Chimeric proteins for use in transport of a selected substance into cells

• US 20010025026A1
• Filed: 01/03/2001
• Published: 09/27/2001
• Est. Priority Date: 06/06/1995
• Status: Active Grant

• Current Assignee: Shire Human Genetic Therapies, Inc. (Shire plc)
• Original Assignee: Transkaryotic Therapies Incorporated Delaware Corporation
• Inventors: Heartlein, Michael W., Concino, Michael F., Lemontt, Jeffrey F.
• Granted Patent: US 6,858,578 B2
• Time in Patent Office: Days
• US Class Current: 514/12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/79 Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C07K 2319/74 containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

Chimeric proteins useful in transporting a selected substance present in extracellular fluids, such as blood or lymph, into cells; quantitative assays for the selected substance using chimeric proteins; DNA encoding the chimeric proteins; plasmids which contain DNA encoding the chimeric proteins; mammalian cells, modified to contain DNA encoding the chimeric proteins, which express and, optionally, secrete the chimeric proteins; a method of producing the chimeric proteins; a method of isolating the chimeric proteins; a method of using the chimeric proteins to assay the selected substance; and a method of reducing extracellular levels of the selected substance through administration of the chimeric protein, which results in transport of the selected substance into cells.

Claims

1. A chimeric protein comprising a functional domain and a carrier domain, wherein the functional domain comprises an amino acid sequence which binds a selected substance and the carrier domain comprises an amino acid sequence which binds a mammalian cell surface receptor other than the mammalian cell surface receptor which normally binds the selected substance in the mammal.

2. The chimeric protein of

claim 1

, wherein the mammalian cell surface receptor is a human cell surface receptor.

3. The chimeric protein of

claim 2

, wherein the human cell surface receptor is selected from the group consisting of:

human low density lipoprotein receptors, human transferrin receptors, human serum albumin receptors, human asialoglycoprotein receptors, human adenovirus receptors, human retrovirus receptors, human CD4, human lipoprotein (a) receptors, human immunoglobulin Fc receptor, human a-fetoprotein receptors, human LDLR-like protein (LRP) receptors, human acetylated LDL receptors, human mannose receptors and human mannose-6-phosphate receptors.

4. The chimeric protein of

claim 2

, wherein the selected substance is LDL cholesterol and the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor.

5. The chimeric protein of

claim 4

, wherein the carrier domain comprises an amino acid sequence which binds a human cell surface receptor other than the low density lipoprotein receptor.

6. The chimeric protein of

claim 5

, wherein the human cell surface receptor is the human transferrin receptor.

34. A pharmaceutical composition comprising the chimeric protein of claims 1, 2, 3, 4, 5, or 6.

7. DNA encoding a chimeric protein, the chimeric protein comprising a functional domain and a carrier domain, wherein the functional domain comprises an amino acid sequence which binds a selected substance and the carrier domain comprises an amino acid sequence which binds a mammalian cell surface receptor other than the mammalian cell surface receptor which normally binds the selected substance in the-mammal.

8. DNA of

claim 7

which encodes a chimeric protein wherein the mammalian cell surface receptor is a human cell surface receptor.

9. DNA of

claim 8

, wherein the human cell surface receptor is selected from the group consisting of:

human low density lipoprotein receptors, human transferrin receptors, human serum albumin receptors, human asialoglycoprotein receptors, human adenovirus receptors, human retrovirus receptors, human CD4, human lipoprotein (a) receptors, human immunoglobulin Fc receptors, human a-fetoprotein receptors, human LDLR-like protein (LRP) receptors, human acetylated LDL receptor, human mannose receptors and human mannose-6-phosphate receptors.

10. DNA encoding a chimeric protein comprising a functional domain and a carrier domain, wherein the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence which binds the human transferrin receptor.

11. An expression plasmid comprising:

a) a promoter;

b) DNA encoding a chimeric protein comprising a functional domain and a carrier domain, wherein the functional domain comprises an amino acid sequence which binds a selected substance and the carrier domain comprises an amino acid sequence which binds a mammalian cell surface receptor other than the mammalian cell surface receptor which normally binds the selected substance in the mammal,

wherein expression of the DNA encoding the chimeric protein is under the control of the promoter.

12. A genetically modified mammalian cell which expresses DNA encoding a chimeric protein, wherein the chimeric protein comprises a functional domain and a carrier domain, wherein the functional domain comprises an amino acid sequence which binds a selected substance and the carrier domain comprises an amino acid sequence which binds a mammalian cell surface receptor other than the mammalian cell surface receptor which normally binds the selected substance in the mammal.

13. The genetically modified mammalian cell of

claim 12

, wherein the mammalian cell surface receptor is a human cell surface receptor.

14. The genetically modified mammalian cell of

claim 13

, wherein the selected substance is LDL cholesterol and the human cell surface receptor is the human transferrin receptor.

15. A genetically modified mammalian cell of

claim 14

, wherein the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor.

16. A genetically modified mammalian cell of

claim 13

, wherein the human cell surface receptor is selected from the group consisting of:

human low density lipoprotein receptors, human transferrin receptors, human serum albumin receptors, human asialoglycoprotein receptors, human adenovirus receptors, human retrovirus receptors, human CD4, human lipoprotein (a) receptors, human immunoglobulin Fc receptors, human a-fetoprotein receptors, human LDLR-like protein (LRP) receptors, human acetylated LDL receptors, human mannose receptors and human mannose-6-phosphate receptors.

17. The genetically modified mammalian cell of

claim 12

, wherein the mammalian cell is selected from the group consisting of:

human cells, porcine cells, hamster cells, bovine cells, canine cells, mouse cells, rat cells, monkey cells, feline cells, rabbit cells, sheep cells and chimpanzee cells.

18. The genetically modified human cell of

claim 17

, wherein the human cell is selected from the group consisting of:

human fibroblasts, human keratinocytes, human epithelial cells, human ovary cells, human endothelial cells, human glial cells, human neural cells, formed elements of the blood, human muscle cells, human hepatocytes and precursors of these human cell types.

19. The genetically modified mammalian cell of

claim 17

, wherein the mammalian cells are Chinese hamster ovary cells.

20. The genetically modified mammalian cell of

claim 17

, wherein the cell is an immortalized cell.

21. The genetically modified human cell of

claim 18

, wherein the cell is a primary cell or a secondary cell.

22. The genetically modified mammalian cell of

claim 12

, wherein the DNA encodes a chimeric protein in which the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence of human transferrin which binds the human transferrin receptor.

23. The genetically modified mammalian cell of

claim 17

, wherein the DNA encodes a chimeric protein in which the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence of human transferrin which binds the human transferrin receptor.

24. The genetically modified mammalian cell of

claim 18

, wherein the DNA encodes a chimeric protein in which the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence of human transferrin which binds the human transferrin receptor.

25. The genetically modified mammalian cell of

claim 19

, wherein the DNA encodes a chimeric protein in which the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence of human transferrin which binds the human transferrin receptor.

26. The genetically modified mammalian cell of

claim 20

, wherein the DNA encodes a chimeric protein in which the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence of human transferrin which binds the human transferrin receptor.

27. The genetically modified mammalian cell of

claim 21

, wherein the DNA encodes a chimeric protein in which the functional domain comprises the amino acid sequence of the ligand-binding domain of the human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence of human transferrin which binds the human transferrin receptor.

35. A pharmaceutical composition comprising the genetically modified cell of claims 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

28. A method of transporting low density lipoprotein into a cell, comprising combining low density lipoprotein with:

a) a chimeric protein which comprises a functional, domain and a carrier domain, wherein the functional domain comprises the amino acid sequence of the ligand-binding domain of human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence which binds a human cell surface receptor other than human low density lipoprotein receptor; and

b) a human cell bearing a surface receptor which binds the amino acid sequence of the carrier domain of the chimeric protein,

under conditions appropriate for binding of low density lipoprotein to the ligand binding domain of low density lipoprotein and for binding of the amino acid sequence of the carrier domain of the chimeric protein with the cell surface receptor on the human cell, whereby a complex of the chimeric protein and low density lipoprotein is formed and the complex is transported into the cell by endocytosis.

29. The method of

claim 28

, wherein the carrier domain binds a human cell surface receptor selected from the group consisting of:

human low density lipoprotein receptors, human transferrin receptors, human serum albumin receptors, human asialoglycoprotein receptors, human adenovirus receptors, human retrovirus receptors, human CD4, human lipoprotein (a) receptors, human immunoglobulin Fc receptor, human a-fetoprotein receptors, human LDLR-like protein (LRP) receptors, human acetylated LDL receptors, human mannose receptors and human mannose-6-phosphate receptors.

30. The method of

claim 28

, wherein the carrier domain comprises an amino acid sequence of human transferrin which binds the cell surface receptor on the human cell.

31. A method of lowering the level of low density lipoprotein in the bloodstream of an individual, comprising administering to the individual a chimeric protein comprising a functional domain and a carrier domain, wherein the functional domain comprises the amino acid sequence of the ligand-binding domain of human low density lipoprotein receptor and the carrier domain comprises an amino acid sequence which binds a human cell surface receptor other than human low density lipoprotein receptor, wherein low density lipoprotein in the bloodstream of the individual binds the ligand binding domain of low density lipoprotein in the chimeric protein and the carrier domain of the chimeric protein binds a human cell surface receptor, whereby the chimeric protein having low density lipoprotein bound thereto is transported into the cell bearing the cell surface receptor, thereby lowering the level of low density lipoprotein in the bloodstream of the individual.

32. The method of

claim 31

, wherein the carrier domain binds a human cell surface receptor selected from the group consisting of:

human low density lipoprotein receptors, human transferrin receptors, human serum albumin receptors, human asialoglycoprotein receptors, human adenovirus receptors, human retrovirus receptors, human CD4, human lipoprotein (a), human immunoglobulin Fc receptor, human a-fetoprotein receptors, human LDLR-like protein (LRP) receptors, human acetylated LDL receptors, human mannose receptors and human mannose-6-phosphate receptors.

33. The method of

claim 31

, wherein the carrier domain comprises an amino acid sequence of human transferrin which binds to the human transferrin receptor.

36. A method of introducing a selected substance into a mammalian cell, comprising contacting a mammalian cell to a chimeric protein which binds the selected substance at a functional domain, and which binds a cell surface receptor on the mammalian cell at a carrier domain, such that the complex of the cell surface receptor, the chimeric protein, and the selected substance forms and the complex is bound to the cell surface receptor and transported into the cell by receptor-mediated endocytosis.

37. The method of

claim 36

, wherein the selected substance is selected from the group consisting of:

acetylated low density lipoprotein, apolipoprotein E4, tumor necrosis factor a, transforming growth factor β

, a cytokine, an immunoglobulin, a hormone, glucose, a bile salt, a glycolipid, and a glycosaminoglycan.

42. The method of claims 36, 37, 38, 39, 40, or 41, in which the mammalian cell is contacted with the chimeric protein in vivo.

38. A method of introducing a functional protein domain into a mammalian cell, comprising contacting a mammalian cell with a chimeric protein which has a functional domain and which binds a cell surface receptor on the mammalian cell at a carrier domain, such that the complex of the cell surface receptor, the chimeric protein, and the selected substance forms and the complex is bound to the cell surface receptor and transported into the cell by receptor-mediated endocytosis.

39. The method of

claim 38

, wherein the functional protein domain comprises an amino acid sequence with enzymatic activity.

40. The method of

claim 38

, wherein the functional protein domain comprises an amino acid sequence which binds to a DNA or RNA sequence and increases or decreases gene expression in the mammalian cell.

41. The method of

claim 38

, wherein the functional protein domain binds to a selected protein, carbohydrate, lipid, or glycolipid target in the mammalian cell.