Receptor-mediated uptake of peptides that bind the human...

Details Receptor-mediated uptake of peptides that bind the human... Receptor-mediated uptake of peptides that bind the human...

2001-11-29

2004-06-01

Kunz, Gary (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C514S002600, C530S350000

Reexamination Certificate

active

06743893

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to use of peptides which target the human transferrin receptor. Peptides of the invention can be used to direct other peptides, proteins and other diagnostic or therapeutic agents into cells for both diagnostic and therapeutic purposes.
BACKGROUND OF THE INVENTION
Previous work relating to redirecting viral vectors in gene therapy by using short peptide ligands to redirect virus particles to specific cell types are known. One of the limitations of this strategy is that short peptide sequences that bind efficiently to cell surface receptors on specific cell types must be identified. One experimental approach to identify such short peptides that holds promise is bacteriophage display.
For more than a decade, phage display has exploited the physical linkage between random peptide sequences expressing on phage and the DNA encoding that sequence. This linkage allows for rapid identification of peptide ligands. A random peptide sequence is expressed as a fusion with a bacteriophage coat protein and is available for testing as a ligand for various targets. Phage display has successfully been used to identify single chain antibodies with specificity for various biological molecules. Phage display strategies can be used to elucidate the amino acids responsible for protein—protein interactions, to find organ-specific phage, and to find substrate recognition sequences for enzymes. The process of using multiple rounds of phage display to enrich for a particular sequence is called biopanning.
The human transferrin receptor (hTfR) has been studied extensively as a model system for receptor-mediated endocytosis, a marker for cellular proliferation, and a target for therapeutics. The hTfR is ubiquitously expressed and over-expressed at least 100 fold in oral, liver, pancreatic, prostate and other cancers. This increase in transferrin receptor (TfR) in cancers has been attributed to the increased metabolism of these transformed cells, making the hTfR a useful diagnostic marker. Because of its expression pattern and pathway characteristics, the hTfR is an attractive target for therapeutics. The TfR is a dimer composed of two identical 95 kDa subunits and is responsible for the majority of cellular iron uptake. The type II cell surface receptor binds 80 kDa transferrin (Tf) and the complex is internalized through clathrin-coated pits. Iron is released from transferrin in the acidic early endosome and the apotransferrin-receptor complex is recycled back to the cell surface where apotransferrin is recycled.
A blast search failed to yield any significant homologies between either HAIYPRH (Seq. ID No. 1) or THRPPMWSPVWP (Seq. ID No. 2) to known proteins, including Tf.
SUMMARY OF THE INVENTION
This invention relates to peptides which are capable of binding to and internalizing with the human transferrin receptor (hTfR). The sequences HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2) are capable of binding to and internalizing with the human transferrin receptor. When these molecules were fused with other molecules, the fusion product was internalized in cells expressing hTfR. The sequences have use for targeting other peptides and proteins into cells expressing hTfR. The phage display system using whole cell selective biopanning could also be applied to find small ligands for other cell surface receptors. This sequence is not found in human transferrin protein. Furthermore, this sequence does not compete with transferrin itself for binding to the hTfR.
DETAILED DESCRIPTION OF THE INVENTION
It is important that easily produced peptides that can facilitate entry of diagnostically and therapeutically useful peptides and proteins into cells having particular characteristics be available. The identification of peptides that will facilitate entry of such peptides into cells which are more likely to be aberrant has particular use. The peptides of the invention are useful for facilitating entry of diagnostically and therapeutically useful agents, including peptides and proteins. Since malignant cells produce increased expression of hTfR, the peptides, HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2), are particularly useful for study and treatment of malignancies.
A phage display selection strategy was utilized that resulted in identification of the peptides. This selection system is based on alternating rounds of negative selection on chicken embryo fibroblast (CEF) cells lacking hTfR and positive selection on chicken embryo fibroblast cells expressing hTfR (CEF+hTfR). Biopanning on whole cells was exploited to select the peptides HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2). These peptides were able to target a macromolecule to and internalize through the hTfR, as was demonstrated by phage binding, competition and immunofluorescence studies. It was also shown that these two peptides bind sites that do not overlap with the native ligand, transferrin, indicating they could be used in vivo for targeting macromolecules to the endocytic pathway in hTfR-positive cells.
The biopanning procedure could be applied to find small peptide ligands for other cell surface receptors. There is a great need to find new epitopes on various cancer cell types for diagnostic purposes. The subtractive method of biopanning disclosed herein would be useful for finding new cell surface markers. Biopanning on whole cells can be especially useful in situations where the receptor can not be purified or does not maintain its native confirmation when isolated.
Materials and Methods:
Cell lines: The two chicken embryo fibroblast cell lines, CEF and CEF+hTfR, used for selective biopanning, were described previously (Collawn, et al,
Cell,
63, 1061-1072 (1990) and Odorizzi, et al.,
J. Cell Biol.,
126, 317-330 (1994)). Chicken embryo fibroblasts have been used extensively for study of hTfR. The native cells express chicken transferrin receptors, but this receptor cannot bind human transferrin. Two cell lines were previously established through stable transduction with retroviral vectors to yield CEF and CEF+hTfR cells. CEF cells do not express the human transferrin receptor. CEF+hTfR cells constitutively express hTfR. Protein expression of hTfR by CEF cells was periodically checked by
125
I-Tf binding. Both cells are grown in monolayer cultures in Dulbecco's Modified Eagle Medium supplemented with 1% chicken serum, 1% bovine calf serum, 1% L-glutamine 200 nM, and 2% tryptose phosphate and maintained at 37° C. in 13% CO
2
.
Antibodies: Monoclonal anti-GFP (green fluorescent protein) antibody (Clontech, Palo Alto, Calif.) was used for Western blot analysis and immunofluorescence at 1:5,000 and 1:250 dilution, respectively. Horse radish peroxidase conjugated goat anti-mouse antibody (Pierce, Rockford, Ill.), Oregon-Green and Texas-Red secondary antibodies (Molecular Probes, Eugene, Oreg.) were used at 1:10,000, 1::250, 1:250 dilution, respectively.
Electrophoretic Methods: Samples were dissolved on SDS-PAGE gels by the methods of Laemmli and transferred to nitrocellulose membrane by electroblotting for Western blot analysis (Laemmli, U.K,
Nature,
227, 680-685). The membranes were blocked with 5% milk in tris buffered saline with 1% Trition X-100 (TBS-TX) (50 mM Tris-HCL, pH 7.5, 0.2 M NaCl, 1% Triton X-100), and incubated with primary antibody in TBS-TX with 2.5% milk overnight at 4° C. The membranes were then washed in TBS-TX and incubated with peroxidase-conjugated secondary antibody and developed with the enhanced chemiluminescence (ECL) kit in accord with the manufacturer's instructions (Amersham Pharmacia Biotech, Buckinghamshire, England).
Biopanning: Ph.D.-7™ or Ph.D.-12™ Phage Display Peptide Library Kit (New England Biolabs, Inc, Bevery, Mass.) was used for biopanning on CEF and CEF+hTfR cells. The Ph.D.™ phage display peptide library is based on a combinatorial library of random 7 or 12 amino acid peptides fused to a minor coat protein of the filamentous coliphage M13. In separate studies, two different phage display pept

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