Selection of single chain antibody fragments against the gm-csf receptor

Abstract

GM-CSF has an important role in the survival and function of cells of the myeloid lineage. The alpha subunit of the GM-CSF receptor (GMR) is expressed on over 95% of AML blasts. This leads to the possibility that GMR can be exploited as a target for leukemia therapy. Our goal is to determine if variable region fragments of monoclonal antibodies against GMR can be be developed as a minimally toxic treatment for patients with AML. The single chain variable region (scFv) cDNAs of four of these unique monoclonal antibodies to GMR have been cloned and inserted into a vector for production in bacteria. Sequence analysis shows similarity in the heavy chain sequence of the monoclonals from the same mouse and a very different heavy chain from the other mouse. The light chain sequences were similar to each other but not derived from the light chain found in the NS1 cells used to made the hybridoma lines. The four unique scFvs and a negative control scFv were expressed as bacteriophage surface gene 3 fusion proteins. Western blot analysis of input phage showed that each scFv phage was made and adequately represented in the mix. The mixture was used to pan cells expressing transfected GMR by three rounds of binding, washing to remove poorly bound scFv phage, elution, and expansion of selectively bound phage. Wild type untransfected cells were used as negative controls. The anti-GMR scFv with the highest affinity was selected.