Methods and compositions for improving recombinant protein production

• US 20090285806A1
• Filed: 10/05/2005
• Published: 11/19/2009
• Est. Priority Date: 10/05/2004
• Status: Abandoned Application

• Current Assignee: Janssen Alzheimer Immunotherapy (Johnson & Johnson), Wyeth LLC (Pfizer Inc.)
• Original Assignee: Wyeth LLC (Pfizer Inc.)
• Inventors: Sinacore, Martin, Rouse, Jason
• Application Number:
  US11/664,865
• Publication Number:
  US 20090285806A1
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• 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 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 2810/859 from immunoglobulins

Abstract

Nucleic acid molecules modified to enhance recombinant protein expression, e.g., that of Aβ peptide binding antibodies, and/or to reduce or eliminate mis-spliced and/or intron read-through (IRT) by-products are disclosed. The invention also provides methods for producing Aβ peptide binding antibodies devoid of mis-spliced and/or intron read-through by-products by expression of such nucleic acid molecules under cell culture conditions suitable for recombinant Aβ peptide binding antibody expression.

Claims

1. A nucleic acid molecule encoding an Aβ

-antibody chain, comprising a nucleotide sequence having one or more intron and exon sequences, wherein at least one intron sequence is deleted compared to the naturally-occurring genomic sequence to reduce a mis-spliced or an intron read-through (IRT) by-product.

3. The nucleic acid molecule of claim 1 or 2, wherein at least three intron sequences are deleted.

4. The nucleic acid molecule of claim 3, wherein the antibody chain is a heavy chain or a fragment thereof.

5. The nucleic acid molecule of claim 4, wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region, a hinge region, a first constant region (CH1), a second constant region (CH2), and third constant region (CH3) of a human immunoglobulin G subtype.

6. The nucleic acid molecule of claim 5, wherein the immunoglobulin G subtype is a human IgG1 or human IgG4.

7. The nucleic acid molecule of claim 6, wherein the human IgG1 or human IgG4 is mutated.

8. The nucleic acid molecule of claim 5, wherein an intron between the CH2 region and the CH3 region of the immunoglobulin heavy chain constant region is deleted.

9. The nucleic acid molecule of claim 8, further comprising a deletion of an intron between the CH1 region and the hinge region.

10. The nucleic acid molecule of claim 8, further comprising a deletion of an intron between the hinge region and the CH2 region.

11. The nucleic acid molecule of claim 5, having a heavy chain that comprises one intron between the heavy chain variable region and the CH1 region.

12. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence at least 95% identical to the nucleotide sequence shown in FIG. 8 (SEQ ID NO:

1).

13. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding the heavy chain hinge region, and a first, second, and third constant region comprises a sequence at least 95% identical to the nucleotide sequence shown in FIG. 9 (SEQ ID NO:

3).

14. The nucleic acid molecule of claim 8, wherein the deletion of the intron between CH2 and CH3 corresponds to about nucleotides 1409 to 1505 of human IgG1 as shown in FIG. 8 (SEQ ID NO:

1).

15. The nucleic acid molecule of claim 8, wherein the deletion of the intron between CH2 and CH3 corresponds to about nucleotides 1401 to 1497 of human IgG4 as shown in FIG. 9 (SEQ ID NO:

3).

16. The nucleic acid molecule of claim 9, wherein the deletion of the intron between CH1 and the hinge region corresponds to about nucleotides 525 to 915 of human IgG1 as shown in FIG. 8 (SEQ ID NO:

1).

17. The nucleic acid molecule of claim 9, wherein the deletion of the intron between CH1 and the hinge region corresponds to about nucleotides 525 to 916 of human IgG4 as shown in FIG. 9 (SEQ ID NO:

3).

18. The nucleic acid molecule of claim 10, wherein the deletion of the intron between the hinge region and CH2 corresponds to about nucleotides 961 to 1078 of human IgG1 as shown in FIG. 8 (SEQ ID NO:

1).

19. The nucleic acid molecule of claim 10, wherein the deletion of the intron between the hinge region and CH2 corresponds to about nucleotides 953 to 1070 of human IgG4 as shown in FIG. 9 (SEQ ID NO:

3).

31. An expression cassette comprising the nucleic acid molecule of claim 5.

32. An expression vector comprising the nucleic acid molecule of claim 5.

33. The expression vector of claim 32, further comprising one or more nucleotide sequences that enhance replication, selection, mRNA transcription, mRNA stability, protein expression or protein secretion in a host cell.

34. A host cell comprising the nucleic acid molecule of claim 5.

35. A host cell comprising the expression cassette of claim 31.

36. A host cell comprising the expression vector of claim 32.

37. The host cell of claim 36, which is a Chinese Hamster Ovary (CHO) cell.

38. A method of expressing a recombinant antibody or fragment thereof which selectively binds an Aβ

peptide and is substantially free of an intron read-through (IRT) product, comprising;

introducing the nucleic acid molecule of claim 5 into a mammalian host cell;

culturing said host cell under conditions that allow expression of the recombinant antibody or fragment thereof, thereby producing a culture of host cells; and

obtaining the recombinant antibody or fragment thereof from the culture of host cells.

39. The method of claim 38, further comprising the step of identifying an IRT product in a nucleic acid sample from the host cell.

40. The method of claim 39, wherein the identification step comprises:

obtaining a nucleic acid sample from the culture of host cells;

contacting said nucleic acid sample with nucleic acid probes complementary to an intron and adjacent exon sequence, under conditions that allow hybridization between the nucleic acid sample and the probes;

detecting the resulting complex, wherein detection in said sample of a complex, using the nucleic acid probe complementary to the intron sequence is indicative of the presence of the IRT product.

41. The method of claim 38, wherein said host cell comprises a nucleotide sequence encoding a light chain variable region and a constant region.

42. A method for enhancing expression of a recombinant antibody or fragment thereof which selectively binds an Aβ

peptide, comprising;

introducing the nucleic acid molecule of claim 5 into a mammalian host cell;

culturing said host cell under conditions that allow expression of the recombinant antibody, thereby producing a culture of host cells; and

obtaining the recombinant antibody from the culture of host cells.

43. The method of claim 42, wherein said host cell comprises a nucleotide sequence encoding a light chain variable region and a constant region.

44. A method for producing a recombinant antibody or fragment thereof which selectively binds a Aβ

peptide and is substantially devoid of intron read-through (IRT) heavy chain by-product, comprising;

culturing a mammalian host cell comprising the nucleic acid molecule of claim 5 and a nucleic acid encoding an antibody light chain of an antibody which selectively binds an Aβ

peptide, under conditions such that the heavy and light chains are expressed.

45. The method of claim 44, further comprising purifying the heavy and light chains form the culture.

46. A method for enhancing expression of a recombinant antibody or fragment thereof which selectively binds an Aβ

peptide, comprising;

culturing a mammalian host cell comprising the nucleic acid molecule of claim 5, and a nucleic acid encoding an antibody light chain of an antibody which selectively binds an Aβ

peptide, under conditions such that the heavy and light chains are expressed.

47. The method of claim 46, further comprising purifying the heavy and light chains form the culture.

49. An antibody or antigen-binding fragment thereof which selectively bind an Aβ

peptide, made by the method comprising the steps of claim 40 under suitable conditions to allow expression and assembly of the antibody or fragment.

50. The antibody of claim 49, which is a chimeric, humanized, CDR-grafted or an in vitro generated antibody.

51. The antibody of claim 50, which is a humanized antibody.

52. The antibody of claim 51, which binds to human 5T4.

53. A pharmaceutical composition comprising the antibody of claim 49, and a pharmaceutically acceptable carrier.

76. The nucleic acid molecule of claim 1 or 2, wherein the antibody chain has the sequence set forth as SEQ ID NO:

12.

77. The nucleic acid molecule of claim 1 or 2, wherein the antibody chain has the sequence set forth as SEQ ID NO:

13.

81. The antibody of any one of claims 77-80, further comprising a corresponding light chain as set forth in one of FIG. 15 or 16A-16B.

2. A nucleic acid molecule encoding an Aβ

-antibody chain, comprising a nucleotide sequence comprising one or more intron and exon sequences, wherein at least one intron sequence is deleted compared to the naturally-occurring genomic sequence to enhance protein expression.

20. A nucleic acid molecule comprising a nucleotide sequence encoding human IgG1, wherein said nucleotide sequence is at least 90% identical to the sequence shown in FIG. 10 (SEQ ID NO:

5).

21. A nucleic acid molecule comprising a nucleotide sequence encoding human IgG4, wherein said nucleotide sequence is at least 90% identical to the sequence shown in FIG. 11 (SEQ ID NO:

6).

22. A genomic nucleotide sequence encoding a human heavy chain constant region, or a mutated form thereof, wherein said nucleotide sequence lacks at least one intron present in the naturally-occurring genomic sequence, and wherein said intron facilitates intron-read through.

24. The nucleotide sequence of either of claims 22 or 23, wherein the at least one intron is the intron between CH2 and CH3 of the constant region.

23. A genomic nucleotide sequence encoding a human IgG1, or a mutated form thereof, wherein said nucleotide sequence lacks at least one intron present in the naturally-occurring genomic sequence, and wherein said intron facilitates intron-read through.

25. A genomic nucleotide sequence encoding a human IgG4, or a mutated form thereof, wherein said genomic sequence lacks three introns present in the naturally-occurring genomic sequence.

26. The nucleotide sequence of claim 25, wherein the introns are the intron between CH1 and hinge region, the intron between the hinge region and CH2, and the intron between CH2 and CH3.

27. A nucleic acid molecule encoding an antibody which selectively binds an Aβ

peptide, comprising a nucleotide sequence represented by the formula;

V_H-Int1-C_H1-Int2-Hinge-Int3-C_H2-C_H3,

wherein V_H is a nucleotide sequence encoding a heavy chain variable region;

C_H1, C_H2, and C_H3 are nucleotide sequences encoding the corresponding heavy chain constant region;

Hinge is a nucleotide sequence encoding a hinge region of a heavy chain constant region; and

Int1, Int2 and Int3 are introns from the heavy chain genomic sequence.

28. The nucleic acid molecule of claim 27, wherein the nucleotide sequence encodes a human immunoglobulin G heavy chain.

29. A nucleic acid molecule encoding an antibody which selectively binds an Aβ

peptide, comprising a nucleotide sequence represented by the formula;

V_H-Int1-C_H1-Hinge-C_H2-C_H3,

wherein V_H is a nucleotide sequence encoding a heavy chain variable region;

C_H1, C_H2, and C_H3 are nucleotide sequences encoding the corresponding heavy chain constant region;

Hinge is a nucleotide sequence encoding a hinge region of a heavy chain constant region; and

Int1 is an intron from the heavy chain genomic sequence.

30. The nucleic acid molecule of claim 29, wherein the nucleotide sequence encodes a human immunoglobulin G heavy chain.

48. A method for detecting an IRT product, in a sample, comprising:

obtaining a nucleic acid sample from a recombinant cell;

contacting said nucleic acid sample with nucleic acid probes complementary to an intron and adjacent exon sequence, under conditions that allow hybridization of the nucleic acid sample and the probes;

detecting the resulting complex, wherein detection in said sample of a complex, using the nucleic acid probe complementary to the intron sequence is indicative of the presence of the IRT product.

54. An isolated nucleic acid molecule encoding an antibody heavy chain which selectively binds a Aβ

peptide, comprising human genomic intron and exon sequences in a modified operative association, wherein modification of the natural operative association of the intron and exon sequences reduces or eliminates expression of an intron read-through heavy chain by-product.

55. The isolated nucleic acid molecule of claim 54, wherein expression of said heavy chain is enhanced relative to a nucleic acid molecule comprising human genomic intron and exon sequences in natural operative association.

56. The nucleic acid molecule of claim 55, wherein the genomic exon sequences encode a variable region, a hinge region, and a first, second and third constant region.

57. The nucleic acid molecule of claim 56, wherein the first, second and third constant regions are IgG1 constant regions.

58. The nucleic acid molecule of claim 56, wherein the first, second and third constant regions are IgG4 constant regions.

59. The nucleic acid molecule of claim 56, wherein the variable region is a humanized variable region.

60. The nucleic acid molecule of claims 56-59, wherein the variable region specifically binds to an Aβ

peptide.

61. The nucleic acid molecule of claim 56-59, wherein the variable region comprises complementarity determining regions (CDRs) from the from the mouse 3D6 antibody.

62. The nucleic acid molecule of claim 54, comprising one intron sequence.

63. The nucleic acid molecule of claim 54, comprising two intron sequences.

64. The nucleic acid molecule of claim 54, comprising three intron sequences.

69. A cell comprising the nucleic acid molecule of any one of claims 54-68.

65. An Aβ

peptide binding antibody heavy chain-encoding nucleic acid molecule, comprising a variable region-encoding exon operably linked to a first, second and third constant region-encoding exon, said nucleic acid molecule further comprising at least one intron sequence, wherein said intron sequence enhances said heavy chain expression from said nucleic acid molecule relative to an Aβ

peptide binding antibody heavy chain-encoding nucleic acid molecule not comprising said intron and wherein said nucleic acid molecule does not lead to the translation of an intron read-through (IRT) heavy chain by-product.

66. An expression cassette comprising the nucleic acid molecule of any one of the proceeding claims.

70. A mammalian cell comprising the cassette of claim 66.

72. The cell of claim 70, which is a Chinese Hamster Ovary (CHO) cell.

75. A method for producing an antibody preparation substantially devoid of intron read-through (IRT) heavy chain by-product, comprising:

culturing a cell of any one of claims 70, 71 or 82, further comprising an Aβ

peptide binding antibody light chain-encoding nucleic acid molecule, under conditions such that the heavy and light chains are expressed and operatively associate in the absence of intron read-through (IRT) heavy chain by-product.

67. An expression vector comprising the nucleic acid molecule of any one of the proceeding claims.

68. The expression vector of claim 67, further comprising a gene encoding a selectable marker and an internal ribosomal entry site sequence (IRES).

71. A mammalian cell comprising the vector of claim 67.

82. The cell of claim 71, which is a Chinese Hamster Ovary (CHO) cell.

83. A mammalian cell comprising the vector of claim 68.

73. A IgG1 heavy chain-encoding nucleotide sequence having the following formula:

V_H-Int1-C_H1-Int2-Hinge-Int3-C_H2-C_H3

wherein V_H is any heavy chain variable region-encoding exon;

C_H1, C_H2 and C_H3 are human heavy chain constant region-encoding exons derived from a naturally-occurring IgG1 heavy chain gene;

Int1, Int2 and Int3 are corresponding introns derived from said gene and Int2 and Int 3 are optionally present; and

Hinge is a hinge-encoding exon derived from said gene.

74. The sequence of claim 73, wherein Int2 and Int3 are present.

78. An Aβ

-antibody comprising a heavy chain comprising constant regions encoded by the nucleic acid of any one of SEQ ID NOs;

6-11 and a variable region encoded by the nucleic acid of SEQ ID NO;

2.

79. An Aβ

-antibody comprising a heavy chain comprising constant regions encoded by the nucleic acid of any one of SEQ ID NOs;

6-11 and a variable region encoded by the nucleic acid of SEQ ID NO;

4.

80. An Aβ

-antibody comprising a heavy chain comprising constant regions encoded by the nucleic acid of any one of SEQ ID NOs;

6-11 and a variable region having the amino acid sequence set forth in any of FIG. 12-14 or 16C-16D.