Immunoproteases

Patent metadata

Field	Value
Publication number	US-9944706-B2
Application number	US-201314440947-A
Country	US
Kind code	B2
Filing date	Nov 4, 2013
Priority date	Nov 7, 2012
Publication date	Apr 17, 2018
Grant date	Apr 17, 2018

Abstract

The technology provided herein relates to novel immunoproteases suitable to induce apotosis in selected diseased target cells, comprising the serine protease granzyme M, and methods for using such cytolytic fusion proteins for the treatment of various diseases, in particular for the treatment of cancer.

First claim

The invention claimed is:

1. A cytolytic fusion protein suitable to induce apoptosis in a target cell comprising at least a first polypeptide comprising a binding structure, wherein the binding structure is an antibody or an antibody binding fragment comprising the amino acid sequence of SEQ ID NO. 8 or SEQ ID NO. 10, which allows binding of the fusion protein to a specific target structure on the surface of a diseased cell, wherein the specific target structure is CD25 or EGFR, and at least a second polypeptide comprising a cytolytic serine protease, wherein the serine protease is granzyme M.

2. The cytolytic fusion protein according to claim 1 , wherein the diseased cell is a cancer cell.

3. The cytolytic fusion protein according to claim 1 , wherein the antibody or antibody fragment is selected from the group consisting of an Fab, a scFv, a bis scFv, an Fab2, an Fab3, a minibody, a diabody, a triabody, a tetrabody, and a tandab.

4. The cytolytic fusion protein according to claim 1 , wherein the cytolytic serine protease comprises the amino acid sequence of SEQ ID NO. 2.

5. The cytolytic fusion protein according to claim 1 , wherein the cytolytic fusion protein comprises the amino acid sequence of SEQ ID NO. 8.

6. The cytolytic fusion protein according to claim 1 , further comprising one or more further polypeptide(s) selected from the group consisting of a leader sequence capable of controlling protein biosynthesis, a protein tag, a translocation domain amphiphatic sequence capable of translocating the fusion protein into the cytosol of the target cell, and a synthetic pro-serine protease amphiphatic sequence capable of intracellular activation of the serine protease.

7. The cytolytic fusion protein according to claim 1 , wherein the cytolytic fusion protein further comprises a leader sequence for secretory expression, an enterokinase cleavage site, and a HIS tag.

8. A nucleic acid molecule encoding the cytolytic fusion protein according to claim 1 .

9. A vector comprising the nucleic acid molecule of claim 8 .

10. A host cell transformed with the vector of claim 9 .

11. A method for preparing a cytolytic fusion protein, which comprises culturing the host cell of claim 10 under conditions for expressing the protein encoded by the nucleic acid molecule; and isolating the cytolytic fusion protein from the culture.

12. A pharmaceutical composition comprising the cytolytic fusion protein according to claim 1 .

13. The pharmaceutical composition according to claim 12 , wherein the composition further comprises a second cytolytic fusion protein suitable to induce apoptosis in a target cell comprising at least a second binding structure embodied as an antibody or an antibody fragment, which allows binding of the second fusion protein to a second specific target structure on the surface of a diseased cell, wherein the second specific structure is selected from the group consisting of CD64, CD25, CD30, and EGFR and at least a second polypeptide comprising a cytolytic serine protease, wherein the serine protease is granzyme B.

14. The pharmaceutical composition according to claim 13 , wherein the second binding structure comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, and SEQ ID NO. 10, further wherein the binding structure and second binding structure bind different specific target structures on the surface of a same diseased cell.

15. A cytolytic fusion protein suitable to induce apoptosis in a target cell comprising at least a first polypeptide comprising a binding structure comprising the amino acid sequence of SEQ ID NO. 6, which allows binding of the fusion protein to CD30 on the surface of a diseased cell, and at least a second polypeptide comprising a cytolytic serine protease, wherein the serine protease is granzyme M.

16. A cytolytic fusion protein suitable to induce apoptosis in a target cell comprising at least a first polypeptide comprising a binding structure comprising the amino acid sequence of SEQ ID NO. 4, which allows binding of the fusion protein to CD64 on the surface of a diseased cell, and at least a second polypeptide comprising a cytolytic serine protease, wherein the serine protease is granzyme M.

17. The cytolytic fusion protein according to claim 15 , wherein the first polypeptide is selected from the group consisting of an Fab, a scFv, a bis scFv, an Fab2, an Fab3, a minibody, a diabody, a triabody, a tetrabody, and a tandab.

18. The cytolytic fusion protein according to claim 16 , wherein the first polypeptide is selected from the group consisting of an Fab, a scFv, a bis scFv, an Fab2,an Fab3, a minibody, a diabody, a triabody, a tetrabody, and a tandab.

19. The cytolytic fusion protein according to claim 1 , wherein the amino acid comprises SEQ ID NO. 10.

Assignees

• Fraunhofer Ges Forschung

Inventors

• Barth Stefan
• Schiffer Sonja

Classifications

• C07K2317/622

  Single chain antibody (scFv) · CPC title

• C12N9/6467

  Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79) · CPC title

• C07K16/283 Primary

  against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851) · CPC title

• C07K16/2863

  against receptors for growth factors, growth regulators · CPC title

• C12N9/6427

  Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4) · CPC title