Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-04-26
2001-09-11
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S006120, C530S866000, C530S867000, C514S012200, C536S023100
Reexamination Certificate
active
06287817
ABSTRACT:
BACKGROUND OF THE INVENTION
The epithelium is the first line of defense of the airways against invading pathogens. Many of the non-specific defenses against such invaders arise from respiratory epithelial cells. Epithelial cells produce low molecular weight antimicrobial peptides, antibacterial enzymes, and antiproteases. In addition, secretory immunoglobulin A, a non-specific immunoglobulin defense, is trafficked into the airway via a specialized receptor, the polymeric immunoglobulin receptor (pIgR), that is expressed only on epithelial cells.
These epithelial defenses are breached early in the life of patients with cystic fibrosis (CF). Once live bacteria reach their surface, the epithelial cells direct the initial inflammatory response by releasing interleukin-8 (IL-8) and interleukin-6 (IL-6) as well as reducing expression of interleukin-10 (IL-10). The chemoattractants, combined with increased expression of adhesin molecules for neutrophils, enhance inflammatory cell migration into the airways. Once there, the neutrophils, in an attempt to clear the bacteria, release lytic enzymes in the process. If the neutrophils remain adherent to the epithelium, these enzymes are released right at the epithelial surface. Both mechanical disruption of cells and even low concentrations of neutrophil elastase (NE) result in the greater release of pro-inflammatory mediators from the respiratory epithelium. Thus, the inflammatory response is further enhanced.
Several strategies to interrupt this cycle have been proposed. Augmenting the antibacterial defenses of the airway at the epithelial surface may be useful. Prevention of the escalation of the inflammatory responses engendered by the neutrophils migrating into the airway could be accomplished by preventing the action of elastase at the airway cell surface. Both antibiotics and antiproteases are available for clinical use. Unfortunately, the results of clinical studies examining the use of the antiprotease in patients with CF have been disappointing. The systemic administration of alpha
1
-antitrypsin (A
1
AT) is inefficient, and the levels achieved by the intravenous administration of the antiprotease are insufficient to inhibit the overwhelming amount NE in the lung of patients with CF. Aerosolized A
1
AT should permit the direct delivery to the airways, but the antiprotease delivered by nebulization has been uneven and deposits the drug atop the mucus blanket rather than the critical site at the surface of the cell.
Thus there is a need in the art for methods to circumvent these difficulties and protect the respiratory epithelial cell surface.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a fusion-protein useful for protein delivery.
It is another object of the invention to provide a method of delivering a therapeutic protein to an epithelial cell.
It is yet another object of the invention to provide a nucleic acid molecule which encodes a fusion protein useful for protein delivery.
These and other objects of the invention are provided by one or more embodiments as described below. In one embodiment a fusion protein is provided. The fusion protein comprises a single chain Fv molecule directed against a human transcytotic receptor covalently linked to a therapeutic protein. The therapeutic protein may be, for example, &agr;,-antitrypsin, a cytokine, such as interleukin-2 or interleukin-10, or a peptide antibiotic. Suitable peptide antibiotics include aerosporin, amphomycin, aspartocin, bacitracins, caperomycins, colistins, dactinomycins, glumamycins, gramicidin D, gramicidin S, mikamycin B, polymixins, pristinamycin, siomycin, staphylomycin S, thiostrepton, tyrocidines, tyrothricin, valinomycin, vancomycin, veramycin B. Any therapeutic protein which one wants delivered to epithelial cells may be used. The fusion protein may further comprise a linker region of less than 50, 40, 30, 20, or 10 amino acid residues. The linker can be covalently linked to and between the single chain Fv molecule and the therapeutic protein.
Also provided according to another aspect of the invention is a method of delivering a therapeutic protein to an epithelial cell. The method comprises: administering a fusion protein as described above to a patient, whereby the therapeutic protein is delivered to an epithelial cell. The eptithelial cell may be an airway epithelial cell or an intestinal lumen cell, for example. The liver may also be targeted. The administration mode may be any known in the art. However, intravenous administration has been found to be both convenient and efficient.
Nucleic acid molecules are also provided by the present invention. These encode a fusion protein comprising a single chain Fv molecule directed against a transcytotic receptor covalently linked to a therapeutic protein. The therapeutic protein may be, for example, &agr;
1
-antitrypsin, a cytokine, such as interleukin-2 or interleukin-10, or a peptide antibiotic. Any therapeutic protein which one wants delivered to epithelial cells may be used. The fusion protein may further comprise a linker region of less than 50, 40, 30, 20, or 10 amino acid residues. The linker can be covalently linked to and between the single chain Fv molecule and the therapeutic protein. Host cells and vectors for replicating the nucleic acid molecules and for expressing the encoded fusion proteins are also provided. Any vectors or host cells may be used, whether prokaryotic or eukaryotic. Many vectors and host cells are known in the art for such purposes. It is well within the skill of the art to select an appropriate set for the desired application.
The present invention thus provides an efficient means of delivering therapeutic proteins to body parts which are often inaccessible or difficult to reliably access.
REFERENCES:
patent: 5108921 (1992-04-01), Low et al.
Ernst Wagner et al. “Transferrin-polycation-DNA complexes: The effect of polycations on the structure of the complex and DNA delivery to cells” Proc. Natl. Acad. Sci. USA vol. 88, pp. 4255-4259, May 1991.
Elizabeth Eckman et al., Pediatric Pulmonology 14 (Suppl): A229 “Targeting the Polymeric Immunoglobulin Receptor as a Means of Directing Therapeutic Proteins to the Airway”, 1997.
Elizabeth Eckman et al. Pediatric Pulmonology 13 (Suppl) A242 “Structure and Function of Anti-Human Secretory Component FV/Human Alpha1-Antitrpsin Fusion Proteins”, 1997.
Davis Pamela B.
Eckman Elizabeth
Ferkol Thomas
Luk John M.
Schreiber John
Banner & Witcoff , Ltd.
Case Western Reserve University
Park Hankyel T.
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