Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha

Hypoxia triggers the transcription of genes responsible for cell survival via the key player transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). Overexpression of this protein has been implicated in cardiovascular disorders, carcinogenesis and cancer progression. For functional and diagnostic studies on the HIF-1α protein, we have identified single-domain antibody fragments directed against this protein by using a llama-derived nonimmune phage display library. This library displays the variable domains of the heavy-chain antibody subclass, found in these animals. Phage display selection with six recombinant HIF-1α proteins yielded five different antibody fragments. By epitope-mapping, we show that all five antibody fragments bind within the functionally important oxygen-dependent degradation domain of the HIF-1α protein. Two of these antibody fragments were engineered into bivalent antibodies that were able to detect human HIF-1α by immunohistochemistry, Western blotting and immunoprecipitation, and mouse HIF-1α by immunofluorescence and immunoprecipitation. These are the first single-domain antibody fragments that may be used in exploration of HIF-1α as a possible therapeutic target through molecular applications.