Mouse Anti-Tn Antigen Recombinant Antibody (clone MLS128) (CAT#: HPAB-0319CQ)

Figure 1 Specificity of VVL and antibodies on large trimer amino acid glycopeptide library, printed on microarray slides.

The library was divided into five groups according to the position of glycosylated (GalNAc) serine (S*) and threonine (T*) in relation to other amino acids (X). VVL, scFv sH1 and G2-D11, mouse monoclonal GOD3-2C4 and MLS128 antibodies were tested in all combinations. Each dot represents a unique sub-library member. Median signals for each group of combinations are marked with a blue line and a red circle is highlighting amino acids (X) affecting binding of the reagents. (A) Group 1 (XT*T*). (B) Group 2 (T*T*X). (C) Group 3 (XT*S*). (D) Group 4 (S*T*X). (E) Group 5a (GT*X3, PT*X3, WT*X3, IT*X3, LT*X3, MT*X3, FT*X3, YT*X3 and VT*X3), where G2-D11 bound to FT*R, WT*K, YT*W and YT*A, and GOD3-2C4 bound to VT*N, VT*M, VT*W, WT*C, WT*D, WT*E, WT*I, WT*M, WT*N, YT*F, YT*M and YT*R. (F) Group 5b (RT*X3, HT*X3, KT*X3, DT*X3, ET*X3, ST*X3, TT*X3, NT*X3 and QT*X3), where GOD3-2C4 bound to HT*R. This figure is available in black and white in print and in colour at Glycobiology online.

Persson, N., Stuhr-Hansen, N., Risinger, C., Mereiter, S., Polónia, A., Polom, K., ... & Danielsson, L. (2017). Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells. Glycobiology, 27(7), 635-645.

Figure 2 Specificity evaluation of Tn-binding antibodies on glycopeptide microarray.

Glycopeptides were printed on a microarray chip. MLS128 were titrated from 20 μg/mL in a series of 10, 5, 1, 0.5, 0.1 and 0.05 μg/mL.

Persson, N., Stuhr-Hansen, N., Risinger, C., Mereiter, S., Polónia, A., Polom, K., ... & Danielsson, L. (2017). Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells. Glycobiology, 27(7), 635-645.

Figure 3 Immunohistochemical staining for Tn antigen in well-differentiated squamous cell carcinoma of the esophagus.

MLS128 was detected in carcinomas with cancer pearls (keratinization). (Immunoperoxidase and methyl green x 460)

Ohshio, G., Imamura, T., Imamura, M., Yamabe, H., Sakahara, H., Nakada, H., & Yamashina, I. (1995). Distribution of Tn antigen recognized by an anti-Tn monoclonal antibody (MLS128) in normal and malignant tissues of the digestive tract. Journal of cancer research and clinical oncology, 121(4), 247-252.

Figure 4 Effects of MLS128 on human colon and breast cancer growth.

Time course of LS 180 cancer cell growth (A) without (PBS: ●) or with 25 μg/mL of control IgG3 (▲) or MLS128 (■). HT-29 (B) and MCF-7 (C) cancer cell growth without (PBS: ●) or with 25 μg/mL of MLS128 (■).

Morita, N., Yajima, Y., Asanuma, H., Nakada, H., & Fujita-Yamaguchi, Y. (2009). Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Bioscience trends, 3(1).

Figure 5 Binding of MLS128 to cancer cells or solubilized glycoproteins. MLS128 binding to LS180 (A), HT-29 (B), and MCF-7 (C) cancer cells was measured by FACS.

Morita, N., Yajima, Y., Asanuma, H., Nakada, H., & Fujita-Yamaguchi, Y. (2009). Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Bioscience trends, 3(1).

Figure 6 Solubilized plasma membranes prepared from various cancer cell lines, i.e. LS180 (lane 1), HT-29 (lane 2), MCF-7 (lane 3), MCF-10A (lane 4), and MDA-MB-231 (lane 5), were subjected to SDS-PAGE and immunoblotting with MLS128.

Molecular markers and the 110 kDa GP are indicated. The 125, 75, and 73 kDa proteins marked with a triangle (◄) are endogenous biotin-containing enzymes.

Morita, N., Yajima, Y., Asanuma, H., Nakada, H., & Fujita-Yamaguchi, Y. (2009). Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Bioscience trends, 3(1).

Figure 7 No cross-reactivity of MLS128 to IGFIR.

LS180 cell lysates and purified human placental IGFIR were compared in triplicate by immunoblotting with MLS128 (A), anti-IGFIR β subunit polyclonal antibody (B), and IGFIR-crossreactive anti-IR polyclonal antibody (C). The positions for IGFIR α and β subunits as well as 110-kDa MLS128-reactive GP are indicated with arrows.

Morita, N., Yajima, Y., Asanuma, H., Nakada, H., & Fujita-Yamaguchi, Y. (2009). Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Bioscience trends, 3(1).

Figure 8 Down-regulation of IGFIR and EGFR in LS180 cells after MLS128 treatment.

LS180 cells were treated with MLS128 (25 μg/mL) for the indicated time. Cell lysates were prepared from live cells and subjected to SDS-PAGE followed by immunoblotting with anti-IGFIR β subunit antibody and anti-EGFR (A). The 125, 75, and 73 kDa proteins marked with a triangle (◄) are endogenous biotin-containing enzymes (14). The amounts of IGFIR β subunits (B) and EGFR (C) after PBS (□) or MLS128 (■) treatment were estimated by densitometric analysis using the 75 and 73 kDa biotin-containing enzymes (◄) as internal controls.

Morita, N., Yajima, Y., Asanuma, H., Nakada, H., & Fujita-Yamaguchi, Y. (2009). Inhibition of cancer cell growth by anti-Tn monoclonal antibody MLS128. Bioscience trends, 3(1).

Figure 9 Addition of MLS128 or 1H7 inhibits growth of three colon cancer cell lines.

Inhibition of growth of three colon cancer cell lines by MLS128 (grey bars) along with anti-IGF-IR 1H7 (black bars) was compared to the control (white bars). Typical results for growth of LS180, LS174T, and HT29 colon cancer cells after 3 days of mAb treatment are shown in A, B, and C, respectively. The relative growth (% control), average ± S.E. from 3 or 4 experiments for each cancer cell line, is summarized in D.

Zamri, N., Masuda, N., Oura, F., Yajima, Y., Nakada, H., & Fujita-Yamaguchi, Y. (2012). Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells. Bioscience trends, 6(6), 303-312.

Figure 10 Effects of 1H7 or MLS128 treatment on cellular levels of IGF-IR (left panel) and 110 kDa GP (right panel).

LS180, LS174T, and HT29 colon cancer cells were cultured in respective media containing 1% FBS in the presence or absence of MLS128 (25 μg/mL) or 1H7 (0.36 μg/mL) for 24 (Day 1), 48 (Day 2) and 72 h (Day 3). Cell lysates (2 μg protein per lane) were subjected to Western blotting using primary antibodies, anti-IGF-IR β subunit, MLS128, and anti-β-actin and then subjected to color development using biotin-labeled secondary antibodies and the alkaline phosphatase kit. Shown are six panels (A~F), each of which consists of a typical immunoblot from 3-4 experiments (upper panel) and average values of IGF-IR or 110 kDa GP levels of 3 or 4 immunoblots (lower panel). The IGF-IR or 110 kDa levels were normalized to β-actin leveles. White bars represent controls, which are IGF-IR or 110 kDa GP levels from cells untreated with mAb. The grey and black bars represent IGF-IR levels (left panel) and 110 kDa GP levels (right panel) in cells treated with MLS128 and 1H7, respectively. Average values of these protein levels on Days 1, 2, and 3 were calculated with respect to Day 0. Average ± S.D. (n = 3-4) of IGF-IR (left panel) and 110 kDa GP (right panel) are shown for LS180 (A and B, respectively), LS174T (C and D, respectively), and HT29 (E and F, respectively). * p < 0.05; ** p < 0.01.

Zamri, N., Masuda, N., Oura, F., Yajima, Y., Nakada, H., & Fujita-Yamaguchi, Y. (2012). Effects of two monoclonal antibodies, MLS128 against Tn-antigen and 1H7 against insulin-like growth factor-I receptor, on the growth of colon cancer cells. Bioscience trends, 6(6), 303-312.