U.S. patent application number 14/358857 was filed with the patent office on 2014-10-23 for variant serum albumin with improved half-life and other properties.
This patent application is currently assigned to ELEVEN BIOTHERAPEUTICS, INC.. The applicant listed for this patent is ELEVEN BIOTHERAPEUTICS, INC.. Invention is credited to Amy Jada Andreucci, Thomas M. Barnes, Eric Steven Furfine, Michael March Schmidt.
Application Number | 20140315817 14/358857 |
Document ID | / |
Family ID | 47295197 |
Filed Date | 2014-10-23 |
United States Patent
Application |
20140315817 |
Kind Code |
A1 |
Schmidt; Michael March ; et
al. |
October 23, 2014 |
VARIANT SERUM ALBUMIN WITH IMPROVED HALF-LIFE AND OTHER
PROPERTIES
Abstract
The invention provides methods and materials for making and
using variant serum albumin amino acid sequences which exhibit
improved properties compared to wild type serum albumin sequences.
The invention further provides methods and materials for making and
using fusion proteins in which the variant serum albumin amino acid
sequences are fused to a therapeutic or diagnostic agent, such as a
therapeutic protein, or a functional fragment or variant thereof
that maintains activity, and exhibits improved properties.
Inventors: |
Schmidt; Michael March;
(Boston, MA) ; Furfine; Eric Steven; (Concord,
MA) ; Andreucci; Amy Jada; (Woburn, MA) ;
Barnes; Thomas M.; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ELEVEN BIOTHERAPEUTICS, INC. |
Cambridge |
MA |
US |
|
|
Assignee: |
ELEVEN BIOTHERAPEUTICS,
INC.
Cambridge
MA
|
Family ID: |
47295197 |
Appl. No.: |
14/358857 |
Filed: |
November 18, 2012 |
PCT Filed: |
November 18, 2012 |
PCT NO: |
PCT/US2012/065733 |
371 Date: |
May 16, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61710476 |
Oct 5, 2012 |
|
|
|
61576491 |
Dec 16, 2011 |
|
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61561785 |
Nov 18, 2011 |
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Current U.S.
Class: |
514/15.2 ;
435/188; 530/351; 530/363 |
Current CPC
Class: |
C07K 16/18 20130101;
A61K 38/385 20130101; C12N 9/6437 20130101; C07K 2319/31 20130101;
C07K 14/755 20130101; C07K 14/55 20130101; C07K 14/765
20130101 |
Class at
Publication: |
514/15.2 ;
530/363; 530/351; 435/188 |
International
Class: |
C07K 14/765 20060101
C07K014/765; C12N 9/64 20060101 C12N009/64; A61K 38/38 20060101
A61K038/38; C07K 16/18 20060101 C07K016/18; C07K 14/55 20060101
C07K014/55; C07K 14/755 20060101 C07K014/755 |
Claims
1. An isolated, recombinant protein comprising a variant serum
albumin polypeptide (VSA) sequence that is a variant of domain III
of a naturally-occurring serum albumin sequence, wherein the
variant comprises a mutation at one or more of the positions
corresponding to V418, T420, V424, E505 and V547.
2. The protein of claim 1 wherein the VSA or the recombinant
protein binds to FcRn at a pH in the range of 5.5 to 6.0, e.g., at
a pH of 5.5 or 6.0, with higher affinity than a corresponding
native serum albumin.
3. The protein of claim 1 or 2, wherein the ratio of the binding
affinity of a serum albumin comprising the VSA sequence at a pH of
5.5 to 6.0 to that at a pH of 7.0 to 7.4 is greater than or equal
to the ratio for a corresponding native human albumin.
4. The protein of any one of claims 1-3, wherein the ratio of the
binding affinity of a serum albumin comprising the VSA sequence at
a pH of 5.5 to 6.0 to that at a pH of 7.0 to 7.4 is 5; 10; 50; 100;
1000; 10,000; 100,000; or 1 million times that of a corresponding
native human albumin.
5. The protein of claim any one of claims 1-4, wherein a serum
albumin comprising the VSA sequence binds to FcRn at a pH in the
range of 7.0 to 7.4 with an affinity not greater than a
corresponding native human albumin.
6. The protein of claim any one of claims 1-5 wherein the VSA
sequence comprises a substitution of V418 with a methionine.
7. The protein of any one of claims 1-6, wherein the VSA sequence
comprises a substitution of T420 with an uncharged amino acid.
8. The protein of claim 7 wherein the VSA sequence comprises a
substitution of T420 with alanine.
9. The protein of any one of claims 1-8, wherein the VSA sequence
comprises a substitution of V424 with an uncharged amino acid.
10. The protein of claim 9, wherein the VSA sequence comprises a
substitution of V424 with isoleucine.
11. The protein of any one of claims 1-10, wherein the VSA sequence
comprises a substitution of E505 with an uncharged amino acid or
positively charged amino acid.
12. The protein of claim 11, wherein the VSA sequence comprises a
substitution of E505 with arginine, lysine or glycine.
13. The protein of any one of claims 1-12, wherein the VSA sequence
comprises a substitution of V547 with an uncharged amino acid.
14. The protein of claim 13, wherein the VSA sequence comprises a
substitution of V547 with alanine.
15. The protein of any one of claims 1-14, wherein the VSA sequence
comprises mutations at two or more of the positions selected from
V418, T420, E505, and V547.
16. The protein of claim 15, wherein the VSA sequence comprises two
or more mutations selected from V418M, T420A, E505(R/K/G) and
V547A.
17. The protein of claim 15, wherein the VSA sequence comprises
mutations at three or more positions selected from V418M, T420A,
E505(R/K/G) and V547A.
18. The protein of claim 15, wherein the VSA sequence comprises
mutations V418M, T420A, E505(R/K/G) and V547A.
19. The protein of any one of claims 1-18, wherein the VSA sequence
comprises at least one mutation selected from V424I, N429D, M446V;
A449V; T467M and A552T.
20. The protein of any one of claims 1-19, wherein the VSA sequence
is at least 80% identical but less than 100% identical to domain
III of a naturally-occurring serum albumin.
21. The protein of claim 20 wherein the VSA sequence is at least
80% identical but less than 100% identical to the corresponding
sequence of human serum albumin.
22. The protein of any one of claims 1-21, wherein the protein
comprises a heterologous sequence.
23. The protein of claim 22, wherein the protein comprises a first
and a second heterologous sequence.
24. The protein of claim 24 wherein the first and the second
heterologous sequence are identical and are positioned in
tandem.
25. The protein of claim 23 wherein the first heterologous sequence
is located N-terminal to the variant sequence and the second
heterologous sequence is located C-terminal to the variant
sequence.
26. The protein of claim 22 wherein the heterologous sequence
comprises a cytokine domain.
27. The protein of claim 26 wherein the cytokine is
interleukin-2.
28. The protein of claim 22, wherein the heterologous sequence
comprises an immunoglobulin variable domain.
29. The protein of claim 22, wherein the heterologous sequence
comprises an Adnectin.TM., a DARPin, or an anti-calin, or a
fragment of an Adnectin.TM., a DARPin, or an anti-calin,
30. The protein of claim 22, wherein the heterologous sequence
comprises a soluble fragment of a cell surface receptor.
31. The protein of claim 22, wherein the heterologous sequence
comprises an enzyme.
32. The protein of claim 22, wherein the heterologous sequence
comprises a functional fragment of a coagulation protein.
33. The protein of claim 32, wherein the heterologous sequence
comprises a functional fragment of FVII.
34. The protein of claim 32, wherein the heterologous sequence
comprises a functional fragment of FVIII.
35. An isolated, recombinant protein comprising a VSA that has
altered binding properties for human FcRn relative to a wild type
human serum albumin and binds to FcRn with a K.sub.D of less than
50 nM at pH 5.5 and optionally an affinity for FcRn at pH 7.4 that
is less than or equal to the affinity for FcRn of a wild type
albumin at pH 7.4.
36. A method of treating a subject, the method comprising
administering to the subject an effective amount of a therapeutic
agent in association with the protein of any one of claim 1-21 or
35, such that the dosage and/or frequency of administration at
which the agent produces a therapeutic effect is reduced relative
to the dosage and/or frequency of administration at which the agent
produces a therapeutic effect when it is not in association with
the albumin protein.
37. The method of claim 36, wherein the agent comprises a
polypeptide component that is fused to the albumin protein.
38. The method of claim 37, wherein the polypeptide component and
the albumin protein are separated by a linker sequence.
39. The method of claim 37, wherein the polypeptide component and
the albumin protein are covalently linked by a non-peptide
bond.
40. The method of claim 37, wherein the polypeptide component and
the albumin protein are non-covalently and stably associated.
41. A method of engineering a VSA associated therapeutic agent, the
method comprising: providing a biologically or pharmaceutically
active agent; and associating the agent with the protein of any one
of claim 1-21 or 35 to provide a VSA associated therapeutic
agent.
42. The method of claim 41, further comprising formulating the VSA
associated therapeutic agent for administration to a subject.
43. A method of engineering a VSA associated diagnostic agent, the
method comprising: providing a diagnostic agent; and associating
the agent with the protein of any one of claim 1-21 or 35 to
provide a VSA associated diagnostic agent.
44. The method of claim 43 further comprising formulating the VSA
associated diagnostic agent for administration to a subject.
45. The method of claim 44 further comprising administering the VSA
associated diagnostic agent to a subject and detecting the VSA
associated diagnostic agent.
46. The method of claim 45, wherein the subject is imaged.
47. A method of treating a subject with a VSA fusion protein, the
fusion protein comprising a therapeutic agent linked to a VSA,
wherein the VSA comprises a sequence that is a variant of domain
III of a naturally-occurring serum albumin that comprises a
mutation at one or more of the positions corresponding to V418,
T420, V424, E505 and V547; the method comprising administering to
the subject a therapeutically effective amount of the VSA fusion
protein, such that the dosage and/or frequency of administration at
which the agent produces a therapeutic effect is reduced relative
to the dosage and/or frequency of administration at which the agent
produces a therapeutic effect when it is not in association with
the VSA.
48. The method of claim 47, wherein the therapeutic agent comprises
a sequence encoding human IL-2 or an active variant of IL-2.
49. The method of claim 48, wherein the subject suffers from, or is
at risk of suffering from, an immune disorder.
50. The method of claim 49, wherein the subject has undergone, or
plans to undergo, a procedure selected from the group consisting of
an organ transplant, or blood transfusion, or bone marrow
transplantation.
51. The method of claim 47, wherein the therapeutic agent comprises
a sequence encoding a urate oxidase, or an active variant of a
urate oxidase.
52. The method of claim 51, wherein the subject suffers from gout.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Application
Ser. No. 61/561,785, filed Nov. 18, 2011; U.S. Application Ser. No.
61/576,491, filed Dec. 16, 2011; and U.S. Application Ser. No.
61/710,476, filed Oct. 5, 2012. The entire content of each of the
foregoing applications is hereby incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 16, 2012, is named D2046703.txt and is 43,129 bytes in
size.
FIELD OF THE INVENTION
[0003] The present invention relates to the area of therapeutic and
diagnostic proteins, materials for producing such proteins, and
methods for their production and use.
BACKGROUND
[0004] Serum albumin is an abundant plasma protein that that is
essential for maintaining oncotic pressure and is useful in
regulating the volume of circulating blood. Serum albumin has
therapeutic uses and is indicated, for example, to treat
hypovolemia and hypoalbuminemia (e.g., hypoalbuminemia associated
with inadequate production, excessive catabolism, hemorrhage,
excessive renal excretion, redistribution within the body e.g., due
to surgery or inflammatory conditions, burns, adult respiratory
distress syndrome, nephrosis), and may be used prior to or during
cardiopulmonary bypass surgery or to treat hemolytic disease of the
newborn. Serum albumin can also be used in association with other
agents.
SUMMARY
[0005] There is a need for improved variant sequences of serum
albumin. Such variant sequences can be used, e.g., to prepare or
provide a protein product (e.g., a therapeutic or diagnostic
protein product). In embodiments the protein product has improved
properties (e.g., higher recycling fraction via an FcRn mechanism
and/or improved pharmacokinetic properties, e.g., increased
half-life and/or reduced clearance), e.g., as compared with a
protein product that is prepared using a corresponding native
(e.g., wild type) serum albumin sequence (e.g., a mammalian serum
albumin, e.g., human serum albumin or bovine serum albumin). As
used herein, a "corresponding native serum albumin" (e.g., a
naturally occurring serum albumin or a wild type serum albumin)
refers to a corresponding full length native serum albumin
sequence, or fragment thereof, of which the variant serum albumin
polypeptide (VSA) is a variant. The disclosure provided herein
includes disclosure of proteins that comprise variant serum albumin
sequences and methods of using such proteins.
[0006] In some aspects, the disclosure provides a variant serum
albumin polypeptide (VSA). In some embodiments, the variant is a
variant of a full-length serum albumin polypeptide sequence (e.g.,
a human serum albumin polypeptide sequence, or a serum albumin
polypeptide sequence from another species, e.g., a bovine serum
albumin polypeptide sequence). In other embodiments, the variant is
a variant of a fragment of a serum albumin polypeptide sequence
(e.g., a fragment of the human serum albumin polypeptide sequence,
or a fragment of a serum albumin polypeptide sequence from another
species, e.g., a fragment of the bovine serum albumin polypeptide
sequence). In some embodiments, the fragment comprises a domain III
of a serum albumin polypeptide sequence (e.g., domain III of the
human serum albumin polypeptide sequence).
[0007] In some aspects, the present disclosure provides an
isolated, recombinant protein that comprises a VSA, e.g., a VSA
that is or comprises a variant of domain III of a
naturally-occurring serum albumin. In some embodiments, the VSA is
in a polypeptide that also includes a heterologous sequence, e.g.,
a heterologous sequence as described herein. For example, a fusion
polypeptide can comprise a VSA and a heterologous sequence.
[0008] In some embodiments, the VSA has or comprises a mutation at
one or more of the positions corresponding to V418, T420, V424,
E505 and V547 of serum albumin.
[0009] In some embodiments, the VSA can bind to an FcRn (e.g.,
human FcRn) at a pH in the range of 5.5 to 6.0 (e.g., at a pH of
5.5, 5.6, 5.7, 5.8, 5.9, or 6.0). In some embodiments, the VSA
binds to an FcRn (e.g., human FcRn) with higher affinity than does
a corresponding native serum albumin. In some embodiments, the
higher affinity binding occurs at a specified pH (e.g., at a pH of
5.5, 5.6, 5.7, 5.8, 5.9, 6.0) or in a specified pH range, e.g., at
a pH in the range of 5.5-6.0 or 6.0-6.5.
[0010] In some embodiments, the ratio of the binding affinity of
the VSA, or of the recombinant protein comprising the VSA, at a pH
of 5.5 to 6.0 to the binding affinity at a pH of 7.0 to 7.4 is
greater than or equal to the ratio for a corresponding native serum
albumin (e.g., a corresponding native human serum albumin). In some
embodiments, the ratio of the binding affinity of the VSA, or of
the recombinant protein comprising the VSA, at a pH of 5.5 to 6.0
to that at a pH of 7.0 to 7.4 is 5; 10; 50; 100; 1000; 10,000;
100,000; or 1 million times that of a corresponding native human
albumin.
[0011] In some embodiments, the VSA, or the recombinant protein
comprising the VSA, binds to FcRn at a pH in the range of 7.0 to
7.4 with an affinity not greater than a corresponding native human
albumin.
[0012] In some embodiments, the disclosure provides an isolated,
recombinant protein comprising a VSA that has altered binding
properties for an FcRn (e.g., a human FcRn) relative to a
corresponding native serum albumin sequence (e.g., the wild-type
human serum albumin sequence). The protein can include a
heterologous sequence as described herein. In some embodiments, the
recombinant protein comprising aVSA binds to FcRn with an affinity
that is altered compared to a recombinant protein comprising a wild
type albumin (e.g., a wild type human albumin) and the heterologous
sequence. In some embodiments, the VSA or recombinant protein
comprising the VSA binds to FcRn with a dissociation constant
(K.sub.D) of less than 50 nM at pH 5.5. In some embodiments, the
VSA or recombinant protein has an affinity for FcRn at pH 7.4 that
is less than or equal to its affinity for FcRn of a wild type
albumin at pH 7.4.
[0013] In some embodiments, the VSA has a K.sub.D that is below 100
nM. In some embodiments, the VSA has a K.sub.D that is below 75 nM,
below 50 nM, below 40 nM, below 30 nM, below 25 nM, below 20 nM,
below 15 nM, below 10 nM, below 9 nM, below 8 nM, below 7 nM, below
6 nM, below 5 nM, or below 4 nM. In some embodiments, the protein
has a K.sub.D that is 3 nM or less.
[0014] In certain embodiments, the VSA comprises a substitution of
V418 with another amino acid, for example, with a methionine. In
certain embodiments, the VSA comprises a substitution of T420 with
another amino acid, for example, an uncharged amino acid, such as
alanine. In certain embodiments, the VSA comprises a substitution
of V424 with another amino acid, for example, an uncharged amino
acid, such as isoleucine. In certain embodiments, the VSA comprises
a substitution of E505 with another amino acid, for example, an
uncharged amino acid, such as glycine, or a positively charged
amino acid, such as lysine or arginine. In certain embodiments, the
VSA comprises a substitution of V547 with another amino acid, for
example, an uncharged amino acid, such as alanine.
[0015] In some embodiments, the VSA comprises substitutions at two
or more (e.g., at two, three, four, or five) of the positions
corresponding to V418, T420, V424, E505 and V547. For example, the
VSA can comprise two, three, four, or five substitutions selected
from V418M, T420A, V424I, E505(R/K/G) and V547A. In a particular
embodiment, the VSA comprises the substitutions V418M, T420A and
E505R. In another particular embodiment, the VSA comprises the
substitutions V418M, T420A, E505G and V547A.
[0016] In some embodiments, the VSA comprises one or more
additional substitutions at positions selected from N429, M446,
A449, T467, and A552. In some embodiments, the substitutions are
selected from N429D, M446V, A449V, T467M, and A552T.
[0017] In some embodiments, a protein provided herein includes a
VSA that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99%, but less than 100% identical to domain
III of a naturally occurring serum albumin, e.g., a human serum
albumin. In certain embodiments, the protein includes a VSA that is
at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99%, but less than 100% identical to domain III of
human serum albumin.
[0018] In some embodiments, a protein provided herein includes a
VSA that differs by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or
20 residues from a corresponding native serum albumin, e.g., a
corresponding human serum albumin sequence. In some embodiments, a
protein provided herein includes a VSA that differs by at least 1,
but no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues
from a corresponding native serum albumin e.g., a corresponding
human serum albumin sequence. In embodiments the differences are in
domain III, e.g., in domain III of human serum albumin.
[0019] In some aspects, the disclosure provides an agent in
association with a VSA, e.g., a VSA as described herein. In some
embodiments, the agent is a therapeutic or diagnostic agent, e.g.,
an agent as described herein. In some embodiments, the agent is
fused to the VSA or to a protein that comprises the VSA, e.g., in
the form of a fusion protein. For example, a VSA can be fused to a
heterologous sequence using recombinant genetic techniques. A
heterologous sequence can comprise, e.g., the agent, a protein that
comprises the agent, or a sequence to which the agent is attached
or can be attached.
[0020] In some aspects, the disclosure provides an isolated,
recombinant protein that comprises a VSA as described herein and a
heterologous sequence, e.g., a heterologous sequence as described
herein (e.g., the heterologous sequence can be a cytokine,
immunoglobulin, cell surface receptor, coagulation protein, or
functional fragment of any of these). The protein can also include
more than one heterologous sequence.
[0021] In some embodiments, the VSA is a variant of domain III of a
naturally-occurring serum albumin. In some embodiments the
recombinant protein is a fusion protein.
[0022] In some embodiments, the heterologous sequence comprises a
cytokine domain, for example, an amino acid sequence originating or
derived from an interleukin-2. In some embodiments, the
heterologous sequence comprises an immunoglobulin single-chain
variable domain, e.g., an scFv. In some embodiments, the
heterologous domain is a scaffold, e.g., an anti-calin, a DARPin, a
Surrobody.TM. Adnectin.TM., a Domain antibody, Affibody, or
fragment of any of the foregoing. In some embodiments, the
heterologous sequence comprises a soluble fragment of a cell
surface receptor. In some embodiments, the heterologous sequence
comprises an enzyme. In still other embodiments, the heterologous
sequence comprises a component of a coagulation protein, such as an
amino acid sequence originating or derived from coagulation factor
VII (FVII) or coagulation factor VIII (FVIII). In some embodiments,
the heterologous sequence comprises two or more of the
foregoing.
[0023] In certain aspects, the protein comprises a first and a
second heterologous sequence. In some embodiments, the first and
the second heterologous sequence are identical. In some
embodiments, the first and second heterologous sequence are in
tandem. In other embodiments, the first heterologous sequence is
located N-terminal to the variant sequence and the second
heterologous sequence is located C-terminal to the variant
sequence.
[0024] In another aspect, the present disclosure provides an
isolated recombinant DNA molecule and/or nucleotide sequence that
encodes a polypeptide, peptide, protein or recombinant protein
disclosed herein. The recombinant DNA molecule and/or nucleotide
sequence comprises a variant nucleotide sequence that encodes a
VSA. In some embodiments, the VSA includes at least a variant of
domain III of a naturally-occurring serum albumin.
[0025] In other aspects, the present disclosure provides a
recombinant host cell (e.g., a recombinant CHO cell, or a
Saccharomyces cerevisiae cell) that has been transformed with a
recombinant DNA molecule or nucleotide sequence that encodes a
polypeptide, peptide, protein or recombinant protein as disclosed
herein. Unless otherwise noted, the term protein includes
polypeptides and peptides.
[0026] In some aspects, the disclosure provides a method of
treatment or diagnosis, the method comprising administering to a
subject (e.g., a human or non-human animal) a VSA (e.g., a VSA as
described herein), or a pharmaceutical composition comprising a
VSA, e.g., a VSA as described herein. In some embodiments, the
subject is suffering from a disease or condition in which albumin
is indicated. In some embodiments the disease or condition is
selected from hypovolemia, hypoalbuminemia, a burn, adult
respiratory distress syndrome, nephrosis, and hemolytic disease of
the newborn.
[0027] In some aspects, the disclosure provides a method of
treatment and/or a method of diagnosis, wherein the method
comprises administering to a subject an agent (e.g., a therapeutic
or diagnostic agent) in association with a VSA (e.g., a VSA as
described herein). In some embodiments, the method is a method of
treatment that comprises administering to a subject a therapeutic
agent in association with a VSA, e.g., a VSA as described herein.
In some embodiments, the method is a method of diagnosis that
comprises administering to a subject a diagnostic agent in
association with a VSA, e.g., a VSA as described herein.
[0028] The agent can be any biologically or pharmaceutically active
moiety (e.g., a biologic, such as a peptide or nucleotide sequence
or a chemical entity such as a small molecule). Typically, the
agent is administered in an effective amount and at an effective
frequency, e.g., in an amount and frequency that is effective for
the desired therapeutic or diagnostic purpose. In some embodiments,
the agent is a known therapeutic or diagnostic agent. In some
embodiments, the agent comprises an entire known protein,
nucleotide sequence or chemical entity. In some embodiments, the
agent comprises a fragment or variant of a known protein,
nucleotide sequence or chemical entity. The fragment or variant can
have the same or similar activity as does the known protein,
nucleotide sequence or chemical entity, or the fragment or variant
can have altered activity, e.g., an increased desirable activity or
a reduced undesirable activity compared with the known protein,
nucleotide sequence or chemical entity.
[0029] In some embodiments, administering the agent in association
with the VSA results in an improved function of the agent. In some
embodiments, the improved function is an improved pharmacokinetic
property (e.g., increased half-life and/or reduced clearance) of
the agent. In some embodiments, the improved function is a reduced
effective dosage and/or a reduced effective frequency of
administration of the agent compared with the agent when it is
administered without the association with VSA. In some embodiments,
the improved function is a more desirable delivery level of the
agent (e.g., a higher, more consistent, and/or less variable level
of delivery). An improved function of the agent can be established,
e.g., by comparing the function of the agent when it is
administered in association with the VSA with the same function of
the agent when it is administered without the association with the
VSA (e.g., when the agent is administered alone or when it is
administered in association with a corresponding native serum
albumin sequence).
[0030] In some embodiments in which the agent (e.g., therapeutic or
diagnostic agent) is in association with the VSA, the agent is
fused to the VSA. In some embodiments, the agent is fused to the
VSA via a covalent bond. In some embodiments, the agent is fused to
the VSA via a peptide bond. In some embodiments, the agent is fused
to the VSA via a non-peptide bond. In some embodiments, the agent
is fused to the VSA via a linker, e.g., a linker as described
herein. In some embodiments, the agent is fused to the VSA using
recombinant genetic methods. In some embodiments, the agent is
fused to the VSA using chemical methods.
[0031] In some embodiments in which the agent (e.g., therapeutic or
diagnostic agent) is in association with the VSA, the agent is not
fused to the VSA. In some embodiments, the agent is not fused to
the VSA and is stably associated with the VSA. "Stably associated"
means that the VSA and the agent can be or remain physically
associated at a pH at which they are intended to function, e.g., at
an endosomal pH. Typically, the agent and VSA also can be or remain
physically associated at a neutral pH. In embodiments, the agent
can be associated with the VSA by any means known in the art. For
example, the agent can be conjugated to a moiety that is capable of
binding the VSA. In some embodiments, the moiety is an albumin
binding protein. In some embodiments the moiety is a fatty acid. In
some embodiments, the agent is non-covalently bound to the VSA. In
embodiments wherein the agent is not fused to the VSA, the agent
can be administered before, after, or concurrently with the VSA. In
some embodiments, the agent is administered concurrently with the
VSA. In some embodiments, the agent is administered at the same
frequency as is the VSA. In some embodiments, the agent is
administered more or less frequently than the VSA. An advantage of
using a VSA can be that a VSA can retain lipophilic features of an
albumin, thereby improving solubility of a molecule (e.g., a
therapeutic agent).
[0032] In some aspects, the disclosure provides a method of
treating a subject, the method comprising administering to the
subject an effective amount of a therapeutic agent in association
with a VSA (e.g., a VSA as described herein), such that the dosage
and/or frequency of administration at which the agent produces a
therapeutic effect is reduced relative to the dosage and/or
frequency of administration at which the agent produces a
therapeutic effect when it is not in association with the albumin
protein. In some embodiments, In some embodiments, the agent
comprises a polypeptide component that is fused to the VSA or to a
protein comprising the VSA. In some embodiments, the polypeptide
component and the VSA or protein that comprises the VSA are
separated by a linker sequence. In some embodiments, the
polypeptide component and the VSA or protein that comprises the VSA
are covalently linked by a non-peptide bond. In some embodiments,
the polypeptide component and the VSA or protein that comprises the
VSA are non-covalently and stably associated.
[0033] In additional aspects, the present disclosure provides a
method of engineering a VSA associated therapeutic agent or a VSA
associated diagnostic agent. The method comprises providing a
biologically or pharmaceutically active agent (e.g., an agent as
described herein) and associating the agent with a VSA as described
herein to provide a VSA associated agent; i.e., a VSA associated
therapeutic agent or a VSA associated diagnostic agent.
"Associating" the agent with the VSA can be done by any means known
in the art or any means described herein, e.g., by fusing the agent
to the VSA using recombinant genetic and/or chemical means, by
conjugating the agent to a moiety that is capable of binding the
VSA, or by non-covalent methods.
[0034] In some embodiments, the method further comprises
formulating the albumin modified agent, or providing a formulation
or pharmaceutical composition, for administration to a subject,
such as a human subject. In embodiments including an albumin
modified diagnostic agent, the method may further comprise
administering the albumin modified diagnostic agent to a subject
and detecting the albumin modified diagnostic agent, for example, a
method wherein the subject is imaged. In embodiments the
formulation is in a storage or delivery device, e.g., a
syringe.
[0035] In some aspects, the present disclosure provides a
formulation that comprises a biologically or pharmaceutically
active agent (e.g., an agent as described herein) in association
with VSA.
[0036] Calculations of "homology" or "sequence identity" between
two sequences (the terms are used interchangeably herein) can be
performed as follows. The sequences are aligned according to the
alignments provided herein, or, in the absence of an appropriate
alignment, the optimal alignment determined as the best score using
the Needleman and Wunsch algorithm as implemented in the Needle
algorithm of the EMBOSS package using a Blosum 62 scoring matrix
with a gap penalty of 10, and a gap extend penalty of 1. (See
Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48:
443-453; Kruskal, J. B. (1983) An overview of sequence comparison
In D. Sankoff and J. B. Kruskal, (Eds.), Time Warps, String Edits
and Macromolecules: the Theory and Practice of Sequence Comparison,
pp. 1-44, Addison Wesley, and tools available from the European
Bioinformatics Institute (Cambridge UK), described in Rice, P. et
al. (2000) Trends in Genetics 16(6): 276-277 and available online
at www.ebi.ac.uk/Tools/emboss/align/index.html and
emboss.open-bio.org/wiki/Appdoc:Needle.) The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences. To determine collective identity of one sequence of
interest to a group of reference sequences, a position is
considered to be identical if it is identical to at least one amino
acid at a corresponding position in any one or more of the group of
reference sequences. With respect to lists of segments, features,
or regions, identity can be calculated collectively for all members
of such list to arrive an overall percentage identity.
[0037] Provided herein are sequences that are at least 60, 65, 70,
75, 80, 82, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or
99% identical to sequences disclosed herein.
[0038] As used herein, the singular forms "a", "an", and "the"
include plural references unless the context clearly dictates
otherwise.
[0039] All patents, patent applications, scientific publications
and other references cited in this specification are hereby
incorporated herein for all purposes, and for the disclosure for
which they have been cited.
DRAWINGS
[0040] FIG. 1 is a set of graphs depicting the results of SPR
experiments determining the binding of the variant serum albumin
polypeptides (VSAs) HSA-5, HSA-11, HSA-7, and HSA-13 to human FcRn
at pH 5.5, pH 6.0, pH 6.5, and pH 7.4.
[0041] FIG. 2 is a table depicting the K.sub.Ds as determined by
ELISA of variant serum albumin polypeptides (VSAs) at pH 5.5.
[0042] FIG. 3 is a set of graphs depicting the results of ELISA
that determining the binding of VSAs and wild type HSA to human
FcRn at pH 5.5, 6.0, and 7.4.
[0043] FIG. 4 is s set of graphs depicting the pharmacokinetics of
VSAs (HSA-5, HSA-11, and HSA-13) and wild type human serum albumin
(wt-HSA) in wild type mice. The left panel shows plasma
concentration over time. The right upper panel shows half-life, and
the right lower panel shows clearance.
[0044] FIG. 5 is s set of graphs that depicting the
pharmacokinetics of VSAs (HSA-5, HSA-11, and HSA-13) and wild type
HSA in human FcRn transgenic mice. The left panel shows plasma
concentration over time. The right upper panel shows half-life, and
the right lower panel shows clearance.
[0045] FIG. 6 is a table depicting the PK parameters of a wild type
HSA (HSA-wt) and a VSA (HSA-7) in a primate.
[0046] FIG. 7 is a graph and a table depicting the pharmacokinetics
of IL-2 fused to wild type HSA and to a VSA (HSA-13) in C57Bl/6J
mice.
DETAILED DESCRIPTION
[0047] One limitation of certain agents (e.g., therapeutic or
diagnostic agents) can be their suboptimal pharmacokinetics (PK),
particularly when administered systemically. Relatively short PKs
(as assessed, e.g., by plasma half-life of the agents) can mean,
for example, that a therapeutic must be administered at a
relatively high frequency. The present disclosure provides VSAs
that have improved functional properties, e.g., improved
pharmacokinetics (e.g., increased half-life, reduced clearance,
and/or reduced beta phase clearance). The VSAs can be useful
themselves, e.g., for therapeutic or diagnostic uses. The VSAs can
be used to improve the functional properties, e.g., the
pharmacokinetics, of other agents.
[0048] The present disclosure provides variant serum albumin
polypeptides (VSAs). As used herein, a variant serum albumin
polypeptide (VSA) can refer to a variant of a full length native
serum albumin or to a variant of a fragment of a native serum
albumin (e.g., a variant of a functional fragment of a native serum
albumin). Typically, a VSA that is a variant of a fragment of a
native serum albumin has a minimal length that provides
functionality (e.g., an ability to bind FcRnat endosomal pH). In
some embodiments, the variant comprises or consists of a sequence
that is a variant of the domain III sequence of a native serum
albumin. In some embodiments, the variant comprises a sequence that
is a variant of the domain III sequence of a native human serum
albumin sequence. In some embodiments, the affinity of a VSA for an
FcRn is increased at an endosomal pH (e.g., pH 5.5 or 6.0) compared
to the affinity of wild type albumin corresponding to the VSA. In
certain embodiments, the affinity of a VSA for an FcRn is increased
at an endosomal pH (e.g., pH 5.5 or 6.0) compared to the affinity
of wild type albumin corresponding to the VSA and the affinity of
the VSA for FcRn at a neutral pH (e.g., pH 7.0 or 7.4) is the same
or less than the affinity of a corresponding wild type albumin for
an FcRn. In some cases, Applicants have found that in selecting a
VSA that is useful, e.g., as a therapeutic, it is necessary to
evaluate the affinity for FcRn at both endosomal and neutral pH,
noting that a VSA with the greatest affinity for FcRn at endosomal
pH may be unsuitable for use because it also has an increased
affinity at a neutral pH.
[0049] In some embodiments, a VSA comprises an amino acid
substitution or deletion at one or more (e.g., 1, 2, 3, 4, 5, or
more) positions disclosed herein, e.g., at the positions that were
mutated in the experiments disclosed in the Examples and Figures.
In some embodiments, a VSA comprises a substitution described
herein, e.g., one or more substitutions of a specific amino acid at
a specific position as described herein, e.g., as disclosed in the
Examples and Figures.
[0050] In some embodiments, a VSA has one or more of the following
mutations (amino acid residue numbers defined in accordance with
the mature human amino acid sequence of SEQ ID NO:2): V418M; T420A;
V424I; N429D; M446V; A449V; T467M; E505(R/K/G); V547A; and A552T,
wherein "(X/Z)" means that the amino acid sequence may
alternatively comprise amino acid X or amino acid Z at that
residue. In certain embodiments, the VSA can include one or more of
the following mutations: V418M; T420A; V424I; E505(R/K/G); and
V547A.
[0051] In some embodiments, a VSA comprises a mutation that is
present in a VSA disclosed herein, e.g., a VSA selected from one or
more of the following variants described herein: HSA-15, HSA-13,
HSA-12, HSA-7, HSA-21, HSA-11, HSA-14, HSA-5, HSA-10, HSA-6, HSA-9,
and HSA-18. In some embodiments, a VSA includes each of the
mutations that are present in a variant described herein, e.g., a
variant selected from HSA-15, HSA-13, HSA-12, HSA-7, HSA-21,
HSA-11, HSA-2, HSA-14, HSA-5, HSA-10, HSA-6, HSA-9, and HSA-18. In
some embodiments, a VSA has the sequence of HSA-15, HSA-13, HSA-12,
HSA-7, HSA-21, HSA-11, HSA-14, HSA-5, HSA-10, HSA-6, HSA-9, or
HSA-18.
[0052] Typically, the VSAs provided herein have improved functional
properties (e.g., higher affinity for FcRn at endosomal pH and/or
extended pharmacokinetics, e.g., increased half-life and/or reduced
clearance) compared with a corresponding native serum albumin
sequence. Such VSAs can be useful for therapeutic or diagnostic
applications. For example, VSAs can be useful for treating, or for
producing formulations that are useful for treating, conditions for
which serum albumin is indicated. A VSA that has improved
properties compared with a corresponding native serum albumin
sequence has therapeutic advantages. For example, a VSA that has
extended PK compared with a corresponding native serum albumin
polypeptide can have therapeutic advantages, such as less frequent
and/or reduced dosing and/or more consistent delivery levels. A VSA
can also be less expensive to administer because fewer doses are
required.
[0053] Native human serum albumin (HSA) binds to human FcRn at pH
6.0 with an affinity of about 1-10 micromolar, and about 400
micromolar affinity at pH 7.4. The VSAs described herein can have
improved binding to FcRn at endosomal pH, e.g., at pH 6.0 compared
with a native serum albumin (e.g., a corresponding native serum
albumin, e.g., the corresponding full length native serum albumin
or fragment thereof of which the VSA is a variant).
[0054] In some embodiments, the VSA binds to FcRn with a K.sub.D
that is less than a value selected from 2 micromolor, 1.5
micromolar, 1 micromolar, and 0.5 micromolar. In some embodiments,
the VSA binds to FcRn with a K.sub.D that is less than a value
selected from 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65
nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM,
15 nM, 10 nM, and 5 nM. The K.sub.D can be determined as described
herein or using any method known in the art.
[0055] The K.sub.D is pH dependent. In some embodiments, the VSA
exhibits improved binding to FcRn at a specified pH, e.g., at pH
5.0, 5.2, 5.5, 5.6, 5.7, 5.8, 5.9, 6.2, 6.0, or 6.2. In some
embodiments, the VSA exhibits improved binding to FcRn at an
endosomal pH, for example at pH 5.0-6.2, pH 5.0-6.2, pH 5.0-6.0, or
pH 5.5-6.0. In some embodiments, the VSA exhibits improved binding
to FcRn at pH 5.5 or 6.0. In some embodiments, the VSA binds to
FcRn at an endosomal pH, e.g., at pH 5.5 or pH 6.0, with a K.sub.D
that is less than a value selected from 2 micromolar, 1.5
micromolar, 1 micromolar, and 0.5 micromolar. In some embodiments,
the VSA binds to FcRn at an endosomal pH, e.g., at pH 5.5 or pH 6.0
with a K.sub.D that is less than a value selected from 100 nM, 95
nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM,
45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, and 5
nM.
[0056] In some embodiments, the serum albumin variants have a
K.sub.D for FcRn at pH 5.5 that is less than 23 nM. In some
embodiments, the serum albumin variants have a K.sub.D for FcRn at
pH 5.5 that is less or equal to 75 nM, 50 nM, 25 nM, 15 nM, 10 nM,
5 nM, or 3 nM.
[0057] In some embodiments, the VSA binds to FcRn at an endosomal
pH, e.g., pH 5.5 or pH 6.0, but does not bind to FcRn at pH 7.4. In
some embodiments, the VSA binds to FcRn at an endosomal pH, e.g.,
pH 5.5 or pH 6.0, and shows reduced binding to FcRn at pH 7.4
compared with its binding to FcRn at pH 5.5 (e.g., the binding to
FcRn at pH 7.4 is less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, or 60% of the level observed at pH 5.5).
[0058] In some embodiments, the VSA binds to FcRn at pH 5.5 and
shows reduced binding to FcRn at pH 7.4 compared with its binding
to FcRn at an endosomal pH, e.g., pH 5.5 or pH 6.0 (e.g., the
binding to FcRn at pH 7.4 is less than 1%, 2%, 3%, 4%, 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the level
observed at pH 5.5), and the VSA has improved pharmacokinetics
(e.g., increased plasma half-life and/or reduced clearance, e.g.,
reduced beta phase clearance) compared with a corresponding native
serum albumin.
[0059] In some embodiments, the VSA has a decreased K.sub.D for
FcRn at an endosomal pH, e.g. pH5.5 or pH 6.0, compared to the
K.sub.D of a wild type albumin (e.g., an HSA) at endosomal pH, and
has the same or increased K.sub.D for FcRn at a neutral pH, e.g.,
pH 7.0 or pH 7.4.
[0060] The ratio of the K.sub.Ds of wild type HSA to FcRn at pH 7.4
and 5.5 is 40-400, depending on the report. In some embodiments,
the ratio of K.sub.Ds of the VSA to FcRn at pH 7.4 and 5.5 is
greater than for wild type HSA (e.g., a ratio of 500; 1000; 5000;
10,000; 100,000; or 1 million). In some embodiments, the binding of
VSA to FcRn is assessed using SPR. In some embodiments, the binding
of VSA to FcRn is assessed using ELISA.
[0061] In some embodiments, the half life (T1/2) of the VSA is
greater than the T1/2 of a corresponding native serum albumin. In
some embodiments, the T1/2 of the VSA is increased by 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared with the T1/2
of a corresponding native serum albumin.
[0062] In some embodiments, the T1/2 of the VSA is at least two
times, three times, or four times as long as the T1/2 of a
corresponding native serum albumin. In some embodiments, the T1/2
of the VSA is at least twice as long as the T1/2 of a corresponding
native serum albumin.
[0063] Wild type HSA has a half-life (T1/2) in plasma of about
15-20 days in humans. A variant serum albumin described herein can
have a longer serum half-life than a corresponding albumin, e.g.,
T1/2 at least about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days.
In some embodiments, the VSA. In some embodiments, the VSA has a
longer serum T1/2 in an animal compared to the corresponding wild
type VSA, e.g., a VSA has a longer serum T1/2 in a monkey than the
T1/2 of a human wild type albumin in a monkey.
[0064] Wild type HSA (UniProt DB Accession No. P02768; SEQ ID NO:1
below) is a 609 amino acid protein, comprising an 18 amino acid
signal peptide; a 6 amino acid propeptide, extending from amino
acid 19 to 24; Domain 1, extending from amino acid 25 to 210;
Domain 2, extending from amino acid 211 to 403; and Domain 3,
extending from amino acid 404 to 601 or amino acid 404 to 609 of
Sequence ID No. 1. An exemplary sequence of a human serum albumin
(including its leader sequence) is as follows:
TABLE-US-00001 [SEQ ID NO: 1] 10 20 30 40 MKWVTFISLL FLFSSAYSRG
VFRRDAHKSE VAHRFKDLGE 50 60 70 80 ENFKALVLIA FAQYLQQCPF EDHVKLVNEV
TEFAKTCVAD 90 100 110 120 ESAENCDKSL HTLFGDKLCT VATLRETYGE
MADCCAKQEP 130 140 150 160 ERNECFLQHK DDNPNLPRLV RPEVDVMCTA
FHDNEETFLK 170 180 190 200 KYLYEIARRH PYFYAPELLF FAKRYKAAFT
ECCQAADKAA 210 220 230 240 CLLPKLDELR DEGKASSAKQ RLKCASLQKF
GERAFKAWAV 250 260 270 280 ARLSQRFPKA EFAEVSKLVT DLTKVHTECC
HGDLLECADD 290 300 310 320 RADLAKYICE NQDSISSKLK ECCEKPLLEK
SHCIAEVEND 330 340 350 360 EMPADLPSLA ADFVESKDVC KNYAEAKDVF
LGMFLYEYAR 370 380 390 400 RHPDYSVVLL LRLAKTYETT LEKCCAAADP
HECYAKVFDE 410 420 430 440 FKPLVEEPQN LIKQNCELFE QLGEYKFQNA
LLVRYTKKVP 450 460 470 480 QVSTPTLVEV SRNLGKVGSK CCKHPEAKRM
PCAEDYLSVV 490 500 510 520 LNQLCVLHEK TPVSDRVTKC CTESLVNRRP
CFSALEVDET 530 540 550 560 YVPKEFNAET FTFHADICTL SEKERQIKKQ
TALVELVKHK 570 580 590 600 PKATKEQLKA VMDDFAAFVE KCCKADDKET
CFAEEGKKLV 609 AASQAALGL
[0065] An exemplary sequence of a human serum albumin (HSA) (in its
mature form) is a 585 amino acid polypeptide as follows in SEQ ID
NO:2. In some embodiments, the VSAs as described herein comprise
variant domain III regions. Typically, such variant domain III
regions retain the three-dimensional fold of domain III of serum
albumin.
[0066] Exemplary variant domain III regions are at least 80%
identical to domain III of a naturally occurring serum albumin,
e.g., at least 90% identical to the domain III of a human serum
albumin (e.g., amino acids 404 to 609 of SEQ ID NO:1 or amino acids
380 to 585 of SEQ ID NO:2 below).
[0067] A variant serum albumin polypeptide can have one or more
(e.g., 1, 2, 3, 4, 5, or more) substitutions, insertions, or
deletions relative to a corresponding native serum albumin. For
example, the variant polypeptide can include a domain III with at
least one, two, three, four or five substitutions relative to a
domain III of HSA.
TABLE-US-00002 [SEQ ID NO: 2] 10 20 30 40 DAHKSEVAHR FKDLGEENFK
ALVLIAFAQY LQQCPFEDHV 50 60 70 80 KLVNEVTEFA KTCVADESAE NCDKSLHTLF
GDKLCTVATL 90 100 110 120 RETYGEMADC CAKQEPERNE CFLQHKDDNP
NLPRLVRPEV 130 140 150 160 DVMCTAFHDN EETFLKKYLY EIARRHPYFY
APELLFFAKR 170 180 190 200 YKAAFTECCQ AADKAACLLP KLDELRDEGK
ASSAKQRLKC 210 220 230 240 ASLQKFGERA FKAWAVARLS QRFPKAEFAE
VSKLVTDLTK 250 260 270 280 VHTECCHGDL LECADDRADL AKYICENQDS
ISSKLKECCE 290 300 310 320 KPLLEKSHCI AEVENDEMPA DLPSLAADFV
ESKDVCKNYA 330 340 350 360 EAKDVFLGMF LYEYARRHPD YSVVLLLRLA
KTYETTLEKC 370 380 390 400 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ
NCELFEQLGE 410 420 430 440 YKFQNALLVR YTKKVPQVST PTLVEVSRNL
GKVGSKCCKH 450 460 470 480 PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS
DRVTKCCTES 490 500 510 520 LVNRRPCFSA LEVDETYVPK EFNAETFTFH
ADICTLSEKE 530 540 550 560 RQIKKQTALV ELVKHKPKAT KEQLKAVMDD
FAAFVEKCCK 570 580 ADDKETCFAE EGKKLVAASQ AALGL
[0068] Typically, a VSA is a polypeptide that has a
three-dimensional fold that is similar or identical to that of a
native serum albumin, e.g., as described in PDB files 1A06 (Sugio
et al. (1999) Protein Eng. Jun; 12(6):439-46); or 1E78
(Bhattacharya et al., (2000) J. Biol. Chem., 275:38731)); or 1E7H
(Bhattacharya et al., (2000) J. Mol. Biol., 303:721).
[0069] Exemplary variant serum albumin polypeptides are at least
70% identical to a naturally occurring serum albumin, e.g., at
least 75%, 80%, 85%, 90%, or 95% identical to a human serum albumin
(e.g., SEQ ID NO:1 above). A variant serum albumin polypeptide can
have one or more substitutions, insertions, or deletions. For
example, the variant polypeptide can have at least one, two, three,
four, or five substitutions relative to HSA.
[0070] The sequences of serum albumins from other species,
particularly mammalian species are also known. Exemplary sequences
include albumin sequences from Bos taurus (CAA76847, P02769,
CAA41735, 229552, AAF28806, AAF28805, AAF28804, AAA51411); Sus
scrofa (P08835, CAA30970, AAA30988); Equus caballus (AAG40944,
P35747, CAA52194); Ovis aries (P14639, CAA34903); Salmo salar
(CAA36643, CAA43187); Gallus gallus (P19121, CAA43098); Felis catus
(P49064, 557632, CAA59279, JC4660); Canis familiaris (P49822,
529749, CAB64867). Variations, e.g., substitutions described herein
in HSA, can also be introduced at corresponding positions into
serum albumins from other species, e.g., into mammalian serum
albumins, including, e.g., bovine serum albumin.
[0071] Serum albumin can be divided into at least three domains,
termed Domain I, Domain II, and Domain III. A serum albumin protein
useful in the present invention typically includes at least a
domain III, and can also include domain I and domain II. Exemplary
domains I, II, and III can have at least 60, 65, 70, 75, 80, 85,
90, 95% identity to respective domains from a mammalian serum
albumin. In some embodiments, the variant serum albumin polypeptide
of the present invention comprises domain III of human HSA, that
is, amino acids 404 to 601 or 404 to 609 of SEQ ID NO:1, or amino
acids 380 to 585 of SEQ ID NO:2, into which at least one, two,
three, four, or five substitutions have been engineered relative to
domain III of HSA.
[0072] In some embodiments, a residue that is within less than 7
angstroms of position H464 is substituted with another amino acid.
For example, the residue is amino acid 415, 418, 458, 459, 460,
461, 462, 463, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474,
475, or 477. In some embodiments, a residue that is within less
than 7 angstroms of position H510 is substituted with another amino
acid. For example, the residue is amino acid 508, 509, 511, 512,
513, 514, 554, 555, 559, 564, 565, 568, or 569. In some
embodiments, a residue that is within less than 7 angstroms of
position H535 is substituted with another amino acid. For example,
the residue is amino acid 412, 497, 498, 499, 500, 501, 502, 503,
507, 529, 530, 531, 532, 533, 534, 536, 537, 538, 580, or 583.
[0073] In some embodiments, residue V418 is altered, e.g., to a
residue other than valine, e.g., to an hydrophobic residue such as
methionine. In some embodiments, a residue that is within less than
7 angstroms of position V418 is substituted with another amino
acid. For example, the residue is amino acid 411, 412, 415, 416,
417, 419, 420, 421, 422, 423, 424, 426, 460, 464, 469, 470, 473,
497, 530, or 534.
[0074] In some embodiments, residue T420 is altered, e.g., to a
residue other than threonine, e.g., to an uncharged residue, e.g.,
a small uncharged residue such as alanine or glycine. In some
embodiments, a residue that is within less than 7 angstroms of
position T420 is substituted with another amino acid. For example,
the residue is amino acid 408, 412, 416, 417, 418, 419, 421, 422,
423, 424, 425, 500, 526, 527, 528, 529, 530, 531, or 534.
[0075] In some embodiments, residue V424 is altered, e.g., to a
residue other than valine, e.g., to an uncharged residue, e.g., to
isoleucine. In some embodiments, a residue that is within less than
7 angstroms of position V424 is substituted with another amino
acid. For example, the residue is amino acid 404, 408, 412, 418,
419, 420, 421, 422, 423, 425, 426, 427, 428, 429, 430, 522, 523,
524, 526, 527, 528, or 530.
[0076] In some embodiments, residue E505 is altered, e.g., to a
residue other than glutamic acid, e.g., to an uncharged residue,
e.g., a small uncharged residue such as alanine or glycine, or to a
positively charged residue, e.g. lysine or arginine. In some
embodiments, a residue that is within less than 7 angstroms of
position E505 is substituted with another amino acid. For example,
the residue is amino acid 502, 503, 504, 506, 507, 508, or 509.
[0077] In some embodiments, residue V547 is altered, e.g., to a
residue other than valine, e.g., to an uncharged residue, e.g., a
small uncharged residue such as alanine or glycine. In some
embodiments, a residue that is within less than 7 angstroms of
position V547 is substituted with another amino acid. For example,
the residue is amino acid 529, 532, 542, 543, 544, 545, 546, 548,
549, 550, 551, or 552.
[0078] Variant serum albumin polypeptides can be incorporated into
a fusion protein, with the N- or C-terminus thereof fused to the N-
or C-terminus of a diagnostic or therapeutic protein, or variant
thereof. In certain embodiments, the variant serum albumin
polypeptide and the diagnostic or therapeutic protein are joined
through a linker moiety, such as a peptide linker Linkers useful in
the present invention are described in greater detail further
herein.
Therapeutic Uses of VSAs
[0079] A VSA can be used as a therapeutic, e.g., in uses for which
albumin such as human albumin is typically used. For such uses, a
VSA as described herein can have the advantage of extended PK,
which can enable less frequent and/or reduced dosing for albumin
replacement or supplementation. Such uses include, for example,
hypovolemia, hypoalbuminemia, burns, adult respiratory distress
syndrome, nephrosis, and hemolytic disease of the newborn.
Hypoalbuminemia can result from, for example, inadequate production
of albumin (e.g., due to malnutrition, burns, major injury, or
infection), excessive catabolism of albumin (e.g., due to burn,
major injury such as cardio-pulmonary bypass surgery, or
pancreatitis), loss through bodily fluids (e.g., hemorrhage,
excessive renal excretion, or burn exudates), deleterious
distribution of albumin within the body (e.g., after or during
surgery or in certain inflammatory conditions). Typically, for such
uses, a VSA for such uses is administered by injection or iv in a
solution that is from 5%-50% VSA (w/v), for example, 10%-40%,
15%-30%, 20%-25%, 20%, or 25%. Typically, administration is
sufficient to produce a total albumin plus VSA concentration in a
treated subject's serum that is 3.4-5.4 grams per deciliter (g/dL).
Methods of assaying albumin concentration are well known in the art
and can generally be used to assay total albumin plus VSA
concentration.
[0080] Also provided herein is a VSA, or a pharmaceutical
composition comprising a VSA, for use in therapy. Also included is
a VSA, or a pharmaceutica composition comprising a VSA, for use in
the treatment of a disease, e.g., a disease or condition for which
albumin is indicated. Other embodiments include the use of a VSA,
or a pharmaceutical composition comprising aVSA, for the
manufacture of a medicament for the treatment of a disease.
Uses of VSAs in Association with Other Agents
[0081] VSAs can also be used in association with other agents,
e.g., therapeutic or diagnostic agents, to confer functional
advantages, e.g., advantages of VSAs as described herein. The agent
can be, e.g., any agent that is useful in the diagnosis or therapy
of a disease or disorder, e.g., a disease or disorder that affects
a human or a non-human animal.
[0082] Advantages of VSAs include, e.g., lack of Fc effector
function, high solubility, potential for high expression, low
immunogenicity, and ability to be fused to another moiety at both
termini to generate bivalent, bispecific, or bifunctional
molecules.
[0083] Disclosed herein are VSAs with improved binding to an FcRn
at endosomal pH, thereby potentiating the ability of the VSA and an
agent associated with that VSA to have improved PK.
[0084] A VSA that has an extended PK can be associated with an
agent (e.g., a therapeutic or diagnostic agent) to extend the PK of
the agent. The extended PK can have advantages; for example, the
agent can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the agent
can be achieved.
[0085] In some embodiments, associating a VSA with an agent
improves the functional properties of the agent. In some
embodiments, the dosage and/or frequency at which the agent is
effective for producing a particular effect (e.g., a desired
therapeutic effect) is reduced when the agent is used in
association with the VSA. In some embodiments, associating a VSA
with an agent improves the pharmacokinetic properties of the agent
(e.g., increases its half-life and/or reduces its clearance). Any
relevant pharmacokinetic parameters that are known in the art can
be used to assess pharmacokinetic properties. The pharmacokinetics
of a VSA or a VSA associated with an agent can be measured in any
relevant biological sample, e.g., in blood, plasma, or serum.
[0086] In some embodiments, associating a VSA with an agent
improves the half life (T1/2) of the agent. In some embodiments,
the T1/2 of the agent is increased by 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 100%. In some embodiments, the T1/2 is at least
two times, three times, or four times as long as the T1/2 of the
agent when it is not associated with the VSA. In some embodiments,
the T1/2 of the VSA is at least twice as long as the T1/2 of the
agent when it is not associated with the VSA.
[0087] In some embodiments, the dose at which the agent is
effective for producing a particular effect (e.g., a desired
therapeutic effect) is reduced when the agent is associated with
the VSA. In some embodiments, the effective dose is reduced to 80%,
70%, 60%, 50%, 40%, 30%, 20%, or 10% of the dose that is required
when the agent is not associated with the VSA.
[0088] In some embodiments, the frequency of dosing of the agent
that is effective for producing a particular effect (e.g., a
desired therapeutic effect) is reduced when the agent is associated
with the VSA. In some embodiments, the frequency of dosing at which
the agent is effective when it is associated with the VSA is
decreased by 10%, 20%, 30%, 40%, 50%, or more compared with the
frequency at which the agent is effective when it is not associated
with the VSA.
[0089] In some embodiments, the frequency of dosing at which the
agent is effective when it is associated with the VSA is decreased
by at least 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 2 weeks, 3
weeks, or 4 weeks compared with the frequency at which the agent is
effective when it is not associated with the VSA.
[0090] The improvement in the properties of the agent can be
assessed relative to any appropriate control. For example, the
improvement in the properties of an agent that is associated with a
VSA can be assessed by comparing the properties of the agent that
is associated with the VSA with the properties of the agent when it
is not in an association with the VSA. Alternatively, the
improvement in the properties of an agent that is associated with a
VSA can be assessed by comparing the properties of the agent that
is associated with the VSA with the properties of the agent when it
is in an association with a corresponding native serum albumin
polypeptide.
[0091] Also provided herein is a VSA, a VSA in association with
another agent, a pharmaceutical composition comprising a VSA, or a
pharmaceutical composition comprising a VSA in association with
another agent, for use in therapy or for use in the treatment of a
disease, e.g., a disease or condition described herein or described
in U.S. Pat. No. 5,766,883, U.S. Pat. No. 5,875,969, U.S. Patent
Publication No. US 2002/0151011, or International Publication No.
WO 2011/103076, the entire contents of each of which are hereby
incorporated herein by reference. Other embodiments include the use
of a VSA, or a pharmaceutical composition comprising aVSA, a VSA in
association with another agent, a pharmaceutical composition
comprising a VSA, or a pharmaceutical composition comprising a VSA
in association with another agent, for the manufacture of a
medicament for the treatment of a disease.
[0092] The agent can be another protein, e.g., a heterologous
protein. In some embodiments, the agent is a diagnostic agent. In
some embodiments, the agent is a therapeutic agent. For example, a
protein that comprises a VSA can be used to extend the PK of a
systemically administered therapeutic agent. The heterologous
protein can be, for example, a therapeutic protein or a diagnostic
protein. The serum albumin polypeptide with altered FcRn binding
properties or a domain thereof (e.g., domain III) can be associated
with (e.g., attached covalently to) the therapeutic protein, or to
an active fragment or variant of the therapeutic protein. The
variant serum albumin or a domain thereof can be in the same
polypeptide chain as is at least a component of the therapeutic
protein.
[0093] A variant serum albumin (VSA) can be associated with another
agent, e.g., a therapeutic agent or a diagnostic agent. The other
agent (e.g., therapeutic agent) can be an entire protein (e.g., an
entire therapeutic protein) or a biologically active fragment
thereof. The activity of the agent (e.g., therapeutic agent) can be
evaluated in an appropriate in vitro or in vivo assay for the
agent's activity. In general, the activity of the agent fused to a
VSA is not reduced, for example, by more than 50%, by more than
40%, by more than 30%, by more than 20%, by more than 10%, by more
than 5%, or by more than 1% compared with the activity of the agent
when it is not in association with the agent. Examples of methods
for assessing the activity of certain agents are provided
herein.
[0094] In some embodiments, the VSA is attached to the agent by one
or more covalent bonds to form a variant serum albumin fusion
molecule. Any agent that can be linked to a VSA described herein
can be used as the agent in a variant serum albumin fusion
molecule. The agent can be a therapeutic or diagnostic agent. For
example, the agent can be any polypeptide or drug known to one of
skill in the art.
[0095] In some embodiments, an agent (e.g., therapeutic or
diagnostic agent) is stably associated with a VSA, but the agent is
not fused to the VSA. In such embodiments, the agent can be
associated with the VSA by any means known in the art. For example,
the agent can be conjugated to a moiety that is capable of binding
the VSA. In some embodiments, the moiety is an albumin binding
protein. In some embodiments the moiety is a fatty acid. In some
embodiments, the agent is non-covalently bound to the VSA,
generally via the affinity of the VSA for small lipophilic
moieties. In embodiments wherein the agent is not fused to the VSA,
the agent can be administered before, after, or concurrently with
the VSA. In some embodiments, the agent is administered
concurrently with the VSA. In some embodiments, the agent is
administered at the same frequency as is the VSA. In some
embodiments, the agent is administered more or less frequently than
the VSA.
[0096] In some embodiments, the agent is a polypeptide consisting
of at least 5, for example, at least 10, at least 20, at least 30,
at least 40, at least 50, at least 60, at least 70, at least 80, at
least 90 or at least 100 amino acid residues. The agent can be
derived from any protein for which an improved property is desired,
e.g., an increase in serum levels and/or serum half-life of the
agent; or a modified tissue distribution and/or tissue-targeting of
the agent.
[0097] In some embodiments, the agent is a cytokine or a variant
thereof. Generally, a cytokine is a protein released by one cell
population that acts on another cell as an intercellular mediator.
Examples of such cytokines include lymphokines, monokines, and
traditional polypeptide hormones. Specific examples include:
interleukins (ILs) such as IL-1 (IL-1.alpha. and IL1.beta.), IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12;
IL-15, IL-18, and IL-37; a tumor necrosis factor such as TNF-alpha
or TNF-beta; growth hormone such as human growth hormone (HGH);
somatotropin; somatrem; N-methionyl human growth hormone, and
bovine growth hormone; parathyroid hormone; thyroxine; insulin;
proinsulin; insulin-like growth factors, such as insulin-like
growth factors-1, -2, and -3 (IGF-1; IGF-2; IGF-3); proglucagon;
glucagon and glucagon-like peptides, such as glucagon-like
peptide-1 and -2 (GLP-1 and GLP-2); exendins, such as exendin-4;
gastric inhibitory polypeptide (GIP); secretin; pancreatic
polypeptide (PP); nicotinamide phosphoribosyltransferase (also
known as visfatin); leptin; neuropeptide Y (NPY); interleukin
IL-1Ra, including (N140Q); ghrelin; orexin; adiponectin;
retinol-binding protein-4 (RBP-4); adropin; relaxin; prorelaxin;
neurogenic differentiation factor 1 (NeuroD1); glicentin and
glicentin-related peptide; cholecystokinin (previously known as
pancreozymin); glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; fibroblast growth factors
(FGF) such as FGF-19, FGF-21 and FGF-23; prolactin; placental
lactogen; tumor necrosis factor-alpha and -beta;
mullerian-inhibiting substance; gonadotropin-associated peptide;
luteinizing-hormone-releasing hormone (LHRH); inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
growth factors (e.g., platelet-derived growth factor, PDGF and its
receptor, EGF and its receptor, nerve growth factors, such as
NGF-beta and its receptor, and KGF, such as palifermin, and its
receptor); platelet-growth factor (PGF); transforming growth
factors (TGFs) such as TGF-alpha and TGF-beta; osteoinductive and
growth and differentiation factors, such as osteocalcin, BMP-2,
BMP-4, BMP-6 and BMP-7; interferons such as interferon-alpha, beta,
and -gamma, including interferon-alpha2B; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
erythropoietin (EPO); darbepoeitin alfa; tissue plasminogen
activator (TPA) or alteplase; tenecteplase; dornase alfa;
entanercept; calcitonin, oxyntomodulin; glucocerebrosidase;
arginine deiminase, Arg-vasopressin, natriuretic peptides,
including A-type natriuretic peptide; B-type natriuretic peptide,
C-type natriuretic peptide and Dendroapsis natriuretic peptide
(DNP); gonadotropin-releasing hormone (GnRH); endostatin;
angiostatin, including (N211Q); Kiss-1; hepcidin; oxytocin;
pancreatic polypeptide; calcitonin gene-related protein (CGRP);
parathyroid hormone (PTH); adrenomedulin; delta-opioids; K-opioids;
mu-opioids; deltorphins; enkephalins; dynorphins; endorphins;
CD276, including (B7-H3); ephrin-B1; tweak-R, cyanovirin, including
cyranovirin-N; gp41 peptides; 5-helix protein; prosaptide;
apolipoprotein A1; BDNF; brain-derived neural protein; CNTF
(Axokine.RTM.); Antithrombin III; FVIII A1 domain; Kringle-5; Apo
A-1 Milano; Kunitz domains; vWF A1 domain; Peptide YY, including
PYY1-36 and PYY3-36; urate oxidase; and other polypeptide factors
including LIF and kit ligand (KL).
[0098] In one embodiment, the agent is BMP peptide analogue (e.g.,
THR-184, Thrasos Therapeutics, Inc.).
[0099] In one embodiment, the agent is GLP-2.
[0100] In some embodiments, the therapeutic protein is a monomeric
protein (such as a monomeric cytokine, e.g., an IL-1Ra, or an
scFv). Albumin fusion proteins are known in the art; the variant
serum albumin polypeptides with altered FcRn binding properties,
described herein, can be used in place of serum albumin and linked
to a therapeutic agent to form fusion proteins with improved
properties, such as increased half-life.
[0101] Examples of other polypeptides useful as the active agent
include, but are not limited to, various types of antibodies,
antibody fragments, such as antigen binding domains (e.g., scFv,
Fv, Fab, F(ab).sub.2, domain antibodies, and the like);
Surrobodies, Adnectins.TM., anti-calin, affibody, or fragments of
the foregoing. Also useful as active agents are receptor
antagonists, such as IL-1Ra (e.g., anakinra), cell adhesion
molecules (e.g., cadherins, such as cadherin-11, CTLA4, CD2, and
CD28); anti-angiogenic factors such as endostatin; receptors, as
well as soluble fragments of tissue-bound receptors.
[0102] The agent, e.g., therapeutic moiety included in a fusion
protein as described herein, can also be a therapeutic moiety such
as a cytotoxin (e.g., a cytostatic or cytocidal agent), a
non-peptide therapeutic agent or a radioactive element (e.g.,
alpha-emitters, gamma-emitters, etc.). Examples of cytostatic or
cytocidal agents include, but are not limited to, paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Non-peptide therapeutic agents
include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU)
and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II)
(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC), and anti-mitotic agents (e.g., vincristine and vinblastine).
The present invention also encompasses fusing the variant serum
albumin (VSA) moiety to an active agent that is a diagnostic agent.
The VSA-diagnostic molecule of the invention can be used
diagnostically to, for example, monitor the development or
progression of a disease, disorder or infection as part of a
clinical testing procedure to, e.g., determine the efficacy of a
given 5 treatment regimen. Detection can be facilitated by coupling
the pharmacologic enhancing molecule to a detectable substance.
Examples of detectable substances include enzymes, prosthetic
groups, fluorescent materials, luminescent materials,
bioluminescent materials, radioactive materials, positron emitting
metals, and nonradioactive paramagnetic metal ions. The detectable
substance can be coupled or conjugated either directly to the
antibody or indirectly, through an intermediate (such as, for
example, a linker known in the art) using techniques known in the
art. See, for example, U.S. Pat. No. 4,741,900 for metal ions that
can be conjugated to antibodies for use as a diagnostic according
to the methods and compositions described herein. Examples of
suitable enzymes include, e.g., horseradish peroxidase, alkaline
phosphatase, O-galactosidase, or acetylcholinesterase; examples of
suitable prosthetic groups include, e.g., complexes including, e.g.
streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; examples of bioluminescent
materials include luciferase, luciferin, and aequorin; and examples
of suitable radioactive material include, e.g., .sup.125I,
.sup.131I, .sup.111In or 99 mTc. See, for example, Plumridge et
al., WO2011/051489; Lubman et al.; WO2010/141329.
[0103] The nucleotide and amino acid sequences and structures for
the above molecules that are useful as, or in preparing the active
moiety to be fused to the variant serum albumin moiety in the
present invention are known, and all of the publications and
websites described as providing such information, such as Genbank,
the protein database and Uniprot (www.ncbi.nlm.nih.gov/genbank;
www.pdb.org; and www.uniprot.org, respectively), are incorporated
herein by reference for this purpose. Non-limiting examples of
therapeutic proteins that can be used in preparing some of the
embodiments of the present invention are described in further
detail below.
[0104] Glucagon-Like Peptide 1 (GLP-1).
[0105] In one embodiment, the therapeutic agent includes a GLP-1 or
a biologically active variant or fragment thereof. GLP-1 is a 37
amino acid peptide that is formed from cleavage of glucagon,
secreted by the L-cells of the intestine in response to food
ingestion (Uniprot accession number: P01275). GLP-1 can stimulate
insulin secretion, causing glucose uptake by cells and decreased
serum glucose levels (e.g., Mojsov, Int. J. Peptide Protein
Research, 40:333-343 (1992)). GLP-1 can be cleaved to produce a
biologically active peptide GLP-1 (7-37)0H. In addition, numerous
GLP-1 analogs and derivatives are known and include, e.g., exendins
which are peptides found in Gila monster venom. Exendins have
sequence homology to native GLP-1 and can bind the GLP-1 receptor
and initiate the signal transduction cascade of GLP-1 (7-37)OH.
GLP-1 compounds can have one or more of the following biological
properties: ability to stimulate insulin release, lower glucagon
secretion, inhibit gastric emptying, and enhance glucose
utilization. (e.g., Nauck et al., Diabetologia 36:741-744 (1993);
Gutniak et al., New England J. of Med. 326:1316-1322 (1992); Nauck
et al., J. Clin. Invest. 91:301-307 (1993)). A therapeutic agent
including GLP-1, GLP-1 (7-37)0H, an exendin, or biologically active
variants or fragments thereof can be used for treating a diabetic
disorder, e.g., non-insulin dependent diabetes mellitus
(NIDDM).
[0106] The amino acid sequence of human GLP-1 is as follows:
TABLE-US-00003 [SEQ ID NO: 3] HDEFERHAEG TFTSDVSSYL EGQAAKEFIA
WLVKGRG
[0107] Advantages of using a VSA in association with a GLP-1 (e.g.,
using a VSA-GLP-1 fusion protein) include, e.g., extending the PK
of the GLP-1. The extended PK can have advantages; for example, the
GLP-1 can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the GLP-1
can be achieved.
[0108] Insulin.
[0109] In one embodiment, the therapeutic agent is an insulin. The
insulin can be a full length insulin polypeptide or a biologically
active variant or fragment thereof. For example, the insulin can be
a glucose sensitive molecule that includes an insulin receptor
agonist. An exemplary insulin polypeptide (110 amino acids) has the
sequence:
TABLE-US-00004 [SEQ ID NO: 4] MALWMRLLPL LALLALWGPD PAAAFVNQHL
CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG
IVEQCCTSIC SLYQLENYCN.
[0110] The B-chain (1-30 or
TABLE-US-00005 [SEQ ID NO: 5] FVNQHL CGSHLVEALY LVCGERGFFY
TPKT)
is present at amino acids 25-54. The A-chain (1-21 or
TABLE-US-00006 [SEQ ID NO: 6] G IVEQCCTSIC SLYQLENYCN)
is present at amino acids 90-110. (See, Uniprot accession no.
P01308). The C-peptide
TABLE-US-00007 [SEQ ID NO: 7] (RREAED LQVGQVELGG GPGAGSLQPL
ALEGSLQKR)
which links the B-chain and A-chain is present at amino acids
55-89. Therapeutic proteins as disclosed herein can include the
B-chain and A-chain of insulin, linked by the C-peptide of insulin
or another natural or artificial sequence, such as a peptide linker
A number of adipose and muscle related cell lines can be used to
test for glucose uptake/transport activity in the absence or
presence of a combination of any one or more of the therapeutic
drugs listed for the treatment of diabetes mellitus. In particular,
the 3T3-L1 murine fibroblast cells and the L6 murine skeletal
muscle cells can be differentiated into 3T3-L1 adipocytes and into
myotubes, respectively, to serve as appropriate in vitro models for
the [3.sup.H]-2-deoxyglucose uptake assay (Urso et al., J Biol
Chem, 274:30864-73 (1999); Wang et al., J Mol Endocrinol, 19:241-8
(1997); Haspel et al., J Membr Biol, 169:45-53 (1999); Tsakiridis
et al., Endocrinology, 136:4315-22 (1995)). Female NOD (non-obese
diabetic) mice are characterized by displaying IDDM with a course
which is similar to that found in humans, although the disease is
more pronounced in female than male NOD mice.
[0111] Advantages of using a VSA in association with an insulin
(e.g., using a VSA-insulin fusion protein) include, e.g., extending
the PK of the insulin. The extended PK can have advantages; for
example, the insulin can be administered less frequently and/or at
reduced concentrations and/or more consistent delivery levels of
the insulin can be achieved.
[0112] Fibroblast Growth Factor 19 (FGF-19).
[0113] In one embodiment, the therapeutic agent is an FGF-19 (e.g.,
human FGF-19) or a biologically active variant or fragment thereof.
Human FGF-19 is a 216 amino acid protein, including a 24 amino acid
N-terminal signal peptide. An exemplary FGF-19 peptide sequence is
the sequence of human FGF-19 (Uniprot accession number:
O95750):
TABLE-US-00008 [SEQ ID NO: 8] MRSGCVVVHV WILAGLWLAV AGRPLAFSDA
GPHVHYGWGD PIRLRHLYTS GPHGLSSCFL RIRADGVVDC ARGQSAHSLL EIKAVALRTV
AIKGVHSVRY LCMGADGKMQ GLLQYSEEDC AFEEEIRPDG YNVYRSEKHR LPVSLSSAKQ
RQLYKNRGFL PLSHFLPMLP MVPEEPEDLR GHLESDMFSS PLETDSMDPF GLVTGLEAVR
SPSFEK
[0114] FGF-19 is active in the suppression of bile acid
biosynthesis and stimulates glucose uptake in adipocytes. Holt et
al., Genes Dev. 17:1581-91 (2003). FGF-19 can be used for treatment
of dietary and leptin-deficient diabetes. Fu et al., Endocrinology,
145:2594-603 (2004).
[0115] Advantages of using a VSA in association with FGF-19 (e.g.,
using a VSA-FGF-19 fusion protein) include, e.g., extending the PK
of the FGF-19. The extended PK can have advantages; for example,
the FGF-19 can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the FGF-19
can be achieved.
[0116] Fibroblast Growth Factor 21 (FGF-21).
[0117] In one embodiment, the therapeutic agent is an FGF-21 (e.g.,
human FGF-21) or a biologically active variant or fragment thereof.
Human FGF-21 is a 209 amino acid protein, including a 28 amino acid
N-terminal signal peptide. An exemplary FGF-21 peptide sequence is
the sequence of human FGF-21 (Uniprot accession number:
Q9NSA1):
TABLE-US-00009 [SEQ ID NO: 9] MDSDETGFEH SGLWVSVLAG LLLGACQAHP
IPDSSPLLQF GGQVRQRYLY TDDAQQTEAH LEIREDGTVG GAADQSPESL LQLKALKPGV
IQILGVKTSR FLCQRPDGAL YGSLHFDPEA CSFRELLLED GYNVYQSEAH GLPLHLPGNK
SPHRDPAPRG PARFLPLPGL PPALPEPPGI LAPQPPDVGS SDPLSMVGPS
QGRSPSYAS
[0118] FGF-21 stimulates glucose uptake in differentiated
adipocytes via the induction of the insulin-independent glucose
transporter GLUT1. In ob/ob mice, FGF-21 has been demonstrated to
have durable glucose control and triglyceride lowering effects with
minimal adverse side effects. Kharitonenkov et al., J. Clin.
Invest. 115:1627-35 (2005). Accordingly, FGF-21 is useful in
treatment of diabetes.
[0119] Advantages of using a VSA in association with FGF-21 (e.g.,
using a VSA-FGF-21 fusion protein) include, e.g., extending the PK
of the FGF-21. The extended PK can have advantages; for example,
the FGF-21 can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the FGF-21
can be achieved.
[0120] Fibroblast Growth Factor 23 (FGF-23).
[0121] In one embodiment, the therapeutic agent is an FGF-23 (e.g.,
human FGF-23) or a biologically active variant or fragment thereof.
Human FGF-23 is a 251 amino acid protein, including a 24 amino acid
N-terminal signal peptide. FGF-23 is cleaved by protein convertases
into an N-terminal peptide of approximately 155 amino acids (amino
acid 25-179); and a C-terminal peptide of approximately 72 amino
acids (amino acids 180-251). An exemplary FGF-23 peptide sequence
is the sequence of human FGF-23 (Uniprot accession number:
Q9GZV9):
TABLE-US-00010 [SEQ ID NO: 10] MLGARLRLWV CALCSVCSMS VLRAYPNASP
LLGSSWGGLI HLYTATARNS YHLQIHKNGH VDGAPHQTIY SALMIRSEDA GFVVITGVMS
RRYLCMDFRG NIFGSHYFDP ENCRFQHQTL ENGYDVYHSP QYHFLVSLGR AKRAFLPGMN
PPPYSQFLSR RNEIPLIHFN TPIPRRHTRS AEDDSERDPL NVLKPRARMT PAPASCSQEL
PSAEDNSPMA SDPLGVVRGG RVNTHAGGTG PEGCRPFAKF I
[0122] FGF-23 is involved in regulating phosphate homeostasis.
FGF-23 has been shown to inhibit renal tubule phosphate transport
(Bowe et al., Biochem. Biophys. Res. Commun., 284:977-81 (2001));
to regulate vitamin D metabolism (Shimada et al., J. Bone Miner.
Res., 19:429-35 (2004)); and to negatively regulate osteoblast
differentiation and matrix mineralization (Wang et al., J. Bone
Miner. Res., 23:939-48 (2008)). Accordingly, FGF-23 can be used in
treatment of autosomal dominant hypophosphatemic rickets (ADHR) and
tumor-induced osteomalacia (Riminucci et al., J. Clin. Invest.,
112:683-92 (2003)).
[0123] Advantages of using a VSA in association with FGF-23 (e.g.,
using a VSA-FGF-23 fusion protein) include, e.g., extending the PK
of the FGF-23. The extended PK can have advantages; for example,
the FGF-23 can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the FGF-23
can be achieved.
[0124] Interleukin-2 (IL-2).
[0125] In one embodiment, the therapeutic agent is an IL-2 (e.g.,
human IL-2) or a biologically active variant or fragment thereof.
Human IL-2 is a 153 amino acid protein, including a 20 amino acid
N-terminal signal peptide. An exemplary IL-2 peptide sequence is
the sequence of human IL-2 (Uniprot accession number: P60568):
TABLE-US-00011 [SEQ ID NO: 11] MYRMQLLSCI ALSLALVTNS APTSSSTKKT
QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL
NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS
TLT
[0126] IL-2 can induce proliferation of antigen-activated T cells
and stimulates natural killer (NK) cells, as well as stimulates
proliferation of regulatory T cells (Tregs). The biological
activity of IL-2 is mediated through a multi-subunit IL-2 receptor
complex (IL-2R) of three polypeptide subunits that span the cell
membrane: p55 (IL-2R.alpha., the alpha subunit, also known as CD25
in humans), p75 (IL-2R.beta., the beta subunit, also known as CD122
in humans) and p64 (IL-2R.gamma., the gamma subunit, also known as
CD132 in humans). IL-2-derived polypeptides can be used in cancer
immunotherapies and to deliver therapeutic agents to CD25-positive
cells in vivo or in cell culture. For example, Proleukin.RTM. is an
approved biologic that contains IL-2. Exemplary agonists are
described by Rao et al., Protein Engineering 16:1081-1087 (2003)
and Rao et al., Biochemistry 44:10696-701 (2005), and in US Patent
Application Publication 2005/0142106; see also U.S. Pat. Nos.
7,569,215 and 7,951,360. The therapeutic agent can also further
include an IL-2 binding fragment of CD25, e.g., the sushi domain of
IL-2R.alpha.. The IL-2 binding fragment of CD25 can be covalently
linked to the biologically active portion of IL-2 or can be
non-covalently linked. Exemplary IL-2 antagonists that bind CD25
but do not activate the IL-2 receptor are described in US
2011/0091412. The IL-2 antagonists can be used to specifically
target T regulatory cells and/or specifically inhibit T regulatory
cell function. The ability of low-dose IL-2 to reduce inflammation
and/or immune response may be particularly surprising in light of
previous publications advocating low doses of IL-2 for stimulation
of immune response. See, U.S. Pat. No. 6,509,313. Low-dose IL-2 can
be used to specifically induce Treg proliferation and promote an
anti-inflammatory state. In particular embodiments, low-dose IL-2
can be administered subcutaneously, for example using a
self-administered pen or cartridge device, such as is used with
other self-administered peptide biotherapeutic drugs, such as
long-acting insulin. Particular indications for which low-dose IL-2
can be appropriate include, for example, graft-vs-host-disease; for
example, in connection with bone marrow transplantation.
Administration of low-dose IL-2 can be advantageous in reducing the
need for pre-graft conditioning and/or post-graft treatment with
immunosuppressant drugs. Ferrara et al., Biology of Blood and
Marrow Transplantation, 5:347-56 (1999). Additional advantages can
include avoiding the adverse effects of short-term, high dose IL-2.
See Sykes et al., Blood, 83:2560-69 (1994).
[0127] Advantages of using a VSA in association with IL-2 (e.g.,
using a VSA-IL-2 fusion protein) include, e.g., extending the PK of
the IL-2. The extended PK can have advantages; for example, the
IL-2 can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the IL-2
can be achieved.
[0128] Interleukin-15 (IL-15).
[0129] In one embodiment, the therapeutic agent is an IL-15 (e.g.,
human IL-15) or a biologically active variant or fragment thereof.
Human IL-15 is a 162 amino acid protein, including a 29 amino acid
N-terminal signal peptide. An exemplary IL-15 peptide sequence is
the sequence of human IL-15 (Uniurot accession number: P409331:
TABLE-US-00012 [SEQ ID NO: 12] MRISKPHLRS ISIQCYLCLL LNSHFLTEAG
IHVFILGCFS AGLPKTEANW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC
FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS
FVHIVQMFIN TS
[0130] IL-15 can induce proliferation of antigen-activated T cells
and stimulate natural killer (NK) cells. The biological activity of
IL-15 is mediated through a multi-subunit receptor complex (IL-15R)
which shares the beta (CD122) and gamma (CD132) subunits of the
IL-2 receptor complex, but has a unique alpha subunit (IL-15Ra,
also known as CD215 in humans). IL-15 derived polypeptides can be
used in cancer immunotherapies. The therapeutic agent can also
further include an IL-15 binding fragment of the IL-15Ra receptor,
e.g., the sushi domain of IL-2R.alpha.. The IL-15 binding fragment
of IL-15Ra can be covalently linked to the biologically active
portion of IL-15 or can be non-covalently linked.
[0131] Advantages of using a VSA in association with IL-15 (e.g.,
using a VSA-IL-15 fusion protein) include, e.g., extending the PK
of the IL-15. The extended PK can have advantages; for example, the
IL-15 can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the IL-15
can be achieved.
[0132] Hepcidin.
[0133] In certain embodiments, the therapeutic agent is a hepcidin
(e.g., human hepcidin), or a biologically active variant of
hepcidin. Human hepcidin is an 84 amino acid protein (Uniprot
Accession number P81172), including a 24 amino acid N-terminal
signal peptide, a thirty amino acid propeptide, and a 25 amino acid
mature peptide (amino acids 60 to 84); hepcidin can be alternately
cleaved to form a 20 amino acid mature peptide (amino acids 65 to
84).
TABLE-US-00013 [SEQ ID NO: 13] MALSSQIWAA CLLLLLLLAS LTSGSVFPQQ
TGQLAELQPQ DRAGARASWM PMFQRRRRRD THFPICIFCC GCCHRSKCGM CCKT
[0134] Hepcidin is known to be involved in iron homeostasis (e.g.,
Laftah et al., Blood, 103:3940-44 (2004); U.S. Pat. No. 7,169,758).
Accordingly, hepcidin can be used an agent for regulation of iron
absorption and homeostasis. It can be used to treat abnormal iron
absorption, e.g., in individuals with .beta.-thalassemia and
related disorders.
[0135] The therapeutic agent can comprise a hepcidin peptide, or a
variant of such peptide containing one or more amino acid
variations from the mature hepcidin peptide. In a particular
embodiment, two mature hepcidin peptides are fused or joined to
each end of an albumin moiety, such as a VSA, such that the
hepcidin molecules are able to interact with the natural binding
partner for hepcidin, ferroportin. Ferroportin is known to exist in
dimers, and both monomers must be capable of binding hepcidin for
Jak2 to bind to ferroportin.
[0136] Advantages of using a VSA in association with a hepcidin
(e.g., using a VSA-hepcidin fusion protein) include, e.g.,
extending the PK of the hepcidin. The extended PK can have
advantages; for example, the hepcidin can be administered less
frequently and/or at reduced concentrations and/or more consistent
delivery levels of the hepcidin can be achieved.
[0137] Coagulation Factor VII (FVII). In certain embodiments, the
therapeutic agent is an FVII (e.g., a human FVII) or a biologically
active variant or fragment thereof. Human FVII is a 466 amino acid
protein, including a 20 amino acid N-terminal signal peptide and a
40 amino acid propeptide, a 152 amino acid light chain and a 254
amino acid heavy chain. Thim et al., Biochemistry, 27:7785-93
(1998); Hagen et al., PNAS USA 83:2412-16 (1986); O'Hara et al.,
PNAS USA 84:5158-62 (1987) and Sabater-Lleal et al., Hum Genet.
118:741-51 (2006); Hagen, U.S. Pat. No. 4,784,950; Uniprot
Accession No. P08709. Thus, the mature FVII is a single chain
glycoprotein (mol. wt. 50,000) of 406 amino acids that is secreted
into the blood where it circulates in a zymogen form. FVII
comprises a 45 amino acid Gla domain (amino acids 61 through 105);
a potentially calcium-binding 37 amino acid EGF-like domain (amino
acids 106 through 142); a 42 amino acid EGF-like domain (amino
acids 147 through 188) and a 240 amino acid serine peptidase domain
(amino acids 213 through 452). In vitro, FVII can be
proteolytically cleaved to form activated FVII, or FVIIa, by the
action of activated coagulation factors Factor X (FXa), Factor IX
(FIXa), Factor XII (FXIIa) or Factor II (FIIa). FVIIa does not
promote coagulation by itself, but can complex with tissue factor
(TF) exposed at the site of injury. The FVIIa/TF complex can
convert FX to FXa, and FIX to FIXa, thereby inducing local
hemostasis at the site of injury. Activation of FVII to FVIIa
involves proteolytic cleavage at a single peptide bond between
Arg-152 and Ile-153, resulting in a two-chain molecule consisting
of a light chain of 152 amino acid residues and a heavy chain of
254 amino acid residues held together by a single disulfide bond.
Persons with hemophilia may have normal levels of FVII. However,
they suffer from a relative deficiency in FVIIa and other activated
clotting factors.
[0138] It is known that basal levels of Factor VIIa in plasma are
greatly reduced in subjects with hemophilia B (Factor IX
deficiency) and, to a lesser extent, subjects with hemophilia A
(Factor VIII deficiency). Wildgoose et al., Blood 1:25-28 (1992).
In the absence of activated FVIIa, the intrinsic blood clotting
pathway involving FVII and FIX, is severely limited in effective
coagulation. Recently, recombinant activated Factor VII (rFVIIa,
NovoSeven.RTM., Novo, Nordisk) has been shown to have therapeutic
value to bypass or correct the coagulation defects in hemophilia A
and B subjects with inhibitors, especially in subjects with
inhibitors who were undergoing surgical procedures. NovoSeven.RTM.
is a 406 amino acid glycoprotein that is structurally similar to
plasma-derived FVIIa. However, recombinant FVIIa is expensive to
manufacture. Another critical problem is the short half-life (2
hours) of recombinant FVIIa. Therefore, recombinant FVIIa therapy
requires an intravenous infusion of high doses of the protein every
2 hours. Accordingly, sequences useful as the therapeutic agent in
the present invention include sequences encoding FVII and
FVIIa.
[0139] In particular embodiments, the agent used herein can be a
Factor VII or Factor VIIa peptide. In particular embodiments, the
Factor VII peptide can contain one or more mutations to provide an
enzymatic cleavage site, such as an enzymatic cleavage site
susceptible to cleavage by SKI-1 or furin (e.g., U.S. Pat. No.
7,615,537).
[0140] Advantages of using a VSA in association with a FVII (e.g.,
using a VSA-FVII fusion protein) include, e.g., extending the PK of
the FVII. The extended PK can have advantages; for example, the
FVII can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the FVII
can be achieved.
[0141] Coagulation Factor VIII (FVIII).
[0142] In certain embodiments, the therapeutic agent is an FVIII
(e.g., human FVIII) or a biologically active variant or fragment
thereof. For example, Toole, U.S. Pat. No. 4,757,006 discloses the
amino acid sequence of the full-length wild-type human FVIII, and
for example, Toole, U.S. Pat. No. 4,868,112 discloses the amino
acid sequence of human FVIII, wherein the B-domain has been
deleted. FVIII, also commonly referred to as antihemophilic factor
(AHF) is a large, 2351 amino acid protein that is processed into
multiple chains in a complex manner, including a 19 amino acid
signal peptide (amino acids 1 to 19); a 1313 amino acid (amino
acids 20 through 1332) or 740 amino acid (amino acids 20 through
759) light chain; a 573 amino acid B chain (amino acids 760 through
1332); and a 684 amino acid light chain (amino acids 1668 through
2351). Wood et al., Nature 312:330-37 (1984); Toole et al., Nature,
312:342-47 (1984); UniProt Accession No. P00451. AHF molecules have
been approved as therapeutic treatments for subjects with
hemophilia A (Recombinate.RTM., Baxter Healthcare/Wyeth BioPharma;
Advate.RTM., Baxter Healthcare; Kogenate.RTM., Bayer Healthcare;
Xyntha.RTM. Wyeth Pharmaceuticals; Monoclate-P.RTM., CSL Behring
LLC). It has been found that the B-domain is not essential to
activity, Pittman et al., Blood, 81:2925-35 (1993), and therefore,
B-domain-deleted Factor VIII molecules have also been approved as a
therapeutic treatment for hemophilia A (ReFacto.RTM.; Genetics
Institute/Wyeth Pharmaceuticals). See, also, U.S. Pat. No.
7,572,619. Accordingly, sequences useful as the therapeutic agent
in the present invention include sequences encoding FVIII and
B-domain deleted FVIII.
[0143] FVIII and B-domain-deleted FVIII may be expressed and/or
administered in conjunction with von Willebrand's Factor (VWF). VWF
is a large (2,813 amino acid) protein comprising multiple
repetitive domains which form multimers and acts as a chaperone
protein for FVIII. Accession number P04275. VWF/FVIII complex has
been approved as a therapeutic treatment for spontaneous or
trauma-induced bleeding episodes in patients with moderate or
severe von Willebrand Disease (Wilate.RTM., Octapharma).
[0144] Advantages of using a VSA in association with a FVIII (e.g.,
using a VSA-FVIII fusion protein) include, e.g., extending the PK
of the FVIII. The extended PK can have advantages; for example, the
FVIII can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the FVIII
can be achieved.
[0145] Coagulation Factor IX (FIX).
[0146] In certain embodiments, the therapeutic agent is an FIX
(e.g., a human FIX) or a biologically active variant or fragment
thereof. Human FIX is a 461 amino acid secreted protein, including
a 28 amino acid N-terminal signal peptide and an 18 amino acid
propeptide. The protein comprises a 415 amino acid chain [amino
acids 47 through 461]; or can be processed by human Factor XIa to
form a 145 amino acid Factor IXa light chain [amino acids 47
through 191]; a 35 amino acid propeptide/activation peptide [amino
acids 192 through 226]; and a 235 amino acid Factor IXa heavy chain
[amino acids 227 through 461]. Kurachi and Davie, PNAS USA
79:6461-64 (1982); Anson et al., Nucleic Acids Res. 11:2325-35
(1983); UniProt Accession No. P00740. Coagulation Factor 1.times.
therapy has been approved as therapeutic treatment for subjects
suffering from hemophilia B, including BeneFIX.RTM. (Wyeth);
Mononine (Behring); and Alphanine (Grifols Biologics). Accordingly,
sequences useful as the therapeutic agent in the present invention
include sequences encoding FIX (below) and FIXa.
TABLE-US-00014 [SEQ ID NO: 14] MQRVNMIMAE SPGLITICLL GYLLSAECTV
FLDHENANKI LNRPKRYNSG KLEEFVQGNL ERECMEEKCS FEEAREVFEN TERTTEFWKQ
YVDGDQCESN PCLNGGSCKD DINSYECWCP FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD
NKVVCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAETVFPDVD YVNSTEAETI
LDNITQSTQS FNDFTRVVGG EDAKPGQFPW QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE
TGVKITVVAG EHNIEETEHT EQKRNVIRII PHHNYNAAIN KYNHDIALLE LDEPLVLNSY
VTPICIADKE YTNIFLKFGS GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI
YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGK YGIYTKVSRY
VNWIKEKTKL T
[0147] Advantages of using a VSA in association with a FIX (e.g.,
using a VSA-FIX fusion protein) include, e.g., extending the PK of
the FIX. The extended PK can have advantages; for example, the FIX
can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the FIX
can be achieved.
[0148] Erythropoietin (EPO).
[0149] In one embodiment, the therapeutic agent is an EPO (e.g., a
human EPO) or a biologically active variant or fragment thereof.
Human EPO is a 193 amino acid protein, including a 27 amino acid
N-terminal signal peptide. (Uniprot Accession Number: P01588). For
example, the therapeutic agent can have the ability to increase red
blood cell production. EPO is an approved therapeutic for treatment
of anemia, and is marketed as Epogen.RTM. (Amgen, Thousand Oaks,
Calif.); Recormon.RTM. (Roche, South San Francisco, Calif.) and
under a number of other marketed names. An exemplary amino acid
sequence encoding human EPO is:
TABLE-US-00015 [SEQ ID NO: 15] MGVHECPAWL WLLLSLLSLP LGLPVLGAPP
RLICDSRVLE RYLLEAKEAE NITTGCAEHC SLNENITVPD TKVNFYAWKR MEVGQQAVEV
WQGLALLSEA VLRGQALLVN SSQPWEPLQL HVDKAVSGLR SLTTLLRALG AQKEAISPPD
AASAAPLRTI TADTFRKLFR VYSNFLRGKL KLYTGEACRT GDR
[0150] Advantages of using a VSA in association with an EPO (e.g.,
using a VSA-EPO fusion protein) include, e.g., extending the PK of
the EPO. The extended PK can have advantages; for example, the EPO
can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the EPO
can be achieved.
[0151] Granulocyte Colony Stimulating Factor (G-CSF).
[0152] In one embodiment, the therapeutic agent is a G-CSF (e.g.,
human G-CSF) or a biologically active variant or fragment thereof.
Human G-CSF is a 207 amino acid protein, including a 29 amino acid
N-terminal signal peptide. (Uniprot Accession No. P09919). For
example, the therapeutic agent can have the ability to increase the
level of neutrophils in the blood. G-CSF is an approved therapeutic
for the treatment of neutropenia, that is low neutrophil levels,
and is marketed as Neupogen.RTM. (Amgen); and as Granocyte.RTM.
(Chugai, Turnham Green, UK). An exemplary amino acid sequence
encoding human G-CSF is:
TABLE-US-00016 [SEQ ID NO: 16] MAGPATQSPM KLMALQLLLW HSALWTVQEA
TPLGPASSLP QSFLLKCLEQ VRKIQGDGAA LQEKLVSECA TYKLCHPEEL VLLGHSLGIP
WAPLSSCPSQ ALQLAGCLSQ LHSGLFLYQG LLQALEGISP ELGPTLDTLQ LDVADFATTI
WQQMEELGMA PALQPTQGAM PAFASAFQRR AGGVLVASHL QSFLEVSYRV LRHLAQP
[0153] Advantages of using a VSA in association with an G-CSF
(e.g., using a VSA-G-CSF fusion protein) include, e.g., extending
the PK of the G-CSF. The extended PK can have advantages; for
example, the G-CSF can be administered less frequently and/or at
reduced concentrations and/or more consistent delivery levels of
the G-CSF can be achieved.
[0154] Interferon.
[0155] In one embodiment, the therapeutic agent is an interferon
(e.g., a human interferon) or a biologically active variant or
fragment thereof. In some embodiments, the interferon is a human
alpha interferon, beta interferon or gamma interferon. For example,
the therapeutic can have antiviral, antibacterial or anticancer
activities, and important immunoregulatory activity, such as
activation of macrophages.
[0156] Alpha interferons are a family of closely related proteins.
Alpha interferon-2 is a 188 amino acid protein, including a 23
amino acid N-terminal signal peptide. (Uniprot Accession No.
P01563). Alpha interferon-2 is an approved therapeutic for the
treatment of various cancers, and is marketed as Roferon-A.RTM.
(Roche); and Intron-A.RTM. (Schering-Plough). An exemplary amino
acid sequence of human alpha interferon-2 is:
TABLE-US-00017 [SEQ ID NO: 17] MALTFALLVA LLVLSCKSSC SVGCDLPQTH
SLGSRRTLML LAQMRKISLF SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF
STKDSSAAWD ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT
LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKE
[0157] Beta interferon is a 187 amino acid protein, including a 21
N-terminal amino acid signal peptide. (Uniprot Accession No.
P01574). Beta interferon is an approved therapeutic for the
treatment of multiple sclerosis, and is marketed as Avonex.RTM.
(Biogen Idec, Cambridge, Mass.); Betaseron.RTM. (Berlex) and
Rebif.RTM. (Ares-Serono). See also, Houghton et al., Nucleic Acids
Res., 8:2885-94 (1980). An exemplary amino acid sequence of human
beta interferon is:
TABLE-US-00018 [SEQ ID NO: 18] MTNKCLLQIA LLLCFSTTAL SMSYNLLGFL
QRSSNFQCQK LLWQLNGRLE YCLKDRMNFD IPEEIKQLQQ FQKEDAALTI YEMLQNIFAI
FRQDSSSTGW NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS LHLKRYYGRI
LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRN
[0158] Gamma interferon is a 166 amino acid protein, including a 23
amino acid N-terminal signal peptide, and a 5 amino acid propeptide
[residues 162-166]. (Uniprot Accession No. P01579). Gamma
interferon is an approved therapeutic for the treatment of serious
infections, such as those associated with chronic granulomatous
disease, and is marketed as Actimmune.RTM. (Genentech). See also,
Rinderknecht et al., J. Biol. Chem., 259:6790-97 (1984). An
exemplary amino acid sequence of human gamma interferon is:
TABLE-US-00019 [SEQ ID NO: 19] MKYTSYILAF QLCIVLGSLG CYCQDPYVKE
AENLKKYFNA GHSDVADNGT LFLGILKNWK EESDRKIMQS QIVSFYFKLF KNFKDDQSIQ
KSVETIKEDM NVKFFNSNKK KRDDFEKLTN YSVTDLNVQR KAIHELIQVM AELSPAAKTG
KRKRSQMLFR GRRASQ
[0159] Advantages of using a VSA in association with an interferon
(e.g., using a VSA-interferon fusion protein) include, e.g.,
extending the PK of the interferon. The extended PK can have
advantages; for example, the interferon can be administered less
frequently and/or at reduced concentrations and/or more consistent
delivery levels of the interferon can be achieved.
[0160] Cytokine Antagonists.
[0161] In some embodiments, the therapeutic agent is an antagonist
of cytokine signaling, e.g., an antagonist of interleukin or
interferon signaling. For example, the therapeutic agent is an IL-1
receptor antagonist (IL-1ra) such as Kineret.RTM. (Amgen) or a
variant thereof (see, e.g., U.S. Pat. No. 6,599,873). Human IL-1Ra
is a 177 amino acid protein, including a 25 amino acid N-terminal
signal peptide (Uniprot Accession No. P018510). IL1-Ra is an
approved therapeutic for rheumatoid arthritis. An exemplary IL-1
receptor antagonist (IL-1ra) includes the following human amino
acid sequence:
TABLE-US-00020 [SEQ ID NO: 20] MEICRGLRSH LITLLLFLFH SETICRPSGR
KSSKMQAFRI WDVNQKTFYL RNNQLVAGYL QGPNVNLEEK IDVVPIEPHA LFLGIHGGKM
CLSCVKSGDE TRLQLEAVNI TDLSENRKQD KRFAFIRSDS GPTTSFESAA CPGWFLCTAM
EADQPVSLTN MPDEGVMVTK FYFQEDE
[0162] In some embodiments, the antagonist is a variant cytokine
can be a cytokine with altered function, e.g., a dominant negative
cytokine. Exemplary variant cytokines include IL-17 molecules
described in WO2011/044563 and IL-23 molecules described in
WO2011/011797.
[0163] Advantages of using a VSA in association with a cytokine
antagonist (e.g., using a VSA-cytokine antagonist fusion protein)
include, e.g., extending the PK of the cytokine antagonist. The
extended PK can have advantages; for example, the cytokine
antagonist can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the
cytokine antagonist can be achieved.
[0164] Urate Oxidase
[0165] In one embodiment, the therapeutic agent is a urate oxidase,
also known as uricase, or a biologically active variant or fragment
thereof. An exemplary urate oxidase sequence is the sequence from
Aspergillus flavus:
TABLE-US-00021 [SEQ ID NO: 21] MSAVKAARYG KDNVRVYKVH KDEKTGVQTV
YEMTVCVLLE GEIETSYTKA DNSVIVATDS IKNTIYITAK QNPVTPPELF GSILGTHFIE
KYNHIHAAHV NIVCHRWTRM DIDGKPHPHS FIRDSEEKRN VQVDVVEGKG IDIKSSLSGL
TVLKSTNSQF WGFLRDEYTT LKETWDRILS TDVDATWQWK NFSGLQEVRS HVPKFDATWA
TAREVTLKTF AEDNSASVQA TMYKMAEQIL ARQQLIETVE YSLPNKHYFE IDLSWHKGLQ
NTGKNAEVFA PQSDPNGLIK CTVGRSSLKS KL
[0166] Both humans and certain other primates lack a naturally
occurring urate oxidase protein, presumably due to an adaptive
evolution. (Wu et al., PNAS USA, 86:9412-16 (1989)). However, many
mammalian species of urate oxidase are known, including pig
(Uniprot Accession No. P16164); cynomogus monkey (Uniprot Accession
No. Q8 MHW6); baboon (Uniprot Accession No. P25689); rabbit
(Uniprot Accession No. P011645); rat (Uniprot Accession No.
P09118); and mouse (Uniprot Accession No. P25688). Urate oxidase
catalyzes the oxidation or uric acid to 5-hydroxyisourate, leading
to increased solubility and renal excretion, which can prevent
symptoms of gout. Urate oxidase is an approved therapeutic for the
treatment of hyperuricaemia, and is marketed as Rasburicase.RTM.
(recombinant uricase from aspergillus flavus produced in
saccharomyces cerevisiae; Sanofi-Aventis) or Krystexxa.RTM.
(recombinant pegylated chimeric uricase, sequence from pig/baboon,
produced in saccharomyces cerevisiae, Savient Pharmaceuticals,
Inc.). These products have been reported to be immunogenic, which
can limit the ability to treat patients repeatedly. For this
reason, the approaches provided herein can have additional
advantages in that the VSA can serve to prevent or impede the
native immune system from readily accessing the biologically active
uricase moiety, and thereby can reduce or prevent the formation of
antibodies to the biologically active uricase moiety after
administration.
[0167] Advantages of using a VSA in association with a urate
oxidase (e.g., using a VSA-urate oxidase fusion protein) include,
e.g., extending the PK of the urate oxidase. The extended PK can
have advantages; for example, the urate oxidase can be administered
less frequently and/or at reduced concentrations and/or more
consistent delivery levels of the urate oxidase can be
achieved.
[0168] C-Natriuretic Peptide (CNP).
[0169] In one embodiment, the therapeutic agent is a CNP (e.g.,
human CNP) or a biologically active variant or fragment thereof.
Human CNP is a 126 amino acid protein, including a 23 amino acid
N-terminal signal peptide; a 50 amino acid propeptide (amino acids
24-73); and a 53 amino acid CNP-53 peptide (amino acids 74-126).
The 53 amino acid CNP-53 peptide may be further processed to a 29
amino acid CNP-29 peptide (amino acids 98-126) or a 22 amino acid
CNP-22 peptide (amino acids 105-126) (Uniprot Accession No.
P23582). An exemplary amino acid sequence encoding human CNP
is:
TABLE-US-00022 [SEQ ID NO: 22] MHLSQLLACA LLLTLLSLRP SEAKPGAPPK
VPRTPPAEEL AEPQAAGGGQ KKGDKAPGGG GANLKGDRSR LLRDLRVDTK SRAAWARLLQ
EHPNARKYKG ANKKGLSKGC FGLKLDRIGS MSGLGC
[0170] CNP has been implicated in a number of physiological
processes, including vasorelaxant with powerful venodilator
effects, exhibiting the ability to reduce mean arterial pressure,
atrial pressure and cardiac output in mammals; dose-dependent
relaxant effects on bronchial smooth muscle and pulmonary arterial
relaxation; and can also have involvement in neurotransmission.
See, Barr et al., Peptides 17:1243-51 (1996).
[0171] Advantages of using a VSA in association with a CNP (e.g.,
using a VSA-CNP fusion protein) include, e.g., extending the PK of
the CNP. The extended PK can have advantages; for example, the CNP
can be administered less frequently and/or at reduced
concentrations and/or more consistent delivery levels of the CNP
can be achieved.
Production
[0172] A variety of molecular biology techniques can be used to
design nucleic acid constructs encoding a protein that includes a
serum albumin or a domain thereof. The nucleic acid sequences can
be any sequences that code for the VSA of interest. For example, a
nucleic acid sequence can be based on a known sequence that
enclodes a corresponding native (e.g., wild type) serum albumin.
For example, a sequence that encodes a wild type albumin can be
that corresponding to UniProt DB Accession No. P02768, e.g., ENI
database sequence V00494.1 (www.ebi.ac.uk/ena/data/viewN00494).
[0173] The coding sequence can include, e.g., a sequence encoding a
protein described herein, a variant of such sequence, or a sequence
that hybridizes to such sequences. An exemplary coding sequence for
mammalian expression can further include an intron. Coding
sequences can be obtained, e.g., by a variety of methods including
direct cloning, PCR, and the construction of synthetic genes.
Various methods are available to construct useful synthetic genes,
see, e.g., the GeneArt.RTM. GeneOptimizer.RTM. from Life
Technologies, Inc. (Carlsbad, Calif.), Sandhu et. al. (2008) In
Silico Biology 8: 0016; Gao et al. (2004) Biotechnol Prog, 20:
443-8; Cai et al. (2010) J Bioinformatics Sequence Analysis 2:
25-29; and Graf et al (2000), J Virol 74: 10822-10826.
[0174] The coding sequence generally employs one or more codons
according to the codon tables for eukaryotic or prokaryotic
expression. A coding sequence can be generated with specific codons
(e.g., preferred codons) and/or one or more degenerate codons. A
possible set of degenerate codons is set forth in the table
below.
TABLE-US-00023 TABLE 1 Amino Degen- acid Code Codons erate Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Stop .cndot. TAA TAG TGA
TRR
[0175] The degenerate codon can be representative of all possible
codons encoding each amino acid, but may not always be unambiguous.
For example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences disclosed
herein. Variant sequences can be readily tested for functionality
as described herein.
[0176] In some embodiments, the coding sequence includes one or
more preferred codons for the cell in which it is to be expressed.
Generally preferred codons are those that are translated most
efficiently and can include the codons that are most frequently
used by cells of the species in question. Each species can exhibit
its own codon preferences. See, e.g. Gustafsson et al. (2004)
Trends in Biotechnol. 22:346-353; Grantham et al. (1980) Nuc. Acids
Res. 8:1893 912; Haas et al. (1996) Curr. Biol. 6:315 24;
Wain-Hobson et al. (1981) Gene 13:355 64; Grosjean and Fiers (1982)
Gene 18:199 209; Holm (1986) Nuc. Acids Res. 14:3075 87; Ikemura
(1982) J. Mol. Biol. 158:573 97. For example, the amino acid
threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT. In
mammalian cells, ACC is the most commonly used Thr codon, whereas
different Thr codons may be preferred in other species. Preferred
codons for a particular species can be introduced into coding
sequences by a variety of methods, including direct cloning, PCR
mutagenesis, and the construction of synthetic genes. Introduction
of preferred codon sequences into recombinant DNA can increase
translational efficiency. In some embodiments, at least 10, 20, 30,
50, 60, 70, or 80% of the codons in a coding sequence are preferred
codons. Sequences containing preferred codons can be constructed,
and tested for expression in various species.
[0177] A protein described herein, such as a protein containing a
serum albumin domain described herein, can be expressed in
bacterial, yeast, plant, insect, or mammalian cells.
[0178] Exemplary mammalian host cells for recombinant expression
include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO
cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci.
USA 77:4216-4220, used with a DHFR selectable marker, e.g., as
described in Kaufman and Sharp (1982) Mol. Biol. 159:601-621),
lymphocytic cell lines, e.g., NS0 myeloma cells and SP2 cells, COS
cells, K562, and a cell from a transgenic animal, e.g., a
transgenic mammal. For example, the cell can be a mammary
epithelial cell.
[0179] Coding nucleic acid sequences can be maintained in
recombinant expression vectors that include additional nucleic acid
sequences, such as a sequence that regulate replications of the
vector in host cells (e.g., origins of replication) and a
selectable marker gene. The selectable marker gene facilitates
selection of host cells into which the vector has been introduced.
Exemplary selectable marker genes appropriate for mammalian cells
include the dihydrofolate reductase (DHFR) gene (for use in dhfr
host cells with methotrexate selection/amplification) and the neo
gene (for G418 selection).
[0180] Within the recombinant expression vector, the coding nucleic
acid sequences can be operatively linked to transcriptional control
sequences (e.g., enhancer/promoter regulatory elements) to drive
high levels of transcription of the genes. Examples of eukaryotic
transcriptional control sequences include the metallothionein gene
promoter, promoters and enhancers derived from eukaryotic viruses,
such as SV40, CMV, adenovirus and the like. Specific examples
include sequences including a CMV enhancer/AdMLP promoter
regulatory element or an SV40 enhancer/AdMLP promoter regulatory
element.
[0181] An exemplary recombinant expression vector also carries a
DHFR gene, which allows for selection of CHO cells that have been
transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are
cultured to allow for expression of the protein.
[0182] An adenovirus system can also be used for protein
production. By culturing adenovirus-infected non-293 cells under
conditions where the cells are not rapidly dividing, the cells can
produce proteins for extended periods of time. For instance, BHK
cells are grown to confluence in cell factories, and exposed to the
adenoviral vector encoding the secreted protein of interest. The
cells are then grown under serum-free conditions, which allows
infected cells to survive for several weeks without significant
cell division. In another method, adenovirus vector-infected 293
cells can be grown as adherent cells or in suspension culture at
relatively high cell density to produce significant amounts of
protein (See Garnier et al., (1994) Cytotechnol. 15:145-55, and Liu
et al., (2009) J. Bioscience and Bioengineering, 107:524-529). The
expressed, secreted heterologous protein can be repeatedly isolated
from the cell culture supernatant, lysate, or membrane fractions
depending on the disposition of the expressed protein in the cell.
Within the infected 293 cell production protocol, non-secreted
proteins can also be effectively obtained.
[0183] Insect cells can be infected with recombinant baculovirus,
commonly derived from Autographa californica nuclear polyhedrosis
virus (AcNPV) according to methods known in the art. Recombinant
baculovirus can be produced through the use of a transposon-based
system described by Luckow et al. (J. Virol. 67:4566-4579, 1993).
This system, which utilizes transfer vectors, is commercially
available in kit form (Bac-to-Bac.TM. kit; Life Technologies,
Rockville, Md.). An exemplary transfer vector (e.g., pFastBac1.TM.
Life Technologies) contains a Tn7 transposon to transfer the DNA
encoding the protein of interest into a baculovirus genome
maintained in E. coli as a bacmid. See, Condreay et al., (2007)
Current Drug Targets 8:1126-1131. In addition, transfer vectors can
include an in-frame fusion with DNA encoding a polypeptide
extension or affinity tag as disclosed above. Using techniques
known in the art, a transfer vector containing nucleic acid
sequence encoding a variant serum albumin fusion is transformed
into E. coli host cells, and the cells are screened for bacmids
which contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is isolated, using common techniques, and used to transfect
Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus
that expresses a protein containing a serum albumin domain is
subsequently produced. Recombinant viral stocks are made by methods
commonly used the art.
[0184] For protein production, the recombinant virus is used to
infect host cells, typically a cell line derived from the fall
armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or
Trichoplusia ni (e.g., High Five.TM. cells; Invitrogen, Carlsbad,
Calif.). See, for example, U.S. Pat. No. 5,300,435. Serum-free
media are used to grow and maintain the cells. Suitable media
formulations are known in the art and can be obtained from
commercial suppliers. The cells are grown up from an inoculation
density of approximately 2-5.times.10.sup.5 cells to a density of
1-2.times.10.sup.6 cells, at which time a recombinant viral stock
is added at a multiplicity of infection (MOI) of 0.1 to 10, more
typically near 3. Procedures used are generally known in the
art.
[0185] Other higher eukaryotic cells can also be used as hosts,
including plant cells and avian cells. Agrobacterium rhizogenes can
be used as a vector for expressing genes in plant cells. See e.g.,
O'Neill et al. (2008) Biotechnol. Prog. 24:372-376.
[0186] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Hansenula polymorpha,
Kluyveromyces lactis, Pichia pastoris, and Pichia methanotica.
Transformed cells are selected by phenotype determined by the
selectable marker, commonly drug resistance or the ability to grow
in the absence of a particular nutrient (e.g., leucine). Production
of recombinant proteins in Pichia methanolica is described, e.g.,
in U.S. Pat. No. 5,716,808, U.S. Pat. No. 5,736,383, U.S. Pat. No.
5,854,039, and U.S. Pat. No. 5,888,768.
[0187] The binding protein is recovered from the culture medium and
can be purified. Various methods of protein purification can be
employed and such methods are known in the art and described for
example in Deutscher, Methods in Enzymology, 182 (1990); and
Scopes, Protein Purification: Principles and Practice,
Springer-Verlag, New York (2010) (ISBN: 1441928332). Purified
variant serum albumin fusion proteins can be concentrated using
standard protein concentration techniques.
[0188] Exemplary of purification procedures include: ion exchange
chromatography, size exclusion chromatography, and affinity
chromatography as appropriate. For example, variant serum albumin
fusion proteins can be purified with a HSA affinity matrix.
[0189] To prepare the pharmaceutical composition a variant serum
albumin fusion protein is typically at least 10, 20, 50, 70, 80,
90, 95, 98, 99, or 99.99% pure and typically free of other proteins
including undesired human proteins and proteins of the cell from
which it is produced. It can be the only protein in the composition
or the only active protein in the composition or one of a selected
set of purified proteins. Purified preparations of a variant serum
albumin fusion protein described herein can include at least 50,
100, 200, or 500 micrograms, or at least 5, 50, 100, 200, or 500
milligrams, or at least 1, 2, or 3 grams of the binding protein.
Accordingly, also featured herein are such purified and isolated
forms of the binding proteins described herein. The term "isolated"
refers to material that is removed from its original environment
(e.g., the cells or materials from which the binding protein is
produced).
Linkers
[0190] In some embodiments described herein, a VSA is associated
with an agent (e.g., a diagnostic or therapeutic agent), e.g., for
the purpose of improving a functional property (e.g., extending the
PK) of the agent. In some embodiments, the VSA is physically
attached to the agent. The VSA can be directly attached to the
agent or it can be attached to the agent via a linker.
[0191] In some embodiments, a heterologous protein that comprises a
VSA and an additional agent (e.g., a diagnostic or therapeutic
agent) is made using recombinant DNA techniques. In some
embodiments, the VSA is produced (e.g., using recombinant DNA
techniques) and subsequently linked to the agent, e.g., by chemical
means.
[0192] A variety of linkers can be used to join a polypeptide
component of an agent to domain III or a variant serum albumin. The
linker can be a molecule or group of molecules (such as a monomer
or polymer) that connects two molecules and optionally to place the
two molecules in a particular configuration. Exemplary linkers
include polypeptide linkages between N- and C-termini of proteins
or protein domains, linkage via disulfide bonds, and linkage via
chemical cross-linking reagents.
[0193] In some embodiments, the linker includes one or more peptide
bonds, e.g., generated by recombinant techniques or peptide
synthesis. The linker can contain one or more amino acid residues
that provide flexibility. In some embodiments, the linker peptide
predominantly includes the following amino acid residues: Gly, Ser,
Ala, and/or Thr. The linker peptide should have a length that is
adequate to link two molecules in such a way that they assume the
correct conformation relative to one another so that they retain
the desired activity. Suitable lengths for this purpose include at
least one and not more than 30 amino acid residues. For example,
the linker is from about 1 to 30 amino acids in length. A linker
can also be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 and 20 amino acids in length.
[0194] Exemplary linkers include glycine-serine polymers
(including, for example, (GS)n, (GSGGS)n, (SEQ ID NO: 23), (GGGGS)n
(SEQ ID NO: 24) and (GGGS)n (SEQ ID NO: 25), where n is an integer
of at least one, e.g., one, two, three, or four), glycine-alanine
polymers, alanine-serine polymers, and other flexible linkers.
Glycine-serine polymers can serve as a neutral tether between
components. Secondly, serine is hydrophilic and therefore able to
solubilize what could be a globular glycine chain. Third, similar
chains have been shown to be effective in joining subunits of
recombinant proteins such as single chain antibodies. Suitable
linkers can also be identified from three-dimensional structures in
structure databases for natural linkers that bridge the gap between
two polypeptide chains. In some embodiments, the linker is from a
human protein and/or is not immunogenic in a human. Thus linkers
can be chosen such that they have low immunogenicity or are thought
to have low immunogenicity. For example, a linker can be chosen
that exists naturally in a human. In certain embodiments the linker
has the sequence of the hinge region of an antibody, that is the
sequence that links the antibody Fab and Fc regions; alternatively
the linker has a sequence that comprises part of the hinge region,
or a sequence that is substantially similar to the hinge region of
an antibody. Another way of obtaining a suitable linker is by
optimizing a simple linker, e.g., (Gly4Ser)n (SEQ ID NO: 24),
through random mutagenesis. Alternatively, once a suitable
polypeptide linker is defined, additional linker polypeptides can
be created to select amino acids that more optimally interact with
the domains being linked. Other types of linkers include artificial
polypeptide linkers and inteins. In another embodiment, disulfide
bonds are designed to link the two molecules. Other examples
include peptide linkers described in U.S. Pat. No. 5,073,627, the
disclosure of which is hereby incorporated by reference. In certain
cases, the diagnostic or therapeutic protein itself can be a linker
by fusing tandem copies of the peptide to a variant serum albumin
polypeptide. In certain embodiments, charged residues including
arginine, lysine, aspartic acid, or glutamic acid can be
incorporated into the linker sequence in order to form a charged
linker.
[0195] In another embodiment, linkers are formed by bonds from
chemical cross-linking agents. For example, a variety of
bifunctional protein coupling agents can be used, including but not
limited to N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Chemical linkers can enable chelation of an isotope. For example,
C.sup.14 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent
for conjugation of radionucleotide to the antibody (see PCT WO
94/11026).
[0196] The linker can be cleavable, facilitating release of a
payload, e.g., in the cell or a particular milieu For example, an
acid-labile linker, peptidase-sensitive linker, dimethyl linker or
disulfide-containing linker (Chari et al., Cancer Research
52:127-131 (1992)) can be used. In some embodiments, the linker
includes a non-proteinaceous polymer, e.g., polyethylene glycol
(PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol.
[0197] In one embodiment, the variant serum albumin fusion of the
present invention is conjugated or operably linked to another
therapeutic compound, referred to herein as a conjugate. The
conjugate can be a cytotoxic agent, a chemotherapeutic agent, a
cytokine, an anti-angiogenic agent, a tyrosine kinase inhibitor, a
toxin, a radioisotope, or other therapeutically active agent.
Chemotherapeutic agents, cytokines, anti-angiogenic agents,
tyrosine kinase inhibitors, and other therapeutic agents have been
described above, and all of these aforementioned therapeutic agents
can find use as variant serum albumin fusion conjugates. In an
alternate embodiment, the variant serum albumin fusion is
conjugated or operably linked to a toxin, including but not limited
to small molecule toxins and enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof. Small molecule toxins include but are not
limited to calicheamicin, maytansine (U.S. Pat. No. 5,208,020),
trichothene, and CC1065. In one embodiment of the invention, the
variant serum albumin fusion is conjugated to one or more
maytansine molecules (e.g. about 1 to about 10 maytansine molecules
per antibody molecule). Maytansine can, for example, be converted
to May-SS-Me which can be reduced to May-SH3 and reacted with a
variant serum albumin fusion (Chari et al., 1992, Cancer Research
52: 127-131) to generate a maytansinoid-antibody or maytansinoid-Fc
fusion conjugate. Another conjugate of interest comprises a variant
serum albumin fusion conjugated to one or more calicheamicin
molecules. The calicheamicin family of antibiotics are capable of
producing double-stranded DNA breaks at sub-picomolar
concentrations. Structural analogues of calicheamicin that can be
used include but are not limited to .gamma..sub.1.sup.1,
.alpha..sub.2.sup.1, .alpha.alpha.sub.3,
N-acetyl-.gamma..sub.1.sup.1, PSAG, and .theta..sub.1.sup.1,
(Hinman et al., 1993, Cancer Research 53:3336-3342; Lode et al.,
1998, Cancer Research 58:2925-2928) (U.S. Pat. No. 5,714,586; U.S.
Pat. No. 5,712,374; U.S. Pat. No. 5,264,586; U.S. Pat. No.
5,773,001). Dolastatin 10 analogs such as auristatin E (AE) and
monomethylauristatin E (MMAE) can find use as conjugates for the
variant serum albumin fusions of the present invention (Doronina et
al., 2003, Nat Biotechnol 21:778-84; Francisco et al., 2003 Blood
102:1458-65). Useful enzymatically active toxins include but are
not limited to diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, PCT WO 93/21232. The present
invention further contemplates a conjugate or fusion formed between
a variant serum albumin fusion of the present invention and a
compound with nucleolytic activity, for example a ribonuclease or
DNA endonuclease such as a deoxyribonuclease (DNase).
[0198] In an alternate embodiment, a variant serum albumin fusion
of the present invention can be conjugated or operably linked to a
radioisotope to form a radioconjugate. A variety of radioactive
isotopes are available for the production of radioconjugate variant
serum albumin fusions. Examples include, but are not limited to,
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32 and radioactive isotopes of
Lu.
Screening Methods
Assays
[0199] Binding of a serum albumin or a domain thereof to FcRn can
be evaluated in vitro, e.g., by surface plasmon resonance, (see,
e.g., Example 2), ELISA (see, e.g., Example 4) or other binding
assay known in the art.
[0200] FcRn can be produced as a single chain molecule, e.g., in
CHO cells. An exemplary method for producing single chain FcRn is
described in Feng et al., Protein Expression and Purification,
79:66-71 (2011).
[0201] The half-life of a protein that includes serum albumin or a
domain thereof in vivo can be evaluated in a mammal, e.g., a murine
model that includes a human FcRn. See e.g., Example 3. For example,
the protein that is evaluated can be a protein that includes serum
albumin or a domain thereof and a therapeutic agent.
Activity Assays
[0202] To assess the activity of an agent (e.g., a therapeutic
agent) that is associated with a VSA as described herein, methods
known in the art for testing the activity of the agent can be used.
The following description provides examples of such methods.
[0203] Assays for Testing the Activity of a FVII
[0204] Examples of assays that can be used to test the activity of
a FVII entity are known in the art. The following exemplary
coagulation assays are adapted from Wildgoose et al., Blood,
80:25-28 (1992). All plasma factor VIIa levels are measured in a
one-stage clotting assay using an ACL300R automated coagulation
instrument which can be purchased from Instrumentation Laboratories
(Ascoli Piceno, Italy). Test samples are diluted fivefold in 0.1
mol/L NaCl10.05 mol/L Tris-HCl/0.1% BSA pH 7.4 (TBSIBSA) and mixed
with an equal volume of hereditary factor VII deficient plasma to
yield a total volume of 100 pL. Each aliquot is subsequently
incubated for 5 minutes at 37.degree. C. with 50 KL of bovine
phospholipids (Thrombofax). Coagulation is then initiated by the
addition of a 100-kL aliquot of 10 nmol/L TF1.218 diluted in 12.5
mmol/L CaCl.sub.2/0.1 mol/L NaCl/0.05 mol/L Tris/1% BSA pH 7.4.
Coagulation times are subsequently converted to factor VIIa
concentration (nanograms per milliliter) by comparison to a
standard curve constructed with varying concentrations (0.05 to 50
ng/mL) of purified recombinant factor VIIa diluted in TBS/BSA.
[0205] Venipunctures are performed atraumatically and blood samples
drawn into citrated vacutainers. The citrated samples are
centrifuged for 15 minutes at 1,200.times.g after which time the
plasma is removed with a plastic pipette and stored at -80.degree.
C. Normal plasma samples are collected from 10 fasting and 10
nonfasting individuals who have a negative history for bleeding as
well as thrombosis and are not taking any medications at the time
of sample collection. Samples are obtained from 13 severe
hemophilia A and seven hemophilia B subjects (<1% FV1II:C and
<1% FIXC). Subjects are excluded from the study if they have
received factor concentrates, cryoprecipitate, and/or
antifibrinolytics within the previous 48 hours.
[0206] The following assay is adapted from Silveira et al.,
Arteriosclerosis and Thrombosis, 14:60-69 (1994). Coagulation
factor VII is assessed in plasma samples drawn before ingestion of
the test meal and 3, 6, and 12 hours thereafter. Factor VIIc is
determined as described in van Deijk et al., Haemostasis, 13:192-97
(1983) in an LODE coagulometer (Groningen, the Netherlands).
Briefly, 100pL of diluted plasma sample, 100/xh of factor
Vll-deficient plasma (Helena Laboratories, Beaumont, Tex.), and 100
.mu.L of human brain thromboplastin (prepared according to Owren
and Aas, Scand J. Clin Lab Invest 3:201-18 (1951) are incubated
together at 37.degree. C. for 30 seconds. Calcium, 100 .mu.L of a
33 mmol/L solution, is added and the clotting time recorded. Factor
VIIa is determined with either Thrombotest (a bovine brain
thromboplastin preparation that also contains adsorbed bovine
plasma; Nyegaard & Co, Oslo, Norway) (VIIa:A) or bovine brain
thromboplastin (Diagnostic Reagents Ltd, Thames, Oxon, UK) (VIIa:B)
in clotting assays that are otherwise essentially as described
above. Factor VII amidolytic activity (VIIam) is determined with a
commercially available kit (Coa-Set FVII; Chromogenix, M61ndal,
Sweden). Factor VII antigen (VII:Ag) concentration is determined
with an enzyme immunoassay kit (Novoclone Factor VII EIA kit, Novo
Nordic A/S, Bagsvaerd, Denmark). Results are expressed in units per
milliliter, one unit being the amount or activity of factor VII
present in 1 mL of a standard pooled plasma. Ratios of VIIa:B to
VIIc and of VIIa:B to VII:Ag are also calculated for evaluation of
the activity state of factor VII in plasma. Control experiments are
performed to rule out the possibility of a direct effect of high
plasma concentrations of TG-rich lipoproteins on the factor VII
levels obtained in the different assays. An Sf 20 to 400
lipoprotein fraction is isolated from blood that had been drawn
from a healthy control subject 3 hours after intake of a fat load.
Addition of this lipoprotein fraction to control plasma to a final
plasma level 500% of normal does not influence factor VII
measurements.
[0207] Additional examples of clotting assays that can be adapted
for use to assess the activity of a FVII in association with a VSA
are described in the following publications: Broze and Majerus, J.
Biol. Chem. 255:1242-47 (1980); Morrissey et al., Blood, 81:734-44
(1993); Herzog et al., Nature Medicine, 5:56-63 (1999); and
Sorensen and Ingerslav, J. Thrombosis and Haemostasis, 2:102-110
(2004).
Assays for Assessing the Activity of a Hepcidin
[0208] Several significant factors can be assayed to evaluate a
subject for iron overload and/or risk of iron overload. First,
serum iron levels can be used as an indicator of iron overload.
Tests include the serum ferritin test. A subject can be considered
to be suffering from iron overload if their serum iron levels are
in excess of about 350 .mu.g/dL (mild iron toxicity); generally in
excess of about 500 .mu.g/dL (serious iron toxicity). A subject is
considered to be at risk of iron overload if their serum iron
levels are high normal or above normal ranges. Normal iron range is
considered to be from about 40 to about 220 .mu.g/dL; for example,
from about 50 to about 160 .mu.g/dL for adult males. Normal iron
ranges for adult females are approximately 5 to 10 percent lower
than that for adult males. `High normal` iron concentration is
considered to be in the upper quarter (25%) of the normal range;
generally in the upper tenth (10%) of the normal range. See, Jacobs
& DeMott, Laboratory Test Handbook, 5.sup.th ed., (LexiComp
Inc, Hudson, Ohio)(2001) at p. 203-205). As is known to those
skilled in the art, `normal ranges` of iron and iron binding
capacity can vary depending upon the specific laboratory and test.
Other parameters that can be used to evaluate a patient for iron
overload and/or risk of overload include: measurement of hepcidin
levels (see U.S. Pat. No. 7,998,691); genetic testing for the
presence of mutations in one or more genes related to
hemochromatosis, such as juvenile hemochromatosis (HFE2A and HFE2B
genes) (see U.S. Pat. No. 8,080,651)
[0209] Assays for Assessing the Activity of a GLP-1
[0210] Culture of BRIN-BD11 Cells.
[0211] BRIN-BD11 cells are cultured in RPMI-1640 tissue culture
medium containing 10% (v/v) foetal calf serum, 1% (v/v) antibiotics
(100 U/ml penicillin, 0.1 mg/ml streptomycin), and 11.1 mM glucose.
BRIN-BD11 cells are produced by electrofusion of a New England
Deaconess Hospital (NEDH) rat pancreatic .beta.-cell with RINm5F
cell to produce and immortal, glucose sensitive cell line which is
described in detail elsewhere. McClenaghan et al., Diabetes (1996)
45:1132-40. All cells are maintained in sterile tissue culture
flasks (Corning Glass Works, Sunderland, UK) at 37.degree. C. in an
atmosphere of 5% CO.sub.2 and 95% air using a LEEC incubator
(Laboratory Technical Engineering, Nottingham, UK). [Green et al.,
Id.]
[0212] Stimulation of Adenylate Cyclase.
[0213] BRIN-BD11 cells are seeded into 24-well plates
(3.times.10.sup.5/well) and cultured for 48 h before being
pre-incubated in media supplemented with tritiated adenine (2
mC.sub.i) for 16 h. The cells are washed twice with cold Hanks'
buffered saline (HBS) and test solution (400 .mu.l; 37.degree. C.)
is added. The cells are then exposed to varying concentrations
(10.sup.-10-10.sup.-5M) of GLP-1 glycopeptides in HBS buffer, in
the presence of 1 mM IBMX and 5.6 mM glucose (20 min; 37.degree.
C.). Following incubation, test solutions are removed and 300 .mu.l
of lysis solution (5% TFA, 3% SDS, 5 mM of unlabelled ATP, and 300
.mu.M of unlabelled cAMP) is added. Dowex and alumina exchange
resins are used to separate tritiated cAMP from tritiated adenine
and ATP in the cell lysate, as described previously. [Miguel et
al., Biochem Pharm. (2003) 65:283-92]. The highest concentration of
GLP-1 (10.sup.-5 M) is used as a maximum. Each peptide is tested by
single experiment (n=3) which incorporated an internal control
incubation of GLP-1 (60 nM) to ensure consistency and accuracy.
[Green et al., Id.]
[0214] Insulin Secretory Responses in the Pancreatic
.beta.-Cell.
[0215] BRIN-BD11 cells are seeded into 24-multiwell plates at a
density of 1.times.10.sup.5/well, and allowed to attach during
overnight culture. Acute studies of insulin release are preceded by
a 40 minute pre-incubation at 37.degree. C. in 1.0 ml Krebs-Ringer
bicarbonate buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM
CaCl.sub.2.2H.sub.2O, 1.2 mM KH.sub.2PO.sub.4, 1.2 mM
MgSO.sub.4.7H.sub.2O, 10 mM NaHCO.sub.3, and 5 g/L bovine serum
albumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations
are performed at 37.degree. C. in the presence of 5.6 mM glucose
with a range of concentrations of GLP-1 glycopeptides
(10.sup.-12-10.sup.-6M). After a 20 minute incubation, the buffer
is removed from each well and aliquots are stored at -20.degree. C.
for measurement of insulin. [Green et al., Id.]
[0216] Glucose-Lowering and Insulin Secretory Activity in Obese
Diabetic (Ob/Ob) Mice.
[0217] The in vivo biological activity of variant serum
albumin/GLP-1 fusion proteins can be assessed in 12-16 week old
obese diabetic (ob/ob) mice. The animals are housed individually in
an air-conditioned room at 22.+-.2.degree. C. with a 12 hour
light:12 hour dark cycle. Animals are allowed drinking water ad
libitum and continuous access to standard rodent maintenance diet
(Trouw Nutrition, Cheshire, UK). Mice are fasted for 18 hours and
intraperitoneally administered 8 ml/kg body weight with saline (9
g/L NaCl), glucose alone (18 mM/kg body weight), or in combination
with GLP-1 or a variant serum albumin/GLP-1 fusion protein (25
nM/kg body weight). Blood samples are collected into chilled
fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht,
Germany) immediately prior to injection and at 15, 30, and 60
minutes post injection, and the plasma obtained is stored at
-20.degree. C. All animal studies are carried out in accord with
the UK Animals (Scientific Procedures) Act 1986 [Green et al., Id.]
or other applicable laws.
[0218] Analyses and Statistics.
[0219] Plasma glucose levels are determined using an Analox glucose
analyser (Hammersmith, London, UK), which employs the glucose
oxidase method. Insulin levels are assayed by dextran-coated
charcoal radioimmunoassay. Incremental areas under plasma glucose
and insulin curves (AUC) are calculated using GraphPad PRISM
version 3.0 (Graphpad Software, San Diego, Calif., USA), which
employs the trapezoidal rule. Results are expressed as means.+-.SEM
and data are compared as appropriate using Student's t test,
repeated measures ANOVA or one-way ANOVA, followed by the
Student-Newman-Keuls post hoc test. Groups of data are considered
significantly different if P<0.05.
[0220] Measurement of Binding Affinity and cAMP Production.
[0221] Binding affinity is assessed by measuring the inhibition of
radiolabeled GLP-1 binding to human GLP-1 receptor-expressing
chinese hamster ovary (CHO) cell membrane. Cell membrane fractions
(5 .mu.g) are incubated with 62 pM [125I]GLP-1 and variant serum
albumin/GLP-1 fusion protein (final conc. 10-11 to 10-6 M) in 25 mM
HEPES (pH 7.4) containing 5 mM MgCl, 1 mM CaCl.sub.2, 0.25 mg/mL
bacitracin, and 0.1% bovine serum albumin (BSA) at room temperature
for 2 hours (100 .mu.L). Membranes are filtered onto a 96-well GF/C
plate (PerkinElmer, Inc.) that had been presoaked in 1%
polyethylenimine containing 0.5% BSA, and then washed with 25 mM
HEPES buffer containing 0.5% BSA (pH 7.4). Radioactivity associated
with the lysates is determined using a gamma counter. Nonspecific
binding is determined by the amount of binding in the presence of 1
.mu.M unlabeled GLP-1. Dose-response curves are plotted for the
individual compounds. IC50 values are calculated using XLfit
software (IDBS Inc.). For measurement of cAMP production, human
GLP-1 receptor expressing CHO cells are passaged into multiwell
plates (4000 cells/well) and cultured for an additional 48 h. The
cells are washed with assay buffer (Hanks balanced salt solution
containing 20 mM HEPES, 0.1% BSA, pH 7.4) and then exposed to
variant serum albumin/GLP-1 fusion proteins (final conc. 10-12 to
10-6 M) in assay buffer containing 0.33 mM isobutylmethylxanthine
and 0.67 mM RO20-1724 at room temperature for 1 h. The cells are
lysed with 1% Triton X-100, and the cAMP formed is measured using a
cAMP femtomolar kit (Cis Bio international). Dose-response curves
are plotted for the individual compounds. EC50 values are
calculated using XLfit software. [Ueda et al., J. ACS, 2009
131:6237-45]
[0222] Characterization of Stability Against Recombinant Human
DPP-IV.
[0223] GLP-1 or variant serum albumin/GLP-1 fusion protein (20-500
.mu.M) is incubated at 37.degree. C. in 100 mM HEPES buffer
containing 0.05% Tween80 and 1 mM EDTA-2Na (pH 7.5) with 0.33
.mu.g/mL, 0.66 .mu.g/mL (19), or 1.32 .mu.g/mL recombinant human
DPP-IV (60 .mu.L). At 5 or 10 min intervals, 7 .mu.L is removed
from the reaction mixture, and the reaction is terminated by the
addition of 28 .mu.L of 8 M GuHCl solution. The reaction products
are subjected to RP-HPLC on a Develosil RPAQUEOUS-AR-3
2.0.degree.--100 mm at 30.degree. C., and the C-terminal
degradation product is quantified by using UV absorption at 210 nm.
The initial rate of the degradation reaction is determined from the
slope of the linear part obtained by plotting product concentration
versus time. The resulting initial rates are plotted versus peptide
concentration, and kinetic parameters (KM and KM/kcat) are
determined using XLfit software based on the Michaelis-Menten
kinetic equation. [Ueda et al., Id.]
[0224] Characterization of Stability Against Recombinant Human NEP
24.11.
[0225] The 125 .mu.M GLP-1 or variant serum albumin/GLP-1 fusion
protein is incubated at 37.degree. C. in 50 mM HEPES buffer
containing 50 mM NaCl, and 0.05% Tween 80 with 4 .mu.g/mL
recombinant human NEP 24.11 (pH 7.4, 84 .mu.L). After 0.5, 1, 2,
3.5, and 5 h, 8 .mu.L is removed from the reaction mixture, and the
reaction is terminated by addition of 32 .mu.L of 8 M GuHCl
solution. The reaction products are subjected to RP-HPLC on a
Develosil ODSHG-54.6.degree.-150 mm at 30.degree. C., and the area
of intact variant serum albumin/GLP-1 fusion protein is measured
using UV absorption at 210 nm. [Ueda et al., Id.]
[0226] Blood Glucose-Lowering Activity in Obese Diabetic Db/Db
Mice.
[0227] Male BKS.Cg-+Leprdb/+Leprdb mice (13-15 weeks of age) are
allowed ad libitum access to food and water until the start of the
experiment. At t) -2 h, access to food is restricted, and the tip
of the tail is cut. At t) 0 min, a 1 .mu.L blood sample is
collected Immediately thereafter, each mouse is injected
subcutaneously with test sample (100 nmol/kg) or vehicle, and
additional blood samples are collected. The vehicle is saline
containing 1% BSA. Blood glucose levels are measured with a glucose
oxidase biosensor (DIAMETERR; Arkray, Inc.). The effects of the
test samples on blood glucose are expressed as % change relative to
the respective pretreatment (t) 0 min) level. The number of mice
tested is 6-7 for each group. Data are presented as means (SEM.
Statistical differences are analyzed using the Dunnett's multiple
comparison test, and P values less than 0.05 are regarded as
significant. [Ueda et al., Id.]
[0228] Assays for Assessing Uric Acid/Gout
[0229] Methods for assessing uric acid levels and/or function as
well as gout are known in the art. Methods for measurements of uric
acid, e.g., in urine can be employed, as disclosed, e.g., in
Ballesta-Clayer et al., Analytica Chimica Acta, 702:254-61 (2011)
and WO 2000/08207.
[0230] Therapeutic Administration of a VSA Composition
[0231] In certain embodiments, a VSA composition (a composition
comprising a VSA or a VSA associated therapeutic agent) is
administered in a therapeutically effective amount to a subject to
treat a disease or condition, or ameliorate one or more symptoms of
a disease or condition. Methods for delivering a therapeutic
composition are known in the art and can be used to administer a
VSA composition e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells that can expressing the VSA
compound, receptor-mediated endocytosis (e.g., Wu and Wu, 1987, J.
Biol. Chem. 262:4429-4432). Methods of introduction can be enteral
or parenteral, including but not limited to, intradermal,
transdermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, pulmonary, intranasal, intraocular, epidural,
topical, intramuscular, subcutaneous, intravenous, intravascular,
and intrapericardial administration and oral routes. A VSA
composition can also be administered, for example, by infusion or
bolus injection, by absorption through epithelial or mucosa (e.g.,
oral mucosa, rectal, or intestinal mucosa) and can be administered
together with other biologically active agents. Administration can
be systemic or local. Pulmonary administration can also be
employed, e.g., by use of an inhaler or nebulizer, and formulation
with an aerosolizing agent. In certain aspects, the disclosure
provides a composition comprising the HSA variant or the chimeric
polypeptide of the disclosure, and a pharmaceutically acceptable
carrier. In certain embodiments, VSA composition is delivered
locally to an area in need of treatment (e.g., muscle); this may be
achieved, for example, by local infusion, topical application, by
injection, by catheter, or by implant (e.g., an implant of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, fibers, or commercial skin
substitutes). In some embodiments, a VSA composition is delivered
in a vesicle such as a liposome (see Langer, 1990, Science 249:
1527-1533), a controlled release system, or with a pump (see
Langer, 1990, supra), or using polymeric materials can be used (see
Howard et al, 1989, J. Neurosurg. 71: 105).
[0232] Further illustration of the invention is provided by the
following non-limiting examples.
EXAMPLES
Example 1
[0233] cDNA encoding mature human serum albumin was cloned into a
modified version of the pYC2/CT yeast expression vector
(Invitrogen) containing a Trp marker, app8 leader peptide (see
Rakestraw et al., Biotechnology and Bioengineering, 103:1192-1201
(2009) for leader sequence), and N-terminal His.sub.6 tag (SEQ ID
NO: 26) and Factor Xa cleavage site. Point mutations, either alone
or in combination, were introduced by Quikchange.RTM. mutagenesis
(Agilent). The vector was transformed into BJ5.alpha. S. cerevisiae
cells using the EZ-yeast kit (Zymo Research) and transformants
selected on SDCAA+ura plates (2% glucose, 0.67% yeast nitrogen
base, 0.5% casamino acids, 0.54% Na.sub.2PO.sub.4, 0.86%
NaH.sub.2PO.sub.4.H.sub.2O, 18.2% sorbitol, 1.5% agar, and 40 mg/L
uracil). Selected colonies were grown in 5 mL liquid SDCAA+ura
overnight at 30.degree. C. with shaking at 250 RPM. The 5 mL
overnight culture was diluted into 50 mL SDCAA+ura in a 250 mL
baffled flask and grown at 30.degree. C./250 RPM to an
OD600.about.5. Cells were then pelleted at 3000 RPM and resuspended
in 50 mL YPG media (2% galactose, 2% peptone, 1% yeast extract,
0.54% Na.sub.2PO.sub.4, 0.86% NaH.sub.2PO.sub.4.H.sub.2O) to induce
albumin expression. After 48 hours in YPG at 20.degree. C./250 RPM,
the cells were pelleted at 3000 RPM and the cleared supernatant
filter sterilized. The secreted human serum albumin was purified by
affinity chromatography using either Ni-NTA resin (Invitrogen),
CaptureSelect HSA affinity resin (BAC), or Vivapure anti-HSA kit
(Sartorius-Stedin). Eluted protein was buffer exchanged into PBS by
several rounds of concentration and dilution using Amicon Ultra-15
spin concentrators with a 10 kDa cutoff (Millipore). Protein purity
was assessed by SDS-PAGE and concentration determined by absorbance
at 280.
[0234] For single-chain FcRn production, DNA encoding human beta-2
microglobulin fused to the extracellular domain of human FcRn heavy
chain through a (G.sub.4S).sub.3 linker (SEQ ID NO: 27) was
synthesized by DNA2.0. The scFcRn DNA was cloned into a modified
version of the pcDNA3.1(+) vector (Invitrogen) containing an IL-2
leader sequence and C-terminal FLAG tag by standard digestion and
ligation. 80 mL of Freestyle-CHO-S cells (Invitrogen) at
1.times.10.sup.6 cells/mL were transiently transfected with
scFcRn-FLAG using the Freestyle.RTM.-MAX reagent (Invitrogen)
according to manufacturer's instructions. Transfected cells were
incubated at 37.degree. C., 8% CO.sub.2, with shaking at 130 rpm
for 6 days. The supernatant was clarified by centrifugation and the
scFcRn-FLAG protein purified on a 0.5 mL M2 anti-FLAG agarose
gravity-flow column (Sigma). Bound protein was eluted with 100 mM
glycine-HCl, pH 3.5 and exchanged into PBS, pH 7.4 by several
rounds of concentration and dilution using Amicon Ultra-15 spin
concentrators with a 10 kDa cutoff (Millipore). Protein purity was
assessed by SDS-PAGE and concentration determined by absorbance at
280.
Example 1A
Methods of Identifying Mutations Modulating FcRn Binding to an
Albumin Moiety
[0235] A library of albumin variants with random mutations in
domain III was generated and displayed on the surface of yeast.
FACS selections were performed to enrich for variants with improved
binding to soluble FcRn at pH 5.6. After three to four rounds of
selection, a population was identified with significantly increased
binding compared to a wild type human serum albumin (HSA). Twelve
clones from the population after sort three and eight clones from
the population after sort four were cloned and sequenced. The
sequence alignments revealed mutations appearing in multiple clones
after sort four: K402E (2/8), V424I (2/8), P447L/S (3/8), E492G
(3/8), E505G (5/8) and V547 (6/8).
[0236] Populations after sorts three, four, five and six were also
sequenced to identify other enriched mutations and the following
mutations were identified: V418M, T420A, V424I, N429D, M446V,
A449V, T467M, E505G/K/R, A552T, V547A.
[0237] After round seven, the library had enriched to a single
clone with the following mutations: V418M, T420A, E505R, V424I, and
N429D.
[0238] These data demonstrate a method for identifying mutations
that can be useful for modulating FcRn binding. Further selection
can be carried out by identifying mutations located near residues
involved in FcRn binding, e.g., near His residues, for example H510
and H535. These data also demonstrate specific sites in an HSA
useful for modulating the PK of an albumin molecule.
Example 2
Characterization of Albumin Variants
[0239] SPR was used to characterize binding of the selected
variants to human FcRn at pH values from 5.5 to 7.5. An exemplary
apparatus that can be used is a Reichert SR7000C.RTM. machine.
FLAG-tagged, single-chain human FcRn was immobilized on a 500,000
Da carboxymethyl dextran chip by NHS/EDC chemistry. Unconjungated
sites were blocked with 1 M ethanolamine. A reference channel was
generated in parallel with no FcRn. HSA variants at concentrations
of 1 nM-100 .mu.M were injected at a flow rate of 50 .mu.L/min and
the difference in signal between the FcRn channel and reference
channel was recorded over time to assess association. Wash buffer
(PBS+0.01% Tween-20) was flowed through the channels to assess
dissociation. Experiments were repeated at pH 5.5, 6.0, 6.5, and
7.4 to assess the pH dependency of binding. The results are shown
in FIG. 1. The VSAs that were used are described in Example 5.
These results demonstrate that affinity of certain VSAs to FcRn at
pH 5.5 had increased, as did the affinity of some of these to FcRn
at pH 7.4. In particular, HSA-5 and HSA-7 bound FcRn at pH 5.5 (a
typical endosomal pH), but not at pH 7.4 (a typical pH of blood).
In each case, the pH dependence of FcRn binding as known for native
albumin was preserved.
[0240] These data demonstrate that VSAs can be generated that have
increased affinity for FcRn at endosomal pH without significantly
altering the affinity for FcRn at a neutral pH (e.g., a pH
associated with blood).
Example 3
[0241] PK studies were performed for selected variants in human
FcRn mice to determine the effect of FcRn affinity on plasma
clearance. The mouse strains 4919 and 14565 from Jackson
Laboratories are homozygous for mouse FcRn knockout and either
hemi- or homozygous for human FcRn knock-in. Selected HSA variants
were injected intravenously into such mice and bleeds were
collected at various time intervals. The plasma concentration of
the HSA at each time point was assessed using a non-mouse
cross-reactive HSA ELISA and plotted to calculate clearance rates
and plasma AUC.
Example 4
[0242] ELISA studies were performed to characterize the binding of
selected HSA variants to human FcRn at pH values of 5.5-7.4.
Purified HSA variants at 1 .mu.g/mL in PBS were immobilized in a 96
well flat bottom EIA plate (Costar 9018) at 4.degree. C. overnight.
Coated wells were then blocked with 200 .mu.L PBS+2% fish gelatin,
pH 7.4 for 2 hours at room temperature. After blocking, wells were
washed 3.times. with 200 .mu.L PBS+0.1% Tween-20 at the appropriate
pH (5.5-7.4). 100 .mu.L of FLAG-tagged single-chain FcRn diluted to
concentrations of 50 pM-200 nM in PBS+0.1% fish gelatin at pH
5.5-7.4 was added to each well and incubated for 2 hours at room
temperature. Wells were then washed 3.times. with 200 .mu.L
PBS+0.1% Tween-20 at the appropriate pH. 100 .mu.L of anti-FLAG-HRP
(Sigma) diluted 1:1000 in PBS+0.1% fish gelatin at pH 5.5-7.4 was
added to each well and incubated for 60 minutes at room
temperature. Wells were washed as above and 100 .mu.L TMB substrate
(Pierce) added to each well. Color development was stopped after
two minutes with 2 M sulfuric acid and the signal read at
absorbance 450-550 on a Spectramax M5 plate reader.
Example 4A
pH Dependent Binding Using Enzyme Linked Immunosorbent Assay
(ELISA)
[0243] Experiments were carried out in which thet binding of
various VSAs and HSA to FLAG-tagged FcRn was assessed at different
pHs. Results demonstrated a range of affinities for the VSAs, which
were tested at pH 5.5, pH 6.0, and pH 7.4 for binding to 0.2 nM to
200 nM FcRn-FLAG. A table summarizing the results of such an
experiment is shown in FIG. 2. The VSAs had K.sub.Ds from 3 nM to
>100 nM at pH 5.5. Wild type HSA is known to have a K.sub.D of
about 1-2 .mu.M at pH 5.5. Many of the VSAs, including HSA-15,
HSA-13, HSA-12, HSA-7, HSA-21, HSA-11, HSA-2, HSA-14, HSA-5,
HSA-10, HSA-6, HSA-9, and HSA-18 had improved affinity for FcRn
compared with wild type HSA. Additional related information,
including the mutations present in the VSAs, is provided in Example
5, infra. The data of FIG. 3 demonstrate that the binding of these
VSAs to FcRn, as assessed by ELISA, preserved the pH dependence of
native albumin (greater binding at pH 5.5 than at pH 7.4).
[0244] These data demonstrate that VSAs with modified affinity
compared to wild type HSA can be generated and provide guidance for
generation of additional VSAs with increased affinity of FcRn at
selected pHs.
Example 5
[0245] The amino acid sequence of Domain 3 of human serum albumin
has the following sequence:
TABLE-US-00024 [SEQ ID NO: 28]
LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSR
NLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCC
TESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQ
TALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL
[0246] Exemplary human serum albumin variants that were prepared
include the sequences in the following table:
TABLE-US-00025 TABLE 2 Human Serum Albumin Variants and FcRn
Binding FcRn BINDING AMINO ACID VARIATIONS FROM [Kd@pH 5.5] VARIANT
DOMAIN 3 OF WILD-TYPE Compared to NUMBER HUMAN SERUM ALBUMIN WT-HSA
hsa-1 WT - Full Length HSA .+-. hsa-2 K573Y +++ hsa-3 WT - Domain 3
of HSA .+-. hsa-4 E492G .+-. hsa-5 V547A ++ hsa-6 E505G + hsa-7
E505G; V547A ++++ hsa-8 V418M .+-. hsa-9 T420A + hsa-10 V418M;
T420A ++ hsa-11 V418M; T420A; E505G +++ hsa-12 V418M; T420A; V547A
++++ hsa-13 V418M; T420A; E505G; V547A +++++ hsa-14 V418M; T420A;
E505G; M446V; +++ A449V; T467M; A552T hsa-15 V418M; T420A; V424I;
N429D E505R +++++ hsa-16 E505R .+-. hsa-17 E505K .+-. hsa-18 V241I
.+-. hsa-19 N429D .+-. hsa-20 M446V; A449V .+-. hsa-21 V418M;
T420A; E505R ++++ hsa-22 A552T .+-. hsa-23 PBS [Negative Control]
.+-.
[0247] All of the variants described in Table 2, with the exception
of HSA-3, are full length HSA (sequence corresponding to SEQ ID
NO:2) with the indicated mutations (numbering of the mutations is
based on SEQ ID NO:2). As indicated in Table 2, HSA-3 is Domain 3
only of HSA with the sequence corresponding to SEQ ID NO:22. In
subsequent experiments, domain 3 only versions of HSA-5 and HSA-13
were also made; these versions also demonstrated improved
affinity.
Example 5A
[0248] The experiments described in this example illustrate
experimental difficulties that were involved in accurately
identifying serum albumin mutants that exhibit improved
pharmacokinetics (e.g., increased half-life and reduced clearance)
and show how these difficulties were effectively overcome.
[0249] The anti-HSA sandwich ELISA utilized in the mouse PK studies
is unable to detect HSA spiked into cynomolgus monkey plasma due to
the high homology of human and monkey albumin. Therefore, we
developed a novel assay utilizing an epitope tag genetically fused
to albumin and an anti-tag capture antibody. HSA variants with a
FLAG tag, His tag, HA tag, or c-myc tag were expressed in yeast,
purified, and titrated in their respective anti-tag ELISA assays in
10% monkey plasma. Both the FLAG tag and HA tag were detected in
their respective assays with high sensitivity (EC50=10 ng/mL for
HA, 50 ng/mL for FLAG).
[0250] In a first cynomolgus monkey PK study, FLAG-tagged HSA-wt,
HSA-5, HSA-7, HSA-11, and HSA-13 were administered into two monkeys
each as a 5 mg/kg iv bolus dose. To control for monkey to monkey
variability and directly test for tag-specific effects on PK, a 1
mg/kg dose of HA-tagged HSA-wt was co-dosed into each animal.
Bleeds were drawn at 15 minutes, 2 hours, 6 hours, 1 day, 2 days, 5
days, 9 days, 12 days, 16 days, and 21 days, and HSA concentrations
were measured by both anti-FLAG and anti-HA ELISAs. A comparison of
the PK traces for the FLAG-tagged HSA's (both wild-type and mutant)
with the HA-tagged HSA-wt internal control indicated that the
FLAG-tagged proteins were cleared at a significantly faster rate,
particularly in the alpha phase. To confirm the more rapid
clearance of the FLAG-tagged protein, a study was performed in
which HA- and FLAG-tagged HSA-wt and HSA-7 were injected into mice.
Bleeds were drawn as before and at each time point, the
concentration of tagged HSA was measured with both an anti-HSA
ELISA and anti-tag ELISA. For HA-HSA-wt and HA-HSA-7, the anti-HSA
and anti-HA ELISA's measured HSA concentrations consistent with
each other and with previous measurement of untagged HSA in mice
suggesting that the HA tag accurately reflected the concentration
of plasma HSA. In contrast, the anti-FLAG ELISA measured a
significantly lower concentration of FLAG-HSA compared to the
anti-HSA ELISA confirming that the tag was cleaved or obscured in a
way that distorted the HSA measurement.
Example 6
[0251] HSA variant binding to mouse FcRn was measured in an ELISA
in which recombinant mouse FcRn (R&D Systems, cat#6775-FC-050)
was directly immobilized on a Costar Maxisorp.RTM. plate. After
blocking for 3 hours with PBS+4% fish gelatin, HSA variants at
concentrations of 500 nM to 685 .mu.M in PBS+0.1% Tween+0.1% fish
gelatin, pH 5.5 were added to each well and incubated for 2.5 hours
at RT. Bound HSA was detected with a HRP conjugated goat anti-HSA
antibody. The rank order of binding affinity was
HSA-13>HSA-7>HSA-11>HSA-5>HSA-wt.
[0252] To test whether the in vitro quality of increased affinity
corresponds to improved pharmacokinetics (PK) in vivo, selected
examples of VSAs, e.g., HSA-5, HSA-11, and HSA-13 and wild type HSA
were injected into wild type mice and the plasma concentration
monitored over time (to about 170 hours after administration).
[0253] In wild type mice, increased affinity in a serum albumin
extended its pharmacokinetics (see FIG. 4). The VSAs exhibited a
longer half-life and a reduced clearance compared with wild type
HSA. The observed improvement in pharmacokinetic properties was
correlated with the affinities observed in ELISA experiments that
tested the affinities of these VSAs for mouse FcRn. In particular,
greater affinity for mouse FcRn was associated with longer
half-life and reduced clearance. The highest affinity variant
HSA-13 had the most extended PK.
[0254] The results obtained in the human-FcRn transgenic mice are
shown in FIG. 5. In these mice, the relationship between affinity
for FcRn and PK was more complex; the VSAs with the highest
affinity for human FcRn did not show the greatest improvement in
pharmacokinetics.
[0255] Taken in combination, the data from experiments in mice with
VSAs indicate that the optimal FcRn affinity for PK extension is
determined by a tradeoff between improved endosomal recycling at pH
5.5 and increased surface binding at pH 7.4. In wild type mice, in
which wild type HSA binds FcRn weakly (Kd .about.80 .mu.M), the
highest affinity variant HSA-13 had the most extended PK. In
human-FcRn transgenic mice, in which wild type HSA binds FcRn with
higher affinity (Kd.about.21.1M), HSA-13 and HSA-11 bound FcRn too
tightly at pH 7.4 (see also FIG. 1), driving rapid clearance and a
robust antibody response (see FIG. 5). Medium affinity variants
HSA-5 and HSA-7 which have minimal pH 7.4 binding (see also FIG. 1)
have extended PK in the FcRn transgenic mouse, as shown in FIG. 5.
It should be noted that mouse albumin has a high affinity for human
FcRn so it is reasonable to expect that a murine model under
predicts the improvement that would be observed in humans.
Therefore, selection of VSAs suitable for use in humans should not
be tested solely in a murine model.
Example 7
VSAs in a Primate Model
[0256] To evaluate the PK of a VSA in a primate model, cynomologus
monkeys were injected with an iv bolus dose of 1 mg/kg or 5 mg/kg
dose of HSA-7 or wild type HSA (HSA-wt). Two monkeys were analyzed
per group by assaying plasma HSA concentration over time using an
anti-epitope tag ELISA.
[0257] FIG. 6 provides the PK parameters of this experiment,
demonstrating that the VSA (HSA-7) has a longer plasma half-life
and reduced beta phase clearance in a primate model.
Example 8
Fusion Proteins
[0258] When fused to a heterologous polypeptide, protein moieties
that extend half-life often do not extend half-life to the same
absolute degree as seen with the unfused moiety. This is usually
due to active clearance mediated by the heterologous partner.
Because it is not clear whether active clearance provides an
absolute maximum to the extension seen, or whether the half-life of
a fusion is a combination in some way of the half-lives of the two
components, we compared wild type HSA fused to IL-2 with a VSA
fused to IL-2. Fusion proteins in which human IL-2 is genetically
fused to the N- or C-terminus of a selected HSA variant were
produced as follows. For N-terminal fusions, cDNA encoding mature
human IL-2 was PCR amplified with a 5' primer that introduces 20-40
by of homology with the app8 yeast leader sequence and a 3' primer
that introduces a (G.sub.4S).sub.3 linker (SEQ ID NO: 27) followed
by 20-40 by of homology with the N-terminus of mature HSA. For
C-terminal fusions, the human IL-2 cDNA was PCR amplified with a 5'
primer that introduces 20-40 by of homology with the C-terminus of
HSA followed by a (G.sub.4S).sub.3 linker (SEQ ID NO: 27) and a 3'
primer that introduces 20-40 by of homology with the stop codon and
downstream sequence of the pYC2/CT vector. In both cases, a
Quikchange.RTM. mutagenesis reaction (Agilent) was then performed
in which a selected HSA variant was used as the template and the
IL-2 PCR product was used in place of the primers. DpnI treated
reactions were transformed into XL-1 Blue E. coli cells (Agilent)
and selected on LB+Amp plates. Selected colonies were miniprepped
and sequenced. In place of wild type human IL-2 cDNA, DNA encoding
a variant of IL-2, such as those described in U.S. Pat. Nos.
7,569,215 and 7,951,360, can be fused to the selected HSA
variant.
[0259] Plasmids encoding the desired fusion sequence were
transformed into BJ5.alpha. S. cerevisiae cells using the EZ-yeast
kit (Zymo Research) and transformants selected on SDCAA+ura plates.
Selected colonies were grown in liquid SDCAA+ura media at
30.degree. C. with shaking at 250 RPM to an OD600-5. Cells were
then pelleted at 3000 RPM and resuspended in YPG media to induce
albumin expression. After 48 hours in YPG at 20.degree. C./250 RPM,
the cells were pelleted at 3000 RPM and the cleared supernatant
filter sterilized. The secreted fusion protein was purified by
affinity chromatography using CaptureSelect.RTM. HSA affinity resin
(BAC).
[0260] Both mouse and human IL-2 fused to the N-terminus of HSA-wt
or HSA-13 through a (G4S).sub.2 linker were also cloned into the
pLVX-Puro mammalian expression plasmid. Fusion sequences were
amplified by PCR using primers that introduced an N-terminal XhoI
site and C-terminal MluI site. The PCR product and pLVX-Puro
plasmid were both double digested with XhoI/MluI in NEBuffer 3+BSA
and ligated with the Quick Ligation kit (New England Biolabs)
according to manufacturer's instructions. Plasmids were transiently
transfected in Hek-293 cells using PEI as a transfection reagent
and cultured for 4-7 days at 37.degree. C./8% CO.sub.2.
Example 9
Fusion Expression
[0261] Transient transfections were performed in Hek-293 cells to
express HSA, IL-2-GS10-HSA, IL-2-GS25-HSA, hepcidin-GS10-HSA, or
factor VII-GS10-HSA. Expressed proteins were evaluated using
non-reducing SDS-PAGE. All of the tested proteins were expressed
and run on the gel in a manner consistent with their expected
molecular weights. Subsequently, products of such transient
transfections were purified using an anti-HSA resin. Purified
products (murine IL-2-HSA wild type and IL-2-HSA-13) were injected
iv into mice at a dose of 0.5 mg/kg. The VSA fusion (i.e., HSA-13
fusion) extended the PK of IL-2 to a greater degree than did the
fusion to wild type HSA (shown in FIG. 7). The IL2-HSA-13 fusion
protein had increased half-life, greater AUC, and reduced
clearance.
[0262] These data demonstrate that a VSA as described herein can be
used to improve the PK of another agent, e.g., a therapeutic
protein, e.g., IL-2.
[0263] The skilled artisan, having read the above disclosure, will
recognize that numerous modifications, alterations of the above,
and additional optimization of the above, may be conducted while
remaining within the scope of the invention. These include but are
not limited to the embodiments that are within the scope of the
following claims.
Sequence CWU 1
1
281609PRTHomo sapiens 1Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp
Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly
Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln
Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val
Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu
Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90
95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala
100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe
Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val
Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn
Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala
Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe
Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala
Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu
Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215
220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val
225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala
Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr
Glu Cys Cys His Gly 260 265 270 Asp Leu Leu Glu Cys Ala Asp Asp Arg
Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile
Ser Ser Lys Leu Lys Glu Cys Cys Glu 290 295 300 Lys Pro Leu Leu Glu
Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met
Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335
Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340
345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val
Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu
Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala
Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro
Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu
Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr
Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460
Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465
470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser
Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg
Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val
Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp
Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln
Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala
Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala
Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585
590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly
595 600 605 Leu 2585PRTHomo sapiens 2Asp Ala His Lys Ser Glu Val
Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala
Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro
Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe
Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55
60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu
65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln
Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp
Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val
Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys
Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr
Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala
Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys
Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185
190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu
195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg
Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr
Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp
Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr
Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala
Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu
Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310
315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala
Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu
Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe
Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn
Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln
Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val
Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430
Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435
440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu
His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys
Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala
Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu
Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu
Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala
Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555
560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val
565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 337PRTHomo
sapiens 3His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser
Asp Val 1 5 10 15 Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu 20 25 30 Val Lys Gly Arg Gly 35 4110PRTHomo sapiens
4Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1
5 10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys
Gly 20 25 30 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu
Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu
Asp Leu Gln Val Gly 50 55 60 Gln Val Glu Leu Gly Gly Gly Pro Gly
Ala Gly Ser Leu Gln Pro Leu 65 70 75 80 Ala Leu Glu Gly Ser Leu Gln
Lys Arg Gly Ile Val Glu Gln Cys Cys 85 90 95 Thr Ser Ile Cys Ser
Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 100 105 110 530PRTHomo sapiens
5Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1
5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20
25 30 620PRTHomo sapiens 6Ile Val Glu Gln Cys Cys Thr Ser Ile Cys
Ser Leu Tyr Gln Leu Glu 1 5 10 15 Asn Tyr Cys Asn 20 735PRTHomo
sapiens 7Arg Arg Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu
Gly Gly 1 5 10 15 Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu
Glu Gly Ser Leu 20 25 30 Gln Lys Arg 35 8216PRTHomo sapiens 8Met
Arg Ser Gly Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10
15 Trp Leu Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro
20 25 30 His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His
Leu Tyr 35 40 45 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu
Arg Ile Arg Ala 50 55 60 Asp Gly Val Val Asp Cys Ala Arg Gly Gln
Ser Ala His Ser Leu Leu 65 70 75 80 Glu Ile Lys Ala Val Ala Leu Arg
Thr Val Ala Ile Lys Gly Val His 85 90 95 Ser Val Arg Tyr Leu Cys
Met Gly Ala Asp Gly Lys Met Gln Gly Leu 100 105 110 Leu Gln Tyr Ser
Glu Glu Asp Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 Asp Gly
Tyr Asn Val Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140
Leu Ser Ser Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145
150 155 160 Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu
Glu Pro 165 170 175 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe
Ser Ser Pro Leu 180 185 190 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu
Val Thr Gly Leu Glu Ala 195 200 205 Val Arg Ser Pro Ser Phe Glu Lys
210 215 9209PRTHomo sapiens 9Met 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 Leu 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 10251PRTHomo sapiens 10Met Leu Gly Ala Arg Leu Arg Leu
Trp Val Cys Ala Leu Cys Ser Val 1 5 10 15 Cys Ser Met Ser Val Leu
Arg Ala Tyr Pro Asn Ala Ser Pro Leu Leu 20 25 30 Gly Ser Ser Trp
Gly Gly Leu Ile His Leu Tyr Thr Ala Thr Ala Arg 35 40 45 Asn Ser
Tyr His Leu Gln Ile His Lys Asn Gly His Val Asp Gly Ala 50 55 60
Pro His Gln Thr Ile Tyr Ser Ala Leu Met Ile Arg Ser Glu Asp Ala 65
70 75 80 Gly Phe Val Val Ile Thr Gly Val Met Ser Arg Arg Tyr Leu
Cys Met 85 90 95 Asp Phe Arg Gly Asn Ile Phe Gly Ser His Tyr Phe
Asp Pro Glu Asn 100 105 110 Cys Arg Phe Gln His Gln Thr Leu Glu Asn
Gly Tyr Asp Val Tyr His 115 120 125 Ser Pro Gln Tyr His Phe Leu Val
Ser Leu Gly Arg Ala Lys Arg Ala 130 135 140 Phe Leu Pro Gly Met Asn
Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg 145 150 155 160 Arg Asn Glu
Ile Pro Leu Ile His Phe Asn Thr Pro Ile Pro Arg Arg 165 170 175 His
Thr Arg Ser Ala Glu Asp Asp Ser Glu Arg Asp Pro Leu Asn Val 180 185
190 Leu Lys Pro Arg Ala Arg Met Thr Pro Ala Pro Ala Ser Cys Ser Gln
195 200 205 Glu Leu Pro Ser Ala Glu Asp Asn Ser Pro Met Ala Ser Asp
Pro Leu 210 215 220 Gly Val Val Arg Gly Gly Arg Val Asn Thr His Ala
Gly Gly Thr Gly 225 230 235 240 Pro Glu Gly Cys Arg Pro Phe Ala Lys
Phe Ile 245 250 11153PRTHomo sapiens 11Met Tyr Arg Met Gln Leu Leu
Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala
Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu
His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn
Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55
60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln
Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn
Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr
Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu
Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser
Thr Leu Thr 145 150 12162PRTHomo sapiens 12Met Arg Ile Ser Lys Pro
His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu
Leu Asn Ser
His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly
Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 Asn Trp Val
Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 Gln
Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70
75 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu
Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp
Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser
Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu
Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser Phe
Val His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser
1384PRTHomo sapiens 13Met Ala Leu Ser Ser Gln Ile Trp Ala Ala Cys
Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Ala Ser Leu Thr Ser Gly Ser
Val Phe Pro Gln Gln Thr Gly 20 25 30 Gln Leu Ala Glu Leu Gln Pro
Gln Asp Arg Ala Gly Ala Arg Ala Ser 35 40 45 Trp Met Pro Met Phe
Gln Arg Arg Arg Arg Arg Asp Thr His Phe Pro 50 55 60 Ile Cys Ile
Phe Cys Cys Gly Cys Cys His Arg Ser Lys Cys Gly Met 65 70 75 80 Cys
Cys Lys Thr 14461PRTHomo sapiens 14Met Gln Arg Val Asn Met Ile Met
Ala Glu Ser Pro Gly Leu Ile Thr 1 5 10 15 Ile Cys Leu Leu Gly Tyr
Leu Leu Ser Ala Glu Cys Thr Val Phe Leu 20 25 30 Asp His Glu Asn
Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn 35 40 45 Ser Gly
Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys 50 55 60
Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn 65
70 75 80 Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly
Asp Gln 85 90 95 Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys
Lys Asp Asp Ile 100 105 110 Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly
Phe Glu Gly Lys Asn Cys 115 120 125 Glu Leu Asp Val Thr Cys Asn Ile
Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140 Cys Lys Asn Ser Ala Asp
Asn Lys Val Val Cys Ser Cys Thr Glu Gly 145 150 155 160 Tyr Arg Leu
Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe 165 170 175 Pro
Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala 180 185
190 Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu
195 200 205 Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn
Asp Phe 210 215 220 Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly
Gln Phe Pro Trp 225 230 235 240 Gln Val Val Leu Asn Gly Lys Val Asp
Ala Phe Cys Gly Gly Ser Ile 245 250 255 Val Asn Glu Lys Trp Ile Val
Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270 Val Lys Ile Thr Val
Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280 285 His Thr Glu
Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn 290 295 300 Tyr
Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu 305 310
315 320 Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys
Ile 325 330 335 Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly
Ser Gly Tyr 340 345 350 Val Ser Gly Trp Gly Arg Val Phe His Lys Gly
Arg Ser Ala Leu Val 355 360 365 Leu Gln Tyr Leu Arg Val Pro Leu Val
Asp Arg Ala Thr Cys Leu Arg 370 375 380 Ser Thr Lys Phe Thr Ile Tyr
Asn Asn Met Phe Cys Ala Gly Phe His 385 390 395 400 Glu Gly Gly Arg
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val 405 410 415 Thr Glu
Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly 420 425 430
Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser 435
440 445 Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 450 455
460 15193PRTHomo sapiens 15Met Gly Val His Glu Cys Pro Ala Trp Leu
Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro
Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val
Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile
Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile
Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80
Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85
90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser
Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala
Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu
Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser
Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg
Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu
Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg
16207PRTHomo sapiens 16Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys
Leu Met Ala Leu Gln 1 5 10 15 Leu Leu Leu Trp His Ser Ala Leu Trp
Thr Val Gln Glu Ala Thr Pro 20 25 30 Leu Gly Pro Ala Ser Ser Leu
Pro Gln Ser Phe Leu Leu Lys Cys Leu 35 40 45 Glu Gln Val Arg Lys
Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 50 55 60 Leu Val Ser
Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 65 70 75 80 Val
Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 85 90
95 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
100 105 110 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu
Gly Ile 115 120 125 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln
Leu Asp Val Ala 130 135 140 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met
Glu Glu Leu Gly Met Ala 145 150 155 160 Pro Ala Leu Gln Pro Thr Gln
Gly Ala Met Pro Ala Phe Ala Ser Ala 165 170 175 Phe Gln Arg Arg Ala
Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 180 185 190 Phe Leu Glu
Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 195 200 205
17188PRTHomo sapiens 17Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu
Leu Val Leu Ser Cys 1 5 10 15 Lys Ser Ser Cys Ser Val Gly Cys Asp
Leu Pro Gln Thr His Ser Leu 20 25 30 Gly Ser Arg Arg Thr Leu Met
Leu Leu Ala Gln Met Arg Lys Ile Ser 35 40 45 Leu Phe Ser Cys Leu
Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu 50 55 60 Glu Phe Gly
Asn Gln Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His 65 70 75 80 Glu
Met Ile Gln Gln Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser 85 90
95 Ala Ala Trp Asp Glu Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
100 105 110 Gln Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Gly Val
Gly Val 115 120 125 Thr Glu Thr Pro Leu Met Lys Glu Asp Ser Ile Leu
Ala Val Arg Lys 130 135 140 Tyr Phe Gln Arg Ile Thr Leu Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro 145 150 155 160 Cys Ala Trp Glu Val Val Arg
Ala Glu Ile Met Arg Ser Phe Ser Leu 165 170 175 Ser Thr Asn Leu Gln
Glu Ser Leu Arg Ser Lys Glu 180 185 18187PRTHomo sapiens 18Met Thr
Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser 1 5 10 15
Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg 20
25 30 Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly
Arg 35 40 45 Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile
Pro Glu Glu 50 55 60 Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp
Ala Ala Leu Thr Ile 65 70 75 80 Tyr Glu Met Leu Gln Asn Ile Phe Ala
Ile Phe Arg Gln Asp Ser Ser 85 90 95 Ser Thr Gly Trp Asn Glu Thr
Ile Val Glu Asn Leu Leu Ala Asn Val 100 105 110 Tyr His Gln Ile Asn
His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125 Lys Glu Asp
Phe Thr Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140 Arg
Tyr Tyr Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser 145 150
155 160 His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe
Tyr 165 170 175 Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn 180 185
19166PRTHomo sapiens 19Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln
Leu Cys Ile Val Leu 1 5 10 15 Gly Ser Leu Gly Cys Tyr Cys Gln Asp
Pro Tyr Val Lys Glu Ala Glu 20 25 30 Asn Leu Lys Lys Tyr Phe Asn
Ala Gly His Ser Asp Val Ala Asp Asn 35 40 45 Gly Thr Leu Phe Leu
Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp 50 55 60 Arg Lys Ile
Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe 65 70 75 80 Lys
Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile 85 90
95 Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg
100 105 110 Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu
Asn Val 115 120 125 Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met
Ala Glu Leu Ser 130 135 140 Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg
Ser Gln Met Leu Phe Arg 145 150 155 160 Gly Arg Arg Ala Ser Gln 165
20177PRTHomo sapiens 20Met Glu Ile Cys Arg Gly Leu Arg Ser His Leu
Ile Thr Leu Leu Leu 1 5 10 15 Phe Leu Phe His Ser Glu Thr Ile Cys
Arg Pro Ser Gly Arg Lys Ser 20 25 30 Ser Lys Met Gln Ala Phe Arg
Ile Trp Asp Val Asn Gln Lys Thr Phe 35 40 45 Tyr Leu Arg Asn Asn
Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn 50 55 60 Val Asn Leu
Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala 65 70 75 80 Leu
Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys 85 90
95 Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp
100 105 110 Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile
Arg Ser 115 120 125 Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala
Cys Pro Gly Trp 130 135 140 Phe Leu Cys Thr Ala Met Glu Ala Asp Gln
Pro Val Ser Leu Thr Asn 145 150 155 160 Met Pro Asp Glu Gly Val Met
Val Thr Lys Phe Tyr Phe Gln Glu Asp 165 170 175 Glu
21302PRTAspergillus flavus 21Met Ser Ala Val Lys Ala Ala Arg Tyr
Gly Lys Asp Asn Val Arg Val 1 5 10 15 Tyr Lys Val His Lys Asp Glu
Lys Thr Gly Val Gln Thr Val Tyr Glu 20 25 30 Met Thr Val Cys Val
Leu Leu Glu Gly Glu Ile Glu Thr Ser Tyr Thr 35 40 45 Lys Ala Asp
Asn Ser Val Ile Val Ala Thr Asp Ser Ile Lys Asn Thr 50 55 60 Ile
Tyr Ile Thr Ala Lys Gln Asn Pro Val Thr Pro Pro Glu Leu Phe 65 70
75 80 Gly Ser Ile Leu Gly Thr His Phe Ile Glu Lys Tyr Asn His Ile
His 85 90 95 Ala Ala His Val Asn Ile Val Cys His Arg Trp Thr Arg
Met Asp Ile 100 105 110 Asp Gly Lys Pro His Pro His Ser Phe Ile Arg
Asp Ser Glu Glu Lys 115 120 125 Arg Asn Val Gln Val Asp Val Val Glu
Gly Lys Gly Ile Asp Ile Lys 130 135 140 Ser Ser Leu Ser Gly Leu Thr
Val Leu Lys Ser Thr Asn Ser Gln Phe 145 150 155 160 Trp Gly Phe Leu
Arg Asp Glu Tyr Thr Thr Leu Lys Glu Thr Trp Asp 165 170 175 Arg Ile
Leu Ser Thr Asp Val Asp Ala Thr Trp Gln Trp Lys Asn Phe 180 185 190
Ser Gly Leu Gln Glu Val Arg Ser His Val Pro Lys Phe Asp Ala Thr 195
200 205 Trp Ala Thr Ala Arg Glu Val Thr Leu Lys Thr Phe Ala Glu Asp
Asn 210 215 220 Ser Ala Ser Val Gln Ala Thr Met Tyr Lys Met Ala Glu
Gln Ile Leu 225 230 235 240 Ala Arg Gln Gln Leu Ile Glu Thr Val Glu
Tyr Ser Leu Pro Asn Lys 245 250 255 His Tyr Phe Glu Ile Asp Leu Ser
Trp His Lys Gly Leu Gln Asn Thr 260 265 270 Gly Lys Asn Ala Glu Val
Phe Ala Pro Gln Ser Asp Pro Asn Gly Leu 275 280 285 Ile Lys Cys Thr
Val Gly Arg Ser Ser Leu Lys Ser Lys Leu 290 295 300 22126PRTHomo
sapiens 22Met His Leu Ser Gln Leu Leu Ala Cys Ala Leu Leu Leu Thr
Leu Leu 1 5 10 15 Ser Leu Arg Pro Ser Glu Ala Lys Pro Gly Ala Pro
Pro Lys Val Pro 20 25 30 Arg Thr Pro Pro Ala Glu Glu Leu Ala Glu
Pro Gln Ala Ala Gly Gly 35 40 45 Gly Gln Lys Lys Gly Asp Lys Ala
Pro Gly Gly Gly Gly Ala Asn Leu 50 55 60 Lys Gly Asp Arg Ser Arg
Leu Leu Arg Asp Leu Arg Val Asp Thr Lys 65 70 75 80 Ser Arg Ala Ala
Trp Ala Arg Leu Leu Gln Glu His Pro Asn Ala Arg 85 90 95 Lys Tyr
Lys Gly Ala Asn Lys Lys Gly Leu Ser Lys Gly Cys Phe Gly 100 105 110
Leu Lys Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly Cys 115 120 125
235PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Gly Ser Gly Gly Ser 1 5
245PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 24Gly Gly Gly Gly Ser 1 5
254PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Gly Gly Gly Ser 1 266PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
6xHis tag" 26His His His His His His 1 5 2715PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 27Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 1 5 10 15 28206PRTHomo sapiens 28Leu Val Glu Glu Pro Gln Asn
Leu Ile Lys Gln Asn Cys Glu Leu Phe 1 5 10 15 Glu Gln Leu Gly Glu
Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr 20 25 30 Thr Lys Lys
Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser 35 40 45 Arg
Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala 50 55
60 Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
65 70 75 80 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val
Thr Lys 85 90 95 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys
Phe Ser Ala Leu 100 105 110 Glu Val Asp Glu Thr Tyr Val Pro Lys Glu
Phe Asn Ala Glu Thr Phe 115 120 125 Thr Phe His Ala Asp Ile Cys Thr
Leu Ser Glu Lys Glu Arg Gln Ile 130 135 140 Lys Lys Gln Thr Ala Leu
Val Glu Leu Val Lys His Lys Pro Lys Ala 145 150 155 160 Thr Lys Glu
Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val 165 170 175 Glu
Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu 180 185
190 Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 195 200
205
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References