Effects of a novel Nodal-targeting monoclonal antibody in melanoma

Abstract

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Keywords: ELISA; Nodal; antibody; cancer; therapy.

Figure 1. Nodal expression in normal human tissue lysates

Commercially available Western blot grade normal human tissue lysates were analyzed for Nodal expression. Lysates from H9 hESCs were used as positive control for Nodal in the first lane. Nodal is not detected in lysates from normal human brain, kidney, liver, pancreas and heart. Low expression was detected in normal skeletal muscle sample 1, but no expression was detected in normal skeletal muscle sample 2. Nodal is highly expressed in C8161 human metastatic melanoma cells.

Figure 2. Characteristics of anti-Nodal 3D1 mAb

A. ELISA-based binding assay of 3D1 mAb to coated hNodal [-67] and hNodal [-67] E49A-E50A. Peptides were coated at 0.18 μg/mL (60 nM). mAb 3D1 was tested at increasing concentrations between 1.0 and 67 nM. B. Overlay plot of SPR sensorgrams showing the interaction between 3D1 mAb and rhNodal immobilized on a CM5 sensor chip. The interaction was monitored at concentrations of mAb ranging between 6.0 and 100 nM, obtaining dose-dependent binding curves. rhNodal was immobilized on a Biacore CM5 sensor chip and 3D1 mAb solutions at increasing concentrations were injected over the chip. C. Inhibition of the rhNodal/rhCripto-1 complex by SPR concentration-dependent competition assay. A plot of %binding versus increasing antibody concentrations is reported. rhNodal was used at the fixed concentration of 5.0 nM whereas 3D1 was used at 1:0.5, 1;1 and 1:2 molar ratio.

Figure 3. In vitro effects of anti-Nodal 3D1 mAb

Results from anchorage independent growth assays A. show a significant reduction in anchorage independent growth of C8161 cells treated with 3D1 mAb compared to control cells. B. Results from vasculogenic network formation assay show a significant reduction in the ability to form junctions and tubules in C8161 cells treated with 3D1 mAb compared to control cells. (*P < 0.05.). Histograms represent mean values +/− SEM.

Figure 4. Effects of anti-Nodal 3D1 antibody on cell signaling and cell cycle related molecules

A. Levels of P-Smad2 and P-ERK1/2 are reduced within 4hr of 3D1 mAb treatment (4 μg/ml) in C8161 human melanoma cells compared to IgG treated control. B. After 72 hr of 3D1 mAb treatment there is a reduction of Nodal, Cyclin B1 and P-H3 with a concomitant increase in p27 in C8161 cells compared to IgG treated control.

Figure 5. In vivo effects of anti-Nodal 3D1 mAb on C8161 human melanoma cells

A. Significantly reduced tumor volumes are observed in C8161 Nude mice orthotopic xenografts treated with direct tumor injections of 3D1 mAb versus control IgG. Histograms represent mean values +/− SD. Representative IHC staining in B. shows reduced nuclear expression (activation) of Smad2 in C8161 orthotopic xenograft Nude mouse treated with 3D1 mAb compared to IgG control (20X original magnification). The potential for lung colonization (shown microscopically with H&E staining) of C8161 cells (after systemic introduction) in Nude mice C. (40X original magnification) is significantly reduced in animals treated with IP administration of 3D1 mAb vs IgG control D. Histograms represent mean values +/− SEM. Representative IHC staining in E. shows reduced nuclear expression (activation) of P-Smad2 in C8161 lung colony of a 3D1 mAb treated Nude mouse versus IgG control (63X original magnification). (*P < 0.05).

Figure 6. In vivo effects of anti-Nodal 3D1 mAb on A375SM human melanoma cells

A. After 8 days, mean tumor volume of A375SM orthotopic Nude mice xenografts was significantly smaller in 3D1 mAb treated than in IgG control treated animals. Tumor volumes in dabrafenib (BRAFi) treated animals also showed a trend towards reduced tumor volumes compared to control. Histograms represent mean values +/− SD. B. Representative IHC of P-Smad2 showing nuclear staining in A375SM orthotopic xenografts in Nude mice treated with control IgG, 3D1 mAb or BRAFi (*P < 0.05).

Figure 7. Sandwich ELISA assay developed to detect Nodal in the serum of breast cancer patients

A. Illustrates a typical calibration curve using 3D1 mAb as the capture antibody for detecting recombinant Nodal; while B. depicts Nodal detected in patient's serum and with a trend for higher Nodal levels in the samples from patients with invasive compared to noninvasive breast cancer. (Dashed line = median level).