Transgenic non-human vertebrate for the in vivo production of dual specificity immunoglobulins or hypermutated heavy chain only immunoglobulins

Abstract

The invention relates, in one aspect, generally to novel concept of guided selection of antibody variable domains, combination and expression entirely in vivo. An application is to produce multivalent polypeptides. The present invention relates to multivalent (eg, multispecific) antibodies, antibody chains and polypeptides, as well as heavy chain-only antibodies (H2 antibodies) that are devoid of light chains. The invention further relates to the selection, maturation and production of these in vivo in non-human vertebrates and non-human vertebrate cells. To this end the invention also relates to such non-human vertebrates and cells. The invention also relates to the provision of means to produce and select heavy chain-only antibodies and heavy chains comprising variable domains that have undergone affinity maturation.

1 . A mouse, wherein the genome of B cells of said mouse comprises:
 (i) an immunoglobulin heavy (IgH) chain locus comprising DNA encoding (in 5′ to 3′ direction) a rearranged or unrearranged variable region, a first switch, a mu constant region, a second switch and a non-mu constant region; wherein the IgH chain locus is capable of undergoing IgM to the non-mu isotype switching; and   
       wherein
 (ii) the non-mu mutant constant region comprises (in 5′ to 3′ direction) a nucleotide sequence encoding a predetermined epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein following isotype switching the mouse expresses multivalent polypeptide chains comprising (in N- to C-terminal direction) an antibody variable domain comprising a heavy chain variable region encoded by said variable region of the heavy chain locus, and an epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes. 
 
     
     
         2 . A mouse cell, the mouse cell genome comprising
 (i) an antibody heavy chain locus comprising (in 5′ to 3′ direction) a rearranged or unrearranged variable region, a first switch, a mu constant region, a second switch and a non-mu constant region; wherein the heavy chain locus of each cell is capable of undergoing IgM to a non-mu isotype switching; and   
       wherein
 (ii) the non-mu constant region comprises (in 5′ to 3′ direction) a nucleotide sequence encoding a predetermined epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab) , for expressing a multivalent polypeptide chain comprising (in N- to C-terminal direction) an antibody variable domain encoded by said variable region of the heavy chain locus, and an epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes. 
 
     
     
         3 . A mouse, wherein the mouse comprises lymphocytic cells whose genomes comprise an antibody heavy or light chain locus comprising (in 5′ to 3′ direction) a rearranged variable region and a non-mu constant region;
 wherein 
 the non-mu constant region comprises (in 5′ to 3′ direction) a nucleotide sequence encoding an and a predetermined epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the mouse expresses multivalent polypeptide chains comprising (in N- to C-terminal direction) an antibody variable domain encoded by said variable region of the chain locus, an and an epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the variable domain and the epitope binding moietyspecifically bind respective first and second epitopes. 
 
     
     
         4 . A mouse cell or a hybridoma, the mouse cell genome comprising an antibody heavy or light chain locus comprising (in 5′ to 3′ direction) a rearranged variable region and a non-mu constant region;
 wherein
 the non-mu constant region comprises (in 5′ to 3′ direction) a nucleotide sequence encoding an and a predetermined epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the mouse cell expresses a multivalent polypeptide chain comprising (in N- to C-terminal direction) an antibody variable domain encoded by said variable region of the locus, an and an epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes. 
 
 
     
     
         5 . A method of producing in a mouse multivalent polypeptide chains comprising (in N- to C-terminal direction) an antibody variable domain, an and an epitope binding moiety comprising a CH3 of an antigen binding Fc fragment (Fcab), wherein the variable domain and the epitope binding moiety specifically bind respective first and second epitopes, the method comprising:
 (a) immunising the mouse with a first predetermined antigen bearing the first epitope, obtaining isotype switching to expression from a non-mu constant region in at least some of the heavy chain loci of the mouse and expression of variable regions that have undergone somatic hypermutation; and   (b) selecting one or more polypeptide chains from said immunised mouse by selection for variable regions that bind said first epitope, wherein each selected chain comprises a somatically hypermutated variable region that specifically binds the epitope and wherein each selected chain binds the first and second epitopes; and   (c) providing the nucleotide sequence of the epitope binding moiety in the non-mu constant region of heavy chain loci in the genome of the mouse, wherein the polypeptide chains selected in step (b) bind the first and second epitopes.   
     
     
         6 . A multispecific polypeptide chain obtained by the method of  claim 5 , that specifically binds the first and second epitopes. 
     
     
         7 . The mouse of  claim 1 , wherein the N-terminal variable domain and/or the 5′-terminal variable region is a human variable domain or region. 
     
     
         8 . The mouse cell of  claim 2 , wherein the N-terminal variable domain and/or the 5′-terminal variable region is a human variable domain or region. 
     
     
         9 . The mouse of  claim 3 , wherein the N-terminal variable domain and/or the 5′-terminal variable region is a human variable domain or region. 
     
     
         10 . The mouse cell or hybridoma of  claim 4 , wherein the N-terminal variable domain and/or the 5′-terminal variable region is a human variable domain or region. 
     
     
         11 . The method of  claim 5 , wherein the N-terminal variable domain and/or the 5′-terminal variable region is a human variable domain or region. 
     
     
         12 . The multispecific polypeptide of  claim 6 , wherein the N-terminal variable domain and/or the 5′-terminal variable region is a human variable domain or region. 
     
     
         13 . The mouse of  claim 1 , wherein the heavy chain variable region and the epitope binding moiety are connected via a linker. 
     
     
         14 . The mouse cell of  claim 2 , wherein the heavy chain variable region and the epitope binding moiety are connected via a linker. 
     
     
         15 . The mouse of  claim 3 , wherein the antibody variable domain and the epitope binding moiety are connected via a linker. 
     
     
         16 . The mouse cell or hybridoma of  claim 4  wherein the antibody variable domain and the epitope binding moiety are connected via a linker. 
     
     
         17 . The method of  claim 5 , wherein the antibody variable domain and the epitope binding moiety are connected via a linker. 
     
     
         18 . The multispecific polypeptide of  claim 6 , wherein the antibody variable domain and the epitope binding moiety are connected via a linker. 
     
     
         19 . The mouse cell of  claim 2 , wherein the non-mu constant region is a gamma constant region and the heavy chain locus is capable of undergoing isotype switching to produce IgG antibodies comprising one or more IgG heavy chains in the absence of light chains, each heavy chain lacking a CH1domain. 
     
     
         20 . The method of  claim 5 , wherein each somatically mutated variable region in step (a) is provided by a non-mu-type heavy chain that lacks a CH1 domain. 
     
     
         21 . The method of  claim 20 , further comprising isolating or synthesizing a nucleic acid comprising a nucleotide sequence encoding a polypeptide chain selected in the method. 
     
     
         22 . The method of  claim 21 , further comprising humanising the nucleotide sequence encoding the polypeptide chain selected.