Compositions comprising AAV expressing dual antibody constructs and uses thereof

Abstract

A recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses two functional antibody constructs in a cell is described. Also described are antibodies comprising a heavy chain and a light chain from a heterologous antibody. In one embodiment, the antibodies are co-expressed from a vector containing: a first expression cassette which encodes at least a first open reading frame (ORF) for a first immunoglobulin under the control of regulatory control sequences which direct expression thereof; and a second expression cassette which comprises a second ORF, a linker, and a third ORF under the control of regulatory control sequences which direct expression thereof, wherein the second and third ORF for a second and third immunoglobulin construct. The vector co-expressing these two antibody constructs is in one embodiment an AAV, in which the 5′ and 3′ ITRs flank the expression cassettes and regulatory sequences.

The invention claimed is: 
     
       1. A method of delivering at least two functional antibodies to a subject, said method comprising administering a recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses at least two functional monospecific antibodies in a cell, and wherein the recombinant AAV comprises:
 a 5′ AAV inverted terminal repeat (ITR); 
 a first expression cassette which encodes a first immunoglobulin chain under the control of regulatory control sequences which direct expression thereof; 
 a second expression cassette which encodes a second immunoglobulin chain, a linker, and a third immunoglobulin chain under the control of regulatory control sequences which direct expression thereof; and 
 a 3′ AAV ITR, and 
 wherein the encoded immunoglobulin chains form the at least two functional monospecific antibodies and wherein the at least two expressed monospecific antibodies have different specificities. 
 
     
     
       2. The method according to  claim 1 , wherein the recombinant AAV comprises a bidirectional enhancer located between the first expression cassette and the second expression cassette. 
     
     
       3. The method according to  claim 1 , wherein at least one of the immunoglobulin chains contain modified Fc coding sequences. 
     
     
       4. The method according to  claim 1 , wherein the linker in the second cassette comprises a linker selected from an IRES or an F2A. 
     
     
       5. The method according to  claim 1 , wherein the regulatory control sequences for the first expression cassette and/or the second cassette comprise a minimal promoter. 
     
     
       6. The method according to  claim 1 , wherein the regulatory control sequences for the first expression cassette and/or the second expression cassette comprise a minimal or synthetic polyA. 
     
     
       7. The method according to  claim 1 , wherein the first expression cassette further encodes a linker and a fourth immunoglobulin chain. 
     
     
       8. The method according to  claim 1 , wherein each of the immunoglobulin chain comprises an scFv. 
     
     
       9. The method according  claim 1 , wherein the vector comprises a bidirectional polyA between the first expression cassette and the second expression cassette. 
     
     
       10. The method according to  claim 1 , wherein the first expression cassette comprises an enhancer and a minimal promoter. 
     
     
       11. The method according to  claim 1 , wherein the second expression cassette comprises an enhancer and a minimal promoter. 
     
     
       12. The method according to  claim 1 , wherein the first and second expression cassettes together express two F(ab') 2 . 
     
     
       13. The method according to  claim 1 , wherein the at least two expressed monospecific antibodies are independently selected from a monoclonal antibody, an immunoadhesin, a Fab, a bifunctional antibody, and combinations thereof. 
     
     
       14. The method according to  claim 1 , wherein the immunoglobulin chains are an immunoglobulin light chain, a first immunoglobulin heavy chain, and a second immunoglobulin heavy chain, wherein the first expressed monospecific antibody comprises the immunoglobulin light chain and the first immunoglobulin heavy chain, and wherein the second expressed monospecific antibody comprises the immunoglobulin light chain and the second immunoglobulin heavy chain. 
     
     
       15. The method according to  claim 14 , wherein the first expression cassette encodes an antibody light chain, the second expression cassette encodes a first antibody heavy chain and a second antibody heavy chain, whereby the expressed functional antibodies have two different heavy chains with different specificities which share the light chain. 
     
     
       16. The method according to  claim 14 , wherein the first expression cassette encodes a first antibody heavy chain, the second expression cassette encodes an antibody light chain and a second antibody heavy chain, whereby the expressed functional antibodies have two different heavy chains with different specificities which share the light chain. 
     
     
       17. The method according to  claim 1 , wherein the recombinant AAV expresses a first monoclonal antibody having a first specificity, a second monoclonal antibody having a specificity different from the first monoclonal antibody, and a bispecific antibody.