Transgenic ungulates capable of human antibody production

• US 20040068760A1
• Filed: 05/19/2003
• Published: 04/08/2004
• Est. Priority Date: 11/19/1999
• Status: Active Grant

• Current Assignee: Kyowa Hakko Kirin Company Limited (Kirin Holdings Company Limited)
• Original Assignee: Kyowa Hakko Kirin Company Limited (Kirin Holdings Company Limited)
• Inventors: Kuroiwa, Yoshimi, Kasinathan, P., Goldsby, Richard A., Tomizuka, Kazuma, Ishida, Isao, Robl, James M., Sullivan, Eddie, Collas, Philippe
• Granted Patent: US 7,414,170 B2
• Time in Patent Office: Days
• US Class Current: 800/6
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2217/00 Genetically modified animals
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0273 Cloned vertebrates
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/0278 Knock-in vertebrates, e.g. humanised vertebrates
• 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 16/42 against immunoglobulins
• CPC Class Codes: C07K 2317/21 from primates, e.g. man
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 15/8771 Bovine embryos
• CPC Class Codes: C12N 2799/025 where the vector is derived from a parvovirus
• CPC Class Codes: C12N 2800/208 of mammalian origin, e.g. minichromosome

Abstract

The invention features novel methods for the production of large quantities of xenogenous antibodies, such as human antibodies. Preferably, this result is effected by inactivation of IgM heavy chain expression and, optionally, by inactivation of Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of xenogenous antibodies (e.g., non-bovine antibodies), preferably human antibodies.

Claims

1. A transgenic ungulate comprising one or more nucleic acids encoding all or part of a xenogenous immunoglobulin (Ig) gene which undergoes rearrangement and expresses more than one xenogenous Ig protein.

2. The ungulate of claim 1, wherein said xenogenous Ig protein is a human Ig protein.

3. The ungulate of claim 1, wherein said nucleic acid is contained within a chromosome fragment.

4. The ungulate of claim 3, wherein said chromosome fragment is a Δ

HAC or Δ

Δ

HAC.

5. The ungulate of claim 1, wherein said ungulate is a bovine, ovine, porcine, or caprine.

6. A transgenic ungulate comprising a mutation that reduces the expression of functional endogenous antibody, wherein said mutation comprises an insertion of a transcription termination sequence into an endogenous immunoglobulin nucleic acid.

7. The ungulate of claim 6, wherein said mutation reduces the expression of functional IgM heavy chain or reduces the expression of functional Ig light chain.

8. The ungulate of claim 6, wherein said mutation substantially eliminates the expression of functional endogenous antibody.

9. The ungulate of claim 6, wherein said mutation is hemizygous.

10. The ungulate of claim 6, wherein said mutation is homozygous.

11. The ungulate of claim 6, wherein said mutation further comprises an insertion of a positive selection marker into an endogenous immunoglobulin gene.

12. The ungulate of claim 11, wherein said positive selection marker is an antibiotic resistance gene.

13. The ungulate of claim 11, wherein said positive selection marker is operably linked to a xenogenous promoter.

14. The ungulate of claim 10, wherein each allele comprises the same antibiotic resistance gene.

15. The ungulate of claim 10, wherein each allele comprises a different antibiotic resistance gene.

16. The ungulate of claim 6, wherein said transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain nucleic acid.

17. The ungulate of claim 6, comprising one or more nucleic acids comprising one or more transgenes and expressing an mRNA or protein encoded by said transgene(s).

18. The ungulate of claim 6, comprising one or more nucleic acids comprising all or part of a xenogenous immunoglobulin (Ig) gene which undergoes rearrangement and expresses more than one xenogenous Ig molecule.

19. The ungulate of claim 18, expressing a xenogenous antibody protein.

20. The ungulate of claim 19, wherein said xenogenous antibody protein is a human antibody protein.

21. The ungulate of claim 6, wherein said ungulate is a bovine, ovine, porcine, or caprine.

22. An ungulate somatic cell comprising one or more nucleic acids encoding all or part of a xenogenous Ig gene, wherein said gene is capable of undergoing rearrangement and expressing one or more xenogenous Ig proteins in B cells.

23. The cell of claim 22, wherein said nucleic acid encodes a xenogenous antibody.

24. The cell of claim 22, wherein said nucleic acid is contained in a chromosome fragment.

25. The cell of claim 22, wherein said cell is a fetal fibroblast or B-cell.

26. The cell of claim 22, wherein said ungulate is a bovine, ovine, porcine, or caprine.

32. A hybridoma formed from the fusion of the B-cell of claim 25 or 30 with a myeloma cell.

43. A method for producing a transgenic ungulate, said method comprising the steps of:

(a) incubating a permeabilized cell of claim 22 in a reprogramming media under conditions that allow the removal of a factor from a nucleus, chromatin mass, or chromosome of said permeabilized cell or the addition of a factor from said reprogramming media to said nucleus, chromatin mass, or chromosome, thereby forming a reprogrammed cell;

(b) inserting said reprogrammed cell into a nucleated or enucleated oocyte, thereby forming a nuclear transfer oocyte; and

(c) transferring said nuclear transfer oocyte or an embryo formed from said nuclear transfer oocyte into the uterus of a host ungulate under conditions that allow said nuclear transfer oocyte or said embryo to develop into a fetus.

44. The method of claim 43, wherein said reprogramming media is a cell extract.

45. The method of claim 43, wherein the nucleus of said permeabilized cell remains membrane-bounded and the chromatin in said nucleus does not condense during incubation in said reprogramming media.

46. The method of claim 43, wherein a chromatin mass is formed from incubation of said permeabilized cell in said reprogramming media.

47. The method of claim 43, wherein said reprogrammed cell is incubated under conditions that allow the membrane of said reprogrammed cell to reseal.

48. The method of claim 43, wherein said reprogrammed cell is purified from said reprogramming media prior to insertion into said nuclear transfer oocyte.

49. The method of claim 43, wherein said fetus develops into a viable offspring.

50. The method of claim 49, further comprising mating said offspring with a transgenic ungulate comprising a mutation in an endogenous immunoglobulin nucleic acid, wherein said mutation comprises an insertion of a transcription termination sequence into an endogenous immunoglobulin nucleic acid.

51. The method of claim 43, wherein said nuclear transfer oocyte from step (b) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times.

52. The method of claim 43, wherein said permeabilized cell and said nuclear transfer oocyte are from the same species.

53. A method for producing a transgenic ungulate, said method comprising the steps of:

(a) contacting a donor nucleus from a cell of claim 22 with a reprogramming media under conditions that allow formation of a chromatin mass;

(b) inserting said chromatin mass into an oocyte, thereby forming a nuclear transfer oocyte; and

(c) transferring said nuclear transfer oocyte or an embryo formed from said nuclear transfer oocyte into the uterus of a host ungulate under conditions that allow said nuclear transfer oocyte or said embryo to develop into a fetus.

54. The method of claim 53, wherein said chromatin mass is formed without DNA replication.

55. The method of claim 53, wherein reprogramming media is a mitotic extract, detergent and salt solution, a detergent solution, a salt solution, or protein kinase solution.

56. The method of claim 53, wherein said chromatin mass from step (a) is purified from said extract prior to insertion into said nuclear transfer oocyte.

57. The method of claim 53, wherein said fetus develops into a viable offspring.

58. The method of claim 53, wherein said nuclear transfer oocyte from step (b) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times.

59. The method of claim 53, wherein said donor nucleus and said nuclear transfer oocyte are from the same species.

60. The method of claim 53, wherein said donor nucleus is diploid.

61. The method of claim 43 or 53, wherein said donor nucleus is from a fibroblast, epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoictic cell, melanocyte, chondrocyte, B-lymphocyte, erythrocyte, macrophage, monocyte, fibroblast, muscle cell, embryonic stem cell, embryonic germ cell, fetal cell, placental cell, a cell of the female reproductive system, or embryonic cell.

62. The method of claim 61, wherein said cell is a fetal fibroblast.

63. A method for producing a transgenic ungulate, said method comprising the steps of:

(a) inserting a cell of claim 22, a nucleus from said cell, or a chromatin mass from said cell into an oocyte, thereby forming a first embryo;

(b) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo;

wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo, and wherein at least one of said first embryo and said second embryo is a compaction embryo; and

(c) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

64. A method for producing a transgenic ungulate, said method comprising the steps of:

(a) inserting a cell of claim 22, a nucleus from said cell, or a chromatin mass from said cell into an oocyte, thereby forming a first embryo;

(b) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo;

wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo, and wherein said first embryo and said second embryo are at different cell-stages; and

(c) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

65. A method for producing a transgenic ungulate, said method comprising the steps of:

(a) contacting a donor nucleus from a cell of claim 22 with a reprogramming media under conditions that allow formation of a chromatin mass;

(b) inserting said chromatin mass into an oocyte, thereby forming a first embryo;

(c) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo;

wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo; and

(d) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

66. The method of claim 65, wherein step (a) involves contacting a donor nucleus that has less than four sets of homologous chromosomes with a reprogramming media under conditions that allow formation of a chromatin mass without causing DNA replication.

67. A method for producing a transgenic ungulate, said method comprising the steps of:

(a) incubating a permeabilized cell of claim 22 in a reprogramming media under conditions that allow the removal of a factor from a nucleus, chromatin mass, or chromosome of said permeabilized cell or the addition of a factor from said reprogramming media to said nucleus, chromatin mass, or chromosome, thereby forming a reprogrammed cell;

(b) inserting said reprogrammed cell into an oocyte, thereby forming a first embryo;

(c) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo, wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo; and

(d) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

68. The method of claim 67, wherein said reprogrammed cell is not incubated under conditions that allow the membrane of said reprogrammed cell to reseal.

69. The method of any one of claims 63-65 and 67, wherein said fetus develops into a viable offspring.

70. The method of any one of claims 63-65 and 67, wherein at least 10% cells in the placenta of said fetus are derived from said second embryo.

71. The method of any one of claims 63-65 and 67, wherein a chromosome fragment comprising a unrearranged human Ig gene is introduced into said cell via microcell fusion prior to step (a).

72. The method of claim 71, wherein said chromosome fragment is an artificially modified chromosome fragment.

73. The method of claim 72, wherein said chromosome fragment comprises both an unrearranged human Ig heavy chain nucleic acid and an unrearranged human Ig light chain nucleic acid.

74. The method of claim 73, wherein said chromosome fragment is Δ

HAC or Δ

Δ

HAC.

27. An ungulate somatic cell comprising a mutation in a nucleic acid encoding an Ig heavy and/or light chain, wherein said mutation comprises an insertion of a transcription termination sequence into said nucleic acid.

28. The cell of claim 27, comprising a mutation in both alleles of said IgM heavy chain or said light chain.

29. The cell of claim 27, further comprising one or more nucleic acids encoding all or part of xenogenous Ig gene, wherein said gene is capable of undergoing rearrangement and expressing one or more xenogenous Ig molecules in B cells.

30. The cell of claim 27, wherein said cell is a fetal fibroblast or a B-cell.

31. The cell of claim 27, wherein said ungulate is a bovine, ovine, porcine, or caprine.

77. A method for producing a transgenic ungulate having reduced expression of functional endogenous antibody protein, said method comprising the steps of:

(a) incubating a permeabilized cell of claim 27 in a reprogramming media under conditions that allow the removal of a factor from a nucleus, chromatin mass, or chromosome of said permeabilized cell or the addition of a factor from said reprogramming media to said nucleus, chromatin mass, or chromosome, thereby forming a reprogrammed cell;

(b) inserting said reprogrammed cell into a nucleated or enucleated oocyte, thereby forming a nuclear transfer oocyte; and

(c) transferring said nuclear transfer oocyte or an embryo formed from said nuclear transfer oocyte into the uterus of a host ungulate under conditions that allow said nuclear transfer oocyte or said embryo to develop into a fetus.

78. The method of claim 77, wherein said reprogramming media is a cell extract.

79. The method of claim 77, wherein the nucleus of said permeabilized cell remains membrane-bounded and the chromatin in said nucleus does not condense during incubation in said reprogramming media.

80. The method of claim 77, wherein a chromatin mass is formed from incubation of said permeabilized cell in said reprogramming media.

81. The method of claim 77, wherein said reprogrammed cell is incubated under conditions that allow the membrane of said reprogrammed cell to reseal.

82. The method of claim 77, wherein said reprogrammed cell is purified from said reprogramming media prior to insertion into said nuclear transfer oocyte.

83. The method of claim 77, wherein said fetus develops into a viable offspring.

84. The method of claim 83, further comprising mating two offspring to produce a transgenic ungulate with a mutation in both alleles of an endogenous immunoglobulin nucleic acid.

85. The method of claim 77, wherein said nuclear transfer oocyte from step (b) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times.

86. The method of claim 77, wherein said permeabilized cell and said nuclear transfer oocyte are from the same species.

87. A method for producing a transgenic ungulate having reduced expression of functional antibody protein, said method comprising the steps of:

(a) contacting a donor nucleus from a cell of claim 27 with a reprogramming media under conditions that allow formation of a chromatin mass;

(b) inserting said chromatin mass into an oocyte, thereby forming a nuclear transfer oocyte; and

(c) transferring said nuclear transfer oocyte or an embryo formed from said nuclear transfer oocyte into the uterus of a host ungulate under conditions that allow said nuclear transfer oocyte or said embryo to develop into a fetus.

88. The method of claim 87, wherein said chromatin mass is formed without DNA replication.

89. The method of claim 87, wherein reprogramming media is a mitotic extract, detergent and salt solution, a detergent solution, a salt solution, or protein kinase solution.

90. The method of claim 87, wherein said chromatin mass from step (a) is purified from said extract prior to insertion into said nuclear transfer oocyte.

91. The method of claim 87, wherein said fetus develops into a viable offspring.

92. The method of claim 87, wherein said nuclear transfer oocyte from step (b) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times.

93. The method of claim 87, wherein said donor nucleus and said nuclear transfer oocyte are from the same species.

94. The method of claim 87, wherein said donor nucleus is diploid.

95. The method of claim 77 or 87, wherein said donor nucleus is from a fibroblast, epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, B-lymphocyte, erythrocyte, macrophage, monocyte, fibroblast, muscle cell, embryonic stem cell, embryonic germ cell, fetal cell, placental cell, a cell of the female reproductive system, or embryonic cell.

96. The method of claim 95, wherein said cell is a fetal fibroblast.

97. A method for producing a transgenic ungulate having reduced expression of functional endogenous antibody protein, said method comprising the steps of:

(a) inserting a cell of claim 27, a nucleus from said cell, or a chromatin mass from said cell into an oocyte, thereby forming a first embryo;

(b) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo;

wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo, and wherein at least one of said first embryo and said second embryo is a compaction embryo; and

(c) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

98. A method for producing a transgenic ungulate having reduced expression of functional endogenous antibody protein, said method comprising the steps of:

(a) inserting a cell of claim 27, a nucleus from said cell, or a chromatin mass from said cell into an oocyte, thereby forming a first embryo;

(b) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo;

wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo, and wherein said first embryo and said second embryo are at different cell-stages; and

(c) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

99. A method for producing a transgenic ungulate having reduced expression of functional endogenous antibody protein, said method comprising the steps of:

(a) contacting a donor nucleus from a cell of claim 27 with a reprogramming media under conditions that allow formation of a chromatin mass;

(b) inserting said chromatin mass into an oocyte, thereby forming a first embryo;

(c) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo;

wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo; and

(d) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

100. The method of claim 99, wherein step (a) involves contacting a donor nucleus that has less than four sets of homologous chromosomes with a reprogramming media under conditions that allow formation of a chromatin mass without causing DNA replication.

101. A method for producing a transgenic ungulate having reduced expression of functional endogenous antibody protein, said method comprising the steps of:

(a) incubating a permeabilized cell of claim 27 in a reprogramming media under conditions that allow the removal of a factor from a nucleus, chromatin mass, or chromosome of said permeabilized cell or the addition of a factor from said reprogramming media to said nucleus, chromatin mass, or chromosome, thereby forming a reprogrammed cell;

(b) inserting said reprogrammed cell into an oocyte, thereby forming a first embryo;

(c) contacting one or more cells from said first embryo with one or more cells from a second embryo, thereby forming a third embryo, wherein said second embryo is an in vitro fertilized embryo, naturally-occurring embryo, or parthenogenetically activated embryo; and

(d) transferring said third embryo into the uterus of a host ungulate under conditions that allow said third embryo to develop into a fetus.

102. The method of claim 101, wherein said reprogrammed cell is not incubated under conditions that allow the membrane of said reprogrammed cell to reseal.

103. The method of any one of claims 97-99 and 101, wherein said fetus develops into a viable offspring.

104. The method of any one of claims 97-99 and 101, wherein at least 10% cells in the placenta of said fetus are derived from said second embryo.

105. The method of any one of claims 33, 39, 97-99, and 101, wherein said antibodies are monoclonal.

106. The method of any one of claims 33, 39, 97-99, and 101, wherein said antibodies are polyclonal.

107. The method of any one of claims 33, 39, 97-99, and 101, wherein said antibodies are recovered from the serum or milk of said ungulate.

108. The method of any one of claims 33, 39, 97-99, and 101, wherein said Igs are directed against a desired antigen.

33. A method of producing antibodies, said method comprising the steps of:

(a) administering one or more antigens of interest to an ungulate comprising nucleic acid encoding a xenogenous antibody gene locus, wherein the nucleic acid segments in said gene locus undergo rearrangement resulting in the production of antibody proteins specific for said antigen; and

(b) recovering said antibodies from said ungulate.

34. The method of claim 33, wherein said ungulate comprises a mutation that reduces the expression of an endogenous antibody, wherein said mutation comprises an insertion of a transcription termination sequence into an endogenous immunoglobulin nucleic acid.

35. The method of claim 34 wherein a transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain nucleic acid.

36. The method of claim 33, wherein said nucleic acid is contained in a chromosome fragment.

37. The method of claim 33, wherein said nucleic acid is a human nucleic acid.

38. The method of claim 33, wherein said ungulate is a bovine, ovine, porcine, or caprine.

39. A method of producing antibodies, said method comprising recovering xenogenous antibodies from an ungulate comprising nucleic acid encoding a xenogenous antibody gene locus, wherein the nucleic acid segments in said gene locus undergo rearrangement resulting in the production of one or more xenogenous antibody proteins.

40. The method of claim 39, wherein said ungulate comprises a mutation that reduces the expression of an endogenous antibody, wherein said mutation comprises an insertion of a transcription termination sequence into an endogenous immunoglobulin nucleic acid.

41. The method of claim 39, wherein said nucleic acid is contained in a chromosome fragment.

42. The method of claim 39, wherein said ungulate is a bovine, ovine, porcine, or caprine.

75. A method of producing a transgenic ungulate, said method comprising the steps of:

(a) inserting a cell, a chromatin mass from a cell, or a nucleus from a cell into an oocyte, wherein said cell comprises a first mutation in an endogenous antibody heavy chain and/or light chain nucleic acid;

wherein said first mutation comprises an insertion of a transcription termination sequence in said nucleic acid; and

(b) transferring said oocyte or an embryo formed from said oocyte into the uterus of a host ungulate under conditions that allow said oocyte or said embryo to develop into a fetus.

76. The method of claim 75, further comprising the steps of:

(c) isolating a cell from said embryo, said fetus, or an offspring produced from said fetus;

(d) introducing a second mutation in an endogenous antibody heavy chain and/or light chain nucleic acid in said cell;

wherein said second mutation comprises an insertion of a transcription termination sequence into an endogenous immunoglobulin nucleic acid;

(e) inserting said cell, a chromatin mass from said cell, or a nucleus from said cell into an oocyte; and

(f) transferring said oocyte or an embryo formed from said oocyte into the uterus of a host ungulate under conditions that allow said oocyte or said embryo to develop into a fetus.