Generation of heavy-chain only antibodies in transgenic animals

Abstract

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

1 - 34 . (canceled) 
     
     
         35 . 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 heterologous V H  heavy chain locus comprises V H  gene segments, 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 V H  gene segments do not encode camelid VHH domains, and 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. 
     
     
         36 . The method of  claim 35 , wherein each heavy chain locus comprises one V H  gene segment. 
     
     
         37 . The method of  claim 35 , wherein each V H  gene segment is different from all other V H  gene segments. 
     
     
         38 . The method of  claim 35 , wherein the V H  gene segments are of human origin. 
     
     
         39 . The method of  claim 35 , wherein the multiple V H  heavy chain loci comprise any number or combination of the 39 functional human V gene segments. 
     
     
         40 . The method of  claim 39 , wherein each different V H  heavy chain locus is present as a single copy in the genome of the transgenic non-human mammal. 
     
     
         41 . The method of  claim 35 , wherein each V H  heavy chain locus comprises from one to forty D gene segments. 
     
     
         42 . The method of  claim 35 , wherein the D gene Segments are human D gene segments. 
     
     
         43 . The method of  claim 35 , wherein each V H  heavy chain locus comprises from one to twenty J gene segments. 
     
     
         44 . The method of  claim 35 , wherein the J gene segments are human J gene segments. 
     
     
         45 . The method of  claim 35 , wherein each V H  heavy chain locus comprises one or more human V H  gene segments, twenty-five functional human D gene segments and 6 human J gene segments. 
     
     
         46 . The method of  claim 35 , 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 at least one of the heavy chain constant region gene segments does not express a C H 1 domain. 
     
     
         47 . The method of  claim 35 , wherein the heavy chain constant region is of human origin. 
     
     
         48 . The method of  claim 45 , wherein the heavy chain constant region is of human origin. 
     
     
         49 . The method of  claim 35 , wherein the transgenic non-human mammal is a rodent. 
     
     
         50 . The method of  claim 45 , wherein the transgenic non-human mammal is a rodent. 
     
     
         51 . The method of  claim 49 , wherein the rodent is a mouse. 
     
     
         52 . A transgenic non-human mammal comprising more than one heterologous V H  heavy chain locus as defined in  claim 35 . 
     
     
         53 . A transgenic non-human mammal comprising more than one heterologous V H  heavy chain locus as defined in  claim 45 . 
     
     
         54 . A method for the production of heavy chain-only antibodies comprising:
 a) immunising a transgenic non-human mammal of  claim 52  with an antigen; and   b) isolating antigen-specific heavy chain-only antibodies.   
     
     
         55 . A method for the production of heavy chain-only antibodies comprising:
 a) immunising a transgenic non-human mammal of  claim 53  with an antigen; and   b) isolating antigen-specific heavy chain-only antibodies.   
     
     
         56 . 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 52  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.   
     
     
         57 . 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 53  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.