FUSION PROTEIN OF THERAPEUTIC POLYPEPTIDE WITH IMPROVED PHARMACOKINETIC PROFILE AND USE THEROF
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Abstract
The present invention provides a fusion protein comprising a therapeutic polypeptide fused to one or more flexible unstructured polypeptides and a trimeric scaffold protein. The flexible unstructured polypeptide sequence within the fusion protein is exhibited as one or more pCloud sequences derived from human fibrinogen alpha chain, and may be flanked by a proteinous connecting moiety of human origin. Also provided are pharmaceutical compositions comprising the fusion protein, nucleic acid molecules encoding the fusion protein, vectors containing the nucleic acids, host cells transformed with the vectors, and methods of making the fusion proteins of the invention, and use thereof.
15 Citations
45 Claims
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1-28. -28. (canceled)
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29. A fusion protein comprising a therapeutic polypeptide fused to one or more flexible un-structured polypeptide linkers and a scaffold protein,
wherein the scaffold protein forms a homo-trimer in solution, the flexible un-structured polypeptide linker contains 1 to 3000 amino acid residues, wherein the sum of G, S, E, A, P and T constitutes more than 90% of the flexible un-structured polypeptide sequence, and the flexible un-structured polypeptide sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm; -
the fusion protein exhibits an improved pharmacokinetic profile when administered to a subject compared with the therapeutic polypeptide by itself; and wherein the fusion protein is configured, from N-terminus to C-terminus, according to the following formula;
(Linker)-TP-(Linker)n-Scaffold,
TP-(Linker)n-Scaffold-(Linker),
(Linker)-TP-(Linker)n-Scaffold-(Linker),
(Linker)-Scaffold-(Linker)n-TP,
Scaffold-(Linker)n-TP-(Linker), or
(Linker)-Scaffold-(Linker)n-TP-(Linker),wherein; (a) Linker is a flexible un-structured polypeptide linker as defined above, and may be identical or different to each other; (b) TP is the therapeutic polypeptide selected from the group consisting of human glucagon-like peptide-1 (GLP-1), Exenatide, GLP-2, C-peptide, Calcitonin, human Parathyroid hormone (PTH), glucagon, G-CSF, GM-CSF, Interferon, interleukin factors, VEGF receptors, TNF alpha receptors, RANK, Growth hormone, Erythropoietin, blood-coagulation factors, single-chain Fv, single domain antibodies and functional variants thereof; (c) Scaffold is the scaffold protein that is selected from the group consisting of human collagen noncollagenous (NC) domains which form stable homo-trimers in solution, C1q-like molecular structures which form homo-trimers in solution, TNF-like molecular structures which form homo-trimers in solution, and proteins with C-type lectin-like domains (CTLD) which form homo-trimers in solution; (d) n is either 0 or 1, wherein 1 indicates the presence of and 0 indicates the absence of the linker. - View Dependent Claims (40, 41, 42, 43, 44, 45)
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33. A fusion protein comprising a therapeutic polypeptide fused to one or more pCloud sequences and a scaffold protein,
wherein the scaffold protein forms a homo-trimer in solution, the pCloud sequence is a flexible un-structured polypeptide, the fusion protein exhibits an improved pharmacokinetic profile when administered to a subject compared with the therapeutic polypeptide by itself, wherein the pCloud polypeptide sequence is characterized in that: -
(a) the total number of pCloud amino acid residues is at least 100 to about 3000; (b) the pCloud polypeptide sequence is generated by use of some or all of the fragments derived from human fibrinogen alpha chain, and in pCloud sequence, the fibrinogen fragments are flanked by flexible loops with various lengths from 1 to 100 residues, the flexible loops are rich in glycine (G) and serine (S), the flexible loops may also contain glutamate (E), alanine (A), proline (P) and threonine (T), the flexible loops have greater than 95% unstructured random coil formation as determined by GOR algorithm, therefore the pCloud polypeptide is primarily human originated and has low immunogenicity when administered to human; (c) the pCloud sequence is rich in glycine (G), serine (S) and Glutamate (E), the pCloud also contains alanine (A), proline (P), arginine (R) and threonine (T), the sum of G, S, E, A, P and T constitutes more than 90% of the pCloud sequence; (d) the pCloud sequence has greater than 90% unstructured random coil formation as determined by GOR algorithm; and (e) the pCloud sequence does not contain any T-cell epitopes as predicted by TEPITOPE algorithm; the pCloud sequence can be placed at either or both of the N-terminal and the C-terminal end of the therapeutic polypeptide, and the pCloud sequences can also be placed at either or both of the N-terminal and the C-terminal end of the scaffold protein, wherein the fusion protein is configured, from N-terminus to C-terminus, using the following formula;
(pCloud)m-TP-(pCloud)n-Scaffold-(pCloud)k Or
(pCloud)m-Scaffold-(pCloud)n-TP-(pCloud)kwherein; (a) the pCloud polypeptide may be identical or different to each other; (b) TP is the therapeutic polypeptide selected from the group consisting of human glucagon-like peptide-1 (GLP-1), Exenatide, GLP-2, C-peptide, Calcitonin, human Parathyroid hormone (PTH), glucagon, G-CSF, GM-CSF, Interferon, interleukin factors, VEGF receptors, TNF alpha receptors, RANK, Growth hormone, Erythropoietin, blood-coagulation factors, single-chain Fv, single domain antibodies and functional variants thereof; (c) Scaffold is the scaffold protein that is selected from the group consisting of human collagen noncollagenous (NC) domains which form stable homo-trimers in solution, C1q-like molecular structures which form homo-trimers in solution, TNF-like molecular structures which form homo-trimers in solution, and proteins with C-type lectin-like domains (CTLD) which form homo-trimers in solution; (d) m is either 0 or 1, n is either 0 or 1, k is either 0 or 1, and m+n+k>
=1, the digits indicate the number of presence of the designated polypeptides. - View Dependent Claims (34, 35, 36, 37, 38, 39)
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Specification