U.S. patent application number 13/469902 was filed with the patent office on 2013-02-14 for compositions and methods for improving production of recombinant polypeptides.
This patent application is currently assigned to Amunix Operationg Inc.. The applicant listed for this patent is Oren Bogin, Nathan C. Geething, Volker Schellenberger, Willem P. Stemmer, Chia-wei Wang, Yong Yin. Invention is credited to Oren Bogin, Nathan C. Geething, Volker Schellenberger, Willem P. Stemmer, Chia-wei Wang, Yong Yin.
Application Number | 20130039884 13/469902 |
Document ID | / |
Family ID | 40351372 |
Filed Date | 2013-02-14 |
United States Patent
Application |
20130039884 |
Kind Code |
A1 |
Bogin; Oren ; et
al. |
February 14, 2013 |
COMPOSITIONS AND METHODS FOR IMPROVING PRODUCTION OF RECOMBINANT
POLYPEPTIDES
Abstract
The present invention relates to biologically active
polypeptides linked to one or more accessory polypeptides. The
present invention also provides recombinant polypeptides including
vectors encoding the subject proteinaceous entities, as well as
host cells comprising the vectors. The subject compositions have a
variety of utilities including a range of pharmaceutical
applications.
Inventors: |
Bogin; Oren; (Sunnyvale,
CA) ; Stemmer; Willem P.; (Los Gatos, CA) ;
Schellenberger; Volker; (Palo Alto, CA) ; Yin;
Yong; (Sunnyvale, CA) ; Wang; Chia-wei; (Santa
Clara, CA) ; Geething; Nathan C.; (Santa Clara,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bogin; Oren
Stemmer; Willem P.
Schellenberger; Volker
Yin; Yong
Wang; Chia-wei
Geething; Nathan C. |
Sunnyvale
Los Gatos
Palo Alto
Sunnyvale
Santa Clara
Santa Clara |
CA
CA
CA
CA
CA
CA |
US
US
US
US
US
US |
|
|
Assignee: |
Amunix Operationg Inc.
Mountain View
CA
|
Family ID: |
40351372 |
Appl. No.: |
13/469902 |
Filed: |
May 11, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12646566 |
Dec 23, 2009 |
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13469902 |
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12228859 |
Aug 15, 2008 |
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12646566 |
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60956109 |
Aug 15, 2007 |
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60981073 |
Oct 18, 2007 |
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60986569 |
Nov 8, 2007 |
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Current U.S.
Class: |
424/85.2 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 31/00 20180101; C07K 2317/626 20130101; C07K 2319/00 20130101;
A61P 1/04 20180101; A61P 31/12 20180101; A61P 31/18 20180101; A61P
43/00 20180101; A61P 27/06 20180101; A61P 7/06 20180101; A61P 7/00
20180101; A61P 19/10 20180101; A61P 3/04 20180101; A61P 11/06
20180101; A61P 29/00 20180101; A61P 37/08 20180101; C07K 2317/569
20130101; C07K 14/61 20130101; A61P 17/06 20180101; A61P 15/10
20180101; A61P 9/12 20180101; C07K 14/56 20130101; A61P 11/00
20180101; C07K 16/40 20130101; A61P 13/12 20180101; A61P 17/02
20180101; C07K 2317/22 20130101; C07K 2319/33 20130101; A61P 7/02
20180101; A61P 21/04 20180101; A61P 19/02 20180101; A61P 25/14
20180101; C07K 2317/55 20130101; C07K 16/2863 20130101; A61P 31/04
20180101; A61P 25/04 20180101; C07K 14/00 20130101; A61P 35/00
20180101; C07K 14/535 20130101; C07K 16/32 20130101; A61P 1/02
20180101; A61P 1/16 20180101; A61P 25/28 20180101; A61P 35/02
20180101; A61P 3/10 20180101; A61P 5/24 20180101; C07K 2317/622
20130101; C07K 16/30 20130101; A61K 38/00 20130101; A61P 5/00
20180101; A61P 9/00 20180101; A61P 9/10 20180101 |
Class at
Publication: |
424/85.2 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under SBIR
grant 1R43GM079873-01 and 2R44GM079873-02 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A biologically active protein comprising at least two domains
wherein (a) a first domain of said at least two domains comprises
an amino acid sequence having and/or mediating said biological
activity; and (b) a second domain of said at least two domains
comprises an amino acid sequence consisting of at least about 100
amino acid residues forming random coil conformation, wherein said
second domains consists of three types of amino acid residues,
whereby said random coil conformation mediates an increased in vivo
and in vitro stability of said biologically active protein.
2. The biologically active protein according to claim 1, wherein
said second domain forming random coil conformation consists of
alanine, serine and proline residues.
3. The biologically active protein according to claim 1, wherein
said second domain forming random coil conformation comprises a
plurality of amino acid repeats, wherein said repeat consist of
Ala, Ser, and Pro residues and wherein no more than 6 consecutive
amino acid residues are identical.
4. The biologically active protein according to claim 2, wherein
said proline residues constitute more than 4% and less than 40% of
the amino acids of said second domain forming random coil
conformation.
5. The biologically active protein according to claim 1, wherein
said second domain of said at least two domains comprises an amino
acid sequence consisting of about 100 to 3000 amino acid residues
forming random coil conformation.
6. The biologically active protein according to claim 1, wherein
said polypeptide with biological activity is selected from the
group consisting of binding molecules, antibody fragments,
cytokines, growth factors, hormones or enzymes.
7. The biologically active protein according to claim 6 wherein
said binding molecule is selected from the group consisting of
antibodies, Fab fragments, F(ab')2 fragments, CDR derived
peptidomimetics, single chain variable fragments (scFv), domain
antibodies and lipocalins.
8. The biologically active protein according to claim 1, wherein
said polypeptide with biological activity is selected from the
group consisting of granulocyte colony stimulating factor, human
growth hormone, alpha_interferon, beta_interferon,
gamma_interferon, tumor necrosis factor, erythropoietin,
coagulation factor VIII, gp120/gp160, soluble tumor necrosis factor
I and II receptor, interleukin 2 and neutrophil gelatinase
associated lipocalin.
9. The biologically active protein according to claim 1, wherein
said increased in vivo stability of said biologically active
protein is a prolonged plasma half life of said biologically active
protein comprising said random coil forming second domain when
compared to said biologically active protein lacking said random
coil forming second domain.
10. A composition comprising the biologically active protein
according to any one of claims 1 to 9.
11. The composition according to claim 10, which is a
pharmaceutical composition, optionally further comprising a
pharmaceutical acceptable carrier.
12. An isolated nucleic acid molecule encoding the biologically
active protein of any one of claims 1 to 9.
13. A vector comprising the nucleic acid of claim 12.
14. An isolated cell comprising the nucleic acid according to claim
12.
15. A method for the preparing a biologically active protein
comprising culturing the cell according to claim 14 and isolating
said biologically active protein from the culture.
16. A method of treating a disease condition selected from the
group consisting of hormone deficiency related disorders,
autoimmune disease, cancer, anaemia, neovascular diseases,
infectious/inflammatory diseases, thrombosis, myocardial
infarction, diabetes, reperfusion injury, and a kidney disease,
comprising administering to a subject in need thereof a composition
according to claim 11.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. Nos. 60/956,109 filed on Aug. 15,
2007; 60/981,073, filed Oct. 18, 2007 and 60/986,569, filed Nov. 8,
2007, pending, which are hereby incorporated herein by reference in
their entirety.
[0003] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Apr. 21,
2010, is named 32808301.txt, and is 280,493 bytes in size.
BACKGROUND OF THE INVENTION
[0004] Recombinant proteins have become very attractive candidates
for the development of novel therapeutics. However, production of
protein pharmaceuticals requires significant optimization of
processes to obtain sufficient yields of specific biologically
active polypeptides. It is well established that the expression of
recombinant proteins in the cytoplasm of Escherichia coli, in
particular mammalian recombinant proteins, frequently results in
the formation of insoluble aggregates known as inclusion bodies.
High cell density fermentation and purification of the recombinant
protein from inclusion bodies of E. coli are two major bottlenecks
for the cost effective production of therapeutic proteins (Panda,
A. K, 2003, Adv. Biochem. Eng. Biotechnol., 85, 43). Similarly, for
research purposes, where hundreds of proteins may need to be
screened for various activities, the expression of soluble, active
protein is desirable, thereby avoiding the step of first purifying
inclusion bodies and then having to denature and refold protein
each separately.
[0005] Examples of the many pharmaceutically important proteins
that form insoluble inclusion bodies when expressed in the
cytoplasmic space of E. coli include human Growth Hormone (hGH)
(Patra, A. K. et al., 2000, Protein Expr. Purif, 18, 182; Khan, R.
H, et al., 1998, Biotechnol. Prog., 14, 722), human
Granulocyte-Colony Stimulating Factor (G-CSF)(Zaveckas, M. et al.
2007, J Chromatogr B Analyt Technol Biomed Life Sci. 852, 409; Lee,
A. Y. et al., 2003, Biotechnol Lett., 25, 205,) and Interferon
alpha (IFN-alpha; Valente, C. A. et al., 2006, Protein. Expr.
Purif. 45, 226). Furthermore, the immunoglobulin domains of
antibodies and their fragments, including domain antibody fragments
(dAb), Fv fragments, single-chain Fv fragments (scFv), Fab
fragments, Fab'2 fragments, and many non-antibody proteins (such as
FnIII domains) generally form inclusion bodies upon expression in
the cytoplasm of bacterial hosts (Kou, G., et al., 2007, Protein
Expr Purif. 52, 131; Cao, P., et al. 2006, Appl Microbiol
Biotechnol., 73, 151; Chen, L. H et al., 2006, Protein Expr Purif.;
46, 495).
[0006] Human proteins typically fold using a hydrophobic core
comprising a large number of hydrophobic amino acids. Research has
shown that proteins can aggregate and form inclusion bodies,
especially when genes from one organism are expressed in another
expression host, such that the protein's native binding partners
are absent, so that folding help is unavailable and hydrophobic
patches remain exposed. This is especially true when large
evolutionary distances are crossed: a cDNA isolated from a
eukaryote for example, when expressed as a recombinant gene in a
prokaryote, has a high risk of aggregating and forming an inclusion
body. While the cDNA may properly code for a translatable mRNA, the
protein that results will emerge in a foreign microenvironment.
This often results in misfolded, inactive protein that generally
accumulates as aggregates if the concentration is high enough.
Other effectors, such as the internal microenvironment of a
prokaryotic cell (pH, osmolarity) may differ from that of the
original source of the gene and affect protein folding. Mechanisms
for folding a protein may also be host-dependent and thus be absent
in a heterologous host, and hydrophobic residues that normally
would remain buried as part of the hydrophobic core instead remain
exposed and available for interaction with hydrophobic sites on
other proteins. Processing systems for the cleavage and removal of
internal peptides of the expressed protein may also be absent in
bacteria. In addition, the fine controls that may keep the
concentration of a protein low will also be missing in a
prokaryotic cell, and over-expression can result in filling a cell
with protein that, even if it were properly folded, would
precipitate by saturating its environment.
[0007] The recovery of biologically active products from the
aggregated state found in inclusion bodies is typically
accomplished by unfolding with chaotropic agents or acids, followed
by dilution or dialysis into optimized refolding buffers. However,
many polypeptides (especially structurally complex oligomeric
proteins and those containing multiple disulfide bonds) do not
easily adopt an active conformation following chemical
denaturation.
[0008] Small changes in primary structure can affect solubility,
presumably by altering folding pathways (Mitraki, A. et al. (1989)
Bio/Technology 7, 690; Baneyx, F, et. al. 2004 Nat Biotechnol, 22,
1399; Ventura, S. 2005 Microb Cell Fact, 4, 11). In order to reduce
the formation of insoluble aggregates during high-density
fermentation, some groups have linked heterologous fusion proteins
to the protein of interest. Examples of such fusion sequences are
Glutathione-S-Transferase (GST), Protein Disulfide Isomerase (PDI),
Thioredoxin (TRX), Maltose Binding Protein (MBP), His6 tag (SEQ ID
NO: 1), Chitin Binding Domain (CBD) and Cellulose Binding Domain
(CBD) (Sahadev, S. et al. 2007, Mol. Cell. Biochem.; Dysom, M. R.
et al. 2004, BMC Biotechnol, 14, 32). In summary, these approaches
were found to be protein-specific, as they do not work for all
proteins.
[0009] While various fusion proteins have been designed to improve
folding, chemical PEGylation of proteins has also been reported to
enhance protein solubility, reduce aggregation, reduce
immunogenicity, and reduce proteolysis. Nonetheless, the proper
folding of overproduced polypeptides remains problematic within the
highly concentrated and viscous environment of the cell cytoplasm,
where aggregation occurs in a concentration-dependent manner.
Another approach for the expression of mammalian proteins in
bacterial hosts avoids leader peptides and expresses the active
protein directly in the cytoplasm of the host. However, this
process tends to result in aggregation and inclusion body
formation.
[0010] One widely used approach for the expression of mammalian
proteins in active form in bacteria is to direct the protein into
the non-reducing environment of the periplasmic space of bacterial
hosts such as E. coli, typically using signal- or leader-peptides
to direct secretion. Secretion into the periplasm (and rarely into
the media) appears to mimic the native eukaryotic process of
protein secretion, folding and disulfide formation and often
results in active protein. This approach has many profound
drawbacks. The periplasm tends to give low yields; the process is
generally limited to smaller proteins; the process tends to be
protein-specific; and also that the procedures for extracting
periplasmic proteins are not as robust as extraction from the
cytoplasm, which contributes to low yields. For these reasons,
expression of proteins in the periplasm of bacteria is not
applicable to most pharmaceutical proteins, which are typically
commercially expressed in yeast or mammalian cell lines.
[0011] Another approach that has been tried to make mammalian
proteins express in the cytoplasm of bacteria without forming
inclusion bodies is to over-express folding-helper proteins, like
the molecular chaperones which play a role in a wide range of
biotechnological applications (Mogk et al. 2002 Chembiochem 3,
807). To date, several different families of chaperones have been
reported. All are characterized by their ability to bind unfolded
or partially unfolded proteins and release correctly folded
proteins into the cytoplasm of bacteria. A well-characterized
example is the heat-shock family of proteins (Hsp), which are
designated according to their relative molecular weight, as
described by Buchner, J., Faseb J. 1996 10, 10 and by Beissinger,
M. and Buchner, 1998. J. Biol. Chem. 379, 245. While many bacterial
and eukaryotic chaperonins have been tried for over-expression of
proteins in bacteria and to a lesser extent mammalian cells, this
approach has generally had little or no effect and this is less
often practiced for expression optimization.
[0012] There therefore remains a significant need for methods and
compositions for production of biologically active proteins and for
improving their solubility to effect large scale production
utilizing host cells, such as prokaryotes.
SUMMARY OF THE INVENTION
[0013] The present invention provides a method of producing a
biologically active polypeptide. The method typically involves the
steps of a) providing a polynucleotide sequence coding for a
modified polypeptide comprising the biologically active polypeptide
linked with an accessory polypeptide such that expression of the
modified polypeptide in a host cell yields a higher quantity of
soluble form of biologically active polypeptide, as compared to
expression of the biologically active polypeptide by itself (e.g.,
free from said accessory polypeptide; and b) causing the modified
polypeptide to be expressed in said host cell, thereby producing
the biologically active polypeptide. In one embodiment, the
expression of the soluble, active form of a biologically active
polypeptide is about 1%, 5%, 25%, 50%, 75%, 95% or 99% of the total
of that protein. In one embodiment, the expression of the modified
polypeptide in a host cell yields at least about 2-fold more
soluble form of biologically active polypeptide as compared to
expression of the biologically active polypeptide by itself. In
another embodiment, the biologically active polypeptide is linked
to the accessory polypeptide via a proteinase cleavage site. Where
desired, the cleavage site can be selected from the group
consisting of TEV protease, enterokinase, Factor Xa, thrombin,
PreScission.TM. protease, 3C protease, sortase A, and granzyme B.
In some embodiments, the expression of the modified polypeptide in
a host cell yields at least about 2-fold, 5-fold, 10-fold, 30-fold,
or 100-fold, or more soluble form of biologically active
polypeptide.
[0014] The present invention also provides a host cell for
expressing the modified polynucleotide sequence. The host cell is
typically prokaryotic including but not limited to E. Coli, and it
may also be eukaryotic such as yeast cells and also mammalian cells
(e.g. CHO cells).
[0015] The present invention also provides a genetic vehicle
comprising the subject polynucleotide sequence that encodes a
biologically active polypeptide linked with or without an accessory
polypeptide.
[0016] Further provided by the present invention is a composition
comprising soluble form of a biologically active polypeptide linked
with an accessory polypeptide, wherein said accessory polypeptide
when linked with the biologically active polypeptide increases
solubility of the biologically active polypeptide in a cytosolic
fraction of a host cell in which the linked biologically active
polypeptide is expressed. Where desired, the biologically active
polypeptide is linked via a protease cleavage site to the accessory
polypeptide. The cleavage site can be selected from the group
consisting of TEV protease, enterokinase, Factor Xa, thrombin,
PreScission.TM. protease, 3C protease, sortase A, and granzyme
B.
[0017] The accessory polypeptide used in the subject methods or
compositions can be characterized in whole or in part by the
following. In one embodiment, the subject accessory polypeptide
provides an average net positive charge density of the modified
biologically active polypeptide of about +0.025, +0.05, +0.075,
+0.1, +0.2, +0.3, +0.4, +0.5, +0.6, +0.7, +0.8, +0.9 or even +1.0
charges per amino acid residue. In another embodiment, the subject
accessory polypeptide provides an average net negative charge
density of the modified biologically active polypeptide of about
-0.25, -0.5, -0.075, -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7,
-0.8, -0.9 or even -1.0 average net charges per amino acid residue.
In one embodiment, the subject accessory polypeptide provides a net
positive charge of the modified biologically active polypeptide of
about +3, +4, +5, +6, +7, +8, +9, +10, +12, +14+16+18+20, +25, +30,
+35, +40, +50 or more. In one embodiment, the subject accessory
polypeptide provides a net negative charge of the modified
biologically active polypeptide of about -3, -4, -5, -6, -7, -8,
-9, -10, -12, -14, -16, -18, -20, -25, -30, -35, -40, -50 or
more.
[0018] In yet another embodiment, the accessory polypeptides of the
invention may comprise more than about 10, 30, 50 or 100
aminoacids. In one embodiment, the accessory polypeptide comprises
at least 40 contiguous amino acids and is substantially incapable
of non-specific binding to a serum protein. In some embodiments,
the sum of glycine (G), aspartate (D), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P) and lysine (K)
residues contained in the accessory polypeptide, constitutes more
than about 80% of the total amino acids of the accessory
polypeptide; and/or at least 50% of the amino acids in the
accessory polypeptide are devoid of secondary structure as
determined by the Chou-Fasman algorithm. In a related embodiment,
the accessory polypeptide comprises at least 40 contiguous amino
acids and the accessory polypeptide has an in vitro serum half-life
greater than about 4 hours, 5 hours, 10 hours, 15 hours or 24
hours. Further wherein (a) the sum of glycine (G), aspartate (D),
alanine (A), serine (S), threonine (T), glutamate (E) and proline
(P) and lysine (K) residues contained in the accessory polypeptide,
constitutes more than about 80% of the total amino acids of the
accessory polypeptide; and/or (b) at least 50% of the amino acids
in the accessory polypeptide are devoid of secondary structure as
determined by Chou-Fasman algorithm. In some embodiments the set of
amino acids from which the 80% (or 50, 60, 70 or 90%) of the total
amino acids are chosen is chosen is G/S/E/D, G/S/K/R, G/S/E/D/K/R,
or G/A/S/T/Q.
[0019] In some embodiments, an accessory polypeptide comprises at
least 50% glycine residues (i.e., 50% of all residues are glycine).
Alternatively, an accessory polypeptide may comprise less than 50%
glycine residues. In some embodiments, accessory polypeptides
comprise at least 50% serine residues. Other embodiments provide
for accessory polypeptides comprising at least 50% serine and
glycine residues. Further embodiments provide for accessory
polypeptides which comprise at least 5% glutamic acid, or
alternatively at least 10, 20 or 30% glutamic acid.
[0020] In one embodiment, an accessory polypeptide may also be
characterized in that (a) it consists of three types of amino
acids, and each type being selected from a group consisting of
alanine (A), aspartic acid (D), glutamic acid (E), glycine (G),
histidine (H), lysine (K), asparagine (N), proline (P), glutamine
(Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and
(b) it comprises 10, 25, 50, 100 or more amino acids. In a related
embodiment, the accessory polypeptide consists of three types of
amino acids, each type being selected from the group consisting of
D, E, G, K, P, R, S, and T. The accessory polypeptide may also
consist of three types of amino acids, each type being selected
from the group consisting of E, G, and S.
[0021] The invention also provides for an accessory polypeptide
characterized in that: (i) it consists of three types of amino
acids, two of which are serine (S) and glycine (G) and the other
type being selected from the group consisting of aspartic acid (D),
glutamic acid (E), lysine (K), proline (P), Arginine (R), Glycine
(G), Threonine (T), alanine (A), histidine (H), asparagine (N),
tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine
(M), phenylalanine (F), isoleucine (I), and cysteine (C); and (ii)
it comprises ten or more amino acid residues, of which 50% or more
are serine or glycine.
[0022] In another embodiment, the accessory polypeptide is
characterized in that: (a) it consists of two types of amino acids,
one of which is glycine (G) and the other type is selected from the
group consisting of aspartic acid (D), glutamic acid (E), lysine
(K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine
(A), histidine (H), asparagine (N), tyrosine (Y), leucine (L),
valine (V), tryptophan (W), methionine (M), phenylalanine (F),
isoleucine (I), and cysteine (C); and (b) it comprises ten or more
amino acid residues, of which 50% or less are glycine.
[0023] Alternatively, the accessory polypeptide consists of two
types of amino acids, wherein 50% or less of the total amino acids
are selected from the group consisting of A, S, T, D, E, K and
H.
[0024] In still another embodiment, the accessory polypeptide is
characterized in that: (a) it comprises 50 or more amino acids; (b)
it consists of two types of amino acids, and (c) 50% or less of the
total amino acids are selected from the group consisting of A, S,
T, D, E, K and H.
[0025] Accessory polypeptides may comprise 1, 2, 5 or 10 or more
repeating motifs, each of which may comprise two to five hundred
amino acids. In some cases, repeating motifs consist of two or
three or more different types of amino acids. Multiple accessory
polypeptides may be used. Accessory polypeptide may also comprise
charged amino acids.
[0026] In some embodiments, the accessory polypeptide comprises an
amino acid sequence (GGEGGS)n (SEQ ID NO: 2), wherein n is an
integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In other
embodiments, the accessory polypeptide comprises an amino acid
sequence (GES)n, wherein G, E, and S can be in any order and n is
an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. Alternatively,
the accessory polypeptide comprises an amino acid sequence
(GGSGGE)n, wherein G, E, and S can be in any order and n is an
integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In yet another
embodiment, the accessory polypeptide comprises an amino acid
sequence (GEGGGEGGE)n (SEQ ID NO: 3), wherein n is an integer of 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In yet another embodiment,
the accessory sequence comprises an minor acid sequence (GE)n,
wherein G and E can be in any order and n is an integer of 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or greater.
[0027] In some embodiments, the accessory polypeptide comprises an
amino acid sequence (S)n (SEQ ID NO: 4), wherein n is an integer of
10, 15 20, 50 or greater. In other embodiments, the accessory
polypeptide comprises an amino acid sequence (SSSSSSE)n, wherein E
and S can be in any order and n is an integer of 2, 3, 4, 5, 6, 7,
8, 9, 10 or greater In other embodiments, the accessory polypeptide
comprises an amino acid sequence (SSSSE)n, wherein E and S can be
in any order and n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or
greater. In yet other embodiments, the accessory polypeptide
comprises an amino acid sequence (SESSSESSE)n, wherein E and S can
be in any order and n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10
or greater. In some embodiments, the accessory polypeptide
comprises an amino acid sequence (SSESSSSESSSE)n, wherein E and S
can be in any order and n is an integer of 3 or greater. In other
embodiments, the accessory polypeptide comprises an amino acid
sequence (SSSESSSSSESSSSE)n, wherein E, and S can be in any order
and n is an integer of 3 or greater. In still other embodiments,
the accessory polypeptide comprises an amino acid sequence
(SSSSESSSSSSESSSSSE)n, wherein E and S can be in any order and n is
an integer of 3 or greater.
[0028] In some embodiments the accessory polypeptide is not
composed of repeating units of a peptide motif of 3, 4, 5, 6 or 7
amino acids, or is not composed of repeating units of any single
polypeptide motif. In some embodiments the accessory polypeptide is
composed of more than 2, 5, 10, or 20 different repeating motifs of
a fixed length. In some embodiments the accessory polypeptide is
composed of more than 2, 5, 10, or 20 different repeating motifs of
any length.
[0029] Additionally, the invention describes a method of making a
pharmaceutical composition, comprising: (a) providing a modified
polypeptide; (b) mixing said modified polypeptide with a polymer
matrix.
[0030] The biologically active polypeptide produced by the subject
methods or present in the subject composition can be human growth
hormone (hGH), glucagon-like peptide-1 (GLP-1), exenatide,
pramlitide, uricase, granulocyte-colony stimulating factor (G-CSF),
interferon-alpha, interferon-beta, interferon-gamma, insulin,
interleukin 1 receptor antagonist (IL-1RA), erythropoietin or tumor
necrosis factor-alpha (TFN-alpha).
[0031] The present invention relates to a pharmaceutical
composition comprising (a) a slow release agent, and (b) a modified
polypeptide comprising a biologically active polypeptide linked to
an accessory polypeptide. The modified polypeptide may yield an
apparent molecular weight factor of greater than 1. The apparent
molecular weight factor may be determined as the apparent molecular
weight of the modified polypeptide as measured by size exclusion
chromatography relative to the predicted molecular weight of the
modified polypeptide. In one embodiment, the apparent molecular
weight factor of the modified polypeptide is greater than 3. In
another embodiment, the apparent molecular weight factor of the
modified polypeptide is greater than 5. In yet another embodiment,
the apparent molecular weight factor of the modified polypeptide is
greater than 7. In still another embodiment, the apparent molecular
weight factor of the modified polypeptide is greater than 9.
[0032] The accessory polypeptide can increase the serum half-life
of a biologically active polypeptide. Alternatively, accessory
polypeptides can increase the protease resistance of a biologically
active polypeptide. In other cases, accessory polypeptides can
increase the solubility of a biologically active polypeptide. In
other cases, accessory polypeptides can decrease the immunogenicity
of a biologically active polypeptide. The accessory polypeptides of
the invention may comprise more than about 10, 30, 50 or 100
aminoacids. In some embodiments, the biologically active
polypeptide can be human growth hormone (hGH), glucagon-like
peptide-1 (GLP-1), exenatide, pramlitide, uricase,
granulocyte-colony stimulating factor (G-CSF), interferon-alpha,
interferon-beta, interferon-gamma, insulin, interleukin 1 receptor
antagonist (IL-IRA), erythropoietin or tumor necrosis factor-alpha
(TNF-alpha).
[0033] In one embodiment, the accessory polypeptide comprises at
least 40 contiguous amino acids and is substantially incapable of
non-specific binding to a serum protein. In some embodiments, the
sum of glycine (G), aspartate (D), alanine (A), serine (S),
threonine (T), glutamate (E) and proline (P) residues contained in
the accessory polypeptide, constitutes more than about 80% of the
total amino acids of the accessory polypeptide; and/or at least 50%
of the amino acids in the accessory polypeptide are devoid of
secondary structure as determined by the Chou-Fasman algorithm. In
a related embodiment, the accessory polypeptide comprises at least
40 contiguous amino acids and the accessory polypeptide has an in
vitro serum half-life greater than about 4 hours, 5 hours, 10
hours, 15 hours or 24 hours. Further wherein (a) the sum of glycine
(G), aspartate (D), alanine (A), serine (S), threonine (T),
glutamate (E) and proline (P) residues contained in the accessory
polypeptide, constitutes more than about 80% of the total amino
acids of the accessory polypeptide; and/or (b) at least 50% of the
amino acids in the accessory polypeptide are devoid of secondary
structure as determined by Chou-Fasman algorithm.
[0034] In some embodiments, an accessory polypeptide comprises at
least 50% glycine residues (i.e., 50% of all residues are glycine).
Alternatively, an accessory polypeptide may comprise less than 50%
glycine residues. In some embodiments, accessory polypeptides
comprise at least 50% serine residues. Other embodiments provide
for accessory polypeptides comprising at least 50% serine and
glycine residues. Further embodiments provide for accessory
polypeptides which comprise at least 5% glutamic acid, or
alternatively at least 10, 20 or 30% glutamic acid.
[0035] In one embodiment, an accessory polypeptide may also be
characterized in that (a) it consists of three types of amino
acids, and each type being selected from a group consisting of
alanine (A), aspartic acid (D), glutamic acid (E), glycine (G),
histidine (H), lysine (K), asparagine (N), proline (P), glutamine
(Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and
(b) it comprises 10, 25, 50, 100 or more amino acids. In a related
embodiment, the accessory polypeptide consists of three types of
amino acids, each type being selected from the group consisting of
D, E, G, K, P, R, S, and T. The accessory polypeptide may also
consist of three types of amino acids, each type being selected
from the group consisting of E, G, and S.
[0036] The invention also provides for an accessory polypeptide
characterized in that: (i) it consists of three types of amino
acids, two of which are serine (S) and glycine (G) and the other
type being selected from the group consisting of aspartic acid (D),
glutamic acid (E), lysine (K), proline (P), Arginine (R), Glycine
(G), Threonine (T), alanine (A), histidine (H), asparagine (N),
tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine
(M), phenylalanine (F), isoleucine (I), and cysteine (C); and (ii)
it comprises ten or more amino acid residues, of which 50% or more
are serine or glycine.
[0037] In another embodiment, the accessory polypeptide is
characterized in that: (a) it consists of two types of amino acids,
one of which is glycine (G) and the other type is selected from the
group consisting of aspartic acid (D), glutamic acid (E), lysine
(K), proline (P), Arginine (R), Serine (S), Threonine (T), alanine
(A), histidine (H), asparagine (N), tyrosine (Y), leucine (L),
valine (V), tryptophan (W), methionine (M), phenylalanine (F),
isoleucine (I), and cysteine (C); and (b) it comprises ten or more
amino acid residues, of which 50% or less are glycine.
[0038] Alternatively, the accessory polypeptide consists of two
types of amino acids, wherein 50% or less of the total amino acids
are selected from the group consisting of A, S, T, D, E, and H.
[0039] In still another embodiment, the accessory polypeptide is
characterized in that: (a) it comprises 50 or more amino acids; (b)
it consists of two types of amino acids, and (c) 50% or less of the
total amino acids are selected from the group consisting of A, S,
T, D, E, and H.
[0040] Accessory polypeptides may comprise 1, 2, 5 or 10 or more
repeating motifs, each of which may comprise two to five hundred
amino acids. In some cases, repeating motifs consist of two or
three or more different types of amino acids. Multiple accessory
polypeptides may be used. Accessory polypeptide may also comprise
charged amino acids.
[0041] In some embodiments, the accessory polypeptide comprises an
amino acid sequence (GGEGGS)n (SEQ ID NO: 2), wherein n is an
integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In other
embodiments, the accessory polypeptide comprises an amino acid
sequence (GES)n, wherein G, E, and S can be in any order and n is
an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. Alternatively,
the accessory polypeptide comprises an amino acid sequence
(GGSGGE)n, wherein G, E, and S can be in any order and n is an
integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In yet another
embodiment, the accessory polypeptide comprises an amino acid
sequence (GEGGGEGGE)n (SEQ ID NO: 3), wherein n is an integer of 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In yet another embodiment,
the accessory sequence comprises an minor acid sequence (GE)n,
wherein G and E can be in any order and n is an integer of 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or greater.
[0042] In some embodiments, the accessory polypeptide comprises an
amino acid sequence (S)n (SEQ ID NO: 4), wherein n is an integer of
10, 15 20, 50 or greater. In other embodiments, the accessory
polypeptide comprises an amino acid sequence (SSSSSSE)n, wherein E
and S can be in any order and n is an integer of 2, 3, 4, 5, 6, 7,
8, 9, 10 or greater In other embodiments, the accessory polypeptide
comprises an amino acid sequence (SSSSE)n, wherein E and S can be
in any order and n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10 or
greater. In yet other embodiments, the accessory polypeptide
comprises an amino acid sequence (SESSSESSE)n, wherein E and S can
be in any order and n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10
or greater. In some embodiments, the accessory polypeptide
comprises an amino acid sequence (SSESSSSESSSE)n, wherein E and S
can be in any order and n is an integer of 3 or greater. In other
embodiments, the accessory polypeptide comprises an amino acid
sequence (SSSESSSSSESSSSE)n, wherein E, and S can be in any order
and n is an integer of 3 or greater. In still other embodiments,
the accessory polypeptide comprises an amino acid sequence
(SSSSESSSSSSESSSSSE)n, wherein E and S can be in any order and n is
an integer of 3 or greater.
[0043] In some embodiments the accessory polypeptide is not
composed of repeating units of a peptide motif of 3, 4, 5, 6 or 7
amino acids, or is not composed of repeating units of any single
polypeptide motif. In some embodiments the accessory polypeptide is
composed of more than 2, 5, 10, or 20 different repeating motifs of
a fixed length. In some embodiments the accessory polypeptide is
composed of more than 2, 5, 10, or 20 different repeating motifs of
any length.
[0044] A slow release agent may include a polymeric matrix. In some
embodiments, the polymeric matrix is charged. In specific
embodiments, the polymeric matrix may be poly-d,l-lactide (PLA),
poly-(d,l-lactide-co-glycolide) (PLGA), PLGA-PEG copolymers,
alginate, dextran and/or chitosan. A slow release agent may also be
packaged including a transdermal patch.
[0045] The present invention also provides a method of producing
modified polypeptides, comprising: a) providing a polynucleotide
sequence encoding the modified polypeptide; b) causing said
modified polypeptide to be expressed in a host cell, thereby
producing said modified polypeptide. A genetic vehicle comprising a
nucleic acid sequence encoding the modified polypeptide is also
provided, as well as host cells expressing the modified
polypeptides of the invention.
[0046] Additionally, the invention describes a method of making a
pharmaceutical composition, comprising: (a) providing a modified
polypeptide; (b) mixing said modified polypeptide with a polymer
matrix.
[0047] Pharmaceutical compositions of the inventions may comprise
a) a slow release agent, b) a modified polypeptide comprising a
biologically active polypeptide linked to a PEG group of greater
than 5 kD in size.
[0048] In yet other embodiments the accessory polypeptide
substantially lacks secondary structure. In still other
embodiments, the accessory polypeptide exhibits a two-fold longer
serum half-life as compared to a corresponding polypeptide lacking
the accessory polypeptide. The biologically active polypeptide and
the accessory polypeptide may be linked via a peptide bond.
[0049] In some embodiments, the modified polypeptide further
comprises at least one depot module. The depot module is at least
10 amino acids in length, preferably at least 100 amino acids in
length. Positively charged depot modules (e.g., lysine rich or
arginine rich polypeptides) may be useful in conjunction with a
negatively charged polymer. Negatively charged depot modules may be
useful in conjunction with a positively charged polymer. A depot
module including poly-His sequences may be used in conjunction with
a chelating hydrogel. In some cases, the depot module can be
protease sensitive, e.g., and without limitation, sensitive to
serum proteases or other proteases. Multiple and/or different depot
modules may be employed. Any combination of depot module,
biologically active polypeptides and accessory polypeptides may be
potentially used to produce a sustained-release therapeutic. In a
particular embodiment, the slow release agent is a depot module
linked to the modified polypeptide.
[0050] Additionally, a genetic vehicle comprising a nucleic acid
sequence encoding an API of the invention is provided. In another
embodiment, a host cell is described expressing the
polypeptides.
[0051] The present invention relates to accessory polypeptides that
may be used to modify the properties of biologically active
polypeptides. In one embodiment, the invention provides for an
isolated polypeptide comprising a biologically active polypeptide
and an accessory polypeptide, wherein the accessory polypeptide is
characterized in that it (i) consists of three types of amino
acids, and each type being selected from a group consisting of
alanine (A), aspartic acid (D), glutamic acid (E), glycine (G),
histidine (H), lysine (K), asparagine (N), proline (P), glutamine
(Q), arginine (R), serine (S), threonine (T) and tyrosine (Y); and
(ii) it comprises ten or more amino acids. In a related embodiment,
the accessory polypeptide consists of three types of amino acids,
and each type being selected from a group consisting of D, E, G, K,
P, R, S, and T. In another related embodiment, the accessory
polypeptide consists of three types of amino acids, and each type
being selected from a group consisting of E, S, G, R, and A. In
another related embodiment, the accessory polypeptide consists of
three types of amino acids, and each type being selected from a
group consisting of E, S, G, R, and A. In yet another embodiment,
the accessory polypeptide consists of three types of amino acids,
and each type being selected from a group consisting of E, G, and
S. The isolated polypeptide may be a therapeutic polypeptide.
[0052] The invention also provides for isolated polypeptides
comprising a biologically active polypeptide and an accessory
polypeptide, wherein the accessory polypeptide is characterized in
that: (i) is poly-serine, and (ii) it comprises ten or more amino
acids. In a related embodiment, the isolated polypeptide (i)
consists of two types of amino acids, the majority of which are
serine, and (ii) it comprises ten or more amino acids.
[0053] In another embodiment, the accessory polypeptide consists of
two types of amino acids, one of which is glycine (G) and the other
type is selected from the group consisting of aspartic acid (D),
glutamic acid (E), lysine (K), proline (P), Arginine (R), Serine
(S), Threonine (T), alanine (A), histidine (H), asparagine (N),
tyrosine (Y), leucine (L), valine (V), tryptophan (W), methionine
(M), phenylalanine (F), isoleucine (I), and cysteine (C); and (ii)
it comprises ten or more amino acid residues, of which 50% or less
are glycine.
[0054] The invention also provides for isolated polypeptides
comprising a biologically active polypeptide and an accessory
polypeptide, wherein the accessory polypeptide is characterized in
that: (i) it consists of two types of amino acids, one of which is
serine (S) and the other type is selected from the group consisting
of aspartic acid (D), glutamic acid (E), lysine (K), proline (P),
Arginine (R), Glycine (G), Threonine (T), alanine (A), histidine
(H), asparagine (N), tyrosine (Y), leucine (L), valine (V),
tryptophan (W), methionine (M), phenylalanine (F), isoleucine (I),
and cysteine (C); and (ii) it comprises ten or more amino acid
residues, of which 50% or more are serine.
[0055] Alternatively, the invention describes an isolated
polypeptide comprising a biologically active polypeptide and an
accessory polypeptide, wherein the accessory polypeptide is
characterized in that: (i) it comprises ten or more amino acids;
(ii) it consists of two types of amino acids, wherein 50% or less
of the total amino acids are selected from the group consisting of
A, S, T, D, E, and H.
[0056] In yet another embodiment, the invention describes an
isolated polypeptide comprising a biologically active polypeptide
and an accessory polypeptide, wherein the accessory polypeptide is
characterized in that: (i) it comprises ten or more amino acids;
(ii) it consists of two types of amino acids, 50% or less of the
total amino acids are selected from the group consisting of A, G,
T, D, E, and H.
[0057] In some embodiments, an isolated polypeptide is provided
comprising a biologically active polypeptide and an accessory
polypeptide, wherein the accessory polypeptide is characterized in
that: (i) it consists of two types of amino acids, one of which is
selected from the group consisting of P, R, L, V, Y, W, M, F, I, K,
and C; and (ii) it comprises ten or more amino acids.
[0058] In other embodiments, an isolated polypeptide is provided
comprising a biologically active polypeptide and an accessory
polypeptide, wherein the accessory polypeptide comprises at least
10 amino acids in length and consists of two different types of
amino acids represented in equal numbers. Alternatively, the two
different types of amino acids are represented in 1:2, 2:3, or 3:4
ratio. The accessory polypeptide may additionally comprise four or
more repeating motifs, each of which comprises two to five hundred
amino acids and is made of two different types of amino acids. The
repeating motif may comprise more than 8 amino acids, and in some
embodiments four or more of the repeating motifs are identical. The
four or more repeating motifs may comprise different amino acid
sequences. In a related embodiment, the accessory polypeptide
comprises at least ten repeating motifs.
[0059] Yet other embodiments provide biologically active
polypeptides modified with accessory polypeptides which
substantially lack secondary structure. Alternatively, the apparent
molecular weight of the isolated polypeptides is greater than that
of a corresponding polypeptide lacking the accessory polypeptide.
In a particular embodiment, the apparent molecular weight of the
accessory polypeptide is at least 3 times greater than its actual
molecular weight. In still other embodiments, the accessory
polypeptide exhibits a two-fold longer serum half-life as compared
to a corresponding polypeptide lacking the accessory polypeptide.
The biologically active polypeptide and the accessory polypeptide
may be linked via a peptide bond.
[0060] In some embodiments, the accessory polypeptide comprises an
amino acid sequence (GGEGGS)n (SEQ ID NO: 5), wherein n is an
integer of 3 or greater. In other embodiments, the accessory
polypeptide comprises an amino acid sequence (GES)n, wherein G, E,
and S can be in any order and n is an integer of 3 or greater.
Alternatively, the accessory polypeptide comprises an amino acid
sequence (GGSGGE)n, wherein G, E, and S can be in any order and n
is an integer of 3 or greater. In yet another embodiment, the
accessory polypeptide comprises an amino acid sequence
(GGEGGEGGES)n (SEQ ID NO: 6), wherein n is an integer of 1 or
greater. In yet another embodiment, the accessory sequence
comprises an amino acid sequence (GE)n, wherein G and E can be in
any order.
[0061] In some embodiments, the accessory polypeptide comprises an
amino acid sequence (S)n (SEQ ID NO: 7), wherein n is an integer of
10 or greater. In other embodiments, the accessory polypeptide
comprises an amino acid sequence (SSSSSSE)n, wherein E and S can be
in any order and n is an integer of 2 or greater In yet other
embodiments, the accessory polypeptide comprises an amino acid
sequence (SESSSESSE)n, wherein E and S can be in any order and n is
an integer of 3 or greater. In some embodiments, the accessory
polypeptide comprises an amino acid sequence (SSESSSSESSSE)n,
wherein E and S can be in any order and n is an integer of 3 or
greater. In other embodiments, the accessory polypeptide comprises
an amino acid sequence (SSSESSSSSESSSSE)n, wherein E, and S can be
in any order and n is an integer of 3 or greater. In still other
embodiments, the accessory polypeptide comprises an amino acid
sequence (SSSSESSSSSSESSSSSE)n, wherein E and S can be in any order
and n is an integer of 3 or greater.
[0062] The present invention also provides a method of producing an
isolated polypeptide, comprising: a). providing a polynucleotide
sequence encoding the isolated polypeptide of any one of claim 1,
6, 7, 8, or 9; b) causing said polypeptide to be expressed in a
host cell, thereby producing said polypeptide.
[0063] Additionally, a genetic vehicle comprising a nucleic acid
sequence encoding the isolated polypeptides of the invention is
provided. In another embodiment, a host cell is described
expressing the subject polypeptides. Libraries of subject
polypeptides are also envisioned. In a particular embodiment,
libraries of polypeptides are displayed on phage particles.
INCORPORATION BY REFERENCE
[0064] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0066] FIG. 1 is an illustrative representation of an accessory
polypeptide modifying a biologically active protein.
[0067] FIGS. 2 and 3 show possible modules for inclusion in
modified polypeptides of the invention: accessory polypeptide(s),
biologically active polypeptide(s), optional depot module(s) and
optional polymeric matrix or matrices.
[0068] FIG. 4 shows examples of various product configurations.
Modules may be used several times in the same product, for example
to increase affinity of the biologically active protein for its
target, to increase half-life by extending the rPEG module, or to
modify the properties of the depot formulation.
[0069] FIG. 5 presents a specific example of a tetrameric modified
polypeptide comprising a depot module that allows for site-specific
biotinylation. The addition of streptavidin induces the formation
of highly stable, yet non-covalent, modified polypeptide tetramers.
Multivalent polypeptides can also be created by combining multiple
modules into a single protein chain or by chemically linking
multiple protein chains containing a specific module.
[0070] FIG. 6 illustrates a lysine- or arginine-rich depot module
(depicted as rectangles) which may be incorporated into the polymer
matrix of an alginate microsphere. The matrix module is depicted as
larger circles. The lysine- or arginine-rich depot will carry a net
positive charge at physiological pH and this property can be
exploited to bind the modified polypeptide to the negatively
charged alginate polymer. Binding may occur in a multivalent
fashion.
[0071] FIG. 7 illustrates a divalent cation chelating hydrogel
(matrix module) exemplified by the divalent cation Cu.sup.2+ bound
to the polymer. The polyhistidine depot module (rectangular module)
binds with high affinity to the Cu.sup.2+ cations.
[0072] FIG. 8 depicts a protease sensitive multimeric modified
polypeptide. The depot module (depicted by a rectangle) connects
individual modified polypeptide units in an extended polymer. The
depot module is designed such that it is specifically sensitive to
serum proteases. Protease cleavage of the depot module releases
individual active modified polypeptides.
[0073] FIG. 9 shows the design of the expression vector pCW0150.
FIG. 9 discloses the "6.times.His-tag" and "H6" sequence as SEQ ID
NO: 1.
[0074] FIG. 10 shows the design and construction of the accessory
polypeptide rPEG(L288) fused to GFP. FIG. 10 discloses the "H6"
sequence as SEQ ID NO: 1.
[0075] FIG. 11 shows the amino acid (SEQ ID NO: 485) and nucleotide
sequence (SEQ ID NO: 484) of the rPEG_L288 polypeptide.
[0076] FIG. 12 shows the design of hGH-rPEG(L288) and
GLP-1-rPEG(L288) constructs.
[0077] FIG. 13 shows examples biologically active proteins
conjugated to accessory polypeptides (SEQ ID NOS 486-489,
respectively, in order of appearance).
[0078] FIGS. 14 and 15 describe exemplary guidelines for sequence
optimization of accessory polypeptides. FIG. 15 discloses SEQ ID
NOS 490-498, respectively, in order or appearance.
[0079] FIG. 16 describes the construction of a vector comprising
the rPEG_J288 accessory polypeptide sequence fused to GFP. FIG. 16
discloses the "H6" sequences as SEQ ID NO: 1.
[0080] FIG. 17 shows the amino acid (SEQ ID NO: 500) and nucleotide
sequence (SEQ ID NO: 499) of the rPEG_J288 polypeptide.
[0081] FIG. 18 shows the design of a stuffer vector suitable for
use in the present invention. FIG. 18 discloses the amino acid
sequences as SEQ ID NOS 502 and 503, respectively, the nucleotide
sequence as SEQ ID NO: 501 and the "6.times.His-tag" as SEQ ID NO:
1.
[0082] FIG. 19 shows the purification of rPEG_J288-modified
GFP.
[0083] FIG. 20 shows the determination of serum stability of
rPEG_J288-modified GFP.
[0084] FIG. 21 shows the interaction of an accessory-modified
polypeptide with a cellular target.
[0085] FIG. 22 illustrates the concept of crosslinked accessory
polypeptides.
[0086] FIG. 23 describes examples of crosslinking components.
[0087] FIG. 24 lists several examples of crosslinked accessory
polypeptides.
[0088] FIG. 25 shows an example wherein streptavidin is used as a
linker
[0089] FIG. 26 describes different modalities of constructing
crosslinked accessory polypeptides.
[0090] FIG. 27 identifies illustrates several possible formats of
crosslinked accessory polypeptides.
[0091] FIG. 28 describes accessory polypeptides additionally
modified with binding domains or other groups
[0092] FIG. 29 illustrates the concept of slow-release accessory
polypeptides.
[0093] FIG. 30 shows universal accessory polypeptides. FIG. 30
discloses the "KKKKKK" sequences as SEQ ID NO: 504.
[0094] FIG. 31 shows an antibody Fc fragment from human IgG1, but
this could also be from IgG2, IgG3, IgG4, IgA, IgD or IgE. This Fc
can have a native hinge from IgG1, IgG2, IgG3, IgG4, IgA, IgD or
IgE. There is natural diversity in the number of hinge disulfides,
but this can also be created by mutation, deletion, or truncation
of the hinge, especially the cysteine residues. The variants that
are useful have either three disulfides (not shown), two
disulfides, one disulfide (choice of first one or second natural
one of IgG1) or no disulfides.
[0095] FIG. 32 illustrates various configurations of modified
polypeptides comprising affinity tags, solubility tags and/or
protease cleavage sites.
[0096] FIG. 33 illustrates improved expression levels of modified
polypeptides using specific accessory polypeptides.
[0097] FIG. 34 illustrates shows activity of an accessory-modified
hGH polypeptide relative to unmodified hGH.
[0098] FIG. 35 shows purification of accessory-modified
polypeptides by anion exchange and size exclusion
chromatography.
[0099] FIG. 36 shows pure product obtained by purification of
rPEG-modified GFP as confirmed by SDS-PAGE.
[0100] FIG. 37 shows the purity of rPEG-linked GLP1 as ascertained
by analytical size exclusion chromatography.
[0101] FIG. 38 shows the purity of rPEG_L288-GFP modified
polypeptide as observed by analytical reverse-phase HPLC.
[0102] FIG. 39 Mass spectrometry of rPEG_J288-GFP
[0103] FIG. 40 demonstrates that little nonspecific binding is
observed between modified polypeptides and serum proteins.
[0104] FIG. 41 describes the increase in apparent molecular weight
observed upon linking a biologically active polypeptide to an
accessory polypeptide.
[0105] FIG. 42 shows the stability of modified polypeptides in rat
and human serum.
[0106] FIG. 43 illustrates a PK profile of rPEG_K288-GFP
polypeptide in rat serum.
[0107] FIG. 44 describes shows the relative lack of immunogenicity
of rPEG polypeptides as determined in animal experiments for
rPEG_J288-GFP, rPEG_K288-GFP and rPEG_L288-GFP.
[0108] FIG. 45 illustrates the advantage of expressing biologically
active polypeptides linked to accessory polypeptides.
[0109] FIG. 46 illustrates sustained release of accessory-modified
polypeptides.
[0110] FIG. 47 shows the purity of rPEG_J288-GLP1 polypeptide as
determined by size exclusion chromatography (multiple injections
per run).
[0111] FIG. 48 shows the purity of rPEG_K288-GLP1 polypeptide as
determined by size exclusion chromatography (multiple injections
per run).
[0112] FIG. 49 describes the increase in apparent molecular weight
observed upon linking a biologically active polypeptide (GLP1) to
rPEG_J288, rPEG_K288, and rPEG_L288 accessory polypeptides.
[0113] FIG. 50 shows the products obtained through protease
cleavage of a polypeptide comprising an affinity tag, an accessory
polypeptide and hGH as a biologically active polypeptide
(rPEG_K288-hGH). The protease removes the Tag, while leaving a
final product which is hGH linked to the rPEG_K288 accessory
polypeptide.
[0114] FIG. 51 shows the purity of rPEG_K288-hGH after protease
cleavage and further purification.
[0115] FIG. 52 shows the structure of a whole IgG1, but IgG2, IgG3,
IgG4, IgE, IgD, IgA and IgM can similarly be used as starting
points. A dAb-dAb-Fc fusion protein is also useful because of its
tetravalency; it is not shown.
[0116] FIG. 53 Constructs are shown with rPEG separating the Fc and
antigen binding domains, and the Fc at the C-terminus:
(dAb/scFv)-rPEG-Fc and (dAb/scFv)-(dAb/scFv)-rPEG-Fc. However,
formats with a different order of the same elements are also
useful, like rPEG-Fc-(dAb/scFv), rPEG-Fc-(dAb/scFv)-(dAb/scFv),
Fc-rPEG-(dAb/scFv), Fc-rPEG-(dAb/scFv)-(dAb/scFv),
Fc-(dAb/scFv)-rPEG, Fc-(dAb/scFv)-(dAb/scFv)-rPEG,
dAb/scFv)-Fc-rPEG, and (dAb/scFv)-(dAb/scFv)-Fc-rPEG. One can also
mix scFv and dAbs, like dAb-scFv or scFv-dAb or combine two scFvs
or two dAbs of different target specificities: scFv1-scFv2 or
dAb1-dAb2.
[0117] FIG. 53a shows a scFv-Fc fusion protein. FIG. 53b shows a
dAb-Fc fusion protein. FIG. 53c shows a scFv-scFv-Fc fusion
protein, which is tetravalent.
[0118] FIG. 54 shows a dimer of a scFv fragment. Both heterodimers
and homodimers can be constructed.
[0119] FIG. 55 single chain diabody
[0120] FIG. 56 shows an example of a single chain Fc fragment.
Optionally, biologically active proteins can be fused to either
terminus of this construct.
[0121] FIG. 57 Products consisting of a single copy of a protein
chain
[0122] FIG. 58: Structure of AFBTs. 58a: Monovalent AFBT; 58b:
Structure of a bispecific AFBT
[0123] FIG. 59: Multivalent binding of an AFBT to a target
antigen
[0124] FIG. 60a: Multivalent AFBT containing antibody fragments
derived from two parent antibodies;
[0125] FIG. 60b: Structure of an AFBT comprising a diabody and a
payload
[0126] FIG. 61: Preparation of a semisynthetic AFBT
[0127] FIG. 62: Purification, characterization and binding activity
of an anti Her-2 scFv fused to rPEG50. 62a: binding activity.
Filled diamonds: binding to coated Her-2; open diamonds: binding to
coated IgG. 62b: Size exclusion chromatography; 62c: Detection of
free SH groups.
[0128] FIG. 63: Purification, characterization and binding activity
of an anti Her-2 diabody, aHer203-rPEG50. 63a: binding activity.
Filled diamonds: binding to coated Her-2; open diamonds: binding to
coated IgG. 63b: Size exclusion chromatography of diabody
aHer203-rPEG50 and scFv aHer230-rPEG50; 63c: SEC of aHer203-rPEG50
over time shows no increase in higher multimers.
[0129] FIG. 64: Construction, sequence, and expression of
scFv-rPEG50 fusion proteins. 64a: Cartoon of the protein
architecture (FIG. 64a discloses the "His6 tag" sequence as SEQ ID
NO: 1); 64b: sequence (SEQ ID NO: 505) of an AFBT with specificity
for Her-2; 64c: SDS/PAGE showing the expression of scFv-rPEG50
fusion proteins; 7d: sequence (SEQ ID NO: 506) of an AFBT with
specificity for EGFR.
[0130] FIG. 65: Construction, sequence, and expression of a
diabody-rPEG50 fusion proteins, aHer203-rPEG. 65a: Cartoon of the
protein architecture (FIG. 65a discloses the "His6 tag" as SEQ ID
NO: 1); 65b: protein sequence (SEQ ID NO: 507); 65c: SDS/PAGE
demonstrating the expression of fusion protein in the cytosol of E.
coli.
[0131] FIG. 66: Codon optimization of an Fc domain for bacterial
expression: 66a: Illustration of the process and oligonucleotide
design. The sequence encoding the human Fc was assembled from
semi-random oligonucleotides and cloned in front of rPEG25 and GFP
that served as reported. 66b: SDS/PAGE of clones that were selected
from the library. The arrow indicates the band of the desired
fusion protein. 66c. Amino acid (SEQ ID NO: 509) and nucleotide
sequence (SEQ ID NO: 508) of and optimized human Fc gene.
[0132] FIG. 67: Cartoon illustrating expression constructs for
Fab-rPEG fusion proteins.
[0133] FIG. 68: Flow chart of the discovery process for AFBTs from
antisera.
[0134] FIG. 69: Amino acid sequence (SEQ ID NO: 510) of GFP-rPEG50.
The sequence of GFP is underlined.
[0135] FIG. 70: Pharmacokinetics of GFP-rPEG50 and Ex4-rPEG50 in
cynomologos monkeys.
[0136] FIG. 71a: Amino acid sequence (SEQ ID NO: 511) of the
CDB-Ex4-rPEG50 fusion protein. FIG. 71b: Illustration of the
process used to liberate Ex4-rPEG50 from the fusion sequence shown
in FIG. 14a.
[0137] FIG. 72: Immunogenicity of Ex4-rPEG50 in mice. FIG. 72a
illustrates the time course of injections and blood sample
analyses. FIG. 72b shows ELISA analyses of blood samples at 1:500
dilution. FIG. 72c shows ELISA analyses of blood samples at
1:12,500 dilution.
[0138] FIG. 73: Size exclusion chromatography of GFP-rPEG25 and
GFP-rPEG50. Grey line indicates molecular weigh standard using
globular proteins.
[0139] FIG. 74: Comparison of the interaction of repetitive and
non-repetitive URPs with B cells. FIG. 74a shows a repetitive URP
that is composed of multiple identical sequence repeats. Such a
repetitive URP can form multivalent contacts with B cells that
recognize the repeating sequence, which can trigger B cell
proliferation. FIG. 74b shows a non-repetitive URP that is composed
of multiple different subsequences. Each subsequence can be
recognized by a particular subset of B-cells with cognate
specificity. However, an individual molecule of a non-repetitive
URP can only form one or few interactions with any particular B
cell, which is unlikely to trigger proliferation.
[0140] FIG. 75: Algorithm to assess the repetitiveness of an amino
acid sequence.
[0141] FIG. 76: Computer algorithm to design nrURPs with very low
repetitiveness.
[0142] FIG. 77: Construction of nrURPs from libraries of URP
segments.
[0143] FIG. 78: Amino acid sequences (SEQ ID NOS 15, 18, 16, 19,
17, 20, 11, 13, 12 and 14, respectively, in order of appearance)
that were used to construct rPEG_Y. The figure also indicates the
relative concentrations of oligonucleotides that were used to
construct the segment libraries.
[0144] FIG. 79: Assembly of URP segments from synthetic
oligonucleotides. FIG. 79a shows the ligation reaction. Repeating
segments are encoded by partially overlapping oligonucleotides that
are phosphorylated. A second pair of annealed oligonucleotides is
added to terminate chain elongation. One of these capping
oligonucleotides is not phosphorylated, which prevents ligation at
one end. FIG. 79b shows an agarose gel of a ligation reaction.
[0145] FIG. 80: Examples of URP_Y144 sequences (SEQ ID NOS 512-521,
respectively, in order or appearance).
[0146] FIG. 81: Amino acid sequence (SEQ ID NO: 522) encoded by
plasmid pCW0279. The open reading frame encodes a fusion protein of
Flag-URP_Y576-GFP. The amino acid sequence of URP_Y576 is
underlined.
[0147] FIG. 82 shows general ways of making `rPEG linked binding
pairs`, which have the advantage of no initial activity and
therefore no burst release effect (increasing the dose that can be
administered without causing toxicity) and reduced initial
receptor-mediated clearance. The general binding pairs can be
receptor-ligand, antibody-ligand, or generally binding protein
1-binding protein 2. The construct can have a cleavage site, which
can be cleaved before injection, after injection (in serum by
proteases) and can be located such that the rPEG stays with the
therapeutic product end (active protein), which can be either the
ligand, the receptor or the antibody.
[0148] FIG. 83a shows a construct with a drug module at the
N-terminus, followed by rPEG, fused to an antibody Fc fragment,
including the hinge. The Fc fragments provides long halflife and
the rPEG allows the Fc fragment to be expressed in the E. coli
cytoplasm in soluble and active form.
[0149] FIG. 83b shows a construct with a drug module at the
N-terminus, followed by rPEG, fused to an antibody Fc fragment, but
without the hinge. The Fc fragments provides long halflife and the
rPEG allows the Fc fragment to be expressed in the E. coli
cytoplasm in soluble and active form.
[0150] FIG. 84a A Diabody is formed when the single chain linker
between the VH and VL domain is shorter than about 10-20 AA,
preventing the formation of a single chain Fv fragment. A diabody
has two protein chains and can have an rPEG at one or both
C-terminal ends, and/or at one or both N-terminal ends. The diabody
has two binding sites, of which zero, one or two may bind to a
pharmaceutical target, or to a halflife target (ie HSA, IgG, Red
Blood Cells, Collagen, etc) or to no target.
[0151] FIG. 84b The diabody may contain zero, one or more drug
modules located at the N-terminal or C-terminal end of zero, one or
both protein chains.
[0152] FIG. 85a shows a single chain Fv fragment, to which a drug
module (like IFNa, hGH, etc) can be fused at one or both of the N-
and/or C-terminal ends. The scFv has one binding site, which may or
may not bind to a pharmaceutical target, or to a halflife target
(ie HSA (see FIG. 85b), IgG, Red Blood Cells, etc)
[0153] FIG. 86 shows the use of rPEG to associate two proteins that
belong to the same complex. The affinity between such proteins is
often insufficient to keep them associated, but the addition of
rPEG stabilizes their interaction and reduces their tendency to
form polymers.
[0154] FIG. 87 shows a Fab fragment binding to a cell-surface
target; the H chain may be fused to Fc (like in whole antibodies)
or to a wide variety of other proteins, domains and peptides.
Extension of the length of the natural linkers from the usual
2-6-amino acids to 4, 5, 6, 7, 8, 9, 10, 11, 1, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 100
or more amino acids, between the VH and the CH domains, and between
the VL and the CL domains, increases the ability of one Fab to
crosslink to another Fab by domain swapping, thereby forming a
binding complex with higher valency, resulting in higher apparent
affinity (avidity). The linker may be rPEG or a different
composition. This `Extended Linker` format allows binding with
increased affinity specifically at sites with a higher density of
target, such as (partially) tumor-specific antigen on tumor
cells.
[0155] FIG. 88 shows how an association peptide, such as SKVILF(E)
(SEQ ID NO: 8) or RARADADA (SEQ ID NO: 9), which bind to another
copy of the same sequence in an antiparallel orientation, can be
used to create a prodrug. In this case the drug is protease-cleaved
in the last manufacturing step, but the cleavage does not activate
the drug since the two chains are still associated by the
association peptides. Only after the drug is injected into the
blood and the concentration is greatly reduced, the small,
non-rPEG-containing protein chain will leave the complex (at a rate
that depends on affinity, especially the off-rate) and is likely to
be cleared via the kidney, thereby activating the r-PEG-containing
drug module.
[0156] FIG. 89 shows the proteolytic cleavage which converts the
manufactured single-chain protein into a complex of two protein
chains. This cleavage can occur as the last manufacturing step
(before injection) or it can occur after injection, by proteases in
the patient's blood.
[0157] FIG. 90a shows an antibody Fc fragment, with a hinge region,
(optionally) fused to a drug module (e.g. IFNa, hGH, etc.) on one
end and (optionally) fused to rPEG on the other end. The sequence
between CH2 and CH3 mediates binding to FcRn, the neonatal Fc
receptor, unless that function is removed by mutation. FIG. 90b
shows a similar construct but without the hinge region.
[0158] FIG. 91a shows a protein construct comprising a paired pair
of CH3 domains; zero, one or both of these chains may be fused to
rPEG on the N-terminal and/or C-terminal end, and to zero, one or
more drug modules at the other end. The FcRn binding sequence can
either be retained or deleted; retention should yield a longer
serum halflife.
[0159] FIG. 91b shows a similar protein, but CH2 was fully removed
so that the binding of the Fc to the FcRn receptor is no longer
functional, reducing halflife.
[0160] FIG. 92a shows a protein that is a full Fc, including a
hinge, CH2 and CH3 domains, fused at the c-terminus to an rPEG,
with the drug/pharmacophore located at the C-terminus.
[0161] FIG. 92b shows a full Fc, but without a hingefused at the
c-terminus to an rPEG, with the drug/pharmacophore located at the
C-terminus; these molecules can chain swap, potentially resulting
in hetero-dimers.
[0162] FIG. 93a shows a partial Fc, without hinge and with a CH2
that is truncated but retains FcRn binding and with the
drug/pharmacophore located at the C-terminus.
[0163] FIG. 93b shows a partial Fc, without hinge and CH2, but
retaining CH3 and with the drug/pharmacophore located at the
C-terminus. This does not bind FcRn but can dimerize via the CH3
domain.
[0164] FIG. 94a shows an rPEG flanked by identical receptor domains
(or domains having the same binding function, or domains that can
bind simultaneously to the same target). If both receptors can bind
the target simultaneously, then the binding of one receptor
stabilizes binding of the second receptor and the
effective/apparent affinity/avidity is increased, typically by
10-100-fold, but at least 3-fold. The rPEG provides serum halflife.
One option is to pre-load the product with a ligand. In that case
the injected product is inactive for as long as it remains bound to
the ligand. This approach reduces peak dose toxicity and also
reduces receptor-mediated clearance and may thus be useful in
application where this is important.
[0165] FIG. 94b shows a product with rPEG flanked by two different
receptors that can bind the ligand simultaneously, which results in
mutual stabilization of the complex and increased apparent affinity
(avidity), with the rPEG serving as a valency bridge that increases
the effective concentration of the receptors.
[0166] FIG. 94c One option is to pre-load the product with a
ligand. In that case the injected product is inactive for as long
as it remains bound to the ligand. When the ligand un-binds, it is
likely to be rapidly cleared via the kidney, resulting in
activation of the product, which has a long halflife because of the
rPEG tail. This approach reduces peak dose toxicity and also
reduces receptor-mediated clearance and may thus be useful in
application where this is important.
[0167] As shown in FIG. 94, some pro-drug formats do not need a
cleavage or other activation site. A single protein chain can
contain two (or more) drug modules separated by rPEG; these modules
can be the same (of a single type) or of two or more different
types. All drug modules are receptor or all are ligand. This rPEG
containing product is complexed with a second, complementary
protein to form a receptor-ligand-receptor interaction. In this
format the ligand is likely to be dimeric or multimeric, but can
also be monomeric, especially if the two drug modules are
different. Both modules bind to a third protein. X and Y can be the
same or different, and X and Y can be the drug module or bind to
the drug module. In each case in FIG. 94, X and Y (and rPEG)
comprise one protein chain, and the molecule they bind to is a
separate molecule, typically protein or small molecule. It is
possible to have more than two binding proteins combined in a
single protein chain. The idea is that the complex of a large
rPEG-containing protein and a non-rPEG containing protein is
inactive when injected, but over 2-24 hours the smaller,
non-rPEG-containing protein leaves the complex and is excreted via
the kidney, thereby activating the drug module(s). The benefit of
this format is that is reduces or removes the initial spike in drug
concentration and the associated safety issues, and that the
complex minimizes the receptor-mediated clearance while it is
complexed, thereby extending the serum secretion halflife.
[0168] FIG. 95 shows an rPEGs flanked on both sides by a
VEGF-receptors. Since VEGF is dimeric, this can be the same
receptor on both sides of the rPEG, or a different receptor
(preferably VEGF-R1 and VEGF-R2, but VEGFR3 can also be used.
[0169] FIG. 96 shows products that are either manufactured (cleaved
before injection) or administered as an inactive pro-drug (cleaved
after injection, in the blood). The inactivation of the drug is
mediated by a binding protein that is linked to the drug by rPEG,
so that all three modules are manufactured as a single protein
chain. If the drug is a receptor, then the binding protein is a
ligand (peptide or protein) of that receptor; if the drug is an
antibody fragment, then the binding site is a peptide or protein
ligand. In these examples, the drug is activated by protease
cleavage of a site between the two binding domains, called X and Y.
If Protein Y is the active product, then Y must retain the rPEG and
the protease cleavage site must be (between X and Y, but) close to
X. If Protein X is the active product, then X must retain the rPEG
and the cleavage site must be close to Y. There can be one or
multiple cleavage sites, as shown by the blue crossbars. The drug
module can be a receptor, a ligand, one or more Ig domains, an
antibody fragment, a peptide, a microprotein, an epitope for an
antibody. The protein that binds to the drug module can be a
binding protein, a receptor, a ligand, one or more Ig domains, an
antibody fragment, a peptide, a microprotein, an epitope for an
antibody. FIG. 96 discloses the "SVILF" sequence as SEQ ID NO: 524
and the "RARADADA" sequence as SEQ ID NO: 9.
[0170] FIG. 97 shows how an inactive pro-drug can be created by
adding a binding peptide to a drug module. The peptide must
neutralize the target binding capacity of the drug and the peptide
is gradually cleared from the blood at a higher rate than the
rPEG-containing drug. Such a peptide can be natural but more
typically it would be obtained by phage panning of random peptide
libraries against the drug module. The peptide would preferably be
made synthetically, but it can be recombinant.
[0171] FIG. 98 shows a single-chain protein drug containing
multiple bio-active peptides, which can be at the same end of rPEG
or at opposite ends of rPEG. These peptides can have the same
activity or different activities. The purpose of having multiple
peptides in a single chain is to increase their effective potency
through binding avidity, without complicating manufacturing.
[0172] FIG. 99 shows how a Pro-drug-rPEG can increase serum
halflife by avoiding receptor-mediated clearance.
[0173] FIG. 100 shows how drug concentration changes over time
after IV injection. The goal in typical therapies is maintain the
drug at a concentration that is higher than the therapeutic does,
but lower than the toxic dose. A typical bolus injection (IV, IM,
SC, IP or similar) of a drug with a short halflife results in a
peak concentration that is much higher than the toxic dose,
followed by an elimination phase that causes the drug concentration
to rapidly drop below the therapeutic dose. This PK profile tends
to cause toxicity and long periods of ineffective treatment, while
the drug is present at therapeutic concentrations for only short
time (blue line). The addition of rPEG to a drug decreases the peak
concentration and thereby decreases toxicity, and increases the
period of time that the drug is present at a therapeutic, non-toxic
dose. The creation of a Pro-drug by addition of rPEG plus a
drug-binding protein can prevent the `burst release` or toxic peak
dose (red line), because the drug is only gradually activated over
several hours. and the length of time between the toxic dose and
the therapeutic dose is increased compared to other formats.
[0174] FIG. 101 shows an N-terminal drug module followed by rPEG
and a C-terminal Fc fragment (with hinge). This is a useful format
for halflife extension of drug modules that can still be
manufactured in the E. coli cytoplasm.
[0175] FIG. 102a shows an alternative format for a Pro-drug
containing an Fc fragment. The format is similar as described in
FIG. 101, with the addition (at the N-terminus) of an inhibitory
sequence (in blue) that binds to and inhibits the drug sequence (in
red). As before, the drug is separated from the inhibitory sequence
by a cleavage site. The N-terminal inhibitory binding sequence is
followed by a cleavage site, which is followed by the drug sequence
(in red). Before cleavage, the drug is bound to the inhibitory
sequence and thus inactive (pro-drug). After cleavage, the
inhibitory binding sequence (blue) is gradually released and
cleared, gradually increasing the amount of time that the drug
(red) is active.
[0176] FIG. 102b. shows an alternative Pro-drug format containing
an Fc fragment. The formats is similar to the format described in
FIG. 101, again with the addition of an inhibitory binding sequence
(peptide or domain, shown in red, typically positioned in or near
the rPEG) which is separated from the drug (shown in blue) by a
cleavage site. Before cleavage, the drug is bound to the inhibitory
sequence and thus inactive (pro-drug). After cleavage, the
inhibitory binding sequence (blue) is gradually released and
cleared, gradually increasing the amount of time that the drug
(blue) is active.
[0177] FIG. 103a-d shows the preferred fusion Sites for rPEG to an
intact, Whole Antibody (incl. IgG1, 2, 3, 4, IgE, IgA, IgD, IgM).
These sites indicated are preferred because they are at the
boundary of structured sequences, such as domains, hinges, etc,
without disturbing the folding of these functional domains. rPEG
can thus be added in 1, 2, 3, 4, 5, 6, 7 or even 8 different
locations to an antibody (and more than 8 for IgM and IgG3) and a
single antibody can have 1, 2, 3, 4, 5, 6, 7, 8 or more rPEGs in
diverse locations and in any combination of the 8 locations
shown.
[0178] FIG. 103e shows the Preferred Fusion Sites for rPEG to
Domains and Fragments of an Antibody (IgG1, 2, 3, 4, IgE, IgA, IgD,
IgM). Fusion sites for N-terminal and/or C-terminal addition of
rPEG are shown with red arrows or red lines.
[0179] FIG. 104 shows assays for correct folding of Fc
fragments.
[0180] FIG. 105 shows the conversion of an inactive protein to an
active protein by a sitespecific protease, either in serum or
before injection. In this example the red sequence is the active
therapeutic.
[0181] FIG. 106 shows the conversion of an inactive drug to an
active drug by a sitespecific protease. In this example the blue
domain (dAb, scFv, other) is the therapeutic entity.
DETAILED DESCRIPTION OF THE INVENTION
[0182] The present invention makes use of the unexpected discovery
that biologically active polypeptides modified with accessory
polypeptides may have the property of remaining soluble in the
cytoplasm and folding into their active form, in conditions in
which a biologically active polypeptide without such a modification
would aggregate and form inclusion bodies. The methods of the
invention may be useful for, among other applications, high
throughput screening of proteins in the design phase, the
manufacturing of proteins that currently require periplasmic
expression, and for manufacturing of proteins that are difficult to
refold from aggregates such as including inclusion bodies. The
invention discloses methods of designing accessory protein
sequences, recombinant DNA molecules encoding modified polypeptide,
expression vectors for such polypeptides, host cells for expression
of such polypeptides and purification processes. For example, the
fusion of a long hydrophilic polypeptide sequence to proteins,
which may include peptides, proteins, antibodies, and vaccines, and
may be eukaryotic or mammalian proteins, results in a soluble
fusion protein showing improved folding in the cytoplasm in active
form.
[0183] Accessory polypeptides of the invention may be linked to
pharmaceutical proteins including GCSF, growth hormone, interferon
alpha and to antibody fragments. These four proteins or classes of
proteins typically form inclusion bodies when expressed in the
cytoplasm of E. coli. However, when linked to a long hydrophilic
accessory polypeptide sequence, the folding properties of the
biologically active polypeptides may be greatly improved, leading
to a greatly increased fraction able to fold correctly into active
protein within the cell, as opposed to immediate and irreversible
aggregation into inclusion bodies which typically occurs for
eukaryotic proteins in the absence of an accessory protein.
Accessory polypeptides may additionally comprise affinity tags for
protein purification by ion exchange, alone or in combination with
other known purification tags, such as chitin binding domain,
cellulose binding domain, MBP, GST or His-tags.
[0184] This and other aspects of the invention will be described in
further detail below.
[0185] General Techniques:
[0186] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of immunology,
biochemistry, chemistry, molecular biology, microbiology, cell
biology, genomics and recombinant DNA, which are within the skill
of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:
A LABORATORY MANUAL, 2.sup.nd edition (1989); CURRENT PROTOCOLS 1N
MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series
METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL
APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds.
(1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY
MANUAL, and ANIMAL CELL CULTURE (R1. Freshney, ed. (1987)).
DEFINITIONS
[0187] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0188] The terms "polypeptide", "peptide", "amino acid sequence"
and "protein" are used interchangeably herein to refer to polymers
of amino acids of any length. The polymer may be linear or
branched, it may comprise modified amino acids, and it may be
interrupted by non-amino acids. The terms also encompass an amino
acid polymer that has been modified, for example, by disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation, such as conjugation with a labeling
component. As used herein the term "amino acid" refers to either
natural and/or unnatural or synthetic amino acids, including but
not limited to glycine and both the D or L optical isomers, and
amino acid analogs and peptidomimetics. Standard single or three
letter codes are used to designate amino acids.
[0189] The term "biologically active polypeptide" refers to a
polypeptide of any length that exhibits binding specificity to a
given target or targets, which can be a therapeutic target and/or
an accessory target, such as for cell-, tissue- or organ targeting.
Alternatively, or in addition, it refers to a polypeptide that
exhibits a desired biological characteristic when used in vitro or
in vivo. By way of example, biologically active polypeptides
include functional therapeutics or in vivo diagnostic proteins that
bind to therapeutic or diagnostic targets. The term "biologically
active polypeptide" and "Binding Module" or "BM" are used
interchangeably herein. Biologically active polypeptides can be,
for example, and without limitation, linear or cyclic peptides,
cysteine-constrained peptides, microproteins, scaffold proteins
like fibronectin, ankyrins, crystalline, streptavidin, antibody
fragments, domain antibodies, peptidic hormones, growth factors,
cytokines, or any type of protein domain, human or non-human,
natural or non-natural, and they may be based on a natural scaffold
or not based on a natural scaffold (i.e. engineered or selected),
or based on combinations or fragments of any of the above.
Optionally, the biologically active polypeptide can be engineered
by adding, removing or replacing one or multiple amino acids in
order to enhance their binding properties, their stability, or
other desired properties. Binding modules can be obtained from
natural proteins, by design or by genetic package display,
including phage display, cellular display, ribosomal display or
other display methods, for example. Binding modules may bind to the
same copy of the same target, which results in avidity, or they may
bind to different copies of the same target (which can result in
avidity if these copies are somehow connected or linked, such as by
a cell membrane), or they may bind to two unrelated targets (which
yields avidity if these targets are somehow linked, such as by a
membrane). Binding modules can be identified by screening or
otherwise analyzing random libraries of peptides or proteins.
[0190] "Recombinant PEG", "rPEG" or "rPEG polypeptides" or
"recombinant PK Enhancing Group" are general terms encompassing a
class of polypeptides that can be used to modify biologically
active polypeptides, whereby the modification results in a
desirable change in biological properties such as serum half-life
or in vivo clearance. In general, rPEG polypeptides lack binding
specificity to the same given target bound by the biologically
active polypeptide. In some aspects, rPEG is a functional analog of
PEG that, may mimic some, but not necessarily all, well-known
properties of PEG. Such properties, described in more detail below,
include enhanced ability to increase hydrodynamic radius, increased
resistance to proteases, decreased immunogenicity and decreased
specific activity. While rPEG molecules may share broad structural
and functional features with PEG, such as linearity or lack of
tertiary structure, strict chemical similarity with PEG is not a
necessary feature of rPEG.
[0191] "Accessory polypeptide" or "accessory protein" refers to a
polypeptide which, when used in conjunction with a biologically
active polypeptide, e.g. by way of linking to the biologically
active polypeptide, renders a desirable change in biological
properties of the entire linked polypeptide. Non-limiting examples
of accessory polypeptides include rPEGs and any other polypeptides
capable of increasing hydrodynamic radius, extending serum
half-life, and/or modifying in vivo clearance rate. When desired,
an accessory polypeptide causes a small increase in predicated
molecular weight, but a much larger increase in apparent molecular
weight. Although the different names emphasize different features,
they refer to the same module and can be used interchangeably.
[0192] The terms "modified polypeptide" and "accessory-modified
polypeptide" are used interchangeably to refer to biologically
active polypeptides which have been modified with the accessory
polypeptides of the invention. These terms may also refer to slow
release or other types or formulations comprising biologically
active polypeptides modified with accessory polypeptides according
to the invention.
[0193] A "repetitive sequence" or "repetitive motif" are used
interchangeably herein and refer to an amino acid sequence that can
be described as an oligomer of repeating peptide sequences
("repeats"), forming direct repeats, or inverted repeats or
alternating repeats of multiple sequence motifs. These repeating
oligomer sequences can be identical or homologous to each other,
but there can also be multiple repeated motifs. Repetitive
sequences are characterized by a very low information content. A
repetitive sequence is not a required feature of an accessory
polypeptide and in some cases a non-repetitive sequence will in
fact be preferred.
[0194] Amino acids can be characterized based on their
hydrophobicity. A number of scales have been developed. An example
is a scale developed by Levitt, M et al. (see Levitt, M (1976) J
Mol Biol 104, 59, #3233, which is listed in Hopp, T P, et al.
(1981) Proc Natl Acad Sci USA 78, 3824, #3232). Examples of
"hydrophilic amino acids" are arginine, lysine, threonine, alanine,
asparagine, and glutamine. Of particular interest are the
hydrophilic amino acids aspartate, glutamate, and serine, and
glycine. Examples of "hydrophobic amino acids" are tryptophan,
tyrosine, phenylalanine, methionine, leucine, isoleucine, and
valine.
[0195] As used herein, the term "cell surface proteins" refers to
the plasma membrane components of a cell. It encompasses integral
and peripheral membrane proteins, glycoproteins, polysaccharides
and lipids that constitute the plasma membrane. An integral
membrane protein is a transmembrane protein that extends across the
lipid bilayer of the plasma membrane of a cell. A typical integral
membrane protein consists of at least one membrane spanning segment
that generally comprises hydrophobic amino acid residues.
Peripheral membrane proteins do not extend into the hydrophobic
interior of the lipid bilayer and they are bound to the membrane
surface via covalent or noncovalent interaction directly or
indirectly with other membrane components.
[0196] The terms "membrane", "cytosolic", "nuclear" and "secreted"
as applied to cellular proteins specify the extracellular and/or
subcellular location in which the cellular protein is mostly,
predominantly, or preferentially localized.
[0197] "Cell surface receptors" represent a subset of membrane
proteins, capable of binding to their respective ligands. Cell
surface receptors are molecules anchored on or inserted into the
cell plasma membrane. They constitute a large family of proteins,
glycoproteins, polysaccharides and lipids, which serve not only as
structural constituents of the plasma membrane, but also as
regulatory elements governing a variety of biological
functions.
[0198] "Non-naturally occurring" as applied to a protein means that
the protein contains at least one amino acid that is different from
the corresponding wildtype or native protein. Non-natural sequences
can be determined by performing BLAST search using, e.g., the
lowest smallest sum probability where the comparison window is the
length of the sequence of interest (the queried) and when compared
to the non-redundant ("nr") database of Genbank using BLAST 2.0.
The BLAST 2.0 algorithm, which is described in Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information.
[0199] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient for the subject vectors. Host
cells include progeny of a single host cell. The progeny may not
necessarily be completely identical (in morphology or in genomic of
total DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a vector of this invention.
[0200] As used herein, the term "isolated" means separated from
constituents, cellular and otherwise, with which the
polynucleotide, peptide, polypeptide, protein, antibody, or
fragments thereof, are normally associated with in nature. As is
apparent to those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody, or
fragments thereof, does not require "isolation" to distinguish it
from its naturally occurring counterpart. In addition, a
"concentrated", "separated" or "diluted" polynucleotide, peptide,
polypeptide, protein, antibody, or fragments thereof, is
distinguishable from its naturally occurring counterpart in that
the concentration or number of molecules per volume is greater than
"concentrated" or less than "separated" than that of its naturally
occurring counterpart. In general, a polypeptide made by
recombinant means and expressed in a host cell is considered to be
"isolated".
[0201] "Conjugated", "linked" and "fused" or "fusion" are used
interchangeably herein. These terms refer to the joining together
of two more chemical elements or components, by whatever means
including chemical conjugation or recombinant means. An "in-frame
fusion" refers to the joining of two or more open reading frames
(ORFs) to form a continuous longer ORF, in a manner that maintains
the correct reading frame of the original ORFs. Thus, the resulting
recombinant fusion protein is a single protein containing two ore
more segments that correspond to polypeptides encoded by the
original ORFs (which segments are not normally so joined in
nature).
[0202] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminus direction in which residues that neighbor each
other in the sequence are contiguous in the primary structure of
the polypeptide. A "partial sequence" is a linear sequence of part
of a polypeptide which is known to comprise additional residues in
one or both directions.
[0203] "Heterologous" means derived from a genotypically distinct
entity from the rest of the entity to which it is being compared.
For example, a glycine rich sequence removed from its native coding
sequence and operatively linked to a coding sequence other than the
native sequence is a heterologous glycine rich sequence. The term
"heterologous" as applied to a polynucleotide, a polypeptide, means
that the polynucleotide or polypeptide is derived from a
genotypically distinct entity from that of the rest of the entity
to which it is being compared.
[0204] The terms "polynucleotides", "nucleic acids", "nucleotides"
and "oligonucleotides" are used interchangeably. They refer to a
polymeric form of nucleotides of any length, either
deoxyribonucleotides or ribonucleotides, or analogs thereof.
Polynucleotides may have any three-dimensional structure, and may
perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: coding or non-coding
regions of a gene or gene fragment, loci (locus) defined from
linkage analysis, exons, introns, messenger RNA (mRNA), transfer
RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, isolated RNA of any sequence, nucleic acid probes, and
primers. A polynucleotide may comprise modified nucleotides, such
as methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component.
[0205] "Recombinant" as applied to a polynucleotide means that the
polynucleotide is the product of various combinations of cloning,
restriction and/or ligation steps, and other procedures that result
in a construct that can potentially be expressed in a host
cell.
[0206] The terms "gene" or "gene fragment" are used interchangeably
herein. They refer to a polynucleotide containing at least one open
reading frame that is capable of encoding a particular protein
after being transcribed and translated. A gene or gene fragment may
be genomic or cDNA, as long as the polynucleotide contains at least
one open reading frame, which may cover the entire coding region or
a segment thereof. A "fusion gene" is a gene composed of at least
two heterologous polynucleotides that are linked together.
[0207] A "vector" is a nucleic acid molecule, preferably
self-replicating, which transfers an inserted nucleic acid molecule
into and/or between host cells. The term includes vectors that
function primarily for insertion of DNA or RNA into a cell,
replication of vectors that function primarily for the replication
of DNA or RNA, and expression vectors that function for
transcription and/or translation of the DNA or RNA. Also included
are vectors that provide more than one of the above functions. An
"expression vector" is a polynucleotide which, when introduced into
an appropriate host cell, can be transcribed and translated into a
polypeptide(s). An "expression system" usually connotes a suitable
host cell comprised of an expression vector that can function to
yield a desired expression product.
[0208] The "target" as used in the context of accessory
polypeptides is a biochemical molecule or structure to which the
biologically active polypeptide can bind and where the binding
event results in a desired biological activity. The target can be a
protein ligand or receptor that is inhibited, activated or
otherwise acted upon by the t protein. Examples of targets are
hormones, cytokines, antibodies or antibody fragments, cell surface
receptors, kinases, growth factors and other biochemical structures
with biological activity.
[0209] "Serum degradation resistance"--Proteins can be eliminated
by degradation in the blood, which typically involves proteases in
the serum or plasma. The serum degradation resistance is measured
by combining the protein with human (or mouse, rat, monkey, as
appropriate) serum or plasma, typically for a range of days (ie
0.25, 0.5, 1, 2, 4, 8, 16 days) at 37 C. The samples for these
timepoints are then run on a Western assay and the protein is
detected with an antibody. The antibody can be to a tag in the
protein. If the protein shows a single band on the western, where
the protein's size is identical to that of the injected protein,
then no degradation has occurred. The timepoint where 50% of the
protein is degraded, as judged by Western Blots or equivalent
techniques, is the serum degradation half-life or "serum half-life"
of the protein.
[0210] "Apparent Molecular Weight Factor" or "Apparent Molecular
Weight" are related terms referring to a measure of the relative
increase or decrease in apparent molecular weight exhibited by a
particular amino acid sequence. The Apparent Molecular Weight is
determined using a size exclusion column that can be calibrated
using globular protein standards and is measured in "apparent kD"
units. The Apparent Molecular Weight Factor is measured as the
ratio between the apparent molecular weight, as determined on a
size exclusion column calibrated with globular proteins and the
actual molecular weight, (i.e., predicted by adding based on amino
acid composition the calculated molecular weight of each type of
amino acid in the amino acid composition). For example, a 20 kD
poly-Glycine sequence has an apparent molecular weight of 200 kD by
size exclusion chromatography, corresponding to an Apparent
Molecular Weight Factor of 10.times.. The `Specific Hydrodynamic
Radius` is the hydrodynamic radius per unit molecular weight (kD),
is a measure for the performance of a halflife extender, which is
measured as the serum secretion halflife per unit mass (hours per
kD). Both of these measurements are correlated with the `Apparent
Molecular Weight Factor`, which is a more intuitive measure.
[0211] The "hydrodynamic radius" of a protein affects its rate of
diffusion in aqueous solution as well as its ability to migrate in
gels of macromolecules. The hydrodynamic radius of a protein is
determined by its molecular weight as well as by its structure,
including shape and compactness. Most proteins have globular
structures, which is the most compact three-dimensional structure a
protein can have with the smallest hydrodynamic radius. Some
proteins adopt a random and open, unstructured, or `linear`
conformation and as a result have a much larger hydrodynamic radius
compared to typical globular proteins of similar molecular
weight.
[0212] "Physiological conditions" refer to a set of conditions
including temperature, salt concentration, pH that mimic those
conditions of a living subject. A host of physiologically relevant
conditions for use in in vitro assays have been established.
Generally, a physiological buffer contains a physiological
concentration of salt and at adjusted to a neutral pH ranging from
about 6.5 to about 7.8, and preferably from about 7.0 to about 7.5.
A variety of physiological buffers is listed in Sambrook et al.
(1989) supra and hence is not detailed herein. Physiologically
relevant temperature ranges from about 25.degree. C. to about
38.degree. C., and preferably from about 30.degree. C. to about
37.degree. C.
[0213] A "reactive group" is a chemical structure that can be
coupled to a second reactive group. Examples for reactive groups
are amino groups, carboxyl groups, sulfhydryl groups, hydroxyl
groups, aldehyde groups, azide groups. Some reactive groups can be
activated to facilitate coupling with a second reactive group.
Examples for activation are the reaction of a carboxyl group with
carbodiimide, the conversion of a carboxyl group into an activated
ester, or the conversion of a carboxyl group into an azide
function.
[0214] A "crosslinking component" includes a chemical structure
that comprises one or more reactive groups. These reactive groups
can be identical in their chemical structure allowing the direct
construction of crosslinked accessory polypeptides. Cross-linking
components can contain reactive groups that have been blocked by
protecting groups. This allows one to conjugate several different
non-cross-linking components to one cross-linking component in
controlled consecutive reactions. Cross-linking components can
contain multiple reactive groups that differ in their structure and
that can be selectively conjugated with different non-cross-linking
components. Proteins that contain multiple high-affinity binding
sites can also serve as cross-linking agents. Examples are
streptavidin, which can bind up to four molecules of a biotinylated
non-cross-linking component. Branched multifunctional polyethylene
glycol (PEG) molecules can serve as cross-linking components. A
variety of reagents with two to eight functional groups and various
lengths of PEG as well as various reactive groups are commercially
available. Suppliers include NOF America Corporation and
SunBio.
[0215] "Non-crosslinking components" include chemical structures
that comprise reactive groups which allow conjugation to a
cross-linking component. Non-cross-linking components can contain a
variety of modules, including one or more biologically active
polypeptides and/or one or more accessory polypeptides. In
addition, non-crosslinking components can contain affinity tags
that facilitate purification and/or detection, such as Flag-tag,
E-tag, Myc-tag, HA-tag, His6-tag (SEQ ID NO: 1), Green Fluorescent
protein, etc.
[0216] A "crosslinked rPEG polypeptide", "crosslinked accessory
polypeptide", "crosslinked rPEG", "CL-rPEG polypeptide", "CL-rPEG"
are terms referring to conjugates of one or more non-crosslinking
components with a crosslinking component.
[0217] "Controlled release agent", "slow release agent", "depot
formulation" or "sustained release agent" are used interchangeably
to refer to an agent capable of extending the duration of release
of a modified polypeptide of the invention relative to the duration
of release when the modified polypeptide is administered in the
absence of agent. Different embodiments of the present invention
may have different release rates, resulting in different
therapeutic amounts.
[0218] "vL domain" refers to the variable domain of the light chain
of an antibody.
[0219] "vH domain" refers to the variable domain of the heavy chain
of an antibody.
[0220] A "variable fragment" (Fv) refers to a portion of an
antibody which comprises two non-covalently associated VL and VH
domains.
[0221] A "single chain variable fragment" (scFv) refers to a
portion of an antibody which comprises one vH linked via a
non-natural peptide linker to one vL domain, as a single chain.
scFvs can have the structure vH-linker-vL or vL-linker-vH where the
linker can be any peptide sequence comprising various numbers of
amino acids. A scFv preferentially occurs under physiological
conditions as a monomeric structure which requires a peptide linker
of preferably more than 12 amino acids.
[0222] Disulfide-stabilized Fv fragments of antibodies (dFv) refer
to molecules in which the V.sub.H-V.sub.L heterodimer is stabilized
by an interchain disulfide bond engineered between structurally
conserved framework positions distant from
complementarity-determining regions (CDRs). This method of
stabilization is applicable for the stabilization of many antibody
Fvs.
[0223] A "variable domain" refers to the domain that forms the
antigen binding site of an antibody. Variable domains can be vH or
vL; Differences, between the variable domains, are located on three
loops known as hypervariable regions (HV-1, HV-2 and HV-3) or CDR1,
CDR2 and CDR3. CDRs are supported within the variable domains by
conserved framework regions.
[0224] A "domain antibody" (dAb) refers to a portion of an antibody
that is capable of binding the target as a monomer. Domain
antibodies correspond to the variable regions of either the heavy
(VH) or light (VL) chains of antibodies. dAbs do not generally
require a second variable domain (vH or vL) for target binding.
dAbs can be generated by phage display or other in vitro methods.
Alternatively, dAb domain can be obtained from immunized camelids
or sharks or other species that generate antibodies that lack a
light chain.
[0225] A "diabody" refers to a recombinant antibody that has two Fv
heads, each consisting of a V.sub.H domain from one polypeptide
paired with the V.sub.I domain from another polypeptide. A diabody
typically contains two vH-vL (or vL-vH) chains. Diabody can be
constructed by joining the vL and vH domains of an antibody by a
peptide linker. The peptide linker lengths comprise various numbers
of amino acids, preferably between 2 and 12 amino acids. A diabody
can be monospecific or bispecific.
[0226] A "triabody" refers to a recombinant antibody that has three
Fv heads, each consisting of a V.sub.H domain from one polypeptide
paired with the V.sub.L domain from a neighboring polypeptide. A
triabody contains three vH-vL (or vL-vH) chains. Triabody can be
constructed by joining the vL and vH domains of an antibody by a
peptide linker. The peptide linker lengths comprise various numbers
of amino acids, preferably between 0 and 2 amino acids. A triabody
can be monospecific, bispecific or trispecific.
[0227] A "tetrabody" comprises four vH-vL (or vL-vH) chains.
Tetrabodies can be constructed by joining the vL and vH domains of
an antibody by a peptide linker. The peptide linker lengths
comprise various numbers of amino acids, preferably between 0 and 2
amino acids. Tetrabodies can be obtained by truncating various
numbers of amino acids, preferably between 1 to 10 amino acids,
from the joined ends of the vL and vH domains.
[0228] A "Fab fragment" refers to a region on an antibody which
binds to antigens. A Fab fragment is composed of one constant and
one variable domain of each of the heavy and the light chain. These
domains shape the paratope--the antigen binding site--at the amino
terminal end of the monomer. The two variable domains bind the
epitope on their specific antigens. A Fab fragment can be linked by
a disulfide bond at the C-terminus. Fab fragments can be generated
in vitro. The enzyme papain can be used to cleave an immunoglobulin
monomer into two Fab fragments and an Fc fragment. The enzyme
pepsin cleaves below the hinge region, so a F(ab')2 fragment and a
Fc fragment is formed. The variable regions of the heavy and light
chains can be fused together to form a single chain variable
fragment (scFv), which retains the original specificity of the
parent immunoglobulin
[0229] The term "antibody fragment" is used herein to include all
of the fragments described in the present invention including any
antigen binding unit as defined in details below, such as dAb, Fv,
Fab, and Fc in any form. Antibody fragments can comprise additional
domains of an antibody. An antibody fragment also encompasses a
complete or full antibody.
[0230] The term "parent antibody" is used herein to refer to the
antibody upon which the construction of an antibody fragment is
based.
[0231] An "antibody fragment based therapeutic" (AFBT) refers to
any therapeutic agent or pharmaceutical composition that is based
on an antibody fragment as described herein. AFBTs can comprise
multiple antibody fragments that can be derived from multiple
different parent antibodies. Multispecific AFBTs may comprise
multiple antibody fragments with specificity against multiple
different epitopes. These epitopes can be part of the same target
antigen or on multiple different target antigens. Bispecific AFBTs
may comprise binding sites (generally two or more, but may be one)
with two different binding specificities.
[0232] The terms "antigen", "target antigen" or "immunogen" are
used interchangeably herein to refer to the structure or binding
determinant that an antibody fragment or an antibody fragment-based
therapeutic binds to or has specificity against.
[0233] The terms "domain reassortment" and "domain swapping" are
used interchangeably herein to refer to a process that changes the
valency of an antibody fragment or an antibody fragment based
therapeutic. For example, single chain variable fragments (scFv)
can reassort to form dimers, trimers etc, as well as diabodies,
triabodies, tetrabodies, and the like. Fabs can exchange whole
chains with other Fabs or even whole antibodies, potentially
yielding mismatched chains that result in loss of one or both
binding activities. The formation of light chain dimers, called
Bence-Jones Protein, is another example. Another example of
reassortment is heavy chain reassortment between IgG4 antibodies,
which do not have a disulfide-bonded hinge that prevents such
exchange, which can lead to bispecific IgG4 antibodies. The rate of
domain reassortment is dependent on the reaction conditions such as
salt concentration, pH, temperature, and the presence of target
antigen.
[0234] The term "payload" as used herein refers to a protein or
peptide sequence that has biological or therapeutic activity,
equivalent to the pharmacophore of small molecules. Examples of
payloads include, but are not limited to, cytokines, enzymes and
growth factors. Payloads can comprise genetically fused or
chemically conjugated moieties. Examples for such chemically
conjugated moieties include, but are not limited to,
chemotherapeutic agents, antiviral compounds, or contrast agents.
These conjugated moieties can be joined to the rest of the AFBT via
a linker which may be cleavable or non-cleavable.
[0235] "Collagen binding domain" (CBD) refers to a protein domain
that binds to or has specificity against collagen. CBDs can be
specific for any particular types of collagen such as collagen I.
Alternatively, CBDs may bind to a variety of collagen types. An
example is fibronectin in which four protein domains are sufficient
for collagen binding.
[0236] The term "repetitiveness" used in the context of a
polypeptide, for example, an accessory polypeptide PEG, refers to
the degree of internal homology in a peptide sequence. A repetitive
sequence may contain multiple identical or homologos copies of an
amino acid sequence. Repetitiveness can be measured by analyzing
the frequency of identical subsequences. For instance, a
polypeptide sequence of interest may be divided into n-mer
sub-sequences and the number of identical subsequences can be
counted. Highly repetitive sequences contain a large fraction of
identical subsequences.
[0237] "Total charge density" as used herein is calculated by
adding the number of negatively charged amino acids with the number
of positively charged amino acids, and dividing the sum by the
total number of amino acids in a polypeptide. For example: hIgG1 Fc
sequence:
(MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSL (SEQ ID NO: 10)) Number of negatively
charged residues: 24; Number of positively charged residues: 22;
Total number of residues: 224; Total charge density of Fc alone:
(22+24)/224=46/224=20.5% "Net charge density" as used herein is
calculated by subtracting the number of positively charged amino
acids from the number of negatively charged amino acids, and
dividing the difference by the total number of amino acids in a
polypeptide. For example: hIgG1 Fc sequence:
(MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSL (SEQ ID NO: 10)) Number of negatively
charged residues: 24; Number of positively charged residues: 22;
Total number of residues: 224; Net charge density of Fc alone:
(24-22)/224=2/224=0.9%.
[0238] "Predicted solubility" as used herein is calculated by
adding the net charge of folded protein to the total charge of an
unstructured protein (e.g. rPEG), and dividing the sum by the total
number of amino acids in the protein. For example, the predicted
solubility of Fc-rPEG50 is (-2+192)/(224+576)=190/800=23.75%
[0239] Design of Accessory Polypeptides for Improving Solubility
During Expression of Biologically Active Polypeptides.
[0240] Expression of soluble modified biologically active
polypeptides may be optimized by modifying the net charge density
of the modified polypeptide. In some cases, the net charge density
is above +0.1 or below -0.1 charges/residue. In other cases, the
charge density is above +0.2 or below -0.2 charges per residue.
Charge density may be controlled by modifying the content of
charged amino acids such as arginine, lysine, glutamic acid and
aspartic acid within accessory polypeptides linked to the
biologically active polypeptide. If desired, the accessory
polypeptide may be composed exclusively of a short stretch of
charged residues. Alternatively, the accessory polypeptide may
comprise charged residues separated by other residues such as
serine or glycine, which may lead to better expression or
purification behavior. Higher expression may be obtained. Use of
serine may lead to higher expression levels.
[0241] The net charge that is required for the accessory protein to
make a fusion protein soluble and fold in the cytoplasm depends on
the biologically active polypeptide, specifically its size and net
charge. The net charge of the modified polypeptide may be positive
or negative. In some applications, accessory polypeptide sequences
rich in negative amino acids such as glutamic acid or aspartic acid
may be desirable. In other applications, accessory polypeptide
sequences rich in positive amino acids such as lysine or arginine
may be preferred. The use of both positively and negatively charged
amino acids may lead to charge neutralization, which could
potentially neutralize the advantage of the invention. For example,
accessory proteins of 288 amino acids with 16%, 25% or 33%
negatively charged residues may provide up to 96 total charges,
which is sufficient to achieve a charge density of 0.1 for a
neutral fusion protein of up to 960 amino acids, or a non-fusion
protein of 672 amino acids. In one specific example, an accessory
polypeptide comprising 33% glutamic acid residues might be used to
make even very large and difficult to express proteins soluble.
[0242] To impart solubility on the binding protein, the net
positive or negative charge of the accessory polypeptide may be
greater than 5, 10, 15 or 20 or even greater than 30, 40, 50, 60,
70, 80, 90 or 100. Charges can be concentrated in a short sequence
of 5, 10, 15, 20, 25, 30, 40, 50 amino acids, or can be spaced out
over a longer sequence of 60, 80, 100, 150, 200, 250, 300, 400, or
500 or more amino acids. The sequence of a negative accessory
polypeptide may contain over 5, 10, 15, 25, 30, 40, 50, 60, 70, 80,
90 or 100 percent of glutamic or aspartic acid, while a positive
accessory polypeptide may contain over 5, 10, 15, 20, 25, 30, 40,
50, 60, 70, 80, 90 or 100 percent of arginine or lysine.
Non-charged residues may be used such as the relatively hydrophilic
residues Serine and Glycine.
[0243] Additional Considerations in the Design of Accessory
Polypeptides:
[0244] One aspect of the present invention is the design of
accessory polypeptides, e.g., rPEG accessory polypeptides and the
like for the modification of biologically active polypeptides (FIG.
1). The accessory polypeptides are particularly useful for
generating recombinant proteins of therapeutic and/or diagnostic
value.
[0245] A variety of accessory polypeptide sequences can be designed
and these may be rich in glycine and/or serine, as well as other
amino acids such as glutamate, aspartate, alanine or proline.
Accessory polypeptide sequences may be rich in hydrophilic amino
acids and contain a low percentage of hydrophobic or aromatic amino
acids. Accessory polypeptide sequences can be designed to have at
least 30, 40, 50, 60, 70, 80, 90 or 100% glycine and/or serine
residues. In some cases, accessory polypeptide sequences contain at
least 50, 55, 60, 65% glycine and/or serine. In other cases,
accessory polypeptide sequences may contain at least 70, 75, 80,
85, 90% glycine and/or serine residues.
[0246] The compositions of the present invention will typically
contain accessory polypeptide sequences consisting of a total of at
least 40 amino acids. However, the products can contain multiple
accessory polypeptide sequences and some or all of these individual
accessory polypeptide sequences may be shorter than 40 amino acids
as long as the combined length of all accessory polypeptide
sequences of a product is at least 40 amino acids. In some
embodiments, the combined length of accessory polypeptide sequences
that are attached to a protein can be 20, 25, 35, 50, 60, 70, 80,
90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 500, 600,
700, 800, 900 or more than 1000 or 2000 amino acids. In some
modified biologically active polypeptides the combined length of
accessory polypeptide sequences exceeds 60, 70, 80, 90 or more
amino acids. In other modified polypeptides the combined length of
accessory polypeptide sequences exceeds 100, 120, 140, 160 or 180
amino acids, and even 200, 250, 300, 350, 400, 5000, 600, 700, 800
or even more than 1000 amino acids.
[0247] One or several accessory polypeptide sequences can be fused
to a biologically active polypeptide, for example to the N- or
C-terminus of the biologically active polypeptide or inserted into
loops of a polypeptide of interest to give the resulting modified
polypeptide improved properties relative to the unmodified
polypeptide. Fusion of accessory sequences to a (therapeutic)
protein leads to a significant increase in the hydrodynamic radius
of the resulting fusion protein relative to the unmodified protein,
which can be detected by ultracentrifugation, size exclusion
chromatography, or light scattering, for example.
[0248] Accessory polypeptide sequences can be designed to avoid one
or more types of amino acids to yield a desired property. For
instance, one can design accessory polypeptide sequences to contain
few or none of the following amino acids: cysteine (to avoid
disulfide formation and oxidation), methionine (to avoid
oxidation), asparagine and glutamine (to avoid desamidation) and
aspartate. Accessory polypeptide sequences can be designed to
contain proline residues that tend to reduce sensitivity to
proteolytic degradation.
[0249] Accessory polypeptide sequences can be designed such as to
optimize protein production. This can be achieved by avoiding or
minimizing repetitiveness of the encoding DNA. Accessory
polypeptide sequences such as poly-glycine or poly-serine may have
very desirable pharmaceutical properties but their manufacturing
can be difficult due to the high GC-content of DNA sequences
encoding for poly-glycine and due to the presence of repeating DNA
sequences that can lead to recombination.
[0250] Accessory polypeptides, including simple sequences composed
of short, repeated motifs rich in sequences rich in G, S and E, may
cause relatively high antibody titers of >1,000 in multiple
species despite the absence of T-cell epitopes in these sequences.
This may be caused be the repetitive nature of the accessory
polypeptides, as it has been shown that immunogens with repeated
epitopes, including protein aggregates, cross-linked immunogens,
and repetitive carbohydrates are highly immunogenic. (Johansson,
J., et al. (2007) Vaccine, 25: 1676-82, Yankai, Z., et al. (2006)
Biochem Biophys Res Commun, 345: 1365-71, Hsu, C. T., et al. (2000)
Cancer Res, 60: 3701-5). B-cells displaying pentavalent IgM
molecules are stimulated by repetitive immunogens even if the
monovalent binding affinity of an immunogen for the IgM is very
low, such as at micromolar concentrations (FIG. 74). Simultaneous
binding of linked repeats to multiple linked IgM domains located on
the same molecule or on the same cell may cause a large (thousand,
million or perhaps even billion-fold) increase in the apparent
(effective) affinity of the interaction, which may stimulate
B-cells. To avoid this type of effect, accessory polypeptides may
be screened for immunogenicity (as well as for effects on halflife
and other properties) in multiple species of animals (such as rats,
rabbits, mice, or guinea pigs. Multiple injections may be
performed, with pharmacokinetic properties being measured in the
same animals before and after immunization). In addition, accessory
polypeptide sequences may be designed to be non-repetitive
(comprising only 1 identical copy of each sequence motif) or to
have a minimal number of copies of each sequence motif. Accessory
polypeptide sequences that are less-repetitive may comprise binding
sites for different IgMs, but they may be less able to bind
multivalently to the same IgM molecule or to the same B-cell, since
each B-cell generally secretes only one type of IgM and each IgM
typically only has one type of binding site. This mechanism is
illustrated in FIGS. 74a and b. In some embodiments, accessory
polypeptides may contain exclusively sequences that occur at 1, 2,
3, 4, 5 or so copies per accessory polypeptide. Polypeptides with a
lower number of repeats, may have a lower expected avidity may be
less likely to induce a substantial immune response. Such sequences
may comprise multiple types of amino acids, such as two types (for
example, G and E or S and E), three types of amino acids (like G, E
and S) or even four or more. Such accessory polypeptides may also
comprise, for example, 30-80% glycine, 10-40% serine and 15-50%
glutamate of the total amino acid composition. Such sequences may
provide an optimal balance of desired properties such as expression
level, serum and E. coli protease resistance, solubility,
aggregation, and immunogenicity.
[0251] FIG. 74 compares the interactions of a repetitive (74a) and
a non repetitive accessory polypeptide sequence (74b) with B cells
that recognize epitopes in said sequences. A repetitive sequence
will be recognized by few B cells in an organism as it contains a
relatively small number of different epitopes. However, a
repetitive sequence can form multivalent contacts with these few B
cells and as a consequence it can stimulate their proliferation as
illustrated in FIG. 74a. A non repetitive sequence can make
contacts with many different B cells as it contains many different
epitopes. However, each individual B cell can only make one or a
small number of contacts with an individual non-repetitive
accessory polypeptide ("nrURP") due to the lack of repetitiveness
as illustrated in FIG. 74b. As a result, non-repetitive accessory
polypeptides may have a much lower tendency to stimulate
proliferation of B cells and thus an immune response.
[0252] An additional advantage of non-repetitive accessory
polypeptides relative to repetitive accessory polypeptides is that
non-repetitive accessory polypeptides form weaker contacts with
antibodies relative to repetitive accessory polypeptides.
Antibodies are multivalent molecules. For instance, IgGs have two
identical binding sites and IgMs contain 10 identical binding
sites. Thus antibodies against repetitive sequences can form
multivalent contacts with such repetitive sequences with high
avidity, which can affect the potency and/or elimination of such
repetitive sequences. In contrast, antibodies against
non-repetitive accessory polypeptides tend to form mostly
monovalent interactions with antibodies as said non-repetitive
accessory polypeptides contain few repeats of each epitope.
[0253] Repetitiveness describes the degree of internal homology in
a peptide sequence. In the extreme case a repetitive sequence can
contain multiple identical copies of an amino acid sequence.
Repetitiveness can be measured by analyzing the frequency of
identical subsequences. For instance one can divide a sequence of
interest into n-mer subsequences and count the number of identical
or homologos subsequences. Highly repetitive sequences will contain
a large fraction of identical or homologos subsequences.
[0254] The repetitiveness of a gene can be measured by computer
algorithms. An example is illustrated in FIG. 75. Based on the
query sequence on can perform a pair wise comparison of all
subsequences of a particular length. These subsequences can be
compared for identity or homology. The example in FIG. 75 compares
subsequences of 4 amino acids for identity. In the example, most
4-mer subsequences occur just once in the query sequence and 3 4mer
subsequences occur twice. One can average the repetitiveness in a
gene. The length of the subsequences can be adjusted. Where
desired, the length of the subsequences can reflect the length of
sequence epitopes that can be recognized by the immune system. Thus
analysis of subsequences of 4-15 amino acids can be performed.
Genes encoding non-repetitive accessory polypeptides can be
assembled from oligonucleotides using standard techniques of gene
synthesis. The gene design can be performed using algorithms that
optimize codon usage and amino acid composition. In addition, one
can avoid amino acid sequences that are protease sensitive or that
are known to be epitopes that can be easily recognized by the human
immune system. Computer algorithms can be applied during sequence
design to minimize the repetitive of the resulting amino acid
sequences. One can evaluate the repetitiveness of large numbers of
gene designs that match preset criteria such as amino acid
composition, codon usage, avoidance of protease sensitive
subsequence, avoidance of epitopes, and chose the least repetitive
sequences for synthesis and subsequent evaluation.
[0255] An alternative approach to the design of non-repetitive
accessory polypeptide genes is to analyze the sequences of existing
collections of non-repetitive accessory polypeptides that show high
level expression, low aggregation tendency, high solubility, and
good resistance to proteases. A computer algorithm can design
non-repetitive accessory polypeptide sequences based on such
pre-existing non-repetitive accessory polypeptide sequences by
re-assembly of sequence fragments. The algorithm generates a
collection of subsequences from these non-repetitive accessory
polypeptide sequences and evaluates multiple ways to assembly
non-repetitive accessory polypeptide sequences from such
subsequences. These assembled sequences can be evaluated for
repetitiveness to identify a non-repetitive accessory polypeptide
sequence that is only composed of subsequences of previously
identified non-repetitive accessory polypeptides.
[0256] Non-repetitive accessory polypeptide-encoding genes can be
assembled from libraries of short accessory polypeptide segments as
illustrated in FIG. 77. One can first generate large libraries of
accessory polypeptide segments. Such libraries can be assembled
from partially randomized oligonucleotides. The randomization
scheme can be optimized to control amino acid choices for each
position as well as codon usage. One may clone the library of
accessory polypeptide segments into an expression vector.
Alternatively, one may clone the library of accessory polypeptide
segments into an expression vector fused to an indicator gene like
GFP. Subsequently, one can screen library members for a number of
properties such as level of expression, protease stability, binding
to antiserum. One can determine the amino acid sequence of the
library members to identify segments that have a particularly
desirable amino acid composition, segment length, or to identify
segments that have a low frequency of internal repeats.
Subsequently, one can assemble non-repetitive accessory polypeptide
sequences from collections of accessory polypeptide segments by
random dimerization or multimerization. Dimerization or
multimerization can be achieved by ligation or PCR assembly. This
process results in a library non-repetitive accessory polypeptide
sequences that can be evaluated for a number of properties to
identify the non-repetitive accessory polypeptide sequences with
the best properties. One can repeat the process of dimerization or
multimerization to further increase the length of non-repetitive
accessory polypeptide sequences.
[0257] In a specific embodiment, an accessory polypeptide comprises
a mixture of the following 8 amino acid motifs: GEGSGEGSE (SEQ ID
NO: 11), GEGGSEGSE (SEQ ID NO: 12), GEGSEGSGE (SEQ ID NO: 13),
GEGSEGGSE (SEQ ID NO: 14), GEGSGEGGE (SEQ ID NO: 15), GEGGSEGGE
(SEQ ID NO: 16), GEGGGEGSE (SEQ ID NO: 17), GEGGEGSGE (SEQ ID NO:
18), GEGGEGGSE (SEQ ID NO: 19), or GEGSEGGGE (SEQ ID NO: 20). This
design has an average of 33% E and 11-22% Serine content, depending
on the ratio of the numbers of motifs relative to each other. In
another specific embodiment, an accessory polypeptide comprises a
mixture of the following 12 amino acid motifs: GXEGSGEGXGXE (SEQ ID
NO: 21), GXEGGSEGXGXE (SEQ ID NO: 22), GXEGSGEGGSGE (SEQ ID NO:
23), GXEGGSEGGSGE (SEQ ID NO: 24), GSGEGXEGXGXE (SEQ ID NO: 25),
GGSEGXEGXGXE (SEQ ID NO: 26), GSGEGXEGGSGE (SEQ ID NO: 27) or
GGSEGXEGGSGE (SEQ ID NO: 28), where X represents either S or E with
equal likelihood. This design has an average of 25% E and around 1%
S, depending on the specific ratios chosen. Suitable specific
ratios may be 1:1:1:1:1:1:1:1 ratio or any other ratio, and may be
to fine-tune the composition.
[0258] Accessory polypeptide sequences can be designed to be highly
repetitive, less repetitive or non-repetitive at the amino acid
level. For example, highly repetitive accessory polypeptide
sequences may contain only a small number of overlapping 9-mer
peptide sequences and in this way the risk of eliciting an immune
reaction can be reduced.
[0259] Examples of single-amino-acid-type accessory polypeptide
sequences are: poly-glycine, poly-glutamic acid, poly-aspartic
acid, poly-serine, poly-threonine, wherein the length is at least
20 residues. Examples of accessory polypeptides with two types of
amino acids are (GX)n (SEQ ID NO: 29), (SX)n (SEQ ID NO: 30), where
G is glycine and S is serine, and X is aspartic acid, glutamic
acid, threonine, or proline and n is at least 10. Another example
is (GGX)n (SEQ ID NO: 31) or (SSX)n (SEQ ID NO: 32), where X is
aspartic acid, glutamic acid, threonine, or proline and n is at
least 7. Another example is (GGGX)n (SEQ ID NO: 33) or (SSSX)n (SEQ
ID NO: 34), where X is aspartic acid, glutamic acid, threonine, or
proline and n is at least 5. Another example is (GGGGX)n (SEQ ID
NO: 35) or (SSSSX)n (SEQ ID NO: 36), where X is aspartic acid,
glutamic acid, threonine, or proline and n is at least 4. Other
examples are (GzX)n (SEQ ID NO: 37) and (SzX)n (SEQ ID NO: 38) and
where X is aspartic acid, glutamic acid, threonine, or proline, n
is at least 10, and z is between 1 and 20.
[0260] The number of these repeats can be any number between 5 and
300 or more. Products of the invention may contain accessory
polypeptide sequences that are semi-random sequences. Examples are
semi-random sequences containing at least 30, 40, 50, 60 or 70%
glycine in which the glycines are well dispersed and in which the
total concentration of tryptophan, phenylalanine, tyrosine, valine,
leucine, and isoleucine is less then 70, 60, 50, 40, 30, 20, or 10%
when combined. A preferred semi-random accessory polypeptide
sequence contains at least 40% glycine and the total concentration
of tryptophan, phenylalanine, tyrosine, valine, leucine, and
isoleucine is less than 10%. A more preferred random accessory
polypeptide sequence contains at least 50% glycine and the total
concentration of tryptophan, phenylalanine, tyrosine, valine,
leucine, and isoleucine is less then 5%. Accessory polypeptide
sequences can be designed by combining the sequences of two or more
shorter accessory polypeptide sequences or fragments of accessory
polypeptide sequences. Such a combination allows one to better
modulate the pharmaceutical properties of the product containing
the accessory polypeptide sequences and it allows one to reduce the
repetitiveness of the DNA sequences encoding the accessory
polypeptide sequences, which can improve expression and reduce
recombination of the accessory polypeptide sequences-encoding
sequences.
[0261] Where high level of solubility is desired, a high fraction
of charged residues, preferably >25% glutamate (E) with the rest
being mostly glycine or serine may be employed. High-level
expression favors 10-50% serine (E), since serine has 6 codons
which generally yields a much higher expression level than glycine
(4 codons). There is generally a trade-off in solubility and rapid
clearance when utilizing high glutamate content in a sequence.
Where desired, a glutamate content of less than 50%, preferably
less than 30%, is used to provide desired solubility and to avoid
rapid clearance in animals.
[0262] Non-Glycine Residues can be Selected to Optimize
Properties
[0263] Of particular interest are accessory polypeptide sequences
that are rich in glycine and/or serine. The sequences of non-gly,
non-ser residues in these gly-rich or ser-rich sequences can be
selected to optimize the properties of the protein. For instance,
one can optimize the sequences of accessory polypeptides to enhance
the selectivity of the biologically active polypeptide for a
particular tissue. Such tissue-selective accessory polypeptide
sequences can be obtained by generating libraries of random or
semi-random accessory polypeptide sequences, injecting them into
animals or patients, and determining sequences with the desired
tissue selectivity in tissue samples. Sequence determination can be
performed by mass spectrometry. Using similar methods one can
select accessory polypeptide sequences that facilitate oral,
buccal, intestinal, nasal, thecal, peritoneal, pulmonary, rectal,
or dermal uptake. Of particular interest are accessory polypeptide
sequences that contain regions that are relatively rich in the
positively charged amino acids arginine or lysine which favor
cellular uptake or transport through membranes; such accessory
polypeptides may be useful for intracellular delivery of
proteins.
[0264] As described in more detail below, accessory polypeptide
sequences can be designed to contain one or several
protease-sensitive sequences. Such accessory polypeptide sequences
can be cleaved once the product of the invention has reached its
target location. This cleavage may trigger an increase in potency
of the pharmaceutically active domain (pro-drug activation) or it
may enhance binding of the cleavage product to a receptor. This is
currently not possible for antibodies. However, in the case of
PEGylated or accessory protein modified biologically active
polypeptides, it is possible to provide a cleavage site for a
foreign protease such as Tomato Etch Virus Protease or a similar
site-specific, non-human protease. If the protease site is between
the accessory protein and the therapeutic protein, or close to the
therapeutic protein, then the injection of the protease will remove
the accessory protein tail from the drug resulting in a shorter
halflife and removal from the patient's system. The concentration
of the drug in the serum will drop 10-100-fold, effectively
terminating treatment. This would be desirable, for example, if
treatment needs to be stopped suddenly, such as due to an infection
during treatment with a TNF-inhibitory microprotein (such as
TNFa-Receptor-rPEG). An example would be to add a protease to the
treatment regime that cleaves off the accessory protein, thereby
sharply reducing the halflife of the active, TNF-inhibitory part of
the protein which is then rapidly cleared. This approach would
allow the infection to be controlled.
[0265] Accessory polypeptide sequences can also be designed to
carry excess negative charges by introducing aspartic acid or
glutamic acid residues. Of particular interest are accessory
polypeptide that contain 8, 10, 15, 20, 25, 30, 40 or even 50%
glutamic acid and less than 2% lysine or arginine. Such accessory
polypeptides carry a high net negative charge and as a result they
have a tendency to adopt open conformations due to electrostatic
repulsion between individual negative charges of the peptide. Such
a net negative charge leads to an effective increase in their
hydrodynamic radius and as a result it can lead to reduced kidney
clearance of such molecules. Thus, one can modulate the effective
net charge and hydrodynamic radius of an accessory polypeptide
sequence by controlling the frequency and distribution of
negatively charged amino acids in the accessory polypeptide
sequences. Most tissues and surfaces in a human or animal have a
net negative charge. By designing accessory polypeptide sequences
to have a net negative charge one can minimize non-specific
interactions between the accessory polypeptide-therapeutic protein
and various surfaces such as blood vessels, healthy tissues, or
various receptors.
[0266] Other accessory polypeptides useful in the present invention
exhibit one or more following features.
[0267] The accessory polypeptide can be characterized by enhanced
hydrodynamic radius, wherein the accessory polypeptide increases
the Apparent Molecular Weight Factor of the biologically active
polypeptide to which it is linked. Because the Apparent Molecular
Weight Factor is a predictor of serum secretion half-life (assuming
the predicted molecular weight is constant), accessory polypeptides
with higher Apparent Molecular Weight Factor are expected to show
longer serum half-lives. In some embodiments, Apparent Molecular
Weight Factors for accessory polypeptides can be greater than 3, 5,
7 or even 9. The Apparent Molecular Weight Factor can be measured
by a variety of methods including but not limited to
ultrafiltration through membranes with controlled pore sizes, or by
size exclusion gel filtration (SEC). The Apparent Molecular Weight
Factor can be affected by the concentration of salts and other
solutes. It should generally be measured under conditions that are
similar to physiological conditions, such as in blood or
saline.
[0268] The accessory polypeptide can also be characterized by the
effect wherein upon its incorporation into a biologically active
polypeptide, the biologically active polypeptide exhibits a longer
serum half-life as compared to the corresponding protein that lacks
the accessory polypeptide. (Methods of ascertaining serum half-life
are known in the art (see e.g., Alvarez, P., et al. (2004) J Biol
Chem, 279: 3375-81). One can readily determine whether the
resulting protein has a longer serum half-life as compared to the
unmodified protein by practicing any methods available in the art
or exemplified herein.
[0269] The accessory polypeptide can also increase the solubility
of the protein to which it is attached. For example, whereas human
Interferon-alpha, human Growth Hormone and human G-CSF typically
form inclusion bodies when expressed in the cytoplasm of E. coli,
attachment of an accessory polypeptide (such as (SSGSSE).sub.48
(SEQ ID NO: 39) or (SSESSSSESSSE).sub.24 (SEQ ID NO: 40),
(GEGGGEGGE).sub.36 (SEQ ID NO: 41), or others) increases the
solubility of the expressed polypeptide such that it no longer
forms inclusion bodies but remains soluble in the cytoplasm from
where it can be easily purified in active form and at high
expression levels and efficiency, avoiding the need for refolding
from inclusion bodies.
[0270] Accessory polypeptides can have a high degree of
conformational flexibility under physiological conditions and they
tend to have large hydrodynamic radii (Stokes' radius) compared to
globular proteins of similar molecular weight, leading to a large
`specific volume` (volume per unit mass). Thus, the accessory
polypeptide can behave like denatured peptide sequences lacking
well defined secondary and tertiary structures under physiological
conditions. Denatured conformation describes the state of a peptide
in solution that is characterized by a large conformational freedom
of the peptide backbone. Most peptides and proteins adopt a
denatured conformation in the presence of high concentrations of
denaturants or at elevated temperature. Peptides in denatured
conformation have characteristic CD spectra and they are
characterized by a lack of long range interactions as determined by
NMR. "Denatured conformation" and "unfolded conformation" are used
synonymously herein. A variety of methods have been established in
the art to discern the presence or absence of secondary and
tertiary structures of a given polypeptide. For example, the
secondary structure of a polypeptide can be determined by CD
spectroscopy in the "far-UV" spectral region (190-250 nm).
Secondary structure elements, such as alpha-helix, beta-sheet, and
random coil structures each give rise to a characteristic shape and
magnitude of CD spectra. Secondary structure can also be
ascertained via certain computer programs or algorithms such as the
Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13:
222-45). For a given accessory sequence, the algorithm can predict
whether there exists some or no secondary structure at all. In many
cases, accessory sequences will have spectra that resemble
denatured sequences due to their low degree of secondary and
tertiary structure. In other cases, accessory sequences can adopt
secondary structure, especially helices such as alpha-helices, or
sheets such as beta-sheets. While unstructured amino acid polymers
are generally preferred for the present invention, it is possible
to use amino acid sequences that adopt some secondary structure,
especially alpha-helices and to a lesser extent beta-sheets.
Tertiary structure is generally undesirable due to its low specific
hydrodynamic radius. Sequences with secondary structure are likely
to have a lower hydrodynamic radius than sequences with less
secondary structure, but they may still be useful. If the accessory
sequence adopts tertiary structure (such as in protein domains),
the hydrodynamic radius is expected to be even smaller. Whereas
polyglycine has the highest ratio of hydrodynamic radius to mass
(glycine is only 70 D), globular proteins have the smallest ratio
of hydrodynamic radius to mass. An exception is the inclusion in
the accessory polypeptide of peptides with 0, 1, 2, 3 or 4
disulfides and varying degrees of secondary and tertiary structure)
that bind to serum-exposed targets and increase the serum secretion
halflife by a different mechanism.
[0271] The accessory polypeptides can be sequences with low
immunogenicity. Low immunogenicity can be a direct result of the
conformational flexibility of accessory sequences. Many antibodies
recognize so-called conformational epitopes in protein antigens.
Conformational epitopes are formed by regions of the protein
surface that are composed of multiple discontinuous amino acid
sequences of the protein antigen. The precise folding of the
protein brings these sequences into a well-defined special
configuration that can be recognized by antibodies. Preferred
accessory polypeptides are designed to avoid formation of
conformational epitopes. For example, of particular interest are
accessory sequences having a low tendency to adapt compactly folded
conformations in aqueous solution. In particular, low
immunogenicity can be achieved by choosing sequences that resist
antigen processing in antigen presenting cells, choosing sequences
that do not bind MHC well and/or by choosing sequences that are
derived from human sequences. Accessory polypeptide sequences can
also reduce the immunogenicity of the biologically active
polypeptide.
[0272] The accessory polypeptides can be sequences with a high
degree of protease resistance. Protease resistance can also be a
result of the conformational flexibility of accessory sequences,
e.g., due to their high entropy. Protease resistance can be
designed by avoiding known protease recognition sites for both
endo- and exo-proteases, and by including a high glycine content.
Alternatively, protease resistant sequences can be selected by
phage display or related techniques from random or semi-random
sequence libraries. Where desired for special applications, such as
slow release from a depot protein, serum protease cleavage sites
can be built into an accessory polypeptide. In such cases, the
compositions of the present invention may dissolve or degrade (or
may be intended to dissolve or degrade) during use. In general,
degradation attributable to biodegradability involves the
degradation of a polymer into its constituents (including, without
limitation, the modified polypeptides and resulting degradation
products). The degradation rate of a polymer often depends in part
on a variety of factors, including the identity of any constituents
that form the polymer (such as a protease sensitive site), the
ratio of any substituents, and how the composition is formed or
treated (e.g. whether substituents are protected). Of interest,
however, are also accessory sequences with high stability (e.g.,
long serum half-life, less prone to cleavage by proteases present
in bodily fluid) in blood or in the bodily tissue that is relevant
for the application. Accessory polypeptides can also improve the
protease resistance of a protein as they shield it from protease
attack. An example of a natural unstructured, repetitive sequence
composed of 3 amino acids is the linker in the pIII protein of M13
phage, which has the repeat (GGGSE)n (SEQ ID NO: 42) and is known
to be exceptionally stable to a vast array of proteases. An
accessory protein with the motif (GGGSE)n (SEQ ID NO: 42) is
predicted to be very useful. For long sequences, one may prefer
(GGSE)n, (SEQ ID NO: 43) to achieve higher solubility which may be
needed at the increased length.
[0273] Accessory polypeptides with good solubility in water, blood
and other bodily fluids under physiological conditions are also
desirable to facilitate bioavailability. Such sequences can be
obtained by designing sequences that are rich in hydrophilic amino
acids such as glycine, serine, aspartate, glutamate, lysine,
arginine, threonine and that contain few hydrophobic amino acids
such as tryptophan, phenylalanine, tyrosine, leucine, isoleucine,
valine, methionine. As a result of their amino acid composition,
accessory polypeptides have a low tendency to form aggregates in
aqueous formulations and the fusion of an accessory polypeptide to
other proteins or peptides tends to enhance their solubility and
reduce their tendency to form aggregates, which is a separate
mechanism to reduce immunogenicity.
[0274] The accessory polypeptide can, in some cases, display
enhanced non-specific binding to tissues or serum proteins (FIG.
28), which can function to prolong their serum half-life. Serum
protein binding can be measured using a variety of methods.
Examples for binding assays are ELISA, Biacore, Kinexa, or Forte
Bio. Since most animal tissue surfaces have a (net) weak negative
charge, proteins with a net negative charge show less non-specific
tissue binding than proteins with a net positive charge. Creating a
net weak negative charge by the addition of negative charges or by
the deletion of positive charges can make a protein bind more
specifically or at least reduce non-specific binding.
[0275] However, if the net negative charge (or the net charge
density) is too high, it can result in non-specific binding to
surfaces with local patches of positive charge, such as parts or
proteins that bind to extracellular matrix, or to DNA or RNA (e.g.
VEGF, histones). In contrast, creating a protein with net positive
charge by the addition of positive charges (such as K, R) or by the
deletion of negative charges can make a protein bind
non-specifically to tissues, which results in an extension of
halflife.
[0276] The charge type and density of the accessory polypeptide
itself can be modified. The negatively charged amino acids are E,
D, (C) and the positively charged amino acids are R, K, (H).
Changes generally involve exchanging one negatively charged residue
for another, such as E for D or vice versa. In some instances, E is
preferred, because D can isomerize leading to chemical instability
that is undesirable for manufacturing. Changes in charge type, from
positive charge to negative charge or vice versa, involve replacing
K or R with E or D (positive replaced by a negative). Changes in
charge also include replacing a non- or weakly charged amino acid
(A,C,F,G,H,I,L,M,N,P,Q,S,T,V,W,Y) with a charged amino acid
(E,D,K,R) or vice versa. "Charge density" is the number of charged
amino acids as a percentage of total residues. Changing the charge
density involves increasing or reducing the number of negatively
charged amino acids (specifically E,D) or positively charged amino
acids (K,R) as a percentage of total amino acids. In contrast, the
`net charge density` is the sum of all positively charged amino
acids minus the sum of all negatively charged amino acids ("net
charge") as a percentage of the total number of residues.
[0277] The "net charge" and the "net charge density (net charge per
AA)" can influence the solubility of the accessory polypeptide and
of the accessory-modified polypeptide, as well as its ability to
bind to other molecules. The accessory polypeptide can modify the
charge type and density of fusion proteins, which can enhance serum
halflife and can be exploited to enhance desirable interactions or
to reduce non-desirable interactions of the fusion protein with
other proteins or materials.
[0278] The accessory polypeptide can, in some cases, display
enhanced non-specific binding to tissues or serum proteins, which
can function to prolong their serum half-life. This can be measured
as an extension of serum halflife compared to an accessory sequence
that does not show non-specific binding, or it can be shown by
ELISA as a weak binding affinity for proteins a high density of the
opposite charge.
[0279] Accessory polypeptides can consist partially or entirely of
a single amino acid, such as (E(n, (G)n or (S)n (also referred to
as poly-E, EEEEE (SEQ ID NO: 44), poly-G, GGGGG (SEQ ID NO: 45), or
poly-S, SSSSS (SEQ ID NO: 46)), or even a homo-polymer of one of
A,C,D,F,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y; ie AAAAA (SEQ ID NO: 47)). The
best single amino acid motifs (E,G,S) are immunologically the least
complex (only one type of 9 amino acid peptide can be created), but
each has some drawbacks. Glycine is weakly hydrophobic and poly-G
has limited solubility. An advantage of glycine is its high
entropy. In some instances, serine may be preferred over glycine
because the corresponding DNA sequence is likely to have a more
balanced GC-ratio and generally provides a higher expression level,
likely due to its 6 codons. The four charged amino acids, including
Glutamic acid (E), have the highest solubility of the 20 natural
amino acids, followed by Glycine and Serine. However, at a high
negative net charge density the proteins start binding
non-specifically to positively charged proteins and surfaces, such
as VEGF (basic exons that bind ECM), histones, DNA/RNA-binding
proteins and also to bone. Others have reported that a long string
of poly-E causes a reduced halflife, instead of the desired
extended halflife.
[0280] Serine and poly-Serine offer high solubility without a risk
of aggregation and with the best codon use and expression level.
The six codons for serine offer a balanced GC content, but more
importantly, they allow poly-S or S-rich sequences to be encoded by
exceptionally diverse DNA sequences that offer a greater degree of
codon usage optimization and expression level optimization than
other amino acids such as poly-E or poly-G (FIGS. 14 and 15).
[0281] The accessory polypeptides can be of any length necessary to
effect the functional changes described above. The length of an
accessory sequence that only contains 1, 2, 3 or more types of
amino acids can have a lower limit of 10, 12, 14, 16, 18, 20, 25,
30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 amino acids and an upper
limit of 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 500, 600 or even 1000 amino acids.
[0282] The amino acid composition of the accessory polypeptide can
be chosen such that the desirable properties of the resulting
polypeptide are maximized. For example, for the extension of serum
half-life a high ratio of apparent molecular weight to predicted
molecular weight is preferred. The unstructured accessory
polypeptides that offer more hydrodynamic radius for the same mass
are constructed with amino acids that do not support structures
such as alpha helices or beta-sheets. According to the rating of
amino acid residues by the Chou-Fasman algorithm, residues A, D, E,
Q, I, L, K, M, F, W, V support alpha-helical structure and residues
C,Q,I,L,M,F,T,W,Y,V support beta-sheet structures. The amino acids
that the Chou-Fasman algorithm considers most unstructured, because
they are turn-forming, are, in order from most to least
unstructured: G, N, P, D, S, C, Y, K. On balance, the residues that
least support structure are G, N, P, S.
[0283] To achieve better fine tuning of the properties of the
polymer, especially solubility and charge density, accessory
polypeptides composed of two or three amino acids are generally
preferred over those composed of a single amino acid. Accessory
polypeptides that are composed of two or three types of amino acids
are preferred because they offer the best balance of immunological
simplicity (yielding only a small number of different 9-mer
peptides can bind MHC complexes or 8-mer peptides that form
epitopes for antibody binding), with the optimization of
solubility, protease resistance, charge type and density, absence
of structure, entropy, and non-specific binding to tissues (which
can be undesirable but can also be used as a half-life mechanism).
In general, the larger the number of non-human 8-mer or 9-mer
peptides that can be created from the accessory protein sequence,
the higher the risk of immunogenicity. Accordingly, in some aspects
the accessory polypeptide comprises a small number of different
8-mer or 9-mer, and wherein all or most of these peptide sequences
occur in the human proteome, preferably with many copies.
[0284] Where desired, a blend of two or three amino acids types can
be optimized for obtaining the desired balance of properties. The
20 natural amino acids (AA) can be separated into groups with
related properties. Residues E, D (and to a lesser extent C) are
negatively charged at physiological (neutral) pH, and residues K, R
and to a lesser extent H are positively charged at neutral pH. The
presence of charged residues E, D, K and R may be desirable for
maximizing the water solubility of long polypeptides. For some
biological applications it is desirable to have a high but equal or
similar frequency of negative and positive residues that result in
an uncharged, or nearly uncharged polypeptide that has high charge
density but low net charge, such polypeptides tend to have low
tendency for non-specific interactions with receptors that bind
charged polymers such as heparin. For some biological applications,
a single charge type (negative) which (unlike D) is chemically
stable; thus favoring E (glutamate). The question is what the
percentage of amino acids should be E, and whether the majority of
non-charges amino acids should be G or S, and whether the sequence
should be highly repetitive or less repetitive.
[0285] A high frequency of negatively charged residues E, D is
likely to make the polymer bind to molecules with a large number of
positive charges, like DNA binding proteins, histones and other
R,K-rich sequences. A high frequency of positively charged residues
K,R is likely to make the polymer bind to surfaces with a large
number of negative charges, which includes most cell surfaces.
Binding to cell surfaces is generally not desirable but a low
degree of such non-specific binding may be useful to increase the
half-life. The polar, hydrophilic amino acids N, Q, S,T,K,R,H,D,E,
and additionally the amino acids C or Y can be useful in making
accessory polypeptides that are relatively water soluble. Q and N
can be glycosylation sites, offering a separate mechanism for
increasing the hydrodynamic radius and thereby halflife. Non-polar,
hydrophobic residues such as A,V,L,I,P,Y,F,W,M,C are less useful
when creating a sequence with high water solubility, but it may be
desirable to incorporate one or more of these residues at a low
frequency, such that they constitute less than 10-20% of total. For
example, a limited number of substitutions of hydrophobic residues
can increase half-life by increasing non-specific binding to
serum-exposed sites. Similarly, free thiols from cysteine residues
may function as a mechanism for half-life extension by binding to
other free thiols, such as the free thiol in human serum albumin.
Also, these less-preferred amino acids can be used to create
peptides that bind to serum-exposed proteins, thereby adding a
second halflife extension mechanism, other than hydrodynamic
radius, to the accessory protein.
[0286] Glycine is a preferred residue that can be used in accessory
polypeptides due to its high ratio of hydrodynamic radius to mass,
or apparent molecular weight to predicated molecular weight.
Glycine does not have a side chain and thus is the smallest
residue, at 70 Da. Because of its small size it provides maximal
rotational freedom and maximum entropy. This makes it difficult for
proteins to bind to sequences with higher frequencies of glycine,
and glycine-rich sequences are highly protease resistant.
[0287] Residues C, W, N, Q, S, T, Y, K, R, H, D, E can form
hydrogen bonds with other residues and thereby support structure
(intermolecular hydrogen bonds) and binding to other proteins
(intermolecular hydrogen bonds). These can be excluded in places
where structure is not desired, or included if some degree of
binding (specific or non-specific) is required for extension of
half-life. The sulfur-containing residues C and M are typically
avoided in accessory polypeptides, but cysteine can be included to
provide half-life via its free thiol and can also be used in cyclic
peptides as low-immunogenicity binding elements to extend half-life
by binding to serum-exposed proteins or to obtain tissue targeting
or modulated biodistribution by binding to tissue specific
sites.
[0288] In one embodiment, the accessory polypeptide contains no or
minimal repetitive sequence. IgM is pentavalent and exhibits
propensity for recognizing repetitive sequences. Even low affinity
contacts with IgM may lead to significant apparent affinity
(avidity) due to the pentavalency of IgM. One way to build
sequences with a reduced degree of repetition and reduced
likelihood of IgM binding is to use repeat sequences that are long
(ie 7, 8, 9, 10, 12, 14, 16, 20, 30, 40, 50, 70, 100, 150, 200
amino acids in repeat length). Examples of sequences with reduced
repetition are (SESSSESSE)n (SEQ ID NO: 48), (SSESSSSESSSE)n (SEQ
ID NO: 49), (SSSESSSSSESSSSE)n (SEQ ID NO: 50), or (SSSSESSSSSSE)n
(SEQ ID NO: 51), whereas the repetitive sequences with similar
overall composition would be (SSE)n, (SSSE)n (SEQ ID NO: 52),
(SSSSE)n (SEQ ID NO: 53), (SSSSSE)n (SEQ ID NO: 54) or (SSSSSSE)n
(SEQ ID NO: 55). Another approach involves the use of multiple
motifs and/or variations of one or more motif intermixed in the
same accessory polypeptide (such as sequence variations of motifs,
spacing variations and variations in the sequences that separate
the motifs). Another aspect of the present invention provides for
the use of long, fully human or humanized sequences that are mostly
non-repetitive and have the desired amino acid composition. In a
related embodiment, other types of amino acids or motifs based on
other types of amino acids can be interspersed. An example would
be: GEGESEGEGEGESEGEGESGE (SEQ ID NO: 56).
[0289] Accessory Polypeptide Sequences Containing Three Different
Types of Aminoacids:
[0290] In one embodiment, the accessory polypeptide comprises a
sequence containing three different types of aminoacids. The
advantage of three amino acids compared to one or two is the
increased ability to fine-tune the properties of the resulting
polymer for the intended commercial applications.
[0291] One particular embodiment of the present invention provides
a non-repetitive sequence containing three different types of
aminoacids. A further embodiment of the invention provides a
non-repetitive sequence containing three different types of
aminoacids, wherein the aminoacids are chosen from the group
consisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y. Exemplary
sequences for this embodiment are shown in Table 1. In a preferred
embodiment, the aminoacids are chosen from the group consisting of
D, E, G, K, P, R, S and T. In a more preferred embodiment, the
aminoacids are chosen from the group consisting of E, S, G, R and
A. In the most preferred embodiment, the aminoacids are E, G and S,
In such proteins, the preferred composition is to have G ranging
from 30-70% (best: 50-60%), E ranging from 20-40% (best) 25-30%)
and S ranging from 10-25%, and preferably with only 1, 2, 3, 4 or 5
copies (repeats) of each sequence with more than 9-15 AA.
[0292] In a separate embodiment, the accessory polypeptide
comprises a sequence containing repeated sequence motifs, wherein
each repeated sequence motif contains three different types of
aminoacids, wherein the aminoacids are chosen from the group
consisting of A, D, E, G, H, K, N, P, Q, R, S, T and Y. Exemplary
sequences for this embodiment are shown in Table 1. In one
embodiment, the aminoacids are chosen from the group consisting of
D, E, G, K, P, R, S and T. In another embodiment, the aminoacids
are chosen from the group consisting of E, S, G, R and A. In yet
another embodiment, the aminoacids are E, G and S (in any
order).
[0293] In a related embodiment, the accessory polypeptide of the
invention contains three different types of aminoacids organized in
repetitive sequence motifs, wherein each repeated sequence motif is
longer than three consecutive aminoacids. Exemplary sequences for
this embodiment are shown in Table 1. Repetitive sequence motifs
can be direct or inverted and 1, 2, 3, 4 or more different types of
motifs can occur separately or intermixed in the same protein. The
repeats can be perfect or imperfect, having 1, 2, 3, 4, 5 or more
mismatched residues, and the repeats can be contiguous or
dispersed, meaning they are separated by other, unrelated sequences
that are not comprised of the same motif. In some embodiments,
repetitive sequences constitute a majority of the accessory
polypeptide, while non-repetitive sequences predominate in other
embodiments. In one particular embodiment, a repetitive sequence
contains interspersed single amino acids which break the strictly
repetitive nature of the sequence. Exemplary sequences for this
embodiment are shown in Table 1. In another related embodiment, the
accessory polypeptide contains primarily three types of aminoacids,
organized in repetitive or non-repetitive sequences, together with
a smaller number of aminoacids of a different type, wherein the
said three types of aminoacid make up for more than 50%, 60%, 70%,
80%, 90%, 95%, 98% or >99% of the entire sequence.
[0294] Another example of a sequence comprising multiple types of
repeated motifs is GGGGGGGGGGEEEEEEEEEEGGGGGGGGGGEEEEEEEEEE (SEQ ID
NO: 57). Other preferred examples are sequences with various
combinations of 2, 3, 4, 5 or more motifs, wherein the motifs are
chosen from E, S, G, GE, GS, SE, GES, GSE, ESG, EGS, SGE, and SEG,
leading to compositions (E)n, (S)n, (G)n, (GE)n, (GS)n, (SE)n,
(GES)n, (GSE)n, (ESG)n, (EGS)n, (SGE)n, and (SEG) as well as many
additional sequences.
[0295] The composition of amino acids in the motif or in the
polymeric sequence can be balanced (for example, 50% G and 50% E;
or 33% G, 33% E and 33% S, and other similar examples) or
unbalanced (ie 75% 5 and 25% E).
[0296] The accessory sequence repeats can be located at the
N-terminus of the protein, at the C-terminus of the protein or 1,
2, 3, 4, 5, 6, 10, 20, 30 or more amino acid residues away from the
N-terminus or C-terminus. The polyamino acid can also lie between
two protein domains.
[0297] The number of repeats of a motif in a polyamino acid can
have a lower limit of 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100 and an upper limit of 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,
150, 200, 250, 300, 400, 500, or even 600.
[0298] A repeated motif can have a major amino acid type and a
minor amino acid type. For a given repeated motif, there are more
residues of the major amino acid type than of the minor type. For
example, in the accessory polypeptide (GGGEE)n (SEQ ID NO: 58), G
is the major and E is the minor amino acid type. These sequences
are by definition not balanced. In such motifs, it is possible to
have 2, 3, 4 or more types of major amino acids. In a preferred
embodiment, the major amino acids are G,E,S, and the minor amino
acids are A,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y, with the
additional limitation that the same amino acid type cannot be in
both the major and minor groups present in the motif. In such
motifs, it is also possible to have two or more types of minor
amino acids; an example is (GGEGGS)n (SEQ ID NO: 59), wherein G is
the major type and E and S are the minor types of amino acids.
Other examples are (EGGSGG)n (SEQ ID NO: 60), (GEGGSG)n (SEQ ID NO:
61), (GGSGGE)n (SEQ ID NO: 62), (SGGEGG)n (SEQ ID NO: 63),
(GSGGEG)n (SEQ ID NO: 64), (GEEGSS)n (SEQ ID NO: 65), (GSSGEE)n
(SEQ ID NO: 66), (SGSEGE)n (SEQ ID NO: 67), (SSGEEG)n (SEQ ID NO:
68).
[0299] Irrespective of the particular sequence, the total number of
amino acid residues in an accessory sequence has a lower limit of
10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 120, 140, 160, 180, 200, 250, or 300 amino acids
and an upper limit of 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
220, 240, 260, 280, 300, 350, 400, 450, 500, 550, or even more than
600, 700, 800, 900 or 1000 amino acids. These numbers can refer to
the length of a single contiguous sequence, or to the cumulative
length total for multiple sequences comprised of multiple motifs
that occur non-contiguously, meaning these repeats are dispersed
and are separated by other sequences including repeats of a
different motif.
TABLE-US-00001 TABLE 1 Accessory polypeptide sequences containing
three different types of amino acids. (DEG)n, (DEK)n, (DEP)n,
(DER)n, (DES)n, (DET)n, (DGK)n, (DGP)n, (DGR)n, (DGS)n, (DET)n,
(DKP)n, (DKR)n, (DKS)n, (DKT)n, (DPR)n, (DPS)n, (DPT)n, (DRK)n,
(DRS)n, (DSE)n, (DSP)n, (DTE)n, (DTG)n, (DTK)n, (DTP)n, (DTR)n,
(DTS)n, (EGD)n, (EGK)n, (EGP)n, (EGR)n, (EGS)n, (EGT)n, (EKD)n,
(EKG)n, (EKP)n, (EKR)n, (EKS)n, (EKT)n, (EPD)n, (EPG)n, (EPK)n,
(EPR)n, (EPS)n, (EPT)n, (ERD)n, (ERG)n, (ERK)n, (ERP)n, (ERS)n,
(ERT)n, (ESD)n, (ESG)n, (ESK)n, (ESP)n, (ESR)n, (EST)n, (ETD)n,
(ETG)n, (ETK)n, (ETP)n, (ETR)n, (ETS)n, (GKD)n, (GKE)n, (GKP)n,
(GKR)n, (GKS)n, (GKT)n, (GPK)n, (GPD)n, (GPE)n, (GPR)n, (GPS)n,
(GPT)n, (GRD)n, (GRE)n, (GRK)n, (DRP)n, (DRS)n, (DRT)n, (GSD)n,
(GSE)n, (GSK)n, (GSP)n, (GST)n, (GTE)n, (GTD)n, (GTK)n, (GTP)n,
(GTR)n, (GTS)n, (KPD)n, (KPE)n, (KPG)n, (KPR)n, (KPS)n, (KPT)n,
(KRD)n, (KRE)n, (KRG)n, (KRP)n, (KRS)n, (KRT)n, (KSD)n, (KSE)n,
(KSG)n, (KSP)n, (KSR)n, (KST)n, (KTD)n, (KTE)n, (KTG)n, (KTP)n,
(KTR)n, (KTS)n, (PRD)n, (PRE)n, (PRG)n, (PRK)n, (PRS)n, (PRT)n,
(PSD)n, (PSE)n, (PSG)n, (PSK)n, (PSP)n, (PSR)n, (PST)n, (PTD)n,
(PTE)n, (PTG)n, (PTK)n, (PTR)n, (PTS)n, (RSD)n, (RSE)n, (RSG)n,
(RSK)n, (RSP)n, (RST)n, (RTD)n, (RTE)n, (RTG)n, (RTK)n, (RTP)n,
(RTS)n, (SED)n, (SEG)n, (SEK)n, (SEP)n, (SER)n, (SET)n, (STD)n,
(STE)n, (STG)n, (STK)n, (STP)n, (STR)n. . . . EEEGGGSSSGEGGSSSGSEE
. . . (SEQ ID NO: 69) . . . ESGGSSEGSSEESGSSEGSE . . . (SEQ ID NO:
70) (EEESSSGGG)n (SEQ ID NO: 71), (EESSGG)n (SEQ ID NO: 72),
(ESGSE)n (SEQ ID NO: 73), (EESGS)n (SEQ ID NO: 74), (ESGGSE)n (SEQ
ID NO: 75) (ESG)n(E)(ESG)n (SEQ ID NO: 76) (ESG)n(P)(ESG)n (SEQ ID
NO: 77)
[0300] Accessory Polypeptides Containing Two Different Types of
Amino Acids:
[0301] In one embodiment, the accessory polypeptide comprises a
sequence containing two different types of aminoacids.
[0302] In a particular embodiment, the accessory polypeptide
comprises a sequence containing two different types of aminoacids,
wherein one of the aminoacids is glycine and the other is D, E, K,
P, R, S, T, A, H, N, Y, L, V, W, M, F, I or C. A more specific
embodiment provides an accessory polypeptide comprising a sequence
containing two different types of aminoacids, wherein one of the
aminoacids is glycine, and wherein glycine makes up 0%, half or
less than half of the entire sequence. In related embodiments, the
accessory polypeptide comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or even 100% glycine residues.
[0303] In different embodiment, the accessory polypeptide comprises
a sequence containing two different types of aminoacids, wherein
one of the aminoacids is serine and the other is D, E, K, P, R, G,
T, A, H, N, Y, L, V, W, M, F, I or C. A more specific embodiment
provides an accessory polypeptide comprising a sequence containing
two different types of aminoacids, wherein one of the aminoacids is
serine, and wherein serine makes up 0%, half or less than half of
the entire sequence. In related embodiments, the accessory
polypeptide comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or even 100% serine residues.
[0304] In a related embodiment, the accessory polypeptide comprises
two different types of amino acids, wherein the amino acids are
represented in equal or about equal amounts (1:1 ratio). In related
embodiments, the two types of amino acids are represented in 1:2,
1:3, 2:3, 3:4 ratios. Example sequences are shown in Table.
[0305] An alternative embodiment of the present invention provides
an accessory polypeptide comprising a sequence containing two
different types of aminoacids, wherein half or less than half of
the total amino acids are A, T, G, D, E or H.
[0306] An alternative embodiment of the present invention provides
an accessory polypeptide comprising a sequence containing two
different types of aminoacids, wherein half or more of the amino
acids are G and half or less than half of the total amino acids are
A, S, T, D, E or H.
[0307] Another embodiment of the present invention provides an
accessory polypeptide comprising a sequence containing two
different types of aminoacids, wherein half or more of the amino
acids are S and half or less than half of the total amino acids are
A, T, G, D, E or H.
[0308] Another embodiment of the present invention provides an
accessory polypeptide comprising a sequence containing two
different types of aminoacids, wherein half or less than half of
the total amino acids are P, R, L, V, Y, W, M, F, I, K or C.
[0309] Accessory polypeptides are also envisioned comprising
repeating sequence motifs, wherein the sequence motifs can consist
of 2, 3, 4, 5, 6, 7, 8, 9 or more aminoacids.
[0310] The composition of amino acids in the motif or in the
polymeric sequence can be balanced (for example, 50% 5 and 50% E),
or unbalanced (i.e., 75% 5 and 25% E).
[0311] The accessory polypeptide repeats can be located at the
N-terminus of the protein, at the C-terminus of the protein or 1,
2, 3, 4, 5, 6, 10, 20, 30 or more amino acid residues away from the
N-terminus or C-terminus. The polyamino acid can also lie between
two protein domains.
[0312] The number of repeats of a motif in a polyamino acid can
have a lower limit of 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 25,
30, 35, 40, 45, 50, 60, 70, 80, 90, 100 and an upper limit of 10,
12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,
150, 200, 250, 300, 400, 500, or even 600.
[0313] Possible motifs comprising two amino acids are
AD,AE,AF,AG,AH,AI,AK,AL,AM,AN,AP,AQ,AR,AS,AT,AV,AW,AY,DA,DE,DF,DG,DH,DI,D-
K,DL,D
M,DN,DP,DQ,DR,DS,DT,DV,DW,DY,EA,ED,EF,EG,EH,EI,EK,EL,EM,EN,EP,EQ,ER-
,ES,ET,EV,EW,E
Y,FA,FD,FE,FG,FH,FI,FK,FL,FM,FN,FP,FQ,FR,FS,FT,FV,FW,FY,GA,GD,GE,GF,GH,GI-
,GK,GL,GM,G
N,GP,GQ,GR,GS,GT,GV,GW,GY,HA,HD,HE,HF,HG,HI,HK,HL,HM,HN,HP,HQ,HR,HS,HT,HV-
,HW,H
Y,IA,ID,IE,IF,IG,IH,IK,IL,IM,IN,IP,IQ,IR,IS,IT,IVIW,IY,KA,KD,KE,KF,K-
G,KH,KI,KL,KM,KN,KP,K
Q,KR,KS,KT,KV,KW,KY,LA,LD,LE,LF,LG,LH,LI,LK,LM,LN,LP,LQ,LR,LS,LT,LV,LW,LY-
,MA,MD,
ME,MF,MG,MH,MI,MK,ML,MN,MP,MQ,MR,MS,MT,MV,MW,MY,NA,ND,NE,NF,NG,NH,-
NI,NK,NL,
NM,NN,NP,NQ,NR,NS,NT,NV,NR,NY,PA,PD,PE,PF,PG,PH,PI,PK,PL,PM,PN,P-
Q,PR,PS,PT,PV,PW,P Y,QA,QD, QE, QF, QG, QH, QI, QK, QL, QM, QN,
QP,QR, QS, QT, QV, QW, QY,RA, RD, RE, RF, RG, RH, RI, RK, RL, RM,
RN, RP, RQ, RR, RS, RT, RV, RW, RY, SA, SD, SE, SF, SG, SH, SI, SK,
SL, SM, SN, SP, SQ, SR, SS, ST, SV, SW, SY, TA,
TD,TE,TF,TG,TH,TI,TK,TL,TM,TN,TP,TQ,TR,TS,TV,TW,TY,VA,VD,VE,VF,VG,VH,VI,V-
K,VL,VM,V
N,VP,VQ,VR,VS,VT,VW,VY,WA,WD,WE,WF,WG,WH,WI,WK,WL,WM,WN,WP,WQ,WR-
,WS,WT,W
V,WY,YA,YD,YE,YF,YG,YH,YI,YK,YL,YM,YN,YP,YQ,YR,YS,YT,YV,YW. Of
these, the preferred 2 amino acid motifs are EG and GE (forming the
polymer EGEGEGEGEGE (SEQ ID NO: 78) and other variants), GS and SG
(forming the polymer GSGSGSGSGSGSGS (SEQ ID NO: 79) and other
variants), ES and SE (forming the polymer SESESESESESESESES (SEQ ID
NO: 80) and other variants). The repeats can also comprise 3, 4, 5,
6 or 7 amino acid residues. It is also possible for the repeats to
comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even
>20 residues. Each such repeat may contain 2, 3, 4, 5 or more
types of amino acids, up to the number of residues present in the
repeat.
[0314] Possible motifs comprising two amino acids are
AD,AE,AF,AG,AH,AI,AK,AL,AM,AN,AP,AQ,AR,AS,AT,AV,AW,AY,DA,DE,DF,DG,DH,DI,D-
K,DL,D
M,DN,DP,DQ,DR,DS,DT,DV,DW,DY,EA,ED,EF,EG,EH,EI,EK,EL,EM,EN,EP,EQ,ER-
,ES,ET,EV,EW,E
Y,FA,FD,FE,FG,FH,FI,FK,FL,FM,FN,FP,FQ,FR,FS,FT,FV,FW,FY,GA,GD,GE,GF,GH,GI-
,GK,GL,GM,G
N,GP,GQ,GR,GS,GT,GV,GW,GY,HA,HD,HE,HF,HG,HI,HK,HL,HM,HN,HP,HQ,HR,HS,HT,HV-
,HW,H
Y,IA,ID,IE,IF,IG,IH,IK,IL,IM,IN,IP,IQ,IR,IS,IT,IV,IW,IY,KA,KD,KE,KF,-
KG,KH,KI,KL,KM,KN,KP,K
Q,KR,KS,KT,KV,KW,KY,LA,LD,LE,LF,LG,LH,LI,LK,LM,LN,LP,LQ,LR,LS,LT,LV,LW,LY-
,MA,MD,
ME,MF,MG,MH,MI,MK,ML,MN,MP,MQ,MR,MS,MT,MV,MW,MY,NA,ND,NE,NF,NG,NH,-
NI,NK,NL,
NM,NN,NP,NQ,NR,NS,NT,NV,NR,NY,PA,PD,PE,PF,PG,PH,PI,PK,PL,PM,PN,P-
Q,PR,PS,PT,PV,PW,P Y,QA,QD, QE, QF, QG, QH, QI, QK, QL, QM, QN,
QP,QR, QS, QT, QV, QW, QY,RA, RD, RE, RF, RG, RH, RI, RK, RL, RM,
RN, RP, RQ, RR, RS, RT, RV, RW, RY, SA, SD, SE, SF, SG, SH, SI, SK,
SL, SM, SN, SP, SQ, SR, SS, ST, SV, SW, SY, TA,
TD,TE,TF,TG,TH,TI,TK,TL,TM,TN,TP,TQ,TR,TS,TV,TW,TY,VA,VD,VE,VF,VG,VH,VI,V-
K,VL,VM,V
N,VP,VQ,VR,VS,VT,VW,VY,WA,WD,WE,WF,WG,WH,WI,WK,WL,WM,WN,WP,WQ,WR-
,WS,WT,W
V,WY,YA,YD,YE,YF,YG,YH,YI,YK,YL,YM,YN,YP,YQ,YR,YS,YT,YV,YW. Of
these, the preferred 2 amino acid motifs are EG and GE (forming the
polymer EGEGEGEGEGE and other variants), GS and SG (forming the
polymer GSGSGSGSGSGSGS and other variants), ES and SE (forming the
polymer SESESESESESESESES and other variants). The repeats can also
comprise 3, 4, 5, 6 or 7 amino acid residues. It is also possible
for the repeats to comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20 or even >20 residues. Each such repeat may contain 2,
3, 4, 5 or more types of amino acids, up to the number of residues
present in the repeat.
[0315] Accessory polypeptide sequences that are closely related to
sequences of human proteins are desirable in some applications as
they carry a diminished risk of inducing an immune reaction in
patients. Such sequences may be used as accessory polypeptides in
some embodiments of the present invention. The relationship of
accessory sequences to human sequences can be assessed by
determining the abundance of partial sequences of said accessory
polypeptide sequences in the human genome. Table 3 shows an example
for the occurrence of 8mer partial sequences. Accessory
polypeptides can be cleaved into a small number of 8mer sequences
as illustrated in Table 3, where the 8mer sequences are underlined.
For each 8mer sequence one can perform a data base search to
identify the number of matches in a data base of human protein
sequences. A similar analysis can be performed for 7mers, 9mers,
10mers, 11 mers, or longer oligomers. One can perform database
analysis searching for complete matches of these partial sequences
or one can search for close homologues. Thus, the stringency of the
search can be tuned to allow a ranking of accessory polypeptides
for their relationship to human proteins. The data in Table 3 shows
several examples of accessory polypeptides which are chosen based
on their close relatedness to human proteins. Of particular
interest are accessory proteins of sequence (SSSSE).sub.n (SEQ ID
NO: 53), (SSSSSSE).sub.n (SEQ ID NO: 55), and (SSSSESSSSSSE).sub.n
(SEQ ID NO: 51) where all 8mer subsequences can be found in several
human proteins.
TABLE-US-00002 TABLE 2 Accessory polypeptides containing two
different types amino acids (DE), (DG), (DK), (DP), (DR), (DS),
(DT), (ED), (EG), (EK), (EP), (ER), (ES), (ET), (GD), (GE), (GK),
(GP), (GR), (GS), (GT), (KD), (KE), (KG), (KP), (KR), (KS), (KT),
(PD), (PE), (PG), (PK), (PR), (PS), (PT), (RD), (RE), (RG), (RK),
(RP), (RS), (RT), (SD), (SE), (SG), (SK), (SP), (SR), (ST), (TD),
(TE), (TG), (TK), (TP, (TR), (TS); (DEE), (DGG), (DKK), (DPP),
(DRR), (DSS), (DTT), (EDD), (EKK), (EPP), (ERR), (ESS), (ETT),
(GDD), (GEE), (GKK), (GPP), (GRR), (GSS), (GTT), (KDD), (KEE),
(KGG), (KPP), (KRR), (KSS), (KTT), (PDD), (PEE), (PGG), (PKK),
(PRR), (PSS), (PTT), (RDD), (REE), (RGG), (RKK), (RPP), (RSS),
(RTT), (SDD), (SEE), (SKK), (SPP), (SRR), (STT), (TDD), (TEE),
(TKK), (TPP), (TRR), (TSS); (DDDEE) (SEQ ID NO: 81), (DDDGG) (SEQ
ID NO: 82), (DDDKK) (SEQ ID NO: 83), (DDDPP) (SEQ ID NO: 84),
(DDDRR) (SEQ ID NO: 85), (DDDSS) (SEQ ID NO: 86), (DDDTT) (SEQ ID
NO: 87), (EEEDD) (SEQ ID NO: 88), (EEEGG) (SEQ ID NO: 89), (EEEKK)
(SEQ ID NO: 90), (EEEPP) (SEQ ID NO: 91), (EEERR) (SEQ ID NO: 92),
(EEESS) (SEQ ID NO: 93), (EEETT) (SEQ ID NO: 94), (GGGDD) (SEQ ID
NO: 95), (GGGEE) (SEQ ID NO: 96), (GGGKK) (SEQ ID NO: 97), (GGGPP)
(SEQ ID NO: 98), (GGGRR) (SEQ ID NO: 99), (KKKDD) (SEQ ID NO: 100),
(KKKEE) (SEQ ID NO: 101), (KKKGG) (SEQ ID NO: 102), (KKKPP) (SEQ ID
NO: 103), (KKKRR) (SEQ ID NO: 104), (KKKSS) (SEQ ID NO: 105),
(KKKTT) (SEQ ID NO: 106), (PPPDD) (SEQ ID NO: 107), (PPPEE) (SEQ ID
NO: 108), (PPPGG) (SEQ ID NO: 109), (PPPKK) (SEQ ID NO: 110),
(PPPRR) (SEQ ID NO: 111), (PPPSS) (SEQ ID NO: 112), (PPPTT) (SEQ ID
NO: 113), (RRRDD) (SEQ ID NO: 114), (RRREE) (SEQ ID NO: 115),
(RRRGG) (SEQ ID NO: 116), (RRRKK) (SEQ ID NO: 117), (RRRPP) (SEQ ID
NO: 118), (RRRSS) (SEQ ID NO: 119), (RRRTT) (SEQ ID NO: 120),
(SSSDD) (SEQ ID NO: 121), (SSSEE) (SEQ ID NO: 122), (SSSGG) (SEQ ID
NO: 123), (SSSKK) (SEQ ID NO: 124), (SSSPP) (SEQ ID NO: 125),
(SSSRR) (SEQ ID NO: 126), (SSSTT) (SEQ ID NO: 127), (TTTDD) (SEQ ID
NO: 128), (TTTEE) (SEQ ID NO: 129), (TTTGG) (SEQ ID NO: 130),
(TTTKK) (SEQ ID NO: 131), (TTTPP) (SEQ ID NO: 132), (TTTRR) (SEQ ID
NO: 133), (TTTSS) (SEQ ID NO: 134). (DDDDEEE) (SEQ ID NO: 135),
(DDDDGGG) (SEQ ID NO: 136), (DDDDKKK) (SEQ ID NO: 137), (DDDDPPP)
(SEQ ID NO: 138), (DDDDRRR) (SEQ ID NO: 139), (DDDDSSS) (SEQ ID NO:
140), (DDDDTTT) (SEQ ID NO: 141), (EEEEDDD) (SEQ ID NO: 142),
(EEEEGGG) (SEQ ID NO: 143), (EEEEKKK) (SEQ ID NO: 144), (EEEEPPP)
(SEQ ID NO: 145), (EEEERRR) (SEQ ID NO: 146), (EEEESSS) (SEQ ID NO:
147), (EEEETTT) (SEQ ID NO: 148), (KKKKDDD) (SEQ ID NO: 149),
(KKKKEEE) (SEQ ID NO: 150), (KKKKGGG) (SEQ ID NO: 151), (KKKKPPP)
(SEQ ID NO: 152), (KKKKRRR) (SEQ ID NO: 153), (KKKSSSS) (SEQ ID NO:
154), (KKKKTTT) (SEQ ID NO: 155), (PPPPDDD) (SEQ ID NO: 156),
(PPPPEEE) (SEQ ID NO: 157), (PPPPGGG) (SEQ ID NO: 158), (PPPKKKK)
(SEQ ID NO: 159), (PPPPRRR) (SEQ ID NO: 160), (PPPPSSS) (SEQ ID NO:
161), (PPPTTT) (SEQ ID NO: 162), (RRRRDDD) (SEQ ID NO: 163),
(RRRREEE) (SEQ ID NO: 164), (RRRRGGG) (SEQ ID NO: 165), (RRRRKKK)
(SEQ ID NO: 166), (RRRRPPP) (SEQ ID NO: 167), (RRRRSSS) (SEQ ID NO:
168), (RRRRTTT) (SEQ ID NO: 169), (SSSSDDD) (SEQ ID NO: 170),
(SSSSEEE) (SEQ ID NO: 171), (SSSSGGG) (SEQ ID NO: 172), (SSSSKKK)
(SEQ ID NO: 173), (SSSSPPP) (SEQ ID NO: 174), (SSSSRRR) (SEQ ID NO:
175), (SSSSTTT) (SEQ ID NO: 176), (TTTTDDD) (SEQ ID NO: 177),
(TTTTEEE) (SEQ ID NO: 178), (TTTTGGG) (SEQ ID NO: 179), (TTTTKKK)
(SEQ ID NO: 180), (TTTTPPP) (SEQ ID NO: 181), (TTTTRRR) (SEQ ID NO:
182), (TTTTSSS) (SEQ ID NO: 183). (DE)n, (DG)n, (DK)n, (DP)n,
(DR)n, (DS)n, (DT)n, (ED)n, (EG)n, (EK)n, (EP)n, (ER)n, (ES)n,
(ET)n, (GD)n, (GE)n, (GK)n, (GP)n, (GR)n, (GS)n, (GT)n, (KD)n,
(KE)n, (KG)n, (KP)n, (KR)n, (KS)n, (KT)n, (PD)n, (PE)n, (PG)n,
(PK)n, (PR)n, (PS)n, (PT)n, (RD)n, (RE)n, (RG)n, (RK)n, (RP)n,
(RS)n, (RT)n, (SD)n, (SE)n, (SG)n, (SK)n, (SP)n, (SR)n, (ST)n,
(TD)n, (TE)n, (TG)n, (TK)n, (TP)n, (TR)n, (TS)n. (DEE)n, (DGG)n,
(DKK)n, (DPP)n, (DRR)n, (DSS)n, (DTT)n, (EDD)n, (EGG)n, (EKK)n,
(EPP)n, (ERR)n, (ESS)n, (ETT)n, (GDD)n, (GEE)n, (GKK)n, (GPP)n,
(GRR)n, (GSS)n, (GTT)n, (KDD)n, (KEE)n, (KGG)n, (KPP)n, (KRR)n,
(KSS)n, (KTT)n, (PDD)n, (PEE)n, (PGG)n, (PKK)n, (PRR)n, (PSS)n,
(PTT)n, (RDD)n, (REE)n, (RGG)n, (RKK)n, (RPP)n, (RSS)n, (RTT)n,
(SDD)n, (SEE)n, (SGG)n, (SKK)n, (SPP)n, (SRR)n, (STT)n, (TDD)n,
(TEE)n, (TGG)n, (TKK)n, (TPP)n, (TRR)n, (TSS)n. (DDE)n, (DDG)n,
(DDK)n, (DDP)n, (DDR)n, (DDS)n, (DDT)n, (EED)n, (EEG)n, (EEK)n,
(EEP)n, (EER)n, (EES)n, (EET)n, (GGD)n, (GGE)n, (GGK)n, (GGP)n,
(GGR)n, (GGS)n, (GGT)n, (KKD)n, (KKE)n, (KKG)n, (KKP)n, (KKR)n,
(KKS)n, (KKT)n, (PPD)n, (PPE)n, (PPG)n, (PPK)n, (PPR)n, (PPS)n,
(PPT)n, (RRD)n, (RRE)n, (RRG)n, (RRK)n, (RRP)n, (RRS)n, (RRT)n,
(SSD)n, (SSE)n, (SSG)n, (SSK)n, (SSP)n, (SSR)n, (SST)n, (TTD)n,
(TTE)n, (TTG)n, (TTK)n, (TTP)n, (TTR)n, (TTS)n. (DDEE)n (SEQ ID NO:
184), (DDGG)n (SEQ ID NO: 185), (DDKK)n (SEQ ID NO: 186), (DDPP)n
(SEQ ID NO: 187), (DDRR)n (SEQ ID NO: 188), (DDSS)n (SEQ ID NO:
189), (DDTT)n (SEQ ID NO: 190), (EEDD)n (SEQ ID NO: 191), (EEGG)n
(SEQ ID NO: 192), (EEKK)n (SEQ ID NO: 193), (EEPP)n (SEQ ID NO:
194), (EERR)n (SEQ ID NO: 195), (EESS)n (SEQ ID NO: 196), (EETT)n
(SEQ ID NO: 197), (GGDD)n (SEQ ID NO: 198), (GGEE)n (SEQ ID NO:
199), (GGKK)n (SEQ ID NO: 200), (GGPP)n (SEQ ID NO: 201), (GGRR)n
(SEQ ID NO: 202), (GGSS)n (SEQ ID NO: 203), (GGTT)n (SEQ ID NO:
204), (KKDD)n (SEQ ID NO: 205), (KKEE)n (SEQ ID NO: 206), (KKGG)n
(SEQ ID NO: 207), (KKPP)n (SEQ ID NO: 208), (KKRR)n (SEQ ID NO:
209), (KKSS)n (SEQ ID NO: 210), (KKTT)n (SEQ ID NO: 211), (PPDD)n
(SEQ ID NO: 212), (PPEE)n (SEQ ID NO: 213), (PPGG)n (SEQ ID NO:
214), (PPKK)n (SEQ ID NO: 215), (PPRR)n (SEQ ID NO: 216), (PPSS)n
(SEQ ID NO: 217), (PPTT)n (SEQ ID NO: 218), (RRDD)n (SEQ ID NO:
219), (RREE)n (SEQ ID NO: 220), (RRGG)n (SEQ ID NO: 221), (RRKK)n
(SEQ ID NO: 222), (RRPP)n (SEQ ID NO: 223), (RRSS)n (SEQ ID NO:
224), (RRTT)n (SEQ ID NO: 225), (SSDD)n (SEQ ID NO: 226), (SSEE)n
(SEQ ID NO: 227), (SSGG)n (SEQ ID NO: 228), (SSKK)n (SEQ ID NO:
229), (SSPP)n (SEQ ID NO: 230), (SSRR)n (SEQ ID NO: 231), (SSTT)n
(SEQ ID NO: 232), (TTDD)n (SEQ ID NO: 233), (TTEE)n (SEQ ID NO:
234), (TTGG)n (SEQ ID NO: 235), (TTKK)n (SEQ ID NO: 236), (TTPP)n
(SEQ ID NO: 237), (TTRR)n (SEQ ID NO: 238), (TTSS)n (SEQ ID NO:
239), (DDDEE)n (SEQ ID NO: 81), (DDDGG)n (SEQ ID NO: 82), (DDDKK)n
(SEQ ID NO: 83), (DDDPP)n (SEQ ID NO: 84), (DDDRR)n (SEQ ID NO:
85), (DDDSS)n (SEQ ID NO: 86), (DDDTT)n (SEQ ID NO: 87), (EEEDD)n
(SEQ ID NO: 88), (EEEGG)n (SEQ ID NO: 89), (EEEKK)n (SEQ ID NO:
90), (EEEPP)n (SEQ ID NO: 91), (EEERR)n (SEQ ID NO: 92), (EEESS)n
(SEQ ID NO: 93), (EEETT)n (SEQ ID NO: 94), (GGGDD)n (SEQ ID NO:
95), (GGGEE)n (SEQ ID NO: 96), (GGGKK)n (SEQ ID NO: 97), (GGGPP)n
(SEQ ID NO: 98), (GGGRR)n (SEQ ID NO: 99), (GGGSS)n (SEQ ID NO:
240), (GGGTT)n (SEQ ID NO: 241), (KKKDD)n (SEQ ID NO: 100),
(KKKEE)n (SEQ ID NO: 101), (KKKGG)n (SEQ ID NO: 102), (KKKPP)n (SEQ
ID NO: 103), (KKKRR)n (SEQ ID NO: 104), (KKKSS)n (SEQ ID NO: 105),
(KKKTT)n (SEQ ID NO: 106), (PPPDD)n (SEQ ID NO: 107), (PPPEE)n (SEQ
ID NO: 108), (PPPGG)n (SEQ ID NO: 109), (PPPKK)n (SEQ ID NO: 110),
(PPPRR)n (SEQ ID NO: 111), (PPPSS)n (SEQ ID NO: 112), (PPPTT)n (SEQ
ID NO: 113), (RRRDD)n (SEQ ID NO: 114), (RRREE)n (SEQ ID NO: 115),
(RRRGG)n (SEQ ID NO: 116), (RRRKK)n (SEQ ID NO: 117), (RRRPP)n (SEQ
ID NO: 118), (RRRSS)n (SEQ ID NO: 119), (RRRTT)n (SEQ ID NO: 120),
(SSSDD)n (SEQ ID NO: 121), (SSSEE)n (SEQ ID NO: 122), (SSSGG)n (SEQ
ID NO: 123), (SSSKK)n (SEQ ID NO: 124), (SSSPP)n (SEQ ID NO: 125),
(SSSRR)n (SEQ ID NO: 126), (SSSTT)n (SEQ ID NO: 127), (TTTDD)n (SEQ
ID NO: 128), (TTTEE)n (SEQ ID NO: 129), (TTTGG)n (SEQ ID NO: 130),
(TTTKK)n (SEQ ID NO: 131), (TTTPP)n (SEQ ID NO: 132), (TTTRR)n (SEQ
ID NO: 133), (TTTSS)n (SEQ ID NO: 134). (DDEEE)n (SEQ ID NO: 242),
(DDGGG)n (SEQ ID NO: 243), (DDKKK)n (SEQ ID NO: 244), (DDPPP)n (SEQ
ID NO: 245), (DDRRR)n (SEQ ID NO: 246), (DDSSS)n (SEQ ID NO: 247),
(DDTTT)n (SEQ ID NO: 248), (EEDDD)n (SEQ ID NO: 249), (EEGGG)n (SEQ
ID NO: 250), (EEKKK)n (SEQ ID NO: 251), (EEPPP)n (SEQ ID NO: 252),
(EERRR)n (SEQ ID NO: 253), (EESSS)n (SEQ ID NO: 254), (EETTT)n (SEQ
ID NO: 255), (GGDDD)n (SEQ ID NO: 256), (GGEEE)n (SEQ ID NO: 257),
(GGKKK)n (SEQ ID NO: 258), (GGPPP)n (SEQ ID NO: 259), (GGRRR)n (SEQ
ID NO: 260), (GGSSS)n (SEQ ID NO: 261), (GGTTT)n (SEQ ID NO: 262),
(KKDDD)n (SEQ ID NO: 263), (KKEEE)n (SEQ ID NO: 264), (KKGGG)n (SEQ
ID NO: 265), (KKPPP)n (SEQ ID NO: 266), (KKRRR)n (SEQ ID NO: 267),
(KKSSS)n (SEQ ID NO: 268), (KKTTT)n (SEQ ID NO: 269), (PPDDD)n (SEQ
ID NO: 270), (PPEEE)n (SEQ ID NO: 271), (PPGGG)n (SEQ ID NO: 272),
(PPKKK)n (SEQ ID NO: 273), (PPRRR)n (SEQ ID NO: 274), (PPSSS)n (SEQ
ID NO: 275), (PPTTT)n (SEQ ID NO: 276), (RRDDD)n (SEQ ID NO: 277),
(RREEE)n (SEQ ID NO: 278), (RRGGG)n (SEQ ID NO: 279), (RRKKK)n (SEQ
ID NO: 280), (RRPPP)n (SEQ ID NO: 281), (RRSSS)n (SEQ ID NO: 282),
(RRTTT)n (SEQ ID NO: 283), (SSDDD)n (SEQ ID NO: 284), (SSEEE)n (SEQ
ID NO: 285), (SSGGG)n (SEQ ID NO: 286), (SSKKK)n (SEQ ID NO: 287),
(SSPPP)n (SEQ ID NO: 288), (SSRRR)n (SEQ ID NO: 289), (SSTTT)n (SEQ
ID NO: 290), (TTDDD)n (SEQ ID NO: 291), (TTEEE)n (SEQ ID NO: 292),
(TTGGG)n (SEQ ID NO: 293), (TTKKK)n (SEQ ID NO: 294), (TTPPP)n (SEQ
ID NO: 295), (TTRRR)n (SEQ ID NO: 296), (TTSSS)n (SEQ ID NO: 297).
(DDDEEE)n (SEQ ID NO: 298), (DDDGGG)n (SEQ ID NO: 299), (DDDKKK)n
(SEQ ID NO: 300), (DDDPPP)n (SEQ ID NO: 301), (DDDRRR)n (SEQ ID NO:
302), (DDDSSS)n (SEQ ID NO: 303), (DDDTTT)n (SEQ ID NO: 304),
(EEEDDD)n (SEQ ID NO: 305), (EEEGGG)n (SEQ ID NO: 306), (EEEKKK)n
(SEQ ID NO: 307), (EEEPPP)n (SEQ ID NO: 308), (EEERRR)n (SEQ ID NO:
309), (EEESSS)n (SEQ ID NO: 310), (EEETTT)n (SEQ ID NO: 311),
(GGGDDD)n (SEQ ID NO: 312), (GGGEEE)n (SEQ ID NO: 313), (GGGKKK)n
(SEQ ID NO: 314), (GGGPPP)n (SEQ ID NO: 315), (GGGRRR)n (SEQ ID NO:
316), (GGGSSS)n (SEQ ID NO: 317), (GGGTTT)n (SEQ ID NO: 318),
(KKKDDD)n (SEQ ID NO: 319), (KKKEEE)n (SEQ ID NO: 320), (KKKGGG)n
(SEQ ID NO: 321), (KKKPPP)n (SEQ ID NO: 322), (KKKRRR)n (SEQ ID NO:
323), (KKKSSS)n (SEQ ID NO: 324), (KKKTTT)n (SEQ ID NO: 325),
(PPPDDD)n (SEQ ID NO: 326),
(PPPEEE)n (SEQ ID NO: 327), (PPPGGG)n (SEQ ID NO: 328), (PPPKKK)n
(SEQ ID NO: 329), (PPPRRR)n (SEQ ID NO: 330), (PPPSSS)n (SEQ ID NO:
331), (PPPTTT)n (SEQ ID NO: 162), (RRRDDD)n (SEQ ID NO: 332),
(RRREEE)n (SEQ ID NO: 333), (RRRGGG)n (SEQ ID NO: 334), (RRRKKK)n
(SEQ ID NO: 335), (RRRPPP)n (SEQ ID NO: 336), (RRRSSS)n (SEQ ID NO:
337), (RRRTTT)n (SEQ ID NO: 338), (SSSDDD)n (SEQ ID NO: 339),
(SSSEEE)n (SEQ ID NO: 340), (SSSGGG)n (SEQ ID NO: 341), (SSSKKK)n
(SEQ ID NO: 342), (SSSPPP)n (SEQ ID NO: 343), (SSSRRR)n (SEQ ID NO:
344), (SSSTTT)n (SEQ ID NO: 345), (TTTDDD)n (SEQ ID NO: 346),
(TTTEEE)n (SEQ ID NO: 347), (TTTGGG)n (SEQ ID NO: 348), (TTTKKK)n
(SEQ ID NO: 349), (TTTPPP)n (SEQ ID NO: 350), (TTTRRR)n (SEQ ID NO:
351), (TTTSSS)n (SEQ ID NO: 352). (DDDDEEE)n (SEQ ID NO: 135),
(DDDDGGG)n (SEQ ID NO: 136), (DDDDKKK)n (SEQ ID NO: 137),
(DDDDPPP)n (SEQ ID NO: 138), (DDDDRRR)n (SEQ ID NO: 139),
(DDDDSSS)n (SEQ ID NO: 140), (DDDDTTT)n (SEQ ID NO: 141),
(EEEEDDD)n (SEQ ID NO: 142), (EEEEGGG)n (SEQ ID NO: 143),
(EEEEKKK)n (SEQ ID NO: 144), (EEEEPPP)n (SEQ ID NO: 145),
(EEEERRR)n (SEQ ID NO: 146), (EEEESSS)n (SEQ ID NO: 147),
(EEEETTT)n (SEQ ID NO: 148), (GGGGDDD)n (SEQ ID NO: 353),
(GGGGEEE)n (SEQ ID NO: 354), (GGGGKKK)n (SEQ ID NO: 355),
(GGGGPPP)n (SEQ ID NO: 356), (GGGGRRR)n (SEQ ID NO: 357),
(GGGGSSS)n (SEQ ID NO: 358), (GGGGTTT)n (SEQ ID NO: 359),
(KKKKDDD)n (SEQ ID NO: 149), (KKKKEEE)n (SEQ ID NO: 150),
(KKKKGGG)n (SEQ ID NO: 151), (KKKKPPP)n (SEQ ID NO: 152),
(KKKKRRR)n (SEQ ID NO: 153), (KKKSSSS)n (SEQ ID NO: 154),
(KKKKTTT)n (SEQ ID NO: 155), (PPPPDDD)n (SEQ ID NO: 156),
(PPPPEEE)n (SEQ ID NO: 157), (PPPPGGG)n (SEQ ID NO: 158),
(PPPKKKK)n (SEQ ID NO: 159), (PPPPRRR)n (SEQ ID NO: 160),
(PPPPSSS)n (SEQ ID NO: 161), (PPPPTTT)n (SEQ ID NO: 360),
(RRRRDDD)n (SEQ ID NO: 163), (RRRREEE)n (SEQ ID NO: 164),
(RRRRGGG)n (SEQ ID NO: 165), (RRRRKKK)n (SEQ ID NO: 166),
(RRRRPPP)n (SEQ ID NO: 167), (RRRRSSS)n (SEQ ID NO: 168),
(RRRRTTT)n (SEQ ID NO: 169), (SSSSDDD)n (SEQ ID NO: 170),
(SSSSEEE)n (SEQ ID NO: 171), (SSSSGGG)n (SEQ ID NO: 172),
(SSSSKKK)n (SEQ ID NO: 173), (SSSSPPP)n (SEQ ID NO: 174),
(SSSSRRR)n (SEQ ID NO: 175), (SSSSTTT)n (SEQ ID NO: 176),
(TTTTDDD)n (SEQ ID NO: 177), (TTTTEEE)n (SEQ ID NO: 178),
(TTTTGGG)n (SEQ ID NO: 179), (TTTTKKK)n (SEQ ID NO: 180),
(TTTTPPP)n (SEQ ID NO: 181), (TTTTRRR)n (SEQ ID NO: 182),
(TTTTSSS)n (SEQ ID NO: 183) (SSSESSESSSSE)n (SEQ ID NO: 361),
(GGEGEGGGE)n (SEQ ID NO: 362)
[0316] Accessory Polypeptide Sequences that are Related to Human
Sequences
[0317] Accessory polypeptide sequences that are closely related to
sequences of human proteins are desirable in some applications as
they carry a diminished risk of inducing an immune reaction in
patients. Such sequences may be used as accessory polypeptides in
some embodiments of the present invention. The relationship of
accessory sequences to human sequences can be assessed by
determining the abundance of partial sequences of said accessory
polypeptide sequences in the human genome. Table 3 shows an example
for the occurrence of 8mer partial sequences. Accessory
polypeptides can be cleaved into a small number of 8mer sequences
as illustrated in Table 3, where the 8mer sequences are underlined.
For each 8mer sequence one can perform a data base search to
identify the number of matches in a data base of human protein
sequences. A similar analysis can be performed for 7mers, 9mers,
10mers, 11mers, or longer oligomers. One can perform database
analysis searching for complete matches of these partial sequences
or one can search for close homologues. Thus, the stringency of the
search can be tuned to allow a ranking of accessory polypeptides
for their relationship to human proteins. The data in Table 3 shows
several examples of accessory polypeptides which are chosen based
on their close relatedness to human proteins. Of particular
interest are accessory proteins of sequence (SSSSE).sub.n,
(SSSSSSE).sub.n, and (SSSSESSSSSSE).sub.n where all 8mer
subsequences can be found in several human proteins.
TABLE-US-00003 TABLE 3 Ranking sequences by their relatedness to
human protein sequences. SEQ hits in ID human Repeating unit 8mers
NOS genome SSESSSSESSSE(SEQ ID SSESSSSESSSESSESSSSESSSE 364 4 NO:
49) SSESSSSESSSESSESSSSESSSE 364 4 SSESSSSESSSESSESSSSESSSE 364 4
SSESSSSESSSESSESSSSESSSE 364 10 SSESSSSESSSESSESSSSESSSE 364 25
SSESSSSESSSESSESSSSESSSE 364 3 SSESSSSESSSESSESSSSESSSE 364 5
SSESSSSESSSESSESSSSESSSE 364 9 SSESSSSESSSESSESSSSESSSE 364 9
SSESSSSESSSESSESSSSESSSE 364 12 SSESSSSESSSESSESSSSESSSE 364 0
SSESSSSESSSESSESSSSESSSE 364 0 SSSSE (SEQ ID NO: 53)
SSSSESSSSESSSSE 365 10 SSSSESSSSESSSSE 365 9 SSSSESSSSESSSSE 365 4
SSSSESSSSESSSSE 365 4 SSSSESSSSESSSSE 365 4 SSSSSE (SEQ ID NO: 54)
SSSSSESSSSSESSSSSE 366 14 SSSSSESSSSSESSSSSE 366 10
SSSSSESSSSSESSSSSE 366 9 SSSSSESSSSSESSSSSE 366 25
SSSSSESSSSSESSSSSE 366 0 SSSSSESSSSSESSSSSE 366 0 SSSSSSE (SEQ ID
NO: 55) SSSSSSESSSSSSE 367 58 SSSSSSESSSSSSE 367 14 SSSSSSESSSSSSE
367 10 SSSSSSESSSSSSE 367 9 SSSSSSESSSSSSE 367 25 SSSSSSESSSSSSE
367 43 SSSSSSESSSSSSE 367 21 SSSSSSESSSSE (SEQ ID
SSSSSSESSSSESSSSSSESSSSE 369 58 NO: 368) SSSSSSESSSSESSSSSSESSSSE
369 14 SSSSSSESSSSESSSSSSESSSSE 369 10 SSSSSSESSSSESSSSSSESSSSE 369
9 SSSSSSESSSSESSSSSSESSSSE 369 4 SSSSSSESSSSESSSSSSESSSSE 369 4
SSSSSSESSSSESSSSSSESSSSE 369 4 SSSSSSESSSSESSSSSSESSSSE 369 10
SSSSSSESSSSESSSSSSESSSSE 369 9 SSSSSSESSSSESSSSSSESSSSE 369 25
SSSSSSESSSSESSSSSSESSSSE 369 43 SSSSSSESSSSESSSSSSESSSSE 369 21
[0318] Unstructured Recombinant Polymers (URPs):
[0319] One aspect of the present invention is the use of
unstructured recombinant polymers (URPs) as accessory polypeptides.
The subject URPs are particularly useful for generating recombinant
proteins of therapeutic and/or diagnostic value. The subject URPs
exhibit one or more following features.
[0320] The subject URPs comprise amino acid sequences that
typically share commonality with denatured peptide sequences under
physiological conditions. URP sequences typically behave like
denatured peptide sequences under physiological conditions. URP
sequences lack well defined secondary and tertiary structures under
physiological conditions. A variety of methods have been
established in the art to ascertain the second and tertiary
structures of a given polypeptide. For example, the secondary
structure of a polypeptide can be determined by CD spectroscopy in
the "far-UV" spectral region (190-250 nm). Alpha-helix, beta-sheet,
and random coil structures each give rise to a characteristic shape
and magnitude of CD spectra. Secondary structure can also be
ascertained via certain computer programs or algorithms such as the
Chou-Fasman algorithm (Chou, P. Y., et al. (1974) Biochemistry, 13:
222-45). For a given URP sequence, the algorithm can predict
whether there exists some or no secondary structure at all. In
general, URP sequences will have spectra that resemble denatured
sequences due to their low degree of secondary and tertiary
structure. Where desired, URP sequences can be designed to have
predominantly denatured conformations under physiological
conditions. URP sequences typically have a high degree of
conformational flexibility under physiological conditions and they
tend to have large hydrodynamic radii (Stokes' radius) compared to
globular proteins of similar molecular weight. As used herein,
physiological conditions refer to a set of conditions including
temperature, salt concentration, pH that mimic those conditions of
a living subject. A host of physiologically relevant conditions for
use in in vitro assays have been established. Generally, a
physiological buffer contains a physiological concentration of salt
and at adjusted to a neutral pH ranging from about 6.5 to about
7.8, and preferably from about 7.0 to about 7.5. A variety of
physiological buffers is listed in Sambrook et al. (1989) supra and
hence is not detailed herein. Physiologically relevant temperature
ranges from about 25.degree. C. to about 38.degree. C., and
preferably from about 30.degree. C. to about 37.degree. C.
[0321] The subject URPs can be sequences with low immunogenicity.
Low immunogenicity can be a direct result of the conformational
flexibility of URP sequences. Many antibodies recognize so-called
conformational epitopes in protein antigens. Conformational
epitopes are formed by regions of the protein surface that are
composed of multiple discontinuous amino acid sequences of the
protein antigen. The precise folding of the protein brings these
sequences into a well-defined special configuration that can be
recognized by antibodies. Preferred URPs are designed to avoid
formation of conformational epitopes. For example, of particular
interest are URP sequences having a low tendency to adapt compactly
folded conformations in aqueous solution. In particular, low
immunogenicity can be achieved by choosing sequences that resist
antigen processing in antigen presenting cells, choosing sequences
that do not bind MHC well and/or by choosing sequences that are
derived from human sequences.
[0322] The subject URPs can be sequences with a high degree of
protease resistance. Protease resistance can also be a result of
the conformational flexibility of URP sequences. Protease
resistance can be designed by avoiding known protease recognition
sites. Alternatively, protease resistant sequences can be selected
by phage display or related techniques from random or semi-random
sequence libraries. Where desired for special applications, such as
slow release from a depot protein, serum protease cleavage sites
can be built into an URP. Of particular interest are URP sequences
with high stability (e.g., long serum half-life, less prone to
cleavage by proteases present in bodily fluid) in blood.
[0323] The subject URP can also be characterized by the effect in
that wherein upon incorporation of it into a biologically active
polypeptide, the modified polypeptide exhibits a longer serum
half-life and/or higher solubility as compared to an unmodified
biologically active polypeptide. The subject URP can be of any
length necessary to effect (a) extension of serum half-life of a
protein comprising the URP; (b) an increase in solubility of the
resulting protein; (c) an increased resistance to protease; and/or
(d) a reduced immunogenicity of the resulting protein that
comprises the URP. Typically, the subject URP has about 30, 40, 50,
60, 70, 80, 90, 100, 150, 200, 300, 400 or more contiguous amino
acids. When incorporated into a protein, the URP can be fragmented
such that the resulting protein contains multiple URPs, or multiple
fragments of URPs. Some or all of these individual URP sequences
may be shorter that 40 amino acids as long as the combined length
of all URP sequences in the resulting protein is at least 40 amino
acids. Preferably, the resulting protein has a combined length of
URP sequences exceeding 40, 50, 60, 70, 80, 90, 100, 150, 200 or
more amino acids.
[0324] URPs may have an isoelectric point (pI) of 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,
9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5 or even 13.0.
[0325] In general, URP sequences are rich in hydrophilic amino
acids and contain a low percentage of hydrophobic or aromatic amino
acids. Suitable hydrophilic residues include but are not limited to
glycine, serine, aspartate, glutamate, lysine, arginine, and
threonine. Hydrophobic residues that are less favored in
construction of URPs include tryptophan, phenylalanine, tyrosine,
leucine, isoleucine, valine, and methionine. URP sequences can be
rich in glycine but URP sequences can also be rich in the amino
acids glutamate, aspartate, serine, threonine, alanine or proline.
Thus the predominant amino acid may be G, E, D, S, T, A or P. The
inclusion of proline residues tends to reduce sensitivity to
proteolytic degradation.
[0326] The inclusion of hydrophilic residues typically increases
URPs' solubility in water and aqueous media under physiological
conditions. As a result of their amino acid composition, URP
sequences have a low tendency to form aggregates in aqueous
formulations and the fusion of URP sequences to other biologically
active polypeptides or peptides tends to enhance their solubility
and reduce their tendency to form aggregates, which is a separate
mechanism to reduce immunogenicity.
[0327] URP sequences can be designed to avoid certain amino acids
that confer undesirable properties to the biologically active
polypeptide. For instance, one can design URP sequences to contain
few or none of the following amino acids: cysteine (to avoid
disulfide formation and oxidation), methionine (to avoid
oxidation), asparagine and glutamine (to avoid desamidation).
[0328] Glycine-Rich URPs:
[0329] In one embodiment, the subject URP comprises a glycine rich
sequence (GRS). For example, glycine can be present predominantly
such that it is the most prevalent residues present in the sequence
of interest. In another example, URP sequences can be designed such
that glycine residues constitute at least about 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the total
amino acids. URPs can also contain 100% glycines. In yet another
example, the URPs contain at least 30% glycine and the total
concentration of tryptophan, phenylalanine, tyrosine, valine,
leucine, and isoleucine is less then 20%. In still another example,
the URPs contain at least 40% glycine and the total concentration
of tryptophan, phenylalanine, tyrosine, valine, leucine, and
isoleucine is less then 10%. In still yet another example, the URPs
contain at least about 50% glycine and the total concentration of
tryptophan, phenylalanine, tyrosine, valine, leucine, and
isoleucine is less then 5%.
[0330] The length of GRS can vary between about 5 amino acids and
200 amino acids or more. For example, the length of a single,
contiguous GRS can contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 240, 280, 320 or
400 or more amino acids. GRS may comprise glycine residues at both
ends.
[0331] GRS can also have a significant content of other amino
acids, for example Ser, Thr, Ala, or Pro. GRS can contain a
significant fraction of negatively charged amino acids including
but not limited to Asp and Glu. GRS can contain a significant
fraction of positively charged amino acids including but not
limited to Arg or Lys. Where desired, URPs can be designed to
contain only a single type of amino acid (i.e., Gly or Glu),
sometimes only a few types of amino acid, e.g., two to five types
of amino acids (e.g., selected from G, E, D, S, T, A and P), in
contrast to typical proteins and typical linkers which generally
are composed of most of the twenty types of amino acids. URPs may
contain negatively charged residues (Asp, Glu) in 30, 25, 20, 15,
12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 percent of the amino acids
positions.
[0332] Typically, the subject GRS-containing URP has about 30, 40,
50, 60, 70, 80, 90, 100, or more contiguous amino acids. When
incorporated into a biologically active polypeptide, the URP can be
fragmented such that the resulting modified polypeptide contains
multiple URPs, or multiple fragments of URPs. Some or all of these
individual URP sequences may be shorter that 40 amino acids as long
as the combined length of all URP sequences in the resulting
polypeptide is at least 30 amino acids. Preferably, the resulting
polypeptide has a combined length of URP sequences exceeding 40,
50, 60, 70, 80, 90, 100, or more amino acids.
[0333] The GRS-containing URPs are of particular interest due to,
in part, the increased conformational freedom of glycine-containing
peptides. Denatured peptides in solution have a high degree of
conformational freedom. Most of that conformational freedom is lost
upon binding of said peptides to a target like a receptor, an
antibody, or a protease. This loss of entropy needs to be offset by
the energy of interaction between the peptide and its target. The
degree of conformational freedom of a denatured peptide is
dependent on its amino acid sequences. Peptides containing many
amino acids with small side chains tend to have more conformational
freedom than peptides that are composed of amino acids with larger
side chains. Peptides containing the amino acid glycine have
particularly large degrees of freedom. It has been estimated that
glycine-containing peptide bonds have about 3.4 times more entropy
in solution as compared to corresponding alanine-containing
sequences (D'Aquino, J. A., et al. (1996) Proteins, 25: 143-56).
This factor increases with the number of glycine residues in a
sequence. As a result, such peptides tend to lose more entropy upon
binding to targets, which reduces their overall ability to interact
with other proteins as well as their ability to adopt defined
three-dimensional structures. The large conformational flexibility
of glycine-peptide bonds is also evident when analyzing
Ramachandran plots of protein structures where glycine peptide
bonds occupy areas that are rarely occupied by other peptide bonds
(Venkatachalam, C. M., et al. (1969) Annu Rev Biochem, 38: 45-82).
Stites et al. studied a database of 12,320 residues from 61
nonhomologous, high resolution crystal structures to determine the
phi, psi conformational preferences of each of the 20 amino acids.
The observed distributions in the native state of proteins are
assumed to also reflect the distributions found in the denatured
state. The distributions were used to approximate the energy
surface for each residue, allowing the calculation of relative
conformational entropies for each residue relative to glycine. In
the most extreme case, replacement of glycine by proline,
conformational entropy changes will stabilize the native state
relative to the denatured state by -0.82+/-0.08 kcal/mol at
20.degree. C. (Stites, W. E., et al. (1995) Proteins, 22: 132).
These observations confirm the special role of glycine among the 20
natural amino acids.
[0334] In designing the subject URPs, natural or non-natural
sequences can be used. For example, a host of natural sequences
containing high glycine content is provided in Table 4, Table 5,
Table 6, and Table 7. One skilled in the art may adopt any one of
the sequences as an URP, or modify the sequences to achieve the
intended properties. Where immunogenicity to the host subject is of
concern, it is preferable to design GRS-containing URRs based on
glycine rich sequences derived from the host. Preferred
GRS-containing URPs are sequences from human proteins or sequences
that share substantial homology to the corresponding glycine rich
sequences in the reference human proteins.
TABLE-US-00004 TABLE 4 Structural analysis of proteins that contain
glycine rich sequences PDB file Protein function Glycine rich
sequences 1K3V Porcine Parvovirus capsid sgggggggggrgagg (SEQ ID
NO: 370) 1FPV Feline Panleukopenia Virus tgsgngsgggggggsgg (SEQ ID
NO: 371) 1IJS CpV strain D, mutant A300d tgsgngsgggggggsgg (SEQ ID
NO: 371) 1MVM Mvm (strain I) virus ggsggggsgggg (SEQ ID NO:
372)
TABLE-US-00005 TABLE 5 Open reading frames encoding GRS with 300 or
more glycine residues Gly GRS Gene Predicted Accession Organism (%)
length length Function NP_974499 Arabidopsis 64 509 579 unknown
thaliana ZP_00458077 Burkholderia 66 373 518 putative cenocopacia
lipoprotein XP_477841 Oryza sativa 74 371 422 unknown NP_910409
Oryza sativa 75 368 400 putative cell-wall precursor NP_610660
Drosophila 66 322 610 transposable melanogaster element
TABLE-US-00006 TABLE 6 Examples of human GRS Gly GRS Gene Predicted
Accession (%) length length Hydrophobics Function NP_000217 62 135
622 yes keratin 9 NP_631961 61 73 592 yes TBP-associated factor 15
isoform 1 NP_476429 65 70 629 yes keratin 3 NP_000418 70 66 316 yes
loricrin, cell envelope NP_056932 60 66 638 yes cytokeratin 2
TABLE-US-00007 TABLE 7 Additional examples of human GRS Accession
Sequences Number of amino acids NP_006228.
GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG 37 NP_787059
GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG 33 NP_009060
GGGSGSGGAGGGSGGGSGSGGGGGGAGGGGGG 32 NP_031393
GDGGGAGGGGGGGGSGGGGSGGGGGGG 27 NP_005850 GSGSGSGGGGGGGGGGGGSGGGGGG
25 NP_061856 GGGRGGRGGGRGGGGRGGGRGGG 23 NP_787059
GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG 33 NP_009060
GGGSGSGGAGGGSGGGSGSGGGGGGAGGGGGG 32 NP_031393
GDGGGAGGGGGGGGSGGGGSGGGGGGG 27 NP_115818 GSGGSGGSGGGPGPGPGGGGG 21
XP_376532 GEGGGGGGEGGGAGGGSG 18 NP_065104 GGGGGGGGDGGG 12
TABLE-US-00008 (Table 7 discloses SEQ ID NOS 373-378, 374-376 and
379-381, respectively, in order of appearance.)
GGGSGSGGAGGGSGGGSGSGGGGGGAGGGGGGSSGGGSGTAGGHSG (SEQ ID NO: 382) POU
domain, class 4, transcription factor 1 [Homo sapiens]
GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG (SEQ ID NO: 373) YEATS domain
containing 2 [Homo sapiens] GGSGAGGGGGGGGGGGSGSGGGGSTGGGGGTAGGG
(SEQ ID NO: 383) AT rich interactive domain 1B (SWI1-like) isoform
3; BRG1-binding protein ELD/OSA1; Eld (eyelid)/Osa protein [Homo
sapiens] GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG (SEQ ID NO: 374) AT rich
interactive domain 1B (SWI1-like) isoform 2; BRG1-binding protein
ELD/OSA1; Eld (eyelid)/Osa protein [Homo sapiens]
GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG (SEQ ID NO: 374) AT rich
interactive domain 1B (SWI1-like) isoform 1; BRG1-binding protein
ELD/OSA1; Eld (eyelid)/Osa protein [Homo sapiens]
GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG (SEQ ID NO: 374) purine-rich
element binding protein A; purine-rich single-stranded DNA-binding
protein alpha; transcrip- tional activator protein PUR-alpha [Homo
sapiens] GHPGSGSGSGGGGGGGGGGGGSGGGGGGAPGG (SEQ ID NO: 384)
regulatory factor X1; trans-acting regulatory factor 1; enhancer
factor C; MHC class II regulatory factor RFX [Homo sapiens]
GGGGSGGGGGGGGGGGGGGSGSTGGGGSGAG (SEQ ID NO: 385) bromo
domain-containing protein disrupted in leukemia [Homo sapiens
GGRGRGGRGRGSRGRGGGGTRGRGRGRGGRG (SEQ ID NO: 386) unknown protein
[Homo sapiens] GSGGSGGSGGGPGPGPGGGGGPSGSGSGPG (SEQ ID NO: 387)
PREDICTED: hypothetical protein XP_059256 [Homo sapiens]
GGGGGGGGGGGRGGGGRGGGRGGGGEGGG (SEQ ID NO: 388) zinc finger protein
281; ZNP-99 transcription factor [Homo sapiens]
GGGGTGSSGGSGSGGGGSGGGGGGGSSG (SEQ ID NO: 389) RNA binding protein
(autoantigenic, hnRNP-associated with lethal yellow) short isoform;
RNA-binding pro- tein (autoantigenic); RNA-binding protein
(autoantigenic, hnRNP-associated with lethal yellow) [Homo sapiens]
GDGGGAGGGGGGGGSGGGGSGGGGGGG (SEQ ID NO: 376) signal recognition
particle 68 kDa [Homo sapiens] GGGGGGGSGGGGGSGGGGSGGGRGAGG (SEQ ID
NO: 390) KIAA0265 protein [Homo sapiens]
GGGAAGAGGGGSGAGGGSGGSGGRGTG (SEQ ID NO: 391) engrailed homolog 2;
Engrailed-2 [Homo sapiens GAGGGRGGGAGGEGGASGAEGGGGAGG (SEQ ID NO:
392) RNA binding protein (autoantigenic, hnRNP-associated with
lethal yellow) long isoform; RNA-binding pro- tein (autoantigenic);
RNA-binding protein (autoantigenic, hnRNP-associated with lethal
yellow) [Homo sapiens] GDGGGAGGGGGGGGSGGGGSGGGGGGG (SEQ ID NO: 376)
androgen receptor; dihydrotestosterone receptor [Homo sapiens]
GGGGGGGGGGGGGGGGGGGGGGGEAG (SEQ ID NO: 393) homeo box D11; homeo
box 4F; Hox-4.6, mouse, homolog of; homeobox protein Hox-D11 [Homo
sapiens] GGGGGGSAGGGSSGGGPGGGGGGAGG (SEQ ID NO: 394) frizzled 8;
frizzled (Drosophila) homolog 8 [Homo sapiens]
GGGGGPGGGGGGGPGGGGGPGGGGG (SEQ ID NO: 395) ocular
development-associated gene [Homo sapiens]
GRGGAGSGGAGSGAAGGTGSSGGGG (SEQ ID NO: 396) homeo box B3; homeo box
2G; homeobox protein Hox-B3 [Homo sapiens]
GGGGGGGGGGGSGGSGGGGGGGGGG (SEQ ID NO: 397) chromosome 2 open
reading frame 29 [Homo sapiens] GGSGGGRGGASGPGSGSGGPGGPAG (SEQ ID
NO: 398) DKFZP564F0522 protein [Homo sapiens]
GGHHGDRGGGRGGRGGRGGRGGRAG (SEQ ID NO: 399) PREDICTED: similar to
Homeobox even-skipped homolog protein 2 (EVX-2) [Homo sapiens
GSRGGGGGGGGGGGGGGGGAGAGGG (SEQ ID NO: 400) ras homolog gene family,
member U; Ryu GTPase; Wnt-1 responsive Cdc42 homolog;
2310026M05Rik; GTP- binding protein like 1; CDC42-like GTPase [Homo
sapiens] GGRGGRGPGEPGGRGRAGGAEGRG (SEQ ID NO: 401) scratch 2
protein; transcriptional repressor scratch 2; scratch (drosophila
homolog) 2, zinc finger pro- tein [Homo sapiens]
GGGGGDAGGSGDAGGAGGRAGRAG (SEQ ID NO: 402) nucleolar protein family
A, member 1; GAR1 protein [Homo sapiens] GGGRGGRGGGRGGGGRGGGRGGG
(SEQ ID NO: 378) keratin 1; Keratin-1; cytokeratin 1; hair alpha
protein [Homo sapiens] GGSGGGGGGSSGGRGSGGGSSGG (SEQ ID NO: 403)
hypothetical protein FLJ31413 [Homo sapiens]
GSGPGTGGGGSGSGGGGGGSGGG (SEQ ID NO: 404) one cut domain, family
member 2; one cut 2 [Homo sapiens] GARGGGSGGGGGGGGGGGGGGPG (SEQ ID
NO: 405) POU domain, class 3, transcription factor 2 [Homo sapiens]
GGGGGGGGGGGGGGGGGGGGGDG (SEQ ID NO: 406) PREDICTED: similar to THO
complex subunit 4 (Tho4) (RNA and export factor binding protein 1)
(REF1-I) (Ally of AML-1 and LEF-1) (Aly/REF) [Homo sapiens]
GGTRGGTRGGTRGGDRGRGRGAG (SEQ ID NO: 407) PREDICTED: similar to THO
complex subunit 4 (Tho4) (RNA and export factor binding protein 1)
(REF1-I) (Ally of AML-1 and LEF-1) (Aly/REF) [Homo sapiens]
GGTRGGTRGGTRGGDRGRGRGAG (SEQ ID NO: 407) POU domain, class 3,
transcription factor 3 [Homo sapiens] GAGGGGGGGGGGGGGGAGGGGGG (SEQ
ID NO: 408) nucleolar protein family A, member 1; GAR1 protein
[Homo sapiens] GGGRGGRGGGRGGGGRGGGRGGG (SEQ ID NO: 378)
fibrillarin; 34-kD nucleolar scleroderma antigen; RNA, U3 small
nucleolar interacting protein 1 [Homo sapiens]
GRGRGGGGGGGGGGGGGRGGGG (SEQ ID NO: 409) zinc finger protein 579
[Homo sapiens] GRGRGRGRGRGRGRGRGRGGAG (SEQ ID NO: 410) calpain,
small subunit 1; calcium-activated neutral proteinase; calpain,
small polypeptide; calpain 4, small subunit (30K);
calcium-dependent protease, small subunit [Homo sapiens]
GAGGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 411) keratin 9 [Homo sapiens]
GGGSGGGHSGGSGGGHSGGSGG (SEQ ID NO: 412) forkhead box D1;
forkhead-related activator 4; Forkhead, drosophila, homolog-like 8;
forkhead (Drosophila)-like 8 [Homo sapiens] GAGAGGGGGGGGAGGGGSAGSG
(SEQ ID NO: 413) PREDICTED: similar to RIKEN cDNA C230094B15 [Homo
sapiens] GGPGTGSGGGGAGTGGGAGGPG (SEQ ID NO: 414)
GGGGGGGGGAGGAGGAGSAGGG (SEQ ID NO: 415) cadherin 22 precursor;
ortholog of rat PB-cadherin [Homo sapiens] GGDGGGSAGGGAGGGSGGGAG
(SEQ ID NO: 416) AT-binding transcription factor 1; AT
motif-binding factor 1 [Homo sapiens] GGGGGGSGGGGGGGGGGGGGG (SEQ ID
NO: 417) eomesodermin; t box, brain, 2; eomesodermin (Xenopus
laevis) homolog [Homo sapiens] GPGAGAGSGAGGSSGGGGGPG (SEQ ID NO:
418) phosphatidylinositol transfer protein, membrane-associated 2;
PYK2 N-terminal domain-interacting receptor 3; retinal degeneration
B alpha 2 (Drosophila) [Homo sapiens] GGGGGGGGGGGSSGGGGSSGG (SEQ ID
NO: 419) sperm associated antigen 8 isoform 2; sperm membrane
protein 1 [Homo sapiens] GSGSGPGPGSGPGSGPGHGSG (SEQ ID NO: 420)
PREDICTED: RNA binding motif protein 27 [Homo sapiens]
GPGPGPGPGPGPGPGPGPGPG (SEQ ID NO: 421) AP1 gamma subunit binding
protein 1 isoform 1; gamma-synergin; adaptor-related protein
complex 1 gamma subunit-binding protein 1 [Homo sapiens]
GAGSGGGGAAGAGAGSAGGGG (SEQ ID NO: 422) AP1 gamma subunit binding
protein 1 isoform 2; gamma-synergin; adaptor-related protein
complex 1 gamma subunit-binding protein 1 [Homo sapiens]
GAGSGGGGAAGAGAGSAGGGG (SEQ ID NO: 422) ankyrin repeat and sterile
alpha motif domain containing 1; ankyrin repeat and SAM domain
containing 1 [Homo sapiens] GGGGGGGSGGGGGGSGGGGGG (SEQ ID NO: 423)
methyl-CpG binding domain protein 2 isoform 1 [Homo sapiens]
GRGRGRGRGRGRGRGRGRGRG (SEQ ID NO: 424) triple functional domain
(PTPRF interacting) [Homo sapiens] GGGGGGGSGGSGGGGGSGGGG (SEQ ID
NO: 425) forkhead box D3 [Homo sapiens GGEEGGASGGGPGAGSGSAGG (SEQ
ID NO: 426) sperm associated antigen 8 iso form 1; sperm membrane
protein 1 [Homo sapiens] GSGSGPGPGSGPGSGPGHGSG (SEQ ID NO: 420)
methyl-CpG binding domain protein 2 testis-specific isoform [Homo
sapiens] GRGRGRGRGRGRGRGRGRGRG (SEQ ID NO: 424) cell death
regulator aven; programmed cell death 12 [Homo sapiens]
GGGGGGGGDGGGRRGRGRGRG (SEQ ID NO: 427) regulator of nonsense
transcripts 1; delta helicase; up-frameshift mutation 1 homolog (S.
cerevisiae); nonsense mRNA reducing factor 1; yeast Upf1p homolog
[Homo sapiens] GGPGGPGGGGAGGPGGAGAG (SEQ ID NO: 428) small
conductance calcium-activated potassium channel protein 2 isoform
a; apamin-sensitive small- conductance Ca2+-activated potassium
channel [Homo sapiens] GTGGGGSTGGGGGGGGSGHG (SEQ ID NO: 429) SRY
(sex determining region Y)-box 1; SRY-related HMG-box gene 1 [Homo
sapiens] GPAGAGGGGGGGGGGGGGGG (SEQ ID NO: 430) transcription factor
20 isoform 2; stromelysin-1 platelet-derived growth
factor-responsive element binding protein; stromelysin 1
PDGF-responsive element-binding protein; SPRE-binding protein;
nuclear factor SPBP [Homo sapiens] GGTGGSSGSSGSGSGGGRRG (SEQ ID NO:
431) transcription factor 20 isoform 1; stromelysin-1
platelet-derived growth factor-responsive element binding protein;
stromelysin 1 PDGF-responsive element-binding protein; SPRE-binding
protein; nuclear
factor SPBP [Homo sapiens] GGTGGSSGSSGSGSGGGRRG (SEQ ID NO: 431)
Ras-interacting protein 1 [Homo sapiens] GSGTGTTGSSGAGGPGTPGG (SEQ
ID NO: 432) BMP-2 inducible kinase isoform b [Homo sapiens]
GGSGGGAAGGGAGGAGAGAG (SEQ ID NO: 433) BMP-2 inducible kinase
isoform a [Homo sapiens] GGSGGGAAGGGAGGAGAGAG (SEQ ID NO: 433)
forkhead box C1; forkhead-related activator 3; Forkhead,
drosophila, homolog-like 7; forkhead (Drosophila)-like 7;
iridogoniodysgenesis type 1 [Homo sapiens] GSSGGGGGGAGAAGGAGGAG
(SEQ ID NO: 434) splicing factor p54; arginine-rich 54 kDa nuclear
protein [Homo sapiens] GPGPSGGPGGGGGGGGGGGG (SEQ ID NO: 435) v-maf
musculoaponeurotic fibrosarcoma oncogene homolog; Avian
musculoaponeurotic fibrosarcoma (MAF) protooncogene; v-maf
musculoaponeurotic fibrosarcoma (avian) oncogene homolog [Homo
sapiens] GGGGGGGGGGGGGGAAGAGG (SEQ ID NO: 436) small nuclear
ribonucleoprotein D1 polypeptide 16 kDa; snRNP core protein Dl;
Sm-D autoantigen; small nuclear ribonucleoprotein D1 polypeptide
(16 kD) [Homo sapiens] GRGRGRGRGRGRGRGRGRGG (SEQ ID NO: 410)
hypothetical protein H41 [Homo sapiens] GSAGGSSGAAGAAGGGAGAG (SEQ
ID NO: 437)
[0335] URPs Containing Non-Glycine Residues (NGR):
[0336] The sequences of non-glycine residues in these GRS can be
selected to optimize the properties of URPs and hence the
biologically active polypeptides that contain the desired URPs. For
instance, one can optimize the sequences of URPs to enhance the
selectivity of the resulting modified polypeptide for a particular
tissue, specific cell type or cell lineage. For example, one can
incorporate protein sequences that are not ubiquitously expressed,
but rather are differentially expressed in one or more of the body
tissues including heart, liver, prostate, lung, kidney, bone
marrow, blood, skin, bladder, brain, muscles, nerves, and selected
tissues that are affected by diseases such as infectious diseases,
autoimmune disease, renal, neuronal, cardiac disorders and cancers.
One can employ sequences representative of a specific developmental
origin, such as those expressed in an embryo or an adult, during
ectoderm, endoderm or mesoderm formation in a multi-cellular
organism. One can also utilize sequence involved in a specific
biological process, including but not limited to cell cycle
regulation, cell differentiation, apoptosis, chemotaxis, cell
motility and cytoskeletal rearrangement. One can also utilize other
non-ubiquitously expressed protein sequences to direct the
resulting protein to a specific subcellular locations:
extracellular matrix, nucleus, cytoplasm, cytoskeleton, plasma
and/or intracellular membranous structures which include but are
not limited to coated pits, Golgi apparatus, endoplasmic reticulum,
endosome, lysosome, and mitochondria.
[0337] URPs can be derived from human sequences. The human genome
contains many subsequences that are rich in one particular amino
acid. Of particular interest are such amino acid sequences that are
rich in a hydrophilic amino acid like serine, threonine, glutamate,
aspartate, or glycine. Of particular interest are such subsequences
that contain few hydrophobic amino acids. Such subsequences are
predicted to be unstructured and highly soluble in aqueous
solution. Such human subsequences can be modified to further
improve their utility. For example, dentin sialophosphoprotein
contains a 670-amino acid subsequence in which 64% of the residues
are serine and most other positions are hydrophilic amino acids
such as aspartate, asparagines, and glutamate. The sequence is
extremely repetitive and as a result it has a low information
content. One can directly use subsequences of such a human protein.
Where desired, one can modify the sequence in a way that preserves
its overall character but which makes it more suitable for
pharmaceutical applications. Examples of sequences that are related
to dentin sialophosphoprotein are (SSD).sub.n (SEQ ID NO: 438),
(SSDSSN).sub.n (SEQ ID NO: 439), (SSE).sub.n (SEQ ID NO: 440),
where n is between about 4 and 200.
[0338] Of particular interest are URP sequences that contain
regions that are relatively rich in the positively charged amino
acids arginine or lysine which favor cellular uptake or transport
through membranes. URP sequences can be designed to contain one or
several protease-sensitive sequences. Such URP sequences can be
cleaved once the product of the invention has reached its target
location. This cleavage may trigger an increase in potency of the
pharmaceutically active domain (pro-drug activation) or it may
enhance binding of the cleavage product to a receptor. URP
sequences can be designed to carry excess negative charges by
introducing aspartic acid or glutamic acid residues. Of particular
interest are URP that contain greater than 5%, greater than 6%, 7%,
8%, 9%, 10%, 15%, 30% or more glutamic acid and less than 2% lysine
or arginine. Such URPs carry an excess negative charge and as a
result they have a tendency to adopt open conformations due to
electrostatic repulsion between individual negative charges of the
peptide. Such an excess negative charge leads to an effective
increase in their hydrodynamic radius and as a result it can lead
to reduced kidney clearance of such molecules. Thus, one can
modulate the effective net charge and hydrodynamic radius of a URP
sequence by controlling the frequency and distribution of
negatively charged amino acids in the URP sequences. Most tissues
and surfaces in a human or animal carry excess negative charges. By
designing URP sequences to carry excess negative charges one can
minimize non-specific interactions between the resulting modified
polypeptide comprising the URP and various surfaces such as blood
vessels, healthy tissues, or various receptors.
[0339] URPs may have a repetitive amino acid sequence of the format
(Motif).sub.x in which a sequence motif forms a direct repeat (ie
ABCABCABCABC) or an inverted repeat (ABCCBAABCCBA) and the number
of these repeats can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 35, 40, 50 or more. URPs or the repeats
inside URPs often contain only 1, 2, 3, 4, 5 or 6 different types
of amino acids. URPs typically consist of repeats of human amino
acid sequences that are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36 or more amino
acids long, but URPs may also consist of non-human amino acid
sequences that are 20, 22, 24, 26, 28, 30, 32, 34 36, 38 40, 42,
44, 46, 48, 50 amino acids long.
[0340] URPs Derived from Human Sequences:
[0341] URPs can be derived from human sequences. The human genome
contains many subsequences that are rich in one particular amino
acid. Of particular interest are such amino acid sequences that are
rich in a hydrophilic amino acid like serine, threonine, glutamate,
aspartate, or glycine. Of particular interest are such subsequences
that contain few hydrophobic amino acids. Such subsequences are
predicted to be unstructured and highly soluable in aqeuous
solution. Such human subsequences can be modified to further
improve their utility. For example, dentin sialophosphoprotein
contains a 670-amino acid subsequence in which 64% of the residues
are serine and most other positions are hydrophilic amino acids
such as aspartate, asparagines, and glutamate. The sequence is
extremely repetitive and as a result it has a low information
content. One can directly use subsequences of such a human protein.
Where desired, one can modify the sequence in a way that preserves
its overall character but which makes it more suitable for
pharmaceutical applications. Examples of sequences that are related
to dentin sialophosphoprotein are (SSD).sub.n, (SSDSSN).sub.n,
(SSE).sub.n, where n is between about 4 and 200.
[0342] The use of sequences from human proteins is particularly
desirable in design of URPs with reduced immunogenicity in a human
subject. A key step for eliciting an immune response to a foreign
protein is the presentation of peptide fragments of said protein by
MHC class II receptors. These MHCII-bound fragments can then be
detected by T cell receptors, which triggers the proliferation of T
helper cells and initiates an immune response. The elimination of T
cell epitopes from pharmaceutical proteins has been recognized as a
means to reduce the risk of eliciting an immune reaction (Stickler,
M., et al. (2003) J Immunol Methods, 281: 95-108). MHCII receptors
typically interact with an epitope having e.g., a 9-amino acid long
region of the displayed peptides. Thus, one can reduce the risk of
eliciting an immune response to a protein in patients if all or
most of the possible 9mer subsequences of the protein can be found
in human proteins and if so, these sequences and repeats of these
sequences will not be recognized by the patient as foreign
sequences. One can incorporate human sequences into the design of
URP sequences by oligomerizing or concatenating human sequences
that have suitable amino acid compositions. These can be direct
repeats or inverted repeats or mixtures of different repeats. For
instance one can oligomerize the sequences shown in table 5. Such
oligomers have reduced risk of being immunogenic. However, the
junction sequences between the monomer units can still contain T
cell epitopes that can trigger an immune reaction. One can further
reduce the risk of eliciting an immune response by designing URP
sequences based on multiple overlapping human sequences. An URP
sequence may be designed as an oligomer based on multiple human
sequences such that each 9mer subsequences of the oligomer can be
found in a human protein. In these designs, every 9-mer subsequence
is a human sequence. For example an URP sequence may be based on
three human sequences. It is also possible to design URP sequences
based on a single human sequences such that all possible 9mer
subsequences in the oligomeric URP sequences occur in the same
human protein. Non-oligomeric URP sequences can be designed based
on human proteins as well. The primary conditions are that all 9mer
sub-sequences can be found in human sequences. The amino acid
composition of the sequences preferably contains few hydrophobic
residues. Of particular interest are URP sequences that are
designed based on human sequences and that contain a large fraction
of glycine residues.
[0343] Utilizing this or similar scheme, one can design a class of
URPs that comprise repeat sequences with low immunogenicity to the
host of interest. Host of interest can be any animals, including
vertebrates and invertebrates. Preferred hosts are mammals such as
primates (e g chimpanzees and humans), cetaceans (e.g. whales and
dolphins), chiropterans (e.g. bats), perrisodactyls (e.g. horses
and rhinoceroses), rodents (e.g. rats), and certain kinds of
insectivores such as shrews, moles and hedgehogs. Where human is
selected as the host, the URPs typically contain multiple copies of
the repeat sequences or units, wherein the majority of segments
comprising about 6 to about 15 contiguous amino acids are present
in one or more native human proteins. One can also design URPs in
which the majority of segments comprising between about 9 to about
15 contiguous amino acids are found in one or more native human
proteins. As used herein, majority of the segments refers to more
than about 50%, preferably 60%, preferably 70%, preferably 80%,
preferably 90%, preferably 100%. Where desired, each of the
possible segments between about 6 to 15 amino acids, preferably
between about 9 to 15 amino acids within the repeating units are
present in one or more native human proteins. The URPs can comprise
multiple repeating units or sequences, for example having 2, 3, 4,
5, 6, 7, 8, 9, 10, or more repeating units.
[0344] Design of URPs that are Substantially Free of Human T-Cell
Epitopes:
[0345] Non-limiting examples of URPs containing repeating amino
acids are: poly-glycine, poly-glutamic acid, poly-aspartic acid,
poly-serine, poly-threonine, (GX).sub.n (SEQ ID NO: 441) where G is
glycine and X is serine, aspartic acid, glutamic acid, threonine,
or proline and n is at least 20, (GGX).sub.n (SEQ ID NO: 442) where
X is serine, aspartic acid, glutamic acid, threonine, or proline
and n is at least 13, (GGGX).sub.n (SEQ ID NO: 443) where X is
serine, aspartic acid, glutamic acid, threonine, or proline and n
is at least 10, (GGGGX).sub.n (SEQ ID NO: 444) where X is serine,
aspartic acid, glutamic acid, threonine, or proline and n is at
least 8, (G.sub.zX).sub.n (SEQ ID NO: 445) where X is serine,
aspartic acid, glutamic acid, threonine, or proline, n is at least
15, and z is between 1 and 20.
[0346] URP sequences can be designed to optimize protein
production. This can be achieved by avoiding or minimizing
repetitiveness of the encoding DNA. URP sequences such as
poly-glycine may have very desirable pharmaceutical properties but
their manufacturing can be difficult due to the high GC-content of
DNA sequences encoding for GRS and due to the presence of repeating
DNA sequences that can lead to recombination.
[0347] As noted above, URP sequences can be designed to be highly
repetitive at the amino acid level. As a result the URP sequences
have very low information content and the risk of eliciting an
immune reaction can be reduced.
[0348] Non-limiting examples of URPs containing repeating amino
acids are: poly-glycine, poly-glutamic acid, poly-aspartic acid,
poly-serine, poly-threonine, (GX).sub.n where G is glycine and X is
serine, aspartic acid, glutamic acid, threonine, or proline and n
is at least 20, (GGX).sub.n where X is serine, aspartic acid,
glutamic acid, threonine, or proline and n is at least 13,
(GGGX).sub.n where X is serine, aspartic acid, glutamic acid,
threonine, or proline and n is at least 10, (GGGGX).sub.n where X
is serine, aspartic acid, glutamic acid, threonine, or proline and
n is at least 8, (G.sub.zX).sub.n where X is serine, aspartic acid,
glutamic acid, threonine, or proline, n is at least 15, and z is
between 1 and 20.
[0349] The number of these repeats can be any number between 10 and
100. Products of the invention may contain URP sequences that are
semi-random sequences. Examples are semi-random sequences
containing at least 30, 40, 50, 60 or 70% glycine in which the
glycines are well dispersed and in which the total concentration of
tryptophan, phenylalanine, tyrosine, valine, leucine, and
isoleucine is less then 70, 60, 50, 40, 30, 20, or 10% when
combined. A preferred semi-random URP sequence contains at least
40% glycine and the total concentration of tryptophan,
phenylalanine, tyrosine, valine, leucine, and isoleucine is less
then 10%. A more preferred random URP sequence contains at least
50% glycine and the total concentration of tryptophan,
phenylalanine, tyrosine, valine, leucine, and isoleucine is less
then 5%. URP sequences can be designed by combining the sequences
of two or more shorter URP sequences or fragments of URP sequences.
Such a combination allows one to better modulate the pharmaceutical
properties of the product containing the URP sequences and it
allows one to reduce the repetitiveness of the DNA sequences
encoding the URP sequences, which can improve expression and reduce
recombination of the URP encoding sequences.
[0350] URP sequences can be designed and selected to possess
several of the following desired properties: a) high genetic
stability of the coding sequences in the production host, b) high
level of expression, c) low (predicted/calculated) immunogenicity,
d) high stability in presence of serum proteases and/or other
tissue proteases, e) large hydrodynamic radius under physiological
conditions. One exemplary approach to obtain URP sequences that
meet multiple criteria is to construct a library of candidate
sequences and to identify from the library the suitable
subsequences. Libraries can comprise random and/or semi-random
sequences. Of particular utility are codon libraries, which is a
library of DNA molecules that contains multiple codons for the
identical amino acid residue. Codon randomization can be applied to
selected amino acid positions of a certain type or to most or all
positions. True codon libraries encode only a single amino acid
sequence, but they can easily be combined with amino acid
libraries, which is a population of DNA molecules encoding a
mixture of (related or unrelated) amino acids at the same residue
position. Codon libraries allow the identification of genes that
have relatively low repetitiveness at the DNA level but that encode
highly repetitive amino acid sequences. This is useful because
repetitive DNA sequences tend to recombine, leading to instability.
One can also construct codon libraries that encode limited amino
acid diversity. Such libraries allow introduction of a limited
number of amino acids in some positions of the sequence while other
positions allow for codon variation but all codons encode the same
amino acid. One can synthesize partially random oligonucleotides by
incorporating mixtures of nucleotides at the same position during
oligonucleotide synthesis. Such partially random oligonucleotides
can be fused by overlap PCR or ligation-based approaches. In
particular, one can multimerize semi-random oligonucleotides that
encode glycine-rich sequences. These oligonucleotides can differ in
length and sequences and codon usage. As a result, one obtains a
library of candidate URP sequences. Another method to generate
libraries is to synthesize a starting sequence and subsequently
subject said sequence to partial randomization. This can be done by
cultivation of the gene encoding the URP sequences in a mutator
strain or by amplification of the encoding gene under mutagenic
conditions (Leung, D., et al. (1989) Technique, 1: 11-15). URP
sequences with desirable properties can be identified from
libraries using a variety of methods. Sequences that have a high
degree of genetic stability can be enriched by cultivating the
library in a production host. Sequences that are unstable will
accumulate mutations, which can be identified by DNA sequencing.
Variants of URP sequences that can be expressed at high level can
be identified by screening or selection using multiple protocols
known to someone skilled in the art. For instance one can cultivate
multiple isolates from a library and compare expression levels.
Expression levels can be measured by gel analysis, analytical
chromatography, or various ELISA-based methods. The determination
of expression levels of individual sequence variants can be
facilitated by fusing the library of candidate URP sequences to
sequence tags like myc-tag, His-tag, HA-tag. Another approach is to
fuse the library to an enzyme or other reporter protein like green
fluorescent protein. Of particular interest is the fusion of the
library to a selectable marker like beta-lactamase or
kanamycin-acyl transferase. One can use antibiotic selection to
enrich for variants with high level of expression and good genetic
stability. Variants with good protease resistance can be identified
by screening for intact sequences after incubation with proteases.
An effective way to identify protease-resistant URP sequences is
bacterial phage display or related display methods. Multiple
systems have been described where sequences that undergo rapid
proteolysis can be enriched by phage display. These methods can be
easily adopted to enrich for protease resistant sequences. For
example, one can clone a library of candidate URP sequences between
an affinity tag and the pIII protein of M13 phage. The library can
then be exposed to proteases or protease-containing biological
samples like blood or lysosomal preparations. Phage that contain
protease-resistant sequences can be captured after protease
treatment by binding to the affinity tag. Sequences that resist
degradation by lysosomal preparations are of particular interest
because lysosomal degradation is a key step during antigen
presentation in dendritic and other antigen presenting cells. Phage
display can be utilized to identify candidate URP sequences that do
not bind to a particular immune serum in order to identify URP
sequences with low immunogenicity. One can immunize animals with a
candidate URP sequence or with a library of URP sequences to raise
antibodies against the URP sequences in the library. The resulting
serum can then be used for phage panning to remove or identify
sequences that are recognized by antibodies in the resulting immune
serum. Other methods like bacterial display, yeast display,
ribosomal display can be utilized to identify variants of URP
sequences with desirable properties. Another approach is the
identification of URP sequences of interest by mass spectrometry.
For instance, one can incubate a library of candidate URP sequences
with a protease or biological sample of interest and identify
sequences that resist degradation by mass spectrometry. In a
similar approach one can identify URP sequences that facilitate
oral uptake. One can feed a mixture of candidate URP sequences to
animals or humans and identify variants with the highest transfer
or uptake efficiency across some tissue barrier (ie dermal, etc) by
mass spectrometry. In a similar way, one can identify URP sequences
that favor other uptake mechanisms like pulmonary, intranasal,
rectal, transdermal delivery. One can also identify URP sequences
that favor cellular uptake or URP sequences that resist cellular
uptake.
[0351] URP sequences can be designed by combining URP sequences or
fragments of URP sequences that were designed by any of the methods
described above. In addition, one can apply semi-random approaches
to optimize sequences that were designed based on the rules
described above. Of particular interest is codon optimization with
the goal of improving expression of the enhanced polypeptides and
to improve the genetic stability of the encoding gene in the
production hosts. Codon optimization is of particular importance
for URP sequences that are rich in glycine or that have very
repetitive amino acid sequences. Codon optimization can be
performed using computer programs (Gustafsson, C., et al. (2004)
Trends Biotechnol, 22: 346-53), some of which minimize ribosomal
pausing (Coda Genomics Inc.). When designing URP sequences one can
consider a number of properties. One can minimize the
repetitiveness in the encoding DNA sequences. In addition, one can
avoid or minimize the use of codons that are rarely used by the
production host (ie the AGG and AGA arginine codons and one Leucine
codon in E. coli) DNA sequences that have a high level of glycine
tend to have a high GC content that can lead to instability or low
expression levels. Thus, when possible it is preferred to choose
codons such that the GC-content of URP-encoding sequence is
suitable for the production organism that will be used to
manufacture the URP.
[0352] URP encoding genes can be made in one or more steps, either
fully synthetically or by synthesis combined with enzymatic
processes, such as restriction enzyme-mediated cloning, PCR and
overlap extension. URP accessory polypeptides can be constructed
such that the URP accessory polypeptide-encoding gene has low
repetitiveness while the encoded amino acid sequence has a high
degree of repetitiveness. As a first step, one constructs a library
of relatively short URP sequences. This can be a pure codon library
such that each library member has the same amino acid sequence but
many different coding sequences are possible. To facilitate the
identification of well-expressing library members one can construct
the library as fusion to a reporter protein. Examples of suitable
reporter genes are green fluorescent protein, luciferase, alkaline
phosphatase, beta-galactosidase. By screening one can identify
short URP sequences that can be expressed in high concentration in
the host organism of choice. Subsequently, one can generate a
library of random URP dimers and repeat the screen for high level
of expression. Dimerization can be performed by ligation, overlap
extension or similar cloning techniques. This process of
dimerization and subsequent screening can be repeated multiple
times until the resulting URP sequence has reached the desired
length. Optionally, one can sequence clones in the library to
eliminate isolates that contain undesirable sequences. The initial
library of short URP sequences can allow some variation in amino
acid sequence. For instance one can randomize some codons such that
a number of hydrophilic amino acids can occur in said position.
During the process of iterative multimerization one can screen
library members for other characteristics like solubility or
protease resistance in addition to a screen for high-level
expression. Instead of dimerizing URP sequences one can also
generate longer multimers. This allows one to faster increase the
length of URP accessory polypeptides.
[0353] Many URP sequences contain particular amino acids at high
fraction. Such sequences can be difficult to produce by recombinant
techniques as their coding genes can contain repetitive sequences
that are subject to recombination. Furthermore, genes that contain
particular codons at very high frequencies can limit expression as
the respective loaded tRNAs in the production host become limiting.
An example is the recombinant production of GRS. Glycine residues
are encoded by 4 triplets, GGG, GGC, GGA, and GGT. As a result,
genes encoding GRS tend to have high GC-content and tend to be
particularly repetitive. An additional challenge can result from
codon bias of the production host. In the case of E. coli, two
glycine codons, GGA and GGG, are rarely used in highly expressed
proteins. Thus codon optimization of the gene encoding URP
sequences can be very desirable. One can optimize codon usage by
employing computer programs that consider codon bias of the
production host (Gustafsson, C., et al. (2004) Trends Biotechnol,
22: 346-53). As an alternative, one can construct codon libraries
where all members of the library encode the same amino acid
sequence but where codon usage is varied. Such libraries can be
screened for highly expressing and genetically stable members which
are particularly suitable for the large-scale production of
URP-containing products.
[0354] Multivalent Unstructured Recombinant Proteins (MURPs):
[0355] As noted above, the subject URPs are particularly useful as
accessory polypeptides for the modification of biologically active
polypeptides. Accordingly, the present invention provides proteins
comprising one or more subject URPs. Such proteins are termed
herein Multivalent Unstructured Recombinant Proteins (MURPs).
[0356] To construct MURPs, one or more URP sequences can be fused
to the N-terminus or C-terminus of a protein or inserted in the
middle of the protein, e.g., into loops of a protein or in between
modules of the biologically active polypeptide of interest, to give
the resulting modified polypeptide improved properties relative to
the unmodified protein. The combined length of URP sequences that
are attached to a protein can be 40, 50, 60, 70, 80, 90, 100, 150,
200 or more amino acids.
[0357] The subject MURPs exhibit one or more improved properties as
detailed below.
[0358] Improved Half-Life:
[0359] Adding a URP sequences to a biologically active polypeptide
can improve many properties of that protein. In particular, adding
a long URP sequence can significantly increase the serum half-life
of the protein. Such URPs typically contain amino acid sequences of
at least about 40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino
acids.
[0360] The URPs can be fragmented such that the resulting protein
contains multiple URPs, or multiple fragments of URPs. Some or all
of these individual URP sequences may be shorter that 40 amino
acids as long as the combined length of all URP sequences in the
resulting protein is at least 30 amino acids. Preferably, the
resulting protein has a combined length of URP sequences exceeding
40, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids. In one
aspect, the fused URPS can increase the hydrodynamic radius of a
protein and thus reduces its clearance from the blood by the
kidney. The increase in the hydrodynamic radius of the resulting
fusion protein relative to the unmodified protein can be detected
by ultracentrifugation, size exclusion chromatography, or light
scattering.
[0361] Improved Tissue Selectivity:
[0362] Increasing the hydrodynamic radius can also lead to reduced
penetration into tissues, which can be exploited to minimize side
effects of a biologically active polypeptide. It is well documented
that hydrophilic polymers have a tendency to accumulate selectively
in tumor tissue which is caused by the enhanced permeability and
retention (EPR) effect. The underlying cause of the EPR effect is
the leaky nature of tumor vasculature (McDonald, D. M., et al.
(2002) Cancer Res, 62: 5381-5) and the lack of lymphatic drainage
in tumor tissues. Therefore, the selectivity of biologically active
polypeptides for tumor tissues can be enhanced by adding
hydrophilic polymers. As such, the therapeutic index of a given
biologically active polypeptide can be increased via incorporating
the subject URPS.
[0363] Protection from Degradation and Reduced Immunogenicity:
[0364] Adding URP sequences can significantly improve the protease
resistance of a protein. URP sequences themselves can be designed
to be protease resistant and by attaching them to a protein one can
shield that protein from the access of degrading enzymes. URP
sequences can be added to biologically active polypeptides with the
goal of reducing undesirable interactions of the protein with other
receptors or surfaces. To achieve this, it can be beneficial to add
the URP sequences to the biologically active polypeptide in
proximity to the site of the protein that makes such undesirable
contacts. In particular, one can add URP sequences to biologically
active polypeptides with the goal of reducing their interactions
with any component of the immune system to prevent an immune
response against the product of the invention. Adding a URP
sequence to a biologically active polypeptide can reduce
interaction with pre-existing antibodies or B-cell receptors.
Furthermore, the addition of URP sequences can reduce the uptake
and processing of the product of the invention by antigen
presenting cells. Adding one or more URP sequence to a protein is a
preferred way of reducing its immunogenicity as it will suppress an
immune response in many species allowing one to predict the
expected immunogenicity of a product in patients based on animal
data. Such species independent testing of immunogenicity is not
possible for approaches that are based on the identification and
removal of human T cell epitopes or sequences comparison with human
sequences.
[0365] Interruption of T Cell Epitopes:
[0366] URP sequences can be introduced into proteins in order to
interrupt T cell epitopes. This is particularly useful for proteins
that combine multiple separate functional modules. The formation of
T cell epitopes requires that peptide fragments of a protein
antigen bind to MHC. MHC molecules interact with a short segment of
amino acids typically 9 contiguous residues of the presented
peptides. The direct fusion of different binding modules in a
protein molecule can lead to T cell epitopes that span two
neighboring domains. By separating the functional modules by URP
accessory polypeptides prevents the generation of such
module-spanning T cell epitopes. The insertion of URP sequences
between functional modules can also interfere with proteolytic
processing in antigen presenting cells, which will lead to an
additional reduction of immunogenicity.
[0367] Improved Solubility:
[0368] Functional modules of a protein can have limited solubility.
In particular, binding modules tend to carry hydrophobic residues
on their surface, which can limit their solubility and can lead to
aggregation. By spacing or flanking such functional modules with
URP accessory polypeptides one can improve the overall solubility
of the resulting product. This is in particular true for URP
accessory polypeptides that carry a significant percentage of
hydrophilic or charged residues. By separating functional modules
with soluble URP modules one can reduce intramolecular interactions
between these functional modules
[0369] Improved pH Profile and Homogeneity of Product Charge:
[0370] URP sequences can be designed to carry an excess of negative
or positive charges. As a result they confer an electrostatic field
to any fusion partner which can be utilized to shift the pH profile
of an enzyme or a binding interaction. Furthermore, the
electrostatic field of a charged URP sequence can increase the
homogeneity of pKa values of surface charges of a protein product,
which leads to sharpened pH profiles of ligand interactions and to
sharpened separations by isoelectric focusing or
chromatofocusing.
[0371] Improved Purification Properties Due to Sharper Product
pKa:
[0372] Each amino acid in solution by itself has a single, fixed
pKa, which is the pH at which its functional groups are half
protonated. In a typical protein you have many types of residues
and due to proximity and protein breathing effects, they also
change each other's effective pKa in variable ways. Because of
this, at a wide range of pH conditions, typical proteins can adopt
hundreds of differently ionized species, each with a different
molecular weight and net charge, due to large numbers of
combinations of charged and neutral amino acid residues. This is
referred to as a broad ionization spectrum and makes the analysis
(eg by mass spectrometry) and purification of such proteins more
difficult.
[0373] PEG is uncharged and does not affect the ionization spectrum
of the protein it is attached to, leaving it with a broad
ionization spectrum. However, an URP with a high content of Gly and
Glu in principle exist in only two states: neutral (--COOH) when
the pH is below the pKa of Glutamate and negatively charged
(--COO.sup.-) when the pH is above the pKa of Glutamate. URP
accessory polypeptides can form a single, homogeneously ionizated
type of molecule and can yield a single mass in mass
spectrometry.
[0374] Where desired, MURPs can be expressed as a fusion with an
URP having a single type of charge (Glu) distributed at constant
spacing through the URP accessory polypeptide. One may choose to
incorporate 25-50 Glu residues per 20 kD of URP and all of these
25-50 residues would have very similar pKa.
[0375] In addition, adding 25-50 negative charges to a small
protein like IFN, hGH or GCSF (with only 20 charged residues) will
increase the charge homogeneity of the product and sharpen its
isoelectric point, which will be very close to the pKa of free
glutamate.
[0376] The increase in the homogeneity of the charge of the protein
population has favorable processing properties, such as in ion
exchange, isoelectric focusing, mass spec, etc. compared to
traditional PEGylation.
[0377] Biologically Active Polypeptides
[0378] Suitable polypeptides that can be linked to the accessory
polypeptide include all biologically active polypeptides exhibiting
a binding specificity to a given target or another desired
biological characteristic when used in vitro or in vivo. In
particular, any protein of therapeutic or diagnostic interest can
be modified by accessory polypeptides. Of particular interest are
polypeptides for which modification of certain properties such as
serum half-life or in vivo clearance is desirable. Such
modification can be envisioned in the context of therapeutic
applications, for example if one desires to prolong the half-life
of an administered protein therapeutic drug. Modification with
accessory polypeptides could also show utility in diagnostic
applications, for example to reduce non-specific binding of a
diagnostic protein or imaging agent to other molecules
[0379] Biologically active polypeptides can include, but are not
limited to cytokines, chemokines, lymphokines, ligands, receptors,
hormones, enzymes, antibodies and antibody fragments, and growth
factors. Examples of receptors include TNF type I receptor, IL-1
receptor type II, IL-1 receptor antagonist, IL-4 receptor and any
chemically or genetically modified soluble receptors. Examples of
enzymes include activated protein C, factor VII, collagenase (e.g.,
marketed by Advance Biofactures Corporation under the name Santyl);
agalsidase-beta (e.g., marketed by Genzyme under the name
Fabrazyme); dornase-alpha (e.g., marketed by Genentech under the
name Pulmozyme); alteplase (e.g., marketed by Genentech under the
name Activase); pegylated-asparaginase (e.g., marketed by Enzon
under the name Oncaspar); asparaginase (e.g., marketed by Merck
under the name Elspar); and imiglucerase (e.g., marketed by Genzyme
under the name Ceredase). Examples of specific polypeptides or
proteins include, but are not limited to granulocyte macrophage
colony stimulating factor (GM-CSF), granulocyte colony stimulating
factor (G-CSF), macrophage colony stimulating factor (M-CSF),
colony stimulating factor (CSF), interferon beta (IFN-.beta.),
interferon gamma (IFN.gamma.), interferon gamma inducing factor I
(IGIF), transforming growth factor beta (TGF-.beta.), RANTES
(regulated upon activation, normal T-cell expressed and presumably
secreted), macrophage inflammatory proteins (e.g., MIP-1-.alpha.
and MIP-1-.beta.), Leishmania elongation initiating factor (LEIF),
platelet derived growth factor (PDGF), tumor necrosis factor (TNF),
growth factors, e.g., epidermal growth factor (EGF), vascular
endothelial growth factor (VEGF), fibroblast growth factor, (FGF),
nerve growth factor (NGF), brain derived neurotrophic factor
(BDNF), neurotrophin-2 (NT-2), neurotrophin-3 (NT-3),
neurotrophin-4 (NT-4), neurotrophin-5 (NT-5), glial cell
line-derived neurotrophic factor (GDNF), ciliary neurotrophic
factor (CNTF), TNF a type II receptor, erythropoietin (EPO),
insulin and soluble glycoproteins e.g., gp120 and gp160
glycoproteins. The gp120 glycoprotein is a human immunodeficiency
virus (HIV) envelope protein, and the gp160 glycoprotein is a known
precursor to the gp120 glycoprotein.
[0380] By way of example, the following are several examples of
biologically active polypeptides which may be suitable for
modification according to the present invention.
[0381] In one embodiment, the biologically active polypeptide is
GLP-1. GLP-1 is an approximately 30 amino acid polypeptide that is
currently being investigated as a possible therapy for diabetes.
GLP-1 suppresses glucagon release and increases insulin release.
Both responses to GLP-1 result in a decrease in the serum
concentration of glucose. GLP-1 is rapidly cleaved by dipeptidyl
peptidase-4 in the body and as a result has an extremely short
serum half-life, .about.2 min. The successful development of GLP-1
as a therapeutic protein requires formulations to increase the
serum half life and delivery of the protein. This example describes
the preparation of an rPEG-GLP-1 fusion protein based on rPEG
(L288) and its encapsulation in a polymer matrix to improve the
half-life of GLP-1 for therapeutic use.
[0382] In another embodiment, the biologically active polypeptide
is nesiritide, human B-type natriuretic peptide (hBNP). Nesiritide
can be manufactured in E. coli using recombinant DNA technology. In
a specific embodiment, nesiritide consists of a 32 amino acid
sequence with a molecular weight of 3464 g/mol.
[0383] In yet another embodiment, the biologically active
polypeptide is secretin, which is a peptide hormone composed of an
amino acid sequence identical to the naturally occurring porcine
secretin consisting of 27 amino acids. After intravenous bolus
administration of 0.4 mcg/kg of unmodified polypeptide, synthetic
human secretin concentration rapidly declines to baseline secretin
levels within 90 to 120 minutes. The elimination half-life of
synthetic human secretin (not modified with accessory polypeptide)
is approximately 45 minutes.
[0384] In an alternative embodiment, the biologically active
polypeptide is enfuvirtide, a linear 36-amino acid synthetic
polypeptide which is an inhibitor of the fusion of HIV-1 with CD4+
cells.
[0385] In an additional embodiment, the biologically active
polypeptide is bivalirudin, a specific and reversible direct
thrombin inhibitor. A more specific embodiment provides for an
biologically active polypeptide which is a synthetic, 20 amino acid
peptide with a molecular weight of 1280 daltons.
[0386] Alternatively, Antihemophilic Factor (AHF) may be selected
as the biologically active polypeptide. AHF is a glycoprotein
amenable to synthesis in a genetically engineered Chinese Hamster
Ovary (CHO) cell line. It is also known as HEMOFIL M.TM. AHF
(Baxter) or Antihemophilic Factor (Human) [AHF (Human)]. The mean
in vivo half-life of HEMOFIL M.TM. AHF is known to be 14.7.+-.5.1
hours (n=61).
[0387] In another embodiment, erythropoietin is the biologically
active polypeptide. Erythropoietin is a 165 amino acid glycoprotein
manufactured by recombinant DNA technology and has the same
biological effects as endogenous erythropoietin. In a specific
embodiment, erythropoietin has a molecular weight of 30,400 daltons
and is produced by mammalian cells into which the human
erythropoietin gene has been introduced. The product may contain
the identical amino acid sequence of isolated natural
erythropoietin. In adult and pediatric patients with chronic renal
failure, the elimination half-life of unmodified plasma
erythropoietin after intravenous administration is known to range
from 4 to 13 hours.
[0388] In still another embodiment, the biologically active
polypeptide is Reteplase. Reteplase is a non-glycosylated deletion
mutein of tissue plasminogen activator (tPA), comprising the
kringle 2 and the protease domains of human tPA. Reteplase contains
355 of the 527 amino acids of native tPA (amino acids 1-3 and
176-527). The polypeptide may be produced by recombinant DNA
technology in E. coli. and may be isolated as inactive inclusion
bodies from E. coli, converted into its active form by an in vitro
folding process and purified by chromatographic separation. The
molecular weight of unmodified Reteplase is 39,571 daltons. Based
on the measurement of thrombolytic activity, the effective
half-life of unmodified Reteplase is known to be approximately 15
minutes.
[0389] A further embodiment provides for a biologically active
polypeptide which is Anakirna, a recombinant, nonglycosylated form
of the human interleukin-1 receptor antagonist (IL-IRa). In one
case, Anakinra consists of 153 amino acids and has a molecular
weight of 17.3 kilodaltons. It may be produced by recombinant DNA
technology using an E. coli bacterial expression system. The in
vivo half-life of unmodified Anakirna is known to range from 4 to 6
hours.
[0390] Becaplermin may also be selected as the biologically active
polypeptide. Becaplermin is a recombinant human platelet-derived
growth factor (rhPDGF-BB) for topical administration. Becaplermin
may be produced by recombinant DNA technology by insertion of the
gene for the B chain of platelet derived growth factor (PDGF) into
the yeast strain Saccharomyces cerevisiae. One form of Becaplermin
has a molecular weight of approximately 25 kD and is a homodimer
composed of two identical polypeptide chains that are bound
together by disulfide bonds.
[0391] The biologically active polypeptide may be Oprelvekin, which
is a recombinant form of interleukin eleven (IL-11) that is
produced in Escherichia coli (E. coli) by recombinant DNA
technology. In one embodiment, the selected biologically active
polypeptide has a molecular mass of approximately 19,000 daltons,
and is non-glycosylated. The polypeptide is 177 amino acids in
length and differs from the 178 amino acid length of native IL-11
only in lacking the amino-terminal proline residue, which is known
not to result in measurable differences in bioactivity either in
vitro or in vivo. The terminal half-life of unmodified Oprelvekin
is known to be approximately 7 hrs.
[0392] Yet another embodiment provides for a biologically active
polypeptide which is Glucagon, a polypeptide hormone identical to
human glucagon that increases blood glucose and relaxes smooth
muscles of the gastrointestinal tract. Glucagon may be synthesized
in a special non-pathogenic laboratory strain of E. coli bacteria
that have been genetically altered by the addition of the gene for
glucagon. In a specific embodiment, glucagon is a single-chain
polypeptide that contains 29 amino acid residues and has a
molecular weight of 3,483. The in vivo half-life is known to be
short, ranging from 8 to 18 minutes.
[0393] G-CSF may also be chosen as a biologically active
polypeptide. Recombinant granulocyte-colony stimulating factor or
G-CSF is used following various chemotherapy treatments to
stimulate the recovery of white blood cells. The reported half life
of recombinant G-CSF is only 3.5 hours.
[0394] Alternatively, the biologically active polypeptide can be
interferon alpha (IFN alpha). Chemically PEG-modified
interferon-alpha 2a is clinically validated for the treatment of
hepatitis C. This PEGylated protein requires weekly injection and
slow release formulations with longer half-life are desirable.
[0395] Additional cellular proteins which may be modified with
accessory polypeptides, or to which biologically active
polypeptides may be targeted are VEGF, VEGF-R1, VEGF-R2, VEGF-R3,
Her-1, Her-2, Her-3, EGF-1, EGF-2, EGF-3, Alpha3, cMet, ICOS,
CD40L, LFA-1, c-Met, ICOS, LFA-1, IL-6, B7.1, B7.2, OX40, IL-1b,
TACI, IgE, BAFF or BLys, TPO-R, CD19, CD20, CD22, CD33, CD28,
IL-1-R1, TNF.alpha., TRAIL-R1, Complement Receptor 1, FGFa,
Osteopontin, Vitronectin, Ephrin A1-A5, Ephrin B1-B3,
alpha-2-macroglobulin, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7,
CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CCL13, CCL14, CCL15, CXCL16,
CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, PDGF, TGFb, GMCSF,
SCF, p40 (IL12/IL23), IL1b, IL1a, IL1ra, IL2, IL3, IL4, IL5, IL6,
IL8, IL10, IL12, IL15, IL23, Fas, FasL, Flt3 ligand, 41BB, ACE,
ACE-2, KGF, FGF-7, SCF, Netrin1,2, IFNa,b,g, Caspase-2,3,7,8,10,
ADAM S1,S5,8,9,15,TS1,TS5; Adiponectin, ALCAM, ALK-1, APRIL,
Annexin V, Angiogenin, Amphiregulin, Angiopoietin-1,2,4, B7-1/CD80,
B7-2/CD86, B7-H1, B7-H2, B7-H3, Bcl-2, BACE-1, BAK, BCAM, BDNF,
bNGF, bECGF, BMP2,3,4,5,6,7,8; CRP, Cadherin-6,8,11; Cathepsin
A,B,C,D,E,L,S,V,X; CD11a/LFA-1, LFA-3, GP2b3a, GH receptor, RSV F
protein, IL-23 (p40, p19), IL-12, CD80, CD86, CD28, CTLA-4,
.alpha.4.beta.1, .alpha.4.beta.7, TNF/Lymphotoxin, IgE, CD3, CD20,
IL-6, IL-6R, BLYS/BAFF, IL-2R, HER2, EGFR, CD33, CD52, Digoxin, Rho
(D), Varicella, Hepatitis, CMV, Tetanus, Vaccinia, Antivenom,
Botulinum, Trail-R1, Trail-R2, cMet, TNF-R family, such as LA
NGF-R, CD27, CD30, CD40, CD95, Lymphotoxin a/b receptor, Wsl-1,
TL1A/TNFSF15, BAFF, BAFF-R/TNFRSF13C, TRAIL R2/TNFRSF10B, TRAIL
R2/TNFRSF10B, Fas/TNFRSF6 CD27/TNFRSF7, DR3/TNFRSF25,
HVEM/TNFRSF14, TROY/TNFRSF19, CD40 Ligand/TNFSF5, BCMA/TNFRSF17,
CD30/TNFRSF8, LIGHT/TNFSF14, 4-1BB/TNFRSF9, CD40/TNFRSF5,
GITR/TNFRSF18, Osteoprotegerin/TNFRSF11B, RANK/TNFRSF11A, TRAIL
R3/TNFRSF10C, TRAIL/TNFSF10, TRANCE/RANK L/TNFSF11, 4-1BB
Ligand/TNFSF9, TWEAK/TNFSF12, CD40 Ligand/TNFSF5, Fas
Ligand/TNFSF6, RELT/TNFRSF19L, APRIL/TNFSF13, DcR3/TNFRSF6B, TNF
RI/TNFRSF1A, TRAIL R1/TNFRSF10A, TRAIL R4/TNFRSF10D, CD30
Ligand/TNFSF8, GITR Ligand/TNFSF18, TNFSF18, TACI/TNFRSF13B, NGF
R/TNFRSF16, OX40 Ligand/TNF SF4, TRAIL R2/TNFRSF10B, TRAIL
R3/TNFRSF10C, TWEAK R/TNFRSF12, BAFF/BLyS/TNFSF13, DR6/TNFRSF21,
TNF-alpha/TNFSF1A, Pro-TNF-alpha/TNFSF1A, Lymphotoxin beta
R/TNFRSF3, Lymphotoxin beta R (LTbR)/Fc Chimera, TNF RI/TNFRSF1A,
TNF-beta/TNFSF1B, PGRP-S, TNF RI/TNFRSF1A, TNF RII/TNFRSF1B,
EDA-A2, TNF-alpha/TNFSF1A, EDAR, XEDAR, TNF RI/TNFRSF1A.
[0396] Of particular interest are human target proteins that are
commercially available in purified form as well as proteins that
bind to these target proteins. Examples are: 4EBP1, 14-3-3 zeta,
53BP1, 2B4/SLAMF4, CCL21/6Ckine, 4-1BB/TNFRSF9, 8D6A, 4-1BB
Ligand/TNFSF9,8-oxo-dG, 4-Amino-1,8-naphthalimide, A2B5,
Aminopeptidase LRAP/ERAP2, A33, Aminopeptidase N/ANPEP, Aag,
Aminopeptidase P2/XPNPEP2, ABCG2, Aminopeptidase P1/XPNPEP1, ACE,
Aminopeptidase PILS/ARTS1, ACE-2, Amnionless, Actin, Amphiregulin,
beta-Actin, AMPK alpha 1/2, Activin A, AMPK alpha 1, Activin AB,
AMPK alpha 2, Activin B, AMPK beta 1, Activin C, AMPK beta 2,
Activin RIA/ALK-2, Androgen R/NR3C4, Activin RIB/ALK-4, Angiogenin,
Activin RIIA, Angiopoietin-1, Activin RIIB, Angiopoietin-2, ADAMS,
Angiopoietin-3, ADAM9, Angiopoietin-4, ADAM10, Angiopoietin-like 1,
ADAM12, Angiopoietin-like 2, ADAM15, Angiopoietin-like 3,
TACE/ADAM17, Angiopoietin-like 4, ADAM19, Angiopoietin-like 7/CDT6,
ADAM33, Angiostatin, ADAMTS4, Annexin A1/Annexin I, ADAMTS5,
Annexin A7, ADAMTS1, Annexin A10, ADAMTSL-1/Punctin, Annexin V,
Adiponectin/Acrp30, ANP, AEBSF, AP Site, Aggrecan, APAF-1, Agrin,
APC, AgRP, APE, AGTR-2, APJ, AIF, APLP-1, Akt, APLP-2, Akt1,
Apolipoprotein AI, Akt2, Apolipoprotein B, Akt3, APP, Serum
Albumin, APRIL/TNFSF13, ALCAM, ARC, ALK-1, Artemin, ALK-7,
Arylsulfatase A/ARSA, Alkaline Phosphatase, ASAH2/N-acylsphingosine
Amidohydrolase-2, alpha 2u-Globulin, ASC, alpha-1-Acid
Glycoprotein, ASGR1, alpha-Fetoprotein, ASK1, ALS, ATM,
Ameloblastin, ATRIP, AMICA/JAML, Aurora A, AMIGO, Aurora B, AMIGO2,
Axin-1, AMIGO3, Axl, Aminoacylase/ACY1, Azurocidin/CAP37/HBP,
Aminopeptidase A/ENPEP, B4GALT1, BIM, B7-1/CD80, 6-Biotin-17-NAD,
B7-2/CD86, BLAME/SLAMF8, B7-H1/PD-L1, CXCL13/BLC/BCA-1, B7-H2,
BLIMP1, B7-H3, Blk, B7-H4, BMI-1, BACE-1, BMP-1/PCP, BACE-2, BMP-2,
Bad, BMP-3, BAFF/TNFSF13B, BMP-3b/GDF-10, BAFF R/TNFRSF13C, BMP-4,
Bag-1, BMP-5, BAK, BMP-6, BAMBI/NMA, BMP-7, BARD1, BMP-8, Bax,
BMP-9, BCAM, BMP-10, Bcl-10, BMP-15/GDF-9B, Bcl-2, BMPR-IA/ALK-3,
Bcl-2 related protein A1, BMPR-IB/ALK-6, Bcl-w, BMPR-II, Bcl-x,
BNIP3L, Bcl-xL, BOC, BCMA/TNFRSF17, BOK, BDNF, BPDE, Benzamide,
Brachyury, Common beta Chain, B-Raf, beta IG-H3, CXCL14/BRAK,
Betacellulin, BRCA1, beta-Defensin 2, BRCA2, BID, BTLA, Biglycan,
Bub-1, Bik-like Killer Protein, c-jun, CD90/Thyl, c-Rel, CD94,
CCL6/C10, CD97, C1q R1/CD93, CD151, C1qTNF1, CD160, C1qTNF4, CD163,
C1qTNF5, CD164, Complement Component C1r, CD200, Complement
Component C1s, CD200 R1, Complement Component C2, CD229/SLAMF3,
Complement Component C3a, CD23/Fc epsilon RII, Complement Component
C3d, CD2F-10/SLAMF9, Complement Component C5a, CD5L,
Cadherin-4/R-Cadherin, CD69, Cadherin-6, CDC2, Cadherin-8, CDC25A,
Cadherin-11, CDC25B, Cadherin-12, CDCP1, Cadherin-13, CDO,
Cadherin-17, CDX4, E-Cadherin, CEACAM-1/CD66a, N-Cadherin,
CEACAM-6, P-Cadherin, Cerberus 1, VE-Cadherin, CFTR, Calbindin D,
cGMP, Calcineurin A, Chem R23, Calcineurin B, Chemerin,
Calreticulin-2, Chemokine Sampler Packs, CaM Kinase II, Chitinase
3-like 1, cAMP, Chitotriosidase/CHIT1, Cannabinoid R1, Chk1,
Cannabinoid R2/CB2/CNR2, Chk2, CAR/NR113, CHL-1/L1CAM-2, Carbonic
Anhydrase I, Choline Acetyltransferase/ChAT, Carbonic Anhydrase II,
Chondrolectin, Carbonic Anhydrase III, Chordin, Carbonic Anhydrase
IV, Chordin-Like 1, Carbonic Anhydrase VA, Chordin-Like 2, Carbonic
Anhydrase VB, CINC-1, Carbonic Anhydrase VI, CINC-2, Carbonic
Anhydrase VII, CINC-3, Carbonic Anhydrase VIII, Claspin, Carbonic
Anhydrase IX, Claudin-6, Carbonic Anhydrase X, CLC, Carbonic
Anhydrase XII, CLEC-1, Carbonic Anhydrase XIII, CLEC-2, Carbonic
Anhydrase XIV, CLECSF13/CLEC4F, Carboxymethyl Lysine, CLECSF8,
Carboxypeptidase A1/CPA1, CLF-1, Carboxypeptidase A2,
CL-P1/COLEC12, Carboxypeptidase A4, Clusterin, Carboxypeptidase B1,
Clusterin-like 1, Carboxypeptidase E/CPE, CMG-2, Carboxypeptidase
X1, CMV UL146, Cardiotrophin-1, CMV UL147, Carnosine Dipeptidase 1,
CNP, Caronte, CNTF, CART, CNTF R alpha, Caspase, Coagulation Factor
II/Thrombin, Caspase-1, Coagulation Factor III/Tissue Factor,
Caspase-2, Coagulation Factor VII, Caspase-3, Coagulation Factor X,
Caspase-4, Coagulation Factor XI, Caspase-6, Coagulation Factor
XIV/Protein C, Caspase-7, COCO, Caspase-8, Cohesin, Caspase-9,
Collagen I, Caspase-10, Collagen II, Caspase-12, Collagen IV,
Caspase-13, Common gamma Chain/IL-2 R gamma, Caspase Peptide
Inhibitors, COMP/Thrombospondin-5, Catalase, Complement Component
C1rLP, beta-Catenin, Complement Component C1qA, Cathepsin 1,
Complement Component C1qC, Cathepsin 3, Complement Factor D,
Cathepsin 6, Complement Factor I, Cathepsin A, Complement MASP3,
Cathepsin B, Connexin 43, Cathepsin C/DPPI, Contactin-1, Cathepsin
D, Contactin-2/TAG1, Cathepsin E, Contactin-4, Cathepsin F,
Contactin-5, Cathepsin H, Corin, Cathepsin L, Cornulin, Cathepsin
O, CORS26/C1qTNF,3, Cathepsin S, Rat Cortical Stem Cells, Cathepsin
V, Cortisol, Cathepsin X/Z/P, COUP-TF I/NR2F1, CBP, COUP-TF
II/NR2F2, CCI, COX-1, CCK-A R, COX-2, CCL28, CRACC/SLAMF7, CCR1,
C-Reactive Protein, CCR2, Creatine Kinase, Muscle/CKMM, CCR3,
Creatinine, CCR4, CREB, CCR5, CREG, CCR6, CRELD1, CCR7, CRELD2,
CCR8, CRHBP, CCR9, CRHR-1, CCR10, CRIM1, CD155/PVR, Cripto, CD2,
CRISP-2, CD3, CRISP-3, CD4, Crossveinless-2, CD4+/45RA-, CRTAM,
CD4+/45RO-, CRTH-2, CD4+/CD62L-/CD44, CRY1, CD4+/CD62L+/CD44,
Cryptic, CD5, CSB/ERCC6, CD6, CCL27/CTACK, CD8, CTGF/CCN2,
CD8+/45RA-, CTLA-4, CD8+/45RO-, Cubilin, CD9, CX3CR1, CD14, CXADR,
CD27/TNFRSF7, CXCL16, CD27 Ligand/TNFSF7, CXCR3, CD28, CXCR4,
CD30/TNFRSF8, CXCR5, CD30 Ligand/TNFSF8, CXCR6, CD31/PECAM-1,
Cyclophilin A, CD34, Cyr61/CCN1, CD36/SR-B3, Cystatin A, CD38,
Cystatin B, CD40/TNFRSF5, Cystatin C, CD40 Ligand/TNFSF5, Cystatin
D, CD43, Cystatin E/M, CD44, Cystatin F, CD45, Cystatin H, CD46,
Cystatin H2, CD47, Cystatin S, CD48/SLAMF2, Cystatin SA, CD55/DAF,
Cystatin SN, CD58/LFA-3, Cytochrome c, CD59, Apocytochrome c, CD68,
Holocytochrome c, CD72, Cytokeratin 8, CD74, Cytokeratin 14, CD83,
Cytokeratin 19, CD84/SLAMF5, Cytonin, D6, DISP1, DAN, Dkk-1, DANCE,
Dkk-2, DARPP-32, Dkk-3, DAX1/NR0B1, Dkk-4, DCC, DLEC, DCIR/CLEC4A,
DLL1, DCAR, DLL4, DcR3/TNFRSF6B, d-Luciferin, DC-SIGN, DNA Ligase
IV, DC-SIGNR/CD299, DNA Polymerase beta, DcTRAIL R1/TNFRSF23,
DNAM-1, DcTRAIL R2/TNFRSF22, DNA-PKcs, DDR1, DNER, DDR2, Dopa
Decarboxylase/DDC, DEC-205, DPCR-1, Decapentaplegic, DPP6, Decorin,
DPPA4, Dectin-1/CLEC7A, DPPA5/ESG1, Dectin-2/CLEC6A,
DPPII/QPP/DPP7, DEP-1/CD148, DPPIV/CD26, Desert Hedgehog,
DR3/TNFRSF25, Desmin, DR6/TNFRSF21, Desmoglein-1, DSCAM,
Desmoglein-2, DSCAM-L1, Desmoglein-3, DSPG3, Dishevelled-1, Dtk,
Dishevelled-3, Dynamin, EAR2/NR2F6, EphA5, ECE-1, EphA6, ECE-2,
EphA7, ECF-L/CHI3L3, EphA8, ECM-1, EphB1, Ecotin, EphB2, EDA,
EphB3, EDA-A2, EphB4, EDAR, EphB6, EDG-1, Ephrin, EDG-5, Ephrin-A1,
EDG-8, Ephrin-A2, eEF-2, Ephrin-A3, EGF, Ephrin-A4, EGF R,
Ephrin-A5, EGR1, Ephrin-B, EG-VEGF/PK1, Ephrin-B1, eIF2 alpha,
Ephrin-B2, eIF4E, Ephrin-B3, Elk-1, Epigen, EMAP-II,
Epimorphin/Syntaxin 2, EMMPRIN/CD147, Epiregulin, CXCL5/ENA,
EPR-1/Xa Receptor, Endocan, ErbB2, Endoglin/CD105, ErbB3,
Endoglycan, ErbB4, Endonuclease III, ERCC1, Endonuclease IV, ERCC3,
Endonuclease V, ERK1/ERK2, Endonuclease VIII, ERK1,
Endorepellin/Perlecan, ERK2, Endostatin, ERK3, Endothelin-1,
ERK5/BMK1, Engrailed-2, ERR alpha/NR3B1, EN-RAGE, ERR beta/NR3B2,
Enteropeptidase/Enterokinase, ERR gamma/NR3B3, CCL11/Eotaxin,
Erythropoietin, CCL24/Eotaxin-2, Erythropoietin R, CCL26/Eotaxin-3,
ESAM, EpCAM/TROP-1, ER alpha/NR3A1, EPCR, ER beta/NR3A2, Eph,
Exonuclease III, EphA1, Exostosin-like 2/EXTL2, EphA2,
Exostosin-like 3/EXTL3, EphA3, FABP1, FGF-BP, FABP2, FGF R1-4,
FABP3, FGF R1, FABP4, FGF R2, FABP5, FGF R3, FABP7, FGF R4, FABP9,
FGF R5, Complement Factor B, Fgr, FADD, FHR5, FAM3A, Fibronectin,
FAM3B, Ficolin-2, FAM3C, Ficolin-3, FAM3D, FITC, Fibroblast
Activation Protein alpha/FAP, FKBP38, Fas/TNFRSF6, Flap, Fas
Ligand/TNFSF6, FLIP, FATP1, FLRG, FATP4, FLRT1, FATP5, FLRT2, Fc
gamma R1/CD64, FLRT3, Fc gamma RIIB/CD32b, Flt-3, Fc gamma
RIIC/CD32c, Flt-3 Ligand, Fc gamma RIIA/CD32a, Follistatin, Fc
gamma RIII/CD16, Follistatin-like 1, FcRH1/IRTA5, FosB/G0S3,
FcRH2/IRTA4, FoxD3, FcRH4/IRTA1, FoxJ1, FcRH5/IRTA2, FoxP3, Fc
Receptor-like 3/CD16-2, Fpg, FEN-1, FPR1, Fetuin A, FPRL1, Fetuin
B, FPRL2, FGF acidic, CX3CL1/Fractalkine, FGF basic, Frizzled-1,
FGF-3, Frizzled-2, FGF-4, Frizzled-3, FGF-5, Frizzled-4, FGF-6,
Frizzled-5, FGF-8, Frizzled-6, FGF-9, Frizzled-7, FGF-10,
Frizzled-8, FGF-11, Frizzled-9, FGF-12, Frk, FGF-13, sFRP-1,
FGF-16, sFRP-2, FGF-17, sFRP-3, FGF-19, sFRP-4, FGF-20, Furin,
FGF-21, FXR/NR1H4, FGF-22, Fyn, FGF-23, G9a/EHMT2, GFR alpha-3/GDNF
R alpha-3, GABA-A-R alpha 1, GFR alpha-4/GDNF R alpha-4, GABA-A-R
alpha 2, GITR/TNFRSF18, GABA-A-R alpha 4, GITR Ligand/TNFSF18,
GABA-A-R alpha 5, GLI-1, GABA-A-R alpha 6, GLI-2, GABA-A-R beta 1,
GLP/EHMT1, GABA-A-R beta 2, GLP-1 R, GABA-A-R beta 3,
Glucagon,GABA-A-R gamma 2, Glucosamine (N-acetyl)-6-Sulfatase/GNS,
GABA-B-R2, GluR1, GAD1/GAD67, GluR2/3, GAD2/GAD65, GluR2, GADD45
alpha, GluR3, GADD45 beta, Glut1, GADD45 gamma, Glut2, Galectin-1,
Glut3, Galectin-2, Glut4, Galectin-3, Glut5, Galectin-3 BP,
Glutaredoxin 1, Galectin-4, Glycine R, Galectin-7, Glycophorin A,
Galectin-8, Glypican 2, Galectin-9, Glypican 3, GalNAc4S-6ST,
Glypican 5, GAP-43, Glypican 6, GAPDH, GM-CSF, Gas1, GM-CSF R
alpha, Gas6, GMF-beta, GASP-1/WFIKKNRP, gp130, GASP-2/WFIKKN,
Glycogen Phosphorylase BB/GPBB, GATA-1, GPR15, GATA-2, GPR39,
GATA-3, GPVI, GATA-4, GR/NR3C1, GATA-5, Gr-1/Ly-6G, GATA-6,
Granulysin, GBL, Granzyme A, GCNF/NR6A1, Granzyme B, CXCL6/GCP-2,
Granzyme D, G-CSF, Granzyme G, G-CSF R, Granzyme H, GDF-1, GRASP,
GDF-3 GRB2, GDF-5, Gremlin, GDF-6, GRO, GDF-7, CXCL1/GRO alpha,
GDF-8, CXCL2/GRO beta, GDF-9, CXCL3/GRO gamma, GDF-11, Growth
Hormone, GDF-15, Growth Hormone R, GDNF, GRP75/HSPA9B, GFAP, GSK-3
alpha/beta, GFI-1, GSK-3 alpha, GFR alpha-1/GDNF R alpha-1, GSK-3
beta, GFR alpha-2/GDNF R alpha-2, EZFIT, H2AX, Histidine, H60,
HM74A, HAI-1, HMGA2, HAI-2, HMGB1, HAI-2A, TCF-2/HNF-1 beta,
HAI-2B, HNF-3 beta/FoxA2, HAND1, HNF-4 alpha/NR2A1, HAPLN1, HNF-4
gamma/NR2A2, Airway Trypsin-like Protease/HAT, HO-1/HMOX1/HSP32,
HB-EGF, HO-2/HMOX2, CCL14a/HCC-1, HPRG, CCL14b/HCC-3, Hrk,
CCL16/HCC-4, HRP-1, alpha HCG, HS6ST2, Hck, HSD-1, HCR/CRAM-A/B,
HSD-2, HDGF, HSP10/EPF, Hemoglobin, HSP27, Hepassocin, HSP60,
HES-1, HSP70, HES-4, HSP90, HGF, HTRA/Protease Do, HGF Activator,
HTRA1/PRSS11, HGF R, HTRA2/Omi, HIF-1 alpha, HVEM/TNFRSF14, HIF-2
alpha, Hyaluronan, HIN-1/Secretoglobulin 3A1,4-Hydroxynonenal, Hip,
CCL1/1-309/TCA-3, IL-10, cIAP (pan), IL-10 R alpha, cIAP-1/HIAP-2,
IL-10 R beta, cIAP-2/HIAP-1, IL-11, IBSP/Sialoprotein II, IL-11 R
alpha, ICAM-1/CD54, IL-12, ICAM-2/CD102, IL-12/IL-23 p40,
ICAM-3/CD50, IL-12 R beta 1, ICAM-5, IL-12 R beta 2, ICAT, IL-13,
ICOS, IL-13 R alpha 1, Iduronate 2-Sulfatase/IDS, IL-13 R alpha 2,
IFN, IL-15, IFN-alpha, IL-15 R alpha, IFN-alpha 1, IL-16, IFN-alpha
2, IL-17, IFN-alpha 4b, IL-17 R, IFN-alpha A, IL-17 R.sup.cC,
IFN-alpha B2, IL-17 RD, IFN-alpha C, IL-17B, IFN-alpha D, IL-17B R,
IFN-alpha F, IL-17C, IFN-alpha G, IL-17D, IFN-alpha H2, IL-17E,
IFN-alpha I, IL-17F, IFN-alpha J1, IL-18/IL-1F4, IFN-alpha K, IL-18
BPa, IFN-alpha WA, IL-18 BPc, IFN-alpha/beta R1, IL-18 BPd,
IFN-alpha/beta R2, IL-18 R alpha/IL-1 R5, IFN-beta, IL-18 R
beta/IL-1 R7, IFN-gamma, IL-19, IFN-gamma R1, IL-20, IFN-gamma R2,
IL-20 R alpha, IFN-omega, IL-20 R beta, IgE, IL-21, IGFBP-1, IL-21
R, IGFBP-2, IL-22, IGFBP-3, IL-22 R, IGFBP-4, IL-22BP, IGFBP-5,
IL-23, IGFBP-6, IL-23 R, IGFBP-L1, IL-24, IGFBP-rp1/IGFBP-7,
IL-26/AK155, IGFBP-rP10, IL-27, IGF-I, IL-28A, IGF-I R, IL-28B,
IGF-II, IL-29/IFN-lambda 1, IGF-II R, IL-31, IgG, IL-31 RA, IgM,
IL-32 alpha, IGSF2, IL-33, IGSF4A/SynCAM, ILT2/CD85j, IGSF4B,
ILT3/CD85k, IGSF8, ILT4/CD85d, IgY, ILT5/CD85a, IkB-beta,
ILT6/CD85e, IKK alpha, Indian Hedgehog, IKK epsilon, INSRR, IKK
gamma, Insulin, IL-1 alpha/IL-1F1, Insulin R/CD220, IL-1
beta/IL-1F2, Proinsulin, IL-1ra/IL-1F3, Insulysin/IDE, IL-1F5/FILL
delta, Integrin alpha 2/CD49b, IL-1F6/FIL1 epsilon, Integrin alpha
3/CD49c, IL-1F7/FIL1 zeta, Integrin alpha 3 beta 1/VLA-3,
IL-1F8/FIL1 eta, Integrin alpha 4/CD49d, IL-1F9/IL-1 H1, Integrin
alpha 5/CD49e, IL-1F10/IL-1HY2, Integrin alpha 5 beta 1, IL-1 RI,
Integrin alpha 6/CD49f, IL-1 RII, Integrin alpha 7, IL-1 R3/IL-1 R
AcP, Integrin alpha 9, IL-1 R4/ST2, Integrin alpha E/CD103, IL-1
R6/IL-1 R rp2, Integrin alpha L/CD11a, IL-1 R8, Integrin alpha L
beta 2, IL-1 R9, Integrin alpha M/CD11b, IL-2, Integrin alpha M
beta 2, IL-2 R alpha, Integrin alpha V/CD51, IL-2 R beta, Integrin
alpha V beta 5, IL-3, Integrin alpha V beta 3, IL-3 R alpha,
Integrin alpha V beta 6, IL-3 R beta, Integrin alpha X/CD11c, IL-4,
Integrin beta 1/CD29, IL-4 R, Integrin beta 2/CD18, IL-5, Integrin
beta 3/CD61, IL-5 R alpha, Integrin beta 5, IL-6, Integrin beta 6,
IL-6 R, Integrin beta 7, IL-7, CXCL10/IP-10/CRG-2, IL-7 R
alpha/CD127, IRAK1, CXCR1/IL-8 RA, IRAK4, CXCR2/IL-8 RB, IRS-1,
CXCL8/IL-8, Islet-1, IL-9, CXCL11/1-TAC, IL-9 R, Jagged 1,
JAM-4/IGSF5, Jagged 2, JNK, JAM-A, JNK1/JNK2, JAM-B/VE-JAM, JNK1,
JAM-C, JNK2, Kininogen, Kallikrein 3/PSA, Kininostatin, Kallikrein
4, KIR/CD158, Kallikrein 5, KIR2DL1, Kallikrein 6/Neurosin,
KIR2DL3, Kallikrein 7, KIR2DL4/CD158d, Kallikrein 8/Neuropsin,
KIR2DS4, Kallikrein 9, KIR3DL1, Plasma Kallikrein/KLKB1, KIR3DL2,
Kallikrein 10, Kirrel2, Kallikrein 11, KLF4, Kallikrein 12, KLF5,
Kallikrein 13, KLF6, Kallikrein 14, Klotho, Kallikrein 15, Klotho
beta, KC, KOR, Keap1, Kremen-1, Kell, Kremen-2, KGF/FGF-7, LAG-3,
LINGO-2, LAIR1, Lipin 2, LAIR2, Lipocalin-1, Laminin alpha 4,
Lipocalin-2/NGAL, Laminin gamma 1,5-Lipoxygenase, Laminin I, LXR
alpha/NR1H3, Laminin S, LXR beta/NR1H2, Laminin-1, Livin,
Laminin-5, LIX, LAMP, LMIR1/CD300A, Langerin, LMIR2/CD300c, LAR,
LMIR3/CD300LF, Latexin, LMIR5/CD300LB, Layilin, LMIR6/CD300LE, LBP,
LMO2, LDL R, LOX-1/SR-E1, LECT2, LRH-1/NR5A2, LEDGF, LRIG1, Lefty,
LRIG3, Lefty-1, LRP-1, Lefty-A, LRP-6, Legumain, LSECtin/CLEC4G,
Leptin, Lumican, Leptin R, CXCL15/Lungkine, Leukotriene B4,
XCL1/Lymphotactin, Leukotriene B4 R1, Lymphotoxin, LIF, Lymphotoxin
beta/TNFSF3, LIF R alpha, Lymphotoxin beta R/TNFRSF3,
LIGHT/TNFSF14, Lyn, Limitin, Lyp, LIMPII/SR-B2, Lysyl Oxidase
Homolog 2, LIN-28, LYVE-1, LINGO-1, alpha 2-Macroglobulin,
CXCL9/MIG, MAD2L1, Mimecan, MAdCAM-1, Mindin, MafB,
Mineralocorticoid R/NR3C2, MafF, CCL3L1/MIP-1 alpha Isoform LD78
beta, MafG, CCL3/MIP-1 alpha, MafK, CCL4L1/LAG-1, MAG/Siglec-4-a,
CCL4/MIP-1 beta, MANF, CCL15/MIP-1 delta, MAP2, CCL9/10/MIP-1
gamma, MAPK, MIP-2, Marapsin/Pancreasin, CCL19/MIP-3 beta, MARCKS,
CCL20/MIP-3 alpha, MARCO, MIP-I, Mash1, MIP-II, Matrilin-2,
MIP-III, Matrilin-3, MIS/AMH, Matrilin-4, MIS RII, Matriptase/ST14,
MIXL1, MBL, MKK3/MKK6, MBL-2, MKK3, Melanocortin 3R/MC3R, MKK4,
MCAM/CD146, MKK6, MCK-2, MKK7, Mc1-1, MKP-3, MCP-6, MLH-1,
CCL2/MCP-1, MLK4 alpha, MCP-11, MMP, CCL8/MCP-2, MMP-1,
CCL7/MCP-3/MARC, MMP-2, CCL13/MCP-4, MMP-3, CCL12/MCP-5, MMP-7,
M-CSF, MMP-8, M-CSF R, MMP-9, MCV-type II, MMP-10, MD-1, MMP-11,
MD-2, MMP-12, CCL22/MDC, MMP-13, MDL-1/CLEC5A, MMP-14, MDM2,
MMP-15, MEA-1, MMP-16/MT3-MMP, MEK1/MEK2, MMP-24/MT5-MMP, MEK1,
MMP-25/MT6-MMP, MEK2, MMP-26, Melusin, MMR, MEPE, MOG, Meprin
alpha, CCL23/MPIF-1, Meprin beta, M-Ras/R-Ras3, Mer, Mrell,
Mesothelin, MRP1 Meteorin, MSK1/MSK2, Methionine Aminopeptidase 1,
MSK1, Methionine Aminopeptidase, MSK2, Methionine Aminopeptidase 2,
MSP, MFG-E8, MSP R/Ron, MFRP, Mug, MgcRacGAP, MULT-1, MGL2,
Musashi-1, MGMT, Musashi-2, MIA, MuSK, MICA, MutY DNA Glycosylase,
MICB, MyD88, MICL/CLEC12A, Myeloperoxidase, beta 2 Microglobulin,
Myocardin, Midkine, Myocilin, MIF, Myoglobin, NAIP NGFI-B
gamma/NR4A3, Nanog, NgR2/NgRH1, CXCL7/NAP-2, NgR3/NgRH2, Nbsl,
Nidogen-1/Entactin, NCAM-1/CD56, Nidogen-2, NCAM-L1, Nitric Oxide,
Nectin-1, Nitrotyrosine, Nectin-2/CD112, NKG2A, Nectin-3, NKG2C,
Nectin-4, NKG2D, Neogenin, NKp30, Neprilysin/CD10, NKp44,
Neprilysin-2/MMEL1/MMEL2, NKp46/NCR1, Nestin, NKp80/KLRF1, NETO2,
NKX2.5, Netrin-1, NMDA R, NR1 Subunit, Netrin-2, NMDA R, NR2A
Subunit, Netrin-4, NMDA R, NR2B Subunit, Netrin-G1a, NMDA R, NR2C
Subunit, Netrin-G2a, N-Me-6,7-diOH-TIQ, Neuregulin-1/NRG1, Nodal,
Neuregulin-3/NRG3, Noggin, Neuritin, Nogo Receptor, NeuroD1,
Nogo-A, Neurofascin, NOMO, Neurogenin-1, Nope, Neurogenin-2,
Norrin, Neurogenin-3, eNOS, Neurolysin, iNOS, Neurophysin II, nNOS,
Neuropilin-1, Notch-1, Neuropilin-2, Notch-2, Neuropoietin,
Notch-3, Neurotrimin, Notch-4, Neurturin, NOV/CCN3, NFAM1, NRAGE,
NF-H, NrCAM, NFkB1, NRL, NFkB2, NT-3, NF-L, NT-4, NF-M,
NTB-A/SLAMF6, NG2/MCSP, NTH1, NGF R/TNFRSF16, Nucleostemin,
beta-NGF, Nun-1/NR4A2, NGFI-B alpha/NR4A1, OAS2, Orexin B, OBCAM,
OSCAR, OCAM, OSF-2/Periostin, OCIL/CLEC2d, Oncostatin M/OSM,
OCILRP2/CLEC21, OSM R beta, Oct-3/4, Osteoactivin/GPNMB, OGG1,
Osteoadherin, Olig 1, 2, 3, Osteocalcin, Olig1, Osteocrin, Olig2,
Osteopontin, Olig3, Osteoprotegerin/TNFRSF11B, Oligodendrocyte
Marker O1, Otx2, Oligodendrocyte Marker O4, OV-6, OMgp,
OX40/TNFRSF4, Opticin, OX40 Ligand/TNFSF4, Orexin A, OAS2, Orexin
B, OBCAM, OSCAR, OCAM, OSF-2/Periostin, OCIL/CLEC2d, Oncostatin
M/OSM, OCILRP2/CLEC21, OSM R beta, Oct-3/4, Osteoactivin/GPNMB,
OGG1, Osteoadherin, Olig 1, 2, 3, Osteocalcin, Olig1, Osteocrin,
Olig2, Osteopontin, Olig3,
Osteoprotegerin/TNFRSF11B, Oligodendrocyte Marker O1, Otx2,
Oligodendrocyte Marker O4, OV-6, OMgp, OX40/TNFRSF4, Opticin, OX40
Ligand/TNFSF4, Orexin A, RACK1, Ret, Rad1, REV-ERB alpha/NR1D1,
Rad17, REV-ERB beta/NR1D2, Rad51, Rex-1, Rae-1, RGM-A, Rae-1 alpha,
RGM-B, Rae-1 beta, RGM-C, Rae-1 delta, Rheb, Rae-1 epsilon,
Ribosomal Protein S6, Rae-1 gamma, RIP1, Raf-1, ROBO1, RAGE, ROBO2,
RalA/RalB, ROBO3, RalA, ROBO4, RalB, ROR/NR1F1-3 (pan),
RANK/TNFRSF11A, ROR alpha/NR1F1, CCL5/RANTES, ROR gamma/NR1F3,
Rap1A/B, RTK-like Orphan Receptor 1/ROR1, RAR alpha/NR1B1, RTK-like
Orphan Receptor 2/ROR2, RAR beta/NR1B2, RP105, RAR gamma/NR1B3,
RPA2, Ras, RSK (pan), RBP4, RSK1/RSK2, RECK, RSK1, Reg 2/PAP, RSK2,
Reg I, RSK3, Reg II, RSK4, Reg III, R-Spondin 1, Reg Ma, R-Spondin
2, Reg IV, R-Spondin 3, Relaxin-1, RUNX1/CBFA2, Relaxin-2,
RUNX2/CBFA1, Relaxin-3, RUNX3/CBFA3, RELM alpha, RXR alpha/NR2B1,
RELM beta, RXR beta/NR2B2, RELT/TNFRSF19L, RXR gamma/NR2B3,
Resistin, S100A10, SLITRK5, S100A8, SLP1, S100A9, SMAC/Diablo,
S100B, Smad1, S100P, Smad2, SALL1, Smad3, delta-Sarcoglycan, Smad4,
Sca-1/Ly6, Smad5, SCD-1, Smad7, SCF, Smad8, SCF R/c-kit, SMC1,
SCGF, alpha-Smooth Muscle Actin, SCL/Tall, SMUG1, SCP3/SYCP3,
Snail, CXCL12/SDF-1, Sodium Calcium Exchanger 1, SDNSF/MCFD2,
Soggy-1, alpha-Secretase, Sonic Hedgehog, gamma-Secretase, SorCS1,
beta-Secretase, SorCS3, E-Selectin, Sortilin, L-Selectin, SOST,
P-Selectin, SOX1, Semaphorin 3A, SOX2, Semaphorin 3C, SOX3,
Semaphorin 3E, SOX7, Semaphorin 3F, SOX9, Semaphorin 6A, SOX10,
Semaphorin 6B, SOX17, Semaphorin 6C, SOX21 Semaphorin 6D, SPARC,
Semaphorin 7A, SPARC-like 1, Separase, SP-D, Serine/Threonine
Phosphatase Substrate I, Spinesin, Serpin A1, F-Spondin, Serpin A3,
SR-AI/MSR, Serpin A4/Kallistatin, Src, Serpin A5/Protein C
Inhibitor, SREC-I/SR-F1, Serpin A8/Angiotensinogen, SREC-II, Serpin
B5, SSEA-1, Serpin C1/Antithrombin-III, SSEA-3, Serpin D1/Heparin
Cofactor II, SSEA-4, Serpin E1/PAI-1, ST7/LRP12, Serpin E2,
Stabilin-1, Serpin F1, Stabilin-2, Serpin F2, Stanniocalcin 1,
Serpin G1/C1 Inhibitor, Stanniocalcin 2, Serpin 12, STAT1, Serum
Amyloid A1, STAT2, SF-1/NR5A1, STAT3, SGK, STAT4, SHBG, STAT5a/b,
SHIP, STAT5a, SHP/NR0B2, STAT5b, SHP-1, STAT6, SHP-2, VE-Statin,
SIGIRR, Stella/Dppa3, Siglec-2/CD22, STRO-1, Siglec-3/CD33,
Substance P, Siglec-5, Sulfamidase/SGSH, Siglec-6, Sulfatase
Modifying Factor 1/SUMF1, Siglec-7, Sulfatase Modifying Factor
2/SUMF2, Siglec-9, SUMO1, Siglec-10, SUMO2/3/4, Siglec-11, SUMO3,
Siglec-F, Superoxide Dismutase, SIGNR1/CD209, Superoxide
Dismutase-1/Cu-Zn SOD, SIGNR4, Superoxide Dismutase-2/Mn-SOD, SIRP
beta 1, Superoxide Dismutase-3/EC-SOD, SKI, Survivin, SLAM/CD150,
Synapsin I, Sleeping Beauty Transposase, Syndecan-1/CD138, Slit3,
Syndecan-2, SLITRK1, Syndecan-3, SLITRK2, Syndecan-4, SLITRK4,
TACI/TNFRSF13B, TMEFF1/Tomoregulin-1, TAO2, TMEFF2, TAPP1,
TNF-alpha/TNFSF1A, CCL17/TARC, TNF-beta/TNFSF1B, Tau, TNF
RI/TNFRSF1A, TC21/R-Ras2, TNF RII/TNFRSF1B, TCAM-1, TOR,
TCCR/WSX-1, TP-1, TC-PTP, TP63/TP73L, TDG, TR, CCL25/TECK, TR
alpha/NR1A1, Tenascin C, TR beta 1/NR1A2, Tenascin R, TR2/NR2C1,
TER-119, TR4/NR2C2, TERT, TRA-1-85, Testican 1/SPOCK1, TRADD,
Testican 2/SPOCK2, TRAF-1, Testican 3/SPOCK3, TRAF-2, TFPI, TRAF-3,
TFPI-2, TRAF-4, TGF-alpha, TRAF-6, TGF-beta, TRAIL/TNFSF10,
TGF-beta 1, TRAIL R1/TNFRSF10A, LAP (TGF-beta 1), TRAIL
R2/TNFRSF10B, Latent TGF-beta 1, TRAIL R3/TNFRSF10C, TGF-beta 1.2,
TRAIL R4/TNFRSF10D, TGF-beta 2, TRANCE/TNFSF11, TGF-beta 3, TfR
(Transferrin R), TGF-beta 5, Apo-Transferrin, Latent TGF-beta bp1,
Holo-Transferrin, Latent TGF-beta bp2, Trappin-2/Elafin, Latent
TGF-beta bp4, TREM-1, TGF-beta RI/ALK-5, TREM-2, TGF-beta RII,
TREM-3, TGF-beta RIIb, TREML1/TLT-1, TGF-beta RIII, TRF-1,
Thermolysin, TRF-2, Thioredoxin-1, TRH-degrading Ectoenzyme/TRHDE,
Thioredoxin-2, TRIMS, Thioredoxin-80, Tripeptidyl-Peptidase I,
Thioredoxin-like 5/TRP14, TrkA, THOP1, TrkB, Thrombomodulin/CD141,
TrkC, Thrombopoietin, TROP-2, Thrombopoietin R, Troponin I Peptide
3, Thrombospondin-1, Troponin T, Thrombospondin-2, TROY/TNFRSF19,
Thrombospondin-4, Trypsin 1, Thymopoietin, Trypsin 2/PRSS2, Thymus
Chemokine-1, Trypsin 3/PRSS3, Tie-1, Tryptase-5/Prss32, Tie-2,
Tryptase alpha/TPS1, TIM-1/KIM-1/HAVCR, Tryptase beta-1/MCPT-7,
TIM-2, Tryptase beta-2/TPSB2, TIM-3, Tryptase epsilon/BSSP-4,
TIM-4, Tryptase gamma-1/TPSG1, TIM-5, Tryptophan Hydroxylase,
TIM-6, TSC22, TIMP-1, TSG, TIMP-2, TSG-6, TIMP-3, TSK, TIMP-4,
TSLP, TL1A/TNFSF15, TSLP R, TLR1, TSP50, TLR2, beta-III Tubulin,
TLR3, TWEAK/TNFSF12, TLR4, TWEAK R/TNFRSF12, TLR5, Tyk2, TLR6,
Phospho-Tyrosine, TLR9, Tyrosine Hydroxylase, TLX/NR2E1, Tyrosine
Phosphatase Substrate I, Ubiquitin, UNC5H3, Ugi, UNC5H4, UGRP1,
UNG, ULBP-1, uPA, ULBP-2, uPAR, ULBP-3, URB, UNC5H1, UVDE, UNC5H2,
Vanilloid R1, VEGF R, VASA, VEGF R1/Flt-1, Vasohibin, VEGF
R2/KDR/Flk-1, Vasorin, VEGF R3/Flt-4, Vasostatin, Versican, Vav-1,
VG5Q, VCAM-1, VHR, VDR/NR1I1, Vimentin, VEGF, Vitronectin, VEGF-B,
VLDLR, VEGF-C, vWF-A2, VEGF-D, Synuclein-alpha, Ku70, WASP, Wnt-7b,
WIF-1, Wnt-8a WISP-1/CCN4, Wnt-8b, WNK1, Wnt-9a, Wnt-1, Wnt-9b,
Wnt-3a, Wnt-10a, Wnt-4, Wnt-10b, Wnt-5a, Wnt-11, Wnt-5b, wnvNS3,
Wnt7a, XCR1, XPE/DDB1, XEDAR, XPE/DDB2, Xg, XPF, XIAP, XPG, XPA,
XPV, XPD, XRCC1, Yes, YY1, EphA4.
[0397] Numerous human ion channels are targets of particular
interest. Non-limiting examples include 5-hydroxytryptamine 3
receptor B subunit, 5-hydroxytryptamine 3 receptor precursor,
5-hydroxytryptamine receptor 3 subunit C, AAD14 protein,
Acetylcholine receptor protein, alpha subunit precursor,
Acetylcholine receptor protein, beta subunit precursor,
Acetylcholine receptor protein, delta subunit precursor,
Acetylcholine receptor protein, epsilon subunit precursor,
Acetylcholine receptor protein, gamma subunit precursor, Acid
sensing ion channel 3 splice variant b, Acid sensing ion channel 3
splice variant c, Acid sensing ion channel 4, ADP-ribose
pyrophosphatase, mitochondrial precursor, Alpha1A-voltage-dependent
calcium channel, Amiloride-sensitive cation channel 1, neuronal,
Amiloride-sensitive cation channel 2, neuronal Amiloride-sensitive
cation channel 4, isoform 2, Amiloride-sensitive sodium channel,
Amiloride-sensitive sodium channel alpha-subunit,
Amiloride-sensitive sodium channel beta-subunit,
Amiloride-sensitive sodium channel delta-subunit,
Amiloride-sensitive sodium channel gamma-subunit, Annexin A7,
Apical-like protein, ATP-sensitive inward rectifier potassium
channel 1, ATP-sensitive inward rectifier potassium channel 10,
ATP-sensitive inward rectifier potassium channel 11, ATP-sensitive
inward rectifier potassium channel 14, ATP-sensitive inward
rectifier potassium channel 15, ATP-sensitive inward rectifier
potassium channel 8, Calcium channel alpha12.2 subunit, Calcium
channel alpha12.2 subunit, Calcium channel alpha1E subunit, delta19
delta40 delta46 splice variant, Calcium-activated potassium channel
alpha subunit 1, Calcium-activated potassium channel beta subunit
1, Calcium-activated potassium channel beta subunit 2,
Calcium-activated potassium channel beta subunit 3,
Calcium-dependent chloride channel-1, Cation channel TRPM4B, cDNA
FLJ90453 fis, clone NT2RP3001542, highly similar to Potassium
channel tetramerisation domain containing 6, cDNA FLJ90663 fis,
clone PLACE1005031, highly similar to Chloride intracellular
channel protein 5, CGMP-gated cation channel beta subunit, Chloride
channel protein, Chloride channel protein 2, Chloride channel
protein 3, Chloride channel protein 4, Chloride channel protein 5,
Chloride channel protein 6, Chloride channel protein ClC-Ka,
Chloride channel protein ClC-Kb, Chloride channel protein, skeletal
muscle, Chloride intracellular channel 6, Chloride intracellular
channel protein 3, Chloride intracellular channel protein 4,
Chloride intracellular channel protein 5, CHRNA3 protein, Clcn3e
protein, CLCNKB protein, CNGA4 protein, Cullin-5, Cyclic GMP gated
potassium channel, Cyclic-nucleotide-gated cation channel 4,
Cyclic-nucleotide-gated cation channel alpha 3,
Cyclic-nucleotide-gated cation channel beta 3,
Cyclic-nucleotide-gated olfactory channel, Cystic fibrosis
transmembrane conductance regulator, Cytochrome B-245 heavy chain,
Dihydropyridine-sensitive L-type, calcium channel alpha-2/delta
subunits precursor, FXYD domain-containing ion transport regulator
3 precursor, FXYD domain-containing ion transport regulator 5
precursor, FXYD domain-containing ion transport regulator 6
precursor, FXYD domain-containing ion transport regulator 7, FXYD
domain-containing ion transport regulator 8 precursor, G
protein-activated inward rectifier potassium channel 1, G
protein-activated inward rectifier potassium channel 2, G
protein-activated inward rectifier potassium channel 3, G
protein-activated inward rectifier potassium channel 4,
Gamma-aminobutyric-acid receptor alpha-1 subunit precursor,
Gamma-aminobutyric-acid receptor alpha-2 subunit precursor,
Gamma-aminobutyric-acid receptor alpha-3 subunit precursor,
Gamma-aminobutyric-acid receptor alpha-4 subunit precursor,
Gamma-aminobutyric-acid receptor alpha-5 subunit precursor,
Gamma-aminobutyric-acid receptor alpha-6 subunit precursor,
Gamma-aminobutyric-acid receptor beta-1 subunit precursor,
Gamma-aminobutyric-acid receptor beta-2 subunit precursor,
Gamma-aminobutyric-acid receptor beta-3 subunit precursor,
Gamma-aminobutyric-acid receptor delta subunit precursor,
Gamma-aminobutyric-acid receptor epsilon subunit precursor,
Gamma-aminobutyric-acid receptor gamma-1 subunit precursor,
Gamma-aminobutyric-acid receptor gamma-3 subunit precursor,
Gamma-aminobutyric-acid receptor pi subunit precursor,
Gamma-aminobutyric-acid receptor rho-1 subunit precursor,
Gamma-aminobutyric-acid receptor rho-2 subunit precursor,
Gamma-aminobutyric-acid receptor theta subunit precursor, GluR6
kainate receptor, Glutamate receptor 1 precursor, Glutamate
receptor 2 precursor, Glutamate receptor 3 precursor, Glutamate
receptor 4 precursor, Glutamate receptor 7, Glutamate receptor B,
Glutamate receptor delta-1 subunit precursor, Glutamate receptor,
ionotropic kainate 1 precursor, Glutamate receptor, ionotropic
kainate 2 precursor, Glutamate receptor, ionotropic kainate 3
precursor, Glutamate receptor, ionotropic kainate 4 precursor,
Glutamate receptor, ionotropic kainate 5 precursor, Glutamate
[NMDA] receptor subunit 3A precursor, Glutamate [NMDA] receptor
subunit 3B precursor, Glutamate [NMDA] receptor subunit epsilon 1
precursor, Glutamate [NMDA] receptor subunit epsilon 2 precursor,
Glutamate [NMDA] receptor subunit epsilon 4 precursor, Glutamate
[NMDA] receptor subunit zeta 1 precursor, Glycine receptor alpha-1
chain precursor, Glycine receptor alpha-2 chain precursor, Glycine
receptor alpha-3 chain precursor, Glycine receptor beta chain
precursor, H/ACA ribonucleoprotein complex subunit 1, High affinity
immunoglobulin epsilon receptor beta-subunit, Hypothetical protein
DKFZp313I0334, Hypothetical protein DKFZp761M1724, Hypothetical
protein FLJ12242, Hypothetical protein FLJ14389, Hypothetical
protein FLJ14798, Hypothetical protein FLJ14995, Hypothetical
protein FLJ16180, Hypothetical protein FLJ16802, Hypothetical
protein FLJ32069, Hypothetical protein FLJ37401, Hypothetical
protein FLJ38750, Hypothetical protein FLJ40162, Hypothetical
protein FLJ41415, Hypothetical protein FLJ90576, Hypothetical
protein FLJ90590, Hypothetical protein FLJ90622, Hypothetical
protein KCTD15, Hypothetical protein MGC15619, Inositol
1,4,5-trisphosphate receptor type 1, Inositol 1,4,5-trisphosphate
receptor type 2, Inositol 1,4,5-trisphosphate receptor type 3,
Intermediate conductance calcium-activated potassium channel
protein 4, Inward rectifier potassium channel 13, Inward rectifier
potassium channel 16, Inward rectifier potassium channel 4, Inward
rectifying K(+) channel negative regulator Kir2.2v, Kainate
receptor subunit KA2a, KCNH5 protein, KCTD17 protein, KCTD2
protein, Keratinocytes associated transmembrane protein 1, Kv
channel-interacting protein 4, Melastatin 1, Membrane protein MLC1,
MGC15619 protein, Mucolipin-1, Mucolipin-2, Mucolipin-3, Multidrug
resistance-associated protein 4, N-methyl-D-aspartate receptor 2C
subunit precursor, NADPH oxidase homolog 1, Nav1.5, Neuronal
acetylcholine receptor protein, alpha-10 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-2 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-3 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-4 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-5 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-6 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-7 subunit precursor,
Neuronal acetylcholine receptor protein, alpha-9 subunit precursor,
Neuronal acetylcholine receptor protein, beta-2 subunit precursor,
Neuronal acetylcholine receptor protein, beta-3 subunit precursor,
Neuronal acetylcholine receptor protein, beta-4 subunit precursor,
Neuronal voltage-dependent calcium channel alpha 2D subunit, P2X
purinoceptor 1, P2X purinoceptor 2, P2X purinoceptor 3, P2X
purinoceptor 4, P2X purinoceptor 5, P2X purinoceptor 6, P2X
purinoceptor 7, Pancreatic potassium channel TALK-1b, Pancreatic
potassium channel TALK-1c, Pancreatic potassium channel TALK-1d,
Phospholemman precursor, Plasmolipin, Polycystic kidney disease 2
related protein, Polycystic kidney disease 2-like 1 protein,
Polycystic kidney disease 2-like 2 protein, Polycystic kidney
disease and receptor for egg jelly related protein precursor,
Polycystin-2, Potassium channel regulator, Potassium channel
subfamily K member 1, Potassium channel subfamily K member 10,
Potassium channel subfamily K member 12, Potassium channel
subfamily K member 13, Potassium channel subfamily K member 15,
Potassium channel subfamily K member 16, Potassium channel
subfamily K member 17, Potassium channel subfamily K member 2,
Potassium channel subfamily K member 3, Potassium channel subfamily
K member 4, Potassium channel subfamily K member 5, Potassium
channel subfamily K member 6, Potassium channel subfamily K member
7, Potassium channel subfamily K member 9, Potassium channel
tetramerisation domain containing 3, Potassium channel
tetramerisation domain containing protein 12, Potassium channel
tetramerisation domain containing protein 14, Potassium channel
tetramerisation domain containing protein 2, Potassium channel
tetramerisation domain containing protein 4, Potassium channel
tetramerisation domain containing protein 5, Potassium channel
tetramerization domain containing 10, Potassium channel
tetramerization domain containing protein 13, Potassium channel
tetramerization domain-containing 1, Potassium voltage-gated
channel subfamily A member 1, Potassium voltage-gated channel
subfamily A member 2, Potassium voltage-gated channel subfamily A
member 4, Potassium voltage-gated channel subfamily A member 5,
Potassium voltage-gated channel subfamily A member 6, Potassium
voltage-gated channel subfamily B member 1, Potassium voltage-gated
channel subfamily B member 2, Potassium voltage-gated channel
subfamily C member 1, Potassium voltage-gated channel subfamily C
member 3, Potassium voltage-gated channel subfamily C member 4,
Potassium voltage-gated channel subfamily D member 1, Potassium
voltage-gated channel subfamily D member 2, Potassium voltage-gated
channel subfamily D member 3, Potassium voltage-gated channel
subfamily E member 1, Potassium voltage-gated channel subfamily E
member 2, Potassium voltage-gated channel subfamily E member 3,
Potassium voltage-gated channel subfamily E member 4, Potassium
voltage-gated channel subfamily F member 1, Potassium voltage-gated
channel subfamily G member 1, Potassium voltage-gated channel
subfamily G member 2, Potassium voltage-gated channel subfamily G
member 3, Potassium voltage-gated channel subfamily G member 4,
Potassium voltage-gated channel subfamily H member 1, Potassium
voltage-gated channel subfamily H member 2, Potassium voltage-gated
channel subfamily H member 3, Potassium voltage-gated channel
subfamily H member 4, Potassium voltage-gated channel subfamily H
member 5, Potassium voltage-gated channel subfamily H member 6,
Potassium voltage-gated channel subfamily H member 7, Potassium
voltage-gated channel subfamily H member 8, Potassium voltage-gated
channel subfamily KQT member 1, Potassium voltage-gated channel
subfamily KQT member 2, Potassium voltage-gated channel subfamily
KQT member 3, Potassium voltage-gated channel subfamily KQT member
4, Potassium voltage-gated channel subfamily KQT member 5,
Potassium voltage-gated channel subfamily S member 1, Potassium
voltage-gated channel subfamily S member 2, Potassium voltage-gated
channel subfamily S member 3, Potassium voltage-gated channel
subfamily V member 2, Potassium voltage-gated channel, subfamily H,
member 7, isoform 2, Potassium/sodium hyperpolarization-activated
cyclic nucleotide-gated channel 1, Potassium/sodium
hyperpolarization-activated cyclic nucleotide-gated channel 2,
Potassium/sodium hyperpolarization-activated cyclic
nucleotide-gated channel 3, Potassium/sodium
hyperpolarization-activated cyclic nucleotide-gated channel 4,
Probable mitochondrial import receptor subunit TOM40 homolog,
Purinergic receptor P2.times.5, isoform A, Putative 4 repeat
voltage-gated ion channel, Putative chloride channel protein 7,
Putative GluR6 kainate receptor, Putative ion channel protein
CATSPER2 variant 1, Putative ion channel protein CATSPER2 variant
2, Putative ion channel protein CATSPER2 variant 3, Putative
regulator of potassium channels protein variant 1, Putative
tyrosine-protein phosphatase TPTE, Ryanodine receptor 1, Ryanodine
receptor 2, Ryanodine receptor 3, SH3 KBP1 binding protein 1, Short
transient receptor potential channel 1, Short transient receptor
potential channel 4, Short transient receptor potential channel 5,
Short transient receptor potential channel 6, Short transient
receptor potential channel 7, Small conductance calcium-activated
potassium channel protein 1, Small conductance calcium-activated
potassium channel protein 2, isoform b, Small conductance
calcium-activated potassium channel protein 3, isoform b,
Small-conductance calcium-activated potassium channel SK2,
Small-conductance calcium-activated potassium channel SK3, Sodium
channel, Sodium channel beta-1 subunit precursor, Sodium channel
protein type II alpha subunit, Sodium channel protein type III
alpha subunit, Sodium channel protein type IV alpha subunit, Sodium
channel protein type IX alpha subunit, Sodium channel protein type
V alpha subunit, Sodium channel protein type VII alpha subunit,
Sodium channel protein type VIII alpha subunit, Sodium channel
protein type X alpha subunit, Sodium channel protein type XI alpha
subunit, Sodium- and chloride-activated ATP-sensitive potassium
channel, Sodium/potassium-transporting ATPase gamma chain,
Sperm-associated cation channel 1, Sperm-associated cation channel
2, isoform 4, Syntaxin-1B1, Transient receptor potential cation
channel subfamily A member 1, Transient receptor potential cation
channel subfamily M member 2, Transient receptor potential cation
channel subfamily M member 3, Transient receptor potential cation
channel subfamily M member 6, Transient receptor potential cation
channel subfamily M member 7, Transient receptor potential cation
channel subfamily V member 1, Transient receptor potential cation
channel subfamily V member 2, Transient receptor potential cation
channel subfamily V member 3, Transient receptor potential cation
channel subfamily V member 4, Transient receptor potential cation
channel subfamily V member 5, Transient receptor potential cation
channel subfamily V member 6, Transient receptor potential channel
4 epsilon splice variant, Transient receptor potential channel 4
zeta splice variant, Transient receptor potential channel 7 gamma
splice variant, Tumor necrosis factor, alpha-induced protein 1,
endothelial, Two-pore calcium channel protein 2, VDAC4 protein,
Voltage gated potassium channel Kv3.2b, Voltage gated sodium
channel beta1B subunit, Voltage-dependent anion channel,
Voltage-dependent anion channel 2, Voltage-dependent
anion-selective channel protein 1, Voltage-dependent
anion-selective channel protein 2, Voltage-dependent
anion-selective channel protein 3, Voltage-dependent calcium
channel gamma-1 subunit, Voltage-dependent calcium channel gamma-2
subunit, Voltage-dependent calcium channel gamma-3 subunit,
Voltage-dependent calcium channel gamma-4 subunit,
Voltage-dependent calcium channel gamma-5 subunit,
Voltage-dependent calcium channel gamma-6 subunit,
Voltage-dependent calcium channel gamma-7 subunit,
Voltage-dependent calcium channel gamma-8 subunit,
Voltage-dependent L-type calcium channel alpha-1C subunit,
Voltage-dependent L-type calcium channel alpha-1D subunit,
Voltage-dependent L-type calcium channel alpha-1S subunit,
Voltage-dependent L-type calcium channel beta-1 subunit,
Voltage-dependent L-type calcium channel beta-2 subunit,
Voltage-dependent L-type calcium channel beta-3 subunit,
Voltage-dependent L-type calcium channel beta-4 subunit,
Voltage-dependent N-type calcium channel alpha-1B subunit,
Voltage-dependent P/Q-type calcium channel alpha-1A subunit,
Voltage-dependent R-type calcium channel alpha-1E subunit,
Voltage-dependent T-type calcium channel alpha-1G subunit,
Voltage-dependent T-type calcium channel alpha-1H subunit,
Voltage-dependent T-type calcium channel alpha-1I subunit,
Voltage-gated L-type calcium channel alpha-1 subunit, Voltage-gated
potassium channel beta-1 subunit, Voltage-gated potassium channel
beta-2 subunit, Voltage-gated potassium channel beta-3 subunit,
Voltage-gated potassium channel KCNA7. The Nav1.x family of human
voltage-gated sodium channels also a particularly promising target.
This family includes, for example, channels Nav1.6 and Nav1.8.
[0398] Many of the microproteins used as scaffolds in this
application have native activity against G-Protein Coupled
Receptors (GPCRs) and offer ideal starting points to create novel
GPCR modulators (including agonists, antagonists and modulators of
any property of the GPCR). Exemplary GPCRs include but are not
limited to Class A Rhodopsin like receptors such as Muscatinic
(Musc.) acetylcholine Vertebrate type 1, Musc. acetylcholine
Vertebrate type 2, Musc. acetylcholine Vertebrate type 3, Musc.
acetylcholine Vertebrate type 4; Adrenoceptors (Alpha Adrenoceptors
type 1, Alpha Adrenoceptors type 2, Beta Adrenoceptors type 1, Beta
Adrenoceptors type 2, Beta Adrenoceptors type 3, Dopamine
Vertebrate type 1, Dopamine Vertebrate type 2, Dopamine Vertebrate
type 3, Dopamine Vertebrate type 4, Histamine type 1, Histamine
type 2, Histamine type 3, Histamine type 4, Serotonin type 1,
Serotonin type 2, Serotonin type 3, Serotonin type 4, Serotonin
type 5, Serotonin type 6, Serotonin type 7, Serotonin type 8, other
Serotonin types, Trace amine, Angiotensin type 1, Angiotensin type
2, Bombesin, Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, APJ like,
Interleukin-8 type A, Interleukin-8 type B, Interleukin-8 type
others, C-C Chemokine type 1 through type 11 and other types, C-X-C
Chemokine (types 2 through 6 and others), C-X3-C Chemokine,
Cholecystokinin CCK, CCK type A, CCK type B, CCK others,
Endothelin, Melanocortin (Melanocyte stimulating hormone,
Adrenocorticotropic hormone, Melanocortin hormone), Duffy antigen,
Prolactin-releasing peptide (GPR10), Neuropeptide Y (type 1 through
7), Neuropeptide Y, Neuropeptide Y other, Neurotensin, Opioid (type
D, K, M, X), Somatostatin (type 1 through 5), Tachykinin (Substance
P(NK1), Substance K (NK2), Neuromedin K (NK3), Tachykinin like 1,
Tachykinin like 2, Vasopressin/vasotocin (type 1 through 2),
Vasotocin, Oxytocin/mesotocin, Conopressin, Galanin like,
Proteinase-activated like, Orexin & neuropeptides FF, QRFP,
Chemokine receptor-like, Neuromedin U like (Neuromedin U,
PRXamide), hormone protein (Follicle stimulating hormone,
Lutropin-choriogonadotropic hormone, Thyrotropin, Gonadotropin type
I, Gonadotropin type II), (Rhod)opsin, Rhodopsin Vertebrate (types
1-5), Rhodopsin Vertebrate type 5, Rhodopsin Arthropod, Rhodopsin
Arthropod type 1, Rhodopsin Arthropod type 2, Rhodopsin Arthropod
type 3, Rhodopsin Mollusc, Rhodopsin, Olfactory (Olfactory II fam 1
through 13), Prostaglandin (prostaglandin E2 subtype EP1,
Prostaglandin E2/D2 subtype EP2, prostaglandin E2 subtype EP3,
Prostaglandin E2 subtype EP4, Prostaglandin F2-alpha, Prostacyclin,
Thromboxane, Adenosine type 1 through 3, Purinoceptors,
Purinoceptor P2RY1-4,6,11 GPR91, Purinoceptor P2RY5,8,9,10
GPR35,92,174, Purinoceptor P2RY12-14 GPR87 (UDP-Glucose),
Cannabinoid, Platelet activating factor, Gonadotropin-releasing
hormone, Gonadotropin-releasing hormone type I,
Gonadotropin-releasing hormone type II, Adipokinetic hormone like,
Corazonin, Thyrotropin-releasing hormone & Secretagogue,
Thyrotropin-releasing hormone, Growth hormone secretagogue, Growth
hormone secretagogue like, Ecdysis-triggering hormone (ETHR),
Melatonin, Lysosphingolipid & LPA (EDG), Sphingosine
1-phosphate Edg-1, Lysophosphatidic acid Edg-2, Sphingosine
1-phosphate Edg-3, Lysophosphatidic acid Edg-4, Sphingosine
1-phosphate Edg-5, Sphingosine 1-phosphate Edg-6, Lysophosphatidic
acid Edg-7, Sphingosine 1-phosphate Edg-8, Edg Other Leukotriene B4
receptor, Leukotriene B4 receptor BLT1, Leukotriene B4 receptor
BLT2, Class A Orphan/other, Putative neurotransmitters, SREB, Mas
proto-oncogene & Mas-related (MRGs), GPR45 like, Cysteinyl
leukotriene, G-protein coupled bile acid receptor, Free fatty acid
receptor (GP40,GP41,GP43), Class B Secretin like, Calcitonin,
Corticotropin releasing factor, Gastric inhibitory peptide,
Glucagon, Growth hormone-releasing hormone, Parathyroid hormone,
PACAP, Secretin, Vasoactive intestinal polypeptide, Latrophilin,
Latrophilin type 1, Latrophilin type 2, Latrophilin type 3, ETL
receptors, Brain-specific angiogenesis inhibitor (BAI),
Methuselah-like proteins (MTH), Cadherin EGF LAG (CELSR), Very
large G-protein coupled receptor, Class C Metabotropic
glutamate/pheromone, Metabotropic glutamate group I through III,
Calcium-sensing like, Extracellular calcium-sensing, Pheromone,
calcium-sensing like other, Putative pheromone receptors, GABA-B,
GABA-B subtype 1, GABA-B subtype 2, GABA-B like, Orphan GPRC5,
Orphan GPCR6, Bride of sevenless proteins (BOSS), Taste receptors
(T1R), Class D Fungal pheromone, Fungal pheromone A-Factor like
(STE2,STE3), Fungal pheromone B like (BAR,BBR,RCB,PRA), Class E
cAMP receptors, Ocular albinism proteins, Frizzled/Smoothened
family, frizzled Group A (Fz 1&2&4&5&7-9), frizzled
Group B (Fz 3 & 6), frizzled Group C (other), Vomeronasal
receptors, Nematode chemoreceptors, Insect odorant receptors, and
Class Z Archaeal/bacterial/fungal opsins.
[0399] Of particular utility is the fusion of accessory sequences
to any of the following active polypeptides: BOTOX, Myobloc,
Neurobloc, Dysport (or other serotypes of botulinum neurotoxins),
alglucosidase alfa, daptomycin, YH-16, choriogonadotropin alfa,
filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleukin,
denileukin diftitox, interferon alfa-n3 (injection), interferon
alfa-n1, DL-8234, interferon, Suntory (gamma-1a), interferon gamma,
thymosin alpha 1, tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab,
nesiritide, abatacept, alefacept, Rebif, eptoterminalfa,
teriparatide (osteoporosis), calcitonin injectable (bone disease),
calcitonin (nasal, osteoporosis), etanercept, hemoglobin glutamer
250 (bovine), drotrecogin alfa, collagenase, carperitide,
recombinant human epidermal growth factor (topical gel, wound
healing), DWP-401, darbepoetin alfa, epoetin omega, epoetin beta,
epoetin alfa, desirudin, lepirudin, bivalirudin, nonacog alpha,
Mononine, eptacog alfa (activated), recombinant Factor VIII+VWF,
Recombinate, recombinant Factor VIII, Factor VIII (recombinant),
Alphanate, octocog alfa, Factor VIII, palifermin, Indikinase,
tenecteplase, alteplase, pamiteplase, reteplase, nateplase,
monteplase, follitropin alfa, rFSH, hpFSH, micafungin,
pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon,
exenatide, pramlintide, imiglucerase, galsulfase, Leucotropin,
molgramostim, triptorelin acetate, histrelin (subcutaneous implant,
Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained
release depot (ATRIGEL), leuprolide implant (DUROS), goserelin,
somatropin, Eutropin, KP-102 program, somatropin, somatropin,
mecasermin (growth failure), enfuvirtide, Org-33408, insulin
glargine, insulin glulisine, insulin (inhaled), insulin lispro,
insulin detemir, insulin (buccal, RapidMist), mecasermin rinfabate,
anakinra, celmoleukin, 99 mTc-apcitide injection, myelopid,
Betaseron, glatiramer acetate, Gepon, sargramostim, oprelvekin,
human leukocyte-derived alpha interferons, Bilive, insulin
(recombinant), recombinant human insulin, insulin aspart,
mecasermin, Roferon-A, interferon-alpha 2, Alfaferone, interferon
alfacon-1, interferon alpha, Avonex' recombinant human luteinizing
hormone, dornase alfa, trafermin, ziconotide, taltirelin,
diboterminalfa, atosiban, becaplermin, eptifibatide, Zemaira,
CTC-111, Shanvac-B, HPV vaccine (quadrivalent), NOV-002,
octreotide, lanreotide, ancestim, agalsidase beta, agalsidase alfa,
laronidase, prezatide copper acetate (topical gel), rasburicase,
ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant house
dust mite allergy desensitization injection, recombinant human
parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin delta,
transgenic antithrombin III, Granditropin, Vitrase, recombinant
insulin, interferon-alpha (oral lozenge), GEM-21S, vapreotide,
idursulfase, omapatrilat, recombinant serum albumin, certolizumab
pegol, glucarpidase, human recombinant C1 esterase inhibitor
(angioedema), lanoteplase, recombinant human growth hormone,
enfuvirtide (needle-free injection, Biojector 2000), VGV-1,
interferon (alpha), lucinactant, aviptadil (inhaled, pulmonary
disease), icatibant, ecallantide, omiganan, Aurograb, pexiganan
acetate, ADI-PEG-20, LDI-200, degarelix, cintredekin besudotox,
FavId, MDX-1379, ISAtx-247, liraglutide, teriparatide
(osteoporosis), tifacogin, AA-4500, T4N5 liposome lotion,
catumaxomab, DWP-413, ART-123, Chrysalin, desmoteplase, amediplase,
corifollitropin alpha, TH-9507, teduglutide, Diamyd, DWP-412,
growth hormone (sustained release injection), recombinant G-CSF,
insulin (inhaled, AIR), insulin (inhaled, Technosphere), insulin
(inhaled, AERx), RGN-303, DiaPep277, interferon beta (hepatitis C
viral infection (HCV)), interferon alfa-n3 (oral), belatacept,
transdermal insulin patches, AMG-531, MBP-8298, Xerecept, opebacan,
AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, lusupultide,
MP52 (beta-tricalciumphosphate carrier, bone regeneration),
melanoma vaccine, sipuleucel-T, CTP-37, Insegia, vitespen, human
thrombin (frozen, surgical bleeding), thrombin, TransMID,
alfimeprase, Puricase, terlipressin (intravenous, hepatorenal
syndrome), EUR-1008M, recombinant FGF-1 (injectable, vascular
disease), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin
(inhaled, cystic fibrosis), SCV-07, OPI-45, Endostatin,
Angiostatin, ABT-510, Bowman Birk Inhibitor Concentrate, XMP-629,
99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix
(extended release), ozarelix, romidepsin, BAY-50-4798,
interleukin-4, PRX-321, Pepscan, iboctadekin, rh lactoferrin,
TRU-015, IL-21, ATN-161, cilengitide, Albuferon, Biphasix, IRX-2,
omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DM1,
ovarian cancer immunotherapeutic vaccine, SB-249553, Oncovax-CL,
OncoVax-P, BLP-25, CerVax-16, multi-epitope peptide melanoma
vaccine (MART-1, gp100, tyrosinase), nemifitide, rAAT (inhaled),
rAAT (dermatological), CGRP (inhaled, asthma), pegsunercept,
thymosin beta-4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1,
AC-100, salmon calcitonin (oral, eligen), calcitonin (oral,
osteoporosis), examorelin, capromorelin, Cardeva, velafermin,
131I-TM-601, KK-220, TP-10, ularitide, depelestat, hematide,
Chrysalin (topical), rNAPc2, recombinant Factor VIII (PEGylated
liposomal), bFGF, PEGylated recombinant staphylokinase variant,
V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, islet cell
neogenesis therapy, rGLP-1, BIM-51077, LY-548806, exenatide
(controlled release, Medisorb), AVE-0010, GA-GCB, avorelin,
AOD-9604, linaclotide acetate, CETi-1, Hemospan, VAL (injectable),
fast-acting insulin (injectable, Viadel), intranasal insulin,
insulin (inhaled), insulin (oral, eligen), recombinant methionyl
human leptin, pitrakinra subcutaneous injection, eczema),
pitrakinra (inhaled dry powder, asthma), Multikine, RG-1068,
MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10 (autoimmune
iseases/inflammation), talactoferrin (topical), rEV-131
(ophthalmic), rEV-131 (respiratory disease), oral recombinant human
insulin (diabetes), RPI-78M, oprelvekin (oral), CYT-99007 CTLA4-Ig,
DTY-001, valategrast, interferon alfa-n3 (topical), IRX-3, RDP-58,
Tauferon, bile salt stimulated lipase, Merispase, alkaline
phosphatase, EP-2104R, Melanotan-II, bremelanotide, ATL-104,
recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174,
CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, malaria
vaccine (virosomes, PeviPRO), ALTU-135, parvovirus B19 vaccine,
influenza vaccine (recombinant neuraminidase), malaria/HBV vaccine,
anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine,
Tat Toxoid, YSPSL, CHS-13340, PTH(1-34) liposomal cream (Novasome),
Ostabolin-C, PTH analog (topical, psoriasis), MBRI-93.02, MTB72F
vaccine (tuberculosis), MVA-Ag85A vaccine (tuberculosis), FAR-404,
BA-210, recombinant plague F1V vaccine, AG-702, OxSODrol, rBetV1,
Der-p1/Der-p2/Der-p7 allergen-targeting vaccine (dust mite
allergy), PR1 peptide antigen (leukemia), mutant ras vaccine,
HPV-16 E7 lipopeptide vaccine, labyrinthin vaccine
(adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5,
CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide,
telbermin (dermatological, diabetic foot ulcer), rupintrivir,
reticulose, rGRF, P1A, alpha-galactosidase A, ACE-011, ALTU-140,
CGX-1160, angiotensin therapeutic vaccine, D-4F, ETC-642, APP-018,
rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828,
ErbB2-specific immunotoxin (anticancer), DT3881L-3, TST-10088,
PRO-1762, Combotox, cholecystokinin-B/gastrin-receptor binding
peptides, 111In-hEGF, AE-37, trastuzumab-DM1, Antagonist G, IL-12
(recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647
(topical), L-19 based radioimmunotherapeutics (cancer),
Re-188-P-2045, AMG-386, DC/1540/KLH vaccine (cancer), VX-001,
AVE-9633, AC-9301, NY-ESO-1 vaccine (peptides), NA17.A2 peptides,
melanoma vaccine (pulsed antigen therapeutic), prostate cancer
vaccine, CBP-501, recombinant human lactoferrin (dry eye), FX-06,
AP-214, WAP-8294A2 (injectable), ACP-HIP, SUN-11031, peptide YY
[3-36] (obesity, intranasal), FGLL, atacicept, BR3-Fc, BN-003,
BA-058, human parathyroid hormone 1-34 (nasal, osteoporosis),
F-18-CCR1, AT-1001 (celiac disease/diabetes), JPD-003, PTH(7-34)
liposomal cream (Novasome), duramycin (ophthalmic, dry eye), CAB-2,
CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528,
AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155,
SUN-E7001, TH-0318, BAY-73-7977, teverelix (immediate release),
EP-51216, hGH (controlled release, Biosphere), OGP-I, sifuvirtide,
TV-4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin (pulmonary
diseases), r(m)CRP, hepatoselective insulin, subalin, L19-IL-2
fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO,
thrombopoietin receptor agonist (thrombocytopenic disorders),
AL-108, AL-208, nerve growth factor antagonists (pain), SLV-317,
CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352,
PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043, S pneumoniae
pediatric vaccine, malaria vaccine, Neisseria meningitidis Group B
vaccine, neonatal group B streptococcal vaccine, anthrax vaccine,
HCV vaccine (gpE1+gpE2+MF-59), otitis media therapy, HCV vaccine
(core antigen+ISCOMATRIX), hPTH(1-34) (transdermal, ViaDerm),
768974, SYN-101, PGN-0052, aviscumine, BIM-23190, tuberculosis
vaccine, multi-epitope tyrosinase peptide, cancer vaccine,
enkastim, APC-8024, G1-5005, ACC-001, TTS-CD3, vascular-targeted
TNF (solid tumors), desmopressin (buccal controlled-release),
onercept, TP-9201.
[0400] Non-Repetitive URPs (nrURPs)
[0401] The present invention also encompasses non-repetitive URPs
(nrURPs). nrURPs are amino acid sequences that are composed mainly
of small hydrophilic amino acids and that have a low tendency to
form secondary structure in vivo. nrURPs possess the
characteristics of URPs including the lack of well defined
secondary and tertiary structures under physiological conditions,
contributing to their conformational flexibility; high degree of
protease resistance; ability to increase the half-life and/or
solubility of a biologically active polypeptide upon incorporation
of the URP sequences into the biologically active polypeptide. A
particular property of nrURPs is their low degree of internal
repetitiveness. nrURPs comprise multiple different peptide
subsequences. These subsequences have URP-like amino acid
composition but differ from each other in their amino acid sequence
and length.
[0402] nrURPs tend to have improved solubility as compared to
repetitive URPs (rURPs) with similar amino acid composition. In
general, repetitive amino acid sequences have a tendency to
aggregate as exemplified by natural repetitive sequences such as
collagens and leucine zippers. Repetitive sequences can form higher
order structures such that identical subsequences from similar
contacts resulting in crystalline or pseudocrystalline structures.
nrURPs have a much lower tendency to form such pseudo-crystalline
structures as they contain multiple different subsequences that
prevent the formation of any repetitive higher order structure. The
low tendency of non-repetitive sequences to aggregate enables the
design URPs with a relatively low frequency of charged amino acids
that would be likely to aggregate in repetitive URPs. The low
aggregation tendency of nrURPs facilitates the formulation of
nrURP-comprising pharmaceutical preparations in particular enabling
preparations containing extremely high drug concentrations
exceeding 100 mg/ml.
[0403] (a) nrURPs have Low Immunogenicity
[0404] The interactions of a repetitive and a non-repetitive URP
sequence with B cells that recognize epitopes in said sequences are
compared and illustrated in FIG. 74. A rURP is recognized by few B
cells in an organism as it contains a relatively small number of
different epitopes. However, a rURP can form multivalent contacts
with these few B cells and as a consequence it can stimulate B cell
proliferation as illustrated in FIG. 74a. In contrast, a nrURP can
make contacts with many different B cells as it contains many
different epitopes. However, each individual B cell can only make
one or a small number of contacts with an individual nrURP due to
the lack of repetitiveness as illustrated in FIG. 74b. As a result,
nrURPs have a much lower tendency to stimulate proliferation of B
cells and thus an immune response.
[0405] An additional advantage of nrURPs relative to rURPs is that
nrURPs form weaker contacts with antibodies relative to rURPs.
Antibodies are multivalent molecules. For instance, IgGs have two
identical binding sites and IgMs contain 10 identical binding
sites. Thus antibodies against repetitive sequences can form
multivalent contacts with such repetitive sequences with high
avidity, which can affect the potency and/or elimination of such
repetitive sequences. In contrast, antibodies against nrURPs form
mainly monovalent interactions with antibodies as said nrURPs
contain few repeats of each epitope.
[0406] (b) Detection of Repetitiveness
[0407] The repetitiveness of a gene can be measured by computer
algorithms. An example is illustrated in FIG. 75. Based on the
query sequence, a pair wise comparison of all subsequences of a
particular length can be performed. These subsequences can be
compared for identity or homology. The example in FIG. 75 compares
subsequences of 4 amino acids for identity. In the example, most
4-mer subsequences occur once in the query sequence and 3 4-mer
subsequences occur twice. The repetitiveness in a gene can be
averaged. The length of the subsequences can be adjusted. The
length of the subsequences reflects the length of sequence epitopes
that can be recognized by the immune system. Thus analysis of
subsequences of 4-15 amino acids may be most useful.
[0408] (c) Design of nrURP Sequences
[0409] Genes encoding nrURPs can be assembled from oligonucleotides
using standard techniques of gene synthesis. The gene design can be
performed using algorithms that optimize codon usage and amino acid
composition. In addition, one can avoid amino acid sequences that
are protease sensitive or that are known to contain epitopes that
can be easily recognized by the human immune system. Computer
algorithms can be applied during sequence design to minimize the
repetitiveness of the resulting amino acid sequences. One can
evaluate the repetitiveness of large numbers of gene designs that
match preset criteria such as amino acid composition, codon usage,
avoidance of protease sensitive subsequence, avoidance of epitopes,
and chose the least repetitive sequences for synthesis and
subsequent evaluation.
[0410] An alternative approach to the design of nrURP genes is to
analyze the sequences of existing collections of nrURPs that show
high level expression, low aggregation tendency, high solubility,
and good resistance to proteases. A computer algorithm can design
nrURP sequences based on such pre-existing nrURP sequences by
re-assembly of sequence fragments as illustrated in FIG. 76. The
algorithm generates a collection of subsequences from these nrURP
sequences and then evaluates multiple ways to assembly nrURP
sequences from such subsequences. These assembled sequences can be
evaluated for repetitiveness to identify nrURP sequences that are
only composed of subsequences of previously identified nrURPs but
that have reduced repetitiveness compared to all parent nrURPs.
[0411] (d) Construction of nrURP Sequences from Libraries
[0412] nrURP-encoding genes can be assembled from libraries of
short URP segments as illustrated in FIG. 77. One can first
generate large libraries of URP segments. Such libraries can be
assembled from partially randomized oligonucleotides. The
randomization scheme can be optimized to control amino acid choices
for each position as well as codon usage and sequence length. In
one embodiment, the library of URP segments is cloned into an
expression vector. In another embodiment, the library of URP
segments is cloned into an expression vector fused to an indicator
gene like GFP. Subsequently, one can screen library members for a
number of properties such as level of expression, protease
stability, binding to serum proteins. One can screen URP segments
for binding to antiserum to eliminate segment with high affinity
for said serum. In particular one can screen library members to
identify and avoid binding to antisera with reactivity to URP
sequences. The amino acid sequence of the library members can be
determined to identify segments that have a particularly desirable
amino acid composition, segment length, or to identify segments
that have a low frequency of internal repeats. Subsequently, nrURP
sequences may be assembled from the collections of URP segments by
random dimerization or multimerization. Dimerization or
multimerization can be achieved by ligation or PCR assembly. This
process results in a library of nrURP sequences that can be
evaluated for a number of properties to identify the nrURP
sequences with the most desirable properties. The process of
dimerization or multimerization can be repeated to further increase
the length of nrURP sequences.
[0413] Design of Crosslinked Accessory Polypeptides
[0414] The present invention also relates to polypeptides with
enhanced properties (such as increased hydrodynamic radius or
extended serum half-life) comprising crosslinked accessory
polypeptides. A crosslinked accessory polypeptide can be generated
by conjugating one or more non-cross-linking components and one or
more cross-linking components.
[0415] The advantage of this approach is that one can use an
accessory polypeptide of moderate length, which is highly
expressed, to efficiently generate larger molecules with desired
properties. For example, using chemical coupling one can create a
molecule comprising five 200 amino acid long units much more
efficiently than a single 1000 amino acid long polypeptide
expressed as a single protein.
[0416] Any number of non-crosslinking components, such as 2, 3, 4,
5, 6, 7, 8, 9, 10 or more components can be linked together. These
components can be identical or of 2, 3, 4, 5, 6, 7, 8, 9 or 10 or
more different kinds. In a preferred embodiment, each component has
a determined binding specificity, which can be the same for each
component or of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more different
types. The sequences of the non-crosslinking components can also be
the same or may comprise 1-10 different sequences.
[0417] A preferred embodiment of the present invention provides for
reacting 1, 2, 3, 4, 5, 6, 7, 8 or more copies of a monoreactive
non-crosslinking component with one copy of a multireactive
cross-linking component, which optionally contains
polyethyleneglycol, an accessory polypeptide or another
water-soluble polymer, resulting in a pre-defined polymer
containing exactly (for example) four copies of the
non-crosslinking component, each copy being linked to the
cross-linking agent. The non-crosslinking component may optionally
comprise a domain with binding specificity.
[0418] A variety of linkage chemistries can be used for
conjugation. In a preferred embodiment, standard amino-carboxyl
coupling, and especially linking via the amino group of a lysine
group or of the N-terminus, or linking via the carboxyl group of
glutamate or of the C-terminus, is especially useful for
cross-linking of crosslinked accessory polypeptides.
[0419] In some embodiments, the cross-linking component can be a
synthetic polypeptide. For example, such a polypeptide may comprise
5 carboxy residues (i.e. 4 glutamates plus the C-terminal carboxy),
optionally spaced by sequences inserted between the carboxyl groups
(`linkage peptide`). The amino-terminus of this linkage peptide can
be blocked, for example by amidation, to prevent the formation of
additional variants (FIG. 27). The second reactive group is the
amino-terminus of the protein that contains accessory polypeptides.
Optionally, one can reserve one or more lysines for coupling to the
carboxyls in the linkage peptide. After exhaustive chemical
linkage, one can obtain a homogeneous single product, which is a
molecule that contains 5 accessory polypeptides (optionally
containing binding domains), as well as the linkage peptide. A
variation is to have the linkage peptide contain the amino groups
and use carboxyls on the other protein, which typically carries the
binding domain.
[0420] In addition to such branched structures, it is also possible
to create linear polymers of 2, 3, 4, 5, 6, 7, 8 or more separately
expressed polypeptides by linking the amino-terminus of one protein
to the carboxy-terminus of another protein. Again, these
polypeptides may be the same or different, as described above.
[0421] The preferred linkage is amino-to-carboxy. The amino group
that is used for coupling is located on the recombinant protein if
the carboxyl group that is used is located on the chemical
crosslinker Alternatively, the amino group that is used for
coupling is located on the chemical cross-linker if the carboxyl
group that is used is located on the recombinant protein.
[0422] The number of coupling sites that is used on the crosslinker
determines whether the product will contain 1, 2, 3, 4, 5, 6, 7, 8,
9 or 10 or more recombinant proteins, each typically containing 1,
2, 3, 4, 5 or more binding domains. The crosslinking component is
typically a small, FDA-approved chemical but can also be a
recombinant polypeptide and optionally contains at least 0, 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100 units of a
repeated motif, and at most 10, 20, 30, 40, 50, 75, 100, 150, 200,
250, 300, 350, 400, 450 or 500 units of a repeated motif.
[0423] Using protection groups that can be differentially removed
by different conditions, it is possible to have several conjugation
steps that each add a different protein to the conjugate. This
allows the creation of conjugates with multiple different protein
chains in a pre-designed stoichiometry. Conjugation of divalent
crosslinker with two protein chains having one linkable position
(like an N-terminus) creates dimers. Crosslinking of proteins with
two linkage sites creates a linear polymer. Crosslinking of a
trivalent crosslinker with a protein containing linkage sites at
both ends creates a dendrimer (FIG. 26).
[0424] In some embodiments, non-crosslinking components may
comprise one or more biologically active polypeptides with affinity
to a target receptor. These biologically active polypeptides can
bind to different target receptors, allowing the generation of
crosslinked accessory polypeptides that bind several copies of
several different target receptors. Alternatively, non-crosslinking
components can comprise multiple biologically active polypeptides
that bind several different epitopes of the same target receptor.
The resulting crosslinked accessory polypeptide can bind multiple
copies of a target receptor while making multiple binding contacts
with each target receptor resulting in very high avidity. Another
option is to use non-crosslinking components that contain multiple
identical binding elements in order to construct crosslinked
accessory polypeptides with a very large number of identical
binding sites.
[0425] In some embodiments, non-cross-linking components can
comprise affinity tags. Examples for useful affinity tags are Flag,
HA-tag, hexa-histidine (SEQ ID NO: 1). These affinity tags
facilitate the purification of the non-cross-linking components as
well as the resulting crosslinked accessory polypeptides. In
addition, affinity tags facilitate the detection of crosslinked
accessory polypeptides in biological samples. In particular,
affinity tags are useful to monitor the serum halflife and/or the
tissue distribution of a crosslinked accessory polypeptide in a
patient or in animals.
[0426] In yet other embodiments, non-cross-linking components can
contain binding elements that increase the serum halflife of the
resulting crosslinked accessory polypeptides. Such binding elements
can bind to one or multiple serum components like HSA, IgG, red
blood cells, or other serum component that is found in high
abundance.
[0427] In still other embodiments, non-cross-linking components can
be conjugated to one or more small molecule drug molecules.
Examples for useful drug molecules are doxorubicin, melphalan,
paclitaxel, maytansines, duocarmycines, calicheamycin, auristatin
and other cytotoxic, cytostatic, antiinfective drugs.
[0428] In some embodiments, non-cross-linking components can
comprise affinity tags. Examples for useful affinity tags are Flag,
HA-tag, hexa-histidine. These affinity tags facilitate the
purification of the non-cross-linking components as well as the
resulting crosslinked accessory polypeptides. In addition, affinity
tags facilitate the detection of crosslinked accessory polypeptides
in biological samples. In particular, affinity tags are useful to
monitor the serum halflife and/or the tissue distribution of a
crosslinked accessory polypeptide in a patient or in animals.
[0429] In other embodiments, non-cross-linking components can
comprise protease sites that allow the slow release of binding
domains, active drugs, or other subsequences with biological
activity.
[0430] Of particular utility are non-cross-linking components that
are free of lysine residues. Such sequences contain a single amino
group at their N-terminus, which can be utilized for conjugation to
the cross-linking component. Non-cross-linking components that
contain a single free cysteine residue are also of utility as there
are many chemistries available that allow the controlled
conjugation to the side chain of free cysteine residues. Another
approach is to utilize the C-terminal carboxyl group of the
non-cross-linking component as reactive group.
[0431] Many molecules that comprise multiple reactive groups can
serve as useful cross-linking components. Many useful cross-linking
agents are commercially available from companies like
Sigma-Aldrich, or Pierce. Of particular utility are cross-linking
components that are available in activated form and can be directly
used for conjugation. Examples are shown in FIG. 22. Cross-linking
components can comprise multiple reactive groups with similar or
identical chemical structure (FIG. 23). Such reactive groups can be
simultaneously activated and coupled to multiple identical
non-cross-linking components resulting in the direct formation of
homomultimeric products. Examples for cross-linking components with
multiple similar reactive groups are citric acid, EDTA, TSAT. Of
particular interest are branched PEG molecules containing multiple
identical reactive groups.
[0432] There are a large number of specific chemical products that
work based on the following small number of basic reaction schemes,
all of which are described in detail at www.piercenet.com. Examples
of useful crosslinking agents are imidoesters, active halogens,
maleimide, pyridyl disulfide, and NHS-esters. Homobifunctional
crosslinking agents have two identical reactive groups and are
often used in a onestep chemical crosslinking procedure. Examples
are BS3 (a non-cleavable water-soluble DSS analog), BSOCOES
(base-reversible), DMA (Dimethyl adipimidate-2HCl), DMP (Dimethyl
pimelimidate-2HCl), DMS (Dimethyl suberimidate-2HCl), DSG (5-carbon
analog of DSS), DSP (Lomant's reagent), DSS (non-cleavable), DST
(cleavable by oxidizing agents), DTBP (Dimethyl
3,3'-dithiobispropionimidate-2HCl), DTSSP, EGS, Sulfo-EGS, THPP,
TSAT, DFDNB (1,5-Difluoro-2,4-dinitrobenzene) is especially useful
for crosslinking between small spacial distances (Kornblatt, J. A.
and Lake, D. F. (1980). Cross-linking of cytochrome oxidase
subunits with difluorodinitrobenzene. Can J. Biochem. 58,
219-224).
[0433] Sulfhydryl-reactive homobifunctional crosslinking agents are
homobifunctional protein crosslinkers that react with sulfhydryls
and are often based on maleimides, which react with --SH groups at
pH 6.5-7.5, forming stable thioether linkages. BM[PEO]3 is an
8-atom polyether spacer that reduces potential for conjugate
precipitation in sulfhydryl-to-sulfhydryl cross-linking
applications. BM[PEO]4 is similar but with an 11-atom spacer. BMB
is a non-cleavable crosslinker with a four-carbon spacer. BMDB
makes a linkage that can be cleaved with periodate. BMH is a widely
used homobifunctional sulfhydryl-reactive crosslinker BMOE has an
especially short linker DPDPB and DTME are cleavable crosslinkers.
HVBS does not have the hydrolysis potential of meleimides. TMEA is
another option. Hetero-bifunctional crosslinking agents have two
different reactive groups. Examples are NHS-esters and
amines/hydrazines via EDC activation, AEDP, ASBA (photoreactive,
iodinatable), EDC (water-soluble carbodiimide). Amine-Sulfhydryl
reactive bifunctional crosslinkers are AMAS, APDP, BMPS, EMCA,
EMCS, GMBS, KMUA, LC-SMCC, LC-SPDP, MBS, SBAP, SIA (extra short),
SIAB, SMCC, SMPB, SMPH, SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS,
Sulfo-KMUS, Sulfo-LC-SMPT, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-SIAB,
Sulfo-SMCC, Sulfo-SMPB Amino-group reactive heterobifunctional
crosslinking agents are ANB-NOS, MSA, NHS-ASA, SADP, SAED, SAND,
SANPAH, SASD, SFAD, Sulfo-HSAB, Sulfo-NHS-LC-ASA, Sulfo-SADP,
Sulfo-SANPAH, TFCS. Arginine-reactive crosslinking agents are, for
example APG, which reacts specifically with arginines at pH
7-8.
[0434] Polypeptides can be designed to serve as cross-linking
components. Such polypeptides can be generated by chemical
synthesis or using recombinant techniques. Examples are
polypeptides that contain multiple aspartate or glutamate residues.
The side chains of these residues as well as the C-terminal
carboxyl group can be used for coupling to the non-crosslinking
component. By adding one or several amino acids between the
aspartate or glutamate residues one can control the distance
between reactive groups, which can affect the efficiency of
conjugation as well as the overall properties of the resulting
crosslinked accessory polypeptide. Of particular utility are
polypeptides that contain multiple aspartate or glutamate residues
and that carry a protection group at their N-terminal amino group.
Examples for suitable protection schemes are acetylation,
succinylation, and other modifications that reduce the reactivity
of the N-terminal amino group of the peptide.
[0435] Of particular utility as cross-linking components are
dendrimeric constructs. Many dendrimeric structures are known in
the art and they can be designed to contain a large number of
reactive groups. Examples of crosslinked accessory polypeptides are
illustrated in FIG. 24.
[0436] Additional Modifications of Accessory Polypeptides
[0437] An additional mechanism may be incorporated into the design
of accessory polypeptides as well as crosslinked accessory
polypeptidesis mediated by peptides with binding affinity to
serum-exposed molecules. By binding to such a target, the halflife
of the polypeptide of the present invention is further increased.
For example, a crosslinked accessory polypeptide may comprise a
non-crosslinking unit that comprises a polypeptide with binding
affinity to a serum-exposed target. Alternatively, an accessory
polypeptide may comprise a sequence coding for a polypeptide with
such binding affinity. Preferred serum-exposed targets that
peptides or protein domains can be made to bind to for halflife
extension are (human, mouse, rat, monkey) serum albumin,
Immunoglobulins such as IgG (IgG1,2,3,4), IgM, IgA, IgE as well as
red blood cells (RBC), or endothelial cells. Accessory polypeptides
may also comprise, by way of example, sequences that target the
extracellular matrix, insert into membranes, or other targeting
peptides and domains (FIG. 28)
[0438] In another embodiment, accessory polypeptides or crosslinked
accessory polypeptides may comprise several biologically active
polypeptides separated as well as sequences that comprise specific
cleavage sites for serum proteases (FIG. 29). Following
administration or exposure to serum, serum proteases act on the
cleavage sites leading to gradual proteolysis and release of
biologically active polypeptides or accessory polypeptides into the
blood.
[0439] Accessory polypeptides or crosslinked accessory polypeptides
may also be modified postsynthetically. In one embodiment,
accessory polypeptides are expressed comprising one or more lysine
residues (FIG. 30). Following expression, the polypeptides are
reacted with a Lys-reactive moiety that is attached to at least one
second functional unit, which may be for example a biologically
active polypeptide. In a related embodiment, the functional unit is
a polypeptide with binding affinity for serum-exposed targets, such
as serum albumin, Immunoglobulins such as IgG (IgG1,2,3,4), IgM,
IgA, IgE as well as red blood cells (RBC) or endothelial cells.
[0440] Accessory Polypeptides Linked to an Antigen-Binding Unit
[0441] The present invention embodies an accessory polypeptide
linked to an antigen-binding unit. The term "antigen-binding units"
collectively refers to immunoglobulin molecules and any form of
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen-binding site which specifically
binds or immunoreacts with an antigen. Structurally, the simplest
naturally occurring antibody (e.g., IgG) comprises four polypeptide
chains, two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. The immunoglobulins represent a
large family of molecules that include several types of molecules,
such as IgD, IgG, IgA, IgM and IgE. The term "immunoglobulin
molecule" includes, for example, hybrid antibodies, or altered
antibodies, and fragments thereof. An antibody binding unit can be
broadly divided into "single-chain" ("Sc") and "non-single-chain"
("Nsc") types, which include, but not limited to, Fv, scFv, dFv,
dAb, diabody, triabody, tetrabody, domain Ab, Fab fragment, Fab',
(Fab').sub.2, bispecific Ab and multispecific Ab.
[0442] Also encompassed within the term "antigen binding unit" are
immunoglobulin molecules of a variety of species origins including
invertebrates and vertebrates. The term "human" as applies to an
antigen binding unit refers to an immunoglobulin molecule expressed
by a human gene or fragment thereof. The term "humanized" as
applies to a non-human (e.g. rodent or primate) antibodies are
hybrid immunoglobulins, immunoglobulin chains or fragments thereof
which contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, rabbit or primate having the desired
specificity, affinity and capacity. In some instances, Fv framework
region (FR) residues of the human immunoglobulin are replaced by
corresponding non-human residues. Furthermore, the humanized
antibody may comprise residues which are found neither in the
recipient antibody nor in the imported CDR or framework sequences.
These modifications are made to further refine and optimize
antibody performance and minimize immunogenicity when introduced
into a human body. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin sequence. The humanized antibody may also comprise
at least a portion of an immunoglobulin constant region (Fc),
typically that of a human immunoglobulin.
[0443] "Non-single-chain antigen-binding unit" are heteromultimers
comprising a light-chain polypeptide and a heavy-chain polypeptide.
Examples of the non-single-chain antigen-binding unit include but
are not limited to (i) a ccFv fragment, which is a dimeric protein
composed of VL and VH regions, which dimerize via the pairwise
affinity of the first and second heterodimerization sequences fused
in-frame with the VL and VH regions; (ii) any other monovalent and
multivalent molecules comprising at least one ccFv fragment; (iii)
an Fab fragment consisting of the VL, VH, CL and CH1 domains; (iv)
an Fd fragment consisting of the VH and CH1 domains; (v) an Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody; (vi) an F(ab')2 fragment, a bivalent fragment comprising
two Fab fragments linked by a disulfide bridge at the hinge region;
(vii) a diabody; and (viii) any other non-single-chain
antigen-binding units that have been described and known in the
art.
[0444] As noted above, a non-single-chain antigen-binding unit can
be either "monovalent" or "multivalent." Whereas the former has one
binding site per antigen-binding unit, the latter contains multiple
binding sites capable of binding to more than one antigen of the
same or different kind. Depending on the number of binding sites, a
non-single-chain antigen-binding unit may be bivalent (having two
antigen-binding sites), trivalent (having three antigen-binding
sites), tetravalent (having four antigen-binding sites), and so on.
Multivalent non-single-chain antigen-binding unit can be further
classified on the basis of their binding specificities. A
"monospecific" non-single-chain antigen-binding unit is a molecule
capable of binding to one or more antigens of the same kind. A
"multispecific" non-single-chain antigen-binding unit is a molecule
having binding specificities for at least two different antigens.
While such molecules normally will only bind two distinct antigens,
antibodies with additional specificities such as trispecific
antibodies are encompassed by the present invention.
[0445] "Single-chain antigen-binding unit" refers to monomeric
antigen-binding unit. Although the two domains of the Fv fragment
are coded for by separate genes, a synthetic linker can be made
that enables them to be made as a single protein chain (i.e. single
chain Fv ("scFv") as described in Bird et al. (1988) Science
242:423-426 and Huston et al. (1988) PNAS 85:5879-5883) by
recombinant methods. Other single-chain antigen-binding units
include antigen-binding molecules stabilized by the subject
heterodimerization sequences, and dAb fragments (Ward et al. (1989)
Nature 341:544-546) which consist of a VH domain and an isolated
complimentarily determining region (CDR). A preferred single-chain
antigen-binding unit contains VL and VH regions that are linked
together and stabilized by a pair of subject heterodimerization
sequences. The scFvs can be assembled in any order, for example,
VH--(first heterodimerization sequence)-(second heterodimerization
sequence)--VL or V.sub.L--(first heterodimerization
sequence)-(second heterodimerization sequence)--VH.
[0446] An antigen-binding unit specifically binds to or
immunoreactive with an antigen if it binds with greater affinity or
avidity than it binds to other reference antigens including
polypeptides or other substances. The antigen-binding unit may be
directly attached to the outer surface of the host cell, or may be
indirectly attached to the host cell via a host cell bound genetic
package such as phage particle.
[0447] The accessory polypeptide which is linked to an
antigen-binding unit includes, but is not limited to, rPEGs,
nrPEGs, and any other polypeptides capable of increasing
hydrodynamic radius, extending serum half-life, and/or modifying in
vivo clearance rate. When desired, an accessory polypeptide causes
a small increase in predicated molecular weight, but a much larger
increase in apparent molecular weight.
[0448] Another embodiment of the present invention includes an
accessory polypeptide such as rPEG linked at both ends to a binding
pair. Such binding pair generically consists of a binding protein 1
and a binding protein 2, linked via rPEG. Examples of such binding
pair include but are not limited to a receptor-ligand pair, an
antibody-antigen pair, or any two polypeptides that are capable of
interacting with each other. FIG. 82 shows the general ways of
making such rPEG linked binding pairs, which have the advantage of
no initial activity and therefore no burst release effect
(increasing the dose that can be administered without causing
toxicity) and reduced initial receptor-mediated clearance. The
general binding pairs can be receptor-ligand, antibody-ligand, or
generally binding protein 1-binding protein 2. The construct can
have a cleavage site, which can be cleaved before injection, after
injection (in serum by proteases) and can be located such that the
rPEG stays with the therapeutic product end (active protein), which
can be the ligand, the receptor or the antibody.
[0449] Antibody Fragment-Based Therapeutics (AFBT)
[0450] Another embodiment of the present invention includes an
antibody fragment-based therapeutic (AFBT). AFBTs comprise at least
one antigen-binding unit or antibody fragment and one accessory
polypeptide such as a rPEG domain. An AFBT may also comprise one or
more payloads, which include moieties that have biological
activities such as cytokines, enzymes and growth factors, as well
as agents that may have therapeutic potentials such as cytotoxic
agents, chemotherapeutic agents, antiviral compounds, or contrast
agents. An AFBT may also include additional domains, for example,
multimerization domains such as an Fc region or leucine zipper.
FIG. 58a shows an example of an AFBT that illustrates the main
components of an AFBT. The antibody fragment provides an AFBT with
specificity for a target antigen (also generically illustrated in
FIG. 21). The rPEG domain provides a variety of benefits to the
antibody fragment as well as to the payload. These benefits
include, but are not limited to, prolonged half-life in vivo,
increased solubility, increased thermal stability, increased
protease stability, improved protein folding, reduced chain
reassortment, reduced immunogenicity of the payload, and avoidance
of preexisting immune responses to chemical PEG. The rPEG domain
also facilitates production and purification. The high solubility
of the rPEG domain renders AFBTs high solubility that can be
formulated at high concentration with a low tendency to form
aggregates. It should be understood that an AFBT may contain
additional components not illustrated in particular in this
figure.
[0451] vH/vL Domain-Based Structures
[0452] In one embodiment of the present invention, an AFBT also
comprises one or more antibody-derived immunoglobulin (Ig) domains
or fragments, including a single-chain variable fragment (scFv).
scFv consists of a vH domain linked to a vL domain via a peptide
linker between the vH and vL domains. The linker in the scFv is
chosen such that it forms a single molecular species, which
includes a scFv, diabody, triabody, or tetrabody (FIGS. 53, 54,
55), as compared to the full-length, i.e. whole antibody (FIG. 52).
Typically the valency of the resulting AFBT is between one and four
although a higher valency is not excluded. Designs that
predominantly form a single, homogeneous species are preferred. An
Fv fragment may include a disulfide bond between contacting vH and
vL domains to reduce the risk of domain reassortment. The fraction
of the desired species that may be achieved ranges from less than
1% to 100% of the antibody fragment mix. The primary controls are
the linker length, which directs the format, and the rPEG, which
reduces antibody fragment chain reassortment. A preferred
embodiment includes the formation of monomeric scFv from a single
vH-vL chain employing linkers of at least 12 amino acids. More
preferred embodiments include a linker length of at least 15, at
least 20, at least 30, at least 50, at least 100, at least 200, or
at least 288 amino acids. Of particular utility are vH-vL chains
that preferentially form diabodies, which require linkers of less
than 10-20 amino acids, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
amino acids (FIG. 84). A diabody has two protein chains and can
have an rPEG at one or both C-terminal ends, and/or at one or both
N-terminal ends. The diabody has two binding sites, of which zero,
one or two may bind to a pharmaceutical target, or to a halflife
target (e.g. HSA, IgG, Red Blood Cells, Collagen, etc) or to no
target. The diabody may contain zero, one or more drug modules
located at the N-terminal or C-terminal end of zero, one or both
protein chains. AFBTs containing diabodies have increased molecular
weight due to their dimeric structure, which slows down renal
elimination. In one embodiment, the degree of antibody fragment
chain reassortment from one species to another species is less than
50%, 40%, 30%, 20% or 10% of the mass of protein per day or per
week at a fixed temperature (e.g. 4.degree. C., 25.degree. C. or
37.degree. C.), preferably less than 5%, 2%, 1% or 0.1%.
[0453] In another embodiment, the AFBTs include triabodies, which
contain three polypeptide chains, each containing a vH and a vL
domain connected via a linker of less than 10 amino acids,
preferably less than 5 amino acids. The frequency of triabodies can
be increased by eliminating one or a few amino acids from one or
both joining ends of the vH and vL domains, shortening the
connecting sequence so that triabodies are favorably formed. The
number of residues removed from one or both of the fused ends of
the antibodies can range from 1 to 10 amino acids.
[0454] In yet another embodiment, the AFBTs include tetrabodies,
which contain four polypeptide chains, each having one vH domain
and a vL domain connected via a short linker of less than 5 amino
acids, or as a result of removal of 1 to 10 residues from one or
both of the fused ends of the antibody. The number of amino acids
to eliminate from one or both joining ends of the vH and vL domains
can be adjusted to ensure the most desirable outcome.
[0455] Examples of various types of single chain (scFv) combination
consisting of a single copy of a polypeptide chain include but are
not limited to scFv-scFv, scFab-scFc, dAb-scFc, scFc-scFc,
scFc-scFab, and scFc-dAb (FIG. 57). A scFv fragment can be fused at
one or both of the N- and/or C-terminal ends to a drug module such
as IFN.alpha., hGH, etc (FIG. 85). The scFv has one binding site,
which may or may not bind to a pharmaceutical target, or to a
halflife target, e.g. HSA (FIG. 85b), IgG, red blood cells,
etc.
[0456] AFBTs that contain Ig domains can have a variety of
architectures. Constructs of particular utility include, but are
not limited to, the following: vL-linker-vH-rPEG,
vH-linker-vL-rPEG, vL-linker-vH-rPEG-payload,
vH-linker-vL-rPEG-payload, vL-linker-vH-payload-rPEG,
vH-linker-vL-payload-rPEG, rPEG-vL-linker-vH, rPEG-vH-linker-vL,
payload-rPEG-vL-linker-vH, payload-rPEG-vH-linker-vL,
rPEG-payload-vL-linker-vH, rPEG-payload-vH-linker-vL. These AFBTs
can contain additional domains that can be inserted between domains
or anywhere into an rPEG domain. There can also be several payload
modules.
[0457] The linker sequence joining vH and vL can be optimized to
achieve optimal protein folding and stability as well as high level
expression and a large fraction of the desired species. A preferred
embodiment includes linker sequences that are rich (e.g. greater
than 50%) in glycine and other small hydrophilic amino acids such
as serine, threonine, glutamic acid, aspartic acid, lysine,
arginine, and alanine. rPEG is particularly suitable as a linker
between vH and vL domains. Linkers with improved properties can be
obtained by selection or screening of libraries.
[0458] scFv with rPEG Linkers
[0459] In yet another embodiment, scFv contain rPEG sequences as
the linker between the vH and vL domains. A preferred embodiment
includes rPEG linkers that contain a significant negative net
charge, which results in improved solubility and folding of the
scFv domains. Preferred embodiments contain linkers with at least
15, at least 20, at least 30, at least 50, at least 100, at least
200, or at least 288 residues.
[0460] Methods to Generate Stable Antibody Fragments of AFBTs
[0461] The present invention also relates to methods of generating
and engineering an antigen binding unit of an AFBT. Many methods
are known to generate antibodies with specificity for a target
antigen. Examples include monoclonal antibodies, in particular in
transgenic animals that produce human antibodies; phage display of
Fab or scFv libraries; ribosomal display; and humanization of
monoclonal antibodies. Multiple methods to engineer the stability
of scFvs have also been described [Worn, A., et al. (2001) J Mol
Biol, 305: 989]. It has been shown that adding a disulfide bond
between the vH and vL domains of scFv can lead to significant
stabilization [Dooley, H, et al. (1998) Biotechnol Appl Biochem 28
(Pt 1), 77, #2802]. An alternative is the introduction of consensus
mutations. The amino acid frequencies at various positions in
antibody framework residues have been analyzed. It has been shown
that the Boltzmann equation can predict the stabilizing effect of
some consensus mutations [Steipe, B, et al. (1994) J Mol Biol 240,
188, #2026]. A combinatorial approach that allows the simultaneous
introduction of multiple consensus mutations into single chain
antibody fragments has been described [Roberge, M., et al. (2006)
Protein Eng Des Sel, 19: 141]. Producing more stable antibody
fragments has resulted in improved in vivo targeting [Worn, A., et
al. (2000) J Biol Chem, 275: 2795].
[0462] Some scFv have been expressed in soluble form in the cytosol
of E. coli. In general, disulfide bonds are not formed in the
cytosol but they can form spontaneously after cell lysis
[Tavladoraki, P., et al. (1999) Eur J Biochem, 262: 617]. In
general, cytosolic expression of an antibody is well correlated
with the antibody stability [Worn, A., et al. (2001) J Mol Biol,
305: 989]. Mutant libraries of antibody fragments can be subjected
to selection for improved cytosolic expression [Martineau, P., et
al. (1998) J Mol Biol, 280: 117]. Redox engineered strains of E.
coli can be used to improve cytosolic expression of Fab fragments
[Levy, R., et al. (2007) J Immunol Methods, 321: 164]. The culture
conditions have been optimized to improve the expression of soluble
scFv in the cytosol of E. coli resulting in expression levels of up
to 35 mg/L of culture [Padiolleau-Lefevre, S., et al. (2007) Mol
Immunol, 44: 1888]. Another approach to improve the cytosolic
expression of scFvs is the screening or selection of genomic
libraries with the goal to identify chaperones or other factors
that facilitate expression. This approach has been evaluated using
lambda phage. Disulfide bonds in scFv have been removed
successfully to form intrabodies. Variants of such intrabodies can
be identified that result in improved cytosolic expression [der
Maur, A. A., et al. (2002) J Biol Chem, 277: 45075]. However,
disulfide bonds are important for the overall stability of most
antibody fragments and in most cases intrabodies have been of
limited utility.
[0463] Complementary Determining Regions (CDR) Grafting
[0464] The binding interactions between antibodies or antibody
fragments and their targets are mainly determined by the
complementary determining regions (CDRs). It has been shown that
CDRs can be grafted between the variable domains of different
antibodies [Jones, P. T., et al. (1986) Nature, 321: 522]. In many
cases other residues in the antibody framework need to be grafted
in addition to CDR residues in order to retain antigen binding. CDR
grafting can be useful to improve the stability of an antibody by
grafting CDRs from a less stable variable domain to a more stable
variable domain. An example is the grafting of CDRs from a
fluorescein-binding scFv into a well-expressed scFv that is used as
a `scaffold`, resulting in improved expression and increased
folding stability [Jung, S., et al. (1997) Protein Eng, 10: 959].
Further examples of CDR grafting into antibody fragments are
described in [Leong, S. R., et al. (2001) Cytokine, 16: 106] and
[Werther, W. A., et al. (1996) J Immunol, 157: 4986]. CDR grafting
can be employed to reduce the immunogenicity of antibodies in
patients by grafting CDRs from murine antibodies to human framework
residues [Winter, G., et al. (1993) Trends Pharmacol Sci, 14:
139].
[0465] Affinity of the Antigen Binding Unit of AFBT
[0466] The present invention also embodies the methods of improving
the affinity of the antigen binding unit of an AFBT. Multiple
approaches have been described that allow the identification of
antibodies and antibody fragments with improved affinity. For
instance Pastan prepared mini libraries of 1000-10000 clones
focused on hot spots that are naturally prone to hypermutation.
Phage panning gave variants with 15-55 fold improvement [Chowdhury,
P S, et al. (1999) Nat Biotechnol 17, 568, #2800]. Phage display
and other display methods can be utilized to identify variants of
antibody fragments with improved affinity. Different vectors are
available for phage display [Corisdeo, S., et al. (2004) Protein
Expr Purif, 34: 270]. Residues that are involved in antigen binding
can be identified using alanine scanning mutagenesis. Subsequently,
these positions can be targeted for mutagenesis to identify
variants with improved affinity [Leong, S. R., et al. (2001)
Cytokine, 16: 106]. Another strategy is CDR walking mutagenesis
that can result in the identification of antibody fragments with
high target-binding affinity [Yang, W. P., et al. (1995) J Mol
Biol, 254: 392]. Improved affinity can result in improved
tumor-selectivity of antibody fragments [Adams, G. P., et al.
(1998) Cancer Res, 58: 485]. High affinity can restrict the tumor
penetration of scFvs [Adams, G. P., et al. (2001) Cancer Res, 61:
4750] [Graff, C. P., et al. (2003) Cancer Res, 63: 1288]. Antibody
fragments with improved affinity can be identified using yeast
display in combination with FACS sorting [Boder, E. T., et al.
(2000) Proc Natl Acad Sci USA, 97: 10701].
[0467] Various IgG Domains
[0468] AFBTs may contain a variety of immunoglobulin domains. These
domains can affect protein expression, multimerization, and can
serve as effectors. The following non-exhaustive list, which
provides examples for illustrating the variety of 1 g domains, is
applicable for fusions to any antibody isotype including IgG1,
IgG2, IgG3, IgG4, IgE, IgM, IgA, and IgD from any species including
humans. Sites for fusion of rPEG to immunoglobulin-family sequences
include but are not limited to the following: [0469] N-terminal to
the CL1 domain, before the interchain cysteine [0470] N-terminal to
the CL1 domain, after the interchain cysteine [0471] C-terminal to
the CL1 domain, before the interchain cysteine [0472] C-terminal to
the CL1 domain, after the interchain cysteine [0473] N-terminal to
the CH1 domain, before the interchain cysteine [0474] N-terminal to
the CH1 domain, after the interchain cysteine [0475] C-terminal to
the CH1 domain, before the interchain cysteine [0476] C-terminal to
the CH1 domain, before the hinge cysteine(s) [0477] C-terminal to
the CH1 domain, after the hinge cysteine(s) [0478] N-terminal to
the hinge cysteine(s) [0479] C-terminal to the hinge cysteine(s),
before CH2 [0480] N-terminal to the CH2 domain [0481] C-terminal to
the CH2 domain [0482] N-terminal to the CH3 domain [0483]
C-terminal to the CH3 domain [0484] N-terminal to the CH4 domain
[0485] C-terminal to the CH4 domain [0486] N-terminal to peptides
derived from CDRH1-3 and/or CDRL1-3 (lambda and kappa) [0487]
N-terminal to peptides derived from CDRH1-3 and/or CDRL1-3 (lambda
and kappa)
[0488] Fab Domain Based AFBTs
[0489] Still another embodiment of the present invention includes a
Fab domain-based AFBT (FIG. 56). Fab domains comprise two peptide
chains, each of which is derived from the heavy and light chains of
an antibody. rPEGs and payloads and other domains can be fused to
either chain of a Fab fragment. Alternatively, rPEGs and payloads
can be fused to both chains of a Fab. Fab domains can be designed
to facilitate the dimerization of the resulting proteins such that
the final protein contains four peptide chains. The following is a
list of AFBTs that comprise at least one Fab domain:
TABLE-US-00009 Light chain Heavy chain vL-CL-rPEG vH-CH1
vL-CL-rPEG-payload vH-CH1 vL-CL-payload-rPEG vH-CH1 rPEG-vL-CL
vH-CH1 payload-rPEG-vL-CL vH-CH1 rPEG-payload-vL-CL vH-CH1
vL-CL-rPEG vH-CH1-rPEG vL-CL-rPEG-payload vH-CH1-rPEG
vL-CL-payload-rPEG vH-CH1-rPEG rPEG-vL-CL vH-CH1-rPEG
payload-rPEG-vL-CL vH-CH1-rPEG rPEG-payload-vL-CL vH-CH1-rPEG
vL-CL-rPEG vH-CH1-rPEG-payload vL-CL-rPEG-payload
vH-CH1-rPEG-payload vL-CL-payload-rPEG vH-CH1-rPEG-payload
rPEG-vL-CL vH-CH1-rPEG-payload payload-rPEG-vL-CL
vH-CH1-rPEG-payload rPEG-payload-vL-CL vH-CH1-rPEG-payload
vL-CL-rPEG vH-CH1-payload-rPEG vL-CL-rPEG-payload
vH-CH1-payload-rPEG vL-CL-payload-rPEG vH-CH1-payload-rPEG
rPEG-vL-CL vH-CH1-payload-rPEG payload-rPEG-vL-CL
vH-CH1-payload-rPEG rPEG-payload-vL-CL vH-CH1-payload-rPEG
vL-CL-rPEG rPEG-vH-CH1 vL-CL-rPEG-payload rPEG-vH-CH1
vL-CL-payload-rPEG rPEG-vH-CH1 rPEG-vL-CL rPEG-vH-CH1
payload-rPEG-vL-CL rPEG-vH-CH1 rPEG-payload-vL-CL rPEG-vH-CH1
vL-CL-rPEG payload-rPEG-vH-CH1 vL-CL-rPEG-payload
payload-rPEG-vH-CH1 vL-CL-payload-rPEG payload-rPEG-vH-CH1
rPEG-vL-CL payload-rPEG-vH-CH1 payload-rPEG-vL-CL
payload-rPEG-vH-CH1 rPEG-payload-vL-CL payload-rPEG-vH-CH1
vL-CL-rPEG rPEG-payload-vH-CH1 vL-CL-rPEG-payload
rPEG-payload-vH-CH1 vL-CL-payload-rPEG rPEG-payload-vH-CH1
rPEG-vL-CL rPEG-payload-vH-CH1 payload-rPEG-vL-CL
rPEG-payload-vH-CH1 rPEG-payload-vL-CL rPEG-payload-vH-CH1
[0490] Full Length Antibodies
[0491] rPEGs and payloads and other domains can be fused to the
light chain or heavy chain of an antibody, or to both chains of an
antibody. The following table illustrates a few examples of AFBTs
that are based on full-length antibodies:
TABLE-US-00010 Light chain Heavy chain Light chain-rPEG Heavy chain
Light chain-rPEG-payload Heavy chain Light chain-payload-rPEG Heavy
chain rPEG-Light chain Heavy chain payload-rPEG-Light chain Heavy
chain rPEG-payload-Light chain Heavy chain Light chain-rPEG Heavy
chain-rPEG Light chain-rPEG-payload Heavy chain-rPEG Light
chain-payload-rPEG Heavy chain-rPEG rPEG-Light chain Heavy
chain-rPEG payload-rPEG-Light chain Heavy chain-rPEG
rPEG-payload-Light chain Heavy chain-rPEG Light chain-rPEG Heavy
chain-rPEG-payload Light chain-rPEG-payload Heavy
chain-rPEG-payload Light chain-payload-rPEG Heavy
chain-rPEG-payload rPEG-Light chain Heavy chain-rPEG-payload
payload-rPEG-Light chain Heavy chain-rPEG-payload
rPEG-payload-Light chain Heavy chain-rPEG-payload Light chain-rPEG
rPEG-Heavy chain Light chain-rPEG-payload rPEG-Heavy chain Light
chain-payload-rPEG rPEG-Heavy chain rPEG-Light chain rPEG-Heavy
chain payload-rPEG-Light chain rPEG-Heavy chain rPEG-payload-Light
chain rPEG-Heavy chain Light chain-rPEG payload-rPEG-Heavy chain
Light chain-rPEG-payload payload-rPEG-Heavy chain Light
chain-payload-rPEG payload-rPEG-Heavy chain rPEG-Light chain
payload-rPEG-Heavy chain payload-rPEG-Light chain
payload-rPEG-Heavy chain rPEG-payload-Light chain
payload-rPEG-Heavy chain Light chain-rPEG rPEG-payload-Heavy chain
Light chain-rPEG-payload rPEG-payload-Heavy chain Light
chain-payload-rPEG rPEG-payload-Heavy chain rPEG-Light chain
rPEG-payload-Heavy chain payload-rPEG-Light chain
rPEG-payload-Heavy chain rPEG-payload-Light chain
rPEG-payload-Heavy chain
[0492] Certain sites on a full-length antibody or an antibody
fragment as defined herein are preferred fusion sites for rPEG to a
full-length antibody (including IgG1, 2, 3, 4, IgE, IgA, IgD, and
IgM) or an antibody fragment. These preferred sites are at the
boundary of structured sequences, such as domains, hinges, etc,
without disturbing the folding of these functional domains. rPEG
can be added in 1, 2, 3, 4, 5, 6, 7 or even 8 different locations
to an antibody (and more than 8 for IgM and IgG3) and a single
antibody can have 1, 2, 3, 4, 5, 6, 7, 8 or more rPEGs in diverse
locations and in any combination of the 8 locations shown in FIG.
103. FIG. 103e shows the preferred fusion sites for rPEG to domains
and fragments of an antibody.
[0493] Domain Antibody-Based AFBTs
[0494] In yet another embodiment, rPEGs and payloads and other
domains can be fused to a domain antibody (dAb). In order to
generate domain antibodies with suitable binding properties, one
can use the naturally monomeric vH domains (called vHH) found in
the immune repertoire of camelids and sharks that naturally lacks a
light chain. [Hamers-Casterman, C., et al. (1993) Nature, 363:
446]. Alternatively, one can engineer the vH-vL interface of a
human vH or vL domain in order to improve solubility and reduce
dimerization and aggregation. Such mutations carry the risk of
increasing immunogenicity of the resulting domain antibody. The
present invention describes fusing human vH or vL Ig domains to
rPEG, which improves solubility and folding, reduces aggregation,
and yet does not induce immune response triggered by the
mutagenesis of human framework residues. Examples of AFBTs which
are based on dAb domains include, but are not limited to, dAb-rPEG,
dAb-rPEG-payload, dAb-payload-rPEG, rPEG-dAb, payload-rPEG-dAb,
rPEG-payload-dAb. dAb domain can be derived from the vH or vL
domain of an antibody molecule.
[0495] Multispecific AFBTs
[0496] The present invention also embodies AFBTs that comprise
fragments derived from multiple different antibodies with different
binding specificities. An example is shown in FIG. 58b. Such AFBTs
combine the binding specificities of two or more parent antibodies.
Parent antibodies can be chosen such that the resulting AFBT binds
to multiple different target antigens. Alternatively, the parent
antibodies can bind to different epitopes of the same target
antigen. AFBTs bind the target very tightly if they can form
multivalent interactions by binding to multiple sites on the same
target antigen as illustrated in FIG. 59. Multispecific AFBTs can
form multimers of the same protein chain. For instance, FIG. 58b
illustrates a multispecific AFBT that is a dimer of two polypeptide
chains that contains two binding sites based on the vH-vL chain A
and two additional binding sites based on the vH-vL chain B. One
skilled in the art can appreciate the possibility of generating a
multispecific AFBT containing many different combinations of
binding domains or binding modules. In addition to different
variable domains, multivalent AFBTs may include one or more payload
domains, rPEG modules and other protein domains that can be chosen
to enhance therapeutic utility or production and purification. One
embodiment includes multispecific AFBTs that interact with multiple
target antigens that are related to the same disease symptoms, the
same pathogen or cause of pathogenesis, or the same physiological
pathway or process. Examples of such multispecific AFBTs include
but are not limited to multispecific AFBTs that block multiple
cytokines which are involved in a related biological process. A
preferred embodiment includes multispecific AFBTs that block
multiple growth factors that are involved in angiogenesis such as
VEGF, PDGF, and PIGF. FIG. 95 shows an rPEGs flanked on both sides
by a VEGF-receptors. Since VEGF is dimeric, it can be the same
receptor on both sides of the rPEG, or a different receptor,
preferably VEGF-R1 and VEGF-R2, but VEGFR3 may also be used.
Another preferred embodiment includes multispecific AFBTs that
block multiple cytokines that are involved in inflammatory diseases
such as TNF-.alpha., IL-1, IL-6, IL12, IL-13, IL17, and IL-23. Yet
another preferred embodiment includes multispecific AFBTs that bind
multiple tumor antigens such as Her1, Her2, Her3, EGFR, TF antigen,
CEA, A33, PSMA, MUC1, .alpha.v/.beta.3 integrin, .alpha.v/.beta.5
integrin, and .alpha.5/.beta.1 integrin. Still another preferred
embodiment includes multispecific AFBTs that bind multiple antigens
that are related to an infectious disease. Said multispecific AFBTs
can form multivalent interactions with an infectious agent
resulting in improved therapeutic efficacy. Multispecific AFBTs can
be engineered to comprise a binding site for a tumor antigen and a
second binding site for an antigen on an immune cell. Examples
include AFBTs that bind tumor antigens and CD3 or CD16, which can
recruit and activate natural killer (NK) cells. To further increase
potency, a cytokine domain such as IL-2 can be included to activate
immune cells in the vicinity of the tumor cells.
[0497] AFBTs Containing Multiple Fragments of the Same Antibody
[0498] AFBTs can be engineered such that each polypeptide chain
contains multiple variable fragments of the same parent antibody.
These fragments can be identical in their sequence or they can be
engineered to facilitate proper domain assembly. An example is
illustrated in FIG. 60a. This AFBT contains a diabody domain and a
monovalent scFv domain based on the same parent antibody. As a
result, the AFBT assembles into a dimeric structure that contains a
total of 4 equivalent target binding sites. Such multivalent AFBTs
can have improved potency due to avidity.
[0499] Bispecific AFBTs Based on Diabodies
[0500] AFBTs can be constructed to combine one diabody and a
variable domain and at least one rPEG domain. The constructs form
dimers and contain a total of 4 antigen binding sites. FIG. 58b
illustrates an example of a bispecific AFBT. The variable domains A
in such a construct can be scFv domains or dAb domains. The
variable domains A can be at the C-terminal side of the diabody
domain B. Alternatively, the variable domains A can be at the
N-terminal side of the diabody domain B. Bispecific AFBTs can
contain additional rPEG domains or other domains such as hormones,
cytokines or enzymes. If the variable domain in a bispecific AFBT
is a scFv domain, the scFv domain can have the configuration
vH-linker-vL or the configuration vL-linker-vH.
[0501] In a preferred embodiment, a bispecific AFBT comprises a
diabody B and a scFv A, in which the diabody and scFv domains are
optimized to reduce incorrect pairing of the 41 g domains in these
constructs. The domains can be optimized such that v.sub.L-A and
v.sub.H-A as well as v.sub.L-B and v.sub.H-B form tighter
interactions than the incorrect pairings v.sub.L-A and v.sub.H-B
and v.sub.L-B and v.sub.H-A. This can be accomplished by choosing
frameworks of both vH and vL domains such that the vH/vL contact
surface of scFv domain A has significant structural differences
form the vH/vL contact surface of diabody domain B. One can further
enhance these differences by engineering the vH/vL contact regions
of scFv domain A and diabody domain B to minimize the chance of
undesired contacts. For instance, one can engineer charge
differences such that an ion pair is formed for correct vH/vL
pairing but the same ion pair can not be formed during incorrect
pairings of vH and vL domains in the bispecific AFBT. Another
approach is to introduce hydrogen bonding partners into the desired
vH/vL contact surfaces that can not be formed in incorrect pairings
of vH and vL domains. Yet another approach is to alter the shape of
the contact vL/vH contact surfaces such that incorrect vL/vH
pairings are destabilized.
[0502] Bispecific AFBTs based on diabodies are of particular
utility as they contain two rPEG domains per divalent complex,
which results in reduced kidney filtration and improved in vivo
half-life. AFBTs can be engineered to contain a diabody domain and
two additional variable domains per polypeptide chain. Such a
protein can form dimeric complexes that comprise a total of 6
antigen binding sites. Further variable fragments or payload
domains can be added to increase potency.
[0503] Dimeric AFBTs Containing Payloads
[0504] FIG. 60b illustrates a dimeric AFBT that contains a diabody
domain and a payload domain. Such proteins form dimeric complexes
such that each complex contains two target binding sites, two
rPEGs, and two payload domains. Additional protein domains can be
added to increase utility. Having two rPEGs per protein complex
reduces kidney filtration and increases in vivo half-life. Having
two payload domains increases potency. The target binding sites of
the diabody domain can be engineered to further increase in vivo
half-life by binding to a component of blood such as red blood
cells, human serum albumin, IgG, collagen or other protein or cell
in the blood.
[0505] Combining Antibody Fragments and Payloads
[0506] The present invention also embodies AFBTs which comprise one
or more payloads. One preferred embodiment includes payloads that
are protein domains and can be directly fused to the other domains
comprising an AFBT. Examples of such payload domains include, but
are not limited to, cytokines, hormones, growth factors, and
enzymes. Such AFBTs combine the specificity of antibodies with the
efficacy of the payload while the rPEG domain provides half-life
and facilitates production and formulation. Another preferred
embodiment includes AFBTs that combine an antibody fragment with
specificity for a particular tissue and a payload that exerts its
activity in the same tissue. One example includes antibody
fragments with specificity for a tumor in combination with
cytostatic or cytotoxic payloads. Another example comprises
antibody fragments with specificity for infected cells or
infectious agents in combination with anti-infective payloads. Yet
another useful combination comprises antibody fragments with
specificity against inflamed tissues in combination with payloads
that have anti-inflammatory activity. Antibodies that can be linked
to an accessory polypeptide include, but are not limited to,
abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab,
cetuximab, daclizumab, eculizumab, efalizumab, ibritumomab,
tiuxetan, infliximab, muromonab-CD3, natalizumab, omalizumab,
palivizumab, panitumumab, ranibizumab, gemtuzumab ozogamicin,
rituximab, tositumomab, trastuzumab, and any antibody fragments
specific for antigens including complement C5, CBL, CD147, IL8,
gp120, VLA4, CD11a, CD18, VEGF, CD40L, anti-Id, ICAM1, CD2, EGFR,
TGF-.beta.2, TNF.alpha., E-selectin, FactII, Her2/neu, F gp,
CD11/18, CD14, CD80, ICAM3, CD4, CD23, .beta.2-integrin,
.alpha.4.beta.7, CD52, CD22, HLA-DR, CD64 (FcR), TCR .alpha..beta.,
CD3, Hep B, CD125, EpCAM, gpIIbIIIa, IgE, CD20, IL5, IL4, CD25,
CD33, HLA, F gp, and VNRintegrin.
[0507] Enzymes can be used as payloads for tumor-specific AFBTs.
Enzymes can be chosen in order to eliminate a required nutrient or
metabolite from the tumor environment, such as asparaginase,
arginase, histidinase, or methioninase. Alternatively, one can
utilize enzymes that exert cytotoxic activity. An example includes
AFBTs that comprise a tumor specific antibody fragment and RNAse
which induces apoptosis upon internalization into cells.
[0508] Payloads that are useful in anti-cancer, anti-microbial,
and/or anti-inflammatory therapeutics include toxins such as
Pseudomonas exotoxin, ricin, botulinum toxin, and other plant or
bacterial toxins. Other biological toxins include, but are not
limited to, abrin, aerolysin, botulinin toxin A, B, C1, C2, D, E,
F, b-bungarotoxin, Caeruleotoxin, Cereolysin, Cholera toxin,
Clostridium difficile enterotoxin A and B, Clostridium perfringens
lecithinase, Clostridium perfringens kappa toxin, Clostridium
perfringens perfringolysin O, Clostridium perfringens enterotoxin,
Clostridium perfringens beta toxin, Clostridium perfringens delta
toxin, Clostridium perfringens epsilon toxin, Conotoxin, Crotoxin,
Diphtheria toxin, Listeriolysin, Leucocidin, Modeccin, Nematocyst
toxins, Notexin, Pertussis toxin, Pneumolysin, Pseudomonas
aeruginosa toxin A, Saxitoxin, Shiga toxin, Shigella dysenteriae
neurotoxin, Streptolysin O, Staphylococcus enterotoxins B and F,
Streptolysin S, Taipoxin, Tetanus toxin, Tetrodotoxin, Viscuminm,
Volkensin, and Yersinia pestis murine toxin.
[0509] Payloads can be chosen to eliminate a toxic metabolite.
Examples are urate oxidase for the treatment of gout and
phenylalanine ammonia lyase for the treatment of phenylketonuria.
Payloads can also comprise chemically conjugated small molecules.
Such payloads can be conjugated to an AFBT resulting in a
semisynthetic AFBT. The protein portion of a semisynthetic AFBT can
be engineered to facilitate controlled chemical conjugation via
exhaustive coupling as illustrated in FIG. 61. The protein portion
can be engineered to have a defined number of coupling sites. This
enables the use of coupling reagent in excess to the concentration
of coupling sites such that coupling efficiency can be close to
completion, which results in a defined coupling product. Useful
coupling sites can be amino groups. The protein portion of such
semisynthetic AFBTs can be engineered such that all or most lysine
residues in the antibody fragments are replaced with other residues
that are compatible with folding and target binding. In many
proteins one can replace lysine residues with arginine, glutamate,
aspartate, serine, threonine or another amino acid. Designated
coupling sites can be incorporated into the rPEG domain or into any
other protein portion of the protein. In addition, the N-terminus
of each protein chain can serve as a conjugation site. Cysteine
residues can also serve as conjugation sites. Example payloads that
can be conjugated to AFBTs include cytotoxic drugs such as
doxorubicin, auristatin, maytansine and related molecules that can
be fused to AFBTs with tumor-specific antibody fragments. Other
payloads of interest for conjugation include antiviral compounds,
imaging reagents, and chelating agents that can be labeled with
radionuclides to generate imaging agents or AFBTs for
radiotherapy.
[0510] Thiols in rPEG Tail
[0511] Another embodiment of the present invention includes AFBTs
comprising rPEG sequences that contain one or multiple cysteine
residues. These cysteine side chains can form disulfide bridges
with other proteins after injection into a patient. These disulfide
bridges can result in increased in vivo half-life. In other
embodiments disulfide bond formation can result in prolonged
retention of AFBTs at the injection site resulting in a
slow-release PK profile. AFBTs that contain free cysteins can also
be engineered for improved bioavailability for oral, intranasal,
and intradermal administered AFBTs. This can be achieved by forming
disulfide bridges with proteins at the surface of epithelial cells
resulting in enhanced uptake of the AFBT.
[0512] RGD-Peptides in rPEG
[0513] AFBTs may also contain one or multiple RGD sequences or
related sequences that are known to interact with integrins as well
as components of the extracellular matrix. These RGD-related
sequences can be flanked by cysteine residues to result in
disulfide-mediated cyclization. Alternatively, the RGD-related
sequences can be flanked by additional amino acids that can be
selected to enhance the affinity and/or specificity of interaction
with a particular integrin. One preferred embodiment includes AFBTs
that contain RGD sequences and interact with integrins
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.5.beta..sub.1, that are overexpressed on a variety of
tumor cells.
[0514] Antibody Fragments that Increase Half-Life
[0515] The present invention also embodies AFBTs that contain
antibody fragments that increase the in vivo half-life of the
AFBTs. This can be achieved by incorporating antibody fragments
that bind to targets that have long in vivo half-lives. Examples of
such targets that increase the in vivo half-life include but are
not limited to serum proteins, in particular, serum albumin,
immunoglobulins, and other highly abundant proteins. AFBTs can also
incorporate antibody fragments with specificity for blood cells or
vessel walls. Of particular interest are red blood cells (RBCs),
which are extremely abundant, have an average life span of
approximately 4 months, and are characterized by minimal metabolic
activity. AFBTs can be engineered to bind any protein on the
surface of an RBC. A preferred embodiment includes AFBTs that bind
to glycophorin A, which is expressed in high abundance on the
surface of RBCs. AFBTs can be engineered to bind to any cell
surface target that can be in contact with an AFBT in vivo
resulting in a prolonged retention of the AFBT. Another embodiment
includes AFBTs that bind to components of the extracellular matrix
(ECM). The ECM contains many proteins including, but not limited
to, agrin, alpha elstin, amisyn, bestrophin, collagens, contactin
1, CRIPT, drebrin, entactin, fetuin A, HAS3, HCAP-G, syndecan,
KAL1, 1 Afadin, laminins, Mint3, MMP24, NCAM, neurocan, nidogen 2,
optimedin, procollagen type 10A, PSCDBP, reelin, SIRP,
synaptotagmin, synCAM, syndecan, syntrophin, TAG1, tenascin C, and
zyxin. Yet another embodiment includes AFBTs which comprise
antibody fragments that bind the FcRn receptor, which results in
recycling of endocytosed AFBTs. Examples include antibody fragments
that show pH-dependent binding to FcRn such that the antibody
fragment binds FcRn with low affinity at around neutral pH but
binds with high affinity at lower pH, e.g. pH 5, which is within
the range of pH predominantly found in lysosomal compartments.
AFBTs that provide increased half-life are illustrated in FIGS. 58a
and 60b. It should be noted that many other configurations can be
designed that comprise a payload domain and an antibody fragment
that provides half-life extension.
[0516] The present invention also embodies fusion proteins
comprising an Fc fragment fused to an rPEG. FIG. 83 shows a
construct with a drug module at the N-terminus, followed by rPEG,
fused to an antibody Fc fragment, with or without the hinge. The Fc
fragment provides a long halflife and the rPEG allows the Fc
fragment to be expressed in the E. coli cytoplasm in a soluble and
active form. In another embodiment, an antibody Fc fragment, with
or without a hinge region, is optionally fused to a drug module
(e.g. IFNa, hGH, etc.) on one end and optionally fused to rPEG on
the other end. The sequence between CH2 and CH3 mediates binding to
FcRn, the neonatal Fc receptor (FIG. 90). Yet another embodiment
includes a protein construct comprising a pair of CH3 domains (FIG.
91). Zero, one or both of the two polypeptide chains may be fused
to rPEG on the N-terminal and/or C-terminal end, and fused to zero,
one or more drug modules at the other end. The FcRn binding
sequence can either be retained or deleted. Retention of the FcRn
binding sequence yields a longer serum halflife. Still another
embodiment describes a protein that is a full Fc, including CH2 and
CH3 domains (with or without a hinge), fused at the C-terminus to
an rPEG with the drug/pharmacophore located at the C-terminus (FIG.
92). There molecules are capable of polypeptide chain swapping,
resulting in heterodimers. Yet another embodiment describes a
partial Fc without a hinge and with a CH2 domain that is truncated
but retains FcRn binding and with a drug/pharmacophore located at
the C-terminus (FIG. 93a). FIG. 93b illustrates a partial Fc
without hinge and CH2 domain, but retaining the CH3 domain and
having a drug/pharmacophore located at the C-terminus. Such Fc
fragment does not bind FcRn but can dimerize via the CH3
domain.
[0517] Still another embodiment employs an N-terminal drug module
followed by rPEG and a C-terminal Fc fragment with hinge (FIG.
101). This is a useful format for halflife extension of drug
modules that can be manufactured in the E. coli cytoplasm. An
alternative format for a pro-drug containing an Fc fragment is
described herein (FIG. 102). The format is similar to that
described in FIG. 101, with the addition of an inhibitory sequence
that binds to and inhibits the drug sequence. The drug is separated
from the inhibitory sequence by a cleavage site. The N-terminal
inhibitory binding sequence is followed by a cleavage site, which
is followed by the drug sequence. Before cleavage, the pro-drug is
bound to the inhibitory sequence and thus it is inactive. Upon
cleavage, the inhibitory binding sequence is gradually released and
cleared, gradually increasing the amount of time that the drug is
active. Assays for assessing correct folding of an Fc fragment
fused to an rPEG, including SDS-PAGE on hinge disulfide formation
and size exclusion chromatography on CH3 dimerization, are depicted
in FIG. 104.
[0518] Antibody Fragments that Result in Slow Release
[0519] AFBTs can be engineered to release slowly from the injection
site resulting in long-term drug exposure. One embodiment of the
present invention includes the incorporation of an antibody
fragment that binds to a molecule expressed in high abundance at
the injection site. For example, such antibody fragments may bind
to target antigens including but not limited to collagen,
hyaluronic acid, heparan sulfate, laminins, elastins, chondroitine
sulfate, keratane sulfate, fibronectin, and integrins. By
engineering the affinity and/or avidity of the antibody fragment
for its target antigen, the rate of AFBT release from the injection
site can be controlled. Another embodiment includes the
introduction of one or several protease sites that can be cleaved
by proteases at the injection site in order to control the rate of
AFBT release at the injection site.
[0520] Antibody Fragments that Affect Tissue Distribution
[0521] The present invention also includes AFBTs that incorporate
antibody fragments that bind to a target antigen present in a
particular cell or tissue or a particular set of tissues. These
constructs can increase the therapeutic window of an active drug by
achieving a local tissue-specific accumulation of the AFBT.
Examples include AFBTs that contain antibody fragments with
specificity for tumor antigens that are overexpressed in tumor
tissues or tumor microenvironment including tumor vasculature. One
can chose tumor antigens that are effectively internalized by cells
as targets for AFBTs that include a payload with intracellular
activity. For instance, AFBTs comprise an antibody fragment with
specificity for a tumor antigen capable of being internalized upon
binding and a cytotoxic payload. Other examples include AFBTs with
specificity for viral targets.
[0522] Collagen Binding Domains (CBDs)
[0523] Another embodiment of the present invention includes the use
of CBDs as domains in AFBTs and other protein drugs. Collagen is
highly abundant in many tissues in particular in the extracellular
space. Protein pharmaceuticals that comprise CBDs can bind to
collagen at the injection site or in the vicinity of the injection
site, forming a depot from which the AFBT is then slowly released.
The release rate can be controlled by introducing protease sites or
by choosing CBDs with a suitable affinity to collagen. By choosing
a CBD with low affinity to collagen, the rate of release of the
AFBT is increased. Alternatively, the rate of AFBT release can be
slowed down by including CBDs that bind to collagen with very high
affinity or by including multiple CBDs into an AFBT to achieve
avidity. CBD sequences can be obtained from naturally occurring
CBDs. Examples of proteins that bind to collagen and comprise CBDs
include, but are not limited to, integrins, in particular
.alpha..sub.1.beta..sub.1 integrin, .alpha..sub.2.beta..sub.1
integrin, .alpha..sub.v.beta..sub.3 integrin, angiogenesis
inhibitor, collagen V, C-proteinase, decorin, fibronectin,
interleukin-2, matrix metalloproteases 1, 2, 9, and 13,
phosphosphoryn, thrombospondin, biglycan, bilirubin, BM40/SPARC,
MRP8, MRP-14, calin from leeches, DDR1, DDR2, fibromodulin, Gla
protein, glycoprotein 46, heat shock protein 47, lumican, myelin
associated glycoprotein, platelet receptors, staphylococcus aureous
surface molecules and other microbial adhesion molecules,
syndecan-1, tenascin-C, vitronectin, von Willebrand factor, and
factor XII. Additional examples of proteins that bind collagen and
contain CBDs are listed in [Di Lullo, G. A., et al. (2002) J Biol
Chem, 277: 4223]. CBDs from natural proteins can be further
engineered to increase their therapeutic utility and improve their
stability. Immunogenicity of the CBD-containing proteins can be
reduced by removing epitopes recognized by B and/or T cells. CBD
sequences can also be optimized to maximize protein production
and/or to improve protein solubility.
[0524] HSA-Binding Peptides in Tail
[0525] AFBTs comprising peptide sequences that increase in vivo
half-life can also be achieved by utilizing peptide sequences that
bind to a serum protein or to the surface of a blood cell. Examples
include peptide sequences that bind to human serum albumin (HSA).
Such sequences can be obtained by phage display of random peptide
sequences or similar selection of screening approaches. AFBTs can
contain one or more, either identical or different, copies of such
peptide sequences.
[0526] Target Antigens
[0527] Yet another embodiment of the invention encompasses an
antibody fragment that binds to a target antigen which is of
therapeutic or diagnostic relevance. FIG. 87 illustrates a Fab
fragment binding to a cell-surface target. Extension of the length
of the natural linkers from the usual 2-6 amino acids to 4 to 100
or more amino acids, between the VH and the CH domains, and between
the VL and the CL domains, increases the ability of one Fab to
crosslink to another Fab by domain swapping, thereby forming a
binding complex with higher valency, resulting in higher apparent
affinity (avidity). The linker may be an rPEG or a different
composition. The extended linker format allows binding with
increased affinity specifically at sites with a higher density of
target. The antibody fragment of an AFBT can bind to a blood
component to increase the half-life of the AFPT in circulation. The
antibody fragment of the AFBT can also bind to a receptor that
facilitates lysosomal recycling. An example is the FcRn receptor
that can re-export proteins after lysosomal uptake. Of particular
interest are antibody fragments that bind with spacially or
temporally-dependent affinity to a receptor that can facilitate
lysosomal recycling such that the antibody fragment binds with high
affinity under conditions found in a lysosome but it binds with
lower affinity to the same receptor under conditions found on the
cell surface. The antibody fragment of an AFBT can bind to a target
antigen that is predominantly found in a disease-relevant tissue.
As a result such AFBTs can accumulate in a particular disease
relevant tissue. Examples include AFBTs that bind to tumor tissue
or virally-infected tissues. The antibody fragment of an AFBT can
bind to a target antigen that facilitates cellular internalization
in a disease-relevant tissue. Antibody fragments of an AFBT can
also bind to a target antigen that facilitates uptake of the AFBT
into a particular compartment of the body, for example, target
antigens that facilitate oral, intranasal, mucosal, or lung uptake
of an AFBT, and target antigens that facilitate the transport of
the AFBT across the blood brain barrier. Examples of target
antigens that are of particular interest include, but are not
limited to, ILL IL4, IL6, Il12, IL13, IL17, IL23, CD22, BAFF, and
TNF.alpha..
[0528] Advantages of rPEG in AFBTs
[0529] AFBTs combine valuable properties of rPEG and antibody
fragments. The rPEG portion of an AFBT results in a low overall
immunogenicity of an AFBT. This is achieved by sterical shielding
of the antibody fragment and other potentially immunogenic portions
of an AFBT by rPEG. rPEGs are highly flexible and as a result they
lack conformational epitopes. Due to their high hydrophilicity and
high entropy, rPEGs have a very low inherent immunogenic
potential.
[0530] The rPEG portions of an AFBT also result in a stabilization
of other AFBT domains. Due to their hydrophilic nature, rPEG
domains reduce aggregation of AFBTs. This greatly simplifies the
formulation development for AFBTs. In addition, steric shielding by
rPEG protects other portions of AFBTs from proteolysis. This is of
particular importance for payloads and antibody fragments that are
prone to proteolytic degradation.
[0531] Another advantage of using AFBT over a full length antibody
is the minimization or elimination of undesirable effector function
associated with a full length antibody. Full length antibody
molecules have a number of effector functions such as
antibody-dependent cell-mediated cytotoxicity (ADCC) as well as
complement activation (CDC) that significantly limit their
therapeutic use for indications where effector function is
undesirable. For instance, many indications require an agent that
binds and sequesters a molecule such as a cytokine or hormone. In
general it is not desirable to utilize antibodies for such
indications as their effector functions results in undesirable
toxicity. Much of the antibody dependent effector function is
mediated through the Fc portion. In many embodiments described
herein, the AFBTs utilize the variable domains of antibodies that
are responsible for target binding while replacing the Fc portion
that is responsible for effector function. AFBTs can be engineered
to bind and activate cell surface receptors such as death receptors
DR4 and DR5. Activation can be achieved by receptor
multimerization. Although full length antibodies are able to
activate such receptors, they also induce toxicity caused by
antibody binding to healthy cells that express the same target
receptor. AFBTs can activate cell surface receptors without
eliciting effector function that would cause toxicity.
[0532] Yet another advantage of rPEG is that it helps associate the
two proteins that belong to the same complex, as illustrated in
FIG. 86. The affinity between such proteins is often insufficient
to keep them associated, but the addition of rPEG stabilizes their
interaction and reduces their tendency to form polymers.
[0533] Manufacture/Production of AFBTs
[0534] The present invention also relates to the production of the
AFBTs. The rPEG domain in AFBTs facilitates protein folding and
reduces protein aggregation. This property facilitates microbial
production of AFBTs. Most antibody fragments described in the
literature require refolding from inclusion bodies or secretion
into the periplasmic space, which results in low production yields.
In contrast, most AFBTs can be produced in soluble form at high
concentration in the cytosol of microbial expression hosts. A
preferred expression host for AFBTs is E. coli (FIG. 45). However,
the properties of AFBTs make them suitable for expression in most
microbial as well as eukaryotic expression systems. The N-terminal
sequence of AFBTs can be optimized to control posttranslational
processing. In particular the amino acid following the start codon
can determine the subsequent processing of the N-terminal
methionine [Hirel, P. H., et al. (1989) Proc Natl Acad Sci USA, 86:
8247]. One embodiment includes N-terminal sequences that result in
uniform products. By choosing gly, ala, pro, ser, thr, or val as
amino acid following the N-terminal met, efficient processing and
removal of the N-terminal met can be achieved. Another embodiment
includes his, gln, glu, phe, met, lys, tyr, trp, or arg as amino
acid following the N-terminal met, which prevents removal of the
N-terminal met and results in homogeneous products. AFBTs also
facilitate refolding under conditions of high protein concentration
where most unmodified proteins yield aggregates. The advantage of
rPEG during manufacturing of AFBTs is crucial as AFBTs contain
multiple protein domains that have a tendency to form aggregates.
Such protein domains can be separated by rPEG sequences in the
AFBTs to minimize aggregation between individual protein domains
during folding.
[0535] Generation and Production of Disease-Associated and/or
Patient-Specific AFBTs
[0536] The present invention also embodies the generation and
production of disease-associated AFBTs, i.e. antibody fragments
fused to an accessory polypeptide such as rPEG. Antibody genes can
be directly isolated from infected or otherwise exposed patients
[Wrammert, J., et al. (2008) Nature]. Various formats of antibody
fragments fused to rPEG can be rapidly generated from such antibody
genes. The resulting fusion proteins can be produced and purified
using standardized protocols, enabling rapid generation of the
disease-associated AFBTs. An example of the process is illustrated
in FIG. 68. The rapid discovery process enables discovery and
preparation of specific treatments in response to an acute disease
outbreak such as a bacterial or viral infection. The rapid
generation of fusion proteins between antibody fragments and rPEG
also enables one to produce patient-specific treatments, which
encompass but are not limited to isolation of immune cells from a
patient; cloning of disease-specific antibody genes from the immune
cells; construction and subsequent manufacturing of antibody
fragment-rPEG fusions (i.e. disease-associated AFBTs); and
treatment of the patient with the disease.
[0537] Polyclonal and Multiclonal AFBTs
[0538] The present invention also relates to a pharmaceutical
composition comprising more than one AFBT. Such composition of AFBT
mixture may have improved performance relative to the individual
AFBTs. AFBT-based product can be multiclonal such that they contain
two, three, or more defined AFBTs. Alternatively, AFBTs can be
polyclonal containing multiple AFBTs. Such polyclonal AFBTs can be
generated by cloning antibody fragments from a source that is
enriched for antibodies or antibody fragments with a useful
specificity. One example is cloning of antibody fragment
repertoires from an infected patient. Another example includes
display libraries that have been enriched by panning against a
target of interest.
[0539] rPEG Fusion Products
[0540] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the human growth hormone (hGH) or human
growth hormone receptor (hGH-R) gene under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system (e.g. Escherichia coli, Pichia
pastoris, CHO--S, etc). Protein expression is induced using
standard techniques well known in the art for the expression system
employed and purified using standard procedures (e.g. ion exchange
chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc).
The purified protein can then be administered to human patients for
therapeutic treatment of indications including, but not limited to:
adult growth hormone deficiency, pediatric growth hormone
deficiency, Turner syndrome, chronic renal failure, idiopathic
short stature, post-transplant growth failure, hypophosphatemic
rickets, inflammatory bowel disease, Noonan syndrome, pediatric
Coeliac disease, AIDS wasting, obesity, aging, or other indications
for which the unmodified protein has been shown to provide
therapeutic benefit. The addition of the rPEG sequence confers the
properties of extended serum half-life, improved patient
exposure/efficacy, and/or improved manufacturing efficiency.
[0541] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the human growth hormone fragment 176-191
or 177-191 gene under control of appropriate transcription and
translation sequences for high level protein expression in a
biological system (e.g. Escherichia coli, Pichia pastoris, CHO--S,
etc). Protein expression is induced using standard techniques for
the expression system employed and purified using standard
procedures (e.g. ion exchange chromatography, size exclusion
chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: adult growth hormone deficiency, pediatric growth
hormone deficiency, Turner syndrome, chronic renal failure,
idiopathic short stature, post-transplant growth failure,
hypophosphatemic rickets, inflammatory bowel disease, Noonan
syndrome, pediatric Coeliac disease, AIDS wasting, obesity, aging,
or other indications for which the unmodified protein has been
shown to provide therapeutic benefit. The addition of the rPEG
sequence confers the properties of extended serum half-life,
improved patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0542] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the exenatide gene under control of
appropriate transcription and translation sequences for high level
protein expression in a biological system (e.g. Escherichia coli,
Pichia pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of the following indications:
type II diabetes, or other indications for which the unmodified
protein has been shown to provide therapeutic benefit. The addition
of the rPEG sequence confers the properties of extended serum
half-life, improved patient exposure/efficacy, and/or improved
manufacturing efficiency. Due to the sensitivity of the N-terminus
of exenatide to maintaining in vivo efficacy, special
considerations may be required to maintain the native N-terminal
structure upon recombinant expression and purification, and
preferred embodiments would comprise fusions of rPEG to the
C-terminus of the exenatide sequence. N-terminal leader sequences
which can be cleaved by proteases either in vitro or in vivo can be
employed to improve manufacturing yield and/or improve delivery of
active molecules in vivo. An alternative strategy would comprise
mutating the internal methionine of exenatide to a compatible amino
acid (eg leucine, which is present at the homologous position in
the GLP-1 sequence) and use cyanogen bromide or similar chemical
methods to remove the N-terminal leader sequence to generate the
native exenatide N-terminus.
[0543] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the GLP-1 gene under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system (e.g. Escherichia coli, Pichia
pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of the following indications:
type II diabetes, or other indications for which the unmodified
protein has been shown to provide therapeutic benefit. The addition
of the rPEG sequence confers the properties of extended serum
half-life, improved patient exposure/efficacy, and/or improved
manufacturing efficiency. Due to the sensitivity of the N-terminus
of GLP-1 to maintaining in vivo efficacy, special considerations
may be required to maintain the native N-terminal structure upon
recombinant expression and purification, and preferred embodiments
would comprise fusions of rPEG to the C-terminus of the GLP-1
sequence. N-terminal leader sequences which can be cleaved by
proteases either in vitro or in vivo can be employed to improve
manufacturing yield and/or improve delivery of active molecules in
vivo.
[0544] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the IL1-RA gene under control of
appropriate transcription and translation sequences for high level
protein expression in a biological system (e.g. Escherichia coli,
Pichia pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: rheumatoid arthritis, psoriatic arthritis,
psoriasis, inflammatory bowel disease, Crohn's disease, or other
indications for which the unmodified protein has been shown to
provide therapeutic benefit. The addition of the rPEG sequence
confers the properties of extended serum half-life, improved
patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0545] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the interferon alpha, beta, or gamma gene
under control of appropriate transcription and translation
sequences for high level protein expression in a biological system
(e.g. Escherichia coli, Pichia pastoris, CHO--S, etc). Protein
expression is induced using standard techniques for the expression
system employed and purified using standard procedures (e.g. ion
exchange chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc)
well known to those skilled in the art. The purified protein can
then be administered to human patients for therapeutic treatment of
indications including, but not limited to: hairy cell leukemia,
AIDS-related Kaposi's syndrome, pH chromosome positive CML, chronic
hepatitis C, condylomata acuminate, chronic hepatitis B, malignant
melanoma, follicular lymphoma, multiple sclerosis, non-Hodgkins
lymphoma, osteopetrosis, chronic granulomatous disease-associated
infections, pulmonary multi-drug resistant tuberculosis, or other
indications for which the unmodified protein has been shown to
provide therapeutic benefit. The addition of the rPEG sequence
confers the properties of extended serum half-life, improved
patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0546] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the G-CSF gene under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system (e.g. Escherichia coli, Pichia
pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: chemotherapy-induced febrile neutropenia,
bone-marrow transplantation, congenital neutropenia, cyclic
neutropenia, idiopathic neutropenia, AIDS-associated neutropenia,
myelodysplastic syndrome, or other indications for which the
unmodified protein has been shown to provide therapeutic benefit.
The addition of the rPEG sequence confers the properties of
extended serum half-life, improved patient exposure/efficacy,
and/or improved manufacturing efficiency.
[0547] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the FGF21 gene under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system (e.g. Escherichia coli, Pichia
pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: diabetes, obesity, or other indications for which
the unmodified protein has been shown to provide therapeutic
benefit. The addition of the rPEG sequence confers the properties
of extended serum half-life, improved patient exposure/efficacy,
and/or improved manufacturing efficiency.
[0548] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the calcitonin gene under control of
appropriate transcription and translation sequences for high level
protein expression in a biological system (e.g. Escherichia coli,
Pichia pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: postmenopausal osteoporosis, Paget's disease,
hypercalcemia or other indications for which the unmodified protein
has been shown to provide therapeutic benefit. The addition of the
rPEG sequence confers the properties of extended serum half-life,
improved patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0549] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the parathyroid hormone (PTH) gene under
control of appropriate transcription and translation sequences for
high level protein expression in a biological system (e.g.
Escherichia coli, Pichia pastoris, CHO--S, etc). Protein expression
is induced using standard techniques for the expression system
employed and purified using standard procedures (e.g. ion exchange
chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc)
well known to those skilled in the art. The purified protein can
then be administered to human patients for therapeutic treatment of
the following indications: osteoporosis, or other indications for
which the unmodified protein has been shown to provide therapeutic
benefit. The addition of the rPEG sequence confers the properties
of extended serum half-life, improved patient exposure/efficacy,
and/or improved manufacturing efficiency.
[0550] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the human chorionic gonadotropin (hCG) gene
under control of appropriate transcription and translation
sequences for high level protein expression in a biological system
(e.g. Escherichia coli, Pichia pastoris, CHO--S, etc). Protein
expression is induced using standard techniques for the expression
system employed and purified using standard procedures (e.g. ion
exchange chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc)
well known to those skilled in the art. The purified protein can
then be administered to human patients for therapeutic treatment of
indications including, but not limited to: infertility, Kaposi's
sarcoma, asthma, artheriopathy, thalassemia, osteopenia, glaucoma,
obesity, or other indications for which the unmodified protein has
been shown to provide therapeutic benefit. The addition of the rPEG
sequence confers the properties of extended serum half-life,
improved patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0551] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the Fuzeon (enfurvitide) gene under control
of appropriate transcription and translation sequences for high
level protein expression in a biological system (e.g. Escherichia
coli, Pichia pastoris, CHO--S, etc). Protein expression is induced
using standard techniques for the expression system employed and
purified using standard procedures (e.g. ion exchange
chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc)
well known to those skilled in the art. The purified protein can
then be administered to human patients for therapeutic treatment of
the following indications: HIV-1 infection, or other indications
for which the unmodified protein has been shown to provide
therapeutic benefit. The addition of the rPEG sequence confers the
properties of extended serum half-life, improved patient
exposure/efficacy, and/or improved manufacturing efficiency.
[0552] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the leptin or leptin receptor gene under
control of appropriate transcription and translation sequences for
high level protein expression in a biological system (e.g.
Escherichia coli, Pichia pastoris, CHO--S, etc). Protein expression
is induced using standard techniques for the expression system
employed and purified using standard procedures (e.g. ion exchange
chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc)
well known to those skilled in the art. The purified protein can
then be administered to human patients for therapeutic treatment of
indications including, but not limited to: breast cancer,
osteoarthritis, osteoporosis, septic arthritis, obesity, or other
indications for which the unmodified protein has been shown to
provide therapeutic benefit. The addition of the rPEG sequence
confers the properties of extended serum half-life, improved
patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0553] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the TNF Binding protein 1 (TNF-BP1; p55)
gene under control of appropriate transcription and translation
sequences for high level protein expression in a biological system
(e.g. Escherichia coli, Pichia pastoris, CHO--S, etc). Protein
expression is induced using standard techniques for the expression
system employed and purified using standard procedures (e.g. ion
exchange chromatography, size exclusion chromatography, affinity
chromatography, differential precipitation, phase extraction, etc)
well known to those skilled in the art. The purified protein can
then be administered to human patients for therapeutic treatment of
indications including, but not limited to: rheumatoid arthritis,
psoriatic arthritis, psoriasis, inflammatory bowel disease, Crohn's
disease, or other indications for which the unmodified protein has
been shown to provide therapeutic benefit. The addition of the rPEG
sequence confers the properties of extended serum half-life,
improved patient exposure/efficacy, and/or improved manufacturing
efficiency.
[0554] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the glucagon gene under control of
appropriate transcription and translation sequences for high level
protein expression in a biological system (e.g. Escherichia coli,
Pichia pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: type II diabetes, juvenile diabetes, or other
indications for which the unmodified protein has been shown to
provide therapeutic benefit. The addition of the rPEG sequence
confers the properties of extended serum half-life, improved
patient exposure/efficacy, and/or improved manufacturing
efficiency. Due to the sensitivity of the N-terminus of glucagon to
maintaining in vivo efficacy, special considerations may be
required to maintain the native N-terminal structure upon
recombinant expression and purification, and preferred embodiments
would comprise fusions of rPEG to the C-terminus of the GLP-1
sequence. N-terminal leader sequences which can be cleaved by
proteases either in vitro or in vivo can be employed to improve
manufacturing yield and/or improve delivery of active molecules in
vivo.
[0555] In one embodiment, an rPEG sequence is genetically fused to
the N- or C-terminus of the IGF-1 gene under control of appropriate
transcription and translation sequences for high level protein
expression in a biological system (e.g. Escherichia coli, Pichia
pastoris, CHO--S, etc). Protein expression is induced using
standard techniques for the expression system employed and purified
using standard procedures (e.g. ion exchange chromatography, size
exclusion chromatography, affinity chromatography, differential
precipitation, phase extraction, etc) well known to those skilled
in the art. The purified protein can then be administered to human
patients for therapeutic treatment of indications including, but
not limited to: IGF-1 deficiency, hGH deficiency caused by gene
deletion or anti-GH antibody formation, or other indications for
which the unmodified protein has been shown to provide therapeutic
benefit. The addition of the rPEG sequence confers the properties
of extended serum half-life, improved patient exposure/efficacy,
and/or improved manufacturing efficiency.
[0556] Depot Modules
[0557] The compositions of the present invention may optionally
include a depot module. The depot module may be a naturally
occurring polypeptide, an artificial polypeptide or one selected by
phage display. In one embodiment, the depot module will bind
directly to the polymeric matrix referred to below. The depot
module can be incorporated at any position within the modified
polypeptide and can be present once or in multiple copies as
indicated in FIGS. 2 and 3.
[0558] The depot module can be attached to the modified polypeptide
in a variety of ways. For example, in one embodiment (FIG. 4), the
modified polypeptide comprises repeating units as follows:
accessory polypeptide-biologically active polypeptide-depot module,
biologically active polypeptide-accessory polypeptide-depot module,
depot module-accessory polypeptide-biologically active polypeptide,
or depot module-biologically active polypeptide-accessory
polypeptide.
[0559] In another aspect of the invention, the depot module
comprises a polypeptide that is specifically sensitive to serum
proteases (FIG. 8). Protease cleavage of the depot module releases
biologically active polypeptide. The protease sites can be
engineered to be sensitive to specific proteases, such as to a
serum protease, or to display different rates of protease cleavage.
Thus the rate or site of release can be controlled through
engineering of the protease cleavage site of the depot module. The
modified polypeptide so engineered can be formulated with a
polymeric matrix as described herein.
[0560] The depot module can also include the use of a "hot
cysteine" to ensure site-specific modification. A "hot cysteine" is
flanked by lysine residues, for example (KCKK) (SEQ ID NO: 446),
where K is lysine and C is cysteine. The proximal lysine residues
shift the pKa of the cysteine, increasing its nucleophilicity and
making this residue more reactive. Several groups have shown that a
"hot cysteine" can be preferentially modified (greater than 90%)
even in the background of 23 other cysteine residues present on the
same protein [Okten, Z., et al. (2004) Nat Struct Mol Biol,
11:884-7]. Thus, the depot module can yield site-specific,
efficient modification of the accessory polypeptide or the
accessory polypeptide-biologically active polypeptide fusion in
vitro. Biotin conjugated to either of these reactive groups is
commercially available.
[0561] In yet a further aspect of the invention, the depot module
is designed to provide a tetravalent accessory protein-biologically
active polypeptide fusion protein, for example, to increase target
avidity and/or for slow release applications. The depot module is
designed to contain an amino acid or amino acids for the
site-specific conjugation of the small molecule biotin. Biotin is a
common vitamin found in over-the-counter nutritional supplements.
It serves as a "co-factor" for several enzymes including those
involved in the biosynthesis of fatty acids. Biotin is also
extensively used in biotechnology applications because it forms a
very high affinity complex with the proteins avidin, neutravidin,
and streptavidin. In this embodiment, avidin, streptavidin, or
neutravidin, which each bind to four molecules of biotin, can be
used to form highly stable accessory polypeptide-biologically
active polypeptide fusion protein tetramers (FIG. 5).
[0562] Lysine (K) and cysteine (C) residues can be modified by
chemical reaction with succidimidyl esters or maleimides,
respectively, under mild conditions with high yield and
specificity. When the accessory polypeptide does not contain any
lysine (K) or cysteine (C) residues, these can be easily
incorporated into the depot module. The depot module can comprise
one, two, or more lysine or cysteine residues.
[0563] The depot module can also include the use of a "hot
cysteine" to ensure site-specific modification. A "hot cysteine" is
flanked by lysine residues, for example (KCKK), where K is lysine
and C is cysteine. The proximal lysine residues shift the pKa of
the cysteine, increasing its nucleophilicity and making this
residue more reactive. Several groups have shown that a "hot
cysteine" can be preferentially modified (greater than 90%) even in
the background of 23 other cysteine residues present on the same
protein [Olden, Z., et al. (2004) Nat Struct Mol Biol, 11:884-7].
Thus, the depot module can yield site-specific, efficient
modification of the accessory polypeptide or the accessory
polypeptide-biologically active polypeptide fusion in vitro. Biotin
conjugated to either of these reactive groups is commercially
available.
[0564] The addition of biotin-binding proteins such as avidin,
streptavidin, or neutravidin can induce the formation of a very
stable accessory polypeptide-binding protein polypeptide tetramer.
The accessory polypeptide-binding protein polypeptide tetramer can
then be formulated with polymeric matrix (e.g., encapsulated into
microspheres) as described below. An accessory polypeptide-binding
protein polypeptide tetramer exhibits a very large hydrodynamic
radius, ensuring slow release from the polymeric matrix, e.g.,
microspheres. An accessory polypeptide-binding protein polypeptide
tetramer will also have an increased avidity towards its biological
target. Because the accessory polypeptide-binding protein
polypeptide tetramer can interact with four target molecules, for
example on the plasma membrane of a cell, the off-rate of the
accessory polypeptide-binding protein polypeptide will be
dramatically reduced. Increased avidity may enhance the biological
activity or reduce the required dose of the accessory
polypeptide-binding protein polypeptide.
[0565] In a further aspect of the invention, the depot module with
the same active residues can be modified with poly-ethylene glycol
instead of the reactive biotin. Of particular interest are four-
and eight-armed PEG molecules. These PEG molecules can be
covalently attached to depot module described herein, thus
generating homogeneous tetramer and octamer species. Protein
therapeutics conjugated in this manner will have a significantly
enhanced avidity towards their biological targets, particularly
toward cell surface proteins.
[0566] Counterions for Making Protein Precipitate
[0567] The present invention also relates to the use of counterions
for regulating the solubility of the protein of interest, i.e.
making protein precipitate for a depot formulation. A counterion is
an ion, the presence of which allows the formation of an overall
neutrally charged species. For example, in the (neutral) species
NaCl the sodium cation is countered by the chloride anion and vice
versa. The mechanism of poorly water-soluble salt formation with a
cation exchanger is depicted by the following formula:
rPEG.sup.n+nC.sup.-.fwdarw.rPEG.C.sub.n (insoluble) in which
rPEG.sup.n+ represents the positively charged peptide ion, whereas
C.sup.- represents a negatively charged counterion. The
participating amino acid residues in this reaction include Arg, Lys
and the N-terminus. The mechanism of poorly water-soluble salt
formation with an anion exchanger is depicted by the following
formula: rPEG.sup.n-+nC.sup.+.fwdarw.rPEG.C.sub.n (insoluble) in
which rPEG represents the negatively charged peptide ion, whereas C
represents a positively charged counterion. The participating amino
acid residues in this reaction include Asp, Glu and the
C-terminus.
[0568] In a preferred embodiment, the counterion displays mixed
hydrophobic and ionic character. Thus, once the charge of the
counterion is neutralized by complex formation with the protein of
interest, the hydrophobic nature of the counterion dominates the
resultant complex, causing its aqueous solubility to decrease
significantly. In addition, the counterions must be compatible with
in vivo administration within the clinical indication intended for
the protein of interest in terms of acute and chronic toxicity,
carcinogenicity, reproductive effects, etc. Non-limiting examples
of mixed counterions suitable for this application are provided
below:
[0569] Anions: [0570] Behenate [0571] Cholesteryl sulfate [0572]
Deoxycholate [0573] Dodecane sulfonate [0574] Epigallocatechin
gallate [0575] Hexadecane sulfonate [0576] Pamoate [0577]
Pentagalloyl Glucose [0578] Stearate [0579] Tannate
[0580] Cations: [0581] Choline derivatives [0582] Peptide
counterions: eg H-Lys-(Leu).sub.n-NH2; H-(Leu)-NH2
[0583] Lipids: [0584] Phosphatidylcholine
[0585] Polymeric Materials: [0586] Chitosan [0587] Collagen [0588]
Hyaluronic Acid [0589] Poly .beta.-amino esters [0590] PLA/PLGA
[0591] Poly(ethylene glycol)bis(2-aminoethyl)
[0592] In one embodiment, a protein of interest is mixed at a
defined ratio with a counterion comprising both hydrophobic and
charged character as described above. Upon interaction, the protein
and counterion form an insoluble complex which precipitates from
the solution. In a preferred embodiment, greater than or equal to
20%, 40%, 60%, or 80% of the total protein is precipitated under
these conditions, which can be assessed by quantitative assay of
the protein remaining in solution. Optimization of the
protein:counterion ratio, inclusion of organic solvents, pH
adjustment, ionic strength, and/or temperature adjustment may be
employed to modulate the efficiency of the precipitation reaction.
The precipitate can be separated from the liquid phase using
standard methods (i.e. filtration, centrifugation), and can be
stored in a dry form or as a suspension in an inert buffer. For a
pharmaceutical composition, protein stability upon storage is a
critical parameter for determining the viability of a given
formulation. In one embodiment, the protein is stable under the
defined storage conditions and formulation for greater than 1, 2,
3, 6, 12, 18, or 24 months.
[0593] The present invention also embodies the method of
administering the above described protein complex into a subject in
vivo. Compounds of the invention may be administered as
pharmaceutical formulations including those suitable for oral
(including buccal and sub-lingual), rectal, nasal, topical,
transdermal patch, pulmonary, vaginal, suppository, or parenteral
(including intramuscular, intraarterial, intrathecal, intradermal,
intraperitoneal, subcutaneous and intravenous) administration or in
a form suitable for administration by aerosolization, inhalation or
insufflation. In a preferred embodiment, the protein complext is
administered to a subject via parenteral injection. As used herein,
the term "parenteral" refers to introduction of the complex into
the body not through the intestines, but rather by injection
through intravenous (i.v.), intraarterial (i.a.), intraperitoneal
(i.p.), intramuscular (i.m.), intraventricular, intrabronchial, and
subcutaneous (s.c.) routes. To be administered via parenteral
injection (e.g. bolus injection or continuous infusion), the
precipitate is resuspended in a buffer compatible with the route of
administration. In the preferred embodiment, the precipitate is
resuspended as a homogeneous suspension capable of passing through
a 18, 22, 25, 26, 27, or 28 gauge needle with minimal occlusion.
Milling or similar processing can be performed in order to improve
the resuspension properties as well as reducing the size of the
particles to enable efficient passage through higher gauge needles.
Detergents or other excipients capable of modifying the surface
tension, viscosity, or wetting properties of the solution can also
be useful in improving the homogeneity of the precipitate
suspension for injection.
[0594] The present invention also relates to the protein release
rate in a depot formulation upon introduction of the precipitate
into an in vivo environment via, for example, parenteral injection.
The protein release rate can be approximated in vitro by suspension
of the protein:counterion precipitate in an isotonic buffer (e.g.
phosphate buffered saline) and measuring the concentration of
soluble protein over time. A preferred embodiment uses
physiological temperatures in order to better mimic the in vivo
conditions, although a higher or lower temperature may be employed
to modify the resolubilization rate depending on the experimental
setup. The optimal release rate for a given protein is dependent
upon its in vivo clearance rate and mechanism, as well as the
required exposure for in vivo efficacy. In order to achieve
significant accumulation of the protein, the resolubilization rate
should be faster than the natural clearance rate. Serum
concentration of the protein is expected to be proportional to the
ratio of the resolubilization rate to the clearance rate. The
kinetics of the protein complex between its soluble and
precipitates states is depicted in the following equation:
##STR00001##
Assuming that the rate of reprecipitation into the complex is
negligible: [Protein.sup.n-] is approximately equal to
k.sub.solubilization/k.sub.clearance (i.e. the ratio of the
resolubilization rate to the clearance rate).
[0595] The actual serum concentration achieved in vivo is also
dependent upon a number of other factors including total amount of
complex injected, surface area of the precipitate particles,
protein absorption rate, binding of the protein to its cognate
receptors, and recycling mechanisms.
[0596] The resolubilization properties of the precipitate may be
modified by various treatments of the precipitate. For example,
heat treatment or ultraviolet crosslinking of the counterion can be
used to modify the chemical (and resolubilization) properties of
the precipitate. The precipitate may also be formed by direct
removal of solvent (e.g. spray drying, lyophilization), followed by
treatment with a counterion or coating material to achieve the
desired depot characteristics.
[0597] Excipients may be included in the complex formation reaction
to control the rate and efficiency of complex formation, as well as
to modulate the rate of resolubilization of the protein:counterion
complex upon transfer to an in vivo environment. Excipients are
typically uncharged, inert molecules which are included in the
complex formation reaction buffer and comprise a varying degree of
the final precipitate mass. Excipients may also comprise a coating
applied to the surface of the precipitate particle which serves to
modulate the surface area of the precipitate particle to solvent
and hence modulate the resolubilization rate and/or stability of
the protein. Examples of excipients include, but are not limited
to, the following:
[0598] Polymers [0599] Polyethylene glycol 500 [0600] Polyethylene
glycol 2000 [0601] Polyethylene glycol 5000 [0602] Polyethylene
glycol 8000 [0603] Polyethylene glycol 20000 [0604] Polylysine
[0605] PLA/PLGA
[0606] Detergents [0607] Polysorbate 20 (Tween 20) [0608]
Polysorbate 80 (Tween 80) [0609] Triton X-100
[0610] Sugars/Polyalcohols [0611] Glucose [0612] Glycerine [0613]
Glycerol [0614] Mannitol [0615] Mannose [0616] Sorbitol [0617]
Sucrose [0618] Trehalose
[0619] Another embodiment of the present invention includes a
formulation such that depot formation occurs in situ upon
injection. For example, the protein and counterion are chosen such
that a precipitate is formed close to physiological pH (i.e. pH
7.4). The protein and counterion are formulated at an optimal
concentration ratio relative to one another, but at a pH
sufficiently different from physiological pH (e.g. pH 4 or pH 10)
such that no complex formation occurs. Upon parenteral injection,
preferably subcutaneous or intramuscular injection, the inherent
buffering capacity of the tissue causes the solution to adjust to
pH 7.4, resulting in the precipitation of the protein:counterion
complex at the site of injection and the resultant slow release
thereof. Temperature change upon injection and complex formation of
the injected protein with a natural counterion found in vivo are
also methods by which a slow releasing protein depot may be formed
in situ.
[0620] Production of Accessory-Linked Polypeptides
[0621] The present invention provides methods of producing
biologically active polypeptide, comprising a) providing a
polynucleotide sequence coding for a modified polypeptide
comprising the biologically active polypeptide linked with an
accessory polypeptide such that expression of the modified
polypeptide in a host cell yields a higher quantity of soluble form
of biologically active polypeptide as compared to expression of the
biologically active polypeptide by itself; and b) causing the
modified polypeptide to be expressed in said host cell, thereby
producing the biologically active polypeptide. Expression of the
modified biologically active polypeptides may yield at least about
100%, 200%, 500% or 1000% more soluble form of biologically active
polypeptide as compared to expression of the biologically active
polypeptide by itself. In some embodiments, the expression of the
modified biologically active polypeptides may yield at least
between 100%, and 1000% more soluble form of biologically active
polypeptide as compared to expression of the biologically active
polypeptide by itself.
[0622] Methods of the invention may involve culturing a cell
transformed with a chimeric DNA molecule encoding an accessory
polypeptide under conditions whereby the DNA is expressed, thereby
producing the accessory-linked polypeptide; and extracting an
expression product of the chimeric DNA molecule from the cell or
culture medium.
[0623] Standard recombinant techniques in molecular biology can be
used to make the accessory-linked polypeptides of the present
invention. In one embodiment, a construct is first prepared
containing the DNA sequence corresponding to the accessory
polypeptide. For example, a gene or polynucleotide encoding the
biologically active protein can be first cloned into a construct,
which can be a plasmid or other vector. In a later step, a second
gene or polynucleotide coding for the accessory polypeptide is
cloned into the construct adjacent and in frame with the gene
coding for the biologically active polypeptide. This second step
can occur through a ligation or multimerization step.
[0624] In this manner, a chimeric DNA molecule coding for a
modified polypeptide is generated within the construct. Optionally,
this chimeric DNA molecule may be transferred or cloned into
another construct that is a more appropriate expression vector. At
this point, a host cell capable of expressing the chimeric DNA
molecule is transformed with the chimeric DNA molecule. The
transformation may occur with or without the utilization of a
carrier, such as an expression vector. Then, the transformed host
cell is cultured under conditions suitable for expression of the
chimeric DNA molecule, resulting in the encoding of the accessory
polypeptide. Methods of ligation or multimerization useful in the
present invention are well known. See, Joseph Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, 2nd ed., 1.53 (Cold Spring
Harbor Laboratory Press 1989).
[0625] Several cloning strategies are envisioned to be suitable for
performing the present invention, many of which can be used to
generate a construct that comprises a gene coding for the accessory
polypeptide of the present invention.
[0626] The vectors containing the DNA segments of interest can be
transferred into the host cell by well-known methods, depending on
the type of cellular host. For example, calcium chloride
transfection is commonly utilized for prokaryotic cells, whereas
calcium phosphate treatment, lipofection, or electroporation may be
used for other cellular hosts. Other methods used to transform
mammalian cells include the use of polybrene, protoplast fusion,
liposomes, electroporation, and microinjection (see, generally,
Sambrook et al., supra). Prokaryotic or eukaryotic cells are
envisioned as hosts. Accessory polypeptides can be produced in a
variety of expression systems including prokaryotic and eukaryotic
systems. Suitable expression hosts are for instance yeast, fungi,
mammalian cell culture, and insect cells.
[0627] Useful expression vectors that can be used include, for
example, segments of chromosomal, non-chromosomal and synthetic DNA
sequences. Suitable vectors include, but are not limited to,
derivatives of SV40 and pcDNA and known bacterial plasmids such as
col EI, pCR1, pBR322, pMal-C2, pET, pGEX as described by Smith, et
al., Gene 57:31-40 (1988), pMB9 and derivatives thereof, plasmids
such as RP4, phage DNAs such as the numerous derivatives of phage I
such as NM98 9, as well as other phage DNA such as M13 and
filamentous single stranded phage DNA; yeast plasmids such as the 2
micron plasmid or derivatives of the 2 m plasmid, as well as
centromeric and integrative yeast shuttle vectors; vectors useful
in eukaryotic cells such as vectors useful in insect or mammalian
cells; vectors derived from combinations of plasmids and phage
DNAs, such as plasmids that have been modified to employ phage DNA
or the expression control sequences; and the like. The requirements
are that the vectors are replicable and viable in the host cell of
choice. Low- or high-copy number vectors may be used as
desired.
[0628] For example in a baculovirus expression system, both
non-fusion transfer vectors, such as, but not limited to pVL941
(BamHI cloning site, available from Summers, et al., Virology
84:390-402 (1978)), pVL1393 (BamHI, SmaI, XbaI, EcoRI, IVotI,
XmaIII, BglII and PstI cloning sites; Invitrogen), pVL1392 (BglII,
PstI, NotI, XmaIII, EcoRI, XbaII, SmaI and BamHI cloning site;
Summers, et al., Virology 84:390-402 (1978) and Invitrogen) and
pBlueBacIII (BamHI, BglII, PstI, NcoI and HindIII cloning site,
with blue/white recombinant screening, Invitrogen), and fusion
transfer vectors such as, but not limited to, pAc7 00 (BamHI and
KpnI cloning sites, in which the BamHI recognition site begins with
the initiation codon; Summers, et al., Virology 84:390-402 (1978)),
pAc701 and pAc70-2 (same as pAc700, with different reading frames),
pAc360 [BamHI cloning site 36 base pairs downstream of a polyhedrin
initiation codon; Invitrogen (1995)) and pBlueBacHisA, B, C (three
different reading frames with BamHI, BglII, PstI, NcoI and HindIII
cloning site, an Nterminal peptide for ProBond purification and
blue/white recombinant screening of plaques; Invitrogen (220) can
be used.
[0629] Mammalian expression vectors can comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements. Mammalian
expression vectors contemplated for use in the invention include
vectors with inducible promoters, such as the dihydrofolate
reductase promoters, any expression vector with a DHFR expression
cassette or a DHFR/methotrexate co-amplification vector such as pED
(PstI, SaiI, SbaI, SmaI and EcoRI cloning sites, with the vector
expressing both the cloned gene and DHFR; Randal J. Kaufman, 1991,
Randal J. Kaufman, Current Protocols in Molecular Biology, 16,12
(1991)). Alternatively a glutamine synthetase/methionine
sulfoximine co-amplification vector, such as pEE14 (HindIII, XbaII,
SmaI, SbaI, EcoRI and SelI cloning sites in which the vector
expresses glutamine synthetase and the cloned gene; Celltech). A
vector that directs episomal expression under the control of the
Epstein Barr Virus (EBV) or nuclear antigen (EBNA) can be used such
as pREP4 (BamHI r SfH, XhoI, NotI, NheI, HindIII, NheI, PvuII and
KpnI cloning sites, constitutive RSV-LTR promoter, hygromycin
selectable marker; Invitrogen), pCEP4 (BamHI, SfH, XhoI, NotI,
NheI, HindIII, NheI, PvuII and KpnI cloning sites, constitutive
hCMV immediate early gene promoter, hygromycin selectable marker;
Invitrogen), pMEP4 (.KpnI, PvuI, NheI, HindIII, NotI, XhoI, SfiI,
BamHI cloning sites, inducible metallothionein H a gene promoter,
hygromycin selectable marker, Invitrogen), pREP8 (BamHI, XhoI,
NotI, HindIII, NheI and KpnI cloning sites, RSV-LTR promoter,
histidinol selectable marker; Invitrogen), pREP9 (KpnI, NheI,
HindIII, NotI, XhoI, SfiI, BamHI cloning sites, RSV-LTR promoter,
G418 selectable marker; Invitrogen), and pEBVHis (RSV-LTR promoter,
hygromycin selectable marker, N-terminal peptide purifiable via
ProBond resin and cleaved by enterokinase; Invitrogen).
[0630] Selectable mammalian expression vectors for use in the
invention include, but are not limited to, pRC/CMV (HindIII, BstXI,
NotI, SbaI and ApaI cloning sites, G418 selection, Invitrogen),
pRc/RSV (HindIII, SpeI, BstXI, NotI, XbaI cloning sites, G418
selection, Invitrogen) and the like. Vaccinia virus mammalian
expression vectors (see, for example, Randall J. Kaufman, Current
Protocols in Molecular Biology 16.12 (Frederick M. Ausubel, et al.,
eds. Wiley 1991) that can be used in the present invention include,
but are not limited to, pSC11 (SmaI cloning site, TK- and beta-gal
selection), pMJ601 (SalI, SmaI, AflI, NarI, BspMII, BamHI, ApaI,
NheI, SacII, KpnI and HindIII cloning sites; TK- and -gal
selection), pTKgptF1S (EcoRI, PstI, SaIII, AccI, HindII, SbaI,
BamHI and Hpa cloning sites, TK or XPRT selection) and the
like.
[0631] Yeast expression systems that can also b e used in the
present include, but are not limited to, the non-fusion pYES2
vector (XJbal, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamHI, Sad,
KpnI and HindIII cloning sites, Invitrogen), the fusion pYESHisA,
B, C (XbaII, SphI, ShoI, NotI, BstXI, EcoRI, BamHI, Sad, KpnI and
HindIII cloning sites, N-terminal peptide purified with ProBond
resin and cleaved with enterokinase; Invitrogen), pRS vectors and
the like.
[0632] In addition, the expression vector containing the chimeric
DNA molecule may include drug selection markers. Such markers aid
in cloning and in the selection or identification of vectors
containing chimeric DNA molecules. For example, genes that confer
resistance to neomycin, puromycin, hygromycin, dihydrofolate
reductase (DHFR), guanine phosphoribosyl transferase (GPT), zeocin,
and histidinol are useful selectable markers. Alternatively,
enzymes such as herpes simplex virus thymidine kinase (tk) or
chloramphenicol acetyltransferase (CAT) may be employed.
Immunologic markers also can be employed. Any known selectable
marker may be employed so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable markers are well known to one of
skill in the art and include reporters such as enhanced green
fluorescent protein (EGFP), beta-galactosidase (.beta.-gal) or
chloramphenicol acetyltransferase (CAT).
[0633] Consequently, mammalian and typically human cells, as well
as bacterial, yeast, fungi, insect, nematode and plant cells can
used in the present invention as host cells and may be transformed
by the expression vector as defined herein.
[0634] Examples of suitable cells include, but are not limited to,
VERO cells, HELA cells such as ATCC No. CCL2, CHO cell lines, COS
cells, WI38 cells, BHK cells, HepG2 cells, 3T3 cells, A549 cells,
PC12 cells, K562 cells, 293 cells, Sf9 cells and CvI cells.
[0635] Other suitable cells that can be used in the present
invention include, but are not limited to, prokaryotic host cells
strains such as Escherichia coli, (e.g., strain DH5-.alpha.),
Bacillus subtilis, Salmonella typhimurium, or strains of the genera
of Pseudomonas, Streptomyces and Staphylococcus. Non-limiting
examples of suitable prokaryotes include those from the genera:
Actinoplanes; Archaeoglobus; Bdellovibrio; Borrelia; Chloroflexus;
Enterococcus; Escherichia; Lactobacillus; Listeria; Oceanobacillus;
Paracoccus; Pseudomonas; Staphylococcus; Streptococcus;
Streptomyces; Thermoplasma; and Vibrio. Non-limiting examples of
specific strains include: Archaeoglobus fulgidus; Bdellovibrio
bacteriovorus; Borrelia burgdorferi; Chloroflexus aurantiacus;
Enterococcus faecalis; Enterococcus faecium; Lactobacillus
johnsonii; Lactobacillus plantarum; Lactococcus lactis; Listeria
innocua; Listeria monocytogenes; Oceanobacillus iheyensis;
Paracoccus zeaxanthinifaciens; Pseudomonas mevalonii;
Staphylococcus aureus; Staphylococcus epidennidis; Staphylococcus
haemolyticus; Streptococcus agalactiae; Streptomyces
griseolosporeus; Streptococcus mutans; Streptococcus pneumoniae;
Streptococcus pyogenes; Thermoplasma acidophilum; Thermoplasma
volcanium; Vibrio cholerae; Vibrio parahaemolyticus; and Vibrio
vulnificus.
[0636] Further suitable cells that can be used in the present
invention include yeast cells such as those of Saccharomyces such
as Saccharomyces cerevisiae.
[0637] A key advantage of using bacterial expression to perform the
present invention is the absence of glycosylation. While
glycosylation of the accessory polypeptide increases its molecular
weight and generally increases its serum half-life, quality control
of glycosylated products is notoriously difficult to perform When
many glycosylation sites are present and the expression level of
the protein is high, the glycosylation machinery may not be able to
keep up and glycosylation is likely to be incomplete due to
incomplete processing, resulting in carbohydrate structures that
are heterogeneous, which greatly complicates purification,
characterization, quality control and reproducibility.
[0638] Depending on how the protein is expressed in bacteria
(secreted to media, to periplasm, soluble in cytoplasm or as
insoluble inclusion bodies in the cytoplasm), the product or
intermediate may contain a formylated N-terminus.
[0639] Additional post-translational modifications to which
accessory polypeptides or the accessory-modified polypeptides of
the invention may be subjected to include, but are not limited to
acylation, acetylation, alkylation, demethylation, amidation,
biotinylation, formylation, gamma-carboxylation, glutamylation,
glycosylation, glycylation, attachment of heme moiety,
hydroxylation, iodination, isoprenylation, lipoylation,
prenylation, myristoylation, farnesylation, geranylgeranylation,
ADP-ribosylation, flavin attachment, oxidation, pegylation,
attachment of phosphatidylinositol, phosphopantetheinylation,
phosphorylation, pyroglutamate formation, racemization of proline
by prolyl isomerase, tRNA-mediation addition of amino acids such as
arginylation, sulfation and selenoylation.
[0640] Host cells containing the polynucleotides of interest can be
cultured in conventional nutrient media (e.g., Ham's nutrient
mixture) modified as appropriate for activating promoters,
selecting transformants or amplifying genes. 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. Cells are
typically harvested by centrifugation, disrupted by physical or
chemical means, and the resulting crude extract retained for
further purification. Microbial cells employed in expression of
proteins can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents, all of which are well known to those skilled in
the art. Embodiments that involve cell lysis may entail use of a
buffer that contains protease inhibitors that limit degradation
after expression of the chimeric DNA molecule. Suitable protease
inhibitors include leupeptin, pepstatin or aprotinin. The
supernatant then may be precipitated in successively increasing
concentrations of saturated ammonium sulfate.
[0641] The accessory polypeptides product may be purified via
methods known to one skilled in the art. Procedures such as gel
filtration, affinity purification, salt fractionation, ion exchange
chromatography, size exclusion chromatography, hydroxylapatite
adsorption chromatography, hydrophobic interaction chromatography
and gel electrophoresis may be used. Some accessory polypeptides
may require refolding. Methods of purification are described in
Robert K. Scopes, Protein Purification: Principles and Practice
(Charles R. Castor, ed., Springer-Verlag 1994) and Joseph Sambrook,
Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring
Harbor Laboratory Press 1989). Multi-step purification separations
are also described in Baron, et al., Crit. Rev. Biotechnol.
10:179-90 (1990) and Below, et al., J. Chromatogr. A. 679:67-83
(1994).
[0642] Production of Crosslinked Accessory Polypeptides
[0643] Crosslinked accessory polypeptides can be produced by a
variety of methods. Both the non-cross-linking and the
cross-linking components can be generated by chemical synthesis or
using recombinant techniques. Of particular utility is the
recombinant manufacture of the non-cross-linking component, which
can be achieved in a variety of microbial as well as eukaryotic
expression systems, for example as described above. The
non-cross-linking component can be purified to remove interfering
or contaminating by-products prior to cross linking. Of particular
utility are chemical crosslinkers that can be activated for
coupling. Examples are shown in FIG. 22. The resulting coupling
products can be further purified by a variety of methods, in
particular size exclusion chromatography and ion exchange
chromatography.
[0644] Multiple different non-crosslinking components can be
conjugated to a crosslinking component using methods that allow the
control of product structure. For instance one can use
cross-linking components that carry several different reactive
groups that allow different conjugation chemistries. Alternatively,
one can use crosslinking components that carry protecting groups on
some of their reactive groups. Such partially-protected
crosslinking components can be coupled to one or more
non-crosslinking components. Subsequently, one can remove the
protecting groups from the crosslinking components and conjugate
additional non-crosslinking components to the crosslinking
component. This process can be repeated by using multiple different
protecting groups that allow selective removal.
[0645] In another embodiment of the present invention, a
recombinant cross-linking component may be used. The cross-linking
component can be amino acids sequences that can be manufactured by
recombinant technology using a variety of expression systems. For
example, fMet amino acids incorporated in the sequence of a
noncross-linking component may be conjugated to amino groups in a
recombinant cross-linking component.
[0646] One preferred embodiment provides for cross-linking
components that comprise one or more glutamate and/or aspartate
residues, which contain side chains that can serve as reactive
groups and can be effectively conjugated to non-cross-linking
components that have a free amino group as reactive group. A
variety of carbodiimides can be used to activate free carboxyl
groups but many more chemistries are suitable. Free amino groups in
the recombinant cross-linking component may be blocked by
acetylation or succinylation.
[0647] Alternatively, the cross-linking component can be a protein
that has multiple high-affinity binding sites. Examples are avidin,
streptavidin, IgGs or IgMs. For instance one can form Crosslinked
accessory polypeptides by contacting biotinylated non-cross-linking
components with streptavidin, which will lead to the formation of a
tetravalent complex. The process is illustrated in FIG. 25. In a
similar way one could use for instance an IgM or IgG with
specificity for a peptide epitope in conjunction with
non-cross-linking components that comprise said peptide
epitope.
[0648] The accessory polypeptides of the present invention may be
assayed in order to determine the effect of which to a biologically
active polypeptide. Methods of assaying biologically active
polypeptides are commonly known in the art. For example, serum
half-life can be measured by combining the protein with human (or
mouse, rat, monkey, as appropriate) serum or plasma, typically for
a range of days (ie 0.25, 0.5, 1, 2, 4, 8, 16 days) at 37.degree.
C. The samples for these timepoints can then be run on a Western
assay and the protein is detected with an antibody. The antibody
can be to a tag in the protein. If the protein shows a single band
on the western, where the protein's size is identical to that of
the injected protein, then no degradation has occurred. The
timepoint where 50% of the protein is degraded, as judged by
Western Blots or equivalent techniques, is determined to be the
serum degradation half-life or "serum half-life" of the
protein.
[0649] The accessory polypeptides of the present invention may be
used to modulate the expression or activity of a variety of
cellular targets, including without limitation those named in the
section "Biologically active polypeptides". In some embodiments,
the expression of a target will be reduced by administration of
accessory polypeptides, while in other embodiments it will be
increased. The accessory polypeptide may interfere with the
activity of a cellular target by interaction with functional sites
on the target.
[0650] Slow Release Agents
[0651] The modified polypeptides of the invention may be
incorporated, encapsulated, formulated or otherwise included into
compositions which allow for controlled release of the polypeptides
in desired applications. Generally, the modified polypeptides of
the invention may interact with the slow release agents of the
invention in various manners, including and not limited to covalent
attachment, ionic interaction, or encapsulation within a polymer or
a formulation.
[0652] Various types of slow release agents suitable for use in the
present invention are described below.
[0653] Polymer Matrices
[0654] In general, microspheres are substantially spherical
colloidal structures having a size ranging from about one or
greater up to about 1000 microns. Microcapsules are generically
described as structures in which a substance, such as a polymeric
formulation, is covered by a coating of some type. The term
"microparticle" may be used to describe structures that may not be
readily placed into either of the above two categories or as a
generic term for both. For structures that are less than about one
micron in diameter the corresponding terms "nanosphere,"
"nanocapsule," and "nanoparticle" may be utilized, but these are
encompassed in the terms "microsphere," microcapsule" and
"microparticle," respectively. In certain embodiments, nanospheres,
nanocapsules or nanoparticles have a size of about 500, 200, 100,
50 or 10 nm.
[0655] The slow release formulations of the invention may also take
the form of microparticles, which may comprise microcapsules or
microspheres.
[0656] In a microparticle, the modified polypeptides may be
centrally located within a membrane formed by the polymer
molecules, or can be dispersed throughout the microparticle. The
internal structure may comprise a matrix of the modified
polypeptide and a polymer excipient. Typically, the outer surface
of the microsphere is permeable to water, which allows aqueous
fluids to enter the microsphere, as well as solubilized modified
polypeptide and polymer to exit the microsphere. In one embodiment,
the polymer membrane comprises a crosslinked polymer. The modified
polypeptide may be released by diffusion and/or by degradation of
the polymer membrane.
[0657] Possible materials for the outer layer of microparticles
include the following categories of polymers: (1)
carbohydrate-based polymers, such as methylcellulose, carboxymethyl
cellulose-based polymers, dextran, polydextrose, chitins, chitosan,
and starch (including hetastarch), and derivatives thereof (2)
polyaliphatic alcohols such as polyethylene oxide and derivatives
thereof including polyethylene glycol (PEG), PEG-acrylates,
polyethyleneimine, polyvinyl acetate, and derivatives thereof (3)
poly(vinyl) polymers such as poly(vinyl) alcohol,
poly(vinyl)pyrrolidone, poly(vinyl)phosphate, poly(vinyl)phosphonic
acid, and derivatives thereof (4) polyacrylic acids and derivatives
thereof (5) polyorganic acids, such as polymaleic acid, and
derivatives thereof; (6) polyamino acids, such as polylysine, and
poly-imino acids, such as polyimino tyrosine, and derivatives
thereof (7) co-polymers and block co-polymers, such as poloxamer
407 or Pluronic L-101.TM.. polymer, and derivatives thereof (8)
tert-polymers and derivatives thereof; (9) polyethers, such as
poly(tetramethylene ether glycol), and derivatives thereof; (10)
naturally occurring polymers, such as zein, chitosan and pullulan,
and derivatives thereof; (11) polyimids, such as poly
n-tris(hydroxymethyl) methylmethacrylate, and derivatives thereof;
(12) surfactants, such as polyoxyethylene sorbitan, and derivatives
thereof; (13) polyesters such poly(ethylene glycol) (n) monomethyl
ether mono(succinimidyl succinate)ester, and derivatives thereof;
(14) branched and cyclo-polymers, such as branched PEG and
cyclodextrins, and derivatives thereof and (15) polyaldehydes, such
as poly(perfluoropropylene oxide-b-perfluoroformaldehyde), and
derivatives thereof as disclosed in U.S. Pat. No. 6,268,053, the
contents of which are incorporated herein by reference. Other
typical polymers known to those of ordinary skill in the art
include poly(lactide-co-glycolide, polylactide homopolymer;
polyglycolide homopolymer; polycaprolactone;
polyhydroxybutyrate-polyhydroxyvalerate copolymer;
poly(lactide-co-caprolactone); polyesteramides; polyorthoesters;
poly .beta.-hydroxybutyric acid; and polyanhydrides as disclosed in
U.S. Pat. No. 6,517,859, the contents of which are incorporated
herein by reference. In some embodiments, the polymer may comprise
alginate polymers, (hydroxyethyl)methacrylated dextran polymers, or
chitosan polymers may be used.
[0658] The modified polypeptides of the invention may be mixed with
physiologically acceptable carriers, excipients, or stabilizers
(Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,
1980), in the form of lyophilized cake or aqueous solutions.
Acceptable carriers, excipients, or stabilizers for the preparation
of microparticles are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobins; hydrophilic polymers such as olyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or non-ionic surfactants such as
Tween, Pluronics, or polyethylene glycol (PEG).
[0659] The microspheres of this invention are manufactured by
standard techniques. For example, in one embodiment, volume
exclusion is performed by mixing the active agent in solution with
a polymer or mixture of polymers in solution in the presence of an
energy source for a sufficient amount of time to form particles as
disclosed in U.S. Pat. No. 6,268,053. The pH of the solution is
adjusted to the desired pH. Next, the solution is exposed to an
energy source, such as heat, radiation, or ionization, alone or in
combination with sonication, vortexing, mixing or stirring, to form
microparticles. The resulting microparticles are then separated
from any unincorporated components present in the solution by
physical separation methods well known to those skilled in the art
and may then be washed.
[0660] In some embodiments, a suspension of microparticles is
prepared by vigorously mixing an aqueous solution containing the
modified polypeptide and an organic solution (typically
dichloromethane) in which the polymer is dissolved. This
water-in-oil suspension is then diluted into aqueous buffer
containing an emulgent (typically poly-vinylalcohol). Finally, the
microspheres are removed from this water-in-oil-in-water (W/O/W)
emulsification and freeze-dried. This well known and tested W/O/W
process generally yields microspheres that are 0.1-100 .mu.m in
diameter. Microspheres of these dimensions are readily prepared as
suspensions for subcutaneous injection. Alternatively, microspheres
can be prepared by the single-emulsion solvent
extraction/evaporation (O/W), the solid/oil/oil methods (S/O/O),
and all variants of these methods described in the literature.
[0661] Known manufacturing procedures are also described in U.S.
Pat. Nos. 6,669,961; 6,517,859; 6,458,387; 6,395,302; 6,303,148;
6,268,053; 6,090,925; 6,024,983; 5,942,252; 5,981,719; 5,578,709;
5,554,730; 5,407,609; 4,897,268; and 4,542,025, the contents of
which are incorporated by reference in their entirety. The
preparation and formulation of microparticles is also described in
the following publications: (Bittner, B., et al. (1998) Eur J Pharm
Biopharm, 45:295-305; Rosa, G. D., et al. (2000) J Control Release,
69:283-95; Kissel, T., et al. (2002) Adv Drug Deliv Rev, 54:99-134;
Kwon, Y. M. and Kim, S W. (2004) Pharm Res, 21:339-43; Lane, M. E.,
et al. (2006) Int J Pharm, 307:16-22; Jackson, J. K., et al. (2007)
Int J Pharm).
[0662] Microparticles are also well known and readily available to
one of ordinary skill in the art from companies experienced in
providing such technologies for extended release drug delivery. For
example, Epic Therapeutics, a subsidiary of Baxter Healthcare
Corp., developed PROMAXX.TM., a protein-matrix drug delivery system
that produces bioerodible protein microspheres in a totally
water-based process; OctoPlus developed OctoDEX.TM., crosslinked
dextran microspheres that release active ingredients based on bulk
degradation of matrix rather than based on surface erosion; and
Brookwood Pharmaceuticals advertises the availability of its
microparticle technologies for drug delivery.
[0663] A search of patents, published patent applications and
related publications will also provide those skilled in the art
reading this disclosure with significant possible microparticle
technologies. For example, U.S. Pat. Nos. 6,669,961; 6,517,859;
6,458,387; 6,395,302; 6,303,148; 6,268,053; 6,090,925; 6,024,983;
5,942,252; 5,981,719; 5,578,709; 5,554,730; 5,407,609; 4,897,268;
and 4,542,025, the contents of which are incorporated by reference
in their entirety, describe microspheres and methods for their
manufacture. One skilled in the art, considering both the
disclosure of this invention and the disclosures of these other
patents could make and use microparticles for the extended release
of the modified polypeptides of the invention.
[0664] Further modifications are provided by the invention. Because
microparticles such as PLGA beads still release significant levels
of drug immediately after administration, the present invention
provides ways of ameliorating this bolus effect by including
accessory polypeptides and optional depot modules as part of the
modified polypeptide, as described hereinabove.
[0665] If desired, release of the therapeutic protein can be
further controlled if microparticles with two or more layers are
used. In one embodiment, the microspheres have an inner layer as
well as an outer layer. The composition or the thickness of the
outer layer may be modified to introduce differences in the time it
takes to expose the modified-polypeptide-containing center of the
bead. In one embodiment, microspheres may have an inner layer
containing the modified polypeptide at high concentration, while
the outer layer may contain a lower concentration of the modified
polypeptide or no modified polypeptide. Alternatively, the outer
layer varies in thickness between different microspheres. The
microspheres with a thin outer layer will release modified
polypeptide earlier (for example, from day 1-5), while the beads
with a medium thickness of outer layer release modified polypeptide
at a later time (for example, from day 4-8), and the beads with a
thicker outer layer release modified polypeptide even later (for
example, from day 7-11). Thus, a more constant rate of release is
obtained in this embodiment.
[0666] The rate of drug release from polymeric matrix formulations
can be dependent on the accessory polypeptide attached to the
biologically active peptide. The accessory polypeptide
significantly increases the hydrodynamic radius of the modified
polypeptide. Thus the accessory polypeptide module provides means
to control the rate of drug release from the microparticles. Any of
the accessory polypeptides described herein can be formulated with
a polymeric matrix to achieve beneficial effects in
controlled-release, serum half-life stability, and other desirable
properties described herein.
[0667] In a further aspect of the invention, the depot modules
described herein can be designed to enhance the non-covalent
interactions between the accessory polypeptide-biologically active
polypeptide and the polymer matrix and to slow down the rate of
release of the modified polypeptide from the matrix beads. For
example, alginate is a polymer consisting of mannuronic and
guluronic acid and alginate microspheres can be prepared via
water/oil emulsion methods [Srivastava, R., et al. (2005) J
Microencapsul, 22: 397-411], similar to the preparation of PLGA
microspheres. Unlike PLGA microspheres, alginate forms highly
porous microspheres from which protein release is usually complete
in days. This present invention provides the use of a depot module
in conjunction with the volume enhancing module and biologically
active polypeptide to increase the retention of the fusion protein
within alginate microspheres.
[0668] Each unit of the alginate polymer matrix contains a carboxyl
group that has a -1 charge at physiological pH. Thus alginate
polymers have a large net negative charge under physiological
conditions. The depot module is designed to have a basic
isoelectric point (that is positively charged at physiological pH)
and will therefore be retained much longer within alginate
microspheres (FIG. 6). This depot module comprises a human
polypeptide containing multiple lysine (K) and/or arginine (R)
residues, for example. At physiological pH the lysine amino acids
will carry a net positive charge, thus increasing its non-covalent
binding to the alginate polymer. The depot module may include
naturally occurring polypeptides or designed/engineered or selected
polypeptides. Potential depot modules can be rapidly evaluated for
their ability to interact with alginate. Additionally, polypeptides
that bind only weakly to alginate can be combined to form repeating
depot module units in order to strengthen the interactions with the
polymer.
[0669] In a further embodiment of the invention, a divalent cation
chelating polymer matrix (e.g. hydrogel; Lin, C. C. and Metters, A.
T. (2007) J Biomed Mater Res A) is used in conjunction with a depot
module that binds to divalent cations. For example, both the depot
module and the chelating polymer matrix binds to Cu.sup.2+,
Co.sup.2+ and Ni.sup.2+ cations and the strong non-covalent
interactions between the depot module and the divalent cations
serve as an efficient mechanism to achieve sustained release of the
therapeutic protein from the hydrogel (FIG. 7). FIG. 46 illustrates
the sustained release of accessory-modified polypeptides. For
example, the depot module can incorporate poly-histidine tagged
protein. Poly-histidine sequences are routinely used as
purification tags, because such sequences bind tightly to Ni.sup.2+
cations on solid support. Alternative depot modules can be
similarly designed in light of the teachings hereinabove. The depot
module can be attached directly to the accessory polypeptide,
instead of the biologically active polypeptide, if the
poly-histidine sequence is otherwise likely to interfere with the
biological activity of the therapeutic polypeptide.
[0670] Thus, any number of variations and choice of polymer matrix,
accessory polypeptide, depot module and/or biologically active
polypeptide can be combined to achieve the desired effect in a
patient.
[0671] The present invention provides pharmaceutical compositions
comprising the modified polypeptide. They can be administered
orally, intranasally, parenterally or by inhalation therapy, and
may take the form of tablets, lozenges, granules, capsules, pills,
ampoules, suppositories or aerosol form. They may also take the
form of suspensions, solutions and emulsions of the active
ingredient in aqueous or nonaqueous diluents, syrups, granulates or
powders. In addition, the pharmaceutical compositions can also
contain other pharmaceutically active compounds or a plurality of
compounds of the invention.
[0672] The compositions of the invention also can be combined with
various liquid phase carriers, such as sterile or aqueous
solutions, pharmaceutically acceptable carriers, suspensions and
emulsions. Examples of non-aqueous solvents include propyl ethylene
glycol, polyethylene glycol and vegetable oils.
[0673] More particularly, the present pharmaceutical compositions
may be administered for therapy by any suitable route including
oral, rectal, nasal, topical (including transdermal, aerosol,
buccal and sublingual), vaginal, parental (including subcutaneous,
intramuscular, intravenous and intradermal) and pulmonary. It will
also be appreciated that the preferred route will vary with the
condition and age of the recipient, and the disease being
treated.
[0674] Extended release formulations useful in the present
invention may be oral formulations comprising a matrix and a
coating composition. Suitable matrix materials may include waxes
(e.g., carnauba, bees wax, paraffin wax, ceresine, shellac wax,
fatty acids, and fatty alcohols), oils, hardened oils or fats
(e.g., hardened rapeseed oil, castor oil, beef tallow, palm oil,
and soya bean oil), and polymers (e.g., hydroxypropyl cellulose,
polyvinylpyrrolidone, hydroxypropyl methyl cellulose, and
polyethylene glycol). Other suitable matrix tabletting materials
are microcrystalline cellulose, powdered cellulose, hydroxypropyl
cellulose, ethyl cellulose, with other carriers, and fillers.
Tablets may also contain granulates, coated powders, or pellets.
Tablets may also be multi-layered. Multi-layered tablets are
especially preferred when the active ingredients have markedly
different pharmacokinetic profiles. Optionally, the finished tablet
may be coated or uncoated.
[0675] The coating composition may comprise an insoluble matrix
polymer and/or a water soluble material. Water soluble materials
can be polymers such as polyethylene glycol, hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone,
polyvinyl alcohol, or monomeric materials such as sugars (e.g.,
lactose, sucrose, fructose, mannitol and the like), salts (e.g.,
sodium chloride, potassium chloride and the like), organic acids
(e.g., fumaric acid, succinic acid, lactic acid, and tartaric
acid), and mixtures thereof. Optionally, an enteric polymer may be
incorporated into the coating composition. Suitable enteric
polymers include hydroxypropyl methyl cellulose, acetate succinate,
hydroxypropyl methyl cellulose, phthalate, polyvinyl acetate
phthalate, cellulose acetate phthalate, cellulose acetate
trimellitate, shellac, zein, and polymethacrylates containing
carboxyl groups. The coating composition may be plasticised by
adding suitable plasticisers such as, for example, diethyl
phthalate, citrate esters, polyethylene glycol, glycerol,
acetylated glycerides, acetylated citrate esters, dibutylsebacate,
and castor oil. The coating composition may also include a filler,
which can be an insoluble material such as silicon dioxide,
titanium dioxide, talc, kaolin, alumina, starch, powdered
cellulose, MCC, or polacrilin potassium. The coating composition
may be applied as a solution or latex in organic solvents or
aqueous solvents or mixtures thereof. Solvents such as water, lower
alcohol, lower chlorinated hydrocarbons, ketones, or mixtures
thereof may be used.
[0676] The modified polypeptides of the invention may be formulated
using a variety of excipients. Suitable excipients include
microcrystalline cellulose (e.g. Avicel PH102, Avicel PH101),
polymethacrylate, poly(ethyl acrylate, methyl methacrylate,
trimethylammonioethyl methacrylate chloride) (such as Eudragit
RS-30D), hydroxypropyl methylcellulose (Methocel K100M, Premium CR
Methocel K100M, Methocel E5, Opadry.RTM.), magnesium stearate,
talc, triethyl citrate, aqueous ethylcellulose dispersion
(Surelease.RTM.). The slow release agent may also comprise a
carrier, which can comprise, for example, solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents. Pharmaceutically acceptable salts can
also be used in these slow release agents, for example, mineral
salts such as hydrochlorides, hydrobromides, phosphates, or
sulfates, as well as the salts of organic acids such as acetates,
proprionates, malonates, or benzoates. The composition may also
contain liquids, such as water, saline, glycerol, and ethanol, as
well as substances such as wetting agents, emulsifying agents, or
pH buffering agents. Liposomes may also be used as a carrier.
[0677] Administration via transdermal formulations can be performed
using methods also known in the art, including those described
generally in, e.g., U.S. Pat. Nos. 5,186,938 and 6,183,770,
4,861,800, 6,743,211, 6,945,952, 4,284,444, and WO 89/09051,
incorporated herein by reference in their entireties. A transdermal
patch is a particularly useful embodiment with polypeptides having
absorption problems. Patches can be made to control the release of
skin-permeable active ingredients over a 12 hour, 24 hour, 3 day,
and 7 day period. In one example, a 2-fold daily excess of a
polypeptide of the present invention is placed in a non-volatile
fluid. The compositions of the invention are provided in the form
of a viscous, non-volatile liquid. The penetration through skin of
specific formulations may be measures by standard methods in the
art (for example, Franz et al., J. Invest. Derm. 64:194-195
(1975)). Examples of suitable patches are passive transfer skin
patches, iontophoretic skin patches, or patches with microneedles
such as Nicoderm.
[0678] In other embodiments, the composition may be delivered via
intranasal, buccal, or sublingual routes to the brain to enable
transfer of the active agents through the olfactory passages into
the CNS and reducing the systemic administration. Devices commonly
used for this route of administration are included in U.S. Pat. No.
6,715,485. Compositions delivered via this route may enable
increased CNS dosing or reduced total body burden reducing systemic
toxicity risks associated with certain drugs. Preparation of a
pharmaceutical composition for delivery in a subdermally
implantable device can be performed using methods known in the art,
such as those described in, e.g., U.S. Pat. Nos. 3,992,518;
5,660,848; and 5,756,115.
[0679] Osmotic Pumps may be used as slow release agents in the form
of tablets, pills, capsules or implantable devices. Osmotic pumps
are well known in the art and readily available to one of ordinary
skill in the art from companies experienced in providing osmotic
pumps for extended release drug delivery. Examples are ALZA's
DUROS.TM.; ALZA's OROS.TM.; Osmotica Pharmaceutical's Osmodex.TM.
system; Shire Laboratories' EnSoTrol.TM. system; and Alzet.TM..
Patents that describe osmotic pump technology are U.S. Pat. Nos.
6,890,918; 6,838,093; 6,814,979; 6,713,086; 6,534,090; 6,514,532;
6,361,796; 6,352,721; 6,294,201; 6,284,276; 6,110,498; 5,573,776;
4,200,0984; and 4,088,864, the contents of which are incorporated
herein by reference. One skilled in the art, considering both the
disclosure of this invention and the disclosures of these other
patents could produce an osmotic pump for the extended release of
the polypeptides of the present invention.
[0680] Syringe Pumps may also be used as slow release agents.
Syringe pumps are known to one skilled in the art and readily
available. Such devices are described in U.S. Pat. Nos. 4,976,696;
4,933,185; 5,017,378; 6,309,370; 6,254,573; 4,435,173; 4,398,908;
6,572,585; 5,298,022; 5,176,502; 5,492,534; 5,318,540; and
4,988,337, the contents of which are incorporated herein by
reference. One skilled in the art, considering both the disclosure
of this invention and the disclosures of these other patents could
produce a syringe pump for the extended release of the polypeptides
of the present invention.
[0681] In another embodiment, the modified polypeptides of the
present invention are encapsulated in liposomes, which have
demonstrated utility in delivering beneficial active agents in a
controlled manner over prolonged periods of time. Liposomes are
closed bilayer membranes containing an entrapped aqueous volume.
Liposomes may also be unilamellar vesicles possessing a single
membrane bilayer or multilamellar vesicles with multiple membrane
bilayers, each separated from the next by an aqueous layer. The
structure of the resulting membrane bilayer is such that the
hydrophobic (non-polar) tails of the lipid are oriented toward the
center of the bilayer while the hydrophilic (polar) heads orient
towards the aqueous phase. In one embodiment, the liposome may be
coated with a flexible water soluble polymer that avoids uptake by
the organs of the mononuclear phagocyte system, primarily the liver
and spleen. Suitable hydrophilic polymers for surrounding the
liposomes include, without limitation, PEG, polyvinylpyrrolidone,
polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,
polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide,
polymethacrylamide, polydimethylacrylamide,
polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,
hydroxymethylcellulose hydroxyethylcellulose, polyethyleneglycol,
polyaspartamide and hydrophilic peptide sequences as described in
U.S. Pat. Nos. 6,316,024; 6,126,966; 6,056,973; 6,043,094, the
contents of which are incorporated by reference in their
entirety.
[0682] Liposomes may be comprised of any lipid or lipid combination
known in the art. For example, the vesicle-forming lipids may be
naturally-occurring or synthetic lipids, including phospholipids,
such as phosphatidylcholine, phosphatidylethanolamine, phosphatidic
acid, phosphatidylserine, phasphatidylglycerol,
phosphatidylinositol, and sphingomyelin as disclosed in U.S. Pat.
Nos. 6,056,973 and 5,874,104. The vesicle-forming lipids may also
be glycolipids, cerebrosides, or cationic lipids, such as
1,2-dioleyloxy-3-(trimethylamino) propane (DOTAP);
N-[1-(2,3,-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium
bromide (DMRIE);
N-[1[(2,3,-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium
bromide (DORIE);
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); 3[N--(N',N'-dimethylaminoethane)carbamoly]cholesterol
(DC-Chol); or dimethyldioctadecylammonium (DDAB) also as disclosed
in U.S. Pat. No. 6,056,973. Cholesterol may also be present in the
proper range to impart stability to the vesicle as disclosed in
U.S. Pat. Nos. 5,916,588 and 5,874,104.
[0683] Liposomes are also well known in the art and readily
available from companies experienced in providing liposomes for
extended release drug delivery. For example, ALZA's (formerly
Sequus Pharmaceuticals') STEALTH.TM. liposomal technology for
intravenous drug delivery uses a polyethylene glycol coating on
liposomes to evade recognition by the immune system; Gilead
Sciences (formerly Nexstar's) liposomal technology was incorporated
into AmBisome.TM., and FDA approved treatment for fungal
infections; and NOF Corp. offers a wide variety of GMP-grade
phospholipids, phospholipids derivatives, and PEG-phospholipids
under the tradenames COATSOME.TM. and SUNBRIGHT.TM..
[0684] Additional possible liposomal technologies are described in
U.S. Pat. Nos. 6,759,057; 6,406,713; 6,352,716; 6,316,024;
6,294,191; 6,126,966; 6,056,973; 6,043,094; 5,965,156; 5,916,588;
5,874,104; 5,215,680; and 4,684,479, the contents of which are
incorporated herein by reference. These describe liposomes and
lipid-coated microbubbles, and methods for their manufacture. Thus,
one skilled in the art, considering both the disclosure of this
invention and the disclosures of these other patents could produce
a liposome for the extended release of the polypeptides of the
present invention.
[0685] Diseases amenable to treatment by administration of the
compositions of the invention include without limitation 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 vain 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.
[0686] Accessory polypeptides may also comprise protease cleavage
sites or other sequences that allow the modified polypeptide to be
cleaved following expression. Such site or sites may be located
anywhere within the modified polypeptide. For example, a protease
cleavage site may be introduced between a sequence that improves
solubility and another sequence comprising an affinity tag, such
that the affinity tag is removed by protease treatment.
Alternatively, the cleavage site may be located between the
biologically active protein and the accessory polypeptide, such
that a specific protease would cleave off the entire accessory
polypeptide sequence. Various enzymatic methods for cleaving
proteins are known. Such methods include enterokinase (DDDK) (SEQ
ID NO: 447), Factor Xa (IDGR) (SEQ ID NO: 448), thrombin (LVPR/GS)
(SEQ ID NO: 449), PreScission.TM. (LEVLFQ/GP) (SEQ ID NO: 450), TEV
protease (EQLYFQ/G) (SEQ ID NO: 451), 3C protease (ETLFQ/GP) (SEQ
ID NO: 452), Sortase A (LPET/G) (SEQ ID NO: 453), Granzyme B (D/X,
N/X, M/N or S/X), inteins, SUMO, DAPase (TAGZyme.TM.), Aeromonas
aminopeptidase, Aminopeptidase M, and carboxypeptidases A and B.
Additional methods are disclosed in Arnau et al, Prot Expr and
Purif (2006) 48, 1-13.
[0687] Optimization of Production of Modified Polypeptide
[0688] Additionally, the accessory polypeptides of the invention
may comprise additional sequences which allow improved folding or
purification during expression. This concept is described generally
in FIG. 32. For example, accessory polypeptides may be linked to
affinity or solubility tags to aid in purification. Non-limiting
examples include His-tag, FLAG, Streptag II, HA-tag, Softagl,
Softag 3, c-myc, T7-tag, S-tag, Elastin-like peptides,
Chitin-binding domain, Thioredoxin, Xylanase 10A, Glutathione
5-transferase (GST), Maltose binding protein (MBP), NusA, and
Cellulose binding protein.
[0689] Accessory polypeptides may also comprise protease cleavage
sites or other sequences that allow the modified polypeptide to be
cleaved following expression. Such site or sites may be located
anywhere within the modified polypeptide. For example, a protease
cleavage site may be introduced between a sequence that improves
solubility and another sequence comprising an affinity tag, such
that the affinity tag is removed by protease treatment.
Alternatively, the cleavage site may be located between the
biologically active protein and the accessory polypeptide, such
that a specific protease would cleave off the entire accessory
polypeptide sequence. Various enzymatic methods for cleaving
proteins are known. Such methods include enterokinase (DDDK),
Factor Xa (IDGR), thrombin (LVPR/GS), PreScission.TM. (LEVLFQ/GP),
TEV protease (EQLYFQ/G), 3C protease (ETLFQ/GP), Sortase A
(LPET/G), Granzyme B (D/X, N/X, M/N or S/X), inteins, SUMO, DAPase
(TAGZyme.TM.), Aeromonas aminopeptidase, Aminopeptidase M, and
carboxypeptidases A and B. Additional methods are disclosed in
Arnau et al, Prot Expr and Purif (2006) 48, 1-13.
[0690] Analysis of Protein Expression
[0691] The activity of the expressed proteins may be measured to
ascertain the degree of correct folding. Such assays are well known
in the art depending on the specific modified polypeptide
expressed. Such assays may include cell based assays, including
assays for proliferation, cell death, apoptosis and cell migration.
Other possible assays may determine receptor binding of expressed
polypeptides, wherein the assay may comprise soluble receptor
molecules, or may determine the binding to cell-expressed
receptors. Additionally, techniques such as flow cytometry or
surface plasmon resonance can be used to detect binding events.
Specific in vivo biological assays may be used to assess the
activity of each biologically active polypeptide of the invention.
For example, the properties of hGH may be determined using an ESTA
bioassay, or alternatively by measuring rhGH induced dose-related
body weight gain and bone growth, or receptor binding. Additional
methods are disclosed in Dattani, M. T., et al. (1996) Horm Res,
46: 64-73; Alam, K. S., et al. (1998) J Biotechnol, 65: 183-90;
Clark, R., et al. (1996) J Biol Chem, 271: 21969-77; Clarg R G et
al, (1996) Endocrinology. 137:4308-15.
[0692] The present invention also relates to the composition and
method of engineering the rPEG fusion products for administration
into a subject. An association peptide, such as SKVILF(E) (SEQ ID
NO: 8) or RARADADA (SEQ ID NO: 9), which bind to another copy of
the same sequence in an antiparallel orientation, can be used to
create a prodrug, as shown in FIG. 88a-c. In one embodiment, the
drug is protease-cleaved in the last step of manufacture, but the
cleavage does not activate the drug since the two chains are still
associated by the association peptides. Only after the drug is
injected into a subject and the concentration is greatly reduced,
the small, non-rPEG-containing protein chain leaves the complex at
a rate that depends on the affinity, and is likely to be cleared
via the kidney, thereby activating the r-PEG-containing drug
module.
[0693] More specifically, cellular localization of expressed
polypeptides of the invention can be determined by any of the
methods named above. For example, a crude lysate obtained from
cells expressing the polypeptide of interest may be centrifuged in
order to separate soluble expressed protein in the cytosolic
fraction from insoluble protein in the inclusion bodies. If
desired, the soluble (cytosolic) and insoluble (inclusion body)
fractions can then be analyzed by Western Blot or similar
techniques to determine the ratio of expression as soluble vs.
insoluble protein.
[0694] Soluble protein in the lysate may be purified further by
techniques such as anion exchange or size exclusion chromatography,
techniques which can be applied preparatively or analytically
(FIGS. 35-39, 47, 48, 50 and 51). Confirmation of the purity of the
final product may be obtained by techniques known in the art such
as SDS-PAGE, HPLC (e.g. reverse phase or size exclusion) or mass
spectrometry. The purification steps may be preceded or followed by
protease cleavage steps to remove affinity/solubility tags and/or
the accessory polypeptide, or both. Further purification steps by
any of the methods outlined above may be needed to remove, for
example, the used protease from digestion mixtures. Such steps
would be well within the grasp of a person skilled in the art.
Several such methods are also described in more detail in the
Examples section.
[0695] Formulation, Pharmacokinetics, and Administration of rPEG
Fusion Products
[0696] The present invention also relates to the composition and
method of engineering the rPEG fusion products for administration
into a subject. An association peptide, such as SKVILF(E) or
RARADADA, which bind to another copy of the same sequence in an
antiparallel orientation, can be used to create a prodrug, as shown
in FIG. 88a-c. In one embodiment, the drug is protease-cleaved in
the last step of manufacture, but the cleavage does not activate
the drug since the two chains are still associated by the
association peptides. Only after the drug is injected into a
subject and the concentration is greatly reduced, the small,
non-rPEG-containing protein chain leaves the complex at a rate that
depends on the affinity, and is likely to be cleared via the
kidney, thereby activating the r-PEG-containing drug module.
[0697] In another embodiment, the rPEG50 contains a proteolytic
site and the proteolytic cleavage converts the manufactured
single-chain protein into a complex of two protein chains (FIG.
89a-c). This cleavage can occur as the last manufacturing step
before injection into a subject or it can occur after injection
into a subject, by proteases present in the subject.
[0698] Another embodiment includes an rPEG flanked by identical
receptor domains or domains having the same binding function, or
domains that can bind simultaneously to the same target (FIG.
94a-c). If both receptors can bind the target simultaneously, then
the binding of one receptor stabilizes binding of the second
receptor, resulting in mutual stabilization of the complex, thereby
increasing the apparent affinity (avidity) typically by 10 to
100-fold, but at least 3-fold, with the rPEG serving as a valency
bridge that increases the effective concentration of the receptors
(FIG. 94b). In one embodiment, the rPEG product is pre-loaded with
a ligand (FIG. 94c). When administered into a subject, the injected
product is inactive for as long as it remains bound to the ligand.
When the ligand dissociates, it is likely to be rapidly cleared via
the kidney, resulting in activation of the product, which has a
long halflife attributed to the rPEG tail. This approach reduces
the peak dose toxicity and receptor-mediated clearance, thereby
extending the serum secretion halflife, as illustrated in FIG.
99.
[0699] As shown in FIG. 94, some pro-drug formats do not need a
cleavage or other activation site. A single protein chain can
contain two or more drug modules separated by rPEG. These modules
can be of a single type or of two or more different types. This
rPEG cotaining product is complexed with a second, complementary
protein to form a receptor-ligand-receptor interaction. In this
format the ligand may be dimeric or multimeric, but may also be
monomeric, especially if the two drug modules are different. Both
modules bind to a third protein. X and Y can be the same or
different, and X and Y can be a drug module or bind to a drug
module. In each case in FIGS. 94a-c, X and Y (and rPEG) comprise
one protein chain, and the molecule they bind to is a separate
molecule, typically protein or small molecule. It is possible to
have more than two binding proteins combined in a single protein
chain.
[0700] It is generally desirable in therapies that the drug be
maintained at a concentration that is higher than the therapeutic
does, but lower than the toxic dose. A typical bolus injection (IV,
IM, SC, IP or similar) of a drug with a short halflife results in a
peak concentration that is much higher than the toxic dose,
followed by an elimination phase that causes the drug concentration
to rapidly drop below the therapeutic dose. FIG. 100 illustrates
the drug concentration changes over time after an i.v. injection of
a drug alone as compared to the drug linked to an rPEG. The drug
alone is present at therapeutic concentrations for only a short
time (blue line). The addition of rPEG to a drug decreases the peak
concentration and thereby decreases toxicity, and increases the
period of time that the drug is present at a therapeutic, non-toxic
dose. The creation of a pro-drug by addition of rPEG plus a
drug-binding protein can prevent the "burst release" or toxic peak
dose (red line), as the drug is only gradually activated over hours
and the length of time between the toxic dose and the therapeutic
dose is increased compared to the other formats.
[0701] In another embodiment, the rPEG fusion products are either
cleaved before administration into a subject or administered as an
inactive pro-drug (i.e. cleaved after administration into a subject
and activated in vivo). The process is illustrated in FIG. 96 a-h.
The inactivation of the drug is mediated by a binding protein that
is linked to the drug by rPEG such that all three modules are
manufactured as a single protein chain. If the drug is a receptor,
then the binding protein may be a ligand of that receptor; if the
drug is an antibody fragment, then the binding site may be an
antigen. In these examples, the drug is activated by protease
cleavage of a site between the two binding domains, herein termed X
and Y. If protein Y is the active product, then Y retains the rPEG
and the protease cleavage site needs to be close to X. If protein Y
is the active product, then X retains the rPEG and the cleavage
site is close to Y. There can be one or multiple cleavage sites, as
shown by the blue crossbars (FIG. 96a-g). The drug module includes,
but is not limited to, a receptor, a ligand, one or more Ig
domains, an antibody fragment, a peptide, a microprotein, or an
epitope for an antibody. The protein that binds to the drug module
includes, but is not limited to, a binding protein, a receptor, a
ligand, one or more Ig domains, an antibody fragment, a peptide, a
microprotein, or an epitope for an antibody. FIGS. 105 and 106
illustrate the conversion of an inactive protein (i.e. pro-drug) to
an active protein (i.e. either an active peptide or a dAb or scFv)
by a site-specific protease, either present in the serum of a
subject or given before administration into a subject.
[0702] The amino acid sequence of hGH used in this experiment
is:
TABLE-US-00011 (SEQ ID NO: 454)
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFELAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQ
QKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRT
GQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF.
A single-chain protein drug may also contain multiple bio-active
peptides, which can be at the same end of rPEG or at an opposite
end of rPEG (FIG. 98). These peptides can have the same activity or
different activities. Having multiple peptides in a single chain
increases their effective potency through binding avidity without
complicating manufacturing.
EXAMPLES
Example 1
Design of Human Growth Hormone (hGH) Fused to Accessory
Polypeptides
[0703] This example describes the preparation of an rPEG-hGH fusion
protein with increased active, cytoplasmic yield and having
improved serum half-life. Human growth hormone products typically
require daily or twice-daily injections because the halflife of hGH
in the serum is only about 30 minutes. Halflife extension through
PEGylation is not feasible as hGH contains multiple lysines that
are required for therapeutic activity and these cannot be used for
conjugation. hGH is typically manufactured by expression in the
cytoplasm of E. coli, where it can aggregate and form inclusion
bodies containing inactive protein. Typically, these inclusion
bodies are solubilized and the protein is refolded to obtain active
protein. In this example, rPEG-hGH is expressed in the cytoplasm in
soluble and active form, avoiding the step of refolding from
inclusion bodies.
[0704] The amino acid sequence of hGH used in this experiment
is:
TABLE-US-00012 FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFELAYIPKEQKYSFLQNP
QTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSV
FANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYS
KFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF.
[0705] hGH contains 191 amino acids, with a pI of 5.27 and a
molecular weight of 22.130 kD. hGH contains 13 Glutamate residues,
11 Aspartate residues (24 total negative residues), 8 Lysine
residues and 11 Arginine residues (19 total positive residues), for
a net charge of -5 and a net charge density of -0.026 (calculated
as -5/191 amino acids). This net charge density correlates with the
experimental pI value of 5.27.
[0706] Various hGH-rPEG fusion proteins are designed as
follows.
[0707] Design 1. Construction of rPEG-modified hGH with Net Charge
Density of -0.1.
[0708] This design describes a polypeptide modified with a
short-length accessory polypeptide and a net charge density of
-0.1.
[0709] The goal of this design is to produce a protein with a net
charge density of -0.1 while adding only a few amino acids. The
number of charges needed to create an hGH protein with a -0.1
charge density is 14.1 (19.1-5=14.1) without accounting for the
increase in total length resulting from the added charged amino
acids. The addition of 16 negatively charged amino acids brings the
net charge density of the modified hGH polypeptide to -0.1
(calculated as (16+5)/(191+16) amino acids).
[0710] Design 2. Construction of rPEG-Modified hGH with Net Charge
Density of -0.2.
[0711] This design describes a polypeptide modified with a
short-length accessory polypeptide and net charge density of
-0.2.
[0712] This design incorporates an accessory protein with 41
negative charges, for a total of 46 combined negatively charged
amino acid residues in the entire polypeptide. The total length of
the modified polypeptide is 232 amino acids (calculated as 191+41
amino acids). Consequently, a charge density of -0.2 requires a
total of 46 negatively charged amino acid residues (calculated as
0.2.times.232 amino acids), which means the accessory protein
contains 41 negatively charged residues (calculated as 46-5).
[0713] Design 3. Construction of rPEG-Modified hGH with Net Charge
Density of +0.1.
[0714] rPEG_J288 has the sequence (GGSGGE).sub.48 (SEQ ID NO: 455)
and contains 48 E residues (FIG. 17). When rPEG_J288 was added to
hGH, the total length of the modified polypeptide became 479 amino
acids (calculated as 191+288) and the net charge became 53
(calculated as 48+5), thus yielding a net charge density of
(calculated as 53/479=0.11). In this design, the accessory
polypeptide itself has a net charge density of 16% due to the
presence of many Glycine and Serine residues, whereas in Design 1
the accessory polypeptide is entirely composed of charged residues.
As the experimental results demonstrated, this design yields highly
soluble and active polypeptide. It appears that a net charge
density of -0.11 can be sufficient to keep the protein in solution
if the charges are spread out by the addition of Serines and/or
Glycines.
[0715] This example describes the construction of a fusion gene
encoding an accessory polypeptide of 144 amino acids and the
sequence (GGSGGE).sub.48 (SEQ ID NO: 455). A stuffer vector pCW0051
is constructed as shown in FIG. 16. The sequence of the expression
cassette in pCW0051 is shown in FIG. 18. An insert is obtained
essentially as described below for rPEG_L288 but by annealing a
synthetic oligonucleotide encoding the rPEG sequence rPEG_J288
(FIG. 11) with a pair of oligonucleotides encoding an adaptor to
the KpnI site. The following oligonucleotides are used as forward
and reverse primers:
TABLE-US-00013 pr_LCW0057for: (SEQ ID NO: 456)
AGGTAGTGGWGGWGARGGWGGWTCYGGWGGAGAAGG, pr_LCW0057rev: (SEQ ID NO:
457) ACCTCCTTCTCCWCCRGAWCCWCCYTCWCCWCCACT,
[0716] The following oligonucleotides are used as stopper
primers:
TABLE-US-00014 (SEQ ID NO: 458) pr_3KpnIstopperFor:
AGGTTCGTCTTCACTCGAGGGTAC, (SEQ ID NO: 459) pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA.
[0717] The following oligonucleotides are used as stopper
primers:
TABLE-US-00015 pr_3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC,
[0718] This design describes a polypeptide modified with a long
hydrophilic accessory polypeptide of 288 amino acids comprising 25%
glutamate residues. rPEG_L288 has the sequence
(SSESSSSESSSE).sub.24 (SEQ ID NO: 40) and contains 72 E residues.
When rPEG_L288 is added to hGH, the total length of the fusion
becomes 479 amino acids (calculated as 191+288 amino acids) and the
net charge becomes 77 (calculated as 72+5), yielding a net charge
density of 0.16 (calculated as 77/479 amino acids). As the
experimental results described below demonstrated, this design with
a net charge density of -0.16 showed excellent solubility and the
protein was active. Some gel formation was observed at low
temperatures but this did not appear to be a problem.
[0719] This section describes the construction of a codon optimized
gene encoding a accessory polypeptide, rPEG_L288 with 288 amino
acids and the sequence (SSSESSESSSSE).sub.24 (SEQ ID NO: 460). A
stuffer vector pCW0150 which is based on a pET vector and includes
a T7 promoter is constructed as shown in FIG. 9. The vector encodes
a Flag sequence followed by a stuffer sequence that is flanked by
BsaI, BbsI, and KpnI sites. The stuffer sequence was followed by a
His6 tag (SEQ ID NO: 1) and the gene of green fluorescent protein
(GFP). GFP was chosen as the biologically active protein and may be
used in imaging applications or as a selection marker. The stuffer
sequence contains stop codons and thus E. coli cells carrying the
stuffer plasmid pCW0150 form non-fluorescent colonies. The stuffer
vector pCW0150 was digested with BsaI and KpnI. A codon library
encoding accessory polypeptides of 36 amino acid length was
constructed. The accessory polypeptide was designated rPEG_L36 and
had the amino acid sequence (SSSESSESSSSE).sub.3 (SEQ ID NO: 461).
The insert was obtained by annealing synthetic oligonucleotide
pairs encoding the amino acid sequence SSESSESSSSES (SEQ ID NO:
462) as well as a pair of oligonucleotides that encode an adaptor
to the KpnI site. The following oligonucleotides were used as
forward and reverse primers:
TABLE-US-00016 pr_LCW0148for: (SEQ ID NO: 463)
TTCTAGTGARTCYAGYGARTCYAGYTCYAGYGAATC, pr_LCW0148rev: (SEQ ID NO:
464) AGAAGATTCRCTRGARCTRGAYTCRCTRGAYTCACT, The following
oligonucleotides are used as stopper primers:
pr_3KpnIstopperForTTCT: (SEQ ID NO: 465) TTCTTCGTCTTCACTCGAGGGTAC,
pr_3KpnIstopperRev: (SEQ ID NO: 459) CCTCGAGTGAAGACGA.
[0720] By varying the ratio of forward/reverse primers to stopper
primers, the size of the resulting PCR products can be controlled.
The annealed oligonucleotide pairs were ligated, which resulted in
a mixture of products with varying length that represents the
varying number of (SSSESSESSSSE) (SEQ ID NO: 362) repeats. The
product corresponding to the length of rPEG_L36 was isolated from
the mixture by agarose gel electrophoresis and ligated into the
BsaI/KpnI digested stuffer vector pCW0150. Cells transformed with
vector showed green fluorescence after induction which shows that
the sequence of rPEG_L36 had been ligated in frame with the GFP
gene. The resulting library was designated LCW0148. Isolates (e.g.,
312 isolates) from library LCW0148 were screened for high level of
fluorescence. Isolates (e.g., 70 isolates) with strong fluorescence
were analyzed by PCR to verify the length of the rPEG_L segment and
34 clones were identified that had the expected length of rPEG_L36.
This process resulted in a collection of 34 isolates of rPEG_L36
showing high expression and differing in their codon usage. A
plasmid mixture was digested with BsaI/NcoI and a fragment
comprising the rPEG_L36 sequence and a part of GFP was isolated.
The same plasmid mixture was also digested with BbsI/NcoI and the
vector fragment comprising rPEG_L36, most of the plasmid vector,
and the remainder of the GFP gene was isolated. Both fragments were
mixed, ligated, and transformed into BL21Gold(DE3) and isolates
were screened for fluorescence. This process of dimerization was
repeated two more rounds. During each round, the length of the
rPEG_L gene was doubled and ultimately a collection of genes that
encode rPEG_L288 were obtained. The rPEG_L288 module contains
segments of rPEG_L36 that differ in their nucleotide sequence
despite having identical amino acid sequence. Thus, internal
homology in the gene is minimized and as a result the risk of
spontaneous recombination is reduced. E. coli BL21Gold(DE3)
harboring plasmids encoding rPEG_L288 were cultured for at least 20
doublings and no spontaneous recombination was observed.
TABLE-US-00017 pr_3KpnIstopperRev: CCTCGAGTGAAGACGA.
[0721] By varying the ratio of forward/reverse primers to stopper
primers, the size of the resulting PCR products can be controlled.
The insert was used to generate a plasmid encoding the
rPEG_J288-modified GFP and cells expressing this plasmid in a
fashion similar to rPEG_L288-modified GFP (FIG. 10). A similar
insert was used to generate a plasmid encoding the
rPEG_J288-modified hGH and rPEG_J288-modified GLP1 and cells
expressing this plasmid in a fashion similar to rPEG_L288-modified
GFP (FIG. 12).
[0722] This design describes a polypeptide modified with a long
hydrophilic accessory polypeptide of 288 amino acids comprising 33%
glutamate residues. rPEG_K288 has the sequence (GEGGGEGGE).sub.32
(SEQ ID NO: 466) and contains 96 E residues. When rPEG_K288 was
added to hGH, the total length of the fusion became 479 amino acids
(calculated as 191+288) and the net charge became 101 (calculated
as 96+5), yielding a net charge density of 0.21 (calculated as
101/479). As predicted and confirmed by the experimental results
described below, this design with a net charge density of -0.21
showed the highest degree of solubility and the protein was active.
No gel formation was observed at the temperature or salt
concentrations tested.
[0723] This section describes the construction of a fusion gene
encoding an accessory polypeptide of the sequence (GEGGGEGGE)32
(SEQ ID NO: 466). An insert is obtained essentially as described
for rPEG_L288 but by annealing a synthetic oligonucleotide encoding
the rPEG sequence rPEG_K288 with a pair of oligonucleotides
encoding an adaptor to the KpnI site. The following
oligonucleotides were used as forward and reverse primers:
TABLE-US-00018 (SEQ ID NO: 467) pr_LCW0147for:
AGGTGAAGGWGARGGWGGWGGWGAAGG (SEQ ID NO: 468) pr_LCW0147rev:
ACCTCCTTCWCCWCCWCCYTCWCCTTC
[0724] The following oligonucleotides are used as stopper
primers:
TABLE-US-00019 (SEQ ID NO: 458) pr_3KpnIstopperFor:
AGGTTCGTCTTCACTCGAGGGTAC (SEQ ID NO: 459) pr_3KpnIstopperRev:
CCTCGAGTGAAGACGA. pr_3KpnIstopperRev: CCTCGAGTGAAGACGA.
[0725] By varying the ratio of forward/reverse primers to stopper
primers, the size of the resulting PCR products can be controlled.
The annealed oligonucleotide pairs were ligated, which resulted in
a mixture of products with varying length that represents the
varying number of (SSSESSESSSSE) repeats. The product corresponding
to the length of rPEG_L36 was isolated from the mixture by agarose
gel electrophoresis and ligated into the BsaI/KpnI digested stuffer
vector pCW0150. Cells transformed with vector showed green
fluorescence after induction which shows that the sequence of
rPEG_L36 had been ligated in frame with the GFP gene. The resulting
library was designated LCW0148. Isolates (e.g., 312 isolates) from
library LCW0148 were screened for high level of fluorescence.
Isolates (e.g., 70 isolates) with strong fluorescence were analyzed
by PCR to verify the length of the rPEG_L segment and 34 clones
were identified that had the expected length of rPEG_L36. This
process resulted in a collection of 34 isolates of rPEG_L36 showing
high expression and differing in their codon usage. A plasmid
mixture was digested with BsaI/NcoI and a fragment comprising the
rPEG_L36 sequence and a part of GFP was isolated. The same plasmid
mixture was also digested with BbsI/NcoI and the vector fragment
comprising rPEG_L36, most of the plasmid vector, and the remainder
of the GFP gene was isolated. Both fragments were mixed, ligated,
and transformed into BL21Gold(DE3) and isolates were screened for
fluorescence. This process of dimerization was repeated two more
rounds. During each round, the length of the rPEG_L gene was
doubled and ultimately a collection of genes that encode rPEG_L288
were obtained. The rPEG_L288 module contains segments of rPEG_L36
that differ in their nucleotide sequence despite having identical
amino acid sequence. Thus, internal homology in the gene is
minimized and as a result the risk of spontaneous recombination is
reduced. E. coli BL21Gold(DE3) harboring plasmids encoding
rPEG_L288 were cultured for at least 20 doublings and no
spontaneous recombination was observed.
[0726] E. coli BL21Gold(DE3) cells harboring plasmids encoding
rPEG_L288 were grown overnight in Terrific Broth (TB) and diluted
200-fold into fresh TB the following day. When the culture reached
an A600 nm=0.6, expression of rPEG_L288-GFP was induced with the
addition of IPTG to 0.2 mM final concentration. The cells were
harvested following 18 hr at 26 C and can be stored at -80C until
further processing. The cells were resuspended in 90 ml of 50 mM
Tris-HCl, 200 mM sodium chloride, 0.1% Tween-20, 10% glycerol, pH
8.0 per liter of bacterial culture. Protease inhibitors, lysozyme
(final 20 ug/ml), and benzonase nuclease were added to the
bacterial suspension prior to lysis. The cells were lysed by
sonication on ice for four minutes followed by heat treatment at
80.degree. C. for 20 min. The lysate was subsequently cooled on ice
and centrifuged for 20 min at 15000 rpm in a Sorvall SS-34 rotor.
The soluble recombinant protein was purified by immobilized metal
ion affinity chromatography (IMAC) of the supernatant. The protein
was further purified by ion exchange chromatography (IEC) and gel
filtration chromatography. Optionally, the protein can be further
purified by a column with immobilized anti-FLAG antibody using
standard techniques. Purity and homogeneity of the protein was
assessed using standard biochemical methods including SDS-PAGE,
native-PAGE, analytical gel filtration chromatography, light
scattering, and mass spectrometry. A purity of at least 90% was
obtained. Additionally, the modified polypeptides rPEG_L288-hGH and
rPEG_L288-GLP1 were obtained in a similar manner.
[0727] The purity of rPEG_L288-modified GFP was confirmed by
SDS-PAGE (FIG. 36), analytical reverse phase HPLC (FIG. 38). The
apparent molecular weight of rPEG_L288-modified GFP was also
measured as previously described (FIG. 41). FIG. 49 illustrates the
increase in apparent molecular weight observed upon linking a
biologically active polypeptide (GLP1) to rPEG_L288 accessory
polypeptide. The in vivo stability in rat and human serum was
determined as shown in FIG. 42. rPEG is stable in rat and human
serum, and rPEG288 has a halflife of about 10 to 20 hours in rats
(FIG. 43). Little immunogenicity in in vivo experiments could be
observed with this polypeptide (FIG. 44).
[0728] Design 6. Construction of rPEG_K288-GFP, rPEG_K288-hGH and
rPEG_K288-GLP1 Accessory Polypeptides.
[0729] This design describes a polypeptide modified with a long
hydrophilic accessory polypeptide of 288 amino acids comprising 33%
glutamate residues. rPEG_K288 has the sequence (GEGGGEGGE).sub.32
and contains 96 E residues. When rPEG_K288 was added to hGH, the
total length of the fusion became 479 amino acids (calculated as
191+288) and the net charge became 101 (calculated as 96+5),
yielding a net charge density of 0.21 (calculated as 101/479). As
predicted and confirmed by the experimental results described
below, this design with a net charge density of -0.21 showed the
highest degree of solubility and the protein was active. No gel
formation was observed at the temperature or salt concentrations
tested.
[0730] This section describes the construction of a fusion gene
encoding an accessory polypeptide of the sequence
(GEGGGEGGE).sub.32. An insert is obtained essentially as described
for rPEG_L288 but by annealing a synthetic oligonucleotide encoding
the rPEG sequence rPEG_K288 with a pair of oligonucleotides
encoding an adaptor to the KpnI site. The following
oligonucleotides were used as forward and reverse primers:
TABLE-US-00020 pr_LCW0147for: AGGTGAAGGWGARGGWGGWGGWGAAGG
pr_LCW0147rev: ACCTCCTTCWCCWCCWCCYTCWCCTTC
[0731] A plasmid harboring hGH, N-terminally fused to 288 amino
acids of rPEG-K288 and, having the repetitive sequence
(GEGGGEGGE).sub.32 (SEQ ID NO: 466) and a TEV protease cleavage
site (ENLYFQ/X) (SEQ ID NO: 469), following the T7 promoter (i.e.
T7 promoter-hGH-TEV-rPEG_K288), is transformed into BL21(DE3)-star
E. coli strain and is grown as described above. Cells are collected
by centrifugation and the cell pellet is resuspended in 50 ml
Buffer containing 50 mM Tris pH=8.0, 100 mM NaCl, Protease
inhibitors, 10% (v/v) glycerol, 0.1% Triton X-100 and DNAse. Cells
are disrupted using an ultrasonic sonicator cell disruptor, and
cell debris is removed by centrifugation at 15000 RPM at 4.degree.
C. Cellular supernatant is applied on an anion-exchanger
(Q-sepharose, Pharmacia), washed with buffer A (25 mM Tris pH=8.0)
and eluted from the column using a linear gradient of the same
buffer with 1M NaCl. Protein elutes at about 500 mM NaCl. The
eluted fusion protein is pooled, dialyzed and TEV digested. The
digestion mixture is reloaded on the anion-exchange (Q-sepharose,
Pharmacia), washed with buffer A (25 mM Tris pH=8.0) and eluted
from the column using a shallow linear gradient of the same buffer
with 1M NaCl. The eluted hGH protein is pooled, dialyzed against
buffer A, concentrated, and purified by size-exclusion
chromatography (SEC) as the final purification. Protein purity is
estimated to be above 98%.
TABLE-US-00021 pr_3KpnIstopperFor: AGGTTCGTCTTCACTCGAGGGTAC
[0732] A pET-series vector was constructed with T7 promoter, which
expresses a protein containing cellulose binding domain (CBD) at
the N-terminus, followed by a Tomato Etch Virus (TEV) protease
cleavage site, followed by the hGH coding sequence, and by the
rPEG_K288 coding sequence: CBD-TEV-rPEG_K288-hGH. The rPEG_K288 has
the repetitive sequence (GEGGGEGGE).sub.32 (SEQ ID NO: 466). The
CBD sequence used is shown in Swissprot file Q06851 and the
purification of CBD fusion proteins is described in Ofir, K. et al.
(2005) Proteomics 5:1806. The sequence of the TEV cleavage site is
ENLYFQ/X (SEQ ID NO: 524); G was used in the X position. This
construct was transformed into BL21(DE3)-star E. coli strain and
grown essentially as described above, except that the CBD sequence
was introduced N-terminal to the rPEG sequence. Cells were
collected and disrupted essentially as described above. The
cellular supernatant was applied on beaded cellulose resin (Perloza
100), washed with buffer A (25 mM Tris pH=8.0) and eluted from the
column with 20 mM NaOH. pH was adjusted by reutilizing the sample
with 1M Tris buffer pH=8.0. Protein purity was estimated to be
above 90%.
[0733] By varying the ratio of forward/reverse primers to stopper
primers, the size of the resulting PCR products can be controlled.
The insert is used to generate a plasmid encoding the
rPEG_K288-modified GFP and cells expressing this plasmid in a
fashion similar to rPEG_L288-modified GFP. Additionally, the
modified polypeptides rPEG_K288-hGH and rPEG_K288-GLP1 were
obtained in a similar manner.
[0734] The purity of rPEG_K288-modified GFP was confirmed by
SDS-PAGE (FIG. 36) and analytical size exclusion chromatography
(see FIG. 37). The apparent molecular weight of rPEG_K288-modified
GFP was also measured as previously described (FIG. 41). FIG. 49
illustrates the increase in apparent molecular weight observed upon
linking a biologically active polypeptide (GLP1) to rPEG_K288
accessory polypeptide. The in vivo stability in rat and human serum
was determined as shown in FIG. 42, and in vivo pharmacokinetic
properties are indicated in FIG. 43. rPEG is stable in rat and
human serum, and rPEG288 has a halflife of about 10 to 20 hours in
rats (FIG. 43). Little immunogenicity in in vivo experiments could
be observed with this polypeptide (FIG. 44).
[0735] Protein Expression
[0736] A plasmid harboring hGH, N-terminally fused to the TEV
protease recognition site and to CBD following the T7 promoter, and
also C-terminally fused to rPEG-K288 having the repetitive sequence
(GEGGGEGGE).sub.32 (SEQ ID NO: 466), resulting in a vector
containing CBD-rPEG_K288-TEV-hGH, is transformed into the
BL21(DE3)-star E. coli strain (Novagen) and grown essentially as
described in Example 3. Cells are collected and disrupted
essentially as described in Example 3 and the cellular supernatant
is applied on beaded cellulose resin (Perloza 100; Iontosorb Inc.),
washed with buffer A (25 mM Tris pH=8.0). After applying the TEV
digest performed essentially as described in Example 3, hGH is
found in the column flow-through, while CBD-rPEG_K288 remains on
the column. The pooled flow-through is loaded on the anion-exchange
(Q-sepharose, Pharmacia), washed with buffer A (25 mM Tris pH=8.0)
and eluted from the column using a shallow linear gradient of same
buffer with 1M NaCl. The eluted hGH protein is pooled, dialyzed
against buffer A, concentrated, and purified by size-exclusion
chromatography (SEC) as the final purification. Protein purity is
estimated to be above 98%. The final protein product is a pure and
active hGH.
[0737] Cells were collected by centrifugation and the cell pellet
was resuspended in 50 ml Buffer containing 50 mM Tris pH=8.0, 100
mM NaCl, Protease inhibitors, 10% (v/v) glycerol, 0.1% Triton X-100
and DNAse. Cells were disrupted using an ultrasonic sonicator cell
disruptor, and cell debris was removed by centrifugation at 15000
RPM at 4.degree. C. Cellular supernatant was applied on an
anion-exchanger (Q-sepharose, Pharmacia), washed with buffer A (25
mM Tris pH=8.0) and eluted from the column using a linear gradient
of the same buffer with 1M NaCl. Protein eluted at about 500 mM
NaCl. The eluted fusion protein was pooled, dialyzed and loaded on
the anion-exchanger (Q-sepharose, Pharmacia), washed with buffer A
(25 mM Tris pH=8.0) and eluted from the column using a shallow
linear gradient of the same buffer with 1M NaCl. The eluted fusion
protein was pooled, dialyzed against buffer A, concentrated, and
purified by size-exclusion chromatography (SEC) as the final
purification. Protein purity was estimated to be above 98%, which
was unexpected considering only ion exchange and SEC had been used
to purify the protein in an rPEG-specific manner from whole cells.
The quantity of eluted fusion protein was determined by SDS-PAGE
analysis and by measurement of total protein concentration. A high
quantity of eluted fusion protein reflects higher solubility of the
fusion protein relative to hGH alone.
[0738] Testing of Accessory Polypeptide-Modified hGH in an hGH
Receptor Binding Assay
[0739] A plasmid harboring hGH, N-terminally fused to 288 amino
acids of rPEG-K, having the repetitive sequence (GEGGGEGGE).sub.32
(SEQ ID NO: 466) following the T7 promoter, is prepared essentially
as described in Example 1 but replacing the hGH coding sequence
with a domain antibody coding sequence. The domain antibody coding
sequence is provided in Dumoulin, M. et al., Protein Science
11:500-505 (2002). Amino acid residues 1-113 of clone dAb-Lys3 are
incorporated into the rPEG construct. This sequence is a domain
antibody that binds to hen egg lysozyme with a Kd of 11 nM. This
domain antibody sequence yields only inclusion bodies composed of
inactive protein when expressed in the cytoplasm of E. coli in the
absence of additional solubility enhancing sequences; alternatively
it can be expressed in active form in the periplasm if guided by a
leader sequence. The VHH dAb sequence is inserted upstream of the
rPEG_K288 sequence and the resulting plasmid is transformed into
BL21(DE3)-star E. coli strain (Novagen). Cells are grown, collected
and disrupted essentially as described above. The cellular
supernatant is applied on an anion-exchange (Q-sepharose,
Pharmacia), washed with buffer A (25 mM Tris pH=8.0) and protein is
eluted from the column using a linear gradient of the same buffer
with 1M NaCl. Protein elutes at about 500 mM NaCl. The eluted
fusion protein is pooled, dialyzed and loaded on the anion-exchange
(Q-sepharose, Pharmacia), washed with buffer A (25 mM Tris pH=8.0)
and eluted from the column using a shallow linear gradient of same
buffer with 1M NaCl. The eluted fusion protein is pooled, dialyzed
against buffer A, concentrated, and purified by size-exclusion
chromatography (SEC) as the final purification. Protein purity is
estimated to be above 98%.
[0740] Designs 1, 2 and 3 are similarly prepared but include 16
negatively charged amino acids (glutamate in all three cases), 41
negatively charged amino acids acids or 27 positively charged amino
acids acids, respectively, instead of the rPEG-J, -K and -L
sequences, and can have improved solubility properties.
[0741] In designing accessory polypeptide sequences, the overall
desired properties of the therapeutic protein may be considered,
including, for example, serum stability, expression level and
immunogenicity, which as described hereinabove, can also be
influenced by the choice of amino acids incorporated into the
accessory polypeptides.
Example 2
Expression of Human Growth Hormone (hGH)-Cleavable rPEG-Modified
Polypeptide
[0742] Interferon alpha 2a has 165 amino acids, a pI of 5.99, and a
molecular weight of 19241.62 corresponding to the sequence:
TABLE-US-00022 (SEQ ID NO: 470)
CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQF
QKAETIPVLHEMIQQIFNLFSTKDSSAAWDETELDKFYTELYQQLND
LEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWE
VVRAEIMRSFSLSTNLQESLRSKE
[0743] A plasmid harboring hGH, N-terminally fused to 288 amino
acids of rPEG-K288 and, having the repetitive sequence
(GEGGGEGGE).sub.32 and a TEV protease cleavage site (ENLYFQ/X),
following the T7 promoter (i.e. T7 promoter-hGH-TEV-rPEG_K288), is
transformed into BL21(DE3)-star E. coli strain and is grown as
described above. Cells are collected by centrifugation and the cell
pellet is resuspended in 50 ml Buffer containing 50 mM Tris pH=8.0,
100 mM NaCl, Protease inhibitors, 10% (v/v) glycerol, 0.1% Triton
X-100 and DNAse. Cells are disrupted using an ultrasonic sonicator
cell disruptor, and cell debris is removed by centrifugation at
15000 RPM at 4.degree. C. Cellular supernatant is applied on an
anion-exchanger (Q-sepharose, Pharmacia), washed with buffer A (25
mM Tris pH=8.0) and eluted from the column using a linear gradient
of the same buffer with 1M NaCl. Protein elutes at about 500 mM
NaCl. The eluted fusion protein is pooled, dialyzed and TEV
digested. The digestion mixture is reloaded on the anion-exchange
(Q-sepharose, Pharmacia), washed with buffer A (25 mM Tris pH=8.0)
and eluted from the column using a shallow linear gradient of the
same buffer with 1M NaCl. The eluted hGH protein is pooled,
dialyzed against buffer A, concentrated, and purified by
size-exclusion chromatography (SEC) as the final purification.
Protein purity is estimated to be above 98%.
Example 3
Expression of Human Growth Hormone (hGH) Fused to CBD and
rPEG_K288
[0744] This example describes the preparation of a
CBD-TEV-rPEG_K288-hGH fusion protein. After digestion with TEV
protease, and purification, the final protein product is
-rPEG_K288-hGH.
[0745] A pET-series vector was constructed with T7 promoter, which
expresses a protein containing cellulose binding domain (CBD) at
the N-terminus, followed by a Tomato Etch Virus (TEV) protease
cleavage site, followed by the hGH coding sequence, and by the
rPEG_K288 coding sequence: CBD-TEV-rPEG_K288-hGH. The rPEG_K288 has
the repetitive sequence (GEGGGEGGE).sub.32. The CBD sequence used
is shown in Swissprot file Q06851 and the purification of CBD
fusion proteins is described in Ofir, K. et al. (2005) Proteomics
5:1806. The sequence of the TEV cleavage site is ENLYFQ/X; G was
used in the X position. This construct was transformed into
BL21(DE3)-star E. coli strain and grown essentially as described
above, except that the CBD sequence was introduced N-terminal to
the rPEG sequence. Cells were collected and disrupted essentially
as described above. The cellular supernatant was applied on beaded
cellulose resin (Perloza 100), washed with buffer A (25 mM Tris
pH=8.0) and eluted from the column with 20 mM NaOH. pH was adjusted
by reutilizing the sample with 1M Tris buffer pH=8.0. Protein
purity was estimated to be above 90%.
[0746] rPEG_J288 has the sequence GGSGGE (SEQ ID NO: 62) and
contains 48 E residues and can therefore be used to increase the
charge density of IFNa2a. When rPEG_J288 is added to IFNa2a, the
total length of the fusion protein is 453 amino acids (calculated
as 165+288 amino acids) and the net charge is 51 (48+3), yielding a
net charge density of 0.11 (calculated as 51/453), which allows
expression of IFNa2a in soluble, active form in the cell cytoplasm.
The constructs, expression and purification methods are prepared
and carried out essentially as described in Example 1. The fusion
protein proved to be soluble and active, although some tendency
towards aggregation could still be observed under some conditions.
This can be overcome by increasing the net charge density to keep
the protein in solution. rPEGs of the same size but with more
charges, such as rPEG_L (288 AA, 25% E) and rPEG_K (288 AA, 33% E),
may be able to make the IFNa2a-rPEG fusion protein completely
soluble and actively folded. For IFNa2a-rPEG_K288 the number of
negatively charged amino acid residues in the accessory polypeptide
is 96, such that the total net charge of the fusion protein is 99
(calculated as 96+3), which means that the net charge density is
0.218 (calculated as 99/(288+165)).
Example 4
Expression of CBD-Human Growth Hormone (hGH) Fused to rPEG_K288
[0747] This example describes the preparation of
CBD-rPEG_K288-TEV-hGH, fusion protein. After TEV protease digest
and purification, the final protein product is pure hGH.
[0748] G-CSF has a length of 174 amino acids, a pI of 5.65 and a
molecular weight of 18672.29 corresponding to the sequence:
TABLE-US-00023 (SEQ ID NO: 471)
TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLCATYKLCHPEELV
LLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGIS
PELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAF
QRRAGGVLVASHLQSFLEVSYRVLRHLAQP.
Example 5
Expression of rPEG_K288-VHH, a Domain Antibody that Binds
Lysozyme
[0749] This example describes the preparation of rPEG_K288 fused to
a VHH domain antibody (dAb).
[0750] A plasmid harboring hGH, N-terminally fused to 288 amino
acids of rPEG-K, having the repetitive sequence (GEGGGEGGE).sub.32
following the T7 promoter, is prepared essentially as described in
Example 1 but replacing the hGH coding sequence with a domain
antibody coding sequence. The domain antibody coding sequence is
provided in Dumoulin, M. et al., Protein Science 11:500-505 (2002)
Amino acid residues 1-113 of clone dAb-Lys3 are incorporated into
the rPEG construct. This sequence is a domain antibody that binds
to hen egg lysozyme with a Kd of 11 nM. This domain antibody
sequence yields only inclusion bodies composed of inactive protein
when expressed in the cytoplasm of E. coli in the absence of
additional solubility enhancing sequences; alternatively it can be
expressed in active form in the periplasm if guided by a leader
sequence. The VHH dAb sequence is inserted upstream of the
rPEG_K288 sequence and the resulting plasmid is transformed into
BL21(DE3)-star E. coli strain (Novagen). Cells are grown, collected
and disrupted essentially as described above. The cellular
supernatant is applied on an anion-exchange (Q-sepharose,
Pharmacia), washed with buffer A (25 mM Tris pH=8.0) and protein is
eluted from the column using a linear gradient of the same buffer
with 1M NaCl. Protein elutes at about 500 mM NaCl. The eluted
fusion protein is pooled, dialyzed and loaded on the anion-exchange
(Q-sepharose, Pharmacia), washed with buffer A (25 mM Tris pH=8.0)
and eluted from the column using a shallow linear gradient of same
buffer with 1M NaCl. The eluted fusion protein is pooled, dialyzed
against buffer A, concentrated, and purified by size-exclusion
chromatography (SEC) as the final purification. Protein purity is
estimated to be above 98%.
[0751] The resulting VHH-rPEG_K288 protein is assayed by ELISA for
the ability to bind to its target, hen egg lysozyme (Sigma). The
protein was shown to bind specifically to lysozyme but not to three
control proteins, demonstrating that the addition of rPEG_K288 to
the VHH caused it to express in soluble and active form in the
cytoplasm of E. coli.
Example 6
Expression of IFNa2a-rPEG
[0752] This example describes the preparation of an IFNa2a-rPEG
fusion protein.
[0753] Interferon alpha 2a has 165 amino acids, a pI of 5.99, and a
molecular weight of 19241.62 corresponding to the sequence:
TABLE-US-00024 CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQ
KAETIPVLHEMIQQIFNLFSTKDSSAAWDETELDKFYTELYQQLNDLE
ACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVR
AEIMRSFSLSTNLQESLRSKE
[0754] rPEG_J288 has the sequence GGSGGE (SEQ ID NO: 62) and
contains 48 E residues. When rPEG_J288 is added to GCSF, the total
length of the fusion becomes 174+288=462 amino acids and the net
charge becomes 48+3=51, yielding a net charge density of 0.11
(calculated as 51/462). This charge is expected to be sufficient to
switch GCSF from >80% aggregation to >80% soluble protein. A
higher charge density of 0.15 or 0.2 can also be used.
[0755] The addition of 15 negatively charged amino acids to
interferon alpha brings the net charge density of the fusion
protein to -0.1 (calculated as (15+3)/(165+15)), which is desirable
for increased solubility. A higher charge density of -0.2
charges/amino acid may be obtained by including about 26 additional
negatively charged amino acid residues in the protein, for a total
of 41 negatively charged amino acid residues. Since the combined
length is 206 amino acids (calculated as 165+41), a charge density
of -0.2 requires 41 total negatively charged amino acid residues
(calculated as 0.2.times.205 amino acids), which means the
accessory protein may include 38 negatively charged residues
(calculated as 41-3).
[0756] By similar reasoning, to reach a net charge density of +0.1,
the accessory polypeptide may include 15+6=21 positively charged
amino acids.
[0757] rPEG_J288-GFP--represents the protein sequence composed of
the repetitive sequence (GSGGEG).sub.48 (SEQ ID NO: 472) fused to
GFP protein sequence.
[0758] rPEG_K288-GFP--represents the protein sequence composed of
the repetitive sequence (GEGEGGGEG).sub.32 (SEQ ID NO: 473) fused
to GFP sequence.
[0759] rPEG_L288-GFP--represents the protein sequence composed of
the repetitive sequence (SSESSESSSSES).sub.24 (SEQ ID NO: 474)
fused to GFP sequence.
[0760] rPEG_O336-GFP--represents the protein sequence composed of
the repetitive sequence (SSSSSESSSSSSES).sub.24 (SEQ ID NO: 475)
fused to GFP sequence.
[0761] rPEG_P320-GFP--represents protein sequence composed of the
repetitive sequence (SSSESSSSES).sub.32 (SEQ ID NO: 476) fused to
GFP sequence.
[0762] For GCSF, the addition of 14 negatively charged amino acids
brings the net charge density of the fusion protein to about -0.1
(calculated as (14+4)/(174+14)), which is typically desirable for
solubility. The preferred charge density of -0.2 would require
about 26 additional negatively charged amino acid residues, for a
total of 41 negatively charged amino acid residues, since combined
length is 217 amino acids (calculated as 174+43). An alternatively
chosen charge density of -0.2 requires 43 total negatively charged
amino acid residues (calculated as 0.2.times.217 amino acids),
which means the accessory protein should contain 39 negatively
charged residues (calculated as 43-4).
[0763] In another alternative design, an accessory protein with
positively charged amino acids to reach a net charge density of
+0.1 is desired, which requires a net positive charge of +21. This
could be achieved by addition of an accessory protein containing 25
positive charges (calculated as 25-4=21), resulting in a combined
fusion protein length of 209 amino acids.
[0764] Experimental Results:
[0765] rPEG_J288 has the sequence GGSGGE and contains 48 E
residues. When rPEG_J288 is added to GCSF, the total length of the
fusion becomes 174+288=462 amino acids and the net charge becomes
48+3=51, yielding a net charge density of 0.11 (calculated as
51/462). This charge is expected to be sufficient to switch GCSF
from >80% aggregation to >80% soluble protein. A higher
charge density of 0.15 or 0.2 can also be used.
[0766] Using standard molecular biological techniques any of the
examples provided hereinabove may be modified to use a different
rPEG module fused to the therapeutic protein. The present inventors
have shown that a net charge density of 0.1 provides improved
solubility of proteins in the cytoplasm (e.g., with GFP, hGH and
IFNa2a), whereas a net charge density of around 0.2 provides highly
soluble proteins with no tendency towards aggregation.
Example 8
Solubility of Different rPEG Sequences Fused to GFP when
Recombinantly-Expressed in the Cytoplasm of E. coli
[0767] The following protein sequences were prepared and tested in
this experiment:
[0768] rPEG with the sequence (GGSGGE).sub.48 (SEQ ID NO: 455) was
fused to green fluorescent protein (GFP) yielding clone LCW0066.
The fusion protein also carried an N-terminal Flag tag and a His6
tag (SEQ ID NO: 1) between rPEG and GFP. The fusion protein was
expressed in E. coli using a standard T7 expression vector. Cells
were cultured in LB medium and expression was induced with IPTG.
After expression, the cells were lysed by heating the pellet to
70.degree. C. for 15 min. Most E. coli proteins denatured during
this heat step and could be removed by centrifugation. The fusion
protein was purified from the supernatant by IMAC chromatography
followed by purification by immobilized anti-Flag (Sigma). The
fusion proteins were analyzed by size exclusion chromatography
(SEC) using 10/30 Superdex-200 (GE, Amersham). The column was
calibrated with globular proteins (diamonds). The fusion protein
comprising rPEG_J288 and GFP eluted significantly earlier from the
column then predicted based on its calculated molecular weight.
Based on the calibration with globular proteins SEC measured an
apparent molecular weight of the fusion protein of 243 kDa, which
is almost 5 times larger than the calculated molecular weight of 52
kDa. A related fusion protein (LCW0057) contained rPEG36 and had an
apparent molecular weight of 55 kDa versus a calculated molecular
weight of 32 kDa. Comparison of the LCW0066 and LCW0057 shows a
difference in apparent molecular weight of 189 kDa which is caused
by the addition of an rPEG chain with a calculated molecular weight
of 20 kDa. Thus, one can calculate that the addition of an rPEG
tail with a calculated molecular weight of 20 kDa lead to an
increase in molecular weight of 189 kDa.
[0769] rPEG_K288-GFP--represents the protein sequence composed of
the repetitive sequence (GEGEGGGEG).sub.32 fused to GFP
sequence.
[0770] rPEG_L288-GFP--represents the protein sequence composed of
the repetitive sequence (SSESSESSSSES).sub.24 fused to GFP
sequence.
[0771] rPEG_O336-GFP--represents the protein sequence composed of
the repetitive sequence (SSSSSESSSSSSES).sub.24 fused to GFP
sequence.
[0772] rPEG_P320-GFP--represents protein sequence composed of the
repetitive sequence (SSSESSSSES).sub.32 fused to GFP sequence.
[0773] rPEG_J288, K288, L288, O336 and P320, each fused to the
N-terminus of GFP, were introduced in to the E. coli strain
BL21-Star, grown on LB-Kan agar plates, and incubated for 16 hours
at 37.degree. C. The next day a single colony of each construct was
inoculated into 2 mL of TB125 growth medium and grown for 5 hr. 100
ul of the each bacterial broth were transferred into 10 ml flasks
with TB125 medium+Kan, and grown until the OD.sub.600 has reached
.about.0.6. The growth flasks were transferred to 26 C, and induced
with 100 uM IPTG for 16 hours with shaking. Bacterial cells were
centrifuged and resuspended in 10 ml of PBS, and later disrupted by
sonication. 100 .mu.l aliquots of each sample were centrifuged and
their supernatant collected. Finally, 100 .mu.l of cellular lysate
and soluble fractions were read and compared for GFP
florescence.
[0774] The results are shown in FIG. 33. GFP modified with rPEG
accessory polypeptides J, K and L had most of the GFP signal in the
soluble form, while a substantial fraction of GFP fluorescence was
retained in the insoluble form in rPEG O and rPEG P fusion
sequences. Furthermore, GFP fused to Ser-rich rPEG sequences
expressed better then Gly-rich sequences, while the Gly-rich rPEG
sequences retained the majority of the GFP fluorescence in the
soluble form.
Example 9
Determination of Properties of Accessory-Linked Polypeptides
[0775] Determination of Serum Stability of an Accessory-Linked
Polypeptide.
[0776] The fusion protein Flag-rPEG_J288-H6-GFP, purified as shown
in FIG. 19, containing an N-terminal Flag tag and the accessory
sequence rPEG_J288 fused to the N-terminus of green fluorescent
protein is incubated in 50% mouse serum at 37 C for 3 days. Samples
are withdrawn at various time points and analyzed by SDS PAGE
followed by detection using Western analysis. An antibody against
the N-terminal flag tag is used for Western detection. FIG. 20
indicates that the accessory protein is stable in serum for at
least three days.
[0777] Determination of Plasma Half-Life of an Accessory-Linked
Polypeptide.
[0778] The plasma half-life of accessory-linked polypeptides can be
measured after i.v. or i.p. injection of the accessory polypeptide
into catheterized rats essentially as described by [Pepinsky, R.
B., et al. (2001) J Pharmacol Exp Ther, 297: 1059-66]. Blood
samples can be withdrawn at various time points (5 min, 15 min, 30
min, 1 h, 3 h, 5 h, 1 d, 2 d, 3 d) and the plasma concentration of
the accessory polypeptide can be measured using ELISA.
Pharmacokinetic parameters can be calculated using WinNonlin
version 2.0 (Scientific Consulting Inc., Apex, N.C.). To analyze
the effect of the rPEG-linked polypeptide one can compare the
plasma half-life of a protein containing the rPEG polypeptide with
the plasma half-life of the same protein lacking the rPEG
polypeptide.
[0779] The in vivo halflife or LCW0057 and LCW0066 was studied in
rats. Both proteins were injected intravenously into rats. Serum
samples were analyzed for the presence of GFP between 5 min and 3
days after injection. For rats injected with LCW0057 no GFP was
detectable 24 h after protein injection. This suggests a halflife
of the protein of 1-3 h. In contrast, LCW0066 was detectable even
48 h after injection and one rat showed detectable GFP even 3 days
after injection. This shows that LCW0066 has a serum halflife in
rats of about 10 hours which is much longer than expected for a
protein with a calculated molecular weight of 52 kDa.
[0780] Solubility Testing of Accessory-Linked Polypeptides.
TABLE-US-00025 (SEQ ID NO: 477)
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEG
GSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSE
GSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGE
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEG
GEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSE
GGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGE LCW0219.068 (SEQ
ID NO: 478) GEGSGEGSEGEGSEGSGEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEG
GEGSGEGEGSGEGSEGEGGGEGSEGEGGSEGSEGEGGSEGSEGEGGEG
SGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSE
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEG
GSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSE
GSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGE LCW0220.038 (SEQ
ID NO: 479) SEGESEESSESGGESSSGGGSEESSEEGSGGGSEGEGEESSGSEGGGG
SGEGSEGGSEEGSEESSEGESEESSESGGESSSGGGSEESSEEGSGGG
SGESGSGSSGSESEGGSEGESEESSGGGGSEGSEGESEESSESGGESS
SGGGSEESSEEGSGGGSEEESGEGSGEGSE GSSGEGSEESSGGSEGGG
SGGSGGEGSGESGSGSSGSESEGGSEGESEESSGGGGSEGSSEESGGS
SEEGSEGSSGGESEESSEGESGGGSGGGSEGS LCW0220.055 (SEQ ID NO: 480)
SEGESEESSESGGESSSGGGSEESSEEGSGGGSEGESEESSESGGESS
SGGGSEESSEEGSGGGSGESGSGSSGSESEGGSEGESEESSGGGGSEG
SESEGEEGSEEGSGEGSGEGGGESSEEGESESSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSGESGSGSSGSESEGGSEGESEESSGGGGSEG
SGESGSGSSGSESEGGSEGESEESSGGGGSEGSSEESGGSSEEGSEGS
SGGESEESSEGESGGGSGGGSEGS LCW0220.064 (SEQ ID NO: 481)
SEGESEESSESGGESSSGGGSEESSEEGSGGGSEGEGEESSGSEGGGG
SGEGSEGGSEEGSEESSEGESEESSESGGESSSGGGSEESSEEGSGGG
SGESGSGSSGSESEGGSEGESEESSGGGGSEGSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSESEGEEGSEEGSGEGSGEGGGESSEEGESES
SEGESEESSESGGESSSGGGSEESSEEGSGGGSSEESGGSSEEGSEGS
SGGESEESSEGESGGGSGGGSEGS LCW0220.093 (SEQ ID NO: 482)
SEGESEESSESGGESSSGGGSEESSEEGSGGGSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSEGESEESSESGGESSSGGGSEESSEEGSGGG
SEEGSGESSGGSESEGSGGESEGGSGGEGGEGSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSSEESGGSSEEGSGGGSESGEESGSGEESEGG
SGGSGGEGSGESGSGSSGSESEGGSEGESEESSGGGGSEGSSGEGEES
SEGEGGESSEEGSGGSSEEGSGEG
[0781] Determination of Serum Concentration of rPEG-GFP Following
Subcutaneous Injection of Encapsulated Protein
[0782] The serum concentration of rPEG-GFP and GFP can be tested by
following a single subcutaneous injection of rPEG-GFP microspheres
or GFP microspheres, respectively, in a model laboratory organism.
Encapsulated rPEG-GFP or encapsulated GFP is injected into mice,
rats, rabbits, or other model organisms (1 mL/kg of body weight) to
evaluate in vivo release rates. Serum samples are collected daily
for one month. Serum concentrations or rPEG-GFP are measured using
the sandwich ELISA assay described above. rPEG-GFP fusion
polypeptides are present at a high concentration much longer than
GFP due to a slower release from the microspheres and a longer
subsequent half life.
Example 11
Polymer Encapsulated Interferon-Alpha (IFN-Alpha) Linked to an
Accessory Polypeptide
[0783] This example describes a depot formulation of rPEG-IFN-alpha
which can extend the dosing interval of this polypeptide. The
rPEG-fused IFN-alpha is constructed essentially as described for
the hGH-rPEG fusion construct in Example 3, except GLP-1 encoding
sequences are replaced by IFN-alpha coding sequence. All other
methodologies and techniques, including encapsulation
methodologies, are essentially as described in Example 10.
[0784] This example describes the construction of scFv-rPEG50
fusions. Two scFvs were made, one that binds Her2 and one that
binds epidermal growth factor receptor (EGFR). Each scFv was
genetically fused to the N-terminus of rPEG50, respectively. The
scFv constructs were cloned into an expression vector with T7
promoter and encoding rPEG50-FLAG-tag-hexahistidine (SEQ ID NO: 1),
resulting in constructs expressing scFv-rPEG50-FLAG-His6 (SEQ ID
NO: 1). The stuffer fragment was removed by restriction digest
using NdeI and BsaI endonucleases. The synthetic scFv fragments
were amplified by polymerase chain reaction (PCR), which introduced
NdeI and BbsI restriction sites that are compatible with the
stuffer construct. Restriction digested scFv fragments and stuffer
construct were ligated using T4 DNA ligase and electrotransformed
into E. coli BL21 (DE3) Gold. The resulting DNA construct is shown
in FIG. 64a, where the light chain (vL) and heavy chain (vH)
variable fragments are separated by rPEGY30, a 30 amino acid
sequence (SGEGSEGEGGGEGSEGEGSGEGGEGEGS) (SEQ ID NO: 483). The
Y30-amino acid-encoding sequence was flanked by AgeI and KpnI
restriction sites for convenient removal or replacement of the
linker sequence between vL and vH. The constructs were confirmed by
DNA sequencing. The protein sequences for the aHer230-rPEG
(M.W.=80,044 Da) and aEGFR30-rPEG (M.W.=80,102 Da) constructs are
shown in FIGS. 64b and d, respectively.
Example 12
Construction of Non-Repetitive Accessory Polypeptides
[0785] This example describes the construction of a library of
accessory polypeptide segments from synthetic oligonucleotides.
FIG. 78 lists the amino acid sequences of that were encoded by
synthetic oligonucleotides. For each amino acid sequence we used
two complementary oligonucleotides. The sequences were designed as
codon libraries, i.e. multiple different codons were allowed but
all sequences encoded just one amino acid sequence. The
complementary oligonucleotides were annealed by heating followed by
cooling. The oligonucleotides were designed to generate 4 base-pair
overlaps during annealing as illustrated in FIG. 79. Two additional
annealed oligonucleotides were also added that acted as terminators
during the multimerization by ligation reaction. FIG. 79 shows the
ligation of annealed oligonucleotides that yielded gene fragments
encoding accessory polypeptide segments of varying length. The
resulting ligation mixture was separated by electrophoresis as
shown in FIG. 79 and the ligation product encoding URP36 was
isolated. This ligation product was ligated into an expression
vector and the library of URP36 segments was expressed as fusion
protein to GFP.
[0786] Accessory polypeptide sequences prepared in this manner are
shown in FIG. 80. Additional sequences are disclosed below:
TABLE-US-00026 LCW0219.040
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEG
GSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSE
GSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGE
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGSEGSGEGEG
GEGSGEGEGSGEGSEGEGGGEGSEGEGSGEGGEGEGSEGGSEGEGGSE
GGEGEGSEGSGEGEGSEGGSEGEGSEGGSEGEGSEGSGEGEGSEGSGE LCW0219.068
GEGSGEGSEGEGSEGSGEGEGSEGGSEGEGSEGSGEGEGSEGSGEGEG
GEGSGEGEGSGEGSEGEGGGEGSEGEGGSEGSEGEGGSEGSEGEGGEG
SGEGEGSEGSGEGEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSE
GEGSGEGSEGEGSEGSGEGEGSEGSGEGEGGSEGSEGEGGSEGSEGEG
GSEGSEGEGSGEGSEGEGGSEGSEGEGSGEGSEGEGSEGGSEGEGGSE
GSEGEGSGEGSEGEGGEGGSEGEGSEGSGEGEGSGEGSEGEGSEGSGE LCW0220.038
SEGESEESSESGGESSSGGGSEESSEEGSGGGSEGEGEESSGSEGGGG
SGEGSEGGSEEGSEESSEGESEESSESGGESSSGGGSEESSEEGSGGG
SGESGSGSSGSESEGGSEGESEESSGGGGSEGSEGESEESSESGGESS
SGGGSEESSEEGSGGGSEEESGEGSGEGSEGSSGEGSEESSGGSEGGG
SGGSGGEGSGESGSGSSGSESEGGSEGESEESSGGGGSEGSSEESGGS
SEEGSEGSSGGESEESSEGESGGGSGGGSEGS LCW0220.055
SEGESEESSESGGESSSGGGSEESSEEGSGGGSEGESEESSESGGESS
SGGGSEESSEEGSGGGSGESGSGSSGSESEGGSEGESEESSGGGGSEG
SESEGEEGSEEGSGEGSGEGGGESSEEGESESSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSGESGSGSSGSESEGGSEGESEESSGGGGSEG
SGESGSGSSGSESEGGSEGESEESSGGGGSEGSSEESGGSSEEGSEGS
SGGESEESSEGESGGGSGGGSEGS LCW0220.064
SEGESEESSESGGESSSGGGSEESSEEGSGGGSEGEGEESSGSEGGGG
SGEGSEGGSEEGSEESSEGESEESSESGGESSSGGGSEESSEEGSGGG
SGESGSGSSGSESEGGSEGESEESSGGGGSEGSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSESEGEEGSEEGSGEGSGEGGGESSEEGESES
SEGESEESSESGGESSSGGGSEESSEEGSGGGSSEESGGSSEEGSEGS
SGGESEESSEGESGGGSGGGSEGS LCW0220.093
SEGESEESSESGGESSSGGGSEESSEEGSGGGSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSEGESEESSESGGESSSGGGSEESSEEGSGGG
SEEGSGESSGGSESEGSGGESEGGSGGEGGEGSGESGSGSSGSESEGG
SEGESEESSGGGGSEGSSEESGGSSEEGSGGGSESGEESGSGEESEGG
SGGSGGEGSGESGSGSSGSESEGGSEGESEESSGGGGSEGSSGEGEES
SEGEGGESSEEGSGGSSEEGSGEG
Example 13
Construction of rPEG_Y576
[0787] This example describes the construction of a library of URP
segments from synthetic oligonucleotides. FIG. 78 lists the amino
acid sequences encoded by the synthetic oligonucleotides. For each
amino acid sequence we used two complementary oligonucleotides. The
sequences were designed as codon libraries, i.e. multiple different
codons were allowed but all sequences encoded only one amino acid
sequence. The complementary oligonucleotides were annealed by
heating followed by cooling. The oligonucleotides were designed to
generate 4 base-pair overlaps during annealing as illustrated in
FIG. 79a. We also added two additional annealed oligonucleotides
that acted as terminators during the multimerization by ligation
reaction. FIG. 79b illustrates the ligation of annealed
oligonucleotides that yielded gene fragments encoding URP segments
of varying lengths. The resulting ligation mixture was separated by
electrophoresis as shown in FIG. 79b and the ligation product
encoding URP36 was isolated. This ligation product was ligated into
an expression vector and the library of URP36 segments was
expressed as fusion protein to GFP (FIG. 81). Library members with
good expression were identified based on their strong fluorescence
intensity.
[0788] The library members of URP36 were dimerized and the
resulting library of URP72 was screened for high level expression.
This process of dimerization and screening was repeated one more
time to generate URP144. FIG. 80 shows a collection of sequences.
The sequences conform to the design of the libraries but most
library members differ in their actual sequences. This collection
of URP_Y144 was dimerized two more times to generate collections of
URP_Y288 and URP_Y576. The amino acid sequence of one isolate of
URP_Y576 is shown in FIG. 80. The resulting isolates were evaluated
for expression, aggregation, and immunogenicity to identify URP
that is most suitable for fusion to a drug protein.
Example 14
Construction of scFv-rPEG50 Fusions
[0789] Construction of Anti-Her2 and Anti-EGFR
[0790] This example describes the construction of scFv-rPEG50
fusions. Two scFvs were made, one that binds Her2 and one that
binds epidermal growth factor receptor (EGFR). Each scFv was
genetically fused to the N-terminus of rPEG50, respectively. The
scFv constructs were cloned into an expression vector with T7
promoter and encoding rPEG50-FLAG-tag-hexahistidine, resulting in
constructs expressing scFv-rPEG50-FLAG-His6. The stuffer fragment
was removed by restriction digest using NdeI and BsaI
endonucleases. The synthetic scFv fragments were amplified by
polymerase chain reaction (PCR), which introduced NdeI and BbsI
restriction sites that are compatible with the stuffer construct.
Restriction digested scFv fragments and stuffer construct were
ligated using T4 DNA ligase and electrotransformed into E. coli
BL21 (DE3) Gold. The resulting DNA construct is shown in FIG. 64a,
where the light chain (vL) and heavy chain (vH) variable fragments
are separated by rPEGY30, a 30 amino acid sequence
(SGEGSEGEGGGEGSEGEGSGEGGEGEGS). The Y30-amino acid-encoding
sequence was flanked by AgeI and KpnI restriction sites for
convenient removal or replacement of the linker sequence between vL
and vH. The constructs were confirmed by DNA sequencing. The
protein sequences for the aHer230-rPEG (M.W.=80,044 Da) and
aEGFR30-rPEG (M.W.=80,102 Da) constructs are shown in FIG. 64b and
d, respectively.
[0791] The anti-Her230-rPEG and aEGFR30-rPEG fusions in E. coli
BL21 (DE3) Gold were expressed by inducing with 0.2 mM isopropyl
.beta.-D-1 thiogalactopyranoside (IPTG) at 20.degree. C. Cells were
harvested by centrifugation and lysed in BugBuster plus Benzonase
in phosphate buffered saline. Lysates were clarified by
centrifugation and supernatants (soluble fractions) loaded onto
4-12% SDS PAGE gels. The scFv-rPEG fusions are overexpressed and
visible in E. coli lysates at approximately 80 kDa (FIG. 64c).
Example 15
Characterization of the scFv-rPEG50 Fusion aHer230-rPEG
[0792] Purification
[0793] A single-chain fragment variable (scFv) antibody fragment
targeting the Her2 receptor and fused to rPEG, to yield
aHer230-rPEG, which was expressed and purified from the cytosol of
E. coli. The aHer230-rPEG plasmid was transformed into
BL21(DE3)-Gold and expression of the recombinant antibody fragment
was induced with 0.2 mM isopropyl .beta.-D-1 thiogalactopyranoside
(IPTG) at 20.degree. C. Cells were harvested by centrifugation and
resuspended in 30 mM sodium phosphate, 0.3 M sodium chloride, 10%
glycerol, and 20 mM imidazole, pH 7.5. Lysis was accomplished by
sonication and the soluble protein was purified by standard
chromatographic methods including, immobilized metal affinity
chromatography (IMAC), hydrophobic interaction chromatography
(HIC), and ion exchange chromatography (IEC).
[0794] Binding
[0795] To evaluate target (Her2) binding, aHer230-rPEG was
expressed in BL21(DE3)-Gold as described above. Cells were lysed by
resuspension in phosphate buffer saline (PBS) containing BugBuster
reagent and 5 U/ml of benzonase (Novagen). The suspension was
incubated for 20 minutes at room temperature prior to
centrifugation at 10000 rpm for 10 minutes. The soluble fraction
was then serially diluted five-fold into PBS containing 1% bovine
serum albumin (BSA) and 0.05% Tween-20. Serially diluted
aHer230-rPEG was added to the wells of a 96-well plate which had
been coated with a Her2-Fc fusion protein (R&D Systems) and
blocked with 1% BSA. The binding reaction was incubated at room
temperature for 2 hours with gentle agitation. The wells were
thoroughly washed with PBS containing 0.05% Tween-20 and the bound
aHer230-rPEG was detected with an HRP-conjugated anti-FLAG antibody
(Sigma). FIG. 62a shows that aHer230-rPEG binds to Her2-Fc fusion
protein and does not non-specifically bind to human IgG. The
binding data are presented as a function of the sample dilution.
The half maximal binding (EC50) is estimated to be achieved at
approximately 10 nM aHer230-rPEG.
[0796] This example describes the construction and bacterial
expression of a Fab-rPEG fusion protein. The fragment, antigen
binding (Fab) of an IgG can be fused to rPEG as a means of
improving soluble Fab expression as well as half-life extension.
The expression construct was designed a bicistronic RNA message
that is under the control of an inducible arabinose promoter (FIG.
67). The bicistronic message is terminated at a hairpin terminator,
such as the T7 terminator sequence. Each cistron or gene has a
ribosomal binding site (RBS) to initiate translation and a stop
codon (TAA, TGA, or TAG) to stop translation. The light chain
(vL/cL) or heavy chain (vH/cH) sequence can be genetically fused to
rPEG and followed by an affinity tag such as HA (hemagglutinin), H
(hexahistidine) (SEQ ID NO: 1), and/or FLAG tag. DNA constructs can
encode the heavy chain first or light chain last (HL) or light
chain first and heavy chain last (LH) as shown in FIG. 67. Protein
expression from this type of construct yields two approximately 50
kDa chains that form a full Fab fragment of approximately 100 kDa
in size, which includes a total of 50 kDa of rPEG sequence.
[0797] SS-Bond Oxidation
[0798] The expression of disulfide containing proteins in the
cytoplasm of E. coli is often unsuccessful due to the highly
reducing nature of the cytoplasm, which inhibits disulfide
formation. However, disulfide bonds may form following cell lysis
when the proteins are exposed to more oxidizing conditions. As
demonstrated above, aHer230-rPEG expressed in E. coli binds to its
target, Her2, suggesting that the protein is properly folded. To
test whether the two disulfide bonds, one each in the vH and vL
domains, of aHer230-rPEG were properly formed in the purified
protein, the number of free sulfhydryls in the denatured, purified
protein was compared to a fully reduced form of the scFv. Purified
aHer230-rPEG was denatured in 6 M urea or in 6 M urea supplemented
with 10 mM Tris[2-carboxyethyl]phosphine (TCEP) for 1 hour at room
temperature. The samples were then desalted on Sephadex G-25 resin
to remove the urea and the TCEP. Immediately, Ellman's reagent
(5,5'-dithio-bis-[2-nitrobenzoic acid]) was added to a final
concentration of 20 mM and the reaction proceeded for 15 minutes.
Finally, the absorbance of each solution was measured at 412 nm.
Denatured aHer230-rPEG exhibits very little absorbance, which
suggests that the purified sample is completely oxidized (FIG.
62c). The denatured and reduced reaction (FIG. 62c) shows the
signal expected if all of the cysteines in aHer230-rPEG were in the
reduced state. Thus, all of the disulfides within the anti-Her2
scFv were properly formed.
Example 16
Construction of the Diabody aHer203-rPEG
[0799] A diabody can be formed by linking the vH and vL domains
with a linker less than 10 amino acids. The short linker does not
allow scFv formation and as a result the vH and vL domains bind to
a complementary, second vH-vL chain, forming a 4-domain, 2 chain 50
kD complex. The diabody was constructed from a single-chain
fragment variable (scFv) antibody fragment that binds Her2, which
was genetically fused to the N-terminus of rPEG50. Constructs were
generated by replacing the Y30 scFv linker sequence from Example 1
with three amino acids (SGE) to allow a diabody format (FIG. 65a).
The SGE sequence was introduced by polymerase chain reaction (PCR),
also introducing NdeI and BbsI restriction sites that are
compatible with the rPEG stuffer construct. Diabody-encoding
fragments were then cloned as in Example 1. The construct was
confirmed by DNA sequencing. The protein sequence for the
aHer203-rPEG diabody (M.W.=156,598 Da as diabody or 78,299 Da
monomer sequence, including rPEG) is shown in FIG. 65b.
[0800] The aHer203-rPEG in BL21 (DE3) Gold was expressed by
inducing with 0.2 mM isopropyl 13-D-1 thiogalactopyranoside (IPTG)
at 20.degree. C. Cells were harvested by centrifugation and lysed
in BugBuster/Benzonase in phosphate buffered saline. Lysates were
clarified by centrifugation and supernatants (soluble fractions)
loaded onto 4-12% SDS PAGE gels. The aHer203-rPEG diabody was
detected in E. coli lysates at approximately 90 kDa (FIG. 65c).
Example 17
Characterization of the Diabody-rPEG50 Fusion aHer203-rPEG
[0801] Purification
[0802] A diabody can be formed by linking the vH and vL domains
with a linker comprising fewer than 10 amino acids. The short
linker does not allow scFv formation and as a result the vH and vL
domains bind to a complementary vH-vL chain. The diabody is a
useful format to generate a bivalent, and possibly bispecific,
therapeutic lacking effector Fc function.
[0803] A diabody that binds to Her2 was designed as described
above. To evaluate target (Her2) binding, recombinant aHer203-rPEG
diabody was expressed and purified as described for aHer230-rPEG.
aHer203-rEPG50 was transformed into BL21(DE3)-Gold and expression
of the recombinant antibody fragment was induced with 0.2 mM
isopropyl .beta.-D-1 thiogalactopyranoside (IPTG) at 20.degree. C.
Cells were harvested by centrifugation and resuspended in 30 mM
sodium phosphate, 0.3 M sodium chloride, 10% glycerol, an 20 mM
imidazole, pH 7.5. Lysis was accomplished by sonication and the
soluble protein was purified by standard chromatographic methods
including, immobilized metal affinity chromatography (IMAC),
hydrophobic interaction chromatography (HIC), and ion exchange
chromatography (IEC).
[0804] Binding
[0805] Binding of the aHer203-rPEG diabody to its target was
performed as described for aHer230-rPEG. Cells were lysed by
resuspension in phosphate buffer saline (PBS) containing BugBuster
reagent and 5 U/ml of benzonase (Novagen). The suspension was
incubated for 20 minutes at room temperature prior to
centrifugation at 10000 rpm for 10 minutes. The soluble fraction
was then serially diluted five-fold into PBS containing 1% bovine
serum albumin (BSA) and 0.05% Tween-20, hereafter referred to as
ELISA binding buffer. Serially diluted aHer203-rPEG diabody was
added to the wells of a 96-well plate which had been coated with a
Her2-Fc fusion protein (R&D Systems) and blocked with 1% BSA.
The binding reaction was incubated at room temperature for 2 hours
with gentle agitation. The wells were thoroughly washed with PBS
containing 0.05% Tween-20 and the bound aHer203-rPEG diabody was
detected with an HRP-conjugated anti-FLAG antibody (M2, Sigma).
FIG. 63a shows that the aHer203-rPEG diabody binds to the Her2-Fc
fusion proteins and does not non-specifically bind to human IgG.
The binding data are presented as a function of the sample
dilution. The half maximal binding (EC50) is estimated to be
achieved at approximately 10 nM aHer203-rPEG diabody. Thus, a
functional aHer203 diabody with an rPEG accessory polypeptide can
be expressed in the cytosol of E. coli.
[0806] SE-HPLC
[0807] Diabodies have been explored as potential bivalent
therapeutics, however, their propensity to reassort into higher
order oligomers--trimers, tetramers, etc.--has limited their
utility. Reassortment is particularly problematic for
manufacturing, because after purifying a monomeric scFv, upon
storage in liquid form it will slowly but predictably reassort to
yield dimers, and higher multimers. This leads not only to large
losses in the amount of protein of the correct format that can
finally be obtained, but it also leads to heterogeneity in the
product upon storage and heterogeneity in pharmacokinetics and in
efficacy. The equilibrium between monomers and multimers of scFv
can be affected by the length of the linker between vH and vL
domains. In general constructs with linkers of more than 12 to 14
amino acids occur predominantly in monomeric form while scFv with
linkers shorter than 12 amino acids occur mostly in multimeric form
[Desplancq, D., et al. (1994) Protein Eng, 7: 1027] [Whitlow, M.,
et al. (1994) Protein Eng, 7: 1017] [Hudson, P. J., et al. (1999) J
Immunol Methods, 231: 177]. Increasing the length of the linker
between vH and vL to 30 amino acids shifts the equilibrium into the
direction of monomers [Desplancq, D., et al. (1994) Protein Eng, 7:
1027]. Linker lengths between 3 and 7 amino acids favor the
formation of diabodies [Dolezal, O., et al. (2000) Protein Eng, 13:
565] [Kortt, A. A., et al. (1997) Protein Eng, 10: 423]. Linkers of
5-10 amino acids give rise to mostly dimer. Antigen presence and
ionic strength can affect monomer-dimer transition [Arndt, K. M.,
et al. (1998) Biochemistry, 37: 12918]. Linkers shorter than 3
amino acids favor the formation of triabodies and tetrabodies [Le
Gall, F., et al. (1999) FEBS Lett, 453: 164] [Dolezal, O., et al.
(2000) Protein Eng, 13: 565] [Kortt, A. A., et al. (1997) Protein
Eng, 10: 423].
[0808] The oligomerization state of the aHer203-rPEG diabody by
SE-HPLC has been evaluated and demonstrated that it does not
reassort. FIG. 63b, shows the size-exclusion chromatograms of
aHer230-rPEG single chain and the aHer203-rPEG diabody. It
demonstrates that the diabody is largely dimeric and,
significantly, it contains less than 3% trimer or tetramer forms.
The oligomerization state of the aHer203-rPEG diabody has also been
monitored during storage at 4.degree. C. and reassortment was not
observed (FIG. 63c). The rPEG accessory polypeptide helps prevent
the reassortment of the diabody, thus enabling the purification and
formulation of a homogenous product.
Example 18
Codon Optimization of an Fc Domain for Bacterial Expression
[0809] The Human IgG1 constant fragment (Fc) was synthesized and
fused to rPEG25-Green Fluorescent Protein (GFP) to yield
Fc-rPEG25-GFP, as shown in FIG. 66a. The DNA encoding the Fc
sequence was constructed in vitro using E. coli optimized codons.
The Fc codon library was assembled using 60-mer oligonucleotides
with 20 nucleotide overlap (annealing) regions. Multiple codons
were introduced in the non-overlapping regions of the synthetic
oligonucleotides. The resulting codon library had a theoretical
size of approximately 10,000 such that all nucleotide sequences
encode the desired Fc sequence. A total of 18 oligonucleotides were
assembled in the presence of dNTPs and DNA polymerase to a final
size of 684 bp. The Fc codon library was amplified by PCR using
primers that create NdeI and BbsI compatible ends. The DNA fragment
was restriction digested and ligated into an rPEG25-GFP vector at
NdeI and BsaI restriction digestion sites. The ligated DNA was
transformed into BL21 (DE3) Gold. A total of 1000 clones were
isolated, grown in 96-well format, and replicated to plates
containing 0.2 mM IPTG to induce expression. Constructs that were
well-expressed showed high levels of fluorescence under ultraviolet
light. A total of 17 clones were characterized as highly
fluorescent. These clones were expressed in 1 ml cultures using 0.2
mM IPTG, cells were harvested by centrifugation, and lysed with
Bugbuster plus Benzonase in phosphate buffered saline. Soluble
fractions were loaded onto 4-12% SDS PAGE gels (FIG. 66b).
Recombinant Fc-rPEG fusions have an observed molecular weight on
SDS-PAGE of approximately 80-90 kDa (predicted MW is about 80 but
the rPEG causes proteins to run high). The DNA sequence of a codon
optimized Fc is shown in FIG. 66c.
Example 19
Expression and Characterization of Fc-rPEG Fusion Proteins
[0810] The Fc fragment of IgG1 was fused to rPEG as detailed in
Example 5 (and variants are illustrated in FIG. 31), and expressed
in the cytoplasm of E. coli. Cells expressing the fusion protein
were resuspended in buffer, in this case 20 mM sodium phosphate pH
7.0, and the cells were lysed by sonication. The insoluble material
was removed by centrifugation and Fc-rPEG-GFP was purified from the
soluble fraction. Intact, folded Fc fragment binds to Protein A and
therefore can be conveniently purified by affinity chromatography
using immobilized recombinant Protein A. Soluble lysate containing
the Fc fusion was applied to a Protein A column (GE Healthcare) and
microbial proteins were removed by extensive washing with phosphate
buffer. The Fc-rPEG-GFP fusion protein was eluted from the Protein
A column using either glycine buffer or sodium citrate buffer pH
3.0. The pH of the elution fractions was immediately adjusted with
and equal amount of Tris buffer pH 8.5. The purified protein was
analyzed by SDS-PAGE under reducing and oxidizing conditions. A
single band of approximately 80 kDa was detected under reducing
conditions, while bands at 160 kDa (hinge oxidized) and 80 kDa
(hinge reduced) were detected under oxidizing conditions. The
addition of either CuSO.sub.4, dehydroascorbic acid, or other
oxidizing reagents was used to catalyze the complete oxidation of
the hinge cysteines.
Example 20
Construction and Bacterial Expression of a Fab-rPEG Fusion
Protein
[0811] This example describes the construction and bacterial
expression of a Fab-rPEG fusion protein. The fragment, antigen
binding (Fab) of an IgG can be fused to rPEG as a means of
improving soluble Fab expression as well as half-life extension.
The expression construct was designed a bicistronic RNA message
that is under the control of an inducible arabinose promoter (FIG.
67). The bicistronic message is terminated at a hairpin terminator,
such as the T7 terminator sequence. Each cistron or gene has a
ribosomal binding site (RBS) to initiate translation and a stop
codon (TAA, TGA, or TAG) to stop translation. The light chain
(vL/cL) or heavy chain (vH/cH) sequence can be genetically fused to
rPEG and followed by an affinity tag such as HA (hemagglutinin), H
(hexahistidine), and/or FLAG tag. DNA constructs can encode the
heavy chain first or light chain last (HL) or light chain first and
heavy chain last (LH) as shown in FIG. 67. Protein expression from
this type of construct yields two approximately 50 kDa chains that
form a full Fab fragment of approximately 100 kDa in size, which
includes a total of 50 kDa of rPEG sequence.
Example 21
PK Analysis of GFP-rPEG50
[0812] The amino acid sequence of GFP-rPEG50 is shown in FIG. 69.
The protein was expressed in BL21(DE3) using a T7 promoter similar
to example 1. The protein was purified by ion exchange
chromatography followed by hydrophobic interaction chromatography.
The pharmacokinetics of GFP-rPEG50 was studied in cynomolgous
macaques monkeys following s.c. and i.v. injection. Three
cynomolgous macaques monkeys were divided into 2 groups, 2 animals
dosed i.v and one dosed s.c. at 0.15 mg/kg with GFP-rPEG50. Serial
blood samples were taken from each monkey, the plasma was
separated, and the test article plasma concentration was measured
by ELISA Assays. The half-life for the i.v. dosed animals was 17.4
hours and 13.8 Hrs for the s.c. dosed animals. The bioavailability
for the test article was approximately 54.6% as shown in FIG.
70.
Example 22
PK Analysis of Ex4-rPEG50
[0813] Ex4-rPEG50 is a fusion protein between exendin-4 and rPEG50.
It was produced as a fusion protein with a cellulose binding domain
(CBD), which was designed to be removed by cleavage with TEV
protease as illustrated in FIG. 71b. The amino acid sequence of the
fusion protein is shown in FIG. 71. The expression plasmid and
purification protein were similar as in Example 1 with the addition
of a step for TEV proteolysis. The cleaved CBD was removed by
incubation with beaded cellulose. The pharmacokinec of Ex4-rPEG50
was studied in cynomologos monkeys. Four cynomolgous macaques
monkeys were divided into 2 groups, 2 animals per group and dosed
s.c. and i.v., at 0.15 mg/kg with Ex4-rPEG50. Serial blood samples
were taken from each monkey and the test article plasma
concentration was measured by ELISA assay. The half-life was 9.5
hours and 9.1 hours for the s.c. and i.v. dosing, respectively as
shown in FIG. 70.
Example 23
PK Analysis of GFP-rPEG50 in Rodents
[0814] This example compares the s.c. and i.v. pharmacokinetics of
GFP-rPEG25 and GFP-rPEG50. 15 rats were divided into 5 groups, 3
rats per group and dosed both s.c. and i.v. at 1.67 mg/kg with
either GFP-rPEGY25 and GFP-rPEG_Y288. GFP-rPEG25 had approximately
an 8-9 t.sub.0.5 when injected s.c versus 11-15 hr t.sub.0.5 for
GFP-rPEG50. GFP-rPEG25 was approximately 25% s.c bioavailability
versus 11% s.c bioavailability for GFP-rPEG50. In mice,
.sup.125I-GFP-rPEG50 was dosed into in nude mice. The half-life was
13.4 hours.
Example 24
PK Analysis of Human Growth Hormone Fused to rPEG50
[0815] rPEG50 was fused to either the C- or N-terminus of human
growth hormone (hGH). Proteins were purified as described in
example 8. The pharmacokinetics was studied in cynomologos monkeys.
Two cynomolgous macaques monkeys were divided into 2 groups, 1
animal per group. Each monkey was i.v. dosed at 0.15 mg/kg with the
one growth hormone construct, either hGH-rPEG50 or rPEG50-hGH. The
two growth hormone constructs had half-life of 7 and 10.5 hrs,
respectively.
Example 25
Mouse Immunogenicity and Toxicology Study of Ex4-rPEG50
[0816] This example describes the immunogenicity and potential
toxicity associated with ten s.c. 50 .mu.g doses of Ex4-rPEG50
(1/week) into a mouse. 20 mice (Swiss Webster) total, each 30-40 g
with 10 mice/group, 5 males and 5 females/group, using 2 groups
dosed weekly with either Ex4-rPEG50 or ELSPAR that served as
control as illustrated in FIG. 72a. Before each dose a blood sample
was taken and the IgG was measured by ELISA Assay as shown in FIGS.
72b and 72c. ELSPAR resulted in a significant immune response that
increased over time. In contrast Ex4-rPEG50 gave a very weak
response that showed a maximum after 6 antigen injections and
decreased in the sample obtained after 10 antigen injections. All
mice gained weight during the study and showed no behavioral signs
of toxicity and necropsy revealed no unusual finding with regard to
organ morphology. After completion of the in life portion blood
samples, blood smears, and plasma and tissue samples were shipped
to RADIL (Columbia, Mo.) for toxicology analysis. Histology
analysis showed that no distinct cytoplasmic vacuolation was
present in the distal or proximal tubules, which is a major concern
for chemical conjugates with PEG. Evaluation of liver histology
showed mild inflammation in all four analyzed samples. This is a
common finding in the livers of apparently healthy animals.
Analysis of the spleen showed that all four mice have moderate to
marked megakaryocytosis and moderate hematopoiesis. Clinical
chemistry revealed ALT and ALP levels that were moderately high for
one of the animal indicating hepatocellular damage/necrosis. It is
not severe or chronic based on the observation. Hematology revealed
that all four mice had at least one slightly elevated blood cell
count, hemoglobin, hematocrit percentage or blood total protein
concentration. Overall, multiple injections of rPEG fusion protein
resulted in very minor immunogenicity and toxicity.
Example 26
Size Exclusion Chromatography of GFP-rPEG Fusion Proteins
[0817] GFP fused to rPEG_Y25 and rPEG_Y50 was expressed as
discussed in Example 8. The proteins were analyzed by analytical
SEC using a TSK G4000 SWXL (Tosoh, Grove City, Ohio) as shown in
FIG. 73. The column was calibrated using a commercial standard of
globular proteins and molecular weights of the controls are shown
in FIG. 73. GFP-rPEG25 eluted at an apparent molecular weight of
500 kDa whereas GFP-rPEG50 eluted at an apparent molecular weight
of 1500 kDa.
Example 27
Formulation and In Vivo Administration of GFP-rPEGY Fusion
Proteins
[0818] A solution of GFP-rPEGY at 10 mg/mL in PBS is mixed with an
equal volume of 5 mg/mL Chitosan in PBS and incubated at room
temperature for 30 minutes. Precipitate is collected by
centrifugation at 5,000.times.g for 10 minutes, and washed quickly
one time with 0.1 volume sterile PBS. The precipitate is then
lyophilized to remove excess fluid and ground to a fine powder. 15
mg of powder is then resuspended in 1 mL sterile PBS and
homogenized by pipetting up and down. The homogenate is stored
rotating at 37.degree. C. for 2 weeks, with 10 uL samples removed
at regular intervals. Samples are prepared immediately by
centrifugation to remove insoluble material, and resolubilized
protein is quantitated in the supernatant by GFP fluorescence,
optical density, and rPEGY ELISA. Supernatant concentration is
plotted as a function of time and fit to a single exponential
process to determine the resolubilization rate. To determine in
vivo release rates, Sprague-Dawly rats are injected subcutaneously
with a freshly prepared suspension of 20 mg powder in 1 mL PBS at a
dosage of 1 mL/kg (5 mg/kg effective dose). Intravenous and
subcutaneous injections of uncomplexed GFP-rPEGY are injected at 5
mg/kg into independent cohorts of animals in parallel. Blood
samples are taken at regular intervals, and serum concentration of
protein is determined by GFP and rPEGY ELISAs. Pharmacokinetic
parameters including clearance rate, C.sub.max, C.sub.ss, V.sub.D,
AUC and serum half-life are determined by standard methods (ie
WinNonLin analysis). Bioavailability and effective dose for
subcutaneous and depot formulations are determined by comparison to
intravenous dosing.
[0819] Thus, while preferred embodiments of the present invention
have been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Sequence CWU 1
1
52416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6x His tag 1His His His His His His1 5260PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly1 5
10 15Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly 20 25 30Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser 35 40 45Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser 50
55 60390PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 3Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly1 5 10 15Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly
Glu Gly Glu Gly Gly Gly 20 25 30Glu Gly Gly Glu Gly Glu Gly Gly Gly
Glu Gly Gly Glu Gly Glu Gly 35 40 45Gly Gly Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly Gly Glu Gly 50 55 60Glu Gly Gly Gly Glu Gly Gly
Glu Gly Glu Gly Gly Gly Glu Gly Gly65 70 75 80Glu Gly Glu Gly Gly
Gly Glu Gly Gly Glu 85 90450PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 4Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser1 5 10 15Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 20 25 30Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 35 40 45Ser Ser
50518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly Gly Glu Gly1 5 10 15Gly Ser610PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Gly Gly Glu Gly Gly Glu
Gly Gly Glu Ser1 5 10710PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser1 5 1087PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 8Ser Lys Val Ile Leu Phe Glu1
598PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Arg Ala Arg Ala Asp Ala Asp Ala1 510224PRTHomo
sapiens 10Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser 35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 100 105 110Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150
155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Ile
Pro 165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 210 215 220119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Gly
Glu Gly Ser Gly Glu Gly Ser Glu1 5129PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Gly
Glu Gly Gly Ser Glu Gly Ser Glu1 5139PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Gly
Glu Gly Ser Glu Gly Ser Gly Glu1 5149PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Gly
Glu Gly Ser Glu Gly Gly Ser Glu1 5159PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Gly
Glu Gly Ser Gly Glu Gly Gly Glu1 5169PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Gly
Glu Gly Gly Ser Glu Gly Gly Glu1 5179PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 17Gly
Glu Gly Gly Gly Glu Gly Ser Glu1 5189PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Gly
Glu Gly Gly Glu Gly Ser Gly Glu1 5199PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 19Gly
Glu Gly Gly Glu Gly Gly Ser Glu1 5209PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gly
Glu Gly Ser Glu Gly Gly Gly Glu1 52112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 21Gly
Xaa Glu Gly Ser Gly Glu Gly Xaa Gly Xaa Glu1 5 102212PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Gly
Xaa Glu Gly Gly Ser Glu Gly Xaa Gly Xaa Glu1 5 102312PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Xaa Glu Gly Ser Gly Glu Gly Gly Ser Gly Glu1 5 102412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 24Gly
Xaa Glu Gly Gly Ser Glu Gly Gly Ser Gly Glu1 5 102512PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Gly
Ser Gly Glu Gly Xaa Glu Gly Xaa Gly Xaa Glu1 5 102612PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Gly
Gly Ser Glu Gly Xaa Glu Gly Xaa Gly Xaa Glu1 5 102712PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Gly
Ser Gly Glu Gly Xaa Glu Gly Gly Ser Gly Glu1 5 102812PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Gly
Gly Ser Glu Gly Xaa Glu Gly Gly Ser Gly Glu1 5 102920PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Gly
Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa1 5 10
15Gly Xaa Gly Xaa 203020PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Ser Xaa Ser Xaa Ser Xaa Ser
Xaa Ser Xaa Ser Xaa Ser Xaa Ser Xaa1 5 10 15Ser Xaa Ser Xaa
203121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Gly Gly Xaa Gly Gly Xaa Gly Gly Xaa Gly Gly Xaa
Gly Gly Xaa Gly1 5 10 15Gly Xaa Gly Gly Xaa 203221PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Ser
Ser Xaa Ser Ser Xaa Ser Ser Xaa Ser Ser Xaa Ser Ser Xaa Ser1 5 10
15Ser Xaa Ser Ser Xaa 203320PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 33Gly Gly Gly Xaa Gly Gly Gly
Xaa Gly Gly Gly Xaa Gly Gly Gly Xaa1 5 10 15Gly Gly Gly Xaa
203420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Ser Ser Ser Xaa Ser Ser Ser Xaa Ser Ser Ser Xaa
Ser Ser Ser Xaa1 5 10 15Ser Ser Ser Xaa 203520PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Gly
Gly Gly Gly Xaa Gly Gly Gly Gly Xaa Gly Gly Gly Gly Xaa Gly1 5 10
15Gly Gly Gly Xaa 203620PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 36Ser Ser Ser Ser Xaa Ser Ser
Ser Ser Xaa Ser Ser Ser Ser Xaa Ser1 5 10 15Ser Ser Ser Xaa
2037210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Xaa Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly Gly
Xaa Gly Gly Gly Gly Gly Gly 35 40 45Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Xaa Gly 50 55 60Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly65 70 75 80Gly Gly Gly Xaa Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 85 90 95Gly Gly Gly Gly
Gly Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly 100 105 110Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly 115 120
125Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
130 135 140Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly145 150 155 160Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly Gly
Gly Gly Gly Gly Gly 165 170 175Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Xaa Gly Gly Gly 180 185 190Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 195 200 205Gly Xaa
21038210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 38Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser1 5 10 15Ser Ser Ser Ser Xaa Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser 20 25 30Ser Ser Ser Ser Ser Ser Ser Ser Ser
Xaa Ser Ser Ser Ser Ser Ser 35 40 45Ser Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Xaa Ser 50 55 60Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser65 70 75 80Ser Ser Ser Xaa Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 85 90 95Ser Ser Ser Ser
Ser Ser Ser Ser Xaa Ser Ser Ser Ser Ser Ser Ser 100 105 110Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Xaa Ser Ser 115 120
125Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
130 135 140Ser Ser Xaa Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser Ser145 150 155 160Ser Ser Ser Ser Ser Ser Ser Xaa Ser Ser Ser
Ser Ser Ser Ser Ser 165 170 175Ser Ser Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Xaa Ser Ser Ser 180 185 190Ser Ser Ser Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser 195 200 205Ser Xaa
21039288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser
Glu Ser Ser Gly Ser1 5 10 15Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser
Gly Ser Ser Glu Ser Ser 20 25 30Gly Ser Ser Glu Ser Ser Gly Ser Ser
Glu Ser Ser Gly Ser Ser Glu 35 40 45Ser Ser Gly Ser Ser Glu Ser Ser
Gly Ser Ser Glu Ser Ser Gly Ser 50 55 60Ser Glu Ser Ser Gly Ser Ser
Glu Ser Ser Gly Ser Ser Glu Ser Ser65 70 75 80Gly Ser Ser Glu Ser
Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu 85 90 95Ser Ser Gly Ser
Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser 100 105 110Ser Glu
Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser 115 120
125Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu
130 135 140Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser
Gly Ser145 150 155 160Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser Gly
Ser Ser Glu Ser Ser 165 170 175Gly Ser Ser Glu Ser Ser Gly Ser Ser
Glu Ser Ser Gly Ser Ser Glu 180 185 190Ser Ser Gly Ser Ser Glu Ser
Ser Gly Ser Ser Glu Ser Ser Gly Ser 195 200 205Ser Glu Ser Ser Gly
Ser Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser 210 215 220Gly Ser Ser
Glu Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu225 230 235
240Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser
245 250 255Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu
Ser Ser 260 265 270Gly Ser Ser Glu Ser Ser Gly Ser Ser Glu Ser Ser
Gly Ser Ser Glu 275 280 28540288PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 40Ser Ser Glu Ser Ser
Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser1 5 10 15Ser Ser Ser Glu
Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu 20 25 30Ser Ser Ser
Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu 35 40 45Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser 50 55 60Ser
Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu65 70 75
80Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu
85 90 95Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu
Ser 100 105 110Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser
Ser Ser Glu 115 120 125Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser Ser Ser Glu 130 135 140Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Glu Ser Ser Glu Ser145 150 155 160Ser Ser Ser Glu Ser Ser
Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu 165 170 175Ser Ser Ser Glu
Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu 180 185 190Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser 195 200
205Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu
210 215 220Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Glu225 230 235 240Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser
Glu Ser Ser Glu Ser 245 250 255Ser Ser Ser Glu Ser Ser Ser Glu Ser
Ser Glu Ser Ser Ser Ser Glu 260 265 270Ser Ser Ser Glu Ser Ser Glu
Ser Ser Ser Ser Glu Ser Ser Ser Glu 275 280 28541324PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
41Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly1
5 10 15Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly
Gly 20 25 30Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly
Glu Gly 35 40 45Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly
Gly Glu Gly 50 55 60Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly
Gly Glu Gly Gly65 70 75 80Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu
Gly Glu Gly Gly Gly Glu 85 90 95Gly Gly Glu Gly Glu Gly Gly Gly Glu
Gly Gly Glu Gly Glu Gly Gly 100 105 110Gly Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly Gly Glu Gly Glu 115 120 125Gly Gly Gly Glu Gly
Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu 130 135 140Gly Glu Gly
Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly145 150 155
160Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly
165 170 175Glu Gly Gly Glu
Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly 180 185 190Gly Gly
Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly 195 200
205Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly
210 215 220Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly
Gly Glu225 230 235 240Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly
Glu Gly Glu Gly Gly 245 250 255Gly Glu Gly Gly Glu Gly Glu Gly Gly
Gly Glu Gly Gly Glu Gly Glu 260 265 270Gly Gly Gly Glu Gly Gly Glu
Gly Glu Gly Gly Gly Glu Gly Gly Glu 275 280 285Gly Glu Gly Gly Gly
Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly 290 295 300Gly Glu Gly
Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly305 310 315
320Glu Gly Gly Glu425PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Gly Gly Gly Ser Glu1
5434PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Gly Gly Ser Glu1445PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Glu
Glu Glu Glu Glu1 5455PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 45Gly Gly Gly Gly Gly1
5465PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Ser Ser Ser Ser Ser1 5475PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Ala
Ala Ala Ala Ala1 5489PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Ser Glu Ser Ser Ser Glu Ser
Ser Glu1 54912PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 49Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Glu1 5 105015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Ser Ser Ser Glu Ser Ser Ser
Ser Ser Glu Ser Ser Ser Ser Glu1 5 10 155112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 51Ser
Ser Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu1 5 10524PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Ser
Ser Ser Glu1535PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Ser Ser Ser Ser Glu1 5546PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Ser
Ser Ser Ser Ser Glu1 5557PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 55Ser Ser Ser Ser Ser Ser
Glu1 55621PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Gly Glu Gly Glu Ser Glu Gly Glu Gly Glu Gly Glu
Ser Glu Gly Glu1 5 10 15Gly Glu Ser Gly Glu 205740PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
57Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Glu Glu Glu Glu Glu1
5 10 15Glu Glu Glu Glu Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu
Glu 20 25 30Glu Glu Glu Glu Glu Glu Glu Glu 35 40585PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Gly
Gly Gly Glu Glu1 5596PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 59Gly Gly Glu Gly Gly Ser1
5606PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 60Glu Gly Gly Ser Gly Gly1 5616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Gly
Glu Gly Gly Ser Gly1 5626PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 62Gly Gly Ser Gly Gly Glu1
5636PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 63Ser Gly Gly Glu Gly Gly1 5646PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 64Gly
Ser Gly Gly Glu Gly1 5656PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 65Gly Glu Glu Gly Ser Ser1
5666PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 66Gly Ser Ser Gly Glu Glu1 5676PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Ser
Gly Ser Glu Gly Glu1 5686PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Ser Ser Gly Glu Glu Gly1
56920PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 69Glu Glu Glu Gly Gly Gly Ser Ser Ser Gly Glu Gly
Gly Ser Ser Ser1 5 10 15Gly Ser Glu Glu 207020PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Glu
Ser Gly Gly Ser Ser Glu Gly Ser Ser Glu Glu Ser Gly Ser Ser1 5 10
15Glu Gly Ser Glu 20719PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 71Glu Glu Glu Ser Ser Ser Gly
Gly Gly1 5726PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 72Glu Glu Ser Ser Gly Gly1
5735PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 73Glu Ser Gly Ser Glu1 5745PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 74Glu
Glu Ser Gly Ser1 5756PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 75Glu Ser Gly Gly Ser Glu1
5767PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 76Glu Ser Gly Glu Glu Ser Gly1 5777PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 77Glu
Ser Gly Pro Glu Ser Gly1 57811PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 78Glu Gly Glu Gly Glu Gly Glu
Gly Glu Gly Glu1 5 107914PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 79Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser1 5 108017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 80Ser
Glu Ser Glu Ser Glu Ser Glu Ser Glu Ser Glu Ser Glu Ser Glu1 5 10
15Ser815PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 81Asp Asp Asp Glu Glu1 5825PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 82Asp
Asp Asp Gly Gly1 5835PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Asp Asp Asp Lys Lys1
5845PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 84Asp Asp Asp Pro Pro1 5855PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Asp
Asp Asp Arg Arg1 5865PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 86Asp Asp Asp Ser Ser1
5875PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 87Asp Asp Asp Thr Thr1 5885PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 88Glu
Glu Glu Asp Asp1 5895PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 89Glu Glu Glu Gly Gly1
5905PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 90Glu Glu Glu Lys Lys1 5915PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 91Glu
Glu Glu Pro Pro1 5925PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 92Glu Glu Glu Arg Arg1
5935PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 93Glu Glu Glu Ser Ser1 5945PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 94Glu
Glu Glu Thr Thr1 5955PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 95Gly Gly Gly Asp Asp1
5965PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 96Gly Gly Gly Glu Glu1 5975PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 97Gly
Gly Gly Lys Lys1 5985PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 98Gly Gly Gly Pro Pro1
5995PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 99Gly Gly Gly Arg Arg1 51005PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 100Lys
Lys Lys Asp Asp1 51015PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 101Lys Lys Lys Glu Glu1
51025PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 102Lys Lys Lys Gly Gly1 51035PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 103Lys
Lys Lys Pro Pro1 51045PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 104Lys Lys Lys Arg Arg1
51055PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 105Lys Lys Lys Ser Ser1 51065PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 106Lys
Lys Lys Thr Thr1 51075PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 107Pro Pro Pro Asp Asp1
51085PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 108Pro Pro Pro Glu Glu1 51095PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 109Pro
Pro Pro Gly Gly1 51105PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 110Pro Pro Pro Lys Lys1
51115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 111Pro Pro Pro Arg Arg1 51125PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 112Pro
Pro Pro Ser Ser1 51135PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 113Pro Pro Pro Thr Thr1
51145PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 114Arg Arg Arg Asp Asp1 51155PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 115Arg
Arg Arg Glu Glu1 51165PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 116Arg Arg Arg Gly Gly1
51175PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 117Arg Arg Arg Lys Lys1 51185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Arg
Arg Arg Pro Pro1 51195PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 119Arg Arg Arg Ser Ser1
51205PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 120Arg Arg Arg Thr Thr1 51215PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 121Ser
Ser Ser Asp Asp1 51225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 122Ser Ser Ser Glu Glu1
51235PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 123Ser Ser Ser Gly Gly1 51245PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 124Ser
Ser Ser Lys Lys1 51255PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 125Ser Ser Ser Pro Pro1
51265PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 126Ser Ser Ser Arg Arg1 51275PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 127Ser
Ser Ser Thr Thr1 51285PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 128Thr Thr Thr Asp Asp1
51295PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 129Thr Thr Thr Glu Glu1 51305PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 130Thr
Thr Thr Gly Gly1 51315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 131Thr Thr Thr Lys Lys1
51325PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 132Thr Thr Thr Pro Pro1 51335PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 133Thr
Thr Thr Arg Arg1 51345PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 134Thr Thr Thr Ser Ser1
51357PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 135Asp Asp Asp Asp Glu Glu Glu1
51367PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 136Asp Asp Asp Asp Gly Gly Gly1
51377PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 137Asp Asp Asp Asp Lys Lys Lys1
51387PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 138Asp Asp Asp Asp Pro Pro Pro1
51397PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 139Asp Asp Asp Asp Arg Arg Arg1
51407PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 140Asp Asp Asp Asp Ser Ser Ser1
51417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 141Asp Asp Asp Asp Thr Thr Thr1
51427PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 142Glu Glu Glu Glu Asp Asp Asp1
51437PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 143Glu Glu Glu Glu Gly Gly Gly1
51447PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 144Glu Glu Glu Glu Lys Lys Lys1
51457PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 145Glu Glu Glu Glu Pro Pro Pro1
51467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 146Glu Glu Glu Glu Arg Arg Arg1
51477PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 147Glu Glu Glu Glu Ser Ser Ser1
51487PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 148Glu Glu Glu Glu Thr Thr Thr1
51497PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 149Lys Lys Lys Lys Asp Asp Asp1
51507PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 150Lys Lys Lys Lys Glu Glu Glu1
51517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 151Lys Lys Lys Lys Gly Gly Gly1
51527PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 152Lys Lys Lys Lys Pro Pro Pro1
51537PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 153Lys Lys Lys Lys Arg Arg Arg1
51547PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 154Lys Lys Lys Ser Ser Ser Ser1
51557PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 155Lys Lys Lys Lys Thr Thr Thr1
51567PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 156Pro Pro Pro Pro Asp Asp Asp1
51577PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 157Pro Pro Pro Pro Glu Glu Glu1
51587PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 158Pro Pro Pro Pro Gly Gly Gly1
51597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 159Pro Pro Pro Lys Lys Lys Lys1
51607PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 160Pro Pro Pro Pro Arg Arg Arg1
51617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 161Pro Pro Pro Pro Ser Ser Ser1
51626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 162Pro Pro Pro Thr Thr Thr1 51637PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 163Arg
Arg Arg Arg Asp Asp Asp1 51647PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 164Arg Arg Arg Arg Glu Glu
Glu1 51657PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 165Arg
Arg Arg Arg Gly Gly Gly1 51667PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 166Arg Arg Arg Arg Lys Lys
Lys1 51677PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 167Arg Arg Arg Arg Pro Pro Pro1
51687PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 168Arg Arg Arg Arg Ser Ser Ser1
51697PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 169Arg Arg Arg Arg Thr Thr Thr1
51707PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 170Ser Ser Ser Ser Asp Asp Asp1
51717PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 171Ser Ser Ser Ser Glu Glu Glu1
51727PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 172Ser Ser Ser Ser Gly Gly Gly1
51737PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 173Ser Ser Ser Ser Lys Lys Lys1
51747PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 174Ser Ser Ser Ser Pro Pro Pro1
51757PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 175Ser Ser Ser Ser Arg Arg Arg1
51767PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 176Ser Ser Ser Ser Thr Thr Thr1
51777PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 177Thr Thr Thr Thr Asp Asp Asp1
51787PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 178Thr Thr Thr Thr Glu Glu Glu1
51797PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 179Thr Thr Thr Thr Gly Gly Gly1
51807PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 180Thr Thr Thr Thr Lys Lys Lys1
51817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 181Thr Thr Thr Thr Pro Pro Pro1
51827PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 182Thr Thr Thr Thr Arg Arg Arg1
51837PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 183Thr Thr Thr Thr Ser Ser Ser1
51844PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 184Asp Asp Glu Glu11854PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 185Asp
Asp Gly Gly11864PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 186Asp Asp Lys Lys11874PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 187Asp
Asp Pro Pro11884PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 188Asp Asp Arg Arg11894PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 189Asp
Asp Ser Ser11904PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 190Asp Asp Thr Thr11914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 191Glu
Glu Asp Asp11924PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 192Glu Glu Gly Gly11934PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 193Glu
Glu Lys Lys11944PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 194Glu Glu Pro Pro11954PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 195Glu
Glu Arg Arg11964PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 196Glu Glu Ser Ser11974PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 197Glu
Glu Thr Thr11984PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 198Gly Gly Asp Asp11994PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 199Gly
Gly Glu Glu12004PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 200Gly Gly Lys Lys12014PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 201Gly
Gly Pro Pro12024PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 202Gly Gly Arg Arg12034PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 203Gly
Gly Ser Ser12044PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 204Gly Gly Thr Thr12054PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 205Lys
Lys Asp Asp12064PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 206Lys Lys Glu Glu12074PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 207Lys
Lys Gly Gly12084PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 208Lys Lys Pro Pro12094PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 209Lys
Lys Arg Arg12104PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 210Lys Lys Ser Ser12114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 211Lys
Lys Thr Thr12124PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 212Pro Pro Asp Asp12134PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 213Pro
Pro Glu Glu12144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 214Pro Pro Gly Gly12154PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 215Pro
Pro Lys Lys12164PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 216Pro Pro Arg Arg12174PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 217Pro
Pro Ser Ser12184PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 218Pro Pro Thr Thr12194PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 219Arg
Arg Asp Asp12204PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 220Arg Arg Glu Glu12214PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 221Arg
Arg Gly Gly12224PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 222Arg Arg Lys Lys12234PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 223Arg
Arg Pro Pro12244PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 224Arg Arg Ser Ser12254PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 225Arg
Arg Thr Thr12264PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 226Ser Ser Asp Asp12274PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 227Ser
Ser Glu Glu12284PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 228Ser Ser Gly Gly12294PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 229Ser
Ser Lys Lys12304PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 230Ser Ser Pro Pro12314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 231Ser
Ser Arg Arg12324PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 232Ser Ser Thr Thr12334PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 233Thr
Thr Asp Asp12344PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 234Thr Thr Glu Glu12354PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 235Thr
Thr Gly Gly12364PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 236Thr Thr Lys Lys12374PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 237Thr
Thr Pro Pro12384PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 238Thr Thr Arg Arg12394PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 239Thr
Thr Ser Ser12405PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 240Gly Gly Gly Ser Ser1
52415PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 241Gly Gly Gly Thr Thr1 52425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 242Asp
Asp Glu Glu Glu1 52435PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 243Asp Asp Gly Gly Gly1
52445PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 244Asp Asp Lys Lys Lys1 52455PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 245Asp
Asp Pro Pro Pro1 52465PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 246Asp Asp Arg Arg Arg1
52475PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 247Asp Asp Ser Ser Ser1 52485PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 248Asp
Asp Thr Thr Thr1 52495PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 249Glu Glu Asp Asp Asp1
52505PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 250Glu Glu Gly Gly Gly1 52515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 251Glu
Glu Lys Lys Lys1 52525PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 252Glu Glu Pro Pro Pro1
52535PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 253Glu Glu Arg Arg Arg1 52545PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 254Glu
Glu Ser Ser Ser1 52555PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 255Glu Glu Thr Thr Thr1
52565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 256Gly Gly Asp Asp Asp1 52575PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 257Gly
Gly Glu Glu Glu1 52585PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 258Gly Gly Lys Lys Lys1
52595PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 259Gly Gly Pro Pro Pro1 52605PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 260Gly
Gly Arg Arg Arg1 52615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 261Gly Gly Ser Ser Ser1
52625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 262Gly Gly Thr Thr Thr1 52635PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 263Lys
Lys Asp Asp Asp1 52645PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 264Lys Lys Glu Glu Glu1
52655PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 265Lys Lys Gly Gly Gly1 52665PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 266Lys
Lys Pro Pro Pro1 52675PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 267Lys Lys Arg Arg Arg1
52685PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 268Lys Lys Ser Ser Ser1 52695PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 269Lys
Lys Thr Thr Thr1 52705PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 270Pro Pro Asp Asp Asp1
52715PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 271Pro Pro Glu Glu Glu1 52725PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 272Pro
Pro Gly Gly Gly1 52735PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 273Pro Pro Lys Lys Lys1
52745PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 274Pro Pro Arg Arg Arg1 52755PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 275Pro
Pro Ser Ser Ser1 52765PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 276Pro Pro Thr Thr Thr1
52775PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 277Arg Arg Asp Asp Asp1 52785PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 278Arg
Arg Glu Glu Glu1 52795PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 279Arg Arg Gly Gly Gly1
52805PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 280Arg Arg Lys Lys Lys1 52815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 281Arg
Arg Pro Pro Pro1 52825PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 282Arg Arg Ser Ser Ser1
52835PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 283Arg Arg Thr Thr Thr1 52845PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 284Ser
Ser Asp Asp Asp1 52855PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 285Ser Ser Glu Glu Glu1
52865PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 286Ser Ser Gly Gly Gly1 52875PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 287Ser
Ser Lys Lys Lys1 52885PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 288Ser Ser Pro Pro Pro1
52895PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 289Ser Ser Arg Arg Arg1 52905PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 290Ser
Ser Thr Thr Thr1 52915PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 291Thr Thr Asp Asp Asp1
52925PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 292Thr Thr Glu Glu Glu1 52935PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 293Thr
Thr Gly Gly Gly1 52945PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 294Thr Thr Lys Lys Lys1
52955PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 295Thr Thr Pro Pro Pro1 52965PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 296Thr
Thr Arg Arg Arg1 52975PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 297Thr Thr Ser Ser Ser1
52986PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 298Asp Asp Asp Glu Glu Glu1 52996PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 299Asp
Asp Asp Gly Gly Gly1 53006PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 300Asp Asp Asp Lys Lys Lys1
53016PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 301Asp Asp Asp Pro Pro Pro1 53026PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 302Asp
Asp Asp Arg Arg Arg1 53036PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 303Asp Asp Asp Ser Ser Ser1
53046PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 304Asp Asp Asp Thr Thr Thr1 53056PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 305Glu
Glu Glu Asp Asp Asp1 53066PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 306Glu Glu Glu Gly Gly Gly1
53076PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 307Glu Glu Glu Lys Lys Lys1 53086PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 308Glu
Glu Glu Pro Pro Pro1 53096PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 309Glu Glu Glu Arg Arg Arg1
53106PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 310Glu Glu Glu Ser Ser Ser1 53116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 311Glu
Glu Glu Thr Thr Thr1 53126PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 312Gly Gly Gly Asp Asp Asp1
53136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 313Gly Gly Gly Glu Glu Glu1
53146PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 314Gly Gly Gly Lys Lys Lys1 53156PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 315Gly
Gly Gly Pro Pro Pro1 53166PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 316Gly Gly Gly Arg Arg Arg1
53176PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 317Gly Gly Gly Ser Ser Ser1 53186PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 318Gly
Gly Gly Thr Thr Thr1 53196PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 319Lys Lys Lys Asp Asp Asp1
53206PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 320Lys Lys Lys Glu Glu Glu1 53216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 321Lys
Lys Lys Gly Gly Gly1 53226PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 322Lys Lys Lys Pro Pro Pro1
53236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 323Lys Lys Lys Arg Arg Arg1 53246PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 324Lys
Lys Lys Ser Ser Ser1 53256PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 325Lys Lys Lys Thr Thr Thr1
53266PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 326Pro Pro Pro Asp Asp Asp1 53276PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 327Pro
Pro Pro Glu Glu Glu1 53286PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 328Pro Pro Pro Gly Gly Gly1
53296PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 329Pro Pro Pro Lys Lys Lys1 53306PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 330Pro
Pro Pro Arg Arg Arg1 53316PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 331Pro Pro Pro Ser Ser Ser1
53326PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 332Pro Pro Pro Thr Thr Thr1 53336PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 333Arg
Arg Arg Asp Asp Asp1 53346PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 334Arg Arg Arg Glu Glu Glu1
53356PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 335Arg Arg Arg Gly Gly Gly1 53366PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 336Arg
Arg Arg Lys Lys Lys1 53376PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 337Arg Arg Arg Pro Pro Pro1
53386PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 338Arg Arg Arg Ser Ser Ser1 53396PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 339Arg
Arg Arg Thr Thr Thr1 53406PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 340Ser Ser Ser Asp Asp Asp1
53416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 341Ser Ser Ser Glu Glu Glu1 53426PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 342Ser
Ser Ser Gly Gly Gly1 53436PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 343Ser Ser Ser Lys Lys Lys1
53446PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 344Ser Ser Ser Pro Pro Pro1 53456PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 345Ser
Ser Ser Arg Arg Arg1 53466PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 346Ser Ser Ser Thr Thr Thr1
53476PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 347Thr Thr Thr Asp Asp Asp1 53486PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 348Thr
Thr Thr Glu Glu Glu1 53496PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 349Thr Thr Thr Gly Gly Gly1
53506PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 350Thr Thr Thr Lys Lys Lys1 53516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 351Thr
Thr Thr Pro Pro Pro1 53526PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 352Thr Thr Thr Arg Arg Arg1
53536PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 353Thr Thr Thr Ser Ser Ser1 53547PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 354Gly
Gly Gly Gly Asp Asp Asp1 53557PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 355Gly Gly Gly Gly Glu Glu
Glu1 53567PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 356Gly Gly Gly Gly Lys Lys Lys1
53577PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 357Gly Gly Gly Gly Pro Pro Pro1
53587PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 358Gly Gly Gly Gly Arg Arg Arg1
53597PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 359Gly Gly Gly Gly Ser Ser Ser1
53607PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 360Gly Gly Gly Gly Thr Thr Thr1
53617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 361Pro Pro Pro Pro Thr Thr Thr1
536212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 362Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu1 5 103639PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 363Gly Gly Glu Gly Glu Gly Gly Gly Glu1
536424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 364Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser
Glu Ser Ser Glu Ser1 5 10 15Ser Ser Ser Glu Ser Ser Ser Glu
2036515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 365Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu1 5 10 1536618PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 366Ser Ser Ser Ser Ser Glu
Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser1 5 10 15Ser
Glu36714PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 367Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser
Ser Ser Glu1 5 1036812PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 368Ser Ser Ser Ser Ser Ser
Glu Ser Ser Ser Ser Glu1 5 1036924PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 369Ser Ser Ser Ser Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser1 5 10 15Ser Ser Glu Ser Ser
Ser Ser Glu 2037015PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 370Ser Gly Gly Gly Gly Gly Gly Gly Gly
Gly Arg Gly Ala Gly Gly1 5 10 1537117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 371Thr
Gly Ser Gly Asn Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly1 5 10
15Gly37212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 372Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly1 5 1037337PRTHomo sapiens 373Gly Pro Gly Gly Gly Gly Gly Pro
Gly Gly Gly Gly Gly Pro Gly Gly1 5 10 15Gly Gly Pro Gly Gly Gly Gly
Gly Gly Gly Pro Gly Gly Gly Gly Gly 20 25 30Gly Pro Gly Gly Gly
3537433PRTHomo sapiens 374Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Ser1 5 10 15Gly Gly Gly Gly Gly Gly Gly Gly Ala
Gly Ala Gly Gly Ala Gly Ala 20 25 30Gly37532PRTHomo sapiens 375Gly
Gly Gly Ser Gly Ser Gly Gly Ala Gly Gly Gly Ser Gly Gly Gly1 5 10
15Ser Gly Ser Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly
20 25 3037627PRTHomo sapiens 376Gly Asp Gly Gly Gly Ala Gly Gly Gly
Gly Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly
Gly Gly Gly 20 2537725PRTHomo sapiens 377Gly Ser Gly Ser Gly Ser
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Ser Gly Gly
Gly Gly Gly Gly 20 2537823PRTHomo sapiens 378Gly Gly Gly Arg Gly
Gly Arg Gly Gly Gly Arg Gly Gly Gly Gly Arg1 5 10 15Gly Gly Gly Arg
Gly Gly Gly 2037921PRTHomo sapiens 379Gly Ser Gly Gly Ser Gly Gly
Ser Gly Gly Gly Pro Gly Pro Gly Pro1 5 10 15Gly Gly Gly Gly Gly
2038018PRTHomo sapiens 380Gly Glu Gly Gly Gly Gly Gly Gly Glu Gly
Gly Gly Ala Gly Gly Gly1 5 10 15Ser Gly38112PRTHomo sapiens 381Gly
Gly Gly Gly Gly Gly Gly Gly Asp Gly Gly Gly1 5 1038246PRTHomo
sapiens 382Gly Gly Gly Ser Gly Ser Gly Gly Ala Gly Gly Gly Ser Gly
Gly Gly1 5 10 15Ser Gly Ser Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly
Gly Gly Gly 20 25 30Ser Ser Gly Gly Gly Ser Gly Thr Ala Gly Gly His
Ser Gly 35 40 4538335PRTHomo sapiens 383Gly Gly Ser Gly Ala Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Ser Gly Ser Gly Gly Gly
Gly Ser Thr Gly Gly Gly Gly Gly Thr Ala 20 25 30Gly Gly Gly
3538432PRTHomo sapiens 384Gly His Pro Gly Ser Gly Ser Gly Ser Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Ser Gly Gly Gly
Gly Gly Gly Ala Pro Gly Gly 20 25 3038531PRTHomo sapiens 385Gly Gly
Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly
Gly Gly Ser Gly Ser Thr Gly Gly Gly Gly Ser Gly Ala Gly 20 25
3038631PRTHomo sapiens 386Gly Gly Arg Gly Arg Gly Gly Arg Gly Arg
Gly Ser Arg Gly Arg Gly1 5 10 15Gly Gly Gly Thr Arg Gly Arg Gly Arg
Gly Arg Gly Gly Arg Gly 20 25 3038730PRTHomo sapiens 387Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Gly Pro Gly Pro Gly Pro1 5 10 15Gly Gly
Gly Gly Gly Pro Ser Gly Ser Gly Ser Gly Pro Gly 20 25
3038829PRTHomo sapiens 388Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Arg Gly Gly Gly Gly1 5 10 15Arg Gly Gly Gly Arg Gly Gly Gly Gly
Glu Gly Gly Gly 20 2538928PRTHomo sapiens 389Gly Gly Gly Gly Thr
Gly Ser Ser Gly Gly Ser Gly Ser Gly Gly Gly1 5 10 15Gly Ser Gly Gly
Gly Gly Gly Gly Gly Ser Ser Gly 20 2539027PRTHomo sapiens 390Gly
Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly1 5 10
15Gly Gly Ser Gly Gly Gly Arg Gly Ala Gly Gly 20 2539127PRTHomo
sapiens 391Gly Gly Gly Ala Ala Gly Ala Gly Gly Gly Gly Ser Gly Ala
Gly Gly1 5 10 15Gly Ser Gly Gly Ser Gly Gly Arg Gly Thr Gly 20
2539227PRTHomo sapiens 392Gly Ala Gly Gly Gly Arg Gly Gly Gly Ala
Gly Gly Glu Gly Gly Ala1 5 10 15Ser Gly Ala Glu Gly Gly Gly Gly Ala
Gly Gly 20 2539326PRTHomo sapiens 393Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly
Gly Glu Ala Gly 20 2539426PRTHomo sapiens 394Gly Gly Gly Gly Gly
Gly Ser Ala Gly Gly Gly Ser Ser Gly Gly Gly1 5 10 15Pro Gly Gly Gly
Gly Gly Gly Ala Gly Gly 20 2539525PRTHomo sapiens 395Gly Gly Gly
Gly Gly Pro Gly Gly Gly Gly Gly Gly Gly Pro Gly Gly1 5 10 15Gly Gly
Gly Pro Gly Gly Gly Gly Gly 20 2539625PRTHomo sapiens 396Gly Arg
Gly Gly Ala Gly Ser Gly Gly Ala Gly Ser Gly Ala Ala Gly1 5 10 15Gly
Thr Gly Ser Ser Gly Gly Gly Gly 20 2539725PRTHomo sapiens 397Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Ser Gly1 5 10
15Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 2539825PRTHomo sapiens
398Gly Gly Ser Gly Gly Gly Arg Gly Gly Ala Ser Gly Pro Gly Ser Gly1
5 10 15Ser Gly Gly Pro Gly Gly Pro Ala Gly 20 2539925PRTHomo
sapiens 399Gly Gly His His Gly Asp Arg Gly Gly Gly Arg Gly Gly Arg
Gly Gly1 5 10 15Arg Gly Gly Arg Gly Gly Arg Ala Gly 20
2540025PRTHomo sapiens 400Gly Ser Arg Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Ala Gly Ala Gly Gly Gly
20 2540124PRTHomo sapiens 401Gly Gly Arg Gly Gly Arg Gly Pro Gly
Glu Pro Gly Gly Arg Gly Arg1 5 10 15Ala Gly Gly Ala Glu Gly Arg Gly
2040224PRTHomo sapiens 402Gly Gly Gly Gly Gly Asp Ala Gly Gly Ser
Gly Asp Ala Gly Gly Ala1 5 10 15Gly Gly Arg Ala Gly Arg Ala Gly
2040323PRTHomo sapiens 403Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser
Ser Gly Gly Arg Gly Ser1 5 10 15Gly Gly Gly Ser Ser Gly Gly
2040423PRTHomo sapiens 404Gly Ser Gly Pro Gly Thr Gly Gly Gly Gly
Ser Gly Ser Gly Gly Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly
2040523PRTHomo sapiens 405Gly Ala Arg Gly Gly Gly Ser Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Pro Gly
2040623PRTHomo sapiens 406Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Asp Gly
2040723PRTHomo sapiens 407Gly Gly Thr Arg Gly Gly Thr Arg Gly Gly
Thr Arg Gly Gly Asp Arg1 5 10 15Gly Arg Gly Arg Gly Ala Gly
2040823PRTHomo sapiens 408Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Ala Gly Gly Gly Gly Gly Gly
2040922PRTHomo sapiens 409Gly Arg Gly Arg Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Arg Gly Gly Gly Gly
2041022PRTHomo sapiens 410Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg
Gly Arg Gly Arg Gly Arg1 5 10 15Gly Arg Gly Gly Ala Gly
2041122PRTHomo sapiens 411Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Gly Gly
2041222PRTHomo sapiens 412Gly Gly Gly Ser Gly Gly Gly His Ser Gly
Gly Ser Gly Gly Gly His1 5 10 15Ser Gly Gly Ser Gly Gly
2041322PRTHomo sapiens 413Gly Ala Gly Ala Gly Gly Gly Gly Gly Gly
Gly Gly Ala Gly Gly Gly1 5 10 15Gly Ser Ala Gly Ser Gly
2041422PRTHomo sapiens 414Gly Gly Pro Gly Thr Gly Ser Gly Gly Gly
Gly Ala Gly Thr Gly Gly1 5 10 15Gly Ala Gly Gly Pro Gly
2041522PRTHomo sapiens 415Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala
Gly Gly Ala Gly Gly Ala1 5 10 15Gly Ser Ala Gly Gly Gly
2041621PRTHomo sapiens 416Gly Gly Asp Gly Gly Gly Ser Ala Gly Gly
Gly Ala Gly Gly Gly Ser1 5 10 15Gly Gly Gly Ala Gly 2041721PRTHomo
sapiens 417Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly
Gly Gly1 5 10 15Gly Gly Gly Gly Gly 2041821PRTHomo sapiens 418Gly
Pro Gly Ala Gly Ala Gly Ser Gly Ala Gly Gly Ser Ser Gly Gly1 5 10
15Gly Gly Gly Pro Gly 2041921PRTHomo sapiens 419Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Ser Ser Gly Gly Gly1 5 10 15Gly Ser
Ser Gly Gly 2042021PRTHomo sapiens 420Gly Ser Gly Ser Gly Pro Gly
Pro Gly Ser Gly Pro Gly Ser Gly Pro1 5 10 15Gly His Gly Ser Gly
2042121PRTHomo sapiens 421Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro
Gly Pro Gly Pro Gly Pro1 5 10 15Gly Pro Gly Pro Gly 2042221PRTHomo
sapiens 422Gly Ala Gly Ser Gly Gly Gly Gly Ala Ala Gly Ala Gly Ala
Gly Ser1 5 10 15Ala Gly Gly Gly Gly 2042321PRTHomo sapiens 423Gly
Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Gly1 5 10
15Gly Gly Gly Gly Gly 2042421PRTHomo sapiens 424Gly Arg Gly Arg Gly
Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg1 5 10 15Gly Arg Gly Arg
Gly 2042521PRTHomo sapiens 425Gly Gly Gly Gly Gly Gly Gly Ser Gly
Gly Ser Gly Gly Gly Gly Gly1 5 10 15Ser Gly Gly Gly Gly
2042621PRTHomo sapiens 426Gly Gly Glu Glu Gly Gly Ala Ser Gly Gly
Gly Pro Gly Ala Gly Ser1 5 10 15Gly Ser Ala Gly Gly 2042721PRTHomo
sapiens 427Gly Gly Gly Gly Gly Gly Gly Gly Asp Gly Gly Gly Arg Arg
Gly Arg1 5 10 15Gly Arg Gly Arg Gly 2042820PRTHomo sapiens 428Gly
Gly Pro Gly Gly Pro Gly Gly Gly Gly Ala Gly Gly Pro Gly Gly1 5 10
15Ala Gly Ala Gly 2042920PRTHomo sapiens 429Gly Thr Gly Gly Gly Gly
Ser Thr Gly Gly Gly Gly Gly Gly Gly Gly1 5 10 15Ser Gly His Gly
2043020PRTHomo sapiens 430Gly Pro Ala Gly Ala Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly 2043120PRTHomo
sapiens 431Gly Gly Thr Gly Gly Ser Ser Gly Ser Ser Gly Ser Gly Ser
Gly Gly1 5 10 15Gly Arg Arg Gly 2043220PRTHomo sapiens 432Gly Ser
Gly Thr Gly Thr Thr Gly Ser Ser Gly Ala Gly Gly Pro Gly1 5 10 15Thr
Pro Gly Gly 2043320PRTHomo sapiens 433Gly Gly Ser Gly Gly Gly Ala
Ala Gly Gly Gly Ala Gly Gly Ala Gly1 5 10 15Ala Gly Ala Gly
2043420PRTHomo sapiens 434Gly Ser Ser Gly Gly Gly Gly Gly Gly Ala
Gly Ala Ala Gly Gly Ala1 5 10 15Gly Gly Ala Gly 2043520PRTHomo
sapiens 435Gly Pro Gly Pro Ser Gly Gly Pro Gly Gly Gly Gly Gly Gly
Gly Gly1 5 10 15Gly Gly Gly Gly 2043620PRTHomo sapiens 436Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Ala1 5 10 15Gly
Ala Gly Gly 2043720PRTHomo sapiens 437Gly Ser Ala Gly Gly Ser Ser
Gly Ala Ala Gly Ala Ala Gly Gly Gly1 5 10 15Ala Gly Ala Gly
20438600PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 438Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser1 5 10 15Ser Asp Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser 20 25 30Asp Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp 35 40 45Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp Ser 50 55 60Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser65 70 75 80Asp Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 85 90 95Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser 100 105 110Ser
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 115 120
125Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
130 135 140Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
Asp Ser145 150 155 160Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
Ser Ser Asp Ser Ser 165 170 175Asp Ser Ser Asp Ser Ser Asp Ser Ser
Asp Ser Ser Asp Ser Ser Asp 180 185 190Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp Ser 195 200 205Ser Asp Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 210 215 220Asp Ser Ser
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp225 230 235
240Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
245 250 255Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
Ser Ser 260 265 270Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
Asp Ser Ser Asp 275 280 285Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser 290 295 300Ser Asp Ser Ser Asp Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser305 310 315 320Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 325 330 335Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser 340 345 350Ser
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 355 360
365Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
370 375 380Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
Asp Ser385 390 395 400Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
Ser Ser Asp Ser Ser 405 410 415Asp Ser Ser Asp Ser Ser Asp Ser Ser
Asp Ser Ser Asp Ser Ser Asp 420 425 430Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp Ser 435 440 445Ser Asp Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 450 455 460Asp Ser Ser
Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp465 470 475
480Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
485 490 495Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp
Ser Ser 500 505 510Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser
Asp Ser Ser Asp 515 520 525Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser 530 535 540Ser Asp Ser Ser Asp Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser545 550 555 560Asp Ser Ser Asp Ser
Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 565 570 575Ser Ser Asp
Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser 580 585 590Ser
Asp Ser Ser Asp Ser Ser Asp 595 6004391200PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
439Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser1
5 10 15Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser
Ser 20 25 30Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn 35 40 45Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser
Ser Asp Ser 50 55 60Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser65 70 75 80Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser Asp Ser Ser Asn 85 90 95Ser Ser Asp Ser Ser Asn Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser 100 105 110Ser Asn Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser 115 120 125Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 130 135 140Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser145 150 155
160Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
165 170 175Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn 180 185 190Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser Asp Ser 195 200 205Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
Asp Ser Ser Asn Ser Ser 210 215 220Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser Asp Ser Ser Asn225 230 235 240Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser 245 250 255Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser 260 265 270Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 275 280
285Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
290 295 300Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser305 310 315 320Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn 325 330 335Ser Ser Asp Ser Ser Asn Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser 340 345 350Ser Asn Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser 355 360 365Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 370 375 380Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser385 390 395
400Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
405 410 415Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn 420 425 430Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser Asp Ser 435 440 445Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
Asp Ser Ser Asn Ser Ser 450 455 460Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser Asp Ser Ser Asn465 470 475 480Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser 485 490 495Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser 500 505 510Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 515 520
525Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
530 535 540Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser545 550 555 560Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn 565 570 575Ser Ser Asp Ser Ser Asn Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser 580 585 590Ser Asn Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser 595 600 605Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 610 615 620Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser625 630 635
640Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
645 650 655Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn 660 665 670Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser Asp Ser 675 680 685Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
Asp Ser Ser Asn Ser Ser 690 695 700Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser Asp Ser Ser Asn705 710 715 720Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser 725 730 735Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser 740 745 750Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 755 760
765Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
770 775 780Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser785 790 795 800Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn 805 810 815Ser Ser Asp Ser Ser Asn Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser 820 825 830Ser Asn Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser 835 840 845Asp Ser Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 850 855 860Ser Ser Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser865 870 875
880Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
885 890 895Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn 900 905 910Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
Ser Ser Asp Ser 915 920 925Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
Asp Ser Ser Asn Ser Ser 930 935 940Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser Asp Ser Ser Asn945 950 955 960Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser 965 970 975Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser 980 985 990Asp
Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 995
1000 1005Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
Asp 1010 1015 1020Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp
Ser Ser Asn 1025 1030 1035Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser Asp 1040 1045 1050Ser Ser Asn Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn 1055 1060 1065Ser Ser Asp Ser Ser Asn
Ser Ser Asp Ser Ser Asn Ser Ser Asp 1070 1075 1080Ser Ser Asn Ser
Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn 1085 1090 1095Ser Ser
Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp 1100 1105
1110Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn
1115 1120 1125Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser
Ser Asp 1130 1135 1140Ser Ser Asn Ser Ser Asp Ser Ser Asn Ser Ser
Asp Ser Ser Asn 1145 1150 1155Ser Ser Asp Ser Ser Asn Ser Ser Asp
Ser Ser Asn Ser Ser Asp 1160 1165 1170Ser Ser Asn Ser Ser Asp Ser
Ser Asn Ser Ser Asp Ser Ser Asn 1175 1180 1185Ser Ser Asp Ser Ser
Asn Ser Ser Asp Ser Ser Asn 1190 1195 1200440600PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
440Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser1
5 10 15Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser 20 25 30Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu 35 40 45Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu Ser 50 55 60Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu Ser Ser65 70 75 80Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu 85 90 95Ser Ser Glu Ser Ser Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu Ser 100 105 110Ser Glu Ser Ser Glu Ser Ser
Glu Ser Ser Glu Ser Ser Glu Ser Ser 115 120 125Glu Ser Ser Glu Ser
Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu 130 135 140Ser Ser Glu
Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser145 150 155
160Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
165 170 175Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu 180 185 190Ser Ser
Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser 195 200
205Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
210 215 220Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu225 230 235 240Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
Glu Ser Ser Glu Ser 245 250 255Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu Ser Ser Glu Ser Ser 260 265 270Glu Ser Ser Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu Ser Ser Glu 275 280 285Ser Ser Glu Ser Ser
Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser 290 295 300Ser Glu Ser
Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser305 310 315
320Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu
325 330 335Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
Glu Ser 340 345 350Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu Ser Ser 355 360 365Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu 370 375 380Ser Ser Glu Ser Ser Glu Ser Ser
Glu Ser Ser Glu Ser Ser Glu Ser385 390 395 400Ser Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser 405 410 415Glu Ser Ser
Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu 420 425 430Ser
Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser 435 440
445Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
450 455 460Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu465 470 475 480Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
Glu Ser Ser Glu Ser 485 490 495Ser Glu Ser Ser Glu Ser Ser Glu Ser
Ser Glu Ser Ser Glu Ser Ser 500 505 510Glu Ser Ser Glu Ser Ser Glu
Ser Ser Glu Ser Ser Glu Ser Ser Glu 515 520 525Ser Ser Glu Ser Ser
Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser 530 535 540Ser Glu Ser
Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser545 550 555
560Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu
565 570 575Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser Glu Ser Ser
Glu Ser 580 585 590Ser Glu Ser Ser Glu Ser Ser Glu 595
60044140PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 441Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly Xaa
Gly Xaa Gly Xaa Gly Xaa1 5 10 15Gly Xaa Gly Xaa Gly Xaa Gly Xaa Gly
Xaa Gly Xaa Gly Xaa Gly Xaa 20 25 30Gly Xaa Gly Xaa Gly Xaa Gly Xaa
35 4044239PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 442Gly Gly Xaa Gly Gly Xaa Gly Gly Xaa Gly Gly
Xaa Gly Gly Xaa Gly1 5 10 15Gly Xaa Gly Gly Xaa Gly Gly Xaa Gly Gly
Xaa Gly Gly Xaa Gly Gly 20 25 30Xaa Gly Gly Xaa Gly Gly Xaa
3544340PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 443Gly Gly Gly Xaa Gly Gly Gly Xaa Gly Gly
Gly Xaa Gly Gly Gly Xaa1 5 10 15Gly Gly Gly Xaa Gly Gly Gly Xaa Gly
Gly Gly Xaa Gly Gly Gly Xaa 20 25 30Gly Gly Gly Xaa Gly Gly Gly Xaa
35 4044440PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 444Gly Gly Gly Gly Xaa Gly Gly Gly Gly Xaa
Gly Gly Gly Gly Xaa Gly1 5 10 15Gly Gly Gly Xaa Gly Gly Gly Gly Xaa
Gly Gly Gly Gly Xaa Gly Gly 20 25 30Gly Gly Xaa Gly Gly Gly Gly Xaa
35 40445315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 445Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly1 5 10 15Gly Gly Gly Gly Xaa Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 20 25 30Gly Gly Gly Gly Gly Gly Gly Gly
Gly Xaa Gly Gly Gly Gly Gly Gly 35 40 45Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Xaa Gly 50 55 60Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly65 70 75 80Gly Gly Gly Xaa
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 85 90 95Gly Gly Gly
Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly 100 105 110Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly 115 120
125Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
130 135 140Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly145 150 155 160Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly Gly
Gly Gly Gly Gly Gly 165 170 175Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Xaa Gly Gly Gly 180 185 190Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 195 200 205Gly Xaa Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 210 215 220Gly Gly Gly
Gly Gly Gly Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly225 230 235
240Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly
245 250 255Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly 260 265 270Xaa Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly 275 280 285Gly Gly Gly Gly Gly Xaa Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly 290 295 300Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Xaa305 310 3154464PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 446Lys Cys Lys
Lys14474PRTHomo sapiens 447Asp Asp Asp Lys14484PRTHomo sapiens
448Ile Asp Gly Arg14496PRTHomo sapiens 449Leu Val Pro Arg Gly Ser1
54508PRTHomo sapiens 450Leu Glu Val Leu Phe Gln Gly Pro1
54517PRTHomo sapiens 451Glu Gln Leu Tyr Phe Gln Gly1 54527PRTHomo
sapiens 452Glu Thr Leu Phe Gln Gly Pro1 54535PRTHomo sapiens 453Leu
Pro Glu Thr Gly1 5454191PRTHomo sapiens 454Phe Pro Thr Ile Pro Leu
Ser Arg Leu Phe Asp Asn Ala Met Leu Arg1 5 10 15Ala His Arg Leu His
Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30Glu Ala Tyr Ile
Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45Gln Thr Ser
Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60Glu Glu
Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu65 70 75
80Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val
85 90 95Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr
Asp 100 105 110Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met
Gly Arg Leu 115 120 125Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe
Lys Gln Thr Tyr Ser 130 135 140Lys Phe Asp Thr Asn Ser His Asn Asp
Asp Ala Leu Leu Lys Asn Tyr145 150 155 160Gly Leu Leu Tyr Cys Phe
Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175Leu Arg Ile Val
Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185
190455288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 455Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser Gly1 5 10 15Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly 20 25 30Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser Gly Gly Glu 35 40 45Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly 50 55 60Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly65 70 75 80Ser Gly Gly Glu
Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu 85 90 95Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly 100 105 110Gly
Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly 115 120
125Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
130 135 140Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser Gly145 150 155 160Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly Gly Glu Gly Gly 165 170 175Ser Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly Gly Glu 180 185 190Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly 195 200 205Gly Glu Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly 210 215 220Ser Gly Gly
Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu225 230 235
240Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
245 250 255Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly 260 265 270Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu 275 280 28545636DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 456aggtagtggw ggwgarggwg
gwtcyggwgg agaagg 3645736DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 457acctccttct ccwccrgawc
cwccytcwcc wccact 3645824DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 458aggttcgtct tcactcgagg gtac
2445916DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 459cctcgagtga agacga 16460288PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
460Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu1
5 10 15Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu
Ser 20 25 30Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser
Ser Glu 35 40 45Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Glu 50 55 60Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu
Ser Ser Glu Ser65 70 75 80Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser
Glu Ser Ser Ser Ser Glu 85 90 95Ser Ser Ser Glu Ser Ser Glu Ser Ser
Ser Ser Glu Ser Ser Ser Glu 100 105 110Ser Ser Glu Ser Ser Ser Ser
Glu Ser Ser Ser Glu Ser Ser Glu Ser 115 120 125Ser Ser Ser Glu Ser
Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu 130 135 140Ser Ser Ser
Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu145 150 155
160Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser
165 170 175Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser
Ser Glu 180 185 190Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Glu 195 200 205Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Glu Ser Ser Glu Ser 210 215 220Ser Ser Ser Glu Ser Ser Ser Glu
Ser Ser Glu Ser Ser Ser Ser Glu225 230 235 240Ser Ser Ser Glu Ser
Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu 245 250 255Ser Ser Glu
Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser 260 265 270Ser
Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu 275 280
28546136PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 461Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser Ser Ser Glu1 5 10 15Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Glu Ser Ser Glu Ser 20 25 30Ser Ser Ser Glu
3546212PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 462Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu
Ser1 5 1046336DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 463ttctagtgar tcyagygart cyagytcyag
ygaatc 3646436DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 464agaagattcr ctrgarctrg aytcrctrga
ytcact 3646524DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 465ttcttcgtct tcactcgagg gtac
24466288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 466Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly
Glu Gly Gly Gly Glu Gly1 5 10 15Gly Glu Gly Glu Gly Gly Gly Glu Gly
Gly Glu Gly Glu Gly Gly Gly 20 25 30Glu Gly Gly Glu Gly Glu Gly Gly
Gly Glu Gly Gly Glu Gly Glu Gly 35 40 45Gly Gly Glu Gly Gly Glu Gly
Glu Gly Gly Gly Glu Gly Gly Glu Gly 50 55 60Glu Gly Gly Gly Glu Gly
Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly65 70 75 80Glu Gly Glu Gly
Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu 85 90 95Gly Gly Glu
Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly 100 105 110Gly
Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu 115 120
125Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu
130 135 140Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly
Glu Gly145 150 155 160Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu
Gly Glu Gly Gly Gly 165 170 175Glu Gly Gly Glu Gly Glu Gly Gly Gly
Glu Gly Gly Glu Gly Glu Gly 180 185 190Gly Gly Glu Gly Gly Glu Gly
Glu Gly Gly Gly Glu Gly Gly Glu Gly 195 200 205Glu Gly Gly Gly Glu
Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly 210 215 220Glu Gly Glu
Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu225 230 235
240Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly
245 250 255Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu
Gly Glu 260 265 270Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly
Glu Gly Gly Glu 275 280 28546727DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 467aggtgaaggw garggwggwg
gwgaagg 2746827DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 468acctccttcw ccwccwccyt cwccttc
274697PRTHomo sapiensMOD_RES(7)..(7)Any amino acid 469Glu Asn Leu
Tyr Phe Gln Xaa1 5470165PRTHomo sapiens 470Cys Asp Leu Pro Gln Thr
His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met
Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe
Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu
Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val
Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110Glu
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120
125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
Glu Ser145 150 155 160Leu Arg Ser Lys Glu 165471174PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
471Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys1
5 10 15Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu
Gln 20 25 30Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu
Leu Val 35 40 45Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu
Ser Ser Cys 50 55 60Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser
Gln Leu His Ser65 70 75 80Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln
Ala Leu Glu Gly Ile Ser 85 90 95Pro Glu Leu Gly Pro Thr Leu Asp Thr
Leu Gln Leu Asp Val Ala Asp 100 105 110Phe Ala Thr Thr Ile Trp Gln
Gln Met Glu Glu Leu Gly Met Ala Pro 115 120 125Ala Leu Gln Pro Thr
Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 130 135 140Gln Arg Arg
Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe145 150 155
160Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165
170472288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 472Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly Gly1 5 10 15Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu Gly Gly Ser 20 25 30Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly Gly Glu Gly 35 40 45Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu Gly Gly Ser Gly Gly 50 55 60Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser65 70 75 80Gly Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly 85 90 95Gly Ser Gly
Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly 100 105 110Glu
Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser 115 120
125Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
130 135 140Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly Gly145 150 155 160Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser 165 170 175Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Ser Gly Gly Glu Gly 180 185 190Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu Gly Gly Ser Gly Gly 195 200 205Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser 210 215 220Gly Gly Glu
Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly225 230 235
240Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly
245 250 255Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser 260 265 270Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly Gly Glu Gly 275 280 285473288PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 473Gly Glu Gly Glu Gly
Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly1 5 10 15Glu Gly Gly Glu
Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly 20 25 30Gly Gly Glu
Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly 35 40 45Glu Gly
Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly 50 55 60Glu
Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu65 70 75
80Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly
85 90 95Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly
Glu 100 105 110Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu
Gly Gly Glu 115 120 125Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly 130 135 140Gly Glu Gly Glu Gly Gly Gly Glu Gly
Gly Glu Gly Glu Gly Gly Gly145 150 155 160Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly 165 170 175Gly Gly Glu Gly
Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly 180 185 190Glu Gly
Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly 195 200
205Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu
210 215 220Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu
Gly Gly225 230 235 240Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu
Gly Gly Glu Gly Glu 245 250 255Gly Gly Gly Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly Gly Glu 260 265 270Gly Glu Gly Gly Gly Glu Gly
Gly Glu Gly Glu Gly Gly Gly Glu Gly 275 280 285474288PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
474Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser1
5 10 15Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser
Ser 20 25 30Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser 35 40 45Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Glu Ser 50 55 60Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser
Ser Glu Ser Ser65 70 75 80Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu
Ser Ser Ser Ser Glu Ser 85 90 95Ser Ser Glu Ser Ser Glu Ser Ser Ser
Ser Glu Ser Ser Ser Glu Ser 100 105 110Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Glu Ser Ser Glu Ser Ser 115 120 125Ser Ser Glu Ser Ser
Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser 130 135 140Ser Ser Glu
Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser145 150 155
160Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser
165 170 175Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser 180 185 190Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Glu Ser 195 200 205Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser
Glu Ser Ser Glu Ser Ser 210 215 220Ser Ser Glu Ser Ser Ser Glu Ser
Ser Glu Ser Ser Ser Ser Glu Ser225 230 235 240Ser Ser Glu Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser 245 250 255Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser 260 265 270Ser
Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser 275 280
285475336PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 475Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser
Ser Ser Glu Ser Ser Ser1 5 10 15Ser Ser Ser Glu Ser Ser Ser Ser Ser
Ser Glu Ser Ser Ser Ser Ser 20 25 30Ser Glu Ser Ser Ser Ser Ser Ser
Glu Ser Ser Ser Ser Ser Ser Glu 35 40 45Ser Ser Ser Ser Ser Ser Glu
Ser Ser Ser Ser Ser Ser Glu Ser Ser 50 55 60Ser Ser Ser Ser Glu Ser
Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser65 70 75 80Ser Ser Glu Ser
Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser Ser 85 90 95Glu Ser Ser
Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser 100 105 110Ser
Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser 115 120
125Ser Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser
130 135 140Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser
Ser Glu145 150 155 160Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser
Ser Ser Glu Ser Ser 165 170 175Ser Ser Ser Ser Glu Ser Ser Ser Ser
Ser Ser Glu Ser Ser Ser Ser 180 185 190Ser Ser Glu Ser Ser Ser Ser
Ser Ser Glu Ser Ser Ser Ser Ser Ser 195 200 205Glu Ser Ser Ser Ser
Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser 210 215 220Ser Ser Ser
Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser225 230 235
240Ser Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser
245 250 255Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser
Ser Glu 260 265 270Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser
Ser Glu Ser Ser 275 280 285Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser
Ser Glu Ser Ser Ser Ser 290 295 300Ser Ser Glu Ser Ser Ser Ser Ser
Ser Glu Ser Ser Ser Ser Ser Ser305 310 315 320Glu Ser Ser Ser Ser
Ser Ser Glu Ser Ser Ser Ser Ser Ser Glu Ser 325 330
335476320PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 476Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu Ser Ser1 5 10 15Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser 20 25 30Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser Ser 35 40 45Glu Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser Ser Glu 50 55 60Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser65 70 75 80Ser Ser Ser Glu
Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser 85 90 95Ser Ser Glu
Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser 100 105 110Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser 115 120
125Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu
130 135 140Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser
Glu Ser145 150 155 160Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Ser Glu Ser Ser 165 170 175Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser 180 185 190Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Ser Glu Ser Ser Ser Ser 195 200 205Glu Ser Ser Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu 210 215 220Ser Ser Ser
Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser225 230 235
240Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
245 250 255Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser 260 265 270Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Ser 275 280 285Glu Ser Ser Ser Ser Glu Ser Ser Ser Ser
Glu Ser Ser Ser Ser Glu 290 295 300Ser Ser Ser Ser Glu Ser Ser Ser
Ser Glu Ser Ser Ser Ser Glu Ser305 310 315 320477288PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
477Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser1
5 10 15Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly
Ser 20 25 30Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly
Glu Gly 35 40 45Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly
Ser Glu Gly 50 55 60Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser
Gly Glu Gly Ser65 70 75 80Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu
Gly Glu Gly Gly Ser Glu 85 90 95Gly Ser Glu Gly Glu Gly Ser Gly Glu
Gly Ser Glu Gly Glu Gly Gly 100 105 110Glu Gly Gly Ser Glu Gly Glu
Gly Ser Glu Gly Ser Gly Glu Gly Glu 115 120 125Gly Ser Gly Glu Gly
Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu 130 135 140Gly Glu Gly
Ser Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser145 150 155
160Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser
165 170 175Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
Glu Gly 180 185 190Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser Gly Glu
Gly Ser Glu Gly 195 200 205Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu
Gly Ser Gly Glu Gly Gly 210 215 220Glu Gly Glu Gly Ser Glu Gly Gly
Ser Glu Gly Glu Gly Gly Ser Glu225 230 235 240Gly Gly Glu Gly Glu
Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser 245 250 255Glu Gly Gly
Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu 260 265 270Gly
Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu 275 280
285478288PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 478Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly Ser Glu Gly Ser1 5 10 15Gly Glu Gly Glu Gly Ser Glu Gly Gly
Ser Glu Gly Glu Gly Ser Glu 20 25 30Gly Ser Gly Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly 35 40 45Gly Glu Gly Ser Gly Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly 50 55 60Glu Gly Gly Gly Glu Gly
Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser65 70 75 80Glu Gly Glu Gly
Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Glu Gly 85 90 95Ser Gly Glu
Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser 100 105 110Gly
Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu 115 120
125Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu
130 135 140Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu
Gly Ser145 150 155 160Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu Gly Gly Ser 165 170 175Glu Gly Ser Glu Gly Glu Gly Gly Ser
Glu Gly Ser Glu Gly Glu Gly 180 185 190Gly Ser Glu Gly Ser Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly 195 200 205Glu Gly Gly Ser Glu
Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser 210 215 220Glu Gly Glu
Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu225 230 235
240Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly
245 250 255Glu Gly Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu 260 265 270Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly
Ser Glu Gly Ser Gly Glu 275 280 285479272PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
479Ser Glu Gly Glu Ser Glu Glu Ser Ser Glu Ser Gly Gly Glu Ser Ser1
5 10 15Ser Gly Gly Gly Ser Glu Glu Ser Ser Glu Glu Gly Ser Gly Gly
Gly 20 25 30Ser Glu Gly Glu Gly Glu Glu Ser Ser Gly Ser Glu Gly Gly
Gly Gly 35 40 45Ser Gly Glu Gly Ser Glu Gly Gly Ser Glu Glu Gly Ser
Glu Glu Ser 50 55 60Ser Glu Gly Glu Ser Glu Glu Ser Ser Glu Ser Gly
Gly Glu Ser Ser65 70 75 80Ser Gly Gly Gly Ser Glu Glu Ser Ser Glu
Glu Gly Ser Gly Gly Gly 85 90 95Ser Gly Glu Ser Gly Ser Gly Ser Ser
Gly Ser Glu Ser Glu Gly Gly 100 105 110Ser Glu Gly Glu Ser Glu Glu
Ser Ser Gly Gly Gly Gly Ser Glu Gly 115 120 125Ser Glu Gly Glu Ser
Glu Glu Ser Ser Glu Ser Gly Gly Glu Ser Ser 130 135 140Ser Gly Gly
Gly Ser Glu Glu Ser Ser Glu Glu Gly Ser Gly Gly Gly145 150 155
160Ser Glu Glu Glu Ser Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Ser
165 170 175Ser Gly Glu Gly Ser Glu Glu Ser Ser Gly Gly Ser Glu Gly
Gly Gly 180 185 190Ser Gly Gly Ser Gly Gly Glu Gly Ser Gly Glu Ser
Gly Ser Gly Ser 195 200 205Ser Gly Ser Glu Ser Glu Gly Gly Ser Glu
Gly Glu Ser Glu Glu Ser 210 215 220Ser Gly Gly Gly Gly Ser Glu Gly
Ser Ser Glu Glu Ser Gly Gly Ser225 230 235 240Ser Glu Glu Gly Ser
Glu Gly Ser Ser Gly Gly Glu Ser Glu Glu Ser 245 250 255Ser Glu Gly
Glu Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Ser 260 265
270480264PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 480Ser Glu Gly Glu Ser Glu Glu Ser Ser Glu
Ser Gly Gly Glu Ser Ser1 5 10 15Ser Gly Gly Gly Ser Glu Glu Ser Ser
Glu Glu Gly Ser Gly Gly Gly 20 25 30Ser Glu Gly Glu Ser Glu Glu Ser
Ser Glu Ser Gly Gly Glu Ser Ser 35 40 45Ser Gly Gly Gly Ser Glu Glu
Ser Ser Glu Glu Gly Ser Gly Gly Gly 50 55 60Ser Gly Glu Ser Gly Ser
Gly Ser Ser Gly Ser Glu Ser Glu Gly Gly65 70 75 80Ser Glu Gly Glu
Ser Glu Glu Ser Ser Gly Gly Gly Gly Ser Glu Gly 85 90 95Ser Glu Ser
Glu Gly Glu Glu Gly Ser Glu Glu Gly Ser Gly Glu Gly 100 105 110Ser
Gly Glu Gly Gly Gly Glu Ser Ser Glu Glu Gly Glu Ser Glu Ser 115 120
125Ser Gly Glu Ser Gly Ser Gly Ser Ser Gly Ser Glu Ser Glu Gly Gly
130 135 140Ser Glu Gly Glu Ser Glu Glu Ser Ser Gly Gly Gly Gly Ser
Glu Gly145 150 155 160Ser Gly Glu Ser Gly Ser Gly Ser Ser Gly Ser
Glu Ser Glu Gly Gly 165 170 175Ser Glu Gly Glu Ser Glu Glu Ser Ser
Gly Gly Gly Gly Ser Glu Gly 180 185 190Ser Gly Glu Ser Gly Ser Gly
Ser Ser Gly Ser Glu Ser Glu Gly Gly 195 200 205Ser Glu Gly Glu Ser
Glu Glu Ser Ser Gly Gly Gly Gly Ser Glu Gly 210 215 220Ser Ser Glu
Glu Ser Gly Gly Ser Ser Glu Glu Gly Ser Glu Gly Ser225 230 235
240Ser Gly Gly Glu Ser Glu Glu Ser Ser Glu Gly Glu Ser Gly Gly Gly
245 250 255Ser Gly Gly Gly Ser Glu Gly Ser 260481264PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
481Ser Glu Gly Glu Ser Glu Glu Ser Ser Glu Ser Gly Gly Glu Ser Ser1
5 10 15Ser Gly Gly Gly Ser Glu Glu Ser Ser Glu Glu Gly Ser Gly Gly
Gly 20 25 30Ser Glu Gly Glu Gly Glu Glu Ser Ser Gly Ser Glu Gly Gly
Gly Gly 35 40 45Ser Gly Glu Gly Ser Glu Gly Gly Ser Glu Glu Gly Ser
Glu Glu Ser 50 55 60Ser Glu Gly Glu Ser Glu Glu Ser Ser Glu Ser Gly
Gly Glu Ser Ser65 70 75 80Ser Gly Gly Gly Ser Glu Glu Ser Ser Glu
Glu Gly Ser Gly Gly Gly 85 90 95Ser Gly Glu Ser Gly Ser Gly Ser Ser
Gly Ser Glu Ser Glu Gly Gly 100 105 110Ser Glu Gly Glu Ser Glu Glu
Ser Ser Gly Gly Gly Gly Ser Glu Gly 115 120 125Ser Gly Glu Ser Gly
Ser Gly Ser Ser Gly Ser Glu Ser Glu Gly Gly 130 135 140Ser Glu Gly
Glu Ser Glu Glu Ser Ser Gly Gly Gly Gly Ser Glu Gly145 150 155
160Ser Glu Ser Glu Gly Glu Glu Gly Ser Glu Glu Gly Ser Gly Glu Gly
165 170 175Ser Gly Glu Gly Gly Gly Glu Ser Ser Glu Glu Gly Glu Ser
Glu Ser 180 185 190Ser Glu Gly Glu Ser Glu Glu Ser Ser Glu Ser Gly
Gly Glu Ser Ser 195 200 205Ser Gly Gly Gly Ser Glu Glu Ser Ser Glu
Glu Gly Ser Gly Gly Gly 210 215 220Ser Ser Glu Glu Ser Gly Gly Ser
Ser Glu Glu Gly Ser Glu Gly Ser225 230 235 240Ser Gly Gly Glu Ser
Glu Glu Ser Ser Glu Gly Glu Ser Gly Gly Gly 245 250 255Ser Gly Gly
Gly Ser Glu Gly Ser 260482264PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 482Ser Glu Gly Glu Ser
Glu Glu Ser Ser Glu Ser Gly Gly Glu Ser Ser1 5 10 15Ser Gly Gly Gly
Ser Glu Glu Ser Ser Glu Glu Gly Ser Gly Gly Gly 20 25 30Ser Gly Glu
Ser Gly Ser Gly Ser Ser Gly Ser Glu Ser Glu Gly Gly 35 40 45Ser Glu
Gly Glu Ser Glu Glu Ser Ser Gly Gly Gly Gly Ser Glu Gly 50 55 60Ser
Glu Gly Glu Ser Glu Glu Ser Ser Glu Ser Gly Gly Glu Ser Ser65 70 75
80Ser Gly Gly Gly Ser Glu Glu Ser Ser Glu Glu Gly Ser Gly Gly Gly
85 90 95Ser Glu Glu Gly Ser Gly Glu Ser Ser Gly Gly Ser Glu Ser Glu
Gly 100 105 110Ser Gly Gly Glu Ser Glu Gly Gly Ser Gly Gly Glu Gly
Gly Glu Gly 115 120 125Ser Gly Glu Ser Gly Ser Gly Ser Ser Gly Ser
Glu Ser Glu Gly Gly 130 135 140Ser Glu Gly Glu Ser Glu Glu Ser Ser
Gly Gly Gly Gly Ser Glu Gly145 150 155 160Ser Ser Glu Glu Ser Gly
Gly Ser Ser Glu Glu Gly Ser Gly Gly Gly 165 170 175Ser Glu Ser Gly
Glu Glu Ser Gly Ser Gly Glu Glu Ser Glu Gly Gly 180 185 190Ser Gly
Gly Ser Gly Gly Glu Gly Ser Gly Glu Ser Gly Ser Gly Ser 195 200
205Ser Gly Ser Glu Ser Glu Gly Gly Ser Glu Gly Glu Ser Glu Glu Ser
210 215 220Ser Gly Gly Gly Gly Ser Glu Gly Ser Ser Gly Glu Gly Glu
Glu Ser225 230 235 240Ser Glu Gly Glu Gly Gly Glu Ser Ser Glu Glu
Gly Ser Gly Gly Ser 245 250 255Ser Glu Glu Gly Ser Gly Glu Gly
26048328PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 483Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly
Gly Glu Gly Ser Glu Gly1 5 10 15Glu Gly Ser Gly Glu Gly Gly Glu Gly
Glu Gly Ser 20 25484864DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 484tct agt gag tcc agt
gaa tcc agc tcc agc gaa tct tct agt gaa tcc 48Ser Ser Glu Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser1 5 10 15agc gag tct agc
tct agc gaa tct tct agt gag tcc agt gag tcc agt 96Ser Glu Ser Ser
Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser 20 25 30tcc agt gaa
tct tct agt gag tcc agt gaa tct agc tcc agt gaa tct 144Ser Ser Glu
Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser 35 40 45tct agt
gag tct agc gaa tct agc tcc agc gaa tct tct agt gaa tcc 192Ser Ser
Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser 50 55 60agc
gaa tcc agc tct agt gaa tct tct agt gaa tct agc gag tcc agc 240Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser65 70 75
80tcc agt gaa tct tct agt gag tcc agt gag tcc agt tct agt gaa tct
288Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser
85 90 95tct agt gaa tct agt gag tcc agc tcc agc gaa tct tct agt gaa
tct 336Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu
Ser 100 105 110agc gag tcc agt tcc agt gaa tct tct agt gaa tct agt
gaa tct agc 384Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser
Glu Ser Ser 115 120 125tct agc gaa tct tct agt gag tcc agc gaa tcc
agt tct agt gaa tct 432Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser
Ser Ser Ser Glu Ser 130 135 140tct agt gag tcc agc gag tcc agc tct
agt gaa tct tct agt gaa tcc 480Ser Ser Glu Ser Ser Glu Ser Ser Ser
Ser Glu Ser Ser Ser Glu Ser145 150 155 160agc gag tcc agt tcc agt
gaa tct tct agt gaa tct agt gag tcc agt 528Ser Glu Ser Ser Ser Ser
Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser 165 170 175tct agt gaa tct
tct agt gag tcc agc gag tcc agc tct agt gaa tct 576Ser Ser Glu Ser
Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser 180 185 190tct agt
gaa tcc agc gag tcc agt tcc agt gaa tct tct agt gaa tct 624Ser Ser
Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser 195 200
205agt gag tcc agt tct agt gaa tct tct agt gaa tct agt gag tcc agt
672Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser
210 215 220tcc agt gaa tct tct agt gaa tct agt gaa tcc agt tct agc
gaa tct 720Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser225 230 235 240tct agt gag tcc agt gag tcc agc tct agt gaa
tct tct agt gaa tcc 768Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Glu Ser 245 250 255agc gaa tcc agc tct agc gaa tct tct
agt gag tcc agc gag tct agt 816Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Glu Ser Ser Glu Ser Ser 260 265 270tcc agt gaa tct tct agt gaa
tcc agc gaa tct agc tcc agc gaa tct 864Ser Ser Glu Ser Ser Ser Glu
Ser Ser Glu Ser Ser Ser Ser Glu Ser 275 280 285485288PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
485Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser1
5 10 15Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser
Ser 20 25 30Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser 35 40 45Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser
Ser Glu Ser 50 55 60Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser
Ser Glu Ser Ser65 70 75 80Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu
Ser Ser Ser Ser Glu Ser 85 90 95Ser Ser Glu Ser Ser Glu Ser Ser Ser
Ser Glu Ser Ser Ser Glu Ser 100 105 110Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Glu Ser Ser Glu Ser Ser 115 120 125Ser Ser Glu Ser Ser
Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser 130 135 140Ser Ser Glu
Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser145 150 155
160Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser
165 170 175Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser 180 185 190Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser
Ser Ser Glu Ser 195 200 205Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser
Glu Ser Ser Glu Ser Ser 210 215 220Ser Ser Glu Ser Ser Ser Glu Ser
Ser Glu Ser Ser Ser Ser Glu Ser225 230 235 240Ser Ser Glu Ser Ser
Glu Ser Ser Ser Ser Glu Ser Ser Ser Glu Ser 245 250 255Ser Glu Ser
Ser Ser Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser 260 265 270Ser
Ser Glu Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser Glu Ser 275 280
28548626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 486Gly Glu Gly Glu Gly Glu Gly Glu Gly Glu Gly
Glu Gly Glu Gly Glu1 5 10 15Gly Glu Gly Glu Gly Glu Gly Glu Gly Glu
20 2548726PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 487Gly Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu
Gly Gly Gly Glu Gly1 5 10 15Gly Glu Gly Glu Gly Gly Gly Glu Gly Gly
20 2548827PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 488Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly1 5 10 15Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser 20 2548929PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 489Ser Glu Ser Ser Ser Glu Ser Ser Glu
Ser Glu Ser Ser Ser Glu Ser1 5 10 15Ser Glu Ser Glu Ser Ser Ser Glu
Ser Ser Glu Ser Glu 20 2549013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 490Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly1 5 1049124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 491Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly1 5 10
15Gly Ser Gly Gly Ser Gly Gly Glu 2049224PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 492Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly1 5 10
15Gly Glu Gly Gly Ser Gly Gly Glu 2049324PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 493Gly
Glu Gly Gly Gly Glu Gly Gly Glu Gly Glu Gly Gly Gly Glu Gly1 5 10
15Gly Glu Gly Glu Gly Gly Gly Glu 2049418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 494Gly
Glu Gly Glu Gly Glu Gly Glu Gly Glu Gly Glu Gly Glu Gly Glu1 5 10
15Gly Glu49513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 495Glu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Glu Glu Glu1 5 1049622PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 496Ser Ser Ser Ser Ser Glu
Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser1 5 10 15Ser Glu Ser Ser Ser
Ser 2049724PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 497Ser Ser Ser Glu Ser Ser Glu Ser Ser Ser Ser
Glu Ser Ser Ser Glu1 5 10 15Ser Ser Glu Ser Ser Ser Ser Glu
2049818PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 498Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Glu
Ser Ser Ser Ser Ser1 5 10 15Ser Glu499864DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
499ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt
48Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly1
5 10 15ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt
ggt 96Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly 20 25 30tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt
ggt gaa 144Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 35 40 45ggt ggt tct
ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt 192Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly 50 55 60ggt gaa
ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt 240Gly Glu
Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly65 70 75
80tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa
288Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
85 90 95ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct
ggt 336Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly 100 105 110ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt
gaa ggt ggt 384Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly 115 120 125tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt
ggt tct ggt ggt gaa 432Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly Glu 130 135 140ggt ggt tct ggt ggt gaa ggt ggt tct
ggt ggt gaa ggt ggt tct ggt 480Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly145 150 155 160ggt gaa ggt ggt tct ggt
ggt gaa ggt ggt tct ggt ggt gaa ggt ggt 528Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly 165 170 175tct ggt ggt gaa
ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa 576Ser Gly Gly Glu
Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu 180 185 190ggt ggt
tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt 624Gly Gly
Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly 195 200
205ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt
672Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
210 215 220tct ggt ggt gaa ggt ggt tct ggt ggt gaa ggt ggt tct ggt
ggt gaa 720Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu225 230 235 240ggt ggt tct ggt ggt gaa ggt ggt tct ggt ggt
gaa ggt ggt tct ggt 768Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly 245 250 255ggt gaa ggt ggt tct ggt ggt gaa ggt
ggt tct ggt ggt gaa ggt ggt 816Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly 260 265 270tct ggt ggt gaa ggt ggt tct
ggt ggt gaa ggt ggt tct ggt ggt gaa 864Ser Gly Gly Glu Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly Gly Glu 275 280 285500288PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
500Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly1
5 10 15Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly 20 25 30Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 35 40 45Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly
Gly Ser Gly 50 55 60Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly65 70 75 80Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Ser Gly Gly Glu 85 90 95Gly Gly Ser Gly Gly Glu Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly 100 105 110Gly Glu Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly Gly Glu Gly Gly 115 120 125Ser Gly Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu 130 135 140Gly Gly Ser
Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly145 150 155
160Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
165 170 175Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu 180 185 190Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu
Gly Gly Ser Gly 195 200 205Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly
Ser Gly Gly Glu Gly Gly 210 215 220Ser Gly Gly Glu Gly Gly Ser Gly
Gly Glu Gly Gly Ser Gly Gly Glu225 230 235 240Gly Gly Ser Gly Gly
Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly 245 250 255Gly Glu Gly
Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly 260 265 270Ser
Gly Gly Glu Gly Gly Ser Gly Gly Glu Gly Gly Ser Gly Gly Glu 275 280
285501126DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 501atg gat tat aaa gac gat gac gat aaa ggg
tct cca ggt tagtaaccta 49Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly
Ser Pro Gly1 5 10ggtgatag gga ggt tcg tct tca ctc gag ggt acc cat
cac cat cac cat 99 Gly Gly Ser Ser Ser Leu Glu Gly Thr His His His
His His 15 20 25cac gag ctc gta ccg gta gaa aaa atg 126His Glu Leu
Val Pro Val Glu Lys Met 30 3550213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 502Met Asp Tyr Lys Asp Asp
Asp Asp Lys Gly Ser Pro Gly1 5 1050323PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 503Gly
Gly Ser Ser Ser Leu Glu Gly Thr His His His His His His Glu1 5 10
15Leu Val Pro Val Glu Lys Met 205046PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 504Lys
Lys Lys Lys Lys Lys1 5505856PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 505Met Glu Gly Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala1 5 10 15Ser Val Gly Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val 20 25 30Asn Thr Ala
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu
Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg 50 55 60Phe
Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser65 70 75
80Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr
85 90 95Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr
Gly 100 105 110Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Gly Glu Gly
Ser Glu Gly 115 120 125Glu Gly Ser Gly Glu Gly Gly Glu Gly Glu Gly
Ser Gly Thr Glu Val 130 135 140Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu145 150 155 160Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile 165 170 175His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg 180 185 190Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly 195 200
205Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
210 215 220Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ser Arg225 230 235 240Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
Trp Gly Gln Gly Thr 245 250 255Leu Val Thr Val Ser Gly Gly Glu Gly
Ser Gly Glu Gly Ser Glu Gly 260 265 270Glu Gly Ser Glu Gly Ser Gly
Glu Gly Glu Gly Ser Glu Gly Ser Gly 275 280 285Glu Gly Glu Gly Gly
Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly 290 295 300Ser Gly Glu
Gly Glu Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser305 310 315
320Gly Glu Gly Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu
325 330 335Gly Ser Gly Glu Gly Gly Glu Gly Glu Gly Ser Glu Gly Gly
Ser Glu 340 345 350Gly Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu Gly
Ser Glu Gly Ser 355 360 365Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser
Glu Gly Glu Gly Ser Glu 370 375 380Gly Gly Ser Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly385 390 395 400Ser Glu Gly Ser Gly
Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly 405 410 415Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser 420 425 430Glu
Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu 435 440
445Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly
450 455 460Gly Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu
Gly Glu465 470 475 480Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly
Ser Glu Gly Ser Glu 485 490 495Gly Glu Gly Gly Glu Gly Ser Gly Glu
Gly Glu Gly Ser Glu Gly Ser 500 505 510Gly Glu Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly Ser Glu 515 520 525Gly Ser Gly Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly 530 535 540Gly Ser Glu
Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly545 550 555
560Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly
565 570 575Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly
Ser Glu 580 585 590Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu
Gly Glu Gly Gly 595 600 605Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu
Gly Ser Gly Glu Gly Glu 610 615 620Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu625 630 635 640Gly Glu Gly Ser Glu
Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly 645 650 655Ser Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Glu 660 665 670Gly
Ser Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 675 680
685Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
690 695 700Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly
Gly Ser705 710 715 720Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly
Glu Gly Ser Glu Gly 725 730 735Gly Ser Glu Gly Glu Gly Ser Glu Gly
Gly Ser Glu Gly Glu Gly Ser 740 745 750Glu Gly Ser Gly Glu Gly Glu
Gly Ser Glu Gly Ser Gly Glu Gly Glu 755 760 765Gly Ser Gly Glu Gly
Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu 770 775 780Gly Glu Gly
Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly785 790 795
800Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Gly Gly
805 810 815Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
Glu Gly 820 825 830Ser Gly Glu Gly Ser Glu Gly Asp Tyr Lys Asp Asp
Asp Asp Lys Gly 835 840 845Gly Ser His His His His His His 850
855506854PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 506Met Glu Asp Ile Leu Leu Thr Gln Ser Pro
Val Ile Leu Ser Val Ser1 5 10 15Pro Gly Glu Arg Val Ser Phe Ser Cys
Arg Ala Ser Gln Ser Ile Gly 20 25 30Thr Asn Ile His Trp Tyr Gln Gln
Arg Thr Asn Gly Ser Pro Arg Leu 35 40 45Leu Ile Lys Tyr Ala Ser Glu
Ser Ile Ser Gly Ile Pro Ser Arg Phe 50 55 60Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Ser Ile Asn Ser Val65 70 75 80Glu Ser Glu Asp
Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp 85 90 95Pro Thr Thr
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Thr Gly Ser 100 105 110Gly
Glu Gly Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu 115 120
125Gly Ser Gly Glu Gly Gly Glu Gly Glu Gly Ser Gly Thr Gln Val Gln
130 135 140Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln Ser
Leu Ser145 150 155 160Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr
Asn Tyr Gly Val His 165 170 175Trp Val Arg Gln Ser Pro Gly Lys Gly
Leu Glu Trp Leu Gly Val Ile 180 185 190Trp Ser Gly Gly Asn Thr Asp
Tyr Asn Thr Pro Phe Thr Ser Arg Leu 195 200 205Ser Ile Asn Lys Asp
Asn Ser Lys Ser Gln Val Phe Phe Lys Met Asn 210 215 220Ser Leu Gln
Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu225 230 235
240Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
245 250 255Thr Val Ser Gly Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly 260 265 270Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly
Ser Gly Glu Gly 275 280 285Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu
Gly Ser Glu Gly Ser Gly 290 295 300Glu Gly Glu Gly Gly Glu Gly Ser
Gly Glu Gly Glu Gly Ser Gly Glu305 310 315 320Gly Ser Glu Gly Glu
Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser 325 330 335Gly Glu Gly
Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu 340 345 350Gly
Gly Ser Glu Gly Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu 355 360
365Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly
370 375 380Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly
Ser Glu385 390 395 400Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser
Gly Glu Gly Glu Gly 405 410 415Ser Glu Gly Ser Gly Glu Gly Glu Gly
Ser Glu Gly Gly Ser Glu Gly 420 425 430Glu Gly Gly Ser Glu Gly Ser
Glu Gly Glu Gly Ser Gly Glu Gly Ser 435 440 445Glu Gly Glu Gly Gly
Ser Glu Gly Ser Glu Gly Glu Gly Gly Gly Glu 450 455 460Gly Ser Glu
Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly465 470 475
480Ser Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu
485 490 495Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser
Gly Glu 500 505 510Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly
Ser Glu Gly Ser 515 520 525Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly
Glu Gly Glu Gly Gly Ser 530 535 540Glu Gly Ser Glu Gly Glu Gly Ser
Gly Glu Gly Ser Glu Gly Glu Gly545 550 555 560Ser Glu Gly Ser Gly
Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly 565 570 575Glu Gly Gly
Ser Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser 580 585 590Glu
Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Glu Gly 595 600
605Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser
610 615 620Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu625 630 635 640Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly
Ser Glu Gly Ser Glu 645 650 655Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu Gly Gly Glu Gly Ser 660 665 670Gly Glu Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly Gly Gly 675 680 685Glu Gly Ser Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly 690 695 700Ser Glu Gly
Ser Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly705 710 715
720Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser
725 730 735Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser
Glu Gly 740
745 750Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly
Ser 755 760 765Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly
Glu Gly Glu 770 775 780Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser
Glu Gly Gly Ser Glu785 790 795 800Gly Glu Gly Gly Glu Gly Ser Gly
Glu Gly Glu Gly Gly Gly Glu Gly 805 810 815Ser Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly Ser Gly 820 825 830Glu Gly Ser Glu
Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Gly Ser 835 840 845His His
His His His His 850507832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 507Met Glu Gly Asp Ile
His Met Glu Asp Ile Gln Met Thr Gln Ser Pro1 5 10 15Ser Ser Leu Ser
Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 20 25 30Ala Ser Gln
Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro 35 40 45Gly Lys
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser 50 55 60Gly
Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr65 70 75
80Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys
85 90 95Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys
Val 100 105 110Glu Ile Lys Ser Gly Glu Glu Val Gln Leu Val Glu Ser
Gly Gly Gly 115 120 125Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly 130 135 140Phe Asn Ile Lys Asp Thr Tyr Ile His
Trp Val Arg Gln Ala Pro Gly145 150 155 160Lys Gly Leu Glu Trp Val
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr 165 170 175Arg Tyr Ala Asp
Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr 180 185 190Ser Lys
Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 195 200
205Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala
210 215 220Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly
Gly Glu225 230 235 240Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu 245 250 255Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu Gly Gly Ser Glu Gly 260 265 270Ser Glu Gly Glu Gly Ser Glu
Gly Ser Gly Glu Gly Glu Gly Gly Glu 275 280 285Gly Ser Gly Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 290 295 300Gly Gly Glu
Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Gly Glu Gly305 310 315
320Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly
325 330 335Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser
Glu Gly 340 345 350Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu
Gly Glu Gly Ser 355 360 365Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu
Gly Ser Gly Glu Gly Glu 370 375 380Gly Ser Glu Gly Ser Gly Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu385 390 395 400Gly Glu Gly Ser Glu
Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly 405 410 415Ser Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser 420 425 430Glu
Gly Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly 435 440
445Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly
450 455 460Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Glu Gly
Ser Gly465 470 475 480Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
Glu Gly Ser Gly Glu 485 490 495Gly Ser Glu Gly Glu Gly Ser Glu Gly
Ser Gly Glu Gly Glu Gly Ser 500 505 510Glu Gly Ser Gly Glu Gly Glu
Gly Gly Ser Glu Gly Ser Glu Gly Glu 515 520 525Gly Ser Gly Glu Gly
Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu 530 535 540Gly Glu Gly
Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly545 550 555
560Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Ser
565 570 575Glu Gly Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly Glu Gly
Glu Gly 580 585 590Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Gly Glu
Gly Ser Glu Gly 595 600 605Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu
Gly Ser Glu Gly Ser Gly 610 615 620Glu Gly Glu Gly Gly Ser Glu Gly
Ser Glu Gly Glu Gly Ser Glu Gly625 630 635 640Ser Gly Glu Gly Glu
Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser 645 650 655Gly Glu Gly
Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu 660 665 670Gly
Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu 675 680
685Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly
690 695 700Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly
Ser Glu705 710 715 720Gly Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser
Gly Glu Gly Glu Gly 725 730 735Ser Glu Gly Ser Gly Glu Gly Glu Gly
Ser Gly Glu Gly Ser Glu Gly 740 745 750Glu Gly Gly Ser Glu Gly Gly
Glu Gly Glu Gly Ser Glu Gly Gly Ser 755 760 765Glu Gly Glu Gly Ser
Glu Gly Gly Ser Glu Gly Glu Gly Gly Glu Gly 770 775 780Ser Gly Glu
Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser785 790 795
800Glu Gly Ser Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Asp
805 810 815Tyr Lys Asp Asp Asp Asp Lys Gly Gly Ser His His His His
His His 820 825 830508684DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 508atg gat aaa act cat
act tgc cct cct tgt cca gcg ccc gaa ctg ctg 48Met Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15ggt ggc ccg tct
gtt ttc ctg ttc cca cca aaa cca aaa gac acc ctg 96Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30atg att tcc
cgt act cct gag gta acc tgt gta gtt gta gac gtt tct 144Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45cac gaa
gat ccg gaa gtt aaa ttc aac tgg tac gtg gat ggt gtt gag 192His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60gtg
cat aac gct aaa acc aaa ccg cgc gag gag caa tat aat tcc acc 240Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75
80tac cgt gtt gtg tct gtt ctg acc gtc ctg cac caa gat tgg ctg aac
288Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
85 90 95ggc aaa gaa tac aag tgt aaa gtg tcc aac aaa gcc ctg cca gcg
ccg 336Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 100 105 110atc gag aaa act att tct aag gcg aaa ggc cag ccg cgc
gaa cca caa 384Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 115 120 125gta tat acc ctg ccg ccg tcc cgt gat gaa ctg
acc aag aac caa gtt 432Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 130 135 140tcc ctg acc tgc ctg gtg aag ggt ttc
tac cca tct gat atc gcc gtc 480Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val145 150 155 160gag tgg gaa tcc aac ggt
cag ccg gag aac aat tat aaa act atc cca 528Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Ile Pro 165 170 175ccg gtt ctg gac
tct gac ggc tcc ttc ttt ctg tat tcc aag ctg acc 576Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190gtt gat
aaa agc cgt tgg cag cag ggc aac gtt ttc tct tgc tct gta 624Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200
205atg cat gaa gca ctg cac aac cat tac acc cag aaa agc ctg tcc ctg
672Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220tcg ccg ggt aag 684Ser Pro Gly Lys225509228PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
509Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1
5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser 35 40 45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu 50 55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro 100 105 110Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155
160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Ile Pro
165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val 195 200 205Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 210 215 220Ser Pro Gly
Lys225510806PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 510Met Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu Val1 5 10 15Glu Leu Asp Gly Asp Val Asn
Gly His Lys Phe Ser Val Ser Gly Glu 20 25 30Gly Glu Gly Asp Ala Thr
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45Thr Thr Gly Lys Leu
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe 50 55 60Ser Tyr Gly Val
Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg65 70 75 80His Asp
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95Thr
Ile Ser Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val 100 105
110Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
115 120 125Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
Tyr Asn 130 135 140Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys
Gln Lys Asn Gly145 150 155 160Ile Lys Ala Asn Phe Lys Ile Arg His
Asn Ile Glu Asp Gly Ser Val 165 170 175Gln Leu Ala Asp His Tyr Gln
Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190Val Leu Leu Pro Asp
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser 195 200 205Lys Asp Pro
Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220Thr
Ala Ala Gly Ile Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu225 230
235 240Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly
Glu 245 250 255Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser
Glu Gly Ser 260 265 270Gly Glu Gly Glu Gly Gly Glu Gly Ser Gly Glu
Gly Glu Gly Ser Gly 275 280 285Glu Gly Ser Glu Gly Glu Gly Gly Gly
Glu Gly Ser Glu Gly Glu Gly 290 295 300Ser Gly Glu Gly Gly Glu Gly
Glu Gly Ser Glu Gly Gly Ser Glu Gly305 310 315 320Glu Gly Gly Ser
Glu Gly Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly 325 330 335Glu Gly
Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Glu Gly 340 345
350Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser
355 360 365Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu 370 375 380Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu
Gly Gly Ser Glu385 390 395 400Gly Glu Gly Gly Ser Glu Gly Ser Glu
Gly Glu Gly Ser Gly Glu Gly 405 410 415Ser Glu Gly Glu Gly Gly Ser
Glu Gly Ser Glu Gly Glu Gly Gly Gly 420 425 430Glu Gly Ser Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 435 440 445Gly Ser Glu
Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 450 455 460Glu
Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly465 470
475 480Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu
Gly 485 490 495Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
Glu Gly Gly 500 505 510Ser Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu
Gly Ser Glu Gly Glu 515 520 525Gly Ser Glu Gly Ser Gly Glu Gly Glu
Gly Ser Glu Gly Ser Gly Glu 530 535 540Gly Glu Gly Gly Ser Glu Gly
Ser Glu Gly Glu Gly Gly Ser Glu Gly545 550 555 560Ser Glu Gly Glu
Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Glu 565 570 575Gly Ser
Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly 580 585
590Ser Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
595 600 605Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu
Gly Ser 610 615 620Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu
Gly Gly Glu Gly625 630 635 640Ser Gly Glu Gly Glu Gly Ser Gly Glu
Gly Ser Glu Gly Glu Gly Gly 645 650 655Gly Glu Gly Ser Glu Gly Glu
Gly Ser Glu Gly Ser Gly Glu Gly Glu 660 665 670Gly Ser Glu Gly Ser
Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu 675 680 685Gly Glu Gly
Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly 690 695 700Ser
Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Glu705 710
715 720Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu
Gly 725 730 735Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly
Gly Glu Gly 740 745 750Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly
Ser Glu Gly Gly Ser 755 760 765Glu Gly Glu Gly Gly Glu Gly Ser Gly
Glu Gly Glu Gly Gly Gly Glu 770 775 780Gly Ser Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly Ser785 790 795 800Gly Glu Gly Ser
Glu Gly 805511797PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 511Met Ala Asn Thr Pro Val Ser Gly
Asn Leu Lys Val Glu Phe Tyr Asn1 5 10 15Ser Asn Pro Ser Asp Thr Thr
Asn Ser Ile Asn Pro Gln Phe Lys Val 20 25 30Thr Asn Thr Gly Ser Ser
Ala Ile Asp Leu Ser Lys Leu Thr Leu Arg 35 40 45Tyr Tyr Tyr Thr Val
Asp Gly Gln Lys Asp Gln Thr Phe Trp Ala Asp 50 55 60His Ala Ala Ile
Ile Gly Ser Asn Gly Ser Tyr Asn
Gly Ile Thr Ser65 70 75 80Asn Val Lys Gly Thr Phe Val Lys Met Ser
Ser Ser Thr Asn Asn Ala 85 90 95Asp Thr Tyr Leu Glu Ile Ser Phe Thr
Gly Gly Thr Leu Glu Pro Gly 100 105 110Ala His Val Gln Ile Gln Gly
Arg Phe Ala Lys Asn Asp Trp Ser Asn 115 120 125Tyr Thr Gln Ser Asn
Asp Tyr Ser Phe Lys Ser Ala Ser Gln Phe Val 130 135 140Glu Trp Asp
Gln Val Thr Ala Tyr Leu Asn Gly Val Leu Val Trp Gly145 150 155
160Lys Glu Pro Gly Gly Ser Val Val Gly Ser Gly Ser Gly Ser Glu Asn
165 170 175Leu Tyr Phe Gln His Gly Glu Gly Thr Phe Thr Ser Asp Leu
Ser Lys 180 185 190Gln Met Glu Glu Glu Ala Val Arg Leu Phe Ile Glu
Trp Leu Lys Asn 195 200 205Gly Gly Pro Ser Ser Gly Ala Pro Pro Pro
Ser Gly Gly Glu Gly Ser 210 215 220Gly Glu Gly Ser Glu Gly Glu Gly
Ser Glu Gly Ser Gly Glu Gly Glu225 230 235 240Gly Ser Glu Gly Ser
Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu 245 250 255Gly Glu Gly
Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Glu Gly Ser 260 265 270Gly
Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Gly 275 280
285Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Gly Glu Gly Glu Gly
290 295 300Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly
Glu Gly305 310 315 320Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly
Ser Glu Gly Gly Ser 325 330 335Glu Gly Glu Gly Ser Glu Gly Gly Ser
Glu Gly Glu Gly Ser Glu Gly 340 345 350Ser Gly Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly Ser 355 360 365Glu Gly Ser Gly Glu
Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu 370 375 380Gly Ser Glu
Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu385 390 395
400Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly
405 410 415Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly
Ser Gly 420 425 430Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser
Glu Gly Glu Gly 435 440 445Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly
Glu Gly Ser Gly Glu Gly 450 455 460Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu Gly Ser Gly Glu Gly Ser465 470 475 480Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly 485 490 495Ser Gly Glu
Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser 500 505 510Gly
Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu 515 520
525Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu
530 535 540Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Ser
Glu Gly545 550 555 560Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly Glu
Gly Glu Gly Ser Glu 565 570 575Gly Ser Gly Glu Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly 580 585 590Ser Glu Gly Ser Gly Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu Gly 595 600 605Glu Gly Gly Ser Glu
Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly 610 615 620Glu Gly Glu
Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser Gly Glu625 630 635
640Gly Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser
645 650 655Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu 660 665 670Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser
Glu Gly Ser Glu 675 680 685Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly
Glu Gly Ser Glu Gly Gly 690 695 700Ser Glu Gly Glu Gly Ser Glu Gly
Ser Gly Glu Gly Glu Gly Ser Glu705 710 715 720Gly Ser Gly Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 725 730 735Gly Ser Glu
Gly Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly 740 745 750Glu
Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly 755 760
765Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly
770 775 780Ser Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly785
790 795512144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 512Gly Glu Gly Ser Gly Glu Gly Ser
Glu Gly Glu Gly Gly Ser Glu Gly1 5 10 15Gly Glu Gly Glu Gly Gly Ser
Glu Gly Ser Glu Gly Glu Gly Ser Glu 20 25 30Gly Gly Ser Glu Gly Glu
Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser Glu Gly Gly
Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu Gly Ser Glu
Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser65 70 75 80Glu Gly
Glu Gly Ser Glu Gly Gly Gly Glu Gly Glu Gly Ser Gly Glu 85 90 95Gly
Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser 100 105
110Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu
115 120 125Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly
Ser Glu 130 135 140513144PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 513Gly Glu Gly Gly Ser
Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly1 5 10 15Gly Glu Gly Glu
Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser 20 25 30Glu Gly Ser
Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser
Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu
Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser65 70 75
80Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu
85 90 95Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly
Ser 100 105 110Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly
Glu Gly Glu 115 120 125Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser
Glu Gly Gly Ser Glu 130 135 140514144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
514Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly1
5 10 15Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly
Ser 20 25 30Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly 35 40 45Gly Ser Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu Gly
Ser Glu Gly 50 55 60Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser
Gly Glu Gly Ser65 70 75 80Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu
Gly Glu Gly Gly Ser Glu 85 90 95Gly Ser Glu Gly Glu Gly Ser Glu Gly
Gly Ser Glu Gly Glu Gly Ser 100 105 110Gly Glu Gly Ser Glu Gly Glu
Gly Gly Ser Glu Gly Gly Glu Gly Glu 115 120 125Gly Gly Ser Glu Gly
Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu 130 135
140515144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 515Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly Gly Ser Glu Gly1 5 10 15Gly Glu Gly Glu Gly Gly Ser Glu Gly
Ser Glu Gly Glu Gly Ser Glu 20 25 30Gly Gly Ser Glu Gly Glu Gly Ser
Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser Glu Gly Gly Glu Gly
Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu Gly Ser Glu Gly Gly
Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser65 70 75 80Glu Gly Glu Gly
Ser Glu Gly Gly Gly Glu Gly Glu Gly Ser Gly Glu 85 90 95Gly Ser Glu
Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser 100 105 110Gly
Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu 115 120
125Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu
130 135 140516144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 516Gly Glu Gly Gly Ser Glu Gly Ser
Glu Gly Glu Gly Ser Glu Gly Gly1 5 10 15Gly Glu Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly Gly Ser 20 25 30Glu Gly Ser Glu Gly Glu
Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser Glu Gly Gly
Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu Gly Ser Glu
Gly Gly Ser Glu Gly Gly Glu Gly Ser Gly Glu Gly65 70 75 80Ser Gly
Ser Glu Gly Ser Gly Glu Gly Ser Glu Gly Ser Gly Gly Glu 85 90 95Gly
Gly Ser Glu Gly Gly Glu Gly Gly Gly Ser Glu Gly Glu Gly Ser 100 105
110Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu
115 120 125Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly
Ser Glu 130 135 140517144PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 517Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly1 5 10 15Ser Glu Gly Glu
Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser Gly 20 25 30Glu Gly Ser
Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser
Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu
Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser65 70 75
80Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly
85 90 95Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Gly Glu
Gly 100 105 110Ser Gly Glu Gly Ser Gly Ser Glu Gly Gly Ser Glu Gly
Gly Glu Gly 115 120 125Gly Gly Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Gly Gly Gly Glu 130 135 140518144PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
518Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly1
5 10 15Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly
Ser 20 25 30Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly 35 40 45Gly Ser Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu Gly
Ser Glu Gly 50 55 60Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser
Gly Glu Gly Ser65 70 75 80Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu
Gly Glu Gly Gly Ser Glu 85 90 95Gly Ser Glu Gly Glu Gly Ser Glu Gly
Gly Ser Glu Gly Glu Gly Ser 100 105 110Gly Glu Gly Ser Glu Gly Glu
Gly Gly Ser Glu Gly Gly Glu Gly Glu 115 120 125Gly Gly Ser Glu Gly
Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu 130 135
140519144PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 519Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly Gly Ser Glu Gly1 5 10 15Ser Glu Gly Glu Gly Gly Gly Glu Gly
Ser Glu Gly Glu Gly Ser Gly 20 25 30Glu Gly Ser Glu Gly Glu Gly Ser
Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser Glu Gly Gly Glu Gly
Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu Gly Ser Glu Gly Gly
Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser65 70 75 80Glu Gly Glu Gly
Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly 85 90 95Gly Ser Glu
Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Ser Glu Gly 100 105 110Ser
Gly Glu Gly Ser Gly Ser Glu Gly Ser Gly Glu Gly Gly Glu Gly 115 120
125Gly Gly Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Ser Gly Gly Glu
130 135 140520144PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 520Gly Glu Gly Gly Ser Glu Gly Ser
Glu Gly Glu Gly Ser Glu Gly Gly1 5 10 15Gly Glu Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly Gly Ser 20 25 30Glu Gly Ser Glu Gly Glu
Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser Glu Gly Gly
Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu Gly Ser Glu
Gly Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser65 70 75 80Glu Gly
Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu 85 90 95Gly
Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser 100 105
110Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu
115 120 125Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly
Ser Glu 130 135 140521144PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 521Gly Glu Gly Gly Ser
Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly1 5 10 15Gly Glu Gly Glu
Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser 20 25 30Glu Gly Ser
Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 35 40 45Gly Ser
Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly 50 55 60Glu
Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Ser Gly Glu Gly Ser65 70 75
80Glu Gly Glu Gly Gly Ser Glu Gly Gly Glu Gly Glu Gly Gly Ser Glu
85 90 95Gly Ser Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly
Ser 100 105 110Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Gly
Glu Gly Glu 115 120 125Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser
Glu Gly Gly Ser Glu 130 135 140522826PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
522Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Pro Gly Glu Gly Ser1
5 10 15Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly
Glu 20 25 30Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly
Ser Glu 35 40 45Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly
Glu Gly Ser 50 55 60Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Glu Gly Gly Gly65 70 75 80Glu Gly Ser Glu Gly Glu Gly Ser Gly Glu
Gly Gly Glu Gly Glu Gly 85 90 95Ser Glu Gly Gly Ser Glu Gly Glu Gly
Gly Ser Glu Gly Gly Glu Gly 100 105 110Glu Gly Ser Glu Gly Ser Gly
Glu Gly Glu Gly Ser Glu Gly Gly Ser 115 120 125Glu Gly Glu Gly Ser
Glu Gly Gly Ser Glu Gly Glu Gly Ser Glu Gly 130 135 140Ser Gly Glu
Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Ser145 150 155
160Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu
165 170 175Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly
Ser Glu 180 185
190Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly
195 200 205Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly
Ser Gly 210 215 220Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser
Glu Gly Glu Gly225 230 235 240Gly Ser Glu Gly Ser Glu Gly Glu Gly
Gly Glu Gly Ser Gly Glu Gly 245 250 255Glu Gly Ser Glu Gly Ser Gly
Glu Gly Glu Gly Ser Gly Glu Gly Ser 260 265 270Glu Gly Glu Gly Ser
Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly 275 280 285Ser Gly Glu
Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser 290 295 300Gly
Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu Gly Glu305 310
315 320Gly Ser Glu Gly Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser
Glu 325 330 335Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly
Ser Glu Gly 340 345 350Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly Glu
Gly Glu Gly Ser Glu 355 360 365Gly Ser Gly Glu Gly Glu Gly Ser Gly
Glu Gly Ser Glu Gly Glu Gly 370 375 380Ser Glu Gly Ser Gly Glu Gly
Glu Gly Ser Glu Gly Ser Gly Glu Gly385 390 395 400Glu Gly Gly Ser
Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly Ser Gly 405 410 415Glu Gly
Glu Gly Gly Glu Gly Ser Gly Glu Gly Glu Gly Ser Gly Glu 420 425
430Gly Ser Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser
435 440 445Glu Gly Ser Gly Glu Gly Glu Gly Ser Glu Gly Ser Gly Glu
Gly Glu 450 455 460Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Ser
Glu Gly Ser Glu465 470 475 480Gly Glu Gly Ser Glu Gly Gly Ser Glu
Gly Glu Gly Ser Glu Gly Gly 485 490 495Ser Glu Gly Glu Gly Ser Glu
Gly Ser Gly Glu Gly Glu Gly Ser Glu 500 505 510Gly Ser Gly Glu Gly
Glu Gly Ser Gly Glu Gly Ser Glu Gly Glu Gly 515 520 525Gly Ser Glu
Gly Gly Glu Gly Glu Gly Ser Glu Gly Gly Ser Glu Gly 530 535 540Glu
Gly Ser Glu Gly Gly Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly545 550
555 560Glu Gly Glu Gly Gly Gly Glu Gly Ser Glu Gly Glu Gly Ser Glu
Gly 565 570 575Ser Gly Glu Gly Glu Gly Ser Gly Glu Gly Ser Glu Gly
Ser Lys Gly 580 585 590Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
Val Glu Leu Asp Gly 595 600 605Asp Val Asn Gly His Lys Phe Ser Val
Ser Gly Glu Gly Glu Gly Asp 610 615 620Ala Thr Tyr Gly Lys Leu Thr
Leu Lys Phe Ile Cys Thr Thr Gly Lys625 630 635 640Leu Pro Val Pro
Trp Pro Thr Leu Val Thr Thr Phe Ser Tyr Gly Val 645 650 655Gln Cys
Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe Phe 660 665
670Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser Phe
675 680 685Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
Glu Gly 690 695 700Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
Asp Phe Lys Glu705 710 715 720Asp Gly Asn Ile Leu Gly His Lys Leu
Glu Tyr Asn Tyr Asn Ser His 725 730 735Asn Val Tyr Ile Thr Ala Asp
Lys Gln Lys Asn Gly Ile Lys Ala Asn 740 745 750Phe Lys Ile Arg His
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp 755 760 765His Tyr Gln
Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro 770 775 780Asp
Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn785 790
795 800Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala
Gly 805 810 815Ile Thr His Gly Met Asp Glu Leu Tyr Lys 820
8255236PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 523Ser Lys Val Ile Leu Phe1 55247PRTHomo sapiens
524Glu Asn Leu Tyr Phe Gln Gly1 5
* * * * *
References