Serine protease molecules and therapies

Patent metadata

Field	Value
Publication number	US-9096840-B2
Application number	US-201314046211-A
Country	US
Kind code	B2
Filing date	Oct 4, 2013
Priority date	Oct 4, 2012
Publication date	Aug 4, 2015
Grant date	Aug 4, 2015

Abstract

Cell-targeted serine protease constructs are provided. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer). In some aspects, recombinant serine proteases, such as Granzyme B polypeptides, are provided that exhibit improved stability and cell toxicity. Methods and compositions for treating lapatinib or trastuzumab-resistant cancers are also provided.

First claim

What is claimed is:

1. A recombinant polypeptide comprising a Granzyme B (GrB) sequence at least 85 identical to SEQ ID NO: 1 wherein the GrB sequence comprises one or more of the following features:
(a) an amino acid substitution or deletion at the position corresponding to Asp 37;
(b) an amino acid substitution or deletion at the position corresponding to Asn 51;
(c) an amino acid substitution or deletion at the position corresponding to Asn 84;
(d) an amino acid substitution or deletion at the position corresponding to Arg 96;
(e) an amino acid substitution or deletion at the position corresponding to Arg 100;
(f) an amino acid substitution or deletion at the position corresponding to Arg 102;
(g) an amino acid substitution or deletion at the position corresponding to Asp 150;
(h) an amino acid substitution or deletion at the position corresponding to Arg 201;
(i) an amino acid substitution or deletion at the position corresponding to Cys 210;
(j) an amino acid substitution or deletion at the position corresponding to Lys 221;
(k) an amino acid substitution or deletion at the position corresponding to Lys 222;
(l) an amino acid substitution or deletion at the position corresponding to Arg 225; or
(m) an amino acid substitution or deletion at the position corresponding to Arg 226.

2. The polypeptide of claim 1 , wherein the polypeptide further comprises an additional amino acid sequence comprising a Cys, wherein the amino acid sequence is positioned C-terminally relative to the GrB coding sequence.

3. The polypeptide of claim 2 , wherein the additional amino acid sequence comprises the sequence SSCSGSA (SEQ ID NO: 12) positioned C-terminally relative to the GrB coding sequence.

4. The polypeptide of claim 1 , wherein the GrB coding sequence comprises two, three, four or five of said features.

5. The polypeptide of claim 4 , comprising an amino acid substitution or deletion at the position corresponding to Asp 37 or Asp 150.

6. The polypeptide of claim 4 , comprising an amino acid substitution or deletion at the position corresponding to Asn 51 or Asn 84.

7. The polypeptide of claim 4 , comprising an amino acid substitution or deletion at the position corresponding to Arg 96, Arg 100, Arg 102, Arg 201 or Arg 226.

8. The polypeptide of claim 4 , comprising an amino acid substitution or deletion at the position corresponding to Lys 221, Lys 222 or Lys 225.

9. The polypeptide of claim 1 , wherein the polypeptide comprises an Ala substitution at the position corresponding to Cys 210.

10. The polypeptide of claim 1 , wherein the polypeptide is conjugated to or fused with a cell binding moiety.

11. The polypeptide of claim 10 , wherein the polypeptide is conjugated to the cell binding moiety by a thioester linkage.

12. The polypeptide of claim 1 , wherein the polypeptide is fused with an additional amino acid sequence comprising a cell binding moiety positioned C-terminally relative to the GrB coding sequence.

13. The polypeptide of claim 10 , wherein the cell binding moiety is a VEGF, BLyS, an antibody or a cell-binding portion of any of the foregoing.

14. The polypeptide of claim 13 , wherein the antibody is a monoclonal, chimeric antibody, Fab′, Fab, F(ab′)2, single domain antibody, Fv, or single chain Fv (scFv) antibody.

15. The polypeptide of claim 13 , wherein the antibody is a human antibody, a humanized antibody or a deimmunized antibody.

16. The polypeptide of claim 13 , wherein the antibody is a 15A8, ZME-018, ScFvMEL, cetuximab or trastuzumab antibody.

17. The polypeptide of claim 10 , wherein the cell binding moiety binds to a protein, carbohydrate or lipid expressed on cancer cells.

18. The polypeptide of claim 10 , wherein the cell binding moiety binds to GP240, 5T4, HER1, HER2, CD-33, CD-38, fltl, Flk-1, CEA, FGFR3, IGFBP2 or IGF-1R.

19. The polypeptide of claim 10 , wherein the polypeptide or cell binding moiety is further conjugated to an imaging agent.

20. A composition comprising a GrB polypeptide in accordance with claim 1 in a pharmaceutically acceptable carrier.

21. The polypeptide of claim 1 , wherein the GrB coding sequence at least 90% identical to SEQ ID NO: 1.

22. The polypeptide of claim 21 , wherein the GrB coding sequence at least 95% identical to SEQ ID NO: 1.

23. The polypeptide of claim 1 , wherein the GrB coding sequence further comprises an amino acid substitution or deletion at the position corresponding to Lys 27 and/or Arg 28.

24. The polypeptide of claim 23 , wherein the GrB coding sequence comprises an amino acid substitution K27E and/or R28A.

Assignees

• Res Dev Foundation

Inventors

• Rosenblum Michael G
• Mohamedali Khalid Amanali
• Cheung Lawrence H

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P35/02

  specific for leukemia · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61P17/00

  Drugs for dermatological disorders · CPC title