NeuroMab™ Anti-HRG Beta 1 BBB Shuttle Antibody, Clone 7E3

Figure 1: Elisa assay of anti-NRG mAb (7E3) specificity.

ExtraCellular Domain of NRG alpha and beta (B) were incubated with purified murine anti-NRG mAb or 35A7 (anti-CEA antibody) or a commercial Ab. After washing, HRP-conjugated anti-mouse IgG was added. 7E3 and 18A10 anti-NRG mAb binds specifically to NRG1b ECD and doesn't cross-react with other ligands and EGF-domain of NRG beta and alpha.

Figure 2: Wound healing assay to study the effect of 7E3 anti-NRG antibody on cell migration.

After growing to confluence, BxPC-3 and MCF7 HER3-positive cells were starved and wounded with pipette tips. Cells were incubated with 25 (BxPC-3) or 12 ng/ml (MCF7) NRG1 and increasing doses of 7E3. 7E3 Ab at 10 and 100 microg/ml reduced repopulation of cleared areas more significantly than irrelevant Ab, even when cells were treated with NRG.

Figure 3: Cell viability assay to study the anti-proliferative efficacy of anti-NRG 7E3 Ab. irrelevant antibody

BxPC-3 and MCF7 cells transfected with the luciferase gene were grown in serum-free medium, and BXPC-3-luc and MCF7-luc were treated with 15 ng/ml or 10 ng/ml NRG1, respectively. Add 10, 50 and 100 μg/ml of 7E3 Ab for 5 days. Irrelevant Ab was added at a concentration of 100 µg/mL.

Figure 4: Western blot detection of the effect of 7E3 antibody on the HER signaling pathway.

BxPC-3, MCF7 and IGROV-1 with NRG1 (15 ng/ml for BxPC-3, 10 ng/ml for MCF7, 25 ng/ml for IGROV-1) and 7E3 or irrelevant antibody (10 or 100 micrograms/ml) 15 min for IGROV-1 and (6 to 100 μg/ml) for BxPC-3 and MCF7. 7E3 Ab induced repression of pHER3, pAKT, and pMAK, whereas no effect on pEGFR and HER2 was observed.

Figure 5: Proliferation assay of 7E3 Ab on NRG1 secreting BxPC-3 cells.

Cells were grown in 1% SVF and incubated with antibodies (10, 50 and 100 μg/ml) for 5 days. 7E3 Ab inhibits the activity of NRG1 expressing BxPC-3 and AsPC1-NRG. It was observed that the viability of MCF7 cells (not expressing NRG1) incubated with CM of BxPC-3 was inhibited.

Figure 6: Effect of 7E3 Ab on NRG non-secreting MCF7 cell line incubated with BxPC-3 conditioned medium.

MCF7 was incubated for 15 min with the supernatant of BxPC-3-secreting NRG1 grown in starvation medium for 48 h. 7E3 Ab was added at a concentration of 10 or 100 μg/ml for 15 minutes. Add irrelevant antibody at a concentration of 100 µg/mL. Inhibition of phosphorylation of HER3, AKT and MAPK was observed.

Figure 7: 7E3 is able to bind NRG already linked to HER3.

Biacore analysis: chip was coated with anti-Fc antibody, then HER3-Fc molecule (25 microg/ml) is injected in the flow and bind anti-Fc antibody. Then NRG (185 nM) is injected and finally 7E3 antibody (200 nM).

Figure 8: ADCC analysis.

Antibody-dependent cellular cytotoxicity (ADCC, Kit Promega LDH release) was studied on BxPC-3 incubated with human PBMC (1/15 ratio) and NRG1 mAb or an irrelevant mAb (10 mg/ml), with or without NRG1 . No ADCC was observed on BxPC-3 shHER3.

Figure 9: In vivo experiments.

BxPC-3 cells were xenografted into athymic nude mice and treated with anti-NRG1 mAb or irrelevant mAb (10 mg/kg, 2 times/week for 1 month) when tumor volume reached 150 mm Handle mice. Significant growth inhibition was observed in the group treated with anti-NRG1 mAb (p=0.064).

Figure 10: Cancer-associated fibroblasts and NRG1.

This conditioned medium is incubated on BxPC-3 and mAbs for 5 days. Cell proliferation is analyzed by SRB assay.