U.S. patent application number 14/623868 was filed with the patent office on 2016-01-14 for proteolytically cleavable fusion proteins with high molar specific activity.
The applicant listed for this patent is CSL Behring GmbH. Invention is credited to Hubert Metzner, Stefan Schulte, Thomas Weimer.
Application Number | 20160010074 14/623868 |
Document ID | / |
Family ID | 46329953 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160010074 |
Kind Code |
A1 |
Metzner; Hubert ; et
al. |
January 14, 2016 |
PROTEOLYTICALLY CLEAVABLE FUSION PROTEINS WITH HIGH MOLAR SPECIFIC
ACTIVITY
Abstract
The invention relates to therapeutic fusion proteins in which a
coagulation factor is fused to a half-life enhancing polypeptide,
and in which both are connected by a linker peptide that is
proteolytically cleavable. The cleavage of such linkers liberates
the coagulation factor from activity-compromising steric hindrance
caused by the half-life enhancing polypeptide and thereby allows
the generation of fusion proteins may show relatively high molar
specific activity when tested in coagulation-related assays.
Furthermore, the fact that the linker is cleavable can enhance the
rates of inactivation and/or elimination after proteolytic cleavage
of the peptide linker compared to the rates measured for
corresponding therapeutic fusion proteins linked by the
non-cleavable linker having the amino acid sequence GGGGGGV.
Inventors: |
Metzner; Hubert; (Marburg,
DE) ; Weimer; Thomas; (Gladenbach, DE) ;
Schulte; Stefan; (Marburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CSL Behring GmbH |
Marburg |
|
DE |
|
|
Family ID: |
46329953 |
Appl. No.: |
14/623868 |
Filed: |
February 17, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13074153 |
Mar 29, 2011 |
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14623868 |
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12000739 |
Dec 17, 2007 |
7939632 |
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13074153 |
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11812016 |
Jun 14, 2007 |
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12000739 |
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60819620 |
Jul 11, 2006 |
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Current U.S.
Class: |
424/94.64 ;
435/226; 435/320.1; 435/358; 514/44R; 536/23.2 |
Current CPC
Class: |
C07K 14/76 20130101;
A61P 7/00 20180101; C07K 14/745 20130101; C07K 2319/31 20130101;
C12N 9/6424 20130101; C07K 2319/50 20130101; C12N 9/6437 20130101;
C12Y 304/21021 20130101; A61K 48/00 20130101; C07K 14/755 20130101;
C07K 14/765 20130101; A61K 38/00 20130101; C12N 9/6443 20130101;
C12Y 304/21022 20130101; C12N 9/6472 20130101; A61P 7/04
20180101 |
International
Class: |
C12N 9/64 20060101
C12N009/64; C07K 14/765 20060101 C07K014/765; C07K 14/76 20060101
C07K014/76 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 14, 2006 |
EP |
06012262.9 |
Claims
1. A fusion protein comprising: a) Factor VII (FVII), Factor VIIa
(FVIIa), or Factor IX (FIX); b) a half-life enhancing polypeptide
(HLEP), and c) a peptide linker which joins the FVII, FVIIa, or FIX
and the half-life enhancing polypeptide; wherein the HELP is an
immunoglobulin without an antigen binding domain, wherein the
peptide linker is cleavable by one or more proteases that can
activate or cleave FVII, FVIIa, or FIX during coagulation, and
wherein the fusion protein has at least one of the following
properties, in comparison to the respective fusion protein linked
by a non-cleavable linker consisting of the amino acid sequence
GGGGGGV (SEQ ID NO: 94): i) an increased molar specific activity in
at least one coagulation-related assay, ii) an increased
inactivation rate after the peptide linker is proteolytically
cleaved in a coagulation-related mode, and iii) an increased
elimination rate after the peptide linker is proteolytically
cleaved in a coagulation-related mode.
2-3. (canceled)
4. The fusion protein of claim 1, wherein said fusion protein has a
higher in vivo recovery compared to the in vivo recovery of FVII,
FVIIa, or FIX when not fused to a half-life enhancing
polypeptide.
5. The fusion protein of claim 1, wherein said fusion protein has
an increased half-life in plasma compared to the half-life in
plasma of FVII, FVIIa, or FIX when not fused to a half-life
enhancing polypeptide.
6. (canceled)
7. The fusion protein of claim 1, wherein the fusion protein
comprises FIX.
8. (canceled)
9. The fusion protein of claim 1, wherein the peptide linker is
cleavable by FXIa and/or FVIIa/TF.
10. The fusion protein of claim 1, wherein the molar specific
activity of the fusion protein is increased at least 25% compared
to that of the respective fusion protein linked by a non-cleavable
linker consisting of the amino acid sequence GGGGGGV (SEQ ID NO:
94) in at least one coagulation-related assays.
11. The fusion protein of claim 1, wherein the inactivation rate of
FVII, FVIIa, or FIX after cleavage of the peptide linker which
links the FVII, FVIIa, or FIX to the half-life enhancing
polypeptide is increased at least 10% as compared to the
inactivation rate of FVII, FVIIa, or FIX in the respective fusion
protein linked by a non-cleavable linker consisting of the amino
acid sequence GGGGGGV (SEQ ID NO: 94).
12. The fusion protein of claim 1, wherein the elimination rate of
FVII, FVIIa, or FIX after cleavage of the peptide linker which
links the FVII, FVIIa, or FIX to the half-life enhancing
polypeptide is increased by at least 10% as compared to the
elimination rate of FVII, FVIIa, or FIX in the respective fusion
protein linked by a non-cleavable linker consisting of the amino
acid sequence GGGGGGV (SEQ ID NO: 94).
13. The fusion protein of claim 1, wherein the peptide linker is
cleavable by a proteases that naturally activates FVII, FVIIa, or
FIX in vivo.
14. The fusion protein of claim 13, wherein the kinetics of the
linker cleavage by the protease is not delayed by more than a
factor of 3 compared to the kinetics of the activation of the FVII,
FVIIa, or FIX.
15. (canceled)
16. The fusion protein of claim 1, wherein the peptide linker is
cleavable by FXIa and/or by FVIIa/TF, and wherein the fusion
protein comprises FIX.
17. The fusion protein of claim 1, wherein the linker is cleavable
by FXIa and/or by FVIIa/TF, and wherein the fusion protein
comprises FVIIa.
18. The fusion protein of claim 1, wherein the linker comprises SEQ
ID NO:113 or SEQ ID NO:114.
19. A polynucleotide encoding the fusion protein of claim 1.
20. A plasmid or vector comprising the polynucleotide of claim
19.
21. The plasmid or vector of claim 20, wherein the plasmid or
vector is an expression vector.
22. The plasmid or vector of claim 20, wherein the vector is a
transfer vector for use in human gene therapy.
23. A host cell comprising a polynucleotide according to claim
19.
24. A method of producing a fusion protein of claim 1, comprising
culturing host cells comprising a polynucleotide encoding the
fusion protein under conditions such that the fusion protein is
expressed.
25. A pharmaceutical composition comprising (a) the fusion protein
of claim 1, (b) a polynucleotide encoding said fusion protein, or
(c) a plasmid or vector comprising a polynucleotide encoding said
fusion protein.
26. A method of administering an effective amount of the fusion
protein of claim 1 to a patient in need thereof, comprising (a)
administering said fusion protein, (b) administering a composition
comprising a polynucleotide encoding said fusion protein via a gene
therapy protocol, or (c) administering a composition comprising a
plasmid or vector comprising a polynucleotide encoding said fusion
protein via a gene therapy protocol.
27. The method of claim 26, wherein the patient suffers from a
blood coagulation disorder.
28. The method of claim 27, wherein the blood coagulation disorder
is hemophilia B.
29. The method of claim 27, wherein the blood coagulation disorder
is FVII and/or FVIIa deficiency.
30. The method of claim 27, wherein the blood coagulation disorder
is hemophilia A.
31. The method of claim 27, wherein the administration comprises
administering via a gene therapy protocol (a) a composition
comprising a polynucleotide encoding said fusion protein or (b) a
composition comprising a plasmid or vector comprising a
polynucleotide encoding said fusion protein.
32. The method according to claim 27, wherein the fusion protein is
effective to act in the patient as a procoagulant.
33. The fusion protein of claim 1, wherein the FVII, FVIIa, or FIX
or immunoglobulin, comprises a sequence that is 95% identical to
the sequence of a wild-type human FVII, FVIIa, or FIX or a
wild-type human immunoglobulin, respectively.
34. The method of claim 24, further comprising recovering the
fusion protein from the host cells or from the culture medium.
35. The method of claim 27, wherein the administration comprises
administering a composition comprising the fusion protein of claim
1.
36. The fusion protein of claim 1, wherein the linker is cleavable
by FVII, FVIIa, or FIX during coagulation.
37. The fusion protein of claim 1, wherein the linker comprises a
sequence from FVII, FVIIa, or FIX.
38. The fusion protein of claim 1, wherein the linker is capable of
being cleaved simultaneously with the activation of FVII, FVIIa, or
FIX, or with comparable kinetics, during coagulation.
39. The fusion protein of claim 1, wherein the fusion protein
comprises FVIIa.
Description
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 11/812,016, filed Jun. 14, 2007, which claims
priority to U.S. Provisional Application No. 60/819,620, filed Jul.
11, 2006. The application also claims priority to European Patent
Application No. 06012262.9, filed Jun. 14, 2006.
[0002] The present invention relates to the field of modified
therapeutic fusion proteins with increased half-life compared to
their non-modified parent therapeutic polypeptides. The invention,
for example, relates to coagulation factors fused to half-life
enhancing polypeptides (HLEPs), which are connected by linker
peptides that are proteolytically cleavable. The cleavage of such
linkers liberates the therapeutic polypeptide from
activity-compromising steric hindrance caused by the HLEP and
thereby allows the generation of fusion proteins, which retain a
high molar specific activity of the coagulation factor. When the
therapeutic fusion proteins are zymogens, one may use linkers that
liberate the therapeutic polypeptide essentially simultaneously
with its activation in vivo upon exposure to the corresponding
protease(s). Embodiments of the present invention may show a faster
inactivation rate of a given coagulation factor once the
coagulation factor is activated and the peptide linker is
proteolytically cleaved in a coagulation-related mode and/or a
faster elimination rate of a given coagulation factor once the
coagulation factor is activated and the peptide linker is
proteolytically cleaved in a coagulation-related mode, compared to
the corresponding fusion protein without cleavable linker.
[0003] One aspect of the invention is demonstrated for example by
human vitamin K-dependent polypeptides Factor IX, Factor VII, and
Factor VIIa. The same concept also may be applied to other
coagulation factors. Any half-life enhancing polypeptide (HLEP) may
be connected to the therapeutic polypeptide by a cleavable linker
peptide. For instance albumin or immunoglobulins or fragments
derived therefrom without an antigen binding domain, such as the Fc
fragment, may serve as HLEPs. The invention also relates to cDNA
sequences coding for the therapeutic polypeptides and derivatives
thereof genetically fused to a cDNA coding for HLEPs, such as human
serum albumin linked by oligonucleotides that code for cleavable,
intervening peptide linkers. Such encoded derivatives may exhibit
improved half-life and molar specific activities that are increased
in comparison to their non-cleavable counterparts. The invention
also relates to recombinant expression vectors containing such cDNA
sequences, host cells transformed with such recombinant expression
vectors, recombinant polypeptides and derivatives which may have
biological activities comparable to the unmodified wild type
therapeutic polypeptide, but having improved half-lifes. The
invention also relates to processes for the manufacture of such
recombinant proteins and their derivatives. The invention also
relates to a transfer vector for use in human gene therapy, which
comprises such modified DNA sequences, which can be useful to
increase half-life in vivo.
[0004] Several recombinant, therapeutic polypeptides are
commercially available for therapeutic and prophylactic use in
humans. The patients in general may benefit from the specific mode
of action of the recombinant active ingredients but a disadvantage
may be their limited availability due to expensive and complex
manufacturing processes. A reduction of the necessary dose or the
frequency of administration of such products could improve this
situation. A reduced frequency of administration could improve the
convenience for the patient and, therefore, also the acceptance of
the therapy. Several ideas have been described to attempt to
achieve the goal of an increased in vivo half-life after
administration. See, e.g. Ballance et al. (WO 01/79271), Sheffield
et al. (Sheffield W. P. et al. (2004), Br. J. Haematol. 126:
565-573), WO 2002/04598, WO 2003/059935, WO 2004/081053, WO
2004/101740, WO 2005/001025, WO 91/09125, and WO 03/068934. Fusions
of coagulation factors to half-life enhancing polypeptides have
been suggested to lengthen the half-life of coagulation factors
administered to patients. However, once a coagulation factor is
activated during coagulation either by proteolytic cleavage of the
zymogen (like FIX) or by contact of an already proteolytically
"pre"-activated factor to a second polypeptide (like FVIIa binding
to Tissue Factor), it may no longer be desirable to maintain the
long half-life of the now activated coagulation factor, as this
might lead to thrombotic complications. This is the case for a wild
type coagulation factor such as FVIIa (Aledort L. M., J Thromb
Haemost 2(10): 1700-1708 (2004)) and may be even more relevant if
the activated factor has an increased half-life. It is therefore
one objective of some embodiments of the present invention to
provide long-lived coagulation factors, which after activation or
after availability of a cofactor have a half-life comparable to
that of an unmodified coagulation factor.
[0005] Fusions of the coagulation factors to half-life enhancing
polypeptides as described in the prior art and as also shown in
examples 6 and 7 may suffer in general from a reduced molar
specific activity of the fused coagulation factor. Another aspect
of the present invention is to provide coagulation factors with
enhanced half-life that show increased molar specific activity
compared to the corresponding therapeutic fusion protein without a
cleavable linker.
[0006] Some embodiments of this invention include therapeutic
fusion proteins comprising: [0007] a) a coagulation factor, [0008]
b) a half-life enhancing polypeptide (HLEP) chosen from albuminand
immunoglobulin, and [0009] c) a peptide linker, which linker joins
the coagulation factor and the half-life enhancing polypeptide;
[0010] wherein the peptide linker is cleavable by proteases
involved in coagulation or proteases activated by coagulation
enzymes, and wherein the therapeutic fusion protein has, in
comparison to the respective therapeutic fusion protein linked by a
non-cleavable linker having the amino acid sequence GGGGGGV, at
least one of the following properties: [0011] i) an increased molar
specific activity in at least one coagulation-related assay, [0012]
ii) an increased inactivation rate of the activated coagulation
factor after the peptide linker is proteolytically cleaved in a
coagulation-related mode, and [0013] iii) an increased elimination
rate of the activated coagulation factor after the peptide linker
is proteolytically cleaved in a coagulation-related mode.
[0014] A "coagulation factor," as used herein, includes variants or
derivatives thereof, such as genetically engineered or chemically
modified variants or active fragments thereof. See below for
additional description of examples of "coagulation factors."
[0015] "Albumin," as used herein, includes polypeptides of the
albumin family of proteins such as human serum albumin and bovine
serum albumin, including variants and derivatives thereof, such as
genetically engineered or chemically modified albumin variants and
fragments of albumin proteins. See below for additional description
of examples of "albumin."
[0016] "Immunoglobulin," as used herein, includes variants and
derivatives of immunoglobulin proteins, such as genetically
engineered or chemically modified immunoglobulin variants and
fragments of immunoglobulin, for example, an Fc fragment or other
fragment not containing an antigen binding domain. See below for
additional description of examples of "immunoglobulin."
[0017] As a consequence of the cleavable linker, after cleavage of
the peptide linker in a coagulation-related mode the coagulation
factor more closely resembles the behaviour of the native,
non-fused factor and does not show an increased half-life of the
active factor with potentially prothrombotic effect.
[0018] "Proteolytic cleavage in a coagulation-related mode," as
used herein, means any proteolytic cleavage that occurs as a
consequence of the activation of at least one coagulation factor or
coagulation cofactor.
[0019] The phrase "activated coagulation factor after the peptide
linker is proteolytically cleaved in a coagulation-related mode,"
as used herein means that the coagulation factor is either
activated almost in parallel to the proteolytic cleavage of the
linker peptide, or that the coagulation factor was already
activated before the proteolytic cleavage of the linker peptide.
Activation may occur, for example by proteolytic cleavage of the
coagulation factor or by binding to a cofactor.
[0020] A further aspect of the present invention is to provide
therapeutic fusion proteins comprising: [0021] a) a coagulation
factor, [0022] b) a half-life enhancing polypeptide (HLEP) chosen
from albumin and immunoglobulin, and [0023] c) a peptide linker
which joins the coagulation factor and the half-life enhancing
polypeptide;
[0024] wherein the peptide linker is cleavable by proteases
involved in coagulation or activated by coagulation enzymes, and
wherein the therapeutic fusion protein has, in comparison to the
respective therapeutic fusion protein linked by a non-cleavable
linker having the amino acid sequence GGGGGGV, at least one of the
following properties: [0025] i) an increased molar specific
activity in at least one coagulation-related assay, [0026] ii) an
increased inactivation rate of the activated coagulation factor
after the peptide linker is proteolytically cleaved in a
coagulation-related mode, [0027] iii) an increased elimination rate
of the activated coagulation factor after the peptide linker is
proteolytically cleaved in a coagulation-related mode, and [0028]
iv) an enhanced in vivo recovery as compared to the in vivo
recovery of the unmodified coagulation factor.
[0029] Some embodiments include therapeutic fusion proteins which
have an enhanced in vivo recovery compared to the unmodified
coagulation factor by at least 10%, for example, by at least 25% or
by 40% or more.
[0030] Exemplary coagulation factors are vitamin-K dependent
coagulation factors, FVIIa, and FIX, and fragments and variants
thereof, such as genetically engineered or chemically modified
variants or fragments, such as are described in more detail
below.
[0031] HLEPs may be albumin and fragments or variants thereof and
immunoglobulins including fragments and variants thereof, as
described above, and in more detail, below.
[0032] The peptide linker in some embodiments may comprise a
sequence of the therapeutic polypeptide to be administered or a
variant thereof, which should result in a decreased risk of
neoantigenic properties (formation of a novel potentially
immunogenic epitope due to the occurrence of a peptide within the
therapeutic antigen which does not exist in human proteins) of the
expressed fusion protein. Also in case the therapeutic protein is a
zymogen (e.g. needs to be proteolytically activated) the kinetics
of the peptide linker cleavage may more closely reflect the
coagulation-related activation kinetics of the zymogen. Thus, in
some embodiments, a zymogen and a corresponding peptide linker are
activated and respectively cleaved, with comparable kinetics. For
this reason, embodiments of the present invention also relate to
fusion proteins of a zymogen and a HLEP, where the kinetics of the
linker cleavage by relevant proteases are not delayed by more than
a factor of 3, such as not by more than a factor of 2, compared to
the kinetics of the zymogen activation.
[0033] In another embodiment, the peptide linker comprises cleavage
sites for more than one protease. This can be achieved, for
example, by a peptide linker that can be cleaved at the same
position by different proteases or by a peptide linker that
provides two or more different cleavage sites. This may be
advantageous circumstances where the therapeutic fusion protein
must be activated by proteolytic cleavage to achieve enzymatic
activity and where different proteases may contribute to this
activation step. This is the case, for example, upon activation of
FIX, which can either be achieved by FXIa or by FVIIa/Tissue Factor
(TF).
[0034] Some embodiments of the invention are therapeutic fusion
proteins wherein the peptide linker is cleavable by the protease
that normally activates the coagulation factor in vivo, thereby
ensuring that the cleavage of the linker is linked to the
activation of the coagulation factor at a site at which coagulation
occurs.
[0035] Other exemplary therapeutic fusion proteins according to the
invention are those wherein the linker is cleavable by the
coagulation factor which is part of the therapeutic fusion protein
once it is activated, thus also ensuring that cleavage of the
fusion protein is connected with a coagulatory event.
[0036] Other exemplary therapeutic fusion proteins according to the
invention are those wherein the linker is cleavable by a protease,
which itself is activated directly or indirectly by the activity of
the coagulation factor which is part of the therapeutic fusion
protein, thus also ensuring that cleavage of the fusion protein is
connected with a coagulatory event.
[0037] One class of therapeutic fusion proteins included in this
invention comprises those wherein the linker is cleavable by FXIa
and/or by FVIIa/TF and the coagulation factor is FIX.
[0038] For example, embodiments of the invention include fusion
proteins comprising the vitamin K-dependent polypeptide Factor IX,
cleavable linkers, and albumin as the HLEP, as well as their
corresponding cDNA sequences. The invention also relates to cDNA
sequences coding for any other coagulation factors which can be
proteolytically activated or that are involved in the activation of
other zymogens or polypeptides. In some embodiments, these cDNAs
are genetically fused to cDNA sequences coding for human serum
albumin or other HLEPs, and are linked by oligonucleotides that
code for intervening, cleavable peptide linkers. The expressed
therapeutic fusion proteins may exhibit molar specific activities
which are increased in comparison to their non-cleavable
counterparts. The invention also relates to recombinant expression
vectors containing such fused cDNA sequences, host cells
transformed with such recombinant expression vectors, recombinant
therapeutic fusion proteins and derivatives that may have
biological activities almost comparable to the unmodified wild type
therapeutic polypeptides but having improved in vivo half-life. The
invention also relates to processes for the manufacture of the
recombinant polypeptides of the invention and their derivatives.
The invention also relates to a transfer vector for use in human
gene therapy, which comprises such modified DNA sequences useful to
increase product levels in vivo.
[0039] Some therapeutic fusion proteins according to the invention
have a molar specific activity, in particular a molar specific
activity in at least one coagulation-related assay, that is at
least 25% increased compared to that of the corresponding
therapeutic fusion protein without a cleavable linker. Other
therapeutic fusion proteins have a molar specific activity that is
increased by at least 50%, or by at least 100%, in at least one of
the different coagulation-related assays available, compared to
that of the corresponding therapeutic fusion protein without a
cleavable linker, such as one with the non-cleavable sequence
GGGGGGV.
[0040] Additional embodiments of the present invention are
therapeutic fusion proteins wherein the inactivation rate of the
activated coagulation factor after cleavage of the peptide linker
which links the coagulation factor to the half-life enhancing
polypeptide is increased by at least 10% as compared to the
inactivation rate of the activated coagulation factor in a
corresponding therapeutic fusion protein without a cleavable
linker. Other embodiments are therapeutic fusion proteins in which
the inactivation rate is increased by at least 25%, or by at least
50% as compared to the inactivation rate of the activated
coagulation factor in a corresponding therapeutic fusion protein
without a cleavable linker, such as one with the non-cleavable
sequence GGGGGGV.
[0041] Additional embodiments of the present invention are
therapeutic fusion proteins wherein the elimination rate of the
coagulation factor after cleavage of the peptide linker that links
the coagulation factor to the half-life enhancing polypeptide is
increased by at least 10% as compared to the elimination rate of
the coagulation factor in a corresponding therapeutic fusion
protein without a cleavable linker. Other embodiments are
therapeutic fusion proteins in which the elimination rate is
increased by at least 25%, or by at least 50% as compared to the
elimination rate of the coagulation factor in a corresponding
therapeutic fusion protein without a cleavable linker, such as one
with the non-cleavable sequence GGGGGGV.
Vitamin K-Dependent Polypeptides
[0042] Vitamin K-dependent polypeptides as one group of the
therapeutic polypeptides are polypeptides that are
.gamma.-carboxylated enzymatically in the liver using vitamin K as
a cofactor. Such vitamin K-dependent polypeptides e.g. include
Factors II, VII, IX, X, Protein C, Protein S, GAS6, and Protein
Z.
Human FIX
[0043] Human FIX, one member of the group of vitamin K-dependent
polypeptides, is a single-chain glycoprotein with a molecular
weight of 57 kDa, which is secreted by liver cells into the blood
stream as an inactive zymogen of 415 amino acids. It contains 12
.gamma.-carboxy-glutamic acid residues localized in the N-terminal
Gla-domain of the polypeptide. The Gla residues require vitamin K
for their biosynthesis. Following the Gla domain there are two
epidermal growth factor domains, an activation peptide, and a
trypsin-type serine protease domain. Further posttranslational
modifications of FIX encompass hydroxylation (Asp 64), N-(Asn157
and Asn167) as well as 0-type glycosylation (Ser53, Ser61, Thr159,
Thr169, and Thr172), sulfation (Tyr155), and phosphorylation
(Ser158). FIX is converted to its active form, Factor IXa, by
proteolysis of the activation peptide at Arg145-Ala146 and
Arg180-Val181 leading to the formation of two polypeptide chains,
an N-terminal light chain (18 kDa) and a C-terminal heavy chain (28
kDa), which are held together by one disulfide bridge. Activation
cleavage of Factor IX can be achieved in vitro e.g. by Factor XIa
or Factor VIIa/TF. Factor IX is present in human plasma in a
concentration of 5-10 .mu.g/ml. Terminal plasma half-life of Factor
IX in humans was found to be about 15 to 18 hours (White G C et al.
1997. Recombinant factor IX. Thromb Haemost. 78: 261-265; Ewenstein
B M et al. 2002. Pharmacokinetic analysis of plasma-derived and
recombinant F IX concentrates in previously treated patients with
moderate or severe hemophilia B. Transfusion 42:190-197).
[0044] Hemophilia B is caused by non-functional or missing Factor
IX and is treated with Factor IX concentrates from plasma or a
recombinant form of Factor IX. As haemophilia B patients often
receive at least biweekly prophylactic administrations of Factor IX
to avoid spontaneous bleedings, it is desirable to increase the
intervals of time between administrations by increasing the
half-life of the Factor IX product applied. An improvement in
plasma half-life may bring significant benefit to the patient. Up
to now no pharmaceutical preparation of a Factor IX with improved
plasma half-life is commercially available nor have any data been
published showing F IX variants with prolonged in vivo half-life
and almost unchanged molar specific activity in coagulation-related
assays. Therefore, a great medical need still exists to develop
forms of Factor IX which have a longer functional half-life in
vivo.
Factor VII and Factor VIIa
[0045] FVII is a single-chain glycoprotein with a molecular weight
of 50 kDa, which is secreted by liver cells into the blood stream
as an inactive zymogen of 406 amino acids. FVII is converted to its
active form Factor VIIa, by proteolysis of the single peptide bond
at Arg152-Ile153 leading to the formation of two polypeptide
chains, a N-terminal light chain (24 kDa) and a C-terminal heavy
chain (28 kDa), which are held together by one disulfide bridge. In
contrast to other vitamin K-dependent coagulation factors, no
activation peptide is cleaved off during activation. Activation
cleavage of Factor VII can be achieved in vitro, for example, by
Factor Xa, Factor IXa, Factor VIIa, Factor XIIa, Factor Seven
Activating Protease (FSAP), and thrombin. Mollerup et al.
(Biotechnol. Bioeng. (1995) 48: 501-505) reported that some
cleavage also occurs in the heavy chain at Arg290 and/or
Arg315.
[0046] Factor VII is present in plasma in a concentration of 500
ng/ml. About 1% or 5 ng/ml of Factor VII is present as activated
Factor VIIa. The terminal plasma half-life of Factor VII was found
to be about 4 hours and that of Factor VIIa about 2 hours.
[0047] By administering supraphysiological concentrations of Factor
VIIa hemostasis can be achieved bypassing the need for Factor Villa
and Factor IXa. The cloning of the cDNA for Factor VII (U.S. Pat.
No. 4,784,950) made it possible to develop activated Factor VII as
a pharmaceutical. Factor VIIa was successfully administered for the
first time in 1988. Ever since the number of indications of Factor
VIIa has grown steadily showing a potential to become an universal
hemostatic agent to stop bleeding (Erhardtsen, 2002). However, the
short terminal half-life of Factor VIIa of approximately 2 hours
and reduced in vivo recovery may be limiting its application.
Therefore, a great medical need still exists to develop forms of
Factor VIIa which have an improved half-life but otherwise almost
uncompromised molar specific activity, inactivation kinetics,
and/or elimination kinetics after start of coagulation.
Therapeutic Fusion Proteins
[0048] "Therapeutic fusion proteins" or "fusion proteins" as used
herein are coagulation factors fused to a half-life enhancing
polypeptide such that, upon administration to a human or other
animal, may produce a prophylactic or therapeutic effect. These
therapeutic fusion proteins may be administered to a human or other
animal via, for example, intravenous, intramuscular, subcutaneous,
oral, topical, parenteral or other routes. In addition, gene
therapy protocols may be used which involve administration of a
polynucleotide encoding the fusion protein or a composition
comprising that polynucleotide, such as a plasmid or vector or host
cell. Specific classes of therapeutic fusion proteins described,
i.e. by the examples below, are coagulation factors, such as
vitamin K-dependent polypeptides, linked to half-life enhancing
polypeptides, such as albumin and immunoglobulin. The expression
"therapeutic fusion protein" is used interchangeably with "fusion
protein".
Half-Life Enhancing Polypeptide (HLEP)
[0049] Albumin and immunoglobulin have been described above as
examples of half-life enhancing polypeptides (HLEPs). The terms
"human serum albumin" (HSA) and "human albumin" (HA) are used
interchangeably in this application. The terms "albumin" and "serum
albumin" are broader, and encompass human serum albumin as well as
albumin from other species, and fragments and variants thereof.
"Albumin," as used herein, includes polypeptides of the albumin
family of proteins. As used herein, "albumin" refers collectively
to albumin polypeptide or amino acid sequence, or an albumin
fragment or variant having one or more functional activities (e.g.,
biological activities) of albumin. Examples include human serum
albumin and bovine serum albumin, including variants and
derivatives thereof, such as genetically engineered or chemically
modified albumin variants and fragments of albumin proteins. For
example, "albumin" refers to human albumin or fragments thereof,
such as the mature form of human albumin as shown in SEQ ID NO:1
herein or albumin from other vertebrates or fragments thereof, or
analogs or variants of these molecules or fragments thereof.
[0050] The albumin portion of the albumin fusion proteins may
comprise the full length of, for instance, the HA sequence as
described above, or may include one or more fragments thereof that
are capable of stabilizing or prolonging the therapeutic activity.
Such fragments may be of 10 or more amino acids in length or may
include about 15, 20, 25, 30, 50, or more contiguous amino acids
from the HA sequence or may include part or all of specific domains
of HA.
[0051] The albumin portion of the albumin fusion proteins of the
invention may comprise a variant or derivative or analog of normal
HA, either natural or artificial. The therapeutic polypeptide
portion of the fusion proteins of the invention may also comprise
variants of the corresponding therapeutic polypeptides as described
herein.
[0052] The terms "variants" "derivatives" and "analogs", throughout
this application, when applied to any protein disclosed herein,
each include, for example, insertions, deletions, and
substitutions, either conservative or non-conservative, either
natural or artificial (i.e. engineered), where such changes do not
substantially alter the active site, or a fragment, such as an
active domain that confers the therapeutic activities of the
therapeutic polypeptides, or chemical modifications that also allow
for therapeutic activity. Examples include, for instance
coagulation factors, albumin, or immunoglobulins that are 80%, 85%,
90%, and 95% identical in sequence to a wild-type, human
coagulation factor, albumin, or immunoglobulin sequence.
[0053] In particular, the albumin fusion proteins of the invention
may include naturally occurring polymorphic variants of human
albumin and fragments of human albumin. The albumin may be derived
from any vertebrate, especially any mammal, for example human, cow,
sheep, or pig. Non-mammalian albumins include, but are not limited
to, hen and salmon. The albumin portion of the albumin-linked
polypeptide may be from a different animal than the therapeutic
polypeptide portion.
[0054] Generally speaking, an albumin fragment or variant
encompassed within the term "albumin" will be at least 10, for
example at least 40, or more than 70 amino acids long. That albumin
variant may comprise at least one whole domain of albumin or
fragments of said domains, for example domains 1 (amino acids 1-194
of SEQ ID NO:1), 2 (amino acids 195-387 of SEQ ID NO:1), 3 (amino
acids 388-585 of SEQ ID NO:1), 1+2 (1-387 of SEQ ID NO:1), 2+3
(195-585 of SEQ ID NO:1) or 1+3 (amino acids 1-194 of SEQ ID
NO:1+amino acids 388-585 of SEQ ID NO:1). Each domain is itself
made up of two homologous subdomains namely 1-105, 120-194,
195-291, 316-387, 388-491 and 512-585, with flexible
inter-subdomain linker regions comprising residues Lys106 to
Glu119, Glu292 to Val315, and Glu492 to Ala511.
[0055] The albumin portion of an albumin fusion protein of the
invention may comprise at least one subdomain or domain of HA or
conservative modifications thereof.
[0056] All fragments and variants of albumin are encompassed by the
invention as fusion partners of a coagulation factor as long as
they lead to a half-life extension of the therapeutic fusion
protein in plasma of at least 25% as compared to the non-fused
coagulation factor.
[0057] The albumin family of proteins, included within the term
"albumin" used herein, comprise evolutionarily related serum
transport proteins, for example, albumin, alpha-fetoprotein (AFP;
Beattie & Dugaiczyk 1982. Gene 20:415-422), afamin (AFM;
Lichenstein et al. 1994. J. Biol. Chem. 269:18149-18154) and
vitamin D binding protein (DBP; Cooke & David 1985. J. Clin.
Invest. 76:2420-2424). Alpha-fetoprotein has been claimed to
enhance the half-life of an attached therapeutic polypeptide in
vivo (WO 2005/024044). Their genes represent a multigene cluster
with structural and functional similarities mapping to the same
chromosomal region in humans, mice and rat. The structural
similarity of those albumin family members suggests their usability
as HLEPs. Some embodiments of the invention, therefore, may use
such albumin family members, or fragments and variants thereof as
defined above, as HLEPs.
[0058] Albumin family members encompassed within the term "albumin"
herein also comprise the full length of the respective proteins
AFP, AFM and DBP, or may include one or more fragments thereof that
are capable of stabilizing or prolonging the therapeutic activity.
Such fragments may be of 10 or more amino acids in length or may
include about 15, 20, 25, 30, 50, or more contiguous amino acids of
the respective protein sequence or may include part or all of
specific domains of the respective protein, as long as the HLEP
fragments provide a half-life extension of at least 25% as compared
to the non-fused coagulation factor. Albumin family members of the
therapeutic fusion proteins of the invention may also include
naturally occurring polymorphic variants of AFP, AFM and DBP.
[0059] The term "immunoglobulin" as used herein, encompasses, for
instance, IgG and IgG-fragments, which may also be used as HLEPs,
as long as the corresponding HLEP fragments provide a half-life
extension of at least 25% as compared to the non-fused coagulation
factor. The therapeutic polypeptide portion may be connected to the
IgG or the IgG fragments via a cleavable linker that allows high
molar specific activities of the fusion protein. For example,
fusion proteins comprising FVII or VIIa and IgG or fragments
thereof may be prepared. A linker sequence of the present invention
liberating FVII (FVIIa) molecules upon cleavage by a protease of
the coagulation cascade such as, e.g., FXIa, FXa, or FIXa could be
able to elevate the clotting activity of the constructs to an
activity level comparable to the monomer/dimer or even higher. A
FIX-Fc fusion protein with cleavable linker is exemplarily shown in
SEQ ID NO:93. Cleavable linkers such as those shown in table 3a and
3b may also be applied in such embodiments.
[0060] The invention also relates to fusion proteins comprising
linking a coagulation factor, including, for example, a fragment or
variant thereof, to the N- or C-terminus of a HLEP, including a
fragment or variant thereof, such that an intervening cleavable
peptide linker is introduced between the therapeutic polypeptide
and the HLEP such that the fusion protein formed has an increased
in vivo half-life compared to the coagulation factor which has not
been linked to a HLEP and that the fusion protein has an at least
25% higher molar specific activity compared to the corresponding
fusion protein with non-cleavable linker in at least one of the
different coagulation-related assays available.
[0061] "Coagulation factor" as used in this application includes,
but is not limited to, polypeptides consisting of Factor IX, Factor
VII, Factor VIII, von Willebrand Factor, Factor V, Factor X, Factor
XI, Factor XII, Factor XIII, Factor I, Factor II (Prothrombin),
Protein C, Protein S, GAS6, or Protein Z as well as their activated
forms. A "coagulation factor," as used herein, also includes
variants or derivatives thereof, such as genetically engineered or
chemically modified variants or active fragments thereof. For
instance, useful therapeutic polypeptides may be wild-type
polypeptides or may contain mutations. Degree and location of
glycosylation or other post-translation modifications may vary
depending on the chosen host cells and the nature of the host
cellular environment. When referring to specific amino acid
sequences, posttranslational modifications of such sequences are
encompassed in this application.
[0062] "Coagulation factor" within the above definition includes
polypeptides that have the natural amino acid sequence including
any natural polymorphisms. It also includes polypeptides with a
slightly modified amino acid sequence, for instance, a modified
N-terminal or C-terminal end including terminal amino acid
deletions or additions, as long as those polypeptides substantially
retain the activity of the respective therapeutic polypeptide.
Variants included may differ in one or more amino acid residues
from the wild type sequence. Examples of such differences may
include truncation of the N- and/or C-terminus by one or more amino
acid residues (e.g. 1 to 30 amino acid residues), or addition of
one or more extra residues at the N- and/or C-terminus, as well as
conservative amino acid substitutions, i.e. substitutions performed
within groups of amino acids with similar characteristics, e.g. (1)
small amino acids, (2) acidic amino acids, (3) polar amino acids,
(4) basic amino acids, (5) hydrophobic amino acids, and (6)
aromatic amino acids. Examples of such conservative substitutions
are shown in the following table.
TABLE-US-00001 TABLE 1 (1) Alanine Glycine (2) Aspartic acid
Glutamic acid (3a) Asparagine Glutamine (3b) Serine Threonine (4)
Arginine Histidine Lysine (5) Isoleucine Leucine Methionine Valine
(6) Phenylalanine Tyrosine Tryptophane
[0063] The in vivo half-life of the fusion proteins of the
invention, in general determined as terminal half-life or
.beta.-half-life, may be at least about 25%, at least about 50%, or
more than 100% higher than the in vivo half-life of the non-fused
polypeptide.
[0064] The fusion proteins of the present invention also have at
least a 25%, and may have at least a 50% or at least 100% increased
molar specific activity compared to the corresponding fusion
proteins without cleavable linkers.
[0065] The "molar specific activity" (or molar specific
coagulation-related activity as considered here in particular) in
this regard is defined for purposes herein as the activity
expressed per mole (or e.g. nmole) of the therapeutic polypeptide
or therapeutic fusion protein of interest. Calculation of the molar
specific activity allows a direct comparison of the activity of the
different constructs that is not affected by the different
molecular weights or optical densities of the polypeptides studied.
The molar specific activity may be calculated as exemplified in
table 2 below for FIX and a FIX-HSA fusion protein.
TABLE-US-00002 TABLE 2 Calculation of molar specific activity as
shown for a purified FIX-HSA fusion protein Molar optical
Calculation of molar Activity/Vol/OD.sub.280 density (OD.sub.(280)
specific activity Product OD.sub.(280 nm. 1%) MW (IU/L/OD.sub.280)
at 1 mol/L) (IU/mol) FIX 13.3.sup.1) 57 000 determined for 75810
(=MW .times. =(Activity/Vol/OD.sub.280) .times. product
OD.sub.(280. 1%)/10) (OD.sub.280 at 1 mol/L) HSA 5.7.sup.2) 66 300
37791 (=MW .times. OD.sub.(280. 1%)/10) FIX-HSA determined for
113601 (=sum =(Activity/Vol/OD.sub.280) .times. product of molar
optical (OD.sub.280 at 1 mol/L) density of FIX and HSA) .sup.1)R.
G. Di Scipio et al., Biochem. 16: 698-706 (1977) .sup.2)C.
Chaudhury et al, J. Exp. Med. 197(3): 315-322 (2003)
[0066] In order to determine a molar specific coagulation-related
activity, any assay may be used that determines enzymatic or
cofactor activities that are relevant to the coagulation
process.
[0067] Therefore "coagulation-related assays" in the sense of the
invention comprise any assay which determines enzymatic or cofactor
activities that are of relevance in the coagulation process or that
are able to determine that either the intrinsic or the extrinsic
coagulation cascade has been activated. The "coagulation-related"
assay thus may be direct coagulation assays like aPTT, PT, or the
thrombin generation assays. However, other assays such as
chromogenic assays applied for specific coagulation factors are
also included. Examples of such assays or corresponding reagents
are Pathromtin.RTM. SL (aPTT assay, Dade Behring) or
Thromborel.RTM. S (Prothrombin time assay, Dade Behring) with
corresponding coagulation factor deficient plasma (Dade Behring),
Thrombin generation assay kits (Technoclone.TM.,
Thrombinoscope.TM.) using e.g. coagulation factor deficient plasma,
chromogenic assays like Biophen.TM. Factor IX (Hyphen BioMed),
Staclot.RTM. FVIIa-rTF (Roche Diagnostics GmbH), Coatest.RTM.
Factor VIII:C/4 (Chromogenix), or others.
[0068] For purposes of this invention, an increase in any one of
the above assays or an equivalent coagulation-related assay is
considered to show an increase in molar specific activity. For
example, a 25% increase refers to a 25% increase in any of the
above or an equivalent assay.
[0069] To determine whether therapeutic fusion proteins fall within
the scope of the present invention, the standard against which the
molar specific activity of these proteins is compared is a
construct in which the respective coagulation factor and the
respective HLEP are linked by a non-cleavable linker having the
amino acid sequence GGGGGGV.
[0070] In the case of FIX, aPTT assays are often used for
determination of coagulation activity. Such a coagulation assay
(aPTT assay) is described in example 5 in more detail. However,
other coagulation-related assays or assay principles may be applied
to determine molar specific activity for FIX.
[0071] Recombinant therapeutic polypeptide drugs are usually
expensive and not all countries can afford costly therapies based
on such drugs. Increasing the in vivo recovery of such drugs could
make the use of these products cheaper and subsequently more
patients would benefit from them. In the case of the fusion
proteins of the present invention an increased in vivo recovery
would also be a desirable advantage. "In vivo recovery" as used
herein in the sense of the invention means the amount of product
found in blood or plasma shortly after administration of the
product. For detection of the in vivo recovery in general the
plasma content is determined a few minutes (e.g. 5 or 15 min) after
administration of the product.
[0072] Although it is desirable to have a high in vivo recovery and
a long half-life for a non-activated coagulation factor, it is
advantageous to limit the half-life of a coagulation factor after
its activation or the activation of its co-factor in order to avoid
a prothrombotic risk. Therefore, after the coagulation process has
been initiated, the half-life of the active coagulation factor
should again be reduced. This can either be achieved by enhancing
inactivation in a coagulation-related mode or by elimination of the
coagulation factor.
[0073] "Inactivation" according to the present invention means the
decrease of activity of the therapeutic polypeptide which can be
caused, for example, by a complex formation of a coagulation factor
and an inhibitor of the corresponding coagulation factor or by
further proteolytic cleavage as known, e.g., in the case of FVIII
and FV.
[0074] The "inactivation rate" of an activated therapeutic fusion
protein is defined as the rate the activity is declining, e.g., by
reaction with inhibitors or by proteolytic inactivation. The
inactivation rate may be measured by following the molar specific
activity of the activated coagulation factor over time in the
presence of physiologic amounts of inhibitors of this coagulation
factor. Alternatively, the inactivation rate may be determined
after administration of the activated product to an animal followed
by testing of plasma samples at an appropriate time frame using
activity and antigen assays.
[0075] To determine whether therapeutic fusion proteins fall within
the scope of the present invention, the standard against which the
molar specific activity of these proteins is compared is a
construct in which the respective coagulation factor and the
respective HLEP are linked by a non-cleavable linker having the
amino acid sequence GGGGGGV.
[0076] The "elimination rate" of an activated therapeutic fusion
protein is defined as the rate the polypeptide is eliminated from
the circulation of humans or other animals. The elimination rate
may be determined by measuring the pharmacokinetics of the
activated, therapeutic fusion protein after intravenous
administration. Using an antigen assay, the elimination by direct
removal from the circulation can be determined. Using an activity
assay in addition, a specific removal and inactivation rate may be
determined.
[0077] To determine whether therapeutic fusion proteins fall within
the scope of the present invention, the standard against which the
molar specific activity of these proteins is compared is a
construct in which the respective coagulation factor and the
respective HLEP are linked by a non-cleavable linker having the
amino acid sequence GGGGGGV.
[0078] According to some embodiments of this invention, the
therapeutic polypeptide moiety is coupled to the HLEP moiety by a
cleavable peptide linker. The linker should be non-immunogenic and
should be flexible enough to allow cleavage by proteases. The
cleavage of the linker should proceed comparably fast as the
activation of the therapeutic polypeptide within the fusion
protein, if the fusion protein is a zymogen.
[0079] The cleavable linker may comprise a sequence derived from
[0080] a) the therapeutic polypeptide to be administered itself if
it contains proteolytic cleavage sites that are proteolytically
cleaved during activation of the therapeutic polypeptide, [0081] b)
a substrate polypeptide of this therapeutic polypeptide, or [0082]
c) a substrate polypeptide cleaved by a protease which is activated
or formed by the direct or indirect involvement of the therapeutic
polypeptide.
[0083] The peptide linker in some embodiments comprises a sequence
of the therapeutic polypeptide to be applied, which should result
in a decreased risk of neoantigenic properties of the expressed
fusion protein. Also in case the therapeutic protein is a zymogen
(e.g. needs to be proteolytically activated) the kinetics of the
peptide linker cleavage will more closely reflect the
coagulation-related activation kinetics of the zymogen in some
embodiments.
[0084] In some embodiments, the therapeutic polypeptide is FIX
zymogen and the HLEP is albumin. In some of those embodiments the
linker sequence is either derived from the sequences of the
activation regions of FIX, from the cleavage region of any
substrate of FIX like FX or FVII or from the cleavage region of any
substrate polypeptide that is cleaved by a protease in whose
activation FIXa is involved.
[0085] In yet other embodiments, the linker peptide is derived from
FIX itself. In other embodiments the linker peptide is derived from
FX or FVII. In other embodiments the linker sequence comprises two
cleavage sequences that can be cleaved by FXIa or FVIIa/TF, two
physiologically relevant activators of FIX.
[0086] Exemplary combinations of therapeutic polypeptide, peptide
linker, and HLEP include the constructs listed in tables 3a and 3b,
but are not limited to them:
TABLE-US-00003 TABLE 3A Examples of possible constructs Linker
derived from (with Coagulation modifications, SEQ factor Linker
HLEP if applicable) ID NO: Linker not cleavable or not sufficiently
rapidly cleavable FIX HSA FIX RI HSA FIX GGGGGGV (Sheffield et al.)
HSA 94 FIX (GGS).sub.nGS HSA FIX SS(GGS).sub.7GS HSA 30 FIX
SSNGS(GGS).sub.3NGS(GGS).sub.3GGNGS HSA 31 Linker with one cleavage
site FIX (1-412) SVSQTSKLTRAETVFPDVD HSA FIX 36 FIX (1-412)
SVSQTSKLTRAETVFPDVDGS HSA FIX 37 FIX SVSQTSKLTRAETVFPDVD HSA FIX 38
FIX SVSQTSKLTRAETVFPDVDGSGGS HSA FIX 95 FIX SVSQTSKLTRAETVFPDVDGS
HSA FIX 39 FIX SVSIDTSKLTRAETVFPDVDNGS HSA FIX 40 FIX
SVSQTSKLTRAETVFPDV HSA FIX 96 FIX QTSKLTRAETVFPDV HSA FIX 97 FIX
SKLTRAETVFPDV HSA FIX 98 FIX SVSQTSKLTRAETVFP HSA FIX 99 FIX
SVSQTSKLTRAETVF HSA FIX 100 FIX QTSKLTRAETVF HSA FIX 101 FIX
SKLTRAETVF HSA FIX 102 FIX SVSQTSKLTRAET HSA FIX 103 FIX QTSKLTRAET
HSA FIX 104 FIX SKLTRAET HSA FIX 105 FIX SVSQTSKLTRGETVFPDVD HSA
FIX 41 FIX SVSQTSKLTRTETVFPDVD HSA FIX 42 FIX SVSQTSKLTRSETVFPDVD
HSA FIX 43 FIX SVSQTSKLTRLETVFPDVD HSA FIX 44 FIX
SVSQTSKLTRTEAVFPDVD HSA FIX 45 FIX SVSQTSKLTRGEAVFPDVD HSA FIX 46
FIX QTSKLTRAETVFPDVDGS HSA FIX 106 FIX SKLTRAETVFPDVDGS NSA FIX 107
FIX SKLTRAETVFPDVD HSA FIX 47 FIX QSFNDFTRVVGGED HSA FIX 48 FIX
QSFNDFTRVVGGEDGS HSA FIX 49 FIX QSFNDFTRVVGGE HSA FIX 108 FIX
QSFNDFTRTVGGED HSA FIX 50 FIX QSFNDFTRLVGGED HSA FIX 51 FIX
QSFNDFTRGVGGED HSA FIX 52 FIX QSFNDFTRVVGGEDNGS HSA FIX 53 FIX
QSFNDFTRVVGGEDN HSA FIX 54 FIX PERGDNNLTRIVGGQEGS HSA FX 109 FIX
PERGDNNLTRIVGGQE HSA FX 61 FIX PERGDNNLTRIVGGQ HSA FX 110 FIX
DNNLTRIVGGQ HSA FX 111 FIX SVSQTSKLTRAETVFPDVD Fc FIX 62 FIX
QSFNDFTRVVGGEDN Fc FIX 63 FIX (1-412) SVSQTSKLTRAETVFPDVD Fc FIX 64
FIX ASKPQGRIVGG HSAdelDAH FVII 112 FIX KRNASKPQGRIVGGKV HSA FVII 65
FIX PEEPQLRMKNNEEAED HSA FVIII 66 FIX DNSPSFIQIRSVAKKHPKT HSA FVIII
67 FIX LSKNNAIEPRSFSQNSRHPS HSA FVIII 68 FIX DEDENQSPRSFQKKTRHYFIA
HSA FVIII 69 FIX SPHVLRNRAQSGSVPQ HSA FVIII 70 FVII or FVIIa
PEEPQLRMKNNEEAEDYDDDLTDS HSA FVIII 71 FVII or FVIIa
DDDNSPSFIQIRSVAKKHPKTWVHYAAEEED HSA FVIII 72 FVII or FVIIa
LSKNNAIEPRSFSQNSRHPSTRQKQFNA HSA FVIII 73 FVII or FVIIa
DEDENQSPRSFQKKTRHYFIAA HSA FVIII 74 FVII or FVIIa
DYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT HSA FVIII 75 FVIII Derived from
cleavage sites HSA FVIII, FIX or of FVIII, FIX, or Fibrinogen Fgn
VWF Derived from cleavage sites of VWF, HSA FIX, FVIII, VWF FVIII,
or FIX VWF DIYDEDENQSPRSFQKKTRHYFIA HSA FVIII 76 VWF
DNSPSFIQIRSVAKKHP HSA FVIII 77 VWF LSKNNAIEPRSFSQNSRHPS HSA FVIII
78 FIX PVSQTSKLTRAETVFPDV HSA FIX 113 FIX PSVSQTSKLTRAETVFPDV HSA
FIX 114
[0087] In the case of linkers derived from the N-terminal region of
the FIX activation peptide, according to the natural polymorphism
T148-A148 the sequences may also contain A instead of T at this
position.
TABLE-US-00004 TABLE 3B Examples of possible constructs with two or
more cleavage sites Coagulation Linker derived from (par- SEQ
factor Linker HLEP tially incl. Modifications) ID NO: Linker with
two cleavage sites FIX SVSOTSKLTRAETVFPDVTQPERGDNNLTRI HSA FIX, FX
79 VGGQE FIX SKLTRAETVFPDNNLTRIVGGQE HSA FIX, FX 80 FIX
RAETVFPDVTQPERGDNNLTRIVGGQE HSA FIX, FX 81 FIX
RAETVFPERGDNNLTRIVGGQE HSA FIX, FX 82 FIX
SVSQTSKLTRAETVFPDVDYVNNLTRIVGGQE HSA FIX, FX 83 FIX
SVSQTSKLTRAETVFPDVDNNLTRIVGGQE HSA FIX, FX 84 FIX
SVSQTSKLTRAETVFPDVDNNLTRIVGGQE HSA FIX, FX 85 FIX
SVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQ HSA FIX 86 STQSFNDFTRVVGGEDA
FIX SVSQTSKLTRAETVFPDVQSFNDFTRVVGGED HSA FIX 87 FIX
SVSQTSKLTRAETVFPDVDSFNDFTRVVGGED HSA FIX 88 FIX
SVSQTSKLTRAETVFPDVNASKPQGRIVGGKV HSA FIX and FVII 89 FIX
SVSQTSKLTRAETVFPDVNASKPQGRLVGGKV HSA FIX and FVII 90 FIX
SVSQTSKLTRAETVFPDVNASKPQGRTVGGKV HSA FIX and FVII 91 FIX
SVSQTSKLTRAETVFPDVD Fc 92
[0088] Variants and fragments of the linkers described in tables 3a
and 3b are also encompassed in the present invention as long as the
linkers can still be cleaved by the protease or the proteases that
cleave the linkers of tables 3a and 3b or by the type of proteases
defined above.
[0089] Other combinations of the cleavage sequences described above
and their variants shall be included in the present invention.
[0090] In another embodiment, amino acid substitutions are included
that change the posttranslational modification pattern of the
peptide linker. These can be, for example, substitutions of amino
acids that are glycosylated, sulphated, or phosphorylated.
[0091] In another embodiment of the invention the peptide linker
between the therapeutic polypeptide and the HLEP moiety contains
consensus sites for the addition of posttranslational
modifications. Such modifications may comprise glycosylation sites.
For example, such modifications may comprise at least one
N-glycosylation site of the structure Asn-X-Ser/Thr, wherein X
denotes any amino acid except proline. Furthermore, such
N-glycosylation sites may be inserted close to the amino and/or
carboxy terminus of the peptide linker such that they are capable
of shielding potential neo-epitopes which might develop at the
sequences where the therapeutic polypeptide moiety is transitioning
into the peptide linker or where the peptide linker is
transitioning into, for example, an albumin moiety sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0092] FIG. 1: In vitro activation of FIX-albumin fusion proteins
by FXIa at 37.degree. C. at a molar ratio of FXIa to fusion protein
of about 1:500. One fusion protein with non-cleavable linker
(1478/797) and two fusion proteins with cleavable linker (1088/797
and 1089/797) were used. Samples were analyzed by SDS-PAGE under
reducing conditions followed by Coomassie blue staining
[0093] FIG. 2: Pharmacokinetics of activated rec FIX and
FIX-albumin fusion proteins with and without cleavable linker in
comparison to non-activated fusion proteins.
[0094] FIG. 3: Inactivation of activated rec FIX or FIX-albumin
fusion protein by AT. Residual FIX activity was determined after
120 min using a non-activated partial thromboplastin time
assay.
EXAMPLES
Example 1
Generation of cDNAs Encoding FIX and FIX-Albumin Fusion
Proteins
[0095] Factor IX coding sequence was amplified by PCR from a human
liver cDNA library (ProQuest, Invitrogen) using primers We1403 and
We1404 (SEQ ID NOS:5 and 6). After a second round of PCR using
primers We1405 and We1406 (SEQ ID NOS:7 and 8) the resulting
fragment was cloned into pCR4TOPO (Invitrogen). From there the FIX
cDNA was transferred as an EcoRI Fragment into the EcoRI site of
pIRESpuro3 (BD Biosciences) wherein an internal XhoI site had been
deleted previously. The resulting plasmid was designated pFIX-496
and was the expression vector for factor IX wild-type.
[0096] For the generation of albumin fusion constructs the FIX cDNA
was reamplified by PCR under standard conditions using primers
We2610 and We2611 (SEQ ID NOS:9 and 10) deleting the stop codon and
introducing an XhoI site instead. The resulting FIX fragment was
digested with restriction endonucleases EcoRI and XhoI and ligated
into an EcoRI/BamH1 digested pIRESpuro3 together with one
XhoI/BamH1 digested linker fragment as described below.
[0097] Two different glycine/serine linker fragments without
internal cleavage sites were generated: Oligonucleotides We2148 and
We2150 (SEQ ID NOS:11 and 12) were annealed in equimolar
concentrations (10 pmol) under standard PCR conditions, filled up
and amplified using a PCR protocol of a 2 min. initial denaturation
at 94.degree. C. followed by 7 cycles of 15 sec. of denaturation at
94.degree. C., 15 sec. of annealing at 55.degree. C. and 15 sec. of
elongation at 72.degree. C., and finalized by an extension step of
5 min at 72.degree. C. The same procedure was performed using
oligonucleotides We2156 and We2157 (SEQ ID NOS:13 and 14). The
resulting linker fragments were digested with restriction
endonucleases XhoI and BamH1 and used separately in the above
described ligation reaction. The resulting plasmids therefore
contained the coding sequence for FIX and a C-terminal extension of
a glycine/serine linker.
[0098] Two different cleavable linker fragments derived from the
activation sites of FIX were generated: Oligonucleotides We2335 and
We2336 (SEQ ID NOS:15 and 16), containing the activation cleavage
site of the FIX light chain/activation peptide border region, were
annealed, filled, and amplified as described above. The resulting
linker fragment was digested with restriction endonucleases XhoI
and BamH1 and used in the above described ligation reaction. The
resulting plasmid therefore contained the coding sequence for FIX
and a C-terminal extension of a cleavable FIX sequence (amino acids
136 to 154 of SEQ ID NO:2). In a subsequent site directed
mutagenesis reaction with a commercially available mutagenesis kit
(QuickChange XL Site Directed Mutagenesis Kit, Stratagene) using
oligonucleotides We2636 and We2637 (SEQ ID NOS:17 and 18) the XhoI
site was deleted.
[0099] For generation of the second cleavable linker fragment
derived from FIX, the same procedure was performed using
oligonucleotides We2337 and We2338 (SEQ ID NOS:19 and 20) for
linker construction. The resulting linker fragment was digested
with restriction endonucleases XhoI and BamH1 and used in the above
described ligation reaction. The resulting plasmid now contained
the coding sequence for FIX and a C-terminal extension of a
cleavable FIX sequence derived from the activation cleavage site of
the FIX activation peptide/heavy chain border region (amino acids
173 to 186 of SEQ ID NO:2). Oligonucleotides We2638 and We 2639
(SEQ ID NOS:21 and 22) were used for deletion of the XhoI site as
described above.
[0100] In the next cloning step the above generated plasmids were
digested with BamH1 and a BamH1 fragment containing the cDNA of
mature human albumin was inserted. This fragment had been generated
by PCR on an albumin cDNA sequence using primers We1862 and We1902
(SEQ ID NOS:23 and 24) under standard conditions.
[0101] The final plasmids with non-cleavable glycine/serine linkers
were designated pFIX-980 (SEQ ID NO:30) and pFIX-986 (SEQ ID
NO:31), respectively. The final plasmids with cleavable linkers
derived from FIX sequences were designated pFIX-1088 (SEQ ID NO:40)
and pFIX-1089 (SEQ ID NO:49), respectively. Their linker sequences
and the C-terminal FIX and N-terminal albumin sequences are
outlined below. Proteolytic cleavage sites within the linkers are
indicated with arrows, the FIX derived linker sequences are
underlined.
TABLE-US-00005 ##STR00001## ##STR00002## ##STR00003##
##STR00004##
[0102] For expression in CHO cells the coding sequences for the FIX
albumin fusion protein were transferred into vectors pIRESneo3 (BD
Biosciences) or pcDNA3.1 (Invitrogen), respectively.
[0103] Using the above protocols and plasmids and by applying
molecular biology techniques known to those skilled in the art (and
as described e.g. in Current Protocols in Molecular Biology,
Ausubel F M et al. (eds.), including Supplement 80, published
October 2007, John Wiley & Sons, Inc.;
http://www.currentprotocols. com/WileyCDA/) other constructs can be
made with insertions of different linker sequences, e.g. as
described in tables 3a and 3b.
[0104] For efficient processing of the propeptide in cells
expressing FIX in high amounts coexpression of furin is required
(Wasley L C et al. 1993. PACE/Furin can process the vitamin
K-dependent pro-factor IX precursor within the secretory pathway.
J. Biol. Chem. 268:8458-8465). Furin was amplified from a liver
cDNA library (Ambion) using primers We1791 and We1792 (SEQ ID
NOS:25 and 26). A second round of PCR using primers We1808 and
We1809 (SEQ ID NOS:27 and 28) yielded a furin fragment where the
carboxyterminal transmembrane domain (TM) was deleted and a stop
codon introduced; this fragment was cloned into pCR4TOPO
(Invitrogen). From there the furin.DELTA.TM cDNA was transferred as
an EcoRI/NotI Fragment into the EcoRI/NotI sites of pIRESpuro3 (BD
Biosciences) wherein an internal XhoI site had been deleted
previously. The resulting plasmid was designated pFu-797. This
plasmid was cotransfected with all FIX constructs in a 1:5
(pFu-797:pFIX-xxx) molar ratio. The amino acid sequence of the
secreted furin encoded by pFu-797 is given as SEQ-ID NO:29.
Example 2
Transfection and Expression of FIX and FIX-Albumin Fusion
Proteins
[0105] Plasmids were grown up in E. coli TOP10 (Invitrogen) and
purified using standard protocols (Qiagen). HEK-293 cells were
transfected using the Lipofectamine 2000 reagent (Invitrogen) and
grown up in serum-free medium (Invitrogen 293 Express) in the
presence of 50 ng/ml Vitamin K and 4 .mu.g/ml Puromycin.
Transfected cell populations were spread through T-flasks into
roller bottles or small-scale fermenters from which supernatants
were harvested for purification.
[0106] Alternatively, CHO K1 or DG44 cells (Invitrogen) were
transfected using the Lipofectamine 2000 reagent (Invitrogen) and
grown up in serum-free medium (Invitrogen CD-CHO) in the presence
of 50 ng/ml Vitamin K and 500-750 ng/ml Geneticin. High expressing
clones were selected and spread through T-flasks into roller
bottles or small-scale fermenters from which supernatants were
harvested for purification.
Example 3
Purification of FIX and FIX-Albumin Fusion Proteins
[0107] Cell culture harvest containing FIX or FIX albumin fusion
protein was applied on a Q-Sepharose FF column previously
equilibrated with 50 mM TrisxHCl/100 mM NaCl buffer pH 8.0.
Subsequently, the column was washed with equilibration buffer
containing 200 mM NaCl. Elution of the bound FIX or FIX fusion
protein was achieved by a salt gradient using 50 mM TrisxHCl/200 mM
NaCl buffer pH 8.0 as a basis. The eluate was further purified by
column chromatography on a hydroxylapatite resin. For this purpose,
the eluate of the Q-Sepharose FF column was loaded on a
hydroxylapatite chromatography column equilibrated with 50 mM
TrisxHCl/100 mM NaCl buffer pH 7.2. The column was washed with the
same buffer and FIX or FIX-HSA were eluted using a potassium
phosphate gradient at pH 7.2. The eluate was dialyzed to reduce the
salt concentration and used for biochemical analysis as well as for
determination of the pharmacokinetic parameters. FIX antigen and
activity were determined as described in example 5.
Example 4
Alternative Purification Scheme of FIX and FIX-Albumin Fusion
Proteins
[0108] As described in example 3, cell culture harvest containing
FIX or FIX albumin fusion protein was purified by chromatography on
Q-Sepharose FF. The Q-Sepharose eluate was further purified by
chromatography on a Heparin-Fractogel column. For this purpose, the
Heparin-Fractogel column was equilibrated using 50 mM
Tris.times.HCl, 50 mM NaCl pH 8.0 buffer (EP), the Q-Sepharose FF
eluate was applied and the column was washed with equilibration
buffer containing 75 mM NaCl. FIX or FIX albumin fusion protein,
respectively, was eluted using EP adjusted to 300 mM NaCl.
[0109] The Heparin-Fractogel eluate was further purified by
chromatography on a hydroxylapatite chromatography column as
described in example 3. The purified FIX resp. FIX albumin fusion
protein concentrate was subjected to FIX activity and antigen
determination according to example 5 and characterized by further
in vitro and in vivo investigations.
Example 5
Determination of FIX Activity and Antigen
[0110] FIX activity was determined as clotting or coagulation
activity (FIX:C) using commercially available aPTT reagents
(Pathromtin SL and FIX depleted plasma, Dade Behring). An internal
substandard calibrated against the WHO International FIX
concentrate Standard (96/854) was used as a reference.
[0111] FIX antigen (FIX:Ag) was determined by an ELISA acc. to
standard protocols known to those skilled in the art. Briefly,
microtiter plates were incubated with 100 .mu.L per well of the
capture antibody (Paired antibodies for FIX ELISA 1:200, Cedarlane,
but other sources of appropriate antibodies may also be applied)
overnight at ambient temperature. After washing plates three times
with washing buffer B (Sigma P3563), each well was incubated with
200 .mu.L blocking buffer C (Sigma P3688) for one hour at ambient
temperature. After another three wash steps with buffer B, serial
dilutions of the test sample in buffer B as well as serial
dilutions of a substandard (SHP) in buffer B (volumes per well: 100
.mu.L) were incubated for two hours at ambient temperature. After
three wash steps with buffer B, 100 .mu.L of a 1:200 dilution of
the detection antibody (Paired antibodies for FIX ELISA, peroxidase
labelled, Cedarlane) in buffer B were added to each well and
incubated for another two hours at ambient temperature. After three
wash steps with buffer B, 100 .mu.L of substrate solution (TMB,
Dade Behring, OUVF) were added per well and incubated for 30
minutes at ambient temperature in the dark. Addition of 100 .mu.L
undiluted stop solution (Dade Behring, OSFA) prepared the samples
for reading in a suitable microplate reader at 450 nm wavelength.
Concentrations of test samples were then calculated using the
standard curve with standard human plasma as reference.
Example 6
Comparison of FIX-Activity/FIX-Antigen Ratio of Different
FIX-Albumin Fusion Proteins in Cell Culture Supernatant
[0112] Cell culture supernatants of HEK cells transfected with DNA
constructs coding for FIX-albumin fusion proteins that contained
different linker peptides were subjected to FIX activity and
antigen testing as described above (see example 5). The ratio of
FIX:C to FIX:Ag was calculated representing a measure directly
proportional to molar specific activity of the different
constructs.
[0113] The results shown in table 4 indicate that there is an
increase in activity/antigen ratio upon introduction of cleavable
linkers into the FIX-HSA molecule. It also shows that the cleavable
linker peptide should have a length of more than two amino acids in
order to provide clearly increased activity/antigen ratios.
TABLE-US-00006 TABLE 4 FIX:C/FIX:Ag ratios of FIX-albumin fusion
proteins containing different linker peptides Fold increase
compared to fusion protein 980/797 with non- cleavable FIX-HSA
FIX:C/ linker construct Linker FIX:Ag (GGGGGGV) 1182/797 None
<0.031 1366/797 RI <0.068 1478/863 GGGGGGV 0.041 --
(Sheffield et al.) 980/797 SS(GGS).sub.7GS 0.070 1.7 986/797
SSNGS(GGS)3NGS 0.076 1.9 (GGS)3GGNGS 1483/863 SVSQTSKLTR AETVFPDVD
0.688 16.8 GSGGS 1088/797 SVSQTSKLTR AETVFPDVD GS 0.832 20.3
1365/797 SVSQTSKLTR AETVFPDVD 0.630 15.4 1482/863 SVSQTSKLTR AETVFP
0.482 11.8 1087/797 SVSQTSKLTR AETVFPDVD GS 0.472 11.5 (FIX
deltaKLT) 1089/797 QSFNDFTR VVGGED GS 0.532 13.0 1091/797
PERGDNNLTR IVGGQE GS 0.111 2.7
Example 7
Comparison of FIX and FIX-Albumin Fusion Proteins in Respect to
Molar Specific Activity, Terminal In Vivo Half-Life and In Vivo
Recovery in Rats or Rabbits
[0114] Purified recombinant wild type FIX (rFIX 496/797) and
FIX-albumin fusion proteins (rFIX 980/797, rFIX 986/797,
rFIX-1088/797 and rFIX 1089/797) were tested for FIX activity in a
clotting assay as described above. In parallel, the difference of
the optical density at 280 and 320 nm was determined as a measure
for protein concentration (OD280-320). The ratios of activity per
OD280-320 were calculated and based on the molar optical densities
the molar specific activities were calculated. In the following
table 5 the results are summarized.
TABLE-US-00007 TABLE 5 Molar specific activities of wt FIX compared
to FIX-albumin fusions Optical FIX Molar density clotting Activity/
specific (OD280- activity Vol/OD activity* Linker 320) (IU/mL)
(IU/mL/OD) (IU/nmol) rFIX.wt (496/797) -- 0.3798 21.2 55.8 4.23
rFIX-HSA (non- GGGGGGV (Sheffield et al.) 2.9189 5.8 2.0 0.23
cleavable, 1478/863 rFIX-HSA (non- SS(GGS)7GS 1.1122 3.4 3.0 0.35
cleavable, 980/797) rFIX-HSA (non- S NGS (GGS)3 NGS (GGS)3 0.8107
3.2 4.0 0.45 cleavable, 986/797) GGN G rFIX-HSA (cleavable, FXIa
cleavable 0.3421 11.9 34.8 3.95 1088/797) rFIX-HSA (cleavable, FXIa
cleavable 0.4512 11.3 25.0 2.84 1089/797) *Molar specific activity
based on activity, optical density and the following molar optical
densities: Molar optical density of FIX: OD(280 nm, 1 mol/L) = 75
810 Molar optical density of albumin: OD(280 nm, 1 mol/L) = 37 791
Molar optical density of FIX-albumin fusion protein: OD(280 nm, 1
mol/L) = 113 601 indicates data missing or illegible when filed
[0115] Taking the results summarized in Table 5 into account, two
constructs that were generated according to the present invention
show highly increased molar specific activities compared to the
fusion proteins with non-cleavable linkers. In addition, the molar
specific activity of these constructs was only moderately decreased
compared to wild type rFIX.
[0116] In vitro investigations of the proteolytic cleavage
reactions by Factor XIa (FXIa) confirmed that FIX-albumin fusion
proteins containing a cleavable linker such as construct no.
1088/797 or 1089/797 are activated and in parallel the linker is
cleaved resulting in release of the albumin moiety (FIG. 1). The
fusion protein with non-cleavable linker did not show a
corresponding release of the albumin moiety.
[0117] In the case of FVIIa as cleaving protease in the presence of
tissue factor, the FIX-albumin fusion proteins 1088/797 or 1089/797
containing a cleavable linker also showed release of the albumin
moiety in parallel to release of the FIX activation peptide (Data
not shown).
[0118] In addition to determination of molar specific coagulation
activity, the polypeptides no. 496/797, 980/797, 986/797, 1088/797
and 1089/797 described above were administered intravenously to
narcotized CD/Lewis rats (6 rats per substance) and/or rabbits (4
rabbits per substance) with a dose of 50 IU/kg body weight. Blood
samples were drawn prior to test substance administration and at
appropriate intervals starting at 5 minutes after administration of
the test substances. FIX antigen content was subsequently
quantified by an ELISA assay specific for human Factor IX (see
above). The mean values of the respective groups were used to
calculate in vivo recovery after 5 min. Half-lives for each protein
were calculated using the time points of the beta phase of
elimination (terminal half-life) according to the formula
t.sub.1/2=ln2/k, whereas k is the slope of the regression line
obtained upon plotting FIX:Ag levels in logarithmic scale and time
in linear scale.
[0119] Calculated in vivo half-lives are summarized in table 6. In
rats as well as in rabbits the in vivo half-lives of the
FIX-albumin fusion proteins were found to be significantly
increased in comparison to non-fused wild-type recombinant FIX
prepared in-house or in comparison to the commercially available
recombinant FIX product BeneFIX.RTM.. The in vivo half-lives of the
albumin fusion proteins compared to BeneFIX.RTM. were increased to
about 200-400%, depending on the animal species or construct used
(Table 6).
[0120] To evaluate the in vivo recovery, the FIX antigen levels
measured per mL of plasma at their maximum concentrations after
intravenous administration (t=5 min) were related to the amount of
product applied per kg. Alternatively, a percentage was calculated
by relating the determined antigen level (IU/mL) 5 min post
infusion to the theoretical product level expected at 100% recovery
(product applied per kg divided by an assumed plasma volume of 40
mL per kg). The in vivo recoveries (IVR) of the FIX-albumin fusion
proteins were significantly higher than the in vitro recoveries of
rFIX (496/797) or BeneFIX.RTM. (Table 7).
TABLE-US-00008 TABLE 6 Terminal in vivo half-lives of FIX
preparations derived from recombinant expression (BeneFIX .RTM.,
rFIX 496/797) and FIX albumin fusion proteins (rFIX 980/797, rFIX
986/797, rFIX 1088/797, and rFIX 1089/797) after intravenous
administration of 50 IU/kg into rats and/or 50 IU/kg into rabbits,
respectively. Rat experiments PSR18-05, Rabbit experiment PSR06-05,
PSR02-05 PSK11-05 relative to relative to Terminal half- BeneFIX
Terminal half- BeneFIX life (h) [%] life (h) [%] rFIX 496/797 4.5*
91 n.t. n.t. rFIX 980/797 11.6* 234 36.9.degree. 410 29.3.degree.
(2.sup.nd exp.) 326 rFIX 986/797 10.5* 212 n.t. n.t. rFIX 1088/797
8.3* 168 30.3.degree. 337 rFIX 1089/797 10.5* 212 n.t. n.t. BeneFIX
4.95* (mean of 100 9.0.degree. 100 5.3 and 4.6) *Determined between
120 and 1440 min .degree.Determined between 4 and 96 h
TABLE-US-00009 TABLE 7 In vivo recoveries (amount of substance 5
minutes post administration) of recombinant FIX preparations
(BeneFIX, rFIX 496/797) and FIX albumin fusion proteins (rFIX
1088/797, rFIX 1089/797) after intravenous administration of 50
IU/kg into rats. The percentage of in vivo recovery was calculated
based on an assumed plasma volume of 40 mL/kg. rat experiment in
vivo recovery IU/dL per relative to IU/kg/[%]* BeneFIX [%] rFIX
0.462/18.5 74.6 496/797 rFIX 1.034/41.4 166.5 1088/797 rFIX
1.063/42.5 171.2 1089/797 BeneFIX 0.621/24.8 100 *Calculated based
on a plasma volume of 40 mL/kg
Example 8
In Vitro Activation of FIX Albumin Fusion Proteins with/without
Cleavable Linker (1088/797 and 980/797) and Determination of
Pharmacokinetics in Rats
[0121] FIX-albumin fusion proteins and rec FIX were activated in
vitro using commercially available Factor XIa (Kordia). Briefly,
identical molar amounts of FIX or FIX-albumin fusion protein
(3.0.times.10.sup.-6 mol/L) were activated at 37.degree. C. in
solution in the presence of FXIa (1.9.times.10.sup.-8 mol/L) and
CaCl.sub.2 (1.5 mmol/L) buffered at pH 6.8. After complete
activation as shown by SDS-PAGE the reaction was stopped by
addition of a 5.times. molar excess of C1-Inhibitor (Berinert.RTM.
P) based on the amount of FXIa. The samples were stored frozen
below -70.degree. C. until start of pharmacokinetic
investigation.
[0122] A pharmacokinetic investigation of the activated FIX and the
FIX-albumin fusion proteins was performed in rats as described in
example 7 and the results were compared to a pharmacokinetic
results covering non-activated fusion proteins. It turned out that
the activated fusion proteins demonstrated significantly reduced
half-lives as well as AUC's compared to the non-activated molecules
(FIG. 2). Upon activation the FIX-fusion protein with cleavable
linker (1088/797) showed a pharmacokinetic behaviour very similar
to activated rec FIX (BeneFIX.RTM.) whereas the activated fusion
protein with non-cleavable linker (980/797) resulted in a higher
initial as well as terminal half-life compared to activated fusion
protein 1088/797 with cleavable linker. Therefore, in this example,
the cleavable linker results in increased elimination of the
coagulation factor after activation and, therefore, avoids
accumulation of potentially thrombogenic, activated fusion proteins
with extended half-lives.
Example 9
Comparison of FIX-Albumin Fusion Proteins with/without Cleavable
Linker in Respect to Inactivation Rate of the Activated Coagulation
Factors by Antithrombin III (AT)
[0123] FIX fusion proteins with (1088/797) and without (980/797)
cleavable linker were activated by incubation with FXIa as
described in example 8. The activated factors were incubated with
AT for 120 min and residual FIXa activity was determined using a
manual FIX clotting assay method without activation (naPTT, see
below) acc. to Schnitger and Gross. As control samples the
activated FIX-albumin fusion proteins were used in presence of the
same amount of AT but without incubation.
[0124] The FIX activity was determined with the aid of a
non-activated partial thromboplastin time assay (naPTT) using FIX
deficient plasma from Dade Behring. The samples were prediluted in
a buffer of pH 6.8 containing His, Gly, Sucrose, and Tween.RTM. 80.
The whole determination was performed using coagulometers acc. to
Schnitger & Gross. A mixture of 0.1 ml F IX deficient plasma,
0.1 ml sample, and 0.1 ml of 0.1% Phospholipids
(Rhone-Poulenc-Nattermann, 1:3 prediluted in imidazole buffer
supplemented with 1% HSA) was incubated for 2 minutes at 37.degree.
C. The coagulation reaction was initiated by adding 0.1 ml 0.025
mol/l CaCl.sub.2 solution and the clotting time was determined.
[0125] FIG. 3 shows the results of a corresponding inactivation
experiment. In the case of the fusion protein with cleavable linker
(1088/797) an increase in clotting time from 210 to 540 sec (factor
of 2.57.times.) demonstrated an accelerated inactivation process of
FIXa activity by AT compared to a fusion protein with non-cleavable
linker (980/797) that only showed an increase from 196 to 411 sec
(factor of 2.10.times.). Most probably, the albumin residue
sterically affects the AT dependent inactivation process in the
case of the fusion protein with non-cleavable linker whereas in the
case of the fusion protein with cleavable linker the albumin
residue is cleaved off resulting in an accelerated inactivation by
AT.
Example 10
Design of FIX-HSA Fusion Proteins with Reduced Immunogenicity
[0126] As there is with any fusion between two proteins a slight
risk associated that a neoepitope is created around the fusion
point it was investigated whether the linker region as described in
table 3a and 3b could be modified in order to decrease this
risk.
[0127] In the course of this investigation all proposed linker
sequences and the adjacent regions of FIX and HSA were analyzed for
potential T-cell epitopes by way of prediction of binding
capabability to multiple MHC-II alleles. One of these approaches
involved the PreDeFT analysis offered by the company EpiVax (146
Clifford St., Providence, R.I. 02903, USA) in which the input
sequences were parsed into overlapping 9-mer frames where each
frame overlaps the last by 8 amino acids. Each frame was then
assessed for its ability to bind with a set of common HLA. These
detailed findings were then summarized producing regional and
overall assessments of immunogenic potential. Finally, any epitope
clusters identified were screened against the non-redundant protein
database at GenBank and EpiVax's own database of known MHC ligands
and T-cell epitopes.
[0128] As a result of these in silico predictions the following FIX
fusion proteins were cloned, expressed and purified:
TABLE-US-00010 SEQ ID NO: 113 FIX- PVSQTSKLTRAETVFPDV-HSA SEQ ID
NO: 114 FIX-PSVSQTSKLTRAETVFPDV-HSA
Example 11
Neoantigenicity Test
[0129] FIX-HSA fusion proteins comprising linkers SEQ ID NO:113 or
114 can be shown to display a reduced immunogenicity compared to
fusion proteins comprising different linkers or to display a
comparable immunogenicity as compared to wild type factor FIX by
the following neoantigenicity test.
[0130] Products to be compared are administered subcutaneously into
rabbits with or in the absence of Freund's adjuvant. The endpoint
assay is a native Western blot.
[0131] A suitable dose of either the FIX-HSA fusion with a linker
with reduced immunogenicity or a FIX-HSA fusion with a linker of
enhanced immunogenicity or FIX wild type can be administered as a
slow bolus.
[0132] A sample can be taken about 30 to 40 days after the start of
the immunization and be assayed by a Western blot method to ensure
that the rabbits developed antibodies against each of the
respective immunogens.
[0133] Antibodies against the test sample for which a potentially
increased immunogenicity is to be measured are blocked by an excess
of a control sample (e.g. wild type FIX) By doing so all of the
antibodies present which formed against native epitopes are unable
to react when that antibody is used as a probe in a Western blot.
The test and the control sample are run on the Western blot
membrane and the blocked antibody is used as a probe.
[0134] If there were epitopes on the test samples (here FIX-HSA
fusion protein with a linker) but not in the control sample (wild
type FIX) that caused antibody formation in the rabbits, these
antibodies would be detected after blocking as residual antibodies
which would react with the test sample, but not the control
sample.
[0135] Likewise FIX-HSA with a linker having predicted reduced
immunogenicity can be used as a control versus a FIX-HSA with a
linker having predicted increased immunogenicity as a test
sample.
[0136] Preferably the same Western blot is assayed with non-blocked
antibodies raised against the test sample as a positive control.
Here it is expected that test sample as well as control sample are
detected in the Western blot assay.
[0137] If with blocked antibodies raised against the test sample
both test and control sample are not detected it can be concluded
that the test sample has no neoepitopes as compared to the control
sample.
[0138] If with blocked antibodies raised against the test sample
only the test sample but not the control sample is detected it can
be concluded that the test sample has neoepitopes as compared to
the control sample.
[0139] For doing the analysis the IgG fraction of the hyper immune
pooled serum can be purified using a protein A column from Pierce
or other suppliers with a bed volume for example greater than 1 ml
according to the instructions of the supplier. Preferably the
rabbit sera are delipidated for example with
trichlorotrifluoroethane.
[0140] Blocking is done for example by mixing 0.1-1.00 mg of the
purified antiserum with 0.1 to 100 mg of the control sample in for
example a 0.5 to 5 ml centrifuge cup, bringing the volume up to 0.5
to 3 ml. The tubes are then rotated slowly at room temperature of a
minimum of 2 hours.
[0141] Nonblocked control antibodies to be used as a positive
control can be prepared in the same way except that no control
sample is used for blocking.
[0142] All blocked and nonblocked antibodies can be added to 3 to
4.5 ml of 5% dry milk solution in TBS+0.1% Tween-20 before
incubation with their respective membrane.
[0143] Preferably Western blots are performed as native Western
blots.
BRIEF DESCRIPTION OF THE FIGURES
[0144] FIG. 1: In vitro activation of FIX-albumin fusion proteins
by FXIa at 37.degree. C. at a molar ratio of FXIa to fusion protein
of about 1:500. One fusion protein with non-cleavable linker
(1478/797) and two fusion proteins with cleavable linker (1088/797
and 1089/797) were used. Samples were analyzed by SDS-PAGE under
reducing conditions followed by Coomassie blue staining.
[0145] FIG. 2: Pharmacokinetics of activated rec FIX and
FIX-albumin fusion proteins with and without cleavable linker in
comparison to non-activated fusion proteins.
[0146] FIG. 3: Inactivation of activated rec FIX or FIX-albumin
fusion protein by AT. Residual FIX activity was determined after
120 min using a non-activated partial thromboplastin time assay.
Sequence CWU 1
1
1141585PRTHomo sapiens 1Asp Ala His Lys Ser Glu Val Ala His Arg Phe
Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile
Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His
Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys
Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His
Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg
Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90
95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu
100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala
Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr
Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu
Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu
Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys
Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys
Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg
Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215
220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys
225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys
Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn
Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys
Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn
Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe
Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala
Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335
Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340
345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His
Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val
Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu
Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr
Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys
Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu
Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460
Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465
470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp
Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr
Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys
Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp
Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp
Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala
Ala Ser Gln Ala Ala Leu Gly Leu 580 585 2415PRTHomo sapiens 2Tyr
Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg 1 5 10
15 Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe
20 25 30 Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val
Asp Gly 35 40 45 Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly
Ser Cys Lys Asp 50 55 60 Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro
Phe Gly Phe Glu Gly Lys 65 70 75 80 Asn Cys Glu Leu Asp Val Thr Cys
Asn Ile Lys Asn Gly Arg Cys Glu 85 90 95 Gln Phe Cys Lys Asn Ser
Ala Asp Asn Lys Val Val Cys Ser Cys Thr 100 105 110 Glu Gly Tyr Arg
Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val 115 120 125 Pro Phe
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr 130 135 140
Arg Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu 145
150 155 160 Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser
Phe Asn 165 170 175 Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys
Pro Gly Gln Phe 180 185 190 Pro Trp Gln Val Val Leu Asn Gly Lys Val
Asp Ala Phe Cys Gly Gly 195 200 205 Ser Ile Val Asn Glu Lys Trp Ile
Val Thr Ala Ala His Cys Val Glu 210 215 220 Thr Gly Val Lys Ile Thr
Val Val Ala Gly Glu His Asn Ile Glu Glu 225 230 235 240 Thr Glu His
Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His 245 250 255 His
Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu 260 265
270 Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile
275 280 285 Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe
Gly Ser 290 295 300 Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys
Gly Arg Ser Ala 305 310 315 320 Leu Val Leu Gln Tyr Leu Arg Val Pro
Leu Val Asp Arg Ala Thr Cys 325 330 335 Leu Arg Ser Thr Lys Phe Thr
Ile Tyr Asn Asn Met Phe Cys Ala Gly 340 345 350 Phe His Glu Gly Gly
Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro 355 360 365 His Val Thr
Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser 370 375 380 Trp
Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys 385 390
395 400 Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr
405 410 415 31386DNAHomo sapiens 3atgcagcgcg tgaacatgat catggcagaa
tcaccaggcc tcatcaccat ctgcctttta 60ggatatctac tcagtgctga atgtacagtt
tttcttgatc atgaaaacgc caacaaaatt 120ctgaatcggc caaagaggta
taattcaggt aaattggaag agtttgttca agggaacctt 180gagagagaat
gtatggaaga aaagtgtagt tttgaagaag cacgagaagt ttttgaaaac
240actgaaagaa caactgaatt ttggaagcag tatgttgatg gagatcagtg
tgagtccaat 300ccatgtttaa atggcggcag ttgcaaggat gacattaatt
cctatgaatg ttggtgtccc 360tttggatttg aaggaaagaa ctgtgaatta
gatgtaacat gtaacattaa gaatggcaga 420tgcgagcagt tttgtaaaaa
tagtgctgat aacaaggtgg tttgctcctg tactgaggga 480tatcgacttg
cagaaaacca gaagtcctgt gaaccagcag tgccatttcc atgtggaaga
540gtttctgttt cacaaacttc taagctcacc cgtgctgaga ctgtttttcc
tgatgtggac 600tatgtaaatt ctactgaagc tgaaaccatt ttggataaca
tcactcaaag cacccaatca 660tttaatgact tcactcgggt tgttggtgga
gaagatgcca aaccaggtca attcccttgg 720caggttgttt tgaatggtaa
agttgatgca ttctgtggag gctctatcgt taatgaaaaa 780tggattgtaa
ctgctgccca ctgtgttgaa actggtgtta aaattacagt tgtcgcaggt
840gaacataata ttgaggagac agaacataca gagcaaaagc gaaatgtgat
tcgaattatt 900cctcaccaca actacaatgc agctattaat aagtacaacc
atgacattgc ccttctggaa 960ctggacgaac ccttagtgct aaacagctac
gttacaccta tttgcattgc tgacaaggaa 1020tacacgaaca tcttcctcaa
atttggatct ggctatgtaa gtggctgggg aagagtcttc 1080cacaaaggga
gatcagcttt agttcttcag taccttagag ttccacttgt tgaccgagcc
1140acatgtcttc gatctacaaa gttcaccatc tataacaaca tgttctgtgc
tggcttccat 1200gaaggaggta gagattcatg tcaaggagat agtgggggac
cccatgttac tgaagtggaa 1260gggaccagtt tcttaactgg aattattagc
tggggtgaag agtgtgcaat gaaaggcaaa 1320tatggaatat ataccaaggt
atcccggtat gtcaactgga ttaaggaaaa aacaaagctc 1380acttaa
138641830DNAHomo sapiens 4atgaagtggg taacctttat ttcccttctt
tttctcttta gctcggctta ttccaggggt 60gtgtttcgtc gagatgcaca caagagtgag
gttgctcatc ggtttaaaga tttgggagaa 120gaaaatttca aagccttggt
gttgattgcc tttgctcagt atcttcagca gtgtccattt 180gaagatcatg
taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat
240gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa
attatgcaca 300gttgcaactc ttcgtgaaac ctatggtgaa atggctgact
gctgtgcaaa acaagaacct 360gagagaaatg aatgcttctt gcaacacaaa
gatgacaacc caaacctccc ccgattggtg 420agaccagagg ttgatgtgat
gtgcactgct tttcatgaca atgaagagac atttttgaaa 480aaatacttat
atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc
540tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga
taaagctgcc 600tgcctgttgc caaagctcga tgaacttcgg gatgaaggga
aggcttcgtc tgccaaacag 660agactcaagt gtgccagtct ccaaaaattt
ggagaaagag ctttcaaagc atgggcagta 720gctcgcctga gccagagatt
tcccaaagct gagtttgcag aagtttccaa gttagtgaca 780gatcttacca
aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac
840agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag
taaactgaag 900gaatgctgtg aaaaacctct gttggaaaaa tcccactgca
ttgccgaagt ggaaaatgat 960gagatgcctg ctgacttgcc ttcattagct
gctgattttg ttgaaagtaa ggatgtttgc 1020aaaaactatg ctgaggcaaa
ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 1080aggcatcctg
attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact
1140ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt
gttcgatgaa 1200tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac
aaaattgtga gctttttgag 1260cagcttggag agtacaaatt ccagaatgcg
ctattagttc gttacaccaa gaaagtaccc 1320caagtgtcaa ctccaactct
tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 1380tgttgtaaac
atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc
1440ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt
caccaaatgc 1500tgcacagaat ccttggtgaa caggcgacca tgcttttcag
ctctggaagt cgatgaaaca 1560tacgttccca aagagtttaa tgctgaaaca
ttcaccttcc atgcagatat atgcacactt 1620tctgagaagg agagacaaat
caagaaacaa actgcacttg ttgagctcgt gaaacacaag 1680cccaaggcaa
caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag
1740aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa
aaaacttgtt 1800gctgcaagtc aagctgcctt aggcttataa
1830521DNAArtificial SequenceSynthetic 5ccactttcac aatctgctag c
21623DNAArtificial SequenceSynthetic 6caattccaat gaattaacct tgg
23721DNAArtificial SequenceSynthetic 7atgcagcgcg tgaacatgat c
21825DNAArtificial SequenceSynthetic 8tcattaagtg agctttgttt tttcc
25921DNAArtificial SequenceSynthetic 9gattcgaatt cgcccttatg c
211032DNAArtificial SequenceSynthetic 10cgctcgaggt gagctttgtt
ttttccttaa tc 321152DNAArtificial SequenceSynthetic 11ctcgagcggg
ggatctggcg ggtctggagg ctctggaggg tcgggaggct ct 521246DNAArtificial
SequenceSynthetic 12ggatccagat cccccagagc ctccagagcc tcccgaccct
ccagag 461356DNAArtificial SequenceSynthetic 13ctcgagcaat
ggatctggcg ggtctggagg ctctggaggg tcgaatggct ctggag
561464DNAArtificial SequenceSynthetic 14ggatccgttc cctccagacc
cgccagatcc cccagagcct ccagagccat tcgaccctcc 60agag
641546DNAArtificial SequenceSynthetic 15cctcgagctc tgtgagccag
acctccaagc tcaccagggc cgagac 461644DNAArtificial SequenceSynthetic
16gggatccgtc cacatcaggg aagacagtct cggccctggt gagc
441733DNAArtificial SequenceSynthetic 17ggaaaaaaca aagctcactt
ctgtgagcca gac 331833DNAArtificial SequenceSynthetic 18gtctggctca
cagaagtgag ctttgttttt tcc 331938DNAArtificial SequenceSynthetic
19cctcgagcag agcttcaatg acttcacccg ggtggtgg 382041DNAArtificial
SequenceSynthetic 20gggatccatc ctccccgccc accacccggg tgaagtcatt g
412134DNAArtificial SequenceSynthetic 21ggaaaaaaca aagctcactc
agagcttcaa tgac 342234DNAArtificial SequenceSynthetic 22gtcattgaag
ctctgagtga gctttgtttt ttcc 342331DNAArtificial SequenceSynthetic
23gtgggatccg atgcacacaa gagtgaggtt g 312435DNAArtificial
SequenceSynthetic 24cacggatccc tataagccta aggcagcttg acttg
352518DNAArtificial SequenceSynthetic 25caaggagacg ggcgctcc
182619DNAArtificial SequenceSynthetic 26gcccaaggag gggattggc
192730DNAArtificial SequenceSynthetic 27gtggaattca tggagctgag
gccctggttg 302838DNAArtificial SequenceSynthetic 28cacgcggccg
ctcactacag ccgttgcccc gcctccac 3829704PRTHomo Sapiens 29Met Glu Leu
Arg Pro Trp Leu Leu Trp Val Val Ala Ala Thr Gly Thr 1 5 10 15 Leu
Val Leu Leu Ala Ala Asp Ala Gln Gly Gln Lys Val Phe Thr Asn 20 25
30 Thr Trp Ala Val Arg Ile Pro Gly Gly Pro Ala Val Ala Asn Ser Val
35 40 45 Ala Arg Lys His Gly Phe Leu Asn Leu Gly Gln Ile Phe Gly
Asp Tyr 50 55 60 Tyr His Phe Trp His Arg Gly Val Thr Lys Arg Ser
Leu Ser Pro His 65 70 75 80 Arg Pro Arg His Ser Arg Leu Gln Arg Glu
Pro Gln Val Gln Trp Leu 85 90 95 Glu Gln Gln Val Ala Lys Arg Arg
Thr Lys Arg Asp Val Tyr Gln Glu 100 105 110 Pro Thr Asp Pro Lys Phe
Pro Gln Gln Trp Tyr Leu Ser Gly Val Thr 115 120 125 Gln Arg Asp Leu
Asn Val Lys Ala Ala Trp Ala Gln Gly Tyr Thr Gly 130 135 140 His Gly
Ile Val Val Ser Ile Leu Asp Asp Gly Ile Glu Lys Asn His 145 150 155
160 Pro Asp Leu Ala Gly Asn Tyr Asp Pro Gly Ala Ser Phe Asp Val Asn
165 170 175 Asp Gln Asp Pro Asp Pro Gln Pro Arg Tyr Thr Gln Met Asn
Asp Asn 180 185 190 Arg His Gly Thr Arg Cys Ala Gly Glu Val Ala Ala
Val Ala Asn Asn 195 200 205 Gly Val Cys Gly Val Gly Val Ala Tyr Asn
Ala Arg Ile Gly Gly Val 210 215 220 Arg Met Leu Asp Gly Glu Val Thr
Asp Ala Val Glu Ala Arg Ser Leu 225 230 235 240 Gly Leu Asn Pro Asn
His Ile His Ile Tyr Ser Ala Ser Trp Gly Pro 245 250 255 Glu Asp Asp
Gly Lys Thr Val Asp Gly Pro Ala Arg Leu Ala Glu Glu 260 265 270 Ala
Phe Phe Arg Gly Val Ser Gln Gly Arg Gly Gly Leu Gly Ser Ile 275 280
285 Phe Val Trp Ala Ser Gly Asn Gly Gly Arg Glu His Asp Ser Cys Asn
290 295 300 Cys Asp Gly Tyr Thr Asn Ser Ile Tyr Thr Leu Ser Ile Ser
Ser Ala 305 310 315 320 Thr Gln Phe Gly Asn Val Pro Trp Tyr Ser Glu
Ala Cys Ser Ser Thr 325 330 335 Leu Ala Thr Thr Tyr Ser Ser Gly Asn
Gln Asn Glu Lys Gln Ile Val 340 345 350 Thr Thr Asp Leu Arg Gln Lys
Cys Thr Glu Ser His Thr Gly Thr Ser 355 360 365 Ala Ser Ala Pro Leu
Ala Ala Gly Ile Ile Ala Leu Thr Leu Glu Ala 370 375 380 Asn Lys Asn
Leu Thr Trp Arg Asp Met Gln His Leu Val Val Gln Thr 385 390 395 400
Ser Lys Pro Ala His Leu Asn Ala Asn Asp Trp Ala Thr Asn Gly Val 405
410 415 Gly Arg Lys Val Ser His Ser Tyr Gly Tyr Gly Leu Leu Asp Ala
Gly 420 425
430 Ala Met Val Ala Leu Ala Gln Asn Trp Thr Thr Val Ala Pro Gln Arg
435 440 445 Lys Cys Ile Ile Asp Ile Leu Thr Glu Pro Lys Asp Ile Gly
Lys Arg 450 455 460 Leu Glu Val Arg Lys Thr Val Thr Ala Cys Leu Gly
Glu Pro Asn His 465 470 475 480 Ile Thr Arg Leu Glu His Ala Gln Ala
Arg Leu Thr Leu Ser Tyr Asn 485 490 495 Arg Arg Gly Asp Leu Ala Ile
His Leu Val Ser Pro Met Gly Thr Arg 500 505 510 Ser Thr Leu Leu Ala
Ala Arg Pro His Asp Tyr Ser Ala Asp Gly Phe 515 520 525 Asn Asp Trp
Ala Phe Thr Thr Thr His Ser Trp Asp Glu Asp Pro Ser 530 535 540 Gly
Glu Trp Val Leu Glu Ile Glu Asn Thr Ser Glu Ala Asn Asn Tyr 545 550
555 560 Gly Thr Leu Thr Lys Phe Thr Leu Val Leu Tyr Gly Thr Ala Pro
Glu 565 570 575 Gly Leu Pro Val Pro Pro Glu Ser Ser Gly Cys Lys Thr
Leu Thr Ser 580 585 590 Ser Gln Ala Cys Val Val Cys Glu Glu Gly Phe
Ser Leu His Gln Lys 595 600 605 Ser Cys Val Gln His Cys Pro Pro Gly
Phe Ala Pro Gln Val Leu Asp 610 615 620 Thr His Tyr Ser Thr Glu Asn
Asp Val Glu Thr Ile Arg Ala Ser Val 625 630 635 640 Cys Ala Pro Cys
His Ala Ser Cys Ala Thr Cys Gln Gly Pro Ala Leu 645 650 655 Thr Asp
Cys Leu Ser Cys Pro Ser His Ala Ser Leu Asp Pro Val Glu 660 665 670
Gln Thr Cys Ser Arg Gln Ser Gln Ser Ser Arg Glu Ser Pro Pro Gln 675
680 685 Gln Gln Pro Pro Arg Leu Pro Pro Glu Val Glu Ala Gly Gln Arg
Leu 690 695 700 3025PRTArtificialLinker 30Ser Ser Gly Gly Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 15 Ser Gly Gly Ser
Gly Gly Ser Gly Ser 20 25 3131PRTArtificialLinker 31Ser Ser Asn Gly
Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Asn Gly 1 5 10 15 Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Asn Gly Ser 20 25 30
3217PRTArtificialLinker 32Arg Ala Glu Thr Val Phe Pro Asp Val Asp
Tyr Val Asn Ser Thr Glu 1 5 10 15 Ala 3314PRTArtificialLinker 33Arg
Ala Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser 1 5 10
3417PRTArtificialLinker 34Arg Ala Glu Ala Val Phe Pro Asp Val Asp
Tyr Val Asn Ser Thr Glu 1 5 10 15 Ala 3519PRTArtificialLinker 35Arg
Ala Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu 1 5 10
15 Ala Gly Ser 3619PRTArtificialLinker 36Ser Val Ser Gln Thr Ser
Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
3721PRTArtificialLinker 37Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp Gly Ser 20
3819PRTArtificialLinker 38Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
3921PRTArtificialLinker 39Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp Gly Ser 20
4022PRTArtificialLinker 40Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp Asn Gly Ser 20
4119PRTArtificialLinker 41Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Gly Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
4219PRTArtificialLinker 42Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Thr Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
4319PRTArtificialLinker 43Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ser Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
4419PRTArtificialLinker 44Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Leu Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
4519PRTArtificialLinker 45Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Thr Glu Ala Val Phe Pro 1 5 10 15 Asp Val Asp
4619PRTArtificialLinker 46Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Gly Glu Ala Val Phe Pro 1 5 10 15 Asp Val Asp
4714PRTArtificialLinker 47Ser Lys Leu Thr Arg Ala Glu Thr Val Phe
Pro Asp Val Asp 1 5 10 4814PRTArtificialLinker 48Gln Ser Phe Asn
Asp Phe Thr Arg Val Val Gly Gly Glu Asp 1 5 10
4916PRTArtificialLinker 49Gln Ser Phe Asn Asp Phe Thr Arg Val Val
Gly Gly Glu Asp Gly Ser 1 5 10 15 5014PRTArtificialLinker 50Gln Ser
Phe Asn Asp Phe Thr Arg Thr Val Gly Gly Glu Asp 1 5 10
5114PRTArtificialLinker 51Gln Ser Phe Asn Asp Phe Thr Arg Leu Val
Gly Gly Glu Asp 1 5 10 5214PRTArtificialLinker 52Gln Ser Phe Asn
Asp Phe Thr Arg Gly Val Gly Gly Glu Asp 1 5 10
5317PRTArtificialLinker 53Gln Ser Phe Asn Asp Phe Thr Arg Val Val
Gly Gly Glu Asp Asn Gly 1 5 10 15 Ser 5415PRTArtificialLinker 54Gln
Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp Asn 1 5 10 15
557PRTArtificialLinker 55Arg Ile Val Gly Gly Gln Glu 1 5
567PRTArtificialLinker 56Arg Leu Val Gly Gly Gln Glu 1 5
577PRTArtificialLinker 57Arg Thr Val Gly Gly Gln Glu 1 5
587PRTArtificialLinker 58Arg Val Val Gly Gly Gln Glu 1 5
597PRTArtificialLinker 59Arg Ala Val Gly Gly Gln Glu 1 5
607PRTArtificialLinker 60Arg Gly Val Gly Gly Gln Glu 1 5
6116PRTArtificialLinker 61Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg
Ile Val Gly Gly Gln Glu 1 5 10 15 6219PRTArtificialLinker 62Ser Val
Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15
Asp Val Asp 6315PRTArtificialLinker 63Gln Ser Phe Asn Asp Phe Thr
Arg Val Val Gly Gly Glu Asp Asn 1 5 10 15 6419PRTArtificialLinker
64Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1
5 10 15 Asp Val Asp 6516PRTArtificialLinker 65Lys Arg Asn Ala Ser
Lys Pro Gln Gly Arg Ile Val Gly Gly Lys Val 1 5 10 15
6616PRTArtificialLinker 66Pro Glu Glu Pro Gln Leu Arg Met Lys Asn
Asn Glu Glu Ala Glu Asp 1 5 10 15 6719PRTArtificialLinker 67Asp Asn
Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His 1 5 10 15
Pro Lys Thr 6820PRTArtificialLinker 68Leu Ser Lys Asn Asn Ala Ile
Glu Pro Arg Ser Phe Ser Gln Asn Ser 1 5 10 15 Arg His Pro Ser 20
6921PRTArtificialLinker 69Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser
Phe Gln Lys Lys Thr Arg 1 5 10 15 His Tyr Phe Ile Ala 20
7016PRTArtificialLinker 70Ser Pro His Val Leu Arg Asn Arg Ala Gln
Ser Gly Ser Val Pro Gln 1 5 10 15 7124PRTArtificialLinker 71Pro Glu
Glu Pro Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp 1 5 10 15
Tyr Asp Asp Asp Leu Thr Asp Ser 20 7231PRTArtificialLinker 72Asp
Asp Asp Asn Ser Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys 1 5 10
15 Lys His Pro Lys Thr Trp Val His Tyr Ala Ala Glu Glu Glu Asp 20
25 30 7328PRTArtificialLinker 73Leu Ser Lys Asn Asn Ala Ile Glu Pro
Arg Ser Phe Ser Gln Asn Ser 1 5 10 15 Arg His Pro Ser Thr Arg Gln
Lys Gln Phe Asn Ala 20 25 7422PRTArtificialLinker 74Asp Glu Asp Glu
Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys Thr Arg 1 5 10 15 His Tyr
Phe Ile Ala Ala 20 7532PRTArtificialLinker 75Asp Tyr Gly Met Ser
Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln 1 5 10 15 Ser Gly Ser
Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr 20 25 30
7624PRTArtificialLinker 76Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser
Pro Arg Ser Phe Gln Lys 1 5 10 15 Lys Thr Arg His Tyr Phe Ile Ala
20 7717PRTArtificialLinker 77Asp Asn Ser Pro Ser Phe Ile Gln Ile
Arg Ser Val Ala Lys Lys His 1 5 10 15 Pro 7820PRTArtificialLinker
78Leu Ser Lys Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser 1
5 10 15 Arg His Pro Ser 20 7936PRTArtificialLinker 79Ser Val Ser
Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp
Val Thr Gln Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg Ile Val 20 25
30 Gly Gly Gln Glu 35 8023PRTArtificialLinker 80Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro Asp Asn Asn Leu Thr 1 5 10 15 Arg Ile Val
Gly Gly Gln Glu 20 8127PRTArtificialLinker 81Arg Ala Glu Thr Val
Phe Pro Asp Val Thr Gln Pro Glu Arg Gly Asp 1 5 10 15 Asn Asn Leu
Thr Arg Ile Val Gly Gly Gln Glu 20 25 8222PRTArtificialLinker 82Arg
Ala Glu Thr Val Phe Pro Glu Arg Gly Asp Asn Asn Leu Thr Arg 1 5 10
15 Ile Val Gly Gly Gln Glu 20 8332PRTArtificialLinker 83Ser Val Ser
Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp
Val Asp Tyr Val Asn Asn Leu Thr Arg Ile Val Gly Gly Gln Glu 20 25
30 8430PRTArtificialLinker 84Ser Val Ser Gln Thr Ser Lys Leu Thr
Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp Asn Asn Leu Thr
Arg Ile Val Gly Gly Gln Glu 20 25 30 8530PRTArtificialLinker 85Ser
Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10
15 Asp Val Asp Asn Asn Leu Thr Arg Ile Val Gly Gly Gln Glu 20 25 30
8652PRTArtificialLinker 86Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp Tyr Val Asn Ser Thr
Glu Ala Glu Thr Ile Leu Asp Asn 20 25 30 Ile Thr Gln Ser Thr Gln
Ser Phe Asn Asp Phe Thr Arg Val Val Gly 35 40 45 Gly Glu Asp Ala 50
8732PRTArtificialLinker 87Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Gln Ser Phe Asn Asp Phe
Thr Arg Val Val Gly Gly Glu Asp 20 25 30 8832PRTArtificialLinker
88Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1
5 10 15 Asp Val Asp Ser Phe Asn Asp Phe Thr Arg Val Val Gly Gly Glu
Asp 20 25 30 8932PRTArtificialLinker 89Ser Val Ser Gln Thr Ser Lys
Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asn Ala Ser
Lys Pro Gln Gly Arg Ile Val Gly Gly Lys Val 20 25 30
9032PRTArtificialLinker 90Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asn Ala Ser Lys Pro Gln
Gly Arg Leu Val Gly Gly Lys Val 20 25 30 9132PRTArtificialLinker
91Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro 1
5 10 15 Asp Val Asn Ala Ser Lys Pro Gln Gly Arg Thr Val Gly Gly Lys
Val 20 25 30 9219PRTArtificialLinker 92Ser Val Ser Gln Thr Ser Lys
Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp
93666PRTHomo Sapiens 93Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val Gln
Gly Asn Leu Glu Arg 1 5 10 15 Glu Cys Met Glu Glu Lys Cys Ser Phe
Glu Glu Ala Arg Glu Val Phe 20 25 30 Glu Asn Thr Glu Arg Thr Thr
Glu Phe Trp Lys Gln Tyr Val Asp Gly 35 40 45 Asp Gln Cys Glu Ser
Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp 50 55 60 Asp Ile Asn
Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys 65 70 75 80 Asn
Cys Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu 85 90
95 Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr
100 105 110 Glu Gly Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro
Ala Val 115 120 125 Pro Phe Pro Cys Gly Arg Val Ser Val Ser Gln Thr
Ser Lys Leu Thr 130 135 140 Arg Ala Glu Thr Val Phe Pro Asp Val Asp
Tyr Val Asn Ser Thr Glu 145 150 155 160 Ala Glu Thr Ile Leu Asp Asn
Ile Thr Gln Ser Thr Gln Ser Phe Asn 165 170 175 Asp Phe Thr Arg Val
Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe 180 185 190 Pro Trp Gln
Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly 195 200 205 Ser
Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu 210 215
220 Thr Gly Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu
225 230 235 240 Thr Glu His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile
Ile Pro His 245 250 255 His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn
His Asp Ile Ala Leu 260 265 270 Leu Glu Leu Asp Glu Pro Leu Val Leu
Asn Ser Tyr Val Thr Pro Ile 275 280 285 Cys Ile Ala Asp Lys Glu Tyr
Thr Asn Ile Phe Leu Lys Phe Gly Ser 290 295 300 Gly Tyr Val Ser Gly
Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala 305 310 315 320 Leu Val
Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys 325 330 335
Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly 340
345 350 Phe His Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly
Pro 355 360 365 His Val Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly
Ile Ile Ser 370 375 380 Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr
Gly Ile Tyr Thr Lys 385 390 395 400 Val Ser Arg Tyr Val Asn Trp Ile
Lys Glu Lys Thr Lys Leu Thr Ser 405 410 415 Val Ser Gln Thr Ser Lys
Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 420 425 430 Val Asp Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 435 440 445 Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 450 455 460
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 465
470 475 480 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp 485 490 495 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 500 505
510 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
515 520 525 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 530 535 540 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 545 550 555 560 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu 565 570 575 Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 580 585 590 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 595 600 605 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 610 615 620 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 625 630
635 640 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 645 650 655 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 660 665
947PRTArtificialLinker 94Gly Gly Gly Gly Gly Gly Val 1 5
9524PRTArtificialLinker 95Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val Asp Gly Ser Gly Gly Ser
20 9618PRTArtificialLinker 96Ser Val Ser Gln Thr Ser Lys Leu Thr
Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val
9715PRTArtificialLinker 97Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr
Val Phe Pro Asp Val 1 5 10 15 9813PRTArtificialLinker 98Ser Lys Leu
Thr Arg Ala Glu Thr Val Phe Pro Asp Val 1 5 10
9916PRTArtificialLinker 99Ser Val Ser Gln Thr Ser Lys Leu Thr Arg
Ala Glu Thr Val Phe Pro 1 5 10 15 10015PRTArtificialLinker 100Ser
Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe 1 5 10 15
10112PRTArtificialLinker 101Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr
Val Phe 1 5 10 10210PRTArtificialLinker 102Ser Lys Leu Thr Arg Ala
Glu Thr Val Phe 1 5 10 10313PRTArtificialLinker 103Ser Val Ser Gln
Thr Ser Lys Leu Thr Arg Ala Glu Thr 1 5 10 10410PRTArtificialLinker
104Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr 1 5 10
1058PRTArtificialLinker 105Ser Lys Leu Thr Arg Ala Glu Thr 1 5
10618PRTArtificialLinker 106Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr
Val Phe Pro Asp Val Asp 1 5 10 15 Gly Ser 10716PRTArtificialLinker
107Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp Val Asp Gly Ser
1 5 10 15 10813PRTArtificialLinker 108Gln Ser Phe Asn Asp Phe Thr
Arg Val Val Gly Gly Glu 1 5 10 10918PRTArtificialLinker 109Pro Glu
Arg Gly Asp Asn Asn Leu Thr Arg Ile Val Gly Gly Gln Glu 1 5 10 15
Gly Ser 11015PRTArtificialLinker 110Pro Glu Arg Gly Asp Asn Asn Leu
Thr Arg Ile Val Gly Gly Gln 1 5 10 15 11111PRTArtificialLinker
111Asp Asn Asn Leu Thr Arg Ile Val Gly Gly Gln 1 5 10
11211PRTArtificialLinker 112Ala Ser Lys Pro Gln Gly Arg Ile Val Gly
Gly 1 5 10 11318PRTArtificialLinker 113Pro Val Ser Gln Thr Ser Lys
Leu Thr Arg Ala Glu Thr Val Phe Pro 1 5 10 15 Asp Val
11419PRTArtificialLinker 114Pro Ser Val Ser Gln Thr Ser Lys Leu Thr
Arg Ala Glu Thr Val Phe 1 5 10 15 Pro Asp Val
* * * * *
References