Processable single chain molecules and polypeptides made using same
The present invention features inter alia nucleic acid molecules which encode polypeptides comprising a single chain Fc region and the polypeptides they encode. The Fc moieties of these constructs are linked by a cleavable scFc linker which is adjacent to at least one enzymatic cleavage site, e.g., an intracellular processing site. The resulting processed molecules comprise two polypeptide chains and substantially lack the extraneous amino acid sequence found in single chain Fc linker molecule. Methods of making and using these dimeric molecules are also described.
1. A nucleic acid molecule encoding a polypeptide, wherein the polypeptide comprises a first biologically active moiety and a second biologically active moiety, wherein the polypeptide comprises a formula selected from the group consisting of: A-F1-B-P1-L-P2-F2 and A-F1-P1-L-P2-B-F2 in linear sequence from amino to carboxy terminus,
wherein A is the first biologically active moiety, which comprises a clotting factor, wherein the clotting factor has pro-clotting activity;
B is the second biologically active moiety;
P1 and P2 are enzymatic cleavage sites;
L is a cleavable single chain Fc (cscFc) linker; and
F1 and F2 are Fc moieties, and
wherein the first biologically active moiety and the second biologically active moiety are different biologically active moieties.
2. The nucleic acid molecule of claim 1 , wherein P1 and P2 are recognized by different enzymes.
3. The nucleic acid molecule of claim 1 , wherein at least one of P1 or P2 comprises an amino acid sequence selected from: RRRR (SEQ ID NO: 40), RKRRKR (SEQ ID NO: 39), RRRRS (SEQ ID NO: 38), TQSFNDFTR (SEQ ID NO: 7), SVSQTSKLTR (SEQ ID NO: 8), DFLAEGGGVR (SEQ ID NO: 9), TTKIKPR (SEQ ID NO: 10), LVPRG (SEQ ID NO:35), and ALRPR (SEQ ID NO: 94).
4. The nucleic acid molecule of claim 1 , wherein the cscFc linker has a length of about 1 to about 50 amino acids.
5. The nucleic acid molecule of claim 1 , wherein the cscFc linker comprises a gly/ser peptide.
6. A nucleic acid molecule encoding a polypeptide comprising two polypeptide chains, wherein the first polypeptide chain comprises a light chain of a clotting factor linked to a first Fc moiety and the second polypeptide chain comprises a heavy chain of the clotting factor linked to a second Fc moiety, wherein the light chain and the heavy chain associate to form an enzymatically active clotting factor.
7. The nucleic acid molecule of claim 6 , wherein the clotting factor is selected from the group consisting of FVII, FVIIa, FIX, FIXa, FX, and FXa.
8. A composition comprising the nucleic acid molecule of claim 6 and a pharmaceutically acceptable carrier.
9. The nucleic acid molecule of claim 1 , wherein the cscFc linker is cleavable by thrombin.
10. The nucleic acid molecule of claim 1 , wherein P1 and P2 are recognized by the same enzyme.
11. A vector comprising the nucleic acid molecule of claim 1 .
12. A host cell comprising the vector of claim 11 , wherein the host cell expresses an enzyme which cleaves the cscFc linker.
13. The host cell of claim 12 , wherein the enzyme is endogenous to the cell.
14. The host cell of claim 12 , wherein the enzyme is exogenous to the cell.