U.S. patent application number 17/096750 was filed with the patent office on 2021-07-01 for anti-cancer fusion polypeptide.
The applicant listed for this patent is Pieris Pharmaceuticals GmbH. Invention is credited to Rachida Siham Bel Aiba, Marlon Hinner, Thomas Jean Jaquin, Ulrich Moebius, Shane Olwill, Christine Rothe, Corinna Schlosser.
Application Number | 20210198380 17/096750 |
Document ID | / |
Family ID | 1000005450968 |
Filed Date | 2021-07-01 |
United States Patent
Application |
20210198380 |
Kind Code |
A1 |
Hinner; Marlon ; et
al. |
July 1, 2021 |
ANTI-CANCER FUSION POLYPEPTIDE
Abstract
The disclosure provides a fusion polypeptide specific for both
CD137 and HER2/neu, which fusion polypeptide can be useful for
directing CD137 clustering and activation to HER2/neu-positive
tumor cells. Such fusion polypeptide can be used in many
pharmaceutical applications, for example, as anti-cancer agents
and/or immune modulators for the treatment or prevention of human
diseases such as a variety of tumors. The present disclosure also
concerns methods of making the fusion polypeptide described herein
as well as compositions comprising such fusion polypeptide. The
present disclosure further relates to nucleic acid molecules
encoding such fusion polypeptide and to methods for generation of
such fusion polypeptide and nucleic acid molecules. In addition,
the application discloses therapeutic and/or diagnostic uses of
such fusion polypeptide as well as compositions comprising one or
more of such fusion polypeptides.
Inventors: |
Hinner; Marlon; (Munich,
DE) ; Rothe; Christine; (Dachau, DE) ; Olwill;
Shane; (Freising, DE) ; Bel Aiba; Rachida Siham;
(Munich, DE) ; Moebius; Ulrich; (Gauting, DE)
; Schlosser; Corinna; (Freising, DE) ; Jaquin;
Thomas Jean; (Freising, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pieris Pharmaceuticals GmbH |
Hallbergmoos |
|
DE |
|
|
Family ID: |
1000005450968 |
Appl. No.: |
17/096750 |
Filed: |
November 12, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15571561 |
Nov 3, 2017 |
10865250 |
|
|
PCT/EP2016/060041 |
May 4, 2016 |
|
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17096750 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/92 20130101;
C07K 2317/73 20130101; C07K 16/32 20130101; C07K 2319/00 20130101;
C07K 14/435 20130101; A61K 2039/505 20130101; C07K 16/2878
20130101; C07K 2317/52 20130101; A61P 35/00 20180101; C07K 2318/20
20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; C07K 14/435 20060101 C07K014/435; C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2015 |
EP |
15166179.0 |
May 18, 2015 |
EP |
15167917.2 |
Sep 17, 2015 |
EP |
15002702.7 |
Nov 4, 2015 |
EP |
15192870.2 |
Jan 11, 2016 |
EP |
16150705.8 |
Apr 15, 2016 |
EP |
16000862.9 |
Claims
1-47. (canceled)
48. A fusion polypeptide that is capable of binding both CD137 and
HER2/neu, wherein the fusion polypeptide comprises at least two
subunits, wherein the first subunit comprises an immunoglobulin
having binding specificity for HER2/neu, and wherein the second
subunit comprises a lipocalin mutein having binding specificity for
CD137, wherein the lipocalin mutein comprises at least 10 of the
following mutated amino acid residues in comparison with the linear
polypeptide sequence of mature human Lipocalin 2 (hNGAL) (SEQ ID
NO: 18): Gln 28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile;
Ile 41.fwdarw.Arg or Lys; Gln 49.fwdarw.Val, Ile, His, Ser or Asn;
Tyr 52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Met, Ala or
Gly; Leu 70.fwdarw.Ala, Lys, Ser or Thr; Arg 72.fwdarw.Asp; Lys
73.fwdarw.Asp; Asp 77.fwdarw.Met, Arg, Thr or Asn; Trp
79.fwdarw.Ala or Asp; Arg 81.fwdarw.Met, Trp or Ser; Phe
83.fwdarw.Leu; Cys 87.fwdarw.Ser; Leu 94.fwdarw.Phe; Asn
96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr
106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr
132.fwdarw.Glu; and Lys 134.fwdarw.Tyr, and wherein the lipocalin
mutein has at least 85% sequence identity to the amino acid
sequence shown in SEQ ID NO: 2.
49. The fusion polypeptide of claim 48, wherein the amino acid
sequence of the lipocalin mutein comprises one of the following
sets of mutated amino acid residues in comparison with the linear
polypeptide sequence of mature hNGAL (SEQ ID NO: 18): (a) Gln
28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Lys; Gln 49.fwdarw.Asn; Tyr 52.fwdarw.Met; Ser
68.fwdarw.Gly; Leu 70.fwdarw.Thr; Arg 72.fwdarw.Asp; Lys
73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp 79.fwdarw.Ala; Arg
81.fwdarw.Ser; Cys 87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr
100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys
125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys
134.fwdarw.Tyr; (b) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln; Ala
40.fwdarw.Ile; Ile 41.fwdarw.Arg; Gln 49.fwdarw.Ile; Tyr
52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Met; Leu
70.fwdarw.Lys; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Met; Trp 79.fwdarw.Asp; Arg 81.fwdarw.Trp; Cys
87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr; (c) Gln
28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Arg; Gln 49.fwdarw.Asn; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Ala; Leu 70.fwdarw.Ala; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp
79.fwdarw.Asp; Arg 81.fwdarw.Trp; Cys 87.fwdarw.Ser; Asn
96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr
106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr
132.fwdarw.Glu; Lys 134.fwdarw.Tyr; (d) Gln 28.fwdarw.His; Leu
36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile 41.fwdarw.Lys; Gln
49.fwdarw.Asn; Tyr 52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser
68.fwdarw.Ala; Leu 70.fwdarw.Ala; Arg 72.fwdarw.Asp; Lys
73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp 79.fwdarw.Asp; Arg
81.fwdarw.Trp; Cys 87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr
100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys
125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys
134.fwdarw.Tyr; (e) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln; Ala
40.fwdarw.Ile; Ile 41.fwdarw.Lys; Gln 49.fwdarw.Ser; Tyr
52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Gly; Leu
70.fwdarw.Ser; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Thr; Trp 79.fwdarw.Ala; Arg 81.fwdarw.Met; Cys
87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr; (f) Gln
28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Lys; Gln 49.fwdarw.Val; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Gly; Leu 70.fwdarw.Thr; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Arg; Trp
79.fwdarw.Asp; Arg 81.fwdarw.Ser; Cys 87.fwdarw.Ser; Leu
94.fwdarw.Phe; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr; (g) Gln
28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Arg; Gln 49.fwdarw.His; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Gly; Leu 70.fwdarw.Thr; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp
79.fwdarw.Ala; Arg 81.fwdarw.Ser; Cys 87.fwdarw.Ser; Asn
96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr
106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr
132.fwdarw.Glu; Lys 134.fwdarw.Tyr; (h) Gln 28.fwdarw.His; Leu
36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile 41.fwdarw.Lys; Gln
49.fwdarw.Asn; Tyr 52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser
68.fwdarw.Gly; Leu 70.fwdarw.Thr; Arg 72.fwdarw.Asp; Lys
73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp 79.fwdarw.Ala; Arg
81.fwdarw.Ser; Phe 83.fwdarw.Leu; Cys 87.fwdarw.Ser; Leu
94.fwdarw.Phe; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr; or (i) Gln
28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Arg; Gln 49.fwdarw.Ser; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Ala; Leu 70.fwdarw.Thr; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Asn; Trp
79.fwdarw.Ala; Arg 81.fwdarw.Ser; Cys 87.fwdarw.Ser; Asn
96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr
106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr
132.fwdarw.Glu; Lys 134.fwdarw.Tyr.
50. The fusion polypeptide of claim 48, wherein the lipocalin
mutein comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2 and 39-46.
51. The fusion polypeptide of claim 48, wherein the mutein
comprises an amino acid sequence having at least 95% sequence
identity to the amino acid sequence shown in SEQ ID NO: 2.
52. The fusion polypeptide of claim 48, wherein the mutein
comprises the amino acid sequence of SEQ ID NO: 2.
53. The fusion polypeptide of claim 48, wherein the first subunit
and the second binding domain are linked via a peptide linker
between the N-terminus of the lipocalin mutein of the second
subunit and the C-terminus of a heavy chain constant region (CH) of
the immunoglobulin of the first subunit.
54. The fusion polypeptide of claim 53, wherein the peptide linker
is a (G4S)3 linker.
55. The fusion polypeptide of claim 48, wherein the fusion
polypeptide comprises the amino acid sequence shown in SEQ ID NO:
19.
56. The fusion polypeptide of claim 48, wherein the immunoglobulin
is a monoclonal antibody.
57. The fusion polypeptide of claim 56, wherein the monoclonal
antibody comprises the antigen-binding domain of trastuzumab or
pertuzumab.
58. The fusion polypeptide of claim 56, wherein the monoclonal
antibody has the heavy and light chains provided by SEQ ID NOs: 3
and 4.
59. The fusion polypeptide of claim 56, wherein the monoclonal
antibody has an IgG4 backbone.
60. The fusion polypeptide of claim 59, wherein the IgG4 backbone
has any one of the following mutations selected from the group
consisting of S228P, N297A, F234A and L235A.
61. The fusion polypeptide of claim 48, wherein the fusion
polypeptide has one or more of the following properties: (i) it is
capable of binding CD137 with an EC.sub.50 value comparable to or
lower than the EC.sub.50 value of the lipocalin mutein specific for
CD137 included in the fusion polypeptide; (ii) it is capable of
binding CD137 with an EC.sub.50 value of about 1 nM or lower; (iii)
it is capable of binding HER2/neu with an EC.sub.50 value
comparable to or lower than the EC.sub.50 value of the
immunoglobulin specific for HER2/neu included in such fusion
polypeptide; (iv) it is capable of binding HER2/neu with an
EC.sub.50 value of about 1 nM or lower; (v) it is capable of
simultaneously binding CD137 and HER2/neu; (vi) it is capable of
simultaneously binding CD137 and HER2/neu with EC.sub.50 values of
about 4 nM or lower.
62. The fusion polypeptide of claim 48, wherein the fusion
polypeptide is capable of co-stimulating T cell responses.
63. The fusion polypeptide of claim 48, wherein the fusion
polypeptide is capable of inducing IL-2 secretion and T cell
proliferation.
64. A nucleic acid molecule comprising a nucleotide sequence
encoding the fusion polypeptide of claim 48.
65. A host cell containing a nucleic acid molecule of claim 64.
66. A method of producing the fusion polypeptide of claim 48,
wherein the fusion polypeptide is produced starting from a nucleic
acid coding for the fusion polypeptide.
67. A pharmaceutical composition comprising the fusion polypeptide
of claim 48.
Description
I. BACKGROUND
[0001] HER2/neu is a member of the human epidermal growth factor
receptor family. Amplification or overexpression of this oncogene
has been shown to play an important role in the development and
progression of a variety of tumors, including certain aggressive
types of breast cancer. HER2/neu has been shown to be highly
differentially expressed on tumor cells with much higher
cell-surface density compared to healthy tissue.
[0002] Trastuzumab (marketed as Herceptin.RTM.), a monoclonal
antibody targeting HER2/neu, is indicated for the treatment of
women with either early stage or metastatic HER2(+) breast cancer.
Despite the promising activity of monoclonal antibodies such as
Trastuzumab in this setting, the response rates among patients with
either refractory or advanced cancer are suboptimal. For example,
while the objective response rate rose significantly in a clinical
trial comparing chemotherapy alone with chemotherapy plus
Trastuzumab--from 32% to 50% --, this still left the other half of
the enrolled patients having no response (Slamon D. J. et al., N
Engl J Med. 2001 Mar. 15; 344(11):783-92). Therefore, better
HER2/neu-targeting therapies with an improved response rate are
required.
[0003] CD137 is a co-stimulatory immune receptor and a member of
the tumor necrosis factor receptor (TNFR) super-family. It is
mainly expressed on activated CD4+ and CD8+ T cells, activated B
cells, and natural killer (NK) cells but can also be found on
resting monocytes and dendritic cells (Li, S. Y. et al., Clin
Pharmacol 2013 5(Suppl 1):47-53), or endothelial cells (Snell, L.
M. et al., Immunol Rev 2011 November; 244(1):197-217). CD137 plays
an important role in the regulation of immune responses and thus is
a target for cancer immunotherapy. CD137 ligand (CD137L) is the
only known natural ligand of CD137, and is constitutively expressed
on several types of APC, such as activated B cells, monocytes, and
splenic dendritic cells, and it can be induced on T
lymphocytes.
[0004] CD137L is a trimeric protein that exists as a membrane-bound
form and as a soluble variant. The ability of soluble CD137L to
activate CD137 e.g. on CD137-expressing lymphocytes is limited,
however, and large concentrations are required to elicit an effect
(Wyzgol, A. et al., J Immunol 2009 Aug. 1; 183(3):1851-1861). The
natural way of activation of CD137 is via the engagement of a
CD137-positive cell with a CD137L-positive cell. CD137 activation
is then thought to be induced by clustering through CD137L on the
opposing cell, leading to signaling via TRAF1, 2 and 3 (Snell, L.
M. et al., Immunol Rev 2011 November; 244(1):197-217, Yao, S. et
al., Nat Rev Drug Disc 2013 February; 12(2):130-146) and further
concomitant downstream effects in the CD137-positive T-cell. In the
case of T-cells activated by recognition of their respective
cognate targets, the effects elicited by costimulation of CD137 are
a further enhanced activation, enhanced survival and proliferation,
the production of pro-inflammatory cytokines and an improved
capacity to kill.
[0005] The benefit of CD137 costimulation for the elimination of
cancer cells has been demonstrated in a number of preclinical
in-vivo models. The forced expression of CD137L on a tumor, for
example, leads to tumor rejection (Melero, I. et al., Eur J Immunol
1998 March; 28(3):1116-1121). Likewise, the forced expression of an
anti-CD137 scFv on a tumor leads to a CD4.sup.+ T-cell and NK-cell
dependent elimination of the tumor (Ye, Z. et al., Nat Med 2002
April; 8(4):343-348, Zhang, H. et al., Mol Canc Ther 2006 January;
5(1):149-155, Yang, Y. et al., Canc Res 2007 Mar. 1;
67(5):2339-2344). A systemically administered anti-CD137 antibody
has also been demonstrated to lead to retardation of tumor growth
(Martinet, O. et al., Gene Ther 2002 June; 9(12):786-792).
[0006] It has been shown that CD137 is an excellent marker for
naturally occurring tumor-reactive T cells in human tumors (Ye, Q.
et al., Clin Canc Res: 2014 Jan. 1; 20(1):44-55), and that
anti-CD137 antibodies can be employed to improve the expansion and
activity of CD8+ melanoma tumor-infiltrating lymphocytes for the
application in adoptive T-cell therapy (Chacon, J. A. et al., PloS
One 2013 8(4):e60031).
[0007] The preclinical demonstration of the potential therapeutic
benefit of CD137 costimulation has spurred the development of
therapeutic antibodies targeting CD137, BMS-663513 (Jure-Kunkel, M.
et al., U.S. Pat. No. 7,288,638) and PF-05082566 (Fisher, T. S. et
al., Canc Immunol Immunother 2012 October; 61(10):1721-1733); both
are currently in early clinical trials.
[0008] However, it has only recently been appreciated that a
bivalent CD137-binder like an antibody may by itself not be
sufficient to cluster CD137 on T-cells or NK-cells and lead to
efficient activation, in analogy to the lack of activity of the
trivalent soluble CD137L. In recent publications utilizing
preclinical mouse models, in-vivo evidence has been presented that
the mode of action of other anti-TNFR antibodies in fact requires
the interaction of the antibodies via their Fc-part with Fc-gamma
receptors on Fc-gamma-receptor expressing cells (Bulliard, Y. et
al., J Exp Med 2013 Aug. 26; 210(9):1685-1693, Bulliard, Y. et al.,
Immunol Cell Biol 2014 July; 92(6):475-480). The mode of action of
the antibodies currently in clinical development may therefore be
dominated by a non-targeted clustering via Fc-gamma receptors which
may be nearly randomly dependent on the presence of
Fc-.gamma.-expressing cells in the vicinity of the tumor.
[0009] Thus, there is unmet need for the generation of therapeutics
that cluster and activate CD137 with a specific tumor-targeted mode
of action.
[0010] To meet this unmet need, the present application, provides a
novel approach of simultaneously engaging CD137 and tumor antigen
HER2/neu via a fusion polypeptide having the following
properties:
(a) binding specificity for CD137; and (b) binding specificity for
HER2/neu;
[0011] This fusion polypeptide is designed to provide a
tumor-target-dependent activation of CD137 on lymphocytes, via HER2
overexpressed on tumor cells. Such a molecule is expected to
further activate T-cells and/or NK cells that are located in the
vicinity of a HER2-positive tumor. Such a bispecific may display
improved therapeutic effects over either anti-HER2 or anti-CD137
antibodies.
II. Definitions
[0012] The following list defines terms, phrases, and abbreviations
used throughout the instant specification. All terms listed and
defined herein are intended to encompass all grammatical forms.
[0013] As used herein, unless otherwise specified, "CD137" means
human CD137. CD137 is also known as "4-1BB" or "tumor necrosis
factor receptor superfamily member 9 (TNFRSF9)" or "induced by
lymphocyte activation (ILA)". Human CD137 means a full-length
protein defined by UniProt Q07011, a fragment thereof, or a variant
thereof.
[0014] As used herein, unless otherwise specified, "HER2" or
"HER2/neu" means human HER2. Her-2 or HER2/neu is also known as
"erbB-2", "c-neu", or "p185". Human Her 2 means a full-length
protein defined by UniProt P04626, a fragment thereof, or a variant
thereof.
[0015] As used herein, "detectable affinity" means the ability to
bind to a selected target with an affinity constant of generally at
least about 10.sup.-5 M or below. Lower affinities are generally no
longer measurable with common methods such as ELISA and therefore
of secondary importance.
[0016] As used herein, "binding affinity" of a protein of the
disclosure (e.g. a mutein of a lipocalin) or a fusion polypeptide
thereof to a selected target (in the present case, CD137 and/or
HER2/neu), can be measured (and thereby KD 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.
[0017] It is also noted that the complex formation between the
respective binder 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.
[0018] Therefore, it is also clear to the skilled person that the
K.sub.D values (dissociation constant of the complex formed between
the respective binder 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 K.sub.D
value was determined by surface plasmon resonance (Biacore), by
competition ELISA, or by "direct ELISA."
[0019] 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,
compared to the naturally occurring (wild-type) nucleic acid or
protein "reference" scaffold. Said term also includes fragments of
a mutein and variants as described herein. Lipocalin muteins of the
present invention, fragments or variants thereof preferably retain
the function of binding to CD137 as described herein.
[0020] The term "fragment" as used herein in connection with the
muteins of the disclosure relates to proteins or peptides derived
from full-length mature human tear lipocalin 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 may
include at least 10, more such as 20 or 30 or more consecutive
amino acids of the primary sequence of the mature lipocalin and are
usually detectable in an immunoassay of the mature lipocalin. In
general, the term "fragment", as used herein with respect to the
corresponding protein ligand CD137 of a lipocalin mutein of the
disclosure or of the combination according to the disclosure or of
a fusion protein described herein, relates to N-terminally and/or
C-terminally shortened protein or peptide ligands, which retain the
capability of the full length ligand to be recognized and/or bound
by a mutein according to the disclosure.
[0021] 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 mature
lipocalin 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 disclosure 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 or deletion may be introduced
independently from each other in any of the peptide segments that
can be subjected to mutagenesis in the disclosure. In one exemplary
embodiment of the disclosure, 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).
[0022] 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.
[0023] "Identity" is a property of sequences that measures their
similarity or relationship. The term "sequence identity" or
"identity" as used in the present disclosure means the percentage
of pair-wise identical residues--following (homologous) alignment
of a sequence of a polypeptide of the disclosure with a sequence in
question--with respect to the number of residues in the longer of
these two sequences. Sequence identity is measured by dividing the
number of identical amino acid residues by the total number of
residues and multiplying the product by 100.
[0024] 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 a polypeptide of the
disclosure (e.g., any lipocalin mutein of the disclosure).
[0025] 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-) 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.
[0026] Specifically, in order to determine whether an amino acid
residue of the amino acid sequence of a lipocalin (mutein)
different from a wild-type lipocalin corresponds to a certain
position in the amino acid sequence of a wild-type lipocalin, 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 wild-type lipocalin
can serve as "subject sequence" or "reference sequence", while the
amino acid sequence of a lipocalin different from the wild-type
lipocalin described herein serves as "query sequence". The terms
"reference sequence" and "wild-type sequence" are used
interchangeably herein. A preferred wild-type lipocalin is shown in
SEQ ID NO: 18 (Tlc) or SEQ ID NO: 17 (NGAL), respectively.
Dependent on whether a lipocalin mutein of the present invention is
based on Tlc or NGAL, respectively, the corresponding wild-type
lipocalin may be used as reference sequence or wild-type
sequence.
[0027] "Gaps" are spaces in an alignment that are the result of
additions or deletions of amino acids. Thus, two copies of exactly
the same sequence have 100% identity, but sequences that are less
highly conserved, and have deletions, additions, or replacements,
may have a lower degree of sequence identity. Those skilled in the
art will recognize that several computer programs are available for
determining sequence identity using standard parameters, for
example Blast (Altschul, et al. (1997) Nucleic Acids Res. 25,
3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol. 215,
403-410), and Smith-Waterman (Smith, et al. (1981) J. Mol. Biol.
147, 195-197).
[0028] The term "variant" as used in the present disclosure relates
to derivatives of a protein or peptide that include modifications
of the amino acid sequence, for example by substitution, deletion,
insertion or chemical modification. Such modifications do in some
embodiments not reduce the functionality of the protein or peptide.
Such variants include proteins, wherein one or more amino acids
have been replaced by their respective D-stereoisomers or by amino
acids other than the naturally occurring 20 amino acids, such as,
for example, ornithine, hydroxyproline, citrulline, homoserine,
hydroxylysine, norvaline. However, such substitutions may also 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. The term "variant", as
used herein with respect to the corresponding protein ligand CD137
of a lipocalin mutein of the disclosure or of the combination
according to the disclosure or of a fusion protein described
herein, relates to CD137 or fragment thereof, respectively, that
has one or more such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more amino acid
substitutions, deletions and/or insertions in comparison to a
wild-type CD137 protein, respectively, such as a CD137 reference
protein as deposited with UniProt as described herein. A CD137
variant, respectively, has preferably an amino acid identity of at
least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type human
CD137, such as a CD137 reference protein as deposited with UniProt
as described herein.
[0029] By a "native sequence" lipocalin is meant a lipocalin that
has the same amino acid sequence as the corresponding polypeptide
derived from nature. Thus, a native sequence lipocalin can have the
amino acid sequence of the respective naturally-occurring lipocalin
from any organism, in particular a mammal. Such native sequence
polypeptide can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native sequence"
polypeptide specifically encompasses naturally-occurring truncated
or secreted forms of the lipocalin, naturally-occurring variant
forms such as alternatively spliced forms and naturally-occurring
allelic variants of the lipocalin. A polypeptide "variant" means a
biologically active polypeptide having at least about 50%, 60%,
70%, 80% or at least about 85% amino acid sequence identity with
the native sequence polypeptide. Such variants include, for
instance, polypeptides in which one or more amino acid residues are
added or deleted at the N- or C-terminus of the polypeptide.
Generally a variant has at least about 70%, including at least
about 80%, such as at least about 85% amino acid sequence identity,
including at least about 90% amino acid sequence identity or at
least about 95% amino acid sequence identity with the native
sequence polypeptide. As an illustrative example, the first 4
N-terminal amino acid residues (His-His-Leu-Leu) and the last 2
C-terminal amino acid residues (Ser-Asp) can be deleted in a tear
lipocalin (Tlc) mutein of the disclosure without affecting the
biological function of the protein, e.g. SEQ ID NOs: 32-38. In
addition, as another illustrative example, certain amino acid
residues can be deleted in a lipocalin 2 (NGAL) mutein of the
disclosure without affecting the biological function of the
protein, e.g. (Lys-Asp-Pro, positions 46-48) as to SEQ ID NO:
42.
[0030] The term "position" when used in accordance with the
disclosure 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. To understand the
term "correspond" or "corresponding" as used herein in the context
of the amino acid sueqnece positions of one or more lipocalin
muteins, a corresponding 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 disclosure
which may be substituted may vary 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 disclosure 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).
[0031] Thus, for a corresponding position in accordance with the
disclosure, it is preferably to be understood that the positions of
nucleotides/amino acids may differ in the indicated number than
similar neighbouring nucleotides/amino acids, but said neighbouring
nucleotides/amino acids, which may be exchanged, deleted, or added,
are also comprised by the one or more corresponding positions.
[0032] In addition, for a corresponding position in a lipocalin
mutein based on a reference scaffold in accordance with the
disclosure, it is preferably to be understood that the positions of
nucleotides/amino acids are structurally corresponding to the
positions elsewhere in a (mutant or wild-type) lipocalin, even if
they may differ in the indicated number, as appreciated by the
skilled in light of the highly-conserved overall folding pattern
among Iipocalins.
[0033] The word "detect", "detection", "detectable" 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.
[0034] A "subject" is a vertebrate, preferably a mammal, more
preferably a human. The term "mammal" is used herein to refer to
any animal classified as a mammal, including, without limitation,
humans, domestic and farm animals, and zoo, sports, or pet animals,
such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as
cynomolgous monkeys and etc., to name only a few illustrative
examples. Preferably, the mammal herein is human.
[0035] An "effective amount" is an amount sufficient to effect
beneficial or desired results. An effective amount can be
administered in one or more administrations.
[0036] A "sample" is defined as a biological sample taken from any
subject. Biological samples include, but are not limited to, blood,
serum, urine, feces, semen, or tissue.
[0037] A "subunit" of a fusion polypeptide disclosed herein is
defined as a stretch of amino acids of the polypeptide, which
stretch defines a unique functional unit of said polypeptide such
as provides binding motif towards a target.
III. DESCRIPTIONS OF FIGURES
[0038] FIG. 1: provides an overview over the design of the
representative fusion polypeptides described in this application,
which are bispecific with regard to the targets, HER2 and CD137.
Representative fusion polypeptides were made based on an antibody
specific for HER2 (SEQ ID NOs: 3 and 4) and a lipocalin mutein
specific for CD137 (SEQ ID NO: 2). Direct fusion of the antibody
with the lipocalin mutein resulted in a fusion polypeptide of SEQ
ID NOs: 5 and 6. Lipocalin muteins were fused to either one of the
four termini of the antibody, using an engineered IgG4 backbone
with the mutations S228P, F234A and L235A (SEQ ID NOs: 9 and 10,
SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and
16). In addition, within the fusion polypeptide SEQ ID NOs: 7 and
8), a N297A mutation was made to remove the glycosylation
motif.
[0039] FIG. 2: depicts the results of an ELISA experiment in which
the affinity of representative fusion polypeptides and the
benchmark antibody against HER2 was determined. Recombinant HER2
was coated on a microtiter plate, and the tested agents were
titrated starting from a concentration of 100 nM. Bound agents
under study were detected via an anti-human IgG Fc antibody as
described in Example 2. The data was fit with a 1:1 binding model
with EC50 value and the maximum signal as free parameters, and a
slope that was fixed to unity. The resulting EC50 values are
provided in Table 2.
[0040] FIG. 3: shows the results of an ELISA experiment in which
the affinity of representative fusion polypeptides and the positive
control lipocalin mutein against CD137 was determined. An Fc-fusion
of human CD137 was coated on a microtiter plate, and the tested
agents were titrated starting from a concentration of 100 nM. Bound
agents under study were detected via an anti-human-IgG-Fc antibody
as described in Example 3. The data was fit with a 1:1 binding
model with EC50 value and the maximum signal as free parameters,
and a slope that was fixed to unity. The resulting EC50 values are
provided in Table 3.
[0041] FIG. 4: illustrates the results of an ELISA experiment in
which the ability of representative fusion polypeptides to
simultaneously bind both targets, HER2 and CD137, was determined.
Recombinant HER2 was coated on a microtiter plate, followed by a
titration of the fusion polypeptides starting from a concentration
of 100 nM. Subsequently, a constant concentration of biotinylated
human CD137-Fc was added, which was detected via extravidin as
described in Example 4.
[0042] FIG. 5: shows the result of a T-cell activation assay in
which the ability of the fusion polypeptide of SEQ ID NOs: 15 and
16 to co-stimulate T-cell responses was assessed. The fusion
polypeptide of SEQ ID NOs: 15 and 16 at different concentrations
was coated onto a plastic dish together with an anti-human CD3
antibody, and purified T-cells were subsequently incubated on the
coated surface. Supernatant interleukin 2 (IL-2) levels were
measured as described in Example 5.
[0043] FIG. 6: provides a representative experiment in which the
ability of the fusion polypeptide of SEQ ID NOs: 9 and 10 to
co-stimulate T-cell activation in a HER2-target-dependent manner
was investigated. As a control, we employed the monospecific,
HER2-binding antibody of SEQ ID NOs: 3 and 4. In the experiment, an
anti-human CD3 antibody was coated on a plastic culture dish, and
subsequently HER2-positive SKBR3 cells were cultured on the dish
overnight. The next day, purified T-cells were incubated on the
coated surface in the presence of various concentrations of the
bispecific fusion polypeptide SEQ ID NOs: 9 and 10 (filled circles)
or the control antibody of SEQ ID NOs: 3 and 4. The values
determined for SEQ ID NOs: 3 and 4 at different concentrations are
provided as the average (dotted line). Supernatant interleukin 2
(IL-2) (A) and IFN-.gamma. (B) were determined by an
Electrochemoluminescence-based assay. The experiment was also
performed in the presence of an excess of SEQ ID NOs: 3 and 4, and
supernatant levels of IL-2 (C) and IFN-.gamma. (D) were measured.
The data was fitted with a 1:1 binding model.
[0044] FIG. 7: provides a representative experiment in which the
ability of the fusion polypeptide of SEQ ID NOs: 11 and 12 to
co-stimulate T-cell activation in a HER2-target-dependent manner
was investigated. For details; see legend of FIG. 6.
[0045] FIG. 8: provides a representative experiment in which the
ability of the fusion polypeptide of SEQ ID NOs: 13 and 14 to
co-stimulate T-cell activation in a HER2-target-dependent manner
was investigated. For details; see legend of FIG. 6.
[0046] FIG. 9: provides a representative experiment in which the
ability of the fusion polypeptide of SEQ ID NOs: 15 and 16 to
co-stimulate T-cell activation in a HER2-target-dependent manner
was investigated. For details; see legend of FIG. 6.
[0047] FIG. 10: provides a representative experiment on the
affinity of polypeptides to FcgRI, FcgRIII and FcRn as described in
Examples 7 and 8.
[0048] FIG. 11: provides a representative experiment in which the
ability of the fusion polypeptides indicated in the Figure to
co-stimulate T-cell activation with different cell lines was
investigated. Cell lines utilized were the highly HER2-positive
cells (SKBR3, BT474) and cell lines expressing HER2 at a level
similar to that of healthy cells (HepG2, MCF7). In the experiment,
an anti-human CD3 antibody was coated on a plastic culture dish,
and subsequently the cell line under study was cultured on the dish
overnight. The next day, purified T-cells were incubated on the
coated surface for three days in the presence of various
concentrations of the bispecific fusion polypeptides as follows:
(A) SEQ ID NOs: 9 and 10 (solid lines) or the control antibody of
SEQ ID NOs: 3 and 4 (broken line). (B) Anti-CD137 antibody SEQ ID
NOs: 32 and 33. (C) Anti-CD137 antibody SEQ ID NOs: 34 and 35.
Supernatant interleukin 2 levels were determined by an
Electrochemoluminescence-based assay. The plotted relative IL-2
response corresponds to the ratio of the responses obtained in the
presence and in the absence ("background") of test articles.
[0049] FIG. 12: provides representative size exclusion
chromatography (SEC) traces of bispecific fusion polypeptides and
the control antibody of SEQ ID NOs: 3 and 4 before (bottom curves)
and after (top curves) incubation for 4 weeks at 40.degree. C. in
PBS, pH 7.4. Sample concentration was 20 mg/mL in each case, fusion
polypeptide identity was as indicated in the figure. SEC curves are
plotted with an offset on the y-axis for better visualization.
[0050] FIG. 13: provides the result of a pharmacokinetic analysis
of the bispecific fusion polypeptides and the control antibody of
SEQ ID NOs: 3 and 4 in mice. Male CD-1 mice (3 mice per timepoint)
were injected intravenously with fusion polypeptides at a dose of
10 mg/kg. Drug levels were detected using a Sandwich ELISA
detecting the full bispecific construct via the targets HER2 and
CD137. Trastuzumab plasma levels were determined using a Sandwich
ELISA with targets HER2 and human Fc. The data were fitted using a
two-compartmental model.
[0051] FIG. 14: provides the result of a pharmacokinetic analysis
of the bispecific fusion polypeptides and the control antibody of
SEQ ID NOs: 3 and 4 in mice. Male, trastuzumab-naive cynomolgus
monkeys received test articles as an intravenous infusion of 60
minutes duration at a dose of 3 mg/kg. Drug levels were detected
using a Sandwich ELISA detecting the full bispecific construct via
the targets HER2 and CD137. Trastuzumab plasma levels were
determined using a Sandwich ELISA with targets HER2 and human Fc.
The data were fitted using a two-compartmental model.
[0052] FIG. 15: provides the result of an in vitro T cell
immunogenicity assessment of the bispecific fusion polypeptides,
the control antibody of SEQ ID NOs: 3 and 4 and the positive
control keyhole limpet hemocyanine (KLH). The assay was performed
using a PBMC-based format as described in Example 13, with 32
donors and human leukocyte antigen (HLA) allotypes reflective of
the distribution in a global population: (A) Stimulation index
(proliferation in the presence vs. absence of test article). The
average responses are indicated as bars. The threshold that defines
a responding donor (stimulation index >2) is indicated as a
dotted line. (B) Number of responders
[0053] FIG. 16: Relative median tumor volume after treatment with
CD137/HER2 bispecifics or controls in humanized mouse tumor model.
NSG mice were engrafted with s.c. SK-OV-3 tumors which were allowed
to grow to an average of 120 mm3. Mice were randomized into
treatment groups and received 7.times.106 fresh human PBMC i.v. and
the molecules and doses indicated 1 hour after PBMC injection on
day 0, and again on day 7 and day 14. Each group contained 10 mice
with the exception of the group studying SEQ ID NOs: 32 and 33
which consisted of 7 mice. Tumor growth was recorded every 3-4
days.
[0054] FIG. 17: provides a representative experiment in which the
ability of the fusion polypeptides indicated in the Figure to
activate the CD137 pathway in dependence of the HER2.sup.high
NCI-N87 target cells was investigated. In the experiment, NCI-N87
tumor target cells were cultured on the dish overnight. The
following day, NF-.kappa.B-uc2P/4-1BB Jurkat reporter cells were
added to the coated target cells in the presence of various
concentrations of the bispecific fusion polypeptides as follows:
(A) SEQ ID NOs: 9 and 10 (solid lines) or the control antibody of
SEQ ID NOs: 3 and 4 (broken line). (B) Anti-CD137 antibody SEQ ID
NOs: 32 and 33. (C) Anti-CD137 antibody SEQ ID NOs: 34 and 35. The
luminescence signal (RLU) represents a relative measurement of
CD137 pathway activation. Four parameter logistic curve analysis
was performed with GraphPad Prism software.
[0055] FIG. 18: (A) shows median tumor volume after treatment with
CD137/HER2 bispecifics or controls in humanized mouse tumor model.
NOG mice were engrafted with s.c. SK-OV-3 tumors which were allowed
to grow to an average of 120 mm.sup.3. Mice were randomized into
treatment groups and received 7.times.10.sup.6 fresh human PBMC
i.v. and the molecules and doses indicated 1 hour after PBMC
injection on day 0, and again on day 7 and day 14. Each group
contained 10 mice. Tumor growth was recorded twice weekly for 20
days. (B) Shows the results of immunohistochemistry for the human
lymphocyte marker CD45 as a marker for infiltration of human T
cells on tumors from two mice that were harvested on day 20 post
treatment.
[0056] FIG. 19: (A) shows CD45, CD3 and CD8 phenotype of PMBCs of
the treatment and control groups of Example 16 taken on day 19 of
that study. FIG. 19A on the left shows the percentage of total
PMBCs expressing human CD45 while the FIG. 19A on the right shows
the percentage of CD45-expressing PMBCs that also express CD3 and
CD8. The figure shows increased CD8.sup.+ human effector T cell
expansion in the anti-CD137 mAb treatment group. (B) shows the
mortality of treatment and control groups of Experiment 16. Plotted
values of FIG. 19B correspond to number of mice per group of ten
that died spontaneously or needed to be sacrificed based on defined
general condition criteria.
[0057] FIG. 20: Mortality of treatment and control groups of
Example 18. Plotted values correspond to number of mice per group
of ten (SEQ ID Nos: 9 and 10, 4 .mu.g: group of nine) that died
spontaneously or needed to be sacrificed based on defined general
condition criteria.
[0058] FIG. 21: Relative median tumor volume after treatment with
CD137/HER2 bispecifics or controls in humanized mouse tumor model.
NSG mice were engrafted with s.c. SK-OV-3 tumors which were allowed
to grow to an average of 110 mm.sup.3. Mice were randomized into
treatment groups and received 7.times.10.sup.6 fresh human PBMC
intravenously and intraperitoneal injections of the molecules and
doses indicated 1 hour after PBMC injection on day 0, and again on
day 7 and day 14. Each group contained 10 mice, except for group 7
(SEQ ID Nos: 9 and 10, 4 .mu.g) which contained only 9 mice. Tumor
growth was recorded every 3-4 days.
[0059] FIG. 22: Immunohistochemistry of human CD45-positive
lymphocytes. Tumors were excised from tumor-bearing mice and up to
six tumors for each group were formalin-fixed, embedded in paraffin
and processed for immunohistochemistry using anti-human CD45
antibodies. CD45-positive cells were identified by
3,3'-diaminobenzidine (DAB) staining. To allow clear visualization
of DAB-positivity in a greyscale image, contrast and brightness of
the images was digitally adjusted.
[0060] FIG. 23: Digital quantitation of DAB positivity of the
images shown in FIG. 22. The Figure illustrates increased
frequencies of hCD45-positive human lymphocytes in the groups
treated with SEQ ID Nos: 9 and 10 compared to various controls (see
Example 17 for details).
[0061] FIG. 24: Phenotype of PMBCs of the treatment and control
groups of Example 18 taken on day (19) of that study. (A)
Percentage of total PMBCs expressing human CD45. (B) Percentage of
CD45-expressing PMBCs that express CD8. The Figure shows increased
CD8.sup.+ human effector T cell expansion in the anti-CD137 mAb
(SEQ ID Nos: 32 and 33) treatment group compared to negative
controls and SEQ ID Nos: 9 and 10 treatment groups.
IV. DETAILED DESCRIPTION OF THE DISCLOSURE
[0062] In some embodiments, the fusion polypeptide contains at
least two subunits in any order: a first subunit that comprises a
full-length immunoglobulin or an antigen-binding domain thereof
specific for HER2/neu, and a second subunit that comprises a
lipocalin mutein specific for CD137.
[0063] In some embodiments, the fusion polypeptide also may contain
a third subunit. For instance, the polypeptide may contain a
subunit specific for CD137. In some embodiments, said third subunit
comprises a lipocalin mutein specific for CD137.
[0064] In some embodiments, one subunit can be linked to another
subunit as essentially described in FIG. 1. For example, one
lipocalin mutein can be linked, via a peptide bond, to the
C-terminus of the immunoglobulin heavy chain domain (VH), the
N-terminus of the VH, the C-terminus of the immunoglobulin light
chain (VL), and/or the N-terminus of the VL (cf. FIG. 1). In some
particular embodiments, a lipocalin mutein subunit can be fused at
its N-terminus and/or its C-terminus to an immunoglobulin subunit.
For example, the lipocalin mutein may be linked via a peptide bond
between the C-terminus of a heavy chain constant region (CH) or the
C-terminus of a light chain constant region (CL) of the
immunoglobulin. In some still further embodiments, the peptide bond
may be an unstructured (G4S)3 linker, for example, as shown in SEQ
ID NO: 19.
[0065] In this regard, one subunit may be fused at its N-terminus
and/or its C-terminus to another subunit. For example, when one
subunit comprises a full-length immunoglobulin, another subunit may
be linked via a peptide bond between the N-terminus of the second
subunit and the C-terminus of a heavy chain constant region (CH) of
said immunoglobulin. In some further embodiments, the third subunit
may be linked via a peptide bond between the N-terminus of the
third binding domain and the C-terminus of a light chain constant
region (CL) of said immunoglobulin. In some still further
embodiments, the peptide bond may be a unstructured (G4S)3 linker,
for example, as shown in SEQ ID NO: 19.
[0066] In some embodiments with respect to a fusion polypeptide of
the disclosure, one of whose subunits comprises a full-length
immunoglobulin, while the polypeptide is simultaneously engaging
HER2/neu and CD137, the Fc function of the Fc region of the
full-length immunoglobulin to Fc receptor-positive cell may be
preserved at the same time.
[0067] In some embodiments, the CD137-specific subunit included in
a fusion polypeptide of the disclosure may be a lipocalin mutein
that is specific for CD137, such as the lipocalin mutein of SEQ ID
NO: 2. In some embodiments, the CD137-specific subunit included in
a fusion polypeptide of the disclosure may be a full-length
immunoglobulin or an antigen-binding domain thereof that is
specific for CD137, such as a monoclonal antibody (e.g. the
antibody of SEQ ID NOs: 3 and 4).
[0068] In some embodiments, the HER2/neu-specific subunit included
in a fusion polypeptide of the disclosure may be a lipocalin mutein
that is specific for HER2/neu. In some embodiments, the
HER2/neu-specific subunit included in a fusion polypeptide of the
disclosure may be a full-length immunoglobulin or an
antigen-binding domain thereof that is specific for HER2/neu.
[0069] In some embodiments, in a fusion polypeptide of the
disclosure, a CD137-specific subunit is fused to a
HER2/neu-specific subunit.
[0070] In some more specific embodiments, the HER2/neu-specific
subunit comprises a full-length immunoglobulin (such as a
monoclonal antibody) or an antigen-binding domain thereof and the
CD137-specific subunit comprises a lipocalin mutein. In some
embodiments, the fusion polypeptide comprises amino acid sequences
selected from the group consisting of SEQ ID NOs of 5 and 6, 7 and
8, 9 and 10, 11 and 12, 13 and 14 or 15 and 16.
[0071] In some other embodiments with respect to a fusion
polypeptide of the disclosure, one of whose subunits comprises a
full-length immunoglobulin, while the polypeptide is simultaneously
engaging HER2/neu and CD137, the Fc function of the Fc region of
the full-length immunoglobulin to Fc receptor-positive cell may be
reduced or fully suppressed by protein engineering. This may be
achieved, for example, by switching from the IgG1 backbone to IgG4,
as IgG4 is known to display reduced Fc-gamma receptor interactions
compared to IgG1. To further reduce the residual binding to
Fc-gamma receptors, mutations may be introduced into the IgG4
backbone such as F234A and L235A. In addition, a S228P mutation may
be introduced into the IgG4 backbone to minimize the exchange of
IgG4 half-antibody. In some still further embodiments, an
additional N297A mutation may be present in the immunoglobulin
heavy chain of the fusion polypeptide in order to remove the
natural glycosylation motif; Example 7 provides evidence that
modifying the subclass of an isotype or engineering an isotype
results in loss of Fc-receptor binding.
[0072] In some other embodiments with respect to a fusion
polypeptide of the disclosure, one of whose subunits comprises a
full-length immunoglobulin, while the polypeptide is simultaneously
engaging HER2/neu and CD137, the Fc function of the Fc region of
the full-length immunoglobulin to neonatal Fc receptor
(FcRn)-positive cells, though the Fc region may be modified, e.g.,
by switching the isotype or subclass of an isotype or by
engineering, e.g. by engineering an isotype as described herein, is
retained. Example 8 provides evidence that Fc-modified or
Fc-engineered Fc regions of a fusion polypeptide of the disclosure
retain binding to FcRn.
[0073] In some embodiments, resulting from the simultaneous binding
to HER2 on tumor cells and CD137 on the surface of effector cells
from the immune system, such as T-cells or NK cells, the fusion
polypeptides of the disclosure may exhibit HER2-dependent
effector-cell activation, whereby the effector cell of the immune
system actively lyses the HER2-expressing tumor cell.
[0074] In some additional embodiments, the fusion polypeptide is
capable of demonstrating comparable or superior level of
HER2-dependent CD137 activation as the immunoglobulin included in
such fusion polypeptide, for example, when measured in an assay
demonstrating target-dependent tumor-infiltrating lymphocyte
expansion ex-vivo as essentially described in Chacon, J. A. et al.,
PloS one 2013 8(4):e60031. In some additional embodiments, the
fusion polypeptide is capable of demonstrating comparable or
superior level of HER2-dependent CD137 activation as the
immunoglobulin included in such fusion polypeptide, for example,
when measured in an in-vivo xenotransplant model of human breast
cancer, as essentially described in Kohrt, H. et al, J Clin Invest.
2012 March; 122(3):1066-75.
[0075] In some embodiments, the Fc portion of the immunoglobulin
included in a fusion polypeptide of the disclosure may contribute
to maintaining the serum levels of the fusion polypeptide, critical
for its stability and persistence in the body. For example, when
the Fc portion binds to Fc receptors on endothelial cells and on
phagocytes, the fusion polypeptide may become internalized and
recycled back to the blood stream, enhancing its half-life within
body.
[0076] In some embodiments, a fusion polypeptide of the disclosure
may be able to bind CD137 with an EC50 value at least as good as or
superior to the EC50 value of the lipocalin mutein specific for
CD137 as included in such fusion polypeptide, such as lipocalin
muteins of SEQ ID NO: 2, for example, when said lipocalin mutein
and the polypeptide are measured in an ELISA assay essentially as
described in Example 3.
[0077] In some embodiments, the fusion polypeptide may be able to
bind CD137 with an EC50 value of at least about 1 nM or even lower,
such as about 0.6 nM, about 0.5 nM, about 0.4 nM or about 0.3 nM,
for example, when the polypeptide is measured in an ELISA assay
essentially as described in Example 3.
[0078] In some embodiments, a fusion polypeptide of the disclosure
may be able to bind HER2/neu with an EC50 value comparable to the
EC50 value of the immunoglobulin specific for HER2/neu as included
in such fusion polypeptide, such as the antibody having the heavy
and light chains provided by SEQ ID NOs: 3 and 4, for example, when
said immunoglobulin and the fusion polypeptide are measured in as
ELISA assay essentially as described in Example 2.
[0079] In another aspect, the fusion polypeptide may be able to
bind HER2/neu to its ligand with an EC50 value of at least about 1
nM or even lower, such as about 0.4 nM, about 0.3 nM or about 0.2
nM, for example, when the polypeptide is measured in an ELISA assay
essentially as described in Example 2.
[0080] In some embodiments, the fusion polypeptides of the
disclosure specific for both CD137 and HER2/neu may be capable of
simultaneously binding of CD137 and HER2/neu, for example, when
said fusion polypeptide is measured in an ELISA assay essentially
described in Example 4.
[0081] In some embodiments, the fusion polypeptides of the
disclosure may be capable of co-stimulating T-cell responses in a
functional T-cell activation assay essentially described in Example
5. In some embodiments, the fusion polypeptides of the disclosure
may be able to induce IL-2 and/or IFN gamma secretion and T cell
proliferation in a functional T-cell activation assay essentially
described in Example 5. In some further embodiments, the fusion
polypeptides of the disclosure may lead to successful T-cell
activation in a functional T-cell activation assay essentially
described in Example 5. In some further embodiments, the fusion
polypeptides of the disclosure may lead to local induction of the
production of IL-2 and/or IFN gamma by T-cells in the vicinity of
HER2/neu-positive tumor cells essentially as described in Example
6. "In the vicinity of HER2/neu-positive cells" when used herein
means a distance between a T-cell bound and a HER2/neu-positive
tumor cell that are both bound, i.e. "linked" by one and the same
fusion polypeptide of the present disclosure.
A. Exemplary Immunoglobulins as Included in the Fusion
Polypeptides.
[0082] In some embodiments, with respect to the fusion polypeptide,
the first binding domain comprises a full-length immunoglobulin or
an antigen-binding domain thereof specific for HER2/neu. The
immunoglobulin, for example, may be IgG1 or IgG4. In further
embodiments, the immunoglobulin is a monoclonal antibody against
HER2/neu. A few illustrative examples for such immunoglobulins
include: Trastuzumab (trade names Herclon, Herceptin) and
Pertuzumab (also called 2C4, trade name Perjeta).
B. Exemplary Lipocalin Muteins as Included in the Fusion
Polypeptides.
[0083] As used herein, a "lipocalin" is defined as a 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.
[0084] As noted above, a lipocalin is a polypeptide defined by its
supersecondary structure, namely 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. The present disclosure is not limited to lipocalin
muteins specifically disclosed herein. In this regard, the
disclosure relates to a lipocalin mutein having a cylindrical
1-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 CD137 with detectable
affinity.
[0085] In one particular embodiment, a lipocalin mutein disclosed
herein is a mutein of human tear lipocalin (TLPC or Tlc), also
termed lipocalin-1, tear pre-albumin or von Ebner gland protein.
The term "human tear lipocalin" or "Tlc" or "lipocalin-1" as used
herein refers to the mature human tear lipocalin with the
SWISS-PROT/UniProt Data Bank Accession Number P31025 (Isoform 1).
The amino acid sequence shown in SWISS-PROT/UniProt Data Bank
Accession Number P31025 may be used as a preferred "reference
sequence", more preferably the amino acid sequence shown in SEQ ID
NO: 18 is used as reference sequence.
[0086] In another particular embodiment, a lipocalin mutein
disclosed herein is a mutein of human lipocalin 2. The term "human
lipocalin 2" or "human Lcn 2" or "human NGAL" as used herein refers
to the mature human neutrophil gelatinase-associated lipocalin
(NGAL) with the SWISS-PROT/UniProt Data Bank Accession Number
P80188. A human lipocalin 2 mutein of the disclosure may also be
designated herein as "an hNGAL mutein". The amino acid sequence
shown in SWISS-PROT/UniProt Data Bank Accession Number P80188 may
be used as a preferred "reference sequence", more preferably the
amino acid sequence shown in SEQ ID NO: 17 is used as reference
sequence.
[0087] In some embodiments, a lipocalin mutein binding CD137 with
detectable affinity may include at least one amino acid
substitution of a native cysteine residue by another amino acid,
for example, a serine residue. In some other embodiments, a
lipocalin mutein binding CD137 with detectable affinity may include
one or more non-native cysteine residues substituting one or more
amino acids of a wild-type lipocalin. In a further particular
embodiment, a lipocalin mutein according to the disclosure includes
at least two amino acid substitutions of a native amino acid by a
cysteine residue, hereby to form one or more cysteine briges. In
some embodiments, said cysteine bridge may connect at least two
loop regions. The definition of these regions is used herein in
accordance with Flower (Flower, 1996, supra, Flower, et al., 2000,
supra) and Breustedt et al. (2005, supra). In a related embodiment,
the disclosure teaches one or more lipocalin muteins that are
capable of activating downstream signaling pathways of CD137 by
binding to CD137.
[0088] Proteins of the disclosure, which are directed against or
specific for CD137, include any number of specific-binding protein
muteins that are based on a defined protein 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, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 being
preferred and 9, 10 or 11 being even more preferred. However, it is
preferred that a lipocalin mutein of the disclosure is still
capable of binding CD137.
[0089] In one aspect, the present disclosure includes various
lipocalin muteins that bind CD137 with at least detectable
affinity. In this sense, CD137 can be regarded a non-natural ligand
of the reference wild-type lipocalin, where "non-natural ligand"
refers to a compound that does not bind to wildtype lipocalins
under physiological conditions. By engineering wildtype lipocalins
with one or more mutations at certain sequence positions, the
present inventors have demonstrated that high affinity and high
specificity for the non-natural ligand, CD137, is possible. In some
embodiments, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more
nucleotide triplet(s) encoding certain sequence positions on
wildtype lipocalins, a random mutagenesis may be carried out
through substitution at these positions by a subset of nucleotide
triplets.
[0090] Further, the lipocalin muteins of the disclosure may have a
mutated amino acid residue at any one or more, including at least
at any one, two, three, four, five, six, seven, eight, nine, ten,
eleven or twelve, of the sequence positions corresponding to
certain sequence positions of the linear polypeptide sequence of
the reference lipocalin.
[0091] A protein of the disclosure 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. In some embodiments, a lipocalin mutein according to the
disclosure may also carry one or more amino acid mutations at a
sequence position/positions as long as such a mutation does, at
least essentially not hamper or not interfere with the binding
activity and the folding of the mutein. Such mutations can be
accomplished very easily on DNA level using established standard
methods (Sambrook, J. et al. (2001) Molecular Cloning: A Laboratory
Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.). Illustrative examples of alterations of the amino
acid sequence are insertions or deletions as well as amino acid
substitutions. Such substitutions may be conservative, i.e. an
amino acid residue is replaced with an amino acid residue of
chemically similar properties, in particular with regard to
polarity as well as size. 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. On 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 the human tear lipocalin as
long as these deletions or insertion result in a stable
folded/functional mutein (for example, Tlc muteins with truncated
N- and C-terminus). In such mutein, for instance, one or more amino
acid residues are added or deleted at the N- or C-terminus of the
polypeptide. Generally such a mutein may have about at least 70%,
including at least about 80%, such as at least about 85% amino acid
sequence identity, with the amino acid sequence of the mature human
tear lipocalin. As an illustrative example, the present disclosure
also encompasses Tlc muteins as defined above, in which the first
four N-terminal amino acid residues of the sequence of mature human
tear lipocalin (His-His-Leu-Leu; positions 1-4) and/or the last two
C-terminal amino acid residues (Ser-Asp; positions 157-158) of the
linear polypeptide sequence of the mature human tear lipocalin have
been deleted (SEQ ID NOs: 32-38). In addition, as another
illustrative example, the present disclosure also encompasses NGAL
muteins as defined above, in which amino acid residues
(Lys-Asp-Pro, positions 46-48) of the linear polypeptide sequence
of the mature human lipocalin 2 (hNGAL) have be deleted (SEQ ID NO:
42).
[0092] The amino acid sequence of a lipocalin mutein disclosed
herein has a high sequence identity to the reference lipocalin when
compared to sequence identities with other lipocalins. In this
general context, the amino acid sequence of a lipocalin mutein of
the disclosure is at least substantially similar to the amino acid
sequence of the reference lipocalin, with the proviso that possibly
there are gaps (as defined below) in an alignment that are the
result of additions or deletions of amino acids. A respective
sequence of a lipocalin mutein of the disclosure, being
substantially similar to the sequences of the reference lipocalin,
has, in some embodiments, at least 70% identity or sequence
homology, at least 75% identity or sequence homology, at least 80%
identity or sequence homology, at least 82% identity or sequence
homology, at least 85% identity or sequence homology, at least 87%
identity or sequence homology, or at least 90% identity or sequence
homology including at least 95% identity or sequence homology, to
the sequence of the reference lipocalin, with the proviso that the
altered position or sequence is retained and that one or more gaps
are possible.
[0093] As used herein, a lipocalin mutein of the disclosure
"specifically binds" a target (for example, CD137) 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.
[0094] In one embodiment, the lipocalin muteins of the disclosure
are fused at its N-terminus and/or its C-terminus to a fusion
partner which is a protein domain that extends the serum half-life
of the mutein. In further particular embodiments, the protein
domain is a Fc part of an immunoglobulin, a CH3 domain of an
immunoglobulin, a CH4 domain of an immunoglobulin, an albumin
binding peptide, or an albumin binding protein.
[0095] In another embodiment, the lipocalin muteins of the
disclosure are 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
immunoglobulin, a CH3 domain of an immoglobulin, a CH4 domain of an
immunoglobulin, an albumin binding peptide, and an albumin binding
protein.
[0096] In yet another embodiment, the current disclosure relates to
a nucleic acid molecule comprising a nucleotide sequence encoding a
lipocalin mutein disclosed herein. The disclosure encompasses a
host cell containing said nucleic acid molecule.
[0097] In one aspect, the present disclosure provides human
lipocalin muteins that bind CD137 and useful applications therefor.
The disclosure also provides methods of making CD137 binding
proteins described herein as well as compositions comprising such
proteins. CD137 binding proteins of the disclosure as well as
compositions thereof may be used in methods of detecting CD137 in a
sample or in methods of binding of CD137 in a subject. No such
human lipocalin muteins having these features attendant to the uses
provided by present disclosure have been previously described.
[0098] 1. Exemplary Lipocalin Muteins Specific for CD137.
[0099] In one aspect, the present disclosure provides CD137-binding
human tear lipocalin muteins.
[0100] In this regard, the disclosure provides one or more Tlc
muteins that are capable of binding CD137 with an affinity measured
by a KD of about 300 nM or lower and even about 100 nM or
lower.
[0101] In some embodiments, such Tlc mutein comprises a mutated
amino acid residue at one or more positions corresponding to
positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85,
94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the
linear polypeptide sequence of the mature human tear lipocalin (SEQ
ID NO: 18).
[0102] In some particular embodiments, such Tlc mutein may contain
a mutated amino acid residue at one or more positions corresponding
to positions 26-34, 55-58, 60-61, 65, 104-106 and 108 of the linear
polypeptide sequence of the mature human tear lipocalin.
[0103] In further particular embodiments, such Tlc mutein may
further include a mutated amino acid residue at one or more
positions corresponding to positions 101, 111, 114 and 153 of the
linear polypeptide sequence of the mature human tear lipocalin.
[0104] In other particular embodiments, the Tlc may contain a
mutated amino acid residue at one or more positions corresponding
to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71,
85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of
the linear polypeptide sequence of the mature human tear
lipocalin.
[0105] In some further embodiments, the Tlc mutein may comprise at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26 or even more, mutated amino acid
residues at one or more sequence positions corresponding to
sequence positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65,
71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153
of the linear polypeptide sequence of the mature human tear
lipocalin and wherein said polypeptide binds CD137, in particular
human CD137.
[0106] In some still further embodiments, the disclosure relates to
a polypeptide, wherein said polypeptide is a Tlc mutein, in
comparison with the linear polypeptide sequence of the mature human
tear lipocalin, comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 or even more, mutated amino acid residues at the sequence
positions 526-34, 55-58, 60-61, 65, 104-106 and 108 and wherein
said polypeptide binds CD137, in particular human CD137.
[0107] In some embodiments, a lipocalin mutein according to the
disclosure may include at least one amino acid substitution of a
native cysteine residue by e.g. a serine residue. In some
embodiments, a Tlc mutein according to the disclosure includes an
amino acid substitution of a native cysteine residue at positions
61 and/or 153 by another amino acid such as a serine residue. In
this context it is noted that it has been found that removal of the
structural disulfide bond (on the level of a respective nave
nucleic acid library) of wild-type tear lipocalin that is formed by
the cysteine residues 61 and 153 (cf. Breustedt, et al., 2005,
supra) may provide tear lipocalin muteins that are not only stably
folded but are also able to bind a given non-natural ligand with
high affinity. In some particular embodiments, the Tlc mutein
according to the disclosure includes the amino acid substitutions
Cys 61.fwdarw.Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn,
Leu, Tyr, Met, Ser, Pro or Trp and Cys 153.fwdarw.Ser or Ala. Such
a substitution has proven useful to prevent the formation of the
naturally occurring disulphide bridge linking Cys 61 and Cys 153,
and thus to facilitate handling of the mutein. However, tear
lipocalin muteins that binds CD137 and that have the disulphide
bridge formed between Cys 61 and Cys 153 are also part of the
present disclosure.
[0108] In some embodiments, the elimination of the structural
disulde bond may provide the further advantage of allowing for the
(spontaneous) generation or deliberate introduction of non-natural
artificial disulfide bonds into muteins of the disclosure, thereby
increasing the stability of the muteins. For example, in some
embodiments, either two or all three of the cysteine codons at
position 61, 101 and 153 are replaced by a codon of another amino
acid. Further, in some embodiments, a Tlc mutein according to the
disclosure includes an amino acid substitution of a native cysteine
residue at position 101 by a serine residue or a histidine
residue.
[0109] In some embodiments, a mutein according to the disclosure
includes an amino acid substitution of a native amino acid by a
cysteine residue at positions 28 or 105 with respect to the amino
acid sequence of mature human tear lipocalin.
[0110] Further, in some embodiments, a mutein according to the
disclosure includes an amino acid substitution of a native arginine
residue at positions 111 by a proline residue. Further, in some
embodiments, a mutein according to the disclosure includes an amino
acid substitution of a native lysine residue at positions 114 by a
tryptophan residue or a glutamic acid.
[0111] In some embodiments, a CD137-binding Tlc mutein according to
the disclosure includes, at one or more positions corresponding to
positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85,
94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the
linear polypeptide sequence of the mature human tear lipocalin (SEQ
ID NO: 18), one or more of the following mutated amino acid
residues: Ala 5.fwdarw.Val or Thr; Arg 26.fwdarw.Glu; Glu
27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro 29.fwdarw.Arg; Glu
30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu 33.fwdarw.Ile; Glu
34.fwdarw.Phe; Thr 42.fwdarw.Ser; Gly 46.fwdarw.Asp; Lys
52.fwdarw.Glu; Leu 56.fwdarw.Ala; Ser 58.fwdarw.Asp; Arg 60 Pro;
Cys 61.fwdarw.Ala; Lys 65.fwdarw.Arg or Asn; Thr 71.fwdarw.Ala; Val
85.fwdarw.Asp; Lys 94.fwdarw.Arg or Glu; Cys 101.fwdarw.Ser; Glu
104.fwdarw.Val; Leu 105.fwdarw.Cys; His 106.fwdarw.Asp; Lys
108.fwdarw.Ser; Arg 111.fwdarw.Pro; Lys 114.fwdarw.Trp; Lys
121.fwdarw.Glu; Ala 133.fwdarw.Thr; Arg 148.fwdarw.Ser; Ser
150.fwdarw.Ile and Cys 153.fwdarw.Ser. In some embodiments, a Tlc
mutein according to the disclosure includes two or more, such as 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, even more such as 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26 or all mutated amino acid
residues at these sequence positions of the mature human tear
lipocalin.
[0112] In some additional embodiments, the Tlc mutein binding CD137
includes one of the following sets of amino acid substitutions in
comparison with the linear polypeptide sequence of the mature human
tear lipocalin:
1. Arg 26.fwdarw.Glu; Glu 27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro
29.fwdarw.Arg; Glu 30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu
33.fwdarw.Ile; Glu 34.fwdarw.Phe; Leu 56.fwdarw.Ala; Ser
58.fwdarw.Asp; Arg 60.fwdarw.Pro; Cys 61.fwdarw.Ala; Cys
101.fwdarw.Ser; Glu 104.fwdarw.Val; Leu 105.fwdarw.Cys; His
106.fwdarw.Asp; Lys 108.fwdarw.Ser; Arg 111.fwdarw.Pro; Lys
114.fwdarw.Trp; Cys 153.fwdarw.Ser; 2. Ala 5.fwdarw.Thr; Arg
26.fwdarw.Glu; Glu 27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro
29.fwdarw.Arg; Glu 30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu
33.fwdarw.Ile; Glu 34.fwdarw.Phe; Leu 56.fwdarw.Ala; Ser
58.fwdarw.Asp; Arg 60.fwdarw.Pro; Cys 61.fwdarw.Ala; Lys
65.fwdarw.Arg; Val 85.fwdarw.Asp; Cys 101.fwdarw.Ser; Glu
104.fwdarw.Val; Leu 105.fwdarw.Cys; His 106.fwdarw.Asp; Lys
108.fwdarw.Ser; Arg 111.fwdarw.Pro; Lys 114.fwdarw.Trp; Lys
121.fwdarw.Glu; Ala 133.fwdarw.Thr; Cys 153.fwdarw.Ser;
157.fwdarw.Pro; 3. Arg 26.fwdarw.Glu; Glu 27.fwdarw.Gly; Phe
28.fwdarw.Cys; Pro 29.fwdarw.Arg; Glu 30.fwdarw.Pro; Met
31.fwdarw.Trp; Leu 33.fwdarw.Ile; Glu 34.fwdarw.Phe; Leu
56.fwdarw.Ala; Ser 58.fwdarw.Asp; Arg 60.fwdarw.Pro; Cys
61.fwdarw.Ala; Lys 65.fwdarw.Asn; Lys 94.fwdarw.Arg; Cys
101.fwdarw.Ser; Glu 104.fwdarw.Val; Leu 105.fwdarw.Cys; His
106.fwdarw.Asp; Lys 108.fwdarw.Ser; Arg 111.fwdarw.Pro; Lys
114.fwdarw.Trp; Lys 121.fwdarw.Glu; Ala 133.fwdarw.Thr; Cys
153.fwdarw.Ser; 4. Ala 5.fwdarw.Val; Arg 26.fwdarw.Glu; Glu
27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro 29.fwdarw.Arg; Glu
30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu 33.fwdarw.Ile; Glu
34.fwdarw.Phe; Leu 56.fwdarw.Ala; Ser 58.fwdarw.Asp; Arg
60.fwdarw.Pro; Cys 61.fwdarw.Ala; Lys 65.fwdarw.Arg; Lys
94.fwdarw.Glu; Cys 101.fwdarw.Ser; Glu 104.fwdarw.Val; Leu
105.fwdarw.Cys; His 106.fwdarw.Asp; Lys 108.fwdarw.Ser; Arg
111.fwdarw.Pro; Lys 114.fwdarw.Trp; Lys 121.fwdarw.Glu; Ala
133.fwdarw.Thr; Cys 153.fwdarw.Ser; 157.fwdarw.Pro; 5. Arg
26.fwdarw.Glu; Glu 27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro
29.fwdarw.Arg; Glu 30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu
33.fwdarw.Ile; Glu 34.fwdarw.Phe; Thr 42.fwdarw.Ser; Leu
56.fwdarw.Ala; Ser 58.fwdarw.Asp; Arg 60.fwdarw.Pro; Cys
61.fwdarw.Ala; Cys 101.fwdarw.Ser; Glu 104.fwdarw.Val; Leu
105.fwdarw.Cys; His 106.fwdarw.Asp; Lys 108.fwdarw.Ser; Arg
111.fwdarw.Pro; Lys 114.fwdarw.Trp; Ser 150.fwdarw.Ile; Cys
153.fwdarw.Ser; 157.fwdarw.Pro; 6. Arg 26.fwdarw.Glu; Glu
27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro 29.fwdarw.Arg; Glu
30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu 33.fwdarw.Ile; Glu
34.fwdarw.Phe; Lys 52.fwdarw.Glu; Leu 56.fwdarw.Ala; Ser
58.fwdarw.Asp; Arg 60.fwdarw.Pro; Cys 61.fwdarw.Ala; Thr
71.fwdarw.Ala; Cys 101.fwdarw.Ser; Glu 104.fwdarw.Val; Leu
105.fwdarw.Cys; His 106.fwdarw.Asp; Lys 108.fwdarw.Ser; Arg
111.fwdarw.Pro; Lys 114.fwdarw.Trp; Ala 133.fwdarw.Thr; Arg
148.fwdarw.Ser; Ser 150.fwdarw.Ile; Cys 153.fwdarw.Ser;
157.fwdarw.Pro; or 7. Ala 5.fwdarw.Thr; Arg 26.fwdarw.Glu; Glu
27.fwdarw.Gly; Phe 28.fwdarw.Cys; Pro 29.fwdarw.Arg; Glu
30.fwdarw.Pro; Met 31.fwdarw.Trp; Leu 33.fwdarw.Ile; Glu
34.fwdarw.Phe; Gly 46.fwdarw.Asp; Leu 56.fwdarw.Ala; Ser
58.fwdarw.Asp; Arg 60.fwdarw.Pro; Cys 61.fwdarw.Ala; Thr
71.fwdarw.Ala; Cys 101.fwdarw.Ser; Glu 104.fwdarw.Val; Leu
105.fwdarw.Cys; His 106.fwdarw.Asp; Lys 108.fwdarw.Ser; Arg
111.fwdarw.Pro; Lys 114.fwdarw.Trp; Ser 150.fwdarw.Ile; Cys
153.fwdarw.Ser; 157.fwdarw.Pro.
[0113] In the residual region, i.e. the region differing from
sequence positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65,
71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and
153, a Tlc mutein of the disclosure may include the wild-type
(natural) amino acid sequence outside the mutated amino acid
sequence positions.
[0114] In still further embodiments, a Tlc mutein according to the
current disclosure has at least 70% sequence identity or at least
70% sequence homology to the sequence of the mature human tear
lipocalin (SEQ ID NO: 18).
[0115] In further particular embodiments, a Tlc mutein of the
disclosure comprises an amino acid sequence as set forth in any one
of SEQ ID NOs: 32-38 or a fragment or variant thereof.
[0116] In further particular embodiments, a Tlc mutein of the
disclosure has at least 75%, at least 80%, at least 85% or higher
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 32-38.
[0117] The disclosure also includes structural homologues of a Tlc
mutein having an amino acid sequence selected from the group
consisting of SEQ ID NOs:32-38, which structural homologues 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 to said Tlc mutein.
[0118] A Tlc mutein according to the present disclosure can be
obtained by means of mutagenesis of a naturally occurring form of
human tear lipocalin. In some embodiments of the mutagenesis, a
substitution (or replacement) is a conservative substitution.
Nevertheless, any substitution--including non-conservative
substitution or one or more from the exemplary substitutions
below--is envisaged as long as the lipocalin mutein retains its
capability to bind to CD137, and/or it has a sequence identity to
the then substituted sequence in that it is at least 60%, such as
at least 65%, at least 70%, at least 75%, at least 80%, at least
85% or higher sequence identity to the amino acid sequence of the
mature human tear lipocalin (SWISS-PROT Data Bank Accession Number
P31025).
[0119] In some particular embodiments, the present disclosure
provides a Tlc mutein that binds CD137 with an affinity measured by
a KD of about 200 nM or lower.
[0120] In some additional embodiments, a Tlc mutein of the
disclosure does not interfere with the binding of CD137L to
CD137.
[0121] In another aspect, the present disclosure relates to novel,
specific-binding human lipocalin 2 (human Lcn2 or hNGAL) muteins
directed against or specific for CD137.
[0122] In this regard, the disclosure provides one or more hNGAL
muteins that are capable of binding CD137 with an affinity measured
by a KD of 200 nM or lower, about 140 nM or lower, about 50 nM or
lower, and even about 10 nM or lower. More preferably, the hNGAL
muteins can have an affinity measured by a KD of about 5 nM or
lower.
[0123] In some embodiments, an hNGAL mutein of the disclosure
includes at one or more positions corresponding to positions 28,
36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96,
100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide
sequence of the mature hNGAL (SEQ ID NO: 17) a substitution.
[0124] In particular embodiments, a lipocalin mutein of the
disclosure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or even more,
substitution(s) at a sequence position corresponding to sequence
position 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83,
87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear
polypeptide sequence of the mature hNGAL (SWISS-PROT Data Bank
Accession Number P80188; SEQ ID NO: 2). Preferably, it is envisaged
that the disclosure relates to a lipocalin mutein which comprises,
in addition to one or more substitutions at positions corresponding
to positions 36, 87 and/or 96 of the linear polypeptide sequence of
the mature human NGAL, at one or more positions corresponding to
positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94,
100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide
sequence of the mature hNGAL a substitution.
[0125] In some still further embodiments, the disclosure relates to
a polypeptide, wherein said polypeptide is an hNGAL mutein, in
comparison with the linear polypeptide sequence of the mature hNGAL
(SWISS-PROT Data Bank Accession Number P80188; SEQ ID NO: 17),
comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, or even more, mutated amino acid
residues at the sequence positions 28, 36, 40-41, 49, 52, 65, 68,
70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and
134, and wherein said polypeptide binds CD137, in particular human
CD137.
[0126] In some embodiments, a CD137-binding hNGAL mutein of the
disclosure includes, at any one or more of the sequence positions
28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94,
96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide
sequence of the mature hNGAL (SEQ ID NO: 17), one or more of the
following mutated amino acid residues: Gln 28.fwdarw.His; Leu
36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile 41.fwdarw.Arg or Lys; Gln
49.fwdarw.Val, Ile, His, Ser or Asn; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Met, Ala or Gly; Leu 70.fwdarw.Ala,
Lys, Ser or Thr; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Met, Arg, Thr or Asn; Trp 79.fwdarw.Ala or Asp; Arg
81.fwdarw.Met, Trp or Ser; Phe 83.fwdarw.Leu; Cys 87.fwdarw.Ser;
Leu 94.fwdarw.Phe; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu and Lys 134.fwdarw.Tyr.
[0127] In some embodiments, an hNGAL mutein of the disclosure
includes two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even
more such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or all
mutated amino acid residues at these sequence positions of the
mature hNGAL.
[0128] In some additional embodiments, an hNGAL mutein of the
disclosure, which binds to CD137 includes the following amino acid
replacements in comparison with the linear polypeptide sequence of
the mature hNGAL: [0129] (a) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln;
Ala 40.fwdarw.Ile; Ile 41.fwdarw.Lys; Gln 49.fwdarw.Asn; Tyr
52.fwdarw.Met; Ser 68.fwdarw.Gly; Leu 70.fwdarw.Thr; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp
79.fwdarw.Ala; Arg 81.fwdarw.Ser; Cys 87.fwdarw.Ser; Asn
96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr
106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr
132.fwdarw.Glu; Lys 134.fwdarw.Tyr; [0130] (b) Gln 28.fwdarw.His;
Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile 41.fwdarw.Arg; Gln
49.fwdarw.Ile; Tyr 52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser
68.fwdarw.Met; Leu 70.fwdarw.Lys; Arg 72.fwdarw.Asp; Lys
73.fwdarw.Asp; Asp 77.fwdarw.Met; Trp 79.fwdarw.Asp; Arg
81.fwdarw.Trp; Cys 87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr
100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys
125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys
134.fwdarw.Tyr; [0131] (c) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln;
Ala 40.fwdarw.Ile; Ile 41.fwdarw.Arg; Gln 49.fwdarw.Asn; Tyr
52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Ala; Leu
70.fwdarw.Ala; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Thr; Trp 79.fwdarw.Asp; Arg 81.fwdarw.Trp; Cys
87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr; [0132] (d)
Gln 28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Lys; Gln 49.fwdarw.Asn; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Ala; Leu 70.fwdarw.Ala; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp
79.fwdarw.Asp; Arg 81.fwdarw.Trp; Cys 87.fwdarw.Ser; Asn
96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr
106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr
132.fwdarw.Glu; Lys 134.fwdarw.Tyr; [0133] (e) Gln 28.fwdarw.His;
Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile 41.fwdarw.Lys; Gln
49.fwdarw.Ser; Tyr 52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser
68.fwdarw.Gly; Leu 70.fwdarw.Ser; Arg 72.fwdarw.Asp; Lys
73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp 79.fwdarw.Ala; Arg
81.fwdarw.Met; Cys 87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr
100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys
125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys
134.fwdarw.Tyr; [0134] (f) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln;
Ala 40.fwdarw.Ile; Ile 41.fwdarw.Lys; Gln 49.fwdarw.Val; Tyr
52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Gly; Leu
70.fwdarw.Thr; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Arg; Trp 79.fwdarw.Asp; Arg 81.fwdarw.Ser; Cys
87.fwdarw.Ser; Leu 94.fwdarw.Phe; Asn 96.fwdarw.Lys; Tyr
100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys
125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys
134.fwdarw.Tyr; [0135] (g) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln;
Ala 40.fwdarw.Ile; Ile 41.fwdarw.Arg; Gln 49.fwdarw.His; Tyr
52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Gly; Leu
70.fwdarw.Thr; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Thr; Trp 79.fwdarw.Ala; Arg 81.fwdarw.Ser; Cys
87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr; [0136] (h)
Gln 28.fwdarw.His; Leu 36.fwdarw.Gln; Ala 40.fwdarw.Ile; Ile
41.fwdarw.Lys; Gln 49.fwdarw.Asn; Tyr 52.fwdarw.Met; Asn
65.fwdarw.Asp; Ser 68.fwdarw.Gly; Leu 70.fwdarw.Thr; Arg
72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp 77.fwdarw.Thr; Trp
79.fwdarw.Ala; Arg 81.fwdarw.Ser; Phe 83.fwdarw.Leu; Cys
87.fwdarw.Ser; Leu 94.fwdarw.Phe; Asn 96.fwdarw.Lys; Tyr
100.fwdarw.Phe; Leu 103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys
125.fwdarw.Phe; Ser 127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys
134.fwdarw.Tyr; or [0137] (i) Gln 28.fwdarw.His; Leu 36.fwdarw.Gln;
Ala 40.fwdarw.Ile; Ile 41.fwdarw.Arg; Gln 49.fwdarw.Ser; Tyr
52.fwdarw.Met; Asn 65.fwdarw.Asp; Ser 68.fwdarw.Ala; Leu
70.fwdarw.Thr; Arg 72.fwdarw.Asp; Lys 73.fwdarw.Asp; Asp
77.fwdarw.Asn; Trp 79.fwdarw.Ala; Arg 81.fwdarw.Ser; Cys
87.fwdarw.Ser; Asn 96.fwdarw.Lys; Tyr 100.fwdarw.Phe; Leu
103.fwdarw.His; Tyr 106.fwdarw.Ser; Lys 125.fwdarw.Phe; Ser
127.fwdarw.Phe; Tyr 132.fwdarw.Glu; Lys 134.fwdarw.Tyr.
[0138] In the residual region, i.e. the region differing from
sequence positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77,
79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134, an
hNGAL mutein of the disclosure may include the wild-type (natural)
amino acid sequence outside the mutated amino acid sequence
positions.
[0139] In another embodiment, the hNGAL mutein has at least 70% or
even higher sequence identity to the amino acid sequence of the
mature human lipocalin 2 (SWISS-PROT Data Bank Accession Number
P80188).
[0140] In further particular embodiments, a lipocalin mutein
according to the current disclosure comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2 and
39-46 or a fragment or variant thereof.
[0141] The amino acid sequence of a CD137-binding hNGAL mutein of
the disclosure may have a high sequence identity, such as 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 sequence selected from the group consisting of SEQ ID NOs: 2
and 39-46.
[0142] The disclosure also includes structural homologues of an
hNGAL mutein having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2 and 39-46, which structural homologues
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 to said hNGAL mutein.
[0143] An hNGAL mutein according to the present disclosure can be
obtained by means of mutagenesis of a naturally occurring form of
human lipocalin 2. In some embodiments of the mutagenesis, a
substitution (or replacement) is a conservative substitution.
Nevertheless, any substitution--including non-conservative
substitution or one or more from the exemplary substitutions
below--is envisaged as long as the lipocalin mutein retains its
capability to bind to CD137, and/or it has an identity to the then
substituted sequence in that it is at least 60%, such as at least
65%, at least 70%, at least 75%, at least 80%, at least 85% or
higher identity to the amino acid sequence of the mature human
lipocalin 2 (SWISS-PROT Data Bank Accession Number P80188).
[0144] In some particular embodiments, the present disclosure
provides an hNGAL mutein that binds CD137 with an affinity measured
by a KD of about 5 nM or lower.
C. Exemplary Uses, Applications and Production of the Fusion
Polypeptides.
[0145] In some embodiments, fusion polypeptides of the disclosure
may produce synergistic effect through dual-targeting CD137 and
HER2. For example, as demonstrated in ex-vivo assays and mouse
models, CD137 stimulation of NK-cells boosts the activity of
Trastuzumab (Kohrt, H. et al, J Clin Invest. 2012 March;
122(3):1066-75) by enhancing NK-cell function and activity.
[0146] Numerous possible applications for the fusion polypeptides
of the disclosure, therefore, exist in medicine.
[0147] In one aspect, the disclosure relates to the use of the
fusion polypeptides disclosed herein for detecting CD137 and HER2
in a sample as well as a respective method of diagnosis.
[0148] In another aspect, the disclosure features the use of one or
more fusion polypeptides disclosed herein or of one or more
compositions comprising such polypeptides for simultaneously
binding of CD137 and HER2/neu.
[0149] The present disclosure also involves the use of one or more
fusion polypeptides as described for complex formation with CD137
and HER2.
[0150] Therefore, in a still further aspect of the disclosure, the
disclosed one or more fusion polypeptides are used for the
detection of CD137 and HER2. Such use may include the steps of
contacting one or more said fusion polypeptides, under suitable
conditions, with a sample suspected of containing CD137 and HER2,
thereby allowing formation of a complex between the fusion
polypeptides and CD137 and HER2, and detecting the complex by a
suitable signal. The detectable signal can be caused by a label, as
explained above, or by a change of physical properties due to the
binding, i.e. the complex formation, itself. One example is surface
plasmon resonance, the value of which is changed during binding of
binding partners from which one is immobilized on a surface such as
a gold foil.
[0151] The fusion polypeptides disclosed herein may also be used
for the separation of CD137 and HER2. Such use may include the
steps of contacting one or more said fusion polypeptides, under
suitable conditions, with a sample supposed to contain CD137 and
HER2, thereby allowing formation of a complex between the fusion
polypeptides and CD137 and HER2, and separating the complex from
the sample.
[0152] In still another aspect, the present disclosure features a
diagnostic or analytical kit comprising a fusion polypeptide
according to the disclosure.
[0153] In addition to their use in diagnostics, in yet another
aspect, the disclosure contemplates a pharmaceutical composition
comprising a fusion polypeptide of the disclosure and a
pharmaceutically acceptable excipient.
[0154] Furthermore, the present disclosure provides fusion
polypeptides that simultaneously bind CD137 and HER2 for use as
anti-cancer agents and immune modulators. As such the fusion
polypeptides of the present disclosure are envisaged to be used in
a method of treatment or prevention of human diseases such as a
variety of tumors including certain aggressive types of breast
cancer. Accordingly, also provided are methods of treatment or
prevention of human diseases such as a variety of tumors including
certain aggressive types of breast cancer in a subject in need
thereof, comprising administering to said subject a therapeutically
effective amount of one or more fusion polypeptides of the
disclosure.
[0155] By simultaneously targeting tumor cells where HER2/neu is
expressed and activating natural killer (NK) cells in the host
innate immune system adjacent to such tumor cells, the fusion
polypeptide of the disclosure may increase targeted anti-tumor T
cells activity, enhance anti-tumor immunity and, at the same time,
have a direct inhibiting effect on tumor growth, thereby produce
synergistic anti-tumor results. In addition, via locally inhibiting
oncogene activity and inducing cell-mediated cytotoxicity by NK
cells, the fusion polypeptide of the disclosure may reduce side
effects of effector lymphocytes towards healthy cells, i.e.
off-target toxicity.
[0156] In T cells CD137-mediated signaling leads to the recruitment
of TRAF family members and activation of several kinases, including
ASK-1, MKK, MAPK3/MAPK4, p38, and JNK/SAPK. Kinase activation is
then followed by the activation and nuclear translocation of
several transcription factors, including ATF-2, Jun, and
NF-.kappa.B. In addition to augmenting suboptimal T cell receptor
(TCR)-induced proliferation, CD137-mediated signaling protects T
cells, and in particular, CD8+ T cells from activation-induced cell
death (AICD).
[0157] The present disclosure encompasses the use of a fusion
polypeptide of the disclosure or a composition comprising such
fusion polypeptide for costimulating T-cells, and/or activating
downstream signaling pathways of CD137 when engaging tumor cells
where HER2/neu is expressed.
[0158] The present disclosure also features a method of
costimulating T-cells and/or activating downstream signaling
pathways of CD137 when engaging tumor cells where HER2/neu is
expressed, comprising applying one or more fusion polypeptide s of
the disclosure or of one or more compositions comprising such
fusion polypeptides.
[0159] Furthermore, the present disclosure involves a method of
activating downstream signaling pathways of CD137 when engaging
tumor cells where HER2/neu is expressed, comprising applying one or
more fusion polypeptides of the disclosure or of one or more
compositions comprising such fusion polypeptides.
[0160] The present disclosure also contemplates a method of
inducing T lymphocyte proliferation when engaging tumor cells where
HER2/neu is expressed, comprising applying one or more fusion
polypeptides of the disclosure or of one or more compositions
comprising such fusion polypeptides.
[0161] The present disclosure encompasses the use of a fusion
polypeptide of the disclosure or a composition comprising such
fusion polypeptide for directing CD137 clustering and activation on
T-cells to tumor cells where HER2/neu is expressed.
[0162] The present disclosure further provides a method of inducing
a local T-cell response in the vicinity of HER2/neu-positive tumor
cells, comprising applying such fusion polypeptides. "Local" means
that upon binding T-cells via CD137 and engaging HER2/neu-positive
tumor cells, T-cells produce cytokines, particularly IL-2 and/or
IFN gamma in vicinity of the HER2/neu-positive cell. Such cytokines
reflect activation of T-cells which may then be able to kill
HER2/neu-positive tumor cells, either directly or indirectly by
attracting other killer cells, such as T-cells or NK cells.
[0163] In another embodiment, the present disclosure also relates
to nucleic acid molecules (DNA and RNA) that include nucleotide
sequences encoding the fusion polypeptides disclosed herein. In yet
another embodiment, the disclosure encompasses a host cell
containing said nucleic acid molecule. Since the degeneracy of the
genetic code permits substitutions of certain codons by other
codons specifying the same amino acid, the disclosure is not
limited to a specific nucleic acid molecule encoding a fusion
polypeptide as described herein but encompasses all nucleic acid
molecules that include nucleotide sequences encoding a functional
polypeptide. In this regard, the present disclosure also relates to
nucleotide sequences encoding the fusion polypeptides of the
disclosure.
[0164] In some embodiments, a nucleic acid molecule encoding a
lipocalin mutein disclosed in this application, such as DNA, may be
"operably linked" to another nucleic acid molecule encoding an
immunoglobulin of the disclosure to allow expression of a fusion
polypeptide disclosed herein. In this regard, an operable linkage
is a linkage in which the sequence elements of one nucleic acid
molecule and the sequence elements of another nucleic acid molecule
are connected in a way that enables expression of the fusion
polypeptide as a single polypeptide.
[0165] The disclosure also relates to a method for the production
of a or a fusion polypeptide of the disclosure is produced starting
from the nucleic acid coding for the polypeptide or any subunit
therein by means of genetic engineering methods. In some
embodiments, the method can be carried out in vivo, the polypeptide
can, for example, be produced in a bacterial or eucaryotic host
organism and then isolated from this host organism or its culture.
It is also possible to produce a fusion polypeptide of the
disclosure in vitro, for example by use of an in vitro translation
system.
[0166] When producing the fusion polypeptide in vivo, a nucleic
acid encoding such polypeptide 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 that includes a
nucleic acid molecule encoding a fusion polypeptide as described
herein using established standard methods. 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.
[0167] In one embodiment of the disclosure, the method includes
subjecting at least one nucleic acid molecule encoding hNGAL to
mutagenesis at nucleotide triplets coding for at least one,
sometimes even more, of the sequence positions corresponding to the
sequence positions 28, 40-52, 60, 68, 65,70,71-81, 87, 89, 96, 98,
100-106, 114, 118, 120, 125-137 and 145 of the linear polypeptide
sequence of hNGAL (SEQ ID NO: 17).
[0168] In addition, in some embodiments, the naturally occurring
disulphide bond between Cys 76 and Cys 175 may be removed in hNGAL
muteins of the disclosure. Accordingly, such muteins can be
produced in a cell compartment having a reducing redox milieu, for
example, in the cytoplasma of Gram-negative bacteria.
[0169] The disclosure also includes nucleic acid molecules encoding
the lipocalin muteins of the disclosure, which include additional
mutations outside the indicated sequence positions of experimental
mutagenesis. Such mutations are often tolerated or can even prove
to be advantageous, for example if they contribute to an improved
folding efficiency, serum stability, thermal stability or ligand
binding affinity of the lipocalin muteins.
[0170] 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.
[0171] 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 se, 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.
[0172] 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.
[0173] Therefore, a nucleic acid molecule of the disclosure can
include a regulatory sequence, such as a promoter sequence. In some
embodiments a nucleic acid molecule of the disclosure 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.
[0174] The nucleic acid molecules of the disclosure 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.
[0175] In one embodiment, the nucleic acid molecule is included in
a phasmid. A phasmid vector denotes a vector encoding the
intergenic region of a temperent phage, such as M13 or f1, or a
functional part thereof fused to the cDNA of interest. After
superinfection of the bacterial host cells with such an phagemid
vector and an appropriate helper phage (e.g. M13K07, VCS-M13 or
R408) intact phage particles are produced, thereby enabling
physical coupling of the encoded heterologous cDNA to its
corresponding polypeptide displayed on the phage surface (see e.g.
Lowman, H. B. (1997) Annu. Rev. Biophys. Biomol. Struct. 26,
401-424, or Rodi, D. J., and Makowski, L. (1999) Curr. Opin.
Biotechnol. 10, 87-93).
[0176] Such cloning vehicles can include, aside from the regulatory
sequences described above and a nucleic acid sequence encoding a
fusion polypeptide as described herein, replication and control
sequences derived from a species compatible with the host cell that
is used for expression as well as selection markers conferring a
selectable phenotype on transformed or transfected cells. Large
numbers of suitable cloning vectors are known in the art, and are
commercially available.
[0177] The DNA molecule encoding a fusion polypeptide as described
herein (for example, SEQ ID NOs: 20 and 31), and in particular a
cloning vector containing the coding sequence of such a polypeptide
can be transformed into a host cell capable of expressing the gene.
Transformation can be performed using standard techniques. Thus,
the disclosure is also directed to a host cell containing a nucleic
acid molecule as disclosed herein.
[0178] The transformed host cells are cultured under conditions
suitable for expression of the nucleotide sequence encoding a
fusion polypeptide of the disclosure. Suitable host cells can be
prokaryotic, such as Escherichia coli (E. coli) or Bacillus
subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia
pastoris, SF9 or High5 insect cells, immortalized mammalian cell
lines (e.g., HeLa cells or CHO cells) or primary mammalian
cells.
[0179] In some embodiments where a lipocalin mutein of the
disclosure, including as comprised in in a fusion polypeptide
disclosed herein, includes intramolecular disulphide 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 favours
the formation of structural disulphide bonds.
[0180] In some embodiments, it is also possible to produce a fusion
polypeptide of the disclosure 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.).
[0181] In some embodiments, a fusion polypeptide of the disclosure
as described herein may be not necessarily generated or produced
only by use of genetic engineering. Rather, such polypeptide 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) mutein or polypeptide in vitro and investigate
the binding activity for a target of interest. Methods for the
solid phase and/or solution phase synthesis of proteins are well
known in the art (see e.g. Bruckdorfer, T. et al. (2004) Curr.
Pharm. Biotechnol. 5, 29-43).
[0182] In another embodiment, a fusion polypeptide of the
disclosure may be produced by in vitro transcription/translation
employing well-established methods known to those skilled in the
art.
[0183] The skilled worker will appreciate methods useful to prepare
fusion polypeptides contemplated by the present disclosure 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 sub-cloning of a polypeptide
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 fusion
polypeptide for its targets (e.g. CD137 and HER2). Furthermore,
mutations can be introduced to modulate certain characteristics of
the polypeptide 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.
[0184] Additional objects, advantages, and features of this
disclosure will become apparent to those skilled in the art upon
examination of the following Examples and the attached Figures
thereof, which are not intended to be limiting. Thus, it should be
understood that although the present disclosure is specifically
disclosed by exemplary embodiments and optional features,
modification and variation of the disclosures 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 disclosure.
V. EXAMPLES
Example 1: Expression and Analysis of Fusion Polypeptides
[0185] To engage HER2 and CD137 at the same time, we generated
several representative antibody-lipocalin mutein fusion
polypeptides, fusing together the antibody having the heavy and
light chains provided by SEQ ID NOs: 3 and 4, and the lipocalin
mutein of SEQ ID NO: 2 via an unstructured (G4S)3 linker (SEQ ID
NO: 19). The different formats that were designed are depicted in
FIG. 1. Such fusion polypeptides (SEQ ID NOs: 9 and 10. SEQ ID NOs:
11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16) were
generated via fusion of the lipocalin mutein of SEQ ID NO: 2 to
either one of the four termini of a mutated variant of the antibody
having an engineered IgG4 backbone, which contains a S228P mutation
to minimize IgG4 half-antibody exchange in-vitro and in-vivo (cf.
Silva 2015) as well as F234A and L235A mutations to reduce Fc-gamma
receptor interactions (Alegre 1992). Furthermore, we generated the
fusion polypeptide of SEQ ID NOs: 7 and 8, which, in comparison
with the fusion polypeptide of SEQ ID NOs: 9 and 10 has an
additional N297A mutation in the antibody heavy chain (cf. Bolt
1993) in order to remove the natural glycosylation motif. This
removal could potentially further reduce the interaction with
Fc-gamma receptors. In addition, we generated the fusion
polypeptide of SEQ ID NOs: 5 and 6, which is a direct fusion of
lipocalin mutein of SEQ ID NO: 2 to C-terminal heavy chain of the
antibody of SEQ ID NOs: 3 and 4 with an IgG1 background and
therefore the fusion polypeptide retains the original Fc-gamma
interaction of the IgG1 antibody.
[0186] The constructs were generated by gene synthesis and cloned
into a mammalian expression vector. They were then transiently
expressed in CHO cells. The concentration of fusion polypeptides in
the cell culture medium was measured using a ForteBio Protein A
sensor (Pall Corp.) and quantified using a human IgG1 standard. The
titers of the constructs were as described in Table 1 below.
TABLE-US-00001 TABLE 1 Expression titers Expression titer Clone
Name [mg/L] SEQ ID NOs: 5 and 6 262 SEQ ID NOs: 9 and 10 156 SEQ ID
NOs: 13 and 14 191 SEQ ID NOs: 7 and 8 181 SEQ ID NOs: 11 and 12
204 SEQ ID NOs: 15 and 16 161
[0187] The fusion polypeptides were purified using Protein A
chromatography followed by size-exclusion chromatography (SEC) in
phosphate-buffered saline (PBS). After SEC purification the
fractions containing monomeric protein were pooled and analyzed
again using analytical SEC. According to this analysis, the fusion
polypeptides were fully monomeric without detectable multimeric
species or aggregates.
Example 2: Specificity of Fusion Polypeptides Towards HER2
[0188] We employed an ELISA assay to determine the affinity of the
fusion proteins to recombinant HER2 (Sino Biological). The target
was dissolved in PBS (5 .mu.g/mL) and coated overnight on
microtiter plates at 4.degree. C. The plate was washed after each
incubation step with 80 .mu.L PBS supplemented with 0.05% (v/v)
Tween 20 (PBS-T) five times. The plates were blocked with 2% BSA
(w/v) in PBS for 1 h at room temperature and subsequently washed.
Different concentrations of the benchmark antibody (SEQ ID NOs: 3
and 4, Trastuzumab or Herceptin.RTM., Roche Diagnostics) or the
fusion polypeptides were added to the wells and incubated for 1 h
at room temperature, followed by a wash step. Bound agents under
study were detected after incubation with 1:5000 diluted anti-human
IgG Fc-HRP (#109-035-098, Jackson Laboratory) in PBS-T. After an
additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo)
was added to each well and the fluorescence intensity was detected
using a fluorescence microplate reader.
[0189] The result of the experiment was plotted in FIG. 2, together
with the fit curves resulting from a 1:1 binding sigmoidal fit,
where the EC50 value and the maximum signal were free parameters,
and the slope was fixed to unity. The resulting EC50 values are
provided in Table 2 below, including the errors of the sigmoidal
fit of the data. The observed EC50 values were in a similar range
for all tested fusion polypeptides (0.22-0.31 nM), and in the same
range as the EC50 value for the benchmark antibody (SEQ ID NOs: 3
and 4), which was at 0.16 nM.
TABLE-US-00002 TABLE 2 ELISA data for HER2 binding EC50 HER2 Agent
Name [nM] SEQ ID NOs: 7 and 8 0.24 .+-. 0.02 SEQ ID NOs: 13 and 14
0.23 .+-. 0.01 SEQ ID NOs: 15 and 16 0.31 .+-. 0.01 SEQ ID NOs: 5
and 6 0.22 .+-. 0.01 SEQ ID NOs: 9 and 10 0.28 .+-. 0.01 SEQ ID
NOs: 11 and 12 0.23 .+-. 0.01 SEQ ID NOs: 3 and 4 0.16 0.01
Example 3: Specificity of Fusion Polypeptides Towards CD137
[0190] We employed an ELISA assay to determine the affinity of the
fusion polypeptides and the positive control lipocalin mutein of
SEQ ID NO: 2 to a recombinant CD137-Fc fusion (#838-4B-100, R&D
Systems). The target was dissolved in PBS (5 .mu.g/mL) and coated
overnight on microtiter plates at 4.degree. C. The plate was washed
after each incubation step with 80 .mu.L PBS-T five times. The
plates were blocked with 2% BSA (w/v) in PBS for 1 h at room
temperature and subsequently washed. Different concentrations of
the CD137-specific lipocalin mutein in monomeric form (SEQ ID NO:
2) or the fusion polypeptides were added to the wells and incubated
for 1 h at room temperature, followed by a wash step. Bound agents
under study were detected after incubation for 1 h at room
temperature with 1:1000 diluted anti-hNGAL antibody conjugated to
HRP in PBS-T. After an additional wash step, fluorogenic HRP
substrate (QuantaBlu, Thermo) was added to each well and the
fluorescence intensity was detected using a fluorescence microplate
reader.
[0191] The result of the experiment is plotted was FIG. 3, together
with the fit curves resulting from a 1:1 binding sigmoidal fit,
where the EC50 value and the maximum signal were free parameters,
and the slope was fixed to unity. The resulting EC50 values are
provided in Table 3, including the errors of the sigmoidal fit of
the data. The observed EC50 values for all tested fusion
polypeptides were nearly identical within the experimental error
and ranged from 0.30 nM to 0.47 nM, slightly superior to the value
obtained for the positive control lipocalin mutein of SEQ ID NO: 2,
which was 0.49 nM.
TABLE-US-00003 TABLE 3 ELISA data for CD137 binding EC50 CD137
Agent Name [nM] SEQ ID NOs: 7 and 8 0.30 .+-. 0.02 SEQ ID NOs: 13
and 14 0.33 .+-. 0.05 SEQ ID NOs: 15 and 16 0.35 .+-. 0.03 SEQ ID
NOs: 5 and 6 0.41 .+-. 0.04 SEQ ID NOs: 9 and 10 0.37 .+-. 0.06 SEQ
ID NOs: 11 and 12 0.47 .+-. 0.06 SEQ ID NO: 2 0.49 .+-. 0.09
Example 4: Demonstration of Simultaneous Target Binding in an
ELISA-Based Setting
[0192] In order to demonstrate the simultaneous binding of the
fusion polypeptides to HER2 and CD137, a dual-binding ELISA format
was used. Recombinant HER2 (Sino Biological) in PBS (5 .mu.g/mL)
was coated overnight on microtiter plates at 4.degree. C. The plate
was washed five times after each incubation step with 80 .mu.L PBS
supplemented with 0.05% (v/v) Tween 20 (PBS-T) using a Biotek
ELx405 select CW washer. The plates were blocked with 2% BSA (w/v)
in PBS for 1 h at room temperature and subsequently washed again.
Different concentrations of the fusion polypeptides were added to
the wells and incubated for 1 h at room temperature, followed by a
wash step. Subsequently, biotinylated human CD137-Fc was added at a
constant concentration of 1 .mu.g/mL in PBS-T for 1 h. After
washing, Extravidin-HRP (Sigma-Adrich, 1:5000 in PBS-T) was added
to the wells for 1 h. After an additional wash step, fluorogenic
HRP substrate (QuantaBlu, Thermo) was added to each well and the
fluorescence intensity was detected using a fluorescence microplate
reader.
[0193] The respective experimental data was plotted in FIG. 4. All
tested fusion polypeptides showed clear binding signals with EC50
values ranging from 1-4 nM, demonstrating that these fusion
polypeptides are able to engage HER2 and CD137 simultaneously.
Example 5: Functional T-Cell Activation Assay Using Coated Fusion
Polypeptides
[0194] We employed a T-cell activation assay to assess the ability
of the fusion polypeptide of SEQ ID NOs: 15 and 16 to co-stimulate
T-cell responses. For this purpose, the fusion polypeptide of SEQ
ID NOs: 15 and 16 at different concentrations was coated onto a
plastic dish together with an anti-human CD3 antibody (OKT3,
eBioscience) and purified T-cells were subsequently incubated on
the coated surface. As readouts, we assessed continued
proliferation of the T-cells after three days incubation using a 4
h BrdU pulse, and measured supernatant interleukin 2 (IL-2))
levels. In the following, we provide a detailed description of the
experiment.
[0195] Human peripheral blood mononuclear cells (PBMC) from healthy
volunteer donors were isolated from buffy coats by centrifugation
through a Polysucrose density gradient (Biocoll 1.077 g/mL from
Biochrom), following Biochrom's protocols. The T lymphocytes were
isolated from the resulting PBMC using a Pan T-cell purification
Kit (Miltenyi Biotec GmbH) and the manufacturer's protocols.
Purified T-cells were resuspended in a buffer consisting of 90% FCS
and 10% DMSO, immediately frozen down using liquid nitrogen and
stored in liquid nitrogen until further use. For the assay, T cells
were thawed for 16 h and cultivated in culture media (RPMI 1640,
Life Technologies) supplemented with 10% FCS and 1%
Penicillin-Streptomycin (Life Technologies).
[0196] The following procedure was performed using triplicates for
each experimental condition. Flat-bottom tissue culture plates were
coated overnight at 4.degree. C. using 200 .mu.L of a mixture of
0.5 .mu.g/mL anti-CD3 antibody and the fusion polypeptide of SEQ ID
NOs: 15 and 16 at a concentration of 3 .mu.g/mL, 10 .mu.g/mL and 30
.mu.g/mL. As a negative control, the anti-CD3 antibody was captured
alone, i.e. without the addition of the fusion polypeptide of SEQ
ID NOs: 15 and 16. The following day, wells were washed twice with
PBS, and 100 .mu.L of the T-cell suspension (corresponding to
5.times.10.sup.4 T cells) in culture media was added to each well.
Plates were covered with a gas permeable seal (4titude) and
incubated at 37.degree. C. in a humidified 5% C02 atmosphere for 3
days. Subsequently, the IL-2 concentration in the supernatant, as
well as cell proliferation, were assessed.
[0197] In order to quantify T-cell proliferation, the
chemiluminescent cell proliferation ELISA kit based on BrdU
incorporation (Roche) was used according to the manufacturer's
instructions. Briefly, on day 3, 10 .mu.L of BrdU labeling solution
were added to each well and proliferation was allowed to proceed
for a further 4 h at 37.degree. C. under a humidified 5% CO.sub.2
atmosphere. Plates were centrifuged at 300 g for 10 min and
supernatants of the triplicates were pooled and immediately stored
at -20.degree. C. for later IL-2 quantification. Plates were
subsequently dried at 60.degree. C. for 1 hour. 200 .mu.L of
"FixDenat" solution were added to each well and the plates were
incubated at room temperature for 30 min. Incorporated BRDU was
labeled with a peroxidase-labelled anti-BrdU antibody by 2 h
incubation at room temperature. BrdU levels were assessed by
quantifying a chemiluminescent peroxidase-catalysed reaction in a
PheraStar FS reader.
[0198] Human IL-2 levels in the cell culture supernatants were
quantified using the IL-2 DuoSet DuoSet kit from R&D Systems.
The procedure is carried out and described in the following. In the
first step, a 384 well plate was coated at room temperature for 2 h
with 1 .mu.g/mL "Human IL-2 Capture Antibody" (R&D System)
diluted in PBS. Subsequently, wells were washed 5 times with 80
.mu.l PBS-T (PBS containing 0.05% Tween20) using a Biotek EL405
select CW washer (Biotek). After 1 h blocking in PBS-T additionally
containing 1% casein (w/w), pooled supernatant and a concentration
series of an IL-2 standard diluted in culture medium were incubated
in the 384-well plate overnight at 4.degree. C. To allow for
detection and quantitation of captured IL-2, a mixture of 100 ng/mL
biotinylated goat anti-hlL-2-Bio detection antibody (R&D
System) and 1 .mu.g/mL Sulfotag-labelled streptavidin (Mesoscale
Discovery) were added in PBS-T containing 0.5% casein and incubated
at room temperature for 1 h. After washing, 25 .mu.L reading buffer
was added to each well and the electrochemiluminescence (ECL)
signal of every well was read using a Mesoscale Discovery reader.
Analysis and quantification were performed using Mesoscale
Discovery software.
[0199] The result for the assessment of T-cell proliferation is
shown in FIG. 5. There is a significant increase in continued
proliferation after 3 d incubation in the wells that were coated
with 10 .mu.g/mL and 30 .mu.g/mL of the fusion polypeptide of SEQ
ID NOs: 15 and 16, compared to the negative control which did not
contain the fusion polypeptide of SEQ ID NOs: 15 and 16 and where
the anti-CD3 antibody was captured alone.
[0200] The result of the IL-2 measurement shows that the fusion
polypeptide of SEQ ID NOs: 15 and 16 can lead to successful T-cell
activation (data not shown).
Example 6: Functional T-Cell Activation Assay Using Tumor Cell
Bound Fusions Polypeptides
[0201] We employed a target-cell dependent T-cell activation assay
to assess the ability of the fusion polypeptides of SEQ ID NOs: 9
and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID
NOs: 15 and 16--capable of binding CD137 and HER2 at the same
time--to co-stimulate T-cell responses when immobilized on a
HER2-positive cell line. As a negative control, we employed the
monospecific, HER2-binding antibody of SEQ ID NOs: 3 and 4. As a
further control, the experiment was performed in the presence of an
excess of the monospecific, HER2-binding antibody of SEQ ID NOs: 3
and 4 in order to displace the bispecific constructs SEQ ID NOs: 9
and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID
NOs: 15 and 16 from binding to HER2-positive cells. In the
experiment, an anti-human CD3 antibody (OKT3, eBioscience) was
coated on a plastic culture dish, and subsequently HER2-positive
SKBR3 cells were cultured on the dish overnight. The next day,
purified T-cells were incubated on the coated surface in the
presence of various concentrations of the fusion polypeptides of
SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14,
and SEQ ID NOs: 15 and 16 or the control antibody of SEQ ID NOs: 3
and 4. As readout, we measured supernatant interleukin 2 (IL-2) and
interferon-.gamma. (IFN-.gamma.) levels. In the following, the
experiment is described in detail.
[0202] Human peripheral blood mononuclear cells (PBMC) from healthy
volunteer donors were isolated from buffy coats by centrifugation
through a Polysucrose density gradient (Biocoll 1.077 g/mL from
Biochrom), following Biochrom's protocols. The T lymphocytes were
isolated from the resulting PBMC using a Pan T-cell purification
Kit (Miltenyi Biotec GmbH) and the manufacturer's protocols.
Purified T-cells were resuspended in a buffer consisting of 90% FCS
and 10% DMSO, immediately frozen down using liquid nitrogen and
stored in liquid nitrogen until further use. For the assay, T cells
were thawed for 16 h and cultivated in culture media (RPMI 1640,
Life Technologies) supplemented with 10% FCS and 1%
Penicillin-Streptomycin (Life Technologies).
[0203] The following procedure was performed using triplicates for
each experimental condition. Flat-bottom tissue culture plates were
pre-coated or not for 1 h at 37.degree. C. using 200 .mu.L of 0.25
.mu.g/mL anti-CD3 antibody. The plates were subsequently washed
twice with PBS. 5.times.10.sup.4 SKBR3 tumor cells per well were
plated and allowed to adhere overnight at 37.degree. C. in a
humidified 5% C02 atmosphere. The SKBR3 cells had before been grown
in culture under standard conditions, detached using Accutase and
resuspended in culture media.
[0204] On the next days, tumor cells were treated 2 hours at
37.degree. C. with mitomycin C (Sigma Aldrich) at a concentration
of 30 .mu.g/ml in order to block their proliferation. Plates were
washed twice with PBS, and 100 .mu.L of the T-cell suspension
(corresponding to 5.times.10.sup.4 T cells) and each of the four
fusion polypeptides, at eleven different concentrations ranging
from 25 .mu.g/mL to 0.4 ng/mL, or the negative control at
concentrations of 25 .mu.g/mL, 0.1 .mu.g/mL and 0.4 ng/mL, were
added to each well. The same setup was performed in parallel, but
with the addition of a final concentration of 50 .mu.g/mL of the
monospecific, HER2-binding antibody SEQ ID NOs: 3 and 4. Plates
were covered with a gas permeable seal (4titude) and incubated at
37.degree. C. in a humidified 5% C02 atmosphere for 3 days.
Subsequently, IL-2 and IFN-.gamma. concentration in the supernatant
were assessed as described below.
[0205] Human IL-2 levels in the cell culture supernatants were
quantified using the IL-2 DuoSet kit and the IFN-.gamma. DuoSet kit
from R&D Systems, respectively. The procedure is carried out
analogously for both cytokines and described in the following for
IL-2 only. In the first step, a 384 well plate was coated at room
temperature for 2 h with 1 .mu.g/mL "Human IL-2 Capture Antibody"
(R&D System) diluted in PBS. Subsequently, wells were washed 5
times with 80 .mu.l PBS-T (PBS containing 0.05% Tween20) using a
Biotek EL405 select CW washer (Biotek). After 1 h blocking in PBS-T
additionally containing 1% casein (w/w), pooled supernatant and a
concentration series of an IL-2 standard diluted in culture medium
were incubated in the 384-well plate overnight at 4.degree. C. To
allow for detection and quantitation of captured IL-2, a mixture of
100 ng/mL biotinylated goat anti-hlL-2-Bio detection antibody
(R&D System) and 1 .mu.g/mL Sulfotag-labelled streptavidin
(Mesoscale Discovery) were added in PBS-T containing 0.5% casein
and incubated at room temperature for 1 h. After washing, 25 .mu.L
reading buffer was added to each well and the
electrochemiluminescence (ECL) signal of every well was read using
a Mesoscale Discovery reader. Analysis and quantification were
performed using Mesoscale Discovery software. The data was fitted
with a 1:1 binding model with EC50 value, background level and
plateau level as free parameters, and a slope that was fixed to
unity. The induction factor was calculated from the fitted values
as the quotient of plateau level and background level.
[0206] The result of a representative experiment for the bispecific
fusion polypeptide SEQ ID NOs: 9 and 10 is depicted in FIG. 6. The
data demonstrates a clear increase of supernatant levels for both
IL-2 (FIG. 6A) and IFN-.gamma. (FIG. 6C) with rising concentrations
of the bispecific fusion polypeptide. At low concentrations of the
bispecific fusion polypeptide, the IL-2 and IFN-.gamma.
concentration measured in the supernatant corresponds to the
background level measured for the negative control SEQ ID NOs: 3
and 4. In the presence of an excess of the HER2-binder SEQ ID NOs:
3 and 4, the concentrations of both IL-2 (FIG. 6B) and IFN-.gamma.
(FIG. 6D) no longer show a SEQ ID NOs: 9 and
10-concentration-dependent increase, but remain invariant at the
background level.
[0207] While the plateau levels reached in the experiment are lower
for SEQ ID NOs: 11 and 12 (FIG. 7) and SEQ ID NOs: 15 and 16 (FIG.
9), overall results regarding concentration dependent induction of
IL-2 and IFN-.gamma. and blockade by an excess of SEQ ID NOs: 3 and
4 are similar. In contrast, there is no discernible increase in
IL-2 or IFN-.gamma. concentration with increasing concentration of
the polypeptide fusion SEQ ID NOs: 13 and 14 (FIG. 8).
[0208] The EC50 values and induction factors resulting from a
sigmoidal fit of the data are provided in Table 4, including data
for an independent repeat of the experiment using a different PBMC
donor. The data indicates that the EC50 values are comparable for
SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12 and SEQ ID NOs: 15 and
16. However, the induction factor decreases in the order SEQ ID
NOs: 9 and 10>SEQ ID NOs: 11 and 12>SEQ ID NOs: 15 and
16.
[0209] The experiment clearly demonstrates a potent functional
activity of SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12 and SEQ ID
NOs: 15 and 16, while SEQ ID NOs: 13 and 14 has no activity. In
light of the nearly identical affinities for all constructs
demonstrated in the Examples 4 and 5, this highlights the important
role of construct geometry.
TABLE-US-00004 TABLE 4 Potency IL-2 Potency IFN-g Efficacy IL-2
Efficacy IFN-g [EC50] [EC50] (max/min) (max/min) SEQ ID NOs: 9 and
10 0.49 nM (0.33/0.65) 0.29 nM (0.25/0.33) 7.1 (7.5/6.7) 2.7
(2.5/2.8) SEQ ID NOs: 11 and 12 0.65 nM (0.72/0.57) 0.55 nM
(0.45/0.64) 5.5 (6.5/4.4) 2.8 (2.9/2.7) SEQ ID NOs: 13 and 14 -- --
-- -- SEQ ID NOs: 15 and 16 0.53 nM (0.55/0.50) 0.38 nM (0.45/0.30)
3.9 (4.1/3.7) 2 (2.1/1.8)
Example 7: Affinity to Fc-Gamma Receptors hFc.gamma. RI/CD64 and
hFc.gamma. RIIIA/CD16a
[0210] To measure the binding affinities of polypeptide fusions
with an engineered, IgG4-based backbone (SEQ ID NOs: 9 and 10, SEQ
ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 15 and
16) to Fc-gamma receptors hFc.gamma. RI/CD64 (R&D Systems) and
hFc.gamma. RIIIA/CD16a (R&D Systems), a Surface Plasmon
Resonance (SPR) based assay was employed. SEQ ID NOs: 3 and 4
served as a control of a monospecific antibody with an IgG1
backbone. SEQ ID NOs: 5 and 6 served as a control of a polypeptide
fusion that was IgG1-based. In the SPR affinity assay, polypeptide
fusions were biotinylated and captured on a sensor chip CAP using
the Biotin CAPture Kit (GE Healthcare). The sensor Chip CAP was
pre-immobilized with an ssDNA oligo. Undiluted Biotin CAPture
Reagent (streptavidin conjugated with the complementary ss-DNA
oligo) was applied at a flow rate of 2 .mu.L/min for 300 s.
Subsequently, 10 .mu.g/mL of biotinylated polypeptide fusion was
applied for 300 s at a flow rate of 5 .mu.L/min. SEQ ID NOs: 3 and
4 and the polypeptide fusions were biotinylated by incubation with
EZ-Link.RTM. NHS-PEG4-Biotin (Thermo Scientific) for two hours at
room temperature. The excess of non-reacted biotin reagent was
removed by loading the reaction mixture onto a Zeba.TM. Spin
Desalting Plate (Thermo Scientific). The reference channel was
loaded with Biotin CAPture Reagent only.
[0211] To determine the affinity, three dilutions of hFc.gamma.
RI/CD64 (at 40, 8 and 1.6 or at 100, 25 and 6 nM) or four to five
dilutions of hFc.gamma. RIIIA/CD16a (at 200, 40, 8 and 1.6 nM or at
1000, 333, 111, 37 and 12 nM) were prepared in running buffer (10
mM HEPES, 150 mM NaCl, 0.05% v/v Surfactant P20, 3 mM EDTA, pH 7.4
(GE Healthcare)) and applied to the chip surface. Applying a flow
rate of 30 .mu.L/min, the sample contact time was 180 s and
dissociation time was 1800/2700 s for hFc.gamma. RI/CD64 or 300 s
hFc.gamma. RIIlA/CD16a. All measurements were performed at
25.degree. C. Regeneration of the Sensor Chip CAP surface was
achieved with an injection of 6 M Gua-HCl with 0.25 M NaOH followed
by an extra wash with running buffer and a stabilization period of
120 s. Prior to the protein measurements three regeneration cycles
were performed for conditioning purposes. Data were evaluated with
Biacore T200 Evaluation software (V 2.0). Double referencing was
used. For hFc.gamma. RI/CD64 the 1:1 binding model was used to fit
the raw data. For hFc.gamma. RIIIA/CD16a the Steady State Affinity
model was used to fit the raw data.
[0212] Table 5 shows the results of the fit of the data for
hFc.gamma. RI/CD64. The IgG1-based test articles SEQ ID NOs: 3 and
4 and SEQ ID NOs: 5 and 6 were both at 0.3 nM, demonstrating that
hFc.gamma. RI/CD64 binding was not affected by fusion of SEQ ID
NOs: 3 and 4 to an Anticalin protein. The polypeptide fusions SEQ
ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and
SEQ ID NOs: 15 and 16 showed no detectable binding to hFc.gamma.
RI/CD64. These data demonstrate that binding to hFc.gamma. RI/CD64
can be reduced below detection limit by switching the isotype from
IgG1 to engineered IgG4.
TABLE-US-00005 TABLE 5 Clone name KD [nM] SEQ ID NOs: 3 and 4 0.3
SEQ ID NOs: 5 and 6 0.3 SEQ ID NOs: 9 and 10 not determinable SEQ
ID NOs: 11 and 12 not determinable SEQ ID NOs: 13 and 14 not
determinable SEQ ID NOs: 15 and 16 not determinable
[0213] Table 6 shows the results of the fit of the data for
hFc.gamma. RIIIA/CD16a. The resulting binding affinities to
hFc.gamma. RIIIA/CD16a of the IgG1-based test articles SEQ ID NOs:
3 and 4 and SEQ ID NOs: 5 and 6 were comparable to each other and
around 350 nM whereas the polypeptide fusions SEQ ID NOs: 9 and 10,
SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 15
and 16 showed no detectable binding to hFc.gamma. RIIIA/CD16a.
These data demonstrate that binding to hFc.gamma. RIIIA/CD16a can
be reduced below detection limit by switching the isotype from IgG1
to engineered IgG4.
TABLE-US-00006 TABLE 6 Name KD [nM] SEQ ID NOs: 3 and 4 335 .+-. 64
SEQ ID NOs: 5 and 6 369 .+-. 76 SEQ ID NOs: 9 and 10 not
determinable SEQ ID NOs: 11 and 12 not determinable SEQ ID NOs: 13
and 14 not determinable SEQ ID NOs: 15 and 16 not determinable
Example 8: Affinity to Neonatal Fc Receptor
[0214] To measure the binding affinities of polypeptide fusions
with an engineered, IgG4-based backbone (SEQ ID NOs: 9 and 10, SEQ
ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, and SEQ ID NOs: 15 and
16) to the neonatal Fc receptor (FcRn, Sino Biologicals,
#CT009-H08H), a Surface Plasmon Resonance (SPR) based assay was
employed. SEQ ID NOs: 3 and 4 served as a control of a monospecific
antibody with an IgG1 backbone. SEQ ID NOs: 5 and 6 served as a
control of a polypeptide fusion that was IgG1-based. In the SPR
affinity assay, FcRn was covalently immobilized on a CM5 sensor
chip (GE Healthcare) according to the manufacturer's instructions.
Briefly, after activating the carboxyl groups of the dextran matrix
with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and
N-hydroxysuccinimide (NHS), the primary amines of the FcRn protein
were allowed to react with the NHS ester on the surface until a
signal of .about.200 RU was reached. Finally, non-reacted
NHS-esters were blocked by passing a solution of 1M ethanolamine
across the surface. The flow rate throughout the immobilization
procedure was 10 .mu.l/min.
[0215] To determine their affinity, six dilutions (1000 nM, 333 nM,
111 nM, 37 nM, 12 nM and 4 nM) of all constructs were prepared in
running buffer (10 mM HEPES, 150 mM NaCl, 0.05% v/v Surfactant P20,
3 mM EDTA, pH 6.0) and applied to the chip surface. Applying a flow
rate of 30 .mu.L/min, the sample contact time was 180 s and
dissociation time was 30 s. All measurements were performed at
25.degree. C. Regeneration of the Sensor Chip CAP surface was
achieved with an injection of 10 mM glycine pH 3.0. Prior to the
protein measurements three regeneration cycles are performed for
conditioning purposes. Data were evaluated with Biacore T200
Evaluation software (V 2.0) with double referencing. The Steady
State Affinity model was used to fit the raw data.
[0216] The resulting binding affinities of all polypeptide fusions
to FcRn were in the range of 1-2 .mu.M which demonstrates that
switching the isotype from IgG1 to IgG4 has no detectable impact on
FcRn binding.
TABLE-US-00007 TABLE 7 Name KD [.mu.M] SEQ ID Nos: 3 and 4 2.0 .+-.
0.2 SEQ ID Nos: 5 and 6 1.1 .+-. 0.03 SEQ ID Nos: 11 and 12 1.3
.+-. 0.07 SEQ ID Nos: 13 and 14 1.8 .+-. 0.04 SEQ ID Nos: 9 and 10
1.7 .+-. 0.05 SEQ ID Nos: 13 and 14 1.8 .+-. 0.01
Example 9: Functional T-Cell Activation Assay Using Tumor Cells
with High and Low HER2 Levels
[0217] We employed a target-cell dependent T-cell activation assay
to assess the ability of the fusion polypeptide of SEQ ID NOs: 9
and 10 to co-stimulate T-cell responses as a function of HER2
expression levels of the target cell. For that purpose, we employed
HER2-high expressing SKBR3 and BT474 cells, as well as cells
expressing HER2 at a level similar to healthy, HER2-expressing
cells, HepG2 and MCF7. For comparison, we investigated the behavior
of reference anti-CD137 monoclonal antibodies of SEQ ID NOs: 47 and
48 and SEQ ID NOs: 49 and 50. As a negative control, we employed
the monospecific, HER2-binding antibody of SEQ ID NOs: 3 and 4. As
a further negative control, the experiment was carried out without
the addition of a test article ("vehicle control"). In the
experiment, an anti-human CD3 antibody (OKT3, eBioscience) was
coated on plastic culture dishes, and subsequently SKBR3, BT474,
HepG2 or MCF7 cells were separately cultured on the dishes
overnight. The next day, purified T cells were incubated on the
coated surface in the presence of various concentrations of the
fusion polypeptide of SEQ ID NOs: 9 and 10, reference antibodies
SEQ ID NOs: 47 and 48 and SEQ ID NOs: 49 and 50, the control
antibody of SEQ ID NOs: 3 and 4, or in the absence of added test
article. As readout, we measured supernatant interleukin 2 (IL-2)
levels. In the following, the experiment is described in
detail.
[0218] Human peripheral blood mononuclear cells (PBMC) from healthy
volunteer donors were isolated from buffy coats by centrifugation
through a Polysucrose density gradient (Biocoll 1.077 g/mL from
Biochrom), following Biochrom's protocols. The T lymphocytes were
isolated from the resulting PBMC using a Pan T cell purification
Kit (Miltenyi Biotec GmbH) and the manufacturer's protocols.
Purified T cells were re-suspended in a buffer consisting of 90%
FCS and 10% DMSO, immediately frozen down using liquid nitrogen and
stored in liquid nitrogen until further use. For the assay, T cells
were thawed for 16 h and cultivated in culture media (RPMI 1640,
Life Technologies) supplemented with 10% FCS and 1%
Penicillin-Streptomycin (Life Technologies).
[0219] The following procedure was performed using triplicates for
each experimental condition. Flat-bottom tissue culture plates were
pre-coated or not for 1 h at 37.degree. C. using 200 .mu.L of 0.25
.mu.g/mL anti-CD3 antibody. The plates were subsequently washed
twice with PBS. 5.times.10.sup.4 target tumor cells per well were
plated and allowed to adhere overnight at 37.degree. C. in a
humidified 5% C02 atmosphere. The target cells had before been
grown in culture under standard conditions, detached using Accutase
and re-suspended in culture media.
[0220] On the next days, tumor cells were treated for 2 hours at
37.degree. C. with mitomycin C (Sigma Aldrich) at a concentration
of 30 .mu.g/ml in order to block their proliferation. Plates were
washed twice with PBS, and 100 .mu.L of the T-cell suspension
(corresponding to 5.times.10.sup.4 T cells) and SEQ ID NOs: 9 and
10, reference antibodies SEQ ID NOs: 47 and 48 and SEQ ID NOs: 49
and 50 or the negative control SEQ ID NOs: 3 and 4 or vehicle, at
concentrations ranging from 0.05 nM to 5 nM (with the exception of
BT474, were concentrations ranged from 0.1 pM to 50 nM), were added
to each well. Plates were covered with a gas permeable seal
(4titude) and incubated at 37.degree. C. in a humidified 5% C02
atmosphere for 3 days. Subsequently, the IL-2 concentration in the
supernatant was assessed as described below.
[0221] Human IL-2 levels in the cell culture supernatants were
quantified using the IL-2 DuoSet kit from R&D Systems. In the
first step, a 384 well plate was coated at room temperature for 2 h
with 1 .mu.g/mL "Human IL-2 Capture Antibody" (R&D System)
diluted in PBS. Subsequently, wells were washed 5 times with 80
.mu.l PBS-T (PBS containing 0.05% Tween20) using a Biotek EL405
select CW washer (Biotek). After 1 h blocking in PBS-T additionally
containing 1% casein (w/w), pooled supernatant and a concentration
series of an IL-2 standard diluted in culture medium were incubated
in the 384-well plate overnight at 4.degree. C. To allow for
detection and quantitation of captured IL-2, a mixture of 100 ng/mL
biotinylated goat anti-hlL-2-Bio detection antibody (R&D
System) and 1 .mu.g/mL Sulfotag-labelled streptavidin (Mesoscale
Discovery) were added in PBS-T containing 0.5% casein and incubated
at room temperature for 1 h. After washing, 25 .mu.L reading buffer
was added to each well and the electrochemiluminescence (ECL)
signal of every well was read using a Mesoscale Discovery reader.
Analysis and quantification were performed using Mesoscale
Discovery software.
[0222] The result of a representative experiment is depicted in
FIG. 11. In this Figure, values are plotted relative to the
background IL-2 production in the absence of test article, and
therefore represent the fold change compared to background. While
the negative control of SEQ ID NOs: 3 and 4 (FIG. 11A) does not
lead to IL-2 induction on T-cells with any of the four cell lines,
rising concentrations of the bispecific fusion polypeptide SEQ ID
NOs: 9 and 10 (FIG. 11A) induce T-cells to produce IL-2 in the
presence of the highly HER2-expressing SKBR3 and BT474 cells.
However, no IL-2 increase due to SEQ ID NOs: 9 and 10 is apparent
for HepG2 and MCF7 cells. This behavior is markedly different from
both the first anti-CD137 antibody SEQ ID NOs: 47 and 48, which
induces IL-2 on T-cells in the presence of all four cell lines
(FIG. 11B), and the second anti-CD137 antibody SEQ ID NOs: 49 and
50, which does not lead to induction of IL-2 on any of the four
cell lines (FIG. 11C).
[0223] The experiment demonstrates that the fusion protein defined
by SEQ ID NOs: 9 and 10 activates T-cells in a manner that is
dependent on the HER2 density of the target cells. While the highly
HER2-expressing SKBR3 and BT474 cells show a clear T-cell
activation as measured by IL-2 production, this effect does not
occur with HepG2 and MCF7 cells, which express HER2 at a
considerably lower level. That this effect is attributable to the
HER2 density and not to a potential inhibition or lack of
costimulation brought about by the cell under study which renders
CD137 signaling ineffective becomes apparent by the fact that the
anti-CD137 antibody SEQ ID NOs: 47 and 48 is capable of activating
T cells via CD137 signaling with all four cell types.
Example 10: 4-Week Stability Study of Fusion Polypeptides in Buffer
at Neutral pH and Elevated Temperature
[0224] To investigate the stability of fusion polypeptide defined
by SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and
14 and SEQ ID NOs: 15 and 16, we employed an experiment where
samples where incubated for 4 weeks at 40.degree. C. in PBS, pH7.4
at a concentration of approximately 20 mg/mL (range 21-23 mg/mL).
For comparison, we investigated the behavior of the polypeptide
defined by SEQ ID NOs: 3 and 4 under identical conditions. Samples
were concentrated from a concentration of around 5 mg/mL to around
20 mg/mL using centrifugal filters (Ultracel-3K, Amicon), and a
part of this concentrated sample was stored at -20.degree. C. as
reference material. 0.1 mL of the concentrated sample in 0.5 mL
tubes (PCR-PT, Sarstedt) were then stored in an incubator (Memmert)
for 4 weeks at 40.degree. C. To investigate the integrity and
monomeric content of the sample, it was then subjected to
analytical size exclusion chromatography (SEC) using a Superdex200
Increase column on an Agilent 1200 Series GPC2 System at a flow
rate of 0.150 mL/min. 20 .mu.g of sample were applied to the
column. Relative protein concentration in the continuous
flow-through was detected by absorption at a wavelength of 280
nm.
[0225] FIG. 12 provides the results of the SEC analysis for all
fusion polypeptides and the control SEQ ID NOs: 3 and 4 as
indicated. The respective bottom and top SEC traces for each fusion
polypeptide correspond to the reference material and the material
incubated for 4 weeks at 40.degree. C., respectively. Comparing
reference material and incubated material reveals that the SEC
trace does not change significantly for either the bispecific
fusion polypeptides or the control SEQ ID NOs: 3 and 4,
demonstrating the stability of the fusion polypeptides against
aggregation.
Example 11: Pharmacokinetics of Fusion Polypeptides in Mice
[0226] An analysis of the pharmacokinetics of fusion polypeptides
defined by SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs:
13 and 14 and SEQ ID NOs: 15 and 16, as well as of SEQ ID NOs: 3
and 4 for reference, was performed in mice. Male CD-1 mice
approximately 5 weeks of age (3 mice per timepoint; Charles River
Laboratories, Research Models and Services, Germany GmbH) were
injected into a tail vein with a fusion polypeptide at a dose of 10
mg/kg. The test articles were administered as a bolus using a
volume of 5 mL/kg. Plasma samples from the mice were obtained at
the timepoints of 5 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 4 d, 8 d,
14 d and 20 d. Sufficient whole blood--taken under isoflurane
anaesthesia--was collected to obtain at least 100 .mu.L Li-Heparin
plasma per animal and time. Drug levels were detected using a
Sandwich ELISA detecting the full bispecific construct via the
targets HER2 and CD137. Trastuzumab plasma levels were determined
using a Sandwich ELISA with targets HER2 and human Fc. The data
were fitted using a two-compartmental model using Prism GraphPad 5
software.
[0227] FIG. 13 shows plots of the plasma concentration over time
for the constructs SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ
ID NOs: 13 and 14 and SEQ ID NOs: 15 and 16, in all cases plotted
together with the values obtained for SEQ ID NOs: 3 and 4 for
reference. The pharmacokinetics looked similar in all cases.
Starting from a plasma concentration of around 200 .mu.g/mL, plasma
levels fell to a level of around 50 .mu.g/mL within 48 hours, and
then further decrease at a much slower rate to a level of around 25
.mu.g/mL at the end of the experiment after 20 days. The
bi-exponential decay of a two-compartmental model was successfully
applied to accurately describe the data, and a fit of the data
(FIG. 13, Table 8) using this model resulted in terminal half-lives
of 15-21 days for the bispecific fusion polypeptides, compared to
13 days for SEQ ID NOs: 3 and 4.
[0228] The data demonstrate that the bispecific fusions have long,
antibody-like terminal half-lives in mice. Because the assay
employed to determine fusion polypeptide plasma concentrations
requires a retained activity both towards HER2 and CD137, the
result also demonstrates that the bispecific molecules remain
intact and active over the time course of 20 days.
TABLE-US-00008 TABLE 8 Terminal half-lives in mice obtained using a
data fit based on a two-compartmental model Construct Terminal
half-life [days .+-. std error of fit] SEQ ID NOs: 3 and 4 13.3
.+-. 1.2 SEQ ID NOs: 9 and 10 20.9 .+-. 3.8 SEQ ID NOs: 11 and 12
19.1 .+-. 2.5 SEQ ID NOs: 13 and 14 14.8 .+-. 1.5 SEQ ID NOs: 15
and 16 15.6 .+-. 1.9
Example 12: Pharmacokinetics of Fusion Polypeptides in Cynomolgus
Monkey
[0229] An analysis of the pharmacokinetics of fusion polypeptides
defined by SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs:
13 and 14 and SEQ ID NOs: 15 and 16, as well as of SEQ ID NOs: 3
and 4 for reference, was performed in cynomolgus monkeys. Male
cynomolgus monkeys received an intravenous infusion over 60
minutes, with a dose of 3 mg/kg test article. Plasma samples from
the cynomolgus monkeys were obtained at the timepoints of 15 min, 2
h, 4 h, 8 h, 24 h, 48 h, 3 d, 4 d, 5 d, 6 d, 7 d, 9 d, 11 d, 14 d,
18 d, and 24 d. Drug levels were detected using a Sandwich ELISA
detecting the full bispecific construct via the targets HER2 and
CD137. Trastuzumab plasma levels were determined using a Sandwich
ELISA with targets HER2 and human Fc. The data were fitted using a
two-compartmental model using Prism GraphPad 5 software.
[0230] FIG. 14 shows semi-logarithmic plots of the plasma
concentration over time for the constructs SEQ ID NOs: 9 and 10,
SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14 and SEQ ID NOs: 15 and
16, in all cases plotted together with the values obtained for SEQ
ID NOs: 3 and 4 for reference. The pharmacokinetics looked similar
in all cases. Starting from a plasma concentration of around 70
.mu.g/mL, plasma levels fall to levels close to zero over the
timecourse of 24 days. The bi-exponential decay of a
two-compartmental model was successfully applied to accurately
describe the data, and a fit of the data (FIG. 14, Table 9) using
this model resulted in terminal half-lives of ranging from
approximately 64 to 99 hours for the bispecific fusion
polypeptides, compared to eighty-six hours for SEQ ID NOs: 3 and
4.
[0231] The data therefore demonstrate that the bispecific fusions
have terminal half-lives in cynomolgus monkeys that are very
similar to the half-life of the reference polypeptide SEQ ID NOs: 3
and 4.
TABLE-US-00009 TABLE 9 Terminal half-lives in male cynomolgus
monkeys obtained using a data fit based on a two-compartmental
model: Construct Terminal half-life [hours .+-. std error of fit]
SEQ ID NOs: 3 and 4 86.0 .+-. 3.1 SEQ ID NOs: 9 and 10 98.7 .+-.
1.5 SEQ ID NOs: 11 and 12 65.2 .+-. 1.4 SEQ ID NOs: 13 and 14 83.9
.+-. 3.0 SEQ ID NOs: 15 and 16 63.7 .+-. 0.7
Example 13: Ex Vivo T Cell Immunogenicity Assessment of Fusion
Polypeptides
[0232] To investigate the risk of the formation of anti-drug
antibodies in man, an in vitro T cell immunogenicity assessment of
the bispecific fusion polypeptides SEQ ID NOs: 9 and 10, SEQ ID
NOs: 11 and 12, SEQ ID NOs: 13 and 14 and SEQ ID NOs: 15 and 16, as
well as of SEQ ID NOs: 3 and 4 for reference, the control antibody
of SEQ ID NOs: 3 and 4 and the positive control keyhole limpet
hemocyanine (KLH) was performed. To perform the experiment, PBMC
from 32 donors selected to cover HLA allotypes reflective of the
distribution in a global population were thawed, washed and seeded
onto 96-well plates at a density of 3.times.10.sup.5 cells per
well. Test articles, diluted in assay media, were added to the
cells at a concentration of 30 .mu.g/mL. Assay medium alone was
used as a blank, and keyhole limpet hemocyanine (KLH) was used as a
naive positive control. PBMC were incubated for 7 days in a
humidified atmosphere at 37.degree. C. and 5% CO.sub.2. On day 7,
PBMCs were labelled for surface phenotypic CD3+ and CD4+ markers
and for DNA-incorporated EdU (5-ethynyl-2'deoxyuridine), used as a
cell proliferation marker. The percentage of
CD3.sup.+CD4.sup.+EdU.sup.+ proliferating cells was measured using
a Guava easyCyte 8HT flow cytometer and analysed using GuavaSoft
InCyte software.
[0233] FIG. 15 provides the results of this assay for all 32 donors
and all test molecules under study. In FIG. 15A, the stimulation
index was plotted, which was obtained by the ratio of proliferation
in the presence vs. absence of test article. The threshold that
defines a responding donor (stimulation index >2) is indicated
as a dotted line. In FIG. 15B, the number of responding donors as
defined by this threshold was plotted. Evidently, the number of
donors responding to the reference SEQ ID NOs: 3 and 4 lies at one
and is therefore small, while all 32 donors respond to the positive
control KLH with strong proliferation above the threshold. For the
bispecific fusion polypeptides, the number of responding donors
ranges from zero (SEQ ID NOs: 9 and 10) via one (SEQ ID NOs: 15 and
16) and two (SEQ ID NOs: 13 and 14) to three (SEQ ID NOs: 11 and
12).
[0234] The experiment therefore demonstrates that the bispecific
fusion polypeptides, in particular SEQ ID NOs: 9 and 10 and SEQ ID
NOs: 15 and 16, induce little response in the in vitro T cell
immunogenicity assessment, which indicates that the risk of
inducing immunogenic responses is low.
Example 14: Tumor Growth Inhibition by CD137/HER2 Bispecifics in
Humanized Mouse Tumor Model
[0235] In order to investigate the activity of SEQ ID NOs: 9 and
10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14 and SEQ ID NOs: 32
and 33 in an in-vivo mouse model, we employed immune deficient NOG
mice (Taconic, NOD/Shi-scid/IL-2R.gamma.null) engrafted with human
SK-OV-3 tumors and human PBMC. 4-6 week old NSG mice were
subcutaneously (s.c.) injected with 5.times.10.sup.6 SK-OV-3 cells
in a matrigel/PBS (1:1) solution. Tumors were allowed to grow to an
average of 120 mm.sup.3 and on day 0 of the experiment mice were
randomized into treatment groups according to tumor size and animal
weight. Mice were given 7.times.10.sup.6 fresh human PBMC
intravenously (i.v.) into a tail vein. Mice received 20 .mu.g or
100 .mu.g of treatment or control into the intraperitoneal cavity 1
hour after PBMC injection on day 0, and again on day 7 and day 14.
The molecules under study were IgG4 isotype control (Cat #DDXCH04P,
Acris Antibodies GmbH), HER2/CD137 bispecifics SEQ ID NOs: 9 and 10
(100 .mu.g or 20 .mu.g), SEQ ID NOs: 11 and 12 (100 .mu.g or 20
.mu.g) and SEQ ID NOs: 13 and 14 (100 .mu.g), or the CD137-binding
benchmark antibody of SEQ ID NOs: 32 and 33 (100 .mu.g). Each group
contained 10 mice with the exception of the group studying SEQ ID
NOs: 32 and 33 which consisted of 7 mice. Tumor growth was recorded
every 3-4 days.
[0236] FIG. 16 shows the median tumor sizes relative to the
starting volume at day 14 of the study. The best responses, ordered
by strength of tumor growth inhibition, were achieved by SEQ ID
NOs: 9 and 10 (100 .mu.g), SEQ ID NOs: 9 and 10 (20 .mu.g) and SEQ
ID NOs: 11 and 12 (100 .mu.g), while SEQ ID NOs: 11 and 12 at the
lower dose of 20 .mu.g, SEQ ID NOs: 13 and 14 (100 .mu.g) as well
as the CD137-binding benchmark antibody of SEQ ID NOs: 32 and 33
has a median response that was similar to that of the isotype
control.
Example 15: Investigating CD137 Pathway Activation Using
NF-.kappa.B-luc2P/4-1BB Jurkat Reporter Cells
[0237] We employed a target-cell based reporter assay to assess the
ability of the fusion polypeptide of SEQ ID NOs: 9 and 10--capable
of binding CD137 and HER2 at the same time--to activate the CD137
pathway in dependence of the HER2 status of the target cell. For
that purpose, we employed highly HER2-expressing NCI-N87 gastric
cancer cells that were mixed with NF-.kappa.B-luc2P/4-1BB Jurkat
cells (Promega, CS196002) engineered to overexpress CD137 and
carrying a NF-.kappa.B Luciferase reporter gene. For comparison, we
investigated the behavior of reference anti-CD137 monoclonal
antibodies of SEQ ID NOs: 32 and 33 and SEQ ID NOs: 34 and 35. As a
negative control, we employed the monospecific, HER2-binding
antibody of SEQ ID NOs: 3 and 4. As further control, we also
assessed the CD137 pathway activation in the absence of NCI-N87
cells for the fusion polypeptide of SEQ ID NOs: 9 and 10 and for
anti-CD137 monoclonal antibodies of SEQ ID NOs: 32 and 33 and SEQ
ID NOs: 34 and 35 as well as for the monospecific, HER2-binding
antibody of SEQ ID NOs: 3 and 4. Finally, the experiment was also
carried out without the addition of a test article ("vehicle
control"). The background signal measured in the presence of
NCI-N87 cells alone was assessed in wells where no
NF-.kappa.B-luc2P/4-1BB Jurkat cells had been added. In the
experiment, NCI-N87 cells were cultured on the dishes overnight.
The next day, freshly thawed out NF-.kappa.B-uc2P/4-1BB Jurkat
cells (Promega, CS196002) were incubated for six hours on the
coated surface in the presence of various concentrations of the
fusion polypeptide of SEQ ID NOs: 9 and 10, the reference
antibodies SEQ ID NOs: 32 and 33 and SEQ ID NOs: 34 and 35, the
control antibody of SEQ ID NOs: 3 and 4, or in the absence of added
test article. As readout, we measured the luminescence induced by
the addition of Bio-GIo.TM. buffer (Promega, G7940) on the Jurkat
reporter cells. In the following, the experiment is described in
detail.
[0238] The following procedure was performed using triplicates for
each experimental condition. Flat-bottom tissue culture plates were
used to coat 5.times.10.sup.4 target NCI-N87 tumor cells per well,
with some wells remaining without target cancer cells as control
wells. The cells were allowed to adhere overnight at 37.degree. C.
in a humidified 5% C02 atmosphere. The target cells had before been
grown in culture under standard conditions, detached using Accutase
and resuspended in culture media.
[0239] On the next day, plates were washed twice with PBS, and 50
.mu.L of the NF-.kappa.B-luc2P/4-1BB Jurkat cells suspension
(corresponding to 1.5.times.10.sup.5 cells) and 25 .mu.L of SEQ ID
NOs: 9 and 10 at concentrations ranging from 0.04 nM to 10 nM,
reference antibodies SEQ ID NOs: 32 and 33 and SEQ ID NOs: 34 and
35 at concentrations ranging from 0.4 nM to 10 nM, the negative
control SEQ ID NOs: 3 and 4 at a concentration of 10 nM, or vehicle
were added to each well. Plates were covered with a gas permeable
seal (4titude) and incubated at 37.degree. C. in a humidified 5%
C02 atmosphere for 6 hours. Subsequently, 75 .mu.L of Bio-GIo.TM.
buffer (Promega, G7940) was added to each well containing cells
(1:1 v/v) and luminescence was measured using a luminescence plate
reader (Pherastar). Analysis, quantification and curve fitting were
performed using Graphpad Prism software.
[0240] The result of a representative experiment is depicted in
FIG. 17. In this Figure, plotted values are provided in relative
luminescence units (RLU). Rising concentrations of the bispecific
fusion polypeptide SEQ ID NOs: 9 and 10 (FIG. 17A) induce CD137
pathway activation in reporter Jurkat cells in the presence of the
highly HER2-expressing NCI-N87 cells, in contrast to the negative
control of SEQ ID NOs: 3 and 4 (FIG. 16A). Furthermore, no increase
luminescence is observed when the target NCI-N87 cells are missing.
This behavior is markedly different to both the first anti-CD137
antibody SEQ ID NOs: 32 and 33, which induces CD137 pathway
activation in the Jurkat reporter cells both in the presence and
absence of NCI-N87 cells (FIG. 17B), and the second anti-CD137
antibody SEQ ID NOs: 34 and 35, which does not lead to CD137
pathway activation in the Jurkat reporter cells at all (FIG.
17C).
[0241] The experiment demonstrates that SEQ ID NOs: 9 and 10
activates the CD137 pathway in a manner that depends upon the
presence of target cells expressing HER2, as no activation occurred
in the absence of NCI-N87 cells. These data validate that the mode
of action of SEQ ID NOs: 9 and 10 in T cell activation is the
activation of the CD137 pathway by crosslinking the CD137 receptor
via engagement of HER2 on cancer cells. The HER2-positive
cell-specific mode of action is further highlighted by comparison
to the data of the anti-CD137 antibody SEQ ID NOs: 32 and 33, which
activates T cells via CD137 signaling whether target cells are
present or not.
Example 16: Tumor Growth Inhibition by CD137/HER2 Bispecifics in
Humanized Mouse Tumor Model
[0242] In a protocol similar to Example 14, immuno-compromised mice
engrafted with HER2-positive tumor cells (SKOV-3) were injected
with human PBMC and treated over 3 weeks with SEQ ID NOs: 9 and 10
at a 100 .mu.g/week or 20 .mu.g/week, anti-CD137 antibody SEQ ID
NOs: 32 and 33, or controls, which were vehicle with PBMC ("PBMC
only"), vehicle without PBMC ("no PBMC") or isotype control with
PBMC. Specifically, NOG mice were subcutaneously (s.c.) injected
with SK-OV-3 cells and tumors were allowed to grow to an average of
120 mm.sup.3 prior to randomization into treatment groups. There
were 10 animals per treatment group. Mice were engrafted with fresh
human PBMC intravenously (i.v.) into a tail vein and treatment
commenced 1 hour later. Mice received 3 weekly intraperitoneal
(i.p.) doses of treatment (20 .mu.g or 100 .mu.g) of SEQ ID NOs: 9
and 10 or SEQ ID NOs: 32 and 33 or control. Tumor growth was
recorded twice weekly. Tumors from two mice were harvested on day
20 post treatment and assessed for infiltration of human T cells by
immunohistochemistry via staining for the human lymphocyte marker
CD45.
[0243] The results of the experiment are reported in FIG. 18. FIG.
18A shows median tumor growth over time. Data points that no longer
represent the full group size of 10 mice are connected by dotted
lines. The best responses, ordered by strength of tumor growth
inhibition, were achieved by SEQ ID NOs: 9 and 10 (100 .mu.g),
followed by the lower dose (20 .mu.g) of the same antibody, while
the CD137-binding benchmark antibody of SEQ ID NOs: 32 and 33 has a
median response that was similar to that of the isotype control.
FIG. 18B shows Immunohistochemistry of tumors after study end.
Sections of formalin-fixed and paraffin-embedded tumors (2 per
group) were stained for the human lymphocyte marker CD45; the
frequency of CD45-positive cells was quantified by dedicated
software as reflected in FIG. 18B. The figure shows that SEQ ID
NOs: 9 and 10 (100 .mu.g) resulted in increased frequency of human
tumor infiltrating lymphocytes (TILs) while SEQ ID NOs: 9 and 10
(20 .mu.g) and controls did not.
[0244] The results reflect that SEQ ID NOs: 9 and 10 treatment at
high dose (100 .mu.g/wk) and low dose (20 .mu.g/wk) resulted in
stronger tumor growth inhibition (TGI) compared to isotype control
or anti-CD137 benchmark. IHC staining for the human lymphocyte
marker CD45 shows increased frequency of human TIL for high dose
(100 .mu.g) SEQ ID NOs: 9 and 10 while low dose (20 .mu.g) SEQ ID
NOs: 9 and 10 does not show this effect. Taken together these data
are consistent with a dual functionality of SEQ ID NOs: 9 and 10:
On the one hand, the tumor-localized targeting of CD137 leads to
expansion of TIL's in the tumor microenvironment and suggests
tumor-localized costimulatory T cell activation by SEQ ID NOs: 9
and 10 while tumor growth inhibition is observed with 20 ug SEQ ID
NOs: 9 and 10 in the absence of TIL expansion, suggesting this
activity may be driven by HER2 antagonism.
Example 17: PBMC Phenotyping and Mortality in Humanized NOG Mouse
SKOV-3 Tumor Model
[0245] In order to assess the safety of SEQ ID Nos: 9 and 10, PBMCs
were isolated from mouse blood samples of the mice of Experiment
16. These samples were taken on day 19 after PBMC engraftment and
analysed by multicolor FACS for human surface markers CD45, CD3 and
CD8. Peripheral blood was resuspended in 10 ml 1.times. erythrocyte
lysis buffer (0.15M NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA) and
lysed for 1-3 minutes at room temperature in a 15-ml tube. Cells
were centrifuged at 300.times.g for 10 minutes at 4.degree. C.,
washed 1.times. with 10 ml FC-buffer (2% fetal calf serum in PBS,
pH7.4) and resuspended in 200 .mu.l of FC-buffer. Cells were
transferred to a 96-well plate at a density of 5.times.10.sup.5
cells/well. Cells were pelleted by centrifugation of the plates at
400.times.g for 3 minutes at 4.degree. C. and the supernatant was
removed. Fc-block antibody (10 .mu.l/well of a 1:100 dilution in
buffer of 2.4G2 antibody, 0.5 mg/ml, #553142--BD Bioscience) was
added to each well and plates were incubated for 15 minutes at room
temperature. Then specific antibodies against human targets hCD45
(Life Technologies), hCD3 and hCD8 (both BD Bioscience) were added
(0.5-1 .mu.g/sample) and plates were incubated at 4.degree. C. for
30 minutes protected from light. Following another washing step
(centrifugation of the plates at 400.times.g for 3 min. at
4.degree. C.), cells were resuspended in 200 .mu.l FC Buffer for
analysis with an Attune Focusing Cytometer (blue (488 nm)/violet
(405 nm) laser configuration). Flow cytometry data were analyzed
with the FlowJo Data Analysis Software.
[0246] FIG. 19A shows the CD45, CD3 and CD8 phenotype of PMBCs from
the treatment and control groups of Example 16 taken on day 19 of
that study. FIG. 19A on the left shows the percentage of total
PMBCs expressing CD45 while the FIG. 19A on the right shows the
fraction of CD3+CD8+T effector cells in the CD45-positive cell
population. Clearly, the results demonstrate that the anti-CD137
antibody SEQ ID NOs: 32 and 33 treatment leads to stronger
expansion of human lymphocytes in the mouse peripheral blood
compared to the control group or SEQ ID NOs: 9 and 10, and that
this expansion correlates with a strong increase in the CD8.sup.+
human effector T cells. FIG. 19B shows the mortality of treatment
and control groups of Experiment 16. Plotted values of FIG. 19B
correspond to number of mice per group of ten that died
spontaneously or needed to be sacrificed based on defined general
condition criteria. The results reflect that Anti-CD137 mAb
treatment led to accelerated graft-versus-host disease with
significant mortality compared to control and SEQ ID NOs: 9 and 10
by the end of the study. Combined with the PBMC phenotyping data,
the results indicate that the accelerated xenograft versus host
disease (xGvHD) induced by anti-CD137 mAb treatment is caused by
strongly increased expansion of CD8.sup.+ human effector T cells in
anti-CD137 group compared to the control or SEQ ID NOs: 9 and 10
groups.
Example 18: Tumor Growth Inhibition by CD137/HER2 Bispecifics in
Humanized Mouse Tumor Model
[0247] In order to investigate the activity of SEQ ID NOs: 9 and 10
in an in-vivo mouse model, we employed immune deficient NOG mice
(Taconic, NOD/Shi-scid/IL-2R.gamma.null) engrafted with human
SK-OV-3 tumors and human PBMC. The monospecific CD137-targeting
antibody SEQ ID NOs: 32 and 33 and the monospecific HER2-targeting
construct (IgG4 backbone) SEQ ID Nos: 51 and 52 were investigated
in parallel to assess the effect of monospecific vs. bispecific
targeting of the receptors HER2 and CD137.
4-6 week old NSG mice were subcutaneously (s.c.) injected with
5.times.10.sup.6 SK-OV-3 cells in a matrigel/PBS (1:1) solution.
Tumors were allowed to grow to an average of 110 mm.sup.3 and on
day 0 of the experiment mice were randomized into treatment groups
according to tumor size and animal weight. Mice were given
7.times.10.sup.6 fresh human PBMC intravenously (i.v.) into a tail
vein. Mice received the HER2/CD137 bispecific SEQ ID NOs: 9 and 10
at four different concentrations (200 .mu.g, 100 .mu.g, 20 .mu.g or
4 .mu.g), isotype control (100 .mu.g, Cat #00004, Crown Bioscience
Inc., CA), monospecific CD137-targeting antibody SEQ ID NOs: 32 and
33 (100 .mu.g) and monospecific HER2-targeting antibody SEQ ID Nos:
51 and 52 (80 .mu.g) into the intraperitoneal cavity 1 hour after
PBMC injection on day 0, and again on day 7 and day 14. Note that
the dose of SEQ ID Nos: 51 and 52 (80 .mu.g) was chosen to be
equimolar to that of the 100 .mu.g SEQ ID NOs: 9 and 10 group. As
negative controls, groups receiving vehicle and PBMC, or vehicle
only ("no PBMC") were also included. Each group contained 10 mice
with the exception of the group SEQ ID NOs: 9 and 10, which
consisted of 9 mice as one mouse succumbed on day 4 of the study
due to treatment-unrelated causes. Tumor growth was recorded every
3-4 days. Statistical significance of tumor growth inhibition
responses was determined by a two-sided student's T-test.
[0248] FIG. 20 shows the mortality across study groups at day 20
after PBMC engraftment. Mortality caused by PBMC
xenograft-vs-host-disease (xGvHD) either led to spontaneous death
or to ethical sacrifice based on predefined criteria. Strikingly,
the monospecific CD137-targeting antibody SEQ ID NOs: 32 and 33 led
to strongly accelerated xGvHD compared to all other groups, with no
mouse surviving to study end. Most other groups also showed
mortality before study end, with up to three mortalities at day 20.
There was no apparent dose-dependency for the four treatment groups
of SEQ ID NOs: 9 and 10, and mortality was similar to the SEQ ID
NOs: 51 and 52 group and the "PBMC only" group, indicating no
impact of SEQ ID NOs: 9 and 10 on onset of xGvHD.
[0249] FIG. 21 shows the absolute median tumor sizes over time.
Note that values are combined by dotted lines for groups that are
no longer complete due to mortality (see above). Strikingly, the
isotype control group led to significant tumor growth inhibition
compared to the vehicle control groups with and without PBMC; in
the following discussion, the relevant control group for the
treatment groups was therefore chosen to be the isotype control
group. Compared to this group, there was strong tumor growth
inhibition for SEQ ID NOs: 9 and 10 at the weekly 200 .mu.g and 100
.mu.g dose as well as for SEQ ID Nos: 51 and 52 at the 80 .mu.g
dose, with all responses being similar and highly statistically
significant (p<0.001). SEQ ID NOs: 9 and 10 at a dose of 20
.mu.g weekly showed a trend towards tumor growth inhibition
compared to the isotype control group, but statistical significance
was borderline (p=0.07). There was no statistically significant
difference at day 20 between the tumor growth of the isotype
control group and that of either the CD137-binding benchmark
antibody of SEQ ID NOs: 32 and 33 or of the lowest dose of the
CD137/HER2 bispecific SEQ ID NOs: 9 and 10 (4 .mu.g). Taken
together, the median tumor growth curves indicate a strong
dose-dependent anti-tumor activity of SEQ ID NOs: 9 and 10, which,
taking the result for SEQ ID NOs: 51 and 52 into account, appear to
be mainly driven by the anti-HER2 activity of SEQ ID NOs: 9 and
10.
Example 19: Phenotyping of Tumor-Infiltrating Lymphocytes by
Immunohistochemistry in Humanized Mouse Tumor Model
[0250] In a follow-up analysis of the in-vivo study described in
Example 18, tumors from five or six tumor-bearing mice from each of
the nine study groups were excised on study end or ethical
sacrifice and assessed for infiltration of human T cells by
immunohistochemistry via staining for the human lymphocyte marker
CD45. For that purpose, tumors were formalin-fixed, embedded in
paraffin and processed for immunohistochemistry using anti-human
CD45 antibodies. CD45-positive cells were identified by
3,3'-diaminobenzidine (DAB) staining. To allow clear visualization
of DAB-positivity in a greyscale image, contrast and brightness of
the images was digitally adjusted.
[0251] An overview over all stained tumor sections is provided in
FIG. 22, while FIG. 23 provides the result of a digital
quantitation of the frequency of CD45-positive cells by dedicated
software. FIG. 22 illustrates an evident qualitative difference
between the SEQ ID NOs: 9 and 10-treated groups at the weekly
dosings of 200 .mu.g, 100 .mu.g or 20 .mu.g compared to all other
groups: Clearly, the DAB-positivity in the corresponding tumor
slices is much stronger. The total absence of staining in the "no
PBMC" group confirms the selectivity of the staining procedure. The
digital quantitation (FIG. 23) confirms this qualitative finding:
With the exception of the 4 .mu.g dosing group, the tumors from SEQ
ID NOs: 9 and 10-treated animals display a strong hCD45-positivity,
indicative of a high frequency of human lymphocytes in the slides.
The hCD45-positivity is statistically significantly higher
(p<0.01) than in the control groups ("PBMC only" or isotype
control) or in the groups treated with the HER2-monospecific SEQ ID
NOs: 51 and 52 or the CD137-monospecific benchmark antibody SEQ ID
NOs: 32 and 33. Notably, the latter group even displays a
statistically significant lower human lymphocyte presence than that
of the control groups, for example compared to the isotype control
(p=0.02). However, this effect may be biased by the earlier
sampling that took place with the mice from this group due to
required ethical sacrifice.
[0252] Taken together, this data illustrates the tumor-localized
costimulatory mode of action of SEQ ID NOs: 9 and 10, leading to an
increased frequency of human lymphocytes in the tumor at weekly
doses of 20 .mu.g or higher. Importantly, this activity is strictly
driven by the bispecific activity of SEQ ID NOs: 9 and 10, because
the monospecific HER2-targeting antibody SEQ ID NOs: 51 and 52 and
the monospecific CD137-targeting antibody SEQ ID NOs: 32 and 33 do
not display this activity. On the contrary, the monospecific
CD137-targeting antibody SEQ ID NOs: 32 and 33 even leads to a
decreased frequency of human lymphocytes compared to the negative
controls.
Example 20: PBMC Phenotyping in Humanized Mouse Tumor Model
[0253] In order to further elucidate the mode of action and assess
the safety of SEQ ID Nos: 9 and 10, PBMCs were isolated from mouse
blood samples of the mice of Experiment 18. These samples were
taken from the final bleeding after sacrifice of the mice at study
end or after ethical sacrifice and analysed by multicolor FACS for
human surface markers CD45 and CD8. Peripheral blood was
resuspended in 10 ml 1.times. erythrocyte lysis buffer (0.15M
NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA) and lysed for 1-3
minutes at room temperature in a 15-ml tube. Cells were centrifuged
at 300.times.g for 10 minutes at 4.degree. C., washed 1.times. with
10 ml FC-buffer (2% fetal calf serum in PBS, pH7.4) and resuspended
in 200 .mu.l of FC-buffer. Cells were transferred to a 96-well
plate at a density of 5.times.10.sup.5 cells/well. Cells were
pelleted by centrifugation of the plates at 400.times.g for 3
minutes at 4.degree. C. and the supernatant was removed. Fc-block
antibody (10 .mu.l/well of a 1:100 dilution in buffer of 2.4G2
antibody, 0.5 mg/ml, #553142--BD Bioscience) was added to each well
and plates were incubated for 15 minutes at room temperature. Then
specific antibodies against human targets hCD45 (Life Technologies)
and hCD8 (BD Bioscience) were added (0.5-1 .mu.g/sample) and plates
were incubated at 4.degree. C. for 30 minutes protected from light.
Following another washing step (centrifugation of the plates at
400.times.g for 3 minutes at 4.degree. C.), cells were resuspended
in 200 .mu.l FC Buffer for analysis with an Attune Focusing
Cytometer (blue (488 nm)/violet (405 nm) laser configuration). Flow
cytometry data were analyzed with the FlowJo Data Analysis
Software.
[0254] FIG. 24 shows the CD45 and CD8 phenotype of PMBCs from the
treatment and control groups of Example 20 after study end. FIG.
24A shows the percentage of total PMBCs expressing CD45 while FIG.
24B shows the fraction of CD45+CD8+T effector cells in the
CD45-positive cell population. Clearly, the results demonstrate
that the anti-CD137 antibody SEQ ID NOs: 32 and 33 treatment leads
to stronger expansion of human lymphocytes in the mouse peripheral
blood compared to the control group or all doses of SEQ ID NOs: 9
and 10, and that this expansion correlates with a stronger increase
in the CD8.sup.+ human effector T cells. Combined with the
mortality data shown in FIG. 20, the results indicate that the
accelerated xGvHD induced by anti-CD137 mAb treatment is caused by
an increased expansion of CD8.sup.+ human effector T cells in the
anti-CD137 group compared to the control or SEQ ID NOs: 9 and 10
groups.
[0255] Combining the evidence of Examples 18, 19 and 20, the
following conclusions can be drawn:
[0256] (i) SEQ ID NOs: 9 and 10 has a dual functionality: It leads
to direct tumor regression due to an anti-HER2 effect, and a
tumor-localized increase in the density of human lymphocytes by
HER2-tumor-target localized CD137 targeting.
[0257] (ii) Surprisingly, the direct anti-HER2 effect is not
dependent on any Fc-gamma receptor mediated effector functionality,
as both the CD137/HER2 bispecific SEQ ID NOs: 9 and 10 and the HER2
monospecific antibody SEQ ID NOs: 51 and 52 should not elicit any
effector functions: Both possess an IgG4 antibody backbone with
additional mutations that essentially eliminates Fc-gamma receptor
interactions.
[0258] (iii) The benefit of bispecific, tumor-localized CD137
targeting regarding both efficacy and safety becomes evident by
comparison with the monospecifically CD137-targeting benchmark
antibody: While the CD137/HER2 bispecific leads to a strong
increase of human lymphocytes in the tumor compared to controls,
the monospecific CD137-targeting antibody even leads to a decrease
compared to controls. On the other hand, the monospecific
CD137-targeting antibody leads to an expansion of human
CD8-positive T cells in the peripheral blood of the mice in the
study, an effect which is not apparent for SEQ ID NOs: 9 and 10.
The peripheral expansion of CD8.sup.+ effector cells correlates
with an accelerated mortality of mice via xGvHD. Such toxicity
brought about by systemic activation of CD137 may also be relevant
for the clinical application of anti-CD137 antibodies such as SEQ
ID NOs: 32 and 33. These observations strongly vouch for both an
improved efficacy and safety of SEQ ID NOs: 9 and 10 compared to
monospecific anti-CD137 benchmarks such as SEQ ID NOs: 32 and
33.
[0259] It is obvious to those skilled in the art that variants of
the in vivo model described in this application can be applied to
show various aspects of the in vivo efficacy of SEQ ID NOs: 9 and
10. Generally, such models will be based on an engraftment with
tumor cells that are positive for the human HER2 receptor or
variants thereof, which is enabled by tumor cells that are either
naturally HER2-receptor positive or made HER2-receptor positive by
methods such as transfection or viral transduction with HER2. Such
cells can be either derived from immortal cancer cell lines or
patient tumors. In addition, the models may rely on alloreactive or
HLA-matched and tumor-reactive T cells of human or murine origin,
for example:
[0260] (I) Humanized models based on HER2-positive tumors and
alloreactive PBMC. Typically, mice employed in such models will be
immunocompromised to a lesser or larger degree. Examples of a model
based on an immortal cell line are provided in this application,
and for example in Sanmamed et al., Cancer Res. 2015 Sep. 1;
75(17):3466-78. The latter publication also provides an example for
a typical model based on a patient-derived tumor cell
xenograft.
[0261] (II) Humanized models based on HER2-positive tumors and
monoclonal or polyclonal T cells that recognize one or more
antigens on the tumor cell line. Typically, mice employed in such
models will be immunocompromised to a lesser or larger degree.
Various combinations of tumor cell specific T cells and tumor cells
are possible. Tumor cell specific T cells may be obtained by
generating partly or fully HLA-matched monoclonal or polyclonal
tumor-reactive T cells via different protocols, for example as
described in Erskine et al., J Vis Exp. 2012 Aug. 8; (66):e3683, or
by transducing T cells with a natural T cell receptor, for example
as described in Wang et al., Cancer Immunol Res. 2016 March;
4(3):204-14, or Hirschhorn-Cymerman et al., J Exp Med. 2012 Oct.
22; 209(11):2113-26. An artificial chimeric antigen receptor may
also be employed to replace the natural TCR.
[0262] (III) Patient-derived tumor cells and autologous
patient-derived PBMC or (expanded) TIL. Typically, mice employed in
such models will be immunocompromised to a lesser or larger
degree.
[0263] (IV) A transgenic mouse model, where the CD137 receptor is
partially or fully humanized, and thus made capable of binding to
SEQ ID NOs: 9 and 10. Transgenic mice will be engrafted with mouse
tumors that were made to express human HER2 by cell biological
methods. To increase the physiological relevance of the model, the
mouse can be additionally made transgenic for the CD137 ligand
and/or human HER2.
[0264] The models described in the above or variants thereof are
expected to be capable of showing one or more of the following
pharmacodynamics effects: an increase in TIL frequency via direct
or indirect enhancement of local proliferation, an increase in TIL
frequency via direct or indirect suppression of lymphocyte cell
death, an increase in TIL activity different from proliferation or
persistence such as the production of proinflammatory cytokines
including but not limited to IL-2, IFN-.gamma. or TNF-.alpha. or an
improved capacity to kill tumor cells as evidenced by a strong
impact on tumor growth that is not due to the anti-HER2 activity of
SEQ ID NOs: 9 and 10 alone. Lymphocytes affected include, but are
not limited to CD4- and CD8-positive T cells, NK cells or NKT
cells. Other cell types may show specific pharmacodynamics effects,
including but not limited to endothelial cells, for example
endothelial cells of the tumor vessels. In the case of tumor
endothelial cells, CD137 targeting by SEQ ID NOs: 9 and 10 may to
an enhancement of trafficking into the tumor (cf. Palazon et al.,
Cancer Res. 2011 Feb. 1; 71(3):801-11.) via expression of targeting
receptors or soluble factors enhancing the targeting.
[0265] The models may be straightforwardly employed to study
additional effects such as specific targeting of lymphocyte
subsets, dose dependency of pharmacodynamic and toxic effects, or
treatment schedules.
[0266] Embodiments 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. 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 embodiments have been specifically disclosed
by preferred embodiments and optional features, modification and
variations thereof 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. All patents, patent
applications, textbooks and peer-reviewed publications described
herein are hereby incorporated by reference in their entirety.
Furthermore, where a definition or use of a term in a reference,
which is incorporated by reference herein, is inconsistent or
contrary to the definition of that term provided herein, the
definition of that term provided herein applies and the definition
of that term in the reference does not apply. Each of the narrower
species and sub-generic groupings falling within the generic
disclosure also forms 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. In addition, where features are described in terms of
Markush groups, those skilled in the art will recognize that the
disclosure is also thereby described in terms of any individual
member or subgroup of members of the Markush group. Further
embodiments will become apparent from the following claims.
[0267] Equivalents: 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
following claims. All publications, patents and patent applications
mentioned in this specification are herein incorporated by
reference into the specification to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference.
Sequence CWU 1
1
521178PRTartificialnegative control 1Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Leu Ala
Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35 40 45Gln Lys Met Tyr
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asn Val Thr
Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile65 70 75 80Arg
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90
95Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val
Ser Gln 115 120 125Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly2178PRTartificialpositive control lipocalin mutein 2Gln Asp Ser
Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu
Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val
Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40
45Ile Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr
50 55 60Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp
Ile65 70 75 80Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr
Leu Gly Lys 85 90 95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val
Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe
Phe Lys Phe Val Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr
Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu
Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu
Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly3450PRTartificialbenchmark antibody heavy chain 3Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 4504214PRTartificialbenchmark antibody light chain 4Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
2105643PRTartificialfusion polypeptide 5Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330
335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 450 455
460Ser Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser
Lys465 470 475 480Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe
His Gly Lys Trp 485 490 495Tyr Val Val Gly Gln Ala Gly Asn Ile Arg
Leu Arg Glu Asp Lys Asp 500 505 510Pro Ile Lys Met Met Ala Thr Ile
Tyr Glu Leu Lys Glu Asp Lys Ser 515 520 525Tyr Asp Val Thr Met Val
Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp 530 535 540Ile Trp Thr Phe
Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly545 550 555 560Lys
Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val 565 570
575Ser Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe
580 585 590Gln Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr
Lys Glu 595 600 605Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe
Ser Lys Ser Leu 610 615 620Gly Leu Pro Glu Asn His Ile Val Phe Pro
Val Pro Ile Asp Gln Cys625 630 635 640Ile Asp
Gly6214PRTartificialfusion polypeptide 6Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 2107640PRTartificialfusion polypeptide 7Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gln Asp 450 455 460Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro
Leu Ser Lys Val Pro Leu465 470 475 480Gln Gln Asn Phe Gln Asp Asn
Gln Phe His Gly Lys Trp Tyr Val Val 485 490 495Gly Gln Ala Gly Asn
Ile Arg Leu Arg Glu Asp Lys Asp Pro Ile Lys 500 505 510Met Met Ala
Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val 515 520 525Thr
Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr 530 535
540Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys Ile
Lys545 550 555 560Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val
Val Ser Thr
Asn 565 570 575Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe
Gln Asn Arg 580 585 590Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr
Lys Glu Leu Thr Ser 595 600 605Glu Leu Lys Glu Asn Phe Ile Arg Phe
Ser Lys Ser Leu Gly Leu Pro 610 615 620Glu Asn His Ile Val Phe Pro
Val Pro Ile Asp Gln Cys Ile Asp Gly625 630 635
6408214PRTartificialfusion polypeptide 8Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser
Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe
Asn Arg Gly Glu Cys 2109640PRTartificialfusion polypeptide 9Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25
30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met
Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gln Asp 450 455 460Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro
Leu Ser Lys Val Pro Leu465 470 475 480Gln Gln Asn Phe Gln Asp Asn
Gln Phe His Gly Lys Trp Tyr Val Val 485 490 495Gly Gln Ala Gly Asn
Ile Arg Leu Arg Glu Asp Lys Asp Pro Ile Lys 500 505 510Met Met Ala
Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val 515 520 525Thr
Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr 530 535
540Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys Ile
Lys545 550 555 560Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val
Val Ser Thr Asn 565 570 575Tyr Asn Gln His Ala Met Val Phe Phe Lys
Phe Val Phe Gln Asn Arg 580 585 590Glu Glu Phe Tyr Ile Thr Leu Tyr
Gly Arg Thr Lys Glu Leu Thr Ser 595 600 605Glu Leu Lys Glu Asn Phe
Ile Arg Phe Ser Lys Ser Leu Gly Leu Pro 610 615 620Glu Asn His Ile
Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly625 630 635
64010214PRTartificialfusion polypeptide 10Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21011447PRTartificialfusion polypeptide
11Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr
Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 44512407PRTartificialfusion polypeptide 12Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 210 215 220Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser
Asp Leu Ile Pro Ala Pro225 230 235 240Pro Leu Ser Lys Val Pro Leu
Gln Gln Asn Phe Gln Asp Asn Gln Phe 245 250 255His Gly Lys Trp Tyr
Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg 260 265 270Glu Asp Lys
Asp Pro Ile Lys Met Met Ala Thr Ile Tyr Glu Leu Lys 275 280 285Glu
Asp Lys Ser Tyr Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys 290 295
300Cys Met Tyr Asp Ile Trp Thr Phe Val Pro Gly Ser Gln Pro Gly
Glu305 310 315 320Phe Thr Leu Gly Lys Ile Lys Ser Phe Pro Gly His
Thr Ser Ser Leu 325 330 335Val Arg Val Val Ser Thr Asn Tyr Asn Gln
His Ala Met Val Phe Phe 340 345 350Lys Phe Val Phe Gln Asn Arg Glu
Glu Phe Tyr Ile Thr Leu Tyr Gly 355 360 365Arg Thr Lys Glu Leu Thr
Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe 370 375 380Ser Lys Ser Leu
Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro385 390 395 400Ile
Asp Gln Cys Ile Asp Gly 40513640PRTartificialfusion polypeptide
13Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1
5 10 15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp
Tyr 20 25 30Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys
Asp Pro 35 40 45Ile Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp
Lys Ser Tyr 50 55 60Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys
Met Tyr Asp Ile65 70 75 80Trp Thr Phe Val Pro Gly Ser Gln Pro Gly
Glu Phe Thr Leu Gly Lys 85 90 95Ile Lys Ser Phe Pro Gly His Thr Ser
Ser Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala
Met Val Phe Phe Lys Phe Val Phe Gln 115 120 125Asn Arg Glu Glu Phe
Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu
Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155
160Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile
165 170 175Asp Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 180 185 190Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly 195 200 205Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asn Ile Lys Asp 210 215 220Thr Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp225 230 235 240Val Ala Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser 245 250 255Val Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 260 265 270Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 275 280
285Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly
290 295 300Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser305 310 315 320Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 325 330 335Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val 340 345 350Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 355 360 365Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 370 375 380Pro Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His385 390 395
400Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
405 410 415Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
Pro Ser 420 425 430Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 435 440 445Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser
Gln Glu Asp Pro 450 455 460Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala465 470 475 480Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val 485 490 495Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 500 505 510Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 515 520 525Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 530 535
540Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys545 550 555 560Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 565 570 575Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp 580 585 590Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser 595 600 605Arg Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala 610 615 620Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys625 630 635
64014214PRTartificialfusion polypeptide 14Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala
Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Arg
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21015447PRTartificialfusion polypeptide
15Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Asp Gly Phe Tyr
Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 44516407PRTartificialfusion polypeptide 16Gln
Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10
15Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr
20 25 30Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp
Pro 35 40 45Ile Lys Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys
Ser Tyr 50 55 60Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met
Tyr Asp Ile65 70 75 80Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu
Phe Thr Leu Gly Lys 85 90 95Ile Lys Ser Phe Pro Gly His Thr Ser Ser
Leu Val Arg Val Val Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met
Val Phe Phe Lys Phe Val Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr
Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu
Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu
Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170
175Asp Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
180 185 190Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val 195 200 205Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Val Asn Thr 210 215 220Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu225 230 235 240Ile Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser Arg Phe Ser 245 250 255Gly Ser Arg Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 260 265 270Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro 275 280 285Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 290 295
300Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser305 310 315 320Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu 325 330 335Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser 340 345 350Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu 355 360 365Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val 370 375 380Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys385 390 395 400Ser
Phe Asn Arg Gly Glu Cys 40517158PRTartificialwildtype hTlc 17His
His Leu Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr1 5 10
15Trp Tyr Leu Lys Ala Met Thr Val Asp Arg Glu Phe Pro Glu Met Asn
20 25 30Leu Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly
Asn 35 40 45Leu Glu Ala Lys Val Thr Met Leu Ile Ser Gly Arg Cys Gln
Glu Val 50 55 60Lys Ala Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr
Thr Ala Asp65 70 75 80Gly Gly Lys His Val Ala Tyr Ile Ile Arg Ser
His Val Lys Asp His 85 90 95Tyr Ile Phe Tyr Cys Glu Gly Glu Leu His
Gly Lys Pro Val Arg Gly 100 105 110Val Lys Leu Val Gly Arg Asp Pro
Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125Asp Phe Glu Lys Ala Ala
Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140Leu Ile Pro Arg
Gln Ser Glu Thr Cys Ser Pro Gly Ser Asp145 150
15518178PRTartificialwildtype hNGAL 18Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30Val Val Gly Leu Ala
Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35 40 45Gln Lys Met Tyr
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asn Val Thr
Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile65 70 75 80Arg
Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90
95Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val
Ser Gln 115 120 125Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly1915PRTartificiallinker 19Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser1 5 10 15201929DNAartificialfusion
polypeptide 20gaagtccagc tggtcgaatc tggtggtggc ctggtccagc
ctggtggatc actgagactg 60tcctgtgctg cttctggttt caacatcaag gacacctaca
tccattgggt cagacaggca 120cctggcaagg gactggaatg ggtcgcccga
atctacccta caaacggcta cactcgctac 180gccgactccg tcaagggacg
ctttaccatc tccgccgaca cctctaaaaa caccgcctac 240ctgcagatga
atagtctgag ggccgaggat actgctgtgt actactgctc acgatgggga
300ggcgacggct tttacgctat ggattactgg ggacagggaa ctctggtcac
tgtgtctagc 360gctagcacaa agggccctag tgtgtttcct ctggctccct
cttccaaatc cacttctggt 420ggcactgctg ctctgggatg cctggtgaag
gattactttc ctgaacctgt gactgtctca 480tggaactctg gtgctctgac
ttctggtgtc cacactttcc ctgctgtgct gcagtctagt 540ggactgtact
ctctgtcatc tgtggtcact gtgccctctt catctctggg aacccagacc
600tacatttgta atgtgaacca caaaccatcc aacactaaag tggacaaaaa
agtggaaccc 660aaatcctgtg acaaaaccca cacctgccca ccttgtcctg
cccctgaact gctgggagga 720ccttctgtgt ttctgttccc accaaaacca
aaagataccc tgatgatctc tagaacccct 780gaggtgacat gtgtggtggt
ggatgtgtct catgaggacc ctgaggtcaa attcaactgg 840tacgtggatg
gagtggaagt ccacaatgcc aaaaccaagc ctagagagga acagtacaat
900tcaacctaca gagtggtcag tgtgctgact gtgctgcatc aggattggct
gaatggcaag 960gaatacaagt gtaaagtctc aaacaaggcc ctgcctgctc
caattgagaa aacaatctca 1020aaggccaagg gacagcctag ggaaccccag
gtctacaccc tgccaccttc aagagaggaa 1080atgaccaaaa accaggtgtc
cctgacatgc ctggtcaaag gcttctaccc ttctgacatt 1140gctgtggagt
gggagtcaaa tggacagcct gagaacaact acaaaacaac cccccctgtg
1200ctggattctg atggctcttt ctttctgtac tccaaactga ctgtggacaa
gtctagatgg 1260cagcagggga atgtcttttc ttgctctgtc atgcatgagg
ctctgcataa ccactacact 1320cagaaatccc tgtctctgtc tcctggcaaa
ggcggcggag gatccggggg tgggggaagc 1380ggcggaggag gtagccagga
ctctactagt gatctgatcc cggcaccgcc actgtcaaaa 1440gtccctctgc
aacaaaactt tcaagacaat cagtttcacg gcaaatggta tgtggtcggc
1500caggccggaa acattaggct gcgggaggac aaggacccca tcaaaatgat
ggctaccatc 1560tacgagctga aggaagacaa atcttatgat gtgacaatgg
tcaagttcga cgataagaaa 1620tgcatgtacg acatctggac cttcgtgccc
ggctcccagc cgggagagtt caccctgggc 1680aagatcaagt ccttccccgg
ccacacttcc agcctggtcc gcgtggtctc gaccaactat 1740aatcagcatg
ctatggtgtt cttcaagttc gtctttcaga atagagagga gttctacatc
1800acactgtatg gacgcaccaa ggagctgaca agcgagctga aagaaaactt
catcaggttt 1860tcaaagtccc tggggctgcc cgaaaatcat atcgtgttcc
cagtccccat cgaccagtgt 1920attgatggt 192921642DNAartificialfusion
polypeptide 21gacatccaga tgacacagtc tccctcttcc ctgtccgctt
ctgtgggcga tcgagtgaca 60atcacctgta gggctagtca ggatgtgaat actgctgttg
cttggtacca gcagaaacca 120ggaaaagccc ctaaactgct gatctactct
gcctcattcc tgtactctgg ggtgccttct 180cgattcagtg gttctagatc
tggcaccgat ttcacactga ccatttcttc actgcaacct 240gaggattttg
ccacctacta ctgtcagcag cactacacaa cacctcccac atttggccag
300ggcacaaaag tggagatcaa acggaccgtg gcggcgcctt ctgtgttcat
tttcccccca 360tctgatgaac agctgaaatc tggcactgct tctgtggtct
gtctgctgaa caacttctac 420cctagagagg ccaaagtcca gtggaaagtg
gacaatgctc tgcagagtgg gaattcccag 480gaatctgtca ctgagcagga
ctctaaggat agcacatact ccctgtcctc tactctgaca 540ctgagcaagg
ctgattacga gaaacacaaa gtgtacgcct gtgaagtcac acatcagggg
600ctgtctagtc ctgtgaccaa atccttcaat aggggagagt gc
642221920DNAartificialfusion polypeptide 22gaagtccagc tggtcgaatc
tggtggtggc ctggtccagc ctggtggatc actgagactg 60tcctgtgctg cttctggttt
caacatcaag gacacctaca tccattgggt cagacaggca 120cctggcaagg
gactggaatg ggtcgcccga atctacccta caaacggcta cactcgctac
180gccgactccg tcaagggacg ctttaccatc tccgccgaca cctctaaaaa
caccgcctac 240ctgcaaatga atagtctgag ggccgaggat actgctgtgt
actactgctc acgatgggga 300ggcgacggct tttacgctat ggattactgg
ggacagggaa ctctggtcac tgtctcgagc 360gctagcacca agggcccctc
cgtgttcccc ctggcccctt gctcccggtc cacctccgag 420tctaccgccg
ctctgggctg cctggtgaaa gactacttcc ccgagcctgt gaccgtgagc
480tggaactctg gcgccctgac ctccggcgtg cacaccttcc ctgccgtgct
gcaatcctcc 540ggcctgtact ccctgtcctc cgtggtgaca gtgccctcct
ccagcctggg caccaagacc 600tacacctgta acgtggacca caagccctcc
aacaccaagg tggacaagcg ggtggaatct 660aaatacggcc ctccctgccc
cccctgccct gcccctgaag cggcgggcgg accttccgtg 720tttctgttcc
ccccaaagcc caaggacacc ctgatgatct cccggacccc cgaagtgacc
780tgcgtggtgg tggacgtgtc ccaggaagat ccagaggtgc agttcaactg
gtatgttgac 840ggcgtggaag tgcacaacgc caagaccaag cccagagagg
aacagttcgc ctccacctac 900cgggtggtgt ccgtgctgac cgtgctgcac
caggactggc tgaacggcaa agagtacaag 960tgcaaggtgt ccaacaaggg
cctgccctcc agcatcgaaa agaccatctc caaggccaag 1020ggccagcccc
gcgagcccca ggtgtacacc ctgcccccta gccaggaaga gatgaccaag
1080aaccaggtgt ccctgacctg tctggtgaaa ggcttctacc cctccgacat
tgccgtggaa 1140tgggagtcca acggccagcc cgagaacaac tacaagacca
ccccccctgt gctggactcc 1200gacggctcct tcttcctgta ctctcggctg
acagtggata agtcccggtg gcaggaaggc 1260aatgtgttct cctgcagcgt
gatgcacgag gccctgcaca accactatac ccagaagtcc 1320ctgtccctga
gcctgggcaa gggcggtgga ggatccgggg gtgggggaag cggcggagga
1380ggtagccagg actctactag tgatctgatc ccggcaccgc cactgtcaaa
agtccctctg 1440caacaaaact ttcaagacaa tcagtttcac ggcaaatggt
atgtggtcgg ccaggccgga 1500aacattaggc tgcgggagga caaggacccc
atcaaaatga tggctaccat ctacgagctg 1560aaggaagaca aatcttatga
tgtgacaatg gtcaagttcg acgataagaa atgcatgtac 1620gacatctgga
ccttcgtgcc cggctcccag ccgggagagt tcaccctggg caagatcaag
1680tccttccccg gccacacttc cagcctggtc cgcgtggtct cgaccaacta
taatcagcat 1740gctatggtgt tcttcaagtt cgtctttcag aatagagagg
agttctacat cacactgtat 1800ggacgcacca aggagctgac aagcgagctg
aaagaaaact tcatcaggtt ttcaaagtcc 1860ctggggctgc ccgaaaatca
tatcgtgttc ccagtcccca tcgaccagtg tattgatggt
192023642DNAartificialfusion polypeptide 23gacatccaga tgacacagtc
tccctcttcc ctgtccgctt ctgtgggcga tcgagtgaca 60atcacctgta gggctagtca
ggatgtgaat actgctgttg cttggtacca gcagaaacca 120ggaaaagccc
ctaaactgct gatctactct gcctcattcc tgtactctgg ggtgccttct
180cgattcagtg gttctagatc tggcaccgat ttcacactga ccatttcttc
actgcaacct 240gaggattttg ccacctacta ctgtcagcag cactacacaa
cacctcccac atttggccag 300ggcacaaaag tggagatcaa acggaccgtg
gcggcgcctt ctgtgttcat tttcccccca 360tctgatgaac agctgaaatc
tggcactgct tctgtggtct gtctgctgaa caacttctac 420cctagagagg
ccaaagtcca gtggaaagtg gacaatgctc tgcagagtgg gaattcccag
480gaatctgtca ctgagcagga ctctaaggat agcacatact ccctgtcctc
tactctgaca 540ctgagcaagg ctgattacga gaaacacaaa gtgtacgcct
gtgaagtcac acatcagggg 600ctgtctagtc ctgtgaccaa atccttcaat
aggggagagt gc 642241920DNAartificialfusion polypeptide 24gaagtccagc
tggtcgaatc tggtggtggc ctggtccagc ctggtggatc actgagactg 60tcctgtgctg
cttctggttt caacatcaag gacacctaca tccattgggt cagacaggca
120cctggcaagg gactggaatg ggtcgcccga atctacccta caaacggcta
cactcgctac 180gccgactccg tcaagggacg ctttaccatc tccgccgaca
cctctaaaaa caccgcctac 240ctgcaaatga atagtctgag ggccgaggat
actgctgtgt actactgctc acgatgggga 300ggcgacggct tttacgctat
ggattactgg ggacagggaa ctctggtcac tgtctcgagc 360gctagcacca
agggcccctc cgtgttcccc ctggcccctt gctcccggtc cacctccgag
420tctaccgccg ctctgggctg cctggtgaaa gactacttcc ccgagcctgt
gaccgtgagc 480tggaactctg gcgccctgac ctccggcgtg cacaccttcc
ctgccgtgct gcaatcctcc 540ggcctgtact ccctgtcctc cgtggtgaca
gtgccctcct ccagcctggg caccaagacc 600tacacctgta acgtggacca
caagccctcc aacaccaagg tggacaagcg ggtggaatct 660aaatacggcc
ctccctgccc cccctgccct gcccctgaag cggcgggcgg accttccgtg
720tttctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc
cgaagtgacc 780tgcgtggtgg tggacgtgtc ccaggaagat ccagaggtgc
agttcaactg gtatgttgac 840ggcgtggaag tgcacaacgc caagaccaag
cccagagagg aacagttcaa ctccacctac 900cgggtggtgt ccgtgctgac
cgtgctgcac caggactggc tgaacggcaa agagtacaag 960tgcaaggtgt
ccaacaaggg cctgccctcc agcatcgaaa agaccatctc caaggccaag
1020ggccagcccc gcgagcccca ggtgtacacc ctgcccccta gccaggaaga
gatgaccaag 1080aaccaggtgt ccctgacctg tctggtgaaa ggcttctacc
cctccgacat tgccgtggaa 1140tgggagtcca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggactcc 1200gacggctcct tcttcctgta
ctctcggctg acagtggata agtcccggtg gcaggaaggc 1260aatgtgttct
cctgcagcgt gatgcacgag gccctgcaca accactatac ccagaagtcc
1320ctgtccctga gcctgggcaa gggcggtgga ggatccgggg gtgggggaag
cggcggagga 1380ggtagccagg actctactag tgatctgatc ccggcaccgc
cactgtcaaa agtccctctg 1440caacaaaact ttcaagacaa tcagtttcac
ggcaaatggt atgtggtcgg ccaggccgga 1500aacattaggc tgcgggagga
caaggacccc atcaaaatga tggctaccat ctacgagctg 1560aaggaagaca
aatcttatga tgtgacaatg gtcaagttcg acgataagaa atgcatgtac
1620gacatctgga ccttcgtgcc cggctcccag ccgggagagt tcaccctggg
caagatcaag 1680tccttccccg gccacacttc cagcctggtc cgcgtggtct
cgaccaacta taatcagcat 1740gctatggtgt tcttcaagtt cgtctttcag
aatagagagg agttctacat cacactgtat 1800ggacgcacca aggagctgac
aagcgagctg aaagaaaact tcatcaggtt ttcaaagtcc 1860ctggggctgc
ccgaaaatca tatcgtgttc ccagtcccca tcgaccagtg tattgatggt
192025642DNAartificialfusion polypeptide 25gacatccaga tgacacagtc
tccctcttcc ctgtccgctt ctgtgggcga tcgagtgaca 60atcacctgta gggctagtca
ggatgtgaat actgctgttg cttggtacca gcagaaacca 120ggaaaagccc
ctaaactgct gatctactct gcctcattcc tgtactctgg ggtgccttct
180cgattcagtg gttctagatc tggcaccgat ttcacactga ccatttcttc
actgcaacct 240gaggattttg ccacctacta ctgtcagcag cactacacaa
cacctcccac atttggccag 300ggcacaaaag tggagatcaa acggaccgtg
gcggcgcctt ctgtgttcat tttcccccca 360tctgatgaac agctgaaatc
tggcactgct tctgtggtct gtctgctgaa caacttctac 420cctagagagg
ccaaagtcca gtggaaagtg gacaatgctc tgcagagtgg gaattcccag
480gaatctgtca ctgagcagga ctctaaggat agcacatact ccctgtcctc
tactctgaca 540ctgagcaagg ctgattacga gaaacacaaa gtgtacgcct
gtgaagtcac acatcagggg 600ctgtctagtc ctgtgaccaa atccttcaat
aggggagagt gc 642261341DNAartificialfusion polypeptide 26gaagtccagc
tggtcgaatc tggtggtggc ctggtccagc ctggtggatc actgagactg 60tcctgtgctg
cttctggttt caacatcaag gacacctaca tccattgggt cagacaggca
120cctggcaagg gactggaatg ggtcgcccga atctacccta caaacggcta
cactcgctac 180gccgactccg tcaagggacg ctttaccatc tccgccgaca
cctctaaaaa caccgcctac 240ctgcaaatga atagtctgag ggccgaggat
actgctgtgt actactgctc acgatgggga 300ggcgacggct tttacgctat
ggattactgg ggacagggaa ctctggtcac tgtctcgagc 360gctagcacca
agggcccctc cgtgttcccc ctggcccctt gctcccggtc cacctccgag
420tctaccgccg ctctgggctg cctggtgaaa gactacttcc ccgagcctgt
gaccgtgagc 480tggaactctg gcgccctgac ctccggcgtg cacaccttcc
ctgccgtgct gcaatcctcc 540ggcctgtact ccctgtcctc cgtggtgaca
gtgccctcct ccagcctggg caccaagacc 600tacacctgta acgtggacca
caagccctcc aacaccaagg tggacaagcg ggtggaatct 660aaatacggcc
ctccctgccc cccctgccct gcccctgaag cggcgggcgg accttccgtg
720tttctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc
cgaagtgacc 780tgcgtggtgg tggacgtgtc ccaggaagat ccagaggtgc
agttcaactg gtatgttgac 840ggcgtggaag tgcacaacgc caagaccaag
cccagagagg aacagttcaa ctccacctac 900cgggtggtgt ccgtgctgac
cgtgctgcac caggactggc tgaacggcaa agagtacaag 960tgcaaggtgt
ccaacaaggg cctgccctcc agcatcgaaa agaccatctc caaggccaag
1020ggccagcccc gcgagcccca ggtgtacacc ctgcccccta gccaggaaga
gatgaccaag 1080aaccaggtgt ccctgacctg tctggtgaaa ggcttctacc
cctccgacat tgccgtggaa 1140tgggagtcca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggactcc 1200gacggctcct tcttcctgta
ctctcggctg acagtggata agtcccggtg gcaggaaggc 1260aatgtgttct
cctgcagcgt gatgcacgag gccctgcaca accactatac ccagaagtcc
1320ctgtccctga gcctgggcaa g 1341271062DNAartificialfusion
polypeptide 27ctgtactctg gggtgccttc tcgattcagt ggttctagat
ctggcaccga tttcacactg 60accatttctt cactgcaacc tgaggatttt gccacctact
actgtcagca gcactacaca 120acacctccca catttggcca gggcacaaaa
gtggagatca aacggaccgt ggcggcgcct 180tctgtgttca ttttcccccc
atctgatgaa cagctgaaat ctggcactgc ttctgtggtc 240tgtctgctga
acaacttcta ccctagagag gccaaagtcc agtggaaagt ggacaatgct
300ctgcagagtg ggaattccca ggaatctgtc actgagcagg actctaagga
tagcacatac 360tccctgtcct ctactctgac actgagcaag gctgattacg
agaaacacaa agtgtacgcc 420tgtgaagtca cacatcaggg gctgtctagt
cctgtgacca aatccttcaa taggggagag 480tgcggcggcg gaggatccgg
gggtggggga agcggcggag gaggtagcca ggactctact 540agtgatctga
tcccggcacc gccactgtca aaagtccctc tgcaacaaaa ctttcaagac
600aatcagtttc acggcaaatg gtatgtggtc ggccaggccg gaaacattag
gctgcgggag 660gacaaggacc ccatcaaaat gatggctacc atctacgagc
tgaaggaaga caaatcttat 720gatgtgacaa tggtcaagtt cgacgataag
aaatgcatgt acgacatctg gaccttcgtg 780cccggctccc agccgggaga
gttcaccctg ggcaagatca agtccttccc cggccacact 840tccagcctgg
tccgcgtggt ctcgaccaac tataatcagc atgctatggt gttcttcaag
900ttcgtctttc agaatagaga ggagttctac atcacactgt atggacgcac
caaggagctg 960acaagcgagc tgaaagaaaa cttcatcagg ttttcaaagt
ccctggggct gcccgaaaat 1020catatcgtgt tcccagtccc catcgaccag
tgtattgatg gt 1062281920DNAartificialfusion polypeptide
28caggactcta ctagtgatct gatcccggca ccgccactgt caaaagtccc tctgcaacaa
60aactttcaag acaatcagtt tcacggcaaa tggtatgtgg tcggccaggc cggaaacatt
120aggctgcggg aggacaagga ccccatcaaa atgatggcta ccatctacga
gctgaaggaa 180gacaaatctt atgatgtgac aatggtcaag ttcgacgata
agaaatgcat gtacgacatc 240tggaccttcg tgcccggctc ccagccggga
gagttcaccc tgggcaagat caagtccttc 300cccggccaca cttccagcct
ggtccgcgtg gtctcgacca actataatca gcatgctatg 360gtgttcttca
agttcgtctt tcagaataga gaggagttct acatcacact gtatggacgc
420accaaggagc tgacaagcga gctgaaagaa aacttcatca ggttttcaaa
gtccctgggg 480ctgcccgaaa atcatatcgt gttcccagtc cccatcgacc
agtgtattga tggaggaggc 540ggaggatccg gcggaggagg aagtggcgga
ggaggaagtg aagtccagct ggtcgaatct 600ggtggtggcc tggtccagcc
tggtggatca ctgagactgt cctgtgctgc ttctggtttc 660aacatcaagg
acacctacat ccattgggtc agacaggcac ctggcaaggg actggaatgg
720gtcgcccgaa tctaccctac aaacggctac actcgctacg ccgactccgt
caagggacgc 780tttaccatct ccgccgacac ctctaaaaac accgcctacc
tgcagatgaa tagtctgagg 840gccgaggata ctgctgtgta ctactgctca
cgatggggag gcgacggctt ttacgctatg 900gattactggg gacagggaac
tctggtcact gtgtctagcg ctagcaccaa gggcccctcc 960gtgttccccc
tggccccttg ctcccggtcc acctccgagt ctaccgccgc tctgggctgc
1020ctggtgaaag actacttccc cgagcctgtg accgtgagct ggaactctgg
cgccctgacc 1080tccggcgtgc acaccttccc tgccgtgctg caatcctccg
gcctgtactc cctgtcctcc 1140gtggtgacag tgccctcctc cagcctgggc
accaagacct acacctgtaa cgtggaccac 1200aagccctcca acaccaaggt
ggacaagcgg gtggaatcta aatacggccc tccctgcccc 1260ccctgccctg
cccctgaagc ggcgggcgga ccttccgtgt ttctgttccc cccaaagccc
1320aaggacaccc tgatgatctc ccggaccccc gaagtgacct gcgtggtggt
ggacgtgtcc 1380caggaagatc cagaggtgca gttcaactgg tatgttgacg
gcgtggaagt gcacaacgcc 1440aagaccaagc ccagagagga acagttcaac
tccacctacc gggtggtgtc cgtgctgacc 1500gtgctgcacc aggactggct
gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc 1560ctgccctcca
gcatcgaaaa gaccatctcc aaggccaagg gccagccccg cgagccccag
1620gtgtacaccc tgccccctag ccaggaagag atgaccaaga accaggtgtc
cctgacctgt 1680ctggtgaaag gcttctaccc ctccgacatt gccgtggaat
gggagtccaa cggccagccc 1740gagaacaact acaagaccac cccccctgtg
ctggactccg acggctcctt cttcctgtac 1800tctcggctga cagtggataa
gtcccggtgg caggaaggca atgtgttctc ctgcagcgtg 1860atgcacgagg
ccctgcacaa ccactatacc cagaagtccc tgtccctgag cctgggcaag
192029642DNAartificialfusion polypeptide 29gacatccaga tgacacagtc
tccctcttcc ctgtccgctt ctgtgggcga tcgagtgaca 60atcacctgta gggctagtca
ggatgtgaat actgctgttg cttggtacca gcagaaacca 120ggaaaagccc
ctaaactgct gatctactct gcctcattcc tgtactctgg ggtgccttct
180cgattcagtg gttctagatc tggcaccgat ttcacactga ccatttcttc
actgcaacct 240gaggattttg ccacctacta ctgtcagcag cactacacaa
cacctcccac atttggccag 300ggcacaaaag tggagatcaa acggaccgtg
gcggcgcctt ctgtgttcat tttcccccca 360tctgatgaac agctgaaatc
tggcactgct tctgtggtct gtctgctgaa caacttctac 420cctagagagg
ccaaagtcca gtggaaagtg gacaatgctc tgcagagtgg gaattcccag
480gaatctgtca ctgagcagga ctctaaggat agcacatact ccctgtcctc
tactctgaca 540ctgagcaagg ctgattacga gaaacacaaa gtgtacgcct
gtgaagtcac acatcagggg 600ctgtctagtc ctgtgaccaa atccttcaat
aggggagagt gc 642301341DNAartificialfusion polypeptide 30gaagtccagc
tggtcgaatc tggtggtggc ctggtccagc ctggtggatc actgagactg 60tcctgtgctg
cttctggttt caacatcaag gacacctaca tccattgggt cagacaggca
120cctggcaagg gactggaatg ggtcgcccga atctacccta caaacggcta
cactcgctac 180gccgactccg tcaagggacg ctttaccatc tccgccgaca
cctctaaaaa caccgcctac 240ctgcaaatga atagtctgag ggccgaggat
actgctgtgt actactgctc acgatgggga 300ggcgacggct tttacgctat
ggattactgg ggacagggaa ctctggtcac tgtctcgagc 360gctagcacca
agggcccctc cgtgttcccc ctggcccctt gctcccggtc cacctccgag
420tctaccgccg ctctgggctg cctggtgaaa gactacttcc ccgagcctgt
gaccgtgagc 480tggaactctg gcgccctgac ctccggcgtg cacaccttcc
ctgccgtgct gcaatcctcc 540ggcctgtact ccctgtcctc cgtggtgaca
gtgccctcct ccagcctggg caccaagacc 600tacacctgta acgtggacca
caagccctcc aacaccaagg tggacaagcg ggtggaatct 660aaatacggcc
ctccctgccc cccctgccct gcccctgaag cggcgggcgg accttccgtg
720tttctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc
cgaagtgacc 780tgcgtggtgg tggacgtgtc ccaggaagat ccagaggtgc
agttcaactg gtatgttgac 840ggcgtggaag tgcacaacgc caagaccaag
cccagagagg aacagttcaa ctccacctac 900cgggtggtgt ccgtgctgac
cgtgctgcac caggactggc tgaacggcaa agagtacaag 960tgcaaggtgt
ccaacaaggg cctgccctcc agcatcgaaa agaccatctc caaggccaag
1020ggccagcccc gcgagcccca ggtgtacacc ctgcccccta gccaggaaga
gatgaccaag 1080aaccaggtgt ccctgacctg tctggtgaaa ggcttctacc
cctccgacat tgccgtggaa 1140tgggagtcca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggactcc 1200gacggctcct tcttcctgta
ctctcggctg acagtggata agtcccggtg gcaggaaggc 1260aatgtgttct
cctgcagcgt gatgcacgag gccctgcaca accactatac ccagaagtcc
1320ctgtccctga gcctgggcaa g 1341311221DNAartificialfusion
polypeptide 31caggactcta ctagtgatct gatcccggca ccgccactgt
caaaagtccc tctgcaacaa 60aactttcaag acaatcagtt tcacggcaaa tggtatgtgg
tcggccaggc cggaaacatt 120aggctgcggg aggacaagga ccccatcaaa
atgatggcta ccatctacga gctgaaggaa 180gacaaatctt atgatgtgac
aatggtcaag ttcgacgata agaaatgcat gtacgacatc 240tggaccttcg
tgcccggctc ccagccggga gagttcaccc tgggcaagat caagtccttc
300cccggccaca cttccagcct ggtccgcgtg gtctcgacca actataatca
gcatgctatg 360gtgttcttca agttcgtctt tcagaataga gaggagttct
acatcacact gtatggacgc 420accaaggagc tgacaagcga gctgaaagaa
aacttcatca ggttttcaaa gtccctgggg 480ctgcccgaaa atcatatcgt
gttcccagtc cccatcgacc agtgtattga tggaggaggc 540ggaggatccg
gcggaggagg aagtggcgga ggaggaagtg acatccagat gacacagtct
600ccctcttccc tgtccgcttc tgtgggcgat cgagtgacaa tcacctgtag
ggctagtcag 660gatgtgaata ctgctgttgc ttggtaccag cagaaaccag
gaaaagcccc taaactgctg 720atctactctg cctcattcct gtactctggg
gtgccttctc gattcagtgg ttctagatct 780ggcaccgatt tcacactgac
catttcttca ctgcaacctg aggattttgc cacctactac 840tgtcagcagc
actacacaac acctcccaca tttggccagg gcacaaaagt ggagatcaaa
900cggaccgtgg cggcgccttc tgtgttcatt ttccccccat ctgatgaaca
gctgaaatct 960ggcactgctt ctgtggtctg tctgctgaac aacttctacc
ctagagaggc caaagtccag 1020tggaaagtgg acaatgctct gcagagtggg
aattcccagg aatctgtcac tgagcaggac 1080tctaaggata gcacatactc
cctgtcctct actctgacac tgagcaaggc tgattacgag 1140aaacacaaag
tgtacgcctg tgaagtcaca catcaggggc tgtctagtcc tgtgaccaaa
1200tccttcaata ggggagagtg c 122132152PRTartificiallipocalin mutein
32Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1
5 10 15Ala Met Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser
Val 20 25 30Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu
Ala Lys 35 40 45Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Lys
Ala Val Leu 50 55 60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp
Gly Gly Lys His65 70 75 80Val Ala Tyr Ile Ile Arg Ser His Val Lys
Asp His Tyr Ile Phe Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro
Val Pro Gly Val Trp Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn
Leu Glu Ala Leu Glu Asp Phe Glu Lys 115 120 125Ala Ala Gly Ala Arg
Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu
Thr Ser Ser Pro Gly145 15033152PRTartificiallipocalin mutein 33Thr
Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10
15Ala Met Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val
20 25 30Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala
Lys 35 40 45Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Arg Ala
Val Leu 50 55 60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly
Gly Lys His65 70 75 80Asp Ala Tyr Ile Ile Arg Ser His Val Lys Asp
His Tyr Ile Phe Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro Val
Pro Gly Val Trp Leu Val 100 105 110Gly Arg Asp Pro Glu Asn Asn Leu
Glu Ala Leu Glu Asp Phe Glu Lys 115 120 125Thr Ala Gly Ala Arg Gly
Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu Thr
Ser Ser Pro Gly145 15034152PRTartificiallipocalin mutein 34Ala Ser
Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala
Met Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val 20 25
30Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys
35 40 45Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Asn Ala Val
Leu 50 55 60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly
Lys His65 70 75 80Val Ala Tyr Ile Ile Arg Ser His Val Arg Asp His
Tyr Ile Phe Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro Val Pro
Gly Val Trp Leu Val 100 105 110Gly Arg Asp Pro Glu Asn Asn Leu Glu
Ala Leu Glu Asp Phe Glu Lys 115 120 125Thr Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu Thr Ser
Ser Pro Gly145 15035152PRTartificiallipocalin mutein 35Val Ser Asp
Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala Met
Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val 20 25 30Thr
Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys 35 40
45Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Arg Ala Val Leu
50 55 60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys
His65 70 75 80Val Ala Tyr Ile Ile Arg Ser His Val Glu Asp His Tyr
Ile Phe Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro Val Pro Gly
Val Trp Leu Val 100 105 110Gly Arg Asp Pro Glu Asn Asn Leu Glu Ala
Leu Glu Asp Phe Glu Lys 115 120 125Thr Ala Gly Ala Arg Gly Leu Ser
Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140Gln Ser Glu Thr Ser Ser
Pro Gly145 15036152PRTartificiallipocalin mutein 36Ala Ser Asp Glu
Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala Met Thr
Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val 20 25 30Thr Pro
Met Thr Leu Ser Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys 35
40 45Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Lys Ala Val
Leu 50 55 60Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly
Lys His65 70 75 80Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His
Tyr Ile Phe Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro Val Pro
Gly Val Trp Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn Leu Glu
Ala Leu Glu Asp Phe Glu Lys 115 120 125Ala Ala Gly Ala Arg Gly Leu
Ser Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140Gln Ile Glu Thr Ser
Ser Pro Gly145 15037152PRTartificiallipocalin mutein 37Ala Ser Asp
Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala Met
Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val 20 25 30Thr
Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Glu 35 40
45Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Lys Ala Val Leu
50 55 60Glu Lys Ala Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys
His65 70 75 80Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His Tyr
Ile Phe Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro Val Pro Gly
Val Trp Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala
Leu Glu Asp Phe Glu Lys 115 120 125Thr Ala Gly Ala Arg Gly Leu Ser
Thr Glu Ser Ile Leu Ile Pro Ser 130 135 140Gln Ile Glu Thr Ser Ser
Pro Gly145 15038152PRTartificiallipocalin mutein 38Thr Ser Asp Glu
Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys1 5 10 15Ala Met Thr
Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val 20 25 30Thr Pro
Met Thr Leu Thr Thr Leu Glu Asp Gly Asn Leu Glu Ala Lys 35 40 45Val
Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val Lys Ala Val Leu 50 55
60Glu Lys Ala Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His65
70 75 80Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His Tyr Ile Phe
Tyr 85 90 95Ser Glu Gly Val Cys Asp Gly Ser Pro Val Pro Gly Val Trp
Leu Val 100 105 110Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu
Asp Phe Glu Lys 115 120 125Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu
Ser Ile Leu Ile Pro Arg 130 135 140Gln Ile Glu Thr Ser Ser Pro
Gly145 15039178PRTartificiallipocalin mutein 39Gln Asp Ser Thr Ser
Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln
Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly
Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45Asn Lys
Met Met Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asn
Val Thr Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile65 70 75
80Ser Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys
85 90 95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val
Ser 100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe
Val Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly
Arg Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile
Arg Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile
Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly40178PRTartificiallipocalin mutein 40Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45Asn Lys Met Met
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asp Val Thr
Ala Val Ala Phe Asp Asp Lys Lys Cys Thr Tyr Asp Ile65 70 75 80Trp
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90
95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val
Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly41178PRTartificiallipocalin muein 41Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45Asn Lys Met Met
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asp Val Thr
Ala Val Ala Phe Asp Asp Lys Lys Cys Thr Tyr Asp Ile65 70 75 80Trp
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90
95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val
Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly42175PRTartificiallipocalin mutein 42Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Lys Leu Arg Glu Asp Ser Lys Met 35 40 45Met Ala Thr Ile
Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr 50 55 60Gly Val Ser
Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile Met Thr Phe65 70 75 80Val
Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys Ile Lys Ser 85 90
95Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr
100 105 110Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn
Arg Glu 115 120 125Glu Phe Tyr Ile Thr Leu Tyr Gly Arg Thr Lys Glu
Leu Thr Ser Glu 130 135 140Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys
Ser Leu Gly Leu Pro Glu145 150 155 160Asn His Ile Val Phe Pro Val
Pro Ile Asp Gln Cys Ile Asp Gly 165 170
17543178PRTartificiallipocalin mutein 43Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45Val Lys Met Met
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asp Val Thr
Gly Val Thr Phe Asp Asp Lys Lys Cys Arg Tyr Asp Ile65 70 75 80Ser
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Phe Gly Lys 85 90
95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val
Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly44178PRTartificiallipocalin mutein 44Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45His Lys Met Met
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asp Val Thr
Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile65 70 75 80Ser
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90
95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val
Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly45178PRTartificiallipocalin mutein 45Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro 35 40 45Asn Lys Met Met
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asp Val Thr
Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile65 70 75 80Ser
Thr Leu Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Phe Gly Lys 85 90
95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val
Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly46178PRTartificiallipocalin mutein 46Gln Asp Ser Thr Ser Asp Leu
Ile Pro Ala Pro Pro Leu Ser Lys Val1 5 10 15Pro Leu Gln Gln Asn Phe
Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30Val Val Gly Gln Ala
Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro 35 40 45Ser Lys Met Met
Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60Asp Val Thr
Ala Val Thr Phe Asp Asp Lys Lys Cys Asn Tyr Ala Ile65 70 75 80Ser
Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Lys 85 90
95Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser
100 105 110Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Phe Val
Phe Gln 115 120 125Asn Arg Glu Glu Phe Tyr Ile Thr Leu Tyr Gly Arg
Thr Lys Glu Leu 130 135 140Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg
Phe Ser Lys Ser Leu Gly145 150 155 160Leu Pro Glu Asn His Ile Val
Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175Asp
Gly47448PRTartificialreference antibody 47Gln Val Gln Leu Gln Gln
Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser Leu Thr
Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp Ser Trp
Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile
Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu 50 55 60Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp
Gly 100 105 110Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
44548216PRTartificialreference antibody 48Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala
Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro
85 90 95Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr
Val 100 105 110Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys 115 120 125Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg 130 135 140Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn145 150 155 160Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200
205Lys Ser Phe Asn Arg Gly Glu Cys 210
21549442PRTartificialreference antibody 49Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Glu1 5 10 15Ser Leu Arg Ile Ser
Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr 20 25 30Trp Ile Ser Trp
Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Lys Ile
Tyr Pro Gly Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe 50 55 60Gln Gly
Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr65 70 75
80Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95Ala Arg Gly Tyr Gly Ile Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu 130 135 140Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly145 150 155 160Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170 175Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe 180 185 190Gly Thr
Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr 195 200
205Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
210 215 220Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe
Pro Pro225 230 235 240Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 245 250 255Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Gln Phe Asn Trp 260 265 270Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285Glu Gln Phe Asn Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val 290 295 300His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn305 310 315
320Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
325 330 335Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu 340 345 350Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 355 360 365Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 370 375 380Asn Tyr Lys Thr Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe385 390 395 400Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 405 410 415Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 420 425 430Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 435
44050214PRTartificialreference antibody 50Ser Tyr Glu Leu Thr Gln
Pro Pro Ser Val Ser Val Ser Pro Gly Gln1 5 10 15Thr Ala Ser Ile Thr
Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45Gln Asp Lys
Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60Asn Ser
Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met65 70 75
80Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu
85 90 95Ala Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
Lys 100 105 110Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
Glu Leu Gln 115 120 125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser
Asp Phe Tyr Pro Gly 130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp
Ser Ser Pro Val Lys Ala Gly145 150 155 160Val Glu Thr Thr Thr Pro
Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu
Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200
205Ala Pro Thr Glu Cys Ser 21051447PRTartificialTrastuzumab-IgG4
S228P FALA (Heavy chain) 51Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr Ile His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Tyr Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ser Arg Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys
Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val225 230 235
240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360
365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys 435 440
44552214PRTartificialTrastuzumab-IgG4 S228P FALA (Light chain)
52Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys 210
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