U.S. patent application number 14/764215 was filed with the patent office on 2015-12-17 for fibronectin based scaffold proteins.
The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY. Invention is credited to Jonathan H. Davis, Dasa LIPOVSEK.
Application Number | 20150361159 14/764215 |
Document ID | / |
Family ID | 50114573 |
Filed Date | 2015-12-17 |
United States Patent
Application |
20150361159 |
Kind Code |
A1 |
LIPOVSEK; Dasa ; et
al. |
December 17, 2015 |
FIBRONECTIN BASED SCAFFOLD PROTEINS
Abstract
Fibronectin type III (.sup.10Fn3) binding domains having novel
designs are provided. Also provided are .sup.10Fn3 domains having
combinations of modified regions that can bind to a desired
target.
Inventors: |
LIPOVSEK; Dasa; (Cambridge,
MA) ; Davis; Jonathan H.; (Auburndale, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRISTOL-MYERS SQUIBB COMPANY |
Princeton |
NJ |
US |
|
|
Family ID: |
50114573 |
Appl. No.: |
14/764215 |
Filed: |
January 30, 2014 |
PCT Filed: |
January 30, 2014 |
PCT NO: |
PCT/US2014/013784 |
371 Date: |
July 29, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61759639 |
Feb 1, 2013 |
|
|
|
Current U.S.
Class: |
506/9 ; 506/18;
530/395 |
Current CPC
Class: |
G01N 33/6845 20130101;
C07K 14/78 20130101; C07K 2317/76 20130101; C07K 2318/20 20130101;
C12N 15/1044 20130101; C07K 2317/92 20130101; C07K 16/244 20130101;
C07K 16/28 20130101; C07K 16/2833 20130101; C07K 16/2857
20130101 |
International
Class: |
C07K 14/78 20060101
C07K014/78 |
Claims
1. A polypeptide comprising a .sup.10Fn3 domain, wherein the
.sup.10Fn3 domain comprises an amino acid sequence wherein the
amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6
are represented by x.sub.3-12, the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15,
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx, or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14, or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxxx.sub.3-12xIx, or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12, or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx, or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14, or the amino acids corresponding to residues 23-29 of
SEQ ID NO: 1 or 6 are represented by x.sub.7, or the amino acids
corresponding to residues 25-29 of SEQ ID NO: 1 or 6 are
represented by x.sub.5, or the amino acids corresponding to
residues 26-29 of SEQ ID NO: 1 or 6 are represented by x.sub.4, or
the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or
6 are represented by x.sub.3, or the amino acids corresponding to
residues 24-28 of SEQ ID NO: 1 or 6 are represented by x.sub.5, or
the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or
6 are represented by x.sub.4, or the amino acids corresponding to
residues 26-28 of SEQ ID NO: 1 or 6 are represented by x.sub.3 or
the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or
6 are represented by xLxIxWx.sub.4, or the amino acids
corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are
represented by x.sub.4YxIx, or the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 or 6 are represented by x.sub.1-9Lx,
wherein x is any amino acid, and wherein the .sup.10Fn3 domain
binds specifically to a target that is not bound by a .sup.10Fn3
domain having an amino acid sequence consisting of SEQ ID NO: 1 or
6.
2-3. (canceled)
4. The polypeptide of claim 1, wherein the .sup.10Fn3 domain
comprises at least one amino acid variation in the amino acid
sequence corresponding to residues 17-30 of SEQ ID NO: 1 or 6.
5. The polypeptide of claim 1, wherein the .sup.10Fn3 domain
comprises at least one amino acid variation in the amino acid
sequence corresponding to residues 1-9 of SEQ ID NO: 1 or 6.
6-7. (canceled)
8. The polypeptide of claim 1, wherein at least 50% of residues x
are not the amino acid at the corresponding position in SEQ ID NO:
1 or 6.
9. The polypeptide of claim 1, wherein the .sup.10Fn3 domain binds
specifically to a target with a Kd of less than 500 nM.
10. A library comprising a plurality of polypeptides comprising a
.sup.10Fn3 domain, wherein the .sup.10Fn3 domain comprises an amino
acid sequence wherein the amino acids corresponding to residues
77-83 of SEQ ID NO: 1 or 6 are represented by x.sub.3-12, the amino
acids corresponding to residues 77-86 of SEQ ID NO: 1 or 6 are
represented by x.sub.6-15, or the amino acids corresponding to
residues 75-86 of SEQ ID NO: 1 or 6 are represented by x.sub.4-8Sx,
or the amino acids corresponding to residues 75-83 of SEQ ID NO: 1
or 6 are represented by x.sub.8-14, or the amino acids
corresponding to residues 73-89 of SEQ ID NO: 1 or 6 are
represented by xAxxxx.sub.3-12xIx, or the amino acids corresponding
to residues 76-86 of SEQ ID NO: 1 or 6 are represented by
xx.sub.3-12, or the amino acids corresponding to residues 76-86 of
SEQ ID NO: 1 or 6 are represented by xx.sub.4-8Sx, or the amino
acids corresponding to residues 76-83 of SEQ ID NO: 1 or 6 are
represented by xx.sub.8-14, or the amino acids corresponding to
residues 23-29 of SEQ ID NO: 1 or 6 are represented by x.sub.7, or
the amino acids corresponding to residues 25-29 of SEQ ID NO: 1 or
6 are represented by x.sub.5, or the amino acids corresponding to
residues 26-29 of SEQ ID NO: 1 or 6 are represented by x.sub.4, or
the amino acids corresponding to residues 27-29 of SEQ ID NO: 1 or
6 are represented by x.sub.3, or the amino acids corresponding to
residues 24-28 of SEQ ID NO: 1 or 6 are represented by x.sub.5, or
the amino acids corresponding to residues 25-28 of SEQ ID NO: 1 or
6 are represented by x.sub.4, or the amino acids corresponding to
residues 26-28 of SEQ ID NO: 1 or 6 are represented by x.sub.3 or
the amino acids corresponding to residues 17-26 of SEQ ID NO: 1 or
6 are represented by xLxIxWx.sub.4, or the amino acids
corresponding to residues 24-35 of SEQ ID NO: 1 or 6 are
represented by x.sub.4YxIx, or the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 or 6 are represented by x.sub.1-9Lx,
wherein x is any amino acid, and wherein the .sup.10Fn3 domain
binds specifically to a target that is not bound by a .sup.10Fn3
domain having an amino acid sequence consisting of SEQ ID NO: 1 or
6.
11-12. (canceled)
13. A method for identifying a polypeptide that binds to a target
comprising screening the library of claim 10 to identify a
polypeptide that binds to the target.
14-274. (canceled)
275. A polypeptide comprising a .sup.10Fn3 domain, wherein the
.sup.10Fn3 domain comprises at least one variable amino acid
sequence, wherein the amino acids corresponding to residues 1-9 of
SEQ ID NO: 1 or 6 are represented by x.sub.1-9Lx; or the amino
acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 consist of
the amino acid sequence GVSDVPGGSG, or a portion thereof, linked to
an amino acid sequence represented by x.sub.1-9Lx; and/or the amino
acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are
represented by x.sub.7; or the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 or 6 are represented by x.sub.5; or
the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or
6 are represented by x.sub.4; or the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 or 6 are represented by x.sub.3; or
the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or
6 are represented by x.sub.5; or the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4; or
the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or
6 are represented by x.sub.3; or the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLxIxWx.sub.4; or the amino acids corresponding to residues 24-35
of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx; and/or the
amino acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6
are represented by x.sub.4; or the amino acids corresponding to
residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx.sub.4;
or the amino acids corresponding to residues 51-60 of SEQ ID NO: 1
or 6 are represented by x.sub.6AxIx; and/or the amino acids
corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.3-12; or the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15;
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx; or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14; or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx; or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12; or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx; or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14; and x is any amino acid; and the .sup.10Fn3 domain
binds specifically to a target that is not bound by a .sup.10Fn3
domain having an amino acid sequence consisting of SEQ ID NO: 1 or
6.
276. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are
represented by x.sub.1-9Lx; or the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid
sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid
sequence represented by x.sub.1-9Lx; and the amino acids
corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are
represented by x.sub.7; or the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 or 6 are represented by x.sub.5; or
the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or
6 are represented by x.sub.4; or the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 or 6 are represented by x.sub.3; or
the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or
6 are represented by x.sub.5; or the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4; or
the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or
6 are represented by x.sub.3; or the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLsIxWx.sub.4; or the amino acids corresponding to residues 24-35
of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx.
277. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are
represented by x.sub.1-9Lx; or the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid
sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid
sequence represented by x.sub.1-9Lx; and the amino acids
corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.3-12; or the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15;
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx; or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14; or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx; or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12; or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx; or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14.
278. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6
are represented by x.sub.7; or the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 or 6 are represented by x.sub.5; or
the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or
6 are represented by x.sub.4; or the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 or 6 are represented by x.sub.3; or
the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or
6 are represented by x.sub.5; or the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4; or
the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or
6 are represented by x.sub.3; or the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLxIxWx.sub.4; or the amino acids corresponding to residues 24-35
of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx; and/or the
amino acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6
are represented by x.sub.3-12; or the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15;
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx; or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14; or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx; or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12; or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx; or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14.
279. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 1-9 of SEQ ID NO: 1 or 6 are
represented by x.sub.1-9Lx; or the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 or 6 consist of the amino acid
sequence GVSDVPGGSG, or a portion thereof, linked to an amino acid
sequence represented by x.sub.1-9Lx; and the amino acids
corresponding to residues 23-29 of SEQ ID NO: 1 or 6 are
represented by x.sub.7; or the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 or 6 are represented by x.sub.5; or
the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or
6 are represented by x.sub.4; or the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 or 6 are represented by x.sub.3; or
the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or
6 are represented by x.sub.5; or the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4; or
the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or
6 are represented by x.sub.3; or the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLxIxWx.sub.4; or the amino acids corresponding to residues 24-35
of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx; and the amino
acids corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.3-12; or the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15;
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx; or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14; or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx; or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12; or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx; or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14.
280. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6
are represented by x.sub.7; or the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 or 6 are represented by x.sub.5; or
the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or
6 are represented by x.sub.4; or the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 or 6 are represented by x.sub.3; or
the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or
6 are represented by x.sub.5; or the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4; or
the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or
6 are represented by x.sub.3; or the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLxIxWx.sub.4; or the amino acids corresponding to residues 24-35
of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx; and the amino
acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6 are
represented by x.sub.4; or the amino acids corresponding to
residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx.sub.4;
or the amino acids corresponding to residues 51-60 of SEQ ID NO: 1
or 6 are represented by x.sub.6AxIx.
281. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6
are represented by x.sub.4; or the amino acids corresponding to
residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx.sub.4;
or the amino acids corresponding to residues 51-60 of SEQ ID NO: 1
or 6 are represented by x.sub.6AxIx; and the amino acids
corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.3-12; or the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15;
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx; or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14; or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx; or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12; or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx; or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14.
282. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at last one variable amino acid sequence, wherein the
amino acids corresponding to residues 23-29 of SEQ ID NO: 1 or 6
are represented by x.sub.7; or the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 or 6 are represented by x.sub.5; or
the amino acids corresponding to residues 26-29 of SEQ ID NO: 1 or
6 are represented by x.sub.4; or the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 or 6 are represented by x.sub.3; or
the amino acids corresponding to residues 24-28 of SEQ ID NO: 1 or
6 are represented by x.sub.5; or the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4; or
the amino acids corresponding to residues 26-28 of SEQ ID NO: 1 or
6 are represented by x.sub.3; or the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLxIxWx.sub.4; or the amino acids corresponding to residues 24-35
of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx; and the amino
acids corresponding to residues 52-55 of SEQ ID NO: 1 or 6 are
represented by x.sub.4; or the amino acids corresponding to
residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx.sub.4;
or the amino acids corresponding to residues 51-60 of SEQ ID NO: 1
or 6 are represented by x.sub.6AxIx; and the amino acids
corresponding to residues 77-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.3-12; or the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15;
or the amino acids corresponding to residues 75-86 of SEQ ID NO: 1
or 6 are represented by x.sub.4-8Sx; or the amino acids
corresponding to residues 75-83 of SEQ ID NO: 1 or 6 are
represented by x.sub.8-14; or the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx; or the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12; or the
amino acids corresponding to residues 76-86 of SEQ ID NO: 1 or 6
are represented by xx.sub.4-8Sx; or the amino acids corresponding
to residues 76-83 of SEQ ID NO: 1 or 6 are represented by
xx.sub.8-14.
283. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
comprises at most 20 additional amino acid variations relative to
SEQ ID NO: 1 or 6.
284. The polypeptide of claim 275, wherein the amino acid sequence
of the .sup.10Fn3 domain that is located outside of the variable
regions is at least 90% identical to the corresponding regions in
SEQ ID NO: 1 or 6.
285. The polypeptide of claim 275, wherein the .sup.10Fn3 domain
does not comprise any other amino acid variation relative to SEQ ID
NO: 1 or 6.
286. The polypeptide of claim 285, wherein the .sup.10Fn3 domain
comprises an amino acid sequence selected from the group of SEQ ID
NOs: 76-131.
287. The polypeptide of claim 283, wherein the .sup.10Fn3 domain
comprises an amino acid sequence that differs from an amino acid
sequence selected from SEQ ID NOs: 76-131 outside of the variable
regions in at most 10 amino acids.
288. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application No. 61/759,639, filed Feb. 1, 2013, the contents of
which are specifically incorporated by reference herein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing,
submitted herewith, which is hereby incorporated by reference in
its entirety.
INTRODUCTION
[0003] Fibronectin is a large protein which plays essential roles
in the formation of extracellular matrix and cell-cell
interactions; it consists of many repeats of three types (types I,
II, and III) of small domains. Fibronectin type III domains (Fn3)
are a large subfamily, members of which are frequently found as
portions of cell adhesion molecules, cell surface hormone and
cytokine receptors, chaperones, and carbohydrate-binding domains.
For reviews see Bork & Doolittle, Proc Natl Acad Sci USA
89(19):8990-4 (1992); Bork et al., J Mol Biol. 242(4):309-20
(1994); Campbell & Spitzfaden, Structure 2(5):333-7 (1994);
Harpez & Chothia, J Mol Biol. 238(4):528-39 (1994)).
[0004] Fibronectin based scaffolds are a family of proteins having
an immunoglobulin like fold. These proteins, which generally make
use of a scaffold derived from a fibronectin type III (Fn3) or
Fn3-like domain, function in a manner characteristic of natural or
engineered antibodies (that is, polyclonal, monoclonal, or
single-chain antibodies) and, in addition, possess structural
advantages. Specifically, the structures of these antibody mimics
have frequently been optimized for optimal folding, stability, and
solubility, even under conditions that normally lead to the loss of
structure and function in antibodies. An example of
fibronectin-based scaffold proteins are Adnectins.TM. (Adnexus, a
wholly owned subsidiary of Bristol-Myers Squibb). It has been shown
that the CDR-like loop regions of the fibronectin based scaffolds
can be modified to evolve a protein capable of binding to any
compound of interest. For example, U.S. Pat. No. 7,115,396
describes Fn3 domain proteins wherein alterations to the BC, DE,
and FG loops result in high affinity TNF.alpha. binders. U.S. Pat.
No. 7,858,739 describes Fn3 domain proteins wherein alterations to
the BC, DE, and FG loops result in high affinity VEGFR2
binders.
[0005] It would be advantageous to obtain further improved
fibronectin domain scaffold proteins bind with high affinity to a
target and that can be used for both therapeutic and diagnostic
purposes.
SUMMARY
[0006] One aspect of the application provides for fibronectin based
scaffold polypeptides comprising combinations of modified loops and
scaffold regions, e.g., the .beta.-strands that are associated with
improved target binding. Another aspect of the application provides
for fibronectin based scaffold polypeptides that are associated
with reduced immunogenicity.
[0007] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises (i) a modification in the
amino acid sequence of at least one north pole loop selected from
the BC, DE and FG loops relative to the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6), and (ii) a
modification in the amino acid sequence of at least one south pole
loop selected from the AB, CD and EF loops relative to the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO: 1 or 6), wherein the at least one modified north pole loop and
the at least one modified south pole loop contribute to binding the
same target. In some embodiments, at least one of the north pole
loops or at least one of the south pole loops of the polypeptide
has the amino acid sequence of the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6).
[0008] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises (i) a modification in the
amino acid sequence of at least one of loops AB, BC, CD, DE, EF, or
FG relative to the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6), and (ii) a modification in
the amino acid sequence of at least one .beta.-strand relative to
the corresponding .beta.-strand of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6), wherein the at least one modified loop
and the at least one modified .beta.-strand contribute to binding
the same target. In some embodiments, the polypeptides may comprise
modified amino acid sequences in at least one .beta.-strand and at
least two loops. In some embodiments, at least one modified loop of
the polypeptide is a north pole loop selected from the BC, DE and
FG loops and at least one modified loop is a south pole loop
selected from the AB, CD and EF loops and both loops contribute to
binding to the target. In some embodiments, at least one loop is
not modified, i.e., at least one loop has the amino acid sequence
of the corresponding loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6).
[0009] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of the CD and FG loops relative to the sequences of
the corresponding loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6), and wherein the CD and FG loops contribute to
binding to the same target. In some embodiments, at least 3, 4, 5,
6, 7, 8, 9, 10 or 11 of the amino acids of the CD loop are modified
relative to the sequence of the CD loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, one or
more amino acid residues of the CD loop corresponding to amino acid
residues 46 or 47 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO: 1 or 6) are the same as the wild-type amino acids at those
positions. In some embodiments, at least 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 or 13 of the amino acids of the FG loop are modified
relative to the sequence of the FG loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, one or
more amino acid residues of the FG loop corresponding to amino acid
residues 75 or 87 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO: 1 or 6) are the same as the wild-type amino acids at those
positions. In some embodiments, the amino acid sequence of the CD
loop, the FG loop, or both are extended in length or reduced in
length relative to the amino acid sequence of the corresponding
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6).
Combinations thereof are also contemplated. For example, the amino
acid sequence of at least one of the CD and FG loops may be
extended in length relative to the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6) and the amino
acid sequence of at least one of the CD and FG loops may be reduced
in length relative to the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments,
polypeptides comprising .sup.10Fn3 domains having modified CD and
FG loops, may further comprise amino acid sequence modifications in
one or more of .beta.-strand C, and .beta.-strand D, .beta.-strand
F and/or .beta.-strand G relative to the sequences of the
corresponding .beta.-strands of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6). In some embodiments, the polypeptides
having modified CD and FG loops may further comprise an amino acid
sequence modification in at least a portion of the BC loop, such
as, for example, modifications in one or more amino acid residues
of the BC loop corresponding to amino acid residues 30 and 31 of
the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some
embodiments, one or more of the modified .beta.-strands, together
with the modified loops, contribute to binding to the same target.
In some embodiments, one or more of the AB, DE and EF loops are not
modified, i.e., the loops have the amino acid sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO: 1 or 6).
[0010] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of the CD and DE loops relative to the sequences of
the corresponding loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6), and wherein the CD and DE loops contribute to
binding to the same target. The polypeptide may further comprise
modifications in the amino acid sequences of one or more of the EF
loop, .beta.-strand C, .beta.-strand D, and/or .beta.-strand F, and
such additional modification may contribute to binding to the same
target together with the CD and DE loops. In some embodiments, at
least 10, 15, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or
all 31 of the residues between the amino acids corresponding to
residues 36 through 66 of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6) have been modified relative to the
corresponding residues in the wild-type sequence. In some
embodiments, the CD loop is extended in length or reduced in length
relative to the CD loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6).
[0011] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of the EF and FG loops relative to the sequences of
the corresponding loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6), and wherein the EF and FG loops contribute to
binding to the same target. In some embodiments, the polypeptides
may further comprise amino acid sequence modifications in one or
more of the AB loop, .beta.-strand A and/or .beta.-strand G, and
such additional modifications may contribute to binding to the
target together with the EF and FG loops. In some embodiments, the
polypeptides may further comprise sequence modifications in the
N-terminus and/or C-terminus. In particular, the amino acid
sequence of the first 7 amino acids or the amino acid sequence of
the amino acids corresponding to residues 93 through 97 of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6) may be
modified relative to the corresponding residues in the wild-type
sequence. These additional modifications at the termini may also
contribute to binding to the target along with the other sequence
modifications. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14 or all 15 of the first 15 amino acid residues
of the .sup.10Fn3 domain may be modified relative to the
corresponding residues in the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6). In some embodiments, at least 3, 4 or 5 of the
amino acid residues of the EF loop may be modified relative to the
corresponding residues in the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6). In some embodiments, at least 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all 18 of the residues
between the amino acids corresponding to residues 80 through 97 of
the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6) may be
modified relative to the corresponding residues in the wild-type
sequence. In some embodiments, the amino acid sequence of the FG
loop is extended in length or reduced in length relative to the FG
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or
6).
[0012] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of .beta.-strand A, loop AB, .beta.-strand B, loop
CD, .beta.-strand E, loop EF, and .beta.-strand F relative to the
sequences of the corresponding .beta.-strands and loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6), and wherein
the modified loops and strands contribute to binding to the same
target. In some embodiments, the amino acid sequence of the CD loop
is extended in length or reduced in length relative to the CD loop
of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). In
some embodiments, the polypeptides may further comprise a
modification in the amino acid sequence of .beta.-strand G and/or
the C terminal tail.
[0013] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises a sequence modification in
the FG loop relative to the sequence of amino acid residues 77-83
of loop FG of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1
or 6), and wherein the .sup.10Fn3 binds to a target with a K.sub.d
of less than 500 nM. In some embodiments, the portion of the FG
loop corresponding to amino acid residues 77-83 of the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1 or 6) is extended in length
or reduced in length relative to the sequence of amino acid
residues 77-83 of the wild-type FG loop. In some embodiments, the
FG loop alone mediates binding to the target. In some embodiments,
one of more of the AB, BC, CD, DE or EF loops has the sequence of
the corresponding loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6).
[0014] In some embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises a sequence modification in
the BC loop and at least one of .beta.-strand B or .beta.-strand C
relative to the sequences of the corresponding loop and
.beta.-strands of the wild-type human .sup.10Fn3 domain (SEQ ID NO:
1 or 6), and wherein the .sup.10Fn3 domain has reduced
immunogenicity relative to an equivalent .sup.10Fn3 domain that
does not have a sequence modification in at least one of
.beta.-strand B or .beta.-strand C relative to wild-type. In some
embodiments, the amino acid sequence of the BC loop is extended in
length or reduced in length relative to the amino acid sequence of
the BC loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1
or 6). In some embodiments, the .sup.10Fn3 domain further comprises
a modification in the amino acid sequence of the first 7 amino acid
residues relative to the amino acid sequence of the first 7 amino
acid residues of the wild-type human .sup.10Fn3 domain (SEQ ID NO:
1 or 6). In some embodiments, the .sup.10Fn3 domain further
comprises a modification in the DE loop, the FG loop, or both,
relative to the sequences of the corresponding loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some
embodiments, the amino acid sequence of the DE loop is extended in
length or reduced in length relative to the amino acid sequence of
the DE loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1
or 6). In some embodiments, the amino acid sequence of the FG loop
is extended in length or reduced in length relative to the amino
acid sequence of the FG loop of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6). In some embodiments, the .sup.10Fn3
domain comprises a sequence modification in the DE loop, and
further comprises a sequence modification in .beta.-strand D,
.beta.-strand E, or both, relative to the sequences of the
corresponding .beta.-strands of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6). In some embodiments, the .sup.10Fn3
domain comprises a sequence modification in the FG loop, and
further comprises a sequence modification in .beta.-strand F,
.beta.-strand G, or both, relative to the sequences of the
corresponding .beta.-strands of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6).
[0015] In some embodiments, at least a portion of the BC loop of
the polypeptides provided herein have the amino acid sequence of
the corresponding loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6). For example, the first 1, 2, 3, 4, 5, 6, 7 or
8 residues of the BC loop may be the same as the corresponding
residues in the BC loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6). In still other embodiments, the entire BC loop
has the amino acid sequence of the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some
embodiments, polypeptides having at least a portion of the BC loop
with the wild-type sequence have reduced immunogenicity relative to
an equivalent polypeptide having additional modifications in the BC
loop.
[0016] In particular embodiments, the polypeptides provided herein
comprise a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises a sequence modification in
a portion of the BC loop and a portion of the FG loop relative to
the sequence of the corresponding loops of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6), and wherein the .sup.10Fn3
domain has reduced immunogenicity relative to an equivalent
.sup.10Fn3 domain having a greater portion of the BC loop modified
relative to the wild-type BC loop. In some embodiments, the portion
of the BC loop that is modified may correspond to residues 28-29,
27-29, 26-29, 25-29, or 24-29 of the BC loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, the
portion of the FG loop that is modified may corresponds to residues
77-79, 77-80, 77-81, 77-82, 77-83, 77-84, 77-85, or 77-86 of the FG
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6).
In some embodiments, the portion of the FG loop that is modified
has an insertion or deletion relative to the corresponding portion
of the FG loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:
1 or 6). In some embodiments, the BC and FG loop contribute to
binding to the target. In some embodiments, the .sup.10Fn3 domain
further comprises a sequence modification in a portion of the DE
loop relative to the sequence of the DE loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some embodiments, the
portion of the DE loop that is modified corresponds to residues 52
and 53 of the BC loop of the wild-type human .sup.10Fn3 domain (SEQ
ID NO: 1). In some embodiments, the .sup.10Fn3 domain has reduced
immunogenicity relative to an equivalent .sup.10Fn3 domain further
comprising modifications in one or more of amino acid residues
23-27, relative to the corresponding positions in the wild-type BC
loop. In some embodiments, the .sup.10Fn3 domain binds to a target
with a K.sub.d of less than 500 nM.
[0017] In some embodiments, the FG loop of the polypeptides
provided herein does not contain an RGD integrin binding site.
[0018] In some embodiments, the hydrophobic core residues of the
polypeptides provided herein have not been modified relative to the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6).
[0019] In some embodiments, the amino acid sequence of at least one
of the modified loops of the polypeptides provided herein has been
extended in length relative to the amino acid sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO: 1 or 6). In other embodiments, the amino acid sequence of at
least one of the modified loops has been reduced in length relative
to the amino acid sequence of the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6).
[0020] In some embodiments, the amino acid sequence of the C
terminal tail of the polypeptides provided herein is modified
relative to the amino acid sequence of the C-terminal tail of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). In some
embodiments, the amino acid sequence of the first 7 amino acid
residues is modified relative to the amino acid sequence of the
first 7 amino acid residues of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6). In other embodiments, the polypeptide
has from 1-7 amino acids truncated from the N-terminus relative to
the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6), from 1-9
amino acids truncated from the C-terminus relative to the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1), or both.
[0021] In some embodiments, the polypeptides provided herein have
at least 50% identity to the amino acid sequence of the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1, 2, 60 or 6). In other
embodiments, the polypeptide has at least 65% identity to the amino
acid sequence of the wild-type human .sup.10Fn3 domain (SEQ ID NO:
1, 2, 60 or 6). In certain embodiments, the .sup.10Fn3 domains
comprise an amino acid sequence that is at least 60, 70, 80 or 90%
identical to the naturally occurring human .sup.10Fn3 domain
represented by SEQ ID NO: 1, 2, 60 or 6.
[0022] In some embodiments, the polypeptides provided herein
comprise a fibronectin type III tenth (.sup.10Fn3) domain, wherein
the .sup.10Fn3 domain comprises an amino acid sequence having at
least 60% identity to SEQ ID NO: 2 or 60 and binds to a target
molecule with a K.sub.d of less than 100 nM, and wherein the
.sup.10Fn3 domain further comprises a C-terminal tail that does not
contain a DK sequence. In some embodiments, the C-terminal tail
comprises the amino acid sequence of SEQ ID NO: 7. In some
embodiments, the C-terminal tail further comprises a cysteine
residue. In other embodiments, the C-terminal tail comprises the
sequence of SEQ ID NO:8. In other embodiments, the C-terminal tail
may comprise the sequence of any one of SEQ ID NOs: 23-31.
[0023] In certain embodiments, the fibronectin based scaffold
proteins bind to a target that is not bound by a wild-type
.sup.10Fn3 domain.
[0024] In some embodiments, the .sup.10Fn3 domains of the
fibronectin based scaffold protein further comprises an N-terminal
extension comprising from 1-10 amino acids. In certain embodiments,
the .sup.10Fn3 domain comprises an M, MG or G N-terminal to first
amino acid of SEQ ID NO: 1 or 6. In other embodiments, the amino
acid residues corresponding to amino acids 1-8 of SEQ ID NO:1 or 6
are replaced with any one of SEQ ID NOs: 9-11 or 16-21.
[0025] In some embodiments, the fibronectin based scaffold proteins
further comprise one or more pharmacokinetic (PK) moieties selected
from: a polyoxyalkylene moiety, a human serum albumin binding
protein, sialic acid, human serum albumin, transferrin, IgG, an IgG
binding protein, and an Fc fragment. In some embodiments, the PK
moiety is the polyoxyalkylene moiety and said polyoxyalkylene
moiety is polyethylene glycol (PEG). In some embodiments, the PEG
moiety is covalently linked to the fibronectin based scaffold
protein via a Cys or Lys amino acid. In some embodiments, the PEG
is between about 0.5 kDa and about 100 kDa.
[0026] In certain embodiments, the application provides
pharmaceutically acceptable compositions comprising the .sup.10Fn3
domains described herein. In some embodiments, the composition is
essentially pyrogen free. In some embodiments, the composition is
substantially free of microbial contamination making it suitable
for in vivo administration. The composition may be formulated, for
example, for intravenous (IV), intraperiotoneal (IP) or
subcutaneous (SubQ) administration. In some embodiments, the
composition comprises a physiologically acceptable carrier. In some
embodiments, the pH of the composition is between 4.0-6.5, between
4.0-5.5, or is equal to 4.0, 4.5, 5.0 or 5.5. In some embodiments,
the concentration of the fibronectin based scaffold protein is 5
mg/ml in the composition.
[0027] In certain embodiments, the application provides a nucleic
acid encoding the .sup.10Fn3 domains as described herein. Vectors
containing polynucleotides for such proteins are included as well.
Suitable vectors include, for example, expression vectors. A
further aspect of the application provides for a cell, comprising a
polynucleotide, vector, or expression vector, encoding a .sup.10Fn3
domain. Sequences are preferably optimized to maximize expression
in the cell type used. In some embodiments, expression is in a
bacterial cell, such as E. coli. In other embodiments, expression
is in a mammalian cell. In one embodiment, the cell expresses a
protein comprising a .sup.10Fn3 domain as described herein. In
certain embodiments, the polynucleotides encoding a .sup.10Fn3
domain are codon optimized for expression in the selected cell
type. Also provided are methods for producing a .sup.10Fn3 domain
as described herein, comprising culturing a host cell comprising a
nucleic acid, vector, or expression vector encoding a .sup.10Fn3
domain and recovering the expressed protein from the culture.
[0028] In certain embodiments, the application provides libraries
of the fibronectin based scaffold proteins described herein. The
libraries provided herein may comprise, for example, at least
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.12, 10.sup.13, or 10.sup.14, or more members. Also provided
are methods for isolating a fibronectin based scaffold protein that
specifically binds to a target of interest from one of the
libraries described herein. For example, a library isolation method
may comprise, for example, contacting a library of fibronectin
based scaffold proteins with a target of interest, and isolating
members of the library that bind to the target (e.g., with a
particular affinity or under suitable wash conditions). The
isolation step may be carried out using any suitable method, such
as phage display or mRNA display. Similarly, target binding may be
conducted using any suitable method such as immobilizing the target
on a solid support (e.g., a column, chip, bead, etc.) and mixing
the immobilized target with the library under conditions suitable
to allow protein binding. The bound library members may then be
separated from unbound library members to yield an isolated
fibronectin based scaffold protein that binds to the target. In
certain embodiments, the isolation method may involve repeated
rounds of target binding and isolation steps.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1: The wildtype .sup.10Fn3 amino acid sequence (SEQ ID
NO: 1) in which the hydrophobic core amino acid residues are
indicated. The beta-strands are in bold, loop regions are
designated by letter pairs, and the hydrophobic core residues are
underlined in bold. Amino acids 95-101 correspond to a tail, which
when absent from a .sup.10Fn3 molecule having SEQ ID NO: 1 forms a
10Fn3 molecule having SEQ ID NO: 6.
[0030] FIG. 2 (A-F): Wildtype .sup.10Fn3 amino acid sequences (SEQ
ID NO: 1 or 6) in which amino acid positions that may be mutated to
provide a representative modified .sup.10Fn3 polypeptide patch
library are indicated in the full-length sequence (SEQ ID NO: 1).
Potential positions that may be modified to generate each of the
respective classes of patch library .sup.10Fn3 polypeptides are in
bold and underlined. Any one of, or combination of, the positions
indicated may be mutated to generate either Northwest Binders (FIG.
2A), Northeast Binders (FIG. 2B), West Side Binders (FIG. 2C),
South-Front Binders (FIG. 2D), AG Strand Binders (FIG. 2E) and
South West Binders (FIG. 2F).
[0031] FIG. 3 (A-F): Crystal structures of the wildtype .sup.10Fn3
domain, in which views of different possible binding interfaces are
shown. Residues that may be varied from wild-type are shown in
black. Sticks are added to varied residues that are not members of
one of the six loops. Crystal structure views of the Northwest
binding interface (FIG. 3A), Northeast binding interface (FIG. 3B),
West Side binding interface (FIG. 3C), South-Front binding
interface (FIG. 3D), AG Strand binding interface (FIG. 3E) and
South West binding interface (FIG. 3F) of the wildtype .sup.10Fn3
domain are shown.
[0032] FIG. 4: HLA-binding data showing the IC.sub.50 binding
affinity (04) of five different HLA allele proteins to five
different peptide segments of the .sup.10Fn3 polypeptide. SEQ ID
NOs: 58, 51 and 52 are loop region clusters of the BC, DE and FG
loops, respectively, with loop region residues underlined. SEQ ID
NOs: 53 and 54 are a wildtype and modified scaffold region segment
of the .sup.10Fn3 polypeptide, respectively. As indicated, the BC
loop region cluster (SEQ ID NO: 58) and the two scaffold region
peptide segments tested (SEQ ID NOs: 53 and 54) were strong binders
(<25 .mu.M) of most of the HLA allele proteins tested. The
predicted immunodominant regions of the scaffold region peptide
segments (SEQ ID NOs: 53 and 54) are underlined.
[0033] FIG. 5: Notch 1:murine DLL4 competition assay results
showing that the WS-LI1 binders of SEQ ID NOs: 3 and 4 were capable
of 100% inhibition, and that the WS-LI1 binder of SEQ ID NO: 5 was
capable of 75% inhibition, of the interaction between Notch 1 and
murine DLL4, as determined by Biacore analysis.
[0034] FIG. 6: Size Exclusion Chromatography results showing that
the WS-LI1 binder of SEQ ID NO: 3 was predominantly monomeric,
while the WS-LI1 binders of SEQ ID NOs: 4 and 5 contained a mixture
of monomeric and aggregated proteins. Bold traces correspond to the
WS-LI1 binder tested, and non-bold traces correspond to molecular
weight markers, with the expected elution of the WS-LI1 binder
monomers to be eluted between the 3.sup.rd and 5.sup.th marker
peaks.
[0035] FIG. 7: Size Exclusion Chromatography results showing that
the WS-LI1 binders of SEQ ID NOs: 45-47 were predominantly
monomeric. Bold traces correspond to the WS-LI1 binders tested, and
non-bold traces correspond to molecular weight markers, with the
expected elution of the WS-LI1 binder monomers to be eluted between
the 3.sup.rd and 5.sup.th marker peaks.
[0036] FIG. 8: Size Exclusion Chromatography results showing that
the WS1 binders of SEQ ID NOs: 48-49 were predominantly monomeric.
Bold traces correspond to the WS1 binders tested, and non-bold
traces correspond to molecular weight markers, with the expected
elution of the WS1 binder monomers to be eluted between the
3.sup.rd and 5.sup.th marker peaks.
[0037] FIG. 9 (A-C): Library designs for non-traditional .sup.10Fn3
binders. FIG. 9A shows library designs in which the BC loop is
substantially or completely unmodified (i.e., all or most of the BC
loop is left as the wild-type sequence). All of the CD and FG loops
of the libraries depicted in 9A may be varied in size, in
particular, loop CD of SP1, WS2', WS2'-CD, Front3 and CD1-loop may
be varied in length. Loop FG of Back3 may also be varied in size,
and the first three amino acids of CD1-loop (i.e., VSD) may be
deleted. All amino acids in loops CD of Front 3 and CD1-loop can be
varied. R93 of SP3 can be varied. FIG. 9B shows library designs in
which varying portions of the N-terminus of the BC loop have been
left as wild-type. FIG. 9C shows library designs in which the BC
loop and one or both of the .beta.-strand regions flanking the BC
loop are modified. Library NP-6 may also be constructed by keeping
the threonine at position 71 constant and/or by keeping the length
of amino acids 1-7 constant. Library NP4-5 may also be constructed
by keeping the threonine at position 71 constant, and/or by keeping
the length of amino acids 1-7 constants, and/or by keeping loop BC
constant. In each of FIGS. 9A-C, the full-length wild-type
.sup.10Fn3 domain (SEQ ID NO: 1) is shown at the top with numbering
from amino acid 1 to amino acid 101 and markings indicating the
loop and strand regions. Below the depiction of the .sup.10Fn3
wild-type domain is a depiction of a classic north pole library
design (i.e., with the BC, DE and FG loops modified). The
non-traditional library designs are shown below the classic north
pole library design. Positions that may be modified by substitution
are indicated in bold and underlined, regions that may be modified
by substitution, insertion and/or deletion are in bold and boxed,
and amino acid residues that are non-wild-type are shaded. In FIG.
9B, all sequences are based on SEQ ID NO: 1. In FIG. 9C, the WT
.sup.10Fn3, Classic NP, NP1, NE1, NP4-5, NP6-1, and NW2 sequences
are disclosed as SEQ ID NO: 1; and all remaining sequences are
disclosed as SEQ ID NO: 59. In FIG. 9A, the WT .sup.10Fn3, Classic
NP, WS1, WS2, WS3, LI-3(a), WS-LI1, WS2' and WS2'-CD are based on
SEQ ID NO: 1; and all remaining sequences are based on SEQ ID NO:
59. All libraries may also be based on SEQ ID NO: 6 or SEQ ID NO:
12, i.e., lacking amino acids 95-101 of SEQ ID NOS 1 and 59,
respectively.
[0038] FIG. 10: Sequence alignment of 8 .sup.10Fn3 polypeptides
(Adnectin-1 to Adnectin-8), which bind specifically to human PXR
ligand binding domain, with the parent .sup.10Fn3 domain (SEQ ID
NO: 1). The location of the .beta.-strands is indicated by the
arrows below the sequence alignment, with corresponding amino acids
indicated in bold. Adnectins-3 (SEQ ID NO: 62) and -4 (SEQ ID NO:
63) correspond to SEQ ID NOs: 48 and 49 with an additional 6xHis
tail (SEQ ID NO: 44). Adnectins-1, -2, -5, -6, -7 and -8 (SEQ ID
NOs: 70-72 and 13-15, respectively) correspond to SEQ ID NOs:
64-69, respectively, with an additional 6xHis tail (SEQ ID NO:
44).
[0039] FIG. 11: Histogram showing the degree of binding of (from
left to right) vitronectin, fibronectin, a non-binding control
adnectin (with RGD changed to RGE), and three different .sup.10Fn3
molecules binding to a specific target and not comprising an RGD
sequence (.sup.10Fn3 A, B and C, respectively) to immobilized
integrin AlphaV-Beta3.
[0040] FIG. 12: Amino acid sequences of wild-type human .sup.10Fn3
(SEQ ID NO: 6) (top line) and those of libraries WS4, WS5, WS6 and
WS7. The underlined bolded and boxed amino acids can be changed by
substitution, deletion and addition. The underlined amino acids can
be changed by substitution.
[0041] FIG. 13 (A-C): Amino acid sequence of .sup.10Fn3 molecules
(SEQ ID NOs: 73-131), wherein x is any amino acid or x is any amino
acid except the amino acid at the corresponding position in the
wild-type .sup.10Fn3 molecule (SEQ ID NO: 1 or 6). The subscript
after "x" indicates the number of amino acid residues. For example,
"x.sub.3-5" refers to 3 to 5 amino acid residues.
[0042] FIG. 14 (A-E): Amino acid sequences of exemplary .sup.10Fn3
molecules having designs shown in FIG. 13, or differing therefrom
in at most a few, e.g., 1, 2, 3, 4 or 5 amino acids.
DETAILED DESCRIPTION
Definitions
[0043] By a "polypeptide" is meant any sequence of two or more
amino acids, regardless of length, post-translation modification,
or function. "Polypeptide," "peptide," and "protein" are used
interchangeably herein. Polypeptides can include natural amino
acids and non-natural amino acids such as those described in U.S.
Pat. No. 6,559,126, incorporated herein by reference. Polypeptides
can also be modified in any of a variety of standard chemical ways
(e.g., an amino acid can be modified with a protecting group; the
carboxy-terminal amino acid can be made into a terminal amide
group; the amino-terminal residue can be modified with groups to,
e.g., enhance lipophilicity; or the polypeptide can be chemically
glycosylated or otherwise modified to increase stability or in vivo
half-life). Polypeptide modifications can include the attachment of
another structure such as a cyclic compound or other molecule to
the polypeptide and can also include polypeptides that contain one
or more amino acids in an altered configuration (i.e., R or S; or,
L or D).
[0044] A "region" of a .sup.10Fn3 domain as used herein refers to
either a loop (AB, BC, CD, DE, EF and FG), a .beta.-strand (A, B,
C, D, E, F and G), the N-terminus (corresponding to amino acid
residues 1-7 of SEQ ID NO: 1), or the C-terminus (corresponding to
amino acid residues 93-101 of SEQ ID NO: 1) of the human .sup.10Fn3
domain.
[0045] A "north pole loop" refers to any one of the BC, DE and FG
loops of a fibronectin human fibronectin type 3 tenth (.sup.10Fn3)
domain.
[0046] A "south pole loop" refers to any one of the AB, CD and EF
loops of a fibronectin human fibronectin type 3 tenth (.sup.10Fn3)
domain.
[0047] A "scaffold region" refers to any non-loop region of a human
.sup.10Fn3 domain. The scaffold region includes the A, B, C, D, E,
F and G .beta.-strands as well as the N-terminal region (amino
acids corresponding to residues 1-7 of SEQ ID NO: 1) and the
C-terminal region (amino acids corresponding to residues 93-101 of
SEQ ID NO: 1).
[0048] "Percent (%) amino acid sequence identity" herein is defined
as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid residues in a selected
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the
full-length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are obtained as
described below by using the sequence comparison computer program
ALIGN-2. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. has been filed with user documentation
in the U.S. Copyright Office, Washington D.C., 20559, where it is
registered under U.S. Copyright Registration No. TXU510087, and is
publicly available through Genentech, Inc., South San Francisco,
Calif. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0049] For purposes herein, the % amino acid sequence identity of a
given amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A.
[0050] As used herein, an amino acid residue in a polypeptide is
considered to "contribute to binding" a target if (1) any of the
non-hydrogen atoms of the residue's side chain or main chain is
found to be within five angstroms of any atom of the binding target
based on an experimentally determined three-dimensional structure
of the complex, and/or (2) mutation of the residue to its
equivalent in wild-type .sup.10Fn3 (e.g., SEQ ID NO: 1 or 6), to
alanine, or to a residue having a similarly sized or smaller side
chain than the residue in question, leads to a measured increase of
the equilibrium dissociation constant to the target (e.g., an
increase in the k.sub.on).
[0051] The "half-life" of a polypeptide can generally be defined as
the time taken for the serum concentration of the polypeptide to be
reduced by 50%, in vivo, for example due to degradation of the
polypeptide and/or clearance or sequestration of the polypeptide by
natural mechanisms. The half-life can be determined in any manner
known per se, such as by pharmacokinetic analysis. Suitable
techniques will be clear to the person skilled in the art, and may,
for example, generally involve the steps of administering a
suitable dose of a polypeptide to a primate; collecting blood
samples or other samples from said primate at regular intervals;
determining the level or concentration of the polypeptide in said
blood sample; and calculating, from (a plot of) the data thus
obtained, the time until the level or concentration of the
polypeptide has been reduced by 50% compared to the initial level
upon dosing. Methods for determining half-life may be found, for
example, in Kenneth et al., Chemical Stability of Pharmaceuticals:
A Handbook for Pharmacists (1986); Peters et al, Pharmacokinete
analysis: A Practical Approach (1996); and "Pharmacokinetics", M
Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev.
edition (1982).
[0052] Half-life can be expressed using parameters such as the
t1/2-alpha, t1/2-beta and the area under the curve (AUC). In the
present specification, an "increase in half-life" refers to an
increase in any one of these parameters, any two of these
parameters, or in all three these parameters. In certain
embodiments, an increase in half-life refers to an increase in the
t1/2-beta, either with or without an increase in the t1/2-alpha
and/or the AUC or both.
Overview
[0053] .sup.10Fn3 domains are structurally and functionally
analogous to antibodies, specifically the variable region of an
antibody. Historically, designs of .sup.10Fn3 binding domains
having relied on the similarity of the .sup.10Fn3 domain structure
to that of the VH domain of an antibody. In particular, .sup.10Fn3
binding domains traditionally have relied on modifications in the
amino acid sequences of the CDR-like loops of the .sup.10Fn3
domain. Each of the AB, BC, CD, DE, EF and FG loops of .sup.10Fn3
domains is analogous to a complementary determining region (CDR)
from an immunoglobulin in that they are flexible and amenable to
modifications in their amino acid sequences without altering the
overall structure of the .sup.10Fn3 domain. Furthermore,
modifications of sets of the CDR-like loops along one face of the
.sup.10Fn3 domain (i.e., the "north pole") have been shown to
permit development of .sup.10Fn3 domains that binds to a desired
target (see e.g., PCT Publication Nos. WO 02/032925, WO
2008/097497, and No. WO 2008/066752). In these traditional
.sup.10Fn3 scaffold designs, the protein sequences between the
loops, i.e. the .beta.-strands, are typically not modified or are
only minimally modified because they play a role in maintaining the
overall structural conformation of the .sup.10Fn3. As further
described herein, it is possible to modify the .sup.10Fn3 domains
in a non-traditional manner to produce proteins that bind to a
desired target while maintaining suitable stability.
[0054] In particular, the present application provides fibronectin
based scaffold polypeptides comprising combinations of modified
loops and scaffold regions and that are associated with improved
properties. The fibronectin based scaffold proteins described
herein comprise one or more human tenth fibronectin type III
domains that have been modified so as to bind to one or more
desired targets. The present application relates, in part, to the
discovery of fibronectin domain loop and/or scaffold region
modifications that are associated with specific target binding. It
has been discovered that scaffold region, e.g. non-loop,
modifications in fibronectin-based scaffold proteins may be
combined with specific loop modifications to obtain specific target
binding. Such scaffold designs provide expanded potential for
designing .sup.10Fn3 based binding proteins. For example, the
non-traditional scaffold designs described herein permit the
creation of libraries with greater diversity by opening up new
areas for sequence modification within the .sup.10Fn3 domain. In
addition, the non-traditional scaffold designs allow for
alternative surface interface geometries as compared to the
interface geometries provided by the traditional CDR-like loop
interface. The additional diversity and alternative surface
geometries provided by these non-traditional binders may facilitate
development of .sup.10Fn3 binding domains with desirable
properties, for example, by providing .sup.10Fn3 binding domains
with higher affinity for a given target, or by providing .sup.10Fn3
binding domains that bind to different epitopes on a given target.
Provided herein are further variations of the traditional and
non-traditional scaffold designs.
[0055] The application also describes fibronectin based scaffold
polypeptides that are associated with reduced immunogenicity. As
described in the examples, the .beta.-strand B/BC
loop/.beta.-strand C region may be an immunogenic `hot spot` based
on strong HLA binding activity. In particular, this region appears
to serve as a strong anchor sequence for HLA binding. The examples
also show that the wild-type sequence for the .beta.-strand B/BC
loop/.beta.-strand C region is recognized as a self-antigen by a
primate host. Therefore, despite strong HLA binding, no immune
response is generated to the wild-type sequence. Described herein
are alternative .sup.10Fn3 scaffolds in which the key areas within
the .beta.-strand B/BC loop/.beta.-strand C region have been left
as wild-type, while modifications in other regions of the sequence
permit high affinity target binding. Such alternative binders will
have an increased chance of generating high affinity .sup.10Fn3
binding domains that avoid undesirable immune responses in a host
organism because the .beta.-strand B/BC loop/.beta.-strand C region
immunogenic hot spot is unaltered and therefore should be
recognized as a self-antigen by the host organism. The application
also provides alternative .sup.10Fn3 binding domains in which the
HLA anchor sequence in the .beta.-strand B/BC loop/.beta.-strand C
region has been destroyed, which should reduce the immunogenic
potential of this region. Such .sup.10Fn3 binding domains with the
anchor sequence removed should allow diversification of all or a
portion of the BC loop, while still avoiding undesirable immune
responses associated with this region. The HLA anchor sequence can
be removed or destroyed by modifying key residues in the
.beta.-strand B and/or .beta.-strand C regions, in conjunction with
modifications to the BC loop region. Exemplary non-traditional
.sup.10Fn3 binding domains having reduced immunogenic potential are
described further below.
[0056] The fibronectin based scaffold polypeptides described herein
may be designed to bind to any target of interest. In exemplary
embodiments, the target is an antigen, a polypeptide or a
therapeutic protein target of interest. Exemplary therapeutically
desirable targets, include, for example, tumor necrosis factor
alpha (TNF-alpha), delta-like protein 4 (DLL4), interleukin 17
(IL-17), and pregnane X receptor (PXR).
Fibronectin Based Scaffolds
[0057] A. General Structure
[0058] Fn3 refers to a type III domain from fibronectin. An Fn3
domain is small, monomeric, soluble, and stable. It lacks disulfide
bonds and, therefore, is stable under reducing conditions. The
overall structure of Fn3 resembles the immunoglobulin fold. Fn3
domains comprise, in order from N-terminus to C-terminus, a beta or
beta-like strand, A; a loop, AB; a beta or beta-like strand, B; a
loop, BC; a beta or beta-like strand, C; a loop, CD; a beta or
beta-like strand, D; a loop, DE; a beta or beta-like strand, E; a
loop, EF; a beta or beta-like strand, F; a loop, FG; and a beta or
beta-like strand, G. The seven antiparallel .beta.-strands are
arranged as two beta sheets that form a stable core, while creating
two "faces" composed of the loops that connect the beta or
beta-like strands. Loops AB, CD, and EF are located at one face
("the south pole") and loops BC, DE, and FG are located on the
opposing face ("the north pole"). Any or all of loops AB, BC, CD,
DE, EF and FG may participate in ligand binding. There are at least
15 different Fn3 modules in human Fibronectin, and while the
sequence homology between the modules is low, they all share a high
similarity in tertiary structure.
[0059] In exemplary embodiments, the ligand binding scaffold
proteins described herein are based on the tenth fibronectin type
III domain, i.e., the tenth module of Fn3, (.sup.10Fn3). The amino
acid sequence of a naturally occurring human .sup.10Fn3 is set
forth in SEQ ID NO: 1:
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVY-
AVTGRGDSPASSKPISINYRTEIDKPSQ (SEQ ID NO: 1) (the AB, CD and EF
loops are underlined; the BC, FG, and DE loops are emphasized in
bold; the .beta.-strands are located between each of the loop
regions; and the N-terminal and C-terminal regions are shown in
italics). SEQ ID NO: 1 is the sequence of a .sup.10Fn3 molecule
that comprises a tail, i.e., amino acids 95-101. SEQ ID NO: 6 is
the amino acid sequence of a wild-type human .sup.10Fn3 molecule
that does not comprise a tail and consists of amino acids 1-94 of
SEQ ID NO: 1.
[0060] Residues involved in forming the hydrophobic core (the "core
amino acid residues") in SEQ ID NO: 1 include the amino acids
corresponding to the following amino acids of SEQ ID NO: 1 or 6:
L8, V10, A13, L18, I20, W22, Y32, I34, Y36, F48, V50, A57, I59,
L62, Y68, I70, V72, A74, I88, I90 and Y92, wherein the core amino
acid residues are represented by the single letter amino acid code
followed by the position at which they are located within SEQ ID
NO: 1. See e.g., Dickinson et al., J. Mol. Biol. 236: 1079-1092
(1994). In some embodiments, the residues involved in forming the
hydrophobic core are used to determine the boundaries of the loop
regions of the polypeptide. For example, the AB loop may be defined
as being the stretch of amino acids between the hydrophobic core
residue A13 of .beta.-strand A and the hydrophobic core residue L18
of the .beta.-strand B. See FIG. 1. In some embodiments, the
hydrophobic core amino acids are not modified relative to the
wild-type sequence. In other embodiments, the following hydrophobic
amino acids may be mutated: A13, which is part of a beta bulge and
can convert to a surface residue; Y32 and A74, either or both of
which can vary to interact differently with the nearby loops; I88,
which is partially solvent-exposed and the corresponding position
is not always a hydrophobic residue in natural fibronectin type III
domains; and Y92, which connects to the C-terminal tail and could
be diversified if the C-terminal region is diversified.
[0061] In some embodiments, the AB loop corresponds to residues
14-17, the BC loop corresponds to residues 23-31, the CD loop
corresponds to residues 37-47, the DE loop corresponds to residues
51-56, the EF loop corresponds to residues 63-67, and the FG loop
corresponds to residues 75-87 of SEQ ID NO: 1. The BC, DE and FG
loops align along one face of the molecule, i.e. the "north pole",
and the AB, CD and EF loops align along the opposite face of the
molecule, i.e. the "south pole". In SEQ ID NO: 1, .beta.-strand A
corresponds to residues 8-13, .beta.-strand B corresponds to
residues 18-22, .beta.-strand C corresponds to residues 32-36, beta
strand D corresponds to residues 48-50, .beta.-strand E corresponds
to residues 57-62, .beta.-strand F corresponds to residues 68-74,
and .beta.-strand G corresponds to residues 88-92. The
.beta.-strands are connected to each other through the
corresponding loop, e.g., strands A and B are connected via loop AB
in the formation .beta.-strand A, loop AB, .beta.-strand B, etc.
The N-terminal and/or C-terminal regions (italicized above), may be
removed or altered to generate a molecule retaining biological
activity and comprising the amino acid sequence of SEQ ID NO: 2 or
SEQ ID NO: 6. In certain embodiments, the first 8 amino acid
residues of SEQ ID NO: 1 and/or the last 7 amino acid residues of
SEQ ID NO: 1 (i.e., amino acid residues 95-101 of SEQ ID NO: 1) may
be removed or altered to generate a polypeptide comprising the
amino acid sequence of SEQ ID NO: 60 or SEQ ID NO: 6 (corresponding
to SEQ ID NO: 1 without the 7 N-terminal amino acids, and
consisting of amino acids 1-94 of SEQ ID NO: 1). The libraries
described herein may comprise the N- or C-terminal region set forth
in SEQ ID NO: 1. In certain embodiments, the libraries comprise the
N-terminal region, but do not comprise the C-terminal region (i.e.,
they are based on SEQ ID NO: 6).
[0062] As described above, amino acid residues corresponding to
residues 14-17, 23-31, 37-47, 51-56, 63-67 and 75-87 of SEQ ID NO:
1 define the AB, BC, CD, DE, EF and FG loops, respectively.
However, it should be understood that not every residue within a
loop region needs to be modified in order to achieve a .sup.10Fn3
binding domain having strong affinity for a desired target. For
example, in some embodiments, only residues corresponding to amino
acids 39-45 of the CD loop and 77-87 of the FG loop were modified
to produce high affinity .sup.10Fn3 binders (see e.g., the murine
DLL4 binding cores having an amino acid sequence of either SEQ ID
NO: 3, 4 or 5 and the murine IL-17 binding cores having an amino
acid sequence of either SEQ ID NO: 45, 46 or 47).
[0063] Additionally, insertions and deletions in the loop regions
may also be made while still producing high affinity .sup.10Fn3
binding domains. For example, the CD loop of the murine DLL4 binder
having SEQ ID NO: 3 has the same length CD loop as the wild-type
.sup.10Fn3 domain, i.e., the seven residues 39-45 of SEQ ID NO: 1
were replaced with the seven residues 41-47 of SEQ ID NO: 3. In
contrast, the FG loop of the murine DLL4 binder having SEQ ID NO: 3
is longer in length than the corresponding FG loop of the wild-type
.sup.10Fn3 domain, i.e., the nine residues 77-85 of SEQ ID NO: 1
were replaced with the nineteen residues 79-98 of SEQ ID NO: 3.
[0064] Accordingly, in some embodiments, one or more loops selected
from AB, BC, CD, DE, EF and FG may be extended or shortened in
length relative to the corresponding loop in wild-type human
.sup.10Fn3. In any given polypeptide, one or more loops may be
extended in length, one or more loops may be reduced in length, or
combinations thereof. In some embodiments, the length of a given
loop may be extended by 2-25, 2-20, 2-15, 2-10, 2-5, 5-25, 5-20,
5-15, 5-10, 10-25, 10-20, or 10-15 amino acids. In some
embodiments, the length of a given loop may be reduced by 1-15,
1-11, 1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. In particular,
the FG loop of .sup.10Fn3 is 13 residues long, whereas the
corresponding loop in antibody heavy chains ranges from 4-28
residues. To optimize antigen binding in polypeptides relying on
the FG for target binding, therefore, the length of the FG loop of
.sup.10Fn3 may be altered in length as well as in sequence to
obtain the greatest possible flexibility and affinity in target
binding.
[0065] In some embodiments, one or more residues of the
integrin-binding motif "arginine-glycine-aspartic acid" (RGD)
(amino acids 78-80 of SEQ ID NO: 1 or 6) may be substituted so as
to disrupt integrin binding. In some embodiments, the FG loop of
the polypeptides provided herein does not contain an RGD integrin
binding site. In one embodiment, the RGD sequence is replaced by a
polar amino acid-neutral amino acid-acidic amino acid sequence (in
the N-terminal to C-terminal direction). In another embodiment, the
RGD sequence is replaced with SGE. In yet another embodiment, the
RGD sequence is replaced with RGE.
[0066] In some embodiments, a fibronectin based scaffold protein
comprises a .sup.10Fn3 domain having at least 40%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, or 90% identity to the human .sup.10Fn3
domain having the amino acid sequence of SEQ ID NO: 1, 2, 60 or 6.
In certain embodiments, the polypeptides provided herein have at
least 50% identity to the amino acid sequence of the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1, 2, 60 or 6). In other
embodiments, the polypeptide has at least 65% identity to the amino
acid sequence of the wild-type human .sup.10Fn3 domain (SEQ ID NO:
1, 2, 60 or 6). In certain embodiments, one or more of the loops
will not be modified relative to the sequence of the corresponding
loop of the wild-type sequence and/or one or more of the
.beta.-strands will not be modified relative to the sequence of the
corresponding .beta.-strand of the wild-type sequence. In certain
embodiments, each of the beta or beta-like strands of a .sup.10Fn3
domain in a fibronectin based scaffold protein may comprise,
consist essentially of, or consist of an amino acid sequence that
is at least 80%, 85%, 90%, 95% or 100% identical to the sequence of
a corresponding beta or beta-like strand of SEQ ID NO: 1 or 6.
Preferably, variations in the .beta.-strand regions will not
disrupt the stability of the polypeptide in physiological
conditions. In exemplary embodiments, the .sup.10Fn3 domain binds
to a desired target with a K.sub.d of less than 500 nM, 100 nM, 50
nM, 1 nM, 500 pM, 100 pM or less. In some embodiments, the
.sup.10Fn3 domain of a fibronectin based protein scaffold binds to
a desired target with a K.sub.d between 1 pM and 1 .mu.M, between
100 pM and 500 nM, between 1 nM and 500 nM, or between 1 nM and 100
nM. In exemplary embodiments, the fibronectin based scaffold
protein binds specifically to a target that is not bound by a
wild-type .sup.10Fn3 domain, particularly the wild-type human
.sup.10Fn3 domain.
[0067] In some embodiments, the disclosure provides polypeptides
comprising a .sup.10Fn3 domain, wherein the .sup.10Fn3 domain
comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop,
EF; and a loop, FG; and has at least one loop selected from loop
AB, BC, CD, DE, EF and FG with an altered amino acid sequence
relative to the sequence of the corresponding loop of the human
.sup.10Fn3 domain of SEQ ID NO: 1. In some embodiments, the BC, DE
and FG loops are altered. In other embodiments, the CD and FG loops
are altered. In other embodiments, the CD, DE and EF loops are
altered. In other embodiments, the EF and FG loops are altered. In
other embodiments, the AB, CD and EF loops are altered. In other
embodiments, the FG loop is the only loop altered. In other
embodiments, the CD and FG loops are both altered. In other
embodiments, the CD and EF loops are altered. In some embodiments,
one or more specific scaffold alterations are combined with one or
more loop alterations. By "altered" is meant one or more amino acid
sequence alterations relative to a template sequence (i.e., the
corresponding wild-type human fibronectin domain) and includes
amino acid additions, deletions, and substitutions.
[0068] In some embodiments, the fibronectin based scaffold protein
comprises a .sup.10Fn3 domain having an amino acid sequence at
least 80, 85, 90, 95, 98, or 100% identical to the non-loop regions
of SEQ ID NO: 1 or 6, wherein at least one loop selected from AB,
BC, CD, DE, EF and FG is altered. For example, in certain
embodiments, the AB loop may have up to 4 amino acid substitutions,
up to 10 amino acid insertions, up to 3 amino acid deletions, or a
combination thereof; the BC loop may have up to 10 amino acid
substitutions, up to 4 amino acid deletions, up to 10 amino acid
insertions, or a combination thereof; the CD loop may have up to 6
amino acid substitutions, up to 10 amino acid insertions, up to 4
amino acid deletions, or a combination thereof; the DE loop may
have up to 6 amino acid substitutions, up to 4 amino acid
deletions, up to 13 amino acid insertions, or a combination
thereof; the EF loop may have up to 5 amino acid substations, up to
10 amino acid insertions, up to 3 amino acid deletions, or a
combination thereof; and/or the FG loop may have up to 12 amino
acid substitutions, up to 11 amino acid deletions, up to 25 amino
acid insertions, or a combination thereof.
[0069] In certain embodiments, the fibronectin based scaffold
protein comprises a .sup.10Fn3 domain that is defined generally by
following the sequence:
VSDVPRDLEVVAA(X).sub.uLLISW(X).sub.vYRITY(X).sub.wFTV(X).sub.xA-
TISGL(X).sub.yYTITVYA(X).sub.zISINYRT (SEQ ID NO: 22)
[0070] In SEQ ID NO: 22, the AB loop is represented by (X).sub.u,
the BC loop is represented by (X).sub.v, the CD loop is represented
by (X).sub.w, the DE loop is represented by (X).sub.x, the EF loop
is represented by (X).sub.y and the FG loop is represented by
X.sub.z. X represents any amino acid and the subscript following
the X represents an integer of the number of amino acids. In
particular, u, v, w, x, y and z may each independently be anywhere
from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15,
6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The sequences of the beta
strands (underlined) may have anywhere from 0 to 10, from 0 to 8,
from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or
from 0 to 1 substitutions, deletions or additions across all 7
scaffold regions relative to the corresponding amino acids shown in
SEQ ID NO: 22. In some embodiments, the sequences of the beta
strands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6,
from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1
conservative substitutions across all 7 scaffold regions relative
to the corresponding amino acids shown in SEQ ID NO: 22. In certain
embodiments, the hydrophobic core amino acid residues (bolded
residues in SEQ ID NO: 22 above) are fixed, and any substitutions,
conservative substitutions, deletions or additions occur at
residues other than the hydrophobic core amino acid residues. In
some embodiments, the hydrophobic core residues of the polypeptides
provided herein have not been modified relative to the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1).
[0071] B. Scaffold Region Modifications
[0072] The non-loop sequences of .sup.10Fn3, i.e., the "scaffold
regions", may be altered provided that the .sup.10Fn3 domain
retains target binding function and/or structural stability. In
some embodiments, one or more of Asp 7, Glu 9, and Asp 23 are
replaced by another amino acid, such as, for example, a
non-negatively charged amino acid residue (e.g., Asn, Lys, etc.).
These mutations have been reported to have the effect of promoting
greater stability of the mutant .sup.10Fn3 at neutral pH as
compared to the wild-type form (See, PCT Publication No. WO
02/04523). A variety of additional alterations in the .sup.10Fn3
scaffold that are either beneficial or neutral have been disclosed.
See, for example, Batori et al., Protein Eng. 2002 15(12):1015-20;
Koide et al., Biochemistry 2001 40(34):10326-33.
[0073] The scaffold regions, e.g., the .beta.-strands and/or N- and
C-termini, of .sup.10Fn3 may be modified to increase binding of the
polypeptide to a desired target or to reduce immunogenicity. In
some embodiments, residues involved in forming the hydrophobic
core, i.e., residues corresponding to residues L8, V10, A13, L18,
I20, W22, Y32, I34, Y36, F48, V50, A57, I59, L62, Y68, I70, V72,
A74, I88, I90 and Y92 of SEQ ID NO: 1 or 6 are not mutated. In some
embodiments, any one of, or a combination of any one of, residues
corresponding to residues 1-7, 9-15, 19, 21, 33, 35, 36, 49, 58,
60, 61, 69, 71 73, 88, 89 and 91-101 of the scaffold region of the
.sup.10Fn3 polypeptide is mutated to a different amino acid as
compared to the corresponding amino acid present in the amino acid
sequence set forth as SEQ ID NO: 1 or 6.
[0074] In some embodiments, mutations may be made to the scaffold
regions of the polypeptides, provided that the one or more of the
following specific mutations are excluded: V1A; S2P; S2T; D3G; D3S;
P5S; R6G; R6S; D7G; D7K; L8P; L8Q; E9D; E9K; E9R; E9V; V10A; V10I;
A12D; A12E; A12V; L18E; L18I; L18P; L18Q; L18R; L19Q; S21C; S21G;
S21N; R29G; 8295; R29Y; Y31H; Y32F; R33G; I34T; I34V; T35A; T35F;
T35I; Y36H; F48L; F48S; T49A; T49I; V50A; V50E; V50M; A57Deletion;
T58A; T58I; T58Deletion; I59V I59Deletion; 560G; S60N; 560R; G61C;
G61R; L62R; D67G; D67K; D67N; Y68A; Y68D; T69I; I70N; I70S; I70V;
T71A; V72A; V72G; Y73C; Y73H; A74G; A74T; I88S; I88T; I88V; S89P;
I90F; I90T; I90V; N91D; N91S; N91T; Y92C; Y92H; Y92L; Y92R;
Y92Deletion; R93Q; R93T and T94A. In certain embodiments, these
specific scaffold mutations are excluded in the context of a
.sup.10Fn3 domain in which the BC, DE and FG loops have been
modified.
[0075] In some embodiments, mutations may be made to the scaffold
regions of the polypeptides, provided that the following mutation
combinations are excluded:
[0076] L18R, S21C and S60G;
[0077] E9D, L18R, V50E and T56I;
[0078] L18R, T49I and N91D;
[0079] F48S and T71A;
[0080] P5S, V10A, S60G and S89P;
[0081] L18R, and Y92C;
[0082] L18R and F48S;
[0083] L18R and V72A;
[0084] L18Q, R33G and F48S;
[0085] Y68D and Y92H;
[0086] R6S, L62R and N91S;
[0087] L8P, E9V, I34V, T71A and Y92Deletion;
[0088] E9K, L18R and F48L;
[0089] E9R, L18R, S60G and I70V;
[0090] L18R, I88V and I90T;
[0091] L18R, N91D and Y92C;
[0092] L18R and I34T;
[0093] L18R and G61C;
[0094] Y32F, T71A and T94A;
[0095] L18R, T58A, Y92L and R93T;
[0096] V50M, T58A, S89P, 190F and Y92R;
[0097] S2T, D7G, E9K, V10I, T58A, S60N and S89P;
[0098] I59V, S60N and T94A;
[0099] R6G, S21G, T35A, T58I and S60G;
[0100] L18P, S21C, T58A, Y73H and Y92C;
[0101] Y31H, R33G and G61R;
[0102] A74G, R93Q and T94A
[0103] S2P and T58I;
[0104] T58I and I88T;
[0105] T58I and I90T;
[0106] G61R and A74T
[0107] A57Deletion, T58Deletion and I59Deletion
[0108] R33G, T35I and V50M
[0109] V1A, R33G and V50M
[0110] R33G and V50M
[0111] R33G, I34V and V50M
[0112] D3G, L18I, R33G, V50M, Y73H and N91T
[0113] R6G, T35F and V72A
[0114] A12V, S21N and T35A
[0115] S21G and T49A
[0116] D3S and D7K
[0117] A12V and L19Q
[0118] A12D, L18I and L19Q
[0119] A12E, L18I and L19Q
In certain embodiments, these specific combinations of scaffold
mutations are excluded in the context of a .sup.10Fn3 domain in
which the BC, DE and FG loops have been modified.
[0120] In some embodiments, polypeptides having mutations at a
position corresponding to position 21 of SEQ ID NO: 1 or 6 are
excluded, unless a mutation at this position is combined with a
mutation or mutations at any one of amino acid positions
corresponding to positions 1-7, 19, 31, 49, 58, 60, 73, 75 and 89
of SEQ ID NO: 1 or 6. In some embodiments, polypeptides having
mutations at a position corresponding to position 60 of SEQ ID NO:
1 are excluded unless a mutation at this position is combined with
a mutation or mutations at an amino acid position corresponding to
any one of positions 1-7, 9-17, 19, 21, 23-31, 33, 35, 49, 51-56,
65-67, 75-87 and 89 of SEQ ID NO: 1 or 6. In some embodiments,
polypeptides having mutations at position 61 of SEQ ID NO: 1 or 6
are excluded, unless a mutation at this position is combined with a
mutation or mutations at an amino acid position corresponding to
any one of positions 11, 12, 19, 46, 66-67, 69 and 91 of SEQ ID NO:
1 or 6. In some embodiments, polypeptides having mutations at
positions 93 or 94 of SEQ ID NO: 1 or 6 are excluded, unless a
mutation at either of these positions is combined with a mutation
or mutations at a position corresponding to any one of amino acid
positions 1-7, 9-14, 65-67, 89 and 91 of SEQ ID NO: 1 or 6. In
certain embodiments, these exclusions apply in the context of a
.sup.10Fn3 domain in which the BC, DE and FG loops have been
modified.
[0121] In certain embodiments, the non-loop region of the
.sup.10Fn3 domain may be modified by one or more conservative
substitutions. As many as 5%, 10%, 20% or even 30% or more of the
amino acids in the .sup.10Fn3 scaffold may be altered by a
conservative substitution without substantially altering the
affinity of the .sup.10Fn3 for a ligand. In certain embodiments,
the scaffold may comprise anywhere from 0-15, 0-10, 0-8, 0-6, 0-5,
0-4, 0-3, 1-15, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2-15, 2-10, 2-8,
2-6, 2-5, 2-4, 5-15, or 5-10 conservative amino acid substitutions.
In exemplary embodiments, the scaffold modification preferably
reduces the binding affinity of the .sup.10Fn3 binder for a ligand
by less than 100-fold, 50-fold, 25-fold, 10-fold, 5-fold, or
2-fold. It may be that such changes will alter the immunogenicity
of the .sup.10Fn3 in vivo, and where the immunogenicity is
decreased, such changes will be desirable. As used herein,
"conservative substitutions" are residues that are physically or
functionally similar to the corresponding reference residues. That
is, a conservative substitution and its reference residue have
similar size, shape, electric charge, chemical properties including
the ability to form covalent or hydrogen bonds, or the like.
Preferred conservative substitutions are those fulfilling the
criteria defined for an accepted point mutation in Dayhoff et al.,
Atlas of Protein Sequence and Structure 5:345-352 (1978 &
Supp.). Examples of conservative substitutions are substitutions
within the following groups: (a) valine, glycine; (b) glycine,
alanine; (c) valine, isoleucine, leucine; (d) aspartic acid,
glutamic acid; (e) asparagine, glutamine; (f) serine, threonine;
(g) lysine, arginine, methionine; and (h) phenylalanine,
tyrosine.
[0122] In some embodiments, a .sup.10Fn3 molecule comprises the
amino acid sequence of any of the library designs set forth herein,
e.g., in FIGS. 2, 9, 12 and 13 (or amino acids 1-94 thereof; SEQ ID
NO: 6) and comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
additional substitutions, additions or deletions in a loop and/or a
strand. In certain embodiments, a .sup.10Fn3 molecule comprises the
amino acid sequence of any of the library designs set forth herein,
e.g., in FIGS. 2, 9, 12 and 13 (or amino acids 1-94 thereof; SEQ ID
NO: 6) with no other amino acid modifications. A .sup.10Fn3
molecule that comprises the amino acid sequence of any of the
library designs set forth herein, e.g., in FIGS. 2, 9, 12 and 13
(or amino acids 1-94 thereof; SEQ ID NO: 6) may comprise any amino
acid at a varied position, and in some instances even that of the
wild-type .sup.10Fn3 molecule. In certain embodiments, a .sup.10Fn3
molecule that comprises the amino acid sequence of any of the
library designs set forth herein, e.g., in FIGS. 2, 9, 12 and 13
(or amino acids 1-94 thereof; SEQ ID NO: 6) comprises only non
wild-type amino acids at each of the positions indicated as varied
(those underlined or boxed, and bolded).
[0123] C. N- and C-Terminal Regions
[0124] In some embodiments, the amino acid sequences of the
N-terminal and/or C-terminal regions of the polypeptides provided
herein may be modified by deletion, substitution or insertion
relative to the amino acid sequences of the corresponding regions
of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). The
.sup.10Fn3 domains generally begin with amino acid number 1 of SEQ
ID NO: 1. However, domains with amino acid deletions are also
encompassed by the invention. In some embodiments, the first eight
(i.e., residues 1-8) and the last seven amino acids (i.e., residues
95-101) of SEQ ID NO: 1 are deleted, generating a .sup.10Fn3 domain
having the amino acid sequence of SEQ ID NO: 60. In certain
embodiments, the last seven amino acids (i.e., residues 95-101) of
SEQ ID NO: 1 are deleted, generating a .sup.10Fn3 domain having the
amino acid sequence of SEQ ID NO: 6. Additional sequences may also
be added to the N- or C-terminus of a .sup.10Fn3 domain having the
amino acid sequence of SEQ ID NO: 1, 2, 6, or 60. For example, in
some embodiments, the N-terminal extension consists of an amino
acid sequence selected from the group consisting of: M, MG, and
G.
[0125] In certain embodiments, the amino acid sequence of the first
1, 2, 3, 4, 5, 6, 7, 8 or 9 residues of SEQ ID NO: 1 or 6 may be
modified or deleted in the polypeptides provided herein relative to
the sequence of the corresponding amino acids in the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). In exemplary
embodiments, the amino acids corresponding to amino acids 1-8 of
SEQ ID NO: 1 or 6 are replaced with an alternative N-terminal
region having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1
amino acids in length. Exemplary alternative N-terminal regions
include (represented by the single letter amino acid code) M, MG,
G, MGVSDVPRDL (SEQ ID NO: 9) and GVSDVPRDL (SEQ ID NO: 11), or
N-terminal truncations of any one of SEQ ID NOs: 9 and 11. Other
suitable alternative N-terminal regions include, for example,
X.sub.nSDVPRDL (SEQ ID NO: 16), X.sub.nDVPRDL (SEQ ID NO: 17),
X.sub.nVPRDL (SEQ ID NO: 18), X.sub.nPRDL (SEQ ID NO: 19),
X.sub.nRDL (SEQ ID NO: 20), X.sub.nDL (SEQ ID NO: 21), or X.sub.nL,
wherein n=0, 1 or 2 amino acids, wherein when n=1, X is Met or Gly,
and when n=2, X is Met-Gly. When a Met-Gly sequence is added to the
N-terminus of a .sup.10Fn3 domain, the M will usually be cleaved
off, leaving a G at the N-terminus. In other embodiments, the
alternative N-terminal region comprises the amino acid sequence
MASTSG (SEQ ID NO: 50).
[0126] In certain embodiments, the amino acid sequence
corresponding to amino acids 93-101, 94-101, 95-101, 96-101,
97-101, 98-101, 99-101, 100-101, or 101 of SEQ ID NO: 1 are deleted
or modified in the polypeptides provided herein relative to the
sequence of the corresponding amino acids in the wild-type human
.sup.10Fn3 domain (SEQ ID NO: 1 or 6). In exemplary embodiments,
the amino acids corresponding to amino acids 95-101 of SEQ ID NO: 1
are replaced with an alternative C-terminal region having from
1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in
length. Specific examples of alternative C-terminal region
sequences include, for example, polypeptides comprising, consisting
essentially of, or consisting of, EIEK (SEQ ID NO: 7), EGSGC (SEQ
ID NO: 23), EIEKPCQ (SEQ ID NO: 24), EIEKPSQ (SEQ ID NO: 25), EIEKP
(SEQ ID NO: 26), EIEKPS (SEQ ID NO: 27), EIEKPC (SEQ ID NO: 8), or
SEQ ID NO: 44. In some embodiments, the alternative C-terminal
region comprises EIDK (SEQ ID NO: 29), and in particular
embodiments, the alternative C-terminal region is either EIDKPCQ
(SEQ ID NO: 31) or EIDKPSQ (SEQ ID NO: 30).
[0127] In certain embodiments, the fibronectin based scaffold
proteins comprise a .sup.10Fn3 domain having both an alternative
N-terminal region sequence and an alternative C-terminal region
sequence.
[0128] When referring herein to molecules comprising a particular
library design, which library design comprises amino acids 1-101
(SEQ ID NO: 1), it is understood that also encompassed herein are
the same molecules comprising amino acids 1-94 (SEQ ID NO: 6) and
not including the 7 N-terminal amino acids, and/or not including
C-terminal amino acids.
[0129] D. Proteins Having Loop and Scaffold Combinations
[0130] A "patch library", as described herein, refers to a library
in which a region on the surface of the scaffold protein is
diversified. Residues to be diversified can be determined by
picking one spot on the surface of the protein, then identifying
all surface and loop residues within some distance (e.g., 8 .ANG.),
and adjusting for shape, sequence connectivity, conservation, etc.
For example, to generate a patch library centering on the
"SouthWest" portion of the scaffold, Asp67, the last amino acid in
the EF loop, which is approximately centered on the SW side was
selected. All residues within an 8 .ANG. distance of Asp67 are then
identified, and the hydrophobic core residues are removed from the
list of residues to be diversified, providing a total of 14 amino
acids to randomize, including G37-G41, K63-D67, T69, and the
C-terminal sequence beginning with N91 (residues positions numbered
in accordance with the sequence of the wild-type human .sup.10Fn3
domain having SEQ ID NO: 1 or 6). Further means of diversifications
can then be incorporated into the scaffold design, for example,
varying the length of the CD loop (to allow greater shape
variation) or modifying or extending the sequence of the N-terminal
region. Examples of possible amino acid residues to be mutated in
order to generate a representative patch library are provided in
FIGS. 2A-F and 9A-C. Three-dimensional structures of .sup.10Fn3
domain peptides illustrating several of the different interfaces
that may be targeted to generate a representative patch library are
shown in FIGS. 3A-F. In some embodiments, amino acids in a
.sup.10Fn3 domain polypeptide are diversified not with regard to
loop definitions, but rather with regard to their physical location
on the surface of the .sup.10Fn3 structure. In some embodiments, a
`patch` of 10 to 30 or more amino acids are diversified, chosen to
form a generally contiguous surface that can span both loops and
strands, or can be solely on strand residues.
[0131] 1. Binders Having a North Pole and South Pole Loop
Modified
[0132] In some embodiments, the polypeptides provided herein
comprise a .sup.10Fn3 domain having (i) a modification in the amino
acid sequence of at least one north pole loop selected from the BC,
DE and FG loops relative to the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1 or 6), and (ii) a
modification in the amino acid sequence of at least one south pole
loop selected from the AB, CD and EF loops relative to the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO: 1 or 6). The modified north pole and south pole loops
contribute to binding to the same target. Various combinations of
modified north pole and south pole loops are contemplated. For
example, a .sup.10Fn3 may comprise one modified north pole loop and
one modified south pole, one modified north pole loop and two
modified south pole loops, two modified north pole loops and one
modified south pole loop, two modified north pole loops and two
modified south pole loops, three modified north pole loops and one
modified south pool loop, etc., wherein each of the modified loops
contributes to binding to the same target. Exemplary combinations
of north pole and south pole loops that may be modified include,
for example, the CD loop (south pole) and the FG loop (north pole),
the CD loop (south pole) and the DE loop (north pole), the EF loop
(south pole) and FG loop (north pole), the AB loop (south pole) and
the FG loop (north pole), or the DE loop (north pole) and the EF
loop (south pole). Another exemplary loop combination is the CD
loop (south pole), the DE loop (north pole) and the EF loop (south
pole). Yet another exemplary loop combination is the DE loop (north
pole) and one of more of the AB, CD and EF loops (south pole). The
modified loops may have sequence modifications across an entire
loop or only in a portion of the loop. Additionally, one or more of
the modified loops may have insertions or deletions such that the
length of the loop is varied relative to the length of the
corresponding loop of the wild-type sequence. In certain
embodiments, additional regions in the .sup.10Fn3 domain (i.e., in
addition to the north and south pole loops), such as .beta.-strand,
N-terminal and/or C-terminal regions, may also be modified in
sequence relative to the wild-type .sup.10Fn3 domain, and such
additional modifications may also contribute to binding to the
target.
[0133] Exemplary .sup.10Fn3 designs having at least one north pole
loop and at least one south pole loop modified include, for
example, the WS1, WS2, WS3, WS2', Front1, Front2, Back1, Back2,
WS-LI1, SouthFront, and AG Strand designs shown in FIG. 9A and the
West Side, South Front and AG Strand designs shown in FIGS.
2C-2E.
[0134] 2. Binders Having Loop and Scaffold Region Modifications
[0135] Also provided herein are .sup.10Fn3 domains having
combinations of loop and scaffold modifications. In particular, the
application provides polypeptides comprising a .sup.10Fn3 domain
comprising (i) a modification in the amino acid sequence of at
least one of loops AB, BC, CD, DE, EF, or FG, and (ii) a
modification in the amino acid sequence of at least one scaffold
region (i.e., a modification in at least one .beta.-strand, the
N-terminal region, and/or the C-terminal region), wherein the
modified loop(s) and modified scaffold region(s) both contribute to
binding the same target. In exemplary embodiments, the scaffold
region modifications are located adjacent to modifications in a
loop region, e.g., if the AB loop is modified, scaffold mutations
may tend to be located in .beta.-strand A and/or .beta.-strand B,
which are adjacent to the AB loop in the linear sequence of the
.sup.10Fn3 domain. In other embodiments, a cluster of modifications
may be found together in loop and scaffold regions that are
adjacent to one another in the linear sequence of the .sup.10Fn3
domain. For example, .sup.10Fn3 binders having both loop and
scaffold modifications, may have clusters of amino acid
modifications in the following combinations of loop and scaffold
regions that are adjacent to each other in the linear sequence of
the .sup.10Fn3 domain: .beta.-strand/loop/.beta.-strand,
loop/.beta.-strand/loop, loop/.beta.-strand/loop/.beta.-strand,
terminal region/.beta.-strand/loop, or loop/.beta.-strand/terminal
region, etc. For example, .sup.10Fn3 domains having combinations of
loop and scaffold modifications may have clusters of modifications
such that over a stretch of 20 contiguous amino acids at least 15
of the amino acids are modified relative to wild-type. In other
embodiments, at least 17 out of 20, 18 out of 20, 17 out of 25, 20
out of 25, or 25 out of 30 residues in a contiguous stretch are
modified relative to the wild-type .sup.10Fn3 domain sequence over
the corresponding stretch of amino acids. In certain embodiments, a
given .sup.10Fn3 domain may have two or three clusters of
modifications separated by stretches of unmodified (i.e.,
wild-type) sequence. For any given region (i.e., a loop,
.beta.-strand or terminal region) that is modified, all or only a
portion of the region may be modified relative to the wild-type
sequence. When a .beta.-strand region is modified, preferably the
hydrophobic core residues remain unmodified (i.e., wild-type) and
one or more of the non-core residues in the .beta.-strand are
modified. Suitable modifications in the loop, .beta.-strand or
terminal regions include amino acid substitutions, deletion and/or
insertions, as well as combinations thereof.
[0136] Exemplary .sup.10Fn3 designs having at least one loop region
and at least one scaffold region modified include, for example, the
WS1, Front1, Front2, Back1, Back2, SP1, SP2, SP3, SouthFront, AG
Strand and SouthWest designs shown in FIG. 9A, the NW3 design shown
in FIG. 9B, the NP1, NE1, NP4-5, NP6-1 and NW2 designs shown in
FIG. 9C, and the designs shown in FIGS. 2A-2F.
[0137] 3. "West-Side" Binders
[0138] In some embodiments, the application provides .sup.10Fn3
domains having a binding face along the "west-side" of the molecule
(See FIG. 3C) and are referred to as "West-side binders" or "WS
binders". WS binders as described herein comprise a .sup.10Fn3
domain that has a modified CD loop and a modified FG loop, as
compared to the corresponding CD and FG loop sequences set forth in
SEQ ID NO: 1 or 6. The CD loop and the FG loop both contribute to
binding to the same target. In certain embodiments, the WS binders
may comprise additional modifications at one or more regions within
the .sup.10Fn3 domain. For example, WS binders may comprise
scaffold modifications in one or more of the .beta.-strand regions
adjacent to the CD and/or FG loops. In particular, WS binders may
comprise sequence modifications in one or more of .beta.-strand C,
.beta.-strand D, .beta.-strand F, and/or .beta.-strand G. Exemplary
scaffold modifications include modifications at one or more
scaffold region positions corresponding to the amino acid
positions: 33, 35, 49, 69, 71, 73, 89 and/or 91 of SEQ ID NO: 1 or
6. The WS binders may also comprise modifications in the BC loop,
particularly in the C-terminal portion of the BC loop. In one
embodiment, the last two residues of the BC loop (i.e.,
corresponding to amino acids 30 and 31 in the wild-type .sup.10Fn3
domain) are modified relative to the wild-type sequence. All or a
portion of the additional loop and scaffold modifications may
contribute to binding to the target in conjunction with the
modified CD and FG loops. Preferably, the hydrophobic core residues
are not modified relative to the wild-type sequence.
[0139] In certain embodiments, a WS binder has a CD loop that is
about 3-11, 4-9 or 5 residues long; an FG loop that is about 1-10,
e.g., 6 or 5, residues long; a C strand that is about 6-14, 8-11,
or 9 residues long; and/or an F strand that is about 9-11 or 10
residues long. Positions 31, 33, 35 and 37-39 of the beta strand C
may be altered relative to the wild-type sequence. Positions 32, 34
and 36 of the beta strand C may be hydrophobic residues. Positions
67, 69, 71 and 73 of the beta strand F may be altered relative to
the wild-type sequence. Positions 68, 70, and 72 of the beta strand
F may be hydrophobic residues. A WS binder may comprise amino acid
substitutions at positions 30, 31, 32, 33, 34, 35, 36, 37, 38
and/or 39, such as positions 31, 33, 35, 37, 38 and/or 39, e.g.,
positions 31 and/or 33, of SEQ ID NO: 1 or 6. A WS binder may
comprise amino acid substitutions at positions 44, 45, 46, 47, 48,
49, 50 and/or 51, such as positions 44, 45, 47 and/or 49, of SEQ ID
NO: 1 or 6. A WS binder may comprise amino acid substitutions at
positions 40, 41, 42, 43, 44 and/or 45 of SEQ ID NO: 1 or 6. A WS
binder may comprise amino acid substitutions at positions 67, 68,
69, 70, 71, 72, 73, 74, 75 and/or 76, such as positions 67, 69, 71,
73 and/or 76 or positions 71, 73, 75 and/or 76, of SEQ ID NO: 1 or
6. A WS binder may comprise amino acid substitutions at positions
76, 77, 78, 79, 81, 82, 83, 84, 85 and/or 86, such as positions 84
and/or 85 of SEQ ID NO: 1 or 6. A WS binder may comprise amino acid
substitutions at positions 85, 86, 87, 88, 89, 90, 91, 92, 93
and/or 94 of SEQ ID NO: 1 or 6. A WS binder may comprise amino acid
substitutions at positions 31, 33, 47, 49, 73 and/or 75 of SEQ ID
NO: 1 or 6. A WS binder may comprise a loop C comprising from 4-9
varied, e.g., non wild-type amino acids; an FG loop comprising from
5-6 varied, e.g., non wild-type amino acids; and wherein amino
acids 31, 33, 35, 37-39, 67, 69, 71, 73 and 76 are not wild-type.
"Not wild-type" amino acids are amino acids that are not those
found at the same position in the wild-type human .sup.10Fn3
molecule (having, e.g., SEQ ID NO: 1 or 6).
[0140] Exemplary .sup.10Fn3 WS binder designs include, for example,
the WS1, WS2, WS3, WS2', and WS-LI1 designs shown in FIG. 9A and
the design shown in FIG. 2C (or amino acids 1-94 thereof). When
referring to a .sup.10Fn3 molecule having a particular design based
on a .sup.10Fn3 sequence comprising amino acids 1-101, the
description is intended to encompass those molecules that do not
comprise "DK" at the end terminus and/or that do not comprise the
N-terminal 7 amino acids, and correspond to amino acids 1-94 of the
sequence shown. Alternatively, when the design shown comprises
amino acids 1-94, the description is intended to encompass the same
design with the N-terminal 7 amino acids, which may devoid of the
sequence "DK." Other modifications that may be made are described
herein.
[0141] Exemplary WS binder designs are provided in FIG. 12. WS3,
for example corresponds to WS1, wherein the length of the loops CD
and FG may be modified, and D67 may also be modified. An example of
a molecule having a WS3 design is PXR binder having SEQ ID NO: 49.
Variants of WS1, WS2, WS3, WS2', WS-LI1, and WS4 include those
having a wild-type or mutated amino acid at positions 30, 31, 33,
35, 37, 38, 46, 47, 49, 50, 67, 69, 71, 73, 75, 76, 84, 85, 86, 87,
89 or 91. For example, a WS binder design may comprise one or more
amino acid modifications in amino acids 39-45 of the CD loop and
one or more amino acid modification in amino acids 77-83 in loop FG
(WS-LI1 design), and wherein a .sup.10Fn3 molecule having that
design binds specifically to a target molecule (and optionally does
not comprise an RGD sequence). A WS binder design may comprise the
design of WS-LI1 and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15,
17, 20 or 25 additional amino acid modifications in the loops or
strands. For example, a WS binder design may comprise the design of
WS-LI1 and at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20 or
25 additional amino acid modifications at amino acid positions such
as at amino acid positions 37, 38, 46, 47, 75, 76, and 85-88. Other
amino acid modifications that may be included are those at
positions 30, 31, 33, 35, 49, 50, 67, 69, 71, 73, 89 and 91. An
exemplary WS design may comprise the amino acid sequence of WS7
(FIG. 12), wherein the loops may vary in length from those in the
wild-type .sup.10Fn3 molecule, and wherein each varied position
(bolded and underlined) may be modified to any other amino acid, or
in certain instances, may be kept unmodified, provided that a
.sup.10Fn3 molecule with such a design binds specifically to a
target molecule (and optionally does not comprise an RGD site). In
certain embodiments, a .sup.10Fn3 molecule comprises the amino acid
sequence of WS7, wherein the length of loops CD and FG may be
varied, wherein no other amino acid may be varied, and wherein
exactly or at most 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20
or 25 amino acid residues that are indicated as variable (those
underlined and bolded) are actually not changed, and are the amino
acids corresponding to those at the same position in the wild-type
human .sup.10Fn3 molecule, i.e., wild-type amino acids (SEQ ID NO:
1 or 6). For example, one or more of amino acids 30, 31, 33, 35,
36, 37, 47, 49, 50, 67, 69, 71, 73, 75 and 87 in WS7 may be the
wild-type amino acid, provided that the WS binder binds
specifically to its target. In certain embodiments, a WS binder
having a WS7 design does not comprise any amino acid modification
other than those indicated. In certain embodiments, a WS binder
having a WS7 design comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 12, 15, 17, 20 or 25 amino acid modifications in addition to
those indicated.
[0142] Also provided are libraries comprising any one of the WS
binder designs described herein. An exemplary library is a library
comprising WS binders having a varied CD and FG loop and further
comprising non wild-type amino acids at positions 30, 31, 33, 47
and 49. An exemplary library is a library having a varied FG loop
and further comprising non wild-type amino acids at positions 30,
31, 33, 47 and 49.
[0143] In certain embodiments, at least or at most 10, 20, 30, 40,
50, or 60 amino acids of a design sequence is not varied, e.g., is
not varied by substitution. For example, one or more of the
following amino acids are retained as the amino acid from the
wild-type human .sup.10Fn3 molecule: amino acids at positions 1-29,
32, 34, 36, 48, 51-66, 68, 70, 72, 88, 90 and 92-101.
[0144] Examples of WS binders that bind specifically to therapeutic
targets are described in the Examples, and include for example
polypeptides having the amino acid sequence of any one of SEQ ID
NOs: 3-5, 45-49, 62-63, 66, and 72.
[0145] In some embodiments, a WS binder comprises the amino acid
sequence of WS1, WS2, WS3, WS2', WS-LI1, WS4, WS5, WS6 or WS7 (or
amino acids 1-94 thereof) and comprises at most 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 additional substitutions, additions or deletions in a
loop and/or a strand. In certain embodiments, a WS binder comprises
the amino acid sequence of WS1, WS2, WS3, WS2', WS-LI1, WS4, WS5,
WS6 or WS7 (or amino acids 1-94 thereof) with no other amino acid
modifications. A WS binder that comprises the amino acid sequence
of WS1, WS2, WS3, WS2', WS-LI1, WS4, WS5, WS6 or WS7 (or amino
acids 1-94 thereof) may comprise any amino acid at a varied
position, and in some instances even that of the wild-type
.sup.10Fn3 molecule. In certain embodiments, a WS binder that
comprises the amino acid sequence of WS1, WS2, WS3, WS2', WS-LI1,
WS4, WS5, WS6 or WS7 (or amino acids 1-94 thereof) comprises non
wild-type amino acids at each of the positions indicated as varied
(those underlined and bolded).
[0146] 4. "Front" Binders
[0147] In some embodiments, the polypeptides provided herein
comprise a .sup.10Fn3 domain having modifications in the CD, DE
and, in some cases, EF loops, wherein the loop modifications all
contribute to target binding. These polypeptides are referred to as
"front binders" herein. The front binders may additionally comprise
modifications in one or more scaffold regions, particularly in
scaffold regions that flank or are adjacent to a modified loop
region. For example, the front binders may comprise a scaffold
modification in one or more of .beta.-strand C, .beta.-strand D,
and/or .beta.-strand E relative to the sequences of the
corresponding .beta.-strands of the wild-type human .sup.10Fn3
domain (SEQ ID NO: 1 or 6). Preferably the hydrophobic core
residues are not modified relative to the wild-type sequence.
Exemplary scaffold modifications that may be present in front
binders, include modifications at one or more positions
corresponding to amino acid positions 36, 49, 58 and/or 60 of SEQ
ID NO: 1 or 6. Such scaffold modifications may contribute to
binding to the target together with the modified loops. In certain
embodiments, the front binders may comprise clusters of
modifications spanning several loop and strand regions of the
.sup.10Fn3 domain. In particular, the front binders may comprise
modifications in at least 15, 20, 24, 25, or 27 of the 31 residues
between the amino acids corresponding to residues 36 through 66 of
the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). The loop
and/or strand modifications may include amino acid substitutions,
deletions and/or insertions, or combinations thereof. In exemplary
embodiments, the CD loop is extended in length or reduced in length
relative to the CD loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO: 1 or 6). Exemplary .sup.10Fn3 front binder designs
include, for example, the Front1 and Front2 designs shown in FIG.
9A.
[0148] 5. "Back" Binders
[0149] In some embodiments, the polypeptides provided herein
comprise a .sup.10Fn3 domain having modifications in the EF and FG
loops, wherein the loop modifications contribute to binding the
same target. These polypeptides are referred to as "back binders"
herein. The back binders may comprise additional modifications in
other loop and/or scaffold regions. For example, a back binder may
contain modifications in at least a portion of the AB loop,
preferably the N-terminal portion of the AB loop. In an exemplary
embodiment, the first two amino acids of the AB loop (i.e.,
corresponding to amino acid residues 14 and 15 of the wild-type
.sup.10Fn3 domain) are modified relative to the wild-type sequence.
In certain embodiments, a back binder may also contain one or more
scaffold modifications, particularly modifications in one or more
scaffold regions that are adjacent to a modified loop region. For
example, back binders may contain one or more modifications in one
or more of .beta.-strand A, .beta.-strand G, the N-terminal region,
and/or the C-terminal region. Preferably the hydrophobic core
residues are not modified relative to the wild-type sequence.
Exemplary scaffold modifications include modifications at one or
more positions corresponding to amino acid positions 1-7, 9-13, 89,
91, 93 and/or 94 of SEQ ID NO: 1 or 6. One or more of the
additional loop and/or scaffold modifications may contribute to
binding to the target along with the modified EF and FG loops.
Suitable loop and/or scaffold region modifications include amino
acid substitutions, deletions and/or insertions, or combinations
thereof. In certain embodiments, the amino acid sequence of the FG
loop is extended in length or reduced in length relative to the FG
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or
6).
[0150] In certain embodiments, a back binder may comprise a cluster
of modified amino acid residues over a contiguous span of several
regions in the .sup.10Fn3 domain. For example, at least 14 of the
first 15 amino acid residues of the .sup.10Fn3 domain may be
modified relative to the corresponding residues in the wild-type
human .sup.10Fn3 domain (SEQ ID NO: 1 or 6), and/or at least 15 of
the 18 residues between the amino acids corresponding to residues
80 through 97 (or 94) of the wild-type human .sup.10Fn3 domain (SEQ
ID NO: 1 or 6) may be modified relative to the corresponding
residues in the wild-type sequence.
[0151] Exemplary .sup.10Fn3 back binder designs include, for
example, the Back1 and Back2 designs shown in FIG. 9A.
[0152] 6. "South Pole" Binders
[0153] In certain embodiments, the application provides
polypeptides comprising a .sup.10Fn3 domain, wherein the .sup.10Fn3
domain comprises modifications in the amino acid sequences of
.beta.-strand A, loop AB, .beta.-strand B, loop CD, .beta.-strand
E, loop EF, and .beta.-strand F, relative to the sequences of the
corresponding regions of the wild-type sequence. These polypeptides
are referred to as "south pole binders" or "SP binders" herein. The
modified loops and strands contribute to binding to the same
target. The amino acid sequence of the CD loop may be extended in
length or reduced in length relative to the CD loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO: 1 or 6). The south
pole binders may comprise additional modifications in .beta.-strand
G and/or the C-terminal region relative to the sequence of the
corresponding region of the wild-type sequence. In exemplary
embodiments, the south pole binders may comprise one or more
modifications at amino acids corresponding to positions 11, 12, 19,
60, 61, 69, 91, 93 and 95-97 of the wild-type sequence. Exemplary
.sup.10Fn3 south pole binder designs include, for example, the SP1,
SP2 and SP3 designs shown in FIG. 9A.
[0154] 7. "Northwest" Binders
[0155] In some embodiments, the application provides polypeptides
comprising a .sup.10Fn3 domain having modified BC, DE and FG loops,
as compared to the corresponding BC, DE and FG loop sequences set
forth in SEQ ID NO: 1 or 6, as well as additional modifications in
one or more of .beta.-strand C, .beta.-strand D, .beta.-strand F
and .beta.-strand G strand residues. The .beta.-strand and loop
region modifications together contribute to binding to the target.
These proteins are referred to as "Northwest binders", or "NW
binders", herein. In exemplary embodiments, the NW binders comprise
one or more scaffold modifications at any one of, or combination
of, amino acid positions corresponding to scaffold region positions
R33, T49, Y73 and S89 of SEQ ID NO: 1 or 6. Suitable modifications
in loop and scaffold regions include amino acid substations,
deletions and/or insertions, or combinations thereof. In certain
embodiments, one or more of the BC, DE and FG loops are extended in
length or reduced in length, or combinations thereof, relative to
the wild-type sequence. In one embodiment, each of the BC, DE and
FG loops are extended in length or reduced in length, or
combinations thereof, relative to the wild-type sequence (e.g., SEQ
ID NO: 1 or 6). In certain embodiments, only a portion of the BC
loop is modified, particularly the C-terminal portion, relative to
the wild-type sequence. For example, the BC loop may be modified
only at amino acid residues corresponding to amino acids 27-31 of
the wild-type BC loop, whereas the rest of the BC loop (i.e.,
corresponding to residues 23-26 of the wild-type loop) are left
unmodified.
[0156] Exemplary .sup.10Fn3 NW binder designs include, for example,
the NW3 design shown in FIG. 9B, the NW2 design shown in FIG. 9C,
and the design shown in FIG. 2A. A model of the NW binder is
depicted in FIG. 3A.
[0157] 8. "Northeast" Binders
[0158] In some embodiments, the application provides polypeptides
comprising a .sup.10Fn3 domain having a modified BC, DE and FG loop
as well as one or more additional modifications in any one of, or
combination of, the N-terminal region, .beta.-strand A,
.beta.-strand B and/or .beta.-strand E. These proteins are referred
to as "Northeast binders", or "NE binders", herein. In exemplary
embodiments, the NE binders are modified at any one of, or
combination of, amino acids corresponding to scaffold region
positions 1-7, E9, L19, S21 and/or T58 of the wild-type sequence
(SEQ ID NO: 1 or 6). The combination of modified loop and scaffold
regions contributes to binding to the target. Exemplary .sup.10Fn3
NE binder designs include, for example, the NE1 design shown in
FIG. 9C and the design shown in FIG. 2B. A model of the NE binder
is depicted in FIG. 3B.
[0159] 9. "South Front" Binders
[0160] In some embodiments, the application provides polypeptides
comprising a .sup.10Fn3 domain having modifications in one or more
of the AB, CD, DE and EF loops, as well as additional modifications
in one or more of .beta.-strand B, .beta.-strand D and/or
.beta.-strand E. These proteins are referred to as "South Front
binders" herein. The combination of modified loop and strand
residues contributes to binding to the target. In exemplary
embodiments, a South Front binder may be modified at one or more
amino acid positions corresponding to scaffold region positions
L19, T49, T58, S60, and/or G61 of SEQ ID NO: 1 or 6 and/or at one
or more amino acid positions corresponding to loop region positions
T14-S17, P51, T56, G40-E47, and/or K63-G65 of SEQ ID NO: 1 or 6. In
exemplary embodiments, a South Front binder may be extended in
length or reduced in length in the AB loop, between amino acids
corresponding to residues 18 and 20 of the wild-type sequence,
and/or in the CD loop. Exemplary .sup.10Fn3 South Front binder
designs include, for example, the SouthFront design shown in FIG.
9A and the design shown in FIG. 2D. A model of the South Front
binder is depicted in FIG. 3D.
[0161] 10. "AG" Binders
[0162] In some embodiments, the application provides polypeptides
comprising a .sup.10Fn3 domain having a modified .beta.-strand A
and .beta.-strand G, as compared to the corresponding strand of SEQ
ID NO: 1 or 6. These proteins are referred to as "AG Binders" or
"AG Strand" binders herein. In certain embodiments, the AG strand
binders comprise clusters of modifications at the N-terminal and
C-terminal portions of the .sup.10Fn3 domain, whereas the middle
portion of the .sup.10Fn3 remains unmodified. For example, an AG
strand binder may comprise modifications at 16 out of 19 of the
first 19 amino acids in the .sup.10Fn3 domain (i.e., corresponding
to amino acid positions 1-19 of SEQ ID NO: 1 or 6) and
modifications at 13-17 out of 18 of the last 18 amino acids in the
.sup.10Fn3 domain (i.e., corresponding to amino acid positions
84-101 of SEQ ID NO: 1) or at 14-18 out of 22 of the last 22 amino
acids in the .sup.10Fn3 domain (i.e., corresponding to amino acid
positions 80-101 of SEQ ID NO: 1). In exemplary embodiments, an AG
binder may comprise modifications at one or more positions
corresponding to positions 1-7, 9, 11-17, 19, 84-89 and 91-97 of
SEQ ID NO: 1. Preferably the modified regions in an AG binder
contribute to binding to the same target. Exemplary .sup.10Fn3 AG
binder designs include, for example, the AG Strand design shown in
FIG. 9A and the design shown in FIG. 2E. A model of the AG binder
is depicted in FIG. 3E.
[0163] 11. "Southwest" Binders
[0164] In some embodiments, the application provides polypeptides
comprising a .sup.10Fn3 domain having a modified CD and EF loop, as
well as additional modifications in any one of, or combination of
residues corresponding to positions 69 or 91-97 of SEQ ID NO: 1.
These proteins are referred to as "Southwest binders", or "SW
binders", herein. The modified loop and scaffold regions contribute
to binding to the target. Exemplary .sup.10Fn3 SW binder designs
include, for example, the SouthWest design shown in FIG. 9A and the
design shown in FIG. 2F. A model of the SW binder is depicted in
FIG. 3F.
[0165] E. Proteins Having Reduced Immunogenicity
[0166] In some embodiments, the polypeptides provided herein are
associated with reduced immunogenicity. As described in the
examples, the region around the BC loop of a .sup.10Fn3 domain
appears to be an immunogenic hot spot. Accordingly, the application
provides two types of .sup.10Fn3 designs having reduced
immunogenicity. In the first type of design, the BC loop is left
entirely or at least partially unmodified such that the host (e.g.,
human) immune response is more likely to recognize the BC region of
the .sup.10Fn3 domain as self thereby avoiding an immune response.
In the second type of design, the strong HLA binding anchor in the
BC region of the .sup.10Fn3 domain is removed or destroyed such
that the BC region should not bind as tightly to the host HLA
receptors thereby decreasing the immunogenic potential of the BC
region of the .sup.10Fn3 binders. These .sup.10Fn3 designs are
described further below.
[0167] In certain embodiments, the application provides
polypeptides having reduced immunogenicity comprising a .sup.10Fn3
domain wherein the entire BC loop is left as wild-type. Preferably
such polypeptides have lower immunogenicity relative to an
equivalent polypeptide with modifications in the BC loop.
Polypeptides with wild-type BC loops have modifications in other
regions of the .sup.10Fn3 domain that are involved in target
binding. Preferably, the modifications outside of the BC loop do
not lead to a strong immune response to the .sup.10Fn3 domain in
the host. Examples of .sup.10Fn3 binders where the entire BC loop
is left as wild-type include, for example, the WS binders, Front
binders, Back binders, South Pole binders, South Front binders, AG
binders and Southwest binders as described herein. Particular
examples of .sup.10Fn3 designs having the BC loop unmodified
relative to the wild-type sequence, include, for example, the WS2,
WS3, Front1, Front2, Back1, Back2, SP1, SP2, SP3, LI-3(a), WS-LI1,
LU-S9, SouthFront, AG Strand and SouthWest designs shown in FIG. 9A
and the designs shown in FIGS. 2D-2F. In .sup.10Fn3 binder designs
having a wild-type BC loop, it may be desirable to leave all or a
portion of .beta.-strand B and/or .beta.-strand C unmodified
relative to the wild-type sequence as well, particularly the
portions of .beta.-strand B and/or .beta.-strand C that are
adjacent to the BC loop (i.e., the C-terminal portion of
.beta.-strand B and/or the N-terminal portion of .beta.-strand C).
In exemplary embodiments, .sup.10Fn3 domains having a wild-type BC
loop and reduced immunogenicity may not have any modifications in
the portion of the .sup.10Fn3 domain that is N-terminal to the CD
loop, i.e., the N-terminal region, .beta.-strand A, AB loop,
.beta.-strand B, BC loop and .beta.-strand C are all left
unmodified relative to the wild-type sequence.
[0168] In certain embodiments, the application provides
polypeptides having reduced immunogenicity comprising a .sup.10Fn3
domains wherein a portion of the BC loop is left as wild-type.
Preferably such polypeptides have lower immunogenicity relative to
an equivalent polypeptide with modifications in a greater portion
of the BC loop. In exemplary embodiments, the N-terminal portion of
the BC loop is left as wild-type. For example, the first 1, 2, 3,
4, 5, or 6 residues of the BC loop may be left as wild-type, while
the remaining C-terminal residues of the BC loop can be modified.
In .sup.10Fn3 designs having at least a portion of the N-terminal
region of the BC loop as wild-type, it may be desirable to leave
all or a portion of .beta.-strand B and/or .beta.-strand C
unmodified relative to the wild-type sequence as well, particularly
the portions of .beta.-strand B and/or .beta.-strand C that are
adjacent to the BC loop (i.e., the C-terminal portion of
.beta.-strand B and/or the N-terminal portion of .beta.-strand C).
In exemplary embodiments, .sup.10Fn3 domains having the wild-type
sequence in an N-terminal portion of the BC loop and reduced
immunogenicity may not have any modifications in the N-terminal
region, .beta.-strand A, AB loop, and .beta.-strand B. In
.sup.10Fn3 designs with a portion of the BC loop as wild-type, the
modified portion of the BC loop may contribute to target binding
along with modifications in other regions of the .sup.10Fn3 domain.
Examples of .sup.10Fn3 binders where an N-terminal portion of the
BC loop is left as wild-type include, for example, the .sup.10Fn3
designs shown in FIG. 9B, the WS1 design shown in FIG. 9A, and the
.sup.10Fn3 design shown in FIG. 2C.
[0169] In certain embodiments, the application provides
polypeptides having reduced immunogenicity comprising .sup.10Fn3
domains, wherein the strong HLA anchor in the region of
.beta.-strand B/BC loop/.beta.-strand C (the "BC anchor") has been
removed or destroyed (e.g., modified relative to the wild-type
sequence in a manner that reduces binding affinity to one or more
HLA receptors). For example, the BC anchor may be removed or
destroyed by modifying the .sup.10Fn3 domain at one or more
positions corresponding to positions L19, S21, R33 and/or T35 of
SEQ ID NO:1 or 6. When the BC anchor has been removed or destroyed,
it is possible to modify the sequence of the BC loop without
significantly increasing the immunogenic potential of the BC
region. Accordingly, many such .sup.10Fn3 designs have
modifications in the BC loop in addition to the modifications in
.beta.-strand B and/or .beta.-strand C. The BC loop may contribute
to target binding, optionally in combination with modifications in
other regions of the .sup.10Fn3 domain. The modifications in
.beta.-strand B and/or .beta.-strand C may or may not contribute to
target binding. Examples of .sup.10Fn3 binders where the BC anchor
has been removed or destroyed include, for example, the .sup.10Fn3
designs shown in FIG. 9C and the .sup.10Fn3 design shown in FIG.
2B.
[0170] In certain embodiments, the polypeptides described herein
have reduced immunogenicity as compared to a polypeptide having SEQ
ID NO: 61, e.g., the immunogenicity of the polypeptide is lower
than the immunogenicity of a polypeptide having SEQ ID NO: 61.
[0171] The immunogenicity of a polypeptide described herein may be
assessed, for example, by one or more of the following methods:
Human Leukocyte Antigen ("HLA") binding, in silico prediction of
HLA binding (for example, with the Epimatrix program), in vitro
activation of human T-cells, in vivo animal immune response, or
other methods for evaluating immunogenicity potential.
[0172] In certain embodiments, immunogenicity may be assessed by
HLA binding experiments. Preferably the polypeptides provided
herein bind to one or more HLA receptors with an IC.sub.50 that is
less than or equal to the IC.sub.50 associated with binding between
an equivalent HLA receptor(s) and a wild-type .sup.10Fn3 domain.
For example, the polypeptides provided herein may bind to an HLA
receptor with an IC.sub.50 of greater than 10 .mu.M, 15 .mu.M, 20
.mu.M, 25 .mu.M, 50 .mu.M, 100 .mu.M, 150 .mu.M or 200 .mu.M. In
some embodiments, the polypeptides may bind to an HLA receptor with
an IC.sub.50 between 10 .mu.M and 1 mM, between 100 .mu.M and 1 mM
or between 500 .mu.M and 1 mM. The HLA allele used for assessing
the polypeptide/HLA IC.sub.50 binding may be one or more of
DRB*0101, DRB*0301, DRB*0401, DRB*0701 and/or DRB*1501.
[0173] In some embodiments, immunogenicity may be assessed by in
silico analysis, such as EpiMatrix. In particular embodiments, the
polypeptides provided herein are associated with an EpiMatrix "Z"
scale score less than or equal to the score associated with a
wild-type .sup.10Fn3 domain. In certain embodiments, the
polypeptides provided herein are associated with an EpiMatrix "Z"
scale score that is no greater than 200% of the score associated
with a wild-type .sup.10Fn3 domain. In some embodiments, the
polypeptides are associated with an EpiMatrix score less than 1.64
on the EpiMatrix "Z" scale (An Z; 2009; Therapeutic Monoclonal
Antibodies: From Bench to Clinic; John Wiley and Sons; New Jersey;
pages 428-429).
[0174] In some embodiments, immunogenicity may be assessed by in
vivo animal immune response experiments. For example, an animal,
e.g. mouse or monkey, may be injected with the polypeptides
provided herein and the IgG and/or IgM immune response measured.
Preferably, the polypeptides described herein display an IgG or IgM
immune response that is no more than 200%, 100%, 50%, 40%, 30%,
25%, 20%, 15%, 10%, 5%, 2% or 1% greater than the IgG or IgM immune
response observed in a mouse or monkey injected with a wild-type
.sup.10Fn3 domain.
[0175] The application also provides libraries of the polypeptides
provided herein and methods of selecting binders to a desired
target from the libraries. There will be a higher likelihood of
isolating a target binding molecule with acceptable immunogenicity
characteristics from the libraries provided herein, relative to
libraries that have not been designed to avoid the immunogenic
potential associated with the BC region. These libraries are useful
for reducing the amount of effort necessary to deimmunize
polypeptide candidates, and to increase the probability of
identifying non-immunogenic polypeptide molecules.
[0176] F. North Pole Variants
[0177] Additional .sup.10Fn3 designs disclosed herein include those
having variations (or variable regions) in the N-terminus; BC loop
and/or amino acids around the BC loop; DE loop and/or amino acids
around the DC loop; and/or FG loop and/or amino acids around the FG
loop. Exemplary .sup.10Fn3 designs are set forth in FIG. 13.
[0178] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 or 6 are represented by x.sub.1-9Lx,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 of the variable (i.e., x) positions. In
certain embodiments, the amino acid sequence x.sub.1-9Lx is not
identical to the amino acid sequence corresponding to amino acids
1-9 of SEQ ID NO: 1. Exemplary .sup.10Fn3 designs having amino
acids represented by x.sub.1-9Lx include, e.g., LI-39(a)-(l),
LI-39B(a)-(j), LI-39F(a)-(h), and LI-59(a)-(f), which are shown in
FIG. 13.
[0179] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 1-9 of SEQ ID NO: 1 are represented by x.sub.1-9Lx, linked
(e.g., covalently linked) at its N-terminus to GVSDVPGGSG (SEQ ID
NO: 250) or 1, 2, 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids
thereof. Exemplary N-terminal amino acid sequences of .sup.10Fn3
domains may comprise: Gx.sub.1-9Lx (SEQ ID NO: 251), SGx.sub.1-9Lx
(SEQ ID NO: 252), GSGx.sub.1-9Lx (SEQ ID NO: 253), GGSGx.sub.1-9Lx
(SEQ ID NO: 254), PGGSGx.sub.1-9Lx (SEQ ID NO: 255),
VPGGSGx.sub.1-9Lx (SEQ ID NO: 256), DVPGGSGx.sub.1-9Lx (SEQ ID NO:
257), SDVPGGSGx.sub.1-9Lx (SEQ ID NO: 258), VSDVPGGSGx.sub.1-9Lx
(SEQ ID NO: 259), GVSDVPGGSGx.sub.1-9Lx (SEQ ID NO: 260),
GVx.sub.1-9Lx (SEQ ID NO: 261), GVSx.sub.1-9Lx (SEQ ID NO: 262),
GVSDx.sub.1-9Lx (SEQ ID NO: 263), GVSDVx.sub.1-9Lx (SEQ ID NO:
264), GVSDVPx.sub.1-9Lx (SEQ ID NO: 265), GVSDVPGx.sub.1-9Lx (SEQ
ID NO: 266), GVSDVPGGx.sub.1-9Lx (SEQ ID NO: 267),
GVSDVPGGSx.sub.1-9Lx (SEQ ID NO: 268), wherein x is either any
amino acid or x is any amino acid except the amino acid that occurs
at the corresponding position in SEQ ID NO: 1. These sequences may
be linked to V10 of a .sup.10Fn3 domain. In certain embodiments,
the amino acid sequence x.sub.1-9Lx is not identical to the amino
acid sequence corresponding to amino acids 1-9 of SEQ ID NO: 1.
Exemplary .sup.10Fn3 designs having amino acids represented by
x.sub.1-9Lx include, e.g., LI-39(a)-(f), LI-39B(a)-(e),
LI-39F(a)-(d), and LI-59(a)-(c), which are shown in FIG. 13.
[0180] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 28-29 of SEQ ID NO: 1 are represented by xx, wherein x is
either any amino acid or x is any amino acid except the amino acid
that occurs at the corresponding position in SEQ ID NO: 1. In
certain embodiments, x is not the amino acid that occurs at the
corresponding position in SEQ ID NO: 1 or 6 in 1 or 2 of the
variable (i.e., x) positions. In certain embodiments, the amino
acid sequence xx is not identical to the amino acid sequence
corresponding to amino acids 28-29 of SEQ ID NO: 1. Exemplary
.sup.10Fn3 designs having amino acids 28-29 represented by xx
include, e.g., LI-3(b), which is shown in FIG. 13.
[0181] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 23-29 of SEQ ID NO: 1 are represented by x.sub.7, wherein
x is either any amino acid or x is any amino acid except the amino
acid that occurs at the corresponding position in SEQ ID NO: 1. In
certain embodiments, x is not the amino acid that occurs at the
corresponding position in SEQ ID NO: 1 or 6 in 1, 2, 3, 4, 5, 6 or
7 of the variable (i.e., x) positions. In certain embodiments, the
amino acid sequence x.sub.7 is not identical to the amino acid
sequence corresponding to amino acids 23-29 of SEQ ID NO: 1.
Exemplary .sup.10Fn3 designs having amino acids 23-29 represented
by x.sub.7 include, e.g., LI-5, LI-51, LI-52, LI-53, LI-54,
LI-39B(d) and LI-39B(i) which are shown in FIG. 13.
[0182] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 25-29 of SEQ ID NO: 1 are represented by x.sub.5, wherein
x is either any amino acid or x is any amino acid except the amino
acid that occurs at the corresponding position in SEQ ID NO: 1 or
6. In certain embodiments, x is not the amino acid that occurs at
the corresponding position in SEQ ID NO: 1 or 6 in 1, 2, 3, 4 or 5
of the variable (i.e., x) positions. In certain embodiments, the
amino acid sequence x.sub.5 is not identical to the amino acid
sequence corresponding to amino acids 25-29 of SEQ ID NO: 1.
Exemplary .sup.10Fn3 designs having amino acids 25-29 represented
by x.sub.5 include, e.g., LI-31(a), LI-32(a), LI-59(a) and
LI-59(d), which are shown in FIG. 13.
[0183] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 26-29 of SEQ ID NO: 1 are represented by x.sub.4, wherein
x is either any amino acid or x is any amino acid except the amino
acid that occurs at the corresponding position in SEQ ID NO: 1 or
6. In certain embodiments, x is not the amino acid that occurs at
the corresponding position in SEQ ID NO: 1 or 6 in 1, 2, 3 or 4 of
the variable (i.e., x) positions. In certain embodiments, the amino
acid sequence x.sub.4 is not identical to the amino acid sequence
corresponding to amino acids 26-29 of SEQ ID NO: 1. Exemplary
.sup.10Fn3 designs having amino acids 26-29 represented by x.sub.4
include, e.g., LI-31(b), LI-32(b), LI-59(b) and LI-59(e), which are
shown in FIG. 13.
[0184] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 27-29 of SEQ ID NO: 1 are represented by x.sub.3, wherein
x is either any amino acid or x is any amino acid except the amino
acid that occurs at the corresponding position in SEQ ID NO: 1 or
6. In certain embodiments, x is not the amino acid that occurs at
the corresponding position in SEQ ID NO: 1 or 6 in 1, 2 or 3 of the
variable (i.e., x) positions. In certain embodiments, the amino
acid sequence x.sub.3 is not identical to the amino acid sequence
corresponding to amino acids 27-29 of SEQ ID NO: 1. Exemplary
.sup.10Fn3 designs having amino acids 27-29 represented by x.sub.3
include, e.g., LI-31(c), LI-32(c), LI-59(c) and LI-59(f), which are
shown in FIG. 13.
[0185] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 24-28 of SEQ ID NO: 1 or 6 are represented by x.sub.5,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4 or 5 of the variable (i.e., x) positions. In certain
embodiments, the amino acid sequence x.sub.5 is not identical to
the amino acid sequence corresponding to amino acids 24-28 of SEQ
ID NO: 1. Exemplary .sup.10Fn3 designs having amino acids 24-28
represented by x.sub.5 include, e.g., LI-38(a), LI-38(d), LI-39(a),
LI-39(d), LI-39(g), LI-39(j), LI-39B(a) and LI-39B(f), which are
represented in FIG. 13.
[0186] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 25-28 of SEQ ID NO: 1 or 6 are represented by x.sub.4,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3 or 4 of the variable (i.e., x) positions. In certain embodiments,
the amino acid sequence x.sub.4 is not identical to the amino acid
sequence corresponding to amino acids 25-28 of SEQ ID NO: 1.
Exemplary .sup.10Fn3 designs having amino acids 25-28 represented
by x.sub.4 include, e.g., LI-38(b), LI-38(e), LI-39(b), LI-39(e),
LI-39(h), LI-39(k), LI-39B(b) and LI-39B(g), which are shown in
FIG. 13.
[0187] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 26-28 of SEQ ID NO: 1 or 6 are represented by x.sub.3,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2
or 3 of the variable (i.e., x) positions. In certain embodiments,
the amino acid sequence x.sub.3 is not identical to the amino acid
sequence corresponding to amino acids 26-28 of SEQ ID NO: 1 or 6.
Exemplary .sup.10Fn3 designs having amino acids 26-28 represented
by x.sub.3 include, e.g., LI-38(c), LI-38(f), LI-39(c), LI-39(f),
LI-39(i), LI-39(l), LI-39B(c) and LI-39B(h), which are shown in
FIG. 13.
[0188] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 17-26 of SEQ ID NO: 1 or 6 are represented by
xLxIxWx.sub.4, wherein x is either any amino acid or x is any amino
acid except the amino acid that occurs at the corresponding
position in SEQ ID NO: 1 or 6. In certain embodiments, x is not the
amino acid that occurs at the corresponding position in SEQ ID NO:
1 or 6 in 1, 2, 3, 4, 5, 6 or 7 of the variable (i.e., x)
positions. In certain embodiments, the amino acid sequence
xLxIxWx.sub.4 is not identical to the amino acid sequence
corresponding to amino acids 17-26 of SEQ ID NO: 1. Exemplary
.sup.10Fn3 designs having amino acids 17-26 represented by x.sub.4
include, e.g., LI39B(e), LI-39B(j), NP4E1, NP4E2 and NP4E3, which
are shown in FIG. 13.
[0189] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 24-35 of SEQ ID NO: 1 or 6 are represented by x.sub.6YxIx,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7 or 8 of the variable (i.e., x) positions. In certain
embodiments, the amino acid sequence x.sub.6YxIx is not identical
to the amino acid sequence corresponding to amino acids 24-35 of
SEQ ID NO: 1. Exemplary .sup.10Fn3 designs having amino acids 24-35
represented by x.sub.6YxIx include, e.g., NP4W, which is shown in
FIG. 13.
[0190] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 52-55 of SEQ ID NO: 1 or 6 are represented by x.sub.4,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3 or 4 of the variable (i.e., x) positions. In certain embodiments,
the amino acid sequence x.sub.4 is not identical to the amino acid
sequence corresponding to amino acids 52-55 of SEQ ID NO: 1.
Exemplary .sup.10Fn3 designs having amino acids 52-55 represented
by x.sub.4 include, e.g., Li-5, LI-52 and LI-54, which are shown in
FIG. 13.
[0191] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 47-55 of SEQ ID NO: 1 or 6 are represented by xFxVx.sub.4,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5 or 6 of the variable (i.e., x) positions. In certain
embodiments, the amino acid sequence xFxVx.sub.4 is not identical
to the amino acid sequence corresponding to amino acids 47-55 of
SEQ ID NO: 1. Exemplary .sup.10Fn3 designs having amino acids 47-55
represented by xFxVx.sub.4 include, e.g., NP4W, which is shown in
FIG. 13.
[0192] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 51-60 of SEQ ID NO: 1 or 6 are represented by x.sub.6AxIx,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7 or 8 of the variable (i.e., x) positions. In certain
embodiments, the amino acid sequence x.sub.6AxIx is not identical
to the amino acid sequence corresponding to amino acids 51-60 of
SEQ ID NO: 1. Exemplary .sup.10Fn3 designs having amino acids 51-60
represented by x.sub.6AxIx include, e.g., NP4E1, NP4E2 and NP4E3,
which are shown in FIG. 13.
[0193] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 77-83 of SEQ ID NO: 1 or 6 are represented by x.sub.3-12,
such as x.sub.7, wherein x is either any amino acid or x is any
amino acid except the amino acid that occurs at the corresponding
position in SEQ ID NO: 1 or 6. In certain embodiments, x is not the
amino acid that occurs at the corresponding position in SEQ ID NO:
1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 of the variable
(i.e., x) positions. In certain embodiments, the amino acid
sequence x.sub.3-12 is not identical to the amino acid sequence
corresponding to amino acids 77-83 of SEQ ID NO: 1 or 6. Exemplary
.sup.10Fn3 designs having amino acids 77-83 represented by
x.sub.3-12 include, e.g., LI-3(a), LI-3(b), LI-5, LI-31(a)-(c),
LI-39F(b), LI-39F(f), LI-51, LI-52, LI-59(a)-(f), which are shown
in FIG. 13.
[0194] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 77-86 of SEQ ID NO: 1 or 6 are represented by x.sub.6-15,
such as x.sub.10, wherein x is either any amino acid or x is any
amino acid except the amino acid that occurs at the corresponding
position in SEQ ID NO: 1 or 6. In certain embodiments, x is not the
amino acid that occurs at the corresponding position in SEQ ID NO:
1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of
the variable (i.e., x) positions. In certain embodiments, the amino
acid sequence x.sub.6-15 is not identical to the amino acid
sequence corresponding to amino acids 77-86 of SEQ ID NO: 1 or 6.
Exemplary .sup.10Fn3 designs having amino acids 77-86 represented
by x.sub.6-15 include, e.g., LI-32(a)-(c), LI-39F(a), LI-39F(e),
LI-53 and LI-54, which are shown in FIG. 13.
[0195] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 75-86 of SEQ ID NO: 1 or 6 are represented by x.sub.4-8Sx,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7, 8 or 9 of the variable (i.e., x) positions. In
certain embodiments, the amino acid sequence x.sub.4-8Sx is not
identical to the amino acid sequence corresponding to amino acids
75-86 of SEQ ID NO: 1 or 6. Exemplary .sup.10Fn3 designs having
amino acids 75-86 represented by x.sub.4-8Sx include, e.g.,
LI-38(a)-(c), LI-39(a)-(c), LI-39(g)-(i) LI-39F(c) and LI-39F(g)
which are shown in FIG. 13.
[0196] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 75-83 of SEQ ID NO: 1 or 6 are represented by x.sub.8-14,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the variable (i.e., x)
positions. In certain embodiments, the amino acid sequence
x.sub.8-14 is not identical to the amino acid sequence
corresponding to amino acids 75-83 of SEQ ID NO: 1 or 6. Exemplary
.sup.10Fn3 designs having amino acids 75-83 represented by
x.sub.8-14 include, e.g., LI-38(d)-(f), LI-39(d)-(f), LI-39(j)-(l),
LI-39F(d) and LI-39F(h), which are shown in FIG. 13.
[0197] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 73-89 of SEQ ID NO: 1 or 6 are represented by
xAxxx.sub.3-12xIx, wherein x is either any amino acid or x is any
amino acid except the amino acid that occurs at the corresponding
position in SEQ ID NO: 1 or 6. In certain embodiments, x is not the
amino acid that occurs at the corresponding position in SEQ ID NO:
1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of
the variable (i.e., x) positions. In certain embodiments, the amino
acid sequence xAxxx.sub.3-12xIx is not identical to the amino acid
sequence corresponding to amino acids 73-89 of SEQ ID NO: 1 or 6.
Exemplary .sup.10Fn3 designs having amino acids 73-89 represented
by xAxxx.sub.3-12xIx include, e.g., NP4W, which is shown in FIG.
13.
[0198] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 76-86 of SEQ ID NO: 1 or 6 are represented by xx.sub.3-12,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the variable (i.e., x)
positions. In certain embodiments, the amino acid sequence
xx.sub.3-12 is not identical to the amino acid sequence
corresponding to amino acids 76-86 of SEQ ID NO: 1 or 6. Exemplary
.sup.10Fn3 designs having amino acids 76-86 represented by
xx.sub.3-12 include, e.g., NP4E1, which is shown in FIG. 13.
[0199] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 76-86 of SEQ ID NO: 1 or 6 are represented by
xx.sub.4-8Sx, wherein x is either any amino acid or x is any amino
acid except the amino acid that occurs at the corresponding
position in SEQ ID NO: 1 or 6. In certain embodiments, x is not the
amino acid that occurs at the corresponding position in SEQ ID NO:
1 or 6 in 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the variable (i.e., x)
positions. In certain embodiments, the amino acid sequence
xx.sub.4-8Sx is not identical to the amino acid sequence
corresponding to amino acids 76-86 of SEQ ID NO: 1 or 6. Exemplary
.sup.10Fn3 designs having amino acids 76-86 represented by
xx.sub.4-8Sx include, e.g., NP4E2, which is shown in FIG. 13.
[0200] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence wherein the amino acids corresponding to
residues 76-83 of SEQ ID NO: 1 or 6 are represented by xx.sub.8-14,
wherein x is either any amino acid or x is any amino acid except
the amino acid that occurs at the corresponding position in SEQ ID
NO: 1 or 6. In certain embodiments, x is not the amino acid that
occurs at the corresponding position in SEQ ID NO: 1 or 6 in 1, 2,
3, 4, 5, 6, 7 or 8 of the variable (i.e., x) positions. In certain
embodiments, the amino acid sequence xx.sub.8-14 is not identical
to the amino acid sequence corresponding to amino acids 76-83 of
SEQ ID NO: 1 or 6. Exemplary .sup.10Fn3 designs having amino acids
76-83 represented by xx.sub.8-14 include, e.g., NP4E3, which is
shown in FIG. 13.
[0201] .sup.10Fn3 domains may also comprise a combination of 2, 3
or 4 of the above described variable regions. Exemplary
combinations of variable regions include (i) an N-terminal variable
region and a BC variable region; (ii) an N-terminal variable region
and a DE variable region; (iii) an N-terminal variable region and
an FG variable region; (iv) a BC variable region and a DE variable
region; (v) a BC variable region and an FG variable region; and
(vi) a DE variable region and an FG variable region.
[0202] An N-terminal variable region may comprise, consist of, or
consist essentially of:
[0203] (1) a variation of the amino acids corresponding to residues
1-9 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.1-9Lx; or
[0204] (2) a variation of the amino acids corresponding to residues
1-9 of SEQ ID NO: 1 or 6, wherein the variation consists of the
amino acid sequence GVSDVPGGSG (SEQ ID NO: 250), or a portion
thereof, linked to an amino acid sequence represented by
x.sub.1-9Lx,
[0205] wherein x is either any amino acid or x is any amino acid
except the amino acid that occurs at the corresponding position in
SEQ ID NO: 1 or 6.
[0206] A BC variable region may comprise, consist of, or consist
essentially of:
[0207] (1) a variation of the amino acids corresponding to residues
23-29 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.7; or
[0208] (2) a variation of the amino acids corresponding to residues
25-29 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.5; or
[0209] (3) a variation of the amino acids corresponding to residues
26-29 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.4; or
[0210] (4) a variation of the amino acids corresponding to residues
27-29 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.3; or
[0211] (5) a variation of the amino acids corresponding to residues
24-28 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.5; or
[0212] (6) a variation of the amino acids corresponding to residues
25-28 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.4; or
[0213] (7) a variation of the amino acids corresponding to residues
26-28 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.3; or
[0214] (8) a variation of the amino acids corresponding to residues
17-26 of SEQ ID NO: 1 or 6, wherein the variation is represented by
xLxIxWx.sub.4; or
[0215] (9) a variation of the amino acids corresponding to residues
24-35 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.6YxIx,
[0216] wherein x is either any amino acid or x is any amino acid
except the amino acid that occurs at the corresponding position in
SEQ ID NO: 1 or 6.
[0217] A DE variable region may comprise, consist of, or consist
essentially of:
[0218] (1) a variation of the amino acids corresponding to residues
52-55 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.4; or
[0219] (2) a variation of the amino acids corresponding to residues
47-55 of SEQ ID NO: 1 or 6, wherein the variation is represented by
xFxVx.sub.4; or
[0220] (3) a variation of the amino acids corresponding to residues
51-60 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.6AxIx,
[0221] wherein x is either any amino acid or x is any amino acid
except the amino acid that occurs at the corresponding position in
SEQ ID NO: 1 or 6.
[0222] An FG variable region may comprise, consist of, or consist
essentially of:
[0223] (1) a variation of the amino acids corresponding to residues
77-83 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.3-12; or
[0224] (2) a variation of the amino acids corresponding to residues
77-86 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.6-15; or
[0225] (3) a variation of the amino acids corresponding to residues
75-86 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.4-8Sx; or
[0226] (4) a variation of the amino acids corresponding to residues
75-83 of SEQ ID NO: 1 or 6, wherein the variation is represented by
x.sub.8-14; or
[0227] (5) a variation of the amino acids corresponding to residues
73-89 of SEQ ID NO: 1 or 6, wherein the variation is represented by
xAxxx.sub.3-12xIx; or
[0228] (6) a variation of the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6, wherein the variation is represented by
xx.sub.3-12; or
[0229] (7) a variation of the amino acids corresponding to residues
76-86 of SEQ ID NO: 1 or 6, wherein the variation is represented by
xx.sub.4-8Sx; or
[0230] (8) a variation of the amino acids corresponding to residues
76-83 of SEQ ID NO: 1 or 6, wherein the variation is represented by
xx.sub.8-14,
[0231] wherein x is either any amino acid or x is any amino acid
except the amino acid that occurs at the corresponding position in
SEQ ID NO: 1 or 6.
[0232] Exemplary combinations of N-terminal, BC, DE and FG variable
regions are shown in FIG. 13. The amino acid sequence of exemplary
.sup.10Fn3 proteins having a design shown in FIG. 13 or a design
that differs therefrom in at most a few amino acids are shown in
FIG. 14. Libraries having these molecular designs, methods of
screening these libraries, as well as .sup.10Fn3 molecules isolated
from such libraries, e.g., using the PROfusion in vitro selection
method, are also encompassed herein. For example, .sup.10Fn3
molecules isolated from a library having a design shown in FIG. 13,
which .sup.10Fn3 molecules have a design that differs from a design
in FIG. 13 in at most 1-3 amino acids, e.g., at most 1, 2 or 3
amino acids, are also encompassed herein.
[0233] In certain embodiments, a .sup.10Fn3 domain comprises an
amino acid sequence set forth in FIG. 13 and does not comprise any
other amino acid variation, such an amino acid substitution,
addition or deletion, outside of the variable regions (i.e.,
outside of the N-terminal, BC, DE and FG variable regions). In
certain embodiments, a .sup.10Fn3 domain comprises an amino acid
sequence set forth in FIG. 13 and at least, at most or exactly 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35 or 40 amino
acid variations, such as amino acid substitutions, additions or
deletions, outside of the variable regions. In certain embodiments,
a .sup.10Fn3 domain comprises an amino acid sequence set forth in
FIG. 13 and at least, at most or exactly 1-25, 1-20, 1-15, 1-10,
1-5, 1-3, 1-2, 3-25, 3-20, 3-15, 3-10, 3-5, 5-25, 5-20, 5-15 or
5-10 amino acid variations, such as amino acid substitutions,
additions or deletions, outside of the variable regions. A
.sup.10Fn3 domain may comprise an amino acid sequence that is at
least 40%, 50%, 60%, 70%, 75%, 80%, 85% or 90% identical to the
amino acid sequence having SEQ ID NO: 1 or 6, and comprising the
amino acid variations of any one of the sequences (SEQ ID NOs:
73-131 or SEQ ID NOs: 76-131) set forth in FIG. 13. A .sup.10Fn3
domain may comprise the amino acid variations of an amino acid
sequence of any one of the sequences set forth in FIG. 13, wherein
the regions indicated as constant in the amino acid sequence
comprise (consist of, or consist essentially of) an amino acid
sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence at
the corresponding location in SEQ ID NO: 1 or 6. The percentage
identity may be calculated as an average number between the
percentage identity of several regions of a .sup.10Fn3 sequence.
For example, the percentage identity of the regions outside the
variable regions of a .sup.10Fn3 molecule may be calculated by
obtaining the average value of the percent identity of each
individual region outside or between the variable regions. A
.sup.10Fn3 domain may comprise a variable region, e.g., an
N-terminal, BC, DE or FG variable region, and at most 50, 40, 35,
30, 25, 20, 15, 10, 7, 5, 3, 2 or 1 additional amino acid changes,
such as amino acid substitutions, deletions or additions relative
to SEQ ID NO: 1 or 6. A .sup.10Fn3 domain may comprise a variable
region, e.g., an N-terminal, BC, DE or FG variable region, wherein
the amino acid sequence of the .sup.10Fn3 domain outside of the
variable region is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
97% or 99% identical to the corresponding sequence in SEQ ID NO: 1
or 6. A .sup.10Fn3 domain may comprise a variable region, e.g., an
N-terminal, BC, DE or FG variable region, wherein the .sup.10Fn3
domain comprises at least one amino acid variation in the amino
acid sequence corresponding to another variable region. The amino
acid sequences of exemplary variants of the designs shown in FIG.
13 are provided in FIG. 14. For example, SEQ ID NOs: 148 and 171
are amino acid sequences of .sup.10Fn3 molecules having a design
that is a variant of that of LI-32, and differ therefrom in 1-3
amino acids. SEQ ID NOs: 133, 143 and 176 are amino acid sequences
of .sup.10Fn3 molecules having a design that is a variant of that
of LI-38, and differ therefrom in 1-3 amino acids. SEQ ID NO: 223
is the amino acid sequence of a .sup.10Fn3 molecule having a design
that is a variant of that of LI-51, and differs therefrom in 1-3
amino acids. SEQ ID NO: 153 and 162 are amino acid sequences of a
.sup.10Fn3 molecule having a design that is a variant of that of
LI-54, and differ therefrom in 1-3 amino acids.
[0234] A .sup.10Fn3 domain, e.g., having a design set forth in one
of the Figures, may bind to a desired target with a K.sub.d of less
than 500 nM, 100 nM, 50 nM, 1 nM, 500 pM, 100 pM or less. In some
embodiments, a .sup.10Fn3 domain binds to a desired target with a
K.sub.d between 1 pM and 1 .mu.M, between 100 pM and 500 nM,
between 1 nM and 500 nM, or between 1 nM and 100 nM. In exemplary
embodiments, a .sup.10Fn3 domain binds specifically to a target
that is not bound by a wild-type Fn3 domain, particularly, the
wild-type human .sup.10Fn3 domain.
[0235] G. Multivalent Proteins
[0236] In certain embodiments, the fibronectin based scaffold
protein is a multivalent protein that comprises two or more
.sup.10Fn3 domains, e.g., .sup.10Fn3 domains described herein. For
example, a multivalent fibronectin based scaffold protein may
comprise 2, 3 or more .sup.10Fn3 domains that are covalently
associated. In exemplary embodiments, the fibronectin based
scaffold protein is a bispecific or dimeric protein comprising two
.sup.10Fn3 domains. In certain embodiments, a multivalent
fibronectin based protein scaffold comprises a first .sup.10Fn3
domain that binds to a first target molecule and a second
.sup.10Fn3 domain that binds to a second target molecule. The first
and second target molecules may be the same or different target
molecules. When the first and second target molecules are the same,
the first and second .sup.10Fn3 domains may bind to the same target
but at different epitopes. Additionally, when the first and second
target molecules are the same, the regions of modification in the
.sup.10Fn3 domain that are associated with target binding may be
the same or different. Furthermore, the first and second .sup.10Fn3
domains may be based on the same or different scaffold designs. For
example, a multivalent fibronectin based protein scaffold may
comprise two .sup.10Fn3 domains, wherein both .sup.10Fn3 are based
on the same non-traditional scaffold design described herein,
wherein one of the .sup.10Fn3 domains is based on a first type of
non-traditional scaffold design and the second .sup.10Fn3 domain is
based on a second type of non-traditional scaffold design, or one
of the .sup.10Fn3 domains is based on a non-traditional scaffold
design and the second is based on a traditional scaffold design
(i.e., the BC, DE and FG loops are modified).
[0237] In exemplary embodiments, each .sup.10Fn3 domain of a
multivalent fibronectin based protein scaffold binds to a desired
target with a K.sub.d of less than 500 nM, 100 nM, 50 nM, 1 nM, 500
pM, 100 pM or less. In some embodiments, each .sup.10Fn3 domain of
a multivalent fibronectin based protein scaffold binds to a desired
target with a K.sub.d between 1 pM and 1 .mu.M, between 100 pM and
500 nM, between 1 nM and 500 nM, or between 1 nM and 100 nM. In
exemplary embodiments, each .sup.10Fn3 domain of a multivalent
fibronectin based protein scaffold binds specifically to a target
that is not bound by a wild-type .sup.10Fn3 domain, particularly
the wild-type human .sup.10Fn3 domain.
[0238] The .sup.10Fn3 domains in a multivalent fibronectin based
scaffold protein may be connected by a polypeptide linker.
Exemplary polypeptide linkers include polypeptides having from
1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 amino acids. Suitable
linkers for joining the .sup.10Fn3 domains are those which allow
the separate domains to fold independently of each other forming a
three dimensional structure that permits high affinity binding to a
target molecule. Specific examples of suitable linkers include
glycine-serine based linkers, glycine-proline based linkers,
proline-alanine based linkers as well as linkers having the amino
acid sequence of SEQ ID NO: 32. In some embodiments, the linker is
a glycine-serine based linker. These linkers comprise glycine and
serine residues and may be between 8 and 50, 10 and 30, and 10 and
20 amino acids in length. Examples of such linkers include SEQ ID
NOs: 39-43. In some embodiments, the linker is a glycine-proline
based linker. These linkers comprise glycine and proline residues
and may be between 3 and 30, 10 and 30, and 3 and 20 amino acids in
length. Examples of such linkers include SEQ ID NOs: 33-35. In some
embodiments, the linker is a proline-alanine based linker. These
linkers comprise proline and alanine residues and may be between 3
and 30, 10 and 30, 3 and 20 and 6 and 18 amino acids in length.
Examples of such linkers include SEQ ID NOs: 36-38. In exemplary
embodiments, the linker does not contain any Asp-Lys (DK)
pairs.
Pharmacokinetic Moieties
[0239] In one aspect, the application provides for fibronectin
based scaffold proteins further comprising a pharmacokinetic (PK)
moiety. Pharmokinetics encompasses properties of a compound
including, by way of example, absorption, distribution, metabolism,
and elimination by a subject. Improved pharmacokinetics may be
assessed according to the perceived therapeutic need. Often it is
desirable to increase bioavailability and/or increase the time
between doses, possibly by increasing the time that a protein
remains available in the serum after dosing. In some instances, it
is desirable to improve the continuity of the serum concentration
of the protein over time (e.g., decrease the difference in serum
concentration of the protein shortly after administration and
shortly before the next administration). The fibronectin based
scaffold proteins may be attached to a moiety that reduces the
clearance rate of the polypeptide in a mammal (e.g., mouse, rat, or
human) by greater than three-fold relative to the unmodified
polypeptide. Other measures of improved pharmacokinetics may
include serum half-life, which is often divided into an alpha phase
and a beta phase. Either or both phases may be improved
significantly by addition of an appropriate moiety. A PK moiety
refers to any protein, peptide, or moiety that affects the
pharmacokinetic properties of a biologically active molecule when
fused to the biologically active molecule.
[0240] PK moieties that tend to slow clearance of a protein from
the blood include polyoxyalkylene moieties, e.g., polyethylene
glycol, sugars (e.g., sialic acid), and well-tolerated protein
moieties (e.g., Fc, Fc fragments, transferrin, or serum albumin).
The fibronectin based scaffold proteins may be fused to albumin or
a fragment (portion) or variant of albumin as described in U.S.
Publication No. 20070048282. In some embodiments, the PK moiety is
a serum albumin binding protein such as those described in U.S.
Publication Nos. 2007/0178082 and 2007/0269422. In some
embodiments, the PK moiety is a serum immunoglobulin binding
protein such as those described in U.S. Publication No.
2007/0178082.
[0241] In some embodiments, the fibronectin based scaffold proteins
may be attached to a PK moiety comprising a nonproteinaceous
polymer. In some embodiments, the polymer is polyethylene glycol
("PEG"), polypropylene glycol, or polyoxyalkylenes, as described in
U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192
or 4,179,337. In exemplary embodiments, the polymer is a PEG
moiety.
[0242] PEG is a well-known, water soluble polymer that is
commercially available or can be prepared by ring-opening
polymerization of ethylene glycol according to methods well known
in the art (Sandler and Karo, Polymer Synthesis, Academic Press,
New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to
encompass any polyethylene glycol molecule, without regard to size
or to modification at an end of the PEG, and can be represented by
the formula: X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1),
where n is 20 to 2300 and X is H or a terminal modification, e.g.,
a C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, European
Published Application No. 473084A and U.S. Pat. No. 5,932,462. One
form of PEGs includes two PEG side-chains (PEG2) linked via the
primary amino groups of a lysine (Monfardini, C., et al.,
Bioconjugate Chem. 6 (1995) 62-69).
[0243] PEG conjugation to peptides or proteins generally involves
the activation of PEG and coupling of the activated
PEG-intermediates directly to target proteins/peptides or to a
linker, which is subsequently activated and coupled to target
proteins/peptides (see Abuchowski, A. et al, J. Biol. Chem., 252,
3571 (1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al.,
and Harris et. al., in: Poly(ethylene glycol) Chemistry:
Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum
Press: New York, 1992; Chap. 21 and 22). It is noted that a
fibronectin based scaffold protein containing a PEG molecule is
also known as a conjugated protein, whereas the protein lacking an
attached PEG molecule can be referred to as unconjugated.
[0244] The size of PEG utilized will depend on several factors
including the intended use of the fibronectin based scaffold
protein. Larger PEGs are preferred to increase half life in the
body, blood, non-blood extracellular fluids or tissues. For in vivo
cellular activity, PEGs of the range of about 10 to 60 kDa are
preferred, as well as PEGs less than about 100 kDa and more
preferably less than about 60 kDa, though sizes greater than about
100 kDa can be used as well. For in vivo imaging applications,
smaller PEGs, generally less than about 20 kDa, may be used that do
not increase half life as much as larger PEGs so as to permit
quicker distribution and less half life. A variety of molecular
mass forms of PEG can be selected, e.g., from about 1,000 Daltons
(Da) to 100,000 Da (n is 20 to 2300), for conjugating to
fibronectin based scaffold proteins. The number of repeating units
"n" in the PEG is approximated for the molecular mass described in
Daltons. It is preferred that the combined molecular mass of PEG on
an activated linker is suitable for pharmaceutical use. Thus, in
one embodiment, the molecular mass of the PEG molecules does not
exceed 100,000 Da. For example, if three PEG molecules are attached
to a linker, where each PEG molecule has the same molecular mass of
12,000 Da (each n is about 270), then the total molecular mass of
PEG on the linker is about 36,000 Da (total n is about 820). The
molecular masses of the PEG attached to the linker can also be
different, e.g., of three molecules on a linker two PEG molecules
can be 5,000 Da each (each n is about 110) and one PEG molecule can
be 12,000 Da (n is about 270). In some embodiments, one PEG moiety
is conjugated to the fibronectin based scaffold protein. In some
embodiments, the PEG moiety is about 20, 30, 40, 50, 60, 70, 80, or
90 KDa. In some embodiments, the PEG moiety is about 40 KDa.
[0245] In some embodiments, PEGylated fibronectin based scaffold
proteins contain one, two or more PEG moieties. In one embodiment,
the PEG moiety(ies) are bound to an amino acid residue which is on
the surface of the protein and/or away from the surface that
contacts the target ligand. In one embodiment, the combined or
total molecular mass of PEG in a pegylated fibronectin based
scaffold protein is from about 3,000 Da to 60,000 Da, or from about
10,000 Da to 36,000 Da. In a one embodiment, the PEG in a pegylated
fibronectin based scaffold protein is a substantially linear,
straight-chain PEG.
[0246] One skilled in the art can select a suitable molecular mass
for PEG, e.g., based on how the pegylated fibronectin based
scaffold protein will be used therapeutically, the desired dosage,
circulation time, resistance to proteolysis, immunogenicity, and
other considerations. For a discussion of PEG and its use to
enhance the properties of proteins, see N. V. Katre, Advanced Drug
Delivery Reviews 10: 91-114 (1993).
[0247] In some embodiments, a fibronectin based scaffold protein is
covalently linked to one poly(ethylene glycol) group of the
formula: --CO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR,
with the --CO (i.e. carbonyl) of the poly(ethylene glycol) group
forming an amide bond with one of the amino groups of the binding
polypeptide; R being lower alkyl; x being 2 or 3; m being from
about 450 to about 950; and n and m being chosen so that the
molecular weight of the conjugate minus the binding polypeptide is
from about 10 to 40 kDa. In one embodiment, a fibronectin based
scaffold protein's .epsilon.-amino group of a lysine is the
available (free) amino group.
[0248] In one specific embodiment, carbonate esters of PEG are used
to form the PEG-fibronectin based scaffold protein conjugates.
N,N'-disuccinimidylcarbonate (DSC) may be used in the reaction with
PEG to form active mixed PEG-succinimidyl carbonate that may be
subsequently reacted with a nucleophilic group of a linker or an
amino group of a fibronectin based scaffold protein (see U.S. Pat.
No. 5,281,698 and U.S. Pat. No. 5,932,462). In a similar type of
reaction, 1,1'-(dibenzotriazolyl)carbonate and
di-(2-pyridyl)carbonate may be reacted with PEG to form
PEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.
5,382,657), respectively.
[0249] Pegylation of a fibronectin based scaffold protein can be
performed according to the methods of the state of the art, for
example by reaction of the fibronectin based scaffold protein with
electrophilically active PEGs (supplier: Shearwater Corp., USA,
world wide web at shearwatercorp.com). Preferred PEG reagents of
the present invention are, e.g., N-hydroxysuccinimidyl propionates
(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or
branched N-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C.,
et al., Bioconjugate Chem. 6 (1995) 62-69). Such methods may used
to pegylate at an .epsilon.-amino group of a lysine of a
fibronectin based scaffold protein or at the N-terminal amino group
of the fibronectin based scaffold protein.
[0250] In another embodiment, PEG molecules may be coupled to
sulfhydryl groups on a fibronectin based scaffold protein (Sartore,
L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991); Morpurgo et
al., Biocon. Chem., 7, 363-368 (1996); Goodson et al.,
Bio/Technology (1990) 8, 343; U.S. Pat. No. 5,766,897). U.S. Pat.
Nos. 6,610,281 and 5,766,897 describes exemplary reactive PEG
species that may be coupled to sulfhydryl groups.
[0251] In some embodiments, the pegylated fibronectin based
scaffold protein is produced by site-directed pegylation,
particularly by conjugation of PEG to a cysteine moiety. In certain
embodiments, the Cys residue may be positioned at the N-terminus,
between the N-terminus and the most N-terminal beta or beta-like
strand, at the C-terminus, or between the C-terminus and the most
C-terminal beta or beta-like strand of the fibronectin based
scaffold protein. A Cys residue may be situated at other positions
as well, particularly any of the loops that do not participate in
target binding or between two binding domains of a multivalent
fibronectin based scaffold protein. A PEG moiety may also be
attached by other chemistry, including by conjugation to
amines.
[0252] In some embodiments where PEG molecules are conjugated to
cysteine residues on a fibronectin based scaffold protein, the
cysteine residues are native to the fibronectin based scaffold
protein, whereas in other embodiments, one or more cysteine
residues are engineered into the fibronectin based scaffold
protein. Mutations may be introduced into a fibronectin based
scaffold protein coding sequence to generate cysteine residues.
This might be achieved, for example, by mutating one or more amino
acid residues to cysteine. Preferred amino acids for mutating to a
cysteine residue include serine, threonine, alanine and other
hydrophilic residues. Preferably, the residue to be mutated to
cysteine is a surface-exposed residue. Algorithms are well-known in
the art for predicting surface accessibility of residues based on
primary sequence or a protein. Alternatively, surface residues may
be predicted by comparing the amino acid sequences of fibronectin
based scaffold proteins, given that the crystal structure of the
tenth fn3 domain framework based on which fibronectin based
scaffold proteins are designed has been solved (see Dickinson, et
al., J. Mol. Biol. 236(4): 1079-92 (1994)) and thus the
surface-exposed residues identified. In one embodiment, cysteine
residues are introduced into fibronectin based scaffold protein at
or near the N- and/or C-terminus, or within loop regions.
Pegylation of cysteine residues may be carried out using, for
example, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, or
PEG-orthopyridyl disulfide.
[0253] In some embodiments, the pegylated fibronectin based
scaffold protein comprises a PEG molecule covalently attached to
the alpha amino group of the N-terminal amino acid. Site specific
N-terminal reductive amination is described in Pepinsky et al.,
(2001) JPET, 297, 1059, and U.S. Pat. No. 5,824,784. The use of a
PEG-aldehyde for the reductive amination of a protein utilizing
other available nucleophilic amino groups is described in U.S. Pat.
No. 4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254, 12579,
and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.
[0254] In another embodiment, pegylated fibronectin based scaffold
proteins comprise one or more PEG molecules covalently attached to
a linker, which in turn is attached to the alpha amino group of the
amino acid residue at the N-terminus of the fibronectin based
scaffold protein. Such an approach is disclosed in U.S. Publication
No. 2002/0044921 and PCT Publication No. WO94/01451.
[0255] In one embodiment, a fibronectin based scaffold protein is
pegylated at the C-terminus. In a specific embodiment, a protein is
pegylated at the C-terminus by the introduction of C-terminal
azido-methionine and the subsequent conjugation of a
methyl-PEG-triarylphosphine compound via the Staudinger reaction.
This C-terminal conjugation method is described in Cazalis et al.,
C-Terminal Site-Specific PEGylation of a Truncated Thrombomodulin
Mutant with Retention of Full Bioactivity, Bioconjug Chem. 2004;
15(5):1005-1009.
[0256] In exemplary embodiments, a fibronectin based scaffold
protein is pegylated in a C-terminal tail region as described
further herein. In exemplary embodiments, the C-terminal contains a
Cys residue, which is used as the site of attachment for the PEG
moiety. Exemplary C-terminal tails include, for example, a
polypeptide having any one of SEQ ID NOs: 23, 24 or 31.
[0257] Conventional separation and purification techniques known in
the art can be used to purify PEGylated fibronectin based scaffold
proteins, such as size exclusion (e.g., gel filtration) and ion
exchange chromatography. Products may also be separated using
SDS-PAGE. Products that may be separated include mono-, di-, tri-
poly- and un-pegylated fibronectin based scaffold proteins, as well
as free PEG. The percentage of mono-PEG conjugates can be
controlled by pooling broader fractions around the elution peak to
increase the percentage of mono-PEG in the composition. About
ninety percent mono-PEG conjugates represents a good balance of
yield and activity. Compositions in which, for example, at least
ninety-two percent or at least ninety-six percent of the conjugates
are mono-PEG species may be desired. In an embodiment of this
invention the percentage of mono-PEG conjugates is from ninety
percent to ninety-six percent.
[0258] In one embodiment of the invention, the PEG in a pegylated
fibronectin based scaffold protein is not hydrolyzed from the
pegylated amino acid residue using a hydroxylamine assay, e.g., 450
mM hydroxylamine (pH 6.5) over 8 to 16 hours at room temperature,
and is thus stable. In one embodiment, greater than 80% of the
composition is stable mono-PEG-fibronectin based scaffold protein,
more preferably at least 90%, and most preferably at least 95%.
[0259] In another embodiment, the pegylated fibronectin based
scaffold proteins will preferably retain at least about 25%, 50%,
60%, 70%, 80%, 85%, 90%, 95% or 100% of the biological activity
associated with the unmodified protein. In one embodiment,
biological activity refers to its ability to bind to one or more
target molecules, as assessed by K.sub.d, k.sub.on or k.sub.off. In
one specific embodiment, the pegylated fibronectin based scaffold
protein shows an increase in binding to one or more target
molecules relative to unpegylated fibronectin based scaffold
protein.
[0260] The serum clearance rate of PEG-modified fibronectin based
scaffold proteins may be decreased by about 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or even 90%, relative to the clearance rate of
the unmodified fibronectin based scaffold protein. The PEG-modified
fibronectin based scaffold protein may have a half-life (t.sub.1/2)
which is enhanced relative to the half-life of the unmodified
fibronectin based scaffold protein. The half-life of PEG-modified
fibronectin based scaffold protein may be enhanced by at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%,
200%, 250%, 300%, 400% or 500%, or even by 1000% relative to the
half-life of the unmodified fibronectin based scaffold protein. In
some embodiments, the protein half-life is determined in vitro,
such as in a buffered saline solution or in serum. In other
embodiments, the protein half-life is an in vivo half life, such as
the half-life of the fibronectin based scaffold protein in the
serum or other bodily fluid of an animal.
Nucleic Acid-Protein Fusion Technology
[0261] In one aspect, the application provides fibronectin based
scaffold proteins comprising a fibronectin type III domain that
bind a human target, such as, for example, TNF-alpha, DLL4, IL-17,
PXR or other proteins. One way to rapidly make and test Fn3 domains
with specific binding properties is the nucleic acid-protein fusion
technology of Adnexus, a Bristol-Myers Squibb Company. Such in
vitro expression and tagging technology, termed PROfusion.TM., that
exploits nucleic acid-protein fusions (RNA- and DNA-protein
fusions) may be used to identify polypeptides and amino acid motifs
that are important for binding to proteins. Nucleic acid-protein
fusion technology is a technology that covalently couples a protein
to its encoding genetic information. For a detailed description of
the RNA-protein fusion technology and fibronectin-based scaffold
protein library screening methods see Szostak et al., U.S. Pat.
Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446;
6,518,018; PCT Publication Nos. WO00/34784; WO01/64942;
WO02/032925; and Roberts and Szostak, Proc Natl. Acad. Sci.
94:12297-12302, 1997, herein incorporated by reference.
Vectors & Polynucleotides Embodiments
[0262] Nucleic acids encoding any of the various fibronectin based
scaffold proteins disclosed herein may be synthesized chemically,
enzymatically or recombinantly. Codon usage may be selected so as
to improve expression in a cell. Such codon usage will depend on
the cell type selected. Specialized codon usage patterns have been
developed for E. coli and other bacteria, as well as mammalian
cells, plant cells, yeast cells and insect cells. See for example:
Mayfield et al., Proc Natl Acad Sci USA. 2003 Jan. 21;
100(2):438-42; Sinclair et al. Protein Expr Purif. 2002 October;
26(1):96-105; Connell N D. Curr Opin Biotechnol. 2001 October;
12(5):446-9; Makrides et al. Microbiol Rev. 1996 September;
60(3):512-38; and Sharp et al. Yeast. 1991 October;
7(7):657-78.
[0263] General techniques for nucleic acid manipulation are
described for example in Sambrook et al., Molecular Cloning: A
Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press,
2 ed., 1989, or F. Ausubel et al., Current Protocols in Molecular
Biology (Green Publishing and Wiley-Interscience: New York, 1987)
and periodic updates, herein incorporated by reference. The DNA
encoding the polypeptide is operably linked to suitable
transcriptional or translational regulatory elements derived from
mammalian, viral, or insect genes. Such regulatory elements include
a transcriptional promoter, an optional operator sequence to
control transcription, a sequence encoding suitable mRNA ribosomal
binding sites, and sequences that control the termination of
transcription and translation. The ability to replicate in a host,
usually conferred by an origin of replication, and a selection gene
to facilitate recognition of transformants are additionally
incorporated.
[0264] The fibronectin based scaffold proteins described herein may
be produced recombinantly not only directly, but also as a fusion
polypeptide with a heterologous polypeptide, which is preferably a
signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by
the host cell. For prokaryotic host cells that do not recognize and
process a native signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, lpp, or
heat-stable enterotoxin II leaders. For yeast secretion the native
signal sequence may be substituted by, e.g., the yeast invertase
leader, a factor leader (including Saccharomyces and Kluyveromyces
alpha-factor leaders), or acid phosphatase leader, the C. albicans
glucoamylase leader, or the signal described in PCT Publication No.
WO90/13646. In mammalian cell expression, mammalian signal
sequences as well as viral secretory leaders, for example, the
herpes simplex gD signal, are available. The DNA for such precursor
regions may be ligated in reading frame to DNA encoding the
protein.
[0265] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2 micron plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter).
[0266] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0267] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0268] Expression and cloning vectors usually contain a promoter
that is recognized by the host organism and is operably linked to
the nucleic acid encoding the fibronectin-based scaffold protein.
Promoters suitable for use with prokaryotic hosts include the phoA
promoter, beta-lactamase and lactose promoter systems, alkaline
phosphatase, a tryptophan (trp) promoter system, and hybrid
promoters such as the tac promoter. However, other known bacterial
promoters are suitable. Promoters for use in bacterial systems also
will contain a Shine-Dalgarno (S.D.) sequence operably linked to
the DNA encoding the fibronectin based scaffold protein.
[0269] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0270] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0271] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
Patent Publication No. 73,657. Yeast enhancers also are
advantageously used with yeast promoters.
[0272] Transcription from vectors in mammalian host cells can be
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and most
preferably Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter or an immunoglobulin promoter,
from heat-shock promoters, provided such promoters are compatible
with the host cell systems.
[0273] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the rous sarcoma
virus long terminal repeat can be used as the promoter.
[0274] Transcription of a DNA encoding fibronectin based scaffold
proteins by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
polypeptide-encoding sequence, but is preferably located at a site
5' from the promoter.
[0275] Expression vectors used in eukaryotic host cells (e.g.,
yeast, fungi, insect, plant, animal, human, or nucleated cells from
other multicellular organisms) will also contain sequences
necessary for the termination of transcription and for stabilizing
the mRNA. Such sequences are commonly available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs
or cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA
encoding the polypeptide. One useful transcription termination
component is the bovine growth hormone polyadenylation region. See
WO94/11026 and the expression vector disclosed therein.
[0276] The recombinant DNA can also include any type of protein tag
sequence that may be useful for purifying the fibronectin based
scaffold protein. Examples of protein tags include but are not
limited to a histidine tag, a FLAG tag, a myc tag, an HA tag, or a
GST tag. Appropriate cloning and expression vectors for use with
bacterial, fungal, yeast, and mammalian cellular hosts can be found
in Cloning Vectors: A Laboratory Manual, (Elsevier, New York,
1985), the relevant disclosure of which is hereby incorporated by
reference.
[0277] The expression construct is introduced into the host cell
using a method appropriate to the host cell, as will be apparent to
one of skill in the art. A variety of methods for introducing
nucleic acids into host cells are known in the art, including, but
not limited to, electroporation; transfection employing calcium
chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or
other substances; microprojectile bombardment; lipofection; and
infection (where the vector is an infectious agent).
[0278] Suitable host cells include prokaryotes, yeast, mammalian
cells, or bacterial cells. Suitable bacteria include gram negative
or gram positive organisms, for example, E. coli or Bacillus spp.
Yeast, preferably from the Saccharomyces species, such as S.
cerevisiae, may also be used for production of polypeptides.
Various mammalian or insect cell culture systems can also be
employed to express recombinant proteins. Baculovirus systems for
production of heterologous proteins in insect cells are reviewed by
Luckow and Summers, (Bio/Technology, 6:47, 1988). Examples of
suitable mammalian host cell lines include endothelial cells, COS-7
monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster
ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK
cell lines. Purified fibronectin based scaffold proteins are
prepared by culturing suitable host/vector systems to express the
recombinant proteins. For many applications, the small size of the
fibronectin based scaffold proteins would make expression in E.
coli the preferred method for expression. The fibronectin based
scaffold protein is then purified from culture media or cell
extracts.
Protein Production
[0279] Host cells are transformed with the herein-described
expression or cloning vectors for protein production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0280] The host cells used to produce the fibronectin based
scaffold proteins may be cultured in a variety of media.
Commercially available media such as Ham's F10 (Sigma), Minimal
Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) are suitable
for culturing the host cells. In addition, any of the media
described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al.,
Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;
4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO87/00195; or U.S.
Pat. No. Re. 30,985 may be used as culture media for the host
cells. Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0281] Fibronectin based scaffold proteins disclosed herein can
also be produced using cell-free translation systems. For such
purposes the nucleic acids encoding the fibronectin based scaffold
protein must be modified to allow in vitro transcription to produce
mRNA and to allow cell-free translation of the mRNA in the
particular cell-free system being utilized (eukaryotic such as a
mammalian or yeast cell-free translation system or prokaryotic such
as a bacterial cell-free translation system).
[0282] Fibronectin based scaffold proteins can also be produced by
chemical synthesis (e.g., by the methods described in Solid Phase
Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co.,
Rockford, Ill.). Modifications to the fibronectin based scaffold
protein can also be produced by chemical synthesis.
[0283] The fibronectin based scaffold proteins disclosed herein can
be purified by isolation/purification methods for proteins
generally known in the field of protein chemistry. Non-limiting
examples include extraction, recrystallization, salting out (e.g.,
with ammonium sulfate or sodium sulfate), centrifugation, dialysis,
ultrafiltration, adsorption chromatography, ion exchange
chromatography, hydrophobic chromatography, normal phase
chromatography, reversed-phase chromatography, gel filtration, gel
permeation chromatography, affinity chromatography,
electrophoresis, countercurrent distribution or any combinations of
these. After purification, fibronectin based scaffold proteins may
be exchanged into different buffers and/or concentrated by any of a
variety of methods known to the art, including, but not limited to,
filtration and dialysis.
[0284] The purified fibronectin based scaffold protein is
preferably at least 85% pure, more preferably at least 95% pure,
and most preferably at least 98% pure. Regardless of the exact
numerical value of the purity, the fibronectin based scaffold
protein is sufficiently pure for use as a pharmaceutical
product.
Exemplary Uses
[0285] In one aspect, the application provides fibronectin based
scaffold proteins labeled with a detectable moiety. The fibronectin
based scaffold proteins may be used for a variety of diagnostic
applications. The detectable moiety can be any one which is capable
of producing, either directly or indirectly, a detectable signal.
For example, the detectable moiety may be a radioisotope, such as
H3, C14, C13, P32, S35, or I131; a fluorescent or chemiluminescent
compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin; or an enzyme, such as alkaline phosphatase,
beta-galactosidase or horseradish peroxidase.
[0286] Any method known in the art for conjugating a protein to the
detectable moiety may be employed, including those methods
described by Hunter, et al., Nature 144:945 (1962); David, et al.,
Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219
(1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982). In
vitro methods, include conjugation chemistry well know in the art
including chemistry compatible with proteins, such as chemistry for
specific amino acids, such as Cys and Lys. In order to link a
detectable moiety to a fibronectin based scaffold protein, a
linking group or reactive group is used. Suitable linking groups
are well known in the art and include disulfide groups, thioether
groups, acid labile groups, photolabile groups, peptidase labile
groups and esterase labile groups. Preferred linking groups are
disulfide groups and thioether groups depending on the application.
For polypeptides without a Cys amino acid, a Cys can be engineered
in a location to allow for activity of the protein to exist while
creating a location for conjugation.
[0287] Fibronectin based scaffold proteins linked with a detectable
moiety are useful for in vitro or in vivo imaging. The polypeptide
may be linked to a radio-opaque agent or radioisotope, administered
to a subject, preferably into the bloodstream, and the presence and
location of the labeled protein in the subject may be assayed. This
imaging technique is useful, for example, in the staging and
treatment of malignancies when the fibronectin based scaffold
protein binds to a target associated with cancer. The fibronectin
based scaffold protein may be labeled with any moiety that is
detectable in a subject, whether by nuclear magnetic resonance,
radiology, or other detection means known in the art.
[0288] Fibronectin based scaffold proteins also are useful as
affinity purification agents. In this process, the fibronectin
based scaffold proteins are immobilized on a suitable support, such
as Sephadex resin or filter paper, using methods well known in the
art.
[0289] Fibronectin based scaffold proteins can be employed in any
known assay method, such as competitive binding assays, direct and
indirect sandwich assays, and immunoprecipitation assays (Zola,
Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC
Press, Inc., 1987)).
[0290] In certain aspects, the disclosure provides methods for
detecting a target molecule in a sample. A method may comprise
contacting the sample with a fibronectin based scaffold protein
described herein, wherein said contacting is carried out under
conditions that allow fibronectin based scaffold protein-target
complex formation; and detecting said complex, thereby detecting
said target in said sample. Detection may be carried out using any
technique known in the art, such as, for example, radiography,
immunological assay, fluorescence detection, mass spectroscopy, or
surface plasmon resonance. The sample will often by a biological
sample, such as a biopsy, and particularly a biopsy of a tumor, or
a suspected tumor, where the fibronectin based scaffold protein
binds to a target associated with cancer. The sample may be from a
human or other mammal. The fibronectin based scaffold protein may
be labeled with a labeling moiety, such as a radioactive moiety, a
fluorescent moiety, a chromogenic moiety, a chemiluminescent
moiety, or a hapten moiety. The fibronectin based scaffold protein
may be immobilized on a solid support.
[0291] In one aspect, the application provides fibronectin based
scaffold proteins useful in the treatment of disorders. The
diseases or disorders that may be treated will be dictated by the
binding specificity of the fibronectin based scaffold protein. As
described herein, fibronectin based scaffold proteins may be
designed to bind to any target of interest. Exemplary targets
include, for example, TNF-alpha, DLL4, IL-17 and PXR. Merely as an
example, fibronectin based scaffold proteins that bind to TNF-alpha
may be used to treat autoimmune disorders such as rheumatoid
arthritis, inflammatory bowel disease, psoriasis, and asthma;
fibronectin based scaffold proteins that bind to IL-17 may be used
to treat asthma; and fibronectin based scaffold proteins that bind
to DLL4 may be used to treat hyperproliferative disorders or
diseases associated with unwanted angiogenesis, such as cancers or
tumors.
[0292] The application also provides methods for administering
fibronectin based scaffold proteins to a subject. In some
embodiments, the subject is a human. In some embodiments, the
fibronectin based scaffold proteins are pharmaceutically acceptable
to a mammal, in particular a human. A "pharmaceutically acceptable"
composition refers to a composition that is administered to an
animal without significant adverse medical consequences. Examples
of pharmaceutically acceptable compositions include compositions
comprising .sup.10Fn3 domains that lack the integrin-binding domain
(RGD) and compositions that are essentially endotoxin or pyrogen
free or have very low endotoxin or pyrogen levels.
Formulation and Administration
[0293] The application further provides pharmaceutically acceptable
compositions comprising the fibronectin based scaffold proteins
described herein, wherein the composition is essentially endotoxin
and/or pyrogen free.
[0294] Therapeutic formulations comprising fibronectin based
scaffold proteins are prepared for storage by mixing the described
proteins having the desired degree of purity with optional
physiologically acceptable carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980)), in the form of aqueous solutions, lyophilized or other
dried formulations. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyidimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrans; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG).
[0295] The formulations herein may also contain more than one
active compounds as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0296] The fibronectin based scaffold proteins may also be
entrapped in microcapsule prepared, for example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsule and
poly-(methylmethacylate) microcapsule, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
[0297] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0298] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the fibronectin
based scaffold proteins described herein, which matrices are in the
form of shaped articles, e.g., films, or microcapsule. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated proteins remain in the
body for a long time, they may denature or aggregate as a result of
exposure to moisture at 37.degree. C., resulting in a loss of
biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0299] While the skilled artisan will understand that the dosage of
each fibronectin based scaffold protein will be dependent on the
identity of the protein, the preferred dosages can range from about
10 mg/square meter to about 2000 mg/square meter, more preferably
from about 50 mg/square meter to about 1000 mg/square meter.
[0300] For therapeutic applications, the fibronectin based scaffold
proteins are administered to a subject, in a pharmaceutically
acceptable dosage form. They can be administered intravenously as a
bolus or by continuous infusion over a period of time, by
intramuscular, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. The protein may
also be administered by intratumoral, peritumoral, intralesional,
or perilesional routes, to exert local as well as systemic
therapeutic effects. Suitable pharmaceutically acceptable carriers,
diluents, and excipients are well known and can be determined by
those of skill in the art as the clinical situation warrants.
Examples of suitable carriers, diluents and/or excipients include:
(1) Dulbecco's phosphate buffered saline, pH about 7.4, containing
about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline
(0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of the
present invention can be practiced in vitro, in vivo, or ex
vivo.
[0301] Administration of fibronectin based scaffold proteins, and
one or more additional therapeutic agents, whether co-administered
or administered sequentially, may occur as described above for
therapeutic applications. Suitable pharmaceutically acceptable
carriers, diluents, and excipients for co-administration will be
understood by the skilled artisan to depend on the identity of the
particular therapeutic agent being co-administered.
[0302] When present in an aqueous dosage form, rather than being
lyophilized, the fibronectin based scaffold protein typically will
be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml,
although wide variation outside of these ranges is permitted. For
the treatment of disease, the appropriate dosage of fibronectin
based scaffold proteins will depend on the type of disease to be
treated, the severity and course of the disease, whether the
fibronectin based scaffold proteins are administered for preventive
or therapeutic purposes, the course of previous therapy, the
patient's clinical history and response to the fibronectin based
scaffold protein, and the discretion of the attending physician.
The fibronectin based scaffold protein is suitably administered to
the patient at one time or over a series of treatments.
[0303] Fibronectin based scaffold proteins may also be used as
crystallization chaperones to generate structures of a protein with
a compound of interest. For example, a fibronectin based scaffold
protein that specifically binds to human pregnane X receptor (PXR)
may be used as a crystallization chaperone to facilitate
crystallization of a compound with PXR, e.g., the ligand binding
domain (LBD) of PXR. Several .sup.10Fn3 molecules that bind to
human PXR are described herein.
[0304] PXR activation upregulates cellular levels of several drug
metabolizing enzymes such as cytochrome P450 enzymes and MDR1. The
increased expression of CYP enzymes can alter the pharmacokinetics
of drug and lead to dangerous drug-drug interactions including loss
of therapeutic efficacy and increased toxicity. To avoid late stage
clinical failures and high costs associated with bringing a new
drug to market, many pharmaceutical companies have adopted
screening assays for early detection of compounds that activate
PXR. Additionally in silico screening using known crystal
structures of PXR are increasingly being used to predict potential
PXR activity. The large and flexible ligand binding pocket of PXR
and the potential of these compounds to bind to different locations
and in multiple orientations within the ligand binding cavity of
PXR complicates reliable prediction of PXR activity. This is
especially true for more advanced compounds/chemotypes with
desirable efficacy, selectivity and bioavailability towards the
intended therapeutic target but with known PXR liability. Given
these limitations, a co-crystal structure is often required to
define exact binding interactions and to suggest specific
modifications that can disturb crucial interactions related to PXR
binding while maintaining activity against the primary target. For
example, in certain embodiments, a method for analyzing the
interaction of a test agent with PXR comprises incubating together
(i) PXR, or a ligand binding domain thereof; (ii) the test agent
and (iii) a .sup.10Fn3 protein specifically binding to PXR, e.g.,
PXR LBD, under conditions suitable for crystallization. Exemplary
.sup.10Fn3 proteins specifically binding to PXR LBD comprise an
amino acid sequence that is at least 70%, 80%, 90%, 95%, 97%, 98%
or 99% identical to one of SEQ ID NOs: 48, 49 and 62-69 and 72. In
certain embodiments, .sup.10Fn3 proteins specifically binding to
PXR LBD comprise an amino acid sequence that differs from any one
of SEQ ID NOs: 48, 49 and 62-69 and 72 in at most 1, 2, 3, 5, 10,
15, 20 or 25 amino acid changes, e.g., substitutions (such as
conservative substitutions), additions or deletions. The method may
further comprise inducing crystallization and determining which
portions (or atoms) of the test agent interacts with PXR (generally
the ligand binding domain of PXR), and optionally modifying the
test agent such that it no longer interacts with PXR or does so
with lower affinity.
EXEMPLARY EMBODIMENTS
[0305] 1. A polypeptide comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises (i) a
modification in the amino acid sequence of at least one north pole
loop selected from the BC, DE and FG loops relative to the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1), and (ii) a modification in the amino acid sequence of at
least one south pole loop selected from the AB, CD and EF loops
relative to the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), wherein the at least one
modified north pole loop and the at least one modified south pole
loop contribute to binding the same target. 2. The polypeptide of
claim 1, wherein at least one of the north pole loops or at least
one of the south pole loops has the amino acid sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 3. The polypeptide of claim 1, wherein at least a
portion of the BC loop has the amino acid sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 4. The polypeptide of claim 3, wherein the first 3
residues of the BC loop are the same as the corresponding residues
in the BC loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 5. The polypeptide of claim 3, wherein the first 4
residues of the BC loop are the same as the corresponding residues
in the BC loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 6. The polypeptide of claim 3, wherein the first 5
residues of the BC loop are the same as the corresponding residues
in the BC loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 7. The polypeptide of claim 3, wherein the BC loop has
the amino acid sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 8. The polypeptide of
any one of claims 3-7, wherein the polypeptide has reduced
immunogenicity relative to an equivalent polypeptide that has
additional modifications in the BC loop. 9. The polypeptide of any
one of claims 1-8, wherein the FG loop does not contain an RGD
integrin binding site. 10. The polypeptide of any of claims 1-9,
wherein the amino acid sequence of at least one .beta.-strand is
modified relative to the amino acid sequence of the corresponding
.beta.-strand of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1
or 6). 11. The polypeptide of claim 10, wherein the modified
.beta.-strand is positioned adjacent to one of the modified loops
in the linear sequence of the .sup.10Fn3 domain. 12. The
polypeptide of claim 10, wherein the modified .beta.-strand is
positioned between the modified north pole loop and the modified
south pole loop in the linear sequence of the .sup.10Fn3 domain.
13. The polypeptide of any one of claims 10-12, wherein the
modified north pole loop, the modified .beta.-strand, and the
modified south pole loop are positioned adjacent to each other in
the linear sequence of the .sup.10Fn3 domain. 14. The polypeptide
of any one of claims 10-13, wherein the modified .beta.-strand
contributes to binding to the target. 15. The polypeptide of any
one of claims 1-13, wherein the hydrophobic core residues have not
been modified relative to the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 16. The polypeptide of any one of claims 1-15,
wherein the amino acid sequence of at least one of the modified
loops has been extended in length relative to the amino acid
sequence of the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 17. The polypeptide of any
one of claims 1-16, wherein the amino acid sequence of at least one
of the modified loops has been reduced in length relative to the
amino acid sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 18. The polypeptide of
any one of claims 1-17, wherein the amino acid sequence of the
C-terminal tail is modified relative to the amino acid sequence of
the C-terminal tail of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6). 19. The polypeptide of any one of claims 1-18,
wherein the amino acid sequence of the first 7 amino acid residues
is modified relative to the amino acid sequence of the first 7
amino acid residues of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6). 20. The polypeptide of any one of claims 1-17,
wherein the polypeptide has from 1-7 amino acids truncated from the
N-terminus relative to the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1), from 1-9 amino acids truncated from the C-terminus
relative to the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6), or both. 21. The polypeptide of any one of claims 1-20, wherein
the polypeptide has at least 50% identity to the amino acid
sequence of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6). 22. The polypeptide of any one of claims 1-21, wherein the
polypeptide has at least 65% identity to the amino acid sequence of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 23. A
library comprising a plurality of polypeptides comprising a human
fibronectin type 3 tenth (.sup.10Fn3) domain, wherein the
.sup.10Fn3 domain comprises (i) a modification in the amino acid
sequence of at least one north pole loop selected from the BC, DE
and FG loops relative to the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1), and (ii) a modification in
the amino acid sequence of at least one south pole loop selected
from the AB, CD and EF loops relative to the corresponding loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 24. The
library of claim 23, wherein the library comprises at least
10.sup.5 polypeptides each comprising a different .sup.10Fn3 domain
sequence. 25. A method for identifying a polypeptide that binds to
a target comprising screening the library of claim 23 or 24 to
identify a polypeptide that binds to the target. 26. An isolated
polypeptide identified by the method of claim 25. 27. The isolated
polypeptide of claim 26, wherein the polypeptide binds to the
target with a K.sub.d of less than 500 nM. 28. A polypeptide
comprising a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises (i) a modification in the
amino acid sequence of at least one of loops AB, BC, CD, DE, EF, or
FG relative to the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), and (ii) a modification in
the amino acid sequence of at least one .beta.-strand relative to
the corresponding .beta.-strand of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6), wherein the at least one modified loop
and the at least one modified .beta.-strand contribute to binding
the same target. 29. The polypeptide of claim 28, wherein the
modified .beta.-strand is adjacent to the modified loop in the
linear sequence of the .sup.10Fn3 domain. 30. The polypeptide of
claim 28 or 29, wherein at least two .beta.-strands have modified
amino acid sequences relative to the corresponding .beta.-strands
of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 31.
The polypeptide of claim 30, wherein the modified .beta.-strands
are adjacent to each side of the modified loop in the linear
sequence of the .sup.10Fn3 domain. 32. The polypeptide of claim 30
or 31, wherein both modified .beta.-strands contribute to binding
to the target. 33. The polypeptide of any one of claims 28-32,
wherein at least two loops have modified amino acid sequences
relative to the corresponding loops of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 34. The polypeptide of claim
33, wherein at least one modified loop is a north pole loop
selected from the BC, DE and FG loops and at least one modified
loop is a south pole loop selected from the AB, CD and EF loops.
35. The polypeptide of claim 33 or 34, wherein both modified loops
contribute to binding to the target. 36. The polypeptide of any one
of claims 28-35, wherein at least one of the north pole loops or at
least one of the south pole loops has the amino acid sequence of
the corresponding loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 37. The polypeptide of any one of claims 28-35,
wherein at least a portion of the BC loop has the amino acid
sequence of the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 38. The polypeptide of claim
37, wherein the first 3 residues of the BC loop are the same as the
corresponding residues in the BC loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 39. The polypeptide of claim
37, wherein the first 4 residues of the BC loop are the same as the
corresponding residues in the BC loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 40. The polypeptide of claim
37, wherein the first 5 residues of the BC loop are the same as the
corresponding residues in the BC loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 41. The polypeptide of claim
37, wherein the BC loop has the amino acid sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 42. The polypeptide of any one of claims 37-41, wherein
the polypeptide has reduced immunogenicity relative to an
equivalent polypeptide that has additional modifications in the BC
loop. 43. The polypeptide of any one of claims 28-42, wherein the
FG loop does not contain an RGD integrin binding site. 44. The
polypeptide of any one of claims 28-43, wherein the hydrophobic
core residues have not been modified relative to the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 45. The polypeptide of
any one of claims 28-44, wherein the amino acid sequence of at
least one modified loop has been extended in length relative to the
amino acid sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 46. The polypeptide of
any one of claims 28-45, wherein the amino acid sequence of at
least one modified loop has been reduced in length relative to the
amino acid sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 47. The polypeptide of
any one of claims 28-46, wherein the amino acid sequence of the
C-terminal tail is modified relative to the amino acid sequence of
the C-terminal tail of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6). 48. The polypeptide of any one of claims 28-47,
wherein the amino acid sequence of the first 7 amino acid residues
is modified relative to the amino acid sequence of the first 7
amino acid residues of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6). 49. The polypeptide of any one of claims 28-46,
wherein the polypeptide has from 1-7 amino acids truncated from the
N-terminus relative to the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6), from 1-9 amino acids truncated from the C-terminus
relative to the wild-type human .sup.10Fn3 domain (SEQ ID NO:1), or
both. 50. The polypeptide of any one of claims 28-49, wherein the
polypeptide has at least 50% identity to the amino acid sequence of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 51. The
polypeptide of any one of claims 28-50, wherein the polypeptide has
at least 65% identity to the amino acid sequence of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 52. A library
comprising a plurality of polypeptides comprising a human
fibronectin type 3 tenth (.sup.10Fn3) domain, wherein the
.sup.10Fn3 domain comprises (i) a modification in the amino acid
sequence of at least one of loops AB, BC, CD, DE, EF, or FG
relative to the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1), and (ii) a modification in the
amino acid sequence of at least one .beta.-strand relative to the
corresponding .beta.-strand of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 53. The library of claim 52, wherein the
library comprises at least 10.sup.5 polypeptides each comprising a
different .sup.10Fn3 domain sequence. 54. A method for identifying
a polypeptide that binds to a target comprising screening the
library of claim 52 or 53 to identify a polypeptide that binds to
the target. 55. An isolated polypeptide identified by the method of
claim 54. 56. The isolated polypeptide of claim 55, wherein the
polypeptide binds to the target with a K.sub.d of less than 500 nM.
57. A polypeptide comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of the CD and FG loops
relative to the sequences of the corresponding loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6), and wherein
the CD and FG loops contribute to binding to the same target. 58.
The polypeptide of claim 57, wherein at least 4 of the 11 amino
acids of the CD loop are modified relative to the sequence of the
CD loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6). 59. The polypeptide of claim 57 or 58, wherein one or more
amino acid residues of the CD loop corresponding to amino acid
residues 46 or 47 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6) are the same as the wild-type amino acids at those
positions. 60. The polypeptide of any one of claims 57-59, wherein
at least 11 of the 13 amino acids of the FG loop are modified
relative to the sequence of the FG loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 61. The polypeptide of claim
60, wherein one or more amino acid residues of the FG loop
corresponding to amino acid residues 75 or 87 of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6) are the same as the
wild-type amino acids at those positions. 62. The polypeptide of
any one of claims 57-61, wherein the amino acid sequence of the CD
loop, the FG loop, or both are extended in length relative to the
amino acid sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 63. The polypeptide of
any one of claims 57-61, wherein the amino acid sequence of the CD
loop, the FG loop, or both are reduced in length relative to the
amino acid sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 64. The polypeptide of
any one of claims 57-61, wherein the amino acid sequence of at
least one of the CD and FG loops is extended in length relative to
the corresponding loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6) and the amino acid sequence of at least one of
the CD and FG loops is reduced in length relative to the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 65. The polypeptide of any one of claims 57-64, wherein
the amino acid sequences of .beta.-strand C and .beta.-strand D are
modified relative to the sequences of the corresponding
.beta.-strands of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 66. The polypeptide of any one of claims 57-65, wherein
at least a portion of the BC loop is modified relative to the
sequence of the BC loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 67. The polypeptide of claim
66, wherein one or more amino acid residues of the BC loop
corresponding to amino acid residues 30 and 31 of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6) are modified relative to
the wild-type amino acids at those positions. 68. The polypeptide
of any one of claims 57-67, wherein the amino acid sequences of
.beta.-strand F and .beta.-strand G are modified relative to the
sequences of the corresponding .beta.-strands of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 69. The polypeptide of
any one of claims 57-68, wherein one or more of the modified
.beta.-strands contribute to binding to the target. 70. The
polypeptide of claim 69, wherein all of the modified .beta.-strands
contribute to binding to the target. 71. The polypeptide of any one
of claims 57-70, wherein one or more of the AB, DE and EF loops
have the amino acid sequence of the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 72. The
polypeptide of any one of claims 57-71, wherein the hydrophobic
core residues have not been modified relative to the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 73. The polypeptide of
any one of claims 57-72, wherein at least a portion of the BC loop
has the amino acid sequence of the corresponding loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 74. The
polypeptide of claim 73, wherein the first 3 residues of the BC
loop are the same as the corresponding residues in the BC loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 75. The
polypeptide of claim 73, wherein the first 4 residues of the BC
loop are the same as the corresponding residues in the BC loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 76. The
polypeptide of claim 73, wherein the first 5 residues of the BC
loop are the same as the corresponding residues in the BC loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 77. The
polypeptide of claim 73, wherein the first 6 residues of the BC
loop are the same as the corresponding residues in the BC loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 78. The
polypeptide of claim 73, wherein the BC loop has the amino acid
sequence of the corresponding loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 79. The polypeptide of any
one of claims 73-78, wherein the polypeptide has reduced
immunogenicity relative to an equivalent polypeptide that has
additional modifications in the BC loop. 80. A library comprising a
plurality of polypeptides comprising a human fibronectin type 3
tenth (.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of the CD and FG loops
relative to the sequences of the corresponding loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 81. The
library of claim 80, wherein the library comprises at least
10.sup.5 polypeptides each comprising a different .sup.10Fn3 domain
sequence. 82. A method for identifying a polypeptide that binds to
a target comprising screening the library of claim 80 or 81 to
identify a polypeptide that binds to the target. 83. An isolated
polypeptide identified by the method of claim 80. 84. The isolated
polypeptide of claim 83, wherein the polypeptide binds to the
target with a K.sub.d of less than 500 nM. 85. A polypeptide
comprising a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of the CD and DE loops relative to the sequences of
the corresponding loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6), and wherein the CD and DE loops contribute to
binding to the same target. 86. The polypeptide of claim 85,
wherein the amino acid sequence of the EF loop is modified relative
to the sequence of EF loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6), and wherein the EF loop contributes to binding
to the target. 87. The polypeptide of claim 85 or 86, wherein the
amino acid sequence of one or more of .beta.-strand C,
.beta.-strand D, and .beta.-strand F are modified relative to the
sequences of the corresponding .beta.-strands of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 88. The polypeptide of
claim 87, wherein one or more of the modified .beta.-strands
contribute to binding to the target. 89. The polypeptide of any one
of claims 85-88, wherein at least 24 of the 31 residues between the
amino acids corresponding to residues 36 through 66 of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6) have been
modified relative to the corresponding residues in the wild-type
sequence. 90. The polypeptide of any one of claims 85-89, wherein
the CD loop is extended in length or reduced in length relative to
the CD loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1
or 6). 91. The polypeptide of any one of claims 85-90, wherein the
hydrophobic core residues have not been modified relative to the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 92. The
polypeptide of any one of claims 85-91, wherein the FG loop does
not contain an RGD integrin binding site. 93. A library comprising
a plurality of polypeptides comprising a human fibronectin type 3
tenth (.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of the CD and DE loops
relative to the sequences of the corresponding loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 94. The
library of claim 93, wherein the library comprises at least
10.sup.5 polypeptides each comprising a different .sup.10Fn3 domain
sequence. 95. A method for identifying a polypeptide that binds to
a target comprising screening the library of claim 93 or 94 to
identify a polypeptide that binds to the target. 96. An isolated
polypeptide identified by the method of claim 95. 97. The isolated
polypeptide of claim 96, wherein the polypeptide binds to the
target with a K.sub.d of less than 500 nM. 98. A polypeptide
comprising a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of the EF and FG loops relative to the sequences of
the corresponding loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1), and wherein the EF and FG loops contribute to
binding to the same target. 99. The polypeptide of claim 98,
wherein the amino acid sequence of the AB loop is modified relative
to the sequence of AB loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6), and wherein the AB loop contributes to binding
to the target. 100. The polypeptide of claim 98 or 99, wherein the
amino acid sequence of one or more of .beta.-strand A and
.beta.-strand G are modified relative to the sequences of the
corresponding .beta.-strands of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 101. The polypeptide of claim 100,
wherein one or more of the modified .beta.-strands contribute to
binding to the target. 102. The polypeptide of any one of claims
98-101, wherein the amino acid sequence of the first 7 amino acids
or the amino acid sequence of the amino acids corresponding to
residues 93 through 97 of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1) have been modified relative to the corresponding
residues in the wild-type sequence. 103. The polypeptide of claim
102, wherein the additional modified amino acids contribute to
binding to the target. 104. The polypeptide of any one of claims
98-103, wherein at least 14 of the first 15 amino acid residues of
the .sup.10Fn3 domain have been modified relative to the
corresponding residues in the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 105. The polypeptide of any one of claims
98-104, wherein at least 4 out of 5 of the amino acid residues of
the EF loop have been modified relative to the corresponding
residues in the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6). 106. The polypeptide of any one of claims 98-105, wherein at
least 15 of the 18 residues between the amino acids corresponding
to residues 80 through 97 of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1) have been modified relative to the corresponding
residues in the wild-type sequence. 107. The polypeptide of any one
of claims 98-106, wherein the amino acid sequence of the FG loop is
extended in length or reduced in length relative to the FG loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 108. The
polypeptide of any one of claims 98-107, wherein the hydrophobic
core residues have not been modified relative to the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 109. The polypeptide of
any one of claims 98-108, wherein the FG loop does not contain an
RGD integrin binding site. 110. A library comprising a plurality of
polypeptides comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of the EF and FG loops
relative to the sequences of the corresponding loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 111. The
library of claim 110, wherein the library comprises at least
10.sup.5 polypeptides each comprising a different .sup.10Fn3 domain
sequence. 112. A method for identifying a polypeptide that binds to
a target comprising screening the library of claim 110 or 111 to
identify a polypeptide that binds to the target. 113. An isolated
polypeptide identified by the method of claim 112. 114. The
isolated polypeptide of claim 113, wherein the polypeptide binds to
the target with a K.sub.d of less than 500 nM. 115. A polypeptide
comprising a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of the AB and FG loops relative to the sequences of
the corresponding loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6), and wherein the AB and FG loops contribute to
binding to the same target. 116. The polypeptide of claim 115,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of .beta.-strand A, loop AB, .beta.-strand B, loop
FG, and .beta.-strand G relative to the sequences of the
corresponding .beta.-strands and loops of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), and wherein the modified
loops and strands contribute to binding to the same target. 117.
The polypeptide of claim 115 or 116, wherein the amino acid
sequence of the first 7 amino acids or the amino acid sequence of
the amino acids corresponding to residues 93 through 97 of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1) have been modified
relative to the corresponding residues in the wild-type sequence.
118. The polypeptide of claim 117, wherein the additional modified
amino acids contribute to binding to the target. 119. The
polypeptide of any one of claims 115-118, wherein the amino acid
sequence of the AB loop is extended in length or reduced in length
relative to the AB loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 120. The polypeptide of any one of claims
115-119, wherein the AB loop contains an insertion or deletion
between the amino acid residues corresponding to amino acid
residues 15 and 18 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 121. The polypeptide of any one of claims 115-120,
wherein the amino acid sequence of the FG loop is extended in
length or reduced in length relative to the FG loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 122. The
polypeptide of any one of claims 115-121, wherein the portion of
the FG loop corresponding to amino acid residues 84-87 of the FG
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6),
has a modified amino acid sequence relative to the same portion of
the wild-type CD loop. 123. The polypeptide of any one of claims
115-122, wherein the FG loop contains an insertion or deletion
between the amino acid residues corresponding to amino acid
residues 83 and 88 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 124. The polypeptide of any one of claims 115-123,
wherein .beta.-strand B contains an insertion or deletion between
the amino acid residues corresponding to amino acid residues 18 and
20 of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6).
125. The polypeptide of any one of claims 115-124, wherein at least
16 of the 19 residues between the amino acids corresponding to
residues 1 through 19 of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6) have been modified relative to the corresponding
residues in the wild-type sequence. 126. The polypeptide of any one
of claims 115-125, wherein at least 13 of the 14 residues between
the amino acids corresponding to residues 84 through 97 of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1) have been modified
relative to the corresponding residues in the wild-type sequence.
127. The polypeptide of any one of claims 115-126, wherein one of
more of the BC, CD, DE or EF loops has the sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 128. The polypeptide of any one of claims 115-127,
wherein the hydrophobic core residues have not been modified
relative to the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6). 129. The polypeptide of any one of claims 115-128, wherein the
FG loop does not contain an RGD integrin binding site. 130. A
library comprising a plurality of polypeptides comprising a human
fibronectin type 3 tenth (.sup.10Fn3) domain, wherein the
.sup.10Fn3 domain comprises modifications in the amino acid
sequences of the AB and FG loops relative to the sequences of the
corresponding loops of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6). 131. The library of claim 130, wherein the library
comprises at least 10.sup.5 polypeptides each comprising a
different .sup.10Fn3 domain sequence. 132. A method for identifying
a polypeptide that binds to a target comprising screening the
library of claim 130 or 131 to identify a polypeptide that binds to
the target. 133. An isolated polypeptide identified by the method
of claim 132. 134. The isolated polypeptide of claim 133, wherein
the polypeptide binds to the target with a K.sub.d of less than 500
nM. 135. A polypeptide comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of loop CD, loop EF,
.beta.-strand F, and .beta.-strand G relative to the sequences of
the corresponding .beta.-strands and loops of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), and wherein the modified
loops and strands contribute to binding to the same target. 136.
The polypeptide of claim 135, wherein the amino acid sequence of
the amino acids corresponding to residues 93 through 97 of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1) have been modified
relative to the corresponding residues in the wild-type
sequence.
137. The polypeptide of claim 136, wherein the additional modified
amino acids contribute to binding to the target. 138. The
polypeptide of any one of claims 135-137, wherein the portion of
the CD loop corresponding to amino acid residues 37-45 of the CD
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6),
has a modified amino acid sequence relative to the same portion of
the wild-type CD loop. 139. The polypeptide of any one of claims
135-138, wherein the portion of the CD loop corresponding to amino
acid residues 37-45 of the CD loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), contains an insertion or a
deletion relative to the same portion of the wild-type CD loop.
140. The polypeptide of any one of claims 135-139, wherein the
amino acid sequence of the EF loop is extended in length or reduced
in length relative to the EF loop of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 141. The polypeptide of any one of
claims 135-140, wherein .beta.-strand F contains an insertion or
deletion between the amino acid residues corresponding to amino
acid residues 68 and 70 of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 142. The polypeptide of any one of claims
135-141, wherein the amino acid sequence corresponding to the
sequence of amino acid residues 91-97 of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1), has a modified amino acid sequence
relative to the same portion of the wild-type sequence. 143. The
polypeptide of any one of claims 135-142, wherein the polypeptide
comprises an insertion or deletion between the amino acid residues
corresponding to amino acid residues 90 and 98 of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1). 144. The polypeptide of any
one of claims 135-143, wherein one of more of the AB, BC or DE
loops has the sequence of the corresponding loop of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 145. The polypeptide of
any one of claims 135-144, wherein the hydrophobic core residues
have not been modified relative to the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 146. The polypeptide of any one of
claims 135-145, wherein the FG loop does not contain an RGD
integrin binding site. 147. A library comprising a plurality of
polypeptides comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of loop CD, loop EF,
.beta.-strand F, and .beta.-strand G relative to the sequences of
the corresponding .beta.-strands and loops of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 148. The library of claim
147, wherein the library comprises at least 10.sup.5 polypeptides
each comprising a different .sup.10Fn3 domain sequence. 149. A
method for identifying a polypeptide that binds to a target
comprising screening the library of claim 147 or 148 to identify a
polypeptide that binds to the target. 150. An isolated polypeptide
identified by the method of claim 149. 151. The isolated
polypeptide of claim 150, wherein the polypeptide binds to the
target with a K.sub.d of less than 500 nM. 152. A polypeptide
comprising a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises modifications in the amino
acid sequences of .beta.-strand A, loop AB, .beta.-strand B, loop
CD, .beta.-strand E, loop EF, and .beta.-strand F relative to the
sequences of the corresponding .beta.-strands and loops of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6), and wherein
the modified loops and strands contribute to binding to the same
target. 153. The polypeptide of claim 152, wherein the amino acid
sequence of the CD loop is extended in length or reduced in length
relative to the CD loop of the wild-type human Fn3 domain (SEQ ID
NO:1 or 6). 154. The polypeptide of claim 152 or 153, wherein the
amino acid sequence of .beta.-strand G is modified relative to the
sequence of .beta.-strand G of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 155. The polypeptide of any one of
claims 152-154, wherein the amino acid sequence of the C-terminal
tail is modified relative to the amino acid sequence of the
C-terminal tail of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1). 156. The polypeptide of any one of claims 152-155, wherein
the hydrophobic core residues have not been modified relative to
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 157. The
polypeptide of any one of claims 152-156, wherein the FG loop does
not contain an RGD integrin binding site. 158. A library comprising
a plurality of polypeptides comprising a human fibronectin type 3
tenth (.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of .beta.-strand A, loop
AB, .beta.-strand B, loop CD, .beta.-strand E, loop EF, and
.beta.-strand F relative to the sequences of the corresponding
.beta.-strands and loops of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 159. The library of claim 158, wherein the
library comprises at least 10.sup.5 polypeptides each comprising a
different .sup.10Fn3 domain sequence. 160. A method for identifying
a polypeptide that binds to a target comprising screening the
library of claim 158 or 159 to identify a polypeptide that binds to
the target. 161. An isolated polypeptide identified by the method
of claim 160. 162. The isolated polypeptide of claim 161, wherein
the polypeptide binds to the target with a K.sub.d of less than 500
nM. 163. A polypeptide comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of loop AB, .beta.-strand
B, loop CD, .beta.-strand D, loop DE, .beta.-strand E, and loop EF
relative to the sequences of the corresponding .beta.-strands and
loops of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6),
and wherein the modified loops and strands contribute to binding to
the same target. 164. The polypeptide of claim 163, wherein the
amino acid sequence of the AB loop is extended in length or reduced
in length relative to the AB loop of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 165. The polypeptide of claim 163 or
164, wherein the portion of the CD loop corresponding to amino acid
residues 40-47 of the CD loop of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6), has a modified amino acid sequence
relative to the same portion of the wild-type CD loop. 166. The
polypeptide of any one of claims 163-165, wherein the portion of
the CD loop corresponding to amino acid residues 40-47 of the CD
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6),
contains an insertion or a deletion relative to the same portion of
the wild-type CD loop. 167. The polypeptide of any one of claims
163-166, wherein .beta.-strand B contains an insertion or deletion
between the amino acid residues corresponding to amino acid
residues 18 and 20 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 168. The polypeptide of any one of claims 163-167,
wherein at least 5 of the 6 residues between the amino acids
corresponding to residues 14 through 19 of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6) have been modified relative to
the corresponding residues in the wild-type sequence. 169. The
polypeptide of any one of claims 163-168, wherein at least 18 of
the 27 residues between the amino acids corresponding to residues
40 through 66 of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1
or 6) have been modified relative to the corresponding residues in
the wild-type sequence. 170. The polypeptide of any one of claims
163-169, wherein the BC loop has the sequence of the corresponding
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6).
171. The polypeptide of any one of claims 163-170, wherein the
hydrophobic core residues have not been modified relative to the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 172. The
polypeptide of any one of claims 163-171, wherein the FG loop does
not contain an RGD integrin binding site. 173. A library comprising
a plurality of polypeptides comprising a human fibronectin type 3
tenth (.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
modifications in the amino acid sequences of loop AB, .beta.-strand
B, loop CD, .beta.-strand D, loop DE, .beta.-strand E, and loop EF
relative to the sequences of the corresponding .beta.-strands and
loops of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6).
174. The library of claim 173, wherein the library comprises at
least 10.sup.5 polypeptides each comprising a different .sup.10Fn3
domain sequence. 175. A method for identifying a polypeptide that
binds to a target comprising screening the library of claim 173 or
174 to identify a polypeptide that binds to the target. 176. An
isolated polypeptide identified by the method of claim 175. 177.
The isolated polypeptide of claim 176, wherein the polypeptide
binds to the target with a K.sub.d of less than 500 nM. 178. A
polypeptide comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises a
sequence modification in the FG loop relative to the sequence of
amino acid residues 77-83 of loop FG of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), and wherein the .sup.10Fn3
binds to a target with a K.sub.d of less than 500 nM. 179. The
polypeptide of claim 178, wherein the portion of the FG loop
corresponding to amino acid residues 77-83 of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6) is extended in length or
reduced in length relative to the sequence of amino acid residues
77-83 of the wild-type FG loop. 180. The polypeptide of claim 178
or 179, wherein the FG loop alone mediates binding to the target.
181. The polypeptide of any one of claims 178-180, wherein one of
more of the AB, BC, CD, DE or EF loops has the sequence of the
corresponding loop of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 182. A library comprising a plurality of polypeptides
comprising a human fibronectin type 3 tenth (.sup.10Fn3) domain,
wherein the .sup.10Fn3 domain comprises a sequence modification in
the FG loop relative to the sequence of amino acid residues 77-83
of loop FG of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6). 183. The library of claim 182, wherein the library comprises at
least 10.sup.5 polypeptides each comprising a different .sup.10Fn3
domain sequence. 184. A method for identifying a polypeptide that
binds to a target comprising screening the library of claim 182 or
183 to identify a polypeptide that binds to the target. 185. An
isolated polypeptide identified by the method of claim 184. 186.
The isolated polypeptide of claim 200, wherein the polypeptide
binds to the target with a K.sub.d of less than 500 nM. 187. A
polypeptide comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises a
sequence modification in a portion of the BC loop and a portion of
the FG loop relative to the sequence of the corresponding loops of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6), and
wherein the .sup.10Fn3 domain has reduced immunogenicity relative
to an equivalent .sup.10Fn3 domain having a greater portion of the
BC loop modified relative to the wild-type BC loop. 188. The
polypeptide of claim 187, wherein the portion of the BC loop that
is modified corresponds to residues 28 and 29 of the BC loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 189. The
polypeptide of claim 187, wherein the portion of the BC loop that
is modified corresponds to residues 27-29 of the BC loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 190. The
polypeptide of claim 187, wherein the portion of the BC loop that
is modified corresponds to residues 24-29 of the BC loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 191. The
polypeptide of any one of claims 187-190, wherein the portion of
the FG loop that is modified corresponds to residues 77-83 of the
FG loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or
6). 192. The polypeptide of any one of claims 187-190, wherein the
portion of the FG loop that is modified corresponds to residues
77-82 of the FG loop of the wild-type human .sup.10Fn3 domain (SEQ
ID NO:1 or 6). 193. The polypeptide of any one of claims 187-190,
wherein the portion of the FG loop that is modified corresponds to
residues 77-86 of the FG loop of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 194. The polypeptide of any one of
claims 187-190, wherein the portion of the FG loop that is modified
corresponds to residues 77-79 of the FG loop of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 195. The polypeptide of any
one of claims 191-194, wherein the portion of the FG loop that is
modified has an insertion or deletion relative to the corresponding
portion of the FG loop of the wild-type human .sup.10Fn3 domain
(SEQ ID NO:1 or 6). 196. The polypeptide of any one of claims
187-195, wherein the BC and FG loop contribute to binding to the
target. 197. The polypeptide of any one of claims 187-196, wherein
the .sup.10Fn3 domain further comprises a sequence modification in
a portion of the DE loop relative to the sequence of the DE loop of
the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 198. The
polypeptide of claim 197, wherein the portion of the DE loop that
is modified corresponds to residues 52 and 53 of the DE loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 199. The
polypeptide of any one of claims 187-198, wherein the .sup.10Fn3
domain has reduced immunogenicity relative to an equivalent
.sup.10Fn3 domain further comprising modifications in one or more
of amino acid residues 23-27, relative to the corresponding
positions in the wild-type BC loop. 200. The polypeptide of any one
of claims 187-199, wherein the .sup.10Fn3 domain binds to a target
with a K.sub.d of less than 500 nM. 201. A library comprising a
plurality of polypeptides comprising a human fibronectin type 3
tenth (.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises
a sequence modification in a portion of the BC loop and a portion
of the FG loop relative to the sequence of the corresponding loops
of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 202.
The library of claim 201, wherein the library comprises at least
10.sup.5 polypeptides each comprising a different .sup.10Fn3 domain
sequence. 203. A method for identifying a polypeptide that binds to
a target comprising screening the library of claim 201 or 202 to
identify a polypeptide that binds to the target. 204. A method for
increasing the likelihood of identifying a polypeptide that binds
to a target and has reduced immunogenicity, comprising screening
the library of claim 201 or 202 to identify a polypeptide that
binds to the target. 205. An isolated polypeptide identified by the
method of claim 203 or 204. 206. The isolated polypeptide of
claim
205, wherein the polypeptide binds to the target with a K.sub.d of
less than 500 nM. 207. A polypeptide comprising a human fibronectin
type 3 tenth (.sup.10Fn3) domain, wherein the .sup.10Fn3 domain
comprises a sequence modification in the BC loop and at least one
of .beta.-strand B or .beta.-strand C relative to the sequences of
the corresponding loop and .beta.-strands of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6), and wherein the .sup.10Fn3
domain has reduced immunogenicity relative to an equivalent
.sup.10Fn3 domain that does not have a sequence modification in at
least one of .beta.-strand B or .beta.-strand C relative to
wild-type. 208. The polypeptide of claim 207, wherein the amino
acid sequence of the BC loop is extended in length or reduced in
length relative to the amino acid sequence of the BC loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 209. The
polypeptide of claim 207 or 208, wherein the .sup.10Fn3 domain
further comprises a modification in the amino acid sequence of the
first 7 amino acid residues relative to the amino acid sequence of
the first 7 amino acid residues of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 210. The polypeptide of any one of
claims 207-209, wherein the .sup.10Fn3 domain further comprises a
modification in the DE loop, the FG loop, or both, relative to the
sequences of the corresponding loops of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 211. The polypeptide of claim
210, wherein the amino acid sequence of the DE loop is extended in
length or reduced in length relative to the amino acid sequence of
the DE loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1
or 6). 212. The polypeptide of claim 210 or 211, wherein the amino
acid sequence of the FG loop is extended in length or reduced in
length relative to the amino acid sequence of the FG loop of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 213. The
polypeptide of any one of claims 210-212, wherein the .sup.10Fn3
domain comprises a sequence modification in the DE loop, and
further comprises a sequence modification in .beta.-strand D,
.beta.-strand E, or both, relative to the sequences of the
corresponding .beta.-strands of the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 214. The polypeptide of any one of
claims 210-212, wherein the .sup.10Fn3 domain comprises a sequence
modification in the FG loop, and further comprises a sequence
modification in .beta.-strand F, .beta.-strand G, or both, relative
to the sequences of the corresponding .beta.-strands of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 215. The
polypeptide of any one of claims 207-214, wherein the portion of
the BC loop corresponding to amino acid residues 27-31 of the BC
loop of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6),
has a modified amino acid sequence relative to the same portion of
the wild-type BC loop. 216. The polypeptide of any one of claims
207-215, wherein the BC loop contains an insertion or deletion
between the amino acid residues corresponding to amino acid
residues 26 and 32 of the wild-type human .sup.10Fn3 domain (SEQ ID
NO:1 or 6). 217. The polypeptide of any one of claims 207-216,
wherein .beta.-strand D contains an insertion or deletion between
the amino acid residues corresponding to amino acid residues 48 and
50 of the wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6).
218. The polypeptide of any one of claims 207-217, wherein
.beta.-strand F contains an insertion or deletion between the amino
acid residues corresponding to amino acid residues 72 and 74 of the
wild-type human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 219. The
polypeptide of any one of claims 207-218, wherein .beta.-strand G
contains an insertion or deletion between the amino acid residues
corresponding to amino acid residues 88 and 90 of the wild-type
human .sup.10Fn3 domain (SEQ ID NO:1 or 6). 220. The polypeptide of
any one of claims 207-219, wherein the hydrophobic core residues
have not been modified relative to the wild-type human .sup.10Fn3
domain (SEQ ID NO:1 or 6). 221. A library comprising a plurality of
polypeptides comprising a human fibronectin type 3 tenth
(.sup.10Fn3) domain, wherein the .sup.10Fn3 domain comprises a
sequence modification in the BC loop and at least one of
.beta.-strand B or .beta.-strand C relative to the sequences of the
corresponding loop and .beta.-strands of the wild-type human
.sup.10Fn3 domain (SEQ ID NO:1 or 6). 222. The library of claim
221, wherein the library comprises at least 10.sup.5 polypeptides
each comprising a different .sup.10Fn3 domain sequence. 223. A
method for identifying a polypeptide that binds to a target
comprising screening the library of claim 221 or 222 to identify a
polypeptide that binds to the target. 224. A method for increasing
the likelihood of identifying a polypeptide that binds to a target
and has reduced immunogenicity, comprising screening the library of
claim 221 or 222 to identify a polypeptide that binds to the
target. 223. An isolated polypeptide identified by the method of
claim 223 or 224. 224. The isolated polypeptide of claim 223,
wherein the polypeptide binds to the target with a K.sub.d of less
than 500 nM.
TABLE-US-00001 SEQUENCES Wild-Type .sup.10Fn3 Sequences: WT
.sup.10Fn3 Domain (SEQ ID NO: 1)
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV
PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS Q WT .sup.10Fn3
Domain Core Sequence version 1 (SEQ ID NO: 2)
LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTA
TISGLKPGVDYTITVYAVTGRGDSPASSKPISINY WT .sup.10Fn3 Domain with D80E
Substitution (SEQ ID NO: 59)
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV
PGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPISINYRTEIDKPS Q WT .sup.10Fn3
Domain Core Sequence version 2 (SEQ ID NO: 60)
EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTAT
ISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT WT .sup.10Fn3 Domain Core
Sequence version 3 (SEQ ID NO: 22)
VSDVPRDLEVVAA(X).sub.uLLISW(X).sub.vYRITY(X).sub.wFTV(X).sub.xATISGL
(X).sub.yYTITVYA(X).sub.zISINYRT WT .sup.10Fn3 Domain Core Sequence
version 4 (SEQ ID NO: 6)
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTV
PGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT DLL4 Binding WS-LI1
Binders: (SEQ ID NO: 3)
MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEQHSKYPHQEF
TVPGSKSTATISGLKPGVDYTITVYAVTIQPQDPEQDYQYHYYETSSKPI SINYRTEIDKPSQ
(SEQ ID NO: 4) MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEHVADHFDHNQ
EFTVPGSKSTATISGLKPGVDYTITVYAVTYQFQDPEEHYYYHFYDSSSK PISINYRTEIDKPSQ
(SEQ ID NO: 5) MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEYHEHYHSPGF
SQKYHYEQEFTVPGSKSTATISGLKPGVDYTITVYAVTGHKHYHYYYYYH
HHSSKPISINYRTEIDKPSQ Exemplary N-terminal Extension Sequences: (SEQ
ID NO: 9) MGVSDVPRDL (SEQ ID NO: 10) VSDVPRDL (SEQ ID NO: 11)
GVSDVPRDL (SEQ ID NO: 16) X.sub.nSDVPRDL, wherein n = 0, 1 or 2
amino acids, wherein when n = 1, X is Met or Gly, and when n = 2, X
is Met- Gly (SEQ ID NO: 17) X.sub.nDVPRDL, wherein n = 0, 1 or 2
amino acids, wherein when n = 1, X is Met or Gly, and when n = 2, X
is Met- Gly (SEQ ID NO: 18) XnVPRDL, wherein n = 0, 1 or 2 amino
acids, wherein when n = 1, X is Met or Gly, and when n = 2, X is
Met- Gly (SEQ ID NO: 19) XnPRDL, wherein n = 0, 1 or 2 amino acids,
wherein when n = 1, X is Met or Gly, and when n = 2, X is Met- Gly
(SEQ ID NO: 20) XnRDL, wherein n = 0, 1 or 2 amino acids, wherein
when n = 1, X is Met or Gly, and when n = 2, X is Met- Gly (SEQ ID
NO: 21) XnDL, wherein n = 0, 1 or 2 amino acids, wherein when n =
1, X is Met or Gly, and when n = 2, X is Met- Gly (SEQ ID NO: 50)
MASTSG Exemplary C-Terminal Tail Sequences: (SEQ ID NO: 7) EIEK
(SEQ ID NO: 8) EIEKPC (SEQ ID NO: 23) EGSGC (SEQ ID NO: 24) EIEKPCQ
(SEQ ID NO: 25) EIEKPSQ (SEQ ID NO: 26) EIEKP (SEQ ID NO: 27)
EIEKPS (SEQ ID NO: 28) EGSGS (SEQ ID NO: 29) EIDK (SEQ ID NO: 30)
EIDKPSQ (SEQ ID NO: 31) EIDKPCQ Exemplary Linker Sequences: (SEQ ID
NO: 32) PSTSTST (SEQ ID NO: 33) GPG (SEQ ID NO: 34) GPGPGPG (SEQ ID
NO: 35) GPGPGPGPGPG (SEQ ID NO: 36) PAPAPA (SEQ ID NO: 37)
PAPAPAPAPAPA (SEQ ID NO: 38) PAPAPAPAPAPAPAPAPA (SEQ ID NO: 39)
GSGSGSGSGS (SEQ ID NO: 40) GSGSGSGSGSGSGSGSGSGS (SEQ ID NO: 41)
GGGGSGGGGSGGGGS (SEQ ID NO: 42) GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID
NO: 43) GGGGSGGGGSGGGSG 6XHis Tag: (SEQ ID NO: 44) HHHHHH IL-17
Binding WS-LI1 Binders: (SEQ ID NO: 45)
MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEYHAFFASNGK
YYFYIQEFTVPGSKSTATISGLKPGVDYTITVYAVTDDTVHHGDSNYHSS KPISINYRTEIDKPSQ
(SEQ ID NO: 46) MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEYSSFFQHQGQ
YYHYIQEFTVPGSKSTATISGLKPGVDYTITVYAVTQHEHSQDSSKPISI NYRTEIDKPSQ (SEQ
ID NO: 47) MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGEFSQFVHSDGE
YYQEYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGQYDQDDEPSSKPI SINYRTEIDKPSQ
PXR Binding WS1 Binders: (SEQ ID NO: 48)
MASTSGVSDVPRDLEVVAATPTSLLISWDAPAVPVSKYVIYYWPGALISS
MQAFKVPGSKSTATISGLKPGVLYSIVVDALTGDGQGSYVWDPITITYRT EGSGS (SEQ ID
NO: 49) MASTSGVSDVPRDLEVVAATPTSLLISWDAPAVTVHSYYITYQELQHHSV
PQGFQVPGSKSTATISGLKPGVAYQIAVYAFTGPGLPPSDAPPIVIYYRT EGSGS .sup.10Fn3
Loop and Scaffold Region Peptides from Figure 4: (SEQ ID NO: 58)
PTSLLISWDAPAVTVRYYRITYG (SEQ ID NO: 51) PVQEFTVPGSKSTATISGLK (SEQ
ID NO: 52) TITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 53)
MGEVVAATPTSLLIS (SEQ ID NO: 54) PHFPTRYYRITYGETGGNS BC Loop
Sequences from Example 2: (SEQ ID NO: 55) PTSLLISWDAPAVTVRYYRITYG
(SEQ ID NO: 56) PTSLLISWSARLKVARYYRITYG (SEQ ID NO: 57)
PTSLLISWRHPHFPTRYYRITYG IGF-1R Binding .sup.10Fn3 Domain with
Modified BC, DE and FG Loops: (SEQ ID NO: 61)
GVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFT
VPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDKPSQ
PXR Binding .sup.10Fn3 Molecules:
[0306] The amino acid sequences of Adnectins 1-8 of FIG. 10
correspond to SEQ ID NOs: 70, 71, 62, 63, 72, 13, 14 and 15,
respectively. Amino acid sequences of Adnectins 1-8 of FIG. 10
without the 6xHis tail (SEQ ID NO: 44) correspond to SEQ ID NOs:
64, 65, 48, 49, 66, 67, 68 and 69, respectively.
EXAMPLES
[0307] The invention is now described by reference to the following
examples, which are illustrative only, and are not intended to
limit the present invention. While the invention has been described
in detail and with reference to specific embodiments thereof, it
will be apparent to one of skill in the art that various changes
and modifications can be made thereto without departing from the
spirit and scope thereof.
Example 1
Expression and Purification of Fibronectin Based Scaffold
Proteins
[0308] Selected binders were cloned into a PET9d vector and
transformed into E. coli BL21 DE3 plysS cells. Transformed cells
were inoculated in 5 ml LB medium containing 50 .mu.g/mL kanamycin
and 34 .mu.g/ml chloromphenicol in a 24-well format and grown at
37.degree. C. overnight (inoculums culture). Production cultures
were established by aspirating 200 .mu.l of the inoculum culture
into 5 ml (in a 24-well format) of TB-Overnight Expression Media
(auto induction) containing 50 .mu.g/ml Kanamycin and 34 .mu.g/ml
chloromphenicol. The cultures were grown at 37.degree. C. for 4
hours at which time the temperature was lowered to 18.degree. C.
and grown for 20 hours. Cultures were harvested by centrifugation
for 10 minutes at 2750 g at 4.degree. C.
[0309] Cell pellets (in 24-well format) were lysed by resuspension
in 450 .mu.l of Lysis buffer (50 mM NaH.sub.2PO.sub.4, 0.5M NaCl,
1.times. Complete.TM. Protease Inhibitor Cocktail-EDTA free
(Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1 mg/ml lysozyme,
30 .mu.g/ml DNAse, 4.1 .mu.g/ml aprotonin, pH 8.0) and shaken at
room temperature for 1-3 hours. Lysates were clarified and
re-racked into a 96-well format by transfer into a 96-well Whatman
GF/D Unifilter fitted with a 96-well, 1.2 ml catch plate and
filtered by positive pressure. The clarified lysates were
transferred to a 96-well HisPur Cobalt Plate that had been
equilibrated with equilibration buffer (50 mM NaH.sub.2PO.sub.4,
0.5M NaCl, 40 mM Imidazole, pH 8.0) and were incubated for 5 min.
Unbound material was removed by positive pressure. The resin was
washed 2.times.0.3 ml/well with Wash buffer #1 (50 mM
NaH.sub.2PO.sub.4, 0.5M NaCl, 5 mM CHAPS, 40 mM Imidazole, pH 8.0)
with each wash removed by positive pressure. Prior to elution each
well was washed with 50 .mu.l Elution buffer (PBS+20 mM EDTA),
incubated for 5 min and this wash was discarded by positive
pressure. Protein was eluted by applying an additional 100 .mu.l of
Elution buffer to each well. After a 30 minute incubation at room
temperature the plate(s) were centrifuged for 5 minutes at 200 g
and eluted protein was collected in 96-well catch plates containing
5 .mu.l of 0.5M MgCl.sub.2 added to the bottom of elution catch
plate prior to elution. Eluted protein was quantified using a total
protein assay with SGE as the protein standard. SGE is a wild-type
.sup.10Fn3 domain in which the RGD sequence in the FG loop is
changed to SGE.
Example 2
Characterization of the Immunogenicity of .sup.10Fn3 Domain
Polypeptides
[0310] The adaptive immune response is initiated by the processing
and digestion of an internalized protein by an antigen-presenting
cell (APC), such as a dendritic cell. The APC clips the
internalized protein into short peptides and then displays the
peptides on its surface MHC Class II molecules. The peptide binding
site of the MHC Class II molecule is long and narrow, like a
hot-dog bun, and holds its peptide in an extended format, with room
for nine amino acids in the primary binding site (and generally
allows for short tails on either side of the peptide). Certain
pockets in the MHC binding site are dominant in determining peptide
binding. These pockets correspond to amino acid positions 1, 4, 6,
and 9 in the anchored portion of the 9-mer peptide. A peptide that
has favorable side chains at each of these four positions will in
general bind to HLA (an MHC Class II molecule) well.
[0311] Position 1 is thought to be the most important `anchor
residue` involved in binding between the peptide and the HLA
molecule. Position 1 generally favors a hydrophobic side
chain--thus, 9-mers that often bind HLA are initiated with V, I, L,
M, F, Y, or W. The other positions are much more variable, with
different HLA alleles favoring different sets of amino acids at
each site. The immunogenicity of the polypeptides described herein
were assessed using both in vitro and in silico methods.
A--In Vitro Determination of Human Leukocyte Antigen ("HLA")
Binding
[0312] In this experiment, synthetic peptides corresponding to
different regions in either a wild-type or engineered .sup.10Fn3
domain sequence were evaluated in HLA binding assays. Similar HLA
binding assays are described in Reijonen H, Kwok W W, Use of HLA
class II tetramers in tracking antigen-specific T cells and mapping
T-cell epitopes, Methods 29(3):282-8 (2003). Each experimental
peptide tested in the HLA binding assay was either a wildtype
.sup.10Fn3 domain north pole loop (BC, DE or FG loop) peptide
segment having additional amino acids flanking the N- and C-termini
of each loop (SEQ ID NOs: 58, 51 and 52), a scaffold region peptide
segment from a wildtype .sup.10Fn3 domain that is positioned
N-terminus to the BC loop (SEQ ID NO: 53) or a scaffold region
peptide segment from an engineered .sup.10Fn3 domain that is
positioned C-terminus to the BC loop (SEQ ID NO: 54). The
experimental peptides were solvated in 100% DMSO at 50.times. the
concentration desired in the assay. Each peptide was then diluted
into reaction buffer and titrated serially from 128 .mu.M to 2
.mu.M.
[0313] In the HLA binding assay, each of five different HLA allele
molecules (either DRB*0101; DRB*0301; DRB*0401; DRB*0701 or
DRB*1501 alleles) were loaded separately into wells of a 96-well
plate along with the unlabeled experimental peptides and a
europium-labeled control (competitor) peptide. The binding mixture
was incubated in the wells for 24 hours so as to reach steady
equilibrium. The HLA molecule complexes then were captured on an
ELISA plate coated with anti-human HLA-DR antibody. Bound
HLA-labeled control peptide was measured by time-resolved
fluorescence and assessed at 615 nm by a Wallac Victor3.TM. unit
(Perkin-Elmer). Binding of experimental peptides was expressed as
the percent inhibition of the labeled control peptide (experimental
fluorescence/control fluorescence multiplied by 100). From the
percent inhibition of labeled control peptide at each
concentration, IC.sub.50 curves were derived for each experimental
peptide against the five alleles tested. The results from these
experiments are illustrated in FIG. 4.
[0314] As shown in FIG. 4, the BC loop peptide was observed to bind
to four of the five alleles tested with high affinity suggesting it
is an immunodominant sequence within the protein. By contrast,
synthetic peptides corresponding to the DE and FG loops bound fewer
HLA alleles and with lower affinity, in general. The DE and FG
loops are, therefore, not predicted to be immunodominant sequences
within the wild-type .sup.10Fn3 protein.
[0315] As shown in FIG. 4, the scaffold region peptide of SEQ ID
NO: 53 was found to bind to five of the HLA alleles with high
affinity, while the scaffold region peptide of SEQ ID NO: 54 was
found to bind to four of the HLA alleles with high affinity. These
results suggest that the scaffold region flanking the BC loop of a
.sup.10Fn3 domain are immunodominant regions. The underlined
portions of the sequences of SEQ ID NOs: 53 and 54 as shown in FIG.
4 are predicted to be the immunodominant portions of these
sequences.
[0316] The BC loop, which was found to be an immunodominant loop,
was further assessed using the same HLA binding assay.
Specifically, three BC loop sequence variant peptides (SEQ ID NOs:
55-57) were examined using the assay and found to show almost
identical patterns of strong binding to the HLA alleles tested. The
peptide sequence of SEQ ID NO: 55 is the human wildtype sequence
and SEQ ID NOs: 56 and 57 are the BC loop regions from .sup.10Fn3
domains that have been engineered to bind to two different targets.
Assuming that a similar binding pattern reflects a shared motif,
the sequences were aligned to aid identification of potential
anchor residues. Potential position 1 residues of the different BC
loop sequences tested are underlined:
TABLE-US-00002 (sequence position based on SEQ ID NO: 1) 15 23 30 |
| | PTSLLISW DAPAVTV RYYRITYG (SEQ ID NO: 55) PTSLLISW SARLKVA
RYYRITYG (SEQ ID NO: 56) PTSLLISW RHPHFPT RYYRITYG (SEQ ID NO:
57)
[0317] The common sequence portions of these three peptides are the
.beta.-strand B, preceding the variable BC loop, and the
.beta.-strand C following the BC loop. Each of these portions has
several hydrophobic residues (potential position 1 anchors), but
those in .beta.-strand C do not have at least 8 more residues
following, and therefore cannot be the anchor residues for MHC
binding. The hydrophobic residues in the variable BC loop are in
different positions in the three peptides, making it unlikely that
a single 9-mer position with shared .beta.-strand C residues can be
anchored in the BC loops. These results therefore suggest that
.beta.-strand B should be useful for designing the peptide
anchors.
[0318] The most likely positions for anchor residues appear to be
"LLI" (positions 18-20 of SEQ ID NO: 1), as these include a stretch
of fixed .beta.-strand B residues preceding the BC loop residues.
If, for instance, a 9-mer beginning at the first L is anchoring the
peptide, the fourth position is always an S, which is favorable for
binding to many HLA alleles.
[0319] It should be noted that while many fully human sequences are
displayed by MHC, the immune system recognizes them as "self" and
does not mount an immune response. Further, in Cyno monkeys, which
have the identical sequence for .sup.10Fn3, an immune response was
generated upon administration of various different .sup.10Fn3
polypeptides, but no immune response was generated upon the
administration of wildtype .sup.10Fn3. This indicates that Cyno
monkeys recognize the human wildtype .sup.10Fn3 sequence as being a
"self" protein, to which an immune response need not be
mounted.
B--In Silico Prediction of HLA Binding
[0320] HLA binding may be predicted in silico, for example, using
EpiMatrix. EpiMatrix is a proprietary computer algorithm developed
by EpiVax, which is used to screen protein sequences for the
presence of putative HLA binding motifs. Input sequences are parsed
into overlapping 9-mer frames where each frame overlaps the last by
8 amino acids. Each of the resulting frames is then scored for
predicted binding affinity with respect to a panel of eight common
Class II HLA alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701,
DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501). Raw scores are
normalized against the scores of a large sample of randomly
generated peptides. The resulting "Z" score is reported. Any 9-mer
peptide with an EpiMatrix Z-score in excess of 1.64 is considered a
putative HLA binding motif.
[0321] Peptide epitopes from a .sup.10Fn3 polypeptide having the
amino acid sequence set forth in SEQ ID NO: 61 were predicted using
the EpiMatrix algorithm. Results showed the BC, DE and FG loops of
the .sup.10Fn3 polypeptide have Z-scores of 21.3, 0.9 and 1.1,
respectively.
[0322] The above in vitro and in silico HLA binding results suggest
that the .beta.-strand B/BC loop segment may be a `hot spot` for
the purposes of HLA binding. The .beta.-strand B/BC loop segment
may be anchored strongly to MHC molecules by the amino acids in
.beta.-strand B, such that at least some variants in BC loop
sequences make little difference in the binding to HLA. The strong
anchor may make it difficult to deimmunize this segment. However,
if the entire stretch is a wildtype sequence, it should be
recognized as self by the immune system, and initiate no immune
response. Therefore, .sup.10Fn3 domain polypeptide libraries may be
designed to have lower immunogenicity by leaving the BC loop as
wildtype, or at the very least, by leaving the residues from D23 to
T28 of SEQ ID NO: 1 or 6 as wildtype in order to retain as wildtype
any 9-mer peptide anchored at L18, L19, or 120. Examples of
libraries in which all or a substantial portion of the BC loop was
left wild-type are shown in FIG. 9A. Examples of libraries in which
varying portions of the N-terminal regions of the BC loop are left
as wild-type are shown in FIG. 9B. Specific examples of .sup.10Fn3
binders in which the residues corresponding to D23 to T28 of SEQ ID
NO: 1 were left wildtype are provided in Example 3. Alternatively,
if the BC loop is modified, reduced immunogenicity may be achieved
by destroying the strong anchor in this region, i.e., by making
modifications in .beta.-strand B, in addition to the modifications
in the BC loop. Examples of libraries in which the anchor is
removed include libraries in which positions L19 and/or S21 have
been diversified thereby increasing the likelihood that members of
the library will be missing the anchor residues. Examples of
libraries in which the anchor residues are removed are shown in
FIG. 9C.
Example 3
Generation of West Side Binders
[0323] Libraries of West Side ("WS") binder polypeptides comprising
a modified .sup.10Fn3 domain were screened using mRNA display (Xu
et al Chem Biol. 2002 August; 9(8):933-42) for binding to murine
IL-17, murine DLL4 or human PXR as targets. The WS binders were
designed such that the BC loop sequence was left as wildtype. The
WS1 library design (see FIG. 9A) was used to identify binders to
the target human PXR and the WS-LI1 library design (see FIG. 9A)
was used to identify binders to the targets murine IL-17 or murine
DLL4. Target binding was monitored by qPCR and populations were
cloned and expressed in E. coli when a specific binding signal was
observed.
Example 4
Disruption of DLL4 and Notch 1 Interaction by WS-LI1 Binders
Capable of Binding Murine DLL4
[0324] DLL4 is a ligand for the Notch 1 protein. The ability of
.sup.10Fn3 polypeptides having the WS-LI1 design to disrupt the
interaction between Notch 1 and murine DLL4 was assessed by
employing a competitive Biacore experiment. Approximately 4500 RU
of Notch1-Fc was immobilized on a CM5 Biacore chip. 2 .mu.M of
WS-LI1 binders were equilibrated with 20 nM murine DLL4 in HBSP
buffer and 5 mM CaCl.sub.2 along with a control in which no
polypeptide was added. Each sample was flowed over the chip and
binding of murine DLL4 was compared to the control in which no
WS-LI1 polypeptide was added, so that a reduction in signal
corresponded to inhibition of the Notch 1:murine DLL4 interaction.
Between each sample run, the chip was regenerated with two
30-second washes in HBSP pH 7.4 and 50 mM EDTA. The results of this
experiment are shown in FIG. 5. The polypeptides having the
sequence of SEQ ID NO: 3 and 4 were each found to result in 100%
competition of the interaction between Notch1 and murine DLL4. The
polypeptides having the sequence of SEQ ID NO: 5 were able to
induce 75% competition of the interaction between Notch 1 and
murine DLL4.
Example 5
Size Exclusion Chromatography Analysis of WS-LI1 Binders Capable of
Binding Murine DLL4
[0325] Size exclusion chromatography was utilized to demonstrate
that the competition results observed in Example 4 were due to
monomeric forms of the WS-LI1 binders tested. The WS-LI1 binders
having amino acid sequences of either SEQ ID NO: 3 or 4 were
predominantly monomeric, whereas the WS-LI1 binder having the amino
acid sequence of SEQ ID NO: 5 contained a mixture of monomeric and
aggregated proteins. The results from this experiment are shown in
FIG. 6 and illustrate that the WS-LI1 binders tested in Example 4
were acting as a monomeric species, suggesting that the binders
exist as stable, well-folded polypeptides.
Example 6
Stability of WS-LI1 Binders Capable of Binding Murine DLL4
[0326] Stability of the polypeptides described in Example 4 was
assessed by a thermal shift fluorescence-based assay (TSF). The
polypeptide having the amino acid sequence of SEQ ID NO: 3 had a
transition at 59.degree. C. The polypeptide having the amino acid
sequence of SEQ ID NO: 4 had a transition at 49.degree. C. No
transition was observed for the polypeptide having the amino acid
sequence of SEQ ID NO: 5.
Example 7
Disruption of the Interaction Between Murine IL-17 and Murine
IL-17RA by WS-LI1 Binders Capable of Binding Murine IL-17
[0327] IL-17 is a ligand for the IL-17 receptor A protein, IL-17RA.
The ability of .sup.10Fn3 domains having the WS-LI1 design to
disrupt the interaction between murine IL-17 and murine IL-17RA was
assessed by employing a competitive Alphascreen experiment.
Streptavidin donor beads, anti-human IgG acceptor beads, 1.5 nM
murine IL-17RA-Fc and 2.5 nM biotinylated murine IL-17 were
combined according to the manufacturer's instructions to give a
robust Alphascreen signal. Polypeptides having the sequences of
either SEQ ID NO: 45, 46 or 47 were assessed for their ability to
inhibit this signal when added to the mixture at 1 .mu.M
concentrations. The polypeptide having the sequence of SEQ ID NO:
45 caused an 83% inhibition, the polypeptide having the sequence of
SEQ ID NO: 46 caused a 94% inhibition and the polypeptide having
the sequence of SEQ ID NO: 47 caused an 81% inhibition of the
Alphascreen signal. These results demonstrate that the WS-LI1
library design produced .sup.10Fn3 domains capable of binding IL-17
and effectively inhibiting the interaction between murine IL-17 and
its receptor.
Example 8
Size Exclusion Chromatography Analysis of WS-LI1 Binders Capable of
Binding Murine IL-17
[0328] Size exclusion chromatography was utilized to demonstrate
that the competition results observed in Example 7 were due to
monomeric forms of the WS-LI1 binders tested. The WS-LI1 binders
having amino acid sequences of either SEQ ID NO: 45, 46 or 47 were
predominantly monomeric. The results from this experiment are shown
in FIG. 7 and illustrate that the WS-LI1 binders tested in Example
7 were acting as a monomeric species, suggesting that the binders
exist as stable, well-folded polypeptides.
Example 9
Stability of WS-LI1 Binders Capable of Binding Murine IL-17
[0329] Stability of the polypeptides described in Example 7 was
assessed by TSF. The polypeptide having the amino acid sequence of
SEQ ID NO: 45 had a transition at 51.degree. C. The polypeptide
having the amino acid sequence of SEQ ID NO: 46 had a transition at
60.degree. C. No transition was observed for the polypeptide having
the amino acid sequence of SEQ ID NO: 47.
Example 10
Characterization of Binding Properties of WS1 Binders Capable of
Binding Human PXR
[0330] WS1 binders that were capable of binding to PXR were
characterized using a Biacore binding assay and GST-tagged PXR.
14000 RU of anti-GST antibody was immobilized on a Biacore chip and
GST-PXR was captured by flowing a 50 nM solution over the chip for
3 min at a rate of 5 .mu.L/min. WS1 binders were flowed over the
chip at 0.5-2 .mu.M concentration to observe binding relative to a
control that lacked PXR. The chip was stripped between each run
using two 30 second washes with 10 mM glycine, pH 2.0 and fresh
GST-PXR was captured. Under these conditions, 146 RU of WS1 binders
having the amino acid sequence of SEQ ID NO: 48 bound the GST-PXR,
and 81 RU of WS1 binders having the amino acid sequence of SEQ ID
NO: 49 bound the GST-PXR. These results demonstrate that the WS1
binders having the amino acid sequence of either SEQ ID NO: 48 or
49 are capable of binding GST-PXR.
Example 11
Size Exclusion Chromatography Analysis of WS1 Binders Capable of
Binding Human PXR
[0331] Size exclusion chromatography was utilized to demonstrate
that the binding results observed in Example 10 were due to
monomeric forms of the WS1 binders tested. The WS1 binders having
amino acid sequences of either SEQ ID NO: 48 or 49 were
predominantly monomeric. The results from this experiment are shown
in FIG. 8 and illustrate that the WS1 binders tested in Example 10
were acting as a monomeric species, suggesting that the binders
exist as stable, well-folded polypeptides.
Example 12
Sequences and Binding Characteristics of .sup.10Fn3 Polypeptides
Binding to Human PXR
[0332] This Example describes 6 additional .sup.10Fn3 polypeptides
that bind to human PXR ligand binding domain (LBD). It also
provides binding characteristics of these 6 polypeptides as well as
the two that are described in Example 10 (and having SEQ ID NOs: 48
and 49).
[0333] .sup.10Fn3 polypeptides having the amino acid sequences set
forth in FIG. 10 were identified by screening various libraries.
For example, Adnectin-1 was isolated from an NP1 library (see FIG.
9C). The .sup.10Fn3 polypeptides were synthesized as follows.
Nucleic acids encoding the .sup.10Fn3 polypeptides were cloned in
pET9D vector and then expressed in Escherichia coli at 20.degree.
C. The lysates were purified in a single step using Ni-agarose
affinity chromatography.
[0334] The K.sub.D values for the .sup.10Fn3 polypeptides were
determined by surface Plasmon resonance (SPR) on a Biacore T100
instrument (GE Healthcare), by injecting a concentration series of
the .sup.10Fn3 polypeptides over human PXR-GST (Invitrogen) that
had been captured on chip immobilized with an anti-GST antibody (GE
Healthcare). Regeneration of the chip surface between kinetic
cycles was performed using 10 mM Glycine, pH 2. Kinetic parameters
for both were calculated using Biacore T100 software. The results
are set forth in Table 1.
TABLE-US-00003 TABLE 1 Binding characteristics of Adnectins-1 to -8
to human PXR Adnectin ka (1/Ms) kd (1/s) KD (nM) Adnectin- 1.04E+05
.+-. 1.20E+04 1.19E-03 .+-. 3.46E-05 11.4 .+-. 1 1 Adnectin-
1.62E+05 .+-. 5.37E+03 4.58E-03 .+-. 3.53E-04 97.8 .+-. 3.8 2
Adnectin- 1.62E+04 .+-. 6.75E+04 3.78E-04 .+-. 2.23E-05 2.5 .+-.
0.9 3 Adnectin- 2.15E+04 .+-. 5.30E+02 1.68E-04 7.8 4 Adnectin-
5.09E+05 .+-. 1.07E+05 2.70E-03 .+-. 2.36E-04 5.4 .+-. 0.7 5
Adnectin- 6.80E+05 .+-. 1.69E+05 1.26E-03 .+-. 1.72E-04 1.9 .+-.
0.2 6 Adnectin- 2.88E+05 .+-. 2.38E+04 8.82E-05 .+-. 1.15E-06 0.31
.+-. 0.03 7 Adnectin- 4.17E+04 .+-. 8.22E+03 1.82E-04 .+-. 2.78E-05
4.4 .+-. 0.2 8
Adnectin-1 was effectively used as a co-crystallization chaperone
of human PXR with a small molecule, and shown to bind to the ligand
binding domain of PXR. The X-ray data provided information on the
interaction between the small molecule and the PXR ligand binding
domain.
Example 13
Lack of RGD in FG Loop Prevents Binding of .sup.10Fn3 Polypeptides
to Fibronectin and Vitronectin
[0335] Recombinant human integrin .alpha.V.beta.3 (R&D Systems,
Minneapolis Minn.) was diluted to 40 ug/mL in Acetate buffer pH5.0
(GE Healthcare, Piscataway N.J.), and. then immobilized on a CM7
chip (GE Healthcare) using standard amine coupling techniques. 500
nM fibronectin (Roche Diagnostics, Indianapolis, Ind.) and
vitronectin (R&D Systems) and 5 .mu.M of either non-binding
control .sup.10Fn3 molecule (consisting of SEQ ID NO: 6 with an
additional MG at the N-terminus and with a single amino acid
substitution that changes RGD to RGE) or targeted .sup.10Fn3
molecules (having a mutated FG loop that does not contain an RGD
motif) were flowed over the top of the immobilized integrin.
Binding RU was collected at the end of the sample injection. The
results indicate that the lack of RGD in the FG loop results in
abolishing binding of .sup.10Fn3 molecules to fibronectin and
vitronectin.
Example 14
Characteristics of Molecules Obtained from Various Libraries
[0336] This Example shows various characteristics of molecules
obtained from 11 different libraries. The following libraries were
made: [0337] LI-1 library, which is a mixture of libraries LI-1
(a), LI-1(b) and LI-1(c), comprising .sup.10Fn3 molecules having
the amino acid sequences that are provided in FIG. 9B. The amino
acid residues that are underlined are those that were varied to any
amino acid by substitution. Residues that are underlined may be the
wild-type residues; [0338] LI-3 library comprising .sup.10Fn3
molecules having the amino acid sequence that is provided in FIG.
9B ("LI-3(b)), wherein the amino acid residues that are boxed were
varied by substitution to any amino acid or deletion or addition.
Residues that are boxed may be the wild-type residues; [0339]
WS-LI1 library, comprising .sup.10Fn3 molecules having the amino
acid sequence set forth in FIG. 9A, wherein the amino acid residues
that are boxed were varied by substitution to any amino acid or
deletion or addition. Residues that are boxed may be the wild-type
residues; [0340] LI-S9 library, comprising .sup.10Fn3 molecules
having the amino acid sequence set forth in FIG. 9A, wherein the
boxed residues were varied by amino acid substitution, addition or
deletion, the underlined residues were varied by substitution, and
the highlighted residue is changed to an E. Residues that are boxed
or underlined may be the wild-type residues; [0341] LI-S8 library,
comprising .sup.10Fn3n3 molecules having the amino acid sequence
set forth in FIG. 9A, wherein the boxed residues were varied by
substitution, addition or deletion, and the highlighted residue is
changed to an E. Residues that are boxed may be the wild-type
residues; [0342] NP4_FG library, comprising .sup.10Fn3 molecules
having the amino acid sequence set forth in FIG. 9C, wherein the
boxed residues were varied by amino acid substitution, addition or
deletion, and the underlined residues were varied by substitution.
Residues that are boxed or underlined may be the wild-type
residues. This library corresponds to the NP4 library, wherein only
residues in FG, F and G were varied. [0343] WS2'_CD library,
comprising .sup.10Fn3 molecules having the amino acid sequence set
forth in FIG. 9A, wherein the boxed residues were varied by amino
acid substitution, addition or deletion, and the underlined
residues were varied by substitution. Residues that are boxed or
underlined may be the wild-type residues. This library corresponds
to the WS2' library, wherein only residues in CD, C and D were
varied; [0344] NP1' library, comprising .sup.10Fn3 molecules having
the amino acid sequence set forth in FIG. 9C, wherein the boxed
residues were varied by amino acid substitution, addition or
deletion, and the underlined residues were varied by substitution.
Residues that are boxed or underlined may be the wild-type
residues; [0345] NP4 library, comprising .sup.10Fn3 molecules
having the amino acid sequence set forth in FIG. 9C, wherein the
boxed residues were varied by amino acid substitution, addition or
deletion, and the underlined residues were varied by substitution.
Residues that are boxed or underlined may be the wild-type
residues; and [0346] WS2' library, comprising .sup.10Fn3 molecules
having the amino acid sequence set forth in FIG. 9A, wherein the
boxed residues were varied by amino acid substitution, addition or
deletion, and the underlined residues were varied by substitution.
Residues that are boxed or underlined may be the wild-type
residues.
[0347] Clones from each of the libraries were expressed and
purified. The resulting .sup.10Fn3 molecules were run on the Perkin
Elmer LabChip GX with the low molecular weight protein ladder per
the manufacturers' recommendations. The concentrations of the
.sup.10Fn3 molecules were extrapolated from a standard curve of SGE
also run on the LabChip GX. Samples with concentrations higher than
that of the standard curve were classified as off-curve-high (OCH).
The percent of OCH wells and the average concentration for each
library was determined. The results indicate that the fraction of
OCH wells and average concentration of measurable samples follow
similar patterns.
[0348] The resistance to aggregation of .sup.10Fn3 molecules from
each of the libraries was also tested. Between 19-200 clones from
each library were assayed for aggregation and retention time by
size exclusion chromatography (SEC) according to manufacturers'
recommendations. The SEC chromatogram was integrated for each clone
and evaluated for peaks that eluted between the times of a 17 kDa
and 1.3 kDa standard. Clones having greater than 90% area in this
retention time window received an SEC score of 1, 50%-90% a score
of 2, 5%-50% a score of 3, and less than 5% a score of 4. Fraction
of SEC score 1 or 2 is measure of resistance to aggregation and
fraction SEC 4 is a measure of load on SEC screening. The results
indicate that fraction of SEC 1 or 2 and fraction of SEC 4 follow
approximately inverse patterns.
[0349] Target binding of clones from each library was determined by
ELISA. The proteins that the clones were directed against were
coated at 2.5 .mu.g/mL on a Nunc Maxisorp Plate. Plates were
blocked with Casein (ThermoScientific #37532) and then incubated
with the .sup.10Fn3 molecules. Binding clones were detected through
a His-tag using an HRP labeled anti-His antibody (R&D Systems
#mAb050H). Hits were determined as anything which gave a signal
greater than 2.times. that of blank wells. The fraction of hits is
a measure of load on screening. All libraries had at least an
expected number of hits.
[0350] The existence of multiple epitopes to which members of each
library bound was also determined. For three targets this involved
competitive Alphascreen assays as referenced in Example 7. For
these targets, multiple epitope coverage was proven by the
existence of competitors and non-competitors from proven target
binders. For one multi-domain target, multiple epitope binding was
demonstrated using an ELISA with recombinant versions of the
individual subdomains. The fraction of targets for which multiple
epitopes are found is a measure of probability of finding diverse
biological activity in a library. The results of multiple epitope
coverage for multiple targets with the different libraries indicate
diversity of blocking
Example 15
Library Selection of .sup.10Fn3 Molecules Having a Design in FIG.
13
[0351] This Example shows that .sup.10Fn3 molecules having certain
designs shown in FIG. 13 bind to their target protein.
[0352] Several libraries having designs referred to as LI-31,
LI-32, LI-38, LI-51, LI-52, LI-53 and LI-54 (see FIG. 13) were
selected for binding to a target protein using PROfusion in vitro
selection method. Several rounds of selection were carried out at
concentrations of target protein ranging from 1 nM to 100 nM.
.sup.10Fn3 molecules binding to the target at a concentration of
100, 10 or 1 nM were selected from each of these libraries. The
amino acid sequences of exemplary .sup.10Fn3 molecules that were
selected are shown in FIG. 14 (6xHis tag is not shown). Some of
these .sup.10Fn3 molecules were isolated in multiple copies. SEQ ID
NOs: 218-249 bound to the target at a concentration of 1 nM during
the selection.
[0353] As can be seen in FIG. 14, some of the .sup.10Fn3 molecules
have a design that differs slightly, e.g., by 1-3 amino acids, from
a design in FIG. 13. As these molecules bind the target protein,
these designs are also encompassed within the scope of the
application. In addition, .sup.10Fn3 molecules that are selected
from a library having a design set forth herein, e.g., in FIG. 13,
are also encompassed herein.
INCORPORATION BY REFERENCE
[0354] All documents and references, including patent documents and
websites, described herein are individually incorporated by
reference to into this document to the same extent as if there were
written in this document in full or in part. The teachings of
published application WO2013/067029 and provisional application
61/553,878, filed Oct. 31, 2011, to which WO2013/067029 claims
priority are also specifically incorporated by reference herein.
Sequence CWU 1
1
2821101PRTHomo sapiens 1Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90
95 Asp Lys Pro Ser Gln 100 285PRTHomo sapiens 2Leu Glu Val Val Ala
Ala Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp 1 5 10 15 Ala Pro Ala
Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr 20 25 30 Gly
Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser 35 40
45 Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr
50 55 60 Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser
Lys Pro 65 70 75 80 Ile Ser Ile Asn Tyr 85 3113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 3Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Gln
His Ser Lys Tyr Pro His Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ile Gln 65 70 75 80 Pro Gln Asp
Pro Glu Gln Asp Tyr Gln Tyr His Tyr Tyr Glu Thr Ser 85 90 95 Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser 100 105
110 Gln 4115PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 4Met Gly Val Ser Asp Val
Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr Tyr
Arg Ile Thr Tyr Gly Glu His Val Ala Asp His Phe Asp His 35 40 45
Asn Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser 50
55 60 Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val
Thr 65 70 75 80 Tyr Gln Phe Gln Asp Pro Glu Glu His Tyr Tyr Tyr His
Phe Tyr Asp 85 90 95 Ser Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg
Thr Glu Ile Asp Lys 100 105 110 Pro Ser Gln 115 5120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 5Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Tyr
His Glu His Tyr His Ser Pro 35 40 45 Gly Phe Ser Gln Lys Tyr His
Tyr Glu Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Gly His Lys His Tyr His Tyr Tyr 85 90 95 Tyr
Tyr Tyr His His His Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 100 105
110 Thr Glu Ile Asp Lys Pro Ser Gln 115 120 694PRTHomo sapiens 6Val
Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10
15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr
20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln
Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser
Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala
Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile
Ser Ile Asn Tyr Arg Thr 85 90 74PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Glu Ile Glu Lys 1 86PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 8Glu Ile Glu Lys Pro Cys 1 5 910PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 9Met Gly Val Ser Asp Val Pro Arg Asp Leu 1 5 10
108PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 10Val Ser Asp Val Pro Arg Asp Leu 1 5
119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 11Gly Val Ser Asp Val Pro Arg Asp Leu 1
5 1294PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 12Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp
Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val
Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Glu 65
70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85
90 13109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 13Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Ser Arg Pro Lys Gly Ser Ile Lys 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Asn Ser Gln Tyr Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Tyr
Gly Tyr 65 70 75 80 Pro Tyr Gln Glu Pro His Tyr Tyr Pro Ile Ser Ile
Asn Tyr Arg Thr 85 90 95 Glu Ile Asp Lys Pro Ser Gln His His His
His His His 100 105 14109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 14Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Thr Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Gly Pro
Lys Tyr Tyr Val Lys 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Arg Thr
Asp His Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Lys Gly Gln 65 70 75 80 Tyr Gly Pro
Tyr Tyr Gly Ser Arg Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln His His His His His His 100 105
15109PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 15Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Ser Gln Pro Gly Arg Tyr Val Lys 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Asn Asp Thr Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Tyr Gly Lys 65
70 75 80 Tyr Gly Pro Tyr Tyr Gly Tyr Asp Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 Glu Ile Asp Lys Pro Ser Gln His His His His His
His 100 105 169PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 16Met Gly Ser Asp Val Pro
Arg Asp Leu 1 5 178PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 17Met Gly Asp Val Pro Arg
Asp Leu 1 5 187PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 18Met Gly Val Pro Arg Asp
Leu 1 5 196PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 19Met Gly Pro Arg Asp Leu 1
5 205PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 20Met Gly Arg Asp Leu 1 5
214PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 21Met Gly Asp Leu 1 22166PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
22Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 20 25 30 Xaa Leu Leu Ile Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Arg Ile Thr Tyr Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Phe Thr Val Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ala Thr Ile Ser Gly Leu 100 105 110 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa
Xaa Xaa Xaa Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa 130 135
140 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile
145 150 155 160 Ser Ile Asn Tyr Arg Thr 165 235PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 23Glu Gly Ser Gly Cys 1 5 247PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 24Glu Ile Glu Lys Pro Cys Gln 1 5 257PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 25Glu Ile Glu Lys Pro Ser Gln 1 5 265PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 26Glu Ile Glu Lys Pro 1 5 276PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 27Glu Ile Glu Lys Pro Ser 1 5 285PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 28Glu Gly Ser Gly Ser 1 5 294PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 29Glu Ile Asp Lys 1 307PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 30Glu Ile Asp Lys Pro Ser Gln 1 5 317PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 31Glu Ile Asp Lys Pro Cys Gln 1 5 327PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 32Pro Ser Thr Ser Thr Ser Thr 1 5 333PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 33Gly Pro Gly 1 347PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 34Gly Pro Gly Pro Gly Pro Gly 1 5 3511PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 35Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 1 5 10
366PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 36Pro Ala Pro Ala Pro Ala 1 5
3712PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 37Pro Ala Pro Ala Pro Ala Pro Ala Pro
Ala Pro Ala 1 5 10 3818PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 38Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala
Pro Ala 1 5 10 15 Pro Ala 3910PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 39Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10
4020PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 40Gly Ser Gly Ser Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 Gly Ser Gly Ser 20
4115PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 41Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 1 5 10 15 4225PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25
4315PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 43Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Ser Gly 1 5 10 15 446PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
6xHis tag" 44His His His His His His 1 5 45116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 45Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Tyr
His Ala Phe Phe Ala Ser Asn 35 40 45 Gly Lys Tyr Tyr Phe Tyr Ile
Gln Glu Phe Thr Val Pro Gly Ser Lys 50 55 60 Ser Thr Ala Thr Ile
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile 65 70 75 80 Thr Val Tyr
Ala Val Thr Asp Asp Thr Val His His Gly Asp Ser Asn 85 90 95 Tyr
His Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp 100 105
110 Lys Pro Ser Gln 115 46111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 46Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Tyr
Ser Ser Phe Phe Gln His Gln 35 40 45 Gly Gln Tyr Tyr His Tyr Ile
Gln Glu Phe Thr Val Pro Gly Ser Lys 50 55 60 Ser Thr Ala Thr Ile
Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile 65 70 75 80 Thr Val Tyr
Ala Val Thr Gln His Glu His Ser Gln Asp Ser Ser Lys
85 90 95 Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser
Gln 100 105 110 47113PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 47Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Phe Ser Gln Phe Val His Ser Asp 35 40
45 Gly Glu Tyr Tyr Gln Glu Tyr Gln Glu Phe Thr Val Pro Gly Ser Lys
50 55 60 Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr
Thr Ile 65 70 75 80 Thr Val Tyr Ala Val Thr Gly Gln Tyr Asp Gln Asp
Asp Glu Pro Ser 85 90 95 Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr
Glu Ile Asp Lys Pro Ser 100 105 110 Gln 48105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 48Met Ala Ser Thr Ser Gly Val Ser Asp Val Pro Arg Asp
Leu Glu Val 1 5 10 15 Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Ala 20 25 30 Val Pro Val Ser Lys Tyr Val Ile Tyr
Tyr Trp Pro Gly Ala Leu Ile 35 40 45 Ser Ser Met Gln Ala Phe Lys
Val Pro Gly Ser Lys Ser Thr Ala Thr 50 55 60 Ile Ser Gly Leu Lys
Pro Gly Val Leu Tyr Ser Ile Val Val Asp Ala 65 70 75 80 Leu Thr Gly
Asp Gly Gln Gly Ser Tyr Val Trp Asp Pro Ile Thr Ile 85 90 95 Thr
Tyr Arg Thr Glu Gly Ser Gly Ser 100 105 49105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 49Met Ala Ser Thr Ser Gly Val Ser Asp Val Pro Arg Asp
Leu Glu Val 1 5 10 15 Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Ala 20 25 30 Val Thr Val His Ser Tyr Tyr Ile Thr
Tyr Gln Glu Leu Gln His His 35 40 45 Ser Val Pro Gln Gly Phe Gln
Val Pro Gly Ser Lys Ser Thr Ala Thr 50 55 60 Ile Ser Gly Leu Lys
Pro Gly Val Ala Tyr Gln Ile Ala Val Tyr Ala 65 70 75 80 Phe Thr Gly
Pro Gly Leu Pro Pro Ser Asp Ala Pro Pro Ile Val Ile 85 90 95 Tyr
Tyr Arg Thr Glu Gly Ser Gly Ser 100 105 506PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 50Met Ala Ser Thr Ser Gly 1 5 5120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 51Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala
Thr Ile 1 5 10 15 Ser Gly Leu Lys 20 5226PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 52Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro
Ala Ser 1 5 10 15 Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 20 25
5315PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 53Met Gly Glu Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile Ser 1 5 10 15 5419PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 54Pro His Phe Pro Thr Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu
Thr Gly 1 5 10 15 Gly Asn Ser 5523PRTHomo sapiens 55Pro Thr Ser Leu
Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg 1 5 10 15 Tyr Tyr
Arg Ile Thr Tyr Gly 20 5623PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 56Pro Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu Lys Val
Ala Arg 1 5 10 15 Tyr Tyr Arg Ile Thr Tyr Gly 20 5723PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 57Pro Thr Ser Leu Leu Ile Ser Trp Arg His Pro His Phe Pro
Thr Arg 1 5 10 15 Tyr Tyr Arg Ile Thr Tyr Gly 20 5823PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 58Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr
Val Arg 1 5 10 15 Tyr Tyr Arg Ile Thr Tyr Gly 20 59101PRTHomo
sapiens 59Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr
Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr
Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr
Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr Ile
Thr Val Tyr Ala Val Thr Gly Arg Gly Glu 65 70 75 80 Ser Pro Ala Ser
Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95 Asp Lys
Pro Ser Gln 100 6086PRTHomo sapiens 60Glu Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile Ser Trp Asp Ala 1 5 10 15 Pro Ala Val Thr Val
Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly 20 25 30 Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr 35 40 45 Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val 50 55
60 Tyr Ala Val Thr Gly Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile
65 70 75 80 Ser Ile Asn Tyr Arg Thr 85 6198PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 61Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala
Ala Thr Pro 1 5 10 15 Thr Ser Leu Leu Ile Ser Trp Ser Ala Arg Leu
Lys Val Ala Arg Tyr 20 25 30 Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu 35 40 45 Phe Thr Val Pro Lys Asn Val
Tyr Thr Ala Thr Ile Ser Gly Leu Lys 50 55 60 Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Arg Phe Arg 65 70 75 80 Asp Tyr Gln
Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro 85 90 95 Ser
Gln 62111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 62Met Ala Ser Thr Ser
Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val 1 5 10 15 Val Ala Ala
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala 20 25 30 Val
Pro Val Ser Lys Tyr Val Ile Tyr Tyr Trp Pro Gly Ala Leu Ile 35 40
45 Ser Ser Met Gln Ala Phe Lys Val Pro Gly Ser Lys Ser Thr Ala Thr
50 55 60 Ile Ser Gly Leu Lys Pro Gly Val Leu Tyr Ser Ile Val Val
Asp Ala 65 70 75 80 Leu Thr Gly Asp Gly Gln Gly Ser Tyr Val Trp Asp
Pro Ile Thr Ile 85 90 95 Thr Tyr Arg Thr Glu Gly Ser Gly Ser His
His His His His His 100 105 110 63111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 63Met Ala Ser Thr Ser Gly Val Ser Asp Val Pro Arg Asp
Leu Glu Val 1 5 10 15 Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Ala 20 25 30 Val Thr Val His Ser Tyr Tyr Ile Thr
Tyr Gln Glu Leu Gln His His 35 40 45 Ser Val Pro Gln Gly Phe Gln
Val Pro Gly Ser Lys Ser Thr Ala Thr 50 55 60 Ile Ser Gly Leu Lys
Pro Gly Val Ala Tyr Gln Ile Ala Val Tyr Ala 65 70 75 80 Phe Thr Gly
Pro Gly Leu Pro Pro Ser Asp Ala Pro Pro Ile Val Ile 85 90 95 Tyr
Tyr Arg Thr Glu Gly Ser Gly Ser His His His His His His 100 105 110
64114PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 64Met Ala Ser Thr Ser Gly Ser Thr
His Tyr Tyr Lys Gly Thr Ala Asp 1 5 10 15 Leu Glu Val Val Ala Ala
Thr Pro Thr Ser Leu Leu Ile Ser Trp Pro 20 25 30 Pro Pro Tyr Tyr
Val Glu Gly Val Thr Val Phe Arg Ile Thr Tyr Gly 35 40 45 Glu Thr
Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Tyr Trp 50 55 60
Thr Glu Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr 65
70 75 80 Ile Thr Val Tyr Ala Glu Met Tyr Pro Gly Ser Pro Trp Ala
Gly Gln 85 90 95 Val Met Asp Ile Gln Pro Ile Ser Ile Asn Tyr Arg
Thr Glu Gly Ser 100 105 110 Gly Ser 65105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 65Met Ala Ser Thr Ser Gly Val Ser Asp Val Pro Arg Asp
Leu Glu Val 1 5 10 15 Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Ala 20 25 30 Val Thr Val Asn Asn Tyr Arg Ile Thr
Tyr Gln Pro Leu Leu Gln Gly 35 40 45 Ser Ser Ile Gln His Phe Asp
Val Pro Gly Ser Lys Ser Thr Ala Thr 50 55 60 Ile Ser Gly Leu Lys
Pro Gly Val Gly Tyr Gln Ile Thr Val Tyr Ala 65 70 75 80 Ser Thr Tyr
Thr His Ser Lys Ala Tyr Tyr Ser Leu Pro Ile Ser Ile 85 90 95 Tyr
Tyr Arg Thr Glu Gly Ser Gly Ser 100 105 66103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 66Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Lys Tyr Pro
Tyr Glu Thr Ile Ser 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Tyr
Arg Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Glu Ala Ser 65 70 75 80 Ala Pro Tyr
Ser Asp Gly Gly Ser Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 67103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 67Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Ser Arg Pro
Lys Gly Ser Ile Lys 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Asn Ser
Gln Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Tyr Gly Tyr 65 70 75 80 Pro Tyr Gln
Glu Pro His Tyr Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 68103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 68Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Thr Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Gly Pro
Lys Tyr Tyr Val Lys 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Arg Thr
Asp His Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Lys Gly Gln 65 70 75 80 Tyr Gly Pro
Tyr Tyr Gly Ser Arg Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 69103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 69Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Ser Gln Pro
Gly Arg Tyr Val Lys 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Asn Asp
Thr Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Tyr Gly Lys 65 70 75 80 Tyr Gly Pro
Tyr Tyr Gly Tyr Asp Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 70120PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 70Met Ala Ser Thr Ser Gly Ser Thr His Tyr Tyr Lys Gly
Thr Ala Asp 1 5 10 15 Leu Glu Val Val Ala Ala Thr Pro Thr Ser Leu
Leu Ile Ser Trp Pro 20 25 30 Pro Pro Tyr Tyr Val Glu Gly Val Thr
Val Phe Arg Ile Thr Tyr Gly 35 40 45 Glu Thr Gly Gly Asn Ser Pro
Val Gln Glu Phe Thr Val Pro Tyr Trp 50 55 60 Thr Glu Thr Ala Thr
Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr Thr 65 70 75 80 Ile Thr Val
Tyr Ala Glu Met Tyr Pro Gly Ser Pro Trp Ala Gly Gln 85 90 95 Val
Met Asp Ile Gln Pro Ile Ser Ile Asn Tyr Arg Thr Glu Gly Ser 100 105
110 Gly Ser His His His His His His 115 120 71111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 71Met Ala Ser Thr Ser Gly Val Ser Asp Val Pro Arg Asp
Leu Glu Val 1 5 10 15 Val Ala Ala Thr Pro Thr Ser Leu Leu Ile Ser
Trp Asp Ala Pro Ala 20 25 30 Val Thr Val Asn Asn Tyr Arg Ile Thr
Tyr Gln Pro Leu Leu Gln Gly 35 40 45 Ser Ser Ile Gln His Phe Asp
Val Pro Gly Ser Lys Ser Thr Ala Thr 50 55 60 Ile Ser Gly Leu Lys
Pro Gly Val Gly Tyr Gln Ile Thr Val Tyr Ala 65 70 75 80 Ser Thr Tyr
Thr His Ser Lys Ala Tyr Tyr Ser Leu Pro Ile Ser Ile 85 90 95 Tyr
Tyr Arg Thr Glu Gly Ser Gly Ser His His His His His His 100 105 110
72109PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 72Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Lys Tyr Pro Tyr Glu Thr Ile Ser 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr Tyr Arg Ser
Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Glu Ala Ser 65 70 75 80 Ala Pro Tyr Ser Asp
Gly Gly Ser Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu Ile Asp
Lys Pro Ser Gln His His His His His His 100 105 7399PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 73Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 7499PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 74Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Asp Ala Pro Ala Val Xaa Xaa Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 7594PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 75Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Xaa Xaa Xaa Xaa
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 7699PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 76Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Xaa Xaa Xaa
Xaa Xaa Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 7799PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 77Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Asp Ala Pro Xaa Xaa Xaa Xaa Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 7899PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 78Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Xaa
Xaa Xaa Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 7999PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 79Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Asp Ala Xaa Xaa Xaa Xaa Xaa Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 8099PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 80Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Xaa Xaa
Xaa Xaa Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 8199PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 81Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Asp Ala Pro Ala Xaa Xaa Xaa Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 8292PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 82Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Xaa Xaa Xaa Xaa
Xaa Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Ser
Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 8392PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 83Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Xaa Xaa Xaa
Xaa Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Ser
Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 8492PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 84Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Xaa Xaa
Xaa Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Ser
Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 8599PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 85Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Xaa Xaa Xaa Xaa
Xaa Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 8699PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 86Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Asp Ala Xaa Xaa Xaa Xaa Val Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 8799PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 87Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Xaa Xaa
Xaa Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 88104PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 88Gly Val Ser Asp Val
Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Leu Xaa Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser
Trp Asp Xaa Xaa Xaa Xaa Xaa Val Arg Tyr Tyr Arg Ile Thr Tyr 35 40
45 Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly
50 55 60 Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val
Asp Tyr 65 70 75 80 Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Ser Xaa 85 90 95 Pro Ile Ser Ile Asn Tyr Arg Thr 100
89104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 89Gly Val Ser Asp Val Pro Gly Gly
Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala
Xaa Xaa Xaa Xaa Val Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60
Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65
70 75 80 Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ser Xaa 85 90 95 Pro Ile Ser Ile Asn Tyr Arg Thr 100
90104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 90Gly Val Ser Asp Val Pro Gly Gly
Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala
Pro Xaa Xaa Xaa Val Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60
Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65
70 75 80 Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Ser Xaa 85 90 95 Pro Ile Ser Ile Asn Tyr Arg Thr 100 91111PRTHomo
sapiensMOD_RES(11)..(19)Any amino acid and this region may
encompass 1-9 residues, wherein some positions may be absent 91Gly
Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile
20 25 30 Ser Trp Asp Xaa Xaa Xaa Xaa Xaa Val Arg Tyr Tyr Arg Ile
Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe
Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu
Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr Val Tyr Ala Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Ser Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 110 92111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 92Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Xaa Xaa Xaa Xaa Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90
95 Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100
105 110 93111PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 93Gly Val Ser Asp Val
Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa
Leu Xaa Val Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser
Trp Asp Ala Pro Xaa Xaa Xaa Val Arg Tyr Tyr Arg Ile Thr Tyr 35 40
45 Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly
50 55 60 Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val
Asp Tyr 65 70 75 80 Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser
Ile Asn Tyr Arg Thr 100 105 110 9494PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 94Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Xaa Xaa
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Ser Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 9594PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Xaa
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Ser Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 9694PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 96Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Ser Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90
97101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 97Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Xaa Xaa Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser 85 90 95 Ile Asn Tyr Arg Thr 100 98101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 98Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Xaa
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr 100 99101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 99Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr 100 100106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 100Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Xaa Xaa Xaa Xaa Xaa Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg Gly Glu Ser Pro Ala Ser 85 90 95 Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 101106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 101Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Xaa Xaa Xaa Xaa Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg Gly Glu Ser Pro Ala Ser 85 90 95 Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 102106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 102Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Pro Xaa Xaa Xaa Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg Gly Glu Ser Pro Ala Ser 85 90 95 Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 103106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 103Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg Gly Glu Ser Pro Ala Ser 85 90 95 Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 104106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 104Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Xaa Leu Xaa Ile 20 25 30 Xaa Trp Xaa Xaa Xaa Xaa Val Thr Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Gly Arg Gly Glu Ser Pro Ala Ser 85 90 95 Ser
Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 10596PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 105Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Xaa Xaa
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Glu Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
10696PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 106Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Xaa Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65
70 75 80 Gly Glu Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 10796PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 107Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Xaa Xaa Xaa Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Glu Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
10896PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 108Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65
70 75 80 Gly Glu Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 10996PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 109Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Xaa Leu Xaa Ile Xaa Trp Xaa Xaa Xaa
Xaa Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg 65 70 75 80 Gly Glu Ser
Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95
110111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 110Gly Val Ser Asp Val Pro Gly Gly
Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala
Pro Ala Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60
Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65
70 75 80 Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Ile Ser Ile Asn
Tyr Arg Thr 100 105 110 111111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 111Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Pro Ala Val Thr Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa
Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 110
112104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 112Gly Val Ser Asp Val Pro Gly Gly
Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala
Pro Ala Val Thr Val Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu
Thr Gly Gly Asn Ser Pro Val
Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala Thr Ile
Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr Val Tyr
Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa 85 90 95 Pro Ile Ser
Ile Asn Tyr Arg Thr 100 113111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 113Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Pro Ala Val Thr Val
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa
Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 110
114101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 114Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Ile Ser 85 90 95 Ile Asn Tyr Arg Thr 100 115101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 115Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr 100 11694PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 116Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Ser Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90
117101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 117Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Val Thr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Xaa Xaa Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser 85 90 95 Ile Asn Tyr Arg Thr 100 11899PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 118Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 11999PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 119Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Xaa Xaa Xaa Xaa Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 12099PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 120Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr 12199PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 121Val Ser Asp Val Pro
Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu
Ile Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Tyr Tyr 20 25 30 Arg
Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40
45 Thr Val Pro Xaa Xaa Xaa Xaa Thr Ala Thr Ile Ser Gly Leu Lys Pro
50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr 122111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 122Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Xaa Xaa Xaa Xaa Xaa
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa
Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 110
123111PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 123Gly Val Ser Asp Val Pro Gly Gly
Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val
Val Ala Ala Thr Pro Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala
Pro Xaa Xaa Xaa Xaa Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu
Thr Gly Gly Asn Ser Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60
Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65
70 75 80 Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg Thr 100 105 110 124111PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 124Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa Val Val Ala Ala Thr Pro
Thr Ser Leu Leu Ile 20 25 30 Ser Trp Asp Ala Pro Ala Xaa Xaa Xaa
Arg Tyr Tyr Arg Ile Thr Tyr 35 40 45 Gly Glu Thr Gly Gly Asn Ser
Pro Val Gln Glu Phe Thr Val Pro Gly 50 55 60 Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly Val Asp Tyr 65 70 75 80 Thr Ile Thr
Val Tyr Ala Val Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa
Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 100 105 110
125101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Xaa Xaa Xaa Xaa Xaa Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser 85 90 95 Ile Asn Tyr Arg Thr 100 126101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 126Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Xaa Xaa Xaa Xaa Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr 100 127101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 127Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Xaa Xaa Xaa Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr 100 12896PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 128Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Xaa Xaa
Xaa Xaa Xaa Xaa Tyr 20 25 30 Xaa Ile Xaa Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Xaa Phe 35 40 45 Xaa Val Xaa Xaa Xaa Xaa Xaa
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Ile Xaa Ile Asn Tyr Arg Thr 85 90 95
12996PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 129Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Xaa Leu Xaa Ile Xaa Trp
Xaa Xaa Xaa Xaa Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val
Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Ile Xaa Gly Leu Lys Pro 50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Xaa Xaa Xaa Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 13094PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 130Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Xaa Leu Xaa Ile Xaa Trp Xaa Xaa Xaa Xaa Val
Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Xaa Xaa Xaa Xaa Xaa
Xaa Ala Xaa Ile Xaa Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa
Xaa Ser Xaa Pro Ile Ser Ile Asn Tyr Arg Thr 85 90
131101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 131Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Xaa Leu Xaa Ile Xaa Trp
Xaa Xaa Xaa Xaa Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val
Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Ile Xaa Gly Leu Lys Pro 50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Xaa Xaa Xaa Xaa Xaa 65
70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Lys Pro
Ile Ser 85 90 95 Ile Asn Tyr Arg Thr 100 132100PRTArtificial
Sequencesource/note="Description of Artificial Sequence
Synthetic
polypeptide" 132Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala His
Ser Tyr Glu Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Pro Phe Pro Asp 65 70 75 80 Gly Pro His
Ser Glu Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp 85 90 95 Lys
Pro Ser Gln 100 133100PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 133Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Lys Ile Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Pro Phe Pro Asp 65 70 75 80 Gly Pro Ser
Ser Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp 85 90 95 Lys
Pro Ser Gln 100 134104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 134Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Thr Ser Trp Asp Ala Pro
Ala Gln Gly Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ala Tyr 65 70 75 80 His Phe Gly
His Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 135102PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 135Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Phe
Lys Gln Ser Ser Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Gly 65 70 75 80 Tyr Tyr Tyr
Leu Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu 85 90 95 Ile
Asp Lys Pro Ser Gln 100 136104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 136Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Tyr His
Gly His Gln Glu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His Gly 65 70 75 80 Tyr Tyr Lys
Tyr Ser Val Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 137104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 137Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Lys
Ile His Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala His Lys Ser Asp 65 70 75 80 His Phe Gly
His Tyr Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 138105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 138Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala His
His Glu Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala His Glu Ser Arg 65 70 75 80 Lys Phe Gly
His Tyr Thr Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 139106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 139Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Pro Glu His Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser Val 65 70 75 80 His Phe Gly
His Tyr Ser Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 140104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 140Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp His Pro
Ile Ser Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser His 65 70 75 80 Tyr Phe Gly
His Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 141105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 141Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Phe His
Asp Glu Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Gly Glu Ser 65 70 75 80 Phe Tyr Phe
Gly Ser Tyr Lys Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 142104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 142Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp His Gly
Phe Tyr His Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala His His Ser Tyr 65 70 75 80 Tyr Phe Gly
Lys Tyr Asp Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 143105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 143Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Glu Gly
Asp Pro Arg His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Gln Gln Ser 65 70 75 80 Tyr Tyr Phe
Gly His Phe Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 144104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 144Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Tyr
Gly Pro Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala His Gln Ser Phe 65 70 75 80 Ile Phe Gly
Asn Tyr Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 145104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 145Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Tyr Phe
His His Lys Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Ser Ser Tyr 65 70 75 80 His Phe Gly
His Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 146104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 146Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Ser Tyr Ala
Asp His His Gln Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65 70 75 80 Ile Phe Gly
His Tyr Thr Glu Glu Asp Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 147103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 147Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Pro Glu Ala
His His Ser Ser Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65 70 75 80 Tyr Phe Gly
His Tyr His His Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 148101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 148Met Gly Val Ser Asp Val Ala Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Leu His
Ala Tyr Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
His Thr Ile Thr Val Tyr Ala Val Thr Ser Phe 65 70 75 80 Tyr Phe Gly
His Tyr His Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95 Asp
Lys Pro Ser Gln 100 149107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 149Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Ile
His Tyr Phe Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Ala Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ala His 65 70 75 80 Tyr Tyr Tyr
Ser His Gln Gln Phe Lys Tyr Phe Tyr Pro Ile Ser Ile 85 90 95 Asn
Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 150106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 150Met Gly Val Ser Asp Val Pro Arg Asp Pro Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp His Tyr Glu
Gln Tyr Tyr Asp Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80 His Tyr Tyr
Ser Tyr Tyr Phe Asp Tyr His Ala Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 151109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 151Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp His Pro Gln
Tyr Lys His Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val
Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile
Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr
Ala Val Thr Lys Ser 65 70 75 80 Tyr Arg Gln His Gln Lys His Tyr Tyr
Arg Asp Arg Tyr His Pro Ile 85 90 95 Ser Ile Asn Tyr Arg Thr Glu
Ile Asp Lys Pro Ser Gln 100 105 152105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 152Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Phe Asn
Tyr Ile Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Ile Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Glu Tyr 65 70 75 80 Tyr Phe His
Arg Pro Gln His Tyr Tyr Asp Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 15397PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 153Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Glu His Phe
Ser Lys Glu Arg Tyr 20 25 30 Tyr Arg Ile Thr Tyr Gly Glu Thr Gly
Gly Asn Ser Pro Val Gln Glu 35 40 45 Phe Thr Val Pro Phe Phe His
Asn Thr Ala Thr Ile Ser Gly Leu Lys 50 55 60 Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Tyr His Tyr 65 70 75 80 Ile Asp Pro
Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser 85 90 95 Gln
154102PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 154Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Tyr Glu Glu Tyr Gln Gln Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr Tyr Asp Phe Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Gly 65
70 75 80 Tyr Tyr Gln Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg
Thr Glu 85 90 95 Ile Asp Lys Pro Ser Gln 100 155103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 155Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Gln Tyr
Asp Gly Thr Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Ser Tyr
Tyr Thr Ala Thr Ile Ser Gly Leu Lys 50 55 60 Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Glu Ala Ser 65 70 75 80 Ser Tyr Tyr
Tyr Gly Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 156105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 156Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Ile Pro Ile
Glu Ser Glu Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro His Ile
Phe Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gln Arg 65 70 75 80 Ser Tyr Tyr
Phe Gly Ser Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 157103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 157Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Phe Glu Tyr
Asp Tyr Thr Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr His
Tyr Phe Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His His 65 70 75 80 Val His Lys
Ser Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 158105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 158Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Thr His
Phe His Tyr Glu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Phe
Pro Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Asn 65 70 75 80 Arg Pro Leu
Tyr Lys Glu Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 159109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 159Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr His Glu
His His Tyr Ser Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Lys His
Lys Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80 Phe Ala Gly
Pro Pro Phe Pro Ser Phe Tyr Glu Ser Ser Lys Pro Ile 85 90 95 Ser
Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
160110PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 160Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Tyr His Tyr Gln Gln His His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro His Tyr Ser Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Val Lys 65
70 75 80 Tyr His Tyr His Gln Asp Glu Val Tyr Ile Lys Asp Ser Ser
Lys Pro 85 90 95 Ile Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro
Ser Gln 100 105 110 161105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 161Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp His Phe Tyr
His Tyr Lys Lys Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro His Ala
Tyr His Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Leu Tyr 65 70 75 80 Phe Ser Glu
His Tyr Asp Glu His Tyr Tyr Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 162106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 162Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Pro His
Glu Tyr His His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Ser Pro
Ser Thr Ala Thr Ile Ser Gly Leu Lys 50 55 60 Pro Gly Val Asp Tyr
Thr Ile Thr Val Tyr Ala Val Thr Tyr Phe Phe 65 70 75 80 Ser Gln Asp
Tyr Gln His Tyr His Gln Arg Glu Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 163106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 163Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Ser Phe
Tyr Ser Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Glu His
Gly Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Gln 65 70 75 80 Phe Tyr Thr
Pro Pro His Val His Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 164105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 164Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Lys Tyr Gln
His Ser Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Tyr
Asp His Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Phe Gln 65 70 75 80 Arg Gln Lys
Phe Asp Ser Glu His Ser Glu Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 165107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 165Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Lys Tyr Gly
Gln Tyr Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Arg His
Tyr Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His Asp 65 70 75 80 Lys Gly Lys
Tyr Phe Pro Tyr His Pro Glu Phe Pro Pro Ile Ser Ile 85 90 95 Asn
Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 166110PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 166Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp His Phe
Gln Tyr Phe Glu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Ile His
Pro Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His Lys 65 70 75 80 Pro Pro His
Phe His His Glu Tyr Asp Gly Glu Tyr Ser Ser Lys Pro 85 90 95 Ile
Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 110
167106PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 167Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Tyr Ser His Lys Tyr His Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Lys Tyr Tyr Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Thr Tyr 65
70 75 80 Gln His His His Asp Tyr Ser Gln Ser Ser Lys Pro Ile Ser
Ile Asn 85 90 95 Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
168104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 168Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Phe Lys Tyr Tyr Tyr Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr Glu Tyr Pro Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 Tyr Phe Gly Lys Ser Ile Val Tyr Gly Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
169106PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 169Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Tyr Lys Arg Tyr Ser Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Ala Ser Gly Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr His 65
70 75 80 Glu Lys Tyr Tyr His Thr Asp Phe Ile Tyr Tyr Pro Ile Ser
Ile Asn 85 90 95 Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
170110PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 170Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Gly Ser Ile Tyr Ile Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val
Gln 35 40 45 Glu Phe Thr Val Pro Ser Glu Val Asn Thr Ala Thr Ile
Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr
Ala Val Thr Phe Glu 65 70 75 80 Phe Phe Tyr His Pro Val Asp Tyr Ser
Asp Pro Tyr His Tyr Tyr Pro 85 90 95 Ile Ser Ile Asn Tyr Arg Thr
Glu Ile Asp Lys Pro Ser Gln 100 105 110 171105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 171Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ile Ile
Asp Tyr Phe Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80 Tyr Tyr Gln
Tyr Ser Tyr His Phe Asp Phe Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 172104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 172Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Ile
Glu Tyr His Thr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65 70 75 80 Tyr Phe Gly
His Tyr Lys Ser Gly Asp Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 173104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 173Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Tyr Gln Tyr Glu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65 70 75 80 Tyr Phe Gly
His Tyr His Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 174105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 174Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Ser
Tyr Lys Asp Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Thr Ser Tyr 65 70 75 80 Tyr Phe Gly
His Phe Ser Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 175104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 175Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Thr Glu
Phe His Lys Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Glu Glu Ser Phe 65 70 75 80 Tyr Phe Gly
Arg Tyr Glu Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 176104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 176Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Asp
Asp Lys Tyr Leu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Thr Ala Ser Tyr 65 70 75 80 Arg Phe Gly
His Tyr Glu Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 177105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 177Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Ile
Ser His Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala His Glu Ser Arg 65 70 75 80 His Phe Gly
His Tyr Asp Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 178108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 178Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Val
Gly Glu Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser Arg 65 70 75 80 His Phe Gly
Lys Tyr Ser Tyr Glu Tyr Phe Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
179105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 179Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Val Lys Tyr Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser Arg 65
70 75 80 Phe Phe Gly His Tyr Ser His Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
180105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 180Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Glu Pro Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Tyr Gln Ser Glu 65
70 75 80 Phe Phe Gly His Tyr Ser Gln Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
181104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 181Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Thr Ile His Asp Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Asp Val Asp Tyr Thr Ile Thr Val Tyr Ala His Gln Ser Ser 65
70 75 80 His Phe Gly His Tyr Gln Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
182105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 182Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp His Ser Thr His Glu Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Phe Glu Pro Ser 65
70 75 80 Gln His Phe Gly Lys Tyr Pro Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
183105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 183Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Pro Ser Gln Gln Asp Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala His Ser Gly Ser 65
70 75 80 Gln His Phe Gly His Phe Pro Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
184105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 184Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Tyr Pro Asn Gly Ser Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Phe Ala Pro Ser 65
70 75 80 Glu Arg Phe Gly His Phe Pro Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
185104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 185Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Phe Tyr Tyr Ala Lys His Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 His Phe Gly His Tyr Gln Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
186105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 186Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Tyr Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Ser 65
70 75 80 Tyr His Phe Gly His Tyr Pro Thr Gly Phe Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
187105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 187Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Tyr Gly Asp Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Lys Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala His Ser Tyr Ser 65
70 75 80 Tyr His Phe Gly Lys Tyr Pro Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
188107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 188Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Tyr Tyr Lys Phe Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Asp Tyr Tyr Val 65
70 75 80 Gly Tyr Ser Tyr Tyr Tyr Gly Ser His Ser Ser Lys Pro Ile
Ser Ile 85 90 95 Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105 189102PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 189Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Asp Asp Phe Tyr Tyr Lys Val Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Pro Phe Pro Asp 65 70 75 80 Gly Pro Leu Tyr Gly
Ser Asp Pro Ile Ser Ile Asn Tyr Arg Thr Glu 85 90 95 Ile Asp Lys
Pro Ser Gln 100 190105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 190Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Asp Gln
Asn Asp Phe Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser Tyr 65 70 75 80 Tyr Phe Gly
Asn Phe Tyr Phe Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 191103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 191Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Ala Asn Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gln Ser 65 70 75 80 Tyr Gly Tyr
Gln Ile Tyr Val Gln Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 192103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 192Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Pro Pro Gln His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Ser 65 70 75 80 Tyr Gly Tyr
Gln Ile Tyr Tyr Gln Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 193101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 193Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Arg Ser His
Pro Tyr Arg Glu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Glu Pro 65 70 75 80 Tyr Phe Lys
Gly Tyr Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95 Asp
Lys Pro Ser Gln 100 194108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 194Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp His Gly Gln
Asp Phe Pro Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gln His
Tyr Gln Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gln Ser 65 70 75 80 Phe Gly Asp
Gly Pro Tyr Tyr Gln Tyr His Tyr Gln Asp Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
195105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 195Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Lys Glu Gln Gln His Tyr Thr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr Pro Phe Gln Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ile Tyr 65
70 75 80 Gly Asp Gly Pro Tyr Tyr Gln Gly Tyr Tyr Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
196104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 196Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp His Pro Lys Pro Asp Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr Glu Tyr Pro Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 Tyr Phe Gly Lys Ser Ile Val Tyr Gly Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
197104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 197Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Phe His Arg Phe Ser Tyr His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Ala Asp His Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 His Phe Gly His Tyr Asp Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
198104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 198Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Pro Glu Asn Ser Asp Tyr Ser Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Ile Ser Tyr His Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 His Phe Gly Ser Tyr His Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Gly Ile Asp Lys Pro Ser Gln 100
199104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 199Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Thr Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp His Ser Pro Gln Gln Tyr Ser Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Asp His Phe Lys Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 Phe Phe Gly His Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
200104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 200Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Tyr His Gly Gln Gln His Pro Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr His Tyr Lys Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 Tyr Phe Gly His Tyr His Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
201105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 201Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Tyr Lys Tyr Lys Phe His Ser Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Lys Glu Ser Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gln Tyr 65
70 75 80 Tyr Tyr Glu His Phe Tyr His Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
202105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 202Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp His Asp Gln Gln Tyr His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Lys Tyr Asp Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Tyr 65
70 75 80 Phe Tyr Leu Ile Ser Gly Tyr Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
203105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 203Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Lys Tyr Pro Gly Glu Ala His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Ser His Glu His Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr His Tyr 65
70 75 80 Phe Gly His Ser Tyr Glu Tyr Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
204107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 204Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Tyr Glu Tyr Tyr Tyr Tyr Gln Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr Tyr His Pro Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr His Glu 65
70 75 80 Tyr Ser Ala His Ser His Glu Gln His Ser Ser Lys Pro Ile
Ser Ile 85 90 95 Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105 205106PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 205Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Tyr Asp Gln His Ser Tyr Phe Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Glu Phe Asp Phe Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Val Ser 65 70 75 80 Tyr Pro His Ser Glu His Tyr Asn Ser Ser Lys Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln
100 105 206104PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 206Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp His Glu Gly His His Tyr Asn Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Ser Gln His His Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Ser Tyr 65 70 75 80 His Phe Gly His Tyr Gln Ser Ser Lys Pro Ile Ser
Ile Asn Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
207104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 207Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Pro Arg Pro Glu His Tyr Gln Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Asp Pro His His Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 His Phe Gly His Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
208104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 208Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Ser His His Glu Lys Tyr Pro Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Pro Gln Val Phe Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70
75 80 His Phe Gly His Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg
Thr 85 90 95 Asp Tyr Lys Asp Asp Asp Asp Lys 100
209109PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 209Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Glu Tyr Glu Ala His Ser His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Tyr His Ser Glu Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Asp Phe 65
70 75 80 Ile Phe His Leu Tyr Tyr Tyr Phe Ser Tyr Gln Tyr Gly Thr
Pro Ile 85 90 95 Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser
Gln 100 105 210101PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 210Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Ser Tyr Ser Ser His Gln His Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Phe Pro His His Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Asp Ile 65 70 75 80 Glu Tyr Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr
Arg Thr Glu Ile 85 90 95 Asp Lys Pro Ser Gln 100
211107PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 211Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Ser Tyr His Tyr His Tyr Glu Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Tyr His Lys Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gln Ala 65
70 75 80 Pro Ile Phe Pro Phe Glu Tyr Pro Tyr Ser Tyr Tyr Pro Ile
Ser Ile 85 90 95 Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100
105 212106PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 212Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Tyr His His Tyr Tyr Tyr Tyr Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser His Val Gln 35 40
45 Glu Phe Thr Val Pro Thr Ser His Gln Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Lys His 65 70 75 80 Gln Tyr Tyr Tyr Tyr Asp Ser Gly Ser His Gln Pro
Ile Ser Ile Asn 85 90 95 Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln
100 105 213109PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 213Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Tyr Gly His Tyr Ser Ser Tyr Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Glu Gln Ser Pro Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Tyr Lys 65 70 75 80 Gln Gly His His Gln Asp Tyr Gln His Tyr Val Gly
His Gly Pro Ile 85 90 95 Ser Ile Asn Tyr Arg Thr Glu Ile Asp Lys
Pro Ser Gln 100 105 214105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 214Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Ser Tyr Pro
Tyr Thr Pro Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Gly
Glu Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65 70 75 80 His Phe Gly
Lys His Tyr Tyr Tyr His Phe Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 215102PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 215Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Pro Tyr Pro
Tyr Tyr Pro Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Ser
Pro Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Phe Tyr 65 70 75 80 Pro Pro Asp
Ser Pro Gly Ala Pro Ile Ser Ile Asn Tyr Arg Thr Glu 85 90 95 Ile
Asp Lys Pro Ser Gln 100 216107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 216Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Ser Tyr Tyr
Gly His Val Ala Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Glu Tyr
Tyr Gln Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His Asp 65 70 75 80 Tyr His Pro
Ala Val Pro Asp Ser Trp Leu Arg Leu Pro Ile Ser Ile 85 90 95 Asn
Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 217104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 217Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Gly Tyr Tyr
Tyr Glu Glu Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Glu Asp
Asp Ile Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Pro Phe 65 70 75 80 Lys Gln Gln
Pro Asp His Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 218108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 218Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Gly Trp Asp Ala Thr
Tyr Ser Ser Val Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Tyr Tyr Gly Tyr 65 70 75 80 Tyr Asn Ser
Tyr Pro Tyr Asp Asp Gln His Ser Ser Lys Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
219104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 219Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Lys Asn Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Gln Gln Ser Tyr 65
70 75 80 His Phe Gly His Tyr Glu Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
220104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 220Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ile Tyr Asp Asn Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Lys Tyr Ser Tyr 65
70 75 80 His Phe Gly His Tyr Tyr Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
221104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 221Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Val Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Tyr Tyr Gly Asp Lys Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala His Glu Ser Tyr 65
70 75 80 Gln Phe Gly His Tyr His Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
222104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 222Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Tyr Pro Gln His His Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala His Glu Ser Tyr 65
70 75 80 Lys Phe Gly His Tyr Gln Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
223104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 223Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Glu Gln Gln Glu Tyr His His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Lys His Ser His 65
70 75 80 Lys Phe Gly Ser Tyr Glu Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
224105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 224Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala His Tyr Gly Tyr Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser Tyr 65
70 75 80 Lys Tyr Gly Ser Tyr Thr Thr Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
225105PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 225Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Tyr Thr Gln Ser Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Tyr Glu Ser His 65
70 75 80 His Phe Gly His Tyr Thr Glu Ser Ser Lys Pro Ile Ser Ile
Asn Tyr 85 90 95 Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
226104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 226Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Phe Tyr Gln Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 Tyr Phe Gly His Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn
Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
227104PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 227Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Thr Ala Tyr Arg Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 His Phe Gly His Tyr His Ser Ser Lys Pro Ile Ser
Ile Asn Tyr Arg 85 90 95 Thr Glu Ile Asp Lys Pro Ser Gln 100
228103PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 228Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Asp Tyr Lys Val Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Tyr 65
70 75 80 Tyr Phe Gly His Tyr His Gln Leu Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 Glu Ile Asp Lys Pro Ser Gln 100
229101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 229Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Tyr Tyr Pro Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Phe 65
70 75 80 Tyr Phe Gly Ser Tyr Thr Pro Ile Ser Ile Asn Tyr Arg Thr
Glu Ile 85 90 95 Asp Lys Pro Ser Gln 100 230108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 230Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Gly His
Tyr Ser Ser Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Pro Tyr 65 70 75 80 Ser His Glu
Glu Tyr Tyr Thr Ser Tyr Tyr His Asp Tyr Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
231102PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 231Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Asp Ala Pro Ala Ile Ser Tyr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Glu His 65
70 75 80 Tyr Tyr Ser Asp Tyr Asn Tyr Pro Ile Ser Ile Asn Tyr Arg
Thr Glu 85 90 95 Ile Asp Lys Pro Ser Gln 100 232103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 232Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Glu
Tyr Tyr Gln Phe Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Lys Ser 65 70 75 80 Tyr Gly Tyr
Gln Ile Tyr Ile Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 233103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 233Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Glu His Gln
Phe Tyr Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Lys Ser 65 70 75 80 Tyr Gly Tyr
Gln Ile Tyr Ile Ser Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 234107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 234Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro
Lys Tyr His Gln Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asn
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Gly 65 70 75 80 Tyr Tyr Lys
His Thr Val Tyr Gly Tyr Phe Tyr Tyr Pro Ile Ser Ile 85 90 95 Asn
Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 235108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 235Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Lys Val Ser
His Glu Tyr Glu Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Ser
Lys Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His Gln 65 70 75 80 Gln Thr Tyr
Gln Asp Gln Ser Asn Lys His His Ile Tyr Pro Ile Ser 85 90 95 Ile
Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105
236101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 236Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Ser His His Lys Tyr Gly His Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Asp Tyr Glu Ile Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Pro 65
70 75 80 Tyr Gly His Gly His Tyr Pro Ile Ser Ile Asn Tyr Arg Thr
Glu Ile 85 90 95 Asp Lys Pro Ser Gln 100 237101PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 237Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp His Glu Gly
His Glu His Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Glu His
Arg Val Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr Tyr 65 70 75 80 Tyr His Lys
Gln His Tyr Pro Ile Ser Ile Asn Tyr Arg Thr Glu Ile 85 90 95 Asp
Lys Pro Ser Gln 100 238104PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 238Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr His His
Lys His His Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Gln
Thr Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Phe 65 70 75 80 His Phe Gly
His Phe Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg 85 90 95 Thr
Glu Ile Asp Lys Pro Ser Gln 100 239105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 239Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Tyr Tyr His
Lys Gln His Gly Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Gly
Gly Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Phe Pro 65 70 75 80 Leu His Tyr
Gly Glu Tyr Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 240106PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 240Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Lys Tyr Leu
Lys Ala Tyr Phe Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Ala Tyr
Phe Lys Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Tyr His 65 70 75 80 Tyr Ile Asp
Tyr Tyr Glu Gln Tyr Ser Ser Lys Pro Ile Ser Ile Asn 85 90 95 Tyr
Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 241102PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 241Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Val Phe Tyr
Leu His Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro His Glu
Thr Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr His Gly 65 70 75 80 Glu Tyr Glu
Tyr Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr Glu 85 90 95 Ile
Asp Lys Pro Ser Gln 100 242103PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 242Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Gln Asp Tyr
Glu His Tyr Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Glu Glu
Tyr Glu Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Gln Gln 65 70 75 80 Thr Glu Val
His Tyr Tyr Tyr Tyr Pro Ile Ser Ile Asn Tyr Arg Thr 85 90 95 Glu
Ile Asp Lys Pro Ser Gln 100 243105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 243Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Phe Ser Tyr
Tyr Ser Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Leu Ser
Glu Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Phe Lys 65 70 75 80 Ala Asp Phe
Val Lys Tyr Ser Ser Ser Lys Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 244107PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 244Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Asp Phe Tyr
Leu Thr Tyr His Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Tyr Val
Glu Glu Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Leu 65 70 75 80 Tyr Tyr Gly
Thr Tyr Ser His Glu Asp Tyr His Tyr Pro Ile Ser Ile 85 90 95 Asn
Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln 100 105 245105PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 245Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Gly Ser Asn
Glu Leu His Gln Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Gly Phe
Gly Asp Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser Gln 65 70 75 80 Tyr Phe Gly
His Tyr Gly Gln Tyr His Asp Pro Ile Ser Ile Asn Tyr 85 90 95 Arg
Thr Glu Ile Asp Lys Pro Ser Gln 100 105 246109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 246Met Gly Val Ser Asp Val Pro Arg Asp Leu Glu Val Val
Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile Ser Trp Gln Ser Tyr
Ser Tyr Glu Tyr Arg 20 25 30 Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr
Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe Thr Val Pro Asn Phe
Glu His Thr Ala Thr Ile Ser Gly Leu 50 55 60 Lys Pro Gly Val Asp
Tyr Thr Ile Thr Val Tyr Ala Val Thr Glu His 65 70 75 80 Arg Thr Tyr
Phe Ser Glu Lys Phe His Leu His Ser Ser Lys Pro Ile 85 90 95 Ser
Ile Asn Tyr Arg Thr Glu Ile Asp Lys Pro Ser Gln
100 105 247103PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 247Met Gly Val Ser Asp
Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser
Leu Leu Ile Ser Trp Tyr Gln Leu Gln Glu Leu Tyr Arg 20 25 30 Tyr
Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40
45 Glu Phe Thr Val Pro Arg Glu Val Tyr Thr Ala Thr Ile Ser Gly Leu
50 55 60 Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr
Ser Tyr 65 70 75 80 Lys Phe Gly Lys Ser His Ile His Pro Ile Ser Ile
Asn Tyr Arg Thr 85 90 95 Glu Ile Asp Lys Pro Ser Gln 100
248103PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 248Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp His Gln Gln Gln Gly Tyr Glu Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Ser Tyr Asp Ser Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser His 65
70 75 80 His Phe Gly His Tyr Ser Glu Tyr Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 Glu Ile Asp Lys Pro Ser Gln 100
249103PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 249Met Gly Val Ser Asp Val Pro Arg
Asp Leu Glu Val Val Ala Ala Thr 1 5 10 15 Pro Thr Ser Leu Leu Ile
Ser Trp Phe Asn Lys Asp Lys Tyr Thr Arg 20 25 30 Tyr Tyr Arg Ile
Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln 35 40 45 Glu Phe
Thr Val Pro Asn Gln His Tyr Thr Ala Thr Ile Ser Gly Leu 50 55 60
Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Ser His 65
70 75 80 His Phe Gly His Tyr Ser Glu Tyr Pro Ile Ser Ile Asn Tyr
Arg Thr 85 90 95 Glu Ile Asp Lys Pro Ser Gln 100 25010PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 250Gly Val Ser Asp Val Pro Gly Gly Ser Gly 1 5 10
25112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 251Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Leu Xaa 1 5 10 25213PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 252Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5
10 25314PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 253Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5 10 25415PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 254Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu
Xaa 1 5 10 15 25516PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 255Pro Gly Gly Ser Gly
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5 10 15
25617PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 256Val Pro Gly Gly Ser Gly Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu 1 5 10 15 Xaa 25718PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 257Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Leu Xaa 25819PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 258Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Leu Xaa 25920PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 259Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Leu Xaa 20 26021PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 260Gly Val Ser Asp Val Pro Gly Gly Ser Gly Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Leu Xaa 20 26113PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 261Gly Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5
10 26214PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 262Gly Val Ser Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5 10 26315PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 263Gly Val Ser Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu
Xaa 1 5 10 15 26416PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 264Gly Val Ser Asp Val
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5 10 15
26517PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 265Gly Val Ser Asp Val Pro Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Leu 1 5 10 15 Xaa 26618PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 266Gly Val Ser Asp Val Pro Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Leu Xaa 26719PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 267Gly Val Ser Asp Val Pro Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Leu Xaa 26820PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 268Gly Val Ser Asp Val Pro Gly Gly Ser Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Leu Xaa 20 2699PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 269Val Ser Asp Val Pro Gly Gly Ser Gly 1 5
2708PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 270Ser Asp Val Pro Gly Gly Ser Gly 1 5
2717PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 271Asp Val Pro Gly Gly Ser Gly 1 5
2726PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 272Val Pro Gly Gly Ser Gly 1 5
2735PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 273Pro Gly Gly Ser Gly 1 5
2744PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 274Gly Gly Ser Gly 1 2759PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 275Gly Val Ser Asp Val Pro Gly Gly Ser 1 5
2768PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 276Gly Val Ser Asp Val Pro Gly Gly 1 5
2777PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 277Gly Val Ser Asp Val Pro Gly 1 5
2786PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 278Gly Val Ser Asp Val Pro 1 5
2795PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 279Gly Val Ser Asp Val 1 5
2804PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 280Gly Val Ser Asp 1
281101PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 281Val Ser Asp Val Pro Arg Asp Leu
Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp
Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr
Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val
Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60
Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65
70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr
Glu Ile 85 90 95 Asp Lys Pro Ser Gln 100 28294PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 282Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala
Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly
Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser
Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr
Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala
Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90
* * * * *