U.S. patent application number 14/974114 was filed with the patent office on 2016-06-23 for single chain fc fusion proteins.
The applicant listed for this patent is Alkermes, Inc.. Invention is credited to Juan Alvarez, Heather R. Brodkin, Leslie A. McSweeney, Demetri T. Moustakas.
Application Number | 20160175458 14/974114 |
Document ID | / |
Family ID | 56127666 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160175458 |
Kind Code |
A1 |
Alvarez; Juan ; et
al. |
June 23, 2016 |
SINGLE CHAIN FC FUSION PROTEINS
Abstract
The present invention provides novel, single chain Fc fusion
proteins having improved properties. The invention provides single
chain fusions of soluble proteins fused to the Fc region of an
immunoglobulin via a novel linker comprising a constant region of
an immunoglobulin light chain linked to a CH1 constant region of an
immunoglobulin heavy chain. This novel linker confers favorable
properties on the Fc fusion proteins of the invention such as
improved bioactivity and increased half-life as compared to non-Fc
fusion counterparts or as compared to prior art Fc fusion proteins.
The novel Fc fusion protein scaffold as described herein may be
designed to include soluble proteins of interest capable of binding
or interacting with any target of interest. Preferably, the Fc
fusion protein of the invention is a dimer. The dimer preferably
forms via a disulfide bond between free cysteine residues in the
hinge region of two monomeric Fc fusion proteins of the
invention.
Inventors: |
Alvarez; Juan; (Chelmsford,
MA) ; Moustakas; Demetri T.; (Belmont, MA) ;
Brodkin; Heather R.; (Warwick, RI) ; McSweeney;
Leslie A.; (Milford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alkermes, Inc. |
Waltham |
MA |
US |
|
|
Family ID: |
56127666 |
Appl. No.: |
14/974114 |
Filed: |
December 18, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62094242 |
Dec 19, 2014 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
424/135.1; 424/85.6; 435/219; 530/351; 530/387.3 |
Current CPC
Class: |
A61K 38/179 20130101;
A61P 13/12 20180101; A61P 1/04 20180101; C07K 14/7151 20130101;
A61P 17/00 20180101; C07K 14/7155 20130101; A61K 38/4846 20130101;
A61P 25/24 20180101; A61K 38/1793 20130101; A61P 27/16 20180101;
A61P 43/00 20180101; A61K 38/2013 20130101; A61K 47/64 20170801;
C07K 14/70578 20130101; C07K 2319/30 20130101; A61P 7/04 20180101;
A61P 37/02 20180101; C07K 14/565 20130101; C07K 2319/70 20130101;
A61K 38/00 20130101; C12N 9/644 20130101; C07K 14/5428 20130101;
A61P 37/06 20180101; C12Y 304/21022 20130101; A61P 17/06 20180101;
C07K 14/55 20130101; A61P 35/00 20180101; A61K 38/2066 20130101;
A61K 38/215 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 14/715 20060101 C07K014/715; C07K 14/55 20060101
C07K014/55; C07K 14/565 20060101 C07K014/565; C12N 9/64 20060101
C12N009/64; A61K 38/17 20060101 A61K038/17; A61K 38/20 20060101
A61K038/20; A61K 38/21 20060101 A61K038/21; A61K 38/48 20060101
A61K038/48; C07K 14/705 20060101 C07K014/705; C07K 14/54 20060101
C07K014/54 |
Claims
1. A single chain fusion protein having the following arrangement
from amino-terminus to carboxy-terminus: X-L1-HINGE-Fc wherein, X
is a soluble protein or any active fragment or derivative thereof;
L1 is a linker having the following arrangement from amino-terminus
to carboxy-terminus: L2-CL-L3-CH1-L4 or L2-CH1-L3-CL-L4 wherein, L2
and L4 are independently polypeptide linkers or are independently
absent; L3 is a polypeptide linker; CL is a constant region
polypeptide of an immunoglobulin light chain; and CH1 is a constant
region polypeptide from a CH1 domain of an immunoglobulin heavy
chain; HINGE is a hinge sequence of an immunoglobulin or is absent
with the proviso that if HINGE is absent, L4 is present; and Fc is
the carboxy-terminus of an immunoglobulin or any active fragment or
derivative thereof.
2. The fusion protein of claim 1, wherein CL, CH1, HINGE and Fc are
at least 90% identical to the CL, CH1, hinge and Fc regions
respectively of human IgG1.
3. The fusion protein of claim 1, wherein Xis Factor IX, TNFR2 or
IL1Ra or any active fragment or derivative thereof.
4. The fusion protein of claim 1, wherein L3 is a polypeptide
linker having the amino acid sequence (GGGGS).sub.n wherein n is
1-5 (SEQ ID NO: 27).
5. The fusion protein of claim 1, wherein L2 is present and is a
polypeptide linker having the amino acid sequence (GGGGS).sub.n
wherein n is 1-5 (SEQ ID NO: 27).
6. The fusion protein of claim 1, wherein L4 is present and is a
polypeptide linker having the amino acid sequence (GGGGS).sub.n
wherein n is 1-5 (SEQ ID NO: 27).
7. The fusion protein of claim 1, wherein HINGE and L2 are present
and L4 is absent.
8. The fusion protein of claim 1, wherein HINGE, L2 and L4 are
present.
9. The fusion protein of claim 1, wherein HINGE is absent and L4 is
present.
10. The fusion protein of claim 1, wherein HINGE is absent and L2
and L4 are present.
11. A dimerized complex comprising the fusion protein of claim
1.
12. The dimerized complex of claim 11, wherein the dimerized
complex is a homodimeric complex.
13. The fusion protein of claim 1, wherein X comprises a soluble
protein that has been modified by circular permutation.
14. The fusion protein of claim 13, wherein X comprises circularly
permuted IL-2.
15. The fusion protein of claim 13, wherein X comprises circularly
permuted IL-2 fused to IL-2R.alpha. via an optional linker.
16. The fusion protein of claim 1, wherein X is IFN.beta..
17. The fusion protein of claim 1, wherein X is IL-10, IL-2,
IL-2R.alpha. or fusions thereof, or IFN.beta. or any active
fragment or derivatives thereof.
18. The fusion protein of claim 1, wherein X is IL-10.
19. A homodimeric complex of the fusion protein of claim 18.
20. A method of treating auditory disorders, renal cell carcinoma,
melanoma, psoriasis, fibrosis, depression, or inflammatory bowel
disease (IBD) in a patient comprising administering to the patient
a therapeutically effective amount of a fusion protein of claim 18
or a homodimeric complex thereof.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/094,242, filed on Dec. 19, 2014. The entire
teachings of the above application are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] One strategy for increasing serum half-life of a therapeutic
protein is to attach the protein to an Fc (fragment crystallizable)
domain of an antibody. Many such fusion proteins are capable of
forming homodimers or heterodimers thereby forming antibody-like
fusion protein molecules. However, many prior art approaches to Fc
fusion protein engineering have limitations including, but not
limited to, immunogenicity and poor pharmacokinetic properties.
[0003] The present invention provides monomers and dimers of Fc
fusion proteins comprising novel linkers having single chain
constant light (CL) and constant heavy (CH1) immunoglobulin
domains. Such novel linkers are also referred to herein as scCLCH1
linkers.
[0004] Without limitation to a particular theory, the novel linkers
of the invention reduce steric hindrance between the protein
"payloads" on each of the single chain Fc fusion protein molecules
when such molecules form dimers. Steric hindrance can result in
losses in bioactivity, inefficient dimerization or reduction in the
half-life of the dimer molecule for example, due to reduced binding
to the FcRn. Thus incorporation of the novel linkers of the
invention may result in improvement in bioactivity, increased dimer
formation, in increased half-life, and the ability to incorporate
larger protein payloads than those possible on prior Fc fusion
proteins. Additionally, in some Fc proteins of the invention are
able to form dimers that provide a more native antibody structure
around the Fc domain that may improve binding of the dimer
molecules to the FcRn receptor and therefore increase the
circulating half-life of the novel Fc fusion proteins of the
invention as compared to prior art fusion proteins.
SUMMARY OF THE INVENTION
[0005] The present invention provides novel, single chain Fc fusion
proteins having improved properties. The invention provides single
chain fusions of soluble proteins fused to the Fc region of an
immunoglobulin via a novel linker comprising a constant region of
an immunoglobulin light chain (CL) linked to a CH1 constant region
of an immunoglobulin heavy chain (scCLCH1 or scCH1CL linkers). This
novel linker confers favorable properties on the Fc fusion proteins
of the invention such as improved bioactivity and increased
half-life as compared to non-Fc fusion counterparts or as compared
to prior art Fc fusion proteins. The novel Fc fusion proteins as
described herein may be designed to include soluble proteins of
interest capable of binding or interacting with any target of
interest with high specificity and affinity.
[0006] Preferably, an Fc fusion protein of the invention is a
dimer. The dimers may be formed via covalent (e.g. disulfide
linkages) or non-covalent interactions between single chain fusion
proteins of the invention resulting in a homodimeric or
heterodimeric protein complex retaining the advantageous properties
of an antibody molecule for use as a therapeutic molecule.
[0007] In another aspect, the invention provides nucleic acids
encoding the Fc fusion proteins provided herein. Also provided are
vectors, including expression vectors, comprise a nucleic acid
encoding any of the Fc fusion proteins described herein. Also
provided are host cells containing such expression vectors and
methods for producing the Fc fusion proteins described herein in
the host cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
[0009] FIG. 1A is a diagram of an Fc fusion protein homodimer
comprising X fused to the Fc region of an IgG1 antibody via the
novel scCLCH1 linker in accordance with the invention.
[0010] FIG. 1B is a diagram of an Fc fusion protein heterodimer of
two polypeptide chains, where the first comprises X fused to the Fc
region of an IgG1 antibody via the novel scCLCH1 linker, and the
second comprises Y, where Y is different from X, fused to the Fc
region of an IgG1 antibody via the novel scCLCH1 linker in
accordance with the invention.
[0011] FIG. 1C is a diagram of an Fc fusion protein homodimer
comprising X fused to the Fc region of an IgG1 antibody via the
novel scCH1CL linker in accordance with the invention.
[0012] FIG. 1D is a diagram of an Fc fusion protein heterodimer of
two polypeptide chains, where the first comprises X fused to the Fc
region of an IgG1 antibody via the novel scCH1CL linker, and the
second comprises Y, where Y is different from X, fused to the Fc
region of an IgG1 antibody via the novel scCH1CL linker in
accordance with the invention.
[0013] FIG. 2 is an SDS-PAGE showing expression of an Fc fusion
protein comprising Factor IX fused to the Fc region of an IgG1
antibody via the novel scCLCH1 linker in accordance with the
invention.
[0014] FIG. 3 is graph showing the clotting activity of an Fc
fusion protein comprising Factor IX fused to the Fc region of an
IgG1 antibody via the novel scCLCH1 linker in accordance with the
invention.
[0015] FIG. 4 is a graph showing the in vivo half-life in rats when
intravenously administered an Fc fusion protein comprising Factor
IX fused to the Fc region of an IgG1 antibody via the novel scCLCH1
linker.
[0016] FIG. 5A is an SDS-PAGE showing expression of an Fc fusion
protein comprising TNFR2 fused to the Fc region of an IgG1 antibody
via the novel scCLCH1 linker under reducing conditions.
[0017] FIG. 5B is an SDS-PAGE showing expression of an Fc fusion
protein comprising TNFR2 fused to the Fc region of an IgG1 antibody
via the novel scCLCH1 linker under non-reducing conditions.
[0018] FIG. 6 is a graph showing the inhibition of the activation
of a reporter gene by the TNFR2 fusion protein of the invention as
compared to a standard TNF direct fusion protein.
[0019] FIG. 7 is a graph showing the in vivo half-life in rats when
intravenously administered an Fc fusion protein comprising TNFR2
fused to the Fc region of an IgG1 antibody via the novel scCLCH1
linker as compared to a standard TNF direct fusion protein.
[0020] FIG. 8A is an SDS-PAGE showing expression of an Fc fusion
protein comprising IL1Ra fused to the Fc region of an IgG1 antibody
via the novel scCLCH1 linker under reducing conditions.
[0021] FIG. 8B is an SDS-PAGE showing expression of an Fc fusion
protein comprising IL1Ra fused to the Fc region of an IgG1 antibody
via the novel scCLCH1 linker under non-reducing conditions.
[0022] FIG. 9 is a graph showing the inhibition of the activation
of a reporter gene by the IL1Ra fusion protein of the
invention.
[0023] FIG. 10 is a graph showing the in vivo half-life in rats
when intravenously administered an Fc fusion protein comprising
IL1Ra fused to the Fc region of an IgG1 antibody via the novel
scCLCH1 linker.
[0024] FIG. 11 is a graph showing the in vivo half-life in rats
when intraocularly administered an Fc fusion protein comprising
IL1Ra fused to the Fc region of an IgG1 antibody via the novel
scCLCH1 linker.
[0025] FIG. 12A is a diagram of an Fc fusion protein homodimer of
two polypeptide chains, wherein in each polypeptide chain comprises
as X, a fusion of IL-2/IL-2R.alpha. which is then fused to the Fc
region of an IgG1 antibody via the novel scCH1CLlinker.
[0026] FIG. 12B is a diagram of an Fc fusion protein homodimer of
two polypeptide chains, wherein in each polypeptide chain comprises
as X, a fusion of IL-2/IL-2R.alpha. which is then fused to the Fc
region of an IgG1 antibody via the novel scCH1CLlinker.
[0027] FIG. 13 is an SDS-PAGE showing expression of an Fc fusion
protein comprising IL-2/IL-2R.alpha. fused to the Fc region of an
IgG1 antibody via the novel scCLCH1 linker (left) or via the novel
scCH1CL linker (right) under reducing and non-reducing
conditions.
[0028] FIG. 14A is a chromatogram showing the characterization of
the IL-2/IL-2R.alpha. fused to the Fc region of an IgG1 antibody
via the novel scCLCH1 linker by analytical gel filtration.
[0029] FIG. 14B is a chromatogram showing the characterization of
the IL-2/IL-2R.alpha. fused to the Fc region of an IgG1 antibody
via the novel scCH1CL linker by analytical gel filtration.
[0030] FIG. 15 is a graph showing activation of pSTAT5 by the
IL-2/IL-2R.alpha. single chain fusion proteins of the invention as
compared to rhIL-2.
[0031] FIG. 16 is a graph showing the in vivo half-life in rats
when intravenously and subcutaneously administered the
IL-2/IL-2R.alpha. single chain fusion proteins of the
invention.
[0032] FIG. 17 is a diagram of an Fc fusion protein homodimer of
two polypeptide chains, wherein in each polypeptide chain comprises
as X, IFN.beta. which is then fused to the Fc region of an IgG1
antibody via the novel scCLCH1 linker of the invention.
[0033] FIG. 18 is an SDS-PAGE showing expression of an Fc fusion
protein comprising IFN.beta. fused to the Fc region of an IgG1
antibody via the novel scCLCH1 linker under reducing and
non-reducing conditions.
[0034] FIG. 19 is a chromatogram showing the characterization of
IFN.beta. fused to the Fc region of an IgG1 antibody via the novel
scCLCH1 linker by analytical gel filtration.
[0035] FIG. 20 is graph showing the activation of a reporter gene
by the IFN.beta. fusion protein of the invention.
[0036] FIG. 21 is graph showing the mean concentration-time profile
after IV (1.4 nMole/Kg and SC (3.6 nMole/kg) administration of the
IFN.beta. fusion protein of the invention.
[0037] FIG. 22A is a diagram of an Fc fusion protein homodimer of
two polypeptide chains, wherein in each polypeptide chain comprises
as X, IL-10 which is then fused to the Fc region of an IgG1
antibody via the novel scCLCH1 linker.
[0038] FIG. 22B is a diagram of an Fc fusion protein homodimer of
two polypeptide chains, wherein in each polypeptide chain comprises
as X, IL-10 which is then fused to the Fc region of an IgG1
antibody via the novel scCH1CL linker.
[0039] FIG. 23 is an SDS-PAGE showing expression of an Fc fusion
protein comprising IL-10 fused to the Fc region of an IgG1 antibody
via the novel scCLCH1 linker (left) or via the novel scCH1CL linker
(right) under reducing and non-reducing conditions.
[0040] FIG. 24A is a chromatogram showing the characterization of
the IL-10 fused to the Fc region of an IgG1 antibody via the novel
scCLCH1 linker by analytical gel filtration.
[0041] FIG. 24B is a chromatogram showing the characterization of
the IL-10 fused to the Fc region of an IgG1 antibody via the novel
scCH1CL linker by analytical gel filtration.
[0042] FIG. 25 is a graph showing stimulation of mouse mast cell
line MC/9 by the IL-10 single chain fusion proteins of the
invention as compared to the scIL-10 direct Fc fusion protein used
as a control.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0043] By "polypeptide" is meant any sequence of two or more amino
acids, regardless of length, post-translation modification, or
function. "Polypeptide," "peptide," and "protein" are used
interchangeably herein. Polypeptides can include natural amino
acids and non-natural amino acids. Polypeptides can also be
modified in any of a variety of standard chemical ways (e.g., an
amino acid can be modified with a protecting group; the
carboxy-terminal amino acid can be made into a terminal amide
group; the amino-terminal residue can be modified with groups to,
e.g., enhance lipophilicity; or the polypeptide can be chemically
glycosylated or otherwise modified to increase stability or in vivo
half-life). Polypeptide modifications can include the attachment of
another structure such as a cyclic compound or other molecule to
the polypeptide and can also include polypeptides that contain one
or more amino acids in an altered configuration (i.e., R or S; or,
L or D).
[0044] As used herein, "antibody" and "immunoglobulin" are used
interchangeably and refer to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof, which specifically bind and recognize an antigen.
Identified immunoglobulin genes include the kappa, lambda, alpha,
gamma, delta, epsilon and mu constant region genes, as well as the
myriad immunoglobulin variable region genes. Light chains are
classified as either kappa or lambda. Heavy chains are classified
as gamma, mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
Terms understood by those in the art of antibody technology are
each given the meaning acquired in the art, unless expressly
defined differently herein. Antibodies are known to have variable
regions, a hinge region, and constant domains. Immunoglobulin
structure and function are reviewed, for example, in Harlow et al,
Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring
Harbor Laboratory, Cold Spring Harbor, 1988).
[0045] "Percent (%) amino acid sequence identity" herein is defined
as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid residues in a selected
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the
full-length of the sequences being compared.
[0046] The notations "mg/kg", or "mg per kg" refer to milligrams
per kilogram. All notations are used interchangeably throughout the
present disclosure.
[0047] The "half-life" of a polypeptide can generally be defined as
the time taken for the serum concentration of the polypeptide to be
reduced by 50%, in vivo, for example due to degradation of the
polypeptide and/or clearance or sequestration of the polypeptide by
natural mechanisms. The half-life can be determined in any manner
known per se, such as by pharmacokinetic analysis. Suitable
techniques will be clear to the person skilled in the art, and may,
for example, generally involve the steps of administering a
suitable dose of a polypeptide to a rodent or primate; collecting
blood samples or other samples from a rodent or primate at regular
intervals; determining the level or concentration of the
polypeptide in said blood sample; and calculating, from (a plot of)
the data thus obtained, the time until the level or concentration
of the polypeptide has been reduced by 50% compared to the initial
level upon dosing. Methods for determining half-life may be found,
for example, in Kenneth et al., Chemical Stability of
Pharmaceuticals: A Handbook for Pharmacists (1986); Peters et al,
Pharmacokinete analysis: A Practical Approach (1996); and
"Pharmacokinetics", M Gibaldi & D Perron, published by Marcel
Dekker, 2nd Rev. edition (1982).
[0048] The half-life of a fusion polypeptide is increased if
presence in a biological matrix (blood, serum, plasma, tissue)
persists, in vivo, for a longer period as compared to an
appropriate control. Half-life may be increased by 10%, 20%, 30%,
40%, 50% or more as compared to an appropriate control.
[0049] Half-life can be expressed using parameters such as the
t.sub.1/2-alpha, t.sub.1/2-beta, and HL_Lambda_z. In the present
specification, an "increase in half-life" refers to an increase in
any one of these parameters, any two of these parameters, or all
three of these parameters. An "increase in half-life" in particular
refers to an increase in the t.sub.1/2-beta and/or HL_Lambda_z,
either with or without an increase in the t.sub.1/2-alpha. Other PK
parameters that can be assessed include volume of distribution
(VD), clearance (CL), and mean residence time (MRT), and the area
under the curve (AUC). In the present specification, a "change in
pharmacokinetics" refers to changes in any one of these parameters,
any two of these parameters, any three of these parameters, or all
four of these parameters, in the presence or absence of changes in
the half-life parameters listed above.
[0050] "Activity" for the purposes herein refers to an action or
effect of a component of a fusion protein consistent with, but not
necessarily identical to, that of the corresponding native active
protein, wherein "biological activity" or "bioactivity" refers to
an in vitro or in vivo biological function or effect, including but
not limited to receptor binding, antagonist activity, agonist
activity, or a cellular or physiologic response.
[0051] As used herein, a "dimer complex" comprises two single chain
X-L1-HINGE-Fc fusion proteins of the invention, wherein the two
single chain polypeptides are associated together under appropriate
conditions via either non-covalent binding or covalent binding, for
example, by a disulfide bridge. A "heterodimeric protein",
"heterodimerized complex", or "heterodimer" as used interchangeably
herein refers to a protein that is made of two single chain
X-L1-HINGE-Fc polypeptides forming a dimer complex, wherein said
two single chain polypeptides have different amino acid sequences,
in particular, X represents different soluble proteins or different
portions of the same soluble protein. A "homodimeric protein"
"homodimerized complex" or "homodimer" as used interchangeably
herein, refers to a protein that is made of two identical or
substantially identical polypeptides forming the dimer complex,
wherein said two single chain polypeptides share 100% identity, or
at least 95% or at least 99% identity, the amino acid differences
consisting of amino acid substitution, addition or deletion which
does not affect the functional and physical properties of the
polypeptide compared to the other one of the homodimer, for example
conservative amino acid substitutions.
[0052] As used herein, a protein is "soluble" when it lacks any
transmembrane domain or protein domain that anchors or integrates
the polypeptide into the membrane of a cell expressing such
polypeptide.
[0053] As used herein, "Fc domain", "Fc region" or "Fc portion" as
those terms may be used interchangeably herein to describe an
X-L1-HINGE-Fc fusion protein of the invention, encompasses domains
derived from the constant region of an immunoglobulin, preferably a
human immunoglobulin, including a fragment, analog, variant, mutant
or derivative of the constant region. Suitable immunoglobulins
include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD,
IgE and IgM. The constant region of an immunoglobulin is defined as
a naturally-occurring or synthetically-produced polypeptide
homologous to the immunoglobulin C-terminal region, and can include
a CH1 domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain,
separately or in combination.
[0054] As used herein, "treatment" or "treating," or "palliating"
or "ameliorating" is used interchangeably herein. These terms refer
to an approach for obtaining beneficial or desired results
including but not limited to a therapeutic benefit and/or a
prophylactic benefit. By therapeutic benefit is meant eradication
or amelioration of the underlying disorder being treated. Also, a
therapeutic benefit is achieved with the eradication or
amelioration of one or more of the physiological symptoms
associated with the underlying disorder such that an improvement is
observed in the subject, notwithstanding that the subject may still
be afflicted with the underlying disorder.
[0055] For prophylactic benefit, the compositions may be
administered to a subject at risk of developing a particular
disease, or to a subject reporting one or more of the physiological
symptoms of a disease, even though a diagnosis of this disease may
not have been made.
[0056] A "therapeutic effect", as used herein, refers to a
physiologic effect, including but not limited to the cure,
mitigation, amelioration, or prevention of disease in humans or
other animals, or to otherwise enhance physical or mental
well-being of humans or animals, caused by a fusion protein of the
invention.
[0057] The terms "therapeutically effective amount" and
"therapeutically effective dose", as used herein, refers to an
amount of an active protein, either alone or as a part of a fusion
protein composition, that is capable of having any detectable,
beneficial effect on any symptom, aspect, measured parameter or
characteristics of a disease state or condition when administered
in one or repeated doses to a subject. Such effect need not be
absolute to be beneficial.
[0058] The term "therapeutically effective dose regimen", as used
herein, refers to a schedule for consecutively administered doses
of an active protein, either alone or as a part of a fusion protein
composition, wherein the doses are given in therapeutically
effective amounts to result in sustained beneficial effect on any
symptom, aspect, measured parameter or characteristics of a disease
state or condition.
Single Chain Fc Fusion Proteins
[0059] Single chain Fc fusion proteins of the invention have the
following arrangement from amino-terminus (N-terminus) to
carboxy-terminus (C-terminus): [0060] X-L1-HINGE-Fc wherein, X is a
soluble protein; L1 is a linker having the following arrangement
from amino-terminus to carboxy-terminus: [0061] L2-CL-L3-CH1-L4 or
L2-CH1-L3-CL-L4 [0062] wherein, [0063] L2 and L4 are independently
polypeptide linkers or are independently absent, [0064] L3 is a
polypeptide linker; [0065] CL is a constant region polypeptide from
an immunoglobulin light chain; and [0066] CH1 a constant region
polypeptide from a CH1 domain of an immunoglobulin heavy chain;
HINGE is a hinge sequence of an immunoglobulin or is absent with
the proviso that if HINGE is absent, L4 is present; and Fc is the
carboxy-terminus of an immunoglobulin or any active fragment or
derivative thereof.
[0067] In accordance with the invention, a soluble protein of
interest is fused to the N-terminal region of an immunoglobulin Fc
region via a novel linker (L1) that is derived from the CL and CH1
domains of an immunoglobulin arranged as a single chain (sc) also
referred to herein as "scCLCH1 linkers".
[0068] The C-terminus of the CL region may be linked to the
N-terminal region of a CH1 region via polypeptide linker L3. The
N-terminus of the CL region may be fused to the C-terminus of the
protein of interest (X) via an optional polypeptide linker L2. The
C-terminus of the CH1 domain is linked to the Fc domain via an
immunoglobulin hinge region (HINGE) or a polypeptide linker (L4) or
both a hinge (HINGE) and a polypeptide linker (L4).
[0069] The C-terminus of the CH1 domain may also be linked to the
N-terminus of a CL region via polypeptide linker L3. The N-terminus
of the CH1 region may be fused to the C-terminus of the protein of
interest (X) via an optional polypeptide linker L2. The C-terminus
of the CL region is linked to the Fc region via an immunoglobulin
hinge region (HINGE) or a polypeptide linker (L4) or both a hinge
(HINGE) and a polypeptide linker (L4).
[0070] Preferably, L3 is selected from artificial flexible domains
comprising amino acids selected from Gly (G), and/or Ser (S).
Preferably, the linker is comprised of polypeptide of the general
formula (Gly-Gly-Gly-Ser (SEQ ID NO: 21))n or (Gly-Gly-Gly-Gly-Ser
(SEQ ID NO: 22))n wherein n is an integer from 1 to 10. Preferably,
each linker is a polypeptide comprising from about 1 to about 100
amino acids, preferably about 1-50 amino acids, preferably about
1-25 amino acids, preferably about 1-15 amino acids preferably
about 1-10 amino acids, preferably about 4-24 amino acids,
preferably about 5-20 amino acids preferably about 5-15 amino acids
and preferably about 5-10 amino acids. Preferably, the linker is
(Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 22)) n wherein n is 2 or 4.
[0071] L2 and L4 are independently selected from artificial
flexible domains comprising amino acids selected from, for example,
Gly (G), and Ser (S). Preferably, the linker is comprised of
polypeptide of the general formula (Gly-Gly-Gly-Ser (SEQ ID NO:
21))n or (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 22))n wherein n is an
integer from 1 to 10. Preferably, each linker is a polypeptide
comprising from about 1 to about 100 amino acids, preferably about
1-50 amino acids, preferably about 1-25 amino acids, preferably
about 1-15 amino acids preferably about 1-10 amino acids,
preferably about 4-24 amino acids, preferably about 5-20 amino
acids preferably about 5-15 amino acids and preferably about 5-10
amino acids. Preferably, the linker is (Gly-Gly-Gly-Gly-Ser(SEQ ID
NO: 22))n wherein n is 2 or 4.
[0072] L2, L3 and L4, may further comprise amino acids such as, for
example, Lys (K), Thr (T), Glu (E), and Asp (D).
[0073] The CL region of the novel scCLCH1 linker (L1) may be
substantially identical to the corresponding CL region of native
immunoglobulins belonging to any of the immunoglobulin classes,
i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody
subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. The CL region (L1)
may have amino acid sequence that is at least 50%, 60%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding
CL region of native immunoglobulins belonging to any of the
immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of
the IgG antibody subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. If
the CL region of L1 is a modified derivative or variant of a native
CL region such modifications include, but are not limited to, amino
acid insertions, deletions, substitutions and rearrangements.
Preferably, the amino acid sequence of the CL region in accordance
with the invention, is at least 80%, more preferably at least 85%,
more preferably at least 90%, and more preferably at least 95%
identical to the corresponding CL region of native immunoglobulins
belonging to any of the immunoglobulin classes, i.e., IgA, IgD,
IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1,
IgG2, IgG3, and IgG4.
[0074] The CH1 region of the novel scCLCH1 linker (L1) may be
substantially identical to the corresponding CH1 region of native
immunoglobulins belonging to any of the immunoglobulin classes,
i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody
subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. The CH1 region of L1
may have amino acid sequence that is at least 50%, 60%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding
CH1 region of native immunoglobulins belonging to any of the
immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM or any of
the IgG antibody subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. If
the CH1 region of the L1 linker is a modified derivative or variant
of a native CH1 immunoglobulin region such modifications include,
but are not limited to, amino acid insertions, deletions,
substitutions and rearrangements. Preferably, the amino acid
sequence of the CH1 region is at least 80%, more preferably at
least 85%, more preferably at least 90%, and more preferably at
least 95% identical to the corresponding CH1 region of native
immunoglobulins belonging to any of the immunoglobulin classes,
i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody
subclasses, i.e., IgG1, IgG2, IgG3, and IgG4.
[0075] The CH1 region and CL regions of L1 do not need to be
identical to or a variant of, the corresponding regions of the same
immunoglobulin class. For example, the CL region may be derived
from the corresponding region of IgE and the CH1 region may be
derived from the corresponding region of IgG.
[0076] Preferably, CL and CH1 of the scCLCH1 linker are derived
from the corresponding CL and CH1 regions of IgG1, preferably human
IgG1.
[0077] An exemplary CL region corresponding to the CL region of a
human IgG1 (hIgG1) includes:
TABLE-US-00001 (SEQ ID NO: 1)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGES.
[0078] An exemplary CH1 region corresponding to the CH1 region of
hIgG1 includes:
TABLE-US-00002 (SEQ ID NO: 2)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKRV.
[0079] The single chain Fc fusion proteins disclosed herein
comprise an Fc region that includes at least a portion of the
carboxy-terminus of an immunoglobulin heavy chain. For example, the
Fc portion may comprise: a CH2 domain, a CH3 domain, a CH4 domain,
a CH2-CH3 domain, a CH2-CH4 domain, a CH2-CH3-CH4 domain, a
hinge-CH2 domain, a hinge-CH2-CH3 domain, a hinge-CH2-CH4 domain,
or a hinge-CH2-CH3-CH4 domain. The Fc domain may be derived from
antibodies belonging any of the immunoglobulin classes, i.e., IgA,
IgD, IgE, IgG, or IgM or any of the IgG antibody subclasses, i.e.,
IgG1, IgG2, IgG3, and IgG4. Preferably, the Fc region is derived
from IgG1 preferably human IgG1.
[0080] The Fc domain may be a naturally occurring Fc sequence
belonging any of the immunoglobulin classes, i.e., IgA, IgD, IgE,
IgG, or IgM or any of the IgG antibody subclasses, i.e., IgG1,
IgG2, IgG3, and IgG4, including natural allelic or splice variants.
Alternatively, the Fc domain may be a hybrid domain comprising a
portion of an Fc domain from two or more different Ig isotypes, for
example, an IgG2/IgG4 hybrid Fc domain. Preferably, the Fc domain
is derived from a human immunoglobulin molecule. Alternatively, the
Fc domain may be a humanized or deimmunized (removal of T cell
epitopes which can activate helper T cells) version of an Fc domain
from a non-human animal, including but not limited to mouse, rat,
rabbit, and monkey.
[0081] The Fc domain may be a variant Fc sequence, e.g., an Fc
sequence that has been modified (e.g., by amino acid substitution,
deletion and/or insertion) relative to a parent Fc sequence (e.g.,
an unmodified Fc polypeptide that is subsequently modified to
generate a variant), to provide desirable structural features
and/or biological activity. For example, one may make modifications
in the Fc region in order to generate an Fc variant that (a) has
increased or decreased antibody-dependent cell-mediated
cytotoxicity (ADCC), (b) increased or decreased complement mediated
cytotoxicity (CDC), (c) has increased or decreased affinity for Clq
and/or (d) has increased or decreased affinity for a Fc receptor
relative to the parent Fc. Such Fc region variants will generally
comprise at least one amino acid modification in the Fc region.
Combining amino acid modifications is thought to be particularly
desirable. For example, the variant Fc region may include two,
three, four, five, etc. substitutions therein, e.g. of the specific
Fc region positions identified herein.
[0082] The hinge region of the Fc fusion proteins of the invention
may be derived from antibodies belonging to any of the
immunoglobulin classes, i.e., IgA, IgD, IgE, IgG, or IgM. The hinge
region may be derived from any of the IgG antibody subclasses,
i.e., IgG1, IgG2, IgG3, and IgG4. The hinge region may naturally
contain a cysteine residue or may be engineered to contain one or
more cysteine residues.
[0083] Preferably, the hinge region may have an amino acid sequence
that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to the corresponding hinge region of native
immunoglobulins belonging to any of the immunoglobulin classes,
i.e., IgA, IgD, IgE, IgG, or IgM or any of the IgG antibody
subclasses, i.e., IgG1, IgG2, IgG3, and IgG4. Preferably, the amino
acid sequence of the hinge region is at least 80%, more preferably
at least 85%, more preferably at least 90%, and more preferably at
least 95% identical to the corresponding hinge region of human
IgG1.
[0084] Shown below is the sequence of a human IgG1 immunoglobulin
constant region, and the relative position of the hinge region is
indicated by solid underlining:
TABLE-US-00003 (SEQ ID NO: 3) ##STR00001##
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK.
The CH1 region is indicated by underlining with a dotted line, and
the CH2 and CH3 regions are indicated by bold lettering. The
C-terminal lysine of an IgG sequence may be removed or replaced
with a non-lysine amino acid, such as alanine, to further increase
the serum half-life of the Fc fusion protein.
[0085] The hinge sequence may include substitutions that confer
desirable pharmacokinetic, biophysical, and/or biological
properties. An exemplary hinge region of the invention comprises an
amino acid sequence that is at least 50%, 60%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the following:
TABLE-US-00004 (SEQ ID NO: 4) EPKSSDKTHTCPPCP.
[0086] The Fc domain and the hinge region may be derived from one
antibody class or subclass. For example, the hinge region and the
Fc domain may be derived from IgG1. The Fc domain and hinge region
may correspond to different antibody classes or subclasses. For
example, the Fc domain may correspond to the Fc region of IgG2 or
IgG4 and the hinge region may correspond to IgG1.
[0087] Preferably, all immunoglobulin domains of the Fc fusion
proteins of the invention are derived from IgG1, preferably human
IgG1. Preferably, the combined hinge region and Fc region of the
fusion proteins of the invention comprise an amino acid sequence
that is at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to:
TABLE-US-00005 (SEQ ID NO: 5)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.
Preferably, the combined hinge region and Fc region of the fusion
proteins of the invention comprise an amino acid sequence that is
at least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to:
TABLE-US-00006 (SEQ ID NO: 6)
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSV
LTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.
[0088] It may be desirable to have a hinge sequence and/or Fc
region of the single chain fusion proteins of the invention
comprising a free cysteine residue in order to permit the formation
of a disulfide bond between the hinge and or Fc regions thereby
forming dimers of the Fc fusion proteins of the invention. It may
be desirable to alter the hinge and/or Fc region sequences to
remove free cysteine residues, e.g., by mutating one or more
cysteine residues in a linker to another residue, such as a serine,
alanine or glycine. The hinge region of the single chain fusion
proteins of the invention may comprise one or more free cysteine
residues capable of forming one or more disulfide bonds with a
second single chain fusion protein of the invention thereby forming
a dimer complex.
[0089] The X-L1-HINGE-Fc fusion proteins described herein contain
an X portion that may be any soluble protein of interest or any
active fragment thereof or any active derivative thereof. Soluble
proteins of interest (X) that may be fused to a single chain
L1-HINGE-Fc scaffold in accordance with the invention include, but
are not limited to: proteins or portions or fragments thereof that
that can bind to, or interact with, a target molecule, cell,
complex and/or tissue, such targets including enzyme substrates,
proteins, nucleic acids, carbohydrates, lipids, low molecular
weight compounds, and fragments thereof.
[0090] Soluble binding proteins of interest (X) include, but are
not limited to, the following list of proteins, as well as active
derivatives, active fragments, subunits, domains, motifs and
epitopes belonging to the following list of proteins: renin; a
growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VII, factor VIIIC, factor IX, tissue factor
(TF), and von Willebrands factor; anti-clotting factors such as
Protein C; atrial natriuretic factor; lung surfactant; a
plasminogen activator, such as urokinase or human urine or
tissue-type plasminogen activator (t-PA); bombesin; thrombin;
hemopoietic growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MIP-1-alpha); a serum albumin such as human serum albumin;
Muellerian-inhibiting substance; relaxin A-chain; relaxin
B-chain;
[0091] prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic
T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors such as, for example, EGFR, VEGFR;
interferons such as alpha interferon (.alpha.-IFN), beta interferon
(.beta.-IFN) and gamma interferon (.gamma.-IFN); protein A or D;
rheumatoid factors; a neurotrophic factor such as bone-derived
neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3,
NT-4, NT-5, or NT-6), or a nerve growth factor; platelet-derived
growth factor (PDGF); fibroblast growth factor such as AFGF and
PFGF; epidermal growth factor (EGF); transforming growth factor
(TGF) such as TGF-alpha and TGF-beta, including TGF-1, TGF-2,
TGF-3, TGF-4, or TGF-5; insulin-like growth factor-I and -II (IGF-I
and IGF-II); des (1-3)-IGF-I (brain IGF-I), insulin-like growth
factor binding proteins; CD proteins such as CD2, CD3, CD4, CD8,
CD11a, CD14, CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40,
CD40L, CD52, CD63, CD64, CD80 and CD147; erythropoietin;
osteoinductive factors; immunotoxins; a bone morphogenetic protein
(BMP); an interferon such as interferon-alpha, -beta, and -gamma;
colony stimulating factors (CSFs), such as M-CSF, GM-CSF, and
G-CSF; interleukins (ILs), e.g., interleukin-1 (IL-1), IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1. IL-12, IL-13,
IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22,
IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31,
IL-32, IL-33, IL-34, IL-35; interleukin receptor antagonists such
as IL1Ra; TNF.alpha., superoxide dismutase; T-cell receptors;
surface membrane proteins; decay accelerating factor; viral antigen
such as, for example, a portion of the AIDS envelope, e.g., gp120;
transport proteins; homing receptors; addressins; regulatory
proteins; cell adhesion molecules such as LFA-1, Mac 1, p150.95,
VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7 integrin, and Xv/p3 integrin
including either a or subunits thereof, integrin alpha subunits
such as CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, alpha7, alpha8,
alpha9, alphaD, CD11a, CD11b, CD51, CD11c, CD41, alphaIIb,
alphaIELb; integrin beta subunits such as, CD29, CD 18, CD61,
CD104, beta5, beta6, beta7 and beta8; Integrin subunit combinations
including but not limited to, .alpha.V.beta.3, .alpha.V.beta.5 and
.alpha.4.beta.7; a member of an apoptosis pathway; IgE; blood group
antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;
CTLA-4; protein C; an Eph receptor such as EphA2, EphA4, EphB2,
etc.; a Human Leukocyte Antigen (HLA) such as HLA-DR; complement
proteins such as complement receptor CR1, C1Rq and other complement
factors such as C3, and C5; a glycoprotein receptor such as
GpIb.alpha., GPIIb/IIIa and CD200; soluble receptors such as
TNFR2.
[0092] Preferably, the soluble protein of interest (X) is Factor
IX, TNFR2 (also known as TNFRSF1B) or IL1Ra. Preferably the soluble
protein of interest (X) is IL-10, IL-2, IL-2R.alpha. or fusions
thereof, or IFN.beta.. Preferably the soluble protein of interest
(X is IL-10, IL-2, IL-2R.alpha. (or fusions thereof), IFN.beta.,
Factor IX, TNFR2 (also known as TNFRSF1B) or IL1Ra.
[0093] Preferably, the fusion protein has the structure of the
homodimer shown in FIG. 1A where X is Factor IX, TNF-R2, or IL-1Ra
or any active fragment or derivative thereof of any of the
foregoing proteins. Preferably the fusion protein has the structure
of the homodimers shown in FIG. 1A where X is IL-10, IL-2,
IL-2R.alpha. (or fusions thereof), or IFN.beta. or any active
fragment or derivative of any of the foregoing proteins.
Preferably, the fusion protein has the structure of the heterodimer
shown in FIG. 1B where X is Factor IX, TNF-R2, or IL-1Ra and Y is
different from X and is Factor IX, TNF-R2, or IL-1Ra. Preferably,
the fusion protein has the structure of the heterodimer shown in
FIG. 1B where X is IL-10, Factor IX, TNFR, 11-2, IL-2R.alpha. (or
fusions thereof), IFN.beta. or IL-1Ra and Y is different from X and
is IL-10, Factor IX, TNF-R2, Il-2, IL-2R.alpha. (or fusions
thereof), IFN.beta. or IL-1Ra. Preferably, the fusion protein has
the structure of the homodimer shown in FIG. 1C where X is Factor
IX, TNF-R2, or IL-1Ra. Preferably, the fusion protein has the
structure of the homodimer shown in FIG. 1C where X is IL-10, IL-2,
IL-2R.alpha. (or fusions thereof), or IFN.beta.. Preferably, the
fusion protein has the structure of the heterodimer shown in FIG.
1D where X is Factor IX, TNF-R2, or IL-1Ra and Y is different from
X and is Factor IX, TNF-R2, or IL-1Ra. Preferably, the fusion
protein has the structure of the heterodimer shown in FIG. 1D where
X is IL-10, Factor IX, TNF-R2, Il-2, IL-2R.alpha. (or fusions
thereof), IFN.beta. or IL-1Ra and Y is different from X and is
IL-10, Factor IX, TNF-R2, Il-2, IL-2R.alpha. (or fusions thereof),
IFN.beta. or IL-1Ra.
[0094] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is factor IX and a single chain fusion protein of the
invention having the formula X-L1-HINGE-Fc comprising an amino acid
sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to:
TABLE-US-00007 (SEQ ID NO: 7)
TVFLDHENANKILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEE
AREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECW
CPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRL
AENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAE
TILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCG
GSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIR
IIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTN
IFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKF
TIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGE
ECAMKGKYGIYTKVSRYVNWIKEKTKLTGGGGSGGGGSRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGESGGGGSGGGGSGGGGSGGGGSASTKGPSVFPLAPSS
KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD
WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
[0095] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is TNFR2 and a single chain fusion protein of the
invention having the formula X-L1-HINGE-Fc comprising an amino acid
sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to:
TABLE-US-00008 (SEQ ID NO: 8)
LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFC
TKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTR
EQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETS
DVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTS
TSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGP
SPPAEGSTGDGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESGGGGSGGG
GSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF
PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH
NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPA
PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS
DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK.
[0096] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is IL1Ra and a single fusion protein of the invention
having the formula X-L1-HINGE-Fc comprises an amino acid sequence
that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to:
TABLE-US-00009 (SEQ ID NO: 9)
RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEK
IDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSE
NRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTN
MPDEGVMVTKFYFQEDEGGGGSGGGGSRTVAAPSVFIFPPSDEQL
KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGESG
GGSGGGGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP
SSSLGTQTYICNVNHKPSNTKVDKRVEPKSSDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.
[0097] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is IFN.beta. and a single fusion protein of the
invention having the formula X-L1-HINGE-Fc comprising an amino acid
sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99% identical to:
TABLE-US-00010 (SEQ ID NO: 18)
MSYNLLGFLQRSSNFQSQKLLWQLNGRLEYCLKDRMNFDIPEEIK
QLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLAN
VYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKA
KEYSHCAWTIVRVEILRNFYFINRLTGYLRNGGGGSGGGGSRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSASTKGPSVFPLA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS
CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK..
[0098] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc comprises a protein that has been modified by
circular permutation as is described in International Publication
Number WO 2013/184942. Circular permutation involves the linking of
the native amino and carboxy ends of a protein, generally with a
linker, and creating new amino and carboxy termini by cleaving at a
new site within the protein sequence, generally a loop; such that
the primary sequence of the resulting protein is reordered, while
the secondary structure (and activity) is retained. Thus, creation
of the new termini may provide better locations for attachment of a
fusion partner relative to the native termini. Circular permutation
of a protein ligand provides a means by which a protein may be
altered to produce new carboxyl and amino termini without
diminishing the specificity and binding affinity of the altered
protein ligand for its target relative to its native form.
Additionally, the new termini can be preferentially moved to a
location preferential for incorporating the circularly permuted
ligand into a fusion polypeptide, and demonstrate better activity
compared with a fusion polypeptide containing the native
(non-circularly permuted) ligand.
[0099] Preferably, the soluble protein X of formula X-L1-HINGE-Fc
comprises a fusion of two different proteins designated as Q-R and
wherein Q and R may be fused via an optional linker L5. Preferably
Q is a soluble ligand which can form a signaling complex with a
membrane associated receptor and R is the extracellular domain of
one receptor chain from the membrane associated receptor.
Preferably, Q-L5-R is IL-2 or circularly permuted IL-2 fused to the
extracellular domain of IL-2R.alpha. via an optional linker.
[0100] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is a fusion of IL-2/IL-2R.alpha. wherein the single
chain protein comprises an amino acid sequence that is 50%, 60%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to:
TABLE-US-00011 (SEQ ID NO: 19)
SKNFHLRPRDLISNINVIVELKGSETTFMCEYADETATIVEFLNR
WITFSQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKN
PKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGG
GSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLY
MLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEM
QSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQ
GYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGSGGGSRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALSG
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS
SPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK.
[0101] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is a fusion of IL-2/IL-2R.alpha. wherein the single
chain protein comprises an amino acid sequence that is 50%, 60%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to:
TABLE-US-00012 (SEQ ID NO: 20)
SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN
RWITFSQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYK
NPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSG
GGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSL
YMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTE
MQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCV
QGYRALHRGPAESVCKMTHGKTRWTQPQLICTGGGGGSGGGGSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKRVGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTAS
VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGGEP
KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK..
[0102] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is IL-10 wherein the single chain protein comprises
an amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to:
TABLE-US-00013 (SEQ ID NO: 23)
MYRMQLLSCIALSLALVTNSSPGQGTQSENSCTHFPGNLPNMLRD
LRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ
FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE
NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN
GGSGGGGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKT
FFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA
ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK
NAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGS
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN
ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT
HQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSGGGGSASTKGPSV
FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV
EPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK.
[0103] Preferably, the soluble protein X of the formula
X-L1-HINGE-Fc is IL-10 wherein the single chain protein comprises
an amino acid sequence that is 50%, 60%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to:
TABLE-US-00014 (SEQ ID NO: 24)
MYRMQLLSCIALSLALVTNSSPGQGTQSENSCTHFPGNLPNMLRD
LRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQ
FYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE
NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN
GGSGGGGSGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKT
FFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA
ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVK
NAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGGSGGGGS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA
LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKRVGGGGSGGGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GGSGGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK.
[0104] Preferably, the X-L1-HINGE-Fc fusion proteins of the
invention are dimer complexes comprising two monomeric single chain
X-L1-HINGE-Fc fusion proteins of the invention linked via a
disulfide bond to the hinge region or in the Fc region of the other
monomer. The dimer complexes may be homodimeric (e.g. both
monomeric fusion proteins are identical) or heterodimeric (e.g. the
protein of interest (X) may be different for each monomeric fusion
protein). Preferably, the dimer complexes are homodimers thereby
forming a homodimeric complex that provides an antibody
configuration that resembles that of a native antibody.
[0105] Without being limited to any one theory, it is believed that
the homodimeric fusion proteins of the invention increase half-life
due to the presence of a dimerized Fc region which more closely
resembles the native antibody structure as compared to traditional
Fc fusion proteins. A more native Fc domain antibody configuration
is believed to enable better binding to the FcRn receptor and
therefore increase the circulating half-life of the of the
X-L1-HINGE-Fc dimer complex.
[0106] Another improved property associated with X-L1-HINGE-Fc
dimer complexes is that bioactivity is increased versus a
traditional Fc fusion proteins based on the use of the scCLCH1
linker which imparts flexibility to relieve steric hindrance caused
by the dimerization through the Fc in the hinge region.
Recombinant Production of X-L1-HINGE-Fc Fusion Proteins
[0107] The invention also provides nucleic acids encoding any of
the various Fc fusion proteins disclosed herein. Codon usage may be
selected so as to improve expression in a cell. Such codon usage
will depend on the cell type selected. Specialized codon usage
patterns have been developed for E. coli and other bacteria, as
well as mammalian cells, plant cells, yeast cells and insect cells.
See for example: Mayfield et al., Proc. Natl. Acad. Sci. USA,
100(2):438-442 (Jan. 21, 2003); Sinclair et al., Protein Expr.
Purif., 26(I):96-105 (October 2002); Connell, N.D., Curr. Opin.
Biotechnol., 12(5):446-449 (October 2001); Makrides et al.,
Microbiol Rev., 60(3):512-538 (September 1996); and Sharp et al.,
Yeast, 7(7):657-678 (October 1991).
[0108] General techniques for nucleic acid manipulation are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition, Vols. 1-3, Cold Spring Harbor
Laboratory Press (1989), or Ausubel, F. et al., Current Protocols
in Molecular Biology, Green Publishing and Wiley-Interscience, New
York (1987) and periodic updates, herein incorporated by reference.
Generally, the DNA encoding the polypeptide is operably linked to
suitable transcriptional or translational regulatory elements
derived from mammalian, viral, or insect genes. Such regulatory
elements include a transcriptional promoter, an optional operator
sequence to control transcription, a sequence encoding suitable
mRNA ribosomal binding sites, and sequences that control the
termination of transcription and translation. The ability to
replicate in a host, usually conferred by an origin of replication,
and a selection gene to facilitate recognition of transformants is
additionally incorporated.
[0109] The Fc fusion proteins described herein may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which is preferably a signal
sequence or other polypeptide having a specific cleavage site at
the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. An exemplary N-terminal leader sequence for
production of polypeptides in a mammalian system is
MYRMQLLSCIALSLALVTNS (SEQ ID NO: 10), which is removed by the host
cell following expression.
[0110] For prokaryotic host cells that do not recognize and process
a native signal sequence, the signal sequence is substituted by a
prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, or heat-stable
enterotoxin II leaders.
[0111] For yeast secretion the native signal sequence may be
substituted by, e.g., the yeast invertase leader, a factor leader
(including Saccharomyces and Kluyveromyces alpha-factor leaders),
or acid phosphatase leader, the C. albicans glucoamylase leader, or
the signal described in U.S. Pat. No. 5,631,144. In mammalian cell
expression, mammalian signal sequences as well as viral secretory
leaders, for example, the herpes simplex gD signal, are available.
The DNA for such precursor regions may be ligated in reading frame
to DNA encoding the protein.
[0112] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2 micron plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0113] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0114] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the nucleic acid encoding the protein disclosed herein, e.g., a
fibronectin-based scaffold protein. Promoters suitable for use with
prokaryotic hosts include the phoA promoter, beta-lactamase and
lactose promoter systems, alkaline phosphatase, a tryptophan (trp)
promoter system, and hybrid promoters such as the tan promoter.
However, other known bacterial promoters are suitable. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno
(S.D.) sequence operably linked to the DNA encoding the protein
disclosed herein. Promoter sequences are known for eukaryotes.
Virtually all eukaryotic genes have an AT-rich region located
approximately 25 to 30 bases upstream from the site where
transcription is initiated. Another sequence found 70 to 80 bases
upstream from the start of transcription of many genes is a CNCAAT
(SEQ ID NO: 16) region where N may be any nucleotide. At the 3' end
of most eukaryotic genes is an AATAAA (SEQ ID NO: 17) sequence that
may be the signal for addition of the poly A tail to the 3' end of
the coding sequence. All of these sequences are suitably inserted
into eukaryotic expression vectors.
[0115] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0116] Transcription from vectors in mammalian host cells can be
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible
with the host cell systems.
[0117] Transcription of a DNA encoding proteins disclosed herein by
higher eukaryotes is often increased by inserting an enhancer
sequence into the vector. Many enhancer sequences are now known
from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature, 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
peptide-encoding sequence, but is preferably located at a site 5'
from the promoter.
[0118] Expression vectors used in eukaryotic host cells (e.g.,
yeast, fungi, insect, plant, animal, human, or nucleated cells from
other multicellular organisms) will also contain sequences
necessary for the termination of transcription and for stabilizing
the mRNA. Such sequences are commonly available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs
or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of mRNA
encoding the protein disclosed herein. One useful transcription
termination component is the bovine growth hormone polyadenylation
region. See WO 94/11026 and the expression vector disclosed
therein.
[0119] The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the protein. Examples of
protein tags include but are not limited to a histidine tag, a FLAG
tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning and
expression vectors for use with bacterial, fungal, yeast, and
mammalian cellular hosts can be found in Cloning Vectors: A
Laboratory Manual, (Elsevier, New York (1985)), the relevant
disclosure of which is hereby incorporated by reference.
[0120] The expression construct is introduced into the host cell
using a method appropriate to the host cell, as will be apparent to
one of skill in the art. A variety of methods for introducing
nucleic acids into host cells are known in the art, including, but
not limited to, electroporation; transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (where the vector is an infectious agent).
[0121] Suitable host cells include prokaryotes, yeast, mammalian
cells, or bacterial cells. Suitable bacteria include gram negative
or gram positive organisms, for example, E. coli or Bacillus spp.
Yeast, preferably from the Saccharomyces species, such as S.
cerevisiae, may also be used for production of polypeptides.
Various mammalian or insect cell culture systems can also be
employed to express recombinant proteins. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow et al. (Bio/Technology, 6:47 (1988)). Examples of suitable
mammalian host cell lines include endothelial cells, COS-7 monkey
kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary
(CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell
lines. Purified polypeptides are prepared by culturing suitable
host/vector systems to express the recombinant proteins. For many
applications, the small size of many of the polypeptides disclosed
herein would make expression in E. coli as the preferred method for
expression. The protein is then purified from culture media or cell
extracts.
[0122] In other aspects, the invention provides host cells
containing vectors encoding the Fc fusion proteins described
herein, as well as methods for producing the Fc fusion proteins
described herein. Host cells may be transformed with the
herein-described expression or cloning vectors for protein
production and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences. Host cells
useful for high-throughput protein production (HTPP) and mid-scale
production include the HMS 174-bacterial strain. The host cells
used to produce the proteins disclosed herein may be cultured in a
variety of media. Commercially available media such as Ham's F10
(Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma))
are suitable for culturing the host cells. In addition, many of the
media described in various scientific literature may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as Gentamycin drug), trace elements
(defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0123] The Fc fusion proteins provided herein can also be produced
using cell-translation systems. For such purposes the nucleic acids
encoding the fusion protein must be modified to allow in vitro
transcription to produce mRNA and to allow cell-free translation of
the mRNA in the particular cell-free system being utilized
(eukaryotic such as a mammalian or yeast cell-free translation
system or prokaryotic such as a bacterial cell-free translation
system).
[0124] The Fc fusion proteins disclosed herein can also be produced
by chemical synthesis (e.g., by the methods described in Solid
Phase Peptide Synthesis, 2nd Edition, The Pierce Chemical Co.,
Rockford, Ill. (1984)). Modifications to the Fc fusion proteins can
also be produced by chemical synthesis.
[0125] The Fc fusion proteins disclosed herein can be purified by
isolation/purification methods for proteins generally known in the
field of protein chemistry. Non-limiting examples include
extraction, recrystallization, salting out (e.g., with ammonium
sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, get filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, polypeptides may be exchanged into
different buffers and/or concentrated by any of a variety of
methods known to the art, including, but not limited to, filtration
and dialysis.
[0126] The purified Fc fusion protein is preferably at least 85%
pure, or preferably at least 95% pure, and most preferably at least
98% pure. Regardless of the exact numerical value of the purity,
the Fc fusion protein is sufficiently pure for use as a
pharmaceutical product.
Uses of X-L1-HINGE-Fc Fusion Proteins
[0127] In one aspect, the invention provides Fc fusion proteins
that are useful as diagnostic or therapeutic agents. In one aspect,
the invention provides Fc fusion proteins useful in the treatment
of disorders. The diseases or disorders that may be treated will be
dictated by the identity of the protein (X) fused to the Fc domain
via the novel L1 linker of the invention and include, but are not
limited to: cancer, inflammatory diseases, arthritis, osteoporosis,
infections in particular hepatitis, bacterial infections, viral
infections, genetic diseases, pulmonary diseases, diabetes,
hormone-related disease, Alzheimer's disease, cardiac diseases,
myocardial infarction, deep vein thrombosis, diseases of the
circulatory system, hypertension, hypotension, allergies, pain
relief, dwarfism and other growth disorders, intoxications, blot
clotting diseases, diseases of the innate immune system, embolism,
wound healing, healing of burns, Crohn's disease, asthma, ulcer,
sepsis, glaucoma, cerebrovascular ischemia, respiratory distress
syndrome, corneal ulcers, renal disease, diabetic foot ulcer,
anemia, factor IX deficiency, factor VIII deficiency, factor VII
deficiency, mucositis, dysphagia, thrombocyte disorder, lung
embolism, infertility, hypogonadism, leucopenia, neutropenia,
endometriosis, Gaucher disease, obesity, lysosome storage disease,
AIDS, premenstrual syndrome, Turners syndrome, cachexia, muscular
dystrophy, Huntington's disease, colitis, SARS, Kaposi sarcoma,
liver tumor, breast tumor, glioma, Non-Hodgkin lymphoma, Chronic
myelocytic leukemia; Hairy cell leukemia; Renal cell carcinoma;
Liver tumor; Lymphoma; Melanoma, multiple sclerosis, Kaposis
sarcoma, papilloma virus, emphysema, bronchitis, periodontal
disease, dementia, parturition, non-small cell lung cancer,
pancreas tumor, prostate tumor, acromegaly, psoriasis, ovary tumor,
Fabry disease, lysosome storage disease.
[0128] Exemplary therapeutic soluble proteins (X) that may be bound
to an Fc domain include, for example, factor IX, IL1Ra, and TNFR.
Exemplary therapeutic soluble proteins (X) that may be bound to an
Fc domain include, for example, IL-10, IL-2, IL-2R.alpha. or
fusions thereof, or IFN.beta.. Exemplary therapeutic soluble
proteins (X) that may be bound to an Fc domain include, for
example, IL-10, IL-2, IL-2R.alpha. or fusions thereof, IFN.beta.,
factor IX, IL1Ra, and TNFR2.
[0129] The invention also provides a method for achieving a
beneficial effect in a subject comprising the step of administering
to the subject a therapeutically or prophylactically-effective
amount of a fusion protein. The effective amount can produce a
beneficial effect in helping to treat a disease or disorder. In
some cases, the method for achieving a beneficial effect can
include administering a therapeutically effective amount of a
fusion protein composition to treat a subject for diseases and
disease categories wherein a therapeutic protein or peptide does
not exist.
[0130] Preferably, the invention provides a fusion protein
X-L1-HINGE-Fc wherein X is factor IX. Preferably, the invention
provides a dimer complex of X-L1-HINGE-Fc wherein X is factor IX.
Preferably the dimer complex is a homodimer complex. Factor IX
fusion proteins in accordance with the invention may be used to
treat patients who are deficient in factor IX and suffer from
hemophilia B for e.g., control and prevention of bleeding episodes,
routine prophylaxis to prevent or reduce the frequency of bleeding
episodes, and perioperative management (surgical prophylaxis).
[0131] A patient in need of control or prevention of bleeding or
bleeding episodes is preferably a human patient. The patient can be
bleeding at the time of administration or be expected to be
bleeding, or can be susceptible to bleeding in minor hemorrhage,
hemarthroses, superficial muscle hemorrhage, soft tissue
hemorrhage, moderate hemorrhage, intramuscle or soft tissue
hemorrhage with dissection, mucous membrane hemorrhage, hematuria,
major hemorrhage, hemorrhage of the pharynx, hemorrhage of the
retropharynx, hemorrhage of the retroperitonium, hemorrhage of the
central nervous system, bruises, cuts, scrapes, joint hemorrhage,
nose bleed, mouth bleed, gum bleed, intracranial bleeding,
intraperitoneal bleeding, minor spontaneous hemorrhage, bleeding
after major trauma, moderate skin bruising, or spontaneous
hemorrhage into joints, muscles, internal organs or the brain. Such
patients also include those in need of perioperative management,
such as management of bleeding associated with surgery or dental
extraction. The patient is preferably in need of prophylaxis of one
or more bleeding episodes. The patient is preferably in need of
individualized interval prophylaxis. The patient is preferably in
need of on-demand treatment of one or more bleeding episodes. The
patient is preferably in need of perioperative management of one or
more bleeding episodes.
[0132] When treating hemophilia with a fusion protein of the
invention comprising factor IX, an "effective dose" reduces or
decreases frequency of bleeding or bleeding disorder. An "effective
dose" preferably stops on-going, uncontrollable bleeding or
bleeding episodes. Preferably an "effective dose" prevents
spontaneous bleeding or bleeding episodes in a subject susceptible
to such spontaneous bleeding or bleeding episodes. A "therapeutic
dose" need not cure hemophilia.
[0133] Preferably, the invention provides a fusion protein
X-L1-HINGE-Fc wherein X is IL-10. Preferably, the invention
provides a dimer complex of X-L1-HINGE-Fc wherein X is IL-10.
Preferably the dimer complex is a homodimer complex. An IL-10
fusion protein and/or a dimerized complex thereof in accordance
with the invention may be used to treat patients who suffer from,
for example, autoimmune disorders, fibrotic diseases, inflammatory
diseases, ischemic diseases, neurodegenerative diseases,
neuropathic diseases, pain disorders, auditory disorders,
psychiatric disorders, cancer and trauma and injury.
[0134] Examples of autoimmune disorders which may be treated by the
IL-10 fusion proteins of the invention include, but are not limited
to: acute disseminated encephalomyelitis (ADEM), acute necrotizing
hemorrhagic leukoencephalitis, Addison's disease,
agammaglobulinemia, alopecia areata, amyloidosis, ankylosing
spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome
(APS), autoimmune angioedema, autoimmune aplastic anemia,
autoimmune dysautonomia, autoimmune hepatitis, autoimmune
hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear
disease (AIED), autoimmune lymphoproliferative syndrome (ALPS),
autoimmune myocarditis, autoimmune oophoritis, autoimmune
pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic
purpura (ATP), autoimmune thyroiditis, autoimmune urticaria, axonal
& neuronal neuropathies, Balo disease, Behcet's disease,
cardiomyopathy, Castleman disease, celiac disease, Chagas disease,
chronic fatigue syndrome, chronic inflammatory demyelinating
polyneuropathy (CIDP), chronic recurrent multifocal ostomyelitis
(CRMO), cicatricial pemphigoid/benign mucosal pemphigoid, Cogans
syndrome, cold agglutinin disease, congenital heart block,
Coxsackie myocarditis, CREST disease, Crohn's disease,
demyelinating neuropathies, dermatitis herpetiformis,
dermatomyositis, Devic's disease (neuromyelitis optica), discoid
lupus, Dressler's syndrome, endometriosis, eosinophilic
esophagitis, eosinophilic fasciitis, erythema nodosum, essential
mixed cryoglobulinemia, Evans syndrome, experimental allergic
encephalomyelitis, fibromyalgia, fibrosing alveolitis, giant cell
arteritis (temporal arteritis), giant cell myocarditis,
glomerulonephritis, Goodpasture's syndrome, granulomatosis with
Polyangiitis (GPA) (formerly called Wegener's Granulomatosis),
Grave's disease, Guillain-Barre syndrome, Hashimoto's encephalitis,
Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein
purpura, herpes gestationis, hypogammaglobulinemia, idiopathic
pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), IgA
nephropathy, IgG4-related sclerosing disease, immunoregulatory
lipoproteins, inclusion body myositis, interstitial cystitis,
juvenile arthritis, juvenile diabetes (Type 1 diabetes), juvenile
myositis, Kawasaki disease, Lambert-Eaton syndrome,
leukocytoclastic vasculitis, lichen planus, lichen sclerosus,
ligneous conjunctivitis, linear IgA disease (LAD), Lupus (systemic
lupus erythematosus), Lyme disease, chronic, Meniere's disease,
microscopic polyangiitis, mixed connective tissue disease (MCTD),
Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis (MS),
myasthenia gravis, myositis, narcolepsy, neuromyelitis optica
(Devic's), neutropenia, ocular cicatricial pemphigoid, optic
neuritis, palindromic rheumatism, PANDAS (Pediatric Autoimmune
Neuropsychiatric Disorders Associated with Streptococcus),
paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome,
Pars planitis (peripheral uveitis), Parsonnage-Turner syndrome,
pemphigus, peripheral neuropathy, perivenous encephalomyelitis,
pernicious anemia, POEMS syndrome, polyarteritis nodosa,
polymyalgia rheumatica, polymyositis, postmyocardial infarction
syndrome, postpericardiotomy syndrome, primary biliary cirrhosis,
primary sclerosing cholangitis, progesterone dermatitis, psoriasis,
psoriatic arthritis, pure red cell aplasia, pyoderma gangrenosum,
Raynauds phenomenon, reactive Arthritis, reflex sympathetic
dystrophy, Reiter's syndrome, relapsing polychondritis, restless
legs syndrome, retroperitoneal fibrosis, rheumatic fever,
rheumatoid arthritis (RA), rheumatoid arthritis, sarcoidosis,
Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm
& testicular autoimmunity, stiff person syndrome, subacute
bacterial endocarditis, Susac's syndrome, sympathetic ophthalmia,
Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,
thrombocytopenic purpura, Tolosa-Hunt syndrome, transverse
myelitis, type 1 diabetes, type I, II, & III autoimmune
polyglandular syndromes, ulcerative colitis, undifferentiated
connective tissue disease (UCTD), uveitis, vasculitis,
vesiculobullous dermatosis, vitiligo, and Wegener's
granulomatosis.
[0135] Examples of fibrotic diseases which may be treated by the
IL-10 fusion proteins of the invention include, but are not limited
to: adhesive capsulitis, arthrofibrosis, atrial fibrosis, chronic
kidney disease, cirrhosis of the liver, cystic fibrosis (CF),
Dupuytren's contracture, endomyocardial fibrosis, glial scar,
idiopathic pulmonary fibrosis, keloid, macular degeneration,
mediastinal fibrosis, myelofibrosis, NAFLD/NASH, nephrogenic
systemic fibrosis, Peyronie's disease, progressive massive fibrosis
(lungs), proliferative vitreoretinopathy, pulmonary fibrosis,
retroperitoneal fibrosis, scar tissue formation resulting from
strokes, scleroderma, systemic sclerosis, tissue adhesion.
[0136] Examples of inflammatory diseases which may be treated by
the IL-10 fusion proteins of the invention include, but are not
limited to: allergic enteritis, alpha-1-antitrypsin deficiency,
ankylosing spondylitis, asthma, Barrett's esophagus, Behcet's
disease, chronic fatigue syndrome (CFS/CFIDS/ME), chronic Lyme
disease (borreliosis), cocaine-associated vasculitis, Crohn's
disease, deficiency of the Interleukin-1 Receptor Antagonist
(DIRA), depression, diabetes, Familial Mediterranean Fever (FMF),
fibromyalgia (FM), gastroesophageal reflux disease (GERD),
glomerulonephritis, graft versus host disease, granulomatous
angiitis, Hashimoto's thyroiditis, hypertension, hyperthyroidism,
hypothyroidism, inflammatory bowel disease (IBD), inflammatory
myopathies (polymyositis, inclusion body myositis,
dermatomyositis), interstitial cystitis (IC), irritable bowel
syndrome (IBS), ischemic colitis, kidney stones, Lofgren's
syndrome, Lupus erythematosis, methamphetamine-associated
vasculitis, migraine headache, Morgellon's, multiple chemical
sensitivity (MCS), multiple sclerosis (MS), neonatal onset
multisystem inflammatory disease (NOMID), optic neuritis,
osteoarthritis, pemphigus vulgaris, polymyalgia rheumatica,
prostatitis, psoriasis, psoriatic arthritis, radiation colitis,
Raynaud's syndrome/phenomenon, reactive arthritis (Reiter
syndrome), reflex sympathetic dystrophy (RSD), restless leg
syndrome, rheumatoid arthritis (RA), sarcoidosis, scleroderma,
seasonal affective disorder (SAD), septic shock, sinusitis,
Sjogren's syndrome, temporal arteritis, tumor necrosis factor (TNF)
receptor-associated periodic syndrome (TRAPS), ulcerative colitis,
uveitis, vasculitis, and vertigo.
[0137] Examples of ischemic diseases which may be treated by the
IL-10 fusion proteins of the invention include, but are not limited
to: acute coronary syndrome, angina pectoris, angor animi,
copeptin, coronary artery disease, coronary ischemia, hibernating
myocardium, ischemic stroke, management of acute coronary syndrome,
meldonium, myocardial infarction, myocardial infarction
complications, myocardial infarction diagnosis, myocytolysis,
post-anoxic encephalopathy, Prinzmetal's angina, Sgarbossa's
criteria, stroke, TIMI, transient ischemic attack (TIA) and
unstable angina.
[0138] Examples of neurodegenerative diseases which may be treated
by the IL-10 fusion proteins of the invention include, but are not
limited to: ataxia telangiectasia, autosomal dominant cerebellar
ataxia, Baggio-Yoshinari syndrome, Batten disease, estrogen and
neurodegenerative diseases, hereditary motor and sensory neuropathy
with proximal dominance, Infantile Refsum disease, JUNQ and IPOD,
locomotor ataxia, Lyme disease, Machado-Joseph disease, mental
retardation and microcephaly with pontine and cerebellar
hypoplasia, multiple system atrophy, neuroacanthocytosis, neuronal
ceroid lipofuscinosis, Niemann-Pick disease, pontocerebellar
hypoplasia, protein aggregation, pyruvate dehydrogenase deficiency,
radiation myelopathy, Refsum disease, retinitis pigmentosa,
Sandhoff disease, Shy-Drager syndrome, spinal muscular atrophy,
spinocerebellar ataxia, subacute combined degeneration of spinal
cord, subacute sclerosing panencephalitis, Tabes dorsalis,
Tay-Sachs disease, toxic encephalopathy, toxic leukoencephalopathy
and Wobbly Hedgehog Syndrome.
[0139] Examples of neuropathic diseases which may be treated by the
IL-10 fusion proteins of the invention include, but are not limited
to: Bell's Palsy, campylobacter-associated motor axonopathies,
Charcot-Marie-Tooth, chronic inflammatory demyelinating
polyneuropathy, diabetic amyotrophy avulsion, diabetic
neuropathies, Guillain Barre Syndrome and vasculitis.
[0140] Examples of pain disorders which may be treated by the IL-10
fusion proteins of the invention include, but are not limited to:
Amplified musculoskeletal pain syndromes, Anterior cutaneous nerve
entrapment syndrome, central pain syndrome, chronic functional
abdominal pain, chronic pain, chronic prostatitis/chronic pelvic
pain syndrome, chronic wound pain, degenerative disc disease,
dentomandibular sensorimotor dysfunction, failed back syndrome,
fibromyalgia, interstitial cystitis, irritable bowel syndrome
(IBS), myofascial pain syndrome, pelvic pain, post-vasectomy pain
syndrome, reflex neurovascular dystrophy, sickle-cell disease,
theramine, and vulvodynia.
[0141] Examples of auditory disorders which may be treated by the
IL-10 fusion proteins of the invention include, but are not limited
to: conductive hearing loss, sensorineural hearing loss (SNHL),
mixed hearing loss.
[0142] Examples of psychiatric disorders which may be treated by
the IL-10 fusion proteins of the invention include, but are not
limited to: major depressive disorder, treatment-refractory
depression, treatment-resistant depression.
[0143] Examples of trauma and injury which may be treated by the
IL-10 fusion proteins of the invention include, but are not limited
to: including central nervous system (CNS) injuries, traumatic
brain injury, spinal cord injury, crush injuries, shock, tendon
damage, wounds to the cornea, wounds to the eye, skin wounds.
[0144] Preferably, an IL-10 dimerized complex in accordance with
the invention may be used to treat patients who suffer from, for
example, autoimmune disorders including autoimmune
lymphoproliferative syndrome (ALPS), autoimmune thyroiditis,
Crohn's disease, Grave's disease, Hashimoto's thyroiditis, Kawasaki
disease, Lupus (systemic lupus erythematosus), multiple sclerosis
(MS), myasthenia gravis, psoriasis, rheumatoid arthritis, Sjogren's
syndrome, type 1 diabetes, ulcerative colitis; fibrotic diseases
including Chronic Kidney Disease, cirrhosis of the liver, macular
degeneration, NAFLD/NASH, proliferative vitreoretinopathy,
pulmonary fibrosis, scar tissue formation resulting from strokes,
tissue adhesion; including inflammatory diseases including allergic
enteritis, alpha-1-antitrypsin deficiency, asthma, Behcet's
disease, cocaine-associated vasculitis, glomerulonephritis, Graft
Versus Host Disease, granulomatous angiitis, inflammatory bowel
disease, inflammatory myopathies (polymyositis, inclusion body
myositis, dermatomyositis), ischemic colitis,
methamphetamine-associated vasculitis, optic neuritis, pemphigus
vulgaris, radiation colitis, sarcoidosis, Septic Shock, temporal
arteritis, vasculitis; ischemic diseases including myocardial
infarction, post-anoxic encephalopathy, stroke; neurodegenerative
diseases including neuronal ceroid lipofuscinosis, radiation
myelopathy, retinitis pigmentosa, spinal muscular atrophy;
neuropathic diseases including campylobacter-associated motor
axonopathies, Charcot-Marie-Tooth, chronic inflammatory
demyelinating polyneuropathy, diabetic amyotrophy avulsion,
diabetic neuropathies, Guillain Barre Syndrome; auditory disorders
including Conductive hearing loss, Sensorineural hearing loss
(SNHL), Mixed hearing loss; psychiatric disorders including major
depressive disorder, treatment-refractory depression,
treatment-resistant depression; trauma and injury including central
nervous system (CNS) injuries, traumatic brain injury, spinal cord
injury, crush injuries, shock, tendon damage, wounds to the cornea,
wounds to the eye, skin wounds.
[0145] Most preferably, an IL-10 dimerized complex in accordance
with the invention may be used to treat patients who suffer from,
for example, autoimmune disorders including autoimmune
lymphoproliferative syndrome (ALPS), autoimmune thyroiditis,
Crohn's disease, Grave's disease, Hashimoto's thyroiditis, Kawasaki
disease, Lupus (systemic lupus erythematosus), multiple sclerosis
(MS), myasthenia gravis, psoriasis, rheumatoid arthritis, Sjogren's
syndrome, type 1 diabetes, ulcerative colitis; fibrotic diseases
including Chronic Kidney Disease, cirrhosis of the liver, macular
degeneration, NAFLD/NASH, proliferative vitreoretinopathy,
pulmonary fibrosis, scar tissue formation resulting from strokes,
tissue adhesion; inflammatory diseases including allergic
enteritis, alpha-1-antitrypsin deficiency, asthma, Behcet's
disease, cocaine-associated vasculitis, glomerulonephritis, Graft
Versus Host Disease, granulomatous angiitis, inflammatory bowel
disease, inflammatory myopathies (polymyositis, inclusion body
myositis, dermatomyositis), ischemic colitis,
methamphetamine-associated vasculitis, optic neuritis, pemphigus
vulgaris, radiation colitis, sarcoidosis, Septic Shock, temporal
arteritis, vasculitis; ischemic diseases including myocardial
infarction, post-anoxic encephalopathy, stroke; neurodegenerative
diseases including neuronal ceroid lipofuscinosis, radiation
myelopathy, retinitis pigmentosa, spinal muscular atrophy;
neuropathic diseases including campylobacter-associated motor
axonopathies, Charcot-Marie-Tooth, chronic inflammatory
demyelinating polyneuropathy, diabetic amyotrophy avulsion,
diabetic neuropathies, Guillain Barre Syndrome; auditory disorders
including Conductive hearing loss, Sensorineural hearing loss
(SNHL), Mixed hearing loss; psychiatric disorders including major
depressive disorder, treatment-refractory depression,
treatment-resistant depression; trauma and injury including central
nervous system (CNS) injuries, traumatic brain injury, spinal cord
injury, crush injuries, shock, tendon damage, wounds to the cornea,
wounds to the eye, skin wounds.
[0146] Preferably an IL-10 fusion protein or dimerized complex
thereof in accordance with the invention may be used to treat
patients who suffer from, for example cancer of the uterus, cervix,
breast, ovaries, prostate, testes, penis, gastrointestinal tract,
esophagus, oropharynx, stomach, small or large intestines, colon,
or rectum, kidney, renal cell, bladder, bone, bone marrow, skin,
head or neck, skin, liver, gall bladder, heart, lung, pancreas,
salivary gland, adrenal gland, thyroid, brain, gliomas, ganglia,
central nervous system (CNS) and peripheral nervous system (PNS),
and immune system, spleen or thymus, papilloma virus-induced
cancers, epithelial cell cancers, endothelial cell cancers,
squamous cell carcinomas, adenocarcinomas, carcinomas, melanomas,
sarcomas, teratocarcinomas, immunogenic tumors, non-immunogenic
tumors, dormant tumors, lymphomas, leukemias, myelomas,
chemically-induced cancers, metastasis, and angiogenesis, and
Tuberous sclerosis.
[0147] Preferably, an IL-10 fusion protein or dimerized complex
thereof in accordance with the invention may be used to treat
patients who suffer from, for example cancer of the uterus, cervix,
breast, ovaries, prostate, testes, penis, gastrointestinal tract,
esophagus, oropharynx, stomach, small or large intestines, colon,
or rectum, kidney, renal cell, bladder, bone, bone marrow, skin,
head or neck, skin, liver, gall bladder, heart, lung, pancreas,
salivary gland, adrenal gland, thyroid, brain, gliomas, ganglia,
central nervous system (CNS) and peripheral nervous system (PNS),
and immune system, spleen or thymus, papilloma virus-induced
cancers, epithelial cell cancers, endothelial cell cancers,
squamous cell carcinomas, adenocarcinomas, carcinomas, melanomas,
sarcomas, teratocarcinomas, immunogenic tumors, non-immunogenic
tumors, dormant tumors, lymphomas, leukemias, myelomas,
chemically-induced cancers, metastasis, and angiogenesis, and
Tuberous sclerosis.
[0148] Preferably, an IL-10 fusion protein or dimerized complex
thereof in accordance with the invention may be used to treat
patients who suffer from, for example cancer of the uterus, cervix,
breast, ovaries, prostate, testes, penis, gastrointestinal tract,
esophagus, oropharynx, stomach, small or large intestines, colon,
or rectum, kidney, renal cell, bladder, bone, bone marrow, skin,
head or neck, skin, liver, gall bladder, heart, lung, pancreas,
salivary gland, adrenal gland, thyroid, brain, gliomas, ganglia,
central nervous system (CNS) and peripheral nervous system (PNS),
and immune system, spleen or thymus, papilloma virus-induced
cancers, epithelial cell cancers, endothelial cell cancers,
squamous cell carcinomas, adenocarcinomas, carcinomas, melanomas,
sarcomas, teratocarcinomas, immunogenic tumors, non-immunogenic
tumors, dormant tumors, lymphomas, leukemias, myelomas,
chemically-induced cancers, metastasis, and angiogenesis, and
Tuberous sclerosis.
[0149] Preferably, an IL-10 fusion protein or dimerized complex
thereof in accordance with the invention may be used to treat
patients who suffer from auditory disorders, renal cell carcinoma,
melanoma, psoriasis, fibrosis, depression, and inflammatory bowel
disease (IBD).
[0150] The invention also provides Fc fusion proteins of the
inventions for use as a medicament. Preferably, the invention
provides a fusion protein X-L1-HINGE-Fc wherein X is a soluble
protein of interest as described earlier for use as a medicament.
Preferably X is factor IX, IL1Ra, or TNFR. Preferably X is IL-10,
IL-2, IL-2R.alpha. (or fusions thereof), or IFN.beta. for use as a
medicament. Preferably the invention provides a dimer complex of
X-L1-HINGE-Fc wherein X is a soluble protein of interest as
described earlier for use as a medicament.
[0151] The invention also provides Fc fusion proteins of the
inventions for use as a medicament to treat disease. Preferably,
the invention provides a fusion protein X-L1-HINGE-Fc wherein X is
a soluble protein of interest as described earlier for use as a
medicament to treat diseases as described earlier. Preferably X is
factor IX, for use as a medicament to treat bleeding. Preferably X
is IL-10 for treatment of Crohn's disease (CD), rheumatoid
arthritis (RA), psoriasis, viral infections such as chronic
hepatitis C and human immunodeficiency virus (HIV).
[0152] Preferably the invention provides a dimer complex of
X-L1-HINGE-Fc wherein X is a soluble protein of interest as
described earlier for use as a medicament to treat disease.
Preferably X is factor IX, IL1Ra, or TNFR, for use as a medicament
to treat cancer, autoimmune disease and bleeding disorders.
Preferably X is IL-10, IL-2, IL-2R.alpha. or fusions thereof, or
IFN.beta. for use in treating, for example, auditory disorders,
renal cell carcinoma, melanoma, psoriasis, fibrosis, depression,
and inflammatory bowel disease (IBD).
[0153] A factor IX dimerized fusion protein complex in accordance
with the invention may also be used in the manufacture of a
medicament to treat patients who are deficient in factor IX and
suffer from hemophilia B for e.g., control and prevention of
bleeding episodes, routine prophylaxis to prevent or reduce the
frequency of bleeding episodes, and perioperative management
(surgical prophylaxis).
[0154] An IL-10 fusion protein or dimerized complex thereof in
accordance with the invention may also be used in the manufacture
of a medicament to treat patients to diseases as set forth above,
auditory disorders, auditory disorders, renal cell carcinoma,
melanoma, psoriasis, fibrosis, depression, and inflammatory bowel
disease (IBD).
[0155] The application further provides pharmaceutically acceptable
compositions comprising the Fc fusion proteins described herein.
Therapeutic formulations comprising Fc fusion proteins are prepared
for storage by mixing the described proteins having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of aqueous solutions,
lyophilized or other dried formulations. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrans; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0156] The formulations herein may also contain more than one
active compounds as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0157] The Fc fusion proteins may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0158] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0159] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the fibronectin
based scaffold proteins described herein, which matrices are in the
form of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl
alcohol)), polylactides, copolymers of lactide and glycolide,
copolymers of L-glutamic acid and y ethyl-L-glutamate,
non-degradable ethylene-vinyl acetate, degradable lactic
acid-glycolic acid copolymers. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable
sustained release of, certain hydrogels release proteins for
shorter time periods. When encapsulated proteins remain in the body
for a long time, they may denature or aggregate as a result of
exposure to moisture at 37.degree. C., resulting in a loss of
biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0160] While the skilled artisan will understand that the dosage of
each Fc fusion protein will be dependent on the identity of the
soluble protein (X), the dosage ranges from about 0.0001 to 100
mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight.
For example dosages can be 0.3 mg/kg body weight, 1 mg/kg body
weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body
weight or within the range of 1-30 mg/kg. An exemplary treatment
regime entails administration once per week, once every two weeks,
once every three weeks, once every four weeks, once a month, once
every 3 months or once every three to 6 months. Dosage regimens
include 1 mg/kg body weight or 3 mg/kg body weight by intravenous
administration, with the protein being given using one of the
following dosing schedules: every four weeks for six dosages, then
every three months; every three weeks; 3 mg/kg body weight once
followed by 1 mg/kg body weight every three weeks. A fusion protein
of the invention is usually administered on multiple occasions.
Intervals between single dosages can be, for example, weekly,
monthly, every three months or yearly. Intervals can also be
irregular as indicated by measuring blood levels of the soluble
protein in the patient. In some methods, dosage is adjusted to
achieve a plasma concentration of soluble protein of about 0.1-1000
pg/ml and in some methods about 5-300 mg/ml.
[0161] For therapeutic applications, the Fc fusion proteins are
administered to a subject, in a pharmaceutically acceptable dosage
form. They can be administered intravenously as a bolus or by
continuous infusion over a period of time, by intramuscular,
subcutaneous, intra-ocular, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. The protein may
also be administered by intratumoral, peritumoral, intralesional,
or perilesional routes, to exert local as well as systemic
therapeutic effects. Suitable pharmaceutically acceptable carriers,
diluents, and excipients are well known and can be determined by
those of skill in the art as the clinical situation warrants.
Examples of suitable carriers, diluents and/or excipients include:
(1) Dulbecco's phosphate buffered saline, pH about 7.4, containing
about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline
(0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of the
present invention can be practiced in vitro, in vivo, or ex
vivo.
[0162] Administration of Fc fusion proteins, and one or more
additional therapeutic agents, whether co-administered or
administered sequentially, may occur as described above for
therapeutic applications. Suitable pharmaceutically acceptable
carriers, diluents, and excipients for co-administration will be
understood by the skilled artisan to depend on the identity of the
particular therapeutic agent being co-administered.
[0163] When present in an aqueous dosage form, rather than being
lyophilized, the Fc fusion protein typically will be formulated at
a concentration of about 0.1 mg/ml to 100 mg/ml, although wide
variation outside of these ranges is permitted. For the treatment
of disease, the appropriate dosage of Fc fusion proteins will
depend on the type of disease to be treated, the severity and
course of the disease, whether the Fc fusion proteins are
administered for preventive or therapeutic purposes, the course of
previous therapy, the patient's clinical history and response to
the Fc fusion protein, and the discretion of the attending
physician. The Fc fusion protein is suitably administered to the
patient at one time or over a series of treatments.
EXAMPLES
Example 1
Factor IX
[0164] Design of Factor IX scC.sub.LC.sub.H1-Fc
[0165] The single chain factor IX molecule contains the factor IX
sequence followed by a 10 residue linker having the amino acid
sequence: GGGGSGGGGS (SEQ ID NO: 11), the CL domain of IgG1
followed by a 20 residue linker having the amino acid sequence:
GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12) followed by the CHL hinge and
Fc portions of human IgG1.
Expression and Characterization of Factor IX
scC.sub.LC.sub.H1-Fc
[0166] The gene, having the following DNA sequence:
TABLE-US-00015 (SEQ ID NO: 13)
ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCC
CTCGTGACCAACAGCACCGTGTTTCTGGACCACGAGAACGCCAAC
AAGATCCTGAACCGGCCCAAGCGGTACAACAGCGGCAAGCTGGAA
GAGTTCGTGCAGGGCAACCTGGAACGCGAGTGCATGGAAGAGAAG
TGCAGCTTCGAAGAGGCCAGAGAGGTGTTCGAGAACACCGAGCGG
ACCACCGAGTTCTGGAAGCAGTACGTGGACGGCGACCAGTGCGAG
AGCAACCCCTGTCTGAATGGCGGCAGCTGCAAGGACGACATCAAC
AGCTACGAGTGCTGGTGCCCCTTCGGCTTCGAGGGCAAGAACTGC
GAGCTGGACGTGACCTGCAACATCAAGAACGGCAGATGCGAGCAG
TTCTGCAAGAACAGCGCCGACAACAAGGTCGTGTGCTCCTGCACC
GAGGGCTACAGACTGGCCGAGAACCAGAAGTCCTGCGAGCCCGCC
GTGCCTTTCCCATGTGGAAGAGTGTCCGTGTCCCAGACCAGCAAG
CTGACCAGAGCCGAGACAGTGTTCCCCGACGTGGACTACGTGAAC
TCCACCGAGGCCGAGACAATCCTGGACAACATCACCCAGAGCACC
CAGTCCTTCAACGACTTCACCAGAGTCGTGGGCGGCGAGGATGCC
AAGCCTGGACAGTTCCCGTGGCAGGTGGTGCTGAACGGAAAGGTG
GACGCCTTTTGCGGCGGCAGCATCGTGAACGAGAAGTGGATCGTG
ACAGCCGCCCACTGCGTGGAAACCGGCGTGAAGATTACAGTGGTG
GCCGGCGAGCACAACATCGAGGAAACCGAGCACACAGAGCAGAAA
CGGAACGTGATCAGAATCATCCCCCACCACAACTACAACGCCGCC
ATCAACAAGTACAACCACGACATTGCCCTGCTGGAACTGGACGAG
CCCCTGGTGCTGAATAGCTACGTGACCCCCATCTGCATTGCCGAC
AAAGAGTACACCAACATCTTTCTGAAGTTCGGCAGCGGCTACGTG
TCCGGCTGGGGCAGAGTGTTTCACAAGGGCAGATCCGCTCTGGTG
CTGCAGTACCTGAGAGTGCCTCTGGTGGACCGGGCCACCTGTCTG
AGAAGCACCAAGTTCACCATCTACAACAACATGTTCTGCGCCGGC
TTCCATGAGGGCGGCAGAGATAGCTGTCAGGGCGATTCTGGCGGC
CCTCACGTGACAGAAGTGGAAGGCACCAGCTTTCTGACCGGCATC
ATCAGCTGGGGCGAGGAATGCGCCATGAAGGGGAAGTACGGCATC
TACACCAAGGTGTCCAGATATGTGAACTGGATCAAAGAAAAGACC
AAGCTGACAGGCGGCGGAGGCTCTGGCGGAGGCGGATCTAGAACA
GTGGCCGCTCCCAGCGTGTTCATCTTCCCACCTAGCGACGAGCAG
CTGAAGTCCGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTC
TACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTG
CAGAGCGGCAACAGCCAGGAAAGCGTGACCGAGCAGGACAGCAAG
GACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCC
GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAG
GGCCTGTCTAGCCCAGTGACCAAGAGCTTCAACCGGGGCGAATCT
GGGGGCGGAGGATCAGGCGGGGGAGGAAGTGGGGGAGGGGGAAGC
GGAGGGGGAGGATCTGCCTCTACAAAGGGCCCTAGCGTGTTCCCC
CTGGCCCCTAGCAGCAAGTCTACAAGCGGAGGCACAGCTGCCCTG
GGCTGCCTCGTGAAGGACTACTTCCCTGAGCCCGTGACCGTGTCC
TGGAACAGCGGAGCACTGACAAGCGGCGTGCACACCTTTCCAGCC
GTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACA
GTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAATACCAAAGTGGACAAGCGGGTGGAACCC
AAGAGCAGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCC
GAACTGCTGGGAGGCCCTTCCGTGTTCCTGTTCCCCCCAAAGCCC
AAGGACACCCTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTG
GTGGTGGATGTGTCCCACGAGGACCCAGAAGTGAAGTTCAATTGG
TATGTGGACGGGGTGGAAGTGCACAACGCCAAGACCAAACCCAGA
GAGGAACAGTACAATAGCACCTACCGGGTGGTGTCCGTGCTGACA
GTGCTGCACCAGGACTGGCTGAATGGCAAAGAGTATAAGTGCAAA
GTGTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGC
AAGGCCAAGGGCCAGCCCCGCGAACCCCAGGTGTACACACTGCCC
CCAAGCCGGGAAGAGATGACCAAGAACCAGGTGTCCCTGACCTGT
CTCGTGAAAGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAG
AGCAACGGCCAGCCCGAGAACAATTACAAGACCACCCCCCCTGTG
CTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAACTGACCGTG
GACAAGAGCCGGTGGCAGCAGGGAAACGTGTTCAGCTGCAGCGTG
ATGCACGAGGCCCTGCACAACCACTACACCCAGAAAAGCCTGAGC CTGTCCCCTGGCAAG;
was synthesized (Genewiz), cloned into pcDNA/UCOE and transiently
expressed in HEK293 cells using the Expi293 expression system (Life
Technologies). Proteins were purified first using Protein A (GE
Healthcare) with low pH elution and dialyzed against 2 L 25 mM TRIS
pH 7.5, 150 mM NaCl 3 times. Following dialysis, the protein was
loaded onto a Q sepharose FF column and eluted with step gradients
of CaCl.sub.2 in 25 mM TRIS pH 7.5, 150 mM NaCl. The most active
fractions were pooled and dialyzed against 1.times.PBS for further
analysis.
[0167] The molecule was analyzed by SDS PAGE gel under reducing and
non-reducing conditions (FIG. 2). For non-reducing conditions, 5 ug
of purified protein was loaded onto a NuPAGE.RTM. NOVEX.RTM. 3-8%
TRIS-Acetate gel (Invitrogen) with a HIMARK.TM. pre-stained protein
standard (Invitrogen) (MW range 31 kD-460 kD). For reducing
conditions, 5 ug of protein was loaded onto an any kD.TM. gel
(Invitrogen) with a PRECISION PLUS PROTEIN.TM. Kaleidoscope
standard (Invitrogen) (MW range 10 kD-250 kD).
Bioactivity Factor IX scC C.sub.LC.sub.H1-Fc (APTT Assay)
[0168] An automated Factor IX activity assay was performed using
the KC-1 Delta.TM. instrument (Tcoag, Wicklow, IRE) to quantify the
ability of the FIX component of the Factor IX scC.sub.LC.sub.H1-Fc
protein to restore the clotting activity of FIX-deficient plasma.
Test samples were mixed with equal volumes of human FIX-deficient
plasma (George King Bio-Medical Inc, Overland Park, Kans.) and
cephalin-containing ellagic acid activator (aPTT-soluble activator,
Helena Laboratories, cat. #5389), and after 4 min incubation, 5 mM
calcium chloride (25 mM stock, VWR) was added and the time to clot
measured. Activity was calculated based on a calibration curve of
clotting times versus activity unit concentration (IU/mL) of serial
dilutions of rHuman Factor IX (FIX) (Haematologic Technologies Inc.
Essex Junction, Vt.) standard for purified proteins. Factors of
intrinsic coagulation systems are activated by incubating the
plasma with the optimal amount of phospholipids and a surface
activator at 37.degree. C. The addition of calcium ions triggers
the coagulation process, and the clotting time is them measured.
The APTT is the time taken for a fibrin clot to form (FIG. 3).
Rat PK of Factor IX scC.sub.LC.sub.H1-Fc
[0169] Single intravenous doses of 51 IU/kg factor IX
scC.sub.LC.sub.H1-Fc were administered into the lateral tail vein
of 3 rats. Blood samples were collected at 0.25, 4, 8, 24, 48, 72,
96, and 168 hours after administration of factor IX
scC.sub.LC.sub.H1-Fc, and citrated plasma (0.32% final) prepared.
Concentrations were measured using standard MSD techniques with
Goat anti-Human factor IX Affinity purified IgG (Enzyme Research
Laboratories, South Bend, Ind.) as the capture antibody and Goat
anti-Human IgG Fc cross-adsorbed antibody biotinylated (Bethyl
Laboratories, Montgomery, Tex.) as the detection antibody.
Pharmacokinetic analysis was performed using non-compartmental
modeling with WINNONLIN.RTM. software (Pharsight Corporation,
Mountain View, Calif.). The pharmacokinetic parameter estimates
derived from MSD data included maximum concentration (Cmax), area
under the time versus concentration curve (AUC), and elimination
half-life (t.sub.1/2) (FIG. 4 and Table 1).
TABLE-US-00016 TABLE 1 Testing Dosing Dose Dose C.sub.max
AUC.sub.0-.infin. t.sub.1/2 Test Article Animal Route (mg/kg)
(IU/kg) (ug/mL) (ug-h/mL) (h) FIXscLCLCH1Fc rat IV 7.9 51 17.0 .+-.
11.4 69.6 .+-. 7.78 53.7 .+-. 12.5 Mono* FIXFc rat IV 200 34.8 .+-.
5.3 rhFIX** rat IV 50 2.6 8.2 5.0 *Peters, R.T. et al. Prolonged
activity of factor IX as a monomeric Fc fusion protein. Blood.
(2013). **Keith, J.C. et al. Evaluation of Recombinant Human Factor
IX: Pharmacokinetic Studies in the Rat and the Dog. Thrombosis and
Haemostasis 73(1): 101-105 (1994).
Example 2
TNF-R2
[0170] Design of TNF-R2 scC.sub.LC.sub.H1-Fc
[0171] The single chain TNFR2 molecule contains the TNFR2 sequence
followed by a 10 residue linker, GGGGSGGGGS (SEQ ID NO: 11), the CL
domain of IgG1 followed by a 20 residue linker GGGGSGGGGSGGGGSGGGGS
(SEQ ID NO: 12) followed by the CH1, hinge and Fc portions of human
IgG1.
Expression of TNF-R2 scC.sub.LC.sub.H1-Fc
[0172] The gene, having the following DNA sequence:
TABLE-US-00017 (SEQ ID NO: 14)
ATGTATAGGATGCAGCTCCTCAGCTGCATCGCTCTGTCCCTCGCC
CTGGTGACCAACAGCCTCCCTGCCCAGGTGGCCTTTACACCCTAC
GCTCCTGAGCCCGGAAGCACCTGCAGGCTCAGGGAGTACTACGAT
CAGACCGCCCAAATGTGTTGCAGCAAGTGCTCCCCTGGCCAGCAC
GCCAAGGTGTTCTGCACCAAGACAAGCGATACCGTGTGCGATAGC
TGTGAGGACAGCACCTACACCCAGCTGTGGAATTGGGTGCCCGAG
TGCCTGAGCTGTGGCAGCAGGTGCAGCAGCGATCAGGTGGAGACA
CAGGCCTGCACCAGAGAGCAGAACAGGATTTGTACCTGCAGGCCC
GGCTGGTATTGCGCCCTGAGCAAGCAGGAGGGATGTAGGCTGTGC
GCCCCTCTGAGGAAATGCAGACCTGGCTTTGGAGTGGCTAGGCCC
GGCACCGAGACATCCGACGTGGTGTGCAAGCCTTGTGCCCCTGGC
ACCTTTTCCAACACCACCAGCTCCACCGACATCTGCAGGCCCCAT
CAGATTTGCAACGTGGTGGCCATCCCCGGAAACGCTAGCATGGAT
GCCGTGTGCACCTCCACCTCCCCTACCAGGAGCATGGCCCCTGGA
GCCGTGCATCTGCCTCAACCCGTCAGCACCAGAAGCCAGCACACA
CAGCCCACCCCCGAACCTAGCACCGCTCCCTCCACCAGCTTCCTG
CTGCCTATGGGACCCTCCCCTCCTGCCGAAGGGAGCACCGGAGAT
GGAGGAGGAGGAAGCGGCGGAGGAGGCTCCAGAACAGTGGCTGCC
CCTAGCGTGTTCATTTTCCCTCCCTCCGACGAGCAGCTCAAGTCC
GGAACCGCTTCCGTGGTCTGCCTGCTGAACAACTTCTACCCCAGA
GAGGCCAAGGTGCAGTGGAAAGTCGACAATGCTCTGCAGAGCGGA
AACTCCCAGGAGTCCGTCACCGAGCAGGACAGCAAGGACTCCACA
TATAGCCTGTCCTCCACCCTGACCCTGAGCAAGGCCGACTATGAG
AAACACAAGGTGTATGCCTGCGAAGTGACCCACCAGGGCCTGTCC
AGCCCCGTCACCAAGTCCTTCAATAGGGGCGAGAGCGGAGGCGGC
GGGAGCGGCGGCGGCGGGAGCGGAGGAGGAGGGAGCGGAGGAGGC
GGAAGCGCTTCCACCAAGGGACCTAGCGTGTTTCCCCTCGCCCCC
AGCTCCAAGAGCACAAGCGGAGGCACAGCCGCTCTGGGCTGTCTG
GTGAAGGATTACTTCCCCGAGCCCGTCACAGTGAGCTGGAACTCC
GGAGCCCTGACCTCCGGAGTGCACACCTTTCCTGCCGTGCTGCAG
AGCAGCGGACTGTACAGCCTGTCCAGCGTGGTCACAGTGCCCTCC
AGCTCCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAG
CCCAGCAACACAAAGGTGGACAAGAGAGTGGAACCTAAGTCCTGT
GACAAAACCCATACCTGCCCTCCCTGCCCTGCCCCTGAGCTGCTG
GGAGGACCTAGCGTGTTTCTGTTTCCCCCCAAACCCAAGGATACC
CTGATGATCAGCAGGACCCCTGAGGTGACATGCGTGGTGGTGGAC
GTGTCCCACGAGGACCCTCAGGTCAAGTTCAACTGGTACGTGGAT
GGCGTCCAGGTGCACAATGCTAAGACCAAGCCCAGGGAGCAGCAA
TACAATTCCACCTACAGGGTGGTGTCCGTGCTCACCGTCCTCCAC
CAGAACTGGCTCGACGGCAAAGAATACAAGTGCAAAGTGAGCAAC
AAGGCTCTCCCCGCCCCTATCGAGAAGACCATTTCCAAAGCCAAG
GGCCAGCCCAGAGAACCTCAAGTCTACACCCTGCCCCCCAGCAGG
GAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTCGTCAAG
GGATTCTATCCCAGCGACATCGCCGTGGAATGGGAGTCCAATGGC
CAGCCCGAGAATAACTACAAGACCACACCCCCCGTGCTGGATTCC
GATGGCAGCTTTTTCCTGTACAGCAAGCTGACAGTGGATAAGAGC
AGGTGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTCATGCACGAA
GCCCTGCACAATCACTACACCCAGAAGAGCCTGTCCCTCAGCCCC GGCAAG;
was synthesized (Genewiz), cloned into pcDNA/UCOE and transiently
expressed in HEK293 cells using the Expi293 expression system (Life
Technologies). Proteins were purified first using Protein A (GE
Healthcare) with low pH elution and dialyzed against 2 L
1.times.PBS 3 times.
[0173] The molecule was analyzed by SDS PAGE gel under reducing and
non-reducing conditions (FIGS. 5A and 5B). For non-reducing
conditions, 5 ug of purified protein was loaded onto a NuPAGE.RTM.
Novex.RTM. 3-8% TRIS-Acetate gel (Invitrogen) with a HiMark.TM.
Pre-stained protein standard (Invitrogen) (MW range 31 kD-460 kD).
For reducing conditions, 5 ug of protein was loaded onto Any kD.TM.
gel (Invitrogen) with a PRECISION PLUS PROTEIN.TM. Kaleidoscope
standard (Invitrogen) (MW range 10 kD-250 kD).
Bioactivity of TNF-R2 scC.sub.LC.sub.H1-Fc
[0174] HEK-Blue.TM. TNF-.alpha. cells (InvivoGen) are human
embryonic kidney cells specifically designed to detect bioactive
TNF-.alpha. in vitro by monitoring the activation of the
NF-.kappa.B/AP-1 pathways. The cell line expresses an inducible
secreted embryonic alkaline phosphatase (SEAP) reporter gene under
control of the IFN-.beta. minimal promoter fused to five NF.kappa.b
and five AP-1 binding sites. For the TNF-.alpha. antagonist assay,
HEK-Blue TNF-.alpha. cells were plated at 50,000 cells/well in DMEM
media containing 2 mM L-glutamine, 4.5 g/l glucose and 10% heat
inactivated FBS (Gibco) and 235 pM TNF-.alpha. 1a (InvivoGen).
Cells were incubated for 20 hours at 37.degree. C., 5% CO.sub.2
with varying concentrations of TNF-R2 direct fusion or TNF-R2
single chain fusion body (TNF-R2 scC.sub.LC.sub.H1-Fc). SEAP
production was detected by adding QUANTI-Blue and incubating for 3
hours at 37.degree. C., 5% CO.sub.2 and read on a plate reader at
630 nm. Activation of the SEAP gene can be inhibited by the
TNF-.alpha. antagonist TNF-R2 in a dose dependent manner. The
TNF-R2 single chain fusion body molecule inhibited activation of
the SEAP gene with an IC.sub.50 of 51 pM vs the direct fusion of
TNF-R2 with an IC.sub.50 of 112 pM (FIG. 6).
Rat PK of TNF-R2 scC.sub.LC.sub.H1-Fc
[0175] Single intravenous doses of 5 mg/kg TNF-R2
scC.sub.LC.sub.H1-Fc were administered into the lateral tail vein
of 3 rats. Blood samples were collected at 0.083, 1, 6, 24, 48 hr,
5, 7, 9, 12, 15, 21, 28 days after administration of TNF-R2
scC.sub.LC.sub.H1-Fc. Concentrations were measured using standard
MSD techniques with Goat anti-Human F(ab')2 IgG Fc (Thermo
Scientific, Rockford, Ill.) as the capture antibody and Goat
anti-Human IgG Fc cross-adsorbed antibody biotinylated (Bethyl
Laboratories, Montgomery, Tex.) as the detection antibody.
Pharmacokinetic analysis was performed using non-compartmental
modeling with WinNonlin.RTM. software (Pharsight Corporation,
Mountain View, Calif.). The pharmacokinetic parameter estimates
derived from MSD data included maximum concentration (Cmax), area
under the time versus concentration curve (AUC), and elimination
half-life (t.sub.1/2) (FIG. 7 and Table 2).
TABLE-US-00018 TABLE 2 Testing Dosing Dose C.sub.max
AUC.sub.0-.infin. t.sub.1/2 Test Article Animal Route (mg/kg) (nM)
(nM-h) (h) TNF-R2 direct rat IV 5 584 .+-. 59.2 3445 .+-. 967 .sup.
24 .+-. 6.5 fusion TNF-R2 rat IV 5 412 .+-. 109 3207 .+-. 157 102
.+-. 33 scC.sub.LC.sub.H1-Fc
Example 3
IL1Ra
[0176] Design of IL1Ra scC.sub.LC.sub.H1-Fc
[0177] The single chain IL1Ra molecule contains the IL1Ra sequence
followed by a 10 residue linker, GGGGSGGGGS (SEQ ID NO: 11), the CL
domain of IgG1 followed by a 20 residue linker,
GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 12) followed by the CH1, hinge and
Fc portions of human IgG1.
Expression of IL1Ra scC.sub.LC.sub.H1-Fc
[0178] The gene having the following DNA sequence:
TABLE-US-00019 (SEQ ID NO: 15)
ATGTACCGGATGCAGCTGCTGTCCTGTATCGCCCTGTCTCTGGCC
CTGGTCACCAACTCCAGACCCTCTGGCCGGAAGTCCTCCAAGATG
CAGGCCTTCCGGATCTGGGACGTGAACCAGAAAACCTTCTACCTG
CGGAACAACCAGCTGGTGGCCGGCTATCTGCAGGGCCCCAACGTG
AACCTGGAAGAGAAGATCGACGTGGTGCCCATCGAGCCCCACGCC
CTGTTTCTGGGAATCCACGGCGGCAAGATGTGCCTGTCCTGCGTG
AAGTCCGGCGACGAGACACGGCTGCAGCTGGAAGCCGTGAACATC
ACCGACCTGTCCGAGAACCGGAAGCAGGACAAGAGATTCGCCTTC
ATCAGATCCGACTCCGGCCCTACCACCTCCTTCGAGTCTGCTGCT
TGCCCCGGCTGGTTCCTGTGCACCGCCATGGAAGCTGACCAGCCC
GTGTCCCTGACCAACATGCCTGACGAGGGCGTGATGGTCACCAAG
TTCTATTTTCAGGAAGATGAGGGCGGAGGCGGCTCTGGCGGCGGA
GGATCTAGAACAGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCT
TCCGACGAGCAGCTGAAGTCTGGCACCGCCTCTGTCGTGTGCCTG
CTGAACAACTTCTACCCTCGCGAGGCCAAGGTGCAGTGGAAGGTG
GACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTCACCGAG
CAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACC
CTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAA
GTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAAC
CGGGGCGAAAGCGGAGGCGGAGGTTCAGGTGGTGGTGGATCAGGT
GGCGGCGGATCTGGCGGTGGTGGCTCTGCTTCTACCAAGGGCCCT
TCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGC
ACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCT
GTGACCGTGTCCTGGAACTCTGGCGCTCTGACATCCGGCGTGCAC
ACCTTCCCTGCTGTGCTGCAGTCCTCCGGCCTGTACAGCCTGTCC
TCCGTCGTGACCGTGCCTTCCAGCTCTCTGGGCACCCAGACCTAC
ATCTGTAACGTGAACCACAAGCCCTCCAACACCAAAGTGGACAAG
CGGGTGGAACCCAAGTCCTCCGACAAGACCCACACCTGTCCTCCC
TGCCCTGCTCCTGAACTGCTGGGCGGACCTAGCGTGTTCCTGTTC
CCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAA
GTGACCTGCGTGGTGGTCGATGTGTCCCACGAGGACCCAGAAGTG
AAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAG
ACCAAGCCCAGAGAGGAACAGTACAACTCCACCTACCGGGTGGTG
TCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAG
TACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCTATCGAA
AAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCTCAGGTG
TACACCCTGCCTCCCAGCCGGGAAGAGATGACCAAGAACCAGGTG
TCACTGACCTGTCTGGTCAAGGGCTTCTACCCCTCCGACATTGCC
GTGGAATGGGAGTCCAACGGCCAGCCCGAGAACAACTACAAGACC
ACCCCTCCCGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCC
AAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTC
TCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG
AAGTCCCTGTCCCTGAGCCCCGGCAAG;
was synthesized (Genewiz, Inc.), cloned into pcDNA/UCOE and
transiently expressed in HEK293 cells using the Expi293 expression
system (Life Technologies). Proteins were purified first using
Protein A (GE Healthcare) with low pH elution and dialyzed against
2 L 1.times.PBS 3 times. The molecule was analyzed by SDS PAGE gel
under reducing and non-reducing conditions (FIGS. 8A and 8B). For
non-reducing conditions, 5 ug of purified protein was loaded onto a
NuPAGE.RTM. NOVEX.RTM. 3-8% TRIS-Acetate gel (Invitrogen) with a
HIMARK.TM. Pre-stained protein standard (Invitrogen) (MW range 31
kD-460 kD). For reducing conditions, 5 ug of protein was loaded
onto an Any kD.TM. gel (Invitrogen) with a PRECISION PLUS
PROTEIN.TM. Kaleidoscope standard (Invitrogen) (MW range 10 kD-250
kD). Bioactivity of IL1Ra scC.sub.LC.sub.H1-Fc
[0179] HEK-Blue.TM. IL-1.beta. cells (InvivoGen) are human
embryonic kidney cells specifically designed to detect bioactive
IL-1.beta. in vitro by monitoring the IL-1.beta.-induced activation
of the NF-.kappa.B/AP-1 pathways. The cell line expresses an
inducible secreted embryonic alkaline phosphatase (SEAP) reporter
gene under control of the IFN-.beta. minimal promoter fused to five
NF.kappa.b and five AP-1 binding sites. For the IL-1.beta.
antagonist assay, HEK-Blue IL-1.beta. cells were plated at 50,000
cells/well in DMEM media containing 2 mM L-glu and 10% heat
inactivated FBS (Gibco) and 57 pM IL-1.beta. (R&D systems).
Cells were incubated for 20 hours at 37.degree. C., 5% CO.sub.2
with varying concentrations of IL1RascC.sub.LC.sub.H1-Fc. SEAP
production was detected by adding QUANTI-Blue and incubating for 3
hours at 37.degree. C., 5% CO.sub.2 and read on a plate reader at
630 nm. IL-1.beta. activation of the SEAP gene can be inhibited by
the IL-1.beta. antagonist IL-1Ra in a dose dependent manner. The
IL-1Ra single chain molecule inhibited IL-1.beta. activation of the
SEAP gene with an IC.sub.50 of 12.5 Nm (FIG. 9).
Rat PK of IL1Ra scC.sub.LC.sub.H1-Fc
[0180] Single intravenous doses of 2 mg/kg IL1Ra
scC.sub.LC.sub.H1-Fc were administered into a jugular vein catheter
of 3 rats. Blood samples were collected at 0.083, 0.25, 1, 2, 6,
24, 48, 72, 96 and 168 hours after administration of IL1Ra
scC.sub.LC.sub.H1-Fc. Single subcutaneous doses of 5 mg/kg IL1Ra
scC.sub.LC.sub.H1-Fc were administered into the interscapular
region of 3 rats. Blood samples were collected at 0.25, 1, 2, 4, 6,
24, 48, 72, 96 and 168 hours after administration of IL1Ra
scC.sub.LC.sub.H1-Fc. Concentrations were measured using standard
MSD techniques with Goat anti-Human F(ab')2 IgG Fc (Thermo
Scientific, Rockford, Ill.) as the capture antibody and Mouse
anti-Human IL1Ra biotin conjugate (Invitrogen, Grand Island, N.Y.)
as the detection antibody. Pharmacokinetic analysis was performed
using non-compartmental modeling with WINNONLIN.RTM. software
(Pharsight Corporation, Mountain View, Calif.). The pharmacokinetic
parameter estimates derived from MSD data included maximum
concentration (Cmax), area under the time versus concentration
curve (AUC), and elimination half-life (t.sub.1/2) (FIG. 10 and
Table 3).
TABLE-US-00020 TABLE 3 Testing Dosing Dose C.sub.max
AUC.sub.0-.infin. t.sub.1/2 Test Article Animal Route (mg/kg) (nM)
(nM-h) (h) IL1Ra- rat IV 2 375 .+-. 7.6 1828 .+-. 139 9.8 .+-. 0.9
scC.sub.LC.sub.H1- Fc IL1Ra- rat SC 5 24.7 .+-. 34.1 363 .+-. 476
9.4 .+-. 1.6 scC.sub.LC.sub.H1- Fc rhIL-1Ra* rat IV 1 448.5 .+-.
134 98.5 .+-. 5.8 1.15 .+-. 0.5 rhIL-1Ra* rat SC 1 25.3 .+-. 3.5
74.1 .+-. 9.3 0.85 .+-. 0.08 *Source: FDA document BLA: 103950/0.
PK parameters were converted to nM concentrations using a MW of
17257.6 g/mole for rhIL-1Ra
Intra-Ocular PK of IL1Ra scC.sub.LC.sub.H1-Fc
[0181] A bolus intravitreal injection of 0.5 mg IL1Ra
scC.sub.LC.sub.H1-Fc was administered into each eye of 8 male
rabbits. Blood samples from two animals were collected at 4, 96,
168 and 336 hours after administration of IL1Ra
scC.sub.LC.sub.H1-Fc. At the time of sacrifice, both eyes from each
animal were collected and flash frozen in liquid nitrogen.
Concentrations were measured using standard MSD techniques.
Pharmacokinetic analysis was performed using non-compartmental
modeling with WINNONLIN.RTM. software (Pharsight Corporation,
Mountain View, Calif.). The pharmacokinetic parameter estimates
derived from MSD data included maximum concentration (Cmax), area
under the time versus concentration curve (AUC), and elimination
half-life (t.sub.112) (Table 4 and FIG. 11).
TABLE-US-00021 TABLE 4 Test Testing C.sub.max AUC.sub.0-.infin.
t.sub.1/2 Article Animal Matrix (ug/mL) (ug/mL-h) (h) IL1Ra rabbit
Aqueous 2.53 369 83 scC.sub.LC.sub.H1- Fc IL1Ra rabbit Vitreous 265
2904 129 scC.sub.LC.sub.H1- Fc
Example 4
IL-2/IL2R.alpha.
[0182] Design of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc
[0183] The IL-2/IL-2R.alpha. single chain fusion body molecule
contains a circularly permuted human IL-2 linked to the
extracellular domain of IL-2R.alpha. fusion protein linked to the
CL-CH1-Fc domain (SEQ ID NO: 19) or the CH1-CL-Fc (SEQ ID NO: 20)
of the IgG1 heavy chain (FIGS. 12A and 12B) referred to herein as
IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and IL-2/IL-2R.alpha.
scC.sub.H1C.sub.L-Fc, respectively. For expression in mammalian
cells, the N-terminal leader sequence of SEQ ID NO: 10 was added to
the protein of SEQ ID NO: 19 and SEQ ID NO: 20).
Expression of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc
[0184] The genes were synthetically synthesized and supplied in
pcDNA3.1 expression vector (GeneArt), and transiently expressed in
HEK293 cells using the Expi293 expression system (Life
Technologies). Proteins were purified using Protein A (GE
Healthcare) with low pH elution and dialyzed against 2 L
1.times.PBS 2 times.
[0185] The molecules were analyzed by SDS PAGE gel under reducing
and non-reducing conditions (FIG. 13). For reducing and
non-reducing conditions, 5 ug of protein was loaded onto an Any kD
gel (Invitrogen) with a Precision Plus Protein Kaleidoscope
standard (Invitrogen) (MW range 10 kD-250 kD). The molecule was
characterized by analytical gel filtration on a BioSuite Ultra High
Resolution SEC column, 250 .ANG., 4 .mu.m, 4.6 mm.times.300 mm
(Waters). The column was equilibrated and run at 0.3 ml/min with
150 mM sodium phosphate pH 7.0 as a running buffer for all
analyses. Purified samples (0.5 mg/ml) were injected (15 ul) and
eluted with a run time of 25 min (FIGS. 14A and 14B).
Bioactivity of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc
[0186] In vitro bioactivity was assessed by evaluating the ability
of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and IL-2/IL-2Ra
scC.sub.H1C.sub.L-Fc to activate pSTAT5 in the human HH T-cell
lymphoma cell line (ATCC CRL-2105) using the Phospho-STAT5A/B
(Tyr694/Tyr699) InstantOne.TM. ELISA kit from eBioscience. For the
assay, HH cells were plated at 2.times.10.sup.5 cells/well in
RPMI1640 media containing 10% FBS. Samples were incubated with
decreasing concentrations of wild-type IL-2 (wtIL-2), IL-2/IL-2Ra
scC1CH1 Fc or IL-2/IL-2Ra scCH2Cl Fc from approximately 50 nM, or
unstimulated, for approximately 25.+-.5 minutes in a 37.degree. C.,
5% CO.sub.2 incubator. Stimulation reaction was terminated by
prompt addition of 25 .mu.L of cell lysis mix (provided in kit) and
incubated at room temperature for 10 minutes with constant shaking
at 300 rpm on a titer plate shaker. 50 .mu.L aliquots of resulting
lysates were added to each well in the assay plate (provided in
kit). After adding 50 .mu.L of antibody cocktail to each well, the
plate was covered and incubated at room temperature for 1 hour with
constant shaking at 300 rpm on a titer plate shaker. Plate was
subsequently washed three times with 300 .mu.L/well of 1.times.
wash buffer. 100 .mu.L of detection reagent was added to each well
and incubated at room temperature for 30 minutes with constant
shaking at 300 rpm. Detection reaction was stopped by addition of
100 .mu.L of stop solution and the absorbance at 450 nM was
measured using a SynergyMx plate reader. IL-2/IL-2Ra scClCH1 Fc
(EC.sub.50=0.97 nM), or IL-2/IL-2Ra scCH2Cl Fc (EC.sub.50=1.1 nM)
and wtIL-2 (EC.sub.50=0.80 nM) were active in a dose dependent
fashion (FIG. 15).
Rat PK of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc
[0187] Single intravenous doses of 1 mg/kg IL-2/IL-2R.alpha.
scC.sub.LC.sub.H1-Fc and IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc
were administered into a tail vein of 3 rats. Blood samples were
collected at 0.083, 0.25, 0.5, 1, 3, 8, 24, 48, 72, 96 and 168 hrs
after administration of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc. Single subcutaneous doses
of 2 mg/kg IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc were administered into the
interscapular region of 3 rats. Blood samples were collected at
0.25, 0.5, 1, 2, 6, 8, 24, 48, 72, 96 and 168 hrs after
administration of IL-2/IL-2R.alpha. scC.sub.LC.sub.H1-Fc and
IL-2/IL-2R.alpha. scC.sub.H1C.sub.L-Fc. Concentrations were
measured using standard MSD techniques. Pharmacokinetic analysis
was performed using non-compartmental modeling with WinNonlin
software (Pharsight Corporation, Mountain View, Calif.). The
pharmacokinetic parameter estimates derived from MSD data included
maximum concentration (Cmax), area under the time versus
concentration curve (AUC), and elimination half-life (t.sub.1/2)
(FIG. 16 and Table 5).
TABLE-US-00022 TABLE 5 Dosing Dose T.sub.max C.sub.max
AUC.sub.0-.infin. t.sub.1/2 MRT CL Vd F Test Article Route
(nmol/kg) (h) (nM) (nM-h) (h) (h) (mL/h/kg) (mL/kg) (%) IL-2/IL-2Ra
scCLCH1 Fc IV 6 0.083 82.9 .+-. 12.0 876 .+-. 16.2 23.4 .+-. 4.4
25.6 .+-. 4.2 6.78 .+-. 0.125 174 .+-. 31.5 IL-2/IL-2Ra scCLCH1 Fc
SC 12 48 6.67 .+-. 0.35 483 .+-. 56.9 15.3 .+-. 3.6 55.0 .+-. 1.7
28 IL-2/IL-2Ra scCH1CL Fc IV 8 0.083 65.5 .+-. 7.6 505 .+-. 66.4
39.5 .+-. 4.7 26.3 .+-. 1.7 11.9 .+-. 1.6 313 .+-. 47.7 IL-2/IL-2Ra
scCH1CL Fc SC 12 (24, 48) 3.36 .+-. 0.84 290 .+-. 1.0 32.9 .+-. 0.3
70.2 .+-. 8.8 29 Note: mean .+-. SD for all parameters except
median (min, max) for Tmax, n = 3 unless otherwise noted; F = %
ratio of dose normalized AUC.sub.0-.infin. after SC vs IV
Example 5
IFN.beta.
[0188] Design of IFN.beta. scC.sub.LC.sub.H1-Fc
[0189] The IFN.beta. single chain fusion body molecule contains
IFN.beta. (C17S) linked to the CL-CH1-Fc domain of the IgG1 heavy
chain (FIG. 17). For expression in mammalian cells, the N-terminal
leader sequence of SEQ ID NO: 10 was added to the protein of SEQ ID
NO: 18.
Expression of IFN.beta. scC.sub.LC.sub.H1-Fc
[0190] The gene was synthetically synthesized and supplied in
pcDNA3.1 expression vector (GeneArt), and transiently expressed in
HEK293 cells using the Expi293 expression system (Life
Technologies). The protein was purified using Protein A (GE
Healthcare) with low pH elution and dialyzed against 2 L
1.times.PBS 2 times.
[0191] The molecule was analyzed by SDS PAGE gel under reducing and
non-reducing conditions (FIG. 18). For reducing and non-reducing
conditions, 5 ug of protein was loaded onto an Any kD gel
(Invitrogen) with a Precision Plus Protein Kaleidoscope standard
(Invitrogen) (MW range 10 kD-250 kD). The molecule was
characterized by analytical gel filtration on a BioSuite Ultra High
Resolution SEC column, 250 .ANG., 4 .mu.m, 4.6 mm.times.300 mm
(Waters). The column was equilibrated and run at 0.3 ml/min with
150 mM sodium phosphate pH 7.0 as a running buffer for all
analyses. Purified samples (0.5 mg/ml) were injected (15 ul) and
eluted with a run time of 60 min (FIG. 19).
Bioactivity of IFN.beta. scC.sub.LC.sub.H1-Fc
[0192] HEK-Blue.TM. IFN.alpha./.beta. cells (InvivoGen) are human
embryonic kidney cells specifically designed to detect bioactive
Type I IFNs in vitro by monitoring the activation of the ISGF3
pathway. The cell line expresses an inducible secreted embryonic
alkaline phosphatase (SEAP) reporter gene under control of the
IFN.alpha./.beta. inducible ISG54 promoter. For the IFN.beta.
agonist assay, HEK-Blue IFN.alpha./.beta. cells were plated at
50,000 cells/well in DMEM media containing 2 mM L-glutamine, 4.5
g/l glucose and 10% heat inactivated FBS (Gibco). Cells were
incubated for 20 hours at 37.degree. C., 5% CO.sub.2 with varying
concentrations of IFN.beta. scC.sub.LC.sub.H1-Fc or wtIFN.beta.
(Peprotech). SEAP production was detected by adding QUANTI-Blue and
incubating for 3 hours at 37.degree. C., 5% CO.sub.2 and read on a
plate reader at 630 nm. IFN.beta. scC.sub.LC.sub.H1-Fc
(EC.sub.50=0.9 pM) and wtIFN.beta. (EC.sub.50=0.6 pM) were active
in a dose dependent fashion (FIG. 20).
Rat PK of IFN.beta. scC.sub.LC.sub.H1-Fc
[0193] A single intravenous dose of 0.5 mg/kg IFN.beta.
scC.sub.LC.sub.H1-Fc was administered into a surgically implanted
jugular vein catheter of 3 rats. Blood samples were collected at
0.083, 0.25, 0.5, 1, 3, 8, 24, 48, 72, 96 and 168 hrs after
administration of IFN.beta. scC.sub.LC.sub.H1-Fc. A Single
subcutaneous dose of 1 mg/kg IFN.beta. scC.sub.LC.sub.H1-Fc was
administered into the interscapular region of 3 rats. Blood samples
were collected at 0.25, 0.5, 1, 2, 6, 8, 24, 48, 72, 96 and 168 hrs
after administration of IFN.beta. scC.sub.LC.sub.H1-Fc.
Concentrations were measured using standard MSD techniques.
Pharmacokinetic analysis was performed using non-compartmental
modeling with WinNonlin software (Pharsight Corporation, Mountain
View, Calif.). The pharmacokinetic parameter estimates derived from
MSD data included maximum concentration (Cmax), area under the time
versus concentration curve (AUC), and elimination half-life
(t.sub.1/2) (FIG. 21 and Table 6).
TABLE-US-00023 TABLE 6 Dose Cmax Tmax Cmax/D AUC.sub..infin.
AUC.sub..infin./D CL V.sub.ss t.sub.1/2 MRT % F Route (nMole/kg)
Animal ID (nM) (h) (kg*nM/nmol) (h*nM) (h*kg*nM/nmol) (mL/h/kg)
(mL/kg) (h) (h) (%) IV 1.4 67363 61.1 0.083 42.7 1320 921 1.09 111
87 100 67364 54.7 0.083 38.2 1320 924 1.08 129 110 120 67366 109
0.083 76.6 1790 1250 0.801 106 120 130 Mean 75.1 0.083 52.5 1480
1030 0.991 115 100 120 SD 30 NA 21 272 190 0.164 12.3 14 15 SC 3.6
67367 16.4 48 4.57 ND ND NA NA ND ND 67369 12.8 24 3.57 2150 600 NA
NA 87 140 Mean 14.6 36 4.07 2150 600 NA NA 87 140 58.3 SD NA NA NA
NA NA NA NA NA NA
Example 6
IL-10
[0194] Design of scIL-10:C.sub.L:C.sub.H1:Fc and
scIL-10:C.sub.H1:C.sub.L:Fc
[0195] The scIL-10 single chain fusion body molecule contains a
covalently linked IL-10 homodimer fusion protein linked to the
CL-CH1-Fc domain or the CH1-CL-Fc of the IgG1 heavy chain (FIGS.
22A and 22B). The amino acid sequences of each molecule synthesized
is found in Table 7.
TABLE-US-00024 TABLE 7 Protein Sequence scIL- ##STR00002##
10:CL:CH1:Fc
LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL
PCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQS
ENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLE
EVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKA
MSEFDIFINYIEAYMTMKIRNGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN
RGECGGGGSGGGGSGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE
QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSPWQQGNVFSCSV
MHEALHNYTQKSLSLSPGK (SEQ ID NO: 23) scIL- ##STR00003## 10:CH1:CL:Fc
LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL
PCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGGGSGGSPGQGTQS
ENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLE
EVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKA
MSEFDIFINYIEAYMTMKIRNGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVGGGGSG
GGGSGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGGEPKSCDK
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE
EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVMHEALHNHYTQKSLSPGK (SEQ ID NO: 24) scIL-10:Fc ##STR00004##
(Control)
LLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHR-
FL
PCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGSGGSPGQGTQSENSCTH
FPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQA
ENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFI
NYIEAYMTMKIRNEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25)
Expression of scIL-10:C.sub.L:C.sub.H1:Fc and
scIL-10:C.sub.H1:C.sub.L:Fc
[0196] The genes were synthetically synthesized and supplied in
pcDNA3.1 expression vector (GeneArt), and transiently expressed in
HEK293 cells using the Expi293 expression system (Life
Technologies). Proteins were purified using Protein A (GE
Healthcare) with low pH elution and dialyzed against 2 L
1.times.PBS 2 times.
[0197] The molecules were analyzed by SDS PAGE gel under reducing
and non-reducing conditions (FIG. 23). For reducing and
non-reducing conditions, 2.5 ug of protein was loaded onto an Any
kD gel (Invitrogen) with a Precision Plus Protein Kaleidoscope
standard (Invitrogen) (MW range 10 kD-250 kD). The molecule was
characterized by analytical gel filtration on an)(Bridge Protein
BEH SEC column, 200 .ANG., 3.5 .mu.m, 7.8 mm.times.150 mm (Waters).
The column was equilibrated and run at 0.9 ml/min with 100 mM
sodium phosphate pH 7.0 as a running buffer for all analyses.
Purified samples (0.5 mg/ml) were injected (15 ul) and eluted with
a run time of 15 min (FIGS. 24A and 24B).
Bioactivity of scIL-10:C.sub.LC.sub.H1:Fc and
scIL-10:C.sub.H1C.sub.L:Fc
[0198] In vitro bioactivity was assessed by evaluating the ability
of scIL-10:C.sub.L:C.sub.H1: Fc and scIL-10:C.sub.H1:C.sub.L:Fc to
stimulate proliferation of the mouse mast cell line MC/9 (ATCC
CRL-8306). The scIL-10 direct Fc fusion protein (scIL-10:Fc) was
used as a control. For the assay, MC/9 cells were plated at 10,000
cells/well in DMEM media containing 10% heat inactivated fetal
bovine serum, 2 mM glutamine and 0.05 mM 2-mercaptoethanol. Cells
were incubated for 72 hours at 37.degree. C., 5% CO.sub.2 with
varying concentrations of human IL-10 (R&D Systems),
scIL-10:C.sub.L:C.sub.H1: Fc, scIL-10:C.sub.H1:C.sub.L:Fc or
scIL-10:Fc. After 72 hours, the cells were stained with
CellTiter-Blue (Promega) for 4 hours at 37.degree. C., 5% CO.sub.2
according to the manufacturer's protocol. Fluorescent measurements
were taken at 560/590 nm. IL-10 (EC.sub.50=75 pM),
scIL-10:C.sub.L:C.sub.H1: Fc (EC.sub.50=79 pM),
scIL-10:C.sub.H1:C.sub.L:Fc (EC.sub.50=93 pM) and scIL-10:Fc
(EC.sub.50=493 pM) were active in a dose dependent fashion (FIG.
25).
Mouse PK of scIL-10:C.sub.L:C.sub.H1:Fc and
scIL-10:C.sub.H1:C.sub.L:Fc
[0199] scIL-10:C.sub.L:C.sub.H1: Fc, scIL-10:C.sub.H1:C.sub.L:Fc,
and scIL-10:Fc pharmacokinetics in mice were evaluated at a single
intravenous doses of 0.5 mg/kg administered into tail vein and a
single subcutaneous doses of 2.5 mg/kg administered into the
interscapular region. Blood samples (n=3 samples/time point/fusion
protein) were collected at 0.083, 0.5, 1, 4, 6, 24, 48, 96, 168,
192 and 216 hours after administration of scIL-10:C.sub.L:C.sub.H1:
Fc, scIL-10:C.sub.H1:C.sub.L:Fc and scIL-10:Fc. For each time
point/fusion protein/route of administration, serum was pooled and
concentrations were measured using standard MSD techniques.
Bioanalytical data was subjected to non-compartmental
pharmacokinetic analysis using Phoenix WinNonlin 6.4 software. The
pharmacokinetic parameter included standard pharmacokinetic
parameters of maximum concentration (C.sub.max), time to maximum
concentration (T.sub.max), area under the time versus concentration
curve (AUC), mean residence time (MRT), elimination half-life
(t1/2), clearance (CL), distribution volume at steady state
(V.sub.ss), and bioavailability (% F) were determined and reported
in Tables 8 and 9.
TABLE-US-00025 TABLE 8 Row Dose Dose Cmax Tmax Cmax/D AUClast ID
Compound (mg/kg) (~nMole/kg) ROA (nM) (h) (nM/D) (h*nM) 1
scIL-10:Fc 0.5 3.93 IV 94.9 0.083 24.2 2080 2 scIL-10:Fc 2.5 19.63
SC 221 24 11.3 12700 3 scIL-10:C.sub.L:C.sub.H1:Fc 0.5 2.85 IV 140
0.083 49.2 2850 4 scIL-10:C.sub.L:C.sub.H1:Fc 2.5 14.25 SC 227 24
15.9 19500 5 scIL-10:C.sub.H1:C.sub.L:Fc 0.5 2.84 IV 115 0.083 40.5
1300 6 scIL-10:C.sub.H1:C.sub.L:Fc 2.5 14.2 SC 120 24 8.48 7570
TABLE-US-00026 TABLE 9 CL Row AUCinf AUCinf/D MRTinf t1/2 (mL/ Vss
ID (h*nM) (h*nM) (h) (h) hr/kg) (mL/kg) % F 1 2170 552 33 21 1.811
59.57 NA 2 12700 649 46 11 NA NA ~100 3 2850 999 30 7.8 1.001 29.56
NA 4 19500 1370 56 8.5 NA NA ~100 5 1300 458 16 9.3 2.183 35.44 NA
6 7570 533 41 9.1 NA NA ~100
[0200] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0201] While this invention has been particularly shown and
described with references to preferred features thereof, it will be
understood by those skilled in the art that various changes in form
and details may be made therein without departing from the scope of
the invention encompassed by the appended claims. It should also be
understood that the various features of the invention described
herein are not mutually exclusive and that features may be combined
in whole or in part in accordance with the invention.
Sequence CWU 1
1
271107PRTHomo sapiens 1Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90
95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 100 105 298PRTHomo
sapiens 2Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val
3330PRTHomo sapiens 3Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Glu
Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15
5232PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 5Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65 70 75 80 Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90
95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215
220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 6232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
6Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1
5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Gln Val Lys
Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Gln Val His Asn Ala Lys
Thr Lys Pro Arg Glu Gln Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asn Trp Leu Asp Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135
140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser
Pro Gly Lys 225 230 7900PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 7Thr Val Phe Leu Asp His
Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro 1 5 10 15 Lys Arg Tyr Asn
Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu 20 25 30 Glu Arg
Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu 35 40 45
Val Phe Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val 50
55 60 Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser
Cys 65 70 75 80 Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe
Gly Phe Glu 85 90 95 Gly Lys Asn Cys Glu Leu Asp Val Thr Cys Asn
Ile Lys Asn Gly Arg 100 105 110 Cys Glu Gln Phe Cys Lys Asn Ser Ala
Asp Asn Lys Val Val Cys Ser 115 120 125 Cys Thr Glu Gly Tyr Arg Leu
Ala Glu Asn Gln Lys Ser Cys Glu Pro 130 135 140 Ala Val Pro Phe Pro
Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys 145 150 155 160 Leu Thr
Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser 165 170 175
Thr Glu Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser 180
185 190 Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro
Gly 195 200 205 Gln Phe Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp
Ala Phe Cys 210 215 220 Gly Gly Ser Ile Val Asn Glu Lys Trp Ile Val
Thr Ala Ala His Cys 225 230 235 240 Val Glu Thr Gly Val Lys Ile Thr
Val Val Ala Gly Glu His Asn Ile 245 250 255 Glu Glu Thr Glu His Thr
Glu Gln Lys Arg Asn Val Ile Arg Ile Ile 260 265 270 Pro His His Asn
Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile 275 280 285 Ala Leu
Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr 290 295 300
Pro Ile Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe 305
310 315 320 Gly Ser Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys
Gly Arg 325 330 335 Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro Leu
Val Asp Arg Ala 340 345 350 Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile
Tyr Asn Asn Met Phe Cys 355 360 365 Ala Gly Phe His Glu Gly Gly Arg
Asp Ser Cys Gln Gly Asp Ser Gly 370 375 380 Gly Pro His Val Thr Glu
Val Glu Gly Thr Ser Phe Leu Thr Gly Ile 385 390 395 400 Ile Ser Trp
Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr 405 410 415 Thr
Lys Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu 420 425
430 Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala
435 440 445 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 450 455 460 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 465 470 475 480 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 485 490 495 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 500 505 510 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 515 520 525 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 530 535 540 Phe
Asn Arg Gly Glu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 545 550
555 560 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys Gly
Pro 565 570 575 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr 580 585 590 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr 595 600 605 Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro 610 615 620 Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 625 630 635 640 Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 645 650 655 His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 660 665 670
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 675
680 685 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 690 695 700 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser 705 710 715 720 His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu 725 730 735 Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr 740 745 750 Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn 755 760 765 Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 770 775 780 Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 785 790 795
800 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
805 810 815 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val 820 825 830 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro 835 840 845 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr 850 855 860 Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val 865 870 875 880 Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 885 890 895 Ser Pro Gly
Lys 900 8 702PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 8Leu Pro Ala Gln Val Ala Phe Thr Pro
Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr
Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser Pro
Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr
Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp
Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70
75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile
Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln
Glu Gly Cys 100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro
Gly Phe Gly Val Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val
Val Cys Lys Pro Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr Thr
Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn
Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val Cys
Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190
His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195
200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met
Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Gly Gly
Gly Gly Ser 225 230 235 240 Gly Gly Gly Gly Ser Arg Thr Val Ala Ala
Pro Ser Val Phe Ile Phe 245 250 255 Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly Thr Ala Ser Val Val Cys 260 265 270 Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala Lys Val Gln Trp Lys Val 275 280 285 Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 290 295 300 Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 305 310 315
320 Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 325 330 335 Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser 340 345 350
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 355
360 365 Gly Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala 370 375 380 Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu 385 390 395 400 Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly 405 410 415 Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser 420 425 430 Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu 435 440 445 Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr 450 455 460 Lys Val
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 465 470 475
480 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
485 490 495 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro 500 505 510 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Gln Val 515 520 525 Lys Phe Asn Trp Tyr Val Asp Gly Val Gln
Val His Asn Ala Lys Thr 530 535 540 Lys Pro Arg Glu Gln Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val 545 550 555 560 Leu Thr Val Leu His
Gln Asn Trp Leu Asp Gly Lys Glu Tyr Lys Cys 565 570 575 Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 580 585 590 Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 595 600
605 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
610 615 620 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly 625 630 635 640 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 645 650 655 Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 660 665 670 Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 675 680 685 Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 690 695 700 9618PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
9Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp 1
5 10 15 Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val
Ala 20 25 30 Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys
Ile Asp Val 35 40 45 Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly
Ile His Gly Gly Lys 50 55 60 Met Cys Leu Ser Cys Val Lys Ser Gly
Asp Glu Thr Arg Leu Gln Leu 65 70 75 80 Glu Ala Val Asn Ile Thr Asp
Leu Ser Glu Asn Arg Lys Gln Asp Lys 85 90 95 Arg Phe Ala Phe Ile
Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu 100 105 110 Ser Ala Ala
Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala Asp 115 120 125 Gln
Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val Thr 130 135
140 Lys Phe Tyr Phe Gln Glu Asp Glu Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160 Gly Ser Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser 165 170 175 Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn 180 185 190 Asn Phe Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala 195 200 205 Leu Gln Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys 210 215 220 Asp Ser Thr Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp 225 230 235 240 Tyr Glu
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu 245 250 255
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser Gly Gly Gly 260
265 270 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 275 280 285 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys 290 295 300 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 305 310 315 320 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 325 330 335 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 340 345 350 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 355 360 365 Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 370 375 380
Arg Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 385
390 395 400 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 405 410 415 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 420 425 430 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 435 440 445 Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu 450 455 460 Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu 465 470 475 480 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 485 490 495 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 500 505
510 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
515 520 525 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr 530 535 540 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 545 550 555 560 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 565 570 575 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 580 585 590 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 595 600 605 Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 610 615 1020PRTUnknownDescription of
Unknown N-terminal leader sequence 10Met Tyr Arg Met Gln Leu Leu
Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20
1110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
1220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 132760DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
13atgtaccgga tgcagctgct gagctgtatc gccctgtctc tggccctcgt gaccaacagc
60accgtgtttc tggaccacga gaacgccaac aagatcctga accggcccaa gcggtacaac
120agcggcaagc tggaagagtt cgtgcagggc aacctggaac gcgagtgcat
ggaagagaag 180tgcagcttcg aagaggccag agaggtgttc gagaacaccg
agcggaccac cgagttctgg 240aagcagtacg tggacggcga ccagtgcgag
agcaacccct gtctgaatgg cggcagctgc 300aaggacgaca tcaacagcta
cgagtgctgg tgccccttcg gcttcgaggg caagaactgc 360gagctggacg
tgacctgcaa catcaagaac ggcagatgcg agcagttctg caagaacagc
420gccgacaaca aggtcgtgtg ctcctgcacc gagggctaca gactggccga
gaaccagaag 480tcctgcgagc ccgccgtgcc tttcccatgt ggaagagtgt
ccgtgtccca gaccagcaag 540ctgaccagag ccgagacagt gttccccgac
gtggactacg tgaactccac cgaggccgag 600acaatcctgg acaacatcac
ccagagcacc cagtccttca acgacttcac cagagtcgtg 660ggcggcgagg
atgccaagcc tggacagttc ccgtggcagg tggtgctgaa cggaaaggtg
720gacgcctttt gcggcggcag catcgtgaac gagaagtgga tcgtgacagc
cgcccactgc 780gtggaaaccg gcgtgaagat tacagtggtg gccggcgagc
acaacatcga ggaaaccgag 840cacacagagc agaaacggaa cgtgatcaga
atcatccccc accacaacta caacgccgcc 900atcaacaagt acaaccacga
cattgccctg ctggaactgg acgagcccct ggtgctgaat 960agctacgtga
cccccatctg cattgccgac aaagagtaca ccaacatctt tctgaagttc
1020ggcagcggct acgtgtccgg ctggggcaga gtgtttcaca agggcagatc
cgctctggtg 1080ctgcagtacc tgagagtgcc tctggtggac cgggccacct
gtctgagaag caccaagttc 1140accatctaca acaacatgtt ctgcgccggc
ttccatgagg gcggcagaga tagctgtcag 1200ggcgattctg gcggccctca
cgtgacagaa gtggaaggca ccagctttct gaccggcatc 1260atcagctggg
gcgaggaatg cgccatgaag gggaagtacg gcatctacac caaggtgtcc
1320agatatgtga actggatcaa agaaaagacc aagctgacag gcggcggagg
ctctggcgga 1380ggcggatcta gaacagtggc cgctcccagc gtgttcatct
tcccacctag cgacgagcag 1440ctgaagtccg gcacagcctc tgtcgtgtgc
ctgctgaaca acttctaccc ccgcgaggcc 1500aaggtgcagt ggaaggtgga
caatgccctg cagagcggca acagccagga aagcgtgacc 1560gagcaggaca
gcaaggactc cacctacagc ctgagcagca ccctgaccct gagcaaggcc
1620gactacgaga agcacaaggt gtacgcctgc gaagtgaccc accagggcct
gtctagccca 1680gtgaccaaga gcttcaaccg gggcgaatct gggggcggag
gatcaggcgg gggaggaagt 1740gggggagggg gaagcggagg gggaggatct
gcctctacaa agggccctag cgtgttcccc 1800ctggccccta gcagcaagtc
tacaagcgga ggcacagctg ccctgggctg cctcgtgaag 1860gactacttcc
ctgagcccgt gaccgtgtcc tggaacagcg gagcactgac aagcggcgtg
1920cacacctttc cagccgtgct gcagagcagc ggcctgtact ctctgagcag
cgtcgtgaca 1980gtgcccagca gctctctggg cacccagacc tacatctgca
acgtgaacca caagcccagc 2040aataccaaag tggacaagcg ggtggaaccc
aagagcagcg acaagaccca cacctgtccc 2100ccttgtcctg cccccgaact
gctgggaggc ccttccgtgt tcctgttccc cccaaagccc 2160aaggacaccc
tgatgatcag ccggacccct gaagtgacct gcgtggtggt ggatgtgtcc
2220cacgaggacc cagaagtgaa gttcaattgg tatgtggacg gggtggaagt
gcacaacgcc 2280aagaccaaac ccagagagga acagtacaat agcacctacc
gggtggtgtc cgtgctgaca 2340gtgctgcacc aggactggct gaatggcaaa
gagtataagt gcaaagtgtc caacaaggcc 2400ctgcctgccc ccatcgagaa
aaccatcagc aaggccaagg gccagccccg cgaaccccag 2460gtgtacacac
tgcccccaag ccgggaagag atgaccaaga accaggtgtc cctgacctgt
2520ctcgtgaaag gcttctaccc ttccgatatc gccgtggaat gggagagcaa
cggccagccc 2580gagaacaatt acaagaccac cccccctgtg ctggactccg
acggctcatt cttcctgtac 2640agcaaactga ccgtggacaa gagccggtgg
cagcagggaa acgtgttcag ctgcagcgtg 2700atgcacgagg ccctgcacaa
ccactacacc cagaaaagcc tgagcctgtc ccctggcaag 2760142166DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
14atgtatagga tgcagctcct cagctgcatc gctctgtccc tcgccctggt gaccaacagc
60ctccctgccc aggtggcctt tacaccctac gctcctgagc ccggaagcac ctgcaggctc
120agggagtact acgatcagac cgcccaaatg tgttgcagca agtgctcccc
tggccagcac 180gccaaggtgt tctgcaccaa gacaagcgat accgtgtgcg
atagctgtga ggacagcacc 240tacacccagc tgtggaattg ggtgcccgag
tgcctgagct gtggcagcag gtgcagcagc 300gatcaggtgg agacacaggc
ctgcaccaga gagcagaaca ggatttgtac ctgcaggccc 360ggctggtatt
gcgccctgag caagcaggag ggatgtaggc tgtgcgcccc tctgaggaaa
420tgcagacctg gctttggagt ggctaggccc ggcaccgaga catccgacgt
ggtgtgcaag 480ccttgtgccc ctggcacctt ttccaacacc accagctcca
ccgacatctg caggccccat 540cagatttgca acgtggtggc catccccgga
aacgctagca tggatgccgt gtgcacctcc 600acctccccta ccaggagcat
ggcccctgga gccgtgcatc tgcctcaacc cgtcagcacc 660agaagccagc
acacacagcc cacccccgaa cctagcaccg ctccctccac cagcttcctg
720ctgcctatgg gaccctcccc tcctgccgaa gggagcaccg gagatggagg
aggaggaagc 780ggcggaggag gctccagaac agtggctgcc cctagcgtgt
tcattttccc tccctccgac 840gagcagctca agtccggaac cgcttccgtg
gtctgcctgc tgaacaactt ctaccccaga 900gaggccaagg tgcagtggaa
agtcgacaat gctctgcaga gcggaaactc ccaggagtcc 960gtcaccgagc
aggacagcaa ggactccaca tatagcctgt cctccaccct gaccctgagc
1020aaggccgact atgagaaaca caaggtgtat gcctgcgaag tgacccacca
gggcctgtcc 1080agccccgtca ccaagtcctt caataggggc gagagcggag
gcggcgggag cggcggcggc 1140gggagcggag gaggagggag cggaggaggc
ggaagcgctt ccaccaaggg acctagcgtg 1200tttcccctcg cccccagctc
caagagcaca agcggaggca cagccgctct gggctgtctg 1260gtgaaggatt
acttccccga gcccgtcaca gtgagctgga actccggagc cctgacctcc
1320ggagtgcaca cctttcctgc cgtgctgcag agcagcggac tgtacagcct
gtccagcgtg 1380gtcacagtgc cctccagctc cctgggcacc cagacctaca
tctgcaacgt gaaccacaag 1440cccagcaaca caaaggtgga caagagagtg
gaacctaagt cctgtgacaa aacccatacc 1500tgccctccct gccctgcccc
tgagctgctg ggaggaccta gcgtgtttct gtttcccccc 1560aaacccaagg
ataccctgat gatcagcagg acccctgagg tgacatgcgt ggtggtggac
1620gtgtcccacg aggaccctca ggtcaagttc aactggtacg tggatggcgt
ccaggtgcac 1680aatgctaaga ccaagcccag ggagcagcaa tacaattcca
cctacagggt ggtgtccgtg 1740ctcaccgtcc tccaccagaa ctggctcgac
ggcaaagaat acaagtgcaa agtgagcaac 1800aaggctctcc ccgcccctat
cgagaagacc atttccaaag ccaagggcca gcccagagaa 1860cctcaagtct
acaccctgcc ccccagcagg gaggagatga ccaagaacca ggtgagcctg
1920acctgcctcg tcaagggatt ctatcccagc gacatcgccg tggaatggga
gtccaatggc 1980cagcccgaga ataactacaa gaccacaccc cccgtgctgg
attccgatgg cagctttttc 2040ctgtacagca agctgacagt ggataagagc
aggtggcagc agggcaacgt gttcagctgc 2100tccgtcatgc acgaagccct
gcacaatcac tacacccaga agagcctgtc cctcagcccc 2160ggcaag
2166151917DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 15atgtaccgga tgcagctgct gtcctgtatc
gccctgtctc tggccctggt caccaactcc 60agaccctctg gccggaagtc ctccaagatg
caggccttcc ggatctggga cgtgaaccag 120aaaaccttct acctgcggaa
caaccagctg gtggccggct atctgcaggg ccccaacgtg 180aacctggaag
agaagatcga cgtggtgccc atcgagcccc acgccctgtt tctgggaatc
240cacggcggca agatgtgcct gtcctgcgtg aagtccggcg acgagacacg
gctgcagctg 300gaagccgtga acatcaccga cctgtccgag aaccggaagc
aggacaagag attcgccttc 360atcagatccg actccggccc taccacctcc
ttcgagtctg ctgcttgccc cggctggttc 420ctgtgcaccg ccatggaagc
tgaccagccc gtgtccctga ccaacatgcc tgacgagggc 480gtgatggtca
ccaagttcta ttttcaggaa gatgagggcg gaggcggctc tggcggcgga
540ggatctagaa cagtggccgc tccctccgtg ttcatcttcc caccttccga
cgagcagctg 600aagtctggca ccgcctctgt cgtgtgcctg ctgaacaact
tctaccctcg cgaggccaag 660gtgcagtgga aggtggacaa cgccctgcag
tccggcaact cccaggaatc cgtcaccgag 720caggactcca aggacagcac
ctactccctg tcctccaccc tgaccctgtc caaggccgac 780tacgagaagc
acaaggtgta cgcctgcgaa gtgacccacc agggcctgtc tagccccgtg
840accaagtctt tcaaccgggg cgaaagcgga ggcggaggtt caggtggtgg
tggatcaggt 900ggcggcggat ctggcggtgg tggctctgct tctaccaagg
gcccttccgt gttccctctg 960gccccttcca gcaagtctac ctctggcggc
acagccgctc tgggctgcct ggtcaaggac 1020tacttccccg agcctgtgac
cgtgtcctgg aactctggcg ctctgacatc cggcgtgcac 1080accttccctg
ctgtgctgca gtcctccggc ctgtacagcc tgtcctccgt cgtgaccgtg
1140ccttccagct ctctgggcac ccagacctac atctgtaacg tgaaccacaa
gccctccaac 1200accaaagtgg acaagcgggt ggaacccaag tcctccgaca
agacccacac ctgtcctccc 1260tgccctgctc ctgaactgct gggcggacct
agcgtgttcc tgttccctcc aaagcccaag 1320gacaccctga tgatctcccg
gacccctgaa gtgacctgcg tggtggtcga tgtgtcccac 1380gaggacccag
aagtgaagtt caattggtac gtggacggcg tggaagtgca caatgccaag
1440accaagccca gagaggaaca gtacaactcc acctaccggg tggtgtccgt
gctgaccgtg 1500ctgcaccagg attggctgaa cggcaaagag tacaagtgca
aggtgtccaa caaggccctg 1560cctgccccta tcgaaaagac catctccaag
gccaagggcc agccccggga acctcaggtg 1620tacaccctgc ctcccagccg
ggaagagatg accaagaacc aggtgtcact gacctgtctg 1680gtcaagggct
tctacccctc cgacattgcc gtggaatggg agtccaacgg ccagcccgag
1740aacaactaca agaccacccc tcccgtgctg gactccgacg gctcattctt
cctgtactcc 1800aagctgaccg tggacaagtc ccggtggcag cagggcaacg
tgttctcctg ctccgtgatg 1860cacgaggccc tgcacaacca ctacacccag
aagtccctgt ccctgagccc cggcaag 1917166DNAUnknownDescription of
Unknown Eukaryotic sequence 16cncaat 6 176DNAUnknownDescription of
Unknown Eukaryotic sequence 17aataaa 6 18633PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln 1
5 10 15 Ser Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys
Leu 20 25 30 Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys
Gln Leu Gln 35 40 45 Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile
Tyr Glu Met Leu Gln 50 55 60 Asn Ile Phe Ala Ile Phe
Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn 65 70 75 80 Glu Thr Ile Val
Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn 85 90 95 His Leu
Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr 100 105 110
Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg 115
120 125 Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp
Thr 130 135 140 Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile
Asn Arg Leu 145 150 155 160 Thr Gly Tyr Leu Arg Asn Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 165 170 175 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu 180 185 190 Gln Leu Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe 195 200 205 Tyr Pro Arg Glu
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 210 215 220 Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 225 230 235
240 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
245 250 255 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser 260 265 270 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly
Gly Gly Gly Ser 275 280 285 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Ala 290 295 300 Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys Ser 305 310 315 320 Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 325 330 335 Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 340 345 350 Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 355 360
365 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
370 375 380 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Arg 385 390 395 400 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro 405 410 415 Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys 420 425 430 Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val 435 440 445 Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 450 455 460 Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 465 470 475 480
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 485
490 495 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 500 505 510 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln 515 520 525 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met 530 535 540 Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 545 550 555 560 Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 565 570 575 Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 580 585 590 Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 595 600 605
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 610
615 620 Lys Ser Leu Ser Leu Ser Pro Gly Lys 625 630
19767PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 19Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu
Ile Ser Asn Ile Asn 1 5 10 15 Val Ile Val Glu Leu Lys Gly Ser Glu
Thr Thr Phe Met Cys Glu Tyr 20 25 30 Ala Asp Glu Thr Ala Thr Ile
Val Glu Phe Leu Asn Arg Trp Ile Thr 35 40 45 Phe Ser Gln Ser Ile
Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser Thr 50 55 60 Lys Lys Thr
Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met 65 70 75 80 Ile
Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met 85 90
95 Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His
100 105 110 Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val
Leu Asn 115 120 125 Leu Ala Gln Gly Ser Gly Gly Gly Ser Glu Leu Cys
Asp Asp Asp Pro 130 135 140 Pro Glu Ile Pro His Ala Thr Phe Lys Ala
Met Ala Tyr Lys Glu Gly 145 150 155 160 Thr Met Leu Asn Cys Glu Cys
Lys Arg Gly Phe Arg Arg Ile Lys Ser 165 170 175 Gly Ser Leu Tyr Met
Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp 180 185 190 Asp Asn Gln
Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys 195 200 205 Gln
Val Thr Pro Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu 210 215
220 Met Gln Ser Pro Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His
225 230 235 240 Cys Arg Glu Pro Pro Pro Trp Glu Asn Glu Ala Thr Glu
Arg Ile Tyr 245 250 255 His Phe Val Val Gly Gln Met Val Tyr Tyr Gln
Cys Val Gln Gly Tyr 260 265 270 Arg Ala Leu His Arg Gly Pro Ala Glu
Ser Val Cys Lys Met Thr His 275 280 285 Gly Lys Thr Arg Trp Thr Gln
Pro Gln Leu Ile Cys Thr Gly Gly Gly 290 295 300 Gly Gly Ser Gly Gly
Gly Ser Arg Thr Val Ala Ala Pro Ser Val Phe 305 310 315 320 Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 325 330 335
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 340
345 350 Lys Val Asp Asn Ala Leu Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu 355 360 365 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu 370 375 380 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val Thr 385 390 395 400 His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu 405 410 415 Cys Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 420 425 430 Gly Gly Gly Gly
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 435 440 445 Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 450 455 460
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 465
470 475 480 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser 485 490 495 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser Ser Ser 500 505 510 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn 515 520 525 Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His 530 535 540 Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 545 550 555 560 Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 565 570 575 Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 580 585
590 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
595 600 605 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser 610 615 620 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys 625 630 635 640 Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile 645 650 655 Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 660 665 670 Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 675 680 685 Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 690 695 700 Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 705 710
715 720 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg 725 730 735 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 740 745 750 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 755 760 765 20770PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 20Ser Lys Asn Phe His
Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn 1 5 10 15 Val Ile Val
Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu 20 25 30 Tyr
Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile 35 40
45 Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Ser Ser Ser
50 55 60 Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp
Leu Gln 65 70 75 80 Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro
Lys Leu Thr Arg 85 90 95 Met Leu Thr Phe Lys Phe Tyr Met Pro Lys
Lys Ala Thr Glu Leu Lys 100 105 110 His Leu Gln Cys Leu Glu Glu Glu
Leu Lys Pro Leu Glu Glu Val Leu 115 120 125 Asn Leu Ala Gln Gly Ser
Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp 130 135 140 Pro Pro Glu Ile
Pro His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu 145 150 155 160 Gly
Thr Met Leu Asn Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys 165 170
175 Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser
180 185 190 Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser Ala Thr Arg Asn
Thr Thr 195 200 205 Lys Gln Val Thr Pro Gln Pro Glu Glu Gln Lys Glu
Arg Lys Thr Thr 210 215 220 Glu Met Gln Ser Pro Met Gln Pro Val Asp
Gln Ala Ser Leu Pro Gly 225 230 235 240 His Cys Arg Glu Pro Pro Pro
Trp Glu Asn Glu Ala Thr Glu Arg Ile 245 250 255 Tyr His Phe Val Val
Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly 260 265 270 Tyr Arg Ala
Leu His Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr 275 280 285 His
Gly Lys Thr Arg Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Gly 290 295
300 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser
305 310 315 320 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala 325 330 335 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val 340 345 350 Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 355 360 365 Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 370 375 380 Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 385 390 395 400 Lys Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Gly Gly Gly Gly Ser 405 410 415
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro 420
425 430 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
Thr 435 440 445 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala Lys 450 455 460 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu 465 470 475 480 Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser Ser 485 490 495 Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 500 505 510 Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 515 520 525 Asn Arg
Gly Glu Cys Gly Gly Ser Gly Gly Glu Pro Lys Ser Cys Asp 530 535 540
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 545
550 555 560 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile 565 570 575 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu 580 585 590 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His 595 600 605 Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg 610 615 620 Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys 625 630 635 640 Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 645 650 655 Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 660 665
670 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
675 680 685 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 690 695 700 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val 705 710 715 720 Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 725 730 735 Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 740 745 750 Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 755 760 765 Gly Lys 770
2140PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly
Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Ser Gly
Gly Gly Ser Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser Gly Gly Gly
Ser 35 40 2250PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 35 40 45 Gly Ser 50
23817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 23Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala
Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ser Pro Gly Gln Gly
Thr Gln Ser Glu Asn Ser Cys 20 25 30 Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu Arg Asp 35 40 45 Ala Phe Ser Arg Val
Lys Thr Phe Phe
Gln Met Lys Asp Gln Leu Asp 50 55 60 Asn Leu Leu Leu Lys Glu Ser
Leu Leu Glu Asp Phe Lys Gly Tyr Leu 65 70 75 80 Gly Cys Gln Ala Leu
Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val 85 90 95 Met Pro Gln
Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn 100 105 110 Ser
Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 115 120
125 His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val
130 135 140 Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met 145 150 155 160 Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu
Ala Tyr Met Thr Met 165 170 175 Lys Ile Arg Asn Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Ser Pro 180 185 190 Gly Gln Gly Thr Gln Ser Glu
Asn Ser Cys Thr His Phe Pro Gly Asn 195 200 205 Leu Pro Asn Met Leu
Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys 210 215 220 Thr Phe Phe
Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu 225 230 235 240
Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser 245
250 255 Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu
Asn 260 265 270 Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly
Glu Asn Leu 275 280 285 Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His
Arg Phe Leu Pro Cys 290 295 300 Glu Asn Lys Ser Lys Ala Val Glu Gln
Val Lys Asn Ala Phe Asn Lys 305 310 315 320 Leu Gln Glu Lys Gly Ile
Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe 325 330 335 Ile Asn Tyr Ile
Glu Ala Tyr Met Thr Met Lys Ile Arg Asn Gly Gly 340 345 350 Gly Gly
Ser Gly Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val 355 360 365
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 370
375 380 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
Gln 385 390 395 400 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu Ser Val 405 410 415 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser Thr Leu 420 425 430 Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala Cys Glu 435 440 445 Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 450 455 460 Gly Glu Cys Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 465 470 475 480 Gly
Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 485 490
495 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
500 505 510 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 515 520 525 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 530 535 540 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser 545 550 555 560 Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 565 570 575 Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 580 585 590 Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 595 600 605 Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 610 615
620 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
625 630 635 640 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn 645 650 655 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val 660 665 670 Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu 675 680 685 Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 690 695 700 Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 705 710 715 720 Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 725 730 735
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 740
745 750 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu 755 760 765 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys 770 775 780 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 785 790 795 800 Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 805 810 815 Lys 24822PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
24Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1
5 10 15 Val Thr Asn Ser Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser
Cys 20 25 30 Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp
Leu Arg Asp 35 40 45 Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met
Lys Asp Gln Leu Asp 50 55 60 Asn Leu Leu Leu Lys Glu Ser Leu Leu
Glu Asp Phe Lys Gly Tyr Leu 65 70 75 80 Gly Cys Gln Ala Leu Ser Glu
Met Ile Gln Phe Tyr Leu Glu Glu Val 85 90 95 Met Pro Gln Ala Glu
Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn 100 105 110 Ser Leu Gly
Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys 115 120 125 His
Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val 130 135
140 Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met
145 150 155 160 Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr
Met Thr Met 165 170 175 Lys Ile Arg Asn Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Ser Pro 180 185 190 Gly Gln Gly Thr Gln Ser Glu Asn Ser
Cys Thr His Phe Pro Gly Asn 195 200 205 Leu Pro Asn Met Leu Arg Asp
Leu Arg Asp Ala Phe Ser Arg Val Lys 210 215 220 Thr Phe Phe Gln Met
Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu 225 230 235 240 Ser Leu
Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser 245 250 255
Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn 260
265 270 Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn
Leu 275 280 285 Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe
Leu Pro Cys 290 295 300 Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys
Asn Ala Phe Asn Lys 305 310 315 320 Leu Gln Glu Lys Gly Ile Tyr Lys
Ala Met Ser Glu Phe Asp Ile Phe 325 330 335 Ile Asn Tyr Ile Glu Ala
Tyr Met Thr Met Lys Ile Arg Asn Gly Gly 340 345 350 Gly Gly Ser Gly
Gly Gly Gly Ser Ala Ser Thr Lys Gly Pro Ser Val 355 360 365 Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 370 375 380
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 385
390 395 400 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val 405 410 415 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 420 425 430 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 435 440 445 Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Gly Gly Gly Gly Ser Gly 450 455 460 Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr 465 470 475 480 Val Ala Ala
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 485 490 495 Lys
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 500 505
510 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
515 520 525 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr 530 535 540 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His 545 550 555 560 Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val 565 570 575 Thr Lys Ser Phe Asn Arg Gly
Glu Cys Gly Gly Ser Gly Gly Glu Pro 580 585 590 Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 595 600 605 Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 610 615 620 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 625 630
635 640 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly 645 650 655 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn 660 665 670 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp 675 680 685 Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro 690 695 700 Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu 705 710 715 720 Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 725 730 735 Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 740 745 750
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 755
760 765 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 770 775 780 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 785 790 795 800 Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu 805 810 815 Ser Leu Ser Pro Gly Lys 820
25577PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 25Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala
Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ser Pro Gly Gln Gly
Thr Gln Ser Glu Asn Ser Cys 20 25 30 Thr His Phe Pro Gly Asn Leu
Pro Asn Met Leu Arg Asp Leu Arg Asp 35 40 45 Ala Phe Ser Arg Val
Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp 50 55 60 Asn Leu Leu
Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu 65 70 75 80 Gly
Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val 85 90
95 Met Pro Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn
100 105 110 Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg
Arg Cys 115 120 125 His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala
Val Glu Gln Val 130 135 140 Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys
Gly Ile Tyr Lys Ala Met 145 150 155 160 Ser Glu Phe Asp Ile Phe Ile
Asn Tyr Ile Glu Ala Tyr Met Thr Met 165 170 175 Lys Ile Arg Asn Gly
Gly Ser Gly Gly Ser Pro Gly Gln Gly Thr Gln 180 185 190 Ser Glu Asn
Ser Cys Thr His Phe Pro Gly Asn Leu Pro Asn Met Leu 195 200 205 Arg
Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Met 210 215
220 Lys Asp Gln Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp
225 230 235 240 Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met
Ile Gln Phe 245 250 255 Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn
Gln Asp Pro Asp Ile 260 265 270 Lys Ala His Val Asn Ser Leu Gly Glu
Asn Leu Lys Thr Leu Arg Leu 275 280 285 Arg Leu Arg Arg Cys His Arg
Phe Leu Pro Cys Glu Asn Lys Ser Lys 290 295 300 Ala Val Glu Gln Val
Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly 305 310 315 320 Ile Tyr
Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu 325 330 335
Ala Tyr Met Thr Met Lys Ile Arg Asn Glu Pro Lys Ser Ser Asp Lys 340
345 350 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro 355 360 365 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 370 375 380 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 385 390 395 400 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 405 410 415 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 420 425 430 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 435 440 445 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 450 455 460
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 465
470 475 480 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr 485 490 495 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 500 505 510 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 515 520 525 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 530 535 540 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 545 550 555 560 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 565 570 575 Lys
2620PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 2725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10
15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
* * * * *