Transgenically produced fusion proteins

• US 20040117863A1
• Filed: 06/27/2003
• Published: 06/17/2004
• Est. Priority Date: 09/18/1998
• Status: Abandoned Application

• Current Assignee: Dan Pollock, Harry M. Meade, Michael D. Edge, Susanna M. Rybak, Yann Echelard
• Original Assignee: Dan Pollock, Harry M. Meade, Michael D. Edge, Susanna M. Rybak, Yann Echelard
• Inventors: Echelard, Yann, Meade, Harry M., Rybak, Susanna M., Pollock, Dan, Edge, Michael D.
• Application Number:
  US10/608,710
• Publication Number:
  US 20040117863A1
• Time in Patent Office: Days
• US Class Current: 800/7
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/206 Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2227/101 Bovine
• CPC Class Codes: A01K 2227/102 Caprine
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• CPC Class Codes: C07K 16/04 from milk
• CPC Class Codes: C07K 16/2881 against CD71
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/54 F(ab')2
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/55 : containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C07K 2319/75 : containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8509 : for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2830/008 : cell type or tissue specific enhancer/promoter combination

Abstract

A method of making a transgenic fusion protein. The method inlcudes providing a transgenic animal which includes a transgene which provides for the expression of the fusion protein; allowing the transgene to be expressed; and, recovering the fusion protein, from the milk of the transgenic animal.

Claims

1. A method of making a transgenic fusion protein comprising providing a transgenic animal which includes a transgene which provides for the expression of the fusion protein;

allowing the transgene to be expressed; and

, recovering the fusion protein, from the milk of the transgenic animal.

2. The method of claim 1, wherein the fusion protein includes an immunoglobulin-subunit and an enzyme.

3. The method of claim 1, wherein the fusion protein includes a first member fused to a second member and the first member includes the subunit of a targeting molecule and the second member encodes a cell toxin.

4. The method of claim 1, wherein the fusion protein includes a subunit of an Ig specific for a tumor antigen.

5. The method of claim 4, wherein the tumor antigen is from the group carcinoembryonic antigen (CEA), a transferring receptor, TAG-72, an epidermal growth factor receptor.

6. The method of claim 1, wherein the fusion protein includes an Rnase.

7. The method of claim 6, wherein the RNase is RnaseA.

8. The method of claim 1, wherein the fusion protein includes angiogenin.

9. The method of claim 1, wherein the fusion protein includes carboxypeptidase B enzyme.

10. The method of claim 1, wherein the fusion protein is made in a mammary gland of the transgenic mammal.

11. The method of claim 1, wherein the fusion protein is secreted into the milk of a transgenic mammal at concentrations of at least about 0.5 mg/ml or higher.

12. The method of claim 1, wherein the fusion protein is secreted into the milk of a transgenic mammal at concentrations of at least about 1.0 mg/ml or higher.

13. The method of claim 1, the immunoglobulin subunit of a fusion protein is a humanized antibody.

14. The method of claim 1, wherein the transgene encoding the transgenic fusion protein is a nucleic acid construct which includes:

(a) optionally, an insulator sequence;

(b) a mammary epithelial specific promoter;

(c) a nucleotide sequence which encodes a signal sequence which can direct the secretion of the fusion protein, e.g. a signal from a milk specific protein;

(d) optionally, a nucleotide sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g. a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of the fusion protein;

(e) one or more nucleotide sequences which encode the fusion protein; and

(f) optionally, a 3′

untranslated region from a mammalian gene.

15. An isolated nucleic acid construct, which includes:

(a) optionally, an insulator sequence;

(b) a mammary epithelial specific promoter;

(c) a nucleotide sequence which encodes a signal sequence which can direct the secretion of the fusion protein, e.g. a signal sequence from a milk specific protein;

(d) optionally, a nucleotide sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein, e.g. a protein secreted into milk, to allow secretion, e.g., in the milk of a transgenic mammal, of fusion protein;

(e) one or more nucleotide sequences which encode a fusion protein as described in claim 1;

and

(f) optionally, a 3′

untranslated region from a mammalian gene, e.g., a mammary epithelial specific gene, (e.g., a milk protein gene).

In another aspect, the invention features, a pharmaceutical or nutraceutical composition having an effective amount of fusion protein, e.g., an immunoglobulin-enzyme fusion protein as described herein, and a pharmaceutically acceptable carrier.

In a preferred embodiment, the composition includes milk.

16. The transgenic animal of claim 15, which can secrete the fusion protein into its milk at concentrations of at least about 0.5 mg/mll or higher.

15-1. A transgenic animal which includes a transgene that encodes a fusion protein described in claim.