Human TNF receptor fusion protein

• US 8,063,182 B1
• Filed: 05/19/1995
• Issued: 11/22/2011
• Est. Priority Date: 09/12/1989
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Brockhaus, Manfred, Schlaeger, Ernst-Jurgen, Lesslauer, Werner, Zlatko, Dembic, Gentz, Reiner, Lotscher, Hansruedi
• Primary Examiner(s): Nickol; Gary
• Assistant Examiner(s): HOWARD, ZACHARY C
• Application Number:
  US08/444,790
• Time in Patent Office: 6,031 Days
• Field of Search: 530/324, 530/350, 424/192.1, 536/23.5
• US Class Current: 530/350
• 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: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/7151 for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C12N 2799/026 where the vector is derived from a baculovirus
• CPC Class Codes: Y10S 930/144 Tumor necrosis factor

Abstract

The present invention is concerned with non-soluble proteins and soluble or insoluble fragments thereof, which bind TNF, in homogeneous form, as well as their physiologically compatible salts, especially those proteins having a molecular weight of about 55 or 75 kD (non-reducing SDS-PAGE conditions), a process for the isolation of such proteins, antibodies against such proteins, DNA sequences which code for non-soluble proteins and soluble or non-soluble fragments thereof, which bind TNF, as well as those which code for proteins comprising partly of a soluble fragment, which binds TNF, and partly of all domains except the first of the constant region of the heavy chain of human immunoglobulins and the recombinant proteins coded thereby as well as a process for their manufacture using transformed pro- and eukaryotic host cells.

Claims

1. A protein comprising

(a) a human tumor necrosis factor (TNF)-binding soluble fragment of an insoluble human TNF receptor, wherein the insoluble human TNF receptor (i) specifically binds human TNF, (ii) has an apparent molecular weight of about 75 kilodaltons on a non-reducing SDS-polyacrylamide gel, and (iii) comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10); and

(b) all of the domains of the constant region of a human immunoglobulin IgG heavy chain other than the first domain of said constant region;

wherein said protein specifically binds human TNF.

2. The protein of claim 1, wherein the soluble fragment comprises the peptides LCAP (SEQ ID NO:

12) and VFCT (SEQ ID NO;

     8).

3. The protein of claim 2, wherein the soluble fragment further comprises the peptide LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10).

4. The protein of claim 1, wherein said human immunoglobulin IgG heavy chain is IgG₁.

5. The protein of claim 4, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by pCD4-Hγ

1 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM

     5314).

6. A pharmaceutical composition comprising the protein of claim 4 and a pharmaceutically acceptable carrier material.

7. The protein of claim 1, wherein the protein is purified.

8. The protein of claim 1, wherein the protein is produced by CHO cells.

9. The protein of claim 1, wherein the protein consists of (a) the soluble fragment of the receptor and (b) all of the domains of the constant region of the human immunoglobulin IgG heavy chain other than the first domain of the constant region.

10. The protein of claim 1, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by pCD4-Hγ

1 vector (deposited at Deutschen Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM

     5314) or by pCD4-Hγ

3 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM

     5523).

11. The protein of claim 1, wherein the protein consists essentially of the extracellular region of the insoluble human TNF receptor and all the domains of the constant region of a human IgG₁ immunoglobulin heavy chain other than the first domain of the constant region.

12. A pharmaceutical composition comprising the protein of claim 11 and a pharmaceutically acceptable carrier material.

29. A pharmaceutical composition comprising the protein of any of claim 1, 18, 26, or 27 and a pharmaceutically acceptable carrier material.

13. A protein comprising

(a) a human tumor necrosis factor (TNF)-binding soluble fragment of an insoluble human TNF receptor, wherein the insoluble human TNF receptor (i) specifically binds human TNF, (ii) has an apparent molecular weight of about 75 kilodaltons on a non-reducing SDS-polyacrylamide gel, and (iii) comprises the amino acid sequences LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10), LCAP (SEQ ID NO;

     12), VFCT (SEQ ID NO;

     8), NQPQAPGVEASGAGEA (SEQ ID NO;

     9) and VPHLPAD (SEQ ID NO;

     13),

wherein the soluble fragment comprises the peptides LCAP (SEQ ID NO;

     12) and VFCT (SEQ ID NO;

     8); and

(b) all of the domains of the constant region of a human IgG₁ heavy chain other than the first domain of the constant region;

wherein said protein specifically binds human TNF.

14. The protein of claim 13, wherein the protein is purified.

15. The protein of claim 13, wherein the protein is produced by CHO cells.

16. The protein of claim 13, wherein the protein consists of (a) the soluble fragment of the receptor and (b) all of the domains of the constant region of the human IgG₁ heavy chain other than the first domain of the constant region.

17. The protein of claim 13, wherein the soluble fragment further comprises the peptide LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10).

18. A protein encoded by a polynucleotide which comprises two nucleic acid subsequences,

(a) one of said subsequences encoding a human TNF-binding soluble fragment of an insoluble human TNF receptor protein having an apparent molecular weight of about 75 kilodaltons on a non-reducing SDS-polyacrylamide gel, said soluble fragment comprising the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10), and

(b) the other of said subsequences encoding all of the domains of the constant region of the heavy chain of a human IgG immunoglobulin other than the first domain of said constant region,

wherein said protein specifically binds human TNF.

19. The protein of claim 18, wherein the soluble fragment comprises the peptides LCAP (SEQ ID NO:

12) and VFCT (SEQ ID NO;

     8).

20. The protein of any one of claim 18 or 19, wherein said human immunoglobulin heavy chain is IgG₁.

21. The protein of claim 20, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by the DNA insert of pCD4-Hγ

1 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM

     5314).

22. The protein of claim 18, wherein the protein is purified.

23. The protein of claim 18, wherein the protein is produced by CHO cells.

24. The protein of claim 18, wherein the protein consists of (a) the soluble fragment of the receptor and (b) all of the domains of the constant region of the human IgG₁ heavy chain other than the first domain of the constant region.

25. The protein of claim 18, wherein said domains of the constant region of the human immunoglobulin heavy chain consist essentially of the immunoglobulin amino acid sequence encoded by pCD4-Hγ

1 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM

     5314) or by pCD4-Hγ

3 vector (deposited at Deutschen Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, FRG under No. DSM

     5523).

26. A protein consisting of

(a) a human tumor necrosis factor (TNF)-binding soluble fragment of an insoluble human TNF receptor, wherein the insoluble human TNF receptor (i) specifically binds human TNF, and (ii) has an apparent molecular weight of about 75 kilodaltons on a non-reducing SDS-polyacrylamide gel and (iii) comprises the amino acid sequence LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10),

wherein the soluble fragment comprises the peptides LCAP (SEQ ID NO;

12) and VFCT (SEQ ID NO;

8), and

(b) all of the domains of the constant region of a human IgG₁ heavy chain other than the first domain of the constant region,

wherein the protein specifically binds human TNF, and

wherein the protein is produced by CHO cells.

27. The protein of claim 26, wherein the soluble fragment comprises the peptide LPAQVAFXPYAPEPGSTC (SEQ ID NO:

10).

28. The protein of claim 26, wherein the protein is purified.

30. A protein comprising

(a) human tumor necrosis factor (TNF) binding soluble fragment of the amino acid sequence encoded by the cDNA insert of the plasmid deposited with the ATCC on Oct. 17, 2006 under accession number PTA 7942,

(b) all of the domains of the constant region of a human immunoglobulin IgG heavy chain other than the first domain of said constant region;

wherein said protein specifically binds human TNF.

31. The protein of claim 30, consisting of the soluble fragment and all the domains of the constant region of the human immunoglobulin IgG heavy chain other than the first domain of said constant region.

32. The protein of claim 30, wherein the protein is expressed by a mammalian host cell.

33. The protein of claim 32, wherein the mammalian host cell is a CHO cell.

34. The protein of claim 32, consisting of the soluble fragment and all the domains of the constant region of the human immunoglobulin IgG heavy chain other than the first domain of said constant region.

35. The protein of claim 30, wherein the protein consists essentially of the extracellular region of the human tumor necrosis factor (TNF) receptor amino acid sequence encoded by the cDNA insert, and all the domains of the constant region of a human IgG₁ immunoglobulin heavy chain other than the first domain of the constant region.

36. A pharmaceutical composition comprising the protein of claim 35 and a pharmaceutically acceptable carrier material.