GENERATION OF HEAVY-CHAIN ONLY ANTIBODIES IN TRANSGENIC ANIMALS

• US 20090307787A1
• Filed: 01/25/2007
• Published: 12/10/2009
• Est. Priority Date: 01/25/2006
• Status: Abandoned Application

• Current Assignee: Harbour Antibodies BV (Harbour BioMed Shanghai Co., Ltd.)
• Original Assignee: Harbour Antibodies BV (Harbour BioMed Shanghai Co., Ltd.)
• Inventors: Grosveld, Franklin Gerardus, Janssens, Richard Wilhelm
• Application Number:
  US12/161,981
• Publication Number:
  US 20090307787A1
• 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 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/072 maintaining or altering function, i.e. knock in
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• 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 16/1009 Picornaviridae, e.g. hepatitis A virus
• CPC Class Codes: C07K 16/1232 from Escherichia (G)
• CPC Class Codes: C07K 16/1282 from Clostridium (G)
• CPC Class Codes: C07K 16/1285 from Corynebacterium (G)
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2015/8518 expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles

Abstract

The present invention relates to a method for the generation of V_H heavy chain-only antibodies in a transgenic non-human mammal. In particular, the present invention relates to a method for the production of a V_H heavy chain-only antibody in a transgenic non-human mammal comprising the step of expressing more than one heterologous V_H heavy chain locus in that mammal.

Claims

1. A method for the production of a V_H heavy chain-only antibody in a transgenic non-human mammal comprising the steps of providing more than one heterologous V_H heavy chain locus in that mammal, wherein each V_H heavy chain locus comprises one or more V gene segments, one or more D gene segments, one or more J gene segments and a gene segment encoding a heavy chain constant region which, when expressed, does not include a C_H1 domain and expressing a V_H heavy chain-only antibody from at least one of said loci.

2. The method of claim 1, wherein each V_H heavy chain locus comprises one or multiple V gene segments.

3. The method of claim 1 or claim 2, wherein each locus comprises only one V gene segment.

4. The method of claim 3, wherein each V gene segment is different from all other V gene segments.

5. The method of claim 3, wherein each V gene segment is identical to all the other V gene segments.

6. The method of claim 5 wherein the remaining gene segments in each locus are the same as those in all the other loci.

7. The method of claim 5 wherein the remaining gene segments in each locus are different from those in all the other loci.

8. The method of claim 1 or claim 2, wherein each locus comprises multiple V gene segments.

9. The method of claim 8, wherein the V gene segments in any one locus are all derived from an organism of the same species.

10. The method of claim 8, wherein the V gene segments in any one locus are derived from organisms of different species.

11. The method of claim 9, wherein the V gene segments are of human origin.

12. The method of claim 1, wherein the multiple heavy chain loci comprise any number or combination of the 39 functional human V gene segments and engineered variants thereof with improved solubility properties distributed across the multiple loci.

13. The method of claim 1, wherein each different heavy chain locus is present as a single copy in the genome of the transgenic non-human mammal.

14. The method of claim 1, wherein each V_H heavy chain locus comprises from one to forty D gene segments.

15. The method of claim 1, wherein the D gene Segments are human D gene segments.

16. The method of claim 1, wherein each V_H heavy chain locus comprises from one to twenty J gene segments.

17. The method of claim 16, wherein the J gene segments are human J gene segments.

18. The method of claim 1, wherein each V_H heavy chain locus contains the same D and J gene segments.

19. The method of claim 1, wherein each V_H heavy chain locus contains different combinations of D and J gene segments.

20. The method of any claim 1, wherein each V_H heavy chain locus comprises one or more V gene segments, twenty-five functional human D gene segments and 6 human J gene segments.

21. The method of claim 1, wherein each V_H heavy chain locus comprises a gene segment encoding at least one heavy chain constant region providing effector functions in vivo wherein the constant region when expressed does not include a C_H1 domain in the antibody.

22. The method of claim 21, wherein each locus contains only one gene segment encoding one particular constant region.

23. The method of claim 21, wherein each locus comprises more than one gene segment, each encoding a different constant region.

24. The method of claim 1, wherein each locus contains the same constant region-encoding gene segment(s).

25. The method of claim 1, wherein each locus has different or a different combination of constant region-encoding gene segment(s).

26. The method of claim 1, wherein each gene segment encoding a heavy chain constant region comprises one or more heavy chain constant region exons of the Cδ

, Cγ

_1-4, Cμ

, Cε

or Cα

_1-2 classes, with the proviso that the heavy chain constant region gene segments do not express a C_H1 domain.

27. The method of claim 1, wherein the heavy chain constant region is of human origin.

28. The method of claim 1, wherein two or more heterologous V_H heavy chain loci in that mammal are present in tandem on the same chromosome.

29. The method of claim 1, wherein one or more heterologous V_H heavy chain loci in that mammal are present on different chromosomes.

30. The method of claim 1, wherein the transgenic non-human mammal is a rodent.

31. The method of claim 30, wherein the rodent is a mouse.

32. A transgenic non-human mammal comprising more than one heterologous V_H heavy chain locus as defined in claim 1.

33. A method for the production of heavy chain-only antibodies by immunising a transgenic non-human mammal of claim 32 with an antigen.

34. A method of producing high affinity, antigen-specific V_H heavy chain-only antibodies comprising:

immunising a transgenic non-human mammal according to claim 32 with an antigen;

generating B-cell hybridomas;

selecting cells expressing antigen-specific heavy chain-only antibody; and

isolating antigen-specific recombined affinity matured V_H heavy chain-only antibody.