Human Anti-CA9 Recombinant Antibody (clone G37) (CAT#: HPAB-0784-CN)

Figure 1 Characterization of selected anti-CAIX scFvFc antibodies.

Flow cytometric analysis of the anti-CAIX scFvFc binding activity. One microgram (1 µg) of each anti-CAIX scFvFc was incubated with 5×105 sk-rc-52 (CAIX positive) or sk-rc-59 (CAIX negative) cells in 50 µl FACS buffer (1% BSA/PBS), followed by staining with PE-conjugated goat anti-human IgG. Binding activity of each antibody was examined by flow cytometric analysis and data is presented as overlapping histograms of the CAIX positive and negative cells as well as the secondary antibody only controls for each cell line. Similar findings were found in two independent experiments.

Xu, C., Lo, A., Yammanuru, A., Tallarico, A. S. C., Brady, K., Murakami, A., ... & Marasco, W. A. (2010). Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology. PloS one, 5(3), e9625.

Figure 2 Characterization of selected anti-CAIX scFvFc antibodies.

Dosage-dependent binding of the anti-CAIX scFvFcs to 293T-CAIX cells. Anti-CAIX scFvFcs or a control of irrelevant anti-CXCR4 scFvFc (X33) at the concentrations indicated on the X-axis were incubated with 5×105 293T-CAIX cells followed by staining with FITC conjugated goat anti-human IgG. Flow cytometric analysis was performed and binding activity of each antibody is presented as both percentage of positive cells (C) and absolute geometric mean fluorescence intensity (GMFI) (D).

Xu, C., Lo, A., Yammanuru, A., Tallarico, A. S. C., Brady, K., Murakami, A., ... & Marasco, W. A. (2010). Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology. PloS one, 5(3), e9625.

Figure 3 Binding specificity of the anti-CAIX antibodies as analyzed by domain mapping studies.

Binding of purified biotinylated CAIX-Fc constructs to the purified anti-CAIX scFvFcs analyzed by ELISA. Average OD450 of each duplicated sample is presented. Similar results were obtained in two independent experiments. Details on the ELISA assays performed are provided in Experimental Procedures.

Xu, C., Lo, A., Yammanuru, A., Tallarico, A. S. C., Brady, K., Murakami, A., ... & Marasco, W. A. (2010). Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology. PloS one, 5(3), e9625.

Figure 4 Inhibition of carbonic anhydrase activity of CAIX by the anti-CAIX scFvFc antibodies.

The ability of the anti-CAIX scFvFc antibodies to inhibit the reaction CO2+H2O → H2CO3 catalyzed by CAIX (CAIX) was assessed using the electrometric assay as described in Experimental Procedures. Different molar ratio of antibody to CAIX enzyme was tested. The anti-CXCR4 scFvFc X33 and carbonic anhydrase inhibitor acetazolamide (AZT) were used as negative and positive control, respectively. Percent inhibition of CAIX enzymatic activity was calculated per formula discussed in the Experimental Procedures. Inhibition of CAIX enzymatic activity by selected anti-CAIX-scFvFc at molar ratio 1∶1, 5∶1, and 25∶1.

Xu, C., Lo, A., Yammanuru, A., Tallarico, A. S. C., Brady, K., Murakami, A., ... & Marasco, W. A. (2010). Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology. PloS one, 5(3), e9625.

Figure 5 Antibody-induced CAIX internalization as analyzed by ImageStream.

Single cells expressing EEA1 and Lamp1 were gated (R2), and the CypHer5E intensity was plotted for cells incubated with anti-CAIX G37, 119, or 36 (left, center, and right plots), with the relative percentage of CypHer5E+ events (R3) displayed in the upper right of each plot. Representative Brightfield, EEA1 (green), LAMP1 (orange), and CypHer5E (red) cell images from the three antibody treatment groups are shown below in the same order.

Xu, C., Lo, A., Yammanuru, A., Tallarico, A. S. C., Brady, K., Murakami, A., ... & Marasco, W. A. (2010). Unique biological properties of catalytic domain directed human anti-CAIX antibodies discovered through phage-display technology. PloS one, 5(3), e9625.

Figure 6 ADCC activity of anti-CAIX scFv-Fc antibodies.

Anti-CAIX scFv-Fc antibodies G10, G36, G37, G39, and G119, were tested for ADCC activity against RCC cells with high CAIX expression. Human PBMC were isolated from healthy donors, and total PBMC counted as effector cells (E) in co-culture with the respective target RCC cell line (T). The concentration of antibodies was 5 μg/ml. Culture supernatant after four hours of co-culture was examined for LDH as a measure of cytotoxicity. Data represent the mean of three independent experimental values, ± S.E.M each experiment performed in triplicate.. * represents p value of two-way ANOVA < 0.05, compared to control antibody.

Chang, D. K., Moniz, R. J., Xu, Z., Sun, J., Signoretti, S., Zhu, Q., & Marasco, W. A. (2015). Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo. Molecular cancer, 14(1), 1-13.

Figure 7 Orthotopic RCC tumor growth following treatment with anti-CAIX mAbs.

Tumor growth was determined in each treatment group by BLI, following injection of antibodies and human PBMC as described in Fig. 4. Data represent measurement of three mice and are representative of two independent experiments. * indicates p value of two-way ANOVA < 0.05, compared to control IgG.

Chang, D. K., Moniz, R. J., Xu, Z., Sun, J., Signoretti, S., Zhu, Q., & Marasco, W. A. (2015). Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo. Molecular cancer, 14(1), 1-13.

Figure 8 Histological analysis of immune infiltrates in orthotopic RCC tumors.

Immunohistochemical detection of Ly6G+, human CD3+, or human PD-1+ cells in the tumor sections at day 14 (upper panels) and day 32 (lower panels) of indicated treatment group. Large panels represent a 10x magnification of the indicated treatment group (rows) for detection of each marker (columns), with the smaller inset of 2x magnification indicating the magnified area. In each magnification, "K" indicates the mouse kidney and "T" represents the orthotopic tumor. Bars scale, 150 μm and 750 μm for 10x and 2x magnification, respectively.

Chang, D. K., Moniz, R. J., Xu, Z., Sun, J., Signoretti, S., Zhu, Q., & Marasco, W. A. (2015). Human anti-CAIX antibodies mediate immune cell inhibition of renal cell carcinoma in vitro and in a humanized mouse model in vivo. Molecular cancer, 14(1), 1-13.