NeuroMab™ Anti-EGFR BBB Shuttle Antibody(NRZP-1022-ZP4062)

Figure 1 Binding of Nanobody 7D12 to EGFR.

Binding assay of unlabeled 7D12 (blue) and [ 13 C, 15 N]-labeled 7D12 (red) to A431 cells.

Figure 2 Binding of Nanobody 7D12 to EGFR.

Confocal microscope images of A431-derived spheres after 4 h

Figure 3 Binding activity of VHH-7D12 variants to EGFR using SPR

Figure 4 SDS-PAGE analysis of 7D12 in Escherichia coli.

Figure 5 IgG and EGFR binding assay.

(A) ELISA assessment of the binding affinity of 7D12, ZZ, 7D12-ZZ, and ZZ-7D12 to IgG from mouse, rabbit, and human. (B) Competitive ELISA assessment of the IC50 of 7D12, 7D12-ZZ and ZZ-7D12 against EGFR. Error bars represent the standard deviation of three parallel experiments. ****: P < 0.0001.

Figure 6 Cell binding and IgG recruitment assays.

Immunofluorescent images of cells treated with Nanobody 7D12, ZZ domain, UEAR Nbs 7D12-ZZ or ZZ-7D12 (scale bar ¼ 20 mm).

Figure 7 Flow cytometry analysis of cells treated with 7D12, ZZ domain, UEAR Nbs 7D12-ZZ or ZZ-7D12. Error bars show SD of three parallel experiments.

Figure 8 Corresponding MFI of cells treated with 7D12, ZZ domain, UEAR Nbs 7D12-ZZ or ZZ-7D12. Error bars show SD of three parallel experiments.

Figure 9 In vitro cytotoxicity test.

(A) ADCC was mediated by different concentrations of 7D12, 7D12-ZZ or ZZ-7D12. (B) CDC was mediated by 50 nmol L 1 of 7D12, 7D12-ZZ or ZZ-7D12 in the presence of RC or HIRC. Error bars represent the standard deviation of three parallel experiments. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 10 In vitro ADCP assay.

(A) Flow cytometry analysis and (B) corresponding phagocytosis of target cells treated with PBS, 7D12, or UEAR Nbs 7D12-ZZ or ZZ-7D12. Error bars show SD of three parallel experiments. ***: P < 0.001.

Figure 11 Plasma concentration and half-life of 7D12-ZZ and ZZ-7D12 in Balb/c mice.

Blood samples collected from the same group of mice at the same time point were pooled and analyzed using ELISA. Error bars represent the standard deviation of three parallel experiments.

Figure 12 Evaluation of in vivo anti-tumor efficacy of UEAR Nbs.

Tumor volume of mice treated with PBS, Nanobody 7D12, 7D12-ZZ or ZZ-7D12. Each arrow represents a treatment. Error bars represent SEM (n = 5).

Figure 13 Evaluation of in vivo antitumor efficacy of UEAR Nbs.

Tumors excised from treated mice at the end of the experiment.

Figure 14 Evaluation of the in vivo antitumor efficacy of UEAR Nbs.

Tumor weight of treated mice at the end point of the experiment.

Figure 15 Evaluation of in vivo anti-tumor efficacy of UEAR Nbs.

Tumor Growth Inhibition (TGI) of Treated Mice at Experimental Endpoints. Tumor growth inhibition was calculated based on tumor weight. Error bars represent SEM (n = 5). **: P < 0.01.