Intrabody Targeting HIF-1? Mediates Transcriptional Downregulation Of Target Genes Related To Solid Tumors

The application of monoclonal antibodies?mAbs?for the diagnosis and tumor therapy has promoted the research and development of different formats of antibodies.Nanobodies?Nbs?,which are derived from the heavy-chain only antibodies?HCAbs?,are thought to be the smallest antibody with full capacity of antigen targeting,and offer in this respect special advantages over conventional antibody fragments.To date,most research involving Nbs focus on extracellular tumor targets,such as signaling receptors and extracellular domain proteins.However,most of the signaling regulatory mechanism connecting to the growth and proliferation of tumor cells is located intracellularly,and targeting of Nbs to intracellular tumor markers might extend diagnostic and tumor therapeutic applications.The current project focuses on the development of specific Nbs against PAS B domain of HIF-1?,which is a notable intracellular tumor marker and renowned for its transcriptional ability to activate more than 100 downstream genes and its role in adaptive response of tumor cells in oxygen deprived environment.Recombinant protein of PAS B domain?rPasB?was produced for the immunization of animals and construction of an immune-library.A synthetic library was designed and constructed based on the scaffold of Nb BcII10.Several specific Nbs have been selected from the immune-library and the synthetic library.The binding specificity of these selected Nbs to rPasB protein and native HIF-1?protein was confirmed.To assess the inhibitory efficiency of selected Nbs to the transcriptional activity of HIF-1 protein,a hypoxia model was established based on HeLa cells.Stimulated by CoCl₂,these cells were demonstrated to increase drastically the endogenous level of HIF-1?protein.Intrabody constructs were prepared with Nb genes and transfected into HeLa cells to express Nbs as intrabodies for targeting endogenous HIF-1?protein.Then,the expression level of HIF-1 regulated target genes was monitored via quantitative real-time PCR as an indicator for the intrabody mediated transcriptional inhibition.The expression and functionality of intrabodies produced in HeLa cells was confirmed.It was demonstrated that these Nb-based intrabodies recongize rPasB protein and native HIF-1?protein.It has been shown that the selected intrabodies inhibit the activation of the selected tumor associated target genes to various extents.Thus,the result demonstrated the efficiency of our selected Nbs to transcriptional inactivate the Hif-1 dependent genes.Remarkably,the most stable Nb with lower affinity for HIF-1?had larger inhibiting capacity than the less stable Nb binding with higher affinity,indicating that stability?and plausibly epitope recognition?are more important for Nb intrabodies than affinity.By serendipity several Nbs were retrieved against an E.coli SlyD from the rPasB immune-library.Purification strategies were exploited for these Nbs to develop easy to eliminate quantitatively SlyD contaminants from recombinant protein preparations.