VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo

Abstract

Objective: We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients.

Methods: Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerase chain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductal adenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues.

Results: VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, and neutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was 42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition.

Conclusions: We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These results suggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

Keywords: HIF-1α inhibitor; Pancreatic cancer; chemosensitizer; gemcitabine; intracellular antibody; nanobody therapeutic.

Figure 1

Graphical abstract of the study. A model summarizing how intrabody VHH212 sensitized the anti-tumor efficacy of gemcitabine by intracellularly targeting HIF-1α.

Figure 2

Engineered nanobody VHH212 has high binding affinity and specifically binds to the HIF-1α PAS-B domain. (A) Schematic representation of the sequence of the intrabody, from N-terminus to C-terminus. (B) Amino acid sequence of VHH212. The framework and complementarity-determining region sequences are defined according to the Kabat numbering scheme using the AbNum program. (C) A surface plasmon resonance sensorgram showing the interactions between the VHH212 and the HIF-1α PAS-B domain. The color lines represent the global fits of the raw data to a 1:1 bimolecular model. (D) Glutathione (GST) and GST-HIF-1α PAS-B fusion proteins were incubated with VHH212 or negative control Nb02, captured on glutathione-Sepharose beads, and analyzed. (E) Three-dimensional structure model of the HIF-1α PAS-B-VHH212 (K_D = 42.7 nM)/HIF-1α PAS-B-ARNT-PAS-B (K_D = 125 nM) interactions. The antigen (HIF-1α PAS-B domain) is shown as the gray translucent molecular surface, with the residues associated with VHH212 binding sites labeled in pink. The VHH212 is in a blue cartoon, with hotspots shown as a green stick model. The ARNT is shown as a yellow translucent molecular surface.

Figure 3

VHH212 shows excellent thermal stability, low cytotoxicity, and the capacity to competitive inhibit the HIF-1 pathway. (A) An experiment to measure the thermal stability of VHH212. The Tm values were determined by melting curves using qPCR, and the value was average from 3 independent experiments. (B) Intrabodies do not have direct cytotoxic properties in vitro. The CCK8 assay was used to determine cell viability, and did not show a statistical difference between intrabodies and the vector control. (C) Western blot analysis of pancreatic cancer cell lines after hypoxic treatment. MIA PaCa-2 and PANC-1 cells were transfected with intrabodies and incubated under hypoxia (36 h), and total protein extracts were processed for Western blot using anti-HIF-1α, anti-VEGF-A, and anti-β-actin antibodies. (D) Quantitative real-time RT-PCR of pancreatic cancer cell lines after hypoxic treatment. MIA PaCa-2 cells were transfected, treated with hypoxia, and lysed in TRIzol for RNA extraction and analyzed for the mRNA expression of HIF-1α and VEGF by quantitative real-time RT-PCR. Data are expressed as the mean ± SEM. Columns, the mean of three experimental determinations; bars, standard deviation. (E) Luciferase analysis of MIA PaCa-2 and PANC-1 cells. The cells were transfected as described above. Relative luciferase analysis used the Dual-Luciferase Reporter Assay System, and the Renila vector was transfected as an internal control. Results are expressed as fold induction relative to cells transfected with the control vector (pcDNA3.1) after normalization to Renila activity. Columns, mean of 3 independent experiments; bars, standard deviation. (*P < 0.05) vs. the control. 3.1, pcDNA-3.1; N1, pEGFP-N1; VHH212, pEGFP-VHH212-2; HIF-OE, pcDNA-HIF-1α-OE. ****P < 0.0001.

Figure 4

VHH212 inhibits cell migration and specific subcellular localization. (A) Confocal images of MIA PaCa-2 cells transfected with pEGFP-N1, pEGFP-VHH212-1, and pEGFP-VHH212-2, then stained with 4′,6-diamidino-2-phenylindole (blue), EGFP (green), and HIF-1α (red) with a magnification of 40×. (B) Inhibition of pancreatic cancer cell migration. Transfected or wild type PANC-1 and MIA PaCa-2 cells were plated in 6-well plates until 90% confluent. The cell layer was scratched and treated with gemcitabine (100 nM) and/or digoxin (40 nM) for 48 h. ***P < 0.001.

Figure 5

VHH212 suppressed the proliferation and metastasis of tumor cells. (A) Inhibition of pancreatic cancer cell invasion. Treated PANC-1 and MIA PaCa-2 cells were seeded into upper Transwell chambers in serum-free Dulbecco’s Modified Eagle’s medium containing gemcitabine (100 nM) and/or digoxin (40 nM) for 72 h. The lower chambers contained 10% fetal bovine serum medium without drugs. After incubation for 72 h, the cells were stained with 0.5% Crystal Violet. (B) Colony formation assay. Five-hundred treated PANC-1 and MIA PaCa-2 cells were plated in 6-well plates and incubated for 14 days. ***P < 0.001.

Figure 6

VHH212 sensitized gemcitabine anti-tumor effects in vivo. (A) The schematic illustration of the establishment of the PANC-1 xenograft model and administration strategy. (B) The tumor volume of mice treated with different treatment strategies. (C) Images of tumor-bearing mice on day 31. (D) An image of tumors isolated from tumor-bearing mice on day 31. (E) Average tumor weights. (F) H&E staining of tumor tissues and the expression of HIF-1α and VEGF-A were confirmed using an immunohistochemistry assay in tumor tissues. *P < 0.05.