Immunoglobulin 2

Abstract

The present invention relates to a method for the generation of single chain immunoglobulins in a mammal. In particular, the present invention relates to a method for the generation of single chain camelid VHH antibodies in a mammal which undergo the process of class-switching and affinity maturation found within antibody producing B cells. Single chain antibodies generated using the method of the present invention and the uses thereof are also described.

1 . A method for the production of a VHH single heavy chain antibody in a mammal comprising the step of expressing a heterologous VHH heavy chain locus in that mammal.  
     
     
         2 . A method according to  claim 1  wherein the VHH heavy chain locus comprises: 
 (a) at least one VHH region each comprising one VHH exon, at least one D region each comprising one D exon and at least one J region each comprising one J exon, wherein the VHH region, the D region and the J region are capable of recombining to form VDJ coding sequence,  
 (b) a constant region heavy chain region comprising at least one Cγ constant heavy chain gene, and which when expressed does not express a functional CH1 domain nor a functional CH4 domain,  
 (c) a recombination sequence (rss) capable of recombining a J region of step (a) directly with a Cγ constant heavy chain gene of step (b),  
 and which locus when expressed is capable of forming a complete single heavy chain IgG molecule (scIgG).  
 
     
     
         3 . A method for the production of a camelised VH single heavy chain antibody in a mammal comprising the step of expressing a camelised VH heavy chain locus in that mammal.  
     
     
         4 . A method according to  claim 3  wherein the camelised VH heavy chain locus comprises: 
 (a) at least one VH region each comprising one VH exon which is mutated such that the nucleic acid sequence is the same as a camelid VHH exon (a “camelised VH exon”), at least one D region each comprising one D exon and at least one J region comprising one J exon, wherein the VH region, the D region and the J region are capable of recombining to form VDJ coding sequence, and  
 (b) a constant region heavy chain region comprising at least one Cγ constant heavy chain gene, and which when expressed does not express a functional CH1 domain nor a functional CH4 domain,  
 (c) a recombination sequence (rss) capable of recombining a J region of step (a) directly with a Cγ constant heavy chain gene of step (b),  
 and which locus when expressed is capable of forming a complete single heavy chain IgG molecule (scIgG).  
 
     
     
         5 . A method according to  claim 1  wherein the locus comprises one or more FRT sites.  
     
     
         6 . A method according to  claim 1  wherein the locus comprises two or more LoxP sites.  
     
     
         7 . A method according to  claim 1  wherein the VHH heavy chain locus comprises at least one D region of human origin and at least one J region of human origin.  
     
     
         8 . A method according to  claim 3  wherein the camelised VH heavy chain locus comprises at least one D region of human origin and at least one J region of human origin.  
     
     
         9 . A method according to  claim 1  wherein the constant heavy chain region comprises at least one constant heavy chain gene which is of camelid origin.  
     
     
         10 . A method according to  claim 1  wherein the constant heavy chain region comprises at least one constant region heavy chain gene which is of non-camelid origin.  
     
     
         11 . A method according to  claim 10  wherein at least one constant region heavy chain gene is of human origin.  
     
     
         12 . A method according to  claim 11  wherein at least one constant region heavy chain gene is of rabbit origin.  
     
     
         13 . A method according to  claim 11  wherein at least one constant region heavy chain gene is of mouse origin.  
     
     
         14 . A method according to claim I wherein the heavy chain locus further comprises one or more cassette sites enabling the cassetting of the locus.  
     
     
         15 . A method according to  claim 14  wherein one or more cassette site is located in the heavy chain locus is in the 5′ leader sequence of the locus.  
     
     
         16 . A method according to  claim 15  wherein one or more cassette site is located in the heavy chain locus is in the 3′ untranslated region of the locus.  
     
     
         17 . A VHH single heavy chain antibody obtainable according to the method of claim I wherein that part of the antibody encoded by a VHH exon is encoded by an exon of camelid origin and the remainder of the antibody molecule are encoded by one or more regions of human origin.  
     
     
         18 . A VHH single heavy chain antibody obtainable according to the method of claim I wherein that part of the antibody encoded by a VHH exon is encoded by an exon of camelid origin and the constant heavy chain region is encoded by one or more regions of rabbit origin.  
     
     
         19 . A VHH single heavy chain antibody obtainable according to the method of  claim 1  wherein that part of the antibody encoded by a VHH exon is encoded by an exon of camelid origin and the constant heavy chain region is encoded by one or more regions of mouse origin.  
     
     
         20 . A camelised VH single heavy chain antibody obtainable according to the method of  claim 3 .  
     
     
         21 . A camelised VH single heavy chain antibody according to  claim 20  wherein the whole of the antibody is encoded by one or more regions of human origin.  
     
     
         22 . A camelised VH single heavy chain antibody according to  claim 20  wherein that part of the antibody encoding the constant heavy chain region is encoded by one or more regions of rabbit origin.  
     
     
         23 . A camelised VH single heavy chain antibody according to  claim 20  wherein that part of the antibody encoding the constant heavy chain region is encoded by one or more regions of mouse origin.  
     
     
         24 . A camelised VH single heavy chain antibody according to  claim 20  which is a monoclonal antibody.  
     
     
         25 . A vector comprising a VHH heavy chain locus described according to  claim 1 .  
     
     
         26 . A vector comprising a camelised VH heavy chain locus described according to  claim 3 .  
     
     
         27 . A host cell transformed with a vector according to  claim 25 .  
     
     
         28 . A transgenic mammal expressing a heterologous VHH heavy chain locus described according to  claim 2 .  
     
     
         29 . A transgenic mammal expressing a camelised VH heavy chain locus described according to  claim 4 .  
     
     
         30 . A transgenic mammal according to  claim 28  which is a mouse.  
     
     
         31 . A method for the production of single chain antibodies by immunising a transgenic mammal according to  claim 28  with an antigen.  
     
     
         32 . The use of a single heavy chain antibody according to  claim 17  in the preparation of a medicament for the prophylaxis and/or treatment of disease.