSC-TEC and TEC treated with TRC105 in combination with different anti-angiogenic drugs.

(A and D) Tube length of CSC-TEC (A) and TEC (D) at increasing doses of TRC (TRC80-TRC320, corresponding to TRC105 from 80 to 320 μg/ml) in combination with Axitinib (AXI), Cabozantinib (CABO), Sorafenib (SOR) (all 1 μM) and Bevacizumab (BEVA) (100 μg/ml). (B and E) Proliferation levels of CSC-TEC (B) and TEC (E) at escalating doses of TRC105 (80-320 μg/ml) in combination with AXI, CABO, SOR and BEVA. Data are represented as mean ± SD of at least three independent experiments normalized to CTL and to 1. (C and F) Percentage of apoptotic CSC-TEC (C) and TEC (F) treated with AXI, CABO, SOR and BEVA alone or in combination with different doses of TRC105 (80-160 μg/ml).

Brossa, A., Buono, L., & Bussolati, B. (2018). Effect of the monoclonal antibody TRC105 in combination with Sunitinib on renal tumor derived endothelial cells. Oncotarget, 9(32), 22680.

Figure 3 TRC105 in combination with Sunitinib inhibits tube formation, proliferation and survival of CSC-TEC and TEC.

(A and D) Tube length of CSC-TEC (A) and TEC (D) at increasing levels of TRC105 (TRC80 and TRC160, corresponding to TRC105 80 and 160 μg/ml) in combination with tyrosine kinase Sunitinib (SUN, 0.1μM). Data are represented as mean ± SD of at least three independent experiments normalized to untreated cells (CTL). **=p<0.001 vs CTL; $=p<0.05 and $=p<0.001 vs SUN. (B and E) Proliferation levels of CSC-TEC (B) and TEC (E) at escalating doses of TRC105 (40-320 μg/ml) in combination with SUN (1μM). Data are represented as mean ± SD of at least three independent experiments normalized to CTL and to 1. *=p<0.05 and **=p<0.001 vs CTL; $=p<0.05 vs SUN. (C and F) Percentage of apoptotic CSC-TEC (C) and TEC (F) treated with SUN alone (1μM) or in combination with two different doses of TRC105 (80-160 μg/ml). Data are represented as mean ± SD of at least three independent experiments. **=p<0.001 vs CTL; $=p<0.05 vs SUN. (G) Representative micrograph of tubular structures formed by CSC-TEC or TEC treated with SUN (0.1μM) in combination with increasing levels of TRC (80-160 μg/ml). Original magnification x100.

Brossa, A., Buono, L., & Bussolati, B. (2018). Effect of the monoclonal antibody TRC105 in combination with Sunitinib on renal tumor derived endothelial cells. Oncotarget, 9(32), 22680.

Figure 4 Molecular analysis of CSC-TEC treated with TRC105 and Sunitinib alone or in combination.

(A) Funrich analysis of regulated genes of CSC-TEC treated with TRC105 (160 μg/ml), Sunitinib 0.1μM (SUN) or the combination of the two drugs (TRC105+SUN). (B) Molecular function analysis of the genes regulated by the single therapy treatment with TRC105 on CSC-TEC. (C) Biological pathway analysis of CSC-TEC genes regulated by the combination of TRC105 and SUN. (D) Real time analysis of KDR mRNA expression in CSC-TEC treated with TRC105 alone, SUN, or the combination of both. **=p<0.001 vs CTL.

Brossa, A., Buono, L., & Bussolati, B. (2018). Effect of the monoclonal antibody TRC105 in combination with Sunitinib on renal tumor derived endothelial cells. Oncotarget, 9(32), 22680.

Figure 5 Western blot analysis on CSC-TEC treated with TRC105 alone or in combination with Axitinib, Sorafenib, Cabozantinib and Sunitinib.

(A) Representative micrographs of western blot on CSC-TEC treated with TRC105 (160 μg/ml) alone or in combination with Sunitinib (1 μM), using antibodies against p-Erk, p-AKT, p-Smad 2/3 and p-Creb. (B) Western blot analysis showing the quantification of the protein levels of CSC-TEC treated with TRC105 (160 μg/ml) alone or in combination with Sunitinib (1 μM), normalized to Vinculin and to CTL. The relative expression of p-Erk and p-Akt were also normalized to basal Erk and Akt.(C) Representative micrographs of western blot on CSC-TEC treated with TRC105 (160 μg/ml) alone or in combination with Axitinib (AXI), Sorafenib (SOR) and Cabozantinib (CABO) (all 1 μM), using antibodies against p-Erk, p-AKT, p-Smad 2/3 and p-Creb. (D) Western blot analysis showing the quantification of the protein levels of CSC-TEC treated with with TRC105 (160 μg/ml) alone or in combination with Axitinib (AXI), Sorafenib (SOR) (all 1 μM), normalized to Vinculin and to CTL. The relative expression of p-Erk and p-Akt were also normalized to basal Erk and Akt.

Brossa, A., Buono, L., & Bussolati, B. (2018). Effect of the monoclonal antibody TRC105 in combination with Sunitinib on renal tumor derived endothelial cells. Oncotarget, 9(32), 22680.

Figure 6 Serum TRC105 concentrations by dose level.

Serum concentrations (with standard deviation) of TRC105 are shown following doses at the 3, 10, and 15 mg/kg dose levels (A) and following multiple doses at the 10 mg/kg and 15 mg/kg dose levels given weekly (B).

Rosen, L. S., Hurwitz, H. I., Wong, M. K., Goldman, J., Mendelson, D. S., Figg, W. D.,... & Theuer, C. P. (2012). A phase I first-in-human study of TRC105 (Anti-Endoglin Antibody) in patients with advanced cancer. Clinical Cancer Research, 18(17), 4820-4829.

Figure 7 Antitumor activity.

Time course of plasma PSA in a patient with castrateresistant prostate cancer receiving ongoing treatment with TRC105 at 0.01 mg/kg (A) and Technetium-99 bone scans before and following normalization of PSA (B).

Rosen, L. S., Hurwitz, H. I., Wong, M. K., Goldman, J., Mendelson, D. S., Figg, W. D.,... & Theuer, C. P. (2012). A phase I first-in-human study of TRC105 (Anti-Endoglin Antibody) in patients with advanced cancer. Clinical Cancer Research, 18(17), 4820-4829.

Figure 8 Synthesis and characterization of TRC105(Fab).

(a) Schematic illustration showing the generation of TRC105(Fab) and its thiolation. (b) SDS-PAGE of molecular weight markers (lane 1), intact TRC105 antibody (lane 2), and TRC105(Fab) after purification on the Sephadex G-75 column (lane 3).

Chen, F., Nayak, T. R., Goel, S., Valdovinos, H. F., Hong, H., Theuer, C. P.,... & Cai, W. (2014). In vivo tumor vasculature targeted PET/NIRF imaging with TRC105 (Fab)-conjugated, dual-labeled mesoporous silica nanoparticles. Molecular pharmaceutics, 11(11), 4007-4014.

Figure 9 Flow cytometry analysis of MSN nanoconjugates in HUVECs (CD105 positive) after 30 min incubation and subsequent washing.

Targeted group: fluorescein conjugated MSN-800CWTRC105(Fab). Non-targeted group: fluorescein conjugated MSN-800CW. Blocking group: fluorescein conjugated MSN-800CWTRC105(Fab) with a blocking dose of TRC105 (500 μg/mL).

Chen, F., Nayak, T. R., Goel, S., Valdovinos, H. F., Hong, H., Theuer, C. P.,... & Cai, W. (2014). In vivo tumor vasculature targeted PET/NIRF imaging with TRC105 (Fab)-conjugated, dual-labeled mesoporous silica nanoparticles. Molecular pharmaceutics, 11(11), 4007-4014.

Figure 10 PET result.

Serial coronal PET images of 4T1 tumor-bearing mice at different time points postinjection of (a) 64Cu-MSN-800CW-TRC105(Fab), (b) 64Cu-MSN-800CW, and (c) 64Cu-MSN-800CW-TRC105(Fab) with a blocking dose of TRC105 (1 mg/mouse). Tumors are indicated by yellow arrowheads.

Chen, F., Nayak, T. R., Goel, S., Valdovinos, H. F., Hong, H., Theuer, C. P.,... & Cai, W. (2014). In vivo tumor vasculature targeted PET/NIRF imaging with TRC105 (Fab)-conjugated, dual-labeled mesoporous silica nanoparticles. Molecular pharmaceutics, 11(11), 4007-4014.

Figure 1 Anti-Human ENG Antibody (TAB-013ML) in WB

Western blot analysis of TAB-013ML was performed with Recombinant Human ENG Protein (lane 1, 0.1 μg; lane 2, 0.3 μg; lane 3, 0.6 μg) onto a 12% Tris-HCl polyacrylamide gel. Proteins were transferred to a CN membrane and blocked with 5% skim milk for at least one hour. Membranes were probed with TAB-013ML and HRP mouse Anti-His tag as a secondary antibody. Chemiluminescent detection was performed.

Figure 2 Anti-Human ENG Antibody (TAB-013ML) in ELISA

ELISA analysis of TAB-013ML was performed by coating with Recombinant Human ENG Protein. Then blocked with BSA, and incubated with Anti-Human ENG Antibody (TAB-013ML). The HRP-conjugated mouse anti-His tag as a secondary antibody. Detection was performed using TMB substrate and stopped with sulfuric acid. The absorbances were read on a spectrophotometer at 450 nm.

Figure 3 Anti-Human ENG Antibody (TAB-013ML) in DB

Antigen: Recombinant human ENG protein
Antibody incubation concentration: 2ng/μL.
The secondary antibody: HRP-conjugated mouse anti-His tag