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 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 H 1 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 H 1 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 H 1 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.