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

Exp Hematol. 1999 May;27(5):936-45. doi: 10.1016/s0301-472x(99)00007-7.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Antigens, CD34 / immunology*
  • Bacteriophages / genetics*
  • Base Sequence
  • DNA Primers
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / immunology*
  • Humans
  • Immunomagnetic Separation
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / immunology*

Substances

  • Antigens, CD34
  • DNA Primers
  • Peptides