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 polypeptide, comprising (i) at least one biologically active moiety, (ii) an Fc region comprising at least two Fc moieties and (iii) a cleavable single chain Fc (cscFc) linker interposed between the two Fc moieties, wherein the cscFc linker is linked to at least one enzymatic cleavage site which results in cleavage of the cscFc linker.
2 . The polypeptide of claim 1 , wherein at least one biologically active moiety comprises a clotting factor selected from the group consisting of FVIII, FIX, FIXa, FX, and FXa.
3 . (canceled)
4 . The polypeptide of claim 1 comprising the formula A-F1-P1-L-P2-B-F2 or A-F1-B-P1-L-P2-F2, each in linear sequence from amino to carboxy terminus, wherein A is optionally present and, if present, is a biologically active moiety, F1 is an Fc moiety or domain, P1 is optionally present and, if present, is an enzymatic cleavage site, L is the cscFc linker, P2 is optionally present and, if present, is an enzymatic cleavage site, B is optionally present and, if present, is a biogically active moiety, and F2 is an Fc moiety, provided that at least one of A and B is present and at least one of P1 and P2 is present.
5 . (canceled)
6 . The polypeptide of claim 4 , wherein A is present and comprises the light chain of a clotting factor and B is present and comprises the heavy chain of the clotting factor which when associated form an active molecule.
7 - 12 . (canceled)
13 . The polypeptide of claim 4 , wherein P1 and P2 are both present and are recognized by the same or by different enzymes.
14 . The polypeptide of claim 13 , wherein at least one of P1 or P2 comprises an amino acid sequence selected from the group consisting of 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).
15 - 18 . (canceled)
19 . The polypeptide of claim 1 , wherein the cscFc linker has a length of about 1 to about 50 amino acids.
20 . (canceled)
21 . The polypeptide of claim 1 , wherein the cscFc linker comprises a Gly/Ser peptide.
22 . The polypeptide of claim 21 , wherein the Gly/Ser peptide is of the formula (Gly 4 Ser) n or Ser(Gly 4 Ser) n , wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
23 - 26 . (canceled)
27 . The polypeptide of claim 1 which further comprises a targeting moiety.
28 . The polypeptide of claim 27 , wherein the targeting moiety binds to resting or activated platelets.
29 - 35 . (canceled)
36 . A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.
37 . (canceled)
38 . A nucleic acid molecule encoding the polypeptide of claim 1 .
39 . A vector comprising the nucleic acid molecule of claim 38 .
40 . (canceled)
41 . A processed polypeptide comprising at least two amino acid chains which polypeptide is encoded by the nucleic acid molecule of claim 38 .
42 . A host cell comprising the vector of claim 39 , wherein the host cell expresses an enzyme which cleaves the cscFc linker.
43 - 44 . (canceled)
45 . A method for producing a polypeptide comprising culturing the host cell of claim 42 in culture such that a mature polypeptide comprising two amino acid chains is produced.
46 - 48 . (canceled)
49 . A method for treating or preventing a disease or disorder in a subject, comprising administering to a subject in need thereof an effective amount of the processed polypeptide of claim 41 .
50 - 51 . (canceled)
52 . The method of claim 49 , wherein the disease or disorder is hemophilia A.
53 . The method of claim 49 , wherein the disease or disorder is hemophilia B.