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

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.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment

“…Significant progresses with nanobody immunoreagents have been achieved in the fields of α-particle radiation and photodynamic therapy as well as in in vivo imaging ( 16 ). Despite no nanobody has been yet approved for cancer treatment ( Table 1 ), nanobody-based multivalent, multispecific and modified constructs have been tested in a multiplicity of cancer applications with therapeutic potential ( 17 , 18 ) ( Table 2 ) and their recombinant nature enables the production of constructs with a wide range of biodistribution and clearance patterns that optimally fit to different applications ( 43 ).…”

Therapeutic Potential of Intrabodies for Cancer Immunotherapy: Current Status and Future Directions

“…The sdAb against F-actin capping protein CapG inhibited breast tumor metastasis in a xenograft tumor mouse model [ 36 ]. Recently an anti-HIF-1α nanobody was developed to decrease gemcitabine resistance in pancreatic cancer patients [ 24 ]. The intrabody competitively inhibited the binding of the transcription factor HIF-1α heterodimer to the aryl hydrocarbon receptor nuclear translocator (ARNT), leading to the inhibition of the HIF-1/VEGF pathway in vitro.…”

“…They can be selected by phage display or ribosomal display from immune, naïve or synthetic single domain antibody repertoires [ 16 , 17 , 18 , 19 ] and inactivate their targets by altering their conformation or interfere with the binding of the target protein to its corresponding binding partner. sdAbs were isolated against intracellular TAAs [ 19 ], including the recent examples of hypoxia-inducible factor-1 (HIF-1) [ 24 ], serine/threonine protein kinase AKT2 [ 25 ], p53 C-terminal region involved in the interaction with Twist1 [ 26 ] and chemokine receptor US28 [ 27 ]. In addition to targeting intracellular TAAs, intrabodies have been selected against intracellular neoantigens, for example against HRASG12V [ 28 , 29 , 30 ] HRASG12V, KRASG12D, KRASG13D, NRASQ61R, KRASG12V, KRASQ61H [ 31 ] H - and K - Ras G12V [ 32 ], p21Ras [ 33 ] and KRASG12V [ 34 ].…”

Pre-Clinical and Clinical Applications of Small Interfering RNAs (siRNA) and Co-Delivery Systems for Pancreatic Cancer Therapy

“…HIF-1α up-regulation occurs in hypoxic conditions. The overexpression of HIF-1α is in favor of PC progression [ 244 ] and can mediate drug resistance [ 245 ]. Co-delivery of HIF-1α siRNA and GEM leads to a four-fold decrease in tumor growth.…”