U.S. patent application number 15/962624 was filed with the patent office on 2018-10-04 for anti-tfpi antibody variants with differential binding across ph range for improved pharmacokinetics.
This patent application is currently assigned to BAYER HEALTHCARE LLC. The applicant listed for this patent is BAYER HEALTHCARE LLC. Invention is credited to John E. Murphy, Zhuozhi Wang, Ruth Winter.
Application Number | 20180282430 15/962624 |
Document ID | / |
Family ID | 50928243 |
Filed Date | 2018-10-04 |
United States Patent
Application |
20180282430 |
Kind Code |
A1 |
Murphy; John E. ; et
al. |
October 4, 2018 |
ANTI-TFPI ANTIBODY VARIANTS WITH DIFFERENTIAL BINDING ACROSS PH
RANGE FOR IMPROVED PHARMACOKINETICS
Abstract
Antibodies are disclosed that bind to and inhibit the
anti-coagulant function of TFPI and have a lower affinity for TFPi
at pH 6.0 than at pH 7.4. The lower affinity at pH 6 improves
circulating half-life (T1/2) due to reduced target mediated
clearance, a process by which an antibody/antigen complex, is
endocytased and trafficked to the lysosome where both components
are degraded. The lower affinity at pH 6.0 results in disruption of
the complex prior to lysosome targeting and allows for
re-circulation of the antibody. Specific modifications to antibody
binding by histidine residue substitution are disclosed along with
methods of use.
Inventors: |
Murphy; John E.; (Boston,
MA) ; Wang; Zhuozhi; (Millbrae, CA) ; Winter;
Ruth; (Oakland, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER HEALTHCARE LLC |
Whippany |
NJ |
US |
|
|
Assignee: |
BAYER HEALTHCARE LLC
Whippany
NJ
|
Family ID: |
50928243 |
Appl. No.: |
15/962624 |
Filed: |
April 25, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14772336 |
Sep 2, 2015 |
|
|
|
PCT/US2014/029048 |
Mar 14, 2014 |
|
|
|
15962624 |
|
|
|
|
61798261 |
Mar 15, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/38 20130101;
C07K 2317/30 20130101; C07K 2317/77 20130101; C07K 2317/55
20130101; C07K 2317/92 20130101; C07K 2317/94 20130101; C07K
2317/56 20130101; C07K 2317/14 20130101; A61P 7/04 20180101 |
International
Class: |
C07K 16/38 20060101
C07K016/38 |
Claims
1. A therapeutic composition comprises an isolated human monoclonal
IgG antibody that binds specifically to human tissue -factor
pathway inhibitor (TFPI) and has increased plasma half-life,
wherein the antibody comprises at least one histidine substitution
in a CDR region in either a human heavy chain or a human light
chain and antibody binds to TFPI at pH 7.4 with at least two fold
higher affinity than at pH 6.0.
2. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEQ ID NO: 5
3. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEQ ID NO: 6
4. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEQ ID NO: 7
5. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEO ID NO: 8
6. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEQ ID NO: 9
7. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEQ ID NO: 10
8. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO: 11
9. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID MO; 12
10. The isolated human antibody of claim 1, wherein the fight chain
comprises SEQ ID NO: 13
11. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO: 14
12. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO: 15
13. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO: 18
14. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO: 17
15. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO 10
16. The isolated human antibody of claim 1, wherein the heavy chain
comprises SEQ ID NO: 10 and the histidine substitution is selected
from the group consisting of Y102H, Y32H and Y100H, and
combinations thereof.
17. The isolated human antibody of claim 1, wherein the light chain
comprises SEQ ID NO: 17 and the histidine substitution is selected
from the group consisting of Y31H, F48HS S50H, Y49R L27H, V45N,
W90H and combinations thereof.
18. The isolated monoclonal antibody of claim 1, having at least
two histidine substitutions selected from the group consisting of
VL-Y31H, VH-Y102H, VH-Y100H, VB-Y32H, VL-F48H, VL-S50H, VL-Y49H,
VL-L27H, VL-Y4SH, VL-W90H and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/772,336, which adopts the
international filing date of Mar. 14, 2014, which is the National
Phase application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2014/029048, filed Mar. 14, 2014, which
claims the benefit of U.S. Provisional Application No. 61/798,261
filed Mar. 15, 2013, the disclosures of each of which are hereby
incorporated by reference in their entireties for all purposes.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
BHC125023PCT-USNSEQLIST.TXT, date recorded: Apr. 25, 2018, size: 37
KB).
BACKGROUND
[0003] Currently the prophylactic management of hemophilia A and B
patients is replacement of either FVIII or FIX (recombinant or
plasma-derived products). These treatments are administered two or
three times per week, placing a heavy burden on patients to comply
with their prophylactic regime. Despite rigorous management and
strict adherence, patients typically experience occasional
breakthrough bleeds and require on-demand treatments. If not
managed properly, frequent and severe bleeding leads to significant
morbidities, especially hemarthropathy. Despite the proven efficacy
of the existing agents used to treat hemophilia A and B patients,
most adolescents, teenagers and older adults decide to lessen the
burden of prophylaxis by reducing the number of injections taken on
a routine basis. This approach further compromises the protection
needed to adequately manage bleeds.
[0004] Consequently, an agent that significantly provides
protection and requires relatively infrequent administration is
most desired. The optimal therapy should provide protection via
weekly or less frequent dosing. Given the current competitive
environment, once a week therapies administered intravenously
(i.v.) or subcutaneously (s.c.) may be a reality over the next 3-4
years. Therefore, an agent that is administered i.v. or s.c. should
provide a superior administration profile with commensurate
protection. In the event subcutaneous administration can be
achieved, once a week dosing could also offer a significant value
to the future treatment landscape due to the reduced invasiveness
of this approach.
[0005] Another major issue for current hemophilia therapy is the
development of inhibitory antibodies against Factor VIII or Factor
IX. Approximately 25% of FVIII treated patients generate
inhibitors, or neutralizing antibodies against FVIII. Inhibitors
are also found in FIX treated patients, although less frequently.
The development of inhibitors significantly reduces the
effectiveness of replacement therapy and provides a challenge for
managing bleeds in hemophilia patients. The current treatment for
bleeds in patients with inhibitors to FVIII or FIX is bypass
therapy with recombinant Factor Vila or plasma derived FEIBA. The
half-life of rFVIIa is quite short (-2 hours) and thus prophylactic
treatment in these patients is uncommon [Blanchette, Haemophilia 16
(supplement 3): 46-51, 2010].
[0006] To address these unmet medical needs, antibodies against
Tissue Factor Pathway Inhibitor (TFPI) as long-acting agents have
been developed. See WO 2010/017196; WO 2011/109452; WO 2012/135671.
TFPI is the major inhibitor of the tissue factor initiated
coagulation pathway, which is intact in Persons with Hemophilia
(PWH), and thus inhibition of TFPI may restore hemostasis in PWH
exhibiting inhibitory antibodies to FVIII or FIX. In addition to
allowing access to this target, monoclonal antibody (mAb)
therapeutics have been shown to have significantly longer
circulating half-lives (up to 3 weeks) than recombinant replacement
factors. Antibodies that inhibit TFPI also have significant
bioavailability following subcutaneous injection. Thus, anti-TFPI
monoclonal antibody therapy would meet an important unmet medical
need for subcutaneous, long acting hemostatic protection for PWH
and PWH with inhibitors.
[0007] However, while inhibition of TFPI has been shown to promote
coagulation in hemophilic plasmas and hemophilic animals,
antibodies against TFPI have relatively short, non-linear
half-lives due to a phenomenon known as target mediated drug
disposition (TMDD) a process by which the antibody is removed from
circulation due to its interaction with a rapidly cleared target or
by being sequestered from the plasma due to its co-localization
with its target, of the antibody:antigen complex. Therefore,
antibodies that avoid TFPI mediated TMDD and have a prolonged
half-life would lead to less frequent dosing and reduce the amount
of material needed per dose. Furthermore, the need for a lower dose
may also make feasible subcutaneous dosing where the dosing volume
becomes a limiting step. For example, an optimized anti-TFPI
antibody 2A8-g200 (WO 2011/109452), has a half-life of 28 hours
when dosed at 5 mg/kg and 67 hrs when dosed at 20 mg/kg in
non-human primates.
[0008] This relatively short half-life, and the need for larger
doses to overcome TMDD, increases the injection burden on patients,
limits formulation for subcutaneous dosing and increases the costs
of goods. Pharmocokinetic analysis of these antibodies in non-human
primates demonstrates that the circulating half-life is not linear
with dose, and, particularly at lower doses, is shorter than is
characteristic of antibody drugs. A similar pharmacokinetic profile
was described in US2011/0318356 AI for another anti-TFPI antibody.
This differential, with a marked shortening of T1/2 at lower doses,
is characteristic of TMDD, where the slower clearance at higher
doses is due to saturation of the target.
[0009] Accordingly, an unmet medical need exists for better
prophylactic treatment for moderate-to-severe hemophilia A and B,
especially for those patients with inhibitors against FVIII or FIX.
This need would be met by an anti-TFPI antibody having improved
characteristics that may be administered intravenously or
subcutaneously, and at a reduced frequency, preferably once weekly
or less.
SUMMARY OF INVENTION
[0010] To increase the half-life of an anti-TFPI antibody and to
reduce the injection burden, a longer acting anti-TFPI antibody was
produced without a loss of efficacy and tested to confirm improved
properties as compared to other anti-TFPI antibodies having
demonstrated TFPI binding characteristics and proven efficacy in
treating coagulation deficiencies. (See WO 2011/109452.)
Specifically, TMDD is reduced by creating a variant anti-TFPI
antibody having reduced affinity at pH 6.0 relative to pH 7.4. An
anti-TFPI antibody may be taken into cells in complex with its
target, TFPI, through receptors involved in TFPI clearance. One
receptor, identified by Narita et al. (JBC 270 (42): 24800-4, 1995)
is LRP (LDL receptor related protein), which targets TFPI for
degradation in the endosome. However, if this antigen:antibody
complex can be disrupted at low pH, which is characteristic of the
endosome, the antibody can be recycled via FcRn binding, thereby
increasing exposure in circulation. This principal has been shown
for an antibody to PSCK9 by Chapparo-Riggers et al. JBC 287 (14):
110-7 (2012).
[0011] One method for disruption of an antigen: antibody complex at
lower pH is to substitute histidine residues near the
antibody:antigen interaction surface. The amino acid histidine
(His) is protonated at low pH, near pH 6.0, and thus, a residue
that is neutral at pH 7.4 acquires a positive charge at pH 6.0.
This can lead to charge repulsion with other amino acids and a
desirable degree of disruption or destabilization at the
antibody:antigen interface.
[0012] To identify pH sensitive residues, the CDR amino acids and
other amino acids involved in antigen:antibody binding of anti-TFPI
antibodies (e.g. 2A8-g200) to TFPI antigen were changed
individually to His. The individual His variants demonstrate
differential binding at pH 7.4 vs. pH 6.0, and combinations of
variants with differential binding have been tested for
optimization.
[0013] Upon endosomal release, these pH sensitive anti-TFPI mAb
variants bind to the neonatal FcRN receptor and are recycled to the
plasma. Thus, a combination between a pH sensitive TFPI-binding
site and a Fc domain with increased affinity for FcRN at low pH
would have a synergistic effect that increases half-life, reduces
the injection burden to patients, and reduces the cost of
goods.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1A and FIG. 1B show alignments of amino acid sequences
for 2A8-g200 and mutated anti-TFPI mAbs suitable for histidine
substitution (SEQ ID NOs for these sequences can be found in Table
1). FIG. 1A shows the Variable Heavy Chain, and FIG. 1B shows the
Variable Light Chain. CDR regions 1-3 are indicated.
[0015] FIGS. 2A-2D show synthesis and subcloning of a 2A8-g200 Fab
Histidine Scanning Library. The CDR1-3 regions are indicated with a
dashed line. Underlined amino acid residues indicate the position
of contact residues to TFPI. An asterisk (*) indicates a proposed
His mutation site. FIG. 2A shows 2A8-g200 heavy chain; FIG. 2B
shows 2A8-g200 light chain; FIG. 2C shows 4B7-gB9.7 heavy chain;
and FIG. 2D shows 4B7-gB9.7 light chain.
[0016] FIGS. 3A and 3B show dissociation constants at two pHs for
the improved antibodies with exemplary histidine mutations: FIG. 3A
shows L-L27H, and FIG. 3B shows L-Y31H. Surface plasmon resonance
(Biacore T200) was used to measure the dissociation rate of the
antibodies.
[0017] FIG. 4 shows pK profiles observed in Hem A mouse plasma over
time for several monoclonal antibodies at concentrations of 2
mg/kg: 2A8-g200 (----), histidine substituted monoclonal antibodies
TPP2256 (L-Y31H/Y49H) (- -- -) and TPP2259 (L-Y31H) (--).
Pharmacokinetic parameters of the antibodies were determined after
intravenous (i.v.) bolus administration to HemA mouse at 2
mg/kg.
DETAILED DESCRIPTION
[0018] The term "tissue factor pathway inhibitor" or "TFPI" as used
herein refers to any variant, isoform and species homolog of human
TFPI that is naturally expressed by cells. In a preferred
embodiment of the invention, the binding of an antibody of the
invention to TFPI reduces the blood clotting time.
[0019] As used herein, an "antibody" refers to a whole antibody and
any antigen binding fragment (i.e., "antigen-binding portion") or
single chain thereof. The term includes a full-length
immunoglobulin molecule (e.g., an IgG antibody) that is naturally
occurring or formed by normal immunoglobulin gene fragment
recombinatorial processes, or an immunologically active portion of
an immunoglobulin molecule, such as an antibody fragment, that
retains the specific binding activity. Regardless of structure, an
antibody fragment binds with the same antigen that is recognized by
the full-length antibody. For example, an anti-TFPI monoclonal
antibody fragment binds to an epitope of TFPI. The antigen-binding
function of an antibody can be performed by fragments of a
full-length antibody. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include
(i) a Fab fragment, a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the V.sub.H and C.sub.H1 domains; (iv) a Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546), which consists of a V.sub.H domain; (vi) an isolated
complementarity determining region (CDR); (vii) minibodies,
diaboidies, triabodies, tetrabodies, and kappa bodies (see, e.g.
Ill et al., Protein Eng 1997; 10:949-57); (viii) camel IgG; and
(ix) IgNAR. Furthermore, although the two domains of the Fv
fragment, V.sub.L and V.sub.H, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic
linker that enables them to be made as a single protein chain in
which the V.sub.L and V.sub.H regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
portion" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are analyzed for utility in the same manner as
are intact antibodies.
[0020] Furthermore, it is contemplated that an antigen binding
fragment may be encompassed in an antibody mimetic. The term
"antibody mimetic" or "mimetic" as used herein is meant a protein
that exhibits binding similar to an antibody but is a smaller
alternative antibody or a non-antibody protein. Such antibody
mimetic may be comprised in a scaffold. The term "scaffold" refers
to a polypeptide platform for the engineering of new products with
tailored functions and characteristics.
[0021] As used herein, the terms "inhibits binding" and "blocks
binding" (e.g., referring to inhibition/blocking of binding of TFPI
ligand to TFPI) are used interchangeably and encompass both partial
and complete inhibition or blocking. Inhibition and blocking are
also intended to include any measurable decrease in the binding
affinity of TFPI to a physiological substrate when in contact with
an anti-TFPI antibody as compared to TFPI not in contact with an
anti-TFPI antibody, e.g., the blocking of the interaction of TFPI
with factor Xa or blocking the interaction of a TFPI-factor Xa
complex with tissue factor, factor VIIa or the complex of tissue
factor/factor VIIa by at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.
[0022] Therapeutic antibodies have been made through hybridoma
technology described by Koehler and Milstein in "Continuous
Cultures of Fused Cells Secreting Antibody of Predefined
Specificity," Nature 256, 495-497 (1975). Fully human antibodies
may also be made recombinantly in prokaryotes and eukaryotes.
Recombinant production of an antibody in a host cell rather than
hybridoma production is preferred for a therapeutic antibody.
Recombinant production has the advantages of greater product
consistency, likely higher production level, and a controlled
manufacture that minimizes or eliminates the presence of
animal-derived proteins. For these reasons, it is desirable to have
a recombinantly produced monoclonal anti-TFPI antibody. The terms
"monoclonal antibody" or "monoclonal antibody composition" as used
herein refer to a preparation of antibody molecules of single
molecular composition. A monoclonal antibody composition displays a
single binding specificity and affinity for a particular epitope.
Generally, therapeutic antibodies for human diseases have been
generated using genetic engineering to create murine, chimeric,
humanized or fully human antibodies. Murine monoclonal antibodies
were shown to have limited use as therapeutic agents because of a
short serum half-life, an inability to trigger human effector
functions, and the production of human anti-mouse-antibodies
(Brekke and Sandlie, "Therapeutic Antibodies for Human Diseases at
the Dawn of the Twenty-first Century," Nature 2, 53, 52-62, January
2003). Chimeric antibodies have been shown to give rise to human
anti-chimeric antibody responses. Humanized antibodies further
minimize the mouse component of antibodies. However, a fully human
antibody avoids the immunogenicity associated with murine elements
completely. Thus, there is a need to develop antibodies that are
human or humanized to a degree necessary to avoid the
immunogenicity associated with other forms of genetically
engineered monoclonal antibodies. In particular, chronic
prophylactic treatment such as would be required for hemophilia
treatment with an anti-TFPI monoclonal antibody has a high risk of
development of an immune response to the therapy if an antibody
with a murine component or murine origin is used due to the
frequent dosing required and the long duration of therapy. For
example, antibody therapy for hemophilia A may require weekly
dosing for the lifetime of a patient. This would be a continual
challenge to the immune system. Thus, the need exists for a fully
human antibody for antibody therapy for hemophilia and related
genetic and acquired deficiencies or defects in coagulation.
Accordingly, the term "human monoclonal antibody" refers to
antibodies displaying a single binding specificity which have at
least portions of variable and constant regions derived from human
germline immunoglobulin sequences. The human antibodies of the
invention may include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). These human antibodies include chimeric antibodies, such
as mouse/human and humanized antibodies that retain non-human
sequences.
[0023] An "isolated antibody," as used herein, is intended to refer
to an antibody which is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that binds to TFPI is substantially free of antibodies
that bind antigens other than TFPI). An isolated antibody that
binds to an epitope, isoform or variant of human TFPI may, however,
have cross-reactivity to other related antigens, e.g., from other
species (e.g., TFPI species homologs). Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals.
[0024] As used herein, "specific binding" refers to antibody
binding to a predetermined antigen. Typically, the antibody binds
with an affinity of at least about 10.sup.5 and binds to the
predetermined antigen with an affinity that is higher, for example
at least two-fold greater, than its affinity for binding to an
irrelevant antigen (e.g., BSA, casein) other than the predetermined
antigen or a closely-related antigen. The phrases "an antibody
recognizing an antigen" and "an antibody specific for an antigen"
are used interchangeably herein with the term "an antibody which
binds specifically to an antigen."
[0025] As used herein, the term "high affinity" for an IgG antibody
refers to a binding affinity of at least about 10.sup.7, in some
embodiments at least about 10.sup.8, in some embodiments at least
about 10.sup.9, 10.sup.10, 10.sup.11 or greater, e.g., up to
10.sup.13 or greater. However, "high affinity" binding can vary for
other antibody isotypes. For example, "high affinity" binding for
an IgM isotype refers to a binding affinity of at least about
1.0.times.10.sup.7. As used herein, "isotype" refers to the
antibody class (e.g., IgM or IgG1) that is encoded by heavy chain
constant region genes.
[0026] "Complementarity-determining region" or "CDR" refers to one
of three hypervariable regions within the variable region of the
heavy chain or the variable region of the light chain of an
antibody molecule that form the N-terminal antigen-binding surface
that is complementary to the three-dimensional structure of the
bound antigen. Proceeding from the N-terminus of a heavy or light
chain, these complementarity-determining regions are denoted as
"CDR1," "CDR2," and "CDR3," respectively. CDRs are involved in
antigen-antibody binding, and the CDR3 comprises a unique region
specific for antigen-antibody binding. An antigen-binding site,
therefore, may include six CDRs, comprising the CDR regions from
each of a heavy and a light chain V region.
[0027] As used herein, except with respect to the individual or
plurality of histidine substitutions described below, "conservative
substitutions" refers to modifications of a polypeptide that
involve the substitution of one or more amino acids for amino acids
having similar biochemical properties that do not result in loss of
a biological or biochemical function of the polypeptide. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolarside
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine), and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). It is
envisioned that the antibodies of the present invention may have
conservative amino acid substitutions and still retain
activity.
[0028] The term "substantial homology" indicates that two
polypeptides, or designated sequences thereof, when optimally
aligned and compared, are identical, with appropriate amino acid
insertions or deletions, in at least about 80% of amino acids,
usually at least about 85%, preferably about 90%, 91%, 92%, 93%,
94%, or 95%, more preferably at least about 96%, 97%, 98%, 99%,
99.1%, 99.2%, 99.3%, 99.4%, or 99.5% of the amino acids. The
invention includes polypeptide sequences having substantial
homology to the specific amino acid sequences recited herein.
[0029] The percent identity between two sequences is a function of
the number of identical positions shared by the sequences (i.e., %
homology=#of identical positions/total #of positions.times.100),
taking into account the number of gaps, and the length of each gap,
which need to be introduced for optimal alignment of the two
sequences. The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm, such as the AlignX.TM. module of
VectorNTI.TM. (Invitrogen Corp., Carlsbad, Calif.). For AlignX.TM.,
the default parameters of multiple alignment are: gap opening
penalty: 10; gap extension penalty: 0.05; gap separation penalty
range: 8; % identity for alignment delay: 40. (further details
found at
http://www.invitrogen.com/site/us/en/home/LINNEA-Online-Guides/LINNEA-Com-
munities/Vector-NTI-Community/Sequence-analysis-and-data-management-softwa-
re-for-PCs/AlignX-Module-for-Vector-NTI-Advance.reg.us.html).
[0030] Another method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the CLUSTALW computer program
(Thompson et al., Nucleic Acids Research, 1994, 2(22): 4673-4680),
which is based on the algorithm of Higgins et al., (Computer
Applications in the Biosciences (CABIOS), 1992, 8(2): 189-191). In
a sequence alignment the query and subject sequences are both DNA
sequences. The result of said global sequence alignment is in
percent identity. Preferred parameters used in a CLUSTALW alignment
of DNA sequences to calculate percent identity via pairwise
alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5,
Gap Penalty=3, Gap Open Penalty=10, Gap Extension Penalty=0.1. For
multiple alignments, the following CLUSTALW parameters are
preferred: Gap Opening Penalty=10, Gap Extension Parameter=0.05;
Gap Separation Penalty Range=8; % Identity for Alignment Delay 32
40.
[0031] Also provided are pharmaceutical compositions comprising
therapeutically effective amounts of anti-TFPI monoclonal antibody
and a pharmaceutically acceptable carrier. As used herein,
"therapeutically effective amount" means an amount of an anti-TFPI
monoclonal antibody variant or of a combination of such antibody
and factor VIII or factor IX that is needed to effectively increase
the clotting time in vivo or otherwise cause a measurable benefit
in vivo to a patient in need. The precise amount will depend upon
numerous factors, including, but not limited to the components and
physical characteristics of the therapeutic composition, intended
patient population, individual patient considerations, and the
like, and can readily be determined by one skilled in the art.
"Pharmaceutically acceptable carrier" is a substance that may be
added to the active ingredient to help formulate or stabilize the
preparation and causes no significant adverse toxicological effects
to the patient. Examples of such carriers are well known to those
skilled in the art and include water, sugars such as maltose or
sucrose, albumin, salts such as sodium chloride, etc. Other
carriers are described for example in Remington's Pharmaceutical
Sciences by E. W. Martin. Such compositions will contain a
therapeutically effective amount of at least one anti-TFPI
monoclonal antibody.
[0032] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. The composition is preferably formulated for
parenteral injection. The composition can be formulated as a
solution, microemulsion, liposome, or other ordered structure
suitable to high drug concentration. The carrier can be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures thereof.
In some cases, it will include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride
in the composition.
[0033] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, some
methods of preparation are vacuum drying and freeze-drying
(lyophilization) that yield a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0034] The human monoclonal antibody can be used for therapeutic
purposes for treating genetic and acquired deficiencies or defects
in coagulation. For example, the human monoclonal antibodies may be
used to block the interaction of TFPI with FXa, or to prevent
TFPI-dependent inhibition of the TF/FVIIa activity. Additionally,
the human monoclonal antibody may also be used to restore the
TF/FVIIa-driven generation of FXa to bypass the insufficiency of
FVIII- or FIX-dependent amplification of FXa.
[0035] The human monoclonal antibodies have therapeutic use in the
treatment of disorders of hemostasis such as thrombocytopenia,
platelet disorders and bleeding disorders (e.g., hemophilia A,
hemophilia B and hemophlia C). Such disorders may be treated by
administering a therapeutically effective amount of the anti-TFPI
monoclonal antibody variant to a patient in need thereof. The human
monoclonal antibodies also have therapeutic use in the treatment of
uncontrolled bleeds in indications such as trauma and hemorrhagic
stroke. Thus, also provided is a method for shortening the bleeding
time comprising administering a therapeutically effective amount of
an anti-TFPI human monoclonal antibody variant of the invention to
a patient in need thereof.
[0036] The antibodies can be used as monotherapy or in combination
with other therapies to address a hemostatic disorder. For example,
co-administration of one or more variant antibodies of the
invention with a clotting factor such as factor VIIa, factor VIII
or factor IX is believed useful for treating hemophilia. In a
separate embodiment, Factor VIII or Factor IX are administered in
the substantial absence of Factor VII. "Factor VII" includes factor
VII and factor VIIa.
[0037] A method for treating genetic and acquired deficiencies or
defects in coagulation comprises administering (a) a first amount
of a variant monoclonal antibody that binds to human tissue factor
pathway inhibitor and (b) a second amount of factor VIII or factor
IX, wherein said first and second amounts together are effective
for treating said deficiencies or defects. Similarly, a method for
treating genetic and acquired deficiencies or defects in
coagulation comprises administering (a) a first amount of a
monoclonal antibody variant that binds to human tissue factor
pathway inhibitor and (b) a second amount of factor VIII or factor
IX, wherein said first and second amounts together are effective
for treating said deficiencies or defects, and further wherein
factor VII is not coadministered. The invention also includes a
pharmaceutical composition comprising a therapeutically effective
amount of the combination of a monoclonal antibody variant of the
invention and factor VIII or factor IX, wherein the composition
does not contain factor VII.
[0038] These combination therapies are likely to reduce the
necessary infusion frequency of the clotting factor. By
co-administration or combination therapy is meant administration of
the two therapeutic drugs each formulated separately or formulated
together in one composition, and, when formulated separately,
administered either at approximately the same time or at different
times, but over the same therapeutic period.
[0039] In some embodiments, one or more antibody variants described
herein can be used in combination to address a hemostatic disorder.
For example, co-administration of two or more of the antibody
variants described herein is believed useful for treating
hemophilia or other hemostatic disorder.
[0040] The pharmaceutical compositions may be parenterally
administered to subjects suffering from hemophilia A or B at a
dosage and frequency that may vary with the severity of the
bleeding episode or, in the case of prophylactic therapy, may vary
with the severity of the patient's clotting deficiency.
[0041] The compositions may be administered to patients in need as
a bolus or by continuous infusion. For example, a bolus
administration of an antibody variant present as a Fab fragment may
be in an amount of from 0.0025 to 100 mg/kg body weight, 0.025 to
0.25 mg/kg, 0.010 to 0.10 mg/kg or 0.10-0.50 mg/kg. For continuous
infusion, an antibody variant present as an Fab fragment may be
administered at 0.001 to 100 mg/kg body weight/minute, 0.0125 to
1.25 mg/kg/min., 0.010 to 0.75 mg/kg/min., 0.010 to 1.0 mg/kg/min.
or 0.10-0.50 mg/kg/min. for a period of 1-24 hours, 1-12 hours,
2-12 hours, 6-12 hours, 2-8 hours, or 1-2 hours. For administration
of an antibody variant present as a full-length antibody (with full
constant regions), dosage amounts may be about 1-10 mg/kg body
weight, 2-8 mg/kg, or 5-6 mg/kg. Such full-length antibodies would
typically be administered by infusion extending for a period of
thirty minutes to three hours. The frequency of the administration
would depend upon the severity of the condition. Frequency could
range from three times per week to once every two weeks to six
months.
[0042] Additionally, the compositions may be administered to
patients via subcutaneous injection. For example, a dose of 10 to
100 mg anti-TFPI antibody can be administered to patients via
subcutaneous injection weekly, biweekly or monthly.
[0043] Variant monoclonal antibodies to human tissue factor pathway
inhibitor (TFPI) are provided. Further provided are the isolated
nucleic acid molecules encoding the same. Pharmaceutical
compositions comprising the variant anti-TFPI monoclonal antibodies
and methods of treatment of genetic and acquired deficiencies or
defects in coagulation such as hemophilia A and B are also
provided. Also provided are methods for shortening the bleeding
time by administering an anti-TFPI monoclonal antibody to a patient
in need thereof. Methods for producing a variant monoclonal
antibody that binds human TFPI according to the present disclosure
are also provided.
[0044] The therapeutic composition comprises antibody having
binding regions that differ from the sequence of a parenteral TFPI
binding antibody by the intentional [illegible] selection or
engineering of one or more substitutions of the amino acid
histidine (H, HIS) for at least one native amino acid as defined in
the parental sequence. The amino acid change confers longer
circulating half-life T1/2 relative to the parental molecule.
[0045] An antibody specific for TFPI that binds to TFPI with at
least 20% lower efficiency at pH 6.0 than at pH 7.0 is disclosed
and that shows an improvement in circulating T1/2 of approximately
400%. The beneficial effect reducing TMDD is demonstrated for an
antibody or antibody binding region that differs from the sequence
of a target antibody such as 2A8-g200 or 4B7-gB9.7 by the
substitution of the amino acid histidine (H, HIS) for at least one
native amino acid as defined relative to the parental sequence.
Specifically, a variant of 2A8-g200 may have any one of the
following substitutions: VL-Y31H, VH-Y102H, VH-Y100H, VH-Y32H,
VL-F48H, VL-S50H, VL-Y49H, VL-L27H, VL-V45H, VL-W90H and
combinations thereof.
[0046] Anti-TFPI antibody 2A8-g200 and 4B7-gB9.7 variants can bind
to TFPI with high affinity and high specificity in vivo (see WO
2011/109452). FIG. 1 shows amino acid sequence information for
2A8-g200 and 4B7-gB9.7, as well as other 2A8and 4B7 variants
described in WO 2011/109452. Table 1 shows the corresponding SEQ ID
NOs for the variable heavy and variable light chains for the 2A8
and 4B7variants shown in FIG. 1.
TABLE-US-00001 TABLE 1 Corresponding SEQUENCE ID NOs of variable
heavy and variable light chains of the human anti-TFPI antibodies
shown in FIG. 1. CHAIN MAb SEQ ID NO: VH 2A8 SEQ ID NO: 1 2A8-127
SEQ ID NO: 5 2A8-143 SEQ ID NO: 6 2A8-200 SEQ ID NO: 7 2A8-216 SEQ
ID NO: 8 2A8-227 SEQ ID NO: 9 2A8-g200 SEQ ID NO: 10 2A8-g216 SEQ
ID NO: 11 4B7 SEQ ID NO: 3 4B7-B18.5 SEQ ID NO: 19 4B7-B2.0 SEQ ID
NO: 20 4B7-B27.1 SEQ ID NO: 21 4B7-B32.5 SEQ ID NO: 22 4B7-B41.2
SEQ ID NO: 23 4B7-B9.7 SEQ ID NO: 24 4B7-gB9.7 SEQ ID NO: 25
4B7-gB9.7-IgG SEQ ID NO: 26 VL 2A8 SEQ ID NO: 2 2A8-127 SEQ ID NO:
12 2A8-143 SEQ ID NO: 13 2A8-200 SEQ ID NO: 14 2A8-216 SEQ ID NO:
15 2A8-227 SEQ ID NO: 16 2A8-g200 SEQ ID NO: 17 2A8-g216 SEQ ID NO:
18 4B7 SEQ ID NO: 4 4B7-B18.5 SEQ ID NO: 27 4B7-B2.0 SEQ ID NO: 28
4B7-B27.1 SEQ ID NO: 29 4B7-B32.5 SEQ ID NO: 30 4B7-B41.2 SEQ ID
NO: 31 4B7-B9.7 SEQ ID NO: 32 4B7-gB9.7 SEQ ID NO: 33 4B7-gB9.7-IgG
SEQ ID NO: 34
[0047] pH sensitive variants of 2A8-g200 and 4B7-gB9.7 were created
by subjecting both the CDR domains and the residues contacting the
TFPI to analysis for binding characteristics upon mutagenesis at
selected sites. FIG. 2 shows the location of possible His mutations
for: A. 2A8-g200 (designated as A200 in FIG. 2A) variable heavy
chain; B. 2A8-g200 (designated as A200 in FIG. 2B) variable light
chain; C. 4B7-gB9.7 variable heavy chain; and D. 4B7-gB9.7 variable
light chain. One histidine residue was substituted for each of the
amino acids in either 1) a contact residue to TFPI as indicated by
an underlined amino acid in FIG. 2, or 2) a CDR 1-3 residue as
indicated by an asterisk in FIG. 2 for the anti-TFPI antibodies
2A8-g200 and 4B7-gB9.7. As shown in FIGS. 2A and 2B, forty (40)
residues from the heavy chain and twenty-nine (29) residues from
the light chain were identified as the positions for mutagenesis in
2A8-g200. As shown in FIGS. 2C and 2D, forty (40) heavy chain and
thirty-two (32) light chain variants were identified in
4B7-gB9.7.
[0048] A 2A8-g200 Fab histidine scanning library was synthesized.
The library contained 69 members. The 2A8-g200 Fab histidine
library was cloned into a bacterial expression vector and the amino
acid sequences were verified.
[0049] Sixty-nine (69) clones from the His scan library were
transformed into E. coli ATCC strain 9637 and grown on selective
media containing carbenecillin (100 .mu.g/ml). Single colonies were
used to inoculate LB-Carbenecillin-100 media. The cultures were
grown to OD600=0.5 at 37.degree. C., induced with 0.25 mM IPTG, and
grown overnight at 30.degree. C. The bacterial expression cultures
were harvested by centrifugation at 5,000.times.g for 15 min at
4.degree. C. The expression media was decanted from the pellet.
Both pellet and cleared expression media were frozen at -20.degree.
C. The His muteins were purified from the expression media with
Protein A. Purified muteins were analysed by SDS-PAGE and a
concentration was obtained by A280.
[0050] Human TFPI, 1 .mu.g/ml, was used to coat Maxisorb.TM.
microtiter plates. Expression media, 100 .mu.l, from each member of
the His scan library, was added to two wells on the plate, in a
pair wise fashion. The plate was incubated on a shaker at room
temperature for 1 hr. The plate was washed 3.times. with PBST. PBS
(pH 7.0) was added to one well of the pair, 100 mM pH6.0 Citrate
buffer was added to the second well of the same pair. The plate was
incubated at 37.degree. C. with shaking for one hr. The plate was
washed 3.times. with PBST and developed using amplex red. A pH
7.0/pH 6.0 ratio was established to rank the sensitive muteins. The
ratio for wild type 2A8-g200 Fab was 1.0. The 10 clones that had a
ratio greater than 1.78 between pH 7.0 and pH 6.0 are shown in
Table 2 below.
TABLE-US-00002 TABLE 2 pH 6.0 TFPI Dissociation ELISA TFPI ELISA
Ratio Rank Mutation pH 7.0 pH 6.0 pH 7.0/pH 6.0 wt gA200Fab 5248
5354 0.98 1 VL-Y31H 913 133 6.84 2 VH-Y102H 3310 1079 3.07 3
VH-Y100H 2545 1431 1.78 4 VH-Y32H 3560 2585 1.38 5 VL-F48H 2068
1551 1.33 6 VH-S50H 2159 1637 1.32 7 VL-Y49H 2661 2044 1.3 8
VL-L27H 3422 2637 1.3 9 VL-V45H 2197 1771 1.24 10 VL-W90H 1833 1509
1.21
[0051] Purified 2A8-g200 variants in Fab format (referred to as wt
gA200Fab in Table 1) were tested using surface plasmon resonance
(Biacore). Surface plasmon resonance (Biacore T200) was used to
measure the dissociation rate of the antibodies. Human TFPI
(American Diagnostica) was amine coupled on a CM4 or CM5 chip using
the method suggested by Biacore, resulting in 100 to 300 RU of
immobilized TFPI. Purified 2A8-g200 variants were injected,
following by 40-minute dissociation either at pH7.4 or pH6.0
buffer. The antibodies were diluted in HBS-P buffer at different
concentrations and the flow rate was set to 50 .mu.l/min. After
each round of antibody injection, the chip was regenerated by
injecting 90 .mu.l of pH 1.5 glycine. The data set was evaluated
using BIAevaluation Software.
[0052] The dissociation constant (kd) for each 2A8-g200 variant
antibody was determined by using a model with the following
equation:
R=R.sub.0e.sup.-kd(t-t.sub.0.sup.)+offset
where R is the response at time t, R.sub.0 is the response at time
t.sub.0.--the start of dissociation, offset allows for a residual
response at the end of complete dissociation. A ratio of kd at pH
6.0 to kd at pH 7.4 was calculated for each 2A8-g200 variant. A
mutation with observed ratio of 2 was considered as pH-sensitive
mutation and could be used for construction of IgG variants of
2A8-g200.
[0053] For example, referring to FIG. 3, observed variations in
dissociation constant responses in the biocore assay at two
different pHs (pH 6.0 and pH 7.4) are shown for two exemplary
2A8-g100 light chain histidine substitution mutations: A. L-L27H
and B. L-Y31H.
[0054] Pharmacokinetic parameters of the antibodies were determined
after intravenous (i.v.) bolus administration to HemA mouse at 2
mg/kg. All the pharmacokinetic parameters were calculated using
WinNonLin software version 5.3.1 (Pharsight Corporation, Mountain
View, Calif.) non-compartmental model. The effect of histidine
mutations on the observed half-life of anti-TFPI monoclonal
antibodies in mouse plasma is shown in FIG. 4. 2A8-g200 with the
histidine mutations TPP2256 (L-Y31H/Y49H) and TPP2259 (L-Y31H)
increased the observed pK profiles over a 500 hour time span as
compared to the corresponding pK profile of 2A8-g200 without any
histidine substitution. Table 3 quantifies the increases in
half-life observed in the data of FIG. 4.
TABLE-US-00003 TABLE 3 pK parameters Dose T1/2 Antibody (mg/kg)
(hr) 2A8-g200 2 71 TTP-2256 2 ~331 TTP-2259 2 ~437
[0055] Therefore, the above designated antibodies that reduce TFPI
mediated TMDD and have a prolonged T1/2 would lead to less frequent
dosing and reduce the amount of material needed per dose.
Furthermore, the need for a lower dose may also make feasible
subcutaneous dosing where the dosing volume becomes a limiting
step, a process by which the antibody is removed from circulation
due to its interaction with a rapidly cleared target or by being
sequestered from the plasma due to its co-localization with its
target.
[0056] There will be various modifications, improvements, and
applications of the disclosed invention that will be apparent to
those of skill in the art, and the present application encompasses
such embodiments to the extent allowed by law. Although the present
invention has been described in the context of certain preferred
embodiments, the full scope of the invention is not so limited, but
is in accord with the scope of the following claims. All
references, patents, or other publications are specifically
incorporated by reference herein.
Sequence CWU 1
1
341117PRTHomo sapiens 1Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Arg Gly
Ser Ser Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Lys Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 2108PRTHomo sapiens 2Asp Ile
Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile
Tyr 35 40 45 Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Trp Asp Asp Gly Val Pro Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 3123PRTHomo sapiens 3Gln Val Gln Leu
Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu
Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30
Ser Ala Ala Trp Ser Trp Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35
40 45 Trp Leu Gly Ile Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Asp Tyr
Ala 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr
Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro
Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp
Lys His Trp Gly Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 4114PRTHomo sapiens 4Asp Ile Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Phe Ser 20 25 30 Asp
Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 Pro Gln Leu Leu Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Gln Gln Tyr 85 90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr
5117PRTArtificialVariant 5Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met Asp Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Arg
Gly Ser Ser Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 6117PRTArtificialVariant
6Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser
Tyr 20 25 30 Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Arg Gly Ser Arg Ser Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Tyr Arg
Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 7117PRTArtificialVariant 7Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25 30 Gly Met Asp
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Arg Gly Ser Arg Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
8117PRTArtificialVariant 8Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Arg Val Tyr 20 25 30 Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Asp Arg
Gly Ser Arg Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Val Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 9117PRTArtificialVariant
9Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser
Tyr 20 25 30 Gly Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Arg Gly Ser Ser Ser Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Tyr Arg
Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 10117PRTArtificialVariant 10Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Asp
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Arg Gly Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Leu Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
11117PRTArtificialVariant 11Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Arg Val Tyr 20 25 30 Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Asp Arg
Gly Ser Arg Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Val Tyr Arg Tyr Trp Phe Asp Tyr Trp Gly Gln Gly Thr
Leu 100 105 110 Val Thr Val Ser Ser 115 12108PRTArtificialVariant
12Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1
5 10 15 Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr
Ala 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val
Val Ile Phe 35 40 45 Tyr Asp Val Asn Arg Pro Ser Asp Ile Pro Glu
Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr
Ile Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys
Gln Ser Trp Asp Asp Gly Val Pro Trp 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Thr Val Leu Gly Gln 100 105 13108PRTArtificialVariant 13Asp
Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10
15 Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val
Ile Phe 35 40 45 Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg
Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile
Ser Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln
Ser Trp Leu Asp Gly Val Pro Trp 85 90 95 Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu Gly Gln 100 105 14108PRTArtificialVariant 14Asp Ile
Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile
Phe 35 40 45 Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe
Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
Gly Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser
Trp Trp Asp Gly Val Pro Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu
Thr Val Leu Gly Gln 100 105 15108PRTArtificialVariant 15Asp Ile Glu
Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr
Ala Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala 20 25
30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe
35 40 45 Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser
Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly
Thr Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp
Leu Asp Gly Val Pro Trp 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu Gly Gln 100 105 16108PRTArtificialVariant 16Asp Ile Glu Leu
Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala
Arg Ile Ser Cys Ser Gly Asp Asn Leu Arg Asn Tyr Tyr Ala 20 25 30
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe 35
40 45 Tyr Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly
Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr
Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Leu
Asp Gly Val Pro Trp 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Gly Gln 100 105 17108PRTArtificialVariant 17Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg
Ile Thr Cys Ser Gly Asp Asn Leu Pro Lys Tyr Tyr Ala 20 25 30 His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe 35 40
45 Tyr Asp Val Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln
Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Trp Ser
Ser Thr Pro Val 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly Gln 100 105 18108PRTArtificialVariant 18Ser Tyr Glu Leu Thr Gln
Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile
Thr Cys Ser Gly Asp Asn Leu Pro Lys Tyr Tyr Ala 20 25 30 His Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Phe 35 40 45
Tyr Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50
55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala
Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Trp Leu Ser Gly
Thr Pro Trp 85 90 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln 100 105 19123PRTArtificialVariant 19Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala
Trp Ser Trp Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp
Leu Gly Ile Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Gln Tyr Ala 50 55
60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn
65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr
Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp
Gly Phe Asp Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 20123PRTArtificialVariant 20Gln Val Gln Leu Gln Gln Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala
Trp Ser Trp Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp
Leu Gly Ile Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55
60 Val Ser Val Lys Ser Arg Ile Thr
Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu
Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala
Arg Trp His Ser Asp Lys His Trp Gly Phe Asp Asp 100 105 110 Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
21123PRTArtificialVariant 21Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala Trp Ser Trp
Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp Leu Gly Ile
Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp Gly Phe Asp
Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
22123PRTArtificialVariant 22Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Ser Trp
Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp Leu Gly Ile
Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp Gly Phe Asp
Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
23123PRTArtificialVariant 23Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala Trp Ser Trp
Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp Leu Gly Ile
Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp Gly Phe Asp
Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
24123PRTArtificialVariant 24Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala Trp Ser Trp
Ile Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp Leu Gly Ile
Ile Tyr Lys Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp Gly Phe Asp
Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
25123PRTArtificialVariant 25Glu Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala Trp Ser Trp
Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Ile
Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp Gly Phe Asp
Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
26123PRTArtificialVariant 26Gln Val Gln Leu Gln Gln Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ala Ala Trp Ser Trp
Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Ile
Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn Arg Tyr Ala 50 55 60 Val Ser
Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80
Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85
90 95 Tyr Tyr Cys Ala Arg Trp His Ser Asp Lys His Trp Gly Phe Asp
Asp 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
27114PRTArtificialVariant 27Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val Ile Ser 20 25 30 Phe Gly Ile Thr Tyr Leu
Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85
90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 Arg Thr 28114PRTArtificialVariant 28Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Phe Arg 20 25 30
Phe Gly Ile Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Gln Gln Tyr 85 90 95 Thr Ser Tyr Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr
29114PRTArtificialVariant 29Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val Phe Ser 20 25 30 Asp Gly Thr Thr Tyr Leu
Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85
90 95 Thr Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 Arg Thr 30114PRTArtificialVariant 30Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Phe Ser 20 25 30
Phe Gly Ile Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Gln Gln Tyr 85 90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr
31114PRTArtificialVariant 31Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val Phe Arg 20 25 30 Phe Gly Ile Thr Tyr Leu
Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85
90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 Arg Thr 32114PRTArtificialVariant 32Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Phe Arg 20 25 30
Asp Gly Ile Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Gln Gln Tyr 85 90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr
33114PRTArtificialVariant 33Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Val Phe Arg 20 25 30 Asp Gly Ile Thr Tyr Leu
Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85
90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105 110 Arg Thr 34114PRTArtificialVariant 34Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Phe Arg 20 25 30
Asp Gly Ile Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Lys Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Gln Gln Tyr 85 90 95 Asp Ser Tyr Pro Leu Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr
* * * * *
References