Bi-specific molecule against EGFR and death receptors simultaneously targets proliferation and death pathways in tumors

Abstract

Developing therapeutics that target multiple receptor signaling pathways in tumors is critical as therapies targeting single specific biomarker/pathway have shown limited efficacy in patients with cancer. In this study, we extensively characterized a bi-functional molecule comprising of epidermal growth factor receptor (EGFR) targeted nanobody (ENb) and death receptor (DR) targeted ligand TRAIL (ENb-TRAIL). We show that ENb-TRAIL has therapeutic efficacy in tumor cells from different cancer types which do not respond to either EGFR antagonist or DR agonist monotherapies. Utilizing pharmacological inhibition, genetic loss of function and FRET studies, we show that ENb-TRAIL blocks EGFR signalling via the binding of ENb to EGFR which in turn induces DR5 clustering at the plasma membrane and thereby primes tumor cells to caspase-mediated apoptosis. In vivo, using a clinically relevant orthotopic resection model of primary glioblastoma and engineered stem cells (SC) expressing ENb-TRAIL, we show that the treatment with synthetic extracellular matrix (sECM) encapsulated SC-ENb-TRAIL alleviates tumor burden and significantly increases survival. This study is the first to report novel mechanistic insights into simultaneous targeting of receptor-mediated proliferation and cell death signaling pathways in different tumor types and presents a promising approach for translation into the clinical setting.

Figure 1

ENb-TRAIL has superior efficacy in cancer cells resistant to EGFR-targeted therapy and DR agonist TRAIL in vitro. (A) Cell viability and caspase 3/7 activity of tumor cells from different cancer types in response to 24 h treatment with TRAIL (50 ng/ml) or ENb-TRAIL (50 ng/ml). (B) Cell viability of HT29, Calu1 and LN229 cells in response to 24 h treatment with Cetuximab, ENb, Erlotinib, TRAIL or ENb-TRAIL. (C) Western blot showing the induction of apoptosis in tumor cells 24 h-post treatment with ENb-TRAIL (50 ng/ml) as compared to ENb (100 nM) or TRAIL (50 ng/ml). (D) Western blot analysis of EGFR signaling in ENb or ENb-TRAIL treated LN229 cells. (E) Western blot analysis showing the induction of apoptosis in ENb-TRAIL vesus the combination of ENb and TRAIL treated tumor cells (post 24 h treatment). *P < 0.05, **P < 0.005 and ***P = 0.0001 determined by unpaired t test. Error bars indicate SD. Western blots were cropped to show specific bands only. For uncropped blots see Fig. S11.

Figure 2

ENb-binding to EGFR is critical for ENb-TRAIL activation of apoptosis. (A) Differential cell membrane EGFR, DR4, and DR5 expression levels in LN229, HT29 and Calu1 cells measured by Flow Cytometry. Left panel: cell membrane EGFR expression. Right panel: cell membrane DR4 and DR5 expression. (B–C) Cells were pretreated with Cetuximab for 30 min and then treated with ENb-TRAIL for 8 h and apoptosis markers were analyzed by Western blotting (B) and caspase 3/7 assay (C). *P < 0.05 determined by unpaired t test. Error bars indicate SD. (D) Co-immunoprecipitation and Western blot analysis showing EGFR and DR5 complex formation in the presence of ENb-TRAIL and the attenuation of complex by Cetuximab. Western blots were cropped to show specific bands only. For uncropped blots see Fig. S12.

Figure 3

TRAIL receptor DR5 plays a major role in ENb-TRAIL-induced apoptosis. (A) Schematic showing DR4/5-CFP and EGFR-YFP fusion protein constructs. (B) Western blot analysis of DR5-CFP/YFP and EGFR-CFP/YFP expression in 293 T cells. (C) Changes in FRET signal between DR4/5-CFP and EGFR-YFP post treatment with ENb-TRAIL in 293 T cells. The left panels show frames from time lapse collection of DR5-CFP or DR4-CFP + EGFR-YFP signals and calculated N-FRET index (Xia); index is not calculated for overexposed signal. Right panel: The quantification of N-FRET index over 1 h period. Cells were treated with ENb-TRAIL at time 0 min. (D) Flow cytometry analysis of membrane DR5 or DR4 level in knocked down (DR5 KD, DR4 KD) HT29 cells. (E) Caspase 3/7 activity assay and (F) cell viability assay analysis of DR5 or DR4 KD effect on ENb-TRAIL induced apoptosis. *P < 0.05, **P < 0.005 and ***P = 0.0001 determined by unpaired t test. Error bars indicate SD. Western blots were cropped to show specific bands only. For uncropped blots see Fig. S12.

Figure 4

Therapeutic stem cell delivered ENb-TRAIL is effective in vitro and in vivo. (A) Plot showing the response of human MSC to different concentrations of ENb-TRAIL. (B) Tumor cells were engineered with FmC and tumor cell viability was visualized by Fluc bioluminescence imaging 48 h post co-culture with MSC-GFP or MSC-ENb-TRAIL-IRES-GFP. Left panel: high magnification fluorescence images of co-culture; Right panel: Fluc bioluminescence images of co-culture and the quantification of Fluc signals. *P < 0.001 determined by unpaired t test. Error bars indicate SD. (C–E) Mice were subcutaneously implanted with HT29-FmC cells (C), Calu1-FmC cells (D) or LN229-FmC (E) cells and ad-mixed with MSC (GFP or ENb-TRAIL-IRES-GFP) and the fate of tumor cells was followed by Fluc bioluminescence imaging. Representative Fluc bioluminescence images and quantitation is shown as fold of 0 h post implantation. Data are mean +/−SD.

Figure 5

MSC delivered ENb-TRAIL has anti-tumor effects in mouse tumor model of GBM resection. (A) Photomicrographs from mice brains on Day 3 post-implantation of GBM31R-FmC (red) and engineered MSC (green). (B) Plot showing changes in tumor volumes of GBM31R-FmC cells as compared to Day 0 following treatment with MSC-GFP or MSC-ENb-TRAIL-IRES-GFP (C) H&E stain from mouse brain sections obtained 21 days post MSC administration. (D) Plot showing changes in cleaved Caspase 3 expression on brain sections harvested 3 days post administration of MSC-GFP or MSC-ENb-TRAIL (inset) representative photomicrographs revealing expression of Cl. Caspase 3 (E) Top: Timeline of GBM in vivo experiment and MSC-ENb-TRAIL efficacy in the GBM31R resection mouse model. Bottom left: Light and fluorescence photomicrographs of an intracranial implanted GBM31R-FmC tumor in a cranial window prior to resection; Bottom right: Light and merged fluorescence photomicrograph of the tumor resection cavity (red; remaining GBM31R-FmC tumor cells) before and after implantation of sECM encapsulated MSC-GFP cells (green). (F) Analysis of pre- and post-resection Fluc intensity (normalized to pre-resection). (G) Plot demonstrating the GBM31R-FmC tumor growth post-resection in different treatment groups over time (Control vs. ENb-TRAIL at day 27, p = 0.066). (H) Kaplan-Meier survival curve analysis of different treatment groups (SC-ENb-TRAIL vs. SC-GFP p < 0.0001; SC-ENb-TRAIL vs. SC-ENb p < 0.0019). *P < 0.05, **P < 0.005 and ***P < 0.0001, determined by unpaired t test.

Figure 6

Model of ENb-TRAIL function. EGFR and DR5 are co-engaged at the surface of tumor cells. Trimerized ENb-TRAIL binds simultaneously to EGFR and DR5 leading to receptor clustering and amplification of apoptotic signal. Blocking EGFR with Cetuximab or ENb abolishes ENb-TRAIL binding to EGFR and subsequent activation of apoptosis. EGFR tyrosine kinase inhibitor, Erlotinib inhibits EGFR and AKT activity but does not influence activation of apoptosis by ENb-TRAIL.