Compositions and methods for production of immunoglobulins

• US 20100239594A1
• Filed: 08/03/2006
• Published: 09/23/2010
• Est. Priority Date: 08/03/2005
• Status: Active Grant

• Current Assignee: iBio, Inc.
• Original Assignee: iBio, Inc.
• Inventors: Hull, Anna, Yusibov, Vidadi, Mett, Vadim
• Granted Patent: US 8,962,278 B2
• Time in Patent Office: Days
• US Class Current: 424/164.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/10 Anthelmintics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 16/1278 from Bacillus (G)
• CPC Class Codes: C07K 2317/13 isolated from plants
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]

Abstract

Provided are oligonucleotides for isolating human antibody cDNAs from cells or cell lines, such as hybridomas. The invention also provides cDNAs that encode at least one provided CDR of a heavy chain or a light chain of a human monoclonal antibody that binds to B. anthracis protective antigen; and cDNAs that encode at least one provided CDR of a heavy chain or a light chain of a human monoclonal antibody that binds to B. anthracis lethal factor. The invention further provides expression vectors that contain one or more cDNAs isolated according to the methods of the invention, host cells expressing one or more inventive cDNAs, and transgenic plants and animals that express one or more inventive cDNAs. In certain embodiments of the invention the expression system is a plant-based expression system. The invention further provides antibody compositions comprising one or more antibodies produced by expressing a cDNA isolated according to the methods of the invention in a suitable expression system. Additionally encompassed in the invention are kits containing one or more of provided compositions, as well as methods of production and use of provided compositions.

Claims

1. An isolated antibody or functional fragment thereof, comprising:

(a) one or more light chain (LC) complementary determining regions (CDRs) selected from the group consisting of;

(i) a light chain CDR1 with at least 90% sequence identity to PA-1CDR1;

RASQSVSYSSLA (SEQ ID NO;

59);

(ii) a light chain CDR2 with at least 90% sequence identity to PA-1CDR2;

GASSRAT (SEQ ID NO;

60); and

(iii) a light chain CDR3 with at least 90% sequence identity to PA-1CDR3;

QHYGNSPYT (SEQ ID NO;

61); and

(b) one or more heavy chain (HC) complementary determining regions (CDRs) selected from the group consisting of;

(iv) a heavy chain CDR1 with at least 90% sequence identity to PA-hCDR1;

GYTFTSNAIQ (SEQ ID NO;

62);

(v) a heavy chain CDR2 with at least 90% sequence identity to PA-hCDR2;

WINGGDGNTKYSQKFQG (SEQ ID NO;

63); and

(vi) a heavy chain CDR3 with at least 90% sequence identity to PA-hCDR3;

HRLQRGGFDP (SEQ ID NO;

64);

wherein the antibody or functional fragment thereof can bind specifically to B. anthracis protective antigen.

2. The antibody or functional fragment of claim 1, comprising:

(a) two or more light chain (LC) complementary determining regions (CDRs) selected from the group consisting of;

(i) a light chain CDR1 with at least 90% sequence identity to PA-1CDR1;

RASQSVSYSSLA (SEQ ID NO;

59);

(ii) a light chain CDR2 with at least 90% sequence identity to PA-1CDR2;

GASSRAT (SEQ ID NO;

60);

(iii) a light chain CDR3 with at least 90% sequence identity to PA-1CDR3;

QHYGNSPYT (SEQ ID NO;

61); and

(b) two or more heavy chain complementary determining regions (CDRs) selected from the group consisting of;

(iv) a heavy chain CDR1 with at least 90% sequence identity to PA-hCDR1;

GYTFTSNAIQ (SEQ ID NO;

62);

(v) a heavy chain CDR2 with at least 90% sequence identity to PA-hCDR2;

WINGGDGNTKYSQKFQG (SEQ ID NO;

63); and

(vi) a heavy chain CDR3 with at least 90% sequence identity to PA-hCDR3;

HRLQRGGFDP (SEQ ID NO;

64);

wherein the antibody or functional fragment thereof can bind specifically to B. anthracis protective antigen.

3. The antibody or functional fragment of claim 1, comprising:

(a) three light chain (LC) complementary determining regions (CDRs) consisting of;

(i) a light chain CDR1 with at least 90% sequence identity to PA-1CDR1;

RASQSVSYSSLA (SEQ ID NO;

59);

(ii) a light chain CDR2 with at least 90% sequence identity to PA-1CDR2;

GASSRAT (SEQ ID NO;

60); and

(iii) a light chain CDR3 with at least 90% sequence identity to PA-1CDR3;

QHYGNSPYT (SEQ ID NO;

61); and

(b) three heavy chain complementary determining regions (CDRs) consisting of;

(iv) a heavy chain CDR1 with at least 90% sequence identity to PA-hCDR1;

GYTFTSNAIQ (SEQ ID NO;

62);

(v) a heavy chain CDR2 with at least 90% sequence identity to PA-hCDR2;

WINGGDGNTKYSQKFQG (SEQ ID NO;

63); and

(vi) a heavy chain CDR3 with at least 90% sequence identity to PA-hCDR3;

HRLQRGGFDP (SEQ ID NO;

64);

wherein the antibody or functional fragment thereof can bind specifically to B. anthracis protective antigen.

7. The antibody, or functional fragment thereof, of any one of claims 1-3, wherein said functional fragment is selected from the group consisting of Fv, Fab, F(ab)₂ and scFV.

8. A nucleic acid encoding the antibody or functional fragment of claim 1.

9. A composition comprising the antibody or functional fragment of claim 1 and a pharmaceutically acceptable carrier.

4. An isolated antibody or functional fragment thereof, comprising:

(a) one or more light chain (LC) complementary determining regions (CDRS) selected from the group consisting of;

(i) a light chain CDR1 with at least 90% sequence identity to LF-1CDR1;

RASQSVGSSLH (SEQ ID NO;

65);

(ii) a light chain CDR2 with at least 90% sequence identity to LF-1CDR2;

YASQSFS (SEQ ID NO;

66); and

(iii) a light chain CDR3 with at least 90% sequence identity to LF-1CDR3;

HQSSSLPLT (SEQ ID NO;

67);

and (b) one or more heavy chain (HC) complementary determining regions (CDRS) selected from the group consisting of;

(iv) a heavy chain CDR1 with at least 90% sequence identity to LF-hCDR1;

GFMFSSYAMS (SEQ ID NO;

68);

(v) a heavy chain CDR2 with at least 90% sequence identity to LF-hCDR2;

GISGSGGTTNYADSVKG (SEQ ID NO;

69); and

(vi) a heavy chain CDR3 with at least 90% sequence identity to LF-hCDR3;

DGVYGRLGGSDY (SEQ ID NO;

70);

wherein the antibody or functional fragment thereof can bind specifically to B. anthracis lethal factor.

5. The antibody or functional fragment thereof of claim 4, comprising:

(a) two or more light chain (LC) complementary determining regions (CDRS) selected from the group consisting of;

(i) a light chain CDR1 with at least 90% sequence identity to LF-1CDR1;

RASQSVGSSLH (SEQ ID NO;

65);

(ii) a light chain CDR2 with at least 90% sequence identity to LF-1CDR2;

YASQSFS (SEQ ID NO;

66); and

(iii) a light chain CDR3 with at least 90% sequence identity to LF-1CDR3;

HQSSSLPLT (SEQ ID NO;

67);

and (b) two or more heavy chain (HC) complementary determining regions (CDRS) selected from the group consisting of;

(iv) a heavy chain CDR1 with at least 90% sequence identity to LF-hCDR1;

GFMFSSYAMS (SEQ ID NO;

68);

(v) a heavy chain CDR2 with at least 90% sequence identity to LF-hCDR2;

GISGSGGTTNYADSVKG (SEQ ID NO;

69); and

(vi) a heavy chain CDR3 with at least 90% sequence identity to LF-hCDR3;

DGVYGRLGGSDY (SEQ ID NO;

70);

wherein the antibody or functional fragment thereof can bind specifically to B. anthracis lethal factor.

6. The antibody or functional fragment thereof, of claim 4, comprising:

(a) three light chain (LC) complementary determining regions (CDRS) consisting of;

(i) a light chain CDR1 with at least 90% sequence identity to LF-1CDR1;

RASQSVGSSLH (SEQ ID NO;

65);

(ii) a light chain CDR2 with at least 90% sequence identity to LF-1CDR2;

YASQSFS (SEQ ID NO;

66); and

(iii) a light chain CDR3 with at least 90% sequence identity to LF-(CDR3;

HQSSSLPLT (SEQ ID NO;

67);

and (b) three heavy chain (HC) complementary determining regions (CDRS) consisting of;

(iv) a heavy chain CDR1 with at least 90% sequence identity to LF-hCDR1;

GFMFSSYAMS (SEQ ID NO;

68);

(v) a heavy chain CDR2 with at least 90% sequence identity to LF-hCDR2;

GISGSGGTTNYADSVKG (SEQ ID NO;

69); and

(vi) a heavy chain CDR3 with at least 90% sequence identity to LF-hCDR3;

DGVYGRLGGSDY (SEQ ID NO;

70);

wherein the antibody or functional fragment thereof can bind specifically to B. anthracis lethal factor.

10. The antibody, or functional fragment thereof, of claim 4, wherein said functional fragment is selected from the group consisting of Fv, Fab, F(ab)₂ and scFV.

11. A nucleic acid encoding the antibody or functional fragment of claim 4.

12. A composition comprising the antibody or functional fragment of claim 4 and a pharmaceutically acceptable carrier.

13. An isolated polypeptide comprising a polypeptide sequence consisting of any of SEQ ID NOs:

46, 48, 50, or 52.

15. A polypeptide of claim 13 or claim 14, wherein said polypeptide is not glycosylated.

16. An isolated nucleic acid that encodes a polypeptide of claim 13.

17. The isolated nucleic acid of claim 16, wherein said nucleic acid sequence is not a naturally occurring sequence.

14. An isolated polypeptide consisting of a polypeptide sequence of any one of SEQ ID NO:

46, 48, 50, or 52.

18. An oligonucleotide primer whose sequence consists of or comprises any of SEQ ID NOs:

1-44.

19. A primer mix containing at least two oligonucleotide primers of claim 18.

20. The primer mix of claim 19, containing at least two primers, wherein the at least two primers are of use in an amplification reaction to amplify an antibody heavy chain or light chain.

21. A nucleic acid amplified from an antibody-producing cell or cell line using a primer of claim 18.

22. An expression vector, cell, transgenic animal, or transgenic plant containing the nucleic acid of claim 21.

23. A plant viral vector or replicon comprising the nucleic acid of claim 21.

24. A method of producing an antibody chain comprising expressing the nucleic acid of claim 21 in a suitable expression system.

25. The method of claim 24, wherein the expression system is a plant-based expression system.

26. A method of isolating a nucleic acid that encodes an antibody heavy chain or light chain comprising amplifying said nucleic acid from a cell or cell line that produces the antibody heavy chain or light chain using one or more primers selected from the group consisting of SEQ ID NOs:

1-44.

27. An isolated nucleic acid comprising a nucleic acid sequence consisting of any of SEQ ID NOs:

45, 47, 49, or 51.

29. An expression vector comprising the nucleic acid of claim 27 or claim 28.

30. A host cell comprising the expression vector of claim 29.

31. The host cell of claim 30, which is a plant cell.

32. A method of producing an antibody or functional fragment thereof, comprising culturing the host cell of claim 30 or claim 31 under conditions sufficient for antibody production, and purifying the produced antibody.

28. An isolated nucleic acid consisting of a nucleic acid sequence of any one of SEQ ID NOs:

45, 47, 49, or 51.