Compositions comprising AAV expressing dual antibody constructs and uses thereof
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 encode 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.
1. 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, wherein the recombinant AAV comprises:
a 5′ AAV inverted terminal repeat (ITR);
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;
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 are for a second and third immunoglobulin construct; and
a 3′ AAV ITR.
2. The recombinant AAV according to claim 1 , wherein the recombinant AAV further expresses a bispecific antibody.
3. The recombinant AAV according to claim 1 , wherein the recombinant AAV comprises a bidirectional enhancer located between the first expression cassette and the second expression cassette.
4. The recombinant AAV according to claim 1 , wherein the first ORF encodes an immunoglobulin light chain, the second ORF encodes a first immunoglobulin heavy chain and third ORF encodes a second heavy chain, whereby the expressed functional antibody constructs have two different heavy chains with different specificities which share a light chain.
5. The recombinant AAV according to claim 1 , wherein at least one of the second and third ORF contain modified Fc coding sequences.
6. The recombinant AAV according to claim 1 , wherein the linker in the second cassette comprises a linker selected from an IRES or an F2A.
7. The recombinant AAV according to claim 1 , wherein the regulatory control sequences for the first expression cassette and/or the second cassette comprise a minimal promoter.
8. The recombinant AAV 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.
9. The recombinant AAV according to claim 1 , wherein the first expression cassette is bicistronic and comprises a further ORF.
10. The recombinant AAV according to claim 9 , wherein each of the ORF comprise an scFv.
11. The recombinant AAV according claim 1 , wherein the vector comprises a bidirectional polyA between the first expression cassette and the second expression cassette.
12. The recombinant AAV according to claim 10 , wherein the first expression cassette comprises an enhancer and a minimal promoter.
13. The recombinant AAV according to claim 12 , wherein the second expression cassette comprises an enhancer and a minimal promoter.
14. The recombinant AAV according to claim 8 , wherein the first and second expression cassettes together express two Fabs.
15. The recombinant AAV according to claim 1 , wherein the at least two antibody constructs have different specificities.
16. The recombinant AAV according to claim 1 , wherein the at least two antibody constructs are independently selected from a monoclonal antibody, an immunoadhesin, a Fab, a bifunctional antibody, and combinations thereof.
17. 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, 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 bifunctional antibody, and wherein the recombinant AAV comprises:
a 5′ AAV inverted terminal repeat (ITR);
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;
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 are for a second and third immunoglobulin construct; and
a 3′ AAV ITR.
18. A pharmaceutical composition comprising a recombinant AAV according to claim 1 and pharmaceutically acceptable carrier.
19. A method of delivering at least two functional antibodies to a subject, said method comprising administering a recombinant AAV according to claim 1 to the subject.
20. The recombinant AAV according to claim 17 , wherein the recombinant AAV comprises a bidirectional enhancer located between the first expression cassette and the second expression cassette.
21. The recombinant AAV according to claim 17 , wherein the first ORF encodes an immunoglobulin light chain, the second ORF encodes a first immunoglobulin heavy chain and third ORF encodes a second heavy chain, whereby the expressed functional antibody constructs have two different heavy chains with different specificities which share a light chain.