Soluble HER2 and HER3 splice variant proteins, splice-switching oligonucleotides, and their use in the treatment of disease

• US 20090105139A1
• Filed: 06/06/2008
• Published: 04/23/2009
• Est. Priority Date: 06/06/2007
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Sazani, Peter, Kole, Ryszard, Wan, Jing
• Granted Patent: US 7,884,194 B2
• Time in Patent Office: Days
• US Class Current: 514/1.100
• 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: C07K 14/71 for growth factors; for growth regulators

Abstract

Soluble epidermal growth factor receptors 2 and 3 (HER2 and HER3) splice variant proteins with HER2 and HER3 antagonist activity and anti-proliferative properties, as well as the corresponding nucleic acids, are provided for treatment of proliferative diseases, in particular cancer. Also provided are compositions and methods for inducing expression of these splice variants, including splice switching oligonucleotides that modulate splicing of pre-mRNA that codes for these receptors.

Claims

1. An isolated, soluble, human epidermal growth factor receptor-2 (HER2) protein lacking the region encoded by exon 15 of the full-length mRNA transcript of the HER2 gene, and truncated, at its C terminus, in the region encoded by exon 16 of the HER2 transcript.

2. The protein of claim 1, wherein the sequence of said protein comprises a sequence having at least 95% sequence homology to the sequence identified by SEQ ID No:

6 or amino acids 23-584 of SEQ ID No;

6.

3. The protein of claim 1, which is modified by protein pegylation.

4. An isolated nucleic acid encoding human epidermal growth factor receptor-2 (HER2) protein, but lacking exon 15 of the normal (HER2) transcript, with exon 14 joined directly to exon 16, and containing a stop codon within exon 16.

5. The nucleic acid of claim 4, having at least 85% sequence homology to the sequence identified by SEQ ID NO:

5 or that portion of the sequence terminating at a stop codon within exon 16.

6. A method of treating a female subject having an ovarian or breast cancer characterized by overexpression of human epidermal growth factor receptor-2 (HER2), comprising

administering to the subject, a pharmaceutically effective amount of a soluble, human epidermal growth factor receptor-2 (HER2) protein lacking the region encoded by exon 15 of the full-length mRNA transcript of the HER2 gene, and truncated, at its C terminus, in the region encoded by exon 16 of the HER2 transcript, and

continuing said administering, at periodic intervals, until a defined end point in the status of the cancer is obtained.

7. The method of claim 6, wherein the sequence of said protein comprises a sequence having at least 95% sequence homology to SEQ ID No:

6 or to amino acids 23-584 of SEQ ID No;

6.

8. The method of claim 7, in which the protein is modified by pegylation.

9. A splice-switching oligonucleotide compound comprising

an oligonucleotide containing between 12-30 bases and at least 12 contiguous bases complementary to an exon-15 acceptor or donor splice site region contained within SEQ ID. NO:

15 of the full-length mRNA transcript of human epidermal growth factor receptor-2 (HER2) protein.

10. The compound of claim 9, wherein the oligonucleotide contains between 12 and 25 bases and a sequence of at least 12 contiguous bases complementary to a region contained with SEQ ID NOS:

44 or 45.

11. The compound of claim 9, wherein the oligonucleotide is selected from the group consisting of a locked nucleic acid, 2′

-O-methoxyethyloligoribonucleotide, and a phosphorodiamidate morpholino oligonucleotide.

12. The compound of claim 9, which further includes, conjugated to the 5′

- or 3′

-end of the oligonucleotide, an arginine-rich polypeptide effective to promote uptake of the compound into cells.

13. The compound of claim 12, wherein the oligonucleotide is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

14. The compound of claim 13, wherein the morpholino subunits in the oligonucleotide compound to which the cells are exposed is administered to the subject are joined by phosphorodiamidate linkages having the structure:

15. A method of treating a female subject having an ovarian or breast cancer characterized by overexpression of human epidermal growth factor receptor-2 (HER2), comprising

administering to the subject, a pharmaceutically effective amount of a compound comprising an oligonucleotide containing between 12-30 bases and at least 12 contiguous bases complementary to an exon-14 acceptor or donor splice site region contained within SEQ ID. NO:

15 of the full-length mRNA transcript of human epidermal growth factor receptor-2 (HER2) protein, and

continuing said administering, at periodic intervals, until a defined end point in the status of the cancer is obtained.

16. The method of claim 15, wherein the oligonucleotide in the compound administered to the subject is selected from the group consisting of a locked nucleic acid, 2′

-O-methoxyethyloligoribonucleotide, and a phosphorodiamidate morpholino oligonucleotide.

17. The method of claim 15, wherein the compound administered to the subject further includes, conjugated to the 5′

- or 3′

-end of the oligonucleotide, an arginine-rich polypeptide effective to promote uptake of the compound into infected host cells.

18. The method of claim 15, wherein the oligonucleotide in the compound administered to the subject is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit.

19. The method of claim 18, wherein the morpholino subunits in the compound to which the host cells are exposed are joined by phosphorodiamidate linkages having the structure:

20. The method of claim 15, which further includes administering to the subject, a pharmaceutically effective amount of a soluble, human epidermal growth factor receptor-2 (HER2) protein lacking the region encoded by exon 15 of the full-length mRNA transcript of the HER2 gene, and truncated, at its C terminus, in the region encoded by exon 16 of the HER2 transcript.