U.S. patent application number 15/007438 was filed with the patent office on 2016-08-25 for fgf21 mutants and uses thereof.
The applicant listed for this patent is Amgen Inc.. Invention is credited to Edward J. Belouski, Murielle Marie Ellison, Colin V. Gegg, JR., Randy I. Hecht, Yue-Sheng Li, Mark L. Michaels, Kenneth W. WALKER, Jing Xu.
Application Number | 20160244497 15/007438 |
Document ID | / |
Family ID | 41571715 |
Filed Date | 2016-08-25 |
United States Patent
Application |
20160244497 |
Kind Code |
A1 |
WALKER; Kenneth W. ; et
al. |
August 25, 2016 |
FGF21 MUTANTS AND USES THEREOF
Abstract
The invention provides nucleic acid molecules encoding FGF21
mutant polypeptides, FGF21 mutant polypeptides, pharmaceutical
compositions comprising FGF21 mutant polypeptides, and methods for
treating metabolic disorders using such nucleic acids,
polypeptides, or pharmaceutical compositions.
Inventors: |
WALKER; Kenneth W.; (Newbury
Park, CA) ; Gegg, JR.; Colin V.; (Camarillo, CA)
; Hecht; Randy I.; (Raleigh, NC) ; Belouski;
Edward J.; (Thousand Oaks, CA) ; Li; Yue-Sheng;
(Cambridge, MA) ; Michaels; Mark L.; (Encino,
CA) ; Xu; Jing; (Thousand Oaks, CA) ; Ellison;
Murielle Marie; (Thousand Oaks, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Amgen Inc. |
Thousand Oaks |
CA |
US |
|
|
Family ID: |
41571715 |
Appl. No.: |
15/007438 |
Filed: |
January 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13123205 |
Apr 7, 2011 |
9279013 |
|
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PCT/US2009/060045 |
Oct 8, 2009 |
|
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15007438 |
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61195761 |
Oct 10, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/60 20170801;
A61P 3/00 20180101; A61P 3/04 20180101; C07K 14/50 20130101; A61P
3/10 20180101; A61K 38/00 20130101 |
International
Class: |
C07K 14/50 20060101
C07K014/50; A61K 47/48 20060101 A61K047/48 |
Claims
1. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding a polypeptide of SEQ ID NO: 4 having at least one
amino acid substitution that is: (a) a lysine residue at one or
more of positions 36, 72, 77, 126 and 175; (b) a cysteine residue
at one or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91,
112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) an arginine
residue at one or more of positions 56, 59, 69, and 122; (d) a
glycine residue at position 170; (e) a glycine residue at position
171; and combinations of (a)-(e).
2. A vector comprising the nucleic acid molecule of claim 1.
3. A host cell comprising the vector of claim 2.
4. The host cell of claim 3 that is a eukaryotic cell.
5. The host cell of claim 3 that is a prokaryotic cell.
6. A process of producing a polypeptide encoded by the vector of
claim 2 comprising culturing a host cell comprising the vector of
claim 2 under suitable conditions to express the polypeptide, and
optionally isolating the polypeptide.
7. A polypeptide produced by the process of claim 6.
8. The polypeptide of claim 7, further comprising a proline or
glycine residue added to the C-terminus of the polypeptide.
9. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO: 4 having at least one amino acid substitution that is:
(a) a lysine residue at one or more of positions 36, 72, 77, 126
and 175; (b) a cysteine residue at one or more of positions 37, 38,
46, 91, 69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170,
and 179; (c) an arginine residue at one or more of positions 56,
59, 69, and 122; (d) a glycine residue at position 170; (e) a
glycine residue at position 171; and combinations of (a)-(e),
10. The polypeptide of claim 9, further comprising a proline or
glycine residue added to the C-terminus of the polypeptide.
11. The isolated polypeptide of claim 9, wherein the polypeptide is
covalently linked to one or more polymers.
12. The isolated polypeptide of claim 11, wherein the polypeptide
is covalently linked to one polymer.
13. The isolated polypeptide of claim 12, wherein the polymer is a
water-soluble polymer.
14. The isolated polypeptide of claim 13, wherein the water-soluble
polymer is polyethylene glycol (PEG), monomethoxy-polyethylene
glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene
glycol, propylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols, or polyvinyl
alcohol.
15. The isolated polypeptide of claim 14, wherein the water-soluble
polymer is PEG.
16. The isolated polypeptide of claim 11, wherein the polymer is a
branched polymer.
17. The isolated polypeptide of claim 9, wherein the polypeptide
has a PEG moiety covalently linked to its amino-terminus.
18. The isolated polypeptide of claim 9, wherein the polypeptide is
covalently linked to two polymers.
19. The isolated polypeptide of claim 18, wherein one if the two
polymers is a water-soluble polymer.
20. The isolated polypeptide of claim 19, wherein the water-soluble
polymer is polyethylene glycol (PEG), monomethoxy-polyethylene
glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene
glycol, propylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols, or polyvinyl
alcohol.
21. The isolated polypeptide of claim 20, wherein the water-soluble
polymer is PEG.
22. The isolated polypeptide of claim 18, wherein one of the
polymers is branched.
23. The isolated polypeptide of claim 18, wherein both of the
polymers are branched.
24. The isolated polypeptide of claim 9, wherein the polypeptide
has a PEG moiety covalently linked to its amino-terminus.
25. A pharmaceutical composition comprising the isolated
polypeptide of claim 9 and a pharmaceutically acceptable
formulation agent.
26. The pharmaceutical composition of claim 25, wherein the
pharmaceutically acceptable formulation agent is a carrier,
adjuvant, solubilizer, stabilizer, or anti-oxidant.
27. A method for treating a metabolic disorder comprising
administering to a human patient in need thereof the pharmaceutical
composition of claim 26.
28. The method of claim 27, wherein the metabolic disorder is
diabetes.
29. The method of claim 27, wherein the metabolic disorder is
obesity.
30. An isolated nucleic acid encoding a polypeptide comprising the
amino acid sequence of SEQ ID NO: 4 having at least one amino acid
substitution that is: (a) a lysine residue at one or more of
positions 36, 72, 77, 126 and 175; (b) a cysteine residue at one or
more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,
120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at
one or more of positions 56, 59, 69, and 122; (d) a glycine residue
at position 170; (e) a glycine residue at position 171; and
combinations of (a)-(e), and which comprises additions, deletions
or further substitutions that make the polypeptide at least 85%
identical to SEQ ID NO:4, provided that the at least one amino acid
substitution of claim 1(a)-(e) is not further modified.
31. A vector comprising the nucleic acid molecule of claim 30.
32. A host cell comprising the vector of claim 31.
33. The host cell of claim 32 that is a eukaryotic cell.
34. The host cell of claim 32 that is a prokaryotic cell.
35. A process of producing a polypeptide encoded by the vector of
claim 30 comprising culturing a host cell comprising the vector of
claim 30 under suitable conditions to express the polypeptide, and
optionally isolating the polypeptide.
36. A polypeptide produced by the process of claim 35.
37. The polypeptide of claim 36, further comprising a proline or
glycine residue added to the C-terminus of the polypeptide.
38. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO: 4 having at least one amino acid substitution that is:
(a) a lysine residue at one or more of positions 36, 72, 77, 126
and 175; (b) a cysteine residue at one or more of positions 37, 38,
46, 91, 69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170,
and 179; (c) an arginine residue at one or more of positions 56,
59, 69, and 122; (d) a glycine residue at position 170; (e) a
glycine residue at position 171; and combinations of (a)-(e), and
which comprises additions, deletions or further substitutions that
make the polypeptide at least 85% identical to SEQ ID NO:4,
provided that the at least one amino acid substitution of claim
1(a)-(e) is not further modified.
39. The polypeptide of claim 38, further comprising a proline or
glycine residue added to the C-terminus of the polypeptide.
40. The isolated polypeptide of claim 38, wherein the polypeptide
is covalently linked to one polymer.
41. The isolated polypeptide of claim 40, wherein the polymer is a
water-soluble polymer.
42. The isolated polypeptide of claim 41, wherein the water-soluble
polymer is polyethylene glycol (PEG), monomethoxy-polyethylene
glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene
glycol, propylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols, or polyvinyl
alcohol.
43. The isolated polypeptide of claim 42, wherein the water-soluble
polymer is PEG.
44. The isolated polypeptide of claim 40, wherein the polymer is
branched.
45. The isolated polypeptide of claim 38, wherein the polypeptide
has a single PEG moiety covalently linked to its
amino-terminus.
46. The isolated polypeptide of claim 38, wherein the polypeptide
is covalently linked to two polymers.
47. The isolated polypeptide of claim 46, wherein one if the two
polymers is a water-soluble polymer.
48. The isolated polypeptide of claim 47, wherein the water-soluble
polymer is polyethylene glycol (PEG), monomethoxy-polyethylene
glycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene
glycol, propylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols, or polyvinyl
alcohol.
49. The isolated polypeptide of claim 48, wherein the water-soluble
polymer is PEG.
50. The isolated polypeptide of claim 46, wherein both of the
polymers are water soluble polymers.
51. The isolated polypeptide of claim 50, wherein the water-soluble
polymers are independently polyethylene glycol (PEG),
monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl
pyrrolidone) polyethylene glycol, propylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols, or polyvinyl alcohol and combinations thereof.
52. The isolated polypeptide of claim 46, wherein both of the water
soluble polymers are PEG.
53. The isolated polypeptide of claim 46, wherein one of the
polymers is branched.
54. The isolated polypeptide of claim 46, wherein both of the
polymers are branched.
55. A pharmaceutical composition comprising the isolated
polypeptide of claim 38 and a pharmaceutically acceptable
formulation agent.
56. The pharmaceutical composition of claim 55, wherein the
pharmaceutically acceptable formulation agent is a carrier,
adjuvant, solubilizer, stabilizer, or anti-oxidant.
57. A method for treating a metabolic disorder comprising
administering to a human patient in need thereof the pharmaceutical
composition of claim 56.
58. The method of claim 57, wherein the metabolic disorder is
diabetes.
59. The method of claim 57, wherein the metabolic disorder is
obesity.
60. A composition comprising a first polypeptide comprising the
amino acid sequence of SEQ ID NO: 4 optionally having at least one
amino acid substitution that is: (a) a lysine residue at one or
more of positions 36, 72, 77, 126 and 175; (b) a cysteine residue
at one or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91,
112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) an arginine
residue at one or more of positions 56, 59, 69, and 122; (d) a
glycine residue at position 170; (e) a glycine residue at position
171; and combinations of (a)-(e), joined by a linker to a second
polypeptide comprising a polypeptide comprising the amino acid
sequence of SEQ ID NO: 4 optionally having at least one amino acid
substitution that is: (a) a lysine residue at one or more of
positions 36, 72, 77, 126 and 175; (b) a cysteine residue at one or
more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,
120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at
one or more of positions 56, 59, 69, and 122; (d) a glycine residue
at position 170; (e) a glycine residue at position 171; and
combinations of (a)-(e).
61. The polypeptide of claim 60, wherein the first, second or both
polypeptides further comprise a proline or glycine residue added to
the C-terminus of the polypeptide.
62. The composition of claim 60, wherein the linker is a
peptide.
63. The composition of claim 60, wherein the linker is a water
insoluble polymer.
64. The composition of claim 63, wherein the water-soluble polymer
is polyethylene glycol (PEG), monomethoxy-polyethylene glycol,
dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,
propylene glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols, or polyvinyl alcohol.
65. The composition of claim 64, wherein the water-soluble polymer
is PEG.
66. The composition of claim 60, wherein the first, second or both
polypeptides are further covalently linked to one polymer, in
addition to the linker.
67. The composition of claim 66, wherein the polymer is a
water-soluble polymer.
68. The composition of claim 67, wherein the water-soluble polymer
is polyethylene glycol (PEG), monomethoxy-polyethylene glycol,
dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,
propylene glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols, or polyvinyl alcohol.
69. The composition of claim 68, wherein the water-soluble polymer
is PEG.
70. The composition of claim 63, wherein the polymer is
branched.
71. The composition of claim 60, wherein the composition has a
single PEG moiety covalently linked to its amino-terminus.
72. The composition of claim 60, wherein the composition is
covalently linked to two polymers.
73. The composition of claim 72, wherein one if the two polymers is
a water-soluble polymer.
74. The composition of claim 73, wherein the water-soluble polymer
is polyethylene glycol (PEG), monomethoxy-polyethylene glycol,
dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,
propylene glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols, or polyvinyl alcohol.
75. The composition of claim 74, wherein the water-soluble polymer
is PEG.
76. The composition of claim 72, wherein both of the polymers are
water soluble polymers.
77. The composition of claim 76, wherein the water-soluble polymers
are independently polyethylene glycol (PEG),
monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl
pyrrolidone) polyethylene glycol, propylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols, or polyvinyl alcohol and combinations thereof.
78. The composition of claim 77, wherein both of the water soluble
polymers are PEG.
79. The isolated polypeptide of claim 72, wherein one of the
polymers is branched.
80. The isolated polypeptide of 72 wherein both of the polymers are
branched.
81. A pharmaceutical composition comprising the composition of
claim 60 and a pharmaceutically acceptable formulation agent.
82. The pharmaceutical composition of claim 81, wherein the
pharmaceutically acceptable formulation agent is a carrier,
adjuvant, solubilizer, stabilizer, or anti-oxidant.
83. A method for treating a metabolic disorder comprising
administering to a human patient in need thereof the pharmaceutical
composition of claim 81.
84. The method of claim 83 wherein the metabolic disorder is
diabetes.
85. The method of claim 83, wherein the metabolic disorder is
obesity.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/123,205 filed Apr. 7, 2011, which is a 35
U.S.C. 371 filing of International Application No.
PCT/US2009/060045 filed Oct. 8, 2009, which claims the benefit of
U.S. Provisional Application No. 61/195,761 filed Oct. 10, 2008,
each of which are incorporated by reference herein in its
entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled "A-1451-WO-PCT_Seq_ListingST25.txt," created Oct. 7,
2009, which is 12 KB in size. The electronic format of the Sequence
Listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates to nucleic acid molecules encoding
FGF21 mutant polypeptides, FGF21 mutant polypeptides,
pharmaceutical compositions comprising FGF21 mutant polypeptides,
and methods for treating metabolic disorders using such nucleic
acids, polypeptides, or pharmaceutical compositions.
[0005] 2. Background of the Invention
[0006] FGF21 is a secreted polypeptide that belongs to a subfamily
of fibroblast growth factors (FGFs) that includes FGF19, FGF21, and
FGF23 (Itoh et al., 2004, Trend Genet. 20: 563-69). FGF21 is an
atypical FGF in that it is heparin independent and functions as a
hormone in the regulation of glucose, lipid, and energy
metabolism.
[0007] FGF21 was isolated from a liver cDNA library as a hepatic
secreted factor. It is highly expressed in liver and pancreas and
is the only member of the FGF family to be primarily expressed in
liver. Transgenic mice overexpressing FGF21 exhibit metabolic
phenotypes of slow growth rate, low plasma glucose and triglyceride
levels, and an absence of age-associated type 2 diabetes, islet
hyperplasia, and obesity. Pharmacological administration of
recombinant FGF21 protein in diabetic rodent models results in
normalized levels of plasma glucose, reduced triglyceride and
cholesterol levels, and improved glucose tolerance and insulin
sensitivity. In addition, FGF21 reduces body weight and body fat by
increasing energy expenditure, physical activity, and metabolic
rate. Experimental research provides support for the
pharmacological administration of FGF21 for the treatment of type 2
diabetes, obesity, dyslipidemia, and other metabolic conditions or
disorders in humans.
[0008] Human FGF21 has a short half-life in vivo. In mice and
cynomolgus monkey, the effective half-life of human FGF21 is 1 to 2
hours. In developing an FGF21 protein for use as a therapeutic in
the treatment of type 2 diabetes, an increase in half-life would be
desirable. FGF21 proteins having an enhanced half-life would allow
for less frequent dosing of patients being administered the
protein.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the present invention provides an
isolated nucleic acid molecule comprising a nucleotide sequence
encoding a polypeptide of SEQ ID NO: 4 having at least one amino
acid substitution that is: (a) a lysine residue at one or more of
positions 36, 72, 77, 126 and 175; (b) a cysteine residue at one or
more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,
120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at
one or more of positions 56, 59, 69, and 122; (d) a glycine residue
at position 170; (e) a glycine residue at position 171; and
combinations of (a)-(e).
[0010] In another embodiment, the present invention provides an
isolated nucleic acid encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 4 having at least one amino acid
substitution that is: (a) a lysine residue at one or more of
positions 36, 72, 77, 126 and 175; (b) a cysteine residue at one or
more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,
120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at
one or more of positions 56, 59, 69, and 122; (d) a glycine residue
at position 170; (e) a glycine residue at position 171; and
combinations of (a)-(e), and which comprises additions, deletions
or further substitutions that make the polypeptide at least 85%
identical to SEQ ID NO:4, provided that the at least one amino acid
substitution of claim 1(a)-(e) is not further modified.
[0011] The present invention also provides vectors and host cells
comprising the nucleic acid molecules of the present invention.
[0012] In a further embodiment, the present invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ ID
NO: 4 having at least one amino acid substitution that is: (a) a
lysine residue at one or more of positions 36, 72, 77, 126 and 175;
(b) a cysteine residue at one or more of positions 37, 38, 46, 91,
69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and
179; (c) an arginine residue at one or more of positions 56, 59,
69, and 122; (d) a glycine residue at position 170; (e) a glycine
residue at position 171; and combinations of (a)-(e),
[0013] In yet another embodiment, the present invention provides an
isolated nucleic acid encoding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 4 having at least one amino acid
substitution that is: (a) a lysine residue at one or more of
positions 36, 72, 77, 126 and 175; (b) a cysteine residue at one or
more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,
120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at
one or more of positions 56, 59, 69, and 122; (d) a glycine residue
at position 170; (e) a glycine residue at position 171; and
combinations of (a)-(e), and which comprises additions, deletions
or further substitutions that make the polypeptide at least 85%
identical to SEQ ID NO:4, provided that the at least one amino acid
substitution of claim 1(a)-(e) is not further modified.
[0014] In still another embodiment, the present invention provides
an isolated polypeptide comprising the amino acid sequence of SEQ
ID NO: 4 having at least one amino acid substitution that is: (a) a
lysine residue at one or more of positions 36, 72, 77, 126 and 175;
(b) a cysteine residue at one or more of positions 37, 38, 46, 91,
69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and
179; (c) an arginine residue at one or more of positions 56, 59,
69, and 122; (d) a glycine residue at position 170; (e) a glycine
residue at position 171; and combinations of (a)-(e), and which
comprises additions, deletions or further substitutions that make
the polypeptide at least 85% identical to SEQ ID NO:4, provided
that the at least one amino acid substitution of claim 1(a)-(e) is
not further modified.
[0015] Additionally, the present invention provides a composition
comprising a first polypeptide comprising the amino acid sequence
of SEQ ID NO: 4 optionally having at least one amino acid
substitution that is: (a) a lysine residue at one or more of
positions 36, 72, 77, 126 and 175; (b) a cysteine residue at one or
more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,
120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at
one or more of positions 56, 59, 69, and 122; (d) a glycine residue
at position 170; (e) a glycine residue at position 171; and
combinations of (a)-(e), joined by a linker to a second polypeptide
comprising a polypeptide comprising the amino acid sequence of SEQ
ID NO: 4 optionally having at least one amino acid substitution
that is: (a) a lysine residue at one or more of positions 36, 72,
77, 126 and 175; (b) a cysteine residue at one or more of positions
37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126,
175, 170, and 179; (c) an arginine residue at one or more of
positions 56, 59, 69, and 122; (d) a glycine residue at position
170; (e) a glycine residue at position 171; and combinations of
(a)-(e).
[0016] The present invention also provides chemically modified
forms of the polypeptides of the present invention. The chemically
modified forms of the polypeptides comprise a polymer attached to
the N-terminus and/or a naturally or non-naturally occurring
polymer attachment site. The present invention further provides
pharmaceutical compositions and methods of treating metabolic
disorders such as obesity and diabetes comprising administering the
pharmaceutical compositions of the present invention to a patient
in need thereof.
[0017] Specific embodiments of the present invention will become
evident from the following more detailed description of certain
embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a cartoon depicting a FGF21 molecule having two
polymers (e.g., PEG molecules) attached to the sequence.
[0019] FIG. 2 comprises four SDS-PAGE gels, showing the degree of
PEGylation of nine FGF21 mutants having a single engineered polymer
attachment site that have been chemically modified with PEG, namely
E37C, R77C and H125C (upper left), D38C, D46C and D79C (upper
right), H87C, E91C, G113C (lower left) and G120C, R126C, N121C
(lower right).
[0020] FIG. 3 comprises an SDS-PAGE gel, showing the degree of
PEGylation of three FGF21 mutants having a single engineered
polymer attachment site that have been chemically modified with a
20 kDa methoxy PEG maleimide molecule, namely K69C, R175C and
Y179C.
[0021] FIG. 4 comprises two plots depicting the results of an
ELK-luciferase assay performed on FGF21 mutant polypeptides having
a single engineered polymer attachment sites that have been
chemically modified by the attachment of a 20 kDa methoxy PEG
maleimide molecule, namely E37C, R77C, E91C, wild-type FGF21 and
N-terminally PEGylated FGF21 (upper plot) and G113C, N121C, D46C,
wild-type FGF21 and N-terminally PEGylated FGF21 (lower plot).
[0022] FIG. 5 comprises two plots depicting the results of an
ELK-luciferase assay performed on FGF21 mutant polypeptides having
a single engineered polymer attachment site, that have been
chemically modified by the attachment of a 20 kDa methoxy-PEG
maleimide, namely H125C, G120C, R126C, wild-type FGF21 and
N-terminally PEGylated FGF21 (upper plot) and D79C, D38C, wild-type
FGF21 and N-terminally PEGylated FGF21 (lower plot).
[0023] FIG. 6 comprises two plots depicting results of an
ELK-luciferase assay performed on wild-type FGF21 and FGF21 mutant
polypeptides having a single engineered polymer attachment site
that has been chemically modified by the attachment of a 20 kDa
methoxy PEG maleimide molecule, namely K69C, D79C, wild-type FGF21
and N-terminally PEGylated FGF21 (upper plot), and R175C, Y179C,
wild-type FGF21 and N-terminally PEGylated FGF21 (lower plot).
[0024] FIG. 7 is a cartoon depicting a Tethered Molecule of the
present invention.
[0025] FIG. 8 is a plot depicting the percent change in blood
glucose levels in mice from time 0 after a single injection of
vehicle (PBS), wild-type FGF21 or N-terminally PEGylated wild-type
FGF21.
[0026] FIG. 9 is a plot depicting the percent change in blood
glucose levels in mice from time 0 after a single injection of
vehicle (PBS), or wild-type FGF21 that was N-terminally PEGylated
with 20, 30 or 40 kDa methoxy PEG maleimide molecules.
[0027] FIG. 10 comprises two plots depicting the percent change in
blood glucose levels in mice over a nine day period from time 0
after a single injection of PBS or N-terminally PEGylated FGF21
mutant polypeptides comprising the mutations R77C or R126K, which
were further PEGylated at these introduced polymer attachment sites
with 20 kDa methoxy PEG maleimide molecules and a fusion comprising
an Fc molecule and a G170E FGF21 mutant polypeptide(upper plot); or
an N-terminally PEGylated FGF21 mutant polypeptide comprising the
mutations R77C, which was further PEGylated at this introduced
polymer attachment site with 20 kDa methoxy PEG maleimide molecule,
and P171G (lower plot).
[0028] FIG. 11 is a plot showing the percent change in blood
glucose levels in mice from time 0 after a single injection of
vehicle (10 mM potassium phosphate, 5% sorbitol, pH 8) or FGF21
mutant polypeptides which were dually PEGylated with 20 kDa methoxy
PEG maleimide molecules at introduced polymer attachment sites,
namely E91C/H125C, E91C/R175C, E37C/G120C, E37C/H125C, and
E37C/R175C; a fusion comprising an Fc molecule and a P171G FGF21
mutant polypeptide was also studied.
[0029] FIG. 12 is a plot showing the percent change in blood
glucose levels in mice from time 0 after a single injection of
vehicle (10 mM potassium phosphate, 5% sorbitol, pH 8) or FGF21
mutant polypeptides which were dually PEGylated with 20 kDa methoxy
PEG maleimide molecules at introduced polymer attachment sites,
namely E91C/H121C, G120C/H125C, or E37C/R77C; a fusion comprising
an Fc molecule and a G170E FGF21 mutant polypeptide was also
studied.
[0030] FIG. 13 is a plot showing the percent change in blood
glucose levels in mice from time 0 after a single injection of
vehicle (10 mM potassium phosphate, 5% sorbitol, pH 8) or FGF21
mutant polypeptides which were dually PEGylated with 20 kDa methoxy
PEG maleimide molecules at introduced polymer attachment sites,
namely E37C/R77C, E91C/R175C, E37C/H125C, E37C/R77C/P171G,
E91C/R77C/P171G and E37C/R125C/P171G.
[0031] FIG. 14 is a plot showing the percent change in blood
glucose levels in mice from time 0 after a single injection of
vehicle (10 mM potassium phosphate, 5% sorbitol, pH 8), FGF21
mutant polypeptides which were dually PEGylated with 20 kDa methoxy
PEG maleimide molecules at introduced polymer attachment sites,
namely E37C/R77C/P171G and E91C/R125C/P171G, or Tethered Molecules
comprising two identical FGF21 mutant polypeptides having the same
introduced mutations, namely R77C/P171G (2.times.) and R78C/P172G
(2.times.), which were joined together via a 20 kDa methoxy PEG
maleimide molecules.
[0032] FIG. 15 is a plot showing the percent change in blood
glucose levels in mice as a function of dose from time 0 after a
single injection of vehicle (10 mM Tris HCl, 150 mM NaCl, pH 8.5),
or an FGF21 mutant polypeptide which was dually PEGylated with 20
kDa methoxy PEG maleimide molecules at introduced polymer
attachment sites, namely E37C/R77C/P171G, and administered at doses
of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg or 1 mg/kg.
[0033] FIG. 16 is a plot showing body weight change in mice from
time 0 after a single injection of vehicle (10 mM potassium
phosphate, 5% sorbitol, pH 8) or FGF21 mutant polypeptides which
were dually PEGylated with 20 kDa methoxy PEG maleimide molecules
at introduced polymer attachment sites, namely E37C/R77C,
E91C/R175C, E37/H125C, E37C/R77C/P171G, E91C/R77C/P171G and
E37C/R125C/P171G.
[0034] FIG. 17 is a plot showing body weight change in mice from
time 0 after a single injection of vehicle (10 mM potassium
phosphate, 5% sorbitol, pH 8), FGF21 mutant polypeptides which were
dually PEGylated with 20 kDa methoxy PEG maleimide molecules at
introduced polymer attachment sites, namely E37C/R77C/P171G and
E91C/R125C/P171G, or Tethered Molecules comprising two FGF21 mutant
polypeptides having the same introduced mutations, namely
R37C/P171G (2.times.) and R77C/P171G (2.times.), which were joined
together via a 20 kDa methoxy PEG maleimide molecule.
[0035] FIG. 18 is a plot showing body weight change in mice as a
function of dose from time 0 after a single injection of vehicle
(10 mM Tris HCl, 150 mM NaCl, pH 8.5), or an FGF21 mutant
polypeptide which was dually PEGylated with 20 kDa methoxy PEG
maleimide molecules at introduced polymer attachment sites, namely
E37C/R77C/P171G, and administered at five different doses.
[0036] FIG. 19A-19F is a series of six plots showing the change in
body weight of mice during an eight week kidney vacuole study using
once weekly dosing of vehicle (squares), 5 mg/kg (triangles) and 25
mg/kg (open circles) PEGylated FGF21 molecules. Mice dosed with
dual cysteine targeted PEG-FGF21 showed a sustained weight loss,
while those dosed with Tethered Molecules showed primarily
transient weight loss.
[0037] FIG. 20 comprises two bar graphs depicting the results of an
eight week kidney vacuole study in mice injected with vehicle or an
FGF21 mutant polypeptide which was dually PEGylated with 20 kDa
methoxy PEG maleimide molecules at introduced polymer attachment
sites, namely E37C/R77C/P171G; E37/H125C/P171G; E91C/H125C/P171G;
E37C/P171G; R77C/P171G; and R77C/P171G; two different doses were
tested.
DETAILED DESCRIPTION OF THE INVENTION
[0038] A human FGF21 protein having enhanced properties such as an
increased half-life can be prepared using the methods disclosed
herein and standard molecular biology methods. It is known that by
binding one or more water soluble polymers, such as PEG molecules,
to a protein the half life of the protein can be extended. Thus, in
various embodiments, the half life of native FGF21 can be extended
by introducing amino acid substitutions into the protein to form
points at which a polymer can be attached to the FGF21 protein.
Such modified proteins are referred to herein as FGF21 mutants and
form embodiments of the present invention. Polymers can also be
introduced at the N-terminus of the FGF21 molecule in conjunction
with the introduction of a non-naturally occurring polymer
attachment site.
[0039] Recombinant nucleic acid methods used herein, including in
the Examples, are generally those set forth in Sambrook et al.,
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory Press, 1989) or Current Protocols in Molecular Biology
(Ausubel et al., eds., Green Publishers Inc. and Wiley and Sons
1994), both of which are incorporated herein by reference for any
purpose.
1. GENERAL DEFINITIONS
[0040] As used herein, the term "a" means one or more unless
specifically indicated otherwise.
[0041] The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule of the invention that (1) has been separated
from at least about 50 percent of proteins, lipids, carbohydrates,
or other materials with which it is naturally found when total
nucleic acid is isolated from the source cells, (2) is not linked
to all or a portion of a polynucleotide to which the "isolated
nucleic acid molecule" is linked in nature, (3) is operably linked
to a polynucleotide which it is not linked to in nature, or (4)
does not occur in nature as part of a larger polynucleotide
sequence. Preferably, the isolated nucleic acid molecule of the
present invention is substantially free from any other
contaminating nucleic acid molecules or other contaminants that are
found in its natural environment that would interfere with its use
in polypeptide production or its therapeutic, diagnostic,
prophylactic or research use.
[0042] The term "isolated polypeptide" refers to a polypeptide of
the present invention that (1) has been separated from at least
about 50 percent of polynucleotides, lipids, carbohydrates, or
other materials with which it is naturally found when isolated from
the source cell, (2) is not linked (by covalent or noncovalent
interaction) to all or a portion of a polypeptide to which the
"isolated polypeptide" is linked in nature, (3) is operably linked
(by covalent or noncovalent interaction) to a polypeptide with
which it is not linked in nature, or (4) does not occur in nature.
Preferably, the isolated polypeptide is substantially free from any
other contaminating polypeptides or other contaminants that are
found in its natural environment that would interfere with its
therapeutic, diagnostic, prophylactic or research use.
[0043] The term "vector" is used to refer to any molecule (e.g.,
nucleic acid, plasmid, or virus) used to transfer coding
information to a host cell.
[0044] The term "expression vector" refers to a vector that is
suitable for transformation of a host cell and contains nucleic
acid sequences that direct and/or control the expression of
inserted heterologous nucleic acid sequences. Expression includes,
but is not limited to, processes such as transcription,
translation, and RNA splicing, if introns are present.
[0045] The term "host cell" is used to refer to a cell which has
been transformed, or is capable of being transformed with a nucleic
acid sequence and then of expressing a selected gene of interest.
The term includes the progeny of the parent cell, whether or not
the progeny is identical in morphology or in genetic make-up to the
original parent, so long as the selected gene is present.
[0046] The term "naturally occurring" when used in connection with
biological materials such as nucleic acid molecules, polypeptides,
host cells, and the like, refers to materials which are found in
nature and are not manipulated by man. Similarly, "non-naturally
occurring" as used herein refers to a material that is not found in
nature or that has been structurally modified or synthesized by
man. When used in connection with nucleotides, the term "naturally
occurring" refers to the bases adenine (A), cytosine (C), guanine
(G), thymine (T), and uracil (U). When used in connection with
amino acids, the term "naturally occurring" refers to the 20 amino
acids alanine (A), cysteine (C), aspartic acid (D), glutamic acid
(E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I),
lysine (K), leucine (L), methionine (M), asparagine (N), proline
(P), glutamine (Q), arginine (R), serine (S), threonine (T), valine
(V), tryptophan (W), and tyrosine (Y).
[0047] The term "FGF21 polypeptide" refers to any naturally
occurring wild-type polypeptide expressed in humans. For purposes
of this application, the term "FGF21 polypeptide" can be used
interchangeably to refer to the full-length FGF21 polypeptide,
which consists of 209 amino acid residues (SEQ ID NO: 2) and which
is encoded by the nucleotide sequence of SEQ ID NO: 1; and the
mature form of the polypeptide, which consists of 181 amino acid
residues (SEQ ID NO: 4), which is encoded by the nucleotide
sequence of SEQ ID NO: 3, and in which the 28 amino acid residues
at the amino-terminal end of the full-length FGF21 polypeptide
(i.e., which constitute the signal peptide) have been removed. An
FGF21 polypeptide can be expressed with or without an N-terminal
Methionine residue; as noted herein, an N-terminal Methionine
residue can be added by design or as a function of a bacterial
expression system.
[0048] The term "biologically active," as applied to an FGF21
polypeptide, including FGF21 mutant polypeptides described herein,
refers to a naturally occurring activity of a wild-type FGF21
polypeptide, such as the ability to lower blood glucose, insulin,
triglyceride, or cholesterol; reduce body weight; and improve
glucose tolerance, energy expenditure, or insulin sensitivity. As
applied to a FGF21 mutant polypeptide, the term is not dependent on
the type or number of modifications that have been introduced into
the FGF21 mutant polypeptide. For example, some FGF21 mutant
polypeptides possess a somewhat decreased level of FGF21 activity
relative to the wild-type FGF21 polypeptide but are nonetheless be
considered to be biologically active FGF21 mutant polypeptides.
Differences in the activity of a particular FGF21 mutant
polypeptide may be observed between in vivo and in vitro assays;
any such differences are related to the particular assays used.
Such an observation, however, does not affect the meaning of the
term "biologically active," and FGF21 mutant polypeptides showing a
naturally occurring activity of a wild-type FGF21 polypeptide, such
as the ability to lower blood glucose, insulin, triglyceride, or
cholesterol; reduce body weight; and improve glucose tolerance,
energy expenditure, or insulin sensitivity, in any in vivo or in
vitro assay are "biologically active."
[0049] The terms "effective amount" and "therapeutically effective
amount" are used interchangeably and refer to the amount of an
FGF21 mutant polypeptide used to support an observable level of one
or more biological activities of the wild-type FGF21 polypeptide,
such as the ability to lower blood glucose, insulin, triglyceride,
or cholesterol levels; reduce body weight; or improve glucose
tolerance, energy expenditure, or insulin sensitivity.
[0050] The term "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier" as used herein refers to one
or more formulation materials suitable for accomplishing or
enhancing the delivery of an FGF21 mutant polypeptide. Examples of
such materials can be found in Remington, supra, incorporated
herein by reference.
[0051] The term "Tethered Molecule" refers to a construct
comprising two or more FGF21 molecules tethered together by a
linker molecule. A Tethered Molecule comprises at least two FGF21
polypeptides, at least one of which is an FGF21 mutant polypeptide
as described herein, but can comprise three, four or more FGF21 or
FGF21 mutant polypeptides joined together by linkers. Thus, the
term Tethered Molecule is not restricted to a molecule comprising
combinations of only one or two FGF21 or FGF21 mutant
polypeptides.
[0052] The term "polymer attachment site" refers to a region of the
primary amino acid sequence of a polypeptide (e.g., an FGF21
polypeptide) that is chemically adaptable to covalent association
with a polymer (e.g., PEG molecules of all molecular weights,
polymeric mannose, glycans, etc). A polymer attachment site can
mean a single amino acid (e.g., cysteine, lysine, arginine or a
suitable non-naturally occurring amino acid) or the term can refer
to two or more amino acids that are adjacent to each other either
in sequence or in space.
[0053] The term "chemically modified," when used in relation to a
FGF21 wild-type or FGF21 mutant polypeptide as disclosed herein,
refers to a FGF21 polypeptide that has been modified from its
naturally occurring state by the covalent attachment of one or more
heterologous molecules. Examples of heterologous molecules include
polyethylene glycol (PEG), monomethoxy-polyethylene glycol,
dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,
propylene glycol homopolymers, polypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols, hydroxyl ethyl starch (HES),
and polyvinyl alcohol. Examples of chemically modified FGF21
polypeptides include PEGylated wild-type FGF21 and FGF21 mutant
polypeptides.
2. FGF21 MUTANT POLYPEPTIDES
[0054] In various aspects, the present invention discloses a series
of methods for the site-directed PEGylation of FGF21 and FGF21
mutant polypeptides, which can enhance the pharmacokinetic
properties of the FGF21 molecule while minimizing the impact on the
in vitro activity. The enhanced pharmacokinetic profile of these
PEGylated FGF21 molecules has an impact on the in vivo efficacy of
the molecule by increasing exposure to the therapeutic agent. In
addition, the strategies described herein are compatible with
creating multiple PEGylation sites, which may both further enhance
the pharmacokinetic properties of the molecule, and lower their
vacuole-forming potential. Two principle strategies were employed
to accomplish this, as described herein.
[0055] In one aspect, the present invention relates to FGF21
sequences into which one or more modifications have been
introduced. Thus, the terms "FGF21 mutant polypeptide" and "FGF21
mutant," which can be used interchangeably, refer to an FGF21
polypeptide in which a wild-type FGF21 amino acid sequence (e.g.,
SEQ ID NOs 2 or 4) has been modified. Such modifications include,
but are not limited to, one or more amino acid substitutions,
including substitutions with non-naturally occurring amino acid
analogs, insertions and truncations. Thus, FGF21 polypeptide
mutants include, but are not limited to, site-directed FGF21
mutants, such as those introducing a non-naturally occurring
polymer attachment site, or which impart a degree of resistance to
proteolysis, as described herein. For the purpose of identifying
the specific amino acid substitutions of the FGF21 mutants of the
present invention, the numbering of the amino acid residues
truncated or mutated corresponds to that of the mature 181-residue
FGF21 polypeptide (i.e., the N terminus of the sequence begins
HPIPD, and these residues are designated as residues 1, 2, 3, 4 and
5, respectively). An N-terminal methionine residue can but does not
need to be present; this N-terminal methionine residue is not
included in the numbering scheme of the protein.
[0056] As stated, FGF21 mutants, including truncated forms of FGF21
comprising one or more substitutions or insertions, which comprise
non-naturally occurring amino acids form an embodiment of the
present invention. Such insertions or substitutions can impart
various properties, including acting as sites for polymer
attachment. In such cases, non-naturally occurring amino acids can
be incorporated into an FGF21 sequence in addition to the various
mutations described herein. Accordingly, an FGF21 mutant can
comprise one or more of the mutations described herein and can
further comprise one or more non-naturally occurring amino acids. A
non-limiting lists of examples of non-naturally occurring amino
acids that can be inserted into an FGF21 sequence or substituted
for a wild-type residue in an FGF21 sequence include .beta.-amino
acids, homoamino acids, cyclic amino acids and amino acids with
derivatized side chains. Examples include (in the L-form or D-form;
abbreviated as in parentheses): para-acetyl-phenylalanine,
para-azido-phenylalanine, para-bromo-phenylalanine,
para-iodo-phenylalanine and para-ethynyl-phenylalanine, citrulline
(Cit), homocitrulline (hCit), N.alpha.-methylcitrulline (NMeCit),
N.alpha.-methylhomocitrulline (N.alpha.-MeHoCit), ornithine (Orn),
N.alpha.-Methylornithine (N.alpha.-MeOrn or NMeOrn), sarcosine
(Sar), homolysine (hLys or hK), homoarginine (hArg or hR),
homoglutamine (hQ), N.alpha.-methylarginine (NMeR),
N.alpha.-methylleucine (N.alpha.-MeL or NMeL), N-methylhomolysine
(NMeHoK), N.alpha.-methylglutamine (NMeQ), norleucine (Nle),
norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),
Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine
(1-Nal), 3-(2-naphthy)alanine (2-Nal),
1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (IgI),
para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or
4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine
(abbreviated "K(N.epsilon.-glycyl)" or "K(glycyl)" or "K(gly)"),
nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or
Amino-Phe), benzylphenylalanine (benzylphe),
.gamma.-carboxyglutamic acid (.gamma.-carboxyglu), hydroxyproline
(hydroxypro), p-carboxyl-phenylalanine (Cpa), .alpha.-aminoadipic
acid (Aad), N.alpha.-methyl valine (NMeVal), N-.alpha.-methyl
leucine (NMeLeu), N.alpha.-methylnorleucine (NMeNle),
cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine
(acetylarg), .alpha., .beta.-diaminopropionoic acid (Dpr), a,
.gamma.-diaminobutyric acid (Dab), diaminopropionic acid (Dap),
cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), .beta.,
.beta.-diphenyl-alanine (BiPhA), aminobutyric acid (Abu),
4-phenyl-phenylalanine (or biphenylalanine; 4Bip),
.alpha.-amino-isobutyric acid (Aib), beta-alanine,
beta-aminopropionic acid, piperidinic acid, aminocaprioic acid,
aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic
acid, N-ethylglycine, N-ethylaspargine, hydroxylysine, all
o-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine,
N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp),
.gamma.-carboxy glutamate, .epsilon.-N,N,N-trimethyllysine,
.epsilon.-N-acetyllysine, 0-phosphoserine, N-acetylserine,
N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,
.omega.-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other
similar amino acids, and derivatized forms of any of those
specifically listed.
[0057] In other embodiments of the present invention, an FGF21
mutant polypeptide comprises an amino acid sequence that is at
least about 85 percent identical to a wild-type FGF21 amino acid
sequence (e.g., SEQ ID NOs: 2 or 4), but wherein the specific
residues introducing non-naturally occurring polymer attachment
sites in the FGF21 mutant polypeptide have not been further
modified. In other words, with the exception of residues in the
FGF21 mutant sequence that have been modified in order to introduce
a non-naturally occurring polymer attachment site or a mutation to
increase resistance to proteolysis, about 15 percent of all other
amino acid residues in the FGF21 mutant sequence may be modified.
For example, in the FGF21 mutant polypeptide G170C, up to 15
percent of all amino acid residues other than the glycine residue
at position 170 could be modified. In still other embodiments, an
FGF21 polypeptide mutant comprises an amino acid sequence that is
at least about 90 percent, or about 95, 96, 97, 98, or 99 percent
identical to a wild-type FGF21 amino acid sequence (e.g., SEQ ID
NO: 2, 4, 6 or 8), but wherein the specific residues that have been
modified to introduce a non-naturally occurring polymer attachment
site or enhance proteolysis resistance have not been further
modified. Such FGF21 mutant polypeptides possess at least one
activity of the wild-type FGF21 polypeptide.
[0058] FGF21 mutant polypeptides can be generated by introducing
amino acid substitutions, either conservative or non-conservative
in nature and using naturally or non-naturally occurring amino
acids, at particular positions of the FGF21 polypeptide. Such
substitutions can be made in addition to substitutions designed or
observed to impart a desirable property to the FGF21 polypeptide.
By way of example, a FGF21 mutant polypeptide can comprise a
substitution designed to achieve a desirable property, such as
introducing a non-naturally occurring polymer attachment site or
enhancing resistance to proteolysis, and can further comprise one
or more conservative or non-conservative substitutions which may,
but need not, maintain the biological activity of the wild-type
FGF21 polypeptide.
[0059] FGF21 mutations can be conservative or non-conservative. A
"conservative amino acid substitution" can involve a substitution
of a native amino acid residue (i.e., a residue found in a given
position of the wild-type FGF21 polypeptide sequence) with a
nonnative residue (i.e., a residue that is not found in a given
position of the wild-type FGF21 polypeptide sequence) such that
there is little or no effect on the polarity or charge of the amino
acid residue at that position. Conservative amino acid
substitutions also encompass non-naturally occurring amino acid
residues that are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems. These
include peptidomimetics, and other reversed or inverted forms of
amino acid moieties.
[0060] Naturally occurring residues can be divided into classes
based on common side chain properties:
[0061] (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0062] (2) neutral hydrophilic: Cys, Ser, Thr;
[0063] (3) acidic: Asp, Glu;
[0064] (4) basic: Asn, Gln, His, Lys, Arg;
[0065] (5) residues that influence chain orientation: Gly, Pro;
and
[0066] (6) aromatic: Trp, Tyr, Phe.
[0067] Conservative substitutions can involve the exchange of a
member of one of these classes for another member of the same
class. Non-conservative substitutions can involve the exchange of a
member of one of these classes for a member from another class.
[0068] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. An exemplary (but not
limiting) list of amino acid substitutions is set forth in Table
1.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Residue
Exemplary Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln
Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln,
Lys, Arg Ile Leu, Val, Met, Ala, Phe Leu Ile, Val, Met, Ala, Phe
Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro
Ala Ser Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser
Val Ile, Met, Leu, Phe, Ala
[0069] 2.A. FGF21 Mutant Polypeptides Comprising a
Proteolysis-Resistant Mutation
[0070] It has been determined that the mature form of FGF21 (i.e.,
the 181 residue form) undergoes in vivo degradation, which was
ultimately determined to arise from proteolytic attack. The in vivo
degradation of mature FGF21 has been found to lead to a shorter
effective half-life, which can adversely affect the therapeutic
potential of the molecule. Accordingly, a directed study was
performed to identify FGF21 mutants that exhibit a resistance to
proteolysis. As a result of this investigation, the sites in the
mature FGF21 polypeptide that were determined to be particularly
susceptible to proteolysis include the peptide bond between the
amino acid residues at positions 4-5, 20-21, 151-152, and
171-172.
[0071] A non-limiting list of exemplary substitutions that
eliminate the proteolytic effect observed in mature FGF21 while not
affecting the biological activity of the protein to an unacceptable
degree that can be employed in the present invention is presented
in Table 2. Table 2 is demonstrative only and other proteolysis
resistant substitutions can be identified and employed in the
present invention. These proteolysis-resistant substitutions can be
made in addition to substitutions that introduce one or more
non-naturally occurring polymer attachment sites, thus generating a
FGF21 mutant polypeptide exhibiting the desirable characteristics
imparted by each type of mutation.
TABLE-US-00002 TABLE 2 Representative Substitutions that Provide
Proteolysis Resistance Amino Acid Native Position Residue Mutations
19 Arg Gln, Ile, Lys 20 Tyr His, Leu, Phe 21 Leu Ile, Phe, Tyr, Val
22 Tyr Ile, Phe, Val 150 Pro Ala, Arg 151 Gly Ala, Val 152 Ile His,
Leu, Phe, Val 170 Gly Ala, Asn, Asp, Cys, Gln, Glu, Pro, Ser 171
Pro Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Lys, Ser, Thr,
Trp, Tyr 172 Ser Leu, Thr 173 Gln Arg, Glu
[0072] Preferably, but not necessarily, FGF21 mutant polypeptides
comprising a proteolysis-resistant mutation have biological
activity essentially the same as, or greater than, the activity of
wild-type FGF21. Therefore, another embodiment of the present
invention is directed to FGF21 mutant polypeptides that comprise
one or more non-naturally occurring polymer attachment sites and
are resistant to proteolysis, yet still retain biological activity
that is the same as, or greater than, wild-type FGF21. Although
less desirable in some cases, FGF21 mutants that comprise one or
more non-naturally occurring polymer attachment sites and are
resistant to proteolysis but exhibit somewhat decreased biological
activity form another embodiment of the present invention. In some
cases it can be desirable to maintain a degree of proteolysis, and
consequently, FGF21 mutants comprising one or more non-naturally
occurring polymer attachment sites and which are resistant to
proteolysis and yet still allow some degree of proteolysis to occur
also form another embodiment of the present invention.
[0073] As with all FGF21 mutant polypeptides of the present
invention, proteolysis-resistant FGF21 mutant polypeptides
comprising one or more non-naturally occurring polymer attachment
sites can be prepared as described herein. Those of ordinary skill
in the art, for example, those familiar with standard molecular
biology techniques, can employ that knowledge, coupled with the
instant disclosure, to make and use the proteolysis-resistant FGF21
mutants comprising one or more non-naturally occurring polymer
attachment sites of the present invention. Standard techniques can
be used for recombinant DNA, oligonucleotide synthesis, tissue
culture, and transformation (e.g., electroporation, lipofection).
See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,
which is incorporated herein by reference for any purpose.
Enzymatic reactions and purification techniques can be performed
according to manufacturer's specifications, as commonly
accomplished in the art, or as described herein. Unless specific
definitions are provided, the nomenclatures utilized in connection
with, and the laboratory procedures and techniques of, analytical
chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques can be used for
chemical syntheses; chemical analyses; pharmaceutical preparation,
formulation, and delivery; and treatment of patients.
[0074] Proteolysis-resistant FGF21 mutants comprising one or more
non-naturally occurring polymer attachment sites which are
resistant to proteolysis can be chemically modified using
methodology known in the art and described herein. Chemically
modifying (e.g., PEGylating) a proteolysis-resistant FGF21 mutant
polypeptide comprising one or more non-naturally occurring polymer
attachment sites can generate molecules that exhibit both
proteolysis resistance and desirable pharmacokinetic and
pharmacodynamic properties.
[0075] 2.B. FGF21 Mutant Polypeptides Comprising a Non-Naturally
Occurring Polymer Attachment Site
[0076] In various aspects of the present invention, FGF21 mutant
polypeptides are disclosed. In another aspect, the FGF21 mutant
polypeptides of the present invention include FGF21 polypeptides
into which a non-naturally occurring polymer (e.g., PEGylation)
attachment site(s) has been introduced. In yet another aspect of
the present invention, truncated forms of FGF21 mutant polypeptides
into which a non-naturally occurring polymer (e.g., PEG) attachment
site(s) has been introduced are disclosed. FGF21 mutant
polypeptides comprising a non-naturally occurring polymer
attachment site and one or more conservative or non-conservative
substitutions, which may but need not maintain the biological
activity of the wild-type FGF21, form another aspect of the
invention. The various FGF21 polypeptide mutants of the present
invention can be prepared as described herein and in references
provided herein.
[0077] In one embodiment, FGF21 polypeptide mutants of the present
invention are modified by introducing a non-naturally occurring
polymer attachment site. Indeed, in one aspect this is a goal of
the FGF21 mutants of the present invention, namely the introduction
of one or more non-naturally occurring polymer attachment sites
such that half life-extending polymers can be attached to the FGF21
polypeptide mutant at desired locations. The polymer selected is
typically, but not necessarily, water-soluble so that the protein
to which it is attached does not precipitate in an aqueous
environment, such as a physiological environment. Included within
the scope of suitable polymers is a mixture of polymers.
Preferably, for therapeutic use of the end-product preparation, the
polymer will be pharmaceutically acceptable, such as PEG of a
suitable molecular weight. Non-water soluble polymers, such as PEG
fatty acid blockcopolymers can also be conjugated to FGF21
polypeptide mutants of the present invention and forms an aspect of
the invention.
[0078] The activity of the FGF21 mutant polypeptides of the present
invention can be assayed in a variety of ways, for example, using
an in vitro ELK-luciferase assay as described herein in Example
10.
[0079] The activity of the FGF21 mutant polypeptides of the present
invention can also be assessed in an in vivo assay, such as with
ob/ob mice as shown in Example 12. Generally, to assess the in vivo
activity of one or more of these polypeptides, the polypeptide can
be administered to a test animal intraperitoneally. After one or
more desired time periods, a blood sample can be drawn, and blood
glucose levels can be measured.
[0080] As with all FGF21 mutant polypeptides of the present
invention, these polypeptides can optionally comprise an
amino-terminal methionine residue, which can be introduced by
directed mutation or as a result of a bacterial expression
process.
[0081] The FGF21 mutant polypeptides of the present invention can
be prepared as described in Example 7. Those of ordinary skill in
the art, familiar with standard molecular biology techniques, can
employ that knowledge, coupled with the instant disclosure, to make
and use the FGF21 mutant polypeptides of the present invention.
Standard techniques can be used for recombinant DNA,
oligonucleotide synthesis, tissue culture, and transformation
(e.g., electroporation, lipofection). See, e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, which is incorporated
herein by reference for any purpose. Enzymatic reactions and
purification techniques can be performed according to
manufacturer's specifications, as commonly accomplished in the art,
or as described herein. Unless specific definitions are provided,
the nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques can be used for chemical syntheses; chemical
analyses; pharmaceutical preparation, formulation, and delivery;
and treatment of patients.
[0082] Following the preparation of a FGF21 mutant polypeptide, the
polypeptide can be chemically modified by the attachment of a
polymer, as described herein in Example 9.
3. Truncated FGF21 Mutant Polypeptides Comprising a Non-Naturally
Occurring Polymer Attachment Site
[0083] One embodiment of the present invention is directed to
truncated forms of a mutant FGF21 polypeptide comprising one or
more non-naturally occurring polymer attachment sites. Such
truncated mutant polypeptides can, but need not, be chemically
modified.
[0084] As used herein, the term "truncated FGF21 mutant
polypeptide" refers to an FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide in which one or more amino acid
residues have been removed from the amino-terminal (or N-terminal)
end of the FGF21 polypeptide, one or more amino acid residues have
been removed from the carboxyl-terminal (or C-terminal) end of the
FGF21 mutant polypeptide or chemically modified FGF21 polypeptide,
or one or more amino acid residues have been removed from both the
N-terminal and C-terminal ends of the FGF21 mutant polypeptide or
chemically modified FGF21 polypeptide.
[0085] The activity of N-terminally truncated mutant FGF21,
C-terminally truncated mutant FGF21 and mutant FGF21 molecules
truncated at both the N- and C-terminal ends of the molecule, as
well as chemically modified forms of these mutants, can be assayed
in a variety of ways, for example, using an in vitro ELK-luciferase
assay as described herein in Example 10.
[0086] The activity of the truncated mutant FGF21 polypeptides and
chemically modified truncated mutant FGF21 polypeptides of the
present invention can also be assessed in an in vivo assay, such as
ob/ob mice as shown in Example 12. Generally, to assess the in vivo
activity of one or more of these polypeptides, the polypeptide can
be administered to a test animal intraperitoneally. After one or
more desired time periods, a blood sample can be drawn, and blood
glucose levels can be measured.
[0087] As with all FGF21 mutants of the present invention,
truncated mutant FGF21 and chemically modified truncated mutant
FGF21 polypeptides can optionally comprise an amino-terminal
methionine residue, which can be introduced by directed mutation or
as a result of a bacterial expression process.
[0088] The truncated FGF21 mutant polypeptides of the present
invention can be prepared as described in Examples 7. Those of
ordinary skill in the art, familiar with standard molecular biology
techniques, can employ that knowledge, coupled with the instant
disclosure, to make and use the truncated mutant FGF21 polypeptides
of the present invention. Standard techniques can be used for
recombinant DNA, oligonucleotide synthesis, tissue culture, and
transformation (e.g., electroporation, lipofection). See, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, which is
incorporated herein by reference for any purpose. Enzymatic
reactions and purification techniques can be performed according to
manufacturer's specifications, as commonly accomplished in the art,
or as described herein. Unless specific definitions are provided,
the nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques can be used for chemical syntheses; chemical
analyses; pharmaceutical preparation, formulation, and delivery;
and treatment of patients.
[0089] Following the preparation of a truncated mutant FGF21
polypeptide, the polypeptide can be chemically modified by the
attachment of a polymer, as described in Example 9.
4. Capped and C-Terminal FGF21 Mutant Polypeptides
[0090] In another embodiment, the present invention is directed to
mutant FGF21 polypeptides comprising one or more non-naturally
occurring polymer attachment sites which have been capped by the
addition of another one or more residues to the C-terminus of the
polypeptide, extending the amino acid sequence beyond that of the
wild-type protein. In yet another embodiment, the present invention
is directed to FGF21 mutant polypeptides comprising one or more
non-naturally occurring polymer attachments sites that further
comprise one or more C-terminal mutations. Such capped and
C-terminally mutated FGF21 mutant polypeptides can, but need not,
be chemically modified.
[0091] As used herein, the term "capped FGF21 mutant polypeptide"
refers to an FGF21 mutant polypeptide or chemically modified FGF21
mutant polypeptide in which one or more amino acid residues have
been added to the C terminus of the FGF21 mutant polypeptide or
chemically modified FGF21 mutant polypeptide. Any naturally or
non-naturally occurring amino acid can be used to cap an FGF21
mutant polypeptide, including one or more proline residues and one
or more glycine residues. Although the wild-type FGF21 sequence is
only 181 residues long, a capped FGF21 mutant polypeptide extends
the length of the polypeptide one residue for each added capping
residue; consistent with the numbering scheme of the present
disclosure, cap residues are numbered beginning with 182. Thus, a
single proline capping residue is indicated as P182. Longer caps
are possible and are numbered accordingly (e.g., X182, Y183, Z184,
where X, Y and Z are any naturally or non-naturally occurring amino
acid). Capping residues can be added to a mutant FGF21 polypeptide
using any convenient method, such as chemically, in which an amino
acid is covalently attached to the C-terminus of the polypeptide by
a chemical reaction. Alternatively, a codon encoding a capping
residue can be added to the FGF21 mutant polypeptide coding
sequence using standard molecular biology techniques. Any of the
mutant FGF21 polypeptides described herein can be capped with one
or more residues, as desired.
[0092] C-terminal mutations form another aspect of the present
invention. As used herein, the term "C-terminal mutation" referes
to one or more changes in the region of residues 91-181 (or longer
if the polypeptide is capped) of a mutant FGF21 polypeptide. A
C-terminal mutation introduced into a FGF21 mutant polypeptide
sequence will be in addition to one or more mutations which
introduce a non-naturally occurring polymer attachment site.
Although C-terminal mutations can be introduced at any point in the
region of 91-181 of the FGF21 mutant polypeptide sequence,
exemplary positions for C-terminal mutations include positions 171,
172, 173, 174, 175, 176, 177, 178, 179, 180 and 181. C-terminal
mutations can be introduced using standard molecular biological
techniques, such as those described herein. Any of the mutant FGF21
polypeptides described herein can comprise a C-terminal
mutation.
[0093] Examples of positions and identities for capped and/or
C-terminally mutations are shown in Table 3:
TABLE-US-00003 TABLE 3 Examples of Capping Positions and/or
C-terminally Mutations E37C, R77C, P171G, P182 P171G, S181P, P182
P171G, S181P P171G, S181T P171G, S181G P171G, S181A P171G, S181L
P171G, A180P P171G, A180G P171G, A180S P171G, Y179P P171G, Y179G
P171G, Y179S P171G, Y179A P171G, L182 P171G, G182 P171G, P182
P171G, G182, G183 P171G, G182, G183, G184, G185, G186
[0094] The activity of capped and/or C-terminally mutated FGF21
mutant polypeptides, as well as chemically modified forms of these
mutants, can be assayed in a variety of ways, for example, using an
in vitro ELK-luciferase assay as described herein in Example
10.
[0095] The activity of the capped and/or C-terminally mutated FGF21
mutant polypeptides, and chemically modified capped and/or
C-terminally mutated FGF21 mutant polypeptides, of the present
invention can also be assessed in an in vivo assay, such as ob/ob
mice as shown in Example 12. Generally, to assess the in vivo
activity of one or more of these polypeptides, the polypeptide can
be administered to a test animal intraperitoneally. After one or
more desired time periods, a blood sample can be drawn, and blood
glucose levels can be measured.
[0096] As with all FGF21 mutants of the present invention, capped
and/or C-terminally mutated FGF21 mutant polypeptides, and
chemically modified capped and/or C terminally mutated FGF21 mutant
polypeptides, can optionally comprise an amino-terminal methionine
residue, which can be introduced by directed mutation or as a
result of a bacterial expression process.
[0097] The capped and/or C-terminally mutated FGF21 mutant
polypeptides of the present invention can be prepared as described
in Example 7. Those of ordinary skill in the art, familiar with
standard molecular biology techniques, can employ that knowledge,
coupled with the instant disclosure, to make and use the capped
and/or C-terminally mutated FGF21 mutant polypeptides of the
present invention. Standard techniques can be used for recombinant
DNA, oligonucleotide synthesis, tissue culture, and transformation
(e.g., electroporation, lipofection). See, e.g., Sambrook et al.,
Molecular Cloning: A Laboratory Manual, which is incorporated
herein by reference for any purpose. Enzymatic reactions and
purification techniques can be performed according to
manufacturer's specifications, as commonly accomplished in the art,
or as described herein. Unless specific definitions are provided,
the nomenclatures utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those well known and commonly used in the art.
Standard techniques can be used for chemical syntheses; chemical
analyses; pharmaceutical preparation, formulation, and delivery;
and treatment of patients.
[0098] Following the preparation of a capped and/or C terminally
mutated FGF21 mutant polypeptide, the polypeptide can be chemically
modified by the attachment of a polymer, as described in Example
9.
5. FGF21 Mutant Polypeptides Containing No Naturally Occurring
Cysteine Residues
[0099] In a further aspect of the present invention, FGF21 mutant
polypeptides can be prepared in which both cysteine residues in the
wild-type FGF21 polypeptide sequence are replaced with residues
that do not form disulfide bonds and do not serve as polymer
attachment sites, such as alanine or serine. Subsequently,
substitutions can be made in the FGF21 mutant polypeptide sequence
that introduce non-naturally occurring polymer attachment sites, in
the form of thiol-containing residues (e.g., cysteine residues or
non-naturally occurring amino acids having thiol groups) or free
amino groups (e.g., lysine or arginine residues or non-naturally
occurring amino acids having free amino groups). Polymers that rely
on thiol or free amino groups for attachment, such as PEG, can then
be targeted to cysteine, lysine or arginine residues that have been
introduced into the FGF21 mutant polypeptide sequence at known
positions. This strategy can facilitate more efficient and
controlled polymer placement.
[0100] In one approach, the two naturally occurring cysteine
residues in the wild-type FGF21 polypeptide, which are located at
positions 75 and 93, can be substituted with non-thiol containing
residues. Subsequently, a cysteine residue can be introduced at a
known location. The FGF21 mutant polypeptide can also comprise
other mutations, which can introduce still more polymer attachments
sites (e.g., cysteine residues) or can be designed to achieve some
other desired property. Examples of such FGF21 mutant polypeptides
include C75A/E91C/C93A/H125C/P171G and C75S/E91C/C93S/H125C/P171G.
In these examples, the naturally occurring cysteines at positions
75 and 93 have been mutated to alanine or serine residues, polymer
attachment sites have been introduced at positions 91 and 125 (in
this case for a thiol-reactive polymer such as PEG) and an
additional mutation has been made at position 171, namely the
substitution of proline 171 with a glycine residue.
[0101] Like all of the FGF21 mutant polypeptides disclosed herein,
the activity of FGF21 mutant polypeptides which contain neither of
the cysteines found in the wild-type FGF21 polypeptide sequence but
instead comprise an introduced polymer attachment site and
optionally one or more additional mutations, as well as chemically
modified forms of these mutants, can be assayed in a variety of
ways, for example, using an in vitro ELK-luciferase assay as
described herein in Example 10. The in vivo activity of these
polypeptides can be assessed in an in vivo assay, such as with
ob/ob mice as shown in Example 12 and as described herein
[0102] As with all FGF21 mutants of the present invention, the
activity of FGF21 mutant polypeptides which contain neither of the
cysteines found in the wild-type FGF21 polypeptide sequence but
instead comprise an introduced polymer attachment site and
optionally one or more additional mutations and chemically modified
forms of these FGF21 mutant polypeptides can optionally comprise an
amino-terminal methionine residue, which can be introduced by
directed mutation or as a result of a bacterial expression
process.
[0103] FGF21 mutant polypeptides which contain neither of the
cysteines found in the wild-type FGF21 polypeptide sequence but
instead comprise an introduced polymer attachment site and
optionally one or more additional mutations can be prepared as
described herein, for example in Example 7. Those of ordinary skill
in the art, familiar with standard molecular biology techniques,
can employ that knowledge, coupled with the instant disclosure, to
make and use these FGF21 mutant polypeptides. Standard techniques
can be used for recombinant DNA, oligonucleotide synthesis, tissue
culture, and transformation (e.g., electroporation, lipofection).
See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,
which is incorporated herein by reference for any purpose.
Enzymatic reactions and purification techniques can be performed
according to manufacturer's specifications, as commonly
accomplished in the art, or as described herein. Unless specific
definitions are provided, the nomenclatures utilized in connection
with, and the laboratory procedures and techniques of, analytical
chemistry, synthetic organic chemistry, and medicinal and
pharmaceutical chemistry described herein are those well known and
commonly used in the art. Standard techniques can be used for
chemical syntheses; chemical analyses; pharmaceutical preparation,
formulation, and delivery; and treatment of patients.
[0104] Following the preparation of FGF21 mutant polypeptides which
contain neither of the cysteines found in the wild-type FGF21
polypeptide sequence but instead comprise an introduced polymer
attachment site and optionally one or more additional mutations,
the polypeptide can be chemically modified by the attachment of a
polymer, as described in Example 9.
6. "TETHERED MOLECULES"
[0105] In still another aspect of the present invention, a
"Tethered Molecule" can be prepared as described herein. A
"Tethered Molecule" is a molecule comprising two FGF21 polypeptides
tethered together by a linker molecule. By joining two FGF21
polypeptides together, the effective half-life and potency of a
Tethered Molecule can be extended beyond the half-life and potency
of a single FGF21 polypeptide.
[0106] A Tethered Molecule of the present invention comprises a
linker and two FGF21 polypeptides, which can be two naturally
occurring FGF21 polypeptides into which no mutations have been
introduced, two FGF21 mutant polypeptides having a linker
attachment site introduced into the FGF21 polypeptides or a
combination of one naturally occurring FGF21 polypeptide and one
FGF21 mutant polypeptide. Tethered Molecules comprising at least
one FGF21 polypeptide having a non-naturally occurring linker
attachment site and one or more additional mutations are also
contemplated and form another aspect of the invention. Such
Tethered Molecules can thus comprise a mutation that forms a site
for the attachment of a linker molecule as well as another mutation
to impart another desirable property to the Tethered Molecule.
[0107] As used herein, the term "linker attachment site" means a
naturally or non-naturally occurring amino acid having a functional
group with which a linker can be associated. In one example, a
linker attachment site is a residue containing a thiol group, which
can be associated with a PEG molecule.
6.A. FGF21 Polypeptides in a Tethered Molecule
[0108] When a Tethered Molecule comprises two FGF21 mutant
polypeptides, the FGF21 mutant polypeptides can comprise one or
more mutations introduced into the sequence, but the mutations need
not be at the same amino acid position in each of the FGF21 mutant
polypeptides. By way of example, if a Tethered Molecule comprises
two FGF21 mutant polypeptides, one FGF21 mutant polypeptide may
contain an H125C mutation, which may form an attachment point for a
linker molecule. In contrast, the other FGF21 mutant polypeptide
can contain a mutation at a position other than H125 which can
serve as an attachment point for the linker tethering the two FGF21
mutant polypeptides together. Even if one or two FGF21 mutant
polypeptides are employed, the linker can be attached at the N
terminal end of the FGF21 mutant polypeptide; introduced attachment
points need not necessarily be used.
[0109] When a Tethered Molecule comprises one or two
naturally-occurring FGF21 polypeptides the linker can be attached
at a point in the FGF21 polypeptide that is amenable to the
attachment chemistry. For example, naturally occurring disulfide
bonds can be reduced and the cysteine residues can serve as
attachment points for a linker, such as PEG. In another embodiment,
a linker can be attached to a FGF21 polypeptide at the N-terminus
or on lysine sidechains.
[0110] One or both of the FGF21 mutant polypeptides of a Tethered
Molecule can comprise a truncated FGF21 mutant polypeptide. As
described herein, a truncated FGF21 mutant polypeptide can be
prepared by removing any number of residues on either the
N-terminus, the C-terminus or both the N- and C-termini.
[0111] Tethered Molecules can also comprise one or both FGF21
polypeptides which comprise a mutation in the polypeptide sequence
that may not be preferred as a linker attachment site, but instead
may impart some other desirable property to the Tethered Molecule.
Thus, Tethered Molecules comprising one or more FGF21 mutant
polypeptides into which a mutation imparting a desirable property
to the Tethered Molecule form a further aspect of the present
invention.
[0112] The activity of Tethered Molecules can be assayed in a
variety of ways, for example, using an in vitro ELK-luciferase
assay as described herein in Example 10.
[0113] The activity of the Tethered Molecules of the present
invention can also be assessed in an in vivo assay, such as with
ob/ob mice as shown in Example 12. Generally, to assess the in vivo
activity of one or more of these polypeptides, the polypeptide can
be administered to a test animal intraperitoneally. After one or
more desired time periods, a blood sample can be drawn, and blood
glucose levels can be measured.
[0114] As with all FGF21 mutants of the present invention, the
FGF21 polypeptides that comprise a Tethered Molecule, which can be
FGF21 mutant polypeptides, wild-type FGF21 polypeptides or a
combination of both, can optionally comprise an amino-terminal
methionine residue, which can be introduced by directed mutation or
as a result of a bacterial expression process.
[0115] Those of ordinary skill in the art, familiar with standard
molecular biology techniques, can employ that knowledge, coupled
with the instant disclosure, to make and use the Tethered Molecules
of the present invention. Standard techniques can be used for
recombinant DNA, oligonucleotide synthesis, tissue culture, and
transformation (e.g., electroporation, lipofection). See, e.g.,
Sambrook et al., Molecular Cloning: A Laboratory Manual, which is
incorporated herein by reference for any purpose. Enzymatic
reactions and purification techniques can be performed according to
manufacturer's specifications, as commonly accomplished in the art,
or as described herein. Processes for associating linkers with
FGF21 polypeptides will depend on the nature of the linker, but are
known to those of skill in the art. Examples of linker attachment
chemistries are described herein. Guidance on how a Tethered
Molecule of the present invention can be formed is provided herein,
for example in Example 9.
[0116] Unless specific definitions are provided, the nomenclatures
utilized in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
can be used for chemical syntheses; chemical analyses;
pharmaceutical preparation, formulation, and delivery; and
treatment of patients.
6.B. Linkers in Tethered Molecules
[0117] Any linker can be employed in a Tethered Molecule to tether
the two FGF21 mutant polypeptides together. Linker molecules can be
branched or unbranched and can be attached to a FGF21 mutant
polypeptide using various known chemistries, such as those
described herein. The chemical structure of a linker is not
critical, since it serves primarily as a spacer. The linker can be
independently the same or different from any other linker, or
linkers, that may be present in a Tethered Molecule (e.g., a
Tethered Molecule comprising three or more FGF21 mutant or
wild-type polypeptides). In one embodiment, a linker can be made up
of amino acids linked together by peptide bonds. Some of these
amino acids can be glycosylated, as is well understood by those in
the art. For example, a useful linker sequence constituting a
sialylation site is X.sub.1X.sub.2NX.sub.4X.sub.5G (SEQ ID NO: 5),
wherein X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are each
independently any amino acid residue. In another embodiment a
linker molecule can be a PEG molecule of any size, such as 20 kDa,
30 kDa or 40 kDa.
[0118] In embodiments in which a peptidyl linker is present (i.e.,
made up of amino acids linked together by peptide bonds) that is
made in length, preferably, of from 1 up to about 40 amino acid
residues, more preferably, of from 1 up to about 20 amino acid
residues, and most preferably of from 1 to about 10 amino acid
residues. In one embodiment, the amino acid residues in the linker
are selected from any the twenty canonical amino acids. In another
embodiment the amino acid residues in the linker are selected from
cysteine, glycine, alanine, proline, asparagine, glutamine, and/or
serine. In yet another embodiment, a peptidyl linker is made up of
a majority of amino acids that are sterically unhindered, such as
glycine, serine, and alanine linked by a peptide bond. It is often
desirable that, if present, a peptidyl linker be selected that
avoids rapid proteolytic turnover in circulation in vivo. Thus,
preferred peptidyl linkers include polyglycines, particularly
(Gly).sub.4 (SEQ ID NO: 6); (Gly).sub.5 (SEQ ID NO: 7);
poly(Gly-Ala); and polyalanines. Other preferred peptidyl linkers
include GGGGS (SEQ ID NO: 8); GGGGSGGGGS (SEQ ID NO: 9);
GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 10) and any linkers used in
the Examples provided herein. The linkers described herein,
however, are exemplary; linkers within the scope of this invention
can be much longer and can include other residues.
[0119] In embodiments of a Tethered Molecule that comprise a
peptide linker moiety, acidic residues, for example, glutamate or
aspartate residues, are placed in the amino acid sequence of the
linker moiety. Examples include the following peptide linker
sequences:
TABLE-US-00004 (SEQ ID NO: 11) GGEGGG; (SEQ ID NO: 12) GGEEEGGG;
(SEQ ID NO: 13) GEEEG; (SEQ ID NO: 14) GEEE; (SEQ ID NO: 15)
GGDGGG; (SEQ ID NO: 16) GGDDDGG; (SEQ ID NO: 17) GDDDG; (SEQ ID NO:
18) GDDD; (SEQ ID NO: 19) GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 20)
WEWEW; (SEQ ID NO: 21) FEFEF; (SEQ ID NO: 22) EEEWWW; (SEQ ID NO:
23) EEEFFF; (SEQ ID NO: 24) WWEEEWW; or (SEQ ID NO: 25)
FFEEEFF.
[0120] In other embodiments, a peptidyl linker constitutes a
phosphorylation site, e.g., X.sub.1X.sub.2YX.sub.3X.sub.4G (SEQ ID
NO: 26), wherein X.sub.1, X.sub.2,X.sub.3 and X.sub.4 are each
independently any amino acid residue;
X.sub.1X.sub.2SX.sub.3X.sub.4G (SEQ ID NO: 27), wherein X.sub.1,
X.sub.2,X.sub.3 and X.sub.4 are each independently any amino acid
residue; or X.sub.1X.sub.2TX.sub.3X.sub.4G (SEQ ID NO: 28), wherein
X.sub.1, X.sub.2,X.sub.3 and X.sub.4 are each independently any
amino acid residue.
[0121] Non-peptide linkers can also be used in a Tethered Molecule.
For example, alkyl linkers such as --NH--(CH.sub.2).sub.s--C(O)--,
wherein s=2 to 20 could be used. These alkyl linkers can further be
substituted by any non-sterically hindering group such as lower
alkyl (e.g., C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br),
CN, NH.sub.2, phenyl, etc.
[0122] Any suitable linker can be employed in the present invention
to from Tethered Molecules. In one example, the linker used to
produce Tethered Molecules described herein were homobifunctional
bis-maleimide PEG molecules having the general structure:
X--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--X
where X is a maleimide group. In other embodiments, X can be an
orthopyridyl-disulphide, an iodoacetamide, a vinylsulfone or any
other reactive moiety known to the art to be specific for thiol
groups. In yet another embodiment X can be an amino-specific
reactive moiety used to tether two mutant polypeptides through
either the N-terminus or an engineered lysyl group. (See, e.g.,
Pasut and Veronese, 2006, "PEGylation of Proteinsas Tailored
Chemistry for Optimized Bioconjugates," Adv. Polym. Sci.
192:95-134).
[0123] In still another embodiment, a linker can have the general
structure:
X--(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2--Y
where X and Y are different reactive moieties selected from the
groups above. Such a linker would allow conjugation of different
mutant polypeptides to generate Tethered heterodimers or
hetero-oligomers.
[0124] In a further embodiment, a linker can be a PEG molecule,
which can have a molecular weight of 1 to 100 kDa, preferably 10 to
50 kDa (e.g., 10, 20, 30 or 40 kDa) and more preferably 20 kDa. The
peptide linkers can be altered to form derivatives in the same
manner as described above.
[0125] Other examples of useful linkers include
aminoethyloxyethyloxy-acetyl linkers as disclosed in International
Publication No. WO 2006/042151, incorporated herein by reference in
its entirety.
[0126] When forming a Tethered Molecule of the present invention,
standard chemistries can be employed to associate a linker with a
wild-type or mutant FGF21 molecule. The precise method of
association will depend on the attachment site (e.g., which amino
acid side chains) and the nature of the linker. When a linker is a
PEG molecule, attachment can be achieved by employing standard
chemistry and a free sufhydryl or amine group, such as those found
on cysteine residues (which can be introduced into the FGF21
polypeptide sequence by mutation or can be naturally occurring) or
on lysine (which can be introduced into the FGF21 polypeptide
sequence by mutation or can be naturally occurring) or N-terminal
amino groups.
7. Chemical Modification of FGF21 Mutants
[0127] In an aspect of the present invention, FGF21 mutant
polypeptides are chemically modified. The term "chemically
modified" refers to a polypeptide (e.g., an FGF21 mutant
polypeptide) that has been modified by the addition of a polymer at
one or more sites on the polypeptide. Examples of chemically
modified forms of a FGF21 mutant polypeptide include PEGylated and
glycosylated forms of an FGF21 mutant polypeptide.
[0128] Chemically modified FGF21 mutant polypeptides of the present
invention can comprise any type of polymer, including water soluble
polymers, such as PEG. Exemplary polymers each can be of any
molecular weight and can be branched or unbranched. The polymers
each typically have an average molecular weight of between about 2
kDa to about 100 kDa (the term "about" indicating that in
preparations of a water-soluble polymer, some molecules will weigh
more and some less than the stated molecular weight). The average
molecular weight of each polymer is preferably between about 5 kDa
and about 50 kDa, more preferably between about 10 kDa and about 40
kDa, and most preferably between about 10 kDa and about 20 kDa.
[0129] Suitable water-soluble polymers or mixtures thereof include,
but are not limited to, carbohydrates, polyethylene glycol (PEG)
(including the forms of PEG that have been used to derivatize
proteins, including mono-(C.sub.1-C.sub.10), alkoxy-, or
aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol,
dextran (such as low molecular weight dextran of, for example,
about 6 kD), cellulose, or other carbohydrate based polymers,
poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol
homopolymers, polypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol.
Also encompassed by the present invention are bifunctional
crosslinking molecules that can be used to prepare covalently
attached FGF21 polypeptide mutant multimers. Also encompassed by
the present invention are FGF21 mutants covalently attached to
polysialic acid.
[0130] In some embodiments of the present invention, an FGF21
mutant polypeptide is covalently, modified to include one or more
water-soluble polymers, including, but not limited to, polyethylene
glycol (PEG), polyoxyethylene glycol, or polypropylene glycol. See,
e.g., U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192; and 4,179,337. In some embodiments of the present
invention, an FGF21 mutant comprises one or more polymers,
including, but not limited to, monomethoxy-polyethylene glycol,
dextran, cellulose, another carbohydrate-based polymer,
poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, or
mixtures of such polymers.
[0131] In yet other embodiments of the present invention, a peptide
or a protein can be conjugated to FGF21 in a site directed manner
though the aforementioned engineered residues in order impart
favorable properties to FGF21 (e.g. potency, stability,
selectivity). Thus the present invention encompasses FGF21 mutant
polypeptides conjugated to a heteroprotein or heteropeptide at an
introduced polymer attachment site. Examples of suitable proteins
include HSA and antibodies that do not bind to FGF21.
7.A. PEGylated FGF21 Mutant Polypeptides
[0132] In some embodiments of the present invention, an FGF21
mutant polypeptide is covalently-modified with PEG subunits. In
some embodiments, one or more water-soluble polymers are bonded at
one or more specific positions (for example, at the N-terminus) of
the FGF21 mutant. In some embodiments, one or more water-soluble
polymers are attached to one or more side chains of an FGF21
mutant; these side chains can be naturally occurring or can form a
component of an engineered polymer attachment site. In some
embodiments, PEG is used to improve the therapeutic capacity of an
FGF21 mutant polypeptide. Certain such methods are discussed, for
example, in U.S. Pat. No. 6,133,426, which is hereby incorporated
by reference for any purpose.
[0133] In embodiments of the present invention wherein the polymer
is PEG, the PEG group can be of any convenient molecular weight,
and can be linear or branched. The average molecular weight of the
PEG group will preferably range from about 2 kD to about 100 kDa,
and more preferably from about 5 kDa to about 50 kDa, e.g., 10, 20,
30, 40, or 50 kDa. The PEG groups will generally be attached to the
FGF21 mutant via acylation or reductive alkylation through a
reactive group on the PEG moiety (e.g., an aldehyde, NHS, or
maleimide, vinylsulfone, alkylhalide) to a reactive group on the
FGF21 mutant (e.g., an amino or thiol group).
[0134] When the polymer(s) attached to a FGF21 mutant polypeptide
is PEG, the PEGylation of a FGF21 mutant polypeptide of the present
invention, can be specifically carried out using any of the
PEGylation reactions known in the art. Such reactions are
described, for example, in the following references: Zalipsky,
1995, Functionalized Poly(ethylene glycol) for Preparation of
Biologically Relevant Conjugates, Bioconjugate Chemistry 6:150-165;
Francis et al., 1992, Focus on Growth Factors 3: 4-10; European
Patent Nos. 0 154 316 and 0 401 384; and U.S. Pat. No. 4,179,337.
For example, when the target residue is a lysine residue (i.e., a
residue with a reactive amine group) PEGylation can be carried out
via an acylation reaction or an alkylation reaction with an
amino-reactive polyethylene glycol molecule (or an analogous
reactive water-soluble polymer) as described herein. For the
acylation reactions, a selected polymer can have a single reactive
ester group. For reductive alkylation, a selected polymer can have
a single reactive aldehyde group. A reactive aldehyde is, for
example, polyethylene glycol propionaldehyde, which is water
stable, or mono C.sub.1-C.sub.10 alkoxy or aryloxy derivatives
thereof (see U.S. Pat. No. 5,252,714). Various reactive PEG
polymers activated with different amino-specific moieties will also
known to those of ordinary skill in the art and can also be
employed as circumstances dictate.
[0135] In another example, when the target residue is a cysteine
residue (i.e., a residue with a reactive sulfhydryl group)
PEGylation can be carried out via standard maleimide chemistry. For
this reaction, the selected polymer can contain one or more
reactive maleimide groups or other thiol reactive moiety such as
vinylsulfone, orthopyridyl-disulphide or iodoacetamide. See, e.g.,
Pasut & Veronese, 2006, "PEGylation of Proteins as Tailored
Chemistry for Optimized Bioconjugates," Adv. Polym. Sci.
192:95-134; Zalipsky, 1995, "Functionalized Poly(ethylene glycol)
for Preparation of Biologically Relevant Conjugates," Bioconjugate
Chemistry 6:150-165, and Hermanson, Bioconjugate Techniques,
2.sup.nd Ed., Academic Press, 2008, each of which is incorporated
herein by reference.
[0136] In some embodiments of the present invention, a useful
strategy for the attachment of the PEG group to a FGF21 mutant
involves combining through the formation of a conjugate linkage in
solution, a FGF21 mutant and a PEG moiety, each bearing a special
functionality that is mutually reactive toward the other. The FGF21
mutant is "preactivated" with an appropriate functional group at a
specific site. The precursors are purified and fully characterized
prior to reacting with the PEG moiety. Ligation of the FGF21 mutant
with PEG usually takes place in aqueous phase and can be easily
monitored by SDS-PAGE or reverse phase analytical HPLC. Detailed
analysis can be done by LC-MS based peptide mapping. Detailed
analysis can be done by LC-MS based peptide mapping.
7.B. Polysaccharide FGF21 Mutant Polypeptides
[0137] Polysaccharide polymers are another type of water-soluble
polymer that can be used for protein modification. Therefore, the
FGF21 mutant polypeptides of the present invention can be attached
to a polysaccharide polymer to form embodiments of the present
invention. Thus, an engineered non-naturally occurring polymer
attachment site in an FGF21 mutant polypeptide can comprise a
residue to which a polysaccharide is attached. Dextrans are
polysaccharide polymers comprised of individual subunits of glucose
predominantly linked by alpha 1-6 linkages. The dextran itself is
available in many molecular weight ranges, and is readily available
in molecular weights from about 1 kD to about 70 kD. Dextran is a
suitable water-soluble polymer for use as a vehicle by itself or in
combination with another vehicle (e.g., Fc). See, e.g.,
International Publication No. WO 96/11953. The use of dextran
conjugated to therapeutic or diagnostic immunoglobulins has been
reported. See, e.g., European Patent Publication No. 0 315 456,
which is hereby incorporated by reference. The present invention
also encompasses the use of dextran of about 1 kDa to about 20
kDa.
7.C. Methods of Chemically Modifying a FGF21 Mutant Polypeptide
[0138] In general, chemical modification (e.g., PEGylation or
glycosylation) can be performed under any suitable condition used
to react a protein with an activated polymer molecule. Methods for
preparing chemically modified FGF21 mutant polypeptides will
generally comprise the steps of: (a) reacting the polypeptide with
the activated polymer molecule (such as a reactive ester, maleimide
or aldehyde derivative of the polymer molecule) under conditions
whereby an FGF21 mutant polypeptide becomes attached to one or more
polymer molecules, and (b) obtaining the reaction products. The
optimal reaction conditions will be determined based on known
parameters and the desired result. For example, the larger the
ratio of polymer molecules to protein, the greater the percentage
of attached polymer molecule. In one embodiment of the present
invention, chemically modified FGF21 mutant polypeptides can have a
single polymer molecule at the amino-terminus, while in other
embodiments an FGF21 mutant polypeptide can have two or more
polymers associated with the primary sequence, for example one
polymer at the N-terminus of the polypeptide and a second at
another residue in the polypeptide. Alternatively, a FGF21 mutant
can have two or more polymers associated at two different residues
in the primary sequence but not at the N-terminus.
[0139] Generally, conditions that can be alleviated or modulated by
the administration of the present chemically modified FGF21 mutant
polypeptides include those described herein for the native FGF21
polypeptide. However, the chemically modified FGF21 mutant
polypeptides disclosed herein can have additional activities, such
as increased half-life, as compared to wild-type FGF21 and FGF21
mutants.
8. THERAPEUTIC COMPOSITIONS OF FGF21 MUTANTS AND ADMINISTRATION
THEREOF
[0140] Therapeutic compositions comprising FGF21 mutant
polypeptides are within the scope of the present invention, and are
specifically contemplated in light of the identification of
Tethered Molecules, FGF21 mutant polypeptides and chemically
modified FGF21 mutant polypeptides that exhibit enhanced
properties. Such Tethered Molecule, FGF21 mutant polypeptide, and
chemically modified FGF21 mutant polypeptide therapeutic
compositions, can comprise a therapeutically effective amount of a
Tethered Molecule, FGF21 mutant polypeptide or chemically modified
FGF21 mutant polypeptide, which can be chemically modified, in a
mixture with a pharmaceutically or physiologically acceptable
formulation agent selected for suitability with the mode of
administration.
[0141] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0142] The pharmaceutical composition can contain formulation
materials for modifying, maintaining, or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption,
or penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine, or lysine), antimicrobials,
antioxidants (such as ascorbic acid, sodium sulfite, or sodium
hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, or other organic acids), bulking agents (such
as mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin, or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,
disaccharides, and other carbohydrates (such as glucose, mannose,
or dextrins), proteins (such as serum albumin, gelatin, or
immunoglobulins), coloring, flavoring and diluting agents,
emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides,
salt-forming counterions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid, or hydrogen peroxide), solvents (such as glycerin,
propylene glycol, or polyethylene glycol), sugar alcohols (such as
mannitol or sorbitol), suspending agents, surfactants or wetting
agents (such as pluronics; PEG; sorbitan esters; polysorbates such
as polysorbate 20 or polysorbate 80; triton; tromethamine;
lecithin; cholesterol or tyloxapal), stability enhancing agents
(such as sucrose or sorbitol), tonicity enhancing agents (such as
alkali metal halides--preferably sodium or potassium chloride--or
mannitol sorbitol), delivery vehicles, diluents, excipients and/or
pharmaceutical adjuvants (see, e.g., Remington's Pharmaceutical
Sciences (18th Ed., A. R. Gennaro, ed., Mack Publishing Company
1990), and subsequent editions of the same, incorporated herein by
reference for any purpose).
[0143] The optimal pharmaceutical composition will be determined by
a skilled artisan depending upon, for example, the intended route
of administration, delivery format, and desired dosage (see, e.g.,
Remington's Pharmaceutical Science). Such compositions can
influence the physical state, stability, rate of in vivo release,
and rate of in vivo clearance of the Tethered Molecule, FGF21
mutant polypeptide (which can be truncated, capped or C-terminally
mutated) or chemically modified FGF21 mutant polypeptide.
[0144] The primary vehicle or carrier in a pharmaceutical
composition can be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier for injection can be water,
physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can
further include sorbitol or a suitable substitute. In one
embodiment of the present invention, Tethered Molecule, FGF21
mutant polypeptide and chemically modified FGF21 mutant polypeptide
compositions can be prepared for storage by mixing the selected
composition having the desired degree of purity with optional
formulation agents (Remington's Pharmaceutical Sciences, supra) in
the form of a lyophilized cake or an aqueous solution. Further, the
Tethered Molecule, FGF21 mutant polypeptide and chemically modified
FGF21 mutant polypeptide product can be formulated as a
lyophilizate using appropriate excipients such as sucrose.
[0145] The pharmaceutical compositions of the present invention can
be selected for parenteral delivery. Alternatively, the
compositions can be selected for inhalation or for delivery through
the digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the
art.
[0146] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at a slightly lower pH, typically within a pH range of from about 5
to about 8.
[0147] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention can be in the
form of a pyrogen-free, parenterally acceptable, aqueous solution
comprising the desired Tethered Molecule, FGF21 mutant polypeptide
or chemically modified FGF21 mutant polypeptide in a
pharmaceutically acceptable vehicle. A particularly suitable
vehicle for parenteral injection is sterile distilled water in
which a Tethered Molecule, FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide is formulated as a sterile,
isotonic solution, properly preserved. Yet another preparation can
involve the formulation of the desired molecule with an agent, such
as injectable microspheres, bio-erodible particles, polymeric
compounds (such as polylactic acid or polyglycolic acid), beads, or
liposomes, that provides for the controlled or sustained release of
the product which can then be delivered via a depot injection.
Hyaluronic acid can also be used, and this can have the effect of
promoting sustained duration in the circulation. Other suitable
means for the introduction of the desired molecule include
implantable drug delivery devices.
[0148] In one embodiment, a pharmaceutical composition can be
formulated for inhalation. For example, Tethered Molecule, FGF21
mutant polypeptide or chemically modified FGF21 mutant polypeptide
can be formulated as a dry powder for inhalation. Such inhalation
solutions can also be formulated with a propellant for aerosol
delivery. In yet another embodiment, solutions can be nebulized.
Pulmonary administration is further described in International
Publication No. WO 94/20069, which describes the pulmonary delivery
of chemically modified proteins.
[0149] It is also contemplated that certain formulations can be
administered orally. In one embodiment of the present invention, a
Tethered Molecule, FGF21 mutant polypeptide (which can be
truncated, capped or C-terminally mutated) or chemically modified
FGF21 mutant polypeptide that is administered in this fashion can
be formulated with or without those carriers customarily used in
the compounding of solid dosage forms such as tablets and capsules.
For example, a capsule can be designed to release the active
portion of the formulation at the point in the gastrointestinal
tract when bioavailability is maximized and pre-systemic
degradation is minimized. Additional agents can be included to
facilitate absorption of the Tethered Molecule, FGF21 mutant
polypeptide (which can be truncated, capped or C-terminally
mutated) or chemically modified FGF21 mutant polypeptide. Diluents,
flavorings, low melting point waxes, vegetable oils, lubricants,
suspending agents, tablet disintegrating agents, and binders can
also be employed.
[0150] Another pharmaceutical composition can involve a
therapeutically effective quantity of a Tethered Molecule, FGF21
mutant polypeptide (which can be truncated, capped or C-terminally
mutated) or chemically modified FGF21 mutant polypeptide in a
mixture with non-toxic excipients that are suitable for the
manufacture of tablets. By dissolving the tablets in sterile water,
or another appropriate vehicle, solutions can be prepared in
unit-dose form. Suitable excipients include, but are not limited
to, inert diluents, such as calcium carbonate, sodium carbonate or
bicarbonate, lactose, or calcium phosphate; or binding agents, such
as starch, gelatin, or acacia; or lubricating agents such as
magnesium stearate, stearic acid, or talc.
[0151] Additional Tethered Molecule, FGF21 mutant polypeptide or
chemically modified FGF21 mutant polypeptide pharmaceutical
compositions will be evident to those skilled in the art, including
formulations involving Tethered Molecule, FGF21 mutant polypeptides
or chemically modified FGF21 mutant polypeptides in sustained- or
controlled-delivery formulations. Techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art (see,
e.g., International Publication No. WO 93/15722, which describes
the controlled release of porous polymeric microparticles for the
delivery of pharmaceutical compositions).
[0152] Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g., films, or microcapsules. Sustained release matrices
can include polyesters, hydrogels, polylactides (U.S. Pat. No.
3,773,919 and European Patent No. 0 058 481), copolymers of
L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,
Biopolymers 22: 547-56), poly(2-hydroxyethyl-methacrylate) (Langer
et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982,
Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al.,
supra) or poly-D(-)-3-hydroxybutyric acid (European Patent No. 0
133 988). Sustained-release compositions can also include
liposomes, which can be prepared by any of several methods known in
the art. See, e.g., Epstein et al., 1985, Proc. Natl. Acad. Sci.
U.S.A. 82: 3688-92; and European Patent Nos. 0 036 676, 0 088 046,
and 0 143 949.
[0153] A Tethered Molecule, FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide pharmaceutical composition to be
used for in vivo administration typically must be sterile. This can
be accomplished by filtration through sterile filtration membranes.
Where the composition is lyophilized, sterilization using this
method can be conducted either prior to, or following,
lyophilization and reconstitution. The composition for parenteral
administration can be stored in lyophilized form or in a solution.
In addition, parenteral compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0154] Once the pharmaceutical composition has been formulated, it
can be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations can be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0155] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
can each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0156] A therapeutically effective amount of a Tethered Molecule,
FGF21 mutant polypeptide (which can be truncated, capped or
C-terminally mutated) or chemically modified FGF21 mutant
polypeptide pharmaceutical composition to be employed
therapeutically will depend, for example, upon the therapeutic
context and objectives. One skilled in the art will appreciate that
the appropriate dosage levels for treatment will thus vary
depending, in part, upon the molecule delivered, the indication for
which the Tethered Molecule, FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide is being used, the route of
administration, and the size (body weight, body surface, or organ
size) and condition (the age and general health) of the patient.
Accordingly, the clinician can titer the dosage and modify the
route of administration to obtain the optimal therapeutic effect. A
typical dosage can range from about 0.1 .mu.g/kg to up to about 100
mg/kg or more, depending on the factors mentioned above. In other
embodiments, the dosage can range from 0.01 mg/kg up to about 10
mg/kg, for example 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4
mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0
mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8
mg/kg, 9 mg/kg or 10 mg/kg.
[0157] The frequency of dosing will depend upon the pharmacokinetic
parameters of the Tethered Molecule, FGF21 mutant polypeptide or
chemically modified FGF21 mutant polypeptide in the formulation
being used. Typically, a clinician will administer the composition
until a dosage is reached that achieves the desired effect. The
composition can therefore be administered as a single dose, as two
or more doses (which may or may not contain the same amount of the
desired molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages can be ascertained through use of
appropriate dose-response data.
[0158] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g., orally; through
injection by intravenous, intraperitoneal, intracerebral
(intraparenchymal), intracerebroventricular, intramuscular,
intraocular, intraarterial, intraportal, or intralesional routes;
by sustained release systems; or by implantation devices. Where
desired, the compositions can be administered by bolus injection or
continuously by infusion, or by implantation device.
[0159] Alternatively or additionally, the composition can be
administered locally via implantation of a membrane, sponge, or
other appropriate material onto which the desired molecule has been
absorbed or encapsulated. Where an implantation device is used, the
device can be implanted into any suitable tissue or organ, and
delivery of the desired molecule can be via diffusion,
timed-release bolus, or continuous administration.
10. THERAPEUTIC USES OF FGF21 POLYPEPTIDE MUTANTS
[0160] The Tethered Molecules, FGF21 mutant polypeptides (which can
be truncated, capped or C-terminally mutated) and chemically
modified FGF21 mutant polypeptides of the present invention can be
used to treat, diagnose, ameliorate, or prevent a number of
diseases, disorders, or conditions, including, but not limited to
metabolic disorders. In one embodiment, the metabolic disorder to
be treated is diabetes. In another embodiment, the metabolic
disorder is obesity. Other embodiments include metabolic conditions
or disorders such as dyslipidimia; hypertension; hepatosteaotosis,
such as non-alcoholic steatohepatitis (NASH); cardiovascular
disease, such as atherosclerosis; and aging.
[0161] In application, a disorder or condition such as diabetes or
obesity can be treated by administering a Tethered Molecule, FGF21
mutant polypeptide or chemically modified FGF21 mutant polypeptide
as described herein to a patient in need thereof in the amount of a
therapeutically effective dose. The administration can be performed
as described herein, such as by IV injection, intraperitoneal
injection, intramuscular injection, or orally in the form of a
tablet or liquid formation. In most situations, a desired dosage
can be determined by a clinician, as described herein, and can
represent a therapeutically effective dose of the Tethered
Molecule, FGF21 mutant polypeptide or chemically modified FGF21
mutant polypeptide. It will be apparent to those of skill in the
art that a therapeutically effective dose of Tethered Molecule,
FGF21 mutant polypeptide or chemically modified FGF21 mutant
polypeptide will depend, inter alia, upon the administration
schedule, the unit dose of antigen administered, whether the
nucleic acid molecule or polypeptide is administered in combination
with other therapeutic agents, the immune status and the health of
the recipient. The term "therapeutically effective dose," as used
herein, means that amount of Tethered Molecule, FGF21 mutant
polypeptide or chemically modified FGF21 mutant polypeptide that
elicits the biological or medicinal response in a tissue system,
animal, or human being sought by a researcher, medical doctor, or
other clinician, which includes alleviation of the symptoms of a
disease or disorder being treated.
11. ANTIBODIES
[0162] Antibodies and antibody fragments that specifically bind to
the Tethered Molecules, FGF21 mutant polypeptides and chemically
modified FGF21 mutant polypeptides of the present invention but do
not specifically bind to wild-type FGF21 polypeptides are
contemplated and are within the scope of the present invention. The
antibodies can be polyclonal, including monospecific polyclonal;
monoclonal (MAbs); recombinant; chimeric; humanized, such as
complementarity-determining region (CDR)-grafted; human; single
chain; and/or bispecific; as well as fragments; variants; or
chemically modified molecules thereof. Antibody fragments include
those portions of the antibody that specifically bind to an epitope
on an FGF21 mutant polypeptide. Examples of such fragments include
Fab and F(ab') fragments generated by enzymatic cleavage of
full-length antibodies. Other binding fragments include those
generated by recombinant DNA techniques, such as the expression of
recombinant plasmids containing nucleic acid sequences encoding
antibody variable regions.
[0163] Polyclonal antibodies directed toward a Tethered Molecule,
FGF21 mutant polypeptide or chemically modified FGF21 mutant
polypeptide generally are produced in animals (e.g., rabbits or
mice) by means of multiple subcutaneous or intraperitoneal
injections of the FGF21 mutant polypeptide and an adjuvant. It can
be useful to conjugate an FGF21 mutant polypeptide to a carrier
protein that is immunogenic in the species to be immunized, such as
keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or
soybean trypsin inhibitor. Also, aggregating agents such as alum
are used to enhance the immune response. After immunization, the
animals are bled and the serum is assayed for anti-Tethered
Molecule, FGF21 mutant polypeptide or chemically modified FGF21
mutant polypeptide antibody titer.
[0164] Monoclonal antibodies directed toward Tethered Molecules,
FGF21 mutant polypeptides or chemically modified FGF21 mutant
polypeptides can be produced using any method that provides for the
production of antibody molecules by continuous cell lines in
culture. Examples of suitable methods for preparing monoclonal
antibodies include the hybridoma methods of Kohler et al., 1975,
Nature 256: 495-97 and the human B-cell hybridoma method (Kozbor,
1984, J. Immunol. 133: 3001; Brodeur et al., Monoclonal Antibody
Production Techniques and Applications 51-63 (Marcel Dekker, Inc.,
1987). Also provided by the invention are hybridoma cell lines that
produce monoclonal antibodies reactive with Tethered Molecules,
FGF21 mutant polypeptides or chemically modified FGF21 mutant
polypeptides.
[0165] The anti-FGF21 mutant antibodies of the invention can be
employed in any known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays (see, e.g., Sola, Monoclonal Antibodies:
A Manual of Techniques 147-158 (CRC Press, Inc., 1987),
incorporated herein by reference in its entirety) for the detection
and quantitation of FGF21 mutant polypeptides. The antibodies will
bind FGF21 mutant polypeptides with an affinity that is appropriate
for the assay method being employed.
[0166] For diagnostic applications, in certain embodiments,
anti-Tethered Molecule, FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide antibodies can be labeled with a
detectable moiety. The detectable moiety can be any one that is
capable of producing, either directly or indirectly, a detectable
signal. For example, the detectable moiety can be a radioisotope,
such as .sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.125I,
.sup.99Tc, .sup.111In, or .sup.67Ga; a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin; or an enzyme, such as alkaline
phosphatase, .beta.-galactosidase, or horseradish peroxidase (Bayer
et al., 1990, Meth. Enz. 184: 138-63).
[0167] Competitive binding assays rely on the ability of a labeled
standard (e.g., an FGF21 mutant polypeptide, or an immunologically
reactive portion thereof) to compete with the test sample analyte
(e.g., a Tethered Molecule, FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide) for binding with a limited
amount of anti-Tethered Molecule, FGF21 mutant polypeptide or
chemically modified FGF21 mutant polypeptide antibody, depending on
the analyte. The amount of Tethered Molecule, FGF21 mutant
polypeptide or chemically modified FGF21 mutant polypeptide in the
test sample is inversely proportional to the amount of standard
that becomes bound to the antibodies. To facilitate determining the
amount of standard that becomes bound, the antibodies typically are
insolubilized before or after the competition, so that the standard
and analyte that are bound to the antibodies can conveniently be
separated from the standard and analyte that remain unbound.
[0168] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected and/or quantitated. In a
sandwich assay, the test sample analyte is typically bound by a
first antibody that is immobilized on a solid support, and
thereafter a second antibody binds to the analyte, thus forming an
insoluble three-part complex. See, e.g., U.S. Pat. No. 4,376,110.
The second antibody can itself be labeled with a detectable moiety
(direct sandwich assays) or can be measured using an
anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assays). For example, one type of
sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in
which case the detectable moiety is an enzyme.
[0169] The anti-Tethered Molecule, FGF21 mutant polypeptide or
chemically modified FGF21 mutant polypeptide antibodies of the
present invention are also useful for in vivo imaging. An antibody
labeled with a detectable moiety can be administered to an animal,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host assayed. The antibody can be
labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0170] The invention also relates to a kit comprising Tethered
Molecules, FGF21 mutant polypeptides or chemically modified FGF21
mutant polypeptide antibodies and other reagents useful for
detecting Tethered Molecule, FGF21 mutant polypeptide or chemically
modified FGF21 mutant polypeptide levels in biological samples.
Such reagents can include a detectable label, blocking serum,
positive and negative control samples, and detection reagents.
EXAMPLES
[0171] The Examples that follow are illustrative of specific
embodiments of the invention, and various uses thereof. They are
set forth for explanatory purposes only, and should not be
construed as limiting the scope of the invention in any way.
Example 1
Preparation of FGF21 Polypeptide Expression Constructs
[0172] A nucleic acid sequence encoding the mature FGF21
polypeptide was obtained by polymerase chain reaction (PCR)
amplification using primers having nucleotide sequences
corresponding to the 5' and 3' ends of the mature FGF21 sequence.
Table 4 lists the primers that were used to amplify the mature
FGF21 sequence.
TABLE-US-00005 TABLE 4 PCR Primers for Preparing FGF21 Construct
SEQ Primer Sequence ID NO: Sense 5'-AGGAGGAATAACATATGCATCCAATT 33
CCAGATTCTTCTCC-3' Antisense 5'-TAGTGAGCTCGAATTCTTAGGAAGCG 34
TAGCTGG-3'
[0173] The primers used to prepare the FGF21 expression construct
incorporated restriction endonuclease sites for directional cloning
of the sequence into a suitable expression vector (e.g., pET30
(Novagen/EMD Biosciences; San Diego, Calif.) or pAMG33 (Amgen;
Thousand Oaks, Calif.)). The expression vector pAMG33 contains a
low-copy number R-100 origin of replication, a modified lac
promoter, and a kanamycin-resistance gene. The expression vector
pET30 contains a pBR322-derived origin of replication, an inducible
T7 promoter, and a kanamycin-resistance gene. While expression from
pAMG33 was found to be higher, pET30 was found to be a more
reliable cloning vector. Thus, the majority of the constructs
described in the instant application were first generated in pET30
and then screened for efficacy. Selected sequences were then
transferred to pAMG33 for further amplification.
[0174] The FGF21 sequence was amplified in a reaction mixture
containing 40.65 .mu.L dH.sub.2O, 5 .mu.L PfuUltra II Reaction
Buffer (10.times.), 1.25 .mu.L dNTP Mix (40 mM-4.times.10 mM), 0.1
.mu.L Template (100 ng/mL), 1 .mu.L Primer1 (10 .mu.M), 1 .mu.L
Primer2 (10 .mu.M), and 1 .mu.L PfuUltra II fusion HS DNA
Polymerase (Stratagene; La Jolla, Calif.). Amplification reactions
were performed by heating for two minutes at 95.degree. C.;
followed by ten cycles at 95.degree. C. for 20 seconds, 60.degree.
C. for 20 seconds (with an additional 1.degree. C. subtracted per
cycle), and 72.degree. C. for 15 seconds/kilobase of desired
product; followed by 20 cycles at 94.degree. C. for 20 seconds,
55.degree. C. for 20 seconds, and 72.degree. C. for 15
seconds/kilobase of desired product; followed by 72.degree. C. for
three minutes. Amplification products were digested with the
restriction endonucleases NdeI and EcoRI; ligated into a suitable
vector; and then transformed into competent cells.
[0175] As a result of the bacterial expression system employed, the
expressed mature FGF21 polypeptide included an N-terminal
methionine residue or a variant methionine residue such as fMet or
gluconylated Met.
Example 2
Purification of Wild-Type FGF21 Polypeptides from Bacteria
[0176] In the Examples that follow, wild-type FGF21 polypeptides
were expressed in a bacterial expression system. After expression,
which is described below, the wild-type FGF21 polypeptides were
purified as described in this Example, unless otherwise
indicated.
[0177] To purify the wild-type FGF21 polypeptide from bacterial
inclusion bodies, double-washed inclusion bodies (DWIBs) were
solubilized in a solubilization buffer containing guanidine
hydrochloride and DTT in Tris buffer at pH 8.5 and then mixed for
one hour at room temperature, and the solubilization mixture was
added to a refold buffer containing urea, arginine, cysteine, and
cystamine hydrochloride at pH 9.5 and then mixed for 24 hours at
5.degree. C. (see, e.g., Clarke, 1998, Curr. Opin. Biotechnol. 9:
157-63; Mannall et al., 2007, Biotechnol. Bioeng. 97: 1523-34;
Rudolph et al., 1997, "Folding proteins," in Protein Function: A
Practical Approach (Creighton, ed., New York, IRL Press), pp 57-99;
and Ishibashi et al., 2005, Protein Expr. Purif. 42: 1-6).
[0178] Following solubilization and refolding, the mixture was
filtered through a 0.45 micron filter. The refold pool was then
concentrated approximately 10-fold with a 10 kD molecular weight
cut-off Pall Omega cassette at a transmembrane pressure (TMP) of 20
psi, and dialfiltered with 3 column volumes of 20 mM Tris, pH 8.0
at a TMP of 20 psi.
[0179] The clarified sample was then subjected to anion exchange
(AEX) chromatography using a Q Sepharose HP resin. A linear salt
gradient of 0 to 250 mM NaCl in 20 mM Tris was run at pH 8.0 at
5.degree. C. Peak fractions were analyzed by SDS-PAGE and
pooled.
[0180] The AEX eluate pool was then subjected to hydrophobic
interaction chromatography (HIC) using a Phenyl Sepharose HP resin.
Protein was eluted using a decreasing linear gradient of 0.7 M to 0
M ammonium sulfate at pH 8.0 and ambient temperature. Peak
fractions were analyzed by SDS-PAGE (Laemmli, 1970, Nature 227:
680-85) and pooled.
[0181] The HIC pool was concentrated with a 10 kD molecular weight
cut-off Pall Omega 0.2 m.sup.2 cassette to 7 mg/mL at a TMP of 20
psi. The concentrate was dialfiltered with 5 volumes of 10 mM
KPO.sub.4, 5% sorbitol, pH 8.0 at a TMP of 20 psi, and the
recovered concentrate was diluted to 5 mg/mL. Finally, the solution
was filtered through a Pall mini-Kleenpac 0.2 .mu.M Posidyne
membrane.
Example 3
Identification of FGF21 Mutants
[0182] Wild-type FGF21 has a relatively short half life, and in
some cases this can be undesirable for the use of wild-type FGF21
as a therapeutic. Additionally, traditional methods of extending
half life, such as PEGylation of the polypeptide are limited by the
number and location of suitable PEGylation sites in the FGF21
sequence. In the wild-type FGF21 polypeptide sequence there are
seven naturally occurring PEGylation sites, namely the alpha amino
group, four lysine residues, and two cysteine residues. These sites
are not ideal for PEGylation because the PEG molecule can adversely
affect the ability of FGF21 to obtain its native structure, or it
can adversely affect the interaction between FGF21 and its receptor
or beta-klotho. In addition, with the exception of the alpha amino
group, these reactive residues do not allow for the site specific
PEGylation at a single targeted location. Accordingly, a directed
and focused study was undertaken to identify residues within the
wild-type FGF21 polypeptide that could be mutated to a residue
suitable for chemical modification. Considerations in the study
included the location of the residue in the FGF21 sequence, as well
as its position on the protein surface.
[0183] Two different strategies were employed in an effort to
identify individual residues in the wild-type FGF21 polypeptide
that would be suitable for mutation to a residue useful for
chemical modification, described herein.
Example 3a
Mutation Candidates Identified Using a Homology Model
[0184] In the first strategy, a homology model was employed in a
systematic rational protein engineering approach to identify
residues with a high probability of having surface exposed
sidechains that were likely to tolerate PEGylation without
interfering with FGF21 activity. Since there are no published X-ray
or NMR structures of FGF21 that could be used to identify such
residues, a high resolution (1.3 .ANG.) X-ray crystal structure of
FGF19 (1PWA) obtained from the Protein Databank (PDB) was used to
create a 3D homology model of FGF21 using MOE (Molecular Operating
Environment; Chemical Computing Group; Montreal, Quebec, Canada)
modeling software. FGF19 was chosen as a template, since among the
proteins deposited in the PDB, FGF19 is the most closely related
protein to FGF21 in terms of the amino acid sequence homology.
[0185] The FGF21 homology model was then extended to represent
FGF-21 bound to an FGF receptor. This model was used to identify
residues that would likely be exposed on the surface of the FGF21
molecule and available for reaction with an activated polymer
(e.g., PEG) moiety, while avoiding those residues that might
interfere with FGF21 interaction with its receptor. Considerations
were also made for the sequence conservation of the residues
between species as well as the biochemical properties of the native
side chain. Residues identified as good candidates by this screen
were ranked according to the estimated probability of successful
PEGylation of this site with minimal disruption of activity.
Residues in Group A reflect the best candidates, and residues in
Group D reflect viable but less preferred candidates. Group A
includes N121, H125, H112, R77, H87, E37 and K69. Group B includes
R126, G113, D79, E91, D38, D46 and G120. Group C includes S71, D89,
L86, T70, G39, T40, R36, P49, S48, 5123, K122, A81, A111, E110 and
R96. Finally, Group D includes Q18, R19, A26, Q28, E34, A44, A45,
E50, L52, Q54, L55, K59, G61, L66, V68, R72, P78, G80, Y83, S85,
F88, P90, A92, S94, L98, E101, D102, Q108, L114, H117, P119, P124,
D127, P128, A129, P130, R131, and P140.
[0186] In addition, potential PEGylation sites within the amino and
carboxy-terminal segments of the molecule were identified based on
sequence alignments and the biochemical properties of the
sidechains, since no three dimensional structural data is available
for these portions of the molecule. Residues that were believed to
be most suitable for mutation were identified and subsequently
grouped according to how well the residues fit a set of selection
criteria. With respect to the N-terminus of the FGF21 polypeptide
sequence, residues 1-13 were evaluated, and D5 was determined to be
the best candidate for mutation, with residues H1, P2, I3, P4 and
S6 also determined to be viable. With respect to the C-terminus of
the FGF21 polypeptide sequence, residues 141-181 were evaluated,
and residues Y179 and R175 were determined to be the best
candidates for mutation, with residues P143, P171, S172, Q173,
G174, S176, P177, S178, A180, and S181 forming another group of
candidates, and residues P144, A145, P147, E148, P149, P150, I152
A154, Q156 and G170 forming a third group of candidates for
mutation.
Example 3B
FGF21 Mutants Generated by Removing Undesired Naturally Occurring
Polymer Attachment Sites
[0187] The second strategy employed an amino-specific conjugation
chemistry for coupling PEG to FGF21. Site-selective dual-PEGylation
of potentially vacuologenic conjugates may significantly reduce
their vacuologenic potential (see, e.g., U.S. Pat. No. 6,420,339).
Accordingly, in one aspect of the present invention, site-selective
dual-PEGylation of FGF21 is accomplished by mutating the protein so
that only two primary amino groups remain for PEGylation.
[0188] The FGF21 protein contains four lysine and ten arginine
residues as highlighted by bold (R and K residues) and underlining
(K residues) in the wild-type FGF21 sequence (SEQ ID NO:4). The two
naturally occurring cysteine residues are highlighted in bold and
italic:
TABLE-US-00006 SEQ ID NO: 4
HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQS
PESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFR
ELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPP
APPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS
[0189] The four naturally occurring lysines were first mutated to
arginines to create a parent molecule containing only one primary
amine group as an .alpha.-amino group at the N-terminus. This was
tested for in vitro activity and found to be fully active. Next,
selected arginines were step-wise replaced with lysine to create up
to ten analogs containing a second primary amino group for
PEGylation at various positions on the FGF21 molecule. An
examination of the homology model of FGF21 bound to its receptor
(as described in Example 3A) allowed ranking of the proposed
conjugation sites as a function of their proximity to the putative
receptor interface. Sites that appeared buried were not
addressed.
[0190] The various positions were ranked and characterized as
follows: (a) Sites that are solvent exposed and distal to the
receptor interface: R36, R77, K122 & R126; (b) Sites that are
solvent exposed and proximal to the receptor interface: K56, K59,
K69, R72 & R175; and (c) Sites that are buried: R17, R19, R131
& R135.
[0191] The final constructs, each containing a single .alpha.-amino
and .epsilon.-amino group were purified, and tested for activity
both before and after conjugation with PEG as described in Examples
9 (conjugation) and 10 (in vitro activity assay).
Example 4
Identification of FGF21 Mutants Comprising a Single Mutation
[0192] A summary of FGF21 mutants comprising a single mutation that
were generated through the rational protein engineering approach
described above in Example 3.A and 3.B is provided in Table 3.
These single mutants provide and incorporate a non-naturally
occurring polymer attachment site, notably a cysteine or lysine
residue. The side chains of these residues are particularly suited
to chemical modification by the attachment of a polymer, such as a
PEG molecule. It was recognized that in addition to the introduced
non-naturally occurring polymer attachment site, a polymer could
optionally be attached to the N-terminus of the protein, as
desired.
[0193] Since the introduced residues were designed to maintain the
wild-type levels of FGF21 biological activity, these FGF21 mutant
polypeptides are expected to maintain FGF21 biological activity and
yet still provide one or more non-naturally occurring polymer
attachment sites. After chemical modification (e.g., PEGylation)
these mutants are expected to have a longer in vivo half life than
wild-type FGF21, while maintaining significant in vivo wild-type
levels of biological activity.
[0194] The numbers of the positions targeted for mutagenesis are
given in Table 5 and correspond to the residue position in the
mature FGF21 protein, which consists of 181 amino acid residues.
Nucleic acid sequences encoding the FGF21 mutant polypeptides
listed in Table 5 below were prepared using the techniques
described below.
TABLE-US-00007 TABLE 5 FGF21 Mutant Polypeptides Comprising a
Single Mutation Residue Number WT Mutation 36 R K R36K 37 E C E37C
38 D C D38C 46 D C D46C 56 K R K56R 60 K R K60R 91 E C E91C 69 K C
K69C 69 K R K69R 72 R K R72K 77 R C R77C 77 R K R77K 79 D C D79C 86
H C H86C 91 E C E91C 112 H C H112C 113 G C G113C 120 G C G120C 121
N C N121C 122 K R K122R 125 H C H125C 126 R C R126C 126 R K R126K
171 P G P171G 175 R C R175C 175 R K R175K 170 G C G170C 179 Y C
Y179C
Example 5
Identification of FGF21 Mutants Comprising Two Mutations
[0195] Further analysis of the homology model and data obtained
from the single cysteine mutants was performed in order to identify
combinations of mutants that would provide two non-naturally
occurring polymer attachment sites. Following chemical
modification, these FGF21 mutants have two polymers (e.g., PEG
molecules) attached to the polypeptide at desired locations. As
with all the FGF21 mutant polypeptides of the present invention, a
polymer can also be attached to the N-terminus of the polypeptide,
depending on the nature of the polymer itself. A cartoon depicting
a dually PEGylated FGF21 mutant is shown graphically in FIG. 1.
[0196] A summary of FGF21 mutants comprising two mutations that
were generated through this rational protein engineering approach
is provided in Table 6. These double mutants incorporate a
non-naturally occurring polymer attachment site, notably a
cysteine. The side chains of these residues are particularly suited
to chemical modification by the attachment of a polymer, such as a
PEG molecule. It was recognized that in addition to the introduced
non-naturally occurring polymer attachment site, a polymer could
optionally be attached to the N terminal of the protein, as
desired.
[0197] Since the introduced residues were designed to maintain
FGF21 biological activity, these FGF21 mutants are expected to
preserve FGF21 biological activity and yet still provide one or
more non-naturally occurring polymer attachment sites. After
chemical modification (e.g., PEGylation) these mutants are expected
to have a longer half life than wild-type FGF21, while maintaining
substantial in vivo potency.
[0198] The numbers of the positions given in Table 6 correspond to
the residue position in the mature FGF21 protein, which consists of
181 amino acid residues. Nucleic acid sequences encoding the FGF21
mutant polypeptides listed in Table 6 below were prepared using the
techniques described below.
TABLE-US-00008 TABLE 6 FGF21 Mutant Polypeptides Comprising Two
Mutations Residue Residue 1 WT Mutation 2 WT Mutation 37 E C 77 R C
E37C, R77C 120 G C 125 H C G120C, H125C 77 R C 91 E C R77C, E91C 77
R C 125 H C R77C, H125C 91 E C 125 H C E91C, H125C 77 R C 120 G C
R77C, G120C 37 E C 91 E C E37C, E91C 91 E C 175 R C E91C, R175C 37
E C 175 R C E37C, R175C 91 E C 120 G C E91C, G120C 37 E C 120 G C
E37C, G120C 77 R C 175 R C R77C, R175C 37 E C 125 H C E37C, H125C
37 E C 69 K C E37C, K69C 69 K C 91 E C K69C, E91C 120 G C 175 R C
G120C, R175C 69 K C 120 G C K69C, G120C 69 K C 125 H C K69C, H125C
69 K C 77 R C K69C, R77C 125 H C 175 R C H125C, R175C 69 K C 175 R
C K69C, R175C 37 E C 170 G C E37C, G170C
Example 6
Identification of FGF21 Mutants Comprising Three Mutations
[0199] As described above, non-naturally occurring polymer
attachment sites can be introduced into the wild-type FGF21
polypeptide sequence. This affords the opportunity for
site-selective chemical modification at desired locations in the
polypeptide. In addition, it has previously been determined that
residue P171 in the wild-type FGF21 sequence is susceptible to
proteolytic degradation. Accordingly, by introducing combinations
of the above modifications, plus a mutation at the P171 position,
FGF21 molecules having both enhanced proteolytic stability and
site-specific polymer attachment sites can be generated.
[0200] Further analysis of the homology model was performed in
order to identify combinations of mutants that would provide two
non-naturally occurring polymer attachment sites. Following
chemical modification, these mutants would have two polymers (e.g.,
PEG molecules) attached to the polypeptide at desired locations. As
with all the FGF21 mutant polypeptides of the present invention, a
polymer can also be attached to the N-terminus of the polypeptide,
depending on the nature of the polymer itself. The analysis
included the evaluation of a mutation at the P171 position in
addition to the two introduced polymer attachment sites, which was
designed to enhance the proteolytic stability of the FGF21 mutant
polypeptide, and provide selected polymer attachment sites and
enhanced proteolytic stability, while at the same time maintaining
or enhancing the in vivo biological activity of wild-type
FGF21.
[0201] In a parallel study, a select subset of single mutations
from Table 5, presented in Example 4, were combined with the
stability enhancing P171 mutation for the purpose of PEGylating
site selectively at both the .alpha.-amino N-terminus and the
introduced cysteine mutation using site-selective mixed chemistries
as previously described (see U.S. Pat. No. 6,420,339, incorporated
herein by reference). These double mutants were designated
R77C/P171G and H125C/P171G and are exemplary of any other
combinations that might be contemplated using the mutations
disclosed in Table 5 of Example 4.
[0202] A summary of FGF21 mutants comprising two mutations, in
addition to a mutation at the P171 site, that were generated
through this rational protein engineering approach is provided in
Table 7. These mutants incorporate two non-naturally occurring
polymer attachment sites, notably cysteine. The side chains of
these residues are particularly suited to chemical modification by
the attachment of a polymer, such as a PEG molecule. It was
recognized that in addition to the introduced non-naturally
occurring polymer attachment site, a polymer could optionally be
attached to the N-terminus of the protein, as desired. The triple
FGF21 mutants described include not only the introduced polymer
attachment sites described above, but also a proteolytic stability
inducing mutation at position 171. Following chemical modification
of the FGF21 triple mutant, this combination of mutations serves
the function of enhancing FGF21 half life via the association of
two polymers (e.g., PEG molecules) with the polypeptide sequence as
well as enhancing the half life via the elimination of a
proteolytic cleavage site.
[0203] Since the introduced residues were designed to maintain the
in vivo wild-type levels of FGF21 biological activity, these FGF21
mutants are expected to maintain FGF21 biological activity and yet
still provide unique non-naturally occurring polymer attachment
sites. After chemical modification (e.g., PEGylation) these mutants
are expected to have a longer half life than wild-type FGF21, while
maintaining significant in vivo wild-type levels of biological
activity.
[0204] The numbers of the positions given in Table 7 correspond to
the residue position in the mature FGF21 protein, which consists of
181 amino acid residues. Nucleic acid sequences encoding the FGF21
mutant polypeptides listed in Table 7 were prepared using the
techniques described herein.
TABLE-US-00009 TABLE 7 FGF21 Mutant Polypeptides Comprising Three
Mutations Residue Residue Residue 1 WT Mutation 2 WT Mutation 3 WT
Mutation 37 E C 77 R C 171 P G E37C, R77C, P171G 91 E C 125 H C 171
P G E91C, H125C, P171G 77 R C 120 G C 171 P G R77C, G120C, P171G 37
E C 91 E C 171 P G E37C, E91C, P171G 91 E C 175 R C 171 P G E91C,
R175C, P171G 37 E C 175 R C 171 P G E37C, R175C, P171G 91 E C 120 G
C 171 P G E91C, G120C, P171G 37 E C 120 G C 171 P G E37C, G120C,
P171G 77 R C 175 R C 171 P G R77C, R175C, P171G 37 E C 125 H C 171
P G E37C, H125C, P171G
Example 7
Preparation and Expression of FGF21 Mutant Polypeptides
[0205] Constructs encoding the FGF21 mutants listed in Tables 3
(single mutations), 4 (double mutations) and 5 (triple mutations,
i.e., double mutations+P171G), collectively "FGF21 mutants" in this
Example, were prepared by PCR amplification of the wild-type FGF21
expression vector as described below (the construction of the
wild-type FGF21 expression vector is described in Example 1). The
goal of these experiments was to generate FGF21 mutants that
comprise one or more non-naturally occurring polymer attachment
sites, and in some cases are resistant to proteolysis. The
chemically modified forms of these FGF21 mutants would be expected
to exhibit longer half lives, and in some cases also resist
proteolysis.
[0206] FGF21 mutant constructs were prepared using primers having
sequences that are homologous to regions upstream and downstream of
a codon (or codons) to be mutated. The primers used in such
amplification reactions also provided approximately 15 nucleotides
of overlapping sequence to allow for recircularization of the
amplified product, namely the entire vector now having the desired
mutant.
[0207] An exemplary FGF21 mutant construct, encoding an FGF21
mutant having a glutamic acid residue at position 170 instead of
the native glycine residue (i.e., a G170E FGF21 mutant polypeptide)
was prepared using the primers shown in Table 8.
TABLE-US-00010 TABLE 8 PCR Primers for Preparing Exemplary FGF21
Mutant SEQ ID Primer Sequence NO: Sense
5'-ATGGTGGAACCTTCCCAGGGCCGAAGC-3' 29
CTCCTCGGACCCTCTGAGCATGGTGGGACCTTCCCA 30 GGGCCGAAGCCCCA
GAGGAGCCTGGGAGACTCGTACCACCCTGGAAGGGT 31 CCCGGCTTCGGGGT Antisense
5'-GGAAGGTTCCACCATGCTCAGAGGGTCCGA-3' 32
[0208] The primers shown in Table 6 allow for the substitution of
the glycine residue with a glutamic acid residue as shown below,
wherein the upper sequence is the sense primer (SEQ ID NO: 29), the
second and third sequences (SEQ ID NOs: 30 and 31) are portions of
an FGF21 expression construct, and the fourth sequence is the
antisense primer (SEQ ID NO: 32):
TABLE-US-00011 5'-ATGGTGGAACCTTCCCAGGGCCGAAGCCTCCTCGGACCCTCT
GAGCATGGTGGGACCTTCCCAGGGCCGAAGCCCCAGAGGAGCCTG
GGAGACTCGTACCACCCTGGAAGGGTCCCGGCTTCGGGGTAGCCT
GGGAGACTCGTACCACCTTGGAAGG-5'
[0209] FGF21 mutant constructs were prepared using essentially the
PCR conditions described in Example 1. Amplification products were
digested with the restriction endonuclease DpnI, and then
transformed into competent cells. The resulting clones were
sequenced to confirm the absence of polymerase-generated
errors.
[0210] FGF21 mutants were expressed by transforming competent BL21
(DE3) or BL21 Star (Invitrogen; Carlsbad, Calif.) cells with the
construct encoding a particular mutant. Transformants were grown
overnight with limited aeration in TB media supplemented with 40
.mu.g/mL kanamycin, were aerated the next morning, and after a
short recovery period, were induced in 0.4 mM IPTG. FGF21 mutant
polypeptides were harvested by centrifugation 18-20 hours after
induction.
[0211] As a result of the bacterial expression system employed, the
FGF21 mutant polypeptides were expressed with an N-terminal
methionine residue.
Example 8
Purification of FGF21 and FGF21 Mutant Polypeptides from
Bacteria
[0212] In the Examples that follow, FGF21 mutant polypeptides were
expressed in a bacterial expression system. After expression, which
is described in Example 7, FGF21 mutant polypeptides were purified
as described in this Example, unless otherwise indicated.
[0213] To purify the FGF21 mutant polypeptide from bacterial
inclusion bodies, double-washed inclusion bodies (DWIBs) were
solubilized in a solubilization buffer containing guanidine
hydrochloride and DTT in Tris buffer at pH 8.5 and then mixed for
one hour at room temperature, and the solubilization mixture was
added to a refold buffer containing urea, arginine, cysteine, and
cystamine hydrochloride at pH 9.5 and then mixed for 24 hours at
5.degree. C. (see, e.g., Clarke, 1998, Curr. Opin. Biotechnol. 9:
157-63; Mannall et al., 2007, Biotechnol. Bioeng. 97: 1523-34;
Rudolph et al., 1997, "Folding Proteins," in Protein Function: A
Practical Approach (Creighton, ed., New York, IRL Press), pp 57-99;
and Ishibashi et al., 2005, Protein Expr. Purif. 42: 1-6).
[0214] Following solubilization and refolding, the mixture was
filtered through a 0.45 micron filter. The refold pool was then
concentrated approximately 10-fold with a 5 kD molecular weight
cut-off Pall Omega cassette at a transmembrane pressure (TMP) of 20
psi, and dialfiltered with 3 column volumes of 20 mM Tris, pH 8.0
at a TMP of 20 psi.
[0215] The clarified sample was then subjected to anion exchange
(AEX) chromatography using a Q Sepharose HP resin. A linear salt
gradient of 0 to 250 mM NaCl in 20 mM Tris was run at pH 8.0 at
5.degree. C. Peak fractions were analyzed by SDS-PAGE and
pooled.
[0216] The AEX eluate pool was then subjected to hydrophobic
interaction chromatography (HIC) using a Phenyl Sepharose HP resin.
Protein was eluted using a decreasing linear gradient of 0.6 M to 0
M ammonium sulfate with 20 mM TRIS at pH 8.0 and ambient
temperature. Peak fractions were analyzed by SDS-PAGE (Laemmli,
1970, Nature 227: 680-85) and pooled.
[0217] The HIC pool was concentrated with a 5 kD molecular weight
cut-off Pall Omega 0.2 m.sup.2 cassette to 7 mg/mL at a TMP of 20
psi. The concentrate was diafiltered with 5 column volumes of 20 mM
TRIS, pH 8.0 at a TMP of 20 psi, and the recovered concentrate was
diluted to about 5 mg/mL. Finally, the solution was filtered
through a 0.22 .mu.m cellulose acetate filter.
Example 9
Chemical Modification of FGF21 Mutants
[0218] Variants of FGF21 having cysteine substituted at the
selected positions shown in Tables 3-5 were produced as described
in Example 7, and the molecules were then subjected to a PEGylation
reaction with a 20 kDa methoxy-PEG-maleimide. Unless indicated
otherwise, all PEGylated FGF21 wild-type and mutant polypeptides
disclosed herein comprise one or more 20 kDa methoxy PEG maleimide
polymers.
[0219] The partially purified FGF21 molecules were then reduced
using 5 molar equivalents of TCEP for 30 minutes at 25.degree. C.
The reduced FGF21 was then buffer exchanged in to 10 mM imidazole,
pH 7.5 using a GE Healthcare Sephadex G25M column. The buffer
exchanged FGF21 was then reacted with 5 molar equivalents of 20 kDa
methoxy-PEG-maleimide for 30 minutes at 25.degree. C. The resulting
reaction mixture was then subjected to ion-exchange chromatography
to isolate the mono-PEGylated species from multi-PEGylated and
un-PEGylated molecules. Most of the FGF21 mutant polypeptides
reacted well with the methoxy-PEG-maleimide and produced
principally mono-PEGylated products in high yield (FIGS. 2 and
3).
[0220] For production of the Tethered Molecules, partially purified
FGF21 molecules were reduced using 5 molar equivalents of TCEP for
30 minutes at 25.degree. C. The reduced FGF21 was then buffer
exchanged in to 10 mM imidazole, pH 7.5 using a GE Healthcare
Sephadex G25M column. The buffer exchanged FGF21 was then reacted
with 0.45 molar equivalents of 20 kDa PEG bis-maleimide for 60
minutes at 4.degree. C. The resulting reaction mixture was then
subjected to ion-exchange chromatography to isolate the Tethered
Molecule from other undesirable reaction products. Frequently an
additional ion-exchange purification step was required, which was
accomplished by diluting the first ion-exchange pool in about 4
volumes of water and reapplying to the ion-exchange column.
[0221] Alternatively amine specific coupling to the N-terminus was
achieved by reductive alkylation using methoxy-PEG-propionaldehyde
as previously described (see U.S. Pat. No. 5,824,784). Briefly,
mutant FGF21 at about 2 mg/ml was reacted overnight at 4 degrees
C., with a 5-fold molar excess of 20 kD mPEG-propionaldehyde in
acetate buffer pH 5.5 and 10 mM sodium cyanoborohydride. N-terminal
PEGylation of any of the FGF21 wild-type and mutant molecules
described herein can be achieved using this strategy.
[0222] When amine specific dual PEGylation was required at both the
N-terminus and a mutant lysine, methoxyPEG-NHS
(N-hydroxysuccinimidyl ester) was used. Briefly, mutant FGF21 at
about 2 mg/ml was reacted for about 2 hours at 4.degree. C. with a
5-fold molar excess (PEG:amino group) of 20 kD mPEG-NHS in bicine
buffer, pH 7.5.
[0223] When applying a mixed chemistry approach to dual PEGylate
FGF21 mutants at both the N-terminus and a mutant Cys position,
both methoxy-PEG-propionaldehyde and methoxy-PEG-maleimide were
used sequentially as previously described (see U.S. Pat. No.
6,420,339). Briefly, mutant FGF21 at about 2 mg/ml was reacted for
about 2 hours with a 1.5-fold molar excess of 20 kD mPEG-maleimide
in phosphate buffer at pH 6.5 at 4.degree. C., then the pH was
adjusted to about pH 5 and a 5-fold excess of 20 kD
mPEG-propionaldehyde added with 10 mM sodium cyanoborohydride. The
final reaction was allowed to continue at 4.degree. C.
overnight.
[0224] Purification of all the different reaction mixtures was by
anion exchange chromatography in Tris buffer at pH 8 as previously
described.
Example 10
In Vitro Activity of FGF21 Mutant Polypeptides and Chemically
Modified FGF21 Mutant Polypeptides
[0225] PEGylated FGF21 mutant polypeptides were then subjected to
an in vitro assay to assess their activity, as compared to the
un-PEGylated form of the FGF21 mutant polypeptide and N-terminally
PEGylated FGF21.
[0226] One goal of these experiments was to identify FGF21 mutant
polypeptides and chemically-modified FGF21 mutant polypeptides that
preserve FGF21 activity in an ELK-luciferase in vitro assay.
ELK-luciferase assays were performed using a recombinant human 293T
kidney cell system, in which the 293T cells overexpress beta-klotho
and luciferase reporter constructs. Beta-klotho is a co-receptor
that is required by FGF21 for activation of its FGF receptors. The
FGF receptors used in this assay are endogenous levels of FGF
receptors expressed in 293T kidney cell. The luciferase reporter
constructs contain sequences encoding GAL4-ELK1 and a luciferase
reporter driven by a promoter containing five tandem copies of the
Gal4 binding site (5xUAS-Luc). Luciferase activity is regulated by
the level of phosphorylated Erk/ELK1, and is used to indirectly
monitor and quantify FGF21 activity.
[0227] ELK-luciferase assays were performed by culturing the 293T
cells in the presence of different concentrations of wild-type
FGF21 or FGF21 mutant polypeptide for 6 hours, and then assaying
the cell lysates for luciferase activity. FIGS. 4-6 and Tables 3-5
summarize the in vitro results obtained for several exemplary FGF21
mutant polypeptides, including those having one, two or three
mutations.
Example 10A
EC50 Values for PEGylated FGF21 Mutant Polypeptides Comprising One
Mutation
[0228] Table 9 below summarizes the EC50 values of various FGF21
mutant polypeptides comprising two mutations, which introduce one
non-naturally occurring polymer attachment sites at a specific,
known location.
[0229] Various FGF21 mutant polypeptides were generated and the
activity determined in the in vitro ELK-luciferase assay described
herein. Table 9 summarizes the data obtained:
TABLE-US-00012 TABLE 9 Summary of EC50 for PEGylated FGF21 Mutant
Polypeptides Comprising One Mutation Mutation EC50 (nM) WT EC50
(nM) N-PEG20 EC50 (nM) H125C 4.5 2.8 36.7 R77C 4.9 3.9 43.3 K69C
5.3 2.8 36.7 G120C 6.0 2.8 36.7 E37C 6.9 3.9 43.3 R175C 7.9 0.9
21.5 E91C 8.3 3.9 43.3 N121C 9.2 3.9 43.3 R126C 10.0 2.8 36.7 G113C
12.0 3.9 43.3 D38C 13.1 2.8 36.7 D79C 20.4 3.4 40.0 D46C 27.8 3.9
43.3 Y179C 287.1 1.1 21.5
[0230] Table 9 summarizes the effect of PEGylation on the in vitro
activity of some of the various FGF21 mutant polypeptides of the
present invention.
[0231] FIGS. 4, 5 and 6 graphically depict the results and EC50
values for several PEGylated FGF21 mutants comprising a single
point mutation. FIGS. 4 and 5 contain data on several of the FGF21
mutants for which data is presented in Table 9. More particularly,
the upper plot of FIG. 4 shows the results of the ERK-luciferase
assay performed on PEGylated E37C, R77C, E91C mutants and
N-terminally PEGylated wild-type FGF21, as well as un-PEGylated
FGF21. In the lower plot in FIG. 4, data is presented on PEGylated
G113C, N121C, D46C mutants and N-terminally PEGylated wild-type
FGF21, as well as un-PEGylated wild-type FGF21.
[0232] Turning to FIG. 5, in the upper plot data is presented for
H125C, G120C, R126C mutants and N-terminally PEGylated wild-type
FGF21, as well as un-PEGylated wild-type FGF21. In the lower plot,
data is presented for D79C, D38C mutants and N-terminally PEGylated
wild-type FGF21, as well as un-PEGylated wild-type FGF21.
[0233] Continuing with the upper plot of FIG. 6, graphical data is
presented for PEGylated K69C and D79C mutants, as well as
N-terminally PEGylated wild-type FGF21, and for un-PEGylated
wild-type FGF21. In the lower plot of FIG. 6, data is presented for
PEGylated R175C and Y179C mutants and N-terminally PEGylated
wild-type FGF21, as well as un-PEGylated wild-type FGF21.
Surprisingly, many of these molecules have in vitro activity close
to that of the unPEGylated molecule.
Example 10B
EC50 Values for Selected Ar.sub.2/Lvs FGF21 Mutants
[0234] As described in Example 3, a series of FGF21 mutant
polypeptides was generated in which naturally-occurring polymer
attachment sites (e.g., PEGylation sites) were removed by
mutagenesis. In these FGF21 mutants reactive Lys groups were first
mutated to arginine, leaving only the N-terminus .alpha.-amino
group available for PEGylation. Next select arginine residues,
either native or mutant, were converted one by one to lysine,
thereby introducing a secondary PEGylation site at defined
positions on the FGF21 surface. One goal of this strategy was to
generate FGF21 mutant polypeptides in which a polymer (e.g., a PEG
molecule) would be conjugated at one or more specific, known
locations.
[0235] Various arginine/lysine FGF21 mutant polypeptides were
generated and the activity determined in the in vitro
ELK-luciferase assay described herein. Table 10 summarizes the data
obtained:
TABLE-US-00013 TABLE 10 Summary of EC50 for PEGylated FGF21 Mutant
Polypeptides Comprising Lysine/Arginine Mutations In Vitro Activity
Construct Type of PEG EC.sub.50 (nM) Native FGF21 No PEG 3 Native
FGF21 (N-terminus) 1 .times. 20k 43.3 Native FGF21 (random) 2
.times. 20k 200 FGF21(all K to all R) 1 .times. 20k nd FGF21(all K
to all R, R36K) 2 .times. 20k 73 FGF21(all K to all R, R77K) 2
.times. 20k 175 FGF21(K56/59/69R) 2 .times. 20k 500 FGF21(K all R,
R126K) 2 .times. 20k 38 FGF21(K59R/K69R/K122R) 2 .times. 20k 499
FGF21(K56R/K69R/K122R) 2 .times. 20k 215 FGF21(K56R/K59R/K122R) 2
.times. 20k 531 FGF21(all K to all R, R72K) 2 .times. 20k >1000
FGF21(all K to all R, R175K) 2 .times. 20k >1000
Example 100
[0236] EC50 Values for Selected PEGylated FGF21 Mutant Polypeptides
Comprising Two Mutations
[0237] The activity of a number of FGF21 mutant polypeptides
comprising two mutations was also examined in the ERK-luciferase
assay described herein. These mutants were engineered to introduce
two non-naturally occurring polymer attachment sites (in the form
of a cysteine residue) and a mutation providing enhanced
proteolytic stability (P171G) into the wild-type FGF21
sequence.
[0238] Various FGF21 mutant polypeptides were generated and the
activity determined in the in vitro ELK-luciferase assay described
herein. EC50 values from those experiments are shown below in Table
11.
TABLE-US-00014 TABLE 11 EC50 Values for Selected Chemically
Modified FGF21 Mutant Polypeptides Comprising Two Mutations EC50
Mutations (nM) Native FGF21 3 N-term PEG20K 37 E37C, R77C 21 G120C,
H125C 23 R77C, E91C 26 R77C, H125C 30 E91, H125C 32 R77C, G120C 34
E37C, E91C 36 E91C, R175C 46 E37C, R175C 50 E91C, G120C 54 E37C,
G120C 55 R77C, R175C 62 E37C, H125C 64 E37C, K69C 67 K69C, E91C 69
G120C, R175C 118 K69C, G120C 119 K69C, H125C 164 K69C, R77C 180
H125C, R175C 200 K69C, R175C 318 E37C, G170C 163
[0239] In parallel, dual-PEGylated FGF21 mutants were prepared
using single cysteine mutants derived from Example 10A, but also
carrying the stability enhancing P171G mutation. These mutants were
site-selectively PEGylated at both the N-terminus and the
engineered cysteine using the mixed PEGylation chemistry as
previously described (see U.S. Pat. No. 6,420,339). Because the
mixed PEGylation chemistry allows the discrimination between the
N-terminus and cysteine conjugation sites, it is also possible to
site-selectively couple different polymers to different sites. In
this case, a series of conjugates were prepared wherein
combinations of 5 kDa, 10 kDa and 20 kDa polymers were coupled to
the N-terminus and a 20 kDa polymer was consistently coupled to the
cysteine mutant. This allowed assessment of the impact of
N-terminal PEGylation on FGF21 in vitro activity. These conjugates
were all tested in the in vitro ELK-luciferase assay described
here. The results are presented in Table 12.
TABLE-US-00015 TABLE 12 EC50 Values for Selected Chemically
Modified FGF21 Mutant Polypeptides Comprising a Cysteine Mutation
and a P171G Mutation EC50 Mutation Type of PEG (nM) R77C, P171G 2
.times. 20k 64 H125C, P171G 2 .times. 20k 152 H125C, P171G 1
.times. 10k, 1 .times. 20k 65 H125C, P171G 1 .times. 5k, 1 .times.
20k 30
Example 10D
EC50 Values for Selected Chemically Modified FGF21 Mutant
Polypeptides Comprising Three Mutations
[0240] The activity of a number of FGF21 mutant polypeptides
comprising three mutations was also examined in the ERK-luciferase
assay described herein. These mutants were engineered to introduce
two non-naturally occurring polymer attachment sites (in the form
of a cysteine residue) and a mutation providing enhanced
proteolytic stability (P171G) into the wild-type FGF21
sequence.
[0241] Various FGF21 mutant polypeptides were generated and the
activity determined in the in vitro ELK-luciferase assay described
herein. Table 13 summarizes the data obtained:
TABLE-US-00016 TABLE 13 EC50 Values for Selected Chemically
Modified FGF21 Mutant Polypeptides Comprising Three Mutations EC50
Mutations Type of PEG (nM) E37C, R77C, P171G 2 .times. 20 kDa 27
E91, H125C, P171G 2 .times. 20 kDa 37 R77C, G120C, P171G 2 .times.
20 kDa 35 E37C, E91C, P171G 2 .times. 20 kDa 30 E91C, R175C, P171G
2 .times. 20 kDa 155 E37C, R175C, P171G 2 .times. 20 kDa 162 E91C,
G120C, P171G 2 .times. 20 kDa 34 E37C, G120C, P171G 2 .times. 20
kDa 35 R77C, R175C, P171G 2 .times. 20 kDa ND E37C, H125C, P171G 2
.times. 20 kDa 37
[0242] Surprisingly, the data indicates that the majority of these
dual-PEGylated FGF21 mutant polypeptides have activity surpassing
the N-terminally mono-PEGylated molecule.
Example 11
EC50 Values for Selected FGF21 Tethered Molecules
[0243] The Tethered Molecules of the present invention comprise two
FGF21 polypeptide sequences tethered together by a linker molecule.
FIG. 7 graphically depicts an example of a Tethered Molecule. As
described herein, it was predicted that these Tethered Molecules
would provide longer half-lives, while still retaining a desirable
level of biological activity.
[0244] Various Tethered Molecules were generated and the activity
determined in the in vitro ELK-luciferase assay described here. The
Tethered Molecules generated comprise FGF21 mutants in which the
mutation introduces a linker attachment site. Several of the FGF21
mutants were double mutants and include the P171G mutation to
enhance proteolytic stability. Conditions and procedures for this
in vitro study were the same as those in Example 10. Table 14
summarizes the data obtained:
TABLE-US-00017 TABLE 14 EC50 Values for Selected FGF21 Tethered
Molecules EC50 Mutations Linker (nM) H125C, P171G 20 kDa PEG 0.21
R77C, P171G 20 kDa PEG 0.25 G120C, P171G 20 kDa PEG 0.51 E37C,
P171G 20 kDa PEG 0.36 R175C, P171G 20 kDa PEG 0.36 E91C, P171G 20
kDa PEG 0.20 G170C 20 kDa PEG 0.47 P171C 20 kDa PEG 0.21
[0245] It can be seem from the data in Table 14 that the Tethered
Molecules possess a surprisingly high in vitro activity equal to or
exceeding that of the unPEGylated molecule which, for reference,
was determined to be 0.63.
Example 12
In Vivo Activity of Chemically-Modified FGF21 Mutant
Polypeptides
[0246] FGF21 possesses a number of biological activities, including
the ability to lower blood glucose, insulin, triglyceride, or
cholesterol levels; reduce body weight; or improve glucose
tolerance, energy expenditure, or insulin sensitivity. Following
the initial in vitro evaluation described in Example 10, PEGylated
FGF21 mutant polypeptides were further analyzed for in vivo FGF21
activity. PEGylated FGF21 polypeptides were introduced into insulin
resistant ob/ob mice, and the ability of a particular PEGylated
FGF21 polypeptide to lower blood glucose was measured. The
procedure for the in vivo work was as follows.
[0247] The PEGylated FGF21 polypeptide to be tested was injected
intraperitoneally into an 8 week old ob/ob mice (Jackson
Laboratory), and blood samples were obtained at various time points
following a single injection, e.g., 0, 6, 24, 72, 120, and 168
hours after injection. Blood glucose levels were measured with a
OneTouch Glucometer (LifeScan, Inc. Milpitas, Calif.), and the
results expressed as a percent change of blood glucose relative to
the baseline level of blood glucose (i.e., at time 0).
[0248] The FGF21 mutants were generated as described herein and
were chemically modified by the addition of two 20 kDa methoxy PEG
maleimide molecules, one at each of the introduced polymer
attachment sites, which were typically cysteine residues. The
mutations were selected so as to provide discrete, known attachment
points for two PEG molecules. PEGylation of the FGF21 mutants was
achieved using the methods described herein.
Example 12a
In Vivo Activity of N-Terminally PEGylated Wild-Type FGF21
[0249] Wild-type FGF21 that was chemically modified by PEGylation
at the N-terminus of the polypeptide was studied in an ob/ob mouse
model. Un-PEGylated wild-type FGF21 was also studied in the same
experiment and PBS was used as a control. A single 20 kDa methoxy
PEG maleimide molecule was used to N-terminally PEGylate wild-type
FGF21. FIG. 8 demonstrates the results of this experiment.
[0250] Both native and N-terminally PEGylated wild-type FGF21
reduced blood glucose levels by 30-40% after a single injection.
However, the blood glucose levels returned to baseline 24 hours
after the injection of native wild-type FGF21. In contrast,
N-terminally PEGylated wild-type FGF21 has sustained blood
glucose-lowering activity for at least 72 hours. The results of
this study indicate PEGylation of wild-type FGF21 prolongs the
pharmacodynamic effects of native molecule.
[0251] Turning to FIG. 9, a dose response study was performed in an
ob/ob mouse model using wild-type FGF21, which was PEGylated at the
N-terminus with a 20, 30 or 40 kDa PEG molecule. FIG. 9
demonstrates that 30 and 40 kDa PEG molecules have greater and
longer glucose lowering efficacy compared with 20 kDa PEG molecule,
suggesting PEG size is positively correlated with in vivo
pharmacodynamic effects.
Example 12.A.1
In Vivo Activity of Selected Chemically Modified FGF21 Mutant
Polypeptides Conjugated at Both the N-Terminus and an Introduced
Polymer Attachment Site
[0252] Several chemically modified mutants of FGF21 that were
site-selectively PEGylated at both the N-terminus and a second
engineered site were studied in a mouse ob/ob model. These
dual-PEGylated FGF21 constructs were all PEGylated at the
N-terminus and a second site comprising either an engineered lysine
as described in Example 4 or an engineered cysteine as described in
Example 6.
[0253] A similar experiment was performed using a different group
of PEGylated FGF21 mutant polypeptides. FIG. 10 comprises two plots
showing the percent change in blood glucose levels of mice injected
with vehicle (PBS), or the PEGylated forms of FGF21 mutants
comprising polymer attachment mutations, namely FGF21 R77C, which
was also N-terminally PEGylated, and FGF21 R126K, which was also
N-terminally PEGylated (upper plot), and N-terminally PEGylated
F77/P171G (lower plot), which were also PEGylated at the introduced
polymer attachment sites. 20 kDa methoxy PEG maleimide molecules
were used. The results of this experiment again confirm that the
dually PEGylated FGF21 mutants demonstrate an enhanced
pharmacodynamics relative to the wild type FGF21 protein with
sustained glucose-lowering for at least 120 hours.
Example 12B
In Vivo Activity of Selected Chemically Modified FGF21 Mutant
Polypeptides Comprising Two or Three Mutations
[0254] A number of chemically modified FGF21 mutant polypeptides
were studied in a mouse ob/ob model. These mutants were chemically
modified by the addition of two 20 kDa methoxy PEG maleimide
molecules, one at each of the introduced cysteine residues, and in
the case of FGF21 mutant polypeptides comprising a single mutation,
at the N terminus of the polypeptide. The mutations were selected
so as to provide discrete known attachment points for one or more
PEG molecules. Some FGF21 mutants also included the P171G mutation
to enhance proteolytic resistance. The PEGylated FGF21 mutant
polypeptides were injected into the mice and the mice bled at
intervals over a 9 day study.
Example 12.B.1
Effect on Glucose Levels
[0255] An experiment was performed using yet another group of
PEGylated FGF21 mutant polypeptides. FIG. 11 is plot showing the
percent change in blood glucose levels of mice injected with
vehicle (PBS), or the dually PEGylated forms of FGF21 mutants
comprising the mutations, namely E91/H125C, E91C/R175C, E37C/G120C,
E37C/H125C, E37C/R175C; unPEGylated Fc-G170E FGF21 was also
studied. 20 kDa methoxy PEG maleimide molecules were used. The
results of this experiment again confirm that the dually PEGylated
FGF21 mutants demonstrate an enhanced pharmacodynamics relative to
the wild type FGF21 protein.
[0256] A similar experiment was performed using yet another group
of PEGylated FGF21 mutant polypeptides. FIG. 12 is a plot showing
the percent change in blood glucose levels of mice injected with
vehicle (PBS), or the dually PEGylated forms of FGF21 mutants
comprising the mutations, namely N-terminally PEGylated R77C which
was also PEGylated at the introduced polymerization site,
N-terminally PEGylated R126K which was also PEGylated at the
introduced polymerization site, E91/G120C, G120C/H125C, and
E37C/R77C; unPEGylated Fc-G170E FGF21 was also studied. 20 kDa
methoxy PEG maleimide molecules were used. The results of this
experiment confirm that the dually PEGylated FGF21 mutants
demonstrate enhanced pharmacodynamics relative to the wild-type
FGF21 protein.
[0257] In another experiment, FIG. 13 shows the effect of the
PEGylated FGF21 mutants on the blood glucose levels of the mice
over the course of a 9 day study. This data demonstrates that the
PEGylated molecules have enhanced in vivo potency compared to that
of the native molecule. In FIG. 13, the results were generated with
PEGylated E37C/R77C, E91C/R175C, E37C/H125C, E37C/R77C/P171G,
E91C/R175C/P171G, and E37C/H125C/P171G FGF21 mutants, which were
dually PEGylated at the introduced polymer attachment sites. The
FGF21 mutants were modified with two 20 kDa methoxy PEG maleimide
molecules in this study, and the vehicle used was 10 mM potassium
phosphate, 5% sorbitol, pH 8.
[0258] As shown in FIG. 13, the three double mutant dually
PEGylated molecules E37C/R77C; E91C/R175C; E37C/H125C reduced blood
glucose levels, and the effects were maintained for 72 hours after
a single injection. Interestingly, introducing P171G mutation
further prolonged the in vivo actions and the maximal
glucose-lowering activities were maintained for additional 2 days
with total duration of action of 120 hours.
[0259] Additionally, the glucose lowering ability of several FGF21
polypeptides comprising three mutations were studied alongside
several Tethered Molecules. The triple mutants used were
E37C/R77C/P171G and E91C/H125C/P171G; the Tethered Molecules
comprised two identical FGF21 mutant polypeptides comprising two
mutations, namely E37C/P171G and R77C/P171G. 20 kDa methoxy PEG
maleimide molecules were used for the dually PEGylated forms of
FGF21, as well as for the linker molecule in the Tethered Molecule.
The results of this experiment are shown in FIG. 14. Compared with
the Tethered FGF21 mutants, the dually PEGylated mutants have
further enhanced pharmacodynamics. The glucose lowering activity of
the dually PEGylated mutants sustained for at least 168 hours as
compared to 120 hours with the Tethered FGF21 mutants.
[0260] FIG. 15 is a plot showing the percent change in blood
glucose as a function of dose in ob/ob mice injected with vehicle
(10 mM TRIS, 150 mM NaCl, pH 8.5) or different doses of a dually
PEGylated E37C/R77C/P171G FGF21 mutant polypeptide over a nine day
period. The results of this experiment demonstrate that the dually
PEGylated triple mutant E37C/R77C/P171G reduced blood glucose
levels in ob/ob mice in a dose-dependent manner. The dose level of
0.3 mg/kg nearly reached maximal glucose-lowering activity and the
effects were maintained for at least 5 days. Further enhanced in
vivo potency and duration of action were observed when the dose
level increased to 1 mg/kg.
Example 12.B.2
Effect on Body Weight
[0261] Type 2 diabetes is often accompanied with increased
adiposity and body weight. A therapeutic molecule would therefore
preferably have the desirable effect of reducing body weight as
well as the desirable effect of lowering blood glucose levels.
Accordingly, many of the PEGylated FGF21 mutants described herein
were evaluated for their effect on mice body weight.
[0262] To study the effect of various FGF21 mutant polypeptides on
body weight, PEGylated wild-type and FGF21 mutant polypeptides were
injected intraperitoneally into 8 week old ob/ob mice (Jackson
Laboratory), and body weight was monitored at various time points
following a single injection, e.g., 0, 24, 72, 120, and 168 hours
after injection.
[0263] FIG. 16 shows the effect of the PEGylated FGF21 mutants on
the weight of the mice over the term of the study. The results were
generated using dually PEGylated E37C/R77C, E91C/R175C, E37C/H125C,
E37C/R77C/P171G, E91C/R175C/P171G, and E37C/H125C/P171G FGF21
mutant polypeptides. The FGF21 mutants were modified with 20 kDa
methoxy PEG maleimide molecules in this study. These FGF21 mutants
were also studied with respect to changes in glucose levels and
those results are shown in FIG. 13. Taken together, this data
demonstrates that the mice receiving the PEGylated FGF21 mutants
gained less weight, relative to the vehicle control and that FGF21
mutant polypeptides comprising the proteolysis resistant mutation
P171G are more efficacious than are their P171 wild-type
counterparts.
[0264] FIG. 17 is plot showing the change in body weight of mice
injected with vehicle, or the dually PEGylated forms of a FGF21
mutant comprising the mutations, namely E37C/R77C/P171G;
E91C/H125C/P171G; R77C/P171G; and Tethered Molecules comprising in
one case two E37C/P171G FGF21 mutant polypeptides and in another
case two R77C/P171G FGF21 mutant polypeptides. 20 kDa methoxy PEG
maleimide molecules were used in the PEGylated forms of FGF21,
while 20 kDa PEG bis-maleimide was used for the Tethered Molecules.
These FGF21 mutants were also studied with respect to changes in
glucose levels and the results are shown in FIG. 14. Taken
together, this data demonstrates that the mice receiving the
PEGylated FGF21 mutants gained less weight, relative to the vehicle
control. It was also observed that the dually PEGylated FGF21
mutant polypeptides were more efficacious in reducing body weight
than the Tethered Molecules studied.
[0265] The effect of a dually PEGylated FGF21 mutant polypeptide on
body weight was also examined in a multi-dose study. FIG. 18 is a
plot showing the change in body weight as a function of dose in
ob/ob mice injected with vehicle or different doses of dually
PEGylated E37C/R77C/P171G over a nine day period. This FGF21 mutant
was also studied with respect to changes in glucose levels and the
results are shown in FIG. 15. Taken together, this data
demonstrates that a dose level of 0.3 mg/kg is sufficient to reduce
body weight gain in ob/ob mice after a single injection.
Example 13
Murine Kidney Vacuole Study
[0266] One consideration for employing PEG and other polymers to
extend the half-life of a therapeutic protein is the possibility
that the PEGylated molecule will form kidney vacuoles. This
property of PEG is well-documented (see, e.g., Bendele, et al 1998,
"Short Communication: Renal Tubular Vacuolation in Animals Treated
with Polyethylene-glycol-conjugated Proteins," Toxicological
Sciences, 42:152-157 and Conover et al., 1996, "Transitional
Vacuole Formation Following a Bolus Infusion of PEG-hemoglobin in
the Rat," Art. Cells, Blood Subs., and Immob. Biotech. 24:599-611)
and can be unpredictable. A PEGylated molecule that induces the
formation of kidney vacuoles typically, but not necessarily,
renders the molecule less desirable as a therapeutic protein. In
some circumstances kidney vacuolization is of less concern and can
be tolerated. Accordingly, a long term kidney vacuole study was
performed in mice.
[0267] FGF21 mutant polypeptides were generated as described
herein. One study was designed and carried out as follows. Multiple
PEGylated FGF21 molecules were administered once daily for 7
consecutive days via subcutaneous injection to female C57BL/6 mice
(3/group) at a dose of 10 mg/kg; a vehicle control (10 mM
KHPO.sub.4, 150 mM NaCl, pH 8) and 2 positive controls were
administered in the same manner. Detailed clinical observations
were performed on all animals prior to each dose administration,
and 1-2 hours post dose on days 1, 3, and 6; cage-side observations
were collected 1-2 hours post dose on all other dosing days. Body
weights were collected prior to each dose administration and at
necropsy. The kidneys and liver were collected from all animals for
histological evaluation. Table 15 summarizes the results of the
study.
TABLE-US-00018 TABLE 15 Summary of Kidney Vacuole Study Molecule
Kidney Liver Vehicle 0.0 0.0 Positive Control 3.7 0.0
FGF21-1X-NT(PEG20K) 2.0 0.0 H126C 1X-PEG20K 2.3 0.0 R78C 1X-PEG20K
2.3 0.0 K70C 1X-PEG20K 2.0 0.0 G121C 1X-PEG20K 2.0 0.0 E38C
1X-PEG20K 2.0 0.0 R176C 1X-PEG20K 3.0 0.0 E92C 1X-PEG20K 2.7 0.0
N122C 1X-PEG20K 2.0 0.0 R127C 1X-PEG20K 3.0 0.0 G114C 1X-PEG20K 2.7
0.0 D80C 1X-PEG20K 2.0 0.0 D47C 1X-PEG20K 3.0 0.0 H112C 1X-PEG20K
2.6 0.0 K56R, K59R, K69R, K122R, 0.0 0.0 R175K 2X-PEG20K K56R,
K59R, K69R, K122R, 0.0 0.0 R77K 2X-PEG20K K56R, K59R, K69R, K122R,
0.0 0.0 R72K 2X-PEG20K
[0268] Table 15 demonstrates the results of a 7-day murine vacuole
study performed using vehicle (10 mM Tris, 150 mM NaCl, pH 8.5),
N-terminally PEGylated form of FGF21, as well as PEGylated forms of
FGF21 mutants comprising the mutations H125C, R77C, K69C, G120C,
E37C, R175, E91C, N121C, R126C, G113C, D79C, D46C and H112C. Either
one or two 20 kDa methoxy PEG maleimide molecules was used, as
indicated in the table. The vacuole indices are a range with 0
being no observed change in vacuoles relative to control and 4
being severe vacuole formation. As is evident from Table 15, all of
the mono-PEGylated FGF21 mutants induced the formation of kidney
vacuoles. However, none of the dual-PEGylated FGF21 mutants induced
vacuole formation. It was also noted that no vacuolization was
observed in the livers of the mice.
[0269] Following the one week study, an 8 week chronic study was
undertaken. In this study, various PEGylated FGF21 molecules were
administered once weekly via subcutaneous injection for 8 weeks to
female C57BL/6 mice (5/group) at doses of either 5 or 25 mg/kg; a
vehicle control (10 mM Tris, 150 mM NaCl, pH 8.5) was administered
in the same manner. All animals were observed for clinical signs
once daily prior to dosing, and 1-2 hours post dose on dosing days;
once daily on non-dosing days. Body weights were collected prior to
each dose administration starting on Day 1 and then on the third
day after each dose, and at necropsy. The kidneys, liver, and
spleen were collected from all animals (moribund sacrifice and
final euthanasia) for histological evaluation.
[0270] FIGS. 19A-19F is a series of plots showing weight change
results observed during the eight week kidney vacuole study
employing a vehicle (squares) and two doses of dual PEGylated,
FGF21 mutants, namely 5 mg/kg (triangles) and 25 mg/kg (open
circles). The FGF21 mutants studied include R77C, P171G (FIG. 19A);
E37C, R77C, P171G (FIG. 19B); E37C, H125C, P171G (FIG. 19C); E91C,
H125C, P171G (FIG. 19D); E37C, P171G (FIG. 19E) and R77C, P171G
(FIG. 19F). In FIG. 19A, a mixed chemistry approach was employed,
leading to N-terminal PEGylation of the FGF21 mutant as well as
PEGylation at the introduced polymer attachment site, the
non-naturally occurring cysteine at 77. In FIGS. 19B-19D, a single
chemistry approach was employed, leading to PEGylation at the
introduced polymer attachment sites, the non-naturally occurring
cysteine residues (cysteines at positions 37 and 77 in FIG. 19B,
positions 37 and 125 in FIG. 19C, and positions 91 and 125 in FIG.
19D). Finally, in FIGS. 19D and 19E, a single bis maleimide PEG was
used to join two FGF21 mutants together at the non-naturally
occurring cysteine residues (cysteine at position 37 in FIG. 21E
and position 77 in FIG. 19F).
[0271] FIG. 20 comprises two bar graphs showing the average score
of kidney vacuoles observed during the eight-week kidney vacuole
study for two doses, 5 and 25 mg/kg, of six singly or dually
PEGylated FGF21 mutants. A score of 0 indicates no more vacuoles
were observed than found in the control animals, while a score of 4
indicates severe vacuole formation relative to the control. The
FGF21 mutants were the same as those described in FIG. 19A-19E.
Thus, for one construct a mixed chemistry approach was employed,
leading to N-terminal PEGylation of the FGF21 mutant as well as
PEGylation at the introduced polymer attachment site, the
non-naturally occurring cysteine at 77. A single chemistry approach
was employed for three other constructs, leading to PEGylation at
the introduced polymer attachment sites, the non-naturally
occurring cysteine residues (cysteines at positions 37 and 77 in
one construct, positions 37 and 125 in a second construct or
positions 91 and 125 in a third construct). Finally, a single bis
maleimide PEG was used to join two FGF21 mutants together at the
non-naturally occurring cysteine residues (cysteine at position 37
or position 77). The P171G mutation was introduced into each of the
six constructs.
[0272] While the present invention has been described in terms of
various embodiments, it is understood that variations and
modifications will occur to those skilled in the art. Therefore, it
is intended that the appended claims cover all such equivalent
variations that come within the scope of the invention as claimed.
In addition, the section headings used herein are for
organizational purposes only and are not to be construed as
limiting the subject matter described.
[0273] All references cited in this disclosure are expressly
incorporated by reference herein.
Sequence CWU 1
1
341630DNAHomo sapiens 1atggactcgg acgagaccgg gttcgagcac tcaggactgt
gggtttctgt gctggctggt 60cttctgctgg gagcctgcca ggcacacccc atccctgact
ccagtcctct cctgcaattc 120gggggccaag tccggcagcg gtacctctac
acagatgatg cccagcagac agaagcccac 180ctggagatca gggaggatgg
gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240ctgcagctga
aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg
300ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga
ccctgaggcc 360tgcagcttcc gggagctgct tcttgaggac ggatacaatg
tttaccagtc cgaagcccac 420ggcctcccgc tgcacctgcc agggaacaag
tccccacacc gggaccctgc accccgagga 480ccagctcgct tcctgccact
accaggcctg ccccccgcac ccccggagcc acccggaatc 540ctggcccccc
agccccccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc
600cagggccgaa gccccagcta cgcttcctga 6302209PRTHomo sapiens 2Met Asp
Ser Asp Glu Thr Gly Phe Glu His Ser Gly Leu Trp Val Ser 1 5 10 15
Val Leu Ala Gly Leu Leu Leu Gly Ala Cys Gln Ala His Pro Ile Pro 20
25 30 Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln Arg
Tyr 35 40 45 Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His Leu
Glu Ile Arg 50 55 60 Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln
Ser Pro Glu Ser Leu 65 70 75 80 Leu Gln Leu Lys Ala Leu Lys Pro Gly
Val Ile Gln Ile Leu Gly Val 85 90 95 Lys Thr Ser Arg Phe Leu Cys
Gln Arg Pro Asp Gly Ala Leu Tyr Gly 100 105 110 Ser Leu His Phe Asp
Pro Glu Ala Cys Ser Phe Arg Glu Leu Leu Leu 115 120 125 Glu Asp Gly
Tyr Asn Val Tyr Gln Ser Glu Ala His Gly Leu Pro Leu 130 135 140 His
Leu Pro Gly Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly 145 150
155 160 Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro Ala Pro Pro
Glu 165 170 175 Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val Gly
Ser Ser Asp 180 185 190 Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg
Ser Pro Ser Tyr Ala 195 200 205 Ser 3546DNAHomo sapiens 3caccccatcc
ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60ctctacacag
atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg
120gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc
cttgaagccg 180ggagttattc aaatcttggg agtcaagaca tccaggttcc
tgtgccagcg gccagatggg 240gccctgtatg gatcgctcca ctttgaccct
gaggcctgca gcttccggga gctgcttctt 300gaggacggat acaatgttta
ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360aacaagtccc
cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca
420ggcctgcccc ccgcaccccc ggagccaccc ggaatcctgg ccccccagcc
ccccgatgtg 480ggctcctcgg accctctgag catggtggga ccttcccagg
gccgaagccc cagctacgct 540tcctga 5464181PRTHomo sapiens 4His Pro Ile
Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val 1 5 10 15 Arg
Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His 20 25
30 Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser
35 40 45 Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val
Ile Gln 50 55 60 Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln
Arg Pro Asp Gly 65 70 75 80 Ala Leu Tyr Gly Ser Leu His Phe Asp Pro
Glu Ala Cys Ser Phe Arg 85 90 95 Glu Leu Leu Leu Glu Asp Gly Tyr
Asn Val Tyr Gln Ser Glu Ala His 100 105 110 Gly Leu Pro Leu His Leu
Pro Gly Asn Lys Ser Pro His Arg Asp Pro 115 120 125 Ala Pro Arg Gly
Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro 130 135 140 Ala Pro
Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 145 150 155
160 Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser
165 170 175 Pro Ser Tyr Ala Ser 180 56PRTARTIFICIAL SEQUENCELinker
Sequence 5Xaa Xaa Asn Xaa Xaa Gly 1 5 64PRTARTIFICIAL
SEQUENCELinker Sequence 6Gly Gly Gly Gly 1 75PRTARTIFICIAL
SEQUENCELinker Sequence 7Gly Gly Gly Gly Gly 1 5 85PRTARTIFICIAL
SEQUENCELinker Sequence 8Gly Gly Gly Gly Ser 1 5 910PRTARTIFICIAL
SEQUENCELinker Sequence 9Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1
5 10 1025PRTARTIFICIAL SEQUENCELinker Sequence 10Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly
Gly Ser Gly Gly Gly Gly Ser 20 25 116PRTARTIFICIAL SEQUENCELinker
Sequence 11Gly Gly Glu Gly Gly Gly 1 5 128PRTARTIFICIAL
SEQUENCELinker Sequence 12Gly Gly Glu Glu Glu Gly Gly Gly 1 5
135PRTARTIFICIAL SEQUENCELinker Sequence 13Gly Glu Glu Glu Gly 1 5
144PRTARTIFICIAL SEQUENCELinker Sequence 14Gly Glu Glu Glu 1
156PRTARTIFICIAL SEQUENCELinker Sequence 15Gly Gly Asp Gly Gly Gly
1 5 167PRTARTIFICIAL SEQUENCELinker Sequence 16Gly Gly Asp Asp Asp
Gly Gly 1 5 175PRTARTIFICIAL SEQUENCELinker Sequence 17Gly Asp Asp
Asp Gly 1 5 184PRTARTIFICIAL SEQUENCELinker Sequence 18Gly Asp Asp
Asp 1 1921PRTARTIFICIAL SEQUENCELinker Sequence 19Gly Gly Gly Gly
Ser Asp Asp Ser Asp Glu Gly Ser Asp Gly Glu Asp 1 5 10 15 Gly Gly
Gly Gly Ser 20 205PRTARTIFICIAL SEQUENCELinker Sequence 20Trp Glu
Trp Glu Trp 1 5 215PRTARTIFICIAL SEQUENCELinker Sequence 21Phe Glu
Phe Glu Phe 1 5 226PRTARTIFICIAL SEQUENCELinker Sequence 22Glu Glu
Glu Trp Trp Trp 1 5 236PRTARTIFICIAL SEQUENCELinker Sequence 23Glu
Glu Glu Phe Phe Phe 1 5 247PRTARTIFICIAL SEQUENCELinker Sequence
24Trp Trp Glu Glu Glu Trp Trp 1 5 257PRTARTIFICIAL SEQUENCELinker
Sequence 25Phe Phe Glu Glu Glu Phe Phe 1 5 266PRTARTIFICIAL
SEQUENCELinker Sequence 26Xaa Xaa Tyr Xaa Xaa Gly 1 5
276PRTARTIFICIAL SEQUENCELinker Sequence 27Xaa Xaa Ser Xaa Xaa Gly
1 5 286PRTARTIFICIAL SEQUENCELinker Sequence 28Xaa Xaa Thr Xaa Xaa
Gly 1 5 2927DNAARTIFICIAL SEQUENCEPrimer Sequence 29atggtggaac
cttcccaggg ccgaagc 273050DNAARTIFICIAL SEQUENCEPrimer Sequence
30ctcctcggac cctctgagca tggtgggacc ttcccagggc cgaagcccca
503150DNAARTIFICIAL SEQUENCEPrimer Sequence 31gaggagcctg ggagactcgt
accaccctgg aagggtcccg gcttcggggt 503230DNAARTIFICIAL SEQUENCEPrimer
Sequence 32ggaaggttcc accatgctca gagggtccga 303340DNAARTIFICIAL
SEQUENCEPrimer Sequence 33aggaggaata acatatgcat ccaattccag
attcttctcc 403433DNAARTIFICIAL SEQUENCEPrimer Sequence 34tagtgagctc
gaattcttag gaagcgtagc tgg 33
* * * * *