Generation of heavy-chain only antibodies in transgenic animals
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.
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 H 1 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 H 1 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.