U.S. patent application number 14/705241 was filed with the patent office on 2015-10-15 for human neutrophil gelatinase-associated lipocalin (hngal) muteins that bind hepcidin and nucleic acid encoding such.
This patent application is currently assigned to PIERIS AG. The applicant listed for this patent is PIERIS AG. Invention is credited to RACHIDA SIHAM BEL AIBA, HANS-JUERGEN CHRISTIAN, HENDRIK GILLE, ANDREAS HOHLBAUM, MARTIN HUELSMEYER, KRISTIAN JENSEN, GABRIELE MATSCHINER, ARNE SKERRA, STEFAN TRENTMANN.
Application Number | 20150291675 14/705241 |
Document ID | / |
Family ID | 44630372 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150291675 |
Kind Code |
A1 |
TRENTMANN; STEFAN ; et
al. |
October 15, 2015 |
HUMAN NEUTROPHIL GELATINASE-ASSOCIATED LIPOCALIN (HNGAL) MUTEINS
THAT BIND HEPCIDIN AND NUCLEIC ACID ENCODING SUCH
Abstract
The present invention relates to novel, specific-binding
therapeutic and/or diagnostic proteins directed against Hepcidin,
which proteins preferably are muteins of lipocalin protein. The
invention also relates to nucleic acid molecules encoding such
proteins and to methods for generation and use of such proteins and
nucleic acid molecules. Accordingly, the invention also is directed
to pharmaceutical and/or diagnostic compositions comprising such a
lipocalin proteins, including uses of these proteins.
Inventors: |
TRENTMANN; STEFAN;
(ALLERSHAUSEN, DE) ; MATSCHINER; GABRIELE;
(MUNICH, DE) ; SKERRA; ARNE; (FREISING, DE)
; HOHLBAUM; ANDREAS; (PAUNZHAUSEN, DE) ;
HUELSMEYER; MARTIN; (WOLFERSDORF, DE) ; GILLE;
HENDRIK; (MUNICH, DE) ; CHRISTIAN; HANS-JUERGEN;
(MOOSBURG, DE) ; JENSEN; KRISTIAN; (LANDSHUT,
DE) ; AIBA; RACHIDA SIHAM BEL; (MUNICH, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIERIS AG |
FREISING-WEIHENSTEPHAN |
|
DE |
|
|
Assignee: |
PIERIS AG
FREISING-WEIHENSTEPHAN
DE
|
Family ID: |
44630372 |
Appl. No.: |
14/705241 |
Filed: |
May 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13816808 |
May 24, 2013 |
9051382 |
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PCT/EP2011/064086 |
Aug 16, 2011 |
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14705241 |
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61374199 |
Aug 16, 2010 |
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Current U.S.
Class: |
514/1.2 ;
435/252.3; 435/69.1; 514/21.2; 530/359; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 2318/20 20130101; C07K 2317/92 20130101; C07K 14/47 20130101;
C07K 16/26 20130101; C07K 14/435 20130101; A61P 3/00 20180101; A61P
29/00 20180101; C07K 2317/76 20130101 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Claims
1-78. (canceled)
79. A method of producing a lipocalin mutein that is capable of
binding hepcidin with an affinity by a KD of about 10 nM or lower,
wherein the lipocalin mutein comprises: (i) a set of mutated amino
acid residues at the sequence positions 96, 100, and/or 106 of the
linear polypeptide sequence of mature human neutrophil
gelatinase-associated lipocalin (hNGAL), selected from the group
consisting of (a) Asn 96.fwdarw.Val, Tyr 100.fwdarw.Gln, and Tyr
106.fwdarw.unchanged (b) Asn 96.fwdarw.Arg, Tyr 100.fwdarw.Glu, and
Tyr 106.fwdarw.Phe, (c) Asn 96.fwdarw.Asp, Tyr 100.fwdarw.Ser, and
Tyr 106.fwdarw.Gly, (d) Asn 96.fwdarw.Gly, Tyr 100.fwdarw.Gly, and
Tyr 106.fwdarw.Gly, (e) Asn 96.fwdarw.Lys, Tyr 100.fwdarw.Ala, and
Tyr 106.fwdarw.Ile, (f) Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Arg, and
Tyr 106.fwdarw.Val, (g) Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Val, and
Tyr 106.fwdarw.Arg, and (h) Asn 96.fwdarw.Thr, Tyr 100.fwdarw.Val,
and Tyr 106.fwdarw.Gly; and (ii) at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated amino acid residues at
any of the sequence positions corresponding to the sequence
positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 103, 125,
127, 132, and 134 of the linear polypeptide sequence of mature
hNGAL, wherein the lipocalin mutein is produced starting from the
nucleic acid coding for the lipocalin mutein by means of genetic
engineering methods.
80. A pharmaceutical or diagnostic composition comprising a
lipocalin mutein that is capable of binding hepcidin with an
affinity by a KD of about 10 nM or lower, wherein the lipocalin
mutein comprises: (i) a set of mutated amino acid residues at the
sequence positions 96, 100, and/or 106 of the linear polypeptide
sequence of mature human neutrophil gelatinase-associated lipocalin
(hNGAL), selected from the group consisting of (a) Asn
96.fwdarw.Val, Tyr 100.fwdarw.Gln, and Tyr 106.fwdarw.unchanged (b)
Asn 96.fwdarw.Arg, Tyr 100.fwdarw.Glu, and Tyr 106.fwdarw.Phe, (c)
Asn 96.fwdarw.Asp, Tyr 100.fwdarw.Ser, and Tyr 106.fwdarw.Gly, (d)
Asn 96.fwdarw.Gly, Tyr 100.fwdarw.Gly, and Tyr 106.fwdarw.Gly, (e)
Asn 96.fwdarw.Lys, Tyr 100.fwdarw.Ala, and Tyr 106.fwdarw.Ile, (f)
Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Arg, and Tyr 106.fwdarw.Val, (g)
Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Arg, and
(h) Asn 96.fwdarw.Thr, Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Gly;
and (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, or 17 mutated amino acid residues at any of the sequence
positions corresponding to the sequence positions 36, 40, 41, 49,
52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the
linear polypeptide sequence of mature hNGAL, and a pharmaceutically
acceptable excipient.
81. A lipocalin mutein that comprises an amino acid sequence as set
forth in any one of SEQ ID NOs: 1-14.
82. A lipocalin mutein that is capable of binding hepcidin with an
affinity by a KD of about 10 nM or lower, wherein the mutein has at
least 90% sequence identity to an amino acid sequence as set forth
in any one of SEQ ID NOs: 1-14.
83. The lipocalin mutein according to claim 81, wherein the
lipocalin mutein is fused at its N-terminus and/or its C-terminus
to a fusion partner which is a protein, or a protein domain or a
peptide.
84. The lipocalin mutein according to claim 81, wherein the mutein
is conjugated to a compound that extends the serum half-life of the
mutein.
85. A nucleic acid molecule comprising a nucleotide sequence
encoding the lipocalin mutein according to claim 81.
86. An isolated host cell containing a nucleic acid molecule of
claim 85.
87. The lipocalin mutein according to claim 82, wherein the
lipocalin mutein is fused at its N-terminus and/or its C-terminus
to a fusion partner which is a protein, or a protein domain or a
peptide.
88. The lipocalin mutein according to claim 82, wherein the mutein
is conjugated to a compound that extends the serum half-life of the
mutein.
89. A nucleic acid molecule comprising a nucleotide sequence
encoding the lipocalin mutein according to claim 82.
90. An isolated host cell containing a nucleic acid molecule of
claim 89.
91. A method of forming a complex with hepcidin and inhibiting the
ability of hepcidin to bind to or interact with ferroportin in a
subject, comprising the step of administrating a lipocalin mutein
that is capable of binding hepcidin with an affinity by a KD of
about 10 nM or lower to the subject, wherein the lipocalin mutein
comprises: (i) a set of mutated amino acid residues at the sequence
positions 96, 100, and/or 106 of the linear polypeptide sequence of
mature human neutrophil gelatinase-associated lipocalin (hNGAL),
selected from the group consisting of (a) Asn 96.fwdarw.Val, Tyr
100.fwdarw.Gln, and Tyr 106.fwdarw.unchanged (b) Asn 96.fwdarw.Arg,
Tyr 100.fwdarw.Glu, and Tyr 106.fwdarw.Phe, (c) Asn 96.fwdarw.Asp,
Tyr 100.fwdarw.Ser, and Tyr 106.fwdarw.Gly, (d) Asn 96.fwdarw.Gly,
Tyr 100.fwdarw.Gly, and Tyr 106.fwdarw.Gly, (e) Asn 96.fwdarw.Lys,
Tyr 100.fwdarw.Ala, and Tyr 106.fwdarw.Ile, (f) Asn 96.fwdarw.Ser,
Tyr 100.fwdarw.Arg, and Tyr 106.fwdarw.Val, (g) Asn 96.fwdarw.Ser,
Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Arg, and (h) Asn
96.fwdarw.Thr, Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Gly; and (ii)
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or
17 mutated amino acid residues at any of the sequence positions
corresponding to the sequence positions 36, 40, 41, 49, 52, 68, 70,
72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the linear
polypeptide sequence of mature hNGAL.
92. A method of treatment or diagnosis of a disease or disorder
involving a disorder of iron homeostasis or an inflammatory
condition associated with an elevated level of hepcidin, comprising
the step of administering to a subject in need thereof a lipocalin
mutein that is capable of binding hepcidin with an affinity by a KD
of about 10 nM or lower to the subject, wherein the lipocalin
mutein comprises: (i) a set of mutated amino acid residues at the
sequence positions 96, 100, and/or 106 of the linear polypeptide
sequence of mature human neutrophil gelatinase-associated lipocalin
(hNGAL), selected from the group consisting of (a) Asn
96.fwdarw.Val, Tyr 100.fwdarw.Gln, and Tyr 106.fwdarw.unchanged (b)
Asn 96.fwdarw.Arg, Tyr 100.fwdarw.Glu, and Tyr 106.fwdarw.Phe, (c)
Asn 96.fwdarw.Asp, Tyr 100.fwdarw.Ser, and Tyr 106.fwdarw.Gly, (d)
Asn 96.fwdarw.Gly, Tyr 100.fwdarw.Gly, and Tyr 106.fwdarw.Gly, (e)
Asn 96.fwdarw.Lys, Tyr 100.fwdarw.Ala, and Tyr 106.fwdarw.Ile, (f)
Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Arg, and Tyr 106.fwdarw.Val, (g)
Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Arg, and
(h) Asn 96.fwdarw.Thr, Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Gly;
and (ii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, or 17 mutated amino acid residues at any of the sequence
positions corresponding to the sequence positions 36, 40, 41, 49,
52, 68, 70, 72, 73, 77, 79, 81, 103, 125, 127, 132, and 134 of the
linear polypeptide sequence of mature hNGAL.
93. A diagnostic or analytical kit comprising a lipocalin mutein
that is capable of binding hepcidin with an affinity by a KD of
about 10 nM or lower, wherein the lipocalin mutein comprises: (i) a
set of mutated amino acid residues at the sequence positions 96,
100, and/or 106 of the linear polypeptide sequence of mature human
neutrophil gelatinase-associated lipocalin (hNGAL), selected from
the group consisting of (a) Asn 96.fwdarw.Val, Tyr 100.fwdarw.Gln,
and Tyr 106.fwdarw.unchanged (b) Asn 96.fwdarw.Arg, Tyr
100.fwdarw.Glu, and Tyr 106.fwdarw.Phe, (c) Asn 96.fwdarw.Asp, Tyr
100.fwdarw.Ser, and Tyr 106.fwdarw.Gly, (d) Asn 96.fwdarw.Gly, Tyr
100.fwdarw.Gly, and Tyr 106.fwdarw.Gly, (e) Asn 96.fwdarw.Lys, Tyr
100.fwdarw.Ala, and Tyr 106.fwdarw.Ile, (f) Asn 96.fwdarw.Ser, Tyr
100.fwdarw.Arg, and Tyr 106.fwdarw.Val, (g) Asn 96.fwdarw.Ser, Tyr
100.fwdarw.Val, and Tyr 106.fwdarw.Arg, and (h) Asn 96.fwdarw.Thr,
Tyr 100.fwdarw.Val, and Tyr 106.fwdarw.Gly; and (ii) at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 mutated
amino acid residues at any of the sequence positions corresponding
to the sequence positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77,
79, 81, 103, 125, 127, 132, and 134 of the linear polypeptide
sequence of mature hNGAL.
Description
[0001] The present invention relates to novel, specific-binding
therapeutic and/or diagnostic proteins directed against Hepcidin,
which proteins preferably are muteins of a lipocalin protein. The
invention also relates to nucleic acid molecules encoding such
proteins and to methods for generation and use of such proteins and
nucleic acid molecules. Accordingly, the invention also is directed
to pharmaceutical and/or diagnostic compositions comprising such a
lipocalin proteins, including uses of these proteins.
BACKGROUND
[0002] Hepcidin, a peptide hormone typically existing in two forms
made of either 20 or 25 amino acids, is expressed and secreted by a
number of cells in response to iron loading and inflammation.
Hepcidin is produced predominantly in hepatocytes of the liver,
plays a central role in the regulation of iron homeostasis, acts as
an antimicrobial peptide and is directly or indirectly involved in
the development of most iron-deficiency/overload syndromes. A major
action of hepcidin is to internalize and degrade the iron exporter
ferroportin, which is expressed on all iron-exporting cells.
Hepcidin directly binds to ferroportin. A high hepcidin level thus
leads to the suppression of intestinal iron absorption and iron
release from macrophages and hepatocytes, while a low concentration
of hepcidin leads to acceleration of iron release from these
cells.
[0003] Hepcidin is also suspected to play role in pathogenesis of
anemia of inflammation and iron-deficiency anemia. Anemia of
inflammation, also known as anemia of chronic disease (ACD) or
anemia of chronic disorders, currently is the most frequent anemia
among hospitalized patients and a common syndrome complicating many
infectious, non-infectious inflammatory and neoplastic disorders.
ACD is a normocytic, normochromic anemia characterized by decreased
iron and iron-binding capacity (transferrin), increased ferritin
and the presence of iron in bone marrow macrophages, indicating
impaired mobilization of iron from its stores. While in anemia of
inflammation hepcidin levels are increased, in iron-deficiency
anemia low hepcidin levels are found. Hence, hepcidin could be used
as a marker to distinguish these diseases. Hepcidin may also be a
useful marker for screening, prognosis and monitoring hereditary
hemochromatosis and iron loading anemias. Hepcidin levels may
further be useful in monitoring EPO treatment and predicting a
response to EPO.
[0004] Methods of isolating, analyzing and quantifying hepcidin as
well as agents for the treatment of diseases and conditions
associated with hepcidin have been described in international
patent applications WO 2008/011158, WO 2008/097461, WO
2009/094551A1, WO 2009/139822, WO 20091058797 and WO 2010/017070.
However, no hepcidin-binding protein having the features attendant
to the proteins provided by present invention has been previously
described.
SUMMARY OF THE INVENTION
[0005] One embodiment of the current relates to a lipocalin mutein
that is capable of binding hepcidin with an affinity measured by a
K.sub.D of about 10 nM or lower. More preferably, the lipocalins
can have an affinity measured by a K.sub.D of about 1 nM or lower.
In another embodiment, the lipocalin mutein is capable of
neutralizing the bioactivity of human hepcidin-25, preferably with
an IC50 value of about 80 nM or lower as determined by a cell-based
assay for hepcidin-induced internalization and degradation of
ferroportin.
[0006] In particular embodiments, a lipocalin mutein according to
the current invention comprises an amino acid sequence selected
from the group consisting of SEQ ID NOs: 1-14. In another
embodiment, the mutein has at least 75% identity to the sequence of
a wild-type human lipocalin, including human Lipocalin 2.
[0007] In another embodiment, the mutein of the current invention
is conjugated to a compound selected from the group consisting of
an organic molecule, an enzyme label, a radioactive label, a
colored label, a fluorescent label, a chromogenic label, a
luminescent label, a hapten, digoxigenin, biotin, a cytostatic
agent, a toxins, a metal complexe, a metal, and colloidal gold. The
mutein can be fused at its N-terminus and/or its C-terminus to a
fusion partner which is a protein, a protein domain, or a
peptide.
[0008] In another embodiment, the mutein is conjugated to a
compound that extends the serum half-life of the mutein. More
preferably, the mutein is conjugated to a compound selected from
the group consisting of a polyalkylene glycol molecule, a
hydroethylstarch, an Fc part of an immunoglubolin, a C.sub.H3
domain of an immoglobulin, a C.sub.H4 domain of an immunoglubolin,
an albumin binding peptide, and an albumin binding protein.
[0009] In another embodiment, the mutein of the current invention
is an antagonist of a Hepcidin. The hepcidin can be mature human
Hepcidin.
[0010] In another embodiment, the current invention relates to a
nucleic acid molecule comprising a nucleotide sequence encoding a
mutein of the current invention.
[0011] In another embodiment, the lipocalin mutein of the current
invention is selected from the group consisting of muteins of
retinol-binding protein (RBP), bilin-binding protein (BBP),
apolipoprotein D (APO D), neutrophil gelatinase associated
lipocalin (NGAL), tear lipocalin (TLPC),
.alpha..sub.2-microglobulin-related protein (A2m), 24p3/uterocalin
(24p3), von Ebners gland protein 1 (VEGP 1), von Ebners gland
protein 2 (VEGP 2), and Major allergen Can f1 precursor (ALL-1). In
related embodiments, the lipocalin mutein is selected from the
group consisting of human neutrophil gelatinase associated
lipocalin (hNGAL), human tear lipocalin (hTLPC), human
apolipoprotein D (APO D) and the bilin-binding protein of Pieris
brassicae.
[0012] In another embodiment, the invention relates to a lipocalin
mutein which prevents human hepcidin-25 induced reduction of serum
iron levels in a subject.
[0013] The invention also includes a method of treating a disease
or disorder associated with an altered level of a Hepcidin, the
method comprising administering a pharmaceutical composition
containing a mutein as described herein to a subject in need
thereof. In related embodiments, the disease or disorder involves a
disorder of iron homeostasis or an inflammatory condition
associated with an elevated level of hepcidin.
DESCRIPTION OF FIGURES
[0014] FIG. 1 illustrates the PCR assembly strategy for the
simultaneous random mutagenesis of the 20 amino acid positions 36,
40, 41, 49, 52, 68, 70, 72, 73, 77, 79 81, 968, 100, 103, 106, 125,
127, 132, and 134 (underlined and numbered) in the amino acid
sequence of the mature Lcn 2. These 20 positions were divided into
four sequence subsets. For randomization of the amino acids in each
subset an oligodeoxynucleotide was synthesized (SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19) wherein NNK mixtures of
the nucleotides were employed at the mutated codons. N means a
mixture of all four bases A, C, G, and T while K means a mixture of
only the two bases G and T; hence such a triplet encodes all 20
natural amino acids as well as the amber stop codon TAG, which is
translated as glutamine in the E. coli supE-strains XL1-blue
(Bullock et al., BioTechniques 5 (1987), 376-378) or TG1 (Sambrook
et al., Molecular Cloning. A Laboratory Manual (1989), Cold Spring
Harbor Press) that were used for phagemid production and gene
expression. Four additional oligodeoxynucleotides (SEQ ID NO: 20,
SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23) with fixed nucleotide
sequences corresponding to the non-coding strand (written below the
DNA double strand sequence in 3'-5' direction) and filling the gaps
between the aforementioned oligodeoxynucleotides were also used in
the assembly reaction. Two shorter flanking oligodeoxynucleotides
(SEQ ID NO. 24 and SEQ ID NO: 25), which were added in excess and
carried biotin groups, served as primers for the PCR amplification
of the assembled, entirely synthetic gene fragment. The two
flanking primers each encompassed a BstXI restriction site
(CCANNNNNNTGG) giving rise to mutually non-compatible overhangs
upon enzyme digestion. This special arrangement of restriction
sites enabled a particularly efficient ligation and cloning of the
synthetic gene. Substitution of the amino acid Gln28 to His with
respect to the original Lcn2 sequence was necessary to introduce
the first BstXI site, while the second one naturally occurs in the
cDNA of Lcn2. Furthermore, the unpaired residue Cys87 was replaced
by Ser during the gene assembly. After one pot PCR the resulting
gene fragment was inserted into a vector providing the missing
parts of the Lcn2 structural gene. This illustration also depicts
two short primers (SEQ ID NO: 32 and SEQ ID NO: 33) upstream and
downstream, respectively, of the cassette flanked by the two BstXI
restriction sites, which served for double stranded DNA
sequencing.
[0015] FIG. 2 illustrates the nucleotide sequence of a library of
synthetic Lcn2 genes (only the central cassette flanked by the two
BstXI restriction sites, as in FIG. 1, is shown). This gene
fragment was prepared by Sloning BioTechnology GmbH. Compared with
the DNA library described in FIG. 1 there are two differences.
First, whenever possible, codons optimized for E. coli expression
were used throughout for the non-mutated amino acid positions.
Second, a mixture of 19 different triplets (GAC, TTC, CTG, CAC,
AAT, AGC, ACC, GCA. ATG, CCT, GTT, TGG, GAG, CAA, ATC, GGA, CGT,
GCA, TAC), each encoding a different amino acid except Cys, was
employed at the 20 randomized positions, which are identical to the
ones depicted in FIG. 1. Numbering of amino acids corresponds here
to an internal scheme employed by Sloning BioTechnology GmbH,
whereby Gly No 1 is the first amino acid codon directly following
the upstream BstX1 restriction site.
[0016] FIG. 3A depicts an alignment of certain amino acid sequences
of hHepcidin-specific, NGAL-based lipocalin muteins in comparison
with the polypeptide sequence of wildtype NGAL lipocalin. The
NGAL-derived, hepdicin binding muteins comprise residues 1 to 178,
meaning they have the length of the mature wildtype proteins.
Residues 179 to 186 are the sequence of a streptavidin binding tag,
Strep-Tag.TM., used in the isolation of generated muteins.
[0017] FIG. 3B depicts an alignment of certain amino acid sequences
of hHepcidin-specific, NGAL-based lipocalin muteins in comparison
with the polypeptide sequence of wildtype NGAL lipocalin. The
NGAL-derived, hepdicin binding muteins comprise residues 1 to 178,
meaning they have the length of the mature wildtype proteins.
Residues 179 to 186 are the sequence of a streptavidin binding tag,
Strep-Tag.TM., used in the isolation of generated muteins.
[0018] FIG. 4A depicts the amino acid sequence of the lipocalin
mutein of SEQ ID NO: 1 fused, via a linker (greyish bold italic) to
an ABD domain (bold) and a streptavidin binding tag, Strep-Tag.TM.
(italic) (SEQ ID NO: 15).
[0019] FIG. 4B depicts the amino acid sequence of the lipocalin
hNGAL, as encoded by the vector phNGAL 98, fused to a streptavidin
binding tag, the Strep-Tag.TM. (italic) and an N-terminal T7 tag
(bold italic) (SEQ ID NO. 34). This polypeptide is encoded by
phNGAL 101.
[0020] FIG. 5 shows the results of a direct ELISA of selected Lcn2
muteins.
[0021] FIG. 6 depicts the results of a competitive binding assay of
selected Lcn2 muteins.
[0022] FIG. 7 depicts the affinities of selected muteins for human
and cynomolgus Hepcidin-25 as determined by
surface-plasmon-resonance (SPR).
[0023] FIG. 8 depicts the in vitro neutralization activity of
anti-Hepcidin-25 lipocalin muteins.
[0024] FIG. 9 demonstrates that a lipocalin mutein directed against
hepcidin neutralizes human hepcidin injected into mice.
[0025] FIG. 10 depicts pharmacokinetic parameters for SEQ ID NO:
14-PEG and SEQ ID NO: 1-ABD (equal to SEQ ID NO: 15).
DETAILED DESCRIPTION OF THE INVENTION
[0026] In one aspect the present invention relates to novel,
specific-binding proteins directed against or specific for
hepcidin. Proteins of the invention may be used for therapeutic
and/or diagnostic purposes. As used herein, a protein of the
invention "specifically binds" a target (here, hepcidin) if it is
able to discriminate between that target and one or more reference
targets, since binding specificity is not an absolute, but a
relative property. "Specific binding" can be determined, for
example, in accordance with Western blots, ELISA-, RIA-, ECL-,
IRMA-tests, FACS, IHC and peptide scans.
[0027] Proteins of the invention, which are directed against or
specific for hepcidin, include any number of specific-binding
protein muteins that are based on a defined protein scaffold. As
used herein, a "mutein," a "mutated" entity (whether protein or
nucleic acid) or "mutant" refers to the exchange, deletion, or
insertion of one or more nucleotides or amino acids, respectively,
compared to the naturally occurring (wild-type) nucleic acid or
protein "reference" scaffold.
[0028] A protein of the invention can be a mutein of a lipocalin,
preferably a lipocalin selected from the group consisting of
retinol-binding protein (RBP), bilin-binding protein (BBP),
apolipoprotein D (APO D), neutrophil gelatinase associated
lipocalin (NGAL), tear lipocalin (TLPC),
.alpha..sub.2-microglobulin-related protein (A2m), 24p3/uterocalin
(24p3), von Ebners gland protein 1 (VEGP 1), von Ebners gland
protein 2 (VEGP 2), and Major allergen Can fi precursor (ALL-1). As
used herein, a "lipocalin" is defined as monomeric protein of
approximately 18-20 kDA in weight, having a cylindrical
.beta.-pleated sheet supersecondary structural region comprising a
plurality of (preferably eight) .beta.-strands connected pair-wise
by a plurality of (preferably four) loops at one end to define
thereby a binding pocket. It is the diversity of the loops in the
otherwise rigid lipocalin scaffold that gives rise to a variety of
different binding modes among the lipocalin family members, each
capable of accommodating targets of different size, shape, and
chemical character (reviewed, e.g., in Flower, D. R. (1996), supra;
Flower, D. R. et al. (2000), supra or Skerra, A. (2000) Biochim.
Biophys. Acta 1482, 337-350). Indeed, the lipocalin family of
proteins have naturally evolved to bind a wide spectrum of ligands,
sharing unusually low levels of overall sequence conservation
(often with sequence identities of less than 20%) yet retaining a
highly conserved overall folding pattern. The correspondence
between positions in various lipocalins is well known to one of
skill in the art. See, for example, U.S. Pat. No. 7,250,297.
[0029] In a preferred embodiment, a protein of the invention is a
mutein of Lipocalin 2 (Lcn 2; also known as human neutrophil
gelatinase-associated lipocalin, hNGAL, or as siderocalin). The
term "human neutrophil gelatinase-associated lipocalin" or "hNGAL"
or "lipocalin 2" or "Lcn2" as used herein to refer to the mature
hNGAL with the SWISS-PROT/UniProt Data Bank Accession Number P80188
or the mature hNGAL shown in SEQ ID NO:35. The mature form of this
protein has amino acids 21 to 198 of the complete sequence, since a
signal peptide of amino acids 1-20 is cleaved off. The protein
further has a disulfide bond formed between the amino acid residues
at positions 76 and 175 of the mature protein.
[0030] In a more preferred embodiment, the invention relates to a
lipocalin mutein having a cylindrical .beta.-pleated sheet
supersecondary structural region comprising eight .beta.-strands
connected pair-wise by four loops at one end to define thereby a
binding pocket, wherein at least one amino acid of each of at least
three of said four loops has been mutated and wherein said
lipocalin is effective to bind a hepcidin as given non-natural
target with detectable affinity. Preferably, said lipocalin mutein
has one or more such as 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 amino acid relacements at a position
corresponding to position 36, 40, 41, 49, 52, 68, 70, 72, 73, 77,
79, 81, 96, 100, 103, 106, 125, 127, 132, and/or 134 of the linear
polypeptide sequence of NGAL.
[0031] In this context, the inventors identified a specific group
of Lipocalin 2 muteins with mutations at specific positions which
show detectable affinity as well as specificity for Hepcidin.
Suitable amino acid positions for mutation include sequence
positions 96, 100, and 106, of the linear polypeptide sequence of
human Lipocalin 2. The present invention also relates to nucleic
acids encoding these proteins.
[0032] Other protein scaffolds that can be engineered in accordance
with the present invention to provide protein muteins that bind
hepcidin with detectable affinity include: an EGF-like domain, a
Kringle-domain, a fibronectin type I domain, a fibronectin type II
domain, a fibronectin type III domain, a PAN domain, a G1a domain,
a SRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain,
tendamistat, a Kazal-type serine protease inhibitor domain, a
Trefoil (P-type) domain, a von Willebrand factor type C domain, an
Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I
repeat, LDL-receptor class A domain, a Sushi domain, a Link domain,
a Thrombospondin type I domain, an immunoglobulin domain or a an
immunoglobulin-like domain (for example, domain antibodies or camel
heavy chain antibodies), a C-type lectin domain, a MAM domain, a
von Willebrand factor type A domain, a Somatomedin B domain, a
WAP-type four disulfide core domain, a F5/8 type C domain, a
Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type
EGF-like domain, a C2 domain, "Kappabodies" (Ill, et al. "Design
and construction of a hybrid immunoglobulin domain with properties
of both heavy and light chain variable regions" Protein Eng
10:949-57 (1997)), "Minibodies" (Martin et al. The
affinity-selection of a minibody polypeptide inhibitor of human
interleukin-6' EMBO J 13-5303-9 (1994)), "Diabodies" (Holliger et
al. "Diabodies": small bivalent and bispecific antibody fragments'
PNAS USA 90:6444-6448 (1993)), "Janusins" (Traunecker et al.
"Bispecific single chain molecules (Janusins) target cytotoxic
lymphocytes on HIV infected cells" EMBO J 10:3655-3659 (1991) and
Traunecker et al. "Janusin: new molecular design for bispecific
reagents" Int J Cancer Suppl 7:51-52 (1992), a nanobody, an
adnectin, a tetranectin, a microbody, an affilin, an affibody an
ankyrin, a crystallin, a knottin, ubiquitin, a zinc-finger protein,
an autofluorescent protein, an ankyrin or ankyrin repeat protein or
a leucine-rich repeat protein, an avimer (Silverman, Lu Q. Bakker
A, To W, Duguay A, Alba B M, Smith R, Rivas A, Li P, Le H.
Whitehorn E, Moore K W, Swimmer C, Perlroth V, Vogt M, Kolkman J,
Stemmer W P 2005, Nat Biotech, December; 23(12):1556-61.
E-Publication in Nat Biotech. 2005 Nov. 20 edition); as well as
multivalent avimer proteins evolved by exon shuffling of a family
of human receptor domains as also described in Silverman J, Lu Q,
Bakker A, To W, Duguay A, Alba B M, Smith R. Rivas A, Li P, Le H,
Whitehorn E, Moore K W, Swimmer C, Perlroth V, Vogt M. Kolkman J,
Stemmer W P, Nat Biotech, December: 23(12):1556-61, E-Publication
in Nat. Biotechnology. 2005 Nov. 20 edition).
[0033] A protein of the invention may include the wild type
(natural) amino acid sequence of the "parental" protein scaffold
(such as a lipocalin) outside the mutated amino acid sequence
positions; alternatively, a lipocalin mutein may also contain amino
acid mutations outside the sequence positions subjected to
mutagenesis that do not interfere with the binding activity and the
folding of the mutein. Such mutations can be accomplished on a DNA
level using established standard methods (Sambrook, J. et al.
(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Possible
alterations of the amino acid sequence are insertions or deletions
as well as amino acid substitutions.
[0034] Such substitutions may be conservative, i.e. an amino acid
residue is replaced with a chemically similar amino acid residue.
Examples of conservative substitutions are the replacements among
the members of the following groups: 1) alanine, serine, and
threonine; 2) aspartic acid and glutamic acid; 3) asparagine and
glutamine: 4) arginine and lysine; 5) isoleucine, leucine,
methionine, and valine; and 6) phenylalanine, tyrosine, and
tryptophan One the other hand, it is also possible to introduce
non-conservative alterations in the amino acid sequence. In
addition, instead of replacing single amino acid residues, it is
also possible to either insert or delete one or more continuous
amino acids of the primary structure of a parental protein
scaffold, where these deletions or insertion result in a stable
folded/functional mutein, which can be readily tested by the
skilled worker.
[0035] The skilled worker will appreciate methods useful to prepare
protein muteins contemplated by the present invention but whose
protein or nucleic acid sequences are not explicitly disclosed
herein. As an overview, such modifications of the amino acid
sequence include. e.g., directed mutagenesis of single amino acid
positions in order to simplify subcloning of a mutated lipocalin
gene or its parts by incorporating cleavage sites for certain
restriction enzymes. In addition, these mutations can also be
incorporated to further improve the affinity of a lipocalin mutein
for a given target. Furthermore, mutations can be introduced to
modulate certain characteristics of the mutein such as to improve
folding stability, serum stability, protein resistance or water
solubility or to reduce aggregation tendency, if necessary. For
example, naturally occurring cysteine residues may be mutated to
other amino acids to prevent disulphide bridge formation.
[0036] Accordingly, the invention also includes functional variants
of proteins disclosed herein, which have a threshold sequence
identity or sequence homology to a reference protein. By "identity"
or "sequence identity" is meant a property of sequences that
measures their similarity or relationship. The term "sequence
identity" or "identity" as used in the present invention means the
percentage of pair-wise identical residues--following (homologous)
alignment of a sequence of a polypeptide of the invention with a
sequence in question--with respect to the number of residues in the
longer of these two sequences. Percent identity is determined by
dividing the number of identical residues by the total number of
residues and multiplying the product by 100. The term "homology" is
used herein in its usual meaning and includes identical amino acids
as well as amino acids which are regarded to be conservative
substitutions (for example, exchange of a glutamate residue by an
aspartate residue) at equivalent positions in the linear amino acid
sequence of two proteins. Most preferred, the amino acid sequence
shown in SEQ ID NO:35 is preferred as a "reference sequence". SEQ
ID NO:35 shows the mature hNGAL. The term "reference sequence" and
"wild type sequence" (of NGAL) is used interchangeably herein.
Alternatively, the amino acid sequence with the SWISS-PROT/UniProt
Data Bank Accession Number P80188 can be used as reference
sequence.
[0037] The percentage of sequence homology or sequence identity
can, for example, be determined herein using the program BLASTP,
version blastp 2.2.5 (Nov. 16, 2002; cf. Altschul, S. F. et al.
(1997) Nucl. Acids Res. 25, 3389-3402). In this embodiment the
percentage of homology is based on the alignment of the entire
polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1, cutoff
value set to 10.sup.-3) including the propeptide sequences,
preferably using the wild type protein scaffold as reference in a
pairwise comparison. It is calculated as the percentage of numbers
of "positives" (homologous amino acids) indicated as result in the
BLASTP program output divided by the total number of amino acids
selected by the program for the alignment.
[0038] It is also possible to deliberately mutate other amino acid
sequence positions to cysteine in order to introduce new reactive
groups, for example, for the conjugation to other compounds, such
as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin,
peptides or proteins, or for the formation of non-naturally
occurring disulphide linkages. With respect to a mutein of human
Lipocalin 2, exemplary possibilities of such a mutation to
introduce a cysteine residue into the amino acid sequence of a
lipocalin including human Lipocalin 2 mutein to include the
introduction of a cysteine (Cys) residue at at least at one of the
sequence positions that correspond to sequence positions 14, 21,
60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild type
sequence of hNGAL. In some embodiments where a human Lipocalin 2
mutein of the invention has a sequence in which, in comparison to
the sequence of the SWISS-PROT/UniProt Data Bank Accession Number
P80188, a cysteine has been replaced by another amino acid residue,
the corresponding cysteine may be reintroduced into the sequence.
As an illustrative example, a cysteine residue at amino acid
position 87 may be introduced in such a case by reverting to a
cysteine as originally present in the sequence of SWISS-PROT
accession No P80188. The generated thiol moiety at the side of any
of the amino acid positions 14, 21, 60, 84, 88, 116, 141, 145, 143,
146 and/or 158 may be used to PEGylate or HESylate the mutein, for
example, in order to increase the serum half-life of a respective
human Lipocalin 2 mutein.
[0039] The term "position" when used in accordance with the
invention means the position of either an amino acid within an
amino acid sequence depicted herein or the position of a nucleotide
within a nucleic acid sequence depicted herein. The term
"corresponding" as used herein also includes that a position is not
only determined by the number of the preceding nucleotides/amino
acids. Accordingly, the position of a given amino acid in
accordance with the invention which may be substituted may very due
to deletion or addition of amino acids elsewhere in a (mutant or
wild-type) lipocalin. Similarly, the position of a given nucleotide
in accordance with the present invention which may be substituted
may vary due to deletions or additional nucleotides elsewhere in a
mutein or wild type lipocalin 5'-untranslated region (UTR)
including the promoter and/or any other regulatory sequences or
gene (including exons and introns).
[0040] Thus, under a "corresponding position" in accordance with
the invention it is preferably to be understood that
nucleotides/amino acids may differ in the indicated number but may
still have similar neighboring nucleotides/amino acids. Said
nucleotides/amino acids which may be exchanged, deleted or added
are also comprised by the term "corresponding position". When used
herein "at a position corresponding to a position" a position in a
"query" amino acid (or nucleotide) sequence is meant that
corresponds to a position in a "subject" amino acid (or nucleotide)
sequence.
[0041] Specifically, in order to determine whether a nucleotide
residue or amino acid residue of the amino acid sequence of a
lipocalin different from a NGAL lipocalin mutein of the invention
corresponds to a certain position in the nucleotide sequence or the
amino acid sequence of a NGAL lipocalin mutein as described, in
particular any of SEQ ID NOs: 1-14 or that having one or more amino
acid substitutions such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19 or 20 at position 36, 40, 41, 49, 52, 68,
70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and/or
134 of the linear polypeptide sequence of NGAL, a skilled artisan
can use means and methods well-known in the art, e.g., alignments,
either manually or by using computer programs such as BLAST2.0,
which stands for Basic Local Alignment Search Tool or ClustalW or
any other suitable program which is suitable to generate sequence
alignments. Accordingly, a lipocalin mutein of any of SEQ ID Nos:
1-14 or that having one or more amino acid substitutions such as 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
at position 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96,
100, 103, 106, 125, 127, 132, and/or 134 or any other position
described herein of the linear polypeptide sequence of NGAL can
serve as "subject sequence", while the amino acid sequence of a
lipocalin different from NGAL serves as "query sequence".
[0042] Given the above, a skilled artisan is thus readily in a
position to determine which amino acid position mutated in Lcn2 as
described herein corresponds to an amino acid of a scaffold other
than Lcn2, preferably such as one of those described herein.
Specifically, a skilled artisan can align the amino acid sequence
of a mutein as described herein, in particular a NGAL mutein (or
anticalin) of the invention with the amino acid sequence of a
different lipocalin to determine which amino acid(s) of said mutein
correspond(s) to the respective amino acid(s) of the amino acid
sequence of said different lipocalin. More specifically, a skilled
artisan can thus determine which amino acid of the amino acid
sequence of said different lipocalin corresponds to the amino acid
at position(s) 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96,
100, 103, 106, 125, 127, 132, and/or 134 or to an amino acid at any
other position as described herein of the linear polypeptide
sequence of NGAL.
[0043] Proteins of the invention, which are directed against or
specific for hepcidin, include any number of specific-binding
protein muteins that are based on a defined protein scaffold.
Preferably, the scaffold is hNGAL. As used herein, a "mutein," a
"mutated" entity (whether protein or nucleic acid) or "mutant"
refers to the exchange, deletion, or insertion of one or more
nucleotides or amino acids, respectively compared to the naturally
occurring (wild-type) nucleic acid or protein "reference" scaffold.
Preferably, the number of nucleotides or amino acids, respectively,
that is exchanged, deleted or inserted is 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25,
30, 35, 40, 45 or 50. However, it is preferred that a mutein of the
invention is still capable of binding hepcidin.
[0044] In some embodiments, a protein according to the invention
binds a hepcidin with a K.sub.D of 100 .mu.M or less, including 5
.mu.M or less, about 500 nM, about 200 nM or less, 100 nM or less,
1 nM or less, or 0.1 nM or less. A protein of the invention may
specifically bind one or more continuous, discontinuous or
conformation epitope(s) of the mature, folded bioactive form of a
hepcidin.
[0045] A protein of the invention is able to bind a hepcidin with
detectable affinity, i.e. with a dissociation constant of at least
200 nM, i.e. K.sub.D of about 200 nM or less. In some embodiments,
a protein of the invention binds a hepcidin with a dissociation
constant of at least about 100 nM, about 50 nM, about 25 nM, about
15 nM, about 5 nM, about 2 nM, about 0.5 nM, about 0.25 nM, about
0.1 nM, about 0.05 nM or even less. A protein of the invention
preferably binds to a mature human hepcidin molecule with an
affinity by a K.sub.D of about 10 nM or stronger. Binding
affinities have been found by the present inventors to often be of
a K.sub.D below about 1 nM and, in some cases, about 0.1 nM and
below.
[0046] The binding affinity of a protein of the invention (e.g. a
mutein of a lipocalin) to a selected target (in the present case,
hepcidin), can be measured (and thereby K.sub.D values of a
mutein-ligand complex be determined) by a multitude of methods
known to those skilled in the art. Such methods include, but are
not limited to, fluorescence titration, competition ELISA,
calorimetric methods, such as isothermal titration calorimetry
(ITC), and surface plasmon resonance (BIAcore). Such methods are
well established in the art and examples thereof are also detailed
below.
[0047] The amino acid sequence of a protein of the invention may
have a high sequence identity to mature human Lipocalin 2 or other
lipocalins. In this context, a protein of the invention may have at
least 70%, at least 75%, at least 80%, at least 82%, at least 85%,
at least 87%, at least 90% identity, including at least 95%
identity to a protein selected from the group consisting of the
sequence of SEQ ID NOS: 1-14. It is preferred that a structural
homolog has still an amino acid replacement at one or more such as
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20 positions corresponding to position 36, 40, 41, 49, 52, 68, 70,
72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and/or 134 of
the linear polypeptide sequence of NGAL.
[0048] The invention also includes structural homologues of the
proteins selected from the group consisting of the sequence of SEQ
ID NOS: 1-14, which have an amino acid sequence homology or
sequence identity of more than about 60%, preferably more than 65%,
more than 70%, more than 75%, more than 80%, more than 85%, more
than 90%, more than 92% and most preferably more than 95% in
relation thereto. It is preferred that a structural homolog has
still an amino acid replacement at one or more such as 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 positions
corresponding to position 36, 40, 41, 49, 52, 68, 70, 72, 73, 77,
79, 81, 96, 100, 103, 106, 125, 127, 132, and/or 134 of the linear
polypeptide sequence of NGAL.
[0049] The term "hepcidin" refers to the protein also termed
liver-expressed antimicrobial peptide 1 or putative liver tumor
regressor, of which the human form has the UniProtKB/Swiss-Prot
accession number P81172. On a general basis the term "hepcidin"
refers to any form of the hepcidin protein known to be present in
vertebrate species, including in mammals. The human unprocessed
protein has a length of 84 amino acids and is encoded by the gene
"HAMP," also known as "HEPC" or "LEAP1." It is cleaved into two
chains, which are herein also included in the term "Hepcidin."
These two chains are of amino acids 60-84, which is Hepcidin-25
(Hepc25) and of amino acids 65-84, which is Hepcidin-20 (Hepc20).
Hepcidin-25 is arranged in the form of a bent hairpin, stabilized
by four disulfide bonds Natural variants also included in the term
"hepcidin" have, for example, the amino acid replacement 59
R.fwdarw.G (VAR.sub.--0425129); the amino acid replacement 70
C.fwdarw.R (VAR.sub.--042513); the amino acid replacement 71
G.fwdarw.D (VAR.sub.--026648) or the amino acid replacement 78
C.fwdarw.Y (VAR.sub.--042514). A further natural variant is
Hepcidin-22, another N-terminally truncated isoform (besides
Hecidin-20) of Hepcidin-25.
[0050] The term "mature hepcidin" as used herein refers to any
mature, bioactive form of the hepcidin protein expressed in a
vertebrate such as a mammal. The term "human hepcidin" refers to
any form of the hepcidin protein present in humans. The expression
"human hepcidin-25" refers to the mature form of human hepcidin
with the amino acid sequence as depicted in SEQ ID NO: 28. In the
present invention lipocalin muteins are provided that are able to
bind each given form of hepcidin including proteolytic fragments
thereof, regardless of whether the respective hepcidin molecule
displays biological/physiological activity. Thus, the hepcidin
molecule may only be present in a biological sample, without having
any measurable physiological relevance. See, for example,
Hepcidin-22 that so far has only been detected in urine found in
urine and that so far is assumed to merely be a urinary degradation
product of Hepcidin-25 (reviewed in Kemna et al., Haematologica,
2008 January; 93:(1)90-97). A mutein of the invention may of course
also bind physiological active species such as the mature,
bioactive Hepcidin-25. Accordingly, a mutein of the invention may
be used as diagnostic and/or pharmaceutical, depending on the
hepcidin form chosen to be recognized.
[0051] In line with the above, a protein of the invention
preferably acts as an antagonist of a hepcidin molecule. In some
embodiments, a protein of the invention (e.g., a human Lipocalin 2
mutein) may act as an antagonist of a hepcidin molecule by
inhibiting the ability of the hepcidin molecule to bind to or
otherwise interact with ferroportin. The hepcidin may be a mature
human hepcidin format such as hepcidin-25 or hepcidin-20. Binding
of a mature hepcidin to ferroportin leads to internalization and
degradation of ferroportin, standard processes of a protein with a
cell surface/membrane location.
[0052] In yet another aspect, the present invention includes
various lipocalin muteins, including muteins of human Lipocalin 2
that specifically bind hepcidin. In this sense, hepcidin can be
regarded a non-natural ligand of wild type human Lipocalin 2, where
"non-natural ligand" refers to a compound that does not bind to
wildtype lipocalins, including human Lipocalin 2 under
physiological conditions. By engineering wildtype lipocalins such
as human Lipocalin 2 with mutations at certain positions, the
present inventors have demonstrated that high affinity and high
specificity for a non-natural ligand is possible. In one aspect at
least at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, and/or 20 nucleotide triplet(s) encoding for any of the
sequence positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81,
96, 100, 103, 106, 125, 127, 132, and/or 134 of the linear
polypeptide sequence of hLcn2, or other parallel sites on
lipocalins, a random mutagenesis can be carried out by allowing
substitution at this positions by a subset of nucleotide
triplets.
[0053] The amino acid replacements in the lipocalin muteins of the
invention as described herein are preferably within one, two, three
or four loop regions of a lipocalin, preferably hNGAL. The loop
regions are from positions 33 to 54 (loop 1), 686 to 83 (loop 2),
94 to 106 (loop 3), and 123 to 136 (loop 4) of hNGAL 24-36, 53-66,
79-84, and 103-110
[0054] Further, the lipocalins can be used to generate muteins that
have a mutated amino acid residue at any one or more, including at
least at any two or all three, of the sequence positions of the
sequence positions corresponding to the sequence positions 96, 100
and 106 of the linear polypeptide sequence of a mature human
Lipocalin 2. A substitution at sequence position 96 may for example
be a substitution Asn 96.fwdarw.Arg, Asp, Gln, Gly, Lys, Ser, Thr
or Val. A substitution at sequence position 100 may for example be
a substitution Tyr 100.fwdarw.Ala, Arg, Glu, Gln, Gly, Ser and Val.
A substitution at sequence position 106 may for example be a
substitution Tyr 106.fwdarw.Ile, Gly, Phe, Val or Arg. A mutein of
the invention may in some embodiments have the set of amino acid
substitutions, relative to the linear polypeptide sequence of a
mature human Lipocalin 2, of Asn 96.fwdarw.Val, and Tyr
100.fwdarw.Gln. In such an embodiment the tyrosine at position 106
may be unchanged. A mutein of the invention may in some embodiments
have the set of amino acid substitutions, relative to the linear
polypeptide sequence of a mature human Lipocalin 2, of Asn
96.fwdarw.Arg, Tyr 100.fwdarw.Glu, and Tyr 106.fwdarw.Phe. In some
embodiments a mutein of the invention may have the set of amino
acid substitutions of Asn 96.fwdarw.Asp, Tyr 100.fwdarw.Ser and Tyr
106.fwdarw.Gly. A mutein of the invention may in some embodiments
have the set of amino acid substitutions of Asn 96.fwdarw.Gly, Tyr
100.fwdarw.Gly and Tyr 106.fwdarw.Gly. A mutein of the invention
may in some embodiments have the set of amino acid substitutions of
Asn 96.fwdarw.Lys, Tyr 100.fwdarw.Ala and Tyr 106.fwdarw.Ile. In
some embodiments a mutein of the invention may have the set of
amino acid substitutions of Asn 96.fwdarw.Ser, Tyr 100.fwdarw.Arg
and Tyr 106.fwdarw.Val. A mutein of the invention may in some
embodiments have the set of amino acid substitutions of Asn
96.fwdarw.Ser, Tyr 100.fwdarw.Val and Tyr 106.fwdarw.Arg. In some
embodiments a mutein of the invention may have the set of amino
acid substitutions of Asn 96.fwdarw.Thr, Tyr 100.fwdarw.Val and Tyr
106.fwdarw.Gly. In some embodiments a mutein of the present
invention further includes a mutated amino acid residue at position
134 within the linear polypeptide sequence of the mature human
Lipocalin 2 In one embodiment this substitution is Lys
134.fwdarw.Trp.
[0055] In some embodiments, a mutein of the present invention
includes, typically in addition to a mutation at one or more of
sequence positions 96, 100 and 106 (supra), a mutated amino acid
residue at any one or more of the sequence positions corresponding
to the sequence positions 52, 68, 81, 127 of the linear polypeptide
sequence of the mature human Lipocalin 2. The mutein may, for
instance, include within the linear polypeptide sequence of the
mature human Lipocalin 2, a substitution Tyr 52.fwdarw.His, Leu,
Phe or Trp. The mutein may also include within the linear
polypeptide sequence of the mature human Lipocalin 2 a substitution
Ser 68.fwdarw.Arg. Gly or Ile. The mutein may also include a
substitution Arg 81.fwdarw.Glu, Gly or Gln. The mutein may, for
instance, include within the linear polypeptide sequence of the
mature human Lipocalin 2 a substitution Ser 127.fwdarw.Thr or Trp.
A mutein of the invention may in some embodiments have the set of
amino acid substitutions, relative to the linear polypeptide
sequence of a mature human Lipocalin 2, of Tyr 52.fwdarw.His, Ser
68.fwdarw.Arg, Arg 81.fwdarw.Ser and Ser 127.fwdarw.Trp. In some
embodiments a mutein of the invention may have the set of amino
acid substitutions of Tyr 52.fwdarw.Leu, Ser 68.fwdarw., Arg
81.fwdarw.Glu and Ser 127.fwdarw.Trp. A mutein of the invention may
in some embodiments have the set of amino acid substitutions of Tyr
52.fwdarw.Phe, Ser 68.fwdarw.Gly, Arg 81.fwdarw.Gly and Ser
127.fwdarw.Trp. A mutein of the invention may in some embodiments
have the set of amino acid substitutions of Tyr 52.fwdarw.Trp. Ser
68.fwdarw.Ile, Arg 81.fwdarw.Gln and Ser 127.fwdarw.Trp. In some
embodiments a mutein of the invention may have the set of amino
acid substitutions of Tyr 52.fwdarw.Trp. Ser 68.fwdarw.Arg, Arg
81.fwdarw.Glu and Ser 127.fwdarw.Trp. A mutein of the invention may
in some embodiments have the set of amino acid substitutions, in
relation to the sequence of a mature human Lipocalin 2, of Tyr
52.fwdarw.Trp, Ser 68.fwdarw.Arg, Arg 81.fwdarw.Glu and Ser
127.fwdarw.Thr. In some embodiments a mutein of the invention may
have the set of amino acid substitutions of Tyr 52.fwdarw.Trp, Ser
68.fwdarw.Arg, Arg 81.fwdarw.Glu and Ser 127.fwdarw.Trp.
[0056] In a further embodiment of the invention, the mutein
includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 mutated amino acid residues at any of the
sequence positions corresponding to the sequence positions 33, 36,
40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59, 65, 68,
70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99, 100, 103,
106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of the linear
polypeptide sequence of hNGAL or the corresponding sites on other
lipocalins. In a further embodiment, the mutein includes at least
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20 mutated amino acid residues at any one of the sequence positions
33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50, 51, 52, 54, 55, 59,
65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86, 87, 98, 96, 99,
100, 103, 106, 107, 110, 111, 125, 127, 132, 134, 136 and 138 of
the linear polypeptide sequence of hNGAL or the corresponding sites
on other lipocalins. In still a further embodiment, the mutein
includes 18, 19 or 20 mutated amino acid residues at any one of the
sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50,
51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86,
87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134,
136 and 138 of the linear polypeptide sequence of human Lipocalin 2
or the corresponding sites on other lipocalins.
[0057] A mutein of the invention may, for example, with respect to
the mature hLcn2 wild type amino acid sequence, include one or more
amino acid replacements such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20 of the group. Leu36.fwdarw.Ala,
Cys, Thr, Val; Ala 40.fwdarw.Arg, Glu, Gly and Ser; Ile
41.fwdarw.Ile, Leu, Met or Val; Gln 49.fwdarw.Leu or Met; Leu
70.fwdarw.Asp, Asn, Gln, Met or Phe; Arg 72.fwdarw.Glu. Gly, Leu or
Val; Lys 73.fwdarw.Ala, Arg, Glu, Gly. Leu. Thr or Tyr; Asp
77.fwdarw.Arg, Glu, Gly. Leu. Ser or Val; Trp 79.fwdarw.Gly, Leu,
Ser, Tyr or Val; Leu 103.fwdarw.Ala, Arg, Gly or Trp; Tyr
106.fwdarw.Gly, Ile, Phe or Val; Lys 125.fwdarw.Arg, Leu, Met, Phe,
Thr, or Val; and Tyr 132.fwdarw.Leu or Val. A mutein of the
invention may, for instance, have the set of amino acid
combinations, in relation to the linear polypeptide sequence of a
mature human Lipocalin 2, of Ala 36, Ser 40, Leu 41, Met 49, Asn
70, Gly 72, Gly 73, Ser 77, Leu 79, Leu 125 and Val 132. A mutein
of the invention may, for example, have the set of amino acid
combinations, in relation to the sequence of a mature human
Lipocalin 2, of Leu 36, Arg 40, Val 41, Gln 49, Asp 70, Arg 72, Thr
73, Leu 77, Ser 79, Thr 125 and Val 132. In some embodiments a
mutein of the invention may have the set of amino acid combinations
of Leu 36, Glu 40, Ile 41, Leu 49, Gln 70, Gly 72, Glu 73, Gly 77,
Gly 79, Phe 125 and Val 132. A mutein of the invention may also
have the set of amino acid combinations of Leu 36, Glu 40, Ile 41,
Met 49, Met 70, Leu 72, Ala 73, Glu 77, Leu 79, Val 125, Val 132 or
the set of amino acid combinations of Leu 36, Glu 40, Val 41, Met
49, Met 70, Leu 72, Ala 73, Glu 77, Leu 79, Thr 125 and Val 132. In
some embodiments a mutein of the invention may have the set of
amino acid combinations of Leu 36, Glu 40, Val 41, Met 49, Met 70,
Leu 72, Ala 73, Glu 77, Leu 79, Val 125 and Val 132 or the set of
amino acid combinations of Thr 36, Ser 40, Ile 41, Gln 49, Phe 70,
Glu 72. Gly 73, Arg 77. Val 79, Val 125 and Leu 132. As a further
example, a mutein of the invention may have the set of amino acid
combinations of Val 36, Glu 40, Met 41, Leu 49, Met 70, Glu 72, Tyr
73, Val 77, Leu 79. Arg 125 and Val 132. A mutein of the invention
may also have the set of amino acid combinations of Val 36, Gly 40,
Leu 41, Leu 49, Leu 70, Val 72, Arg 73, Arg 77, Tyr 79, Met 125 and
Val 132.
[0058] In one embodiment of the present invention, the mutein
includes mutated amino acid residues at at least any 10, 14, 15,
20, 22, 24, 26, 28, 29, 30, 31, 32, 33, 35 or all 45 of the
above-listed sequence positions.
[0059] A mutein of the invention, which binds to Hepcidin, can
include with respect to the mature human Lipocalin 2 wild type
amino acid sequence (Lcn2) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16 or 17 amino acid replacements which include, but
are not limited to, Leu36.fwdarw.Val or Cys; Ala40.fwdarw.Tyr or
Lys or Val; Ile41.fwdarw.Thr or Ser or Leu; Gln49.fwdarw.Leu or
Trp; Leu70.fwdarw.Gly; Arg72.fwdarw.Gly or Asp; Lys73.fwdarw.Leu or
Thr or Asp; Asp77.fwdarw.Asn or His or Leu; Trp79.fwdarw.Lys;
Asn96.fwdarw.Ile or Arg; Tyr100.fwdarw.Gln or Arg or Glu;
Leu103.fwdarw.Met or Arg or Gly; Tyr106.fwdarw.Tyr or Ala or Trp;
Lys125.fwdarw.Thr or Val or Glu; Ser127.fwdarw.Gly or Gln or Ala;
Tyr132.fwdarw.Met or Ser or Thr, and Lys134.fwdarw.Asn
[0060] In one embodiment, a mutein of the invention, which binds to
Hepcidin includes the following amino acid replacements:
Leu36.fwdarw.Val; Ala40.fwdarw.Tyr; Ile41.fwdarw.Thr;
Gln49.fwdarw.Leu; Leu70.fwdarw.Gly; Lys73.fwdarw.Leu;
Asp77.fwdarw.Asn; Trp79.fwdarw.Lys; Asn96.fwdarw.Ile;
Tyr100.fwdarw.Gln; Leu103.fwdarw.Met; Lys125.fwdarw.Thr;
Ser127.fwdarw.Gly; Tyr132.fwdarw.Met; and Lys134.fwdarw.Asn. In a
further embodiment, a mutein of the invention, which binds to
Hepcidin, includes the following amino acid replacements
Leu36.fwdarw.Val; Ala40.fwdarw.Lys; Ile41.fwdarw.Ser;
Gln49.fwdarw.Trp; Leu70.fwdarw.Gly; Arg72.fwdarw.Gly;
Lys73.fwdarw.Thr, Asp77.fwdarw.His; Trp79.fwdarw.Lys;
Asn96.fwdarw.Arg; Tyr100.fwdarw.Arg; Leu103.fwdarw.Arg;
Tyr106.fwdarw.Ala; Lys125.fwdarw.Val; Ser127.fwdarw.Gln.
Tyr132.fwdarw.Ser; and Lys134.fwdarw.Asn. In another embodiment, a
mutein of the invention, which binds to Hepcidin, includes the
following amino acid replacements Leu36.fwdarw.Cys;
Ala40.fwdarw.Val; Ile41.fwdarw.Leu; Gln49.fwdarw.Leu;
Leu70.fwdarw.Gly; Arg72.fwdarw.Asp; Lys73.fwdarw.Asp;
Asp77.fwdarw.Leu; Trp79.fwdarw.Lys; Asn96.fwdarw.Arg;
Tyr100.fwdarw.Glu; Leu103.fwdarw.Gly; Tyr106.fwdarw.Trp;
Lys125.fwdarw.Glu; Ser127.fwdarw.Ala; Tyr132.fwdarw.Thr; and
Lys134.fwdarw.Asn.
[0061] A mutein according to the present invention may further
include, with respect to the mature hLcn2 wild type amino acid
sequence, the amino acid replacement Gln 28.fwdarw.His. A mutein
according to the invention may also include, relative to the mature
hLcn2 wild type amino acid sequence, the amino acid replacement Lys
62.fwdarw.Arg. Further, a mutein according to the present invention
may include, relative to the mature hLcn2 wild type amino acid
sequence, the amino acid replacement Phe 71.fwdarw.Pro or Ser. A
further amino acid replacement that may be present in a mutein of
the present invention, relative to the mature hLcn2 wild type amino
acid sequence, is the replacement Lys 74.fwdarw.Glu. Yet a further
amino acid replacement that may be included in a mutein of the
invention is the replacement Lys 75.fwdarw.Glu. A mutein of the
invention may also include, with respect to the mature hLcn2 wild
type amino acid sequence, the amino acid replacement Cys
87.fwdarw.Ser. A mutein of the invention may also include the amino
acid replacement Ser 146.fwdarw.Pro. A further amino acid
replacement that may be present in a mutein of the present
invention, relative to the mature hLcn2 wild type amino acid
sequence, is the replacement Glu 147.fwdarw.Gly. A mutein of the
invention may include further amino acid replacements. The muteins
can further include amino acid replacements, such as
Tyr52.fwdarw.Gln or Val; Ser68.fwdarw.Lys or Asn; or
Arg81.fwdarw.Trp, Asn or His.
[0062] A mutein of the invention typically exists as monomeric
protein. However, it is also possible that a lipocalin mutein of
the invention is able to spontaneously dimerise or oligomerise.
Although the use of lipocalin muteins that form stable monomers may
be preferred for some applications, e.g. because of faster
diffusion and better tissue penetration, the use of lipocalin
muteins that form stable homodimers or multimers may be
advantageous in other instances, since such multimers can provide
for a (further) increased affinity and/or avidity to a given
target. Furthermore, oligomeric forms of the lipocalin mutein may
have slower dissociation rates or prolonged serum half-life.
[0063] It is also noted that the complex formation between the
respective mutein and its ligand is influenced by many different
factors such as the concentrations of the respective binding
partners, the presence of competitors, pH and the ionic strength of
the buffer system used, and the experimental method used for
determination of the dissociation constant K.sub.D (for example
fluorescence titration, competition ELISA or surface plasmon
resonance, just to name a few) or even the mathematical algorithm
which is used for evaluation of the experimental data.
[0064] Therefore, it is also clear to the skilled person that the
K.sub.D values (dissociation constant of the complex formed between
the respective mutein and its target/ligand) may vary within a
certain experimental range, depending on the method and
experimental setup that is used for determining the affinity of a
particular lipocalin mutein for a given ligand. This means that
there may be a slight deviation in the measured K.sub.D values or a
tolerance range depending, for example, on whether the KO value was
determined by surface plasmon resonance (Biacore), by competition
ELISA, or by "direct ELISA."
[0065] In one embodiment, the muteins disclosed herein can be
linked, either N- or C-terminal to an affinity tag such as
pentahistidine tag, a hexahistidine tag or a steptavidin tag (e.g.
Streptag.RTM.). Thus, the present application encompasses also all
explicitly and generic described muteins equipped with such
tags.
[0066] The term "fragment" as used in the present invention in
connection with the feature lipocalin mutein fragment relates to
proteins or peptides derived from full-length mature Lcn 2 that are
N-terminally and/or C-terminally shortened, i.e. lacking at least
one of the N-terminal and/or C-terminal amino acids. Such fragments
include preferably at least 10, more preferably 20, most preferably
30 or more consecutive amino acids of the primary sequence of
mature Lcn 2 and are usually detectable in an immunoassay of mature
Lcn 2. The word "detect" or "detecting" as used herein is
understood both on a quantitative and a qualitative level, as well
as a combination thereof. It thus includes quantitative,
semi-quantitative and qualitative measurements of a molecule of
interest. Accordingly, the presence or absence of a molecule such
as a hepcidin, e.g. in a sample, as well as its concentration or
level may be determined.
[0067] Also included in the scope of the present invention are the
above muteins, which have been altered with respect to their
immunogenicity, to reduce any detected immunogenicity by employing
methods known to the skilled worker in the field.
[0068] Cytotoxic T-cells recognize peptide antigens on the cell
surface of an antigen-presenting cell in association with a class I
major histocompatibility complex (MHC) molecule. The ability of the
peptides to bind to MHC molecules is allele specific and correlates
with their immunogenicity. To reduce the immunogenicity of a given
protein, the ability to predict which peptides in a protein have
the potential to bind to a given MHC molecule is of great value.
Approaches that employ a computational threading approach to
identify potential T-cell epitopes have been previously described
to predict the binding of a given peptide sequence to MHC class I
molecules (Altuvia et al. (1995) J. Mol. Biol. 249: 244-250). Such
an approach may also be utilized to identify potential T-cell
epitopes in the muteins of the invention and to make, depending on
its intended use, a selection of a specific mutein on the basis of
its predicted immunogenicity. It may be furthermore possible to
subject peptide regions that have been predicted to contain T-cell
epitopes to additional mutagenesis to reduce or eliminate these
T-cell epitopes and thus minimize immunogenicity. The removal of
amphipathic epitopes from genetically engineered antibodies has
been described (Mateo et al. (2000) Hybridoma 19(6):463-471) and
may be adapted to the muteins of the present invention. The muteins
thus obtained may possess a minimized immunogenicity, which is
desirable for their use in therapeutic and diagnostic applications,
such as those described below.
[0069] For some applications, it is also useful to employ the
muteins of the invention in a conjugated form. Accordingly, the
invention is also directed to lipocalin muteins which are
conjugated to a compound which can include, but is not limited to
organic molecules, an enzyme label, a colored label, a cytostatic
agent, a toxin, a label that can be photoactivated and which is
suitable for use in photodynamic therapy, a fluorescent label, a
radioactive label, a chromogenic label, a luminescent label, metal
complexes, metal, such as colloidal gold, haptens, digoxigenin,
biotin, a chemotherapeutic metal, or a chemotherapeutic metal, to
name only a few evocative examples. The mutein may also be
conjugated to an organic drug molecule. The conjugation can be
carried out using any conventional coupling method known in the
art.
[0070] The term "organic molecule" or "small organic molecule" as
used herein for the non-natural target denotes an organic molecule
comprising at least two carbon atoms, but preferably not more than
7 or 12 rotatable carbon bonds, having a molecular weight in the
range between 100 and 2000 Dalton, preferably between 100 and 1000
Dalton, and optionally including one or two metal atoms.
[0071] In general, it is possible to label a lipocalin mutein
described herein with any appropriate chemical substance or enzyme,
which directly or indirectly generates a detectable compound or
signal in a chemical, physical, optical, or enzymatic reaction. An
example for a physical reaction and at the same time optical
reaction/marker is the emission of fluorescence upon irradiation.
Alkaline phosphatase, horseradish peroxidase or
.beta.-galactosidase are examples of enzyme labels (and at the same
time optical labels) which catalyze the formation of chromogenic
reaction products. In general, all labels commonly used for
antibodies (except those exclusively used with the sugar moiety in
the Fc part of immunoglobulins) can also be used for conjugation to
the muteins of the present invention. The muteins of the invention
may also be conjugated with any suitable therapeutically active
agent, e.g., for the targeted delivery of such agents to a given
cell, tissue or organ or for the selective targeting of cells,
e.g., of tumor cells without affecting the surrounding normal
cells. Examples of such therapeutically active agents include
radionuclides, toxins, small organic molecules, and therapeutic
peptides (such as peptides acting as agonists/antagonists of a cell
surface receptor or peptides competing for a protein binding site
on a given cellular target). Examples of suitable toxins include,
but are not limited to pertussis-toxin, diphtheria toxin, ricin,
saporin, pseudomonas exotoxin, calicheamicin or a derivative
thereof, a taxoid, a maytansinoid, a tubulysin or a dolastatin
analogue. The dolastatin analogue may be auristatin E,
monomethylauristatin E, auristatin PYE and auristatin PHE. Examples
of cytostatic agent include, but are not limited to Cisplatin,
Carboplatin, Oxaliplatin, 5-Fluorouracil. Taxotere (Docetaxel),
Paclitaxel, Anthracycline (Doxorubicin), Methotrexate, Vinblastin,
Vincristine. Vindesine, Vinorelbine, Dacarbazine, Cyclophosphamide,
Etoposide. Adriamycine, Camptotecine. Combretatastin A-4 related
compounds, sulfonamides, oxadiazolines, benzo[b]thiophenessynthetic
spiroketal pyrans, monotetrahydrofuran compounds, curacin and
curacin derivatives, methoxyestradiol derivatives and Leucovorin.
The lipocalin muteins of the invention may also be conjugated with
therapeutically active nucleic acids such as antisense nucleic acid
molecules, small interfering RNAs, micro RNAs or ribozymes. Such
conjugates can be produced by methods well known in the art.
[0072] In one embodiment, the muteins of the invention may also be
coupled to a targeting moiety that targets a specific body region
in order to deliver the inventive muteins to a desired region or
area within the body. One example wherein such modification may be
desirable is the crossing of the blood-brain-barrier. In order to
cross the blood-brain barrier, the muteins of the invention may be
coupled to moieties that facilitate the active transport across
this barrier (see Gaillard P J, et al. (2005) International
Congress Series. 1277.185-198 or Gaillard P J, et al. (2005) Expert
Opin Drug Deliv. 2(2), 299-309). Such compounds are for example
available under the trade name 28-Trans.TM. (to-BBB technologies
BV, Leiden, NL). Other exemplary targeting molecules to which the
muteins of the present invention may be coupled include antibodies,
antibody fragments or lipocalin muteins with affinity for a desired
target molecule. The target molecule of the targeting moieties may,
for example, be a cell-surface antigen. Cell-surface antigens may
be specific for a cell or tissue type, such as, for example, cancer
cells. Illustrative examples of such cell surface proteins are
HER-2 or proteoglycans such as NEU-2.
[0073] As indicated above, a mutein of the invention may in some
embodiments be conjugated to a compound that extends the serum
half-life of the mutein (in this regard see also PCT publication WO
2006/56464 where such conjugation strategies are described with
references to muteins of human neutrophil gelatinase-associated
lipocalin with binding affinity for CTLA-4). The compound that
extends the serum half-life may be a polyalkylene glycol molecule,
such as polyethylene (PEG) or an activated derivative thereof;
hydroxyethyl starch, fatty acid molecules, such as palmitic acid
(Vajo & Duckworth (2000) Pharmacol. Rev. 52, 1-9), an Fc part
of an immunoglobulin, a C.sub.H3 domain of an immunoglobulin, a
C.sub.H4 domain of an immunoglobulin, albumin or a fragment
thereof, an albumin binding peptide, an albumin binding protein,
transferrin, or the tag Pro-Ala-Ser, to name only a few. The
albumin binding protein may be a bacterial albumin binding protein,
an antibody, an antibody fragment including domain antibodies (see
U.S. Pat. No. 6,696,245, for example), or a lipocalin mutein with
binding activity for albumin. Accordingly, suitable conjugation
compounds for extending the half-life of a lipocalin mutein of the
invention include albumin (Osborn et al. (2002) J. Pharmacol. Exp.
Ther. 303, 540-548), or an albumin binding protein, for example a
bacterial albumin binding domain, such as the one of streptococcal
protein G (Konig, T. and Skerra, A. (1998) J. Immunol. Methods 218,
73-83). Other examples of albumin binding peptides that can be used
as conjugation partner are, for instance, those having a
Cys-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Cys consensus sequence,
wherein Xaa.sub.1 is Asp, Asn, Ser, Thr, or Trp; Xaa.sub.2 is Asn,
Gln. His, Ile, Leu, or Lys; Xaa.sub.3 is Ala, Asp, Phe, Trp, or
Tyr; and Xaa.sub.4 is Asp, Gly, Leu, Phe, Ser, or Thr as described
in US patent application 2003/0069395 or Dennis et al (Dennis et
al. (2002) J. Biol. Chem. 277, 35035-35043).
[0074] In other embodiments, albumin itself or a biological active
fragment of albumin can be used as compound of a lipocalin mutein
of the invention that extends the serum half-life of the mutein.
The term "albumin" includes all mammal albumins such as human serum
albumin or bovine serum albumin or rat albumin. The albumin or
fragment thereof can be recombinantly produced as described in U.S.
Pat. No. 5,728,553 or European patent applications EP 0 330 451 and
EP 0 361 991. Recombinant human albumin (Recombumin.RTM.) for use
as a protein stabilizer is for example available from Novozymes
Delta Ltd. (Nottingham, UK).
[0075] If the albumin-binding protein is an antibody fragment it
may be a domain antibody Domain Antibodies (dAbs) are engineered to
allow precise control over biophysical properties and in vivo
half-life to create the optimal safety and efficacy product
profile. Domain Antibodies are for example commercially available
from Domantis Ltd. (Cambridge, UK and MA. USA).
[0076] Using transferrin as a moiety to extend the serum half-life
of the muteins of the invention, the muteins can be genetically
fused to the N or C terminus, or both, of non-glycosylated
transferrin. Non-glycosylated transferrin has a half-life of 14-17
days, and a transferrin fusion protein will similarly have an
extended half-life. The transferrin carrier also provides high
bioavailability, biodistribution and circulating stability. This
technology is commercially available from BioRexis (BioRexis
Pharmaceutical Corporation. PA, USA). Recombinant human transferrin
(DeltaFerrin.TM.) for use as a protein stabilizer is also
commercially available from Novozymes Delta Ltd. (Nottingham,
UK).
[0077] If an Fc part of an immunoglobulin is used for the purpose
to prolong the serum half-life of the muteins of the invention, the
SynFusion.TM. technology, commercially available from Syntonix
Pharmaceuticals, Inc (MA, USA), may be used. The use of this
Fc-fusion technology allows the creation of longer-acting
biopharmaceuticals and may for example comprise two copies of the
mutein linked to the Fc region of an antibody to improve
pharmacokinetics, solubility, and production efficiency.
[0078] Yet another alternative to prolong the half-life of a mutein
of the invention is to fuse the N- or C-terminus of a mutein of the
invention to long, unstructured, flexible glycine-rich sequences
(for example poly-glycine with about 20 to 80 consecutive glycine
residues). This approach disclosed in WO2007/038619, for example,
has also been term "rPEG" (recombinant PEG).
[0079] If polyalkylene glycol is used as compound that extends the
half-life of the mutein, the polyalkylene glycol can be substituted
or unsubstituted. It can also be an activated polyalkylene
derivative. Examples of suitable compounds are polyethylene glycol
(PEG) molecules as described in WO 99/84016, in U.S. Pat. No.
6,177,074 or in U.S. Pat. No. 6,403,564 in relation to interferon,
or as described for other proteins such as PEG-modified
asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide
dismutase (see for example. Fuertges et al. (1990) "The Clinical
Efficacy of Poly(Ethylene Glycol)-Modified Proteins" J. Control.
Release 11, 139-148). The molecular weight of such a polymer,
preferrably polyethylene glycol, may range from about 300 to about
70.000 Dalton, including for example, polyethylene glycol with a
molecular weight of about 10 000, of about 20.000, of about 30.000
or of about 40.000 Dalton. Moreover. e.g. as described in U.S. Pat.
Nos. 6,500,930 or 6,620,413, carbohydrate oligo- and polymers such
as starch or hydroxyethyl starch (HES) can be conjugated to a
mutein of the invention for the purpose of serum half-life
extension.
[0080] In another embodiment, in order to provide suitable amino
acid side chains for conjugating one of the above compounds to the
muteins of the invention artificial amino acids may be introduced
by mutagenesis. Generally, such artificial amino acids are designed
to be more reactive and thus to facilitate the conjugation to the
desired moiety. One example of such an artificial amino acid that
may be introduced via an artificial tRNA is
para-acetyl-phenylalanine.
[0081] For several applications of the muteins disclosed herein, it
may be advantageous to use them in the form of fusion proteins. In
some embodiments, the inventive mutein is fused at its N-terminus
and/or it's C-terminus to a protein, a protein domain or a peptide
such as a signal sequence and/or an affinity tag.
[0082] For pharmaceutical applications, a mutein of the invention
may be fused to a fusion partner that extends the in vivo serum
half-life of the mutein (see again PCT publication WO 2006/56464
where suitable fusion partner are described with references to
muteins of human neutrophile gelatinase-associated lipocalin with
binding affinity for CTLA-4). Similar to the conjugated compounds
described above, the fusion partner may be an Fc part of an
immunoglobulin, a C.sub.H3 domain of an immunoglobulin, a C.sub.H4
domain of an immunogloubulin, albumin, an albumin binding peptide
or an albumin binding protein, to name only a few. Again, the
albumin binding protein may be a bacterial albumin binding protein
or a lipocalin mutein with binding activity for albumin.
Accordingly, suitable fusion partners for extending the half-life
of a lipocalin mutein of the invention include albumin (Osbom, B.
L. et al. (2002) supra J. Pharmacol. Exp. Ther. 303, 540-548), or
an albumin binding protein, for example, a bacterial albumin
binding domain, such as streptococcal protein G (Konig, T, and
Skerra, A. (1998) supra J. Immunol. Methods 218, 73-83). The
albumin binding peptides described in Dennis et al. supra (2002) or
US patent application 2003/0069395 having a
Cys-Xaa.sub.1-Xaa.sub.2-Xaa.sub.3-Xaa.sub.4-Cys consensus sequence,
wherein Xaa.sub.1 is Asp. Asn. Set, Thr, or Trp; Xaa.sub.2 is Asn,
Gln, His, Ile, Leu, or Lys; Xaa.sub.3 is Ala, Asp, Phe, Trp, or
Tyr, and Xaa.sub.4 is Asp, Gly, Leu. Phe, Ser, or Thr can also be
used as fusion partner. It is also possible to use albumin itself
or a biological active fragment of albumin as fusion partner of a
lipocalin mutein of the invention. The term "albumin" includes all
mammal albumins such as human serum albumin or bovine serum albumin
or rat serum albumin. The recombinant production of albumin or
fragments thereof is well known in the art and for example
described in U.S. Pat. No. 5,728,553, European patent application
EP 0 330 451 or EP 0 361 991
[0083] The fusion partner may confer new characteristics to the
inventive lipocalin mutein such as enzymatic activity or binding
affinity for other molecules. Examples of suitable fusion proteins
are alkaline phosphatase, horseradish peroxidase,
gluthation-S-transferase, the albumin-binding domain of protein G,
protein A, antibody fragments, oligomerization domains, lipocalin
muteins of same or different binding specificity (which results in
the formation of "duocalins," cf. Schlehuber, S., and Skerra, A.
(2001), Duocalins, engineered ligand-binding proteins with dual
specificity derived from the lipocalin fold (Biol. Chem. 382,
1335-1342), or toxins.
[0084] In particular, it may be possible to fuse a lipocalin mutein
of the invention with a separate enzyme active site such that both
"components" of the resulting fusion protein together act on a
given therapeutic target. The binding domain of the lipocalin
mutein attaches to the disease-causing target, allowing the enzyme
domain to abolish the biological function of the target.
[0085] Affinity tags such as the Strep-Tag.RTM. or Strep-Tag.RTM.
II (Schmidt. T. G. M. et al. (1996) J. Mol. Biol. 255, 753-766),
the myc-tag, the FLAG-tag, the His.sub.6-tag or the HA-tag or
proteins such as glutathione-S-transferase also allow easy
detection and/or purification of recombinant proteins are further
examples of preferred fusion partners. Finally, proteins with
chromogenic or fluorescent properties such as the green fluorescent
protein (GFP) or the yellow fluorescent protein (YFP) are suitable
fusion partners for a lipocalin mutein of the invention as
well.
[0086] The term "fusion protein" as used herein also includes
lipocalin muteins according to the invention containing a signal
sequence. Signal sequences at the N-terminus of a polypeptide
direct this polypeptide to a specific cellular compartment, for
example the periplasm of E. coli or the endoplasmatic reticulum of
eukaryotic cells. A large number of signal sequences is known in
the art. A preferred signal sequence for secretion a polypeptide
into the periplasm of E. coli is the OmpA-signal sequence.
[0087] The present invention also relates to nucleic acid molecules
(DNA and RNA) comprising nucleotide sequences coding for muteins as
described herein. Since the degeneracy of the genetic code permits
substitutions of certain codons by other codons specifying the same
amino acid, the invention is not limited to a specific nucleic acid
molecule encoding a mutein of the invention but includes all
nucleic acid molecules comprising nucleotide sequences encoding a
functional mutein.
[0088] A nucleic acid molecule disclosed in this application may be
"operably linked" to a regulatory sequence (or regulatory
sequences) to allow expression of this nucleic acid molecule.
[0089] A nucleic acid molecule, such as DNA, is referred to as
"capable of expressing a nucleic acid molecule" or capable "to
allow expression of a nucleotide sequence" if it includes sequence
elements which contain information regarding to transcriptional
and/or translational regulation, and such sequences are "operably
linked" to the nucleotide sequence encoding the polypeptide. An
operable linkage is a linkage in which the regulatory sequence
elements and the sequence to be expressed are connected in a way
that enables gene expression. The precise nature of the regulatory
regions necessary for gene expression may vary among species, but
in general these regions include a promoter which, in prokaryotes,
contains both the promoter per so, i.e. DNA elements directing the
initiation of transcription, as well as DNA elements which, when
transcribed into RNA, will signal the initiation of translation.
Such promoter regions normally include 5' non-coding sequences
involved in initiation of transcription and translation, such as
the -35/-10 boxes and the Shine-Dalgarno element in prokaryotes or
the TATA box, CAAT sequences, and 5'-capping elements in
eukaryotes. These regions can also include enhancer or repressor
elements as well as translated signal and leader sequences for
targeting the native polypeptide to a specific compartment of a
host cell.
[0090] In addition, the 3' non-coding sequences may contain
regulatory elements involved in transcriptional termination,
polyadenylation or the like. If, however, these termination
sequences are not satisfactory functional in a particular host
cell, then they may be substituted with signals functional in that
cell.
[0091] Therefore, a nucleic acid molecule of the invention can
include a regulatory sequence, preferably a promoter sequence. In
another preferred embodiment, a nucleic acid molecule of the
invention includes a promoter sequence and a transcriptional
termination sequence. Suitable prokaryotic promoters are, for
example, the tet promoter, the lacUV5 promoter or the T7 promoter.
Examples of promoters useful for expression in eukaryotic cells are
the SV40 promoter or the CMV promoter.
[0092] The nucleic acid molecules of the invention can also be part
of a vector or any other kind of cloning vehicle, such as a
plasmid, a phagemid, a phage, a baculovirus, a cosmid or an
artificial chromosome.
[0093] The DNA molecule encoding lipocalin muteins of the
invention, and in particular a cloning vector containing the coding
sequence of such a lipocalin mutein can be transformed into a host
cell capable of expressing the gene. Transformation can be
performed using standard techniques (Sambrook, J et al. (1989),
supra).
[0094] Thus, the invention is also directed to a host cell
containing a nucleic acid molecule as disclosed herein.
[0095] The invention also relates to a method for the production of
a mutein of the invention, wherein the mutein, a fragment of the
mutein or a fusion protein of the mutein and another polypeptide is
produced starting from the nucleic acid coding for the mutein by
means of genetic engineering methods. The method can be carried out
in vivo, the mutein can for example be produced in a bacterial or
eucaryotic host organism and then enriched, purified or isolated
from this host organism or its culture. It is also possible to
produce a protein in vitro, for example by use of an in vitro
translation system. The term "enriched" means that the mutein or a
functional fragment thereof constitutes a significantly higher
fraction of the total protein present in a sample or solution of
interest than in a sample or solution from which it was taken.
Enrichment may for instance include the isolation of a certain
fraction from a cell extract. This may be obtained by standard
techniques such as centrifugation. Examples of other means of
enrichment are filtration or dialysis, which may for instance be
directed at the removal of undesired molecules below a certain
molecular weight or a precipitation using organic solvents or
ammonium sulphate. Purification may for instance include a
chromatographic technique, for example gel filtration, ion exchange
chromatography, affinity purification, hydrophobic interaction
chromatography or hydrophobic charge induction chromatography.
Another example for a purification is an electrophoretic technique,
such as preparative capillary electrophoresis. Isolation may
include the combination of similar methods. As used herein,
"substantially pure" or "substantially purified" means a compound
or species that is the predominant species present (i.e., on a
molar basis it is more abundant than any other individual species
in the composition). In some embodiments, a substantially purified
composition is a composition in which the species includes at least
about 50 percent (on a molar basis) of all molecular or, as
applicable, all macromolecular species present. In certain
embodiments, a substantially pure composition will have more than
about 80%, about 85%, about 90%, about 95%, or about 99% of all
molecular or, as applicable, all macromolar species present in the
composition.
[0096] When producing the mutein in vivo, a nucleic acid encoding a
mutein of the invention is introduced into a suitable bacterial or
eukaryotic host organism by means of recombinant DNA technology (as
already outlined above). For this purpose, the host cell is first
transformed with a cloning vector comprising a nucleic acid
molecule encoding a mutein of the invention using established
standard methods (Sambrook, J. et al. (1989), supra). The host cell
is then cultured under conditions, which allow expression of the
heterologous DNA and thus the synthesis of the corresponding
polypeptide. Subsequently, the polypeptide is recovered either from
the cell or from the cultivation medium.
[0097] In one aspect, the present invention relates to a method for
the generation of a mutein which binds hepcidin, comprising:
[0098] subjecting a nucleic acid molecule encoding a lipocalin to
mutagenesis, resulting in one or more mutein nucleic acid
molecule(s).
[0099] The method can further include:
[0100] expressing the one more mutein nucleic acid molecule(s)
obtained in (a) in a suitable expression system,
[0101] bringing the plurality of muteins into contact with at least
a fragment or a mature form of hepcidin, and
[0102] enriching the one or more mutein(s) having a detectable
binding affinity for a given target by means of selection and/or
isolation.
[0103] The term "mutagenesis" as used herein means that the
experimental conditions are chosen such that the amino acid
naturally occurring at a given sequence position of the lipocalin,
including Lcn 2 (hNGAL, Swiss-Prot data bank entry P80188) can be
substituted by at least one amino acid that is not present at this
specific position in the respective natural polypeptide sequence.
The term "mutagenesis" also includes the (additional) modification
of the length of sequence segments by deletion or insertion of one
or more amino acids. Thus, it is within the scope of the invention
that, for example, one amino acid at a chosen sequence position is
replaced by a stretch of three random mutations, leading to an
insertion of two amino acid residues compared to the length of the
respective segment of the wild type protein. Such an insertion of
deletion may be introduced independently from each other in any of
the peptide segments that can be subjected to mutagenesis in the
invention. In one exemplary embodiment of the invention, an
insertion of several mutations may be introduced into the loop AB
of the chosen lipocalin scaffold (cf. International Patent
Application WO 2005/019256 which is incorporated by reference its
entirety herein). The term "random mutagenesis" means that no
predetermined single amino acid (mutation) is present at a certain
sequence position but that at least two amino acids can be
incorporated with a certain probability at a predefined sequence
position during mutagenesis.
[0104] In one non-limiting approach, the coding sequence of human
Lipocalin 2 can be used as a starting point for the mutagenesis of
the peptide segments selected in the present invention. For the
mutagenesis of the recited amino acid positions, the person skilled
in the art has at his disposal the various established standard
methods for site-directed mutagenesis (Sambrook, J. et al. (1989),
supra). A commonly used technique is the introduction of mutations
by means of PCR (polymerase chain reaction) using mixtures of
synthetic oligonucleotides, which bear a degenerate base
composition at the desired sequence positions. Other similar
techniques are well known to those of skill in the art.
[0105] The nucleic acid molecules defined above can be connected by
ligation with the missing 5'- and 3'-sequences of a nucleic acid
encoding a lipocalin polypeptide and/or the vector, and can be
cloned in a known host organism. A multitude of established
procedures are available for ligation and cloning (Sambrook, J. et
al. (1989), supra). For example, recognition sequences for
restriction endonucleases also present in the sequence of the
cloning vector can be engineered into the sequence of the synthetic
oligonucleotides. Thus, after amplification of the respective PCR
product and enzymatic cleavage the resulting fragment can be easily
cloned using the corresponding recognition sequences.
[0106] Longer sequence segments within the gene coding for the
protein selected for mutagenesis can also be subjected to random
mutagenesis via known methods, for example by use of the polymerase
chain reaction under conditions of increased error rate, by
chemical mutagenesis or by using bacterial mutator strains. Such
methods can also be used for further optimization of the target
affinity or specificity of a lipocalin mutein Mutations possibly
occurring outside the segments of experimental mutagenesis are
often tolerated or can even prove to be advantageous, for example
if they contribute to an improved folding efficiency or folding
stability of the lipocalin mutein.
[0107] In a further embodiment, the method includes subjecting the
nucleic acid molecule to mutagenesis at nucleotide triplets coding
for at least any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 or 20 of the sequence positions corresponding to the
sequence positions 33, 36, 40, 41, 42, 43, 44, 46, 47, 48, 49, 50,
51, 52, 54, 55, 59, 65, 68, 70, 72, 73, 75, 77, 78, 79, 80, 81, 86,
87, 98, 96, 99, 100, 103, 106, 107, 110, 111, 125, 127, 132, 134,
136 and/or 138 of the linear polypeptide sequence of the lipocalin,
or, for example, human Lipocalin 2. Such a nucleic acid may
subjected to mutagenesis and introduced into a suitable bacterial
or eukaryotic host organism by using recombinant DNA technology.
Obtaining a nucleic acid library of a lipocalin can be carried out
using any suitable technique that is known in the art for
generating lipocalin muteins with antibody-like properties, i.e.
muteins that have affinity towards a given target. Examples of such
combinatorial methods are described in detail in the international
patent applications WO 99/16873, WO 00/75308, WO 03/029471, WO
03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255, WO
2005/019256, or WO 2006/56464 for instance. The content of each of
these patent applications is incorporated by reference herein in
their entirety. After expression of the nucleic acid sequences that
were subjected to mutagenesis in an appropriate host, the clones
carrying the genetic information for the plurality of respective
lipocalin muteins, which bind a given target can be selected from
the library obtained. Well known techniques can be employed for the
selection of these clones, such as phage display (reviewed in Kay,
B. K. et al (1996) supra; Lowman, H. B (1997) supra or Rodi, D. J.,
and Makowski, L. (1999) supra), colony screening (reviewed in Pini.
A. et al. (2002) Comb. Chem. High Throughput Screen. 5, 503-510),
ribosome display (reviewed in Amstutz, P. et al. (2001) Curr. Opin.
Biotechnol. 12, 400-405) or mRNA display as reported in Wilson, D.
S. et al. (2001) Proc. Natl. Acad. Sci. USA 98, 3750-3755 or the
methods specifically described in WO 99/16873, WO 00/75308, WO
03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO
2005/019255, WO 20051019256, or WO 2006/56464.
[0108] In accordance with this disclosure, another embodiment of
the above methods comprises:
[0109] (i) providing at least a fragment of hepcidin as a given
target/ligand for example,
[0110] contacting the plurality of muteins with said target/ligand
in order to allow formation of complexes between said ligand and
muteins having binding affinity for said target/ligand, and
[0111] removing muteins having no or no substantial binding
affinity.
[0112] In one embodiment of the methods of the invention, the
selection binding affinity is carried out under competitive
conditions. Competitive conditions as used herein means that
selection of muteins encompasses at least one step in which the
muteins and the fragment of hepcidin or a mature hepcidin such as
Hepcidin-25 (target) are brought in contact in the presence of an
additional ligand, which competes with binding of the muteins to
the target (hepcidin). This additional target may be another form
of hepcidin, for example Hepcidin-20 (in case muteins are to be
selected that selectively bind Hepcidin-25 or even the five
N-terminal residues of Hepcidin-25 (as indicated earlier, it is
presently assumed that the iron-regulating bioactivity is almost
exclusively due to the 25 amino acid form Hepcidin-25, indicating
that the five N-terminal amino acids are essential for this
activity, Kenma et al., supra), an excess of the target itself or
any other non-physiological ligand of the hepcidin that binds at
least an overlapping epitope to the epitope recognized by the
muteins of the invention and thus interferes with target (hepcidin)
binding of the muteins. Alternatively, the additional ligand
competes with binding of the muteins by complexing an epitope
distinct from the binding site of the muteins to the target by
allosteric effects. Accordingly, any fragment, precursor or mature
form of Hepcidin can be used in the generation of muteins of the
invention.
[0113] A further embodiment of the methods of the invention
involves operably fusing a nucleic acid coding for the plurality of
muteins of of the invention and resulting from mutagenesis at the
3' end with a gene coding for the coat protein pill of a
filamentous bacteriophage of the M13-family or for a fragment of
this coat protein, in order to select at least one mutein for the
binding of a given ligand.
[0114] The fusion protein may include additional components such as
an affinity tag, which allows the immobilization, detection and/or
purification of the fusion protein or its parts. Furthermore, a
stop codon can be located between the sequence regions encoding the
lipocalin or its muteins and the phage capsid gene or fragments
thereof, wherein the stop codon, preferably an amber stop codon, is
at least partially translated into an amino acid during translation
in a suitable suppressor strain.
[0115] For example, the phasmid vector pTLPC27, now also called
pTlc27 that is described here can be used for the preparation of a
phagemid library encoding muteins of the invention. The inventive
nucleic acid molecules coding for muteins of the invention can be
inserted into the vector using the two BstXI restriction sites.
After ligation a suitable host strain such as E. coli XL1-Blue is
transformed with the resulting nucleic acid mixture to yield a
large number of independent clones. A respective vector can be
generated for the preparation of a hyperphagemid library, if
desired.
[0116] Once a mutein with affinity to a given target has been
selected, it is additionally possible to subject such a mutein to
another mutagenesis in order to subsequently select variants of
even higher affinity or variants with improved properties such as
higher thermostability, improved serum stability, thermodynamic
stability, improved solubility, improved monomeric behavior,
improved resistance against thermal denaturation, chemical
denaturation, proteolysis, or detergents etc. This further
mutagenesis, which in case of aiming at higher affinity can be
considered as in vitro "affinity maturation," can be achieved by
site specific mutation based on rational design or a random
mutation. Another possible approach for obtaining a higher affinity
or improved properties is the use of error-prone PCR, which results
in point mutations over a selected range of sequence positions of
the lipocalin mutein. The error-prone PCR can be carried out in
accordance with any known protocol such as the one described by
Zaccolo at al. (1996) J. Mol. Biol. 255, 589-603. Other methods of
random mutagenesis that are suitable for such purposes include
random insertion/deletion (RID) mutagenesis as described by
Murakami et al. (2002) Nat. Biotechnol. 20, 76-81 or nonhomologous
random recombination (NRR) as described by Bittker et al. (2002)
Nat. Biotechnol. 20, 1024-1029. If desired, affinity maturation can
also be carried out according to the procedure described in WO
00/75308 or Schlehuber et al. (2000) J. Mol. Biol. 297, 1105-1120,
where muteins of the bilin-binding protein having high affinity to
digoxigenin were obtained. A further approach for improving the
affinity is to carry out positional saturation mutagenesis. In this
approach "small" nucleic acid libraries can be created in which
amino acid exchanges/mutations are only introduced at single
positions within any of the four loop segments. These libraries are
then directly subjected to a selection step (affinity screening)
without further rounds of panning. This approach allows the
identification of residues that contribute to improved binding of
the desired target and allows identification of "hot spots" that
are important for the binding.
[0117] In one embodiment, the above method for modifying a mutein
further includes introducing a Cys residue at at least one of any
of the sequence positions that correspond to sequence positions 14,
21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild type
sequence of human Lipocalin 2 and coupling a moiety that is able to
modify the serum half time of said mutein via the thiol group of a
Cys residue introduced at at least one of any of the sequence
positions that correspond to sequence positions 14, 21, 60, 84, 88,
116, 141, 145, 143, 146 or 158 of the wild type sequence of hNGAL.
The moiety that is able to modify the serum half time of said
mutein may be selected from the group consisting of a polyalkylene
glycol molecule and hydroxyethylstarch.
[0118] Where a protein of the invention is a human Lipocalin 2
mutein of the invention, the naturally occurring disulfide bond
between Cys 76 and Cys 175 may be removed. Accordingly, such
muteins (or any other human Lipocalin 2 mutein that does not
include an intramolecular disulfide bond) can be produced in a cell
compartment having a reducing redox milieu, for example, in the
cytoplasma of Gram-negative bacteria.
[0119] In case a lipocalin mutein of the invention includes
intramolecular disulfide bonds, it may be preferred to direct the
nascent polypeptide to a cell compartment having an oxidizing redox
milieu using an appropriate signal sequence. Such an oxidizing
environment may be provided by the periplasm of Gram-negative
bacteria such as E. coli, in the extracellular milieu of
Gram-positive bacteria or in the lumen of the endoplasmatic
reticulum of eukaryotic cells and usually favors the formation of
structural disulfide bonds.
[0120] It is, however, also possible to produce a mutein of the
invention in the cytosol of a host cell, preferably E. coli. In
this case, the polypeptide can either be directly obtained in a
soluble and folded state or recovered in form of inclusion bodies,
followed by renaturation in vitro. A further option is the use of
specific host strains having an oxidizing intracellular milieu,
which may thus allow the formation of disulfide bonds in the
cytosol (Venturi et al. (2002) J. Mol. Biol. 315, 1-8).
[0121] However, a mutein of the invention may not necessarily be
generated or produced only by use of genetic engineering. Rather, a
lipocalin mutein can also be obtained by chemical synthesis such as
Merrifield solid phase polypeptide synthesis or by in vitro
transcription and translation. It is for example possible that
promising mutations are identified using molecular modeling and
then to synthesize the wanted (designed) polypeptide in vitro and
investigate the binding activity for a given target. Methods for
the solid phase and/or solution phase synthesis of proteins are
well known in the art (reviewed, e.g., in LLoyd-Williams et al.
(1997) Chemical Approaches to the Synthesis of Peptides and
Proteins. CRC Press, Boca Raton, Fields, GB, and Colowick (1997)
Solid-Phase Peptide Synthesis. Academic Press, San Diego, or
Bruckdorfer et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).
[0122] In another embodiment, the muteins of the invention may be
produced by in vitro transcription/translation employing
well-established methods known to those skilled in the art.
[0123] The invention also relates to a pharmaceutical composition
that includes at least one inventive mutein referred to in the
claims or a fusion protein or conjugates thereof and, optionally, a
pharmaceutically acceptable excipient.
[0124] The lipocalin muteins according to the invention can be
administered via any parenteral or non-parenteral (e.g. enteral)
route that is therapeutically effective for proteinaceous
drugs.
[0125] Accordingly, the muteins of the present invention can be
formulated into compositions using pharmaceutically acceptable
ingredients as well as established methods of preparation (Gennaro
and Gennaro (2000) Remington: The Science and Practice of Pharmacy,
20th Ed., Lippincott Williams & Wilkins. Philadelphia. Pa.). To
prepare the pharmaceutical compositions, pharmaceutically inert
inorganic or organic excipients can be used.
[0126] A protein of the invention of the invention may also be used
to target a compound to a pre-selected site. In one such
embodiment, a protein of the invention is used for the targeting of
a pharmaceutically active compound to a pre-selected site in an
organism or tissue, comprising:
[0127] a) conjugating the protein with said compound, and
[0128] b) delivering the protein/compound complex to the
pre-selected site.
[0129] For such a purpose the mutein is contacted with the compound
of interest in order to allow complex formation. Then the complex
comprising the mutein and the compound of interest are delivered to
the pre-selected site. This may, for example, be achieved by
coupling the mutein to a targeting moiety, such as an antibody,
antibody fragment or lipocalin mutein or lipocalin mutein fragment
with binding affinity for the selected target.
[0130] This use is in particular suitable, but not restricted to,
for delivering a drug (selectively) to a pre-selected site in an
organism, such as an infected body part, tissue or organ which is
supposed to be treated with the drug. Besides formation of a
complex between mutein and compound of interest, the mutein can
also be reacted with the given compound to yield a conjugate of
mutein and compound. Similar to the above complex, such a conjugate
may be suitable to deliver the compound to the pre-selected target
site.
[0131] Such a conjugate of mutein and compound may also include a
linker that covalently links mutein and compound to each other.
Optionally, such a linker is stable in the bloodstream but is
cleavable in a cellular environment.
[0132] The muteins disclosed herein and its derivatives can thus be
used in many fields similar to antibodies or fragments thereof. In
addition to their use for binding to a support, allowing the target
of a given mutein or a conjugate or a fusion protein of this target
to be immobilized or separated, the muteins can be used for
labeling with an enzyme, an antibody, a radioactive substance or
any other group having biochemical activity or defined binding
characteristics. By doing so, their respective targets or
conjugates or fusion proteins thereof can be detected or brought in
contact with them. For example, muteins of the invention can serve
to detect chemical structures by means of established analytical
methods (e.g. ELISA or Western Blot) or by microscopy or
immunosensorics. Here, the detection signal can either be generated
directly by use of a suitable mutein conjugate or fusion protein or
indirectly by immunochemical detection of the bound mutein via an
antibody.
[0133] Numerous possible applications for the inventive muteins
also exist in medicine. In addition to their use in diagnostics and
drug delivery, a mutant polypeptide of the invention, which binds,
for example, tissue- or tumor-specific cellular surface molecules
can be generated. Such a mutein may, for example, be employed in
conjugated form or as a fusion protein for "tumor imaging" or
directly for cancer therapy.
[0134] In a further aspect, the present invention also encompasses
the use of a mutein according to the invention for the manufacture
of a pharmaceutical composition. The pharmaceutical composition
thus obtained may be suited for reducing the level of a Hepcidin.
The pharmaceutical composition may be used as monotherapy or as
combination therapy. Accordingly, the invention also relates to a
mutein as defined above for the treatment of a disease or disorder
associated with an altered, e.g. increased or reduced level of a
Hepcidin.
Diseases Associated with Hepcidin
[0135] Anemia is a disease associated with serum iron depletion
leading to a decrease of hematological parameters such as red blood
cell (RBC) counts, hematocrit (Ht), hemoglobin (Hb), serum iron
level and transferrin (Tf) saturation. This results in a decreased
oxygen level in the blood and is associated with a declined quality
of life (QOL) described by weakness, poor concentration, shortness
of breath and dyspnea. Severe anemia can lead to a fast heart rate,
cardiac enlargement and heart failure. Anemia is often associated
with chronic kidney disease/established chronic kidney disease
(CKD), anemia of cancer (AC), chemotherapy induced anemia (CIA) and
anemia of chronic disease (ACD).
[0136] The effective management of anemia has a major impact on
quality of life and may influence the survival of patients. The
declined quality of life can be described by weakness, fatigue,
poor concentration, shortness of breath up to dyspnea. Severe
anemia is associated with a fast heart rate, and can lead to
cardiac enlargements and heart failure.
[0137] The standard treatment of care are transfusions and the
administration of ESAs and iron. Nevertheless, new therapeutic
approaches are desired since the standard treatments are associated
with the following disadvantages or potential draw backs.
Transfusion bears the risk of hemolysis, infections and allergic
reactions due to an incompatible blood type. Iron treatment can
lead to iron overload in long term treatments and is not
recommended for the treatment of anemia of inflammation since iron
contributes to inflammatory responses (e.g. inflammatory joint
disease). As far as ESAs are concerned, about 40-50% of anemic
patients are ESA non-responder with no or delayed Hb-response only
after high dose ESA-treatment that are associated with safety
concerns like poorer survival and shorter progression free survival
time in cancer patients.
[0138] Iron deficiency anemia is a disorder of iron homeostasis
that is easily cured by iron administration in contrast to anemia
associated with inflammatory disease. Hepcidin is a parameter that
allows distinguishing between these two disorders since the
hepcidin level is only upregulated in combination with
inflammation.
[0139] Anemias associated with chronic inflammatory disease like
chronic infections, rheumatologic and systemic autoimmune disorders
and inflammatory bowel disease are called anemia of inflammation
(AI) or anemia of chronic disease (ACD). Hepcidin expression is
induced by the inflammatory cytokine IL-6, as part of the
inflammatory response, resulting in iron deficiency induced anemia
and a blunted response to ESAs.
[0140] Patients with established chronic kidney disease (chronic
renal failure (CRF)) develop uremic anemia as one of the most
obvious signs of the disease. This symptom is caused by impeded
renal production of erythropoietin (EPO). EPO controls red blood
cell (RBC) production by promoting survival, proliferation and
differentiation of erythroid progenitors in the bone marrow.
Effective management of anemia in chronic renal failure (CRF) has a
major impact on quality of life and may influence survival.
Supplementation with recombinant human erythropoietin (rhEPO) is
currently the standard treatment for anemia in those patients. A
response rate of 70-90% to various ESA's (erythropoesis stimulating
agents) has been observed in clinical trials with CRF patients.
Only in patients with additional inflammatory disease hepcidin
plays a prominent role in the anemia associated with CKD.
[0141] Anemia is common in patients with cancer and has a
multifactorial aetiology. It may be related to the malignancy
itself and its extent, as well as to the type, duration and
intensity of myelosuppressive chemotherapy. Moreover, most patients
with cancer have been shown to have inappropriately low levels of
circulating EPO for their degree of anemia, reflecting a change in
this homeostatic mechanism. The incidence of anemia severe enough
to result in blood transfusions may be as high as 60% in certain
tumor types. Anemic patients with cancer may experience symptoms as
fatigue, dizziness, shortness of breath, and cardiovascular
symptoms such as palpitations and cardiac failure. Such clinical
sequelae may decrease the quality of life of these patients.
Furthermore, a potential relationship between the correction of
anemia and increased survival in patients receiving chemotherapy
has recently been discussed. Currently, therapeutic options for
anemia in cancer patients are RBC transfusions or ESA's.
Transfusion of RBCs can be associated with non-hemolytic and
hemolytic transfusion reactions, iron overload in heavily
transfused patients, or the transmission of infections. Safety and
screening requirements in transfusion therapy have increased the
logistics and cost of transfusion therapy thus restricting
transfusions to cases of severe and/or symptomatic anemia. ESA's
have provided an alternative to blood transfusions in the treatment
of symptomatic anemia which is still not severe enough to merit
transfusions with current policies. However, a clear dose response
relationship for ESA's has not been established, and 40% to 50% of
patients show no Hb response at all or a delayed response. During
the last years important concerns have emerged regarding the impact
of ESAs on cancer patients' survival as well as their potential to
increase the risk of thromboembolism (in march 2007 the FDA
instituted a black-box warning about the possible association of
ESAs with tumor promotion and thromboembolic events). There is
raising evidence from the literature that ESA-resistance of cancer
patients is not only predicted by a missing increase in Hb-response
within 4 weeks of ESA administration but also by an elevated
hepcidin level--presumably as part of an inflammatory response.
[0142] As explained above, Hepcidin is the central negative
regulator of iron homeostasis. Hepcidin production increases with
iron loading and inflammation and decreases under low iron
conditions and hypoxia. Hepcidin acts via binding to the only known
mammalian cellular iron exporter, ferroportin, and induces its
internalization and degradation. Since ferroportin is expressed in
the duodenal enterocytes, spleen, and liver, hepcidin increase, and
the subsequent decrease of ferroportin, results in the inhibition
of duodenal iron absorption, release of recycled iron from
macrophages, and mobilization of iron stores in the liver. Hepcidin
is thought to play a critical role in the development of anemia
associated with inflammatory disease. Acute or chronic inflammatory
conditions result in the up-regulation of hepcidin expression,
leading to iron deficiency, which can cause anemia associated with
inflammatory disease (ACD), cancer (AC, CIA) and Chronic Kidney
Disease (CKD) (anemia of CKD).
[0143] A lipocalin mutein according to the invention may be used as
an antagonist of a hepcidin (supra). In this regard a lipocalin
mutein according to the invention, typically an isolated lipocalin
mutein, may be used in therapy, such as human therapy. A respective
mutein is capable of forming a complex with a hepcidin, e.g. a
human hepcidin, typically with high affinity. Thereby the lipocalin
mutein typically blocks the interaction with the hepcidin receptor
ferroportin. As a result internalization and degradation of
ferroportin are prevented. The lipocalin mutein thereby supports
erythropoiesis by allowing mobilization of stored iron and improved
enteral iron absorption. An illustrative example of a subject in
need of application of a respective antagonist of a hepcidin
according to the invention is a subject hyporesponsive to
ESA-therapy (about 40-50% of patients) which is thought to be
caused by the decreased availability of iron for the synthesis of
hemoglobin due to upregulated hepcidin. The term "subject" refers
to a vertebrate animal, including a mammal, and in particular a
human, in which case the term "patient" can also be used. In some
embodiments, the subject may have a disorder that would benefit
from a decreased level of a hepcidin such as hepcidin-25, a
decrease in bioactivity of a hepcidin (e.g. hepcidin-25
bioactivity), and/or an increase in serum iron level, reticulocyte
count, red blood cell count, hemoglobin, and/or hematocrit.
[0144] A lipocalin mutein according to the invention may be used to
increase iron levels in a body fluid such as serum. It may also be
used to increase reticulocyte count, red blood cell count,
hemoglobin, and/or hematocrit in a subject, e.g. a human. A
pharmaceutical composition comprising a lipocalin mutein of the
invention may be used in this regard.
[0145] Another aspect of the present invention relates to a method
of treating a subject suffering from a disease or disorder that is
associated with an altered level of a Hepcidin, such as an
increased or a decreased level of a Hepcidin. A respective disease
or disorder may include a genetic or a non-genetic disease/disorder
causing iron deficiency or overload. A disease state or disorder
may include an infectious disease involving e.g. bacteria, fungi,
yeast or viruses. As explained above, in some embodiments the
disease or disorder is anemia, including, but not limited to,
anemia resulting from infection, inflammation, chronic disease,
and/or cancer. It may in some embodiments include an inflammatory
disease such as arthritis and certain cancer types, a liver disease
or a haematological disease. In some embodiments of the disease
associated with an increased level of a Hepcidin is an anemia or a
chronic kidney disease or an anemia associated with chronic kidney
disease. As already explained above, such a method involves
administering a respective mutein of the invention or a
pharmaceutical composition comprising a mutein of the invention to
a subject in need thereof.
[0146] A lipocalin mutein of the invention may for instance be used
to treat a subject having an elevated level of hepcidin, a
hepcidin-related disorder, a disorder of iron homeostasis, anemia
or inflammatory condition associated with an elevated level of
hepcidin. The subject may, for example, be a mammal such as a human
suffering from African iron overload, alpha thalassemia,
Alzheimer's disease, anemia, anemia of cancer, anemia of chronic
disease, anemia of inflammation, arteriosclerosis or
atherosclerosis (including coronary artery disease, cerebrovascular
disease or peripheral occlusive arterial disease), ataxias, ataxias
related to iron, atransferrinemia, cancer, ceruloplasmin
deficiency, chemotherapy-induced anemia, chronic renal/kidney
disease (in particular anemia associated with chronic kidney
disease), including end stage renal disease or chronic renal/kidney
failure, cirrhosis of liver, classic hemochromatosis,
collagen-induced arthritis (CIA), a condition involving hepcidin
excess (elevated hepcidin), congenital dyserythropoietic anemia,
congestive heart failure, Crohn's disease, diabetes, a disorder of
iron biodistribution, a disorder of iron homeostasis, a disorder of
iron metabolism, ferroportin disease, ferroportin mutation
hemochromatosis, folate deficiency. Friedrich's ataxia, funicular
myelosis, gracile syndrome, a bacterial infection such as H.
pyelori infection, Hallervordan Spatz disease, hemochromatosis,
hemochromatosis resulting from mutations in transferrin receptor 2,
hemoglobinopathies, hepatitis, hepatitis (Brock), hepatitis C,
hepatocellular carcinoma, hereditary hemochromatosis a viral
infection such as HIV, Huntington's disease, hyperfernitinemia,
hypochromic microcytic anemia, hypoferremia, insulin resistance,
iron deficiency anemia, an iron deficiency disorder, an iron
overload disorder, an iron-deficiency condition with hepcidin
excess, juvenile hemochromatosis (HFE2), multiple sclerosis, a
mutation of a gene involved in iron metabolism, for instance
expressing a protein involved therein such as transferrin receptor
2, HFE, hemojuvelin or ferroportin, neonatal hemochromatosis, a
neurodegenerative disease related to iron, osteopenia, osteoporosis
pancreatitis, Pantothenate kinase-associated neurodegeneration,
Parkinson's disease, pellagra, pica, porphyria, porphyria cutanea
tarda, pseudoencephalitis, pulmonary hemosiderosis, a red blood
cell disorder, rheumatoid arthritis, sepsis, sideroblastic anemia,
systemic lupus erythematosus, thalassemia, thalassemia intermedia,
transfusional iron overload, a tumor, vasculitis, vitamin B6
deficiency, vitamin B12 deficiency Wilson's disease, or
inflammatory condition associated with an elevated level of
hepcidin.
[0147] As a further illustrative example a mutein according to the
present invention can in some embodiments be used in combination
with erythropoietin. Anemia in patients with cancer (AC) and anemia
of chronic disease (ACD) are associated with high concentrations of
hepcidin (about 30 nmol/L) leading to serum iron deficiency and
thus to reduced erythropoiesis. Subjects with baseline hepcidin
concentrations below 13 nmol/L in serum have been reported to show
a better response to erythropoietin (EPO) therapy than subjects
with concentrations above 13 nmol/L. Therefore the treatment of
anemic cancer patients with a hepcidin antagonist can improve their
response to erythropoietin.
[0148] Furthermore a widespread phenomenon among anemic subjects is
resistance to recombinant erythropoietin (rhEPO), a therapeutic
problem that can be overcome by combinatorial therapy with a mutein
according to the present invention. Hepcidin likely plays a major
role in this rhEPO resistance. Sasu et al. (Blood (2010) 115, 17,
3816-3624) have shown a distinct correlation between increased
hepcidin level and resistance to erythropoiesis-stimulating agents
(ESAs) in mice. They also were able to restore ESA-responsiveness
by the administration of a hepcidin-specific antibody.
[0149] In yet another aspect the invention relates to the use of a
mutein according to the invention in diagnosis. The use of a mutein
according to the invention is typically for the diagnosis of a
disease or disorder associated with an altered level of a Hepcidin
as well as a respective method of diagnosis. The use may in some
embodiments involve assessing the level of a hepcidin in a body
fluid of a subject. For this purpose body fluid may have been taken
from the respective subject. The level of the Hepcidin may be
compared to a control sample, which is known to include a normal
level of the Hepcidin. It may thereby be determined whether
non-physiological levels of the Hepcidin are present in the
subject.
[0150] Accordingly, the invention also relates to a mutein as
defined above for the diagnosis of a disease or disorder associated
with an altered, e.g. increased or reduced, level of a Hepcidin. In
some embodiments the disease is an anemia, including, but not
limited to, anemia resulting from infection, inflammation, chronic
disease, and/or cancer. The disease or disorder may for instance be
associated with decreased levels of a Hepcidin, such as hereditary
hemochromatosis, an iron-loading anemia or Hepatitis C. The disease
or disorder may also be associated with increased levels of a
Hepcidin, e.g. anemia of inflammation, iron-refractory iron
deficiency anemia or a chronic kidney disease. Hepatitis C for
instance typically involves a hepatic iron overload, generally via
hepcidin synthesis suppression. In the context of diagnosis a
mutein according to the invention can be used to assess hepcidin
levels in body fluid of a subject. Since anemic cancer patients
with low hepcidin concentrations (<13 nmol/L) have been observed
to show a better response to erythropoietin therapy than patients
with high hepcidin concentrations (>13 nmol/L) hepcidin serum
concentrations can for instance be used for predicting the response
to epoetin therapy (about 50% of the patients are EPO
resistant).
[0151] In still another aspect, the present invention features a
diagnostic or analytical kit comprising a mutein according to the
present invention.
[0152] The subject in need of such a treatment may be a mammal,
such as a human, a dog, a mouse, a rat, a pig, an ape such as
cynomolgous monkeys to name only a few illustrative examples.
[0153] In still another aspect, the present invention features a
method for in vivo imaging in a subject, including administering to
said subject a mutein of the invention or a pharmaceutical
composition comprising a mutein of the invention. The subject may
be defined as above.
[0154] The invention is further illustrated by the following
non-limiting Examples and the attached drawings.
[0155] Unless otherwise indicated, established methods of
recombinant gene technology were used, for example, as described in
Sambrook at al. (supra).
Example 1
Construction of a Mutant Lcn2 Phage Display Library
[0156] A combinatorial library of Lcn2 variants was generated on
the basis of the cloned cDNA (Breustedt et al. (2006) Biochim.
Biophys. Acta 1764, 161-173), which carried the amino acid
substitutions Cys87Ser, to remove the single unpaired thiol side
chain (Goetz et al. (2000) Biochemistry 39, 1935-1941), as well as
Gln28His to introduce a second BstXI restriction site. Mutagenesis
and polymerase chain reaction (PCR) assembly of this region was
essentially performed according to a published strategy (Beste at
al. (1999) Proc. Natl. Acad. Sci. USA 96, 1898-1903; Skerra (2001)
J. Biotechnol. 74, 257-275), this time using a one pot
amplification reaction with oligodeoxynucleotides (sequences of SEQ
ID NO: 16 to SEQ ID NO: 25) as illustrated in FIG. 1.
Oligodeoxynucleotides were designed such that the primers with
sequences of SEQ ID NO: 16 to SEQ ID NO: 19 corresponded to the
coding strand and carried degenerate codons at the amino acid
positions 36, 40, 41, 49, 52, or 68, 70, 72, 73, 77, 79, 81, or 96,
100, 103, 106, or 125, 127, 132, 134 respectively, while primers
with sequences of SEQ ID NO: 20 to SEQ ID NO: 23 corresponded to
the non-coding strand and did not carry degenerate codons or
anticodons. The two flanking primers with SEQ ID NO: 24 and SEQ ID
NO: 25 were used in excess and served for the amplification of the
assembled randomized gene fragment. All PCR steps were performed
using Go-Taq Hot Start DNA polymerase (Promega, Mannheim, Germany)
as described (Schlehuber et al. (2000) J. Mol. Biol. 297,
1105-1120).
[0157] Oligodeoxynucleotides that did not carry degenerate codons
were purchased in HPLC grade from Metabion (Munich, Germany).
NNK-containing oligodeoxynucleotides were purchased desalted from
the same vendor and further purified by urea PAGE. The resulting
DNA library was cut with BstXI (Promega. Mannheim, Germany) and
cloned on the phagemid vector phNGAL102 (SEQ ID NO: 26), which is
based on the generic expression vector pASK111 (Vogt and Skerra
(2001) J. Mol. Recognit. 14 (1), 79-86) and codes for a fusion
protein composed of the OmpA signal peptide, the modified mature
Lcn2, followed by an amber codon, and the C-terminal fragment of
the gene III coat protein of the filamentous bacteriophage M13,
i.e. similar as previously described for the bilin-binding protein
(Beste et al., supra; Skerra, supra). After electroporation of E.
coli XL1-Blue (Bullock et al. (1987) Biotechniques 5, 376-378) with
the ligation mixture of 8.4 .mu.g digested PCR product and 94 .mu.g
digested plasmid DNA, 1.times.10.sup.10 transformants were
obtained.
[0158] Alternatively, a cloned synthetic Lcn2 random library, which
is described in FIG. 2, was obtained from Sloning BioTechnology
GmbH (Puchheim, Germany). The central gene cassette flanked by the
two BstXI restriction sites was amplified via PCR in 20 cycles
using appropriate primers (SEQ ID NO: 24 and SEQ ID NO: 25) and
subcloned on phNGAL108 (SEQ ID NO: 27), which is based on the
generic expression vector pASK75 (Skerra (1994) Gene 151, 131-135)
and carries essentially the same features as phNGAL102 (SEQ ID NO:
26) but mediates ampicillin resistance instead of chloramphenicol
resistance, in the same way, yielding a library with a complexity
corresponding to 1.7.times.10.sup.10 independent transformants.
[0159] The following steps in library generation were performed
identically for both Lcn2 libraries. 100 ml of the culture,
containing the cells which were transformed with the phasmid
vectors on the basis of phNGAL102 or phNGAL108, respectively,
coding for the library of the lipocalin muteins as phage pIII
fusion proteins, were transferred to a sterile Erlenmeyer flask and
incubated for one hour at 37.degree. C., 160 rpm in 2YT medium
without antibiotic selection pressure. Before infection with
VCS-M13 helper phage the culture was diluted in 2YT medium to an
OD550 of 0.1 with the corresponding antibiotic added and further
grown under identical conditions until an OD550 of 0.6 was reached.
After infection with VCS-M13 helper phage (Agilent Technologies, La
Jolla, USA) at a multiplicity of infection of approximately 10 the
culture was shaken for additional 30 min at 37.degree. C., 100 rpm.
Then the incubator temperature was lowered to 26.degree. C. and the
shaker speed was increased again to 160 rpm, after 10 min kanamycin
(70 .mu.g/ml) was added, followed by induction of gene expression
via addition of anhydrotetracycline (ACROS Organics, Geel, Belgium)
at 25 .mu.g/l (125 .mu.l of a 200 .mu.g/ml stock solution in
dimethylformamide. DMF per liter of culture). Incubation continued
for another 12-15 h at 26 C, 160 rpm.
[0160] Cells from the complete culture were sedimented by
centrifugation (30 min. 18000 g, 4.degree. C.). The supernatant
containing the phagemid particles was sterile-filtered (0.45
.mu.m), mixed with 1/4 volume 20% w/v PEG 8000, 15% w/v NaCl, and
incubated on ice for at least 2 h. After centrifugation (30 min.
18000 g, 4.degree. C.) the precipitated phagemid particles from 1
liter of culture were dissolved in 30 ml of cold BBS/E (200 mM
Na-borate, 160 mM NaCl, 1 mM EDTA pH 8.0) containing 50 mM
benzamidine (Sigma) and Pefabloc 1 .mu.g/ml (Roth, Karlsruhe,
Germany). The solution was incubated on ice for 1 h. After
centrifugation of undissolved components (10 min. 43000 g,
4.degree. C.) each supernatant was transferred to a new reaction
vessel.
[0161] Addition of 1/4 volume 20% w/v PEG 8000, 15% w/v NaCl and
incubation for 60 min on ice served to reprecipitate the phagemid
particles until the phagemids were aliquoted and frozen at
-80.degree. C. for storage. For the first selection cycle phagemids
were thawed and centrifuged (30 min. 34000 g, 4.degree. C.), the
supernatant was removed, and the precipitated phagemid particles
were dissolved and combined in a total of 400 .mu.l PBS containing
50 mM benzamidine. After incubation for 30 min on ice the solution
was centrifuged (5 min, 18500 g, 4.degree. C.) in order to remove
residual aggregates and the supernatant was used directly for the
phage display selection.
Example 2
Procurement of Soluble Hepcidin 25 Peptides
[0162] Synthetic non-modified Hepcidin-25 (human
DTHFPICIFCCGCCHRSKCGMCCKT, SEQ ID NO: 28, 2789.4 g/mol; mouse
DTNFPICIFCCKCCNNSQCGICCKT, SEQ ID NO: 29, 2754.2 g/mol; rat
DTNFPICLFCCKCCKNSSCGLCCIT, SEQ ID NO: 30, 2711.9 g/mol) and the
C-terminal biotinylated rat Hepcidin-25 (DTNFPICLFCCKCCKNSSCGLCCIT
(SEQ ID NO: 30)-Mini-PEG-linker-K-Biotin, 3210.5 g/mol) was
obtained from PeptaNova GmbH (Sandhausen, GE).
[0163] The human and mouse C-terminal biotinylated Hepcidin-25 was
obtained from Bachem AG (Bubendorf, CH). Analogous to the rat
Hepcidin-25 these targets were biotinylated via a Lysine residue
coupled to the C-terminus via a Mini-PEG linker.
Example 3
Generation of a Library with 10 Billion Independent NGAL
Muteins
[0164] A random library of NGAL lipocalin (Lcn2) with high
complexity was prepared essentially as described in Example 1
above. The amplification reaction is illustrated in FIG. 1, the
phagemid vector phNGAL102 is of SEQ ID NO: 26.
Example 4
Phagemid Presentation and Selection of NGAL Muteins with Affinity
for Human Hepcidin
[0165] Phagemid display and selection was performed employing the
phagemids obtained from Example 1 essentially as described in
international patent application WO/20051019256. The library was
subjected to 3 cycles of phage display selection against the
soluble. C-terminal biotinylated human Hepcidin-25 target
peptide.
[0166] 2.times.10.sup.12 to 1.times.10.sup.13 phagemids of the
library obtained in Example 1 were used. In brief, the phagemids
were centrifuged (21460.times.g, 4.degree. C., 20 min) and
resuspended 1 ml PBS (4 mM KHaPO.sub.4, 16 mM Na.sub.2BPO.sub.4,
115 mM NaCl, pH 7.4) containing 50 mM benzamidine. PBS containing
1% w/v Casein (Sigma) and 0.1% Tween 20.RTM. was used as blocking
buffer. Prior to the incubation with the target protein, phagemids
from the library were incubated with casein-blocked Streptavidin
beads for 30 minutes for the depletion of phagemids representing
multi-reactive or misfolded lipocalin mutein or Streptavidin
bead-specific muteins.
[0167] In different Panning approaches a 1 .mu.M solution of target
was either captured on Streptavidin.TM.-coated, 1% Casein-blocked
magnetic beads prior to the incubation with phagemids (solid in
solution approach) or 500 nM Hepcidin-25 was incubated in solution
with 310.sup.12 phagemids from the NGAL library blocked with 1%
Casein (solution approach). In the solution approach peptide bound
phagemids were captured via Streptavidin.TM.-coated magnetic beads
(Invitrogen/Dynal) within 20 min, followed by 8 wash cycles and
elution with either 300 .mu.L 70 mM Triethylamin for 10 min, and
neutralization with an appropriate amount of 1 M Tris/HCl, pH 7.4
(basic elution) or with 300 .mu.L 0.1 M Glycin/HCl pH 2.2 for 10
min, and neutralization with an appropriate amount of 0.5 M
Tris-Base (acidic elution).
[0168] In the solid in solution approach blocked phagemids were
incubated with the Streptavidin bead-coated target followed by 8
wash cycles and elution as described above [0199]. Beginning with
the second enrichment cycle, only half of the combined phagemid
solutions were used for phagemid amplification.
[0169] Phagemid amplification between each panning cycle was
performed as described in Schlehuber, S. et al. (J. Mol. Biol.
(2000), 297, 1105-1120).
[0170] Two further selection rounds against Hepcidin-25 were
carried out in this way employing the preparation of amplified
phagemids from the respective previous enrichment cycle with the
exception that only about 1.times.10.sup.I1 phagemids were utilized
beginning with the second enrichment cycle.
Example 5
Identification of hHepcidin-Specific Muteins Using High-Throughput
ELISA Screening
[0171] Screening of the muteins selected according to Example 4 was
performed essentially as described in Example 3 of international
patent application WO 2006/56464.
[0172] Lipocalin muteins were selected in a HT-screening ELISA.
Therein, NGAL variants equipped with a T7 detection tag (Novagen)
as well as a Strep-tag II affinity tag (IBA) were soluble expressed
in a 96 well microtiter plate using the E. coli strain TG1/F with
phNGAL 101. This vector corresponds to phNGAL 98 (SEQ ID NO: 31)
with an N-terminal T7 tag consisting of 11 amino acids
(MASMTGGQQMG) (SEQ ID NO: 34, see also FIG. 4B). Lipocalin mutein
expression was induced overnight at 22.degree. C. at 700 rpm with
anhydrotetracycline (0.2 .mu.g/ml) at an OD.sub.550 of 0.6.
Afterwards, cells were lysed (100 mM Na-borate, pH 8.0, 80 mM NaCl,
1 mM EDTA, 0.025% w/v lysozyme) for 1 h under agitation. To
minimize non-specific binding in the subsequent ELISA screen, the
crude cell lysates were supplemented with 2% w/v BSA and 0.1% v/v
Tween 20 and tested in ELISA for binding to human Hepcidin-25.
Therefore, soluble C-terminal biotinylated human Hepcidin-25 was
immobilized on wells of black Fluotrac 600 ELISA plates (Greiner;
384 well) with 1 .mu.g/ml via capturing by Neutravidin (5 .mu.g/ml,
Thermo Scientific). Neutravidin, Streptavidin, 5 .mu.g/ml each, and
3% milk were used as negative control. Plates were blocked with
PBST/0.1 containing 2% w/v BSA, and subsequently incubated with the
bacterial cell extract for 1 h at room temperature plates were
washed five times and bound Lipocalin muteins were detected via an
anti-T7 monoclonal antibody-HRP conjugate (Novagen), diluted
1:10.000 in PBST/O 1. Therefore, QuantaBlu.TM. (Pierce; 1:2 diluted
in PBS/T 0.1%) was used as fluorogenic HRP substrate. After 45 min
of signal development at room temperature fluorescence was excited
at a wavelength of 320 nm (.+-.12.5 nm) and measured at 430 nm
(.+-.17.5 nm) in a GENiosPlus plate reader (Tecan).
[0173] In a reverse ELISA approach soluble expressed muteins from
the crude cell lysate were captured in ELISA plates via their
T7-tag following incubation with varying amounts of C-terminal
biotinylated hHepcidin to reach target-limiting conditions in order
to differentiate the muteins by their affinity. Binding of the
target was detected via Extravidin-HRP conjugate (Sigma). One could
compete for mutein binding by the addition of 100 nM
non-biotinylated human Hepcidin-25 indicating, that the muteins
bind the non-modified hHepcidin-25 as well.
[0174] Screening of 2160 clones, selected as described in Example
4, led to the identification of more then 1000 primary hits
indicating the successful isolation of target-specific muteins. The
reverse ELISA approach under target-limiting conditions and the
competition ELISA allowed for a differentiation of
hepcidin-specific muteins in terms of their target affinity. Using
these ELISA approaches the clones with SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4. SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:
7 SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ
ID NO: 12 were identified. The sequences of these muteins are
depicted in FIG. 3.
Example 6
Production of Hepcidin-Binding Muteins (NGAL)
[0175] The recombinant Lcn2 and the hHepcidin-specific Lcn2
variants were produced by periplasmic secretion in E. coli K12
strain JM83 (Yanisch-Perron et al. (1985) Gene 33, 103-119), the E.
coli supE strain TG1.about.F.about. (a derivative of E. coli K12
TG1 [Kim et al. (2009) J. Am. Chem. Soc. 131, 3565-3576] that was
cured from its episome using acridinium orange), E. coli BL21
(Studier and Moffat (1986) J. Mol. Biol. 189, 113-130), or E. coli
W3110 (Bachmann (1990) Microbiol. Rev. 54, 130-197).
[0176] For a small scale soluble protein expression the plasmid
phNGAL98 (SEQ ID NO: 31) was used, encoding a fusion of the OmpA
signal peptide with the respective mutein and the C-terminal
Strep-tag II, whereby the plasmid carries the two non-compatible
BstXI restriction sites for unidirectional subcloning of the
mutated gene cassette. Growth was allowed to occur in a 2 L shaking
flask culture in the presence of LB-Ampicillin medium according to
the protocol described in Schlehuber, S. et al. (J. Mol. Biol.
(2000), 297, 1105-1120). For larger amounts of protein the
periplasmatic production was performed with the same vector
expressed in the E. coli strain W3110 via bench top fermenter
cultivation in a 1 l or 10 l vessel based on the protocol described
in Schiweck, W., and Skerra, A. Proteins (1995) 23, 581-565).
[0177] In order to increase the in vivo half-life, selected
lipocalin muteins were exemplarily modified by the following
procedures.
[0178] An ABD-fusion protein was constructed and periplasmatically
expressed for the mutein of SEQ ID NO: 1 The albumin binding domain
from the streptococcal protein G was fused to the C-terminus of the
mutein via the original linker derived from streptococcal protein G
as described in SEQ ID NO: 15.
[0179] In the case of site-directed PEGylation the hNGAL muteins
having a free cystein residue at amino acid position 87 (SEQ ID NO
13, SEQ ID NO: 14) were used for PEGylation with branched 40 k
PEG-maleimide. To this aim, the Serine at position 87 was
back-mutated to a Cysteine that originally occurs in hNGAL wildtype
by site-directed mutagenesis (Quick-change mutagenesis Kit,
Stratagene). Prior to the PEGylation reaction the free cysteine
residue was reduced in a 1:1 molar ratio of Anticalin with TCEP for
3 h at RT. Thereafter, PEGylation was performed by mixing the
protein with >2 molar excess of PEG40-maleimide reagent for 1.5
h at RT.
[0180] The Lcn2 variants were purified from the periplasmic
fraction in a single step via streptavidin affinity chromatography
(Strep-Tactin.TM. Superflow, IBA) using a column of appropriate bed
volume according to the procedure described by Skerra, A. &
Schmidt, T. G. M. (2000) (Use of the Strep-tag and streptavidin for
detection and purification of recombinant proteins. Methods
Enzymol. 326A, 271-304). To achieve higher purity and to remove any
aggregated recombinant protein, a gel filtration of the muteins was
finally carried out on a Superdex 75 HR 10/30 column (24-ml bed
volume, Amersham Pharmacia Biotech, Freiburg, Germany) in the
presence of PBS buffer. The monomeric protein fractions were
pooled, analysed for purity by SDS-PAGE (Fling and Gregerson (1986)
Anal. Biochem. 155, 83-88), and used for further biochemical
characterization.
[0181] The pegylated versions of hNGAL muteins were purified by
chromatography and, where necessary, a further reduction of
bacterial endotoxins was achieved by MustangE membrane (Pail
Corporation, US) filtration.
Example 7
Affinity Measurement Using ELISA Techniques
[0182] A "direct" ELISA was performed to verify the binding
affinity and specificity of the selected Lcn2 muteins. Therefore, a
constant concentration of 1 .mu.g/ml C-terminal biotinylated
hepcidin (Bachem AG, CH) was captured on the surface of a
polystyrol plate (Greiner, GE) via Neutravidin (Thermo Scientific,
5 .mu.g/ml). Two step dilution series of purified Lcn2 muteins were
incubated with the captured hepcidin for h at room temperature and
detected either via the Strep-tag II using a rabbit anti-strep-tag
II polyconal antibody (GenScript, USA) or by using a
scaffold-specific polyclonal rabbit antibody. In both cases an anti
rabbit IgG-HRP conjugate (Abcam, UK) was employed as secondary
detection antibody.
[0183] The absorption .DELTA.A at 320 nm was measured in an ELISA
reader (Tecan, GE) and the data were fitted with Graphpad Prism
software (Statcom, USA).
[0184] Results from measurements employing the muteins of the
sequences of SEQ ID NO: 1 to SEQ ID NO: 12, as well as of SEQ ID
NO: 14, linked to PEG40, of SEQ ID NO: 13, linked to PEG40 and of
SEQ ID NO: 1, linked to albumine-binding domain (ABD) (SEQ ID NO
15) are summarized in FIG. 5.
[0185] K.sub.D values of the selected Lcn2 muteins vary from 220 pM
up to 8.8 nM. All muteins bound human and cynomolgus hepcidin-25
with comparable affinity. Serum half-life extension of the
lipocalin mutein of SEQ ID NO: 1 via C-terminal fusion of the
albumine-binding domain had no significant effect on the binding
affinity of the mutein whereas pegylation reduced the binding
affinities in this ELISA format significantly by a factor of 5 for
SEQ ID NO: 8 and a factor of 8 for the mutein of SEQ ID NO: 1.
[0186] The binding affinity of the Lcn2 muteins to non-modified
hepcidin-25 in solution was evaluated in a competition ELISA
approach. Therefore, a constant concentration of 1 .mu.g/ml
C-terminal biotinylated human hepcidin (Bachem AG, CH) was captured
on the surface of a polystyrol plate (Greiner, GE) via Neutravidin
(Thermo Scientific, 5 .mu.g/ml, GE). In parallel a two step
dilution series of non-biotinylated human hepcidin starting from 1
.mu.M was incubated with a constant concentration of
hepcidin-specific mutein for 1 h at room temperature in a
non-protein binding 96 well polypropylene plate (Nunc, GE). The
constant concentration of lipocalin muteins corresponds to the
EC.sub.50 of the respective muteins as determined in a direct ELISA
as described above in this example. In the following the mixture of
non-modified human hepcidin and lipocalin mutein was transferred
onto the hepcidin-captured Neutravidin plate. The C-terminal
biotinylated hepcidin was allowed to compete with the non-modified
hepcidin for Anticalin binding for 20 min at room temperature.
During these 20 min. free lipcocalin mutein was bound to the
captured hepcidin and detected via a rabbit anti-strep-tag II
polyconal antibody (GenScript, USA). A goat anti-rabbit IgG-HRP
conjugate (Abcam, UK) was employed as secondary detection antibody.
Parallel to the competition assay, anticalin binding was determined
on the same plate in a "direct" ELISA, in order to obtain a
standard curve linking the RFU values to anticalin concentration.
This curve was then used to standardize competition data to the
level of anticalins bounds to the plate and fitted with Graphpad
software IC.sub.50 values correspond to the half-maximum amount of
lipocalin mutein bound to the plate.
[0187] Results from measurements employing the muteins of the
sequences of SEQ ID NO: 1 to SEQ ID NO: 12, as well as of SEQ ID
NO: 14, linked to PEG40, of SEQ ID NO: 13, linked to PEG40 and of
SEQ ID NO: 1, linked to ABD (SEQ ID NO: 15), are summarized in FIG.
6.
[0188] IC.sub.50 values of the selected Lcn2 muteins vary from 100
pM up to 10.8 nM. Serum half-life extension via the
albumine-binding domain had no effect on the binding affinity of
the mutein of SEQ ID NO: 1, whereas pegylation reduced the binding
affinities by a factor of 2 for SEQ ID NO: 13-PEG40 and by a factor
of 4 for SEQ ID NO: 14-PEG40, respectively.
Example 8
Affinity Measurement Using Surface-Plasmon-Resonance (SPR)
[0189] Surface plasmon resonance was used to measure binding
kinetics and affinity of the lipocalin muteins disclosed
herein.
[0190] Lipocalin muteins were immobilized to a CM5 sensor chip
using standard amine chemistry. The surface of the chip was
activated using EDC and NHS. Subsequently, 20 .mu.g/mL lipocalin
mutein solutions in 10 mM sodium acetate pH 4.5 (60 .mu.g/mL in 10
mM sodium acetate pH 4.0 for peglyted lipocalin mutein) were
applied at a flow rate of 5 .mu.L/min until a surface density of
500-700 resonance units (RU) for non-modified lipocalin muteins and
of approximately 1600 RU for pegylated lipocalin with the sequence
of SEQ ID NO: 13 was achieved. Residual activated groups were
saturated with ethanolamine. The reference channels were treated
with EDC/NHS following ethanolamine (blank immobilization). All
reagents and materials were purchased from GE Healthcare.
[0191] Serial dilutions of human and cynomolgus Hepcidin-25 in
running buffer (HBS-EP+, GE Healthcare, BR-1006-68) were applied to
the prepared surface. The following parameters were used for the
binding assay, contact time 60 s, dissociation time 180-360 s, flow
rate 30 .mu.L/min. All measurements were performed on a Biacore
T100 instrument (GE Healthcare) at 25.degree. C. Regeneration of
the surfaces having lipocalins immobilized thereon were achieved
with subsequent injections of 2 M/4 M Guanidinium-HCl (120-600 s)
and 10 mM glycine-HCl pH 1.5/2.0 (40-240 s) followed by an extra
wash with running buffer and a stabilization period of 120 s.
[0192] Data were evaluated with Biacore T100 Evaluation software (V
2.0.1). Double referencing was used. The 1:1 Binding model
(Langmuir) was used to fit the raw data.
[0193] Dublicates were reproducible and no binding to the reference
channel was detected. The binding parameters of the lipocalin
muteins with the sequences of SEQ ID NO: 1. SEQ ID NO: 8 as well as
of SEQ ID NO: 13, linked to PEG40, to human and cynomolgus
Hepcidin-25 are summarized in FIG. 7.
[0194] Cynomolgus Hepcidin-25 was binding to immobilized lipocalin
muteins with an approximately 2-fold higher affinity compared to
the human target. Kinetic analysis of hHepcidin-25 on the
immobilized pegylated variant having the sequence of SEQ ID NO. 13
revealed a high affinity of 40 pM.
Example 9
Cell-Based Assay for Hepcidin-Induced Internalization and
Degradation of Ferroportin
[0195] An in vitro cell-based assay was used to measure the
neutralization activity of the lipocalin muteins of the present
invention that are directed against human hepcidin. The assay is
based on hepcidin-induced internalisation and degradation of its
receptor, ferroportin and was implemented basically as described
(Nemeth et al, 2004, 2006).
[0196] Briefly, a HEK-293 stable cell line was prepared that
allowed for the inducible expression of murine ferroportin (FPN)
carboxy-terminally fused with green fluorescent protein (GFP). The
inducible expression of the FPN-GFP fusion protein was controlled
by Doxycyclin using the commercially available
tetracycline-regulated T-REx expression system (Invitrogen,
Karlsruhe, Germany). The FPN-GFP coding sequence was cloned into
pcDNA 4/TO vector, which contains an inducible promoter and a
Zeocin resistance marker. The resulting construct was stably
transfected into T-REx-293 cells which express the regulatory
protein required for doxycycline-inducible expression.
[0197] The assay for hepcidin-induced internalisation of the
hepcidin receptor was performed as follows: Cells of the
T-REx-293::FPN-GFP stable line were seeded in T75 cell culture
flasks at 80% confluence. In the evening FPN-GFP expression was
induced with 4 ng/ml Doxycyclin and stabilized with 10 .mu.M
Ammonium iron (III) citrate for 16 h at 37.degree. C. On the next
morning cells were trypsinized and seeded in a 24-well plate at 0.3
million cells/well in a volume of 450 .mu.l. Cells were allowed to
attach for 1 h at 37.degree. C. prior to the addition of hepcidin.
Cells were incubated at 37.degree. C. for 24 h and GFP fluorescence
of the detached cell suspension was analyzed by flow cytometry.
[0198] The EC80 (40 nM) of hepcidin-mediated degradation of the
Fpn-GFP fusion protein was used in neutralization assays. For this
purpose Anticalins were incubated with hepcidin at room temperature
for 30 min prior to addition to the cells. Following the 24 h
incubation period fluorescence was quantified as described
above.
[0199] The anti-hepcidin lipocalin muteins having the sequences of
SEQ ID NO. 1, SEQ ID NO. 2. SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO:
7, SEQ ID NO: 8, SEQ ID NO. 10, SEQ ID NO: 11, as well as of SEQ ID
NO: 14 linked to PEG40 and SEQ ID NO: 13 linked to PEG40 and SEQ ID
NO: 1 linked to ABD (SEQ ID NO: 15) neutralized the bioactivity of
human hepcidin-25 with IC50 values shown in FIG. 8.
Example 10
Anti-Hepcidin Lipocalin Muteins Neutralize Human Hepcidin in
Mice
[0200] The activity of anti-human hepcidin lipocalin muteins was
evaluated in vivo in mice that were administered human hepcidin in
an amount sufficient to generate a hypoferremic response as
described (Nemeth et al (2006) Blood, 107:328-333).
[0201] Two weeks before the experiment C57BL/6 mice were switched
to an iron deficient diet to suppress endogenous hepcidin. Prior to
the experiment a 3-fold molar excess of lipocalin mutein was
allowed to bind synthetic human hepcidin-25 for 30 minutes. In
parallel wildtype lipocalin (NGAL 98) was pre-incubated with human
hepcidin-25 in the same molar ratio as an isotypic control. Mice
received a single intraperitoneal (i.p.) injection of either PBS
(vehicle) or 2 mg/kg hepcidin or 2 mg hepcidin/Kg pre-incubated
with either lipocalin mutein or wild type lipocalin (negative
control). Two hours later, blood was collected under isoflurane
anesthesia and total serum iron levels were determined using a
colorimetric assay on a KoneLab XTi clinical analyzer.
[0202] The results are depicted in FIG. 9 as total serum iron
levels in .mu.M concentrations. Hepcidin treatment induced a
significant drop of serum iron levels in iron-starved mice.
Hepcidin pre-incubated with the wildtype lipocalin also exhibited
hypoferremia. The pre-complexation of human hepcidin with the
lipocalin mutein protected the animals from the hypoferremic
response.
Example 11
Determination of Pharmacokinetic (PK) Parameter for
Anti-Hepcidin-25 Lipocalin Muteins
[0203] Pharmacokinetic (PK) parameters (half-life plasma
concentration) for the Lcn 2 mutein having the sequence of SEQ ID
NO: 14 linked to PEG40 and of SEQ ID NO: 1 linked to ABD (SEQ ID
NO: 15) were determined following i.v. single bolus administration
in NMRI mice and Cynomolgus (Macacca fascicularis) at doses
depicted in FIG. 10. Plasma was prepared from terminal blood
samples taken at pre-determined timepoints and the concentrations
of the lipocalin mutein were determined by ELISA. The elimination
rate constant was calculated by least squares linear regression of
the terminal portion of the log transformed plasma
concentration-time curve. The start of the terminal elimination
phase for each individual profile was defined by visual inspection
and was the first point at which there was no systematic deviation
from the log-linear decline in serum concentrations. T1/2 was
calculated according to the following formula:
t 1 / 2 = ln ( 2 ) .lamda. x ##EQU00001##
T1/2 SEQ ID NO: 14-PEG (mouse): 27.9 h; T1/2 SEQ ID NO: 1-ABD
(mouse): 30 h: T1/2 SEQ ID NO: 14-PEG (Cyno): 88 h.
[0204] One skilled in the art would readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. Further, it will be readily apparent to one skilled in the
art that varying substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention. The compositions, methods, procedures,
treatments, molecules and specific compounds described herein are
presently representative of preferred embodiments are exemplary and
are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in the
art which are encompassed within the spirit of the invention are
defined by the scope of the claims. The listing or discussion of a
previously published document in this specification should not
necessarily be taken as an acknowledgement that the document is
part of the state of the art or is common general knowledge.
[0205] It must be noted that as used herein, the singular forms
"a". "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "an
antibody" includes one or more of such different antibodies and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein.
[0206] All publications and patents cited in this disclosure are
incorporated by reference in their entirety. To the extent the
material incorporated by reference contradicts or is inconsistent
with this specification, the specification will supersede any such
material.
[0207] Unless otherwise indicated, the term "at least" preceding a
series of elements is to be understood to refer to every element in
the series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention.
[0208] As used herein, the conjunctive term "and/or" between
multiple recited elements is understood as encompassing both
individual and combined options. For instance, where two elements
are conjoined by "and/or", a first option refers to the
applicability of the first element without the second. A second
option refers to the applicability of the second element without
the first. A third option refers to the applicability of the first
and second elements together. Any one of these options is
understood to fall within the meaning, and therefore satisfy the
requirement of the term "and/or" as used herein. Concurrent
applicability of more than one of the options is also understood to
fall within the meaning, and therefore satisfy the requirement of
the term "and/or" as used herein.
[0209] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising. "including," containing," etc.
shall be read expansively and without limitation. In each instance
herein any of the terms "comprising", "consisting essentially of"
and "consisting of" may be replaced with either of the other two
terms.
[0210] Additionally, the terms and expressions employed herein have
been used as terms of description and not of limitation, and there
is no intention in the use of such terms and expressions of
excluding any equivalents of the features shown and described or
portions thereof, but it is recognized that various modifications
are possible within the scope of the invention claimed. Thus, it
should be understood that although the present invention has been
specifically disclosed by exemplary embodiments and optional
features, modification and variation of the inventions embodied
therein herein disclosed may be resorted to by those skilled in the
art, and that such modifications and variations are considered to
be within the scope of this invention.
[0211] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0212] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
TABLE-US-00001 <160> NUMBER OF SEQ ID NOS: 48 <210> SEQ
ID NO 1 <211> LENGTH: 178 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutein of hNGAL <400> SEQUENCE: 1 Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn
Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly
Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro 35 40 45 Met
Lys Met Trp Ala Thr Ile Tyr Glu Leu Glu Glu Asp Lys Ser Tyr 50 55
60 Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe
65 70 75 80 Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu
Gly Asp 85 90 95 Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val
Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe
Phe Lys Thr Val Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile Thr
Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu
Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu
Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp
Gly <210> SEQ ID NO 2 <211> LENGTH: 178 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic Mutein of hNGAL <400> SEQUENCE: 2 Gln Asp
Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20
25 30 Val Val Gly Thr Ala Gly Asn Ser Ile Leu Arg Glu Asp Lys Asp
Pro 35 40 45 Gln Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp
Lys Ser Tyr 50 55 60 Asn Val Thr Arg Val Phe Phe Glu Gly Lys Lys
Cys Arg Tyr Val Ile 65 70 75 80 Glu Thr Phe Val Pro Gly Ser Gln Pro
Gly Glu Phe Thr Leu Gly Lys 85 90 95 Ile Lys Ser Ala Pro Gly Gly
Thr Ser Ile Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln
His Ala Met Val Phe Phe Lys Val Val Trp Gln 115 120 125 Asn Arg Glu
Leu Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr
Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150
155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys
Ile 165 170 175 Asp Gly <210> SEQ ID NO 3 <211> LENGTH:
178 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutein of hNGAL <400>
SEQUENCE: 3 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser
Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His
Gly Lys Trp Tyr 20 25 30 Val Val Gly Val Ala Gly Asn Gly Leu Leu
Arg Glu Asp Lys Asp Pro 35 40 45 Leu Lys Met His Ala Thr Ile Tyr
Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Arg Val Leu
Phe Val Arg Lys Lys Cys Arg Tyr Tyr Ile 65 70 75 80 Ser Thr Phe Val
Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Arg 85 90 95 Ile Lys
Ser Glu Pro Gly Arg Thr Ser Phe Leu Val Arg Val Val Ser 100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Met Val Trp Gln 115
120 125 Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu
Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys
Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val
Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> SEQ ID NO 4
<211> LENGTH: 178 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic Mutein
of hNGAL <400> SEQUENCE: 4 Gln Asp Ser Thr Ser Asp Leu Ile
Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Val
Ala Gly Asn Glu Met Leu Arg Glu Asp Lys Asp Pro 35 40 45 Leu Lys
Met Leu Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60
Asn Val Thr Arg Val Met Phe Glu Tyr Lys Lys Cys Val Tyr Leu Ile 65
70 75 80 Glu Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu
Gly Thr 85 90 95 Ile Lys Ser Val Pro Gly Leu Thr Ser Gly Leu Val
Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe
Phe Lys Arg Val Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile Thr
Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu
Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu
Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp
Gly <210> SEQ ID NO 5 <211> LENGTH: 178 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic Mutein of hNGAL <400> SEQUENCE: 5 Gln Asp
Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15
Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20
25 30 Val Val Gly Ala Ala Gly Asn Ser Leu Leu Arg Glu Asp Lys Asp
Pro 35 40 45 Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp
Lys Ser Tyr 50 55 60 Asn Val Thr Arg Val Asn Phe Gly Gly Lys Lys
Cys Ser Tyr Leu Ile 65 70 75 80 Glu Thr Phe Val Pro Gly Ser Gln Pro
Gly Glu Phe Thr Leu Gly Ser 85 90 95 Ile Lys Ser Arg Pro Gly Ala
Thr Ser Val Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln
His Ala Met Val Phe Phe Lys Leu Val Thr Gln 115 120 125 Asn Arg Glu
Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr
Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150
155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys
Ile 165 170 175 Asp Gly <210> SEQ ID NO 6 <211> LENGTH:
178 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutein of hNGAL <400>
SEQUENCE: 6
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5
10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp
Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Glu Ile Leu Arg Glu Asp
Lys Asp Pro 35 40 45 Leu Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys
Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Arg Val Gln Phe Gly Glu
Lys Lys Cys Gly Tyr Gly Ile 65 70 75 80 Glu Thr Phe Val Pro Gly Ser
Gln Pro Gly Glu Phe Thr Leu Gly Ser 85 90 95 Ile Lys Ser Val Pro
Gly Gly Thr Ser Arg Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr
Asn Gln His Ala Met Val Phe Phe Lys Phe Val Trp Gln 115 120 125 Asn
Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135
140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp
Gln Cys Ile 165 170 175 Asp Gly <210> SEQ ID NO 7 <211>
LENGTH: 178 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic Mutein of hNGAL
<400> SEQUENCE: 7 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro
Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn
Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn
Arg Val Leu Arg Glu Asp Lys Asp Pro 35 40 45 Gln Lys Met Phe Ala
Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr
Gly Val Asp Phe Arg Thr Lys Lys Cys Leu Tyr Ser Ile 65 70 75 80 Gly
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Val 85 90
95 Ile Lys Ser Gln Pro Gly Trp Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Thr Val
Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210>
SEQ ID NO 8 <211> LENGTH: 178 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Mutein of hNGAL <400> SEQUENCE: 8 Gln Asp Ser Thr
Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu
Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30
Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro 35
40 45 Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser
Tyr 50 55 60 Asn Val Thr Ile Val Met Pro Leu Ala Glu Lys Cys Glu
Tyr Leu Phe 65 70 75 80 Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu
Phe Thr Leu Gly Gly 85 90 95 Ile Lys Ser Gly Pro Gly Arg Thr Ser
Gly Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala
Met Val Phe Phe Lys Val Val Trp Gln 115 120 125 Asn Arg Glu Val Phe
Trp Val Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu
Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165
170 175 Asp Gly <210> SEQ ID NO 9 <211> LENGTH: 178
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutein of hNGAL <400>
SEQUENCE: 9 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser
Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His
Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Glu Val Leu
Arg Glu Asp Lys Asp Pro 35 40 45 Met Lys Met Trp Ala Thr Ile Tyr
Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ile Val Met
Ser Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65 70 75 80 Gln Thr Phe Val
Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp 85 90 95 Ile Lys
Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser 100 105 110
Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln 115
120 125 Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu
Leu 130 135 140 Thr Ser Gly Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys
Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val
Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> SEQ ID NO
10 <211> LENGTH: 179 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutein of hNGAL <400> SEQUENCE: 10 Gln Asp Ser Thr Ser Asp
Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln
Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val
Gly Leu Ala Gly Asn Glu Ile Leu Arg Glu Asp Lys Asp Pro 35 40 45
Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Arg Ser Tyr 50
55 60 Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu
Phe 65 70 75 80 Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr
Leu Gly Asp 85 90 95 Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu
Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val
Phe Phe Lys Val Val Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile
Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Pro Glu Leu Lys
Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro
Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175
Asp Gly Phe <210> SEQ ID NO 11 <211> LENGTH: 178
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutein of hNGAL <400>
SEQUENCE: 11 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu
Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe
His Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Glu Ile
Leu Arg Glu Asp Lys Asp Pro 35 40 45 Met Lys Met Trp Ala Thr Ile
Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ile Val
Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65 70 75 80
Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asp 85
90 95 Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val
Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val
Val Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile Thr Leu Tyr Gly
Arg Thr Lys Glu Leu 130 135 140 Thr Ser Gly Leu Lys Glu Asn Phe Ile
Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile
Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly
<210> SEQ ID NO 12 <211> LENGTH: 178 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic Mutein of hNGAL <400> SEQUENCE: 12 Gln Asp Ser Thr
Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu
Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30
Val Val Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro 35
40 45 Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser
Tyr 50 55 60 Asn Val Thr Ile Val Met Phe Leu Ala Glu Glu Cys Glu
Tyr Leu Phe 65 70 75 80 Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu
Phe Thr Leu Gly Asp 85 90 95 Ile Lys Ser Ser Pro Gly Arg Thr Ser
Gly Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala
Met Val Phe Phe Lys Val Val Trp Gln 115 120 125 Asn Arg Glu Val Phe
Trp Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu
Leu Lys Lys Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165
170 175 Asp Gly <210> SEQ ID NO 13 <211> LENGTH: 178
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutein of hNGAL <400>
SEQUENCE: 13 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu
Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe
His Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Glu Val
Leu Arg Glu Asp Lys Asp Pro 35 40 45 Met Lys Met Trp Ala Thr Ile
Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ile Val
Met Pro Leu Ala Glu Lys Cys Glu Tyr Leu Phe 65 70 75 80 Gln Thr Phe
Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Gly 85 90 95 Ile
Lys Ser Gly Pro Gly Arg Thr Ser Gly Leu Val Arg Val Val Ser 100 105
110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Val Val Trp Gln
115 120 125 Asn Arg Glu Val Phe Trp Val Thr Leu Tyr Gly Arg Thr Lys
Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser
Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro
Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> SEQ ID
NO 14 <211> LENGTH: 178 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Mutein of hNGAL <400> SEQUENCE: 14 Gln Asp Ser Thr Ser Asp
Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln
Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val
Gly Leu Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro 35 40 45
Met Lys Met Trp Ala Thr Ile Tyr Glu Leu Glu Glu Asp Lys Ser Tyr 50
55 60 Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu
Phe 65 70 75 80 Gln Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr
Leu Gly Asp 85 90 95 Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu
Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val
Phe Phe Lys Thr Val Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile
Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys
Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro
Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175
Asp Gly <210> SEQ ID NO 15 <211> LENGTH: 243
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Mutein of hNGAL with ABD domain and
STREP tag <400> SEQUENCE: 15 Gln Asp Ser Thr Ser Asp Leu Ile
Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu
Ala Gly Asn Glu Val Leu Arg Glu Asp Lys Asp Pro 35 40 45 Met Lys
Met Trp Ala Thr Ile Tyr Glu Leu Glu Glu Asp Lys Ser Tyr 50 55 60
Asn Val Thr Ile Val Met Phe Leu Ala Lys Lys Cys Glu Tyr Leu Phe 65
70 75 80 Gln Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu
Gly Asp 85 90 95 Ile Lys Ser Ser Pro Gly Arg Thr Ser Gly Leu Val
Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe
Phe Lys Val Val Trp Gln 115 120 125 Asn Arg Glu Val Phe Trp Ile Thr
Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu
Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu
Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp
Gly Ser Ala Gly Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys 180 185
190 Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr
195 200 205 Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys
Ala Leu 210 215 220 Ile Asp Glu Ile Leu Ala Ala Leu Pro Ser Ala Trp
Ser His Pro Gln 225 230 235 240 Phe Glu Lys <210> SEQ ID NO
16 <211> LENGTH: 88 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
NNK oligomer for positions 36, 40, 41, 49, 52 <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(35)..(36) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(59)..(60) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(68)..(69) <223> OTHER INFORMATION: a, c, g, or t <400>
SEQUENCE: 16
gaagtggtat gtggtaggtn nkgcagggaa tnnknnkctc agagaagaca aagacccgnn
60 kaagatgnnk gccaccatct atgagctg 88 <210> SEQ ID NO 17
<211> LENGTH: 79 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic NNK
oligomer for positions 68, 70, 72, 73, 77, 79, 81 <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(26)..(27) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(32)..(33) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(35)..(36) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(47)..(48) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(53)..(54) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(59)..(60) <223> OTHER INFORMATION: a, c, g, or t <400>
SEQUENCE: 17 caagagctac aatgtcaccn nkgtcnnktt tnnknnkaag aagtgtnnkt
acnnkatcnn 60 kacttttgtt ccaggttcc 79 <210> SEQ ID NO 18
<211> LENGTH: 68 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic NNK
oligomer for positions 96, 100, 103, 106 <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(19)..(20) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(31)..(32) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(40)..(41) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(49)..(50) <223> OTHER INFORMATION: a, c, g, or t <400>
SEQUENCE: 18 ggcgagttca cgctgggcnn kattaagagt nnkcctggan nkacgagtnn
kctcgtccga 60 gtggtgag 68 <210> SEQ ID NO 19 <211>
LENGTH: 68 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic NNK oligomer for
positions 125, 127, 132, 134 <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (19)..(20)
<223> OTHER INFORMATION: a, c, g, or t <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(25)..(26) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(40)..(41) <223> OTHER INFORMATION: a, c, g, or t <220>
FEATURE: <221> NAME/KEY: modified_base <222> LOCATION:
(46)..(47) <223> OTHER INFORMATION: a, c, g, or t <400>
SEQUENCE: 19 gctatggtgt tcttcaagnn kgttnnkcaa aacagggagn nkttcnnkat
caccctctac 60 gggagaac 68 <210> SEQ ID NO 20 <211>
LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic PCR-Primer with fixed
nucleotide sequences corresponding to the non-coding strand
<400> SEQUENCE: 20 ggtgacattg tagctcttgt cttctttcag
ctcatagatg gtggc 45 <210> SEQ ID NO 21 <211> LENGTH: 42
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic PCR-Primer with fixed nucleotide
sequences corresponding to the non-coding strand <400>
SEQUENCE: 21 gcccagcgtg aactcgcctg gctgggaacc tggaacaaaa gt 42
<210> SEQ ID NO 22 <211> LENGTH: 54 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic PCR-Primer with fixed nucleotide sequences corresponding
to the non-coding strand <400> SEQUENCE: 22 cttgaagaac
accatagcat gctggttgta gttggtgctc accactcgga cgag 54 <210> SEQ
ID NO 23 <211> LENGTH: 64 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
PCR-Primer with fixed nucleotide sequences corresponding to the
non-coding strand <400> SEQUENCE: 23 ggagaagcgg atgaagttct
cctttagttc cgaagtcagc tccttggttc tcccgtagag 60 ggtg 64 <210>
SEQ ID NO 24 <211> LENGTH: 40 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic 5' flanking PCR-Oligo biotinylated <400> SEQUENCE:
24 ccaggacaac caattccatg ggaagtggta tgtggtaggt 40 <210> SEQ
ID NO 25 <211> LENGTH: 40 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic 3'
flanking PCR-Oligo biotinylated <400> SEQUENCE: 25 ttcagggagg
cccagagatt tggagaagcg gatgaagttc 40 <210> SEQ ID NO 26
<211> LENGTH: 4746 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Phage display vector phNGAL102 with CamR used as backbone for
NNK-Library <400> SEQUENCE: 26 ccataacgct cggttgccgc
cgggcgtttt ttattggcca gatgattaat tcctaatttt 60 tgttgacact
ctatcattgg tagagttatt ttaccactcc ctatcagtga tagagaaaag 120
tgaaatgaat agttcgacaa aaatctagat aacgagggca aaaaatgaaa aagacagcta
180 tcgcgattgc agtggctctg gctggcttcg ctaccgtagc gcaggcccag
gactccacct 240 cagacctgat cccagcccca cctctgagca aggtccctct
gcagcagaac ttccaggaca 300 accaattcca tgggaagtgg tatgtggtag
gtctcgcagg gaatgcaatt ctcagagaag 360 acaaagaccc gcaaaagatg
tatgccacca tctatgagct gaaagaagac aagagctaca 420 atgtcacctc
cgtcctgttt aggaaaaaga agtgtgacta ctggatcagg acttttgttc 480
caggttccca gccaggcgag ttcacgctgg gcaacattaa gagttaccct ggattaacga
540 gttacctcgt ccgagtggtg agcaccaact acaaccagca tgctatggtg
ttcttcaaga 600 aagtttctca aaacagggag tacttcaaga tcaccctcta
cgggagaacc aaggagctga 660 cttcggaact aaaggagaac ttcatccgct
tctccaaatc tctgggcctc cctgaaaacc 720 acatcgtctt ccctgtccca
atcgaccagt gtatcgacgg cagcgctggt ggggcctaga 780 ctgttgaaag
ttgtttagca aaaccccata cagaaaattc atttactaac gtctggaaag 840
acgacaaaac tttagatcgt tacgctaact atgagggctg tctgtggaat gctacaggcg
900 ttgtagtttg tactggtgac gaaactcagt gttacggtac atgggttcct
attgggcttg 960 ctatccctga aaatgagggt ggtggctctg agggtggcgg
ttctgagggt ggcggttctg 1020 agggtggcgg tactaaacct cctgagtacg
gtgatacacc tattccgggc tatacttata 1080
tcaaccctct cgacggcact tatccgcctg gtactgagca aaaccccgct aatcctaatc
1140 cttctcttga ggagtctcag cctcttaata ctttcatgtt tcagaataat
aggttccgaa 1200 ataggcaggg ggcattaact gtttatacgg gcactgttac
tcaaggcact gaccccgtta 1260 aaacttatta ccagtacact cctgtatcat
caaaagccat gtatgacgct tactggaacg 1320 gtaaattcag agactgcgct
ttccattctg gctttaatga ggatccattc gtttgtgaat 1380 atcaaggcca
atcgtctgac ctgcctcaac ctcctgtcaa tgctggcggc ggctctggtg 1440
gtggttctgg tggcggctct gagggtggtg gctctgtggg tggcggttct gagggtggcg
1500 gctctgaggg aggcggttcc ggtggtggct ctggttccgg tgattttgat
tatgaaaaga 1560 tggcaaacgc taataagggg gctatgaccg aaaatgccga
tgaaaacgcg ctacagtctg 1620 acgctaaagg caaacttgat tctgtcgcta
ctgattacgg tgctgctatc gatggtttca 1680 ttggtgacgt ttccggcctt
gctaatggta atggtgctac tggtgatttt gctggctcta 1740 attcccaaat
ggctcaagtc ggtgacggtg ataattcacc tttaatgaat aatttccgtc 1800
aatatttacc ttccctccct caatcggttg aatgtcgccc ttttgtcttt ggcgctggta
1860 aaccatatga attttctatt gattgtgaca aaataaactt attccgtggt
gtctttgcgt 1920 ttcttttata tgttgccacc tttatgtatg tattttctac
gtttgctaac atactgcgta 1980 ataaggagtc ttaataagct tgacctgtga
agtgaaaaat ggcgcacatt gtgcgacatt 2040 ttttttgtct gccgtttacc
gctactgcgt cacggatctc cacgcgccct gtagcggcgc 2100 attaagcgcg
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 2160
agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
2220 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
ggcacctcga 2280 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
ccatcgccct gatagacggt 2340 ttttcgccct ttgacgttgg agtccacgtt
ctttaatagt ggactcttgt tccaaactgg 2400 aacaacactc aaccctatct
cggtctattc ttttgattta taagggattt tgccgatttc 2460 ggcctattgg
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 2520
ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc aactttcacc ataatgaaat
2580 aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta
aggaagctaa 2640 aatggagaaa aaaatcactg gatataccac cgttgatata
tcccaatggc atcgtaaaga 2700 acattttgag gcatttcagt cagttgctca
atgtacctat aaccagaccg ttcagctgga 2760 tattacggcc tttttaaaga
ccgtaaagaa aaataagcac aagttttatc cggcctttat 2820 tcacattctt
gcccgcctga tgaatgctca tccggaattc cgtatggcaa tgaaagacgg 2880
tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
2940 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
tacacatata 3000 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
ttccctaaag ggtttattga 3060 gaatatgttt ttcgtctcag ccaatccctg
ggtgagtttc accagttttg atttaaacgt 3120 ggccaatatg gacaacttct
tcgcccccgt tttcactatg ggcaaatatt atacgcaagg 3180 cgacaaggtg
ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca 3240
tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
3300 ataggaatta atgatgtctc gtttagataa aagtaaagtg attaacagcg
cattagagct 3360 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa
ctcgcccaga agctaggtgt 3420 agagcagcct acattgtatt ggcatgtaaa
aaataagcgg gctttgctcg acgccttagc 3480 cattgagatg ttagataggc
accatactca cttttgccct ttagaagggg aaagctggca 3540 agatttttta
cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg 3600
agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
3660 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg
cactcagcgc 3720 agtggggcat tttactttag gttgcgtatt ggaagatcaa
gagcatcaag tcgctaaaga 3780 agaaagggaa acacctacta ctgatagtat
gccgccatta ttacgacaag ctatcgaatt 3840 atttgatcac caaggtgcag
agccagcctt cttattcggc cttgaattga tcatatgcgg 3900 attagaaaaa
caacttaaat gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg 3960
atggtaactt cactagttta aaaggatcta ggtgaagatc ctttttgata atctcatgac
4020 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
aaaagatcaa 4080 aggatcttct tgagatcctt tttttctgcg cgtaatctgc
tgcttgcaaa caaaaaaacc 4140 accgctacca gcggtggttt gtttgccgga
tcaagagcta ccaactcttt ttccgaaggt 4200 aactggcttc agcagagcgc
agataccaaa tactgttctt ctagtgtagc cgtagttagg 4260 ccaccacttc
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 4320
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
4380 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
ccagcttgga 4440 gcgaacgacc tacaccgaac tgagatacct acagcgtgag
ctatgagaaa gcgccacgct 4500 tcccgaaggg agaaaggcgg acaggtatcc
ggtaagcggc agggtcggaa caggagagcg 4560 cacgagggag cttccagggg
gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 4620 cctctgactt
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 4680
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgac
4740 ccgaca 4746 <210> SEQ ID NO 27 <211> LENGTH: 4963
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic Phage display vector phNGAL108 with
AmpR used for Sloning Library <400> SEQUENCE: 27 ccatcgaatg
gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt 60
tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct
120 agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc
tctggctggc 180 ttcgctaccg tagcgcaggc ccaggactcc acctcagacc
tgatcccagc cccacctctg 240 agcaaggtcc ctctgcagca gaacttccag
gacaaccaat tccatgggaa gtggtatgtg 300 gtaggtctcg cagggaatgc
aattctcaga gaagacaaag acccgcaaaa gatgtatgcc 360 accatctatg
agctgaaaga agacaagagc tacaatgtca cctccgtcct gtttaggaaa 420
aagaagtgtg actactggat caggactttt gttccaggtt cccagccagg cgagttcacg
480 ctgggcaaca ttaagagtta ccctggatta acgagttacc tcgtccgagt
ggtgagcacc 540 aactacaacc agcatgctat ggtgttcttc aagaaagttt
ctcaaaacag ggagtacttc 600 aagatcaccc tctacgggag aaccaaggag
ctgacttcgg aactaaagga gaacttcatc 660 cgcttctcca aatctctggg
cctccctgaa aaccacatcg tcttccctgt cccaatcgac 720 cagtgtatcg
acggcagcgc ttggcgtcac ccgcagttcg gtggggccta gactgttgaa 780
agttgtttag caaaacccca tacagaaaat tcatttacta acgtctggaa agacgacaaa
840 actttagatc gttacgctaa ctatgagggc tgtctgtgga atgctacagg
cgttgtagtt 900 tgtactggtg acgaaactca gtgttacggt acatgggttc
ctattgggct tgctatccct 960 gaaaatgagg gtggtggctc tgagggtggc
ggttctgagg gtggcggttc tgagggtggc 1020 ggtactaaac ctcctgagta
cggtgataca cctattccgg gctatactta tatcaaccct 1080 ctcgacggca
cttatccgcc tggtactgag caaaaccccg ctaatcctaa tccttctctt 1140
gaggagtctc agcctcttaa tactttcatg tttcagaata ataggttccg aaataggcag
1200 ggggcattaa ctgtttatac gggcactgtt actcaaggca ctgaccccgt
taaaacttat 1260 taccagtaca ctcctgtatc atcaaaagcc atgtatgacg
cttactggaa cggtaaattc 1320 agagactgcg ctttccattc tggctttaat
gaggatccat tcgtttgtga atatcaaggc 1380 caatcgtctg acctgcctca
acctcctgtc aatgctggcg gcggctctgg tggtggttct 1440 ggtggcggct
ctgagggtgg tggctctgag ggtggcggtt ctgagggtgg cggctctgag 1500
ggaggcggtt ccggtggtgg ctctggttcc ggtgattttg attatgaaaa gatggcaaac
1560 gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc
tgacgctaaa 1620 ggcaaacttg attctgtcgc tactgattac ggtgctgcta
tcgatggttt cattggtgac 1680 gtttccggcc ttgctaatgg taatggtgct
actggtgatt ttgctggctc taattcccaa 1740 atggctcaag tcggtgacgg
tgataattca cctttaatga ataatttccg tcaatattta 1800 ccttccctcc
ctcaatcggt tgaatgtcgc ccttttgtct ttggcgctgg taaaccatat 1860
gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta
1920 tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg
taataaggag 1980 tcttaataag cttgacctgt gaagtgaaaa atggcgcaca
ttgtgcgaca ttttttttgt 2040 ctgccgttta ccgctactgc gtcacggatc
tccacgcgcc ctgtagcggc gcattaagcg 2100 cggcgggtgt ggtggttacg
cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2160 ctcctttcgc
tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 2220
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
2280 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
gtttttcgcc 2340 ctttgacgtt ggagtccacg ttctttaata gtggactctt
gttccaaact ggaacaacac 2400 tcaaccctat ctcggtctat tcttttgatt
tataagggat tttgccgatt tcggcctatt 2460 ggttaaaaaa tgagctgatt
taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc 2520 ttacaatttc
aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 2580
tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
2640 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
attccctttt 2700 ttgcggcatt ttgccttcct gtttttgctc acccagaaac
gctggtgaaa gtaaaagatg 2760 ctgaagatca gttgggtgca cgagtgggtt
acatcgaact ggatctcaac agcggtaaga 2820 tccttgagag ttttcgcccc
gaagaacgtt ttccaatgat gagcactttt aaagttctgc 2880 tatgtggcgc
ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 2940
actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
3000 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
actgcggcca 3060 acttacttct gacaacgatc ggaggaccga aggagctaac
cgcttttttg cacaacatgg 3120 gggatcatgt aactcgcctt gatcgttggg
aaccggagct gaatgaagcc ataccaaacg 3180 acgagcgtga caccacgatg
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 3240 gcgaactact
tactctagct tcccggcaac aattgataga ctggatggag gcggataaag 3300
ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
3360 gagccggtga gcgtggctct cgcggtatca ttgcagcact ggggccagat
ggtaagccct 3420
cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
3480 agatcgctga gataggtgcc tcactgatta agcattggta ggaattaatg
atgtctcgtt 3540 tagataaaag taaagtgatt aacagcgcat tagagctgct
taatgaggtc ggaatcgaag 3600 gtttaacaac ccgtaaactc gcccagaagc
taggtgtaga gcagcctaca ttgtattggc 3660 atgtaaaaaa taagcgggct
ttgctcgacg ccttagccat tgagatgtta gataggcacc 3720 atactcactt
ttgcccttta gaaggggaaa gctggcaaga ttttttacgt aataacgcta 3780
aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat ttaggtacac
3840 ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta
tgccaacaag 3900 gtttttcact agagaatgca ttatatgcac tcagcgcagt
ggggcatttt actttaggtt 3960 gcgtattgga agatcaagag catcaagtcg
ctaaagaaga aagggaaaca cctactactg 4020 atagtatgcc gccattatta
cgacaagcta tcgaattatt tgatcaccaa ggtgcagagc 4080 cagccttctt
attcggcctt gaattgatca tatgcggatt agaaaaacaa cttaaatgtg 4140
aaagtgggtc ttaaaagcag cataaccttt ttccgtgatg gtaacttcac tagtttaaaa
4200 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
cgtgagtttt 4260 cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4320 ttctgcgcgt aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg gtggtttgtt 4380 tgccggatca agagctacca
actctttttc cgaaggtaac tggcttcagc agagcgcaga 4440 taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 4500
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
4560 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
cagcggtcgg 4620 gctgaacggg gggttcgtgc acacagccca gcttggagcg
aacgacctac accgaactga 4680 gatacctaca gcgtgagcta tgagaaagcg
ccacgcttcc cgaagggaga aaggcggaca 4740 ggtatccggt aagcggcagg
gtcggaacag gagagcgcac gagggagctt ccagggggaa 4800 acgcctggta
tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 4860
tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
4920 ggttcctggc cttttgctgg ccttttgctc acatgacccg aca 4963
<210> SEQ ID NO 28 <211> LENGTH: 25 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<223> OTHER INFORMATION: Hepcidin-25 <400> SEQUENCE: 28
Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg 1 5
10 15 Ser Lys Cys Gly Met Cys Cys Lys Thr 20 25 <210> SEQ ID
NO 29 <211> LENGTH: 25 <212> TYPE: PRT <213>
ORGANISM: Mus musculus <220> FEATURE: <223> OTHER
INFORMATION: Hepcidin-25 <400> SEQUENCE: 29 Asp Thr Asn Phe
Pro Ile Cys Ile Phe Cys Cys Lys Cys Cys Asn Asn 1 5 10 15 Ser Gln
Cys Gly Ile Cys Cys Lys Thr 20 25 <210> SEQ ID NO 30
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Rattus norvegicus <220> FEATURE: <223> OTHER
INFORMATION: Hepcidin-25 <400> SEQUENCE: 30 Asp Thr Asn Phe
Pro Ile Cys Leu Phe Cys Cys Lys Cys Cys Lys Asn 1 5 10 15 Ser Ser
Cys Gly Leu Cys Cys Ile Thr 20 25 <210> SEQ ID NO 31
<211> LENGTH: 3745 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
Expression vector phNGAL98 with AmpR encoding wild type Lcn2 with
the C-terminal Strep-tagII <400> SEQUENCE: 31 ccatcgaatg
gccagatgat taattcctaa tttttgttga cactctatca ttgatagagt 60
tattttacca ctccctatca gtgatagaga aaagtgaaat gaatagttcg acaaaaatct
120 agataacgag ggcaaaaaat gaaaaagaca gctatcgcga ttgcagtggc
tctggctggc 180 ttcgctaccg tagcgcaggc ccaggactcc acctcagacc
tgatcccagc cccacctctg 240 agcaaggtcc ctctgcagca gaacttccag
gacaaccaat tccatgggaa gtggtatgtg 300 gtaggtctcg cagggaatgc
aattctcaga gaagacaaag acccgcaaaa gatgtatgcc 360 accatctatg
agctgaaaga agacaagagc tacaatgtca cctccgtcct gtttaggaaa 420
aagaagtgtg actactggat caggactttt gttccaggtt cccagccagg cgagttcacg
480 ctgggcaaca ttaagagtta ccctggatta acgagttacc tcgtccgagt
ggtgagcacc 540 aactacaacc agcatgctat ggtgttcttc aagaaagttt
ctcaaaacag ggagtacttc 600 aagatcaccc tctacgggag aaccaaggag
ctgacttcgg aactaaagga gaacttcatc 660 cgcttctcca aatctctggg
cctccctgaa aaccacatcg tcttccctgt cccaatcgac 720 cagtgtatcg
acggcagcgc ttggtctcac ccgcagttcg aaaaataata agcttgacct 780
gtgaagtgaa aaatggcgca cattgtgcga catttttttt gtctgccgtt taccgctact
840 gcgtcacgga tctccacgcg ccctgtagcg gcgcattaag cgcggcgggt
gtggtggtta 900 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
cgctcctttc gctttcttcc 960 cttcctttct cgccacgttc gccggctttc
cccgtcaagc tctaaatcgg gggctccctt 1020 tagggttccg atttagtgct
ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 1080 gttcacgtag
tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 1140
cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
1200 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
aatgagctga 1260 tttaacaaaa atttaacgcg aattttaaca aaatattaac
gtttacaatt tcaggtggca 1320 cttttcgggg aaatgtgcgc ggaaccccta
tttgtttatt tttctaaata cattcaaata 1380 tgtatccgct catgagacaa
taaccctgat aaatgcttca ataatattga aaaaggaaga 1440 gtatgagtat
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 1500
ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
1560 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
agttttcgcc 1620 ccgaagaacg ttttccaatg atgagcactt ttaaagttct
gctatgtggc gcggtattat 1680 cccgtattga cgccgggcaa gagcaactcg
gtcgccgcat acactattct cagaatgact 1740 tggttgagta ctcaccagtc
acagaaaagc atcttacgga tggcatgaca gtaagagaat 1800 tatgcagtgc
tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 1860
tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
1920 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
gacaccacga 1980 tgcctgtagc aatggcaaca acgttgcgca aactattaac
tggcgaacta cttactctag 2040 cttcccggca acaattgata gactggatgg
aggcggataa agttgcagga ccacttctgc 2100 gctcggccct tccggctggc
tggtttattg ctgataaatc tggagccggt gagcgtggct 2160 ctcgcggtat
cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 2220
acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
2280 cctcactgat taagcattgg taggaattaa tgatgtctcg tttagataaa
agtaaagtga 2340 ttaacagcgc attagagctg cttaatgagg tcggaatcga
aggtttaaca acccgtaaac 2400 tcgcccagaa gctaggtgta gagcagccta
cattgtattg gcatgtaaaa aataagcggg 2460 ctttgctcga cgccttagcc
attgagatgt tagataggca ccatactcac ttttgccctt 2520 tagaagggga
aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt 2580
tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt
2640 atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca
ctagagaatg 2700 cattatatgc actcagcgca gtggggcatt ttactttagg
ttgcgtattg gaagatcaag 2760 agcatcaagt cgctaaagaa gaaagggaaa
cacctactac tgatagtatg ccgccattat 2820 tacgacaagc tatcgaatta
tttgatcacc aaggtgcaga gccagccttc ttattcggcc 2880 ttgaattgat
catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcttaaaagc 2940
agcataacct ttttccgtga tggtaacttc actagtttaa aaggatctag gtgaagatcc
3000 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
tgagcgtcag 3060 accccgtaga aaagatcaaa ggatcttctt gagatccttt
ttttctgcgc gtaatctgct 3120 gcttgcaaac aaaaaaacca ccgctaccag
cggtggtttg tttgccggat caagagctac 3180 caactctttt tccgaaggta
actggcttca gcagagcgca gataccaaat actgtccttc 3240 tagtgtagcc
gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 3300
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
3360 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
gggggttcgt 3420 gcacacagcc cagcttggag cgaacgacct acaccgaact
gagataccta cagcgtgagc 3480 tatgagaaag cgccacgctt cccgaaggga
gaaaggcgga caggtatccg gtaagcggca 3540 gggtcggaac aggagagcgc
acgagggagc ttccaggggg aaacgcctgg tatctttata 3600 gtcctgtcgg
gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 3660
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
3720 ggccttttgc tcacatgacc cgaca 3745 <210> SEQ ID NO 32
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 32
cccaggactc cacctcagac c 21 <210> SEQ ID NO 33 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 33 actgcgggtg ggaccaagcg ctgccgt 27 <210> SEQ ID NO
34 <211> LENGTH: 197 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
hNGAL with T7 tag encoded by phNGAL 101 <400> SEQUENCE: 34
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5
10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp
Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp
Lys Asp Pro 35 40 45 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys
Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ser Val Leu Phe Arg Lys
Lys Lys Cys Asp Tyr Trp Ile 65 70 75 80 Arg Thr Phe Val Pro Gly Ser
Gln Pro Gly Glu Phe Thr Leu Gly Asp 85 90 95 Ile Lys Ser Tyr Pro
Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr
Asn Gln His Ala Met Val Phe Phe Lys Thr Val Ser Gln 115 120 125 Asn
Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135
140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp
Gln Cys Ile 165 170 175 Asp Gly Ser Ala Trp Ser His Pro Gln Phe Met
Ala Ser Met Thr Gly 180 185 190 Gly Gln Gln Met Gly 195 <210>
SEQ ID NO 35 <211> LENGTH: 178 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic wild type hNGAL <400> SEQUENCE: 35 Gln Asp Ser Thr
Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu
Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30
Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35
40 45 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser
Tyr 50 55 60 Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp
Tyr Trp Ile 65 70 75 80 Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu
Phe Thr Leu Gly Asn 85 90 95 Ile Lys Ser Tyr Pro Gly Leu Thr Ser
Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala
Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125 Asn Arg Glu Tyr Phe
Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu
Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160
Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165
170 175 Asp Gly <210> SEQ ID NO 36 <211> LENGTH: 12
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION: (4)..(9)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<400> SEQUENCE: 36 ccannnnnnt gg 12 <210> SEQ ID NO 37
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic 5xHis
tag <400> SEQUENCE: 37 His His His His His 1 5 <210>
SEQ ID NO 38 <211> LENGTH: 6 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic 6xHis tag <400> SEQUENCE: 38 His His His His His
His 1 5 <210> SEQ ID NO 39 <211> LENGTH: 79 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(20)..(21) <223> OTHER INFORMATION: a, c, t, g, unknown or
other <220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (26)..(27) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (32)..(33)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (35)..(36) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (47)..(48)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (53)..(54) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (59)..(60)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<400> SEQUENCE: 39 caagagctac aatgtcacan nkgtcnnktt
tnnknnkaag aagtgtnnkt acnnkatcnn 60 kacttttgtt ccaggttcc 79
<210> SEQ ID NO 40 <211> LENGTH: 45 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 40 ggtgacattg
tagctcttat cttctttcag ctcatagatg gtggc 45 <210> SEQ ID NO 41
<211> LENGTH: 64 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 41 ggagaagcgg atgaagttct
cctttagttc cgaagccagc tccttggttc tcccgtagag 60 ggtg 64 <210>
SEQ ID NO 42 <211> LENGTH: 567 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <220> FEATURE: <221> NAME/KEY:
CDS <222> LOCATION: (1)..(564) <400> SEQUENCE: 42
cag gac tcc acc tca gac ctg atc cca gcc cca cct ctg agc aag gtc 48
Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5
10 15 cct ctg cag cag aac ttc cag gac aac caa ttc cat ggg aag tgg
tat 96 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp
Tyr 20 25 30 gtg gta ggt ctc gca ggg aat gca att ctc aga gaa gac
aaa gac ccg 144 Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp
Lys Asp Pro 35 40 45 caa aag atg tat gcc acc atc tat gag ctg aaa
gaa gac aag agc tac 192 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys
Glu Asp Lys Ser Tyr 50 55 60 aat gtc acc tcc gtc ctg ttt agg aaa
aag aag tgt gac tac tgg atc 240 Asn Val Thr Ser Val Leu Phe Arg Lys
Lys Lys Cys Asp Tyr Trp Ile 65 70 75 80 agg act ttt gtt cca ggt tcc
cag cca ggc gag ttc acg ctg ggc aac 288 Arg Thr Phe Val Pro Gly Ser
Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95 att aag agt tac cct
gga tta acg agt tac ctc gtc cga gtg gtg agc 336 Ile Lys Ser Tyr Pro
Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 acc aac tac
aac cag cat gct atg gtg ttc ttc aag aaa gtt tct caa 384 Thr Asn Tyr
Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125 aac
agg gag tac ttc aag atc acc ctc tac ggg aga acc aag gag ctg 432 Asn
Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135
140 act tcg gaa cta aag gag aac ttc atc cgc ttc tcc aaa tct ctg ggc
480 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly
145 150 155 160 ctc cct gaa aac cac atc gtc ttc cct gtc cca atc gac
cag tgt atc 528 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp
Gln Cys Ile 165 170 175 gac ggc agc gct tgg tcc cac ccg cag ttc gaa
aaa taa 567 Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 180 185
<210> SEQ ID NO 43 <211> LENGTH: 188 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 43 Gln Asp Ser Thr Ser
Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln
Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val
Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35 40
45 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60 Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr
Trp Ile 65 70 75 80 Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe
Thr Leu Gly Asn 85 90 95 Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr
Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met
Val Phe Phe Lys Lys Val Ser Gln 115 120 125 Asn Arg Glu Tyr Phe Lys
Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu
Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu
Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175 Asp Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys 180 185
<210> SEQ ID NO 44 <211> LENGTH: 567 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 44 ttatttttcg
aactgcgggt gggaccaagc gctgccgtcg atacactggt cgattgggac 60
agggaagacg atgtggtttt cagggaggcc cagagatttg gagaagcgga tgaagttctc
120 ctttagttcc gaagccagct ccttggttct cccgtagagg gtgatcttga
agtactccct 180 gttttgagaa actttcttga agaacaccat agcatgctgg
ttgtagttgg tgctcaccac 240 tcggacgagg taactcgtta atccagggta
actcttaatg ttgcccagcg tgaactcgcc 300 tggctgggaa cctggaacaa
aagtcctgat ccagtagtca cacttctttt tcctaaacag 360 gacggaggtg
acattgtagc tcttgtcttc tttcagctca tagatggtgg catacatctt 420
ttgcgggtct ttgtcttctc tgagaattgc attccctgcg agacctacca cataccactt
480 cccatggaat tggttgtcct ggaagttctg ctgcagaggg accttgctca
gaggtggggc 540 tgggatcagg tctgaggtgg agtcctg 567 <210> SEQ ID
NO 45 <211> LENGTH: 404 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <220> FEATURE: <221> NAME/KEY: CDS
<222> LOCATION: (2)..(403) <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (32)..(34)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (44)..(49) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (71)..(73)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (80)..(82) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (128)..(130)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (134)..(136) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (140)..(145)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (155)..(157) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (161)..(163)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (167)..(169) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (212)..(214)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (224)..(226) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (233)..(235)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (242)..(244) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (299)..(301)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (305)..(307) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (320)..(322)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (326)..(328) <223> OTHER INFORMATION:
a, c, t, g, unknown or other <400> SEQUENCE: 45 c caa ttc cat
ggg aaa tgg tat gtc gtg ggc nnn gcc gga aat nnn nnn 49 Gln Phe His
Gly Lys Trp Tyr Val Val Gly Xaa Ala Gly Asn Xaa Xaa 1 5 10 15 ctg
cgt gag gat aag gat ccg nnn aaa atg nnn gcg acc att tac gag 97 Leu
Arg Glu Asp Lys Asp Pro Xaa Lys Met Xaa Ala Thr Ile Tyr Glu 20 25
30 ttg aaa gaa gat aaa tca tat aac gtc acc nnn gtg nnn ttt nnn nnn
145 Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Xaa Val Xaa Phe Xaa Xaa
35 40 45 aag aaa tgc nnn tac nnn att nnn acc ttt gtg ccg ggg agc
cag ccg 193 Lys Lys Cys Xaa Tyr Xaa Ile Xaa Thr Phe Val Pro Gly Ser
Gln Pro 50 55 60 ggc gag ttt act tta ggc nnn att aaa agt nnn ccg
ggc nnn aca tca 241 Gly Glu Phe Thr Leu Gly Xaa Ile Lys Ser Xaa Pro
Gly Xaa Thr Ser 65 70 75 80 nnn ttg gtc cgc gtc gtg agc acc aac tac
aac cag cat gcc atg gtg 289 Xaa Leu Val Arg Val Val Ser Thr Asn Tyr
Asn Gln His Ala Met Val 85 90 95 ttc ttc aag nnn gtg nnn cag aac
cgc gag nnn ttt nnn atc aca ctg 337 Phe Phe Lys Xaa Val Xaa Gln Asn
Arg Glu Xaa Phe Xaa Ile Thr Leu 100 105 110 tac ggg cgc acg aaa gaa
ctg aca agc gag ctg aag gaa aat ttt atc 385 Tyr Gly Arg Thr Lys Glu
Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile 115 120 125 cgc ttt tcc aaa
tct ctg g 404 Arg Phe Ser Lys Ser Leu
130 <210> SEQ ID NO 46 <211> LENGTH: 134 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic polypeptide <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (11)..(11) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (15)..(15) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (16)..(16) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (24)..(24) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (27)..(27) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (43)..(43) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (45)..(45) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (47)..(47) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (48)..(48) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (52)..(52) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (54)..(54) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (56)..(56) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (71)..(71) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (75)..(75) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (78)..(78) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (81)..(81) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (100)..(100) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (102)..(102) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (107)..(107) <223>
OTHER INFORMATION: Any amino acid <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (109)..(109) <223>
OTHER INFORMATION: Any amino acid <400> SEQUENCE: 46 Gln Phe
His Gly Lys Trp Tyr Val Val Gly Xaa Ala Gly Asn Xaa Xaa 1 5 10 15
Leu Arg Glu Asp Lys Asp Pro Xaa Lys Met Xaa Ala Thr Ile Tyr Glu 20
25 30 Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Xaa Val Xaa Phe Xaa
Xaa 35 40 45 Lys Lys Cys Xaa Tyr Xaa Ile Xaa Thr Phe Val Pro Gly
Ser Gln Pro 50 55 60 Gly Glu Phe Thr Leu Gly Xaa Ile Lys Ser Xaa
Pro Gly Xaa Thr Ser 65 70 75 80 Xaa Leu Val Arg Val Val Ser Thr Asn
Tyr Asn Gln His Ala Met Val 85 90 95 Phe Phe Lys Xaa Val Xaa Gln
Asn Arg Glu Xaa Phe Xaa Ile Thr Leu 100 105 110 Tyr Gly Arg Thr Lys
Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile 115 120 125 Arg Phe Ser
Lys Ser Leu 130 <210> SEQ ID NO 47 <211> LENGTH: 404
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic polynucleotide <400> SEQUENCE:
47 ccagagattt ggaaaagcgg ataaaatttt ccttcagctc gcttgtcagt
tctttcgtgc 60 gcccgtacag tgtgatctta aagtactcgc ggttctggga
cactttcttg aagaacacca 120 tggcatgctg gttgtagttg gtgctcacga
cgcggaccaa gtatgatgtc aggcccgggt 180 aacttttaat gttgcctaaa
gtaaactcgc ccggctggct ccccggcaca aaggtacgaa 240 tccagtagtc
gcatttcttt ttgcgaaaca acacggaggt gacgttatat gatttatctt 300
ctttcaactc gtaaatggtc gcatacattt tctgcggatc cttatcctca cgcagaatgg
360 catttccggc caggcccacg acataccatt tcccatggaa ttgg 404
<210> SEQ ID NO 48 <211> LENGTH: 197 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polypeptide <400> SEQUENCE: 48 Met Ala Ser Met Thr
Gly Gly Gln Gln Met Gly Gln Asp Ser Thr Ser 1 5 10 15 Asp Leu Ile
Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn 20 25 30 Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly Leu Ala 35 40
45 Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro Gln Lys Met Tyr Ala
50 55 60 Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr
Ser Val 65 70 75 80 Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile Arg
Thr Phe Val Pro 85 90 95 Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly
Asn Ile Lys Ser Tyr Pro 100 105 110 Gly Leu Thr Ser Tyr Leu Val Arg
Val Val Ser Thr Asn Tyr Asn Gln 115 120 125 His Ala Met Val Phe Phe
Lys Lys Val Ser Gln Asn Arg Glu Tyr Phe 130 135 140 Lys Ile Thr Leu
Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys 145 150 155 160 Glu
Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly Leu Pro Glu Asn His 165 170
175 Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly Ser Ala Trp
180 185 190 Ser His Pro Gln Phe 195
* * * * *