Afuco™ Anti-Human Lewis Y ADCC Recombinant Antibody (MB 311), ADCC Enhanced (CAT#: AFC-468CL)

Figure 1 Binding activity MB314 and MB311.

(A) Binding of MB311 and MB314 to the Lewis-Y positive tumor cell line SKBR-3 was determined by fl ow cytometry. Geometric Mean fl uorescence intensity (MFI) was plotted vs. the logarithm of the antibody concentration and fi tted using a sigmoidal four parameter fi t using GraphPad Prism 4 software for calculation of EC 50 values. Mean and SD of triplicates are shown. Binding of 100 μg/ml MB311 (dark gray line) or MB314 (black line) to purifi ed human NK-cells expressing the FcγRIII variant V/F (B), to the FcγRIII variant F/F (C) and the FcγRIII variant V/V (D). Results obtained with medium only are shown as light gray lines.

Kircheis, R., Halanek, N., Koller, I., Jost, W., Schuster, M., Gorr, G., ... & Nechansky, A. (2012, July). Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. In MAbs (Vol. 4, No. 4, pp. 532-541). Taylor & Francis.

Figure 2 MB314 and MB311 mediated effector functions.

(A and B) CDC reactivity against two Lewis Y positive tumor cell lines was tested using a FACS based approach. Tumor cells were incubated with diff erent concentrations of antibodies in the presence of human complement for 1 h. As complement source frozen human serum was used. Cytotoxicity against SKBR-3 cells (A) or OVCAR-3 cells (B) was quantifi ed by FACS TM analysis using a live/ dead cell stain. Gated 7-AAD + population (dead cells) as percentage of the whole population are plotted vs. the logarithm of the antibody concentration and fi tted using a sigmoidal four parameter fi t using GraphPad Prism 4 software. Mean and SD of triplicates are shown. (C and D) ADCC activity against two Lewis Y positive tumor cell lines was tested in a 51 Cr release cell lysis assay. 51 Cr labeled target cells (T) were incubated with eff ector cells (PBMCs from a healthy volunteer donor, E) at indicated E:T ratios for 16 h. Supernatants were counted for released 51 Cr using a γ-counter. Specifi c lysis (%) was plotted against the logarithm of the antibody concentration (ng/ml) and fitted using a sigmoidal four parameter fi t using GraphPad Prism 4 software.

Kircheis, R., Halanek, N., Koller, I., Jost, W., Schuster, M., Gorr, G., ... & Nechansky, A. (2012, July). Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. In MAbs (Vol. 4, No. 4, pp. 532-541). Taylor & Francis.

Figure 3 Competition of the ADCC reaction with excess human IgG.

̶⁵¹Cr-labeled SKBR-3 cells (T) were incubated with eff ector cells (E) at an E:T ratio of 30:1 for 16 h. Specifi c lysis (%) induced by 400 ng/ml of MB 314 or MB 311, respectively, in the presence or absence of excess of human IgG is shown. Horizontal line indicates lysis of target cells by eff ector cells without antibody.

Kircheis, R., Halanek, N., Koller, I., Jost, W., Schuster, M., Gorr, G., ... & Nechansky, A. (2012, July). Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314. In MAbs (Vol. 4, No. 4, pp. 532-541). Taylor & Francis.

Figure 4 Pharmacokinetics (Titer) and Pharmacodynamics (CDC and ADCC) of MB311.

Pharmacokinetics: Sera obtained from the patients at different time points after infusion of MB311 were incubated in 96-well plates coated with the anti-idiotypic antibody MMA383, followed by incubation with mouse anti-human IgG1 HRP conjugate. Color was developed using OPD as substrate. A standard curve of MB311 spiked into normal human serum was used for calibration. CDC: ̶⁵¹Cr-labelled SKBR3 cells were incubated for 60 minutes at 37˚C with serial dilutions of patient's sera, thereby using patient's complement. Released 51Cr in the supernatants was counted using a Packard γ-counter. The percentage of cytotoxicity was plotted against the logarithm of the antibody concentration (ng/ml). ADCC: ̶⁵¹Cr-labelled SKBR3 cells (target cells) were incubated for 2 hours with freshly prepared effector cells (PBMC from healthy volunteers) at a E:T ratio of 40:1, followed by addition of heat inactivated patient sera (diluted 1:20) and incubation for another 16 hours. Released 51Cr in the supernatants was counted using a Packard γ-counter. The percentage of cytotoxicity was plotted against the logarithm of the antibody concentration (ng/ml).

Bauernhofer, T., Samonigg, H., Regitnik, P., Weitzer, W., Lileg, B., Waxenecker, G., ... & Fido, M. (2014). Safety and therapeutic efficacy of the Lewis Y carbohydrate specific humanized antibody MB311 in patients with malignant effusion. J Cancer Ther, 5, 28-37.

Figure 5 Comparison of ADCC against LeY positive tumor cells in serum and effusion before treatment and at the end of study.

⁵¹Cr-labelled SKBR3 cells (target cells) were incubated for 2 hours with freshly prepared effector cells (PBMC from healthy volunteers) at a E:T ratio of 40:1, followed by addition of heat inactivated patient's serum or patient's effusion sample (diluted 1:20, respectively) and incubated for another 16 hours. Released ⁵¹Cr in the supernatants was counted using a Packard γ-counter. CDC activity in the serum compared to the CDC activity in the effusion samples before (white bars: serum day 1, black bars: effusion day 1) and after treatment (light gray bars: serum day 21, dark gray bars: effusion day 21) with MB311, respectively, is shown. No material from pt 5 was available.

Bauernhofer, T., Samonigg, H., Regitnik, P., Weitzer, W., Lileg, B., Waxenecker, G., ... & Fido, M. (2014). Safety and therapeutic efficacy of the Lewis Y carbohydrate specific humanized antibody MB311 in patients with malignant effusion. J Cancer Ther, 5, 28-37.

Figure 6 IHC stainings of cell blocks prepared from effusion aspirates of patient 4 before and after treatment with MB311.

Effusion specimens were embedded in agarose gel and subsequently formalin fixed and paraffin embedded. Serial 4 μm sections were cut, and incubated with antiEpCAM, anti-LeY, anti-calretinin, anti-CD45, anti-HER2/ neu and mouse-anti-human IgG mAbs followed by detection using a streptavidin biotin method on an autostainer (DAKO, Denmark). Hematoxilin Eosin Staining (magnification 100x) before (a) and after (b) treatment. LeY staining before ((c), magnification 100×) and after ((d), magnification 200×) treatment. CD45 staining before ((e), magnification 400×) and after ((f), magnification 200×) treatment. HER-2/neu staining (magnification 40×) before (g) and after (h) treatment.

Bauernhofer, T., Samonigg, H., Regitnik, P., Weitzer, W., Lileg, B., Waxenecker, G., ... & Fido, M. (2014). Safety and therapeutic efficacy of the Lewis Y carbohydrate specific humanized antibody MB311 in patients with malignant effusion. J Cancer Ther, 5, 28-37.