Design And Optimization Of An Anti-HIF-1α Nanobody And Its Application In Pancreatic Cancer Therapy

Although pancreatic cancer only contributes to 3%of all cancers,it is the fourth leading cause of cancer-related death in developed countries.Due to the lack of effective treatment options,to date,the 5-year overall survival rate of pancreatic cancer patients only reaches 10%.Therefore,resistance to first-line gemcitabine chemotherapy is an urgent problem that needs to be solved.In this study,an adenoviral vector encoding anti-HIF-1αintrabody was constructed to explore its intervention on the HIF-1 pathway in a xenograft pancreatic cancer model.Further,the effect of anti-HIF-1αintrabody on enhancing the sensitivity of pancreatic cancer tumors to gemcitabine chemotherapy was identified.It is in order to provide a safe and effective regimen and pre-clinical trial data for pancreatic cancer patients with gemcitabine resistance.Initially,VHH14,VHH67,and VHH212 with higher binding affinity were screened out from the nine anti-HIF-1αcandidate nanobody sequences keep in our research group by enzyme-linked immunosorbent assay.VHH212 was stand out during the analysis of the binding hotspots between the candidate nanobody and the HIF-1α-PAS-B domain,which competitively inhibits the formation of HIF-1heterodimer complex.After that,VHH212 was affinity-matured in vitro by VIMAS strategy,while a mutant VHH212^M3 with 11.5-fold enhancement in binding affinity（3.72 n M）was obtained.Multiple intrabodies were designed and constructed based on the mutant VHH212^M3,which were stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The intrabody VHH212-2 can effectively inhibit the transcription and protein expression of HIF-1downstream cytokines and inhibit the migration,invasion,and proliferation of pancreatic cancer cells.Further,an adenovirus vector encoding intrabody VHH212-2 was designed and constructed,while the PANC-1 pancreatic cancer xenograft model was established.When tumors grew to an average of 100 mm³,30 mice were randomly distributed into five groups,normal saline group,mock adenovirus group,intrabody encoding adenovirus treatment group,gemcitabine treatment group,and combined treatment group.Compared with the normal saline group,the tumor inhibition rate was 12.56%,41.58%,64.89%,and 80.44%,respectively.HE staining and immunohistochemical analysis showed that adenovirus encoding intrabody could effectively inhibit vascular endothelial growth factor expression and inhibit angiogenesis in the tumor.In vitro and in vivo studies have demonstrated that targeting HIF-1αcan effectively enhance the sensitivity of pancreatic cancer cells and tumor tissues to gemcitabine chemotherapy.In conclusion,anti-HIF-1αintrabody VHH212-2 combined with first-line gemcitabine chemotherapy has potential as a regimen for advanced pancreatic cancer.