Transgenic ungulates capable of human antibody production

• US 20080040821A1
• Filed: 06/20/2007
• Published: 02/14/2008
• Est. Priority Date: 11/19/1999
• Status: Active Grant

• Current Assignee: SAB LLC (W.P. Carey, Inc. (Real Estate))
• Original Assignee: Kirin Pharma KK
• Inventors: Goldsby, Richard, Kuroiwa, Yoshimi, Tomizuka, Kazuma, Ishida, Isao, Kasinathan, Poothappillai, Osborne, Barbara, Sullivan, Eddie, Collas, Philippe, Robl, James
• Granted Patent: US 7,803,981 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 whose genome comprises a mutation in an endogenous immunoglobulin (Ig) heavy chain or Ig light chain locus, said mutation reducing the expression of said Ig heavy chain or Ig light chain.

2. The ungulate of claim 1, wherein said mutation reduces the expression of functional IgM heavy chain.

3. The ungulate of claim 1, wherein said mutation substantially eliminates the expression of said Ig heavy chain or said Ig light chain.

4. The ungulate of claim 1, wherein said mutation is hemizygous.

5. The ungulate of claim 1, wherein said mutation is homozygous.

6. The ungulate of claim 1, wherein said mutation is an insertion of a positive selection marker into said endogenous locus.

7. The ungulate of claim 6, wherein said positive selection marker is an antibiotic resistance gene.

8. The ungulate of claim 7, wherein each allele comprises the same antibiotic resistance gene.

9. The ungulate of claim 7, wherein each allele comprises a different antibiotic resistance gene.

10. The ungulate of claim 6, wherein said positive selection marker is operably linked to a xenogenous promoter.

11. The ungulate of claim 1, wherein said mutation is an insertion of a transcription termination sequence into said endogenous loci.

12. The ungulate of claim 11, wherein said transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain locus.

13. The ungulate of claim 1, comprising one or more nucleic acids comprising one or more transgenes and expressing an mRNA or protein encoded by said transgene(s).

14. The ungulate of claim 1, the cells of said ungulate further comprising one or more chromosomal fragments comprising one or more, all or part of an unrearranged xenogenous Ig locus which undergoes rearrangement and expresses one or more xenogenous Ig molecules in B-cells.

15. The ungulate of claim 14, wherein said molecule is an antibody protein.

16. The ungulate of claim 15, wherein said antibody protein is a human antibody protein.

17. The ungulate of claim 1, wherein said ungulate is a pig.

18. An isolated ungulate somatic cell comprising a mutation in an endogenous immunoglobulin (Ig) heavy chain or Ig light chain locus, said mutation reducing the expression of said Ig heavy chain or Ig light chain.

19. The cell of claim 18, comprising a mutation in both alleles of said IgM heavy chain or said light chain.

20. The cell of claim 18, wherein said mutation is a transcription termination sequence.

21. The cell of claim 20, wherein said transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain locus.

22. The cell of claim 18, further comprising one or more chromosomal fragments comprising one or more, all or part of a xenogenous Ig locus.

23. The cell of claim 18, wherein said cell is a fetal fibroblast or a B-cell.

24. The cell of claim 18, wherein said ungulate is a pig.

35. A method for producing a transgenic ungulate having reduced expression of an endogenous Ig heavy chain or Ig light chain locus, said method comprising the steps of:

(a) incubating a permeabilized cell of claim 18 in an extract from a mitotic somatic cell or oocyte under conditions that allow chromatin condensation and nuclear envelope breakdown in said permeabilized cell;

(b) inserting said cell formed in step (a) into a nucleated or enucleated ungulate oocyte, thereby forming a reconstituted oocyte; and

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

36. The method of claim 35, wherein, prior to step (b), said cell is incubated under conditions that allow the membrane of said cell to reseal.

37. The method of claim 35, wherein said cell is purified from said extract prior to insertion into said oocyte.

38. The method of claim 35, wherein said fetus develops into a viable offspring.

39. The method of claim 38, further comprising mating two offspring to produce a transgenic ungulate whose genome comprises mutations in both alleles of an endogenous immunoglobulin (Ig) heavy chain or Ig light chain locus.

40. The method of claim 35, wherein said oocyte from step (b) is cultured under conditions that allow cell division and one of the resulting cells is recloned one or more times.

41. The method of claim 35, wherein said permeabilized cell and said oocyte are from the same species.

42. The method of claim 35, wherein said permeabilized cell is a fibroblast, epithelial cell, neural cell, epidermal cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, 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.

43. The method of claim 35, wherein said mutation in said endogenous locus of said ungulate somatic cell of claim 18 is an insertion of a transcription termination sequence into said endogenous locus.

44. The method of claim 43, wherein said transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain locus.

45. The method of claim 35, wherein said ungulate is a pig.

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

(a) administering one or more antigens of interest to an ungulate whose genome comprises a mutation in an endogenous immunoglobulin (Ig) heavy chain or Ig light chain locus and the cells of said ungulate further comprising a chromosomal fragment or fragments comprising an unrearranged human light chain locus and an unrearranged human heavy chain locus, wherein the loci undergo rearrangement resulting in the production of human antibody proteins specific for said one or more antigens; and

(b) recovering said human antibodies from said ungulate.

26. The method of claim 25, wherein said mutation is an insertion of a transcription termination sequence into said endogenous locus.

27. The method of claim 26, wherein said transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain locus.

31. The method of claims 25 or 28, wherein said antibodies are directed against a desired antigen.

32. The method of claims 25 or 28, wherein said antibodies are polyclonal.

33. The method of claims 25 or 28, wherein said antibodies are recovered from the serum of said ungulate.

34. The method of claims 25 or 28, wherein said ungulate is a pig.

28. A method of producing antibodies, said method comprising recovering human antibodies from an ungulate whose genome comprises a mutation in an endogenous immunoglobulin (Ig) heavy chain or Ig light chain locus and the cells of said ungulate further comprising a chromosomal fragment comprising a human antibody gene locus, wherein said antibody gene locus undergoes rearrangement resulting in the production of human antibodies.

29. The method of claim 28, wherein said mutation is a transcription termination sequence into said endogenous locus.

30. The method of claim 29, wherein said transcription termination sequence is inserted downstream of the initial ATG codon in exon 2 of an endogenous mu heavy chain locus.