Immunoglobulin DNA cassette molecules, monobody constructs, methods of production, and methods of use therefor

• US 20040033561A1
• Filed: 10/17/2002
• Published: 02/19/2004
• Est. Priority Date: 10/19/2001
• Status: Active Grant

• Current Assignee: Takeda Pharmaceutical Company Limited
• Original Assignee: Millennium Pharmaceuticals Incorporated (Takeda Pharmaceutical Company Limited)
• Inventors: Healey, Judith Jacques, OʼKeefe, Theresa L., Keyt, Bruce A., Newman, Walter, Ponath, Paul D.
• Granted Patent: US 7,053,202 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Featured are DNA molecules, expression vectors, and host cells useful for creation of immunoglobulins, as well as novel immunoglobulin molecules termed monobodies. Additionally provided are methods of production of immunoglobulins, including monobodies, as well as methods of using the disclosed immunoglobulin and monobody constructs, expression vectors and host cells containing DNA encoding molecules for production of immunoglobulin and monobody proteins.

Claims

1. An isolated nucleic acid molecule comprising an immunoglobulin cassette construct, wherein the immunoglobulin cassette comprises an immunoglobulin leader molecule operably linked to a stable immunoglobulin domain region.

2. The isolated nucleic acid molecule of claim 1 wherein the immunoglobulin cassette is a light chain immunoglobulin cassette.

3. The isolated nucleic acid molecule of claim 1 wherein the immunoglobulin cassette is a heavy chain immunoglobulin cassette.

4. The isolated nucleic acid molecule of claim 1 wherein the stable immunoglobulin domain comprises a constant region.

6. The isolated nucleic acid molecule of claim 1 further comprising a transcriptional promoter region operably linked to the leader molecule.

7. The isolated nucleic acid molecule of claim 1 further comprising a polyadenylation sequence operably linked to the stable immunoglobulin domain.

8. The isolated nucleic acid molecule of claim 1 wherein the operable linkage of the leader molecule to the stable immunoglobulin domain comprises a multiple cloning region.

9. The isolated nucleic acid molecule of claim 1 wherein the operable linkage of the immunoglobulin domain to the polyadenylation site comprises a multiple cloning site.

10. An isolated nucleic acid molecule of claim 1 comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:

65, SEQ ID NO;

67, SEQ ID NO;

69, SEQ ID NO;

71, SEQ ID NO;

73, SEQ ID NO;

75, SEQ ID NO;

77, SEQ ID NO;

79, SEQ ID NO;

81, SEQ ID NO;

66, SEQ ID NO;

68, SEQ ID NO;

70, SEQ ID NO;

72, SEQ ID NO;

74, SEQ ID NO;

76, SEQ ID NO;

78, SEQ ID NO;

80, SEQ ID NO;

82, or a sequence at least 85%, identical thereto.

12. An isolated DNA cassette expression vector comprising one or more DNA immunoglobulin cassette molecules of claim 1.

13. The isolated DNA cassette expression vector of claim 12 wherein the vector contains two immunoglobulin cassette molecules.

14. The isolated DNA cassette expression vector of claim 13 wherein a first immunoglobulin cassette comprises a light chain immunoglobulin and a second immunoglobulin cassette comprises a heavy chain immunoglobulin.

15. A recombinant host cell comprising the expression vector of claim 12.

16. The recombinant host cell of claim 15 wherein the host cell is any one of a bacterial cell, a yeast cell or a mammalian cell.

5. The isolated nucleic acid molecule of claim I wherein the stable immunoglobulin domain comprises a variable region.

11. An isolated nucleic acid molecule comprising immunoglobulin cassette insert sequences selected from the group consisting of SEQ ID NO:

83, SEQ ID NO;

84, SEQ ID NO;

85, SEQ ID NO;

86, SEQ ID NO 87, SEQ ID NO;

88, or a sequence at least 85% identical thereto.

17. A method of synthesizing an immunoglobulin cassette construct comprising:

isolating a nucleic acid comprising an immunoglobulin leader sequence;

isolating a nucleic acid comprising an immunoglobulin domain sequence selected from a variable region or a constant region; and

operably linking the leader sequence nucleic acid and the immunoglobulin domain, wherein the operable linkage comprises a cloning site available for addition of additional immunoglobulin nucleic acid sequences.

18. A method of synthesizing a DNA vector capable of encoding an immunoglobulin comprising:

synthesizing one or more immunoglobulin DNA cassettes wherein an immunoglobulin leader sequence is operably linked to a stable immunoglobulin domain, wherein the operable linkage includes a cloning site;

introducing one or more immunoglobulin DNA cassettes into an expression vector; and

incorporating insert sequences into the cloning site of one or more immunoglobulin DNA cassettes;

wherein incorporation of insert sequences results in formation of DNA sequences encoding a desired immunoglobulin molecule.

19. The method of claim 18, wherein two immunoglobulin DNA cassettes are incorporated into the expression vector, wherein a first immunoglobulin DNA cassette comprises a heavy chain immunoglobulin and a second immunoglobulin DNA cassette comprises a light chain immunoglobulin.

20. A DNA expression vector comprising nucleic acid sequences encoding one or more immunoglobulin molecules wherein the DNA vector is synthesized by the method of claim 18.

21. A transfected cell line capable of producing an antibody comprising a DNA expression vector of claim 20.

25. The method of claim 21, wherein the mammalian cell is a cultured cell or a cell line.

26. The method of claim 25, wherein the mammalian cell is selected from the group consisting of a lymphocytic cell line, CHO, and COS cells.

22. An improved method of antibody production, comprising:

providing a first nucleic acid encoding a light chain immunoglobulin comprising a light chain leader sequence operably linked to a light chain variable region operably linked to a light chain constant region; and

providing a second nucleic acid encoding a heavy chain immunoglobulin comprising a heavy chain leader sequence operably linked to a heavy chain variable region operably linked to a heavy chain constant region immunoglobulin; and

introducing the nucleic acids into a host cell under conditions that allow expression and assembly of said light and heavy chain immunoglobulins to produce a functional antibody preparation.

23. The method of claim 22, wherein the first and second nucleic acids are linked.

24. The method of claim 22, wherein the host cell is any one of a bacterial, yeast or mammalian cell.

27. A method of production of a battery of antibodies having identical antigen specificity and variable effector functions comprising:

isolating immunoglobulin light and heavy chain DNA variable domain insert sequences having the desired antigen specificity;

providing a selection of expression vectors comprising immunoglobulin DNA cassette vectors, wherein the selection includes cassette vectors having different stable immunoglobulin light and heavy chain constant domains having variable effector functions;

incorporating the immunoglobulin DNA variable domain insert sequences into the cloning site of the DNA cassettes; and

introducing the resulting DNA expression vectors into a suitable host cell under conditions that allow expression and assembly of resulting light and heavy chain immunoglobulins to produce functional antibody preparation;

wherein resultant expressed antibodies comprises a battery of antibodies having identical antigen specificity and variable effector function.

28. The method of claim 27, wherein the first and second nucleic acids are linked.

29. The method of claim 27, wherein the host cell is any one of a bacterial yeast or mammalian cell.

30. The method of claim 27, wherein the mammalian cell is a cultured cell or a cell line.

Attorney Docket No.:

MPIO 1-244P2RM

31. The method of claim 31, wherein the mammalian cell is selected from the group consisting of a lymphocytic cell line, CHO, and COS cells.

32. A method of production of a battery of antibodies having identical effector function and variable antigen specificity comprising:

isolating immunoglobulin light and heavy chain DNA variable domain insert sequences having variable antigen specificity;

providing expression vector comprising immunoglobulin DNA cassette vectors, wherein the vector comprises stable immunoglobulin light and heavy chain constant domains having a desired effector function;

incorporating the immunoglobulin DNA variable domain insert sequences into the cloning site of the DNA cassettes; and

introduction of resulting DNA expression vectors into a suitable host cell under conditions that allow expression and assembly of resulting light and heavy chain immunoglobulins to produce functional antibody preparation;

wherein resultant expressed antibodies comprises a battery of antibodies having variable antigen specificity and identical effector function.

33. The method of claim 32, wherein the first and second nucleic acids are linked.

34. The method of claim 32, wherein the host cell is any one of a bacterial yeast or mammalian cell.

35. The method of claim 32, wherein the mammalian cell is a cultured cell or a cell line.

36. The method of claim 35, wherein the mammalian cell is selected from the group consisting of a lymphocytic cell line, CHO, and COS cells.