Method of improving the pharmacokinetic profile of a therapeutic polypeptide and the use thereof

• US 10,246,503 B2
• Filed: 12/19/2013
• Issued: 04/02/2019
• Est. Priority Date: 12/24/2012
• Status: Active Grant

• Current Assignee: Beijing Anxinhuaide Biotech. Co., Ltd.
• Original Assignee: Beijing Anxinhuaide Biotech. Co., Ltd.
• Inventors: Qian, Xinguo, Hong, Wei, Ma, Xiaoyu
• Primary Examiner(s): Chandra, Gyan
• Application Number:
  US14/655,282
• Publication Number:
  US 20160194370A1
• Time in Patent Office: 1,930 Days
• CPC Class Codes: A61K 38/179 for growth factors; for growth regulators
• CPC Class Codes: A61K 38/1816 Erythropoietin [EPO]
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61K 38/21 Interferons [IFN]
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: A61K 38/29 Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• CPC Class Codes: A61P 3/08 for glucose homeostasis pancreatic hormones A61P5/48
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/605 Glucagons
• CPC Class Codes: C07K 14/635 Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• CPC Class Codes: C07K 14/70578 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 14/7151 for tumor necrosis factor [TNF], for lymphotoxin [LT]
• CPC Class Codes: C07K 14/7156 for interferons [IFN]
• CPC Class Codes: C07K 14/78 Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
• CPC Class Codes: C07K 2319/31 fusions, other than Fc, for prolonged plasma life, e.g. albumin

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.

Claims

1. A fusion protein comprising a therapeutic polypeptide (TP) fused to one or more pCloud sequences and a scaffold protein,

wherein TP is human glucagon-like peptide or a functional variant thereof,

the scaffold protein forms a homo-trimer in solution and is selected from the group consisting of human collagen noncollagenous (NC) domains which form stable homo-trimers in solution, and

the pCloud sequence is a flexible un-structured polypeptide comprising some or all of the fragments of the human fibrinogen alpha chain;

and

wherein the pCloud polypeptide sequence;

(a) comprises at least 100 to about 3000 amino acid residues;

(b) comprises some or all of the fragments derived from human fibrinogen alpha chain;

wherein the fibrinogen alpha chain fragments are flanked by flexible loops with various lengths from 1 to 100 amino acid residues;

wherein 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;

(c) is rich in glycine (G), serine (S) and glutamate (E), and optionally further comprising alanine (A), proline (P), arginine (R) and threonine (T), and wherein the sum of G, S, E, A, P and T amino acid residues constitutes more than 90% of the pCloud amino acid sequence;

(d) has greater than 90% unstructured random coil formation as determined by GOR algorithm; and

(e) does not contain a T-cell epitope as predicted by TEPITOPE algorithm;

wherein the pCloud sequence can be at either or both of the N-terminal and the C-terminal end of the therapeutic polypeptide, and the pCloud sequences can optionally be placed at either or both of the N-terminal and the C-terminal end of the scaffold protein; and

wherein the pCloud polypeptides can be identical or different to each other;

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)k

wherein 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;

wherein 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; and

wherein the fusion protein exhibits an improved pharmacokinetic profile when administered to a subject compared with the TP by itself.

2. The fusion protein of claim 1, wherein the human fibrinogen fragments are selected from

3. The fusion protein of claim 1, wherein the TP is connected with the pCloud sequence via a proteinous connecting moiety (PCM) of human origin, wherein a flexible loop can be utilized to fuse the TP and the PCM, the flexible loop is as defined in claim 1, the PCM can be fused at either the N-terminal or the C-terminal end of the TP;

wherein the fusion protein is configured, from N-terminus to C-terminus, according to the following formula;

(pCloud)_m-TP-Loop-PCM-(pCloud)_n-Scaffold-(pCloud)_k or

(pCloud)_m-Scaffold-(pCloud)_n-PCM-Loop-TP-(pCloud)_k

wherein;

(a) pCloud is the pCloud polypeptide as defined in claim 1 and can be identical or different to each other;

(b) TP is GLP-1 or a functional variant thereof;

(c) Scaffold indicates the scaffold protein as defined in claim 1;

(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;

(e) Loop is a flexible loop as defined in claim 1; and

(f) PCM is the proteinous connecting moiety of human origin.

4. The fusion protein of claim 3, wherein the human fibrinogen alpha chain fragments are selected from

5. The fusion protein of claim 4, wherein the PCM is a human Fibronectin type III domain.

6. The fusion protein of claim 4, wherein the fusion protein comprises:

(i) a TP selected from the group consisting of GLP-1, GLP-1(A8G/G22E), GLP-1(A8G/G22E/R36G) and GLP-1(A8G/G22E/R36S);

(ii) a flexible loop;

(iii) a PCM selected from the group consisting of human fibronectin type III domain 7 (Fn7), and human fibronectin type III domain 8 (Fn8);

(iv) a pCloud polypeptide sequence as defined in claim 1; and

(v) the scaffold protein is the human collagen XVIII NC1 domain (COL18NC1).

7. The fusion protein of claim 5, wherein the fusion protein comprises:

(i) GLP-1(A8G/G22E/R36S) or GLP-1(A8G/G22E/R36G);

(ii) a flexible loop;

(iii) the PCM is Fn8;

(iv) a pCloud polypeptide; and

(v) the scaffold protein is the human collagen XVIII NC1 domain COL18NC1.