Aav vector compositions and methods for enhanced expression of immunoglobulins using the same

Abstract

Single AAV vector constructs for expression of an immunoglobulin molecule or fragment thereof and methods of making and using the same are described. The AAV vectors comprise a self-processing cleavage sequence between a first and second immunoglobulin coding sequence allowing for expression of a functional antibody molecule using a single promoter. The vector constructs may further include an additional proteolytic cleavage sequence which provides a means to remove the self processing peptide sequence from an expressed immunoglobulin molecule or fragment thereof. The vector constructs find utility in enhanced production of biologically active immunoglobulins or fragments thereof in vitro and in vivo.

1 . An AAV vector for expression of a recombinant immunoglobulin, comprising: in the 5′ to 3′ direction, a promoter operably linked to the coding sequence for a first chain of an immunoglobulin molecule or a fragment thereof, a sequence encoding a self-processing cleavage site and the coding sequence for a second chain of an immunoglobulin molecule or a fragment thereof, wherein the sequence encoding the self-processing cleavage site is inserted between the coding sequence for the first chain and the coding sequence for the second chain of said immunoglobulin molecule. 
     
     
         2 . An AAV vector according to  claim 1 , wherein the sequence encoding said self-processing cleavage site comprises a 2A sequence. 
     
     
         3 . An AAV vector according to  claim 2 , wherein said 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence. 
     
     
         4 . An AAV vector according to  claim 3 , wherein the 2A sequence encodes a peptide comprising amino acid residues LLNFDLLKLAGDVESNPGP (SEQ ID NO:1) or TLNFDLLKLAGDVESNPGP (SEQ ID NO:2). 
     
     
         5 . An AAV vector according to  claim 2 , wherein the coding sequence for the first chain of said immunoglobulin molecule or a fragment thereof encodes an immunoglobulin heavy chain. 
     
     
         6 . An AAV vector according to  claim 2 , wherein the coding sequence for the first chain of said immunoglobulin molecule or a fragment thereof encodes an immunoglobulin light chain. 
     
     
         7 . An AAV vector according to  claim 2 , further comprising an additional proteolytic cleavage site between the coding sequence for said first chain of an immunoglobulin molecule or a fragment thereof and the coding sequence for said second chain of an immunoglobulin molecule or a fragment thereof. 
     
     
         8 . An AAV vector according to  claim 7 , wherein said additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXK(R)R (SEQ ID NO:10). 
     
     
         9 . An AAV vector according to  claim 2 , wherein the promoter is selected from the group consisting of an elongation factor 1-alpha promoter (EF1-alpha) promoter, a phosphoglycerate kinase-1 promoter (PGK) promoter, a cytomegalovirus immediate early gene promoter (CMV), a chimeric liver-specific promoter (LSP), a cytomegalovirus enhancer/chicken beta-actin promoter (CAG), a tetracycline responsive promoter (TRE), a transthyretin promoter (TTR), a simian virus 40 promoter (SV40) and a CK6 promoter. 
     
     
         10 . An AAV vector according to  claim 9 , wherein said promoter is a CAG hybrid promoter/enhancer. 
     
     
         11 . An AAV vector according to  claim 9 , wherein said promoter is an elongation factor 1-alpha promoter (EF1a) promoter. 
     
     
         12 . An AAV vector according to  claim 2 , wherein said heavy and light chain immunoglobulin coding sequences are expressed in an equimolar ratio or close to equimolar ratio. 
     
     
         13 . An AAV vector according to  claim 2 , wherein vector said AAV vector is an AAV6 vector. 
     
     
         14 . An AAV vector according to  claim 2 , wherein vector said AAV vector is an AAV8 vector. 
     
     
         15 . A recombinant immunoglobulin molecule produced by a cell transduced with a vector of  claim 10 . 
     
     
         16 . A recombinant immunoglobulin molecule produced by a cell transduced with a vector of  claim 12 . 
     
     
         17 . A host cell transduced with a vector of  claim 10 . 
     
     
         18 . A host cell transduced with a vector of  claim 12 . 
     
     
         19 . A method for producing a recombinant immunoglobulin molecule, comprising the steps of:
 a. transducing a host cell with a vector according to  claim 2 ; and   b. expressing said recombinant immunoglobulin in said transduced host cell, wherein said first immunoglobulin coding sequence and said second immunoglobulin coding sequence are expressed in a substantially equimolar ratio.   
     
     
         20 . The method according to  claim 19 , wherein the sequence encoding said self-processing cleavage site comprises a 2A sequence. 
     
     
         21 . The method according to  claim 19 , wherein said 2A sequence is a Foot and Mouth Disease Virus (FMDV) sequence. 
     
     
         22 . The method according to  claim 21 , wherein the 2A sequence encodes a peptide comprising amino acid residues LLNFDLLKLAGDVESNPGP (SEQ ID NO:1) or TLNFDLLKLAGDVESNPGP (SEQ ID NO:2). 
     
     
         23 . The method according to  claim 19 , wherein said vector further comprises an additional proteolytic cleavage site between the coding sequence for said first protein or polypeptide chain and the sequence encoding said self-processing cleavage site. 
     
     
         24 . The method according to  claim 23 , wherein said additional proteolytic cleavage site is a furin cleavage site with the consensus sequence RXK(R)R (SEQ ID NO:10). 
     
     
         25 . The method according to  claim 23 , further comprising treating said expressed immunoblobulin with a carboxypeptidase. 
     
     
         26 . The method according to  claim 19 , wherein said AAV is administered in vivo by a route selected from the group consisting of portal vein injection, intramuscular injection, intratumoral injection and intraperitoneal injection. 
     
     
         27 . The method according to  claim 26 , wherein said full length recombinant immunoglobulin is expressed in vivo for at least 3 months. 
     
     
         28 . The method according to  claim 26 , wherein said full length recombinant immunoglobulin is expressed at a level of at least 1 mg/ml.