Identification of a peptide directed against the anti-CD34 antibody, 9C5, by phage display and its use in hematopoietic stem cell selection

A peptide sequence was identified by phage display technology that could be used as an alternative to chymopapain for the release of hematopoietic pro genitor cells captured by anti-CD34 monoclonal antibodies. This was achieve d by affinity selection screening (biopanning) of a random hexapeptide sequ ence phage display library. Four rounds of biopanning were performed to enr ich for phage clones with specific affinity for anti-CD34 monoclonal antibo dy, 9C5, DNA sequence analyses of these phage clones revealed an enrichment of two predominant sequences, QQCWFP and TQGSFW. These two clones also sha red a consensus sequence motif, QGxF, that exhibited 50% and 67% homology w ith a region spanning amino acids 14-19 of the mature CD34 antigen. Based o n these data, synthetic peptides were generated and assessed for their abil ity to release 9C5 from CD34(+) cells. Using a flow cytometric assay, it,va s found that the synthetic peptide, 9069N, effectively released 9C5 from th e CD34-expressing cell line, KG1a, in a concentration-dependent manner (77% and 99% release of 9C5 at 0.14 and 0.70 mM peptide concentrations, respect ively). In the Isolex(R) 300i immunomagnetic selection system, this peptide was shown to be effective at releasing 9C5 sensitized CD34(+) hematopoieti c progenitors from sheep anti-mouse Ige Dyna-beads(R), Thus, a synthetic pe ptide, which specifically and efficiently released immunomagnetically selec ted hematopoietic progenitor cells from paramagnetic beads, was identified. This reagent is a significant advance in the selection of hematopoietic pr ogenitors in that it does not alter cell surface antigens, As such, further phenotypic characterization or immunoselection can be performed. (C) 1999 international Society for Experimental Hematology. Published by Elsevier Sc ience Inc.