B-cell malignancies like Acute Lymphoblastic Leukemia (B-ALL), which often have high numbers of malignant cells in circulation in the blood would be an excellent model system for the early preclinical development and testing of targeted small particle therapeutics. We selected the transmembrane protein CD19 (Cluster of Differentiation 19) as one potential promising target due to its remarkably common over-expression on nearly every case of B-ALL, and on many other types of leukemias and lymphomas with varying frequency. We attempted to create a panel of appropriate CD19-specific targeting reagents for attachment to the surface of perhaps many different small particle platforms currently in development worldwide. We first utilized an M13 bacteriophage random peptide display library to attempt to isolate a short CD19-binding peptide using both a cell-based and a purified protein-based strategy. Despite multiple attempts, neither of these approaches yielded the discovery of such a peptide. We have, however, in process, developed novel phage purification procedures that we demonstrate are particularly important when validating the binding of clones recovered from the library using two cell-based assays; these consist of a simple flow cytometry method and a complex, but higher throughput, assay done in 96-well plates that can measure phage binding to cells with a qPCR approach. As a second strategy for making targeting reagents, we expressed and refolded large quantities of two known anti-CD19 Single-chain Fv fragments from cytoplasmic inclusion bodies in E. coli. We here demonstrate that one of them, HD37 (originally isolated from a mouse hybridoma cell line as an IgG1 subtype) could be refolded into a somewhat active form by using a previously published procedure. A second antibody fragment, FMC 63 (also originally from mouse hybridoma, an IgG2 subtype) did not refold properly by this procedure and work is ongoing to find a suitable refolding strategy for this antibody in particular. Thus, we proffer the recombinant HD37 antibody fragment as the simplest strategy for the production of large quantities of CD19-targeting reagent suitable for attachment to small particle therapeutics, as we find that CD19 is particularly challenging to use as a target for phage display technologies in the cell context.
leukemia, CD19, nanoparticle, phage display, ScFv antibody
University of New Mexico Cancer Nanotechnology Training Center
Level of Degree
Biomedical Sciences Graduate Program
First Committee Member (Chair)
Second Committee Member
Rogers, Jason Hugh. "Development of CD19 binding reagents for targeted nanoparticles." (2013). http://digitalrepository.unm.edu/biom_etds/73