# US 10,246,503 B2 · Method of improving the pharmacokinetic profile of a therapeutic polypeptide and the use thereof

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- Number: US 10,246,503 B2
- Document ID: US10246503B2
- Status: Granted
- Kind: B2
- Application number: 14/655,282
- Filed: 2013-12-19
- Published: 2016-07-07
- Granted: 2019-04-02
- Earliest claimed: 2012-12-24
- Assignees: Beijing Anxinhuaide Biotech. Co., Ltd.
- Applicants: Beijing Anxinhuaide Biotech. Co., Ltd
- Inventors: Xinguo Qian; Wei Hong; Xiaoyu Ma
- Web page: https://patentgrep.com/patent/US10246503B2

## CPC

- C07K 14/605: Glucagons
- A61K 38/26: Glucagons
- A61P 3/08: for glucose homeostasis (pancreatic hormones A61P5/48)
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 35/00: Antineoplastic agents
- C07K 14/635: Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 14/71: for growth factors; for growth regulators
- C07K 14/7151: {for tumor necrosis factor [TNF], for lymphotoxin [LT]}
- C07K 14/7156: {for interferons [IFN]}
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- A61K 38/179: {for growth factors; for growth regulators}
- A61K 38/1816: {Erythropoietin [EPO]}
- A61K 38/193: {Colony stimulating factors [CSF]}
- A61K 38/21: Interferons {[IFN]}
- A61K 38/27: Growth hormone [GH], i.e. somatotropin
- A61K 38/29: Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
- C07K 2319/31: fusions, other than Fc, for prolonged plasma life, e.g. albumin
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## 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

### Claim 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.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the human fibrinogen fragments are selected from
GSTSSGTGSETESP;
SEQ ID NO: 32
PSSAGS;
SEQ ID NO: 33
SGSSGPGSTG;
SEQ ID NO: 34
PGSSGTGGTAT;
SEQ ID NO: 35
PGSSGPGSTGS;
SEQ ID NO: 36
SGSSGTGSTG;
SEQ ID NO: 37
PGSPRPGSTGT;
SEQ ID NO: 38
PGSSERGSAG;
SEQ ID NO: 39
TSESSASGSTG;
SEQ ID NO: 40
SESGS;
SEQ ID NO: 41
PESPGSG;
SEQ ID NO: 42
or
TSGST,
SEQ ID NO: 43
and the flexible loops are selected from
(G2S)n, (G3S)n SEQ ID NO: 91, (G4S)n SEQ ID NO:
92, (G5S)n SEQ ID NO: 93,(GS)n, (G2S2)n SEQ ID NO:
94, (GS2)n, (GS3)n SEQ ID NO: 95, (S2G)n, (S3G)n
SEQ ID NO: 96, (S4G)n SEQ ID NO: 97, (S5G)n SEQ
ID NO: 98, (SG)n, (S2G2)n SEQ ID NO: 99, (SG2)n,
(SG3)n SEQ ID NO: 100 where n is an integer;
SEQ ID NO: 77
GGGSGGGG;
SEQ ID NO: 90
GGGSGGGGS;
SEQ ID NO: 101
GSGG;
SEQ ID NO: 102
GGGSGGG;
SEQ ID NO: 103
GGGGSGGG;
SEQ ID NO: 89
GGGSGG;
SEQ ID NO: 76
GGGGSGG;
SEQ ID NO: 104
GGGGSGGGGSGGGGSGGGGS;
SEQ ID NO: 105
GGGGSGGGGSASSASTGGPSGGGGSGGGGS;
SEQ ID NO: 106
GGGGSGGGGSTASSASTGGPSGGGGSGGGGSAPSSGSTSGGTAAGGGGSG
GGGS;
or
SEQ ID NO: 107
GGGSGGGSGGGSTASSASTKGPSGGGSGGGSGGGSAPSSKSTSGGTAAGG
GSGGGSGGGS.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 3)

The fusion protein of claim 3, wherein the human fibrinogen alpha chain fragments are selected from
GSTSSGTGSETESP;
SEQ ID NO: 32
PSSAGS;
SEQ ID NO: 33
SGSSGPGSTG;
SEQ ID NO: 34
PGSSGTGGTAT;
SEQ ID NO: 35
PGSSGPGSTGS;
SEQ ID NO: 36
SGSSGTGSTG;
SEQ ID NO: 37
PGSPRPGSTGT;
SEQ ID NO: 38
PGSSERGSAG;
SEQ ID NO: 39
TSESSASGSTG;
SEQ ID NO: 40
SESGS;
SEQ ID NO: 41
PESPGSG;
SEQ ID NO: 42
or
TSGST,
SEQ ID NO: 43
and
the flexible loops are selected from
(G2S)n, (G3S)n SEQ ID NO: 91, (G4S)n SEQ ID NO:
92, (G5S)n SEQ ID NO: 93, (GS)n, (G2S2)n SEQ ID
NO: 94, (GS2)n, (GS3)n SEQ ID NO: 95, (S2G)n,
(S3G)n SEQ ID NO: 96, (S4G)n SEQ ID NO: 97, (S5G)n
SEQ ID NO: 98, (SG)n, (S2G2)n SEQ ID NO: 99,
(SG2)n, (SG3)n SEQ ID NO: 100 where n is an
integer;
SEQ ID NO: 77
GGGSGGGG;
SEQ ID NO: 90
GGGSGGGGS;
SEQ ID NO: 101
GSGG;
SEQ ID NO: 102
GGGSGGG;
SEQ ID NO: 103
GGGGSGGG;
SEQ ID NO: 89
GGGSGG;
SEQ ID NO: 76
GGGGSGG;
SEQ ID NO: 104
GGGGSGGGGSGGGGSGGGGS;
SEQ ID NO: 105
GGGGSGGGGSASSASTGGPSGGGGSGGGGS;
SEQ ID NO: 106
GGGGSGGGGSTASSASTGGPSGGGGSGGGGSAPSSGSTSGGTAAGGGGSG
GGGS;
or
SEQ ID NO: 107
GGGSGGGSGGGSTASSASTKGPSGGGSGGGSGGGSAPSSKSTSGGTAAGG
GSGGGSGGGS.

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 1, 4)

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).

### Claim 7 (depends on 5)

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.