U.S. patent application number 12/499710 was filed with the patent office on 2010-02-18 for method of improving efficacy of biological response-modifying proteins and the exemplary muteins.
This patent application is currently assigned to MEDEXGEN, CO., LTD. Invention is credited to Yong-Hoon Chung, Youn-Hwa Heo, Jae-Youn Kim, Hak-sup Lee, Ki-Wan Yi.
Application Number | 20100041586 12/499710 |
Document ID | / |
Family ID | 36284112 |
Filed Date | 2010-02-18 |
United States Patent
Application |
20100041586 |
Kind Code |
A1 |
Chung; Yong-Hoon ; et
al. |
February 18, 2010 |
METHOD OF IMPROVING EFFICACY OF BIOLOGICAL RESPONSE-MODIFYING
PROTEINS AND THE EXEMPLARY MUTEINS
Abstract
Disclosed is a protein variant which substitutes valine for
phenylalanine residue in a binding domain having a biological
response-modifying function by binding to a receptor, ligand or
substrate. Also, the present invention discloses a DNA encoding the
protein variant, a recombinant expression vector to which the DNA
is operably linked, a host cell transformed or transfected with the
recombinant expression vector, and a method of preparing the
protein variant comprising cultivating the host cell and isolating
the protein variant from the resulting culture. Further, the
present invention discloses a pharmaceutical composition comprising
the protein variant and a pharmaceutically acceptable carrier.
Inventors: |
Chung; Yong-Hoon; (Seoul,
KR) ; Lee; Hak-sup; (Gangwon-do, KR) ; Yi;
Ki-Wan; (Gyeonggi-do, KR) ; Heo; Youn-Hwa;
(Seoul, KR) ; Kim; Jae-Youn; (Seoul, KR) |
Correspondence
Address: |
SHERIDAN ROSS PC
1560 BROADWAY, SUITE 1200
DENVER
CO
80202
US
|
Assignee: |
MEDEXGEN, CO., LTD
Seoul
KR
|
Family ID: |
36284112 |
Appl. No.: |
12/499710 |
Filed: |
July 8, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10519390 |
Jul 29, 2005 |
7569361 |
|
|
PCT/KP2004/001246 |
May 27, 2004 |
|
|
|
12499710 |
|
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 25/14 20180101;
A61P 7/00 20180101; C07K 14/715 20130101; C07K 14/52 20130101; C07K
14/524 20130101; C07K 14/535 20130101; A61K 38/00 20130101; A61P
7/06 20180101 |
Class at
Publication: |
514/8 |
International
Class: |
A61K 38/16 20060101
A61K038/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2003 |
KR |
10-2003-0051846 |
Claims
1. A method of treating a patient having an anemia associated
disease comprising administering to the patient a therapeutic
effective amount of a pharmaceutical composition comprising a
pharmaceutical acceptable carrier and a thrombopoietin (TPO)
variant, wherein the variant comprises an amino acid substitution
of a valine residue for a phenylalanine residue at position 141 of
SEQ ID NO:25 thereby treating the anemia associated disease in the
patient.
2. The method of claim 1, wherein the anemia associated disease is
selected from the group consisting of anemia, anemia in
inflammatory bowel disease, anemia in progressive kidney disease,
anemia of renal failure, anemia associated with HIV infection in
zidovudine treated patients, anemia associated with cancer
chemotherapy, anemia associated with Huntington's disease, anemia
associated with sickle cell anemia, and anemia associated with Late
Hyporegenerative anemia in neonates with Rh hemolytic disease after
in-utero exchange transfusion.
3. The method of claim 1, wherein the therapeutic effective amount
of the variant is administered in a smaller amount than wild-type
TPO.
4. The method of claim 3, wherein the therapeutic effective amount
of the variant is administered in an amount of 0.01-1000
.mu.g/kg/day.
5. The method of claim 3, wherein the therapeutic effective amount
of the variant is administered in amount of 0.1-5000
.mu.g/kg/day.
6. The method of claim 3, wherein the therapeutic effective amount
of the variant is administered in an amount of 1-100
.mu.g/kg/day.
7. The method of claim 1, wherein administering the therapeutic
effective amount is selected from the group consisting of
topically, orally, parenterally, intraocularly, transdermally,
intrarectally and intraluminally.
8. The method of claim 1, wherein the pharmaceutical composition is
in a form selected from the group consisting of solutions,
suspensions, tablets, pills, capsules and sustained released
preparations.
9. The method of claim 1, wherein the pharmaceutical acceptable
carrier is selected from the group consisting of ion exchange,
alumina, aluminum stearate, lecithin, serum proteins, buffering
agents, water, salts, electrolytes, colloidal silica, magnesium,
trisilicate, polyvinylpyrrolidone, cellulose-based substrates,
polyethylene glycol, sodium carboxymethylcellulose, polyarylate,
waxes, polyethylene-polyoxypropylene-block copolymers, polyethelene
glycol and wool fat.
Description
TECHNICAL FIELD
[0001] The present invention relates to a protein variant which
substitutes valine for phenylalanine residue in a binding domain
having a biological response-modifying function by binding to a
receptor, ligand or substrate. More particularly, the present
invention relates to a protein variant which substitutes valine for
phenylalanine residue in an .alpha.-helix domain participating in
the binding of a human cytokine protein to a corresponding
receptor.
BACKGROUND ART
[0002] Many human diseases are caused by the loss of protein
function due to defects or an insufficient amount of a protein To
treat such diseases, related proteins have been directly
administered to patients. However, many physiologically active
proteins used as medicines are easily degraded in serum before they
arrive at target tissues and act therein. For this reason, most
physiologically active proteins having therapeutic value are
excessively or frequently administered to patients to maintain an
appropriate concentration capable of offering satisfactory
therapeutic effects.
[0003] An approach to solve they e problems is to conjugate with
polyethylene glycol (PEGylation) or microencapsulate
physiologically active proteins. However, these methods are
cumbersome because target proteins are primarily produced in
microorganisms and purified, and are then PEGlyated or
microencapsulated. In addition, cross-linking may occur at
undesired positions, which may negatively affect the homogeneity of
final products.
[0004] Another approach involves glycosylation. Cell surface
proteins and secretory proteins produced by eukaryotic cells are
modified by a glycosylation process. Glycosylation is known to
influence in vivo stability and function of proteins, as well as
their physiological properties. However, since glycosylated
proteins can be produced only by eukaryotic cells capable of
performing glycosylation, their production process is complicated,
and it is difficult to attain homogeneous final products which are
glycosylated at all desired positions.
[0005] In addition, the conventional techniques all improve the
problems associated with administration frequency, but do not
increase the physiological efficacy of proteins, leading to
excessive dosage. For example, NESP developed by the Amgen Company
(see U.S. Pat. No. 6,586,398) improves the frequent administration
by extending the half-lives of proteins in the blood, but does not
increase the efficacy of proteins, leading to excessive dosage that
may induce the production of blocking antibodies.
[0006] An approach used to improve the efficacy of physiologically
active proteins is to mutagenize some amino acid residues of a
wild-type protein to improve biological activity of the protein.
Related protein variants are disclosed in the following patent
publications: (1) U.S. Pat. No. 5,457,089: human erythropoietin
(EPO) variants where the carboxyl terminal region was altered to
increase binding affinity of EPO to its receptor, (2) International
Pat. Publication No. 02/077034: human granulocyte colony
stimulating factor (G-CSF) variants where a T-cell epitope was
altered to reduce immunogenicity of human G-CSF in humans; (3)
International Pat Publication No. 99/57147: human thrombopoietin
(TPO) variants prepared by substring glutaminic acid at the 115
position with lysine, arginine or tyrosine in a TOP protein having
an amino acid sequence corresponding to 7th to 151st amino acid
residues of human mature TPO; and (4) U.S. Pat. Nos. 6,136,563 and
6,022,711 that disclose human growth hormone variants having
alanine substitutions at the 18, 22, 25, 26, 29, 65, 168 and 174
positions.
[0007] However, the aforementioned protein variants are altered
forms made for improving only therapeutic efficacy regardless of
changes in in vivo antigenicity. Thus, the scale, degree and
position of these alterations have high potential to induce immune
responses in humans. Antigenicity in humans may cause serious
adverse effects (Casadevall et al. N. Eng. J. Med. 2002, vol. 346,
p. 469).
DISCLOSURE OF THE INVENTION
[0008] It is therefore an object of the present invention to
provide biological response-modifying protein variants having
improved pharmacological action, which are capable of maximizing
biological response modifying effects upon administration and
preventing the formation of blocking antibodies through an
improvement in efficacy of conventional biological
response-modifying proteins, and methods of preparing such
variants.
[0009] In one aspect, the present invention provides a protein
variant which substitutes valine for phenylalanine residue in a
binding domain of a protein having a biological response-modifying
function by binding to a receptor, ligand or substrate.
[0010] In another aspect, the present invention provides a DNA
encoding a protein variant which substitutes valine for
phenylalanine residue in a binding domain of a protein having a
biological response-modifying function by binding to a receptor,
ligand or substrate.
[0011] In a further aspect, the present invention provides a
recombinant expression vector to which a DNA encoding a protein
variant which substitutes valine for phenylalanine residue in a
binding domain of a protein having a biological response-modifying
function by binding to a receptor, ligand or substrate is operably
linked.
[0012] In yet another aspect, the present invention provides a host
cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a protein variant which substitutes
valine for phenylalanine residue in a binding domain of a protein
having a biological response-modifying function by binding to a
receptor, ligand or substrate is operably linked.
[0013] In still another aspect, the present invention provides a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a protein variant which substitutes
valine for phenylalanine residue in a binding domain of a protein
having a biological response-modifying function by binding to a
receptor, ligand or substrate is operably linked, and isolating the
protein variant from a resulting culture.
[0014] In still another aspect, the present invention provides a
pharmaceutical composition comprising a protein variant which
substitutes valine for phenylalanine residue in a binding domain of
a protein having a biological response-modifying function by
binding to a receptor, ligand or substrate, and a pharmaceutically
acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The above and other objects, features and other advantages
of the present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0016] FIG. 1A is a multiple alignment of amino acid sequences of
domains participating in the binding of 4-helix bundle cytokines to
corresponding receptors;
[0017] FIG. 1B is a multiple alignment of amino acid sequences of
domains participating in the binding of interferons to
corresponding receptors;
[0018] FIG. 2A shows the results of Western blotting of TPO
variants according to the present invention, (from the leftmost
lane: marker, wild-type TPO; TPO-[F46V]; TPO-[F128V]; TPO[F131V];
and TPO-[F141V]);
[0019] FIG. 2B shows the results of Western blotting of EPO
variants according to the present invention, (from the leftmost
lane: marker, wild-type EPO; EPO-[F48V]; EPO-[F138V]; EPO-[F142V];
and EPO-[F148V]);
[0020] FIG. 2C shows the results of Western blotting of G-CSF
variants according to the present invention, (from the leftmost
lane: marker, wild-type G-CSF; G-CSF-[F13V]; G-CSF-[F83V];
G-CSF-[F113V]; G-CSF-[F140V]; G-CSF-[F144V]; and
G-CSF-[F160V]);
[0021] FIG. 3A is a graph showing the relative expression levels of
TPO variants according to the present invention, compared to a
wild-type TPO;
[0022] FIG. 3B is a graph showing the relative expression levels of
EPO variants according to the present invention, compared to a
wild-type EPO;
[0023] FIG. 3C is a graph showing the relative expression levels of
G-CSF variants according to the present invention, compared to a
wild-type G-CSF;
[0024] FIG. 4A shows the results of an ELISA assay for binding
affinity of TPO variants according to the present invention to TPO
receptors;
[0025] FIG. 4B shows the results of an ELISA assay for binding
affinity of EPO variants according to the present invention to EPO
receptors;
[0026] FIG. 4C shows the results of an ELISA assay for binding
affinity of G-CSF variants according to the present invention to
G-CSF receptors;
[0027] FIG. 4D shows the results of an ELISA assay for binding
affinity of GH variants according to the present invention to GH
receptors;
[0028] FIG. 5A shows the results of an SPR assay for binding
affinity of TPO variants according to the present invention to TPO
receptors;
[0029] FIG. 5B shows the results of an SPR assay for binding
affinity of EPO variants according to the present invention to EPO
receptors;
[0030] FIG. 6A shows the results of a FACS analysis for binding
affinity of a TPO variant according to the present invention to TPO
receptors;
[0031] FIG. 6B shows the results of a FACS analysis for binding
affinity of an EPO variant according to the present invention to
EPO receptors;
[0032] FIG. 7A is a graph showing the proliferation rates of
TF-1/c-Mp1 cells according to the concentration of TPO variants
according to the present invention;
[0033] FIG. 7B is a graph showing the proliferation rates of TF-1
cells according to the concentration of EPO variants according to
the present invention;
[0034] FIG. 7C is a graph showing the proliferation rates of HL60
cells according to the concentration of G-CSF variants according to
the present invention;
[0035] FIG. 7D is a graph showing the proliferation rates of Nb2
cells according to the concentration of GH variants according to
the present invention;
[0036] FIG. 8A is a graph showing the results of a pharmacokinetic
assay of a TPO variant according to the present invention, in which
the TPO variant was intravenously injected into rabbits, and serum
levels of the TPO variant were measured;
[0037] FIG. 8B is a graph showing the results of a pharmacokinetic
assay of an EPO variant according to the present invention, in
which the EPO variant was intravenously injected into rabbits, and
serum levels of the EPO variant were measured;
[0038] FIG. 8C is a graph showing the results of a pharmacokinetic
assay of an EPO variant according to the present invention, in
which the EPO variant was intraperitoneally injected into mice, and
serum levels of the EPO variant were measured;
[0039] FIGS. 9A, 9B and 9C are graphs showing the proliferation
rates of erythrocytes, proliferation rates of reticulocytes, and
changes in hematocrit, respectively, as results of tests to
evaluate in vivo activity of EPO variants according to the present
invention, in mice intraperitoneally injected with the EPO
variants; and
[0040] FIGS. 10A, 10B and 10C are graphs showing proliferation
rates of platelets, leukocytes and neutrophils, respectively, as
results of tests to evaluate the in vivo activity of TPO variants
according to the present invention, in rats intraperitoneally
injected with the TPO variants.
BEST MODE FOR CARRYING OUT THE INVENTION
[0041] Single capital letters standing for amino acids, as used
herein, represent the following amino acids according to the
standard abbreviations defined by the International Union of
Biochemistry:
[0042] A: Alanine; B: Asparagine or Aspartic acid;
[0043] C: Cysteine; D: Aspartic acid; E: Glutamic acid;
[0044] F: Phenylalanine; G: Glycine; H: Histidine;
[0045] I: Isoleucine; K: Lysine; L: Leucine;
[0046] M: Methionine; N: Asparagine; P: Proline;
[0047] Q: Glutamine; R: Arginine; S: Serine;
[0048] T: Threonine; V: Valine; W: Tryptophan;
[0049] Y: Tyrosine; and Z: Glutamine or Glutamic acid.
[0050] The designation "(one capital for an amino acid)(amino acid
position)(one capital for another amino acid)", as used herein,
means that the former amino acid is substituted by the latter amino
acid at the designated amino acid position of a certain protein.
For example, F48V indicates that the phenylalanine residue at the
48th position of a certain protein is substituted by valine. The
amino acid position is numbered from the N terminus of a mature
wild-type protein
[0051] The term "protein variant", as used herein, refers to a
protein that has an amino acid sequence different from a wild-type
form by a substitution of valine for phenylalanine residue in a
protein having physiological function by binding to a receptor,
ligand or substrate, in particular, in a domain participating in
the binding to a receptor, ligand or substrate. In the present
invention, a protein variant is designated for convenience as
"protein name-[(one capital for an amino acid)(amino acid
position)(one capital for another amino acid)]". For example,
TPO-[F131V] indicates a TPO variant in which the phenylalanine
residue at position 131 of wild-type TPO is substituted by
valine.
[0052] The term "biological response-modifying proteins", as used
herein, refers to proteins involved in maintaining homeostasis in
the body by inducing the initiation or stop of various biological
responses occurring in the multicellular body and regulating the
responses to be organically connected to each other. These proteins
typically act by binding to receptors, ligands or substrates.
[0053] Proteins capable of being altered according to the present
invention include all proteins that have innate function to
modulate biological responses by binding receptors, ligands or
substrates. Non-limiting examples of the proteins include
cytokines, cytokine receptors, adhesion molecules, tumor necrosis
factor (TNF) receptors, enzymes, receptor tyrosine kinases,
chemokine receptors, other cell surface proteins, and soluble
ligands. Non-limiting examples of the cytokines include CNTF
(cytoneurotrophic factor), GH (growth hormone), IL,1, IL-1Ra
(interleukin-1 receptor antagonist), placental lactogen (PL),
cardioliphin, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12,
IL-17, TNF, TGF (transforming growth factor), IFN (interferon),
GM-CSF (granulocyte-monocyte colony stimulating factor), G-CSF
(granulocyte colony stimulating factor), EPO (erythropoietin), TPO
(thrombopoietin), M-CSF (monocyte colony stimulating factor), LIF
(leukemia inhibitory factor), OSM (oncostatin-M), SCF (stem cell
factor), HGF (hepatocyte growth factor), FGF (fibroblast growth
factor), IGF (insulin-like growth factor), and LPT (Leptin).
Non-limiting examples of the cytokine receptors include growth
hormone receptor (GHR), IL-13R, IL-1R, IL-1R, IL-2R, IL-3R, IL-4R,
IL-5R, IL-6R, IL-7R, IL-9R, IL-15R, TNFR, TGFR, IFNR (e.g.,
IFN-.gamma.R .alpha.-chain, IFN-.gamma.R .beta.-chain),
interferon-.alpha.R, -.beta.R and -.gamma.R, GM-CSFR, G-CSFR, EPOR,
cMp1, gp130, and Fas (Apo 1). Examples of the chemokine receptors
include CCR1 and CXCR1-4. Examples of the receptor tyrosine kinases
include TrkA, TrkB, TrkC, Hrk, REK7, Rse/Tyro-3, hepatocyte growth
factor R, platelet-derived growth factor R, and Flt-1. Examples of
other cell surface proteins include CD2, CD4, CD5, CD6, CD22, CD27,
CD28, CD30, CD31, CD40, CD44, CD100, CD137, CD150, LAG-3, B7, B61,
.beta.-neurexin, CTLA-4, ICOS, ICAM-1, complement R-2(CD21), IgER,
lysosomal membrane gp-1, .alpha.2-microglobulin receptor-related
protein, and natriuretic peptide receptor.
[0054] To improve the efficacy of modulating biological responses
for the aforementioned numerous proteins having biological
response-modulating function, the present invention intends to
provide protein variants capable of binding to receptors, ligands
or substrates having a higher hydrophobic force than that of wild
types. For this purpose, the present invention is characterized by
substituting valine for phenylalanine residue in a binding domain
of each of the proteins.
[0055] Phenylalanine is a relatively non-polar amino acid that has
an aromatic side chain and a known hydrophobicity index of 3.0.
Valine is a non-polar hydrophobic amino acid that has an aliphatic
side chain and a known hydrophobicity index of 4.0. In addition,
since valine is smaller than phenylalanine, a protein substituting
valine for phenylalanine residue becomes more deeply depressed in a
pocket binding to a corresponding receptor, ligand or substrate.
Thus, a protein substituting valine for phenylalanine residue in a
binding domain has increased hydrophobic force and a more deeply
depressed space so that it has increased binding affinity to a
receptor, ligand or substrate, leading to a desired increase in
biological response-modulating efficiency.
[0056] In addition, the valine substitution for phenylalanine
residue, as a conservative substitution, has a minimal influence on
the secondary or tertiary structure of a protein, and thus rarely
affects the function of the protein (Argos, EMBO J. 1989, vol. 8,
pp 779-85). Further, because phenylalanine is mainly present in a
highly hydrophobic region, it is rarely exposed to the exterior.
When such phenylalanine residue is substituted by valine, a protein
becomes more deeply depressed from the surface due to the higher
hydrophobicity of valine. Thus, this substitution has a lower
potential to induce antibody production. A certain protein should
primarily bind a corresponding receptor, ligand or substrate to
modulate a specific biological response. In the case that the
stronger this binding is, the efficacy of modulating a biological
response is improved, related proteins all may be altered according
to the present invention, and the present invention includes all of
the resulting protein variants.
[0057] The fact that such a substitution of valine for
phenylalanine residue leads to increased binding affinity is
supported by the finding of a mutation of Fc.gamma.RIIIa(CD16)
expressed on NK cells in human autoimmune diseases. The human
receptor protein has a genetic polymorphism That is, individuals
are divided into two groups: at position 176 in a region
participating in recognizing Fc of an antibody ligand, one group
has phenylalanine, and the other group has valine. Individuals
having phenylalanine at position 176 of the receptor have weakened
binding affinity to the Fc region of the antibody ligand and are
highly susceptible to systemic lupus erythematosus (SLE) (Jianming
Wu et al. J. Clin. Invest. 1997, vol. 100, pp. 1059-70).
[0058] On the other hand, as noted above, the present invention is
characterized by substituting valine for phenylalanine residue in a
binding domain of a biological response-modulating protein. The
term "binding domain", as used herein, refers to a portion (that
is, domain) of a protein performing its biological function by
binding to a receptor, ligand or substrate, and has relatively high
hydrophobicity and low antigenicity compared to other regions of
the protein. Binding domains of proteins are well known in the art.
For example, some 4-.alpha. helix bundle cytokines and interferons,
which are used in an embodiment of the present invention, are known
to have a D-.alpha. helix structure and an A-.alpha. helix
structure, respectively, that serve as binding domains for
corresponding receptors.
[0059] However, a binding domain altered according to the present
invention is not limited to binding domains known in the art. This
is because the binding of a biological response-modulating protein
to a receptor, ligand or substrate is influenced by, in addition to
amino acid residues involved in direct binding, other several amino
acid residues. A "binding domain" of a biological
response-modulating protein, altered according to the present
invention, further includes about 50 amino acid residues,
preferably about 25 amino acid residues, and more preferably about
10 amino acid residues, from both ends of a binding domain known in
the art.
[0060] One aspect of the present invention involves cytokines that
typically contain several .alpha. helix structures. Among them, the
first and last helices from the N-termius are known as binding
domains participating in binding of cytokines to corresponding
cytokine receptors (see FIG. 1). .alpha. helices responsible for
binding of cytokines to corresponding receptors differ according to
the type of cytokines, and are well known in the art. For example,
in IL-2, the second and fifth helices bind to the p55.alpha.
receptor among IL-2 receptors, the first helix binds to the
p75.gamma. receptor among IL-2 receptors, and the sixth helix binds
to gamma receptor (Fernando Bazan, Science J. 1992, vol. 257, pp.
410-2). As described above, cytokines each have particular helices
participating in binding, but the helices have highly conserved
amino acid sequences. The present invention provides a cytokine
variant that is capable of binding to a cytokine receptor with
higher affinity than a wild-type cytokine by substituting valine
for phenylalanine residue in an alpha helix corresponding to a
binding domain of a cytokine.
[0061] One aspect related to the cytokines involves the 4-helix
bundle family of cytokines. Such cytokines include CNTF, EPO,
Flt3L, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF,
M-CSF, OSM, PL, SCF, TPO G-CSF, GHR and IFN. These cytokines all
have four alpha helices, which are designated as A-alpha helix,
B-alpha helix, C-alpha helix and D-alpha helix, respectively. The
D- and A-alpha helices mainly participate in binding to receptors
(Fernando Bazan, Immunology today, 1990, vol. 1 pp. 350-4, The
Cytokine Facts Book, 1994, pp. 104-247).
[0062] Among the aforementioned 4helix bundle cytokines, CNTF, EPO,
Flt3L, GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF,
M-CSF, OSM, PL, SCF, TPO, G-CSF and GHR have binding domains which
each include a D-alpha helix and a region linking a C-alpha helix
and the D-alpha helix. More particularly, the binding domains
include amino acid residues between positions 110 and 180 among
amino acid residues of the 4helix bundle cytokines. Therefore, in
an aspect, the present invention provides a 4-helix bundle cytokine
variant that is capable of binding to a corresponding receptor with
higher affinity than a wild type by substituting valine for
phenylalanine among amino acid residues between positions 110 and
180 of a 4-helix bundle cytokine.
[0063] Of the aforementioned 4-helix bundle cytokines, interferons
(e.g., IFN-.alpha.2A, IFN-.alpha.2B, IFN-.beta., IFN-.gamma.,
IFN-.omega., IFN-.tau.) have a binding domain that contains an
"A-alpha helix". More particularly, the binding domain of
interferons includes amino acid residues between positions 1 and
50. Therefore, in another aspect, the present invention provides an
interferon variant that is capable of binding to an interferon
receptor having higher affinity than a wild type by substituting
valine for phenylalanine among amino acid residues between
positions 1 and 50 of an interferon.
[0064] On the other hand, the binding domain altered according to
the present invention may include two or more phenylalanine
residues. The two or more phenylalanine residues may all be
substituted by valine. However, because this case leads to a great
reduction in protein expression levels, preferably only one
phenylalanine residue is substituted by valine. In this regard, the
present inventors found that, when phenylalanine residue present in
a highly hydrophobic region is substituted by valine, the
biological response-modulating protein has much improved efficacy.
Therefore, in the present invention, the phenylalanine residue to
be substituted by valine is preferably selected in a highly
hydrophobic region present in the binding domain specified
according to the present invention. Hydrophobicity for a specific
region of an amino acid sequence comprising a protein may be
determined by a method known in the art (Kyte, J. et al. J. Mol.
Biol. 1982, vol. 157, pp. 105-132, Hopp, T. P. et al. Proc. Nat
Acad. Sci. USA, 1981, vol. 78(6), pp. 3824-3828).
[0065] The variant of a biological response-modulating protein
according to the present invention may be prepared by chemical
synthetic methods generally known in the art (Creighton, Proteins:
Structures and Molecular Principles, W.H. Freeman and Co., NY
1983). Representative methods, but are not limited to, include
liquid or solid phase synthesis, fragment condensation, and F-MOC
or T-BOC chemical synthesis (Chemical Approaches to the Synthesis
of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca
Raton Fla., 1997; A Practical Approach, Atherton & Sheppard,
Eds., IRL Press, Oxford, England, 1989).
[0066] Alternatively, the protein variant according to the present
invention may be prepared by recombinant DNA techniques. These
techniques include a process of preparing a DNA sequence encoding
the protein variant according to the present invention. Such a DNA
sequence may be prepared by altering a DNA sequence encoding a
wild-type protein. In brief after a DNA sequence encoding a
wild-type protein is synthesized, a codon for phenylalanine is
changed to another codon for valine by site-directed mutagenesis,
thus generating a desired DNA sequence.
[0067] Also, the preparation of a DNA sequence encoding the protein
variant according to the present invention may be achieved by a
chemical method. For example, a DNA sequence encoding the protein
variant may be synthesized by a chemical method using an
oligonucleotide synthesizer. An oligonucleotide is made based on an
amino acid sequence of a desired protein variant, and preferably by
selecting a appropriate codon used by a host cell producing a
protein variant. The degeneracy in the genetic code, which means
that one amino acid is specified by more than one codon, is well
known in the art Thus, there is a plurality of DNA sequences with
degeneracy encoding a specific protein variant, and they all fall
into the scope of the present invention.
[0068] A DNA sequence encoding the protein variant according to the
present invention may or may not include a DNA sequence encoding a
signal sequence. The signal sequence, if present, should be
recognized by a host cell selected for the expression of the
protein variant. The signal sequence may have a prokaryotic or
eukaryotic origin or a combinational origin, and may be a signal
sequence of a native protein. The employment of a signal sequence
may be determined according to the effect of expression of a
protein variant as a secretory form in a recombinant cell producing
the protein variant. If a selected cell is a prokaryotic cell, a
DNA sequence typically does not encode a signal sequence but
instead contains preferably an N-terminal methionine for direct
expression of a desired protein, and most preferably, a signal
sequence derived from a wild type protein is used.
[0069] Such a DNA sequence as prepared above is operably linked to
another DNA sequence encoding the protein variant of the present
invention, and is inserted into a vector including one or more
expression control sequences regulating the expression of the
resulting DNA sequence. Then, a host is transformed or transfected
with the resulting recombinant expression vector. The resulting
tansformant or transfectant is cultured in a suitable medium under
suitable conditions for the expression of the DNA sequence. A
substantially pure variant of a biological response-modulating
protein coded by the DNA sequence is recovered from the resulting
culture.
[0070] The term "vector", as used herein, means a DNA molecule
serving as a vehicle capable of stably carrying exogeneous genes
into host cells. For useful application, a vector should be
replicable, have a system for introducing itself into a host cell,
and possess selectable markers. In addition, the term "recombinant
expression vector", as used herein, refers to a circular DNA
molecule carrying exogeneous genes operably linked thereto to be
expressed in a host cell. When introduced into a host cell, the
recombinant expression plasmid has the ability to replicate
regardless of host chromosomal DNA at a high copy number and to
produce heterogeneous DNA As generally known in the art in order to
increase the expression level of a transfected gene in a host cell,
the gene should be operably linked to transcription and translation
regulatory sequences functional in a host cell selected as an
expression system. Preferably, the expression regulation sequences
and the exogeneous genes may be carried in a single expression
vector containing bacteria-selectable markers and a replication
origin. In the case that eukaryotic cells are used as an expression
system, the expression vector should further comprise expression
markers useful in the eukaryotic host cells.
[0071] The term "expression control sequences", as used herein in
connection with a recombinant expression vector, refers to
nucleotide sequences necessary or advantageous for expression of
the protein variant according to the present invention. Each
control sequence may be native or foreign to the nucleotide
sequence encoding the protein variant Non-limiting examples of the
expression control sequences include leader sequences,
polyadenylation sequences, propeptide sequences, promoters,
enhancers or upstream activating sequences, signal peptide
sequences, and transcription terminators. The expression control
sequence contains at least one promoter sequence.
[0072] The term "operably linked" refers to a state in which a
nucleotide sequence is arranged with another nucleotide sequence in
a functional relationship. The nucleotide sequences maybe a gene
and control sequences, which are linked in such a manner that gene
expression is induced when a suitable molecule (for example,
transcription-activating protein) binds to the control sequence(s).
For example, when a pre-sequence or secretory leader facilitates
secretion of a mature protein, it is referred to as "operably
linked to the protein". A promoter is operably linked with a coding
sequence when it regulates transcription of the coding sequence. A
ribosome-binding site is operably linked to a coding sequence when
it is present at a position allowing translation of the coding
sequence. Typically, the term "operably linked" means that linked
nucleotide sequences are in contact with each other. In the case of
a secretory leader sequence, the term means that it contacts a
coding sequence and is present within a leading frame of the coding
sequence. However, an enhancer need not necessarily contact a
coding sequence. Linkage of the nucleotide sequences may be
achieved by ligation at convenient restriction enzyme recognition
sites. In the absence of restriction enzyme recognition sites,
oligonucleotide adaptors or linkers may be used, which are
synthesized by the conventional methods.
[0073] In order to express a DNA sequence encoding the protein
variant according to the present invention, a wide variety of
combinations of host cells and vectors as an expression system may
be used. Expression vectors useful for transforming eukaryotic host
cells contain expression regulation sequences from, for example,
SV40, bovine papillomavirus, adenovirus, adeno-associated viruses,
cytomegalovirus and retroviruses. Expression vectors useful in
bacterial host cells include bacterial plasmids from E. coli, which
are exemplified by pET, pRSET, pBluescript, pGEX2T, pUC, pBR322,
pMB9 and derivatives thereof, plasmids having a broad range of host
cells, such as RP4, phage DNAs, exemplified by a wide variety of
.lamda. phage derivatives including .lamda. gt10, .lamda. gt11 and
NM989, and other DNA phages, exemplified by filamentous
single-stranded DNA phages such as M13. Expression vectors useful
in yeast cells include 2.mu. plasmid and derivatives thereof
Expression vectors useful in insect cells include pVL 941.
[0074] To express a DNA sequence encoding the protein variant
according to the present invention, any of a wide variety of
expression control sequences may be used by these vectors. Such
useful expression control sequences include those associated with
structral genes of the aforementioned expression vectors. Examples
of useful expression control sequences include the early and later
promoters of SV40 or adenoviruses, the lac system, the trp system,
the TAC or TRC system, T3 and T7 promoters, the major operator and
promoter regions of phage .lamda., the control regions for fd coat
protein, the promoter for 3-phosphoglycerate kinase or other
glycolytic enzymes, the promoters of phosphatases, for example,
Pho5, the promoters of the yeast alpha-mating system and other
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells or their viruses, and various combinations
thereof In particular, T7 RNA polymerase promoter .PHI.10 is useful
for expressing a polypeptide in E. coli.
[0075] Host cells transformed or transfected with the
aforementioned recombinant expression vector comprise another
aspect of the present invention. A wide range of mononuclear host
cells may be used for expressing a DNA sequence encoding the
protein variant of the present invention Examples of the host cells
include prokaryotic and eukaryotic cells such as E. coli,
Pseudomonas sp., Bacillus sp., Streptomyces sp., fungi or yeasts,
insect cells such as Spodoptera frugiperda (Sf9), animal cells such
as Chinese hamster ovary cells (CHO) or mouse cells, African green
monkey cells such as COS 1, COS 7, BSC 1, BSC 40 or BMT 10, and
tissue-cultured human and plant cells. Preferred hosts include
bacteria such as E. coli and Bacillus subtilis, and tissue-cultured
mammalian cells.
[0076] The transformation and transfection may be performed by the
methods described in basic experimental guidebooks (Davis et al.,
Basic Methods in Molecular Biology, 1986; Sambrook, J., et al.,
Basic Methods in Molecular Biology, 1989). The preferred methods
for introducing a DNA sequence encoding the protein variant
according to the present invention into a host cell include, for
example, calcium phosphate transfection, DEAE-dextran mediated
transfection, transvection, microinjection, cationic lipid-mediated
transfection, electroporation, transduction, scrape loading,
ballistic introduction, and infection.
[0077] Also, it will be understood that all vectors and expression
control sequences do not function equally in expressing the DNA
sequence of the present invention. Likewise, all hosts do not
function equally for an identical expression system. However, those
skilled in the art are able to make a suitable selection from
various vectors, expression control sequences and hosts, within the
scope of the present invention, without a heavy experimental
burden. For example, a vector may be selected taking a host cell
into consideration because the vector should be replicated in the
host cell. The copy number of a vector, ability to control the copy
number, and expression of other proteins encoded by the vector, for
example, an antibiotic marker, should be deliberated. Also, an
expression control sequence may be selected taking several factors
into consideration. For example, relative strength, control
capacity and compatibility with the DNA sequence of the present
invention of the sequence, particularly with respect to possible
secondary structures, should be deliberated. Further, the selection
of a host cell may be made under consideration of compatibility
with a selected vector, toxicity of a product encoded by a
nucleotide sequence, secretory nature of the product, ability to
correctly fold a polypeptide, fermentation or cultivation
requirements, ability to ensure easy purification of a product
encoded by a nucleotide sequence, or the like.
[0078] In the method of preparing the protein variant according to
the present invention, the host cells are cultivated in a nutrient
medium suitable for production of a polypeptide using methods known
in the art. For example, the cells may be cultivated by shake flask
cultivation, small-scale or large-scale fermentation in laboratory
or industrial fermenters performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or isolated
The cultivation takes place in a suitable nutrient medium
containing carbon and nitrogen sources and inorganic salts, using
procedures known in the art Suitable media are commercially
available from commercial suppliers and may be prepared according
to published compositions (for example, the catalog of American
Type Culture Collection). If the polypeptide is secreted into the
nutrient medium, the polypeptide can be recovered directly from the
medium. If the polypeptide is not secreted, it can be recovered
from cell lysates.
[0079] The biological response-modulating protein variant according
to the present invention may be recovered by methods known in the
art For example, the protein variant may be recovered from the
nutrient medium by conventional procedures including, but not
limited to, centrifugation, filtration, extraction, spray drying,
evaporation, or precipitation. Further, the protein variant may be
purified by a variety of procedures known in the art including, but
not limited to, chromatography (e.g., ion exchange, affinity,
hydrophobicity, and size exclusion), electrophoresis, differential
solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or
extraction.
[0080] The present invention provides a pharmaceutical composition
comprising a variant of a biological response-modulating protein
and a pharmaceutically acceptable carrier. In the pharmaceutical
composition according to the present invention, the biological
response-modulating protein variant is preferably contained in a
therapeutically effective amount.
[0081] The carrier used in the pharmaceutical composition of the
present invention includes the commonly used carriers, adjuvants
and vehicles, in the pharmaceutical field, which are as a whole
called "pharmaceutically acceptable carriers". Non-limiting
pharmaceutically acceptable carriers useful in the pharmaceutical
composition of the present invention include ion exchange, alumina,
aluminum stearate, lecithin, serum proteins (e.g., human serum
albumin), buffering agents (e.g., sodium phosphate, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of vegetable
saturated fatty acids), water, salts or electrolytes (e.g.,
protamine sulfate, disodium hydrophosphate, potassium
hydrophoshate, sodium chloride, and zinc salts), colloidal silica,
magnesium trisilicate, polyvinylpyrrolidone, cellulose-based
substrates, polyethylene glycol, sodium carboxymethylcellulose,
polyarylate, waxes, polyethylene-polyoxypropylene-block copolymers,
polyethylene glycol, and wool fat.
[0082] The pharmaceutical composition of the present invention may
be administered via any of the common routes, if it is able to
reach a desired tissue. Therefore, the pharmaceutical composition
of the present invention may be administered topically, orally,
parenterally, intraocularly, transdermally, intrarectally and
intraluminally, and may be formulated into solutions, suspensions,
tablets, pills, capsules and sustained release preparations. The
term "parenteral", as used herein, includes subcutaneous,
intranasal, intravenous, intraperitoneal, intramuscular,
intra-articular, intra-synovial, intrastemal, intracardial
intrathecal, intralesional and intracranial injection or infusion
techniques.
[0083] In an aspect, the pharmaceutical composition of the present
invention may be formulate as aqueous solutions for parenteral
administration. Preferably, a suitable buffer solution, such as
Hank's solution, Ringer's solution or physiologically buffered
saline, may be employed Aqueous injection suspensions may be
supplemented with substances capable of increasing viscosity of the
suspensions, which are exemplified by sodium
carboxymethylcellulose, sorbitol and dextran. In addition,
suspensions of the active components, such as oily injection
suspension, include lipophilic solvents or carriers, which are
exemplified by fatty oils such as sesame oil, and synthetic fatty
acid esters such as ethyl oleate, triglycerides or liposomes.
Polycationic non-lipid amino polymers may also be used as vehicles.
Optionally, the suspensions may contain suitable stabilizers or
drugs to increase the solubility of protein variants and obtain
high concentrations of the protein variants.
[0084] The pharmaceutical composition of the present invention is
preferably in the form of a sterile injectable preparation, such as
a sterile injectable aqueous or oleaginous suspension Such
suspension may be formulated according to the methods known in the
art, using suitable dispersing or wetting agents (e.g., Tween 80)
and suspending agents. The sterile injectable preparations may also
be a sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, such as a solution in
1,3-butanediol. The acceptable vehicles and solvents include
mannitol, water, Ringer's solution and isotonic sodium chloride
solution. In addition, sterile fixed oils may conventionally be
employed as a solvent or suspending medium. For this purpose, any
bland fixed oil may be employed, including synthetic mono- or
di-glycerides. In addition, fatty acids, such as oleic acid and
glyceride derivatives thereof may be used in the preparation of
injectable preparations, like the pharmaceutically acceptable
natural oils (e.g., olive oil or castor oil), and particularly,
polyoxyethylated derivatives thereof.
[0085] The aforementioned aqueous composition is sterilized manly
by filtration using a filter to remove bacteria, mixing with
disinfectants or in combination with radiation. The sterilized
composition can be hardened, for example, by freeze-drying to
obtain a hardened product, and for practical use, the hardened
product is dissolved in sterilized water or a sterilized diluted
solution.
[0086] The term "therapeutically effective amount", as used herein
in connection with the pharmaceutical composition of the present
invention, means an amount in which an active component shows an
improved or therapeutic effect toward a disease to which the
pharmaceutical composition of the present invention is applied The
therapeutically effective amount of the pharmaceutical composition
of the present invention may vary according to the patient's age
and sex, application sites, administration frequency,
administration duration, formulation types and adjuvant types.
Typically, the pharmaceutical composition of the present invention
is administered in smaller amounts than a wild-type protein, for
example, 0.01-1000 .mu.g/kg/day, more preferably 0.1-500
.mu.g/kg/day, and most preferably 1-100 .mu.g/kg/day.
[0087] On the other hand, it will be apparent to those skilled in
the art that diseases to which the present composition is applied
may vary according to the protein type. The EPO and TPO altered as
in an embodiment of the present invention may be used for treating,
in addition to anemia itself, anemia as a complication associated
with other diseases (e.g., anemia in inflammatory bowel disease,
Progressive Kidney Disease, anemia of renal failure, the anemia
associated with HIV infection in zidovudine (AZT) treated patients,
anemia associated with cancer chemotherapy, Huntington's disease
(HD), sickle cell anemia, Late Hyporegenerative Anemia in Neonates
with Rh Hemolytic Disease after in utero Exchange Transfusion). In
addition, the G-CSF altered according to the present invention may
be used for treating neutropenia itself and neutropenia developed
after bone marrow transplantation or cancer chemotherapy, the GH
variants may be used for treating pituitary dwarfism and paediatric
chronic renal failure. However, the present invention is not
limited to these applications.
[0088] Hereinafter, the present invention provides interferon
variants which each substitute valine for specific phenylalanine
residue of 4helix bundle cytokines, in detail, CNTF, EPO, Flt3L,
G-CSF, GM-CSF, GH, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT,
LIF, M-CSF, OSM, PI, SCF, TPO, IFN-.alpha.2A, IFN-.alpha.2B,
IFN-.beta., IFN-.gamma., IFN-.omega. and IFN-.tau..
[0089] In one specific aspect, the present invention provides the
following protein variants: (1) a CNTF variant that substitutes
valine for the phenylalanine residue at the position 3, 83, 98,
105, 119, 152 or 178 of an amino acid sequence (SEQ ID NO.: 1) of a
wild-type CNTF; (2) an EPO variant that substitutes valine for the
phenylalanine residue at the position 48, 138, 142 or 148 of an
amino acid sequence (SEQ ID NO.: 2) of a wild-type EPO; (3) a Flt3L
variant that substitutes valine for the phenylalanine residue at
the position 6, 15, 81, 87, 96 or 124 of an amino acid sequence
(SEQ ID NO.: 3) of a wild-type Flt3L; (4) a G-CSF variant that
substitutes valine for the phenylalanine residue at the position
13, 83, 113, 140, 144 or 160 of an amino acid sequence (SEQ ID NO.:
4) of a wild-type G-CSF; (5) a GM-CSF variant that substitutes
valine for the phenylalanine residue at the position 47, 103, 106,
113 or 119 of an amino acid sequence (SEQ ID NO.: 5) of a wild-type
GM-CSF; (6) a GH variant that substitutes valine for the
phenylalanine residue at the position 1, 10, 25, 31, 44, 54, 92,
97, 139, 146, 166, 176 or 191 of an amino acid sequence (SEQ ID
NO.: 6) of a wild-type GH; (7) an IFN-.alpha.2A variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid
sequence (SEQ ID NO.: 7) of a wild-type IFN-.alpha.2A; (8) an
IFN-.alpha.2B variant that substitutes valine for the phenylalanine
residue at the position 27, 36, 38, 43, 47, 64, 67, 84, 123 or 151
of an amino acid sequence (SEQ ID NO.: 8) of a wild-type
IFN-.alpha.2B; (9) an IFN-.beta. variant that substitutes valine
for the phenylalanine residue at the position 8, 38, 50, 67, 70,
111 or 154 of an amino acid sequence (SEQ ID NO.: 9) of a wild-type
IFN-.beta.; (10) an IFN-.gamma. variant that substitutes valine for
the phenylalanine residue at the position 18, 32, 55, 57, 60, 63,
84, 85, 95 or 139 of an amino acid sequence (SEQ ID NO.: 10) of a
wild-type IFN-.gamma.; (11) an IFN-.omega. variant that substitutes
valine for the phenylalanine residue at the position 27, 36, 38,
65, 68, 124 or 153 of an amino acid sequence (SEQ ID NO.: 11) of a
wild-type IFN-.omega.; (12) an IFN-.tau. variant that substitutes
valine for the phenylalanine residue at the position 8, 39, 68, 71,
88, 127, 156, 157, 159 or 183 of an amino acid sequence (SEQ ID
NO.: 12) of a wild-type IFN-.tau.; (13) an IL-2 variant that
substitutes valine for the phenylalanine residue at the position
42, 44, 78, 103, 117 or 124 of an amino acid sequence (SEQ ID NO.:
13) of a wild-type IL-2; (14) an IL-3 variant that substitutes
valine for the phenylalanine residue at the position 37, 61, 107,
113 or 133 of an amino acid sequence (SEQ ID NO.: 14) of a
wild-type IL-3; (15) an IL-4 variant that substitutes valine for
the phenylalanine residue at the position 33, 45, 55, 73, 82 or 112
of an amino acid sequence (SEQ ID NO.: 15) of a wild-type IL-4;
(16) an IL-5 variant that substitutes valine for the phenylalanine
residue at the position 49, 69, 96 or 103 of an amino acid sequence
(SEQ ID NO.: 16) of a wild-type IL-5; (17) an IL-6 variant that
substitutes valine for the phenylalanine residue at the position
73, 77, 93, 104, 124, 169 or 172 of an amino acid sequence (SEQ ID
NO.: 17) of a wild-type IL-6; (18) an IL-12p35 variant that
substitutes valine for the phenylalanine residue at the position
13, 39, 82, 96, 116, 132, 150, 166 or 180 of an amino acid sequence
(SEQ ID NO.: 18) of a wild-type IL-12p35; (19) a LPT variant that
substitutes valine for the phenylalanine residue at the position 41
or 92 of an amino acid sequence (SEQ ID NO.: 19) of a wild-type
LPT; (20) a LIF variant that substitutes valine for the
phenylalanine residue at the position 41, 52, 67, 70, 156 or 180 of
an amino acid sequence (SEQ ID NO.: 20) of a wild-type LIF; (21) a
M-CSF variant that substitutes valine for the phenylalanine residue
at the position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255,
311, 439, 466 or 485 of an amino acid sequence (SEQ ID NO.: 21) of
a wild-type M-CSF; (22) an OSM variant that substitutes valine for
the phenylalanine residue at the position 56, 70, 160, 169, 176 or
184 of an amino acid sequence (SEQ ID NO.: 22) of a wild-type OSM;
(23) a PL variant that substitutes valine for the phenylalanine
residue at the position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176
or 191 of an amino acid sequence (SEQ ID NO.: 23) of a wild-type
PL; (24) a SCF variant that substitutes valine for the
phenylalanine residue at the position 63, 102, 110, 115, 116, 119,
126, 129, 158, 199, 205, 207 or245 of an amino acid sequence (SEQ
ID NO.: 24) of a wild-type SCF; and (25) a TPO variant that
substitutes valine for the phenylalanine residue at the position
46, 128, 131, 141, 186, 204, 240 or 286 of an amino acid sequence
(SEQ ID NO.: 25) of a wild-type TPO.
[0090] In another specific aspect, the present invention provides
the following DNA molecules: (1) a DNA encoding a CNTF variant that
substitutes valine for the phenylalanine residue at the position 3,
83, 98, 105, 119, 152 or 178 of an amino acid sequence (SEQ ID NO.:
1) of a wild-type CNTF; (2) a DNA encoding an EPO variant that
substitutes valine for the phenylalanine residue at the position
48, 138, 142 or 148 of an amino acid sequence (SEQ ID NO.: 2) of a
wild-type EPO; (3) a DNA encoding a Flt3L variant that substitutes
valine for the phenylalanine residue at the position 6, 15, 81, 87,
96 or 124 of an amino acid sequence (SEQ ID NO.: 3) of a wild-type
Flt3L; (4) a DNA encoding a G-CSF variant that substitutes valine
for the phenylalanine residue at the position 13, 83, 113, 140, 144
or 160 of an amino acid sequence (SEQ ID NO.: 4) of a wild-type
G-CSF; (5) a DNA encoding a GM-CSF variant that substitutes valine
for the phenylalanine residue at the position 47, 103, 106, 113 or
119 of an amino acid sequence (SEQ ID NO.: 5) of a wild-type
GM-CSF; (6) a DNA encoding a-GH variant that substitutes valine for
the phenylalanine residue at the position 1, 10, 25, 31, 44, 54,
92, 97, 139, 146, 166, 176or191 of an amino acid sequence (SEQ ID
NO.: 6) of a wild-type GH; (7) a DNA encoding an IFN-.alpha.2A
variant that substitutes valine for the phenylalanine residue at
the position 27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino
acid sequence (SEQ ID NO.: 7) of a wild-type IFN-.alpha.2A; (8) a
DNA encoding an IFN-.alpha.2B variant that substitutes valine for
the phenylalanine residue at the position 27, 36, 38, 43, 47, 64,
67, 84, 123 or 151 of an amino acid sequence (SEQ ID NO.: 8) of a
wild-type IFN-.alpha.2B; (9) a DNA encoding an IFN-.beta. variant
that substitutes valine for the phenylalanine residue at the
position 8, 38, 50, 67, 70, 111 or 154 of an amino acid sequence
(SEQ ID NO.: 9) of a wild-type IFN-.beta.; (10) a DNA encoding an
IFN-.gamma. variant that substitutes valine for the phenylalanine
residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95 or 139
of an amino acid sequence (SEQ ID NO.: 10) of a wild-type
IFN-.gamma.; (11) a DNA encoding an IFN-.omega. variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 65, 68, 124 or 153 of an amino acid sequence (SEQ ID
NO.: 11) of a wild-type IFN-.omega.; (12) a DNA encoding an
IFN-.tau. variant that substitutes valine for the phenylalanine
residue at the position 8, 39, 68, 71, 88, 127, 156, 157, 159 or
183 of an amino acid sequence (SEQ ID NO.: 12) of a wild-type
IFN-.tau.; (13) a DNA encoding an IL-2 variant that substitutes
valine for the phenylalanine residue at the position 42, 44, 78,
103, 117 or 124 of an amino acid sequence (SEQ ID NO.: 13) of a
wild-type IL-2; (14) a DNA encoding an IL-3 variant that
substitutes valine for the phenylalanine residue at the position
37, 61, 107, 113 or 133 of an amino acid sequence (SEQ ID NO.: 14)
of a wild-type IL-3; (15) a DNA encoding an IL-4 variant that
substitutes valine for the phenylalanine residue at the position
33, 45, 55, 73, 82 or 112 of an amino acid sequence (SEQ ID NO.:
15) of a wild-type IL-4; (16) a DNA encoding an IL-5 variant that
substitutes valine for the phenylalanine residue at the position
49, 69, 96 or 103 of an amino acid sequence (SEQ ID NO.: 16) of a
wild-type IL-5; (17) a DNA encoding an IL-6 variant that
substitutes valine for the phenylalanine residue at the position
73, 77, 93, 104, 124, 169 or 172 of an amino acid sequence (SEQ ID
NO.: 17) of a wild-type IL-6; (18) a DNA encoding an IL-12p35
variant that substitutes valine for the phenylalanine residue at
the position 13, 39, 82, 96, 116, 132, 150, 166 or 180 of an amino
acid sequence (SEQ ID NO.: 18) of a wild-type IL-12p35; (19) a DNA
encoding a LPT variant that substitutes valine for the
phenylalanine residue at the position 41 or 92 of an amino acid
sequence (SEQ ID NO.: 19) of a wild-type LPT; (20) a DNA encoding a
LIF variant that substitutes valine for the phenylalanine residue
at the position 41, 52, 67, 70, 156 or 180 of an amino acid
sequence (SEQ ID NO.: 20) of a wild-type LIF; (21) a DNA encoding a
M-CSF variant that substitutes valine for the phenylalanine residue
at the position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255,
311, 439, 466 or 485 of an amino acid sequence (SEQ ID NO.: 21) of
a wild-type M-CSF; (22) a DNA encoding an OSM variant that
substitutes valine for the phenylalanine residue at the position
56, 70, 160, 169, 176 or 184 of an amino acid sequence (SEQ ID NO.:
22) of a wild-type OSM; (23) a DNA encoding a PL variant that
substitutes valine for the phenylalanine residue at the position
10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191 of an amino acid
sequence (SEQ ID NO.: 23) of a wild-type PL; (24) a DNA encoding a
SCF variant that substitutes valine for the phenylalanine residue
at the position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199,
205, 207 or 245 of an amino acid sequence (SEQ ID NO.: 24) of a
wild-type SCF; and (25) a DNA encoding a TPO variant that
substitutes valine for the phenylalanine residue at the position
46, 128, 131, 141, 186, 204, 240 or 286 of an amino acid sequence
(SEQ ID NO.: 25) of a wild-type TPO.
[0091] In a further specific aspect, the present invention provides
the following recombinant expression vectors: (1) a recombinant
expression vector to which a DNA encoding a CNTF variant that
substitutes valine for the phenylalanine residue at the position 3,
83, 98, 105, 119, 152 or 178 of an amino acid sequence (SEQ ID NO.:
1) of a wild-type CNTF is operably linked; (2) a recombinant
expression vector to which a DNA encoding an EPO variant that
substitutes valine for the phenylalanine residue at the position
48, 138, 142 or 148 of an amino acid sequence (SEQ ID NO.: 2) of a
wild-type EPO is operably linked; (3) a recombinant expression
vector to which a DNA encoding a Flt3L variant that substitutes
valine for the phenylalanine residue at the position 6, 15, 81, 87,
96 or 124 of an amino acid sequence (SEQ ID NO.: 3) of a wild-type
Flt3L is operably linked; (4) a recombinant expression vector to
which a DNA encoding a G-CSF variant that substitutes valine for
the phenylalanine residue at the position 13, 83, 113, 140, 144 or
160 of an amino acid sequence (SEQ ID NO.: 4) of a wild-type G-CSF
is operably linked; (5) a recombinant expression vector to which a
DNA encoding a GM-CSF variant that substitutes valine for the
phenylalanine residue at the position 47, 103, 106, 113 or 119 of
an amino acid sequence (SEQ ID NO.: 5) of a wild-type GM-CSF is
operably linked; (6) a recombinant expression vector to which a DNA
encoding a GH variant that substitutes valine for the phenylalanine
residue at the position 1, 10, 25, 31, 44, 54, 92, 97, 139, 146,
166, 176 or 191 of an amino acid sequence (SEQ ID NO.: 6) of a
wild-type GH is operably linked; (7) a recombinant expression
vector to which a DNA encoding an IFN-.alpha.2A variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid
sequence (SEQ ID NO.: 7) of a wild-type IFN-.alpha.2A is operably
linked; (8) a recombinant expression vector to which a DNA encoding
an IFN-.alpha.2B variant that substitutes valine for the
phenylalanine residue at the position 27, 36, 38, 43, 47, 64, 67,
84, 123 or 151 of an amino acid sequence (SEQ ID NO.: 8) of a
wild-type IFN-.alpha.2B is operably linked; (9) a recombinant
expression vector to which a DNA encoding an IFN-.beta. variant
that substitutes valine for the phenylalanine residue at the
position 8, 38, 50, 67, 70, 111 or 154 of an amino acid sequence
(SEQ ID NO.: 9) of a wild-type IFN-.beta. is operably linked; (10)
a recombinant expression vector to which a DNA encoding an
IFN-.gamma. variant that substitutes valine for the phenylalanine
residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95 or 139
of an amino acid sequence (SEQ ID NO.: 10) of a wild-type
IFN-.gamma. is operably linked; (11) a recombinant expression
vector to which a DNA encoding an IFN-.omega. variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 65, 68, 124 or 153 of an amino acid sequence (SEQ ID
NO.: 11) of a wild-type IFN-.omega. is operably linked; (12) a
recombinant expression vector to which a DNA encoding an IFN-.tau.
variant that substitutes valine for the phenylalanine residue at
the position 8, 39, 68, 71, 88, 127, 156, 157, 159 or 183 of an
amino acid sequence (SEQ ID NO.: 12) of a wild-type IFN-.tau. is
operably linked; (13) a recombinant expression vector to which a
DNA encoding an IL-2 variant that substitutes valine for the
phenylalanine residue at the position 42, 44, 78, 103, 117 or 124
of an amino acid sequence (SEQ ID NO.: 13) of a wild-type IL-2 is
operably linked; (14) a recombinant expression vector to which a
DNA encoding an IL-3 variant that substitutes valine for the
phenylalanine residue at the position 37, 61, 107, 113 or 133 of an
amino acid sequence (SEQ ID NO.: 14) of a wild-type IL-3 is
operably linked; (15) a recombinant expression vector to which a
DNA encoding an IL-4 variant that substitutes valine for the
phenylalanine residue at the position 33, 45, 55, 73, 82 or 112 of
an amino acid sequence (SEQ ID NO.: 15) of a wild-type IL-4 is
operably linked, (16) a recombinant expression vector to which a
DNA encoding an IL-5 variant that substitutes valine for the
phenylalanine residue at the position 49, 69, 96 or 103 of an amino
acid sequence (SEQ ID NO.: 16) of a wild-type IL-5 is operably
linked, (17) a recombinant expression vector to which a DNA
encoding an IL-6 variant that substitutes valine for the
phenylalanine residue at the position 73, 77, 93, 104, 124, 169 or
172 of an amino acid sequence (SEQ ID NO.: 17) of a wild-type W-6
is operably linked; (18) a recombinant expression vector to which a
DNA encoding an IL-12p35 variant that substitutes valine for the
phenylalanine residue at the position 13, 39, 82, 96, 116, 132,
150, 166 or 180 of an amino acid sequence (SEQ ID NO.: 18) of a
wild-type IL-12p35 is operably linked; (19) a recombinant
expression vector to which a DNA encoding a LPT variant that
substitutes valine for the phenylalanine residue at the position 41
or 92 of an amino acid sequence (SEQ ID NO.: 19) of a wild-type LPT
is operably linked; (20) a recombinant expression vector to which a
DNA encoding a LIF variant that substitutes valine for the
phenylalanine residue at the position 41, 52, 67, 70, 156 or 180 of
an amino acid sequence (SEQ ID NO.: 20) of a wild-type LIF is
operably linked; (21) a recombinant expression vector to which a
DNA encoding a M-CSF variant that substitutes valine for the
phenylalanine residue at the position 35, 37, 54, 67, 91, 106, 121,
135, 143, 229, 255, 311, 439, 466 or 485 of an amino acid sequence
(SEQ ID NO.: 21) of a wild-type M-CSF is operably linked; (22) a
recombinant expression vector to which a DNA encoding an OSM
variant that substitutes valine for the phenylalanine residue at
the position 56, 70, 160, 169, 176 or 184 of an amino acid sequence
(SEQ ID NO.: 22) of a wild-type OSM is operably linked; (23) a
recombinant expression vector to which a DNA encoding a PL variant
that substitutes valine for the phenylalanine residue at the
position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191 of an
amino acid sequence (SEQ ID NO.: 23) of a wild-type PL is operably
linked; (24) a recombinant expression vector to which a DNA
encoding a SCF variant that substitutes valine for the
phenylalanine residue at the position 63, 102, 110, 115, 116, 119,
126, 129, 158, 199, 205, 207 or 245 of an amino acid sequence (SEQ
ID NO.: 24) of a wild-type SCF is operably linked; and (25) a
recombinant expression vector to which a DNA encoding a TPO variant
that substitutes valine for the phenylalanine residue at the
position 46, 128, 131, 141, 186, 204, 240 or 286 of an amino acid
sequence (SEQ ID NO.: 25) of a wild-type TPO is operably
linked.
[0092] In yet another specific aspect, the present invention
provides the following host cells: (1) a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a CNTF variant that substitutes valine for the
phenylalanine residue at the position 3, 83, 98, 105, 119, 152 or
178 of an amino acid sequence (SEQ ID NO.: 1) of a wild-type CNTF
is operably linked; (2) a host cell transformed or transfected with
a recombinant expression vector to which a DNA encoding an EPO
variant that substitutes valine for the phenylalanine residue at
the position 48, 138, 142 or 148 of an amino acid sequence (SEQ ID
NO.: 2) of a wild-type EPO is operably linked, (3) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding a Flt3L variant that substitutes valine for
the phenylalanine residue at the position 6, 15, 81, 87, 96 or 124
of an amino acid sequence (SEQ ID NO.: 3) of a wild-type Flt3L is
operably linked; (4) a host cell transformed or transfected with a
recombinant expression vector to which a DNA encoding a G-CSF
variant that substitutes valine for the phenylalanine residue at
the position 13, 83, 113, 140, 144 or 160 of an amino acid sequence
(SEQ ID NO.: 4) of a wild-type G-CSF is operably linked; (5) a host
cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a GM-CSF variant that substitutes
valine for the phenylalanine residue at the position 47, 103, 106,
113 or 119 of an amino acid sequence (SEQ ID NO.: 5) of a wild-type
GM-CSF is operably linked; (6) a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a GH variant that substitutes valine for the phenylalanine
residue at the position 1, 10, 25, 31, 44, 54, 92, 97, 139, 146,
166, 176 or 191 of an amino acid sequence (SEQ ID NO.: 6) of a
wild-type GH is operably linked; (7) a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding an IFN-.alpha.2A variant that substitutes valine for the
phenylalanine residue at the position 27, 36, 38, 43, 47, 64, 67,
84, 123 or 151 of an amino acid sequence (SEQ ID NO.: 7) of a
wild-type IFN-.alpha.2A is operably linked; (8) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding an IFN-.alpha.2B variant that substitutes
valine for the phenylalanine residue at the position 27, 36, 38,
43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ ID
NO.: 8) of a wild-type IFN-.alpha.2B is operably linked; (9) a host
cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IFN-.beta. variant that
substitutes valine for the phenylalanine residue at the position 8,
38, 50, 67, 70, 111 or 154 of an amino acid sequence (SEQ ID NO.:
9) of a wild-type IFN-.beta. is operably led; (10) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding an IFN-.gamma. variant that substitutes valine
for the phenylalanine residue at the position 18, 32, 55, 57, 60,
63, 84, 85, 95 or 139 of an amino acid sequence (SEQ ID NO.: 10) of
a wild-type IFN-.gamma. is operably linked; (11) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding an IFN-.omega. variant that substitutes valine
for the phenylalanine residue at the position 27, 36, 38, 65, 68,
124 or 153 of an amino acid sequence (SEQ ID NO.: 11) of a
wild-type IFN-.omega. is operably linked; (12) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding an IFN-.tau. variant that substitutes valine
for the phenylalanine residue at the position 8, 39, 68, 71, 88,
127, 156, 157, 159 or 183 of an amino acid sequence (SEQ ID NO.:
12) of a wild-type IFN-.tau. is operably linked; (13) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding an IL-2 variant that substitutes valine for
the phenylalanine residue at the position 42, 44, 78, 103, 117 or
124 of an amino acid sequence (SEQ ID NO.: 13) of a wild-type IL-2
is operably linked; (14) a host cell transformed or transfected
with a recombinant expression vector to which a DNA encoding an
IL-3 variant that substitutes valine for the phenylalanine residue
at the position 37, 61, 107, 113 or 133 of an amino acid sequence
(SEQ ID NO.: 14) of a wild-type IL-3 is operably linked; (15) a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IL-4 variant that substitutes
valine for the phenylalanine residue at the position 33, 45, 55,
73, 82 or 112 of an amino acid sequence (SEQ ID NO.: 15) of a
wild-type IL-4 is operably linked; (16) a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding an IL-5 variant that substitutes valine for the
phenylalanine residue at the position 49, 69, 96 or 103 of an amino
acid sequence (SEQ ID NO.: 16) of a wild-type IL-5 is operably
linked; (17) a host cell transformed or transfected with a
recombinant expression vector to which a DNA encoding an IL-6
variant that substitutes valine for the phenylalanine residue at
the position 73, 77, 93, 104, 124, 169 or 172 of an amino acid
sequence (SEQ ID NO.: 17) of a wild-type IL-6 is operably linked;
(18) a host cell transformed or transfected with a recombinant
expression vector to which a DNA encoding an IL-12p35 variant that
substitutes valine for the phenylalanine residue at the position
13, 39, 82, 96, 116, 132, 150, 166 or 180 of an amino acid sequence
(SEQ ID NO.: 18) of a wild-type IL-12p35 is operably linked; (19) a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a LPT variant that substitutes
valine for the phenylalanine residue at the position 41 or 92 of an
amino acid sequence (SEQ ID NO.: 19) of a wild-type LPT is operably
linked; (20) a host cell transformed or transfected with a
recombinant expression vector to which a DNA encoding a LIF variant
that substitutes valine for the phenylalanine residue at the
position 41, 52, 67, 70, 156 or 180 of an amino acid sequence (SEQ
ID NO.: 20) of a wild-type LIF is operably linked; (21) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding a M-CSF variant that substitutes valine for
the phenylalanine residue at the position 35, 37, 54, 67, 91, 106,
121, 135, 143, 229, 255, 311, 439,466 or 485 of an amino acid
sequence (SEQ ID NO.: 21) of a wild-type M-CSF is operably linked;
(22) a host cell transformed or transfected with a recombinant
expression vector to which a DNA encoding an OSM variant that
substitutes valine for the phenylalanine residue at the position
56, 70, 160, 169, 176 or 184 of an amino acid sequence (SEQ ID NO.:
22) of a wild-type OSM is operably linked; (23) a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding a PL variant that substitutes valine for the
phenylalanine residue at the position 10, 31, 44, 52, 54, 92, 97,
146, 166, 176 or 191 of an amino acid sequence (SEQ ID NO.: 23) of
a wild-type PL is operably linked; (24) a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a SCF variant that substitutes valine for the
phenylalanine residue at the position 63, 102, 110, 115, 116, 119,
126, 129, 158, 199, 205, 207 or 245 of an amino acid sequence (SEQ
ID NO.: 24) of a wild-type SCF is operably linked; and (25) a host
cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a TPO variant that substitutes
valine for the phenylalanine residue at the position 46, 128, 131,
141, 186, 204, 240 or 286 of an amino acid sequence (SEQ ID NO.:
25) of a wild-type TPO is operably linked.
[0093] In still another specific aspect, the present invention
provides the following methods of preparing a protein variant: (1)
a method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a CNTF variant that substitutes
valine for the phenylalanine residue at the position 3, 83, 98,
105, 119, 152 or 178 of an amino acid sequence (SEQ ID NO.: 1) of a
wild-type CNTF is operably linked, and isolating the protein
variant from a resulting culture; (2) a method of preparing a
protein variant, comprising cultivating a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding an EPO variant that substitutes valine for the
phenylalanine residue at the position 48, 138, 142 or 148 of an
amino acid sequence (SEQ ID NO.: 2) of a wild-type EPO is operably
linked, and isolating the protein variant from a resulting culture;
(3) a method of preparing a protein variant, comprising cultivating
a host cell transformed or transfected with a recombinant
expression vector to which a DNA encoding a Flt3L variant that
substitutes valine for the phenylalanine residue at the position 6,
15, 81, 87, 96 or 124 of an amino acid sequence (SEQ ID NO.: 3) of
a wild-type Flt3L is operably linked, and isolating the protein
variant from a resulting culture; (4) a method of preparing a
protein variant, comprising cultivating a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a G-CSF variant that substitutes valine for the
phenylalanine residue at the position 13, 83, 113, 140, 144 or 160
of an amino acid sequence (SEQ ID NO.: 4) of a wild-type G-CSF is
operably linked, and isolating the protein variant from a resulting
culture; (5) a method of preparing a protein variant, comprising
cultivating a host cell transformed or transfected with a
recombinant expression vector to which a DNA encoding a GM-CSF
variant that substitutes valine for the phenylalanine residue at
the position 47, 103, 106, 113 or 119 of an amino acid sequence
(SEQ ID NO.: 5) of a wild-type GM-CSF is operably linked, and
isolating the protein variant from a resulting culture; (6) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a GH variant that substitutes valine
for the phenylalanine residue at the position 1, 10, 25, 31, 44,
54, 92, 97, 139, 146, 166, 176 or 191 of an amino acid sequence
(SEQ ID NO.: 6) of a wild-type GH is operably linked, and isolating
the protein variant from a resulting culture; (7) a method of
preparing a protein variant, comprising cultivating a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding an IFN-.alpha.2A variant that substitutes
valine for the phenylalanine residue at the position 27, 36, 38,
43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ ID
NO.: 7) of a wild-type IFN-.alpha.2A is operably linked, and
isolating the protein variant from a resulting culture; (8) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IFN-.alpha.2B variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid
sequence (SEQ ID NO.: 8) of a wild-type IFN-.alpha.2B is operably
linked, and isolating the protein variant from a resulting culture;
(9) a method of preparing a protein variant, comprising cultivating
a host cell transformed or transfected with a recombinant
expression vector to which a DNA encoding an IFN-.beta. variant
that substitutes valine for the phenylalanine residue at the
position 8, 38, 50, 67, 70, 111 or 154 of an amino acid sequence
(SEQ ID NO.: 9) of a wild-type IFN-.beta. is operably linked, and
isolating the protein variant from a resulting culture; (10) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IFN-.gamma. variant that
substitutes valine for the phenylalanine residue at the position
18, 32, 55, 57, 60, 63, 84, 85, 95 or 139 of an amino acid sequence
(SEQ ID NO.: 10) of a wild-type IFN-.gamma. is operably linked, and
isolating the protein variant from a resulting culture; (11) a
method of preparing a protein variant) comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IFN-.omega. variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 65, 68, 124 or 153 of an amino acid sequence (SEQ ID
NO.: 11) of a wild-type IFN-.omega. is operably linked, and
isolating the protein variant from a resulting culture; (12) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IFN-.tau. variant that
substitutes valine for the phenylalanine residue at the position 8,
39, 68, 71, 88, 127, 156, 157, 159 or 183 of an amino acid sequence
(SEQ ID NO.: 12) of a wild-type IFN-.tau. is operably linked, and
isolating the protein variant from a resulting culture; (13) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IL-2 variant that substitutes
valine for the phenylalanine residue at the position 42, 44, 78,
103, 117 or 124 of an amino acid sequence (SEQ ID NO.: 13) of a
wild-type IL-2 is operably linked, and isolating the protein
variant from a resulting culture; (14) a method of preparing a
protein variant, comprising cultivating a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding an IL-3 variant that substitutes valine for the
phenylalanine residue at the position 37, 61, 107, 113 or 133 of an
amino acid sequence (SEQ ID NO.: 14) of a wild-type IL-3 is
operably linked, and isolating the protein variant from a resulting
culture; (15) a method of preparing a protein variant, comprising
cultivating a host cell transformed or transfected with a
recombinant expression vector to which a DNA encoding an IL-4
variant that substitutes valine for the phenylalanine residue at
the position 33, 45, 55, 73, 82 or 112 of an amino acid sequence
(SEQ ID NO.: 15) of a wild-type IL-4 is operably linked, and
isolating the protein variant from a resulting culture; (16) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IL-5 variant that substitutes
valine for the phenylalanine residue at the position 49, 69, 96 or
103 of an amino acid sequence (SEQ ID NO.: 16) of a wild-type IL-5
is operably linked, and isolating the protein variant from a
resulting culture; (17) a method of preparing a protein variant,
comprising cultivating a host cell transformed or transfected with
a recombinant expression vector to which a DNA encoding an IL-6
variant that substitutes valine for the phenylalanine residue at
the position 73, 77, 93, 104, 124, 169 or 172 of an amino acid
sequence (SEQ ID NO.: 17) of a wild-type IL-6 is operably linked,
and isolating the protein variant from a resulting culture; (18) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an IL-12p35 variant that substitutes
valine for the phenylalanine residue at the position 13, 39, 82,
96, 116, 132, 150, 166 or 180 of an amino acid sequence (SEQ ID
NO.: 18) of a wild-type IL-12p35 is operably linked, and isolating
the protein variant from a resulting culture; (19) a method of
preparing a protein variant, comprising cultivating a host cell
transformed or transfected with a recombinant expression vector to
which a DNA encoding a LPT variant that substitutes valine for the
phenylalanine residue at the position 41 or 92 of an amino acid
sequence (SEQ ID NO.: 19) of a wild-type LPT is operably linked,
and isolating the protein variant from a resulting culture; (20) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding a LIF variant that substitutes
valine for the phenylalanine residue at the position 41, 52, 67,
70, 156 or 180 of an amino acid sequence (SEQ ID NO.: 20) of a
wild-type LIF is operably linked, and isolating the protein variant
from a resulting culture; (21) a method of preparing a protein
variant, comprising cultivating a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a M-CSF variant that substitutes valine for the
phenylalanine residue at the position 35, 37, 54, 67, 91, 106, 121,
135, 143, 229, 255, 311, 439, 466 or 485 of an amino acid sequence
(SEQ ID NO.: 21) of a wild-type M-CSF is operably linked, and
isolating the protein variant from a resulting culture; (22) a
method of preparing a protein variant, comprising cultivating a
host cell transformed or transfected with a recombinant expression
vector to which a DNA encoding an OSM variant that substitutes
valine for the phenylalanine residue at the position 56,70, 160,
169, 176 or 184 of an amino acid sequence (SEQ ID NO.: 22) of a
wild-type OSM is operably linked, and isolating the protein variant
from a resulting culture; (23) a method of preparing a protein
variant, comprising cultivating a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a PL variant that substitutes valine for the phenylalanine
residue at the position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176
or 191 of an amino acid sequence (SEQ ID NO.: 23) of a wild-type PL
is operably linked, and isolating the protein variant from a
resulting culture; (24) a method of preparing a protein variant,
comprising cultivating a host cell transformed or transfected with
a recombinant expression vector to which a DNA encoding a SCF
variant that substitutes valine for the phenylalanine residue at
the position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199, 205,
207 or 245 of an amino acid sequence (SEQ ID NO.: 24) of a
wild-type SCF is operably linked, and isolating the protein variant
from a resulting culture; and (25) a method of preparing a protein
variant, comprising cultivating a host cell transformed or
transfected with a recombinant expression vector to which a DNA
encoding a TPO variant that substitutes valine for the
phenylalanine residue at the position 46, 128, 131, 141, 186, 204,
240 or 286 of an amino acid sequence (SEQ ID NO.: 25) of a
wild-type TPO is operably linked, and isolating the protein variant
from a resulting culture.
[0094] In still another specific aspect the present invention
provides the following pharmaceutical compositions: (1) a
pharmaceutical composition comprising a CNTF variant that
substitutes valine for the phenylalanine residue at the position 3,
83, 98, 105, 119, 152 or 178 of an amino acid sequence (SEQ ID NO.:
1) of a wild-type CNTF and a pharmaceutically acceptable carrier,
(2) a pharmaceutical composition comprising an EPO variant that
substitutes valine for the phenylalanine residue at the position
48, 138, 142 or 148 of an amino acid sequence (SEQ ID NO.: 2) of a
wild-type EPO and a pharmaceutically acceptable carrier, (3) a
pharmaceutical composition comprising a Flt3L variant that
substitutes valine for the phenylalanine residue at the position 6,
15, 81, 87, 96 or 124 of an amino acid sequence (SEQ ID NO.: 3) of
a wild-type Flt3L and a pharmaceutically acceptable carrier, (4) a
pharmaceutical composition comprising a G-CSF variant that
substitutes valine for the phenylalanine residue at the position
13, 83, 113, 140, 144 or 160 of an amino acid sequence (SEQ ID NO.:
4) of a wild-type G-CSF and a pharmaceutically acceptable carrier,
(5) a pharmaceutical composition comprising a GM-CSF variant that
substitutes valine for the phenylalanine residue at the position
47, 103, 106, 113 or 119 of an amino acid sequence (SEQ ID NO.: 5)
of a wild-type GM-CSF and a pharmaceutically acceptable carrier,
(6) a pharmaceutical composition comprising a GH variant that
substitutes valine for the phenylalanine residue at the position 1,
10, 25, 31, 44, 54, 92, 97, 139, 146, 166, 176 or 191 of an amino
acid sequence (SEQ ID NO.: 6) of a wild-type GH and a
pharmaceutically acceptable carrier, (7) a pharmaceutical
composition comprising an IFN-.alpha.2A variant that substitutes
valine for the phenylalanine residue at the position 27, 36, 38,
43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ ID
NO.: 7) of a wild-type IFN-.alpha.2A and a pharmaceutically
acceptable carrier, (8) a pharmaceutical composition comprising an
IFN-.alpha.2B variant that substitutes valine for the phenylalanine
residue at the position 27, 36, 38, 43, 47, 64, 67, 84, 123 or 151
of an amino acid sequence (SEQ ID NO.: 8) of a wild-type
IFN-.alpha.2B and a pharmaceutically acceptable carrier, (9) a
pharmaceutical composition comprising an IFN-.beta. variant that
substitutes valine for the phenylalanine residue at the position 8,
38, 50, 67, 70, 111 or 154 of an amino acid sequence (SEQ ID NO.:
9) of a wild-type IFN-.beta. and a pharmaceutically acceptable
carrier; (10) a pharmaceutical composition comprising an
IFN-.gamma. variant that substitutes valine for the phenylalanine
residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95 or 139
of an amino acid sequence (SEQ ID NO.: 10) of a wild-type
IFN-.gamma. and a pharmaceutically acceptable carrier, (11) a
pharmaceutical composition comprising an IFN-.omega. variant that
substitutes valine for the phenylalanine residue at the position
27, 36, 38, 65, 68, 124 or 153 of an amino acid sequence (SEQ ID
NO.: 11) of a wild-type IFN-.omega. and a pharmaceutically
acceptable carrier, (12) a pharmaceutical composition comprising an
IFN-.tau. variant that substitutes valine for the phenylalanine
residue at the position 8, 39, 68, 71, 88, 127, 156, 157, 159 or
183 of an amino acid sequence (SEQ ID NO.: 12) of a wild-type
IFN-.tau. and a pharmaceutically acceptable carrier, (13) a
pharmaceutical composition comprising an IL-2 variant that
substitutes valine for the phenylalanine residue at the position
42, 44, 78, 103, 117 or 124 of an amino acid sequence (SEQ ID NO.:
13) of a wild-type IL-2 and a pharmaceutically acceptable carrier;
(14) a pharmaceutical composition comprising an IL-3 variant that
substitutes valine for the phenylalanine residue at the position
37, 61, 107, 113 or 133 of an amino acid sequence (SEQ ID NO.: 14)
of a wild-type IL-3 and a pharmaceutically acceptable carrier, (15)
a pharmaceutical composition comprising an IL-4 variant that
substitutes valine for the phenylalanine residue at the position
33, 45, 55, 73, 82 or 112 of an amino acid sequence (SEQ ID NO.:
15) of a wild-type IL-4 and a pharmaceutically acceptable carrier,
(16) a pharmaceutical composition comprising an IL-5 variant that
substitutes valine for the phenylalanine residue at the position
49, 69, 96 or 103 of an amino acid sequence (SEQ ID NO.: 16) of a
wild-type IL-5 and a pharmaceutically acceptable carrier, (17) a
pharmaceutical composition comprising an IL-6 variant that
substitutes valine for the phenylalanine residue at the position
73, 77, 93, 104, 124, 169 or 172 of an amino acid sequence (SEQ ID
NO.: 17) of a wild-type IL-6 and a pharmaceutically acceptable
carrier, (18) a pharmaceutical composition comprising an IL-12p35
variant that substitutes valine for the phenylalanine residue at
the position 13, 39, 82, 96, 116, 132, 150, 166 or 180 of an amino
acid sequence (SEQ ID NO.: 18) of a wild-type IL-12p35 and a
pharmaceutically acceptable carrier, (19) a pharmaceutical
composition comprising a LPT variant that substitutes valine for
the phenylalanine residue at the position 41 or 92 of an amino acid
sequence (SEQ ID NO.: 19) of a wild-type LPT and a pharmaceutically
acceptable carrier, (20) a pharmaceutical composition comprising a
LIF variant that substitutes valine for the phenylalanine residue
at the position 41, 52, 67, 70, 156 or 180 of an amino acid
sequence (SEQ ID NO.: 20) of a wild-type LIF and a pharmaceutically
acceptable carrier, (21) a pharmaceutical composition comprising a
M-CSF variant that substitutes valine for the phenylalanine residue
at the position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255,
311, 439, 466 or 485 of an amino acid sequence (SEQ ID NO.: 21) of
a wild-type M-CSF and a pharmaceutically acceptable carrier, (22) a
pharmaceutical composition comprising an OSM variant that
substitutes valine for the phenylalanine residue at the position
56, 70, 160, 169, 176 or 184 of an amino acid sequence (SEQ ID NO.:
22) of a wild-type OSM and a pharmaceutically acceptable carrier,
(23) a pharmaceutical composition comprising a PL variant that
substitutes valine for the phenylalanine residue at the position
10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191 of an amino acid
sequence (SEQ ID NO.: 23) of a wild-type PL and a pharmaceutically
acceptable carrier, (24) a pharmaceutical composition comprising a
SCF variant that substitutes valine for the phenylalanine residue
at the position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199,
205, 207 or 245of an amino acid sequence (SEQ ID NO.: 24) of a
wild-type SCF and a pharmaceutically acceptable carrier, and (25) a
pharmaceutical composition comprising a TPO variant that
substitutes valine for the phenylalanine residue at the position
46, 128, 131, 141, 186, 204, 240 or 286 of an amino acid sequence
(SEQ ID NO.: 25) of a wild-type TPO and a pharmaceutically
acceptable carrier.
[0095] The present purpose to improve the efficacy in modulating
biological responses was accomplished in the following examples
using TPO, EPO, G-CSF and GH. It will be apparent to those skilled
in the art that the following examples are provided only to
illustrate the present invention, and the scope of the present
invention is not limited to the examples.
Example 1
Construction of DNA Coding Wild Type TPO/EPO/G-CSF/GH
[0096] A. Construction of DNA Coding Wild Type TPO
[0097] 750 .mu.l of TRIzol reagent (MRC., USA) was added to bone
marrow tissue in a microcentrifuge tube and incubated at room
temperature for 5 minutes. 200 .mu.l of chloroform was added into
the tube and then the tube was shaken vigorously for 15 seconds.
After incubating the tube at room temperature for 2-3 minutes, it
was centrifuged at 15,000 rpm for 15 minutes at 4.degree. C. The
upper phase was transferred to a 1.5 ml tube and 500 .mu.l of
isopropanol was added. The sample was incubated at -70.degree. C.
for 30 minutes and centrifuged at 15,000 rpm for 15 minutes at
4.degree. C. After discarding supernatant, RNA pellet was washed
once with 75% DEPCethanol by vortexing and centrifuged at 15,000
rpm for 15 minutes at 4.degree. C. The supernatant was removed and
the RNA pellet was dried for 5 minutes at room temperature and then
the pellet was dissolved in 50 .mu.l of DEPC-treated 3.degree.
distilled water.
[0098] 2 .mu.g of mRNA purified as above and 1 .mu.l of oligo dT30
primer (10 .mu.M, Promega, USA) were mixed and heated at 70.degree.
C. for 2 minutes and then it was immediately cooled on ice for 2
minutes. After that, this reaction mixture was added with 200 U
M-MLV reverse transcriptase (Promega, USA), 10 .mu.l of 5.times.
reaction buffer(250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM
MgCl.sub.2, 50 nM DTT), 1 .mu.l of dNTP (10 mM dATP, 10 mM dTTP, 10
mM dGTP, 10 mM dCTP) and DEPC-treated 3.degree. water was added to
make the total volume of 50 .mu.l. After mixing gently, the
reaction mixture was incubated at 42.degree. C. for 60 minutes.
[0099] To amplify cDNA coding wild type TPO, the first strand cDNA
as template, primer 1 and primer 2 (Table 1) were added into a PCR
tube including 2 U of pfu DNA polymerase (Stratagene, USA), 10
.mu.l of 10.times. reaction buffer, 1% Triton X-100, 1 mg/ml BSA,
3.mu.l of primer 1(10 .mu.M), 3 .mu.l of primer 2 (10 .mu.M), 2
.mu.l of dNTP (10 mM dATP, 10 mM dTTP, 10 mM dGTP, 10 mM dCTP), and
distilled water was added to make the total volume of 100 .mu.l.
The PCR reaction condition was as follows; 1 cycle at 95.degree. C.
for 3 minutes, and then 30 cycles at 95.degree. C. for 30 seconds,
at 52.degree. C. for 1 minute, and at 72.degree. C. for 1.5
minutes, and finally 1 cycle at 72.degree. C. for 10 minutes to
make PCR product with completely blunt end.
[0100] The PCR product obtained was separated in 0.8% agarose gel
(BMA, USA) and was purified with Qiaex II gel extraction kit
(Qiagen, USA). After the isolated DNA was mixed with 15U of EcoRI,
10 U of NotI, 3 .mu.l of 10.times. reaction buffer and 3.degree.
distilled water was added to make the total volume of 30 .mu.l, DNA
was restricted by incubation at 37.degree. C. for 2 hours. The PCR
product was separated in 0.8% agarose gel and was purified with
Qiaex II gel extraction kit.
[0101] After 5 .mu.g of pBluescript KS II(+) vector was mixed with
15 U of EcoRI, 10 U of NotI, 3 .mu.l of 10.times. reaction buffer
and 3.degree. distilled water was added to make the total volume of
30 .mu.l, DNA was restricted by incubation at 37.degree. C. for 2
hours. The restricted pBluescript KS II(+) vector was separated in
0.8% agarose gel and was purified with Qiaex II gel extraction
kit.
[0102] 100 ng of the digested pBluescript KS II(+) vector was
ligated with 20 ng of the PCR product which was digested with same
enzymes. This ligation mixture was incubated at 16.degree. C. water
bath for 16 hours, thus producing a recombinant vector comprising
cDNA coding wild type TPO. Then, it was transformed into a E. coli
Top 10 (Invitroger, USA) which was made to a competent cell by
rubidium chloride method. The transformed bacteria was cultured on
LB agar plate containing 50 .mu.g/ml of ampicillin (Sigma, USA).
After overnight incubation, colonies were transferred into tubes
with 3 ml of LB medium containing 50 .mu.g/ml ampicillin and then
they were cultured at 37.degree. C. for 16 hours. Plasmid was
isolated from the cultured bacteria with alkaline lysis method and
the restriction of EcoRI/NotI was used to detect inclusion of
cloned gene in the plasmid.
[0103] B. Construction of DNA Coding Wild Type EPO
[0104] Procedure of cloning DNA coding wild type EPO was basically
same to that used for cloning DNA coding wild type TPO.
[0105] The first strand cDNA as template, primer 11 and primer 12
(Table 2) were used for PCR amplification of DNA coding wild type
EPO. The PCR product and cloning vector, pBluescript KS II(+) were
digested with both EcoRI and BamHI endonucleases. The digested PCR
product and cloning vector were ligated and transformed into
competent cell, E. coli Top 10 (Invitrogen, USA). Plasmid was
isolated from the cultured bacteria with alkaline lysis method and
the restriction of EcoRI/BamHI was used to detect existence of
cloned gene in the plasmid
[0106] C. Construction of DNA Coding Wild Type G-CSF
[0107] Construction procedure of DNA coding wild type G-CSF was
similar to that used for DNA coding wild type TPO.
[0108] Leukocytes from healthy people were used for the mRNA
extraction, and primers 21 and 22 (Table 3) were used for PCR
amplification of cDNA coding wild type G-CSF. Both the PCR product
and cloning vector, pBluescript KS II(+) were digested with SmaI
and EcoRI endonuclease. The digested PCR product and cloning vector
were ligated and transformed into competent cell, E. coli Top 10
(Invitrogen, USA). Plasmid was isolated from the cultured bacteria
with alkaline lysis method and the restriction of SmaI/EcoRI was
used to detect existence of cloned gene in the plasmid.
[0109] D. Construction of DNA Coding Wild Type GH
[0110] DNA coding wild type GH was purchased from ATCC (ATCC No.
67097). To add leader sequence to N-terminal end of this cDNA,
primer 35 and 36 (Table 4) were used for PCR. In order to make
complete cDNA coding wild type GH linked to the leader sequence,
secondary PCR was carried out using primers 37 and 38 (Table 4).
The PCR product and cloning vector, pBluescript KS II(+) were
digested with EcoRI and HindIII endonuclease. Plasmid was isolated
from the cultured bacteria with alkaline lysis method and the
restriction of EcoRI/HindIII was used to detect existence of cloned
gene in the plasmid.
Example 2
Construction of cDNA Coding TPO/EPO/G-CSF/GH Muteins
[0111] A. Construction of cDNAs Coding TPO Muteins
[0112] Four muteins of TPO, TPO-[F46V], TPO-[F128V], TPO-[F131V]
and TPO-[F141V] were constructed according to procedures as follows
to have a single amino acid-substitution from phenylalanine to
valine at each positions, respectively.
TABLE-US-00001 TABLE 1 Primers used in constructing cDNAs coding
TPO- wild type and muteins Primer No. Nucleotide sequence Sequence
No. 1 Wild type TPO Sense 5'-CGGAATTCCGATGGAGCTGACTGAATTG-3' 26 2
Antisense 5'-TTTACCGGCCGCATTCTTACCCTTCCTGAG- 27 3' 3 TPO-[F46V]
Sense T3 4 Antisense 5'-CCAAGCTAACGTCCACAGCAG-3' 28 5 TPO-[F128V]
Sense T3 6 antisense 5'-GCTCAGGACGATGGCAT-3' 29 7 TPO-[F131V] Sense
T3 8 antisense 5'-GGTGTTGGACGCTCAGGAAGATG-3' 30 9 TPO-[F141V] Sense
T3 10 antisense 5'-CATCAGGACACGCACCTTTCC-3' 31
[0113] cDNA which code TPO-[F46V], TPO-[F128V], TPO-[F131V] and
TPO-[F141V] was constructed by primary PCR using specific primers
(Table 1) and universal primer T3 and secondary PCR using the
primary PCR product and universal primer T7. The template for these
reactions was the cDNA coding wild type TPO cloned in pBluescript
KS II(+) obtained from Example 1.
[0114] The primary PCR was performed by adding 2.5 U Ex taq
(Takara, Japan), 5 .mu.l of 10.times. buffer, 1 mM MgCl.sub.2, 2.5
mM dNTP and D.W was added to make the total volume of 50 .mu.l. The
PCR condition consisted of 1 cycle at 94.degree. C. for 3 minutes
followed by 30 cycles at 95.degree. C. for 30 seconds, at
60.degree. C. for 30 seconds and at 72.degree. C. for 30 seconds
and then linked to 1 cycle at 72.degree. C. for 7 minutes. The
primary PCR product was used as a megaprimer in the secondary PCR
together with universal primer T7 (10 pmole). The cDNA coding wild
type TPO cloned in pBluescript KS II(+) was used as the template in
the secondary PCR. The secondary PCR was performed by adding 2.5 U
Ex taq, 5 .mu.l of 10.times. buffer, 2.5 mM dNTP and D.W was added
to make the total volume of 50 .mu.l. The PCR condition consisted
of 1 cycle at 94.degree. C. for 3 minutes followed by 30 cycles at
94.degree. C. for 1 minute, at 58.degree. C. for 1 minute, and at
72.degree. C. for 1.5 minutes and finally lied to 1 cycle at
72.degree. C. for 7 minutes prior to termination.
[0115] To minimize errors derived form DNA synthesis, Mg.sup.2+
concentration was reduced to 1 mM in the primary PCR. Sizes of
megaprimers amplified were 280 b.p for TPO-[F46V], 520 b.p for
TPO-[F128V], 530 b.p for TPO-[F131V] and 560 b.p for TPO-[F141V].
In the secondary PCR using megaprimers, cDNA coding each muteins
produced showed the same size of 1062 b.p. Substitution from
phenylalanine to valine at nucleotide sequence of the individual
TPO mutein was verified by direct sequencing.
[0116] Each PCR product of 1062 b.p was separated in 0.8% agarose
gel and purified with Qiaex II gel extraction kit. The PCT product
was digested with 15 U EcoRI and 10 U NotI at 37.degree. C.
for2hours. The digested PCR product was separated in 0.8% agarose
gel and purified with Qiaex II gel extraction kit and ligated with
pBluescript KS II(+) as described above. The recombinant expression
vector containing DNA which codes TPO-[F141V] was named
Tefficacin-4 and was deposited at the KCCM (Korean Culture Center
of Microorganisms) under the Budapest Treaty on Jun. 9, 2003.
Accession number given by international depositary authority was
KCCM-10500.
[0117] B. Construction of cDNAs Coding EPO Muteins
[0118] Four muteins of EPO, EPO-[F48V], EPO-[F138V], EPO-[F142V]
and EPO-[F148V] were constructed according to procedures as follows
to have a single amino acid-substitution from phenylalanine to
valine at each positions, respectively.
TABLE-US-00002 TABLE 2 Primers used in constructing cDNAs coding
EPO- wild type and muteins Primer No. Nucleotide sequence Sequence
No. 11 Wild EPO Sense 5'-GGCGCGGAGATGGGGGT-3' 32 12 Antisense
5'-TGGTCATCTGTCCCCTGTCCTG-3' 33 13 EPO-[F48V] Sense T3 14 Antisense
5'-GACATTAACTTTGGTGTCTGGGAC-3' 34 15 EPO-[F138V] Sense
5'-CTGTCCGCAAACTCTTCCGAG-3' 35 16 Antisense T7 17 EPO-[F142V] Sense
5'-CGCAAACTCGTCCGAGTCTACT-3' 36 18 Antisense T7 19 EPO-[F148V]
Sense 5'-GAGTCTACTCCAATGTGGTGGG-3' 37 20 Antisense T7
[0119] Construction procedure of cDNA coding EPO muteins was
basically similar to that of TPOs. cDNAs which code EPO-[F48V],
EPO-[F138V], EPO-[F142V], and EPO-[F148V] were constructed by
primary PCR using specific primers (Table 2) and universal primer
T3 and secondary PCR using the primary PCR product and universal
primer T7. The template for these reactions was the cDNA coding
wild type EPO cloned in pBluescript KS II(+) obtained from Example
1.
[0120] Mg.sup.2+ concentration was adjusted to 1 mM in the primary
PCR Sizes of amplified megaprimers were 300 b.p for EPO-[F48V], 550
b.p for EPO-[F138V], 550 b.p for EPO-[F142V] and 550 b.p for
EPO-[F148V]. In the secondary PCR using the megaprimers, cDNAs
coding the individual muteins were amplified as the same size of
580 b.p. Substitution from phenylalanine to valine at nucleotide
sequence of the individual EPO mutein was verified by direct
sequencing.
[0121] Each PCR product of 580 b.p was separated in 0.8% agarose
gel and was purified with Qiaex II gel extraction kit. The PCR
product was digested with 15 U EcoRI and 10 U BamHI at 37.degree.
C. for 2 hours. The digested PCR product was ligated into
pBluescript KS II(+) as described above and was used for
constructing the expression vector. The recombinant expression
vector containing DNA which codes TPO-[F141V] was named
Refficacin-4 and was deposited at the KCCM (Korean Culture Center
of Microorganisms) under the Budapest Treaty on Jun. 9, 2003.
Accession number given by international depositary authority was
KCCM-10501.
[0122] C. Construction of cDNAs Coding G-CSF Muteins
[0123] Muteins of G-CSF, G-CSF[F13V], G-CSF[F83V], G-CSF[F113V],
G-CSF[F140V], G-CSF[F144V] and G-CSF[F160V] were constructed
according to procedures as follows to have a single amino
acid-substitution from phenylalanine to valine at each positions,
respectively.
TABLE-US-00003 TABLE 3 Primers used in constructing cDNAs coding
G-CSF- wild type and muteins Primer No. Nucleotide sequence
Sequence No. 21 wild G-CSF Sense
5'-CCCCGGGACCATGGCTGGACCTGCCACCCAG- 38 3' 22 Antisense
5'-CGAATTCGCTCAGGGCTGGGCAAGGAG-3' 39 23 G-CSF-[F13V] Sense T7 24
Antisense 5'-ACTTGAGCAGGACGCTCT-3' 40 25 G-CSF-[F83V] Sense
5'-AGCGGCCTTGTCCTCTA-3' 41 26 Antisense T3 27 G-CSF-[F113V] Sense
5'-GACGTTGCCACCACCAT-3' 42 28 Antisense T3 29 G-CSF-[F140V] Sense
5'-GCCGTCGCCTCTGCTTT-3' 43 30 Antisense T3 31 G-CSF-[F144V] Sense
5'-TCGCCTTCTGCTGTCCAG-3' 44 32 Antisense T3 33 G-CSF-[F160V] Sense
5'-TCTGCAAGACGTCCTGG-3' 45 34 Antisense T3
[0124] Construction procedure of cDNA coding G-CSF muteins was
basically similar to that of TPOs. cDNAs which code G-CSF-[F13V,
G-CSF-[F83V], G-CSF-[F113V], G-CSF-[F140V], G-CSF-[F144V], and
G-CSF-[F160V] were constructed by primary PCR using specific
primers (Table 3) and universal primer T3 and secondary PCR using
the primary PCR product and universal primer 17. The template for
these reactions was the cDNA coding wild type G-CSF cloned in
pBluescript KS II(+) obtained from the Example 1.
[0125] Mg.sup.2+ concentration was adjusted to 1 mM in the primary
PCR. Sizes of amplified megaprimers were 600 b.p for G-CSF-[F13V],
390 b.p for G-CSF-[F83V], 300 b.p for G-CSF-[F113V], 200 b.p for
G-CSF-[F140V], 200 b.p for G-CSF-[F144V], and 150 b.p for
G-CSF[F160V]. In the secondary PCR using the megaprimers, cDNAs
coding each muteins were amplified as the same size of 640 b.p.
Substitution from phenylalanine to valine at nucleotide sequence of
the individual G-CSF mutein was verified by direct sequencing.
[0126] Each PCR product of 640 b.p was separated in 0.8% agamse gel
and purified with Qiaex II gel extraction kit. The PCR product was
digested with 15 U SmaI and 10 U EcoRI at 37.degree. C. for 2 hours
and separated in 0.8% agarse gel and purified with Qiaex II gel
extraction kit. The digested PCR product was ligated into
pBluescript KS II(+) as described above. The recombinant expression
vector containing DNA which codes G-CSF-[F140V] was named
Grefficacin4 and was deposited at the KCCM (Korean Culture Center
of Microorganisms) under the Budapest Treaty on May 17, 2004.
Accession number given by international depositary authority was
KCCM-10571.
[0127] D. Construction of cDNAs Coding GH Muteins
[0128] Four muteins of GH, GH-[F44V], GH-[F97V], GH-[F139V],
GH-[F146V], GH-[F166V], and GH-[F176V] were constructed according
to procedures as follows to have a single amino acid-substitution
from phenylalanine to valine at each positions, respectively.
TABLE-US-00004 TABLE 4 Primers used in constructing cDNAs coding
GH- wild type and muteins Sequence Primer No. Nucleolide Sequence
No. 35 Leader Sense-1 5'-CTTTTGGCCTGCTCTGCCTGTCCTGGCTTCAA 46
sequence GAGGGCAGTGCCTTCCAACCATTCCCTTATC-3' 36 addition Antisense
T3 37 Sense-2 5'-GGAATTCATGGCTGCAGGCTCCCGGACGTCC 47
CTGCTCCTGGCTTTTGGCCTGCTCTGCCT-3' 38 Antisense T3 39 GH- Sense T7 40
[F44V] Antisense 5'-GGGGTTCTGCAGGACTGAATACTTC-3' 48 41 GH- Sense T7
42 [F97V] Antisense 5'-GGCTGTTGGCGACGATCCTG-3' 49 43 GH- Sense T7
44 [F139V] Antisense 5'-GTAGGTCTGCTTGACGATCTGCCCAG-3' 50 45 GH-
Sense T7 46 [F146V] Antisense 5'-GAGTTTGTGTCGACCTTGCTGTAG-3' 51 47
GH- Sense T7 48 [F166V] Antisense 5'-GTCCTTCCTGACGCAGTAGAGCAG-3' 52
49 GH- Sense T7 50 [F176V] Antisense
5'-CGATGCGCAGGACTGTCTCGACCTTGTC-3' 53
[0129] Construction procedure of cDNA coding GH muteins was
basically similar to that of TPOs. cDNAs which code muteins
GH-[F44V), GH-[F97V], GH-[F139V], GH-[F146l7, GH-[F166V] and
GH-[F176V] were constructed by primary PCR using specific primers
(Table 4) and universal primer T3 and secondary PCR using the
primary PCR product and universal primer T7. The template for these
reactions was the cDNA coding wild type GH cloned in pBluescript KS
II(+) obtained from Example 1.
[0130] Mg.sup.2+ concentration was adjusted to 1 mM in the primary
PCR. Sizes of each amplified megaprimers were 130 b.p for
GH-[F44V], 300 b.p for GH-[F97V], 420 b.p for GH-[F139V], 450 b.p
for GH-[F146V], 500 b.p for GH-[F166V] and 530 b.p for GH-[F176V]
PCRs. Substitution from phenylalanine to valine at nucleotide
sequence of the individual GH mutein was verified by direct
sequencing.
[0131] Each PCR product of 650 b.p was separated in 0.8% agarose
gel and purified with Qiaex II gel extraction kit. The PCR product
was digested with 15 U EcoRI and 10 U HindIII at 37.degree. C. for
2 hours and separated in 0.8% agarose gel and purified with Qiaex
II gel extraction kit. The digested PCR product was ligated into
pBluescript KS II(+) as described above.
Example 3
Expression and Purification of TPO Muteins
[0132] A. TPO Muteins
[0133] a. Establishments of Transfected Cell Lines by Using
Lipofection Method
[0134] Chinese hamster ovary ("CHO-K1")(ATCC, CCL61) cells were
prepared at a density 1.5.times.10.sup.5 cells per 35 mm dish
containing Dulbecco's modified Eagle's medium ("DMEM") [Gibco BRL,
USA] supplemented with 10% fetal bovine serum ("FBS"). The cells
were grown at 37.degree. C. in a 5% CO.sub.2 for 18-24 hrs. 6 .mu.l
of Lipofectanine was added to 1.5 .mu.g of the recombinant
expression vector comprising DNA coding TPO mutein in a sterile
tube. Volume of this mixture was adjusted to 100 .mu.l by adding
serum-free DMEM. The tube was incubated at room temperature for 45
min. The cells grown in 35 mm dish were washed twice with
serum-free DMEM and 800 .mu.l of serum-free DMEM was added to the
dish. The washed cells were gently overlaid on the
lipofectamine-DNA complex and then incubated for 5 hrs at
37.degree. C. in 5% CO.sub.2. After 5 hrs incubation, 1 ml of DMEM
containing 20% FBS was added to transfected cells and then the
cells were incubated for 18-24 hrs at 37.degree. C., 5% CO.sub.2.
After the incubation, the cells were washed twice with serum-free
DMEM and then 2 ml of DMEM containing 10% FBS was added to the
culture. These cells were incubated for 72 hrs at 37.degree. C., 5%
CO.sub.2.
[0135] b. Analysis of Expression Level of TPO Muteins Using
ELISA
[0136] The cells transfected with plasmid containing cDNA coding
TPO-wild type or muteins were analyzed on their protein expression
level by using ELISA assay. An goat anti-human TPO polyclonal
antibody (R&D, U.S.A) diluted to 10 .mu.g/ml with coating
buffer [0.1M Sodium bicarbonate, (pH 9.6)] was added into each
wells of 96 well plate (Falcon, USA) up to 100 .mu.l per well and
incubated for 1 hour at room temperature. The plate was washed with
0.1% Tween-20 in 1.times. PBS (PBST) three times. After washing,
the plate was incubated with 200 .mu.l of blocking buffer (1% FBS,
5% sucrose, 0.05% sodium azide) for 1 hour at room temperature and
then washed three times with PBST. The cultured superuatants
(icluding the transfected cells) and dilution buffer [0.1% BSA,
0.05% Tween-20, 1.times. PBS] were mixed with serial dilutions. 25
ng/ml of recombinant human TPO [Calbiocher, USA] as a positive
control and untransfected CHO-K1cultured supcnatants as a negative
control were equally diluted. These controls and samples were
incubated for 1 hr at room temperature. Then, the plate was washed
with PBST three times. A biotinylated goat anti-human TPO antibody
(R&D, USA) diluted to 0.2 .mu.g/ml with dilution buffer was
added to the 96 well plate up to 100 .mu.l per well and incubated
for 1 hr at room temperature. The plate was washed with PBST three
times. Streptavidin-HRP (R&D, USA) diluted to 1:200 in dilution
buffer was added 100 .mu.l per well to the 96 well plate and
incubated for 1 hr at room temperature. After 1 hour, the plates
was washed three times with PBST, and then coloring reaction was
performed by using TMB microwell peroxidase substrate system (KPL,
USA) and O.D was read at 630 nm with microplate reader [BIO-RAD,
Model 550].
[0137] c. Analysis of Expression Level and Molecular Weight of
Muteim TPO using Western Blotting
[0138] In order to exclude FBS in medium, CHOS-S-SFM II (Gibco BRL,
USA) was used for culture of the above-transfected cell. Culture
medium from CHOS-SFM II was filtrated with 0.2 .mu.m syringe filter
and concentrated with centricon (Mol. 30,000 Millipore, USA). To
perform the reduced SDS-PAGE, sample-loading buffer containing 5%
.beta.-mercaptoethanol was added to the sample and heated for 5
minutes. Stacking gel and running gel were used for this SDS-PAGE.
The stacking gel was composed of 3.5% acrylamide, 0.375 M Tris
(pH6.8), 0.4% SDS and the running gel was composed of 10%
acrylamide gel, 1.5 M Tris (pH8.8), 0.4% SDS. After SDS-PAGE gel
runing treatment, protein samples were transferred to Westran (PVDF
transfermembbrane, S&S) having 4 .mu.m pore at 350 mA for 2 hrs
in a 25 mM Tris-192 mM glycine (pH 8.3) -20% methanol
buffer-containg reservoir. After transferring, it was blocked three
times for 10 minutes with 5% fat free milk powder in PBST. The
biotinylated goat anti-human TPO antibody (R&D, USA) was
diluted to 0.25 .mu.g/ml in blocking buffer and 3 ml of this
solution was added and shaken for 6 hrs. The membrane was washed
with washing solution three times. Streptavidin-HRP (R&D, USA)
was diluted to 1:100 in blocking buffer and incubated for 1 hr. The
membrane was washed three times with washing solution. Protein
bands were visualized by incubating with DAB substrate (VECTOR
LABORATORIES, USA) for 10 minutes. This reaction was stopped with
soaking the membrane in deionized water.
[0139] In FIG. 2a, wild type and mutein forms of TPOs had the same
molecular weight (55 kD).
[0140] Relative expression level of wild type and muteins of TPO
was shown in FIG. 3a. Expression level of each TPO mutein was
compared to that of wild type TPO as a control. Expression level of
TPO-[F128V] was increased 1.4 times more than that of wild type
TPO. But expressions of TPO-[F46V], -[F113V] and -[F141V] were deed
to 20%, 40% , and 40% of that of wild type, respectively.
[0141] B. EPO Muteins
[0142] Expression vectors containing cDNAs coding EPO muteins were
transfected to CHO-K1 cell and expression level of each of EPO
mutein was detected by using ELISA assay. And molecular weight of
each of wild type and mutein of EPO was analyzed by western
blotting.
[0143] In FIG. 2b, wild type and mutein forms of EPO had the same
molecular weight (45 kD).
[0144] Relative expression level of wild type and muteins of EPO
was shown in FIG. 3b. Expressions level of EPO-[F48V] and -[F138V]
was increased 1.4 and 1.2 times more than that of the wild type
EPO, respectively. But expression level of EPO-[F142V] and -[F148V]
was deceased to 20% and 30% of that of wild type EPO,
respectively.
[0145] C. G-CSF Muteins
[0146] Expression vectors containing cDNAs coding G-CSF muteins
were transfected to CHO-K1 cell and expression level of each G-CSF
mutein was detected by using ELISA assay. And molecular weight of
each of wild type and muteins of G-CSF was analyzed by western
blotting.
[0147] In FIG. 2c, wild type and mutein forms of G-CSF had the same
molecular weight (50 kD).
[0148] Relative expression level of wild types and muteins of G-CSF
was shown in FIG. 3c. Expression levels of rest of G-CSF muteins
were similar to that of wild type G-CSF. Expression level of G-CSF
mutein-[F83V] was increased 1.9 times than that of wild-type. But
expression levels of G-CSF muteins -[F140V] and -[F144V] were
decreased to 50% and 70% of that of wild type G-CSF,
respectively.
[0149] D. GH Muteins
[0150] Expression vectors containing cDNAs coding GH muteins were
transfected to CHO-K1 cell. Method for the expression of each of
the GH muteins was the same as those used for TPO production.
Example 4
Construction of DNA Coding EPO, TPO, G-CSF, and GH Receptors
[0151] A. Construction of DNA Coding EPO and TPO Receptors
[0152] DNAs coding EPO and TPO receptors were constructed to
analyze binding affinities of each of EPO muteins and TPO muteins.
DNA coding extracellular domain of each receptor was linked to DNA
coding Fc domain of IgG1 such that the C-terminal region of
extracellular domain of each receptor was fused to N-terminal
region of human IgG1 Fc domain. cDNA coding EPO receptor was
constructed by PCR using sense primer (primer 51) with restriction
sites of EcoRI and leader sequence of EPO receptor and antisense
primer (primer 52) with the sequence coding 3' end of EPO receptor
and the sequence coding 5' end Fc domain of IgG. cDNA coding TPO
receptor linked to Fc domain of IgG1 was constructed by PCR using
sense primer (primer 53) with restriction sites of HindIII and
leader sequence of TPO receptor and antisense primer (primer 54)
with the sequence coding 3' end of TPO receptor and the sequence
coding 5' end of Fc domain of IgG.
[0153] cDNA coding EPO receptor produced as described above and DNA
coding Fc domain of IgG1 were mixed in the same tube, complementary
binding between the common sequences was induced. Using this
mixture, cDNA coding EPO receptor linked to Fc domain of IgG1 was
constructed by PCR using sense primer (primer 51) with restriction
sites of EcoRI and leader sequence of EPO receptor and antisense
primer (primer 55) with restriction sites of XbaI and 3' end of Fc
domain of IgG. The PCR product was cut with EcoRI and XbaI and
inserted into PCR-3 expression vector for production of EPO
receptor-Fc fusion protein.
[0154] cDNA coding TPO receptor produced as described above and DNA
coding Fc domain of IgG1 were mixed in the same tube, thus
complementary binding between the common sequences was induced.
Using this mixture, cDNA coding TPO receptor linked to Fc domain of
IgG1 was constructed by PCR using sense primer (primer 53) with
restriction sites of EcoRI and leader sequence of EPO receptor and
antisense primer (primer 55) with restriction sites of XbaI and 3'
end of Fc domain of IgG. The PCR product was cut with HindIII and
XbaI and inserted into PCR-3 expression vector for production of
TPO receptor-Fc fusion protein.
TABLE-US-00005 TABLE 5 A List of primers used in constructing TPO
and EPO receptors fused to immunoglobulin Primer Sequence No.
Nucleotide sequence No. EPO 51 Sense
5'-CGGAATTCATGGACCACCTCGGGGCG-3' 54 receptor 52 Antisense
5'-GCTCTAGACTAAGAGCAAGCCACATAGCTGGG-3' 55 TPO 53 Sense
5'-CCCAAGCTTATGGAGCTGACTGAATTGCTCCTC-3' 56 receptor 54 Antisense
5'-GGAATTCTTACCCTTCCTGAGACAGATTCTGG-3' 57 IgG1-R- 55
5'-GCTCTAGAGCTCATTTACCCGGAGACAGGGAGAG-3' 58 XbaI
[0155] B. Construction of DNA Coding G-CSF and GH Receptors
[0156] cDNA coding G-CSF receptor was constructed by PCR using
sense primer (primer 56) with restriction site of HindIII and
leader sequence of G-CSF receptor and antisense primer (primer 57)
with restriction site of EcoRI and the sequence coding 3' end of
G-CSF receptor. cDNA coding GH receptor was constructed by PCR
using sense primer (primer 58) with restriction site of EcoRI and
leader sequence of G-CSF receptor and antisense primer (primer 59)
with restriction site of SpeI and the sequence coding 3' end of
G-CSF receptor.
[0157] The PCR product encoding G-CSF receptor was digested with
HindIII and EcoRI, and was cloned by inserting into a commercially
available cloning vector, pBluescript KS II(+) at HindIII/EcoRI
site. The PCR product encoding GH receptor was digested with EcoRI
and SpeI, and cloned by inserting into a commercially available
cloning vector, pBluescript KS II(+) at EcoRI/SpeI site.
[0158] Fc domain of human IgG was constructed by PCR using sense
primer (primer 60 for G-CSF, primer 61 for GH) with sequence coding
5' end part of hinge region of human IgG and antisense primer
(primer 62). For G-CSF receptor, the PCR product coding Fc domain
of human IgG was digested with EcoRI and XbaI, and cloned by
inserting into a commercially available cloning vector, pBluescript
KS II(+) at EcoRI/XbaI site. For GH receptor, the PCR product
coding Fc domain of human IgG was digested with SpeI and XbaI, and
cloned by inserting into a commercially available cloning vector,
pBluescript KS II(+) at SpeI site/XbaI.
[0159] Both of the cloned cDNA coding G-CSF receptor and the cloned
Fc domain of human IgG were digested with EcoRI/XbaI and then
ligated to prepare DNA coding G-CSF receptor linked to Fc domain of
human IgG. This DNA construct was cut with HindIII and XbaI and
inserted into PCR-3 expression vector. Both of the cloned cDNA
coding GH receptor and the cloned Fc domain of human IgG were
digested with SpeI/XbaI and then ligated to prepare DNA coding
G-CSF receptor linked to Fc domain of human IgG. This DNA construct
was cut with EcoRI and XbaI and inserted into PCR-3 expression
vector.
TABLE-US-00006 TABLE 6 A List of primers used in constructing G-CSF
and GH receptors fused to Immunoglobulin Primer Sequence No.
Nucleotide sequence No. G-CSF 56 Sense
5'-CCCAAGCTTATGGCTGGACCTGCCACCC-3' 59 receptor 57 Antisense
5'-GGAATTCGCAACAGAGCCAGGCAGTTCCA-3 60 GH 58 Sense
5'-CGGAATTCATGGATCTCTGGCAGCTG-3' 61 receptor 59 Antisense
5'-GGACTAGTTTGGCTCATCTGAGGAAGTG-3' 62 IgG1-F- 60 Sense
5'-GGAATTCGCAGAGCCCAAATCTTGTGACAAAACTC-3' 63 EcoR I IgG1-F- 61
Sense 5'-GACTAGTGCAGAGCCCAAATCTTGTGA-3' 64 Spe I IgG1-R- 62
Antisense 5'-GCTCTAGAGCTCATTTACCCGGAGACAGGGAGAG-3' 65 XbaI
Example 5
Measurement of Binding Affinity of Cytokines and their Muteins to
Each of Their Receptors by Using ELISA
[0160] A. Binding of TPO and TPO Muteins to TPO Receptor
[0161] Culture supernatants of CHO cell transfected with expression
vectors caring genes for TPO muteins were used for measuring
cytokine-receptor interactions.
[0162] TPO receptor-Ig fusion protein was purified from culture
supernatant of CHO cell transfected with recombinant expression
vector carrying gene coding for TPO receptor-Fc fusion protein by
using Protein A Sepharose-4B column (Pharmacia, Sweden). The
purified fusion protein diluted to 10 .mu.g/ml with coating buffer
[0.1M Sodium bicarbonate, (pH 9.6)] was added into each wells of 96
well plate (Falcon, USA) up to 100 .mu.l per well and incubated for
1 hour at room temperature. The plate was washed with 0.1% Tween-20
in 1.times. PBS[PBST] three times. After washing, the plate was
incubated with 200 .mu.l of blocking buffer (1% FBS, 5% sucrose,
0.05% sodium azide) for 1 hour at room temperature and then washed
three times with PBST.
[0163] After washing, culture supematants consising of four TPO
muteins and one TPO wild type, respectively were diluted serially
with dilution buffer [0.1% BSA, 0.05% Tween-20, 1.times. PBS] and
was added to 96 well plate coated with the TPO receptor-Fc fusion
protein and incubated for 1 br. The washing was repeated three
times with PBST. A recombinant human TPO [Calbiochem, USA] as a
positive control and untransfected CHO-K1 cultured supernatants as
a negative control were equally diluted. The plates were washed
with PBST three times. A biotinylated goat anti-human TPO antibody
(R&D, USA) diluted to 0.2 .mu.g/ml in dilution buffer was added
to the 96 well plate to 100 .mu.l per well and incubated for 1 hr
at room temperature. The plate was washed with PBST three times.
Streptavidin-HRP (R&D, USA) diluted to 1:200 in dilution buffer
was added 100 .mu.l per well to 96 well plate and incubated for 1
hr at room temperature. The plate was washed three times with PBST
after 1 hour. Coloring reaction was performed using TMB microwell
peroxidase substrate system (KPL, USA) and O.D was read at 630 nm
with microplate reader [BIO-RAD, Model 550].
[0164] The binding affinity of TPO-[F141V] and TPO-[F131V] to the
TPO receptor was increased compared to that of wild type TPO (FIG.
4a). And the former mutein had the strongest binding affinity among
all TPO muteins.
[0165] B. Binding of EPO and EPO Muteins to EPO Receptor
[0166] Measurement of binding affinity of EPO wild type and muteims
to the receptor was basically similar to that of binding affinity
of TPO and TPO muteins to TPO Receptor.
[0167] The binding affinity of EPO-[F148V] and EPO-[F142V] to the
EPO receptor was increased compared to that of wild type EPO (FIG.
4b). And the former mutein had the strongest binding affinity among
all EPO muteins.
[0168] C. Binding of G-CSF and G-CSF Muteins to G-CSF Receptor
[0169] Measurement of binding affinity of G-CSF wild type and
muteins to the receptor was basically similar to that of binding
affinity of TPO and TPO muteins to TPO Receptor.
[0170] Results (FIG. 4c) showed binding affinity of G-CSF-[F140V],
G-CSF-[F144V], and G-CSF-[F160V] to the G-CSF receptor was
increased compared to that of wild type G-CSF. And the first mutein
(G-CSF-[F140V]) had the strongest binding affinity among all G-CSF
muteins.
[0171] D. Binding of GH and GH Muteins to GH Receptor
[0172] Measurement of binding affinity of GH wild type and muteins
to the receptor was basically similar to that of binding affinity
of TPO and TPO muteins to TPO Receptor.
[0173] Results (FIG. 4d) showed that GH-[F139V] had the strongest
binding affinity to the GH receptor.
Example 6
Measurement of Bindings of Cytokines and their Muteins to Each of
Their Receptors by Using SPR
[0174] A. Binding of TPO and TPO Muteins to TPO Receptor
[0175] To measure the binding affinity of TPO-[F141V] and
TPO-[F131V] to TPO receptor, SPR was performed on a BIAcore 3000
instrument containing CM5 sensor chip. Anti-human IgG antibody was
immobilized onto each flow cells 1 and 2 using amine-coupling
chemistry. To inactivate any active group, surfaces were blocked
with 1 M ethanolamine. TPO receptor-Fc fusion protein was added to
bind to the anti-human IgG antibody for 2 mm at 30 .mu.l/min and
then TPO and TPO muteins were reacted to bind to the TPO
receptor.
[0176] At the same density of ligand, increased resonance unit (RU)
means higher binding affinities. In FIG. 5a, wild TPO, TPO-[F141V]
and TPO-[F131V] were 10RU, 30RU and 20RU, respectively. This result
showed that TPO-[F141V] had the strongest binding affinity. In
addition, K.sub.D values of wild type and mutein TPO were shown in
Table 7.
TABLE-US-00007 TABLE 7 Changes of Binding-kinetic rate constant of
wild type and mutein TPO Relative Binding
K.sub.on(M.sup.-1s.sup.-1) .times. 10.sup.5 K.sub.off(S.sup.-1)
.times. 10.sup.-2 K.sub.D(.mu.M) = K.sub.off/K.sub.on Chi.sup.2
affinity Wild type TPO 2.42 13.7 5.66 5.81 1 TPO-[F141] 12.8 0.51
0.04 6.03 141
[0177] B. EPO Muteins
[0178] SPR was performed to measure binding affinities of EPO
mutein-[F148V] and EPO-[F142V] with EPO receptor. Experimental
procedure was similar to that for TPOs.
[0179] FIG. 5b was the SPR result of EPO wild type and muteins. In
FIG. 5b, EPO[F148V] showed 40RU, EPO-[F142V] 30RU. These results
show that EPO-[F148V] had the strongest binding affinity. In
addition, K.sub.D values of EPO muteins were shown in Table 8.
TABLE-US-00008 TABLE 8 Changes of Binding-kinetic rate constant of
wild type and mutein EPO Relative Binding
K.sub.on(M.sup.-1s.sup.-1) .times. 10.sup.5 K.sub.off(S.sup.-1)
.times. 10.sup.-2 K.sub.D(.mu.M) = K.sub.off/K.sub.on Chi.sup.2
affinity Wild type EPO 1.84 8.83 4.80 4.55 1 EPO-[F148] 14.0 0.64
0.05 2.26 105
Example 7
Measurement of Binding Affinities of Wild Type- and Muteins of
Cytokine by Using FACS
[0180] A. Establishment of TF-1/c-Mpl Cell Line
[0181] TF-1/c-Mpl cell line was established by transfecting cDNA
coding c-Mpl into TF-1 cell. Expression of c-Mpl was verified by
using FACS analysis. The 1.times.10.sup.6/ml of the TF-1/c-Mpl
cells was washed with PBS buffer and purified c-Mpl mouse
anti-human monoclonal antibody (BD PharMingen, USA) was incubated
with the TF-1/c-Mpl cells. And then FITC-conjugated anti-mouse IgG
(whole molecule; Sigma, USA) was added to verify expression of
c-Mpl on surface of the TF-1/c-Mpl cells. As a result, graph of the
TF-1/c-Mpl cell was shifted rightward from that of TF-1 cells. This
result showed that c-Mpl, TPO receptor, was expressed on the
TF-1/c-Mpl cell.
[0182] B. FACS Analysis of TPO Muteins
[0183] The 1.times.10.sup.6/ml of TF-1/c-Mpl cell was suspended in
PBS buffer and TPO wild type and -[F141V] was added to the
suspension and incubated at 4.degree. C. for 30-60 minutes,
respectively. Biotinylated goat anti-human TPO polyclonal antibody
(R&D, USA) was added to the cells above and incubated at
4.degree. C. for 30-60 minutes. Streptavidin-FITC (Sigma, USA) was
added to the cells above and incubated at 4.degree. C. for 30-60
minutes. The cells were washed twice with PBS buffer to remove
non-reacted Streptavidin-FITC. The cells were suspended in PBS
buffer and flow cytometric analysis was performed at 488 nm using
EXCALIBUR (BD, U.S.A).
[0184] In FIG. 6a, a binding curve of TPO-[F141V] was shifted
rightward from that of wild type TPO. This result showed that
TPO-[F141V] had much stronger receptor-binding affinity than the
wild type TPO.
[0185] C. FACS Analysis of EPO Muteins
[0186] FACS procedure of EPO muteins was carried out similarly to
that of TPO.
[0187] In FIG. 6b, a binding curve of EPO-[F148V] was shifted
rightward from that of wild type TPO. This result showed that
TPO-[F141V] is much stronger in receptor-binding affinity than the
wild type EPO.
Example 8
Measurement of Biological Activities of TPO, EPO, G-CSF and GH
Muteins
[0188] A. Cell Proliferation Assay of TPO Muteins
[0189] To investigate differences of cell proliferation and
biological activities between TPO-wild type and muteins, TF-1/c-Mpl
cell line produced above was used. TF-1/c-Mpl cells were grown in
DMEM medium supplemented with 10% fetal bovine serum, 1 ng/ml
GM-CSF at 37.degree. C., 5% CO.sub.2. 0.4, 1,5, 10, 20, 40, 75
ng/ml of each of TPO-wild type and muteins in RPMI-1640 were seeded
in 96-well tissue-culture plates (FALCON, USA). 1.times.10.sup.4
cell of the TF-1/c-Mpl cells in RPMI-1640 containing 10% fetal
bovine serun was added to each wells of the 96-well plate. After 4
days cultivation at 37.degree. C., 5% CO.sub.2, 20 .mu.l of MTS
solution
[3-(4,5-dimethyl-2-yl)-5-3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetr-
azolium, inner salt, MTS] and the phenazine ethosulfate (PES;
promega) was added and incubated for 4 hours. O.D. was measured
with microplate reader (BIO-RAD Model 550) at 490 nm.
[0190] FIG. 7a showed differences of TPO wild type and muteins in
stimulating TF-1/c-Mpl cell proliferation. TPO was applied to the
TF-1/c-Mpl from 0.4 ng/ml to 75 ng/ml. Cell proliferation was
increased up to 50 ng/ml of TPO concentration. TF-1/c-Mpl cell
proliferation potential of TPO-[F141V] was much stronger than that
of wild type and was the first in biological activity among TPO
muteins. Biological activity of TPO-[F131V] was the second
strongest among TPO muteins. Activity of TPO-[F46V] was similar to
that of wild type.
[0191] B. Cell Proliferation Assay of EPO Muteins
[0192] Biological activity for EPO muteins was examined by cell
proliferation assay using EPO-dependent TF-1 cell. Experimental
procedure of cell proliferation assay of EPO muteins was similar to
that of TPO muteins.
[0193] FIG. 7b showed differences of EPO wild type and muteins in
stimulating TF-1 cell proliferation EPO was applied to the TF-1
Cell from 0.011 IU/ml to 7 IU/ml. TF-1 cell proliferation potential
of EPO-[F148V] was much stronger than that of the wild type and was
the fist in biological strength among EPO muteins. Biological
activities of EPO-[F142V] and EPO[F138V] were the second and the
third strongest among EPO muteins, respectively.
TABLE-US-00009 TABLE 9 Biological activities of TPOs The maximum
activity TPO comparison (%) Wild type 100 Muteins TPO-[F46V] 107
TPO-[F128V] 63 TPO-[F131V] 119 TPO-[F141V] 146
TABLE-US-00010 TABLE 10 Biological activities of EPOs The maximum
activity EPO comparison (%) Wild type 100 Muteins EPO-[F48V] 84
EPO-[F138V] 57 EPO-[F142V] 122 EPO-[F148V] 137
[0194] C. G-CSF Muteins
[0195] Biological activity for G-CSF muteins was examined by cell
proliferation assay using G-CSF dependent HL-60 cell. Experimental
procedure of cell proliferation assay of G-CSF muteins was similar
to that of TPO muteins.
[0196] FIG. 7c showed differences of G-CSF wild type and muteins in
stimulating HL-60 cell proliferation. G-CSF was applied to the
HL-60 Cell from 0.4 ng/ml to 75 ng/ml. HL-60 cell proliferation
potential of G-CSF-[F140V] was much stronger than Mat of the wild
type and was the first in biological strength among G-CSF
muteins.
[0197] D. GH Muteins
[0198] Biological activity for GH muteins was examined by cell
proliferation assay using GH dependent NB2 cell. Experimental
procedure of cell proliferation assay of GH muteins was similar to
that of GH muteins.
[0199] FIG. 7d showed differences of GH wild type and muteins in
stimulating NB2 cell proliferation. GH was applied to the NB2 Cell
from 0.4 ng/ml to 75 ng/ml. NB2 cell proliferation potential of
GH-[F139V] was much stronger than that of the wild type and was the
first in biological strength among GH muteins.
Example 9
Pharmacokinetic Profiles of EPO- and TPO-Wild Types and Muteins
[0200] Difference of pharmacokinetic profiles of each EPO- and
TPO-muteins between their wildtype was investigated. TPO or TPO
muteins was injected intravenously into rabbits New Zealand White,
3 kg). And then blood samples were collected serially. EPO and TPO
concentrations from each samples were detected by using
quantitative ELISA assay as described above. Injection of EPOs into
mice (12 weeks, Balb/c, 30 g) was performed by both
intraperitonealy and intravenously. Blood samples in
heparin-containing tubes were separated by centrifugation at 3,000
rpm for 10 minutes. Supernatant containing plasma was used to
detect blood concentrations of EPO and TPO by using ELISA.
[0201] After intravenous injection of 5 .mu.g/kg of TPO wild type
and -[F141V] into rabbit, plasma concentration profiles of TPO wild
type and -[F141V] were shown in FIG. 8a. Concentration of
TPO-[F141V] was decreased more rapidly than that of wild type TPO.
TPO-[F141V] was shined from blood to peripheral target tissues more
rapidly, due to its stronger binding affinity to receptor.
[0202] After intravenous injection of 1000 I.U/kg of wild type EPO
and EPO-[F148V] into rabbit, plasma concentration profiles of wild
type EPO and EPO-F148V] in blood were shown in FIG. 8b.
Concentration of EPO-[F148V] was decreased more rapidly than that
of EPO wild type.
[0203] After intraperitoneal injection of 20 I.U/g of wild type EPO
and EPO-[F148V] into mice, plasma concentration profiles were shown
in FIG. 8c. The diffusion velocity of EPO wild type was higher than
that of EPO-[F148V] at early stage and maximum concentration in
blood (Cmax) of wild type EPO was also higher than that of
EPO-[F148V]. Cmax of EPO-[F48V] remained longer than wild type EPO.
These results suggested that EPO-[F148V] was more hydrophobic and
had higher binding affinity to receptor than the wild type EPO. And
these results lead to the conclusion that EPO-[F148V] was diffused
into blood more slowly and shifted from blood to peripheral target
tissues more quickly than those of wild type EPO.
TABLE-US-00011 TABLE 11 Pharmacokinetic parameters of EPO wild type
and EPO-[F148V] mutein Mouse Rabbit Wild type EPO-mutein Wild type
EPO-mutein EPO [F148V] EPO [F148V] T.sub.1/2(Half life) 1.9 1.4 3.8
2.4 AUC 100 78 100 80
Example 10
In vivo Activities of EPO Muteins
[0204] Difference of biological activities between EPO-wild type
and muteins was verified in mice. Mice (12 weeks Balb/c, 20 g,
Jungang Lab Animal Inc., Korea) were .gamma.-irradiated at 700 Rad.
250 ng of purified EPO wild type and muteins in 50 .mu.l of PBS
were injected intraperitoneally 3 times everyday. Blood samples
were collected from their tail vein. And then hematologic
parameters were tested according to ordinary CBC test. Wild type
EPO was used as a positive control and CHO cell culture supernatant
was used as a negative control. Blood was collected into tubes
containing EDTA at 0, 1st, 2nd, 4th, 7th, 10th, 15th,20th, 25th,
and 30th days after the injection.
[0205] FIG. 9 showed that CBC results in mice injected
intraperitoneally with EPO-wild type and muteins to verify change
in count of RBC and reticulocyte. Increase of RBC count (FIG. 9a)
was much more remarkable in EPO[F148V]-injected mice than mice
injected with wild type EPO. And the RBC increase of in EPO[F48V]-
and EPO[138V]-injected mice was weaker than that of mice injected
with wild type EPO. Increase of reticulocyte count (FIG. 9b) and
hematocrit was similar to the result of RBC count change in mice
injected with EPO-[F148V].
Example 11
In vivo Activities of TPO Muteins
[0206] Difference of biological activities between TPO-wild type
and muteins was studied in mice. Mice (12 weeks Balb/c, 20 g,
Jungang Lab Animal Inc., Korea) were .gamma.-irradiated at 700 Rad.
250 ng of purified TPO wild type and muteins in 50 .mu.l of PBS
were injected intraperitoneally 3 times everyday. Blood samples
were collected from their tail vein. And then hematologic
parameters were tested according to ordinary CBC test. Wild type
TPO was used as a positive control and CHO cell culture supernatant
was used as a negative control. Blood was collected into tubes
containing EDTA at 0, 1st, 4th, 7th, 10th, 14th, 18th 23rd, 28th,
and 32nd days after injection.
[0207] FIG. 10 showed the changes of platelet count (FIG. 10a),
leukocyte count (FIG. 10b), and neutrophil count (FIG. 10c) in mice
injected intraperitoneally with TPO-wild type and muteins. Increase
of platelet count was the most remarkable in mice injected with
TPO-[F141V]. And mice injected with TPO-[F131V] was the second
highest. Mice injected with TPO-[F46V] was similar to those
injected with wild type TPO. And mice injected with TPO-[F128V]
showed platelet count similar to that of negative controls injected
with PBS (FIG. 10a). Increase of leukocyte count (FIG. 10b) and
neutrophil count (FIG. 10c) showed similar patterns as those seen
in platelet change.
INDUSTRIAL APPLICABILITY
[0208] As apparent from the above results of the present invention,
valine substitution for phenylalanine residue, which is present in
a domain participating in the binding of conventional wild-type
biological response-modulating proteins to corresponding receptors,
ligands or substrates, leads to an increase in binding affinity and
biological activity, and reduces the production of autoantibodies
to conventional protein variants, thereby making it possible to
produce improved protein drugs.
Sequence CWU 1
1
651200PRTArtificial SequenceCNTF 3rd, 83rd, 98th, 105th, 119th,
152nd or 178th Phe is replaced by Val. 1Met Ala Phe Thr Glu His Ser
Pro Leu Thr Pro His Arg Arg Asp Leu 1 5 10 15Cys Ser Arg Ser Ile
Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr 20 25 30Ala Leu Thr Glu
Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile 35 40 45Asn Leu Asp
Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp 50 55 60Ser Glu
Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr 65 70 75
80Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr
Leu 100 105 110Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu
Leu Met Ile 115 120 125Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala
Asp Gly Met Pro Ile 130 135 140Asn Val Gly Asp Gly Gly Leu Phe Glu
Lys Lys Leu Trp Gly Leu Lys145 150 155 160Val Leu Gln Glu Leu Ser
Gln Trp Thr Val Arg Ser Ile His Asp Leu 165 170 175Arg Phe Ile Ser
Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His 180 185 190Tyr Ile
Ala Asn Asn Lys Lys Met 195 2002166PRTArtificial SequenceEPO 48th,
138th, 142nd or 148th Phe is replcaced by Val. 2Ala Pro Pro Arg Leu
Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15Leu Glu Ala
Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30Cys Ser
Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45Tyr
Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55
60Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His
Val Asp 85 90 95Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu
Arg Ala Leu 100 105 110Arg Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp
Ala Ala Ser Ala Ala 115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr
Phe Arg Lys Leu Phe Arg Val 130 135 140Tyr Ser Asn Phe Leu Arg Gly
Lys Leu Lys Leu Tyr Thr Gly Glu Ala145 150 155 160Cys Arg Thr Gly
Asp Arg 1653209PRTArtificial SequenceFlt3L 6th, 15th, 81st, 87th,
96th or 124th Phe is replaced by Val. 3Thr Gln Asp Cys Ser Phe Gln
His Ser Pro Ile Ser Ser Asp Phe Ala 1 5 10 15Val Lys Ile Arg Glu
Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val 20 25 30Thr Val Ala Ser
Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp 35 40 45Arg Leu Val
Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala 50 55 60Gly Ser
Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His 65 70 75
80Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln
Leu 100 105 110Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser
Arg Cys Leu 115 120 125Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu
Pro Pro Pro Trp Ser 130 135 140Pro Arg Pro Leu Glu Ala Thr Ala Pro
Thr Ala Pro Gln Pro Pro Leu145 150 155 160Leu Leu Leu Leu Leu Leu
Pro Val Gly Leu Leu Leu Leu Ala Ala Ala 165 170 175Trp Cys Leu His
Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly 180 185 190Glu Gln
Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu Val Glu 195 200
205His4174PRTArtificial SequenceG-CSF 13rd, 83rd, 113rd, 140th,
144th or 160th Phe is replaced by Val. 4Thr Pro Leu Gly Pro Ala Ser
Ser Leu Pro Gln Ser Phe Leu Leu Lys 1 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 Ser 65 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 1705127PRTArtificial
SequenceGM-CSF 47th, 103rd, 106th, 113rd or 119th Phe is replaced
by Val. 5Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu
His Val 1 5 10 15Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu
Ser Arg Asp Thr 20 25 30Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile
Ser Glu Met Phe Asp 35 40 45Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg
Leu Glu Leu Tyr Lys Gln 50 55 60Gly Leu Arg Gly Ser Leu Thr Lys Leu
Lys Gly Pro Leu Thr Met Met 65 70 75 80Ala Ser His Tyr Lys Gln His
Cys Pro Pro Thr Pro Glu Thr Ser Cys 85 90 95Ala Thr Gln Ile Ile Thr
Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp 100 105 110Phe Leu Leu Val
Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120
1256191PRTArtificial SequenceGH 1st, 10th, 25th, 31st, 44th, 54th,
92th, 97th, 139th, 146th, 166th, 176th or 191st Phe is replaced by
Val. 6Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu
Arg 1 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 Leu 65 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 1907165PRTArtificial SequenceIFN-alpha 2A 27th,
36th, 38th, 43rd, 47th, 64th, 67th, 84th, 123rd or 151st Phe is
replaced by Val. 7Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg
Arg Thr Leu Met 1 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 Leu 65 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 1658165PRTArtificial
SequenceIFN-alpha 2B 27th, 36th, 38th, 43rd, 47th, 64th, 67th,
84th, 123rd or 151st Phe is replaced by Val. 8Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15Leu Leu Ala
Gln Met Arg Arg 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 Leu
65 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 1659166PRTArtificial SequenceIFN-Beta 8th, 38th, 50th, 67th,
70th, 111st or 154th Phe is replaced by Val. 9Met Ser Tyr Asn Leu
Leu Gly Phe Leu Gln Arg Ser Ser Asn Cys Gln 1 5 10 15Cys Gln Lys
Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu 20 25 30Lys Asp
Arg Arg Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln 35 40 45Gln
Phe Gln Lys Glu Asp Ala Ala Val Thr Ile Tyr Glu Met Leu Gln 50 55
60Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn
65 70 75 80Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln
Arg Asn 85 90 95His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu
Asp Phe Thr 100 105 110Arg Gly Lys Arg Met Ser Ser Leu His Leu Lys
Arg Tyr Tyr Gly Arg 115 120 125Ile Leu His Tyr Leu Lys Ala Lys Glu
Asp Ser His Cys Ala Trp Thr 130 135 140Ile Val Arg Val Glu Ile Leu
Arg Asn Phe Tyr Val Ile Asn Arg Leu145 150 155 160Thr Gly Tyr Leu
Arg Asn 16510146PRTArtificial SequenceIFN-gamma 18th, 32nd, 55th,
57th, 60th, 63rd, 84th, 85th, 95th or 139th Phe is replaced by Val.
10Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys 1
5 10 15Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu
Phe 20 25 30Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys
Ile Met 35 40 45Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys
Asn Phe Lys 50 55 60Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile
Lys Glu Asp Met 65 70 75 80Asn Val Lys Phe Phe Asn Ser Asn Lys Lys
Lys Arg Asp Asp Phe Glu 85 90 95Lys Leu Thr Asn Tyr Ser Val Thr Asp
Leu Asn Val Gln Arg Lys Ala 100 105 110Ile His Glu Leu Ile Gln Val
Met Ala Glu Leu Ser Pro Ala Ala Lys 115 120 125Thr Gly Lys Arg Lys
Arg Ser Gln Met Leu Phe Gln Gly Arg Arg Ala 130 135 140Ser
Gln14511172PRTArtificial SequenceIFN-omega 27th, 36th, 38th, 65th,
68th, 124th or 153rd Phe is replaced by Val. 11Cys Asp Leu Pro Gln
Asn His Gly Leu Leu Ser Arg Asn Thr Leu Val 1 5 10 15Leu Leu His
Gln Met Arg Arg Ile Ser Pro Phe Leu Cys Leu Lys Asp 20 25 30Arg Arg
Asp Phe Arg Phe Pro Gln Glu Met Val Lys Gly Ser Gln Leu 35 40 45Gln
Lys Ala His Val Met Ser Val Leu His Glu Met Leu Gln Gln Ile 50 55
60Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala Ala Trp Asn Met Thr
65 70 75 80Leu Leu Asp Gln Leu His Thr Gly Leu His Gln Gln Leu Gln
His Leu 85 90 95Glu Thr Cys Leu Leu Gln Val Val Gly Glu Gly Glu Ser
Ala Gly Ala 100 105 110Ile Ser Ser Pro Ala Leu Thr Leu Arg Arg Tyr
Phe Gln Gly Ile Arg 115 120 125Val Tyr Leu Lys Glu Lys Lys Tyr Ser
Asp Cys Ala Trp Glu Val Val 130 135 140Arg Met Glu Ile Met Lys Ser
Leu Phe Leu Ser Thr Asn Met Gln Glu145 150 155 160Arg Leu Arg Ser
Lys Asp Arg Asp Leu Gly Ser Ser 165 17012187PRTArtificial
SequenceIFN-tau 8th, 39th, 68th, 71st, 88th, 127th, 156th, 157th,
159th or 183rd Phe is replaced by Val. 12Leu Asp Leu Lys Leu Ile
Ile Phe Gln Gln Arg Gln Val Asn Gln Glu 1 5 10 15Ser Leu Lys Leu
Leu Asn Lys Leu Gln Thr Leu Ser Ile Gln Gln Cys 20 25 30Leu Pro His
Arg Lys Asn Phe Leu Leu Pro Gln Lys Ser Leu Ser Pro 35 40 45Gln Gln
Tyr Gln Lys Gly His Thr Leu Ala Ile Leu His Glu Met Leu 50 55 60Gln
Gln Ile Phe Ser Leu Phe Arg Ala Asn Ile Ser Leu Asp Gly Trp 65 70
75 80Glu Glu Asn His Thr Glu Lys Phe Leu Ile Gln Leu His Gln Gln
Leu 85 90 95Glu Tyr Leu Glu Ala Leu Met Gly Leu Glu Ala Glu Lys Leu
Ser Gly 100 105 110Thr Leu Gly Ser Asp Asn Leu Arg Leu Gln Val Lys
Met Tyr Phe Arg 115 120 125Arg Ile His Asp Tyr Leu Glu Asn Gln Asp
Tyr Ser Thr Cys Ala Trp 130 135 140Ala Ile Val Gln Val Glu Ile Ser
Arg Cys Leu Phe Phe Val Phe Ser145 150 155 160Leu Thr Glu Lys Leu
Ser Lys Gln Gly Arg Pro Leu Asn Asp Met Lys 165 170 175Gln Glu Leu
Thr Thr Glu Phe Arg Ser Pro Arg 180 18513133PRTArtificial
SequenceIL-2 42nd, 44th, 78th, 103rd, 117th or 124th Phe is
replaced by Val. 13Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu
Gln Leu Glu His 1 5 10 15Leu Leu Leu Asp Leu Gln Met Ile Leu Asn
Gly Ile Asn Asn Tyr Lys 20 25 30Asn Pro Lys Leu Thr Arg Met Leu Thr
Phe Lys Phe Tyr Met Pro Lys 35 40 45Lys Ala Thr Glu Leu Lys His Leu
Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60Pro Leu Glu Glu Val Leu Asn
Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80Arg Pro Arg Asp Leu
Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95Lys Gly Ser Glu
Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110Thr Ile
Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120
125Ile Ser Thr Leu Thr 13014133PRTArtificial SequenceIL-3 37th,
61st, 107th, 113rd or 133rd Phe is replaced by Val. 14Ala Pro Met
Thr Gln Thr Thr Pro Leu Lys Thr Ser Trp Val Asn Cys 1 5 10 15Ser
Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu 20 25
30Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu
35 40 45Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg
Ala 50 55 60Val Lys Ser Leu
Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn 65 70 75 80Leu Leu
Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro 85 90 95Ile
His Ile Lys Asp Gly Asp Trp Asn Glu Phe Arg Arg Lys Leu Thr 100 105
110Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu
115 120 125Ser Leu Ala Ile Phe 13015129PRTArtificial SequenceIL-4
33rd, 45th, 55th, 73rd, 82nd or 112nd Phe is replaced by Val. 15His
Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser 1 5 10
15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile
20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg
Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp
Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys
Gln Leu Ile 65 70 75 80Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp
Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro Val Lys Glu Ala Asn Gln
Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu Arg Leu Lys Thr Ile Met
Arg Glu Lys Tyr Ser Lys Cys Ser 115 120 125Ser16115PRTArtificial
SequenceIL-5 49th, 69th, 96th or 103rd Phe is replaced by Val.
16Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1
5 10 15Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu
Arg 20 25 30Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr Glu
Glu Ile 35 40 45Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln
Gly Gly Thr 50 55 60Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys
Lys Tyr Ile Asp 65 70 75 80Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg
Arg Arg Val Asn Gln Phe 85 90 95Leu Asp Tyr Leu Gln Glu Phe Leu Gly
Val Met Asn Thr Glu Trp Ile 100 105 110Ile Glu Ser
11517183PRTArtificial SequenceIL-6 73rd, 77th, 93rd, 104th, 124th,
169th or 172nd Phe is replaced by Val. 17Val Pro Pro Gly Glu Asp
Ser Lys Asp Val Ala Ala Pro His Arg Gln 1 5 10 15Pro Leu Thr Ser
Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu 20 25 30Asp Gly Ile
Ser Ala Leu Arg Lys Glu Thr Cys Asn Lys Ser Asn Met 35 40 45Cys Glu
Ser Ser Lys Glu Ala Leu Ala Glu Asn Asn Leu Asn Leu Pro 50 55 60Lys
Met Ala Glu Lys Asp Gly Cys Phe Gln Ser Gly Phe Asn Glu Glu 65 70
75 80Thr Cys Leu Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val
Tyr 85 90 95Leu Glu Tyr Leu Gln Asn Arg Phe Glu Ser Ser Glu Glu Gln
Ala Arg 100 105 110Ala Val Gln Met Ser Thr Lys Val Leu Ile Gln Phe
Leu Gln Lys Lys 115 120 125Ala Lys Asn Leu Asp Ala Ile Thr Thr Pro
Asp Pro Thr Thr Asn Ala 130 135 140Ser Leu Leu Thr Lys Leu Gln Ala
Gln Asn Gln Trp Leu Gln Asp Met145 150 155 160Thr Thr His Leu Ile
Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser 165 170 175Leu Arg Ala
Leu Arg Gln Met 18018197PRTArtificial SequenceIL-12p35 13rd, 39th,
82nd, 96th, 116th, 132nd, 150th, 166th or 180th Phe is replaced by
Val. 18Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys
Leu 1 5 10 15His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met
Leu Gln Lys 20 25 30Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser
Glu Glu Ile Asp 35 40 45His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr
Val Glu Ala Cys Leu 50 55 60Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys
Leu Asn Ser Arg Glu Thr 65 70 75 80Ser Phe Ile Thr Asn Gly Ser Cys
Leu Ala Ser Arg Lys Thr Ser Phe 85 90 95Met Met Ala Leu Cys Leu Ser
Ser Ile Tyr Glu Asp Leu Lys Met Tyr 100 105 110Gln Val Glu Phe Lys
Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys 115 120 125Arg Gln Ile
Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu 130 135 140Met
Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser145 150
155 160Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile
Leu 165 170 175Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg
Val Met Ser 180 185 190Tyr Leu Asn Ala Ser 19519146PRTArtificial
SequenceLPT 41st or 92nd Phe is replaced by Val. 19Val Pro Ile Gln
Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 15Ile Val
Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser 20 25 30Lys
Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 35 40
45Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile
50 55 60Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp
Leu 65 70 75 80Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser
Lys Ser Cys 85 90 95His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp
Ser Leu Gly Gly 100 105 110Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu
Val Val Ala Leu Ser Arg 115 120 125Leu Gln Gly Ser Leu Gln Asp Met
Leu Trp Gln Leu Asp Leu Ser Pro 130 135 140Gly
Cys14520180PRTArtificial SequenceLIF 41st, 52nd, 67th, 70th, 156th
or 180th Phe is replaced by Val. 20Ser Pro Leu Pro Ile Thr Pro Val
Asn Ala Thr Cys Ala Ile Arg His 1 5 10 15Pro Cys His Asn Asn Leu
Met Asn Gln Ile Arg Ser Gln Leu Ala Gln 20 25 30Leu Asn Gly Ser Ala
Asn Ala Leu Phe Ile Leu Tyr Tyr Thr Ala Gln 35 40 45Gly Glu Pro Phe
Pro Asn Asn Leu Asp Lys Leu Cys Gly Pro Asn Val 50 55 60Thr Asp Phe
Pro Pro Phe His Ala Asn Gly Thr Glu Lys Ala Lys Leu 65 70 75 80Val
Glu Leu Tyr Arg Ile Val Val Tyr Leu Gly Thr Ser Leu Gly Asn 85 90
95Ile Thr Arg Asp Gln Lys Ile Leu Asn Pro Ser Ala Leu Ser Leu His
100 105 110Ser Lys Leu Asn Ala Thr Ala Asp Ile Leu Arg Gly Leu Leu
Ser Asn 115 120 125Val Leu Cys Arg Leu Cys Ser Lys Tyr His Val Gly
His Val Asp Val 130 135 140Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp
Val Phe Gln Lys Lys Lys145 150 155 160Leu Gly Cys Gln Leu Leu Gly
Lys Tyr Lys Gln Ile Ile Ala Val Leu 165 170 175Ala Gln Ala Phe
18021522PRTArtificial SequenceM-CSF 35th, 37th, 54th, 67th, 91st,
106th, 121st, 135th, 143rd, 229th, 255th, 311st, 439th, 466th or
485th Phe is replaced by Val. 21Glu Glu Val Ser Glu Tyr Cys Ser His
Met Ile Gly Ser Gly His Leu 1 5 10 15Gln Ser Leu Gln Arg Leu Ile
Asp Ser Gln Met Glu Thr Ser Cys Gln 20 25 30Ile Thr Phe Glu Phe Val
Asp Gln Glu Gln Leu Lys Asp Pro Val Cys 35 40 45Tyr Leu Lys Lys Ala
Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr 50 55 60Met Arg Phe Arg
Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu 65 70 75 80Gln Glu
Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu 85 90 95Glu
His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln 100 105
110Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu
115 120 125Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser
Phe Ala 130 135 140Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro Asp
Cys Asn Cys Leu145 150 155 160Tyr Pro Lys Ala Ile Pro Ser Ser Asp
Pro Ala Ser Val Ser Pro His 165 170 175Gln Pro Leu Ala Pro Ser Met
Ala Pro Val Ala Gly Leu Thr Trp Glu 180 185 190Asp Ser Glu Gly Thr
Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro 195 200 205Leu His Thr
Val Asp Pro Gly Ser Ala Lys Gln Arg Pro Pro Arg Ser 210 215 220Thr
Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser225 230
235 240Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser Val Gly Ala Phe
Asn 245 250 255Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met Gly Thr
Asn Trp Val 260 265 270Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile
Pro Val Pro Gln Gly 275 280 285Thr Glu Leu Ser Pro Ser Arg Pro Gly
Gly Gly Ser Met Gln Thr Glu 290 295 300Pro Ala Arg Pro Ser Asn Phe
Leu Ser Ala Ser Ser Pro Leu Pro Ala305 310 315 320Ser Ala Lys Gly
Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu Pro 325 330 335Arg Val
Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn His Thr Pro 340 345
350Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro
355 360 365Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln Gly Leu Ser
Asn Pro 370 375 380Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Arg Ser
His Ser Ser Gly385 390 395 400Ser Val Leu Pro Leu Gly Glu Leu Glu
Gly Arg Arg Ser Thr Arg Asp 405 410 415Arg Arg Ser Pro Ala Glu Pro
Glu Gly Gly Pro Ala Ser Glu Gly Ala 420 425 430Ala Arg Pro Leu Pro
Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly 435 440 445His Glu Arg
Gln Ser Glu Gly Ser Ser Ser Pro Gln Leu Gln Glu Ser 450 455 460Val
Phe His Leu Leu Val Pro Ser Val Ile Leu Val Leu Leu Ala Val465 470
475 480Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu
Pro 485 490 495Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser
Pro Leu Thr 500 505 510Gln Asp Asp Arg Gln Val Glu Leu Pro Val 515
52022227PRTArtificial SequenceOSM 56th, 70th, 160th, 169th, 176th
or 184th Phe is replaced by Val. 22Ala Ala Ile Gly Ser Cys Ser Lys
Glu Tyr Arg Val Leu Leu Gly Gln 1 5 10 15Leu Gln Lys Gln Thr Asp
Leu Met Gln Asp Thr Ser Arg Leu Leu Asp 20 25 30Pro Tyr Ile Arg Ile
Gln Gly Leu Asp Val Pro Lys Leu Arg Glu His 35 40 45Cys Arg Glu Arg
Pro Gly Ala Phe Pro Ser Glu Glu Thr Leu Arg Gly 50 55 60Leu Gly Arg
Arg Gly Phe Leu Gln Thr Leu Asn Ala Thr Leu Gly Cys 65 70 75 80Val
Leu His Arg Leu Ala Asp Leu Glu Gln Arg Leu Pro Lys Ala Gln 85 90
95Asp Leu Glu Arg Ser Gly Leu Asn Ile Glu Asp Leu Glu Lys Leu Gln
100 105 110Met Ala Arg Pro Asn Ile Leu Gly Leu Arg Asn Asn Ile Tyr
Cys Met 115 120 125Ala Gln Leu Leu Asp Asn Ser Asp Thr Ala Glu Pro
Thr Lys Ala Gly 130 135 140Arg Gly Ala Ser Gln Pro Pro Thr Pro Thr
Pro Ala Ser Asp Ala Phe145 150 155 160Gln Arg Lys Leu Glu Gly Cys
Arg Phe Leu His Gly Tyr His Arg Phe 165 170 175Met His Ser Val Gly
Arg Val Phe Ser Lys Trp Gly Glu Ser Pro Asn 180 185 190Arg Ser Arg
Arg His Ser Pro His Gln Ala Leu Arg Lys Gly Val Arg 195 200 205Arg
Thr Arg Pro Ser Arg Lys Gly Lys Arg Leu Met Thr Arg Gly Gln 210 215
220Leu Pro Arg22523191PRTArtificial SequencePL 10th, 31st, 44th,
52nd, 54th, 92nd, 97th, 146th, 166th, 176th or 191st Phe is
replaced by Val. 23Val Gln Thr Val Pro Leu Ser Arg Leu Phe Asp His
Ala Met Leu Gln 1 5 10 15Ala His Arg Ala His Gln Leu Ala Ile Asp
Thr Tyr Gln Glu Phe Glu 20 25 30Glu Thr Tyr Ile Pro Lys Asp Gln Lys
Tyr Ser Phe Leu His Asp Ser 35 40 45Gln Thr Ser Phe Cys Phe Ser Asp
Ser Ile Pro Thr Pro Ser Asn Met 50 55 60Glu Glu Thr Gln Gln Lys Ser
Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80Leu Leu Ile Glu Ser
Trp Leu Glu Pro Val Arg Phe Leu Arg Ser Met 85 90 95Phe Ala Asn Asn
Leu Val Tyr Asp Thr Ser Asp Ser Asp Asp Tyr His 100 105 110Leu Leu
Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120
125Glu Asp Gly Ser Arg Arg Thr Gly Gln Ile Leu Lys Gln Thr Tyr Ser
130 135 140Lys Phe Asp Thr Asn Ser His Asn His 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 Met Val Gln Cys Arg Ser Val
Glu Gly Ser Cys Gly Phe 180 185 19024248PRTArtificial SequenceSCF
63rd, 102nd, 110th, 115th, 116th, 119th, 126th, 129th, 158th,
199th, 205th, 207th or 245th Phe is replaced by Val. 24Glu Gly Ile
Cys Arg Asn Arg Val Thr Asn Asn Val Lys Asp Val Thr 1 5 10 15Lys
Leu Val Ala Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys Tyr 20 25
30Val Pro Gly Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu Met
35 40 45Val Val Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe
Ser 50 55 60Asn Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys
Leu Val 65 70 75 80Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu
Asn Ser Ser Lys 85 90 95Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro
Arg Leu Phe Thr Pro 100 105 110Glu Glu Phe Phe Arg Ile Phe Asn Arg
Ser Ile Asp Ala Phe Lys Asp 115 120 125Phe Val Val Ala Ser Glu Thr
Ser Asp Cys Val Val Ser Ser Thr Leu 130 135 140Ser Pro Glu Lys Asp
Ser Arg Val Ser Val Thr Lys Pro Phe Met Leu145 150 155 160Pro Pro
Val Ala Ala Ser Ser Leu Arg Asn Asp Ser Ser Ser Ser Asn 165 170
175Arg Lys Ala Lys Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala
180 185 190Met Ala Leu Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala
Phe Gly 195 200 205Ala Leu Tyr Trp Lys Lys Arg Gln Pro Ser Leu Thr
Arg Ala Val Glu 210 215 220Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu
Ile Ser Met Leu Gln Glu225 230 235 240Lys Glu Arg Glu Phe Gln Glu
Val 24525332PRTArtificial SequenceTPO 46th, 128th, 131st, 141st,
186th, 204th, 240th or 286th Phe is replaced by Val. 25Ser Pro Ala
Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu 1 5 10 15Arg
Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val 20 25
30His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu
35 40 45Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile
Leu 50 55 60Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg
Gly Gln 65 70 75 80Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln
Leu Ser Gly Gln 85 90 95Val Arg Leu Leu Leu Gly Ala Leu Gln Ser
Leu Leu Gly Thr Gln Leu 100 105 110Pro Pro Gln Gly Arg Thr Thr Ala
His Lys Asp Pro Asn Ala Ile Phe 115 120 125Leu Ser Phe Gln His Leu
Leu Arg Gly Lys Val Arg Phe Leu Met Leu 130 135 140Val Gly Gly Ser
Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr Ala145 150 155 160Val
Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn 165 170
175Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr
180 185 190Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala
Lys Ile 195 200 205Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp
Gln Ile Pro Gly 210 215 220Tyr Leu Asn Arg Ile His Glu Leu Leu Asn
Gly Thr Arg Gly Leu Phe225 230 235 240Pro Gly Pro Ser Arg Arg Thr
Leu Gly Ala Pro Asp Ile Ser Ser Gly 245 250 255Thr Ser Asp Thr Gly
Ser Leu Pro Pro Asn Leu Gln Pro Gly Tyr Ser 260 265 270Pro Ser Pro
Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu 275 280 285Pro
Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro 290 295
300Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn
Thr305 310 315 320Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly
325 3302628DNAArtificial Sequenceprimer 1 26cggaattccg atggagctga
ctgaattg 282730DNAArtificial Sequenceprimer 2 27tttagcggcc
gcattcttac ccttcctgag 302821DNAArtificial Sequenceprimer 4
28ccaagctaac gtccacagca g 212917DNAArtificial Sequenceprimer 6
29gctcaggacg atggcat 173023DNAArtificial Sequenceprimer 8
30ggtgttggac gctcaggaag atg 233121DNAArtificial Sequenceprimer 10
31catcaggaca cgcacctttc c 213217DNAArtificial Sequenceprimer 11
32ggcgcggaga tgggggt 173322DNAArtificial Sequenceprimer 12
33tggtcatctg tcccctgtcc tg 223424DNAArtificial Sequenceprimer 14
34gacattaact ttggtgtctg ggac 243521DNAArtificial Sequenceprimer 15
35ctgtccgcaa actcttccga g 213622DNAArtificial Sequenceprimer 17
36cgcaaactcg tccgagtcta ct 223722DNAArtificial Sequenceprimer 19
37gagtctactc caatgtggtg gg 223831DNAArtificial Sequenceprimer 21
38ccccgggacc atggctggac ctgccaccca g 313927DNAArtificial
Sequenceprimer 22 39cgaattcgct cagggctggg caaggag
274018DNAArtificial Sequenceprimer 24 40acttgagcag gacgctct
184117DNAArtificial Sequenceprimer 25 41agcggccttg tcctcta
174217DNAArtificial Sequenceprimer 27 42gacgttgcca ccaccat
174317DNAArtificial Sequenceprimer 29 43gccgtcgcct ctgcttt
174418DNAArtificial Sequenceprimer 31 44tcgccttctg ctgtccag
184517DNAArtificial Sequenceprimer 33 45tctgcaagac gtcctgg
174664DNAArtificial Sequenceprimer 35 46cttttggcct gctctgcctg
tcctggcttc aagagggcag tgccttccca accattccct 60tatc
644760DNAArtificial Sequenceprimer 37 47ggaattcatg gctgcaggct
cccggacgtc cctgctcctg gcttttggcc tgctctgcct 60 604825DNAArtificial
Sequenceprimer 40 48ggggttctgc aggactgaat acttc 254920DNAArtificial
Sequenceprimer 42 49ggctgttggc gacgatcctg 205026DNAArtificial
Sequenceprimer 44 50gtaggtctgc ttgacgatct gcccag
265124DNAArtificial Sequenceprimer 46 51gagtttgtgt cgaccttgct gtag
245224DNAArtificial Sequenceprimer 48 52gtccttcctg acgcagtaga gcag
245328DNAArtificial Sequenceprimer 50 53cgatgcgcag gactgtctcg
accttgtc 285426DNAArtificial Sequenceprimer 51 54cggaattcat
ggaccacctc ggggcg 265532DNAArtificial Sequenceprimer 52
55gctctagact aagagcaagc cacatagctg gg 325633DNAArtificial
Sequenceprimer 53 56cccaagctta tggagctgac tgaattgctc ctc
335732DNAArtificial Sequenceprimer 54 57ggaattctta cccttcctga
gacagattct gg 325834DNAArtificial Sequenceprimer 55 58gctctagagc
tcatttaccc ggagacaggg agag 345928DNAArtificial Sequenceprimer 56
59cccaagctta tggctggacc tgccaccc 286029DNAArtificial Sequenceprimer
57 60ggaattcgca acagagccag gcagttcca 296126DNAArtificial
Sequenceprimer 58 61cggaattcat ggatctctgg cagctg
266228DNAArtificial Sequenceprimer 59 62ggactagttt ggctcatctg
aggaagtg 286335DNAArtificial Sequenceprimer 60 63ggaattcgca
gagcccaaat cttgtgacaa aactc 356427DNAArtificial Sequenceprimer 61
64gactagtgca gagcccaaat cttgtga 276534DNAArtificial Sequenceprimer
62 65gctctagagc tcatttaccc ggagacaggg agag 34
* * * * *