Recombinant bi-functional fusion proteins, preparations and methods for treating disease

• US 9,527,901 B2
• Filed: 03/24/2015
• Issued: 12/27/2016
• Est. Priority Date: 03/24/2014
• Status: Active Grant

• Current Assignee: Macroimmune Inc.
• Original Assignee: Macroimmune Inc.
• Inventors: Jing, Deqiang
• Primary Examiner(s): LANDSMAN, ROBERT S
• Application Number:
  US14/666,328
• Publication Number:
  US 20150266942A1
• Time in Patent Office: 644 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 16/1063 env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
• CPC Class Codes: C07K 16/1253 from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
• CPC Class Codes: C07K 2317/10 characterized by their source of isolation or production
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/30 characterized by aspects of specificity or valency
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

A recombinant bi-functional fusion protein, comprising an Ig region of an extracellular domain of a signal-regulator protein (SIRP), linked via a Fc fragment of an Ig, to an Ig region of an extracellular domain of VEGFR, wherein the protein can bind to CD47 and VEGF simultaneously, blocking the binding of CD47 with the SIRP on the cell surface of macrophages to stimulate the phagocytosis of tumor cells by macrophages, and inhibiting the growth of vascular endothelial cells induced by VEGF. The present application also provides a nucleic acid molecule encoding the recombinant bi-functional fusion protein and an expression vector expressing the protein, a method for producing the protein and a method for treating a disease over-expressing CD47 or VEGF.

Claims

1. A recombinant bi-functional fusion protein, comprising an Ig-like region of an extracellular domain of a signal-regulatory protein alpha (SIRPα

), linked via a linker, to an Ig-like region of an extracellular domain of vascular endothelial growth factor (VEGF) Receptor (VEGFR), wherein the protein can bind to Cluster of Differentiation 47 (CD47) and YEW blocking the binding of CD47 to the SIRP on the cell surface of macrophages to stimulate the phagocytosis of tumor cells by macrophages, and inhibiting the growth of vascular endothelial cells induced by VEGF.

2. The recombinant bi-functional fusion protein according to claim 1, wherein the Ig-like region of the extracellular domain of the signal-regulatory protein is SIRPα

D1.

3. The recombinant bi-functional fusion protein according to claim 1, wherein the VEGFR is VEGFR1.

4. The recombinant bi-functional fusion protein according to claim 1, wherein the Ig-like region of VEGFR1 is a second Ig-like region of the extracellular domain of VEGFR1 (VEGFR1D2).

5. The recombinant bi-functional fusion protein according to claim 1, wherein the linker is an Fc fragment of an Ig.

6. The recombinant bi-functional fusion protein according to claim 1, wherein the Fc fragment is an Fc fragment of IgG1.

7. The recombinant bi-functional fusion protein according to claim 1, comprising a second Ig-like region of an extracellular domain of human VEGFR1, linked via Fc fragment of human IgG1 to the first Ig-like region of the extracellular domain of SIRPα

(SIRPα

D1).

8. The recombinant bi-functional fusion protein according to claim 1, comprising an amino acid sequence which is at least 95% identical to SEQ ID NO:

8.

9. The recombinant bi-functional fusion protein according to claim 8, comprising the amino acid sequence of SEQ ID NO:

8.

10. The recombinant bi-functional fusion protein according to claim 8, comprising an amino acid sequence which is at least 98% identical to SEQ ID NO:

8.

11. The recombinant bi-functional fusion protein according to claim 10, comprising an amino acid sequence which is at least 99% identical to SEQ ID NO:

8.

12. A polynucleotide encoding the recombinant bi-functional fusion protein according to claim 8.

13. A polynucleotide encoding the recombinant bi-functional fusion protein according to claim 9.

14. An expression vector comprising a polynucleotide of claim 12.

15. An expression vector comprising a polynucleotide of claim 13.

16. An isolated host cell comprising the expression vector of claim 14.

17. The isolated host cell of claim 16, wherein the host cell is a prokaryotic cell.

18. The isolated host cell of claim 16, wherein the host cell is a yeast cell.

19. The isolated host cell of claim 16, wherein the host cell is a yeast cell.

20. The isolated host cell of claim 16, wherein the host cell is a mammalian cell.

21. The isolated host cell of claim 20, wherein the host cell is CHO cell.

22. The isolated host cell of claim 20, wherein the host cell is a human cell.

23. A pharmaceutical composition, comprising recombinant bi-functional fusion protein of claim 1, and at least one pharmaceutical adjuvant.

24. A method for treating a disease caused by over-expression of CD47, comprising administering to a patient or a subject a therapeutically effective amount of the pharmaceutical composition according to claim 23.

25. The method according to claim 24, wherein the disease is selected from the group of acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), acute lymphoblastic leukemia (ALL), non-Hodgkin'"'"'s lymphoma (NHL), multiple myeloma (MM), Bladder cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, breast cancer, pancreatic cancer, and renal cell carcinoma.

26. The method according to claim 24, wherein the disease is selected from the group of Crohn'"'"'s disease, allergic asthma and rheumatoid arthritis.

27. A method for treating a disease caused by enhanced VEGF function and activity, comprising administering to a patient or a subject a therapeutically effective amount of the pharmaceutical composition according to claim 23.

28. The method according to claim 24, wherein the disease is selected from age-related macular degeneration, AMD, diabetic retinopathy, liver fibrosis and angiosarcoma.