Afuco™ Anti-Human Integrin αvβ6 ADCC Recombinant Antibody (264RAD), ADCC Enhanced (CAT#: AFC-431CL)

Figure 1 is a line graph showing the ability of the purified monoclonal antibodies to bind to αVβ6 and block its binding to a GST-LAP peptide.

Figure 2 shows affinity data for mAb 264 RAD.

Figure 3 is bar graph showing the ability of the purified monoclonal antibody to mediate complement-dependent cytotoxicity in 293 cells stably expressing αVβ6 integrin.

Figure 4 is a bar graph showing the ability of antibodies 264RAD, 133 and 188 SDM to inhibit tumour growth using the Detroit-562 nasophayngeal cell line.

Figure 5 is a bar chart showing comparison of the activity of 264 RAD with 264 RAD/ADY.

Figure 6 The effect of 264RAD in combination with trastuzumab on human breast cancer xenograft growth in SCID mice.

A) Mice bearing human BT-474 tumors were treated with IgG (square, solid line), 264RAD (triangle, dashed line, in line with TRA treatment), trastuzumab (TRA) (square, dashed line), or 264RAD+TRA (triangle on lower dashed line) (10 mg/kg; ip) twice weekly for two consecutive weeks (start of treatment indicated by arrow, day 0). Data are presented as mean tumor volume (error bars represent 95% confidence interval, n ≥ 4 mice/group). Treatment commenced when tumors reached 100 mm 3. B) Mice bearing human HER2-18 tumors were treated as in (A). (C) Photographic images of representative BT-474 and HER2-18 xenografts posttreatment outlined in (A). Magnification bar = 5 mm. D) BT-474 xenograft protein expression. Xenografts were treated as in (A), harvested, protein extracted, and subjected to immunoblotting. Blots were probed for indicated proteins. E) Histograms of relative protein expression from blots shown in (D) determined by optical density (n = 3 individual tumors, error bars represent 95% confidence interval). *P = .05, **P = .01, ***P < .001 (relative to IgG or to treatment indicated by corresponding lines to the side of growth curves and above histograms, as determined by plotting individual growth curves and then applying a linear mixed model to test for differences between treatments [A and B], and two-sided, one-way analysis of variance with Bonferroni's Multiple Comparison Test [E and G]). F and G) HER2-18 xenograft protein expression and quantification as outlined in (D and E). HER2 = Human Epidermal Growth Factor Receptor 2; IgG = immunoglobulin; TRA = trastuzumab.

Moore, K. M., Thomas, G. J., Duffy, S. W., Warwick, J., Gabe, R., Chou, P., ... & Saha, A. (2014). Therapeutic targeting of integrin αvβ6 in breast cancer. JNCI: Journal of the National Cancer Institute, 106(8).

Figure 7 The effect of 264RAD in combination with trastuzumab on human xenograft BT-474 tumor growth and stroma.

A) Micrographs of BT-474 tumor xenografts from mice treated with IgG or 264RAD+TRA (10 mg/kg; ip) twice weekly for two consecutive weeks (tumors harvested after two weeks of treatment from Figure 4A). Tumors were harvested, fixed, parafin embedded, and sections subjected to immunohistochemical staining for the indicated molecules of interest, including pancytokeratin (CK, epithelial marker), Ki67 (proliferation), endomucin (vasculature), α-sma (myofibroblast), cleaved caspase 3 (apoptosis), as well as αvβ6 and HER2 expression. Representative images are shown of the three tumors harvested for IgG and the combination treatment, where the greatest effect was observed. Scale bar in whole tumor images = 1000 μM, magnified images (x60) are of indicated region of interest (CK+ cells). Scale bar in x60 magnification images = 20 μM. B) Bar graph of composition of xenografts (% CK+ cells = white bar, % necrotic area in black, and % stroma is in gray or blue) and histograms of specific marker expression of xenografts shown in (A). Assessed and scored by two individuals (n = 3 individual tumors, error bars represent 95% confidence interval). *P = .05, **P = .01, ***P < .001 (relative to IgG, as determined by two-sided, one-way analysis of variance with Bonferroni Multiple Comparison Test [B, upper panel] and two-sided Student's t-test [B, lower panel]). CK = pancytokeratin; HER2 = Human Epidermal Growth Factor Receptor 2; IgG = immunoglobulin; TRA = trastuzumab.

Moore, K. M., Thomas, G. J., Duffy, S. W., Warwick, J., Gabe, R., Chou, P., ... & Saha, A. (2014). Therapeutic targeting of integrin αvβ6 in breast cancer. JNCI: Journal of the National Cancer Institute, 106(8).

Figure 8 The effect of 264RAD in combination with trastuzumab on human xenograft MCF-7/HER2-18 tumor growth and stroma.

A) Micrographs of MCF-7/HER2-18 tumor xenografts from mice treated with IgG, or 264RAD+TRA (10 mg/kg; ip) twice weekly for two consecutive weeks (tumors harvested after two weeks treatment from Figure 4B). Tumors were harvested, fixed, parafin embedded, and sections subjected to immunohistochemical staining for the indicated molecules of interest including cytokeratin (CK, epithelial marker), Ki67 (proliferation), endomucin (vasculature), α-sma (myofibroblasts), as well as αvβ6 and HER2 expression. Representative images are shown of the three tumors harvested for IgG and the combination treatment, where the greatest effect was observed. Scale bar in whole tumor images = 2000 μM, magnified images (x60) are of indicated region of interest (CK+ cells). Scale bar in x60 magnification images = 20 μM. B) Bar graph of composition of xenografts (% CK+ cells = white bar, % necrotic area in black, and % stroma is in gray or blue) and histograms of specific marker expression of xenografts shown in (A). Assessed and scored by two individuals (n = 3 individual tumors, error bars represent 95% confidence interval). *P = .05, **P = .01, ***P < .001 (relative to IgG, as determined by two-sided, one-way analysis of variance with Bonferroni Multiple Comparison Test [B, upper panel] and two-sided Student's t-test [B, lower panel]). CK = pancytokeratin; HER2 = Human Epidermal Growth Factor Receptor 2; IgG = immunoglobulin; TRA = trastuzumab.

Moore, K. M., Thomas, G. J., Duffy, S. W., Warwick, J., Gabe, R., Chou, P., ... & Saha, A. (2014). Therapeutic targeting of integrin αvβ6 in breast cancer. JNCI: Journal of the National Cancer Institute, 106(8).

Figure 9 The effect of long-term (six-week) treatment of 264RAD in combination with trastuzumab on human xenograft MCF-7/HER2-18 cell growth in SCID mice.

Mice bearing human MCF-7/HER2-18 tumors were treated with IgG (square, solid line), 264RAD (triangle, dashed line, in line with TRA), trastuzumab (TRA) (square, dashed line), or 264RAD+TRA (triangle on lower dashed line) (10 mg/kg; ip) twice weekly for six consecutive weeks. Data are presented as mean tumor volume (error bars represent 95% confidence interval, n> 5 mice/ group). Treatment commenced (indicated by arrows) when tumors were 4 mm in any one dimension (A), and when tumors reached 200 mm 3 (n> 6 mice/group) (B). C) Kaplan-Meier survival plot shows survival of mice from study of larger tumors shown in (B). D) Tumors from treated mice in (A) were analyzed by immunoblotting for indicated targets.

Moore, K. M., Thomas, G. J., Duffy, S. W., Warwick, J., Gabe, R., Chou, P., ... & Saha, A. (2014). Therapeutic targeting of integrin αvβ6 in breast cancer. JNCI: Journal of the National Cancer Institute, 106(8).