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Kathlynn C Brown

Publications and source records attributed to Kathlynn C Brown.

12 recordsLinked to original sources
PubMed2006

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries.

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.

Amino Acid Sequence↗
PubMed2006

Novel ligands for cancer diagnosis: selection of peptide ligands for identification and isolation of B-cell lymphomas.

OBJECTIVE: Lymphoma and leukemia account for nearly 8% of cancer fatalities each year. Present treatments do not differentiate between normal and malignant cells. New reagents that distinguish malignant cells and enable the isolation of these cells from the normal background will enhance the molecular characterization of disease and specificity of treatment. METHODS: Peptide ligands were selected from a phage-displayed peptide library by biopanning on the B-cell lymphoma line, A20. The isolated peptides were assessed as reagents for identification and isolation of lymphoma cells by flow cytometry and cell capture with magnetic beads. RESULTS: Two novel peptides and one obtained previously on cardiomyocytes were selected. A20 cells bind phage displaying these peptides 250- to 450-fold over control phage. These phage bind to other bone marrow-derived cancel lines including some macrophage and T cells but do not bind to normal splenocytes. Synthetic constructs of these peptides have binding affinities comparable to B-cell-specific antibodies. Similar to antibodies, these peptides can be used in flow cytometry and magnetic bead capture to distinguish lymphoma cells from normal splenocytes. CONCLUSION: Bone marrow-derived malignant cells express cell surface markers that can be used to distinguish them from normal cells. These results demonstrate the ability to use an unbiased screen to rapidly generate high-affinity peptide ligands for identification and isolation of lymphoma cells.

Animals↗
PubMed2005

Isolation of a mycoplasma-specific binding peptide from an unbiased phage-displayed peptide library.

An important goal in medicine is the development of methods for cell-specific targeting of therapeutic molecules to pathogens or pathogen-infected cells. However, little progress has been made in cell-specific targeting of bacterially infected cells. Using a phage display approach, we have isolated a 20-mer peptide that binds to Mycoplasma arginini infected pancreatic beta-cells in tissue culture. This peptide binds to M. arginini infected beta-cells 200 times better than a control phage and is specific for the infected cells. Furthermore, transferring the M. arginini contamination to another cell line renders the newly infected cell line susceptible to peptide binding. Immunolocalization experiments suggest that the peptide is binding to M. arginini adhered to the cell surface. The free synthetic peptide retains its binding in the absence of the phage vehicle and tetramerization of the peptide increases its affinity for the infected cells. Efforts have been made to use this peptide to eliminate Mycoplasma from infected cell lines using ferromagnetic beads coated with the selected peptide. A ten-fold reduction of infection was accomplished with one fractionation via this approach. Our results suggest that this peptide, isolated from an unbiased selection, may be of utility for the detection and reduction of Mycoplasma infection in cultured cells. Furthermore, a general implication of our findings is that phage display methods may be useful for identifying peptides that target a broad array of other biological pathogens in a specific fashion.

Bacteriophages↗
PubMed2005

Peptide-mediated targeting of the islets of Langerhans.

Strategies for restoring beta-cell function in diabetic patients would be greatly aided by the ability to target genes, proteins, or small molecules specifically to these cells. Furthermore, the ability to direct imaging agents specifically to beta-cells would facilitate diagnosis and monitoring of disease progression. To isolate ligands that can home to beta-cells in vivo, we have panned a random phage-displayed 20-mer peptide library on freshly isolated rat islets. We have isolated two 20-mer peptides that bind to islets ex vivo. One of these peptides preferentially homes to the islets of Langerhans in a normal rat with clear differentiation between the endocrine and exocrine cells of the pancreas. Furthermore, this peptide does not target beta-cells in a type 2 diabetes animal model, suggesting that the peptide can discriminate between glucose-stimulated insulin secretion-functional and -dysfunctional beta-cells.

Amino Acid Sequence↗
PubMed2004

Cell-specific delivery of a chemotherapeutic to lung cancer cells.

We report that lung cancer-targeting peptides isolated from a peptide library can be used to deliver an active chemotherapeutic in a cell-specific fashion. The peptides were removed from the context of the phage and placed on a pegylated tetrameric scaffold. The tetrameric peptides were shown to block uptake of their cognate phage. The tetrameric peptides were coupled to doxorubicin, and their cytotoxicity against a panel of different cell lines was tested. Our data demonstrate that these targeting peptides can deliver an active anticancer agent in a cell-specific fashion, resulting in an increase of the therapeutic index of the targeted drug compared to systemic delivery. The efficacy of the peptide conjugate correlates to the affinity of the targeting peptide for a particular cell line. As such, we have demonstrated that cell-specific targeted drugs can be synthesized, even when the cell surface target is unknown.

Amino Acid Sequence↗
PubMed2004

In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo.

One approach to targeted therapies for cardiovascular disease relies on isolating ligands that enhance the tissue-specific uptake of genes or drugs by heart cells. To obtain heart-targeting ligands, phage display biopanning was used to isolate a 20-mer peptide that binds to isolated primary cardiomyocytes. The isolated phage, PCM.1, displays the peptide WLSEAGPVVTVRALRGTGSW, and binds these cells 180 times better than a control phage from the library. Furthermore, phage displaying this peptide preferentially bind to cardiomyocytes when compared with a panel of other cell types. A BLAST search revealed that this peptide contains a 12 amino acid segment with sequence identity to a peptide in tenascin-X, an extracellular matrix protein. Synthetic peptides containing the complete 20-mer or a 12-mer tenascin peptide partially blocked phage binding to the cardiomyocytes. We developed a quantitative real-time PCR assay to assess uptake of this phage by tissues in vivo. Using this assay, preferential localization of the PCM.1 phage in heart was observed compared to the uptake of this phage by other tissues or other phage by heart. Furthermore, PCM.1 phage was associated with cardiomyocytes isolated from mice treated with a phage in vivo. These results demonstrate the utility of biopanning on isolated cells for identifying specific binding peptides that can target a tissue in vivo.

Amino Acid Sequence↗
PubMed2004

Patterning adhesion of mammalian cells with visible light, tris(bipyridyl)ruthenium(II) chloride, and a digital micromirror array.

Patterns of cellular adhesion were created on a surface using novel photochemistry that is stimulated with visible light. A glass surface coated with polyethylene glycol is nonadhesive to a variety of adherent mammalian cell types. Treatment of that surface with a mixture of tris(bipyridyl)ruthenium(II) chloride, ammonium persulfate, and a tryptophan derivative or tryptophan-bearing peptide in conjunction with irradiation with visible light (447 nm) made the surface adhesive to several cell types including mouse fibroblasts, human myoblasts, and human lung tumor cells. Immunostaining data suggest that tryptophan-containing peptides are crosslinked intact to the surface by this chemistry, which enables patterning of peptides containing only naturally occurring amino acids. Microscopic patterns of cellular adhesion were created with this chemistry by projecting microscopic patterns of visible light with a digital micromirror array. Using this method, regions of cellular adhesion were patterned with single-cell resolution.

Animals↗
PubMed2004

Hitting the target with bifunctional phage.

Peptide libraries are rich sources of cell-targeting peptides. In this issue of Chemistry & Biology, Pasqualini and coworkers have developed a chimeric cell-targeting phage system that can easily be modified to delivery of a variety of reagents without need for chemical conjugation.

Animals↗
PubMed2004

A library-selected, Langerhans cell-targeting peptide enhances an immune response.

The ability to deliver antigens and immunomodulators specifically to Langerhans cells (LCs) in the skin could impact vaccine development. However, cell-specific targeting of therapeutic molecules remains a challenge in biomedicine. Using phage display technologies, we have developed a protocol that identifies peptides that mediate uptake into target cell types. Employing this approach, we have isolated a 20-mer peptide that mediates specific uptake by immunopotent LCs. The peptide is functional outside the context of the phage and is able to deliver a nanoparticle to LCs in vitro. Although selected on cells in vitro, the peptide is able to direct antigens and genes to LCs in vivo. Liposomes bearing the LC targeting peptide are able to deliver a transcriptionally active gene to LCs in a mouse model. Furthermore, we demonstrate that a low-dose injection into mice of phage bearing the LC-targeting peptide yields faster and higher immune responses against phage-associated antigens than control-phage injections.

Amino Acid Sequence↗
PubMed2003

Isolation of lung tumor specific peptides from a random peptide library: generation of diagnostic and cell-targeting reagents.

Discovery of ligands specific to receptor(s) on a surface of a cancer cell could impact clinical issues including functional diagnosis and cell-specific drug delivery. Using a phage display approach, we have isolated 20-mer peptide ligands that bind to 3 different human lung tumor cell lines, NCI-H1299, NCI-H2009, and A549. The panning protocol is unbiased with no selection pressure towards binding a particular cellular receptor. The isolated phage bind to their target cells 24-300 times better than a control phage. Furthermore, the isolated peptides display remarkable cell-specificities and are able to discriminate between normal and cancerous cells as well as different lung tumor cells. The cell-specificities are not coincident with tumor classes indicating that the peptides are able to recognize cell-surface features that are not represented within the classification of tumor type. The isolated peptides are functional outside of the context of the phage and multimerization of the peptide increases its affinity for its given cell type, thus expanding their utility in clinical situations.

Biomarkers, Tumor↗
PubMed2002

Chemically mediated site-specific proteolysis. Alteration of protein-protein interaction.

The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine. Allylglycine-containing ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest. A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites. The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (K(i)s) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84(A)Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site. Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84(A)Gly. This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent.

Amino Acid Sequence↗