Generation of heavy-chain only antibodies in transgenic animals

• US 10,638,735 B2
• Filed: 12/08/2015
• Issued: 05/05/2020
• Est. Priority Date: 01/25/2006
• Status: Active Grant

• Current Assignee: Harbour Antibodies BV (Harbour BioMed Shanghai Co., Ltd.)
• Original Assignee: Erasmus University Medical Center Rotterdam
• Inventors: Grosveld, Franklin Gerardus, Janssens, Richard Wilhelm, Craig, Roger Kingdon
• Primary Examiner(s): Hill, Kevin K
• Application Number:
  US14/962,335
• Publication Number:
  US 20160295843A1
• Time in Patent Office: 1,610 Days
• 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 increasing the diversity of V_H heavy chain-only antibodies in a transgenic non-human mammal comprising the steps of

a) providing a transgenic non-human mammal whose genome comprises more than one heterologous V_H heavy chain locus, wherein each V_H heavy chain locus comprises naturally occurring V_H gene segments of human origin, D gene segments, and J gene segments, and a gene segment encoding a heavy chain constant region which, when expressed, does not include a functional C_H1 domain and

b) expressing a V_H heavy chain-only antibody from at least one of said loci in B-cells,

wherein said heterologous V_H heavy chain loci are present on the same or different chromosomes, and the expression of a heterologous V_H locus is determined by allelic exclusion.

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

3. The method of claim 1, wherein each V gene segment is different from all other V gene segments.

4. The method of claim 1, wherein the multiple V_H heavy chain loci comprise any number or combination of the 39 functional human V gene segments.

5. The method of claim 4, wherein each different V_H heavy chain locus is present as a single copy in the genome of the transgenic non-human mammal.

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

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

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

9. The method of claim 1, wherein the J gene segments are human J gene segments.

10. The method of 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.

11. 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.

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

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

14. The method of claim 13, wherein the rodent is a mouse.

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

16. A method for the production of heavy chain-only antibodies comprising:

a) immunising a transgenic non-human mammal of claim 15 with an antigen; and

b) isolating antigen-specific heavy chain-only antibodies.

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

a) immunising a transgenic non-human mammal according to claim 15 with an antigen;

b) generating B-cell hybridomas;

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

d) isolating antigen-specific heavy chain-only antibody.