Publication Date

2012

Document Type

Thesis

Committee Members

Cheryl Conley (Committee Member), David Cool (Committee Member), Osvaldo Lopez (Advisor)

Degree Name

Master of Science (MS)

Abstract

Peptides with high affinity to the B-cell receptor (BCR) fused to a toxin could be an effective therapy for Chronic Lymphocytic Leukemia (CLL) patients. We screened captured BCR of a CLL patient with peptides from 7 and 12-mer phage display libraries, using two strategies. Lymphocytes from two patients diagnosed with CLL expressing two different unmutated VH genes (VH 1-3 and VH4-34, respectively) were used. Membrane BCRs were obtained from patient CLL cells by lysis, identified by western blot, semi-quantified and screened with phage libraries. The first strategy involved using patient VH4-34 BCRs which were captured using goat anti-human IgM to deplete bound phages. Unbound-phages were positively screened for those binding to patient VH 1-3 BCRs. Several clones were randomly selected and a sequence consisting of "LLPPAR_" peptide was found in both libraries. A phage clone displaying peptide "LLPPARE" was identified to bind to goat anti-human IgM. By including more goat anti-human IgM negative selections, we identified 3 different phages displaying peptides "GFTFMPA", "QSRPLLP" and "GLPCCSS". Clones "GFTFMPA" and "GLPCCSS" showed binding to goat anti-human IgM, while "QSRPLLP" did not. Phage clone "QSRPLLP" showed no binding to human serum IgM but showed binding to both patients' BCRs. "QSRPLLP" peptide binds a common BCR molecule region present in both patients but not present in human serum IgM. This data suggests that it is possible to use a peptide-phage display library to select peptides unique for the BCRs of CLL patients. However, the critical component in making this process patient-specific resides in enhanced discrimination in phage selection.

Page Count

134

Department or Program

Department of Pharmacology and Toxicology

Year Degree Awarded

2012

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