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 progenitor cells captured by anti-CD34 monoclonal antibodies. This was achieved by affinity selection screening (biopanning) of a random hexapeptide sequence phage display library. Four rounds of biopanning were performed to enrich for phage clones with specific affinity for anti-CD34 monoclonal antibody, 9C5. DNA sequence analyses of these phage clones revealed an enrichment of two predominant sequences, QQGWFP and TQGSFW. These two clones also shared a consensus sequence motif, QGxF, that exhibited 50% and 67% homology with a region spanning amino acids 14-19 of the mature CD34 antigen. Based on these data, synthetic peptides were generated and assessed for their ability to release 9C5 from CD34+ cells. Using a flow cytometric assay, it was found that the synthetic peptide, 9069N, effectively released 9C5 from the 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, respectively). In the Isolex 300i immunomagnetic selection system, this peptide was shown to be effective at releasing 9C5 sensitized CD34+ hematopoietic progenitors from sheep anti-mouse IgG Dynabeads. Thus, a synthetic peptide, which specifically and efficiently released immunomagnetically selected hematopoietic progenitor cells from paramagnetic beads, was identified. This reagent is a significant advance in the selection of hematopoietic progenitors in that it does not alter cell surface antigens. As such, further phenotypic characterization or immunoselection can be performed.
Tseng-Law
J
J
Nexell Therapeutics Inc., Irvine, CA 92618, USA.
Szalay
P
P
Guillermo
R
R
Kobori
J
J
Van Epps
D
D
Schneidkraut
M J
MJ
Deans
R
R
D000595:Amino Acid Sequence; D018952:Antigens, CD34 / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology*; D006801:Humans; D018189:Immunomagnetic Separation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D018952:Antigens, CD34; D017931:DNA Primers; D010455:Peptides