Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-08
2001-07-17
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S069700, C530S391100, C530S391700, C530S402000, C530S807000, C530S866000, C514S012200, C536S023100
Reexamination Certificate
active
06261787
ABSTRACT:
BACKGROUND OF THE INVENTION
The epithelium is the first line of defense against a variety of pathogens. Epithelial cells produce low molecular weight antimicrobial peptides, antibacterial enzymes, and antiproteases. However, optimal methods of specifically targeting therapeutic molecules to epithelial cells have been lacking in the art. There is a continuing need in the art for methods of providing therapeutic agents to respiratory epithelia cells in diseases such as cyptic fibrosis, asthma, and emphysema, and to intestinal epithelial cells, for example, in inflammatory bowel diseases.
SUMMARY OF THE INVENTION
It is an object of the invention to provide bifunctional molecules useful for delivery of therapeutic molecules and methods for delivering therapeutic molecules to cells. These and other objects of the invention are provided by one or more embodiments as described below.
In one embodiment the invention provides a fusion protein. 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;
1
-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 epithelial 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, inhalation and intravenous administration have 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 invention also provides a bifunctional molecule comprising a ligand coupled to a non-protein therapeutic molecule. The ligand specifically binds to a transcytotic receptor, such as the human secretory component of polymeric immunoglobulin receptor. The coupling is carried out such that the ligand can bind to the transcytotic receptor.
Another embodiment of the invention provides a method of delivering a therapeutic molecule to an epithelial cell. A bifunctional molecule is administered to a patient. The bifunctional molecule comprises a ligand coupled to a non-protein therapeutic molecule. The ligand specifically binds to a transcytotic receptor, such as the human secretory component of polymeric immunoglobulin receptor. The therapeutic molecule is thereby delivered to an epithelial cell. The epithelial 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, inhalation and intravenous administration have been found to be both convenient and efficient.
The present invention thus provides an efficient means of delivering therapeutic molecules to body parts which are often inaccessible or difficult to access reliably.
REFERENCES:
patent: 5100788 (1992-03-01), Lofdahl et al.
patent: 5108921 (1992-04-01), Low et al.
patent: 5521291 (1996-05-01), Curiel et al.
patent: 5763192 (1998-06-01), Kauffman et al.
patent: 5871974 (1999-02-01), Huse
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-Antitrypsin Fusion Proteins” 1996.
Amedeo Caflisch “Computational combinatorial ligand design: Application to human a-thrombin” Journal of Computer-Aided Molecular Design 10 (1996) 372-396.
Moon and Howe “Computer Design of Bioactive Molecules: A Method for Receptor-Based de Novo Ligand Design” Proteins: Structure, Function and Genetics 11:314-328 (1991).
Aya Jakobovits “Production of fully human antibodies by transgenic mice” Current Opinion in Biotechnology 1995 6:561-566.
Wagneret al. “The diversity of antigen-specific monoclonal antibodies from transgenic mice bearing human immunoglobulin gene miniloci” Eur. J. Immunol. 1994, 24:2672-2681.
Lonberg et al. “Antigen-specific human antibodies from mice comprising four distinct genetic modifications” Nature vol. 368 Apr. 28, 1994 pp. 856-859.
Neuberger & Bruggemann “Mice perform a human repertoire” Nature, vol. 386, Mar. 1997 pp. 25-26.
Mendez et al. “Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice” Nature Genetics vol. 15, Feb. 1997 pp. 146-156.
Aya Jakobovits Production of Antigen-Specific Human Antibodies from Mice Engineered with Human Heavy and Light Chain YACs, Annals of the New York Academy of Sciences, 1995 vol. 764 pp. 525-535.
L.L. Green “Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs” Nature Genetics 1994 vol. 7 (1) pp. 13-21.
Powers et al. “Specificity of Porcine Pancreatic Elastase, Human Leukocyte Elastase and Cathepsin G” Biochimica et Biophysica Acta, 485 (1977) pp. 156-166.
Bode et al. “Human Leukocyte and Porcine Pancreatic Elastase: X-ray Crystal Structures, Mechanism, Substrate Specifcity, and Mechanism-Based Inhibitors” Biochemistry, vol. 28, No. 5 Mar. 7, 1989 pp. 1951-1963.
Nakajima et al. “Mapping the Extended Substrate Binding Site of Cathepsin G and Human Leukocyte Elastase” The Journal of Biological Chemistry, vol. 254, No. 10, May 25, 1979 pp 4027-4032.
Zimmerman and Ashe “Substrate Specificity of the Elastase and the Chymotrypsin-Like Enzyme of the Human Granulocyte” Biochmicia et Biophysica Acta, 480 (1977) pp. 241-245.
Ferkol et al. Receptor-mediated gene transfer into macrophages, Proc. Natl. Acad. Sci. USA. Jan. 1996, vol. 93, No. 1, pp. 101-105.
Davis Pamela B.
Eckman Elizabeth
Ferkol, Jr. Thomas W.
Banner & Witcoff Ltd
Case Western Reserve University
Park Hankyel T.
LandOfFree
Bifunctional molecules for delivery of therapeutics does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Bifunctional molecules for delivery of therapeutics, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Bifunctional molecules for delivery of therapeutics will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2554404