Figure 1.
The fine specificity of binding of anti-Lewisy and Lewisb mAbs.
The binding of 692/29 (A), 2-25 LE (B) and BR96 (C) to the Consortium for Functional Glycomics glycan array. Where a = Lewisa, b = Lewisb, y = Lewisy, x-Lewisx, D- = Di, T- = Tri, S- = sialyl, Ex- = extended, φ denotes a mannose containing glycan, and y×x = Lewisy-Lewisx.
Table 1.
Lewisy/b mAbs have differing fine specificity. 692/29, 2-25 LE and BR96 were screened on the Consortium for Functional Glycomics glycan array.
Figure 2.
Binding of anti-Lewisy and Lewisb mAbs to a panel of tumour cell lines.
A range of cancer cell lines were stained by indirect immunofluorescence with mAbs 692/29, BR96 and 2-25 LE and analysed by flow cytometry. An IgG isotype control was used as a negative control. This assay was repeated 6 times and one representative result is shown.
Figure 3.
ADCC and CDC assays with anti-Lewisy and Lewisb mAbs.
Anti-Lewisy and Lewisb mAbs, 692/29 2-25 LE and BR96 were screened by chromium release assay for ADCC with human PBMCs on Colo 205 (A), OVCAR-3 (B), OAW28 (C) and for CDC lysis with human serum (D). All data points have error bars, but may be obscured by marker. The assay was repeated 3 times and one representative result is shown.
Figure 4.
Direct killing assay with anti-Lewisy and Lewisb mAbs.
Anti-Lewisy and Lewisb mAbs, 692/29 2-25 LE and BR96 were screened for direct killing by uptake of PI and flow cytometric analysis. (A) A range of cell lines were incubated with 692/29, BR96, 2-25 LE or an isotype control overnight at 37°C. (B) C170 cells were incubated with 30 µg/ml 692/29, 692/42 and 2-25 LE either alone, or with rabbit anti-mouse or rabbit anti-mouse and goat anti-rabbit mAbs overnight at 37°C. (C) C170 cells were incubated with 30 µg/ml 692/29, 2-25 LE and BR96 with and without a pan caspase inhibitor overnight at 37°C. Jurkat cells were incubated with 500 µg/ml anti-Fas as a positive control. (D) C170 cells were incubated with 30 µg/ml mAb and 3, 400 and 500 K MW dextran-FITC beads or PI overnight at 37°C before measuring uptake using flow cytometry. Treatment with 0.4% saponin was used as a positive control for bead uptake. Anti-Fas treatment of Jurkat cells was used as a control for apoptosis (negative control for 500 kMW dextran uptake). This assay was repeated 3 times and one representative result is shown.
Figure 5.
Binding of 692/29, BR96 and 2-25 LE to normal tissues.
692/29, BR96 and 2-25 LE were assessed for normal tissue binding by IHC. Normal staining to placenta, oesophagus, rectum, gallbladder, bladder, ileum, spleen, jejunum, stomach, breast, kidney, thymus, lung, colon, tonsil, pancreas and duodenum are represented for 692/29 (A), BR96 (B) and 2-25 LE (C).
Table 2.
Binding of 692/29 to normal tissues compared to BR96 and 2-25 LE. No binding, weak, moderate or strong binding represented by 0, 1, 2, 3 respectively.
Table 3.
Binding of mAbs to a colorectal tumour microarray. Binding of mAbs to a colorectal tumour array was assessed. Levels of expression are shown as percentage of tumours bound.
Figure 6.
(A) The effect of 10 µg or 100 µg 692/29 mAb, or the vehicle control, on the final liver tumour weights (g) in the C170HM2 liver metastases nude mouse model. The median values for 10 µg and 100 µg of 692/29 mAb (Groups 2 and 3) were found to be significant (p<0.001) from vehicle control (Group 1). (B) The effect of 692/29 mAb, 5-FU/leucovorin or a combination of 692/29 mAb and 5-FU/leucovorin on the growth of C170 xenografts growing in nude mice. Growth of C170 xenografts was measured at days 12, 16, 19 and 23 by measurement of cross-sectional area (mm2) when animals were treated with either 692/29 mAb ip (0.2 mg), control antibody ip (0.2 mg) and 5-FU/leucovorin (12.5 mg/kg; iv). Analysis of variance of the results from day 23 showed the significant values of p<0.004 when comparing 692/29 mAb to the untreated control group and p<0.020 when comparing 692/29 mAb plus 5-FU/leucovorin to 5-FU/leucovorin with control antibody but due to the variability in tumour growth this did not reach significance when compared to 692/29 mAb alone. (C) Termination tumour weights of mice not treated (group 1), immunised with 692/29 mAb (group 2) alone, 5-FU/leucovorin alone (group 3) or the combination of 692/29 mAb and 5-FU/leucovorin (group 4). The combination of 692/29 mAb and 5-FU/leucovorin showed significantly lower tumour weights when compared to control (P>0.01) or to either 692/29 mAb or 5-FU/leucovorin on their own (p<0.05).