NeuroMab™ Anti-P2X7 BBB Shuttle Antibody(NRZP-1022-ZP3305)

Figure 1. In vitro cell inhibition assay.

The IgM form of the original antibody to the trimer form of the non-functional P2X7 receptor expressed on cancer cells was found to inhibit cell growth using the Cell Titer Blue Assay. An example is shown in which the control IgM antibody is seen to have no effect on cell growth (left columns) for increasing concentrations from 2.5 to 40 ug/rriL while the 2F6 inhibited cell growth (right columns) over the same dose range in a 3 day growth assay.

Figure 2. In vitro cell inhibition assay.

The IgM form of the original antibody to the trimer form of the non-functional P2X7 receptor expressed on cancer cells was found to inhibit cell growth using the Cell Titer Blue Assay. An example is shown in which the control IgM antibody is seen to have no effect on cell growth (left columns) for increasing concentrations from 2.5 to 40 ug/rriL while the 2F6 inhibited cell growth (right columns) over the same dose range in a 3 day growth assay.

Figure 3. In vitro cell inhibition assay.

The IgM form of the original antibody to the trimer form of the non-functional P2X7 receptor expressed on cancer cells was found to inhibit cell growth using the Cell Titer Blue Assay. An example is shown in which the control IgM antibody is seen to have no effect on cell growth (left columns) for increasing concentrations from 2.5 to 40 ug/rriL while the 2F6 inhibited cell growth (right columns) over the same dose range in a 3 day growth assay.

Figure 4. In vitro cell inhibition assay.

The IgM form of the original antibody to the trimer form of the non-functional P2X7 receptor expressed on cancer cells was found to inhibit cell growth using the Cell Titer Blue Assay. An example is shown in which the control IgM antibody is seen to have no effect on cell growth (left columns) for increasing concentrations from 2.5 to 40 ug/rriL while the 2F6 inhibited cell growth (right columns) over the same dose range in a 3 day growth assay.

Figure 5. Blocking response in cell killing assay.

The Cell Titer Blue cell growth inhibition assay is used over three day cell growth With MCF-7 breast cancer cell line.

Figure 6. Mechanism of 2F6-induced cell death, caspase 3/7 activation associated with reactivation of apoptosis.

In this experiment the effect of the Gemcitibine control drug is shown at the left, known to activate caspases through induction of apoptosis in C0L0205 cancer cells. In contrast, the absence of drug or antibody has no effect (cells only column). The presence of control IgM at doses up to 40 ug/mL similarly has no effect on caspase activation while increasing amounts of 2F6 antibody shows a steady increase in Caspase 3f7 activation associated with apoptosis induction by the antibody over the 3 day time course of the experiment.

Figure 7. Inhibition of 2F6hIgG1 on the number of lung metastases at day 14 in the 4T1 syngeneic xenograft model.

Figure 8. The number of lung metastases was suppressed in the Lewis lung (LL) syngeneic xenograft model by day 11.

The five groups are the untreated control (Group 1), sheep polyclonal E200-300 at 10 mg/kg (Group 2), 2F6hIgG1 at 1 mg/kg (Group 3) and at 10 mg/kg (Group 4) and Sorafenib at 5 mUkg daily (Group 5). Both sheep polyclonal and 2F6 hIgG1 were equipotent with Sorafenib with 96% inhibition.

Figure 9. ELISA for IgM, IgG2a and Fab Leads. Lead Affinity Matured 2F6-Derived Fab ELISA (ratio 0.01-12.5ug/mL for IgM and IgG2a; 0.1-100 ug/mL for Fab).

Figure 10. Flow cytometry results of binding of recombinant Fabs to live colorectal C0L0205 tumor cells.

Figure 11. Comparison with various preparations of recombinant 2F6 IgG2a to determine the relative binding strength of the WT form of the antibody to PC3 cells compared to the affinity matured Fab.