Generation of heavy-chain only antibodies in transgenic animals

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.

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.